Activation of the IGF-II gene by HBV-X protein requires PKC and p44/p42 map kinase signalings.

We have recently shown that HBx protein, one of the causative agents of hepatocellular carcinomas, regulates Sp1 mediated transcription of insulin-like growth factor II promoter 4 (Lee et al. (1998) Oncogene 16, 2367-2380). Here we show that PKC and p44/p42MAPK signalings are required for the HBx-induced Sp1-mediated IGF-II P4 transcriptional activity since (i) PKC activation by PMA or PKC expression vector increases Sp1 phosphorylation and P4 activity in HBx-transfected HepG2 cells; (ii) PKC inhibition by PKC inhibitor Gö6976 reduces Sp1 phosphorylation, P4 activity, and IGF-II mRNA in HBx-transfected HepG2 cells; and (iii) the inhibition of MEK activation by U0126 reduces Sp1 phosphorylation, P4 activity and IGF-II mRNA in HBx-transfected HepG2 cells. These results demonstrate that PKC and p44/p42 MAPK cascades are the essential signaling pathways in Sp1-mediated IGF-II gene activation by HBx.

The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade.

The hepatitis B virus-X (HBx) protein is known as a multifunctional protein that not only coactivates transcription of viral and cellular genes but coordinates the balance between proliferation and programmed cell death, by inducing or blocking apoptosis. In this study the role of the HBx protein in activation of phosphatidylinositol 3-kinase (PI3K) was investigated as a possible cause of anti-apoptosis in liver cells. HBx relieved serum deprivation-induced and pro-apoptic stimuli-induced apoptosis in Chang liver (CHL) cells. Treatment with 1-d-3-deoxy-3-fluoro-myo-inositol, an antagonist to PI3K, which blocks the formation of 3'-phosphorylated phosphatidyl inositol in CHL cells transformed by HBx (CHL-X) but not normal Chang liver (CHL) cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI3K, stimulated apoptosis in HBx-transformed CHL cells but not in normal cells, confirming that HBx blocks apoptosis through the PI3K pathway. The serine 47 threonine kinase, Akt, one of the downstream effectors of PI3K-dependent survival signaling was 2-fold higher in HBx-transformed CHL (CHL-X) cells than CHL cells. Phosphorylation of Akt at serine 473 and Bad at serine 136 were induced by HBx, which were specifically blocked by wortmannin and dominant negative mutants of Akt and Bad, respectively. We also demonstrated that HBx inhibits caspase 3 activity and HBx down-regulation of caspase 3 activity was blocked by the PI3K inhibitor. Regions required for PI3K phosphorylation on the HBx protein overlap with the known transactivation domains. HBx blocks apoptosis induced by serum withdrawal in CHL cells in a p53-independent manner. The results indicate that, unlike other DNA tumor viruses that block apoptosis by inactivating p53, the hepatitis B virus achieves protection from apoptotic death through a HBx-PI3K-Akt-Bad pathway and by inactivating caspase 3 activity that is at least partially p53-independent in liver cells. Moreover, these data suggest that modulation of the PI3K activity may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in human hepatocellular carcinoma.

Role for PKC alpha and PKC epsilon in down-regulation of CFTR mRNA in a human epithelial liver cell line.

BACKGROUND/AIMS: In the liver, intrahepatic biliary cells are the sole site of expression of the cystic fibrosis transmembrane conductance regulator, the product of the cystic fibrosis gene. We examined the regulation of cystic fibrosis transmembrane conductance regulator gene expression by protein kinase C in the recently characterized human liver epithelial BC1 cell line which expresses, at early confluence, both biliary (cystic fibrosis transmembrane conductance regulator, cytokeratin 19) and hepatocytic (albumin) specific markers. METHODS: Expression of the cystic fibrosis transmembrane conductance regulator was examined at the mRNA level by Northern blot, reverse transcription-polymerase chain reaction and nuclear run-on assays and at the protein level by Western blotting. The functionality of this protein was tested by measurement of chloride efflux. Protein kinase C isotype expression and cytosol-to-membrane translocation were analysed by Western blotting. RESULTS: 1) Phorbol ester down-regulated cystic fibrosis transmembrane conductance regulator mRNA expression in a time- and dose-dependent manner through a post-transcriptional mechanism with concomitant inhibition of stimulated chloride efflux. 2) Phorbol ester also activated protein kinase C as indicated by the cytosol-to-membrane translocation of both protein kinase C alpha and epsilon the two major protein kinase C isotypes expressed by BC1 cells. 3) Further, maximal down-regulation of the cystic fibrosis transmembrane conductance regulator mRNA by the phorbol ester was inhibited by H7 and by GF 109203X, two known protein kinase C inhibitors. CONCLUSIONS: These findings provide the first evidence for phorbol ester-induced down-regulation of cystic fibrosis transmembrane conductance regulator mRNA expression in a human liver epithelial cell line and point to a role for the classical protein kinase C alpha and the novel protein kinase C epsilon in this process.

Evidence for a role of insulin in hepatocytic differentiation of human hepatoma BC1 cells.

To examine the effect of insulin on hepatocytic differentiation, we took advantage of the properties of the newly established human hepatoma BC1 cell line to maintain quiescence after confluency and to progressively acquire in culture (3 weeks after confluency) an hepatocytic phenotype, as assessed by expression of specific hepatic genes (Le Jossicet al., 1995). In BC1 cells cultured in the presence of insulin (1 µM: ), expression of albumin and transferrin mRNA and protein occurs earlier than in cells cultured in its absence (1 weekvs 2 weeks). Moreover, at any time considered, the level of the two hepatic markers was higher (2- to 3-fold) in the former than in untreated cells. The beneficial effect of insulin on hepatocytic differentiation of BC1 cells was paralleled by: i) modest increases in insulin receptor (IR) mRNA level and IR binding activity, and ii) a 6-fold increase in sensitivity to insulin for stimulation of glycogenesis. These results provide the first evidence for insulin's ability to exert a positive effect on hepatocytic differentiation. The beneficial effect of insulin probably results both from increased IR expression and binding activity and from alteration at post-receptor levels.