Identification of new mutations of hepcidin and hemojuvelin in patients with HFE C282Y allele

HFE-hemochromatosis is the most common form of hereditary hemochromatosis. The disorder is associated with the homozygous C282Y mutation and has variable phenotype, being modulated by environmental and genetic factors. Candidate modifier genes are hemojuvelin and hepcidin, which are responsible for juvenile hemochromatosis. We used DHPLC to scan mutations in these genes in a cohort of unrelated patients with C282Y mutation. They consisted of 136 C282Y homozygous, 43 heterozygous, and 42 C282Y/H63D compound heterozygous, plus 62 controls subjects. Mutations and polymorphisms were found in 16 patients and 4 controls. Abnormally high indices of iron status were found in subjects C282Y/H63D heterozygous for the N196K hemojuvelin mutation and the -72C > T hepcidin substitution. The already described G71D mutation of hepcidin did not induce evident modification of the C282Y/H63D phenotype. The data show that heterozygous mutations of the hemojuvelin gene contribute like those of hepcidin to the phenotypic heterogeneity of hemochromatosis. However, they are rare and explain only a minor portion of the variable penetrance of the disorder.     

Iron transferrin regulates hepcidin synthesis in primary hepatocyte culture through hemojuvelin and BMP2/4

The peptide hormone hepcidin is the principal regulator of systemic iron homeostasis. We examined the pathway by which iron stimulates the production of hepcidin. In humans who ingested 65 mg of iron, the increase in transferrin saturation preceded by hours the increase in urinary hepcidin excretion. Increases in urinary hepcidin concentrations were proportional to the increment in transferrin saturation. Paradoxically, in previous studies in primary hepatocytes and cell lines, hepcidin response to iron or iron transferrin was not observed. We now report that freshly isolated murine primary hepatocytes responded to holotransferrin but not apotransferrin by increasing hepcidin mRNA. Hepcidin increase was not due to contamination of the transferrin preparations by endotoxin, a potent pathologic stimulus of hepcidin synthesis. Using this culture system, we showed that holotransferrin concentrations regulate hepcidin mRNA concentrations through a hemojuvelin/BMP2/4-dependent pathway. Although BMP9 is known to be expressed in the liver and potently increased the basal concentrations of hepcidin mRNA, it did not interact with hemojuvelin, and interference with its signaling pathway did not affect iron regulation. Fresh primary hepatocytes constitute a sufficient system for the regulation of hepcidin by physiologic iron stimuli and will greatly facilitate studies of major disorders of iron homeostasis.     

Furin-mediated release of soluble hemojuvelin: a new link between hypoxia and iron homeostasis

The liver peptide hepcidin regulates iron absorption and recycling. Hemojuvelin (HJV) has a key role in hepcidin regulation, and its inactivation causes severe iron overload both in humans and in mice. Membrane HJV (m-HJV) acts as a coreceptor for bone morphogenetic proteins (BMPs), whereas soluble HJV (s-HJV) may down-regulate hepcidin in a competitive way interfering with BMP signaling. s-HJV is decreased by iron in vitro and increased by iron deficiency in vivo. However, the mechanisms regulating the 2 HJV isoforms remain unclear. Here we show that s-HJV originates from a furin cleavage at position 332-335. s-HJV is reduced in the cleavage mutant R335Q as well as in cells treated with a furin inhibitor, and increased in cells overexpressing exogenous furin, but not in cells overexpressing an inactive furin variant. Furin is up-regulated by iron deficiency and hypoxia in association with the stabilization of HIF-1alpha. Increased s-HJV in response to HIF-1alpha occurs during differentiation of murine muscle cells expressing endogenous Hjv. Our data are relevant to the mechanisms that relate iron metabolism to the hypoxic response. The release of s-HJV might be a tissue-specific mechanism, signaling the local iron requests of hypoxic skeletal muscles independently of the oxygen status of the liver.     

核因子-κB参与小鼠Hepcidin基因的转录调控

目的探讨核因子-κB（NFKB）参与Hepcidin基因急性时相表达转录调控的可能性及其分子机制。方法腹腔注射脂多糖（LPS）建立小鼠急性时相反应模型，检测Hepcidin表达与LPs的剂量效应和时间效应关系；电泳迁移率变动实验（EMSA）初步探讨NF-κB参与Hepcidin基因转录调控的可能性；构建NFicBp65亚基特异的小干扰RNA（siRNA）表达载体pAVU6＋27-NF-κB，DOTAP脂质体法瞬时转染小鼠原代肝细胞，观察NF-κB p65基因沉默后Hepcidin表达改变以及LPS诱导Hepcidin表达情况。结果Hepcidin表达与LPS注射剂量和时间呈正相关，50μg LPS注射后l0h，Hepcidin表达达到峰值。EMSA显示53～64bp位点出现明显的滞后带。NF-κBP65特异的SiRNA表达载体瞬时转染原代肝细胞后，基因沉默效率为50％～67％，转染细胞Hepcidin的表达水平明显下降，且并不能被LPS显著诱导。结论NF-κB参与Hepcidin转录调控，p65亚基是其上调Hepcidin基因表达的关键组分，53～64bp是p65亚基的作用位点。     

Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading in mice

In response to iron loading, hepcidin synthesis is homeostatically increased to limit further absorption of dietary iron and its release from stores. Mutations in HFE, transferrin receptor 2 (Tfr2), hemojuvelin (HJV), or bone morphogenetic protein 6 (BMP6) prevent appropriate hepcidin response to iron, allowing increased absorption of dietary iron, and eventually iron overload. To understand the role each of these proteins plays in hepcidin regulation by iron, we analyzed hepcidin messenger RNA (mRNA) responsiveness to short and long-term iron challenge in iron-depleted Hfe, Tfr2, Hjv, and Bmp6 mutant mice. After 1-day (acute) iron challenge, Hfe(-/-) mice showed a smaller hepcidin increase than their wild-type strain-matched controls, Bmp6(-/-) mice showed nearly no increase, and Tfr2 and Hjv mutant mice showed no increase in hepcidin expression, indicating that all four proteins participate in hepcidin regulation by acute iron changes. After a 21-day (chronic) iron challenge, Hfe and Tfr2 mutant mice increased hepcidin expression to nearly wild-type levels, but a blunted increase of hepcidin was seen in Bmp6(-/-) and Hjv(-/-) mice. BMP6, whose expression is also regulated by iron, may mediate hepcidin regulation by iron stores. None of the mutant strains (except Bmp6(-/-) mice) had impaired BMP6 mRNA response to chronic iron loading. CONCLUSION: TfR2, HJV, BMP6, and, to a lesser extent, HFE are required for the hepcidin response to acute iron loading, but are partially redundant for hepcidin regulation during chronic iron loading and are not involved in the regulation of BMP6 expression. Our findings support a model in which acute increases in holotransferrin concentrations transmitted through HFE, TfR2, and HJV augment BMP receptor sensitivity to BMPs. A distinct regulatory mechanism that senses hepatic iron may modulate hepcidin response to chronic iron loading.     

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.     

Positive regulation of human thyrotropin receptor mRNA by thyrotropin

We have assessed the influence of natural and recombinant thyrotropin (TSH) on mRNA specific for the human TSH receptor (TSHR) in normal and abnormal adult human thyroid monolayer cells. Using physiological concentrations of TSH (less than 100 mU/L), a marked increase in the level of TSHR mRNA was observed within 12 hours and reached a greater than 1000% increase after 24 hours exposure. At high TSH concentrations (greater than 1000 mU/L), this increase in TSHR-specific mRNA was markedly reduced. However, at no time were basal TSHR mRNA levels suppressed even with 100 U/L of TSH for 48 hours. These observations demonstrate ligand-induced up-regulation of the human TSHR mRNA by physiologically relevant concentrations of TSH.     

Gender and hormonal status affect the regulation of hepatic cholesterol 7alpha-hydroxylase activity and mRNA abundance by dietary soluble fiber in the guinea pig

Dietary soluble fiber (SF) consistently lowers plasma LDL cholesterol (LDL-C) concentrations, however, secondary mechanisms governing this reduction are not completely defined. Moreover, these mechanisms appear to differ with gender. Male, female and ovariectomized (to mimic menopause) guinea pigs were used to assess effects of gender, hormonal status and SF on activity and expression of hepatic cholesterol 7alpha-hydroxylase (CYP7). Diets were identical except for fiber source (control 10% cellulose, SF 5% psyllium/5% pectin). SF intake resulted in 44% lower plasma total cholesterol, 51% lower plasma LDL-C and 22% lower plasma triacylglycerol (TAG) concentrations. However, ovariectomized guinea pigs fed either the control or SF diets, had the highest plasma LDL-C and TAG levels (P<0.01). SF altered hepatic cholesterol metabolism by effectively reducing hepatic free cholesterol, TAG and microsomal free cholesterol, while activity of CYP7, the rate-limiting enzyme of cholesterol catabolism, was up-regulated. Hepatic CYP7 mRNA abundance paralleled the increase in enzyme activity. Ovariectomized guinea pigs had lowest activity and expression of hepatic CYP7 even after intervention with SF. These results suggest that induction of hepatic CYP7 activity may account, in large part, for the hypocholesterolemic effect of SF. Gender and hormonal status influence metabolic responses to dietary SF with estrogen deprivation leading to the most detrimental lipid profile.     

Hypoxic regulation of VEGF mRNA stability by RNA-binding proteins

Vascular endothelial growth factor (VEGF), is a potent angiogenic factor whose expression is dramatically induced by hypoxia. We have previously demonstrated that the induction of VEGF by hypoxia is in large part the result of an increased stability of VEGF mRNA. The stabilization of VEGF mRNA by hypoxia is mediated by the binding of sequence-specific RNA-binding proteins. This review focuses on one such protein, HuR, an RNA-binding protein which we have recently shown is critical for the hypoxic stabilization of VEGF mRNA.