SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta

The human type VII collagen gene (COL7A1) recently has been identified as a n immediate-early response gene for transforming growth factor beta (TGF-be ta)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4-/- bre ast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-beta response element in the region -496/-444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assa ys with nuclear extracts from COS-1 cells transfected with expression vecto rs for SMADs 1-5 indicate that SMAD3 forms a complex with a migration simil ar to that of the endogenous TGF-beta-specific complex observed in fibrobla st extracts. Electrophoretic mobility-shift assays using recombinant glutat hione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-ter minally truncated SMAD3 but not SMAD2, can bind the COL7A1 SBS. Coexpressio n of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes : a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being dete cted in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant o verexpression of SMAD3 and SMAD4. These data may represent the first identi fication of a functional homomeric SMAD3 complex regulating a human gene.