Inhibitory guanine nucleotide regulatory protein activation of mitogen-activated protein kinase in experimental hepatocellular carcinoma in vitro.

OBJECTIVE: Hepatocellular carcinoma (HCC) is associated with altered expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the interaction between Gi-proteins and the extracellular regulated kinase (ERK) component of the mitogen activated protein kinase (MAPK) cascade in regulating mitogenesis in an experimental model of HCC. DESIGN: Pharmacological agents which selectively interact with specific target proteins involved in signal transduction through a Gi-MAPK pathway have recently become available. These agents in combination with scientific assays allow us to address the role of individual components of this cascade in the regulation of mitogenesis in HCC. METHODS: These studies were performed using rat hepatic tumorigenic cells (H4IIE) and isolated cultured hepatocytes in vitro in conjunction with pharmacological agents which interact with Gi-protein or MAPK components of intracellular signalling. RESULTS: Direct activation of Gi-proteins with mastoparan M7 (M7) significantly increased nuclear thymidine incorporation in hepatic tumorigenic H4IIE cells in a dose-dependent manner (10-1000 nM, n = 4, P < 0.05), an effect that was abolished by treatment with either pertussis toxin (PTx) or the selective mitogen-activated ERK-regulated kinase (MEK) inhibitor PD098059. In contrast, M7 inhibited nuclear thymidine incorporation in serum-stimulated isolated hepatocytes. ERK2 activity was then determined as the ability of immunoprecipitated ERK2 proteins to phosphorylate the ERK substrate myelin basic protein. These studies demonstrated a time- and dose-dependent increase in ERK2 activity in H4IIE cells following Gi-protein activation with M7, a maximal response being attained at 20 min. In contrast, M7 failed to significantly alter ERK2 activity in isolated cultured hepatocytes at any of the doses or time points assayed (10-5000 nM, 10-120 min). Gi-stimulated ERK activation was completely blocked in tumorigenic cells following treatment with PTx. CONCLUSIONS: These data demonstrate for the first time a Gi-linked MAPK cascade in experimental HCC, activation of which stimulates cellular mitogenesis.     

Interleukin-22 promotes human hepatocellular carcinoma by activation of STAT3

Interleukin-22 (IL-22), one of the cytokines secreted by T helper 17 (Th17) cells, was recently reported to be a novel inflammation driver through STAT3 signaling activation. We aimed to investigate the role of IL-22 expression in hepatocellular carcinoma (HCC). We demonstrated significant up-regulation of IL-22 in human HCC tumor infiltrated leukocytes (TILs) compared to peripheral lymphocytes. Moreover, IL-22 expression was significantly higher in Edmondson Grade III-IV HCC patients versus Grade I-II, confirmed by both real-time polymerase chain reaction and immunohistochemistry. Both IL-22 receptor α and IL-23 were highly expressed in HCC and adjacent cirrhotic tissues compared to normal controls. Enhanced tumor growth and metastasis was found in mice that underwent subrenal transplantation of MHCC-97H cells cotransplanted with IL-22+ TILs cells. STAT3 phosphorylation and up-regulation of downstream genes Bcl-2, Bcl-XL, CyclinD1, and vascular endothelial growth factor (VEGF) promoted tumor growth and metastasis. In vitro studies confirmed the tumor-promoting and antiapoptotic effect of IL-22, as well as IL-6. In the mouse chronic hepatitis and HCC model, sustained and increased IL-22 expression and STAT3 activation were found in liver tissues. A linear correlation was demonstrated between IL-22 expression and hepatic complementary proliferation. An in vivo diethyl-nitrosamine-induced mouse HCC model verified that tumor formation was significantly decreased in IL-22 knockout mice. Conclusion: Excessive IL-22 can be found in the HCC microenvironment, leading to tumor growth, inhibition of apoptosis, and promotion of metastasis due to STAT3 activation.     

Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat.

Upon liver injury, hepatic stellate cells (HSC) show increased proliferation, motility, and extracellular matrix (ECM) production. The extracellular signal-regulated kinases (ERK) control different functions in a cell-specific manner. In this study, we evaluated the role of ERK activation in cultured HSC stimulated with platelet-derived growth factor (PDGF) and after induction of liver injury in vivo. HSC were isolated from normal human liver tissue, cultured on plastic, and used in their myofibroblast-like phenotype. In in vivo experiments, HSC were isolated from normal rats or at different time points after a single intragastric administration of CCl(4). Nontoxic concentrations of PD98059, a specific inhibitor of ERK activation, reduced PDGF-induced activation of ERK in a dose-dependent fashion. Suppression of ERK activation was associated with complete inhibition of HSC proliferation and with a 57% reduction in chemotaxis. In the presence of the ERK inhibitor, binding of the AP-1 complex and of STAT1 to the related regulatory elements was inhibited. The inhibition of the DNA binding activity of STAT1 was mediated by a reduction in PDGF-induced tyrosine phosphorylation. Expression of c-fos in response to PDGF was also reduced, but not suppressed, by treatment with PD98059. In HSC isolated from CCl(4)-treated rats, ERK activity increased as early as 6 hours following liver damage, and declined thereafter. The results of this study indicate that ERK activation regulates proliferation and chemotaxis of HSC, and modulates nuclear signaling. Acute liver damage in vivo leads to activation of ERK in HSC.     

IL-37b suppresses epithelial mesenchymal transition in hepatocellular carcinoma by inhibiting IL-6/STAT3 signaling

Background: Interleukin-37 b(IL-37 b), a vital negative regulator of the innate immune system, has been reported to be a tumor inhibitor in different type of cancers. However, little is known about the relationship between IL-37 b and hepatocellular carcinoma(HCC). The present study aimed to investigate the potential roles of IL-37 b in HCC progression. Methods: Subjects( n = 237) were recruited, and serum IL-37 b was measured using ELISA. The tumorsuppressive capacity and underlying mechanisms of IL-37 b in HCC were investigated in vitro and in vivo. Results: Compared to healthy controls, serum IL-37 b levels were elevated in chronic hepatitis B(CHB) patients but decreased significantly in HBV-HCC patients, especially for those with portal venous tumor thrombus. Low level serum IL-37 b in HBV-HCC patients correlated with high HCC stage and poor overall survival and disease-free survival. In vitro and in vivo, recombinant human IL-37 b inhibited proliferation and metastasis in HCC cells. Furthermore, IL-37 b inhibited epithelial mesenchymal transition in HCC cells in vitro by downregulating IL-6, pSTAT3(Y705), N-cadherin, and vimentin expression and by upregulating E-cadherin expression. These effects were partially reversed by transfection of adenovirus encoding human IL-6. Conclusions: IL-37 b inhibits HCC growth, metastasis and epithelial mesenchymal transition by regulating IL-6/STAT3 signaling. Serum IL-37 b may be a biomarker for HBV-HCC and its staging.     

Chronic ethanol consumption enhances interleukin-1-mediated signal transduction in rat liver and in cultured hepatocytes

BACKGROUND: Interleukin-1 (IL-1) is a central mediator of the inflammatory process. Increased serum levels of IL-1 have been reported in alcoholics with liver damage, but it remains unknown whether chronic ethanol intake, in the presence or absence of lipopolysaccharide (LPS), activates IL-1 release and signaling in the hepatocyte. METHODS: IL-1beta and IL-10 release, expression of their receptors (IL-1RI and IL-10R), and the IL-1RI signal transduction response were evaluated in livers and cultured hepatocytes from ethanol-fed or pair-fed rats exposed in vivo or in vitro to LPS, ethanol, or both. RESULTS: Chronic ethanol intake increased both the serum levels of IL-1beta and IL-10 and the expression of IL-1RI, but not of IL-10R, in the liver microsomal fraction. In vivo LPS administration potentiated the ethanol-induced release of plasma cytokines. It is interesting to note that ethanol, either given in a single dose or chronically fed, stimulated IL-1beta and IL-10 release from cultured hepatocytes. Stimulation of hepatocytes with IL-1beta caused a higher activation of IL-1-associated kinase, extracellular receptor-activated kinases 1 and 2, and nuclear factor-kappaB (NF-kappaB) in hepatocytes from alcohol-fed animals than from controls. Furthermore, in the absence of any stimulation, hepatocytes from alcohol-fed animals showed an activation of both kinases, as well as an increase in NF-kappaB binding. Our results suggest the participation of the extracellular signal-regulated kinase (ERK)1/2 pathway in ethanol-induced NF-kappaB activation, because treatment with PD-98059, an ERK1/2 inhibitor, partially suppressed IL-1beta-induced NF-kappaB expression. CONCLUSIONS: Chronic ethanol intake potentiates the action of the proinflammatory cytokine IL-1beta, enhancing the release and signaling response of IL-1beta in the hepatocyte, which in conjunction with other cytokines or LPS may exacerbate the inflammatory damage associated with alcoholic liver disease.     

Lack of gp130 expression in hepatocytes promotes liver injury

BACKGROUND & AIMS: Interleukin 6 (IL-6) contributes via its signal transducer gp130 to the acute phase response (APR) in hepatocytes. Recent studies indicated that IL-6 is involved in the regulation of different pathophysiologic conditions of the liver. To define the IL-6-dependent intracellular pathways more specifically, we generated a hepatocyte-specific gp130 knockout mouse. METHODS: Hepatocyte-specific gp130-deficient mice were generated using the Cre-loxP system. Expression of the Cre recombinase was under the control of a hepatocyte-specific control element. Adult mice were challenged with IL-6, oncostatin M (OSM), and LPS. RESULTS: Cre expression started at day 10.5 postconception, and a complete deletion of gp130 in hepatocytes was found at day 14 during liver development. The adult liver of these mice showed no abnormalities; however, after IL-6 and OSM stimulation, gp130-dependent pathways (STAT3, APR gene expression) were completely blocked in the liver of these animals. Additionally, challenging hepatocyte-specific gp130 knockout animals with lipopolysaccharides (LPS) lead to an onset of acute liver injury with an increase of hepatocyte apoptosis associated with elevated tumor necrosis factor alpha (TNF-alpha) serum levels and reduced nuclear factor kappaB (NF-kappaB) activation in hepatocytes. CONCLUSIONS: Our findings demonstrate that gp130 is of minor relevance for embryonal development of hepatocytes. However, the molecule has an essential role in controlling acute phase gene expression and provides hepatocellular protection after LPS challenge.     

Ghrelin对脓毒症小鼠肺脏炎症及JAK/STAT通路的影响

目的 通过观察生长素释放肽(ghrelin)对脓毒症小鼠肺组织炎症反应、肺组织janus激酶/信号转导通路和转录激活因子mRNA、STAT3蛋白及肺组织炎细胞因子肿瘤细胞α(TNF-α)、IL-6的表达水平的影响,探讨ghrelin在脓毒症炎症反应中的调节作用及可能的分子机制.方法 选用腹腔注射脂多糖的方法制作小鼠脓毒性模型:选取雌性小鼠54只,随机分为对照组、模型组及ghrelin干预组,对照组经腹腔注射等量生理盐水;模型组经腹腔内注射脂多糖(6 mg/kg);干预组先经腹腔注射ghrelin(200 μg/kg),30 min后再经腹腔注射脂多糖(6 mg/kg);各组分别于3、9、18h随机处死小鼠各6只,光镜下观察肺组织炎症改变;反转录聚合酶链反应(RT-PCR)法检查肺组织STAT3mRNA的基因转录水平;酶联免疫吸附测定(ELISA)法测定肺组织STAT3、TNF-α、IL-6的表达量.结果 Ghrelin可抑制肺组织STAT3mRNA基因转录活性,从而降低STAT3蛋白表达、减少炎症因子TNF-α、IL-6的分泌(P值均＜0.05);改善脓毒症小鼠肺组织病理结构损伤(充血、水肿、炎症细胞浸润).结论 ghrelin可减轻脓毒症小鼠肺脏炎症反应,其作用机制可能与抑制了janus激酶/信号转导和转录激活因子(JAK/STAT通路)、下调TNF-α和IL-6的表达有关.     

Insulin-like growth factor I is a comitogen for hepatocyte growth factor in a rat model of hepatocellular carcinoma

Hepatocyte growth factor-scatter factor (HGF-SF) is a potent hepatic mitogen yet inhibits hepatocellular carcinoma (HCC) cell growth in vitro. Insulin-like growth factor I (IGF-I) is a pleiotropic growth factor shown to be important in cell growth and differentiation in other tumors. We hypothesized that IGF-I may play a role in regulating HGF-SF activity and HCC progression. Using an in vivo model of HCC, we showed elevated IGF-I messenger RNA (mRNA) expression in normal liver from tumor-burdened animals in the absence of changes in circulating IGF-I levels. Analysis of IGF-I receptor (IGF-IR) and HGF-SF (c-met) receptor expression showed significantly higher expression of both receptors in normal liver compared with an HCC specimen. Using cultured HCC cells from this model, we next showed that treatment with IGF-I led to significant increases in mitogen-activated protein kinase (MAPK) activity. Furthermore, we observed significant time-dependent increases in the expression of the c-fos and c-jun proto-oncogenes after addition of IGF-I (n = 5 per group, P <.05). Despite activation of a MAPK pathway and increased proto-oncogene expression, IGF-I failed to significantly affect cell mitogenesis. In contrast, HGF significantly inhibited cell mitogenesis in HCC lines (68.4% +/- 9.4% vs. control, n = 4, P <.05). Pretreatment of HCC cells with IGF-I (60 minutes) led to significant HGF-SF stimulation of total cell mitogenesis dependent on both IGF-I and HGF-SF dose (194% +/- 8% increase vs. control, n = 4, P <.05). In conclusion, tumor burden is important in altering intrahepatic growth factor synthesis. Signal cooperation between multiple cytokine pathways is an important factor in the progression of HCC.