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Cardiovascular Research 2006 71(1):108-117; doi:10.1016/j.cardiores.2006.02.032
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Copyright © 2006, European Society of Cardiology

JunD attenuates phenylephrine-mediated cardiomyocyte hypertrophy by negatively regulating AP-1 transcriptional activity

Denise Hilfiker-Kleinera,*, Andres Hilfikerb, Marc Castellazzic, Kai C. Wollerta, Christian Trautweind, Heribert Schunkerte and Helmut Drexlera

aDepartments of Cardiology and Angiology, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany
bCardiovascular Surgery, Hannover Medical School, Germany
cInserm U-758, IFR128 Lyon-Gerland, France
dUniversitätsklinikum RWTH Aachen, Germany
eMedical University Luebeck, Luebeck, Germany

* Corresponding author. Tel.: +49 511 532 2531; fax: +49 511 532 3263. Email address: hilfiker.denise{at}mh-hannover.de

Received 11 November 2005; revised 11 February 2006; accepted 28 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
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 {alpha}-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 {alpha}-adrenergic 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-{delta}162), or c-Jun. After stimulation with PE (10– 5mol/L), hypertrophic growth of cardiomyocytes (cross-sectional area and [3H]-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.

KEYWORDS Adrenergic agonists; Hypertrophy; Signal transduction; Cardiomyocyte


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
It has previously been reported that human and murine adult myocardium constitutively expresses high levels of the AP-1 transcription factor JunD in cardiomyocytes and non-myocytes [1-3]. Under pathophysiologic stress, such as chronic pressure overload or high levels of tumor necrosis factor-{alpha} cardiac JunD protein levels are reduced [3,4]. Moreover, JunD mRNA and protein levels are also reduced in the failing human myocardium [1,4]. We have recently shown that the lack of JunD in mice is associated with enhanced cardiac hypertrophy and cardiomyocyte growth in response to chronic pressure overload [4], suggesting that JunD may act as an endogenous inhibitor of cardiomyocyte hypertrophy.

The pro-hypertrophic effect of the {alpha}-adrenergic agonist phenylephrine (PE) on cardiomyocytes, is mediated, in part, via c-Jun NH2-terminal kinases (JNK), which phosphorylate c-Jun, thus allowing the formation of transcriptionally active AP-1 complexes, which in turn promote cardiomyocyte growth and induction of a fetal gene expression program [5-7]. AP-1 transcriptional complexes are composed of Jun proteins (c-Jun, JunB and JunD) that can form homo- or heterodimers with Fos and ATF proteins [8]. Jun proteins share a high degree of sequence homology, but they often exert opposing effects on cell growth, division, and survival. In this regard, c-Jun has been shown to promote proliferation, dedifferentiation and hypertrophy, while JunD has been associated with anti-proliferative effects, differentiation, and inhibition of hypertrophy [4,9].

In the present study the expression of JunD, its role in AP-1 transcriptional regulation, and its function during cardiomyocyte hypertrophy in response to {alpha}-adrenergic receptor stimulation with PE was investigated in cardiomyocytes in vitro.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
Cell culture media were obtained from Biochrome, all other chemicals from Sigma.

2.1 Cell culture and transfection methods
Cardiomyocytes were isolated from 1- to 3-day-old Sprague–Dawley rats as described [10]. Cells were first cultured for 24h in DMEM/medium 199 (4:1), supplemented with 10% horse serum, 5% FCS, glutamine, and antibiotics. Transfections with expression vectors containing LacZ or wild-type JunD, JunD-{delta}162 [11] or JunDeb1 [12], all sub-cloned in the p-Shuttle CMV expression vector (Quantum Appligene), the full-length c-jun cDNA [8] and luciferase-reporter plasmids (AP-1 7 x, Stratagene; ANP luc [13]; pANPluc-3418mutAP-1 [14]) were carried out with Lipofectamin (Invitrogen). Subsequently, cardiomyocytes were switched to DMEM/medium 199 without serum and were stimulated 24h later as indicated. We have tested the expression efficiency for each plasmid by transfecting 293 HEK cells with 2µg of each expression plasmid. Quantitative assessments using Western blot analysis confirmed a 20-fold increase in JunD and JunDeb1 protein levels and a 10-fold increase in c-Jun protein compared to the baseline expression of JunD, respectively c-Jun.

For adenoviral infections, cardiomyocytes (DMEM/M199 supplemented with 5% FCS) were infected for 3h at 37°C at the indicated p.f.u. After infection, cells were washed three times with DMEM/M199 supplemented with 5% FCS and then cultured in serum-free DMEM/M199 for additional 24h to 48h as indicated, before morphological or biochemical analysis.

All animal studies were in compliance with the Guide for the Care and Use of Laboratory Animals as published by the U.S. National Institutes of Health and were approved by the local animal care committees.

2.2 Assessment of cardiomyocyte hypertrophy and survival
Cultured cardiomyocytes were seeded in 6-well plates and transfected with the indicated adenovirus. After 48h, cardiomyocyte surface area was determined by planimetry with a phase contrast microscope and digital image analyzer (Leica Q500 MC). [3H] leucine incorporation was measured as an index of total protein accumulation. Cardiomyocytes survival was analyzed 72h after transfection by the ability of cells to exclude trypan blue (1:1 diluted in 1 x PBS). Number of blue and white cardiomyocytes were counted and the ratio of blue to white cardiomyocytes in AdLacZ-CM were set at 100%. Experiments were repeated with three different cell preparations.

2.3 Recombinant adenoviruses
The entire coding region of the wild-type rat JunD gene was amplified by RT-PCR using the primer pair: 5'-TCTGTACGGGCAGCGGAC-3' and 5'-CAGAGTAAAGGGGGGTCCAGC-3' (GenBank D26307 [GenBank] ), resulting in the amplification of a 1233bp fragment which was subcloned and sequenced. For construction of recombinant adenoviruses expressing JunD protein (AdJunD) the AdEasy Kit (Quantum Appligene) was used. An adenovirus expressing the bacterial LacZ gene (AdLacZ, Quantum Appligene) served as control. Plaque forming units (p.f.u.) were determined in agarose covered QBI-293A cells. Infections of cardiomyocytes with 10p.f.u. AdLacZ resulted in almost 99% of cardiomyocytes positive for LacZ expression (data not shown) confirming high infection rates. Infection of cardiomyocytes with 10p.f.u. to 20p.f.u. of AdJunD resulted in a 10- to 20-fold increase in JunD protein content when compared with similar amount of p.f.u. AdLacZ transfected cardiomyocytes. Maximal JunD protein expression was reached between 24h and 48h after infection (Fig. 2A). Therefore, all following infection experiments were carried out with 10p.f.u. AdLacZ or 10p.f.u. AdJunD and stimulations were started 48h after adenoviral infection.


Figure 2
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  		Fig. 2 Over-expression of JunD in cardiomyocytes in vitro. (A) Western blot showing JunD protein levels in cardiomyocytes 24h after infection with increasing p.f.u. of AdLacZ or AdJunD adenovirus. Actin staining indicates equal loading. Western blot showing time course analysis of JunD protein levels in cardiomyocytes transfected with AdJunD in comparison with time matched non-transfected cardiomyocytes. (B) Cardiomyocytes transfected with AdLacZ (LacZ) or AdJunD (JunD) for 24h, 48h or 72h. (C) Cell surface area of at least 200 cardiomyocytes and [3H] leucine incorporation of three individual cell preparations are summarized in the bar graphs. (D) Northern blot and bar graph showing ANP mRNA expression of AdLacZ-CM or AdJunD-CM, without (control) or with addition of PE (10– 5mol/L) for 48h. Experiments shown in A, B, and D were performed in triplicate using at least three different cardiomyocyte preparations. *P<0.05, **P<0.01 versus control AdLacZ-CM; #P<0.05, PE stimulated AdJunD-CM versus PE stimulated AdLacZ-CM.

 
2.4 RT-PCR, Northern, and immunoblot analyses
RT-PCR, Northern blotting and immunoblotting were performed according to standard procedures [15]. cDNA probes for atrial natriuretic peptide (ANP) [13] and for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) [3] have been described previously. Antibodies against JunD and c-Jun were from Santa Cruz [4]. Primer pairs used for RT-PCR of JunD, c-Jun, c-Fos, and G3PDH were:
JunD: 5'-ACACGCAGGAACGCATC-3' 5'-ATCCAGAGTAAAGGGGGGTC-3'
c-Jun: 5'-CTTCTACGACGATGCCCTC-3' 5'-GTGCCACCTGTTCCCTG-3'
c-Fos: 5'-GTTCTCGGGTTTCAACGC-3' 5'-AGTCCAGGGAGGTCACAGAC-3'
G3PDH: 5'-ACCACCATGGAGAAGGCTGG-3' 5'-CTCAGTGTAGCCCAGGATGC-3'

2.5 Nuclear extracts and gel shift experiments
Preparation of protein extracts from cardiomyocytes and gel shift experiments were performed essentially as described [3]. An AP-1 consensus oligonucleotide (Promega) was end-labeled with [{gamma}32P] ATP using T4 polynucleotide kinase (NEB). For supershift assays, 1µg/µl antibodies against JunD, c-Jun, or unspecific IgG (Santa Cruz) were added to the reactions. Protein–DNA complexes were resolved on 4% nondenaturing polyacrylamide gels and visualized by autoradiography using a PhosphoImager (BAS 1000, Fuji).

2.6 Statistical analysis
All data are given as mean±S.D. Differences between groups were evaluated by Student's t-test or ANOVA followed by Bonferoni as appropriate. Statistical significance was defined as P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 {alpha}-Adrenergic agonist phenylephrine lowers expression of JunD in cardiomyocytes
Cardiomyocyte hypertrophy can be induced in cultured cardiomyocytes by treatment with a number of trophic effectors including peptide growth factors and the {alpha}-adrenergic agonist phenylephrine (PE) [7,16]. We have investigated the expression of AP-1 transcription factors JunD, c-Jun, and c-Fos in cardiomyocytes exposed to PE (10– 5mol/L). As described by others [17], transcript levels of c-Jun and c-Fos were rapidly and markedly induced in cardiomyocytes following stimulation with PE (Fig. 1A and B). In contrast, JunD transcript levels were not altered 30min and 1h after PE stimulation and were slightly but significantly decreased (PE 3h: – 39%±25% versus unstimulated control, P<0.05) 3h after PE treatment (Fig. 1C).


Figure 1
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  		Fig. 1 Expression analysis of c-Jun, c-Fos and JunD. Time course analysis of c-Jun (A), c-Fos (B) and JunD (C) expression at the mRNA level assessed by RT-PCR in cardiomyocytes exposed to phenylephrine (PE: 10– 5mol/L). Experiments shown in A, B, and C were performed in triplicate from three individual cell preparations (*P<0.05 versus 0h).

 
3.2 Over-expression of JunD has no effect on survival of cardiomyocytes
Infection of cardiomyocytes with AdJunD resulted in a 10- to 20-fold increase in JunD protein content between 24h and 48h after infection when compared with AdLacZ (Fig. 2A). Since we observed just one type of JunD mRNA by Northern blot we assume that the profile of proteins detected by the JunD antibodies may arise from posttranslational modifications. In fact, posttranslational modifications of JunD protein, generating two or more proteins of different migration properties, have been described by other investigators for example in JURKAT cells [18]. It is not known, however, whether these different JunD isoforms have distinguished functions. Adenoviral over-expression of JunD had no effect on the survival of cardiomyocytes since the number of cells staining positive for trypan blue as a marker for cell death was similar in cardiomyocytes 72h after transfection with AdJunD or with AdLacZ (data not shown). In addition, AdJunD had no significant effect on cardiomyocyte hypertrophy and ANP expression in unstimulated cardiomyocytes (Fig. 2B to D).

3.3 Over-expression of JunD attenuates PE mediated cardiomyocyte hypertrophy
In order to investigate the role of JunD for PE mediated cardiomyocyte hypertrophy, cardiomyocytes were transfected with AdJunD (AdJunD-CM) or AdLacZ (AdLacZ-CM) 24h prior stimulation with PE (10– 5mol/L, for 48h). PE induced an increase in cardiomyocytes cell surface area and [3H]-leucine incorporation in AdLacZ-CM while in AdJunD-CM the PE mediated increase in cell surface area (Fig. 2B) and [3H]-leucine incorporation was attenuated (Fig. 2C), indicating a lower rate of protein synthesis in AdJunD-CM as compared with AdLacZ-CM. In addition, the PE mediated increase in ANP mRNA expression was attenuated in AdJunD-CM compared with AdLacZ-CM (Fig. 2D).

3.4 Over-expressed JunD protein binds to the AP-1 motif in cardiomyocytes independent of PE stimulation
Nuclear extracts from AdJunD-CM but not from AdLacZ-CM showed enhanced binding activities to the AP-1 consensus oligonucleotide under basal conditions (Fig. 3A). The DNA–protein complex contained JunD, but not c-Jun protein as shown by supershift experiments with their respective antibodies (Fig. 3B). PE strongly increased AP-1 DNA–protein binding in AdLacZ-CM, while in AdJunD-CM PE did not further enhance DNA–protein binding (Fig. 3A). c-Jun, but not JunD was supershifted from AP-1 DNA–protein complex in PE stimulated AdLacZ-CM (Fig. 3A). In contrast, JunD, but not c-Jun, was supershifted from the AP-1 DNA–protein complex in PE stimulated AdJunD-CM (Fig. 3A).


Figure 3
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  		Fig. 3 Adenoviral over-expression of JunD induces DNA–protein binding to an AP-1 consensus oligonucleotide. (A) Representative gel shifts showing DNA–protein binding activities to a consensus AP-1 oligonucleotide in protein extracts from AdLacZ-CM or AdJunD-CM without (control, C) or with addition of PE for 24h. Supershifts were performed with anti-JunD, anti-c-Jun antibodies or unspecific IgG. For competitions, 25 x, 50 x or 100 x excess of unlabeled AP-1 oligonucleotide were added to protein extracts. (B) Supershifts were performed with anti-JunD or anti-c-Jun antibodies with protein extracts from AdJunD-CM without stimulation (AdJunD/C). Arrows point at specific bands, asterisks mark super-shifted bands. Experiments shown in A and B were performed in triplicate and repeated with three different cardiomyocyte preparations.

 
3.5 Over-expression of JunD homodimers block PE-mediated ANP promoter activity in cardiomyocytes
Kovacic-Milivojevic et al. [19] have shown in various cells that over-expression of c-Jun induces expression from the ANP promoter while over-expression of JunD had no effect. In order to test whether JunD affects transcriptional activities from the native ANP promoter, we co-transfected cardiomyocytes with an ANP luciferase reporter plasmid (ANP luc) [13] and with plasmids encoding LacZ, wild-type JunD protein (JunD), or a mutated JunD protein where the C-terminal dimerization domain was replaced by the homodimerization domain of the EB1 transcription factor and therefore allows only the formation of JunD homodimers (JunDeb1) [12]. While neither LacZ nor JunD nor JunDeb1 had a significant effect on ANP promoter activity in unstimulated cardiomyocytes, exposure to PE enhanced ANP promoter activity in LacZ transfected cardiomyocytes (5- to 11-fold). PE mediated ANP promoter activity was moderately attenuated in JunD transfected cardiomyocytes (Fig. 4A, JunD-PE: 441%±211% versus LacZ-PE: 659±299%, n.s.) and was significantly lower in cardiomyocytes transfected with JunDeb1 (Fig. 4A, JunDeb1-PE: 241%±69% versus LacZ-PE: 659±299%, P<0.05) suggesting that JunD homodimers attenuate PE-mediated ANP-promoter activity.


Figure 4
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  		Fig. 4 JunD attenuates PE mediated ANP promoter activities. (A) Bar graphs summarizing luciferase activities in protein extracts of cardiomyocytes (control, or stimulated with PE) co-transfected with wild-type ANP promoter luciferase reporter plasmid (ANP luc) and plasmids expressing LacZ, JunD or JunDeb1. Luciferase activity for control LacZ was set at 100% (*P<0.05, **P<0.01 versus control LacZ; #P<0.05 versus PE stimulated LacZ). (B) Luciferase activities in cardiomyocytes (control, or stimulated with PE) transfected with ANP luc or mutated ANP promoter luciferase reporter plasmid with deleted AP-1 site at position – 3418 (ANPluc-3418mutAP-1, ANPmut) (*P<0.05, **P<0.01 versus control ANP luc; ##P<0.01, PE stimulated ANPmut versus PE stimulated ANP luc). Experiments shown in A and B were performed in triplicate and repeated with at least three different cell preparations.

 
3.6 An AP-1 site is critically involved in PE mediated ANP promoter activity
Next we investigated whether the AP-1 motif is crucial for PE mediated ANP promoter activation by transfecting cardiomyocytes with an ANP-luciferase reporter plasmid with a point mutation at the relevant AP-1 motif at position – 3418 (ANPluc-3418mutAP-1) [14]. As shown in Fig. 4B, basal and PE mediated induction of luciferase activities was blunted in cardiomyocytes transfected with ANPluc-3418mutAP-1 compared with ANP luc.

3.7 JunD homodimers attenuate PE mediated AP-1 activity in cardiomyocytes
In order to analyze the regulation of AP-1 transcriptional activity in cardiomyocytes by JunD and c-Jun, we measured luciferase activity from a luciferase reporter plasmid with seven AP-1 consensus sites, which was co-transfected with LacZ, c-Jun, JunD, or JunDeb1 expression plasmids. The expression efficiency of JunD, JunDeb1 and c-Jun expression plasmids were analyzed in a human embryonic kidney cell line (293 HEK) and showed a 20-fold increase in JunD wild-type protein or JunDeb1 protein after transfection compared with LacZ or non-transfected cells (Fig. 5A). The increase in c-Jun protein obtained after transfection was 10-fold compared with LacZ or non-transfected cells (Fig. 5A).


Figure 5
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  		Fig. 5 Regulation of AP-1 promoter activities by JunD. (A) Analysis of Jun protein levels generated by expression plasmids transfected into 293 HEK cells show an increase of 20-fold of wild-type JunD protein (arrows point at three isoforms with different migration properties), an increase of 20-fold of JunDeb1 (arrows point at two different isoforms) and an increase of 10-fold of c-Jun compared with respective protein levels in non-transfected cells or in cells transfected with LacZ 48h after transfection. (B) Luciferase activity of cardiomyocytes co-transfected with an AP-1 luciferase reporter plasmid (AP-1 luc) and with plasmids expressing LacZ, JunD, JunD{delta}162, JunDeb1, or c-Jun proteins without (control) or with addition of PE. *P<0.05, **P<0.01 versus control LacZ, #P<0.05, ##P<0.01 versus PE stimulated LacZ; §P<0.05, §§P<0.01, PE stimulated JunD, JunD{delta}162, or JunDeb1 versus PE stimulated c-Jun. All experiments were performed in triplicate and repeated with at least three different cell preparations.

 
In non-stimulated cardiomyocytes c-Jun prominently induced luciferase activity, while only a slight increase in luciferase activity was observed in cardiomyocytes transfected with JunD (not significant) and JunDeb1 (P<0.05) compared with LacZ (Fig. 5B). Stimulation of cardiomyocytes with PE augmented luciferase activity in LacZ cardiomyocytes (Fig. 5B). c-Jun strongly enhanced PE induced luciferase activity, while JunDeb1 attenuated PE induced luciferase activity compared with LacZ (Fig. 5B). In cardiomyocytes transfected with wild-type JunD PE induced luciferase activity tended to be lower compared with LacZ, but due to high variations between individual cell preparations, differences in PE induced luciferase activity between JunD and LacZ were not significant (Fig. 5B).

3.8 JNK activation site is not required for inhibitory effect of JunD in response to PE
The N-terminal end of the JunD protein contains a phosphorylation site for JNK. In order to test whether this site is required for the effects of JunD on PE mediated AP-1 activity, we used a mutated JunD expression plasmid, with a deficiency at the N-terminus deleting the JNK phosphorylation site (JunD-{delta}162) [11]. There was no significant difference in luciferase activity between cardiomyocytes transfected with JunD-{delta}162, JunD or JunDeb1 plasmids in unstimulated cardiomyocytes or after stimulation with PE (Fig. 5B), indicating that the JNK activation site of JunD is not required for the inhibitory effect of JunD.

3.9 JunD homodimers attenuate c-Jun mediated AP-1 activity in cardiomyocytes
Cardiomyocyte hypertrophy induced by PE is mediated, in part, via JNK, which phosphorylates c-Jun, a major component of transcriptionally active AP-1 complexes [7,20,21]. Transfection of cardiomyocytes with wild-type c-Jun [8] enhanced AP-1 promoter activity under basal conditions and strongly accelerated PE mediated AP-1 activity (Fig. 6). In order to analyze whether JunD homodimers can interfere with c-Jun mediated AP-1 transcriptional activation cardiomyocytes were co-transfected with c-Jun alone, or with c-Jun in combination with LacZ (c-Jun/LacZ) or in combination with JunDeb1 (c-Jun/JunDeb1). JunDeb1 blunted c-Jun mediated AP-1 activity under basal conditions and after PE stimulation (Fig. 6), indicating that JunD homodimers attenuate c-Jun induced AP-1 activity in cardiomyocytes at baseline or after PE stimulation.


Figure 6
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  		Fig. 6 JunDeb1 attenuates c-Jun mediated AP-1 activity. Bar graph summarizing luciferase activities of cardiomyocytes co-transfected with AP-1 luc and the following expression plasmid combinations: c-Jun/LacZ, c-Jun alone, or c-Jun/JunDeb1 without (control) or with addition of PE (**P<0.01 versus c-Jun/LacZ; ##P<0.01, PE stimulated c-Jun or PE stimulated c-Jun/JunDeb1 versus PE stimulated c-Jun/LacZ; §§P<0.01, PE stimulated c-Jun/JunDeb1 versus PE stimulated c-Jun). Control LacZ transfected cardiomyocytes were set at 100%. All experiments were performed in triplicate and repeated with at least three different cell preparations.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The present study indicates that JunD counteracts PE induced cardiomyocyte hypertrophy in vitro by directly interfering with AP-1 transcriptional activity. Moreover, we present evidence that JunD plays a crucial role in modulating PE mediated JNK–c-Jun effects on cardiomyocyte hypertrophy.

We have recently shown that cardiomyocyte hypertrophy following chronic pressure overload is enhanced in mice lacking JunD (JunD– / –), suggesting that JunD is a negative regulator of cardiomyocyte hypertrophy under pathophysiological conditions [4]. In contrast, JunD– / – mice have no obvious cardiac phenotype at baseline [4]. Consistent with this finding, we did not observe any effects of over-expressed wild-type JunD protein on cardiomyocyte survival or growth. In addition, we did not observe effects of JunD on the expression of the hypertrophy marker gene ANP in cardiomyocytes at baseline, which is in line with previous reports showing that JunD alone is not promoting ANP transcription [19].

Catecholamines inducing {alpha}-adrenergic and β-adrenergic receptor mediated signaling have been implicated in the neurohumoral response to pressure overload and the development of left ventricular hypertrophy [22]. Moreover, cardiomyocyte hypertrophy can be induced in isolated neonatal rat cardiomyocytes by treatment with the {alpha}-adrenergic agonist phenylephrine (PE) [7,16]. In addition, it has been shown that the AP1 transcription complex mediates {alpha}- but not β-adrenergic hypertrophic growth responses in adult cardiomyocytes [23]. The pro-hypertrophic effects of PE have been shown to depend, at least in part, on the activation of JNK–c-Jun and subsequently of AP-1, which in turn promotes cardiomyocyte growth and induction of a fetal gene program [5-7]. Here we show that over-expression of JunD protein in cardiomyocytes prevents PE mediated hypertrophy. The up-regulation of left ventricular ANP mRNA is a highly conserved marker of cardiac hypertrophy and is induced by PE [7,24]. Our data show that the over-expression of wild-type JunD moderately and over-expression of a mutant JunD protein forming only homodimeric AP-1 complexes (JunDeb1) [12] strongly blunted the promoter activity of ANP suggesting that JunD is interfering with PE mediated transcriptional regulation in cardiomyocytes.

Using a luciferase reporter plasmid with a mutated ANP promoter lacking the first AP-1 site [14], we demonstrated that this AP-1 site is crucial for PE mediated ANP promoter activity. Taimor et al. [23] have recently shown that PE is promoting the formation of c-Jun/c-Fos AP-1 transcription complexes and is enhancing AP-1 transcriptional activation in cardiomyocytes. We show that over-expression of c-Jun enhances baseline and strongly accelerates PE mediated transcriptional activation of the AP-1 promoter element. Oumra et al. [7] have shown that PE mediated cardiomyocyte hypertrophy is inhibited by a dominant negative mutant of c-Jun. Together, these data support the notion that c-Jun, via activating AP-1, is a necessary regulator for PE mediated cardiomyocyte hypertrophy. JunD, however, promoted a 3 times lower induction of AP-1 activity at baseline compared with c-Jun. Moreover, wild-type JunD protein slightly and the homodimeric JunDeb1 [12] protein strongly attenuated PE induced AP-1 activation. The observation, that over-expression of JunDeb1 strongly attenuated PE mediated AP-1 activation, suggests that AP-1 complexes consisting mainly of JunD homodimers have only low transcriptional activation on AP-1. As a limitation of this experiment, we cannot directly monitor the amount of JunD homodimers formed in cardiomyocytes over-expressing the wild-type JunD protein. However, the observation that almost the entire AP-1 DNA–protein complex could be supershifted with JunD, but not with c-Jun antibodies in AdJunD transfected cardiomyocytes at baseline and after PE stimulation suggests that AP-1 complexes in cells which over-express JunD contain a high amount of JunD proteins, most likely JunD homodimers.

Interestingly, attenuation of PE induced AP-1 transcriptional activity in JunD over-expressing cardiomyocytes occurred despite strongly enhanced AP-1 complex-DNA binding. Furthermore, in LacZ over-expressing cardiomyocytes, PE strongly induced AP-1 complex-DNA binding, while baseline AP-1 complex-DNA binding was not further enhanced by PE in JunD over-expressing cardiomyocytes. Supershift experiments showed that AP-1 DNA–protein complexes in JunD over-expressing cardiomyocytes consisted mainly of JunD, while in PE stimulated LacZ over-expressing cardiomyocytes AP-1 DNA–protein complexes contained c-Jun, but no detectable JunD. Thus, c-Jun containing AP-1 complexes are highly active, while AP-1 complexes consisting mainly of JunD have a low transcriptional activation potential.

Compared with the strong attenuation of PE mediated cardiomyocyte hypertrophy and the complete inhibition of PE induced increase in ANP mRNA in cardiomyocytes transfected with the JunD adenovirus, the JunD wild-type expression plasmid was less efficient in attenuating PE mediated ANP and AP-1 transcriptional activity. We assume that in cardiomyocytes transfected with the JunD expression plasmid lower amounts of JunD protein have been synthesized compared to cardiomyocytes infected with the JunD adenovirus. In a situation where high levels of c-Jun proteins are present, it has been shown that JunD can heterodimerize with c-Jun [12] and form AP-1 complexes, that are transcriptionally more active as JunD homodimers. This feature may explain the lower inhibitory effect of the wild-type JunD expression plasmid in PE stimulated cardiomyocytes. The transfection with the JunDeb1 plasmid, which does not dimerize with other AP-1 transcription factors and forms only JunD homodimers, however, efficiently attenuates PE induced transcriptional activation of the ANP promoter or the AP-1 motif. Therefore, the effects of the JunD adenovirus are similar to the effects of the JunDeb1 expression plasmid, supporting the notion that JunD homodimers are attenuating PE induced cardiomyocyte hypertrophy. Moreover, the observation that the co-over-expression of c-Jun with JunDeb1 lowered c-Jun mediated AP-1 activity further supports the hypothesis that JunD homodimers can lower c-Jun signaling by directly competing for complex binding to AP-1 promoter elements.

In contrast to c-Jun, JunD contains the JNK phosphorylation site, but no JNK docking site and, therefore, JNK is not efficiently activating JunD homodimers [25]. Our observations, that mutant JunD proteins either lacking the JNK docking site (JunD-{delta}162) [11] or forming only JunD homodimers (JunDeb1) [12] attenuated PE induced AP-1 transcriptional activity efficiently, indicate that JunD can translocate to the nucleus and bind there to AP-1 sites independent of activation by JNK. Moreover, JunD does not need activation by JNK for its inhibitory effect on PE mediated AP-1 activity. Interestingly, a recent report showed that ANP itself acts as a negative regulator of cardiomyocyte hypertrophy. The mechanism behind this appears to rely on the ANP induced up-regulation of the MAPK phosphatase-1 (MKP-1), a phosphatase known to attenuate activation of MAPK and induction of immediate early response genes and fetal type genes [26], suggesting that multiple negative regulatory mechanism exist to control gene expression related to cardiac hypertrophy.

For embryonic and fetal heart development multiple cases of functional redundancy and combinatorial interactions that reflect the complex networks of gene expression have been reported [27,28]. However, little is known concerning combinatorial aspects of signaling pathways and downstream effectors in the pathophysiology of the adult heart. AP-1 transcription factors are downstream nuclear effectors of multiple stress induced signaling pathways involving PKC, the small G protein Ras, and several MAP kinases such as JNK and ERK. The Jun proteins are essential to form homo- and heterodimeric AP-1 transcription factors complexes. Our observations demonstrate that individual Jun transcription factors such as JunD and c-Jun can have opposing effects in postnatal cardiomyocytes in response to stress and suggest that the hypertrophic response of cardiomyocytes can be influenced by the relative subunit composition of the prevailing AP-1 complexes depending directly on the ratio of individual Jun proteins present in response to pathophysiologic stimuli. Thus, the transcriptional and posttranslational regulation of Jun transcription factors sheds some light on the complex combinatorial interactions of signaling pathways orchestrating the stress response in cardiomyocytes. This idea is summarized schematically in Fig. 7.


Figure 7
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  		Fig. 7 Hypothesis for modulation of cardiomyocyte hypertrophy by JunD. In the presence of low levels of JunD protein {alpha}-adrenergic activation with phenylephrine (PE) induces the formation of transcriptionally highly active AP-1 complexes composed of c-Jun and c-Fos, which bind to AP-1 promoter elements strongly inducing hypertrophic gene expression programs and resulting in accelerated hypertrophic growth. In contrast, in the presence of high JunD protein levels (right side), transcriptionally highly active AP-1 complexes induced by PE compete with transcriptionally low active JunD complexes for binding to AP-1 promoter elements, which results in an attenuation of the hypertrophic gene expression program and subsequently in moderate hypertrophic growth.

 
It has previously been reported that JunD protein levels are reduced in the heart exposed to pathophysiological stress [1,3,4]. {alpha}-Adrenergic receptor activation has been implicated in the neurohumoral response of the heart to pressure overload [22]. Our observation that the {alpha}-adrenergic agonist PE reduces the expression of JunD suggests that {alpha}-adrenergic receptor activation may, at least in part, account for the down-regulation of JunD in the heart exposed to pressure overload or other stresses. Furthermore, it has been demonstrated that PE induces the expression of c-Jun and c-Fos in cardiomyocytes in vitro and in vivo [6,29,30]. Thus, PE mediated hypertrophy may be achieved not only by inducing c-Jun and c-Fos thereby allowing the formation of highly active AP-1 transcription complexes, but also by attenuating the expression of less efficient or even inhibiting factors like JunD (Fig. 7).

In conclusion, JunD attenuates cardiomyocyte hypertrophy by inhibiting AP-1 transcriptional activity and might effectively function as an endogenous dominant negative regulator, buffering against extensive hypertrophic growth of cardiomyocytes in response to pathophysiological stress.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We are grateful to Birgit Brandt, Silvia Gutzke and Dr. Beate Fiedler for excellent technical assistance. This study was supported by the Jean Leducq Foundation.


   Notes
 
Time for primary review 17 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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