PAX9 mutations disrupt the Pax9-Msx1-Bmp4 signaling pathway

Pax9 and Msx1 have essential functions in tooth development. Mutations in both genes are associated with nonsyndromic tooth agenesis. Previous studies have shown that these two transcription factors interact with each other and regulate the expression of Bmp4.

Objective: In this study, we focused on PAX9 mutations in human tooth agenesis to investigate the functional consequences of the mutations on the Pax9-Msx1-Bmp4 signaling pathway.

Methods: Mouse Pax9 was cloned into pCMV3b mammalian expression vector. Site-direct mutagenesis was used to generate expression constructs encoding eight human missense mutations causing tooth agenesis. Two luciferase reporter constructs containing either a -3.5kb Msx1 promoter or -2.4kb Bmp4 promoter were used to evaluate the transcriptional activity of mutant Pax9. Co-immunoprecipitation assay was used to assess protein-protein interaction between Msx1 and mutant Pax9 proteins.

Results: All eight missense mutations in Pax9 gene are predicted to change evolutionarily conserved residues in the paired domain, the DNA binding domain of Pax9. Luciferase assay showed that, with both Msx1 and Bmp4 promoters, the Pax9 mutants decreased transcription activity to different extents. G6R and G51S, the two mutations with mildest phenotypes, retained more transcription activation ability when compared to the other mutations. When we co-transfected the mutants with wild-type Pax9, no dominant-negative effect was observed. Our co-immunoprecipitation assay showed that all of the mutant proteins were able to physically interact with Msx1, but with different affinities.

Conclusion: The ability of Pax9 to transcriptionally activate Msx1 and Bmp4 was affected by missense mutations in the paired domain. The ability of physically interact with Msx1 was also impaired by the mutations. Thus, our results suggest a disturbance of Pax9-Msx1-BMP4 signal pathway may be responsible for the observed tooth phenotypes and may explain genotype-phenotype correlations in Pax9 mutations.

This study was supported by NIH U24 (DE16472), TAMHSC-VPR grant, NIH K08 (DE16346).