Growth Hormone Signaling Pathway Leading to the Induction of DNA Synthesis and Proliferation in Primary Cultured Hepatocytes of Adult Rats

Background: We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes. Methods: Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams’ medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting. Results: Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10−6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10−6 M), RTK (AG538, 10−6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10−6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10−6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis. Conclusions: The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

Effects of medicinal plant ipe on expression of inducible nitric oxide synthase in inerleukin-1β-stimulated Hepatocytes

Background: The traditional medicine ipe is thought to have various pharmacological actions including anticancer and anti-inflammatory activities. However, there is little scientific evidence to demonstrate the organ-protective effects of ipe. The prevention of nitric oxide (NO) production in inflamed livers by inducible NO synthase (iNOS) is an indicator of liver protection. We examined proinflammatory cytokine-stimulated hepatocytes as a simple “in vitro liver injury model” to determine ipe’s liver-protective effects of ipe and clarify its mechanisms. Methods: Primary cultured hepatocytes were treated with interleukin (IL)-1β in the presence or absence of ipe. The induction of iNOS and its signal pathway were analyzed. Results: Ipe inhibited the production of NO stimulated by IL-1β and showed the greatest effect (more than 90% inhibition) at 2 mg/ml. Ipe decreased iNOS protein and mRNA expression. Ipe decreased NF-κB activation (its translocation to the nucleus and DNA binding), although there was no effect on IκBα degradation. Ipe inhibited Akt activation, followed by decreased the type I IL-1 receptor mRNA and protein levels. Transfection experiments revealed that ipe decreased both activities of iNOS promoter transactivation and mRNA stability. In support of the latter observation, ipe inhibited the expression of the antisense transcript of the iNOS gene. Conclusion: Ipe blocked IκB kinase and phosphatidylinositol 3-kinase/Akt signal pathways, which caused the reduction of iNOS mRNA synthesis and its stability. This resulted in the inhibition of iNOS induction and NO production. Ipe may have a potent beneficial effect against NO-mediated injury in organs including the liver. Key words: ipe, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, nuclear factor-κB, the type I interleukin-1 receptor, iNOS antisense transcript

Genetic dissection of Rift Valley fever pathogenesis:Rfvs2on mouse chromosome 11 enables survival to acute-onset hepatitis

The systemic inoculation of mice with Rift Valley fever virus (RVFV) reproduces major pathological features of severe human disease, notably the acute-onset hepatitis and delayed-onset encephalitis. We previously reported that a genomic interval(Rvfs2)derived from the susceptible MBT/Pas strain is associated with reduced survival time after RVFV infection. In this study, we investigated the pathophysiological mechanisms by whichRvfs2confers increased susceptibility to BALB/c mice that are congenic forRvfs2(C.MBT-Rvfs2) after infection with virulent RVFV. Clinical traits, biochemical parameters, and histopathological features indicated similar liver damage in BALB/c and C.MBT-Rvfs2mice between the third and fifth days after infection. However, C.MBT-Rvfs2mice died at that point from acute liver injury while most BALB/c mice recovered from this condition but eventually died of encephalitis. We observed that hepatocytes proliferated actively within the infected liver of BALB/c mice on the sixth day after infection, promoting organ regeneration on the eighth day after infection and recovery from liver damage. We found that the production of infectious virions was up to 100-fold lower in the peripheral blood and liver of BALB/c compared to C.MBT-Rvfs2mice. Likewise, RVFV protein amounts were much lower in BALB/c liver compared to C.MBT-Rvfs2liver. Primary cultured hepatocytes showed higher viral replication rate in C.MBT-Rvfs2which could contribute to the susceptibility conferred byRvfs2. Using bone marrow chimera experiments, we uncovered that both hematopoietic and non-hematopoietic cells are required for the BALB/c allele ofRvfs2to exert its protective effects against the RVFV-induced acute liver disease. Taken together, we have established thatRvfs2acts as an important RVFV restriction factor by limiting virus multiplication in mice.Author SummaryRift Valley fever (RVF) is a mosquito-borne viral disease with potential to generate a public health emergency. The wide variation in RVF symptoms and severity observed within patient population suggests that natural host genetic determinants, among other factors, can influence the disease outcome. Infection of mice mimics several features of the pathology in humans, including acute-onset hepatitis and delayed-onset encephalitis. BALB/c inbred mice bearing the BALB/c haplotype at theRvfs2locus survive longer than those bearing the MBT haplotype. In this study, we investigated clinical traits, biochemical parameters, virological evidence, and histological features to characterize the pathogenesis of RVF in early and late susceptible mice. We show that animals of both groups develop acute liver disease shortly after infection. We demonstrate that, by comparison with early susceptible mice, BALB/c mice exhibit significantly reduced replication of RVF virusin vivoin the blood and liver andin vitroin primary cultured hepatocytes, and eventually self-recover from the liver damages. We use reciprocal transplantations of bone marrow cells between early and late susceptible mice to show that survival to severe liver disease requires both hematopoietic and non-hematopoietic cells. Taken together, we establishRvfs2as a single locus that enables mice to survive RVF virus-induced liver disease.