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Contribution of STAT3 and SMAD4 pathways to the regulation of hepcidin by opposing stimuli

Hua Huang, Marco Constante, Antonio Layoun and Manuela M. Santos

Article Figures & Data

Figures

  • Figure 1

    Factors affecting mRNA hepcidin expression in the liver. Hepcidin levels are regulated by iron levels (store regulator), immune mediators (inflammatory regulator), hypoxia (hypoxia regulator), and erythropoietic demand (erythroid regulator). Pointed arrows indicate up-regulation of hepcidin, and blunt arrows inhibition of its mRNA expression. For each regulator, the treatments used in this study to stimulate or suppress hepcidin expression are shown.

  • Figure 2

    Erythropoietin but not hypoxia inhibits hepcidin induction through the inflammatory pathway. (A) Hepcidin mRNA levels in the liver of mice treated with saline (control [CTL]), EPO, LPS, and mice with combined treatments (EPO + LPS). (B) Hepcidin mRNA levels in the liver of mice treated with saline (CTL), mice subjected to 10% oxygen (hypoxia [Hpx]), LPS, and mice with combined treatments (Hpx + LPS). Hepatic hepcidin expression was quantified by real-time RT-PCR and normalized to β-actin. The hepcidin/β-actin ratios are shown, each symbol representing 1 mouse. Statistical analysis was performed by 1-way ANOVA; **P < .001 for comparison with control mice. n.s. indicates not significant.

  • Figure 3

    EPO inhibits LPS-mediated hepcidin induction independently of IL-6 production. (A) IL-6 mRNA levels in liver, (B) IL-6 levels in serum, and (C) intrahepatic IL-6 protein levels in mice treated with saline (CTL), EPO, LPS, and mice with combined treatments (EPO + LPS). (D) Hepcidin mRNA levels in the liver of mice treated with saline (CTL), EPO, mouse recombinant IL-6 (IL-6), and mice with combined treatments (EPO + IL-6). IL-6 and hepcidin mRNA levels were quantified by real-time RT-PCR and normalized to β-actin. The IL-6/β-actin and hepcidin/β-actin ratios are shown. IL-6 protein levels were measured by ELISA. Statistical analysis was performed by 1-way ANOVA; *P < .01 and **P < .001 for comparison with control mice. Data are presented as means plus or minus SD with n = 6 mice per group in panels A through C and as individual mice in panel D. n.s. indicates not significant.

  • Figure 4

    Stat3 phosphorylation induced by LPS is partially inhibited by EPO but not by hypoxia. (A,C) Liver nuclear extracts from mice treated with saline (CTL), EPO, hypoxia, LPS, and mice with combined treatments (EPO + LPS and hypoxia + LPS) were analyzed by Western blotting with an antibody to phosphorylated Stat3 and total Stat3. Blots were stripped and reprobed with an antibody to β-actin as loading control. A representative Western blot is shown. Lane “+” is a positive control consisting of total cell extracts from serum-starved HeLa cells prepared with interferon-α treatment. (B,D) Quantification of chemiluminescence to calculate the ratio of phosphorylated Stat3 relative to β-actin (pStat3/β-actin). This experiment was repeated twice, and the combined results are shown as means plus or minus SD with n = 7. Statistical analysis was performed by 1-way ANOVA; *P < .01, **P < .001; and ***P < .0001 for comparison with control mice. n.s. indicates not significant.

  • Figure 5

    EPO but not hypoxia inhibits hepcidin induction through the iron-sensing pathway. (A) Hepcidin mRNA levels in the liver of mice treated with saline (CTL), EPO, LPS, carbonyl iron–supplemented diet (2.5% CI), and mice with combined treatments (EPO + CI). (B) Hepcidin mRNA levels in the liver of mice treated with saline (CTL), mice subjected to 10% oxygen (Hpx), 2.5% CI, and mice with combined treatments (Hpx + CI). Hepatic hepcidin expression was quantified by real-time RT-PCR and normalized to β-actin. The hepcidin/β-actin ratios are shown, each symbol representing one mouse. Statistical analysis was performed by 1-way ANOVA; **P < .001 for comparison with control mice. n.s. indicates not significant.

  • Figure 6

    Smad1/5/8 phosphorylation induced by dietary iron-loading is partially inhibited by erythropoietin, but not by hypoxia. (A,C) Liver nuclear extracts from mice treated with saline (CTL), EPO, 2.5% CI, hypoxia, and mice with combined treatments (EPO + CI and Hypoxia + CI) were analyzed by Western blotting with an antibody to phosphorylated Smad1/5/8 and total Smad1/5/8. Blots were stripped and reprobed with an antibody to β-actin as loading control. A representative Western blot is shown. (B,D) Quantification of chemiluminescence to calculate the ratio of phosphorylated Smad1/5/8 relative to β-actin (pSmad/β-actin). This experiment was repeated twice, and the combined results are shown as means plus or minus SD with n = 7. Statistical analysis was performed by 1-way ANOVA; *P < .01; and **P < .001 for comparison with control mice. n.s. indicates not significant.

  • Figure 7

    Relationship between erythropoiesis rate and hepatic hepcidin expression. (A) Spleen weight–body weight ratio in control, hypoxic, and EPO-treated mice (50 U for 4 days). (B) Spleen weight–body weight ratio and (C) hepatic hepcidin expression in mice treated with increasing amounts of EPO (total dosage over 4 days is shown) alone (CTL) and in combination with LPS or CI-supplemented diet (CI). (A-C) Data are presented as means plus or minus SD with n = 5 to 6 mice per group. Statistical analysis was performed by 1-way ANOVA; *P < .01 and **P < .001 for comparison with control mice. (D-F) Negative correlation between spleen weight–body weight ratio and hepatic hepcidin expression in mice treated with increasing amounts of EPO: (D) alone (EPO); (E) in combination with LPS (EPO + LPS); and (F) in combination with CI-supplemented diet (EPO + CI). Hepatic hepcidin expression was quantified by real-time RT-PCR and normalized to β-actin. The hepcidin/β-actin ratios are shown.

  • Figure 8

    Additive effect of LPS and dietary iron on EPO-mediated hepcidin suppression. Hepatic hepcidin expression in control mice (CTL), mice treated with EPO alone (50 U for 4 days; EPO) and in combination with LPS (EPO + LPS), with CI-supplemented diet (EPO + CI), with both LPS and CI (EPO + LPS + CI), with LPS alone (LPS), and CI-supplemented diet alone (CI). Data are presented as means plus or minus SD with n = 6 mice per group. Statistical analysis was performed by 1-way ANOVA; *P < .05 for comparison with control mice.