Yuzo Kodama

Toll-Like Receptor 9 Promotes Steatohepatitis by Induction of Interleukin-1β in Mice

BACKGROUND & AIMS Development of nonalcoholic steatohepatitis (NASH) involves the innate immune system and is mediated by Kupffer cells and hepatic stellate cells (HSCs). Toll-like receptor 9 (TLR9) is a pattern recognition receptor that recognizes bacteria-derived cytosine phosphate guanine (CpG)-containing DNA and activates innate immunity. We investigated the role of TLR9 signaling and the inflammatory cytokine interleukin-1beta (IL-1beta) in steatohepatitis, fibrosis, and insulin resistance. METHODS Wild-type (WT), TLR9(-/-), IL-1 receptor (IL-1R)(-/-), and MyD88(-/-) mice were fed a choline-deficient amino acid-defined (CDAA) diet for 22 weeks and then assessed for steatohepatitis, fibrosis, and insulin resistance. Lipid accumulation and cell death were assessed in isolated hepatocytes. Kupffer cells and HSCs were isolated to assess inflammatory and fibrogenic responses, respectively. RESULTS The CDAA diet induced NASH in WT mice, characterized by steatosis, inflammation, fibrosis, and insulin resistance. TLR9(-/-) mice showed less steatohepatitis and liver fibrosis than WT mice. Among inflammatory cytokines, IL-1beta production was suppressed in TLR9(-/-) mice. Kupffer cells produced IL-1beta in response to CpG oligodeoxynucleotide. IL-1beta but not CpG-oligodeoxynucleotides, increased lipid accumulation in hepatocytes. Lipid accumulation in hepatocytes led to nuclear factor-kappaB inactivation, resulting in cell death in response to IL-1beta. IL-1beta induced fibrogenic responses in HSCs, including secretion of tissue inhibitor of metalloproteinase-1. IL-1R(-/-) mice had reduced steatohepatitis and fibrosis, compared with WT mice. Mice deficient in MyD88, an adaptor molecule for TLR9 and IL-1R signaling, also had reduced steatohepatitis and fibrosis. TLR9(-/-), IL-1R(-/-), and MyD88(-/-) mice had less insulin resistance than WT mice on the CDAA diet. CONCLUSIONS In a mouse model of NASH, TLR9 signaling induces production of IL-1beta by Kupffer cells, leading to steatosis, inflammation, and fibrosis.

Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis

During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element–binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)—two key transcriptional regulators of this process. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A–mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein–coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with Gsα. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.

Hepatocytes do not undergo epithelial‐mesenchymal transition in liver fibrosis in mice

The origin of fibrogenic cells in liver fibrosis remains controversial. We assessed the emerging concept that hepatocytes contribute to production of extracellular matrix (ECM) in liver fibrosis through epithelial‐mesenchymal transition (EMT). We bred triple transgenic mice expressing ROSA26 stop β‐galactosidase (β‐gal), albumin Cre, and collagen α1(I) green fluorescent protein (GFP), in which hepatocyte‐derived cells are permanently labeled by β‐gal and type I collagen‐expressing cells are labeled by GFP. We induced liver fibrosis by repetitive carbon tetrachloride (CCl4) injections. Liver sections and isolated cells were evaluated for GFP and β‐gal as well as expression of α‐smooth muscle actin (α‐SMA) and fibroblast‐specific protein 1 (FSP‐1). Upon stimulation with transforming growth factor β‐1, cultured hepatocytes isolated from untreated liver expressed both GFP and β‐gal with a fibroblast‐like morphological change but lacked expression of other mesenchymal markers. Cells from CCl4‐treated livers never showed double‐positivity for GFP and β‐gal. All β‐gal‐positive cells exhibited abundant cytoplasm, a typical morphology of hepatocytes, and expressed none of the mesenchymal markers including α‐SMA, FSP‐1, desmin, and vimentin. In liver sections of CCl4‐treated mice, GFP‐positive areas were coincident with fibrotic septa and never overlapped X‐gal‐positive areas. Conclusion: Type I collagen‐producing cells do not originate from hepatocytes. Hepatocytes in vivo neither acquire mesenchymal marker expression nor exhibit a morphological change clearly distinguishable from normal hepatocytes. Our results strongly challenge the concept that hepatocytes in vivo acquire a mesenchymal phenotype through EMT to produce the ECM in liver fibrosis. (HEPATOLOGY 2009.)

Hepatitis C virus–induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity

Chronic hepatitis C is characterized by iron accumulation in the liver, and excessive iron is hepatotoxic. However, the mechanism by which hepatitis C virus (HCV) regulates iron metabolism is poorly understood. Hepcidin plays a pivotal role as a negative regulator of iron absorption. The aim of the current study was to elucidate the mechanisms that govern hepcidin expression by HCV. Huh 7 cells, Huh7.5 cells, full‐length HCV replicon cells established from Huh7.5 cells, and adenoviruses expressing HCV‐core or HCV nonstructural proteins 3 through 5 (NS3‐5) were used. Hepcidin expression was significantly lower in HCV replicon cells and in HCV core–expressing Huh7 cells. The expression was inversely correlated with the amount of reactive oxygen species (ROS) production. Anti‐oxidants restored hepcidin expression in HCV replicon cells and Huh7 cells expressing HCV core. In HCV replicon cells, histone deacetylase (HDAC) activity was elevated at baseline and after exposure to hydrogen peroxide. Anti‐oxidants reduced HDAC activity in a dose‐dependent manner. HDAC inhibition increased hepcidin expression without affecting ROS production in HCV replicon cells. HCV‐induced ROS stabilized the expression of two negative hepcidin regulators, HIF1α and HIF2α, and its expression was decreased by a HDAC inhibitor or an anti‐oxidant. HCV‐induced ROS also caused hypoacetylation of histones and inhibited binding of two positive regulators, C/EBPα and STAT3, to the hepcidin promoter, whereas anti‐oxidant treatment of cells recovered C/EBPα and STAT3 binding to the hepcidin promoter. In addition, an HDAC inhibitor restored their binding to the hepcidin promoter via acetylation of histones. Conclusion: HCV‐induced oxidative stress suppresses hepcidin expression through increased HDAC activity. (HEPATOLOGY 2008.)

Disruption of TAK1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis

TGF-β–activated kinase 1 (TAK1) is a MAP3K family member that activates NF-κB and JNK via Toll-like receptors and the receptors for IL-1, TNF-α, and TGF-β. Because the TAK1 downstream molecules NF-κB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1-deficient (Tak1ΔHEP) mice. The Tak1ΔHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1ΔHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including α-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-α. TNF-α increased caspase-3 activity but activated neither NF-κB nor JNK in Tak1-deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1ΔHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1ΔHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver.

Hepatic Stellate Cells Secrete Angiopoietin 1 That Induces Angiogenesis in Liver Fibrosis

BACKGROUND & AIMS Although angiogenesis is closely associated with liver fibrosis, the angiogenic factors involved in liver fibrosis are not well characterized. Angiopoietin 1 is an angiogenic cytokine indispensable for vascular development and remodeling. It functions as an agonist for the receptor tyrosine kinase with immunoglobulin G-like and endothelial growth factor-like domains 2 (Tie2) and counteracts apoptosis, promotes vascular sprouting or branching, and stabilizes vessels. METHODS Liver samples from patients with liver fibrosis were evaluated for mRNA expression of angiogenic cytokines. Liver fibrosis was induced in BALB/c mice by either carbon tetrachloride (CCl(4)) or bile duct ligation (BDL). Hepatic stellate cells (HSCs) were isolated from BALB/c mice. We used an adenovirus expressing the extracellular domain of Tie2 (AdsTie2) to block angiopoietin signaling in mice and evaluated its effect on liver fibrosis. RESULTS mRNA expression level of angiopoietin 1 was increased in human fibrotic livers and correlated with the expression level of CD31, an endothelial cell marker. During experimental models of murine liver fibrosis, angiopoietin 1 was expressed by activated HSCs. In primary cultures, activated HSCs express and secrete angiopoietin 1 more abundantly than quiescent HSCs, and the inflammatory cytokine tumor necrosis factor-alpha stimulates its expression in an nuclear factor-kappaB-dependent manner. AdsTie2 inhibits angiogenesis and liver fibrosis induced by either CCl(4) or BDL. CONCLUSIONS These results reveal an angiogenic role of HSCs mediated by angiopoietin 1, which contributes to development of liver fibrosis. Thus, angiogenesis and hepatic fibrosis are mutually stimulatory, such that fibrosis requires angiogenesis and angiogenesis requires angiopoietin 1 from activated HSCs.

CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury

CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a key component in endoplasmic reticulum (ER) stress-mediated apoptosis. The goal of the study was to investigate the role of CHOP in cholestatic liver injury. Acute liver injury and liver fibrosis were assessed in wild-type (WT) and CHOP-deficient mice following bile duct ligation (BDL). In WT livers, BDL induced overexpression of CHOP and Bax, a downstream target in the CHOP-mediated ER stress pathway. Liver fibrosis was attenuated in CHOP-knockout mice. Expression levels of alpha-smooth muscle actin and transforming growth factor-beta1 were reduced, and apoptotic and necrotic hepatocyte death were both attenuated in CHOP-deficient mice. Hepatocytes were isolated from WT and CHOP-deficient mice and treated with 400 microM glycochenodeoxycholic acid (GCDCA) for 8 h to examine bile acid-induced apoptosis and necrosis. GCDCA induced overexpression of CHOP and Bax in isolated WT hepatocytes, whereas CHOP-deficient hepatocytes had reduced cleaved caspase-3 expression and a lower propidium iodide index after GCDCA treatment. In conclusion, cholestasis induces CHOP-mediated ER stress and triggers hepatocyte cell death, and CHOP deficiency attenuates this cell death and subsequent liver fibrosis. The results demonstrate an essential role of CHOP in development of liver fibrosis due to cholestatic liver damage.

c-Jun N-terminal kinase-1 from hematopoietic cells mediates progression from hepatic steatosis to steatohepatitis and fibrosis in mice.

BACKGROUND & AIMS c-Jun N-terminal kinase (JNK) plays a pivotal role in the development of the metabolic syndrome including nonalcoholic fatty liver disease. However, the mechanism underlying the contribution of JNK to the progression from simple steatosis to steatohepatitis and liver fibrosis is unresolved. METHODS Hepatic steatosis, inflammation, and fibrosis were examined in wild-type, jnk1(-/-), or jnk2(-/-) mice fed a choline-deficient L-amino acid-defined (CDAA) diet for 20 weeks. The functional contribution of JNK isoforms in Kupffer cells was assessed in vitro and in vivo using chimeric mice in which the hematopoietic compartment including Kupffer cells was replaced by wild-type, jnk1(-/-), or jnk2(-/-) cells. RESULTS CDAA diet induced significantly less hepatic inflammation and less liver fibrosis despite a similar level of hepatic steatosis in jnk1(-/-) mice as compared with wild-type or jnk2(-/-) mice. CDAA diet-induced hepatic inflammation was chronic and mediated by Kupffer cells. Pharmacologic inhibition of JNK or gene deletion of jnk1 but not jnk2 repressed the expression of inflammatory and fibrogenic mediators in primary Kupffer cells. In vivo, CDAA diet induced less hepatic inflammation and liver fibrosis despite an equivalent level of hepatic steatosis in chimeric mice with jnk1(-/-) hematopoietic cells as compared with chimeric mice with wild-type or jnk2(-/-) hematopoietic cells. CONCLUSIONS jnk1(-/-) mice are resistant to diet-induced steatohepatitis and liver fibrosis. JNK1 in hematopoietic cells, especially in Kupffer cells, contributes to the development of liver fibrosis by inducing chronic inflammation.

Protection from liver fibrosis by a peroxisome proliferator-activated receptor δ agonist

Peroxisome proliferator-activated receptor delta (PPARδ), a member of the nuclear receptor family, is emerging as a key metabolic regulator with pleiotropic actions on various tissues including fat, skeletal muscle, and liver. Here we show that the PPARδ agonist KD3010, but not the well-validated GW501516, dramatically ameliorates liver injury induced by carbon tetrachloride (CCl4) injections. Deposition of extracellular matrix proteins was lower in the KD3010-treated group than in the vehicle- or GW501516-treated group. Interestingly, profibrogenic connective tissue growth factor was induced significantly by GW501516, but not by KD3010, following CCl4 treatment. The hepatoprotective and antifibrotic effect of KD3010 was confirmed in a model of cholestasis-induced liver injury and fibrosis using bile duct ligation for 3 wk. Primary hepatocytes treated with KD3010 but not GW501516 were protected from starvation or CCl4-induced cell death, in part because of reduced reactive oxygen species production. In conclusion, our data demonstrate that an orally active PPARδ agonist has hepatoprotective and antifibrotic effects in animal models of liver fibrosis, suggesting a possible mechanistic and therapeutic approach in treating patients with chronic liver diseases.

Antiapoptotic Effect of c-Jun N-terminal Kinase-1 through Mcl-1 Stabilization in TNF-Induced Hepatocyte Apoptosis

BACKGROUND & AIMS c-Jun N-terminal Kinase (JNK) is a key regulator in tumor necrosis factor (TNF)-mediated liver injury. However, distinct roles for JNK1 and JNK2 in hepatocyte apoptosis are still unresolved. Although myeloid cell leukemia-1 (Mcl-1) has been reported as a substrate of JNK, the role of Mcl-1 and its functional regulation by JNK in TNF-induced hepatocyte apoptosis and liver injury remain to be elucidated. METHODS TNF-induced hepatocyte apoptosis was investigated in wild-type, jnk1-/- and jnk2-/- mice in vitro and in the galactosamine/TNF (GalN/TNF) liver injury model. For further analysis, we used adenoviruses expressing wild-type Mcl-1 or its substitution mutant, and the Cre/loxP system (mcl-1f/f) to delete mcl-1. RESULTS jnk2-/- Hepatocytes showed increased Mcl-1 expression and were more resistant to TNF-induced apoptosis compared with wild-type or jnk1-/- hepatocytes. Increased Mcl-1 expression in jnk2-/- hepatocytes correlated with their JNK activity, which is mediated by residual JNK1 and higher than in wild-type or jnk1-/- hepatocytes. JNK activation led to phosphorylation of Mcl-1 in hepatocytes, and this increased the half-life of the Mcl-1 protein. Overexpression of Mcl-1 confirmed its antiapoptotic effect in TNF-induced hepatocyte apoptosis in vitro and in vivo. Deletion of mcl-1 in jnk2-/- hepatocytes increased TNF-induced hepatocyte apoptosis both in vitro and in GalN/TNF-induced liver injury model. CONCLUSIONS jnk2-/- Hepatocytes are resistant to TNF-induced apoptosis. Activated JNK1 contributes to this antiapoptotic phenotype of jnk2-/- hepatocytes through phosphorylation-mediated stabilization of Mcl-1.