Choline-deficient Amino Acid-defined Research Articles

PARD3 drives tumorigenesis through activating Sonic Hedgehog signalling in tumour-initiating cells in liver cancer

BackgroundPar-3 Family Cell Polarity Regulator (PARD3) is a cellular protein essential for asymmetric cell division and polarized growth. This study aimed to study the role of PARD3 in hepatic tumorigenesis.MethodsThe essential role of PARD3 in mediating hepatic tumorigenesis was assessed in diet-induced spontaneous liver tumour and syngeneic tumour models. The mechanism of PARD3 was delineated by bulk and single-cell RNA sequencing. The clinical significance of PARD3 was identified by tissue array analysis.ResultsPARD3 was overexpressed in tumour tissues and PARD3 overexpression was positively correlated with high tumour stage as well as the poor prognosis in patients. In models of spontaneous liver cancer induced by choline-deficient, amino acid-defined (CDAA) and methionine-choline-deficient (MCD) diets, upregulation of PARD3 was induced specifically at the tumorigenesis stage rather than other early stages of liver disease progression. Site-directed knockout of PARD3 using an adeno-associated virus 8 (AAV8)-delivered CRISPR/Cas9 single-guide RNA (sgRNA) plasmid blocked hepatic tumorigenesis, while PARD3 overexpression accelerated liver tumour progression. In particular, single-cell sequencing analysis suggested that PARD3 was enriched in primitive tumour cells and its overexpression enhanced tumour-initiating cell (TICs). Overexpression of PARD3 maintained the self-renewal ability of the CD133+ TIC population within hepatocellular carcinoma (HCC) cells and promoted the in vitro and in vivo tumorigenicity of CD133+ TICs. Transcriptome analysis revealed that Sonic Hedgehog (SHH) signalling was activated in PARD3-overexpressing CD133+ TICs. Mechanistically, PARD3 interacted with aPKC to further activate SHH signalling and downstream stemness-related genes. Suppression of SHH signalling and aPKC expression attenuated the in vitro and in vivo tumorigenicity of PARD3-overexpressing CD133+ TICs. Tissue array analysis revealed that PARD3 expression was positively associated with the phosphorylation of aPKC, SOX2 and Gli1 and that the combination of these markers could be used to stratify HCC patients into two clusters with different clinicopathological characteristics and overall survival prognoses. The natural compound berberine was selected as a potent suppressor of PARD3 expression and could be used as a preventive agent for liver cancer that completely blocks diet-induced hepatic tumorigenesis in a PARD3-dependent manner.ConclusionThis study revealed PARD3 as a potential preventive target of liver tumorigenesis via TIC regulation.

MiR-21-5p promotes NASH-related hepatocarcinogenesis.

The mechanisms governing the progression of non-alcoholic fatty liver disease (NAFLD) towards steatohepatitis (NASH) and hepatocellular carcinoma (HCC) remain elusive. Here, we evaluated the role of hsa-miRNA-21-5p in NASH-related hepatocarcinogenesis. Hepatic hsa-miR-21-5p expression was evaluated in two cohorts of patients with biopsy-proven NAFLD (n = 199) or HCC (n = 366 HCC and n = 11 NAFLD-HCC). Serum/liver metabolomic profiles were correlated with hsa-miR-21-5p in NAFLD obese patients. Wild-type (WT) and Mir21 KO mice were fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks to induce NASH and NASH-HCC, respectively. In obese individuals, hsa-miR-21-5p expression increased with NAFLD severity and associated with a hepatic lipotoxic profile. CDAA-fed WT mice displayed increased hepatic mmu-miR-21-5p levels and progressively developed NASH and fibrosis, with livers presenting macroscopically discernible pre-neoplastic nodules, hyperplastic foci and deregulated cancer-related pathways. Mir21 KO mice exhibited peroxisome-proliferator-activated receptor α (PPARα) activation, augmented mitochondrial activity, reduced liver injury and NAS below the threshold for NASH diagnosis, with the pro-inflammatory/fibrogenic milieu reversing to baseline levels. In parallel, Mir21 KO mice displayed reduced number of pre-neoplastic nodules, hepatocyte proliferation and activation of oncogenic signalling, being protected from NASH-associated carcinogenesis. The hsa-miRNA-21-5p/PPARα pathway was similarly deregulated in patients with HCC- or NASH-related HCC, correlating with HCC markers and worse prognosis. Hsa-miR-21-5p is a key inducer of whole-spectrum NAFLD progression, from simple steatosis to NASH and NASH-associated carcinogenesis. The inhibition of hsa-miR-21-5p, leading to a pro-metabolic profile, might constitute an appealing therapeutic approach to ameliorate NASH and prevent progression towards HCC.

Myeloid-specific blockade of Notch signaling ameliorates nonalcoholic fatty liver disease in mice.

Rationale: Macrophages play a central role in the development and progression of nonalcoholic fatty liver disease (NAFLD). Studies have shown that Notch signaling mediated by transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBP-J), is implicated in macrophage activation and plasticity. Naturally, we asked whether Notch signaling in macrophages plays a role in NAFLD, whether regulating Notch signaling in macrophages could serve as a therapeutic strategy to treat NAFLD. Methods: Immunofluorescence staining was used to detect the changes of macrophage Notch signaling in the livers of human patients with NAFLD and choline deficient amino acid-defined (CDAA) diet-fed mice. Lyz2-Cre RBP-Jflox or wild-type C57BL/6 male mice were fed with CDAA or high fat diet (HFD) to induce experimental steatohepatitis or steatosis, respectively. Liver histology examinations were performed using hematoxylin-eosin (H&E), Oil Red O staining, Sirius red staining and immunohistochemistry staining for F4/80, Col1α1 and αSMA. The expression of inflammatory factors, fibrosis or lipid metabolism associated genes were evaluated by quantitative reverse transcription (qRT)-PCR, Western blot or enzyme-linked immunosorbent assay (ELISA). The mRNA expression of liver samples was profiled by using RNA-seq. A hairpin-type decoy oligodeoxynucleotides (ODNs) for transcription factor RBP-J was loaded into bEnd.3-derived exosomes by electroporating. Mice with experimental NAFLD were treated with exosomes loading RBP-J decoy ODNs via tail vein injection. In vivo distribution of exosomes was analyzed by fluorescence labeling and imaging. Results: The results showed that Notch signaling was activated in hepatic macrophages in human with NAFLD or in CDAA-fed mice. Myeloid-specific RBP-J deficiency decreased the expression of inflammatory factors interleukin-1 beta (IL1β) and tumor necrosis factor alpha (TNFα), attenuated experimental steatohepatitis in mice. Furthermore, we found that Notch blockade attenuated lipid accumulation in hepatocytes by inhibiting the expression of IL1β and TNFα in macrophages in vitro. Meanwhile, we observed that tail vein-injected exosomes were mainly taken up by hepatic macrophages in mice with steatohepatitis. RBP-J decoy ODNs delivered by exosomes could efficiently inhibit Notch signaling in hepatic macrophages in vivo and ameliorate steatohepatitis or steatosis in CDAA or HFD mice, respectively. Conclusions: Combined, macrophage RBP-J promotes the progression of NAFLD at least partially through regulating the expression of pro-inflammatory cytokines IL1β and TNFα. Infusion of exosomes loaded with RBP-J decoy ODNs might be a promising therapy to treat NAFLD.

RIPK3 dampens mitochondrial bioenergetics and lipid droplet dynamics in metabolic liver disease.

Receptor-interacting protein kinase 3 (RIPK3) mediates NAFLD progression, but its metabolic function is unclear. Here, we aimed to investigate the role of RIPK3 in modulating mitochondria function, coupled with lipid droplet (LD) architecture in NAFLD. Functional studies evaluating mitochondria and LD biology were performed in wild-type (WT) and Ripk3-/- mice fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks and in CRISPR-Cas9 Ripk3 -null fat-loaded immortalized hepatocytes. The association between hepatic perilipin (PLIN) 1 and 5, RIPK3, and disease severity was also addressed in a cohort of patients with NAFLD and in PLIN1 -associated familial partial lipodystrophy. Ripk3 deficiency rescued impairment in mitochondrial biogenesis, bioenergetics, and function in CDAA diet-fed mice and fat-loaded hepatocytes. Ripk3 deficiency was accompanied by a strong upregulation of antioxidant systems, leading to diminished oxidative stress upon fat loading both in vivo and in vitro. Strikingly, Ripk3-/- hepatocytes displayed smaller size LD in higher numbers than WT cells after incubation with free fatty acids. Ripk3 deficiency upregulated adipocyte and hepatic levels of LD-associated proteins PLIN1 and PLIN5. PLIN1 upregulation controlled LD structure and diminished mitochondrial stress upon free fatty acid overload in Ripk3-/- hepatocytes and was associated with diminished human NAFLD severity. Conversely, a pathogenic PLIN1 frameshift variant was associated with NAFLD and fibrosis, as well as with increased hepatic RIPK3 levels in familial partial lipodystrophy. Ripk3 deficiency restores mitochondria bioenergetics and impacts LD dynamics. RIPK3 inhibition is promising in ameliorating NAFLD.

Homocysteine supplementation ameliorates steatohepatitis induced by a choline-deficient diet in mice.

High concentrations of homocysteine are believed to induce lipid synthesis and cell injury through endoplasmic reticulum (ER) stress in metabolic syndrome. However, homocysteine could be used to improve steatohepatitis induced by choline deficiency, in which methyl donors are decreased. The aim of the present study was to clarify the role of the physiological concentration of homocysteine in the development of steatohepatitis induced by choline deficiency. Wild-type mice were fed a choline-deficient amino acid-defined (CDAA) diet with or without homocysteine supplementation for 24weeks. Liver cells isolated from mice were exposed to homocysteine under choline-deficient conditions. Wild-type mice fed the CDAA diet developed steatohepatitis with increased ER stress and decreased S-adenosylmethionine (SAM), a methyl donor. Homocysteine supplementation reduced ER stress and restored hepatic SAM, leading to the improvement of steatohepatitis. In in vitro experiments using primary cultured hepatocytes, the physiological concentration of homocysteine decreased the lipid accumulation and ER stress induced by the choline-deficient conditions. However, hepatocyte death was not induced by a physiological concentration of homocysteine or in choline-deficient medium. Interestingly, tumor necrosis factor (TNF)α promoted hepatocyte death under choline-deficient conditions, which was suppressed by homocysteine supplementation. Hepatic macrophages increased the production of TNFα under choline-deficient conditions whereas supplementation of SAM reduced the TNFα production. Homocysteine supplementation ameliorates steatohepatitis by reducing ER stress and increasing SAM in mice fed a CDAA diet. These results were opposite to those of previous reports, which showed that homocysteine induced cell injury.

Lansoprazole prevents the progression of liver fibrosis in non-alcoholic steatohepatitis model rats

We previously demonstrated that lansoprazole provided hepatoprotection in a drug-induced hepatitis animal model partially through the Nrf2/HO-1 pathway. Here, we examined whether lansoprazole could also provide hepatoprotection in a rat model of non-alcoholic steatohepatitis (NASH). Six-week-old rats were fed a normal chow or a choline-deficient amino acid-defined (CDAA) diet to establish a rat model of NASH. The groups fed a CDAA diet for 5 weeks were subcutaneously administered either a vehicle or a lansoprazole suspension for 4 weeks beginning the second week of the experiment. Bridging fibrosis was observed in the livers of almost all the NASH model rats (six of seven), but it was not always observed in NASH model rats (one of seven) continuously administered lansoprazole. The serum aspartate aminotransferase level elevated by the CDAA diet was significantly decreased following lansoprazole administration. Lansoprazole also increased the expression of Nrf2, but not HO-1, in the liver of NASH model rats. Lansoprazole decreased the level of activated TGF-β protein. Furthermore, interleukin-6 gene and protein expression were decreased. Lansoprazole inhibits hepatic fibrogenesis, at least during the early stages, in CDAA diet-induced NASH model rats. The mechanisms might be associated with cytokine suppression but not the inhibition of reactive oxygen species.

Atg9b Deficiency Suppresses Autophagy and Potentiates Endoplasmic Reticulum Stress-Associated Hepatocyte Apoptosis in Hepatocarcinogenesis.

The aim of this study was to investigate the mechanism underlying autophagy deficiency during hepatic carcinogenesis. For this purpose, we used choline-deficient, amino acid-defined (CDAA) hepatocarcinogenesis model in mice. miRNA microarrays combined with computational target predictions and GO analysis were used to identify molecular processes involved in carcinogenesis. PCR profiler array was employed to detect the dysregulated autophagy-related genes during carcinogenesis. We observed induction of hepatic tumours with increased inflammation, DNA damage, and cell death. These cellular processes were particularly detected upon oncogenic transformation of hepatocytes in which ER stress was excessively induced. Microarray combined with GO analysis showed that transformation of hepatocytes resulted in dysregulated events associated with cytoplasmic vesicle formation, which, in turn, was related to ER stress-induced autophagy. Defects of autophagy were observed in livers harbouring tumours and suffered a loss of expression of autophagy-related protein 9b (Atg9b). Hepatocytes lacking Atg9b were vulnerable to cell death induced by ER stress stimulus mainly caused by accumulation of ubiquitinated proteins. Loss of Atg9b also blocked recruitment of p62-associated ubiquitinated protein for autophagosome-lysosome degradation as Atg9b-driven phagophores may facilitate docking of both LC3 and p62 to initiate autophagy-associated degradation. miR-3091-3p from tumour-derived exosomes, which were internalised by hepatocytes, could suppress Atg9b expression. Observations from this study advance our knowledge about the regulation of autophagy during hepatocarcinogenesis.

Altered Gut Microbiota Composition and Immune Response in Experimental Steatohepatitis Mouse Models.

Although several types of diet have been used in experimental steatohepatitis models, comparison of gut microbiota and immunological alterations in the gut among diets has not yet been performed. We attempted to clarify the difference in the gut environment between mice administrated several experimental diets. Male wild-type mice were fed a high-fat (HF) diet, a choline-deficient amino acid-defined (CDAA) diet, and a methionine-choline-deficient (MCD) diet for 8weeks. We compared the severity of steatohepatitis, the composition of gut microbiota, and the intestinal expression of interleukin (IL)-17, an immune modulator. Steatohepatitis was most severe in the mice fed the CDAA diet, followed by the MCD diet, and the HF diet. Analysis of gut microbiota showed that the composition of the Firmicutes phylum differed markedly at order level between the mice fed the CDAA and HF diet. The CDAA diet increased the abundance of Clostridiales, while the HF diet increased that of lactate-producing bacteria. In addition, the CDAA diet decreased the abundance of lactate-producing bacteria and antiinflammatory bacterium Parabacteroides goldsteinii in the phylum Bacteroidetes. In CDAA-fed mice, IL-17 levels were increased in ileum as well as portal vein. In addition, the CDAA diet also elevated hepatic expression of chemokines, downstream targets of IL-17. The composition of gut microbiota and IL-17 expression varied considerably between mice administrated different experimental diets to induce steatohepatitis.

Hepatocyte glutathione peroxidase-1 deficiency improves hepatic glucose metabolism and decreases steatohepatitis in mice.

In obesity oxidative stress is thought to contribute to the development of insulin resistance, non-alcoholic fatty liver disease and the progression to non-alcoholic steatohepatitis. Our aim was to examine the precise contributions of hepatocyte-derived H2O2 to liver pathophysiology. Glutathione peroxidase (GPX) 1 is an antioxidant enzyme that is abundant in the liver and converts H2O2 to water. We generated Gpx1 lox/lox mice to conditionally delete Gpx1 in hepatocytes (Alb-Cre;Gpx1 lox/lox) and characterised mice fed chow, high-fat or choline-deficient amino-acid-defined (CDAA) diets. Chow-fed Alb-Cre;Gpx1 lox/lox mice did not exhibit any alterations in body composition or energy expenditure, but had improved insulin sensitivity and reduced fasting blood glucose. This was accompanied by decreased gluconeogenic and increased glycolytic gene expression as well as increased hepatic glycogen. Hepatic insulin receptor Y1163/Y1163 phosphorylation and Akt Ser-473 phosphorylation were increased in fasted chow-fed Alb-Cre;Gpx1 lox/lox mice, associated with increased H2O2 production and insulin signalling in isolated hepatocytes. The enhanced insulin signalling was accompanied by the increased oxidation of hepatic protein tyrosine phosphatases previously implicated in the attenuation of insulin signalling. High-fat-fed Alb-Cre;Gpx1 lox/lox mice did not exhibit alterations in weight gain or hepatosteatosis, but exhibited decreased hepatic inflammation, decreased gluconeogenic gene expression and increased insulin signalling in the liver. Alb-Cre;Gpx1 lox/lox mice fed a CDAA diet that promotes non-alcoholic steatohepatitis exhibited decreased hepatic lymphocytic infiltrates, inflammation and liver fibrosis. Increased hepatocyte-derived H2O2 enhances hepatic insulin signalling, improves glucose control and protects mice from the development of non-alcoholic steatohepatitis.

Ectopic expression of N-acetylglucosaminyltransferase V accelerates hepatic triglyceride synthesis.

Glycosylation changes induce various types of biological phenomena in human diseases. N-Acetylglucosaminyltransferase V (GnT-V) is one of the most important glycosyltransferases involved in cancer biology. Recently, many researchers have challenged studies of lipid metabolism in cancer. To elucidate the relationships between cancer and lipid metabolism more precisely, we investigated the effects of GnT-V on lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on hepatic triglyceride production. We compared lipid metabolism in GnT-V transgenic (Tg) mice with that of wild-type (WT) mice fed with normal chow or a choline-deficient amino acid-defined (CDAA) diet in vivo. HepG2 cells and GnT-V transfectants of Hep3B cells were used in an in vitro study. Serum triglyceride levels and hepatic very low-density lipoprotein (VLDL) secretion in Tg mice were significantly elevated compared with that of WT mice. Hepatic lipogenic genes (Lxrα, Srebp1, Fas and Acc) and VLDL secretion-related gene (Mttp1) were significantly higher in Tg mice. Expression of these genes was also significantly higher in GnT-V transfectants than in mock cells. Knockdown of GnT-V decreased, while both epidermal growth factor and transforming growth factor-β1 stimulation increased LXRα gene expression in HepG2 cells. Finally, we found that the blockade of VLDL secretion by CDAA diet induced massive hepatic steatosis in Tg mice. Our study demonstrates that enhancement of hepatic GnT-V activity accelerates triglyceride synthesis and VLDL secretion. Glycosylation modification by GnT-V regulation could be a novel target for a therapeutic approach to lipid metabolism.

NLRP3 inflammasome activation is required for fibrosis development in NAFLD.

NLR inflammasomes, caspase 1 activation platforms critical for processing key pro-inflammatory cytokines, have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). As the direct role of the NLRP3 inflammasome remains unclear, we tested effects of persistent NLRP3 activation as a contributor to NAFLD development and, in particular, as a modulator of progression from benign hepatic steatosis to steatohepatitis during diet-induced NAFLD. Gain of function tamoxifen-inducible Nlrp3 knock-in mice allowing for in vivo temporal control of NLRP3 activation and loss of function Nlrp3 knockout mice were placed on short-term choline-deficient amino acid-defined (CDAA) diet, to induce isolated hepatic steatosis or long-term CDAA exposure, to induce severe steatohepatitis and fibrosis, respectively. Expression of NLRP3 associated proteins was assessed in liver biopsies of a well-characterized group of patients with the full spectrum of NAFLD. Nlrp3(-/-) mice were protected from long-term feeding CDAA-induced hepatomegaly, liver injury, and infiltration of activated macrophages. More importantly, Nlrp3(-/-) mice showed marked protection from CDAA-induced liver fibrosis. After 4 weeks on CDAA diet, wild-type (WT) animals showed isolated hepatic steatosis while Nlrp3 knock-in mice showed severe liver inflammation, with increased infiltration of activated macrophages and early signs of liver fibrosis. In the liver samples of patients with NAFLD, inflammasome components were significantly increased in those patients with nonalcoholic steatohepatitis (NASH) when compared to those with non-NASH NAFLD with mRNA levels of pro-IL1 beta correlated to levels of COL1A1. Our study uncovers a crucial role for the NLRP3 inflammasome in the development of NAFLD. These findings may lead to novel therapeutic strategies aimed at halting the progression of hepatic steatosis to the more severe forms of this disease. Key message: Mice with NLRP3 inflammasome loss of function are protected from diet-induced steatohepatitis. NLRP3 inflammasome gain of function leads to early and severe onset of diet-induced steatohepatitis in mice. Patients with severe NAFLD exhibit increased levels of NLRP3 inflammasome components and levels of pro-IL1β mRNA correlate with the expression of COL1A1.

Deletion of nardilysin prevents the development of steatohepatitis and liver fibrotic changes.

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1+/+ and Nrd1−/− mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1+/+ and Nrd1−/− mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1−/− mice was lower than that in Nrd1+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1+/+ mice but not in Nrd1−/− mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1−/− mice than in Nrd1+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1+/+ and Nrd1−/− mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1−/− mice was significantly less than that in Nrd1+/+ mice, indicating the decreased TNF-α shedding in Nrd1−/− mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1+/+ mice but not in Nrd1−/− mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1−/− mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH.

133 CYTOGLOBIN-DEFICIENCY PROMOTES THE DEVELOPMENT OF LIVER CANCER IN MOUSE MODELS VIA THE ACTIVATION OF LOCAL FIBROSIS, INFLAMMATION AND OXIDATIVE STRESS

Chronic liver damage may eventually progress to end-stage liver cirrhosis and hepatocellular carcinoma. Cytoglobin (Cygb) is a 21 kDa globin expressed in hepatic stellate cells and functions as a hypoxia sensor and a gas carrier. It serves as local peroxidase by degrading H2O2. However, its pathophysiological role in vivo remains undetermined. Here, we report the promotion of liver cancer development in Cygb-deficient (KO) mice administrated with either diethylnitrosamine (DEN) or choline-deficient amino acid-defined (CDAA) diet that induces hepatosteatosis. Methods: Cygb KO mice and corresponding wild-type (WT) mice at 8 week-old were treated with either DEN, CDAA, or CSAA diet for 8–32 weeks. Macroscopic and microscopic observations were performed. Gene expressions and intracellular signaling pathways were analyzed. Oxidative stress was determined by the formation of 8-OHdG, DHE, and nitrotyrosine. Results: Model 1; 25 or 0.05 ppm DEN treatment for 25 or 36 weeks induced liver tumor formation in 100% or 44%, respectively, in Cygb KO mice compared to 44% or 0%, respectively, in WT mice. Background liver developed fibrosis together with the augmented expression of mRNA of TGF-beta 3, collagen 1a1, and TIMP-1. Inflammatory gene expressions and augmented oxidative stress formation were evident in KO mice. Model 2; as early as 8 weeks of CDAA treatment, Cygb KO mice exhibited dominant steatohepatitis, which resulted in advanced fibrosis at 16 week-point, compared with WT as assessed by pathological NASH scores, collagen deposition, alpha-smooth muscle actin expression, and hepatic hydroxyproline content. Surprisingly, after 32 weeks under CDAA administration, 100% of both male and female Cygb KO mice developed liver cancers, compared to 0% in corresponding WT mice. Analyses at 32 week-point showed histologically severe inflammatory reactions concomitant with increased mRNA expression of Tnfa, Tgfb1, Il-1b and Il-6 in Cygb KO mice compared with WT mice. Cygb KO mice showed increased hepatocyte proliferation (Ki67 staining) and expression of AFP. Oxidative stress and antioxidant defense PCR array identified altered expression of 31 genes involved in the metabolism of reactive oxygen species in Cygb KO mice. Conclusion: Deficiency of Cygb promotes liver cancer development through activating inflammatory reaction and oxidative stress pathway.

135 Jnk1 IN HEPATIC STELLATE CELLS MODULATES LIVER FIBROGENESIS IN VIVO AND IN VITRO

Chronic liver damage may eventually progress to end-stage liver cirrhosis and hepatocellular carcinoma. Cytoglobin (Cygb) is a 21 kDa globin expressed in hepatic stellate cells and functions as a hypoxia sensor and a gas carrier. It serves as local peroxidase by degrading H2O2. However, its pathophysiological role in vivo remains undetermined. Here, we report the promotion of liver cancer development in Cygb-deficient (KO) mice administrated with either diethylnitrosamine (DEN) or choline-deficient amino acid-defined (CDAA) diet that induces hepatosteatosis. Methods: Cygb KO mice and corresponding wild-type (WT) mice at 8 week-old were treated with either DEN, CDAA, or CSAA diet for 8–32 weeks. Macroscopic and microscopic observations were performed. Gene expressions and intracellular signaling pathways were analyzed. Oxidative stress was determined by the formation of 8-OHdG, DHE, and nitrotyrosine. Results: Model 1; 25 or 0.05 ppm DEN treatment for 25 or 36 weeks induced liver tumor formation in 100% or 44%, respectively, in Cygb KO mice compared to 44% or 0%, respectively, in WT mice. Background liver developed fibrosis together with the augmented expression of mRNA of TGF-beta 3, collagen 1a1, and TIMP-1. Inflammatory gene expressions and augmented oxidative stress formation were evident in KO mice. Model 2; as early as 8 weeks of CDAA treatment, Cygb KO mice exhibited dominant steatohepatitis, which resulted in advanced fibrosis at 16 week-point, compared with WT as assessed by pathological NASH scores, collagen deposition, alpha-smooth muscle actin expression, and hepatic hydroxyproline content. Surprisingly, after 32 weeks under CDAA administration, 100% of both male and female Cygb KO mice developed liver cancers, compared to 0% in corresponding WT mice. Analyses at 32 week-point showed histologically severe inflammatory reactions concomitant with increased mRNA expression of Tnfa, Tgfb1, Il-1b and Il-6 in Cygb KO mice compared with WT mice. Cygb KO mice showed increased hepatocyte proliferation (Ki67 staining) and expression of AFP. Oxidative stress and antioxidant defense PCR array identified altered expression of 31 genes involved in the metabolism of reactive oxygen species in Cygb KO mice. Conclusion: Deficiency of Cygb promotes liver cancer development through activating inflammatory reaction and oxidative stress pathway.

134 HEPCIDIN KNOCKOUT MICE DEVELOP CHRONIC LIVER INJURY AND LIVER FIBROSIS AS A CONSEQUENCE OF LYSOSOMAL IRON OVERLOAD

Chronic liver damage may eventually progress to end-stage liver cirrhosis and hepatocellular carcinoma. Cytoglobin (Cygb) is a 21 kDa globin expressed in hepatic stellate cells and functions as a hypoxia sensor and a gas carrier. It serves as local peroxidase by degrading H2O2. However, its pathophysiological role in vivo remains undetermined. Here, we report the promotion of liver cancer development in Cygb-deficient (KO) mice administrated with either diethylnitrosamine (DEN) or choline-deficient amino acid-defined (CDAA) diet that induces hepatosteatosis. Methods: Cygb KO mice and corresponding wild-type (WT) mice at 8 week-old were treated with either DEN, CDAA, or CSAA diet for 8–32 weeks. Macroscopic and microscopic observations were performed. Gene expressions and intracellular signaling pathways were analyzed. Oxidative stress was determined by the formation of 8-OHdG, DHE, and nitrotyrosine. Results: Model 1; 25 or 0.05 ppm DEN treatment for 25 or 36 weeks induced liver tumor formation in 100% or 44%, respectively, in Cygb KO mice compared to 44% or 0%, respectively, in WT mice. Background liver developed fibrosis together with the augmented expression of mRNA of TGF-beta 3, collagen 1a1, and TIMP-1. Inflammatory gene expressions and augmented oxidative stress formation were evident in KO mice. Model 2; as early as 8 weeks of CDAA treatment, Cygb KO mice exhibited dominant steatohepatitis, which resulted in advanced fibrosis at 16 week-point, compared with WT as assessed by pathological NASH scores, collagen deposition, alpha-smooth muscle actin expression, and hepatic hydroxyproline content. Surprisingly, after 32 weeks under CDAA administration, 100% of both male and female Cygb KO mice developed liver cancers, compared to 0% in corresponding WT mice. Analyses at 32 week-point showed histologically severe inflammatory reactions concomitant with increased mRNA expression of Tnfa, Tgfb1, Il-1b and Il-6 in Cygb KO mice compared with WT mice. Cygb KO mice showed increased hepatocyte proliferation (Ki67 staining) and expression of AFP. Oxidative stress and antioxidant defense PCR array identified altered expression of 31 genes involved in the metabolism of reactive oxygen species in Cygb KO mice. Conclusion: Deficiency of Cygb promotes liver cancer development through activating inflammatory reaction and oxidative stress pathway.

Toll-like receptor 2 and palmitic acid cooperatively contribute to the development of nonalcoholic steatohepatitis through inflammasome activation in mice

Innate immune signaling associated with Toll-like receptors (TLRs) is a key pathway involved in the progression of nonalcoholic steatohepatitis (NASH). Here we show that both TLR2 and palmitic acid are required for activation of the inflammasome, interleukin (IL)-1α, and IL-1β, resulting in the progression of NASH. Wild-type (WT) and TLR2(-/-) mice were fed a choline-deficient amino acid-defined (CDAA) diet for 22 weeks to induce NASH. Bone marrow-transplanted TLR2 chimeric mice were generated after the recipient mice were lethally irradiated. Kupffer cells and hepatic stellate cells (HSCs) were isolated from WT mice and stimulated with TLR2 ligand and/or palmitic acid. WT mice on the CDAA diet developed profound steatohepatitis and liver fibrosis. In contrast, TLR2(-/-) mice had suppressed progression of NASH. Although both Kupffer cells and HSCs respond to TLR2 ligand, TLR2 bone marrow chimeric mice demonstrated that Kupffer cells were relatively more important than HSCs in TLR2-mediated progression of NASH. In vitro, palmitic acid alone did not increase TLR2 signaling-target genes, including cytokines and inflammasome components in Kupffer cells and HSCs. The TLR2 ligand increased Nod-like receptor protein 3, an inflammasome component, in Kupffer cells but not in HSCs. In the presence of TLR2 ligand, palmitic acid did induce caspase-1 activation and release of IL-1α and IL-1β in Kupffer cells; however, these effects were not observed in HSCs. In vivo, WT mice on the CDAA diet showed increased caspase-1 activation in the liver and elevated serum levels of IL-1α and IL-1β levels, which were suppressed in TLR2(-/-) mice. TLR2 and palmitic acid cooperatively activate the inflammasome in Kupffer cells and/or macrophages in the development of NASH.

Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2

Inflammatory cell infiltration in the liver is a hallmark of nonalcoholic steatohepatitis (NASH). The chemokine-chemokine receptor interaction induces inflammatory cell recruitment. CC-chemokine receptor (CCR)2 is expressed on hepatic macrophages and hepatic stellate cells. This study aims to investigate the therapeutic potential of CCR2 to NASH. Twenty-two weeks on a choline-deficient amino acid-defined (CDAA) diet induced steatosis, inflammatory cell infiltration, and liver fibrosis with increased CCR2 and monocyte chemoattractant protein (MCP)-1 expression in the wild-type livers. The infiltrated macrophages expressed CD68, CCR2, and a marker of bone marrow-derived monocytes, Ly6C. CCR2(-/-) mice had less steatosis, inflammatory cell infiltration, and fibrosis, and hepatic macrophages expressing CD68 and Ly6C were decreased. Toll-like receptor (TLR)4(-/-), TLR9(-/-), and MyD88(-/-) mice had reduced hepatic macrophage infiltration with decreased MCP-1 and CCR2 expression because TLR signaling is a potent inducer of MCP-1. To assess the role of Kupffer cells at the onset of NASH, Kupffer cells were depleted by liposomal clodronate. The Kupffer cell depletion ameliorated steatohepatitis with a decrease in the MCP-1 expression and recruitment of Ly6C-expressing macrophages at the onset of NASH. Finally, to test the therapeutic potential of targeting CCR2, a CCR2 inhibitor was administered to mice on a CDAA diet. The pharmaceutical inhibition of CCR2 prevented infiltration of the Ly6C-positive macrophages, resulting in an inhibition of liver inflammation and fibrosis. We concluded that CCR2 and Kupffer cells contribute to the progression of NASH by recruiting bone marrow-derived monocytes.

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

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. 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. 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. In a mouse model of NASH, TLR9 signaling induces production of IL-1beta by Kupffer cells, leading to steatosis, inflammation, and fibrosis.