Abstract
Fibrotic diseases such as scleroderma (systemic sclerosis, SSc) are characterized by an excessive production of extracellular matrix and profibrotic proteins such as connective tissue growth factor (CTGF). In normal dermal fibroblasts, CTGF is not expressed unless induced by proteins such as tumor growth factor-beta (TGFbeta). Conversely, in fibroblasts cultured from fibrotic lesions CTGF mRNA and protein are constitutively expressed, even in the absence of exogenously added TGFbeta. Thus, studying the mechanism underlying CTGF overexpression in SSc fibroblasts is likely to yield valuable insights into the basis of the fibrotic phenotype of SSc and possibly other scarring disease. CTGF overexpression is mediated primarily by sequences in the CTGF promoter. In this report, we identify the minimal promoter element involved with the overexpression of CTGF in SSc fibroblasts. This element is distinct from the element necessary and sufficient for the induction of CTGF expression by TGFbeta in normal fibroblasts. Within this region is a functional Sp1 binding site. Blocking Sp1 activity reduces the elevated, constitutive levels of CTGF promoter activity and protein expression observed in SSc fibroblasts. Relative to those prepared from normal dermal fibroblasts, nuclear extracts prepared from SSc fibroblasts possess increased Sp1 binding activity. Removal of phosphate groups from nuclear extracts enhanced Sp1 binding activity, suggesting that phosphorylation of Sp1 normally reduces Sp1 binding to DNA. Thus, the constitutive overexpression of CTGF in SSc fibroblasts seems to be independent of TGFbeta signaling but dependent at least in part on Sp1.
Highlights
Wound healing requires the de novo synthesis of connective tissue
The Region Lying between Nucleotides Ϫ86 and ϩ17 of the connective tissue growth factor (CTGF) Promoter Is Required for Its Overexpression in SSc Fibroblasts—To identify regions of the CTGF promoter important for its overexpression in SSc fibroblasts, we transfected a series of CTGF promoter deletion constructs into normal and SSc dermal fibroblasts
All constructs contained abundant CTGF promoter expression, suggesting that the elevated level of CTGF expression observed in SSc fibroblasts was controlled by the first 86 base pairs upstream of the transcription initiation start site (Fig. 1)
Summary
Wound healing requires the de novo synthesis of connective tissue. If this process is not appropriately terminated, excessive matrix deposition occurs, resulting in pathological fibrosis [1, 2]. Because TGF1 promotes fibroblast proliferation and matrix synthesis, attention has long been devoted to the potential role of this factor in initiation and maintenance of fibrosis Whereas subcutaneous injection of TGF into neonatal rats results only in a transient fibrotic response, coinjection of CTGF and TGF results in sustained fibrosis [18] This result could possibly be because CTGF can bind TGF and may enhance the activity of TGF, at low concentrations, to bind its receptors [19]. Analyzing the relative contribution of TGF-dependent and -independent mechanisms to the elevated level of CTGF promoter activity in lesional SSc fibroblasts should yield valuable insights into the molecular basis of the maintenance of the SSc phenotype
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