Molecular and metabolic retinoid pathways in the human ocular surface

To maintain its integrity, the human ocular surface (cornea and conjunctiva) has an absolute requirement for vitamin A and its active derivatives, the retinoic acids. These retinoids regulate transcriptional levels of target genes through the activation of members of a super-family of ligand-dependant nuclear receptors that feature retinoic acid receptors (RAR) alpha, beta, and gamma as well as retinoid X receptors (RXR) alpha, beta, and gamma. The expression patterns of these receptors have been partial characterized in rabbit, mouse, and human cornea and conjunctiva, but systematic tissue and cellular expression of the three RARs and three RXRs had to be completed at the adult human ocular surface. The first objective of our work was to define their expression patterns in term of genes and proteins for total human conjunctiva, cornea, and the major cell types comprising the adult human ocular surface. The second objective was to demonstrate the presence of different enzymes transforming vitamin A to retinoic acid and the functionality of this metabolic pathway in the corneal epithelium.

Total mRNA was extracted from human total cornea, conjunctiva, corneal epithelial cells (primary culture and established cell line), corneal keratocytes (primary culture), corneal endothelial cells (established cell line), and conjunctival epithelial cells (established cell line) and was submitted to reverse transcription-polymerase chain reaction (RT-PCR) analysis to determine the expression patterns of the RARs and RXRs using specific primers. Immunological staining (via histochemistry and cellular chemistry) experiments were performed to better localize RAR and RXR proteins in the ocular surface at tissue and cellular levels. We also checked mRNA expression of cellular retinol binding proteins (CRBPs) and cellular retinoic acid binding proteins (CRABPs) with the enzymes involved in retinoic acid generation, i.e., alcohol dehydrogenases (ADHs) and retinal dehydrogenases (RALDHs) or degradation (Cyp26 family members). The enzymatic generation of functional retinoids was confirmed using epithelial corneal cells treated with specific inhibitors of retinol metabolism.

RAR alpha, RAR gamma, and RXR alpha are expressed in the cornea, conjunctiva, and all of their constitutive cells, whereas RXR gamma and RXR beta were never detected in the cornea or conjunctiva. RAR beta was absent in primary cultures of corneal keratinocytes. ADH3, ADH4, dehydrogenase/reductase (SDR family) 4 (DHRS4), dehydrogenase/reductase (SDR family) 9 (DHRS9), RALDH1, and RALDH3 are expressed in the ocular surface, as were the retinoid-binding proteins CRBP1, CRABP1, and CRABP2. Retinol dehydrogenase 4 (RODH4) was only detected in the conjunctiva. Corneal epithelial cells convert retinol into retinoic acid using an enzymatic pathway.

For the first time, we have established an exhaustive description of the expressions patterns of RARs, RXRs, ADHs, RALDHs, CRBP, and CRABPs in the human ocular surface. Our results for the human ocular surface demonstrated the presence of all the metabolic and molecular actors of the retinoic acid signaling pathway. We also demonstrated the enzymatic conversion of retinol into active retinoids in the corneal environment.