Capra Valérie, Ravasi Saula, Accomazzo Maria Rosa, Parenti Marco, Rovati G Enrico
Laboratory of Molecular Pharmacology, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy.
Biochem Pharmacol. 2004 Apr 15;67(8):1569-77. doi: 10.1016/j.bcp.2003.12.027.
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+)) 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+) 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+) 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+) 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).
我们研究了白三烯D4（LTD4）在人原单核细胞U937细胞中的信号转导途径，该细胞系已知组成性表达半胱氨酰白三烯1（CysLT1）受体。在此，我们证明LTD4特异性作用于CysLT1受体，通过G(i/o)蛋白以剂量依赖性方式增加（比基础水平高3至5倍）RasGTP。事实上，虽然胞质Ca2+（[Ca2+]i）增加仅部分对百日咳毒素（PTx）敏感，但Ras激活几乎完全被同一毒素抑制。此外，磷脂酶C（PLC）抑制剂U73122完全抑制了[Ca2+]i和RasGTP的增加，表明在这些细胞中PLC是导致[Ca2+]i释放和Ras激活的PTx不敏感和敏感途径的汇聚点。确实，螯合细胞内Ca2+强烈（>70%）阻止了LTD4诱导的Ras激活，表明该离子在分化的U937（dU937）细胞中对CysLT1诱导的下游信号传导起重要作用。此外，虽然Src似乎未大量参与CysLT1诱导的信号传导，但染料木黄酮能够部分抑制LTD4诱导的[Ca2+]i瞬变（约34%）并几乎完全阻止Ras激活（>90%），表明其他Ca2+依赖性酪氨酸激酶在LTD4诱导的信号传导中可能起作用。最后，激动剂诱导的CysLT1刺激后伴随着特异性细胞外调节激酶（ERK）1/2磷酸化，这一事件的药理学特征与Ras激活相似，部分（约40%）对索氏梭菌致死毒素敏感并完全被PTx阻断。总之，dU937细胞中LTD4诱导的CysLT1受体激活主要通过PTx敏感的G(i/o)蛋白、PLC和Ca2+依赖性酪氨酸激酶导致Ras激活和ERK磷酸化。
