Study On Effect Of HNF-4alpha On Hepcidin Expression In Vitro

BackgroudHepcidin is a liver-derived peptide hormone and plays a key role in ironhomeostasis by negatively regulating intestinal iron absorption and iron release frommacrophages and hepatocytes. Hepatic iron overload (HIO) is frequently observed inadult NAFLD patientsand has been confirmed to promote the progression ofnon-alcoholic fatty liver disease (NAFLD) by increasing hepatic fbrosis and livercholesterol synthesis. Dysregulation of hepcidin was frequently observed in patientswith NAFLD with HIO. Although serum or liver hepcidin was found to be increasedin NAFLD patients, the production of hepcidin was still insufficient with theelevation of body iron stores when normalized to serum ferritin or hepatic iron score(HIS). Impaired hepcidin response was considered to be closely related with thesevere form of HIO in NAFLD and has also been proved to play an important role inhepatic iron overload in some other metabolic diseases, such as type2diabetes.However, the mechanism underlying hepcidin deficiency in NAFLD patientsremains elusive.Hepatocyte nuclear factor-4α (HNF-4α) is a liver-enriched transcription factor thatis critical in lipid and glucose metabolism. Courselaud and his colleague foundHEPC transcripts increased in liver-specific HNF4α-null mice. However, themechanism underlying HNF-4α regulates hepcidin remains elusive.The aim of the present study was to systematically discuss the effect of HNF-4αon hepcidin expression in vitro in HepG2cells, and further clarify its molecularmechanisms. Our study gained deeper insight into the etiology of hepcidindeficiency in NAFLD patients with HIO.ObjectiveThe aim of the present study was to systematically discuss the effect of HNF-4αon hepcidin expression in vitro in HepG2cells, and further clarify its molecularmechanisms. Our study gained deeper insight into the etiology of hepcidindeficiency in NAFLD patients with HIO and thus supplied proof. Methods1. Cell culture and transfectionThe human hepatoma cell line HepG2was grown at37°C in a mixture of highglucose DMEM medium supplemented with10%FBS and100IU/ml penicillin,50μg/ml streptomycin sulfatev. For HNF-4α or/and BMPR1A silence, cells weretransferred to6-well plates and transfected with HNF-4α or/and BMPR1A siRNAproducts or negative control oligos in the presence of lipofectamine RNAiMAX,according to the manufacturer’s instructions. After12-h transfection, the mediumwas replaced with penicillin/streptomycin containing growth medium, and cells wereincubated for additional36h. For HNF-4α over-expression, HepG2cells wereseeded in6-well plates and transfected with2μg HNF-4α or nonsense plasmids inthe presence of FuGENE HD transfection reagent for48h.2. Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) assay was performed using acommercially available kit (Upstate Biotechnology) as described by themanufacturer’s instructions.3. Generation of hepcidin promoter plasmid constructs, celltransfection and luciferase reporter assaysGeneration of pGL3-basic luciferase reporter with full-length human hepcidinpromoter was performed by obtaining genomic DNA from HepG2cells and cloningthe proximal942bp of human HAMP promoter into the pGL3-basic luciferasereporter vector. Site-directed mutagenesis on putative BMPs responsive elementwere as described by Matak et aland depletion on putative HNF-4α responsiveelement was according to Brice Courselaud et al. HepG2cells were transfected withempty pGL3-basic vectors, certain HAMP reporter constructs, and HNF-4α plasmidor HNF-4α siRNA, using FuGENE HD Transfection Reagent according to themanufacturer’s instructions. To normalize for the transfection efficiency, an internalcontrol pRL-SV40Renilla luciferase plasmid was co-transfected alongside theHAMP constructs in serum-free medium. After48-h equilibration, luciferase activitywas determined in triplicate in at least three independent experiments using the DualLuciferase ReporterAssay, according to the manufacturer’s instructions.4. RNA isolation and real-time quantitative PCR Real-time qPCR was used to analyze HNF-4α and hepcidin mRNA levels inHepG2cells after transfection. Total RNA was carried out from HepG2cells using asingle step extraction method TRIzol reagent. Total RNA was reverse-transcribedusing RT regent Kit. Real-time qPCR was performed in SYBR GREEN withgene-specific primers by the ABI Prism7300Sequence Detection System, andmRNA levels of specific genes were normalized to the β-actin levels of the samesample.5. Western BlottingHepG2cells were transfected with HNF-4α siRNA or nonsense oligos, HNF-4αover-expression or nonsense plasmids. After48-h equilibration, KeyGene Protein kitwas used to extract nuclear proteins and cytosolic proteins according to themanufacturer’s instructions. The protein levels of HNF-4α, hepcidin, SMAD1,pSMAD1/5/8, SMAD4, STAT3, pSTAT3, SMAD6, SMAD7, BMP2, BMP4,BMPR1A, BMPR1B, BMPR2, ActR2A, ActR2B and HJV were detected by WesternBlotting. Signals quantitated by densitometry were normalized to β-actin levels or, inthe case of phosphoproteins, to the total levels of the same protein.6. StatisticsValues are represented as Mean±SEM. Statistical analysis was performed usingthe Statview software (SPSS). Statistical difference between two groups wasassessed by the Independent-t test. One way ANOVA, followed by LSD-t andDunnett post-hoc test, was performed to analyze the difference between the three ormore groups.Results1. Effect of HNF-4α on hepcidin expression in HepG2cellsHNF-4α siRNA or plasmids were transfected into HepG2cells, and hepcidinmRNA and protein levels were detected by real-time RT-PCR and Western Blotting.Compared with the blank control group, hepcidin mRNA and protein levels wereboth markedly increased when HNF-4α was knocked downand significantly reducedwhen HNF-4α was over expressed. 2. Effect of HNF-4α on its potential responsive elements on humanhepcidin promoterKnowing that two potential response elements were reported on the flankingregion of human HEPC gene, we at first verified whether HNF-4α could bind tohuman HEPC promoter through chromatin immunoprecipitation (chIP) experiments.Our data showed that HNF-4α bound to human hepcidin promoter through theproximal binding site but not the distal one. Next, we observed the role of thechIP-verified response element in the HNF4α-induced reduction of hepcidin throughexperiments of reporter genes. HepG2cells were co-transfected with humanhepcidin promoter with or without the HNF-4α binding sites and plasmids encodingHNF-4α. HNF-4α plasmids showed an obvious inhibitory effect on the luciferaseactivities of the wild full-length hepcidin promoter. Furthermore, depletion of theHNF-4α binding sites significantly suppressed the basal level of the hepcidinpromoter activity, but showed no block effect on the HNF-4α-induced reduction.3. Pathway may mediated HNF-4α regulates hepcidinSTAT3and SMADs are known as critical regulators of hepcidn and mediatedhepcidin response to IL-6and BMPs. To determine whether these two novelpathways were involved in HNF4α-induced hepcidin repression, we observed theeffect of HNF-4α on the total and (or) phosphorylated protein levels of STAT3,SMAD1and SMAD4. It was found that the phosphorylation of SMAD1wasmarkedly inhibited by HNF-4α plasmids and markedly enhanced when HNF-4α wasknocked down by specific siRNA, without change on the total level of SMAD1andSMAD4. Besides, no significant alteration was found in the total or phosphorylatedprotein levels of STAT3when HNF-4α was interfered with or over-expressed.In order to further verify the role of the BMP signaling pathway onHNF4α-mediated hepcidin suppression, chIP and reporter gene assay was performed.The results of chIP showed that the amount of SMAD4binding to hepcidin promotermarkedly increased in HepG2cells transfected with HNF-4α siRNA and obviouslydecreased when HNF-4α was over expressed by encoding plasmids, but the amountof STAT3remained unchanged under both of above conditions. Luciferase activityof full length hepcidin promoter was significantly reduced when HepG2cells wasco-transfected with plasmids encoding HNF-4α. However, the luciferase activity ofhepcidin promoter with mutation SMAD4binding site was no longer stimulated byHNF-4α interference and even increased when HNF-4α plasmids were co-transfected.4. Neither inhibitory SMADs, SMAD6or SMAD7, nor BMPRligands, BMP2or BMP4, was regulated by HNF-4α in HepG2cells.SMAD6and SMAD7are both served as inhibitory SMADs in negative feedbackloops of BMP pathway, for they can form stable complexes with activated type IBMPRs and therefore block the phosphorylation of SMADs. To further clarify themolecular mechanisms underlying the repression of phosphorylation of SMADs byHNF-4α, we detected the protein levels of SMAD6and SMAD7, and also theintracellular protein levels of BMP2and BMP4, when HNF-4α was silenced orover-expressed, respectively. No change was found in SMAD6, SMAD7, BMP2orBMP4when HNF-4α expression was elevated or interfered.5. BMPR1A mediates HNF4α-induced suppression on the BMPpathway and hepcidin expression.Protein levels of type I BMP receptors (BMPR1A, BMPR1B), type II BMPreceptors (BMPR2, ActR2A, ActR2B) and BMPs co-receptor HJV were analyzed byWestern Blotting to further clarify how HNF-4α affected the activation of the BMPpathway, or more precisely, the phosphorylation of SMAD1. It was demonstratedthat silencing of HNF-4α increased the level of BMPR1A, while over-expressiondecreased it without significantly altering BMPR1B, BMPR2, ActR2A, ActR2B andHJV.To better investigate the role of BMPR1A in the suppressive effect of HNF-4α onhepcidin, we observed the hepcidin expression in HepG2cells co-transfected withHNF-4α and BMPR1A siRNA. It was found that the up-regulation of hepcidin byHNF-4α silencing was entirely blocked by simultaneous knockdown of BMPR1A.