Phagocytosis of tips of rod outer segments (ROS) by retinal pigment epithelial (RPE) cells is vitally important for maintaining structural and functional integrity of the retina. We previously reported that receptor-mediated specific phagocytosis of ROS induces expression of early response genes coding for transcription factors. Here we study the expression of peroxisome proliferator-activated receptors (PPAR) -alpha, -delta (beta) and -gamma during ROS phagocytosis of rat RPE cells in primary cell culture, using competitive quantitative RT-PCR. During phagocytosis of ROS (but not of latex particles) by RPE cells, RT-PCR revealed a transient increase in PPARgamma mRNA expression, that peaked at 4-6 hr. We sequenced and described two alternatively spliced variants of rat PPARgamma: rPPARgamma1a and rPPARgamma1b. Both of these, along with the recently described rPPARgamma2 were induced by ROS phagocytosis. PPARalpha and PPARdelta mRNA expression was also detected in RPE cells, but the level of expression did not change during ROS phagocytosis. All-trans-retinoic acid and prostaglandin E(2) (PGE(2)) selectively potentiated both basal and ROS-phagocytosis-induced PPARgamma expression. All-trans-retinoic acid had the opposite inhibitory effect on PPARalpha and PPARdelta expression. Cycloheximide had a dual action on PPARgamma expression in RPE cells: it enhanced expression under basal conditions but repressed expression induced by ROS phagocytosis. It also stimulated expression of PPARalpha but had no effect on PPARdelta. Selective activation of PPARgamma may play an important role in regulating the expression of target genes that are involved in lipid and fatty acid metabolism in the photoreceptor renewal process.

Expression of early response genes during rod outer segment phagocytosis by normal Long Evans and Royal College of Surgeons-rdy

Retinal pigment epithelial cells (RPE) actively retrieve and recycle docosahexaenoic acid (DHA, 22:6n-3) from phagosomal phospholipids back to photoreceptor cells. Here we studied the fate of DHA in primary culture rat RPE cells after feeding with a suspension of rod outer segments (ROS) for 4 hr. Phospholipids (PLs), triacylglycerols (TAG), and free fatty acids were isolated from cells and media by thin layer chromatography (TLC), and their acyl groups quantified by gas liquid chromatography (GLC). In RPE cells, DHA-PLs increased 3. 5-fold by 4 hr, decreasing thereafter to 1.6-fold above basal by 24 hr. In contrast, 18:1-PLs were decreased by 13%-18% below RPE basal values by 8-24 hr, respectively. DHA-TAG showed the highest increase (21-fold) by 8 hr. Free DHA displayed a small increase in the cells with a preferential release and accumulation into the media by 24 hr. These results show that in rat RPE cells, photoreceptor cell DHA is transiently incorporated into TAG prior to its release and uptake into 18:1-PLs. These metabolic pathways and remodeling may be critical in the conservation of this essential, photoreceptor cell fatty acid.

No abstract