Calcium in the regulation of osteoclast formation

钙对破骨细胞形成的调节

• 批准号: 6725184
• 负责人: Mone Zaidi
• 金额: $ 46.11万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6725184
• 关键词: Adenoviridae; CD38 molecule; biological signal transduction; bone imaging /visualization /scanning; calcium channel; calcium ion; cell differentiation; densitometry; genetically modified animals; laboratory mouse; osteoblasts; osteoclasts; osteoporosis; pathologic process; phenotype; physiologic bone resorption; protein localization; transfection

CD38 (ADP-ribosyl cyclase) catalyses the cyclization of NAD+ to cyclic ADP-ribose (cADPr), a second messenger that releases Ca2+ from ryanodine receptor-gated Ca2+ stores. During the current funding period, we have made several key observations. Firstly, we showed that CD38 is expressed in abundance in the osteoclast, and its stimulation by an activating antibody triggers Ca2+ release via ryanodine receptors resulting resorption inhibition. Second, we found that the CD38-/- mouse displays profound osteoporosis characterized by excessive bone loss due to increased osteoclast formation and resorptive function. Finally, to study the topological requirements for NAD+-induced Ca2+ signaling, we made several mutated CD38 constructs that did not localize the plasma membrane. We demonstrated that microsomal membrane, rather that the plasma membrane CD38 is necessary for NAD+-induced Ca2+ release in NIH3T3 cells. Our hypothesis is that CD38 negatively regulates osteoclast formation and function by acting as an intracellular ?NAD? receptor? that couples intermediary metabolism for Ca2+ signaling. We will explore this hypothesis in two specific aims. Specific aim 1 will focus on the function of CD38 as a negative regulation of osteoclast formation. We will further characterize the bone phenotype of CD38-/- mice using densitometry, 3-dimensional pQCT imaging, histomorphometry, and biomechanical testing. We will also examine ex vivo osteoclast formation in CD38-/- mice, and more importantly, determine whether the cellular phenotype (a) is mediated via supporting osteoblasts, and (b) can be rescued by adenoviral CD38 transfer. Specific aim 2 will investigate whether CD38, as a putative NAD+ regulator, negatively regulates the resorptive function of mature osteoclasts. We will first examine the localization and function of endogenous and recombinant full-length CD38 and each CD38 mutant in the osteoclast by confocal microscopy, immunoblotting and cyclase assays. We will next characterize NAD+-induced cytosolic Ca2+ responses in osteoclasts infected with full length CD38 or its mutated constructed. We will also investigated whether CD38 inhibits osteoclastic bone resorption in the pit assay by sensitizing both microsomal and plasma membrane ryanodine receptors. Finally, we will determine whether CD38 over-expression in transgenic mice results in an osteopetrotic phenotype and dysfunctional osteoclasts.

CD38（ADP-核糖基环化酶）将NAD+的环化催化为环状ADP-ribose（CADPR），这是一种从ryanodine受体门控的Ca2+存储中释放Ca2+的第二信使。在当前的资金期间，我们进行了几个关键观察。首先，我们表明CD38在破骨细胞中以丰度表达，并且通过激活抗体触发Ca2+​​通过ryanodine受体释放而刺激，从而导致抑制作用。其次，我们发现CD38 - / - 小鼠表现出严重的骨质疏松症，其特征是由于骨细胞的形成和吸收功能增加而导致骨质流失过多。最后，为了研究NAD+诱导的Ca2+信号传导的拓扑要求，我们制作了几个突变的CD38构建体，这些构建体没有定位质膜。我们证明了微粒体膜，而是质膜CD38对于NAD+诱导的NIH3T3细胞中的Ca2+释放是必需的。我们的假设是，CD38通过充当细胞内的NAD来负调节破骨细胞的形成和功能？受体？该伴侣将CA2+信号传导中介代谢。我们将以两个具体目标探讨这一假设。特定的目标1将集中于CD38的功能，作为破骨细胞形成的负调节。我们将使用光密度测定法，3维PQCT成像，组织形态测定法和生物力学检测进一步表征CD38 - / - 小鼠的骨表型。我们还将检查CD38 - / - 小鼠中的体内破骨细胞形成，更重要的是，确定是否通过支持成骨细胞介导细胞表型（a），并且可以通过腺病毒CD38转移来挽救（b）。具体的目标2将调查CD38作为推定的NAD+调节剂是否对成熟破骨细胞的吸收功能进行负调节。我们将首先通过共聚焦显微镜，免疫印迹和循环酶测定法检查全源性和重组全长CD38和每个CD38突变体的定位和功能。接下来，我们将表征由全长CD38或其突变构建的破骨细胞中NAD+诱导的胞质Ca2+反应。我们还将研究CD38是否通过敏化微粒体和质膜ryanodine受体来抑制凹坑测定中的破骨骨吸收。最后，我们将确定转基因小鼠中的CD38过表达是否会导致骨质术表型和功能障碍的整骨细胞。