TLR4 signaling and the inhibition of liver hepcidin expression by alcohol.
TLR4 signaling and the inhibition of liver hepcidin expression by alcohol.
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DOI：
10.3748/wjg.v20.i34.12161
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发表时间：
2014-09
影响因子：
4.3
通讯作者：
Emily M Zmijewski;Sizhao Lu;D. Harrison-Findik
中科院分区：
文献类型：
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作者：
Emily M Zmijewski;Sizhao Lu;D. Harrison-Findik
目的了解Toll样受体4(TLR4)信号在慢性饮酒对铁调节激素海普西丁调节中的作用。方法在慢性酒精摄入研究中，将C3 H/HeJ背景下的TLR4突变小鼠和C3 H/HeOuJ背景下的野生型TLR4突变小鼠配对喂养正常对照和含乙醇的Lieber de Carli液体饲料。实时荧光定量聚合酶链式反应检测基因表达。免疫共沉淀法和免疫印迹法检测蛋白质间相互作用和蛋白质表达。用染色质免疫沉淀法测定海普西丁基因启动子的占有率。结果长期饮酒可抑制野生型小鼠肝脏中海普西丁的表达，但不能抑制TLR4突变型小鼠肝脏中海普西丁的表达。在酒精喂养的野生型小鼠中观察到核因子-κB p65亚基蛋白的磷酸化和核转位，而在酒精喂养的突变小鼠中未观察到该蛋白的磷酸化和核转位。同样，在野生型小鼠中，酒精诱导了NF-κB p50亚单位蛋白与海普西丁基因启动子的结合，而在突变型小鼠中则不能。相反，与酒精处理的野生型小鼠相比，酒精处理的TLR4突变小鼠肝脏中STAT3的磷酸化程度更强。在酒精喂养的突变小鼠中观察到STAT3基因启动子的占位，而在野生型小鼠中没有观察到。免疫共沉淀研究表明，野生型和κ突变小鼠肝脏中均存在核因子-TLR4P65亚单位蛋白和小分子二聚体伙伴蛋白的相互作用。酒精摄入增加了胞质SHP的表达，但减弱了其与肝脏中的NF-κB的相互作用，与TLR4突变小鼠相比，这在野生型小鼠的肝脏中更为突出。结论TLR4信号通路和NF-кB的激活参与了乙醇对肝脏炎症状态下Hepsidin基因转录的抑制作用。
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.