Hypoxia up-regulates the activity of novel erythropoietin mRNA binding protein (original) (raw)
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Journal of Biological Chemistry, 1997
We have previously identified a sequence in the 3untranslated region (3-UTR) of erythropoietin (Epo) mRNA which binds a protein(s), erythropoietin mRNA-binding protein (ERBP). A mutant lacking the ERBP binding site (Epo M) was generated. Hep3B cells were stably transfected with a wild-type Epo (Epo WT) cDNA or Epo M cDNA construct located downstream of a promoter of cytomegalovirus. Following inhibition of transcription, the half-lives of Epo WT and Epo M mRNAs were 7 h and 2.5 h in normoxia, respectively. The Epo M mRNA half-life remained unchanged in hypoxia. Epo WT mRNA half-life increased ϳ40% in response to a 6-h hypoxic pre-exposure and an additional ϳ50% when preexposed to 12 h hypoxia. The steady-state level of Epo WT mRNA was 4-fold that of Epo M mRNA reflecting the difference in mRNA decay rates in normoxia. The Epo protein level expressed from exogenous Epo M was unchanged in both normoxia and hypoxia. In contrast, the Epo protein level expressed from exogenous Epo WT increased 50% in hypoxia when compared with normoxia. These observations were further supported by chimeric chloramphenicol acetyltransferase and Epo-3-UTR constructs. We have demonstrated that Epo mRNA stability was modulated in normoxia and further by hypoxia, therefore, providing evidence that Epo is regulated at the post-transcriptional level through ERBP complex formation.
Changes in redox affect the activity of erythropoietin RNA binding protein
FEBS Letters, 1995
We have previously identified a cytosolic protein, erythropoietin RNA binding protein (ERBP), which is up-regulated in certain tissues in response to hypoxia. To further characterize the interaction of ERBP and erythropoietin (EPO) mRNA, we have examined the role of reduction-oxidation in the EPO mRNA binding mechanism of ERBP isolated from human hepatoma cells (Hep3B). Reducing agents dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) increased ERBP binding activity in a concentration-dependent manner, whereas the oxidizing agent, diamide, abolished ERBP binding activity. In addition, treatment of Hep3B cell lysates with the irreversible suifhydryl alkylating agent N-ethylmaleimide resulted in inhibition of the EPO mRNA-ERBP complex. Taken together, these findings suggest that sulfhydryi groups may play a role in vivo in the regulation of EPO production through the modulation of ERBP binding activity.
Advances in Experimental Medicine and Biology
Hypoxia induces tissue-specific gene products such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF), which improve the peripheral O2 supply, and glucose transporters and glycolytic enzymes, which adapt cells to reduced O2 availability. EPO has been the fountainhead in research on pO2-dependent synthesis of proteins. The EPO gene enhancer (like the flanking DNA-elements of several other pO2-controlled genes) contains a consensus sequence (CGTG) that binds the trans-acting dimeric hypoxia-inducible factor 1 (HIF-1alpha/beta). The alpha-subunit of HIF-1 is rapidly degraded by the proteasome under normoxic conditions, but it is stabilized on occurrence of hypoxia. HIF-1 DNA-binding is also increased by insulin, and by interleukin-1 and tumor necrosis factor. Thus, in some aspects there is synergy in the cellular responses to hypoxia, glucose deficiency and inflammation. In viewing clinical medicine recombinant human EPO (rHu-EPO) has become the mainstay of treatment...
Proceedings of the National Academy of Sciences, 1991
Human erythropoietin gene expression in liver and kidney is inducible by anemia or hypoxia. DNase I-hypersensitive sites were identified 3' to the human erythropoietin gene in liver nuclei. A 256-base-pair region of 3' flanking sequence was shown by DNase I protection and electrophoretic mobility-shift assays to bind four or more different nuclear factors, at least two of which are induced by anemia in both liver and kidney, and the region functioned as a hypoxia-inducible enhancer in transient expression assays. These results provide insight into the molecular basis for the regulation of gene expression by a fundamental physiologic stimulus, hypoxia.
Regulation of the erythropoietin gene
Blood, 1999
Erythropoietin (Epo), the hormone that stimulates red blood cell production, is induced by hypoxia. We have utilized the human hepatoma cell line, Hep3B, to investigate the regulation of the Epo gene. We present evidence that the oxygen sensor in Hep3B cells is a heme protein. Hypoxic and cobalt induction of Epo protein is paralleled by a 50-to 100-fold increase in Epo mRNA which we have accurately quantified hy means of an assay based on competitive polymerase chain reaction. This increase in Epo mRNA is due primarily to increased transcription. lkansfection experiments utilizing the sensitive luciferase reporter gene show that the minimal portions of the Epo gene required for hypoxic induction include a 53 bp promoter element and a 43 bp enhancer located downstream from the polyadenylation site. Gel shift experiments show that these two regions cross-compete for specific DNA binding proteins. The enhancer contains a hexanucleotide direct repeat with a two bp insert which footprints with nuclear extracts from Hep3B cells and, when mutated, results in loss of hypoxic induction. This sequence is likely to bind to a member of the steroid thyroid hormone receptor family of DNA bmding proteins. These enhancer and promoter elements appear to cooperate in enabling the Epo gene to respond to hypoxia in a physiologically appropriate manner.
Erythropoietin Receptor in Human Tumor Cells: Expression and Aspects Regarding Functionality
2001
Aims and background: Recombinant human erythropoietin (Epo) and granu l o cy t e-c o l o ny-s t i mulating factor (G-CSF) are used to stimulate hematopoiesis in patients with malignant dise a s e s. These cytokines transduce their biological signal via the Epo receptor (EpoR) and G-CSF receptor (G-CSF-R) into the cell. We therefore investigated in human tumor cell lines the expression of these receptors in tumor cells as well as their response to Epo and G-CSF. Methods and study design: The expression of EpoR and G-CSF-R mRNA was analy zed with rev e rse transcription-poly m e r a s e chain reaction (RT-PCR). EpoR protein expression was further monitored with Western blot and immunocytochemistry analys i s. The cellular response to various concentrations of Epo was evaluated using 3 [H]-thymidine uptake, Northern blot of c-fos expression and tyrosine kinase activity assay. The proliferation after G-CSF incubation was analyzed with the MTS assay. Results: In this study EpoR mRNA and protein were detected in various human tumor cell lines. Treatment with Epo did not influence the pro l i feration rate of examined EpoR-positive tumor cell lines.Epo did not stimulate the tyrosine kinase activity nor did it affect the c-fos mRNA in these cell lines.G-CSF-R mRNA was only detected in two myeloid cell lines. Treatment with G-CSF did not increase the proliferation of these cells. C o n c l u s i o n s : These results demonstrate that Epo and G-CSF did not modulate the growth rate of examined receptor-positive tumor cell lines; the presence of the Epo receptor seems not essential for cell growth of these tumor cells in cell culture.
Oxygen-regulated expression of the erythropoietin gene in the human renal cell line REPC
Blood, 2011
Erythropoietin (EPO), the key hormone in red blood cell renewal, is mainly produced in the adult kidney. Anemia and hypoxia substantially enhance EPO expression to increase erythropoiesis. Investigations of the cellular physiology of renal EPO production have been hampered by the lack of an adequate human cell line. In the present study, we present the human kidney cell line REPC (for renal Epo–producing cells), established from an explanted human kidney exhibiting EPO gene expression and release of the EPO protein in an oxygen-dependent manner. Hypoxic induction of EPO mRNA showed the typical transient increase and peak in expression after 36 hours under continuous conditions of hypoxia. Bioactive EPO protein accumulated in the culture supernatant. The induction of EPO gene expression in REPCs critically depended on the activation of hypoxia-inducible transcription factors (HIFs). SiRNA treatment revealed that the expression of EPO was largely dependent on the activation of the tra...
Molecular mechanism and systemic response of erythropoietin:A Review
This paper reviews the molecular mechanism and systemic response of erythropoietin (Epo).Erythropoietin(Epo) is a glycoprotein hormone that causes the production of red blood cell.This helps to increase the level of haemoglobin and packed cell volume.It is produced mainly in the interstitial fibroblast of the renal cortex.It has additional nonhaematopoietic effects.Erythropoietin enhancer is activated by hypoxia-inducible transcription factors (HIFs) which is composed of an O 2 -labile α-subunit and a constitutive subunit.Erythropoietin synthesis follows a negative feedback mechanism and is regulated by hypoxia.There is an inverse relationship between erythropoietin (Epo) level and haematocrit (Hct) or haemaoglobin (Hb) concentration.The level of erythropoietin varies greatly between individuals and there is no serious sex or age differences.This diurnal fluctuation with a nadir occuring in the morning.Therefore,the kidneys shoud be kept in good health conditions always to maintain the daily requirement of erythropoietin level to prevent anaemia and apoptosis of the red blood cells.
Oncology letters, 2013
Erythropoietin (Epo) is a potent inducer of erythropoiesis that is mainly produced in the kidney. Epo is expressed not only in the normal kidney, but also in renal cell carcinomas (RCCs). The aim of the present study was to gain insights into the roles of Epo and its receptor (EpoR) in RCC cells. The study used two RCC cell lines, Caki-1 and SKRC44, in which Epo and EpoR are known to be highly expressed. The proliferation rate and expression level of hypoxia-inducible factor-1α (HIF-1α) were measured prior to and following Epo treatment and under normoxic and hypoxic conditions. To examine whether HIF-1α or Epo were involved in cellular proliferation during hypoxia, these proteins were knocked down using small interfering RNA (siRNA) in Caki-1 and SKRC44 cells. The results demonstrated that Epo enhanced the proliferation of the Caki-1 and SKRC44 cells. HIF-1α expression was increased upon the induction of hypoxia in the Caki-1 cells, but remained unaffected in the SKRC44 cells. The ...
The Influence of Erythropoietin (EPO) on Cancer Cells and its Role in the Cancer Treatment
The hormone erythropoietin (EPO) is essential for the survival, proliferation and differentiation of the erythrocytic progenitors. The EPO receptor (EPO-R) of erythrocytic cells belongs to the cytokine class I receptor family and signals through various protein kinases and STAT transcription factors. The EPO-R is also expressed in many organs outside the bone marrow, suggesting that EPO is a pleiotropic anti-apoptotic factor. The controversial issue as to whether the EPO-R is functional in tumor tissue is critically reviewed. Importantly, most studies of EPO-R detection in tumor tissue have provided falsely positive results because of the lack of EPO-R specific antibodies. However, endogenous EPO appears to be necessary to maintain the viability of endothelial cells and to promote tumor angiogenesis. This review paper reviews EPO use in cancer patients and its management of anemia. While the findings promise beneficial effects of endogenous EPO and its therapeutic analogues as tissue-protective factors, for example in ischemic and degenerative heart and brain diseases, fear has also arisen that EPO may promote tumor cell survival and stimulate tumor growth. If the cancer patient is being treated with curative intent, the use of ESAs should be avoided. If the treatment plan is more conservative or palliative, ESA should be considered for anemia treatment, but the treatment should be controlled.