Liver hemojuvelin protein levels in mice deficient in matriptase-2 (Tmprss6).

Mutations of the TMPRSS6 gene, encoding the serine protease matriptase-2, lead to iron-refractory iron deficiency anemia. Matriptase-2 is a potent negative regulator of hepcidin. Based on in vitro data, it has recently been proposed that matriptase-2 decreases hepcidin synthesis by cleaving membrane hemojuvelin, a key protein of the hepcidin-regulatory pathway. However, in vivo evidence for this mechanism of action of matriptase-2 is lacking. To investigate the hemojuvelin-matriptase-2 interaction in vivo, an immunoblot assay for liver membrane hemojuvelin was optimized using hemojuvelin-mutant mice as a negative control. In wild-type mice, two hemojuvelin-specific bands of 35kDa and 20kDa were detected in mouse liver membrane fraction under reducing conditions; under non-reducing conditions, a single band of approximately 50kDa was seen. Phosphatidylinositol-specific phospholipase C treatment confirmed binding of the detected protein to the cell membrane by a glycosylphosphatidylinositol anchor, indicating that the major form of mouse liver membrane hemojuvelin is a glycosylphosphatidylinositol-bound heterodimer. Unexpectedly, comparison of liver homogenates from Tmprss6+/+ and Tmprss6-/- mice revealed significantly decreased, rather than increased, hemojuvelin heterodimer content in Tmprss6-/- mice. These data do not provide direct support for the concept that matriptase-2 cleaves membrane hemojuvelin and may indicate that, in vivo, the role of matriptase-2 in the regulation of hepcidin gene expression is more complex.

Liver and muscle hemojuvelin are differently glycosylated.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

The effect of nitric oxide on vaccinia virus-encoded ribonucleotide reductase.

Growth inhibition of the DNA virus vaccinia (VACV) by NO is known to occur at the level of DNA synthesis. This inhibition is partially reversed by addition of deoxyribonucleosides, suggesting that NO or NO-related species inhibit viral ribonucleotide reductase (RR). However, the effect of NO on VACV-encoded RR or other DNA-synthesizing enzymes has not been demonstrated. In order to study the effects of NO on VACV-encoded RR, DNA polymerase (DNA pol) and thymidine kinase (TK), we generated a VACV recombinant expressing murine macrophage iNOS under control of a VACV early/late promoter p7.5. Using this recombinant, we demonstrate that expression of iNOS and the resulting production of NO inhibit activity of the viral RR, but not of viral DNA pol and TK. This NO-mediated inhibition of viral RR occurred around the same time as the increase of ADP levels, while it preceded the block in VACV DNA synthesis and the decrease of ATP levels. In addition, we tested the effects of DPTA/NONOate on the growth of different VACV mutants. Fold-inhibition of the growth of VACV deletion mutant for TK was comparable to that of wild-type VACV. VACV containing amplification of the gene for the small subunit of RR appeared to be least sensitive to DPTA/NONOate, while VACV deletion mutant for the large subunit of RR was most sensitive. The results provide a direct evidence for NO-mediated inhibition of VACV-encoded RR.

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.