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EGFR in Enamel Matrix Derivative-induced Gingival Fibroblast Mitogenesis

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

View larger version (52K): [in this window] [in a new window]   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).

View larger version (32K): [in this window] [in a new window]   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).

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

View larger version (17K): [in this window] [in a new window]   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.

Journal of Dental Research, Vol. 87, No. 9, 850-855 (2008)
DOI: 10.1177/154405910808700902


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