Culture of Highly Differentiated Human Retinal Pigment Epithelium for Analysis of the Polarized Uptake, Processing, and Secretion of Retinoids
The retinal pigment epithelium (RPE) occupies a strategic position within the eye, given its location between the neurosensory retina and the vascular bed (choroid) that nourishes the photoreceptor cells (rods and cones). Among the many attributes of this versatile monolayer of cells is its unique ability to convert vitamin A (retinol) into the prosthetic group (11-cis-retinal) for the rod and cone opsins, the photopigments essential for vision. It does so by absorbing retinol via a receptor-mediated process that involves the interaction of a carrier protein secreted by the liver, retinol-binding protein (RBP), and a receptor/channel that is the gene product of STRA6 (stimulated by retinoic acid 6). Following its uptake through the basolateral plasma membrane of the RPE, retinol encounters a brigade of binding proteins, membrane-bound receptors, and enzymes that mediate its multi-step conversion to 11-cis-retinal and the transport of this visual chromophore to the light-sensitive photoreceptor cell outer segment, the portion of the cell that houses the phototransduction cascade. This process is iterative, repeating itself via the retinoid visual cycle. Most of the human genes that code for this cohort of proteins carry disease-causing mutations in humans. The consequences of these mutations range in severity from relatively mild dysfunction such as congenital stationary night blindness to total blindness. The RPE, although post-mitotic in situ, is capable of proliferation when removed from its native milieu. This offers one the opportunity to study the retinoid visual cycle in modular form, providing insights into this intriguing process in health and disease. This chapter describes a cell culture method whereby the entire visual cycle can be created in vitro.
Key wordsRetinoid visual cycle retinal pigment epithelium 11-cis-retinal all-trans-retinal inherited retinal disease Stargardt macular dystrophy Leber congenital amaurosis age-related macular degeneration
- 6.Gehrs, K.M., Jackson, J.R., Brown, E.N., Allikmets, M., Hageman, G. (2009) Complement, age-related macular degeneration and a vision of the future. Arch. Ophthalmol. 128, 349–358.Google Scholar
- 13.Pasutto, F., Sticht, H., Hammersen, G., et al. (2007) Mutations in STRA6 cause a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia and mental retardation. Am. J. Hum. Genet. 80, 550–560.PubMedCrossRefGoogle Scholar
- 21.Bok, D. (1994) The retinal pigment epithelium; a versatile partner in vision. J. Cell Sci. 17(Suppl), 189–195.Google Scholar
- 33.Carlson, A., Bok, D. Promotion of the release of 11-cis-retinal from cultured retinal pigment epithelium by interphotoreceptor retinoid-binding protein. Biochemistry 31, 9056–9062.Google Scholar
- 34.Radu, R.A., Hu, J., Peng, J., Bok, D., Mata, N., Travis, G.H. (2008) Retinal pigment epithelium-retinal G protein receptor-opsin mediates light-dependent translocation of all-trans-retinyl esters for synthesis of visual chromophore in retinal pigment epithelial cells. J. Biol. Chem. 283, 19730–19738.PubMedCrossRefGoogle Scholar