The muscle segment homeogenes Msx1 and Msx2 play a major role in tooth and bone formation. Periodontal osteoclast impairment also occurs in Msx2 null mutant mice, which is restored by overexpression of the receptor activator of NF-κB targeted in osteoclast lineage. Here, we investigated the role of Msx2 in dentinogenesis. Experiments were performed on Msx2(-/-) mice and the MDPC-23 odontoblastic cell line. After Msx2 gene silencing, real-time quantitative RT-PCR data showed significant overexpression of Runx2, Bglap, Dspp, and Alpl. Of three inhibitors of Wnt/β-catenin signaling (Dkk1, SostDc1, and Sost/Sclerostin), only Sost was expressed in postnatal teeth and overexpressed in Msx2(-/-) tooth samples. Initial crown dentin formation-primary dentinogenesis-occurred fairly normally in Msx2(-/-) teeth, albeit with distorted cusp patterns. Later stages of tooth development were characterized by a deviation from secondary toward tertiary dentinogenesis with osteodentin formation and impaired dentin deposition leading to limited root elongation. In Msx2(-/-)/receptor activator of NF-κB-transgenic double mutants, the dentin phenotype, notably in the roots, was rescued and sclerostin levels were normalized. These data suggest that Msx2 may act indirectly on dentinogenesis by controlling osteoclast activity and the signaling network related to eruption, supporting and further extending the concept that Msx2 controls formation of mineralized tissues by inhibition of the Wnt/β-catenin pathway; Sost in dentin and Dkk1 in bone, as previously demonstrated.

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling.