HNF-4alpha Negatively Regulates Hepcidin Expression Through BMPR1A in HepG2 Cells
Hepcidin synthesis is reported to be inadequate according to the body iron store in patients with non-alcoholic fatty liver disease (NAFLD) undergoing hepatic iron overload (HIO). However, the underlying mechanisms remain unclear. We hypothesize that hepatocyte nuclear factor-4α (HNF-4α) may negatively regulate hepcidin expression and contribute to hepcidin deficiency in NAFLD patients. The effect of HNF-4α on hepcidin expression was observed by transfecting specific HNF-4α small interfering RNA (siRNA) or plasmids into HepG2 cells. Both direct and indirect mechanisms involved in the regulation of HNF-4α on hepcidin were detected by real-time PCR, Western blotting, chromatin immunoprecipitation (chIP), and reporter genes. It was found that HNF-4α suppressed hepcidin messenger RNA (mRNA) and protein expressions in HepG2 cells, and this suppressive effect was independent of the potential HNF-4α response elements. Phosphorylation of SMAD1 but not STAT3 was inactivated by HNF-4α, and the SMAD4 response element was found essential to HNF-4α-induced hepcidin reduction. Neither inhibitory SMADs, SMAD6, and SMAD7 nor BMPR ligands, BMP2, BMP4, BMP6, and BMP7 were regulated by HNF-4α in HepG2 cells. BMPR1A, but not BMPR1B, BMPR2, ActR2A, ActR2B, or HJV, was decreased by HNF-4α, and HNF4α-knockdown-induced stimulation of hepcidin could be entirely blocked when BMPR1A was interfered with at the same time. In conclusion, the present study suggests that HNF-4α has a suppressive effect on hepcidin expression by inactivating the BMP pathway, specifically via BMPR1A, in HepG2 cells.
KeywordsHNF-4α·hepcidin pSMAD1/5/8 BMPR1A
This study was supported by the funds from the National Natural Science Foundation of China (81273053). There are no potential conflicts of interest relevant to this article.
Min Li, who designed the concept of the study, made critical revisions of the manuscript and was responsible for obtaining the funding. Wencai Shi and Heyang Wang, who designed the study, provided technical or material support, participated in data acquisition, analysis and interpretation, drafted the manuscript, and performed the statistical analysis. Xuan Zheng, Xin Jiang, Zheng Xu, and Hui Shen participated in data acquisition and technical support. Min Li is the guarantor of this article and, as such, has full access to all the data in the study and is responsible for the integrity of the data and the accuracy of the data analysis.
We also thank Shunxing Zhang, the professor of English Department of Second Military Medical University, for his work on the modifications on our revision manuscript.
- 1.Fleming MD (2008) The regulation of hepcidin and its effects on systemic and cellular iron metabolism. Hematology Am Soc Hematol Educ Program:151–158Google Scholar
- 13.Sam AH, Busbridge M, Amin A, Webber L, White D, Franks S, Martin NM, Sleeth M, Ismail NA, Mat Daud N, Papamargaritis D, Le Roux CW, Chapman RS, Frost G, Bloom SR, Murphy KG (2013) Hepcidin levels in diabetes mellitus and polycystic ovary syndrome. Diabetic MED 30:1495–1499CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Souza Pauli LS, Ropelle EC, de Souza CT, Cintra DE, da Silva AS, de Almeida Rodrigues B, de Moura LP, Marinho R, de Oliveira V, Katashima CK, Pauli JR, Ropelle ER (2014) Exercise training decreases mitogen-activated protein kinase phosphatase-3 expression and suppresses hepatic gluconeogenesis in obese mice. J Physiol 592:1325–1340CrossRefPubMedGoogle Scholar
- 23.Courselaud B, Pigeon C, Inoue Y, Inoue J, Gonzalez FJ, Leroyer P, Gilot D, Boudjema K, Guguen-Guillouzo C, Brissot P, Loréal O, Ilyin G (2002) C/EBPalpha regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism. Cross-talk between C/EBP pathway and iron metabolism. J Biol Chem 277:41163–41170CrossRefPubMedGoogle Scholar
- 25.Ling C, Wang Y, Zhang Y, Ejjigani A, Yin Z, Lu Y, Wang L, Wang M, Li J, Hu Z, Aslanidi GV, Zhong L, Gao G, Srivastava A, Ling C (2014) Selective in vivo targeting of human liver tumors by optimized AAV3 vectors in a murine xenograft model. Hum Gene Ther 25:1023–1034CrossRefPubMedPubMedCentralGoogle Scholar
- 26.Wang LN, Wang Y, Lu Y, Yin ZF, Zhang YH, Aslanidi GV, Srivastava A, Ling CQ, Ling C (2014) Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo. J Intern Med 12:20–34Google Scholar
- 29.Hirota K, Sakamaki J, Ishida J, Shimamoto Y, Nishihara S, Kodama N, Ohta K, Yamamoto M, Tanimoto K, Fukamizu A (2008) A combination of HNF-4 and Foxo1 is required for reciprocal transcriptional regulation of glucokinase and glucose-6-phosphatase genes in response to fasting and feeding. J Biol Chem 283:32432–32441CrossRefPubMedGoogle Scholar
- 32.Gaussin V, Van de Putte T, Mishina Y, Hanks MC, Zwijsen A, Huylebroeck D, Behringer RR, Schneider MD (2002) Endocardial cushion and myocardial defects after cardiac myocyte-specific conditional deletion of the bone morphogenetic protein receptor ALK-3. Proc Natl Acad Sci U S A 99:2878–2883CrossRefPubMedPubMedCentralGoogle Scholar
- 40.Talianidis I, Tambakaki A, Toursounova J, Zannis VI (1995) Complex interactions between SP1 bound to multiple distal regulatory sites and HNF-4 bound to the proximal promoter lead to transcriptional activation of liver-specific human APOCIII gene. Biochemistry 34:10298–10309CrossRefPubMedGoogle Scholar
- 44.Castoldi M, Vujic Spasic M, Altamura S, Elmén J, Lindow M, Kiss J, Stolte J, Sparla R, D'Alessandro LA, Klingmüller U, Fleming RE, Longerich T, Gröne HJ, Benes V, Kauppinen S, Hentze MW, Muckenthaler MU (2011) The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice. J Clin Invest 121:1386–1396CrossRefPubMedPubMedCentralGoogle Scholar