SMAD3/4-dependent transcriptional activation of the human type VII collagen gene ( COL 7A1 ) promoter by transforming growth factor β

Abstract

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor ␤ (TGF-␤)͞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 SMADmediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-␤ response element in the region ؊496͞؊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-␤-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 DNA͞protein 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.

Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice

“…Aside from the reduction of lung fibrosis, we found that loss of Smad3 inhibited bleomycin-induced collagen accumulation in the lung interstitium. Recent evidences have shown that Smad3 is involved in the transcriptional activation of TGF-␤-mediated stimulation of collagen expression (5,32,33). Smad3 is necessary for the formation of transcriptional activation complex in the stimulation of type I collagen gene expression by TGF-␤ ligands in human skin fibroblasts (6,9).…”

Regulation of cell proliferation by Smad proteins

“…Multimers of SBE when placed in front of a minimal promoter reporter construct provide a strong enhancer function for TGF-b family members. SBE-like sequences have been shown to be critically important for TGF-binducibility of multiple TGF-b responsive genes (Dennler et al, 1998;Vindevoghel et al, 1998;Hanai et al, 1999;Nagarajan et al, 1999;Brodin et al, 2000;Chen et al, 2000;Taylor and Khachigian, 2000). The crystal structure of Smad3 MH1 domain with SBE showed that the b-hairpin loop in Smad3 is in contact region with the SBE (Shi et al, 1998).…”

Functional consequences of tumorigenic missense mutations in the amino-terminal domain of Smad4

“…Smad2 is unable to bind directly to DNA because it contains an extra exon just N-terminal of the b-hairpin loop (Dennler et al, 1998;Yagi et al, 1999). SBE-like sequences have been identi®ed in the promoters of multiple TGF-b responsive genes, including plasminogen activator inhibitor-1 (PAI-1) (Dennler et al, 1998;Luo et al, 1999), junB (Jonk et al, 1998), type VII collagen (Vindevoghel et al, 1998) and the germline immunoglobulin Ia region (Hanai et al, 1999;Pardali et al, 2000a;Zhang and Derynck, 2000). Mutation of SBEs attenuate TGF-b inducibility.…”