The long term goal of this project is to understand the signal transduction mechanisms through which growth factors control hematopoietic proliferation and differentiation. This knowledge is essential to understand disorders of hematopoietic regulation including aplastic anemia and leukemia. The major goal of this grant 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. This system is a model to delineate the immediate signaling events which follow interaction of Epo with its receptor on normal cells. The following specific aims will be addressed:
Specific Aim 1 : Identification of the signaling mechanisms through which Epo regulates calcium channels. We have characterized the Epo-modulated calcium channel with patch-clamp methodology and have determined that tyrosine phosphorylation and the G protein subunit Gialpha2 are required. We have also shown that Jak2 is involved. (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. If Ras is required, the role of other transducers in the Ras pathway will be examined. (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 functions of the NF-kappaB 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-kappaB 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 JNK activation and the role of PI 3-kinase in this pathway.
|Cheung, Joseph Y; Miller, Barbara A (2017) Transient Receptor Potential-Melastatin Channel Family Member 2: Friend or Foe. Trans Am Clin Climatol Assoc 128:308-329|
|Bao, Lei; Chen, Shu-Jen; Conrad, Kathleen et al. (2016) Depletion of the Human Ion Channel TRPM2 in Neuroblastoma Demonstrates Its Key Role in Cell Survival through Modulation of Mitochondrial Reactive Oxygen Species and Bioenergetics. J Biol Chem 291:24449-24464|
|Hoffman, Nicholas E; Miller, Barbara A; Wang, JuFang et al. (2015) Ca²? entry via Trpm2 is essential for cardiac myocyte bioenergetics maintenance. Am J Physiol Heart Circ Physiol 308:H637-50|
|Miller, Barbara A; Hoffman, Nicholas E; Merali, Salim et al. (2014) TRPM2 channels protect against cardiac ischemia-reperfusion injury: role of mitochondria. J Biol Chem 289:7615-29|
|Chen, Shu-jen; Hoffman, Nicholas E; Shanmughapriya, Santhanam et al. (2014) A splice variant of the human ion channel TRPM2 modulates neuroblastoma tumor growth through hypoxia-inducible factor (HIF)-1/2?. J Biol Chem 289:36284-302|
|Chen, Shu-jen; Zhang, Wenyi; Tong, Qin et al. (2013) Role of TRPM2 in cell proliferation and susceptibility to oxidative stress. Am J Physiol Cell Physiol 304:C548-60|
|Miller, Barbara A; Wang, JuFang; Hirschler-Laszkiewicz, Iwona et al. (2013) The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 304:H1010-22|
|Hirschler-Laszkiewicz, Iwona; Zhang, Wenyi; Keefer, Kerry et al. (2012) Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis. Exp Hematol 40:71-83|
|Hirschler-Laszkiewicz, Iwona; Tong, Qin; Waybill, Kathleen et al. (2011) The transient receptor potential (TRP) channel TRPC3 TRP domain and AMP-activated protein kinase binding site are required for TRPC3 activation by erythropoietin. J Biol Chem 286:30636-46|
|Hirschler-Laszkiewicz, Iwona; Tong, Qin; Conrad, Kathleen et al. (2009) TRPC3 activation by erythropoietin is modulated by TRPC6. J Biol Chem 284:4567-81|
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