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）催化 NAD+ 环化为环状 ADP-核糖 (cADPr)，后者是从兰尼碱受体门控 Ca2+ 库中释放 Ca2+ 的第二信使。在当前的资助期间，我们做出了几项重要的观察。首先，我们发现 CD38 在破骨细胞中大量表达，并且激活抗体的刺激会通过兰尼碱受体触发 Ca2+ 释放，从而抑制吸收。其次，我们发现CD38-/-小鼠表现出严重的骨质疏松症，其特征是由于破骨细胞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，我们制作了几种不定位于质膜的突变 CD38 构建体。我们证明微粒体膜（而不是质膜 CD38）对于 NIH3T3 细胞中 NAD+ 诱导的 Ca2+ 释放是必需的。我们的假设是 CD38 通过充当细胞内“NAD”来负向调节破骨细胞的形成和功能。受体？耦合 Ca2+ 信号传导的中间代谢。我们将在两个具体目标中探讨这一假设。具体目标 1 将重点关注 CD38 作为破骨细胞形成负调节的功能。我们将使用密度测定、3 维 pQCT 成像、组织形态测定和生物力学测试进一步表征 CD38-/- 小鼠的骨表型。我们还将检查 CD38-/- 小鼠的离体破骨细胞形成，更重要的是，确定细胞表型 (a) 是否是通过支持成骨细胞介导的，以及 (b) 可以通过腺病毒 CD38 转移来挽救。具体目标 2 将研究 CD38 作为假定的 NAD+ 调节剂是否负向调节成熟破骨细胞的再吸收功能。我们将首先通过共聚焦显微镜、免疫印迹和环化酶测定来检查破骨细胞中内源性和重组全长 CD38 以及每个 CD38 突变体的定位和功能。接下来我们将描述感染全长 CD38 或其突变构建体的破骨细胞中 NAD+ 诱导的胞质 Ca2+ 反应。我们还将研究 CD38 是否通过使微粒体和质膜兰尼碱受体敏感而在坑试验中抑制破骨细胞骨吸收。最后，我们将确定转基因小鼠中 CD38 过度表达是否会导致骨硬化表型和破骨细胞功能障碍。