NF-kB SUBUNITS p65 AND RelB IN OSTEOCLASTS

描述(申请人提供)：破骨细胞(OCs)对钙稳态和骨结构完整性至关重要，但其不适当的激活导致许多疾病的显著发病率，包括骨质疏松症、类风湿性关节炎和癌症骨转移。RANKL介导巨噬细胞向OCS分化，并诱导核因子-kappaB。核因子-kappaB家族有5个亚基(p65、RelB、c-Rel、p50和p52)，每个亚基都具有DNA结合的Rel同源结构域，转录由同源和异源二聚体介导。核因子-kappaB的激活有两条不同的途径。经典途径包括依赖于IkappaB激酶β(IKKbeta)的IkappaBalpha的降解和活性的NF-kappaB二聚体的快速移位，主要是p65/p50。此外，p65的磷酸化和乙酰化以信号依赖的方式发生，并进一步调节NF-kappaB的活性。另一条途径是由Nik控制的，它激活IKKalpha，促使从其前体P100产生活性的NF-kappaB亚单位p52，它与RelB一起在几个小时内到达细胞核，并保持数天。因此，RANKL暴露通过分别刺激经典途径和替代途径，导致NF-kappaB的急性和长期激活，每条途径控制着不同的异源二聚体。我们发现，经典途径的关键转导蛋白LASK P65在体外可导致OC前体细胞凋亡，在体内可降低OC对RANKL的反应性。相反，缺乏替代途径的独特亚单位RelB，在体外会阻止OC的分化，并在体内减少病理性的骨再分配，而不是在没有细胞凋亡的情况下。此外，一个亚基的过度表达不能弥补另一个亚基的缺失，这表明这些核因子-kappaB亚基具有不同的功能。我们假设：1)在体内，P65控制OC的分化和存活，而RelB控制OC的分化；2)P65和RelB转录激活(TA)和REL同源(RH)结构域的特定成分在OC的存活和分化中具有不同的功能；3)P65的翻译后修饰(乙酰化和磷酸化)对其在RANKL下游的功能至关重要。因此，我们建议：1)通过研究缺乏p65或RelB的小鼠的基础和RANKL刺激的骨表型，确定p65和RelB在OC分化和存活中的需求；2)确定p65和RelB的TA和RH结构域的哪些成分对OC的存活和分化具有不同的功能；3)定义翻译后的p65乙酰化和磷酸化事件，这些事件对其RANKL下游的功能至关重要。由于核因子-kappaB是现有药物的靶点，如蛋白酶体抑制剂Bortezomib和其他目前正在开发的药物，因此了解其在生理环境中的特定途径至关重要，如骨骼。我们期望这项研究将有助于更好地合理设计针对特定病理生理学的药物。

DESCRIPTION (provided by applicant): Osteoclasts (OCs) are critical for calcium homeostasis and structural bone integrity, but their inappropriate activation is responsible for significant morbidity in many diseases, including osteoporosis, rheumatoid arthritis, and cancer metastasis to bone. RANKL mediates differentiation of macrophage lineage cells into OCs and induces NF-kappaB. There are 5 subunits in the NF-kappaB family (p65, RelB, c-rel, p50, and p52) each bearing the DNA-binding Rel homology domain, and transcription is mediated by homo-and -heterodimers. Activation of NF-kappaB occurs by two distinct routes. The classical pathway involves IkappaB kinase beta (IKKbeta)-dependent degradation of IkappaBalpha and rapid translocation of active NF-kappaB dimers, primarily p65/p50, into the nucleus. Additionally, phosphorylation and acetylation of p65 occur in a signal-dependent manner, and further modulate NF-kappaB activity. The alternative pathway is controlled by NIK which activates IKKalpha, prompting generation, from its precursor p100, of the active NF-kappaB subunit p52, which travels to the nucleus with RelB over several hours and remains for days. Thus, RANKL exposure leads to both acute and prolonged activation of NF-kappaB by stimulating the classical and alternative pathways, respectively, with each controlling different heterodimers. We find that lack p65, the key transducer of the classical pathway, leads to OC precursor apoptosis, in vitro, and a decreased OC response to RANKL, in vivo. In contrast, lack of RelB, the unique subunit of the alternative pathway, blocks OC differentiation, in vitro, and diminishes pathological bone resporption, in vivo, in the absence of apoptosis. Additionally, overexpression of one subunit cannot compensate for the absence of the other subunit, indicating distinct functions for these NF-kappaB subunits. We hypothesize that: 1) in vivo, p65 controls OC differentiation and survival, and RelB controls OC differentiation, 2) specific components of the transcriptional activation (TA) and Rel homology (RH) domains of p65 and RelB confer their distinct functions in OC survival and differentiation, 3) post- translational modifications of p65 (acetylation and phosphorylation) are critical for its function downstream of RANKL. We therefore propose to: 1) define the requirements for p65 and RelB in OC differentiation and survival, in vivo, by examining the basal and RANKL-stimulated bone phenotype in mice lacking p65 or RelB, 2) determine which components of the TA and RH domains of p65 and RelB confer their distinct functions on OC survival and differentiation, 3) define the post-translational p65 acetylation and phosphorylation events that are critical for its function downstream of RANKL. Because NF-kappaB is a target of existing drugs such as the proteosome inhibitor bortezomib, and other drugs currently in development, it is critically important to understand its specific pathways in physiological contexts such as bone. We expect that this study will contribute to better rational design of drugs to target specific pathophysiology.