Wnt -4 Stimulation

of Human Bone

Marrow Stromal

Stem Cells and

Mesenchymal

Stem Cells of the

Maxilla

Gayathri Vijayakumar

Poly ID# 0408513

Guided Studies- Fall 2010

Nicola C. Partridge, Ph.D.

Professor, Chair

Basic Science & Craniofacial

Biology E-mail: ncp234@nyu.edu

Michael D Turner, D.D.S., M.D.

Assistant Professor

Oral and Maxillofacial Surgery E-mail: mdt4@nyu.edu

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TABLE OF CONTENTS

INTTRODUCTION ....................................................................................................................... 3

OVERVIEW ................................................................................................................................... 4

WNT PROTEINS ...................................................................................................................................... 4

WNT SECRETION & EXTRACELLULAR TRANSPORT ..................................................................... 7

WNT RECEPTION.................................................................................................................................... 8

WNT SIGNAL TRANSDUCTION PATHWAYS .................................................................................... 9

REGULATION OF WNT SIGNALING.................................................................................................. 14

WNT SIGNALING IN BONE FORMATION ......................................................................................... 15

WNT-4 ..................................................................................................................................................... 17

PTH & WNT PROTEINS (WNT-4) ........................................................................................................ 20

WNT PROTEINS & STEM CELLS ................................................................................................................. 24

ROLE OF WNT SIGNALING IN MESENCHYMAL STEM CELLS ....................................................................... 26

CONCLUSION ............................................................................................................................ 30

REFERENCE ............................................................................................................................... 31

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1. INTRODUCTION

Wnt proteins are palmitoylated and glycosylated ligands that play a well established role in

embryonic patterning, cell proliferation and cell determination [1][2], besides playing a

significant role in cell cycle arrest, differentiation, apoptosis and tissue homeostasis. As a result,

aberrations in the Wnt signaling pathway are associated with birth defects as well as a multitude

of diseases, most notably cancer [3]. Wnt proteins can be categorized into two types: the

canonical and non-canonical.

Wnts have important roles in regulating many aspects of skeletal development,

from limb formation to chondrogenesis and osteoblast maturation [4]. In addition to

promoting osteoblast maturation, Wnts may play a role in lineage commitment of mesenchymal

precursor cells by preventing adipogenesis is the hypothesized default pathway for mesenchymal

stem cells that do not receive proper inductive signals to

become osteoblasts, chondrocytes, myocytes, or other mesodermal cells [5].

Parathyroid hormone (PTH) regulates calcium homeostasis in addition to stimulating bone

formation [6]. Human and experimental animal studies have shown that PTH

increases osteoblast activity and number via increased differentiation and survival, increases

bone mass and bone strength, and decreases fracture rate [7]. Due to its anabolic actions, PTH is

currently the only clinically available anabolic agent used to effectively treat osteoporosis. [8][9].

In vivo microarray analysis has shown that PTH regulates molecules of the Wnt signaling

pathway in bone [10]. PTH has been demonstrated to stimulate bone lining osteoblasts to

produce non-canonical Wnt-4 in vivo, and exogenous Wnt-4 has been shown to regulate

osteoblast differentiation through non-canonical Wnt signaling pathway [11].

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Wnt signaling has been implicated in the control over various types of stem cells and may act as

a niche factor to maintain stem cells in a self-renewing state [12]. Recent studies have shown that

Wnt-3a stimulates proliferation while inhibiting osteogenic differentiation of hMSCs [17], while

non-canonical Wnt-5a enhanced osteogenic differentiation but had no effect on hMSC

proliferation [18]. Most significantly, Wnt-4 has been shown to stimulate osteogenesis in hMSCs

isolated from craniofacial tissue through a novel p38 non-canonical Wnt signaling pathway that

is a known pathway associated with osteogenic differentiation [19][20].

Preliminary findings from Dr. Nicola C. Partridge’s lab at the Department of Basic Science and

Craniofacial Biology at NYU-CD have shown that Wnt-4 is stimulated by PTH in vivo [11], and

that Wnt-4 enhances proliferation and osteogenesis of mouse bone marrow stromal stem cells

(BMSSC) in vitro. From this it is hypothesized that Wnt-4 has an important function in

proliferation and differentiation of BMSSC.

The specific aims of this study are to:

1) Assess the Wnt-4 effect on proliferation on human bone marrow stromal stem cells

2) Assess the Wnt-4 effect on differentiation of human bone marrow stromal stem cells

3) Assess the Wnt-4 effect on the proliferation and differentiation of human dental pulp

stem cells.

2. Overview

2.1 Wnt Protiens

Together with the other families of secreted factors such as FGF, TGF-beta, and Hedgehog

proteins,Wnt proteins are implicated in a wide variety of biological processes. The first Wnt gene

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mouse Wnt-1, was discovered in 1982 as a proto-oncogene activated by integration of mouse

mammary tumor virus in mammary tumors [21]. With the molecular identification of the

Drosophila segment polarity gene wingless (wg) as the orthologue of Wnt-1 [22][23] and the

phenotypic analysis of Wnt-1 mutations in the mouse [24], it became clear that Wnt genes are

important regulators of many developmental decisions [25][26].

Table 1 [21] Phenotypes of Wnt mutations in mouse, Drosophila, and C. elegans

Gene Organism Phenotype Organism Phenotype

Wnt-1 Mouse Loss of midbrain and

cerebellum

Wnt-2 Mouse Placental defects

Wnt-3A Mouse Lack of caudal somites and

tailbud

Wnt-4 Mouse Kidney defects

Wnt-7A Mouse Ventralization of limbs

Wingless Drosophila Segment polarity, limb

development, many others

Dwnt-2 Drosophila Muscle defects, testis

development

lin-44 C.elegans Defects in asymmetric cell

divisions

mom-2 C. elegans Defects in endoderm induction

and spindle orientation

The Wnts comprise a large family of protein ligands that affect diverse processes such as

embryonic induction, generation of cell polarity and the specification of cell fate. In addition to

influencing developmental processes, recent studies point to a key role for Wnt signaling during

adult homeostasis in the maintenance of stem cell pluripotency [27]. Wnts are defined by amino

acid sequence rather than by functional properties [28-29]. As many as 19 mammalian Wnt

homologues are known and are expressed in temporal spatial patterns. Shared features of all

Wnts include a signal sequence for secretion, several highly charged amino acid residues, and

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many glycosylation sites. Wnt proteins also display a characteristic distribution of 22 cysteine

residues. overexpression in tissue culture cells, several different N-linked glycosylated

intermediate Wnt protein products are observed in cell lysates [30-32], suggesting that Wnt

protein processing and secretion are highly regulated processes.

Even though the primary amino acid sequence of Wnts suggests that they should be soluble,

secreted Wnt proteins are hydrophobic and are mostly found associated with cell membranes and

extracellular matrix (ECM), the reason being that Wnt proteins are lipid modified by the

attachment of a palmitate on the first conserved cysteine residue within the protein family and on

a serine in the middle of the protein which may be necessary for:

Wnt signaling as well as secretion

May be necessary for their glycosylation.

Might also aid in Wnt transport between cells as glycosylation might increase Wnt

interactions with heparin sulfate proteoglycans (HSPGs) present on the surface of Wnt

responding cells.

Potentially anchoring Wnt proteins into the membrane for sustained signaling. [33-34].

There are a total of 7 related Wnt genes in the Drosophila genome and 19 in the human genome

(some with multiple isoforms) that generally have close orthologues in mice. Orthologous Wnt

gene products (proteins with the same function in different species) are often very highly

conserved. [35] Based on their ability to induce secondary body axis in Xenopus embryos, the

Wnt family of proteins is grouped into two functional classes. The Wnt-1 class (Wnts-1, 2, 3, 3a,

7b, 8, 8b, 10b) is able and Wnt-5a (Wnts-4, 5a, 5b, 6, 7a, 11) is unable to induce a secondary

body axis [36].

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2.2 Wnt Secretion and Extracellular Transport

Two recent genetic screens have identified the multipass transmembrane protein Wntless

(Wls)/Evenness interrupted (Evi)/Mom-3 as acting in the secretory pathway to promote the

release of Wnts from producing cells [37]. A model of Wnt secretion and realease is shown in

figure 1.Exogenously derived lipoproteins termed ―argosomes‖ are implicated in moving Wnts

and other lipid-modified proteins such as Hedgehogs [38]. A model is proposed wherein

palmitoylated proteins associate with lipoprotein particles on the extracellular face of cells.

Traffic of Wnt proteins from one cell to the next requires this association [38]. In addition,

transcytosis may regulate Wnt movement. It has been proposed that the retromer complex

promotes the association of secreted Wnts with other proteins required for ligand transport, such

as lipoprotein particles. [39]

Figure 1 [40]: Model of Wnt secretion and release. Wnt is lipid modified in the ER and is transported to the Golgi,

where it binds to Wls. Next, the Wnt–Wls complex is transported to the plasma membrane and Wnt is released (1).

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The hydrophobic Wnt protein either remains associated with the plasma membrane or binds directly to lipoprotein

particles or HSPGs to facilitate spreading and gradient formation (2). After the release of Wnt, Wls is internalized

through AP-2/clathrin-mediated endocytosis (3) and is transported back to the TGN through a retromer-dependent

trafficking step (4). An alternative possibility is that Wnt is not released at the plasma membrane, but is

reinternalized together with Wls (5). Dissociation of Wnt from Wls may take place in endosomes, after which Wnt

is released (possibly in association with lipoprotein particles) through a recycling endosomal pathway (6). Also in

this scenario, Wls is recycled back to the TGN through a retromer-dependent transport step (7). Wnt may also be

reinternalized through a Reggie-1/Flotillin-2 dependent pathway (8), which may lead to the release of a more

mobile, micelle-like form of Wnt or to the association of Wnt with lipoprotein particles (6).

2.3 Wnt Reception

The seven-pass transmembrane protein

Frizzled (Fz) protein was first receptor found

to transduce a Wnt signal [41]. . Fz proteins

contain a large extracellular domain

containing a conserved motif comprised of

10 cysteine residues called the cysteine-rich

domain (CRD) that has been shown to bind

to multiple Wnts with high affinity. At the

cytoplasmic side, Fzs may interact directly

with the Dishevelled protein, a known

mediator of Wnt signaling [42-44].

Following Wnt binding, it is thought that Fzs form a co-receptor complex with single-pass

transmembrane proteins of the low-density lipoprotein (LDL) family called Lrp5 and -6 to

transduce the canonical Wnt signal. Lrp5 and -6 proteins have a relatively small intracellular

domain and a large extracellular domain containing several potential protein interaction

domains [45].

Figure 2 [21]: Structure of Frizzleds and FRPs. Frizzled

receptors are characterized by an N-terminal signal peptide,

a cysteine-rich ligand-binding domain (CRD) followed by a

hydrophilic linker,seven transmembrane regions, and a

cytoplasmic tail. FRPs are secreted proteins with a CRD

similar to Frizzleds. In addition, they contain a region with

similarity to netrins, secreted proteins involved in axon

guidance.

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There are other proteins with known Wnt-binding

domains that can serve as receptors for Wnt ligands.

The single-pass tyrosine kinase Ror2, although

structurally distinct from Fz receptors, is involved

in other forms of Wnt signaling. Another well-

characterized Wnt-binding domain is the Wnt

inhibitory factor (WIF) module, which is also found

in the cell surface atypical receptor tyrosine kinase

Ryk [46][47].

2.4 Wnt Signal Transduction Pathways

Wnt proteins signal through the canonical and the non-canonical pathways which are composed

of three independent signal transduction pathways (the Wnt/β-catenin pathway, the

Wnt/Ca+2

pathways, or the Wnt/planar polarity pathway) that are used to regulate the expression

of different genes. The Wnt/β-catenin pathway is commonly referred to as the canonical

pathway. It promotes cell fate determination, proliferation, and survival by increasing β-

catenin levels and altering gene expression through Lef/Tcf transcription factors [48]. The non-

canonical Wnt/Ca+2

pathway stimulates heterotrimeric G proteins, increases intracellular

calcium levels, decreases cyclic GMP levels, and activates protein kinase C to induce NF-

AT and other transcription factors [49]. The non-canonical Wnt/planar polarity pathway activates

Rho/Rac GTPases and Jun N-terminal kinase to modulate cytoskeletal organization and gene

expression. Distinct Wnt ligands probably act through specific Frizzled (Fzd) receptors to initiate

each [49].

Figure 3: Different receptors that Wnt proteins

can bind to.

http://www.stanford.edu/group/nusselab/cgi-

bin/wnt/receptors

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The canonical Wnt signaling pathway (see Figure 5) is activated when Wnts interact with

Lrp/Fzd receptor complexes as shown in the middle portion of figure 4. Receptor engagement

activates an unknown kinase(s) (K) that phosphorylates the cytoplasmic tail of Lrp5/6. These

phosphorylated residues (P) serve as docking sites for Axin and the APC, Dsh, β-catenin

complex. A GSK3β binding protein (GBP) is also mobilized after receptor ligation and excludes

GSK3β from the proximal receptor complex. β-Catenin thereby escapes phosphorylation events

that normally promote its ubiquitination (U) by E2 ligases and degradation by the proteosome

(right side of this figure). As β-catenin levels rise above those needed to bridge cadherins to the

Figure 4: Wnt Signaling Pathways and their Implications

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actin cytoskeleton (lower left of this figure), some β-catenin molecules travel to the nucleus

where they interact with Lef1/Tcf transcription factors to either increase or decrease the

expression of specific target genes. β-Catenin displaces nuclear co-repressors (CoR) from

Lef1/Tcf to facilitate the expression of genes involved in cell cycle progression (e.g. cyclin D1)

and survival (e.g. c-myc). β-Catenin and Lef1/Tcf suppress other genes, such as osteocalcin

(OCN) and E-cadherin, through unknown mechanisms, but that may involve interactions with

other transcription factors (TF). Soluble antagonists block the canonical Wnt signaling pathway

and promote β-catenin degradation via two mechanisms.

Soluble frizzled related proteins (Sfrp) bind free Wnt molecules and compete with surface

receptors (top right of figure 5). In contrast, Dkks interact with extracellular domains in Lrp5/6

Figure 5 [49]: The Canonical Wnt Signaling Pathway

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and recruit them to complexes containing Krm (top left of figure 5). This trimolecular complex is

internalized to lysosomes where Lrp5/6 are either degraded or recycled to the surface. Dkk

therefore decreases Lrp5/6 cell surface expression to regulate Wnt signaling [49].

Accumulated ß-catenin then translocates to the nucleus, replaces Groucho from TCF, and

activates target genes. ß-catenin forms a complex with TCF and the transcription factors Brg1

and CBP. Lgs and Pygo also bind to -catenin, possibly driving its nuclear localization in addition

to playing a direct role in transcriptional activation. Negative regulation of signaling is provided

by NLK (Nemo-like kinase) which phosphorylates TCF, and ICAT (inhibitor of catenin) and

Chibby, which are antagonists of ß-catenin. In addition to TCF, two other DNA-binding proteins

have been shown to associate with ß-catenin: Pitx2 and Prop1. In the case of Prop1, ß-catenin

can act as a transcriptional activator or repressor of specific genes, depending on the co-factors

Figure 6 [55]: Nuclear activity of ß-catenin

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present. The participation of any particular ß-catenin complex in transcriptional regulation is

highly cell type-dependent.[55]

The non-canonical pathway can be broadly classified into 2 branches based upon phenotypic

response; the Planar Cell Polarity (PCP) pathway and the Wnt/ Ca2+

pathway [50]. However

some authors have classified the pathways as Wnt/calcium signaling, Wnt/PCP signaling,

Wnt/JNK signaling and Wnt/Rho signaling [56]. The PCP pathway is involved in cellular

asymmetry, and it is this cellular asymmetry that controls the rigid architectural orientation of

epithelial tissues and sensory organs (e.g. inner ear cochlea), as well as the morphology and the

migratory processes of mesodermal cells undergoing gastrulation. Activation of PCP signaling

occurs basically through the binding of Wnts to Frizzled (Fz) receptors alone, without LRP co-

receptor involvement. These signals activate Dishevelled (Dvl), which in turn leads to the

activation of the GTPases Rho and Rac. Activated Rac subsequently stimulates JNK activation

[50].

The second branch of non-canonical signaling – the Wnt/Ca2+

pathway – is characterized by

Wnt-Fzd-induced PLC (phospholipase C) activation and the resultant increase of cytoplasmic

Ca2+

levels. These Ca2+

fluxes activate several Ca2+

-responsive proteins, such as PKC (protein

kinase C) and CaMKII(calcium/calmodulin-dependent kinase II). CaMKII has been shown to

activate the transcription factor NFAT, TAK1 (TGF-beta activated kinase), and NLK (Nemo-like

kinase, all of which have the net effect of decreasing intracellular cGMP and consequently,

antagonizing Wnt/beta-catenin/TCF signaling [51-52]. The Wnt/Ca2+

signaling pathway is

involved in regulating cellular adhesion, cytoskeletal rearrangements, and other developmental

processes, such as dorsoventral patterning and tissue separation in embryos [53].

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2.5 Regulation of Wnt Signaling:

Wnt signaling is tightly regulated by members of several families of secreted antagonists

1. Wnt signaling requires interaction with frizzled receptors (Fz) and the presence of a

single-pass transmembrane molecule of the Lrp family (Lrp5 or 6) which can be inhibited

by members of the secreted frizzled-related protein (Sfrp) family and Wnt inhibitory

factor 1 (WIF-1).

Figure 7 [54]: Non- canonical pathways

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2. Dkk1 binds to Lrp with high affinity and to another class of transmembrane molecules,

the Kremens. Dkk1 promotes the internalization of Lrp and makes it unavailable for Wnt

reception by forming a complex with Lrp and Kremen [57].

3. Lrp coreceptor activity is also inhibited by members of sclerostin (SOST gene product).

4. Chibby is a nuclear antagonist that binds to the C terminus of β-catenin [58].

5. Another β-catenin binding protein, ICAT, can block the binding of β-catenin to TCF and

also can lead to the dissociation of complexes between β-catenin, LEF,

and CBP/p300 [56].

6. TCF can be phosphorylated by the mitogen-activated protein (MAP) kinase-related

protein kinase NLK/Nemo.

7. The phosphorylation of TCF/LEF by activated Nemo is thought to diminish the DNA-

binding affinity of the β-catenin/TCF/LEF complex, thereby affecting transcriptional

regulation of Wnt target genes [56].

2.6 Wnt Signaling in Bone formation:

The Wnt signal transduction pathway has been implicated in bone formation: patients suffering

from osteoporosis–pseudoglioma syndrome have an inactivating mutation in the Wnt co-

receptor LRP5 [13], whereas an activating LRP5 mutation is associated with high bone mass

syndrome [14] [15]. Analysis of LRP5-deficient mice revealed a decreased number

of osteoblasts suggesting that Wnt signaling stimulates bone formation at the level of

osteoprogenitor proliferation [16].

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The effects of Wnt signaling on chondrogenesis are complex and have been implicated in the

regulation of chondrogenic differentiation and hypertrophy [56]. Chondrocyte-specific

inactivationof β-catenin using Col2a1-Cre transgene leads to decreased chondrocyte proliferation

and delayed hypertrophic chondrocyte differentiation [59]. In another loss-of-function

model, Dermo 1-Cre transgene was used to delete the β-catenin gene in the mesenchymal

precursors of both chondrocytes and osteoblasts. It was shown that there is a significant delay

in chondrocyte maturation in conditional knockout embryos [60]. At the same time Activation

of β-cateninin limb and head mesenchyme repressed the expression of Sox9, a factor essential

for chondrogenesis, thereby preventing mesenchymal cells from differentiating into skeletal

precursors [59]. In the absence of β-catenin, the expression of early osteoblast markers, such

as collagen I, osterix, and osteocalcin was greatly diminished [60].

Figure 8 [56]: Role of canonical Wnt signaling in skeletal development. Mesenchymal stem cells have the ability

to differentiate into chondrocytes or osteoblasts, depending on the environmental cues. Canonical Wnt signaling

is regulated to control the lineage progression between chondrocyte and osteoblast. If there is inadequate

canonical Wnt signaling, differentiation towards chondrocyte lineage is encouraged. However, the maturation of

chondrocytes requires the presence of canonical Wnt signaling. Canonical Wnt signaling is also required for the

progression of osteoblast progenitor cells toward osterix positive osteoblasts and then osteocalcin-positive

osteoblasts. Canonical Wnt signaling represses osteoclastogenesis by increasing the osteoprotegrin (OPG)

expression, therefore the OPG/RANKL ratio. Green plus signs indicate positive effects of Wnt; red circle minus

signs indicate inhibitory effects of physiological canonical Wnt signaling

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Wnt-4:

Wnt-4 is a signaling factor with multiple roles in organogenesis, a deficiency that leads to

abnormal development of the kidney, pituitary gland, female reproductive system, and mammary

gland. Wnt4 is conserved throughout vertebrates and was originally studied in non-mammalian

vertebrates such as zebrafish, chicken and xenopus [61]. Several studies have tried to gauge the

varied functions of this protein, giving the impression that this is indeed a highly conserved

protein essential for proper organ formation and development. Some of the findings are as

follows:

1. It is essential for nephrogenesis. The Wnt-4 gene is expressed in the

assembling nephrons and the medullary stromal cells during kidney development The

Figure 9 [49]: Effects of Wnt signaling on osseous cells. The canonical Wnt signaling pathway

promotes the proliferation, expansion and survival of pre- and immature osteoblasts. Dkks,

Sfrps, and Wif-1 antagonize Wnt signaling in osteoblasts to facilitate death of immature cells,

but they may also downregulate the pathway in mature cells to induce terminal differentiation.

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role of Wnt-4 signaling in controlling mesenchyme to epithelium transformation

and kidney tubule induction is well established [63].

2. Wnt-4 signaling plays also a role in the determination of the fate of smooth muscle

cells in the medullary stroma of the developing kidney. Smooth muscle α-actin (α-SMA)

is markedly reduced in the absence of its signaling [62].

3. Wnt-4 plays an important role in female sexual development and ovarian function. Wnt-4

deficiency leads disturbed development of the internal genitalia in mouse and human, and

to a dramatic reduction of mouse oocytes. The expression of Wnt-4 protein

in human fetal ovaries was high during mid-pregnancy, when new follicles are also

rapidly being formed. This implies that Wnt-4 may have a substantial role in regulation

of the follicle formation in human ovaries [64].

4. In the reproductive system, Wnt-4 is specifically involved in Müllerian duct formation,

sex-specific blood vessel formation, oocyte maintenance and repression of

steroidogenesis. Partial XY male-to-female sex-reversal is observed in mice lacking Wnt-

4 as well as in one human patient carrying a Wnt-4 point mutation [61].

5. During mammalian embryogenesis, Wnt-4 is expressed in the gonads of both sexes

before sex determination events take place and is subsequently down-regulated in the

male gonad [61].

6. Wnt4 regulates mammary gland development in response to hormonal changes that take

place during pregnancy and may also play a part in mammary gland tumorigenesis.

Expression of Wnt4 is inversely correlated with cell proliferation in the mouse mammary

C57 mg cell line, suggesting that Wnt4 participates in restricting cell proliferation. On the

other hand, the elevated expression of theWNT4 gene in fibroadenomas, and occasionally

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also in malignant tumor tissue, implies that changes in WNT4 signaling are associated

with abnormal cell proliferation in human breast cells [68].

7. Screening of the multiple adult human tissues

revealed three human WNT4 mRNA bands of 1.5,

2.4, and 4.3 kb in size (Figure 8). The most

common 1.5 kb transcript was detected in a

number of tissues: the adrenal

gland, placenta, liver, mammary

gland, prostate, spinalcord, stomach, thyroid, trach

ea, skeletal muscle and small intestine, the signal

being strongest in the adrenal gland and placental

samples.

8. The involvement of Wnt-4 in normal mammary

gland and ovary development suggests that Wnt-

4 germline mutations may be associated with

the human cancer predisposition [65].

9. Wnt-4 is critical for development of the adrenal gland, the zona glomerulosa of which is

incomplete at birth in Wnt-4-deficient mice, probably as a result of significantly reduced

aldosterone production. In addition, Wnt-4 represses the migration of steroidogenic

adrenal precursors into the gonad [66]. In mouse spleen, Wnt1and Wnt-4 signaling

regulates differentiation of the thymocytes, the number of which is decreased in

compound mutants [67].

Figure 10 [65]. WNT4 transcripts in human adult

(A) and embryonic (B) tissues. Three mRNA bands,

1.5, 2.4 and 4.3 kb in size, are seen in the adult

tissues and one, 2.4 kb, in the embryonic liver and

kidney tissues. The ages of the embryos from 6 to

12 weeks are indicated below the sample rows. The

arrows show the positions of ribosomal

RNA.

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2.7 PTH and Wnt Proteins (WNT-4)

PTH is a single chain polypeptide with 84 amino acids and is the principle regulator of

calcium homeostasis in vertebrates. However, although continuous infusion of PTH induces

bone loss, intermittent administration of PTH results in bone formation [69]. Binding

of PTH and PTHrP to PTH1R activates two signaling pathways in osteoblasts: the PKA pathway

which is responsible for the majority of the calciotropic and skeletal actions of PTH, the PKC

pathway leading to accumulation of 1, 4, 5-inositol triphosphate and increased intracellular

calcium. This pathway has been found to regulate IGF-binding protein-5 [70].

A number of studies suggest that the PTH and Wnt signaling pathways do indeed overlap.

Following treatment of osteoblastic cells with N-terminal PTH, an increase in β-catenin that was

measured in whole cell lysates by Western blot was seen [71].

Figure 11 [65]. Mapping of the human WNT4 locus to chromosome region 1p36.12 by prometaphase FISH. (A)

Hybridization signals for WNT4 (green signals) and BAC RP11-285H13 (red signals). (B) Chromosomal

location of BAC RP11-285H13 to the region 1p36.11a-1p36.11b and BAC RP11-145C4 to the region 1p36.13c-

1p36.13d (C) Hybridization signals for WNT4 (green signals) and BAC RP11-145C4 (red signals).

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Furthermore, PTH increased the level of β-catenin expression in mouse osteoblastic cells

(MC3T3-E1) via both PKA and PKC signaling pathways [74]. More recently, PTH was shown to

activate β-catenin signaling in osteoblasts in vitro and in vivo by direct recruitment of LRP6 to

PTH/PTH1R complex. In vivo studies confirmed that intermittent PTH treatment led to an

increase in amount of β catenin in osteoblasts (immunohistochemical analysis with antibody

to β-catenin) with a concurrent increase in bone formation in rat [75].

Partridge et al. also demonstrated a link between PTH and Wnt4 expression in bone [72]. In

vivo microarray analysis of intermittent and continuous PTH 1–34 showed

Figure 12: PTH regulation of Wnt-4

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that PTH regulated Wnt4 in bone. PTH was shown to stimulate Wnt4 primarily through

the PKA pathway and Wnt4 treatment in osteoblasts induced early expression of bone marker

genes and stimulated key canonical Wnt pathway genes. This finding suggested cross-talk

between the Wnt signaling cascades [73].

Previous studies at Dr. Partridge’s lab at the Department of Basic Science & Craniofacial

Biology at NYU-CD has showed that Wnt-4 is significantly regulated by PTH in vivo after

anabolic and catabolic protocols in bone lining osteoblasts. As pointed out earlier Partridge et

al. demonstrated that PTH stimulated Wnt-4 expression in cultured osteoblastic cells and that

this stimulation is PKA dependent and is a primary response to PTH. It has also been shown

that exogenous Wnt-4 enhances bone marker gene expression during osteoblast

differentiation by activating the non-canonical Wnt/Ca2+ and Wnt/PCP signaling pathways

but does not significantly stimulate the canonical Wnt/ß-catenin pathway. Most significantly,

Wnt-4 has been shown to stimulate osteogenesis in hMSCs isolated from craniofacial tissue

through a novel p38 non-canonical Wnt signaling pathway that is a known pathway

associated with osteogenic differentiation [19][20]. Therefore it can be hypothesized that non-

canonical Wnt signaling plays a significant role in bone and that Wnt-4 may be an important

molecule in PTH’s anabolic effect.[72]

Real time TR-PCR results have shown that PTH stimulates Wnt-4 mRNA expression in all

phases of osteoblastic differentiation but is greatest in the mineralization phase and maximal

stimulation occurs 8h after PTH treatment. Partridge et al. has demonstrated that non-

canonical Wnt-4 does not increase pre-osteoblastic proliferation or the expression of bone

marker genes at proliferation day 7, but enhances bone marker gene expression (runx2,

osterix, osteocalcin, alkaline phosphatase, MMP-13) significantly in the absence of

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differentiation-promoting factors such as ascorbate. Continuous treatment of rat primary

osteobalsts with Wnt-4 in an osteogenic environment showed that there was increased

osteocalcin mRNA and alkaline phosphatase expression in late stage differentiation with

increased mineralized nodules. In addition there was increased relative expression of runx2

and osterix mRNA during osteoblast differentiation. It was also found that Wnt-4 acute

treatment in the early stages of primary cell differentiation was much more effective at

stimulating the relative gene expression of runx2, osterix, osteocalcin, alkaline phosphatase,

and collagen-1a mRNA expression. Together these suggest that Wnt-4 may promote the

differentiation of osteoblasts as well as uncommitted cells in the bone environment as part of

PTH’s anabolic effect.[72]

Figure 13: Effect of WNT-4 on osteoblast proliferation & differentiation

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2.8 Wnt Proteins and Stem Cells

The Wnt Signaling pathway has been found to play a very important role in maintaining the

potency of stem cells and stem cell fate determination. Stem cells from different locations

interpret Wnt in different ways which reflects an activation of distinct genetic programs in

response to the same signal. In addition, the time point during development at which a stem cell

is challenged by Wnt signals determines whether the cell responds by self-renewal or

differentiation. Besides the cell-intrinsic cues that influence the biological activity of Wnt in

distinct stem and progenitor cell types, the same type of stem cell might respond in different

ways to Wnts, depending on its extracellular microenvironment (illustrated in Figure 14 and 15)

[88].

Figure 14. [88] Cell-intrinsic differences among stem cells

influence the biological function of Wnts. (a) Different stem cell

types differentially respond to canonical Wnt signaling and

undergo either self-renewal or lineage commitment. The

differential responsiveness of stem cells is presumably due to

distinct cell-intrinsic determinants (indicated in the figure by

differently colored cells). (b) A stem cell type of a given cell

lineage (indicated in various grades of green) can integrate

canonical Wnt signaling in different ways, depending on its cell-

intrinsic properties which change over time. At different stages of

development, Wnt promotes stem cell self-renewal or lineage

commitment, or the cell loses its ability to respond to Wnt.

Figure 15. [88] The effect of canonical Wnt on a particular

type of stem cell is context-dependent. (a) In a

microenvironment X, Wnt activity is modulated by the

factor(s) X. In this context, Wnt signaling elicits self-

renewal. (b) However, the very same stem cell in

microenvironment Y, in which Wnts are modulated by the

factor(s) Y, responds to Wnt signaling by adopting a specific

cell fate rather than by self-renewing. Thus, the biological

activity of Wnt in a particular microenvironment is influenced

by the convergence of Wnt signaling with other signal

transduction pathways.

25 | P a g e

Some of the findings regarding the role of Wnt signaling in stem cells are as follows:

1. In embryonic stem cells, over expression of Wnt1 or of stabilized β-catenin results in the

inhibition of neural differentiation [77].

2. Treatment of haematopoietic stem cells with Wnt proteins and sustained expression of β-

catenin promotes self-renewal in long-term cultures and increases the reconstitution of

haematopoietic lineages in vivo, which could have been mediated by Notch1 and

the transcription factor HoxB4. However, conditional ablation of β-catenin in

haematopoietic stem cells did not impair haematopoiesis and lymphopoiesis, suggesting

that β-catenin is not required for self-renewal and development of haematopoietic stem

cells under physiological conditions [78].

3. Wnts play major roles during central nervous system (CNS) development. Ablation

of wnt1 results in severe defects of the midbrain, the cerebellum and the

developing spinal cord, while ablation of wnt3A results in a total loss of

the hippocampus. The defects observed in Wnt mutants are possibly explained by

perturbed proliferation of stem or progenitor cells in the ventricular zone [78] [79] [80].

4. Wnt/β-catenin signaling is not only essential for the homeostasis of the intestinal

epithelium; sustained β-catenin activity has also been implicated in the formation

of colon carcinoma [78] [81].

5. In vivo manipulation of genes encoding Wnt signaling components indicates that ß–

catenin deficient stem cells fail to differentiate into follicular keratinocytes and instead

adopt an epidermal fate and thus play a very important role in the fate determination

process of epidermal stem cells [82].

26 | P a g e

6. Wnt signaling has been implicated in the early stages of neural crest development, such

as neural crest induction and melanocyte formation. In neural crest stem cells, the genetic

ablation of β-catenin results in lack of melanocytes and sensory neural cells in dorsal root

ganglia. In fact, NCSCs lacking β-catenin emigrate and proliferate normally but are

unable to acquire a sensory neuronal fate. Constitutive expression of β-catenin in neural

stem/progenitor cells results in the expansion of the entire neural tube, supporting a role

of β-catenin in progenitor proliferation [83].

7. In a recent study from Nusse's laboratory, Axin2 gain-of-function mice were used to

demonstrate that Wnt3A functions as a rate-limiting, self-renewal factor to clonally

expand mammary stem cells (MaSC) [84].

2.9 Role of Wnt Signaling in Mesenchymal Stem Cells:

Mesenchymal stem cells (MSCs), otherwise termed as mesenchymal progenitor cells or marrow

stromal cells are adherent, fibroblast-like population with the potential for extensive self-renewal

and multilineage differentiation. Under appropriate culture conditions, MSCs are capable of

giving rise to osteoblasts, adipocytes, chondrocytes and myoblasts. Their multipotency, ease of

isolation and ready availability make MSCs particularly suited for tissue engineering and gene

therapy applications [76].

MSCs express a number of Wnt ligands such as Wnt-2, Wnt-4, Wnt-5a, Wnt-11 and several Wnt

receptors as well as various co-receptors and Wnt inhibitors [85]. Exogenous application of Wnt-

3a to cell cultures expands the multi-potential population of MSCs and this proliferative is

presumably achieved by the up-regulation of cyclin D1 and c-myc both of which drive cell cycle

progression to promote growth [17] [86].

27 | P a g e

Figure 16: Wnt-4 signaling in the bone as part of PTH’s anabolic effect

28 | P a g e

In previous studies done in Dr. Partridge’s lab by Bergenstock et al., it was found that Wnt-4

increased cell proliferation and CFU-F numbers by inhibiting mouse BMSSC apoptosis and

stimulating their rate of growth. mBMSSCs treated with Wnt-4 in osteogenic and non-

osteogenic cultures showed a significant increase in osteocalcin bone marker gene expression

and a significant reduction of adipocyte marker genes (ap2, pparϒ and C/EBPα). It was found

that Wnt-4 stimulated the Wnt/ß-catenin pathway in proliferating mBMSSCs after Wnt-4

treatment. Wnt-4 did not alter ß-catenin expression but stimulated the phosphorylation of

CamKII (non-canonical Wnt/Ca2+) in differentiating mBMSSCs while inhibiting JNK

phophorylation (non-canonical Wnt/PCP) in proliferating mBMSSCs. This suggests the

divergent effect of Wnt-4 depending on the developmental stage of the cell. Since Wnt-4 is

regulated by PTH in bone, this suggests two functions for Wnt-4 in the stem cell environment

as part of PTH’s anabolic effects. The first being to act on stem cells and still uncommitted

osteoprogenitor cells to expand cell numbers which would eventually be released into

osteogenic differentiation.

The mechanism of signaling switch, that determines whether canonical or non-canonical Wnts

increase stem cell proliferation or stimulate differentiation in certain tissues is not well

understood. Now the question arises how Wnt-4 stimulates the canonical and non-canonical

pathways. One paper suggests that the balance and coordination between

nuclear/transcriptionally active beta-catenin and cytoplasmic/cytoskeletal beta-catenin couples

canonical and non-canonical Wnt signaling. Another suggestion is that the coactivators CBP and

p300, part of the Wnt signaling network of proteins, plays the integrator role [87].

29 | P a g e

CREB binding protein (CBP) and p300 are

key regulators of RNA polymeraseII-mediated

transcription that encode highly related protein

accetlytransferases that bind a variety of

transcriptional regulators and other proteins.

As pointed out earlier, research done in this

lab has pointed out that continuous treatment

of mBMSSCs with Wnt-4 activates Wnt/beta-

catenin pathway in the proliferation stage in a

significant manner and then the Wnt/Ca2+

pathway is activated in differentiation, but

beta-catenin continues to be produced albeit

not in a significant manner as in the

proliferation stage. Perhaps the co-activators

CBP and p300 along with nuclear beta-catenin

plays a role in making the switch from

proliferation to differentiation.

A recent paper has described that that CBP/beta-catenin mediated transcription is critical for

stem cell/ progenitor cell maintenance and proliferation, whereas a switch to p300/beta-

catenin mediated transcription is the initial critical step to initiate differentiation and a decrease

in cellular potency. A subset of the gene expression cassette (e.g. Oct4, surviving, etc.) is critical

for the maintenance of potency and proliferation, other genes such as hNkd and axin2 that are

regulated in this manner are negative regulators that stop proliferation, exit cell cycle and initiate

Figure 17 [87]: A. model of coactivator usage. Antagonizing the

CBP/beta-catenin interaction leads to the downregulation of genes

that are critical for stem cell/progenitor cell maintenance and

proliferation (left arm of pathway). This also pushes the cell to

utilize p300 as its coactivator. The switch to p300/beta-catenin-

mediated transcription is the first critical step to initiate a

differentiative program. B. a subset of the gene expression cassette

that is regulated by the CBP/beta-catenin arm is critical for the

maintenance of potency and proliferation (e.g. Oct4, survivin, etc.).

Other genes that are similarly regulated by CBP/beta-catenin (e.g.

hNkd and axin2) are involved in the negative feedback of this arm of

the pathway, and initiate differentiation via a switch to the

p300/beta-catenin arm.

30 | P a g e

the process of differentiation [87]. The shift from the canonical to the non-canonical pathway

relies on the activation of the PKC pathway. PKC phosphorylation of Ser89 of p300 increases

the affinity of p300 for beta-catenin both in vivo an in vitro and thus the switch occurs and the

differentiation pathway is initiated (shown in Figure 17) [87]. If this is true in the case of Wnt-4

is still to be investigated.

3. Conclusion:

Wnt-4 is significantly regulated by PTH as part of its anabolic effect in the bone environment.

Preliminary studies have revealed that Wnt-4 expression is stimulated in osteoblasts in a PKA

dependent manner and it is a primary response to PTH. It has been found to promote osteoblast

differentiation by enhancing bone marker gene expression by activating the non-canonical

pathways.

In mBMSSCs, Wnt-4 has been found to stimulate proliferation by activating the canonical

pathway and then induce differentiation along the osteoblast lineage by stimulating the non-

canonical pathway.

Next we are going to assess if the Wnt-4 can induce proliferation and differentiation along the

osteoblast lineage in human BMSSCs and human dental pulp stem cells (hDPSCs).

31 | P a g e

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