A novel integrin α5β1 binding domain in module 4 of connective tissue growth factor (CCN2/CTGF) promotes adhesion and migration of activated pancreatic stellate cells

Background: Connective tissue growth factor (CCN2) is upregulated in pancreatic fibrosis and desmoplastic pancreatic tumours. CCN2 interacts with integrin alpha(5)beta(1) on pancreatic stellate cells (PSC) in which it stimulates fibrogenesis, adhesion, migration, and proliferation. Aim: To determine the structural domain(s) in CCN2 that interact with integrin alpha(5)beta(1) to regulation PSC functions. Methods: Primary activated rat PSC were tested for their adherence to isoforms of CCN2 comprising modules 1-4 (CCN2(1-4)), modules 3-4 (CCN2(3-4)), module 3 alone (CCN2(3)), or module 4 alone (CCN2(4)). Adhesion studies were performed in the presence of EDTA, divalent cations, anti-integrin alpha(5)beta(1) antibodies, CCN2 synthetic peptides, or heparin, or after pretreatment of the cells with heparinase, chondroitinase, or sodium chlorate. CCN2 integrin alpha(5)beta(1) binding was analysed in cell free systems. The ability of CCN2(1-4), CCN2(3-4), or CCN2(4) to stimulate PSC migration was evaluated in the presence of anti-integrin alpha(5)beta(1) or heparin. Results: PSC adhesion was stimulated by CCN2(1-4), CCN2(3-4), or CCN2(4) and supported by Mg2+ but not Ca2+. CCN2(4) supported PSC adhesion or migration were blocked by anti-integrin alpha(5)beta(1) antibodies or by treatment of cells with heparinase or sodium chlorate. A direct interaction between CCN2(4) and integrin alpha(5)beta(1) was demonstrated in cell free assays. The sequence GVCTDGR in module 4 mediated the binding between CCN2(4) and integrin alpha(5)beta(1) as well as CCN2(4) mediated PSC adhesion and migration. Conclusions: A GVCTDGR sequence in module 4 of CCN2 is a novel integrin alpha(5)beta(1) binding site that is essential for CCN2 stimulated functions in PSC and which represents a new therapeutic target in PSC mediated fibrogenesis.