Under inflammatory conditions, bone destruction can be linked to excessive activity of bone-resorbing osteoclasts (OCs), which results not only from the differentiation of too many OCs, but also from over- maturation of OCs. While most current bone loss treatments prevent bone loss by reducing OC numbers, it may be better if future therapeutic strategies focus on targeting OC maturation rather than early OC differentiation to avoid inhibiting coupled bone formation that depends on interactions between bone-forming osteoblasts and OCs. In an effort to target maturation, we previously identified immunoglobulin superfamily member 11 (IgSF11) as a novel cell surface receptor that regulates OC differentiation but not new bone formation. To characterize IgSF11 signaling, we analyzed, by mass spectrometry, proteins phosphorylated after IgSF11 activation and identified Pyruvate kinase M2 (PKM2), the enzyme that catalyzes the last step of glycolysis, as a downstream target. This finding highlights a potentially greater than previously known determinative role for metabolic regulation during OC differentiation and inflammatory bone loss. We therefore propose the following specific aims: 1. Examine the role of IgSF11-PKM2 signaling in inflammatory bone loss. We will investigate OC-expressed IgSF11 in the context of inflammatory bone loss by using an LPS-induced model of bone loss. To test the contribution of PKM2-dependent effects, we will treat LPS-induced IgSF11-/- mice with small molecule modulators (TEPP-46, shikonin) of PKM2. Our preliminary data suggests that TEPP- 46 activation of PKM2 reduces DSS-induced bone loss. To examine whether IgSF11 expression affects colitis- associated bone loss, we will perform DSS-induced colitis experiments using IgSF11-deficient mice. We will also perform DSS-induced colitis experiments using IgSF11-/- mice treated with TEPP-46 or shikonin. These studies will be critical to establishing the intersection of IgSF11 and PKM2 contributions to clinically relevant inflammatory bone loss. 2. Characterization of IgSF11-PKM2 signaling mechanisms in osteoclast differentiation. We have formulated a four-step model of IgSF11-PKM2 function during OC differentiation, which we will test with the aid of hCD3-iFL, a retroviral (RV) construct to directly activate intracellular IgSF11 in differentiating OCs. We will first investigate possible crosstalk between RANK and IgSF11-PKM2, which we speculate is mediated by TRAF6-dependent K63-linked polyubiquitination of the IgSF11 scaffold protein PSD- 95. Second, we aim to identify kinases proximal to the IgSF11-PSD-95 complex that phosphorylate PKM2. Third, we will use RV mutants to confirm the importance of various PKM2 modifications, PKM2 allosteric confirmation, and PKM2 subcellular localization to OC differentiation. Finally, PKM2 is a well-characterized enzymatic regulator of glycolysis, so we will employ metabolic assays and inhibitors to confirm the significance of this aspect of PKM2 ...