This study was undertaken to delineate a possible role for tissue transglutaminase (tTG), an enzyme that catalyzes protein cross-linking, in hepatic fibrogenesis. Rats were treated with CCl4 solution and then killed at different stages of liver injury and fibrogenesis. Liver tTG mRNA levels were markedly increased as early as 6 h after the first injection, peaked at 4 days and 1 wk, and remained increased for 8 wk. The enzymatic activity of tTG was increased in livers of rats treated with CCl4, in a fashion that paralleled the Northern blot results. Cell isolation experiments indicated that all hepatic cell types synthesize tTG mRNA. Increased binding to the nuclear factor-kappaB (NF-kappaB) motif of the tTG promoter was found in the nuclear extracts prepared from CCl4-treated samples. These data demonstrate an increase in tTG gene expression during hepatic injury and fibrosis, suggesting a possible role for this enzyme in stabilizing the fibrotic bands during hepatic fibrogenesis. Moreover, increased NF-kappaB binding to the tTG promoter may represent one of the mechanisms by which cell injury induces tTG transcription and thus potentiates the process of fibrogenesis.
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.
SPARC (secreted protein, acidic and rich in cysteine)--also known as osteonectin, BM-40, and 43K glycoprotein--is secreted by endothelial cells and fibroblasts in response to culture shock. SPARC has been found in association with tissues undergoing cell proliferation, migration, and extracellular matrix remodeling. We demonstrate that normal livers from humans, rats, and mice express substantial levels of SPARC messenger RNA (mRNA). Moreover, when compared with control specimens, significantly increased levels of SPARC mRNA were found in fibrotic livers from two animal models of liver disease: murine schistosomiasis and carbon tetrachloride-induced fibrosis in rats. Fibrotic human livers also had markedly increased levels of SPARC mRNA in comparison with normal livers. We also detected an increased production of SPARC protein in the liver of animals treated with carbon tetrachloride. By immunocytochemical analysis, SPARC protein was apparent in freshly isolated Ito cells. Hybridization studies showed Ito cells to be the main source of SPARC mRNA. Extracts from a Kupffer-endothelial cell fraction exhibited traces of SPARC transcript, but expression of SPARC mRNA was absent in extracts from freshly isolated hepatocytes. These studies demonstrate the increased expression of SPARC--a protein that modulates cell shape and disrupts cell-matrix interactions--during the initial stages of hepatic fibrosis.
The mechanisms by which ethanol inhibits hepatocyte proliferation have been a source of some considerable investigation. Our studies have suggested a possible role for tissue transglutaminase (tTG) in this process. Others have shown that tTG has two distinctly different functions: it catalyzes protein cross-linking, which can lead to apoptosis and enhancement of extracellular matrix stability, and it can function as a G protein (G␣ h ). Under that circumstance, we speculated that the cross-linking activity would be decreased and that it would function to enhance hepatocyte proliferation in response to adrenergic stimulation. Ethanol treatment inhibited hepatocyte proliferation and led to enhanced tTG crosslinking activity, whereas treatment of hepatocytes with an ␣1 adrenergic agonist, phenylephrine, enhanced hepatocyte proliferation while decreasing tTG cross-linking. However, phenylephrine treatment of several hepatoma cell lines had no effect on cellular proliferation or tTG cross-linking activity, and of note, Northern blot analysis demonstrated that whereas primary hepatocytes had high levels of the ␣1 adrenergic receptor (␣1BAR) mRNA, the hepatoma cell lines did not have this mRNA. When the Hep G 2 cell line was stably transduced with an expression vector containing the ␣1BR cDNA, the cell line responded to phenylephrine treatment with enhanced proliferation and with decreased tTG cross-linking activity. Ethanol treatment of the ␣1BAR-transfected cells suppressed the phospholipase C-mediated signaling pathways, as detected in the phenylephrine-induced Ca 2؉ response. These results suggest that phenylephrine stimulation of hepatocyte proliferation appears to be occurring through the ␣1BAR, which is known to be coupled with the tTG G protein moiety, G␣ h , and that tTG appears to play a significant role in either enhancing or inhibiting hepatocyte proliferation, depending on its cellular location and on whether it functions as a cross-linking enzyme or a G protein.
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