Previous studies have shown that transforming growth factor- (TGF-) and tumor necrosis factor-␣ (TNF-␣) modulate type I collagen gene expression in fibroblasts. To fine-map the corresponding response elements in the human ␣2(I) collagen (COL1A2) promoter, we have generated a series of 5 deletion promoter/ chloramphenicol acetyltransferase (CAT) reporter gene constructs. Transient cell transfection assays using human dermal fibroblasts and stable transfection experiments using NIH 3T3 fibroblasts identified the region located between residues ؊265 and ؊241, as critical for TGF- response. Specifically, we demonstrate that this 25-base pair region mediates the up-regulatory effect of TGF- on COL1A2 promoter activity and allows antagonistic activity of TNF-␣ on the TGF- effect. Gel mobility shift assays indicate that nuclear factor binding to this 25-base pair region of COL1A2 promoter is competed by AP-1, but not NF-1 or NF-B, oligonucleotides. Transient cell transfection experiments with plasmid constructs in which the potential AP-1-binding site located within this short region of promoter was modified by sitedirected mutagenesis indicated that this element plays a significant role in the basal activity of the promoter. Furthermore, this sequence is essential for TGF- response and does not require the presence of the three Sp-1-binding sites located further upstream, between nucleotides ؊273 and ؊304. In addition, overexpression of c-jun in co-transfection experiments with COL1A2 promoter/CAT constructs blocks the TGF- response, further implicating AP-1 in the regulation of COL1A2 gene expression. Our results clarify the molecular mechanisms involved in the regulation of type I collagen gene expression and further emphasize the importance of AP-1 in mediating some of the TGF- effects on gene transcription.Recently, significant progress has been made in understanding the expression of the human ␣2(I) collagen (COL1A2) gene and its transcriptional regulation by cytokines and growth factors. In particular, it has been shown that a GC-rich region located between residues Ϫ303 and Ϫ271, containing Sp-1-binding sites, is important for high basal promoter activity (Tamaki et al., 1995). This region is comprised within a larger segment of the COL1A2 promoter which has been shown to confer both TGF- 1 (Inagaki et al., 1994) and TNF-␣ responsiveness (Inagaki et al., 1995). However, despite extensive analyses, these studies did not allow precise characterization of TGF- or TNF-␣-response element(s) within the COL1A2 promoter. It was suggested that the TGF--responsive element (TbRE) is located within a 131-bp region, between nucleotides Ϫ378 and Ϫ255, and consists of at least two cis-elements which act in a concerted manner to mediate the effect of TGF-. Once inserted upstream of the thymidine kinase promoter, the TbRE confers TGF- inducibility to this heterologous promoter. Two protein binding sequences within the TbRE, box 3A between residues Ϫ313 and Ϫ286 which contains Sp-1 binding sites, and box B between r...
Transforming growth factor- (TGF-) plays a major role in regulating connective tissue deposition by controlling both extracellular matrix production and degradation. In this study, we show that TGF- transcriptionally represses both basal and tumor necrosis factor-␣-induced collagenase (matrix metalloprotease-1) gene expression in dermal fibroblasts in culture, whereas it activates its expression in epidermal keratinocytes. We demonstrate that this differential effect of TGF- on collagenase gene expression is due to a cell type-specific induction of distinct oncogenes of the Jun family, which participate in the formation of AP-1 complexes with different trans-activating properties. Specifically, our data indicate that the inhibitory effect of TGF- in fibroblasts is likely to be mediated by jun-B, based on the following observations: (a) TGF- induces high levels of jun-B expression and (b) over-expression of jun-B mimics TGF- effect in inhibiting basal collagenase promoter activity and preventing tumor necrosis factor-␣-induced trans-activation of the collagenase promoter. In contrast, TGF- induction of collagenase gene expression in keratinocytes is preceded by transient elevation of c-jun proto-oncogene expression. Over-expression of c-jun leads to trans-activation of the collagenase promoter in both cell types, suggesting that c-jun is a ubiquitous inducer of collagenase gene expression. Transfection of keratinocytes with an antisense c-jun construct together with a collagenase promoter/reporter gene construct inhibits basal and TGF--induced up-regulation of the collagenase promoter activity, implying that c-jun mediates TGF- effect in this cell type. Collectively, our data suggest differential signaling pathways for TGF- in dermal fibroblasts and epidermal keratinocytes, leading to cell type-specific induction of two AP-1 components with opposite transcriptional activities.
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