Tiam1 expression is required for the second wave of EGF-induced Rac1 activation in A431 cells.
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.
Thus, our study is the first to demonstrate the involvement of CysLT1 signaling in the mechanism of the second wave of EGF-induced Rac1 activation.
These two waves of lamellipodia formation were reflected by two waves of EGF-induced Rac1 activation, which were observed at 2 5 min and 6 12 h, respectively.
These results indicate that cysteinyl leukotrienes such as LTC4 and LTD4 produced by EGF stimulation mediated the second EGF-induced wave of lamellipodia formation in A431 cells.
However, we cannot yet exclude the possibility that the first EGF-induced wave of Rac1 activation is also involved in the mechanism underlying EGF-induced upregulation of CysLT1.
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.
This conclusion is supported by our findings that not only 5-LO Xinhibitors but also CysLT1 antagonists inhibited the EGF-induced increase of Tiam1 expression, as well as the second EGF-induced wave of Rac1 activation (Figs 4a,b and 5).
It was shown that even 100 ng/ml of EGF did not cause any wave of internalized caveolae from the plasma membrane, or any increased turnover of caveolae at the plasma membrane.
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).
This blockage indicates that LTC4-mediated Cysignalingaling regulates the second EGF-induced wave of Rac1 activation.
