ZONAB and ZO–1 Are Involved in the Differentiation of Retinal Pigment Epithelium

Abstract

Abstract: : Purpose: The retinal pigment epithelium (RPE) establishes tight junctions, which regulate selective diffusion of solutes along the paracellular space. Tight junctions are also involved in the control of cell proliferation and gene expression. ZONAB, a Y–box transcription factor functionally interacts with the tight junction protein ZO–1 to regulate gene expression and cell cycle progression (2000, EMBO J. 19, 2004;2003, J Cell Biol 160, 423). Our hypothesis is that ZO–1 and ZONAB regulate epithelial cell proliferation and differentiation in a cell–density dependent manner . Therefore, we wish to determine the role of ZONAB and tight junctions in the control of RPE proliferation and differentiation. Methods: We have generated ARPE–19 cell lines that overexpress either ZO–1 or ZONAB to assess RPE differentiation by fluorescence microscopy and cell and molecular biological assays. We analysed ZONAB expression in RPE cells in situ of the Royal College of Surgeons (RCS) rat, a model of retinal degeneration, and control animals. We developed lentiviruses to overexpress ZONAB or downregulate ZO–1 in RPE cells in mice at different postnatal days. Results: In ARPE–19 cells, overexpression of ZONAB causes dedifferentiation manifested by disorganisation of cell–cell junctional and cytoskeletal proteins (ZO–1, beta–catenin, actin and alpha–smooth muscle actin). Overexpression of ZO–1 or downregulation of ZONAB arrests proliferation of ARPE–19 cells . In RPE cells of degenerated retinas of RCS rats, ZONAB and alpha–smooth muscle actin are upregulated. Overexpression of ZONAB and/or downregulation of ZO–1 by lentiviral vectors induce retinal dedifferentiation in mice. In reporter gene assays, ZONAB upregulates cyclin D1 and PCNA transcription, two important cell cycle regulators. Conclusions: These results indicate that the ZONAB signal transduction pathway regulates cell proliferation and differentiation of ARPE–19 cells, ZONAB overexpression correlates with increases in alpha–smooth muscle actin expression in cells in culture as well as RPE degeneration in vivo. Our data suggest that ZO–1/ZONAB signalling is important for RPE differentiation and/or maintenance and that manipulation of ZONAB function might enable us to manipulate RPE proliferation and differentiation in vivo. The Wellcome Trust and the Medical Research Council UK support this work.

Keywords: retinal pigment epithelium • cell adhesions/cell junctions • transcription factors