Autocrine Overexpression of CTGF Maintains Fibrosis: RDA Analysis of Fibrosis Genes in Systemic Sclerosis

Abstract

We have used representational difference analysis (RDA) to identify up-regulated genes in skin fibroblasts from fibrotic lesions obtained from patients with systemic sclerosis (scleroderma). RDA of cDNA libraries derived from fibroblasts from involved and uninvolved skin detected several differentially expressed genes. One such gene consistently up-regulated in scleroderma cells coded for human connective tissue growth factor (CTGF). Other studies described here show that the CTGF protein is readily detected in cultures of systemic sclerosis fibroblasts but was not detected in comparable normal cells. High levels of CTGF are also evident in biological fluids from patients with systemic sclerosis. TGFβ stimulates CTGF production in both normal and systemic sclerosis fibroblasts with the latter found to be higher producers. Moreover, an analysis of constitutive and TGFβ-induced CTGF gene activation showed altered and elevated transcriptional responses in systemic sclerosis cells compared with controls. CTGF stimulated a two- to threefold increase in proα1(I) collagen and fibronectin synthesis by both dermal and lung fibroblasts in culture and promoted significant matrix remodeling of fibroblast-populated three-dimensional collagen lattices. A direct relation between the overexpression of CTGF and elevated collagen synthesis was suggested by the observation that transfection of a CMV–CTGF cDNA construct and protein expression in fibroblasts increased the transcription of a Col 1α2 promoter–reporter construct to levels seen in systemic sclerosis fibroblasts. Using Col 1α2 promoter deletion constructs the CTGF responsive element was localized to the first 379 bp upstream of the transcriptional start site. These data indicate that there is an overexpression of CTGF in the systemic sclerosis cells, probably due to increased gene transcription, and suggest that the dysregulation of CTGF production is an important factor in fibroblast activation and the excessive deposition of collagen in systemic sclerosis.