Estrogen regulates hepcidin expression via GPR30-BMP6-dependent signaling in hepatocytes

Abstract

Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E(2)) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E(2)-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E(2) and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E(2)-induced hepcidin expression. BMP6 expression induced by E(2) was abolished by GPR30 silencing. Finally, both E(2) and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E(2) and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Effect of estrogen deprivation via an ovariectomy on hepcidin expression and iron absorption in vivo.

The expression of hepcidin (A) and bone morphological protein (BMP6) (B) was reduced in the livers of the OVX mice. The expression of hepcidin and BMP6 in liver tissues at 3 months after the sham operation or ovariectomy was analyzed. The expression values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 6–10 in each group. (C) The expression of ferroportin (FPN) was augmented in the duodena of the OVX mice. The protein expression of FPN in duodenal tissues at 3 months after the sham operation or ovariectomy was analyzed. Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 6 in each group. (D) Immunohistochemical analysis of FPN expression in the duodena of sham-operated mice (upper) and OVX mice (lower). (E) Serum (µg⋅dl−1 ) and hepatic (ng⋅g−1 ) iron concentrations in sham-operated and OVX mice. Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 8–12 in each group.

Figure 2. The effect of estrogen on hepcidin expression in HepG2 cells.

(A) E2 treatment upregulated hepcidin expression in a concentration-dependent manner. HepG2 cells were treated with E2 for 24 h. Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 4–8 in each group. (B) E2-induced hepcidin expression was not abolished by the ER inhibitor ICI 182720. HepG2 cells were pretreated with ICI 182720 (1×10−6 M) for 1 h before E2 treatment. Subsequently, the cells were treated with E2 (1×10−7 M) or vehicle for 24 h. Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 4 in each group. (C) E2 and ICI 182780 increased hepcidin expression in HuH-7 cells. HuH-7 cells were pretreated with ICI 182720 (1×10−6 M) for 1 h before E2 treatment. Subsequently, the cells were treated with E2 (1×10−7 M) or vehicle for 24 h. Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 5 in each group.

Figure 3. Silencing of GPR30 reduced E2-induced hepcidin expression in HepG2 cells.

(A) HepG2 cells were transfected with 40 nM GPR30 siRNA. GPR30 mRNA levels were reduced after treatment with GPR30 siRNA; n = 4 in each group. (B) Treatment with GPR30 siRNA suppressed E2-induced hepcidin upregulation in HepG2 cells. Forty-eight hours after siRNA transfection, HepG2 cells were treated with E2 (1×10−7 M) or vehicle for 24 h; n = 10 in each group. (C) Treatment with GPR30 siRNA decreased ICI 182720-induced hepcidin upregulation in HepG2 cells. Forty-eight hours after siRNA transfection, HepG2 cells were treated with ICI 182720 (1×10−6 M) or vehicle for 24 h; n = 4 in each group. (D) The effect of the GPR30 antagonist G1 in HepG2 cells. G1 upregulated hepcidin expression in HepG2 cells in a concentration-dependent manner. Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 4–8 in each group.

Figure 4. BMP6 signaling is involved in estrogen-induced hepcidin expression via GPR30 in HepG2 cells.

(A) Effects of E2 (1×10−7 M), ICI 182720 (1×10−6 M), and G1 (1×10−8 M) on BMP6 expression in HepG2 cells. HepG2 cells were incubated for 24 h with each reagent; n = 10 in each group. (B) E2-induced upregulation of BMP6 expression was inhibited by GPR30 silencing in HepG2 cells. Forty-eight hours after GPR30 siRNA transfection, HepG2 cells were treated with E2 (1×10−7 M) or vehicle for 24 h; n = 8 in each group. (C) BMP6 silencing decreased hepcidin expression in HepG2 cells. Left panel: HepG2 cells transfected with 40 nM BMP6 siRNA. BMP6 mRNA levels were reduced after treatment with BMP6 siRNA. Values are expressed as mean ± SEM; *P<0.05, n = 4 in each group. Right panel: Treatment with BMP6 siRNA decreased hepcidin expression in HepG2 cells. Hepcidin downregulation induced by BMP6 silencing was not restored by E2 treatment. Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 8 in each group.

Figure 5. The mRNA and protein expression of hepcidin in the liver.

(A) The mRNA expression of hepcidin in the liver of OVX mice was restored by E2 or G1 supplementation. Hepcidin expression was decreased in OVX mice (black bar); and its expression was restored by administration of E2 (light gray bar) or G1 (dark gray bar). Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 7 in each group. (B) The protein expression of hepcidin in the liver of OVX mice was restored by E2 or G1 supplementation. Hepcidin expression was decreased in the OVX mice (black bar); its expression was restored by administration of E2 (light gray bar) or G1 (dark gray bar). Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 6–8 in each group. The mRNA and protein expression of BMP6 in the liver. (C) The mRNA expression of BMP6 in the livers of OVX mice was recovered by E2 or G1 supplementation. BMP6 expression was diminished in OVX mice (black bar); its expression was restored by the administration of E2 (light gray bar) or G1 (dark gray bar). Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 7 in each group. (D) The protein expression of BMP6 in the liver of OVX mice was recovered by E2 or G1 supplementation. BMP6 expression was diminished in thr OVX mice (black bar); and its expression was restored by administration of E2 (light gray bar) or G1 (dark gray bar). Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 6–8 in each group. (D).

Figure 6. The changes of FPN expression in the duodenum, serum and liver iron, and serum hepcidin.

(A) OVX-induced duodenal FPN upregulation was ameliorated in the OVX mice by E2 or G1 administration. FPN expression was augmented in OVX mice (black bar); its expression was diminished by treatment with E2 (light gray bar) or G1 (dark gray bar). Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 6 in each group. (B) Immunohistochemical analysis of FPN expression in the duodenal tissue of sham-operated mice, OVX mice, and OVX mice treated with E2 or G1. (C) Serum iron levels were increased in the OVX mice (black bar); levels were diminished by the administration of E2 (light gray bar) or G1 (dark gray bar). Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 8–13 in each group. (D) Liver iron concentration in the liver was elevated in OVX mice (black bar); its concentration was reduced by the administration of E2 (light gray bar) or G1 (dark gray bar). Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 8–11 in each group. (E) Serum hepcidin-1 levels were augmented in the OVX mice (black bar) compared to the sham- operated mice (white bar); its levels was reduced by the administration of E2 (light gray bar) or G1 (dark gray bar). Values are expressed as mean ± SEM; *P<0.05, **P<0.01, n = 8–9 in each group.