Erythropoietin Gene Expression Research Articles

Induction of erythropoietin gene expression in epithelial cells by chemicals identified in GATA inhibitor screenings.

Erythropoietin (EPO) is a hormone that promotes proliferation, differentiation and survival of erythroid progenitors. EPO gene expression is regulated in a tissue-specific and hypoxia-inducible manner and is mainly restricted to renal EPO-producing cells after birth. Chronic kidney disease (CKD) confers high risk for renal anemia due to lower EPO production from injured kidneys. In transgenic reporter lines of mice, disruption of a GATA-binding motif within the Epo gene promoter-proximal region restores constitutive reporter expression in epithelial cells. Here, mitoxantrone and its analogues, identified as GATA factor inhibitors through high-throughput chemical library screenings, markedly induce EPO/Epo gene expression in epithelium-derived cell lines and mice regardless of oxygen levels. In contrast, mitoxantrone interferes with hypoxia-induced EPO gene expression in Hep3B cells. Cryptic promoters are created for the EPO/Epo gene expression in epithelial cells upon mitoxantrone treatment, and consequently, unique 5'-untranslated regions are generated. The mitoxantrone-induced aberrant transcripts contribute to the reporter protein production in epithelial cells that carry the reporter gene in the proper reading frame of mouse Epo gene. Thus, EPO production in uninjured adult epithelial cells may be a therapeutic approach for renal anemia in patients with CKD.

The role of indoxyl sulfate in renal anemia in patients with chronic kidney disease.

Renal anemia is a common complication in patients with advanced chronic kidney disease. In vitro studies have shown that indoxyl sulfate decreases erythropoietin production. Whether this effect is seen in vivo remains unclear. Our goal was to explore the role of indoxyl sulfate in renal anemia. We found serum indoxyl sulfate levels are significantly and negatively associated with erythropoietin levels in human. A multiple stepwise linear regression analyses after adjustment for other independent parameters revealed that free indoxyl sulfate, and total indoxyl sulfate were significantly associated with erythropoietin levels. In animal studies, erythropoietin gene and protein expression were markedly inhibited in rats with chronic kidney disease; however, this effect was significantly reversed by lowering serum indoxyl sulfate with AST-120. Indoxyl sulfate may also inhibit erythropoietin expression in animal models with chronic kidney disease. These findings further support the role of indoxyl sulfate in the development of renal anemia.

Iron suppresses erythropoietin expression via oxidative stress-dependent hypoxia-inducible factor-2 alpha inactivation

Renal anemia is a major complication in chronic kidney disease (CKD). Iron supplementation, as well as erythropoiesis-stimulating agents, are widely used for treatment of renal anemia. However, excess iron causes oxidative stress via the Fenton reaction, and iron supplementation might damage remnant renal function including erythropoietin (EPO) production in CKD. EPO gene expression was suppressed in mice following direct iron treatment. Hypoxia-inducible factor-2 alpha (HIF-2α), a positive regulator of the EPO gene, was also diminished in the kidney of mice following iron treatment. Anemia-induced increase in renal EPO and HIF-2α expression was inhibited by iron treatment. In in vitro experiments using EPO-producing HepG2 cells, iron stimulation reduced the expression of the EPO gene, as well as HIF-2α. Moreover, iron treatment augmented oxidative stress, and iron-induced reduction of EPO and HIF-2α expression was restored by tempol, an antioxidant compound. HIF-2α interaction with the Epo promoter was inhibited by iron treatment, and was restored by tempol. These findings suggested that iron supplementation reduced EPO gene expression via an oxidative stress-HIF-2α-dependent signaling pathway.

Episomal lentiviral vectors confer erythropoietin expression in dividing cells

Lentiviral vectors are now widely considered as one of the most common gene delivery tools for dividing and non-dividing cells. However, insertional mutagenesis has been found in clinical trials with retroviral vectors, which poses a safety risk. The use of non-integrating lentiviral (NIL) vectors, which avoid integration, eliminates the insertional mutagenesis problem. These NIL vectors are unable to mediate stable gene delivery into dividing cells, which makes them of limited use in the clinical practice of gene therapy. In this study, we constructed a NIL vector which harbors the scaffold/matrix attachment region (S/MAR) sequence and a therapeutic gene. NIL retained episomal erythropoietin (EPO) gene expression for 74days in dividing cells both with and without selection. Furthermore, Southern blot analysis showed that the NIL vector was retained extrachromosomally in CHO cells. In conclusion, the NIL vector based on an S/MAR sequence retained the extrachromosomal expression of a therapeutic gene in dividing cells. Our results show that NIL vectors maybe a safe and effective means of gene delivery, which is of potential clinical significance.

Multi-omic profiling of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production.

Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO-K1 cells under growth-limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO-producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)+, adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT-PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)+ and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post-translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time-course analysis of high- and low-producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Skeletal Muscle Erythropoietin Expression Is Responsive to Hypoxia and Exercise.

Erythropoietin is responsible for regulating the growth and development of red blood cells. Reports conflict on whether skeletal muscle is able to produce erythropoietin and release it into circulation and if exercise affects this. We set out to determine how erythropoietin is regulated in skeletal muscle and to determine whether skeletal muscle-derived erythropoietin can stimulate erythropoiesis. Using an in vitro approach, we exposed proliferating and differentiated skeletal muscle cells to various forms of exercise-induced physiological stimuli and measured erythropoietin gene expression. To understand if skeletal muscle cells were able to stimulate erythropoiesis, independent of other cell types found in skeletal muscle, we used myoblast-conditioned media to treat bone marrow and to measure erythropoiesis through flow cytometry. We also measured erythropoietin expression and hypoxia in mice subjected to an exercise protocol designed to induce skeletal muscle oxygen stress. Hypoxia increased erythropoietin expression in C2C12 myoblasts, myotubes, and primary myoblasts in vitro by 50% to 130%. Bone marrow treated with media conditioned with hypoxic myoblasts for 24 h increased the number of Ter-119-positive cells by 32%. An erythropoietin-neutralizing antibody prevented this increase. Compared with unexercised controls, exhaustive exercise increased skeletal muscle HIF1α levels by 50% and HIF2α levels by 20%. Moreover, exercised skeletal muscle erythropoietin expression was 70% higher. These results demonstrate that skeletal muscle produces erythropoietin in a hypoxia and HIF-dependent manner and that hypoxia-treated muscle is capable of stimulating erythropoiesis in vitro.

The Involvement of Iron on Erythropoietin Expression

BackgroundRenal anemia is often seen in the development of chronic renal failure (CRF). Therefore, Iron supplementation, as well as erythropoiesis‐stimulating agents, are used for treatment of renal anemia. Meanwhile, iron is shown to cause oxidative stress through toxic hydroxyl radical production via iron catalyzed‐Fenton reaction. Therefore, iron supplementation might damage remnant renal function including erythropoietin (EPO) production in CRF. In the present study, we studied iron action on erythropoietin expression.Methods and ResultsIn mice with unilateral ureter obstruction (UUO), EPO gene was reduced, and renal iron content was elevated in kidney of UUO side. Similarly, EPO gene was suppressed in mice with iron treatment. The expression of hypoxia‐inducible factor‐2 alpha (HIF‐2a), a positive regulator of EPO gene, was also diminished in both UUO‐ and iron‐treated kidney. In in vitro experiments using HepG2 cells, iron stimulation reduced the expression of EPO gene, as well as HIF‐2a, in a dose‐dependent manner. Iron also suppressed CoCl2‐induced EPO gene upregulation under hypoxia‐mimicked condition. Moreover, iron treatment augmented oxidative stress production, and iron‐induced reduction of EPO and HIF‐2a expression was restored by an anti‐oxidant agent treatment.ConclusionThese findings suggested that Iron supplementation reduced EPO gene expression through oxidative stress‐HIF‐2a‐dependent manner.

Progression of anemia and its relationship with renal function, blood pressure, and erythropoietin in rats with chronic kidney disease.

In chronic kidney disease (CKD), anemia and hypertension are significant co-morbidities that contribute to cardiovascular and renal disease progression. The purpose of the study was to identify correlations between changes in hematologic variables against markers of renal function, blood pressure, and erythropoietin (EPO) in a naturally occurring hypertensive model of CKD, the Lewis polycystic kidney (LPK) rat. Complete blood count, systolic blood pressure, urea and creatinine concentration, urinary protein to creatinine ratio, and plasma EPO concentration were determined in control Lewis (n = 51) and LPK rats (n = 56) aged 6-24 weeks. Renal EPO gene expression and RBC osmotic fragility were also documented. Hematopoiesis in spleen and bone marrow were assessed. Lewis polycystic kidney rats had increasing urea and creatinine concentrations, concurrent with the development of a nonregenerative normocytic/normochromic anemia and hypertension, with a significant negative correlation between both HGB and HCT with urea concentration and blood pressure (P < .01). HCT was also significantly negatively correlated with creatinine concentration (P = .014). WBC was significantly negatively correlated with urea (P < .01). Plasma EPO concentration was increased and renal EPO mRNA expression was significantly upregulated in LPK animals. The former was significantly positively correlated with blood pressure and platelet count (P < .05). RBC osmotic fragility was normal in LPK rats and there was no evidence for increased RBC elimination or extramedullary hematopoiesis. Marked anemia in the LPK CKD rodent model in the presence of elevated EPO suggests inefficient erythropoiesis that is correlated with plasma urea concentration and blood pressure.

Multi‐omic profiling ­of EPO‐producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

ABSTRACTChinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi‐omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO‐K1 cells under growth‐limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO‐producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)+, adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT‐PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)+ and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post‐translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time‐course analysis of high‐ and low‐producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Iron-hepcidin dysmetabolism, anemia and renal hypoxia, inflammation and fibrosis in the remnant kidney rat model.

Anemia is a common complication of chronic kidney disease (CKD) that develops early and its severity increases as renal function declines. It is mainly due to a reduced production of erythropoietin (EPO) by the kidneys; however, there are evidences that iron metabolism disturbances increase as CKD progresses. Our aim was to study the mechanisms underlying the development of anemia of CKD, as well as renal damage, in the remnant kidney rat model of CKD induced by 5/6 nephrectomy. This model of CKD presented a sustained degree of renal dysfunction, with mild and advanced glomerular and tubulointerstitial lesions. Anemia developed 3 weeks after nephrectomy and persisted throughout the protocol. The remnant kidney was still able to produce EPO and the liver showed an increased EPO gene expression. In spite of the increased EPO blood levels, anemia persisted and was linked to low serum iron and transferrin levels, while serum interleukin (IL)-6 and high sensitivity C-reactive protein (hs-CRP) levels showed the absence of systemic inflammation. The increased expression of duodenal ferroportin favours iron absorption; however, serum iron is reduced which might be due to iron leakage through advanced kidney lesions, as showed by tubular iron accumulation. Our data suggest that the persistence of anemia may result from disturbances in iron metabolism and by an altered activity/function of EPO as a result of kidney cell damage and a local inflammatory milieu, as showed by the increased gene expression of different inflammatory proteins in the remnant kidney. In addition, this anemia and the associated kidney hypoxia favour the development of fibrosis, angiogenesis and inflammation that may underlie a resistance to EPO stimuli and reduced iron availability. These findings might contribute to open new windows to identify putative therapeutic targets for this condition, as well as for recombinant human EPO (rHuEPO) resistance, which occurs in a considerable percentage of CKD patients.

Oligomeric TTR V30M aggregates compromise cell viability, erythropoietin gene expression and promoter activity in the human hepatoma cell line Hep3B

Familial amyloidotic polyneuropathy, ATTRV30M (p. TTRV50M) amyloidosis, is a neurodegenerative disease characterized by systemic extracellular amyloid deposition of a mutant transthyretin, TTR V30M. Anemia, with low erythropoietin (EPO) levels and spared kidney function, affects about 25% of symptomatic patients, suggesting a blockage of EPO-producing cells. Early non-fibrillar TTR aggregates are highly cytotoxic, inducing oxidative stress, the expression of apoptosis-related molecules and secretion of pro-inflammatory cytokines, factors capable of inhibiting EPO production. Low EPO levels in these patients are not related to renal amyloid deposition or the presence of circulating TTR V30M. However, the role of early non-fibrillar TTR aggregates remains unexplored. We used the EPO producing Hep3B human hepatoma cell line to study the effect of TTR oligomeric aggregates on EPO expression. Hep3B cells were incubated with soluble and oligomeric TTR V30M, and cell proliferation as well as caspase 3/7 activation was evaluated. Relative quantification of EPO mRNA transcripts was performed by real-time PCR. Significant reductions in cell viability (13 ± 7.3%) and activation of caspases 3/7 were seen after 24 h in the presence of oligomeric TTR V30M. Also, EPO expression was significantly reduced (50 ± 2.8%), in normoxic conditions. A reporter assay was constructed with a PCR fragment of the EPO promoter linked to the luciferase gene to evaluate the role of transcription factors targeting the promoter. A significant reduction of EPO promoter activity (53 ± 6.5%) was observed in transfected cells exposed to TTR oligomers. Our results show that oligomeric TTR V30M reduces EPO expression, at least in part through inhibition of promoter activity.

Erythropoietin gene expression: developmental-stage specificity, cell-type specificity, and hypoxia inducibility.

Erythrocytes play an essential role in the delivery of oxygen from the lung to every organ; a decrease in erythrocytes (anemia) causes hypoxic stress and tissue damage. To maintain oxygen homeostasis in adult mammals, when the kidney senses hypoxia, it secretes an erythroid growth factor, erythropoietin (Epo), which stimulates erythropoiesis in the bone marrow. Recently, studies using genetically modified mice have shown that the in vivo expression profile of the Epo gene changes dramatically during development. The first Epo-producing cells emerge in the neural crest and neuroepithelium of mid-stage embryos and support primitive erythropoiesis in the yolk sac. Subsequently, Epo from the hepatocytes stimulates erythropoiesis in the fetal liver of later stage embryos in a paracrine manner. In fact, erythroid lineage cells comprise the largest cell population in the fetal liver, and hepatocytes are distributed among the erythroid cell clusters. Adult erythropoiesis in the bone marrow requires Epo that is secreted by renal Epo-producing cells (REP cells). REP cells are widely distributed in the renal cortex and outer medulla. Hypoxia-inducible Epo production both in hepatocytes and REP cells is controlled at the gene transcription level that is mainly mediated by the hypoxia-inducible transcription factor (HIF) pathway. These mouse studies further provide insights into the molecular mechanisms of the cell-type specific, hypoxia-inducible expression of the Epo gene, which involves multiple sets of cis- and trans-regulatory elements.

EPO gene expression induces the proliferation, migrationand invasion of bladder cancer cells through the p21WAF1‑mediated ERK1/2/NF-κB/MMP-9 pathway.

Erythropoietin(EPO) is a cytokine that modulates the production of red blood cells. Previous studies have contradicted the assumed role of EPO in tumor cell proliferation. In the present study, we investigated the effect of EPO in the proliferation, migration and invasion that is involved in the signaling pathways and cell-cycle regulation of bladder cancer 5637 cells. The results showed that an overexpression of the EPO gene has a potent stimulatory effect on DNA synthesis, migration and invasion. EPO gene expression increased the expression of matrix metalloproteinase(MMP)-9 via the binding activity of NF-κB, AP-1 and Sp-1 in 5637 cells. The transfection of 5637 cells with the EPO gene induced the phosphorylation of ERK1/2. Treatment with ERK1/2 inhibitor U0126 significantly inhibited the increased proliferation, migration and invasion of EPO gene-transfected cells. U0126 treatment suppressed the induction of MMP-9 expression through NF-κB binding activity in EPO gene transfectants. In addition, EPO gene expression was correlated with the upregulation of cyclins/CDKs and the upregulation of the CDK inhibitor p21WAF1 expression. Finally, the inhibition of p21WAF1 function by siRNA blocked the proliferation, migration, invasion and phosphorylation of ERK1/2 signaling, as well as MMP-9 expression and activation of NF-κB in EPO gene-transfected cells. These novel findings suggest that the molecular mechanisms of EPO contribute to the progression and development of bladder tumors.

An acetate switch regulates stress erythropoiesis.

Endocrine erythropoietin (Epo), which is synthesized in the kidney or liver of adult mammals, controls erythrocyte production and is regulated by the stress-responsive transcription factor Hypoxia Inducible Factor 2 (HIF-2). We previously reported that the lysine acetyltransferase Cbp is required for HIF-2α acetylation and efficient HIF-2 dependent Epo induction during hypoxia. We now show these processes require acetate-dependent acetyl CoA synthetase 2 (Acss2). In Hep3B hepatoma cells and in Epo-generating organs of hypoxic or acutely anemic mice, acetate levels increase and Acss2 is required for HIF-2α acetylation, Cbp/HIF-2α complex formation and recruitment to the Epo enhancer, and efficient Epo induction. In acutely anemic mice, acetate supplementation augments stress erythropoiesis in an Acss2-dependent manner. In acquired and genetic chronic anemia mouse models, acetate supplementation also increases Epo expression and resting hematocrits. Thus, a mammalian stress-responsive acetate switch controls HIF-2 signaling and Epo induction during pathophysiological states marked by tissue hypoxia.

Translation of the human erythropoietin transcript is regulated by an upstream open reading frame in response to hypoxia

Erythropoietin (EPO) is a key mediator hormone for hypoxic induction of erythropoiesis that also plays important nonhematopoietic functions. It has been shown that EPO gene expression regulation occurs at different levels, including transcription and mRNA stabilization. In this report, we show that expression of EPO is also regulated at the translational level by an upstream open reading frame (uORF) of 14 codons. As judged by comparisons of protein and mRNA levels, the uORF acts as a cis-acting element that represses translation of the main EPO ORF in unstressed HEK293, HepG2, and HeLa cells. However, in response to hypoxia, this repression is significantly released, specifically in HeLa cells, through a mechanism that involves processive scanning of ribosomes from the 5' end of the EPO transcript and enhanced ribosome bypass of the uORF. In addition, we demonstrate that in HeLa cells, hypoxia induces the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) concomitantly with a significant increase of EPO protein synthesis. These findings provide a framework for understanding that production of high levels of EPO induced by hypoxia also involves regulation at the translational level.

A novel distal upstream hypoxia response element regulating oxygen-dependent erythropoietin gene expression

Erythropoietin (Epo) is the main humoral regulator of erythropoiesis. During development, Epo production switches in a species-specific manner from the liver to the kidneys which account for approximately 90% of total Epo synthesis in the adult. Renal Epo is produced by peritubular interstitial

Erythropoietin promotes breast tumorigenesis through tumor-initiating cell self-renewal

Erythropoietin (EPO) is a hormone that induces red blood cell production. In its recombinant form, EPO is the one of most prescribed drugs to treat anemia, including that arising in cancer patients. In randomized trials, EPO administration to cancer patients has been associated with decreased survival. Here, we investigated the impact of EPO modulation on tumorigenesis. Using genetically engineered mouse models of breast cancer, we found that EPO promoted tumorigenesis by activating JAK/STAT signaling in breast tumor-initiating cells (TICs) and promoted TIC self renewal. We determined that EPO was induced by hypoxia in breast cancer cell lines, but not in human mammary epithelial cells. Additionally, we demonstrated that high levels of endogenous EPO gene expression correlated with shortened relapse-free survival and that pharmacologic JAK2 inhibition was synergistic with chemotherapy for tumor growth inhibition in vivo. These data define an active role for endogenous EPO in breast cancer progression and breast TIC self-renewal and reveal a potential application of EPO pathway inhibition in breast cancer therapy.

Effect of ghrelin on renal erythropoietin production in chronic hypoxic rats

Ghrelin, a 28 amino acid peptide, has diverse effects in body organs. Erythropoietin is a key mediator in increasing the red blood cells during hypoxia. Previously, we have shown that ghrelin has a polycythemic effect. In the present study, we evaluated the effect of ghrelin on erythropoietin gene expression with the aim to find out the mechanism of its effect. Thirty two adult male Wistar rats were divided randomly into four groups. The hypoxic condition was induced by placing the rats into the hypoxic chamber with 11% oxygen for two weeks. Saline- and ghrelin-treated control rats remained in room with a regular air conditions. Erythropoietin gene expression was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). Plasma erythropoietin was measured by enzyme linked immunosorbent assay (ELISA). After 2-weeks of hypoxia, erythropoietin transcripts and erythropoietin plasma levels were significantly increased in hypoxic animals compared with control animals. Ghrelin treatment decreased both plasma erythropoietin and erythropoietin gene expression only in the hypoxic rats. Our data indicate that ghrelin might induce polycythemia through an erythropoietin-independent manner. However, to confirm this hypothesis and to find out the precise mechanism of this phenomenon further investigations are needed.

Aging is Associated with Impaired Renal Function, INF-gamma Induced Inflammation and with Alterations in Iron Regulatory Proteins Gene Expression.

Our aim was to contribute to a better understanding of the pathophysiology of anemia in elderly, by studying how aging affects renal function, iron metabolism, erythropoiesis and the inflammatory response, using an experimental animal model. The study was performed in male Wistar, a group of young rats with 2 months age and an old one with 18 months age. Old rats presented a significant higher urea, creatinine, interferon (INF)-gamma, ferritin and soluble transferrin receptor serum levels, as well as increased counts of reticulocytes and RDW. In addition, these rats showed significant lower erythropoietin (EPO) and iron serum levels. Concerning gene expression of iron regulatory proteins, old rats presented significantly higher mRNA levels of hepcidin (Hamp), transferrin (TF), transferrin receptor 2 (TfR2) and hemojuvelin (HJV); divalent metal transporter 1 (DMT1) mRNA levels were significantly higher in duodenal tissue; EPO gene expression was significantly higher in liver and lower in kidney, and the expression of the EPOR was significantly higher in both liver and kidney. Our results showed that aging is associated with impaired renal function, which could be in turn related with the inflammatory process and with a decline in EPO renal production. Moreover, we also propose that aging may be associated with INF-gamma-induced inflammation and with alterations upon iron regulatory proteins gene expression.

Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation

Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance.