losartan-potassium and cobaltous-chloride

The kidney is sensitive to changes in oxygen delivery. This sensitivity has the merit of facilitating the kidneys in their adjustment of erythropoietin (EPO) production to changes in oxygen supply. The main determinant of EPO synthesis is the transcriptional activity of its gene in kidneys, which is related to local oxygen tensions. Regulation of EPO production is mediated by hypoxia-inducible factor (HIF). When local oxygen tension decreases, accumulated HIF binds to the key sequence of the EPO gene, the hypoxia-responsive element (HRE), and activates transcription of EPO. HIF consists of a constitutive beta-subunit and one of alternative oxygen-regulated HIF alpha-subunits (HIF-1alpha, HIF-2alpha, and HIF-3alpha), and HIF-2alpha is responsible for erythropoietin production. However, the high sensitivity to changes in oxygen tension also makes the kidney prone to hypoxic injury. Severe energy depletion and subsequent activation of a number of critical alterations in metabolism occurs under hypoxic conditions. Hypoxia is also a profibrogenic stimulus. In addition to ischemic acute renal failure, hypoxia can also play a crucial role in the development of nephrotoxic acute kidney injury, radiocontrast nephropathy, and acute glomerulonephritis. Furthermore, accumulating evidence suggests that chronic hypoxia is a final common pathway to end-stage kidney failure in chronic kidney disease. Given that renal hypoxia has pivotal roles on the development and progression of both acute and chronic kidney disease, hypoxia can be a valid therapeutic target for chronic kidney disease. Activation of HIF leads to expression of a variety of adaptive genes in a coordinated manner. Studies utilizing HIF-stimulating agents proved efficacy in various kidney disease models, suggesting that HIF activation is an ideal target of future therapeutic approaches.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cobalt; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Iron Chelating Agents; Kidney; Kidney Diseases; Oxygen; Signal Transduction

Topics: Animals; Cell Line; Cobalt; Erythropoietin; Exons; Gene Expression Regulation; Humans; Mice; Oxygen; Regulatory Sequences, Nucleic Acid; RNA Processing, Post-Transcriptional; RNA, Messenger; Transcription, Genetic; Transfection

Topics: Adenine; Animals; Autophagosomes; Autophagy; Cell Line; Chemical and Drug Induced Liver Injury; Chromones; Cobalt; Disease Models, Animal; Erythropoietin; Humans; Liver; Liver Function Tests; Mice; Mice, Inbred C57BL; Morpholines; Peptide Fragments; Proto-Oncogene Proteins c-akt; Random Allocation; Reperfusion Injury; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

It is well known that cobalt chloride (CoCl2) can enhance the stability of hypoxia-inducible factor (HIF)-1α. The aim of this study is to detect the effect of CoCl2 on the hypoxia tolerance of mice which were repeatedly exposed to autoprogressive hypoxia. Balb/c mice were randomly divided into groups of chemical pretreatment and normal saline (NS), respectively injected with CoCl2 and NS 3 h before exposure to hypoxia for 0 run (H0), 1 run (H1), and 4 runs (H4). Western Blot, electrophoretic mobility shift assay (EMSA), extracellular recordings population spikes in area cornus ammonis I (CA 1) of mouse hippocampal slices and real-time were used in this study. Our results demonstrated that the tolerance of mice to hypoxia, the changes of HIF-1α protein level and HIF-1 DNA binding activity in mice hippocampus, the mRNA level of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the disappearance time of population spikes of hippocampal slices were substantially different between the control group and the CoCl2 group. Over-induction of HIF-1α by pretreatment with CoCl2 before hypoxia did not increase the hypoxia tolerance.

Topics: Animals; Cobalt; DNA; Erythropoietin; Hippocampus; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Models, Animal; Protein Binding; RNA, Messenger; Vascular Endothelial Growth Factor A

Understanding the endogenous survival pathways induced by ischemic tolerance may yield targets for neuroprotection from stroke. One well-studied pathway reported to be evoked by preconditioning stimuli is the transcription factor HIF (hypoxia-inducible factor). However, whether HIF induction by ischemic insults is neuroprotective or toxic is still unclear. We examined the ability of three prolyl-hydroxylase inhibitors, which induce HIF, to protect hippocampal cultures from oxygen-glucose deprivation. Hippocampal cultures were exposed to ischemic preconditioning or various concentrations of cobalt chloride, deferoxamine (DFO) or dimethyloxylalyglycine (DMOG), prior to lethal oxygen-glucose deprivation (OGD). Cell survival of neurons and astrocytes was determined with dual-label immunocytochemistry. The induction of HIF targets was assessed in mixed as well as astrocyte-enriched cultures. Ischemic preconditioning, as well as low concentrations of cobalt and DFO, enhanced the survival of neurons following OGD. However, DMOG exacerbates OGD-induced neuronal death. At low concentrations, all three prolyl-hydroxylase (PHD) inhibitors increased the survival of astrocytes. Neuroprotective concentrations of cobalt induced the transcription of the cytokine erythropoietin (EPO) in astrocyte cultures. In addition, pretreatment with recombinant human erythropoietin (rH-EPO) also protected neurons from OGD. Our data suggest that HIF-induced EPO, released from astrocytes, protects neurons from OGD. However, the three PHD inhibitors each exhibited different neuroprotective profiles at low concentrations, suggesting that not all PHD inhibitors are created equal. The protective effects at low doses is reminiscent of HIF involvement in ischemic tolerance, in which sub-lethal insults induce HIF pathways resulting in neuroprotection, whereas the high-dose toxicity suggests that over-activation of HIF is not always protective. Therefore, the choice of inhibitor and dose may determine the clinical utility of these compounds. Deferoxamine exhibited little toxicity even at higher doses, and therefore appears a promising candidate for clinical use.

Topics: Amino Acids, Dicarboxylic; Animals; Astrocytes; Brain Ischemia; Cell Survival; Cells, Cultured; Cobalt; Deferoxamine; Erythropoietin; Hippocampus; Hypoxia-Inducible Factor 1; Immunohistochemistry; Ischemic Preconditioning; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Polymerase Chain Reaction; Prolyl-Hydroxylase Inhibitors

Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen. Although HIF-1 is usually considered as the principal mediator of hypoxic adaptation, several tissues and different cell types express both HIF-1 and HIF-2 isoforms under hypoxia or when treated with hypoxia mimetic chemicals such as cobalt. However, the similarities or differences between HIF-1 and HIF-2, in terms of their tissue- and inducer-specific activation and function, are not adequately characterized. To address this issue, we investigated the effects of true hypoxia and hypoxia mimetics on HIF-1 and HIF-2 induction and specific gene transcriptional activity in two hepatic cancer cell lines, Huh7 and HepG2. Both hypoxia and cobalt caused rapid induction of both HIF-1α and HIF-2α proteins. Hypoxia induced erythropoietin (EPO) expression and secretion in a HIF-2-dependent way. Surprisingly, however, EPO expression was not induced when cells were treated with cobalt. In agreement, both HIF-1- and HIF-2-dependent promoters (of PGK and SOD2 genes, respectively) were activated by hypoxia while cobalt only activated the HIF-1-dependent PGK promoter. Unlike cobalt, other hypoxia mimetics such as DFO and DMOG activated both types of promoters. Furthermore, cobalt impaired the hypoxic stimulation of HIF-2, but not HIF-1, activity and cobalt-induced HIF-2α interacted poorly with USF-2, a HIF-2-specific co-activator. These data show that, despite similar induction of HIF-1α and HIF-2α protein expression, HIF-1 and HIF-2 specific gene activating functions respond differently to different stimuli and suggest the operation of oxygen-independent and gene- or tissue-specific regulatory mechanisms involving additional transcription factors or co-activators.

Topics: Amino Acids, Dicarboxylic; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Cell Line, Tumor; Cell Nucleus; Cobalt; Deferoxamine; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Phosphoglycerate Kinase; Promoter Regions, Genetic; Propanolamines; Protein Binding; Pyrrolidines; RNA, Messenger; Superoxide Dismutase; Transcription, Genetic; Upstream Stimulatory Factors

Hypoxia upregulates the hypoxia-inducible factor (HIF) pathway and the endoplasmic reticulum (ER) stress signal, unfolded protein response (UPR). The cross talk of both signals affects the pathogenic alteration by hypoxia. Here we showed that ER stress induced by tunicamycin or thapsigargin suppressed inducible (CoCl(2) or hypoxia) transcription of erythropoietin (EPO), a representative HIF target gene, in HepG2. This suppression was inversely correlated with UPR activation, as estimated by expression of the UPR regulator glucose-regulated protein 78, and restored by an ER stress inhibitor, salubrinal, in association with normalization of the UPR state. Importantly, the decreased EPO expression was also observed in HepG2 overexpressing UPR activating transcription factor (ATF)4. Overexpression of mutated ATF4 that lacks the transcriptional activity did not alter EPO transcriptional regulation. Transcriptional activity of the EPO 3'-enhancer, which is mainly regulated by HIF, was abolished by both ER stressors and ATF4 overexpression, while nuclear HIF accumulation or expression of other HIF target genes was not suppressed by ER stress. Chromatin immunoprecipitation analysis identified a novel ATF4 binding site (TGACCTCT) within the EPO 3'-enhancer region, suggesting a distinct role for ATF4 in UPR-dependent suppression of the enhancer. Induction of ER stress in rat liver and kidney by tunicamycin decreased the hepatic and renal mRNA and plasma level of EPO. Collectively, ER stress selectively impairs the transcriptional activity of EPO but not of other HIF target genes. This effect is mediated by suppression of EPO 3'-enhancer activity via ATF4 without any direct effect on HIF, indicating that UPR contributes to oxygen-sensing regulation of EPO.

Topics: 3' Untranslated Regions; Activating Transcription Factor 4; Animals; Basic Helix-Loop-Helix Transcription Factors; Binding Sites; Cell Hypoxia; Cell Nucleus; Cobalt; Endoplasmic Reticulum Stress; Enhancer Elements, Genetic; Erythropoietin; Hep G2 Cells; Humans; Kidney; Liver; Male; Protein Binding; Protein Transport; Rats; Rats, Wistar; Transcription, Genetic; Transcriptional Activation; Unfolded Protein Response

Inflammation is a physiological defense response, but may also represent a potential pathological process in neurological diseases. In this regard, microglia have a crucial role in either progression or amelioration of degenerative neuronal damage. Because of the role of hypoxia in pro-inflammatory mechanisms in the nervous system, and the potential anti-inflammatory protective effect of erythropoietin (Epo), we focused our investigation on the role of this factor on activation of microglia and neuroprotection. Activation of microglial cells (EOC-2) was achieved by chemical hypoxia induced by cobalt chloride (CoCl2 ) and characterized by increased levels of nitrite, tumor necrosis factor-α and reactive oxygen species production, as well as up-regulation of inducible nitric oxide synthase expression. Under these conditions, cell proliferation data and proliferating cell nuclear antigen (PCNA) staining demonstrated a mitogenic effect of chemical hypoxia. Even though pre-treatment with Epo did not prevent nitrite production, inducible nitric oxide synthase protein expression or tumor necrosis factor-α secretion, it prevented the oxidative stress induced by CoCl2 as well as cell proliferation. Neuronal cells (SH-SY5Y) cultured in the presence of conditioned medium from activated EOC-2 cells or macrophages (RAW 264.7) developed significant apoptosis, an effect that was abolished by Epo via Epo/Epo receptor activation. The results show that even though Epo did not exert a direct anti-inflammatory effect on microglia activation, it did increase the resistance of neurons to subsequent damage from pro-inflammatory agents. In addition to its anti-apoptotic ability, the Epo antioxidant effect may have an indirect influence on neuronal survival by modulation of the pro-inflammatory environment.

Topics: Animals; Cell Hypoxia; Cell Proliferation; Cells, Cultured; Cobalt; Culture Media, Conditioned; Erythropoietin; Humans; Inflammation; Mice; Microglia; Neurons; Neuroprotective Agents; Neurotoxicity Syndromes; Nitric Oxide Synthase Type II; Nitrites; Proliferating Cell Nuclear Antigen; Reactive Oxygen Species; Receptors, Erythropoietin; Tumor Necrosis Factor-alpha

It is known that bone marrow-derived mesenchymal stem cells (BM-MSCs) are able to improve neuronal function through secretion of trophic factors in animal models of middle cerebral artery occlusion (MCAo). In this study, we demonstrated that incubation of BM-MSCs protects PC12 cells against apoptosis induced by CoCl(2) via the production of erythropoietin (EPO). Addition of CoCl(2) to BM-MSCs cultures induced the expression of EPO in a time-dependent manner. Additionally, BM-MSCs co-culture protected PC12 cells against apoptosis induced by CoCl(2) in a ratio-dependent manner. To explore whether expression of EPO induced by CoCl(2) is required for BM-MSCs-mediated cytoprotection, we transfected BM-MSCs with EPO small interfering RNA (siRNA). Knocking-down EPO abrogated increases in EPO expression induced by CoCl(2), and the cytoprotective effect of BM-MSCs. Reverse transcriptase polymerase chain reaction results showed that EPO siRNA reversed upregulation of Bcl-2, Bcl-X(L) expression and downregulation of Bax, Bak, caspase-9, and caspase-3 expression. Our results revealed that the protective effect of BM-MSCs against PC12 cell apoptosis induced by CoCl(2) might be dependent on EPO expression, at least in part, via the regulation of Bcl-2 family members and caspases.

Topics: Animals; Apoptosis; Bone Marrow Cells; Caspases; Cell Hypoxia; Cells, Cultured; Cobalt; Cytoprotection; Erythropoietin; Gene Knockdown Techniques; Male; Mesenchymal Stem Cells; Neurons; PC12 Cells; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Up-Regulation

Stroke is a major human health problem inducing long-term disability without any efficient therapeutic option being currently available. Under hypoxia, hypoxia-inducible factor-1α (HIF-1α) activates several genes as erythropoietin receptor (Epo-R) related with O(2) supply, and the multidrug-resistance gene (MDR-1) related with drug-refractory phenotype. Brain cortical injection of CoCl(2) produces focal hypoxia-like lesion with neuronal and glial alterations, as well as HIF-1α stabilization and MDR-1 overexpression. Intranasal (IN) drug delivery can by-pass blood-brain barrier (BBB) where MDR-1 is normally expressed. We evaluated the effects of IN-rHu-Epo administration on spontaneous motor activity (SMA) and the brain pattern expression of HIF-1α, MDR-1, and Epo-R in our cobalt-induced hypoxia model. Adult male Wistar rats were injected by stereotaxic surgery in frontoparietal cortex, with CoCl(2) (2 μl-50 mM; n = 20) or saline (controls; n = 20). Ten rats of each group were treated with IN-rHu-Epo 24 U or IN-saline. In addition, erythropoietic stimulation was evaluated by reticulocytes (Ret) account during three consecutive days, after intraperitoneal (i.p.)-recombinant-human Epo (rHu-Epo) (950 U; n = 6) or IN-rHu-Epo (24 U; n = 6) administration. SMA was evaluated by open field and rotarod tests, before and after surgical procedures during five consecutive days. Histological and immunostaining studies of HIF-1α, MDR-1, and Epo-R were performed on brain slides. A significant difference in SMA was observed in the hypoxic rats of IN-rHu-Epo-administered group as compared with Co-Saline-treated subjects and controls (p < 0.001). HIF-1α, EPO-R, and MDR-1 were overexpressed in the hypoxic cortex areas, while in contralateral hemisphere or controls, they were negatives. Reticulocytes were only increased in intraperitoneal (i.p.)-rHu-Epo-administered group. In spite of MDR-1 overexpression being detected in neurons, the coexpression of Epo-R could explain the positive effects observed on SMA of IN-rHu-Epo-administered group.

Topics: Administration, Intranasal; Analysis of Variance; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cobalt; Disease Models, Animal; Erythropoietin; Exploratory Behavior; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia, Brain; Male; Motor Activity; Rats; Rats, Wistar; Receptors, Erythropoietin; Recombinant Proteins; Recovery of Function; Reticulocytes; Rotarod Performance Test

Advanced chronic kidney disease (CKD) patients encounter anemia through insufficient erythropoietin (EPO) production by peritubular fibroblasts. Recent studies showed an increase in EPO production by pharmacological activation of hypoxia-inducible transcription factors (HIFs) in dialysis patients, suggesting that desensitization of the oxygen-sensing mechanism is responsible for the development of renal anemia. Our recent work demonstrated that indoxyl sulfate (IS), a uremic toxin, dysregulates oxygen metabolism in tubular cells. Here we provide evidence of an additional property that IS impairs oxygen sensing in EPO-producing cells. HepG2 cells were stimulated with cobalt chloride (CoCl(2)) or hypoxia under varying concentrations of IS. EPO mRNA was evaluated by quantitative PCR. Nuclear accumulation of HIF-α was evaluated by western blotting. Transcriptional activity of HIF was checked by hypoxia-responsive element (HRE)-luciferase reporter assay. The impact of IS was further evaluated in vivo by administering rats with indole, a metabolic precursor of IS, and subjecting them to CoCl(2) stimulation, in which renal EPO mRNA as well as plasma EPO levels were measured by quantitative PCR and enzyme-linked immunosorbent assay, respectively. Although IS induced cellular toxicity at relatively high concentrations (2.5 mM), EPO mRNA expression was significantly suppressed by IS at concentrations below cytotoxic ranges. In HepG2 cells, IS treatment decreased nuclear accumulation of HIF-α proteins and suppressed HRE-luciferase activity following hypoxia. Furthermore, administration of rats with indole suppressed renal EPO mRNA expression and plasma EPO levels, corroborating in vitro findings. Results of the present study provide a possible connection between a uremic toxin and the desensitization of the oxygen-sensing mechanism in EPO-producing cells, which may partly explain inadequate EPO production in hypoxic kidneys of CKD patients.

Topics: Animals; Blotting, Western; Cobalt; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression Regulation; Hep G2 Cells; Humans; Hypoxia-Inducible Factor 1; Indican; Indoles; Kidney Failure, Chronic; Luciferases; Oxygen; Rats; Uremia

Acute systemic hypoxia induces delayed cardioprotection against ischaemia-reperfusion injury in the heart. As cobalt chloride (CoCl₂) is known to elicit hypoxia-like responses, it was hypothesized that this chemical would mimic the preconditioning effect and facilitate acclimatization to hypobaric hypoxia in rat heart.. Male Sprague-Dawley rats treated with distilled water or cobalt chloride (12.5 mg Co/kg for 7 days) were exposed to simulated altitude at 7622 m for different time periods (1, 2, 3 and 5 days).. Hypoxic preconditioning with cobalt appreciably attenuated hypobaric hypoxia-induced oxidative damage as observed by a decrease in free radical (reactive oxygen species) generation, oxidation of lipids and proteins. Interestingly, the observed effect was due to increased expression of the antioxidant proteins hemeoxygenase and metallothionein, as no significant change was observed in antioxidant enzyme activity. Hypoxic preconditioning with cobalt increased hypoxia-inducible factor 1α (HIF-1α) expression as well as HIF-1 DNA binding activity, which further resulted in increased expression of HIF-1 regulated genes such as erythropoietin, vascular endothelial growth factor and glucose transporter. A significant decrease was observed in lactate dehydrogenase activity and lactate levels in the heart of preconditioned animals compared with non-preconditioned animals exposed to hypoxia.. The results showed that hypoxic preconditioning with cobalt induces acclimatization by up-regulation of hemeoxygenase 1 and metallothionein 1 via HIF-1 stabilization.

Topics: Acclimatization; Animals; Cardiotonic Agents; Cobalt; DNA-Binding Proteins; Erythropoietin; Heart; Heme Oxygenase-1; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Metallothionein; Myocardium; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Up-Regulation; Vascular Endothelial Growth Factor A

In the present study, we report the molecular mechanisms of action by cobalt in facilitating acclimatization to hypobaric hypoxia using male Sprague-Dawley rats as the model system. We determined hypoxic gasping time and survival time as a measure to assess the degree of tolerance of animals to hypobaric hypoxia by exposing the animals to an altitude of 10,668 m. Oral administration of cobalt chloride (12.5 mg Co/kg body weight, BW, for 7 days) increased gasping time and hypoxic survival time by 3 to 4 times compared to the control animals. This could be attributed to an increased expression and the DNA binding activity of hypoxia inducible transcriptional factor (HIF-1alpha) and its regulated genes, that is, erythropoietin (EPO), vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1), and nitric oxide synthase (NOS) levels. This in turn leads to better oxygenation, oxygen delivery, glucose transport, and maintenance of vascular tone, respectively, under oxygen-limited conditions. This was further confirmed by lower levels of lactate dehydrogenase (LDH) activity and lactate in the brain of cobalt + hypoxia group compared with animals exposed to hypoxia. Glucose levels also increased after cobalt supplementation. The findings of the study provide a basis for the possible use of cobalt for facilitating acclimatization to hypoxia and other conditions involving oxygen deprivation.

Topics: Acclimatization; Adaptation, Physiological; Altitude Sickness; Animals; Cobalt; Erythropoietin; Glucose Transporter Type 1; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia, Brain; Male; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Species Specificity; Vascular Endothelial Growth Factor A

Here, we demonstrate by chromatin immunoprecipitation that the binding of hypoxia-inducible factors to gene regulatory regions is differentially influenced in cancer cells. Binding of HIF-2alpha varies depending on hypoxic conditions, although HIF-1alpha is constantly bound to these regions. We found by RNA interference experiments that HIF-2alpha plays a minor role in VEGF gene upregulation under hypoxia or CoCl(2) treatment, even when both HIFs are similarly bound to the promoter region. HIF-2alpha activated or suppressed the ENO1 gene under various conditions, irrespective of promoter binding. We additionally found that HIF dependence on EPO gene induction could be altered depending on the conditions, irrespective of the binding pattern of HIFs. These results demonstrate that, unlike HIF-1alpha, HIF-2alpha differentially binds and regulates transcription under hypoxia.

Topics: Anaerobiosis; Basic Helix-Loop-Helix Transcription Factors; Cell Line, Tumor; Cobalt; Erythropoietin; Gene Expression; Gene Expression Regulation; Humans; Oxygen; Promoter Regions, Genetic; Transcriptional Activation

Erythropoietin (Epo) and the epo-receptor (EpoR) have been implicated in tumor growth, invasion and metastasis. We previously demonstrated Epo and EpoR expression in a small group of archived papillary thyroid cancers (PTC), but were unable to examine functional integrity using formalin-fixed tissues. In the present study, we examined the in vitro expression, induction and function of Epo and EpoR in papillary (NPA), follicular (WRO) and anaplastic (ARO-81) thyroid cancer cells. We found that all three cell lines expressed Epo and EpoR mRNA and that the hypoxia-mimetic cobalt induced Epo expression in all cell lines. None of the growth factors we examined (thyrotropin, vascular endothelial growth factor, IGF-I, or human Epo) altered Epo or EpoR gene expression. Importantly, however, administration of Epo to NPA but not WRO cells resulted in significant alterations in the expression of several mitogenic genes including cyclooxygenase-2 (COX-2), beta-casein (CSN2), wild type p53-induced gene-1 (WIG1) and cathepsin D (CTSD). Epo treated ARO-81 cells only had an increase in CSN2 expression. We conclude that Epo and EpoR are expressed by thyroid cancers and that stimulation of the Epo/EpoR signal pathway results in changes that could impact on the clinical behavior of thyroid cancers.

Topics: Carrier Proteins; Caseins; Cathepsin D; Cell Line, Tumor; Cobalt; Cyclooxygenase 2; Erythropoietin; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Nuclear Proteins; Proto-Oncogene Proteins; Receptors, Erythropoietin; RNA-Binding Proteins; RNA, Messenger; Thyroid Neoplasms; Tumor Suppressor Protein p53

Although erythropoietin (Epo) is a known stimulator of erythropoiesis, recent evidence suggests that its biological functions are not confined to hematopoietic cells. To elucidate the role of Epo and erythropoietin receptor (EpoR) in melanoma, we examined the expression and function of these proteins in melanocytes and melanoma cells. We found increased expression of Epo in melanoma cells compared to melanocyte in vitro. EpoR was also strongly expressed in all of the melanoma cell lines and two of the three melanocyte cell lines examined. Epo expression was significantly higher in melanoma than in benign nevi as determined by immunohistochemistry. Although melanoma cells secreted Epo in normoxic condition in vitro, hypoxia and CoCl(2) treatment increased Epo secretion. EpoR in melanoma cells was functional, because exogenous Epo increased melanoma resistance to hypoxic stress, pretreatment of melanoma cells with Epo significantly increased resistance to dacarbazine treatment, and Epo increased the phosphorylation of EpoR, RAF, and MEK. In conclusion, we demonstrated constitutive expression of Epo and EpoR as well as autonomous secretion of Epo by melanoma cells, indicating a novel autocrine loop of Epo in melanoma. The results suggest that the autocrine and paracrine functions of Epo might play a role in malignant transformation of melanocytes and in the survival of melanoma cells in hypoxia and other adverse conditions.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Cobalt; Coloring Agents; Culture Media, Conditioned; Disease Progression; Dose-Response Relationship, Drug; Erythropoietin; Humans; Hypoxia; Immunohistochemistry; MAP Kinase Signaling System; Melanocytes; Melanoma; Membrane Potentials; Mitochondria; Phosphorylation; Receptors, Erythropoietin; Tetrazolium Salts; Thiazoles; Trypan Blue

We previously demonstrated that chronic hypoxia has pivotal roles in the progression of tubulointerstitial injury from the early stage of the uninephrectomized Thy1 nephritis model. We have also shown that pretreatment of cobalt confers renoprotection in the ischemia/reperfusion (I/R) injury, in association with the up-regulation of hypoxia-inducible factor (HIF)-regulated genes. Here, we tested the hypothesis that cobalt administration not only attenuates acute ischemic insult, but also ameliorates tubulointerstitial injury secondary to chronic hypoxia.. We applied sustained cobalt treatment to the uninephrectomized Thy1 nephritis model at 3 to 5 weeks, when tubular hypoxia appeared. Histologic evaluation, including glomerular and peritubular capillary networks, was made at 8 weeks. HIF activation was confirmed by real-time polymerase chain reaction (PCR) analyses for HIF-regulated genes, such as erythropoietin (EPO), vascular endothelial growth factor (VEGF), and heme oxygenase 1 (HO-1). Up-regulation of HIF-1alpha and HIF-regulated genes was also verified by Western blotting analysis. To elucidate responsible mechanisms of cobalt in the amelioration of tubuloniterstitial injury, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining was conducted at 5 weeks. A combination therapy with angiotensin receptor blocker (ARB), olmesartan, was also challenged.. Although the intervention did not change glomerular structural damage or urinary protein excretion rate, tubulointerstitial injury was improved in cobalt-treated animals when compared with the vehicle-treated group. The amelioration was associated with the parallel up-regulation of renoprotective, HIF-regulated gene expression. TUNEL staining revealed that the number of apoptotic cells was reduced in the cortex by cobalt administration, suggesting that renoprotection was achieved partly through its antiapoptotic properties. Furthermore, it was demonstrated that cobalt treatment exerts additional renoprotective effects with the ARB treatment in this model.. Maneuvers to activate HIF in the ischemic tubulointerstitium will be a new direction to future therapeutic strategies.

Topics: Animals; Antibodies; Antimutagenic Agents; Apoptosis; Capillaries; Cobalt; Erythropoietin; Gene Expression Regulation; Glomerulonephritis; Glomerulosclerosis, Focal Segmental; Heme Oxygenase-1; Hypoxia; Hypoxia-Inducible Factor 1; In Situ Nick-End Labeling; Isoantibodies; Kidney Tubules; Male; Nephrectomy; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System; Reperfusion Injury; Vascular Endothelial Growth Factor A

Quinolinic acid (QA) is a potent endogenous excitotoxin; elevation of its concentration in an organism has been implicated in the pathogenesis of various disorders. The purpose of this study was the assessment of QA impact on the process of erythropoiesis. Marked increase of QA concentration was observed in plasma and peripheral tissues of uremic rats. These changes were proportional to the amount of the removed renal tissue and positively correlated with the concentration of creatinine but negatively correlated with hematological parameters, i.e., hematocrit and Hb red blood cells count. The changes were accompanied by a slight decrease in the concentration of endogenic erythropoietin (EPO) in the plasma of animals with uremia. Chronic treatment with QA diminished the increase in EPO concentration after introduction of cobalt in rats. These changes were associated with the decrease in all hematological parameters after QA administration. The in vitro study in the conditions of hypoxia showed that QA inhibited the EPO release from HepG2 cells to the culture base. Additionally, in HepG2 cells QA had a dose-dependent inhibitory effect on hypoxia- and cobalt-induced EPO gene expression without any cell toxicity. In conclusion, the erythropoiesis in chronic renal failure could be attributed to the influence of QA on EPO synthesis. Thus we propose that QA can be a uremic toxin responsible for anemia in animals or patients with renal failure.

Topics: Anemia; Animals; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Survival; Cobalt; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Humans; Kidney Failure, Chronic; Male; Neurotoxins; Organ Specificity; Quinolinic Acid; Rats; Rats, Wistar; RNA, Messenger; Tumor Cells, Cultured

Hypoxic preconditioning induces tolerance to hypoxic-ischemic injury in neonatal rat brain and is associated with changes in gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that is strongly induced by hypoxia or the hypoxia-mimetic compound cobalt chloride (CoCl(2)). Hypoxia-inducible factor-1 modulates the expression of several target genes including the glycolytic enzymes, glucose transporter-1 (GLUT-1), and erythropoietin. Recently, HIF-1 expression was shown to increase after hypoxic and CoCl(2) preconditioning in newborn rat brain. To study the involvement of HIF-1 target genes in neonatal hypoxia-induced ischemic tolerance, the authors examined the brains of newborn rats after exposure to hypoxia (8% O(2) for 3 hours) or injection of CoCl(2) (60 mg/kg). Preconditioning with hypoxia or CoCl(2) 24 hours before hypoxia-ischemia afforded a 96% and 76% brain protection, respectively, compared with littermate control animals. Hypoxic preconditioning increased the expression of GLUT-1 mRNA and protein, and of aldolase, phosphofructokinase, and lactate dehydrogenase proteins but not mRNA. This suggests that the modulation of glucose transport and glycolysis by hypoxia may contribute to the development of hypoxia-induced tolerance. In contrast, preconditioning with CoCl(2) did not produce any change in HIF-1 target gene expression suggesting that different molecular mechanisms may be involved in the induction of tolerance by hypoxia and CoCl(2) in newborn brain.

Topics: Animals; Animals, Newborn; Blotting, Northern; Brain; Cobalt; DNA-Binding Proteins; Erythropoietin; Female; Fructose-Bisphosphate Aldolase; Gene Expression; Glucose Transporter Type 1; Glycolysis; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia, Brain; Ischemic Preconditioning; L-Lactate Dehydrogenase; Male; Monosaccharide Transport Proteins; Nuclear Proteins; Phosphofructokinase-1; Pyruvate Kinase; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription Factors

The cellular site of erythropoietin (epo) production within the mammalian kidney is still not completely understood. In the present study, we examined the expression of epo mRNA in microdissected rat nephron segments by RT-PCR after induction of epo expression with cobalt chloride. Erythropoietin mRNA was not detected in nephron segments from saline injected rats. In cobalt chloride injected animals, epo mRNA was found in the majority of samples from the cortical region of the nephron, PCT, and CAL. Medullary tubule preparations (MCT and MAL) were mostly negative for epo mRNA, and glomeruli were uniformly negative. The induction of epo transcripts in tubular cells by cobalt chloride was paralleled by stimulation of the major transport enzyme in the kidney, namely, Na-K ATPase in a tubular profile similar to that of induction of epo transcripts. These results support some earlier findings that epo gene expression in response to cobalt salt stimulation of rat kidney occurs in transporting tubular epithelial cells.

Topics: Adenosine Triphosphatases; Animals; Antimutagenic Agents; Blotting, Southern; Cobalt; Erythropoietin; Kidney Tubules; Male; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium-Potassium-Exchanging ATPase

The aryl hydrocarbon receptor (AHR) and the alpha-class hypoxia inducible factors (HIF1alpha, HIF2alpha, and HIF3alpha) are basic helix-loop-helix PAS (bHLH-PAS) proteins that heterodimerize with ARNT. In response to 2,3,7,8-tetrachlorodibenzo-p-dioxin, the AHR. ARNT complex binds to "dioxin responsive enhancers" (DREs) and activates genes involved in the metabolism of xenobiotics, e.g. cytochrome P4501A1 (Cyp1a1). The HIF1alpha.ARNT complex binds to "hypoxia responsive enhancers" and activates the transcription of genes that regulate adaptation to low oxygen, e.g. erythropoietin (Epo). We postulated that activation of one pathway would inhibit the other due to competition for ARNT or other limiting cellular factors. Using pathway specific reporters in transient transfection assays, we observed that DRE driven transcription was markedly inhibited by hypoxia and that hypoxia responsive enhancer driven transcription was inhibited by AHR agonists. When we attempted to support this cross-talk model using endogenous loci, we observed that activation of the hypoxia pathway inhibited Cyp1a1 up-regulation, but that activation of the AHR actually enhanced the induction of Epo by hypoxia. To explain this unexpected additivity, we examined the Epo gene and found that its promoter harbors DREs immediately upstream of its transcriptional start site. These experiments outline conditions where inhibitory and additive cross-talk occur between the hypoxia and dioxin signal transduction pathways and identify Epo as an AHR-regulated gene.

Topics: Base Sequence; Binding, Competitive; Cobalt; Cytochrome P-450 CYP1A1; Dioxins; DNA; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation, Enzymologic; Molecular Sequence Data; Promoter Regions, Genetic; Receptor Cross-Talk; Receptors, Aryl Hydrocarbon; RNA, Messenger; Signal Transduction; Transcription Factors; Tumor Cells, Cultured

Iron chelators are pluripotent neuronal antiapoptotic agents that have been shown to enhance metabolic recovery in cerebral ischemia models. The precise mechanism(s) by which these agents exert their effects remains unclear. Recent studies have demonstrated that iron chelators activate a hypoxia signal transduction pathway in non-neuronal cells that culminates in the stabilization of the transcriptional activator hypoxia-inducible factor-1 (HIF-1) and increased expression of gene products that mediate hypoxic adaptation. We examined the hypothesis that iron chelators prevent oxidative stress-induced death in cortical neuronal cultures by inducing expression of HIF-1 and its target genes. We report that the structurally distinct iron chelators deferoxamine mesylate and mimosine prevent apoptosis induced by glutathione depletion and oxidative stress in embryonic cortical neuronal cultures. The protective effects of iron chelators are correlated with their ability to enhance DNA binding of HIF-1 and activating transcription factor 1(ATF-1)/cAMP response element-binding protein (CREB) to the hypoxia response element in cortical cultures and the H19-7 hippocampal neuronal cell line. We show that mRNA, protein, and/or activity levels for genes whose expression is known to be regulated by HIF-1, including glycolytic enzymes, p21(waf1/cip1), and erythropoietin, are increased in cortical neuronal cultures in response to iron chelator treatment. Finally, we demonstrate that cobalt chloride, which also activates HIF-1 and ATF-1/CREB in cortical cultures, also prevents oxidative stress-induced death in these cells. Altogether, these results suggest that iron chelators exert their neuroprotective effects, in part, by activating a signal transduction pathway leading to increased expression of genes known to compensate for hypoxic or oxidative stress.

Topics: Activating Transcription Factor 1; Animals; Apoptosis; Cells, Cultured; Cerebral Cortex; Chelating Agents; Cobalt; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Deferoxamine; DNA-Binding Proteins; Enzyme Induction; Erythropoietin; Fetus; Fructose-Bisphosphate Aldolase; Gene Expression Regulation; Glutathione; Glycolysis; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; L-Lactate Dehydrogenase; Mimosine; Neurons; Neuroprotective Agents; Nuclear Proteins; Oxidative Stress; Rats; Rats, Sprague-Dawley; Transcription Factors

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Topics: Adult; Antigens, CD34; Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Cells, Cultured; Cobalt; Culture Media, Serum-Free; Enzyme-Linked Immunosorbent Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hematopoietic Stem Cells; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Liver Neoplasms; Molecular Sequence Data; Peptides; Receptors, Erythropoietin; RNA, Messenger; Tumor Cells, Cultured

Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1. 5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (rho0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) rho0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in rho degrees cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

Topics: Adenosine Triphosphate; Antioxidants; Azoles; Cell Hypoxia; Cell Line; Cobalt; DNA-Binding Proteins; DNA, Mitochondrial; Endothelial Growth Factors; Erythropoietin; Glycolysis; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Isoindoles; Lymphokines; Mitochondria, Liver; Nuclear Proteins; Organoselenium Compounds; Pyrrolidines; Reactive Oxygen Species; RNA, Messenger; Thiocarbamates; Transcription Factors; Transcriptional Activation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

In response to hypoxia, mammalian cells express multiple gene products [including erythropoietin (EPO) and vascular endothelial growth factor (VEGF)] that serve to increase O2 delivery, as well as glucose transporters and glycolytic enzymes (such as enolase 1) that allow metabolic adaptation to decreased O2 availability. Increased transcription of the genes encoding these proteins in hypoxic cells is mediated by hypoxia-inducible factor 1 (HIF-1), a basic helix-loop-helix transcription factor. Expression of HIF-1 and downstream genes can also be induced by exposure of cells to divalent metals (such as CoCl2) or iron chelators [such as desferrioxamine (DFO)]. We report here that the organomercurial compound mersalyl induced expression of VEGF and enolase 1 mRNA, as well as HIF-1 activity, in cultured cells. Expression of reporter genes containing hypoxia response elements from the EPO and VEGF genes was also induced by mersalyl treatment. However, mersalyl inhibited endogenous EPO mRNA expression induced by hypoxia, CoCl2, or DFO. In cells lacking expression of the insulin-like growth factor-1 receptor, mersalyl did not induce HIF-1 activity or VEGF mRNA expression, whereas induction by hypoxia, CoCl2, or DFO was unaffected. The mitogen-activated protein kinase kinase inhibitor PD098059 markedly reduced induction of HIF-1 by mersalyl but not by hypoxia. These results indicate that mersalyl induces expression of HIF-1 and a subset of hypoxia-inducible genes by a mechanism, involving the insulin-like growth factor-1 receptor and mitogen-activated protein kinase activity, that is distinct from mechanisms of induction by hypoxia, CoCl2, or DFO.

Topics: Animals; Antimutagenic Agents; Base Sequence; Cell Hypoxia; Chelating Agents; Cobalt; Deferoxamine; DNA; DNA-Binding Proteins; Endothelial Growth Factors; Enzyme Inhibitors; Erythropoietin; Fibroblasts; Gene Expression Regulation, Neoplastic; Hepatoblastoma; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Lymphokines; Mersalyl; Nuclear Proteins; Phosphopyruvate Hydratase; Rats; Receptor, IGF Type 1; RNA, Messenger; Transcription Factors; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Hypoxia is thought to be a common precursor of coronary artery disease and malignant tumors, both diseases representing the leading causes of death in industrial nations. So far, investigations of oxygen-regulated erythropoietin (EPO) gene expression in the human hepatoma cell lines Hep3B and HepG2 allowed many important insights into the mechanisms of oxygen-sensing, signalling and regulation of an increasing number of oxygen-responsive genes. To differentiate the various signalling pathways involved in EPO production by these two cell lines, we examined several factors that positively influenced EPO expression. The results demonstrate a keen differential effect of cell density and oxygen concentration on EPO induction in Hep3B compared to HepG2 cells. Using optimized cell culture conditions, EPO production rates as high as 1 U EPO per 10(6) Hep3B cells in 24 h could be achieved. We also found a moderate but reproducible positive effect of CoCl2 on hypoxia-induced EPO expression in Hep3B but a negative CoCl2 effect on hypoxic induction in HepG2 cells. CoCl2 inhibited cell growth in a concentration-dependent manner. Interleukin-6 was synergistic with hypoxia on EPO induction in Hep3B as well as HepG2 cells, and dexamethasone enhanced this effect in Hep3B but not in HepG2 cells. The moderate CoCl2-dependent increase of EPO production observed in hypoxic Hep3B cells might indicate that CoCl2 and hypoxia do not necessarily act via, identical signalling pathways.

Topics: Carcinoma, Hepatocellular; Cell Count; Cell Hypoxia; Cobalt; Dexamethasone; Erythropoietin; Humans; Interleukin-6; Liver Neoplasms; Oxygen; Signal Transduction

The erythropoietin (EPO) gene is one of the best examples of a mammalian gene controlled by oxygen tension. The DNA elements responsible for hypoxia-induced transcription consist of a short region of the proximal promoter and a <50-bp 3' enhancer. The elements act cooperatively to increase the transcriptional initiation rate approximately 100-fold in response to low oxygen tension in Hep3B cells. Two distinct types of transactivating proteins have been demonstrated to bind the response elements in the human EPO enhancer in vitro: one shows hypoxia-inducible DNA binding activity, while the other activity binds DNA under normoxic and hypoxic conditions. We have investigated the DNA-protein interactions on the human EPO enhancer in living tissue culture cells that produce EPO in a regulated fashion (Hep3B) and in cells that do not express EPO under any conditions tested (HeLa). We have identified in vivo DNA-protein interactions on the control elements in the human EPO enhancer by ligation-mediated PCR technology. We show that the putative protein binding sites in the EPO enhancer are occupied in vivo under conditions of normoxia, hypoxia, and cobalt exposure in EPO-producing cells. These sites are not occupied in cells that do not produce EPO. We also provide evidence for a conformational change in the topography of the EPO enhancer in response to hypoxia and cobalt exposure.

Topics: Base Sequence; Binding Sites; Carcinoma, Hepatocellular; Cell Hypoxia; Cobalt; DNA; DNA Footprinting; DNA Methylation; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Genes; Humans; Molecular Sequence Data; Nucleic Acid Conformation; Polymerase Chain Reaction; Trans-Activators; Tumor Cells, Cultured

We have recently proposed a H2O2-generating b-type cytochrome as part of the cellular oxygen sensor that controls O2-dependent erythropoietin (Epo) production in the human hepatocellular carcinoma cell line HepG2. H2O2 could act as an intracellular signaling molecule because its production in HepG2 cells is strictly dependent on the pericellular PO2. High cellular levels of H2O2 inhibit hypoxia-induced Epo production while low levels-as under hypoxic conditions-allow full expression of the Epo gene. Since cobalt chloride (CoCl2) and the iron chelator desferrioxamine (DSF) both mimic the hypoxic induction of Epo production we studied the influence of CoCl2 and DSF on the formation and on the action of reactive O2-species with respect to Epo production. Both chemicals reduced the H2O2-dependent 123-dihydrorhodamine fluorescence in HepG2 cells. The inhibition of Epo production by exogenous H2O2 was completely antagonized by DSF. This might indicate that H2O2 exerts its inhibition through a Fenton type reaction. On the other hand, NADPH and pyrogallol which stimulate the production of O2- inhibited Epo production. CoCl2 antagonized their effects. From our results we propose different sites of interaction with the putative signaling chain for DSF and CoCl2. While DSF appears to reduce the action of the H2O2 molecule, CoCl2 might act further upstream through the induction of H2O2-scavenger systems or by interfering with its production.

Topics: Cell Line; Cobalt; Deferoxamine; Erythropoietin; Humans; Hydrogen Peroxide; NADP; Reactive Oxygen Species; Signal Transduction

Diphenylene iodonium chloride suppresses the cobaltous chloride-induced expression of erythropoietin by Hep3B cells to about 50% at a concentration of 30 nM. At that concentration, it has no effect on the response to low oxygen. The related compound iodonium diphenyl chloride acts similarly but is a much less effective inhibitor. If, as reported, diphenylene iodonium chloride is a specific inhibitor of cytochrome b, it follows that the response to CoCl2 is dependent on that enzyme but the response to hypoxia is not.

Topics: Biphenyl Compounds; Cobalt; Erythropoietin; Onium Compounds; Tumor Cells, Cultured

Fanconi anemia (FA) is a recessively inherited disease characterized by bone marrow failure, congenital anomalies, chromosomal instability and hypersensitivity to crosslinking agents. Some of the cellular defects of FA are known to be responsive to the ambient oxygen concentration. We examined the responsiveness of the FA complementation group C (FAC) gene to changes in oxygen concentration using two types of human cell lines, hypoxia-responsive Hep3B hepatoma cells and Epstein-Barr virus-immortalized lymphoblasts (normal and FA complementation groups B and C). Although the expression of erythropoietin in Hep3B cells was induced in response to the hypoxia-mimicking agent CoCl₂, there was no concomitant induction in FAC expression as assessed by mRNA levels and immunoprecipitable protein, and no detectable change in the cytoplasmic location of the FAC polypeptide as determined by indirect immunofluorescence. In human lymphoblasts we examined the effect of oxygen (0.1% -95% O₂) on cell proliferation and FAC expression. FA lymphoblasts had a normal sensitivity to the cytostatic effect of hyperoxia, while in both control and FA lymphoblasts FAC mRNA levels were unaffected by oxygen. Our results indicate that ambient oxygen is not a regulator of the FAC gene.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Hypoxia; Cell Line, Transformed; Cobalt; DNA-Binding Proteins; Erythropoietin; Fanconi Anemia; Fanconi Anemia Complementation Group C Protein; Fanconi Anemia Complementation Group Proteins; Gene Expression Regulation; Herpesvirus 4, Human; Humans; Liver Neoplasms; Lymphocytes; Nuclear Proteins; Oxygen; Protein Biosynthesis; Proteins; RNA, Messenger; Tumor Cells, Cultured

Production of the glycoprotein hormone erythropoietin (Epo) in response to hypoxic stimuli is almost entirely restricted to particular cells within liver and kidney, yet the transcriptional enhancer lying 3' to the Epo gene shows activity inducible by hypoxia after transfection into a wide variety of cultured cells. The implication of this finding is that many cells which do not produce Epo contain a similar, if not identical, oxygen-regulated control system, suggesting that the same system is involved in the regulation of other genes. We report that the human phosphoglycerate kinase 1 and mouse lactate dehydrogenase A genes are induced by hypoxia with characteristics which resemble induction of the Epo gene. In each case expression is induced by cobalt, but not by cyanide, and hypoxic induction is blocked by the protein-synthesis inhibitor cycloheximide. We show that the relevant cis-acting control sequences are located in the 5' flanking regions of the two genes, and we define an 18-bp element in the 5' flanking sequence of the phosphoglycerate kinase 1 gene which is both necessary and sufficient for the hypoxic response, and which has sequence and protein-binding similarities to the hypoxia-inducible factor 1 binding site within the Epo 3' enhancer.

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line; Cloning, Molecular; Cobalt; Cyanides; Cycloheximide; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Isoenzymes; L Cells; L-Lactate Dehydrogenase; Liver Neoplasms; Mice; Molecular Sequence Data; Oxygen; Phosphoglycerate Kinase; Promoter Regions, Genetic; Sequence Deletion; Sequence Homology, Nucleic Acid; TATA Box; Transfection; Tumor Cells, Cultured

The hypoxia-induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 microM) for 18-36 h. The redox state of cytochrome c and cytochrome aa3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.

Topics: Cells, Cultured; Cobalt; Cytochromes; Erythropoietin; Hydrogen Peroxide; Oxidation-Reduction; Signal Transduction

Erythropoietin (EPO) gene transcription is activated in kidney cells in vivo and in Hep3B cells exposed to hypoxia or cobalt chloride. Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor that binds to the hypoxia-inducible enhancer of the EPO gene at a site that is required for transcriptional activation. HIF-1 DNA-binding activity is induced by hypoxia or cobalt chloride treatment of Hep3B cells. We report that treatment of Hep3B cells with desferrioxamine (DFX) induced HIF-1 activity and EPO RNA expression with kinetics similar to the induction of HIF-1 by hypoxia or cobalt chloride. Induction by each of these stimuli was inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. DFX appears to induce HIF-1 by chelating iron as induction was inhibited by coadministration of ferrous ammonium sulfate. DFX administration to mice transiently increased EPO RNA levels in the kidney. As previously shown for hypoxia and cobalt treatment, DFX also induced HIF-1 activity in non-EPO-producing cells, suggesting the existence of a common hypoxia signal-transduction pathway leading to HIF-1 induction in different cell types.

Topics: Base Sequence; Cell Hypoxia; Cell Line; Cell Nucleus; Cobalt; Deferoxamine; DNA-Binding Proteins; Erythropoietin; Gene Expression; Hepatoblastoma; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Molecular Sequence Data; Nuclear Proteins; Oligonucleotide Probes; RNA, Neoplasm; Signal Transduction; Transcription Factors; Tumor Cells, Cultured

Exposure of Hep3B cells to metalloporphyrins (tinprotoporphyrin and heme) or cobalt chloride resulted in the production of a significant number of heme oxygenase transcripts, erythropoietin transcripts or both, as indicated by in situ hybridization. Exposure to heme 10 mumol/L resulted in a 30-fold to 40-fold increase in cells expressing erythropoietin messenger RNA (erythropoietin-positive cells) by 6 hr; this increased level remained elevated for 24 hr. Tin-protoporphyrin (10 mumol/L) produced an eightfold to 10-fold increase in erythropoietin RNA within 40 min. This value then returned to control levels by 60 min. Exposure to cobalt chloride (100 mumol/L) resulted in a 20-fold to 30-fold increase in erythropoietin expression for 5 to 20 min, returning to control by 40 min. Additionally, nuclear runoff assays demonstrated that the increase in heme oxygenase or erythropoietin messenger RNA accumulation by cobalt chloride appeared to be a result of stimulated transcription of the heme oxygenase and erythropoietin genes. However, the pattern for heme oxygenase messenger RNA induction was different from that for erythropoietin expression. Heme produced an immediate expression of heme oxygenase RNA (50-fold within 5 min) and a second sustained response during the next 24 hr. Tin-protoporphyrin also produced an immediate response (40-fold within 5 min) and remained elevated (20-fold) for 6 hr. Cobalt chloride produced a 22-fold increase within 20 min and returned to the control value by 1 hr. Thus both erythropoietin and heme oxygenase genes appear to be expressed after treatment with tin-protoporphyrin, heme or cobalt chloride; however, the time and patterns of expression are different.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carcinoma, Hepatocellular; Cobalt; Erythropoietin; Gene Expression; Heme; Heme Oxygenase (Decyclizing); Humans; Liver Neoplasms; Metalloporphyrins; Protoporphyrins; RNA, Messenger; Tumor Cells, Cultured

The mechanism responsible for the accumulation of heme oxygenase and erythropoietin (epo) transcripts due to cobalt chloride (CoCl2) administration was investigated in rat kidney using a rat heme oxygenase and mouse epo probes. We found an increase of heme oxygenase transcripts in kidney in response to CoCl2. Quantitative evaluation of the heme oxygenase mRNA changes, by scanning densitometry, indicated that the levels of mRNA encoding heme oxygenase were increased by about fiftyfold in rat kidney after administration of CoCl2. That the increase in heme oxygenase mRNA levels resulted from enhanced transcription of the heme oxygenase gene was confirmed by nuclear runoff using isolated rat kidney nuclei after CoCl2 administration. Transcription of the heme oxygenase gene is greatly increased in rat kidney within 1 hr of administration of CoCl2 as evidenced from the levels of 32P-UTP incorporation into the specific transcript. Time course studies showed that stimulation of transcription was increased about fortyfold 3 hr after CoCl2 administration. This stimulation is the most rapid transcriptional response to heavy metals yet described. In addition, Northern blot analysis demonstrated that epo mRNA was first detected 4 hr following CoCl2 administration and reached a maximum at 5 hr. On the other hand, PCR analysis indicated that epo mRNA was increased as early as 1 hr following CoCl2 administration. The fact that CoCl2 caused increased transcription of both the epo and heme oxygenase genes suggests that a common mechanism may be involved in the regulation of these two genes by the heavy metal ion.

Topics: Animals; Base Sequence; Blotting, Northern; Cobalt; Erythropoietin; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Heme Oxygenase (Decyclizing); Kidney; Kinetics; Male; Molecular Sequence Data; Polydeoxyribonucleotides; Polymerase Chain Reaction; Rats; Rats, Inbred Strains; RNA, Messenger; Transcription, Genetic

Effects of agents affecting cytochrome P450 were studied on the production of erythropoietin (Epo) in cultures of the human hepatoma cell line HepG2. Epo was measured by radioimmunoassay of the culture media after 24 h of incubation. The addition of phenobarbital or 3-methylcholanthrene, which induce cytochrome P450, significantly enhanced the formation of Epo. Likewise, the thyroid hormones T3 and T4 stimulated the rate of the production of Epo. On the other hand, the formation of Epo was lowered following the addition of diethyldithiocarbamate or cysteamine chloride, which inhibit cytochrome P450. These findings support the idea that O2 sensitive hemoproteins of the microsomal mixed-functional oxidases play a role in the control of the synthesis of Epo.

Topics: Carcinoma, Hepatocellular; Cobalt; Cysteamine; Cytochrome P-450 Enzyme System; Ditiocarb; Erythropoietin; Humans; Liver Neoplasms; Methylcholanthrene; Metyrapone; Microsomes; Oxygen; Phenobarbital; Thyroxine; Triiodothyronine; Tumor Cells, Cultured

The promoter regions of the mouse and human erythropoietin genes have regions of identity within 130 base pairs upstream of the cap site, suggesting a cis-acting regulatory role for the conserved sequences. We have used a double-stranded deoxyoligonucleotide corresponding to the -61 to -45 region relative to the start site of transcription of the mouse gene in DNA mobility shift assays. Nuclear extracts from kidneys of both control and cobalt-stimulated mice contain factors that bind to this oligonucleotide in a specific manner. One factor is a 47-kDa protein, whereas the others may be one or more ribonucleoproteins. Under denaturing conditions, four RNA species which show specific binding to the oligonucleotide were observed, suggesting that recognition of the oligonucleotide by ribonucleoprotein is mediated by the RNA component. In nuclear extracts of kidneys from stimulated animals, the amount of the two largest RNA species that bind to the oligonucleotide was reduced relative to that of control, whereas the other RNA species as well as the 47-kDa protein remained relatively unaffected. These results suggest that the ribonucleoprotein containing the down-regulated RNA species may be a negative transcriptional factor and that activation of the erythropoietin gene by cobalt salts may involve, in part, decreased binding of this factor, thus allowing transcription to proceed.

Topics: Animals; Base Sequence; Cell Nucleus; Cobalt; Erythropoietin; Female; Gene Expression Regulation; Humans; Kidney; Mice; Mice, Inbred Strains; Molecular Sequence Data; Oligonucleotide Probes; Reference Values; Ribonucleoproteins; Sequence Homology, Nucleic Acid

The hemin regulation of L-alanine:4,5-dioxovalerate transaminase, the enzyme proposed for an alternate route of delta-aminolevulinic acid (ALA) biosynthesis in mammalian system was studied in different conditions: phenylhydrazine induced anemia, polycythemia by erythropoietin to anemic rats, treatment with cobalt chloride, a porphyrogenic drug. The activity of L- alanine:4,5-dioxovalerate transaminase in liver and kidney is stimulated in phenylhydrazine, whereas, erythropoietin injection to anemic rats prevents such stimulation. Further treatment with cobalt chloride to erythropoietin treated anemic rats stimulates the enzyme activity. Actinomycin D, however, inhibits the stimulation of L-alanine:4,5-dioxovalerate transaminase by phenylhydrazine suggesting that induction is at the level of transcription. Induced level of this enzyme in anemic condition was estimated quantitatively by radial immunodiffusion using antibody raised against L-alanine:4,5-dioxovalerate transaminase. Moreover, our studies reveal that stimulation of L-alanine:4,5-dioxovalerate transaminase in anemic condition is dependent on depletion of heme level. The regulatory role of intracellular heme pool on the induction of this enzyme suggests its physiological importance in heme biosynthesis.

Topics: Anemia; Animals; Cobalt; Dactinomycin; Erythropoietin; Hematocrit; Heme; Immune Sera; Immunodiffusion; Kidney; Kinetics; Liver; Male; Phenylhydrazines; Rats; Rats, Inbred Strains; Transaminases

The balance of erythropoietin production by the dog kidney and liver was studied during controlled normoxic perfusion. The hormone production was stimulated by acute posthemorrhagic anemia (bloodletting of 25% total blood volume) combined with subcutaneous injection of cobaltous chloride (250 microM/kg body weight). The increase in erythropoietin level was revealed in posthypoxic animal perfusate after 6 hours of perfusion. The amount of hepatic erythropoietin was shown to be 2.5 times higher than that excreted by kidneys.

Topics: Animals; Cobalt; Dogs; Erythropoiesis; Erythropoietin; Hemorrhage; Kidney; Liver; Male; Mice; Mice, Inbred CBA; Perfusion; Polycythemia