Abstract
A critical component of the epidermal basement membrane, collagen type VII, is produced by keratinocytes and fibroblasts, and its production is stimulated by the cytokine transforming growth factor-beta (TGF-beta). The gene, COL7A1, is activated by TGF-beta via Smad transcription factors in cooperation with AP1. Here we report a previously unsuspected level of complexity in this regulatory process. We provide evidence that TGF-beta may activate the COL7A1 promoter by two distinct inputs operating through a common region of the promoter. One input is provided by TGF-beta-induced Smad complexes via two Smad binding elements that function redundantly depending on the cell type. The second input is provided by relieving the COL7A1 promoter from chicken ovalbumin upstream promoter transcription factor (COUP-TF)-mediated transcriptional repression. We identified COUP-TFI and -TFII as factors that bind to the TGF-beta-responsive region of the COL7A1 promoter in an expression library screening. COUP-TFs bind to a site between the two Smad binding elements independently of Smad or AP1 and repress the basal and TGF-beta-stimulated activities of this promoter. We provide evidence that endogenous COUP-TF activity represses the COL7A1 promoter. Furthermore, we show that TGF-beta addition causes a rapid and profound down-regulation of COUP-TF expression in keratinocytes and fibroblasts. The results suggest that TGF-beta signaling may exert tight control over COL7A1 by offsetting the balance between opposing Smad and COUP-TFs.
Highlights
Transforming growth factor- (TGF-)1 in particular is a potent inducer of COL7A1 expression in fibroblast and keratinocytes (9 –11)
TGF- addition strongly stimulated the expression of the wild type COL7A1 promoter in mouse NIH 3T3 fibroblasts, and this response was diminished by mutations targeting the 5Ј-Smad binding element (SBE) (Fig. 1B)
The simultaneous mutation of the 5Ј-SBE and 3Ј-SBE sites completely eliminated the TGF- response in fibroblasts and in keratinocytes (Fig. 1B). These results suggested that the 5Ј-SBE and 3Ј-SBE in the COL7A1 promoter are important for the TGF- response in both cell types, acting redundantly in keratinocytes but not in fibroblasts
Summary
Transforming growth factor- (TGF-)1 in particular is a potent inducer of COL7A1 expression in fibroblast and keratinocytes (9 –11). We identified COUP-TFI and -TFII as factors that bind to the TGF--responsive region of the COL7A1 promoter in an expression library screening. Role of Two Smad Binding Elements in the COL7A1 Promoter—To investigate the transcriptional activation of COL7A1 by TGF-, we generated luciferase reporter constructs driven by the Ϫ496/ϩ92 region of the human COL7A1 [9] (Fig. 1A).
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