Foxo1 and Smad proteins coregulate hepcidin transcription. (A) Analysis of Foxo binding sites (red) and Smad binding sites/BMP-responsive elements (green) in the 3000 bp upstream region of hepcidin gene transcription start site in different mammals. (B) In the murine Hamp1 promoter, a scheme summarizing the 2 predicted Foxo binding sites and their corresponding mutant forms. WT, wild-type Hamp1 promoter; Mut, Hamp1 promoter with mutated Foxo binding site (red). (C-D) ChIP-qPCR analysis of the interaction between Foxo1 and 2 Foxo binding sites in the murine Hamp1 promoter region under basal condition (C) or holo-Tf/BMP6/AS1842856–treated condition (D). Cells were treated with 10 μM AS1842856 1 hour before the addition of 20 ng/mL BMP6 and/or 30 μM holo-Tf (AS1842856, 7 hours; BMP6, 6 hours; holo-Tf, 6 hours). (E) As indicated in panel B, Hamp1 promoter luciferase reporter plasmids containing wild-type (WT) or mutated (Mut) Foxo binding sites were transfected into primary hepatocytes and assayed for their luciferase reporter activities (holo-Tf, 30 μM; BMP6, 20 ng/mL; 7 hours). (F) Immunoprecipitation (IP) and western blot analysis of the Foxo1-pSmad1/5/8 interaction in primary hepatocytes from 8-week-old male Foxo1LKO and Foxo1flox/flox mice (left) and 8-week-old male WT 129S2/SvPasCrl mice (right). (G) IP/western blot analysis of the Foxo1-pSmad1/5/8 interaction in the cytosol of primary hepatocytes from 8-week-old male Foxo1LKO and Foxo1flox/flox mice. (H) IP/western blot analysis of the Foxo1-pSmad1/5/8 interaction in the primary hepatocytes treated with holo-Tf and/or BMP6 (7 hours; holo-Tf, 30 μM; BMP6, 20 ng/mL). (I) Quantifications of Foxo1-pSmad1/5/8 interaction from results displayed in panel H. (J) A working model to elucidate the role of Foxo1 in hepatic hepcidin regulation. Primary hepatocytes were isolated from 8-week-old male WT 129S2/SvPasCrl mice. Results from ChIP-qPCR, luciferase assay, IP/western blot, and corresponding quantification analysis were all obtained from 3 independent biological replicates. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. The P value was calculated by the Student t test for 2 group comparison or the 1-way ANOVA with the Sidak post hoc test for multiple comparison. For panels D-E, multiple comparison test was separately performed in different Foxo binding sites or their mutations.
Figure 6.

Foxo1 and Smad proteins coregulate hepcidin transcription. (A) Analysis of Foxo binding sites (red) and Smad binding sites/BMP-responsive elements (green) in the 3000 bp upstream region of hepcidin gene transcription start site in different mammals. (B) In the murine Hamp1 promoter, a scheme summarizing the 2 predicted Foxo binding sites and their corresponding mutant forms. WT, wild-type Hamp1 promoter; Mut, Hamp1 promoter with mutated Foxo binding site (red). (C-D) ChIP-qPCR analysis of the interaction between Foxo1 and 2 Foxo binding sites in the murine Hamp1 promoter region under basal condition (C) or holo-Tf/BMP6/AS1842856–treated condition (D). Cells were treated with 10 μM AS1842856 1 hour before the addition of 20 ng/mL BMP6 and/or 30 μM holo-Tf (AS1842856, 7 hours; BMP6, 6 hours; holo-Tf, 6 hours). (E) As indicated in panel B, Hamp1 promoter luciferase reporter plasmids containing wild-type (WT) or mutated (Mut) Foxo binding sites were transfected into primary hepatocytes and assayed for their luciferase reporter activities (holo-Tf, 30 μM; BMP6, 20 ng/mL; 7 hours). (F) Immunoprecipitation (IP) and western blot analysis of the Foxo1-pSmad1/5/8 interaction in primary hepatocytes from 8-week-old male Foxo1LKO and Foxo1flox/flox mice (left) and 8-week-old male WT 129S2/SvPasCrl mice (right). (G) IP/western blot analysis of the Foxo1-pSmad1/5/8 interaction in the cytosol of primary hepatocytes from 8-week-old male Foxo1LKO and Foxo1flox/flox mice. (H) IP/western blot analysis of the Foxo1-pSmad1/5/8 interaction in the primary hepatocytes treated with holo-Tf and/or BMP6 (7 hours; holo-Tf, 30 μM; BMP6, 20 ng/mL). (I) Quantifications of Foxo1-pSmad1/5/8 interaction from results displayed in panel H. (J) A working model to elucidate the role of Foxo1 in hepatic hepcidin regulation. Primary hepatocytes were isolated from 8-week-old male WT 129S2/SvPasCrl mice. Results from ChIP-qPCR, luciferase assay, IP/western blot, and corresponding quantification analysis were all obtained from 3 independent biological replicates. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. The P value was calculated by the Student t test for 2 group comparison or the 1-way ANOVA with the Sidak post hoc test for multiple comparison. For panels D-E, multiple comparison test was separately performed in different Foxo binding sites or their mutations.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal