Molecular regulation of osteoclast maturation

 描述（由申请方提供）：骨稳态由骨形成成骨细胞（OB）和骨吸收破骨细胞（OC）维持。过度的OC活性可导致致病性骨丢失，因此了解控制OC生物学大致三个阶段的分子信号传导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，双膦酸盐和抗RANKL，可能针对早期OC承诺和/或后期活力。虽然抗吸收，长期使用这些治疗可能会导致骨强度受损。这种副作用可能是由于抑制偶联骨形成，这需要OC和OB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OC，而不是早期OC分化。最近针对OC再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再吸收。对缺乏晚期OC因子DC-STAMP或Atp 6v 0 d2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，我们设计了一个筛选方案，以确定与OC成熟相关的基因。最符合我们标准的基因被鉴定为P2 X5，它是嘌呤能受体P2 X亚家族的一员，对此知之甚少。我们现在使用P2 X5-/-小鼠，并初步显示P2 X5-/- OC体外成熟是有缺陷的。这一新的数据可能将P2 X5确定为据报道影响OC功能的P2 X亚家族抑制剂的潜在靶点，并且P2 X5可能代表进一步追求潜在的OC成熟相关治疗策略的必要遗传联系。因此，我们提出以下具体目标：1.研究P2 X5缺乏对破骨细胞发育和功能的影响。为了开始定义OC生物学本身对P2 X5的要求，我们将首先离体评估早期P2 X5-/- OC定型，然后通过基因表达和细胞生物学方法离体询问P2 X5-/- OC成熟。为了检查P2 X5缺乏对体内骨稳态、OC功能和成熟的影响，我们将对在正常、OVX、PTH处理或炎症条件下获得的P2 X5-/-骨和骨切片进行高分辨率显微计算机断层扫描和组织形态测定以及TRAP染色。2.确定破骨细胞生物学背景下P2 X5功能的机制。为了开始确定P2 X 5在OC中通过何种机制发挥作用，我们将评估用各种P2 X亚家族成员以及TM 1和TM 2结构域杂交体逆转录病毒拯救的P2 X 5-/-OC的分化/成熟。此外，我们将研究OC特异性P2 X5与其他P2 X成员的异源寡聚化的发生和潜在重要性。最后，为了确定靶点特异性和开始评估治疗潜力，我们将 检查P2 X5缺乏对OC对P2 X配体ATP和已知P2 X化学抑制剂的敏感性的影响。总之，这些研究应该大大提高我们对OC成熟及其与P2 X5功能关系的理解。

 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.