A novel integrin 5 1 binding domain in module 4 of connective tissue growth factor (CCN2/CTGF) promotes adhesion and migration of activated pancreatic stellate cells

Abstract

Background: Connective tissue growth factor (CCN2) is upregulated in pancreatic fibrosis and desmoplastic pancreatic tumours. CCN2 interacts with integrin α₅β₁ 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 α₅β₁ 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 (CCN2₃), or module 4 alone (CCN2₄). Adhesion studies were performed in the presence of EDTA, divalent cations, anti-integrin α₅β₁ antibodies, CCN2 synthetic peptides, or heparin, or after pretreatment of the cells with heparinase, chondroitinase, or sodium chlorate. CCN2 integrin α₅β₁ binding was analysed in cell free systems. The ability of CCN2_1–4, CCN2_3–4, or CCN2₄ to stimulate PSC migration was evaluated in the presence of anti-integrin α₅β₁ or heparin. Results: PSC adhesion was stimulated by CCN2_1–4, CCN2_3–4, or CCN2₄ and supported by Mg²⁺ but not Ca²⁺. CCN2₄ supported PSC adhesion or migration were blocked by anti-integrin α₅β₁ antibodies or by treatment of cells with heparinase or sodium chlorate. A direct interaction between CCN2₄ and integrin α₅β₁ was demonstrated in cell free assays. The sequence GVCTDGR in module 4 mediated the binding between CCN2₄ and integrin α₅β₁ as well as CCN2₄ mediated PSC adhesion and migration. Conclusions: A GVCTDGR sequence in module 4 of CCN2 is a novel integrin α₅β₁ binding site that is essential for CCN2 stimulated functions in PSC and which represents a new therapeutic target in PSC mediated fibrogenesis.