Effect of Iron Overload and Iron Deficiency on Liver Hemojuvelin Protein

INTRODUCTION: Hemojuvelin (Hjv) is a key component of the signaling cascade that regulates liver hepcidin (Hamp) expression. The purpose of this study was to determine Hjv protein levels in mice and rats subjected to iron overload and iron deficiency. METHODS: C57BL/6 mice were injected with iron (200 mg/kg); iron deficiency was induced by feeding of an iron-deficient diet, or by repeated phlebotomies. Erythropoietin (EPO)-treated mice were administered recombinant EPO at 50 U/mouse. Wistar rats were injected with iron (1200 mg/kg), or fed an iron-deficient diet. Hjv protein was determined by immunoblotting, liver samples from Hjv-/- mice were used as negative controls. Mouse plasma Hjv content was determined by a commercial ELISA kit. RESULTS: Liver crude membrane fraction from both mice and rats displayed a major Hjv-specific band at 35 kDa, and a weaker band of 20 kDa. In mice, the intensity of these bands was not changed following iron injection, repeated bleeding, low iron diet or EPO administration. No change in liver crude membrane Hjv protein was observed in iron-treated or iron-deficient rats. ELISA assay for mouse plasma Hjv did not show significant difference between Hjv+/+ and Hjv-/- mice. Liver Hamp mRNA, Bmp6 mRNA and Id1 mRNA displayed the expected response to iron overload and iron deficiency. EPO treatment decreased Id1 mRNA, suggesting possible participation of the bone morphogenetic protein pathway in EPO-mediated downregulation of Hamp mRNA. DISCUSSION: Since no differences between Hjv protein levels were found following various experimental manipulations of body iron status, the results indicate that, in vivo, substantial changes in Hamp mRNA can occur without noticeable changes of membrane hemojuvelin content. Therefore, modulation of hemojuvelin protein content apparently does not represent the limiting step in the control of Hamp gene expression

Effect of iron status on rat liver membrane hemojuvelin.

<p>Immunoblots of Hjv protein in rat liver crude membrane fractions. Panel A: Detection with primary anti-Hjv antibody AF3634. Panel B: Identical samples detected with primary anti-Hjv antibody AF3720. Panel C: Densitometric quantification of the 35 kDa Hjv band. Liver samples from <i>Hjv+/+</i> and <i>Hjv−/−</i> mice are included for comparison. C: Control; Fe: Iron injection (1200 mg/kg); ID: Iron-deficient diet. WT: <i>Hjv</i>+/+ mice, KO: <i>Hjv</i>−/− mice. 60 µg of protein was loaded per lane. Arrows indicate Hjv-specific bands. Connexin 43 was used as loading control. For densitometry, the optical density of control samples was set at 100%, n = 3.</p

Effect of experimental protocols on iron status in rats.

<p>Iron was administered to male Wistar rats as iron-dextran in three weekly injections (100 mg iron/rat), total administered dose was 300 mg iron/rat. Low iron diet was fed to female Wistar rats for four weeks after weaning. Asterisk denotes significant difference from controls (p<0.05, n = 3).</p

Effect of iron status on mouse liver GPI-bound membrane hemojuvelin.

<p>Panel A: Immunoblot of supernatants from Pi-PLC-treated samples. Panel B: Densitometric quantification of the 35 kDa Hjv band. Pi-PLC treatment was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037391#s4" target="_blank">Materials and Methods</a>. C: Control, Fe: Iron injection (200 mg/kg), Bl: Bleeding (0.6 ml of blood once weekly for 3 weeks), ID: Iron-deficient diet, Epo: Erythropoietin (50 U/day) administration for 4 days, WT: <i>Hjv</i>+/+ mice, KO: <i>Hjv</i>−/− mice. Arrows indicate Hjv-specific bands. 60 µg of protein was loaded per lane, equal protein loading was verified by staining of the membrane with Amido Black dye. Primary anti-Hjv antibody: AF3634. For densitometry, the optical density of control samples was set at 100%, n = 3.</p

Effect of iron status on mouse liver membrane hemojuvelin.

<p>Immunoblots of Hjv protein in liver crude membrane fractions. Panel A: Detection with primary anti-Hjv antibody AF3634. Panel B: Identical samples detected with primary anti-Hjv antibody AF3720. Panel C: Densitometric quantification of the 35 kDa Hjv band. C: Control, Fe: Iron injection (200 mg/kg), Bl: Bleeding (0.6 ml of blood once weekly for 3 weeks), ID: Iron-deficient diet, Epo: Erythropoietin (50 U/day) administration for 4 days, WT: <i>Hjv</i>+/+ mice, KO: <i>Hjv</i>−/− mice. 60 µg of protein was loaded per lane. Arrows indicate Hjv-specific bands. Connexin 43 was used as loading control. For densitometry, the optical density of control samples was set at 100%, n = 4.</p

Effect of iron status on mouse liver plasma mebrane hemojuvelin.

<p>Immunoblots of hemojuvelin in samples enriched in plasma membrane fraction. Samples were obtained with a commercial plasma membrane extraction kit. KO: <i>Hjv</i>−/− mice, C: Control, ID: Iron deficient diet, Fe: Iron injection (200 mg/kg). Arrows indicate Hjv-specific bands. Connexin 43 was used as loading control, 40 µg of protein was loaded per lane. Primary anti-Hjv antibody: AF3720.</p

Effect of iron status on mouse liver <i>Hamp</i>, <i>Bmp6</i>, <i>Tmprss6</i> and <i>Id1</i> mRNA.

<p><i>Hamp</i>, <i>Bmp6</i>, <i>Tmprss6</i> and <i>Id1</i> mRNA was determined by real-time PCR and expressed relative to β-actin mRNA. C: Control, Fe: Iron injection (200 mg/kg), ID: Iron-deficient diet, Bl: Bleeding (0.6 ml of blood once weekly for 3 weeks), Epo: Erythropoietin (50 U/day) administration for 4 days, WT: <i>Hjv</i>+/+ mice, KO: <i>Hjv</i>−/− mice. Asterisks indicate statistically significant difference (n = 4, p<0.05).</p

Effect of experimental protocols on iron status in mice.

<p>Iron was administered to male C57BL/6 mice as iron polyisomaltosate at 200 mg/kg, erythropoietin (EPO) was injected at 50 U/mouse on days 1–4, mice were sacrificed on day 5. Low iron diet was fed to female C57BL/6 mice for four weeks after weaning, bleeding was performed once weekly (0.6 ml of blood) by retrobulbar puncture in halothane anesthesia for four weeks. Asterisk denotes significant difference from controls (p<0.05, n≥4).</p

Hemojuvelin protein in mouse whole liver homogenates.

<p>C: Control, Bl: Bleeding (0.6 ml of blood once weekly for 3 weeks), Fe: Iron injection (200 mg/kg), KO: <i>Hjv</i>−/− mice. 80 µg of protein was loaded per lane. Arrows indicate Hjv-specific bands. Gapdh was used as loading control. Primary anti-Hjv antibody: AF3634.</p