Published online Sep 14, 2014. doi: 10.3748/wjg.v20.i34.12161
Revised: April 12, 2014
Accepted: May 19, 2014
Published online: September 14, 2014
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, 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.
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.
Core tip: Chronic alcohol intake induces inflammation and also alters iron homeostasis by inhibiting the expression of hepcidin in the liver. Besides being the key iron-regulatory hormone, hepcidin also acts as an acute phase protein, and is induced by endotoxin and inflammation. The mechanisms by which chronic alcohol consumption can lead to the suppression of hepcidin expression despite the presence of inflammatory processes in the liver are unknown. This study investigates these mechanisms by using wildtype and toll-like receptor 4 mutant mice, and chronic alcohol administration as a model.