Signal Transduction Mechanisms of Erythropoietin

描述（由申请人提供）：促红细胞生成素（Epo）受体是理解细胞因子受体超家族信号转导机制的模型。钙信号在细胞增殖和凋亡中起着重要作用，但造血生长因子对离子通道的调控却知之甚少。使用定量荧光显微镜和电生理学，我们的实验室是第一个证明，Epo调节细胞内钙浓度（[Ca 2 +]i）通过激活TRPC离子通道家族的成员。瞬时受体电位（TRP）超家族是一组不同的电压非依赖性钙渗透通道，它们参与非兴奋细胞中持续的钙离子进入。TRPC 3和TRPC 6是TRPC亚家族的成员，其在人红系细胞中表达。TRPC 3被Epo激活，而TRPC 6抑制TRPC 3激活。TRPC 2在鼠红系细胞中也被Epo激活，但在人类中是假基因。在这项资助中，具体目标1将研究Epo通过激活TRPC 3调节[Ca 2 +]i的机制。我们将研究（1）通过鉴定磷酸化和通道激活所需的TRPC 3上的相关激酶和关键酪氨酸位点，在Epo刺激后调节TRPC 3酪氨酸磷酸化的生理重要性和机制;（2）磷脂酶C和IP 3R在Epo激活TRPC 3中的作用;（3）Epo调节TRPC 3细胞表面定位的机制。我们将利用原代人红系祖细胞/前体细胞的两相液体培养系统来研究TRPC 3的调节机制及其在人红系生成中的作用。具体目标2将检查TRPC 6通过Epo抑制TRPC 3激活的机制及其生理重要性。TRPC 3和TRPC 6的表达在正常红细胞分化过程中受到调节。我们将确定TRPC 6是否抑制TRPC 3膜定位或酪氨酸磷酸化，并确定TRPC 6结构域参与抑制TRPC 3。具体目标3将使用TRPC 3全动物和组织特异性敲除小鼠以及TRPC 2-/-TRPC 3-/-双敲除来检查TRPC 3在红细胞生成中的功能重要性。初步结果显示，TRPC 3基因敲除动物在应激条件下红细胞生成有缺陷。将在TRPC 3-/-和TRPC 2-/-TRPC 3-/-小鼠中表征红细胞生成，这些小鼠在人类环境中复制TRPC 3缺陷。了解TRP通道在红系细胞增殖和分化中的作用具有重要的生物学意义。此外，TRPC 3的调节可能在红细胞生成疾病的未来治疗中具有重要应用，所述红细胞生成疾病包括红细胞增多症和某些贫血症，例如慢性肾衰竭。公共卫生相关性：这项研究是第一个研究造血生长因子促红细胞生成素对离子通道TRPC 3的调节的研究，我们对敲除小鼠的研究表明TRPC 3在红细胞的产生中很重要。我们提出的工作在理解和治疗继发于许多原因的红细胞生成减少的疾病方面具有广泛的相关性，包括慢性肾衰竭贫血，以及红细胞生成过多的疾病，如红细胞增多症或红白血病。TRPC 3活性的治疗性操作可能在调节血细胞产生方面具有广泛的适用性，因此在改善公众健康方面具有直接相关性。

DESCRIPTION (provided by applicant): 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.