Laminin is a basement-membrane
protein that increases in
liver fibrosis. To study the role of oxidative stress on
laminin expression, hepatic stellate cells (HSC) were co-cultured with HepG2 cells that do or do not express (E47 or C34 cells, respectively)
CYP2E1, a potent generator of
oxygen radicals. Co-incubation of HSC with E47 cells increased
laminin beta1 and gamma1
proteins compared with co-incubation with C34 cells; this increase was prevented by
antioxidants and
CYP2E1 inhibitors. Similar results were observed in co-culture with primary hepatocytes from saline- or
pyrazole-treated (with high levels of
CYP2E1) rats.
Laminin alpha1 chain was not detectable in the HSC in any of the systems; however,
laminin alpha2 chain increased in HSC co-cultured with E47 cells. Synthesis but not turnover of
laminin beta1 and gamma1
proteins was increased in HSC in the E47 co-culture.
Laminin beta1 and gamma1 mRNAs were up-regulated in HSC in the E47 co-culture because of transcriptional activation of both genes. Transfection experiments in HSC with reporter constructs driven by the
laminin gamma1 promoter showed maximal responsiveness with the -230/+106 and the -1400/+106 constructs in the E47 system. Gel-shift assays demonstrated an increase in Sp1 binding to the
laminin gamma1 promoter in HSC when co-incubated with E47 cells, which was blocked by an anti-Sp1 antibody. Co-transfection of a Sp1 expression vector further increased the responsiveness of the -330LAMgamma1-CAT reporter vector in HSC in the HSC/E47 system. These results show that diffusable CYP2E1-derived oxidative-stress mediators induce synthesis of laminins by a transcriptional mechanism in HSC. Such interactions between hepatocytes and HSC may be important during
liver fibrosis.