EPO Modulation of Thioredoxin Interacting Protein (TXNIP), and Key Roles for TXNIP during EPO-Dependent Human Erythroid Progenitor Cell Growth and Development

Via contemporary investigatory approaches, important new EPO action mechanisms continue to be discovered. As recent illustrating examples, lineage tracking studies have demonstrated guiding effects of EPO on the developmental fate of myeloerythroid progenitors [J Exp Med. 211:181-8]. Profiling of EPO response genes in primary erythroid progenitor cells (EPCs) has uncovered a pathway of EPO cytoprotection against leached lysosomal cathepsins in stressed erythroblasts [J Exp Med. 210:225-32]. And in a context of iron metabolism, Erythroferrone has been identified as an EPO-induced inhibitor of hepcidin [Nat Genet. 46:678-84]. In a unique approach to defining EPO's cell and molecular actions, our laboratory has launched post-translational modification based LC-MS/MS proteomic analyses in human erythroid precursor cell systems. In interrogations of novel EPO/EPOR targets modified within a category of "p-TPP" motifs, TXNIP proved to be rapidly phosphorylated >10 fold due to EPO at novel C-terminal p-T349 and p-S358 sites. In parallel, multi-fold increases in TXNIP levels also were observed within 20 minutes of EPO exposure. To gain functional insight into TXNIP's roles during EPO-dependent erythropoiesis, LOF studies were performed via lentiviral shRNA mediated inhibition of TXNIP. As analyzed first in UT7epo cells, TXNIP knockdown (>90% efficiency) attenuated cell proliferation ~2.5-fold with EPO-dose dependency. This was reflected further in selective attenuation of cell cycle progression at S-phase (with only limited effects of TXNIP knockdown on cell survival observed). LOF experiments were also performed in human bone marrow derived CD34+ primary hematopoietic progenitor cells. Following initial plating, hematopoietic progenitors transduced with TXNIP shRNA exhibited a limited transient lag in growth (as compared directly with shRNA-NT transduced controls), but by day 4 of culture, and thereafter, expanded at essentially normal levels. Subsequently, erythroid progenitors with inhibited TXNIP expression prematurely committed to a program of late erythroblast differentiation. Specifically, TXNIP knockdown resulted in elevated frequencies of GPA-high erythroblasts (33.4+/-1.3% vs controls at 6.9+/-1.1%, p = 0.0001) and decreased KIT-high expression (6.4+/-3.0% vs controls at 62.5+/-7.1%, p = 0.0002). In addition, visibly obvious increases in erythroblast hemoglobinization due to TXNIP knockdown also were observed. The present investigations thus employ a unique PTM LC-MS/MS approach to identify TXNIP as a new EPO/EPOR target modified at novel C-terminal sites upon EPO expression, and in association with an indicated stabilization of TXNIP. LOF studies further reveal pro-erythropoietic roles for TXNIP as a novel mediator of EPO-dependent erythropoiesis with important effects exerted on erythroid precursor cell proliferation, together with a key requirement for TXNIP in governing a transition to a program of late erythroblast differentiation. Significance is further underlined by the nature of TXNIP as an emerging target within beta cells for small molecule inhibition in type-2 diabetics, a group highly representative among anemic chronic renal disease patients [J Diabetes Res Apr 7 2015:801348]. In particular, based on our present findings, such inhibition of TXNIP would be predicted to further compromise erythropoiesis in this CKD patient population. Disclosures No relevant conflicts of interest to declare.

Enhanced expression of interleukin-3 and granulocyte-macrophage colony- stimulating factor receptor subunits in murine hematopoietic cells stimulated with hematopoietic growth factors

AIC2A and AIC2B are closely related genes encoding components of the receptors for murine interleukin-3 (IL-3) (AIC2A) and granulocyte- macrophage colony-stimulating factor (GM-CSF) and IL-5 (AIC2B). We have studied the parallel regulation of expression of these genes in erythroid and myeloid progenitor cell lines. AIC2A and AIC2B transcription was transiently induced in these cells in response to a variety of hematopoietic growth factors, including erythropoietin (EPO), monocyte-CSF, IL-3, GM-CSF, and stem cell factor (SCF or kit ligand). Run-on assays established that the increase occurred mainly at the transcriptional level. Immunoprecipitation experiments confirmed that the increase in messenger RNA expression resulted in augmented synthesis of both AIC2A and AIC2B proteins, and binding studies further showed these proteins to be functional. We observed a fourfold increase in low-affinity IL-3 sites in an erythroid precursor cell line stimulated with EPO, and a threefold increase in GM-CSF high-affinity sites in a myeloid cell line stimulated with IL-3. In addition, we showed that the increase in the IL-3 receptor chain AIC2A in the erythroid precursor cell line correlated with the ability of IL-3 to exert a cooperative effect with EPO in the induction of beta-globin in these cells.

Enhanced expression of interleukin-3 and granulocyte-macrophage colony- stimulating factor receptor subunits in murine hematopoietic cells stimulated with hematopoietic growth factors

AIC2A and AIC2B are closely related genes encoding components of the receptors for murine interleukin-3 (IL-3) (AIC2A) and granulocyte- macrophage colony-stimulating factor (GM-CSF) and IL-5 (AIC2B). We have studied the parallel regulation of expression of these genes in erythroid and myeloid progenitor cell lines. AIC2A and AIC2B transcription was transiently induced in these cells in response to a variety of hematopoietic growth factors, including erythropoietin (EPO), monocyte-CSF, IL-3, GM-CSF, and stem cell factor (SCF or kit ligand). Run-on assays established that the increase occurred mainly at the transcriptional level. Immunoprecipitation experiments confirmed that the increase in messenger RNA expression resulted in augmented synthesis of both AIC2A and AIC2B proteins, and binding studies further showed these proteins to be functional. We observed a fourfold increase in low-affinity IL-3 sites in an erythroid precursor cell line stimulated with EPO, and a threefold increase in GM-CSF high-affinity sites in a myeloid cell line stimulated with IL-3. In addition, we showed that the increase in the IL-3 receptor chain AIC2A in the erythroid precursor cell line correlated with the ability of IL-3 to exert a cooperative effect with EPO in the induction of beta-globin in these cells.

Relationship of Erythropoietin Effectiveness to the Generative Cycle of Erythroid Precursor Cell

Hydroxyurea, a cytotoxic agent that kills cells in DNA synthesis, was used to study the relationship between erythropoietin and the generative cycle of the immediate erythroid precursor cell. When OHU and EP were administered simultaneously to hypertransfused mice, the resultant erythroid response was diminished relative to EP treated controls. OHU given at intervals after EP resulted in a progressively greater diminution of erythroid response. From these studies, then, we would suggest that in the suppressed animal the committed stem cell compartment is in cycle but with a prolonged G1. After EP there is a shortening of the generation time and an increase in the rate of turnover of the committed stem cells. The data also indicate that cells in cycle are differentiated into the pronormoblast compartment. It further may be suggested that erythropoietin is effective throughout the bulk of the generative cycle although it seems unlikely that differentiation is accomplished during the mitotic phase. Whether erythropoietin must be present in both G1 and S as suggested by Kretchmar cannot be answered by the present studies. The data also indicate that cells of the pluripotential compartment are normally in G0 or perhaps a prolonged G1. Damage to the committed compartment appears to be in part repaired by the influx of cells from the pluripotential compartment.