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 maintainedbybone-formingosteoblasts(OBs)andbone-resorbingOCsareperturbedbyinflammatorystimuli. 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.WewillemployPcdh7-/-bonemarrow(BM)cellstoexamineexpressionofknownbiologicalmarkersand cell biological functions, including adhesion, motility, actin ring formation, ruffled border formation, and vesicle trafficking.Pcdh7floxedmiceandBMchimeraswillbegeneratedforthepurposeofmorepreciselyinterrogating OC-versusOB-specific(orother)Pcdh7functionsinthecontextofbonehomeostasis.Thesemicewillalsobe 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 mechanismsofPcdh7molecularfunctionwithinOCbiology.ToinvestigatehowOC-expressedPcdh7protein 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- activatedandhCD3-inducibleretroviral(RV)Pcdh7constructs.First,wewilltestwhetherPcdh7mediatescell- 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 whetherand,ifso,howPcdh7mediatesintracellularsignalingviatheoncoproteinSET.Fourth,wewillemploy siRNA and chemical inhibitors to test the relative contributions of Pcdh7-dependent activation of various signalingpathwaystoPcdh7-mediatedOCadhesionandmaturation.Together,thesestudieswillimproveour understandingofthefunctionofPcdh7proteingenerally,andmorespecifically,howitcontrolsOCmaturation.
Inflammatory bone loss can be induced by excessive activation of bone-resorbing osteoclasts. Osteoclast activation is significantly affected by multiple membrane receptors and their intracellular signaling pathways. Understanding the molecular pathways that control osteoclast maturation and activation is required in order to properly treat inflammatory bone pathologies.
