Cell Adhesion Regulation of Osteoclast Maturation

 描述(申请人提供)：骨平衡由骨形成成骨细胞(OBS)和骨吸收破骨细胞(OCS)的平衡功能维持。过多的OC活性会导致致病性骨丢失，因此了解控制OC发育的承诺、成熟和吸收阶段的分子信号和遗传程序是很重要的。目前的骨丢失治疗针对的是早期OC承诺和/或后期生存能力。长期使用这些治疗方法可能会导致骨强度下降，这可能是由于抑制了偶联骨形成，这需要OCS和OBS之间的积极相互作用。更好的治疗策略可能是以晚期OC生物学为靶点，有利于早期OC分化，事实上，最近的一些临床试验已经成功地采用了靶向抑制吸收的方法，但没有观察到OC数量或偶联骨形成的减少。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。特别是，越来越多的细胞黏附分子现在与骨稳态有关，它们是临床干预的良好潜在靶点。因此，我们设计了一种筛选方案--在本提案中描述--以确定与OC成熟和细胞黏附双重相关的基因。我们现在发现，IgSF11是破骨细胞分化的成熟阶段所必需的，它以前在骨骼中没有报道过的功能。我们已经建立和使用了IGSF11-/-小鼠，初步表明IGSF11-/-OC的体外成熟是有缺陷的，表明可能存在潜在的遗传联系，以进一步寻求与OC成熟相关的治疗策略。因此，我们提出以下具体目标：1.研究IgSF11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IGSF11的要求，我们将首先评估IGSF11-/-OC的体外早期承诺，然后通过基因表达和细胞生物学方法在体外询问IGSF11-/-OC的成熟。为了研究IGSF11缺乏对体内骨稳态、OC功能和成熟度的影响，我们将IGSF11-/-骨和在正常、去卵巢、甲状旁腺素处理或炎症条件下获得的骨切片进行高分辨率显微计算机断层扫描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探讨IgSF11的作用机制。我们将通过混合表达IgSF11和缺失IgSF11的OC培养来确定在OC成熟过程中细胞间的黏附是否需要IgSF11。我们将检测OC-IgSF11缺乏对已知OC相关细胞黏附因子表达和功能的影响。通过突变分析，将分析OCS中逆转录病毒表达的特定IgSF11结构域对同型和异型相互作用和细胞信号的调节。这些实验将不仅探讨IgSF11是否是OC细胞黏附所必需的，而且还将探讨在OC成熟的背景下，IgSF11介导的黏附的特异性和潜在的信号机制。

 DESCRIPTION (provided by applicant): Bone homeostasis is maintained by the balanced functions of 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 commitment, maturation, and resorption phases of OC development. Current bone loss treatments target early OC commitment and/or later viability. Long-term use of these treatments may cause compromised bone strength, possibly due to inhibition of coupled bone formation, which requires positive interplay between OCs and OBs. A better treatment strategy may be to target late-stage OC biology in favor of early OC differentiation, and in fact some recent clinical trials have successfully employed targeted inhibition of resorption without observed diminution of either OC numbers or coupled bone formation. Therefore, better understanding the regulatory factors and mechanisms of OC maturation may be extremely useful for providing better therapeutic targets. In particular, a growing number of cell adhesion molecules are now implicated in bone homeostasis and they are good potential targets for clinical intervention. Therefore, we designed a screening protocol -- described in this proposal -- to identify genes dually associated with OC maturation and cell adhesion. We now identify IgSF11, which has no previously reported function in bone, as being required specifically for progression from the commitment to the mature stages of osteoclast differentiation. We have generated and employed IGSF11-/- mice to preliminarily show that IGSF11-/- OC maturation in vitro is defective, demonstrating a potential genetic link to further pursue OC maturation-related therapeutic strategies. We therefore propose the following specific aims: 1. Investigate the effect of IgSF11 deficiency on osteoclast development and function. To begin to define the requirement for IGSF11 in OC biology per se, we will first assess early IGSF11-/- OC commitment ex vivo, and then interrogate IGSF11-/- OC maturation through gene expression and cell biologic approaches ex vivo. To examine the effects of IGSF11 deficiency on bone homeostasis, OC function and maturation in vivo, we will subject IGSF11-/- 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 IgSF11 function in the context of osteoclast biology. We will determine whether IgSF11 is required for adhesion between cells during OC maturation by mixing IgSF11-expressing and IgSF11- deficient OC cultures. The effects of OC IgSF11 deficiency on expression and function of known OC- associated cell adhesion factors will be assayed. Via mutational analysis, regulation of homo- and hetero-typic interactions and cell signaling by specific IgSF11 domains retrovirally expressed in OCs will be analyzed. These experiments will address not only whether IgSF11 is required for OC cell adhesion, but also the specificity of IgSF11-mediated adhesion and potential signaling mechanisms in the context of OC maturation.