The hypoxia inducible factor/erythropoietin (EPO)/EPO receptor pathway is disturbed in a rat model of chronic kidney disease related anemia.

OBJECTIVES: Anemia is a known driver for hypoxia inducible factor (HIF) which leads to increased renal erythropoietin (EPO) synthesis. Bone marrow (BM) EPO receptor (EPOR) signals are transduced through a JAK2-STAT5 pathway. The origins of anemia of chronic kidney disease (CKD) are multifactorial, including impairment of both renal EPO synthesis as well as intestinal iron absorption. We investigated the HIF- EPO- EPOR axis in kidney, BM and proximal tibia in anemic juvenile CKD rats. METHODS: CKD was induced by 5/6 nephrectomy in young (20 days old) male Sprague-Dawley rats while C group was sham operated. Rats were sacrificed 4 weeks after CKD induction and 5 minutes after a single bolus of IV recombinant human EPO. An additional control anemic (C-A) group was daily bled for 7 days. RESULTS: Hemoglobin levels were similarly reduced in CKD and C-A (11.4 ± 0.3 and 10.8±0.2 Vs 13.5±0.3 g/dL in C, p<0.0001). Liver hepcidin mRNA was decreased in CA but increased in CKD. Serum iron was unchanged while transferrin levels were mildly decreased in CKD. Kidney HIF2α protein was elevated in C-A but unchanged in CKD. Kidney EPO protein and mRNA levels were unchanged between groups. However, BM EPO protein (which reflects circulating EPO) was increased in C-A but remained unchanged in CKD. BM and proximal tibia EPOR were unchanged in C-A but decreased in CKD. Proximal tibial phospho-STAT5 increased after the EPO bolus in C but not in CKD. CONCLUSIONS: Compared to blood loss, anemia in young CKD rats is associated with inappropriate responses in the HIF-EPO-EPO-R axis: kidney HIF2α and renal EPO are not increased, BM and bone EPOR levels, as well as bone pSTAT5 response to EPO are reduced. Thus, anemia of CKD may be treated with additional therapeutic avenues beyond iron and EPO supplementation.

EPOR2/ßcR2-independendent effects of low-dose epoetin-α in porcine liver transplantation.

Ischemia-reperfusion injury (IRI) remains a key component of graft damage during transplantation. Erythropoietin (EPO) induces anti-inflammatory and anti-apoptotic effects via the EPOR2/ßcR2 complex, with a potential risk of thrombosis. Previous work indicates that EPO has EPOR2/ßcR2-independent protective effects via direct effects on the endothelium. As the EPOR2/ßcR2 receptor has a very low affinity for EPO, we aimed to test the hypothesis that EPO doses below the level that stimulate this receptor elicit cytoprotective effects via endothelial stimulation in a porcine liver transplantation model. Landrace pigs underwent allogenic liver transplantation (follow-up: 6 h) with a portojugular shunt. Animals were divided into two groups: donor and recipient treatment with low-dose EPO (65 IU/kg) or vehicle, administered 6 h before cold perfusion and 30 min after warm reperfusion. Fourteen of 17 animals (82.4%) fulfilled the inclusion criteria. No differences were noted in operative values between the groups including hemoglobin, cold or warm ischemic time. EPO-treated animals showed a significantly lower histopathology score, reduced apoptosis, oxidative stress, and most important a significant up-regulation of endothelial nitric oxide (NO) synthase (eNOS). Donor and recipient treatment with low-dose EPO reduces the hepatic IRI via EPOR2/ßcR2-independent cytoprotective mechanisms and represents a clinically applicable way to reduce IRI.

Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL.

OBJECTIVE: The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. MATERIALS AND METHODS: hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. RESULTS: During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. CONCLUSION: This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.

Optimization and utilization of the SureFire phospho-STAT5 assay for a cell-based screening campaign.

The family of signal transducers and activators of transcription (STATs) consists of seven transcription factors that respond to a variety of cytokines, hormones, and growth factors. STATs are activated by tyrosine phosphorylation, which results in their dimerization and translocation into the nucleus where they exert their effect on transcription of regulated target genes. The phosphorylation of STATs is mediated mainly by Janus kinases (JAKs). The JAK/STAT pathway plays a critical role in hematopoietic and immune cell function. Here we focus on one member of the STAT family, STAT5. STAT5 is phosphorylated by several JAKs, including Jak3, Jak2, and Tyk2, in response to interleukin-2, erythropoietin (EPO), and interleukin-22, respectively. Activation of STAT5 is essential to T cell development and has been associated with hematologic malignancies. Therefore, the ability to assess STAT5 phosphorylation is important for discovery efforts targeting these indications. The assay formats available to detect phosphorylated STAT5 (pSTAT5) are relatively low throughput and involve lengthy protocols. These formats include western blot analysis, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The SureFire (Perkin Elmer, Waltham, MA) pSTAT5 assay is a homogeneous assay that utilizes AlphaScreen (Perkin Elmer) technology to detect pSTAT5 in cell lysates. We have used this assay format to evaluate EPO-induced STAT5 phosphorylation in HEL cells and successfully complete a small-scale screening campaign to identify inhibitors of this event. The results obtained in these studies demonstrate that the SureFire pSTAT5 assay is a robust, reliable assay format that is amenable to high-throughput screening (HTS) applications.

Jak2 FERM domain interaction with the erythropoietin receptor regulates Jak2 kinase activity.

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement.

[Possible role of soluble erythropoietin receptors in renal anemia].

Recombinant human erythropoietin(rHuEpo) is effective for the treatment of renal anemia associated with chronic renal failure(CRF). However, we have encountered some patients with CRF who have sometimes developed a resistance to rHuEpo. This resistance can be due to iron or folate deficiency, aluminum toxicity, hyperparathyroidism, or auto-antibodies for rHuEpo. In this study, we focused on the soluble erythropoietin receptor(sEpoR), which can bind to rHuEpo. To demonstrate the possibility that the sweeping of rHuEpo by sEpoR results in resistance to rHuEpo, we performed a bioassay using the rHuEpo-dependent cell line, UT7/EPO. The results showed that recombinant mouse sEpoR(rmsEpoR) can reduce the proliferation of UT7/EPO induced by rHuEpo in a dose-dependent manner. We consider that this cell line could be a useful tool in a bioassay to detect the inhibitory factor(s) against Epo. We selected sera from three groups of patients with renal anemia associated with CRF who were receiving hemodialysis three times a week: the first was a patient group that needed a high dose of rHuEpo(7,500-9,000 unit/dialysis), the second was a patient group that needed an intermediate dose of rHuEpo (4,500 unit/dialysis), the third was a patient group that needed a low dose of rHuEpo(below 1,500 unit/dialysis). Interestingly, the proliferation of UT7/EPO determined with [3H]-thymidine incorporation was reduced by the addition of sera from the first group, but not by the addition of sera from the third group. These results suggested that serum sEpoR may play an important role in signal transduction via EpoR on erythroid progenitor in CRF patients.

Identification of the human erythropoietin receptor region required for Stat1 and Stat3 activation.

Signal transducers and activators of transcription (Stat) proteins play important roles in the regulation of hematopoiesis as downstream molecules of cytokine signal transduction. It was previously demonstrated that erythropoietin (EPO), a major regulator of erythropoiesis, activates 3 different Stat members, Stat1, Stat3, and Stat5, in a human EPO-dependent cell line, UT-7/EPO. To clarify the mechanism by which EPO activates Stat1 and Stat3 via the EPO receptor (EPOR), a series of chimeric receptors was constructed bearing the extracellular domain of the granulocyte colony-stimulating factor receptor linked to the transmembrane domain of EPOR and the full length or several mutants of the cytoplasmic domain of EPOR, and these chimeric receptor complementary DNAs were introduced into UT-7/EPO cells. Tyr432 on human EPOR was important for activation of Stat1 and Stat3 and c-myc gene induction. In addition, Jak2 and Fes tyrosine kinases were involved in EPO-induced activation of Stat1 and Stat3. These results indicate that Stat1 and Stat3 are activated by EPO via distinct mechanisms from Stat5.

Characterization of the domains of the erythropoietin receptor necessary for induction of cell growth and differentiation.

To define the cytoplasmic region(s) of the erythropoietin receptor (EpoR) necessary for promotion of growth and induction of differentiation, mutated EpoR cDNAs containing truncations and conversions of tyrosine residues to phenylalanines were generated. Constructs were introduced into IL-3 dependent Ba/F3 cells by electroporation, and individual transfectants were propagated in methylcellulose-containing medium in the presence of erythropoietin (Epo). Truncated receptor at amino acid 350 was partially deficient in promoting cellular growth and the receptor lacking the box 2 region was not capable of inducing cellular growth. When a large number of cells were screened in Epo-containing liquid medium, clones arose in which Epo-dependent growth was due to activation of the endogenous EpoR gene. Analysis of the degree of Epo-dependent differentiation of the transfectants, based on the steady-state levels of beta major-globin mRNA, showed that the carboxyl terminal 133 amino acids and tyrosyl residues located at positions 429/431 and 460/464 were not necessary for the induction of differentiation. Examination of clones from diverse origins demonstrated that an inverse relationship existed between the rate of Epo-induced cellular replication and the degree of Epo-induced differentiation.