Glaucoma Targeted Overexpression of TGF-a in the Corneal Epithelium of Adult Transgenic Mice Induces Changes in Anterior Segment Morphology and Activates Noncanonical Wnt Signaling Yong Yuan,* ,1,4 Lung-Kun Yeh, 2,4 Hongshan Liu, 1 Osamu Yamanaka, 1 William D. Hardie, 3 Winston W.-Y. Kao, 1 and Chia-Yang Liu* ,1 PURPOSE. Transforming growth factor-alpha (TGF-a) transduces its signal through the epidermal growth factor receptor and is essential for corneal epithelial homeostasis. Previous studies have demonstrated that overexpression of TGF-a in the developing eye leads to anterior segment dysgenesis. However, the underlying mechanisms remain unclear. Here we examined the effects of TGF-a overexpression on adult ocular surface homeostasis. METHODS. Binary Tet-On transgenic Krt12 rtTA /tet-O-TGF-a mice were subjected to doxycycline (Dox) induction to overexpress TGF-a in the corneal epithelium. Intraocular pressure (IOP) was measured by noninvasive tonometry. The enucleated eyes of the experimental mice were subjected to histopathology, immunohistochemistry, and biochemistry examination. RESULTS. Histologic and immunofluorescent examination showed that double-transgenic mice overexpressing TGF-a manifested peripheral anterior synechiae. Elevation of IOP, activation of glial cells, and loss of retinal ganglion cells were also observed. Quantitative real-time PCR revealed that the expressions of genes (RXRa, PITX2, and FOXC1) related to anterior segment dysgenesis were downregulated. Canonical Wnt signaling was suppressed, whereas noncanonical Wnt ligands (Wnt4 and Wnt5a) were upregulated. Increased myosin light chain phosphorylation suggested that noncanonical Wnt signaling is activated in affected eyes. CONCLUSIONS. Overexpression of TGF-a in the corneal epithe- lium induces changes in anterior segment morphology. Corneal endothelial abnormalities are associated with the activation of the noncanonical Wnt and RhoA/ROCK signaling axis, indicating a potential application of RhoA/ROCK inhibitors as a therapeutic strategy for certain types of secondary angle- closure glaucoma. (Invest Ophthalmol Vis Sci. 2013;54:1829– 1837) DOI:10.1167/iovs.12-11477 T ransforming growth factor alpha (TGF-a) belongs to the epidermal growth factor (EGF) family of mitogens. Both TGF-a and EGF exert their biological activity by binding to the EGF receptor (EGFR). TGF-a was first discovered in the medium of virus-transformed cells. 1 TGF-a and EGFR also coexpress in many types of tumors, including breast carcino- mas, 2 renal carcinomas, 3 and melanomas. 4 TGF-a was also found in wound fluid from skin graft donor site wounds. 5 However, the expression of TGF-a is not restricted to pathologic conditions. It also plays a pivotal role in embryonic development and adult homeostasis. TGF-a null mice display pronounced waviness of whiskers and fur, suggesting the role of TGF-a in hair follicle development. This animal also has subtle eye abnormalities. Some TGF-a null mice were born with their eyes partially open. The corneal epithelium appeared uniformly thinner and corneal inflammation was observed in affected eyes. 6,7 In addition to its role in hair and eye development, both EGF and TGF-a are also components of human tear fluid, indicating their role in corneal epithelial homeostasis. 8 Although TGF-a is essential for normal eye development, excess TGF-a has detrimental effects on ocular surface morphogenesis during development. For example, it has been reported that a-crystallin promoter-driven human TGF- a overexpression in the lens manifested multiple eye defects including corneal opacities, cataracts, and microphthalmia due to altered cell fate of mesenchymal cells during embryonic eye development. 9 Interestingly, phenotypes of transgenic mice appeared associated with the level of TGF-a. For example, a milder phenotype was also noted in a similar transgenic mouse line expressing a lower level of TGF-a. In mildly affected transgenic eyes, the corneal endothelium did not differentiate properly and the iris directly attached to the cornea, manifesting a typical anterior segment dysgenesis (ASD) phenotype. 10 ASD is usually caused by mutation of genes critical for anterior segment morphogenesis, including Pitx2, 11 FoxC1, 12 FoxE3, 13 BMP4, 14 Cyp1b1, 15 RXRa, 16 and DKK. 17 From the 1 Crawley Vision Research Laboratory, Department of Ophthalmology, College of Medicine, University of Cincinnati, Cincinnati, Ohio; the 2 Department of Ophthalmology, Chang-Gung Memorial Hospital, Chang-Gung University College of Medicine, Linko, Taiwan; and 3 Divisions of Pulmonary Biology and Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio. 4 These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors. Supported in part by National Eye Institute/National Institutes of Health Grants EY21501 (C-YL) and EY013755 (WW-YK); Research to Prevent Blindness; Ohio Lions Eye Research Foundation; National Science Council (Taiwan) Grant 1012314B182A056MY3; and Grant CMRPG3A1291 from Chang-Gung Memorial Hospital, Linko (L-KY). Submitted for publication December 12, 2012; accepted February 7, 2013. Disclosure: Y. Yuan, None; L.-K. Yeh, None; H. Liu, None; O. Yamanaka, None; W.D. Hardie, None; W.W.-Y. Kao, None; C.-Y. Liu, None *Each of the following is a corresponding author: Yong Yuan, Department of Ophthalmology, University of Cincinnati, 3230 Eden Avenue, Cincinnati, OH 45267; yuany@ucmail.uc.edu. Chia-Yang Liu, Department of Ophthalmology, University of Cincinnati, 3230 Eden Avenue, Cincinnati, OH 45267; liucg@ucmail.uc.edu. Investigative Ophthalmology & Visual Science, March 2013, Vol. 54, No. 3 Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc. 1829