Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways

Abstract

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.

Figures

Fig. 1.

Effect of sphingosine-1-phosphate (S1P) and caged S1P on [Ca2+]i. Endothelial cells (ECs) grown on 35-mm glass-bottom dishes were loaded with calcium fluorescent indicator Fluor-4 AM, and intracellular ionic Ca2+ was monitored by confocal microscopy as described in materials and methods. A: ECs were challenged with S1P (1 μM). B: caged S1P (1 μM) was added to the incubation media. C: ECs were preloaded with caged S1P for 15 min, cells were washed, and intracellular calcium was monitored. D: same as C, but cells were treated with 5 μM thapsigargin (TG) before UV flash. E–G: same as C, but cells were pretreated with Ga3+ (1 μM, 15 min) (E), or incubated in Ca-free media (F), or pretreated with BAPTA (25 μM, 1 h) (G) before UV flash. As expected, treatment with TG resulted in the depletion of intracellular Ca2+ stores, and further exposure to UV had no additional effect, demonstrating that intracellular S1P targeted intracellular stored [Ca2+]i. Representative tracings from 3 independent experiments are shown.

Fig. 2.

Effect of pertussis toxin (PTx) on S1P-mediated [Ca2+]i. A: ECs were pretreated with PTx (100 ng/ml) for different time intervals, loaded with Fura-2 AM, and treated with S1P (1 μM), and changes in intracellular Ca2+ were monitored by spectrofluorimetry. B: ECs were pretreated with PTx (100 ng/ml, 6 h), loaded with Fura-2 AM, and then stimulated with 1 μM S1P or caged S1P as indicated, and intracellular Ca2+ was monitored. Values are means ± SE. *Significantly different from control (P < 0.05).

Fig. 3.

Effect of PTx on S1P- and caged S1P-mediated tyrosine phosphorylation of MAPKs. ECs grown on 100-mm dishes were preincubated with PTx (100 ng/ml, for 6 h) and treated with 1 μM S1P or caged S1P as indicated. Cell lysates (20–40 μg of protein) were subjected to 10% SDS-PAGE and probed with anti-phospho-JNK or total JNK, anti-phospho-p38 MAPK and total p38, or anti-phospho-ERK and total ERK antibodies. Fold change in phospho-JNK/JNK, phospho-p38/p38, or phospho-ERK/ERK MAPKs determined from the respective Western blots by image analysis were normalized to total JNK, p38 MAPK, or ERK. Representative blots from 3 different experiments are shown. Values are means ± SE. *Significantly different from vehicle control (P < 0.05); **significantly different from PTx-treated cells for caged S1P added outside and S1P (P < 0.05).

Fig. 4.

Effect of BAPTA on S1P- and caged S1P-mediated tyrosine phosphorylation of MAPKs. ECs grown on 100-mm dishes were preincubated with BAPTA (25 μM) for 1 h and treated with 1 μM S1P or caged S1P as indicated. Cell lysates (20–40 μg of protein) were subjected to 10% SDS-PAGE, blotted, and probed with anti-phospho-JNK or total JNK, anti-phospho-p38 MAPK and total p38, or anti-phospho-ERK and total ERK antibodies. Fold changes in phospho-JNK/JNK, phospho-p38/p38 MAPK, or phospho-ERK/ERK were calculated from the respective Western blots by image analysis, and data were normalized to total JNK, p38 MAPK, or ERK. Representative blots from 3 different experiments are shown. Values are means ± SE. *Significantly different from vehicle control (P < 0.05); **significantly different from BAPTA untreated cells with caged S1P added outside (P < 0.05).

Fig. 5.

Effect of PTx on S1P- and caged S1P-mediated cortactin redistribution. ECs grown on coverslips were preincubated with PTx (100 ng/ml, 6 h) and then stimulated with 1 μM S1P or caged S1P as indicated. Cortactin reorganization was visualized by immunocytochemistry. Shown are representative immunofluorescence images from several independent experiments.

Fig. 6.

Intracellular S1P mediates reorganization of adherens and tight junction proteins and activation Rac1 and IQGAP1. Human pulmonary artery ECs (HPAECs) were preloaded with caged S1P (1 μM), and S1P was released by UV flash. Alternately, cells were stimulated with S1P added outside. Reorganization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens (ZO)-1 (A) and activation of Rac1 and IQGAP1 (B) was analyzed by immunocytochemistry. Representative immunofluorescence images from several independent experiments are shown.

Fig. 7.

Intracellular S1P induces barrier function in ECs through cell-to-cell adhesion contacts. HPAECs grown on gold electrodes were loaded with caged S1P so as to generate intracellular S1P after photolysis with UV (A) or treated directly with extracellular caged S1P and transendothelial electrical resistance (TER) measured. Cell-to-cell (Rb) and cell-matrix (α) components (B) were resolved from A using manufacturer's software. Shown are representative tracings from 3 independent experiments.

Fig. 8.

Downregulation of sphingosine kinase 1 (SphK1) attenuates S1P-induced intracellular calcium and transendothelial barrier enhancement. A: HPAECs grown on glass coverslips infected with 5 pfu/ml of vector control or SphK1-flag (Dn) for 24 h and intracellular Ca2+ were monitored by spectrofluorimetry as described in materials and methods. Cell lysates were probed by Western blotting as indicated. B: HPAECs were transfected with 50 nM scrambled siRNA or SphK1 siRNA for 72 h, and cell lysates were analyzed for SphK1 expression by real-time PCR and immunoblotting (IB) as indicated. HPAECs grown on gold electrodes or 35-mm dishes were transfected with 50 nM scrambled siRNA or SphK1 siRNA for 72 h or pretreated with 10 μM CII (SphK1 inhibitor) as indicated and were then challenged with S1P (1 μM), and TER was measured. Values are mean ± SE. *Significantly different from vector control or control (P < 0.05). **Significantly different from S1P-treated cells in scrambled RNA (P < 0.05).

Fig. 9.

The role of S1P1 receptor and Rac1 activation in the intracellular S1P-mediated barrier function in ECs. HPAECs grown on gold electrodes were pretreated with S1P1 receptor inhibitor SB649146 (1 μM, 3 h) (A) or Rac1 inhibitor NSC23766 (50 μM, 30 min) (B) and were then loaded with caged S1P so as to generate intracellular S1P after photolysis with UV and TER measured. Values are means ± SE. *Significantly different from control (P < 0.05).