CysLT1 signal transduction in differentiated U937 cells involves the activation of the small GTP-binding protein Ras.
We investigated the signal transduction pathway(s) of leukotriene D(4) (LTD(4)) in the human promonocytic U937 cells, a cell line known to constitutively express CysLT(1) receptors. Herein, we demonstrate that LTD(4) specifically acts on a CysLT(1) receptor to dose-dependently increase (three to five-fold over basal) RasGTP through a G(i/o) protein. In fact, while cytosolic Ca(2+) ([Ca(2+)](i)) increase was only partially sensitive to pertussis toxin (PTx), Ras activation was almost completely inhibited by the same toxin. Furthermore, the phospholipase C (PLC) inhibitor U73122 completely inhibited both [Ca(2+)](i) and RasGTP increase, suggesting that in these cells PLC is the point of convergence for both PTx insensitive and sensitive pathways leading to [Ca(2+)](i) release and Ras activation. Indeed, chelating intracellular Ca(2+) strongly (>70%) prevented LTD(4)-induced Ras activation, indicating that this ion plays an essential role for CysLT(1)-induced downstream signaling in differentiated U937 (dU937) cells. In addition, while Src did not appear to be substantially involved in CysLT(1)-induced signaling, genistein was able to partially inhibit LTD(4)-induced [Ca(2+)](i) transient ( approximately 34%) and almost completely prevented Ras activation (>90%), suggesting a potential role for other Ca(2+)-dependent tyrosine kinases in LTD(4)-induced signaling. Finally, agonist-induced CysLT(1) stimulation was followed by a specific extracellular regulated kinase (ERK) 1/2 phosphorylation, an event with a pharmacological profile similar to that of Ras activation, partially ( approximately 40%) sensitive to Clostridium sordellii lethal toxin and totally blocked by PTx. In conclusion, LTD(4)-induced CysLT(1) receptor activation in dU937 cells leads to Ras activation and ERK phosphorylation mostly through a PTx-sensitive G(i/o) protein, PLC, and Ca(2+)-dependent tyrosine kinase(s).
Capra
Valérie
V
Laboratory of Molecular Pharmacology, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. GEnrico.Rovati@unimi.it
Ravasi
Saula
S
Accomazzo
Maria Rosa
MR
Parenti
Marco
M
Rovati
G Enrico
GE
D002118:Calcium / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation; D004963:Estrenes / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology*; D017998:Leukotriene D4 / Q000494:pharmacology; D015289:Leukotrienes / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D018077:Receptors, Leukotriene / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D020298:U937 Cells; D018631:ras Proteins / Q000378:metabolism*
C412373:AG 1879; D004963:Estrenes; D018836:Inflammation Mediators; D015289:Leukotrienes; D008565:Membrane Proteins; D011743:Pyrimidines; D011760:Pyrrolidinones; D018077:Receptors, Leukotriene; C112381:cysteinyl-leukotriene; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D004533:Egtazic Acid; D017998:Leukotriene D4; D019833:Genistein; D037342:Pertussis Toxin; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; D018631:ras Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D003545:Cysteine; C083169:leukotriene D4 receptor; D002118:Calcium