Mechanisms of RANKL Mediated Osteoclast Activation

描述（由申请人提供）：炎性骨溶解，发生在类风湿性关节炎和骨科植入物松动中，反映了破骨细胞（OC）的加速募集和激活。因此，发现局部炎症招募和激活OCs的方法对于预防这种致残性并发症至关重要。我们的首要目标是详细说明细胞因子，如TNF， RANKL和M-CSF介导骨质流失的机制。我们通过描述各种TNF靶细胞对破骨细胞生成过程的贡献，并详细描述TNF促进破骨细胞生成的许多细胞内事件，实现了我们之前应用的目标。在目前的建议中，我们转向所有病理性骨吸收所必需的细胞因子，即RANKL，其晶体结构与其受体RANK复杂，我们在当前的资助期内解决了。RANKL对OC的分化至关重要，它也增强了成熟OC的骨吸收活性，我们发现这一过程依赖于细胞因子对细胞骨架适配器蛋白paxillin和小GTPase Rac的调节。此外，我们对RANKL/RANK晶体结构的解析，以及RANKL与诱饵受体OPG的富含半胱氨酸结构域的解析，使我们能够开发基于结构的RANKL拮抗剂来阻止OC中的RANK信号传导。鉴于抑制RANK信号通路已被证明具有治疗益处，这些新的RANKL抑制剂可能会影响病理性骨溶解的治疗。因此，我们假设：1)RANKL的OC激活是通过paxillin介导的；2)RANKL的OC激活是通过Rac介导的；3)基于结构的RANKL拮抗剂会阻止OC的发育和功能。因此，我们的具体目标是：1)确定paxillin介导OC中RANKL激活的机制；2)确定RANKL激活OC中Rac的机制；3)设计RANKL和OPG的变体，破坏OC中RANK信号传导。公共卫生相关性。破骨细胞是破坏骨骼的细胞，因此，它们活动的增加导致了大多数形式的病理性骨质流失，如骨质疏松症或类风湿关节炎。激活破骨细胞的关键分子是RANK配体（RANKL）。本提案的目的是深入了解RANKL激活破骨细胞的机制，并设计候选药物来抑制这一事件，从而防止病理性骨质流失。

DESCRIPTION (provided by applicant): Inflammatory osteolysis, as occurs in rheumatoid arthritis and orthopedic implant loosening, reflects accelerated osteoclast (OC) recruitment and activation. Hence, discovering the means by which local inflammation recruits and activates OCs is central to preventing this crippling complication. Our overriding goal has been to detail the mechanisms by which cytokines, such as TNF, RANKL and M-CSF mediate bone loss. We have achieved the aims of our previous application by characterizing the contributions of various TNF target cells to the osteoclastogenic process and detailing many of the intracellular events by which TNF promotes osteoclastogenesis. In the present proposal we turn to the cytokine essential for all pathological bone resorption, namely RANKL, whose crystal structure, in complex with its receptor, RANK, we resolved in the current funding period. While RANKL is essential for OC differentiation, it also enhances the bone resorptive activity of the mature OC, a process we find depends upon the cytokine's regulation of the cytoskeletal adaptor protein, paxillin and the small GTPase, Rac. Furthermore, our resolution of the RANKL/RANK crystal structure and that of RANKL with the cysteine-rich domains of OPG, the decoy receptor, positions us to develop structure-based RANKL antagonists to arrest RANK signaling in the OC. Given that inhibition of the RANK signaling pathway has proven therapeutic benefit, these novel RANKL inhibitors may impact the treatment of pathological osteolysis. Thus, we hypothesize that: 1) RANKL activation of the OC is mediated via paxillin 2) RANKL activation of the OC is mediated via Rac and 3) Structure-based RANKL antagonists will arrest OC development and function. Our Specific Aims are therefore to 1) Determine the mechanisms by which paxillin mediates RANKL activation of the OC 2) Determine the mechanisms by which RANKL activates Rac in the OC and 3) Engineer variants of RANKL and OPG that disrupt RANK signaling in the OC. PUBLIC HEALTH RELEVANCE. Osteoclasts are the cells which destroy bone, and thus, their increased activity is responsible for most forms of pathological bone loss such as osteoporosis or that attending rheumatoid arthritis. The key molecule which activates ostoclasts is known as RANK ligand (RANKL). The purpose of this proposal is to gain insight into the mechanism by which RANKL activates osteoclasts and to design candidate drugs which will inhibit this event and consequently, prevent pathological bone loss.