Intestinal calcium absorption: molecular mechanisms

描述（由申请人提供）：肠道钙吸收的控制在两个方面对人体健康很重要。首先，钙吸收效率低的女性发生骨质疏松性髋部骨折的风险较高，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 D，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刺激肠钙吸收并引起高钙血症。我们的长期目标是阐明1，25（OH）2 D促进钙吸收的机制，并利用这一信息改善钙吸收分数低的人的钙吸收，并帮助设计可用作非钙依赖性癌症治疗剂的维生素D类似物。新的研究表明，1，25（OH）2 D快速激活第二信使和激酶途径，包括MAP激酶及其上游激活剂;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。拟议研究的目标是确定1，25（OH）2 D介导的肠钙吸收的转录激活如何受到MAP激酶ERK 1和2的基础或诱导活性的影响。本项目的具体目标是：（1）确定nVDR介导的控制肠钙吸收的基因组途径，这些途径由1，25（OH）2诱导的ERK 1和2激活调节，以及（2）建立1，25（OH）2 D介导的基因表达所必需的蛋白质-蛋白质相互作用，这些基因表达由1，25（OH）2 D诱导的ERK 1和2活性促进。我们将通过研究1，25（OH）2 D在一个良好表征的细胞培养模型（Caco-2细胞）和小鼠小肠中的作用来实现这些目标。将在存在蛋白激酶激活剂和抑制剂（药理学抑制剂、显性负性激酶）的情况下研究1，25（OH）2 D的生物学作用，并将通过细胞成像、报告基因、多杂交试验和染色质免疫沉淀（CHIP）试验研究维生素D（维生素D类似物）的快速作用、nVDR作用和功能。阐明这种维生素D信号通路串扰的机制将为肠钙吸收的受控调节提供基础，例如当存在与衰老或雌激素缺乏相关的维生素D抗性时。

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.