Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-β responsiveness

Background. The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological Fibrosis, but its physiologic role remains elusive. in vitro, transforming growth factor-beta (TGF-beta) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-beta, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-beta. To explore this notion, we characterized TGF-beta-induced activation of fibroblasts from CCN2-null (CCN2(-/-)) mouse embryos. Methods. The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-beta signal transduction and regulation of Collagen gene expression were examined in CCN2(-/-) MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results. Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2(-/-) MEFs was markedly reduced compared to wild type MEFs, TGF-beta-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2(-/-) MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion. Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-beta-induced myofibroblast trans differentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts. (c) 2007 Elsevier Inc. All rights reserved.