MEK1 Inhibits Cardiac PPARα Activity by Direct Interaction and Prevents Its Nuclear Localization

BACKGROUND: The response of the postnatal heart to growth and stress stimuli includes activation of a network of signal transduction cascades, including the stress activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK) and the extracellular signal-regulated kinase (ERK1/2) pathways. In response to increased workload, the mitogen-activated protein kinase kinase (MAPKK) MEK1 has been shown to be active. Studies embarking on mitogen-activated protein kinase (MAPK) signaling cascades in the heart have indicated peroxisome-proliferators activated-receptors (PPARs) as downstream effectors that can be regulated by this signaling cascade. Despite the importance of PPARα in controlling cardiac metabolism, little is known about the relationship between MAPK signaling and cardiac PPARα signaling. METHODOLOGY/PRINCIPAL FINDING: Using co-immunoprecipitation and immunofluorescence approaches we show a complex formation of PPARα with MEK1 and not with ERK1/2. Binding of PPARα to MEK1 is mediated via a LXXLL motif and results in translocation from the nucleus towards the cytoplasm, hereby disabling the transcriptional activity of PPARα. Mice subjected to voluntary running-wheel exercise showed increased cardiac MEK1 activation and complex formation with PPARα, subsequently resulting in reduced PPARα activity. Inhibition of MEK1, using U0126, blunted this effect. CONCLUSION: Here we show that activation of the MEK1-ERK1/2 pathway leads to specific inhibition of PPARα transcriptional activity. Furthermore we show that this inhibitory effect is mediated by MEK1, and not by its downstream effector kinase ERK1/2, through a mechanism involving direct binding to PPARα and subsequent stimulation of PPARα export from the nucleus

The MEF2 transcriptional target DMPK induces loss of sarcomere structure and cardiomyopathy

Aims The pathology of heart failure is characterized by poorly contracting and dilated ventricles. At the cellular level, this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres. While it is known that the transcription factor myocyte enhancer factor-2 (MEF2) is involved in this cardiomyocyte remodelling, the underlying mechanism remains to be elucidated. Here, we aim to mechanistically link MEF2 target genes with loss of sarcomeres during cardiomyocyte remodelling. Methods Neonatal rat cardiomyocytes overexpressing MEF2 elongated and lost their sarcomeric structure. We identified and results myotonic dystrophy protein kinase (DMPK) as direct MEF2 target gene involved in this process. Adenoviral overexpression of DMPK E, the isoform upregulated in heart failure, resulted in severe loss of sarcomeres in vitro, and transgenic mice overexpressing DMPK E displayed disruption of sarcomere structure and cardiomyopathy in vivo. Moreover, we found a decreased expression of sarcomeric genes following DMPK E gain-of-function. These genes are targets of the transcription factor serum response factor (SRF) and we found that DMPK E acts as inhibitor of SRF transcriptional activity. Conclusion Our data indicate that MEF2-induced loss of sarcomeres is mediated by DMPK via a decrease in sarcomeric gene expression by interfering with SRF transcriptional activity. Together, these results demonstrate an unexpected role for DMPK as a direct mediator of adverse cardiomyocyte remodelling and heart failure

Conditional dicer gene deletion in the postnatal myocardium provokes spontaneous cardiac remodeling

BACKGROUND: Dicer, an RNAse III endonuclease critical for processing of pre-microRNAs (miRNAs) into mature 22-nucleotide miRNAs, has proven a useful target to dissect the significance of miRNAs biogenesis in mammalian biology. METHODS AND RESULTS: To circumvent the embryonic lethality associated with germline null mutations for Dicer, we triggered conditional Dicer loss through the use of a tamoxifen-inducible Cre recombinase in the postnatal murine myocardium. Targeted Dicer deletion in 3-week-old mice provoked premature death within 1 week accompanied by mild ventricular remodeling and dramatic atrial enlargement. In the adult myocardium, loss of Dicer induced rapid and dramatic biventricular enlargement, accompanied by myocyte hypertrophy, myofiber disarray, ventricular fibrosis, and strong induction of fetal gene transcripts. Comparative miRNA profiling revealed a set of miRNAs that imply causality between miRNA depletion and spontaneous cardiac remodeling. CONCLUSIONS: Overall, these results indicate that modifications in miRNA biogenesis affect both juvenile and adult myocardial morphology and functio

Cooperative Synergy between NFAT and MyoD Regulates Myogenin Expression and Myogenesis*S⃞

Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD, myogenin, Myf5, and MRF4 specify the myogenic lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T cells) signaling is required for primary myogenesis by transcriptional cooperation with the basic helix-loop-helix transcription factor MyoD. Calcineurin/NFAT signaling is involved in myogenin expression in differentiating myoblasts, where the myogenic regulatory factor MyoD synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel shift and chromatin immunoprecipitation assays, we identified two conserved NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon skeletal muscle differentiation. The transcriptional integration between NFATc3 and MyoD is crucial for primary myogenesis in vivo, as myogenin expression is weak in myod:nfatc3 double null embryos, whereas myogenin expression is unaffected in embryos with null mutations for either factor alone. Thus, the combined findings provide a novel transcriptional paradigm for the first steps of myogenesis, where a calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with MyoD during myogenesis

MEK1 interaction with PPARα induces nuclear export.

<p>(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.</p

Inhibition of PPARα by MEK1 relies on the nuclear export of MEK1 and not on MEK1 kinase activity.

<p>(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 <i>mCPT</i> 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 <i>mCPT</i> 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 <i>mCPT</i> 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).</p

MEK1 interacts with PPARα via the LxxLL motif.

<p>(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.</p

Voluntary running-wheel exercise stimulates cardiac MEK1 activation.

<p>(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).</p

MEF2 activates a genetic program promoting chamber dilation and contractile dysfunction in calcineurin-induced heart failure

BACKGROUND: Hypertrophic growth, a risk factor for mortality in heart disease, is driven by reprogramming of cardiac gene expression. Although the transcription factor myocyte enhancer factor-2 (MEF2) is a common end point for several hypertrophic pathways, its precise cardiac gene targets and function in cardiac remodeling remain to be elucidated. METHODS AND RESULTS: We report the existence of synergistic interactions between the nuclear factor of activated T cells and MEF2 transcription factors triggered by calcineurin signaling. To circumvent the embryonic lethality and mitochondrial deficiency associated with germ-line null mutations for MEF2C and MEF2A respectively, we used conditional transgenesis to express a dominant-negative form of MEF2 in the murine postnatal heart and combined this with magnetic resonance imaging to assess MEF2 transcriptional function in Ca2+/calcineurin-induced cardiac remodeling. Surprisingly, end-diastolic and end-systolic ventricular dimensions and contractility were normalized in the presence of severely hypertrophied left ventricular walls on MEF2 inhibition in calcineurin transgenic mice. In line, we generated lines of transgenic mice expressing MEF2A in the heart, which displayed primarily chamber dilation. Microarray profiling indicated that MEF2 promotes a gene profile functioning primarily to or at the nucleus, cytoskeletal and microtubular networks, and mitochondria. CONCLUSIONS: These findings assign a novel function to MEF2 transcription factors in the postnatal heart, where they activate a genetic program that minimally affects cardiac growth yet promotes chamber dilation, mechanical dysfunction, and dilated cardiomyopath

Self-Assembling Peptide Epitopes as Novel Platform for Anticancer Vaccination

The aim of the present study was to improve the immunogenicity of peptide epitope vaccines using novel nanocarriers based on self-assembling materials. Several studies demonstrated that peptide antigens in nanoparticulate form induce stronger immune responses than their soluble forms. However, several issues such as poor loading and risk of inducing T cell anergy due to premature release of antigenic epitopes have challenged the clinical success of such systems. In the present study, we developed two vaccine delivery systems by appending a self-assembling peptide (Ac-AAVVLLLW-COOH) or a thermosensitive polymer poly­(<i>N</i>-isopropylacrylamide (pNIPAm) to the N-terminus of different peptide antigens (OVA_250–264, HPV-E7_43–57) to generate self-assembling peptide epitopes (SAPEs). The obtained results showed that the SAPEs were able to form nanostructures with a diameter from 20 to 200 nm. The SAPEs adjuvanted with CpG induced and expanded antigen-specific CD8⁺ T cells in mice. Furthermore, tumor-bearing mice vaccinated with SAPEs harboring the HPV E7_43–57 peptide showed a delayed tumor growth and an increased survival compared to sham-treated mice. In conclusion, self-assembling peptide based systems increase the immunogenicity of peptide epitope vaccines and therefore warrants further development toward clinical use