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

• 批准号: 9231367
• 负责人: Jae-Hyuck Shim
• 金额: $ 36.85万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 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/9231367
• 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; Hypersensitivity; In Vitro; Laboratories; Mass Spectrum Analysis; Mediating; Molecular; Multivesicular Body; Mus; Osteitis Deformans; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Pathogenesis; Pathologic; Pathway interactions; Phenotype; Process; Property; Protein Sortings; Proteins; Proteomics; Recombinant Proteins; Regulation; Secondary to; Signal Transduction; Stem cells; 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; transcriptome

 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）：骨重建平衡需要成骨细胞（OB）和破骨细胞（OC）活性的时空耦合。然而，这种偶联活性可能限制目前治疗骨质疏松症的有效性，因为增加骨形成的治疗（例如，四氢叶酸）也增加骨吸收，而阻断骨吸收的治疗（例如，二膦酸盐）阻止新骨形成。这种偶联过程的失调也有助于骨量的病理变化，例如骨质疏松症和佩吉特骨疾病（PDB）。PDB是一种影响约5%的老年人的普遍疾病，其特征在于高度夸大的骨重建的局灶性区域。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建疾病的新治疗方法至关重要。在此，我们提出扩展我们先前的发现，即晚期内体分选蛋白CHMP 5是OC中NF-κ B B信号传导和OC/OB偶联的新损伤，目的如下。在目的1中，我们将通过检查CHMP 5缺陷型造血干细胞（HSC）的转移是否导致辐照WT小鼠中的PDB样表型以及WT HSC的转移是否逆转辐照CHMP 5缺陷型小鼠中的PDB样表型来确定CHMP 5的OC特异性缺失对PDB样表型的贡献。此外，为了证实CHMP 5与人类疾病的相关性，我们将检查CHMP 5缺陷是否会导致人类OC中的Pagetic表型。在目标2中，我们将通过进行生物化学研究以确定CHMP 5复合物的失调如何有助于OC中的Pagetic表型，从而建立在我们的初步数据CHMP 5是OC中NF-κ B B信号传导和泛素介导的蛋白酶体降解的关键调节剂的基础上。首先，我们将检查抑制增强的NF-κ B B活性是否可以逆转CHMP 5缺陷小鼠的PDB样表型。此外，我们将确定由CHMP 5复合物在OC使用泛素化蛋白质组学和亲和纯化的质谱相结合的调节蛋白。最后，将在OC中验证所鉴定的蛋白质在NF-κ B B信号传导和破骨细胞生成中的功能.在目标3中，鉴于我们的初步数据，即从CHMP 5缺陷型OC获得的条件培养基增强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/或分化的影响来进一步验证。在完成这些目标后，我们将更好地了解OC中CHMP 5缺失如何有助于PDB的发病机制。由于这种疾病显示出继发于OC活性增强的OB活性的显著增加，因此利用这种机制促进骨形成将是治疗低骨量疾病的有吸引力的方法。