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Regulation of ferroportin degradation
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CDE
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Figure 4. ARIH1 regulates BMP6-mediated induction of hepcidin. (A) Transfection with siARIH1 successfully depleted ARIH1 in HepG2-FPN-GFP cells, as determined by quantitative polymerase chain reaction (qPCR) (mRNA expression relative to control; ***P<0.001; Student’s t-test). (B) HepG2-FPN-GFP cells were transfected with siControl or siARIH1 and were treated with doxycycline (Dox) or Dox followed by hepcidin (4 ng/mL for 18 hours [h]) as indicated. In the presence of Dox, the expression of the ferroportin-green fluorescent protein (FPN-GFP) fusion protein was induced. Treatment with hepcidin caused FPN-GFP localization to lysosomes and its subsequent degradation in siControl-treated cells as well as in siARIH1-treated cells. White bar indicates 100 μm. (C) Treatment of HepG2 cells with siARIH1 reduced the basal expression of hepcidin mRNA, as determined by qPCR (mRNA expression relative to control; ***P≤0.001; Student’s t-test). (D) Pretreatment of HepG2 cells with small interfering RNA (siRNA) directed against ARIH1 reduced BMP6-mediated hepcidin mRNA expression (relative to control), as determined by qPCR (***=P≤0.001; One-way ANOVA). (E) BMP6 (10 ng/mL for 18 h) induced the expression of ID1 in siControl transfected cells. Pretreatment of HepG2 cells with siRNA directed against ARIH1 blunted BMP6 induced expression of ID1, as determined by qPCR (mRNA expression relative to control; **P<0.01; ***P<0.001; One- way ANOVA). (F) Immunoblot showing levels of FPN-GFP and phosphorylated SMAD1/5/8 in siARIH1- or siARIH2-transfected cells in the presence (+) or absence (-) of BMP6 (10 ng/mL for 18 h). The level of pSMAD 1/5/8 was increased in all BMP6-treated cells. siRNA directed against ARIH1, but not ARIH2, prevented BMP6- induced degradation of the FPN-GFP fusion protein. GAPDH was used as a loading control. The immunoblot is representative of 4 separate experiments.
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