Roles of ENPP1-Catalyzed ATP Hydrolysis in Cementogenesis

Objectives: Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) is a multi-domain transmembrane protein known to regulate cementogenesis through generation of pyrophosphate (PPi), a mineralization inhibitor. Previous studies observed hypercementosis in ENPP1-asj (features total loss of ENPP1 function) mice, but the underlying mechanisms are poorly understood. The phosphodiesterase (PDE) domain of ENPP1 includes a catalytic site, which enables ENPP1 to hydrolyze ATP. This catalytic site is ablated by the T238A point mutation, preventing ATP hydrolysis. This study aims to evaluate the periodontal phenotype resulting from the T238A mutation to determine the extent to which ATP hydrolysis contributes to hypercementosis.
Methods: Mandibles and paws harvested from WT, ENPP1-asj, and ENPP1-T238A (n=3-5) mice at 60-70 days postnatal (dpn) were analyzed using microcomputed tomography (microCT) and histology. Gene expression of progressive ankylosis protein (Ank), alkaline phosphatase (Alpl), and osteopontin (Spp1) was evaluated via qPCR using RNA harvested from the periodontal ligament (PDL) and bone. Osteopontin (OPN) expression was evaluated using immunohistochemistry.
Results: Histology and microCT confirmed hypercementosis in ENPP1-T238A mice to be less severe than ENPP1-asj mice. Mandibular 1st molar dentin/cementum volumes in ENPP1-T238A and ENPP1-asj were increased by 14.6% and 23.7% compared to WT. Degree of hypercementosis paralleled severity of ectopic calcification in the paws. In PDL samples, ENPP1-T238A and ENPP1-asj mice exhibited higher Ank (3 and 4 fold, respectively) expression compared to WT. Spp1 expression was upregulated by 30 fold in ENPP1-asj, whereas ENPP1-T238A was upregulated by 2 fold. These trends were not observed in bone samples. IHC revealed increased expression of OPN along cementum and bone surfaces in ENPP1-T238A and ENPP1-asj compared to WT.
Conclusions: ENPP1-T238A mice exhibited hypercementosis and Spp1 upregulation in PDL to a lesser extent compared to ENPP1-asj mice. The data suggests that ATP hydrolysis is not the only mechanism responsible for cementogenesis but is a significant contributor.