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

The Separation of Gastric Aspartic Proteases Using Affinity Chromatography

Human gastric juice contains mainly aspartic proteases - pepsin A and pepsin C. Both pepsins are produced by gastric mucosa as inactive pepsinogens (pepsinogen A and pepsinogen C) that differ in their physico-chemical and immunological properties. Both pepsinogens consist of molecular variants, isozymogens. Pepsinogens are activated to the corresponding pepsins in the acidic environment of the gastric lumen. The levels of pepsinogens in serum reflect the morphological and functional status of gastric mucosa. The levels of pepsinogens are associated with gastric diseases (e.g. gastric cancer, gastritis). For a diagnostic purpose, the ratio of pepsinogen A to pepsinogen C not only in the gastric mucosa but also in serum is relevant. The gastric cancer patients or the atrophic gastritis patients have significantly lower ratio of serum pepsinogen A to pepsinogen C in comparison with the healthy subjects. In addition, proportion of individual isozymogens reflects the type of the gastric disease. Determination of changes in pepsinogen levels could be useful for early diagnostics of gastric diseases mentioned above. A subject of this Ph.D. thesis is a part of a long-term investigation that focuses on the elaboration of methods for the separation of gastric aspartic proteases that would be suitable for..

The Separation of Gastric Aspartic Proteases Using Affinity Chromatography

Human gastric juice contains mainly aspartic proteases - pepsin A and pepsin C. Both pepsins are produced by gastric mucosa as inactive pepsinogens (pepsinogen A and pepsinogen C) that differ in their physico-chemical and immunological properties. Both pepsinogens consist of molecular variants, isozymogens. Pepsinogens are activated to the corresponding pepsins in the acidic environment of the gastric lumen. The levels of pepsinogens in serum reflect the morphological and functional status of gastric mucosa. The levels of pepsinogens are associated with gastric diseases (e.g. gastric cancer, gastritis). For a diagnostic purpose, the ratio of pepsinogen A to pepsinogen C not only in the gastric mucosa but also in serum is relevant. The gastric cancer patients or the atrophic gastritis patients have significantly lower ratio of serum pepsinogen A to pepsinogen C in comparison with the healthy subjects. In addition, proportion of individual isozymogens reflects the type of the gastric disease. Determination of changes in pepsinogen levels could be useful for early diagnostics of gastric diseases mentioned above. A subject of this Ph.D. thesis is a part of a long-term investigation that focuses on the elaboration of methods for the separation of gastric aspartic proteases that would be suitable for..

Marketing Communication in Pharmaceutical Industry: GS CONDRO FORTE

The aim of this thesis is to present the theoretical attributes of marketing communications, as part of the marketing mix and in particular in relation to specific areas of the pharmaceutical industry. The practical part is focused on pharmaceutical product marketing communication towards the ultimate consumer. For this goal I chose Green pharmaceutical company - Swan Pharmaceuticals CR, as a nutritional supplement for her joints Condro Forte GS

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

Heart Ferroportin Protein Content Is Regulated by Heart Iron Concentration and Systemic Hepcidin Expression

The purpose of the study was to investigate the expression of ferroportin protein following treatments that affect systemic hepcidin. Administration of erythropoietin to C57BL/6J mice decreased systemic hepcidin expression; it also increased heart ferroportin protein content, determined by immunoblot in the membrane fraction, to approximately 200% of control values. This increase in heart ferroportin protein is very probably caused by a decrease in systemic hepcidin expression, in accordance with the classical regulation of ferroportin by hepcidin. However, the control of heart ferroportin protein by systemic hepcidin could apparently be overridden by changes in heart non-heme iron content since injection of ferric carboxymaltose to mice at 300 mg Fe/kg resulted in an increase in liver hepcidin expression, heart non-heme iron content, and also a threefold increase in heart ferroportin protein content. In a separate experiment, feeding an iron-deficient diet to young Wistar rats dramatically decreased liver hepcidin expression, while heart non-heme iron content and heart ferroportin protein content decreased to 50% of controls. It is, therefore, suggested that heart ferroportin protein is regulated primarily by the iron regulatory protein/iron-responsive element system and that the regulation of heart ferroportin by the hepcidin-ferroportin axis plays a secondary role

Effect of Erythropoietin, Iron Deficiency and Iron Overload on Liver Matriptase-2 (TMPRSS6) Protein Content in Mice and Rats.

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

Matriptase-2 and Hemojuvelin in Hepcidin Regulation: In Vivo Immunoblot Studies in Mask Mice

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