Inactivating including Ligament early-onset mutations in human ENPP1 results in aberrant soft tissue and skeletal mineralization disorders, Autosomal Recessive Hypophosphatemic Rickets (ARHR2) Ossification of the Posterior Longitudinal (OPLL), and Generalized Arterial Calcification of Infancy (GACI) in homozygous deficiency, and osteoporosis (EOOP) in , ENPP1 haploinsufficiency. ENPP1 deficienct patients therefore exhibit paradoxical mineralization, with concurrent low bone mass and progressive calcifications in kidneys, tendons, and vasculature. Paradoxical mineralization is also present in the general medical population in aging patients, and in patients with chronic kidney disease mineral and bone disorder (CKD-MBD). Fracture risk and high mortality in CKD-MBD patients has not changed in the last 20 years despite significant progress in other skeletal disorders, illustrating continued serious limitations in the understanding and treatment of CKD-MBD. The study of ENPP1 deficiency and its inherent paradoxical mineralization, will serve to identify and validate signaling pathways by which ENPP1 regulates bone mass; we strongly believe that this approach will inform longstanding issues hampering our understanding of paradoxical mineralization, enabling better therapeutic agents for these patients. ENPP1 is the only human enzyme which generates PPi, a strong inhibitor of accrual of bone mineral in the extant bone matrix. One would anticipate, therefore, that disorders inducing low PPi would result in increased bone mass and volume, and not the low bone mass observed in humans and mice. Therefore, the mechanism by which ENPP1 induces low bone mass is not apparent based on an understand of the enzyme's catalytic activity alone. In response to this paradox, we hypothesize the presence of catalytically independent ENPP1 signaling pathways regulating mammalian bone mass. This proposal seeks to (a) establish the pathways involved, (b) define the catalytically dependent and independent genetic and protein signal transduction pathways by which ENPP1 regulates bone mass, and (c) quantitate their effect on bone fragility, microarchitecture, and growth, as well as on biomarkers associated with bone mineralization. To accomplish these Aims, we will use novel animal models which uncouple ENPP1 protein signaling from ENPP1 catalysis and novel and proprietary biologics we have designed and engineered to activate ENPP1 catalytic and catalytic-independent signaling in vivo. The investigative team has a strong history of success as evidenced by several recent publications supporting the overall hypothesis, the specific aims, and the bench to bedside development of a novel biologics treating GACI and ARHR2 that have entered the clinic, thus validating the scientific rigor, experimental approach, and scientific impact of this proposal.