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dc.contributor.advisorWill, Britta
dc.contributor.authorKao, Yun-Ruei Christine
dc.date.accessioned2020-04-02T19:54:14Z
dc.date.available2020-04-02T19:54:14Z
dc.date.issued2018
dc.identifier.citationSource: Dissertations Abstracts International, Volume: 80-09, Section: B.;Publisher info.: Dissertation/Thesis.;Advisors: Will, Britta.en_US
dc.identifier.isbn978-1-392-00050-2
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:13855621en_US
dc.identifier.urihttps://hdl.handle.net/20.500.12202/5381
dc.description.abstractIron homeostasis-regulatory pathways mediate hematopoietic stem cell fate Hematopoiesis is a highly regulated, step-wise process in which hematopoietic stem cells (HSCs) residing at the top of the hematopoietic hierarchy are capable of self-renewing to maintain the stem cell pool, and differentiating to give rise to blood cells of all lineages. Inefficient hematopoiesis is a frequent and critical clinical problem in aplastic anemia, myelodysplastic syndromes (MDS), immune thrombocytopenia, as well as chemotherapy-induced pancytopenia. Eltrombopag (EP), a small molecule initially designed as a thrombopoietin receptor (TPO-R) agonist, has emerged as a potent platelet-stimulating agent and has also shown remarkable efficacy in stimulating sustained multilineage hematopoiesis, suggesting an effect at the level of primitive HSCs. Apart from stimulating TPO signaling, EP has been reported to trigger TPO-R independent pathways involving iron chelation. Nevertheless, it remains to be determined whether EP exerts its effect at the HSC level, and whether the iron-chelating property is functionally relevant to the HSC stimulation by EP. We found that EP significantly enhanced not only multilineage differentiation, but also serial replating capacity of purified human HSCs. In addition, comparative analysis of stem cells in the bone marrow of patients receiving EP showed a marked increase in the number of functional stem cells compared to patients treated with romiplostim, another TPO-R agonist lacking iron-chelating ability. Microarray analysis of human HSCs also confirmed iron-associated molecular changes in EP-treated HSCs that were absent in TPO-treated HSCs. This cellular and molecular evidence strongly suggests a role of iron-mediated pathways in regulating HSC function that is distinct from TPO stimulation. Therefore, we utilized separation-of-function mouse models, including wild type and TPO receptor (TPOR) knockout models, to examine TPO-R independent effects of EP on HSC function ex vivo and in vivo. In both mouse models, we observed a significant increase of HSC self-renewal upon EP treatment, which was also consistently observed with two other clinically available iron chelators, Deferoxamine (DFO) and Deferasirox (DFX). Importantly, the increase of HSC self-renewal upon iron chelation was abrogated by preloading with ferric ammonium citrate (FAC), demonstrating the causative role of intracellular iron levels in the modulation of HSC self-renewal. Gene expression profiling of mouse HSCs treated ex vivo with DFO or EP revealed alterations in molecular pathways that are consistent with reduction of intracellular labile iron pools (LIP), including the activation of transferrin receptor (TfrciCD71) and Nuclear receptor coactivator 4 (encoded by Ncoa4). Intriguingly, simultaneous inhibition of CD71 and NOCA4 abrogated the increase of HSC self-renewal by iron chelators, suggesting the activation of iron-regulatory pathways following iron reduction mediated the HSC stimulatory effects. Further gene expression and metabolite profiling of cells exposed to iron chelators also revealed alterations in metabolic pathways associated with fatty acid oxidation (FAO), which was validated by Seahorse, an assay that directly measures the extracellular fluxes of oxygen consumption. Furthermore, iron chelation-mediated increase in HSC number was rescued by pharmacologic inhibition of CPT-1, a mitochondrial enzyme involved in the conjugation of fatty acids to carnitine for subsequent transfer inside mitochondria. Together, our data demonstrates the integral role of FAO in governing HSC fate transitions following reduction of LIP. Further molecular interrogation revealed an increase in free arachidonic acid (AA) following iron chelator treatment ex vivo, which we hypothesized could be partially regulated by NCOA4-mediated ferritinophagy. We designed short hairpin RNA (shRNA) constructs to knockdown Acsl4 , a member of the long-chain acyl-CoA synthetases that preferentially utilizes AA as substrates, to selectively inhibit the increase in FAO contributed by AA. We found that Acsl4 knockdown abrogated the increase in FAO rate stimulated by DFO, indicating that iron chelation increases the rate of FAO through the mobilization of intracellular AA stores. It has been previously described that upon nutrient deprivation, fatty acids packaged in lipid droplets mobilize to mitochondria and induce β-oxidation of the fatty acids. Inhibition of lipolysis by diethylumbelliferyl phosphate (DEUP) abrogated FAO stimulation upon iron chelation, suggesting the contribution of lipid droplets in fueling mitochondrial oxidation. Interestingly, simultaneous inactivation of AA and inhibition of lipolysis did not further decrease FAO. These findings indicate that AA fuels FAO by a mechanism that is predominantly dependent on lipid droplet mobilization and lipolysis. In conclusion, our data has provided proof-of-concept that experimental reduction of the intracellular labile iron pool, the most readily chelatable form of iron within cells, leads to an array of metabolic reprogramming and an increase in HSC numbers. In-depth investigation on the molecular underpinnings demonstrate that the intracellular labile iron pool reinforces stem cell-maintaining metabolic programs and acts as a rheostat in dividing HSCs. *Please refer to dissertation for diagrams.en_US
dc.language.isoen_USen_US
dc.publisherProQuest Dissertations & Theses Globalen_US
dc.subjectBiologyen_US
dc.subjectCellular biologyen_US
dc.titleIron Homeostasis-Regulatory Pathways mediate Hematopoietic Stem Cell Fateen_US
dc.typeDissertationen_US
dc.typeThesisen_US


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