RANK-Associated Inhibitor (RAIN) in Osteoclast Formation

描述（由申请人提供）：NF-κ B受体激活剂（RANK）及其配体（RANKL，也称为TRANCE/ODF/OPGL）是破骨细胞生成的重要介质，并与各种疾病有关，包括类风湿性关节炎、骨质疏松症、骨巨细胞瘤、佩吉特病、转移性乳腺癌、多发性骨髓瘤和家族性膨胀性骨质溶解。骨保护素（OPG，也称为OCIF/TR 1）是一种可溶性诱饵受体，可抑制RANKL与其细胞表面受体RANK结合。RANK通过与肿瘤坏死因子受体相关因子（TRAF）的相互作用介导信号通路的激活。RANKL、RANK或TRAF 6缺失的小鼠缺乏破骨细胞并发生严重的骨硬化症，而缺乏OPG的小鼠发生骨质疏松症。因此，RANKL和OPG是调节正常骨稳态的主导因子。 RANK的胞质结构域与TRAF 1、2、3、5和6相互作用，我们的实验室描述了RANK与TRAF 2、5和6相互作用的不同区域。为了鉴定与RANK胞质结构域相互作用的其他因子，我们使用酵母双杂交方法鉴定了一种新的蛋白质，我们将其命名为RAIN，即RANK相关抑制剂，因为其能够抑制RANKL介导的破骨细胞形成。克隆了小鼠和人cDNA，它们分别含有241和242个残基的开放阅读框。RAIN是一种新的蛋白质，没有可识别的结构域或基序。RAIN与RAW 264.7（RAW）细胞中的内源性RANK共沉淀。此外，RAIN与RAW细胞中的TRAF 2、TRAF 5和TRAF 6相互作用。为了理解RAIN的功能，稳定表达RAIN的RAW细胞不干扰早期RANKL信号传导，如NF-κ B、JNK、ERK或p38 MAPK激活。然而，当用RANKL刺激时，RAIN表达细胞未形成多核破骨细胞，尽管细胞为TRAP+且细胞周期抑制剂p27上调。因此，RAIN似乎在破骨细胞分化过程中充当融合事件的负调节剂。为了支持该模型，我们建立了稳定表达反义RAIN的RAW细胞。令人惊讶的是，我们观察到破骨细胞数量增加，这早在第2天就观察到了。此外，RAW细胞的RANKL处理引起RAIN mRNA和蛋白的诱导，其从第2天开始并持续至第5天。生化证据表明，RAIN可能通过隔离或阻止F-肌动蛋白聚合而起作用。因此，我们已经确定了一种新的蛋白质，与RANK和TRAF相互作用，并可能控制多核破骨细胞的形成。 我们建议扩展这些研究，以进一步了解RAIN在破骨细胞生成中的功能，通过追求以下具体目标：（1）确定RAIN和TRAFs的分子相互作用;（2）确定RAIN与肌动蛋白聚合有关的生化蛋白;（3）通过靶向基因破坏和表达RAIN的转基因小鼠来确定RAIN的生理作用。RAIN的鉴定及其在控制破骨细胞形成中的功能将为破骨细胞形成机制提供新的见解，并可能为开发旨在预防与代谢性骨病相关的不必要的骨破坏和与溶骨性病变相关的癌症的药物提供新的靶点。

DESCRIPTION (provided by applicant): Receptor activator of NF-kappaB (RANK) and its ligand (RANKL, also known as TRANCE/ODF/OPGL) are essential mediators of osteoclastogenesis and have been implicated in various diseases, which include rheumatoid arthritis, osteoporosis, giant cell tumor of bone, Paget's disease, metastatic breast cancer, multiple myeloma, and familial expansile osteolysis. Osteoprotegerin (OPG, also known as OCIF/TR1) is a soluble, decoy receptor that inhibits RANKL from binding to its cell surface receptor RANK. Activation of signaling pathways by RANK is mediated through its interaction with tumor necrosis factor receptor-associated factors (TRAFs). Mice deleted of RANKL, RANK, or TRAF6 lack osteoclasts and develop severe osteopetrosis while mice lacking OPG develop osteoporosis. Thus, RANKL and OPG are the governing factors that regulate normal bone homeostasis. The cytoplasmic domain of RANK interacts with TRAF1, 2, 3, 5, and 6, and our laboratory described the distinct regions of RANK that interact with TRAF2, 5, and 6. In an effort to identify other factors that interact with the cytoplasmic domain of RANK, we used a yeast two-hybrid approach and identified a novel protein, which we termed RAIN, for RANK-Associated Inhibitor, for its ability to inhibit RANKL-mediated osteoclast formation Both mouse and human cDNAs were cloned and contain an open reading frame of 241 and 242 residues, respectively. RAIN is a novel protein with no identifiable domains or motifs. RAIN coprecipitates with endogenous RANK in RAW264.7 (RAW) cells. Furthermore, RAIN interacts with TRAF2, TRAF5, and TRAF6 in RAW cells. To understand the function of RAIN, RAW cells stably expressing RAIN did not interfere with early RANKL signaling such as NF-kappaB, JNK, ERK, or p38 MAPK activation. However, RAIN expressing cells did not form multinucleated osteoclasts when stimulated with RANKL, although the cells were TRAP+ and the cell cycle inhibitor p27 was upregulated. Thus, it appears RAIN acts as a negative regulator of the fusion event during osteoclast differentiation. In support of this model, we established RAW cells stably expressing anti-sense RAIN. Surprisingly, we observed increased osteoclast number, which was observed as early as day 2. Additionally, RANKL treatment of RAW cells caused induction of RAIN mRNA and protein, which begins on day 2 and continues through day 5. Biochemical evidence suggests that RAIN may function by sequestering or preventing F-actin polymerizatio. Thus, we have identified a novel protein that interacts with RANK and TRAFs, and presumably controls the formation multi-nucleated osteoclasts. We propose to extend these studies to further our understanding of RAIN's function in osteoclastogenesis by pursing the following specific aims: (1) define the molecular interactions of RAIN and TRAFs; (2) determine the biochemical proteins of RAIN with respect to actin polymerization; and (3) determine the physiological role of RAIN by targeted gene disruption and transgenic mice expressing RAIN. The identification of RAIN and it function in controlling osteoclast formation will provide new insights into the mechanism of osteoclast formation and may provide a novel target for the development of pharmaceutical agents aimed at preventing unwanted bone destruction associated with metabolic bone disorders and cancers associated with osteolytic lesions.