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. 2006 Aug;169(2):405–415. doi: 10.2353/ajpath.2006.060049

Figure 4-6929.

Figure 4-6929

αvβ8-mediated activation of TGF-β is regulated by PMA inducible metalloprotease activity. A: The level of surface MT1-MMP correlates with the relative abilities of fetal tracheal epithelial cells and fibroblasts to active TGF-β. Immunoprecipitations of cell surface biotinylated cell lysates using either no primary antibody (lane 1) or anti-MT1-MMP (lanes 2 to 4) were resolved by 10% SDS-PAGE under reducing conditions. Lanes 1 and 2: fetal tracheal epithelial cells; lane 3: fetal tracheal fibroblasts; lane 4: HT1080 fibrosarcoma cells. Shown is the immunoprecipitation product migrating at the expected kd for MT1-MMP. B: PMA markedly up-regulates the surface expression of MT1-MMP by fetal tracheal epithelial cells. Immunoprecipitations of cell surface biotinylated cell lysates of HT1080 fibrosarcoma cells (top) or fetal tracheal epithelial cells (FTEC, bottom) using either no primary antibody (control, lanes 1 and 3) or anti-MT1-MMP (lanes 2 and 4) were resolved by 10% SDS-PAGE under reducing conditions. The bands shown migrated at the size expected for MT1-MMP. C: PMA treatment of fetal tracheal epithelial cells leads to increased activity of MT1-MMP. Gelatin zymography was performed using conditioned media harvested from HT1080 cells (top) or fetal tracheal epithelial cells (FTEC, bottom) treated with pro-MMP with no inhibitor (none, lane 1), 1 nmol/L PMA (lane 2), 10 nmol/L PMA (lane 3), or PMA with GM6001 (lane 4). The migration of recombinant pro-MMP-2 is shown (pro-MMP-2, lane 5). A–C show representative experiments of at least three independent experiments giving similar results. D: Fetal tracheal epithelial cell αvβ8-dependent activation of TGF-β is markedly enhanced by PMA treatment. Fetal tracheal epithelial cells (n = 5) either not treated or treated with PMA (1 nmol/L) were co-cultured with TMLC TGF-β reporter cells in the presence of nothing (filled bar), neutralizing anti-β8 (horizontal crosshatched bar), or the metalloprotease inhibitor GM6001 (diagonal hatched bars). *P < 0.05, **P < 0.001.