TNF-α modulates expression of the tissue transglutaminase gene in liver cells

Abstract

One of several postulated roles for tissue transglutaminase (tTG) is the stabilization and assembly of extracellular matrix via peptide cross-linking. We previously determined that tTG activity increased in an animal model of hepatic fibrogenesis and in human liver disease. To further study the role of tTG in liver disease, we initiated investigations into the effect of a proinflammatory mediator, tumor necrosis factor (TNF)-α, on tTG activity in cultured liver cells. Treatment of human Hep G2 cells with 1 ng/ml TNF-α increased [14C]putrescine cross-linking to cellular proteins. An increase in tTG mRNA content was observed 1 h after addition of TNF-α, and levels of tTG mRNA remained elevated after 24 h. Hep G2 cells, transiently transfected with a luciferase reporter containing 1.67 kb of the human tTG promoter, showed an increase in reporter activity after addition of TNF-α. Gel shift experiments using nuclear extracts from TNF-α-treated cells and oligonucleotides containing the tTG nuclear factor (NF)-κB motif revealed increased binding, concordant with mRNA data. Transient transfections with a truncated reporter construct lacking the tTG NF-κB sequence showed an attenuated response to TNF-α 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 β-galactosidase reporter, show similar time-dependent increases in promoter activity when treated with TNF-α. Furthermore, Hep G2 cells are incapable of upmodulating tTG promoter reporter activity in the presence of TNF-α when those cells overexpress a transdominant, negative mutant NF-κB subunit. Because TNF-α expression is upregulated in hepatic inflammation, the data suggest TNF-α-mediated increases in tTG expression may play an important role in the process of hepatic fibrogenesis.

Roles of Tissue Transglutaminase in Ethanol-induced Inhibition of Hepatocyte Proliferation and α1-Adrenergic Signal Transduction

“…We determined previously that tTG cross-linking activity is increased in animal models of acute liver injury and during fibrogenesis (7), and that it appears that TNF-␣ enhances the expression of tTG (8). In the present report, our results suggest that tTG may manifest two distinct and somewhat conflicting activities in hepatocytes depending on the cellular milieu.…”

“…The adrenergic agonist had no effect on proliferation or tTGase cross-linking activity in hepatoma-derived cell lines, despite the fact that the cell lines contained tTG mRNA (8). In an attempt to explain this result, we demonstrated that whereas hepatocytes had substantial amounts of ␣1BAR mRNA, the hepatoma cell lines lacked the mRNA for the receptors as determined by Northern blot analysis.…”

“…The hepatocytes seeded on 6-well Costar® plates were used for determination of tTGase activity. The method is based on incorporation of [1,4-14 C]putrescine dihydrocholoride (Amersham Pharmacia Biotech) into proteins and was described previously (8). The isotope was added to reach a concentration of 0.5 Ci/100 l of incubation buffer and the cell extracts were incubated with the isotope for 30 min.…”

“…Although its physiological significance has not been proven, tTG has been implicated to be involved in extracellular matrix organization (5) and in inhibition of cell growth and proliferation (6). We have demonstrated that its transglutaminase cross-linking activity is increased in humans with acute liver injury and in model systems of hepatic injury and fibrosis (7) and that tumor necrosis factor-␣ (TNF-␣) up-regulated tTG gene expression in the Hep G 2 cell line (8). In addition to its tissue transglutaminase cross-linking (tTGase) activity, tTG is unique in that it also is a GTP-binding protein, with GTPase activity referred to as G␣ h (9).…”

Post-translational Modification of the Hepatitis C Virus Core Protein by Tissue Transglutaminase

“…tTG Assay-The tTG activity was measured by analyzing the incorporation of [1,[4][5][6][7][8][9][10][11][12][13][14] C]putrescine dihydrochloride into proteins (31)(32)(33). Cells were treated with CalC or serum-deprived and then lysed in Tris-buffered saline containing 0.5% Nonidet P-40 and 1 mM phenylmethylsulfonyl fluoride.…”