Sphingosine-1-Phosphate Receptor 1 Is Involved in Non-Obese Diabetic Mouse Thymocyte Migration Disorders.

NOD (non-obese diabetic) mice spontaneously develop type 1 diabetes following T cell-dependent destruction of pancreatic β cells. Several alterations are observed in the NOD thymus, including the presence of giant perivascular spaces (PVS) filled with single-positive (SP) CD4⁺ and CD8⁺ T cells that accumulate in the organ. These cells have a decreased expression of membrane CD49e (the α5 integrin chain of the fibronectin receptor VLA-5 (very late antigen-5). Herein, we observed lower sphingosine-1-phosphate receptor 1 (S1P1) expression in NOD mouse thymocytes when compared with controls, mainly in the mature SP CD4⁺CD62Lhi and CD8⁺CD62Lhi subpopulations bearing the CD49e− phenotype. In contrast, differences in S1P1 expression were not observed in mature CD49e⁺ thymocytes. Functionally, NOD CD49e− thymocytes had reduced S1P-driven migratory response, whereas CD49e⁺ cells were more responsive to S1P. We further noticed a decreased expression of the sphingosine-1-phosphate lyase (SGPL1) in NOD SP thymocytes, which can lead to a higher sphingosine-1-phosphate (S1P) expression around PVS and S1P1 internalization. In summary, our results indicate that the modulation of S1P1 expression and S1P/S1P1 interactions in NOD mouse thymocytes are part of the T-cell migratory disorder observed during the pathogenesis of type 1 diabetes.

Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization.

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P1) phosphorylation was studied. Activation of S1P1 receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P1 phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P1 phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P1 phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and ß isoforms in S1P1 immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or ß and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P1 phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P1.