Iron, erythropoietin, and inflammation regulate hepcidin in Bmp2-deficient mice, but serum iron fails to induce hepcidin in Bmp6-deficient mice

Abstract

The bone morphogenetic protein (BMP)-SMAD signaling pathway is a key transcriptional regulator of hepcidin in response to tissue iron stores, serum iron, erythropoietic drive and inflammation to increase the iron supply when needed for erythropoiesis, but to prevent the toxicity of iron excess. Recently, BMP2 was reported to play a non-redundant role in hepcidin regulation in addition to BMP6. Here, we used a newly validated BMP2 ELISA assay and mice with a global or endothelial conditional knockout (CKO) of Bmp2 or Bmp6 to examine how BMP2 is regulated and functionally contributes to hepcidin regulation by its major stimuli. Erythropoietin (EPO) did not influence BMP2 expression in control mice, and still suppressed hepcidin in Bmp2 CKO mice. Lipopolysaccharide (LPS) reduced BMP2 expression in control mice, but still induced hepcidin in Bmp2 CKO mice. Chronic dietary iron loading that increased liver iron induced BMP2 expression, whereas acute oral iron gavage that increased serum iron without influencing liver iron did not impact BMP2. However, hepcidin was still induced by both iron loading methods in Bmp2 CKO mice, although the degree of hepcidin induction was blunted relative to control mice. Conversely, acute oral iron gavage failed to induce hepcidin in Bmp6 ^-/- or CKO mice. Thus, BMP2 has at least a partially redundant role in hepcidin regulation by serum iron, tissue iron, inflammation and erythropoietic drive. In contrast, BMP6 is absolutely required for hepcidin regulation by serum iron.

Figure 1:. BMP2 is not regulated by EPO and is not required for hepcidin suppression by EPO.

(A-B) Bmp2 endothelial CKO (Bmp2^fl/fl;Tek-Cre+) and littermate Cre- control male (blue circles) and female mice (red triangles) were sacrificed at 8 weeks of age (n=3–5 per sex per group). Levels of (A) serum and (B) liver BMP2 were quantitated by ELISA. (C-G) Bmp2^fl/fl;Tek-Cre+ and littermate Cre- control male (blue circles, n=3–5 per group) and female (red triangles, n=6–8 per group) mice at 6 weeks of age were injected with PBS or EPO (200 U per mouse) and tissues were collected after 15 hours to determine relative (C) bone marrow erythroferrone (Erfe), (D) liver Bmp2, and (G) liver hepcidin (Hamp) mRNA levels by qRT-PCR. Transcript levels were normalized to Rpl19. (E) Liver and (F) serum BMP2 protein levels in female Cre- animals were determined by ELISA. Values represent mean ± SEM. **P<0.01, ***P<0.001 relative to the respective Cre- controls or PBS-treated controls of the same genotype by Student’s t test. Fold-change relative to PBS treated controls of the same genotype as calculated by 2^-ΔΔCt are reported in panel G.

Figure 2:. BMP2 is suppressed by LPS and is not required for hepcidin induction by LPS.

Bmp2^fl/fl;Tek-Cre+ and littermate Cre- control male (blue circles, n=6–8 per group) and female (red triangles, n=4–5 per group) mice at 6 weeks of age were injected with PBS or LPS (1 mg per kg body weight) and sacrificed after 6 hours. Livers were collected to determine (A) Il6, (B) Bmp2, (E) Bmp6 and (F) Hamp mRNA levels. Transcripts measured by qRT-PCR were normalized to Rpl19. (C) Liver and (D) serum BMP2 protein levels in Cre- animals were determined by ELISA. Values represent mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 relative to the PBS-treated controls of the same genotype by Student’s t test. Fold-change relative to PBS treated controls of the same genotype as calculated by 2^-ΔΔCt are reported in panel F.

Figure 3:. BMP2 is regulated concordantly with hepcidin by chronic changes in dietary iron, but BMP2 is partially redundant for hepcidin induction by chronic dietary iron loading.

(A-E) 4-week-old C57BL/6 male mice were treated with a low (2–6 ppm), sufficient (48 ppm, Control) or high iron (2% carbonyl iron) diet for 3 weeks (n=8 per group). (F-I) 3-week-old male (blue circles) and female (red triangles) Bmp2^fl/fl;Tek-Cre+ and littermate Cre- controls were weaned to a low iron diet for 3 weeks and then either switched to the house diet (380 ppm iron) or kept on a low iron diet for 1 more week (n=6 per group, 2–4 of each sex). (A) Serum iron and (B,F) liver iron levels were quantified by colorimetric assay. (C) Liver Bmp2, (G) Bmp6, and (H) Hamp mRNA levels were measured by qRT-PCR and normalized to Rpl19. (D) Liver and (E) serum BMP2 protein levels were quantified by ELISA. (I) Liver Hamp normalized to Rpl19 mRNA relative to liver iron content was calculated for mice switched to the house diet. Values represent mean ± SEM. *P<0.05, **P<0.01, *** P<0.001 relative to mice treated with a control diet by one-way ANOVA with Tukey’s post-hoc test (Panels A-E), a low iron diet of the same genotype by Student’s t test (Panels F-H) or Cre- mice switched to a house diet by Student’s t test (Panel I). Fold-change relative to low iron diet controls of the same genotype as calculated by 2^-ΔΔCt are reported in panel H.

Figure 4:. Neither BMP2 nor BMP6 are regulated by acute serum iron loading, but BMP2 has a partially redundant role, whereas BMP6 is required for hepcidin induction by acute serum iron loading.

3-week-old male (blue circles) and female (red triangles) Bmp2^fl/fl;Tek-Cre+ and littermate Cre- controls (n=13–17 per group, 5–10 of each sex) and Bmp6^fl/fl;Tek-Cre+ mice (n=12 per group, 3–4 males and 8–9 females) were weaned to a low iron (2–6 ppm) diet for 3 weeks before treated with 2 mg/kg ferrous sulfate in 0.5 M ascorbic acid or equivalent volume of distilled water by oral gavage. Mice were sacrificed 6 hours after gavage. (A) Serum transferrin saturation and (B) liver iron levels were determined by colorimetric assays. (C) Liver Bmp6, (D) Bmp2 and (G) Hamp mRNA levels were quantified and normalized to Rpl19 by qRT-PCR. (E) Serum and (F) liver BMP2 protein concentrations were measured by ELISA. Values represent mean ± SEM. **P<0.01, ***P<0.001 relative to mice treated with distilled water of the same genotype by Student’s t test. Fold-change relative to vehicle treated controls of the same genotype as calculated by 2^-ΔΔCt are reported in panel G.