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
• anemie z nedostatku železa genetika   metabolismus   MeSH
• buněčná membrána genetika   metabolismus   MeSH
• dimerizace MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fosfolipasa C fosfoinositidové signalizace metabolismus   MeSH
• glykosylfosfatidylinositoly chemie   metabolismus   MeSH
• játra metabolismus   patologie   MeSH
• kationické antimikrobiální peptidy genetika   metabolismus   MeSH
• membránové proteiny nedostatek   genetika   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• polymerázová řetězová reakce MeSH
• regulace genové exprese MeSH
• serinové endopeptidasy nedostatek   genetika   MeSH
• signální transdukce genetika   MeSH
• tkáňové extrakty chemie   MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• aktivinové receptory typu II metabolismus   MeSH
• deficit železa MeSH
• down regulace MeSH
• inhibitor diferenciace 1 genetika   metabolismus   MeSH
• injekce intraperitoneální MeSH
• játra účinky léků   metabolismus   MeSH
• membránové proteiny nedostatek   genetika   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• receptory morfogenetických kostních proteinů typu I metabolismus   MeSH
• receptory transferinu metabolismus   MeSH
• serinové endopeptidasy nedostatek   genetika   MeSH
• železo-dextranový komplex aplikace a dávkování   MeSH
• železo MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity (mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.

• MeSH
• deficit železa MeSH
• dietní železo farmakologie   MeSH
• erythropoetin farmakologie   MeSH
• GPI-vázané proteiny biosyntéza   nedostatek   genetika   fyziologie   MeSH
• hepcidiny biosyntéza   genetika   MeSH
• inhibitor diferenciace 1 biosyntéza   genetika   MeSH
• játra metabolismus   MeSH
• kostní morfogenetický protein 6 biosyntéza   genetika   MeSH
• membránové proteiny nedostatek   genetika   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• orgánová specificita MeSH
• přetížení železem metabolismus   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein hemochromatózy biosyntéza   nedostatek   genetika   fyziologie   MeSH
• proteinové domény MeSH
• regulace genové exprese účinky léků   MeSH
• rekombinantní proteiny metabolismus   MeSH
• serinové endopeptidasy nedostatek   genetika   fyziologie   MeSH
• slezina metabolismus   MeSH
• železo MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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
• anemie z nedostatku železa metabolismus   MeSH
• deficit železa MeSH
• erythropoetin metabolismus   farmakologie   MeSH
• játra účinky léků   metabolismus   MeSH
• kostní morfogenetický protein 6 genetika   MeSH
• krysa rodu rattus MeSH
• membránové proteiny metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• mutace MeSH
• myši knockoutované MeSH
• myši MeSH
• přetížení železem metabolismus   MeSH
• serinové endopeptidasy metabolismus   MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• anemie z nedostatku železa genetika   metabolismus   MeSH
• deficit železa MeSH
• játra metabolismus   MeSH
• krysa rodu rattus MeSH
• membránové proteiny genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• potkani Wistar MeSH
• protein hemochromatózy genetika   metabolismus   MeSH
• proteolýza MeSH
• serinové endopeptidasy genetika   metabolismus   MeSH
• železo MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.

• MeSH
• adaptivní imunita účinky léků   MeSH
• aktivace lymfocytů účinky léků   MeSH
• antikoagulancia izolace a purifikace   farmakologie   MeSH
• cytokiny metabolismus   MeSH
• hemokoagulace účinky léků   MeSH
• hmyzí proteiny izolace a purifikace   farmakologie   MeSH
• imunologické faktory izolace a purifikace   farmakologie   MeSH
• inhibitory proteas izolace a purifikace   farmakologie   MeSH
• klíště metabolismus   MeSH
• králíci MeSH
• kultivované buňky MeSH
• lidé MeSH
• lymfocyty účinky léků   imunologie   metabolismus   MeSH
• morčata MeSH
• myši inbrední C3H MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• proliferace buněk účinky léků   MeSH
• slezina účinky léků   imunologie   metabolismus   MeSH
• slinné proteiny a peptidy izolace a purifikace   farmakologie   MeSH
• sliny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• morčata MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Serpins are widely distributed and functionally diverse inhibitors of serine proteases. Ticks secrete serpins with anti-coagulation, anti-inflammatory, and immunomodulatory activities via their saliva into the feeding cavity to modulate host's hemostatic and immune reaction initiated by the insertion of tick's mouthparts into skin. The suppression of the host's immune response not only allows ticks to feed on a host for several days but also creates favorable conditions for the transmission of tick-borne pathogens. Herein we present the functional and structural characterization of Iripin-1 (Ixodes ricinus serpin-1), whose expression was detected in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Of 16 selected serine proteases, Iripin-1 inhibited primarily trypsin and further exhibited weaker inhibitory activity against kallikrein, matriptase, and plasmin. In the mouse model of acute peritonitis, Iripin-1 enhanced the production of the anti-inflammatory cytokine IL-10 and chemokines involved in neutrophil and monocyte recruitment, including MCP-1/CCL2, a potent histamine-releasing factor. Despite increased chemokine levels, the migration of neutrophils and monocytes to inflamed peritoneal cavities was significantly attenuated following Iripin-1 administration. Based on the results of in vitro experiments, immune cell recruitment might be inhibited due to Iripin-1-mediated reduction of the expression of chemokine receptors in neutrophils and adhesion molecules in endothelial cells. Decreased activity of serine proteases in the presence of Iripin-1 could further impede cell migration to the site of inflammation. Finally, we determined the tertiary structure of native Iripin-1 at 2.10 Å resolution by employing the X-ray crystallography technique. In conclusion, our data indicate that Iripin-1 facilitates I. ricinus feeding by attenuating the host's inflammatory response at the tick attachment site.

• MeSH
• antiflogistika farmakologie   MeSH
• chemokiny MeSH
• endoteliální buňky metabolismus   MeSH
• klíště * metabolismus   MeSH
• monocyty metabolismus   MeSH
• myši MeSH
• serpiny * metabolismus   MeSH
• trypsin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH