Mechanisms and function of the microtubule podosome connection in osteoclasts

描述(申请人提供)：破骨细胞吸收增加导致多种骨骼疾病，无论是全身性(例如骨质疏松症、Paget病、甲状旁腺功能亢进症)还是局部(类风湿性关节炎、牙周病、多发性骨髓瘤、肿瘤骨溶解等)。尽管存在几种抗吸收药物，但有证据表明存在有害的效果。因此，更好地了解破骨细胞(OC)和骨吸收的生物学仍然是临床上最重要的。我们的实验室在OC生物学和骨吸收的详细分子理解方面投入了大量资金。这项工作最显著的特点之一，得到了许多其他实验室的支持，证明了OC与骨附着的关键重要性，这种附着是由一种特殊的黏附结构--含有肌动蛋白的足体--介导的，在OCS中，该足体专门组织在外围带(肌动蛋白环)中，当OCS在骨骼上并主动吸收时，它最终组织在密封区中。足体带的形成是OC特有的特征。一个重要的突破是证明了微管(MTS)在将足体组织成肌动蛋白环和密封区从而在骨吸收中发挥的关键作用(Destaing等人)。2004年；Gil-Henn等人，2007年；Purev等人，2009年)。这一应用的主要目的是进一步阐明MTS连接和调节足小体的分子机制，特别是从簇状到外周带的转变和骨吸收。我们的初步数据揭示了MTS、TiP蛋白EB1、皮质蛋白和足体之间的功能相互作用，这是形成足体带所必需的。我们的建议侧重于MTS末端的分子调控，以及它们与OCS中的肌动蛋白和足体肌动蛋白调节蛋白的相互作用。这将有助于理解调控足小体的定位和周转的机制及其在外周带中的组织，这是OC特有的必要步骤，建立密封区本身是骨吸收所必需的。我们建议：1-确定TIPS蛋白EB1在足体中将MTS与肌动蛋白动力学联系起来的作用。2-确定Cortactin(及其近的同源物HS1)及其与EB1的相互作用在足体MTS和肌动蛋白动力学之间的联系中的作用。3-分析Src和HDAC6，即酪氨酸磷酸化和乙酰化，在足体中MTS和肌动蛋白动态之间的联系中的作用。这些研究可能会为调节骨吸收开辟新的治疗途径，并可能对骨质疏松症、溶骨性疾病以及癌症的治疗产生影响，因为足体和内侧足骨之间存在相似之处。 公共卫生相关性：破骨细胞吸收增加导致多种骨骼疾病，无论是全身性(例如骨质疏松、Paget病、甲状旁腺功能亢进症)还是局部(类风湿性关节炎、牙周病、多发性骨髓瘤、原发或转移性肿瘤骨溶解等)。本应用程序的主要目标是进一步阐明 破骨细胞附着和吸收骨的分子机制，以更好地了解破骨细胞和骨吸收的生物学，以设计新的和更安全的药物。

DESCRIPTION (provided by applicant): Increased osteoclastic resorption is responsible for multiple bone diseases, whether systemic (osteoporosis, Paget's disease, hyper-parathyroidism for instance) or local (rheumatoid arthritis, periodontal disease, multiple myeloma, tumor osteolysis etc.). Despite the existence of several anti- resorptive drugs, evidence of unwanted effects is emerging. It therefore remains of the utmost clinical importance to better understand the biology of the osteoclast (OC) and of bone resorption. Our laboratory has been heavily invested in the detailed molecular understanding of OC biology and bone resorption. One of the most salient features of this work, supported by many other laboratories, is the demonstration of the critical importance of the attachment of the OC to bone, mediated by a specialized adhesion structure, the actin-containing podosome, which in OCs is specifically organized in a peripheral belt (the actin ring) and ultimately in a sealing zone when OCs are on bone and actively resorbing. The formation of a podosome belt is an OC-specific feature. An important breakthrough has been the demonstration of the critical role played by microtubules (MTs) in the organization of podosomes into an actin ring and a sealing zone and consequently in bone resorption (Destaing et al. 2004; Gil-Henn et al., 2007; Purev et al., 2009). The primary goal of this application is to further elucidate the molecular mechanisms by which MTs are connected to and regulate the podosomes, and in particular the transition from clusters to a peripheral belt, and bone resorption. Our preliminary data revealed a functional interaction between MTs, the +Tip protein EB1, cortactin and podosomes, required for the formation of podosome belts. Our proposal focuses on the molecular regulation of the + ends of MTs and of their interaction with actin and actin-regulating proteins in podosomes in OCs. This will help understand the mechanisms that regulate the localization and turnover of podosomes and their organization in a peripheral belt, an OC-specific and necessary step in the establishment of the sealing zone, itself required for bone resorption. We propose to: 1- Determine the role of the +TIPs protein EB1 in linking MTs to actin dynamics in podosomes. 2- Determine the role of cortactin (and its close homolog HS1) and its interaction with EB1 in the link between MTs and actin dynamics in podosomes. 3- Analyze the role of Src and HDAC6, i.e. Tyrosine phosphorylation and acetylation, in the link between MTs and actin dynamics in podosomes. The studies may open new therapeutic avenues to regulate bone resorption, with potential impact on the treatment of osteoporosis, osteolytic diseases and also cancer, given the similarities between podosomes and invadopodia. PUBLIC HEALTH RELEVANCE: Increased osteoclastic resorption is responsible for multiple bone diseases, whether systemic (osteoporosis, Paget's disease, hyper-parathyroidism for instance) or local (rheumatoid arthritis, periodontal disease, multiple myeloma, primary or metastatic tumor osteolysis etc.). The primary goal of this application is to further elucidate the molecular mechanisms by which osteoclast attach to and resorb bone to better understand the biology of the osteoclast and of bone resorption to design new and safer drugs.