Anabolic effects of nitric oxide on osteoblast metabolism revealed by deficiency of argininosuccinate lyase

Previous studies have shown that nitric oxide (NO) supplements may prevent bone loss and fractures in preclinical models of estrogen deficiency. However, the mechanisms by which NO modulates bone anabolism remain largely unclear. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing arginine, the sole precursor for nitric oxide synthase (NOS)-dependent NO synthesis. Moreover, ASL is also required for channeling extracellular arginine to NOS for NO production. ASL deficiency (ASLD) is thus a model to study cell-autonomous, NOS-dependent NO deficiency. Here, we report that loss of ASL leads to decreased NO production and impairment of osteoblast differentiation in osteoblastic lineage cells and primary osteoblasts derived from a hypomorphic mouse model of ASLD (AslNeo/Neo). Osteoblast-lineage specific Asl knockout mice (Osteocalcin Cre; Aslflox/flox) have decreased bone mass and reduced bone formation. Mechanistically, we show that the bone phenotype is at least in part driven by the loss of NO-mediated activation of the glycolysis pathway in osteoblasts that leads to decreased osteoblast differentiation and function. Heterozygous deletion of Caveolin-1, a negative regulator of NO synthesis, restores NO production, osteoblast differentiation, glycolysis, and bone mass in AslNeo/Neo mice. The central role of ASL in bone anabolism and the translational significance of these preclinical studies were further reiterated by studies conducted in induced pluripotent stem cells (iPSCs) from an individual with ASLD. The glycolytic gene expression and the ability to differentiate into osteoblasts which were impaired in iPSCs generated from an individual with ASLD was restored in corrected, isogenic cells. Finally, in a convenient sample of ASLD subjects, the proportion with areal bone mineral density Z-score lower than -2.0 at the lumbar spine was higher as compared to the expected proportions from age- and sex-matched data from the general population. Taken together, our findings suggest that ASLD is a unique genetic model for studying NO-dependent osteoblast function and that the NO-glycolysis pathway may be a new target to modulate bone anabolism.