TGFβ3 inhibits E-cadherin gene expression in palate medial-edge epithelial cells through a Smad2-Smad4-LEF1 transcription complex

Abstract

Dissociation of medial-edge epithelium (MEE) during palate development is essential for mediating correct craniofacial morphogenesis. This phenomenon is initiated by TGFβ3 upon adherence of opposing palatal shelves, because loss of E-cadherin causes the MEE seam to break into small epithelial islands. To investigate the molecular mechanisms that cause this E-cadherin loss, we isolated and cultured murine embryonic primary MEE cells from adhered or non-adhered palates. Here, we provide the first evidence that lymphoid enhancer factor 1 (LEF1), when functionally activated by phosphorylated Smad2 (Smad2-P) and Smad4 (rather than β-catenin), binds with the promoter of the E-cadherin gene to repress its transcription in response to TGFβ3 signaling. Furthermore, we found that TGFβ3 signaling stimulates epithelial-mesenchymal transformation (EMT) and cell migration in these cells. LEF1 and Smad4 were found to be necessary for up-regulation of the mesenchymal markers vimentin and fibronectin, independently of β-catenin. We proved that TGFβ3 signaling induces EMT in MEE cells by forming activated transcription complexes of Smad2-P, Smad4 and LEF1 that directly inhibit E-cadherin gene expression.

Reprogramming during epithelial to mesenchymal transition under the control of TGFβ

“…28 During endothelial-mesenchymal transition (EndoMT) that forms the valves in the heart in response to TGFb2, EMT induced by TGFb3 in the palate, or EMT developed during lung fibrosis, b-catenin/TCF complexes are critical, together with Smads for the induction of mesenchymal genes in the transforming endothelial cells or for the repression of E-cadherin in the regressing epithelial cells of the palate. [29][30][31] These examples of EndoMT will be discussed further later. In the case of lung EMT after inflammation, a specific protein complex between Smad2 and phosphorylated b-catenin on tyrosine 654 that is mediated by activation of integrin-a3b1 is a crucial determinant that controls E-cadherin repression.…”

“…TGFb3 is the key player in this process, which causes the EMT by repressing E-cadherin via a nuclear Smad-TCF4 transcriptional complex on the E-cadherin promoter region as explained above. 31 Not only does TGFb3 promote the formation of the Smad-b-catenin-TCF4 complexes, but also induces the levels of TCF4 at the transcriptional level. 54 In addition to Smads, non-Smad signaling contributes to the stabilization and activation of Snail1 and ZEB2 during palatal EMT, and additionally, the small GTPase Rho and its downstream Rho kinase contribute to mesenchymal phenotypic changes in the palate, including actin cytoskeleton remodeling.…”

Epithelial—mesenchymal and mesenchymal—epithelial transitions in carcinoma progression

“…EMT-like changes are thought to be a feature of palatal formation, which is the coming together of ectodermal layers and fusion of underlying branchial arch mesenchymal tissues, resulting in a separation of the nasal and oral cavities. Whether this fusion is a result of EMT is speculative, as some have argued that apoptosis or epithelial cell migration occurs, or that all of these processes occur simultaneously (Shuler, 1995;Dudas et al, 2007;Nawshad et al, 2007). Other examples of regulated EMT in the adult is placental formation (Vicovac and Aplin, 1996), and the production of fibroblasts during inflammation (Iwano et al, 2002) and wound healing (Desmouliere, 1995).…”

Interactions Between β-Catenin and Transforming Growth Factor-β Signaling Pathways Mediate Epithelial-Mesenchymal Transition and Are Dependent on the Transcriptional Co-activator cAMP-response Element-binding Protein (CREB)-binding Protein (CBP)

“…Direct interactions between components of these pathways in transcriptional regulation of EMT target genes has focused largely on regulation of epithelial genes and little is known of direct effects on regulation of mesenchymal genes. In this regard, TGF-␤3 stimulates transcription of LEF-1, which interacts with Smad2 and Smad4 to inhibit E-cadherin and induce EMT (38). Kim et al (39) demonstrated formation of a cytoplasmic complex between tyrosine-phosphorylated ␤-catenin and Smad2 in the context of EMT but did not directly demonstrate transcriptional regulation of target genes by this complex.…”