sphingosine-1-phosphate and rottlerin

We investigated the effects of sphingosine 1-phosphate and histamine on the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin, and their signaling pathways in human umbilical vein endothelial cells. Sphingosine 1-phosphate increased the mRNA and protein level of VCAM-1, and the mRNAs of E-selectin and ICAM-1. The effects of sphingosine 1-phosphate were inhibited by the pertussis toxin and the respective inhibitors (10 microM 1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) for phosphoinositide-specific phospholipase C; 10 microM 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) for p38 mitogen-activated protein kinase (MAPK); 1 microM 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976) for the alpha form of protein kinase C (PKC-alpha)), but not by a PKC-delta inhibitor (1 microM rottlerin). Histamine, which alone showed no effect, enhanced the sphingosine 1-phosphate-induced expressions via histamine H(1) receptor. The histamine response decreased by U73122 and rottlerin, but not by SB203580 and Gö6976. The effects of sphingosine 1-phosphate with and without histamine were abolished by the higher concentrations of PKC inhibitors and in the PKC-depleted cells. Sphingosine 1-phosphate and histamine alone stimulated phosphorylation of p38 MAPK in a phosphoinositide-specific phospholipase C-dependent but not in a PKCs-independent manner. These findings suggest that sphingosine 1-phosphate-induced expression of adhesion molecules was mediated by phosphoinositide-specific phospholipase C and preferentially by PKC-alpha and p38 MAPK, and the histamine response was mediated by phosphoinositide-specific phospholipase C and PKC-delta in human umbilical vein endothelial cells.

Topics: Acetophenones; Benzopyrans; Blotting, Western; Carbazoles; Cell Adhesion Molecules; Cells, Cultured; E-Selectin; Endothelium, Vascular; Estrenes; Histamine; Histamine Agonists; Humans; Imidazoles; Indoles; Intercellular Adhesion Molecule-1; Lysophospholipids; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Pyridines; Pyrrolidinones; RNA, Messenger; Signal Transduction; Sphingosine; Type C Phospholipases; Umbilical Veins; Vascular Cell Adhesion Molecule-1

The regulatory role of protein kinase C (PKC) delta isoform in the stimulation of phospholipase D (PLD) by sphingosine-1-phosphate (SPP) in a human-airway epithelial cell line (CFNPE9o(-)) was revealed by using antisense oligodeoxynucleotide to PKCdelta, in combination with the specific inhibitor rottlerin. Cell treatment with antisense oligodeoxynucleotide, but not with sense oligodeoxynucleotide, completely eliminated PKCdelta expression and resulted in the strong inhibition of SPP-stimulated phosphatidic acid formation. Indeed, among the PKCalpha, beta, delta, epsilon and zeta isoforms expressed in these cells, only PKCdelta was activated on cell stimulation with SPP, as indicated by translocation into the membrane fraction. Furthermore, pertussis toxin and genistein eliminated both PKCdelta translocation and PLD activation. In particular, a significant reduction in phosphatidylbutanol formation by SPP was observed in the presence of 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP1), an inhibitor of Src tyrosine kinase. Furthermore, the activity of Src kinase was slightly increased by SPP and inhibited by PP1. However, the level of PKCdelta tyrosine phosphorylation was not increased in SPP-stimulated cells, suggesting that Src did not directly phosphorylate PKCdelta. Finally, the level of serine phosphorylation of PLD1 and PLD2 isoforms was not changed, whereas the PLD1 isoform alone was threonine-phosphorylated in SPP-treated cells. PLD1 threonine phosphorylation was strongly inhibited by rottlerin, by anti-PKCdelta oligodeoxynucleotide and by PP1. In conclusion, in CFNPE9o(-) cells, SPP interacts with a membrane receptor linked to a G(i) type of G-protein, leading to activation of PLD, probably the PLD1 isoform, by a signalling pathway involving Src and PKCdelta.

Topics: Acetophenones; Benzopyrans; Cell Line; Cell Membrane; Cytosol; Enzyme Inhibitors; Epithelial Cells; Genistein; Glycerophospholipids; Humans; Isoenzymes; Lysophospholipids; Nasal Cavity; Oligonucleotides, Antisense; Phosphatidic Acids; Phospholipase D; Phosphorylation; Protein Binding; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Proto-Oncogene Proteins pp60(c-src); Respiratory Mucosa; Serine; Sphingosine; Subcellular Fractions; Threonine; Time Factors

Rho GTPases participate in various important signaling pathways and have been implicated in myogenic differentiation. Here the first evidence is provided that in C2C12 myoblasts sphingosine 1-phosphate (SPP) rapidly and transiently induced membrane association of Rho A in a pertussis toxin-insensitive manner. The bioactive lipid preferentially relocalized the GTPase to Golgi-enriched membrane. Translocation of Rho A was abolished by inhibition or down-regulation of protein kinase C (PKC). Notably, treatment with Gö6976, an inhibitor of conventional PKCs, which selectively blocked PKC alpha in these cells, prevented SPP-induced Rho A translocation. Conversely rottlerin, a selective inhibitor of PKC delta, was without effect, demonstrating that SPP signaling to Rho A involves PKC alpha but not PKC delta activation. This novel functional relationship between the two proteins may have a role in SPP-mediated regulation of downstream effectors.

Topics: Acetophenones; Animals; Benzopyrans; Carbazoles; Cell Fractionation; Cell Line; Cell Membrane; Endoplasmic Reticulum; Endosomes; Enzyme Inhibitors; Golgi Apparatus; Indoles; Isoenzymes; Lysophospholipids; Maleimides; Mice; Muscle, Skeletal; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Protein Transport; rhoA GTP-Binding Protein; Sphingosine; Tetradecanoylphorbol Acetate