Induction of palate epithelial mesenchymal transition by transforming growth factor β3 signaling

Abstract

TGFβ3 is essential for palate development, particularly during the late phase of palatogenesis when the disintegration of the palatal medial edge seam (MES) occurs resulting in mesenchymal confluence. The MES is composed of medial-edge epithelium (MEE) of opposite palatal shelves; its complete disintegration is essential for mediating correct craniofacial morphogenesis. This phenomenon is initiated by TGFβ3 upon adherence of opposing palatal shelves, and subsequently epithelial-mesenchymal transition (EMT) instigates the loss of E-Cadherin, causing the MES to break into small epithelial islands forming confluent palatal mesenchyme, however, apoptosis and cell migration or in combination of all are other established mechanisms of seam disintegration. To investigate the molecular mechanisms that cause this E-Cadherin loss, we isolated and cultured murine embryonic primary MES cells from adhered palates and employed several biological approaches to explore the mechanism by which TGFβ3 facilitates palatal seam disintegration. Here, we demonstrate that TGFβ3 signals by activating both Smad-dependent and Smad-independent pathways. However, activation of the two most common EMT related transcription factors, Snail and SIP, was facilitated by Smad-independent pathways, contrary to the commonly accepted Smad-dependent pathway. Finally, we provide the first evidence that TGFβ3-activated Snail and SIP1, combined with Smad4, bind to the E-Cadherin promoter to repress its transcription in response to TGFβ3 signaling. These results suggest that TGFβ3 uses multiple pathways to activate Snail and SIP1 and these transcription factors repress the cell-cell adhesion protein, E-Cadherin, to induce palatal epithelial seam EMT. Manipulation and intervention of the pathways stimulated by TGFβ3 during palate development may have a significant therapeutic potential.

Strain-dependent effects of transforming growth factor-β1 and 2 during mouse secondary palate development

“…Consistent with biochemical studies [18], the function of TGF-␤3 in palate fusion is mediated by Smad2, since the overexpression of Smad2 in Tgf-ˇ3 mutant palate epithelial cells rescues the fusion defect [29]. Interestingly, studies with palatal epithelium cells suggested that TGF-␤3 induces cell cycle arrest and epithelial to mesenchymal trans-differentiation (EMT), which could be a mechanism underlying TGF-␤3 in mediating palate fusion [30,31]. In addition, a recent study revealed that TGF-␤3 may also promote MEE fusion by activating the p38 MAPK pathway [32].…”

Microtubule disassembly prevents palatal fusion and alters regulation of the E-cadherin/catenin complex

“…Quantitative real-time PCR also revealed that NDZ inhibited the transcription factors, Snail and Zeb in the MEE. TGF-b/SMAD promotes EMT via induction of transcriptional repressors of E-cadherin including Snail and Zeb (Dave et al, 2011, Jalali et al, 2012, Pirozzi et al, 2011, Shirakihara et al, 2007. These findings indicated that microtubule disruption in the MEE led to a failure to down-regulate E-cadherin transcription via inhibition of the transcriptional repressors secondary to blockage of signaling from the TGF-b/SMAD2 pathway.…”

“…Transcriptional repression of E-cadherin is a key molecular event in EMT (Peinado et al, 2004). Down regulation of E-cadherin by transcriptional repressors such as the zinc finger factor Snail (Snai1 and Snai2), two-handed zinc finger factor Zeb (Zeb1 and Zeb2) is an essential mechanism by which TGF-b elicits EMT (Dave et al, 2011, Jalali et al, 2012, Pirozzi et al, 2011, Shirakihara et al, 2007. The signaling pathway is well known to induce EMT in MEE during palatal fusion (Kaartinen et al, 1997, Sun et al, 1998.…”

“…Epithelial mesenchymal transition (EMT) plays a crucial role in MES degradation (Greene and Pisano, 2010, Griffith and Hay, 1992, Jalali et al, 2012, Jin and Ding, 2006, Shuler et al, 1992, Yu W, 2009. It is characterized by loss of epithelial cell morphology and acquisition of mesenchymal properties.…”

Reprogramming during epithelial to mesenchymal transition under the control of TGFβ

“…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. 55,56 Some additional examples of EMT can be listed based on their sensitivity to different TGFb isoforms. Similar to the heart, pulmonary valve morphogenesis requires EndoMT, which is driven by TGFb2 and is antagonized by the pro-angiogenic factor VEGF-A.…”