The prime research goal of this application is to define roles of TSAD (T-cell specific adaptor protein) in a novel context of EPOR/JAK2 dependent erythropoiesis. This NRSA fellowship opportunity will foster advanced skills and knowledge in erythropoiesis, and will provide me with tailored training to advance my objective to emerge as an independent investigator in hematology. EPO is a key regulator of erythroid cell formation in response to hypoxia and anemia. The binding of EPO to its cell surface receptor (EPOR) on pro-erythroblasts activates EPOR/JAK2 kinase complexes. Downstream signaling pathways are then stimulated, in part via 8 phospho- tyrosine (PY) docking sites in the EPOR cytoplasmic region. Dysregulation can lead to either anemia or myeloproliferative disease. These issues, together with the emergence of novel EPO mimetics and apparent EPO effects on cancer progression, underscore a need to better understand EPOR/JAK2 effects and action mechanisms. To this end, our laboratory has employed a global phospho-proteomic approach to discover over 70 EPOR-regulated proteins that are rapidly and highly EPO/EPOR- modulated in their tyrosine phosphorylation. Proposed investigations focus on a novel EPO/EPOR-regulated factor, TSAD. This is based on: 30-fold EPO-induced PY-modulation of TSAD;TSAD's essential roles in T-cell signaling, proliferation, migration, and cell death;our observation of strong stage-specific expression of TSAD in mouse primary bone marrow erythroid progenitor cells;and TSAD's associations with juvenile arthritis and chronic inflammatory polyradiculoneuropathy (each with associated anemia). I hypothesize that TSAD associates with activated EPO/EPOR/JAK2 complexes or functional docking complexes, and predominately regulates late-stage erythroblast development.
Specific Aims are to: 1) Identify functional roles of TSAD during EPO-mediated erythroid progenitor cell growth, survival and development using TSAD retroviral and shRNA lentiviral gain- and loss- of function approaches;2) Determine how TSAD couples to EPOR/JAK2 complexes via the use of primary erythroblasts expressing knocked-in PY-deficient EPOR alleles;3) Determine the effects of TSAD- deficiency on steady-state erythropoiesis, EPO-responsiveness, and anemia in Tsad- null mice using hematological analyses, flow cytometry, and ex vivo pro-erythroblast culture. For career development, training will advance via active participation (and presentation) at institutional data review forums, national meetings and workshops, our postdoctoral co-mentorship trainee program;co-development of a focused journal club in hematopoiesis;mentored construction of impacting scientific manuscripts;direct interactions with scientific collaborators;and interactions with outstanding institute and center scientific advisory committees.
Erythropoietin (EPO) is used effectively to treat the anemia of chronic renal disease, and can protect damaged cardiac, renal, and neural tissues against cell death post-injury. Adversely, EPO treatment is also associated with the acceleration of cancer progression and with increased thrombolytic events. To better understand how EPO (and emerging EPO-like therapeutics) exert such effects, the proposed research will identify the role of a newly discovered EPO-activated molecular adaptor, TSAD, in the novel context of EPO-dependent erythropoiesis.
