Forkhead Box M1 Transcription Factor Drives Liver Inflammation Linking to Hepatocarcinogenesis in Mice.

BACKGROUND & AIMS: Liver inflammation has been recognized as a hallmark of hepatocarcinogenesis. Although Forkhead Box M1 (FoxM1) is a well-defined oncogenic transcription factor that is overexpressed in hepatocellular carcinoma (HCC), its role in liver inflammation has never been explored. METHODS: We generated hepatocyte-specific FoxM1 conditional transgenic (TG) mice by using the Cre-loxP and Tetracycline (Tet)-on systems to induce FoxM1 expression in a hepatocyte-specific and time-dependent manner. RESULTS: After treatment of Tet-derivatives doxycycline (DOX) to induce FoxM1, TG mice exhibited spontaneous development of hepatocyte death with elevated serum alanine aminotransferase levels and hepatic infiltration of macrophages. The removal of DOX in TG mice completely removed this effect, suggesting that spontaneous inflammation in TG mice occurs in a hepatocyte FoxM1-dependent manner. In addition, liver inflammation in TG mice was associated with increased levels of hepatic and serum chemokine (C-C motif) ligand 2 (CCL2). In vitro transcriptional analysis confirmed that CCL2 is a direct target of FoxM1 in murine hepatocytes. After receiving FoxM1 induction since birth, all TG mice exhibited spontaneous HCC with liver fibrosis at 12 months of age. Hepatic expression of FoxM1 was significantly increased in liver injury models. Finally, pharmacologic inhibition of FoxM1 reduced liver inflammation in models of liver injury. CONCLUSIONS: Hepatocyte FoxM1 acts as a crucial regulator to orchestrate liver inflammation linking to hepatocarcinogenesis. Thus, hepatocyte FoxM1 may be a potential target not only for the treatment of liver injury but also for the prevention toward HCC.

Pivotal role of STIM2, but not STIM1, in IL-4 production by IL-3-stimulated murine basophils.

Basophils have nonredundant roles in various immune responses that require Ca2+ influx. Here, we examined the role of two Ca2+ sensors, stromal interaction molecule 1 and 2 (STIM1 and STIM2), in basophil activation. We found that loss of STIM1, but not STIM2, impaired basophil IL-4 production after stimulation with immunoglobulin E (IgE)-containing immune complexes. In contrast, when basophils were stimulated with IL-3, loss of STIM2, but not STIM1, reduced basophil IL-4 production. This difference in STIM proteins was associated with distinct time courses of Ca2+ influx and transcription of the Il4 gene that were elicited by each stimulus. Similarly, basophil-specific STIM1 expression was required for IgE-driven chronic allergic inflammation in vivo, whereas STIM2 was required for IL-4 production after combined IL-3 and IL-33 treatment in mice. These data indicate that STIM1 and STIM2 have differential roles in the production of IL-4, which are stimulus dependent. Furthermore, these results illustrate the vital role of STIM2 in basophils, which is often considered to be less important than STIM1.

Targeting the mevalonate pathway is a novel therapeutic approach to inhibit oncogenic FoxM1 transcription factor in human hepatocellular carcinoma.

Dysregulation of cell metabolism is a hallmark of cancer. The mevalonate pathway in lipid metabolism has been implicated as a potential target of cancer therapy for hepatocellular carcinoma (HCC). The role of the Forkhead Box M1 (FoxM1) transcription factor in HCC development has been well documented, however, its involvement in cancer metabolism of HCC has not been fully determined. Here, we hypothesized that FoxM1 is involved in the mevalonate pathway of cholesterol biosynthesis in HCC. Inhibition of the mevalonate pathway by statins, inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), resulted in reduced expression of FoxM1 and increased cell death in human hepatoma cells. Re-exposure of mevalonate, a product of HMGCR, restored these effects. Likewise, knockdown of HMGCR reduced FoxM1 expression, indicating that FoxM1 expression was regulated by the mevalonate pathway in HCC. Mechanistically, protein geranylgeranylation was found to be responsible for FoxM1 expression and geranylgeranylated proteins, including RhoA, Rac1 or Cdc42, were shown to be involved in this process. In surgically resected human HCC tissues, the gene expression of FoxM1 had a positive correlation with that of the mevalonate pathway-related genes, such as HMGCR or sterol regulatory element-binding protein 2 (SREBP2). Furthermore, the gene expression of FoxM1 along with that of HMGCR or SREBP2 defined prognosis of HCC patients, suggesting the clinical significance of the mevalonate-FoxM1 pathway in human HCC. Our data indicate that FoxM1 links the mevalonate pathway to oncogenic signals in HCC. Thus, we propose a novel therapeutic approach to inhibit FoxM1 by targeting the mevalonate pathway for HCC.

Increased expression of Forkhead box M1 transcription factor is associated with clinicopathological features and confers a poor prognosis in human hepatocellular carcinoma.

AIM: Forkhead Box M1 (FoxM1) is a proliferation-specific transcription factor. In this study, we aimed to elucidate the clinicopathological and prognostic values of FoxM1 expression in human hepatocellular carcinoma (HCC) and correlate FoxM1 expression with various etiologies of liver diseases. We also investigated its therapeutic value in HCC. METHODS: We investigated the expression of FoxM1 in tumor tissues and adjacent non-tumor tissues of 79 Japanese HCC patients by quantitative real-time reverse transcription-polymerase chain reaction analysis. Depletion by siRNA or specific inhibition by siomycin A were also used to investigate the effect of FoxM1 inhibition on stem-like features of human HCC cells. RESULTS: Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that tumor tissues displayed an approximately 14-fold increase in FoxM1 expression compared with adjacent non-tumor tissues. Interestingly, the expression levels of FoxM1in tumor tissues did not depend on the etiology of liver disease. The expression of FoxM1 in tumor tissues was associated with serum α-fetoprotein level, maximum tumor size, histological grade, TNM staging, and portal involvement. Kaplan-Meier analysis indicated that the high FoxM1 expression (≥median) group had a poor prognosis compared with the low FoxM1 expression (

Gab1 adaptor protein acts as a gatekeeper to balance hepatocyte death and proliferation during acetaminophen-induced liver injury in mice.

UNLABELLED: Acetaminophen (APAP) overdose is the leading cause of drug-induced acute liver failure. In APAP-induced acute liver failure, hepatocyte death and subsequent liver regeneration determines the prognosis of patients, making it necessary to identify suitable therapeutic targets based on detailed molecular mechanisms. Grb2-associated binder 1 (Gab1) adaptor protein plays a crucial role in transmitting signals from growth factor and cytokine receptors to downstream effectors. In this study, we hypothesized that Gab1 is involved in APAP-induced acute liver failure. Hepatocyte-specific Gab1 conditional knockout (Gab1CKO) and control mice were treated with 250 mg/kg of APAP. After APAP treatment, Gab1CKO mice had significantly higher mortality and elevated serum alanine aminotransferase levels compared to control mice. Gab1CKO mice had increased hepatocyte death and increased serum levels of high mobility group box 1, a marker of hepatocyte necrosis. In addition, Gab1CKO mice had reduced hepatocyte proliferation. The enhanced hepatotoxicity in Gab1CKO mice was associated with increased activation of stress-related c-Jun N-terminal kinase (JNK) and reduced activation of extracellular signal-regulated kinase and AKT. Furthermore, Gab1CKO mice showed enhanced mitochondrial translocation of JNK accompanied by an increase in the release of mitochondrial enzymes into the cytosol, which is indicative of increased mitochondrial dysfunction and subsequent nuclear DNA fragmentation. Finally, in vitro experiments showed that Gab1-deficient hepatocytes were more susceptible to APAP-induced mitochondrial dysfunction and cell death, suggesting that hepatocyte Gab1 is a direct target of APAP-induced hepatotoxicity. CONCLUSION: Our current data demonstrate that hepatocyte Gab1 plays a critical role in controlling the balance between hepatocyte death and compensatory hepatocyte proliferation during APAP-induced liver injury.

Loss of Gab1 adaptor protein in hepatocytes aggravates experimental liver fibrosis in mice.

Grb2-associated binder 1 (Gab1) adaptor protein amplifies signals downstream of a broad range of growth factors/receptor tyrosine kinases. Although these signals are implicated in liver fibrogenesis, the role of Gab1 remains unclear. To elucidate the role of Gab1, liver fibrosis was examined in hepatocyte-specific Gab1-conditional knockout (Gab1CKO) mice upon bile duct ligation (BDL). Gab1CKO mice developed exacerbated liver fibrosis with activation of hepatic myofibroblasts after BDL compared with control mice. The antifibrotic role of hepatocyte Gab1 was further confirmed by another well-established mouse model of liver fibrosis using chronic injections of carbon tetrachloride. After BDL, Gab1CKO mice also displayed exacerbated liver injury, decreased hepatocyte proliferation, and enhanced liver inflammation. Furthermore, cDNA microarray analysis was used to investigate the potential molecular mechanisms of the Gab1-mediated signal in liver fibrosis, and the fibrosis-promoting factor chemokine (C-C motif) ligand 5 (Ccl5) was identified as upregulated in the livers of Gab1CKO mice following BDL. Interestingly, in vitro studies using primary hepatocytes isolated from control and Gab1CKO mice revealed that the loss of Gab1 resulted in increased hepatocyte CCL5 synthesis upon lipopolysaccharide stimulation. Finally, pharmacological antagonism of CCL5 reduced BDL-induced liver fibrosis in Gab1CKO mice. In conclusion, our results demonstrate that hepatocyte Gab1 is required for liver fibrosis and that hepatocyte CCL5 could be an important contributor to this process. Thus, we present a novel antifibrotic function of hepatocyte Gab1 in liver fibrogenesis.

Secreted frizzled-related protein 5 (Sfrp5) decreases hepatic stellate cell activation and liver fibrosis.

BACKGROUND & AIMS: Obesity-related adipocytokine dysregulation is known to accelerate liver fibrosis progression. Recently, a natural Wnt5a inhibitor, secreted frizzled-related protein 5 (Sfrp5), was identified as a novel adipocytokine that has reduced expression in obese adipose tissue in both rodents and human. In addition, hepatic gene expression of Wnt5a and its receptor frizzled 2 (Fz2) is elevated during fibrosis progression. Therefore, Sfrp5 could have biological significance in liver fibrosis. METHODS: We first investigated the effects of Sfrp5 on primary cultured mouse hepatic stellate cells (HSCs) in vitro. Next, to elucidate the roles of Sfrp5 in liver fibrosis, we investigated a carbon-tetrachloride (CCl4 )-induced liver fibrosis model using Sfrp5 knockout (KO) and wild type (WT) mice in vivo. Each mouse was injected intraperitoneally with CCl4 (0.5 ml/kg) or olive oil as a single dose (acute liver injury model), or twice a week for 6 weeks (liver fibrosis model). RESULTS: In in vitro studies, Wnt5a enhanced both proliferation and migration of HSCs, and these effects could be completely blocked by Sfrp5. Moreover, siRNA knockdown of Fz2 in HSCs could block the effects of Wnt5a on both HSC proliferation and migration. In in vivo studies, there were no differences in the CCl4 -induced liver injury between KO and WT mice. Hepatic Wnt5a gene expression and plasma Wnt5a levels significantly increased after a single CCl4 injection in both mice. Sfrp5 knockout significantly enhanced CCl4 -induced liver fibrosis. CONCLUSIONS: Our findings demonstrate that Sfrp5 may ameliorate mouse liver fibrosis through inhibition of Wnt5a/Fz2 signalling.

Lipid overloading during liver regeneration causes delayed hepatocyte DNA replication by increasing ER stress in mice with simple hepatic steatosis.

BACKGROUND AND AIM: Impaired fatty liver regeneration has already been reported in many genetic modification models. However, in diet-induced simple hepatic steatosis, which showed similar phenotype with clinical pathology, whether liver regeneration is impaired or not remains unclear. In this study, we evaluated liver regeneration in mice with diet-induced simple hepatic steatosis, and focused on excess lipid accumulation occurring during liver regeneration. METHODS: Mice were fed high fat diet (HFD) or control diet for 9-10 weeks. We analyzed intrahepatic lipid accumulation, DNA replication, and various signaling pathways including cell proliferation and ER stress during liver regeneration after partial hepatectomy. In addition, some of mice were pretreated with tauroursodeoxycholic acid (TUDCA), a chemical chaperone which alleviates ER stress, and then we estimated TUDCA effects on liver regeneration. RESULTS: The peak of hepatocyte BrdU incorporation, the expression of proliferation cell nuclear antigen (PCNA) protein, and the expressions of cell cycle-related genes were observed in delayed time in HFD mice. The expression of phosphorylated Erk1/2 was also delayed in HFD mice. The amounts of liver triglyceride were at least twofold higher in HFD mice at each time point. Intrahepatic palmitic acid was increased especially in HFD mice. ER stress induced during liver regeneration was significantly higher in HFD mice. In HFD mice, pretreatment with TUDCA reduced ER stress and resulted in improvement of delayed liver regeneration. CONCLUSION: In simple hepatic steatosis, lipid overloading occurring during liver regeneration might be caused ER stress and results in delayed hepatocyte DNA replication.

Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice.

Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-ß-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-ß-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

Inflammatory monocytes recruited to allergic skin acquire an anti-inflammatory M2 phenotype via basophil-derived interleukin-4.

Monocytes and macrophages are important effectors and regulators of inflammation, and both can be divided into distinct subsets based on their phenotypes. The developmental and functional relationship between individual subsets of monocytes and those of macrophages has not been fully elucidated, although Ly6C(+)CCR2(+) inflammatory and Ly6C(-)CCR2(-) resident monocytes are generally thought to differentiate into M1 (classically activated) and M2 (alternatively activated) macrophages, respectively. Here we show that inflammatory monocytes recruited to allergic skin acquired an M2-like phenotype in response to basophil-derived interleukin-4 (IL-4) and exerted an anti-inflammatory function. CCR2-deficient mice unexpectedly displayed an exacerbation rather than alleviation of allergic inflammation, in spite of impaired recruitment of inflammatory monocytes to skin lesions. Adoptive transfer of inflammatory monocytes from wild-type but not IL-4 receptor-deficient mice dampened the exacerbated inflammation in CCR2-deficient mice. Thus, inflammatory monocytes can be converted from being proinflammatory to anti-inflammatory under the influence of basophils in allergic reactions.

Pitavastatin ameliorated the progression of steatohepatitis in ovariectomized mice fed a high fat and high cholesterol diet.

AIM: Many studies indicate an accelerated progression of non-alcoholic steatohepatitis (NASH) in postmenopausal women. Very recently, we reported that estrogen deficiency enhanced the progression of steatohepatitis in mice fed a high fat and high cholesterol (HFHC) diet. Hypercholesterolemia is often observed in postmenopausal women, and recent studies indicate it to be an important risk factor for the progression of NASH. Statins can slow NASH progression in the estrogen-deficient state but the precise mechanisms of their effects are still unclear. METHODS: We investigated the effects of pitavastatin on steatohepatitis progression using ovariectomized (OVX) mice fed a HFHC diet or HFHC + pitava diet (containing 5 p.p.m. pitavastatin) for 6 weeks. RESULTS: Serum alanine aminotransferase and cholesterol levels significantly decreased in mice fed the HFHC + pitava diet compared with mice fed the HFHC diet. Real-time reverse transcription polymerase chain reaction representing hepatic inflammatory gene expressions significantly decreased in mice fed the HFHC + pitava diet compared with the HFHC-fed mice. Pitavastatin treatment also decreased both hepatic macrophage infiltration and hepatocyte chemokine (C-C motif) ligand 2 expression and improved the liver fibrosis condition when compared with the mice fed the HFHC diet. In addition, the enhanced spleen monocyte chemokine (C-C motif) receptor 2 expression in ovariectomized mice fed the HFHC diet was also decreased by pitavastatin administration. CONCLUSION: Our study demonstrated that the exacerbated steatohepatitis progression in OVX mice fed a HFHC diet could be attenuated by pitavastatin treatment at least through inhibition of hepatic macrophage infiltration. We concluded that statins should be useful for treating NASH in postmenopausal women.

Conditional knockout of heparin-binding epidermal growth factor-like growth factor in the liver accelerates carbon tetrachloride-induced liver injury in mice.

AIM: We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced in response to several liver injuries. Because the HB-EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver-specific HB-EGF conditional knockout mice using the interferon-inducible Mx-1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. METHODS: We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl4 ) in HB-EGF KO mice and wild-type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB-EGF-dependent anti-apoptosis and wound-healing process of the liver in vitro. RESULTS: HB-EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl4 injection. We also demonstrated that HB-EGF treatment inhibited tumor necrosis factor-α-induced apoptosis of AML12 mouse hepatocytes and promoted the wound-healing response of these cells. CONCLUSION: This study showed that HB-EGF plays a protective role during acute liver injury.

Adiponectin negatively correlates with alcoholic and non-alcoholic liver dysfunction: Health check-up study of Japanese men.

AIM: Central obesity, insulin resistance and alcohol consumption are thought to be major risk factors for fatty liver formation. Adiponectin (APN) prevents fatty liver formation, and its serum levels are lower in subjects with central obesity and/or insulin resistance. The aim of this study was to explore the association among serum APN levels, central obesity, insulin resistance and liver dysfunction with or without fatty liver classified by alcohol consumption in healthy subjects. METHODS: A total of 5588 Japanese male subjects who underwent a health check-up were classified into three groups according to alcohol consumption: non- or light drinkers (15 g/day ≥ ethanol); mild drinkers (15 g/day < ethanol ≤ 30 g/day); and moderate- or heavy drinkers (30 g/day < ethanol). Central obesity and insulin resistance were assessed by waist circumference (WC) and Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR), respectively. RESULTS: WC was significantly increased, while HOMA-IR was significantly decreased according to the extent of alcohol consumption. Serum alanine aminotransferase levels were significantly lower and serum APN levels were significantly higher in mild drinkers than in the other two groups. Multiple linear regression analysis showed that serum APN level served as the significant and independent determinant for liver dysfunction in the subjects with fatty liver, irrespective of alcohol consumption. However, WC became a non-significant determinant of liver dysfunction as alcohol consumption increased. CONCLUSION: Hypoadiponectinemia is a significant determinant for steatotic dysfunction for all levels of alcohol consumption, but central obesity was not a significant determinant for alcoholic fatty liver-induced liver dysfunction.