The effects of erythropoetic activity and iron burden on hepcidin expression in patients with thalassemia major

Hepcidin production is homeostatically regulated by iron stores, anemia and hypoxia. We evaluated the effect of iron overload and of ineffective erythropoeisis on hepcidin expression in patients with thalassemia major. Liver hepcidin mRNA levels correlated with hemoglobin concentration and inversely correlated with serum transferrin receptor, erythropoietin and non-transferrin-bound iron. They did not correlate with indices of iron load. Urinary hepcidin levels were disproportionably suppressed in regards to iron burden. We conclude that hepcidin expression is regulated mainly by increased erythropoietic activity rather than by iron load and that hepcidin plays a central regulatory role in iron circulation and iron toxicity in patients with thalassemia.     

Hypoxia-inducible nuclear factors bind to an enhancer element located 3'' to the human erythropoietin gene.

Human erythropoietin gene expression in liver and kidney is inducible by anemia or hypoxia. DNase I-hypersensitive sites were identified 3' to the human erythropoietin gene in liver nuclei. A 256-base-pair region of 3' flanking sequence was shown by DNase I protection and electrophoretic mobility-shift assays to bind four or more different nuclear factors, at least two of which are induced by anemia in both liver and kidney, and the region functioned as a hypoxia-inducible enhancer in transient expression assays. These results provide insight into the molecular basis for the regulation of gene expression by a fundamental physiologic stimulus, hypoxia.     

Suppression of hepcidin during anemia requires erythropoietic activity

Hepcidin, the principal iron regulatory hormone, regulates the absorption of iron from the diet and the mobilization of iron from stores. Previous studies indicated that hepcidin is suppressed during anemia, a response that would appropriately increase the absorption of iron and its release from stores. Indeed, in the mouse model, hepcidin-1 was suppressed after phlebotomy or erythropoietin administration but the suppression was reversed by inhibitors of erythropoiesis. The suppression of hepcidin necessary to match iron supply to erythropoietic demand thus requires increased erythropoiesis and is not directly mediated by anemia, tissue hypoxia, or erythropoietin.     

Increased adipose tissue expression of hepcidin in severe obesity is independent from diabetes and NASH

BACKGROUNDS & AIMS: Hepcidin is an acute-phase response peptide. We have investigated the possible involvement of hepcidin in massive obesity, a state of chronic low-grade inflammation. Three groups of severely obese patients with or without diabetes or nonalcoholic steatohepatitis were investigated. METHODS: Hepcidin expression was studied in liver and adipose tissue of these patients. Hepcidin regulation was investigated in vitro by adipose tissue explant stimulation studies. RESULTS: Hepcidin was expressed not only in the liver but also at the messenger RNA (mRNA) and the protein levels in adipose tissue. Moreover, mRNA expression was increased in adipose tissue of obese patients. The presence of diabetes or NASH did not modify the hepcidin expression levels in liver and adipose tissue. In adipose tissue, mRNA expression correlated with indexes of inflammation, interleukin-6, and C-reactive protein. Interleukin-6 also promoted in vitro hepcidin expression. A low transferrin saturation ratio was observed in 68% of the obese patients; moreover, 24% of these patients presented with anemia. The observed changes in iron status could be due to the role of hepcidin as a negative regulator of intestinal iron absorption and macrophage iron efflux. Interestingly, a feedback control mechanism on hepcidin expression related to low transferrin saturation occurred in the liver but not in the adipose tissue. CONCLUSIONS: Hepcidin is a proinflammatory adipokine and may play an important role in hypoferremia of inflammation in obese condition.     

The effects of iron deficiency on estradiol-induced suppression of erythropoietin induction in rats: implications of pregnancy-related anemia

Pregnant women often develop anemia concomitant with the increase in serum erythropoietin levels, which are actually lower than those of nonpregnant anemic women due to the possible suppressive effect of endogenous estradiol on erythropoietin induction. The anemia, derived from hemodilution, does not act as a drive for erythropoietin induction, but iron deficiency, often observed during pregnancy, might. In order to demonstrate this, we investigated the effects of iron deficiency on estradiol-induced suppression of erythropoietin induction in rats. Single doses of estradiol suppressed hypoxia-, cobalt-, and bleeding-stimulated elevation of plasma erythropoietin levels and renal erythropoietin mRNA expression. Repeated administration of estradiol at 0.1 and 1 mg/kg for 2 months induced a slight anemic trend without elevation of plasma erythropoietin. Feeding an iron-deficient diet for 2 months induced plasma erythropoietin elevation without obvious anemia, but the simultaneous repeated administration of estradiol suppressed it and reversed the iron deficiency. Plasma erythropoietin levels had distinct negative correlations with plasma iron, plasma ferritin, and iron concentrations in the organs, but not with plasma hemoglobin level. These results suggest that iron deficiency would significantly stimulate erythropoietin induction during pregnancy, although estradiol might suppress it through iron restoration.     

The iron regulatory peptide hepcidin is expressed in the heart and regulated by hypoxia and inflammation

The peptide hormone hepcidin plays a central role in iron homeostasis. It is predominantly expressed in the liver and regulated by iron, hypoxia, and inflammation. Although it has been shown that iron plays a key pathophysiological role in cardiac diseases, including iron-overload cardiomyopathy, myocardial ischemia-reperfusion injury, and atherosclerosis, very little is known about the putative expression and the role of hepcidin in the heart. In the present study, expression and regulation of hepcidin in rat heart were analyzed. Basal cardiac expression of hepcidin was demonstrated on mRNA and protein level in vivo in a rat model and compared with its regulation in the liver. The cellular localization was analyzed by immunofluorescence microscopy. Sixteen hours after a single injection of turpentine, a more than 2-fold increase of cardiac hepcidin mRNA and a more than 3-fold increase of hepatic hepcidin mRNA was observed. In response to hypoxia, expression of hepcidin in the liver decreased. In contrast, hypoxia resulted in a strong up-regulation of hepcidin expression on mRNA and protein level in the heart, accompanied by an increased immunoreactivity of hepcidin pronounced at the myocardial intercalated disc area. The finding of a regulated expression of the iron-regulatory peptide hormone hepcidin in the heart suggests that hepcidin may have an important role in cardiac diseases.     

STAT3 mediates hepatic hepcidin expression and its inflammatory stimulation

Hepcidin is a key iron-regulatory hormone produced by the liver. Inappropriately low hepcidin levels cause iron overload, while increased hepcidin expression plays an important role in the anemia of inflammation (AI) by restricting intestinal iron absorption and macrophage iron release. Its expression is modulated in response to body iron stores, hypoxia, and inflammatory and infectious stimuli involving at least in part cytokines secreted by macrophages. In this study we established and characterized IL6-mediated hepcidin activation in the human liver cell line Huh7. We show that the proximal 165 bp of the hepcidin promoter is critical for hepcidin activation in response to exogenously administered IL6 or to conditioned medium from the monocyte/macrophage cell line THP-1. Importantly, we show that hepcidin activation by these stimuli requires a STAT3 binding motif located at position -64/-72 of the promoter. The same STAT binding site is also required for high basal-level hepcidin mRNA expression under control culture conditions, and siRNA-mediated RNA knockdown of STAT3 strongly reduces hepcidin mRNA expression. These results identify a missing link in the acute-phase activation of hepcidin and establish STAT3 as a key effector of baseline hepcidin expression and during inflammatory conditions.     

Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease

The anemia of chronic disease is a prevalent, poorly understood condition that afflicts patients with a wide variety of diseases, including infections, malignancies, and rheumatologic disorders. It is characterized by a blunted erythropoietin response by erythroid precursors, decreased red blood cell survival, and a defect in iron absorption and macrophage iron retention, which interrupts iron delivery to erythroid precursor cells. We noted that patients with large hepatic adenomas had severe iron refractory anemia similar to that observed in anemia of chronic disease. This anemia resolved spontaneously after adenoma resection or liver transplantation. We investigated the role of the adenomas in the pathogenesis of the anemia and found that they produce inappropriately high levels of hepcidin mRNA. Hepcidin is a peptide hormone that has been implicated in controlling the release of iron from cells. We conclude that hepcidin plays a major, causative role in the anemia observed in our subgroup of patients with hepatic adenomas, and we speculate that it is important in the pathogenesis of the anemia of chronic disease in general.     

Cyclooxygenase 2 and intermittent hypoxia-induced spatial deficits in the rat

Intermittent hypoxia (IH) during sleep, a critical feature of sleep apnea, induces significant neurobehavioral deficits in the rat. Cyclooxygenase (COX)-2 is induced during stressful conditions such as cerebral ischemia and could play an important role in IH-induced learning deficits. We therefore examined COX-1 and COX-2 genes and COX-2 protein expression and activity (prostaglandin E2 [PGE2] tissue concentration) in cortical regions of rat brain after exposure to either IH (10% O2 alternating with 21% O2 every 90 seconds) or sustained hypoxia (10% O2). In addition, the effect of selective COX-2 inhibition with NS-398 on IH-induced neurobehavioral deficits was assessed. IH was associated with increased COX-2 protein and gene expression from Day 1 to Day 14 of exposure. No changes were found in COX-1 gene expression after exposure to hypoxia. IH-induced COX-2 upregulation was associated with increased PGE2 tissue levels, neuronal apoptosis, and neurobehavioral deficits. Administration of NS-398 abolished IH-induced apoptosis and PGE2 increases without modifying COX-2 mRNA expression. Furthermore, NS-398 treatment attenuated IH-induced deficits in the acquisition and retention of a spatial task in the water maze. We conclude that IH induces upregulation and activation of COX-2 in rat cortex and that COX-2 may play a role in IH-mediated neurobehavioral deficits.     

Lipocalin 2 deficiency alters estradiol production and estrogen receptor signaling in female mice

We have previously characterized lipocalin 2 (Lcn2) as a new adipokine having a critical role in energy and lipid metabolism in male mice. Previous studies by others have suggested that Lcn2 is a putative target gene of estrogens. In this study, we reported the effect of Lcn2 deficiency on estradiol biosynthesis and estrogen receptor signaling in female Lcn2-deficient (Lcn2-/-) mice. We found that Lcn2 expression in white adipose tissue is gender, depot, and age dependent. In female mice, Lcn2 is predominantly expressed in inguinal adipose tissue but at relatively very low levels in perigonadal depot and ovary. After 22 wk of high-fat diet (HFD) feeding or at old age, Lcn2-/- female mice had significantly reduced levels of serum 17β-estradiol and down-regulated expression of estrogen receptor α in multiple metabolic tissues. Consistently, the expression of estrogen-regulated genes involved in cholesterol homeostasis, such as liver X receptor β and low-density lipoprotein receptor was also down-regulated in the adipose tissue of Lcn2-/- mice. These changes were in line with the development of atherogenic dyslipidemia in response to HFD feeding; female Lcn2-/- mice had significantly elevated levels of total cholesterol and low-density lipoprotein cholesterol, whereas reduced high-density lipoprotein cholesterol levels compared with wild-type female mice. Interestingly, when compared with wild-type controls, HFD-fed female Lcn2-/- mice had significantly reduced expression levels of aromatase, a key enzyme regulating estradiol biosynthesis, in adipose tissue. Moreover, Lcn2 deficiency markedly blunted age-related increase in adipose aromatase expression but had no significant impact on age-related reduction in ovarian aromatase expression. Our findings suggest that Lcn2 has a tissue-specific role in adipose estradiol biosynthesis, which may link Lcn2 to obesity- and age-related estradiol production and metabolic complications in females.     

Hepcidin is a Potential Regulator of Iron Status in Chronic Kidney Disease

Hepcidin is a small defensin‐like peptide produced primarily by hepatocytes, but also by other cells, including macrophages. In addition to hepcidin's antimicrobial properties, it is the main regulator of iron metabolism and controls both the amount of dietary iron absorbed in the duodenum and the iron release by reticuloendothelial cells. Hepcidin expression is upregulated by a variety of stimuli, including inflammation and iron overload, and downregulated by anemia, hypoxia, and iron deficiency. Chronic kidney disease (CKD) is associated with increased serum hepcidin levels, and the increased levels may contribute to the development and severity of anemia and to resistance to erythropoiesis‐stimulating agents (ESAs). Elevated serum hepcidin levels contribute to the dysregulation of iron homeostasis in CKD patients. Although parenteral iron supplementation can bypass some of the iron‐blocking effects of hepcidin in CKD patients with anemia, and free iron and iron stores increase as a result, the anemia is only partially corrected, and the ESA dose requirements remain significantly higher than needed for physiological replacement. Treatment with agents that lower serum hepcidin levels or inhibit its actions may be an effective strategy for restoring normal iron homeostasis and improving anemia in CKD patients. The aim of this article was to review the regulation of hepcidin levels and the role of hepcidin in CKD‐related anemia, and to discuss hepcidin's potential as a clinical biomarker and several investigational treatments designed to lower serum hepcidin levels.     

HIF-2alpha regulates murine hematopoietic development in an erythropoietin-dependent manner

Erythropoiesis in the adult mammal depends critically on erythropoietin, an inducible cytokine with pluripotent effects. Erythropoietin gene expression increases under conditions associated with lowered oxygen content such as anemia and hypoxia. HIF-1alpha, the founding member of the hypoxia-inducible factor (HIF) alpha class, was identified by its ability to bind and activate the hypoxia-responsive enhancer in the erythropoietin regulatory region in vitro. The existence of multiple HIF alpha members raises the question of which HIF alpha member or members regulates erythropoietin expression in vivo. We previously reported that mice lacking wild-type HIF-2alpha, encoded by the EPAS1 gene, exhibit pancytopenia. In this study, we have characterized the etiology of this hematopoietic phenotype. Molecular studies of EPAS1-null kidneys reveal dramatically decreased erythropoietin gene expression. EPAS1-null as well as heterozygous mice have impaired renal erythropoietin induction in response to hypoxia. Treatment of EPAS1-null mice with exogenous erythropoietin reverses the hematopoietic and other defects. We propose that HIF-2alpha is an essential regulator of murine erythropoietin production. Impairments in HIF signaling, involving either HIF-1alpha or HIF-2alpha, may play a prominent role in conditions involving altered hematopoietic or erythropoietin homeostasis.     

Iron metabolism in the hemoglobin-deficit mouse: correlation of diferric transferrin with hepcidin expression

The iron requirements of the erythroid compartment modulate the expression of hepcidin in the liver, which in turn alters intestinal iron absorption and iron release from the reticuloendothelial system. We have taken advantage of an inherited anemia of the mouse (hemoglobin deficit, or hbd) to gain insights into the factors regulating hepcidin expression. hbd mice showed a significant anemia but, surprisingly, their iron absorption was not increased as it was in wild-type animals made anemic to a similar degree by dietary iron depletion. In wild-type mice hepatic hepcidin levels were decreased but in hbd animals a significant and unexpected increase was observed. The level of absorption was appropriate for the expression of hepcidin in each case, but in hbd mice did not reflect the degree of anemia. However, this apparent inappropriate regulation of hepcidin correlated with increased transferrin saturation and levels of diferric transferrin in the plasma, which in turn resulted from the reduced capacity of hbd animals to effectively use transferrin-bound iron. These data strengthen the proposal that diferric transferrin is a key indicator of body iron requirements.     

Role of erythropoietin in the brain

Multi-tissue erythropoietin receptor (EPO-R) expression provides for erythropoietin (EPO) activity beyond its known regulation of red blood cell production. This review highlights the role of EPO and EPO-R in brain development and neuroprotection. EPO-R brain expression includes neural progenitor cells (NPC), neurons, glial cells and endothelial cells. EPO is produced in brain in a hypoxia sensitive manner, stimulates NPC proliferation and differentiation, and neuron survival, and contributes to ischemic preconditioning. Mice lacking EPO or EPO-R exhibit increased neural cell apoptosis during development before embryonic death due to severe anemia. EPO administration provides neural protection in animal models of brain ischemia and trauma, reducing the extent of injury and damage. Intrinsic EPO production in brain and EPO stimulation of endothelial cells contribute to neuroprotection and these are of particular importance since only low levels of EPO appear to cross the blood-brain barrier when administered at high dose intravenously. The therapeutic potential of EPO for brain ischemia/trauma and neurodegenerative diseases has shown promise in early clinical trial and awaits further validation.