To further investigate the signaling pathway leading to KLF2 upregulation by TA, the role of the two known upstream signaling molecules in KLF2 regulatory network, namely, ERK5 and MEF2 were assessed7, 25. Previous studies have shown that the transcription factor MEF2 is necessary for the induction of KLF2 by biomechanical and other types of stimuli7, 21, 25, therefore, we first investigated whether MEF2 was required for the TA-mediated increase in KLF2. Using the KLF2 -221 WT Luc promoter fragment which contains a single consensus MEF binding site, the KLF2 inductive effect of TA was totally maintained (Fig. 3A). To assess the importance of this site in TA-mediated induction of the KLF2 luciferase activity, we used the mutant in which MEF2 binding sites was disrupted in the context of the KLF2 -221 WT Luc construct. As shown in Fig. 3A, mutation of the MEF binding site almost completely abolished the TA-mediated induction of the KLF2 luciferase activity. In this experiment, simvastatin was used as a positive control, which could strongly induce KLF2 activity in KLF2 -221 WT Luc promoter plasmid but totally lost in KLF2 -221 mutant Luc promoter plasmid (Fig. 3A). These data implicate that TA induces the KLF2 promoter activity through MEF2 binding.Figure 3The ERK5/MEF2 pathway is involved in TA-induces KLF2 expression. (A) COS-7 cells were transfected with KLF2 -221 WT or KLF2 -221 mutant plasmids, treated with TA (10.0 µM), DMSO (vehicle) or simvastatin (1.0 µM, positive control) for 24 h, and luciferase activities were then detected. *P < 0.05. n = 3. (B–D) HCAECs were pretreated with TA (10.0 µM) for 0, 5, 10, 15, 30 and 60 min, respectively, then protein expression of phospho-ERK5, ERK5 and GAPDH were determined by Western blotting. *P < 0.05. n = 3.