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EGFR in Enamel Matrix Derivative-induced Gingival Fibroblast Mitogenesis
E. Zeldich1,
R. Koren2,
M. Dard3,
C. Nemcovsky4 and
M. Weinreb1,*
1 Departments of Oral Biology and
4 Periodontology, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel;
2 Department of Physiology and Pharmacology, Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel; and
3 Pre-clinical Research, Institut Straumann, Basel, Switzerland
Correspondence: * corresponding author, weinreb{at}post.tau.ac.il
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ABSTRACT
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We previously reported that EMD (Enamel Matrix Derivative) induces proliferation of human gingival fibroblasts via activation of Extracellular Regulated Kinase (ERK), and this study assessed the possible mediatory role of EGFR (Epidermal Growth Factor Receptor) in this effect. Treatment of gingival fibroblasts with EMD resulted in tyrosine phosphorylation of the EGFR, as assessed by immunoblotting and ELISA, while EMD-induced ERK activation and thymidine incorporation were markedly inhibited (~ 40–50%) by a specific EGFR tyrosine kinase inhibitor. Using appropriate inhibitors, we established that EMD-induced EGFR activation is largely due to shedding of HB-EGF (Heparin-binding EGF) from the cell membrane via a metalloproteinase-mediated process. Finally, the addition of PP1, a Src family inhibitor, abrogated both EGFR phosphorylation and ERK activation. Taken together, these results indicate that, at least in human gingival fibroblasts, EMD-induced ERK activation and proliferation are partially due to a Src-dependent, metalloproteinase-mediated transactivation of EGFR.
Key Words: Emdogain • enamel matrix derivative • gingival fibroblasts • ERK • EGFR
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INTRODUCTION
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The discovery of the profound biological role of Enamel Matrix Proteins (EMPs) in cementogenesis and the formation of the periodontal supporting tissues (Hammarström, 1997) prompted extensive research into the possible usage of EMPs in periodontal regeneration, which culminated in the development of the current EMP product (EMDOGAIN®), which contains EMD (Enamel Matrix Derivative) and a PGA (propylene glycol alginate) carrier. It consists of hydrophobic enamel matrix proteins, of which amelogenin is the major protein, extracted from porcine developing enamel. The proliferative effect of EMD was demonstrated previously on periodontal ligament (PDL) cells in vitro (Van der Pauw et al., 2000; Matsuda et al., 2002; Cattaneo et al., 2003), but several recent clinical studies (Hagewald et al., 2002; Cueva et al., 2004; Tonetti et al., 2004) suggested that Emdogain may promote gingival health by direct effect on gingival fibroblasts. We have recently shown that EMD enhances the proliferation of rat (Keila et al., 2004) and human (Zeldich et al., 2007a) gingival fibroblasts and promotes the survival of these cells in culture following an apoptotic stimulus (Zeldich et al., 2007b).
Despite significant clinical use and a multitude of in vitro effects, the molecular mechanisms underlying the documented effects of EMD are largely unknown. We demonstrated that EMD-induced gingival fibroblast proliferation is dependent on ERK activation (Zeldich et al., 2007a), which was observable as early as 5–15 min after exposure to EMD. Such rapid ERK phosphorylation is usually caused by upstream activation of receptors of the tyrosine kinase family (RTKs). Activation of RTKs can be caused by their respective ligands or by transactivation mediated by another receptor (Harris et al., 2003; Edwin et al., 2006). Past studies of EMD preparations failed to demonstrate the presence of any known growth factors that could potentially account for their mitogenic action (Gestrelius et al., 1997; Zeichner-David, 2001).
Transactivation of RTKs, particularly EGFR, with subsequent ERK activation has been well-documented in different cell types, including fibroblasts (Saito and Berk, 2001; Pierce et al., 2001; Kim et al., 2002; Xu et al., 2006; Matsuo at al., 2006). Recent findings suggest two independent mechanisms of EGFR transactivation: One is an intracellular pathway, which is mediated by Src family non-receptor tyrosine kinases (Biscardi et al., 1999); the other is an extracellular pathway, which is mediated by the shedding of a transmembrane pro-form of EGFR ligands by metalloproteinases (Prenzel et al., 1999; Edwin et al., 2006).
Therefore, the purpose of the present study was to examine whether a specific RTK can be implicated in EMD-induced ERK phosphorylation and proliferation of human gingival fibroblasts, and to elucidate the molecular mechanisms involved in its (trans)activation.
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MATERIALS & METHODS
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Materials
EMD (Enamel Matrix Derivative) was generously donated by the Straumann Institute (Basel, Switzerland) and was added in all experiments in a soluble form.
All chemicals and reagents for tissue culture were from Biological Industries (Beit Haemek, Israel). Tissue culture dishes were from Nunc (Rosekilde, Denmark). Trichloroacetic acid (TCA), lauryl sulfate (SDS), trypsin, Tris, trisodium citrate, phenanthroline, leupeptin, aprotonin, and CRM197, a non-toxic diphtheria toxin mutant that selectively binds and inactivates HB-EGF (heparin-binding EGF-like growth factor), were from Sigma Chemical Co. (St. Louis, MO, USA). EGF was from PeproTech Inc. (Rocky Hill, NJ, USA).
AG1478, AG1295, and AG1024 (EGFR, PDGFR, and IGFR phosphorylation inhibitors, respectively), and Alk-5 inhibitor 1 (TGF-β R1 kinase inhibitor) were purchased from Alexis Biochemicals (Lausen, Switzerland); GM 6001 (a broad-spectrum inhibitor of MMPs) and TAPI1 (a broad-spectrum inhibitor of ADAMs), PP-1 (Src kinase inhibitor), and H-Gly-Arg-Gly-Asp-Ser-Pro-OH (GRGDSP) were from Calbiochem (San Diego, CA, USA). [3H] Thymidine was from Perkin Elmer (Boston, MA, USA), and the BCA protein determination kit was from Pierce (Rockford, IL, USA). The following antibodies were used: Phospho-ERK and ERK (Sigma), phosphotyrosine (4G10) (Upstate Biotechnology Inc., Lake Placid, NY, USA), and peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG (Jackson, West Grove, PA, USA). All other reagents are of analytical grade.
Cell Isolation and Culture
The experiments were approved by the Helsinki committee of the Tel-Aviv University, and informed consent was obtained from all donors. Gingival fibroblasts were isolated from gingival connective tissue, excised during periodontal or implant surgery procedures, as described previously (Zeldich et al., 2007a) and grown in  -MEM medium + 10% FBS (fetal bovine serum). Cells from more than 10 donors, between the fourth and eighth passages, having a typical fibroblastic morphology, were used.
Thymidine Incorporation
Thymidine incorporation was assayed as described previously (Zeldich et al., 2007a). Cells were starved for 24 hrs and then challenged with various agents in serum-free conditions. [3H] Thymidine was added after 20 hrs at a final concentration of 1 µCi/ mL for 4 hrs, and cells were washed 3 times with PBS. DNA was precipitated with 5% TCA for 45 min on ice, and solubilized with 0.5 N NaOH for 90 min at room temperature. The radioactivity in the cell lysate was determined in a Beckman® LS-6000SC Liquid Scintillation Counter (Beckman Instruments, Ramsey, MN, USA).
ELISA for Phospho-EGFR
The amount of tyrosine-phosphorylated EGFR in cell lysates was measured with the use of a specific Human Phospho-EGFR ELISA kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Briefly, cell lysates were extracted and protein content was measured. Ninety-six-well plates were coated with an EGFR-capture antibody, which binds both phosphorylated and non-phosphorylated EGFR. After saturation of wells with PBS containing 1% BSA, cell lysates were added in duplicate and incubated for 2 hrs at RT, after which the unbound material was washed away. Tyrosine-phosphorylated EGFR was detected with a peroxidase-conjugated secondary antibody, and optical density of the product was read at 450 nm by means of a microplate reader (Spectramax Plus; Molecular Devices, Sunnyvale, CA, USA). Standard curves were used to translate optical density to the amount of phosphorylated EGFR.
Western Blot Analysis
Cells were washed with ice-cold PBS, subjected to lysis with SDS-sample buffer, and boiled for 15 min. Samples were subjected to SDS-PAGE under reducing conditions with 10% polyacrylamide gels (10–30 µg protein per lane) on a TransBlot SD device (Bio-Rad, Hercules, CA, USA). Proteins were transferred to nitrocellulose membranes and probed for 2 hrs at room temperature with specific primary antibodies. For negative controls, the primary antibodies were omitted. Bound antibodies were visualized with peroxidase-conjugated secondary antibodies, enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA), and BioMax light film (Kodak, Rochester, NY, USA) (Zeldich et al., 2007a). Protein content in the samples was measured by the BCA Protein Assay Kit (product no. 23227, Pierce), which allows for protein determination in the presence of detergent.
Statistical Analysis
All assays were performed in triplicate/quadruplicate for each condition, and each experiment was repeated at least twice. The results are presented as mean ± standard deviation (SD). Statistical analysis was performed by the t test.
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RESULTS
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Incubation of gingival fibroblasts with EMD stimulated DNA synthesis and ERK activation maximally at 50 µg/mL (Figs. 1A, 1B ), and this concentration was used in subsequent experiments. Since EMD-induced mitogenesis is dependent on ERK activation (Zeldich et al., 2007a), we used this assay as the biological endpoint in this study. We first examined the involvement of three well-known members of the RTK family (PDGFR, IGFR, and EGFR) in EMD-induced ERK activation, using their specific inhibitors (AG1295, AG1024, and AG1478, respectively). Only inhibition of EGFR tyrosine kinase (EGFR-TK) resulted in significant decreases in the levels of phospho-ERK, by ~ 40% (Figs. 1C, 1E). A possible mediatory role of TGF-βR was ruled out by the use of its specific inhibitor (Fig. 1D).
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Figure 1. The mitogenic signal of EMD is reduced by an EGFR tyrosine kinase inhibitor. (A,B) Dose response of EMD on thymidine incorporation (A) and ERK activation (B). (A) Quiescent cells were stimulated with EMD in serum-free medium for 24 hrs. [3H] Thymidine was added for the last 4 hrs (A). (B-E) Western blot analysis of the effect of EMD on ERK1/2 phosphorylation (p-ERK) in human gingival fibroblasts in the absence (B) or in the presence (45-minute pre-treatment) of AG1295, AG1024 (C), AG1478 (C,E), or TGF-βR1 inhibitor (each labeled with its target receptor) (D). Cells were analyzed 15 (C,D) or 0–60 (E) min after the addition of EMD to quiescent cells. Lower bands show the abundance of total ERK as loading control. The data represent one of 2 or 3 similar experiments. The ratio between p-ERK and total ERK, obtained by densitometric analysis, is displayed under each lane. None of the inhibitors used altered basal p-ERK levels by itself. (F) Quiescent cells were stimulated with EMD (after pre-treatment with AG1478 for 45 min) for 24 hrs. [3H] Thymidine was added for the last 4 hrs. In A and F, each bar represents the mean ± SD of 3–6 wells. *p < 0.05; ***p < 0.005 (effect of EMD); ### p < 0.005 (effect of AG1478).
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The inhibition of ERK signaling by AG1478 translated into a significant reduction in EMD-induced thymidine incorporation [44% at 24 hrs (Fig. 1F )], providing strong evidence for the involvement of EGFR both in EMD-induced ERK activation and in the subsequent proliferation of gingival fibroblasts. EGFR signaling accounted for ~ 40–50% of both effects.
Further support for the role of the EGFR was gained by the direct assessment of EGFR activation (tyrosine phosphorylation) by Western blotting and pEGFR ELISA. An elevation in tyrosine phosphorylation (at 175 kD) was detected within 5 min of stimulation with EMD. The phosphorylated protein co-migrated with a tyrosine phosphorylated protein derived from EGF-treated cultures, and its phosphorylation was abolished by treatment with the EGFR-TK inhibitor (Fig. 2A). The EMD-induced increase in EGFR tyrosine phosphorylation was verified and quantified by ELISA and shown to equal approximately 50% (Fig. 2B). Analysis of these combined data indicates that signaling through the EGFR is partially responsible for the mitogenic effect of EMD on gingival fibroblasts.
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Figure 2. Quiescent cells were stimulated with EMD (A,B) or EGF (A) in serum-free medium for 5–15 min. (A) Western blot analysis of the effects of EMD, AG1478 (45-minute pre-treatment), and EGF on tyrosine phosphorylation in human gingival fibroblasts. EGF was used for the location of EGFR. (B) Quantitation of EGFR phosphorylation in cell lysates stimulated with EMD after a 45-minute pre-treatment with AG1478 by ELISA. Each bar represents the mean ± SD of 3 biological replicates. ***p < 0.005 (effect of EMD); ### p < 0.005 (effect of AG1478).
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Since previous studies failed to detect EGFR ligands in EMD (Gestrelius et al., 1997; Zeichner-David, 2001), we investigated the possibility that EGFR transactivation results from a metalloproteinase-dependent shedding of membrane-bound EGFR ligands (Harris et al., 2003; Edwin et al., 2006). Both phenanthroline, a non-specific MMP inhibitor, and GM6001, a broad-spectrum MMP inhibitor, decreased the amount of p-ERK induced by EMD by ~ 40–50%; however, TAPI1, a broad-spectrum ADAMs inhibitor, did not affect ERK activation (Fig. 3A). None of these inhibitors altered ERK activation induced by EGF itself. Furthermore, addition of CRM 197, a diphtheria toxin mutant that selectively binds to and inactivates HB-EGF (a membrane-bound EGF-like growth factor known to be shed by MMP activity), markedly inhibited ERK activation induced by EMD, but not by EGF (Fig. 3B). Quantitative analysis of EGFR phosphorylation showed that GM6001, phenanthroline, and CRM197 almost completely inhibited EMD-induced EGFR phosphorylation (Fig. 3C). Analysis of these data indicates that almost all of the EGFR activation can be explained by the shedding of HB-EGF in gingival fibroblasts, which is induced by EMD.
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Figure 3. Quiescent cells were stimulated with EMD in serum-free medium for 15 min after pre-treatment with the indicated inhibitors for 45 min. (A,B) Western blot analysis of the effects of MMP/ADAM/HB-EGF inhibitors on EMD-induced ERK1/2 phosphorylation (p-ERK) in human gingival fibroblasts. Lower bands show the abundance of total ERK as loading control. (C) ELISA measurements of EGFR phosphorylation in cell lysates after stimulation with EMD and a 45-minute pre-treatment with the inhibitors. Each bar represents the mean ± SD of 3 biological replicates. ***p < 0.005 (effect of EMD treatment). Phenanth = phenanthroline. In A-B, the ratio between p-ERK and total ERK, obtained by densitometric analysis, is displayed under each lane. None of the inhibitors altered basal p-ERK levels by itself.
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We demonstrated the involvement of Src family protein kinases in EMD-induced EGFR transactivation and ERK phosphorylation by showing that PP1, a Src family inhibitor, totally abrogated both effects (Figs. 4A, 4B). In contrast, the integrin antagonist (RGD peptide: GRGDSP) only marginally inhibited ERK activation (Fig. 4C), indicating that integrin signaling does not contribute significantly to the mitogenic effect of EMD.
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Figure 4. Quiescent cells were stimulated with EMD in serum-free medium for 15 min after a 45-minute pre-treatment with PP1 (A,B) or a three-hour pre-treatment with an RGD-containing peptide (C). (A) ELISA measurements of EGFR phosphorylation by EMD in cell lysates. Each bar represents the mean ± SD of 3 biological replicates. ***p < 0.005 (effect of EMD); ## p < 0.01 (effect of PP1). (B,C) Western blot analysis of the effect of PP1 or GRGDSP on EMD-induced ERK1/2 phosphorylation (p-ERK) in human gingival fibroblasts. Lower bands show the abundance of total ERK as loading control. The data represent one of 2 similar experiments. In B-C, the ratio between p-ERK and total ERK, obtained by densitometric analysis, is displayed under each lane. None of the inhibitors used altered basal p-ERK levels by itself.
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DISCUSSION
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This is the first demonstration of the mediatory role of the EGF receptor in EMD-induced mitogenesis. This conclusion is based on two lines of evidence: First, the inhibition of EGFR signaling, but not that of PDGFR, IGFR, or TGFβR1, markedly reduced both ERK activation and thymidine incorporation. The possible general involvement of RTKs in EMD-induced mitogenesis has been proposed in only one study (Matsuda et al., 2002), which showed that EMD-induced DNA synthesis and ERK activation in PDL cells were diminished by pre-treatment with suramin, a general inhibitor of RTK, and suggested that EMD elicits ERK activation through an EMD-specific RTK. We also used a TGF-βR inhibitor in response to other reports that suggested the presence, in EMD, of constituents with TGF-β-like properties (Kawase et al., 2001) that could induce fibroblast mitogenesis (Strutz et al., 2001). Second, we provided direct evidence of EGFR phosphorylation upon EMD treatment.
Because earlier studies failed to demonstrate any defined growth factors (such as EGF) or other EGFR ligands (e.g., TGF-) in the commercial preparation of EMD (Gestrelius et al., 1997; Zeichner-David, 2001), we hypothesized that EGFR is transactivated by another stimulus. Furthermore, the rapid (from five- to 15-minute) activation of ERK in our experimental system excluded the possibility that it was caused by de novo synthesis of endogenous growth factors in response to EMD treatment, which was documented previously (Van der Pauw et al., 2000; Lyngstadaas et al., 2001; Brett et al., 2002). Rapid EGFR transactivation can occur either by cleavage of a membrane-bound ligand, such as HB-EGF, by metalloproteinases (Prenzel et al., 1999), or by a metalloproteinase-independent, intracellular pathway (Saito and Berk, 2001; Stirnweiss et al., 2006). Using the MMP inhibitors phenanthroline and GM6001, we established that EMD-induced activation of EGFR is MMP-dependent, in agreement with studies in other systems demonstrating transactivation of EGFR by MMPs such as MMP-2 and MMP-9 (Lucchesi et al., 2004; Shah et al., 2004; Matsuo et al., 2006). The involvement of metalloproteinases of the ADAM family in EGFR transactivation, also documented in other systems (Matsuo et al., 2006), was ruled out, since ERK activation was insensitive to inhibition by TAPI-1, an ADAMs family inhibitor. Finally, since ERK activation was inhibited by the HB-EGF antagonist, CRM197, we conclude that, within the limitation of the specificity of the inhibitors used, EMD treatment of human gingival fibroblasts results in an autocrine/paracrine EGFR transactivation, due to MMP-mediated HB-EGF release. Future experiments will test whether such a mechanism exists in other cell types, such as osteoblasts or PDL cells.
Two additional observations are worth mentioning. First, the inhibition of EGFR phosphorylation by MMP inhibitors and CRM 197 was dramatic (about 70%), but incomplete, in contrast to complete inhibition with AG1478. Analysis of these data suggests that there is another, MMP-independent, mechanism that could contribute to EMD-induced EGFR activation from inside the cell. Since EGFR activation was completely abrogated by PP1, we conclude that both the MMP-dependent (HB-EGF) and MMP-independent processes are mediated by Src, which is known to be involved in these two pathways of EGFR transactivation in other experimental systems, including human fibroblasts (Biscardi et al., 1999; Kim et al., 2002; Xu et al., 2006).
Second, even when EGFR signaling was completely inhibited by treatment with AG1478, ERK phosphorylation and thymidine incorporation were reduced by only 40–50%. This observation indicates that EMD induces gingival fibroblast proliferation via at least two mechanisms involving Src signaling, one of which is EGFR transactivation. Notably, PP1 abrogated ERK phosphorylation completely, suggesting that EGFR-independent ERK activation by EMD also involves Src activation. Indeed, Src kinase is known to activate ERK independently of the EGFR (Pierce et al., 2001; Kim et al., 2002; Zhou et al., 2004). The nature of the stimuli emanating from EMD that activated Src is yet to be determined. Crosstalk between integrins and EGFR has been suggested by numerous studies (Moro et al., 2002; Cabodi et al., 2004, Matsuo et al., 2006), and the association of integrin signaling with Src kinase leading to ERK activation is well-documented (Zhou et al., 2004; Cox et al., 2006). However, treatment of gingival fibroblasts with an RGD-containing peptide (an integrin inhibitor) in our study had a negligible effect on EMD-induced ERK activation. Therefore, activation of the integrin pathway by EMD cannot be considered the major upstream event leading to the transmission of the mitogenic signal.
In conclusion, this is the first study to demonstrate a role for EGFR in the EMD-induced mitogenesis of human gingival fibroblasts through the shedding of a transmembrane EGFR ligand. In view of the many known cellular effects of EGFR activation, it remains to be seen whether the same mechanism underlies additional actions of EMD on other target cells, in addition to the mitogenesis of gingival fibroblasts.
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ACKNOWLEDGMENTS
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The authors thank Dr. G. Weingarten for technical advice. This work was performed in partial fulfillment of the requirements for the PhD degree for Ella Zeldich, Sackler Faculty of Medicine, Tel Aviv University (Tel Aviv, Israel), and was funded by an internal fund of the Faculty of Medicine. M. Dard is an employee of Straumann Institute.
Received for publication October 11, 2007.
Revision received June 2, 2008.
Accepted for publication June 5, 2008.
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Journal of Dental Research, Vol. 87, No. 9,
850-855 (2008)
DOI: 10.1177/154405910808700902
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ABSTRACT
INTRODUCTION