Fig. 3.

MKK6, but not MKK3, contributes to high NaCl-induced increase of TonEBP/OREBP transcriptional activity. (A) HEK293 cells stably expressing an ORE-X luciferase reporter of TonEBP/OREBP activity were transfected with control siRNA or siRNA against MKK3 for 32 h. Then the medium was changed, either maintaining osmolality at 290 mosmol/kg or increasing it to 500 mosmol/kg (NaCl added) for 16 h before measuring luciferase activity. (B) As in A, except siRNA was against MKK6 (*P < 0.05 compared with control at 500 mosmol/kg, n = 3). (C) A conditional (Tet-On) vector expressing dominant-negative MKK6-FLAG (dnMKK6) was cotransfected together with an ORE-X reporter of TonEBP/OREBP transcriptional activity into HEK293 cells for 16 h. Then expression of dnMKK6 was induced by adding doxycycline for 24 h before changing the medium, either maintaining osmolality at 290 mosmol/kg or increasing it to 500 mosmol/kg (NaCl added) for 16 h (*P < 0.05 compared with control at 500 mosmol/kg, n = 3). (D) MKK6 contributes to high NaCl-induced increase of TonEBP/OREBP transactivating activity. As in B, except that TonEBP/OREBP transactivating activity was measured in HEK293 cells stably expressing a yeast binary GAL4 reporter assay system (*P < 0.05 compared with respective control, n = 3). (E) MKK6 does not contribute to high NaCl-induced nuclear localization of TonEBP/OREBP. As in B, except that NaCl was increased for 30 min before extracting cytoplasmic and nuclear proteins separately for Western blot analysis of TonEBP/OREBP and calculating its nuclear to cytoplasmic ratio. (F) p38 does not contribute to high NaCl-induced nuclear localization of TonEBP/OREBP. HEK293 cells were preincubated with 0.1% DMSO (Control) or 10 μM SB203580 in 0.1% DMSO for 60 min. Then the medium was changed, either maintaining osmolality at 290 mosmol/kg with the inhibitor or increasing it to 500 mosmol/kg with the inhibitor (NaCl added) for 30 min before measuring the TonEBP/OREBP nuclear to cytoplasmic ratio.