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 (LTD4) in the human promonocytic U937 cells, a cell line known to constitutively express CysLT(1) receptors. Herein, we demonstrate that LTD4 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 Ca2+ ([Ca2+](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 [Ca2+](i) and RasGTP increase, suggesting that in these cells PLC is the point of convergence for both PTx insensitive and sensitive pathways leading to [Ca2+](i) release and Ras activation. Indeed, chelating intracellular Ca2+ strongly (>70%) prevented LTD4-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 LTD4-induced [Ca2+](i) transient (similar to34%) and almost completely prevented Ras activation (>90%), suggesting a potential role for other Ca2+-dependent tyrosine kinases in LTD4-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 (similar to40%) sensitive to Clostridium sordellii lethal toxin and totally blocked by PTx. In conclusion, LTD4-induced CysLT(1) receptor activation in dU937 cells leads to Ras activation and ERK phosphorylation mostly through a PTx-sensitive G(i/0), protein, PLC, and Ca2+-dependent tyrosine kinase(s). (C) 2004 Elsevier Inc. All rights reserved.