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Mouse SPNS2 functions as a sphingosine-1-phosphate transporter in vascular endothelial cells.

Hisano Y, Kobayashi N, Yamaguchi A, Nishi T - PLoS ONE (2012)

Bottom Line: However, little is known about the molecular mechanism by which S1P is supplied to extracellular environments such as blood plasma.Here, we show that SPNS2 functions as an S1P transporter in vascular endothelial cells but not in erythrocytes and platelets.Moreover, the plasma S1P concentration of SPNS2-deficient mice was reduced to approximately 60% of wild-type, and SPNS2-deficient mice were lymphopenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan.

ABSTRACT
Sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is produced inside the cells, regulates a variety of physiological and pathological responses via S1P receptors (S1P1-5). Signal transduction between cells consists of three steps; the synthesis of signaling molecules, their export to the extracellular space and their recognition by receptors. An S1P concentration gradient is essential for the migration of various cell types that express S1P receptors, such as lymphocytes, pre-osteoclasts, cancer cells and endothelial cells. To maintain this concentration gradient, plasma S1P concentration must be at a higher level. However, little is known about the molecular mechanism by which S1P is supplied to extracellular environments such as blood plasma. Here, we show that SPNS2 functions as an S1P transporter in vascular endothelial cells but not in erythrocytes and platelets. Moreover, the plasma S1P concentration of SPNS2-deficient mice was reduced to approximately 60% of wild-type, and SPNS2-deficient mice were lymphopenic. Our results demonstrate that SPNS2 is the first physiological S1P transporter in mammals and is a key determinant of lymphocyte egress from the thymus.

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Thymocytes of SPNS2-deficient mice can mature and migrate towardS1P.(A and B) Expression profiles for CD4 and CD8. Thymus-derivedCD4+ and CD8+ cells from wild-type(A, WT, n = 5) and SPNS2-deficient (B, Δ,n = 7) mice were analyzed by flow cytometry. Eachplot is representative of multiple experiments. Numbers show the percentof total lymphocytes, identified by their size. (C) The percentagecorresponding to CD4+ CD8+ (DP),CD4− CD8+ (CD8SP) orCD4+ CD8− (CD4SP) populations. (D)Quantitative analysis of S1p1 mRNA in maturethymocytes. CD4 or CD8 single positive cells were purified from thethymus of wild-type (WT, n = 3) or SPNS2-deficient(Δ, n = 5) mice with MACS. The amount ofS1p1 mRNA is normalized to that ofHprt. The primers and probes used for PCR areindicated in Table S2. TheP-values from comparisons between WT and Δ samplesare indicated. (E, F) Chemotaxis assays of mature thymocytes ofwild-type (WT, n = 10) or SPNS2-deficient (Δ,n = 5) mice. The percentage of input cells of theCD62Lhi and CD4 (E) or CD8 (F) single positive phenotypethat migrated toward S1P is shown. open square, wild-type CD4 singlepositive; closed square, SPNS2-deficient CD4 single positive; opencircle, wild-type CD8 single positive; closed circle, SPNS2-deficientCD8 single positive. The graphs show the average values from multipleexperiments, with error bars representing the standard error.
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pone-0038941-g005: Thymocytes of SPNS2-deficient mice can mature and migrate towardS1P.(A and B) Expression profiles for CD4 and CD8. Thymus-derivedCD4+ and CD8+ cells from wild-type(A, WT, n = 5) and SPNS2-deficient (B, Δ,n = 7) mice were analyzed by flow cytometry. Eachplot is representative of multiple experiments. Numbers show the percentof total lymphocytes, identified by their size. (C) The percentagecorresponding to CD4+ CD8+ (DP),CD4− CD8+ (CD8SP) orCD4+ CD8− (CD4SP) populations. (D)Quantitative analysis of S1p1 mRNA in maturethymocytes. CD4 or CD8 single positive cells were purified from thethymus of wild-type (WT, n = 3) or SPNS2-deficient(Δ, n = 5) mice with MACS. The amount ofS1p1 mRNA is normalized to that ofHprt. The primers and probes used for PCR areindicated in Table S2. TheP-values from comparisons between WT and Δ samplesare indicated. (E, F) Chemotaxis assays of mature thymocytes ofwild-type (WT, n = 10) or SPNS2-deficient (Δ,n = 5) mice. The percentage of input cells of theCD62Lhi and CD4 (E) or CD8 (F) single positive phenotypethat migrated toward S1P is shown. open square, wild-type CD4 singlepositive; closed square, SPNS2-deficient CD4 single positive; opencircle, wild-type CD8 single positive; closed circle, SPNS2-deficientCD8 single positive. The graphs show the average values from multipleexperiments, with error bars representing the standard error.

Mentions: As shown in Figure 5, T cellsexamined in the thymus of SPNS2-deficient mice, and the population of mature Tcells (CD4 or CD8 positive; single positive) in the thymus was increased whilethat of immature T cells (CD4 and CD8 positive; double positive) was decreased.To examine the ability of mature thymocytes to migrate in response to S1P, theamount of S1p1 mRNA in mature thymocytes was quantified andtheir migration activity was measured in a transwell assay. The amount ofS1p1 mRNA in CD4 single positive cells was two to threetimes higher level than in CD8 single positive cells, and mature thymocytes ofSPNS2-deficient mice have more S1p1 mRNA than wild-type mice,in both CD4 and CD8 single positive cells (Figure 5D). Moreover, CD4 and CD8 singlepositive cells from SPNS2-deficient mice showed a high migration activity at alower S1P dose (1 to 10 nM) compared to that of wild-type (10 to 100 nM),presumably due to the increased expression of S1P1 (Figure 5E and 5F). It is possible that thehigher amount of S1p1 mRNA and the increased S1P sensitivity insingle positive cells from SPNS2-deficient mice might be caused by compensationfor the depletion of S1P required for thymocyte egress. These results indicatethat thymocytes of SPNS2-deficient mice can mature and migrate toward S1Pnormally, but they could not emigrate from the thymus into blood in the absenceof SPNS2. Consequently, the number of circulating T cells in the peripheralblood was dramatically reduced in SPNS2-deficient mice.


Mouse SPNS2 functions as a sphingosine-1-phosphate transporter in vascular endothelial cells.

Hisano Y, Kobayashi N, Yamaguchi A, Nishi T - PLoS ONE (2012)

Thymocytes of SPNS2-deficient mice can mature and migrate towardS1P.(A and B) Expression profiles for CD4 and CD8. Thymus-derivedCD4+ and CD8+ cells from wild-type(A, WT, n = 5) and SPNS2-deficient (B, Δ,n = 7) mice were analyzed by flow cytometry. Eachplot is representative of multiple experiments. Numbers show the percentof total lymphocytes, identified by their size. (C) The percentagecorresponding to CD4+ CD8+ (DP),CD4− CD8+ (CD8SP) orCD4+ CD8− (CD4SP) populations. (D)Quantitative analysis of S1p1 mRNA in maturethymocytes. CD4 or CD8 single positive cells were purified from thethymus of wild-type (WT, n = 3) or SPNS2-deficient(Δ, n = 5) mice with MACS. The amount ofS1p1 mRNA is normalized to that ofHprt. The primers and probes used for PCR areindicated in Table S2. TheP-values from comparisons between WT and Δ samplesare indicated. (E, F) Chemotaxis assays of mature thymocytes ofwild-type (WT, n = 10) or SPNS2-deficient (Δ,n = 5) mice. The percentage of input cells of theCD62Lhi and CD4 (E) or CD8 (F) single positive phenotypethat migrated toward S1P is shown. open square, wild-type CD4 singlepositive; closed square, SPNS2-deficient CD4 single positive; opencircle, wild-type CD8 single positive; closed circle, SPNS2-deficientCD8 single positive. The graphs show the average values from multipleexperiments, with error bars representing the standard error.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3379171&req=5

pone-0038941-g005: Thymocytes of SPNS2-deficient mice can mature and migrate towardS1P.(A and B) Expression profiles for CD4 and CD8. Thymus-derivedCD4+ and CD8+ cells from wild-type(A, WT, n = 5) and SPNS2-deficient (B, Δ,n = 7) mice were analyzed by flow cytometry. Eachplot is representative of multiple experiments. Numbers show the percentof total lymphocytes, identified by their size. (C) The percentagecorresponding to CD4+ CD8+ (DP),CD4− CD8+ (CD8SP) orCD4+ CD8− (CD4SP) populations. (D)Quantitative analysis of S1p1 mRNA in maturethymocytes. CD4 or CD8 single positive cells were purified from thethymus of wild-type (WT, n = 3) or SPNS2-deficient(Δ, n = 5) mice with MACS. The amount ofS1p1 mRNA is normalized to that ofHprt. The primers and probes used for PCR areindicated in Table S2. TheP-values from comparisons between WT and Δ samplesare indicated. (E, F) Chemotaxis assays of mature thymocytes ofwild-type (WT, n = 10) or SPNS2-deficient (Δ,n = 5) mice. The percentage of input cells of theCD62Lhi and CD4 (E) or CD8 (F) single positive phenotypethat migrated toward S1P is shown. open square, wild-type CD4 singlepositive; closed square, SPNS2-deficient CD4 single positive; opencircle, wild-type CD8 single positive; closed circle, SPNS2-deficientCD8 single positive. The graphs show the average values from multipleexperiments, with error bars representing the standard error.
Mentions: As shown in Figure 5, T cellsexamined in the thymus of SPNS2-deficient mice, and the population of mature Tcells (CD4 or CD8 positive; single positive) in the thymus was increased whilethat of immature T cells (CD4 and CD8 positive; double positive) was decreased.To examine the ability of mature thymocytes to migrate in response to S1P, theamount of S1p1 mRNA in mature thymocytes was quantified andtheir migration activity was measured in a transwell assay. The amount ofS1p1 mRNA in CD4 single positive cells was two to threetimes higher level than in CD8 single positive cells, and mature thymocytes ofSPNS2-deficient mice have more S1p1 mRNA than wild-type mice,in both CD4 and CD8 single positive cells (Figure 5D). Moreover, CD4 and CD8 singlepositive cells from SPNS2-deficient mice showed a high migration activity at alower S1P dose (1 to 10 nM) compared to that of wild-type (10 to 100 nM),presumably due to the increased expression of S1P1 (Figure 5E and 5F). It is possible that thehigher amount of S1p1 mRNA and the increased S1P sensitivity insingle positive cells from SPNS2-deficient mice might be caused by compensationfor the depletion of S1P required for thymocyte egress. These results indicatethat thymocytes of SPNS2-deficient mice can mature and migrate toward S1Pnormally, but they could not emigrate from the thymus into blood in the absenceof SPNS2. Consequently, the number of circulating T cells in the peripheralblood was dramatically reduced in SPNS2-deficient mice.

Bottom Line: However, little is known about the molecular mechanism by which S1P is supplied to extracellular environments such as blood plasma.Here, we show that SPNS2 functions as an S1P transporter in vascular endothelial cells but not in erythrocytes and platelets.Moreover, the plasma S1P concentration of SPNS2-deficient mice was reduced to approximately 60% of wild-type, and SPNS2-deficient mice were lymphopenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan.

ABSTRACT
Sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is produced inside the cells, regulates a variety of physiological and pathological responses via S1P receptors (S1P1-5). Signal transduction between cells consists of three steps; the synthesis of signaling molecules, their export to the extracellular space and their recognition by receptors. An S1P concentration gradient is essential for the migration of various cell types that express S1P receptors, such as lymphocytes, pre-osteoclasts, cancer cells and endothelial cells. To maintain this concentration gradient, plasma S1P concentration must be at a higher level. However, little is known about the molecular mechanism by which S1P is supplied to extracellular environments such as blood plasma. Here, we show that SPNS2 functions as an S1P transporter in vascular endothelial cells but not in erythrocytes and platelets. Moreover, the plasma S1P concentration of SPNS2-deficient mice was reduced to approximately 60% of wild-type, and SPNS2-deficient mice were lymphopenic. Our results demonstrate that SPNS2 is the first physiological S1P transporter in mammals and is a key determinant of lymphocyte egress from the thymus.

Show MeSH
Related in: MedlinePlus