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Cardiovascular Research 2006 70(3):555-565; doi:10.1016/j.cardiores.2006.02.010
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Copyright © 2006, European Society of Cardiology

Cross talk between corticosteroids and alpha-adrenergic signalling augments cardiomyocyte hypertrophy: A possible role for SGK1

Kerrie Listera, Dominic J. Autelitanoa,1, Anna Jenkinsb, Ross D. Hannanb,c,d and Karen E. Sheppardb,e,*

aBaker Heart Research Institute, Prahran, Victoria, 3181 Australia
bDivision of Research, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, Australia
cDepartment of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia
dDepartment of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
eDepartment of Immunology, Monash University, AMREP, Prahran, Melbourne, Vic., Australia

* Corresponding author. Growth Control Laboratory, Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, St Andrew's Place, East Melbourne 3002, Victoria, Australia. Tel.: +61 3 9656 1283; fax: +61 3 9656 1411. Email address: karen.sheppard{at}petermac.org

Received 18 October 2005; revised 18 January 2006; accepted 7 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective Mineralocorticoids and glucocorticoids have been implicated in the pathogenesis of cardiac diseases; however, both in vivo and in vitro studies indicate that changes in the cellular milieu of either the cardiomyocyte and/or cells of the vasculature is required for corticosteroid signalling to be pathological. The aim of the current study was to directly address whether signalling pathways that are activated during myocyte hypertrophy alter corticosteroid signalling and thus enable these steroids to significantly impact on the hypertrophic response.

Methods Neonatal rat ventricular cardiomyocytes were treated with phenylephrine or phorbol ester for 48 h to induce myocyte hypertrophy. Following treatment, the expression of glucocorticoid receptor, mineralocorticoid receptor, and 11β-hydroxysteroid dehydrogenase were determined by ribonuclease protection assay. In addition, the activity of 11β-hydroxysteroid dehydrogenase and the ability of glucocorticoid and mineralocorticoid receptors to induce serum- and glucocorticoid-induced kinase 1 (SGK1) gene transcription were assessed. Corticosteroid effects on phenylephrine and phorbol ester-induced hypertrophy were determined by measuring atrial natriuretic peptide (ANP) mRNA expression, protein synthesis, or induction of rDNA transcription.

Results Incubation of cardiomyocytes with phenylephrine and phorbol ester for 48 h led to a hypertrophic response with an associated 8- to 12-fold increase in ANP mRNA and 2-fold increase in rDNA transcription. Cardiomyocyte hypertrophy led to a significant 2-fold increase in glucocorticoid receptor and mineralocorticoid receptor expression that resulted in enhanced receptor signaling as judged via the ability of corticosterone and aldosterone to induce SGK1 gene transcription. 11β-Hydroxysteroid dehydrogenase2 was not detected in normal or hypertrophied cardiomyocytes, and 11β-hydroxysteroid dehydrogenase exclusively demonstrated reductase activity, converting the inactive 11-ketometabolite back to active glucocorticoid. 11β-Hydroxysteroid dehydrogenase1 expression and reductase activity were increased with phorbol ester-induced hypertrophy but not phenylephrine-induced hypertrophy. In basal cardiomyocytes, either aldosterone or corticosterone induced only a minor increase in ANP mRNA and protein synthesis; however, in cardiomyocytes primed with phenylephrine, both corticosteroids significantly potentiated phenylephrine-mediated effects via activation of the glucocorticoid receptor.

Conclusion In the present study we demonstrate that significant cross talk exists in the cardiomyocyte between corticosteroid receptor-activated pathways and both protein kinase C and alpha-adrenergic signalling. Cellular changes associated with the hypertrophic response promote corticosteroid signalling and allow for corticosteroid-mediated potentiation of alpha-adrenergic receptor signalling. Such augmentation of cardiomyocyte hypertrophy may in part explain the role that corticosteroid hormones play in the pathophysiological progression of heart disease.

KEYWORDS Hypertrophy; Myocytes; Hormones


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac hypertrophy involves both an increase in cardiomyocyte size and proliferation of cardiac fibroblasts and represents an initial compensatory mechanism of the heart in response to increases in hemodynamic load. However, under pathological conditions such as systemic arterial hypertension and myocardial infarction, compensatory hypertrophy often progresses to heart failure. Recently, both experimental and clinical studies have suggested direct effects of aldosterone in cardiac disease. In animal models, high dose aldosterone in conjunction with high salt promotes cardiac hypertrophy and both perivascular and interstitial fibrosis [1,2], in a manner that is independent of blood pressure elevation [3]. Furthermore, mineralocorticoid receptor antagonists given in conjunction with existing therapies reduced morbidity and mortality in heart failure patients [4,5]. These effects also appear to be independent of changes in blood pressure [6], suggesting that aldosterone directly induces cardiac pathology.

Aldosterone effects are predominantly mediated by intracellular mineralocorticoid receptors (MRs), which have high affinity for both aldosterone and endogenous glucocorticoids. The enzyme 11β-hydroxysteroid dehydrogenase 2 (11βHSD2) converts endogenous glucocorticoids to receptor inactive 11-ketometabolites and in doing so enables aldosterone to access MR in vivo where glucocorticoid concentrations are 2–3 orders of magnitude higher [7,8]. In the absence of 11βHSD2, MR binds endogenous glucocorticoid which then can act as either an antagonist [9] or an agonist [10–13]. In contrast to 11βHSD2, which only has the capacity to act as a dehydrogenase and inactivate glucocorticoids, 11βHSD1 can inter-convert hormonally active corticosterone and inactive 11-dehydrocorticosterone (DHB), although the predominant activity is oxoreductase (DHB to corticosterone) in intact cells [14]. Cardiomyocytes express little if any 11βHSD2 but do express MR, GR, and 11βHSD1, which converts the 11-ketometabolite back to an active glucocorticoid [15]. In vivo, heart MR has equivalent affinity for aldosterone and corticosterone [16], thus cardiomyocyte MR would primarily be occupied by endogenous glucocorticoids. When 11βHSD2 is over-expressed in cardiomyocytes of transgenic mice, which effectively inhibits binding of corticosterone to both MR and GR and thus allows aldosterone access to cardiomyocyte MR, animals are normotensive but spontaneously developed cardiac hypertrophy, fibrosis and die prematurely of heart failure. This phenotype is only partially antagonized by the aldosterone antagonist eplerenone, suggesting that aldosterone activation of cardiac MR was in part causative of the cardiac hypertrophy, and in addition that loss of glucocorticoid signalling also contributes to this phenotype [17]. In another transgenic mouse model in which the animals are normotensive but exhibit chronic hyperaldosteronism there is no evidence of cardiac hypertrophy, fibrosis or remodelling [18], indicating that high plasma aldosterone alone does not induce cardiac remodelling.

The role of glucocorticoid signalling in cardiac hypertrophy is less clear. Studies on neonates suggest that glucocorticoids induce myocardial hypertrophy in vivo [19,20] and can directly induce hypertrophy in rat cardiomyocytes in vitro [21,22]. In addition, treatment with high-dose glucocorticoids is associated with an increase risk for cardiovascular disease [23,24]. In contrast, beneficial effects of glucocorticoids are indicated by the observation that in the myocyte 11βHSD2 transgenic mouse in which myocyte glucocorticoid signalling would be ablated, cardiac hypertrophy is only partially reversed by MR blockade suggesting myocyte glucocorticoid signalling is also required for complete reversal.

In the current studies we directly address whether aldosterone or corticosterone induce myocyte hypertrophy, if corticosteroid receptor signalling and/or 11βHSD dehydrogenase activity is altered in the hypertrophied myocyte and the impact of corticosteroid signalling on the hypertrophic response. These studies demonstrate cross talk between corticosteroid signalling and hypertrophic signalling pathways that result in potentiation of myocyte hypertrophy. Such potentiation of cardiomyocyte growth responses may in part explain the role that corticosteroid hormones play in the pathophysiological progression of heart disease.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Culture of neonatal cardiomyocytes
Neonatal rat ventricular myocyte cultures were prepared from 1- to 2-day-old Sprague–Dawley rats as previously described [25]. Cells were pre-plated twice for 30 min each to remove non-myocytes and left to attach for 18 h in DMEM/Hepes, 10% fetal calf serum, 0.1 mM bromodeoxyuridine (BrdU), 50 U/ml penicillin G, 50 µg/ml streptomycin sulfate and 125 µg/ml fungizone. Medium was then replaced with a defined serum-free medium consisting of DMEM/Hepes, 10 mg/ml human insulin, 10 mg/ml bovine apo-transferrin, 0.1 mM BrdU, 50 U/ml penicillin, 50 µg/ml streptomycin sulfate and 125 µg/ml fungizone. For all experiments cardiomyocytes were plated at low density (400 cells/mm2) in 6 well plates and hypertrophy induced by treating cells for 48 h with either 25 µM phenylephrine (PE) plus 1 µM propranolol or 100 µM phorbol-12-myristate-13-acetate (PMA). All procedures conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996) and to the Australian National Health and Medical Research Council code for the care and use of animals for medical research.

2.2 RNase protection analysis
[32P]-labelled riboprobes were generated from cDNAs corresponding to rat 11βHSD1, 11βHSD2, GR, MR, serum and glucocorticoid induced kinase 1 (SGK1), glyceraldehyde phosphate dehydrogenase (GAPDH) and atrial naturetic peptide (ANP) as previously described [15,25,26]. A [32P]-labelled riboprobe was also generated from a cDNA corresponding to a 508 nucleotide fragment of the 5' external transcribed spacer (5'ETS) of the rat 45S ribosomal RNA (rRNA) precursor. The 5'ETS cDNA was PCR cloned from the vector pU5.1E/X, which contains the rat 45S rDNA (– 236 to +690) promoter [27]. The 508 nucleotide fragment was subcloned into pGEM4Z and linearized with EcoR1 for riboprobe synthesis. The 5' external transcribed spacer (5'ETS) of the 45S ribosomal RNA precursor was used to assess rDNA transcription. Ribosomal RNA's are transcribed as a single pre-rRNA transcript. Following synthesis the ETS regions are rapidly processed and degraded to give rise to 28S, 5.8S and 18S rRNA. Thus, steady state rRNA levels of the 5'ETS region of the 45S rRNA precursor reflect rates of rDNA transcription [28]. Cardiomyocytes were plated at 3.8 x 105 cells (400 cells/mm2) per well of a 6 well plate and total RNA was prepared from cells by the guanidinium isothiocyanate method [29]. Extracted RNA from each well was resuspended in 40 µl 0.1% SDS of which 5 µl was assayed for ANP, and 35 µl for either SGK1, or GR and MR. For the simultaneous measurement of ANP mRNA and 5'ETS rRNA, half the total RNA extracted from a well went into each assay. Thus, for each sample in a particular RNase protection assay RNA corresponding to an equivalent cell number was assessed, thus for each protection assay samples are normalized to cell number. As previously described [26] a solution hybridization/RNase protection analysis of total RNA was used to simultaneously quantify the various mRNA and rRNA levels. Nuclease protection was performed by addition of 300 µL of digestion buffer containing RNase-T1 (250 U, Boehringer Mannheim) and RNase A (10 µg, Boehringer Mannheim). RNase-protected RNA hybrids were then detected and quantified by phosphorimage analysis (BAS system, Fuji) after electrophoresis on non-denaturing 5% polyacrylamide gels.

2.3 11βHSD Activity
For 11βHSD activity studies, cells were incubated with DMEM/HEPES containing 10 nM [3H]11-dehydrocorticosterone (DHB) or 10 nM [3H]corticosterone, and media was sampled at 2 and 6 h. As previously described [26], following incubation steroids were ethyl acetate extracted from medium, separated by thin layer chromatography, and [3H]labelled steroids visualized and quantified by phosphorimage analysis.

2.4 Phalloidin staining of actin filaments
Control and treated cardiomyocytes were fixed in 4% phosphate buffered formalin. Permalized with 0.2% Triton-X-100 and blocked in PBS containing 1% BSA . The cells were stained with tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin prior to visualization by fluorescence microscopy.

2.5 Protein assay
After treatment media were aspirated and cells lysed by the addition of 200 µl of lysis buffer (0.1 M K2HPO4, 1.0% Triton X-100 and 1.0 mMM dithithreitol). The lysate was then removed and protein measured by Lowry assay [30].

2.6 Statistics
Data were compared by one-way analysis of variance followed by Fisher's PLSD test. Differences of P<0.05 were considered significant. All data are expressed as mean±SEM.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 MR, GR and 11βHSD1 expression are increased in myocyte hypertrophy
Changes in the expression of 11βHSD1, 11βHSD2, GR and/or MR in cardiomyocytes may potentially alter the ability of aldosterone and corticosterone to induce a functional response. Thus to initially address if corticosteroid signalling is altered as a consequence of the phenotypic change associated with cardiomyocyte hypertrophy, we determined the levels of these receptors and 11βHSD isoforms in response to two distinct hypertrophic signalling agents. Cardiomyocyte hypertrophy was induced in rat neonatal myocytes by treating cells for 48 h with the alpha-adrenergic receptor agonist, phenylephrine (PE, 25 µM, plus propranolol 1 µM) or phorbol myristate acetate (PMA), a protein kinase C activator. ANP mRNA expression, a well-established marker for cardiomyocyte hypertrophy was used to confirm hypertrophic signalling. RNA was extracted following treatment and ANP mRNA quantified by RNase protection analysis. Both PE and PMA induced myocyte hypertrophic signalling as indicated by a significant 8–12-fold increase in steady state ANP mRNA levels (Fig. 1A). To assess if corticosteroid receptor levels and/or 11βHSD isoform expression were altered, mRNA for MR, GR, 11βHSD2 and 11βHSD1 was also measured. Both MR and GR mRNA levels demonstrated a significant (2-fold) increase in response to the hypertrophic agents (Fig. 1B) and 11βHSD2 was undetectable in all groups (data not shown). Interestingly, PMA- but not PE-induced hypertrophy increased 11βHSD1 mRNA levels (Fig. 1C) suggesting that hypertrophy per se does not induce 11βHSD1 expression but the change is selective for the intracellular signalling pathway involved.


Figure 1
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  		Fig. 1 Solution hybridization/RNase protection of ANP, GR, MR and 11βHSD1 following in vitro induction of hypertrophy in neonatal ventricular cardiomyocytes. Cardiomyocytes were treated with 25 µM phenylephrine (PE) in the presence of 1 µM propranolol or 100 µM phorbol-12-myristate (PMA) for 48 h. Cells were harvested, RNA extracted and the specific mRNA's were measured by ribonuclease protection analysis and both visualized and quantitated by phosphorimage analysis. The specific mRNA's were measured in RNA extracted from an equivalent cell number, thus all samples are normalized to cell number. (A) Typical phosphorimage of ANP and GAPDH protected [32P]hydrids following nuclease protection analysis and relative expression of ANP mRNA. (B) Typical phosphorimage of GR and MR protected [32P]hydrids following nuclease protection analysis and relative expression of the mRNA's. (C) Typical phosphorimage of 11βHSD1 [32P]hydrids following nuclease protection analysis and relative expression of the 11βHSD1 mRNA. Values shown are mean±SEM, n=8. *P<0.05 relative to control (Con).

 
3.2 11βHSD1 activity is increased in myocyte hypertrophy
11βHSD1 can either act as a reductase or dehydrogenase depending on the state of the cell [31,32]. In normal cardiomyocytes 11βHSD1 acts as a reductase converting inactive 11-ketometabolites to active glucocorticoid [15]. To determine whether the hypertrophic phenotype results in a shift in enzyme activity, 11βHSD activity was assessed in intact cardiomyocytes following induction of hypertrophy. Cells were treated for 48 h with PE or PMA and then either [3H]11-dehydrocorticosterone (DHB) or [3H]corticosterone was added. After 2 or 6 h media was collected and steroids extracted using ethyl acetate and separated by thin layer chromatography. As illustrated in Fig. 2A, DHB was converted to corticosterone in control and both PE and PMA induced hypertrophic cardiomyocytes. Activity of 11βHSD1 was not changed in response to PE induced hypertrophy but increased 100% in response to PMA, consistent with the increase in 11βHSD1 mRNA. In contrast to reductase activity there is very little dehydrogenase activity in control and hypertrophied myocytes. Conversion of corticosterone to DHB after 24 h in control, PE and PMA treated cells was 6.8+0.2%, 7.6+0.5% and 4.9+0.1%, respectively. We have previously demonstrated that 11βHSD1-mediated conversion of DHB to corticosterone allows this steroid to activate GR and induce SGK1 gene transcription [15]. To assess if the PMA induced increase in 11βHSD1 activity enhances the ability of DHB to induce gene transcription, cardiomyocytes were pretreated for 24 h with 100 µM PMA followed by a 6 h incubation with 30 nM DHB+4 µM carbenoxolone, an inhibitor of 11βHSD activity. As illustrated in Fig. 2B, addition of exogenous DHB increased SGK1 gene transcription 2.4-fold while in PMA pre-treated cells, DHB increased SGK gene transcription by 4.2-fold. These effects were totally abolished by the 11βHSD1 inhibitor carbenoxolone, demonstrating that DHB per se is inactive in this context, but acts as a substrate for 11βHSD conversion to corticosterone that then is able to induce SGK1 gene transcription (Fig. 2B).


Figure 2
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  		Fig. 2 (A) 11βHSD activity in control and hypertrophied cardiomyocytes. Cardiomyocytes were treated with 25 µM phenylephrine (PE) in the presence of 1 µM propranolol or 100 µM phorbol-12-myristate (PMA) for 48 h. Following treatment cells were incubated with [3H]dehydrocorticosterone (DHB 20 nM) and media was sampled at 2 (open bars) or 6 h (solid bars). Control (Con) is untreated cells incubated with [3H]dehydrocorticosterone. A typical phosphorimage of the TLC profile of extracted [3H]steroids after 6 h of incubation is shown. Bands were quantitated by phosphorimage analysis and the percentage of [3H]DHB converted to [3H]Corticosterone (Cort) is illustrated in the bar graph. Values shown are mean±SEM. n=4. *P<0.05 relative to the appropriate control. (B) Dehydrocorticosterone induction of SGK1 gene transcription. Cardiomyocytes were pretreated for 24 h with PMA (100 µM) followed by a 6 h incubation with 30 nM dehydrocorticosterone (DHB)+4 µM carbenoxolone (CBX) the 11βHSD inhibitor. Following incubation cells were harvested, RNA extracted and the SGK1 mRNA was measured by ribonuclease protection analysis and both visualized and quantitated by phosphorimage analysis. Illustrated is a typical phosphorimage of SGK1 protected [32P]hydrids following nuclease protection analysis. Graphs represent the relative expression of SGK1 mRNA when expressed as percent of untreated cells (control). Values shown are mean±SEM. n=4. *P<0.05 compared to control. +P<0.05 compared to control and DHB alone.

 
3.3 Myocyte hypertrophy potentiates aldosterone and corticosterone induced gene transcription
We further assessed whether the increase in corticosteroid receptors in the hypertrophied cardiomyocyte led to an increased functional response by mediating enhanced MR and GR signalling. Cardiomyocytes were treated for 48 h with the hypertrophic agents followed by a 2 h treatment with either aldosterone or corticosterone, both of which have the capacity to activate SGK1 gene transcription. Following treatment RNA was isolated and both ANP and SGK1 mRNA induction was assessed by RNase protection analysis. As illustrated in Fig. 3A, both PMA and PE induced myocyte hypertrophy as indicated by the greater than 8-fold increase in ANP mRNA expression, however, neither treatment alone altered SGK1 mRNA expression (Fig. 3B). At the concentration used (30 nM), which would saturate MR but only partially occupy GR, corticosterone but not aldosterone induced SGK1 mRNA expression. Pre-treatment of cells with the hypertrophic agents for 48 h prior to steroid addition potentiated corticosterone induced gene transcription. Corticosterone treatment of PE and PMA pre-treated cells induced a 120% and 40% increase in SGK1 mRNA, respectively, above corticosterone alone. Although 30 nM aldosterone had no ability to activate SGK1 transcription in control myocytes, this sub-threshold concentration led to a 130% increase in SGK1 gene expression in cells primed by the hypertrophic agents PE and PMA. The GR antagonist RU38486 blocked the aldosterone effect indicating that aldosterone was mediating its transcriptional effect on the SGK1 gene via GR (Fig. 3B).


Figure 3
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  		Fig. 3 Corticosteroid induction of SGK1 mRNA in hypertrophied cardiomyocytes. Cardiomyocytes were treated with 25 µM phenylephrine (PE) in the presence of 1 µM propranolol or 100 µM phorbol-12-myristate (PMA) for 48 h followed by a 2 h treatment with either aldosterone (30 nM) or corticosterone (30 nM) in the presence or absence of RU38486 (300 nM). Following treatment cells were harvested, RNA isolated and either ANP or SGK1 mRNA measured by ribonuclease protection analysis. Specific mRNA bands were visualized and quantitated by phosphorimage analysis. Graphs represent the relative expression of ANP and SGK1 mRNA when expressed as percent of untreated cells (control). Values shown are mean±SEM, n=5. *P<0.05 relative to untreated control cells. +P<0.05 compared to both untreated cells and to corticosterone or aldosterone alone.

 
3.4 Corticosterone and aldosterone potentiate the hypertrophic response
To assess if aldosterone and/or corticosterone can potentiate myocyte hypertrophy, we treated cardiomyocytes for 48 h with either PE or PMA together with steroid. Myocyte hypertrophy was assessed by measuring ANP mRNA expression, ribosomal gene (rDNA) transcription, cellular protein levels and reorganization of actin filaments into sarcomeric units, all of which are well-established markers for cardiomyocyte hypertrophy. rDNA transcription is one of the rate limiting steps in protein synthesis and accelerated rates of rRNA synthesis have been demonstrated to accompany increased rates of protein synthesis in all models of cardiomyocyte hypertrophy tested [28,33–35]. Thus, rDNA transcription is an accurate marker for myocyte hypertrophy and directly reflects changes in protein synthetic capacity. To assess rDNA transcription a ribonuclease protection assay that measured rRNA complementary to the 5' external transcribed spacer (5'ETS) of the 45S ribosomal RNA precursor was used [28,36]. As illustrated in Fig. 4 PE, PMA, corticosterone and aldosterone all increased ANP mRNA expression 7.4-fold, 12-fold, 4.4-fold and 2.5-fold, respectively. Corticosterone synergized with PE and induced ANP mRNA expression 20-fold above control and 3-fold above PE alone (Fig. 4A). In contrast, corticosterone did not further increase PMA induced ANP mRNA (Fig. 4C). Similarly, aldosterone synergized with PE but not PMA in the induction of ANP mRNA expression (Fig. 4A and C). Both corticosterone and aldosterone action were inhibited by RU38486 indicating that both these steroids were inducing a response via the GR. Similar to the effects on ANP expression, PE, PMA and corticosterone increased rDNA transcription by 80+19%, 122+19% and 34+8%, respectively (Fig. 4B, D), and when added in combination, corticosterone and PE synergized, increasing rDNA transcription by 146+24%, while the response to combined PMA and corticosterone was equivalent to PMA alone. Protein levels assessed by Lowry assay, demonstrated that corticosterone increased protein by 7.9+3% and PE increased levels by 17.7+2.3%, while there was an additive effect with combined PE and corticosterone treatment (27.1+3.5%: Fig. 5).


Figure 4
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  		Fig. 4 Corticosterone and aldosterone potentiates cardiomyocyte hypertrophy. Cardiomyocytes were treated for 48 h with aldosterone (100 nM) or corticosterone (100 nM)+RU38486 (1 µM) together with either 25 µM phenylephrine (PE) plus 1 µM propranolol (A,B) or 100 µM phorbol-12-myristate (PMA; C,D). Following treatment cells were harvested, RNA isolated and ANP mRNA (A,B) and 5'ETS rRNA (B,D) were measured simultaneously by ribonuclease protection analysis. Specific RNA bands were visualized and quantitated by phosphorimage analysis. Quantitative data represents expression of RNA relative to untreated cells (Control). (A) Quantitative analysis of ANP mRNA in response to PE treatment and expressed relative to untreated cells. (B) Quantitative analysis of 5'ETS rRNA in response to PE treatment and expressed relative to untreated cells. (C) Quantitative analysis of ANP mRNA expressed in response to PMA treatment and expressed relative to untreated cells. (D) Quantitative analysis of 5'ETS rRNA in response to PMA treatment and expressed relative to untreated cells. Values shown are mean±SEM, n=5. *P<0.05 relative to untreated control cells. +P<0.05 relative to PMA alone.

 

Figure 5
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  		Fig. 5 Corticosterone and phenylephrine increase myocyte protein levels. Cardiomyocytes were treated for 48 h with corticosterone (100 nM), 25 µM phenylephrine (PE) plus 1 µM propranolol or a combination of both. Following treatment cells were harvested, and protein levels determined by Lowry assay. Values shown are mean±SEM, n=6. *P<0.05 relative to untreated control cells and to each of the other treatments.

 
The hypertrophic effect of PE, PMA and corticosterone on cardiomyocytes was confirmed by fluorescence microscopy (Fig. 6). Cardiomyocytes (400 cells/mm2) stained with TRITC-labelled phalloidin displayed a morphology typified by small, irregularly shaped cells (Fig. 6A), in contrast PE (Fig. 6C) and PMA (Fig. 6E) and to a lesser degree corticosterone (Fig. 6B) resulted in both an increase in cell size and reorganization of actin filaments into sarcomeric units.


Figure 6
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  		Fig. 6 Reorganization of cardiomyocyte actin filaments into sarcomeric units induced by corticosterone, PE and PMA. Cardiomyocytes were untreated (A) or treated for 48 h with corticosterone (B), phenylephrine (C), phenylephrine plus corticosterone (D) or phorbol-12-myristate (E). Following treatment cells were stained with TRITC-labeled phalloidin and actin filaments visualized by fluorescence microscopy. Left panel shows the change in cell size and the right panel illustrates the reorganization of the actin filaments.

 
In addition to inhibiting corticosterone and aldosterone induced responses, RU38486 partially inhibited both PMA induced ANP mRNA and rDNA transcription (Fig. 4C, D). Further analysis of RU38486 inhibition of PMA action demonstrated a dose dependent inhibition in PMA induced ANP mRNA and rDNA transcription (Fig. 7). This effect of RU38486 was not mediated via antagonism of GR or MR action because high dose spironolactone that blocks both MR and GR responses in these cells [15] did not mimic the RU38486 effect (Fig. 7).


Figure 7
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  		Fig. 7 RU38486 inhibition of PMA induced ANP mRNA and 5'ETS rRNA. Cardiomyocytes were treated for 48 h with 100 µM phorbol-12-myristate (PMA) plus either RU38486 (RU) or 10 µM spironolactone (spiro). Following treatment cells were harvested, RNA isolated and ANP mRNA and 5'ETS rRNA were measured simultaneously by ribonuclease protection analysis. Specific RNA bands were visualized and quantitated by phosphorimage analysis. (A) Typical phosphorimage of ANP and 5'ETS protected [32P]hydrids following nuclease protection analysis. (B) Graphs represent the relative expression of ANP mRNA and 5'ETS rRNA when expressed as percent of untreated cells. Quantitative data represents expression of RNA relative to untreated cells. Values shown are mean±SEM, n=5. *P<0.05 relative to PMA alone.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study we demonstrate that there is significant cross talk between corticosteroid receptor activated pathways and both PKC and alpha-adrenergic signalling in cardiomyocytes. Cellular changes associated with the hypertrophic response promote corticosteroid signalling and allow for corticosteroid-mediated potentiation of alpha-adrenergic signalling. Such amplification of alpha-adrenergic induced cardiomyocyte hypertrophy may in part explain the role that corticosteroid hormones play in the pathophysiological progression rather than the initiation of heart disease.

Both animal models and clinical studies have implicated mineralocorticoids and glucocorticoids as important factors in the pathogenesis of cardiac disease. Studies in the mineralocorticoid/salt model of cardiac hypertrophy suggest that the initial pathological event is an inflammatory response at the level of the cardiac vasculature [37] and that this is mediated via activation of the mineralocorticoid receptor by either mineralocorticoids or glucocorticoids [38]. In addition, studies in the cardiomyocyte specific 11βHSD2 transgenic mouse suggest aldosterone access to the cardiomyocyte in the absence of glucocorticoid signalling also induces cardiac hypertrophy and fibrosis [17]. Thus, corticosteroid induced cardiac pathology potentially can involve a direct action on either the cardiomyocytes or vasculature, however, a change in the cellular milieu is required for signalling to be pathological. Studies on the cardiomyocyte specific 11βHSD2 transgenic mouse suggest that induction of 11β-dehydrogenase activity in cardiomyocytes is one mechanism by which aldosterone could gain access to the MR and exert its pathological effect. In the present study, induction of 11β-dehydrogenase activity in cardiomyocytes was not associated with PE or PMA induced myocyte hypertrophy suggesting that activation of PKC or alpha-adrenergic receptors alone does not allow aldosterone access to myocyte MR. In contrast, myocyte hypertrophy induced by PMA increased 11βHSD1 reductase activity which would allow cardiomyocytes to utilize circulation 11-ketoglucocorticoid more effectively, thus making it less likely that aldosterone could access cardiomyocyte MR. Cardiac hypertrophy is a function of the activation of numerous intracellular signalling pathways [39], which appear to be stimulus specific and differential activated during the progression to heart failure [40]. Thus, although we did not observe changes in 11β-dehydrogenase activity (glucocorticoid inactivation) we cannot rule out the possibility that induction of 11β-dehyrogenase activity can occur in the diseased heart.

Compared to GR, MR is expressed at low levels in cardiomyocytes and our previous in vitro studies have demonstrated that neither corticosterone nor aldosterone could sufficiently activate endogenous MR to induce a measurable change in gene transcription [15]. Since the cellular concentration of corticosteroid receptors can impact on their functional response, we tested whether hypertrophy increased the expression of MR and thus led to functional changes in MR signalling. We used SGK1 gene transcription as a functional readout of MR and GR activation as this serine threonine kinase contains a functional glucocorticoid response element in its promoter region [41] and is transcriptionally induced by glucocorticoid-activated GR and aldosterone-activated MR [42–44]. In adrenergic- and PKC-stimulated myocyte hypertrophy the expression of both MR and GR were increased, and corticosterone and aldosterone induced SGK1 gene transcription was augmented, however the response to both steroids was inhibited by the specific GR antagonist RU38486, indicating that both corticosterone and aldosterone responses were mediated via GR not MR. Several studies have demonstrated that aldosterone can directly affect cardiac cells, although whether this occurs via MR or GR remains equivocal. Specifically, high concentrations of aldosterone appear to be required for a maximum response [14], suggesting that the effect is via GR, not MR. Spironolactone has been used as a specific MR antagonist [14], although interpretation of these results is problematic since it can antagonize both MR and GR [15,45]. Moreover aldosterone can act with a relatively short latency [46,47] suggesting nongenomic action of MR or alternatively action via a putative membrane receptor. The aldosterone-induced effects that have been reported can be phenocopied by glucocorticoids [48–52], further suggesting that the aldosterone effects may be mediated via GR. In one study, however, high glucose concentrations potentiated aldosterone effects via MR in cardiomyocytes [53], suggesting that under pathological conditions, these cells may become more sensitive to aldosterone-activated MR.

GR activation induces cardiomyocyte hypertrophy, increases SGK1 gene expression and potentiates adrenergic- but not PKC-induced hypertrophy. Aldosterone at a concentration that would saturate MR and partially bind to GR is unable to induce either hypertrophy or SGK1 gene expression indicating that MR activation alone does not cause myocyte hypertrophy, however aldosterone did potentiated adrenergic-induced hypertrophy albeit to a lesser degree than corticosterone. The ability of RU38486 to block the aldosterone effect together with the less robust response of this steroid compared to corticosterone is good evidence that aldosterone was mediating its effects via GR not MR. However, a caveat in this interpretation, is that RU38486 was also able to dose dependently inhibited PMA induced hypertrophy in the absence of steroid, indicating that RU38486 inhibits PKC signalling. Studies in neurons have also indicated that RU38486 has inhibitory effects on PKC that are independent of its antagonistic properties at GR [54,55]. Given that aldosterone can stimulate PKC epsilon in rabbit cardiomyocytes [56], we cannot rule out the possibility that RU38486 induced inhibition of aldosterone effects in rat cardiomyocytes is via its ability to inhibit PKC activity rather than block binding to GR.

The mechanism by which GR activation potentiates adrenergic induced hypertrophy is not clear, although a number of observations suggest that SGK1 is a likely candidate. Over expression of constitutively active SGK1 in cardiomyocytes induces myocyte hypertrophy and potentiates the alpha-adrenergic response, however SGK1 is not mandatory for the hypertrophic response to phenylephrine [57]. Similarly, in our studies, GR activation induced SGK1 expression, caused weak hypertrophic responses in cardiomyocytes and significantly potentiated the alpha-adrenergic response, although phenylephrine did not induce SGK1 expression. In addition to SGK1 being transcriptionally regulated, it is activated by phosphorylation through the phosphoinositide 3-kinase (PI3kinase) signalling pathway [58], ERK5 [59] and possibly other kinases. Phenylephrine activates the PI3kinase pathway [60] and ERK5 [61] in cardiomyocytes, both of which phosphorylate and activate SGK1. Therefore, a mechanism for corticosteroid potentiation of alpha-adrenergic induced hypertrophy may involve GR-mediated transcriptional induction of SGK1 followed by its post-translational activation via phenylephrine-induced PI3kinase and/or ERK5. This mechanism would explain why GR activation did not potentiate PKC induced hypertrophy, as PKC has not been shown to phosphorylate and activate SGK1.

The present data demonstrate that increased glucocorticoid metabolism as a mechanism by which aldosterone could gain access to myocyte MR does not occur in these in vitro models of cardiomyocyte hypertrophy. In contrast we have demonstrated that glucocorticoid and aldosterone signalling is increased in the hypertrophied cardiomyocyte as a result of increased GR levels and potentiation of GR induced gene transcription. The enhanced corticosteroid signalling leads to significant up regulation of SGK1 which may play a role in functionally enhancing alpha-adrenergic induced hypertrophy. These data suggest that a positive feed back loop exists between glucocorticoid and alpha-adrenergic signalling which results in augmentation of cardiomyocyte hypertrophy. Initially this augmentation of cardiac hypertrophy may aid in the early compensatory mechanism of the heart in response to increases in hemodynamic load, however under pathological conditions it may be deleterious which is consistent with the beneficial effects of corticosteroid blockade in heart failure.


   Acknowledgements
 
This work was supported by a grant from the National Health and Medical Research Council of Australia to K. E. Sheppard and a NHMRC fellowship to R. Hannan. We thank Julia Zebol and Marissa Boyd for the technical assistance.


   Notes
 
1 Current address: Cryptome Pharmaceuticals Ltd. PO Box 6492, St. Kilda Road Central, Melbourne VIC 8008, Australia. Back

Time for primary review 14 days


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

  1. Brilla C.G., Weber K.T. Mineralocorticoid excess, dietary sodium, and myocardial fibrosis. J Lab Clin Med (1992) 120:893–901.[ISI][Medline]
  2. Young M., Fullerton M., Dilley R., Funder J. Mineralocorticoids, hypertension, and cardiac fibrosis. J Clin Invest (1994) 93:2578–2583.[ISI][Medline]
  3. Young M., Head G., Funder J. Determinants of cardiac fibrosis in experimental hypermineralocorticoid states. Am J Physiol (1995) 269:E657–E662.[ISI][Medline]
  4. Pitt B., Remme W., Zannad F., Neaton J., Martinez F., Roniker B., et al. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med (2003) 348:1309–1321.[Abstract/Free Full Text]
  5. Pitt B., Zannad F., Remme W.J., Cody R., Castaigne A., Perez A., et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized aldactone evaluation study investigators. N Engl J Med (1999) 341:709–717.[Abstract/Free Full Text]
  6. Zannad F., Dousset B., Alla F. Treatment of congestive heart failure: interfering the aldosterone–cardiac extracellular matrix relationship. Hypertension (2001) 38:1227–1232.[Abstract/Free Full Text]
  7. Funder J.W., Pearce P.T., Smith R., Smith A.I. Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science (1988) 242:583–585.[Abstract/Free Full Text]
  8. Edwards C.R.W., Stewart P.M., Burt D., Brett L., McIntyre M.A., Sutanto W.S., et al. Localisation of 11β-hydroxysteroid dehydrogenase-tissue specific protector of the mineralocorticoid receptor. Lancet (1988) 2:986–989.[CrossRef][ISI][Medline]
  9. Gomez-Sanchez E.P., Fort C.M., Gomez-Sanchez C.E. Intracerebroventricular infusion of RU28318 blocks aldosterone-salt hypertension. Am J Physiol (1990) 258:E482–E484.[ISI][Medline]
  10. Tedde R., Pala A., Melis A., Ulick S. Evidence for cortisol as the mineralocorticoid in the syndrome of apparent mineralocorticoid excess. J Endocrinol Invest (1992) 15:471–474.[ISI][Medline]
  11. Mune T., Rogerson F.M., Nikkila H., Agarwal A.K., White P.C. Human hypertension caused by mutations in the kidney isozyme of 11beta-hydroxysteroid dehydrogenase. Nat Genet (1995) 10:394–399.[CrossRef][ISI][Medline]
  12. Wilson R.C., Krozowski Z.S., Li K., Obeyesekere V.R., Razzaghy-Azar M., Harbison M.D., et al. A mutation in the HSD11B2 gene in a family with apparent mineralocorticoid excess. J Clin Endocrinol Metab (1995) 80:2263–2276.[Abstract]
  13. Stewart P.M., Krozowski Z.S., Gupta A., Milford D.V., Howie A.J., Sheppard M.C., et al. Hypertension in the syndrome of apparent mineralocorticoid excess due to mutation of the 11 beta-hydroxysteroid dehydrogenase type 2 gene. Lancet (1996) 347:88–91.[CrossRef][ISI][Medline]
  14. Sheppard K.E. Corticosteroid Receptors, 11β-Hydroxysteroid Dehydrogenase and the Heart. Vitam Horm (2003) 66:77–112.[ISI][Medline]
  15. Sheppard K.E., Autelitano D.J. 11Beta-hydroxysteroid dehydrogenase 1 transforms 11- dehydrocorticosterone into transcriptionally active glucocorticoid in neonatal rat heart. Endocrinology (2002) 143:198–204.[Abstract/Free Full Text]
  16. Pearce P., Funder J.W. High affinity aldosterone binding sites (type I receptors) in rat heart. Clin Exp Pharmacol Physiol (1987) 14:859–866.[ISI][Medline]
  17. Qin W., Rudolph A.E., Bond B.R., Rocha R., Blomme E.A., Goellner J.J., et al. Transgenic model of aldosterone-driven cardiac hypertrophy and heart failure. Circ Res (2003) 93:69–76.[Abstract/Free Full Text]
  18. Wang Q., Clement S., Gabbiani G., Horisberger J.D., Burnier M., Rossier B.C., et al. Chronic hyperaldosteronism in a transgenic mouse model fails to induce cardiac remodeling and fibrosis under a normal-salt diet. Am J Physiol Renal Physiol (2004) 286:F1178–F1184.[Abstract/Free Full Text]
  19. de Vries W.B., van der Leij F.R., Bakker J.M., Kamphuis P.J., van Oosterhout M.F., Schipper M.E., et al. Alterations in adult rat heart after neonatal dexamethasone therapy. Pediatr Res (2002) 52:900–906.[CrossRef][ISI][Medline]
  20. La Mear N.S., MacGilvray S.S., Myers T.F. Dexamethasone-induced myocardial hypertrophy in neonatal rats. Biol Neonate (1997) 72:175–180.[CrossRef][ISI][Medline]
  21. Gardner D.G., Wu J.P., LaPointe M.C., Hedges B.K., Deschepper C.F. Expression of the gene for the atrial natriuretic peptide in cardiac myocytes in vitro. Cardiovasc Drugs Ther (1988) 2:479–486.[CrossRef][ISI][Medline]
  22. Whitehurst R.M. Jr., Zhang M., Bhattacharjee A., Li M. Dexamethasone-induced hypertrophy in rat neonatal cardiac myocytes involves an elevated L-type Ca(2+)current. J Mol Cell Cardiol (1999) 31:1551–1558.[CrossRef][ISI][Medline]
  23. Wei L., MacDonald T.M., Walker B.R. Taking glucocorticoids by prescription is associated with subsequent cardiovascular disease. Ann Intern Med (2004) 141:764–770.[Abstract/Free Full Text]
  24. Souverein P.C., Berard A., Van Staa T.P., Cooper C., Egberts A.C., Leufkens H.G., et al. Use of oral glucocorticoids and risk of cardiovascular and cerebrovascular disease in a population based case–control study. Heart (2004) 90:859–865.[Abstract/Free Full Text]
  25. Autelitano D.J., Woodcock E.A. Selective activation of alpha1A-adrenergic receptors in neonatal cardiac myocytes is sufficient to cause hypertrophy and differential regulation of alpha1-adrenergic receptor subtype mRNAs. J Mol Cell Cardiol (1998) 30:1515–1523.[CrossRef][ISI][Medline]
  26. Sheppard K.E., Li K.X., Autelitano D.J. Corticosteroid receptors and 11beta-hydroxysteroid dehydrogenase isoforms in rat intestinal epithelia. Am J Physiol (1999) 277:G541–G547.[ISI][Medline]
  27. Hannan R.D., Cavanaugh A., Hempel W.M., Moss T., Rothblum L. Identification of a mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system. Nucleic Acids Res (1999) 27:3720–3727.[Abstract/Free Full Text]
  28. Brandenburger Y., Arthur J.F., Woodcock E.A., Du X.J., Gao X.M., Autelitano D.J., et al. Cardiac hypertrophy in vivo is associated with increased expression of the ribosomal gene transcription factor UBF. FEBS Lett (2003) 548:79–84.[CrossRef][ISI][Medline]
  29. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem (1987) 162:156–159.[ISI][Medline]
  30. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin phenol reagent. J Biol Chem (1951) 193:265–275.[Free Full Text]
  31. Ge R.S., Hardy D.O., Catterall J.F., Hardy M.P. Developmental changes in glucocorticoid receptor and 11beta-hydroxysteroid dehydrogenase oxidative and reductive activities in rat Leydig cells. Endocrinology (1997) 138:5089–5095.[Abstract/Free Full Text]
  32. Bujalska I.J., Walker E.A., Hewison M., Stewart P.M. A switch in dehydrogenase to reductase activity of 11 beta-hydroxysteroid dehydrogenase type 1 upon differentiation of human omental adipose stromal cells. J Clin Endocrinol Metab (2002) 87:1205–1210.[Abstract/Free Full Text]
  33. Hannan R.D., Rothblum L.I. Regulation of ribosomal DNA transcription during neonatal cardiomyocyte hypertrophy. Cardiovasc Res (1995) 30:501–510.[CrossRef][ISI][Medline]
  34. Hannan R.D., Luyken J., Rothblum L.I. Regulation of rDNA transcription factors during cardiomyocyte hypertrophy induced by adrenergic agents. J Biol Chem (1995) 270:8290–8297.[Abstract/Free Full Text]
  35. Luyken J., Hannan R.D., Cheung J.Y., Rothblum L.I. Regulation of rDNA transcription during endothelin-1-induced hypertrophy of neonatal cardiomyocytes. Hyperphosphorylation of upstream binding factor, an rDNA transcription factor. Circ Res (1996) 78:354–361.[Abstract/Free Full Text]
  36. Poortinga G., Hannan K.M., Snelling H., Walkley C.R., Jenkins A., Sharkey K., et al. MAD1 and c-MYC regulate UBF and rDNA transcription during granulocyte differentiation. EMBO J (2004) 23:3325–3335.[CrossRef][ISI][Medline]
  37. Rocha R., Rudolph A.E., Frierdich G.E., Nachowiak D.A., Kekec B.K., Blomme E.A., et al. Aldosterone induces a vascular inflammatory phenotype in the rat heart. Am J Physiol Heart Circ Physiol (2002) 283:H1802–H1810.[Abstract/Free Full Text]
  38. Young M.J., Moussa L., Dilley R., Funder J.W. Early inflammatory responses in experimental cardiac hypertrophy and fibrosis: effects of 11 beta-hydroxysteroid dehydrogenase inactivation. Endocrinology (2003) 144:1121–1125.[Abstract/Free Full Text]
  39. Molkentin J.D., Dorn I.G. II. Cytoplasmic signaling pathways that regulate cardiac hypertrophy. Annu Rev Physiol (2001) 63:391–426.[CrossRef][ISI][Medline]
  40. Haq S., Choukroun G., Lim H., Tymitz K.M., del Monte F., Gwathmey J., et al. Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation (2001) 103:670–677.[Abstract/Free Full Text]
  41. Maiyar A.C., Phu P.T., Huang A.J., Firestone G.L. Repression of glucocorticoid receptor transactivation and DNA binding of a glucocorticoid response element within the serum/glucocorticoid-inducible protein kinase (SGK) gene promoter by the p53 tumor suppressor protein. Mol Endocrinol (1997) 11:312–329.[Abstract/Free Full Text]
  42. Webster M.K., Goya L., Ge Y., Maiyar A.C., Firestone G.L. Characterization of SGK, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol (1993) 13:2031–2040.[Abstract/Free Full Text]
  43. Naray-Fejes-Toth A., Canessa C., Cleaveland E.S., Aldrich G., Fejes-Toth G. SGK is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial Na+ channels. J Biol Chem (1999) 274:16973–16978.[Abstract/Free Full Text]
  44. Chen S.Y., Bhargava A., Mastroberardino L., Meijer O.C., Wang J., Buse P., et al. Epithelial sodium channel regulated by aldosterone-induced protein SGK. Proc Natl Acad Sci U S A (1999) 96:2514–2519.[Abstract/Free Full Text]
  45. Couette B., Marsaud V., Baulieu E.E., Richard-Foy H., Rafestin-Oblin M.E. Spironolactone, an aldosterone antagonist, acts as an antiglucocorticosteroid on the mouse mammary tumor virus promoter. Endocrinology (1992) 130:430–436.[Abstract]
  46. Mihailidou A.S., Buhagiar K.A., Rasmussen H.H. Na+ influx and Na(+)-K+ pump activation during short-term exposure of cardiac myocytes to aldosterone. Am J Physiol (1998) 274:C175–C181.[ISI][Medline]
  47. Sato A., Liu J.P., Funder J.W. Aldosterone rapidly represses protein kinase C activity in neonatal rat cardiomyocytes in vitro. Endocrinology (1997) 138:3410–3416.[Abstract/Free Full Text]
  48. Muto S., Nemoto J., Ohtaka A., Watanabe Y., Yamaki M., Kawakami K., et al. Differential regulation of Na+-K+-ATPase gene expression by corticosteriods in vascular smooth muscle cells. Am J Physiol (1996) 270:C731–C739.[ISI][Medline]
  49. Takimoto K., Li D., Nerbonne J.M., Levitan E.S. Distribution, splicing and glucocorticoid-induced expression of cardiac alpha 1C and alpha 1D voltage-gated Ca2+ channel mRNAs. J Mol Cell Cardiol (1997) 29:3035–3042.[CrossRef][ISI][Medline]
  50. Barreto-Chaves M.L., Aneas I., Krieger J.E. Glucocorticoid regulation of angiotensin-converting enzyme in primary culture of adult cardiac fibroblasts. Am J Physiol Regul Integr Comp Physiol (2001) 280:R25–R32.[Abstract/Free Full Text]
  51. Fishel R.S., Eisenberg S., Shai S.Y., Redden R.A., Bernstein K.E., Berk B.C. Glucocorticoids induce angiotensin-converting enzyme expression in vascular smooth muscle. Hypertension (1995) 25:343–349.[Abstract/Free Full Text]
  52. Friedland J., Setton C., Silverstein E. Angiotensin converting enzyme: induction by steroids in rabbit alveolar macrophages in culture. Science (1977) 197:64–65.[Abstract/Free Full Text]
  53. Sato A., Funder J.W. High glucose stimulates aldosterone-induced hypertrophy via type I mineralocorticoid receptors in neonatal rat cardiomyocytes. Endocrinology (1996) 137:4145–4153.[Abstract]
  54. Ghoumari A.M., Dusart I., El-Etr M., Tronche F., Sotelo C., Schumacher M., et al. Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum. Proc Natl Acad Sci U S A (2003) 100:7953–7958.[Abstract/Free Full Text]
  55. Ghoumari A.M., Wehrle R., De Zeeuw C.I., Sotelo C., Dusart I. Inhibition of protein kinase C prevents Purkinje cell death but does not affect axonal regeneration. J Neurosci (2002) 22:3531–3542.[Abstract/Free Full Text]
  56. Mihailidou A.S., Mardini M., Funder J.W. Rapid, nongenomic effects of aldosterone in the heart mediated by epsilon protein kinase C. Endocrinology (2004) 145:773–780.[Abstract/Free Full Text]
  57. Aoyama T., Matsui T., Novikov M., Park J., Hemmings B., Rosenzweig A. Serum and glucocorticoid-responsive kinase-1 regulates cardiomyocyte survival and hypertrophic response. Circulation (2005) 111:1652–1659.[Abstract/Free Full Text]
  58. Park J., Leong M.L., Buse P., Maiyar A.C., Firestone G.L., Hemmings B.A. Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3- kinase-stimulated signaling pathway. EMBO J (1999) 18:3024–3033.[CrossRef][ISI][Medline]
  59. Hayashi M., Tapping R.I., Chao T.H., Lo J.F., King C.C., Yang Y., et al. BMK1 mediates growth factor-induced cell proliferation through direct cellular activation of serum and glucocorticoid-inducible kinase. J Biol Chem (2001) 276:8631–8634.[Abstract/Free Full Text]
  60. Shioi T., Kang P.M., Douglas P.S., Hampe J., Yballe C.M., Lawitts J., et al. The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J (2000) 19:2537–2548.[CrossRef][ISI][Medline]
  61. Nicol R.L., Frey N., Pearson G., Cobb M., Richardson J., Olson E.N. Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy. EMBO J (2001) 20:2757–2767.[CrossRef][ISI][Medline]

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