Noelynn A. Oliver

Cooperative interaction of CTGF and TGF-β in animal models of fibrotic disease

BackgroundConnective tissue growth factor (CTGF) is widely thought to promote the development of fibrosis in collaboration with transforming growth factor (TGF)-β; however, most of the evidence for its involvement comes from correlative and culture-based studies. In this study, the importance of CTGF in tissue fibrosis was directly examined in three murine models of fibrotic disease: a novel model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-β2; the unilateral ureteral obstruction (UUO) renal fibrosis model; and an intratracheal bleomycin instillation model of pulmonary fibrosis.ResultsIntraperitoneal coadministration of CTGF and TGF-β2 elicited a profound fibrotic response that was inhibited by the human anti-CTGF antibody FG-3019, as indicated by the ability of FG-3019 to ameliorate the histologic signs of fibrosis and reduce the otherwise increased hydroxyproline:proline (Hyp:Pro) ratios by 25% in kidney (P < 0.05), 30% in liver (P < 0.01) and 63% in lung (P < 0.05). Moreover, administration of either cytokine alone failed to elicit a fibrotic response, thus demonstrating that CTGF is both necessary and sufficient to initiate fibrosis in the presence of TGF-β and vice versa. In keeping with this requirement for CTGF function in fibrosis, FG-3019 also reduced the renal Hyp:Pro response up to 20% after UUO (P < 0.05). In bleomycin-injured animals, a similar trend towards a FG-3019 treatment effect was observed (38% reduction in total lung Hyp, P = 0.056). Thus, FG-3019 antibody treatment consistently reduced excessive collagen deposition and the pathologic severity of fibrosis in all models.ConclusionCooperative interactions between CTGF and TGF-β signaling are required to elicit overt tissue fibrosis. This interdependence and the observed anti-fibrotic effects of FG-3019 indicate that anti-CTGF therapy may provide therapeutic benefit in different forms of fibroproliferative disease.

Regulation of Fibronectin-EDA through CTGF Domain–Specific Interactions with TGFβ2 and Its Receptor TGFβRII

PURPOSE To investigate the role of fibronectin containing extra domain A (FN-EDA) in the pathogenesis of proliferative vitreoretinopathy (PVR) and the regulation of FN-EDA by transforming growth factor (TGF)-β and connective tissue growth factor (CTGF) in retinal pigment epithelial (RPE) cells. METHODS Expression of FN-EDA in normal human retinas and PVR membranes was evaluated by immunohistochemistry. The effects of TGFβ and CTGF on FN-EDA mRNA and protein expression in primary cultures of human RPE cells were analyzed at different time points by real-time PCR and Western blot, respectively. The interaction of CTGF with TGFβ2 or with its type II receptor TGFβRII was examined by ELISA, immunoprecipitation, and solid-phase binding assays. RESULTS FN-EDA was abundantly expressed in PVR membranes but absent from the RPE monolayer in normal human retinas. Treatment of RPE cells with TGFβ2 induced FN-EDA expression in a time- and dose-dependent manner, but CTGF alone had no effect. However, CTGF, through its N-terminal half fragment, augmented TGFβ2-induced expression of FN-EDA at the protein level. This effect was blocked by antibodies against TGFβ2 or TGFβRII. Interaction of TGFβ2 or TGFβRII with CTGF was dose dependent and specific. CTGF directly bound TGFβ2 and TGFβRII at its N- and C-terminal domains, respectively. CONCLUSIONS These findings suggest that CTGF promotes the profibrotic activities of TGFβ acting as a cofactor through direct protein interactions and complex regulatory mechanisms.

TGF-β–Stimulated CTGF Production Enhanced by Collagen and Associated with Biogenesis of a Novel 31-kDa CTGF Form in Human Corneal Fibroblasts

PURPOSE Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-β) after corneal wounding. This study addressed the role of the extracellular matrix in the induction of CTGF by TGF-β. METHODS Human corneal fibroblasts (HCFs) were grown on fibronectin (FN), vitronectin (VN), or collagen (CL) in supplemented serum-free media alone or with TGF-β1 or fibroblast growth factor plus heparin. CTGF mRNA was analyzed by qPCR and protein expression by Western blot analysis of Triton X-100 (TX-100)-soluble and TX-100-insoluble cell lysates using antibodies to N-terminal, mid, and C-terminal CTGF regions. Immunocytochemistry was performed on nonconfluent or scrape-wounded confluent HCFs. RESULTS TGF-β-treated HCFs grown on CL produced five times more 38-kDa CTGF than untreated controls (72 hours). TGF-β-treated HCFs on CL secreted twofold more CTGF than those on FN or VN. Furthermore, a 31-kDa CTGF form, lacking the N-terminal domain, was detected in Triton X-100 insoluble fractions in Western blot analysis. Immunodetectable extracellular CTGF formed linear arrays parallel to, but not colocalized with, CL or FN. It also did not colocalize with FAK, vinculin, or integrins α(v)β(3) and α(5)β(1). Intracellular CTGF was detected in the Golgi apparatus and vesicles, including endosomes. CONCLUSIONS Enhanced CTGF secretion induced by TGF-β in CL-grown cells may contribute to positive feedback in which CL is overexpressed in CTGF-induced fibrosis. N-terminal CTGF fragments in the plasma of patients with severe fibrotic disease may be a product of CTGF proteolysis that also produces the newly identified 31-kDa CTGF that remains cell associated and may have its impact by non-integrin signaling pathways.