Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue growth factor (CCN2)

Production of connective tissue growth factor (CCN2, also known as CTGF) is a hallmark of hepatic fibrosis. This study examined early primary cultures of hepatic stellate cells (HSC) for (i) CCN2 regulation of its cognate receptor integrin subunits; and (ii) interactions between CCN2 and integrin alpha(5)beta(1), heparan sulphate proteoglycans (HSPG) or fibronectin (FN) in supporting cell adhesion. HSC were isolated from healthy male Balb/c mice. mRNA levels of CCN2 or alpha(5), beta(1), alpha v or beta(3) integrin subunits were measured in days 1-7 primary culture HSC, and day 3 or day 7 cells treated with recombinant CCN2 or CCN2 small interfering RNA. Interactions between CCN2 and integrin alpha(5)beta(1), HSPG or FN were investigated using an in vitro cell adhesion assay. Co-incident with autonomous activation over the first 7 days, primary culture HSC increasingly expressed mRNA for CCN2 or integrin subunits. Addition of exogenous CCN2 or knockdown of endogenous CCN2 differentially regulated integrin gene expression in day 3 versus day 7 cells. Either full length CCN2 ('CCN2(1-4)') or residues 247-349 containing module 4 alone ('CCN2(4)') supported day 3 cell adhesion in an integrin alpha(5)beta(1)- and HSPG-dependent fashion. Adhesion of day 3 cells to FN was promoted in an integrin alpha(5)beta(1)-dependent manner by CCN2(1-4) or CCN2(4), whereas FN promoted HSPG-dependent HSC adhesion to CCN2(1-4) or CCN2(4). These findings suggest CCN2 regulates integrin expression in primary culture HSC and supports HSC adhesion via its binding of cell surface integrin alpha(5)beta(1), a novel CCN2 receptor in primary culture HSC which interacts co-operatively with HSPG or FN.