Overlapping functions of bone sialoprotein and pyrophosphate regulators in directing cementogenesis.

Abstract

Although acellular cementum is essential for tooth attachment, factors directing its development and regeneration remain poorly understood. Inorganic pyrophosphate (PP_i), a mineralization inhibitor, is a key regulator of cementum formation: tissue-nonspecific alkaline phosphatase (Alpl/TNAP) null mice (increased PP_i) feature deficient cementum, while progressive ankylosis protein (Ank/ANK) null mice (decreased PP_i) feature increased cementum. Bone sialoprotein (Bsp/BSP) and osteopontin (Spp1/OPN) are multifunctional extracellular matrix components of cementum proposed to have direct and indirect effects on cell activities and mineralization. Studies on dentoalveolar development of Bsp knockout (Bsp^-/-) mice revealed severely reduced acellular cementum, however underlying mechanisms remain unclear. The similarity in defective cementum phenotypes between Bsp^-/- mice and Alpl^-/- mice (the latter featuring elevated PP_i and OPN), prompted us to examine whether BSP is operating by modulating PP_i-associated genes. Genetic ablation of Bsp caused a 2-fold increase in circulating PP_i, altered mRNA expression of Alpl, Spp1, and Ank, and increased OPN protein in the periodontia. Generation of a Bsp knock-out (KO) cementoblast cell line revealed significantly decreased mineralization capacity, 50% increased PP_i in culture media, and increased Spp1 and Ank mRNA expression. While addition of 2μg/ml recombinant BSP altered Spp1, Ank, and Enpp1 expression in cementoblasts, changes resulting from this dose were not dependent on the integrin-binding RGD motif or MAPK/ERK signaling pathway. Decreasing PP_i by genetic ablation of Ank on the Bsp^-/- mouse background reestablished cementum formation, allowing >3-fold increased acellular cementum volume compared to wild-type (WT). However, deleting Ank did not fully compensate for the absence of BSP. Bsp^-/-; Ank^-/- double-deficient mice exhibited mean 20-27% reduced cementum thickness and volume compared to Ank^-/- mice. From these data, we conclude that the perturbations in PP_i metabolism are not solely driving the cementum pathology in Bsp^-/- mice, and that PP_i is more potent than BSP as a cementum regulator, as shown by the ability to override loss of BSP by lowering PP_i. We propose that BSP and PP_i work in concert to direct mineralization in cementum and likely other mineralized tissues.