Development of R-Spondin 1 as an anabolic agent for the treatment of estrogen deficiency-induced bone loss A major challenge in the elderly is osteoporosis and the associated high risk of fracture. One in two women and one in four men >50 years will have an osteoporosis-related fracture in their lifetimes. Age-related bone conditions cause significant morbidity worldwide, particularly among the elderly, and include systemic bone loss (e.g., postmenopausal osteoporosis), focal bone loss, and fractures. Bone is continuously remodeled, and this remodeling process couples osteoclastic bone resorption and osteoblastic bone formation. Various therapeutics are currently used for the treatment of low bone mass (osteopenia or osteoporosis), with the vast majority being antiresorptive agents (e.g., bisphosphonates) that act by limiting osteoclast-mediated bone resorption. However, these agents cannot replace bone that has been lost, and the successful development of therapeutics that can stimulate bone formation would have tremendous implications for achieving increased bone mass and bone strength. Such bone-rebuilding anabolic agents could thus provide important treatment options, not only for conditions of low-bone-mass, but also for fracture healing and other orthopedic applications. Bone tissue adapts to its functional environment by optimizing its morphology for mechanical demand. Among the mechanosensitive cells that recognize and respond to forces in the skeleton are osteocytes, osteoblasts, and mesenchymal progenitor cells (MPCs). Therefore, the ability to use mechanical signals to improve bone health through exercise and devices that deliver mechanical signals is an attractive approach to age- related bone loss; however, the extracellular or circulating mediators of such signals are largely unknown. Using SDS- PAGE separation of proteins secreted by MPCs in response to low magnitude mechanical signals and in-gel trypsin digestion followed by HPLC and mass spectroscopy, we identified secreted proteins up-regulated by vibratory stimulation. We exploited a cell senescence-associated secretory phenotype screen, and reasoned that a subset of vibration-induced proteins with diminished secretion by senescent MPCs will have the capacity to promote bone formation in vivo. We identified one such vibration-induced bone-enhancing (vibe) gene as R-Spondin 1, a Wnt pathway modulator, and demonstrated that it has the capacity to promote bone formation in three mouse models of age-related bone loss. By virtue of their secretory status, some vibe proteins, including R-Spondin 1, may be candidates for pre- clinical development as anabolic agents for the treatment of osteoporosis. The goals of this application are to establish R-Spondin 1 as a pivotal regulator of bone formation in the aging skeleton, and provide evidence that R-spondin 1 represents a promising new therapeutic approach for the anabolic treatment of bone-related disorders, such as post- menopausal osteoporosis. The specific aims of this proposal are to (1) demonstrate a reversal of estrogen deficiency- induced bone loss after treatment with R-Spondin 1; and (2) determine possible short-term adverse effects of R-Spondin 1 on organ function.