Mutations of hepcidin (HAMP)and IntroductionDuring the past few years, a number of new genes participating in iron metabolism have been identified. Mutations in 2 genes, hepcidin (HAMP) 1 and hemojuvelin (HJV) 2,3 have been shown to result in juvenile hemochromatosis. Hepcidin, a small peptide synthesized predominantly in hepatocytes, is emerging as an important regulator of iron homeostasis, which inhibits iron absorption from the intestine and iron release from macrophages. Hepcidin expression is controlled by iron status and erythropoietic activity, as well as by inflammatory stimuli 4 ; inappropriate expression of hepcidin probably plays a role in the pathophysiology of hereditary hemochromatosis and anemia of inflammation. 5 On the other hand, the function and regulation of hemojuvelin are at present unknown. Prior to identification of the HJV gene, it was speculated that its product could function in the hepcidin signaling pathway, possibly as a hepcidin receptor, 5 whereas a current concept proposes that hemojuvelin could modulate hepcidin expression. 2,6 Orthologs of the HJV gene have been identified in zebrafish, mice, and rats 2 ; the mouse HJV ortholog Rgmc is, like HJV, expressed mainly in skeletal muscle, heart, and liver. 7 The aim of the present study was to examine whether experimental conditions known to influence hepatic Hamp expression in mice will also change hepatic Rgmc mRNA levels and to compare possible similarities or discrepancies in the regulation of these 2 genes. Study designAll animal experiments were approved by the Animal Care Committee of the First Faculty of Medicine. Male C57BL/6N mice (Charles River, Sulzfeld, Germany) were treated with lipopolysaccharide (LPS, serotype 0111:B4, 1 mg/kg intraperitoneally; Sigma Aldrich, Prague, Czech Republic) and humanely killed by cervical dislocation after 90 minutes or 6 hours. Iron overload (600 mg/kg) was induced by a single subcutaneous injection of iron polyisomaltosate (Ferrum Lek; Lek, Ljubljana, Slovenia); mice were humanely killed 1 week or 3 weeks after application. Erythropoietin (EPREX 10 000, Cilag AG, Schaffhausen, Switzerland) was administered at 50 U/mouse for 4 days, and mice were killed on day 5.Liver RNA was extracted using RNABlue (Top-Bio, Prague, Czech Republic), treated with DNase I (Gibco, Life Technologies, Gaithersburg, MD), and 1 g total RNA was reverse transcribed by the RevertAid First-Strand cDNA synthesis kit (Fermentas, Vilnius, Lithuania).Levels of Hamp and Rgmc mRNA were determined by real-time polymerase chain reaction (PCR) on a Roche LightCycler instrument, using LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Mannheim, Germany). Primer sequences were: ␤-actin forward 5Ј-GACATGGAGAAGATCTGGCA-3Ј, reverse 5Ј-GGTCTTTACGGATGT-CAACG-3Ј; Hamp forward 5Ј-CTGAGCAGCACCACCTATCTC-3Ј, Hamp reverse 5Ј-TGGCTCTAGGCTATGTTTTGC-3Ј; Rgmc forward 5-CCCA-GATCCCTGTGACTATGA -3, Rgmc reverse 5-CAGGAAGATTGTCCAC-CTCAG -3. Rgmc primers were designed to amplify a sequence from exons 3 and 4 of Rgmc DNA. 2 Beca...

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.

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Hepatic Hamp2 expression displays considerable strain- and sex-dependent variation. Lipopolysaccharide increases expression of Hamp both in the liver and pancreas, but Hamp2 does not respond to lipopolysaccharide treatment. The significance of the amino acid substitutions in hepcidin peptides in DBA/2N mice is at present unknown.

Tmprss6-mutated mask mice display iron deficiency anemia and high expression of hepcidin. The aim of the study was to determine the effect of erythropoietin administration on proteins participating in the control of iron homeostasis in the liver and spleen in C57BL/6 and mask mice. Administration of erythropoietin for four days at 50 IU/mouse/day increased hemoglobin and hematocrit in C57BL/6 mice, no such increase was seen in mask mice. Erythropoietin administration decreased hepcidin expression in C57BL/6 mice, but not in mask mice. Erythropoietin treatment significantly increased the spleen size in both C57BL/6 and mask mice. Furthermore, erythropoietin administration increased splenic Fam132b, Fam132a and Tfr2 mRNA content. At the protein level, erythropoietin increased the amount of splenic erythroferrone and transferrin receptor 2 both in C57BL/6 and mask mice. Splenic ferroportin content was decreased in erythropoietin-treated mask mice in comparison with erythropoietin-treated C57BL/6 mice. In mask mice, the amount of liver hemojuvelin was decreased in comparison with C57BL/6 mice. The pattern of hemojuvelin cleavage was different between C57BL/6 and mask mice: In both groups, a main hemojuvelin band was detected at approximately 52 kDa; in C57BL/6 mice, a minor cleaved band was seen at 47 kDa. In mask mice, the 47 kDa band was absent, but additional minor bands were detected at approximately 45 kDa and 48 kDa. The results provide support for the interaction between TMPRSS6 and hemojuvelin in vivo; they also suggest that hemojuvelin could be cleaved by another as yet unknown protease in the absence of functional TMPRSS6. The lack of effect of erythropoietin on hepcidin expression in mask mice can not be explained by changes in erythroferrone synthesis, as splenic erythroferrone content increased after erythropoietin administration in both C57BL/6 and mask mice.

Matriptase-2 (TMPRSS6) is an important negative regulator of hepcidin expression; however, the effects of iron overload or accelerated erythropoiesis on liver TMPRSS6 protein content in vivo are largely unknown. We determined TMPRSS6 protein content in plasma membrane-enriched fractions of liver homogenates by immunoblotting, using a commercial antibody raised against the catalytic domain of TMPRSS6. Plasma membrane-enriched fractions were obtained by centrifugation at 3000 g and washing. TMPRSS6 was detected in the 3000 g fraction as a 120 kDa full-length protein in both mice and rats. Feeding of iron-deficient diet as well as erythropoietin treatment increased TMPRSS6 protein content in rats and mice by a posttranscriptional mechanism; the increase in TMPRSS6 protein by erythropoietin was also observed in Bmp6-mutant mice. Administration of high doses of iron to mice (200, 350 and 700 mg/kg) decreased TMPRSS6 protein content. Hemojuvelin was detected in the plasma membrane-enriched fractions of control animals as a full length protein of approximately 52 kDa; in iron deficient animals, the full length protein was partially cleaved at the N-terminus, resulting in an additional weak band of approximately 47 kDa. In livers from hemojuvelin-mutant mice, TMPRSS6 protein content was strongly decreased, suggesting that intact hemojuvelin is necessary for stable TMPRSS6 expression in the membrane. Overall, the results demonstrate posttranscriptional regulation of liver TMPRSS6 protein by iron status and erythropoietin administration, and provide support for the interaction of TMPRSS6 and hemojuvelin proteins in vivo.

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Biochemical markers, specifically enzymes of the first phase of xenobiotic transformation - cytochrome P450 and ethoxyresorufin-O-deethylase (EROD) - were used to determine the quantities of persistent organic pollutants (POPs) in fish muscle (PCB, HCB, HCH, OCS, DDT). Eight rivers were monitored (Orlice, Chrudimka, Cidlina, Jizera, Vltava, Ohře and Bílina; and the River Blanice was used as a control). The indicator species selected was the chub (Leuciscus cephalus L.). There were no significant differences in cytochrome P450 content between the locations monitored. The highest concentration of cytochrome P450 in fish liver was in the Vltava (0.241 nmol mg-1 protein), and the lowest was in the Orlice (0.120 nmol mg-1 protein). Analysis of EROD activity showed a significant difference between the Blanice and the Vltava (P< 0.05), and also between the Orlice and the Vltava (P< 0.01), the Orlice and the Bílina (P< 0.01), and the Orlice and the Ohře (P< 0.05). The highest EROD activity in fish liver was in the Vltava (576.4 pmol min-1 mg-1 protein), and the lowest was in the Orlice (63.05 pmol min-1 mg-1 protein). In individual locations, results of chemical monitoring and values of biochemical markers were compared. A significant correlation (P< 0.05) was found between biochemical markers and OCS, and PCB. Among the tributaries studied those that contaminated the Elbe most were the Vltava and the Bílina. These tributaries should not be considered the main sources of industrial contamination of the River Elbe, because the most important contamination sources were along the river Elbe itself.