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 fulllength CCN2 molecule comprises four structural modules (CCN2 1-4 ) and is susceptible to proteolysis by HSC yielding isoforms comprising essentially modules 3 and 4 (CCN2 3-4 ) or module 4 alone (CCN2 4 ). 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 ␣ v ␤ 3 , 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 ␣ v ␤ 3 was detected in HSC lysates by immunoprecipitation and Western blot, and CCN2 4 -mediated HSC adhesion was blocked by anti-integrin ␣ v ␤ 3 antibody. Second, as assessed by immunoprecipitation and solid phase binding assay, CCN2 4 bound directly to integrin ␣ v ␤ 3 in cell-free systems. Third, destruction or inhibition of synthesis of cell surface HSPGs with, respectively, heparinase or sodium chlorate abrogated HSC adhesion to CCN2 4 . Fourth, prior occupancy of heparin-binding sites on CCN2 4 with soluble heparin completely blocked HSC adhesion. These findings indicate that integrin ␣ v ␤ 3 functions as a co-receptor with HSPGs for CCN2 4 -mediated HSC adhesion. Furthermore, by peptide mapping and site-directed mutagenesis we demonstrated that the sequence IRTPKISKPIKFELSG within CCN2 4 is a unique binding domain for integrin ␣ v ␤ 3 that is sufficient to mediate integrin ␣ v ␤ 3 -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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A GVCTDGR sequence in module 4 of CCN2 is a novel integrin alpha5beta1 binding site that is essential for CCN2 stimulated functions in PSC and which represents a new therapeutic target in PSC mediated fibrogenesis.

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