TGF-β3-mediated suppression of SOX2 and OCT4 drives a predominantly non-canonical profile of epithelial-mesenchymal transition during palatal fusion.

Abstract

Epithelial-mesenchymal transition (EMT) of medial edge epithelial cells is a critical mechanism underlying embryonic palatal fusion, driven by transforming growth factor beta 3 (TGF-β3). However, its regulatory interplay with pluripotency factors (SOX2/OCT4) and reliance on downstream cascades remain poorly understood. This study investigates these networks to elucidate the mechanisms of cellular reprogramming during palatogenesis. Embryonic day 15 C57BL6/J mouse palatal epithelial cells were isolated to eliminate mesenchymal interference. Cells were subjected to recombinant TGF-β3 (1, 3, 10 ng/mL) or targeted siRNA knockdowns of Tgfb3, Sox2, Oct4, and Smad2/3, and evaluated via RNA sequencing, qRT-PCR, immunofluorescence, TEER, and flow cytometry. Treatment with 1-3 ng/mL TGF-β3 successfully induced EMT in vitro, characterized by decreased E-cadherin, increased fibronectin, diminished membrane resistance, and a fibroblast-like morphological shift. This transition was predominantly associated with non-canonical pathways; phosphorylated Smad2/3 levels remained unchanged while Smad-independent mediators (p38 MAPK and Mmp13) were upregulated (p<0.05). Furthermore, TGF-β3 downregulated the pluripotency markers Sox2 and Oct4 (p<0.05). Combined Sox2/Oct4 knockdown with TGF-β3 yielded the most pronounced EMT characteristic. While Tgfb3 knockdown heavily attenuated EMT, partial mesenchymal transition persisted, indicating redundant compensatory networks. In conclusion, optimal TGF-β3 concentrations (1-3 ng/mL) shift EMT toward Smad-independent pathways during palatogenesis. This reprogramming requires TGF-β3 to suppress SOX2 and OCT4, underscoring their roles as negative regulators of EMT during palatal fusion.