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Phospho-proteomic discovery of novel signal transducers including thioredoxin-interacting protein as mediators of erythropoietin-dependent human erythropoiesis.

ABSTRACT: Erythroid cell formation critically depends on signals transduced via erythropoietin (EPO)/EPO receptor (EPOR)/JAK2 complexes. This includes not only core response modules (e.g., JAK2/STAT5, RAS/MEK/ERK), but also specialized effectors (e.g., erythroferrone, ASCT2 glutamine transport, Spi2A). By using phospho-proteomics and a human erythroblastic cell model, we identify 121 new EPO target proteins, together with their EPO-modulated domains and phosphosites. Gene ontology (GO) enrichment for "Molecular Function" identified adaptor proteins as one top EPO target category. This includes a novel EPOR/JAK2-coupled network of actin assemblage modifiers, with adaptors DLG-1, DLG-3, WAS, WASL, and CD2AP as prime components. "Cellular Component" GO analysis further identified 19 new EPO-modulated cytoskeletal targets including the erythroid cytoskeletal targets spectrin A, spectrin B, adducin 2, and glycophorin C. In each, EPO-induced phosphorylation occurred at pY sites and subdomains, which suggests coordinated regulation by EPO of the erythroid cytoskeleton. GO analysis of "Biological Processes" further revealed metabolic regulators as a likewise unexpected EPO target set. Targets included aldolase A, pyruvate dehydrogenase α1, and thioredoxin-interacting protein (TXNIP), with EPO-modulated p-Y sites in each occurring within functional subdomains. In TXNIP, EPO-induced phosphorylation occurred at novel p-T349 and p-S358 sites, and was paralleled by rapid increases in TXNIP levels. In UT7epo-E and primary human stem cell (HSC)-derived erythroid progenitor cells, lentivirus-mediated short hairpin RNA knockdown studies revealed novel pro-erythropoietic roles for TXNIP. Specifically, TXNIP's knockdown sharply inhibited c-KIT expression; compromised EPO dose-dependent erythroblast proliferation and survival; and delayed late-stage erythroblast formation. Overall, new insight is provided into EPO's diverse action mechanisms and TXNIP's contributions to EPO-dependent human erythropoiesis.

SUBMITTER: Held MA

PROVIDER: S-EPMC7480993 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimsThioredoxin-interacting protein (TXNIP) contributes to cellular redox-state homeostasis via binding and inhibiting thioredoxin (TRX). Increasing evidence suggests that cellular redox homeostasis regulates vascular endothelial growth factor (VEGF)-mediated signaling. This study aims to examine the redox-dependant role of TXNIP in regulating VEGF-mediated S-glutathionylation and angiogenic signaling. TXNIP-knockout mice (TKO) or wild-type (WT) treated with the reduced glutathione (GSH)-precursor, N-acetyl cysteine (WT-NAC, 500?mg/kg) were compared to WT using hypoxia-induced neovascularization model.ResultsIn response to hypoxia, retinas from TKO and WT-NAC mice showed significant decreases in reparative revascularization and pathological neovascularization with similar VEGF expression compared with WT. VEGF failed to stimulate vascular sprouting from aortic rings of TKO compared to WT mice. TKO mice or WT+NAC experienced reductive stress as indicated by twofold increase in TRX reductase activity and fourfold increase in reduced-GSH levels compared with WT. In human microvascular endothelial (HME) cells, VEGF stimulated co-precipitation between vascular endothelial growth factor receptor 2 (VEGFR2) with low molecular weight protein tyrosine phosphatase (LMW-PTP). Silencing TXNIP expression blunted VEGF-induced oxidation of GSH and S-glutathionylation of the LMW-PTP in HME cells. These effects were associated with impaired VEGFR2 phosphorylation that culminated in inhibiting cell migration and tube formation. Overexpression of TXNIP restored VEGFR2 phosphorylation and cell migration in TKO-endothelial cells.InnovationTXNIP expression is required for VEGF-mediated VEGFR2 activation and angiogenic response in vivo and in vitro. TXNIP expression regulates VEGFR-2 phosphorylation via S-glutathionylation of LMW-PTP in endothelial cells.ConclusionOur results provide novel mechanistic insight into modulating TXNIP expression as a potential therapeutic target in diseases characterized by aberrant angiogenesis.

Project description:Anaemia is a common haematologic side effect of dose-dense multi-cycle cytotoxic polychemotherapy requiring erythrocyte transfusions or erythropoietin (EPO) administration. To simulate the effectiveness of different EPO application schedules, we performed both modelling of erythropoiesis under chemotherapy and pharmacokinetic and dynamic modelling of EPO applications in the framework of a single comprehensive biomathematical model. For this purpose, a cell kinetic model of bone marrow erythropoiesis was developed that is based on a set of differential compartment equations describing proliferation and maturation of erythropoietic cell stages. The system is regulated by several feedback loops comprising those mediated by EPO. We added a model of EPO absorption after injection at different sites and a pharmacokinetic model of EPO derivatives to account for the effects of external EPO applications. Chemotherapy is modelled by a transient depletion of bone marrow cell stages. Unknown model parameters were determined by fitting the predictions of the model to data sets of circulating erythrocytes, haemoglobin, haematocrit, percentage of reticulocytes or EPO serum concentrations derived from the literature or cooperating clinical study groups. Parameter fittings resulted in a good agreement of model and data. Depending on site of injection and derivative (Alfa, Beta, Delta, Darbepoetin), nine groups of EPO applications were distinguished differing in either absorption kinetics or pharmacokinetics. Finally, eight different chemotherapy protocols were modelled. The model was validated on the basis of scenarios not used for parameter fitting. Simulations were performed to analyze the impact of EPO applications on the risk of anaemia during chemotherapy. We conclude that we established a model of erythropoiesis under chemotherapy that explains a large set of time series data under EPO and chemotherapy applications. It allows predictions regarding yet untested EPO schedules. Prospective clinical studies are needed to validate model predictions and to explore the feasibility and effectiveness of the proposed schedules.

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Phospho-proteomic discovery of novel signal transducers including thioredoxin-interacting protein as mediators of erythropoietin-dependent human erythropoiesis.

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