Transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) are ubiquitously expressed in various forms of tissue fibrosis, including fibrotic diseases of the kidney. To clarify the common and divergent roles of these growth factors in the cells responsible for pathological extracellular matrix (ECM) deposition in renal fibrosis, the effects of TGF-beta and CTGF on ECM expression in primary human mesangial (HMCs) and human proximal tubule epithelial cells (HTECs) were studied. Both TGF-beta and CTGF significantly induced collagen protein expression with similar potency in HMCs. Additionally, alpha(2)(I)-collagen promoter activity and mRNA levels were similarly induced by TGF-beta and CTGF in HMCs. However, only TGF-beta stimulated collagenous protein synthesis in HTECs. HTEC expression of tenascin-C (TN-C) was increased by TGF-beta and CTGF, although TGF-beta was the more potent inducer. Thus both growth factors elicit similar profibrogenic effects on ECM production in HMCs, while promoting divergent effects in HTECs. CTGF induction of TN-C, a marker of epithelial-mesenchymal transdifferentiation (EMT), with no significant induction of collagenous protein synthesis in HTECs, may suggest a more predominant role for CTGF in EMT rather than induction of excessive collagen deposition by HTECs during renal fibrosis.

Objective. Aberrant transforming growth factor ␤ (TGF␤) signaling has been implicated in the pathogenesis of scleroderma (systemic sclerosis [SSc]), but the contribution of specific components in this pathway to SSc fibroblast phenotype remains unclear. This study was undertaken to delineate the role of TGF␤ receptor type I (TGF␤RI) and TGF␤RII in collagen overexpression by SSc fibroblasts.Methods. Primary dermal fibroblasts from SSc patients and healthy adults were studied (n ‫؍‬ 10 matched pairs). Adenoviral vectors were generated for TGF␤RI (AdTGF␤RI), TGF␤RII (AdTGF␤RII), and kinase-deficient TGF␤RII (Ad⌬kRII). TGF␤RI basal protein levels were analyzed by 35 S-methionine labeling/ immunoprecipitation and immunohistochemistry. Type I collagen and TGF␤RII basal protein levels were analyzed by Western blot and newly secreted collagen by 3 H-proline incorporation assay.Results. Analysis of endogenous TGF␤RI and TGF␤RII protein levels revealed that SSc TGF␤RI levels were increased 1.7-fold (P ‫؍‬ 0.008; n ‫؍‬ 7) compared with levels in healthy controls, while TGF␤RII levels were decreased by 30% (P ‫؍‬ 0.03; n ‫؍‬ 7). This increased TGF␤RI:TGF␤RII ratio correlated with SSc collagen overexpression. To determine the consequences of altered TGF␤RI:TGF␤RII ratio on collagen expression, healthy fibroblasts were transduced with AdTGF␤RI or AdTGF␤RII. Forced expression of TGF␤RI in the range corresponding to elevated SSc TGF␤RI levels increased basal collagen expression in a dose-dependent manner, while similar TGF␤RII overexpression had no effect, although transduction of fibroblasts at higher multiplicities of infection led to a marked reduction of basal collagen levels. Blockade of TGF␤ signaling via Ad⌬kRII resulted in ϳ50% inhibition of basal collagen levels in healthy fibroblasts and in 5 of 9 SSc cell lines. A subset of SSc fibroblasts (4 of 9 cell lines) was resistant to this treatment. SSc fibroblasts with the highest levels of TGF␤RI were the least responsive to collagen inhibition via ⌬kRII.Conclusion. This study indicates that an increased TGF␤RI:TGF␤RII ratio may underlie aberrant TGF␤ signaling in SSc and contribute to elevated basal collagen production, which is insensitive to TGF␤ signaling blockade via ⌬kRII.Scleroderma (systemic sclerosis [SSc]) is characterized by excessive deposition of extracellular matrix

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 culturemedium 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 C␦ (PKC␦) inhibitor.Results. Collagen levels in both SSc and normal fibroblasts were increased after treatment with transforming growth factor ␤ 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 PKC␦ 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 PKC␦ is a downstream signaling molecule necessary for CTGF stimulation of collagen synthesis.