Identification of novel regulators governing osteoclast-osteoblast coupling

 描述(由申请人提供)：成骨细胞(OB)和破骨细胞(OC)活动的时空耦合是骨重建平衡所必需的。然而，这种偶联作用可能会限制目前治疗骨质疏松症的方法的有效性，因为增加骨形成的治疗方法(例如Terparatide)也会增加骨吸收，而阻止骨吸收的治疗方法(例如双膦酸类)会阻止新骨形成。这种偶联过程的失调也导致了骨量的病理变化，如骨质疏松症和帕吉特氏病(PDB)。PDB是一种流行的疾病，影响大约5%的老年人，其特点是局部高度夸大的骨重塑。因此，了解OC/OB偶联的分子基础对于开发骨丢失和其他骨重塑障碍的新治疗方法至关重要。在此，我们建议扩展我们先前的发现，即晚期内体分选蛋白CHMP5是一种新的抑制OCS中的NF-B信号和OC/OB偶联的因子，其目的如下。在目标1中，我们将通过检测CHMP5缺陷的造血干细胞(HSCs)的转移是否导致辐射后WT小鼠的PDB样表型，以及WT HSCs的转移是否逆转辐射CHMP5缺陷小鼠的PDB样表型，来确定OC特异性缺失CHMP5对PDB样表型的贡献。此外，为了确认CHMP5与人类疾病的相关性，我们将检查CHMP5缺乏是否会导致人类OCS的寻呼表型。在目标2中，我们将在我们的初步数据的基础上，通过执行生化研究来确定CHMP5复合体的失调如何影响OCS的寻呼表型，从而对B信号和泛素介导的OCS蛋白酶体降解起关键调节作用。首先，我们将检验抑制增强的NF-CHMP5B活性是否可以逆转缺陷小鼠的PDB样表型。此外，我们将结合泛素化蛋白质组学和亲和纯化质谱仪鉴定OCS中受CHMP5复合体调控的蛋白质。最后，鉴定的蛋白质在NF-B信号和破骨细胞形成中的功能将在嗅鞘细胞中得到验证。在目标3中，根据我们的初步数据，从CHMP5缺乏的OCS获得的条件培养液可以增强OB活性，我们将利用高效液相色谱-质谱法鉴定促进OB活性的OC衍生偶联因子(S)。这些假定的成骨因子将通过检测过度表达和/或敲除在促进OB迁移和/或分化方面的作用而进一步得到验证。完成这些目标后，我们将更好地了解CHMP5在OCS中缺失是如何在PDB的发病机制中起作用的。由于这种疾病表现出继发于OC活性增强的OB活性的显著增加，利用这一机制来促进骨形成将是治疗低骨量疾病的一种有吸引力的方法。

 DESCRIPTION (provided by applicant): Spatiotemporal coupling of the activity of osteoblasts (OB) and osteoclasts (OC) is required for balance in bone remodeling. However, this coupling activity can limit the effectiveness of current therapies to treat osteoporosis, as therapies that increase bone formation (e.g. teraparatide) also increase bone resorption, and treatments that block bone resorption (e.g. bisphosphonates) arrest new bone formation. Dysregulation of this coupling process also contributes to pathological changes in bone mass, such as osteoporosis and Paget's disease of bone (PDB). PDB is a prevalent disorder affecting approximately 5% of elderly adults and is characterized by focal regions of highly exaggerated bone remodeling. Hence, understanding the molecular basis of OC/OB coupling is central to developing new treatments for bone loss and other disorders of bone remodeling. Here, we propose to expand our prior discovery that the late endosomal sorting protein CHMP5 is a novel dampener of NF-B signaling and OC/OB coupling in OCs with the following aims. In Aim 1, we will determine the contribution of OC-specific deletion of CHMP5 to PDB-like phenotypes by examining whether transfer of CHMP5-deficient hematopoetic stem cells (HSCs) results in PDB-like phenotypes in irradiated WT mice and whether transfer of WT HSCs reverses PDB-like phenotypes in irradiated CHMP5-deficient mice. Additionally, to confirm relevance of CHMP5 to human disease, we will examine whether CHMP5 deficiency can result in Pagetic phenotypes in human OCs. In Aim 2, we will build upon our preliminary data that CHMP5 is a key regulator of NF-B signaling and ubiquitin-mediated proteasomal degradation in OCs by performing biochemical studies to determine how dysregulation of the CHMP5 complex contributes to Pagetic phenotypes in OCs. First, we will examine whether inhibition of enhanced NF-B activity can reverse PDB-like phenotypes of CHMP5-deficient mice. Additionally, we will identify the proteins regulated by the CHMP5 complex in OCs using a combination of ubiquitination proteomics and affinity purification-based mass spectrometry. Finally, functions of the identified proteins in NF-B signaling and osteoclastogenesis will be validated in OCs. In Aim 3, given our preliminary data that the conditioned medium obtained from CHMP5-deficient OCs enhances OB activity, we will identify the OC-derived coupling factor(s) that promote OB activity using HPLC-based mass spectrometry. These putative osteogenic factors will be further validated by examining effects of overexpression and/or knockdown on promoting OB migration and/or differentiation. Upon completion of these aims, we will better understand how CHMP5 deletion in OCs contributes to the pathogenesis of PDB. As this disorder displays dramatic increases in OB activity that occur secondary to enhanced OC activity, harnessing this mechanism to promote bone formation would be an attractive approach for the treatment of disorders of low bone mass.