ABSTRACT The overall goal of this supplemental research project is to support the career development of Rebecca Peters, who is a PhD candidate at the University of Maine Graduate School of Biomedical Sciences and Engineering. The sympathetic nervous system (SNS) is known to promote osteoclastogenesis via osteoblast-mediated RANKL production. However, recent work from our laboratory has demonstrated that β-blockers can limit osteoclast activity directly, without influencing RANKL production, in vitro and in mice treated with anabolic parathyroid hormone (PTH) therapy. Furthermore, the efficacy of the cardio-selective β-blocker atenolol to prevent osteoporosis is currently being tested in humans in the Atenolol to Prevent Osteoporosis (APO) trial, but mechanisms of efficacy with relationship to osteoclasts have not been studied in humans or mouse models. In the parent award, we are using genetic mouse models to test the hypotheses that β1AR and β2AR signaling in osteoclasts specifically promote osteoclast differentiation and function. However, the award does not examine the pharmacological properties of β-blockers that allow them to limit osteoclast differentiation directly, nor does it test the translatability of the findings to human osteoclast models. In this supplemental award, we will study the function of β1AR and β2AR in response to β-blockers in human and murine osteoclasts. We will also test whether our in vitro findings of atenolol-mediated suppression of osteoclast differentiation and bone resorption can be recapitulated in vivo. The proposed studies will not overlap with ongoing work on the parent award, but will complement and add translational significance to the findings. Supplement Specific Aim 1: Test the function of βARs in human osteoclasts in vitro. We have found that propranolol potentiates PTH-induced Ca2+ signaling in osteoblasts, however, β-blockers may have a distinct function in osteoclasts. Specifically, we hypothesize that β1AR and β2AR promote intracellular Ca2+-induced NFATc1 activity to support osteoclast differentiation, and that this is prevented by β-blockers. In this aim, we will use primary human peripheral blood mononuclear cell (PBMC) and murine bone marrow-derived osteoclasts to test this hypothesis. Supplement Specific Aim 2: Test the efficacy of atenolol to prevent bone resorption in vivo. We have preliminary data that atenolol also prevents human osteoclast differentiation in vitro, but whether it could be a potential co-therapy for osteoporosis treatment with PTH is unknown. Furthermore, whether atenolol would limit sympathetic nervous system-mediated resorption in vivo is unknown. We hypothesize that atenolol will limit resorption in both cases, indicating it could be clinically useful as an anti- resorptive therapy, whether or not SNS activity is high. To test whether atenolol will prevent bone resorption in the context of PTH therapy and elevated SNS tone, we will treat mice with atenolol and PTH or sal...