CALCIUM REGULATION OF cAMP-DEPENDENT PROLIFERATION

营依赖性增殖的钙调节

• 批准号: 7070151
• 负责人: DARREN P. WALLACE
• 金额: $ 20.21万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7070151
• 关键词: autosomal dominant trait; autosomal recessive trait; calcium flux; calcium metabolism; cell growth regulation; cell proliferation; cyclic AMP; enzyme activity; gene expression; gene mutation; genetic disorder; hormone inhibitor; hormone receptor; human tissue; kidney pharmacology; mitogen activated protein kinase; molecular pathology; phenotype; podocyte; polycystic kidney; protein kinase; renal tubule; stimulant /agonist; tissue /cell culture; vasopressins

Polycystic kidney diseases (PKDs) are lethal, hereditary disorders characterized by hyperplasia of the tubule epithelium, cyst formation and massive kidney enlargement. cAMP agonists, including arginine vasopressin, accelerate the proliferation of epithelial cells from PKD cysts but not from normal human kidneys (NHK). We discovered that cAMP activates extracellular signal-regulated kinases1/2[2] (ERK) in human PKD, but inhibits ERK activation in NHK cells. The molecular mechanisms underlying this phenotypic difference between PKD and NHK cells are linked to cAMP/ protein kinase A-dependent B-Raf activation of MEK and ERK, leading to increased cell proliferation. Recent studies in animals with four different genetic types of PKD showed that inhibition of renal cAMP production by vasopressin V2 receptor antagonist OPC-31260 dramatically halted cyst and kidney enlargement, demonstrating a central role for cAMP in renal cystic disease. Mutated gene products of hereditary cystic disorders are thought to cause abnormal Ca2+ levels in renal tubule cells. Recently, we found that inhibition of Ca2+ entry in mouse cortical collecting duct cells (M-1) with channel blockers or reduced extracellular [Ca2+] caused a phenotypic switch in the proliferation response to cAMP. cAMP inhibited the proliferation of M-1 cells with normal intracellular [Ca2+]; however, in M-1 cells with reduced [Ca2+] cAMP stimulated B-Raf, the MEK-ERK pathway and cell proliferation, mimicking the PKD phenotype. The central hypothesis is that in human ADPKD and ARPKD, dysfunctional Ca2+ metabolism by renal epithelial cells induces and maintains a "phenotypic switch" that uncovers a common cellular pathway leading to cAMP-dependent activation of B-Raf and ERK, and increased cell proliferation. The strength of this proposal is the use of cyst epithelial cells from two different types of human hereditary disease, ADPKD and ARPKD, to address the following specific aims: Aim 1: Determine if [Ca2+]i modifies cAMP-dependent B-Raf signaling through the MEK-ERK pathway and contributes to the phenotypic difference in between PKD and NHK cells in the cAMP mitogenic response. Aim 2. Elucidate mechanisms by which vasopressin V2 receptor agonists and antagonists adjust intracellular Ca2+ and modulate cAMP-dependent B-Raf activation and the proliferation of human PKD cells. Aim 3. Determine if selective reduction of B-Raf abundance and inhibition of B-Raf kinase activity diminish cAMP-dependent ERK activation and cell proliferation in PKD cells. The results from these studies will provide fundamentally new opportunities for developing novel small molecule therapies to slow, and possibly halt the progression of PKD in patients.

多囊肾是一种致死性的遗传性疾病，其特征是肾小管上皮细胞增生、囊肿形成和巨大的肾脏肿大。cAMP激动剂，包括精氨酸加压素，加速PKD囊肿上皮细胞的增殖，但不加速正常人肾脏上皮细胞的增殖（NHK）。我们发现cAMP激活人PKD中的细胞外信号调节激酶1/2[2]（ERK），但抑制NHK细胞中的ERK激活。PKD和NHK细胞之间这种表型差异的分子机制与MEK和ERK的cAMP/蛋白激酶A依赖性B-Raf活化有关，导致细胞增殖增加。最近在四种不同遗传类型PKD动物中的研究表明，加压素V2受体拮抗剂OPC-31260抑制肾cAMP的产生可显著阻止囊肿和肾脏增大，这证明了PKD在治疗PKD中的核心作用。 cAMP在肾囊肿中的作用遗传性囊性疾病的突变基因产物被认为会导致肾小管细胞中钙离子水平异常。最近，我们发现，抑制钙离子进入小鼠皮质集合管细胞（M-1）与通道阻滞剂或减少细胞外[Ca 2 +]引起的表型开关的增殖反应cAMP。cAMP抑制具有正常细胞内[Ca 2 +]的M-1细胞的增殖;然而，在具有降低的[Ca 2 +] cAMP的M-1细胞中，刺激B-Raf、MEK-ERK途径和细胞增殖，模拟PKD表型。中心假设是在人类ADPKD和ARPKD中，肾上皮细胞的功能失调的Ca 2+代谢诱导并维持“表型转换”， 揭示了一个共同的细胞途径，导致cAMP依赖性激活B-Raf和ERK，并增加细胞增殖。该建议的优势在于使用来自两种不同类型的人类遗传性疾病ADPKD和ARPKD的囊肿上皮细胞来解决以下特定目标：目的1：确定[Ca 2 +]i是否通过MEK-ERK途径修饰cAMP依赖性B-Raf信号传导，并导致PKD和NHK细胞在cAMP促有丝分裂反应中的表型差异。目标2.阐明加压素V2受体激动剂和拮抗剂调节细胞内Ca 2+和调节cAMP依赖性B-Raf激活和人PKD细胞增殖的机制。目标3。确定B-Raf丰度的选择性降低和B-Raf激酶活性的抑制是否减少PKD细胞中cAMP依赖性ERK活化和细胞增殖。这些研究的结果将为开发新型小分子疗法提供新的机会，以减缓甚至可能阻止PKD患者的进展。