The search for molecular targets and pathways that can be manipulated to improve bone properties is a highly active area of investigation. Recently, particular interest has been expressed in targeting biomolecules that can augment mechanical signaling in bone; the next generation of osteoporosis drugs is likely to work in conjunction with physical activity/loading in order to disproportionately direct new bone formation to the skeletal loci that need it most (i.e., those loci that endure the greatest strains and are at the greatest risk of failure). The WNT signaling pathway has emerged as a key regulator of bone mass and strength, but also of bone cell mechanotransduction. Recent clinical approval of the osteoporosis therapy Evenity™ (i.e., Romosozumab-aqqg) has highlighted the utility and potency of targeting the WNT pathway to improve skeletal properties and fracture resistance. However, the FDA issued a black-box label to the product because of its side effects on cardiovascular function and health. Recent work suggests that the effects of WNT stimulation on bone formation can be isolated from those on cardiovascular function, but the molecular mechanisms driving these disparate processes are completely unknown. The goal of the present is application is to understand precisely how LRP5 and LRP6--two structurally related cell surface proteins that function as WNT co-receptors--differ in their skeletal and non-skeletal contexts, with the goal of exploiting signaling differences to improve clinically based WNT therapeutic outcomes (e.g., sclerostin inhibition). Among the key questions addressed are: (1) Do LRP5 and LRP6 exhibit different predilection, or selectivity, to different WNT ligands in bone cells in vivo? (2) Does LRP5 activation trigger different downstream signaling networks than activated LRP6? (3) Is LRP6 involved in mechanotransduction in bone (overuse and/or disuse)? (4) Can activation of LRP5 alone (through sclerostin antibody-mediated inhibition or by gain-of-function mutation in the receptor) improve bone properties to a greater extent than activation of LRP6 alone? and (5) Are cardiovascular effects differentially affected by LRP5 vs LRP6 signaling (in isolation)? We will use cutting-edge mouse models, microscopic techniques, single cell and spatial transcriptomic approaches, mechanotransduction models, and radiographic/ histologic/biochemical approaches to reveal the underlying biology and therapeutic potential/differences of LRP5 and LRP6 in bone tissue. The project is a continuation of the close collaboration between the Robling (Indiana University) and Warman (Harvard University) labs, an extremely fruitful partnership for more than 18 yrs. We have assembled a unique combination of expertise, resources, biological models and tools, and technical innovation to elucidate, distinguish, and exploit the differing roles of LRP5 and LRP6 in bone biology and disease. Project Summary/Abstract