Arthur Veis1,4, Kevin Tompkins1, Keith Alvares1, Kuiru ... · A rat incisor tooth odontoblast-pulp...

Specific Amelogenin Gene Splice Products have Signaling Effects on Cells in

Culture and in Implants In Vivo.

Arthur Veis1,4, Kevin Tompkins1, Keith Alvares1, Kuiru Wei1, Lin Wang2, Xue Song

Wang1, Anna G. Brownell3, Shure-Min Jengh1 and Kevin E. Healy1.

1 Department of Basic and Behavioral Sciences, Northwestern University Dental School,

303 E. Chicago Ave., Chicago, IL 60611, USA.

2Present Address: Department of Orthodontics, Stomatological Hospital, Nanjing

Medical University, Nanjing, Jiangsu, China (PRC), 21029.

3 Present Address: Chapman University, Department of Biological Sciences

Orange, CA 92866

We are pleased to acknowledge that this work has been supported by grants from the

NIH-NIDCR, DE-01374 and DE-08525, and from Osiris Therapeutics, Inc.

Running Title: Small amelogenins: Signaling Molecules

4 Corresponding Author:

Dr. Arthur Veis

Department of Basic and Behavioral Sciences,

Northwestern University Dental School,

303 E. Chicago Ave.,

Chicago, IL 60611, USA.

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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 20, 2000 as Manuscript M002308200 by guest on A

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Phone: 312-503-1355; Fax: 312-503-2544; E-mail: [email protected]

ABSTRACT

Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin

have the ability to affect the differentiation pathway of embryonic muscle fibroblasts

(EMFs) in culture and lead to the formation of mineralized matrix in in vivo implants.

The objective of the present study was to determine if the bioactive peptides could have

been amelogenin protein degradation products or specific amelogenin gene splice

products. Thus, the splice products were prepared and their activities were determined in

vitro and in vivo. A rat incisor tooth odontoblast-pulp cDNA library was screened using

probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised

from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d,7 were identified. The

corresponding recombinant proteins, designated r[A+4] (8.1 kDa) and r[A-4] (6.9 kDa),

were produced. Both peptides enhanced in vitro sulfate incorporation into proteoglycan

and the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivo

implant assays demonstrated implant mineralization accompanied by vascularization and

the presence of the bone matrix proteins, BSP and BAG-75. We postulate that during

tooth development these specific amelogenin gene splice products, [A+4] and [A-4],

may have a role in preodontoblast maturation. The [A+4] and [A-4] may thus be tissue-

specific epithelial-mesenchymal signaling molecules.

Key Words: Amelogenin, Differentiation, Odontogenesis, Chondrogenesis, Osteogenesis,

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Transcription factors

Members of the BMP/VGR family of proteins have the ability to induce

osteogenesis when implanted in appropriate carriers at non-bone sites in vivo [1, 2].

Demineralized bone matrix was the initial source of the BMPs. Addition of the proteins

extracted from bone to non-bone cells in vitro led to the expression of proteins

characteristic of the chondrogenic and/or osteogenic phenotype [3-10]. Surprisingly,

demineralized dentin matrix implants exhibited a stronger osteogenic inductive activity

[3-6]. Fractionation studies showed that the principal activity of rat incisor dentin matrix

resided in a fraction with molecular mass in the range of 6 – 10 kDa, with pI 5.4-5.5, and

a composition devoid of cysteine: properties distinctly different from the members of the

BMP-TGFβ family [8-10]. It was thus likely that the dentin matrix activity was not

related to the BMP/VGR family.

Unfortunately, the final peptide fractions obtained by Amar et al. [8] were not

pure, and the amino-terminal sequence and composition data obtained could not be

related to a single protein component. The very low content of the active peptides in rat

incisor dentin made it impractical to continue using rat incisor dentin as the peptide

source. With some modifications in isolation procedure, but using the same in vitro assay

systems [11] the comparable fraction was isolated from bovine dentin in essentially

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homogeneous form and its activity was verified in in vitro and in vivo assays. The

amino-terminal sequence and one internal tryptic peptide sequence were determined.

Both sequences proved to be derived from the amino terminal portion of bovine

amelogenin [11, 12]. This was a surprising result for two reasons. First, the active

peptides had been isolated from both rat and bovine dentin cleaned as well as possible

from enamel contamination. Second, the principal function of the amelogenins and their

degradation products have been assigned to structural roles in creating the space and

milieu for promoting enamel mineralization [13]. Recently, however, a mixture of

porcine enamel proteins has been used clinically [14] to induce cementogenesis along the

tooth root surface, and the activity was attributed to amelogenin. Thus, it appeared to be

of interest to explore the cell signaling activity of the amelogenin peptides.

The amelogenins present in the tooth at any stage are a complex mixture of gene

isoforms and degradation products [13]. The two peptides partially sequenced by Nebgen

et al. [11] were the products of exon 2-3 and exon 5 transcription, respectively, both

from the amino terminal region of amelogenin. Every intact amelogenin molecule, most

alternatively spliced isoforms, and the major amino-terminal region degradation product

known as TRAP (Tyrosine Rich Amelogenin Peptide) would have yielded these

sequences. Amelogenin amino acid sequences are highly conserved across all species

although the human and bovine have amelogenin genes on the X and Y chromosomes

whereas rat and murine amelogenin genes reside only on the Y chromosome. These genes

yield distinct sets of splice product isoforms, [12,15,16]. However, the larger

amelogenins are specifically degraded in step-wise fashion and also yield a variety of

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smaller peptides during the process of enamel mineralization [17].

The “active” peptide described by Nebgen et al. [11] was characterized only by

amino-terminal sequencing. It was not determined if it was an amelogenin degradation

product or an intact polypeptide transcribed and translated as a specific enamel gene

splice product. This is a very important distinction relative to the function and regulation

of the potential in vivo activity of the peptide. Thus, the objective of the work reported

here was to determine if the message corresponding to the specific gene splice product

was present and, if so, to prepare the peptide and determine if it could express the cell

inductive activities equivalent to the peptide isolated by Nebgen et al. [11].

Since the protein isolation work [7,8 10,11] had focused on dentin extracts, our

approach was to examine a rat incisor odontoblast-pulp based cDNA library for the

presence of an amelogenin-related cDNA. The rationale for choosing the rat incisor

cDNA library was three fold. First, there is high conservation of the amelogenin

sequences between rat and bovine species [13]. Second, our cDNA library has been

verified [18,19,20] to contain the cDNAs for the dentin matrix proteins, DMP1, DMP2

and DMP3 (DSPP). Third, the mRNAs for these three dentin proteins are transiently

expressed in mouse molar enamel organs during fetal and immediately post-natal tooth

development [21,22,23], suggesting the possibility that there might be a reciprocal

transient expression of particular splice products of the amelogenin gene in developing

odontoblasts.

MATERIALS AND METHODS

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Cloning and Sequencing of the Amelogenin Peptides. Freshly extracted rat incisors were

cleaned to remove the soft enamel. The odontoblasts and pulp cells were retained. Poly

A+ RNA was isolated from these cells using the Oligotex mRNA kit (Quiagen, Valencia,

CA). The mRNA was converted to first strand cDNA using an 18mer oligo(dT) and

Superscript II reverse transcriptase (Life Technologies, GIBCO BRL, Grand Island, NY).

The first strand cDNA was then used in a PCR reaction. The forward primer (P1)

ATGCCTCTACCACCT was based on the amelogenin amino terminal peptide sequence

MPLPP and the reverse primer (P2) TATCATGCTCTGGTACCA corresponded to the

tryptic peptide sequence WYQSMI [11]. Fig. 1A shows the rat amelogenin gene intron-

exon organization and the specific location of the primers. The PCR conditions were 25

cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Two PCR product bands,

differing in size by 42 nucleotides, were obtained. These were further amplified by

another round of PCR, then cloned in pGEMT vector (Promega, Madison, WI) and

sequenced.

Each of the amplified bands was used to screen a previously prepared λgt11 rat

incisor odontoblast cDNA library [18]. Positive clones were picked and plaque purified

through three successive rounds of screening. Finally, pure plaques were then amplified

and phage DNA was prepared [24], digested with EcoR1, then cloned into the EcoR1 site

of pBluescript KS (Stratagene, La Jolla, CA) and sequenced.

Since two amelogenin amino-terminal domain PCR products were obtained

initially, two new primers were designed to examine the possibility of differentially

spliced products. Forward primer (P3) TTCCCGAATTCCATGCCCCTACCACCTCA

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contained a unique EcoR1 site (underlined) and included the first fifteen nucleotides of

the secreted form of the protein. The reverse primer (P4)

GGCCGCTCGAGTTAATCCACTTCTTCCCG contained a unique Xho1 site (underlined) and included the

nucleotides and the stop codon TAA. These primers (Fig. 1A) were used in a PCR

reaction, under the conditions described above, using the phage DNA obtained from

amplification of the same λgt11 odontoblast library as template. The PCR amplified

bands were cloned in pGEMT vector and sequenced.

Expression of the Cloned Amelogenins. The cloned amelogenins were expressed as the

GST-fusion proteins. The inserts in pGEMT were re-amplified by PCR, using the

primers P3 and P4 and conditions described above. The PCR products were digested with

EcoR1 and Xho1, purified on a 1% agarose gel and cloned in frame into the EcoR1/Xho1

site of the GST expression vector pGEXT4 (Pharmacia, Piscataway, NJ). The resulting

plasmid was introduced into the E. coli strain BL21(DE3). For preparation of the fusion

protein, a single colony was inoculated into 100 ml of LB and grown overnight. An

additional 900 ml of LB was added and growth continued for 4 h, after which isopropyl

β-D thiogalactoside (IPTG) (Pharmacia) was added to a final concentration of 1 µM.

Incubation was carried on for an additional 4h. The expressed protein was then passed

over and collected on a Glutathione-Sepharose affinity column (Pharmacia) according to

the manufacturer’s instructions. For different purposes the fusion proteins were either

directly eluted from the column with reduced glutathione or the bound protein was treated

with thrombin to release the recombinant peptide.

Isolation of the Recombinant Peptides. In most preparations, the thrombin released

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peptides were a heterogeneous mixture. Therefore the eluted thrombin cleaved protein

was passed over a C-18 reverse phase column (Vydac, Sep/a/ra/tions Group, Hesperia,

CA) developed by an increasing gradient of acetonitrile - 1% trifluoroacetic acid as

described [11] for the final step of purification of the protein extracted from dentin

matrix.

Assays for Biological Activity. In vitro [35S]- SO4 assay for chondrogenic activity. The

purified recombinant proteins were tested for biological activity by the assay for

enhanced incorporation of 35[S]-SO4 into proteoglycan [8,11] by embryonic rat muscle

fibroblasts (EMF). Recombinant human BMP2 (A kind gift from Genetics Institute,

Boston, MA) and the bioactive crude S100 fractions from rat incisor dentin [8] and/or

bovine dentin [11] were used as the positive controls. Bovine serum albumin (BSA) in

phosphate buffered saline (PBS) was the negative control. A commercial preparation of

purified porcine amelogenins, known as Emdogain® (BIORA AB, Malmö, Sweden) [14]

was also tested.

In vitro assay for expression of chondrogenic/osteogenic activity via production

of marker mRNAs. The expression of SOX9 protein is necessary, but not sufficient, for

the induction of chondrogenesis and type II collagen [25-32], while expression of Cbfa1

protein is necessary, but not sufficient, for osteoblast differentiation [33-41]. EMF

cultures at passage 2 were seeded into type I collagen-coated T-150 flasks (Corning,

Corning, NY) according to Nebgen et al. (11) and grown to ~ 80 % confluence in 10%

fetal bovine serum (FBS), 1% pen/strep. The cells were trypsinized and passed into T75

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flasks and grown again to ~ 80 % confluence. The media was removed and the cells

washed with PBS. Conditioning media, 0.5% FBS in αMEM, 1% pen/strep, was added

and the cells were held for 24 h. The conditioning media was replaced with fresh

conditioning media containing various concentrations of the test factors, or no additions

for the controls. At selected time periods of incubation the cells were washed in PBS,

detached with trypsin. An equal volume of 10% FBS was added and the cells were

pelleted. The pelleted cells were suspended in PBS, repelleted and stored at -80° C.

RNA was isolated from the cells using the Rneasy Mini kit (Qiagen) according to

the manufacturers instructions. Reverse transcription was carried out using the Promega

RT system with reaction at 49°C for 50 min. The gene-specific primers were used in

every case for the RT reaction, except for the type I collagen. In that case a non-specific

oligo-dT primer was used, as well as the gene-specific primer noted below. PCR was

carried out using 45 µl Gibco PCR Platinum Tag Supermix to which 1 µl of each primer

(40 mM) and 3 µl cDNA template was added. The primers and conditions were: Sox9

[42] - F - CGGAACAGACTCACATCTCTCCTAATGC (nt #878-906); R -

CGAAGG TCTCAATGTTGGAGATGACGTC (nt #1142-1170), denaturation 3 min at

94°C, followed by 30 cycles: 30 s at 94°C, 30 s at 60°C, 50 s at 72°C, followed by

extension at 72°C for 10 min; product, 292 bp. Cbfa1 [38] F - CCGCACGAC

AACCGCACCAT (nt #511-530); R - CGCTCCGGCCCACAAATCTC (nt #781-800),

denaturation 3 min at 94°C, followed by 30 cycles: 30 s at 94°C, 30 s at 60°C, 50 s at

72°C, followed by extension at 72°C for 10 min; product, 289 bp. Collagen II (Rat type II,

Gene Bank Accession Number L48440) F - CACACCGGT AAGTGGGGCAAGACC

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(nt #4258-4281), R - CTGCGGTTAGAAAGTATTTGGGTC (nt #4444-4468),

denaturation 3 min at 94°C, followed by 30 cycles: 30 s at 94°C, 30 s at 65°C; 50 s at

72°C, followed by extension for 10 min at 72°C ; product, 210 bp. Collagen I (Rat type I, pro

α2(I), Gene Bank Accession Number AF121217 ), F –

GCTCAGCTTTGTGGATACGCG (nt #3-24), R – GTCAGAATACTGAGCAGCAAA

(nt #243-267), denaturation 3 min at 94°C, followed by 30 cycles: 30 s 94°C, 30 s at

58°C, 50 s at 72°C, followed by extension for 10 min at 72°C; product, 264 bp.

Glyceraldehyde phosphate dehydrogenase (GAPDH) [43,44], F

–CTTCACCACCATGGAGAAGG (nt #276-293), R- CTT ACTCCTTGGAGGCCAT

(nt #944-963), denaturation 3 min at 94°C, followed by 30 cycles: 30 s 94°C, 30 s at

58°C, 50 s at 72°C, followed by extension for 10 min at 72°C; product, 687 bp.

All PCR products were run on ethidium bromide-containing 3% agarose gels at

75 volts for 60 min.

In vivo activity. Implant protocols. The recombinant proteins, Emdogain and

BSA controls were each included in a bioabsorbable polymer matrix of poly(D,L-

lactide-co-glycolide) [45]. The polymer scaffolds were cast as 2.5 cm discs containing a

total of 1 mg recombinant protein, 1 mg BSA, 1 mg rhBMP2 or 1.5 mg Emdogain. Each

disc was cut into six equal wedges. A wedge was then placed into the right hind thigh

muscle of a 4 week old, ∼100 g Long-Evans rat. A negative control wedge of bovine

serum albumin in PBS was placed in the contra-lateral left thigh. Four animals were used

for each test condition. All surgical implant protocols and animal care procedures were

reviewed and approved by the Northwestern University Animal Care and Use

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Committee. The implants were followed radiographically with a measurement every

week.

Vascularization, Mineralization. The matrices were removed at four weeks or six

weeks after implantation, and processed for histology. The implant blocks were fixed in

10% formalin, radiographed, then embedded in paraffin. Serial sections were cut and

examined following staining with standard hematoxylin-eosin (H&E), von Kossa,

Alizarin Red and Goldner’s Trichrome stains.

Immunodetection of Bone-Specific Matrix Proteins. The sections were

deparaffinized with xylene washes, 3 times, for 3 min each, and rehydrated by passage

through decreasing concentrations of alcohol. The tissue was then fixed in 10% formalin

for 15 min, and washed 1 min with PBS. The cells were permeabilized by exposure to

acetone for 5 min, washed 1 min with PBS, blocked for 1 h in phosphate buffered saline

plus 0.5% BSA, then washed with PBS. Primary antibody was added to each section

directly without dilution from stock, 10-20 µl/section. Sections were incubated in the

dark for 1 h, then washed 3 times for 1 min with PBS. The secondary antibody was

applied at 1:50 dilution, 10-20 µl/section. The sections were incubated for 1 h in the

dark, then washed 3x with PBS, 1 min/wash. The sections were mounted and viewed

immediately using either a Zeiss Axiovert 100 microscope with a ZVS-3C75DE digital

camera, or a Leitz Dialux 20 microscope with a RT SPOT slider camera (Diagnostic

Instruments, Inc., Sterling Heights, MI).

The primary antibodies were anti-bone sialoprotein (BSP, Antibody

WVID1(9C5), Developmental Studies Hybridoma Bank, University of Iowa, Iowa City,

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Iowa), and anti-bone acidic glycoprotein 75 (BAG-75), a generous gift from Dr. Jeffrey

P. Gorski, University of Missouri, Kansas City. Secondary antibodies were Texas Red

conjugated to anti-mouse IgG for BSP and fluorescein (FITC)-conjugated to anti-rabbit

IgG for BAG-75. These antibodies were all from Jackson ImmunoResearch

Laboratories, West Grove, PA. Nuclei were labeled with DAPI reagent (Pierce,

Rockford, IL)

RESULTS

Preparation of amelogenin peptides from rat incisor cDNA.

When the PCR primers P1 and P2 were used to probe the mRNA isolated from

fresh rat incisor odontoblast-pulp complex (Fig. 1A), two PCR products were detected,

Fig. 2A, and sequenced. Their nucleotide sequences corresponded to the amino acid

sequences, MPLPPHPGHPGYINFSYEVLTPLKWYQSMI (PCR90) (primers P1 and P2

underlined), and MPLPPHPGHPGYINFSYEKSHSQAINTDRTALVLTPLKWYQSMI

(PCR132). The band corresponding to PCR90 was much more intense than that for

PCR132. PCR90 corresponded exactly to the secreted protein amino terminal sequence

encoded by rat amelogenin gene exons 2,3,5. PCR132 included exon 4 (sequence in

Italics, above) [46]. These data established that differentially spliced amelogenin

mRNAs, containing exons 2,3,5 and 2,3,4,5, respectively, were indeed present in the

presumed odontoblast-pulp tissue. Based on the higher intensity of PCR90 on the gels it

is likely that there was a higher concentration of its mRNA than for the PCR132

transcript containing exon 4, although this could also signify that the two mRNAs require

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different conditions for optimal reverse transcription.

When the established odontoblast-pulp rat incisor λgt11 cDNA library [18] was

screened with forward and reverse primers P3 and P4, four PCR product bands were

amplified from the template phage DNA. The PCR bands at approximately 600 and 200

bp were strong, PCR 650 and PCR250 were weak. All four bands were reamplified,

Figure 2B, and cloned in pGEMT vector and sequenced. These data showed that mRNAs

for four specific amelogenin gene splice products had been present when the rat incisor

odontoblast-pulp cDNA library was created: [PCR650] Exons 2,3,4,5,6,7; [PCR600] Ex.

2,3,5,6,7; [PCR250] Ex. 2,3,4,5,6d,7 (73 amino acids, M=8135); and [PCR200] Ex.

2,3,5,6d,7 (59 amino acids, M=6697). These are shown diagrammatically in Figure 1A,

and designated as [B+4], [B-4], [A+4] and [A-4], in order of decreasing size.

Screening of the λgt11 cDNA library [18] using PCR132 as probe, identified

several plaques. Two positive clones were picked and plaque purified through three

successive rounds of screening. The phage DNA was digested with EcoR1. The inserts

were cloned into the EcoR1 site of pBluescript KS and sequenced. The nucleotide and

derived amino acid sequence of one proved to be those of rat incisor amelogenin [B+4],

from the signal peptide through to the poly-A+ tail, corresponding in detail to the rat

incisor amelogenin data of Bonass et al. [44] except for the inclusion of the exon 4

sequence. The second clone corresponding to the splice product [A+4] with the deletion

of exons 6a,b,c, yielded the sequence shown in Fig. 1B.

Since the in vitro chondrogenic activity of the dentin extract correlated with rat

and bovine peptides in the Mr 6,000-10,000 range [7,8,11], attention was focused on the

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plasmids corresponding to [A+4] and [A-4] (Figure 1A). These were amplified by PCR,

using the primers described above. The PCR products were digested with Eco R1 and

Xho1 and cloned into the Eco R1/Xho1 sites of the GST expression vector pGEX4T. The

resulting plasmids were transfected into E. coli BL21. Following IPTG induction the

expressed fusion proteins were collected on Glutathione-Sepharose affinity columns. The

[A+4] and [A-4] were cleaved from the bound GST with thrombin and eluted. Gel

electrophoresis showed the eluted proteins to be rich in the desired full-length

polypeptides in both cases, but some lower mass, incompletely elongated peptides were

present along with other protein impurities. The eluted proteins were therefore

fractionated by reverse phase HPLC using the same system as the final step in the

isolation of the tissue extracted peptides [11], yielding the pure recombinant peptides, as

illustrated for both r[A+4] and r[A-4] in Figure 3.

In vitro activity of the recombinant peptides.

Since the basic assay that led to the isolation of the amelogenin peptides was their

ability to induce an enhancement of sulfate incorporation into proteoglycan by the EMF

cells, the in vitro 35[S]-SO4 incorporation assay was used to determine if the

recombinant peptides had comparable activities. The parameters of this assay were

developed on the basis of the activity of the crude S-100 fraction at 100 µg/ml, which

produces a maximal 4-fold increase in sulfate incorporation/cell. The second positive

control, rhBMP2, yields a 3-fold increase at 10 ng/ml. [A+4] and [A-4] at 10 ng/ml were

comparable in activity to rhBMP2, Figure 4. The [A-4] showed a maximum in activity

between 1 and 5 ng/ml, ~ 140 – 700 pM, as compared to concentrations > 10 ng/ml. The

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r[A+4] did not show the low concentration maximum seen with [A-4]. A distinct

difference in behavior was that in vitro the r[A-4] did not act as a growth factor, whereas

r[A+4] and rhBMP2 did. Even after 5 days in culture, following a 24 h exposure to r[A-

4], the cell number did not increase as it did in the presence of rhBMP2 and r[A+4].

Thus, while similar, the effects of r[A-4] and r[A+4] were distinguishable. The

commercial preparation of porcine amelogenins known as Emdogain® was not effective

in this assay at such low concentrations but activity could be seen at concentrations

greater than 500 µg/ml (data not shown). The standard deviations shown in Figure 4 were

based on 5 independent assays in each case.

The transcription factor Sox9 [25-32,48,49] is a regulator of the type II collagen

gene and required for the expression of the chondrogenic phenotype. The transcription

factor Cbfa1 [33] is similarly required for induction of the osteogenic phenotype, but it

has wider functions. It is expressed in the early stages of tooth formation in the dental

mesenchyme and, later, in the maturation phase ameloblasts, clearly having a role in the

epithelial-mesenchymal interactions involved in tooth morphogenesis [41]. Cbfa1 also

plays a role in chondrocyte differentiation and maturation [50,51]. PCR was used to

determine the appearance of these messages in EMF cultures treated with r[A+4] and

r[A-4] for several time periods. PCR was also used to determine the induction of the

messages for type II collagen [52], as well as changes in the level of type I collagen

message. These data are shown in Figure 5, along with the expression of the message for

housekeeping gene GAPDH (Fig. 5A) which remained essentially constant for all

cultures, indicating that comparable amounts of total mRNA had been used. Sox9

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message was detected only in the r[A+4] treated cultures, induced at between 8 and 24 h

(Fig. 5B, lanes 12,13). Type II collagen (COL2) message (Fig. 5C, lanes 12,13) appeared

in concert with the SOX9 message in the r[A+4] treated cultures. The COL2 message

also appeared very early after addition of r[A-4], at 1-4 h, and then diminished, but

persisted through 48 h (Fig, 5C, lanes 14-18). Cbfa1 transcription also rose sharply

immediately after addition of r[A-4] to the cultures, but then diminished over the 48 h

period examined (Fig. 5D, lanes 14,15). The EMFs expressed a background of COL1

transcription at all conditions (Fig. 5E). These data support the sulfate incorporation data

noted above in showing that the two amelogenin peptides do not act identically on the

cells.

In vivo implants.

The in vivo assay for activity was the ectopic induction of mineralization in

implants of the recombinant protein in bioabsorbable matrices in muscle. As shown in

Figure 6, after 4 weeks, implants containing r[A-4] stained strongly with Alizarin Red

and von Kossa, showing the presence of mineral deposits. The in vivo assay also

distinguished between r[A+4] and r[A-4]. The r[A+4] implants were mineralized to a

lesser extent, with restricted and more focal mineral deposits than seen with r[A-4], but

they were clearly more strongly mineralized than the BSA negative control. Treatment of

the r[A+4]and r[A-4] sections with EGTA eliminated the Alizarin Red and von Kossa

staining in the implants (Figure 6, micrographs 3 & 6), verifying that the radio-opaque

areas seen in panel 9, Figure 6, represented calcium phosphate deposits in the implants.

In data not shown, the r[A-4] implants were positive for alkaline phosphatase, another

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marker of mineralizing systems. Emdogain® implants were virtually identical to the BSA

implants.

H & E staining showed that r[A-4] and r[A+4] implants became vascularized and

filled with extracellular matrix within 4 weeks, Figure 7. Relative to the BSA control

implants, Fig.7-1, capillary invasion was most prominent in the r[A-4] implants (Fig 7-

2), as was the formation of extracellular matrix. The formation of islands of

osteoid/bone-like extracellular matrix surrounding the capillaries was clearly revealed by

both H & E and Goldner’s Trichrome stains, and was especially prominent in the focal

mineralization regions of the r[A+4] implants (Fig. 7-3,4).

The matrices of the r[A+4] and r[A-4] implants showed the presence of typical

bone matrix proteins, BSP and BAG-75, Fig. 8, upon staining with their respective

antibodies. Fig. 8-1 shows a r[A+4] matrix containing region comparable to that in

Fig.7-3 and 7- 4, stained with anti-BAG75 (green) and DAPI (blue) to show the cell

nuclei. The intense green marked the red blood cells within the capillaries. Regions

immediately surrounding the cell nuclei in areas where the matrix had not yet formed

showed abundant BAG-75 staining. A typical area of BAG-75 staining, shown in Fig.

8-2 at higher magnification, also showed the presence of BSP (red, Fig. 8-3). The BAG-

75 and BSP were co-localized, Fig. 8-4. The r[A-4] implants, Figs. 8–5,6,7,8, which

had been more heavily mineralized, showed a more abundant cellularity, but similar co-

localization of BSP and BAG-75. The control BSA loaded implants did not show the

presence of these proteins, and the sections stained only with the second antibodies were

also negative (data not shown).

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The in vitro and in vivo assays for biological activity thus showed that the two

specific recombinant small amelogenin splice products had the same type of activities

that had been attributed to the amelogenin peptides of comparable molecular size isolated

from crude S-100 dentin extracts [11]. In contrast, the full-length amelogenins and their

major degradation products, as represented by the porcine amelogenin of EMDOGAIN®,

were orders of magnitude less active. These data indicate that the specific amelogenin

splice products, [A+4] and [A-4], have cell signaling activity leading to an altered

phenotypic expression by the affected cells. Under the conditions used, r[A+4] and r[A-

4] induce EMF cells in vitro to produce products phenotypic of chondrocytes and/or

osteoblasts, while in in vivo implants the induction leads to the production of

extracellular matrix, matrix vascularization and matrix mineralization. The typical bone

matrix proteins, bone acidic glycoprotein -75 and bone sialoprotein, accumulate within

the [A+4] and [A-4] treated implant matrix. These components do not appear within the

control BSA implants.

DISCUSSION

Two distinctly different points can be made from the data presented above. First,

the specific low molecular mass amelogenin gene splice products, [A+4] and [A-4], have

the ability to interact with immature mesenchymal cells, both in culture and in in vivo

implants, and initiate a change in cell phenotype and maturation pathway. In the EMF

culture system interaction [A-4] upregulates transcription factor Cbfa1, whereas [A+4]

more prominently upregulates Sox9. Both amelogenins, at concentrations of 10 ng/ml,

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induce a 3-fold enhancement of sulfate incorporation into proteoglycan, and lead to the

subsequent production of type II collagen, markers of the chondrogenic phenotype. In

vivo [A-4] containing implants become profusely mineralized within a 4-week period,

[A+4] implants are mineralized more focally, but both types of implants are infiltrated by

cells, become vascularized and form islands of extracellular matrix. The matrix

developed after four weeks shows the presence of BSP and BAG-75, proteins

characteristic of mineralized tissues. Thus, the cell signaling activities of the amelogenin

peptides relate to the formation of mineralized tissues.

The second point to be made, based on the demonstration of their mRNAs in the

odontoblast-pulp complex cells, is that the messages for the amelogenins may be

transiently expressed within the odontoblasts during tooth morphogenesis, just as the

messages for several supposedly dentin specific proteins are transiently expressed by

ameloblasts [21,22,23].

During the embryonic period of organ development, complex sets of signals are

passed in both directions between epithelial tissues and their adjacent mesenchyme.

These inductive, regulatory signals determine the course of tissue differentiation and can

lead to highly specialized meristic structures such as hair follicles, kidney tubules and

teeth [53]. A key aspect of such interactions is that they take place as a chain of

sequential and reciprocal events [54] throughout the course of development.

Odontogenesis is a particularly interesting process since individual tooth epithelium and

mesenchyme can be separated at specific stages of embryonic development and then

recombined with tissues at other stages or from other organs [55-57]. The stage-specific

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progress of development can then be observed in the recombined tissues. In tooth

development, the oral epithelium first thickens then forms a bud growing into the

underlying neural crest mesenchyme. The bud grows to form a “cap” that enfolds part of

the mesenchyme. Sox9 [42] and Cbfa1 [33,41] exert their actions on such mesenchymal

cell condensations. In the tooth, those mesenchymal cells condense to form the dental

papilla. The papillary cells immediately in contact with the inner enamel epithelium

differentiate to become odontoblasts and form dentin. Subsequently the epithelial cells in

contact with the mesenchyme differentiate to ameloblasts and produce enamel. In

heterotypic recombination experiments, Mina and Kollar [57] showed that in the mouse

embryo, the mandibular arch epithelia at <E12 could elicit formation of a dental papilla

in non-odontogenic neural crest-derived cells. Conversely, the cells of the dental papilla

could induce non-odontogenic epithelia to become committed to odontogenesis, but only

after the papilla cells had become odontogenic, at E>12. Thus, signals instructive or

permissive for differentiation pass between the two tissues at different developmental

stages [2,54,58,59] during tooth morphogenesis. The elements of specificity that direct

the programming of the differentiating cells remain undefined at this time.

We believe that the data presented here are pertinent to this problem of epithelial-

mesenchymal signaling. Our earlier biochemical studies [8,11] showed that dentin does

indeed contain small amounts of amelogenin related protein closely associated with the

dentin matrix. Others [60,61,62] have shown by immunostaining that the mantle dentin

contains amelogenin-related peptides. Sawada and Nanci [62] postulated that low-

molecular-size amelogenin degradation products diffuse through the basement

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membrane separating preameloblasts and preodontoblasts and become trapped between

odontoblasts in the forming dentin. Karg et al. [63] found amelogenin immunostaining in

developing hamster teeth in the early predentin and adjacent partially polarized

preameloblasts before any overt deposition of enamel. Young odontoblasts stained

weakly with anti-amelogenin antibodies before they formed the first layer of dentin.

Wurtz et al. [64] specifically examined the presence of amelogenin mRNA in growing rat

molars using in situ hybridization with a probe encoding the exon 5-6d boundary (as in

[A+4] and [A-4]). They reported that the mRNA was exclusively limited to cells of the

inner enamel epithelium. Inspection of Figure 3 in their paper, in the light of our present

results, suggests that there was specific DIG-labeling in the preodontoblast layer.

However, the labeling was substantially weaker than that in the adjacent preameloblasts,

and hence was treated as background. It is worth noting, as well, that in that study there

was a clear difference in the pattern of expression of the mRNAs for the “short” and “full

length” amelogenins in teeth of the same age.

Our data provide direct evidence that the r[A+4] and r[A-4] have specific

biological activities, equivalent in many ways to rBMP2, in directing the change in

phenotype of the embryonic rat muscle fibroblasts in vitro and inducing development of a

mineralized matrix in muscle implants in vivo. The induction may operate via

upregulation of Sox9 and/or Cbfa1. Cbfa1 is required for induction of the osteogenic

phenotype [33], but it is expressed in the early stages of tooth formation in the dental

mesenchyme and, later, in the maturation phase ameloblasts. Clearly, Cbfa1 has a role in

the epithelial-mesenchymal interactions involved in tooth morphogenesis [41]. Cbfa1 is

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also involved in chondrocyte differentiation and maturation [50,51]. Sox9 [25-32,48,49]

is a regulator of the type II collagen gene and required for the expression of the

chondrogenic phenotype. Thus, one may postulate that these specific amelogenin gene

splice products may be among the sought after epigenetic signaling factors operating

during odontogenesis. Their effect may depend upon the local environment and the

presence of additional cytokines and growth factors. As suggested by the literature cited

above, the [A-4] and [A+4] peptides could originate in the preameloblast layer of the

inner enamel epithelium, and, because of their small size, diffuse into the preodontoblast

layer. The peptides could then trigger the maturation of the preodontoblasts and initiate

dentinogenesis. However, it is likely that the appearance of the amelogenin peptides in

dentin is a programmed event since the epithelial-mesenchymal signaling process is such

a crucial aspect of tooth development. If that is the case, it is also likely that the

amelogenin peptides in dentin are probably the specific gene splice products, rather than

degradation peptides. An alternative scenario to the diffusion of the peptides into the

mantle dentin comparable to the transient, very early expression of dentin matrix proteins

in preameloblasts [21,22] is the transient expression of [A+4]/[A-4] in the

preodontoblasts. A study of that possibility is underway. We are now in position to

evaluate the cell-specific expression and mechanisms of action of these hitherto

unrecognized differentiation-instructive/permissive agents.

ACKNOWLEDGEMENT

We thank Dr. Ada Cole, Rush University Medical School, and Dr. Lili Yue, University

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of Illinois Dental School, for their advice concerning the histology and immuno-

fluorescence procedures. We are indebted to Mr. Thomas Dahl for his assistance with the

figures.

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FIGURE LEGENDS

Figure 1. The organization of rat incisor amelogenin cDNA and its specific splice

products. A. The exon distribution in the rat amelogenin cDNA. The designation of the

exons is given above the line, the number of amino acids in each exon is given below the

line. The primers P1 and P2 used initially to verify the presence of amelogenins in the

library, and the primers, P3 and P4, used to determine the specific splice products

present, are indicated. The EcoR1 and Xho1 restriction sites added to these primers are

indicated by the jagged lines. The exon designations follow the system of Simmer [13].

The exon compositions, and numbers of amino acids in each exon, of the PCR products,

[B+4], [B-4], [A+4], [A-4], correspond to the bands shown in Figure 2B, lanes 3, 5, 4

and 2, respectively. B. The nucleotide and amino acid compositions of cloned [A+4].

These sequences were determined from the λgt11cDNA expression library from a plaque

detected by the cDNA giving rise to the PCR product of band 4, Figure 2B. They are

compatible with the sequences presented for rat amelogenin by Bonass et al. [47] except

for the inclusion of the exon 4 sequence and deletion of the exon 6a,b,c sequence

segment. The sequence of [A-4] was determined to be identical to that shown for [A+4],

except for the exclusion of exon 4 nucleotides and amino acids.

Figure 2. Identification of the amelogenin-related products obtained by PCR from rat

incisor tooth cDNA. A. Demonstration that amelogenin mRNA was present in the

odontoblast-pulp derived mRNA, using the primers P1 and P2 for detection of the N-

terminal message sequence common to all amelogenin gene splice products. These data

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show the unequivocal presence of two messages (right arrow heads), with nucleotide

sizes 90 and 132 bases. B. Amelogenins detected by screening the λgt11 rat odontoblast-

pulp cDNA library with primers P3 and P4 to obtain all potential splice products. The

products obtained initially were reamplified by PCR using the same primers. The

amplified products are [A-4] (PCR200, Lane 2), A4 (PCR250, Lane 4), [B-4] (PCR600,

Lane 5) and B4 (PCR650, Lane3). The PCR products were run on a 1% agarose gel and

visualized by staining with ethidium bromide. Lane 1 was loaded with a 1 kb DNA

ladder. In both panels, the marker DNA sizes are indicated on the left.

Figure 3. HPLC purification of thrombin cleaved GST-fusion proteins containing r[A-4]

and r[A+4]. The HPLC conditions were those described by Nebgen et al. [11]. The inset

shows the Coomassie stained gel of the final recovered peptides. r[A+4]. ∗ r[A-

4]. The homogeneous r[A+4] protein fraction at 20.3 min, and the r[A-4] fraction at 21.3

min were used for the bioassays. In the inset, lane 1 – molecular weight markers, lane 2 –

r[A+4], lane 3 – r[A-4], denoted by the double arrows.

Figure 4. In vitro assay for incorporation of 35S-SO4 into rat embryonic muscle

fibroblasts (EMF). The EMF were seeded onto type I collagen coated 96 well plates,

with an initial loading of 104 cells/well. The cells were grown to near confluence in alpha

MEM, 10% FBS, 1% Pen/Strep in 5 days. At day 5 the growth media was replaced with

conditioning media (CM), alpha MEM, 0.5% FBS, 1% Pen/Strep, and grown for an

additional 24 h. Fresh CM containing the factors to be tested, was added at the

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concentrations noted (per mL). Four hours later, 1 µCi 35S-SO4 in 10 µL sterile PBS

was added per well, and incubation was continued for 20 h. The 35S-SO4 incorporated

into secreted proteoglycan was determined by precipitation of the proteoglycan with cetyl

pyridinium chloride [11], followed by scintillation counting of the precipitate. The cell

layer was trypsinized and the number of cells counted. Incorporation is presented as

counts per minute per cell x 103. The standard deviations shown are based on n=5 in each

case. The S-100 is a semi-purified fraction of the bovine dentin extract containing the

sulfate incorporating activity [8]. The S-100 and recombinant BMP2 were used as

positive controls. PBS/BSA was the negative control.

Figure 5. PCR detection of messenger RNA for selected cartilage/bone phenotypic

proteins following EMF culture. A. GAPDH control. B. Sox 9. C. COL2. D. Cbfa1. E.

COL1. Lane 1, DNA ladder. Lane 2, EMF, 10 % FCS Growth media. Lane 3, EMF, after

24 hr conditioning media (CM), T0. Lanes 4, 9,14 1hr. Lanes 5,10,15 4 hr. Lanes 6,11,16

8hr. Lanes 7,12,17 24 hr. Lane 8,13,18 48 hr. Lanes 4-8 CM. Lane 9-13 r[A+4] + CM.

Lane 14-18 r[A-4]+ CM. The number of bases expected for each PCR product is

indicated on the right. r[A+4] or r[A+4] was added to the cells at 10 ng/ml for each case

in lanes 9 through 18.

Figure 6. In vivo assay for the implant activity of r[A+4] and r[A-4]. Implants were

placed in the quadriceps of the hind legs of 100 g, 4 week old, male Long-Evans rats.

The implants were prepared in poly (lactide)-poly(glycolide) scaffolds by the procedure

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of Whang et al.[45] with 167 µg per implant. Each animal received a negative control

implant containing 167 µg of BSA in PBS. The implants were removed after 28 days

and radiographed. Following fixation in 4% paraformaldehyde, they were dehydrated in

graded ethanol and embedded in paraffin. Sections (7 µ thick) were cut and stained with

either Von Kossa or Alizarin Red dyes, both of which can indicate the presence of

mineralized deposits containing divalent cations. To assure that the stains seen were

calcific deposits, serial sections were treated with 5 % EGTA for 10 min before staining.

In the figure, Numbers 1, 2 and 3 were taken from r[A-4] treated implants; 4,5 and 6

were from r[A+4] treated implants; and 7 and 8 were BSA implants. Numbers 1, 4 and 7

were Von Kossa stained; 2, 5 and 8 were Alizarin Red stained. Numbers 3 and 6 were

EGTA- treated sections. These data show that the [A-4] implants were highly positive

for deposition of mineral, comparable to BMP2 implants [Whang et al., 45]. [A+4]

yielded more sparsely focal deposits of mineral, and the BSA implant controls were

negative. Number 9 shows radiographs of the [A-4] and [A+4] implants immediately

after excision and before processing for histology. Note the heavier mineralization around

the periphery of the [A-4] implant in contrast to the more punctate deposition of mineral

within [A+4].

Figure 7. Hematoxylin and Eosin, Goldners Trichrome stained sections of implants

after 4 weeks. 1. Control negative, implant of poly (lactide)-poly(glycolide) scaffold

containing BSA, H & E stained. The dense tissue at the upper right corner of the

micrograph is the connective tissue encapsulating the implant. Some of this tissue grows

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into the implant at the implant interface. 2. H & E stained implant containing r[A-4] after

4 weeks. The intense vascularization of the implant is obvious. The scaffold has been

infiltrated by many cells, and an abundant network of capillaries and a dense extracellular

matrix has begun to form. 3,4. Implants containing r[A+4] after 4 weeks. The implants

are vascularized but more sparsely than the r[A-4] implants. There is nevertheless,

copious cellular infiltration and matrix production. Micrograph 4, stained with Goldner’s

Trichrome stain, shows, in green, the forming extracellular matrix. Capillaries are

obvious.

Figure 8. Immunofluoresence identification of bone matrix proteins, BAG75 and BSP,

cell nuclei in implants. 1. The interior of a r[A+4] implant stained with anti-BAG75 and

DAPI. The dense matrix areas are green-fluorescent, but are much less intense than the

areas deeper into the implant, at 25x. 2. Selected area within implant stained with anti-

BAG-75, 100x. 3. Same area stained with anti-BSP, 100x. 4. Merged 2-3, showing

colocalization of BAG-75 and BSP in most areas. 5. The interior of an r[A-4] implant

stained with anti-BAG75 and DAPI (25x). 6. Selected area within r[A-4] implant stained

with anti-BAG-75, 100x. 7. Same area stained with anti-BSP, 100x. 8. Merged 2-3,

showing colocalization of BAG-75 and BSP in most areas with r[A-4].

34


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Anna G. Brownell, Shure-Min Jengh and Kevin E. HealyArthur Veis, Kevin Tompkins, Keith Alvares, Kuiru Wei, Lin Wang, Xue Song Wang,

and in Implants In VivoSpecific Amelogenin Gene Splice Products have Signaling Effects on Cells in Culture

published online September 20, 2000J. Biol. Chem.

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