5-Lipoxygenase and cysteinyl leukotriene receptor 1 regulate epidermal growth factor-induced cell migration through Tiam1 upregulation and Rac1 activation

Abstract

Cell migration is an essential step for tumor metastasis. The small GTPase Rac1 plays an important role in cell migration. Previously, we reported that epidermal growth factor (EGF) induced two waves of Rac1 activation; namely, at 5 min and 12 h after stimulation. A second wave of EGF-induced Rac1 activation was required for EGF-induced cell migration, however, the spatiotemporal regulation of the second wave of EGF-induced Rac1 activation remains largely unclear. In this study, we found that 5-lipoxygenase (5-LOX) is activated in the process of EGF-induced cell migration, and that leukotriene C4 (LTC4 ) produced by 5-LOX mediated the second wave of Rac1 activation, as well as cell migration. Furthermore, these effects caused by LTC4 were found to be blocked in the presence of the antagonist of cysteinyl leukotriene receptor 1 (CysLT1). This blockage indicates that LTC4 -mediated CysLT1 signaling regulates the second EGF-induced wave of Rac1 activation. We also found that 5-LOX inhibitors, CysLT1 antagonists and the knockdown of CysLT1 inhibited EGF-induced T cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1) expression. Tiam1 expression is required for the second wave of EGF-induced Rac1 activation in A431 cells. Therefore, our results indicate that the 5-LOX/LTC4 /CysLT1 signaling pathway regulates EGF-induced cell migration by increasing Tiam1 expression, leading to a second wave of Rac1 activation. Thus, CysLT1 may serve as a new molecular target for antimetastatic therapy. In addition, the CysLT1 antagonist, montelukast, which is used clinically for allergy treatment, might have great potential as a novel type of antimetastatic agent.

Keywords: 5-Lipoxygenase; T-cell lymphoma invasion and metastasis-inducing protein 1; cell migration; cysteinyl leukotriene receptor 1; epidermal growth factor signaling.

Figure 1

5-lipoxygenase inhibitors suppress cell migration in A431 cells. (a) Inhibitory activity of BU-4664L (left panel) and AA-861 (right panel) on epidermal growth factor-induced cell migration, monitored using a transwell chamber. The data represent the mean ± SD (n = 5). (b) Evaluation of cytotoxicity of BU-4664L (left panel) and AA-861 (right panel). The data represent the mean ± SD (n = 3).

Figure 2

5-lipoxygenase (5-LOX) inhibitors decrease the second epidermal growth factor (EGF)-induced wave of lamellipodia formation and leukotriene synthesis in A431 cells. (a, b) A431 cells were pretreated with the indicated concentrations of compounds for 15 min and stimulated with EGF. After 5 min (first wave; a) or 12 h (second wave; b), the cells were observed by confocal microscopy. The data represent the mean ± SD (n = 3). (c) A431 cells were pretreated with various concentrations of BU-4664L in the presence or absence of LTB₄, LTC₄ or LTD₄ for 15 min, and stimulated with 30 ng/mL of EGF. After 12 h incubation, cells were observed by confocal microscopy. The data represent the mean ± SD (n = 3). (d) Time course experiments of LTC₄ production induced by EGF. A431 cells were stimulated with EGF for the indicated periods, and then extracellular levels of LTC₄ were measured. The data represent the mean ± SD (n = 3). (e) The effect of 5-LOX inhibitors on the EGF-induced production of LTC₄. A431 cells were pretreated with each inhibitor for 15 min and stimulated with EGF for 12 h. Subsequently, the extracellular LTC₄ were measured. The data represent the mean ± SD (n = 3).

Figure 3

5-lipoxygenase (5-LOX) inhibitors and cysteinyl leukotriene receptor 1 (CysLT1) antagonists inhibit the second epidermal growth factor (EGF)-induced wave of Rac1 activation. The effect of 5-LOX inhibitors (a, b) and CysLT1 antagonists (c, d) on EGF-induced Rac1 activation. A431 cells were pretreated with the indicated concentrations of compounds for 15 min and stimulated with EGF. After 5 min (first wave; a, c) or 12 h (second wave; b, d), the cells were examined for active Rac1 by pull-down assay.

Figure 4

Cysteinyl leukotriene receptor 1 (CysLT1) signaling regulates expression levels of T cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1) in A431 cells. (a, b) Time-course data of epidermal growth factor (EGF)-induced Tiam1 (a) and mRNA (b) in A431 cells. (c) The effect of CysLT1 antagonists on the expression level of Tiam1. A431 cells were treated with the indicated concentrations of inhibitors and stimulated with EGF. After 6 h, the cells were subjected to western blotting using the indicated antibodies. (d) The effect of CysLT1 antagonists on the mRNA level of Tiam1. A431 cells were treated with the indicated concentrations of inhibitors and stimulated with EGF. After 6 h, the cells were subjected to real-time RT-PCR analysis. (e) The effect of 5-lipoxygenase inhibitors and leukotrienes on the expression level of Tiam1. A431 cells were treated with the indicated concentrations of inhibitors and stimulated with EGF. After 6 h, the cells were subjected to western blotting using the indicated antibodies.

Figure 5

Knockdown of cysteinyl leukotriene receptor 1 (CysLT1) inhibits cell migration, as well as lamellipodia formation and Rac1 activation. (a) A431 cells were transfected with control or CysLT1 siRNA and then cultured for 72 h. The cells were collected and subjected to western blotting using the indicated antibodies. (b) Control or CysLT1 siRNA-transfected A431 cells were incubated in the upper chamber and stimulated with or without epidermal growth factor (EGF) for 24 h, and then the migrated cells were counted. The data represent the mean ± SD (n = 5). (c, d) The effect of silencing CysLT1 on lamellipodia formation. Control or CysLT1 siRNA-transfected A431 cells were stimulated with EGF for 5 min (c) or 12 h (d). The cells were then observed under a confocal microscope. (e, f) The effect of silencing CysLT1 on Rac1 activation. Control or CysLT1 siRNA-transfected A431 cells were stimulated with EGF for 2 min (e) or 12 h (f). Subsequently, Rac1-GTP was analyzed by pull-down assay. (g) The effect of silencing CysLT1 on the protein level of T cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1). Control or CysLT1 siRNA-transfected A431 cells were stimulated with EGF for 6 h. The cells were then subjected to western blotting using the indicated antibodies. (h) The effect of silencing CysLT1 on the mRNA level of Tiam1. Control or CysLT1 siRNA-transfected A431 cells were stimulated with EGF for 6 h. The cells were then subjected to real-time RT-PCR analysis.

Figure x6

Schematic illustration of suggested model for the mechanism regulating the second epidermal growth factor (EGF)-induced wave of Rac1 activation. Details are described in the text.