Neuroscience Letters 424 (2007) 190–193 Media conditioned by retinal pigment epithelial cells suppress the canonical Wnt pathway  Toshihiro Inoue a,b,1 , Takahiro Kawaji a,∗,1 , Miyuki Inoue-Mochita b , Tetsuya Taga b , Hidenobu Tanihara a a Department of Ophthalmology and Visual Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan b Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Kumamoto, Japan Received 7 May 2007; received in revised form 13 June 2007; accepted 17 July 2007 Abstract Retinal pigment epithelial (RPE) cells play critical roles in the maintenance of visual function, partly by secreting various biologically active factors that modulate the intraocular environment. Recent studies suggest involvement of Wnt proteins secreted by RPE cells in the pathogenesis of photoreceptor degeneration. In the present study, we examined, via the luciferase assay, the effect of media conditioned by RPE cells (RPE-CM) on activity of the canonical Wnt pathway in vitro. We isolated primary RPE cells from Long–Evans rats at P6–P9. In culture, these cells formed a monolayer with polygonal cell morphology and demonstrated repigmentation at confluency and immunoreactivity for ZO-1, a marker for tight junctions. To evaluate the effect of RPE-CM on the canonical Wnt pathway, we replaced the culture media of COS-7 cells transfected with (Tcf) 7 LUC, a multimeric Tcf-responsive element luciferase reporter construct, with RPE-CM and measured luciferase activity with or without Wnt3a or SB216763, a specific GSK3 inhibitor. RPE-CM did not enhance basal or Wnt3a-induced (Tcf) 7 LUC activity; instead, this activity decreased by 60%. RPE-CM also reduced SB216763-induced (Tcf) 7 LUC activity by 65%, which suggests that the inhibitory effect of RPE-CM is probably due to intracellular crosstalk rather than extracellular antagonism. RPE cells may thus be able to modulate the intraocular environment by regulating the canonical Wnt pathway. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Retinal pigment epithelial cell; Wnt pathway; Conditioned media; SB216763 Retinal pigment epithelium (RPE) cells are important types of cells in ocular tissues to maintain visual function, because they play critical roles in this maintenance of visual function: absorp- tion of light energy, transport of ions and metabolic endproducts, isomerization of retinal, and phagocytosis of photoreceptor outer segments. In addition, RPE cells can secret various biologi- cally active factors that modulate the intraocular environment [16]. Therefore, RPE cell dysfunction disrupts the intraocular environment and can lead to loss of visual function.  This work was supported in part by a grant-in-aid for the 21st Century COE Research from Ministry of Education, Science and Culture ‘Cell Fate Regulation Research and Education Unit’; Scientific Research in Priority Areas Research from the Ministry of Education, Culture, Science, Sports and Technology. ∗ Corresponding author. Tel.: +81 96 373 5247; fax: +81 96 373 5249. E-mail address: kawag@white.plala.or.jp (T. Kawaji). 1 These authors contributed equally to this article. Wnt proteins are secreted signaling molecules that activate different intracellular signaling cascades, including the canon- ical pathway, the c-Jun N-terminal kinase pathway, the Ca 2+ pathway, and the focal adhesion kinase pathway [14]. In the canonical pathway, binding of Wnt proteins to its receptors – Frizzled and low-density-lipoprotein receptor-related proteins – causes inactivation of glycogen synthase kinase (GSK) 3 and axin, respectively. Inactivation of these proteins stabilizes - catenin, which then accumulates in the cell nucleus and activates the transduction of target genes that are crucial in the G 1 -S-phase transition, such as cyclin D1 and c-Myc [20]. The canonical Wnt pathway thereby regulates cell proliferation in various vertebrate tissues. Recent studies suggest, in addition to the role for Wnt signaling in ocular development, a role in pathogenesis of photoreceptor degeneration. In an animal model of human retinitis pigmentosa – mice with rd6 – the membrane-type frizzled-related protein gene was highly expressed in RPE cells, 0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.07.022