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Am J Physiol Heart Circ Physiol 275: H906-H916, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 3, H906-H916, September 1998

Role of AT1 and AT2 receptors in regulation of MAPKs and MKP-1 by ANG II in adult cardiac myocytes

Thomas A. Fischer, Krishna Singh, Donald S. O'Hara, David M. Kaye, and Ralph A. Kelly

Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

ANG II has been implicated in the hypertrophic response in ventricular myocytes by acting at the angiotensin type 1 (AT1) receptor. However, the role of the angiotensin type 2 (AT2) receptor in the adult heart is not as clearly understood. In adult rat ventricular myocytes (ARVM) and cardiac microvascular endothelial cells (CMEC), we examined the role of ANG II signaling, via AT1 and AT2 receptors, on the activation of the extracellular signal-regulated protein kinases (ERKs) and on the expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1. ANG II caused no detectable increase in ERK activity or in c-fos mRNA abundance in ARVM but increased ERK activity within 5 min in CMEC and increased c-fos mRNA levels. However, in the presence of the selective phosphoprotein phosphatase (PP-2A/PP-1) inhibitor okadaic acid (OA), a sustained increase in ERK activity, as well as in c-jun NH2-terminal protein kinase activity, in ARVM was observed. ANG II increased MKP-1 mRNA levels within 15 min in ARVM and CMEC. In contrast to the response in endothelial cells, however, ANG II activation of MKP-1 in ARVM was mediated by AT2-receptor activation. Thus there is constitutive as well as inducible suppression of ERKs and c-jun NH2-terminal protein kinases by MKP and PP-2A/PP-1 in the adult cardiac myocyte phenotype.

cardiac hypertrophy; angiotensin type 2 receptor; mitogen-activated protein kinases; mitogen-activated protein kinase phosphatase 1; okadaic acid

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

AN INCREASING NUMBER of biologic mediators have been implicated in initiating or regulating the growth response of cardiac muscle to physiological stress (24, 25, 35, 42, 46, 50, 59). Although the role of signal transduction cascades leading to tyrosine and threonine phosphorylation and activation of mitogen-activated protein kinases (MAPKs) by cytokines, peptide growth factors, and norepinephrine has been well described (5, 26, 37, 41, 42, 44, 45, 52, 55), their role in regulating the expression of inactivating phosphatases in the heart has not been as extensively studied. Neither the expression of the nuclear dual-specificity tyrosine/threonine protein phosphatase MKP-1, a principal inactivating phosphatase of extracellular signal-regulated protein kinase (ERK)1/ERK2 in many cell types, nor the activities of the serine/threonine phosphatases PP-2A and PP-1 have been studied in detail in cellular constituents of cardiac muscle. MKP-1 is one of a family of related MAPK phosphatases (MKPs), which, along with MKP-2, is known to be abundantly expressed in cardiac muscle (30).

Much attention has been focused on the role of angiotensins in mediating cardiac hypertrophy and the ventricular remodeling that follows myocardial injury. This is because agents that inhibit the activity or the generation of ANG II limit myocardial growth in response to increased hemodynamic load in experimental animals and in humans (39). In addition, Sadoshima and colleagues (42-46) demonstrated that increased mechanical stretch of neonatal rat ventricular myocytes causes the release of ANG II, which, acting at angiotensin type 1 (AT1) receptors in these cells, triggers several intracellular signaling cascades that result in cell growth. These include the activation of ERK1/ERK2 and the phosphorylation by apparently independent pathways of p90RSK and p70S6K (7, 17). However, the function of the angiotensin type 2 (AT2) receptor in the adult cardiac myocyte is not fully understood. In cultured smooth muscle cells, AT2-receptor transfection reduced proliferation and inhibited MAPK activity (33), whereas in a rat pheochromocytoma cell line and in mouse fibroblasts that express abundant AT2 receptors, the regulation of MKP-1 was associated with AT2-receptor activation (58).

However, there appear to be important differences between the phenotypic responses of neonatal ventricular myocytes and the responses of late postnatal and adult ventricular myocytes to mechanical stress or to ANG II itself (20, 21, 23-25). Although neonatal rat ventricular myocytes have been reported to contain renin, angiotensinogen, and angiotensin-converting enzyme (10, 11, 27, 35, 36), ANG II release in response to mechanical activity, if it occurs, appears to be at a lower rate than that observed in neonatal cells, since the increased protein synthesis and growth that occur in adult feline ventricular myocytes in vitro, induced to contract repetitively by continual uniform electric field pacing, was not affected by the presence of an AT1- or AT2-receptor antagonist (56).

Other cell types within the adult myocardium could also be a source of angiotensin generation. Coronary microvascular endothelial cells (CMEC) isolated from ventricular muscle do synthesize and secrete angiotensins, and this endothelial cell phenotype and late postnatal and adult cardiac myocytes express AT1 and AT2 receptors (15, 16, 35). In rat coronary endothelial cells, inhibition of the AT2 receptor by PD-123177 is followed by proliferation in response to ANG II, an effect that is reversed by pretreatment with losartan and highlights a possible antiproliferative function of this receptor (53).

In this report we examined the time course of activation of ERK1/ERK2 in adult rat ventricular myocyte (ARVM) primary isolates and serum-starved confluent CMEC primary cultures in vitro in response to ANG II and to the PP-2A/PP-1 inhibitor okadaic acid. MKP-1 gene expression also was examined in both cell types after exposure to ANG II receptor subtype-selective activation.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Isolation of AVRM. ARVM were isolated as previously described, with minor modifications (1). Briefly, hearts from male Sprague-Dawley rats (200-225 g) were excised under deep ether anesthesia and perfused with oxygenated modified Krebs-Henseleit bicarbonate buffer (KH) containing (in mM) 118 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 12 dextrose at 37°C. After 5 min the buffer was changed to calcium-free KH, and the hearts were digested with 200 ml of freshly prepared and recirculated calcium-free KH containing 0.35 mg/ml collagenase (Worthington) and 0.2 mg/ml hyaluronidase (Sigma Chemical) for 25 min. After removal of the atria, minced ventricular muscle was incubated in 20 ml of fresh digestion buffer including 0.02 mg/ml trypsin (Sigma Chemical) and 0.02 mg/ml DNase I (Worthington) in a shaking water bath at 37°C. The homogenate was agitated with a sterile pipette, filtered through 80-µm nylon mesh, resuspended in DMEM (GIBCO) containing 10% FCS (Sigma Chemical), and centrifuged at 50 g for 2 min. The pellet was washed with DMEM twice and sedimented through a 6% BSA gradient cushion. The final pellet was resuspended in a serum-free medium (DMEM) including 100 IU/ml penicillin and 100 µg/ml streptomycin (GIBCO). Purified ARVM were plated in DMEM on laminin-coated culture dishes (1 µg/cm2) at a density of 5 × 105-1 × 106 cells/dish. This preparation contains <5% contaminating cells, especially fibroblasts (14). The medium was changed after 1 h to remove loosely attached residual nonmyocyte cells. Rod-shaped myocytes were cultured in defined serum-free medium as previously described overnight at 37°C in 5% CO2, and all experiments were performed 12-16 h after isolation.

Isolation of adult CMEC. Rat CMEC were isolated as described previously (34), with minor modifications. Briefly, hearts from male Sprague-Dawley rats (200-225 g) were perfused with DMEM containing 100 IU/ml penicillin and 100 µg/ml streptomycin for 15 min. The atria, visible connective tissue, valvular tissue, and right ventricle were cut away, and the remaining left ventricle was briefly immersed in 70% ethanol to devitalize epicardial mesothelial and endocardial endothelial cells. The outer one-third of the free left ventricular wall was dissected and removed, and the remaining tissue was washed in calcium-free Hanks' medium (GIBCO). The tissue was minced finely and incubated for 15 min in 25 ml of Hanks' medium containing 30 mg of collagenase (Worthington) in a shaking water bath at 37°C. After the addition of 3 mg of trypsin (Sigma Chemical), the crude homogenate was agitated with a sterile pipette and incubated for another 15 min under the same conditions. The resulting cell suspension was filtered through 80-µm nylon mesh and centrifuged at 100 g for 5 min. The pellet was washed in 25 ml of Hanks' medium and resuspended in DMEM containing 20% heat-inactivated FCS (Sigma Chemical). Cells were plated on laminin-coated dishes for 1 h and then tapped briefly and washed to remove traces of nonattached contaminating nonendothelial cells. After cells were washed twice with DMEM, they were cultured in DMEM supplemented with 20% FCS. The medium was changed every 3 days, and the cells typically became confluent 7-10 days after isolation. Confluent CMEC primary cultures were serum starved 24 h before each experiment.

Northern blot analyses for MKP-1. Total RNA was isolated following the acid guanidinium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi (8), with some minor modifications. Fifteen micrograms of total RNA were lyophilized at 4°C and dissolved in 1× MOPS, 6% formaldehyde, and 50% formamide. The RNA samples were size fractionated on a 1% agarose gel containing 0.66 M formaldehyde at 80 mA. The integrity of the 18S and 28S tRNA bands was documented, and the gel was soaked in 50 mM NaOH for 30 min at room temperature. RNA was blotted onto a Gene Screen Plus membrane (Du Pont-NEN) in 10× saline-sodium citrate (SSC) transfer buffer using a vacuum blotter (Bio-Rad Laboratories, Hercules, CA), and the transferred RNA was ultraviolet cross-linked (Stratagene UV Crosslinker). Prehybridization was carried out in 50% formamide, 10% dextran sulfate, 1% SDS, 1 M NaCl, and 200 µg/ml salmon sperm DNA for 3 h to overnight at 42°C. Membranes were hybridized to a [32P]dCTP random primed-labeled 1-kb PST-1 restricted cDNA fragment encoding for mouse 3CH134 (MKP-1; gift of Dr. Lester F. Lau, Dept. of Genetics, University of Chicago) and c-fos (gift of Drs. J. Belasco and M. Greenberg, Dept. of Microbiology and Molecular Genetics, Harvard Medical School) overnight and washed twice in 2× SSC for 20 min at room temperature, 2× SSC-1% SDS for 60 min at 55°C (MKP-1) and 65°C (c-fos), and 0.1% SSC-0.1% SDS for 60 min at room temperature. The membrane was exposed to X-ray film (Kodak Xomat AR, NEN-Du Pont Reflection, or Kodak Biomax) for 12-48 h at -70°C. Equivalent loading was verified by reprobing of the same filters with an 18S oligonucleotide using the T4-kinase-labeling technique.

MAPK assay. Total protein from ARVM or CMEC primary culture cell lysates was extracted in ice-cold lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, 1 µM EGTA, pH 9.0, 0.4 mM phenylmethylsulfonyl fluoride, and 0.036% sodium vanadate, vortexed twice with intermittent chilling, and centrifuged at 4°C for 12 min to pellet the debris. Total protein was determined using the Bio-Rad Bradford assay or the Bio-Rad DC assay using microtiter plates. Fifty micrograms of total protein for the CMEC experiments or 100 µg of total protein for the ARVM experiments were boiled in equal volumes of 2× sample buffer containing 2% SDS, 0.062 M Tris (pH 6.8), 10% glycerol, 5% beta -mercaptoethanol, and 0.001% bromphenol blue. Samples were loaded on a 4.5% SDS-PAGE stacking gel and then separated by size in a 10% SDS-PAGE resolving gel containing 200 µg/ml myelin basic protein (MBP; Sigma Chemical). After electrophoresis, gels were washed in 50 mM Tris containing 20% 2-propanol at room temperature for 60 min with two washes to remove the SDS. The gel was soaked in 5 mM beta -mercaptoethanol freshly prepared in 50 mM Tris, pH 8.0 (buffer A), at room temperature for 60 min, and the proteins were denatured by addition of 6 M guanidinium to the buffer for an additional 60 min. Gels were then incubated overnight at 4°C in buffer containing 0.04% Tween 40, and the solution was changed every 20 min over the next 3 h. After it was washed, the gel was preincubated in kinase buffer (in mM: 20 HEPES, 1 MgCl2, 5 mM beta -mercaptoethanol) for 45 min, and the kinase reaction was started by addition of a mixture of gamma -[32P]ATP and unlabeled ATP for an additional 60 min. The reaction was terminated by washing the gel in 5% TCA containing 1% sodium pyrophosphate until the activity of the buffer became negligible. The gel was transferred to 3 MM Whatman paper, dried under vacuum, and exposed to autoradiography at room temperature and -70°C. Identity of extracellular regulated protein kinases (ERKs) in the in-gel kinase assays was previously described (52).

Immunoprecipitation of c-jun NH2-terminal protein kinases. After stimulation with okadaic acid (100 nM), ARVM were extracted in ice-cold lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, 1 µM EGTA, pH 9.0, 0.4 mM phenylmethylsulfonyl fluoride, and 0.036% sodium vanadate, vortexed twice with intermittent chilling, and centrifuged at 4°C for 12 min to pellet the debris. Total cell lysate (400 µg) was immunoprecipitated with 1 g of anti-c-jun NH2-terminal protein kinase type 1 (JNK1, FL) antibodies (Santa Cruz Biotechnology). This antibody immunoprecipitates JNK1 (p46) and JNK (p54). The immunoprecipitates were analyzed by in-gel kinase assay using MBP as substrate, as described above.

Chemicals and receptor antagonists. Purified rat [Sar1]ANG II was obtained from Peninsula (Belmont, CA). Losartan was a gift of Dr. Ronald D. Smith (Du Pont Merck Pharmaceutical, Wilmington, DE), and PD-123319 was purchased from Research Biochemicals (Natick, MA). Okadaic acid was purchased from GIBCO-BRL (Gaithersburg, MD). All other chemicals were obtained from Sigma Chemical (St. Louis, MO), unless otherwise stated.

Statistics. Values are means ± SE unless otherwise stated. The data were analyzed with the PC Scan Image System (Bio-Rad Molecular Analyst Software). Student's t-test or an appropriate nonparametric analogon (Mann-Whitney rank sum test), as well as one-way ANOVA and an appropriate nonparametric analogon (Kruskal-Wallis one-way ANOVA on ranks), was used for final data calculation. P < 0.05 was considered statistically significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

ANG II stimulates ERK1/ERK2 phosphorylation in isolated CMEC but not in ARVM. ANG II (100 nM) stimulated phosphorylation of ERK1 and ERK2 in confluent serum-starved primary cultures of microvascular endothelial cells. A characteristic time course of ANG II-induced increase in ERK phosphorylation in this cell type is shown in Fig. 1, and the data are summarized in Fig. 5B. Activation was rapid, resulting in a 4.0-fold increase in p42MAPK phosphorylation and a 2.3-fold increase in p44MAPK phosphorylation with a maximal response within 5-10 min after exposure to ANG II. The phosphorylation signal subsequently declined, returning to baseline within 20 min. Inhibition of the AT1 receptor with losartan (1 µM) significantly reduced activation of these MAPKs by 67% (Fig. 2) and contrasts with the effects of PD-123319, which had no statistically significant effect on ANG II-mediated activation of either MAPK, although these data cannot exclude a modest AT2-receptor effect. In contrast, ANG II activation of ERK1/ERK2 could not be demonstrated consistently in ARVM primary isolates (see below and Fig. 5).


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  		Fig. 1.   ANG II activates p42MAPK (ERK2) and p44MAPK (ERK1) in cardiac microvascular endothelial cells (CMEC). Confluent, serum-starved CMEC were treated with 100 nM ANG II for 5-60 min. Proteins were separated by SDS-PAGE, and an in-gel kinase assay was performed. Positions of extracellular-regulated protein kinases (ERKs) are indicated by arrows at left. p42MAPK phosphorylation increased by 4.0 ± 0.5-fold and p44MAPK phosphorylation by 2.3 ± 0.3-fold at 5 min (P < 0.05 vs. control, by t-test). Experiment is representative of 3 independent experiments with similar results.


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  		Fig. 2.   ANG II activation of ERKs is mediated by ANG II type 1 (AT1), rather than type 2 (AT2), receptor in CMEC. A: confluent, serum-starved CMEC were treated with 100 nM ANG II for 5 min in presence or absence of specific AT1-receptor antagonist losartan (1 µM) and AT2-receptor antagonist PD-123319 (1 µM). Proteins were separated by SDS-PAGE and in-gel kinase assay. Experiment is representative of 3 independent experiments. 12-O-tetradecanoylphorbol-13-acetate (TPA, 20 ng/ml) serves as a positive control. B: data for relative phosphorylation intensities of ERKs normalized to baseline activity of p42MAPK and p44MAPK in control CMEC cultures. Inhibition of AT1 receptor with losartan reduced activation of these mitogen-activated protein kinases (MAPKs) by 67% (* P < 0.05 vs. ANG II, ** P < 0.01 vs. control; by t-test). ANG II + PD-123319 was not significantly different from ANG II.

ANG II increases MKP-1 mRNA levels in ARVM and CMEC: role of AT1 and AT2 receptors. Cells were treated with 100 nM ANG II, and total RNA was isolated at successive time points. Baseline expression of MKP-1 mRNA was low in unstimulated CMEC and ARVM primary cultures (Fig. 3, A-D). ANG II increased mRNA abundance for MKP-1 in cardiac microvascular endothelial cells and myocytes in a time-dependent manner. In CMEC, incubation with ANG II was followed by a rapid induction of MKP-1 mRNA, with a 4.5-fold increase after 30 min of stimulation, then a rapid decline by 60 min. In ARVM a comparable 2.2-fold increase of MKP-1 mRNA was observed that was more sustained, and maximal induction was still obvious at 60 min with a delayed decline at 90 min (Fig. 3E). At 30 min, losartan and PD-123319 effectively suppressed the ANG II-mediated increase in MKP-1 mRNA levels in microvascular endothelial cell primary cultures. In contrast, as shown in Fig. 3B, the ANG II-mediated increase in MKP-1 mRNA abundance in cardiac myocytes was blocked by PD-123319 (1 µM), whereas losartan had no consistent effect. Neither losartan nor PD-123319 had an effect on MKP-1 mRNA levels in the absence of ANG II.


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  		Fig. 3.   ANG II increases type 1 MAPK phosphatase (MKP-1) mRNA levels in CMEC and adult rat ventricular myocytes (ARVM). CMEC (A) and ARVM (B) were treated with 100 nM ANG II for 30 min in presence or absence of AT1 (losartan)- and AT2 (PD-123319)-receptor antagonists; 15 µg of total RNA were isolated for each experimental condition. C and D: MKP-1 mRNA hybridization signals after correction with 18S rRNA for loading and normalized to those from control (untreated) CMEC or ARVM cultures. E: representative time course of activation of MKP-1 mRNA in CMEC and ARVM by 100 nM ANG II. ANG II increased MKP-1 mRNA in CMEC 4.5 ± 0.4-fold (P < 0.01 vs. control, t-test). In CMEC, AT1-receptor antagonist losartan and AT2-receptor antagonist PD-123319 significantly reduced this stimulation by 55 and 57%, respectively (P < 0.05, t-test). In ARVM, MKP-1 mRNA was induced 2.2 ± 0.18-fold (P < 0.01, t-test), and pretreatment with PD-123319 reduced this response by 51% (P < 0.05, t-test); losartan remains uneffective. Data are from 4 independent experiments. ** P < 0.01 vs. control; * P < 0.05 vs. ANG II.

Inhibition of PP-2A and PP-1 increases ERK1/ERK2 and p54JNK kinase activity in CMEC and ARVM. Okadaic acid is known to promote phosphorylation of ERK1/ERK2 by inhibiting the activity of PP-2A and PP-1 serine/threonine phosphatases in a number of cell types (28). Okadaic acid (100 nM) alone modestly increased ERK1/ERK2 activity within 5-15 min in serum-starved confluent CMEC primary cultures (1.7 ± 0.2- and 1.6 ± 0.3-fold vs. control at 5 min, respectively), and coadministration with ANG II increased the activities of these MAPKs to levels fourfold higher than with ANG II or okadaic acid alone (Fig. 4A). However, okadaic acid had no sustained effects on ERK activation after 20 min, nor did it affect the decline of the ANG II response in this cell type (Fig. 4B). In ARVM, okadaic acid alone caused a detectable increase in activation of ERKs within 5 min that was sustained over 2 h, but this response was not modified by ANG II at any time point in these cells, in contrast to CMEC (Fig. 5).


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  		Fig. 4.   Inhibition of phosphoprotein phosphatases PP-2A and PP-1 with okadaic acid (OA) potentiates ANG II activation of ERK1/ERK2 in CMEC. A: confluent, serum-starved CMEC primary cultures were treated with 100 nM ANG II for 5 min in presence or absence of okadaic acid (100 nM). Proteins were separated by SDS-PAGE, and in-gel kinase assay was performed. Positions of ERKs are indicated by arrows at left. Experiment is representative of 4 independent experiments with similar results. B: at 5 min of stimulation, ANG II increased p42MAPK 4.0 ± 0.5-fold. In presence of phosphoprotein phosphatase (PP-2A/PP-1) inhibitor okadaic acid, a 7.2 ± 1.8-fold increase in MAPK phosphorylation in response to ANG II was observed. This further 80% increase of ANG II activation of MAPKs in presence of okadaic acid was limited to first 10 min of stimulation (** P < 0.05 vs. control; * P < 0.05 vs. ANG II, Kruskal-Wallis ANOVA on ranks with post hoc test).


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  		Fig. 5.   PP-2A/PP-1 inhibition in ARVM induces delayed activation of ERK1/ERK2 and of p46JNK and p54JNK in cardiac myocytes. ARVM primary isolates were treated with 100 nM ANG II in absence or presence of okadaic acid (100 nM). Proteins from myocyte lysates were separated by SDS-PAGE, and in-gel kinase assays were performed. A, B, and C: pooled data from 3 independent experiments for p42MAPK, p44MAPK, and p54JNK, respectively. Values are means ± SD. A 2.4 ± 0.8-fold increase in p42MAPK and a 4.9 ± 1.1-fold increase in p44MAPK phosphorylation were detected at 120 min of stimulation only in presence of okadaic acid (P < 0.05, Kruskal-Wallis 1-way ANOVA on ranks with post hoc test). a.u., Arbitrary units. D, E, and F: time- and dose-dependent effect of okadaic acid on activation of ERKs and p46JNK and p54JNK, respectively. Positions of ERKs and p54JNK in gels containing myelin basic protein are indicated by arrows at left.

Longer-term incubations with okadaic acid revealed a time- and concentration-dependent increase in ERK1/ERK2 activity. Increased phosphorylation of the ERK1/ERK2 substrate MBP in these assays could be consistently demonstrated with okadaic acid at 2 h in cardiac myocytes, at concentrations (<= 50 nM) that are relatively selective for PP-2A, suggesting that this protein phosphatase could be a predominant regulatory phosphatase for these MAPKs in adult cardiac myocytes (4.9 ± 1.1- and 2.4 ± 0.8-fold vs. control at 120 min, respectively). In addition, as shown in Fig. 5, inhibition of PP-2A and PP-1 by okadaic acid in ARVM promoted a marked increase of a kinase phosphorylating the MBP substrate in the in-gel assays at ~55 kDa. Immunoprecipitation of ARVM lysates with a JNK-specific antibody identified this kinase as likely being p54JNK2 (Fig. 5F). As little as 10 nM okadaic acid, a concentration relatively specific for PP-2A, was followed by an increase in MBP phosphorylation. The magnitude of the increase in p54JNK activity at 120 min was 10-fold greater than that observed for ERK1/ERK2 activation.

Inhibition of PP-2A and PP-1 increases c-fos mRNA abundance in ARVM and potentiates the response of c-fos by ANG II in ARVM. In CMEC, ANG II alone induced an increase in c-fos that was not accentuated by okadaic acid (5.2 ± 0.2- vs. 4.7 ± 0.2-fold; Fig. 6, A and C), paralleling the actions of these agents on ERK1/ERK2 activation in these cells. Consistent with the absence of activation of ERK1/ERK2 in ARVM by ANG II at physiologically relevant concentrations, only a minor elevation in c-fos mRNA levels (1.3 ± 0.3-fold vs. control) could be detected with ANG II in this cardiac myocyte phenotype (Fig. 6, B and D). In addition, an increase in mRNA levels of this immediate early gene in ARVM exposed to okadaic acid alone could only be detected after a minimum of 30 min and was not maximal until 180 min, consistent with the time course of activation of the MAPKs by this phosphatase inhibitor (Fig. 6E). However, coincubation with okadaic acid and ANG II resulted in an obvious accentuation of the increase in c-fos mRNA. As little as 10 nM okadaic acid, a concentration relatively specific for PP-2A, was followed by an increase in MBP phosphorylation. As shown in Fig. 5F, densitometric analysis revealed a 0.5-fold increase in p46 (JNK1) activity and a 2-fold increase in p54 (JNK2) activity after 3 h of okadaic acid treatment. The magnitude of the increase in JNK2 activity was 10-fold higher than that observed for ERK1/ERK2 activation.


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  		Fig. 6.   Inhibition of PP-2A and PP-1 with okadaic acid (OA) potentiates ANG II activation of c-fos mRNA abundance in ARVM but not in CMEC. Confluent, serum-starved CMEC primary cultures (A) and ARVM primary isolates (B) were treated with 100 nM ANG II for 30 min in presence or absence of 100 nM okadaic acid or with okadaic acid alone (90 min). c-fos mRNA hybridization signals were corrected for loading differences using 18S rRNA and normalized to mean corrected hybridization signals for c-fos mRNA from control CMEC (C) and ARVM (D). Pooled data are from 3 independent experiments. ANG II increased c-fos 5.2 ± 0.2-fold in CMEC (P < 0.01 vs. control, t-test), but a similar increase was not seen in ARVM (1.3 ± 0.3-fold, P = NS). In presence of okadaic acid, c-fos mRNA could not be induced further by ANG II (4.7 ± 0.2-fold vs. control) in CMEC. However, in ARVM, there was a significant increase in c-fos mRNA with okadaic acid (2.3 ± 0.4-fold vs. control, * P < 0.05 vs. control, ** P < 0.01 vs. control, Mann-Whitney rank sum test). E: time course of c-fos mRNA induction in ARVM by okadaic acid.

Inhibition of PP-2A and PP-1 increases c-fos mRNA abundance in ARVM and potentiates the response of c-fos by ANG II in ARVM. In CMEC, ANG II alone induced an increase in c-fos that was not accentuated by okadaic acid (5.2 ± 0.2- vs. 4.7 ± 0.2-fold; Fig. 6, A and C), paralleling the actions of these agents on ERK1/ERK2 activation in these cells. Consistent with the absence of activation of ERK1/ERK2 in ARVM by ANG II at physiologically relevant concentrations, only a minor elevation in c-fos mRNA levels (1.3 ± 0.3-fold vs. control) could be detected with ANG II in this cardiac myocyte phenotype (Fig. 6, B and D). In addition, an increase in mRNA levels of this immediate early gene in ARVM exposed to okadaic acid alone could only be detected after a minimum of 30 min and was not maximal until 180 min, consistent with the time course of activation of the MAPKs by this phosphatase inhibitor (Fig. 6E). However, coincubation with okadaic acid and ANG II resulted in an obvious accentuation of the increase in c-fos mRNA abundance in ARVM (2.3 ± 0.4-fold vs. control; Fig. 6, B and D).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The cellular constituents responsible for local myocardial synthesis and secretion of angiotensins within the heart and the specific cellular signal transduction pathways utilized by ANG II are only now becoming more clear (2, 3, 10, 11, 16, 29, 45, 46). In neonatal ventricular myocytes, a model system used by many laboratories to investigate induction and regulation of myocyte growth, an array of growth stimuli activate p44/p42 MAPKs (i.e., ERK1/ERK2), which are thought to be involved in the hypertrophic response of cardiac muscle (5, 40). These include 12-O-tetradecanoylphorbol-13-acetate and cytokines such as ANG II, endothelin-1, basic fibroblast growth factor, and interleukin-1beta (IL-1beta ), as well as biogenic amines such as alpha 1-adrenergic agonists, among other agents (37, 38, 42, 47, 50, 51, 59). However, in late postnatal or adult ventricular myocyte primary cultures, the activation of ERK1/ERK2 in response to a number of these stimuli and the associated cellular growth response become much less robust or undetectable (5). In contrast to neonatal myocytes, for example, ARVM exhibit only minimal activation of ERK1/ERK2 in response to endothelins in vitro, and, as demonstrated here, no activation of these MAPKs by ANG II, but continue to respond to IL-1beta or interferon-gamma with a rapid increase in phosphorylation and activation of both MAPKs (52).

However, in confluent serum-starved CMEC in the adult rat heart, as in neonatal ventricular myocyte primary cultures, ANG II-induced rapid phosphorylation and activation of ERK1/ERK2 could be significantly reduced by losartan, suggesting a predominant influence of the AT1 receptor mediating this response. Activation of ERK1/ERK2 in cardiac myocytes is mediated by phosphorylation of threonine and tyrosine residues by MAPK kinases (MEKs). MEKs are a family of dual-specificity kinases that are activated by ras- and raf-coupled signaling pathways and by other MEK kinases, some of which appear to be activated directly or indirectly by diacylglycerol-regulated protein kinase C and tyrosine kinases such as lyn and syk (4, 57). Interferon-gamma , for example, acting at type II interferon receptors on adult cardiac myocytes, mediates activation of ERK1/ERK2 in these cells through a diacylglycerol-sensitive protein kinase C.

ANG II also appears to mediate growth in neonatal rat ventricular myocytes by initiating several, perhaps parallel, kinase cascades that result in activation of the 40S ribosomal S6 kinase p70S6K and of p90RSK (7, 43, 44, 47). Interestingly, Post et al. (40) demonstrated that ERK1/ERK2 activation in neonatal myocytes was not sufficient and may not be necessary for hypertrophic growth or atrial natriuretic peptide transcriptional activation by the alpha -adrenergic agonist phenylephrine in neonatal rat ventricular myocytes.

Despite the absence of evidence for ERK signaling by ANG II in the adult phenotype of cardiac myocytes in this report, these pathways are intact in adult cardiac myocytes, since inhibition of PP-2A/PP-1 was followed by an increase in ERK activity in adult cardiac myocytes. Thus activities of MAPKs in cardiac myocytes and microvascular endothelial cells are also regulated, as in other cell types, by inactivating phosphatases (19, 49). In CMEC, for example, okadaic acid, a potent shellfish-derived toxin and tumor promoter and a relatively selective inhibitor of PP-2A/PP-1 (28), rapidly enhanced ERK1/ERK2 activation by IL-1beta (52) and by ANG II but only modestly increased ERK activation when given alone. However, in adult cardiac myocytes, okadaic acid induced a sustained activation of ERK1/ERK2, indicating that these cytosolic phosphatases tonically suppress activation of these MAPKs directly or by acting on upstream signaling kinases such as MEKs. Along with activation of ERKs in the adult cardiac myocytes, okadaic acid simultaneously resulted in a substantial increase in activity of other protein kinases that can use MBP as a substrate. Although the identity of these kinase(s) has yet to be determined, a likely candidate is p54JNK. Indeed, in our experiments, okadaic acid induced an increase in p54JNK in adult cardiac myocytes (Fig. 5F). Concurrent activation of the JNK/stress-activated protein kinase (SAPK) pathway by a Ras-dependent MAPK and ERK kinase kinase (MEKK) with ERK1/ERK2 may serve to regulate or limit the proliferative response to growth stimuli. In support of this paradigm, Bokemeyer et al. (6) showed that selective activation of a p54JNK in NIH/3T3 cells stably transfected with an activated MEKK construct was accompanied by increased MKP-1 expression. Other regulatory mechanisms may also operate to attenuate cytokine-induced increases in ERK1/ERK2 activation. Atrial natriuretic peptide, for example, the expression of which is increased by many growth stimuli in neonatal and adult ventricular phenotypes, has been shown recently to induce MKP-1 expression in a specialized vascular smooth muscle phenotype, glomerular mesangial cells (54). Consistent with the stimulation of ERKs by okadaic acid in the differentiated adult cardiac myocyte is the observation that this phosphatase inhibitor also caused a sustained increase in c-fos transcript levels in ARVM, a transcription factor proposed to be regulated by MAPKs. Finally, although ANG II (100 nM) caused no significant rise in c-fos mRNA levels at 30 min in ARVM, Kent and McDermott (25) recently reported that the same concentration of ANG II did induce a significant threefold increase in c-fos mRNA in adult feline ventricular myocytes at 1 h. However, in isolated perfused rat hearts the response to ANG II infusion on c-fos expression in cardiac myocytes was less robust (48).

In addition to serine/threonine kinases, the dual-specificity phosphatase MKP-1 has also been shown to regulate ERK1/ERK2 activity (9, 12, 13, 30, 31, 32). Transcript levels of this phosphatase are elevated rapidly and in parallel to c-fos expression in ARVM and in CMEC by ANG II, as has been described in vascular smooth muscle in vitro (12). Unexpectedly, however, elevation of MKP-1 mRNA levels was clearly mediated by different receptor subtypes in CMEC and ARVM. Although AT1- and AT2-receptor antagonists influenced MKP-1 mRNA levels in CMEC, only the AT2-receptor antagonist consistently suppressed the rise of MKP-1 levels in response to ANG II in adult cardiac myocytes. This role of the AT2 receptor in suppressing MAPK activities is supported by the observation that transfection of adult rat aortic vascular smooth muscle cells with an AT2-receptor construct was followed by a decrease in MAPK activity and decreased DNA synthesis (18).

Our data suggest that stimulation of the AT2 receptor in adult cardiac myocytes is followed by an increase in MKP-1 expression that might be associated with an antiproliferative response in this cardiac cell type, a finding that is supported by parallel results in reporter cell lines abundantly expressing the AT2 receptor (58). Although not addressed by us in this report, the site of regulation of MKP-1 mRNA abundance by ANG II has been shown to be at the transcriptional level in vascular smooth muscle cells by Duff et al. (13), although these authors did not address which ANG II receptor subtypes were responsible for mediating the induction of MKP-1 in these cells. Kambayashi et al. (22) reported that ANG II inhibited one or more phosphotyrosine phosphatases in plasma membrane preparations of COS cells that had been transfected with the rat AT2 receptor. We could not demonstrate conclusively in this report that increased MKP-1 mRNA levels in CMEC or ARVM primary cultures were accompanied by increased phosphatase activity in these cells. However, Duff et al. (13) reported, in a carefully controlled study, that ERK1/ERK2 phosphorylation and activation of ANG II could be enhanced by actinomycin D or antisense oligodeoxynucleotides to MKP-1 in rat aortic vascular smooth muscle cells.

These data indicate that ANG II alone does not activate the ERK1/ERK2 kinase cascade in primary isolates of adult ventricular myocytes. Although this may be due in part to a decline in the levels of these MAPKs in the late postnatal or adult myocyte phenotype (5), ERK1/ERK2 kinase activities, as well as p54JNK activity, can be rapidly increased by inhibiting constitutive PP-2A/PP-1 serine/threonine phosphatases in these cells. Interestingly, ANG II, through AT2-receptor activation, increases mRNA levels of the inducible MAPK phosphatase MKP-1 in adult ventricular myocytes. Although we have not addressed in this report the cellular mechanisms by which ANG II regulates MKP-1 expression, one possibility is that activation of a JNK or another SAPK is involved, although this was not the focus of the work reported here. Deactivation of kinase cascades is crucial to normal and pathological control of signaling and cellular growth. These results emphasize the potential importance of these phosphatases in mediating the integrated cellular response to growth-promoting agents in cardiac myocytes as in other cells.

    ACKNOWLEDGEMENTS

We thank Lester F. Lau (Dept. of Genetics, University of Chicago, Chicago, IL) for the 3CH134 cDNA, Janice M. Pfeffer for excellent help throughout the study, and Marc A. Pfeffer for advice and support of this work.

    FOOTNOTES

T. A. Fischer was the recipient of a postdoctoral fellowship award from the Deutsche Forschungsgemeinschaft (Bonn, Germany). D. M. Kaye was supported by a Ralph Reader Overseas Research Fellowship of the National Heart Foundation of Australia. This work was supported in part by National Heart, Lung, and Blood Institute Grant R37-HL-36141 to R. A. Kelly.

Present addresses: T. A. Fischer, Dept. of Medicine, University of Mainz, Langenbeckstr. 1, 55101 Mainz, Germany; K. Singh, Boston University Medical Center, R-408, 88 E. Newton St., Boston, MA 02118; D. M. Kaye, Baker Medical Research Institute, PO Box 348, Prahan 3181, Victoria, Australia.

Address for reprint requests: R. A. Kelly, Cardiovascular Div., Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.

Received 9 July 1997; accepted in final form 6 May 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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