cyclin-d1 and Carbon-Tetrachloride-Poisoning

Plasminogen activator inhibitor-1 (PAI-1) is an acute phase protein that has been shown to play a role in experimental fibrosis caused by bile duct ligation (BDL) in mice. However, its role in more severe models of hepatic fibrosis (e.g., carbon tetrachloride; CCl(4)) has not been determined and is important for extrapolation to human disease. Wild-type or PAI-1 knockout mice were administered CCl(4) (1 ml/kg body wt ip) 2x/wk for 4 wk. Plasma (e.g., transaminase activity) and histological (e.g., Sirius red staining) indexes of liver damage and fibrosis were evaluated. Proliferation and apoptosis were assessed by PCNA and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively, as well as by indexes of cell cycle (e.g., p53, cyclin D1). In contrast to previous studies with BDL, hepatic fibrosis was enhanced in PAI-1(-/-) mice after chronic CCl(4) administration. Indeed, all indexes of liver damage were elevated in PAI-1(-/-) mice compared with wild-type mice. This enhanced liver damage correlated with impaired hepatocyte proliferation. A similar effect on proliferation was observed after one bolus dose of CCl(4), without concomitant increases in liver damage. Under these conditions, a decrease in phospho-p38, coupled with elevated p53 protein, was observed; these results suggest impaired proliferation and a potential G(1)/S cell cycle arrest in PAI-1(-/-) mice. These data suggest that PAI-1 may play multiple roles in chronic liver diseases, both protective and damaging, the latter mediated by its influence on inflammation and fibrosis and the former via helping maintain hepatocyte division after an injury.

Topics: Animals; Apoptosis; Carbon Tetrachloride Poisoning; Cell Proliferation; Cyclin D1; Hepatocytes; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Plasminogen Activator Inhibitor 1; Tissue Inhibitor of Metalloproteinase-1; Tumor Suppressor Protein p53

Liver injury initiated by non-lethal doses of CCl(4) and thioacetamide (TA) progresses to hepatic failure and death of type 2 diabetic (DB) rats due to failed advance of liver cells from G(0)/G(1) to S-phase and inhibited tissue repair. Objective of the present study was to investigate cellular signaling mechanisms of failed cell division in DB rats upon hepatotoxicant challenge. In CCl(4)-treated non-diabetic (non-DB) rats, increased IL-6 levels, sustained activation of extracellular regulated kinases 1/2 (ERK1/2) MAPK, and sustained phosphorylation of retinoblastoma protein (p-pRB) via cyclin D1/cyclin-dependent kinase (cdk) 4 and cyclin D1/cdk6 complexes stimulated G(0)/G(1) to S-phase transition of liver cells. In contrast to the non-DB rats, CCl(4) administration led to lower plasma IL-6, decreased ERK1/2 activation, lower cyclin D1, and cdk 4/6 expression resulting in decreased p-pRB and inhibition of liver cell division in the DB rats. Furthermore, higher TGFbeta1 expression and p21 activation may also contribute to decreased p-pRB in DB rats compared to non-DB rats. Similarly, after TA administration to DB rats, down-regulation of cyclin D1 and p-pRB leads to markedly decreased advance of liver cells from G(0)/G(1) to S-phase and tissue repair compared to the non-DB rats. Hepatic ATP levels did not differ between the DB and non-DB rats obviating its role in failed tissue repair in the DB rats. In conclusion, decreased p-pRB may contribute to blocked advance of cells from G(0)/G(1) to S-phase and failed cell division in DB rats exposed to CCl(4) or TA, leading to progression of liver injury and hepatic failure.

Topics: Adenosine Triphosphate; Animals; Carbon Tetrachloride Poisoning; Cell Cycle; Chemical and Drug Induced Liver Injury; Cyclin D1; Cyclin-Dependent Kinases; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Immunoblotting; Interleukin-6; Liver Diseases; Male; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Rats; Rats, Sprague-Dawley; Retinoblastoma Protein; Thioacetamide; Transforming Growth Factor beta1

Smad family proteins Smad2 and Smad3 are activated by transforming growth factor beta (TGF-beta)/activin/nodal receptors and mediate transcriptional regulation. Although differential functional roles of Smad2 and Smad3 are apparent in mammalian development, the relative functional roles of Smad2 and Smad3 in postnatal systems remain unclear. We used Cre/loxP-mediated gene targeting for hepatocyte-specific deletion of Smad2 (S2HeKO) in adult mice and generated hepatocyte-selective Smad2/Smad3 double knockouts by intercrossing AlbCre/Smad2(f/f) (S2HeKO) and Smad3-deficient Smad3ex8/ex8 (S3KO) mice. All strains were viable and had normal adult liver. However, necrogenic CCL4-induced hepatocyte proliferation was significantly increased in S2HeKO compared to Ctrl and S3KO livers, and transplanted S2HeKO hepatocytes repopulated recipient liver at dramatically increased rates compared to Ctrl hepatocytes in vivo. Using primary hepatocytes, we found that TGF-beta-induced G1 arrest, apoptosis, and epithelial-to-mesenchymal transition in Ctrl and S2HeKO but not in S3KO hepatocytes. Interestingly, S2HeKO cells spontaneously acquired mesenchymal features characteristic of epithelial-to-mesenchymal transition (EMT). Collectively, these results demonstrate that Smad2 suppresses hepatocyte growth and dedifferentiation independent of TGF-beta signaling. Smad2 is not required for TGF-beta-stimulated apoptosis, EMT, and growth inhibition in hepatocytes.

Topics: Animals; Apoptosis; Carbon Tetrachloride Poisoning; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cyclin D1; Hepatocytes; Liver; Mesoderm; Mice; Mice, Knockout; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta