Hereditary hemochromatosis, characterized by iron overload in multiple organs, is one of the most common genetic disorders among Caucasians. Hepcidin, which is synthesized in the liver, plays important roles in iron overload syndromes. Here, we show that a Cre-loxP-mediated liver-specific disruption of SMAD4 results in markedly decreased hepcidin expression and accumulation of iron in many organs, which is most pronounced in liver, kidney, and pancreas. Transcript levels of genes involved in intestinal iron absorption, including Dcytb, DMT1, and ferroportin, are significantly elevated in the absence of hepcidin. We demonstrate that ectopic overexpression of SMAD4 activates the hepcidin promoter and is associated with epigenetic modification of histone H3 to a transcriptionally active form. Moreover, transcriptional activation of hepcidin is abrogated in SMAD4-deficient hepatocytes in response to iron overload, TGF-beta, BMP, or IL-6. Our study uncovers a novel role of TGF-beta/SMAD4 in regulating hepcidin expression and thus intestinal iron transport and iron homeostasis.

In mammalian cells, regulation of the expression of proteins involved in iron metabolism is achieved through interactions of iron-sensing proteins known as iron regulatory proteins (IRPs), with transcripts that contain RNA stem-loop structures referred to as iron responsive elements (IREs). Two distinct but highly homologous proteins, IRP1 and IRP2, bind IREs with high affinity when cells are depleted of iron, inhibiting translation of some transcripts, such as ferritin, or turnover of others, such as the transferrin receptor (TFRC). IRPs sense cytosolic iron levels and modify expression of proteins involved in iron uptake, export and sequestration according to the needs of individual cells. Here we generate mice with a targeted disruption of the gene encoding Irp2 (Ireb2). These mutant mice misregulate iron metabolism in the intestinal mucosa and the central nervous system. In adulthood, Ireb2(-/-) mice develop a movement disorder characterized by ataxia, bradykinesia and tremor. Significant accumulations of iron in white matter tracts and nuclei throughout the brain precede the onset of neurodegeneration and movement disorder symptoms by many months. Ferric iron accumulates in the cytosol of neurons and oligodendrocytes in distinctive regions of the brain. Abnormal accumulations of ferritin colocalize with iron accumulations in populations of neurons that degenerate, and iron-laden oligodendrocytes accumulate ubiquitin-positive inclusions. Thus, misregulation of iron metabolism leads to neurodegenerative disease in Ireb2(-/-) mice and may contribute to the pathogenesis of comparable human neurodegenerative diseases.

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Iron-regulatory proteins (IRPs) 1 and 2 posttranscriptionally regulate expression of transferrin receptor (TfR), ferritin, and other iron metabolism proteins. Mice with targeted deletion of IRP2 overexpress ferritin and express abnormally low TfR levels in multiple tissues. Despite this misregulation, there are no apparent pathologic consequences in tissues such as the liver and kidney. However, in the central nervous system, evidence of abnormal iron metabolism in IRP2 ؊/؊ mice precedes the development of adult-onset progressive neurodegeneration, characterized by widespread axonal degeneration and neuronal loss. Here, we report that ablation of IRP2 results in ironlimited erythropoiesis. TfR expression in erythroid precursors of IRP2 ؊/؊ mice is reduced, and bone marrow iron stores are absent, even though transferrin saturation levels are normal. Marked overexpression of 5-aminolevulinic acid synthase 2 (Alas2) results from loss of IRPdependent translational repression, and markedly increased levels of free protoporphyrin IX and zinc protoporphyrin are generated in IRP2 ؊/؊ erythroid cells. IRP2 IntroductionIron functions as an indispensable cofactor for numerous enzymes and proteins in mammals, and regulation of iron uptake and distribution within animals is accordingly highly regulated. 1,2 Intestinal iron absorption and tissue iron storage are optimized to deliver the iron needed for numerous metabolic processes, including heme synthesis. The recently identified peptide hormone, hepcidin, is responsible for appropriately coordinating intestinal iron uptake and macrophage iron release to meet the needs of the organism and to maintain normal serum transferrin saturation levels. 3 In most tissues, the circulating pool of diferric transferrin serves as the major source of iron for individual cells. When diferric transferrin (Tf) binds to transferrin receptors (TfRs), the Tf-TfR complex internalizes in endosomes, where acidification facilitates release of free iron, 4 and the membrane iron transporter divalent metal transporter 1 (DMT1) (SLC11A2) transports iron into the cytosol. 5,6 In the cytosol, iron is incorporated into iron proteins or transported to cellular organelles, and excess cytosolic iron is sequestered and stored by ferritin. 6,7 Cells regulate expression of ferritin and TfR to optimize cytosolic iron levels. When cells are iron depleted, they increase TfR expression and uptake of transferrinbound iron, while they simultaneously decrease expression of ferritin and iron sequestration. Proteins known as iron regulatory proteins (IRPs) coordinately regulate expression of TfR, ferritin, and numerous other iron metabolism proteins. IRP1 and IRP2 are homologous genes that monitor cytosolic iron levels. When cells are iron depleted, IRPs bind to RNA motifs known as iron-responsive elements (IREs) within transcripts that encode iron metabolism proteins (reviewed in Rouault and Klausner 1 ; and Hentze et al 2 ). IREs are found in numerous transcripts, including ferritin H-and L-chains, TfR1, 7 erythrocyti...

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To characterize the precursor of mammalian thyrotropin-releasing hormone (TRH), a rat hypothalamic lambda gt11 library was screened with an antiserum directed against a synthetic peptide representing a portion of the rat TRH prohormone. The nucleotide sequence of the immunopositive complementary DNA encoded a protein with a molecular weight of 29,247. This protein contained five copies of the sequence Gln-His-Pro-Gly flanked by paired basic amino acids and could therefore generate five TRH molecules. In addition, potential cleavage sites in the TRH precursor could produce other non-TRH peptides, which may be secreted. In situ hybridization to rat brain sections demonstrated that the pre-proTRH complementary DNA detected neurons concentrated in the parvocellular division of the paraventricular nucleus, the same location as cells detected by immunohistochemistry. These findings indicate that mammalian TRH arises by posttranslational processing of a larger precursor protein. The ability of the TRH prohormone to generate multiple copies of the bioactive peptide may be an important mechanism in the amplification of hormone production.

Cells derived from a rat pheochromocytoma (PC12 cells) can generate an action potential only upon treatment with nerve growth factor. Using electrophysiological methods, we found that the appearance of action potentials in nerve growth factor-treated PC12 cells can be explained by an increase in the density of Na' channels. The functional properties of Na' channels in PC12 cells are similar to those described for peripheral nerves but appear to be different from Na' channels synthesized in Xenopus oocytes injected with brain type II Na'-channel mRNA. To determine if PC12 cells express the brain type II Na+ -channel gene, we performed RNase-protection analyses using probes that can distinguish between the brain type I and type II Na'-channel mRNAs. The results from these studies indicate that undifferentiated PC12 cells express the type II but not the type I Na+ -channel gene. Treatment with nerve growth factor increases expression of the type II Na+-channel gene but has no effect on type I gene expression. Our findings suggest that N+ -channel excitability in PC12 cells is due to the specific induction of the brain type II gene by nerve growth factor.Neurons can be distinguished from inexcitable types of cells by their capacity to generate an action potential. The appearance of excitability occurs early in neuronal differentiation (1-3) and results from the insertion of voltage-dependent ion channels into the membrane (4). PC12 cells, derived from a tumor of adrenal tissue (5), have proven useful for studying the factors that regulate excitability. Treatment with nerve growth factor (NGF), for example, enables PC12 cells to generate action potentials. Electrophysiological studies have indicated that the action potential in NGF-treated PC12 cells is due to activation of voltage-dependent Na' channels (6, 7). It has also been shown that the number of saxitoxin binding sites on PC12 cells increases after treatment with NGF (8). Therefore, even though voltage-dependent K+ and Ca2" channels also appear during differentiation of PC12 cells (9, 10), it is the expression of Na+ channels that best accounts for the development of the action potential. The effect of NGF on the density of functional Na+ channels has not been examined in detail. Previous electrophysiological studies in PC12 cells have been limited either to measurement of action potentials (6, 7) or to single-channel Na' currents (11) and have not provided quantitative estimates of the density of functional Na+ channels. Therefore, in this study we utilized the whole-cell voltage-clamp technique in order to quantitate the effect of NGF on Na+-channel density. These experiments were performed in concert with molecular studies designed to measure changes in Na+-channel gene expression in response to treatment with NGF.Molecular studies have indicated that excitable cells express different Na'-channel mRNAs (12)(13)(14). cDNA clones coding for two structurally distinct voltage-dependent Na' channels in rat brain (termed type I and type II) have been ide...