The erythropoietin (Epo) receptor is a model for understanding signal transduction mechanisms of the cytokine receptor superfamily. Calcium signaling plays an important role in cell proliferation and apoptosis, yet little is known about regulation of ion channels by hematopoietic growth factors. Using quantitative fluorescence microscopy and electrophysiology, our laboratory was the first to demonstrate that Epo modulates the intracellular calcium concentration ([Ca2+]i) through activation of members of the TRPC ion channel family. The transient receptor potential (TRP) superfamily is a diverse group of voltage-independent calcium permeable channels which are involved in sustained calcium entry in nonexcitable cells. TRPC3 and TRPC6 are members of the TRPC subfamily which are expressed in human erythroid cells. TRPC3 is activated by Epo, while TRPC6 inhibits TRPC3 activation. TRPC2 is also activated by Epo in murine erythroid cells but is a pseudogene in humans. In this grant, Specific Aim 1 will study the mechanisms through which Epo regulates [Ca2+]i by activation of TRPC3. We will examine (1) the physiological importance and mechanisms regulating TRPC3 tyrosine phosphorylation after Epo stimulation by identifying the involved kinase and key tyrosine sites on TRPC3 which are phosphorylated and required for channel activation;(2) the role of phospholipase C and IP3R in TRPC3 activation by Epo;and (3) the mechanisms through which Epo regulates TRPC3 cell surface localization. We will utilize a two phase liquid culture system of primary human erythroid progenitors/precursors to study mechanisms of regulation of TRPC3 and their role in human erythropoiesis.
Specific Aim 2 will examine the mechanisms through which TRPC6 inhibits TRPC3 activation by Epo and the physiological importance. TRPC3 and TRPC6 expression are regulated during normal erythroid differentiation. We will determine whether TRPC6 inhibits TRPC3 membrane localization or tyrosine phosphorylation, and identify TRPC6 domains which are involved in inhibition of TRPC3.
Specific Aim 3 will examine the functional importance of TRPC3 in erythropoiesis using TRPC3 whole animal and tissue specific knockout mice, and TRPC2-/-TRPC3-/- double knockouts. Preliminary results show that TRPC3 knockout animals have a defect in red cell production under stress. Erythropoiesis will be characterized in TRPC3-/- and TRPC2-/-TRPC3-/- mice, which replicate TRPC3 deficiency in the human environment. Understanding the role of TRP channels in erythroid proliferation and differentiation is of fundamental biological importance. In addition, modulation of TRPC3 may have important applications in future therapy of diseases of red cell production including polycythemia and certain anemias, such as that of chronic renal failure.

Public Health Relevance

This research is among the first to study regulation of an ion channel, TRPC3, by a hematopoietic growth factor, erythropoietin, and our studies with knockout mice suggest that TRPC3 is important in the production of red blood cells. The work that we propose has broad relevance in understanding and treating disease of decreased red cell production secondary to many causes including anemia of chronic renal failure, as well as diseases of overproduction such as polycythemia or erythroleukemia. Therapeutic manipulation of TRPC3 activity may have wide applicability in regulating blood cell production, and hence have direct relevance in improving public health.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Erythrocyte and Leukocyte Biology Study Section (ELB)
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Bishop, Terry Rogers
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Pennsylvania State University
Schools of Medicine
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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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