Progesterone receptor membrane component-1 regulates hepcidin biosynthesis
Xiang Li, … , Donald B. Bloch, Randall T. Peterson
Xiang Li, … , Donald B. Bloch, Randall T. Peterson
Published December 14, 2015
Citation Information: J Clin Invest. 2016;126(1):389-401. https://doi.org/10.1172/JCI83831.
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Research Article Hematology

Progesterone receptor membrane component-1 regulates hepcidin biosynthesis

Abstract

Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism.

Authors

Xiang Li, David K. Rhee, Rajeev Malhotra, Claire Mayeur, Liam A. Hurst, Emily Ager, Georgia Shelton, Yael Kramer, David McCulloh, David Keefe, Kenneth D. Bloch, Donald B. Bloch, Randall T. Peterson

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Figure 4

PGRMC1 is required for progesterone, but not BMP6 or IL-6, to increase hepcidin gene expression.

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PGRMC1 is required for progesterone, but not BMP6 or IL-6, to increase h...
(A–C) qPCR for PGRMC1 gene expression in HepG2 cells transfected with scrambled siRNAs (siControl) or siRNAs directed against PGRMC1 (siPGRMC1) and exposed to vehicle, (A) progesterone (30 μM for 8 hours), (B) BMP6 (20 ng/ml for 3 hours), or (C) IL-6 (100 ng/ml for 3 hours. *P < 0.01, #P < 0.001 compared with siControl cells treated with vehicle, 2-way ANOVA (n = 4 per group). (D–F) qPCR for hepcidin gene expression in HepG2 cells transfected with siControl or siPGRMC1 siRNAs and treated with vehicle, (D) progesterone, (E) BMP6, or (F) IL-6 (identical concentrations and incubation times as above). (G–I) qPCR for hepcidin gene expression in HepG2 cells preincubated with control IgG or anti-PGRMC1 antibodies (PGRMC1 Ab) for 3 hours and then exposed to vehicle or (G) progesterone, (H) BMP6, or (I) IL-6 (identical concentrations and incubation times as above). #P < 0.001, *P < 0.01, compared with siControl cells (D–F) or IgG-treated cells (G–I) exposed to vehicle; **P < 0.001 compared with siControl cells (D) or IgG-treated cells (G) exposed to progesterone, 2-way ANOVA (n = 4 per group). (J–L) A plasmid containing the pgrmc1 morpholino oligonucleotide target site upstream of an EGFP coding sequence was injected (J) alone, (K) with control morpholino oligonucleotides (control MO), or (L) with pgrmc1 morpholino oligonucleotides (pgrmc1 MO) into 1-cell stage zebrafish embryos, and fluorescence was examined after 8 hours (original magnification, ×2.5). (M) qPCR for hepcidin gene expression in 3-day-old wild-type zebrafish embryos injected with control morpholino oligonucleotides or pgrmc1 morpholino oligonucleotides and treated with vehicle or progesterone (5 μM for 12 hours). #P < 0.001 compared with control morpholino oligonucleotides embryos treated with vehicle, **P < 0.001 compared with control morpholino oligonucleotides embryos treated with progesterone, 2-way ANOVA (n = 5 per group). qPCR results expressed as mean ± SEM.