title: SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta.
abstract: The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor beta (TGF-beta)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4-/- breast 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 assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-beta-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression 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 detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.
authors.affiliation: Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: L
authors.initials: L
authors.lastname: Vindevoghel
authors.forename: R J
authors.initials: RJ
authors.lastname: Lechleider
authors.forename: A
authors.initials: A
authors.lastname: Kon
authors.forename: M P
authors.initials: MP
authors.lastname: de Caestecker
authors.forename: J
authors.initials: J
authors.lastname: Uitto
authors.forename: A B
authors.initials: AB
authors.lastname: Roberts
authors.forename: A
authors.initials: A
authors.lastname: Mauviel
mesh_terms: D000818:Animals; D019556:COS Cells; D003094:Collagen / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D006801:Humans; D008233:Lymphotoxin-alpha / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D008233:Lymphotoxin-alpha; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D003094:Collagen