OBJECTIVES: The acute respiratory syndrome, known as COVID-19, is characterised by high morbidity and increased mortality. Genetic factors may partially explain the differences in susceptibility to and severity of COVID-19. METHODS: We have analysed common functional polymorphisms within the OAS1 (rs4767027), TMPRSS6 (rs855791), DPP4 (rs3788979), and ZNF335 (rs3848719) genes in SARS-CoV-2 positive subjects (n = 521, different disease severity) and in population controls (n = 2,559 subjects, COVID-19 status unknown). RESULTS: Neither DPP4 nor ZNF335 were associated with disease susceptibility or severity in the Czech population in any of the models used for calculation. T allele carriers of the OAS1 polymorphism seem to be protective against symptomatic COVID-19 (p = 0.002 calculated for trend; asymptomatic, symptomatic, hospitalised). Similarly, within the TMPRSS6, minor TT homozygotes associated with lower plasma Fe concentrations were underrepresented in the overall patient group (p = 0.044; OR = 0.77, 95% CI: 0.59-0.99), and the difference was mainly driven by the severe COVID-19 subjects. In general, risky homozygotes of these two polymorphisms were less frequent than expected in the group of hospitalised COVID-19 survivors. CONCLUSIONS: Common variants within OAS1 (rs4767027) and TMPRSS6 (rs855791) play some role in COVID-19 pathology in the Czech Caucasian population. Whether the depletion of minor allele carriers of these two variants is associated with increased COVID-19 mortality, needs to be analysed in an external confirmatory study.

• MeSH
• 2',5'-Oligoadenylate Synthetase MeSH
• COVID-19 * genetics   MeSH
• Dipeptidyl Peptidase 4 MeSH
• DNA-Binding Proteins MeSH
• Polymorphism, Single Nucleotide MeSH
• Humans MeSH
• Membrane Proteins MeSH
• SARS-CoV-2 MeSH
• Serine Endopeptidases genetics   MeSH
• Transcription Factors MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

Although the relationship between hereditary hemochromatosis and mutations in the HFE gene was discovered more than 20 years ago, information on the in vivo regulation of HFE protein expression is still limited. The purpose of the study was to determine the response of liver HFE protein content to iron deficiency in mice and rats by immunoblotting. Attempts to visualize the HFE protein in whole liver homogenates were unsuccessful; however, HFE could be detected in liver microsomes or in plasma membrane-enriched fractions. Five-week-old male C57BL/6 mice fed an iron-deficient diet for 4 wk presented with a significant decrease in liver iron content and liver Hamp expression, as well as with a significant decrease in liver HFE protein content. Rats fed an iron-deficient diet for 4 wk also displayed significant decrease in liver Hamp expression and liver HFE protein content. These results suggest that the downregulation of HFE-dependent signaling may contribute to decreased Hamp gene expression in states of prolonged iron deficiency. It has recently been proposed that HFE protein could be a potential target of matriptase-2, a hepatocyte protease mutated in iron-refractory iron deficiency anemia. However, immunoblot analysis of HFE protein in the livers from Tmprss6-mutated mask mice did not show evidence of matriptase-2-dependent HFE protein cleavage. In addition, no indication of HFE protein cleavage was seen in iron-deficient rats, whereas the full-length matriptase-2 protein content in the same animals was significantly increased. These results suggest that HFE is probably not a major physiological target of matriptase-2. NEW & NOTEWORTHY Feeding of iron-deficient diet for 4 wk decreased liver HFE protein content in both mice and rats, suggesting that decreased HFE-dependent signaling may contribute to hepcidin downregulation in iron deficiency. There was no difference in HFE protein band appearance between matriptase-2-mutated mask mice and wild-type mice, indicating that HFE is probably not a major physiological substrate of matriptase-2-mediated protease activity in vivo.

• MeSH
• Anemia, Iron-Deficiency genetics   metabolism   MeSH
• Iron Deficiencies MeSH
• Liver metabolism   MeSH
• Rats MeSH
• Membrane Proteins genetics   metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Rats, Wistar MeSH
• Hemochromatosis Protein genetics   metabolism   MeSH
• Proteolysis MeSH
• Serine Endopeptidases genetics   metabolism   MeSH
• Iron MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• MeSH
• Cytokines genetics   metabolism   MeSH
• Erythropoietin genetics   pharmacology   MeSH
• Hepcidins genetics   metabolism   MeSH
• Liver metabolism   pathology   MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Mice, Mutant Strains MeSH
• Mice MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Gene Expression Regulation drug effects   genetics   MeSH
• Serine Endopeptidases genetics   metabolism   MeSH
• Spleen metabolism   pathology   MeSH
• Muscle Proteins genetics   metabolism   MeSH
• Organ Size drug effects   genetics   MeSH
• Iron metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Erythroferrone (ERFE) and TMPRSS6 are important proteins in the regulation of iron metabolism. The objective of the study was to examine splenic ERFE and liver TMPRSS6 synthesis in rats treated with a combination of iron and erythropoietin (EPO). EPO was administered to female Wistar rats at 600U/day for four days, iron-pretreated rats received 150mg of iron before EPO treatment. Content of ERFE and TMPRSS6 proteins was determined by commercial antibodies. Iron pretreatment prevented the EPO-induced decrease in hepcidin expression. Content of phosphorylated SMAD 1,5,8 proteins was decreased in the liver by both EPO and iron plus EPO treatment. Fam132b expression in the spleen was increased both by EPO and iron plus EPO treatments; these treatments also significantly induced splenic Fam132a expression. ERFE protein content in the spleen was increased both by EPO and iron plus EPO to a similar extent. EPO administration increased TMPRSS6 content in the plasma membrane-enriched fraction of liver homogenate; in iron-pretreated rats, this increase was abolished. The results confirm that iron pretreatment prevents the EPO-induced decrease in liver Hamp expression. This effect probably occurs despite high circulating ERFE levels, since EPO-induced ERFE protein synthesis is not influenced by iron pretreatment.

• MeSH
• Erythropoietin pharmacology   MeSH
• Liver metabolism   MeSH
• Rats MeSH
• Peptide Hormones metabolism   MeSH
• Rats, Wistar MeSH
• Serine Endopeptidases metabolism   MeSH
• Spleen metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

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.

• MeSH
• Anemia, Iron-Deficiency metabolism   MeSH
• Iron Deficiencies MeSH
• Erythropoietin metabolism   pharmacology   MeSH
• Liver drug effects   metabolism   MeSH
• Bone Morphogenetic Protein 6 genetics   MeSH
• Rats MeSH
• Membrane Proteins metabolism   MeSH
• Disease Models, Animal MeSH
• Mutation MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Iron Overload metabolism   MeSH
• Serine Endopeptidases metabolism   MeSH
• Sodium-Potassium-Exchanging ATPase metabolism   MeSH
• Iron metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Matriptase-2, a membrane protein encoded by the Tmprss6 gene, is a negative regulator of hepcidin expression. Although matriptase-2 has been proposed to cleave membrane hemojuvelin, we have recently found decreased hemojuvelin protein levels in Tmprss6 -/- mice. The purpose of this study was to confirm this observation by determining hemojuvelin protein levels in another strain of mice with disrupted Tmprss6 gene, and to determine the effect of matriptase-2 deficiency on the expression of other membrane proteins participating in the bone morphogenetic protein signal transduction. Mask mice, which lack the proteolytic domain of matriptase-2, displayed decreased liver hemojuvelin protein content, while Id1 mRNA level, an indicator of hemojuvelin-dependent signal transduction, was increased. Protein levels of bone morphogenetic protein receptors Alk3 and Acvr2a were unchanged, and transferrin receptor 2 and neogenin protein levels were slightly decreased. The results confirm that the loss of matriptase-2 increases bone morphogenetic protein-dependent signaling, while paradoxically decreasing liver hemojuvelin protein content. The regulation of transferrin receptor 2 protein levels by transferrin saturation was not affected in mask mice. How the loss of matriptase-2 proteolytic activity leads to decreased hemojuvelin protein levels is at present unclear.

• MeSH
• Activin Receptors, Type II metabolism   MeSH
• Iron Deficiencies MeSH
• Down-Regulation MeSH
• Inhibitor of Differentiation Protein 1 genetics   metabolism   MeSH
• Injections, Intraperitoneal MeSH
• Liver drug effects   metabolism   MeSH
• Membrane Proteins deficiency   genetics   metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Bone Morphogenetic Protein Receptors, Type I metabolism   MeSH
• Receptors, Transferrin metabolism   MeSH
• Serine Endopeptidases deficiency   genetics   MeSH
• Iron-Dextran Complex administration & dosage   MeSH
• Iron MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

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.

• MeSH
• Anemia, Iron-Deficiency genetics   metabolism   MeSH
• Cell Membrane genetics   metabolism   MeSH
• Dimerization MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Phosphoinositide Phospholipase C metabolism   MeSH
• Glycosylphosphatidylinositols chemistry   metabolism   MeSH
• Liver metabolism   pathology   MeSH
• Antimicrobial Cationic Peptides genetics   metabolism   MeSH
• Membrane Proteins deficiency   genetics   metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Polymerase Chain Reaction MeSH
• Gene Expression Regulation MeSH
• Serine Endopeptidases deficiency   genetics   MeSH
• Signal Transduction genetics   MeSH
• Tissue Extracts chemistry   MeSH
• Iron metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Hepcidin is a key regulator of iron homeostasis, while hemojuvelin is an important component of the hepcidin regulation pathway. It has been recently proposed that soluble hemojuvelin, produced from hemojuvelin by the protease furin, decreases hepcidin expression. The aim of the presented study was to examine the downregulation of hepcidin by chronic bleeding in hemojuvelin-mutant mice. Male mice with targeted disruption of the hemojuvelin gene (Hjv-/- mice) and wild-type littermates were maintained on an iron-deficient diet and subjected to weekly phlebotomies for 7 weeks. Gene expression was examined by real-time PCR. In wild type mice, repeated bleeding decreased hepcidin mRNA by two orders of magnitude. In Hjv-/- mice, basal hepcidin expression was low; however, repeated bleeding also decreased hepcidin mRNA content by an order of magnitude. Phlebotomies reduced hepatic iron overload in Hjv-/- mice by 80 %. Liver and muscle furin mRNA content was not significantly changed. No effect on hepatic Tmprss6 mRNA content was observed. Results from the study indicate that soluble hemojuvelin is not the sole factor responsible for hepcidin downregulation. In addition, the presented data suggest that, under in vivo conditions, tissue hypoxia does not transcriptionally regulate the activity of furin or TMPRSS6 proteases.

• MeSH
• Time Factors MeSH
• Iron Deficiencies MeSH
• Iron, Dietary administration & dosage   MeSH
• Down-Regulation MeSH
• Erythropoiesis MeSH
• Financing, Organized MeSH
• Phlebotomy MeSH
• Furin metabolism   MeSH
• Cell Hypoxia MeSH
• Liver metabolism   MeSH
• Antimicrobial Cationic Peptides genetics   metabolism   MeSH
• Muscle, Skeletal metabolism   MeSH
• Hemorrhage etiology   genetics   metabolism   MeSH
• Membrane Proteins genetics   metabolism   deficiency   MeSH
• RNA, Messenger metabolism   MeSH
• Disease Models, Animal MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Iron Overload metabolism   prevention & control   MeSH
• Serine Endopeptidases metabolism   MeSH
• Iron MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH