Intestinal calcium absorption: molecular mechanisms

描述（由申请人提供）：控制肠道钙吸收对人体健康的重要性体现在两个方面。首先，骨质疏松性髋部骨折的风险在钙吸收效率低的女性中更高，这可能是由于年龄相关的钙吸收不良或肠道对1,25(OH)2维生素D3 （1,25(OH)2，肠道钙吸收的主要调节剂）的抵抗。其次，在癌症治疗中使用维生素D类似物作为促分化剂的一个重要障碍是，它们会刺激肠道钙吸收，导致高钙血症。我们的长期目标是阐明125 (OH) 2d促进钙吸收的机制，并利用这些信息来改善钙吸收分数低的人的钙吸收，并帮助设计维生素D类似物，可作为非钙缺乏性癌症治疗药物。新的研究表明，1,25(OH) 2d可快速激活第二信使和激酶途径，包括MAP激酶及其上游激活因子；抑制这些激酶会减弱1,25(OH)2 D介导的基因转录，表明经典和非经典维生素D信号通路相互作用。拟议研究的目的是确定1,25(OH) 2d介导的肠钙吸收转录激活如何受到MAP激酶ERK1和2的基础或诱导活性的影响。该项目的具体目标是：(1)确定由1,25(OH)2诱导的ERK1和2激活调节的nvdr介导的控制肠道钙吸收的基因组途径；(2)建立由1,25(OH)2诱导的ERK1和2活性促进的1,25(OH)2介导的基因表达所必需的蛋白质-蛋白质相互作用。我们将通过研究1,25(OH) 2d在具有良好特征的细胞培养模型（Caco-2细胞）和小鼠小肠中的作用来实现这些目标。1,25(OH) 2d的生物作用将在蛋白激酶的激活剂和抑制剂（药理学抑制剂，显性负激酶）和维生素D（维生素D类似物）的快速作用下进行研究，nVDR的作用和功能将通过细胞成像、报告基因、多杂交试验和染色质免疫沉淀（CHIP）试验进行研究。阐明这种维生素D信号通路串导的机制将为肠道钙吸收的控制调节提供基础，例如当维生素D抵抗与衰老或雌激素缺乏相关时。

DESCRIPTION (provided by applicant): The control of intestinal calcium absorption is important for human health in two ways. First, the risk of osteoporotic hip fracture is higher in women with low calcium absorption efficiency and this may be due to age-associated calcium malabsorption or intestinal resistance to 1,25(OH)2 vitamin D3 (1,25(OH)2 D, the primary regulator of intestinal calcium absorption). Second, a significant barrier to the use of vitamin D analogs as pro-differentiating agents in cancer treatment is that they stimulate intestinal calcium absorption and cause hypercalcemia. Our long-term objective is to clarify the mechanisms used by 1,25(OH)2 D to promote calcium absorption and to utilize this information to improve calcium absorption in people with low fractional calcium absorption and to aid in the design of vitamin D analogs that can be used as non-calcemic cancer therapeutics. New research shows that 1,25(OH)2 D rapidly activates second messenger and kinases pathways including the MAP kinases and their upstream activators; inhibition of these kinases blunts 1,25(OH)2 D-mediated gene transcription indicating the classical and non-classical vitamin D signaling pathways interact. The goal of the proposed research is to determine how the 1,25(OH)2 D-mediated transcriptional activation of intestinal calcium absorption is influenced by the basal or induced activity of the MAP kinases ERK1 and 2. The specific aims of this project are to: (1) Identify the nVDR-mediated genomic pathways controlling intestinal calcium absorption that are modulated by 1,25(OH)2-induced activation of ERK1 and 2, and (2) Establish the protein-protein interactions necessary for 1,25(OH)2 D-mediated gene expression that are promoted by 1,25(OH)2 D-induced ERK1 and 2 activity. We will accomplish these aims by studying the effect of 1,25(OH)2 D in a well-characterized cell culture model (Caco-2 cells) and in the small intestine of mice. Biological actions of 1,25(OH)2 D will be studied in the presence of activators and inhibitors of protein kinases (pharmacologic inhibitors, dominant negative kinases) and the rapid actions of vitamin D (vitamin D analogs), nVDR action and function will be studied with cellular imaging, reporter genes, multi-hybrid assays, and chromatin immunoprecipitation (CHIP) assays. Elucidating the mechanism of this vitamin D signal pathway cross-talk will provide the foundation for controlled modulation of intestinal calcium absorption, e.g. when vitamin D resistance associated with aging or estrogen deficiency is present.