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

Healing without scarring is essential to achieve complete repair of corneal function. One factor that is upregulated in both fibroblasts and epithelium after corneal wounding is connective tissue growth factor (CTGF; CCN2).1,2 Although it participates in the regulation of diverse biological processes related to growth and development, the overexpression of CTGF is correlated with severe fibrotic disorders, including fibrosis in skin, kidney, liver, lung, and vasculature (atherosclerosis),3–7 and several forms of cancer in which it may contribute to tumor angiogenesis and anchorage-independent growth.8–10 CTGF was initially identified as a growth factor, then classified as a matricellular protein, and most recently appreciated as a matrix component.3,11 It is a member of the CCN (Cyr61, CTGF, Nov) family of secreted, cell surface, and extracellular matrix (ECM)–associated 35- to 40-kDa proteins.3,12 Early studies of cells in culture media with serum showed that CTGF is produced in response to transforming growth factor-β (TGF-β) as an immediate early gene product.12,13 In the present study, we took advantage of the opportunity to perform in vitro studies with human corneal fibroblasts (HCFs) grown in the absence of serum to clarify the role of growth factors and matrix on CTGF secretion induced by TGF-β. This was of special interest because during wound healing, the matrix composition changes from one that is initially collagen rich to a provisional matrix that is rich in fibronectin and vitronectin. Experiments using enzymatic dissection of CTGF into its four distinct structural motifs/domains have yielded individual domains of CTGF whose application has helped resolve the source(s) of its great variety of effects in embryogenesis, implantation, angiogenesis, chondrogenesis, tumorigenesis, differentiation, and wound healing.4,14 Thus CTGF domains, individually and in combination, have been reported to be associated with the induction of cell proliferation, chemotaxis, cell adhesion, collagen synthesis, and myofibroblast differentiation.3,4,15–18 Although many of these studies have been performed using CTGF domains expressed in prokaryotic systems, the likelihood that individual domains have a physiological role is supported by the finding of bioactive cleaved forms of CTGF in situ19–22; in particular an N-terminal fragment of CTGF has been detected in the plasma of scleroderma patients.23 We show here that the amount of CTGF secreted by HCFs in response to TGF-β is matrix dependent and that a novel 31-kDa form of CTGF lacking the N terminus is enriched in the detergent-insoluble fraction that includes the matrix and in the conditioned media. Furthermore we present the first evidence that CTGF N-terminal and C-terminal domains are detected in separate vesicles distal from the Golgi apparatus, only some of which have endosomal markers. Our data support the hypothesis that fibroblasts generate different forms of CTGF and that each form may be responsible for a particular profibrotic effect. In addition, since we demonstrated that extracellular CTGF does not overlap with focal adhesions, we suggest that CTGF signaling in HCFs may be initiated through interaction with other surface molecules not primarily localized in focal adhesions.