Regulation of c-jun mRNA expression in adult cardiocytes by MAP kinase interacting kinase-1 (MNK1) (original) (raw)
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Circulation Research
The increased expression of immediate-early genes is a key feature of the myocardial response to hypertrophic stimuli. In this study, we investigated whether pressure overload or phenylephrine treatment stimulated myocyte enhancer factor 2 (MEF2)-dependent transcriptional activation of c-jun in cardiac myocytes. Western blotting and immunohistochemical analysis of rat myocardium demonstrated that p70 MEF2 is highly expressed in the rat heart and is predominantly located at the nuclei of cardiac myocytes. Electrophoretic mobility shift assays of myocardial nuclear extracts revealed a consistent DNA binding activation of MEF2 after 1 and 2 hours of pressure overload. We further showed that pressure overload induced a progressive nuclear translocation and activation of extracellular signalregulated kinase 5 (ERK5). Coimmunoprecipitation and in vitro kinase assays indicated that the activation of ERK5 was paralleled by increased association of ERK5/p70 MEF2 and by enhanced ability of ERK5 to phosphorylate p70 MEF2. Experiments with in vivo transfection of the left ventricle with the c-jun promoter reporter gene showed that pressure overload induced a consistent increase of c-jun transcriptional activity in the rat myocardium. Rendering the MEF2 site of the c-jun plasmid inactive by mutation abolished the load-induced activation of the c-jun promoter reporter gene. Mutation of the MEF2 site also abolished the phenylephrine-induced c-jun promoter activation in neonatal rat ventricular myocytes. In addition, we demonstrated that neonatal rat ventricular myocyte transfection with ERK5antisense oligodeoxynucleotide inhibited the phenylephrine-induced c-jun promoter activation. These findings identify MEF2 as a potential regulator of c-jun transactivation and suggest that ERK5 might be an important mediator of MEF2 and c-jun promoter activation in response to hypertrophic stimuli in cardiac myocytes. (Circ Res. 2003;92:243-251.
The increased expression of immediate-early genes is a key feature of the myocardial response to hypertrophic stimuli. In this study, we investigated whether pressure overload or phenylephrine treatment stimulated myocyte enhancer factor 2 (MEF2)-dependent transcriptional activation of c-jun in cardiac myocytes. Western blotting and immunohistochemical analysis of rat myocardium demonstrated that p70 MEF2 is highly expressed in the rat heart and is predominantly located at the nuclei of cardiac myocytes. Electrophoretic mobility shift assays of myocardial nuclear extracts revealed a consistent DNA binding activation of MEF2 after 1 and 2 hours of pressure overload. We further showed that pressure overload induced a progressive nuclear translocation and activation of extracellular signalregulated kinase 5 (ERK5). Coimmunoprecipitation and in vitro kinase assays indicated that the activation of ERK5 was paralleled by increased association of ERK5/p70 MEF2 and by enhanced ability of ERK5 to phosphorylate p70 MEF2 . Experiments with in vivo transfection of the left ventricle with the c-jun promoter reporter gene showed that pressure overload induced a consistent increase of c-jun transcriptional activity in the rat myocardium. Rendering the MEF2 site of the c-jun plasmid inactive by mutation abolished the load-induced activation of the c-jun promoter reporter gene. Mutation of the MEF2 site also abolished the phenylephrine-induced c-jun promoter activation in neonatal rat ventricular myocytes. In addition, we demonstrated that neonatal rat ventricular myocyte transfection with ERK5antisense oligodeoxynucleotide inhibited the phenylephrine-induced c-jun promoter activation. These findings identify MEF2 as a potential regulator of c-jun transactivation and suggest that ERK5 might be an important mediator of MEF2 and c-jun promoter activation in response to hypertrophic stimuli in cardiac myocytes. (Circ Res. 2003;92:243-251.) Key Words: pressure overload Ⅲ transcription factors Ⅲ myocyte enhancer factor 2 Ⅲ c-jun Ⅲ extracellular signal-regulated kinase 5
Cardiovascular Research, 2006
Objective: Mice deficient for the AP-1 transcription factor JunD, the only Jun protein constitutively expressed and clearly detectable in the mammalian heart, develop enhanced cardiac hypertrophy in response to chronic pressure overload. Catecholamines inducing a-adrenergic receptor-mediated signaling have been implicated in the neurohumoral response to pressure overload and the development of left ventricular hypertrophy. In the present study we analyzed the mechanistic role of JunD in cardiomyocyte hypertrophy in vitro in response to aadrenergic agonist phenylephrine (PE). Methods: Cardiomyocytes were isolated from 1-to 3-day-old rats and transfected with adenoviruses expressing LacZ or wild-type JunD, or with expression vectors encoding LacZ, wild-type JunD, mutated JunD forming only JunD homodimers (JunDeb1), mutated JunD lacking the JNK site (JunD-D162), or c-Jun. After stimulation with PE (10 À 5 mol/L), hypertrophic growth of cardiomyocytes (cross-sectional area and [ 3 H]-leucine incorporation) and mRNA expression of JunD, c-Jun, c-Fos, and atrial natriuretic peptide (ANP) were analyzed. Transcriptional activation was determined by luciferase activity in cardiomyocytes transfected with AP-1 or ANP luciferase reporter plasmids. Gel shift assays with an AP-1 consensus oligonucleotide were performed to analyze AP-1 DNA binding activities. Results: PE augmented mRNA levels of c-Jun and c-Fos, but decreased JunD transcript levels. Adenoviral over-expression of wild-type JunD blunted PE-induced hypertrophic growth and expression of ANP mRNA. Over-expression of JunD in cardiomyocytes caused enhanced AP-1 protein -DNA binding, without increasing the transcriptional response from AP-1 or ANP luciferase reporter plasmids at baseline or upon PE stimulation. Moreover, over-expression of JunDeb1 attenuated transcription from AP-1 or ANP luciferase reporter plasmids and blunted c-Jun-mediated acceleration of AP-1 transcriptional activity at baseline and in response to PE. Conclusions: Our observations establish a novel role for JunD as a negative regulator of cardiomyocyte hypertrophy in response to hypertrophic stimuli by inhibiting AP-1 transcriptional activity.
American Journal of Physiology-Heart and Circulatory Physiology, 2004
The transient increase in the expression of transcription factors encoded by immediate-early genes has been considered to play a critical role in the coordination of early gene expression during the hypertrophic growth of cardiac myocytes. Here, we investigated the regulation of c-Jun and its upstream activators JNKs in the myocardium of rats subjected to acute pressure overload induced by transverse aortic constriction. Western blotting and immunohistochemistry analysis demonstrated that both JNK1 and JNK2 were transiently activated by pressure overload, but only JNK1 was activated at the nuclei of cardiac myocytes. JNK1 activation was paralleled by phosphorylation of c-Jun at serine-63 in the myocardial nuclear fraction and by an increase in c-Jun expression in cardiac myocytes. A consistent increase in DNA binding of activator protein-1 (AP-1) complex was observed after 10 and 30 min of pressure overload and Supershift assays confirmed that c-Jun was a major component of activate...
Journal of Biological Chemistry, 2006
Microsomal prostaglandin (PG) E 2 synthase-1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE 2 , a key proinflammatory mediator. The purpose of this study was to elucidate the regulation of mPGES-1 mRNA expression in cardiomyocytes, define the role of JNK enzymes in this process, and characterize the role of mPGES-1 in cardiomyocyte PGE 2 biosynthesis. In neonatal cardiomyocytes, interleukin-1 and lipopolysaccharide (LPS) both stimulated mPGES-1 mRNA expression and increased mPGES-1 mRNA stability and protein synthesis but failed to increase mPGES-1 mRNA transcription. Treatment with the JNK1/2 inhibitor, SP600125, abrogated the increases in mPGES-1 mRNA stability, mPGES-1 protein synthesis, and PGE 2 release induced by interleukin-1 or LPS. mPGES-1 protein synthesis was observed in LPS-stimulated neonatal cardiomyocytes from jnk1 ؊/؊ or jnk2 ؊/؊ mice. In contrast, infection of jnk1 ؊/؊ cardiomyocytes with an adenovirus encoding phosphorylation-resistant JNK2 (ad-JNK2-DN), or of jnk2 ؊/؊ cardiomyocytes with ad-JNK1-DN, significantly decreased LPS-stimulated mPGES-1 protein synthesis. Similarly, co-infection with ad-JNK1-DN and ad-JNK2-DN attenuated LPS-stimulated mPGES-1 protein synthesis in cardiomyocytes from wild type mice. Targeted deletion of the gene encoding mPGES-1 led to a 3.2-fold decrease in LPS-stimulated PGE 2 release by cardiomyocytes in comparison with wild type cells but had no effect on COX-1, COX-2, mPGES-2, or cytosolic PGES mRNA levels. These studies provide direct evidence that mPGES-1 mRNA levels in cardiomyocytes are augmented by stabilization of mPGES-1 mRNA, that JNK1 or JNK2 can participate in the regulation of mPGES-1 protein synthesis in these cells, and that mPGES-1 catalyzes the majority of LPS-induced PGE 2 biosynthesis by cardiomyocytes.
Cardiovascular Toxicology, 2014
Sunitinib (SUN) is a multi-targeted tyrosine kinase inhibitor used for the treatment of gastrointestinal stromal tumors and renal cell carcinoma. Cardiotoxicity has been reported as a significant side effect associated with the SUN treatment, yet the mechanism is poorly understood. The main purpose of this study was to investigate the potential effects of SUN on cardiac hypertrophic genes and the role of mitogen-activated protein kinases (MAPKs) signaling pathway in rat cardiomyocyte H9c2 cell line. In the present study, real-time quantitative polymerase chain reaction showed that the treatment of H9c2 cells with increasing concentrations of SUN (0, 1, 2.5, and 5 lM) significantly induced hypertrophic gene markers, such as brain natriuretic peptides (BNP) and myosin heavy chain (b-MHC and a-MHC) in concentration-and timedependent manners. The onset of mRNA induction was observed as early as 9 h and remained elevated for at least 18 h after treatment with SUN 5 lM. At the protein level, Western blot analysis showed that SUN increased BNP and b-MHC, while it inhibited a-MHC protein levels in a concentration-dependent manner. These SUN-mediated effects were associated with increase in cell size and hypertrophy by approximately 70 % at the highest concentration, 5 lM. Importantly, inhibition of the MAPK signaling pathway using SB203580 (p38 MAPK inhibitor), U0126 (extracellular signal-regulated kinase inhibitor), and SP600125 (c-Jun NH 2 -terminal kinase inhibitor) significantly potentiated the SUN-induced BNP and b-MHC mRNA levels, but did alter the a-MHC level. Whereas at the protein level, MAPK inhibitors generally decreased the SUN-induced BNP, whereas only SB and U0 increased b-MHC protein levels with no effect on a-MHC, which were associated with a significant decrease in cell size. Together, these results indicate that SUN induced hypertrophic gene expression through MAPK-dependent mechanisms.
Induction and nuclear accumulation of fos and jun proto-oncogenes in hypoxic cardiac myocytes
Journal of Biological Chemistry, 1993
Hypoxic and ischemic stresses cause a series of well documented changes in myocardial cells and tissues, including increased anaerobic glycolysis, loss of contractility, changes in lipid and fatty acid metabolism, and eventual irreversible membrane damage and cell death. In this article we describe changes in the expression and regulation of the proto-oncogenes fos and jun in cardiac myocytes exposed to severe hypoxia. The mRNAs encoding C-Fos, c-Jun, Jun-D, and Jun-B were induced within 1 h of exposure to hypoxia, increased 6-10-fold between 1 and 4 h and then declined. These inductions coincided with loss in myocyte contractility but occurred before there was irreversible cell damage or significant ATP loss. Immunostaining with anti-Fos and anti-Jun antibodies revealed the accumulation of these proteins in hypoxic cell nuclei. Pre-treatment of cells with protein kinase inhibitors significantly repressed the response at the mRNA level. We propose that hypoxic stress in these cells activates signal transduction pathways, possibly involving protein kinases, that result in the inductions of fos and jun gene families. Therefore APl may regulate myocardial adaptive responses to hypoxia in advance of energy depletion, cell damage, or reoxygenation. The proto-oncogenes fos and jun are members of multigene families that are induced rapidly, and usually transiently, in many cell types in response to a range of diverse stimuli (reviewed in Refs. 1-3). The c-Fos and c-Jun proteins are components of the AP-1 transcription factor complex that is involved in the transcriptional regulation of a number of genes (reviewed in Refs. 4-6). It is assumed that transcription regulation, associated with adaptive responses, is an integral part of the cellular roles of these proteins. Transcriptional regulation of fos and jun genes themselves is complex. Fos/ Jun proteins have been referred to as third messengers that respond to the second messenger of a primary, usually receptor associated stimulus, perhaps the best understood pathway being the phorbol ester or diacylglycerol-protein kinase C (PKC)' pathway (7,8).