Stress-activated protein kinases in the activation of rat hepatic stellate cells in culture.

BACKGROUND/AIMS: The signal cascades involved in the activation of hepatic stellate cells (HSC) are largely unknown. Factors initiating activation include tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, endothelin, and oxidative stress. In other cell types some of these have been reported to stimulate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). We have therefore investigated the role of these kinases in HSC activation. METHODS: HSC were isolated from male Wistar rats. Quiescent experiments were performed on day 2 HSC and transformed experiments on day 15 passage 1 HSC. Kinase activities were determined by immunoprecipitation and phosphorylation of specific substrate proteins and alpha-smooth muscle actin (SMA) expression by immunoblotting. RESULTS: The constitutive activity of p38 MAP kinase was higher in transformed versus quiescent cells. In quiescent cells TNFalpha stimulated p38 MAP kinase and JNK activities 12- and 4-fold respectively and this was halved by 2-mercaptoethanol, an indirect antioxidant. Endothelin-1 activated both kinases in quiescent cells via the endothelin-B receptor, while TGFbeta had no effect. Both 2-mercaptoethanol and a p38 inhibitor (SB202190) inhibited alpha-SMA expression by day 5 cells. CONCLUSIONS: The activation of p38 MAP kinase and JNK by TNFalpha and endothelin, together with the inhibition of this activation by 2-mercaptoethanol, provides indirect evidence supporting their role in HSC transformation. Direct evidence for a role for p38 MAP kinase is provided by the observations that its constitutive activity is higher in transformed versus quiescent cells and that its inhibitor reduces HSC activation in culture as assessed by alpha-SMA expression.

The role of phosphatidic acid in platelet-derived growth factor-induced proliferation of rat hepatic stellate cells.

Platelet-derived growth factor (PDGF) is the most potent mitogen for hepatic stellate cells (HSCs) in vitro. The aim of this study was to investigate the role of the lipid-derived second messenger phosphatidic acid (PA) in mediating this effect and, in particular, to determine its interaction with the extracellular signal-regulated kinase (ERK) cascade. HSCs were isolated from rat livers. PA production was determined by lipid extraction and thin-layer chromatography (TLC) after prelabeling cells with [(3)H]myristate. ERK activity was measured by an in vitro kinase assay after immunoprecipitation. Mitogenic concentrations of PDGF, but not those of the relatively less potent mitogen, transforming growth factor alpha (TGF-alpha), stimulated the sustained production of PA from HSCs. Exogenous PA stimulated HSC proliferation and a sustained increase in ERK activity, and proliferation was completely blocked by the inhibition of ERK activation with PD98059. The stimulation of ERK by PDGF was of a similar magnitude but more sustained than that caused by TGF-alpha. These results suggest that the potent mitogenic effect of PDGF in HSCs may be caused, in part, by the generation of PA and subsequently by a more sustained activation of ERK than occurs with less potent mitogens that do not induce the production of this lipid second messenger.

Macrophage and hepatic stellate cell responses during experimental hepatocarcinogenesis.

The aim of the study was to assess the monocyte/macrophage and hepatic stellate cell responses during experimental diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Diethylnitrosamine (50mg/L) was administered to 39 rats for 10 weeks; liver tissue was obtained at weeks 10, 16 and 19. In this model, necroinflammatory damage occurs during the period of DEN administration but thereafter subsides; dysplastic nodules and carcinomas subsequently develop. Monocytes/ macrophages were detected immunohistochemically using ED1 and ED2 monoclonal antibodies; hepatic stellate cells (HSC) were detected using antibodies to alpha-smooth muscle actin (alpha-SMA) (activated HSC) and glial fibrillary acidic protein (GFAP). Parenchymal ED1- and ED2-positive monocytes/macrophages and alpha-SMA-positive HSC increased at week 10 when there was ongoing DEN-induced necroinflammatory activity. ED1- and ED2-positive cells were also prominent at weeks 16 and 19, particularly around the periphery of dysplastic and carcinomatous nodules, with occasional macrophages between dysplastic hepatocytes. alpha-SMA-positive HSC were present within sinusoids between dysplastic cells and were more abundant at weeks 16 and 19 than in control or week 10 animals. Activated HSC were prominent in fibrous septa around and within dysplastic and carcinomatous nodules at weeks 16 and 19. In contrast, GFAP-positive HSC did not accumulate in developing septa or within dysplastic and carcinomatous nodules. We have demonstrated changes in the monocyte/ macrophage and HSC populations during the development of hepatocellular dysplasia and carcinoma at time points when there is little necroinflammatory activity; this may therefore represent a host response to hepatocyte dysplasia. The HSC activation may be mediated, in part, by monocyte/ macrophage-derived factors, but we speculate that it may also result from direct stimulation by factors released from dysplastic hepatocytes.