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

Magi S, Takemoto Y, Kobayashi H, Kasamatsu M, Akita T, Tanaka A, Takano K, Tashiro E, Igarashi Y, Imoto M - Cancer Sci. (2014)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama, Japan.

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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.
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fig05: 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.

Mentions: Finally, we confirmed the involvement of CysLT1 signaling in EGF-induced Tiam1 expression by using RNAi experiments. The successful knockdown of CysLT1 by siRNA was confirmed by western blotting (Fig.5a). Silencing of CysLT1 expression consequently suppressed not only EGF-induced Tiam1 expression at both mRNA and protein levels, but also the second EGF-induced wave of Rac1 activation, lamellipodia formation and cell migration (Fig.5b,d,f–h). In contrast, silencing of CysLT1 had little effect on the first EGF-induced wave of Rac1 activation and the corresponding lamellipodia formation (Fig.5c,e). These results indicate that 5-LOX/LTC4(D4)/CysLT1 signaling regulated EGF-induced expression of Tiam1, resulting in the second EGF-induced wave of Rac1 activation and cell migration.


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

Magi S, Takemoto Y, Kobayashi H, Kasamatsu M, Akita T, Tanaka A, Takano K, Tashiro E, Igarashi Y, Imoto M - Cancer Sci. (2014)

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.
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fig05: 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.
Mentions: Finally, we confirmed the involvement of CysLT1 signaling in EGF-induced Tiam1 expression by using RNAi experiments. The successful knockdown of CysLT1 by siRNA was confirmed by western blotting (Fig.5a). Silencing of CysLT1 expression consequently suppressed not only EGF-induced Tiam1 expression at both mRNA and protein levels, but also the second EGF-induced wave of Rac1 activation, lamellipodia formation and cell migration (Fig.5b,d,f–h). In contrast, silencing of CysLT1 had little effect on the first EGF-induced wave of Rac1 activation and the corresponding lamellipodia formation (Fig.5c,e). These results indicate that 5-LOX/LTC4(D4)/CysLT1 signaling regulated EGF-induced expression of Tiam1, resulting in the second EGF-induced wave of Rac1 activation and cell migration.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama, Japan.

Show MeSH
Related in: MedlinePlus