Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-D-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heatdenatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca 2؉ , but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.Mammals respond to oxygen deficiency in many different ways (1). One strategy for survival of the individual cells under hypoxic conditions is the induction of glycolytic enzymes, facilitating ATP production by glycolysis rather than mitochondrial oxidative phosphorylation. In response to the systemic oxygen deficiency due to anemia or decreasedenvironmental oxygen concentration, erythropoietin (EPO) production is stimulated. EPO is a glycoprotein that stimulates differentiation and proliferation of erythroid precursor cells, and hypoxic induction of EPO production increases red blood cells, leading to better oxygen supply to tissues (2, 3). The action of EPO is mediated by binding to the specific receptor that belongs to a new family of cytokine receptors that have no tyrosine kinase domain (4). EPO regulating erythropoiesis is mainly produced by the kidney in adults and by the liver at fetal stages (2, 3).Stimulation of red blood cell formation was thought to be the sole physiological function of EPO, but a different function in the central nervous system has been proposed (5-7). Neuronal cell lines such as PC12 and SN6 express EPO receptor (EPOR), and binding of EPO to PC12 cells increases the intracellular concentration of monoamines (8). Immunochemical staining with anti-EPOR antibody showed that EPOR is expressed in murine hippocampal and cerebral cortical areas, and also in primary cultured hippocampal and cortical neurons (6, 9). With the use of radioiodinated EPO, specific EPO binding sites were found in some defined areas of the murine brain including the hippocampus and cerebral cortex (10). Because the blood-brain barrier prevents neurons fr...

No abstract

In recent years, a number of mammalian zinc transporters have been identified, and candidate genes are rapidly growing. These transporters are classified into two families: ZIP (ZRT, IRT-like protein) and CDF (cation diffusion facilitator). ZIP members facilitate zinc influx into the cytosol, while CDF members facilitate its efflux from the cytosol. Molecular characterization of the transporters has brought about major advances in our understanding of their physiological functions. Zinc metabolism is regulated primarily through zinc-dependent control of transcription, translation, and intracellular trafficking of transporters. Analyses of mice whose zinc transporter genes have been genetically disrupted and of the naturally occurring mutant mice with symptoms related to abnormal zinc metabolism have provided compelling evidence that some zinc transporters play critical roles in zinc homeostasis. In this review, we review the literature of mammalian zinc transporters with emphasis on very recent findings and elicit integrative knowledge of zinc homeostasis.

Intracellular homeostasis for zinc is achieved through the coordinate regulation of specific transporters engaged in zinc influx, efflux, and intracellular compartmentalization. We have identified a novel mammalian zinc transporter, zinc transporter 5 (ZnT-5), by virtue of its similarity to ZRC1, a zinc transporter of Saccharomyces cerevisiae, a member of the cation diffusion facilitator family. Human ZnT-5 (hZnT-5) cDNA encodes a 765-amino acid protein with 15 predicted membranespanning domains. hZnT-5 was ubiquitously expressed in all tested human tissues and abundantly expressed in the pancreas. In the human pancreas, hZnT-5 was expressed abundantly in insulin-containing ␤ cells that contain zinc at the highest level in the body. The hZnT-5 immunoreactivity was found to be associated with secretory granules by electron microscopy. The hZnT-5-derived zinc transport activity was detected using the Golgi-enriched vesicles prepared from hZnT-5-induced HeLa/hZnT-5 cells in which exogenous hZnT-5 expression is inducible by the Tet-on gene regulation system. This activity was dependent on time, temperature, and concentration and was saturable. Moreover, zinc at a high concentration (10 mM) inhibited the growth of yeast expressing hZnT-5. These results suggest that ZnT-5 plays an important role for transporting zinc into secretory granules in pancreatic ␤ cells.

No abstract

Although erythropoietin (Epo) has been shown to possess in vitro angiogenic activity, its physiological significance has not been demonstrated. Normally angiogenesis does not occur actively in adults but an exception is the female reproductive organ. In the uterine endometrium, angiogenesis takes place actively for supporting the endometrial growth that occurs during transition from the diestrus to estrous stage. This transition is under control of 17␤-estradiol (E 2 ), an ovarian hormone, and can be mimicked by injection of E 2 to ovariectomized (OVX) mouse. Thus, the uterus is a pertinent site to examine the Epo function in angiogenesis. We found that Epo protein and its mRNA were produced in an E 2 -dependent manner, when the uterus from OVX mouse was cultured in vitro. The de novo protein synthesis was not needed for E 2 induction of Epo mRNA. Administration of E 2 to OVX mouse induced a rapid and transient increase in Epo mRNA in the uterus. Injection of Epo into the OVX mouse uterine cavity promoted blood vessel formation in the endometrium. Furthermore, injection of the soluble Epo receptor capable of binding with Epo into the uterine cavity of non-OVX mouse in diestrus stage inhibited the endometrial transition to proestrus stage, whereas heat-inactivated soluble Epo receptor allowed the transition to occur. These results, combined with our finding that the endothelial cells in uterine endometrium express Epo receptor, strongly suggest that Epo is an important factor for the E 2 -dependent cyclical angiogenesis in uterus.

Lysine specific demethylase 1 (LSD1) plays a key role in the regulation of gene expression by removing the methyl groups from methylated Lys4 of histone H3 (H3K4). Here we report the identification of the first small-molecule LSD1-selective inhibitors. These inhibitors show in vivo H3K4-methylating activity and antiproliferative activity and should be useful as lead structures for anticancer drugs and as tools for studying the biological roles of LSD1.

The Aft1 transcription factor regulates the iron regulon in response to iron availability in Saccharomyces cerevisiae. Aft1 activates a battery of genes required for iron uptake under iron-starved conditions, whereas Aft1 function is inactivated under iron-replete conditions. Previously, we have shown that iron-regulated DNA binding by Aft1 is responsible for the controlled expression of target genes. Here we show that this iron-regulated DNA binding by Aft1 is not due to the change in the total expression level of Aft1 or alteration of DNA binding activity. Rather, nuclear localization of Aft1 responds to iron status, leading to iron-regulated expression of the target genes. We identified the nuclear export signal (NES)-like sequence in the AFT1 open reading frame. Mutation of the NES-like sequence causes nuclear retention of Aft1 and the constitutive activation of Aft1 function independent of the iron status of the cells. These results suggest that the nuclear export of Aft1 is critical for ensuring iron-responsive transcriptional activation of the Aft1 regulon and that the nuclear import/ export systems are involved in iron sensing by Aft1 in S. cerevisiae.Iron is an essential nutrient for virtually all organisms, working as a cofactor for critical proteins that mediate diverse processes such as cellular respiration and synthesis of metabolic intermediates (1). On the other hand, excess iron is extremely toxic, being capable of generating free radicals that damage macromolecules such as proteins, lipids, and DNA (2). The uptake and metabolism of iron must therefore be strictly regulated. In all organisms studied, the rates of iron uptake are tightly coupled to the levels of available iron and the needs of cells (3). In metazoans, the mechanisms that underlie regulation of iron metabolism including uptake, sequestration, and utilization have been well characterized. Two RNA-binding proteins called "iron regulatory protein," IRP1 1 and IRP2, are responsible for this regulation by controlling mRNA translation or mRNA stability by iron regulated mRNA-protein interactions (4). Although these proteins are closely related, IRP1 is a relatively stable protein (5, 6), and its RNA binding activity is inactivated by iron-sulfur cluster formation in iron-replete cells (7). By contrast, IRP2 is degraded by the ubiquitin-proteasome system in iron-replete cells (8).In Saccharomyces cerevisiae, iron deprivation induces activities of a high affinity iron uptake system (9) and siderophoremediated iron uptake system (10, 11). A transcription factor, Aft1, plays a critical role in this process (12). Aft1 protein binds to a regulatory region in the DNA of the genes required for iron uptake and induces the transcription of these genes in ironstarved cells (13). Conversely, Aft1 is not bound to its regulatory target element in iron-replete cells, and the expression of its target genes is not induced. An increasing number of Aft1 target genes have been implicated in iron metabolism, including a putative transporter complex located i...