RANK-Associated Inhibitor (RAIN) in Osteoclast Formation

描述(申请人提供)：核因子-kappaB受体激活剂(RANK)及其配体(RANKL，也称为TRANCE/ODF/OPGL)是破骨细胞形成的重要介质，与多种疾病有关，包括类风湿性关节炎、骨质疏松症、骨巨细胞瘤、Paget病、转移性乳腺癌、多发性骨髓瘤和家族性扩张性骨溶解。骨保护素(OPG，又称OCIF/TR1)是一种可溶性诱骗受体，可抑制RANKL与其细胞表面受体RANK的结合。RANK通过与肿瘤坏死因子受体相关因子(TRAF)的相互作用来激活信号通路。缺失RANKL、RANK或TRAF6的小鼠缺乏破骨细胞，出现严重的骨化生，而缺乏OPG的小鼠出现骨质疏松。因此，RANKL和OPG是调节正常骨稳态的控制因子。 RANK的细胞质结构域与TRAF1、2、3、5和6相互作用，我们的实验室描述了与TRAF2、5和6相互作用的RANK的不同区域。为了确定与RANK细胞质结构域相互作用的其他因素，我们使用酵母双杂交方法鉴定了一种新的蛋白质，我们称之为RAIN，用于RANK相关抑制物，因为它具有抑制RANKL介导的破骨细胞形成的能力。我们克隆了小鼠和人的cDNA，并分别含有241和242个残基的开放阅读框架。RAIN是一种新的蛋白质，没有可识别的结构域或基序。RAW264.7(RAW)细胞中RAIN与内源性RANK共沉淀。此外，RAIN与RAW细胞中的TRAF2、TRAF5和TRAF6相互作用。为了了解RAIN的功能，稳定表达RAIN的原始细胞不干扰早期RANKL信号，如NF-kappaB、JNK、ERK或p38MAPK的激活。RANKL刺激后，RAIN表达的细胞呈TRAP+，细胞周期抑制因子p27表达上调，但不能形成多核破骨细胞。因此，雨似乎在破骨细胞分化过程中对融合事件起到了负面调节作用。为了支持这一模型，我们建立了稳定表达反义RAIN的原始细胞。令人惊讶的是，我们观察到破骨细胞数量的增加，这早在第二天就观察到了。此外，RANKL处理原始细胞引起RAIN mRNA和蛋白的诱导，从第二天开始一直持续到第五天。生化证据表明，RAIN可能通过隔离或阻止F-肌动蛋白聚合来发挥作用。因此，我们已经确定了一种新的蛋白质，它与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在破骨细胞形成中的作用，其具体目的如下：(1)明确RAIN和TRAF的分子相互作用；(2)确定RAIN与肌动蛋白聚合相关的生化蛋白；(3)通过靶向基因干扰和表达RAIN的转基因小鼠来确定RAIN的生理作用。RAIN及其在控制破骨细胞形成中的作用的鉴定将为破骨细胞的形成机制提供新的见解，并可能为药物的开发提供新的靶点，旨在防止与代谢性骨疾病相关的有害骨破坏和与溶骨性病变相关的癌症。

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.