Molecular regulation of hepatic expression of iron regulatory hormone hepcidin

Iron is a micronutrient that is an essential component that drives many metabolic reactions. Too little iron leads to anemia and too much iron increases the oxidative stress of body tissues leading to inflammation, cell death, and system organ dysfunction, including cancer. Maintaining normal iron balance is achieved by rigorous control of the amount absorbed by the intestine, that released from macrophages following erythrophagocytosis of effete red cells and by either release or uptake from hepatocytes. Hepcidin is a recently characterized molecule that appears to play a key role in the regulation of iron efflux from enterocytes, macrophages, and hepatocytes. It is produced by hepatocytes under basal conditions, in response to alterations in increased iron stores or reduced requirement for erythropoiesis and by inflammation. The proteins that regulate hepcidin expression are presently being defined, albeit that our present understanding is still far from complete. This review focuses on the molecules which regulate hepcidin expression. The subsequent characterization of these proteins using molecular, cellular, and physiological approaches also is discussed along with inflammatory signals and receptors involved in hepcidin expression.     

Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice

Bone morphogenetic protein (BMP) signaling induces hepatic expression of the peptide hormone hepcidin. Hepcidin reduces serum iron levels by promoting degradation of the iron exporter ferroportin. A relative deficiency of hepcidin underlies the pathophysiology of many of the genetically distinct iron overload disorders, collectively termed hereditary hemochromatosis. Conversely, chronic inflammatory conditions and neoplastic diseases can induce high hepcidin levels, leading to impaired mobilization of iron stores and the anemia of chronic disease. Two BMP type I receptors, Alk2 (Acvr1) and Alk3 (Bmpr1a), are expressed in murine hepatocytes. We report that liver-specific deletion of either Alk2 or Alk3 causes iron overload in mice. The iron overload phenotype was more marked in Alk3- than in Alk2-deficient mice, and Alk3 deficiency was associated with a nearly complete ablation of basal BMP signaling and hepcidin expression. Both Alk2 and Alk3 were required for induction of hepcidin gene expression by BMP2 in cultured hepatocytes or by iron challenge in vivo. These observations demonstrate that one type I BMP receptor, Alk3, is critically responsible for basal hepcidin expression, whereas 2 type I BMP receptors, Alk2 and Alk3, are required for regulation of hepcidin gene expression in response to iron and BMP signaling.     

Melatonin induces the expression of gonadotropin-inhibitory hormone in the avian brain

We recently identified a novel hypothalamic neuropeptide inhibiting gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). Cell bodies and terminals containing the dodecapeptide GnIH are localized in the paraventricular nucleus (PVN) and median eminence, respectively. To understand the physiological role of GnIH, we investigated the mechanisms that regulate GnIH expression. In this study, we show that melatonin originating from the pineal gland and eyes induces GnIH expression in the quail brain. Pinealectomy (Px) combined with orbital enucleation (Ex) (Px plus Ex) decreased the expression of GnIH precursor mRNA and content of mature GnIH peptide in the diencephalon, which includes the PVN and median eminence. Melatonin administration to Px plus Ex birds caused a dose-dependent increase in expression of GnIH precursor mRNA and production of mature peptide. The expression of GnIH was photoperiodically controlled and increased under short-day photoperiods, when the duration of melatonin secretion increases. To identify the mode of melatonin action on GnIH induction, we investigated the expression of Mel(1c), a melatonin receptor subtype, in GnIH neurons. In situ hybridization of Mel(1c) mRNA combined with immunocytochemistry for GnIH revealed that Mel(1c) mRNA was expressed in GnIH-immunoreactive neurons in the PVN. Melatonin receptor autoradiography further revealed specific binding of melatonin in the PVN. These results indicate that melatonin is a key factor for GnIH induction. Melatonin appears to act directly on GnIH neurons through its receptor to induce GnIH expression. This is the first demonstration, to our knowledge, of a direct action of melatonin on neuropeptide induction in any vertebrate class.     

Autocrine formation of hepcidin induces iron retention in human monocytes

Hepcidin, a master regulator of iron homeostasis, is produced in small amounts by inflammatory monocytes/macrophages. Chronic immune activation leads to iron retention within monocytes/macrophages and the development of anemia of chronic disease (ACD). We questioned whether monocyte-derived hepcidin exerts autocrine regulation toward cellular iron metabolism. Monocyte hepcidin mRNA expression was significantly induced within 3 hours after stimulation with LPS or IL-6, and hepcidin mRNA expression was significantly higher in monocytes of ACD patients than in controls. In ACD patients, monocyte hepcidin mRNA levels were significantly correlated to serum IL-6 concentrations, and increased monocyte hepcidin mRNA levels were associated with decreased expression of the iron exporter ferroportin and iron retention in these cells. Transient transfection experiments using a ferroportin/EmGFP fusion protein construct demonstrated that LPS inducible hepcidin expression in THP-1 monocytes resulted in internalization and degradation of ferroportin. Transfection of monocytes with siRNA directed against hepcidin almost fully reversed this lipopolysaccharide-mediated effect. Using ferroportin mutation constructs, we found that ferroportin is mainly targeted by hepcidin when expressed on the cell surface. Our results suggest that ferroportin expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin, thus contributing to iron sequestration within monocytes as found in ACD.     

Delayed hepcidin response explains the lag period in iron absorption following a stimulus to increase erythropoiesis

INTRODUCTION: The delay of several days between an erythropoietic stimulus and the subsequent increase in intestinal iron absorption is commonly believed to represent the time required for body signals to programme the immature crypt enterocytes and for these cells to migrate to the villus. Recent data however suggest that signals from the body to alter absorption are mediated by circulating hepcidin and that this peptide exerts its effect on mature villus enterocytes. METHODS: We have examined the delay in the absorptive response following stimulated erythropoiesis using phenylhydrazine induced haemolysis and correlated this with expression of hepcidin in the liver and iron transporters in the duodenum. RESULTS: There was a delay of four days following haemolysis before a significant increase in iron absorption was observed. Hepatic hepcidin expression did not decrease until day 3, reaching almost undetectable levels by days 4 and 5. This coincided with the increase in duodenal expression of divalent metal transporter 1, duodenal cytochrome b, and Ireg1. CONCLUSION: These results suggest that the delayed increase in iron absorption following stimulated erythropoiesis is attributable to a lag in the hepcidin response rather than crypt programming, and are consistent with a direct effect of the hepcidin pathway on mature villus enterocytes.     

Stimulated erythropoiesis with secondary iron loading leads to a decrease in hepcidin despite an increase in bone morphogenetic protein 6 expression

The BMP/SMAD signalling pathway plays an important role in iron homeostasis, regulating hepcidin expression in response to body iron levels. However, the role of this pathway in the reduction in hepcidin associated with increased erythropoiesis (and secondary iron loading) is unclear. To investigate this, we established a mouse model of chronic stimulated erythropoiesis with secondary iron loading using the haemolytic agent phenylhydrazine. We then examined the expression of components of the BMP6/SMAD signalling pathway in these animals. We also examined this pathway in the Hbb(th3/+) mouse, a model of the iron loading anaemia β-thalassaemia intermedia. Increasing doses of phenylhydrazine led to a progressive increase in both liver iron levels and Bmp6 mRNA expression, but, in contrast, hepatic Hamp expression declined. The increase in Bmp6 expression was not associated with a corresponding change in the phosphorylation of hepatic SMAD1/5/8, indicating that stimulated erythropoiesis decreases the ability of BMP6 to alter SMAD phosphorylation. Increased erythropoiesis also reduces the capacity of phosphorylated SMAD (pSMAD) to induce hepcidin, as Hamp levels declined despite no changes in pSMAD1/5/8. Similar results were seen in Hbb(th3/+) mice. Thus the erythroid signal probably affects some components of BMP/SMAD signalling, but also may exert some independent effects.     

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.     

Activation and inactivation of the iron hormone hepcidin: Biochemical characterization of prohepcidin cleavage and sequential degradation to N-terminally truncated hepcidin isoforms

The hormone hepcidin is produced mainly in the liver in response to iron loading and inflammation and secreted into the circulation as a 25-amino acid peptide. The 84-amino acid prohormone undergoes limited proteolytic cleavage at a conserved proprotein convertase (PC) recognition site. In addition to the 25-amino acid hepcidin, N-terminally truncated isoforms of lower biological activity are found in plasma and urine.Here we show that a redundant system of proprotein convertases cleaves prohepcidin at the predicted site releasing active hepcidin-25 from the proprotein. In addition to furin mediated cleavage of prohepcidin, we found prohepcidin peptidase activity of proprotein convertases PC5/6, PC7/LPC, PC1/3 and PC2 which was specific for the release of hepcidin-25 from prohepcidin as shown by mass spectrometry. In native tissue extracts, a calcium-dependent prohepcidin peptidase activity is present specifically releasing the 25-mer hepcidin isoform from the recombinant prohormone. In contrast, the 20-mer isoform of hepcidin is generated by a calcium-independent tissue activity which cleaves the 25-mer peptide but has no activity on the entire prohormone. This finding demonstrates the presence of an additional peptidase in this inactivation mechanism for hepcidin. An inhibitor of prohepcidin cleavage was designed and synthesized from d-amino acids (QRRRRR). Biochemical studies indicated that this is a potent and generic inhibitor of prohepcidin cleavage.Biochemical and inhibitor studies of endogenous tissue peptidase activities support the implication of proprotein convertases in the activation of hepcidin. Inactivation of the peptide hormone by N-terminal truncation is mediated by other distinct peptidases, which appear to act sequentially to initial release of hepcidin-25 from the proprotein.     

Anemia in beta-thalassemia patients targets hepatic hepcidin transcript levels independently of iron metabolism genes controlling hepcidin expression

Thalassemia associates anemia and iron overload, two opposite stimuli regulating hepcidin gene expression. We characterized hepatic hepcidin expression in 10 thalassemia major and 13 thalassemia intermedia patients. Hepcidin mRNA levels were decreased in the thalassemia intermedia group which presented both lower hemoglobin and higher plasma soluble transferrin receptor levels. There was no relationship between hepcidin mRNA levels and those of genes controlling iron metabolism, including HFE, hemojuvelin, transferrin receptor-2 and ferroportin. These results underline the role of erythropoietic activity on hepcidin decrease in thalassemic patients and suggest that mRNA modulations of other studied genes do not have a significant impact.     

The role of iron and copper molecules in the neuronal vulnerability of locus coeruleus and substantia nigra during aging

In this study, a comparative analysis of metal-related neuronal vulnerability was performed in two brainstem nuclei, the locus coeruleus (LC) and substantia nigra (SN), known targets of the etiological noxae in Parkinson's disease and related disorders. LC and SN pars compacta neurons both degenerate in Parkinson's disease and other Parkinsonisms; however, LC neurons are comparatively less affected and with a variable degree of involvement. In this study, iron, copper, and their major molecular forms like ferritins, ceruloplasmin, neuromelanin (NM), manganese-superoxide dismutase (SOD), and copper/zinc-SOD were measured in LC and SN of normal subjects at different ages. Iron content in LC was much lower than that in SN, and the ratio heavy-chain ferritin/iron in LC was higher than in the SN. The NM concentration was similar in LC and SN, but the iron content in NM of LC was much lower than SN. In both regions, heavy- and light-chain ferritins were present only in glia and were not detectable in neurons. These data suggest that in LC neurons, the iron mobilization and toxicity is lower than that in SN and is efficiently buffered by NM. The bigger damage occurring in SN could be related to the higher content of iron. Ferritins accomplish the same function of buffering iron in glial cells. Ceruloplasmin levels were similar in LC and SN, but copper was higher in LC. However, the copper content in NM of LC was higher than that of SN, indicating a higher copper mobilization in LC neurons. Manganese-SOD and copper/zinc-SOD had similar age trend in LC and SN. These results may explain at least one of the reasons underlying lower vulnerability of LC compared to SN in Parkinsonian syndromes.     

The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney

It is generally accepted that iron homeostasis is mainly controlled in the gastrointestinal tract by absorption of dietary iron. However, recent studies have shown that the kidneys are also involved in iron metabolism. Since the iron-regulatory and antimicrobial peptide hormone hepcidin was originally isolated from human urine we have investigated the expression as well as the zonal and cellular localization of hepcidin in the mammalian kidney and developed an ELISA assay to analyze hepcidin concentrations in serum and urine. The expression of hepcidin was shown by RT-PCR and immunoblot experiments; its cellular localization was studied by immunocytochemistry in human, mouse and rat kidney, which revealed similar patterns of immunoreactivity. Hepcidin expression was absent from the proximal tubule and descending and ascending thin limbs. There was strong expression in the thick ascending limb of the cortex and in connecting tubules. Moderate expression was noted in the thick ascending limb and collecting ducts of the medulla and in collecting ducts of the papilla. Importantly, the cells of the macula densa were unstained. At the cellular level, hepcidin was localized to the apical cell pole of the renal epithelial cells. Based on its presence in urine, hepcidin may be released apically into the urine. Enhanced levels of hepcidin were determined in patients with chronic renal insuffciency (156.8 ng/ml, controls 104.2 ng/ml) indicating that the kidneys may metabolize and/or eliminate the circulating peptide. From the expression of hepcidin in the mammalian kidney, we have concluded that the iron-regulatory hormone is an intrinsic renal peptide which is not only eliminated by the kidney but is also synthesized in the kidney tubular system. Localization of hepcidin in the kidney implicates an iron-regulatory role of this peptide hormone in the renal tubular system, possibly in connection with the iron transporter divalent metal transporter-1.     

Exam anxiety induces significant blood pressure and heart rate increase in college students

To investigate the relationship between the anxiety and blood pressure (BP) and heart rate (HR) increase in peri-exam period. Sixty-four college students(20.0 ± 0.1 year old) were included in this study. The BP and HR were measured in the morning and in the evening for 3 days during the prereview (ba), review, and exam periods. The BP and HR increase amplitudes (HRIA) of review and exam periods were from the difference of corresponding values and basic values, and the BPIA/baBP and HRIA/baHR were calculated. All of the students completed the Self-Rating Anxiety score (SAS) questionnaire the first day of the exam period. Scores over 50 points were used as the standard for anxiety. From the prereview to exam periods, the BP and HR increased gradually. The exam SBPIA (4.3 ± 1.3 vs. 0.3 ± 0.5 mmHg, P < 0.05) and DBPIA (4.4 ± 1.5 vs. 1.0 ± 0.5 mmHg, P < 0.05) were significantly higher in the anxiety group than in the no-anxiety group. The SBPIA/DBPIA and HRIA showed a similar profile also(9.7 ± 2.1 vs. 1.9 ± 0.9 bpm, P < 0.05). Strong positive correlations were found between the SAS score and BPIA and HRIA both in the review and exam period. The smoking group and family hypertension group had higher anxiety score; meanwhile, their exam BPIAs and HRIAs were significantly higher than their corresponding group. The BP and HR increase in the review and exam period, anxiety is an important factor of BP and HR increase.     

The streptozotocin-induced rat model of diabetes mellitus evidences significant reduction of myosin-Va expression in the brain

Diabetes mellitus is a disease characterized by increased glucose levels in the blood. Hyperglycemia causes damage to the brain tissue, and induces significant changes in synaptic transmission. In this investigation, we have found a significant alteration in the expression of the molecular motor involved in the synaptic vesicles transport, myosin-Va, and its distribution in rat brains of streptozotocin-induced diabetes model. Brains were removed after 20 days, homogenized and analysed by Western blotting, qRT-PCR and immunohistochemistry. Myosin-Va presented significantly lower levels of both mRNA and protein in diabetic than those observed in non-diabetic animals. Moreover, neuronal and glial cells of the occipital and frontal cortex exhibited decreased myosin-Va immunostaining in diabetic rat brains. In conclusion, diabetic rat brains displayed altered expression and distribution of myosin-Va, and these finding may contribute to the basic understanding about this myosin role in brain function related to diabetes.