TNF-ALPHA MODULATES EXPRESSION OF THE TISSUE TRANSGLUTAMINASE GENE INLIVER-CELLS

One of several postulated roles for tissue transglutaminase (tTG) is t he stabilization and assembly of extracellular matrix via peptide cros s-linking. We previously determined that tTG activity increased in an animal model of hepatic fibrogenesis and in human liver disease. To fu rther study the role of tTG in liver disease, we initiated investigati ons into the effect of a proinflammatory mediator, tumor necrosis fact or (TNF)-alpha, on tTG activity in cultured liver cells. Treat ment of human Hep G2 cells with 1 ng/ml TNF-alpha increased [C-14]putrescine cross-linking to cellular proteins. An increase in tTG mRNA content wa s observed 1 h after addition of TNF-alpha, and levels of tTG mRNA rem ained elevated after 24 h. Hep G2 cells, transiently transfected with a luciferase reporter containing 1.67 kb of the human tTG promoter, sh owed an increase in reporter activity after addition of TNF-alpha. Gel shift experiments using nuclear extracts from TNF-alpha-treated cells and oligonucleotides containing the tTG nuclear factor (NF)-kappa B m otif revealed increased binding, concordant with mRNA data. Transient transfections with a truncated reporter construct lacking the tTG NF-k appa B sequence showed an attenuated response to TNF-alpha treatment. Similar responses were seen in stably transfected HeLa cells. Primary hepatocytes isolated from a trangenic mouse Line containing the mouse tTG promoter driving the beta-galactosidase reporter, show similar tim e-dependent increases in promoter activity when treated with TNF-alpha . Furthermore, Hep G2 cells are incapable of upmodulating tTG promoter reporter activity in the presence of TNF-alpha when those cells overe xpress a transdominant, negative mutant NF-kappa B subunit. Because TN F-alpha expression is upregulated in hepatic inflammation, the data su ggest TNF-alpha-mediated increases in tTG expression may play an impor tant role in the process of hepatic fibrogenesis.