SUMMARY. Bone loss is accelerated in menopausal women, and more generally in both men and women with aging. Current anti-resorptive and osteoanabolic therapies are generally prescribed only after pronounced bone loss, and all can have adverse clinical effects. Dual-acting agents that are both osteoanabolic and anti-resorptive may offer novel approaches to increase bone mass. The Pyk2 tyrosine kinase is dual-acting, both decreasing osteoblast (OB) activity and increasing osteoclast (OC) resorption. Despite a similar increase in cortical microarchitecture in male and female Pyk2-/- mice, only females exhibit increased cancellous bone mass and mechanical properties, suggesting sex-specific differences in tissue level properties. In preliminary studies, we found that ovariectomized Pyk2-/- female mice supplemented with 17β-estradiol (E2) have additively higher bone mass than WT, and that ulnar loaded Pyk2-/- males (females not tested) exhibit higher bone formation rate, suggesting enhanced osteogenic responses in vivo. Pyk2-/- OBs have enhanced mineralizing activity in vitro, which is further stimulated by 17β-estradiol (E2) and raloxifene, a clinically-approved selective estrogen receptor modulator (SERM). Pyk2-/- OBs exhibit reduced levels of estrogen receptor, ERα, due to its ubiquitin-proteasome degradation, and we found that ERβ antagonists prevent the E2-stimulated increase in Pyk2-/- OB activity, suggesting an increase in E2-ERβ signaling. On the other hand, Pyk2-/- OCs exhibit defective OC resorption activity, and in the presence of E2, OC formation and survival are reduced compared to WT OCs. Our Specific Aims will test the hypothesis that Pyk2 acts via distinct E2-ERα/ERβ-regulated mechanisms to exert dual- modulating effects that repress OB activity and promote OC resorption. In Aim 1, we will demonstrate the role of Pyk2 in the anabolic versus catabolic arms of the bone remodeling cycle by examining 4-, 12- and 21-month mice in which Pyk2-deletion is targeted to either OBs with collagen type 1a, 2.3-Cre (Pyk2Col2.3) or mature OCs with cathepsin K-Cre (Pyk2CatK). In addition, Pyk2Col2.3 mice will be subject to tibial anabolic loading, while Pyk2CatK mice will be examined after hindlimb unloading-induced bone loss. In Aim 2, we will determine if bone mass is preserved after sex steroid depletion (gonadectomy) of 4-month male/female Pyk2Col2.3 mice (due to elevated osteogenesis) and Pyk2CatK mice (due to reduced resorption), and if E2 or raloxifene will additively improve bone mass. We will dissect the Pyk2 mechanisms that regulate ERα degradation/stability, and effects on ERα/ERβ dimerization, and E2-ERβ signaling in OBs and OBs in vitro. In Aim 3, we will establish the non-inferiority of a Pyk2-selective inhibitor in preventing/reversing age-associated decline of bone mass and mechanical properties in 12-month C57BL/6 female mice, compared to a broad-acting inhibitor of focal adhesion kinases, and raloxifene. Clinical translational impact ...