International Journal of Molecular Sciences
Article Characterizing Sphingosine Kinases and Sphingosine 1-Phosphate Receptors in the Mammalian Eye and Retina
Hunter Porter 1,†, Hui Qi 1,†, Nicole Prabhu 1,†, Richard Grambergs 2, Joel McRae 1, Blake Hopiavuori 1 and Nawajes Mandal 1,2,* 1 Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA; [email protected] (H.P.); [email protected] (H.Q.); [email protected] (N.P.); [email protected] (J.M.); [email protected] (B.H.) 2 Departments of Ophthalmology, Anatomy and Neurobiology, University of Tennessee Health Sciences Center, Memphis, TN 38163, USA; [email protected] * Correspondence: [email protected]; +1-901-448-7740 † Equally contributing author.
Received: 19 November 2018; Accepted: 27 November 2018; Published: 5 December 2018
Abstract: Sphingosine 1-phosphate (S1P) signaling regulates numerous biological processes including neurogenesis, inflammation and neovascularization. However, little is known about the role of S1P signaling in the eye. In this study, we characterize two sphingosine kinases (SPHK1 and SPHK2), which phosphorylate sphingosine to S1P, and three S1P receptors (S1PR1, S1PR2 and S1PR3) in mouse and rat eyes. We evaluated sphingosine kinase and S1P receptor gene expression at the mRNA level in various rat tissues and rat retinas exposed to light-damage, whole mouse eyes, specific eye structures, and in developing retinas. Furthermore, we determined the localization of sphingosine kinases and S1P receptors in whole rat eyes by immunohistochemistry. Our results unveiled unique expression profiles for both sphingosine kinases and each receptor in ocular tissues. Furthermore, these kinases and S1P receptors are expressed in mammalian retinal cells and the expression of SPHK1, S1PR2 and S1PR3 increased immediately after light damage, which suggests a function in apoptosis and/or light stress responses in the eye. These findings have numerous implications for understanding the role of S1P signaling in the mechanisms of ocular diseases such as retinal inflammatory and degenerative diseases, neovascular eye diseases, glaucoma and corneal diseases.
Keywords: S1P; sphingosine 1-phosphate; S1PR1; sphingosine 1-phosphate receptor 1; S1PR2; S1PR3; sphingosine kinase 1; SPHK2; retina; eye
1. Introduction Sphingolipids play diverse roles in the biology of cells and tissues [1]. As structural lipids, they are essential to maintaining membrane fluidity and organizing lipid rafts [2,3]. However, their role is far from limited to membrane dynamics. The various species of sphingolipids are found throughout the cell including in the nucleus, cytoplasm, and in the extracellular space as signaling molecules [3–6]. Bioactive sphingolipids such as sphingosine 1-phosphate (S1P) and ceramide (Cer) are now recognized as important mediators of many basic cellular processes including cell migration, survival, contraction, proliferation, gene expression and various cell-cell interactions [1,5,7–15]. The scope of the sphingolipid system’s impact on mammalian biology has proven to be impressive due to the complexity of the “sphingolipid rheostat” and the many signaling pathways that sphingolipids are involved in. The sphingolipid rheostat is comprised of dozens of enzymes that constantly shift concentrations of sphingolipids such as sphingomyelin (SM), Cer and sphingosine (Sph) by metabolizing them into
Int. J. Mol. Sci. 2018, 19, 3885; doi:10.3390/ijms19123885 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 3885 2 of 18 other sphingolipid species. Ceramides and S1P are potent signaling molecules, which the sphingolipid rheostat can generate to respond to a stimulus or use as intermediates toward the degradation pathway. S1P is generated from sphingosine by sphingosine kinases 1 and 2 (SPHK1/SPHK2). Mice deficient in both SPHK1 and SPHK2 have many vascular and neuronal deficits during the development and are not viable past E13.5 [16]. SPHK1 and SPHK2 single knockout animals generally have no phenotype and are thought to compensate with the remaining kinase. However, knocking out SPHK2 is sufficient to reduce resilience to oxygen-induced retinopathy [17]. Meanwhile, the S1P produced by the two kinases has shown myriad roles in retinal development and function. S1P has shown to be essential for photoreceptor development [18] and Muller glia migration [19]. After development, modulating S1P has shown benefits in preventing immune cell migration and neovascularization after insult. The biological effects of S1P are largely attributed to its high-affinity association with receptors present on various cell types. S1P receptors are divided into five subgroups (S1PR1-5), which are G-protein-coupled receptors with unique expression patterns and roles in cell signaling [20–24]. S1PR1 is the most well studied and is present on endothelial cells, B-cells and T-cells. S1PR1 is also an essential receptor for influencing blood vessel development and maturation [21,25,26]. S1PR1 knockout animals are nonviable. S1PR2 and 3 are also ubiquitously expressed [20–24] and important for proper vascular development [27,28]. S1PR2 is important for cell migration [29] and S1PR2 mutations induce hearing loss by disrupting endocochlear potential gradients [30]. S1PR3 has demonstrated an important role in a bacterial infection response [31], nociception and itching sensation [32]. S1PR4 and S1PR5 expression is more restricted and they are mostly found on the cells of the immune and nervous systems, respectively [33,34]. S1PR4 is essential to differentiation of dendritic cells [35] and is important to immune cell chemoattraction [36]. S1PR5 is important in cell migration and, by selectively modulating its activity, can ameliorate demyelination [37] and alter the integrity of the blood brain barrier [38]. In summary, S1P receptors are essential for immune cell response modulation, cell migration, and vascular development and maintenance. These functions are critical to retinal development and survival, but their role in the eye is poorly understood. In the eye, S1P can protect neural cells from apoptosis by inhibiting synthesis of its precursor ceramide, which is a pro-death signaling molecule [5,39–43]. We have previously shown that targeting S1P receptors with FTY-720, which is marketed as a drug for multiple sclerosis, can delay retinal degeneration in light damage and genetic models [44,45]. Ocular tissues outside the retina such as the cornea and retinal pigment epithelium (RPE) have shown S1P receptor expression with functions important to controlling fibrotic responses [46]. Modulating the S1P receptor signaling with FTY-720 has also shown to be beneficial in ocular autoimmune conditions such as uveitis [47]. In the retina proper, S1P is critical to photoreceptor development [18], axonal guidance [48] and survival of retinal ganglion cells in glaucoma [49]. Given the numerous effects of S1P receptors showing translation into ocular development and pathology, we have identified a critical need to understand the distributions of both S1P receptors and sphingosine kinases in the eye. As S1P receptor modulation has shown benefits in counteracting retinal degeneration, we also studied how the expression of S1P-producing sphingosine kinases and S1P receptors changes over time in cases of retinal damage induced by intense light stress. Characterizing S1P signaling in ocular development and stress responses will deepen our understanding of eye health and diseases and potentially identify novel pathways for therapeutic intervention.
2. Results
2.1. Distribution and Expression of Sphingosine Kinase (Sphk) and Sphingosine 1-Phosphate Receptor (S1pr) mRNA in Rat Tissue To understand the roles of S1P signaling in the eye, we first explored the expression of relevant protein coding transcripts throughout the body. Using qRT-PCR, we assayed the expression and distribution of S1P-producing enzymes and the major S1P receptors in various tissues from adult SD rats. SPHK2 is corroborated as a major S1P-producing enzyme with high expression across all Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 18
Int.distribution J. Mol. Sci. 2018 of S1P-producing, 19, 3885 enzymes and the major S1P receptors in various tissues from adult3 of SD 18 rats. SPHK2 is corroborated as a major S1P-producing enzyme with high expression across all assayed tissues. At the same time, SPHK1 was expressed primarily in ovary and kidney tissue with assayedminimal tissues.expression At thein most same other time, tissues—n SPHK1 wasotably expressed the brain primarily and retina in ovary(Figure and 1A). kidney S1pr1 tissuewas highly with minimalexpressed expression in the hindbrain, in most othermoderately tissues—notably expressed thein the brain forebrain, and retina retina, (Figure heart,1A). lungsS1pr1 andwas spleen, highly expressedand minimally in the hindbrain,expressed moderatelyin the liver, expressed skin and in testes. the forebrain, S1pr3 was retina, moderately heart, lungs expressed and spleen, in andthe minimallyforebrain, retina, expressed liver, in heart, the liver, lungs, skin testes and testes.and kidneyS1pr3 wasand moderatelyin retinal pigment expressed epithelium-choroid in the forebrain, retina,(RPE-choroid) liver, heart, tissue. lungs, S1pr2 testes and and S1pr5 kidney showed and inminimal retinal pigmentexpression epithelium-choroid in the tissues analyzed (RPE-choroid) (Figure tissue.1B). S1pr2 and S1pr5 showed minimal expression in the tissues analyzed (Figure1B).
Figure 1. The expression and distribution of sphingosine kinases and S1P receptors in adult (2–7 Figure 1. The expression and distribution of sphingosine kinases and S1P receptors in adult (2–7 months old) SD rat tissues. For each feature, the mean of three independent qRT-PCR experiments months old) SD rat tissues. For each feature, the mean of three independent qRT-PCR experiments (± (±SE) is shown and normalized to the expression of the ribosomal protein Rpl19. (A) Sphingosine SE) is shown and normalized to the expression of the ribosomal protein Rpl19. (A) Sphingosine kinase kinase (Sphk) 1 and 2 mRNA expression in the tissue. (B) Sphingosine 1-phosphate receptor (S1pr) 1, 2, (Sphk) 1 and 2 mRNA expression in the tissue. (B) Sphingosine 1-phosphate receptor (S1pr) 1, 2, 3 and 3 and 5 mRNA expression in tissue. FB, forebrain. HB, hindbrain. R, retina. Li, liver. H, heart. Lu, lung. 5 mRNA expression in tissue. FB, forebrain. HB, hindbrain. R, retina. Li, liver. H, heart. Lu, lung. Sk, Sk, skin. T, testes. O, ovary. Sp, spleen. K, kidney. RPE-Ch, retinal pigment epithelium-choroids. skin. T, testes. O, ovary. Sp, spleen. K, kidney. RPE-Ch, retinal pigment epithelium-choroids. 2.2. Distribution and Expression of Sphingosine Kinase and Major S1P Receptor mRNA in Mouse Eye Tissues 2.2. Distribution and Expression of Sphingosine Kinase and Major S1P Receptor mRNA in Mouse Eye TissuesNext, we determined the expression and distribution of Sphk1 and Sphk2 as well as the major S1P receptors S1pr1, S1pr2 and S1pr3 in various mouse eye tissues. RNA was prepared from cleanlyNext, dissected we determined mouse cornea, the expression lens, iris-ciliary and distribution body, optic of nerve, Sphk1 eye and cups Sphk2 and as retina, well as describedthe major previouslyS1P receptors [50 S1pr1–52]., Using S1pr2 qRT-PCR,and S1pr3 wein various observed mouse high eye expression tissues. RNA of Sphk2 wasthroughout prepared from the cornea,cleanly iris/ciliarydissected mouse body, opticcornea, nerve, lens, eye iris-ciliary cup and retinabody, whileoptic Sphk1nerve,expression eye cups wasand minimalretina, as in described all ocular tissuespreviously and [50–52]. highest inUsing the retinaqRT-PCR, and opticwe observed nerve (Figure high 2expressionA), which reflectsof Sphk2 our throughout previous findingsthe cornea, in ratiris/ciliary tissue. Webody, observed optic nerve, high expression eye cup and of S1pr1retina mRNAwhile Sphk1 in mouse expression retinas, was which minimal is also in in all line ocular with ourtissues prior and findings, highest and in the noted retina high andS1pr1 opticexpression nerve (Fig inure the 2A), iris/ciliary which reflects body, opticour previous nerve and findings eye cups in (Figurerat tissue.2B). WeS1pr1 observedis highly high expressed expression in of vascular S1pr1 mRNA tissues in [21 mouse,53,54 ].retinas, Thus, which the expression is also in observedline with may,our prior to some findings, extent, and reflect noted the high vascular S1pr1 componentexpression in of the each iris/ciliary specific eyebody, tissue. optic Compared nerve and toeyeS1pr1 cups, S1pr2(Figureexpression 2B). S1pr1 was is highly very low expressed in the various in vascular eye tissues. tissues Yet,[21,53,54]. we did Thus, detect the some expression expression observed in the iris-ciliary body, optic nerve, eye cup and retina (Figure2B). S1pr3, on the other hand, was ubiquitously Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 18
Int.may, J. Mol. to some Sci. 2018 extent,, 19, 3885 reflect the vascular component of each specific eye tissue. Compared to S1pr14 of 18, S1pr2 expression was very low in the various eye tissues. Yet, we did detect some expression in the iris-ciliary body, optic nerve, eye cup and retina (Figure 2B). S1pr3, on the other hand, was expressedubiquitously in allexpressed of the eye in all tissues of the examined eye tissues and ex displayedamined and very displayed high expression very high in expression the optic nervein the (Figureoptic nerve2B). (Figure 2B). We furtherfurther determineddetermined thethe expressionexpression levelslevels ofof sphingosinesphingosine kinases and S1P receptors in whole eyes of mice during embryonic development (E15 and E18) and adulthood (at 2 and 7 months) by using qRT-PCR. As seenseen inin ourour previousprevious ratrat data,data, Sphk2Sphk2 waswas highlyhighly expressedexpressed throughout early development and adulthood while Sphk1 expression remained relatively low at all stages stages (Figure (Figure 22C).C). S1pr1 expressionexpression was was approximately approximately two-fold two-fold higher high iner eyes in isolatedeyes isolated from adult from mice adult when mice compared when tocompared embryonic to embryonic eyes (Figure eyes2D). (Figure In contrast, 2D). In the contrast, levels of theS1pr2 levelsmRNA of S1pr2 were mRNA lower were in the lower developed in the eyesdeveloped than the eyes embryonic than the eyes.embryonic In addition, eyes. InS1pr3 addition,mRNA S1pr3 were mRNA relatively were constant relatively during constant embryonic during developmentembryonic development and in adulthood and in (Figureadulthood2D). (FigureS1pr3 was2D). moreS1pr3 highlywas more expressed highly thanexpressedS1pr2 thanat all S1pr2 ages (Figureat all ages2D). (Figure 2D). In the developing mouse retina, both Sphk1 and Sphk2 expression appear to increase during early retinal development andand peakpeak atat adulthood levelslevels byby postnatal dayday 30 (P30) (Figure2 2E).E). RetinalRetinal S1pr1 receptor expression gradually increases from P1 to P15 (Figure2 2F),F), whichwhich couldcould bebe relatedrelated toto retinalretinal vascular development in this tissue. The retina is initially avascular when a mouse is born. However, However, starting at at P1, P1, the the vasculature vasculature grows grows radially radially from from the the center center of the of retina the retina towards towards the periphery the periphery until untilthe retina the retinareaches reaches its mature its mature size at around size at aroundP15 [55,56]. P15 S1pr1 [55,56 expression]. S1pr1 expression in the retina in themay, retina therefore, may, therefore,reflect the reflectgradual the increase gradual of increasethe vascular of the volume vascular in the volume retina. in S1pr2 the retina.had lowerS1pr2 levelshad of lower expression levels ofthrough expression all stages through of retinal all stages development of retinal while development S1pr3 expression while S1pr3 wasexpression relatively high was and relatively constant high at andall time constant points at (Figure all time 2F). points To monitor (Figure 2stagesF). To of monitor retinal stagesdevelopment, of retinal we development, evaluated the we expression evaluated of the expressionmarkers Elovl4 of the and markers RhodopsinElovl4 andand foundRhodopsin that theyand follow found the that expected they follow pattern theexpected (Supplementary pattern (SupplementaryFigure S1). Figure S1).
Figure 2. Cont. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 18 Int. J. Mol. Sci. Sci. 20182018,, 19,, x 3885 FOR PEER REVIEW 5 of 18
Figure 2. The expression and distribution of sphingosine kinase (Sphk) 1 and 2 and sphingosine 1- Figure 2. TheThe expression expression and and distribution distribution of of sphingosine sphingosine kinase kinase (Sphk (Sphk) 1) and 1 and 2 and 2 and sphingosine sphingosine 1- phosphate receptor (S1pr) 1, 2 and 3 mRNA in mouse eye tissues. Each bar shows the mean of three 1-phosphatephosphate receptor receptor (S1pr (S1pr) 1,) 2 1, and 2 and 3 mRNA 3 mRNA in mouse in mouse eye eyetissues tissues.. EachEach bar shows bar shows the mean the meanof three of independent qRT-PCR experiments (±SE) normalized with the expression of the ribosomal protein threeindependent independent qRT-PCR qRT-PCR experiments experiments (±SE) normalized (±SE) normalized with the with expression the expression of the ribosomal of the ribosomal protein Rpl19. (A) Sphk1 and Sphk2 expression profile in mouse eye tissues. (B) S1pr1-3 expression profile in proteinRpl19. (A Rpl19.) Sphk1 (A and) Sphk1 Sphk2and expressionSphk2 expression profile in profile mouse in eye mouse tissues. eye (B tissues.) S1pr1-3 (B expression) S1pr1-3 expression profile in mouse eye tissues. (C) Sphk1-2 expression profile in embryonic and adult whole mouse eyes. (D) profilemouse ineye mouse tissues. eye (C tissues.) Sphk1-2 (C expression) Sphk1-2 expression profile in profileembryoni inc embryonic and adult andwhole adult mouse whole eyes. mouse (D) S1pr1-3 expression profile in embryonic and adult whole mouse eyes. (E) Sphk1-2 expression profile eyes.S1pr1-3 (D expression) S1pr1-3 expression profile in profile embryoni in embryonicc and adult and whole adult mouse whole eyes. mouse (E) eyes. Sphk1-2 (E) Sphk1-2expressionexpression profile in developing mouse retinas from postnatal day 1 (P1) to P30. (F) S1pr1-3 expression profile in profilein developing in developing mouse mouse retinas retinas from frompostnatal postnatal day day1 (P1) 1 (P1) to toP30. P30. (F ()F S1pr1-3) S1pr1-3 expressionexpression profile profile in developing mouse retinas from P1 to P30. Cor, cornea. I-CB, iris/ciliary body. ON, optic nerve. EC, developing mousemouse retinasretinas from from P1 P1 to to P30. P30. Cor, Cor, cornea. cornea. I-CB, I-CB, iris/ciliary iris/ciliary body. body. ON, ON, optic optic nerve. nerve. EC, EC, eye eye cup. Ret, retina. E15, 15 day embryo. E18, 18 day embryo. 2M, 2 months aged. 7M, 7 months aged. cup.eye cup. Ret, Ret, retina. retina. E15, E15, 15 15 day da embryo.y embryo. E18, E18, 18 18 day day embryo. embryo. 2M, 2M, 2 2 months months aged.aged. 7M, 7 months aged. aged. P1, P7, P15 and P30 are postnatal day 1, 7, 15 and 30, respectively. P1, P7, P15 and P30 are postnatal day 1, 7, 15 and 30, respectively. 2.3. Localization of Major S1P Receptors and SphingosineSphingosine Kinases in Rat Eye Tissues andand RetinaRetina 2.3. Localization of Major S1P Receptors and Sphingosine Kinases in Rat Eye Tissues and Retina 2.3.1. Localization of SPHK1 and SPHK2 2.3.1. Localization of SPHK1 and SPHK2 To observeobserve thethe localizationlocalization of SPHK1SPHK1 and SPHK2SPHK2 in thethe retinaretina andand cornea,cornea, we performedperformed To observe the localization of SPHK1 and SPHK2 in the retina and cornea, we performed immunohistochemistry (IHC) (IHC) on on sections sections of of rat rat eyes eyes taken taken through through the vertical the vertical meridian meridian and the and optic the immunohistochemistry (IHC) on sections of rat eyes taken through the vertical meridian and the optic opticnerve nervehead headusing using anti-SPHK1 anti-SPHK1 and and anti-SPHK2 anti-SPHK2 antibodies. antibodies. IHC IHC localization localization of of SPHK1 SPHK1 showed nerve head using anti-SPHK1 and anti-SPHK2 antibodies. IHC localization of SPHK1 showed expression throughout the retina with strong stainingstaining in the ganglion cell bodies, large puncta in the expression throughout the retina with strong staining in the ganglion cell bodies, large puncta in the IPL and extranuclearextranuclear stainingstaining inin photoreceptorphotoreceptor cellcell bodiesbodies (Figure(Figure3 B—arrowheads).3B—arrowheads). SPHK1SPHK1 alsoalso IPL and extranuclear staining in photoreceptor cell bodies (Figure 3B—arrowheads). SPHK1 also showed expression in thethe cornealcorneal epitheliumepithelium (Figure(Figure3 C—arrowheads).3C—arrowheads). No-primaryNo-primary controlscontrols forfor showed expression in the corneal epithelium (Figure 3C—arrowheads). No-primary controls for corneal staining is shown in supplemental figuresfigures (Figure S2A). SPHK2 expression was observedobserved corneal staining is shown in supplemental figures (Figure S2A). SPHK2 expression was observed throughout thethe retina retina notably notably in in the the RPE, RPE, ONL, ONL, INL INL and and GCL GCL (Figure (Figure3E—arrowheads). 3E—arrowheads). We also We noted also throughout the retina notably in the RPE, ONL, INL and GCL (Figure 3E—arrowheads). We also somenoted SPHK2some SPHK2 expression expression in the cornealin the corn epitheliumeal epithelium (Figure (Figure3F—arrowheads). 3F—arrowheads). noted some SPHK2 expression in the corneal epithelium (Figure 3F—arrowheads).
Figure 3. Cont. Int. J. Mol. Sci. 2018, 19, 3885 6 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 18
FigureFigure 3. Localization 3. Localization of ofSPHK1 SPHK1 and and SPHK2 SPHK2 inin adultadult (2–7 months months old) old) rat rat ocular ocular tissues. tissues. SPHK1 SPHK1 and and SPHK2SPHK2 localization localization was was detected detected by by anti-SPHK1 anti-SPHK1 (green) and and anti-SPHK2 anti-SPHK2anti-SPHK2 (red) (red) antibodies. antibodies.antibodies. (A) ( RatA) Rat retinalretinal sectionsection section inin which inwhich which the the anti-SPHK1the anti-SPHK1 anti-SPHK1 antibodies antibodiesantibodies were werewere replaced replacedreplaced with with with normal normal normal rabbit rabbit rabbit serum. serum. serum. The The layers The werelayerslayers marked were were marked and marked served and and asserved aserved negative as as a anegativecontrol. negative( Bcontrol.control.) Retinal ((BB section)) RetinalRetinal of section SDsection rat of eye ofSD andSD rat rat SPHK1eye eye and and localizationSPHK1 SPHK1 localizationin differentlocalization layersin different in different is shown layers layers by is arrowheads. isshown shown by by arrowheads. arrowheads. (C) Corneal (( sectionCC)) Corneal Corneal of SD section section rat eye of ofSD and SD rat SPHK1rat eye eye and and localization SPHK1 SPHK1 localizationin differentlocalization layersin differentin isdifferent shown layers layers by arrowheads. is is shown shown byby (D arrowheads.arrowheads.) Rat retinal ( section(DD) )Rat Rat inretinal retinal which section section the anti-SPHK2 in inwhich which the antibodies theanti- anti- SPHK2 antibodies were replaced with normal rabbit serum. The layers were marked and served as a wereSPHK2 replaced antibodies with were normal replaced rabbit wi serum.th normal The rabbit layers serum. were marked The layers and were served marked as a negative and served control. as a negative control. (E) Retinal section of SD rat eye and SPHK2 localization in different layers is shown (negativeE) Retinal control. section (E of) Retinal SD rat eyesection and of SPHK2 SD rat localizationeye and SPHK2 in different localization layers in isdifferent shown bylayers arrowheads. is shown by arrowheads. (F) Corneal section of SD rat eye and SPHK2 localization in different layers is shown (byF) arrowheads. Corneal section (F) ofCorneal SD rat section eye and of SPHK2 SD rat eye localization and SPHK2 in different localization layers in isdifferent shown layers by arrowheads. is shown by arrowheads. CB, ciliary body. RPE, retinal pigment epithelium. PR, photoreceptor. ONL, outer CB, ciliary body. RPE, retinal pigment epithelium. PR, photoreceptor. ONL, outer nuclear layer. OPL, by arrowheads.nuclear layer. CB, OPL, ciliary outer plexiformbody. RPE, layer. retinal INL, pigmentinner nuclear epithelium. layer. IPL, PR, inner photoreceptor. plexiform layer. ONL, GCL, outer outer plexiform layer. INL, inner nuclear layer. IPL, inner plexiform layer. GCL, ganglion cell layer. nuclearganglion layer. cell OPL, layer. outer Endo, plexiform endothelium. layer. Epi, INL, epithelium. inner nuclear Scale =layer. 50 µm. IPL, inner plexiform layer. GCL, ganglionEndo, endothelium. cell layer. Endo, Epi, epithelium. endothelium. Scale Epi, = epithelium. 50 µm. Scale = 50 µm. 2.3.2.2.3.2. Localization Localization of of S1PR1 S1PR1 2.3.2. Localization of S1PR1 LittleLittle is knownis known about about thethe localization of of S1PR1 S1PR1 in the in theneural neural retina retina and other and eye other tissues. eye We tissues. detected prominent labeling on retinal pigment epithelial (RPE) cells and photoreceptor cells with an We detectedLittle is prominentknown about labeling the localization on retinal pigment of S1PR1 epithelial in the neural (RPE) cellsretina and and photoreceptor other eye tissues. cells with We anti-S1PR1 receptor antibody but not an IgG control (Figure 4A and Figure 4B, arrowheads). However, detectedan anti-S1PR1 prominent receptor labeling antibody on re buttinal not pigment an IgG epithelial control (Figure (RPE)4 A,B,cells arrowheads).and photoreceptor However, cells with we did an we did not detect expression of S1PR1 in other parts of the eye such as the iris, lens (data not shown), anti-S1PR1not detect expression receptor antibody of S1PR1 but in othernot an parts IgG contro of the eyel (Figure such 4A as theand iris, Figure lens 4B, (data arrowheads). not shown), However, or ciliary we didor ciliary not detect body (Figureexpression 4C and of FigureS1PR1 4D). in other In genera partsl, no of localization the eye such of S1PR1as the was iris, noticed lens (data in the not cornea. shown), bodyCorneal (Figure stromal4C,D). labeling In general, could no be localization non-specific ofbindin S1PR1g of wasthe fluorescent noticed in antibody the cornea. to the Corneal collagenous stromal or ciliary body (Figure 4C and Figure 4D). In general, no localization of S1PR1 was noticed in the cornea. labelingstroma could (Figure be non-specific 4C, arrows), bindingas we did of not the detect fluorescent this labeling antibody in no to-primary the collagenous controls (Supplementary stroma (Figure 4C, Corneal stromal labeling could be non-specific binding of the fluorescent antibody to the collagenous arrows),Figure as S2B). we did not detect this labeling in no-primary controls (Supplementary Figure S2B). stroma (Figure 4C, arrows), as we did not detect this labeling in no-primary controls (Supplementary Figure S2B).
FigureFigure 4. Localization 4. Localization of of S1PR1 S1PR1 in in adult adult(2–7 (2–7 months old) old) rat rat ocular ocular tissues. tissues. S1PR1 S1PR1 localization localization (green) (green) waswas detected detected with with anti-S1PR1 anti-S1PR1 antibodies. antibodies. ((AA)) RatRat retinal section section in in which which the the anti-S1PR1 anti-S1PR1 antibodies antibodies
werewere replaced replaced with with normal normal rabbit rabbit serum. serum. TheThe layers were were marked marked and and served served as asa negative a negative control. control. (FigureB) Retinal(B) 4.Retinal Localization section section of ofSDof SD S1PR1 rat rat eye eye in and adultand S1PR1 S1PR1 (2–7 months localizationlocalization old) in inrat differentdifferent ocular tissues.layers layers is S1PR1 shown is shown localization by arrowheads. by arrowheads. (green) (C) Corneal section of SD rat eye with S1PR1 labeling. (D) Partial anterior segment of eye containing (wasC) Corneal detected section with anti-S1PR1 of SD rat eye antibodies. with S1PR1 (A) labeling. Rat retinal (D )section Partial in anterior which segmentthe anti-S1PR1 of eye containingantibodies ciliary epithelial cells, trabecular meshwork and Schlemm’s canal. S1PR1 localization is shown by wereciliary replaced epithelial with cells, normal trabecular rabbit meshworkserum. The and layers Schlemm’s were marked canal. and S1PR1 served localization as a negative is shown control. by arrowheads. RPE, retinal pigment epithelium. PR, photoreceptor. ONL, outer nuclear layer. OPL, (arrowheads.B) Retinal section RPE, retinal of SD pigmentrat eye and epithelium. S1PR1 localization PR, photoreceptor. in different ONL, layers outer is nuclear shown layer. by arrowheads. OPL, outer plexiform(C) Corneal layer. section INL, of innerSD rat nuclear eye with layer. S1PR1 IPL, labeling. inner plexiform (D) Partial layer. anterior GCL, segme ganglionnt of cell eye layer. containing Endo, ciliaryendothelium. epithelial Epi, cells, epithelium. trabecular CB, meshwork ciliary body. and Scale Schlemm’s = 25 µm. canal. S1PR1 localization is shown by arrowheads. RPE, retinal pigment epithelium. PR, photoreceptor. ONL, outer nuclear layer. OPL, Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 18
Int. J. Mol.outer Sci. plexiform2018, 19, 3885layer. INL, inner nuclear layer. IPL, inner plexiform layer. GCL, ganglion cell layer.7 of 18 Endo, endothelium. Epi, epithelium. CB, ciliary body. Scale = 25 µm.
2.3.3. Localization of S1PR2 Using IHC, we found that an anti-S1PR2 antibody instead of IgG is labeled the very top layer of cells of the inner nuclear layer (INL) of rat retinal sectionssections (Figure5 5A,B,D,E).A,B,D,E). SinceSince thethe INLINL consistsconsists primarily of bipolar cells, we sought to determine if the S1PR2 receptor colocalizes with PKCα, which is a markermarker of bipolarbipolar cellcell dendrites.dendrites. We We found thatthat S1PR2 was present on the cell body of bipolar cells but did not overlap with the dendrites (Figure5 5D,D, arrowheads).arrowheads). WeWe alsoalso foundfound thatthat anti-S1PR2anti-S1PR2 antibodies labeled the corneal epithelium but not the corneal endothelium or or stroma stroma (Figure 55C).C). Lastly, punctatepunctate labelinglabeling of S1PR2 was prominent around the large vacuoles and Schlemm’s canal in the outflowoutflow pathway (Figure(Figure5 5F,F, arrowheads). arrowheads).
Figure 5. Localization of S1PR2 in adult (2–7 months old) rat ocular tissues. S1PR2 localization (red) was detecteddetected byby anti-S1PR2 anti-S1PR2 antibodies. antibodies. (A ()A Rat) Rat retinal retinal section section in which in which the anti-S1PR2the anti-S1PR2 antibodies antibodies were replacedwere replaced with normalwith normal goat serum. goat se Therum. layers The layers were marked were marked and served and served as a negative as a negative control. control. (B) Retinal (B) sectionRetinal ofsection SD rat of eye SD andrat eye S1PR2 and localization S1PR2 localization in different in different layers is layers shown is by shown arrowheads. by arrowheads. (C) Corneal (C) sectionCorneal of section SD rat of eye SD and rat S1PR2eye and localization S1PR2 localization in different in layersdifferent is shown layers by is arrowheads.shown by arrowheads. (D) Magnified (D) viewMagnified of SD view rat retinal of SD sectionrat retinal and section S1PR2 and localization S1PR2 localization with respect with to PKCrespectα in to the PKC INLα andin the OPL INL layer, and whichOPL layer, is shown which by is arrowheads.shown by arrowheads. (E) Magnified (E) Magnified view of SD view rat retinalof SD rat section retinal with section emphasis with emphasis on inner nuclearon inner layer nuclear (INL) layer localization (INL) localization of S1PR2 of shown S1PR2 by shown arrowheads. by arrowheads. (F) Partial (F anterior) Partial segment anterior ofsegment SD rat eyeof SD containing rat eye containing ciliary epithelial ciliary cells, epithelial Trabecular cells,meshwork Trabecular and meshwork Schlemm’s and canal. Schlemm’s S1PR2 localizationcanal. S1PR2 is shownlocalization by arrowheads. is shown by CB, arrowheads. ciliary body. RPE,CB, ciliary retinal body. pigment RPE, epithelium. retinal pigment PR, photoreceptor. epithelium. ONL, PR, outerphotoreceptor. nuclear layer. ONL, OPL, outer outer nuclear plexiform layer. OPL, layer. outer INL, innerplexiform nuclear layer. layer. INL, IPL, inner inner nuclear plexiform layer. layer. IPL, GCL,inner ganglionplexiform cell layer. layer. GCL, Endo, ganglion endothelium. cell layer. Epi, Endo, epithelium. endothelium. Scale =Epi, 25 µepithelium.m. Scale = 25 µm.
2.3.4. Localization of S1PR3 Int. J. Mol. Sci. 2018, 19, 3885 8 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 18 2.3.4. Localization of S1PR3 Using IHC on SD rat retinal sections, we detected expression of S1PR3 in the ganglion cells and photoreceptorUsing IHC cells on SDalong rat retinalwith some sections, expression we detected in the expression photoreceptor of S1PR3 inner in segment the ganglion region cells (inner and photoreceptornuclear layer) (Figure cells along 6A withand Figure some expression6B, arrowheads in the). photoreceptorIn the other parts inner ofsegment the eye, regionwe observed (inner nuclearstrong anti-S1PR3 layer) (Figure receptor6A,B, arrowheads). staining of Inthe the corn othereal partsepithelium of the eye,and we some observed staining strong of the anti-S1PR3 corneal receptorendothelium staining (Figure of the 6C, corneal arrowheads). epithelium Although and some the S1PR3 staining receptor of the corneal was not endothelium detected in the (Figure corneal6C, arrowheads).stroma (FigureAlthough 6C), it was the observed S1PR3 receptor in the pigmented was not detected epithelial in cells the cornealof the ciliary stroma body (Figure and6 C),iris it(Figure was observed 6D, arrowheads). in the pigmented Focal clustered epithelial labeling cells of was the observed ciliary body around and iristhe (FigureSchlemm’s6D, arrowheads).canal (Figure Focal6D, arrowheads). clustered labeling was observed around the Schlemm’s canal (Figure6D, arrowheads).
Figure 6. LocalizationLocalization ofof S1PR3 S1PR3 in adult in adult (2–7 (2–7 months months old) old)rat ocular rat ocular tissues. tissues. S1PR3 localization S1PR3 localization (green) (green)was detected was detected by anti-S1PR3 by anti-S1PR3 antibodies. antibodies. (A) The rat (A retinal) The rat section retinal in sectionwhich the in whichanti-S1PR3 the anti-S1PR3 antibodies antibodieswere replaced were with replaced normal with rabbit normal serum. rabbit The serum. layers The were layers marked were and marked served and as served a negative as a negative control. control.(B) The retinal (B) The section retinal of sectionSD rat eye of SD and rat S1PR3 eye andlocalization S1PR3 localization in different layers in different is shown layers by arrowheads. is shown by C arrowheads.(C) The corneal ( ) Thesection corneal of sectionSD rat ofeye SD and rat eyeS1PR3 and S1PR3localization localization in different in different layers layers is shown is shown by by arrowheads. (D) Partial anterior segment of SD rat eye containing ciliary epithelial cells, trabecular arrowheads. (D) Partial anterior segment of SD rat eye containing ciliary epithelial cells, trabecular meshwork and Schlemm’s canal. The S1PR3 localization is shown by arrowheads. CB, ciliary body. meshwork and Schlemm’s canal. The S1PR3 localization is shown by arrowheads. CB, ciliary body. RPE, retinal pigment epithelium. PR, photoreceptor. ONL, outer nuclear layer. OPL, outer plexiform RPE, retinal pigment epithelium. PR, photoreceptor. ONL, outer nuclear layer. OPL, outer plexiform layer. INL, inner nuclear layer. IPL, inner plexiform layer. GCL, ganglion cell layer. Endo, endothelium. layer. INL, inner nuclear layer. IPL, inner plexiform layer. GCL, ganglion cell layer. Endo, Epi, epithelium. Scale = 25 µm. endothelium. Epi, epithelium. Scale = 25 µm.
2.4. Expression and Distribution of Sphingosine Kinases and S1P Receptors in Light Stressed Retinas Int. J. Mol. Sci. 2018, 19, 3885 9 of 18
2.4.Int. ExpressionJ. Mol. Sci. 2018 and, 19 Distribution, x FOR PEER ofREVIEW Sphingosine Kinases and S1P Receptors in Light Stressed Retinas 9 of 18
InIn order order to to examine examine the the expression expression of Sphk1-2 of Sphk1-2and theand three the three major major S1P receptors S1P receptors under lightunder stress, light westress, used ourwe light-damagedused our light-damaged rat retina model rat retina in which model photoreceptor in which cellphotoreceptor death occurs cell by apoptosis death occurs mainly by dueapoptosis to oxidative mainly stress due inducedto oxidative by intense stress induced light exposure. by intense Apoptosis light exposure. starts after Apoptosis 8–12 h andstarts by after 24 h8– almost12 hours all ofand the by photoreceptor 24 hours almost cells all in of the the central photorecep retinator enter cells into in the apoptosis central [retina57–60 ].enterUsing into qRT-PCR, apoptosis we[57–60]. observed Using a significant qRT-PCR, and we observed sustained a increase significant in Sphk1 and sustainedmRNA expression increase in following Sphk1 mRNA light expression damage. Notably,followingSphk1 lightmRNA damage. was Notably, elevated Sphk1 by four-fold, mRNAseven-fold was elevated and by six-fold four-fold, at LD seven-fold + 0, LD + 3and and six-fold LD + 6, at respectively,LD + 0, LD and+ 3 and began LD trending + 6, respectively, back toward and baseline began trending at 12 h (p back< 0.01, toward Figure baseline7A). However, at 12 hours only ( ap < slight,0.01, non-significantFigure 7A). However, elevation only in Sphk2a slight,expression non-significant was recorded elevation following in Sphk2 light expression damage was (Figure recorded7A). S1pr2followingand S1pr3 lightmRNA damage expression (Figure increased7A). S1pr2 significantly and S1pr3 immediatelymRNA expression after light increased damage significantly (LD+0) by five-foldimmediately (p < 0.05) after andlight twenty-fold damage (LD+0) (p < 0.01),by five-fold respectively (p < 0.05) (Figure and 7twenty-foldB). S1pr2 and (p Figure 7. Light stress modulates sphingosine kinase (Sphk) 1 and 2 and sphingosine 1-phosphate receptorFigure (7.S1pr Light) 1, 2stress and 3modulates mRNA expression sphingosine in adult kinase (2–4 (Sphk months) 1 and old) rat2 and retinas. sphingosine Adult SD 1-phosphate rats were exposedreceptor to (S1pr damaging) 1, 2 and light 3 mRNA at an intensity expression of 2700 in ad luxult for (2–4 6 hmonths and their old) retinas rat retinas. were Adult then harvested SD rats were at differentexposed time to damaging points after light light at damage:an intensity 0 h of (LD 2700 + 0), lu 3x hfor (LD 6 hours + 3), 6and h (LD their + retinas 6), 12 h were (LD +then 12) andharvested 24 h (LDat different + 24). Harvested time points samples after werelight prepareddamage: 0 for hour RNA (LD extraction + 0), 3 hours and (LD subsequent + 3), 6 hours qRT-PCR. (LD + ( A6),) mRNA12 hours expression(LD + 12) of andSphk1 24 andhoursSphk2 (LDwere + 24). presented Harvested as foldsamp overles control.were prepared (B) mRNA for expressionRNA extraction of S1pr1 and, S1pr2subsequentand S1pr3 qRT-PCR.presented (A) as mRNA fold over expression control. of The Sphk1 expression and Sphk2 data were were presented calculated as byfold dCt over methods control. and(B) compared mRNA expression to the expression of S1pr1, of S1pr2 control and housekeeping S1pr3 presented genes. as fold The over data control. presented The are expression the mean data of threewere independent calculated by experiments dCt methods (± SE).and compared to the expression of control housekeeping genes. The data presented are the mean of three independent experiments (± SE). We further examined protein localization in the retina immediately after light damage (LD + 0). No labelingWe further of S1PR2 examined was detected protein in localization the photoreceptor in the re nucleitina immediately (ONL) in the after no-light-damaged light damage (LD retina + 0). No labeling of S1PR2 was detected in the photoreceptor nuclei (ONL) in the no-light-damaged retina (NLD, Figure 8A). However, at LD + 0, we detected abundant expression of S1PR2 in the photoreceptor cells, which may be undergoing apoptosis (Figure 8B). S1PR2 labeling increased in the ganglion cells after light damage as well (Figure 8B, arrowhead). S1PR3 labeling also increased and Int. J. Mol. Sci. 2018, 19, 3885 10 of 18 (NLD,Int. J. Mol. Figure Sci. 20188A)., 19 However,, x FOR PEER at REVIEW LD + 0, we detected abundant expression of S1PR2 in the photoreceptor 10 of 18 cells, which may be undergoing apoptosis (Figure8B). S1PR2 labeling increased in the ganglion intenselycells after co-localized light damage with as well S1PR2 (Figure in photoreceptor8B, arrowhead). cells S1PR3 after labelinglight damage also increased (Figure 8D and and intensely Figure 8E).co-localized with S1PR2 in photoreceptor cells after light damage (Figure8D,E). Figure 8. Localization of S1PR2, S1PR3 and PKC α in light damaged (LD) andand non-lightnon-light damageddamaged (NLD) adult (2–4 months old) rat retinal tissues.tissues. ( A) Retinal section of NLD SD rat eye with S1PR2 localization in different layers shown byby arrowheads. (B) Retinal section of LD SD rat eye with S1PR2 localization in different layers shown by arrowheads.arrowheads. ( (CC)) Retinal Retinal section section of of NLD SD rat eye with S1PR3 localization in different layers shown by arrowheads. (D) Retinal section of LD SD rat eye with S1PR3 localization in different layers shown by arrowheads. ( (EE)) Magnified Magnified view view of of LD SD rat eye showing localization of S1PR2 and S1PR3 in the ON ONLL and INL with arrowheads. RPE, retinal pigment epithelium. PR, PR, photoreceptor. photoreceptor. ONL, ONL, outer outer nuclear nuclear layer. OPL, OPL, outer plexiform layer. INL, inner µ nuclear layer. IPL, inner plexiform layer. GCL, ganglionganglion cellcell layer.layer. ScaleScale == 25 µm.m. 3. Discussion 3. Discussion G-protein-mediated signaling via S1P receptors has recently been shown to be involved in various G-protein-mediated signaling via S1P receptors has recently been shown to be involved in cellular and biochemical processes including cell migration, inflammation, protection from apoptosis, various cellular and biochemical processes including cell migration, inflammation, protection from etc. [5,11,13,21,43,61]. S1P receptors have been shown to function in neurogenesis, inflammation, apoptosis, etc. [5,11,13,21,43,61]. S1P receptors have been shown to function in neurogenesis, neovascularization and tissue fibrosis, which are all associated with many blinding eye diseases. It is, inflammation, neovascularization and tissue fibrosis, which are all associated with many blinding therefore, of great interest and importance to understand the function of sphingolipid metabolism and eye diseases. It is, therefore, of great interest and importance to understand the function of signaling in the eye. However, very limited information is available on the expression and function of sphingolipid metabolism and signaling in the eye. However, very limited information is available on sphingosine kinases and S1P receptors in the mammalian retina and other ocular tissues. To fill this the expression and function of sphingosine kinases and S1P receptors in the mammalian retina and gap in knowledge, we determined the retinal expression levels of the sphingosine kinases (SPHK1 other ocular tissues. To fill this gap in knowledge, we determined the retinal expression levels of the and SPHK2) and major S1P receptors (S1PR1, S1PR2, S1PR3 and S1PR5) relative to other rat tissues. sphingosine kinases (SPHK1 and SPHK2) and major S1P receptors (S1PR1, S1PR2, S1PR3 and S1PR5) We then evaluated the expression and localization of these kinases and the three major S1P receptors relative to other rat tissues. We then evaluated the expression and localization of these kinases and the three major S1P receptors in specific ocular tissues from rats and mice and measured changes in their expression and localization following light-induced eye damage (LD). S1P receptor expression in mammalian eye tissues has been reported previously and mouse S1pr2 expression in the retina has also been reported in the context of neovascularization. S1PR1-3, 5 Int. J. Mol. Sci. 2018, 19, 3885 11 of 18 in specific ocular tissues from rats and mice and measured changes in their expression and localization following light-induced eye damage (LD). S1P receptor expression in mammalian eye tissues has been reported previously and mouse S1pr2 expression in the retina has also been reported in the context of neovascularization. S1PR1-3, 5 also had expression demonstrated in human RPE, conjunctival fibroblasts and corneal fibroblasts [46,54,62]. The trend of S1P receptor expression in eye tissue follows the general pattern of other tissues [63,64] such as S1pr1 > S1pr3 > S1pr2. The optic nerve has very high expression of S1pr3 and also the highest relative Sphk1 expression levels (Figure2A,B), which could be of importance in terms of understanding the roles of S1P signaling in many forms of human optic nerve degeneration diseases and glaucoma. The S1P receptor expression has been studied in detail across different cardiac tissues and reveals similar patterns for many we observed. In both cardiac myocytes and vascular endothelial cells, S1pr1 has the highest expression, followed by S1pr3, and then followed by S1pr2. In cardiac fibroblasts, S1pr3 has higher expression than S1pr1 and, in aortic muscle smooth cells, S1pr2 dominates S1pr1 and S1pr3 [63]. It should be noted that the expression pattern of S1P receptors changes during development and differentiation, which is shown by Figure2D,F and leads to different combinations on cells and tissues [33,53,65,66]. This diversity in receptor expression and activation of divergent signaling pathways may explain how S1P signals have such pleiotropic responses. We focused here on sphingosine kinase and S1P receptor expression in eye tissues to help elucidate their roles in development, differentiation of ocular tissues, and pathogenesis of various eye diseases. We observed high expression of S1pr1 mRNA in mouse and rat retinas (Figures1 and2). As mentioned earlier, S1pr1 is highly expressed in the vasculature [21,53,54]. Therefore, S1pr1 mRNA expression might reflect the amount of vasculature present in that tissue. qRT-PCR revealed increasing Sphk1, Sphk2 and S1pr1 expression with the age in mouse retinas (Figure2E,F), which could be due to the development of vasculature and/or the development of the outer segments. Using IHC, we found that the neural cells of the mouse eye express negligible levels of S1PR1. However, prominent expression was detected in the RPE-choroid interface and the photoreceptor outer segment (Figure4). Future studies using mouse models that do not develop outer segments could prove useful in determining the role that S1PR1 plays in the outer segments. S1PR2, on the other hand, has expression in some inner retinal neurons, which could be rod bipolar cells since those cells can also be labeled with PKCα. This specifically labels the dendrites of rod bipolar cells. However, the PKCα and S1PR2 labels do not overlap, which indicates the presence of S1PR2 on the cell membrane of the bipolar cells but not on the dendrites (Figure5D). This bipolar localization of S1PR2 suggests that this receptor may participate in some form of neurotransmission of bipolar cells through Gi, Gq, or G12/13 pathways [16,20,67]. S1PR2 was also found to localize to the photoreceptor inner segments, which is specifically very close to the outer nuclear layer (Figure5E). Photoreceptors are primary cilia and the other related primary ciliary structure are the hair cells of the mammalian cochlea. Knocking out S1PR2 causes hair cell degeneration and deafness in mice [67,68]. It will be very interesting to study photoreceptor development and functioning in the S1pr2 knock-out mice to help understand the role of S1PR2 photoreceptor development and function. Exogenous S1P has been shown to decrease the outflow facility in the entire eyes of porcine, human and mouse models [69–71]. Using several S1P receptor specific agonists and antagonists, S1PR2 activation by exogenous S1P was found to cause an increase in outflow resistance and higher intraocular pressure [69–71]. This could be a potential target for developing therapeutics for regulating intraocular pressure. For the first time, we detected labeling of S1PR2 in the trabecular meshwork cells and higher on the endothelial cells close to the Schlemm’s canal (Figure5F). We also detected intense, focal localization of S1PR3 in some of the proximal endothelial cells to the Schlemm’s canal (Figure6D). S1P signaling is gaining importance with regards to their role in the human aqueous humor outflow pathway and glaucoma [69–71]. Determination of the presence of these receptors in the outflow pathway may bear significant potential to discover novel pathways and mechanisms for glaucoma development. Int. J. Mol. Sci. 2018, 19, 3885 12 of 18 We noticed S1PR3 expression also localized to other retinal cells besides the photoreceptor cells (Figure6B). In the photoreceptor cells, S1PR3 localizes to the cell body but not to the retinal outer segment (Figure6D,G). S1pr3 has higher expression in the retina and it increases significantly in retinal light-stress (Figures1,2 and7). However, nothing is known on its role in retinal physiology and diseases. There is a potential for discovering novel pathways and mechanisms related to retinal development and function by studying S1PR3 in the retina. In the light-stressed retina, we observed Sphk1, S1pr2 and S1pr3 expression increases significantly [39] (Figure7). Light stress especially affects the photoreceptor cells [ 57,59,72–76]. An increase in the expression of a gene in this condition suggests its association with photoreceptor cell physiology. We found, in the light-stressed retina, that S1P receptors specifically and intensely localized to the pycnotic photoreceptor nuclei, which are entering into apoptosis (Figure8E). This could be a cellular mechanism to up-regulate cytoprotective S1P signaling to counter the apoptosis, which might even be a feedback regulation of ceramide induced apoptosis of photoreceptor cells. S1P has previously been proposed as a key regulator in photoreceptor development as a stimulator of proliferation. Inhibiting S1P synthesis was shown to block mitogenic effects of glial-derived neurotrophic factor (GDNF) [77]. We have shown intense light-induced ceramide generation can induce apoptosis in photoreceptor cells [39]. We reported the S1P level also increases with light stress, which intensifies the expression of the photoreceptor specific S1P receptors and suggests a definitive signaling in the stressed photoreceptor cells. This could also be related to the G-protein coupled action of S1P receptors to affect channel functioning in the stressed photoreceptor cells. Our light damage model showing upregulation of SPHK2 and S1P receptors may implicate a signal for the immune response [78]. In conclusion, our results unveiled novel patterns of sphingosine kinase and S1P receptor expression and localization in the mammalian eye and retina. In line with previous literature and as discussed above, our results suggest that S1P receptor signaling is involved in numerous key processes in the eye ranging from light-stress and apoptotic responses to retinal and vascular development. The combination of the background high expression of S1pr1, high S1pr1 and S1pr3 expression in the developing retina and increased expression of S1pr2 and S1pr3 in the retina immediately after light-induced stress suggests that sphingolipid signaling plays a role in both vision and apoptosis. S1PR20s presence in the outflow pathway follows logically from past studies on S1P receptor antagonist drugs and S1PR2 specific agonists and has great implications for understanding and treating conditions that include elevated IOP in glaucoma. S1PR2 is also likely involved in neurotransmission in the eye based on its localization to cell membranes in bipolar cells. S1pr30s presence in the retina and upregulation during light damage pose interesting questions for the receptor’s poorly understood functions in the eye. These data allow future research to elaborate on not only mechanisms behind the receptors’ function in the respective tissues but also identifies potential markers and therapeutic targets for numerous ocular diseases. 4. Materials and Methods 4.1. Animal Care and Tissues All procedures were performed, according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the University of Oklahoma Health Sciences Center (OUHSC) Guidelines for Animals in Research. All protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the OUHSC and the Dean A. McGee Eye Institute (DMEI) (IACUC approval #: 15-015-B, approval date: 24 February 2015; IACUC approval # 13-060-T, approval date 16 June 2015). All the tissues used in this study were from albino Sprague Dawley (SD) rats, C57Bl6J mice and bovine retina. Mouse and rat retinal tissues used in this study for gene expression were harvested after overnight dark adaptation. To study tissue distribution of S1P receptor expression, we prepared RNA from adult (2–7 months old) rat tissue. For every tissue harvest, the rats were first euthanized by carbon dioxide asphyxiation. Int. J. Mol. Sci. 2018, 19, 3885 13 of 18 The harvested forebrain, hindbrain, liver, heart, lungs, skin, testes, ovary, spleen and kidney was then snap frozen in liquid N2. Light-adapted and dark-adapted rat retinas were harvested under room-light and red-light, respectively, snap-frozen, and used for preparing rod outer segments (ROS). The whole eyes were harvested and fixed in Prefer fixative (Anatech Ltd., Battle Creek, MI, USA) for 20 to 40 min and embedded in paraffin. Additionally, 5 µm-thick sections were prepared from the fixed rat eyes and used for immunohistochemistry. To study the distribution of gene expression in eye tissues, six to eight eyeballs were obtained from adult mice (2–3 months old) after euthanization and the distal-most 3-mm optic nerve cut from the scleral surface of the eyeballs (ON), posterior segment (PS), retina, iris-ciliary body (I-CB), lens and corneal tissues were collected after dissection. Eyeballs (6 to 8) from embryonic (E15 and E18) and adult (2 and 7 months) mice were collected to study the expression profile of S1P receptors in the whole eye during embryonic and adult stages. To study retinal expression during development, we collected 8 to 10 retinas from four to five C57Bl6J mice at postnatal days P1, P7, P15 and P30. 4.2. Light Damage of Albino Rat Retina Adult (2–4 months old) SD rats were light damaged by being subjected to 2700 lux of white light for 6 h in specially fabricated light boxes, which was described in previous publications [39,59,60]. The retinas were harvested at various time points following the light exposure (0 h to 24 h). Eyes were also harvested at various time points, fixed in Prefer fixative (Anatech Ltd., Battle Creek, MI, USA) for 20 to 40 min, embedded in paraffin, and sectioned for conducting immunohistochemistry. 4.3. Gene Expression Analysis by Quantitative RT-PCR (qRT-PCR) The RNA was isolated and purified from frozen rat tissues using PureLink® Micro-to Midi Total RNA Purification System from Invitrogen (Carlsbad, CA, USA) and following the manufacturer’s protocol. Equal quantities (1.0 µg) of total RNA from each tissue were converted to first-strand cDNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA, USA) for RT-PCR. First-strand cDNA was used for qRT-PCR. Primers for qRT-PCR were intron-spanning (Supplemental Table S1). Quantitative PCR and melt-curve analyses were performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and an iCycler machine. Relative quantities of expression of the genes of interest in different samples were calculated by the comparative Ct (threshold cycle) value method [39,79,80]. 4.4. Immunohistochemistry Immunolabeling on paraffin sections was performed as described previously [39,51,52,80]. The sections were deparaffinized, rehydrated in PBS (phosphate-buffered saline), and nonspecific labeling was blocked using 10% normal horse serum in PBS for 2 h. Excess blocker was removed and the primary antibody was applied overnight at 4 ◦C. IHC antibodies were used as follows: SphK1 rabbit polyclonal [1:100–1:200] (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Sphk2 goat polyclonal [1:100–1:200] (Santa Cruz Biotechnology, Santa Cruz, CA), S1PR1 rabbit polyclonal [1:200] (Cayman, Ann Arbor, MI, USA), S1PR2 goat polyclonal [1:100] (Santa Cruz Biotechnology, Santa Cruz, CA, USA), S1PR3 rabbit polyclonal [1:200] (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PKCα rabbit polyclonal [1:200] (Cell Signaling, Danvers, MA, USA), and Rhodopsin mouse monoclonal [1:200] (Abcam, Cambridge, MA, USA). Sections were rinsed and incubated with appropriate secondary antibodies conjugated to fluorescence dye (Alexa flour, Invitrogen, Carlsbad, CA, USA) @ 1:1000 dilution for 1 h at room temperature to visualize labeling. Sections were rinsed and cover-slipped with ProLong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA). Confocal microscopy was performed using an Olympus FluoView FV500 confocal microscopy system (Olympus Microsystems, Center Valley, PA, USA). To ensure quantitative image quality, laser power, pinhole settings, photomultiplier tube settings, and intensity thresholds were kept constant for the used antibody. Int. J. Mol. Sci. 2018, 19, 3885 14 of 18 4.5. Statistical Analyses Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc.; La Jolla, CA, USA). The quantitative data are expressed as mean ± SE for each group. One-way or two-way ANOVA and Student’s and paired t-tests were performed to assess differences between means. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/12/ 3885/s1. Author Contributions: Conceptualization, N.M. Data curation, H.P., H.Q., N.P. and J.M. Formal analysis, H.P. and N.M. Funding acquisition, N.M. Investigation, N.M. Methodology, H.P., H.Q., N.P. and J.M. Project administration, N.M. Resources, B.H. Supervision, N.M. Validation, N.M. Visualization, H.Q. and N.M. Writing—original draft, H.P. and N.M. Writing—review & editing, R.G. and N.M. Funding: This work was supported by the grants from the National Eye Institute, the National Institute of Health (R01 EY-022071, P30 EY-021725), and the National Center for Research Resources, the National Institute of Health (RR-17703), the Foundation Fighting Blindness Inc., USA, and Research to Prevent Blindness, Inc., USA. Acknowledgments: Robert E. Anderson, Richard S. Brush, Mark Dittmar, Louisa J. Williams, Carolina Abrahan (Fulbright scholar), and Linda S. Boone (from the Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA) are gratefully acknowledged by the authors for providing research reagents and technical support. Conflicts of Interest: The authors declare no conflict of interest. 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