TGF-beta type III receptor signaling for fate MEE in palatogenesis

The molecular mechanisms regulating palatogenesis remain incompletely characterized but are most likely to be involved in the molecular etiology for cleft palate. We previously identified a role of the TGF-beta type III receptor (TβR-III) during palatal fusion using small interfering RNA (siRNA). The treatment with siRNA specific for TβR-III decreased the amount of protein by approximately 75% and delayed palatal fusion. Objective: The goal of this study was to investigate TGF-β downstream signaling, and identify the fate of the MEE when TβR-III was knocked down during palatogenesis. Methods: E13 palatal shelves were dissected and organ cultured in BGjb medium with siRNA. Western blot analysis was performed to analyze Smad2 and phospho-Smad2 when TβR-III was knocked down by siRNA treatment. We also examined the effects of exogenous TGF-β3 (10 and 50 ng/ml of rhTGF-β3) on the process of palatal fusion in organ culture following exposure to TβR-III siRNA. The fate of the MEE was examined at E13+72h using BrdU incorporation for cell proliferation and in situ TUNEL to assess apoptosis. Results: The amount of phospho-Smad2 protein was decreased in the TβR-III siRNA treated palatal shelves. Both BrdU-positive and TUNEL-positive cells were identified in the remaining MEE cells at E13+72h. Palatal organ cultures treated with TβR-III siRNA+rhTGF-β3 completely fused by 72 hours in vitro. Conclusion: Our results demonstrated knocking down TβR-III could affect the down stream signaling pathway of TGF-β by reducing the levels of phospho-Smad2. The effects of exogenous rhTGF-β3 demonstrated that the palatal fusion phenotype could be rescued in organ cultures treated with TβR-III siRNA. We provided evidence that TβR-III siRNA treatment resulted in persistent MEE cell proliferation, which has been shown to be linked to a failure to complete palatal fusion events. (Supported by NIDCR grants PO1DE-12941, RO1DE-16296, MEXT Grant-in-Aid for Scientific Research (C) and Nihon University Research Grant).