Epithelial-msenchymal transition (EMT) contributes to posterior capsule opacification (PCO) type of cataract. Transcription factors Snail is a key trigger of EMT activated by transforming growth factor β (TGFβ). This study was done to investigate the effect of Snail targeting siRNA on TGFβ2-induced EMT in human lens epithelial cells. TGFβ2 treatment of cultured human epithelial cell line (HLEB3) upregulated the expression of Snail and the EMT relevant molecules such as vimentin and α-SMA but downregulated the expression of keratin and E-cadherin. After the stimulation of TGFβ2, the HLEB3 cells became fibroblast-like in morphology, and the junctions of cell-cell disappeared. TGFβ2 treatment also enhanced migration ability of HLEB3 cells. TGFβ2-induced Snail expression and EMT were significantly inhibited by Snail siRNA. By analyzing the response characteristics of HLEB3 in TGFβ2-induced EMT model with/without Snail-specific siRNA, we concluded that Snail is an element in the EMT of HLEB3 cells induced by TGFβ2. Snail siRNA targeting can block the induced EMT and therefore has the potential to suppress the development of PCO. 1. Introduction Epithelial-mesenchymal transition (EMT) is a programmed development of biological cells characterized by loss of cell adhesion, repression of E-cadherin expression, increased cell mobility, and change of morphology. EMT is a highly conserved and fundamental process not only in development, but also in fibrosis, metastasis of tumor cells, and wound healing [1–4]. In cataract surgery, where entire lens content is removed, lens epithelial cells (LECs) can undergo EMT, migrate to the posterior capsular surface, and result in fibrosis of the posterior capsule as well as the residual anterior capsule [4–6]. Clinically, the EMT of LECs after cataract lens removal usually results in secondary cataract that can present as anterior polar cataracts and/or posterior capsular opacification [7, 8]. During EMT, epithelium cells undergo transdifferentiation toward a myofibroblastic phenotype. The two cell types have different skeletal proteins, keratin for epithelium and vimentin for myofibroblastic. The cells derived from surface ectoderm always express E-cadherin to form adherence to each other. The EMT process involves transcriptional reprogramming of a series of genes that include α-SMA known as a maker of myofibroblast cells. Therefore, except for the distinct expression of keratin and vimentin, the α-SMA expression is considered as the feature of LECs transdifferentiation as well as the loss of E-cadherin production [9–11].
References
[1]
E. D. Hay, “An overview of epithelio-mesenchymal transformation,” Acta Anatomica, vol. 154, no. 1, pp. 8–20, 1995.
[2]
P. Savagner, “Leaving the neighborhood: molecular mechanisms involved during epithelial-mesenchymal transition,” BioEssays, vol. 23, no. 10, pp. 912–923, 2001.
[3]
J. P. Thiery, “Epithelial-mesenchymal transitions in cancer onset and progression,” Bulletin de l'Academie Nationale de Medecine, vol. 193, no. 9, pp. 1969–1979, 2009.
[4]
D. Sun, S. Baur, and E. D. Hay, “Epithelial-mesenchymal transformation is the mechanism for fusion of the craniofacial primordia involved in morphogenesis of the chicken lip,” Developmental Biology, vol. 228, no. 2, pp. 337–349, 2000.
[5]
S. Saika, Y. Okada, T. Miyamoto, Y. Ohnishi, A. Ooshima, and J. W. McAvoy, “Smad translocation and growth suppression in lens epithelial cells by endogenous TGFβ2 during wound repair,” Experimental Eye Research, vol. 72, no. 6, pp. 679–686, 2001.
[6]
S. Saika, T. Miyamoto, S. Tanaka et al., “Response of lens epithelial cells to injury: role of lumican in epithelial-mesenchymal transition,” Investigative Ophthalmology and Visual Science, vol. 44, no. 5, pp. 2094–2102, 2003.
[7]
S. Saika, S. Kono-Saika, Y. Ohnishi et al., “Smad3 signaling is required for epithelial-mesenchymal transition of lens epithelium after injury,” American Journal of Pathology, vol. 164, no. 2, pp. 651–663, 2004.
[8]
R. U. de Iongh, E. Wederell, F. J. Lovicu, and J. W. McAvoy, “Transforming growth factor-β-induced epithelial-mesenchymal transition in the lens: a model for cataract formation,” Cells Tissues Organs, vol. 179, no. 1-2, pp. 43–55, 2005.
[9]
M. B. Vaughan, E. W. Howard, and J. J. Tomasek, “Transforming growth factor-β1 promotes the morphological and functional differentiation of the myofibroblast,” Experimental Cell Research, vol. 257, no. 1, pp. 180–189, 2000.
[10]
G. Serini, M. Bochaton-Piallat, P. Ropraz et al., “The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-β1,” Journal of Cell Biology, vol. 142, no. 3, pp. 873–881, 1998.
[11]
A. Vernon and C. LaBonne, “Tumor metastasis: a new twist on epithelial-mesenchymal transitions,” Current Biology, vol. 14, no. 17, pp. R719–R721, 2004.
[12]
J. Massague, “TGF-beta signal transduction,” Annual Review of Biochemistry, vol. 67, pp. 753–791, 1998.
[13]
C. M. Zimmerman and R. W. Padgett, “Transforming growth factor β signaling mediators and modulators,” Gene, vol. 249, no. 1-2, pp. 17–30, 2000.
[14]
K. Miyazono, P. Ten Dijke, and C. Heldin, “TGF-β signaling by Smad proteins,” Advances in Immunology, vol. 75, pp. 115–157, 2000.
[15]
A. Moustakas, K. Pardali, A. Gaal, and C. Heldin, “Mechanisms of TGF-β signaling in regulation of cell growth and differentiation,” Immunology Letters, vol. 82, no. 1-2, pp. 85–91, 2002.
[16]
P. Ten Dijke, M. J. Goumans, F. Itoh, and S. Itoh, “Regulation of cell proliferation by Smad proteins,” Journal of Cellular Physiology, vol. 191, no. 1, pp. 1–16, 2002.
[17]
A. B. Roberts and M. B. Sporn, “Differential expression of the TGF-β isoforms in embryogenesis suggests specific roles in developing and adult tissues,” Molecular Reproduction and Development, vol. 32, no. 2, pp. 91–98, 1992.
[18]
C. Gordon-Thomson, R. U. de Iongh, A. M. Hales, C. G. Chamberlain, and J. W. McAvoy, “Differential cataractogenic potency of tgf-β1, β2, and -β3 and their expression in the postnatal rat eye,” Investigative Ophthalmology and Visual Science, vol. 39, no. 8, pp. 1399–1409, 1998.
[19]
T. Ashish, C. K. T. Jonathan, S. Ajay, G. Rangan, and R. M. Rajiv, “Role of transforming growth factor beta in corneal function, biology and pathology,” Current Molecular Medicine, vol. 10, no. 6, pp. 565–578, 2010.
[20]
K. Ohta, S. Yamagami, A. W. Taylor, and J. W. Streilein, “IL-6 antagonizes TGF-β and abolishes immune privilege in eyes with endotoxin-induced uveitis,” Investigative Ophthalmology and Visual Science, vol. 41, no. 9, pp. 2591–2599, 2000.
[21]
H. D. Jampel, N. Roche, W. J. Stark, and A. B. Roberts, “Transforming growth factor-β in human aqueous humor,” Current Eye Research, vol. 9, no. 10, pp. 963–969, 1990.
[22]
S. W. Cousins, M. M. McCabe, D. Danielpour, and J. W. Streilein, “Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor,” Investigative Ophthalmology and Visual Science, vol. 32, no. 8, pp. 2201–2211, 1991.
[23]
T. Kita, Y. Hata, R. Arita et al., “Role of TGF-β in proliferative vitreoretinal diseases and ROCK as a therapeutic target,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 45, pp. 17504–17509, 2008.
[24]
T. Kokudo, Y. Suzuki, Y. Yoshimatsu, T. Yamazaki, T. Watabe, and K. Miyazono, “Snail is required for TGFβ-induced endothelial-mesenchymal transition of embryonic stem cell-derived endothelial cells,” Journal of Cell Science, vol. 121, no. 20, pp. 3317–3324, 2008.
[25]
C. C？me, V. Arnoux, F. Bibeau, and P. Savagner, “Roles of the transcription factors Snail and slug during mammary morphogenesis and breast carcinoma progression,” Journal of Mammary Gland Biology and Neoplasia, vol. 9, no. 2, pp. 183–193, 2004.
[26]
E. Rosivatz, I. Becker, K. Specht et al., “Differential expression of the epithelial-mesenchymal transition regulators Snail, SIP1, and twist in gastric cancer,” American Journal of Pathology, vol. 161, no. 5, pp. 1881–1891, 2002.
[27]
E. Batlle, E. Sancho, C. Franci et al., “The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells,” Nature Cell Biology, vol. 2, no. 2, pp. 84–89, 2000.
[28]
H. G. Pálmer, M. J. Larriba, J. M. García et al., “The transcription factor Snail represses vitamin D receptor expression and responsiveness in human colon cancer,” Nature Medicine, vol. 10, no. 9, pp. 917–919, 2004.
[29]
S. A. Murray and T. Gridley, “Snail1 gene function during early embryo patterning in mice,” Cell Cycle, vol. 5, no. 22, pp. 2566–2570, 2006.
[30]
W. A. Paznekas, K. Okajima, M. Schertzer, S. Wood, and E. W. Jabs, “Genomic organization, expression, and chromosome location of the human Snail gene (SNAI1) and a related processed pseudogene (SNAI1P),” Genomics, vol. 62, no. 1, pp. 42–49, 1999.
[31]
K. Aomatsu, T. Arao, K. Sugioka et al., “TGF-β induces sustained upregulation of SNAI1 and SNAI2 through smad and non-smad pathways in a human corneal epithelial cell line,” Investigative Ophthalmology and Visual Science, vol. 52, no. 5, pp. 2437–2443, 2011.
[32]
K. Shirai, S. Saika, T. Tanaka et al., “A new model of anterior subcapsular cataract: involvement of TGFβ/Smad signaling,” Molecular Vision, vol. 12, pp. 681–691, 2006.
[33]
A. M. Abu El-Asrar, L. Missotten, and K. Geboes, “Expression of myofibroblast activation molecules in proliferative vitreoretinopathy epiretinal membranes,” Acta Ophthalmologica, vol. 89, no. 2, pp. e115–e121, 2011.
[34]
A. Cano, M. A. Pérez-Moreno, I. Rodrigo et al., “The transcription factor Snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression,” Nature Cell Biology, vol. 2, no. 2, pp. 76–83, 2000.
[35]
E. Batlle, E. Sancho, C. Franci et al., “The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells,” Nature Cell Biology, vol. 2, no. 2, pp. 84–89, 2000.
[36]
H. J. Cho, K. E. Baek, S. Saika, M. Jeong, and J. Yoo, “Snail is required for transforming growth factor-β-induced epithelial-mesenchymal transition by activating PI3 kinase/Akt signal pathway,” Biochemical and Biophysical Research Communications, vol. 353, no. 2, pp. 337–343, 2007.
[37]
S. M. Elbashir, W. Lendeckel, and T. Tuschl, “RNA interference is mediated by 21- and 22-nucleotide RNAs,” Genes and Development, vol. 15, no. 2, pp. 188–200, 2001.
[38]
C. M. Sax, F. X. Farrell, Z. E. Zehner, and J. Piatigorsky, “Regulation of vimentin gene expression in the ocular lens,” Developmental Biology, vol. 139, no. 1, pp. 56–64, 1990.
[39]
D. S. Clark, “Posterior capsule opacification,” Current Opinion in Ophthalmology, vol. 11, no. 1, pp. 56–64, 2000.
[40]
D. A. Schaumberg, M. R. Dana, W. G. Christen, and R. J. Glynn, “A systematic overview of the incidence of posterior capsule opacification,” Ophthalmology, vol. 105, no. 7, pp. 1213–1221, 1998.
[41]
P. J. McDonnell, M. A. Zarbin, and W. R. Green, “Posterior capsule opacification in pseudophakic eyes,” Ophthalmology, vol. 90, no. 12, pp. 1548–1553, 1983.
[42]
S. Dewey, “Posterior capsule opacification,” Current Opinion in Ophthalmology, vol. 17, no. 1, pp. 45–53, 2006.
[43]
L. M. Cobo, E. Ohsawa, and D. Chandler, “Pathogenesis of capsular opacification after extracapsular cataract extraction. An animal model,” Ophthalmology, vol. 91, no. 7, pp. 857–863, 1984.
[44]
I. M. Wormstone, “Posterior capsule opacification: a cell biological perspective,” Experimental Eye Research, vol. 74, no. 3, pp. 337–347, 2002.
[45]
R. Frezzotti, A. Caporossi, D. Mastrangelo et al., “Pathogenesis of posterior capsular opacification. Part II: histopathological and in vitro culture findings,” Journal of Cataract and Refractive Surgery, vol. 16, no. 3, pp. 353–360, 1990.
[46]
J. P. Kappelhof and G. F. Vrensen, “The pathology of after-cataract. A minireview,” Acta ophthalmologica, supplement 205, pp. 13–24, 1992.
[47]
N. Awasthi and B. J. Wagner, “Suppression of human lens epithelial cell proliferation by proteasome inhibition, a potential defense against posterior capsular opacification,” Investigative Ophthalmology and Visual Science, vol. 47, no. 10, pp. 4482–4489, 2006.
[48]
I. M. Wormstone, C. S. C. Liu, J. Rakic, J. M. Marcantonio, G. F. J. M. Vrensen, and G. Duncan, “Human lens epithelial cell proliferation in a protein-free medium,” Investigative Ophthalmology and Visual Science, vol. 38, no. 2, pp. 396–404, 1997.
[49]
J. L. Walker, I. M. Wolff, L. Zhang, and A. S. Menko, “Activation of Src kinases signals induction of posterior capsule opacification,” Investigative Ophthalmology and Visual Science, vol. 48, no. 5, pp. 2214–2223, 2007.
[50]
D. Javelaud and A. Mauviel, “Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-β: implications for carcinogenesis,” Oncogene, vol. 24, no. 37, pp. 5742–5750, 2005.
[51]
M. A. Nieto, “The Snail superfamily of zinc-finger transcription factors,” Nature Reviews Molecular Cell Biology, vol. 3, no. 3, pp. 155–166, 2002.
[52]
R. Kalluri and E. G. Neilson, “Epithelial-mesenchymal transition and its implications for fibrosis,” Journal of Clinical Investigation, vol. 112, no. 12, pp. 1776–1784, 2003.
[53]
M. A. Huber, N. Azoitei, B. Baumann et al., “NF-κB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression,” Journal of Clinical Investigation, vol. 114, no. 4, pp. 569–581, 2004.
[54]
J. M. Lee, S. Dedhar, R. Kalluri, and E. W. Thompson, “The epithelial-mesenchymal transition: new insights in signaling, development, and disease,” Journal of Cell Biology, vol. 172, no. 7, pp. 973–981, 2006.
[55]
M. A. Nieto, “Epithelial-Mesenchymal Transitions in development and disease: old views and new perspectives,” International Journal of Developmental Biology, vol. 53, no. 8–10, pp. 1541–1547, 2009.
[56]
G. A. Barrallo and M. A. Nieto, “The Snail genes as inducers of cell movement and survival: implications in development and cancer,” Development, vol. 132, no. 14, pp. 3151–3161, 2005.
[57]
B. De Craene, F. Van Roy, and G. Berx, “Unraveling signalling cascades for the Snail family of transcription factors,” Cellular Signalling, vol. 17, no. 5, pp. 535–547, 2005.
[58]
J. Choi, Y. P. Sun, and C. Joo, “Transforming growth factor-β1 represses E-cadherin production via Slug expression in lens epithelial cells,” Investigative Ophthalmology and Visual Science, vol. 48, no. 6, pp. 2708–2718, 2007.
[59]
M. T. McManus and P. A. Sharp, “Gene silencing in mammals by small interfering RNAs,” Nature Reviews Genetics, vol. 3, no. 10, pp. 737–747, 2002.
