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

描述(申请人提供)：RANKL诱导的[Ca~(2+)]i振荡通过NFAT2激活途径在破骨细胞分化中起启动作用，然而，关于RANKL如何引起[Ca~(2+)]j振荡-NFAT2途径的问题仍然存在。有证据表明，细胞内[Ca~(2+)]_i振荡主要由细胞外Ca~(2+)通过多个通道产生，其中包括L通道和N-型通道，而Ca~(2+)内流是维持振荡所必需的。希夫等人。(Schiff et al.，2000)首次报道RGS12能够通过其PTB结构域与原代培养背根神经节神经元的酪氨酸磷酸化钙通道直接相互作用。通过差异筛选，我们发现G蛋白信号转导调控基因12(RGS12)主要在破骨细胞样细胞(OCL)中表达。RNA干扰(RNAi)下调RGS12的表达可抑制RANKL诱导的破骨细胞分化。破骨细胞分化失败的原因是缺乏[Ca~(2+)]i振荡和NFAT2的表达。我们的研究进一步表明，RGS12直接与N型钙通道相互作用，可能调节[Ca~(2+)]i振荡，并且RGS12与钙敏感受体(CAR)结合。这些体外实验数据表明，RGS12是RGS蛋白家族中最大的成员，是一个多结构域的RGS蛋白，可能在许多信号调节元件中发挥作用。到目前为止，对RGS12的体内功能及其多个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。根据我们的初步数据和引用的研究，我们假设RGS12与钙通道、CAR、G-蛋白和其他异源二聚体伙伴相互作用，调节[Ca2+]i振荡的产生，并触发破骨细胞分化。为了验证这一假说，我们提出了以下三个具体目标：目的1.利用慢病毒RNAi和过表达系统研究RGS12在区分破骨细胞和成熟破骨细胞中的作用。目的2.通过对RGS12异二聚体的鉴定，探讨RGS12在破骨细胞分化过程中的作用机制。目的3.利用Cre/loxP技术，通过破骨细胞特异性靶向阻断RGS12基因，明确RGS12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!