Metabolic and Molecular Signaling Pathways of Retinoids on Human Ocular Surface

Abstract

Abstract: : Purpose: The integrity of the human ocular surface has an absolute requirement of vitamin A and its active derivatives, the retinoic acids. These retinoids regulate transcriptional levels of target genes through the activation of ligand–dependant nuclear receptors: Retinoic Acid Receptors and Retinoid X Receptors. Partial characterization of these receptors have been realised in rabbit, mouse and human cornea, but systematic tissue and cellular expression of the 3 RARs and RXRs had to be completed at the human ocular surface. The first objective of this work was to define their expression patterns in term of genes and proteins for total human conjunctiva, cornea and the major cell types present constituting human ocular surface. The second objective was to demonstrate the presence of differents enzymes transforming vitamin A to retinoic acid and the activity of this retinoids pathway in the ocular surface. Methods: Total mRNA was extracted from human total cornea, conjunctiva, corneal epithelial cells, corneal keratocytes, corneal endothelial cells and conjunctival epithelial cell and submitted to RT–PCR experiments in order to determine the expression patterns of the RARs and RXRs using specific primers. Immunological staining (histo– and cellular chemistry) experiments were performed to better define the RARs and RXRs protein localization in ocular surface at tissular and cellular levels. The expression patterns of retinal deshydrogenase (RALDHs) and alcohol deshydrogénase (ALDHs) was also determined in corneal epithelial cells. The metabolic pathway is tested by targeted disruption of different enzyme step who transforms retinol to active derivatives. Results: RAR alpha, gamma and RXR alpha are expressed in cornea, conjunctiva and all of their constitutive cells, checked in our study. RXR gamma was never detected in cornea and conjunctiva. RXR beta was only present in primary cultures of corneal keratinocytes, which don't express RAR beta in contrast of the other corneal and conjunctival cells.ALDH3, ALDH4, RALDH1, RALDH 3 are expressed in ocular surface. Conclusions: For the first time, we established an exhaustive description of RARs and RXRs, ALDHs and RALDHs expression patterns, in the human ocular surface. Our results demonstrated at the human ocular surface, the presence of all the actors of retinoic acid signalling pathway and their activation.

Keywords: retinoids/retinoid binding proteins • transcription factors • cornea: basic science