Retinoid processing proteins in the ocular ciliary epithelium

• Type of Material: Article
• Authors: Mercedes Salvador-Silva, Yale University; Sikha Ghosh, Yale University; Rubens Bertazolli-Filho, Yale University; Jeffrey Boatright, Emory University; John Nickerson, Emory University; Gregory G. Garwin, University of Washington; John C. Saari, University of Washington; Miguel Coca-Prados, Yale University
• Language: English
• Date: 2005-05-18
• Publisher: Molecular Vision
• Publication Version: Final Published Version
• Copyright Statement: ©2005 Molecular Vision
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://www.molvis.org/molvis/v11/a42/
• Title of Journal or Parent Work: Molecular Vision
• ISSN: 1090-0535
• Volume: 2005
• Issue: 11
• Start Page: 356
• End Page: 365
• Grant/Funding Information: This work was supported by NIH grants EY04873 (MC-P), EY00785 (for core facilities), EY02317, EY01730 (JCS) and Research to Prevent Blindness, Inc. RB-F was a recipient of a Molecular Vision 2005; 11:356-65 <http://www.molvis.org/molvis/v11/a42> ©2005 Molecular Vision 363fellowship (1998-05443-6) of the Fundaçao de Amparo a Pesquisa do Estado de São Paulo (Brasil).
• Research Categories: Health Sciences, Opthamology

Abstract

PURPOSE: To identify retinoids and retinoid processing proteins in the ocular ciliary epithelium (CE), and to compare in cultured ciliary epithelial cell lines promoter activities of the cellular retinaldehyde binding protein (CRALBP) and interphotoreceptor retinoid binding protein (IRBP). METHODS: Retinoid processing proteins were detected by RT-PCR, western analysis and immunocytochemistry. PCR products were verified by DNA sequence analysis. Retinoids were measured by normal phase HPLC and UV visible spectroscopy. Reporter product from CRALBP and IRBP promoter fragments was measured following transient transfection in bovine pigmented and nonpigmented CE cells. RESULTS: CRALBP, IRBP, cellular retinol binding protein (CRBP), 11-cis-retinol dehydrogenase (11-cis-RDH), lecithin:retinol acyltransferase (LRAT), and ATP binding cassette receptor (ABCR) were detected in human CE tissue by RT-PCR. Retinal pigment epithelium specific protein 65 kDa (RPE65) mRNA and protein were also detected. CRALBP and IRBP were detected by western analysis in tissue extracts from bovine CE and were localized to the PE and NPE cell layers, respectively, by immunocytochemistry. IRBP immunoreactivity was also detected in aqueous humor. Retinoids identified in the bovine CE include retinyl esters (7.4+/-3.5 pMol/mg of protein) and all-trans-retinol (14.9+/-1.1 pMol/mg of protein). Betacarotene was also tentatively identified. 11-cis-Retinoids were not detected. In CE cell cultures, the CRALBP p2.1-kb promoter construct exhibited reporter activity 15-30 fold above basal level, with 2 fold more activity in pigmented than nonpigmented CE cells. IRBP promoter constructs exhibited low level reporter activities in vitro in both CE cell layers. CONCLUSIONS: The ocular CE expresses genes encoding components of the rod visual cycle. The differential localization of CRALBP and IRBP along the bilayer of the CE suggests a potential role in retinoid transport and/or retinoid metabolism. However, the absence of 11-cis-retinoids suggests that the function of retinoid processing proteins in the CE differs from that of the retina.