Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

Abstract

Sphingosine 1-phosphate (S1P), produced by Sphks (sphingosine kinases), is a multifunctional lipid mediator that regulates immune cell trafficking and vascular development. Mammals maintain a large concentration gradient of S1P between vascular and extravascular compartments. Mechanisms by which S1P is released from cells and concentrated in the plasma are poorly understood. We recently demonstrated [Ancellin, Colmont, Su, Li, Mittereder, Chae, Stefansson, Liau and Hla (2002) J. Biol. Chem. 277, 6667-6675] that Sphk1 activity is constitutively secreted by vascular endothelial cells. In the present study, we show that among the five Sphk isoforms expressed in endothelial cells, the Sphk-1a isoform is selectively secreted in HEK-293 cells (human embryonic kidney cells) and human umbilical-vein endothelial cells. In sharp contrast, Sphk2 is not secreted. The exported Sphk-1a isoform is enzymatically active and produced sufficient S1P to induce S1P receptor internalization. Wild-type mouse plasma contains significant Sphk activity (179 pmol x min(-1) x g(-1)). In contrast, Sphk1-/- mouse plasma has undetectable Sphk activity and approx. 65% reduction in S1P levels. Moreover, human plasma contains enzymatically active Sphk1 (46 pmol x min(-1) x g(-1)). These results suggest that export of Sphk-1a occurs under physiological conditions and may contribute to the establishment of the vascular S1P gradient.

Figure 1. Isoforms of hSphk1

(A) Amino acid sequence alignment of hSphk1 isoforms. Sphk isoform 1a has 384 amino acids, isoform 1b has 398 amino acids and isoform 1c has 470 acids. A short stretch of the N-terminus is shown. Identical amino acid residues are indicated in upper-case, while similar amino acid residues are shown in lower-case. (B) RT–PCR analysis of Sphk1 transcripts in HUVEC. Bars a, b and c represent the expression of 384-, 398- and 470-amino-acid isoforms. Results are normalized to GAPDH mRNA levels. Transcripts were not detected in the absence of reverse transcription (results not shown). (C) Immunoblot analysis of the expression of endogenous Sphk1 polypeptides in HUVEC cytosolic fraction is shown. Cytosolic fraction (50 μg) was electrophoresed and probed with rabbit polyclonal anti-Sphk1 antibody.

Figure 2. Pulse–chase analysis of Sphk1 isoforms in HEK-293 cells

HEK-293 cells transiently transfected with pcDNA 3.0 (M), Sphk-1a (1a), Sphk-1b (1b) and Sphk-1c (1c) plasmids were pulsed with [³⁵S]methionine/cysteine. (A) Total cellular extracts (intracellular) and (B) CM (extracellular) were subjected to immunoprecipitation with anti-Sphk1 antibody.

Figure 3. Expression and enzymatic activity of transiently transfected Sphk1 isoforms in HEK-293 cells

(A) Immunoblot analysis of Sphk1 isoforms in HEK-293 cells. HEK-293 cells were transfected with 3 μg of pcDNA 3.0 (M), hSphk-1a (1a), hSphk-1b (1b) and hSphk-1c (1c) plasmids and 10 μg of the total homogenate was subjected to immunoblot analysis with rabbit polyclonal Sphk1 antibody. (B, C) Phosphorylation of sphingosine by Sphk1 isoforms. Total cellular extract (10 μg) (B) and 0.25 ml of CM (C) from corresponding isoforms were directly assayed for Sphk activity in vitro by addition of sphingosine and [³²P]ATP, and the formation of [³²P]S1P was analysed by TLC. TLC bands corresponding to [³²P]S1P are shown in (B, C). Results shown are the means±S.E.M. (n=3).

Figure 4. Pulse–chase analysis of endogenous Sphk1 polypeptides in HUVEC

(A) [³⁵S]methionine/cysteine-labelled HUVEC extracts (intracellular) and (B) CM (extracellular) were immunoprecipitated with anti-Sphk1 antibody or NRS for the indicated time and ³⁵S-labelled Sphk1 was quantified by a phosphoimager. Decay of intracellular Sphk-1a is plotted on a semi-logarithmic scale. Results shown are from a representative experiment that was repeated three times.

Figure 5. Isoforms of hSphk2

(A) Amino acid sequence alignment of hSphk isoforms. Sphk isoform 2a has 618 amino acids and isoform 2b has 654 amino acids. Conserved residues are shown in upper-case. (B) RT–PCR analysis of Sphk2 transcripts in HUVEC. Bars a and b represent the expression of 618- and 654-amino-acid isoforms. Results are normalized to GAPDH mRNA levels. (C) Immunoblot analysis of overexpressed Sphk2 isoforms in HEK-293 cells. HEK-293 cells were transfected with 3 μg of pcDNA 3.0 (M), hSphk-2a (2a) and hSphk-2b (2b) cDNA encoding plasmids. Total homogenate (10 μg) was subjected to immunoblot analysis with rabbit polyclonal Sphk2 antibody. (D) Phosphorylation of sphingosine by Sphk2 isoforms was evaluated with 10 μg of total cellular extract (intracellular) and 0.25 ml of CM (extracellular) for corresponding isoforms in an in vitro kinase assay by addition of sphingosine and [³²P]ATP, and the formation of [³²P]S1P was analysed by TLC. TLC bands corresponding to [³²P]S1P are shown. Results shown are the means±S.E.M. (n=4).

Figure 6. Subcellular localization of Sphk1 and Sphk2 isoenzymes in HEK-293 cells

Representative confocal images of HEK-293 cells transiently transfected with Mock (pcDNA 3.0), Sphk-1a, Sphk-1b, Sphk-1c, Sphk-2a and Sphk-2b plasmids. Confocal images were collected from fixed cells that were immunostained with polyclonal Sphk1 or Sphk2 antibody. For each construct, the x–y image (centre), x–z (box below, section indicated with horizontal arrowhead) and y–z (right box, section indicated with vertical horizontal arrowhead) images are shown. Note that among the Sphk1 constructs, the b and the c isoforms are more associated with the membrane and the c isoform exhibits a granular localization in the cytoplasm. Sphk-2a is present in the nucleus, whereas the Sphk-2b is not.

Figure 7. Immunolocalization of endogenously expressed Sphk1 and Sphk2 isoenzymes in HUVEC

HUVEC were immunostained with polyclonal Sphk1 and Sphk2 antibodies and visualized in a confocal immunofluorescence microscope as described. Sphk1 isoforms are localized to cytosol, membranous compartments and vesicle-like structures. In contrast, Sphk2 is localized to nuclear and perinuclear region, in addition to cytosol.

Figure 8. Production of S1P in the CM from Sphk-1a-expressing cells

(A) HEK-293 cells were transfected with various isoforms of hSphk1 and hSphk2 plasmids, CM (at 2 h) was collected, and incubated with 1 μM sphingosine and 500 μM ATP to allow the Sphk enzymatic activity to occure. Subsequently, CM was added to HEK-293 cells stably expressing GFP–S1PR1. Internalization of GFP-tagged S1PR1 was monitored after 1 h on fixed cells by confocal microscopy. (B) Intensity of GFP–S1PR1 on the plasma membrane is quantified. Results represent means±S.E.M.; n=7–10 cells from a representative experiment that was repeated at least twice. (C) Formation of S1P was determined in the respective CM as described in the Materials and methods section. Results represent the means±S.E.M. (n=3).

Figure 9. TLC analysis of Sphk activity in whole blood, PRP and PPP fractions from Sphk1^+/+ and Sphk1^−/− mice

(A) A volume of 25 μl of whole blood and PRP and (B) PPP was incubated in the presence or absence of 20 μM sphingosine with 10 μCi of [³²P]ATP in the final concentration 500 μM ATP. Formation of [³²P]S1P was determined by TLC analysis.