Functional consequences of protein-protein interaction between Pax9 and Msx1

Recent findings from in vivo mouse and human genetic studies suggest a functional relationship between Pax9 and Msx1 in tooth development. Based on studies in the mouse, the expression patterns of Pax9 and Msx1 in dental mesenchyme are closely overlapping and consistent with a role in epithelial-mesenchymal interactions. Independent association studies have provided evidence of a statistically significant interaction between PAX9 and MSX1. However, classic biochemical and molecular experiments to elucidate the molecular basis for the interactions between these key molecules have not been performed. Objectives: The goals of this study were to clarify the interaction between Pax9 and Msx1 on the protein level and to assess the functional consequences of this interaction. Methods: To assess the formation of Pax9-Msx1 complex in vivo, we transiently expressed full-length mouse Pax9 together with mouse full-length Msx1 in COS7 cells and performed co-immunoprecipitation. Transient co-transfection assays with CD19-TK-Luc reporter, a classical paired domain recognition sequence, in COS7 cells were also performed. Furthermore, we examined the DNA binding activity of Pax9 in the presence or absence of Msx1 using nuclear protein after transfection with Pax9 and/or Msx1 in COS7 cells. Results: Western blot analysis of immunoprecipitation showed that Pax9 is able to form a protein complex with Msx1 in vivo. Co-transfection of a constant amount of Pax9 expression plasmid with increasing amounts of Msx1 plasmid alleviates repression of transcriptional activities of CD19-TK-Luc reporter. Gel retardation assays demonstrate that Msx1 inhibited DNA binding by Pax9. Conclusion: Pax9 interact Msx1 in mammalian cells, forming transcriptionally inactive complexes that cannot bind to DNA. These data increase our understanding of the relationship between the two gene products during early stages of odontogenesis. Supported by NIH (DE011663) to R.D.S. and NIH (K08-DE14237) to H.K.