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HNF-4alpha Negatively Regulates Hepcidin Expression Through BMPR1A in HepG2 Cells

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Abstract

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.

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Acknowledgments

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.

Author Contributions

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.

Author information

Authors and Affiliations

  1. Military Hygiene Department, Faculty of Naval Medicine, Second Military Medical University, No. 800 Xiangyin Road, Shanghai, 200433, China

    Wencai Shi, Heyang Wang, Xin Jiang, Zheng Xu, Hui Shen & Min Li

  2. Department of Clinical Nutrition, Changhai Hospital, Second Military Medical University, Shanghai, China

    Wencai Shi & Xuan Zheng

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  1. Wencai Shi
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Correspondence to Min Li.

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Wencai Shi and Heyang Wang contribute equally to this work.

Electronic supplementary material

ESM 1

HNF-4α reduces Id1 expression in HepG2 cells. HepG2 cells were transferred to 6-well plates and incubated with growth medium containing negative invalid control siRNA (Con) or specific HNF-4α siRNA (si-HNF4α) (A), empty control plasmids (Con) or plasmids encoding HNF-4α (oe-HNF4α) (B) for 12 h and normal growth medium for additional 36 h. Id1 protein was measured by Western blotting. ***, significantly different from control, p < 0.001. Values are expressed as mean ± S.E.M., determined in three independent experiments. Statistical difference between two groups was assessed by the Independent-t test. (DOCX 219 kb)

ESM 2

HNF-4α blocks BMP2-induced stimulation of hepcidin in HepG2 cells. HepG2 cells were transfected with 2 μg HNF-4α or empty plasmids, and then incubated for 48 h, with or without 2 μg/ml recombinant human BMP2 in the last 8 h. Protein levels of HNF-4α, hepcidn and β-actin were detected by Western blotting. ***, significantly different from control, p < 0.001. Values are expressed as mean ± S.E.M., determined in three independent experiments. Statistical difference between groups was assessed by one-way ANOVA followed by LSD t post hoc test. (DOCX 83 kb)

ESM 3

HNF-4α has no effect on the synthesis of novel BMPR1A ligands. Protein levels of BMP2, BMP4, BMP6, and BMP7 were detected by Western blotting (BMP2: Proteintech, USA; 1:1000 dilution; BMP4: BBI, China; 1:1000 dilution; BMP6: BBI, China; 1:1000 dilution; BMP7: Proteintech, USA; 1:1000 dilution) in HepG2 cells transfected with invalid negative control siRNA (Con) or specific HNF-4α siRNA (si-HNF4α), or in empty HepG2 cells transfected with empty control plasmids (Con) or plasmids encoding HNF-4α (oe-HNF4α). Values are expressed as mean ± S.E.M., determined in three independent experiments. Statistical difference between two groups was assessed by the Independent-t test. (DOCX 278 kb)

ESM 4

HNF-4α has no effect on the expression of intracellular inhibitory SMADs. Protein levels of SMAD6 and SAMD7 were detected by Western blotting (SMAD6: Bioworld, USA; 1:500 dilution; SMAD7: BBI, China; 1:1000 dilution) in HepG2 cells transfected with invalid negative control siRNA(Con) or specific HNF-4α siRNA (si-HNF4α), or in empty HepG2 cells transfected with empty control plasmids (Con) or plasmids encoding HNF-4α (oe-HNF4α). Values are expressed as mean ± S.E.M., determined in three independent experiments. Statistical difference between the two groups was assessed by the Independent-t test. (DOCX 165 kb)

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Shi, W., Wang, H., Zheng, X. et al. HNF-4alpha Negatively Regulates Hepcidin Expression Through BMPR1A in HepG2 Cells. Biol Trace Elem Res 176, 294–304 (2017). https://doi.org/10.1007/s12011-016-0846-5

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