ALDOSTERONE AND IL-18 EXPRESSION Aldosterone induces ...

Articles in PresS. Am J Physiol Heart Circ Physiol (July 25, 2008). doi:10.1152/ajpheart.00148.2008 1

ALDOSTERONE AND IL-18 EXPRESSION

Aldosterone

induces

Interleukin-18

through

Endothelin-1,

Angiotensin-II,

Rho/Rho-kinase, and PPARs in cardiomyocytes.

Takashi Doi1, Tsuyoshi Sakoda1, Takafumi Akagami1, Toshio Naka1, Yoshitomo Mori1, Takeshi Tsujino2, Tohru Masuyama2, Mitsumasa Ohyanagi1

1

Department of Internal Medicine, Division of Coronary Heart Disease

2

Department of Internal Medicine, Cardiovascular Division

Hyogo College of Medicine, Nishinomiya, Japan

Address for correspondence; Tsuyoshi Sakoda, Department of Internal Medicine, Division of Coronary Heart Disease, 1-1, Mukogawa-Cho, Nishinomiya-City, Hyogo, Japan Telephone number: ＋81-798-45-6553 Fax number: ＋81-798-45-6551 [email protected]

Running head: Aldosterone and IL-18 expression.

Copyright © 2008 by the American Physiological Society.

2


Abstract Aldosterone (Aldo) is recognized as an important risk factor for cardiovascular diseases.

IL-18 induces myocardial hypertrophy, loss of contractility of

cardiomyocytes and apoptosis leading myocardial dysfunction.

However, so far, there

have been few reports concerning the interaction between Aldo and IL-18. The present study examined the effects and mechanisms of Aldo on IL-18 expression and the roles of Peroxisome proliferator-activated receptors (PPAR) agonists in rat cardiomyocytes. We used cultured rat neonatal cardiomyocytes stimulated with Aldo in order to measure IL-18 mRNA and protein expression, and Rho-kinase and NF-kB activity.

We also

investigated the effects of PPAR agonists on these actions. Aldo, endothelin-1 (ET-1), and angiotensin-II (Ang-II) increased IL-18 mRNA and protein expression. Mineralocorticoid receptor antagonists, endothelin A receptor antagonist, and angiotensin-II receptor antagonist inhibited Aldo-induced IL-18 expression. induced ET-1 and Ang-II production in cultured media.

Aldo

Moreover, Rho/Rho-kinase

inhibitor and statin inhibited Aldo-induced IL-18 expression. On the other hand, Aldo up-regulated the activities of Rho-kinase and NF-kB.

PPAR agonists attenuated the

Aldo-induced IL-18 expression and NF-kB activity but not the Rho-kinase activity. Our findings indicate that Aldo induces IL-18 expression through a mechanism that involves, at a minimum, ET-1 and Ang-II acting via the Rho/Rho-kinase and PPAR/NF-kB pathway. The induction of IL-18 in cardiomyocytes by Aldo, ET-1, and Ang-II, might therefore cause a deterioration of the cardiac function in an autocrine and paracrine fashion.

The inhibition of the IL-18 expression by PPAR agonists might be

one of the mechanisms whereby the beneficial cardiovascular effects are exerted. Key words: Interleukin-18, Aldosterone, Angiotensin-II, Rho/Rho-kinase, PPARs

3


Introduction Interleukin-18 (IL-18), a member of the IL-1 family, is a proinflammatory cytokine with multiple biologic functions (36, 38).

IL-18, originally identified as an

interferon γ-inducing factor (IGIF) (34), can induce TNFα and IL-6 expression in murine macrophages (34).

IL-18 is expressed by both immune and non-immune cells,

and plays a critical role in the pathophysiology of various diseases including myocardial ischemia, myocardial infarction, and myocarditis.

In particular, IL-18 is thought to

induce a proinflammatory response in the myocardium through different mechanisms including increased expression of endothelial cell adhesion molecules (55) and production of proinflammatory cytokines, such as TNFα, IL-1β, and IL-8. It also brings bout the expression of inducible nitric oxide synthase (37, 41).

These

molecules are implicated in the modulation of myocardial contractile function (7, 26) and myocyte apoptosis (29).

Myocardial hypertrophy is a major cause of myocardial

dysfunction and cardiac remodeling.

It has been shown that IL-18 induces cardiac

myocyte hypertrophy through activation of phosphatidylinositol 3-kinase (PI3K), Akt, and GATA-4 signaling pathway (6).

Furthermore, atrial natriuretic peptide (ANP)

expression is upregulated in the myocardium of patients with congestive heart failure leading to myocardial hypertrophy (49).

In addition, the effect of IL-18 on left

ventricular (LV) function was examined.

Daily intraperitoneal injection of IL-18

increased LV end-diastolic pressure and reduced β-adrenergic responsiveness to isoproterenol (57).

We and others have reported that there are increased levels of

circulating IL-18 in patients with acute coronary syndromes and congestive heart failure (24, 25, 34). Furthermore, an epidemiologic study suggested that IL-18 can predict cardiovascular death in patients with stable and unstable angina (24, 61).

IL-18

4


signaling has been studied in depth in immune cells where it activates NF-kB and p38 mitogen activated protein kinase (p38 MAPK), this signaling involves IL-1 receptor associated kinases (IRAKs 1 and 4), and adaptor proteins like MyD88 and TRAF-6 (45). In recent studies it was shown that IL-18 could activate AP-1 and NF-kB dependent inflammatory genes in vascular smooth muscle cells (VSMCs).

However, there have

so far been few studies that have precisely addressed how IL-18 expression is regulated under both physiological and pathophysiological conditions. Aldosterone is an important mediator of the renin-angiotensin-aldosterone system (RAAS) that is involved in a variety of pathophysiological processes associated with cardiovascular events.

Aldosterone has been reported to induce vascular inflammation

(44), endothelial dysfunction (12), cardiac fibrosis (43), and cardiac hypertrophy (33). These results indicate that aldosterone is an important risk factor for cardiovascular diseases. Endothelin-1 (ET-1) and angiotensin-II (Ang-II) also contribute to pathophysiological conditions including cardiac hypertrophy and remodeling (8, 21).

Furthermore,

aldosterone has been reported to upregulate ET-1 (14, 58) and angiotensin converting enzyme (ACE) activity and Ang-II (18).

These findings suggest that aldosterone is

closely related to ET-1- and Ang-II-induced cardiovascular disorders. However, the cellular mechanism whereby aldosterone contributes to cardiovascular disorders is still not well understood. A small GTPase, RhoA, and Rho binding protein, Rho-kinase, participate in cytoskeletal organization, smooth muscle contraction and gene expression.

Ang-II

increases the TNFα gene expression through RhoA and Rho-kinase in cardiomyocytes (23).

Recently, RhoA was reported to be one of the targets of the pleiotropic effect of

5


statins (53). In dilated cardiomyopathy patients, simvastatin improved functional class assessed by New York Heart Association (NYHA) and decreased the concentration of TNFα in blood (35).

ET-1, similar to Ang-II, has been reported to induce myocyte

hypertrophy through RhoA.

Accumulating evidence has shown that Rho-kinase also

plays an important role in such pathophysiological conditions including hypertension (16), coronary vasospasm (30), inflammation (28), and atherosclerosis (27). Nuclear factor kappa B (NF-kB) is the one of the most important transcription factors related to the mechanism of inflammation. NF-kB has been known to be activated by proinflammatory cytokine, lipopolysaccharide, and reactive oxygen species (1).

The

activation of NF-kB has been reported to play an important role in the pathogenesis of cardiac remodeling and heart failure (40).

The IL-18 gene sequence contains the

NF-kB binding site (13). Furthermore, it has been reported that TNFα induces IL-18 expression in cardiomyocytes via NF-kB activation (5). However, there have so far been few reports concerning the relationship and mechanism between NF-kB activation and aldosterone-, ET-1-, and Ang-II-induced IL-18 expression in cardiomyocytes. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate gene expression by binding with the retinoid X receptor (RXR) to PPAR-responsive elements (PPREs).

PPARs have three independent isoforms; PPARα, β/δ, and γ.

PPARs have been reported to regulate lipid metabolism (50) and possess pronounced anti-inflammatory activities (32).

Furthermore, clinical trials have shown fibrates

(PPARα/γ agonist) have a beneficial effect on cardiovascular disease and stroke (2) while pioglitazone (PPARγ agonist) reduces the composite of all-cause mortality, non-fatal myocardial infarction, and stroke in patients with type 2 diabetes who have a high risk of macrovascular events (PROactive study) (10). Recent studies have shown

6


PPARs inhibit cardiac hypertrophy, have an antifibrotic effect, and suppress cardiac remodeling by inhibition of NF-kB activation (4, 11, 19, 20). However, there have so far been few reports addressing the interaction of aldosterone and IL-18, and the effect of PPAR agonists on these molecules.

This study was designed to clarify the

mechanism of the effect of aldosterone on IL-18 expression and the role of the Rho/Rho-kinase pathway and PPAR agonists, pioglitazone, and bezafibrate in rat cardiomyocytes.

Materials and methods

Materials Sprague-Dawley rats were purchased from Japan SLC Co. Ltd (Shizuoka, Japan).

All procedures involving animals conformed 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 were approved by the Animal Research Committee of Hyogo College of Medicine.

The standard culture media were

Dulbecco's modified Eagle medium (DMEM) and DMEM/Nutrient Mixture F-12 (DMEM/F-12, 1:1 (v/v) from GIBCO-BRL.

Olmesartan (Daiichi-Sankyo, Co, Tokyo,

Japan), an Angiotensin-II type I receptor blocker, simvastatin (Banyu, Co, Tokyo, Japan), an HMG-CoA reductase inhibitor, pioglitazone (Takeda, Co, Osaka, Japan), a PPARγ agonist, and bezafibrate (Kissei, Co, Nagano, Japan), a PPARα/γ agonist were kindly donated by Daiichi-Sankyo Co, Banyu Co, Takeda Co, and Kissei, Co, respectively.

All other materials and chemicals were obtained from commercial

7


sources.

Cell culture Primary cultures of neonatal rat cardiomyocytes were prepared as previously described (46, 54).

The culture medium was DMEM/F-12 supplemented with 5% calf

serum. 5-Bromodeoxyuridine (BrdU) (100mM) was added during the first 24 hours to prevent proliferation of Non Myocyte Cells (NMCs).

The cells were seeded at 37℃

in an atmosphere of 5% CO2. Two days after removal from serum-containing medium, the cultures were used for further experiments. The cells were cultured with or without various concentrations of aldosterone for various times. cardiomyocytes

were

treated

with

mineralocorticoid

For some experiments, receptor

inhibitors,

spironolactone and eplerenone, a protein synthesis inhibitor, cycloheximide, pioglitazone, bezafibrate, endothelin A receptor antagonist, BQ123, endothelin B receptor antagonist, BQ788, olmesartan, RhoA inhibitor, C3 toxin, Rho-kinase inhibitor, fasudil, simvastatin, mevalonolactone, specific NF-kB inhibitor, PDTC (pyrrolidine dithiocarbamate), SN50 which is characterized cell-permeable peptide that acts as a specific inhibitor of translocation of the NF-kB active complex into the nucleus and a nonfunctional mutant of SN50, SN50M (39) simultaneously with aldosterone stimulation.

For each experimental condition, six myocyte cultures from three

separate isolations were included in the study.

The cell viability after various

stimulations or exposure to drugs at different times and concentrations exceeded 95% as assessed by trypan blue exclusion (data not shown).

Design of Primers and Probes

8


Oligonucleotide primers and TaqMan probes for rat IL-18 were designed based on the cDNA sequences reported in the GenBank database. The forward primer was 5’-AAACCCGCCTGTGTTCGA-3’,

the

5’-TCAGTCTGGTCTGGGATTCGT-3’,

and

5’-ACATGCCTGATATCGACCGAACAGCC-3’.

reverse the

primer

TaqMan

was

probe

was

The primers and the TaqMan probe

for rat β-actin were purchased from Applied Biosystems (Tokyo, Japan).

Isolation of Total RNA and PCR Total RNA was extracted from cardiomyocytes by using TRIzol (Invitrogen) according

to

the

manufacturer’s

protocol.

Real-time

quantitative

reverse

transcription-polymerase chain reaction (RT-PCR) was performed to screen for the expression of Interleukin-18 and β-actin. RT-PCR reactions were prepared with the TaqMan One-Step RT-PCR Master Mix Reagents Kit with an ABI Prism 7900 HT Detection System (Applied Biosystems) according to the manufacturers’ protocol.

The

same amount of reagents, primers, and probes were used for every reaction.

To

obtain a calibration curve, serial dilutions of stock standard RNA (total RNA extracted from rat cardiomyocytes; 100, 50, 25, 12.5, and 6.25 ng) were used. The threshold cycle (Ct) is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold value above baseline. The target message in the unknown samples is quantified by measuring the Ct and by using a calibration curve to determine the starting target message quantity.

Cts ranging from

27 to 30 in the assays for β-actin, and from 33 to 36 for IL-18 were adopted.

The

reaction mixtures were subjected to the following amplification scheme: one cycle at 48℃ for 30 min (reverse transcription) and one cycle at 95℃ for 10 min (AmpliTaq

9


Gold activation), followed by 40 cycles each consisting of denaturation at 95℃ for 15 s and extension/ annealing at 60℃ for 1 min.

The relative amount of each mRNA was

normalized by comparison with the quantity of mRNA of a housekeeping gene, β-actin.

IL-18 Protein synthesis Cell lysates were prepared from cardiomyocytes suspended in phosphate buffered saline, pH 7.4 (PBS), by sonication (59).

IL-18 was measured using a human

IL-18 ELISA Kit (Medical & Biological Laboratories, Aichi, Japan) according to the manufacturer’s protocol. The assay uses a sandwich ELISA method that employs two monoclonal antibodies against two different epitopes of human IL-18 in a 96-well ELISA format.

The concentrations of ET-1 and Ang-II secreted in response to

aldosterone stimulation were measured by SRL Co (Tokyo, Japan).

Rho-kinase activity Rho-kinase activity was measured using the Rho-Kinase Assay Kit (CycLex, Nagano, Japan) according to the manufacturer’s protocol. peroxidase

coupled

anti-phospho-MBS

(Myosin-Binding

This assay uses a Subunit

of

myosin

phosphatase) threonine 696 monoclonal antibody as a reporter molecule in a 96-well ELISA format.

NF-kB activity The NF-kB activity was measured using the NF-kB/p65 ActivELISA Kit (IMGENEX, San Diego, USA) according to the manufacturer’s protocol. This kit uses a sandwich ELISA with an anti-p65 antibody.

10


Statistical Analysis Values are reported as the mean ± standard error of mean (SEM).

The

statistical analysis was performed using an ANOVA followed by the Bonferroni test (Statview version 5, Abacus Concepts).

Differences were considered to be statistically

significant when the probability value, P, was ＜0.05.

Results

Aldosterone induced IL-18 expression in a time- and dose-dependent manner via the mineralocorticoid receptor (MR) by a genomic reaction. The ability of aldosterone to induce IL-18 mRNA expression was first examined in cultured rat neonatal cardiomyocytes.

Aldosterone increased IL-18

mRNA expression in a time- and dose-dependent manner in rat neonatal cardiomyocytes (Fig. 1A, 1B).

A significant increase was found which peaked at 48 hr

after stimulation by a concentration of 500 nM aldosterone (5-fold increase; Fig. 1A). Fig. 1B shows a significant increase using 500 nM aldosterone at 48 hr (5-fold increase). With these results, we determined IL-18 mRNA expression at 48 hr after stimulation with 500 nM aldosterone using various antagonists. To determine whether the aldosterone-induced IL-18 mRNA expression was mediated via the MR, the effects of MR antagonists were examined.

Addition of an

MR antagonists, spironolactone (1 µM) or eplerenone (10 µM) resulted in a significant reduction in aldosterone-induced IL-18 mRNA expression.

Spironolactone and

11


eplerenone alone did not significantly affect the basal levels of IL-18 mRNA expression (Fig. 1C).

Therefore, aldosterone-induced IL-18 mRNA expression in cardiomyocytes

is likely to be mediated via the MR. Aldosterone increased IL-18 mRNA expression with a peak induction at 48 hr after addition.

This was a considerably long time for mRNA expression. Therefore, to

determine whether aldosterone-induced IL-18 mRNA expression requires de novo protein synthesis, the effect of a protein synthesis inhibitor, cycloheximide, was assessed on mRNA expression. Aldosterone-induced IL-18 mRNA expression was completely inhibited by treatment with cycloheximide (100 µM).

Cycloheximide

alone did not significantly affect the basal levels of IL-18 mRNA expression (Fig. 1C). These results indicate that IL-18 expression requires de novo protein synthesis via an MR-mediated genomic reaction.

Aldosterone increases IL-18 mRNA and protein expression via the endothelin A receptor (ETAR) and Ang-II receptor (AT-IIR). Aldosterone has been reported to up-regulate ET-1 and Ang-II production (14, 18, 58).

To determine whether aldosterone-induced IL-18 mRNA expression is

mediated by ET-1 and Ang-II production, the effects of ET-1 receptor and AT-IIR antagonists on IL-18 mRNA expression were assessed.

The ETAR antagonist, BQ123

(1 µM) and the AT-IIR antagonist, olmesartan (10 µM) led to a significant reduction in aldosterone-induced IL-18 mRNA expression. However, the endothelin B receptor (ETBR) antagonist BQ788 (1 µM) did not inhibit these effects.

BQ123, BQ788, and

olmesartan alone did not significantly affect the basal levels of IL-18 mRNA expression (Fig. 2A).

Furthermore, ET-1 (10 nM) and Ang-II (100 nM) induced IL-18 mRNA

12


expression with peak inductions at 4 hr and 8 hr after stimulation, respectively (Fig. 2B, 2C).

These results indicate that IL-18 is likely produced in response to aldosterone

through a pathway involving ET-1 and Ang-II production via ETAR and AT-IIR. Aldosterone (500 nM) increased the ET-1 and Ang-II production in the conditioned media, and concentration of ET-1 and Ang-II peaked at 24 hr after stimulation (Fig. 3A, 3B).

Without aldosterone stimulation ET-1 and Ang-II were not detected in the

conditioned media.

Moreover, ET-1 (10 nM) and Ang-II (100 nM) increased the

production of IL-18 protein measured at 12 hr and the concentration peaked at 24 hr after stimulation (Fig. 3C).

IL-18 protein expression is represented as the fold

increases compared with the beginning of various stimulations (at 0 hr).

Subsequently,

aldosterone (500 nM) increased IL-18 protein expression with a peak induction at 60 hr after stimulation (Fig. 3D).

These results indicate that aldosterone induces IL-18

expression through intermediates, ET-1 and Ang-II, via ETAR and AT-IIR.

The Rho/Rho-kinase pathway is involved in aldosterone-induced IL-18 expression. The Rho/Rho-kinase pathway is associated with ET-1- and Ang-II-induced gene expression in cardiomyocytes.

To determine whether the Rho/Rho-kinase

pathway is involved in the aldosterone-induced IL-18 expression, the effects of RhoA and Rho-kinase inhibitors and statin were evaluated on IL-18 mRNA expression. Treatment with RhoA inhibitor C3 toxin (1 µM) and the Rho-kinase inhibitor fasudil (100 µM) led to a significant reduction in aldosterone-induced IL-18 mRNA expression. C3 toxin and fasudil alone did not significantly affect the basal levels of IL-18 mRNA expression (Fig. 4A).

Futhermore, HMG-CoA reductase inhibitor, simvastatin (1 µM)

which is known to inhibit the isoprenilation of small G protein including RhoA also

13


inhibited the aldosterone-induced IL-18 mRNA expression.

Mevalonate (100 µM) in

combination with simvastatin reversed the inhibitory effects of simvastatin on IL-18 mRNA expression. Simvastatin and mevalonate alone did not significantly affect the basal levels of IL-18 mRNA expression (Fig. 4B).

Rho-kinase activity, determined by

phosphorylation of MBS (Myosin-Binding Subunit of myosin phosphatase), which is known to be one of the target molecules of Rho-kinase, was observed with a peak induction at 24 hr after stimulation by aldosterone (500 nM) (Fig. 5A).

Rho-kinase

activity is represented as the fold increases compared with the beginning of various stimulations (at 0 hr).

These effects were prevented by the AT-IIR antagonist,

olmesartan (10 µM) and ETAR antagonist, BQ123 (1 µM).

However, the PPARγ

agonist, pioglitazone (10 µM) and PPARα/γ agonist, bezafibrate (100 µM) did not inhibit these reactions (Fig. 5B).

Furthermore, Rho-kinase activity was up-regulated

also by ET-1 (10 nM) and Ang-II (100 nM) with a peak induction at 2 hr (Supplemental Figs. S1A and S1C).

(Supplemental data for this article is available

online at the American Journal of Physiology Heart and Circulatory Physiology website: http://ajpheart.physiology.org).

BQ123 and olmesartan inhibited the ET-1-

and Ang-II-induced Rho-kinase activity, respectively.

However, pioglitazone and

bezafibrate did not inhibit these effects (Supplemental Figs. S1B and S1D).

These

results indicate that aldosterone induces IL-18 expression via the Rho/Rho-kinase pathway through ET-1 and Ang-II production.

PPAR agonists, pioglitazone and bezafibrate attenuated the aldosterone-induced IL-18 expression. PPAR agonists inhibit inflammatory responses and suppress the production of

14


inflammatory cytokines in several tissues and cells (22).

Cardiomyocytes were treated

with aldosterone in the presence or absence of PPARγ agonist, pioglitazone (10 µM) or PPARα/γ agonist bezafibrate (100 µM) (Fig. 6).

Addition of pioglitazone or

bezafibrate led to a significant reduction in the aldosterone-induced IL-18 mRNA expression. Pioglitazone and bezafibrate also inhibited the ET-1 and Ang-II-induced IL-18 mRNA expression.

Pioglitazone and bezafibrate alone did not significantly

affect the basal levels of IL-18 mRNA expression (Supplemental Figs. S2A and S2B). However, pioglitazone and bezafibrate did not inhibit the aldosterone-induced ET-1 and Ang-II secretion (data not shown) and aldosterone-, ET-1-, and Ang-II-induced Rho-kinase phosphorylation.

Furthermore, pioglitazone and bezafibrate did not induce

Rho-kinase phosphorylation by themselves (Fig. 5B, Supplemental Figs. S1B and S1D).

These results indicate that PPAR agonists attenuate the aldosterone-induced

IL-18 expression at a point downstream from Rho-kinase.

Aldosterone induced NF-kB activity, and pioglitazone and bezafibrate inhibited this action. NF-kB is a key transcription factor that regulates inflammatory processes. The activation of NF-kB has been reported to increase proinflammatory proteins. Lastly, we examined whether aldosterone induced NF-kB activity.

Aldosterone (500

nM) up-regulated NF-kB activity with a peak at 24 hr after start of stimulation (Fig.7A). NF-kB activity is represented as the fold increases compared with the beginning of various stimulations (at 0 hr). activity.

PPAR agonists have been shown to inhibit NF-kB

The aldosterone-induced NF-kB activity was inhibited by pioglitazone (10

µM) and bezafibrate (100 µM) (Fig. 7B).

ET-1 (10 nM) and Ang-II (100 nM) also

15


induced NF-kB activity with a peak at 2 hr after start of stimulation and pioglitazone and bezafibrate inhibited the ET-1- and Ang-II-induced NF-kB activity (Supplemental Figs. S3A-S3D).

Specific NF-kB inhibitor, PDTC (100 µM) and SN50 (10 µM)

inhibited the aldosterone-, ET-1-, and Ang-II-induced NF-kB activity, but not SN50M (10 µM), a nonfunctional mutant of SN50 (Fig.7B, Supplemental Figs. S3B and S3D). Likewise, PDTC and SN50 inhibited the aldosterone-induced IL-18 mRNA expression, but not SN50M (Fig.7C). These results indicate that PPAR agonists attenuate the aldosterone-induced NF-kB activity.

Discussion In the present study, we demonstrated for the first time that aldosterone, a proinflammatory cytokine, induces IL-18 mRNA expression in cultured neonatal rat cardiomyocytes.

This effect was blocked by an MR antagonist.

Furthermore, the

aldosterone-induced IL-18 mRNA expression was blocked by a protein synthesis inhibitor, suggesting that the aldosterone induced IL-18 mRNA expression proceeds via an MR-mediated genomic reaction. The maximal induction of IL-18 was 48 hr at the mRNA level and 60 hr at the protein level after aldosterone stimulation. These results indicate the possibility that some molecules might act as an intermediate in aldosterone-induced IL-18 expression.

ET-1 and Ang-II have been reported to be

produced in response to aldosterone stimulation (14, 18, 58).

This study examined the

effect of the ET-1 and Ang-II receptor on aldosterone-induced IL-18 expression. The ETAR antagonist, BQ123 and AT-IIR antagonist, olmesartan inhibited the aldosterone-induced IL-18 mRNA expression. However, the ETBR antagonist, BQ788

16


did not inhibit the aldosterone-induced IL-18 mRNA expression.

Furthermore, ET-1

and Ang-II induced IL-18 mRNA expression maximally at 4 hr and 8 hr, respectively. Moreover, the ET-1 and Ang-II protein concentrations which peaked at 24 hr were up-regulated by aldosterone stimulation.

These results suggest that initially,

aldosterone stimulates ET-1 and Ang-II synthesis in cardiomyocytes, and then ET-1 and Ang-II up-regulate the IL-18 expression. Rho-kinase and RhoA are involved in the ET-1- and Ang-II- induced signal transduction pathway.

Rho-kinase has been reported to be involved in the signal

transduction pathway in ET-1- and Ang-II-induced cardiovascular hypertrophy in vivo and in vitro (17, 30).

Rho-kinase has also been reported to be involved in the

pathogenesis of LV remodeling after a myocardial infarction and is associated with upregulation of proinflammatory cytokines (15).

These reports suggest that

Rho-kinase is involved in the ET-1- and Ang-II-induced IL-18 expression.

In fact,

fasudil, a Rho-kinase inhibitor, significantly reduced the IL-18 mRNA expression. Furthermore, Rho-kinase was phosphorylated by ET-1 or Ang-II stimulation in this study. An HMG-CoA reductase inhibitor, a statin, prevents the development of cardiac hypertrophy in a cholesterol-independent manner. part,

to

the

inhibition

of

isoprenilation

of

small

This mechanism is due, in G

proteins

geranylgeranylation of Rho and Rac, and farnesylation of Ras (53).

including

A recent study

demonstrated that short-term statin therapy has a beneficial effect in patients with nonischemic, dilated cardiomyopathy.

Statin decreases the markers of inflammation

while improving neurohormonal imbalance and the cardiac function (35).

Simvastatin,

an HMG-CoA reductase inhibitor, decreases the myocardial TNFα expression in heart

17


transplant recipients (56).

RhoA has been reported to be involved in the ET-1- and

Ang-II-induced gene expression. Furthermore, this molecule is involved in ET-1- and Ang-II-induced cardiac hypertrophy (3).

In this study, simvastatin led to a significant

reduction in the IL-18 mRNA expression.

Furthermore, the specific RhoA inhibitor C3

toxin inhibited the aldosterone-induced IL-18 mRNA expression. There is thus, a possibility that simvastatin might inhibit the participation of the small G protein RhoA in combination with Rho-kinase that are involved the signal transduction pathway of ET-1- and Ang-II-induced IL-18 expression. The inhibition of the IL-18 expression by simvastatin might therefore be a novel mechanism for the pleiotropic effects of HMG-CoA reductase inhibitors. PPARα activators inhibit the inflammatory responses in aortic smooth muscle cells (51), and PPARγ activators suppress the production of inflammatory cytokines in activated macrophages (22, 42).

Furthermore, in cardiomyocytes, PPARγ inhibits

Ang-II-, and mechanical stretch-induced cardiac hypertrophy and PPARα, and γ inhibit lipopolysaccharide-induced TNFα expression (52, 60).

On the other hand, angiotensin

type-1 receptor blockers induce PPARγ activity and statins activate PPARα activity via the Rho signal cascade (31, 48). Consequently, the activation of PPARs is thought to have a beneficial effect on cardiovascular diseases. In this study, the effects of PPARγ agonist, pioglitazone, and PPARα/γ agonist, bezafibrate, on aldosterone-induced IL-18 expression were examined.

Pioglitazone and bezafibrate significantly reduced the

aldosterone-induced IL-18 mRNA expression.

Furthermore, pioglitazone and

bezafibrate significantly reduced ET-1-, and Ang-II-induced IL-18 mRNA expression. However, pioglitazone and bezafibrate did not inhibit the aldosterone-induced ET-1 and Ang-II

induction

and

aldosterone-,

ET-1-,

and

Ang-II-induced

Rho-kinase

18


phosphorylation.

These results indicate that pioglitazone and bezafibrate inhibit

aldosterone-, ET-1-, and Ang-II-induced IL-18 expression downstream of Rho-kinase. The mechanism by which PPARs inhibit the IL-18 expression is unknown. However, ligand-activated PPARs are known to positively regulate the gene expression through binding to a specific DNA sequence (PPAR response element) (47) or by inhibiting other types of gene expression, in part, through antagonism of the activities of other transcription factors, such as NF-kB (42).

A recent study revealed that aldosterone

activates NF-kB and PPAR agonists inhibit NF-kB activation (9). In this study, the effects of PPAR agonists, pioglitazone and bezafibrate on aldosterone-, ET-1-, and Ang-II-induced NF-kB activity were also examined.

Pioglitazone and bezafibrate

attenuated the aldosterone-, ET-1-, and Ang-II-induced NF-kB activity.

From these

results, aldosterone appears to induce the IL-18 expression at least through ET-1 and Ang-II via the Rho/Rho-kinase and PPAR/NF-kB pathway (Fig. 8). Further studies are needed to fully elucidate this mechanism. In conclusion, the induction of IL-18 in cardiomyocytes might cause a deterioration of cardiac function in an autocrine and paracrine fashion.

The inhibition

of IL-18 expression by PPAR agonists might provide some beneficial cardiovascular effects. The MR antagonist, AT-IIR antagonist, ETAR antagonist, Rho/Rho-kinase inhibitor, statin, and PPAR agonists may therefore play a critical role in the aldosterone-induced cardiovascular diseases, such as heart failure, ischemia reperfusion, and myocardial infarction. These effects have already been reported in our clinical studies related to the serum concentrations of IL-18 (24, 25, 34).

19


Acknowledgments We are grateful to N. Kumon for her skillful secretarial assistance.

Grants This study was supported by a Grant-in-Aid for Researchers, Hyogo College of Medicine (T. S.), and a Grant-in-Aid for the promotion of technological SEEDS in advanced medicine, Hyogo College of Medicine (M. O.).

Disclosures None.

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Figure Legends

Figure 1.

The effects of aldosterone on IL-18 mRNA expression and spironolactone,

eplerenone, and cycloheximide on aldosterone-induced IL-18 mRNA expression. Aldosterone at 500 nM increased the IL-18 mRNA expression in a time-dependent manner (A).

IL-18 mRNA expression after 48 hr stimulation by aldosterone increased

in a dose-dependent manner (B) and the maximal effect was observed at an aldosterone concentration of 500 nM in rat neonatal cardiomyocytes.

Cultured cardiomyocytes

from neonatal rats were stimulated by aldosterone (Aldo, 500 nM), and incubated with or without spironolactone (Spi, 1 µM), eplerenone (Eple, 10 µM), and cycloheximide (Cyc, 100 µM) for 48 hr.

Addition of spironolactone, eplerenone, or cycloheximide

led to a significant reduction in aldosterone-induced IL-18 mRNA expression (C). The ratio of IL-18/β-actin was determined using the RT-PCR as described in the Materials and Methods.

The values are means±standard errors (n=6).

versus the control group (C).

Figure 2.

*P