Mechanisms of IKK Regulation of Basal and Inflammatory Osteoclastogenesis

描述(申请人提供)：转录因子核因子(B)对于破骨细胞的形成是必不可少的，被认为是炎症反应的关键调节因子。核因子-B的激活需要诱导一个由IKK1、IKK2和IKK(/Nemo)组成的大I(B)激酶(IKK)复合体。IKK2和IKK1分别激活典型的(p50/p65)和非规范的(p52/relB)核因子-B通路。最近的研究表明，IKK2和IKK1是破骨细胞形成所必需的，但其作用机制尚不清楚。此外，IKK2被认为是由肿瘤坏死因子、内毒素和其他促炎剂(通过经典的核因子-B途径)诱导的炎症事件的关键介质，而对于IKK1在这些反应中的作用则知之甚少。在过去的几年里，我们研究了在溶骨反应中调节破骨细胞活性的分子途径，如炎性骨溶解和关节炎骨侵蚀，并揭示了调节破骨细胞前体中核因子-B激活的关键调控步骤。在这一点上，我们发现，在多个水平上破坏核因子-B的激活可以减少破骨细胞的形成和炎症性骨侵蚀。具体地说，我们已经证明，使用显性-阴性形式的I(B，抵抗IKK2的磷酸化；以及最近使用诱饵Nemo结合结构域(NBD)抑制IKK与Nemo的组装，在体外和体内都是抑制破骨细胞生成和骨侵蚀的成功方法。最重要的是，我们有初步的证据表明，1)IKK1缺失的OCPs对RANKL诱导的破骨细胞分化的失败可以通过重新引入IKK1的cDNA构建物来挽救，以及2)组织特异性缺失的OCPs(使用CD11b-cre)导致骨骼畸形的小鼠。很明显，在缺乏IKK1或IKK2的情况下，破骨细胞的生成受到损害，然而，IKKS在基础和炎症性破骨细胞生成中的确切信号转导机制(S)仍然缺乏。此外，任何一种蛋白质对这些反应的相对贡献是不确定的。众所周知，IKK1和IKK2有相当大的序列和结构域相似之处，尽管它们保持着不同的和不重叠的功能。因此，我们假设IKK1和IKK2通过替代的和经典的核因子-B激活途径来不同地调节基础和炎症性破骨细胞的形成。因此，通过研究IKKS在破骨细胞中分子信号转导的细节，利用相关基因的种系和特定组织的缺失，阐明IKKS在破骨细胞形成中的单独作用，将使我们能够设计针对IKK1和IKK2的策略和选择性抑制剂，这可能有助于调节破骨细胞的形成和减轻各种骨吸收疾病的炎性骨溶解。因此，我们建议研究以下特定目标：1)确定IKK1和IKK2在基础和炎症性破骨细胞形成中的分子作用。2)确定酪氨酸磷酸化调控破骨细胞系中IKK2的命运和活性的机制及其对破骨细胞生成的影响。3)体内测定IKK1和IKK2在炎性骨溶解中的作用。与公共健康相关：转录因子NF-B对破骨细胞的形成是必不可少的，并调节炎症反应。核因子-B被丝氨酸激酶、I(B激酶(Ikk)-1、ikk-2和ikk(/Nemo))激活。IKK1或IKK2基因缺失导致破骨细胞缺陷和炎症反应。利用IKK1和IKK2缺失的小鼠，我们建议研究IKK1和IKK2的分子结构域在破骨细胞形成和炎性骨溶解中的作用，就像炎症性关节炎的情况一样。我们的提案有望找到新的选择性抗骨溶解疗法。

DESCRIPTION (provided by applicant): The transcription factor NF-(B is essential for osteoclastogenesis and is considered a key modulator of inflammatory responses. Activation of NF-(B entails induction of a large I(B kinase (IKK) complex that comprises IKK1, IKK2, and IKK(/NEMO. IKK2 and IKK1 activate the canonical (p50/p65) and non- canonical (p52/relB) NF-(B pathways, respectively. Recent studies implicate IKK2 and IKK1 as essential for osteoclastogenesis, yet the mechanisms underling this function, remain unclear. In addition, IKK2 is considered as a key mediator of inflammatory events induced by TNF, LPS, and other pro-inflammatory agents (through the classical NF-(B pathway), whereas less is known about the role of IKK1 is such responses. In the past few years we have investigated molecular pathways regulating osteoclast activity in osteolytic responses, such as inflammatory osteolysis and arthritic bone erosion, and unveiled critical regulatory steps modulating NF-(B activation in osteoclast precursors. In this regard, we find that disruption of NF-(B activation at multiple levels attenuates osteoclastogenesis and inflammatory bone erosion. Specifically, we have shown that administration of dominant-negative forms of I(B(, that resist phosphorylation by IKK2; and most recently inhibition of IKK assembly with NEMO, using a decoy NEMO binding domain (NBD) peptide, all are successful approaches to inhibit osteoclastogenesis and bone erosion, in vitro and in vivo. Most importantly, we have preliminary evidence that, 1) failure of RANKL-induced osteoclastogenesis by IKK1-null OCPs is rescued by re-introduction of IKK1 cDNA constructs, and that 2) tissue-specific deletion of IKK2 in OCPs (using CD11b-cre) results with skeletally deformed mice. It is evident that osteoclastogenesis is impaired in the absence of IKK1 or IKK2, however, the precise mechanism(s) underlying signaling of IKKs in basal and inflammatory osteoclastogenesis remain scarce. Moreover, the relative contribution of either protein to these responses is indefinite. It is also known that IKK1 and IKK2 share considerable sequence and domain similarities, albeit they maintain distinct and non-overlapping functions. Thus, we hypothesize that IKK1 and IKK2 differentially regulate basal and inflammatory osteoclastogenesis through the alternative and classical NF-(B activation pathways. Hence, clarifying the individual roles of IKKs in osteoclastogenesis by investigating the details of their molecular signaling in osteoclasts, utilizing germ-line and specific tissue deletions of the relevant genes, will enable us to design strategies and selective inhibitors directed against IKK1 and IKK2 that may be useful for regulating osteoclastogenesis and alleviating inflammatory osteolysis in various bone resorptive disorders. Thus, we propose to investigate the following specific aims: 1) Determine the molecular role of IKK1 and IKK2 in basal and inflammatory osteoclastogenesis. 2) Determine the mechanism by which tyrosine phosphorylation regulates the fate and activity of IKK2 in the osteoclast lineage and it's impact on osteoclastogenesis. 3) Determine the role of IKK1 and IKK2 in inflammatory osteolysis, in vivo. PUBLIC HEALTH RELEVANCE: The transcription factor NF-(B is essential for osteoclastogenesis and modulates inflammatory responses. NF-(B is activated by the serine kinases, I(B kinase (IKK)-1, IKK-2, and IKK(/NEMO. Gene deletions of IKK1 or IKK2 led to defects in osteoclasts and inflammatory responses. Using IKK1 and IKK2-null mice, we propose to investigate the molecular domain contributions of IKK1 and IKK2 to osteoclastogenesis and inflammatory osteolysis, as is the case in inflammatory arthritis. Our proposal holds promise to identify novel selective anti-osteolytic therapies.