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Biological

Heparan Sulfate Interacting Protein (HIP/L29) Negatively Regulates Growth Responses to Basic Fibroblast Growth Factor in Gingival Fibroblasts

T.-V. Ta1, D. Baraniak1, J. Julian1, J. Korostoff2, D.D. Carson1 and M.C. Farach-Carson1,*

1 Department of Biological Sciences, University of Delaware, Newark, DE 19716; and
2 Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104;

Correspondence: *corresponding author, farachca{at}udel.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Basic fibroblast growth factor (bFGF) modulates gingival growth, and its release from heparan sulfate (HS) in the extracellular matrix (ECM) governs local tissue bioavailability. We identified a heparin/HS interacting protein (HIP/L29) that recognizes specific HS sequences. We hypothesize that HIP/L29, by modulating the interactions of bFGF with HS chains on proteoglycans, could regulate bFGF bioavailability. To investigate interactions between bFGF and HIP/L29, we isolated and cultured fibroblasts from normal gingiva and overgrown gingiva from patients on cyclosporine (CSA). bFGF significantly stimulated gingival fibroblast proliferation with or without heparin. Recombinant human HIP/L29 dramatically decreased bFGF-induced proliferation, but did not alter responses to insulin-like growth factor-1 (IGF-1). Analysis of mitogen-activated protein kinase (MAPK) phosphorylation patterns showed that bFGF stimulation of p44 (Erk-1), but not p42 (Erk-2), also was inhibited by HIP/L29 in a dose-dependent manner. Together, these results support our hypothesis that HIP/L29 modulates the bioavailability and action of bFGF.

Key Words: HIP/L29 • basic fibroblast growth factor • gingiva, cyclosporine A • heparin/heparan sulfate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Basic fibroblast growth factor (bFGF), implicated in gingival overgrowth (Saito et al., 1996), is sequestered in the ECM, where it is captured and released in a regulated fashion by tissue proteases and endoglycosidases. Release, rather than synthesis rates, governs bFGF bioavailablility. Sequestration of bFGF occurs through specific binding to proteoglycans, specifically those bearing heparan sulfate (HS) glycosaminoglycan chains (Chang et al., 2000). Recent models include HS as part of the ternary complex containing bFGF and its receptor (McKeehan et al., 1999).

We previously identified a novel heparin/HS interacting protein (HIP/RPL29) (Wilson et al., 1990; Raboudi et al., 1992) expressed by human epithelia and epithelial-derived cells, including gingiva (Rohde et al., 1996). The ability of HIP/RPL29 to inhibit heparanase and support cell attachment is retained in simple peptide sequences found within the intact protein (Marchetti et al., 1997; Liu et al., 1998). HIP/RPL29 recognizes specific HS sequences and is a candidate to modulate the interactions of bFGF with HS chains in the gingival ECM. We hypothesize that HIP/RPL29 can modulate bFGF bioavailability through the regulation of ECM sequestration and release. To test this hypothesis, we performed studies to investigate the ability of HIP/RPL29 to inhibit gingival fibroblast proliferation stimulated by bFGF and a non-heparin-binding growth factor, insulin-like growth factor 1 (IGF-1). The involvement of the mitogen-activated protein kinases (MAPK) Erk-1 (p44) and Erk-2 (p42), both known to stimulate cell cycle progression, also was examined. Gingival fibroblasts obtained from both normal gingiva and overgrown gingiva from patients receiving the immunosuppressive drug cyclosporine A (CsA) were examined under various treatment conditions. Gingival overgrowth associated with CsA treatment is thought to involve fibroplasia with hypertrophic growth, excess production of extracellular matrix, and increased fibroblast numbers (Seymour et al., 1996; Murata et al., 1997). Because overgrowth tissue displays increased expression of growth factors including bFGF, its receptor, and also glycosaminoglycans (Saito et al., 1996; Murata et al., 1997), gingival fibroblasts from this tissue provide a good in vitro model for testing bFGF responses that occur in vivo.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Heparin (sodium salt), imidazole, IGF-1 (human recombinant) secondary antibodies, and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). bFGF was obtained from Becton Dickinson Labware (Bedford, MA, USA). Minimal essential medium (MEM), penicillin/streptomycin, Fungizone_, fetal bovine serum (FBS), tissue culture supplements, trypsin, and phosphate-buffered saline (PBS) were obtained from Gibco/BRL (Grand Island, NY, USA). Maleimide-activated BSA and the enhanced chemiluminescence detection kit were obtained from Pierce (Rockford, IL, USA). Cyclosporine A was obtained from Calbiochem (LaJolla, CA, USA). MAPK kits were obtained from Cell Signaling Technologies (Beverly, MA, USA).

Gingival Explants
All procedures complied with approved human subjects protocols at the participating universities. Tissue was removed during routine periodontal surgery. Gingival fibroblasts were prepared by explant culture from healthy and CsA-enlarged gingiva. Tissue was minced, then seeded into 24-well plates in MEM supplemented with 10% (v/v) heat-inactivated FBS, penicillin (100 U/mL), streptomycin (100 µg/mL), and 1% (v/v) Fungizone® at 37°C in a humidified atmosphere of 95% (v/v) air and 5% (v/v) CO2. At confluence (14-18 days), gingival fibroblasts were passaged into 75-cm2 flasks with MEM plus 10% (v/v) FBS without Fungizone®. Cells were harvested, fast-frozen as stocks at passage 3, and stored. Using specific antibodies to human marker proteins [goat anti-vimentin, rabbit anti-desmin, rabbit anti-fibronectin (Sigma), rabbit anti-factor VIII (Dakopatts, Glostrup, Denmark), and rabbit anti-cytokeratin 14 (from Dennis Roop, Baylor College of Medicine, Houston, TX, USA)], we confirmed cultures to be free from epithelia, endothelia, and smooth muscle. Cells were used prior to passage 8.

Peptide Synthesis, Conjugation to Maleimide-activated BSA
Peptide synthesis and conjugation to maleimide-activated BSA were described previously (Liu et al., 1998). Briefly, a synthetic HIP-derived peptide, CRPKAKAKAKAKDQTK, was synthesized on a Vega 250 peptide synthesizer by means of FMOC [N-(9-fluorenyl)methoxycarbonyl] methodology (Chang and Meienhofer, 1978) and conjugated to maleimide-activated BSA with the use of sulfhydryl groups and the coupling protocol provided by the manufacturer.

Recombinant HIP/L29 Purification
Human HIP/L29 containing an oligo-histidine tag at the N-terminal was expressed as described (Liu et al., 1997a). The extract was applied to a cobalt resin that was pre-blocked with non-transformed bacterial extract. The resin was rinsed with 10 bed volumes of buffer (20 mM Tris HCl, pH 8.0, containing 0.5 M NaCl and 5 mM imidazole). The cobalt-binding fraction was eluted with 100 mM imidazole in the same buffer, then diluted two-fold prior to being loaded onto heparin agarose equilibrated with PBS. The column was rinsed with 5 bed volumes of 0.5 M NaCl in 20 mM phosphate buffer, pH 7.4 (PB), then eluted with 1.5 M NaCl/PB. All steps were performed at 4°C. SDS-PAGE and Western blot analysis with an antibody to HIP/L29 peptide (Rohde et al., 1996) monitored elution profiles. HIP purity was greater than 95%. Non-specific proteins were purified as negative controls from non-transformed bacterial extracts under identical conditions.

Proliferation Assays
To measure the effects of CsA, bFGF, and HIP/L29 on proliferation, we plated cells onto 24-well plates at a density of 3500 cells/well and allowed them to attach and spread overnight in MEM containing 10% (v/v) FBS. Cells were rinsed twice with PBS (Ca2+/Mg2+) and replaced with MEM containing 0.2% (v/v) FBS (500 µL/well) for an additional 24 hrs. Media were replaced with media containing test reagents, including CsA, bFGF, IGF-1, and HIP/L29 with or without heparin. All test media contained 0.2% (v/v) FBS. The synthetic HIP/L29-derived peptide and a BSA-conjugated HIP/L29 peptide were used at concentrations of 0.5, 5.0, and 50 µg/mL, with or without bFGF. Control extracts from non-transformed bacteria were used at a concentration of 0.5 µg/mL, equivalent to 1/10th the amount of purified HIP/L29 added to cells, calculated based on maximal percentage of contaminating proteins in HIP/L29 preparations. At days 0, 3, 5, 9, and 13, cells were trypsinized and counted by means of a hemocytometer. Experiments were repeated 4 to 6 times. Differences in cell growth rates were compared by analysis of variance (ANOVA) followed by the Tukey-Kramer secondary multiple-comparisons test. Results are presented as mean and standard deviation (SD) or standard error of the mean (SEM).

MAPK Assays
Cells between passages 3 and 8 were plated in four-well dishes (Nalge Nunc International, Rochester, NY, USA) at a concentration of 30,000 cells/well and allowed to attach in the presence of 10% (v/v) serum. Cells were rinsed with culture-grade PBS, then cultured in serum-free MEM for 24 hrs. Various combinations of bFGF, HIP/L29, and appropriate controls were added to the cells in the wells for 30 and 60 min. Cells underwent lysis and were sonicated on ice in 50 µL of sample buffer [62.5 mM Tris-HCl, pH 6.8, containing 2% (w/v) SDS, 25% (v/v) glycerol, 0.01% (w/v) bromophenol blue], then separated by 10% (w/v) SDS-PAGE. Proteins were transferred to nitrocellulose, then blocked with TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.1% [v/v] Tween 20) plus 5% (w/v) non-fat dry milk. Blots were washed with TBST 3 times for 5 min each, then incubated overnight at 4°C with p44/42 MAPK antibody that recognizes both MAPK forms (Erk-1 and Erk-2) or primary antibodies (E10 monoclonal) specific for phosphorylated forms (phospho-p44/42 MAPK, Thr202/Tyr204). Blots were washed with TBST before the addition of goat anti-rabbit antibody conjugated to horseradish peroxidase. Blots were developed after 1 hr by means of enhanced chemiluminescence.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HIP/L29 Inhibits bFGF-induced Gingival Fibroblast Proliferation
No significant differences in the responses of gingival fibroblasts derived from normal or CsA-treated patients to bFGF, heparin, or CsA were observed (not shown). Therefore, further experiments used CsA-patient gingival fibroblasts, because tissue could be obtained in greater amounts. We first determined if bFGF required exogenous heparin to stimulate proliferation. Heparin (10 µg/mL) was incubated with bFGF for 1 hr with agitation prior to addition. Exogenous heparin did not affect proliferation in the presence of 10 ng/mL bFGF (Fig. 1AGo) or at 50 µg/mL (not shown). Dose-responses with the use of bFGF indicated that 10 ng/mL bFGF stimulated proliferation (p < 0.05); a 50 ng/mL dose of bFGF more significantly (p < 0.001) induced proliferation by day 9 (Fig. 1BGo).


Figure 1
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  		Figure 1. bFGF stimulates gingival fibroblast proliferation. (A) Cells were plated in 24-well plates at an initial density of 3500 cells/well and allowed to attach overnight in MEM containing 10% (v/v) FBS. The following day, cells were rinsed twice with PBS, and the media were changed to 0.2% (v/v) FBS with the addition of either 10 ng/mL bFGF or 10 µg/mL heparin or both compounds. In the control experiment, cells were cultured in MEM with 0.2% (v/v) FBS. Cell number was counted by means of a hemocytometer at days 0, 3, and 9 after treatments as described in MATERIALS & METHODS. Data are presented as the mean ± standard deviation (SD) of 6 experiments. (B) Cells were plated in 24-well plates as in (A). Subsequently, cells were rinsed twice with PBS and exposed to the indicated concentrations of bFGF in media containing 0.2% (v/v) FBS only. In the control experiment, cells were cultured in MEM with 0.2% (v/v) FBS. Cell number was counted on days 0, 3, and 9. Data are presented as the means ± SD of 6 experiments. *p < 0.05, **p < 0.001, ***p < 0.0001 relative to day 9 control.

 
Parallel experiments demonstrated proliferative responses to IGF-1 (100 ng/mL) as well over a similar time course (data not shown). A typical experiment with this growth factor gave a 35-40% increase in cell number compared with untreated controls over a seven-day period. The addition of recombinant HIP/L29 at 5 µg/mL to the medium decreased bFGF-induced proliferation in the presence or absence of 10-9 g/mL CsA with p < 0.001 at day 9 (Fig. 2AGo). During early time points, no significant difference in the cell number between groups with and without HIP/L29 was seen. Therefore, experiments were taken to day 9, where the inhibition of bFGF-dependent proliferation was evident. The addition of HIP/L29 without bFGF had no effect on proliferation. We analyzed dose-responses with native HIP/L29 to determine the minimal concentration required for inhibition of bFGF-stimulated proliferation. As shown in Fig. 2BGo, at 5 µg/mL, HIP/L29 maximally inhibited bFGF-induced cell proliferation (p < 0.05). No inhibitory activity of HIP/L29 on proliferation was found at a 10-fold-lower concentration (0.5 µg/mL). Cultures treated with IGF-1 (100 ng/mL) in the presence of HIP/L29 grew at the same rate as control cultures (not shown). To determine if CsA alone stimulated proliferation, we used CsA at concentrations similar to that in serum of patients taking this drug (10-9 g/mL), 1000-fold higher (10-6 g/mL) and 1000-fold lower (10-12 g/mL). CsA had no consistent effect on proliferation in vitro at any concentration tested (not shown). Contaminants co-purifying with HIP/L29 analyzed by SDS-PAGE and silver staining revealed a similar minor band protein profile seen in typical HIP/L29 preparations. Control extracts had no effect on proliferation (not shown).


Figure 2
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  		Figure 2. HIP/L29 antagonizes bFGF-induced proliferation. (A) Cells were plated as in Fig. 1Go. HIP/L29 (5 µg/mL) and bFGF (50 ng/mL) were added to the medium with or without 10-9 g/mL CsA. In the control experiment, cells were cultured in MEM with 0.2% (v/v) FBS. On days 0, 3, 9, and 13, cells were trypsinized and counted. Data are presented as ± the SD of 6 experiments. *p < 0.05, **p < 0.001, ***p < 0.0001 relative to bFGF only or bFGF + CsA at day 13. (B) Cells were plated as above. The following day, cells were rinsed twice with PBS and the medium was changed to 0.2% (v/v) FBS. Cells were exposed to HIP/L29 at the indicated concentrations in the presence of 50 ng/mL bFGF. In the control experiment, gingival fibroblasts were cultured in MEM with 0.2% (v/v) FBS. Cell number was counted on days 0, 3, and 9. Data are presented as the means ± SD of 6 experiments. *p < 0.05 relative to bFGF alone at day 9.

 
We added synthetic HIP/L29 peptide to culture media to determine if it displayed bFGF antagonism similar to that of intact recombinant HIP/L29. As shown in Fig. 3AGo, HIP/L29 peptide at concentrations up to 50 µg/mL had no effect on bFGF-stimulated proliferation. However, a multivalent form of HIP/L29 peptide, conjugated to BSA, at 5 µg/mL significantly antagonized proliferation (p < 0.001; Fig. 3BGo). Higher concentrations (50 µg/mL) of BSA-conjugated HIP/L29 peptide not only prevented cell proliferation, but also gradually reduced cell numbers over 9 days of culture. BSA alone had no effect. Collectively, these results demonstrate that both intact HIP/L29 and a multivalent form of a HIP/L29 peptide motif strongly antagonize bFGF-stimulated, but not IGF-1-stimulated, proliferation.


Figure 3
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  		Figure 3. Effect of HIP/L29 peptide on bFGF-induced proliferation. Gingival fibroblasts were plated in 24-well plates at an initial density of 3500 cells/well and allowed to attach as above. Subsequently, cells were rinsed twice with PBS and the medium was changed to 0.2% (v/v) FBS. HIP/L29 peptide (CRPKAKAKAKAKDQTK) (A) and BSA-conjugated peptide (AKAK-BSA) (B) were added to medium at the indicated concentrations in the presence of 50 ng/mL bFGF. On the indicated days, cells were trypsinized and counted. While peptide conjugated to BSA was very effective in reducing cell growth (bottom panel), unconjugated peptide had little effect (top panel). Data are presented as the means ± SD of 6 experiments. ***p < 0.0001 relative to corresponding day of group receiving bFGF only.

 
Phosphorylation of MAPK in Gingival Fibroblasts
The addition of bFGF stimulated phosphorylation of the MAPK Erk-1 (p44) within 30 min, and persisted after 60 min (Fig. 4AGo). HIP/L29 addition (5 or 50 µg/mL) consistently reduced Erk-1 (p44) phosphorylation at the 30-minute and particularly at the 60-minute timepoint. In contrast, little effect on ERK-2 (p42) phosphorylation was observed. Antibodies recognizing epitopes present in phosphorylated and non-phosphorylated ERK forms (Fig. 4BGo) showed that all lanes contained comparable amounts of both MAPK forms.


Figure 4
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  		Figure 4. Western blot analysis of MAPK in human gingival fibroblast. Cells were plated in 24-well plates at an initial density of 30,000 cells/well and allowed to attach overnight in MEM containing 10% (v/v) FBS. Subsequently, cells were rinsed twice with PBS, serum-starved for 24 hrs, treated as indicated, and then subjected to lysis on the plates. Antibodies were used for the detection of MAPK and phosphorylated MAPK as described in MATERIALS & METHODS. (A) Effects of bFGF and HIP on the phosphorylation of Erk-1 (p44) and Erk-2 (p42). (B) We verified equal loading by re-probing with antibody recognizing both phosphorylated and non-phosphorylated forms of p44 and p42.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Various heparin/HS-binding growth factors, including bFGF, contribute to gingival overgrowth. These growth factors, their receptors, and HS glycosaminoglycans increase during gingival overgrowth and contribute to altered tissue homeostasis (Brunius and Modéer, 1989; Modéer et al., 1990; Dill et al., 1993; Saito et al., 1996; Murata et al., 1997). Increases in fibroblast numbers and matrix production both contribute to increased tissue mass (Wysocki et al., 1983; Friskopp et al., 1986; Rostock et al., 1987). CsA stimulates DNA synthesis of fibroblasts derived from normal human healthy gingiva (Kataoka et al., 2000), decreases degradation of type I collagen, and increases type I collagen accumulation (Saito et al., 1996; Sugano et al., 1998), phenomena that likely involve bFGF-mediated pathways.

An HS-interacting protein (HIP/L29) that promotes cell adhesion and modulates heparin activity (Liu et al., 1997b) and a 17-amino-acid peptide within the C-terminus of human HIP/L29 (HIP peptide) that binds subsets of heparan sulfate and supports cell adhesion (Liu et al., 1998) were used in these studies. HIP/L29 peptide recognizes the same motifs within HS chains at cell surfaces and in the ECM as heparanase, an enzyme involved in bFGF release from the ECM (Marchetti et al., 1997). Multiple domains within human HIP/L29 cooperate to generate high-affinity HP/HS binding (Hoke et al., 2000). We hypothesized that HIP/L29 can regulate bFGF responsiveness in gingival fibroblasts.

HS supports growth and proliferation by forming ternary complexes with heparin-binding growth factors and their receptors (McKeehan et al., 1999; Ornitz, 2000). Both proteoglycans and glycosaminoglycans, essential components of the ECM, are hydrolyzed in the periodontium (Yamalik et al., 1998). Thus, low-molecular-weight fragments can bind bFGF and form active complexes to promote proliferation. We considered that exogenous heparin might modulate bFGF and HIP/L29 activities. Heparin addition had no effect on proliferation with or without bFGF, indicating that gingival fibroblasts synthesized sufficient glycosaminoglycans to support bFGF responses (Barber et al., 1992; Saito et al., 1996). The bFGF dose-response indicates that, at 50 ng/mL, bFGF significantly stimulates proliferation, which became highly significant during treatment in vitro (day 9; p < 0.001).

The direct effect of CsA on cell proliferation remains controversial (Bartold, 1989; Barber et al., 1992). Reported differences may represent patient variability. Thirty-five percent of gingival fibroblasts do not bind CsA, while 41% avidly bind the drug and are more responsive in terms of synthetic and proliferative behavior (Hassell et al., 1988; Mariotti et al., 1998). This heterogeneity may explain the lack of consistent effect of CsA on gingival fibroblast proliferation at various concentrations (10-6-10-12 g/mL) in our studies.

Results of the HIP/L29 dose-responses indicated that HIP/L29 (5 µg/mL or higher) strongly inhibited cell proliferation in the presence of 50 ng/mL bFGF, but not IGF-1 (100 ng/mL). Without bFGF, HIP/L29 had no effect on proliferation, indicating that HIP/L29 can attenuate the bFGF response, but alone has little or no growth-inhibitory effect. A previous study (Hill and Ebersole, 1996) found that LPS inhibited DNA synthesis, particularly in non-quiescent cells. In general, non-quiescent cells are more responsive to growth factors and LPS/growth factor combinations than quiescent cells (Newell and Irwin, 1997). Similarly, our results showed that HIP/L29 did not affect proliferation in low-serum medium without bFGF (quiescent state). Finally, HIP/L29 had no effect on proliferation in the presence of CsA unless bFGF was also present. Thus, we propose that HIP/L29 is a novel antagonist of bFGF-stimulated gingival fibroblast proliferation. The lack of effect on IGF-1-stimulated proliferation indicates that the mechanism must be heparin/HS-dependent.

No inhibitory activity of HIP/L29 peptide occurred, even at a concentration 100-fold higher than native HIP/L29. In contrast, when a multivalent form of HIP/L29 peptide was added, it significantly inhibited bFGF-stimulated proliferation. We propose that, consistent with the known heparin/HS-binding properties of HIP/L29 (Hoke et al., 2000), multiple heparin-binding domains within human HIP/L29 cooperate to produce the inhibitory activity. HIP/L29 can disrupt the formation of heparin/HS/bFGF/receptor complexes and slow proliferation (Raboudi et al., 1992). HIP/L29 also may competitively displace bFGF from HS in ECM or cell surfaces.

HIP/L29 Modulation of bFGF Bioactivity
Competition for HS binding sites in the ECM between HIP/L29 and bFGF can occur. Previous studies indicated that bFGF interacts with specific sequences in HS chains (Raboudi et al., 1992). Thus, the presence of competitors for these sequences (Liu et al., 1997b) could block formation of active growth factor complexes. In cases where CsA leads to increased GAG production and depolymerization (Tipler and Embery, 1985; Barber et al., 1992; Newell and Irwin, 1997), enzymatic cleavage of HS proteoglycans could release active bFGF (Whitelock et al., 1996). In the ECM surrounding human endothelial cells, bFGF is released by the concerted action of proteases that degrade proteoglycan core proteins and heparanases that digest HS polysaccharides (Whitelock et al., 1996). HIP/L29, by competing for free HS chains, could interfere with formation of functional bFGF/bFGF-receptor complexes.

Consistent with a previous report (Komaki et al., 2000), the spreading of gingival fibroblasts activated the MAPK cascade, resulting in increases in levels of phosphorylated p42 (Erk-2). Such changes in MAPK phosphorylation are likely to signal cell-cycle progression (Lavoie et al., 1996). Phosphorylation was not increased by the addition of bFGF, nor was it sensitive to the addition of HIP/L29. In contrast, bFGF addition rapidly stimulated phosphorylation of p44 (Erk-1), a process that was antagonized by the addition of HIP/L29. This differential effect on Erk-1, but not Erk-2, phosphorylation implies that the Erk-1 pathway is the target of bFGF activation in the gingival fibroblast. We conclude that HIP/L29 antagonizes time-dependent MAPK activation associated with BFGF-stimulated proliferation.

These exciting possibilities challenge us to pursue the question of HIP/L29 distribution in overgrowth compared with normal gingiva. These studies are currently under way in our laboratory and will further our understanding of the role of HIP/L29 in the periodontium.


   ACKNOWLEDGMENTS
 
We thank Drs. C. Kirn-Safran, R. Gomes, and R. Liu for helpful comments and critical reading of the manuscript. We thank Sharron Kingston and Ginger Moore for excellent secretarial assistance. These studies were supported by NIH grants DE12950 (to MCF-C) and HD25235 (to DDC).

Received for publication April 26, 2001. Revision received February 6, 2002. Accepted for publication February 7, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Dental Research, Vol. 81, No. 4, 247-252 (2002)
DOI: 10.1177/154405910208100405
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