Abstract
The pleiotropic cytokine transforming growth factor-beta (TGF-beta) is a potent inducer of collagen synthesis and is implicated in the pathogenesis of fibrosis. Acting in concert with transcriptional coactivators p300/CBP, the Smads mediate TGF-beta stimulation of collagen synthesis in human dermal fibroblasts. Little information exists regarding positive and negative modulation of physiological TGF-beta responses. Because the tumor suppressor p53 is implicated in connective tissue homeostasis, here we examined the regulation of collagen gene expression by p53. Forced expression of ectopic p53 in dermal fibroblasts repressed basal and TGF-beta-stimulated collagen gene expression, whereas the absence of cellular p53 was associated with significantly enhanced transcriptional activity of the Type I collagen gene (COL1A2) and collagen synthesis. Ectopic expression of p53 also repressed TGF-beta stimulation of promoter activity driven by minimal Smad-binding elements, suggesting that p53 modulated Smad-dependent intracellular signaling. Inhibition was not due to altered levels, phosphorylation, or nuclear translocation of cellular Smads. Treatment of fibroblasts with etoposide, a potent inducer of cellular p53, abrogated TGF-beta stimulation of COL1A2 promoter activity and collagen synthesis in a p53-dependent manner. Overexpression of the transcriptional coactivator p300 rescued TGF-beta stimulation of COL1A2 promoter activity in fibroblasts overexpressing p53. Furthermore, the ligand-induced interaction of cellular Smad3 with p300 or with its cognate Smad-binding DNA element and recruitment of p300 to the DNA-protein complex assembled on the Smad-binding element were markedly reduced in p53-overexpressing fibroblasts. Collectively, these results indicate, for the first time, that p53 is a potent and selective endogenous repressor of TGF-beta-regulated collagen gene expression in dermal fibroblasts. The ligand-dependent interaction of Smad3 with p300 may be one of the targets of p53-mediated inhibition of TGF-beta responses. These findings suggest that a novel and important physiologic function for the tumor suppressor p53 is the regulation of fibrotic cellular responses.
Highlights
Transforming growth factor- (TGF-)1 is the prototype of a large superfamily of multifunctional cytokines that control cellular growth and differentiation
Because the tumor suppressor p53 is implicated in connective tissue homeostasis, here we examined the regulation of collagen gene expression by p53
To characterize the effects of p53 on collagen synthesis, normal dermal fibroblasts were transiently transfected with expression vector for p53 or empty vector, followed by incubation with TGF- (12.5 ng/ml)
Summary
Transforming growth factor- (TGF-) is the prototype of a large superfamily of multifunctional cytokines that control cellular growth and differentiation. The tumor suppressor p53 is a short lived nuclear phosphoprotein that is mutated in a majority of human cancers It plays important roles in the regulation of cell growth, apoptosis, differentiation, and senescence [9, 10]. These activities of p53 are mediated through inhibition or stimulation of target gene expression during cell cycle [11, 12]. The transcription of the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene was repressed by p53 [27] These results, indicating that p53 can positively or negatively modulate the expression of multiple extracellular matrix genes, suggest a novel function for p53 in physiological regulation of connective tissue homeostasis. It has been shown that p53 modulates TGF--mediated stimulation or inhibition of cellular proliferation [30, 31]
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