Figure 1.
MEK1 expression inhibits PPARα transcriptional activity in an ERK1/2 phosphorylation independent manner.
(a) Luciferase measurements on NkL-Tag cells transiently transfected with a mCPT promoter driven reporter as a functional readout for PPARα activity after co-transfection with PPARα-V5 and MEK1 for 24 hr, and stimulated with Wy-14643 (1 µM) or U0126 (5 µM) as indicated, for 2 hours. (b) Western blot analysis using anti-phosphorylated ERK1/2 (p-ERK1/2) antibody on lysates of NkL-Tag cells transiently transfected with MEK1 for 24 hr, indicating enhanced activation of ERK1/2 after MEK1 expression. (c) Schematic representation of the trans-activating domain of PPARα along with the three putative phosphorylation target sites for ERK1/2 and the LXXLL motif. (d) Luciferase measurements of NkL-Tag cells transiently transfected with a mCPT promoter driven reporter and co-transfected with mutants of PPARα-V5 and MEK1 for 24 hr, and stimulated 2 hours with Wy-14643 (1 µM), indicating MEK1 induced inhibition of PPARα to be ERK1/2 phosphorylation-independent. pGL3-luc construct was transiently transfected as a negative control (white bar).
Figure 2.
Inhibition of PPARα by MEK1 relies on the nuclear export of MEK1 and not on MEK1 kinase activity.
(a) Western blot analysis using anti-phosphorylated ERK1/2 (p-ERK1/2) antibody on lysates of NkL-Tag cells transiently transfected with MEK1 and MKP1, indicating decreased activation of ERK1/2 after co-expression of MKP1. (b) Luciferase measurements of NkL-Tag cells transiently transfected with a mCPT promoter driven reporter and co-transfected with PPARα-V5, MEK1 and MKP1, and stimulated 2 hours with Wy-14643 (1 µM) as indicated. (c) Western blot analysis using anti-ERK1/2 (ERK1/2) antibody on lysates of NkL-Tag cells transiently transfected with siRNAs against ERK1 and ERK2, or scrambled siRNA as a negative control (scr), indicating decreased levels of ERK1/2 after co-transfection of siRNAs targeting ERK1/2. (d) Luciferase measurements of NkL-Tag cells transiently transfected with a mCPT promoter driven reporter and co-transfected with PPARα-V5, MEK1 and siRNAs, and stimulated 2 hours with Wy-14643 (1 µM), as indicated. (e) Luciferase measurements of NkL-Tag cells transiently transfected with a mCPT promoter driven reporter and co-transfected with PPARα-V5, MEK1, MEK1-KD and MEK1-LL, indicating that the inhibition of PPARα by MEK1 relies on the nuclear export of MEK1 and not on MEK1 kinase activity. pGL3-luc construct was transiently transfected as a negative control (white bar).
Figure 3.
MEK1 interaction with PPARα induces nuclear export.
(a) Western blot analysis on precipitates of HEK293 cells transiently co-transfected with PPARα-V5, MEK1 and treated with U0126 (5 µM) or not for 2 hr and immunoprecipitated using an anti-MEK1 antibody. (b) Western blot analysis on precipitates of HEK293 cells transiently co-transfected with PPARα-V5, MEK1 and treated with U0126 (5 µM) as indicated for 2 hr and co-immunoprecipitated using an anti- PPARα antibody. (c) Western blot analysis on precipitates of HEK293 cells transiently transfected with a mutant PPARα-GFPΔ(LxxLL)-V5 expression vector (lacking the LxxLL motif) with or without a MEK1 expression vector and stimulation with Wy-14643 (1 µM) for 2 hr, and immunoprecipitated using an anti-PPARα antibody. (d) Fluorescence immunocytochemistry images of HEK293 cells transiently co-transfected with a PPARα-GFP expression vector, with or without a MEK1 expression vector and stimulation with or without Wy-14643 for 2 hr (1 µM), showing co- cytoplasmic translocation of PPARα and co-localization with MEK1 after co-transfection with MEK1. Addition of U0126 (5 µM) inhibited the MEK1 induced translocation (lower panels). (e) Bar graph indicates mean ± SEM of the percentage of nuclear GFP, showing decreased nuclear PPARα-GFP after co-transfection with MEK1. Addition of U0126 inhibited the MEK1 induced translocation of PPARα-GFP.
Figure 4.
MEK1 interacts with PPARα via the LxxLL motif.
(a) Fluorescence immunocytochemistry images of HEK293 cells transiently co-transfected with a mutant PPARα-GFPΔLxxLL (lacking the LxxLL motif) with or without a MEK1 expression vector and stimulation with or without Wy-14643 (1 µM) for 2 hr. (b) Bar graph indicates mean ± SEM of the percentage of nuclear GFP, showing no significant changes in nuclear PPARα-GFPΔLxxLL after co-transfection with MEK1. (c) Fluorescence immunocytochemistry images of HEK293 cells transiently co-transfected with a PPARβ/δ-GFP with or without a MEK1 expression vector and stimulation with or without the PPARβ/δ-selective agonist GW-510516 (1 µM) for 2 hr. (d) Bar graph indicates mean ± SEM of the percentage of nuclear GFP, showing no significant changes in nuclear PPARβ/δ-GFP after co-transfection with MEK1, indicating that MEK1 does not interact with this PPAR isoform.
Figure 5.
Voluntary running-wheel exercise stimulates cardiac MEK1 activation.
(a) Western blot analysis using anti-PPAR antibodies on lysates of heart samples of indicated experimental procedure, showing reduced PPAR expression of mice hearts subjected to transverse aortic constriction (TAC). (b) Quantification of PPAR protein levels of in hearts from sedentary or exercised mice (n = 6 per group). (c) Quantification of PPAR protein levels of in hearts from sham or transverse aortic constricted mice ( = 6 per group). (d) Average daily distance that mice ran voluntarily. (e) Representative images of hearts from mice that remained sedentary or were subjected to voluntary wheel exercise for 4 weeks. Note the increase in size of the exercised heart. (f) Heart weight to body weight (HW/BW) ratios of mice that remained sedentary or were subjected to voluntary wheel exercise (n = 8 per group). (g) Western blot analysis using anti-phosphorylated ERK1/2 (p-ERK1/2) antibody on lysates of indicated heart samples, demonstrating enhanced MEK1-ERK1/2 activity following exercise-induced cardiac hypertrophy. (h) Quantification of the phosphorylation status of ERK1/2 in hearts from sedentary or exercised mice (n = 6 per group).
Figure 6.
Activation of MEK1 during physiological cardiac hypertrophy inhibits PPARα activity.
(a) Heart weight to body weight (HW/BW) ratios of mice treated with vehicle or U0126, and subjected to voluntary wheel exercise or not (n = 8 per group). (b) Western blot analysis using anti-phosphorylated ERK1/2 (p-ERK1/2) antibody on lysates of heart samples of indicated experimental procedure, showing reduced MEK1-ERK1/2 activity of exercised mice treated with U0126. (c) Western blot analysis using anti-PPARα antibody on nuclear and cytosolic fractions of lysates of heart samples, indicating decreased nuclear PPARα levels and increased cytosolic PPARα levels following exercise-induced cardiac hypertrophy. Treatment with U0126 reduced the MEK1 induced cytoplasmic translocation of PPARα. (d) Western blot analysis using anti- PPARα antibody on precipitates of heart lysates of exercised or sedentary mice, treated with U0126 or vehicle and immunoprecipitated using an anti-MEK1 antibody. (e) Quantification of the co-immunoprecipitated PPAR protein levels in hearts from sedentary or exercised mice (n = 6 per group), treated with U0126 or vehicle. (f) Western blot analysis using anti-MEK1 antibody on precipitates of heart lysates of exercised or sedentary mice, treated with U0126 or vehicle and immunoprecipitated using an anti-PPARα antibody. (g) Quantification of the co-immunoprecipitated MEK1 protein levels in hearts from sedentary or exercised mice, treated with U0126 or vehicle (n = 6 per group). (h) RT-PCR analyses of PPARα target genes expression in sedentary and exercised hearts, treated with vehicle or U0126, indicating decreased PPARα activity during physiological hypertrophy.