Erythropoietin Stimulation of Mitochondrial Protein Content - A Potential Mechanism through Direct Binding of Erythropoietin Receptor and AMP-Activated Protein Kinase

Abstract

Proliferating cells have unique metabolic requirements beyond those of quiescent cells. Specifically, blood forming hematopoietic stem cells, during periods of severe blood loss, switch from a quiescent glycolytic state to a state dependent on mitochondrial metabolism during differentiation and proliferation. This dissertation attempts to define some of the signaling details of this switch by using erythropoietin receptor signaling as a model. In cytokine-dependent Ba/F3 cell line expressing the receptor for erythropoietin (EpoR) (Ba/F3-EpoR), chemical inhibition of mitochondrial function by rotenone decreases in erythropoietin(Epo)-stimulated proliferation. This observation led to the examination of whether Epo could stimulate mitochondrial function. To further assess the role of mitochondria in cell proliferation and the metabolic functions of Epo, levels of oxidative phosphorylation markers and signaling molecules important for mitochondrial biogenesis were measured. Western blotting scans showed increased protein levels of cytochrome oxidase subunit IV (CoxIV) and Complex III core protein 2 following 24 hours of Epo treatment. Interestingly, inhibition of Janus Kinase 2 (Jak2), the tyrosine kinase associated with Epo receptor, by AG490 elicited a similar decrease in CoxIV to Epo withdrawal even in the presence of Epo. In addition, Epo increased the levels of the mitochondrial biogenesis regulator AMP-activated protein kinase α (AMPKα) in a Jak2-dependent manner within Ba/F3 cells. Both total and phosphorylated (activated) AMPKα were increased following Epo stimulation. Treatment with the AMPK inhibitor Compound C decreased Epo stimulation of CoxIV, suggesting a linear signaling cascade from Jak2 to mitochondrial biogenesis through AMPKα. Examining potential mechanisms, direct binding of AMPKα to (EpoR) and Jak2 were observed through immunoprecipitations of transfected lysates in a manner exclusive to AMPK regulator subunits β and γ. Furthermore AMPKα was found to be tyrosine phosphorylated in an Epo and Jak2 dependent manner. Taken together, data in this dissertation suggests a role for Epo in regulating mitochondrial biogenesis in cytokine dependent cells through a potential mechanism of forming a signaling complex between EpoR, Jak2, and AMPKα. This signaling complex may provide intersection between Epo's signaling in cell proliferation and metabolism through AMPKα.