LYSYL OXIDASESCloning and characterization of the fourth and the fifth human lysyl oxidase isoenzymes, and the consequences of a targeted inactivation of the first described lysyl oxidase isoenzyme in mice

JONIMÄKI

Collagen Research Unit,Biocenter Oulu and

Department of Medical Biochemistry andMolecular Biology,University of Oulu

OULU 2002

JONI MÄKI

LYSYL OXIDASESCloning and characterization of the fourth and the fifth human lysyl oxidase isoenzymes, and the consequences of a targeted inactivation of the first described lysyl oxidase isoenzyme in mice

Academic Dissertation to be presented with the assent ofthe Faculty of Medicine, University of Oulu, for publicdiscussion in the Auditorium L101 of the Department ofMedical Biochemistry, on July 5th, 2002, at 13 noon.

OULUN YLIOPISTO, OULU 2002

Copyright © 2002University of Oulu, 2002

Reviewed byAssociate Professor Helena KuivaniemiAssociate Professor Philip C. Trackman

ISBN 951-42-6739-7 (URL: http://herkules.oulu.fi/isbn9514267397/)

ALSO AVAILABLE IN PRINTED FORMATActa Univ. Oul. D 682, 2002ISBN 951-42-6738-9ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/)

OULU UNIVERSITY PRESSOULU 2002

Mäki, Joni, Lysyl oxidases Cloning and characterization of the fourth and the fifthhuman lysyl oxidase isoenzymes, and the consequences of a targeted inactivation ofthe first described lysyl oxidase isoenzyme in miceCollagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and MolecularBiology, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland Oulu, Finland2002

Abstract

Lysyl oxidases (EC 1.4.3.13, protein-lysine 6-oxidases) are extracellular copper enzymes that initiatethe cross-linking of collagens and elastin by catalyzing oxidative deamination of the ε-amino groupin certain lysine and hydroxylysine residues. The cross-links formed are responsible for the tensilestrength of collagen fibers and the unique elastic properties of elastin.

Three human lysyl oxidase isoenzymes, lysyl oxidase (LOX), lysyl oxidase-like protein (LOXL),and lysyl oxidase-like 2 protein (LOXL2), have been identified and characterized so far. Twoadditional human lysyl oxidase isoenzymes, lysyl oxidase-like 3 (LOXL3) and lysyl oxidase-like 4(LOXL4), proteins were identified, cloned, and partially characterized in this study. Bothpolypeptides showed a high degree of overall similarity to each other and to the LOXL2 polypeptide,whereas the two polypeptides showed a significant similarity to LOX and LOXL only in the C-terminal region, which contains all amino acid residues thought to be needed for the catalytic activityof the LOX enzyme. The LOXL3 gene is expressed in several tissues, the highest expression levelsbeing in the placenta, heart, ovary, testis, small intestine, and spleen. The LOXL4 gene is likewiseexpressed in most human tissues studied, the highest levels being seen in the skeletal muscle, testis,and pancreas. Both polypeptides were shown to be secreted extracellular proteins.

The role of the first described LOX isoenzyme was studied by inactivating its gene in mice. MostLox-/- embryos died at the end of gestation, and the few live-born pups were cyanotic and died withina few hours, autopsy revealing large aortic aneurysms. Light microscopy demonstrated structuralabnormalities in the aortic walls of Lox-/- embryos, and further analysis by electron microscopyshowed highly fragmented elastic fibers, discontinuity in the smooth muscle cell layers, andendothelial cell damage. Doppler ultrasonography of Lox-/- embryos in utero revealed multiple signsof cardiovascular dysfunction, which contributed to the early death of the Lox-/- mice. The resultsindicate that Lox has an essential role in the development and function of the cardiovascular systemand that this role cannot be replaced to any significant extent by other lysyl oxidase isoenzymes.

Keywords: collagen, connective tissue, copper enzyme, elastin, cardiovascular system

To Michaela

Acknowledgements

This work was carried out at the Collagen Research Unit and the Department of Medical Biochemistry and Molecular Biology, University of Oulu, and Biocenter Oulu during the years 1994-2002.

I wish to express my deepest gratitude to my supervisor, Proferssor of the Academy of Finland Kari Kivirikko, for his inspiring attitude, never-ending optimism and continuous encouragement throughout this work. I would like to express my sincere thanks to Professor Taina Pihlajaniemi for providing excellent research facilities during these years, and to Professor Ilmo Hassinen for the stimulating atmosphere in our Department. Docent Johanna Myllyharju deserves my warmest thanks for her helpful advice and the never-ending optimism that she has shown towards my work in these years. I am also truly grateful to Docent Leena Ala-Kokko for her optimism towards my work, and for the opporturnity to spend ‘some serious quality time’ in Philadelphia during the summer of 1998.

Associate Professors Helena Kuivaniemi and Philip Trackman are acknowledged for their thorough reviewing and valuable comments on the manuscript of this thesis.

My kindest thanks belongs to my colleague, Hilkka Tikkanen, for her valuable contribution to this study. I would like to express my thanks to Eija-Riitta Hämäläinen and Ritva Kemppainen for their advice during my first steps in the Department. I wish to thank my co-authors Raija Soininen and Docent Juha Räsänen for their extremely valuable contribution to the last manuscript in this thesis, and their helpful advice in field of research. I also would like to thank my other co-authors Docent Raija Sormunen and Kaarin Mäkikallio for their essential contribution to this work.

I wish to express my most warmest thanks to Lauri Eklund and Harri Elamaa for their friendship, support, and many unforgettable moments both at work and outside the lab for the past ten years. Anne Latvanlehto and Jaana Väisänen are acknowledged for all their support and for all those scientific and non-scientific discussions during these years. I also want to thank Merja Nieminen, Kati Rautavuoma, Laura Silvennoinen, and Ari-Pekka Kvist for the support they have shown to me. I wish to acknowledge Ritva Nissi and Jussi Vuoristo for the cheerful, and sometimes even desperate, moments during this work and when organizing the dissertation party. Most of all, every single member of our

group deserves my warmest thanks for creating a good scientific, but also quite relaxing, atmosphere in our Department.

I wish to thank the staff of the Department, especially Riitta Polojärvi and Jaana Jurvansuu, for their excellent and thorough technical assistance at all stages during this work, Juha Näpänkangas for helping with computers, and Pertti Vuokila, Marja-Leena Kivelä, Auli Kinnunen, and Seppo Lähdesmäki for their kind help in many problems during these years. I would also like to thank Sandra Barkoczy for the careful revision of the language of this thesis.

I owe my greatest gratitude to my parents, Marja-Leena and Tuomo, and to my brother Jari, for the support and love that they have always shown to me. I also wish to thank all my friends outside the lab, especially Maarit Mäkitalo, who always has such a positive attitude towards life. I want to express my special thanks to the members of the Canyon band, who have made it possible to share and do the most pleasurable and relaxing thing in life – music.

Finally, and above all, I owe my most sincere thanks to my wife Michaela for her never-ending support. The true spirit and force of my life has always been your love and friendship, and will always be. I dedicate this thesis to you.

This work has been supported by the Finnish Cultural Foundation and the Finnish Center of Excellence Programme (2000-2005) of the Academy of Finland.

Oulu, May 2002 Joni Mäki

Abbreviations

ATP7A Menkes disease gene

βAPN β-aminopropionitrile

BMP-1 bone morphogenetic protein-1

bp basepair(s)

C carboxy

cDNA complementary deoxyribonucleic acid

ES embryonic stem cell

kb kilobase(s)

kDa kilodalton(s)

LO lysyl oxidase

LOX the first described lysyl oxidase enzyme

LOXL lysyl oxidase-like protein

LTQ lysine tyrosylquinone

mRNA messenger RNA

N amino

OHS occipital horn syndrome

PCR polymerase chain reaction

rrg ras recision gene

TGF-β1 transforming growth factor-β 1

SRCR scavenger receptor cysteine-rich region

List of orginal articles

This thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I Mäki JM & Kivirikko KI (2001) Cloning and characterization of a fourth human lysyl oxidase isoenzyme. Biochem J 355: 381-387.

II Mäki JM, Tikkanen H & Kivirikko KI (2001) Cloning and characterization of a fifth human lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains. Matrix Biol 20: 493-496.

III Mäki JM, Räsänen J, Tikkanen H, Sormunen R, Mäkikallio K, Kivirikko KI & Soininen R (2002) Inactivation of the lysyl oxidase gene leads to aortic aneurysms, cardiovascular dysfunction and perinatal death in mice. Submitted for publication.

Contents

Acknowledgements Abbreviations List of orginal articles Contents 1 Introduction................................................................................................................... 15 2 Review of the literature................................................................................................. 16

2.1 Collagens and elastin ............................................................................................. 16 2.1.1 The collagen family of proteins ...................................................................... 16 2.1.2 Elastin ............................................................................................................. 17

2.2 Cross-links in collagens and elastin ....................................................................... 18 2.3 The lysyl oxidase isoenzyme LOX ........................................................................ 20

2.3.1 Molecular properties ....................................................................................... 20 2.3.1.1 Biosynthesis and processing of the LOX precursor ................................. 21

2.3.2 Catalytic properties ......................................................................................... 23 2.3.2.1 Cofactors .................................................................................................. 23 2.3.2.2 Reaction mechanism ................................................................................ 24 2.3.2.3 Inhibitors .................................................................................................. 25

2.3.3 Regulation of LOX ......................................................................................... 26 2.3.3.1 Expression in different cell types and during development ..................... 26 2.3.3.2 LOX in fibrotic tissues ............................................................................. 27 2.3.3.3 Response to different effectors in various cell types ................................ 28

2.3.4 Novel biological roles ..................................................................................... 31 2.3.4.1 LOX in tumor suppression ....................................................................... 31 2.3.4.2 Intracellular and intranuclear activities .................................................... 32

2.4 Recently identified lysyl oxidase isoenzymes........................................................ 33 2.4.1 Lysyl oxidase-like protein (LOXL)................................................................. 33 2.4.2 Lysyl oxidase-like 2 protein (LOXL2)............................................................ 34

2.5 Consequences of reduced lysyl oxidase activities.................................................. 35 2.5.1 Nutritional copper deficiency.......................................................................... 35 2.5.2 Menkes disease and occipital horn syndrome ................................................. 36

2.5.2.1 Clinical, biochemical and pathological findings ...................................... 36

2.5.2.2 Animal models of Menkes disease and occipital horn syndrome............. 37 2.5.3 Lathyrism ........................................................................................................ 37

3 Outlines of the present research .................................................................................... 38 4 Materials and methods .................................................................................................. 39

4.1 Cloning and characterization of the fourth and the fifth human lysyl oxidase isoenzymes (I, II) ......................................................................................................... 39

4.1.1 Search for expressed sequence tags and high throughput genomic sequences (I, II)........................................................................................................ 39 4.1.2 Isolation of cDNA clones (I, II) ...................................................................... 39 4.1.3 Characterization of the exon-intron organization of the LOXL3 gene (I)....... 40 4.1.4 Northern blot analysis (I, II) ........................................................................... 40 4.1.5 Sequence analyses (I-III) ................................................................................ 40 4.1.6 Recombinant expression of the LOXL3 and LOXL4 polypeptides (I, II) ...... 41 4.1.7 Immunofluorescence staining of the recombinant LOXL3 protein (I)............ 41

4.2 Generation and analysis of a mouse strain lacking the Lox gene (III) ................... 42 4.2.1 Generation of the mutant mice (III) ................................................................ 42 4.2.2 Light microscopy (III)..................................................................................... 42 4.2.3 Transmission electron microscopy (III) .......................................................... 43 4.2.4 Ultrasonographic examination of the embryos (III)........................................ 43

5 Results........................................................................................................................... 44 5.1 The fourth and the fifth lysyl oxidase isoenzymes (I, II) ....................................... 44

5.1.1 Molecular cloning of the cDNAs (I, II)........................................................... 44 5.1.2 Molecular characterization of the cDNAs (I, II) ............................................. 45 5.1.3 Exon-intron organization of the LOXL3 gene (I) ........................................... 46 5.1.4 Expression of the mRNAs in various human tissues (I, II)............................. 46 5.1.5 Recombinant expression of the LOXL3 and LOXL4 polypeptides in human HT-1080 cells (I, II) ................................................................................................. 47

5.2 Inactivation of the Lox gene leads to cardiovascular dysfunction in mice (III) ..... 47 5.2.1 Generation of mice with an inactivated Lox gene (III) ................................... 47 5.2.2 Histological findings (III) ............................................................................... 48 5.2.3 Ultrasonographic findings (III) ....................................................................... 48

6 Discussion..................................................................................................................... 50 6.1 Cloning and characterization of the fourth and the fifth lysyl oxidase isoenzymes (I, II) ......................................................................................................... 50

6.1.1 Primary structures of the LOXL3 and LOXL4 polypeptides (I, II) ................ 50 6.1.2 The structure of the LOXL3 gene, and expression of LOXL3 and LOXL4 mRNAs in various tissues (I, II) .............................................................................. 51 6.1.3 Recombinant expression of the LOXL3 and LOXL4 polypeptides (I, II) ...... 52

6.2 Lack of Lox activity leads to a severe cardiovascular dysfunction and perinatal death in mice (III) ........................................................................................................ 53

7 Future perspectives ....................................................................................................... 57 References..................................................................................................................... 58

1 Introduction

Cells are the basic units of all living organisms. In multicellular organisms, cells are surrounded by several structural and functional proteins which are arranged as a large heterogenous network, the extracellular matrix. It consists of numerous compounds including insoluble fibers, microfibrils, soluble proteins, and glycoproteins. The extracellular matrix is essential for providing mechanical and physiological properties for various tissues, and it also provides attachment sites for the anchoring of cells and support for their migration in tissues.

Collagens are the most abundant proteins in the human body and they provide most of the tensile strength needed in several tissues. However, proper functioning of numerous tissues such as the skin, lungs, and vessels also requires elastic properties. This resiliency is provided mainly by elastic fibers, in which the main component is a protein called elastin. The mechanical and physiological properties of the insoluble fibers formed by the two proteins, collagens and elastin, are highly dependent on covalent cross-links, which stabilize their fibrillar structures and enhance their resistance to proteolysis. These cross-links are the final products of a reaction which is initiated by the oxidation of certain lysine and hydroxylysine residues. This reaction is accomplished by extracellular copper-dependent enzymes, lysyl oxidases.

In the present study, two of the five known human lysyl oxidase isoenzymes were identified and partially characterized at the molecular level. These new enzymes were named lysyl oxidase-like 3 protein (LOXL3) and lysyl oxidase-like 4 protein (LOXL4) based on their high degree of similarity to the already known three lysyl oxidase isoenzymes. The LOXL3 and LOXL4 gene products were shown to be secreted extracellular proteins. In addition to these new isoenzymes, mice lacking the first described lysyl oxidase isoenzyme (Lox) were generated to obtain information on the function of this isoenzyme. Lox was found to be essential for proper cardiovascular function, and depletion of its activity resulted in perinatal death in this mouse model.

2 Review of the literature

2.1 Collagens and elastin

2.1.1 The collagen family of proteins

Collagens form a multigene family with at least 39 members, the genes for which are known to be dispersed throughout at least 15 chromosomes. Collagens are structural proteins of the extracellular matrix, and are the most abundant proteins in the mammalian body. Collagen molecules consist of three identical or dissimilar polypeptide chains, called α chains, and contain at least one triple-helical collagenous domain with repeating (Gly-X-Y)n sequences, i.e. a glycine residue as every third amino acid, with the frequent presence of proline and 4-hydroxyproline in the X and Y positions, respectively. In addition to the actual collagenous domains, all collagens also contain noncollagenous domains. Most collagen molecules form supramolecular assemblies, and the superfamily can be divided into nine different families based on their polymeric structures or other features: collagens that forms fibrils (types I-III, V and XI); collagens that are located on the surface of fibrils and are called fibril-associated collagens with interupted triple helices (FACIT and structurally related collagens, types IX, XII, XIV, XVI, XIX, XX and XXI); collagens that form hexagonal networks (types VIII and X); the family of type IV collagens found in basement membranes; type VI collagen that forms beaded filaments; type VII collagen that forms anchoring fibrils for basement membranes; collagens with transmembrane domains (types XIII and XVII); and the family of type XV and XVIII collagens. For references and more thorough reviews, see Byers (2001) and Myllyharju & Kivirikko (2001). For detailed information about recently found type XX and XXI collagens, see Koch et al. (2001) and Fitzgerald & Bateman (2001), respectively.

The biosynthesis of the precursors of fibrillar collagens, called procollagens, involves a number of posttranslational modifications. The intracellular modifications require five specific enzymes: three collagen hydroxylases (Kivirikko & Pihlajaniemi 1998, Kivirikko & Myllyharju 1998) and two collagen glycosyltransferases (Kivirikko & Myllylä 1979,

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Kivirikko 1993, Prockop & Kivirikko 1995). Once outside the cells, the procollagen molecules undergo proteolytic conversion to collagen molecules. The collagen molecules form fibrils and interact with noncollagenous and collagenous proteins, and the fibrils are stabilized by the formation of intra- and intermolecular cross-links. These extracellular modifications of collagens require two specific proteinases to cleave the N- and C-terminal propeptides (Prockop et al. 1998) and specific oxidases to convert certain lysine and hydroxylysine residues to their reactive aldehydes. These oxidases will be discussed in detail in the next sections.

2.1.2 Elastin

Elastin is the most important component of the elastic fibers found in the extracellular matrix and provides elasticity and resilience to tissues requiring the ability to deform repetitively and reversibly. Ultrastructurally, elastin fibers consist of two major elements: an amorphous component made up of elastin and fibrillar components called microfibrils, which serve as a scaffold for the incorporation of elastin into the amorphous component (Cleary & Gibson 1996). Elastin is an extremely insoluble protein due to extensive cross-linking at lysine residues, and it is amongst the most hydrophobic proteins known. In higher vertebrates, including humans, over 30% of amino acid residues in elastin are glycine residues, and approximately 75 % of the entire sequence is made up of just four hydrophobic amino acids - glycine, valine, alanine, and proline. Tissues rich in elastin include the aorta and large blood vessels (28-32% of the dry mass), lungs (3-7%), elastic ligaments (50%), tendons (4%), and skin (2%) (Vrhovski & Weiss 1998, Debelle & Tamburro 1999).

Tropoelastin, the precursor of elastin, is encoded by a single gene located on chromosome 7q11 in humans and has at least 11 variants due to alternative splicing of the transcripts (Vrhovski & Weiss 1998). Tropoelastin undergoes only minor post-translational modifications, and there is no evidence of glycosylation. Hydroxylation of proline residues occurs to a variable extent, 0-20%, but this hydroxylation is not necessary for the synthesis of the elastic fibre (Vrhovski & Weiss 1998). After translation of tropoelastin, a 67 kDa elastin-binding protein becomes bound to tropoelastin, acting as a chaperone and preventing premature intracellular aggregation of tropoelastin molecules. This association lasts until the complex has been secreted into the extracellular space, where the elastin-binding protein interacts with galactosugars of the microfibrils. At this point the affinity of the elastin-binding protein for elastin decreases dramatically, leading to the release of the tropoelastin molecule into the microfibrilar scaffold (Hinek & Rabinovitch 1994). Before tropoelastin is assembled into the amorphous component of the elastic fiber, it becomes covalently cross-linked by lysyl oxidase, which has been found to be associated with microfibrils (Kagan et al. 1986, Cleary & Gibson 1996). For references and more thorough reviews of elastin and the elastic fiber, see Rosenbloom (1993), Cleary & Gibson (1996), Vrhovski & Weiss (1998) and Debelle & Tamburro (1999).

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2.2 Cross-links in collagens and elastin

In spite of the major structural differences between collagens and elastin, both proteins contain lysine or hydroxylysine-derived covalent cross-links, the formation of which is catalyzed by the family of lysyl oxidase enzymes. The locations of these cross-links depend on the amino acid sequence and quaternary structural arrangements, but may also be quite flexible (Siegel 1974, Kagan et al. 1984, Nagan & Kagan 1994, for reviews, see Kielty et al. 1993 and Smith-Mungo & Kagan 1998). Lysyl oxidases oxidatively deaminate certain lysine and hydroxylysine residues in collagens and lysine residues in elastin to form the corresponding α-aminoadipic-δ-semialdehydes, usually referred to by their trivial names as allysine and hydroxyallysine (Figure 1) (Pinnell & Martin 1968, Kagan 1986, Reiser et al. 1992, Kielty et al. 1993, Kagan 1994, Bateman et al. 1996, Cleary & Gibson 1996).

Fig. 1. Lysyl oxidase oxidatively deaminates a peptidyl lysine to generate a peptidyl αααα-aminoadipic-δδδδ-semialdehyde (allysine), which spontaneously reacts with corresponding aldehydes to form various di-, tri-, or tetrafunctional cross-links (Kagan 1986).

In collagens, the lysine and hydroxylysine-derived cross-links are essential in providing the tensile strength and mechanical stability of the collagen fibrils and other supramolecular assemblies (Light & Bailey 1980, Bateman et al. 1996, Bailey 2001). Two related cross-linking pathways can be distinguished for collagens because the oxidations are restricted to two specific lysine and hydroxylysine residues, one in each of the N- and C-terminal telopeptide sequence (see Figure 2A) (Eyre et al. 1984, Kagan 1986, 1994, Reiser et al. 1992, Kielty et al. 1993). The lysine aldehyde pathway occurs primarily in the adult skin, cornea, and sclera (Eyre 1987), whereas the hydroxylysine aldehyde pathway occurs primarily in the bone, cartilage, ligament, tendons, embryonic skin, and most major internal connective tissues of the body (Kielty et al. 1993). Oxidative deamination is followed by spontaneous condensation reactions that result in various bi-, tri-, and tetrafunctional cross-links (Figure 2A). Lysine- and hydroxylysine-derived aldehydes can react with corresponding aldehydes on adjacent polypeptide chains forming aldol condensation products, or with unmodified lysine and hydroxylysine residues, forming bifunctional cross-links, such as lysinonorleucine and

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hydroxylysinonorleucine. The aldol condensation products can also react with a histidine residue to form aldol histidine, which may further react with an additional lysine residue to form the tetrafunctional cross-link histidinohydroxymerodesmosine (Reiser et al. 1992). In the hydroxylysine pathway, the bifunctional cross-links spontaneously undergo an Amadori rearrangement, resulting in ketoimine cross-links, which then mature further into trifunctional 3-hydroxypyridinium and lysyl pyridinium cross-links (Reiser et al. 1992, Bailey et al. 1998, Byers 2001).

Fig. 2. Reactions of lysine and hydroxylysine in the biosynthesis of the cross-links in collagens (A) and elastin (B). After oxidative deamination to form reactive aldehydes, subsequent condensation reactions result in various bi-, tri-, and tetrafunctional cross-links. To simplify, intermediates in the reactions are not shown.

20 The most important feature of elastin, crucial for its proper functioning in elastic

fibers, is the high degree of cross-linking of the individual polypeptide chains (Kielty et al. 1993). Approxymately 30 of the 40 lysine residues per 1000 amino acids present in tropoelastin, the precursor of elastin, are oxidated to aldehydes (Eyre et al. 1984, Kagan 1986, 1994, Reiser et al. 1992, Rosenbloom 1993, Rosenbloom et al. 1993). Tropoelastin differs from collagens in that it has no hydroxylysine or histidine residues, and therefore only lysine-derived cross-links are found in mature elastin (Rosenbloom 1993). In addition, certain condensation products, such as desmosines and isodesmosines, are specific for elastin and are not found in collagens (Reiser et al. 1992, Kielty et al. 1993, Rosenbloom 1993). The cross-links in elastin are otherwise formed in a similar manner as those in collagens (see Figure 2B). Two allysines can form an aldol condensation product, and one allysine can form lysinonorleucine with an unmodified lysine residue. Aldol condensation of the product with an unmodified lysine residue results in the trifunctional merodesmosine. Further condensation of a dehydrated aldol condensation product with dehydrolysinonorleucine, or reaction of merodesmosine with allysine results in the tetrafunctional desmosine and isodesmosine cross-links (Reiser et al 1992, Rosenbloom 1993).

2.3 The lysyl oxidase isoenzyme LOX

2.3.1 Molecular properties

Lysyl oxidase enzyme (LOX, protein-lysine 6-oxidase, EC 1.4.3.13) was initially believed to be of one type only. Studies on this enzyme were seriously limited for a long time by its marked insolubility and aggregation tendency. A major breakthrough for studies on lysyl oxidase was the discovery that the enzyme can be subsequently solubilized by using buffers containing 4-6 M urea, and its activity can be recovered by the removal of the urea (Narayanan et al. 1974). Lysyl oxidase was then purified from several different tissues and organisms, including chick cartilage (Stassen 1976), bovine aorta (Kagan et al. 1979, Cronlund & Kagan 1986) and lung (Cronlund & Kagan 1986), human placenta (Kuivaniemi et al. 1984), and piglet skin (Shackleton & Hulmes 1990). The molecular mass of the enzyme from all those sources was found to be approximately 32 kDa. Interestingly, lysyl oxidase extracted from several tissues resolved upon DEAE chromatography into multiple forms (Kagan 1986). Furthermore, at least four forms obtained from chick cartilage (Stassen 1976) and human placenta (Kuivaniemi et al. 1984) had similar substrate specifities, amino acid compositions, and molecular masses of approximately 32 kDa (Kagan 1986). These findings suggested the existence of alternatively spliced or other forms of a single lysyl oxidase, or the presence of isoenzymes with similar but not identical properties.

The lysyl oxidase isoenzyme LOX has now been cloned from rat (Trackman et al. 1990, 1991), human (Hämäläinen et al. 1991, 1993, Mariani et al. 1992), chick (Wu et al. 1992), and mouse (Contente et al. 1993) tissues. The mRNA for human LOX is found as

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multiple species with sizes of 5.5, 4.3, 2.4, and 2.0 kb due to the use of alternate polyadenylation sites, and partially to the existence of multiple transcription initiation sites (Hämäläinen et al. 1991, Mariani et al. 1992, Boyd et al 1995). The biological relevance of these transcripts, however, is unknown. The human LOX gene is located on chromosome 5q23.3-31.2 (Hämäläinen et al. 1991, 1993, Mariani et al. 1992, Boyd et al. 1995) and encodes a 417 amino acid polypeptide, of which the first 21 residues correspond to the signal peptide (Hämäläinen et al. 1991, Mariani et al. 1992). The mouse Lox gene has been mapped to chromosome 18 (Mock et al. 1992, Chang et al. 1993, Contente et al. 1993).

2.3.1.1 Biosynthesis and processing of the LOX precursor

The LOX protein is known to be secreted into the extracellular space (Layman et al. 1972, Byers et al. 1980, Royce et al. 1980, Peltonen et al. 1983, Kuivaniemi et al. 1986a). The transport through the Golgi and to the cell membrane probably takes place in vesicles formed by budding of, and fusion with, the subcellular membranes (Kosonen et al. 1997, Rucker et al. 1998). The first evidence for the biosynthesis of a LOX proenzyme was obtained by immunoprecipitation of a 46 kDa cell-free translation product of a rat smooth muscle cell LOX mRNA, using an antibody directed against the 32 kDa bovine enzyme (Trackman et al. 1990, 1991), and a 48 kDa product, using mRNA isolated from fibrotic rat liver (Wakasaki & Ooshima 1990a). Three forms of LOX with molecular masses of 50, 45, and 32 kDa were identified by immunoprecipation of media and cell layer extracts of cultured rat smooth muscle cells, which were pulse-labelled with [35S]methionine (Trackman et al. 1992). The 50 kDa fragment was found both as a secreted protein in the cell medium and in the intracellular fraction, and pulse-chase studies revealed that it is converted to a 32 kDa protein in the medium. This 50 kDa fragment was found to be an N-glycosylated derivative of the 45 kDa proprotein (Trackman et al. 1992). Procollagen C-proteinase cleaves the 50 kDa precursor between Gly-168 and Asp-169 (numbered according to the human sequence) to yield the non-glycosylated 32 kDa mature enzyme (Figure 3) (Cronshaw et al. 1995, Panchenko et al. 1996). In mammals, procollagen C-proteinase activity is provided by products of the BMP1 gene, which encodes the alternatively spliced mRNA species for bone morphogenetic protein 1 (BMP-1), and mammalian tolloid (mTLD) (Takahara et al. 1994). The BMP1 gene is a member of a multigene family including two genetically distinct mammalian tolloid-related proteinases, tolloid-like 1 (mTLL-1) and tolloid-like 2 (mTLL-2). BMP-1, mTLD, and mTLL-1 all have procollagen C-proteinase activity, however, their substrate specificites vary. Recently, Uzel et al. (2001) demonstrated that BMP-1, mTLD, mTLL-1, and mTLL-2 all process the 50 kDa LOX precursor and control its activation in mouse embryo fibroblasts. In vitro assays with purified recombinant enzymes showed that all four proteinases productively cleaved the LOX precursor at the correct physiological site. However, BMP-1 was 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively (Uzel et al. 2001). Fibroblasts from Bmp1 and Tll1 null mice produced both the 50 kDa LOX precursor and the processed LOX

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enzyme at similar levels, indicating apparently normal processing. In contrast, double null Bmp1/Tll1 cells produced predominantly the unprocessed 50 kDa precursor and had a 70% reduction in lysyl oxidase activity when compared to wild-type, Bmp1-null and Tll1-null fibroblasts. These results suggest that BMP-1/mTLD and mTLL-1 are responsible for the majority of LOX precursor processing into the active LOX enzyme, at least in murine embryonic fibroblasts. In addition, BMP-1 may be able to regulate the LOX activity through the processing event (Uzel et al. 2000).

Fig. 3. Pathway for LOX biosynthesis. The LOX precursor enters into the rough endoplasmic reticulum where its signal peptide is cleaved. The precursor is glycosylated within the endoplastic reticulum at an Asn residue located in the propeptide region. The addition of copper (Cu2+) and the formation of lysine tyrosylquinone (LTQ) cofactor (which are discussed in Section 2.3.2.1) may occur in the endoplastic reticulum or during protein trafficking through the Golgi elements. After secretion of the LOX precursor into the extracellular space, the propeptide region is cleaved by procollagen C-proteinase (PCP) between Gly-168 and Asp-169 to obtain an active 32 kDa enzyme. Molecular masses of LOX polypeptide are indicated according to rat LOX enzyme (see Trackman et al. 1990, 1991, 1992).

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2.3.2 Catalytic properties

2.3.2.1 Cofactors

Direct evidence that copper is a component of LOX was obtained from enzymes isolated from chick bone (Iguchi & Sano 1985, Iguchi et al. 1990), chick aorta (Harris et al. 1974), and bovine aorta (Gacheru et al. 1990). Atomic absorption spectroscopic studies have demonstrated that approximately one tightly bound copper atom is present in the 32 kDa LOX monomer. Removal of the copper ion leads to a catalytically inactive apoenzyme. Electron spin resonance studies have indicated that the copper in the resting enzyme is in the Cu(II) state, and is bound in a tetragonally distorted, octahedrally coordinated ligand field (Gacheru et al. 1990). The sequence WEWHSCHQHYH in human has been suggested to be the actual copper-binding region, which provides four histidine residues that are involved in the copper-binding coordination complex (Krebs & Krawetz 1993). In addition to the tightly bound Cu ion, purified LOX preparations have been shown to contain 5-9 atoms of loosely bound copper per enzyme molecule (Gacheru et al. 1990). Experiments in a cell free transcription/translation system in vitro have shown that the unprocessed 50 kDa LOX precursor binds copper (Kosonen et al. 1997). Further information of a post-translational incorporation of copper into LOX was obtained using skin fibroblasts incubated with different inhibitors of post-translational modifications, together with carrier-free 67Cu. When protein synthesis was inhibited with cycloheximide, only minor amounts of copper (bound to LOX) were found in the medium of cultured skin fibroblasts, indicating the importance of protein synthesis for the incorporation of copper into the enzyme. In contrast, inhibition of N-linked glycosylation with tunicamycin had no effect on secretion of the copper that was bound to LOX. Inhibition of processing of the 50 kDa LOX precursor into its 32 kDa active form with a procollagen C-proteinase inhibitor had no apparent effect on the amount of copper that was bound to LOX in the cell media (Kosonen et al. 1997). This observation was in agreement with earlier data by Kagan et al. (1995), which demonstrated that the propeptide region is not essential to the folding and secretion of the functional enzyme. However, when the assembly of secretory vesicles was inhibited with brefeldin A, the secretion of copper bound to LOX was inhibited (Kosonen et al. 1997), thus indicating that copper is incorporated into lysyl oxidase in the endoplastic reticulum or during protein trafficking through the Golgi elements (see Figure 3) (Kosonen et al. 1997, Rucker et al. 1998).

In additon to copper, LOX was long known to contain a covalently bound carbonyl prosthetic group (Levene 1961, Williamson et al 1986a, Williamson et al. 1986b, see Kagan 1986, 1994, Smith-Mungo & Kagan 1998 for reviews). However, the nature of this carbonyl group remained unknown for several decades. Knowledge obtained from other members of copper amine oxidases and their cofactors played an essential role in studies leading to the identification of the carbonyl cofactor (Klinman 1996). The cofactor of one of these family members similar to LOX, namely bovine plasma amine oxidase, was initially identified as pyrrolequinoline (PQQ) (Lobenstein-Verbeek et al.

24

1984). Comparison of the resonance Raman spectra of a proteolytic peptide obtained from LOX with the corresbonding spectrum of bovine plasma amine oxidase suggested that the cofactor in lysyl oxidase resembles PQQ (Williamson et al 1986b). However, strong evidence was presented later, indicating that the true carbonyl cofactor in bovine plasma amine oxidase is in fact topa quinone (TPQ) (Janes et al. 1990). Because TPQ is found in a wide range of copper amine oxidases, the presence of TPQ in LOX was regarded as plausible, but the large size difference between LOX and other amine oxidases (Klinman & Mu 1994), together with the absence of the consensus sequence for TPQ in LOX (Janes et al. 1990), raised the possibility of a different cofactor. This different and previously unknown cofactor was indeed found to occupy the active site of bovine LOX (Wang et al. 1996, Wang et al. 1997). Its structure is derived from the cross-linking of the ε amino group of a peptidyl lysine (Lys-314 in bovine LOX) with the modified side chain of a tyrosyl residue (Tyr-349 in bovine LOX), and it has been named lysine tyrosylquinone (LTQ). LTQ could be formed in the endoplasmic reticulum or during protein trafficking through the Golgi elements (see Figure 3) (Rucker et al. 1998). In this pathway, copper may play a structural role in stabilizing the LTQ (Tang & Klinman 2001).

2.3.2.2 Reaction mechanism

LOX catalyses primary amine oxidation through a ping pong bi ter kinetic mechanism (Williamson & Kagan 1986), which is illustrated in Figure 4. The first step in this reaction is the formation of a Schiff base with the lysine tyrosylquinone (LTQ) cofactor (I → II). While the LTQ is bound to the substrate, it undergoes a rate-limiting, general base-facilitated α proton abstraction (Williamson & Kagan 1987a). A histidine residue has been suggested to act as the general base in LOX (Gacheru et al. 1988). In the following reaction, stereospecific abstraction of the pro-S α-proton takes place (Shah et al. 1993a), and the electrons migrating from the substrate carbanion reduce the LTQ cofactor (II → III). Hydrolysis of the imine intermediate releases the reactive aldehyde product, which can then react spontaneously to form lysine- or hydroxylysine-derived cross-links. After the release of the aldehyde product, the reduced enzyme is reoxidized by molecular oxygen with the aid of Cu(II) to produce hydrogen peroxide and ammonia (Akagawa & Suyama 2001). By this way, the oxidized enzyme is regenerated and the catalytic cycle is completed (IV → V → I) (Kagan 1994, Smith-Mungo & Kagan 1998, Akagawa & Suyama 2001).

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Fig. 4. Reaction mechanism of LOX. The RNH substituent represents peptidyl lysine 314 in bovine LOX, the letter B represents a general base, LTQ represents lysine tyrosylquinone cofactor, and LysAld represents the end product of the reaction, a lysine-derived aldehyde (modified from Smith-Mungo & Kagan 1998, and Akagawa & Suyama 2001).

2.3.2.3 Inhibitors

Administration of β-aminopropionitrile (βAPN) to growing animals results in a molecular disease known as lathyrism, which is characterized by an increased fragility of all connective tissues and an elevation of solubility of collagen from tissues due to diminished cross-linking (see Section 2.5.3). Therefore, the ability of βAPN to inhibit cross-link formation was recognized even before identification of lysyl oxidase activity in vitro (Kagan 1986). βAPN was later found to be a potent irreversible LOX inhibitor with a Ki of about 3-5 µM (Narayanan et al. 1972). βAPN binds to the active site of LOX and is enzymatically processed to a reactive form, which derivatizes the enzyme (Tang et al.

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1983). βAPN has therefore been used to specifically inhibit activities of all lysyl oxidase isoenzymes in numerous studies. The structure of βAPN was used in a directed search for other inhibitors. β-Haloethylamines and β-nitroethylamine were identified as additional active site-directed irreversible inhibitors, with inhibition constants similar to that of βAPN (Tang et al. 1984, Williamson & Kagan 1987a). Benzylamines substituted in the para position with electron-withdrawing functions behave as ground-state inhibitors, presumably by forming LOX enzyme-bound intermediates that are not completely processed into the aldehyde (Williamsson & Kagan 1987b). Vicinal diamines like cis-1,2-diaminocyclohexane and ethylenediamine are also potent irreversible LOX inhibitors (Gacheru et al. 1989). LOX is further inhibited by heparin (Gavriel & Kagan 1988), N-(5-aminopentyl)aziridine (Nagan et al. 1998), and trans-2-phenylcyclopropylamine (Shah et al. 1993b) with the latter functioning as a noncompetetive, reversible inhibitor (Shah et al. 1993b). LOX is additionally inhibited by homocysteine thiolactone and its selenium and oxygen analogs in an active-site-directed and irreversible manner (Liu et al. 1997a).

2.3.3 Regulation of LOX

2.3.3.1 Expression in different cell types and during development

LOX is expressed in several different cells, including fibroblasts, aortic and lung smooth muscle cells, osteoblasts, osteosarcoma cells (Csiszar et al. 1996, Uzel et al. 2000), myofibroblasts (Peyrol et al. 1997), corneal endothelial cells (Fujimaki et al. 1999), and chondrocytes (Gregory et al. 1999). Moreover, a secreted form of LOX is associated with tracheal chondrocytes, endothelial cells, basal cells, biliary epithelial cells, liver parenchymal cells, and spleen reticulum cells (Di Donato et al. 1997a). In kidney, LOX is detected in glomeruli, medulla, renal cell lines, and tubular epithelial cells (Di Donato et al. 1997a). At the tissue level, LOX is abundantly expressed and immunologically detected in fetal and adult aorta (Kagan et al. 1986, Baccarani-Contri et al. 1989), human placenta, skin, and lung (Baccarani-Contri et al. 1989).

During the development of different organisms, most observations of LOX expression patterns have been made in association with the assembly of collagen and elastin fibers in different tissues. With the aid of polyclonal antibodies, LOX has been localized in association with collagen fibers despite the age of the subjects, at least in skin and aorta. In contrast, only small amounts of aortic elastin fiber-associated LOX was observed in a 24-week-old human fetus, and by the age of 16 weeks, positive signals were completely diminished (Baccarani-Contri et al. 1989). In human amnion, the highest level of lysyl oxidase activity, as well the highest expression of LOX, were detected in gestational weeks 12-14, and the lowest values were seen at weeks 20-24 (Casey & MacDonald 1997). In adulthood, elastin fiber-associated LOX staining is mostly negative (Baccarani-Contri et al. 1989).

27 Between days 8 and 16 of chick embryonic development, the steady-state levels of

LOX mRNA are increased together with its substrates, tropoelastin, and type I collagen. In situ hybridizations showed that the spatial expressions of the LOX and tropoelastin transcripts differ, suggesting that the enzyme and substrate genes may be regulated differentially within cells of the arterial wall (Wu et al. 1992). In a similar manner, the LOX mRNA level exhibited a fourfold increase from gestational day 9 to day 15 in rat embryos, the highest expression level being seen in day 13 (Tchaparian et al. 2000). However, the corresponding enzyme activity level was found to be relatively constant during those days. A measurable enzyme activity level was observed in rat embryos at gestational day 9, which corresponds to the transition of the embryo from the postblastocyst stage to preembryonic stage (Tchaparian et al. 2000). Moreover, in the embryonic development of rat, a pattern of temporal expression for a copper transporting P-type ATPase gene (ATP7A) was consistent with its possible role in copper delivery to LOX (Tchaparian et al. 2000).

During the development of sea urchin embryos, lysyl oxidase activity is increased severalfold, the activity being peaked during gastrulation and larva formation. Treatment of developing sea urchin embryos with βAPN resulted in developmental arrest at the mesenchymal blastula stage, suggesting a critical role for LOX in mesenchyme migration, gastrulation, and morphogenesis during development (Butler et al. 1987). However, at the time of this study, it was supposed that only one enzyme is responsible for the cross-linking of elastin and collagens, and therefore affected by βAPN (see Section 2.4 for details). Di Donato et al. (1997b) studied the role of LOX in Xenopus laevis oocytes, which are useful models in unraveling the signal transduction involved in mitogenesis and other oncogene-dependent processes. After the coinjection of LOX and the oncogenic p21-Ha-rasval12 into maturing oocytes, the ras-dependent maturation of these oocytes was inhibited as an effect of the intracellular LOX. Treatment of these coinjected oocytes with βAPN abolished this inhibition of maturation. Results from this study suggest that the LOX protein is actually able to antagonize oncogenic Ras-dependent meiotic maturation of Xenopus laevis oocytes.

2.3.3.2 LOX in fibrotic tissues

Fibrosis is characterized by an extensive accumulation of the essentially insoluble collagen fibers. Therefore, it should be possible to treat this condition by selective suppression of key events in collagen biosynthesis, including the reaction catalyzed by the lysyl oxidases. Inhibition of lysyl oxidases would likely decrease the amount of cross-links in collagens, which may enhance its degradation by proteases (see Kagan 1986, 1994, 2000, Franklin 1995, 1997, Myllyharju & Kivirikko 2001 for reviews).

In the past decades, several reports have suggested a strong association between organ fibrosis and increased lysyl oxidase activity. Such observations have been made in experimental hepatic fibrosis in rat (Siegel et al. 1978, Carter et al. 1982, Wakasaki & Ooshima 1990a), in models of lung (Counts et al. 1981, Riley et al. 1982, Almassian et al. 1991), arterial (Kagan et al. 1981) and dermal fibrosis (Chanoki et al. 1995), in

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chronic human liver fibrosis (Murawaki et al. 1991), adriamycin-induced kidney fibrosis in rat (Di Donato et al. 1997a), and in other pathological conditions leading to fibrosis (Sommer et al. 1993, Jourdan-Le Saux et al. 1994, Ma et al. 1995, Decitre et al. 1998, Trivedy et al. 1999). Among these, probably the most striking increases in enzyme activity are seen in the rat model of CCl4-induced liver fibrosis (Siegel et al. 1978, Carter et al. 1982). In this model, an increase of lysyl oxidase activity reflects increases in the steady-state levels of LOX mRNA (Wakasaki & Ooshima 1990a). A significantly higher level of lysyl oxidase activity was also seen in the media of mesenchymal cells cultured from cirrhotic human livers when compared to that in the media of cells from normal liver or from livers of patients with chronic hepatitis (Konishi et al. 1985). In these studies, the low level of enzyme activity in the healthy liver increased 15- to 30-fold in fibrotic livers. Because the lysyl oxidase activity level is normally negligible in the serum of healthy subjects, but significantly increased in experimental liver fibrosis, it has been suggested that lysyl oxidase might serve as a marker of internal fibrosis (Siegel et al. 1978, McPhie 1981, Carter et al. 1982, Sakamoto et al. 1987, Murawaki et al. 1991).

Interestingly, changes in the expression level of type III collagen often precede or parallel changes in the level of lysyl oxidase activity (Krzyzosiak et al. 1992, Sharma et al. 1997, Kim et al. 1999). To clarify these findings, Giampuzzi et al. (2001) investigated the effects of LOX overexpression on the activity of the human COL3A1 gene promoter in monkey renal fibroblasts (COS-7) and human primary skin fibroblasts. Overexpression of the recombinant LOX led to a dramatic increase in the COL3A1 promoter activity. The data further suggested that induction of the COL3A1 promoter was dependent on the catalytic activity of LOX, as it was completely suppressed by βAPN. Moreover, a sequence similar to the nuclear regulatory element 1 (NRE1) was found in the COL3A1 promoter and was shown to be able to bind Ku antigen, which is known to be involved in some of the main DNA repair and recombination processes (Jin & Weaver 1997, Jeggo 1998, Jeggo et al. 1999). However, the authors of this study had great difficulties in explaining and defining any possible mechanism by which LOX increased the Ku binding into its target sequence on the COL3A1 promoter (Giampuzzi et al. 2001).

In addition to the association between the expression of LOX and type III collagen, LOX expression also seems to be associated with type I collagen expression. In developing granulomas, LOX was transiently up-regulated at the transcriptional level in parallel with the α1 chain of type I procollagen (Sommer et al. 1993). Jourdan-Le Saux et al. (1997) subsequently demonstrated by transfection studies that the LOX and COL1A1 promoters may be regulated by similar negative and positive cis-acting elements. These regulatory regions include a potential TGF-β response element, which was also found in the rat Col1a1 (Ritzenthaler et al. 1991) and mouse Col1a2 (Rossi et al. 1988) promoters.

2.3.3.3 Response to different effectors in various cell types

LOX expression is highly responsive to a variety of physiological states, such as growth, wound repair, ageing, genetic diseases involving altered copper metabolism, and tumorigenesis. Therefore, it is obvious that expression of LOX is regulated by several

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different specific cytokines, growth factors, and related intercellular molecular messengers (see Smith-Mungo & Kagan 1998, Csiszar 2001 for reviews). The factors contributing to LOX expression and its activity in different cell lines, tissues, and physiological situations, and in several disorders, include specific transcription factors like the interferon response factor-1 (IRF-1) (Tan et al. 1996), metal ions (Li et al. 1995), cytokines and growth factors, such as transforming growth factor-β1 (TGF-β1) (Shibanuma et al. 1993, Boak et al. 1994, Feres-Filho et al. 1995, Roy et al. 1996, Gacheru et al. 1997, Hong et al. 1999, Shanley et al. 1997, Bose et al. 2000), basic fibroblast growth factor (bFGF) (Feres-Filho et al. 1996), fibroblast growth factor-2 (FGF-2), insulin-like growth factor (IGF) (Trackman et al. 1998), and platelet-derived growth factor (PDGF) (Green et al. 1995), hormones, such as testosterone (Bronson et al. 1987) and prostaglandin E2 (PGE2) (Boak et al. 1994, Roy et al. 1994, Choung et al. 1998), and signaling molecules like ras (Contente et al. 1990, Hajnal et al. 1993, Kryzosiak et al. 1992, Csiszar et al. 1996, Di Donato et al. 1997b) and cAMP (Choung et al. 1998, Ravid et al. 1999). The growth factors and other effectors that are known to affect LOX expression and activity are listed in Table 1. Because of cell type differences and experiments carried out under a variety of conditions, it is difficult to draw any general conclusions concerning the transcriptional and post-translational regulation of LOX.

One of these effectors, TGF-β1, is a fibrogenic growth factor known to regulate the synthesis of collagens (Chambers & Laurent 1996, Wight 1996) and elastin (Cleary & Gibson 1996). TGF-β1 has been found to strongly promote expression of LOX in fibroblasts from neonatal rat lungs (Boak et al. 1994) and human embryos (Roy et al. 1996), and in rat vascular smooth muscle cells (Gacheru et al. 1997, Shanley et al. 1997). In human lung fibroblasts, the increase of LOX expression induced by TGF-β1 is effectively prevented by PGE2 (Roy et al. 1996). PGE2 is known to induce the expression of cyclooxygenase-1, the catalyst leading to the production of PGE2, and therefore it has been suggested that a coordinated and autocrine-like mechanism could limit LOX expression in inflammatory responses to connective tissue injury (Roy et al. 1996). Most of the regulatory effects of TGF-β1 on LOX expression in cultured rat aorta smooth muscle cells probably occurs at a post-transcriptional level by an enhancement of the stabilization of the LOX mRNA (Gacheru et al. 1997). In MC3T3-E1 osteoblastic cells, TGF-β1 also stimulated LOX expression both at the mRNA and protein levels. However, proteolytic prosessing of the LOX precursor was proportionately less stimulated by TGF-β1, which may account for the delay and slightly lower magnitude of the increase in the corresponding enzyme activity when compared to the peak of the increase in the mRNA level (Feres-Filho et al. 1995). TGF-β1 also regulates BMP-1 and mTLD (Lee et al. 1997), which are responsible for the proteolytic processing of the LOX precursor (Uzel et al. 2001; see also Section 2.3.1.1), and therefore serves as one of the possible regulatory levels in the activation of the enzyme. In contrast, this processing event did not seem to be affected by the elevation of LOX expression by TGF-β1 in cultured neonatal rat lung fibroblasts (Boak et al. 1994).

In adult rat vascular smooth muscle cells, the LOX mRNA level and the secreted enzyme activity were low in quiescent cells from adult rats, but were markedly induced by PDGF, angiotensin II, and by increasing the serum concentration to 10 % (Green et al.

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1995). However, in quiescent neonatal rat vascular smooth muscle cells, the expression of LOX mRNA and the secreted enzyme activity were elevated, but were markedly decreased by stimulation of the proliferation by serum enrichment (Gacheru et al. 1997), suggesting age-specific mechanisms of regulation in these cells. Interferon γ (IFN- γ) has been shown to downregulate LOX gene expression in rat aortic smooth muscle cells by transcriptional and posttranscriptional mechanisms (Song et al. 2000). This agrees with the finding that an IFN-sensitive motif is present in the promoter of the LOX gene (Tan et al. 1996). Table 1. Transcriptional and posttranscriptional regulation of LOX.

Effector Cell or tissue Effect

IFN-γ1 Rat aortic smooth muscle cells Downregulation of mRNA; decreased mRNA half-life

bFGF2 Mouse osteoblastic cells Decreased mRNA level (1-10 nM); upregulation of mRNA (0.01-0.1 nM)

FGF-2 and IGF-13 Inflamed oral tissues, rat fibroblastic mesenchymal cells

Increase of mRNA

PGE24-6 Rat lung fibroblasts Unchanged mRNA level; reduction in

enzyme activity Human embryonic lung fibroblasts Downregulation of mRNA TGF-β1

4,5,7−12 Rat aortic smooth muscle cells Increased mRNA level and enzyme activity Human gingival, flexor reticulum

cells, renal cell lines Increased mRNA level and enzyme activity

Murine osteoblast-like cells Increased mRNA level and enzyme activity Cadmium13 Mouse fibroblasts Decreased mRNA level Mouse cadmium-resistant fibroblasts Increased mRNA level Testosterone14 Calf aortic smooth muscle cells Increased enzyme activity Bleomycin15 Human lung fibroblasts Increased mRNA level Human dermal fibroblasts Decreased mRNA level Hydralazine15 Human dermal fibroblasts Increased mRNA level Minoxidil15 Human dermal fibroblasts Increased mRNA level Adriamycin16 Rat kidney glomeruli, medula Increased mRNA level cAMP6,17 Rat and human vascular smooth

muscle cells Upregulation of transcription

PDGF18 Rat vascular smooth muscle cells Upregulation of mRNA Dexamethasone19 Cultured fetal murine lungs Upregulation of mRNA Retinoic acid20 Adipocytes in early adipogenesis Prevents downregulation of mRNA and

enzyme activity References: 1Song et al. 2000, 2Feres-Filho et al. 1996, 3Trackman et al. 1998, 5Roy et al. 1996, 4Boak et al. 1994, 6Choung et al. 1998, 7Shibanuma et al. 1993, 8Feres-Filho et al. 1995, 9Gacheru et al. 1997, 10Hong et al. 1999, 11Shanley et al. 1997, 12Bose et al. 2000, 13Li et al. 1995, 14Bronson et al. 1987. 15Yeowell et al. 1994, 16Di Donato et al. 1997a, 17Ravid et al. 1999, 18Green et al. 1995, 19Chinoy et al. 2000, 20Dimaculangan et al. 1994.

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2.3.4 Novel biological roles

LOX has been reported not only to be involved in the cross-linking of collagens and elastin, but to also act as a tumor suppressor and to have intracellular and even intranuclear activities (for reviews, see Smith-Mungo & Kagan 1998 and Csiszar 2001).

2.3.4.1 LOX in tumor suppression

The first direct evidence of the tumor suppressor activity of LOX was demonstrated by Contente et al. (1990). They identified a markedly down-regulated cDNA species upon transformation of mouse NIH 3T3 cells with LTR-c-H-ras. The expression level of this cDNA was restored when the transformed cell line (RS485) was treated with interferon to obtain a persistent revertant cell line (PR4). This cDNA species was named as ras recision gene (rrg). These persistent revertant cells are phenotypically non-transformed and non-tumorgenic. However, when a rrg antisense RNA was used to block the expression of the corresponding mRNA in PR4 cells, the reappearance of the tumorgenic and transformed phenotype was observed. Furthermore, subcutaneous injection of these antisense cells into athymic mice induced tumor formation. The cDNA sequences of the mouse rrg and rat Lox were subsequently found to be identical, thus indicating that rrg encodes Lox (Kenyon et al. 1991), the activity of which was already known to be markedly low in the medium of malignantly transformed cultured human cell lines (Kuivaniemi et al. 1986b). These results have been supported by several other groups. Expression of LOX was down-regulated in immortalized rat 208F fibroblasts after transformation by activated H-ras (Hajnal et al. 1993, Oberhuber et al. 1995) and up-regulated in spontaneous phenotypic revertants that continued to express the ras oncogene (Hajnal et al. 1993). In addition, LOX expression was found to be irreversibly and coordinately regulated with type I collagen expression in spontaneous phenotypic revertants (Hajnal et al. 1993). A putative tumor suppressor role for LOX was also demonstrated by stable transfection of a LOX cDNA in antisense orientation to normal rat kidney fibroblasts (NRK-49F). The transfected cells exhibited a loose attachment to plate, anchorage-independent growth, and high tumorgenicity in nude mice, and also showed an impaired response of the PDGF and IGF-1 receptors to their ligands (Giampuzzi et al. 2001). Hämäläinen et al. (1995) demonstrated that the low lysyl oxidase activity level in several malignantly transformed human cell lines is due to low quantities of the LOX mRNA and a low transcription level of the corresponding gene. A similar transcriptional silencing of LOX expression was detected in H-ras-transformed rat embryo fibroblasts (CREF) by using nuclear run-on assays (Su et al. 1995). The LOX gene was also identified as a target for the anti-oncogenic transcription factor (IRF-1), which manifests tumor suppressor activity and contributes to the development of human hematopoietic malignancies (Tan et al. 1996).

In malignant human breast carcinomas, LOX was highly expressed in myofibroblasts and myoepithelial cells surrounding the in situ tumor and in the reactive fibrosis facing

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the invasion front of infiltrating tumors (Peyrol et al. 1997). Type I, III, and IV collagens and elastin were found to be co-distributed with LOX in this study, thus resulting in the stabilization of a scar-like peritumor barrier. In contrast, LOX was found to be absent from the carcinoma cells. These findings suggested a possible host defense mechanism, while a late stromal reaction lacking LOX favors tumor dispersion (Peyrol et al. 1997).

A similar phenomenon was observed in experimental mouse prostate cancer, where LOX was expressed in normal epithelium, but progressively lost in primary prostate cancer and associated metastatic lesions (Ren et al. 1998). In the stromal reaction of different types of broncho-pulmonary carcinoma, a strong expression of LOX is associated with the hypertrophic scar-like stromal reaction found at the front of tumor progression. In contrast, little or no expression was found within the stromal reaction of invasive carcinomas, small cell carcinomas, and neuro-endocrine carcinomas (Peyrol et al. 2000). Very recently, it was demonstrated that a loss or reduction of LOX function during tumor development may be a direct consequence of somatic mutations and may be associated with the pathogenesis of colon cancer (Csiszar et al. 2002). The LOX gene is located in chromosomal region 5q23 (Hämäläinen et al. 1991, 1993, Mariani et al. 1992, Boyd et al. 1995), which is known to be deleted at a high frequency in many different types of cancer (Tamura et al. 1996, Wieland et al. 1996).

2.3.4.2 Intracellular and intranuclear activities

LOX has been localized within fibroblasts, chondrocytes, smooth muscle cells, and a variety of non-fibroblastic cells. In cultured fibroblasts, LOX was immunologically detected in association with filamentous structures in the cytoplasm consistent with cytoskeletal proteins (Wakasaki & Ooshima 1990b). However, since the molecular weight of this intracellularly observed enzyme was not assessed, it was unknown wheter it represented the proenzyme and/or mature LOX form. It was recently suggested that an intracellularly expressed recombinant mature 32 kDa LOX form may be able to regulate the activity of the COL3A1 gene promoter, as discussed in Section 2.3.3.2 (Giampuzzi et al. 2001). In addition, coinjection of LOX with oncogenic p21-rasval12 into Xenopus laevis oocytes suggests one possible intracellular role for LOX in antagonizing a Ras-induced meiotic maturation of these cells. The authors of this study suggested that a LOX-dependent block in oocyte maturation may be downstream of Erk2, a member of the mitogen-activated protein kinases (Di Donato et al. 1997b).

In addition, a 32 kDa active form of LOX has been localized by immunocytochemistry and Western blot analysis to the nucleus of rat vascular smooth muscle cells and 3T3 fibroblasts (Li et al. 1997). Moreover, the nucleus of the vascular smooth muscle cells contained lysinonorleucine, which is the adduct formed during the LOX catalyzed cross-link reaction. Formation of lysinonorleucine was prevented by administration of βAPN, thus confirming a role of LOX in this reaction (Li et al. 1997). More recently, it was demonstrated by using cultured smooth muscle cells that a purified bovine 32 kDa LOX polypeptide fluorescently labeled with rhodamine was able to enter into the cytosol and become rapidly concentrated into the nuclei of these cells. The intracellular uptake and

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distribution were not altered by βAPN, and therefore were independent of the catalytic activity of LOX (Nellaiappan et al. 2000). The identity of a nuclear substrate of LOX is currently unknown (Li et al. 1997).

2.4 Recently identified lysyl oxidase isoenzymes

Isoenzymes are distinct forms with the same catalytic activity, differing in their physical and chemical properties. They can be present in different tissues, cell types, subcellular compartments, or developmental stages of the same organism, or in different organisms. In past decade, four lysyl oxidase isoenzymes have been characterized in addition to LOX; lysyl oxidase-like protein (LOXL) (Kenyon et al. 1993, Kim et al. 1995), lysyl oxidase-like 2 protein (LOXL2) (Saito et al. 1997, Jourdan-Le Saux et al. 1999), lysyl oxidase-like 3 protein (LOXL3) (paper I of the present study and Jang et al. 1999, Huang et al. 2001, Jourdan-Le Saux et al. 2001), and lysyl oxidase-like 4 protein (LOXL4) (paper II of the present study and Ito et al. 2001, Asuncion et al. 2001). Polypeptides of all these isoenzymes are highly conserved within their C-terminal ends, which include the copper-binding sites, cytokine receptor-like domains, and lysine tyrosylquinone cofactor sites (see Csiszar 2001 for review).

The emerging members of the lysyl oxidase gene family raise many questions concerning the role of each member with respect to altered lysyl oxidase activities in nutritional copper-deficiency, Menkes and occipital horn diseases, lathyrism, tumor suppression, and fibrotic tissues. The substrate or tissue specificity may vary among these five family members, although no strong evidence for this has been presented so far. The isoenzymes LOXL3 and LOXL4 identified in the present study will be discussed in detail in Chapters 4, 5, and 6.

2.4.1 Lysyl oxidase-like protein (LOXL)

Kenyon et al. (1993) characterized a novel human cDNA with a predicted protein similar to LOX. This protein was named lysyl oxidase-like protein (LOXL). The LOXL cDNA corresponds to a single polyadenylated mRNA species of 2.5 kb, which shows a concomitant expression with LOX mRNA in several human tissues (Kim et al. 1995). The LOXL polypeptide consists of 574 amino acids, with a calculated molecular mass of 63 kDa. It shows 76% identity with LOX at its C-terminal region (Kenyon et al. 1993, Kim et al. 1995), which includes the copper-binding site and the four histidine residues within the conserved sequence (WEWHSCHQHYH) that are involved in the copper binding coordination complex. Furthermore, a growth factor and cytokine receptor-like sequence are present in this region in both the LOX and LOXL polypeptides. This highly homologous region encompasses the mature 32 kDa form of LOX (Kim et al. 1995). The

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LOXL gene contains 7 exons and has been mapped to chromosome 15q24 in humans (Szabo et al. 1997) and chromosome 9 in mice (Tchernev et al 1997, Wydner et al. 1997).

The LOXL protein has been identified as a secreted protein that is expressed in the extracellular matrix in active fibrotic diseases and in the early stromal reaction of breast cancer (Decitre et al. 1998). Coincident appearance of increased steady-state levels of LOXL and COL3A1 mRNAs was detected in the early development of liver fibrosis, suggesting that the LOXL protein is involved in the development of lysine-derived cross-links in collagenous substrates. In contrast, steady-state levels of LOX mRNA were increased throughout the onset of hepatic fibrosis and appeared in parallel with increased stedy-state levels of COL1A1 mRNA (Kim et al. 1999). A specific antibody against LOXL has been used to identify proteins immunochemically distinct from LOX in various cells and in bovine aorta. These proteins had molecular weights of approximately 68, 52, 42, and 30 kDa (Decitre et al. 1998). Recently, Borel et al. (2001) isolated and characterized a 56 kDa LOXL protein from bovine aorta. This LOXL precursor was cleaved by a furin-like activity and was largely inactive. However, further processing by BMP-1 in vitro led to an enzyme that was active on elastin and collagen substrates (Borel et al. 2001).

2.4.2 Lysyl oxidase-like 2 protein (LOXL2)

Lysyl oxidase-like 2 protein (LOXL2) was orginally cloned, characterized, and named WS9-14 by Saito et al. (1997) for its possible association with Werner syndrome, which is characterized by premature aging (Murano et al. 1991). However, it was subsequently reported that WS9-14 mRNA corresponds to LOXL2 mRNA, with the exception that the WS9-14 transcript encodes one additional scavenger receptor cysteine-rich domain (SRCR) in its 5’end region (Jourdan-Le Saux et al. 1999, Csiszar 2001).

The LOXL2 mRNA encodes an 87 kDa polypeptide, which contains four SRCR domains that are found in several secreted and cell surface proteins (Saito et al. 1997). The LOXL2 polypeptide shares a 48% identity with the LOX polypeptide in its amino acid sequence from residue 546 to 751 in its C-terminal region. This region contains all conserved amino acid sequences needed for the proper function of the mature 32 kDa form of the LOX enzyme (Saito et al. 1997, Jourdan-Le Saux et al. 1999). The LOXL2 gene has been mapped to chromosome 8p21 in humans (Jourdan Le Saux et al. 1998) and 14 in mice (Jourdan-Le Saux et al. 2000), and it consists of at least 11 exons (Jourdan-Le Saux et al. 1999).

LOXL2 is abundantly expressed in senescent fibroblasts and several adherent tumour cell lines, but is down-regulated in several nonadherent tumour cells. This suggests that it may be involved in cell adhesion and that a loss of this protein may be associated with the loss of tumour cell adhesion. LOXL2 may, therefore, play a role in metastasis (Saito et al. 1997). LOXL2 shows similar spatial expression with LOX and LOXL in human placenta and fetal tissues in early pregnancy. However, this pattern diverges during gestation (Hein et al. 2000). In full-term human placenta, LOX is expressed

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predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL shows the highest expression in the placenta and lowest expression in the amnion. LOXL2 expression differs in that it is detected predominantly in chorionic cytotrophoblasts of the membranes with only low expression levels in the amnion and placenta (Hein et al. 2000, see also Casey & MacDonald 1997, Jourdan-Le Saux 1999).

2.5 Consequences of reduced lysyl oxidase activities

Reduced lysyl oxidase activities are found in two X-linked recessively inherited disorders, Menkes disease and occipital horn syndrome (OHS), and in nutritional copper deficiency and lathyrism (see Kagan 1986, Steinmann et al. 1993, Smith-Mungo & Kagan 1998, Kaler 1998 for reviews). In nutritional copper deficiency, as well as the Menkes disease and OHS, reduced availability of copper for lysyl oxidase leads to diminished number cross-links in collagens and elastin, which is manifested by several connective tissue defects. However, several other copper-dependent enzymes are also affected, as will be discussed in Section 2.5.2. In contrast, lathyrism is caused by β-aminopropionitrile, a potent irreversible inhibitor of lysyl oxidase (see Section 2.3.2.3), and therefore connective tissue abnormalities in this disease are due to direct effects of decreased lysyl oxidase activity (Kagan 1986, Steinmann et al. 1993). Abnormalities in copper availability and administration of β-aminopropionitrile may in principle decrease activities of all known lysyl oxidase isoenzymes.

2.5.1 Nutritional copper deficiency

Copper may be an essential metal cofactor of all lysyl oxidase isoenzymes. The functional activity of lysyl oxidase has been shown to vary up to 10-fold in response to variations in the availability of dietary copper (Harris 1976, Rayton & Harris 1979, Harris 1986, Opsahl et al. 1982). Reduced lysyl oxidase activity due to copper deficiency has been detected in the chick aorta (Harris 1976), tendon (Opsahl et al. 1982, Rucker et al. 1999), and bone (Opsahl et al. 1982), as well as in rat skin (Romero-Chapman et al. 1991, Rucker et al. 1996). In these situations, reduced lysyl oxidase activity leads to a reduction in the cross-link formation in vivo, and therefore to connective tissue defects in growing animals, including osteoporosis, joint deformaties, bone fragility, internal hemorrhages, and aortic aneurysms (Rucker et al. 1975, Harris 1976, Rayton & Harris 1979, Opsahl et al. 1982).

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2.5.2 Menkes disease and occipital horn syndrome

Lysyl oxidase activity is reduced in two X-linked recessively inherited disorders, the Menkes disease and its less severe variant occipital horn syndrome (OHS, also known as X-linked cutis laxa) (see Kagan 1986, Smith-Mungo & Kagan 1998, Kaler 1998 for reviews). Both diseases are caused by mutations in the ATP7A gene that encodes a copper-transporting P-type ATPase. As a result, copper accumulates in connective tissue cells and does not enter secretory vesicles (Chelly et al. 1993, Mercer et al. 1993, Vulpe et al. 1993, Vulpe & Packman 1995). Therefore, the reduced lysyl oxidase activity is secondary to the copper deficiency, as are activities of at least 12 different copper-dependent enzymes (Kaler 1998).

2.5.2.1 Clinical, biochemical and pathological findings

The findings in both diseases include abnormal neural development, connective tissue abnormalities, and often premature death. Manifestations in OHS include bladder diverticula with spontaneus ruptures, inguinal hernias, slight skin laxity and hyperextensibility, skeletal changes, such as occipital horn-like exostoses, and vascular tortuoisity. Manifestations in Menkes disease also include bladder diverticula, hyperextensibility and laxity of skin, and skeletal abnormalities. Unlike OHS, Menkes disease further involves neurological degeneration, mental retardation, and generalized arterial disease with grossly abnormal elastic lamellaes. These complications are often so severe that the disease leads to premature death by the age of 3 years (see Kivirikko & Kuivaniemi 1987, Danks 1993, Kaler 1998 for reviews).

The reduced lysyl oxidase activity level may significantly reduce the strength of connective tissues in many tissues, and therefore probably has major clinical consequences in both diseases. The vascular tortuoisity, bladder diverticula, and castric polyps are all likely to be results of a decreased lysyl oxidase activity (Royce et al. 1980, Harcke et al. 1977, Daly & Rabinovitch 1981, Kaler et al. 1993). Because OHS patients exhibit mainly connective tissue manifestations with only mild neurological changes, lysyl oxidase may be one of the most sensitive copper-dependent enzymes to defects in the ATP7A gene product (Das et al. 1995, Levinson et al. 1996). It has been hypothesized that OHS results from the presence of low levels of a functional ATP7A, whereas no normal ATP7A activity exists in Menkes disease (Das et al. 1995). Møller et al. (2000) recently demonstrated that ATP7A-transcript levels as low as 2-5% of normal are sufficient to result in the less severe OHS, thus supporting the earlier hypothesis.

In Menkes and OHS patients, a reduction in lysyl oxidase activity level has been demonstrated in skin specimens (Byers et al. 1980, Peltonen et al. 1983) and in the medium of cultured fibroblasts (Byers et al. 1980, Peltonen et al. 1983, Royce et al. 1980, Kuivaniemi et al. 1982, Kuivaniemi et al. 1985, Kemppainen et al. 1996). Cultured skin fibroblasts from Menkes and OHS patients have also been found to contain and secrete reduced amounts of the LOX protein, indicating that transcription or translation

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may be impaired (Kuivaniemi et al. 1985, Gacheru et al. 1993). Kemppainen et al. (1996) studied potential transcriptional abnormalities in fibroblasts from Menkes and OHS fibroblasts by using quantitative PCR. Their work demonstrated a decreased amount of LOX mRNA in cultured fibroblasts from patients with Menkes and OHS diseases when compared to control cell lines.

2.5.2.2 Animal models of Menkes disease and occipital horn syndrome

Mouse mutants involving the X-linked locus mottled (Mo) serve as animal models of the Menkes disease and OHS (Danks 1993, Mercer 1998). This locus represents a murine homologue of the human ATP7A locus. In mouse, various Atp7a gene mutations lead to phenotypes similar to those found in patients with the Menkes disease and OHS (Mercer 1998). The mottled brindle (Mobr) mouse is phenotypically and biochemically similar to patients with Menkes disease, exhibiting severe neurological impairment and death at an early age (Danks 1993). Likewise, the mottled blotchy (Moblo) mouse represents a phenotype similar to OHS with predominant connective tissue manifestations (Rowe et al. 1977, Mercer 1998). Interestingly, Moblo males and Moblo/+ females have a significantly higher incidence of spontaneous aortic aneurysms when compared to the other mouse lines. Histologically, the Moblo aortas exhibit disrupted elastic lamellae and thickening of the interlamellar spaces (Andrews et al. 1975, Brophy et al. 1988). Similar findings have also been made in other mottled mice (Fry et al. 1967, Rowe et al. 1974). However, in Moblo males, the incidence of aneurysms increased progressively, reaching 100% by 6 months of age (Brophy et al. 1988).

2.5.3 Lathyrism

Lathyrism is caused by chronic ingestion of the sweet pea Lathyrys odoratus, which contains β-(γ-glutamyl)aminopropionitrile, a potent irreversible inhibitor of lysyl oxidase when metabolized to β-aminopropionitrile (βAPN), as discussed in Section 2.3.2.3 (see Kagan 1986, 1994, Smith-Mungo & Kagan 1998 for reviews). Lysyl oxidase inhibition leads to the diminished cross-linking of collagens and elastin, and consequently, to connective tissue defects (Kagan 1986, Steinmann et al. 1993). The connective tissue manifestations include kyphoscoliosis, bone deformities, weakening of tendons and ligament attachments, dislocation of joints, weakening of skin and cartilage, hernias, and dissecting or saccular aneurysms of the aorta (Steinmann et al. 1993). The connective tissue manifestations seen in lathyrism resemble closely those observed in the Menkes and OHS diseases and also manifestations observed in dietary copper-deficient animals discussed in previous sections. This fact supports the suggestion that abnormalities in the functioning of LOX or its isoenzymes have a central role in the connective tissue defects seen in these conditions.

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3 Outlines of the present research

Lysyl oxidases play an important role in the formation of lysine-derived cross-links in collagens and elastin. These cross-links in turn stabilize the structures of these proteins in the extracellular matrix. Moreover, lysyl oxidases may have additional unknown intracellular or even intranuclear functions. The critical role of lysyl oxidase activity is well demonstrated by the defects in the collagen and elastin fibers that are seen in the Menkes and OHS diseases, their animal models, and also in lathyrism.

To date, little is known about the tissue, developmental stage, or substrate specifities of the emerging lysyl oxidase isoenzymes. Identification and characterization of these new isoenzymes and homozygous inactivation of one of them can be expected to give valuable information about their functions in a living organism. Therefore, the goals of this study were: 1. To attempt to identify, clone, and characterize additional human lysyl oxidase

isoenzymes. Subsequent goals were to study expression of the mRNAs for these isoenzymes in human tissues and to produce a recombinant protein in a mammalian expression system to study whether this protein is located in the extracellular space or whether it is found intracellularly or even intranuclearly.

2. To produce mice lacking the activity of the first characterized lysyl oxidase

isoenzyme (Lox) using homologous recombination in embryonic stem cells, and to analyse the consequence of the lack of this activity in the homozygous Lox-/- mice.

4 Materials and methods

Detailed descriptions of the material and methods are presented in the orginal papers I-III.

4.1 Cloning and characterization of the fourth and the fifth human lysyl oxidase isoenzymes (I, II)

4.1.1 Search for expressed sequence tags and high throughput genomic sequences (I, II)

The BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST/) was used to search all available databases for sequences that are similar to those of the coding regions of the human LOX (Hämäläinen et al. 1991), LOXL (Kenyon et al. 1993), and LOXL2 (Saito et al. 1997, Jourdan-Le Saux et al. 1999) cDNAs. Three high-throughput genomic sequences (AC005033, AC005041, AL139241) and one expressed sequence tag (AL751493) similar to LOXL2 sequence were found. Oligonucleotides for PCR were synthesized based on these sequences.

4.1.2 Isolation of cDNA clones (I, II)

To identify cDNA clones for LOXL3, the PCR primer pairs HLO406F/HLO406R and HLO402F/HLO404R were used to generate 204 bp and 527 bp products from a human placenta λgt11 cDNA library (Clontech). These PCR products were used to screen the same library, and two positive clones were obtained and characterized. To obtain the 5’end of the cDNA, the primers LO4cDNA1F and HLO406R were used for PCR with human placenta and fetus cDNA pools (Marathon Ready cDNA, Clontech). The PCR was performed using the Advantage 2 PCR Enzyme System kit (Clontech). Rapid

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amplification of the 5’ cDNA ends (5’RACE) was performed using the same cDNA pools as templates, and the Advantage 2 PCR Enzyme System kit with primer pairs AP1/HLO406R and AP1/LO4RNA1R. These PCR products were subcloned into the plasmid pUC18 (Amersham Pharmacia Biotech). All products were sequenced using standard methods.

To obtain cDNA clones for LOXL4, oligonucleotides HLO52F and HLO52R were used for PCR with a human kidney λgt11 cDNA library (Clontech). The PCR product was then used for screening the same library, and a 1611 bp clone was identified. To obtain the 5’end of the cDNA, the oligonucleotide LO5E1AR was used with AP2 according to the Marthon-Ready cDNA User Manual (Clontech) and a 347 bp product was obtained from a human fetus cDNA pool.

4.1.3 Characterization of the exon-intron organization of the LOXL3 gene (I)

The high throughput genomic sequences were compared with the cDNA sequences obtained using the exon prediction softwares GRAIL 1.3, FGENES, and Genie (http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html). To verify the position of the first intron, PCR was performed using the placenta and fetus cDNA pools and Advantage 2 PCR Enzyme System kit (Clontech) with primer pairs HLO4prom4F/HLO406R and HLO4prom4F/LO4RNA1R, yielding products of the sizes 971 bp and 371 bp, respectively.

4.1.4 Northern blot analysis (I, II)

Human multi-tissue Northern blots I and II were used for analysis of the expression of LOXL3 and LOXL4 mRNA species. For LOXL3 mRNA, a 971 bp PCR product from the 5’end of the cDNA was used as a probe. This probe was obtained by PCR with primers HLO4prom4F and HLO406R. For LOXL4 mRNA, the corresponding full-length cDNA was used as a probe.

4.1.5 Sequence analyses (I-III)

DNA sequencing was performed using an automated sequencer (ABI Prism 377, Applied Biosystems). DNASIS and PROSIS version 6.0 softwares (Pharmacia) were used to analyse the raw sequence data.

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4.1.6 Recombinant expression of the LOXL3 and LOXL4 polypeptides (I, II)

Recombinant expression of the LOXL3 and LOXL4 cDNAs was carried out using the mammalian expression vector pcDNA3.1/V5-HisA (Invitrogen). Expression constructs corresponding to the open reading frames of both cDNAs were generated by PCR with the human fetus cDNA pool as the template. These constructs contained, at their 3’ end, sequences encoding the V5 epitope and a His tag to be used for antibody staining of the recombinant protein. The plasmids were then transfected into human HT-1080 fibrosarcoma cells (American Type Culture Collection) using the Fugene6- transfection reagent (Boehringer Mannheim). The transfected HT-1080 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Inc.) containing 10% fetal bovine serum (FBS), 400 µg/ml G-418 sulphate and 100 units/ml penicillin and streptomycin at 37°C in 5 % CO2. Lysates were prepared from confluent plates by removing the medium, rinsing the cell layers with PBS, and suspending the cells at 37°C in Hank’s balanced salt solution containing 0.05 % trypsin and 0.53 mM EDTA. After addition of an equal volume of 10 % FBS/DMEM, the cells were sedimented, and the cell pellet was resuspended and washed in PBS. The resulting pellet was suspended in PBS, and aliquots from the cells were lysed by adding a solution of 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 5% β-mercaptoethanol, followed by heating at 100°C. For medium samples, confluent plates were cultured for 16 h, as above except that the medium contained no FBS. The samples for SDS/PAGE were concentrated using MicroSep centrifugal 10 kDa concentrators (Filtron). All samples were analyzed by 8% SDS/PAGE and immunoblotting with a monoclonal anti-V5 antibody (Invitrogen).

4.1.7 Immunofluorescence staining of the recombinant LOXL3 protein (I)

LOXL3 transfected HT-1080 cells seeded on glass coverslips and grown to the desired density were fixed in pre-cooled methanol at -20°C and incubated in 2% BSA-PBS, pH 7.2, to reduce nonspecific staining. A monoclonal anti-V5 antibody was applied at an appropriate dilution, and the samples were incubated for 1 h at room temperature, followed by extensive washing with PBS. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody was allowed to bind to the specimens for 1 h at room temperature. After extensive washing with PBS, the coverslips were mounted on microscope slides with Immu-Mount (Shandon) and viewed and photographed using a light microscope.

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4.2 Generation and analysis of a mouse strain lacking the Lox gene (III)

4.2.1 Generation of the mutant mice (III)

A genomic clone containing murine Lox gene sequences was isolated from the 129SV library using human LOX cDNA (Hämäläinen et al. 1991) as a probe. A 10.2 kb fragment containing the 5’end of the gene was subcloned into BlueScript plasmid (Promega), and the loxP sequence was inserted into a NsiI site located about 600 bp upstream of the translation start codon in front of the predicted transcription start sites (Contente et al. 1993). A HSV-tk-neor selection cassette flanked by loxP sites was inserted into a BamHI site located 90 bp downstream of the first exon (see Figure 1A in III).

The linearized targeting construct was electroporated into R1 embryonic stem (ES) cells (Nagy et al. 1993). Genomic DNA from the G418-resistant ES clones was digested with SpeI and hybridized with a B1/SpeI external probe. Cells from correctly targeted ES clones were electroporated with a pIC-Cre plasmid and grown in the presence of Gancyclovir. DNA from clones that had survived selection was digested with NsiI and hybridized with the B1-E1/E1 probe. Cells in which the selection cassette and the first exon of the Lox gene had been deleted were injected into C57BL/6 blastocysts, which were then implanted into pseudopregnant females. The resulting chimeras were bred with C57BL/6 mice to produce heterozygous mutant mice. By using heterozygous siblings, mutant embryos were obtained. Embryos were genotyped by Southern blotting, and total RNA was extracted from them. Northern blots from extracted total RNA were prepared by routine methods (see Figure 1 in III).

4.2.2 Light microscopy (III)

Tissue samples were fixed overnight in 10% buffered formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin and Masson’s trichrome. The hematoxylin-eosin-stained aorta sections were examined under ultraviolet light. Processing and sectioning of Lox+/+ and Lox-/- samples were performed parallelly and were inspected without knowledge of the genotype. The diameter of the aortic lumen and wall thickness were measured from the proximal part of the descending aorta. Student’s t-test were used for establishing significant differences among groups.

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4.2.3 Transmission electron microscopy (III)

Biopsies from the descending aorta of E18.5 embryos were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in Epon LX112. Thin sections were cut with a Reichert Ultracut ultramicrotome and examined in a Phillips CM100 transmission electron microscope.

4.2.4 Ultrasonographic examination of the embryos (III)

Anesthesized embryos were examined using an Acuson Sequoia 512 Doppler ultrasonograph with a 13 MHz linear probe. The embryonic heart was identified by color Doppler, and the sample volume of the pulsed Doppler was placed over it. The length of the sample volume was adjusted to cover the entire heart, and the high pass filter was set at its minimum. Different views of the heart were examined to minimize the angle between the Doppler beam and the inflow and outflow regions and to obtain their maximal velocities. The maximal inflow and outflow velocities were recorded using a sweep speed of 100 mm/s. Different vessels were located on the sagittal view of the embryo with the help of color Doppler, and blood velocity waveforms were obtained by the pulsed Doppler method (see Figure 4A in III). Immediately after the ultrasonographic examination, the dam was sacrificed, the abdomen carefully opened, and the embryos identified according to their location in ultrasonography.

The ultrasonographic data were analysed using the cardiovascular measurement package included with the ultrasound equipment. Time-velocity-integrals were measured from the inflow and outflow blood velocity waveforms by planimetry of the area underneath the Doppler spectrum. The fetal heart rate was obtained, and the inflow and outflow mean velocities were calculated. Pulsatility index values were obtained from the descending aorta, intracranial arteries, and umbilical artery blood velocity waveforms, and pulsatility index values for veins were calculated from the ductus venosus blood velocity waveforms. The Doppler ultrasonographic parameters were analyzed statistically using Student’s t-test.

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5 Results

5.1 The fourth and the fifth lysyl oxidase isoenzymes (I, II)

5.1.1 Molecular cloning of the cDNAs (I, II)

To find additional genes for human lysyl oxidase isoenzymes, the available databases were searched using sequences of all known human lysyl oxidase isoenzymes. This search resulted in the identification of three high-throughput genomic sequences (AC005033, AC005041 and AL139241) and one expressed tag sequence (AL751493), which were highly similar to the LOX (Hämäläinen et al. 1991), LOXL (Kenyon et al. 1993), and LOXL2 (Saito et al. 1997, Jourdan-Le Saux et al. 1999) sequences. Based on the information obtained from high-throughput genomic sequences AC005033 and AC005041 two primer pairs were synthesized, and then used to generate PCR products of 240 and 527 bp in lenght from a human placenta cDNA library. These PCR products were used as probes to screen the same library. Two LOXL3 cDNA clones covering nucleotides 825-2574 were obtained and sequenced. Based on the information obtained from these two cDNA clones and two high-throughput genomic clones, several primers were tested to obtain the 5’end of the cDNA. By doing this, a 913 bp PCR product including an overlapping 5’region of the cDNA was obtained. To obtain a 5’RACE product, the primers AP1 and HLO406R were used in the first round of PCR, and AP1 and LO4RNA1R in the second round, yielding a product of 388 bp.

Based on the information obtained from sequences AL139241 and AL751493 the full-length LOXL4 cDNA was obtained using several primers, as described in Material and Methods.

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5.1.2 Molecular characterization of the cDNAs (I, II)

The full-length LOXL3 cDNA obtained was 2574 bp in size, and contained an open reading frame of 2226 bp. The 5’ noncoding region was 74 bp in length and the 3’ noncoding region 238 bp. The cloned cDNA had no polyadenylation signal, which indicated that the actual lenght of the 3’ noncoding region is longer than 238 bp. The open reading frame encoded a polypeptide of 753 amino acids (Figure 1 in I). A putative signal peptide was present in the N-terminus, and was likely to be cleaved between amino acids 25 and 26. The size of the processed polypeptide was, therefore, 728 residues, with predicted molecular mass of 80.3 kDa. The nonprocessed LOXL3 polypeptide shared a 55% overall amino acid sequence identity with the LOXL2 polypeptide, whereas its residues 1-528 shared no significant identity with the LOX and LOXL polypeptides. However, the C-terminal residues from 529 to 729 showed a high degree of identity with the LOX, LOXL, and LOXL2 polypeptides, namely 51%, 53%, and 69%, respectively.

The LOXL4 mRNA is at least 2736 bp in size and contains an open reading frame of 2271 bp. The LOXL4 gene is located on chromosome 10 (based on the sequence AL139241) and encodes a polypeptide of 756 amino acids, including a signal peptide of 24 residues (Figure 1 in II). The LOXL4 polypeptide shows a high overall identity with the LOXL2 and LOXL3 polypeptides, the degrees of identity being 63.9% and 65.5%, respectively. The highest identity is found within the lysyl oxidase (LO) domain, being 85% for LOXL2 and 87.4% for LOXL3 (Figure 1 in II). An extensive similarity of the LOXL4 polypeptide to the LOX and LOXL polypeptides is found only within the LO domain, with identities of 51.1% and 50.2%, respectively. In addition, the human LOXL4 polypeptide shows a 93.7% overall identity with a recently reported murine Loxc polypeptide (Ito et al. 2001), indicating that these two cDNAs encode the same polypeptide in different species.

As mentioned above, LOXL3 and LOXL4 polypeptides showed a high degree of overall similarity to each other and to the LOXL2 polypeptide, whereas the two polypeptides showed a significant similarity to LOX and LOXL only in the C-terminal region. This region contains a putative copper-binding sequence (Krebs & Krawets 1993) (WVWHECHGHYH, residues 601-611 in the LOXL3, and WVWHQCHRHYH, residues 605-615 in the LOXL4 seqeuence), a putative collagen-related copper-affinity site (Krebs & Krawetz 1993) (GHK, residues 632-634 in the LOXL3, and residues 636-638 in the LOXL4 sequence), as well as lysine and tyrosine residues (Lys-634 and Tyr-670 in the LOXL3, and Lys-638 and Tyr-674 in the LOXL4 sequence) (note an error in Figure 1 and text in I and II), which are involved in the formation of the lysine tyrosylquinone cofactor (Wang et al. 1996), and a growth factor and cytokine binding sequence (Bazan 1989, 1990, Kim et al. 1995) (C-X9-C-X-W-X26-32-C-X10-15-C, residues 666-727 in the LOXL3, and residues 670-731 in the LOXL4 sequence). All these residues are highly conserved in all lysyl oxidase isoenzymes (Figure 1 in I and II). In addition, the putative sites for proteolytic processing by procollagen C-proteinase, Gly-447 and Asp-448, were found in LOXL3 sequence (Cronshaw et al. 1995, Panchenko et al. 1996), but not in the LOXL4 sequence. The 10 cysteine residues present in LOX are entirely conserved in both polypeptides (Figure 1 in I and II).

46 The N-terminal regions of the LOXL3 and LOXL4 polypeptides contains four repeats

of scavenger receptor cysteine-rich (SRCR) repeats (Resnick et al. 1994) that are also found in the LOXL2 polypeptide (Saito et al. 1997, Jourdan-Le Saux et al. 1999) but not in the LOX and LOXL polypeptides (Figure 1 in I and II). In this N-terminal region, all 35 cysteine residues are totally conserved between the processed LOXL2, LOXL3 and LOXL4 polypeptides. In addition, this region of LOXL3 polypeptide contain three putative O-glycosylation and five putative N-glycosylation sites, and the corresponding region of LOXL4 polypeptide contain three putative O-glycosylation and two putative N-glycosylation sites. A bipartite nuclear localization signal (Dingwall et al. 1991, Boulikas 1994) was found in LOXL3 sequence (KKQQQSKPQGEARVRLKG, in residues 293-311), but was not found in any other lysyl oxidase isoenzymes.

5.1.3 Exon-intron organization of the LOXL3 gene (I)

The LOXL3 gene has 14 exons and is located on chromosome 2p13 based on the information obtained from the high-throughput sequences AC005033 and AC005041. As suggested by 5’RACE experiments, this gene may have its first intron within the 5’ untranslated region. To verify the existence of this intron, PCR was performed with pools of human placental and fetal cDNAs using two pairs of primers. These reactions gave products with the expected sizes of 971 and 371 bp, thus indicating the presence of this intron.

The sizes of exons 2-13 vary from 112 to 247 bp, and the sizes of the introns vary from 79 to 12,440 bp, intron 4 being the longest (Table 1 in I). All the exon-intron boundaries show the consensus sequence (C/T)AG-exon-GT(A/G), except those of exons 3 and 7, in which the sequence at the 5’ splice donor site is GTC.

The 3’ untranslated region of the LOXL3 gene has a TTATATAAAAA sequence 213 bp downstream of the 3’ end of the cloned cDNA, a TAAATATAT sequence 837 bp downstream of the 3’ end, and a GCAATAAAGT sequence 904 bp downstream of the 3’ end, all of which may act as polyadenylation signals.

5.1.4 Expression of the mRNAs in various human tissues (I, II)

Northern analyses indicated the presence of only a single LOXL3 mRNA of about 3.1 kb, and a single LOXL4 mRNA of about 4.0 kb in size. The highest expression levels of LOXL3 mRNA were seen in the placenta, heart, ovary, testis, small intestine, and spleen. However, distinct hybridization signals were obtained from all the tissues examined (Figure 2 in I). The highest expression levels of LOXL4 mRNA among the tissues studied were seen in the skeletal muscle, testis, and pancreas, whereas no signal was detected in the brain or in blood leukocytes (Figure 2a in II).

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5.1.5 Recombinant expression of the LOXL3 and LOXL4 polypeptides in human HT-1080 cells (I, II)

Recombinant expression of the LOXL3 and the LOXL4 polypeptides were studied by transfecting human HT-1080 cells with a construct that also contained sequences encoding the V5 epitope and a six-histidine tag at its 3’ end. In addition, recombinant LOXL4 polypeptide was also studied by transfecting CHO cells and mouse embryonic fibroblasts with the same construct. Western blots of crude lysates from the transfected cells by antibodies to the V5 epitope showed the expression of a 97 kDa LOXL3 and LOXL4 polypeptides, whereas no staining was seen in lysates from nontransfected cells (Figure 3 in I, and Figure 2b in II). Analysis of concentrated medium samples showed the presence of two major LOXL3 and LOXL4 polypeptides of slightly larger sizes than the polypeptides present in the cell lysates, as well as a very minor 97 kDa polypeptides (Figure 3 in I, and Figure 2b in II). No intracellular or extracellular processing of the polypeptides were detected in HT-1080 (Figure 3 in I, and Figure 2b in II) or CHO cells, or in mouse embryonic fibroblasts (data not shown).

Immunofluorescence staining of the LOXL3 transfected HT-1080 cells by a monoclonal antibody specific for the V5 epitope gave a strong cellular signal, but no signal could be detected in the nuclei (Figure 4 in I).

5.2 Inactivation of the Lox gene leads to cardiovascular dysfunction in mice (III)

5.2.1 Generation of mice with an inactivated Lox gene (III)

ES cells carrying an inactivated Lox gene were generated by a two-step targeting method comprising homologous recombination followed by cre recombination (Figure 1A-D in III). The targeted ES cells were used to generate chimeras, of which one chimeric male transmitted the mutation to his offspring when mated with wild-type females. Deletion of the first exon, including the transcription start sites, led to complete inactivation of the Lox gene, as seen in the Northern blots (Figure 1E in III). Heterozygous Lox+/- mice, which are viable and fertile, were crossbred, and the offspring were genotyped at two weeks of age. The numbers of Lox+/+ and Lox+/- pups (n = 111) were in perfect balance, comprising 32.4% and 67.6%, respectively, but no Lox-/- pups were obtained.

To study the time of death of the Lox-/- mice, pregnant Lox+/- females were sacrificed 18.5 days after breeding with Lox+/- males, and the embryos were genotyped. Here, a normal Mendelian ratio of all three genotypes was obtained (26.7% Lox+/+, 45% Lox+/-, 28.3% Lox-/-, n = 127). Pregnant females were then carefully observed during the assumed day of delivery, and in this way four live-born Lox-/- pups were obtained for

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analysis and three were found dead. In addition, the remnants of three Lox-/- pups were recovered, which were used for DNA analysis. The gross appearance of the four newborn Lox-/- pups was normal, but they had cyanotic skin and their condition was poor. They were breathing, but were sluggish and did not suck milk (Figure 2A in III). Autopsy revealed large aneurysms (Figure 2B in III) in the aortas of three of the four. A narrowed lumen was observed in paraffin sections of the abdominal aorta of all four pups, and a large aneurysmal dilatation, of a diameter approximately 3 times larger than in the aortas of their wild-type littermates, in sections from two pups (Figure 2C and D in III). The three neonatal Lox-/- pups that had died immediately after birth had a congenital diaphragmatic hernia, and two of them also had a large hemorrhage in the upper chest region.

5.2.2 Histological findings (III)

Since preliminary analyses indicated defects in blood vessels, aortas from embryos at different stages were taken for detailed analysis. Alterations in the structure of the aortic wall were already seen at E14.5, which was the earliest time point analyzed, but were most obvious in the E18.5 embryos. Light microscopy revealed hazy and unruffled elastin lamellae in the Lox-/- samples, whereas the lamellae in the wild-type aorta were well defined and ruffled. The wall of the aorta in the Lox-/- embryos was significantly thicker (P<0.005; see Table 1 in III) and the diameter of the aortic lumen was significantly smaller (P<0.001; see Table 1 in III) than in the Lox+/+ aortas. Most of the endothelial cells on the internal elastic lamellae in the Lox-/- aortic wall were rounded in appearance, whereas those in the Lox+/+ aorta were flat (Figure 3A and B in III). No diaphragmatic hernias were found in the E16.5-18.5 Lox-/- embryos, even though these were seen in three out of the seven Lox-/- neonates.

Electron microscopy of the Lox-/- aortas indicated that the elastic laminae were fragmented, having only remnants of their elastic fibers, and the smooth muscle cell layer was disorganized. The endothelial cells showed degenerative changes such as vacuolization, blebbing, and even detachment from the elastic lamina (Figure 3C and D in III). No major differences in the collagen fibers of the aortic adventitia were seen between the Lox-/- and Lox+/+ embryos by electron microscopy.

5.2.3 Ultrasonographic findings (III)

Since the Lox+/- adult mice seemed to be fertile and have a normal life-span, we continued to compare the data from Lox-/- and Lox+/+ embryos only. No difference in fetal heart rate was found between the two groups, but the pulsatility indices for the descending aorta, umbilical artery, and intracranial arteries were significantly higher, and the mean outflow and inflow velocities were significantly lower in the Lox-/- embryos

49

(Table 2 in III). In addition, embryos with the highest pulsatility indices in the descending aorta had semilunar valve regurgitation. The pulsatility indices for veins in the ductus venosus were found to be significantly higher in the Lox-/- embryos (Table 2 in III), and were diagnosed as a sign of congestive heart failure. In addition, embryos with the highest pulsatility indices in the descending aorta had semilunar valve regurgitation (see Figure 4B-C in III).

6 Discussion

6.1 Cloning and characterization of the fourth and the fifth lysyl oxidase isoenzymes (I, II)

6.1.1 Primary structures of the LOXL3 and LOXL4 polypeptides (I, II)

We identified two additional human lysyl oxidase isoenzymes; lysyl oxidase-like 3 protein (LOXL3) and lysyl oxidase-like 4 protein (LOXL4). Our findings were largely supported by Huang et al. (2001) and Jourdan-Le Saux et al. (2001), concerning the characterization of LOXL3 isoenzyme, and by Asuncion et al. (2001), concerning the characterization of LOXL4 isoenzyme. Furthermore, the mouse lysyl oxidase-related protein 2 identified by Jang et al. (1999) was found to be orthologous to human LOXL3 (Huang et al. 2001, Jourdan-Le Saux et al. 2001).

The LOXL3 and LOXL4 polypeptides have a high overall similarity to each other, but also to the LOXL2 polypeptide, and each of them has four conserved SRCR domains at their N-terminal region (see Figure 5, and Figure 1 in II). LOXL3 and LOXL4, however, share a significant similarity to isoenzymes LOX and LOXL only in the C-terminal region, which contains the putative copper-binding region (Krebs & Krawetz 1993), the lysine and tyrosine residues that form the lysine tyrosylquinone cofactor (Wang et al. 1996), and a growth factor and cytokine binding domain (Bazan 1989, 1990, Kim et al. 1995). These features of the catalytic i.e. LO domain are highly conserved within all lysyl oxidase isoenzymes (Figure 5).

The existence of a putative signal sequence and four SRCR domains suggests that the LOXL3 and LOXL4 isoenzymes are extracellular proteins (Figure 5). SRCR domains have been reported to be able to interact with other proteins (Resnick et al. 1994, Yamada et al. 1998), and therefore LOXL3 and LOXL4 may be involved in the binding and cross-linking of cell surface and extracellular matrix proteins. Interestingly, LOXL2 has been suggested to be involved in cell adhesion based on its significantly higher expression levels in adherent than nonadherent cell lines (Saito et al. 1997). Based on these data,

51

Saito et al. (1997) proposed that the LOXL2 function may involve posttranslational modification of extracellular matrix components or other cell membrane proteins. This would be also a plausible function for LOXL3 and LOXL4 because of their SRCR domains.

Fig. 5. The lysyl oxidase family. The predicted signal peptides are represented by black boxes, and four scavenger receptor cysteine-rich regions (SRCR) in LOXL2, LOXL3, and LOXL4, the propeptide region in LOX, and the proline-rich region in LOXL are also indicated. The sites of the putative copper-binding region (Cu), the lysine tyrosylquinone cofactor formation (LTQ), and the cytokine receptor-like domain (CRL) are highly conserved between all lysyl oxidase proteins. The number of amino acids in each protein is indicated on the right.

6.1.2 The structure of the LOXL3 gene, and expression of LOXL3 and LOXL4 mRNAs in various tissues (I, II)

According to the information gained from HTGS sequences, the LOXL3 gene has 14 exons, and it is located on chromosome 2p13. The gene encodes a single 3.1 kb mRNA identified in Northern blots from several tissues. The size of the mRNA corresponds well to the size of 2.8 kb for an mRNA lacking the poly(A)+ tail, assuming that the first of the three potential polyadenylation signals is used. Northern analyses indicated the highest expression levels of the LOXL3 mRNA in the placenta, heart, ovary, testis, small intestine, and spleen. Distinct hybridization signals were, however, obtained from all tissues examined.

Based on the HTGS sequence, the LOXL4 gene is located on chromosome 10. It codes for a single mRNA of approxymately 4.0 kb in size, which is expressed in most human tissues studied, the highest expression levels were seen in skeletal muscles, testis, and pancreas. No expression, however, was detected in the brain or blood leukocytes. Our findings disagree with those made by Ito et al. (2001) concerning the corresponding

52

mouse gene, Loxc, that was reported to show a cartilage specific expression. Because the corresponding human gene is expressed in many different human tissues, it would seem appropriate to call this new polypeptide LOXL4 rather than LOXC.

6.1.3 Recombinant expression of the LOXL3 and LOXL4 polypeptides (I, II)

Western analyses of lysates from both the LOXL3- and LOXL4-transfected HT-1080 cells showed the expression of a 97 kDa polypeptide. This size is slightly larger than the estimated overall molecular masses of 83.6 kDa for LOXL3 and 84.5 kDa for LOXL4. The differences are probably due to the V5 epitope and the histidine tag located at the C-terminal end of the recombinant polypeptides. Analysis of concentrated medium samples showed the presence of at least two major LOXL3 polypeptides and at least one LOXL4 polypeptide slightly larger in size than the corresponding polypeptides present in the cell lysates, as well as a very minor 97 kDa polypeptide. Since both recombinant polypeptides have several putative glycosylation sites, it is possible that these differences in sizes are due to utilization of the glycosylation sites.

The 50 kDa precursor of human LOX is cleaved by procollagen C-proteinase between Gly-168 and Asp-169 to yield a non-glycosylated, 32 kDa mature enzyme (Cronshaw et al. 1995, Panchenko et al. 1996). Two putative Gly-Asp processing sites are found in the bovine LOXL polypeptide (Borel et al. 2001), while one such site is present in the human LOXL2 polypeptide (Jourdan-Le Saux et al. 1999), but the possible utilization of none of these sites has been demonstrated experimentally. The bovine LOXL protein was found to be largely inactive, but was activated by processing with procollagen C-proteinase, suggesting that one or both of these sites may be involved in the processing event (Borel et al. 2001). A similar potential cleavage site for procollagen C-proteinase was also found in the LOXL3 polypeptide. If proteolytic processing of the LOXL3 polypeptide occurred between Gly-447 and Asp-448, the predicted size of the cleaved product would be 306 amino acids, with a theoretical molecular mass of 34.8 kDa. However, no intracellular or extracellular processing of the LOXL3 polypeptide was detected during recombinant expression in the transfected HT-1080 cells or by the addition of conditioned medium of human primary skin fibroblasts (data not shown).

The consensus sequence for the procollagen C-proteinase cleavage site may not be highly conserved (see Borel et al. 2001 for details). Therefore, despite of the lack of any potential procollagen C-proteinase cleavage site in the LOXL4 polypeptide, this isoenzyme was also tested for possible processing by recombinant expression in HT-1080 and CHO cells and in mouse embryonic fibroblasts (data not shown). No intracellular or extracellular processing of the LOXL4 polypeptide was detected in these cells. The results do not, however, exlude the possibility that the nonrecombinant LOXL3 and LOXL4 polypeptides may be processed in some other cell types, and thus further studies are needed to exclude or demonstrate the processing event.

53 The N-terminal region of the LOXL3 polypeptide contains a bipartite nuclear

localization signal (KKQQQSKPQGEARVRLKG), which is not found in any other lysyl oxidase isoenzyme. Despite the existence of the nuclear localization signal, our results suggest that LOXL3 may not contribute to the lysyl oxidase activity identified in the nucleus, which has been shown to be due at least in part to the LOX polypeptide (Li et al. 1997). The present data do not exclude the possibility that the nonrecombinant LOXL3 polypeptide may be found in the nuclei of cells of some other type, and thus further studies are needed to either exclude or identify a nuclear location.

6.2 Lack of Lox activity leads to a severe cardiovascular dysfunction and perinatal death in mice (III)

The consequence of inactivation of the Lox gene, neonatal lethality, was highly surprising, considering that Lox has at least four additional isoenzymes, as discussed in previous sections, mice lacking elastin survive for up to four days (Li et al. 1998), and mice totally lacking type III collagen can survive for up six months (Liu et al. 1997b). Our results show that Lox has an essential function in the development of the embryonic cardiovascular system, and particulary in the maintenance of the structure and mechanical properties of the aortic wall. Deficiency in lysyl oxidase activity is likely to affect several aortic wall components, including its major components elastin and collagen types I and III (see Jacob et al. 2001, Silver et al. 2001 for review), but may also affect components found in minor amounts. The early development of the aorta, and the translation and secretion of the aortic wall extracellular matrix components, are likely to occur normally in Lox-/- embryos, as evidenced by the normal number of elastic lamellae and smooth muscle cell layers. Elastin exists in elastic fibers in two forms: as tropoelastin, a soluble precursor of elastin, and as a highly cross-linked elastin in the amorphous component (Cleary & Gibson 1996, Vrhovski & Weiss 1998). Assembly of tropoelastin into the amorphous component involves covalent cross-linking into a highly insoluble form (Cleary & Gibson 1996, Vrhovski & Weiss 1998). Lox has been localized immunologically to the mature elastic fibers (Kagan et al. 1986, Baccarani-Contri et al. 1989), and the formation of lysyl oxidase-catalysed cross-linking has been suggested to play a critical role in the nucleation of the elastin assembly (Brown-Augsburger et al. 1995). Our results, therefore, suggest that other lysyl oxidase isoezymes may also play a role in elastin cross-linking, since the amorphous and insoluble component of the elastic fibers exists in Lox-/- embryos, as seen in Figure 6. It is possible that the diminished amount of cross-links in Lox-/- embryos increases the susceptibility of elastin to degradation by matrix metalloproteinases (Tinker et al. 1990, Grange et al. 1997), which would lead to the fragmentation of the elastic fibers. As a result, the fragmented elastic fibers are probably unable to bear the hemodynamic stress, which leads to dilatation and rupture of the aortic wall. A similar destruction of the elastic fibers, as is seen in the aortic walls of the Lox-/- embryos, has been hypothesized to play a key role in the formation of an aneurysmal aortic dilatation, thus shifting the load produced by the blood pressure

54

onto collagens, with consequent dilatation and eventually rupture of the aortic wall (MacSweeney et al. 1992, 1994). Disintegration of the intimal and medial elastic lamellaes, to which the endothelial cells and smooth muscle cells attach, also results in the degeneration of endothelial cells and disorganization of smooth muscle cells. In elastin null mice, smooth muscle cells proliferate and thereby stabilize the arterial structure, but finally obliterate the arterial lumen (Li et al. 1998). We did not observe any significant compensatory smooth muscle cell proliferation in the Lox-/- aortas, probably because normal amounts of elastin were produced, at least during the early stages. It has been suggested that elastin may control the proliferation of vascular smooth muscle cells (Li et al. 1998), but polymerization does not seem to be necessary for this regulatory function.

Fig. 6. Structure of the aortic wall of the Lox+/+ and Lox-/- E18.5 embryos. Upper panels: Sections from the descending aorta at the site of the right crus of the diaphragm, stained with hematoxylin-eosin and illuminated in UV light. The lumen of the aorta is marked with an asterisk, and the single intimal elastic lamella is boxed. Lower panels: Electron micrographs from the site of a single elastic fiber from the boxed sections in upper panels: Amorphous elastin (aEL) in a continuous elastic fiber is seen in the Lox+/+ aortic wall (left). Despite the high degree of fragmentation of the elastic fiber, the remnants of the amorphous elastin (arrows) are still seen in the Lox-/- aortic wall (right).

55 To study the actual physiological consequences of structural abnormalities found in

the Lox-/- aortic walls, we decided to use ultrasonographic studies. Increased pulsatility indices were measured in the umbilical artery, descending aorta, and intracranial arteries in Doppler ultrasonography of the E18.5 Lox-/- embryos. By comparison, although increased impedance of the umbilical artery and descending aorta is also found in human pregnancies with placental insufficiency, it is connected with a concomitant decrease in pulsatility indices in the cerebral circulation, as the fetus aims to maintain an adequate oxygen supply to the coronary and cerebral circulation (Reed 1997). Therefore, the universally increased arterial pulsatility indices observed in the Lox-/- embryos are likely to be due to abnormal elastic properties of the arterial walls, as discussed above, and not due to placental insufficiency.

The decreased mean velocities in the inflow and outflow regions suggest a lower cardiac output in Lox-/- than in Lox+/+ embryos. In order to maintain adequate blood supply and tissue perfusion, arterial blood pressure should be increased, as suggested in fetuses with intrauterine growth restriction and lower relative pulse amplitude in the descending aorta (Stale et al. 1991). The increase in systolic blood pressure in the Lox-/-

embryos, in combination with aortic wall fragility, would then lead to the formation of aortic aneurysms and ruptures, which are otherwise rare in newborns. A decrease in the resilience of the arterial walls will lead to an increased afterload on the heart, as evidenced by the fact that the two Lox-/- embryos with the highest pulsatility indices in the descending aorta, and thus the greatest rise in the afterload, also had semilunar valve regurgitations. Furthermore, the increased pulsatility indices for veins in the ductus venosus imply an increase in systemic venous pressure, indicating congestive heart failure (Reed et al. 1997, Kiserud et al. 1999), which may contribute to the death of Lox-/-

mice at the end of gestation or during delivery. Reduced lysyl oxidase activity is found in animals with copper deficiency, in

lathyrism, and in two X-linked, recessively inherited human disorders, Menkes syndrome and OHS, as discussed in Section 2.5. Lathyrism is caused by administration of βAPN, a potent irreversible inhibitor of the LOX enzyme, while the low lysyl oxidase activity levels in Menkes disease, OHS, and copper-deficiency are secondary to abnormalities in copper metabolism. In all these conditions, all lysyl oxidase isoenzymes are thought to be affected, but their activities are not completely abolished. The present study provides the first evidence indicating that the lack of activity of the single isoenzyme Lox leads to major defects in the vascular elastic fibers. Mottled blotchy mice, a mouse model for OHS, die of aortic ruptures before six months of age, and the histological findings of early changes in their aortas (Brophy et al. 1988) are similar to those seen here in the Lox-/- embryonic aortas and closely resemble the pathological alterations in elastic fibers found in human aneurysms. In blotchy mice, the level of total lysyl oxidase activity in the aortic tissue is decreased by 59% (Moursi et al. 1995), but since these mice are born without aneurysms or other cardiovascular abnormalities, this level of activity must be sufficient during embryogenesis. Our data suggest that significantly reduced activity of the human LOX enzyme may also contribute to cardiovascular symptoms, and therefore alterations in LOX activity may play a critical role in human cardiovascular diseases.

All lysyl oxidase isoenzymes are likely to be able to use both elastin and collagens as their substrates, as has so far been shown for three of the five isoenzymes (Kagan 1986,

56

Borel et al. 2001, Ito et al. 2001). Our data indicate, however, that the Lox functions were not compensated for by other isoenzymes, at least not to any significant extent. Thus, Lox clearly has a central role among the isoenzymes in the development of the cardiovascular system during embryogenesis, and the functions and significance of the other isoenzymes remain to be explored.

In conclusion, the pathophysiological sequence of events in the cardiovascular system of Lox-/- embryos suggests that deficient cross-linking of elastin and collagens causes a decline in the resilience and tensile strength of the arterial walls that leads to endothelial cell damage and disorganization of the smooth muscle cells, and causes severe abnormalities in the cardiovascular functions, finally contributing to perinatal death. In some of the cases, a diaphragmatic hernia may contribute to neonatal lethality. Our findings suggest that alterations in LOX activity may also play a critical role in human cardiovascular diseases. Therefore, this mouse model will be a useful tool for further studies.

7 Future perspectives

To this date, five human lysyl oxidase isoenzymes have been characterized (Hämäläinen et al. 1991, Mariani et al. 1992, Kenyon et al. 1993, Saito et al. 1997, Jourdan-Le Saux et al. 1999, Asuncion et al. 2001, Huang et al. 2001, Jourdan-Le Saux et al. 2001, papers I and II in present study). All these isoenzymes are likely to be able to use both elastin and collagens as their substrates, as has so far been shown for LOX (Kagan 1986), LOXL (Borel et al. 2001), and LOXL4 (Ito et al. 2001). The primary structures of the LOX and LOXL polypeptides differ dramatically from those of the most recently characterized isoenzymes LOXL2, LOXL3, and LOXL4. The differences in the structural properties of these isoenzymes may indicate differences in their functions or their location in the extracellular matrix. This would be plausible since LOX is known to have a very broad substrate specifity (see Smith-Mungo & Kagan 1998 for review) and may have unidentified intracellular (Wakasaki & Ooshima 1990b) or intranuclear substrates (Li et al. 1997). The use of monoclonal and polyclonal antibodies prepared against the recently identified isoenzymes should provide more information about their tissue distribution, their expression in various cell types, and even their location within the cell. Further purification of the new isoenzymes would provide a proper tool for their extensive functional and biochemical characterization.

Since the lack of Lox activity leads to highly fragmented elastin fibers in the aorta, it can be assumed that abnormalities in elastic fibers may be found also in other tissues. The manifestations in lathyrism, Menkes disease, and OHS include several defects associated with abnormal elastic fibers, but also defects in collagens in various tissues. Although no major abnormalities were observed in collagen fibers of Lox-/- embryos by electron microscopy, further immunological and biochemical analyses of collagens are needed. Therefore, further detailed and extensive analyses of Lox-/- embryos may provide essential information about possible specific roles of Lox in various cell and tissue types, and in certain developmental stages of the growing embryo.

The heterozygous adult Lox+/- mice may turn out to be valuable animal models for research on certain cardiovascular diseases. By using the Cre/loxP-method, a conditional or tissue-specific knockout mouse model could be produced in the future, thus giving the opporturnity to analyze the consequences of the lack of Lox activity in adult mice.

8 References

Akagawa M & Suyama K. (2001) Characterization of a model compound for the lysine tyrosylquinone cofactor of lysyl oxidase. Biochem Biophys Res Commun 281: 193-199.

Almassian B, Trackman PC, Iguchi H, Boak A, Calvaresi D & Kagan HM. (1991) Induction of lung lysyl oxidase activity and lysyl oxidase protein by exposure of rats to cadmium chloride: properties of the induced enzyme. Connect Tissue Res. 25: 197-208.

Andrews EJ, White WJ & Bullock LP (1975) Spontaneus aortic aneurysms in blotchy mice. Am J Pathol 78: 199-210.

Asuncion L, Fogelgren B, Fong KS, Fong SF, Kim Y & Csiszar K (2001) A novel human lysyl oxidase-like gene (LOXL4) on chromosome 10q24 has an altered scavenger receptor cysteine rich domain. Matrix Biol 20: 487-491.

Baccarani-Contri M, Vincenzi D, Quaglino D Jr, Mori G & Pasquali-Ronchetti I. (1989) Localization of human placenta lysyl oxidase on human placenta, skin and aorta by immunoelectronmicroscopy. Matrix 9: 428-36.

Bailey AJ, Paul RG & Knott L (1998) Mechanisms of maturation and ageing of collagen. Mech Ageing Dev 106: 1-56.

Bailey AJ (2001) Molecular mechanisms of ageing in connective tissues. Mech Ageing Dev 122: 735-55.

Bateman JF, Lamandé SR & Ramshaw JAM (1996) Collagen Superfamily. In: Comper WD (ed) Extracellular Matrix, Vol. 2. Harvood Academic Publishers, Amsterdam, pp. 22-67.

Bazan JF (1989) A novel family of growth factor receptors: a common binding domain in the growth hormone, prolactin, erythropoietin and IL-6 receptors, and the p75 IL-2 receptor β-chain. Biochem Biophys Res Commun 164: 788-795.

Bazan JF (1990) Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci USA 87: 6934-6938.

Boak AM, Roy R, Berk J, Taylor L, Polgar P, Goldstein RH & Kagan HM (1994) Regulation of lysyl oxidase expression in lung fibroblasts by transforming growth factor-β1 and prostaglandin E2. Am J Respir Cell Mol Biol 11: 751-5.

Borel A, Eichenberger D, Farjanel J, Kessler E, Gleyzal C, Hulmes DJ, Sommer P & Font B (2001) Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1. J Biol Chem 276: 48944-48949.

59

Bose KK, Chakraborty J, Khuder S, Smith-Mensah WH & Robinson J (2000) Lysyl oxidase activity in the cells of flexor retinaculum of individuals with carpal tunnel syndrome. J Occup Environ Med 42: 582-587.

Boulikas T (1994) Putative nuclear localization signals (NLS) in protein transcription factors. J Cell Biochem 55: 32-58.

Boyd CD, Mariani TJ, Kim Y & Csiszar K (1995) The size heterogeneity of human lysyl oxidase mRNA is due to alternate polyadenylation site and not alternate exon usage. Mol Biol Rep 21: 95-103.

Bronson RE, Calaman SD, Traish AM & Kagan HM (1987) Stimulation of lysyl oxidase (EC 1.4.3.13) activity by testosterone and characterization of androgen receptors in cultured calf aorta smooth-muscle cells. Biochem J 244: 317-323.

Brophy CM, Tilson JE, Braverman IM & Tilson MD (1988) Age of onset, pattern of distribution, and histology of aneurysm development in a genetically predisposed mouse model. J Vasc Surg 8: 45-48.

Brown-Augsburger P, Tisdale C, Broekelmann T, Sloan C & Mecham RP (1995) Identification of an elastin cross-linking domain that joins three peptide chains. Possible role in nucleated assembly. J Biol Chem 270: 17778-17783.

Butler E, Hardin J & Benson S (1987) The role of lysyl oxidase and collagen cross-linking during sea urchin development. Exp Cell Res 173: 174-182.

Byers PH, Siegel RC, Holbrook KA, Narayanan AS, Bornstein P & Hall JG (1980) X-linked cutis laxa: defective cross-link formation in collagen due to decreased lysyl oxidase activity. N Engl J Med 303: 61-65.

Byers PH (2001) Disorders of Collagen Biosynthesis and Structure. In: Scriver CR, Beaudet AL, Sly WS & Valle D (eds) The Metabolic & Molecular Bases of Inherited Disease, Vol. IV. McGraw-Hill Medical Publishing Division, pp. 5241-5285.

Carter EA, McCarron MJ, Alpert E & Isselbacher KJ (1982) Lysyl oxidase and collagenase in experimental acute and chronic liver injury. Gastroenterology 82: 526-34.

Casey ML & MacDonald PC (1997) Lysyl oxidase (ras recision gene) expression in human amnion: ontogeny and cellular localization. J Clin Endocrinol Metab 82: 167-172.

Chambers RC & Laurent GJ (1996) The Lung. In: Comper WD (ed) Extracellular Matrix, Vol. 1. Harvood Academic Publishers, Amsterdam, pp. 378-409.

Chang YS, Svinarich DM, Yang TP & Krawetz SA (1993 The mouse lysyl oxidase gene (Lox) resides on chromosome 18. Cytogenet Cell Genet 63: 47-49.

Chanoki M, Ishii M, Kobayashi H, Fushida H, Yashiro N, Hamada T & Ooshima A (1995) Increased expression of lysyl oxidase in skin with scleroderma. Br J Dermatol 133: 710-715.

Chelly J, Tumer Z, Tonnesen T, Petterson A, Ishikawa-Brush Y, Tommerup N, Horn N & Monaco AP (1993) Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat Genet 3: 14-19.

Chinoy MR, Zgleszewski SE, Cilley RE & Krummel TM (2000) Dexamethasone enhances ras-recision gene expression in cultured murine fetal lungs: role in development. Am J Physiol Lung Cell Mol Physiol 279: 312-318.

Choung J, Taylor L, Thomas K, Zhou X, Kagan H, Yang X & Polgar P (1998) Role of EP2 receptors and cAMP in prostaglandin E2 regulated expression of type I collagen α1, lysyl oxidase, and cyclooxygenase-1 genes in human embryo lung fibroblasts. J Cell Biochem 71: 254-263.

60

Cleary EG & Gibson MA (1996) Elastic Tissue, Elastin and Associated Microfibrils. In: Comper WD (ed) Extracellular Matrix, Vol. 2. Harvood Academic Publishers, Amsterdam, pp. 95-140.

Contente S, Kenyon K, Rimoldi D & Friedman RM (1990) Expression of gene rrg is associated with reversion of NIH 3T3 transformed by LTR-c-H-ras. Science 249: 796-798.

Contente S, Csiszar K, Kenyon K & Friedman RM (1993) Structure of the mouse lysyl oxidase gene. Genomics 16: 395-400.

Cronlund AL & Kagan HM (1986) Comparison of lysyl oxidase from bovine lung and aorta. Connect Tissue Res 15: 173-185.

Counts DF, Evans JN, Dipetrillo TA, Sterling KM Jr & Kelley J (1981) Collagen lysyl oxidase activity in the lung increases during bleomycin-induced lung fibrosis. J Pharmacol Exp Ther 219: 675-678.

Cronshaw AD, Fothergill-Gilmore LA & Hulmes DJ (1995) The proteolytic processing site of the precursor of lysyl oxidase. Biochem J 306: 279-284.

Csiszar K, Entersz I, Trackman PC, Samid D & Boyd CD (1996) Functional analysis of the promoter and first intron of the human lysyl oxidase gene. Mol Biol Rep 23: 97-108.

Csiszar K (2001) Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol 70: 1-32.

Csiszar K, Fong SF, Ujfalusi A, Krawetz SA, Salvati EP, Mackenzie JW & Boyd CD (2002) Somatic mutations of the lysyl oxidase gene on chromosome 5q23.1 in colorectal tumors. Int J Cancer 97: 636-642.

Daly WJ & Rabinovitch HH (1981) Urologic abnormalities in Menkes' syndrome. J Urol 126: 262-264.

Danks DM (1993) Disorders of copper transport: Menkes disease and the occipital horn syndrome. In Royce PM & Steinmann B (ed) Connective Tissue and Its Heritable Disorders: Molecular, Genetic and Medical Aspects. Wiley-Liss, New York, pp. 487-506.

Das S, Levinson B, Vulpe C, Whitney S, Gitschier J & Packman S (1995) Similar splicing mutations of the Menkes/mottled copper-transporting ATPase gene in occipital horn syndrome and the blotchy mouse. Am J Hum Genet 56: 570-576.

Danks DM (1993) Disorders of copper transport: Menkes disease and the occipital horn syndrome. In Royce PM & Steinmann B (ed) Connective Tissue and Ist Heritable Disorders. Molecular, Genetics, and Medical Aspects. Wiley-Liss, Inc., New York. pp. 487-506.

Decitre M, Gleyzal C, Raccurt M, Peyrol S, Aubert-Foucher E, Csiszar K & Sommer P (1998) Lysyl oxidase-like protein localizes to sites of de novo fibrinogenesis in fibrosis and in the early stromal reaction of ductal breast carcinomas. Lab Invest 78: 143-151.

Debelle L & Tamburro AM (1999) Elastin: molecular description and function. Int J Biochem Cell Biol 31: 261-272.

Di Donato A, Ghiggeri GM, Di Duca M, Jivotenko E, Acinni R, Campolo J, Ginevri F & Gusmano R (1997a) Lysyl oxidase expression and collagen cross-linking during chronic adriamycin nephropathy. Nephron 76: 192-200.

Di Donato A, Lacal JC, Di Duca M, Giampuzzi M, Ghiggeri G & Gusmano R (1997b) Micro-injection of recombinant lysyl oxidase blocks oncogenic p21-Ha-Ras and progesterone effects on Xenopus laevis oocyte maturation. FEBS Lett 419: 63-68.

Dimaculangan DD, Chawla A, Boak A, Kagan HM & Lazar MA (1994) Retinoic acid prevents downregulation of ras recision gene/lysyl oxidase early in adipocyte differentiation. Differentiation 58: 47-52.

61

Dingwall C & Laskey RA. Nuclear targeting sequences--a consensus ? (1991) Trends Biochem Sci 16: 478-481.

Eyre DR, Paz MA & Gallop PM (1984) Cross-linking in collagen and elastin. Annu Rev Biochem 53: 717-748.

Eyre D (1987) Collagen cross-linking amino acids. Methods Enzymol 144: 115-139. Feres-Filho EJ, Choi YJ, Han X, Takala TE & Trackman PC (1995) Pre- and post-translational

regulation of lysyl oxidase by transforming growth factor-β1 in osteoblastic MC3T3-E1 cells. J Biol Chem 270: 30797-30803.

Feres-Filho EJ, Menassa GB & Trackman PC (1996) Regulation of lysyl oxidase by basic fibroblast growth factor in osteoblastic MC3T3-E1 cells. J Biol Chem 271: 6411-6416.

Fitzgerald J & Bateman JF (2001) A new FACIT of the collagen family: COL21A1. FEBS Lett 505: 275-280.

Franklin TJ (1995) Current approaches to the therapy of fibrotic diseases. Biochem Pharmacol 49: 267-273.

Franklin TJ (1997) Therapeutic approaches to organ fibrosis. Int J Biochem Cell Biol 29: 79-89. Fry RJ, Slaughter B, Grahn D, Wassermann F, Manelis F, Hamilton KF & Staffeldt E (1967)

Inherited connective tissue defect in tortoise mice. 7409: 114-117. Fujimaki T, Hotta Y, Sakuma H, Fujiki K & Kanai A (1999) Large-scale sequencing of the rabbit

corneal endothelial cDNA library. Cornea 18: 109-114. Gacheru SN, Trackman PC & Kagan HM (1988) Evidence for a functional role for histidine in

lysyl oxidase catalysis. J Biol Chem 263: 16704-16708. Gacheru SN, Trackman PC, Calaman SD, Greenaway FT & Kagan HM (1989) Vicinal diamines as

pyrroloquinoline quinone-directed irreversible inhibitors of lysyl oxidase. J Biol Chem 264: 12963-12969.

Gacheru SN, Trackman PC, Shah MA, O'Gara CY, Spacciapoli P, Greenaway FT & Kagan HM (1990) Structural and catalytic properties of copper in lysyl oxidase. J Biol Chem 265: 19022-19027.

Gacheru S, McGee C, Uriu-Hare JY, Kosonen T, Packman S, Tinker D, Krawetz SA, Reiser K, Keen CL & Rucker RB (1993) Expression and accumulation of lysyl oxidase, elastin, and type I procollagen in human Menkes and mottled mouse fibroblasts. Arch Biochem Biophys 301: 325-329.

Gacheru SN, Thomas KM, Murray SA, Csiszar K, Smith-Mungo LI & Kagan HM (1997) Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: effects of TGF-β1 and serum deprivation. J Cell Biochem 65: 395-407.

Gavriel P & Kagan HM (1988) Inhibition by heparin of the oxidation of lysine in collagen by lysyl oxidase. Biochemistry 27: 2811-2815.

Giampuzzi M, Botti G, Cilli M, Gusmano R, Borel A, Sommer P & Di Donato A (2001) Down-regulation of lysyl oxidase-induced tumorigenic transformation in NRK-49F cells characterized by constitutive activation of ras proto-oncogene. J Biol Chem 276: 29226-29232.

Green RS, Lieb ME, Weintraub AS, Gacheru SN, Rosenfield CL, Shah S, Kagan HM & Taubman MB (1995) Identification of lysyl oxidase and other platelet-derived growth factor-inducible genes in vascular smooth muscle cells by differential screening. Lab Invest 73: 476-482.

Gregory KE, Marsden ME, Anderson-MacKenzie J, Bard JB, Bruckner P, Farjanel J, Robins SP & Hulmes DJ (1999) Abnormal collagen assembly, though normal phenotype, in alginate bead cultures of chick embryo chondrocytes. Exp Cell Res 246: 98-107.

62

Grange JJ, Davis V & Baxter BT (1997) Pathogenesis of abdominal aortic aneurysm: an update and look toward the future. Cardiovasc Surg 5: 256-265.

Hajnal A, Klemenz R & Schafer R (1993) Up-regulation of lysyl oxidase in spontaneous revertants of H-ras-transformed rat fibroblasts. Cancer Res 53: 4670-4675.

Harcke HT Jr, Capitanio MA, Grover WD & Valdes-Dapena M (1977) Bladder diverticula and Menkes' syndrome. Radiology 124: 459-461.

Harris ED, Gonnerman WA, Savage JE & O'Dell BL (1974) Connective tissue amine oxidase. II. Purification and partial characterization of lysyl oxidase from chick aorta. Biochim Biophys Acta 341: 332-344.

Harris ED (1976) Copper-induced activation of aortic lysyl oxidase in vivo. Proc Natl Acad Sci USA 73: 371-374.

Harris ED (1986) Biochemical defect in chick lung resulting from copper deficiency. J Nutr 116: 252-258.

Hein S, Yamamoto SY, Okazaki K, Jourdan-LeSaux C, Csiszar K & Bryant-Greenwood GD (2001) Lysyl oxidases: expression in the fetal membranes and placenta. Placenta 22: 49-57.

Hinek A & Rabinovitch M (1994) 67-kD elastin-binding protein is a protective "companion" of extracellular insoluble elastin and intracellular tropoelastin. J Cell Biol 126: 563-574.

Hong HH, Uzel MI, Duan C, Sheff MC & Trackman PC (1999) Regulation of lysyl oxidase, collagen, and connective tissue growth factor by TGF-β1 and detection in human gingiva. Lab Invest 79: 1655-1667.

Huang Y, Dai J, Tang R, Zhao W, Zhou Z, Wang W, Ying K, Xie Y & Mao Y (2001) Cloning and characterization of a human lysyl oxidase-like 3 gene (hLOXL3). Matrix Biol 20: 153-157.

Hämäläinen ER, Jones TA, Sheer D, Taskinen K, Pihlajaniemi T & Kivirikko KI (1991) Molecular cloning of human lysyl oxidase and assignment of the gene to chromosome 5q23.3-31.2. Genomics 11: 508-516.

Hämäläinen ER, Kemppainen R, Pihlajaniemi T & Kivirikko KI (1993) Structure of the human lysyl oxidase gene. Genomics 17: 544-548.

Hämäläinen ER, Kemppainen R, Kuivaniemi H, Tromp G, Vaheri A, Pihlajaniemi T & Kivirikko KI (1995) Quantitative polymerase chain reaction of lysyl oxidase mRNA in malignantly transformed human cell lines demonstrates that their low lysyl oxidase activity is due to low quantities of its mRNA and low levels of transcription of the respective gene. J Biol Chem 270: 21590-21593.

Iguchi H & Sano S (1985) Cadmium- or zinc-binding to bone lysyl oxidase and copper replacement. Connect Tissue Res 14: 129-139.

Iguchi H, Kasai R, Okumura H, Yamamuro T & Kagan HM (1990) Effect of dietary cadmium and/or copper on the bone lysyl oxidase in copper-deficient rats relative to the metabolism of copper in the bone. Bone Miner 10:51-59.

Ito H, Akiyama H, Iguchi H, Iyama K, Miyamoto M, Ohsawa K & Nakamura T (2001) Molecular cloning and biological activity of a novel lysyl oxidase-related gene expressed in cartilage. J Biol Chem 276: 24023-24029.

Jacob MP, Badier-Commander C, Fontaine V, Benazzoug Y, Feldman L & Michel JB (2001) Extracellular matrix remodeling in the vascular wall. Pathol Biol 49: 326-332.

Janes SM, Mu D, Wemmer D, Smith AJ, Kaur S, Maltby D, Burlingame AL & Klinman JP (1990) A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. Science 248: 981-987.

63

Jang W, Hua A, Spilson SV, Miller W, Roe BA & Meisler MH (1999) Comparative sequence of human and mouse BAC clones from the mnd2 region of chromosome 2p13. Genome Res 9: 53-61.

Jeggo PA (1998) Identification of genes involved in repair of DNA double-strand breaks in mammalian cells. Radiat Res 150: 80-91.

Jeggo P, Singleton B, Beamish H & Priestley A (1999) Double strand break rejoining by the Ku-dependent mechanism of non-homologous end-joining. C R Acad Sci III 322: 109-112.

Jin S & Weaver DT (1997) Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions. EMBO J 16: 6874-6885.

Jourdan-Le Saux C, Gleyzal C, Garnier JM, Peraldi M, Sommer P & Grimaud JA (1994) Lysyl oxidase cDNA of myofibroblast from mouse fibrotic liver. Biochem Biophys Res Commun 199: 587-592.

Jourdan-Le Saux C, Gleyzal C, Raccurt M & Sommer P (1997) Functional analysis of the lysyl oxidase promoter in myofibroblast-like clones of 3T6 fibroblast. J Cell Biochem 64: 328-341.

Jourdan-Le Saux C, Le Saux O, Donlon T, Boyd CD & Csiszar K (1998) The human lysyl oxidase-related gene (LOXL2) maps between markers D8S280 and D8S278 on chromosome 8p21.2-p21.3. Genomics 51: 305-307.

Jourdan-Le Saux C, Tronecker H, Bogic L, Bryant-Greenwood GD, Boyd CD & Csiszar K (1999) The LOXL2 gene encodes a new lysyl oxidase-like protein and is expressed at high levels in reproductive tissues. J Biol Chem 274: 12939-12944.

Jourdan-Le Saux C, Le Saux O, Gleyzal C, Sommer P & Csiszar K (2000) The mouse lysyl oxidase-like 2 gene (mLOXL2) maps to chromosome 14 and is highly expressed in skin, lung and thymus. Matrix Biol 19: 179-183.

Jourdan-Le Saux C, Tomsche A, Ujfalusi A, Jia L & Csiszar K (2001) Central nervous system, uterus, heart, and leukocyte expression of the LOXL3 gene, encoding a novel lysyl oxidase-like protein. Genomics 74: 211-218.

Kagan HM, Sullivan KA, Olsson TA 3rd & Cronlund AL (1979) Purification and properties of four species of lysyl oxidase from bovine aorta. Biochem J 177: 203-214.

Kagan HM, Raghavan J & Hollander W (1981) Changes in aortic lysyl oxidase activity in diet-induced atherosclerosis in the rabbit. Arteriosclerosis. 1: 287-291.

Kagan HM, Williams MA, Williamson PR & Anderson JM (1984) Influence of sequence and charge on the specificity of lysyl oxidase toward protein and synthetic peptide substrates. J Biol Chem 259: 11203-11207.

Kagan HM (1986) Characterization and regulation of lysyl oxidase. In: Mecham, R.P. (ed) Biology of Extracellular Matrix. 1 Academic Press, Orlando, pp. 321-389.

Kagan HM, Vaccaro CA, Bronson RE, Tang SS & Brody JS (1986) Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue. J Cell Biol 103: 1121-1128.

Kagan HM (1994) Lysyl oxidase: mechanism, regulation and relationship to liver fibrosis. Pathol Res Pract 190: 910-919.

Kagan HM, Reddy VB, Panchenko MV, Nagan N, Boak AM, Gacheru SN & Thomas KM (1995) Expression of lysyl oxidase from cDNA constructs in mammalian cells: the propeptide region is not essential to the folding and secretion of the functional enzyme. J Cell Biochem 59: 329-338.

Kagan HM (2000) Intra- and extracellular enzymes of collagen biosynthesis as biological and chemical targets in the control of fibrosis. Acta Trop 77: 147-152.

64

Kaler SG, Westman JA, Bernes SM, Elsayed AM, Bowe CM, Freeman KL, Wu CD & Wallach MT (1993) Gastrointestinal hemorrhage associated with gastric polyps in Menkes disease. J Pediatr 122: 93-95.

Kaler SG (1998) Metabolic and molecular bases of Menkes disease and occipital horn syndrome. Pediatr Dev Pathol 1: 85-98.

Kemppainen R, Hämäläinen ER, Kuivaniemi H, Tromp G, Pihlajaniemi T & Kivirikko KI (1996) Expression of mRNAs for lysyl oxidase and type III procollagen in cultured fibroblasts from patients with the Menkes and occipital horn syndromes as determined by quantitative polymerase chain reaction. Arch Biochem Biophys 328: 101-106.

Kenyon K, Contente S, Trackman PC, Tang J, Kagan HM & Friedman RM (1991) Lysyl oxidase and rrg messenger RNA. Science 253: 802.

Kenyon K, Modi WS, Contente S & Friedman RM (1993) A novel human cDNA with a predicted protein similar to lysyl oxidase maps to chromosome 15q24-q25. J Biol Chem 268: 18435-18437.

Kielty CM, Hopkinson I & Grant ME (1993) Collagen. The Collagen Family: Structure, assembly, and organization in the extracellular matrix. In Royce PM & Steinmann B (ed) Connective Tissue and Ist Heritable Disorders. Molecular, Genetics, and Medical Aspects. Wiley-Liss, Inc., New York. pp. 103-147.

Kim Y, Boyd CD & Csiszar K (1995) A new gene with sequence and structural similarity to the gene encoding human lysyl oxidase. J Biol Chem 270: 7176-7182.

Kim Y, Peyrol S, So CK, Boyd CD & Csiszar K (1999) Coexpression of the lysyl oxidase-like gene (LOXL) and the gene encoding type III procollagen in induced liver fibrosis. J Cell Biochem 72: 181-188.

Kiserud T, Hellevik LR, Eik-Nes SH, Angelsen BA & Blaas HG (1994) Estimation of the pressure gradient across the fetal ductus venosus based on Doppler velocimetry. Ultrasound Med Biol 20: 225-232.

Kivirikko KI & Myllylä R (1979) Collagen glycosyltransferases. Int Rev Connect Tissue Res 8: 23-72.

Kivirikko KI (1993) Collagens and their abnormalities in a wide spectrum of diseases. Ann Med 25: 113-126.

Kivirikko KI & Kuivaniemi H (1987) Posttranslational modifications of collagens. In: Uitto J & Perejda AJ (ed) Connective Tissue Diseases. Marcel Dekker, New York. pp. 263-292.

Kivirikko KI & Myllyharju J (1998) Prolyl 4-hydroxylases and their protein disulfide isomerase subunit. Matrix Biol 16: 357-368.

Kivirikko KI & Pihlajaniemi T (1998) Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4-hydroxylases. Adv Enzymol Relat Areas Mol Biol 72: 325-398.

Klinman JP & Mu D (1994) Quinoenzymes in biology. Annu Rev Biochem. 63: 299-344. Klinman JP (1996) New quinocofactors in eukaryotes. J Biol Chem 271: 27189-27192. Koch M, Foley JE, Hahn R, Zhou P, Burgeson RE, Gerecke DR & Gordon MK (2001) α1(Xx)

collagen, a new member of the collagen subfamily, fibril-associated collagens with interrupted triple helices. J Biol Chem 276: 23120-23126.

Konishi A, Iguchi H, Ochi J, Kinoshita R, Miura K & Uchino H (1985) Increased lysyl oxidase activity in culture medium of nonparenchymal cells from fibrotic livers. Gastroenterology 89: 709-715.

65

Kosonen T, Uriu-Hare JY, Clegg MS, Keen CL & Rucker RB (1997) Incorporation of copper into lysyl oxidase. Biochem J 327: 283-289.

Krebs CJ & Krawetz SA (1993) Lysyl oxidase copper-talon complex: a model. Biochim Biophys Acta 1202: 7-12.

Krzyzosiak WJ, Shindo-Okada N, Teshima H, Nakajima K & Nishimura S (1992) Isolation of genes specifically expressed in flat revertant cells derived from activated ras-transformed NIH 3T3 cells by treatment with azatyrosine. Proc Natl Acad Sci USA 89: 4879-4883.

Kuivaniemi H, Peltonen L, Palotie A, Kaitila I & Kivirikko KI (1982) Abnormal copper metabolism and deficient lysyl oxidase activity in a heritable connective tissue disorder. J Clin Invest 69: 730-733.

Kuivaniemi H, Savolainen ER & Kivirikko KI (1984) Human placental lysyl oxidase. Purification, partial characterization, and preparation of two specific antisera to the enzyme. J Biol Chem 259: 6996-7002.

Kuivaniemi H, Peltonen L & Kivirikko KI (1985) Type IX Ehlers-Danlos syndrome and Menkes syndrome: the decrease in lysyl oxidase activity is associated with a corresponding deficiency in the enzyme protein. Am J Hum Genet 37: 798-808.

Kuivaniemi H, Ala-Kokko L & Kivirikko KI (1986a) Secretion of lysyl oxidase by cultured human skin fibroblasts and effects of monensin, nigericin, tunicamycin and colchicine. Biochim Biophys Acta 883: 326-334.

Kuivaniemi H, Korhonen RM, Vaheri A & Kivirikko KI (1986b) Deficient production of lysyl oxidase in cultures of malignantly transformed human cells. FEBS Lett 195: 261-264.

Layman DL, Narayanan AS & Martin GR (1972) The production of lysyl oxidase by human fibroblasts in culture. Arch Biochem Biophys 149: 97-101.

Lee S, Solow-Cordero DE, Kessler E, Takahara K & Greenspan DS (1997) Transforming growth factor-β regulation of bone morphogenetic protein-1/procollagen C-proteinase and related proteins in fibrogenic cells and keratinocytes. J Biol Chem 272: 19059-19066.

Levene CI (1961) Structural requirements for lathyrogenic agents. J Exp Med 114: 295-310. Levinson B, Conant R, Schnur R, Das S, Packman S & Gitschier J (1996) A repeated element in

the regulatory region of the MNK gene and its deletion in a patient with occipital horn syndrome. Hum Mol Genet 5: 1737-1742.

Li W, Chou IN, Boak A & Kagan HM (1995) Downregulation of lysyl oxidase in cadmium-resistant fibroblasts. Am J Respir Cell Mol Biol 13: 418-425.

Li W, Nellaiappan K, Strassmaier T, Graham L, Thomas KM & Kagan HM (1997) Localization and activity of lysyl oxidase within nuclei of fibrogenic cells. Proc Natl Acad Sci USA 94: 12817-12822.

Li DY, Brooke B, Davis EC, Mecham RP, Sorensen LK, Boak BB, Eichwald E & Keating MT (1998) Elastin is an essential determinant of arterial morphogenesis. Nature 393: 276-280.

Light ND & Bailey AJ (1980) The chemistry of the collagen cross-links. Purification and characterization of cross-linked polymeric peptide material from mature collagen containing unknown amino acids. Biochem J 185: 373-381.

Liu G, Nellaiappan K & Kagan HM (1997a) Irreversible inhibition of lysyl oxidase by homocysteine thiolactone and its selenium and oxygen analogues. Implications for homocystinuria. J Biol Chem 272: 32370-32377.

66

Liu X, Wu H, Byrne M, Krane S & Jaenisch R (1997b) Type III collagen is crucial for collagen I fibrillogenesis and for normal cardiovascular development. Proc Natl Acad Sci USA 94: 1852-1856.

Lobenstein-Verbeek CL, Jongejan JA, Frank J & Duine JA (1984) Bovine serum amine oxidase: a mammalian enzyme having covalently bound PQQ as prosthetic group. FEBS Lett 170: 305-309.

Ma RH, Tsai CC & Shieh TY (1995) Increased lysyl oxidase activity in fibroblasts cultured from oral submucous fibrosis associated with betel nut chewing in Taiwan. J Oral Pathol Med 24: 407-412.

MacSweeney ST, Young G, Greenhalgh RM & Powell JT (1992) Mechanical properties of the aneurysmal aorta. Br J Surg 79: 1281-1284.

MacSweeney ST, Powell JT & Greenhalgh RM (1994) Pathogenesis of abdominal aortic aneurysm. Br J Surg 81: 935-941.

Mariani TJ, Trackman PC, Kagan HM, Eddy RL, Shows TB, Boyd CD & Deak SB (1992) The complete derived amino acid sequence of human lysyl oxidase and assignment of the gene to chromosome 5 (extensive sequence homology with the murine ras recision gene). Matrix 12: 242-248.

McPhie JL (1981) The activity of lysyl oxidase in experimental hepatic fibrosis. Hepatogastroenterology 28: 240-241.

Mercer JF, Livingston J, Hall B, Paynter JA, Begy C, Chandrasekharappa S, Lockhart P, Grimes A, Bhave M, Siemieniak D & Glover TW (1993) Isolation of a partial candidate gene for Menkes disease by positional cloning. Nat Genet 3: 20-25.

Mercer JF (1998) Menkes syndrome and animal models. Am J Clin Nutr 67: 1022-1028. Mock BA, Contente S, Kenyon K, Friedman RM & Kozak CA (1992) The gene for lysyl oxidase

maps to mouse chromosome 18. Genomics 14: 822-823. Moursi MM, Beebe HG, Messina LM, Welling TH & Stanley JC (1995) Inhibition of aortic

aneurysm development in blotchy mice by beta adrenergic blockade independent of altered lysyl oxidase activity. J Vasc Surg 21: 792-799.

Møller LB, Tumer Z, Lund C, Petersen C, Cole T, Hanusch R, Seidel J, Jensen LR & Horn N (2000) Similar splice-site mutations of the ATP7A gene lead to different phenotypes: classical Menkes disease or occipital horn syndrome. Am J Hum Genet 66: 1211-1220.

Murano S, Thweatt R, Shmookler Reis RJ, Jones RA, Moerman EJ & Goldstein S (1991) Diverse gene sequences are overexpressed in Werner syndrome fibroblasts undergoing premature replicative senescence. Mol Cell Biol. 11: 3905-3914.

Murawaki Y, Kusakabe Y & Hirayama C (1991) Serum lysyl oxidase activity in chronic liver disease in comparison with serum levels of prolyl hydroxylase and laminin. Hepatology 14: 1167-1173.

Myllyharju J & Kivirikko KI (2001) Collagens and collagen-related diseases. Ann Med 33: 7-21. Nagan N & Kagan HM (1994) Modulation of lysyl oxidase activity toward peptidyl lysine by

vicinal dicarboxylic amino acid residues. Implications for collagen cross-linking. J Biol Chem 269: 22366-22371.

Nagan N, Callery PS & Kagan HM (1998) Aminoalkylaziridines as substrates and inhibitors of lysyl oxidase: specific inactivation of the enzyme by N-(5-aminopentyl)aziridine. Front Biosci 3:A23-26.

67

Narayanan AS, Siegel RC & Martin GR (1972) On the inhibition of lysyl oxidase by -aminopropionitrile. Biochem Biophys Res Commun 46: 745-751.

Narayanan AS, Siegel RC & Martin GR (1974) Stability and purification of lysyl oxidase. Arch Biochem Biophys 162: 231-237.

Nagy A, Rossant J, Nagy R, Abramow-Newerly W & Roder JC (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 90: 8424-8428.

Nellaiappan K, Risitano A, Liu G, Nicklas G & Kagan HM (2000) Fully processed lysyl oxidase catalyst translocates from the extracellular space into nuclei of aortic smooth-muscle cells. J Cell Biochem 79: 576-582.

Oberhuber H, Seliger B & Schafer R (1995) Partial restoration of pre-transformation levels of lysyl oxidase and transin mRNAs in phenotypic ras revertants. Mol Carcinog 12: 198-204.

Opsahl W, Zeronian H, Ellison M, Lewis D, Rucker RB & Riggins RS (1982) Role of copper in collagen cross-linking and its influence on selected mechanical properties of chick bone and tendon. J Nutr 112: 708-716.

Panchenko MV, Stetler-Stevenson WG, Trubetskoy OV, Gacheru SN & Kagan HM (1996) Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase. J Biol Chem 271: 7113-7119.

Peltonen L, Kuivaniemi H, Palotie A, Horn N, Kaitila I & Kivirikko KI (1983) Alterations in copper and collagen metabolism in the Menkes syndrome and a new subtype of the Ehlers-Danlos syndrome. Biochemistry 22: 6156-6163.

Peyrol S, Raccurt M, Gerard F, Gleyzal C, Grimaud JA & Sommer P (1997) Lysyl oxidase gene expression in the stromal reaction to in situ and invasive ductal breast carcinoma. Am J Pathol 150: 497-507.

Peyrol S, Galateau-Salle F, Raccurt M, Gleyzal C & Sommer P (2000) Selective expression of lysyl oxidase (LOX) in the stromal reactions of broncho-pulmonary carcinomas. Histol Histopathol 15: 1127-1135.

Pinnell SR & Martin GR (1968) The cross-linking of collagen and elastin: enzymatic conversion of lysine in peptide linkage to α-aminoadipic-δ-semialdehyde (allysine) by an extract from bone. Proc Natl Acad Sci USA 61: 708-716.

Prockop DJ & Kivirikko KI (1995) Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem 64: 403-434.

Prockop DJ, Sieron AL & Li SW (1998) Procollagen N-proteinase and procollagen C-proteinase. Two unusual metalloproteinases that are essential for procollagen processing probably have important roles in development and cell signaling. Matrix Biol 16: 399-408.

Ravid K, Smith-Mungo LI, Zhao Z, Thomas KM & Kagan HM (1999) Upregulation of lysyl oxidase in vascular smooth muscle cells by cAMP: role for adenosine receptor activation. J Cell Biochem 75: 177-185.

Rayton JK & Harris ED (1979) Induction of lysyl oxidase with copper. Properties of an in vitro system. J Biol Chem 254: 621-626.

Reed KL (1997) Doppler-the fetal circulation. Clin Obstet Gynecol 40: 750-754. Reed KL, Chaffin DG, Anderson CF & Newman AT (1997) Umbilical venous velocity pulsations

are related to atrial contraction pressure waveforms in fetal lambs. Obstet Gynecol 89: 953-956. Reiser K, McCormick RJ & Rucker RB (1992) Enzymatic and nonenzymatic cross-linking of

collagen and elastin. FASEB J 6: 2439-2449.

68

Ren C, Yang G, Timme TL, Wheeler TM & Thompson TC (1998) Reduced lysyl oxidase messenger RNA levels in experimental and human prostate cancer. Cancer Res 58: 1285-1290.

Resnick D, Pearson A & Krieger M (1994) The SRCR superfamily: a family reminiscent of the Ig superfamily. Trends Biochem Sci 19: 5-8.

Riley DJ, Kerr JS, Berg RA, Ianni BD, Pietra GG, Edelman NH & Prockop DJ (1982) β-Aminopropionitrile prevents bleomycin-induced pulmonary fibrosis in the hamster. Am Rev Respir Dis 125: 67-73.

Ritzenthaler JD, Goldstein RH, Fine A, Lichtler A, Rowe DW & Smith BD (1991) Transforming-growth-factor-β activation elements in the distal promoter regions of the rat α1 type I collagen gene. Biochem J 280: 157-162.

Romero-Chapman N, Lee J, Tinker D, Uriu-Hare JY, Keen CL & Rucker RR (1991) Purification, properties and influence of dietary copper on accumulation and functional activity of lysyl oxidase in rat skin. Biochem J 275: 657-662.

Rosenbloom J, Abrams WR & Mecham R (1993) Extracellular matrix 4: The elastic fiber. FASEB J 7: 1208-1218.

Rosenbloom J (1993) Elastin. In Royce PM & Steinmann B (ed) Connective Tissue and Its Heritable Disorders. Molecular, Genetics, and Medical Aspects. Wiley-Liss, Inc., New York. pp. 103-147.

Rossi P, Karsenty G, Roberts AB, Roche NS, Sporn MB & de Crombrugghe B (1988) A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-β. Cell 52: 405-414.

Rowe DW, McGoodwin EB, Martin GR, Sussman MD, Grahn D, Faris B & Franzblau C (1974) A sex-linked defect in the cross-linking of collagen and elastin associated with the mottled locus in mice. J Exp Med 139: 180-192.

Rowe DW, McGoodwin EB, Martin GR & Grahn D (1977) Decreased lysyl oxidase activity in the aneurysm-prone, mottled mouse. J Biol Chem 252: 939-942.

Roy R, Polgar P, Wang Y, Goldstein RH, Taylor L & Kagan HM (1996) Regulation of lysyl oxidase and cyclooxygenase expression in human lung fibroblasts: interactions among TGF-β, IL-1β, and prostaglandin E. J Cell Biochem 62: 411-417.

Royce PM, Camakaris J & Danks DM (1980) Reduced lysyl oxidase activity in skin fibroblasts from patients with Menkes' syndrome. Biochem J 192: 579-586.

Rucker RB, Riggins RS, Laughlin R, Chan MM, Chen M & Tom K (1975) Effects of nutritional copper deficiency on the biomechanical properties of bone and arterial elastin metabolism in the chick. J Nutr 105: 1062-1070.

Rucker RB, Romero-Chapman N, Wong T, Lee J, Steinberg FM, McGee C, Clegg MS, Reiser K, Kosonen T, Uriu-Hare JY, Murphy J & Keen CL (1996) Modulation of lysyl oxidase by dietary copper in rats. J Nutr 126: 51-60.

Rucker RB, Kosonen T, Clegg MS, Mitchell AE, Rucker BR, Uriu-Hare JY & Keen CL (1998) Copper, lysyl oxidase, and extracellular matrix protein cross-linking. Am J Clin Nutr 67: 996-1002.

Rucker RB, Rucker BR, Mitchell AE, Cui CT, Clegg M, Kosonen T, Uriu-Adams JY, Tchaparian EH, Fishman M & Keen CL (1999) Activation of chick tendon lysyl oxidase in response to dietary copper. J Nutr 129: 2143-2146.

Saito H, Papaconstantinou J, Sato H & Goldstein S (1997) Regulation of a novel gene encoding a lysyl oxidase-related protein in cellular adhesion and senescence. J Biol Chem 272: 8157-8160.

69

Sakamoto M, Murawaki Y & Hirayama C (1987) Serum lysyl oxidase activity in patients with various liver diseases. Gastroenterol Jpn 22: 730-736.

Shackleton DR & Hulmes DJ (1990) Purification of lysyl oxidase from piglet skin by selective interaction with Sephacryl S-200. Biochem J 266: 917-919.

Shah MA, Scaman CH, Palcic MM & Kagan HM (1993a) Kinetics and stereospecificity of the lysyl oxidase reaction. J Biol Chem 268: 11573-11579.

Shah MA, Trackman PC, Gallop PM & Kagan HM (1993b) Reaction of lysyl oxidase with trans-2-phenylcyclopropylamine. J Biol Chem 268: 11580-11585.

Shanley CJ, Gharaee-Kermani M, Sarkar R, Welling TH, Kriegel A, Ford JW, Stanley JC & Phan SH (1997) Transforming growth factor-β1 increases lysyl oxidase enzyme activity and mRNA in rat aortic smooth muscle cells. J Vasc Surg 25: 446-452.

Sharma R, Kramer JA & Krawetz SA (1997) Lysyl oxidase, cellular senescence and tumor suppression. Biosci Rep 17: 409-414.

Shibanuma M, Mashimo J, Mita A, Kuroki T & Nose K (1993) Cloning from a mouse osteoblastic cell line of a set of transforming-growth-factor-β1-regulated genes, one of which seems to encode a follistatin-related polypeptide. Eur J Biochem 217: 13-19.

Siegel RC (1974) Biosynthesis of collagen crosslinks: increased activity of purified lysyl oxidase with reconstituted collagen fibrils. Proc Natl Acad Sci USA 71: 4826-4830.

Siegel RC, Chen KH, Greenspan JS & Aguiar JM (1978) Biochemical and immunochemical study of lysyl oxidase in experimental hepatic fibrosis in the rat. Proc Natl Acad Sci USA 75: 2945-2949.

Silver FH, Horvath I & Foran DJ (2001) Viscoelasticity of the vessel wall: the role of collagen and elastic fibers. Crit Rev Biomed Eng 29: 279-301.

Smith-Mungo LI & Kagan HM (1998) Lysyl oxidase: properties, regulation and multiple functions in biology. Matrix Biol 16: 387-398.

Sommer P, Gleyzal C, Raccurt M, Delbourg M, Serrar M, Joazeiro P, Peyrol S, Kagan H, Trackman PC & Grimaud JA (1993) Transient expression of lysyl oxidase by liver myofibroblasts in murine schistosomiasis. Lab Invest 69: 460-470.

Song YL, Ford JW, Gordon D & Shanley CJ (2000) Regulation of lysyl oxidase by interferon-γ in rat aortic smooth muscle cells. Arterioscler Thromb Vasc Biol 20: 982-988.

Stale H, Marsal K, Gennser G, Benthin M, Dahl P & Lindstrom K (1991) Aortic diameter pulse waves and blood flow velocity in the small, for gestational age, fetus. Ultrasound Med. Biol 17: 471-478.

Stassen FL (1976) Properties of highly purified lysyl oxidase from embryonic chick cartilage. Biochim Biophys Acta 438: 49-60.

Steinmann B, Royce PM & Superti-Furga A (1993) The Ehlers-Danlos syndrome. In Royce PM & Steinmann B (ed) Connective Tissue and Its Heritable Disorders: Molecular, Genetic and Medical Aspects. Wiley-Liss, New York, pp. 351-407.

Su Z-Z, Yemul S, Estabrook A, Friedman RM, Zimmer SG & Fisher PB (1995) Transcriptional switching model for the regulation of tumorigenesis and metastasis by the Ha-ras oncogene: transcriptional changes in the Ha-ras tumor suppressor gene lysyl oxidase. Int J Oncol 7: 1279-1284.

Szabo Z, Light E, Boyd CD & Csiszar K (1997) The human lysyl oxidase-like gene maps between STS markers D15S215 and GHLC.GCT7C09 on chromosome 15. Hum Genet 101: 198-200.

70

Takahara K, Lyons GE & Greenspan DS (1994) Bone morphogenetic protein-1 and a mammalian tolloid homologue (mTld) are encoded by alternatively spliced transcripts which are differentially expressed in some tissues. J Biol Chem 269: 32572-32578.

Tamura G, Ogasawara S, Nishizuka S, Sakata K, Maesawa C, Suzuki Y, Terashima M, Saito K & Satodate R (1996) Two distinct regions of deletion on the long arm of chromosome 5 in differentiated adenocarcinomas of the stomach. Cancer Res 56: 612-615.

Tan RS, Taniguchi T & Harada H (1996) Identification of the lysyl oxidase gene as target of the antioncogenic transcription factor, IRF-1, and its possible role in tumor suppression. Cancer Res 56: 2417-2421.

Tang C & Klinman JP (2001) The catalytic function of bovine lysyl oxidase in the absence of copper. J Biol Chem 276: 30575-30578.

Tang SS, Trackman PC & Kagan HM (1983) Reaction of aortic lysyl oxidase with β-aminopropionitrile. J Biol Chem 258: 4331-4338.

Tang SS, Simpson DE & Kagan HM (1984) β-substituted ethylamine derivatives as suicide inhibitors of lysyl oxidase. J Biol Chem 259: 975-979.

Tchaparian EH, Uriu-Adams JY, Keen CL, Mitchell AE & Rucker RB (2000) Lysyl oxidase and P-ATPase-7A expression during embryonic development in the rat. Arch Biochem Biophys 379: 71-77.

Tchernev VT, Yang TP & Kingsmore SF (1997) Genetic mapping of lysyl oxidase-2 (Loxl) on mouse chromosome 9. Mamm Genome 8: 621-622.

Tinker D, Romero-Chapman N, Reiser K, Hyde D & Rucker R (1990) Elastin metabolism during recovery from impaired crosslink formation. Arch Biochem Biophys 278: 326-332.

Trackman PC, Pratt AM, Wolanski A, Tang SS, Offner GD, Troxler RF & Kagan HM (1990) Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence. Biochemistry 29: 4863-4870.

Trackman PC, Pratt AM, Wolanski A, Tang SS, Offner GD, Troxler RF & Kagan HM (1991) Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence. Biochemistry 30: 8282.

Trackman PC, Bedell-Hogan D, Tang J & Kagan HM (1992) Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor. J Biol Chem 267: 8666-8671.

Trackman PC, Graham RJ, Bittner HK, Carnes DL, Gilles JA & Graves DT (1998) Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1. Histochem Cell Biol 110: 9-14.

Trivedy C, Warnakulasuriya KA, Hazarey VK, Tavassoli M, Sommer P & Johnson NW (1999) The upregulation of lysyl oxidase in oral submucous fibrosis and squamous cell carcinoma. J Oral Pathol Med 28: 246-251.

Uzel MI, Shih SD, Gross H, Kessler E, Gerstenfeld LC & Trackman PC (2000) Molecular events that contribute to lysyl oxidase enzyme activity and insoluble collagen accumulation in osteosarcoma cell clones. J Bone Miner Res 15: 1189-1197.

Uzel MI, Scott IC, Babakhanlou-Chase H, Palamakumbura AH, Pappano WN, Hong HH, Greenspan DS & Trackman PC (2001) Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures. J Biol Chem 276: 22537-22543.

Vrhovski B & Weiss AS (1998) Biochemistry of tropoelastin. Eur J Biochem 258: 1-18.

71

Vulpe C, Levinson B, Whitney S, Packman S & Gitschier J (1993) Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat Genet 3: 7-13.

Vulpe CD & Packman S (1995) Cellular copper transport. Annu Rev Nutr 15: 293-322. Wakasaki H & Ooshima A (1990a) Synthesis of lysyl oxidase in experimental hepatic fibrosis.

Biochem Biophys Res Commun 166: 1201-1204. Wakasaki H & Ooshima A. Immunohistochemical localization of lysyl oxidase with monoclonal

antibodies (1990b) Lab Invest 63: 377-384. Wang SX, Mure M, Medzihradszky KF, Burlingame AL, Brown DE, Dooley DM, Smith AJ,

Kagan HM & Klinman JP (1996) A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains. Science 273: 1078-1084.

Wang SX, Nakamura N, Mure M, Klinman JP & Sanders-Loehr J (1997) Characterization of the native lysine tyrosylquinone cofactor in lysyl oxidase by Raman spectroscopy. J Biol Chem 272: 28841-28844.

Wieland I, Bohm M, Arden KC, Ammermuller T, Bogatz S, Viars CS & Rajewsky MF (1996) Allelic deletion mapping on chromosome 5 in human carcinomas. Oncogene 12: 97-102.

Wight TN (1996) Arterial Wall. In: Comper WD (ed) Extracellular Matrix, Vol. 1. Harvood Academic Publishers, Amsterdam, pp. 175-202.

Williamson PR & Kagan HM (1986) Reaction pathway of bovine aortic lysyl oxidase. J Biol Chem 261: 9477-9482.

Williamson PR, Kittler JM, Thanassi JW & Kagan HM (1986a) Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5'-phosphate. Biochem J 235: 597-605.

Williamson PR, Moog RS, Dooley DM & Kagan HM (1986b) Evidence for pyrroloquinolinequinone as the carbonyl cofactor in lysyl oxidase by absorption and resonance Raman spectroscopy. J Biol Chem 261: 16302-16305.

Williamson PR & Kagan HM (1987a) α-proton abstraction and carbanion formation in the mechanism of action of lysyl oxidase. J Biol Chem 262: 8196-8201.

Williamson PR & Kagan HM (1987b) Electronegativity of aromatic amines as a basis for the development of ground state inhibitors of lysyl oxidase. J Biol Chem 262: 14520-14524.

Wu Y, Rich CB, Lincecum J, Trackman PC, Kagan HM & Foster JA (1992) Characterization and developmental expression of chick aortic lysyl oxidase. J Biol Chem 267: 24199-24206.

Wydner KS, Kim Y, Csiszar K, Boyd CD & Passmore HC (1997) An intron capture strategy used to identify and map a lysyl oxidase-like gene on chromosome 9 in the mouse. Genomics 40: 342-345.

Yamada Y, Doi T, Hamakubo T & Kodama T (1998) Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system. Cell Mol Life Sci 54: 628-640.

Yeowell HN, Marshall MK, Walker LC, Ha V & Pinnell SR (1994) Regulation of lysyl oxidase mRNA in dermal fibroblasts from normal donors and patients with inherited connective tissue disorders. Arch Biochem Biophys 308: 299-305.