ENPP1 Roles and ATP Metabolism in Cementogenesis

Proper pyrophosphate regulation is critical in mineralization processes. Pyrophosphate (PPi) is a direct inhibitor of hydroxyapatite crystal growth, thus inhibiting mineralization. A main source of PPi is ATP, which is hydrolyzed by ectonucleotide pyrophosphatase phosphodiesterases (ENPP), including ENPP1. ENPP1 is expressed in bone and cementum, and when mutated or lost, results in skeletal and dental abnormalities. In particular, ENPP1 has been linked with Generalized Arterial Calcification of Infancy (GACI), which is characterized by vascular and joint calcifications. Notably, hypercementosis has been observed in individuals with GACI. Understanding ENPP1 domains and roles will allow us to identify aspects of ENPP1 modulation to promote cementum formation. The studies proposed here were developed to elucidate functions of ENPP1, focusing on ENPP1 modulation of cementogenesis through local regulation of ATP and PPi. Periodontal disease, a global burden with significant psychosocial and financial consequences, features destruction of the periodontium (cementum, periodontal ligament, alveolar bone). Abnormal PPi regulation (as exhibited by individuals with ENPP1 mutations) can lead to alterations in the periodontium, specifically cementum. Low extracellular PPi conditions lead to hypercementosis, e.g, when ENPP1 is mutated. This observation led us to pursue ENPP1 modulation as a potential strategy for promoting cementogenesis. The primary substrate of ENPP1 is ATP, although little is known about ATP’s direct role on cementogenesis. In collaborative studies, we demonstrated the importance of ENPP1 mediated ATP hydrolysis in cementogenesis. Specifically, ENPP1 mutant mice with targeted loss of ATP hydrolysis (ENPP1T238A) exhibited hypercementosis and ectopic joint calcifications, albeit to a lesser extent compared to ENPP1asj mice. To further understand contributions of ENPP1 to cementogenesis, we will also employ a model with ENPP1- mediated cGAMP hydrolysis (ENPP1H362A), which features low plasma PPi without ectopic joint calcifications. Because ENPP1T238A and ENPP1H362A exhibit similar low plasma PPi levels, in vitro studies are expected to aid in the understanding of ENPP1-mediated ATP hydrolysis at the cellular level. In preliminary studies, ENPP1KO cementoblasts exhibited differences in ATP levels measured in cells versus media, suggesting that ENPP1-mediated ATP hydrolysis can affect intracellular ATP. ENPP1T238A and ENPP1H362A cementoblasts will be generated, and evaluation of resultant impact on mineralization as well as metabolism of extracellular/ intracellular ATP will be conducted. ATP dynamics will be investigated using genetically-encoded ATP biosensors, which have not been used before to investigate mineralization. Results from studies proposed here are expected to further define ENPP1 roles in periodontal development and maintenance, specifically in regulating PPi levels in context of ATP metabolism. Ultimately, the studies proposed here are aimed toward identifying targets that can be used to promote cementogenesis. Project Number: 1R03DE034020-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Emily Chu | Institution: UNIVERSITY OF MARYLAND BALTIMORE, BALTIMORE, MD | Award Amount: $155,500 | Activity Code: R03 | Study Section: Special Emphasis Panel[ZDE1 TO (04)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11139168