Increased Sp1-dependent transactivation of the LAMgamma 1 promoter in hepatic stellate cells co-cultured with HepG2 cells overexpressing cytochrome P450 2E1.
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.
Nieto
Natalia
N
Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029, USA. ny2000@hotmail.com
Cederbaum
Arthur I
AI
D000818:Animals; D018920:Coculture Techniques; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D007797:Laminin / Q000096:biosynthesis* / Q000235:genetics; D008103:Liver Cirrhosis / Q000209:etiology; D008297:Male; D018384:Oxidative Stress / Q000235:genetics; D019899:Paracrine Communication; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D016329:Sp1 Transcription Factor / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D007797:Laminin; D016329:Sp1 Transcription Factor; C089141:laminin gamma 1; D019392:Cytochrome P-450 CYP2E1