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 SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells : Implications for COVID-19 vascular research

Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is impor-tant to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs, pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type II cells. The onset of ACE2 expression is organ specific: in bronchial epithelium already at birth, in brain pericytes before, andin heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modeling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.Peer reviewe

HypoxamiRs : Regulators of cardiac hypoxia and energy metabolism

Hypoxia and its intricate regulation are at the epicenter of cardiovascular research. Mediated by hypoxia-inducible factors as well as by several microRNAs, recently termed 'hypoxamiRs', hypoxia affects several cardiac pathophysiological processes. Hypoxia is the driving force behind the regulation of the characteristic metabolic switch from predominant fatty acid oxidation in the healthy heart to glucose utilization in the failing myocardium, but also instigates reactivation of the fetal gene program, induces the cardiac hypertrophy response, alters extracellular matrix composition, influences mitochondrial biogenesis, and impacts upon myocardial contractility. HypoxamiR regulation adds a new level of complexity to this multitude of hypoxia-mediated effects, rendering the understanding of the hypoxic response a fundamental piece in solving the cardiovascular disease puzzle

HypoxamiRs : Regulators of cardiac hypoxia and energy metabolism

Hypoxia and its intricate regulation are at the epicenter of cardiovascular research. Mediated by hypoxia-inducible factors as well as by several microRNAs, recently termed 'hypoxamiRs', hypoxia affects several cardiac pathophysiological processes. Hypoxia is the driving force behind the regulation of the characteristic metabolic switch from predominant fatty acid oxidation in the healthy heart to glucose utilization in the failing myocardium, but also instigates reactivation of the fetal gene program, induces the cardiac hypertrophy response, alters extracellular matrix composition, influences mitochondrial biogenesis, and impacts upon myocardial contractility. HypoxamiR regulation adds a new level of complexity to this multitude of hypoxia-mediated effects, rendering the understanding of the hypoxic response a fundamental piece in solving the cardiovascular disease puzzle

Epitranscriptomics of cardiovascular diseases (Review)

RNA modifications have recently become the focus of attention due to their extensive regulatory effects in a vast array of cellular networks and signaling pathways. Just as epigenetics is responsible for the imprinting of environmental conditions on a genetic level, epitranscriptomics follows the same principle at the RNA level, but in a more dynamic and sensitive manner. Nevertheless, its impact in the field of cardiovascular disease (CVD) remains largely unexplored. CVD and its associated pathologies remain the leading cause of death in Western populations due to the limited regenerative capacity of the heart. As such, maintenance of cardiac homeostasis is paramount for its physiological function and its capacity to respond to environmental stimuli. In this context, epitranscriptomic modifications offer a novel and promising therapeutic avenue, based on the fine-tuning of regulatory cascades, necessary for cardiac function. This review aimed to provide an overview of the most recent findings of key epitranscriptomic modifications in both coding and non-coding RNAs. Additionally, the methods used for their detection and important associations with genetic variations in the context of CVD were summarized. Current knowledge on cardiac epitranscriptomics, albeit limited still, indicates that the impact of epitranscriptomic editing in the heart, in both physiological and pathological conditions, holds untapped potential for the development of novel targeted therapeutic approaches in a dynamic manner

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

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

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