Friday, May 9, 2025
5/9/2025
Protocadherin 7 and Osteoclast Maturation - Inflammation is known to cause bone destruction by excessive osteoclast (OC) activity in patients with inflammatory diseases, such as periodontitis. To address the underlying causes of such inflammation-related bone loss, it is important to understand how the cellular and molecular mechanisms of bone homeostasis maintained by bone-forming osteoblasts (OBs) and bone-resorbing OCs are perturbed by inflammatory stimuli. Targeting OC maturation rather than differentiation is of particular interest and provides an added benefit of avoiding unintentionally inhibiting new bone formation. However, identifying promising therapeutic targets of OC maturation will require greater understanding of its mechanisms of regulation. Cell adhesion is a physiologic process critical to both OC maturation and its hallmark feature, multinucleation. In the course of screening potential genes that regulate OC maturation in vitro, we identified a cell adhesion-related gene, Pcdh7, a protocadherin member of the cadherin superfamily. We have now generated Pcdh7-/- mice for the purpose of further studying Pcdh7 in OC maturation and inflammatory responses, and therefore propose the following specific aims: 1. Investigate the role of Pcdh7 in OC differentiation, function, and inflammatory bone loss. We will employ Pcdh7-/- bone marrow (BM) cells to examine expression of known biological markers and cell biological functions, including adhesion, motility, actin ring formation, ruffled border formation, and vesicle trafficking. Pcdh7floxed mice and BM chimeras will be generated for the purpose of more precisely interrogating OC- versus OB-specific (or other) Pcdh7 functions in the context of bone homeostasis. These mice will also be employed to confirm the importance of OC-expressed Pcdh7 in the context of inflammatory bone loss and immune responses that occur after LPS treatment or ligature-induced periodontitis. Together, these studies should elucidate the cell-specific roles of Pcdh7 in OC maturation and pathologic bone loss. 2. Investigate mechanisms of Pcdh7 molecular function within OC biology. To investigate how OC-expressed Pcdh7 protein regulates cell adhesion and/or signal transduction, we will test a four-step model. For each step, we will test OC maturation, cell adhesion, and activation of signaling pathways, and will employ both physiologically- activated and hCD3-inducible retroviral (RV) Pcdh7 constructs. First, we will test whether Pcdh7 mediates cell- cell interactions that activate Pcdh7 intracellular signaling by separately track WT and Pcdh7-/- OCs in mixed heterotypic OC cultures. Second, we will test the effects of cytoplasmic domain truncation isoforms of Pcdh7 by assaying physiologic expression in OCs and then by RV-expressing isoforms in OCs. Third, we will test whether and, if so, how Pcdh7 mediates intracellular signaling via the oncoprotein SET. Fourth, we will employ siRNA and chemical inhibitors to test the relative contributions of Pcdh7-dependent activation of various signaling pathways to Pcdh7-mediated OC adhesion and maturation. Together, these studies will improve our understanding of the function of Pcdh7 protein generally, and more specifically, how it controls OC maturation.
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