Suppression of iron-regulatory hepcidin by vitamin D

Abstract

The antibacterial protein hepcidin regulates the absorption, tissue distribution, and extracellular concentration of iron by suppressing ferroportin-mediated export of cellular iron. In CKD, elevated hepcidin and vitamin D deficiency are associated with anemia. Therefore, we explored a possible role for vitamin D in iron homeostasis. Treatment of cultured hepatocytes or monocytes with prohormone 25-hydroxyvitamin D or active 1,25-dihydroxyvitamin D decreased expression of hepcidin mRNA by 0.5-fold, contrasting the stimulatory effect of 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D on related antibacterial proteins such as cathelicidin. Promoter-reporter and chromatin immunoprecipitation analyses indicated that direct transcriptional suppression of hepcidin gene (HAMP) expression mediated by 1,25-dihydroxyvitamin D binding to the vitamin D receptor caused the decrease in hepcidin mRNA levels. Suppression of HAMP expression was associated with a concomitant increase in expression of the cellular target for hepcidin, ferroportin protein, and decreased expression of the intracellular iron marker ferritin. In a pilot study with healthy volunteers, supplementation with a single oral dose of vitamin D (100,000 IU vitamin D2) increased serum levels of 25D-hydroxyvitamin D from 27±2 ng/ml before supplementation to 44±3 ng/ml after supplementation (P<0.001). This response was associated with a 34% decrease in circulating levels of hepcidin within 24 hours of vitamin D supplementation (P<0.05). These data show that vitamin D is a potent regulator of the hepcidin-ferroportin axis in humans and highlight a potential new strategy for the management of anemia in patients with low vitamin D and/or CKD.

Figure 1.

Vitamin D suppresses expression of hepcidin (HAMP) in human monocytes and hepatocytes. Effect of in vitro treatment of PBMC monocytes (PBMCm), monocytic THP1 cells, and HepG2 hepatocytic cells with vehicle, 25D (100 nM), or 1,25D (5 nM) for 6 hours on expression of mRNA for HAMP (A), CAMP (B), and CYP24A1 (C). Data are shown as mean fold-change in gene expression (±SD) relative to vehicle (0.1% ethanol) controls. *P<0.05; **P<0.01; ***P<0.001, statistically different from vehicle-treated cells. For RT-PCR data, n=8 separate donors for PBMC monocytes, n=4 separate cultures of THP1 cells, and n=5 separate cultures of HepG2 cells.

Figure 2.

VDR-mediated suppression of hepcidin (HAMP) gene expression by 1,25D. (A) ChIP analysis of VDR and RNA Pol II interaction with the HAMP gene promoter. PBMC monocytes are treated with 1,25D (5 nM, 24 hours), chromatin extracts are prepared, and ChIP-grade antibodies are used to detect VDR and RNA Pol II interactions. The resulting enriched genomic DNA is qPCR amplified using primers for CYP24A1 (−1 kb from the transcription start site and −2 kb from the transcription start site), CAMP (−1 kb from the transcription start site), HAMP (−1 kb from the transcription start site). A negative control sequence for VDR and RNA Pol II (IGX1A) is also used. Data are shown first as mean arbitrary units for PCR amplification of DNA associated with RNA Pol II or VDR in cells treated with vehicle or 1,25D (mean of two separate chromatin preparations). In addition, fold-change values are presented showing the change in RNA Pol II or VDR binding to DNA after treatment with 1,25D relative to vehicle-treated cells. (B) Effect of 25D (100 nM) and 1,25D (5 nM) on HAMP promoter-reporter activity in VDR-expressing MC3T3 mouse osteoblastic cells. Data are shown as HAMP target gene firefly/Renilla housekeeping luciferase activity (×10⁻³). *P<0.05, statistically different from vehicle-treated cells.

Figure 3.

Effect of vitamin D metabolites on ferroportin and ferritin expression in human monocytes and hepatocytes. (A) Effect of in vitro treatment of PBMC monocytes (PBMCm), monocytic THP1 cells, and HepG2 hepatocytic cells with vehicle, 25D (100 nM) or 1,25D (5 nM) for 6 hours on ferroportin mRNA expression. Data are shown as mean fold-change in gene expression (±SD relative to vehicle [0.1% ethanol] controls). (B) Western blot analysis of protein for ferroportin protein in HepG2 cells treated with vehicle, 25D (100 nM), or 1,25D (5 nM) for 24 hours. Loading is normalized by analysis of the housekeeping protein β-actin. (C) Immunohistochemical analysis of ferroportin protein in PBMC monocytes after treatment with vehicle or 1,25D (5 nM) for 24 hours. Ferroportin protein is shown in red with 4′,6-diamidino-2-phenylindole (DAPI) staining of nuclei shown in blue. (D) Effect of in vitro treatment of PBMC monocytes, THP1 cells, and HepG2 cells with vehicle, 25D (100 nM), or 1,25D (5 nM) for 6 hours on ferritin mRNA expression. Data are shown as mean fold-change in gene expression (±SD relative to vehicle controls). (E) Immunohistochemical analysis of ferritin protein in HepG2 cells after treatment with vehicle, 25D (100 nM), or 1,25D (5 nM) for 24 hours. Ferritin protein is shown in red with DAPI staining of nuclei in blue. *P<0.05; **P<0.01; ***P<0.001, statistically different from vehicle-treated cells. For RT-PCR data, n=8 separate donors for PBMC monocytes, n=4 separate cultures of THP1 cells, and n=5 separate cultures of HepG2 cells.

Figure 4.

Effects of supplementation with vitamin D₂ on circulating hepcidin levels in healthy humans. A single-arm pharmacokinetic study is performed in seven healthy volunteers (four men; median age 42 years; range, 27–63) to assess changes in serum levels of hepcidin after a single dose of oral vitamin D₂ (100,000 IU). Two blood samples are drawn before supplementation and two are drawn after supplementation. Serum concentrations of 25D (ng/ml) (A), 1,25D (pg/ml) (B), and hepcidin (ng/ml) (C). Data are shown as the mean±SEM. Experimental means were compared statistically using a paired t test.*P<0.05; **P<0.01; ***P<0.001, statistically different from baseline values.

Figure 5.

Vitamin D and the hepcidin-ferroportin iron-regulatory axis. Schematic representation of a proposed mechanism for vitamin D regulation of hepcidin/HAMP – ferroportin (Fp) interaction in hepatocytes and monocytes. Under conditions of vitamin D deficiency, elevated synthesis of hepcidin by hepatocytes or monocytes may increase intracellular and systemic concentrations of hepcidin and decrease membrane expression of Fp in these cells. The resulting suppression of iron export will, in turn, lead to intracellular accumulation, increased cellular ferritin, and decreased systemic levels of iron. Under conditions of vitamin D sufficiency, decreased transcription of HAMP may lead to decreased intracellular and systemic concentrations of hepcidin and concomitant increased membrane expression of Fp. The resulting enhancement of iron export may then lead to decreased intracellular iron and ferritin and increased systemic levels of iron.