Limits...
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.

Show MeSH

Related in: MedlinePlus

SPNS2 releases S1P from human vascular ECs.HUVECs and HPAECs were treated with two siRNAs targetingSPNS2 mRNA or with a negative control siRNA. (A andB) Relative amount of SPNS2 mRNA in cells treated withsiRNA. Total RNA was isolated, and SPNS2 andGAPDH mRNA levels were determined by quantitativereal-time PCR. The amount of SPNS2 mRNA is normalizedto GAPDH mRNA. (C and D) Intracellular S1P. (E and F)The cells were incubated with 1% BSA for 4 hr at 37 °C, andthe released S1P was measured by UPLC-MS/MS. The cells were collected,and the intracellular S1P content was measured by HPLC.C17-S1P was used as the internal standard. The graphs showthe average values from three (C and E) or four (A, B, D and F)experiments, with error bars representing the standard error.*P<0.005 compared to‘control’.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3379171&req=5

pone-0038941-g011: SPNS2 releases S1P from human vascular ECs.HUVECs and HPAECs were treated with two siRNAs targetingSPNS2 mRNA or with a negative control siRNA. (A andB) Relative amount of SPNS2 mRNA in cells treated withsiRNA. Total RNA was isolated, and SPNS2 andGAPDH mRNA levels were determined by quantitativereal-time PCR. The amount of SPNS2 mRNA is normalizedto GAPDH mRNA. (C and D) Intracellular S1P. (E and F)The cells were incubated with 1% BSA for 4 hr at 37 °C, andthe released S1P was measured by UPLC-MS/MS. The cells were collected,and the intracellular S1P content was measured by HPLC.C17-S1P was used as the internal standard. The graphs showthe average values from three (C and E) or four (A, B, D and F)experiments, with error bars representing the standard error.*P<0.005 compared to‘control’.

Mentions: We also examined whether SPNS2 functions as an S1P transporter in human vascularECs, such as HUVECs and human pulmonary artery ECs (HPAECs), derived from venousand arterial endothelia, respectively. When HUVECs or HPAECs were treated withSPNS2-specific siRNAs, the expression ofSPNS2 mRNA decreased to less than 20% of the control(Figure 11A and 11B). Toinvestigate the off-target effect of these siRNAs, the expression of four ABCtransporters was examined because ABCA1, ABCB1, ABCC1 and ABCG2 have beenreported to play a role in S1P release from the cells [25], [27], [37], [38], [39], [40]. We confirmed that theexpression of these ABC transporters was not changed between the siRNAstargeting SPNS2 and the negative control (Figure S4).The amount of secreted S1P was significantly decreased, while the amount ofintracellular S1P was not altered, in HUVECs or HPAECs treated with siRNAstargeting SPNS2 (Figure 11C–11F). Because intracellular S1P should berigorously controlled by various sphingolipid metabolizing enzymes such asSPHKs, SPL and SPPs, the intracellular S1P concentration should show nosignificant change regardless of the deletion of S1P secretion activity. Theseresults indicate that SPNS2 plays a central role in releasing S1P from ECs inmice and humans.


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

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

SPNS2 releases S1P from human vascular ECs.HUVECs and HPAECs were treated with two siRNAs targetingSPNS2 mRNA or with a negative control siRNA. (A andB) Relative amount of SPNS2 mRNA in cells treated withsiRNA. Total RNA was isolated, and SPNS2 andGAPDH mRNA levels were determined by quantitativereal-time PCR. The amount of SPNS2 mRNA is normalizedto GAPDH mRNA. (C and D) Intracellular S1P. (E and F)The cells were incubated with 1% BSA for 4 hr at 37 °C, andthe released S1P was measured by UPLC-MS/MS. The cells were collected,and the intracellular S1P content was measured by HPLC.C17-S1P was used as the internal standard. The graphs showthe average values from three (C and E) or four (A, B, D and F)experiments, with error bars representing the standard error.*P<0.005 compared to‘control’.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038941-g011: SPNS2 releases S1P from human vascular ECs.HUVECs and HPAECs were treated with two siRNAs targetingSPNS2 mRNA or with a negative control siRNA. (A andB) Relative amount of SPNS2 mRNA in cells treated withsiRNA. Total RNA was isolated, and SPNS2 andGAPDH mRNA levels were determined by quantitativereal-time PCR. The amount of SPNS2 mRNA is normalizedto GAPDH mRNA. (C and D) Intracellular S1P. (E and F)The cells were incubated with 1% BSA for 4 hr at 37 °C, andthe released S1P was measured by UPLC-MS/MS. The cells were collected,and the intracellular S1P content was measured by HPLC.C17-S1P was used as the internal standard. The graphs showthe average values from three (C and E) or four (A, B, D and F)experiments, with error bars representing the standard error.*P<0.005 compared to‘control’.
Mentions: We also examined whether SPNS2 functions as an S1P transporter in human vascularECs, such as HUVECs and human pulmonary artery ECs (HPAECs), derived from venousand arterial endothelia, respectively. When HUVECs or HPAECs were treated withSPNS2-specific siRNAs, the expression ofSPNS2 mRNA decreased to less than 20% of the control(Figure 11A and 11B). Toinvestigate the off-target effect of these siRNAs, the expression of four ABCtransporters was examined because ABCA1, ABCB1, ABCC1 and ABCG2 have beenreported to play a role in S1P release from the cells [25], [27], [37], [38], [39], [40]. We confirmed that theexpression of these ABC transporters was not changed between the siRNAstargeting SPNS2 and the negative control (Figure S4).The amount of secreted S1P was significantly decreased, while the amount ofintracellular S1P was not altered, in HUVECs or HPAECs treated with siRNAstargeting SPNS2 (Figure 11C–11F). Because intracellular S1P should berigorously controlled by various sphingolipid metabolizing enzymes such asSPHKs, SPL and SPPs, the intracellular S1P concentration should show nosignificant change regardless of the deletion of S1P secretion activity. Theseresults indicate that SPNS2 plays a central role in releasing S1P from ECs inmice and humans.

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