Erythropoietin receptor signal is crucial for periodontal ligament stem cell-based tissue reconstruction in periodontal disease.

Alveolar bone loss caused by periodontal disease eventually leads to tooth loss. Periodontal ligament stem cells (PDLSCs) are the tissue-specific cells for maintaining and repairing the periodontal ligament, cementum, and alveolar bone. Here, we investigated the role of erythropoietin receptor (EPOR), which regulates the microenvironment-modulating function of mesenchymal stem cells, in PDLSC-based periodontal therapy. We isolated PDLSCs from patients with chronic periodontal disease and healthy donors, referred to as PD-PDLSCs and Cont-PDLSCs, respectively. PD-PDLSCs exhibited reduced potency of periodontal tissue regeneration and lower expression of EPOR compared to Cont-PDLSCs. EPOR-silencing suppressed the potency of Cont-PDLSCs mimicking PD-PDLSCs, whereas EPO-mediated EPOR activation rejuvenated the reduced potency of PD-PDLSCs. Furthermore, we locally transplanted EPOR-silenced and EPOR-activated PDLSCs into the gingiva around the teeth of ligament-induced periodontitis model mice and demonstrated that EPOR in PDLSCs participated in the regeneration of the periodontal ligament, cementum, and alveolar bone in the ligated teeth. The EPOR-mediated paracrine function of PDLSCs maintains periodontal immune suppression and bone metabolic balance via osteoclasts and osteoblasts in the periodontitis model mice. Taken together, these results suggest that EPOR signaling is crucial for PDLSC-based periodontal regeneration and paves the way for the development of novel options for periodontal therapy.

Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry.

BACKGROUND: Calcium (Ca2+) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN. METHODS: Intracellular Ca2+ levels were measured over a time frame of 0-1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca2+ concentrations were assessed from 0-108 s. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution, measuring Ca2+ levels from 109-594 s (store depletion). Then, 2 mM of Ca2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca2+ levels were measured from 595-1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann-Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test. RESULTS: Following EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells. CONCLUSIONS: This report highlights the impact of JAK2 and CALR mutations on Ca2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells.

Molecular basis of JAK2 activation in erythropoietin receptor and pathogenic JAK2 signaling.

Janus kinase 2 (JAK2) mediates type I/II cytokine receptor signaling, but JAK2 is also activated by somatic mutations that cause hematological malignancies by mechanisms that are still incompletely understood. Quantitative superresolution microscopy (qSMLM) showed that erythropoietin receptor (EpoR) exists as monomers and dimerizes upon Epo stimulation or through the predominant JAK2 pseudokinase domain mutations (V617F, K539L, and R683S). Crystallographic analysis complemented by kinase activity analysis and atomic-level simulations revealed distinct pseudokinase dimer interfaces and activation mechanisms for the mutants: JAK V617F activity is driven by dimerization, K539L involves both increased receptor dimerization and kinase activity, and R683S prevents autoinhibition and increases catalytic activity and drives JAK2 equilibrium toward activation state through a wild-type dimer interface. Artificial intelligence-guided modeling and simulations revealed that the pseudokinase mutations cause differences in the pathogenic full-length JAK2 dimers, particularly in the FERM-SH2 domains. A detailed molecular understanding of mutation-driven JAK2 hyperactivation may enable novel therapeutic approaches to selectively target pathogenic JAK2 signaling.

Hemolysis-driven IFNα production impairs erythropoiesis by negatively regulating EPO signaling in sickle cell disease.

ABSTRACT: Disordered erythropoiesis is a feature of many hematologic diseases, including sickle cell disease (SCD). However, very little is known about erythropoiesis in SCD. Here, we show that although bone marrow (BM) erythroid progenitors and erythroblasts in Hbbth3/+ thalassemia mice were increased more than twofold, they were expanded by only ∼40% in Townes sickle mice (SS). We further show that the colony-forming ability of SS erythroid progenitors was decreased and erythropoietin (EPO)/EPO receptor (EPOR) signaling was impaired in SS erythroid cells. Furthermore, SS mice exhibited reduced responses to EPO. Injection of mice with red cell lysates or hemin, mimicking hemolysis in SCD, led to suppression of erythropoiesis and reduced EPO/EPOR signaling, indicating hemolysis, a hallmark of SCD, and could contribute to the impaired erythropoiesis in SCD. In vitro hemin treatment did not affect Stat5 phosphorylation, suggesting that hemin-induced erythropoiesis suppression in vivo is via an indirect mechanism. Treatment with interferon α (IFNα), which is upregulated by hemolysis and elevated in SCD, led to suppression of mouse BM erythropoiesis in vivo and human erythropoiesis in vitro, along with inhibition of Stat5 phosphorylation. Notably, in sickle erythroid cells, IFN-1 signaling was activated and the expression of cytokine inducible SH2-containing protein (CISH), a negative regulator of EPO/EPOR signaling, was increased. CISH deletion in human erythroblasts partially rescued IFNα-mediated impairment of cell growth and EPOR signaling. Knocking out Ifnar1 in SS mice rescued the defective BM erythropoiesis and improved EPO/EPOR signaling. Our findings identify an unexpected role of hemolysis on the impaired erythropoiesis in SCD through inhibition of EPO/EPOR signaling via a heme-IFNα-CISH axis.

Erythropoietin receptor is a risk factor for prognosis: A potential biomarker in lung adenocarcinoma.

Lung cancer has the highest mortality rate of all cancers, and LUAD's survival rate is particularly poor. Erythropoietin receptor (EPOR) can be detected in lung adenocarcinoma (LUAD), however, the expression levels and prognostic value of EPOR in LUAD are still unclear. In our study, clinicopathological data of 92 LUAD patients between January 2008 and June 2016, multiple bioinformatics databases and immunohistochemistry were used to explore the EPOR expression, the mutant genes affecting EPOR expression, and the correlation of EPOR expression with oxidative stress - related genes, prognosis, immune microenvironment. All statistical analyses were performed in the R version 4.1.1. The study found that EPOR expression might be down-regulated at the mRNA levels and significantly up-regulated at the protein levels in LUAD, which indicates that the mRNA and protein levels of EPOR are inconsistent. The muTarget showed that the expression of EPOR was significantly different between the mutant group and the wild group of 15 genes, including DDX60L and C1orf168. Importantly, we found that EPOR was associated with VEGF and HIF family members, and had significant positive correlation with oxidative stress - related genes such as CCS, EPX and TXNRD2. This suggests that EPOR may be involved in the regulation of oxidative stress. The Kaplan-Meier Plotter and PrognoScan databases consistently concluded that EPOR was associated with prognosis in LUAD patients. Our clinicopathological data showed that high EPOR expression was associated with poorer overall survival (29.5 vs 46 months) and had a good predictive ability for 4-year and 5-year survival probability. EPOR is expected to be a potential new prognostic marker for LUAD.

Elevated erythroferrone distinguishes erythrocytosis with inherited defects in oxygen-sensing pathway from primary familial and congenital polycythaemia.

Congenital erythrocytoses represent a heterogenous group of rare defects of erythropoiesis characterized by elevated erythrocyte mass. We performed molecular-genetic analysis of 21 Czech patients with congenital erythrocytosis and assessed the mutual link between chronic erythrocyte overproduction and iron homoeostasis. Causative mutations in erythropoietin receptor (EPOR), hypoxia-inducible factor 2 alpha (HIF2A) or Von Hippel-Lindau (VHL) genes were detected in nine patients, including a novel p.A421Cfs*4 EPOR and a homozygous intronic c.340+770T>C VHL mutation. The association and possible cooperation of five identified missense germline EPOR or Janus kinase 2 (JAK2) variants with other genetic/non-genetic factors in erythrocytosis manifestation may involve variants of Piezo-type mechanosensitive ion channel component 1 (PIEZO1) or Ten-eleven translocation 2 (TET2), but this requires further research. In two families, hepcidin levels appeared to prevent or promote phenotypic expression of the disease. No major contribution of heterozygous haemochromatosis gene (HFE) mutations to the erythrocytic phenotype or hepcidin levels was observed in our cohort. VHL- and HIF2A-mutant erythrocytosis showed increased erythroferrone and suppressed hepcidin, whereas no overproduction of erythroferrone was detected in other patients regardless of molecular defect, age or therapy. Understanding the interplay between iron metabolism and erythropoiesis in different subgroups of congenital erythrocytosis may improve current treatment options.

Protection of insect neurons by erythropoietin/CRLF3-mediated regulation of pro-apoptotic acetylcholinesterase.

Cytokine receptor-like factor 3 (CRLF3) is a conserved but largely uncharacterized orphan cytokine receptor of eumetazoan animals. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin. To identify mechanisms of CRLF3-mediated neuroprotection we studied the expression and proapoptotic function of acetylcholinesterase in insect neurons. We exposed primary brain neurons from Tribolium castaneum to apoptogenic stimuli and dsRNA to interfere with acetylcholinesterase gene expression and compared survival and acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increased apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in normal and apoptogenic conditions. Pharmacological inhibition of acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevented hypoxia-induced apoptosis. Activation of CRLF3 with protective concentrations of erythropoietin prevented the increased expression of acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of erythropoietin that cause neuronal death induced ace-1 expression and hence promoted apoptosis. Our study confirms the general proapoptotic function of AChE, assigns a role of both ace-1 and ace-2 in the regulation of apoptotic death and identifies the erythropoietin/CRLF3-mediated prevention of enhanced acetylcholinesterase expression under apoptogenic conditions as neuroprotective mechanism.

Erythropoietin Receptor (EPOR) Signaling in the Osteoclast Lineage Contributes to EPO-Induced Bone Loss in Mice.

Erythropoietin (EPO) is a pleiotropic cytokine that classically drives erythropoiesis but can also induce bone loss by decreasing bone formation and increasing resorption. Deletion of the EPO receptor (EPOR) on osteoblasts or B cells partially mitigates the skeletal effects of EPO, thereby implicating a contribution by EPOR on other cell lineages. This study was designed to define the role of monocyte EPOR in EPO-mediated bone loss, by using two mouse lines with conditional deletion of EPOR in the monocytic lineage. Low-dose EPO attenuated the reduction in bone volume (BV/TV) in Cx3cr1Cre EPORf/f female mice (27.05%) compared to controls (39.26%), but the difference was not statistically significant. To validate these findings, we increased the EPO dose in LysMCre model mice, a model more commonly used to target preosteoclasts. There was a significant reduction in both the increase in the proportion of bone marrow preosteoclasts (CD115+) observed following high-dose EPO administration and the resulting bone loss in LysMCre EPORf/f female mice (44.46% reduction in BV/TV) as compared to controls (77.28%), without interference with the erythropoietic activity. Our data suggest that EPOR in the monocytic lineage is at least partially responsible for driving the effect of EPO on bone mass.

Identification of Two Novel EPOR Gene Variants in Primary Familial Polycythemia: Case Report and Literature Review.

Primary familial and congenital polycythemia is a rare disease characterized by an increase in red cell mass that may be due to pathogenic variants in the EPO receptor (EPOR) gene. To date, 33 genetic variants have been reported to be associated. We analyzed the presence of EPOR variants in two patients with polycythemia in whom JAK2 pathogenic variants had been previously discarded. Molecular analysis of the EPOR gene was performed by Sanger sequencing of the coding regions and exon/intron boundaries of exon 8. We performed in vitro culture of erythroid progenitor cells. Segregation studies were done whenever possible. The two patients studied showed hypersensitivity to EPO in in vitro cultures. Analysis of the EPOR gene unveiled two novel pathogenic variants. Genetic testing of asymptomatic relatives could guarantee surveillance and proper management.

Effect of Erythropoietin on Mononuclear Cells of the Bone Marrow and Spleen.

We studied the effect of preconditioning of human bone marrow mononuclear cells with erythropoietin on the immunophenotype of immunocompetent cells and paracrine activity of mouse splenocytes. The expression of erythropoietin receptors on immunocompetent human bone marrow cells was shown to change after a short-term (60 min) exposure to erythropoietin. The number of T helpers carrying erythropoietin receptors decreased and the number of T suppressors, B lymphocytes, and monocytes carrying erythropoietin receptors increased. The presence of 30% conditioned medium from human bone marrow mononuclear cells or 33.4 U/ml of erythropoietin reduced apoptosis/necrosis, increased intracellular activity of NADPH-dependent oxidoreductases of splenocytes, and did not affect oxidative phosphorylation (did not enhance lactate production and glucose uptake by cells).

Epo-IGF1R cross talk expands stress-specific progenitors in regenerative erythropoiesis and myeloproliferative neoplasm.

We found that in regenerative erythropoiesis, the erythroid progenitor landscape is reshaped, and a previously undescribed progenitor population with colony-forming unit-erythroid (CFU-E) activity (stress CFU-E [sCFU-E]) is expanded markedly to restore the erythron. sCFU-E cells are targets of erythropoietin (Epo), and sCFU-E expansion requires signaling from the Epo receptor (EpoR) cytoplasmic tyrosines. Molecularly, Epo promotes sCFU-E expansion via JAK2- and STAT5-dependent expression of IRS2, thus engaging the progrowth signaling from the IGF1 receptor (IGF1R). Inhibition of IGF1R and IRS2 signaling impairs sCFU-E cell growth, whereas exogenous IRS2 expression rescues cell growth in sCFU-E expressing truncated EpoR-lacking cytoplasmic tyrosines. This sCFU-E pathway is the major pathway involved in erythrocytosis driven by the oncogenic JAK2 mutant JAK2(V617F) in myeloproliferative neoplasm. Inability to expand sCFU-E cells by truncated EpoR protects against JAK2(V617F)-driven erythrocytosis. In samples from patients with myeloproliferative neoplasm, the number of sCFU-E-like cells increases, and inhibition of IGR1R and IRS2 signaling blocks Epo-hypersensitive erythroid cell colony formation. In summary, we identified a new stress-specific erythroid progenitor cell population that links regenerative erythropoiesis to pathogenic erythrocytosis.

Developmental changes in iron metabolism and erythropoiesis in mice with human gain-of-function erythropoietin receptor.

Iron availability for erythropoiesis is controlled by the iron-regulatory hormone hepcidin. Increased erythropoiesis negatively regulates hepcidin synthesis by erythroferrone (ERFE), a hormone produced by erythroid precursors in response to erythropoietin (EPO). The mechanisms coordinating erythropoietic activity with iron homeostasis in erythrocytosis with low EPO are not well defined as exemplified by dominantly inherited (heterozygous) gain-of-function mutation of human EPO receptor (mtHEPOR) with low EPO characterized by postnatal erythrocytosis. We previously created a mouse model of this mtHEPOR that develops fetal erythrocytosis with a transient perinatal amelioration of erythrocytosis and its reappearance at 3-6 weeks of age. Prenatally and perinatally, mtHEPOR heterozygous and homozygous mice (differing in erythrocytosis severity) had increased Erfe transcripts, reduced hepcidin, and iron deficiency. Epo was transiently normal in the prenatal life; then decreased at postnatal day 7, and remained reduced in adulthood. Postnatally, hepcidin increased in mtHEPOR heterozygotes and homozygotes, accompanied by low Erfe induction and iron accumulation. With aging, the old, especially mtHEPOR homozygotes had a decline of erythropoiesis, myeloid expansion, and local bone marrow inflammatory stress. In addition, mtHEPOR erythrocytes had a reduced lifespan. This, together with reduced iron demand for erythropoiesis, due to its age-related attenuation, likely contributes to increased iron deposition in the aged mtHEPOR mice. In conclusion, the erythroid drive-mediated inhibition of hepcidin production in mtHEPOR mice in the prenatal/perinatal period is postnatally abrogated by increasing iron stores promoting hepcidin synthesis. The differences observed in studied characteristics between mtHEPOR heterozygotes and homozygotes suggest dose-dependent alterations of downstream EPOR stimulation.

Amplified EPOR/JAK2 Genes Define a Unique Subtype of Acute Erythroid Leukemia.

Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia characterized by prominent erythroid proliferation whose molecular basis is poorly understood. To elucidate the underlying mechanism of erythroid proliferation, we analyzed 121 AEL using whole-genome, whole-exome, and/or targeted-capture sequencing, together with transcriptome analysis of 21 AEL samples. Combining publicly available sequencing data, we found a high frequency of gains and amplifications involving EPOR/JAK2 in TP53-mutated cases, particularly those having >80% erythroblasts designated as pure erythroid leukemia (10/13). These cases were frequently accompanied by gains and amplifications of ERG/ETS2 and associated with a very poor prognosis, even compared with other TP53-mutated AEL. In addition to activation of the STAT5 pathway, a common feature across all AEL cases, these AEL cases exhibited enhanced cell proliferation and heme metabolism and often showed high sensitivity to ruxolitinib in vitro and in xenograft models, highlighting a potential role of JAK2 inhibition in therapeutics of AEL. SIGNIFICANCE: This study reveals the major role of gains, amplifications, and mutations of EPOR and JAK2 in the pathogenesis of pure erythroleukemia. Their frequent response to ruxolitinib in patient-derived xenograft and cell culture models highlights a possible therapeutic role of JAK2 inhibition for erythroleukemia with EPOR/JAK2-involving lesions. This article is highlighted in the In This Issue feature, p. 369.

Liver kinase B1 (LKB1) in murine erythroid progenitors modulates erythropoietin setpoint in association with maturation control.

Erythropoiesis is a tightly regulated process. It is stimulated by decreased oxygen in circulation, which leads to the secretion of the hormone erythropoietin (Epo) by the kidneys. An additional layer of control involves the coordinated sensing and use of nutrients. Much cellular machinery contributes to sensing and responding to nutrient status in cells, and one key participant is the kinase LKB1. The current study examines the role of LKB1 in erythropoiesis using a murine in vivo and ex vivo conditional knockout system. In vivo analysis showed erythroid loss of LKB1 to be associated with a robust increase in serum Epo and mild reticulocytosis. Despite these abnormalities, no evidence of anemia or hemolysis was found. Further characterization using an ex vivo progenitor culture assay demonstrated accelerated erythroid maturation in the LKB1-deficient cells. Based on pharmacologic evidence, this phenotype appeared to result from impaired AMP-activated protein kinase (AMPK) signaling downstream of LKB1. These findings reveal a role for LKB1 in fine-tuning Epo-driven erythropoiesis in association with maturational control.

Erythropoietin promotes the differentiation of fetal neural stem cells into glial cells via the erythropoietin receptor-ß common receptor/Syne-1/H3K9me3 pathway.

AIMS: To investigate the effect of erythropoietin (EPO) on the differentiation of neural stem cells (NSCs)/neural progenitors (NPs) in the treatment of hypoxic-ischemic injury and its potential mechanisms. METHODS: Fetal NSCs/NPs were treated with EPO after oxygen and glucose deprivation/reoxygenation (OGD/R). Cell viability, proliferation, and differentiation of NSCs/NPs were detected by CellTiter-Glo, Edu assay, flow cytometry, and quantitative real-time PCR (qPCR). Immunofluorescence staining, co-immunoprecipitation (Co-IP), and western blotting were used to test the existence of EPO receptor/ß common receptor (EPOR/ßCR) heterodimer on NSCs/NPs and the possible pathway. RESULTS: EPO treatment at different time points increased cell viability without affecting proliferation. EPO treatment immediately after OGD/R promoted oligodendrocyte and astrocyte differentiation, while decreasing neuronal differentiation of NSCs/NPs. EPOR/ßCR heterodimer existed on the cell surface of the fetal cortical NSCs/NPs, EPO treatment significantly increased the mRNA expression of ßCR and elevated the correlation between EPOR and ßCR levels. In addition, mass spectrometry analysis identified Syne-1 as a downstream signaling molecule of the EPOR/ßCR heterodimer. Immunofluorescence staining and western blotting indicated that the ßCR/Syne-1/H3K9me3 pathway was possibly involved in the differentiation of fetal neural stem cells into the glial cell effect of EPO. CONCLUSION: EPO treatment immediately after OGD/R could not facilitate fetal NSCs/NPs neurogenesis but promoted the formation of the EPOR/ßCR heterodimer on fetal NSCs/NPs, which mediates its function in glial differentiation.

Novel mutations in EPO-R and oxygen-dependent degradation (ODD) domain of EPAS1 genes-a causative reason for Congenital Erythrocytosis.

Congenital Erythrocytosis (CE) can be primary or secondary due to the mutations in genes involved in the erythropoietin receptor and oxygen sensing pathway. In this study, 42 patients with 38 unrelated patients and one family (4 patients) who were JAK-2 mutation (both exon 12 and exon 14) negative with high haematocrit values were investigated. The Endogenous Erythroid colony (EEC) assay was performed in all patients, interestingly EEC colonies were high in EPAS1 and EPOR mutated patients compared to non-mutated patients. The sequence analysis of EPAS1 (exon 12), EPO-R (exon-8), VHL (exon-3), and EGLN1 (exon-1) genes in all these patients showed 19% of patients (8/42) had mutations, in exon12 of EPAS1 and exon 8 of EPO-R genes. Two novel missense mutations MW_600850:c.1183G>C, MW_600851:c.1028A>C in EPO-R gene were observed in the study group. One new MW_600849:c.1969C>T nonsense mutation and five MW_619914:c.1715A>G, MW_619915:c.1694G>T, MW_619916:c.1634T>C, MW_600852:c.1771C>G, MW_600848:c.1859G>A novel missense mutations were observed in the EPAS1 gene. Among them, 4 mutations p. (Gln572Arg), p. (Ser565Ile), p. (Ile545Thr), p. (Gln591Glu) in the ODD (Oxygen-dependent degradation) domain of HIF2α, all these variations contributed to the formation of non-functional HIF2α. No mutations were observed in VHL and EGLN1 genes. Using in silico analysis we observed that these mutations contributed to major conformational changes in the HIF2α protein making it non-functional. The mutations in the EPAS1 gene were heterozygous and show autosomal dominant inheritance patterns and we observed in one family. These novel mutations in the EPAS1 (75% (6/8)) and 25% (2/8) EPO-R genes correlating with EEC positivity were observed for the first time in India in CE patients.

Erythroid-specific inactivation of Slc12a6/Kcc3 by EpoR promoter-driven Cre expression reduces K-Cl cotransport activity in mouse erythrocytes.

Investigation of erythrocytes from spontaneous or engineered germ-line mutant mice has been instrumental in characterizing the physiological functions of components of the red cell cytoskeleton and membrane. However, the red blood cell expresses some proteins whose germline loss-of-function is embryonic-lethal, perinatal-lethal, or confers reduced post-weaning viability. Promoter regions of erythroid-specific genes have been used to engineer erythroid-specific expression of Cre recombinase. Through breeding with mice carrying appropriately spaced insertions of loxP sequences, generation of erythroid-specific knockouts has been carried out for signaling enzymes, transcription factors, peptide hormones, and single transmembrane span signaling receptors. We report here the use of Cre recombinase expression driven by the erythropoietin receptor (EpoR) promoter to generate EpoR-Cre;Kcc3f/f mice, designed to express erythroid-specific knockout of the KCC3 K-Cl cotransporter encoded by Kcc3/Slc12A6. We confirm KCC3 as the predominant K-Cl cotransporter of adult mouse red cells in mice with better viability than previously exhibited by Kcc3-/- germline knockouts. We demonstrate roughly proportionate preservation of K-Cl stimulation by hypotonicity, staurosporine, and urea in the context of reduced, but not abrogated, K-Cl function in EpoR-Cre;Kcc3f/f mice. We also report functional evidence suggesting incomplete recombinase-mediated excision of the Kcc3 gene in adult erythroid tissues.

An inherited gain-of-function risk allele in EPOR predisposes to familial JAK2V617F myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are mainly sporadic but inherited variants have been associated with higher risk development. Here, we identified an EPOR variant (EPORP488S ) in a large family diagnosed with JAK2V617F -positive polycythaemia vera (PV) or essential thrombocytosis (ET). We investigated its functional impact on JAK2V617F clonal amplification in patients and found that the variant allele fraction (VAF) was low in PV progenitors but increase strongly in mature cells. Moreover, we observed that EPORP488S alone induced a constitutive phosphorylation of STAT5 in cell lines or primary cells. Overall, this study points for searching inherited-risk alleles affecting the JAK2/STAT pathway in MPN.