Autocrine overexpression of CTGF maintains fibrosis: RDA analysis of fibrosis genes in systemic sclerosis

We have used representational difference analysis (RDA) to identify up-regu lated genes in skin fibroblasts from fibrotic lesions obtained from patient s with systemic sclerosis (scleroderma). RDA of cDNA libraries derived from fibroblasts from involved and uninvolved skin detected several differentia lly expressed genes. One such gene consistently up-regulated in scleroderma cells coded for human connective tissue growth factor (CTGF). Other studie s described here show that the CTGF protein is readily detected in cultures of systemic sclerosis fibroblasts but was not detected in comparable norma l cells. High levels of CTGF are also evident in biological fluids from pat ients with systemic sclerosis, TGF ss stimulates CTGF production in both no rmal and systemic sclerosis fibroblasts with the latter found to be higher producers. Moreover, an analysis of constitutive and TGF ss-induced CTGF ge ne activation showed altered and elevated transcriptional responses in syst emic sclerosis cells compared with controls. CTGF stimulated a two-to three fold increase in proal(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 rela tion between the overexpression of CTGF and elevated collagen synthesis was suggested by the observation that transfection of a CMV-CTGF cDNA construc t and protein expression in fibroblasts increased the transcription of a Co l 1 alpha 2 promoter-reporter construct to levels seen in systemic sclerosi s fibroblasts. Using Col 1 alpha 2 promoter deletion constructs the CTG;F r esponsive element was localized to the first 379 bp upstream of the transcr iptional start site. These data indicate that there is an overexpression of CTGF in the systemic sclerosis cells, probably due to increased gene trans cription, and suggest that the dysregulation of CTGF production is an impor tant factor in fibroblast activation and the excessive deposition of collag en in systemic sclerosis, (C) 2000 academic Press.