Hypoxia-induced Erythropoietin Expression Research Articles

Deficiency of lysophosphatidic acid receptor 3 decreases erythropoietin production in hypoxic mouse kidneys.

Lysophosphatidic acid (LPA) is a lipid mediator with diverse biological functions through its receptors on the cell membrane. As one of the six LPA receptors, LPA receptor 3 (LPAR3) is highly expressed in mouse kidneys, but its physiological function in the kidney has been poorly explored. Wild-type (WT) and Lpar3-/- mice were used to investigate the renal physiological function of LPAR3 under hypoxia. The expression levels of LPA receptors in the kidneys of WT mice with or without exposure to hypoxia (8% O2) were detected by RT‒qPCR. RNA sequencing analysis was performed to identify differences in gene expression profiles between the hypoxic kidneys of WT and Lpar3-/- mice. The effects of LPAR3 deficiency and treatment with the LPAR1/3 inhibitor Ki16425 or the LPAR3 selective agonist 2S-OMPT on erythropoietin (EPO) production in the kidneys of hypoxic mice were determined by RT‒qPCR and ELISAs. The mechanism of LPAR3-mediated regulation of EPO expression was further studied in vivo with mouse models and in vitro with cultured human cells. LPAR3 is the major LPA receptor in mouse kidneys, and its expression is significantly upregulated under hypoxic conditions. RNA sequencing analysis revealed that, compared with WT mice, Lpar3-/- mice presented a significant decrease in hypoxia-induced EPO expression in the kidney, together with reduced plasma EPO levels and lower hematocrit and hemoglobin levels. Hypoxic renal EPO expression in WT mice was diminished by the administration of the LPAR1/3 inhibitor Ki16425 and increased by 2S-OMPT, a selective agonist of LPAR3. Hypoxia-induced HIF-2α accumulation in mouse kidneys was impaired by LPAR3 deficiency. Further studies revealed that the PI3K/Akt pathway participated in the regulation of HIF-2α accumulation and EPO expression by LPAR3 under hypoxic conditions. Our study revealed the role of LPAR3 in promoting the HIF-2α‒EPO axis in hypoxic mouse kidneys, suggesting that the LPA receptor may serve as a novel potential pharmaceutical target to regulate renal EPO production in hypoxia-related situations, such as chronic kidney disease and altitude disease.

Nuclear receptor Rev-erbα role in fine-tuning erythropoietin gene expression

AbstractThe regulation of red blood cell (RBC) homeostasis by erythropoietin (EPO) is critical for O2 transport and maintaining the adequate number of RBCs in vertebrates. Therefore, dysregulation in EPO synthesis results in disease conditions such as polycythemia in the case of excessive EPO production and anemia, which occurs when EPO is inadequately produced. EPO plays a crucial role in treating anemic patients; however, its overproduction can increase blood viscosity, potentially leading to fatal heart failure. Consequently, the identification of druggable transcription factors and their associated ligands capable of regulating EPO offers a promising therapeutic approach to address EPO-related disorders. This study unveils a novel regulatory mechanism involving 2 pivotal nuclear receptors (NRs), Rev-ERBA (Rev-erbα, is a truncation of reverse c-erbAa) and RAR-related orphan receptor A (RORα), in the control of EPO gene expression. Rev-erbα acts as a cell-intrinsic negative regulator, playing a vital role in maintaining erythropoiesis at the correct level. It accomplishes this by directly binding to newly identified response elements within the human and mouse EPO gene promoter, thereby repressing EPO production. These findings are further supported by the discovery that a Rev-erbα agonist (SR9011) effectively suppresses hypoxia-induced EPO expression in mice. In contrast, RORα functions as a positive regulator of EPO gene expression, also binding to the same response elements in the promoter to induce EPO production. Finally, the results of this study revealed that the 2 NRs, Rev-erbα and RORα, influence EPO synthesis in a negative and positive manner, respectively, suggesting that the modulating activity of these 2 NRs could provide a method to target disorders linked with EPO dysregulation.

Exploiting moderate hypoxia to benefit patients with brain disease: Molecular mechanisms and translational research in progress.

Hypoxia is increasingly recognized as an important physiological driving force. A specific transcriptional program, induced by a decrease in oxygen (O2) availability, for example, inspiratory hypoxia at high altitude, allows cells to adapt to lower O2 and limited energy metabolism. This transcriptional program is partly controlled by and partly independent of hypoxia-inducible factors. Remarkably, this same transcriptional program is stimulated in the brain by extensive motor-cognitive exercise, leading to a relative decrease in O2 supply, compared to the acutely augmented O2 requirement. We have coined the term "functional hypoxia" for this important demand-responsive, relative reduction in O2 availability. Functional hypoxia seems to be critical for enduring adaptation to higher physiological challenge that includes substantial "brain hardware upgrade," underlying advanced performance. Hypoxia-induced erythropoietin expression in the brain likely plays a decisive role in these processes, which can be imitated by recombinant human erythropoietin treatment. This article review presents hints of how inspiratory O2 manipulations can potentially contribute to enhanced brain function. It thereby provides the ground for exploiting moderate inspiratory plus functional hypoxia to treat individuals with brain disease. Finally, it sketches a planned multistep pilot study in healthy volunteers and first patients, about to start, aiming at improved performance upon motor-cognitive training under inspiratory hypoxia.

HIF-α Prolyl Hydroxylase Inhibitors and Their Implications for Biomedicine: A Comprehensive Review.

Oxygen is essential for the maintenance of the body. Living organisms have evolved systems to secure an oxygen environment to be proper. Hypoxia-inducible factor (HIF) plays an essential role in this process; it is a transcription factor that mediates erythropoietin (EPO) induction at the transcriptional level under hypoxic environment. After successful cDNA cloning in 1995, a line of studies were conducted for elucidating the molecular mechanism of HIF activation in response to hypoxia. In 2001, cDNA cloning of dioxygenases acting on prolines and asparagine residues, which play essential roles in this process, was reported. HIF-prolyl hydroxylases (PHs) are molecules that constitute the core molecular mechanism of detecting a decrease in the partial pressure of oxygen, or hypoxia, in the cells; they can be called oxygen sensors. In this review, I discuss the process of molecular cloning of HIF and HIF-PH, which explains hypoxia-induced EPO expression; the development of HIF-PH inhibitors that artificially or exogenously activate HIF by inhibiting HIF-PH; and the significance and implications of medical intervention using HIF-PH inhibitors.

JTZ-951, an HIF prolyl hydroxylase inhibitor, suppresses renal interstitial fibroblast transformation and expression of fibrosis-related factors.

Some preceding studies have provided evidence that hypoxia-inducible factor (HIF)-prolyl hydroxylase (PH) inhibitors have therapeutic potential against tubular interstitial fibrosis (TIF). Recently, transformation of renal interstitial fibroblasts (RIFs) into α-smooth muscle actin-positive myofibroblasts with loss of their hypoxia-inducible erythropoietin (EPO) expression has been hypothesized as the central mechanism responsible for TIF with renal anemia (the RIF hypothesis). These reports have suggested that HIF-PH inhibitors may suppress TIF via suppressing transformation of RIFs. However, the direct effect of HIF-PH inhibitors on transformation of RIFs has not been demonstrated because there has been no appropriate assay system. Here, we established a novel in vitro model of the transformation of RIFs. This model expresses key phenotypic changes such as transformation of RIFs accompanied by loss of their hypoxia-inducible EPO expression, as proposed by the RIF hypothesis. Using this model, we demonstrated that JTZ-951, a newly developed HIF-PH inhibitor, stabilized HIF protein in RIFs, suppressed transformation of RIFs, and maintained their hypoxia-inducible EPO expression. JTZ-951 also suppressed the expression of FGF2, FGF7, and FGF18, which are upregulated during transformation of RIFs. Furthermore, expression of Fgf2, Fgf7, and Fgf18 was correlated with TIF in an animal model of TIF. We also demonstrated that not only FGF2, which is a well-known growth-promoting factor, but also FGF18 promoted proliferation of RIFs. These data suggest that JTZ-951 has therapeutic potential against TIF with renal anemia. Furthermore, FGF2, FGF7, and FGF18, which faithfully reflect the anti-TIF effects of JTZ-951, have potential as TIF biomarkers.

Hypoxia and inflammation

Uncontrolled or non-resolving inflammation is central to the pathophysiology of clinically important conditions including inflammatory bowel disease (IBD), psoriasis, atherosclerosis and arthritis. A combination of increased oxygen demand and decreased supply renders the local microenvironment of chronically inflamed tissues oxygen deprived (hypoxic), leading to the expression of a programme of genes that promote adaptation to the hypoxic challenge. This ancient and ubiquitous adaptive transcriptional pathway is governed by a transcription factor termed the hypoxia-inducible factor (HIF). Originally identified in the search for regulators of hypoxia-induced erythropoietin expression and adaptation to high altitude, HIF has been more recently recognized as a major regulator of immune cell function, which is central to the control of immunity and inflammation. Indeed, recent studies have demonstrated that the use of drugs targeting the HIF pathway may be of benefit in the treatment of chronic inflammatory disease.

Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia-inducible factor-2α.

Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood-brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side-effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia-induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12h, TNFα suppressed the hypoxia-induced increase in EPO mRNA expression in a concentration-dependent manner. TNFα inhibition of hypoxia-induced EPO expression was mediated primarily by hypoxia-inducible factor (HIF)-2α rather than HIF-1α. The effects of TNFα in reducing hypoxia-induced upregulation of EPO mRNA expression probably involve destabilization of HIF-2α, which is regulated by the nuclear factor (NF)-κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response.

The volatile anesthetic isoflurane differentially suppresses the induction of erythropoietin synthesis elicited by acute anemia and systemic hypoxemia in mice in an hypoxia-inducible factor-2-dependent manner

Erythropoietin (EPO) is a glycoprotein hormone essential for the regulation of erythroid homeostasis. Although EPO production is prominent in the kidney and liver, its production in the central nervous system has also been detected. Tissue hypoxia due to systemic or local hypoxemia and acute anemia due to blood loss occurs frequently during various clinical settings, leading to a high possibility of elevated plasma EPO levels. However, it is largely unknown whether volatile anesthetic agents affect EPO production elicited by acute hypoxia in vivo. Male C57BL/6N CrSlc mice were exposed to a hypoxic insult as a result of bleeding-related anemia or hypoxemia while they were under anesthetized using various concentrations of isoflurane. EPO protein concentrations were assessed by enzyme-linked immunosorbent assay and mRNA levels were measured by quantitative real-time reverse transcriptase-polymerase chain reaction. Plasma EPO concentration was induced as early as 3h following acute anemic and hypoxemic hypoxia and suppressed by clinically relevant doses of isoflurane in a dose-dependent manner. Anemic hypoxia induced EPO mRNA and protein synthesis in the kidney. In the kidney, isoflurane inhibited EPO induction caused by anemia but not that caused by hypoxemia. On the other hand, in the brain hypoxemia-induced EPO production was suppressed by isoflurane. Finally, qRT-PCR studies demonstrate that isoflurane differentially inhibit HIF-1α and HIF-2α mRNA expression in brain and kidney, indicating the involvement of HIF-2 in the hypoxia-induced EPO expression and inhibition of the induction by isoflurane.

Induction of SENP1 in Endothelial Cells Contributes to Hypoxia-driven VEGF Expression and Angiogenesis

SENP1 (SUMO-specific protease 1) has been shown to be essential for the stability and activity of hypoxia-inducible factor 1 (HIF-1α) under hypoxia conditions. However, it is unknown how SENP1 activation and hypoxia signaling are coordinated in the cellular response to hypoxia. Here, we report the essential role of SENP1 in endothelial cells as a positive regulator of hypoxia-driven VEGF production and angiogenesis. SENP1 expression is increased in endothelial cells following exposure to hypoxia. Silencing of HIF-1α blocks SENP1 expression in cell response to hypoxia. Mutation of the hypoxia response element (HRE) on the Senp1 promoter abolishes its transactivation in response to hypoxia. Moreover, silencing of SENP1 expression decreases VEGF production and abrogates the angiogenic functions of endothelial cell. We also find that the elongated endothelial cells in embryonic brain section and vascular endothelial cells in embryonic renal glomeruli in Senp1(-/-) mice are markedly reduced than those in wild-type. Thus, these results show that hypoxia implies a positive feedback loop mediated by SENP1. This feedback loop is important in VEGF production, which is essential for angiogenesis in endothelial cells.

A Chinese Herbal Decoction Prepared from Radix Astragali and Radix Angelicae Sinensis Induces the Expression of Erythropoietin in Cultured Hep3B Cells

Danggui Buxue Tang (DBT), a Chinese medicinal decoction used commonly for treating women's ailments, contains Radix Astragali (RA) and Radix Angelicae Sinensis (RAS). According to Chinese medicinal theory, this decoction is to nourish the blood function; this, however, has not been demonstrated on the molecular level. In order to reveal the hematopoietic effect of this decoction, DBT was applied to cultured Hep3B human hepatocellular carcinoma cells. The treatment of DBT induced mRNA expression of erythropoietin (EPO) in a dose-dependent manner and peaked at approximately 2.5-fold induction. The secreted EPO in cultured Hep3B cells was quantified by ELISA: the treatment of DBT potentiated the effect of hypoxia-induced EPO expression in the cultured cells. In addition, the DBT-induced EPO expression could be abolished by pre-treatment with U0126, a mitogen-activated kinase inhibitor. The current results verified the hematopoietic function of this ancient herbal decoction.

Interleukin-1beta inhibits the hypoxic inducibility of the erythropoietin enhancer by suppressing hepatocyte nuclear factor-4alpha.

The suppression of hypoxia-induced erythropoietin (EPO) expression by inflammatory cytokines like interleukin-1 (IL-1) contributes to the development of the anemia of chronic disease (ACD). However, the precise mechanism of this suppression is unclear. The 3'-EPO enhancer mediates the transcriptional response to hypoxia by binding several transcription factors, including hypoxia-inducible factor, hepatocyte nuclear factor-4alpha (HNF-4alpha) and chicken ovalbumin upstream promoter transcription factor. We investigated whether IL-1beta inhibits the activity of the 3'-EPO enhancer via HNF-4alpha. IL-1beta inhibited HNF-4alpha mRNA expression and caused proteasome-dependent degradation of HNF-4alpha protein, which resulted in a strongly reduced DNA-binding activity of HNF-4alpha. Reporter gene assays revealed that IL-1beta caused a complete suppression of the hypoxic inducibility of the 3' enhancer via inhibition of HNF-4alpha. We conclude that IL-1beta, at least partially, reduces hypoxia-induced EPO expression by down-regulation of HNF-4alpha.

Erythropoietin receptor signalling is required for normal brain development.

Erythropoietin, known for its role in erythroid differentiation, has been shown to be neuroprotective during brain ischaemia in adult animal models. Although high levels of erythropoietin receptor are produced in embryonic brain, the role of erythropoietin during brain development is uncertain. We now provide evidence that erythropoietin acts to stimulate neural progenitor cells and to prevent apoptosis in the embryonic brain. Mice lacking the erythropoietin receptor exhibit severe anaemia and defective cardiac development, and die at embryonic day 13.5 (E13.5). By E12.5, in addition to apoptosis in foetal liver, endocardium and myocardium, the erythropoietin receptor null mouse shows extensive apoptosis in foetal brain. Lack of erythropoietin receptor affects brain development as early as E10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells from Epor(-/-) mice also exhibited decreases in neuron generation compared with normal controls and increased sensitivity to low oxygen tension with no surviving neurons in Epor(-/-) cortical cultures after 24 hour exposure to hypoxia. The viability of primary Epor(+/+) rodent embryonic cortical neurons was further increased by erythropoietin stimulation. Exposure of these cultures to hypoxia induced erythropoietin expression and a tenfold increase in erythropoietin receptor expression, increased cell survival and decreased apoptosis. Cultures of neuronal progenitor cells also exhibited a proliferative response to erythropoietin stimulation. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain.

Molecular basis of hypoxia-induced erythropoietin expression.

Erythropoietin gene (EPO) expression is activated by tissue hypoxia in renal peritubular interstitial fibroblasts and, to a lesser extent, in hepatocytes and ito cells of the liver. A hypoxia-inducible enhancer spanning approximately 50 bp within the 3'-flanking region of the EPO gene is required for transcriptional activation in hypoxic cells. Hypoxia-inducible factor 1 is a basic helix-loop-helix protein that binds at the 5' end of the enhancer. The binding of hypoxia-inducible factor 1 is absolutely required for enhancer function. Hepatocyte nuclear factor 4 is an orphan receptor that binds at the 3' end of the enhancer. The binding of hepatocyte nuclear factor 4 augments hypoxia-inducible transcription mediated by the enhancer but is not absolutely required for enhancer function. Factors binding to the enhancer may interact synergistically with factors binding to the EPO promoter to activate transcription in hypoxic cells. Indirect evidence suggests that oxygen tension may be sensed by a hemoprotein. In one model, the putative hemoprotein adopts different conformational states depending on whether O2 is bound. Another model proposes that the hemoprotein converts O2 to H2O2. The protein tyrosine kinase c-Src, GTP-binding protein Ras, and MAP kinase signal pathways have been implicated in hypoxia signal transduction, but no direct evidence links these pathways to EPO transcriptional activation.