Selective stimulation of collagen synthesis in the presence of costimulatory insulin signaling by connective tissue growth factor in scleroderma fibroblasts

Objective. To examine the mechanism of collagen induction by connective tissue growth factor (CTGF), a profibrotic cytokine overexpressed in the skin of patients with systemic sclerosis (SSc). Methods. Dermal fibroblasts from 7 SSc patients and 7 matched healthy adult donors were stimulated with CTGF in the presence or absence of the culture-medium supplement, insulin-transferrin-selenium (ITS). Expression of collagen protein was analyzed by a 3 H-proline incorporation assay. To identify the signaling pathways mediating CTGF induction of collagen, pharmacologic inhibitors were used, including rottlerin, a protein kinase CS (PKCdelta) inhibitor. Results. Collagen levels in both SSc and normal fibroblasts were increased after treatment with transforming growth factor beta in serum-free medium, whereas no stimulation was observed following addition of CTGF. In the presence of ITS, CTGF (2.5 ng/ml) potently stimulated collagenous protein levels in SSc cell lines (n = 5); however, CTGF was not stimulatory in the majority of normal fibroblasts (n = 6). ITS alone induced collagen levels in normal fibroblasts to the levels observed in SSc skin fibroblasts, thereby diminishing the hallmark difference in basal collagen levels in these cell types. Insulin was the ITS component responsible for promoting the basal and CTGF stimulation of collagenous proteins. Rottlerin, the PKCS inhibitor, down-regulated collagen synthesis in normal and SSc fibroblasts cultured in ITS, and inhibited the stimulatory effects of CTGF in cooperation with insulin or of insulin (500 ng/ml) alone. Conclusion. Increased responsiveness of SSc fibroblasts to CTGF-mediated collagen synthesis requires the costimulatory activation of insulin signaling pathways to induce matrix production. Blockade of this effect via rottlerin may suggest that PKCdelta is a downstream signaling molecule necessary for CTGF stimulation of collagen synthesis.