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| 1 | TLR4 signaling and the inhibition of liver hepcidin expression by alcohol显示文摘AIM:To understand the role of toll-like receptor 4(TLR4)signaling in the regulation of iron-regulatory hormone,hepcidin by chronic alcohol consumption.METHODS:For chronic alcohol intake studies,TLR4mutant mice on C3H/HeJ background and wildtype counterpart on C3H/HeOuJ background were pair-fed with regular(control)and ethanol-containing Lieber De Carli liquids diets.Gene expression was determined by real-time quantitative PCR.Protein-protein interactions and protein expression were determined by co-immunoprecipitation and western blotting.The occupancy of hepcidin gene promoter was determined by chromatin immunoprecipitation assays.RESULTS:Chronic alcohol intake suppressed hepcidin mRNA expression in the livers of wildtype,but not TLR4 mutant,mice.The phosphorylation and nuclear translocation of nuclear factor(NF)-κB p65 subunit protein was observed in alcohol-fed wildtype,but not in alcohol-fed TLR4 mutant,mice.Similarly,alcohol induced the binding of NF-κB p50 subunit protein to hepcidin gene promoter in wildtype,but not in TLR4mutant,mice.In contrast,the phosphorylation of Stat3in the liver was stronger in alcohol-treated TLR4 mutant mice compared to alcohol-treated wildtype mice.The occupancy of hepcidin gene promoter by Stat3was observed in alcohol-fed mutant,but not in wildtype,mice.An interaction between NF-κB p65 subunit protein and small heterodimer partner protein(SHP)was observed in the livers of both wildtype and TLR4mutant mice fed with the control diet,as shown by coimmunoprecipitation studies.Alcohol intake elevated cytosolic SHP expression but attenuated its interaction with NF-κB in the liver,which was more prominent in the livers of wildtype compared to TLR4 mutant mice.CONCLUSION:Activation of TLR4 signaling and NF-кB are involved in the suppression of hepcidin gene transcription by alcohol in the presence of inflammation in the liver. | Emily Zmijewski Sizhao Lu Duygu Dee Harrison-Findik | 2014 | World Journal of Gastroenterology2014，20，34： | 6 |
| 2 | Effect of alcohol exposure on hepatic superoxide generation and hepcidin expression显示文摘AIM: To understand the role of mitochondrial-produced superoxide(O 2 ?) in the regulation of iron-regulatory hormone, hepcidin by alcohol in the liver. METHODS: For alcohol experiments, manganese superoxide dismutase knockout mice heterozygous for Sod2 gene expression(Sod2 +/) and age-matched littermate control mice(LMC), expressing Sod2 gene on both alleles, were exposed to either 10%(w/v) ethanol in the drinking water or plain water(control) for 7 d. Total cellular O 2 ? levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, O 2 ?-sensitive fluorogenic probe, MitoSOX Red and flow cytometry were utilized to measure O 2 ? in mitochondria. Gene and protein expression were determined by Taqman Real-time quantitative PCR and Western blotting, respectively. RESULTS: Sod2 +/- mice expressed 40% less MnSOD protein(SOD2) in hepatocytes compared to LMC mice. The deletion of Sod2 allele did not alter the basal expression level of hepcidin in the liver. 10% ethanol exposure for 1 wk inhibited hepatic hepcidin mRNA expression three-fold both in Sod2 +/ and LMC mice. O 2 ? levels in hepatocytes of untreated Sod2 +/ mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O 2 ? levels in mitochondria of Sod2 +/ mice were four-fold higher than in mitochondria of untreated LMC mice, as measured by MitoSOX Red fluorescence and flow cytometry. Alcohol induced a two-fold higher increase in O 2 ? levels in hepatocytes of LMC mice than in Sod2 +/ mice compared to respective untreated counterparts. In contrast, 1 wk alcohol exposure did not alter mitochondrial O 2 ? levels in both Sod2 +/- and control mice. CONCLUSION: Mitochondrial O2 ? is not involved in the inhibition of liver hepcidin transcription and thereby regulation of iron metabolism by alcohol. These findings also suggest that short-term alcohol consumption significantly elevates O 2 ? levels in hepatocytes, which appears not to originate from mitochondria. | Duygu Dee Harrison-Findik Sizhao Lu Emily M Zmijewski Jocelyn Jones Matthew C Zimmerman | 2013 | World Journal of Biological Chemistry2013，4，4： | 2 |
| 3 | Apoptosis induced by Fas signaling does not alter hepatic hepcidin expression显示文摘AIM: To determine the regulation of human hepcidin(HAMP) and mouse hepcidin(hepcidin-1 and hepcidin-2) gene expression in the liver by apoptosis using in vivo and in vitro experimental models. METHODS: For the induction of the extrinsic apoptotic pathway, HepG2 cells were treated with various concentrations of CH11, an activating antibody for human Fas receptor, for 12 h. Male C57BL/6NCR and C57BL/6J strains of mice were injected intraperitoneally with sublethal doses of an activating antibody for mouse Fas receptor, Jo2. The mice were anesthetized and sacrificed 1 or 6 h after the injection. The level of apoptosis was quantified by caspase-3 activity assay. Liver injury was assessed by measuring the levels of ALT/AST enzymes in the serum. The acute phase reaction in the liver was examined by determining the expression levels of IL-6 and SAA3 genes by SYBR green quantitative real-time PCR(qPCR). The phosphorylation of transcription factors, Stat3, Smad4 and NF-κB was determined by western blotting. Hepcidin gene expression was determined by Taqman qPCR. The binding of transcription factors to hepcidin-1 promoter was studied using chromatin immunoprecipitation(ChIP) assays.RESULTS: The treatment of HepG2 cells with CH11 induced apoptosis, as shown by the significant activation of caspase-3(P < 0.001), but did not cause any significant changes in HAMP expression. Short-term(1 h) Jo2 treatment(0.2 μg/g b.w.) neither induced apoptosis and acute phase reaction nor altered mRNA expression of mouse hepcidin-1 in the livers of C57BL/6NCR mice. In contrast, 6 h after Jo2 injection, the livers of C57BL/6NCR mice exhibited a significant level of apoptosis(P < 0.001) and an increase in SAA3(P < 0.023) and IL-6(P < 0.005) expression in the liver. However, mRNA expression of hepcidin-1 in the liver was not significantly altered. Despite the Jo2-induced phosphorylation of Stat3, no occupancy of hepcidin-1 promoter by Stat3 was observed, as shown by ChIP assays. Compared to C57BL/6NCR mice, Jo2 treatment(0.2 μg/g b.w.) of C57BL/6J strain mice for 6 h induced a more prominent activation of apoptosis, liver injury and acute phase reaction. Similar to C57BL/6NCR mice, the level of liver hepcidin-1 mRNA expression in the livers of C57BL/6J mice injected with a sublethal dose of Jo2(0.2 μg/g b.w.) remained unchanged. The injection of C57BL/6J mice with a higher dose of Jo2(0.32 μg/g b.w.) did not also alter hepatic hepcidin expression.CONCLUSION: Our findings suggest that human or mouse hepcidin gene expression is not regulated by apoptosis induced via Fas receptor activation in the liver. | Sizhao Lu Emily Zmijewski John Gollan Duygu Dee Harrison-Findik | 2014 | World Journal of Biological Chemistry2014，5，3： | 1 |
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