The effect of transforming growth factor (TGF)-beta in palate growth and development

Cleft palate is a common craniofacial deformity in human due to abnormality of growth, elevation or fusion of the two palatal shelves. Failure of palatal shelves to grow after elevation is the most common reason to cause cleft palate in mouse models. Palatal growth is caused by cell proliferation of the palatal mesenchyme. The expression of all three isoforms of TGFbeta in palatal mesenchyme and the knockout of TGFbeta3 always cause cleft palate in mouse models indicating the important role of TGFbeta in palatal mesenchyme cell proliferation. Furthermore, maternal biotin deficiency induces cleft palate in mouse models. On this basis, we hypothesized that TGFbeta and biotin can promote palatal mesenchymal cell proliferation and palatal growth. To test this hypothesis, we designed and performed experiments using murine heads and human embryonic palatal mesenchymal (HEPM) cells. After 24 hours' TGFbeta or biotin treatment, HEPM cell proliferation was studied by FAGS, RT-PCR and western blot analyses. Our results showed that all isoforms of TGFbeta, especially TGFbeta3, increased HEPM cell proliferation by up-regulating the expression of cyclins and CDKs as well as c-Myc proto-oncogene. Both Smad-dependent and Smad-independent pathways were activated by TGFbeta to induce c-Myc gene expression. Microarray analysis and RNA sequencing were performed to study the gene expression profile. Microarray analysis of TGFR treated HEPM cells showed that 234 of the 566 differentially expressed genes were clustered in network with specific cellular biofunctions. The altered TGFbeta canonical pathway genes and genes with high fold changes were demonstrated with a possible role in causing cleft palate. The potential causative cleft palate genes in TGFbeta3 knockout mice were identified by RNA sequencing. Microarray analysis of gene expression in CF1 wildtype palatal shelves showed that ACC2 is the only up-regulated biotin dependent carboxylase in palatal growth stage. Further studies indicated ACC2 is downstream of smad-dependent pathway. These data highlights the potential role of TGbeta3 and biotin as therapeutic molecules to correct cleft palate by promoting palatal growth.