FIGURE 6.

EGFR-AKT signaling is involved in KLF11-mediated cPLA2α promoter repression.A, FLO cells were co-transfected with the cPLA2α promoter reporter construct along with either EV or KLF11, with or without vErbB (constitutively active EGFR), or CA-AKT. KLF11 overexpression reduced the cPLA2α promoter activity by 63 ± 4.3% (p < 0.05), however, this repression was released in the presence of vErbB and CA-AKT (6 ± 0.9 and 31 ± 7% repression, respectively, p < 0.05 compared with KLF11). B, FLO cells co-transfected with cPLA2α along with either EV or KLF11 were treated with either vehicle or the blockers of EGFR-AKT pathway (10 μm PD168393, 100 μm LY294002, or 1 μm KP372–1). 48 h later, KLF11 decreased the cPLA2α promoter activity in the presence of vehicle by 3.7-fold (100 ± 1.3 versus 27 ± 4.3%), with PD168393 by 10.7-fold (43 ± 6.5 versus 4 ± 1%, p < 0.05 compared with KLF11 with vehicle), with LY294002 by 9.5-fold (29.7 ± 1.6 versus 3 ± 0.9%, p < 0.05 compared with KLF11 with vehicle), and with KP372-1 by 10-fold (10 ± 1 versus 1 ± 0.05%, p < 0.05 compared with KLF11 with vehicle). C, FLO cells were co-transfected with cPLA2α along with either EV or KLF11 and siRNA against AKT or scramble RNA. Cells were maintained in 10% FBS for 48 h. KLF11 decreased cPLA2α promoter activity by 12-fold (73 ± 1 versus 6 ± 1%) in the presence of AKT siRNA compared with a 3.7-fold reduction (100 ± 1.3 versus 27 ± 4.3%) with scramble siRNA (p < 0.05). D, FLO cells transfected with either cPLA2α-WT promoter or cPLA2α-SDM2 (mutation in GC-rich sequence to which KLF11 binds). Cells were treated with vehicle, 10 μm PD168393, or 100 μm LY294002 or both PD168393 and LY294002 for 24 h in 10% FBS. PD168393 decreased the cPLA2α-WT promoter activity by 66% (100 ± 10 versus 44 ± 6%, p < 0.05) but had no significant effect on the cPLA2α-SDM2 promoter activity (100 ± 33 versus 89 ± 35%). A similar pattern was also noted with LY294002 alone or with both PD168393 and LY294002. E, KLF11-transfected FLO cells were treated with either vehicle or 10 μm PD168393 plus 100 μm LY294002 for 24 h in the presence of 10% FBS. Chromatin immunoprecipitation with anti-Sin3a antibody showed a slight increase in cPLA2α promoter enrichment in the blocker-treated group. To compliment this, FLO cells were either co-transfected with KLF11 and AKT siRNA (or scramble RNA control) or treated with 10 μm PD168393 plus 100 μm LY294002 (or vehicle control). Western blots after immunoprecipitation of His-tagged KLF11 followed by probing with the anti-Sin3a antibody shows that compared with control there was increased KLF11-Sin3a complexing in AKT blockers as well as AKT-siRNA-treated cells compared with the control. DMSO, dimethyl sulfoxide.