Protein Structure and Folding
Connective Tissue Growth Factor (CCN2) Induces Adhesion of Rat Activated Hepatic Stellate Cells by Binding of Its C-terminal Domain to Integrin αvβ3 and Heparan Sulfate Proteoglycan*

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Connective tissue growth factor (CCN2, also known as CTGF) is a matricellular protein that appears to play an important role in hepatic stellate cell (HSC)-mediated fibrogenesis. After signal peptide cleavage, the full-length CCN2 molecule comprises four structural modules (CCN21–4) and is susceptible to proteolysis by HSC yielding isoforms comprising essentially modules 3 and 4 (CCN23–4) or module 4 alone (CCN24). In this study we show that rat activated HSC are capable of adhesion to all three CCN2 isoforms via the binding of module 4 to integrin αvβ3, a process that is dependent on interactions between module 4 and cell surface heparan sulfate proteoglycans (HSPGs). These findings are based on several lines of evidence. First, integrin αvβ3 was detected in HSC lysates by immunoprecipitation and Western blot, and CCN24-mediated HSC adhesion was blocked by anti-integrin αvβ3 antibody. Second, as assessed by immunoprecipitation and solid phase binding assay, CCN24 bound directly to integrin αvβ3 in cell-free systems. Third, destruction or inhibition of synthesis of cell surface HSPGs with, respectively, heparinase or sodium chlorate abrogated HSC adhesion to CCN24. Fourth, prior occupancy of heparin-binding sites on CCN24 with soluble heparin completely blocked HSC adhesion. These findings indicate that integrin αvβ3 functions as a co-receptor with HSPGs for CCN24-mediated HSC adhesion. Furthermore, by peptide mapping and site-directed mutagenesis we demonstrated that the sequence IRTPKISKPIKFELSG within CCN24 is a unique binding domain for integrin αvβ3 that is sufficient to mediate integrin αvβ3- and HSPG-dependent HSC adhesion. These findings offer the possibility of developing novel antifibrotic therapies that target the integrin-binding domain.

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This work was supported by National Institutes of Health Grant R01 AA12817 (to D. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.