The long term goal of studies proposed here is to determine the signal transduction mechanisms through which growth factors stimulate hematopoietic proliferation and differentiation. The major goal of this project is to understand the mechanisms through which erythropoietin (Epo) regulates ion channels during erythroid differentiation and to determine the functional role of calcium influx in erythropoiesis. Blood will be drawn and bone marrow aspirates will be performed in the CRC on healthy volunteers. The following specific aims will be addressed:
Specific Aim 1 : Identification of the signaling mechanism through which Epo regulates calcium channels. (A) Here we will determine the Epo signaling pathways which link Jak2 to calcium channel activation. Involvement of STAT, Ras, or the IRS-2/PI 3 kinase pathways will be examined using microinjection of single BFU-E derived erythroblasts and quantitative fluorescence microscopy coupled digital video imaging. (B) We will determine the domains of erythropoietin receptor required for Epo modulation of calcium channel opening.
Specific Aim 2 :Determination of the role of [Cai] in regulation of transcription factor activation in erythropoiesis. The function of the NF-kB and bHLH transcription factors and the c-Jun N-terminal kinases (JNK) are modulated by calcium. We will determine if the amplitude or duration of the Ca++ response in erythroid cells affects NF-kB transcription factor activation, or if calcium/calmodulin levels influence DNA binding of bHLH proteins, particularly SCL. We will also determine the effect of [Cai] on JNLK activation on the role of PI 3- kinase in this pathway.
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