Vitamin A And Ocular Tissues

National Eye Institute

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Abstract

Retinoic acid, a natural derivative of vitamin A, and its synthetic analogues have a profound effect on many cellular functions including cell growth, differentiation, and apoptosis. Among them, N?(4?hydroxyphenyl)retinamide (4HPR, fenretinide), an amide analog of retinoic acid, exerts its chemotherapeutic effects on cancer cells through induction of apoptosis. We have found that 4HPR induces apoptosis in human RPE (ARPE?19) cells. Earlier, we demonstrated that retinoic acid regulates the expression of stearoyl?CoA desaturase (SCD) in these cells. SCD, a microsomal enzyme catalyzing the initial desaturation of long chain fatty acids into monounsaturated fatty acids, is thought to play an important role in cell growth, differentiation and apoptosis by its ability to control the ratio of saturated to unsaturated fatty acids in cells. In addition certain fatty acid analogues, such as thia?fatty acids, are known to exert antineoplastic activity by inhibiting SCD. We investigated the effect of 9?thiastearate, a specific inhibitor of SCD, on 4HPR?induced apoptosis of human RPE cells and found that 9-thiastearate effectively blocked the apoptosis. 4HPR?induced apoptosis was accompanied by a marked increase in the activities of caspase?2 and caspase?3, and the 4HPR?induced activation of caspases was effectively inhibited by 9?thiastearate. NORPEG (novel retinal pigment epithelial cell gene, RAI14) is a gene that we originally characterized from the human retinal pigment epithelial (RPE) cell line ARPE?19 and showed to be regulated by retinoic acid. The developmentally regulated gene encodes a protein consisting of 980 amino acid residues containing an ankyrin repeats region towards the N?terminal and a coiled?coil domain towards the C?terminal.The N?terminal 286 amino acid residues of NORPEG containing the ankyrin repeats region was expressed as a HIS tag fusion protein and used to generate antibodies against the NORPEG protein. The antibody recognized a110 kDa protein band in ARPE?19 cell extracts analyzed by Western blotting and immunoprecipitation. Western blot analysis also showed the presence of the 110 kDa immunoreactive band in mouse eye extracts. NORPEG expression was detected in mouse eyes throughout the early postnatal period. Confocal immunolocalization studies detected NORPEG expression in the ganglion cell layer and the outer margin of the outer nuclear layer, in the lens epithelium, ciliary body, iris, cornea and ocular muscles. The expression of the protein encoded by NORPEG, a gene regulated by retinoic acid, in the mouse eye during postnatal development suggests that this protein may play an important role in eye development and function. Interphotoreceptor retinoid?binding protein (IRBP) is a component of the interphotoreceptor matrix (IPM) and is known to bind visual cycle retinoids. Serum albumin, a protein capable of binding retinoids, has also been reported to be a component of the IPM. Collaborative studies with the laboratories of M. Carter Cornwall and David R. Pepperberg on the role of IRBP on the visual cycle of rhodopsin demonstrated an apparent saturation in the clearance of all-trans retinol by IRBP, but not by serum albumin, from both bleached salamander red rods and from toad retinas, suggesting the involvement of specific binding sites for IRBP in the retina. Immunofluorescence analysis using a highly specific monoclonal antibody to bovine serum albumin demonstrated that in bovine retinas obtained shortly after slaughter there was no detectable albumin in the IPM, so it appears unlikely that albumin plays a physiological role in retinoid transport in the retina.