Response of TGF-β isoforms in epithelial-mesenchymal transition of enamel epithelial cells

Objective: During enamel formation, transforming growth factor-beta (TGF-beta) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF beta isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells. Design: Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-beta isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-beta pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-beta receptor gene in response to the binding affinity of the TGF-beta isoform were analyzed. Results: EMT was observed in mHAT9d cultured in the presence of TGF-beta 1 and beta 3 but not TGF-beta 2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-beta signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-beta 1 or beta 3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-beta signaling in mHAT9d cells reduced following stimulation with each TGF-beta isoform. In contrast, endoglin levels increased following TGF-beta 1 or beta 3 stimulation, but no change was noted in response to TGF-beta 2. Conclusions: We propose that in TGF-beta-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-beta isoforms were due to their endoglin-mediated binding affinity for the TGF-beta receptor.