Identification of novel regulators governing osteoclast-osteoblast coupling

鉴定控制破骨细胞-成骨细胞偶联的新型调节剂

基本信息

批准号：
9385624
负责人：
Jae-Hyuck Shim
金额：
$ 26.46万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-01 至 2021-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9385624
关键词：
Adult Affect Affinity Chromatography Biochemical Bioinformatics Biology Biomechanics Bone Diseases Bone Matrix Bone Resorption Bone remodeling Cells Charge Complex Conditioned Culture Media Core Facility Coupled Coupling Data Deposition Deubiquitinating Enzyme Development Disease Effectiveness Elderly Equilibrium Forteo Generations Grant High Pressure Liquid Chromatography Human In Vitro Laboratories Mass Spectrum Analysis Mediating Molecular Multivesicular Body Mus Osteitis Deformans Osteoblasts Osteoclasts Osteogenesis Osteoporosis Pathogenesis Pathway interactions Phenotype Process Property Proteins Proteomics Recombinant Proteins Regulation Secondary to Signal Transduction Sorting - Cell Movement TNFSF11 gene Therapeutic Ubiquitin Ubiquitination base bisphosphonate bone bone loss bone mass bone turnover early onset human disease in vivo inhibitor/antagonist knock-down microCT migration mouse model novel osteoblast differentiation osteoclastogenesis osteogenic overexpression public health relevance screening skeletal spatiotemporal stem cells transcriptome

项目摘要

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.

描述（由申请人提供）：骨重塑的平衡需要成骨细胞（OB）和破骨细胞（OC）活性的时空耦合，然而，这种耦合活性可能限制当前治疗骨质疏松症的疗法的有效性，因为该疗法会增加骨质疏松症的治疗效果。骨形成（例如特拉帕肽）也会增加骨吸收，而阻止骨吸收的治疗（例如双磷酸盐）会阻止新骨形成这种耦合的失调。该过程还会导致骨量发生病理变化，例如骨质疏松症和佩吉特骨病 (PDB)，PDB 是一种影响约 5% 老年人的常见疾病，其特点是局部区域高度夸张的骨重塑。 OC/OB 偶联的分子基础对于开发骨丢失和其他骨重塑疾病的新疗法至关重要。在这里，我们建议扩展我们先前的发现，即晚期内体分选蛋白 CHMP5 是一种新型的抑制剂。 OC 中的 NF-κB 信号传导和 OC/OB 耦合具有以下目标：在目标 1 中，我们将通过检查 CHMP5 缺陷型造血干细胞是否转移来确定 OC 特异性删除 CHMP5 对 PDB 样表型的贡献。 HSC）在受辐射的 WT 小鼠中产生 PDB 样表型，以及 WT HSC 的转移是否会逆转受辐射的 CHMP5 缺陷小鼠中的 PDB 样表型此外，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 OC 中的 Pagetic 表型。在目标 2 中，我们将基于 CHMP5 是 NF-κB 的关键调节因子的初步数据。通过进行生化研究来确定 CHMP5 复合物的失调如何影响 OC 中的 Pagetic 表型，从而研究 OC 中的信号传导和泛素介导的蛋白酶体降解。增强的 NF-κB 活性可以逆转 CHMP5 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基于亲和纯化的质谱分析来鉴定 OC 中受 CHMP5 复合物调节的蛋白质。考虑到我们从条件培养基中获得的初步数据，在目标 3 中，将在 OC 中验证 NF-κB 信号传导和破骨细胞生成中所鉴定的蛋白质。 CHMP5 缺陷的 OC 增强 OB 活性，我们将使用基于 HPLC 的质谱法鉴定促进 OB 活性的 OC 衍生的偶联因子，这些假定的成骨因子将通过检查过度表达和/或敲低对促进的影响来进一步验证。完成这些目标后，我们将更好地了解 OC 中的 CHMP5 缺失如何促进 PDB 的发病机制，因为这种疾病表现出继发于 OB 活性的显着增加。增强OC活性，利用这种机制促进骨形成将是治疗低骨量疾病的一种有吸引力的方法。