Molecular regulation of osteoclast maturation

破骨细胞成熟的分子调控

• 批准号: 9220645
• 负责人: YONGWON CHOI
• 金额: $ 35.2万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9220645
• 关键词: Actins; Address; Adhesions; Adverse effects; Affect; Biological Process; Biology; Bone Marrow Cells; Bone Resorption; Cells; Chemicals; Clinical Trials; Complex; Coupled; Data; Development; Differentiation Antigens; Disease; Eligibility Determination; Exhibits; FOS gene; Family member; Gene Expression; Generations; Genes; Genetic; Homeostasis; Hybrids; In Vitro; Individual; Inflammation; Inflammatory; Ions; Ligands; Link; Molecular; Mus; Mutant Strains Mice; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis prevention; Ovariectomy; P2X-receptor; Pathogenicity; Pathologic; Pathway interactions; Pattern; Phase; Physiological; Play; Proteins; Purinoceptor; Regulation; Reporting; Resolution; Role; Signal Transduction; Specificity; Staining method; Stains; TNFSF11 gene; Testing; Therapeutic; Therapeutic Agents; Tissues; Vesicle; X-Ray Computed Tomography; bisphosphonate; bone; bone loss; bone strength; cathepsin K; cell motility; design; differential expression; experimental study; improved; in vivo; inhibitor/antagonist; member; novel; programs; public health relevance; receptor; therapeutic target; trafficking; treatment strategy; vacuolar H+-ATPase; virtual

 DESCRIPTION (provided by applicant): Bone homeostasis is maintained by bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCs). Excessive OC activity can cause pathogenic bone loss, so it is important to understand the molecular signaling and genetic programs controlling the roughly three phases of OC biology: commitment, maturation, and resorption. Current bone loss treatments, bisphosphonates and anti-RANKL, may target early OC commitment and/or late viability. While anti-resorptive, long-term use of these treatments may cause compromised bone strength. This side effect may be due to inhibition of coupled bone formation, which requires positive interplay between OCs and OBs. Therefore, a better treatment strategy may be to target late-stage OCs rather than early OC differentiation. Recent clinical trials targeting OC resorption support this idea, inhibiting resorption without diminutionof either OC numbers or coupled bone formation. Studies in mice lacking late-stage OC factors DC-STAMP or Atp6v0d2 further support this idea. Therefore, better understanding the regulatory factors and mechanisms of OC maturation may be extremely useful for providing better therapeutic targets. For this reason, we designed a screening protocol to identify genes associated with OC maturation. The gene that best fit our criteria was identified as P2X5, a member of the P2X subfamily of purinergic receptors about which little is known. We are now employing P2X5-/- mice and preliminarily show P2X5-/- OC maturation in vitro is defective. This new data may identify P2X5 as a potential target of P2X subfamily inhibitors reported to affect OC function, and P2X5 may represent the necessary genetic link to further pursue potential OC maturation- related therapeutic strategies. We therefore propose the following specific aims: 1. Investigate the effect of P2X5 deficiency on osteoclast development and function. To begin to define the requirement for P2X5 in OC biology per se, we will first assess early P2X5-/- OC commitment ex vivo, and then interrogate P2X5-/- OC maturation through gene expression and cell biologic approaches ex vivo. To examine the effects of P2X5 deficiency on bone homeostasis, OC function and maturation in vivo, we will subject P2X5-/- bones and bone sections obtained under normal, OVX, PTH-treated, or inflammatory conditions to high-resolution micro- computed tomography and histomorphometry, as well as TRAP staining. 2. Determine mechanisms of P2X5 function in the context of osteoclast biology. To begin to determine by what mechanism(s) P2X5 functions in OCs, we will assess differentiation/maturation of P2X5-/- OCs retrovirally-rescued with various P2X subfamily members as well as TM1 and TM2 domain hybrids. Further, we will examine the occurrence and potential importance of OC-specific P2X5 hetero-oligomerization with other P2X members. Finally, to both determine target specificity and to begin to assess therapeutic potential, we will examine the effects of P2X5 deficiency on OC sensitivity to the P2X ligand ATP and to known P2X chemical inhibitors. Together, these studies should greatly improve our understanding of OC maturation and its relationship to P2X5 function.

 描述（由适用提供）：骨骼体内抑制作用是通过成骨成骨细胞（obs）和骨质骨细胞（OCS）维持的。过度的OC活性会导致致病性骨质流失，因此了解控制OC生物学大约三个阶段的分子信号传导和遗传程序很重要：承诺，成熟和分辨率。当前的骨丢失处理，双膦酸盐和抗裂殖剂，可能针对早期的OC承诺和/或晚期生存能力。虽然反应性，但长期使用这些治疗可能会导致骨骼强度受损。这种副作用可能是由于抑制耦合的骨形成，这需要OC和OBS之间的正相互作用。因此，更好的治疗策略可能是针对后期OC而不是早期的OC分化。针对OC解决方案的最新临床试验支持了这一想法，抑制了分辨率，而没有OC数字或耦合骨形成的尺寸。缺乏晚期OC因子DC-Stamp或ATP6V0D2的小鼠的研究进一步支持了这一想法。因此，更好地理解OC成熟的调节因素和机制对于提供更好的治疗靶标可能非常有用。因此，我们设计了一种筛选协议来识别与OC成熟相关的基因。最适合我们的标准的基因被鉴定为P2X5，P2X5是嘌呤能受体的P2X亚家族的成员，几乎没有知道。我们现在使用P2X5 - / - 小鼠，并初步显示P2X5 - / - OC在体外成熟是有缺陷的。该新数据可以将P2X5识别为报告影响OC功能的P2X亚家族抑制剂的潜在靶标，而P2X5可能代表了进一步购买潜在的OC成熟 - 相关理论策略的必要遗传联系。因此，我们提出以下特定目的：1。研究P2X5缺乏对破骨细胞开发和功能的影响。为了开始定义OC生物学本身的P2X5的要求，我们将首先评估P2X5 - / - OC的早期承诺，然后通过基因表达和细胞生物学方法在体内询问P2X5 - / - OC成熟。为了检验P2X5缺乏对体内骨骼稳态，OC功能和成熟的影响，我们将对在正常，OVX，PTH，PTH治疗或炎症条件下获得的P2X5 - / - 骨骼和骨切片对高分辨率的微分辨率微分辨率的微分造影术和组织型和组织力学以及TRAP染色。 2。确定在破骨细胞生物学背景下P2X5功能的机理。为了通过OC中的机制开始确定哪些机制的功能，我们将评估P2X5 - / - OC的分化/成熟，与各种P2X亚家族成员以及TM1和TM2和TM2结构域的逆转录病毒反应。此外，我们将研究OC特异性P2X5杂音异构化与其他P2X成员的发生和潜在的重要性。最后，为了确定目标特异性并开始评估治疗潜力，我们将 检查P2X5缺乏对OC对P2X配体ATP和已知P2X化学抑制剂的影响。总之，这些研究应大大提高我们对OC成熟的理解及其与P2X5功能的关系。