Program: Oral and Poster Abstracts
Session: 101. Red Cells and Erythropoiesis, Structure and Function, Metabolism, and Survival, Excluding Iron: Poster II
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.
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