Bi Zhang

Transforming growth factor beta signaling in hepatocytes participates in steatohepatitis through regulation of cell death and lipid metabolism in mice

Transforming growth factor beta (TGF‐β) signaling activates Smad‐ and TGF‐β‐activated kinase 1 (TAK1)‐dependent signaling to regulate cell survival, proliferation, fibrosis, and tumorigenesis. The effects of TGF‐β signaling on metabolic syndrome, including nonalcoholic fatty liver disease, remain elusive. Wild‐type (WT) and hepatocyte‐specific TGF‐β receptor type II‐deficient (Tgfbr2ΔHEP) mice were fed a choline‐deficient amino acid (CDAA)‐defined diet for 22 weeks to induce NASH. WT mice fed a CDAA diet displayed increased activation of Smad2/3 and had marked lipid accumulation, inflammatory cell infiltration, hepatocyte death, and fibrosis; in comparison, Tgfbr2ΔHEP mice fed a CDAA diet had suppressed liver steatosis, inflammation, and fibrosis. Both palmitate‐induced steatotic hepatocytes and hepatocytes isolated from WT mice fed a CDAA diet had increased susceptibility to TGF‐β‐mediated death. TGF‐β‐mediated death in steatotic hepatocytes was inhibited by silencing Smad2 or blocking reactive oxygen species (ROS) production and was enhanced by inhibiting TAK1 or nuclear factor kappa B. Increased hepatic steatosis in WT mice fed a CDAA diet was associated with the increased expression of lipogenesis genes (Dgat1 and Srebp1c), whereas the decreased steatosis in Tgfbr2ΔHEP mice was accompanied by the increased expression of genes involved in β‐oxidation (Cpt1 and Acox1). In combination with palmitate treatment, TGF‐β signaling promoted lipid accumulation with induction of lipogenesis‐related genes and suppression of β‐oxidation‐related genes in hepatocytes. Silencing Smad2 decreased TGF‐β‐mediated lipid accumulation and corrected altered gene expression related to lipid metabolism in hepatocytes. Finally, we confirmed that livers from patients with nonalcoholic steatohepatitis (NASH) displayed phosphorylation and nuclear translocation of Smad2/3. Conclusions: TGF‐β signaling in hepatocytes contributes to hepatocyte death and lipid accumulation through Smad signaling and ROS production that promote the development of NASH. (Hepatology 2014;59:483–495)

TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis

The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.

TLR2 and TLR9 contribute to alcohol-mediated liver injury through induction of CXCL1 and neutrophil infiltration

Although previous studies reported the involvement of the TLR4-TRIF pathway in alcohol-induced liver injury, the role of TLR2 and TLR9 signaling in alcohol-mediated neutrophil infiltration and liver injury has not been elucidated. Since alcohol binge drinking is recognized to induce more severe form of alcohol liver disease, we used a chronic-binge ethanol-feeding model as a mouse model for early stage of alcoholic hepatitis. Whereas a chronic-binge ethanol feeding induced alcohol-mediated liver injury in wild-type mice, TLR2- and TLR9-deficient mice showed reduced liver injury. Induction of neutrophil-recruiting chemokines, including Cxcl1, Cxcl2, and Cxcl5, and hepatic neutrophil infiltration were increased in wild-type mice, but not in TLR2- and TLR9-deficient mice. In vivo depletion of Kupffer cells (KCs) by liposomal clodronate reduced liver injury and the expression of Il1b, but not Cxcl1, Cxcl2, and Cxcl5, suggesting that KCs are partly associated with liver injury, but not neutrophil recruitment, in a chronic-binge ethanol-feeding model. Notably, hepatocytes and hepatic stellate cells (HSCs) produce high amounts of CXCL1 in ethanol-treated mice. The treatment with TLR2 and TLR9 ligands synergistically upregulated CXCL1 expression in hepatocytes. Moreover, the inhibitors for CXCR2, a receptor for CXCL1, and MyD88 suppressed neutrophil infiltration and liver injury induced by chronic-binge ethanol treatment. Consistent with the above findings, hepatic CXCL1 expression was highly upregulated in patients with alcoholic hepatitis. In a chronic-binge ethanol-feeding model, the TLR2 and TLR9-dependent MyD88-dependent pathway mediates CXCL1 production in hepatocytes and HSCs; the CXCL1 then promotes neutrophil infiltration into the liver via CXCR2, resulting in the development of alcohol-mediated liver injury.

TRIF Differentially Regulates Hepatic Steatosis and Inflammation/Fibrosis in Mice

Background & Aims Toll-like receptor 4 (TLR4) signaling is activated through 2 adaptor proteins: MyD88 and TIR-domain containing adaptor-inducing interferon-β (TRIF). TLR4 and MyD88 are crucial in nonalcoholic steatohepatitis (NASH) and fibrosis. However, the role of TRIF in TLR4-mediated NASH and fibrosis has been elusive. This study investigated the differential roles of TRIF in hepatic steatosis and inflammation/fibrosis. Methods A choline-deficient amino acid defined (CDAA) diet was used for the mouse NASH model. On this diet, the mice develop hepatic steatosis, inflammation, and fibrosis. TLR4 wild-type and TLR4-/- bone marrow chimeric mice and TRIF-/- mice were fed CDAA or a control diet for 22 weeks. Hepatic steatosis, inflammation, and fibrosis were examined. Results In the CDAA diet–induced NASH, the mice with wild-type bone marrow had higher alanine aminotransferase and hepatic tumor necrosis factor levels than the mice with TLR4-/- bone marrow. The nonalcoholic fatty liver disease activity score showed that both wild-type and TLR4-/- bone marrow chimeras had reduced hepatic steatosis, and that both types of chimeras had similar levels of inflammation and hepatocyte ballooning to whole-body wild-type mice. Notably, wild-type recipients showed more liver fibrosis than TLR4-/- recipients. Although TRIF-/- mice showed reduced hepatic steatosis, these mice showed more liver injury, inflammation, and fibrosis than wild-type mice. TRIF-/- stellate cells and hepatocytes produced more C-X-C motif chemokine ligand 1 (CXCL1) and C-C motif chemokine ligand than wild-type cells in response to lipopolysaccharide. Consistently, TRIF-/- mice showed increased CXCL1 and CCL3 expression along with neutrophil and macrophage infiltration, which promotes liver inflammation and injury. Conclusions In TLR4-mediated NASH, different liver cells have distinct roles in hepatic steatosis, inflammation, and fibrosis. TRIF promotes hepatic steatosis but it inhibits injury, inflammation, and fibrosis.

Neurotropin Suppresses Inflammatory Cytokine Expression and Cell Death through Suppression of NF-κB and JNK in Hepatocytes

Inflammatory response and cell death in hepatocytes are hallmarks of chronic liver disease, and, therefore, can be effective therapeutic targets. Neurotropin® (NTP) is a drug widely used in Japan and China to treat chronic pain. Although NTP has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the action mechanism of NTP remains elusive. We hypothesize that NTP functions to suppress inflammatory pathways, thereby attenuating disease progression. In the present study, we investigated whether NTP suppresses inflammatory signaling and cell death pathways induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) in hepatocytes. NTP suppressed nuclear factor-κB (NF-κB) activation induced by IL-1β and TNFα assessed by using hepatocytes isolated from NF-κB-green fluorescent protein (GFP) reporter mice and an NF-κB-luciferase reporter system. The expression of NF-κB target genes, Il6, Nos2, Cxcl1, ccl5 and Cxcl2 induced by IL-1β and TNFα was suppressed after NTP treatment. We also found that NTP suppressed the JNK phosphorylation induced by IL-1β and TNFα. Because JNK activation contributes to hepatocyte death, we determined that NTP treatment suppressed hepatocyte death induced by IL-1β and TNFα in combination with actinomycin D. Taken together, our data demonstrate that NTP attenuates IL-1β and TNFα-mediated inflammatory cytokine expression and cell death in hepatocytes through the suppression of NF-κB and JNK. The results from the present study suggest that NTP may become a preventive or therapeutic strategy for alcoholic and non-alcoholic fatty liver disease in which NF-κB and JNK are thought to take part.

647 TLR7 Signaling As Potential Therapeutic Target for Mouse Model of Alcoholic Hepatitis

Background: The activation of classical complement pathway contributes to ethanol-induced liver injury. Ethanol feeding in mice activates complement pathway via C1q binding to apoptotic cells in the liver and increases downstream activation of C3b. However, the mechanism by which ethanol activates C1q is unclear. This knowledge is critical to understanding liver injury-repair mechanisms. Molecules targeting the C1q pathway to decrease inflammation would be expected to preserve complement activation via the lectin and alternative pathways, thus maintaining the role of complement in resistance to infections. Secretory IgM (sIgM) is the primary isotype of natural antibodies that arise in the absence of exposure to foreign antigens. In addition to the anti-microbial functions, there is accumulating evidence that sIgM has critical housekeeping functions by facilitating clearance of apoptotic cells through activation of C1q. Therefore, we hypothesized that ethanol exposure activates C1q through sIgM binding to apoptotic hepatocytes. The presence of sIgM-C1q complex further exacerbates apoptosis in a feed forward mechanism. Further, C1INH (C1 esterase inhibitor) targeted to decrease the sIgM-C1q activation may prevent complement activation and decrease apoptosis in ethanol-induced liver injury.Methods: Two experimental protocols were used: 1) Female C57BL/6J wild type mice and sIgM-/mice were fed ethanol containing diets for 2 weeks, at a maximal concentration of 4% (vol/vol) ethanol (22% calories) or pair-fed control diets. 2) C57BL/6 mice were fed ethanol containing diets for 4 days, at a maximal concentration of 6%(vol/vol) ethanol (32% of calories). Mice were treated with C1INH or vehicle via tail vein injection at 24 and 48 hours prior to euthanasia. Results: Ethanol exposure increased hepatic IgM, C3b and C1q deposition as measured by IHC. C1q co-localized with IgM in liver. sIgM-/mice had decreased hepatic C1q, C3b deposition and TUNEL positive nuclei after ethanol exposure. AST/ALT, as well as MCP-1 mRNA, was decreased in sIgM-/mice compared to wild type mice after ethanol feeding. Importantly, C1INH decreased hepatic C3b deposition and TUNEL positive nuclei after ethanol feeding. Conclusion: The data indicate that sIgM contributes to activation of classical complement pathway in ethanol-induced liver injury via binding of sIgM to C1q. The presence of sIgMC1q complex is associated with cell death and may further enhance cell death and injury. Blocking of the activation via C1INH decreased C3b deposition and apoptosis. Taken together, the data suggest that sIgM is critical in ethanol-induced complement activation and that therapies targeting the classical complement pathway may be beneficial in ethanolinduced liver injury. The study is supported in part by ABMRF/The Foundation for Alcohol Research to DJC and NIH grant 5R37 AA011876 to LEN.

802 Tgfb Signaling Participates in Steatohepatitis Through the Induction of Cell Death and Suppression of Beta-Oxidation in Mice

Background: Alcohol abuse leads to alcoholic liver disease (ALD). In ALD hepatic steatosis is a prerequisite of disease progresses to steatohepatitis (SH) at which stage the liver injury becomes evident. The mechanisms of steatosis in ALD are noit fully understood. Calciumdependent signaling is activated in ALD in mice. The Aim of our study was to evaluate the role of calcium-dependent signaling in development of steatosis in ALD. Methods: We fed alcohol (Lieber-deCarli) or control diet to control C57Bl6 and NFAT-KO mice or cyclosporine-treated C57Bl6 mice. Results: Alcohol, but not control, diet led to significant ALD, revealed by elevated liver Tg content and significant OilRedO liver tissue staining suggestive of steatosis, increased serum ALT suggestive of liver injury, and serum cytokines TNFa, IL1, IL6, suggestive of inflammation, in C57Bl6 mice. There was significant elevation of calcium signaling in livers of alcohol-fed animals compared to control diet, as revealed by higher expression of Calcineurin, PLC, PKC, and MAPKp38 and elevated NFAT activity. Alcohol, but not control, diet lead to significant induction ACS, SCD1, ELOV16, GPAT and DGAT, LDLR HMG-CoA reductase mRNA in the livers of ethanol-fed animals. Further, the amount of mature SREBP-1protein, suggestive of SREBP activation, was increased in liver of alcoholfed animals. Inhibition of calcium signaling by either Cyclosporine treatment (at the level of Calcineurin) or by genetic NFAT deficiency partially prevented alcohol diet-induced upregulation of ACS, SCD1, ELOV16, GPAT and DGAT; more important, inhibition of calcium signaling led to partial protective against alcohol diet-induced liver injury and steatosis. NFAT protein was detected in both KCs and Hpt. In vitro, palmitic acid-exposed Hepa1.6 cells, used as surrogate of Hpt, developed steatosis; this process was significantly impaired when the cells were treated with cyclosporine and in cells made NFAT deficient by specific siRNA treatment. Co-culture of Hepa 1.6 cells+palmitic acid with inflammatory (LPS-pretreated) KCs lead to further upregulation of lipid uptake; sole exposure of KCs to cyclosporine did not prevent steatosis in co-culture. These data suggested that calciumdependent signaling mechanisms are involved in lipid synthesis in hepatocytes at different levels, including lipogenesis and lipolysis, in a KC-dependent manner. In conclusion, we report novel finding that calcium signaling is, in part, responsible for development of steatosis component of ALD in mice. It remains to be determined if inhibition of calcium signaling may be beneficial for delaying of steatosis and/or blunting of progression from HS to SH phases of ALD.

383 Ablation of TGFbeta Receptor II Inhibits Liver Injury, Inflammation, Fibrosis and Carcinogenesis in Hepatocyte Specific TAK1 Deleted Mice

Background and Aims: Smad7 has been reported to inhibit inflammation. The role of Smad7 in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. We investigated the functional significance of Smad7 in HCC both In Vivo and In Vitro. Methods: Smad7 knockout (KO) and wildtype (WT) littermates were single injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days to induce HCC. The biological function of Smad7 was determined In Vitro in human HCC cell line (Hep3B) and primary cultures established from resected HCCs from KO and WT mice. Cell growth was evaluated by cell viability and colony formation, cell apoptosis by Annexin V/7-AAD double staining, cell cycle distribution by flow cytometry and cell invasion ability by Matrigel invasion assay. Signaling pathways and downstream effectors of Smad7 in HCC was evaluated by luciferase reporter activity assay and pathway-PCR-array. Results: Smad7 KO mice were more susceptible to DENinduced HCC than WT mice (78% vs 22%, P=0.018). HCCs from KO mice displayed a greater proliferative activity (P<0.01), and a reduced apoptotic index compared with the WT littermates (P<0.05). Smad7 was down-regulated in human HCC tissues compared to their adjacent non-tumor tissues (n=11, P<0.05). These In Vivo results indicated that Smad 7 may function as a potential tumor suppressor, which was further functionally tested In Vitro. Ectopic expression of Smad7 in Hep3B cells suppressed cell growth as evidenced by retarded cell growth curve (P<0.0001) and colony formation assay (P<0.01). Conversely, Smad7 increased cell apoptosis (P<0.05), consistent with enhanced cleaved caspase-9, caspase-3 and PARP, indicative of Smad7-induced apoptosis through the intrinsic caspase dependent pathway. Cell cycle analysis in Heb3B transfected with Smad7 revealed an increased proportion of cells arrested at G0/G1 phase (P<0.001), concomitant with a reduction in the proportion of cells in S-phase (P<0.001) as compared with control. Smad7 also repressed cell invasive ability (P<0.05). Moreover, primary culture cells form HCCs of KO mice showed a increased cell growth curve (P<0.01) and colony formation (P<0.01) compared with the cells generated from HCCs of WT mice. Moreover, pathway luciferase reporter assay indicated that Smad7 inhibited NF-κB and TGF-β signalings. Co-immunoprecipitation analysis showed that Smad7 directly interacts with TAB2, an upstream activator of NF-κB. The downstream targets of Smad7 was further revealed by TGF-β and NF-κB pathway PCR array as shown in Figure 1, which contribute to the tumor suppressive effect. Conclusion: Loss of Smad7 is sufficient to enhance susceptibility to HCC. Smad7 suppresses HCC growth through reducing cell proliferation and invasion, inducing G0/G1 phase arrest and apoptosis by inhibiting NF-κB and TGF-β signalings. Thus, Smad7 acts as a tumor-suppressor in the liver.