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.
A better understanding of a novel molecular pathway regulating osteoclast/osteoblast coupling will take advantage of our mouse model to identify novel osteoclast-derived coupling factor(s) that promote osteoblast activity. Such factors may be of use in the development of anabolic therapeutics for bone loss.
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