IRF6 is the mediator of TGFβ3 during regulation of the epithelial mesenchymal transition and palatal fusion

www.nature.com/scientificreports OPEN received: 07 July 2015 accepted: 09 July 2015 Published: 04 August 2015 IRF6 is the mediator of TGFβ3 during regulation of the epithelial mesenchymal transition and palatal fusion Chen-Yeh Ke1, Wen-Lin Xiao2, Chun-Ming Chen1, Lun-Jou Lo2 & Fen-Hwa Wong1 Mutation in interferon regulatory factor 6 (IRF6) is known to cause syndromic and non-syndromic cleft lip/palate in human. In this study, we investigated the molecular mechanisms related to IRF6 during palatal fusion using palatal shelves organ culture. The results showed that ablation of Irf6 resulted in a delay in TGFβ3-regulated palatal fusion. Ectopic expression of IRF6 was able to promote palatal fusion and rescue shTgfβ3-induced fusion defect. These findings indicate that IRF6 is involved in TGFβ3-mediated palatal fusion. Molecular analysis revealed that ectopic expression of IRF6 increased the expression of SNAI2, an epithelial mesenchymal transition (EMT) regulator, and diminished the expression of various epithelial markers, such as E-cadherin, Plakophilin and ZO-1. In addition, knockdown of Irf6 expression decreased SNAI2 expression, and restored the expression of ZO-1 and Plakophilin that were diminished by TGFβ3. Blocking of Snai2 expression delayed palatal fusion and abolished the IRF6 rescuing effect associated with shTgfβ3-induced fusion defect. These findings indicate that TGFβ3 increases IRF6 expression and subsequently regulates SNAI2 expression, and IRF6 appears to regulate EMT during palatal fusion via SNAI2. Taken together, this study demonstrates that IRF6 is a mediator of TGFβ3, which regulates EMT and fusion process during the embryonic palate development. In mammals, the palatal tissue contains primary and secondary palates. The primary palate builds the anterior palate up to the incisive foramen, while the secondary palate forms the hard and soft palates. Secondary palate development initially starts from two vertical palatal shelves, which subsequently grow and reorient horizontally over the tongue and eventually touch each other1. Following this, the epithelial cells covering the edges adhere and form the midline epithelial seam (MES). The medial edge epithelium (MEE) cells then intercalate with each other and gradually disappear2. Finally, mesenchymal cells fill the midline, forming an intact palate. Degeneration of MES is important for palatal fusion3,4. If MEE cells fail to disappear, this results in a cleft palate. Three mechanisms have been proposed for MES degeneration; these are cell migration, apoptosis, and epithelial mesenchymal transition (EMT)5–9. These mechanisms are regulated by transforming growth factor beta3 (TGFβ 3) during palate development10–16. In the palate of mice, Tgfβ 3 mRNA is largely expressed in the MEE cells17,18. Knockout of Tgfβ 3 gene has been shown to result in cleft palate19,20. TGFβ 3 activates both SMAD-dependent and SMAD-independent pathways through TGFβ R1, TGFβ R2, and/or TGFβ R3, and these in turn regulate the palatal fusion during mouse palate development16,21–25. Enhancement of Lef1, Snai1, Snai2, Twist, and Gemin2 expression in MEE by TGFβ 3 has been reported to promote EMT during palatal fusion12,15,26,27. TGFβ 3 also regulates MEE apoptosis through activating TGFBI expression, the FasL-Fas-Caspase pathway, and the IRF6/Δ Np63/ 1 Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, 11221, Taiwan. 2Department of Plastic and Reconstructive Surgery, and Craniofacial Research Center, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, 333, Taiwan. Correspondence and requests for materials should be addressed to L.-J.L. (email: ) or F.-H.W. (email: ) Scientific Reports | 5:12791 | DOI: 10.1038/srep12791 1 www.nature.com/scientificreports/ Figure 1. Schematic illustration of virus infection and TGFβ3 treatment on the palatal shelves organ culture. Palatal shelves were dissected from E13.5 C57BL/6 mouse embryos and cultured for 3 hours. The palatal shelves were then infected with lentivirus, adenovirus, or treated with 20 ng/ml TGFβ 3 for indicated time interval. Then the palatal shelves were harvested for RNA extraction or fixed with 4% PFA or had a change of medium and then were cultured for another 6, 12, 18, or 24 hours, after which they were fixed with 4% PFA. p21 pathway14,16,28. Moreover, TGFβ 3 participates in MEE specification and periderm desquamation by downregulating JAG2 or Δ Np6329,30. In organ culture system, treatment with TGFβ 3 recombinant protein promotes the fusion of palatal shelves31–33. These studies indicate that expression of TGFβ 3 is important and required in palatal fusion. Cleft lip and palate are common congenital craniofacial disorders that occur once in every 600 new births34,35. Orofacial cleft can be categorized into syndromic or non-syndromic cleft according to the presence or absence of associated anomalies. Van der Woude syndrome (VWS) is the most common form of syndromic cleft and is an autosomal dominant disorder. Mutations in the interferon regulatory factor 6 (IRF6) gene lead to VWS36–38. In addition to VWS, IRF6 mutations are also known to cause popliteal pterygium syndrome (PPS) and non-syndromic cleft lip/palate37,39–41. IRF6 is a transcription factor that regulates cell proliferation, cell cycle, periderm formation, and keratinocyte differentiation42–45. Irf6 null and Irf6R84C mutant mice have abnormal skin, limb, and craniofacial development46,47. In addition, Irf6clft1 mutant mice, with an ENU-induced P39L mutation in Irf6, show abnormal adhesion between the palate and tongue resulting in cleft palate48. Recently, an interaction between TGFβ signaling and IRF6 activity has been reported. TGFβ increases Irf6 expression through both SMAD-dependent pathway and p38 MAPK pathway; during the palatal fusion this effect regulates MEE apoptosis through IRF6/Δ Np63/ p21 signaling cascade16. These studies suggest that IRF6 is important to MEE apoptosis and palate development. In addition to apoptosis, IRF6 regulates EMT and cellular migration. It was reported that IRF6 regulated N-cadherin, an EMT related gene, in human breast cancer cells49. Loss of Irf6 in mouse embryonic keratinocytes leads to a delay in cellular migration and wound healing via RhoA pathway50. These findings suggest that IRF6 may regulate EMT and cellular migration. However, whether IRF6 is involved in TGFβ 3-regulated EMT during palatal fusion remains poorly understood. Irf6-null and Irf6-mutant homozygous embryos showed a phenotype involving intraoral adhesions that inhibited shelf elevation and eventually resulted in cleft palate. However, it is not known whether and how IRF6 is involved in palatal fusion. In this study, we investigate the role of IRF6 in TGFβ 3-regulated palatal fusion using palatal shelves organ culture, and find that IRF6 regulates EMT during palatal fusion via SNAI2. Results Knockdown of Irf6 delays TGFβ3 mediated palata (...truncated)