Molecular regulation of osteoclast maturation

 描述(申请人提供)：骨稳态由骨形成成骨细胞(OBS)和骨吸收破骨细胞(OCS)维持。过多的OC活性会导致致病性骨丢失，因此了解控制OC生物学大约三个阶段的分子信号和遗传程序是很重要的：承诺、成熟和吸收。目前的骨丢失治疗，如双磷酸盐和抗RANKL，可能针对早期OC承诺和/或晚期生存能力。虽然抗吸收，但长期使用这些治疗可能会导致骨骼强度下降。这种副作用可能是由于抑制了偶联的骨形成，这需要OCS和OBS之间的积极相互作用。因此，更好的治疗策略可能是针对晚期OCS而不是早期OC分化。最近针对OC吸收的临床试验支持这一观点，在不减少OC数量或偶联骨形成的情况下抑制吸收。对缺乏晚期OC因子DC-STAMP或Atp6v0d2的小鼠的研究进一步支持了这一观点。因此，更好地了解OC成熟的调控因素和机制可能对提供更好的治疗靶点非常有用。为此，我们设计了一种筛选方案来识别与OC成熟相关的基因。最符合我们标准的基因被确定为P2X5，它是嘌呤能受体P2X亚家族的成员，人们对此知之甚少。我们现在使用的是P2X5-/-小鼠，初步表明P2X5-/-OC的体外成熟是有缺陷的。这一新的数据可能表明，P2X5可能是已报道的影响OC功能的P2X亚家族抑制剂的潜在靶点，而P2X5可能代表着进一步寻求潜在的OC成熟相关治疗策略的必要遗传联系。因此，我们提出了以下具体目标：1.研究P2X5缺乏对破骨细胞发育和功能的影响。为了开始确定卵巢癌生物学本身对P2X5的需求，我们将首先评估早期的P2X5-/-OC体外承诺，然后通过基因表达和细胞生物学方法在体外询问P2X5-/-OC的成熟。为了研究P2X5缺乏对体内骨稳态、OC功能和成熟度的影响，我们将对正常、去卵巢、PTH处理或炎症条件下获得的P2X5-/-骨和骨切片进行高分辨率显微CT和组织形态计量学以及TRAP染色。2.从破骨细胞生物学的角度确定P2X5的作用机制。为了开始确定P2X5在OCS中的作用机制(S)，我们将评估逆转录病毒拯救的P2X5-/-OCS与不同的P2X亚家族成员以及TM1和TM2结构域杂交体的分化/成熟。此外，我们将研究OC特异性的P2X5与其他P2X成员的异源齐聚的发生及其潜在的重要性。最后，为了确定靶点特异性并开始评估治疗潜力，我们将 研究P2X5缺乏对OC对P2X配体ATP和已知的P2X化学抑制剂的敏感性的影响。总之，这些研究将极大地提高我们对OC成熟及其与P2X5功能的关系的理解。

 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.