EXPRESSION AND REGULATION OF COMPONENTS OF THE VISUAL CYCLE CRALBP AND IRBP IN CILIARY EPITHELIAL CELLS

Abstract

Abstract: : Purpose: The cellular retinaldehyde–binding protein (CRALBP), and the interphotoreceptor binding protein (IRBP) are soluble proteins involved in the regeneration of photopigment in the visual cycle. CRALBP has been shown to be expressed in the ocular ciliary epithelium (CE), a bilayer of non–pigmented (NPE) and pigmented (PE) epithelial cells. The purpose of this work was to determine the transcriptional activity of the CRALBP and IRBP genes in PE and NPE cells in vitro. Methods: Expression of IRBP and CRALBP were determined by PCR on cDNA templates synthesized in vitro from poly A⁺ mRNA isolated from the human or bovine CE. Two human CRALBP luciferase reporter gene constructs (p2.1–kb and p0.2–kb), and three murine IRBP CAT reporter gene constructs (p45CAT; p70CAT and p156CAT) were used in transient transfections assays of PE and NPE cells cultures. Antibodies to CRALBP and IRBP were also used by indirect immunofluorescence to determine their cellular localization along the CE and by Western blotting to determine their protein expression on bovine CE extracts. Results: By RT–PCR and DNA sequencing we verified that the human and bovine CE express CRALBP and IRBP mRNA. Transfection with the CRALBP p2.1–kb construct induced a 2–fold increase in its transcriptional activity on PE cells over the NPE cells, and 30–fold over the basal activity from the p0.2–kb construct. The IRBP promoter p70CAT induced a 2.5–fold increase in its transcriptional activity on NPE cells over PE cells and a 4–fold over the basal activity observed with the control vector psV0CAT. By indirect immunofluorescence IRBP is preferentially restricted to the NPE cells layer, whereas CRALBP is along the PE cells. By Western blot the anti–IRBP sera cross–reacted with a 145–kDa protein, and the anti–CRALBP cross–reacted with a 36–kDa protein respectively on whole tissue extracts from the CE. Conclusions: These results together with earlier observations that the CE contains many components of phototransduction supports the view that the CE may be is photosensitive. Further experiments are needed to demonstrate this possibility.

Keywords: ciliary body • gene/expression • retinoids/retinoid binding proteins