Epidermal Growth Factor Impairs Palatal Shelf Adhesion and Fusion in the T gf-β3 Null Mutant

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The cleft palate presented by transforming growth factor-β3 (Tgf-β3 ) null mutant mice is caused by altered palatal shelf adhesion, cell proliferation, epithelial-to-mesenchymal transformation and cell death. The expression of epidermal growth factor (EGF), transforming growth factor-β1 ( Tgf-β1 ) and muscle segment homeobox-1 (Msx-1) is modified in the palates of these knockout mice, and the cell proliferation defect is caused by the change in EGF expression. In this study, we aimed to determine whether this change in EGF expression has any effect on the other mechanisms altered in Tgf-β 3 knockout mouse palates. We tested the effect of inhibiting EGF activity in vitro in the knockout palates via the addition of Tyrphostin AG 1478. We also investigated possible interactions between EGF, Tgf-β 1 and Msx-1 in Tgf-β 3 null mouse palate cultures. The results show that the inhibition of EGF activity in Tgf-β 3 null mouse palate cultures improves palatal shelf adhesion and fusion, with a particular effect on cell death, and restores the normal distribution pattern of Msx-1 in the palatal esenchyme. Inhibition of TGF-β 1 does not affect either EGF or Msx-1 expression.
Accepted after revision: March 14, 2014 Published online: May 23, 2014
1- Abbott, B.D. (1997) Developmental toxicity of dioxin: searching for the cellular and molecular basis of morphological responses; in Kavlock, R., G. Daston (eds): Handbook of Experimental Pharmacology: Section III: Pathogenesis and Mechanisms of Drug Toxicity in Development. New York, Springer, vol 124, pp 407-433. 2- Abbott, B.D., D.S. Best, M.G. Narotsky (2005a) Teratogenic effects of retinoic acid are modulated in mice lacking expression of epidermal growth factor and transforming growth factor-alpha. Birth Defects Res A Clin Mol Teratol 73: 204-217. 3- Abbott, B.D., A.R. Buckalew, M.J. DeVito, D. Ross, P.L. Bryant, J.E. Schmid (2003) EGF and TGF-α expression influence the developmental toxicity of TCDD: dose response and AhR phenotype in EGF, TGF-α, and EGF + TGF-α knockout mice. Toxicol Sci 71: 84-95. 4- Abbott, B.D., A.R. Buckalew, K.E. Leffler (2005b) Effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), and 2,3,7,8-tetrachlorodibenzo-p-dioxin on fusion of embryonic palates in serum-free organ culture using wild-type, EGF knockout, and TGFalpha knockout mouse strains. Birth Defects Res A Clin Mol Teratol 73: 447-454. 5- Abud, H.E., N. Watson, J.K. Heath (2005) Growth of intestinal epithelium in organ culture is dependent on EGF signalling. Exp Cell Res 303: 252-262. 6- Brinkley, L. (1984) Changes in cell distribution during mouse secondary palate closure in vivo and in vitro. I. Epithelial cells. Dev Biol 102: 216-227. 7- Bryant, P.L., J.E. Schmid, S.E. Fenton, A.R. Buckalew, B.D. Abbott (2001) Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the expression of EGF and/or TGF-α. Toxicol Sci 62: 103-114. 8- Bush, J.O., R. Jiang (2012) Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development. Development 139: 231-243. 9- Carette, M.J., M.W. Ferguson (1992) The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labeling and confocal microscopy. Development 114: 379-388. 10- Cuervo, R., L. Covarrubias (2004) Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis. Development 131: 15-24. 11- Cuervo, R., C. Valencia, R.A.S. Chandaratna (2002) Programmed cell death is required for palate shelf fusion and is regulated by retinoic acid. Dev Biol 245: 145-156. 12- Cui, X.M., Y. Chai, J. Chen, T. Yamamoto, Y. Ito, P. Bringas, C.F. Shuler (2003) TGF-beta3-dependent SMAD2 phosphorylation and inhibition of MEE proliferation during palatal fusion. Dev Dyn 227: 387-394. 13- D'Amaro, R., R. Scheidegger, S. Blumer, P. Pazera, C. Katsaros, D. Graf, M. Chiquet (2012) Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis. Front Physiol 3: 377. 14- Del Río, A., M.C. Barrio, J. Murillo, E. Maldonado, Y. López-Gordillo, E. Martínez-Sanz, M.L. Martínez, C. Martínez-Álvarez (2011) Analysis of the presence of cell proliferation-related molecules in the Tgf-beta3 null mutant mouse palate reveals misexpression of EGF and Msx-1. Cells Tissues Organs 193: 135-150. 15- Dixon, M.J., M.W. Ferguson (1992) The effects of epidermal growth factor, transforming growth factors alpha and beta and platelet-derived growth factor on murine palatal shelves in organ culture. Arch Oral Biol 37: 395-410. 16- Dudas, M., J. Kim, W.Y. Li, A. Nagy, J. Larsson, S. Karlsson, Y. Chai, V. Kaartinen (2006) Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion. Dev Biol 296: 298-314. 17- Ferguson, M.W. (1988) Palate development. Development 103(suppl): 41-60. 18- Fitchett, J.E., E.D. Hay (1989) Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse. Dev Biol 131: 455-474. 19- Foreman, D.M., P.M. Sharpe, M.W. Ferguson (1991) Comparative biochemistry of mouse and chick secondary-palate development in vivo and in vitro with particular emphasis on extracellular matrix molecules and the effects of growth factors on their synthesis. Arch Oral Biol 36: 457-471. 20- Furukawa, S., K. Usuda, M. Abe, I. Ogawa (2004) Histopathological findings of cleft palate in rat embryos induced by triamcinolone acetonide. J Vet Med Sci 66: 397-402. 21- Gato, A., M.L. Martínez, C. Tudela, I. Alonso, J.A. Moro, M.A. Formoso, M.W. Ferguson, C. Martínez-Álvarez (2002) TGF beta(3)-induced chondroitin sulphate proteoglycan mediates palatal shelf adhesion. Dev Biol 250: 393-405. 22- Griffith, C.M., E.D. Hay (1992) Epithelial-mesenchymal transformation during palatal fusion: carboxyfluorescein traces cells at light and electron microscopic levels. Development 116: 1087-1099. 23- Hassell, J.R. (1975) The development of rat palatal shelves in vitro: an ultrastructural analysis of the inhibition of epithelial cell death and palate fusion by the epidermal growth factor. Dev Biol 45: 90-102. 24- Ho, E., L. Dagnino (2012) Epidermal growth factor induction of front-rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2. Mol Biol Cell 23: 492-502. 25- Jin, J.Z., J. Ding (2006) Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion. Development 133: 3341-3347. 26- Kaartinen, V., X.M. Cui, N. Heisterkamp, J. Groffen, C.F. Shuler (1997) Transforming growth factor-beta3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane. Dev Dyn 209: 255-260. 27- Kaartinen, V., J.W. Voncken, C. Shuler, D. Warburton, D. Bu, N. Heisterkamp, J. Groffen (1995) Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects of epithelial-mesenchymal interaction. Nat Genet 11: 415-421. 28- Martínez-Álvarez, C., M.J. Blanco, R. Perez, M.A. Rabadán, M. Aparicio, E. Resel, T. Martínez, M.A. Nieto (2004) Snail family members and cell survival in physiological and pathological cleft palates. Dev Biol 265: 207-218. 29- Martínez-Álvarez, C., R. Bonelli, C. Tudela, A. Gato, J. Mena, S. O'Kane, M.W. Ferguson (2000a) Bulging medial edge epithelial cells and palatal fusion. Int J Dev Biol 44: 331-335. 30- Martínez-Álvarez, C., C. Tudela, J. Pérez-Miguelsanz, S. O'Kane, J. Puerta, M.W. Ferguson (2000b) Medial edge epithelial cell fate during palatal fusion. Dev Biol 220: 343-357. 31- Martínez-Sanz, E., A. Del Rio, C. Barrio, J. Murillo, E. Maldonado, B. Garcillan, M. Amorós, T. Fuerte, A. Fernández, E. Trinidad, M.A. Rabadán, Y. López, M.L. Martínez, C. Martínez-Álvarez (2008) Alteration of medial-edge epithelium cell adhesion in two Tgfbeta3 null mouse strains. Differentiation 76: 417-430. 32- Medici D., E.D. Hay, B.R. Olsen (2008) Snail and Slug promote epithelial-mesenchymal transition through beta-catenin-T-cell factor-4-dependent expression of transforming growth factor-beta3. Mol Biol Cell 19: 4875-4887. 33- Mori, C., N. Nakamura, Y. Okamoto, M. Osawa, K. Shiota (1994) Cytochemical identification of programmed cell death in the fusing fetal mouse palate by specific labelling of DNA fragmentation. Anat Embryol 190: 21-28. 34- Morris, Z.S., A.I. McClatchey (2009) Aberrant epithelial morphology and persistent epidermal growth factor receptor signaling in a mouse model of renal carcinoma. Proc Natl Acad Sci USA 106: 9767-9772. 35- Mu, Z., Z. Yang, D. Yu, Z. Zhao, J.S. Munger (2008) TGFbeta1 and TGFbeta3 are partially redundant effectors in brain vascular morphogenesis. Mech Dev 125: 508-516. 36- Murillo, J., E. Maldonado, M.C. Barrio, A. Del Río, Y. López, E. Martínez-Sanz, I. González, C. Martín, I. Casado, C. Martínez-Álvarez (2009) Interactions between TGF-beta1 and TGF-beta3 and their role in medial edge epithelium cell death and palatal fusion in vitro. Differentiation 77: 209-220. 37- Nawshad, A., D. LaGamba, E.D. Hay (2004) Transforming growth factor beta (TGFbeta) signalling in palatal growth, apoptosis and epithelial mesenchymal transformation (EMT). Arch Oral Biol 49: 675-689. 38- Nugent, P., R.M. Greene (1998) MSX-1 gene expression and regulation in embryonic palatal tissue. In Vitro Cell Dev Biol Anim 34: 831-835. 39- Peplow, P.V., M.P. Chatterjee (2013) A review of the influence of growth factors and cytokines in in vitro human keratinocyte migration. Cytokine 62: 1-21. 40- Proetzel, G., S.A. Pawlowski, M.V. Wiles, M. Yin, G.P. Boivin, P.N. Howles, J. Ding, M.W. Ferguson, T. Doetschman (1995) Transforming growth factor-beta 3 is required for secondary palate fusion. Nat Genet 11: 409-414. 41- Pullar, C.E., B.S. Baier, Y. Kariya, A.J. Russell, B.A. Horst, M.P. Marinkovich, R.R. Isseroff (2006) beta4 integrin and epidermal growth factor coordinately regulate electric field-mediated directional migration via Rac1. Mol Biol Cell 17: 4925-4935. 42- Rowe, R.G., Weiss, S.J. (2008) Breaching the basement membrane: who, when and how? Trends Cell Biol 18: 560-574. 43- Sasaki, T., H. Kuniyasu, Y. Luo, D. Kato, S. Shinya, K. Fujii, H. Ohmori, Y. Yamashita (2012) Significance of epithelial growth factor in the epithelial-mesenchymal transition of human gallbladder cancer cells. Cancer Sci 103: 1165-1171. 44- Shin, J.O., J.M. Lee, K.W. Cho, S. Kwak, H.J. Kwon, M.J. Lee, S.W. Cho, K.S. Kim, H.S. Jung (2012) MiR-200b is involved in Tgf-β signaling to regulate mammalian palate development. Histochem Cell Biol 137: 67-78. 45- Shintani, Y., M. Maeda, N. Chaika, K.R. Johnson, M.J. Wheelock (2008) Collagen I promotes epithelial-to-mesenchymal transition in lung cancer cells via transforming growth factor-beta signaling. Am J Respir Cell Mol Biol 38: 95-104. 46- Shuler, C.F., D.E. Halpern, Y. Guo, A.C. Sank (1992) Medial edge epithelium fate traced by cell lineage analysis during epithelial-mesenchymal transformation in vivo. Dev Biol 154: 318-330. 47- Silver, M.H., J.C. Murray, R.M. Pratt (1984) Epidermal growth factor stimulates type-V collagen synthesis in cultured murine palatal shelves. Differentiation 27: 205-208. 48- Singh, G.D., B.J. Moxham, M.S. Langley, G. Embery (1997) Glycosaminoglycan biosynthesis during 5-fluoro-2-deoxyuridine-induced palatal clefts in the rat. Arch Oral Biol 42: 355-363. 49- Smith, A., T.N. Teknos, Q. Pan (2013) Epithelial to mesenchymal transition in head and neck squamous cell carcinoma. Oral Oncol 49: 287-292. 50- Song, K., T.L. Krebs, D. Danielpour (2006) Novel permissive role of epidermal growth factor in transforming growth factor beta (TGF-beta) signaling and growth suppression. Mediation by stabilization of TGF-beta receptor type II. J Biol Chem 281: 7765-7774. 51- Sun, D., C.R. Vanderburg, G.S. Odierna, E.D. Hay (1998) TGFbeta3 promotes transformation of chicken palate medial edge epithelium to mesenchyme in vitro. Development 125: 95-105. 52- Takigawa, T., K. Shiota (2004) Terminal differentiation of palatal medial edge epithelial cells in vitro is not necessarily dependent on palatal shelf contact and midline epithelial seam formation. Int J Dev Biol 48: 307-317. 53- Taniguchi, K., N. Sato, Y. Uchiyama (1995) Apoptosis and heterophagy of medial edge epithelial cells of the secondary palatine shelves during fusion. Arch Histol Cytol 58: 191-203. 54- Taya, Y., S. O'Kane, M.W.J. Ferguson (1999) Pathogenesis of cleft palate in TGF-b3 knockout mice. Development 126: 3869-3879. 55- Trowell, O.A. (1959) The culture of mature organs in a synthetic medium. Exp Cell Res 16: 118-147. 56- Tudela, C., M.A. Formoso, T. Martínez, R. Pérez, M. Aparicio, C. Maestro, A. Del Río, E. Martínez, M.W.J. Ferguson, C. Martínez-Álvarez (2002) TGF-b3 is required for the adhesion and intercalation of medial edge epithelial cells during palate fusion. Int J Dev Biol 46: 333-336. 57- Vaziri Sani, F., K. Hallberg, B.D. Harfe, A.P. McMahon, A. Linde, A. Gritli-Linde (2005) Fate-mapping of the epithelial seam during palatal fusion rules out epithelial-mesenchymal transformation. Dev Biol 285: 490-495. 58- Xu, X., J. Han, Y. Ito Jr, P. Bringas, M.M. Urata, Y. Chai (2006) Cell autonomous requirement for Tgfbr2 in the disappearance of medial edge epithelium during palatal fusion. Dev Biol 297: 238-248. 59- Yamamoto, T., X.M. Cui, C.F. Shuler (2003) Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion. Dev Biol 260: 512-521. 60- Yang, L.T., V. Kaartinen (2007) Tgfb1 expressed in the Tgfb3 locus partially rescues the cleft palate phenotype of Tgfb3 null mutants. Dev Biol 312: 384-395. 61- Zhang, Z., Y. Song, X. Zhao, X. Zhang, C. Fermin, Y. Chen (2002) Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis. Development 129: 4135-4146.