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（由适用提供）：成骨细胞（OB）和破骨细胞（OC）活性的时空耦合是骨骼重塑的平衡所必需的。然而，这种耦合活性可以限制当前疗法治疗骨质疏松症的有效性，因为增加骨形成的疗法（例如teraparatide）也会增加骨骼的分辨率，以及阻止骨骼分辨率（例如双磷酸盐）的治疗方法。这种耦合过程的失调也有助于骨骼质量的病理变化，例如骨质疏松症和骨骼疾病（PDB）。 PDB是一种流行的疾病，影响了大约5％的基本成年人，其特征是高度夸张的骨骼重塑的焦点区域。因此，了解OC/OB偶联的分子基础对于开发新的骨质流失和其他骨骼重塑疾病的疗法至关重要。在这里，我们建议扩大我们先前的发现，即晚期的内体分类蛋白CHMP5是NF-B信号传导和OC/OB偶联的新型阻尼器，其目标是以下目标。在AIM 1中，我们将通过检查CHMP5缺乏的造血干细胞的转移（HSCS）是否会导致PDB样表型在辐射WT小鼠中导致PDB样表型的转移以及在WT HSC的转移中是否会导致pdb样现场的转移是否会导致PDB样表型在irrAD-CHM中是否会导致PDB样表型在IRRAD-CHM中是否会导致PDB样表型在irrad chm的转移中是否会导致PDB样表型在irrAD-pdb的转移中是否会导致PDB样表型在irrad chm的转移中，从而导致PDB样表型在irrAD-pdb中的转移是否会导致PDB样表型在irrAD的转移中，从而确定OC特异性删除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缺乏症是否会导致人类OC中的雌蕊表型。在AIM 2中，我们将基于我们的初步数据，即CHMP5是NF-B信号传导和泛素介导的OC中OC中蛋白酶体降解的关键调节剂，通过进行生物化学研究来确定CHMP5复合物的失调如何对OCS中的Pagetic表型有效。首先，我们将检查抑制增强的NF-B活性是否可以逆转CHMP5缺乏小鼠的PDB样表型。此外，我们将使用泛素化蛋白质组学和基于亲和力纯化的质谱法的组合来鉴定OC中CHMP5复合物调节的蛋白质。最后，将在OC中验证已鉴定蛋白在NF-B信号传导和破骨细胞生成中的功能。在AIM 3中，鉴于我们的初步数据，从CHMP5缺乏性OCS获得的条件培养基增强了OB活性，我们将确定使用基于HPLC的质谱法促进OB活性的OC衍生耦合因子。这些推定的成骨因子将通过检查过表达和/或敲低对促进OB迁移和/或分化的影响进一步验证。完成这些目标后，我们将更好地了解OC中CHMP5缺失如何促进PDB的发病机理。由于这种疾病显示出急剧活动的急剧增加，而OB活性则是增强的OC活性，因此利用该机制促进骨骼形成将是一种治疗低骨骼质量疾病的有吸引力的方法。