NF-kB SUBUNITS p65 AND RelB IN OSTEOCLASTS

破骨细胞中的 NF-kB 亚基 p65 和 RelB

• 批准号: 7196934
• 负责人: DEBORAH J VEIS
• 金额: $ 30.18万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7196934
• 关键词: acetylation; apoptosis; bone; lead; nuclear factor kappa beta; phosphorylation

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.

描述（由申请人提供）：破骨细胞（OC）对于钙稳态和结构骨完整性至关重要，但其不适当的激活是导致许多疾病（包括骨质疏松症、类风湿性关节炎和癌症骨转移）显著发病的原因。RANKL介导巨噬细胞谱系细胞分化为OC并诱导NF-κ B。在NF-κ B家族中有5个亚基（p65、RelB、c-rel、p50和p52），每个亚基携带DNA结合Rel同源结构域，并且转录由同源和异源二聚体介导。NF-κ B的激活通过两种不同的途径发生。经典途径涉及IkappaB激酶β（IKK β）依赖性的IkappaB α降解和活性NF-κ B二聚体（主要是p65/p50）快速易位到细胞核中。此外，p65的磷酸化和乙酰化以信号依赖性方式发生，并进一步调节NF-κ B活性。旁路途径由NIK控制，NIK激活IKK α，促使其前体p100产生活性NF-κ B亚基p52，p52与RelB一起在数小时内进入细胞核并保留数天。因此，RANKL暴露通过分别刺激经典和旁路途径导致NF-κ B的急性和长期激活，每种途径控制不同的异源二聚体。我们发现，缺乏p65，经典途径的关键转换器，导致OC前体细胞凋亡，在体外，和降低OC响应RANKL，在体内。相反，缺乏RelB，替代途径的独特亚基，阻断OC分化，在体外，并减少病理性骨吸收，在体内，在细胞凋亡的情况下。此外，一个亚基的过表达不能补偿另一个亚基的缺失，表明这些NF-κ B亚基的不同功能。我们假设：1）在体内，p65控制OC分化和存活，RelB控制OC分化，2）p65和RelB的转录激活（TA）和Rel同源（RH）结构域的特定组分赋予其在OC存活和分化中的独特功能，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）通过检查缺乏p65或RelB的小鼠中的基础和RANKL刺激的骨表型，确定体内OC分化和存活中对p65和RelB的要求，2）确定p65和RelB的TA和RH结构域的哪些组分赋予OC存活和分化其不同的功能，3）定义翻译后p65乙酰化和磷酸化事件，这对其RANKL下游的功能至关重要。由于NF-κ B是现有药物（如蛋白体抑制剂硼替佐米）和目前正在开发的其他药物的靶点，因此了解其在生理环境（如骨骼）中的特定途径至关重要。我们希望这项研究将有助于更好地合理设计药物，以针对特定的病理生理。