Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue...

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 α(5)β(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 α(5), β(1), αv or β(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 α(5)β(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 α(5)β(1) - and HSPG-dependent fashion. Adhesion of day 3 cells to FN was promoted in an integrin α(5) β(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 α(5)β(1), a novel CCN2 receptor in primary culture HSC which interacts co-operatively with HSPG or FN.