PROJECT SUMMARY/ABSTRACT – PROJECT 3 Nitric oxide (NO) is an important signaling molecule that influences a wide range of biological processes, including bone metabolism. Recently, we utilized argininosuccinate lyase deficiency (ASLD) as a model to study cell-autonomous, nitric oxide synthase (NOS)-dependent NO deficiency. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing arginine, the sole precursor for NOS-dependent NO synthesis. Moreover, ASL is also required for channeling extracellular arginine to NOS for NO production. Using ASLD as a model, we reported that NO promotion of bone formation is associated with stimulation of glycolysis during osteoblast differentiation. Accumulating evidence suggests that osteoblasts utilize glucose metabolism via glycolysis as their main energy source. Importantly, some of the anabolic agents used to increase bone mass, such as Wnt proteins have also been shown to stimulate glycolysis during osteoblast differentiation, thus suggesting an essential role of glycolysis in bone anabolism. The overall goal of this project is to identify the mechanisms by which NO stimulates bone formation in physiological conditions and during mechanical loading. We also will examine whether NO effects on glycolysis are responsible for effects on bone mass. We reported that NO-deficient cells had downregulated mRNAs encoding components of the glycolysis pathway, suggesting that NO may modulate transcription factors that regulate expression of genes in the glycolysis pathway. Moreover, NO-induced stimulation of glycolysis may be involved in load-induced bone formation, a process in which abundant work has implicated NO as a critical mediator. The central hypothesis of this project is that NO production by osteoblast-lineage cells promotes osteoblast differentiation and bone formation by stimulating glycolysis. We will test this hypothesis with three specific aims. Aim 1 will determine whether NO promotes bone formation and glycolysis in osteoblasts by activating cAMP-responsive element binding protein (CREB). Aim 2 will determine whether genetic upregulation of glycolysis is sufficient to stimulate bone formation or rescue the bone mass deficiency in osteoblast-specific Asl knockout mice. Aim 3 will determine whether NO produced from osteoblasts and osteocytes is required for load-induced anabolism. Successful completion this project should advance knowledge of the molecular mechanisms underlying the effects of NO on bone. Furthermore, it may provide new insights into therapeutic targets for bone anabolism.