Hemojuvelin is essential for transferrin-dependent and transferrin-independent hepcidin expression in mice

Here we investigate the regulation of hepcidin, a hormone that inhibits dietary iron absorption and macrophage iron recycling, by the serum iron-binding protein transferrin. Mice deficient in transferrin (Tf(hpx/hpx)) and hemojuvelin (Hjv(-/-)), a gene mutated in juvenile hemochromatosis, a disease of hepcidin deficiency and iron overload, were generated. While Tf(hpx/hpx) Hjv(+/+) and Tf(hpx/hpx) Hjv(-/-) phenotypes did not differ markedly, transferrin treatment and RBC transfusions robustly increased hepcidin levels in Tf(hpx/hpx) Hjv(+/+) but not Tf(hpx/hpx) Hjv(-/-)mice. These results suggest that, while hemojuvelin is not essential for the establishment or maintenance of hepcidin deficiency in transferrin-deficient mice, hemojuvelin is essential for transferrin-dependent and transferrin-independent hepcidin expression in conditions of iron overload.     

Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice

As a central regulator of iron metabolism, hepcidin inhibits dietary iron absorption and macrophage iron recycling. Its expression is regulated by multiple factors including iron availability and erythropoietic activity. To investigate the role of transferrin (Tf) in the regulation of hepcidin expression by these factors in vivo, we employed the hypotransferrinemic (hpx) mouse. These Tf-deficient mice have severe microcytic anemia, tissue iron overload, and hepcidin deficiency. To determine the relationship of Tf levels and erythropoiesis to hepcidin expression, we subjected hpx mutant and control mice to a number of experimental manipulations. Treatment of hpx mice with Tf injections corrected their anemia and restored hepcidin expression. To investigate the effect of erythropoiesis on hepcidin expression, we suppressed erythropoiesis with blood transfusions or myeloablation with chemotherapeutic drugs. Transfusion of hpx animals with wild-type red blood cells led to increased hepcidin expression, while hepcidin expression in myeloablated hpx mice increased only if Tf was administered postablation. These results suggest that hepcidin expression in hpx mice is regulated both by Tf-restricted erythropoiesis and by Tf through a mechanism independent of its role in erythropoiesis.     

Intestinal damage mediated by Kupffer cells in rats with endotoxemia

AIM：To determine the in vivo effects of phagocytic blockade of Kupffer cell(KC)on the release of proinflammatory cytokines in small intestinal lesion and on the integrity of intestinal tract by using gadolinium chloride(GdCl3)during early endotoxemia.METHODS:Wistar rats were divided into three groups:GropA,rats were injected with endotoxin(E.coliO111:B4,adose of 12mg·kg^-1)only;GroupB,rats were pretreated intravenously with 25mg of GdCl3per kg24hare given endotoxin;and Group C,sham operation only.All animals were sacrificed 4h after endotoxin injecton.In portion of the rats of three groups.bile duct was cannulated,which the bile was collected externally.Morphological changes of ileum were observed under light microscopy and electronic microscopy.The KC were isolated rfrom rats by collagenase perfusion and inKC,expression of TNF-αand IL-6mRNA were determined by RT-PCRanalysis.Plasma and bile TNF-αandIL-6Levels were determined by enzyme-linked immunosorbent assay(EISA)>RESULTS:In group A,there were neutrophil infiltration and superficial epitelial necrosis of the ilealvvilli,sloughing of mucosal epithelium.and disappear ance of somevilli.In groupB.the ileal mucosal damage was much reduced.which in groupC,no significant morphological changes were seen,GdCl3pretreatment decreased significantly the expression of TNF-αand IL-6mRNA ingroupB(4.32±0.47and4.05±0.43)when compared to groupA(9.46±1.21and9.04±1.09)(P<0.05).There was no significant expression of TNF-αand IL-6mRNA in groupC(1.03±0.14and10.4±0.13).In rats of groupA,the levels of TNF-αand IL-6in bile and plasma were 207±29ng·L^-1,1032±107ng·L^-1,213±33ng·L^-1,and 1185±127ng·L^-1,respectively.In groupB,they were113±18ng·L^-1,repectively.In groupC,they were 67±10ng·L^-1,72±13ng·L^-1,109±18ng·L^-1,and 118±22ng·L^-1respectively.There were significant difference between the three group(P<0.05).CONCLUSION:KC release cytokinesTNF-αand IL-6causing damage to the integrity of intestinal epithelium and play a crucial role in the initiation and progression of intestinal mucosal damage during early endotoxemia.     

Resistance of bcr-abl-positive acute lymphoblastic leukemia to daunorubicin is not mediated by mdr1 gene expression

In vitro resistance to anthracyclines is thought to be a poor prognosis in achieving long-term remission in patients with acute lymphoblastic leukemia (ALL). Expression of a multidrug resistance gene (mdr1) that codes for 170 Kd transmembrane glycoprotein is responsible for conferring resistance to malignant cells to anthracyclines. The t(9:22) translocation, resulting in bcr-abl fusion gene, is commonly found in B-lineage ALL and is known to be a poor prognostic factor for long-term remission. To investigate whether resistance to anthracyclines contributes to poor prognosis in bcr-abl-positive ALL, we studied daunorubicin sensitivity by an in vitro colorimetric methyl tetrazolium (MTT) assay in B-lineage ALL patients who were bcr-abl-positive and compared them with the B-lineage, age-matched bcr-abl-negative group. We also looked for and compared the presence of mdr1 gene expression in these two groups of patients by RT-PCR. Of the 46 patients included in the study, 16 (34.7%) were positive for the bcr-abl fusion gene. mdr1 gene expression was seen in 14 of these 46 patients (30.4%). However, the expression of the mdr1 gene was relatively lower in the bcr-abl-positive group (3 out of 16, 18.7%) compared to the bcr-abl-negative group (11 out of 30, 36.6%). The median LD(50) of daunorubicin (concentration lethal to 50% of the leukemic blasts) differed significantly between bcr-abl-positive and -negative patients (P = 0.018). This in vitro study suggests that bcr-abl-positive ALL is relatively resistant to daunorubicin, but this resistance is not mediated through mdr1 gene expression.     

Superoxide produced by Kupffer cells is an essential effector in concanavalin A-induced hepatitis in mice

Although concanavalin A (Con-A)-induced experimental hepatitis is thought to be induced by activated T cells, natural killer T (NKT) cells, and cytokines, precise mechanisms are still unknown. In the current study, we investigated the roles of Kupffer cells, NKT cells, FasL, tumor necrosis factor (TNF), and superoxide in Con-A hepatitis in C57BL/6 mice. Removal of Kupffer cells using gadolinium chloride (GdCl(3)) from the liver completely inhibited Con-A hepatitis, whereas increased serum TNF and IFN-gamma levels were not inhibited at all. Unexpectedly, anti-FasL antibody pretreatment did not inhibit Con-A hepatitis, whereas it inhibited hepatic injury induced by a synthetic ligand of NKT cells, alpha-galactosylceramide. Furthermore, GdCl(3) pretreatment changed neither the activation-induced down-regulation of NK1.1 antigens as well as T cell receptors of NKT cells nor the increased expression of the CD69 activation antigen of hepatic T cells. CD68(+) Kupffer cells greatly increased in proportion in the early phase after Con-A injection; this increase was abrogated by GdCl(3) pretreatment. Anti-TNF antibody (Ab) pretreatment did not inhibit the increase of Kupffer cells, but it effectively suppressed superoxide/reactive oxygen production from Kupffer cells and the resulting hepatic injury. Conversely, depletion of NKT cells in mice by NK1.1 Ab pretreatment did suppress both the increase of CD68(+) Kupffer cells and Con-A hepatitis. Consistently, the diminution of oxygen radicals produced by Kupffer cells by use of free radical scavengers greatly inhibited Con-A hepatitis without suppressing cytokine production. However, adoptive transfer experiments also indicate that a close interaction/cooperation of Kupffer cells with NKT cells is essential for Con-A hepatitis. Conclusion: Superoxide produced by Kupffer cells may be the essential effector in Con-A hepatitis, and TNF and NKT cells support their activation and superoxide production.     

Adiponectin receptor gene expression in human skeletal muscle cells is not regulated by fibrates and thiazolidinediones

BACKGROUND: Thiazolidinediones as PPARgamma agonists and fibrates as PPARalpha agonists improve insulin sensitivity in insulin-responsive tissues. Recent data show an induction of adiponectin receptor 2 (AdipoR2) by PPARalpha and PPARgamma agonists in human macrophages. OBJECTIVE: In this study, we examined the effects of thiazolidinediones and fibrates on the expression of adiponectin receptors in human skeletal muscle cells, an important cell type in the context of insulin resistance. RESULTS AND METHODS: In vitro differentiated human myotubes treated with troglitazone or rosiglitazone (20 h) showed no significant changes in AdipoR1 and AdipoR2 mRNA expression. PPARgamma activation was controlled by determination of PPARgamma mRNA induction. Likewise, differentiated myotubes treated with Wy-14,643 or fenofibrate (20 h) revealed no significant regulation of AdipoR1 and AdipoR2 mRNA. PPARalpha activation was assessed by measuring PDHK4 mRNA expression. CONCLUSION: Induction of AdipoR gene expression in human skeletal muscle cells is not involved in the insulin-sensitizing effects of thiazolidinediones or fibrates.     

Atheroprotection via cannabinoid receptor-2 is mediated by circulating and vascular cells in vivo

Low-dose oral tetrahydrocannabinol (THC) reduces progression of atherosclerosis in mice. THC activates central cannabinoid-1 receptors (CB1) with subsequent psychoactive effects as well as peripheral cannabinoid-2 receptors (CB2). In order to dissect the underlying mechanisms, we performed experiments under selective CB2 stimulation as well as after genetic disruption of the CB2 receptor. Atherosclerosis prone apolipoprotein E-deficient mice were crossed with cannabinoid receptor-2 deficient mice to obtain ApoE -/- CB2 -/- double knockout mice. After 8weeks of a high-cholesterol diet, immunohistochemical stainings of the aortic root revealed that vascular leukocyte infiltration in atherosclerotic plaques was accelerated in ApoE -/- CB2 -/- mice compared with ApoE -/- mice. This was accompanied by increased release of reactive oxygen species as measured using L012-enhanced chemiluminescence, and by decreased endothelial function as assessed in isolated aortic rings in organ chamber experiments. ApoE -/- mice treated with the selective CB2 agonist JWH 133 during a high-cholesterol diet showed decreased atherosclerotic lesion formation, improved endothelial function and reduced levels of reactive oxygen species. To assess whether CB2 expression in circulating cells influences atherosclerosis, irradiated ApoE -/- mice were repopulated with bone marrow-derived cells from ApoE -/- and ApoE -/- CB2 -/- mice and were fed a high-cholesterol diet for 8weeks. CB2 deficiency in bone marrow-derived cells increased leukocyte infiltration into the vessel wall, but had no impact on plaque formation. Cell culture experiments revealed that CB2 activation diminishes ROS generation in vascular cells. Selective CB2 receptor stimulation modulates atherogenesis via impact on both circulating proinflammatory and vascular cells.     

Atheroprotection via cannabinoid receptor-2 is mediated by circulating and vascular cells in vivo

Low-dose oral tetrahydrocannabinol (THC) reduces progression of atherosclerosis in mice. THC activates central cannabinoid-1 receptors (CB1) with subsequent psychoactive effects as well as peripheral cannabinoid-2 receptors (CB2). In order to dissect the underlying mechanisms, we performed experiments under selective CB2 stimulation as well as after genetic disruption of the CB2 receptor. Atherosclerosis prone apolipoprotein E-deficient mice were crossed with cannabinoid receptor-2 deficient mice to obtain ApoE −/− CB2 −/− double knockout mice. After 8 weeks of a high-cholesterol diet, immunohistochemical stainings of the aortic root revealed that vascular leukocyte infiltration in atherosclerotic plaques was accelerated in ApoE −/− CB2 −/− mice compared with ApoE −/− mice. This was accompanied by increased release of reactive oxygen species as measured using L012-enhanced chemiluminescence, and by decreased endothelial function as assessed in isolated aortic rings in organ chamber experiments. ApoE −/− mice treated with the selective CB2 agonist JWH 133 during a high-cholesterol diet showed decreased atherosclerotic lesion formation, improved endothelial function and reduced levels of reactive oxygen species. To assess whether CB2 expression in circulating cells influences atherosclerosis, irradiated ApoE −/− mice were repopulated with bone marrow-derived cells from ApoE −/− and ApoE −/− CB2 −/− mice and were fed a high-cholesterol diet for 8 weeks. CB2 deficiency in bone marrow-derived cells increased leukocyte infiltration into the vessel wall, but had no impact on plaque formation. Cell culture experiments revealed that CB2 activation diminishes ROS generation in vascular cells. Selective CB2 receptor stimulation modulates atherogenesis via impact on both circulating proinflammatory and vascular cells.     

C-kit gene is expressed by skin mast cells in embryos but not in puppies of Wsh/Wsh mice: age-dependent abolishment of c-kit gene expression

The Wsh is a mutant allele at the W (c-kit) locus of mice, but no significant abnormalities are found at the coding region of the Wsh allele. Since cultured mast cells derived from the spleen of Wsh/Wsh mice do not express messenger RNA (mRNA) of c-kit, we studied the interrelation between the number of mast cells and the magnitude of c- kit mRNA expression in the skin of Wsh/Wsh mice of various ages. The number of mast cells in the skin of Wsh/Wsh embryos of 18 days postcoitum (pc) was approximately 40% that of normal control (+/+) embryos, but the number of mast cells decreased exponentially after birth; the number dropped to 0.6% that of +/+ mice at day 150 after birth. A weak but apparent signal of c-kit mRNA was detectable in the skin of 18-day pc Wsh/Wsh embryos by RNase protection assay but not in the skin of 5-day-old Wsh/Wsh mice. The number of c-kit protein- containing cells was significantly greater in the skin of 18-day pc Wsh/Wsh embryos than in the skin of 5-day-old Wsh/Wsh mice. The abolishment of c-kit mRNA expression appeared to be specific, because the expression of mast cell carboxypeptidase A mRNA but not of c-kit mRNA was detectable by in situ hybridization in skin mast cells of 5- day-old Wsh/Wsh mice. Taken together, the expression of c-kit mRNA was abolished first, then the content of c-kit protein dropped to undetectable levels, and then the disappearance of Wsh/Wsh mast cells themselves followed.     

Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin

Hepcidin is encoded as an 84 amino acid prepropeptide containing a typical N-terminal 24 amino acid endoplasmic reticulum targeting signal sequence, and a 35 amino acid proregion (pro) with a consensus furin cleavage site immediately followed by the C-terminal 25 amino acid bioactive iron-regulatory hormone (mature peptide). We performed pulse-chase studies of posttranslational processing of hepcidin in human hepatoma HepG2 cells and in primary human hepatocytes induced with bone morphogenic protein (BMP-9). In some experiments, the cells were treated with the furin protease inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) or furin siRNA. In the absence of furin inhibitor, hepcidin was found to be processed in less than 1 h and secreted as a 3 kDa form reactive with anti-mature but not anti-pro antibody. In the presence of furin inhibitors or furin siRNA, a 6 kDa form reactive with both anti-pro and anti-mature antibody was rapidly secreted into the medium. Processing was not affected by inhibitors of the hypoxia inducible factor (HIF) pathway, or by treatment with 30 microM holo- or apo-transferrin. In conclusion, the hepatic prohormone convertase furin mediates the posttranslational processing of hepcidin. The proteolytic cleavage of prohepcidin to hepcidin is not regulated by iron-transferrin or the HIF pathway.     

Skeletal muscle glucose transporter gene expression is not affected by injecting growth-hormone-secreting cells in young rats

Summary To elucidate the diabetogenic effect of growth hormone on glucose metabolism the regulation of glucose transporter (GLUT) gene expression was examined in rat skeletal muscles. Female Wistar-Furth rats were implanted subcutaneously with growth-hormone-producing pituitary tumour (GH₃) cells. Animals were killed 4 or 9 weeks after GH₃ cell injection. Although body weight, serum growth hormone and insulin-like growth factor I levels were remarkably elevated during the 4–9 week period, serum blood glucose levels were within normal range. Muscles were obtained from the quadriceps muscle, diaphragm and heart, respectively. Northern blot analysis and Western blot analysis were performed using specific cDNA probes and antibodies. During the 4–9 week period, the levels of muscle GLUT 1 and 4 mRNA (corrected by β-actin mRNA level) in each muscle from the rats injected with tumour cells were not significantly different from those of control rats. Chronic elevation of growth hormone in these rats did not cause any change in GLUT1 and 4 expression compared to the controls during the euglycaemic period. These results provide the first evidence that chronic growth hormone elevation itself does not affect a key gene of in vivo glucose metabolism.     

Erythropoietin stimulation decreases hepcidin expression through hematopoietic activity on bone marrow cells in mice

Erythropoiesis-stimulating agents (ESA) are now central to the treatment of renal anemia and are associated with improved clinical outcomes. It is well known that erythropoietin (EPO) is a key regulator of erythropoiesis through its promotion of red blood cell production. In order to investigate the role of ESA on iron metabolism, we analyzed the regulation of the iron regulatory hormone hepcidin by ESA treatment in a bone marrow transplant model in mouse. After treating C57BL/6 mice with continuous erythropoietin receptor activator (C.E.R.A.), recombinant human epoetin-β (rhEPO), or recombinant human carbamylated epoetin-β (rhCEPO), we investigated serum hepcidin concentrations and parameters of erythropoiesis. Serum hepcidin concentrations after rhEPO treatment were analyzed in mice subjected to total body irradiation followed by bone marrow transplantation. C.E.R.A. administration caused long-term downregulation of serum hepcidin levels. Serum hepcidin levels in rhEPO-treated mice decreased significantly, whereas there was no change in rhCEPO-treated mice. The reduction in circulating hepcidin levels after rhEPO administration was not observed in irradiated mice. Finally, bone marrow transplantation recovered the response to rhEPO administration that downregulates hepcidin concentration in irradiated mice. These results indicate that ESA treatment downregulates serum hepcidin concentrations, mainly by indirect mechanisms affecting hematopoietic activity in bone marrow cells.     

Effect of phlebotomy on hepcidin expression in hemojuvelin-mutant mice

Hemojuvelin (Hjv) is an essential component of the pathway regulating hepcidin (Hamp1) gene expression. Mice with targeted disruption of the Hjv gene (Hjv-/- mice) fail to upregulate hepatic Hamp1 expression following iron overload. The main aim of the study was to determine whether the Hjv protein is also necessary for Hamp1 downregulation. In addition, sex differences in Hamp1 expression in Hjv-/- mice were also examined. Male and female Hjv-/- mice (129SvJ background) were used for the experiments, tissue Hamp1 and Hamp2 mRNA content was determined by real-time PCR. Hepatic Hamp1 mRNA content in male Hjv-/- mice was low (0.6% of Hjv+/+ males), however, female Hjv-/- mice displayed only moderately reduced (to 17%) Hamp1 mRNA levels. Hepatic non-heme iron concentration was similar in Hjv-/- mice of both sexes. Disruption of the Hjv gene did not affect Hamp1 mRNA content in the myocardium or Hamp2 mRNA content in the pancreas. Single phlebotomy resulted in significant reduction of Hamp1 mRNA in both male and female Hjv+/+ mice (to 17% and 27% of controls respectively), measured 20 h after treatment. In Hjv-/- mice, phlebotomy decreased Hamp1 mRNA content to 46% in males and to 11% in females. Bleeding also significantly decreased (to 16%) hepatic Hamp2 mRNA levels in Hjv-/- females. The obtained results indicate that the pathway mediating hepcidin downregulation by phlebotomy does not require functional hemojuvelin protein. In addition, they confirm a significant effect of sex on hepcidin gene expression.