Mechanisms of transforming growth factor β induced cell cycle arrest in palate development.

Immaculate and complete palatal seam disintegration, which takes place at the last phase of palate development, is essential for normal palate development. And in absence of palatal midline epithelial seam (MES) disintegration, cleft palate may arise. It has been established that transforming growth factor (TGF) β induces both epithelial mesenchymal transition (EMT) and/or apoptosis during MES disintegration. It is likely that MES might cease cell cycle to facilitate cellular changes prior to undergoing transformation or apoptosis, which has never been studied before. This study was designed to explore whether TGFβ, which is crucial for palatal MES disintegration, is capable of inducing cell cycle arrest. We studied the effects of TGFβ1 and TGFβ3, potent negative regulators of the cell cycle, on p15ink4b activity in MES cells. We surprisingly found that TGFβ1, but not TGFβ3, plays a major role in activation of the p15ink4b gene. In contrast, following successful cell cycle arrest by TGFβ1, it is TGFβ3 but not TGFβ1 that causes later cellular morphogenesis, such as EMT and apoptosis. Since TGFβ signaling activates Smads, we analyzed the roles of three Smad binding elements (SBEs) on the p15ink4b mouse promoter by site specific mutagenesis and found that these binding sites are functional. The ChIP assay demonstrated that TGFβ1, not TGFβ3, promotes Smad4 binding to two 5' terminal SBEs but not the 3' terminal site. Thus, TGFβ1 and TGFβ3 play separate yet complimentary roles in achieving cell cycle arrest and EMT/apoptosis and cell cycle arrest is a prerequisite for later cellular changes.