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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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Tissue distribution of Spns2 mRNAs.Quantitative real-time PCR was performed with first strand cDNAsynthesized from mRNAs of various mouse tissues. The amount ofSpns2 mRNA in each tissue is shown relative to thatof Hprt. The primers and probes used for PCR are givenin TableS2. The graph shows the average values from four experiments,with error bars representing the standard error.
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pone-0038941-g007: Tissue distribution of Spns2 mRNAs.Quantitative real-time PCR was performed with first strand cDNAsynthesized from mRNAs of various mouse tissues. The amount ofSpns2 mRNA in each tissue is shown relative to thatof Hprt. The primers and probes used for PCR are givenin TableS2. The graph shows the average values from four experiments,with error bars representing the standard error.

Mentions: We examined whether vascular ECs utilize SPNS2 for S1P secretion because it wasreported that human umbilical vein ECs (HUVECs) are able to release S1P into theculture medium [19]. In fact, Spns2 mRNA was detected inECs of peripheral blood vessels in the thymus and kidney (Figure S3).The transcription of Spns2 mRNA was limited to ECs and was notdetected at all in other cells, including blood cells. AlthoughSpns2 mRNA was detected in the aorta by quantitativereal-time PCR, it was not detected by in situ hybridization inECs of the aorta or the cava (Figure 7 and Figure S3K). Because the targeting vector forthe SPNS2-deficient mice was designed to replace Spns2 with thelacZ gene, tissues from Spns2-heterozygousmice were stained with X-gal to identify the SPNS2-expressing cells.CD31-positive ECs were clearly stained with X-gal in the thymus, similar to thein situ hybridization results. Furthermore, the signalswere observed in ECs of the cava (although not detected by insitu hybridization) but not in the aorta (Figure 8). These results suggest that therewere enough Spns2 transcripts present in the aorta fordetection with quantitative real-time PCR, but the level of transcript was belowthe sensitivity of in situ hybridization and X-gal staining. Toconfirm the transcription of Spns2 mRNA in aortic ECs,quantitative real-time PCR was performed using the mRNAs from whole aorta andfrom ECs-depleted aorta. The amount of Spns2 mRNA wassignificantly decreased in the ECs-depleted aorta (Figure 9). The relative transcription ofSpns2 mRNA was higher in the ECs prepared from aorta. Whenthe amount of Spns2 mRNA was normalized to that ofCdh5, an EC marker gene, the expression level was nearlyequivalent, suggesting that the amount of Spns2 mRNA isdependent on that of ECs (Figure9). These results indicate that Spns2 mRNA istranscribed in aortic ECs, although at a low level.


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

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

Tissue distribution of Spns2 mRNAs.Quantitative real-time PCR was performed with first strand cDNAsynthesized from mRNAs of various mouse tissues. The amount ofSpns2 mRNA in each tissue is shown relative to thatof Hprt. The primers and probes used for PCR are givenin TableS2. The graph shows the average values from four experiments,with error bars representing the standard error.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038941-g007: Tissue distribution of Spns2 mRNAs.Quantitative real-time PCR was performed with first strand cDNAsynthesized from mRNAs of various mouse tissues. The amount ofSpns2 mRNA in each tissue is shown relative to thatof Hprt. The primers and probes used for PCR are givenin TableS2. The graph shows the average values from four experiments,with error bars representing the standard error.
Mentions: We examined whether vascular ECs utilize SPNS2 for S1P secretion because it wasreported that human umbilical vein ECs (HUVECs) are able to release S1P into theculture medium [19]. In fact, Spns2 mRNA was detected inECs of peripheral blood vessels in the thymus and kidney (Figure S3).The transcription of Spns2 mRNA was limited to ECs and was notdetected at all in other cells, including blood cells. AlthoughSpns2 mRNA was detected in the aorta by quantitativereal-time PCR, it was not detected by in situ hybridization inECs of the aorta or the cava (Figure 7 and Figure S3K). Because the targeting vector forthe SPNS2-deficient mice was designed to replace Spns2 with thelacZ gene, tissues from Spns2-heterozygousmice were stained with X-gal to identify the SPNS2-expressing cells.CD31-positive ECs were clearly stained with X-gal in the thymus, similar to thein situ hybridization results. Furthermore, the signalswere observed in ECs of the cava (although not detected by insitu hybridization) but not in the aorta (Figure 8). These results suggest that therewere enough Spns2 transcripts present in the aorta fordetection with quantitative real-time PCR, but the level of transcript was belowthe sensitivity of in situ hybridization and X-gal staining. Toconfirm the transcription of Spns2 mRNA in aortic ECs,quantitative real-time PCR was performed using the mRNAs from whole aorta andfrom ECs-depleted aorta. The amount of Spns2 mRNA wassignificantly decreased in the ECs-depleted aorta (Figure 9). The relative transcription ofSpns2 mRNA was higher in the ECs prepared from aorta. Whenthe amount of Spns2 mRNA was normalized to that ofCdh5, an EC marker gene, the expression level was nearlyequivalent, suggesting that the amount of Spns2 mRNA isdependent on that of ECs (Figure9). These results indicate that Spns2 mRNA istranscribed in aortic ECs, although at a low level.

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