PROJECT SUMMARY Bone responds to many signals and cues to regulate its mass and quality. During the last funding period, we identified a pathway by which several bone anabolic agents regulate an important bone effector protein, sclerostin. This regulation of sclerostin occurred at a surprisingly rapid time scale (minutes) in bone cells and occurred via post- translational control of sclerostin protein destruction by the lysosome, prior to changes in gene expression. A central integrator of these bone anabolic cues that converged on sclerostin protein destruction was the serine/threonine kinase, CaMKII. To test the role of CaMKII in vivo in bone anabolism, we generated osteoblast-lineage (OCN-Cre) conditional deletion mice of the two Camk2 genes expressed in the osteoblast-lineage, Camk2d and Camk2g. Prior to these deletions, very little was known about CaMKII signaling in bone cells, and what data did exist suggested only a relatively subtle role in osteoblast differentiation. To our surprise, these conditional knockout mice revealed a severe, post-natal skeletal phenotype that included a large decrease in bone mass in the trabecular and cortical compartments, spanning the appendicular and axial skeleton as well as the skull. In addition to low bone mass, these mice exhibited skeletal fragility, markedly reduced mechanical properties, and a surprising defect in phosphate homeostasis. Given the severity of this phenotype, we were unable to challenge this mouse with bone anabolic cues in vivo, although we did demonstrate defective sclerostin control ex vivo in response to some bone anabolic cues. The severity of this skeletal phenotype on this mouse is on par with the deletion of beta-catenin, a key signal integrator for bone formation, in bone cells, and establishes CaMKII as a fundamentally important pathway controlling bone homeostasis. In this application, our goal is to understand the unexpected role of CaMKII in osteoblast and osteocyte physiology. We will test the hypothesis that CaMKII is a node of convergence that plays an essential role in bone physiology, including phosphate homeostasis, the degradation of sclerostin in response to bone anabolic cues, and the post-translational control of key effector proteins in bone. In three aims, we will examine the independent roles of Camk2d and Camk2g in the osteoblast/osteocyte and their individual and combined impacts on osteoblast/osteocyte protein and gene expression; disentangle the molecular contributions to the severe skeletal phenotypes of the Camk2d and Camk2g conditional knockout mice; and resolve the role of CaMKII as a signaling node regulating sclerostin degradation for bone anabolic signals in vivo. In total, these experiments will provide much greater insight into the unexpectedly severe post-natal skeletal phenotype in Camk2d and Camk2g in bone, clarify their respective contributions, provide insight into the major effector proteins driving this multifactorial phenotype, identify t...