Retinoid processing proteins in the ocular ciliary epithelium.
OBJECTIVE
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.
Salvador-Silva
Mercedes
M
Department of Ophthalmology and Visual Science, Yale University, New Haven, CT 06510, USA. Mercedes_Salvador-Silva@bausch.com
Ghosh
Sikha
S
Bertazolli-Filho
Rubens
R
Boatright
Jeffrey H
JH
Nickerson
John M
JM
Garwin
Gregory G
GG
Saari
John C
JC
Coca-Prados
Miguel
M
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002924:Ciliary Body / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012176:Retinoids / Q000235:genetics / Q000378:metabolism*; D012177:Retinol-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D054840:Retinol-Binding Proteins, Cellular; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019745:cis-trans-Isomerases; D000099214:Retinol-Binding Proteins, Interstitial; D000099178:Retinoid Isomerohydrolase
D018528:ATP-Binding Cassette Transporters; D000217:Acyltransferases; D000429:Alcohol Oxidoreductases; D002352:Carrier Proteins; D005136:Eye Proteins; D012333:RNA, Messenger; D012176:Retinoids; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D019745:cis-trans-Isomerases; D000099214:Retinol-Binding Proteins, Interstitial; D000099178:Retinoid Isomerohydrolase; C020932:11-cis-retinal-binding protein; C022520:retinol dehydrogenase; C056942:lecithin-retinol acyltransferase