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J. Biol. Chem., Vol. 278, Issue 17, 15360-15372, April 25, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/17/15360    most recent M206790200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nieto, N. Articles by Cederbaum, A. I. Search for Related Content PubMed PubMed Citation Articles by Nieto, N. Articles by Cederbaum, A. I. Social Bookmarking                      What's this?

Increased Sp1-dependent Transactivation of the LAM1 Promoter in Hepatic Stellate Cells Co-cultured with HepG2 Cells Overexpressing Cytochrome P450 2E1^*

Natalia Nieto and Arthur I. Cederbaum

From the Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029

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 1 and 1 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 1 chain was not detectable in the HSC in any of the systems; however, laminin 2 chain increased in HSC co-cultured with E47 cells. Synthesis but not turnover of laminin 1 and 1 proteins was increased in HSC in the E47 co-culture. Laminin 1 and 1 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 1 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 1 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 330LAM1-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.

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