Calcium in the regulation of osteoclast formation

CD 38（ADP-核糖基环化酶）催化NAD+环化为环状ADP-核糖（cADPr），cADPr是从兰尼碱受体门控的Ca 2+储存中释放Ca 2+的第二信使。在本报告所述期间，我们提出了几项重要意见。首先，我们发现，CD 38在破骨细胞中大量表达，其激活抗体的刺激通过兰尼碱受体触发Ca 2+释放，导致再吸收抑制。其次，我们发现CD 38-/-小鼠表现出严重的骨质疏松症，其特征在于由于破骨细胞形成和吸收功能增加而导致的过度骨丢失。最后，为了研究NAD+诱导的Ca 2+信号传导的拓扑要求，我们制备了几种不定位于质膜的突变的CD 38构建体。我们证明，微粒体膜，而不是质膜CD 38是必需的NAD+诱导的Ca 2+释放在NIH 3 T3细胞。我们的假设是，CD 38负调控破骨细胞的形成和功能，作为细胞内？NAD？受体？连接中间代谢和Ca 2+信号传导。我们将在两个具体目标中探讨这一假设。具体目标1将集中于CD 38作为破骨细胞形成的负调节的功能。我们将使用密度测定、三维pQCT成像、组织形态测定和生物力学测试进一步表征CD 38-/-小鼠的骨表型。我们还将检查CD 38-/-小鼠中的体外破骨细胞形成，更重要的是，确定细胞表型（a）是否通过支持成骨细胞介导，以及（B）是否可以通过腺病毒CD 38转移来拯救。具体目标2将研究CD 38作为一种假定的NAD+调节剂是否负调节成熟破骨细胞的吸收功能。我们将首先研究的本地化和功能的内源性和重组全长CD 38和每个CD 38突变体的破骨细胞共聚焦显微镜，免疫印迹和环化酶测定。我们接下来将描述用全长CD 38或其突变构建体感染的破骨细胞中NAD+诱导的胞浆Ca 2+反应。我们还将研究CD 38是否通过致敏微粒体和质膜ryanodine受体来抑制骨陷窝试验中的骨吸收。最后，我们将确定转基因小鼠中CD 38的过度表达是否会导致骨硬化表型和破骨细胞功能障碍。

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.