Smad6 and Smad7 are co-regulated with hepcidin in mouse models of iron overload

Abstract

The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe-/- and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe.

Fig. 1.

Iron supplementation increases Bmp6, Smad6, Smad7 and hepcidin mRNA expression in Wt mice maintained on different genetic backgrounds. Wt mice raised on C57BL/6J genetic background were injected with iron-dextran (n=5) or dextran as control (n=4) (A). In addition, Wt mice maintained on FVB genetic background were fed an iron-rich (4 mice/group) or iron-balanced diet (6 mice/group) (B). Hepatic non-heme iron concentrations (LIC) were measured and expressed as μg of iron per gram of dried liver tissue. Bmp6, Smad6, Smad7 and hepcidin mRNA was quantified by real-time PCR and normalized to the expression of Gapdh. Results are shown as the mean ± S.D.

Fig. 2.

Deregulated Smad6, Smad7 and hepcidin mRNA expression in Hfe −/− mice. The liver iron content (LIC; μg of iron per gram of dried liver tissue) was measured in Hfe −/− (n=19) and Wt (n=12) mice maintained on C57BL/6J genetic background and fed standard chow until the age of 8–12 weeks (A). Steady-state mRNA expression of Bmp6, Smad6, Smad7 and hepcidin in Hfe −/− was quantified by real-time PCR and normalized to the expression of Gapdh. Results are shown as the mean ± S.D. (A, B). To provide the relationship between iron mediatedregulation of Smad6, Smad7, hepcidin and Bmp6, we used iron-dextran injected Wt mice (n=5) in comparison to dextran-injected Hfe −/− (n=4) and Wt (n=4) mice (C). The ratios between Smad6, Smad7,or hepcidin to Bmp6 mRNA expression were determined individually for each sample and expressed as the mean ± S.D. relative to Wt, dextran-injected mice.

Fig. 3.

Deregulated Smad6, Smad7 and hepcidin mRNA expression in Hfe −/−, TfR2 −/− and Hfe/TfR2 mutant mice. The liver iron content (LIC; μg of iron per gram of dried liver tissue) was analyzed in Wt (n=6) and each of the HH mouse models: Hfe −/− (n=5), TfR2 −/− (n=6), and Hfe/TfR2 (n=5) mice (A), all raised on FVB genetic background. Steady-state mRNA expression of Bmp6, Smad6, Smad7 and hepcidin was quantified by real-time PCR and normalized to the expression of Gapdh. Results are shown as the mean ± S.D. (A, B). The ratios between Smad6, Smad7 or hepcidin to Bmp6 mRNA expression were determined individually for each sample and expressed as the mean ± S.D. relative to Wt mice (C).

Fig. 4.

Deregulated Smad6, Smad7 and hepcidin mRNA expression in Hjv −/− but not in hepcidin1 −/− mutant mice. The liver iron content (LIC; μg of iron per gram of dried liver tissue) was analyzed in Hjv +/+ (n=8) and Hjv −/− (n=6) mice (A). Steady-state mRNA expression of Bmp6, Smad6, Smad7 and hepcidin in Hjv −/− (A, B), hepcidin1 −/− (n=6) and hepcidin1 +/+ control (n=6) mice (D) was quantified by real-time PCR and normalized to the expression of Gapdh. Results are shown as the mean ± S.D. (A, B, D). The ratios between Smad6, Smad7 or hepcidin to Bmp6 mRNA expression were determined individually for each sample and expressed as the mean ± S.D. relative to control mice (C, E).

Fig. 5.

Systemic iron injections in Hfe −/− mice cause increased hepatic iron levels and induction of Bmp6, Smad6, Smad7 and hepcidin mRNA expression. Wt (n=5) and Hfe −/− (n=5) mice were subjected to repeated iron-dextran injections over a period of 3 weeks. In parallel 4 Wt and 4 Hfe −/− mice were injected with dextran solution only. Mice were sacrificed at the age of 8 weeks and analyzed for hepatic iron content (LIC; indicated as μg of iron per g of dried liver tissue) (A, C). The mRNA expression of Bmp6 (A, C), hepcidin, Smad7 and Smad6 mRNA (B, D) and Id1 (E) was quantified by real-time PCR and expressed relative to Gapdh mRNA expression as the mean ± S.D.

Fig. 6.

Smad6, Smad7 and hepcidin mRNA expression in Hfe/TfR2 mutant mice upon dietary iron loading. Mice with combined loss of Hfe and TfR2 (Hfe/TfR2 mice) were maintained on iron balanced (n=5) or on a high-iron diet (n=3) containing 25,000 ppm iron as previously described [13]. Four Wt mice were maintained on the same iron-rich diet. All mice were raised on FVB genetic background. Liver Smad6, Smad7 and hepcidin mRNA expression were quantified by real time PCR and normalized to Gapdh mRNA. Data are expressed as the mean ± S.D. in Hfe/TfR2 mice fed an iron-rich diet in regard to (A) Hfe/TfR2 mice fed an iron-balanced diet or (B) Wt mice fed an iron-rich diet.

Fig. 7.

Smad6 overexpression suppresses hepcidin mRNA levels in primary hepatocytes. Primary hepatocytes from C57/BL6 mice were transduced with adenoviral vectors expressing the full length murine Smad6 gene under the control of the CMV promoter (AdSmad6) or with adenoviral vectors encoding β-galactosidase as a control (AdLacZ). Cells were left untreated or treated with TGFβ and harvested for RNA and protein isolation. (A, B) mRNA expression of hepcidin, Id1 and Smad7 was analyzed by quantitative real-time PCR and normalized to the expression of Gapdh. Results are shown as the mean ± S.D. (C) The levels of pSmad1, pSmad2 and pSmad3 and β-actin proteins were analyzed by Western blot analysis.