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J Dent Res 86(1):41-46, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Enamel Matrix Derivative Stimulates Human Gingival Fibroblast Proliferation via ERK

E. Zeldich1, R. Koren2, C. Nemcovsky3, and M. Weinreb1,*

1 Departments of Oral Biology and
3 Periodontology, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel; and
2 Department of Physiology and Pharmacology, Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel;

* corresponding author, weinreb{at}post.tau.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Emdogain®, a formulation of Enamel Matrix Proteins, is used clinically for periodontal regeneration to stimulate PDL (periodontal ligament), cementum, and bone formation. Its effects on gingival fibroblasts and tissue have not been thoroughly studied. Therefore, we investigated the mechanisms by which Emdogain affects the cell cycle of human gingival fibroblasts. Without serum, Emdogain (50 µg/mL) induced human gingival fibroblast entry into the S phase and DNA synthesis, but not completion of the cell cycle. With low serum concentrations (0.2–0.5%), Emdogain synergistically induced completion of the cell cycle, resulting in increased cell numbers. The mitogenic response to Emdogain depended on Extracellular Regulated Kinase (ERK) activation, which occurred in two waves, peaking after 15 min and 4 to 6 hrs, since it was abolished by U0126, a specific MAPK inhibitor. Inhibition of the second wave was sufficient to abrogate mitogenesis. This study characterized the mitogenic effect of Emdogain on primary human gingival fibroblasts, its cooperation with serum growth factors, and the key mediatory role of the ERK cascade.

KEY WORDS: enamel proteins • in vitro • gingival fibroblasts • ERK • cell cycle


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Enamel matrix proteins are secreted by ameloblasts and regulate enamel mineralization (Fincham et al., 1992; Simmer and Fincham, 1995), but are also secreted by epithelial cells during root formation, and affect cementogenesis and the formation of the periodontal supporting tissues (Slavkin and Boyd, 1975; Hammarström, 1997). Consequently, Emdogain® (enamel matrix derivative) is being used successfully in the regeneration of periodontal tissues (cementum, alveolar bone, and PDL [periodontal ligament]) of individuals with periodontal diseases (Sculean et al., 2000; Giannobile and Somerman, 2003).

In the search for mechanisms whereby Emdogain induces periodontal regeneration, many in vitro studies used various cell systems, showing that Emdogain promotes differentiation of osteogenic precursors (Ohyama et al., 2002; Hagewald et al., 2004; Keila et al., 2004), and enhances the proliferation and matrix production of PDL cells (Gestrelius et al., 1997; Van der Pauw et al., 2000; Matsuda et al., 2002; Cattaneo et al., 2003), but inhibits the proliferation of epithelial cells (Kawase et al., 2000, 2001).

However, the possible effects of Emdogain on gingival fibroblasts and tissue have not been studied in depth. This research topic is prompted by clinical observations that the surgical application of Emdogain onto root surfaces has a beneficial effect on gingival tissue (Hagewald et al., 2002; Nemcovsky et al., 2004).

Only a few studies have suggested that Emdogain is mitogenic for human gingival fibroblasts (Kawase et al., 2000, 2001; Van der Pauw et al., 2000; and Rincon et al., 2003), and a recent study in our laboratory extended this observation to rat gingival fibroblasts (Keila et al., 2004). Furthermore, very little is known about the cellular pathways that participate in the mitogenic effect of Emdogain on these cells. One study (Kawase et al., 2001) proposed the participation of the mitogen-activated protein kinase (MAPK) cascade in this mitogenic effect, but no functional correlation between the two phenomena was shown.

Therefore, we examined, in greater depth, the mitogenic effect of Emdogain on cultured primary human gingival fibroblasts, its cooperation with serum, and the important role of proliferation-related intracellular signaling molecules, like ERK.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Emdogain® was generously donated by the Straumann Institute (Basel, Switzerland). All reagents for tissue culture were from Biological Industries (Beit Haemek, Israel). Tissue culture dishes were from Nunc (Roskilde, Denmark). Crystal violet, trichloracetic acid (TCA), lauryl sulfate (SDS), trypsin, propidium iodide, Tris, trisodium citrate, collagenase type VII, ß-aminopropionitrile, ascorbic acid, and neutral red were from Sigma (St. Louis, MO, USA). U0126 (MAPK inhibitor) was from ALEXIS Biochemicals, Lausen, Switzerland. [3H]Thymidine was from Perkin Elmer (Boston, MA, USA), [3H]L-proline was from Amersham (Little Chalfont, England), and the BCA protein determination kit was from Pierce (Rockford, IL, USA). The following antibodies were used: phospho-ERK and ERK (Sigma), cyclin B1 (GNS-1) and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cyclin D1 (Serotec, Oxford, UK), and peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Jackson, West Grove, USA).

Cell Isolation and Culture
The experiments were approved by the Helsinki committee of the Tel-Aviv University, and informed consent was obtained from all participants. Gingival tissue was removed during periodontal or implant procedures, the epithelium was removed, and connective tissue fragments were cut into small pieces and placed in culture medium ({alpha}-MEM supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 12.5 U/mL nystatin, 0.11 mg/mL sodium pyruvate, and non-essential amino acids) at 37°C in a humidified atmosphere of 5% CO2 and 95% air, to allow for cell outgrowth. The medium was replaced every 3 days until confluence was reached. Cells between the 4th and 8th passages, having a typical fibroblastic morphology, were used.

Cell Number
Cell number was determined colorimetrically by crystal violet (Saati et al., 1997) or neutral red staining (Borenfreund and Puerner, 1985). Cells were plated at 50,000 cells/well in 24-well plates in replicates (4 and more), and allowed to attach and spread for 24 hrs in a medium containing 10% fetal calf serum. Cells were then starved for 24 hrs in a serum-free medium and further incubated with different medium combinations (Emdogain at 50 µg/mL and serum at 0–10%) or with the Emdogain diluent (0.1% acetic acid), and cell numbers were determined 48 hrs later. Cells were washed with PBS, fixed in 70% ethanol, and stained with 1% crystal violet. Unincorporated stain was removed by washing, cells were air-dried, and the dye was extracted with 70% ethanol and its absorbance (550 nm) was measured in a Microplate Reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA). For neutral red staining, cells were incubated in a 1:100 (in DMEM) dye solution at 37°C for 2 hrs and washed twice with PBS. The dye was extracted with Sorrenson solution (0.07 M trisodium citrate, 0.03M citric acid, and 0.1 HCl). Optical density was determined at 550 nm. Calibration curves showed that cell number was linearly correlated to optical density of the 2 dyes.

Thymidine Incorporation
Thymidine incorporation was assayed as described previously (Koren et al., 1981). Twenty hrs after cell challenge, [3H] thymidine was added at a final concentration of 1 µCi/mL for 4 hrs, and cells were washed 3x with PBS. DNA was precipitated with 5% TCA for 45 min on ice, and solubilized with 0.5 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).

Flow Cytometric Analysis
Cells were seeded into six-well plates at 250,000–300,000 cells/well, attached, starved, and challenged as described above. After 25 hrs, cells were trypsinized, centrifuged, and washed with PBS. Cells were stained with propidium iodine (PI), according to Vindelov et al.(1983), and analyzed by a FacScan sorter (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed with the WinMDI software (http://facs.scripps.edu).

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 (20 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. We performed negative controls by omitting the primary antibody. Bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA) and BioMax light film (Kodak, Rochester, NY, USA) (Ravid et al., 1994). 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.

Proline Incorporation
Cells were seeded and starved as described, then challenged with Emdogain (50 or 100 µg/mL) in serum-free medium containing ascorbic acid (50 µg/mL), ß-aminopropio-nitrile (50 µg/mL), and 2 µCi of [3H]-proline for 24 hrs. The medium was collected and incubated with or without collagenase for 18 hrs, followed by TCA precipitation. The amount of radiolabeled collagen was estimated as the difference between total proline [3H]-containing proteins and those left after collagenase digestion (Granot et al., 1993).

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 t tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In the absence of fetal calf serum, Emdogain (50 µg/mL) had no significant effect on human gingival fibroblast cell numbers (Fig. 1AGo). Low fetal calf serum concentrations (0.2–2%), caused a marginal increase in cell numbers, but Emdogain exerted a further significant increase. However, in the presence of an optimal concentration (10%) of fetal calf serum, Emdogain failed to increase cell numbers further. Therefore, all subsequent experiments were performed with 0 or 0.5% fetal calf serum. Similar results were obtained when cell numbers were measured with 2 different methods, crystal violet and neutral red (Figs. 1B, 1CGo). The results of comparable experiments with cells from seven different donors showed the predicted increase in cell numbers, assuming an additive mitogenic action of Emdogain and fetal calf serum, as well as the observed increase (Fig. 1DGo). Although the extent of mitogenesis varied between donors, actual cell numbers were invariably higher than the predicted cell numbers, indicating that Emdogain acted synergistically with low concentrations of fetal calf serum to increase human gingival fibroblast cell numbers.


Figure 1
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  		Figure 1. Effect of Emdogain (EMD) and serum on human gingival fibroblast cell numbers, measured by crystal violet (A,B,D) or neutral red staining (C). Emdogain and serum were added to quiescent cells for 48 hrs. Each bar represents the mean ± SD of 3–5 wells. Dye uptake in serum-free medium without Emdogain (= control) was set to 100%. (D) Calculation of predicted (assuming an additive effect of Emdogain and fetal calf serum) and actual cell numbers in 7 different experiments and their means. For calculation of predicted cell numbers, the number of cells without serum or Emdogain was set to 100%, and the effects of serum (0.5% without Emdogain) and Emdogain (50 µg/mL without serum) were added and compared with the actual cell numbers with both agents. *p < 0.05, **p < 0.005, Emdogain 50 vs. Emdogain 0 µg/mL, or actual vs. predicated cell number.

 
Next we examined the association between increased human gingival fibroblast cell numbers and stimulation of DNA synthesis as assessed by thymidine incorporation. Whereas thymidine incorporation into serum-starved cells was below detection, it was markedly stimulated by a 24-hour treatment with Emdogain alone (Fig. 2AGo). The addition of 0.5% fetal calf serum increased thymidine incorporation by itself, and Emdogain stimulated it further. At a high concentration of fetal calf serum (10%), there was no significant additional effect of Emdogain on thymidine incorporation (data not shown).


Figure 2
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  		Figure 2. Effects of Emdogain (EMD) and fetal calf serum on human gingival fibroblast proliferation. (A) Quiescent cells were stimulated with Emdogain and fetal calf serum (0 and 0.5%) for 24 hrs. [3H] thymidine was added for the last 4 hrs. Each bar represents the mean ± SD of 3–6 wells. (B) Representative histogram plots of flow cytometric analysis. Quiescent cells were stimulated with Emdogain for 25 hrs in serum-free conditions and stained with propidium iodide (PI) for DNA content. (C) Summary of cell-cycle kinetics of human gingival fibroblasts (% of total) in the presence of 0 or 0.5% fetal calf serum ± Emdogain, 25 hrs following stimulation. Each bar represents the mean ± SD of 3–6 wells. (D) Western blot analysis of cyclin D1 and cyclin B1, 12 and 24 hrs, respectively, after quiescent cells were treated with Emdogain. Lower bands show the abundance of ß-actin in these samples for loading control. In A and C, *p < 0.05, **p < 0.005, Emdogain 50 vs. Emdogain 0 µg/mL.

 
To investigate why Emdogain, acting alone, increased DNA synthesis but not cell numbers in serum-free conditions, we analyzed the human gingival fibroblast cell-cycle progression by flow cytometric analysis. In serum-free conditions, Emdogain significantly increased the percentage of cells in the S phase (p < 0.05), but not in the G2/M phase (Figs. 2B, 2CGo). However, in the presence of 0.5% fetal calf serum, Emdogain significantly increased both the fraction of cells in the S phase (7.2% vs. 4.5% in Emdogain-treated vs. untreated cells) and the fraction of cells in the G2/M phase (6.1% vs. 3.8%) (Fig. 2CGo). Further, the abundance of cyclin D1 (controlling cell-cycle entry) 12 hrs following exposure to Emdogain increased in both serum-free medium and 0.5% fetal calf serum, while that of cyclin B1 (controlling G2/M entry) increased at 24 hrs only in the presence of 0.5% fetal calf serum (Fig. 2DGo).

To explore the intracellular mechanisms mediating Emdogain-induced mitogenesis, we examined the activation of the MAPK cascade. Emdogain induced 2 waves of the dual phosphorylation of ERK1/2 in human gingival fibroblasts (Fig. 3AGo). The first wave peaked at 15 min and returned to basal levels by 60 min. A second wave peaked between 4 and 6 hrs, with low residual activity after 18 hrs.


Figure 3
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  		Figure 3. Western blot analysis of the effects of Emdogain (EMD) and U0126 on ERK1/2 phosphorylation in human gingival fibroblasts. (A) Quiescent cells were stimulated with Emdogain in serum-free medium for either 15–60 min or 4–18 hrs. (B) Dose-dependent inhibition of Emdogain-induced ERK phosphorylation by U0126. Lower bands show the abundance of total ERK as loading control.

 
We established the cause/effect relationship between Emdogain-induced ERK phosphorylation and stimulation of proliferation by showing that pre-incubation of human gingival fibroblasts with U0126, a specific MEK1/2 inhibitor, dose-dependently abrogated Emdogain-induced ERK phosphorylation (Fig. 3BGo), increase in cell numbers (Fig. 4AGo), and stimulation of DNA synthesis (Fig. 4BGo), indicating that Emdogain induced human gingival fibroblast proliferation in an ERK1/2-dependent manner.


Figure 4
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  		Figure 4. Effect of inhibiting ERK phosphorylation on Emdogain-induced increase in human gingival fibroblast cell numbers and DNA synthesis. (A) Cells were pre-treated with U0126 (0, 0.5, 5 µM) before the addition of Emdogain (EMD) in the presence of 0.5% serum and stained with crystal violet. Dye uptake without Emdogain and U0126 (= control) was set to 100%. (B) Thymidine incorporation of human gingival fibroblasts after pre-treatment with different concentrations of U0126. (C) Effects of U0126 (5 µM) on Emdogain-induced DNA synthesis, when the inhibitor was added 1 hr before or 3 hrs after the addition of Emdogain. Each bar represents the mean ± SD of 3–6 wells. (D) Effect of Emdogain on collagen production by 3H proline incorporation. *p < 0.05, **p < 0.005.

 
Finally, blocking only the second wave with U0126 was as effective as blocking both waves in preventing the Emdogain-induced DNA synthesis by human gingival fibroblasts (Fig. 4CGo), suggesting that the second wave of the ERK cascade activation was critical for the Emdogain-proliferative effect, with the reservation that selective inhibition of the first wave of ERK activation is technically impossible, since removing the medium from the culture may also partially remove Emdogain and any autocrine mediators secreted by the cells.

Last, we found that Emdogain treatment (100 µg/mL) increased collagen production by human gingival fibroblasts (Fig. 4DGo), indicating that Emdogain stimulated, in human gingival fibroblasts, both proliferation and extracellular matrix production.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Analysis of our data clearly demonstrated that Emdogain stimulated the proliferation of primary human gingival fibroblasts in cooperation with fetal calf serum. In the absence of fetal calf serum, Emdogain stimulated cells to enter into the S phase and synthesize DNA, but not to progress into the G2/M phase and complete the cell cycle, an effect which did not alter cell numbers. In contrast, with high fetal calf serum concentrations (10%), cell proliferation was very high, with 40–50% of the cells entering into the S and G2/M phases (data not shown), and Emdogain failed to increase DNA synthesis or cell numbers further. However, at low fetal calf serum concentrations (0.2–2%), human gingival fibroblast proliferation was low (especially between 0.2 and 0.5%), and Emdogain exerted a significant (up to 1.5-fold) increase in cell numbers within 48 hrs. Our flow cytometric analysis confirmed that the addition of low concentrations of fetal calf serum enabled Emdogain-stimulated cells to enter into the G2/M phase and complete the cell cycle, resulting in an increase in cell numbers. This differential effect of Emdogain was well-reflected in the expression of cyclins, which control cell-cycle progression (Buolamwini, 2000). While cyclin D1 (which controls entry into the cell cycle) was induced by Emdogain regardless of fetal calf serum concentration, cyclin B1 (which controls entry into the G2/M phase) was induced by Emdogain only in the presence of fetal calf serum. Furthermore, we found that Emdogain and 0.5% serum had a synergistic, rather than additive, effect on human gingival fibroblast cell numbers, presumably due to one or more factors present in fetal calf serum. Previous studies have suggested possible candidates for this role, namely, PDGF-BB (Haase et al., 1998; Marcopoulou et al., 2003) and IGF1 (Haase et al., 1998). Future experiments with selective agonists or antagonists may clarify if any of these growth factors plays a major role in the cooperative mitogenic effect. Growth factors such as those found in fetal calf serum are naturally present at the site of periodontal injury and surgery; therefore, such a synergistic action could occur in vivo. Our data establishing the mitogenic effect of Emdogain on human gingival fibroblasts are in agreement with those from previous studies performed with different cell systems, such as primary human gingival fibroblasts (Van der Pauw et al., 2000; Rincon et al., 2003), a human gingival fibroblast cell line (Gin-1, Kawase et al., 2000, 2001), and our own recently published study using primary rat gingival fibroblasts (Keila et al., 2004).

Our study identified the activation (phosphorylation) of ERK as a crucial signaling event for the mitogenic effect of Emdogain, since its inhibition completely abolished the proliferative effect. Previous studies have implicated MAPK signaling in the mitogenic effect of Emdogain, but with variable results: Kawase et al.(2001) reported Emdogain-induced phosphorylation of ERK, p38, and JNK in a human gingival fibroblast-like cell line, but did not show whether any of these cascades was crucial. In contrast, Matsuda et al.(2002) reported Emdogain-induced phosphorylation of ERK but not p38 or JNK in PDL fibroblasts, but again, without a functional analysis. The biological significance of ERK activation by Emdogain is also manifested by the increased abundance of cyclin D1, a well-documented outcome of activation of the ERK cascade.

We also found that ERK phosphorylation proceeds in 2 waves: an immediate one, occurring within minutes after exposure; and a later one, peaking at 4–6 hrs. Previous studies of ERK phosphorylation in response to Emdogain examined only a short period of time, immediately post-stimulation. We believe that the second of the two waves is critical, since its selective inhibition was sufficient to abrogate the mitogenic effect of Emdogain. This observation is in agreement with the finding that extracellular calcium induces osteoblast proliferation via ERK, and that it is the second, sustained, ERK activation that is critical for the mitogenic effect (Huang et al., 2001). Future experiments will aim to identify downstream targets of the "later" ERK activation in this model.

Finally, the emerging role of Emdogain as a human gingival fibroblast mitogen, together with its stimulatory effect on collagen production, can help explain the reported observations that Emdogain application onto root surfaces promotes soft-tissue healing and density (e.g., Tonetti et al., 2004).


   ACKNOWLEDGMENTS
 
The authors thank Dr. Michel Dard, the Straumann Institute, Basel, for the generous gift of Emdogain, Dr. N. Kamin-Belsky and Dr. Y. Oshri for technical assistance, and Dr. A. Ravid for helpful discussions. This work was supported by the Lefcoe Fund for Oral Biology of the Goldschleger School of Dental Medicine and was carried out in the Rosenberg Bone Research Laboratory of the Goldschleger School of Dental Medicine in partial fulfillment of the requirements for the PhD degree of Ella Zeldich, Sackler Faculty of Medicine, Tel Aviv University (Tel Aviv, Israel).

Received November 9, 2005; Last revision September 5, 2006; Accepted September 18, 2006


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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
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