Degradation of DQ gelatin was visualized in the optical section as a green fluorescent signal. Al though unstimulated MG 63 cells did not produce any visible signals, EMD pro tein significantly www.selleckchem.com/products/CP-690550.html enhanced degradation of gelatin. Because gelatin is a substrate for MMPs, it is possible that TIMP 2 could inhibit cell mediated degradation of gelatin. To test this hypothesis, recombinant TIMP 2 was incubated with EMD and MG 63 cells and then cultured on DQ gelatin. TIMP 2 almost completely eliminated degradation by EMD stimulated MG 63 cells, indicating that degradation of gelatin is mediated by the catalytic activity of MMP, which is a key step in the promotion of peri odontal connective tissue remodeling. EMD protein enhanced MMP 2 activity on MG 63 cells We previously showed that MG 63 cells spontaneously produced MMP 2, but not MMP 9.

As shown in this study, EMD protein enhanced degradation of gelatin in MG 63 cells. Therefore, we also tested whether EMD protein affected the production of MMP 2 on 100 ug/ml gelatin coated plates of MG 63 cells. EMD protein enhanced the production of 66 kDa, 68 kDa and 46 kDa MMP 2, which correspond to the pro, intermediate and active forms of this enzyme, respectively. Importantly, when EMD protein activated cells were cultured on gelatin coated plates, generation of the active form of MMP 2 was also observed. These results were confirmed by RT PCR studies to demonstrate that the transcription level of MMP 2 mRNA was augmented in the presence of EMD compared to unstimulated MG 63 cells.

P38 MAPK pathway is involved in EMD protein stimulated MMP 2 activity on MG 63 cells Previous studies have shown that p38 MAPK regulated MMP 2 production induced by various cytokines. Thus, it is possible that EMD protein also stimulates MAP kinases in osteoblasts cells to induce MMP 2 pro duction. To test this hypothesis, we first tested whether EMD protein is able to activate p38 MAPK using Western blotting analysis. When MG63 cells were cultured in the presence of 100 ug/ml EMD protein on gelatin coated plates, p38 MAPK activation was observed in a time dependent Anacetrapib manner, with the maximum phosphoryl ation at 5 min. Total amount of p38 MAPK protein was not affected. A synthetic specific inhibitor for p38 MAPK, SB203580, eliminated the activation of this kinase in EMD protein stimulated MG 63 cells, further indicating the activation of p38 MAPK in MG 63 cells in the presence of EMD protein. In order to further characterize the role of EMD protein induced p38 MAPK pathway activation, MG 63 cells were pretreated with SB203580, followed by EMD protein stimulation. SB203580 significantly inhibited both production and activation of MMP 2 by EMD protein on MG 63 cells.