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M. A. Mandal, M.-P. Agbaga, J.-T. Tran, K. Henry, L. Zheng, R. S. Brush; Comprehensive Evaluation of Sphingolipid Gene Expression in Normal and Light-Stressed Retina and Oxidant-Stressed 661W Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5173.
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Sphingolipids/ceramides are essential components of every cell membrane and are especially important in neural tissues. Many sphingolipid metabolites, such as ceramide, sphingosine and sphingosine-1-phosphate (S-1-P), are bioactive lipids that act as second messengers and regulate cellular functions. Retina is a neural tissue and recent evidences suggest ceramide metabolism plays an important role in retinal photoreceptor cell survival and apoptosis. The purpose of this study was to profile the sphingolipids in the retina and perform a comprehensive assessment of the expression of sphingolipid metabolic genes in normal and light-stressed retina. We also assessed the expression profile of these genes in mouse retina-derived 661W cells stressed with H2O2.
Total sphingolipids were extracted from rat brain, skin, and retina as well as bovine retina, purified and analyzed on HPTLC plates. Expression and tissue distribution of a set of 16 genes (Spt1, Spt2, Gcs, Bcgb, Ck, Sphk1, Sphk2, Ss1, Sp1, Sp2, Sp3, Lass2, Lass4, Asah1, Asah2, and Asah3), which are involved in biosynthesis of ceramides were determined in different rat tissues by qRT-PCR and compared with retina. Expression of these genes in the intense light-stressed (2700 lux for 6 h) SD rat retina was determined by microarray and qRT-PCR. Similarly, the expression profile of these genes was also determined in H2O2-stressed 661W cells.
We detected at least 2 species of polar, 3 species of glyco-, 2 species of α-hydroxy-, and 3 species of non-hydroxy ceramides in the retina. Normal retina expresses high levels of Spt2, Gcs, Bcgb, Ck, Sphk2, Ss1, Sp1, Sp2, Lass2, Lass4, and Asah1genes. The presence of Spt1, Bcgb, Ck, Sphk1, Sphk2, and Sp2 was detected in photoreceptor-cell-derived cDNA. Retinal expression of Sphk1, Ck and Lass4 is higher than in brain. Intense light stress significantly up-regulates the expression of Sphk1 and its receptor Edg5. Other genes altered in light stress include Gcs, Ck, Lass4, Lass2 and Asah1. Significant up-regulation of Sphk1 was also detected in 661W cells stressed with H2O2.
Retina has a unique profile of sphingolipids. Expression of most of the sphingolipid biosynthetic genes in retina is comparable to that in brain. Sphk1 and Ck, which are involved in production of the bio-active ceramides S-1-P and ceramide-1-phosphate, increases in the retina under stress conditions. Our data establishes ceramides as integral components of the retina that play important roles in retinal structure and homeostasis.
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