Activation of the p38 SAPK and NF-κB pathways by S1PR2 in endothelial cells. (A) Western blot of phospho-p38 (p-p38) and total VE-cadherin (VE-cad) levels in adenovirus control (β-gal) and Ad-S1PR2–transduced HUVEC (S1PR2) stimulated with S1P for 10 minutes (lanes 1 to 8), and naïve HUVEC treated with vehicle or 10 ng/mL TNF-α for 10 minutes (lanes 9 and 10). (B) Phospho-p38 SAPK (p-p38) and total p38 SAPK levels in adenovirus control (β-gal) and Ad-S1PR2–transduced HUVEC (S1PR2) stimulated with 100 nM S1P for the times indicated. (C) Western blot of phospho-p38 SAPK and total p38 SAPK from SVEC cells stimulated with 100 nM S1P in the absence or the presence of JTE013. (D) The activation of the NF-κB pathway by S1PR2 is dependent on ROCK activity. Lanes 1 to 6: adenovirus control (β-gal) and Ad-S1PR2–transduced HUVEC (S1PR2) were pretreated with vehicle (C) or 10 μM Y-27632 (Y) and treated with 100 nM S1P for 10 minutes when indicated (+). Lanes 7 to 8: naïve HUVEC were treated with vehicle (C) or 10 ng/mL TNF-α for 10 minutes. (A-D) Fold induction vs non-treated cells is plotted. Values are mean ± SEM of 3 to 5 independent experiments. One representative Western blot is shown. *P < .05 treated vs non-treated cells. (E) Upregulation of S1PR2 in HUVEC induces U937 monocyte adhesion in a ROCK-, p38-, and NF-κB–dependent way. Twenty hours after transduction, adenovirus control (β-gal) and Ad-S1PR2 HUVEC (S1PR2) were incubated with 10 μM Y-27632 (Y), 10 μM SB203580 (SB), 5 μM BAY 11-7085 (BAY), or 10 μM JTE013 (JTE) for 4 hours, and monocyte adhesion assays were conducted as described. Adhesion to naïve HUVEC stimulated with 2 ng/mL TNF-α is shown on the right. Results are mean ± SEM (n = 4) of 1 representative experiment of 3 experiments with similar results. *P < .05 β-gal vs S1PR2 and, when indicated, between vehicle-treated and inhibitor-treated Ad-S1PR2 HUVEC. (F) Diagram summarizing our findings (black) together with other data published by our laboratory and other groups (blue). Upon endothelial cell activation by proinflammatory stimuli, SPHK-1 is activated^44,46  and S1P is released.⁴⁵  Blockade of S1PR2 signaling results in inhibition of the expression of proinflammatory and procoagulant molecules by TNF-α. Upregulation of S1PR2 increases endothelial permeability, which is dependent on the Rho-ROCK pathway.¹⁷  In addition, S1PR2 induces endothelial inflammation and the activation of the Rho-ROCK-NF-κB and p38 SAPK pathways. Both pathways are activated in parallel by S1PR2 and play a critical role in the induction of proinflammatory molecules.