title: TLR4 signaling and the inhibition of liver hepcidin expression by alcohol.
abstract: OBJECTIVE
  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, TLR4 mutant 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 TLR4 mutant, mice. In contrast, the phosphorylation of Stat3 in the liver was stronger in alcohol-treated TLR4 mutant mice compared to alcohol-treated wildtype mice. The occupancy of hepcidin gene promoter by Stat3 was 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 TLR4 mutant mice fed with the control diet, as shown by co-immunoprecipitation 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Emily Zmijewski, Sizhao Lu, Duygu Dee Harrison-Findik, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198-5820, United States.
authors.forename: Emily
authors.initials: E
authors.lastname: Zmijewski
authors.affiliation: Emily Zmijewski, Sizhao Lu, Duygu Dee Harrison-Findik, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198-5820, United States.
authors.forename: Sizhao
authors.initials: S
authors.lastname: Lu
authors.affiliation: Emily Zmijewski, Sizhao Lu, Duygu Dee Harrison-Findik, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198-5820, United States.
authors.forename: Duygu Dee
authors.initials: DD
authors.lastname: Harrison-Findik
mesh_terms: D000818:Animals; D001665:Binding Sites; D004195:Disease Models, Animal; D015536:Down-Regulation; D000431:Ethanol; D064451:Hepcidins / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008108:Liver Diseases, Alcoholic / Q000209:etiology / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008809:Mice, Inbred C3H; D008817:Mice, Mutant Strains; D009154:Mutation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: C579599:Hamp protein, mouse; D064451:Hepcidins; D012333:RNA, Messenger; C498144:Rela protein, mouse; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA; D000431:Ethanol
keywords: Alcoholic liver disease; Inflammation; Iron; Nuclear factor-κB; Small heterodimer partner protein