Liver and muscle hemojuvelin are differently glycosylated

<p>Abstract</p> <p>Background</p> <p>Hemojuvelin (HJV) is one of essential components for expression of hepcidin, a hormone which regulates iron transport. HJV is mainly expressed in muscle and liver, and processing of HJV in both tissues is similar. However, hepcidin is expressed in liver but not in muscle and the role of the muscle HJV is yet to be established. Our preliminary analyses of mouse tissue HJV showed that the apparent molecular masses of HJV peptides are different in liver (50 kDa monomer and 35 and 20 kDa heterodimer fragments) and in muscle (55 kDa monomer and a 34 kDa possible large fragment of heterodimer). One possible explanation is glycosylation which could lead to difference in molecular mass.</p> <p>Results</p> <p>We investigated glycosylation of HJV in both liver and muscle tissue from mice. PNGase F treatment revealed that the HJV large fragments of liver and muscle were digested to peptides with similar masses, 30 and 31 kDa, respectively, and the liver 20 kDa small fragment of heterodimer was digested to 16 kDa, while the 50 kDa liver and 55 kDa muscle monomers were reduced to 42 and 48 kDa, respectively. Endo H treatment produced distinct digestion profiles of the large fragment: a small fraction of the 35 kDa peptide was reduced to 33 kDa in liver, while the majority of the 34 kDa peptide was digested to 33 kDa and a very small fraction to 31 kDa in muscle. In addition, liver HJV was found to be neuraminidase-sensitive but its muscle counterpart was neuraminidase-resistant.</p> <p>Conclusions</p> <p>Our results indicate that different oligosaccharides are attached to liver and muscle HJV peptides, which may contribute to different functions of HJV in the two tissues.</p

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 stimulated erythropoiesis on liver SMAD signaling pathway in iron-overloaded and iron-deficient mice.

Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload and decreased by accelerated erythropoiesis or iron deficiency. The purpose of the study was to examine the effect of these stimuli, either alone or in combination, on the main signaling pathway controlling hepcidin biosynthesis in the liver, and on the expression of splenic modulators of hepcidin biosynthesis. Liver phosphorylated SMAD 1 and 5 proteins were determined by immunoblotting in male mice treated with iron dextran, kept on an iron deficient diet, or administered recombinant erythropoietin for four consecutive days. Administration of iron increased liver phosphorylated SMAD protein content and hepcidin mRNA content; subsequent administration of erythropoietin significantly decreased both the iron-induced phosphorylated SMAD proteins and hepcidin mRNA. These results are in agreement with the recent observation that erythroferrone binds and inactivates the BMP6 protein. Administration of erythropoietin substantially increased the amount of erythroferrone and transferrin receptor 2 proteins in the spleen; pretreatment with iron did not influence the erythropoietin-induced content of these proteins. Erythropoietin-treated iron-deficient mice displayed smaller spleen size in comparison with erythropoietin-treated mice kept on a control diet. While the erythropoietin-induced increase in splenic erythroferrone protein content was not significantly affected by iron deficiency, the content of transferrin receptor 2 protein was lower in the spleens of erythropoietin-treated mice kept on iron-deficient diet, suggesting posttranscriptional regulation of transferrin receptor 2. Interestingly, iron deficiency and erythropoietin administration had additive effect on hepcidin gene downregulation in the liver. In mice subjected both to iron deficiency and erythropoietin administration, the decrease of hepcidin expression was much more pronounced than the decrease in phosphorylated SMAD protein content or the decrease in the expression of the SMAD target genes Id1 and Smad7. These results suggest the existence of another, SMAD-independent pathway of hepcidin gene downregulation

Cell cycle and differentiation of Sca-1⁺ and Sca-1⁻ hematopoietic stem and progenitor cells

<p>Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analyzed the cell cycle and cell production rate in HSPCs in murine hematopoiesis. The labeling of DNA-synthesizing cells by two thymidine analogues, optimized for <i>in-vivo</i> use, enabled determination of the cell cycle flow rate into G2-phase, the duration of S-phase and the average cell cycle time in Sca-1⁺ and Sca-1⁻ HSPCs. Determination of cells with 2n DNA content labeled in preceding S-phase was then used to establish the cell flow rates in G1-phase. Our measurements revealed a significant difference in how Sca-1⁺ and Sca-1⁻ myeloid progenitors self-renew and differentiate. Division of the Sca-1⁺ progenitors led to loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. Sca-1⁻ cells arising from cell division entered a new round of the cell cycle, corresponding to symmetric self-renewing cell division. The novel data also enabled the estimation of the cell production rates in Sca-1⁺ and in three subtypes of Sca-1⁻ HSPCs and revealed Sca-1 negative cells as the major amplification stage in the blood cell development.</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 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