Regulation of (Ca2+)i oscillation and osteoclast differentiation by RGS12

描述（由申请人提供）：RANKL诱发的[Ca 2 +]i振荡通过NFAT 2活化途径在破骨细胞分化中发挥开启作用，然而，关于RANKL如何诱发[Ca 2 +] i振荡-NFAT 2途径仍存在问题。有证据表明，胞浆内[Ca ~（2+）]i振荡主要是由细胞外Ca ~（2+）通过多种通道（包括L型和N型通道）内流引起的，而Ca ~（2+）内流是维持振荡的必要条件。Schiff等人（Schiff等人，2000）首次报道RGS 12能够通过其PTB结构域与原代背根神经节神经元培养物中的酪氨酸磷酸化钙通道直接相互作用。RGS蛋白向G蛋白效应物的募集可能代表了G蛋白偶联途径中信号终止的另一种机制。利用差异筛选，我们发现G蛋白信号传导调节因子12基因（regulator of G-protein signaling 12 gene，RGS 12）主要表达于破骨细胞样细胞（osteoclast like cell，OCL）中。使用RNA干扰（RNAi）敲低RGS 12表达抑制RANKL诱导的破骨细胞分化。破骨细胞分化的失败是由于缺乏[Ca 2 +]i振荡和NFAT 2表达。我们的研究进一步揭示了RGS 12直接与N型钙通道相互作用，可能调节[Ca 2 +]i振荡，并且RGS 12与钙敏感受体（CaR）结合。这些体外数据表明，RGS 12，RGS蛋白家族的最大成员和多结构域RGS蛋白，可能在许多信号调节元件中发挥作用。目前，关于RGS 12在体内的功能及其多结构域在破骨细胞分化和骨吸收中的作用和机制还知之甚少。基于我们的初步数据和引用的研究，我们假设RGS 12与钙通道、CaR、G蛋白和其他异源二聚化伴侣相互作用，以调节[Ca 2 +]i振荡的产生并触发破骨细胞分化。为了验证这一假设，我们提出了以下三个具体目标：目标1。利用慢病毒RNAi和过表达系统研究RGS 12在破骨细胞和成熟破骨细胞分化中的作用。目标2.通过对RGS 12异源二聚化伴侣的表征，确定RGS 12在破骨细胞分化过程中的作用机制。目标3。通过Cre/loxP技术对RGS 12基因进行破骨细胞特异性靶向破坏，明确RGS 12在体内骨重建中的作用和机制！

DESCRIPTION (provided by applicant): RANKL-evoked [Ca2+]i oscillations play a switch-on role in osteoclast differentiation through the NFAT2 activation pathway, however, the question remains as to how RANKL evokes the [Ca2+]j oscillation-NFAT2 pathway. Some evidence has shown that cytosolic [Ca2+]i oscillations are generated mainly by influx of extracellular Ca2+ through multiple channels, which include L- and N-type channels, and Ca2+ influx is necessary for maintenance of oscillations. Schiff et al. (Schiff et al., 2000) first reported that RGS12 is capable of direct interaction with the tyrosine-phosphorylated calcium channel in culture of primary dorsal root ganglion neurons through its PTB domain. Recruitment of RGS proteins to G-protein effectors may represent an additional mechanism for signal termination in G-protein-coupled pathways/Using differential screening, we have found that regulator of G-protein signaling 12 gene (RGS12) is predominantly expressed in osteoclast like cells (OCLs). Knockdown of RGS12 expression using RNA interference (RNAi) inhibited the osteoclast differentiation induced by RANKL. The failure of osteoclast differentiation results from the absence of [Ca2+]i oscillations and NFAT2 expression. Our studies further revealed that RGS12 directly interacted with the N type calcium channel to likely regulate [Ca2+]i oscillations and that RGS12 binds with calcium sensing receptor (CaR). These in vitro data suggested that RGS12, the largest member of the RGS protein family and a multi-domain RGS protein, may play roles in numerous signaling regulatory elements. So far, little is known about the in vivo function of RGS12 and the role and mechanism of its multi-domains in osteoclast differentiation and bone resorption. Based on our preliminary data and the cited research, we hypothesize that RGS12 interacts with calcium channels, CaR, G-protein and other heterodimerization partners, to regulate generation of [Ca2+]i oscillations and trigger osteoclast differentiation. To test this hypothesis, we propose the following three Specific Aims: Aim 1. To characterize the role of RGS12 in differentiating osteoclasts and mature osteoclasts by using lentivirus RNAi and overexpression systems. Aim 2. To identify the mechanism of RGS12 action during osteoclast differentiation by characterization of RGS12 heterodimerization partners. Aim 3. To define the role and mechanism of RGS12 in vivo in bone remodeling by osteoclast-specific targeted disruption of the RGS12 gene using Cre/loxP technology!