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Enamel Matrix Derivative Stimulates Human Gingival Fibroblast Proliferation via ERK

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;

View larger version (16K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   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.

View larger version (39K): [in this window] [in a new window]   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.

View larger version (15K): [in this window] [in a new window]   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.