VITAMIN D-REGULATED EXOCYTOSIS IN OSTEOBLASTS

描述（由申请人提供）：类固醇1 α，25（OH）2维生素D3（1，25 D）是一种全身激素，具有骨合成代谢作用。1，25 D通过作用于成骨细胞促进骨基质的合成及其矿化。这通过与维生素D受体（VDR）的相互作用和基因转录的调节而发生。此外，1，25 D在质膜水平上快速作用（秒-分钟），在那里它激活细胞质信号传导途径和离子通道功能。我最近证明，1，25 D-增强氯电流耦合到刺激分泌基质蛋白的成骨细胞表达功能性VDR。然而，这些1，25 D效应的精确分子机制仍然只是部分理解。该提案的长期目标是阐明成骨细胞中骨形成的1，25 D非基因组机制。我的工作假设，我提出作为一个新的研究者，是由1，25 D触发的信号转导作用于膜相关的VDR导致非基因组快速胞吐反应，这是耦合到成骨细胞中的氯离子通道激活。这在一定程度上解释了这种激素在骨骼中的合成代谢作用。该提议的第一个具体目的是研究成骨细胞中由膜相关VDR募集的两个平行信号转导途径：a）Ga q/cAMP/PKA/CI通道磷酸化/胞吐，和B）Ga q/PLC/IP 3/钙/胞吐。第二个具体目标研究成骨细胞CIC-3基因，其蛋白质产物，并耦合到胞吐。这将在从VDR WT和KO小鼠获得的成骨细胞中进行研究，后者表达佝偻病表型。虽然该提案的主要重点是基础研究，但其目标是确定以骨量和矿化减少为特征的骨病理治疗中的分子靶点。这分别代表骨骼疾病，如骨质疏松症和骨软化症。骨质疏松症特别影响美国老龄人口的大部分，并构成重大的经济负担。

DESCRIPTION (provided by applicant): The steroid 1alpha,25(OH)2 vitamin D3 (1,25D) is a systemic hormone with bone anabolic effects. 1,25D promotes the synthesis of bone matrix and its mineralization by acting on osteoblasts. This occurs via interaction with the vitamin D receptor (VDR) and modulation of gene transcription. In addition, 1,25D acts rapidly (sec-min) at the plasma membrane level, where it activates cytoplasmic signaling pathways and ion channel functions. I recently demonstrated that 1,25D-potentiation of chloride currents is coupled to a stimulation of secretion of matrix proteins in osteoblasts expressing a functional VDR. However, the precise molecular mechanisms of these 1,25D effects remain only partially understood. The long-term goal of this proposal is to elucidate 1,25D non-genomic mechanisms of bone formation in osteoblasts. My working hypothesis, which I propose as a new investigator, is that signal transduction triggered by 1,25D acting at a membrane-associated VDR leads to a non-genomic rapid exocytotic response, which is coupled to chloride channel activation in osteoblasts. This explains in part the anabolic effects of the hormone in bone. The first specific aim of this proposal investigates two parallel signal transduction pathways recruited by a membrane-associated VDR in osteoblasts: a) Galpha q/cAMP/PKA/CI- channel phosphorylation/exocytosis, and b) Galpha q/PLC/IP3/calcium/exocytosis. The second specific aim studies the osteoblastic CIC-3 gene, its protein product, and coupling to exocytosis. This will be studied in osteoblasts obtained from VDR WT and KO mice, the latter expressing a rachitic phenotype. Although the primary focus of this proposal is on basic research, the objective is to identify molecular targets in the treatment of bone pathologies characterized by decreased bone mass and mineralization. This typifies skeletal diseases such as osteoporosis and osteomalacia, respectively. Osteoporosis in particular affects a large sector of the aging American population and constitutes a significant economic burden.