Functional Role of CaT1 and Calbindin D in Ca Transport

CaT1 和 Calbindin D 在 Ca 转运中的功能作用

• 批准号: 6605377
• 负责人: RICHARD James WOOD
• 金额: $ 26.06万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6605377
• 关键词: 1,25 dihydroxycholecalciferol; apical membrane; basolateral membrane; biological signal transduction; brush border membrane; calbindin; calcium binding protein; calcium channel; calcium flux; calcium ion; enzyme linked immunosorbent assay; gastrointestinal absorption /transport; gene expression; membrane transport proteins; phospholipase D; polymerase chain reaction; protein kinase C; protein structure function; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): Absorbing too little calcium (Ca) in the intestine can be a risk factor for negative Ca balance and the development of osteoporosis. An important gap in our knowledge is that the molecular determinants of Ca influx into the enterocyte and transcellular Ca transport have not been unambiguously identified. This lack of knowledge limits the development of specifically targeted therapeutic agents to manipulate Ca absorption. The long-range goal of our research program is to identify the molecular factors responsible for age-related calcium malabsorption, and to target the development of safe and effective therapeutic agents that can modulate active calcium absorption. The recent discovery of the CaT1 (also known as ECaC2 and TRPV6) gene and the demonstration that its protein product functions in non-intestinal cells as a membrane Ca transporter, and our recent report that the expression of CaT1 in the Caco-2 human intestinal cell line is regulated by 1,25-dihydroxyvitamin D, offers an exciting new opportunity to further knowledge of the molecular mechanisms of vitamin D-mediated intestinal Ca absorption. We have two specific aims in this proposal (1) to establish the role of CaT1 and calbindin D in vitamin D-mediated calcium transport and (2) to identify to what extent CaT1 expression is a modulator of the rapid non-genomic vitamin D response in the enterocyte. We will determine the functional role of these proteins in the enterocyte by using Caco-2 cells transfected with sense and antisense CaT1 and calbindin D cDNA to alter the level of these proteins independently of vitamin D. The central hypothesis of our first research aim is that CaT1 acts as the primary gatekeeper of intestinal Ca absorption by controlling, under regulation by 1,25-dihydroxyvitamin D, the rate of Ca entry across the apical brush border membrane of the enterocyte. The cytosolic vitamin D-dependent calbindin D acts in concert with CaT1 as both intracellular buffer and transporter, delivering Ca that has crossed the brush border membrane to the basolateral membrane of the enterocyte for Ca exit into the blood. We hypothesize in our second research aim that the CaT1 molecule either is responsible for or modulates the rapid 'non-genomic' response to 1,25-dihydroxyvitamin D in Caco-2 cells by determining the rate of Ca entry into the enterocyte following 1,25-dihydroxyvitamin D treatment and thereby modulating the rise of free intracellular Ca and subsequent downstream activation of protein kinase C and phospholipase D.

描述（由申请人提供）：肠道吸收钙 (Ca) 过少可能是钙负平衡和骨质疏松症发生的危险因素。我们知识中的一个重要差距是，Ca 流入肠上皮细胞和跨细胞 Ca 转运的分子决定因素尚未明确确定。这种知识的缺乏限制了控制钙吸收的特异性靶向治疗剂的开发。我们研究计划的长期目标是确定导致与年龄相关的钙吸收不良的分子因素，并致力于开发能够调节主动钙吸收的安全有效的治疗药物。最近发现了 CaT1（也称为 ECaC2 和 TRPV6）基因，并证明其蛋白产物在非肠细胞中作为膜 Ca 转运蛋白发挥作用，并且我们最近的报告表明 CaT1 在 Caco-2 人肠道中的表达细胞系受 1,25-二羟基维生素 D 调节，为进一步了解维生素 D 介导的肠道 Ca 吸收的分子机制提供了令人兴奋的新机会。我们在此提案中有两个具体目标 (1) 确定 CaT1 和钙结合蛋白 D 在维生素 D 介导的钙转运中的作用，以及 (2) 确定 CaT1 表达在多大程度上调节快速非基因组维生素 D 反应在肠上皮细胞中。我们将使用转染正义和反义 CaT1 和钙结合蛋白 D cDNA 的 Caco-2 细胞来独立于维生素 D 改变这些蛋白质的水平，从而确定这些蛋白质在肠上皮细胞中的功能作用。我们第一个研究目标的中​​心假设是CaT1 作为肠道 Ca 吸收的主要看门人，在 1,25-二羟基维生素 D 的调节下，控制 Ca 穿过肠上皮细胞顶端刷状缘膜的进入速率。胞质维生素 D 依赖性钙结合蛋白 D 与 CaT1 协同作用，既作为细胞内缓冲剂又作为转运蛋白，将穿过刷状缘膜的 Ca 递送至肠上皮细胞的基底外侧膜，以便 Ca 进入血液。我们在第二个研究目标中假设，CaT1 分子通过确定 Ca 进入肠上皮细胞的速率，负责或调节 Caco-2 细胞中对 1,25-二羟基维生素 D 的快速“非基因组”反应 1， 25-二羟基维生素 D 处理，从而调节细胞内游离 Ca 的升高以及随后下游蛋白激酶 C 和磷脂酶 D 的激活。