Transforming growth factor beta1 induces alphavbeta3 integrin expression in human lung fibroblasts via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway

In response to transforming growth factor beta1 (TGFbeta) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFbeta has been shown to increase the expression of specific alphav integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFbeta1 increased both beta3 integrin subunit mRNA and protein levels as well as surface expression of alphavbeta3 in human lung fibroblasts. TGFbeta1-induced alphavbeta3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of beta3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or alphavbeta3 blocking antibody prevented the increase in beta3 but not beta5 integrin expression. In addition, echistatin inhibited TGFbeta1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFbeta1-induced expression of alphavbeta3 and p38 MAPK phosphorylation but not beta5 integrin expression and Smad3 activation. The TGFbeta1-induced alphavbeta3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-kappaB-dependent pathways. Our results demonstrate that TGFbeta1 induces alphavbeta3 integrin expression via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFbeta1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.     

Constitutive connective tissue growth factor expression in scleroderma fibroblasts is dependent on Sp1

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.