Evidence for a lack of a direct transcriptional suppression of the iron regulatory peptide hepcidin by hypoxia-inducible factors.

BACKGROUND: Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation. METHODOLOGY/PRINCIPAL FINDINGS: Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1alpha or HIF-2alpha knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased. CONCLUSIONS/SIGNIFICANCE: Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression

Tumor Necrosis Factor α Inhibits Expression of the Iron Regulating Hormone Hepcidin in Murine Models of Innate Colitis

Background: Abnormal expression of the liver peptide hormone hepcidin, a key regulator of iron homeostasis, contributes to the pathogenesis of anemia in conditions such as inflammatory bowel disease (IBD). Since little is known about the mechanisms that control hepcidin expression during states of intestinal inflammation, we sought to shed light on this issue using mouse models. Methodology/Principal Findings: Hepcidin expression was evaluated in two types of intestinal inflammation caused by innate immune activation—dextran sulfate sodium (DSS)-induced colitis in wild-type mice and the spontaneous colitis occurring in T-bet/Rag2-deficient (TRUC) mice. The role of tumor necrosis factor (TNF) $\alpha$ was investigated by in vivo neutralization, and by treatment of a hepatocyte cell line, as well as mice, with the recombinant cytokine. Expression and activation of Smad1, a positive regulator of hepcidin transcription, were assessed during colitis and following administration or neutralization of TNF$\alpha$. Hepcidin expression progressively decreased with time during DSS colitis, correlating with changes in systemic iron distribution. TNF$\alpha$ inhibited hepcidin expression in cultured hepatocytes and non-colitic mice, while TNF$\alpha$ neutralization during DSS colitis increased it. Similar results were obtained in TRUC mice. These effects involved a TNF$\alpha$-dependent decrease in Smad1 protein but not mRNA. Conclusions/Significance: TNF$\alpha$ inhibits hepcidin expression in two distinct types of innate colitis, with down-regulation of Smad1 protein playing an important role in this process. This inhibitory effect of TNF$\alpha$ may be superseded by other factors in the context of T cell-mediated colitis given that in the latter form of intestinal inflammation hepcidin is usually up-regulated

Hepcidin Is Involved in Iron Regulation in the Ischemic Brain

Oxidative stress plays an important role in neuronal injuries caused by cerebral ischemia. It is well established that free iron increases significantly during ischemia and is responsible for oxidative damage in the brain. However, the mechanism of this ischemia-induced increase in iron is not completely understood. In this report, the middle cerebral artery occlusion (MCAO) rat model was performed and the mechanism of iron accumulation in cerebral ischemia-reperfusion was studied. The expression of L-ferritin was significantly increased in the cerebral cortex, hippocampus, and striatum on the ischemic side, whereas H-ferritin was reduced in the striatum and increased in the cerebral cortex and hippocampus. The expression level of the iron-export protein ferroportin1 (FPN1) significantly decreased, while the expression of transferrin receptor 1 (TfR1) was increased. In order to elucidate the mechanisms of FPN1 regulation, we studied the expression of the key regulator of FPN1, hepcidin. We observed that the hepcidin level was significantly elevated in the ischemic side of the brain. Knockdown hepcidin repressed the increasing of L-ferritin and decreasing of FPN1 invoked by ischemia-reperfusion. The results indicate that hepcidin is an important contributor to iron overload in cerebral ischemia. Furthermore, our results demonstrated that the levels of hypoxia-inducible factor-1α (HIF-1α) were significantly higher in the cerebral cortex, hippocampus and striatum on the ischemic side; therefore, the HIF-1α-mediated TfR1 expression may be another contributor to the iron overload in the ischemia-reperfusion brain

Primate beta-defensins--structure, function and evolution.

Host defense peptides (HDPs) are endogenous antibiotics that play a multifunctional role in the innate immunity of mammals. Among these, \u3b2-defensins contribute to mucosal and epithelial defense, also acting as signal molecules for cellular components of innate and adaptive immunity. Numerous members of this family have been identified in mammalian and avian species, and genomic studies in human and mouse indicate a considerable complexity in their gene organization. Recent reports on the evolution of primate and rodent members of this family indicate quite a complex pattern of variation. In this review we briefly discuss the evolution of mammalian \u3b2-defensins in relation to other types of defensins, and then concentrate on the evolution of \u3b2-defensins 1 to 4 in primates. In particular, the surprisingly varied patterns of evolution, which range from neutral or weakly purifying, to positive selection to a high level of conservation are analyzed in terms of possible genetics, structural or functional implications, as well as to observed variations on the antimicrobial activity in vitro. The role of polymorphisms in the genes encoding for these host defense peptides in determining susceptibility to human diseases are also briefly considered. \ua9 2005 Bentham Science Publishers Ltd

SINGLE-AGENT INHIBITION OF CHECKPOINT KINASE 1 (CHK1) AND 2 (CHK2) BY PF-0477736 (PFIZER) AS A NEW PROMISING THERAPY IN B-ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

Introduction. Chk1 and Chk2 are serine/threonine kinases that play a critical role in determining cellular responses to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. We explored the cellular effects of single-agent inhibition of Chk1/2 by PF-0477736 and its potential use as a therapeutic strategy for the treatment of B-ALL. Methods. Cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Results. BCR-ABL1-positive (BV-173, SUPB-15) and negative cell lines (NALM6, NALM19, REH) were incubated with increasing concentrations of PF-0477736 (0.005-2 M) for 24, 48 and 72 hours. Inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1-1.5 \u3bcM, with BV-173 being the most sensitive, while NALM6 the most resistant. All cell lines were TP53 wild-type, CDKN2A deleted. Consistent with the viability results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in all cell lines. Functionally, PF-0477736 decreased the inhibitory phosphorylation of Cdc25c Ser216 which is inactivated by Chk1 to prevent mitotic entry and increased the number of H2AX foci, a markers of DNA damage, that culminated in a proportion of cells developing intense staining for H2AX together with nuclear morphological characteristics of apoptosis as demonstrated by immunofluorescence analysis. The efficacy of PF-0477736 was thereafter confirmed in primary blasts from 11 B-ALL patients. Based on the viability results, three groups of patients were identified: very good responders, 46% (IC50: 0.1-0.5 \u3bcM at 24 hours); good responders, 36% (IC50: 0.6-1 \u3bcM at 24 hours); poor responders, 18% (IC50 > 1 \u3bcM at 24 hours). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis was performed on treated cell lines and their untreated counterparts (DMSO 0.1%) after 24 hours. Consistent with a specific Chk1- mechanism of action, treatment resulted in differential expression (p < 0.05) of genes involved in apoptosis and cell cycle (e.g. CEBPB, CUL1, Histone H1-H2A, 2B family clusters) and DNA damage (DDIT3, GADD34 and GADD45a), suggesting that PF-0477736 contributes to accumulation of DNA damage and subsequent apoptosis in B-ALL cells. Conclusions. For the first time we demonstrated the efficacy of PF-0477736 in vitro models of B- ALL, suggesting that single-agent Chk1/2 inhibition may be further evaluated in clinical trials. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, FIRB2006, PRIN2009, PIO program, Programma Ricerca Regione-Universit\ue0 2007\u20132009. PF-0477736 provided by Pfizer

Effect of body mass index reduction on serum hepcidin levels and iron status in obese children.

Item does not contain fulltextIron deficiency has been linked to obesity. Hepcidin is the main regulator of iron homeostasis and is higher in obese children compared to controls. To gain insight into the link between obesity and hepcidin, we performed an intervention study in 15 obese children. These children were subjected to a 6-month weight loss program and underwent physical examination and iron status and absorption as well as hepcidin, interleukin-6 and leptin serum levels evaluation at baseline and after the weight loss program. After the program all children reduced their body mass index standard deviation score (BMI SDS) of at least 0.5. We observed a significant decrease in hepcidin (P=0.003) and leptin levels (P=0.005), and a significant increase in iron absorption (P=0.02). A direct correlation between the measure of hepcidin and leptin reduction was observed and this correlation appeared significant (r(2)=0.33, P=0.003) when adjusted for interleukin-6 and BMI SDS variations. In conclusion, we have shown that, in obese children, BMI reduction is associated with hepcidin reduction, potentially improving iron status and absorption. Implications of these findings could be considered in the management of obese children with poor iron status.1 december 201