sphingosine-1-phosphate and prolinedithiocarbamate

We showed previously that TNF-α down-regulates the Na+/K+ ATPase in HepG2 cells. This work was undertaken to study the role of ceramide and its metabolites in TNF-α action. Treating HepG2 cells with the cytokine in presence of an inhibitor of sphingomyelinase, abrogated the effect of TNF-α on the ATPase. To confirm the involvement of ceramide or its metabolites, cells were incubated with exogenous ceramide. Ceramide reduced time-dependently the activity of the ATPase and its effect disappeared in presence of CAY 10466 or SHKI, respective inhibitors of ceramidase and spingosine kinase, suggesting that ceramide acts via sphingosine or sphingosine-1-phosphate (S1P). However, HepG2 cells treated with exogenous sphingosine showed a higher Na+/K+ ATPase activity inferring that S1P is the one responsible for the down-regulatory effect of TNF-α and ceramide. This hypothesis was confirmed by the observed inhibitory effect of exogenous S1P on the pump, which was maintained when JNK and NF-κB were inhibited separately or simultaneously. The concurrent, but not individual inhibition of the kinase and transcription factor in the absence of S1P imitated the effect of S1P. It was concluded that S1P down-regulates the ATPase by inhibiting both JNK and NF-κB. This conclusion was supported by the observed decrease in the phosphorylation of c-jun and the enhanced protein expression of IκB and lower NK-KB activity.

Topics: Aniline Compounds; Anthracenes; Apoptosis; Benzylidene Compounds; Cell Line; Ceramides; Hep G2 Cells; Humans; I-kappa B Proteins; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Proline; Sodium-Potassium-Exchanging ATPase; Sphingomyelin Phosphodiesterase; Sphingosine; Thiocarbamates; Tumor Necrosis Factor-alpha

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low-molecular-weight lysophospholipid (LPL) ligands which are recognized by the Edg family of G protein-coupled receptors (GPCRs). In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. Interleukin-8 (IL-8) is a C-X-C chemokine and acts as a chemoattractant of neutrophils, whereas monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine and functions mainly as a chemoattractant of monocytes/macrophages. Both factors are secreted from endothelial cells and have been implicated in the processes leading to atherosclerosis. We examined the effects of LPLs on the expression of IL-8 and MCP-1, key regulators of leukocyte recruitment in human umbilical cord vein endothelial cells (HUVECs). Work illustrated in this article showed that LPA and S1P enhanced IL-8 and MCP-1 mRNA expressions, and protein secretions in dose- and time-dependent fashions. Maximal mRNA expression appeared at 16 hr post-ligand treatment. Using prior treatments with chemical inhibitors, LPLs enhanced IL-8 and MCP-1 expressions through a Gi-, Rho-, and NFkappaB-dependent mechanism. In a chemotaxis assay system, LPL treatments of endothelial cells enhanced monocyte recruitment through upregulating IL-8 and MCP-1 protein secretions. Pre-incubation with AF12198, an IL-1 receptor antagonist or IL-1 functional blocking antibody both suppressed the enhanced effects elicited by LPLs of IL-8 and MCP-1 mRNA expressions in HUVECs. These results suggest that LPLs released by activated platelets might enhance the IL-8- and MCP-1-dependent chemoattraction of monocytes toward the endothelium through an IL-1-dependent mechanism, which may play an important role in facilitating wound-healing and inflammation processes.

Topics: Carrier Proteins; Chemokine CCL2; Chemotaxis; Endothelial Cells; Humans; Interleukin-1; Interleukin-8; Lysophospholipids; Pertussis Toxin; Proline; RNA, Messenger; Sphingosine; Thiocarbamates; Time Factors; Umbilical Veins; Up-Regulation; Vesicular Transport Proteins