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.

多囊肾病 (PKD) 是一种致命的遗传性疾病，其特征是肾小管上皮增生、囊肿形成和肾脏大量肿大。 cAMP 激动剂，包括精氨酸加压素，可加速 PKD 囊肿上皮细胞的增殖，但不会加速正常人肾脏 (NHK) 的上皮细胞增殖。我们发现 cAMP 在人 PKD 中激活细胞外信号调节激酶 1/2[2] (ERK)，但在 NHK 细胞中抑制 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 激活有关，从而导致细胞增殖增加。最近对具有四种不同遗传类型 PKD 的动物进行的研究表明，加压素 V2 受体拮抗剂 OPC-31260 抑制肾脏 cAMP 的产生，可显着阻止囊肿和肾脏增大，这证明了 肾囊性疾病中的 cAMP。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 Ca2+ 水平异常。最近，我们发现用通道阻滞剂或减少细胞外 [Ca2+] 抑制小鼠皮质集合管细胞 (M-1) 中的 Ca2+ 进入，导致 cAMP 增殖反应发生表型转变。 cAMP抑制细胞内[Ca2+]正常的M-1细胞增殖；然而，在 M-1 细胞中，[Ca2+] cAMP 减少会刺激 B-Raf、MEK-ERK 通路和细胞增殖，模拟 PKD 表型。中心假设是，在人类 ADPKD 和 ARPKD 中，肾上皮细胞功能失调的 Ca2+ 代谢诱导并维持“表型转换”， 揭示了导致 B-Raf 和 ERK 的 cAMP 依赖性激活以及细胞增殖增加的常见细胞途径。该提案的优点是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具体目标： 目标 1：确定 [Ca2+]i 是否通过改变 cAMP 依赖性 B-Raf 信号传导MEK-ERK 通路并导致 PKD 和 NHK 细胞在 cAMP 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 Ca2+ 并调节 cAMP 依赖性 B-Raf 激活和人 PKD 细胞增殖的机制。目标 3. 确定选择性降低 B-Raf 丰度和抑制 B-Raf 激酶活性是否会减少 PKD 细胞中 cAMP 依赖性 ERK 激活和细胞增殖。这些研究的结果将为开发新型小分子疗法提供全新的机会，以减缓甚至可能阻止患者 PKD 的进展。