Sphingosine kinase-dependent directional migration of leukocytes in response to phorbol ester

Abstract

Syndecan-4 participates in focal adhesion by non-G protein-dependent activation of protein kinase C. Ligation of syndecan-4 with antithrombin elicits pertussis toxin-sensitive chemotaxis of leukocytes. As activation of protein kinase C stimulates release of sphingosine-1-phosphate, a chemoattracting G protein-coupled receptor agonist, we studied directional migration of leukocytes in response to phorbol myristate acetate (PMA), a direct activator of protein kinase C. Human peripheral blood neutrophils, monocytes, and lymphocytes were purified and tested for chemotactic migration in micropore filter assays in response to PMA. Dose-dependent stimulation of migration was seen only when leukocytes were exposed to concentration gradients of PMA; in the absence of such a gradient, inhibition of random migration was induced. Dimethylsphingosine inhibited PMA-induced leukocyte chemotaxis, indicating that activation of sphingosine kinase for enhanced production of sphingosine-1-phosphate mediates the chemotactic response to PMA. Pertussis toxin abrogated the chemotactic response to PMA, suggesting involvement of G protein-coupled sphingosine-1-phosphate receptor. Dimethylsphingosine also inhibited leukocyte chemotaxis toward antithrombin, indicating that similar mechanisms may be involved upon syndecan-4 ligation. Data show that protein kinase C-dependent activation of sphingosine kinase may play a central role in leukocyte chemotaxis toward non-G protein-coupled receptor agonists.