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RESEARCH REPORT |
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;
*corresponding author, farachca{at}udel.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: HIP/L29 • basic fibroblast growth factor • gingiva, cyclosporine A • heparin/heparan sulfate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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) 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. 1B).
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). 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. 2B, 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).
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, 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. 3B). 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.
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). 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. 4B) showed that all lanes contained comparable amounts of both MAPK forms.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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Received April 26, 2001; Last revision February 6, 2002; Accepted February 7, 2002
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