Sphingolipid Signaling in Mammalian Photoreceptor Cells

Abstract

Purpose: : Sphingolipids are essential components of every cell membrane. Recently, a signaling role of bioactive sphingolipids such as ceramide and sphingosine-1-phosphate (S1P) has been established. Ceramide induces apoptosis and S1P plays an anti-apoptotic role. S1P signals through G-protein coupled receptors. Here we investigated ceramide and S1P signaling in photoreceptor cells.

Methods: : Expression of S1P biosynthetic enzymes sphingosine kinase (SPHK1 and 2) and their receptors S1P1-5 in rat retina was tested by quantitative RT-PCR, Western blotting (WB) and by Immunohistochemistry (IHC). Developing rat photoreceptors were isolated and used for immunocytochemistry. Rod outer segments (ROS) were prepared from light-adapted and dark-adapted rat retinas by sucrose density gradient centrifugation and subjected to WB with antibodies of the above proteins. Further, rat retinas were light stressed in vivo at 2700 lux for 6 h, which specifically affects the photoreceptor cells, and the signaling of ceramide and S1P was measured by assaying gene expression, activity of bio-synthetic enzymes, and levels of bio-synthetic intermediates of ceramide and S1P.

Results: : We detected expression of both SPHK1 and 2 in photoreceptor cells by RT-PCR; we detected localization of both the proteins in different layers of the retina; SPHK1 was not detected in ROS. Photoreceptor cells expressed S1P receptors S1P1, S1P2 and S1P3 with very prominent expression of S1P2 in ROS. S1P1 expression in ROS was reduced in light-adaptation. In immunohistochemical sections, S1P1 had intense labeling at RPE apical membrane. In isolated rod photoreceptors S1P3 was located on plasma membrane and S1P2 on developing ROS. Intense light exposure increased retinal ceramide and S1P levels well before the onset of apoptosis. In the light-damage model, ceramide increased by de novo biosynthesis whereas increased S1P blocked ceramide formation from acid sphingomyelinase (aSMase). Intense light also increased expression of S1P2 and S1P3 indicating S1P signaling in photoreceptors.

Conclusions: : Ceramide and S1P play a critical role in photoreceptor apoptosis induced by light stress. Expression and localization of SPHKs and G-protein-coupled S1P receptors on photoreceptor and RPE cells suggest their functions in these cells, which may include visual signal transduction, membrane turnover and transport or trafficking of proteins in the RPE and ROS membrane, and stress response.

Keywords: photoreceptors • retinal degenerations: cell biology • retina