Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ζ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ζ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ζ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1.

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.

Mild acidification caused by transcutaneous administration of carbon dioxide (CO₂) has been reported to improve some epidermal skin impairments, such as desquamation and inflammation; however, its effects on dermal tissue remain unclear. Here, we examined the effect and mechanism of mild acidity on extracellular matrix (ECM) protein production in normal human dermal fibroblasts (NHDFs). To achieve this, the skin permeability of CO₂ and its effect on intradermal pH were evaluated by treating reconstructed human skin equivalents (HSEs) with a CO₂‐containing formulation. Additionally, NHDFs were cultured in a pH‐adjusted medium (pH 6.5). CO₂ successfully permeated HSEs and reduced the intradermal pH. Decreased extracellular pH activated CREB, upregulated TGF‐β1 expression, promoted the production of elastic and collagen fibres, and increased hyaluronan concentration in NHDFs. Additionally, the low pH‐induced increase in TGF‐β1 expression was attenuated via the RNAi‐mediated suppression of the expression of CREB1 and proton‐sensing G protein‐coupled receptors (GPCRs), including GPR4 and GPR65. Moreover, low pH‐induced CREB activation was suppressed by the inhibition of the cAMP/PKA and PLC/PKC signalling pathways. Taken together, a CO₂‐induced decrease in intradermal pH may promote ECM production in NHDFs via the upregulation of TGF‐β1 expression, which was mediated by the activation of the GPCR signalling pathway and CREB, indicating that CO₂ could be used to treat ultraviolet radiation‐induced photoaging, intrinsic ageing and ECM deterioration.