Calcium in the regulation of osteoclast formation

钙对破骨细胞形成的调节

• 批准号: 7262485
• 负责人: Mone Zaidi
• 金额: $ 44.99万
• 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/7262485
• 关键词: 3-Dimensional; ADP-ribosyl Cyclase; Antibodies; Biological Assay; Biomechanics; Bone Resorption; Calcium; Cell membrane; Cells; Confocal Microscopy; Cyclic ADP-Ribose; Cyclization; Data; Defect; Densitometry; Funding; Image; Immunoblotting; In Vitro; Length; Localized; Mediating; Membrane; Metabolism; Mus; Mutate; Nicotinamide adenine dinucleotide; Osteoblasts; Osteoclasts; Osteoporosis; Phenotype; Protein Overexpression; Recombinants; Regulation; Role; Ryanodine Receptor Calcium Release Channel; Second Messenger Systems; Signal Transduction; Slice; Testing; Transgenic Mice; base; bone; bone loss; in vivo; mutant; novel; osteoclastogenesis; receptor; receptor coupling; research study; response; second messenger

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+环化成环adp -核糖（cADPr），这是第二个信使，从ryanodine受体控制的Ca2+储存中释放Ca2+。在目前的资助期内，我们进行了几项重要观察。首先，我们发现CD38在破骨细胞中大量表达，激活抗体刺激CD38可通过ryanodine受体触发Ca2+释放，从而导致吸收抑制。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再吸收功能增加而导致骨质过度流失。最后，为了研究NAD+诱导的Ca2+信号传导的拓扑要求，我们制作了几个不定位质膜的突变CD38构建体。我们证明了微粒体膜，而不是质膜CD38是NIH3T3细胞中NAD+诱导的Ca2+释放所必需的。我们的假设是，CD38通过作为细胞内的调控因子负调控破骨细胞的形成和功能。NAD吗?受体?结合Ca2+信号的中间代谢。我们将在两个具体目标中探讨这一假设。具体目标1将关注CD38作为破骨细胞形成的负调控的功能。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CD38-/-小鼠的骨表型。我们还将研究CD38-/-小鼠体内破骨细胞的形成，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CD38转移来拯救。特异性目的2将研究CD38作为一种假定的NAD+调节剂是否负向调节成熟破骨细胞的吸收功能。我们将首先通过共聚焦显微镜、免疫印迹和环化酶检测来检测内源性和重组全长CD38以及每个CD38突变体在破骨细胞中的定位和功能。接下来，我们将在全长CD38或其突变构建体感染的破骨细胞中表征NAD+诱导的细胞质Ca2+反应。我们还将研究CD38是否通过增敏微粒体和质膜良嘌呤受体来抑制破骨细胞骨吸收。最后，我们将确定CD38在转基因小鼠中的过度表达是否会导致骨质疏松表型和破骨细胞功能障碍。