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Am J Physiol Heart Circ Physiol 286: H2195-H2203, 2004. First published February 19, 2004; doi:10.1152/ajpheart.00475.2003
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PKC-{epsilon} regulation of extracellular signal-regulated kinase: a potential role in phenylephrine-induced cardiocyte growth

Vijay U. Rao, Hirokazu Shiraishi, and Paul J. McDermott

Cardiology Division, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, and the Ralph H. JohnsonVeterans Affairs Medical Center, Charleston, South Carolina 29425-2221

Submitted 22 May 2003 ; accepted in final form 9 February 2004


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypertrophic growth of cardiac muscle is dependent on activation of the PKC-{epsilon} isoform. To define the effectors of PKC-{epsilon} involved in growth regulation, recombinant adenoviruses were used to overexpress either wild-type PKC-{epsilon} (PKC-{epsilon}/WT) or dominant negative PKC-{epsilon} (PKC-{epsilon}/DN) in neonatal rat cardiocytes. PKC-{epsilon}/DN inhibited acute activation of PKC-{epsilon} produced in response to phorbol ester and reduced ERK1/2 activity as measured by the phosphorylation of p42 and p44 isoforms. The inhibitory effects were specific to PKC-{epsilon} because PKC-{epsilon}/DN did not prevent translocation of either PKC-{alpha} or PKC-{delta}. Overexpression of PKC-{epsilon}/DN blunted the acute increase in ERK1/2 phorphorylation induced by the {alpha}1-adrenergic agonist phenylephrine (PE ). Inhibition of PKC-{delta} with rottlerin potentiated the effects of PE on ERK1/2 phosphorylation. PKC-{epsilon}/DN adenovirus also blocked cardiocyte growth as measured after 48 h of PE treatment, although the multiplicity of infection was lower than that required to block acute ERK1/2 activation. PE activated p38 mitogen-activated protein kinase as measured by its phosphorylation, but the response was not blocked by PKC inhibitors or by overexpression of PKC-{epsilon}/DN. Taken together, these studies show that the hypertrophic agonist PE regulates ERK1/2 activity in cardiocytes by a pathway dependent on PKC-{epsilon} and that PE-induced growth is mediated by PKC-{epsilon}.

heart; hypertrophy; cell signaling; mitogen-activated protein kinase

CARDIAC HYPERTROPHY is a cellular growth response that normalizes increased hemodynamic load produced during pathophysiological states such as hypertension, valvular disease, and remodeling after myocardial infarction (12). Understanding the molecular mechanisms that are involved in controlling growth is of critical importance given that prolonged cardiac hypertrophy leads to cardiovascular morbidity and mortality (3). Hypertrophic growth is characterized by several phenotypic changes that include an increase in sarcomere content of individual cardiac muscle cells (cardiocytes), an acceleration of the rate of protein synthesis, and an altered pattern of gene expression (7, 16, 22, 31). Although a diverse array of signaling pathways are involved in regulating cardiocyte growth, mounting evidence indicates that activation of one or more isoenzymes of protein kinase C (PKC) is an essential step (4, 11, 18, 24, 34, 36).

PKCs are a family of lipid-dependent serine/threonine kinases that consist of at least 12 mammalian isoforms (32). By convention, each PKC isoform is assigned to one of three subfamilies (classical, novel, and atypical) according to its requirements for activation by Ca2+ and diacylglycerol (13). The activation of most PKC isoforms also are dependent on translocation from the cytosol to subcellular compartments such as the cell membrane (15, 29). PKC isoforms are targeted to specific sites by a family of intracellular receptors referred to as receptors for activated C-kinase. Consequently, activation of PKC isoforms is spatially regulated because it usually occurs at distinct subcellular locations (28).

Although multiple PKC isoforms are expressed in the heart, studies using cardiocytes in vitro indicate that the Ca2+-insensitive, novel PKC-{epsilon} isoform has a primary role in regulating cardiocyte hypertrophy (19, 36, 37). This conclusion is supported by murine transgenic models in which cardiac-restricted PKC-{epsilon} activation has been directly linked to hypertrophic growth (30, 38, 41). The effectors of PKC-{epsilon} that are relevant to hypertrophy have not been fully characterized, but recent studies point to the extracellular signal-regulated kinases 1 and 2 (ERK1/2) of the MAPK family (19, 37). In accordance with these studies, we have shown that activation of PKC in adult cardiocytes regulates ERK1/2 through the c-Raf/MEK/ERK signaling pathway (21). The potential importance of this signaling pathway is underscored by the finding that cardiocyte growth produced by agonists of G protein-coupled receptors such as phenylephrine (PE) and endothelin-1 (ET-1) can be blocked by direct inhibition of ERK1/2 (43). Furthermore, causality between ERK1/2 activation and cardiac growth has been established (6, 39, 42). It is likely that ERK1/2 signaling regulates cardiac growth through convergent mechanisms that ultimately control the transcription of genes associated with hypertrophy and modify the activity enzymes that control translation such as p70S6 kinase and MAPK signal-integrating kinases (Mnk-1 and -2) (16, 21, 24, 40).

Given that activation of both PKC-{epsilon} and ERK1/2 are involved in regulating growth of neonatal rat cardiocytes, we hypothesized that ERK1/2 functions as a downstream effector of PKC-{epsilon}. To directly test this hypothesis, adenoviral gene transfer was used as an approach for overexpression of either wild-type (PKC-{epsilon}/WT) or dominant negative PKC-{epsilon} (PKC-{epsilon}/DN) in neonatal rat cardiocytes. The corresponding effects on ERK signaling were determined by measuring changes in phosphorylation state of ERK1/2 produced in response to the {alpha}1-adrenergic agonist phenylephrine (PE). These studies, which were done in conjunction with selective PKC inhibitors, demonstrate that ERK1/2 is activated in cardiocytes by a pathway dependent on PKC-{epsilon}.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. Cell culture media and reagents were purchased from GIBCO-BRL (Grand Island, NY). PE and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma Chemical (St. Louis, MO). The broad-spectrum PKC inhibitor bisindolylmaleimide (BIM) and the PKC-{delta} inhibitor rottlerin were obtained from Calbiochem (La Jolla, CA). The restriction enzymes BglII, KpnI, NheI, and NarI were obtained from Promega (Madison, WI). PacI was obtained from New England Biolabs (Beverly, MA).

Antibodies. Rabbit polyclonal antibodies for PKC-{epsilon}, PKC-{delta}, and Na+/K+-ATPase and a monoclonal antibody for PKC-{alpha} were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies for p38 MAPK, phospho-p38 MAPK, ERK1/2, and phospho-ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA).

Adenoviral constructs. Full-length, human PKC-{epsilon} cDNA was obtained from American Type Culture Collection (Manassas, VA). PKC-{epsilon}/DN (L-436 to R) was made by site-directed mutagenesis using the QuikChange method from Stratagene (La Jolla, CA). The forward primer was 5'-GAAGTATATGCTGTGAGGGTCTTAAAGAAGGAC-3' and the reverse primer was 5'-GTCCTTCTTTAAGACCCTCACAGCATATACTTC-3'. The mutation was confirmed by DNA sequencing. Replication-defective recombinant adenoviruses were generated by homologous recombination using a bacterial system as before (35). Recombinant adenoviruses were screened for protein expression by Western blotting, and the positives were plaque-purified (17). After large-scale propagation, adenoviruses were purified by centrifugation on cesium chloride gradients and titered by plaque assay (17). An adenovirus expressing the {beta}-galactosidase ({beta}-Gal) gene was used as a control (35).

Cell culture and isolation. The procedure for isolation of neonatal rat cardiocytes was approved by the Institutional Animal Care and Use Committee. Ventricular cardiocytes were isolated from the hearts of 1- to 4-day-old Sprague-Dawley rats by enzymatic digestion in combination with mechanical dissociation as described previously (27). The cardiocytes were plated at a cell density of 2 x 106 cells/dish. After an overnight incubation in media containing 10% newborn calf serum, cardiocytes were maintained in serum-free media for 48 h before infection with adenovirus.

PKC translocation assay. Cardiocytes were washed twice with PBS and lysed in (0.5 ml/2.5 x 106 cells) ice-cold homogenization buffer [20 mmol/l Tris·HCl (pH 7.5), 2 mmol/l EDTA, 2 mmol/l EGTA, 6 mmol/l {beta}-mercaptoethanol, 50 µg/ml aprotinin, 48 µg/ml leupeptin, 5 µmol/l pepstatin A, 1 mmol/l phenylmethylsulfonyl fluoride, 0.1 mmol/l sodium vanadate, and 50 mmol/l NaF]. The homogenate was sonicated and then centrifuged for 10 min at 500 g. The supernatant was centrifuged for 1 h at 100,000 g. The resulting supernatant was designated as the soluble fraction. The pellet was solubilized in homogenization buffer containing 1% Triton X-100 and centrifuged at 12,000 g. The supernatant was designated as the particulate (membrane) fraction.

PKC-{epsilon} kinase assay. Cardiocytes were infected with {beta}-Gal, PKC-{epsilon}/WT, or PKC-{epsilon}/DN adenoviruses and treated with 100 nM/l PMA for 30 min before being harvested. Particulate fractions were prepared as described above and chromatographed on DE-52 cellulose columns. Aliquots were used to measure protein concentration by the bicinchoninic acid (BCA) method (Pierce; Rockford, IL). The activity of PKC was measured using the PKC assay kit from Upstate Biotechnology (Lake Placid, NY). Briefly, the extract was incubated for 15 min at 30°C with a reaction mixture containing substrate cocktail (PKC-{epsilon}-specific substrate peptide, protein kinase inhibitor cocktail, assay dilution buffer, lipid activator, and [{gamma}-32P]ATP). Aliquots of the reaction mixture were spotted onto P81 paper, washed with phosphoric acid, and measured for radioactivity by scintillation counting. Enzyme activity was calculated as picomoles of phosphate per minute per microgram of protein.

Western blotting. Cardiocytes were scraped in lysis buffer [30 mmol/l Tris·HCl (pH 7.4), 2% (vol/vol) Triton X-100, 10 mmol/l {beta}-glycerophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µmol/l E-64, 0.5 mmol/l PMSF, 1 mmol/l sodium orthovanadate, 0.2 µmol/l okadaic acid, and 1 mmol/l EGTA]. The lysate was passed 10 times through a 26-gauge needle and centrifuged at 12,000 g for 10 min. Protein concentrations were measured by the BCA method and equal amounts of protein were analyzed by Western blotting as described before (21). In the PKC membrane translocation experiments, an initial set of Western blots was probed with anti-Na+/K+-ATPase antibody to correct for differences in relative purity of the particulate fractions.

Quantitation of protein, RNA, and DNA. Cardiocytes were harvested in standard sodium citrate buffer (SSC) [300 mmol/l NaCl, 30 mmol/l sodium citrate, and 0.25% (wt/vol) SDS]. After a freeze-thaw cycle, the lysates were vortexed extensively. Protein concentrations were assayed by the BCA method. The concentrations RNA and DNA were measured as described before (27).

Immunofluorescence. Cardiocytes were washed twice with PBS and fixed in buffer containing 4% (vol/vol) paraformaldehyde in PBS for 15 min at room temperature. The cardiocytes were rinsed again and incubated for 2 min in PBS containing 0.2% Triton X-100. The cardiocytes were stained for 20 min with a 1:100 dilution of rhodamine phalloidin in PBS containing 1% BSA (Molecular Probes).

Data analysis. Autoradiograms of Western blots were quantified by digital image analysis using NIH Image software. Data were analyzed by ANOVA followed by a Student-Newman-Keuls test. P values <=0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of PKC-{epsilon} by adenoviral gene transfer. To determine the possible functions of PKC-{epsilon}, adenoviruses were used for high-efficiency gene transfer into neonatal rat cardiocytes. The first step was to infect the cardiocytes with adenovirus expressing {beta}-Gal at differing multiplicities of infection (MOIs) to optimize the efficiency of gene transfer. Figure 1 shows that 90% of the cardiocytes were infected using a MOI of 2, whereas essentially 100% of the cardiocytes were infected by increasing the MOI to 20. The micrographs further show that the intensity of {beta}-Gal staining in individual cardiocytes was markedly increased between 2 and 20. Next, cardiocytes were infected with adenovirus expressing either PKC-{epsilon}/WT or PKC-{epsilon}/DN at a MOI of 2, followed by a 48-h incubation period. Western blot analysis showed that expressions of PKC-{epsilon}/DN and PKC-{epsilon}/WT were increased by approximately eightfold and sevenfold above endogenous levels, respectively (Fig. 1C).



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  		Fig. 1. Adenoviral gene transfer into neonatal rat cardiocytes. A: cardiocytes were infected with {beta}-galactosidase ({beta}-Gal) adenovirus at increasing multiplicities of infection (MOIs) as indicated. After a 48-h incubation period, cardiocytes were fixed and stained for {beta}-Gal activity. Representative light micrographs are shown. B: percentages of cardiocytes positive for {beta}-Gal activity were quantified by light microscopy. The values at each MOI are means ± SE of 10 independent fields. C: cardiocytes were infected with the indicated adenoviruses at a MOI of 2 and incubated for 48 h. NI, noninfected; PKC-{epsilon}/WT, wild-type PKC-{epsilon}; PKC-{epsilon}/DN, dominant negative PKC-{epsilon}. Western blot analysis was performed on equivalent amounts of protein derived from whole cell lysates using PKC-{epsilon} antibody. The results from duplicate infections are shown, and the size of protein standards (in kDa) are indicated.

 
The activation of PKC isoforms can be determined by translocation of the enzyme from the cytosol to membrane fraction in response to acute stimulation by phorbol esters. To establish that the exogenous pool of PKC-{epsilon} was capable of translocation and therefore functional, cardiocytes were treated with PMA for 30 min and the soluble and membrane fractions were prepared for Western blot analysis (Fig. 2A). Similar to the endogenous pool of PKC-{epsilon} expressed in the {beta}-Gal controls, PKC-{epsilon}/WT and PKC-{epsilon}/DN translocated from the soluble to corresponding membrane fraction. PMA increased the amount of PKC-{epsilon}/WT associated with the membrane fraction by 33 ± 9% (mean ± SE, n = 4 experiments) and caused a reciprocal decrease of 51 ± 11% in the soluble fraction (n = 3 experiments). PMA increased the amount of membrane-associated PKC-{epsilon}/DN by 32 ± 2% in association with a decrease in soluble PKC-{epsilon}/DN (mean ± range of 2 experiments). Thus the single point mutation in the kinase domain did not affect translocation of PKC-{epsilon}/DN to the membrane.



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  		Fig. 2. Effects of either PKC-{epsilon}/WT or PKC-{epsilon}/DN on the activity of individual PKC isoforms. A: cardiocytes were infected with each of the indicated adenoviruses at a MOI of 2. After a 48-h incubation, cardiocytes were either treated for 30 min with 200 nmol/l phorbol 12-myristate 13-acetate (PMA; +) or not treated (–). Soluble (Sol) and membrane (Mem) fractions were prepared immediately. The relative purity of the membrane fractions was determined by Western blotting with an anti-Na+/K+-ATPase antibody. Equivalent amounts of membrane protein were used for Western blot analysis with an anti-PKC-{epsilon} antibody. Summary data are provided in the text. B: cardiocytes were infected with either {beta}-Gal or PKC-{epsilon}/DN adenovirus at a MOI of 2. After a 48-h incubation, cardiocytes were treated for 30 min with 100 nmol/l PMA and membrane fractions were prepared immediately. Nontreated (NT) cardiocytes were used as an indicator of basal activity. PKC-{epsilon}-specific kinase activity was measured by adding equivalent amount of membrane protein to the assay cocktail. The data were normalized to PKC-{epsilon} activity in nontreated cardiocytes infected with {beta}-Gal adenovirus and are means + SE of 3 experiments. *P < 0.05 vs. the nontreated {beta}-Gal group as determined by one-way ANOVA followed by the Student-Newman-Keuls test. C: cardiocytes were infected with the indicated adenoviruses at a MOI of 2. After a 48 h incubation, cardiocytes were either treated for 30 min with 200 nmol/l PMA (+) or not treated (–). Cardiocytes were fractionated immediately, and equivalent amounts of protein from the soluble fractions were analyzed by Western blotting with antibodies specific for individual PKC isoforms. The loss of PKC signal from the soluble fraction was taken to represent translocation and activation of PKC isoforms. Data are representative of 2 experiments.

 
To directly measure the activity of exogenous PKC-{epsilon}, kinase assays were done using a selective PKC-{epsilon} substrate peptide (Fig. 2B). Membrane fractions derived from cardiocytes overexpressing {beta}-Gal exhibited a statistically significant increase in PKC-{epsilon} activity after 30 min of PMA treatment. PKC-{epsilon} activity did not increase in cardiocytes overexpressing PKC-{epsilon}/DN, nor was basal PKC-{epsilon} activity affected compared with the {beta}-Gal control. These results demonstrate that PKC-{epsilon}/DN specifically inhibited PKC-{epsilon} activity in cardiocytes. When cardiocytes overexpressing PKC-{epsilon}/WT were assayed as a positive control, PKC-{epsilon} activity was increased markedly and could not be further enhanced by PMA (data not shown).

To determine whether overexpression of either PKC-{epsilon}/WT and PKC-{epsilon}/DN had nonspecific effects on endogenous PKC isoforms in cardiocytes, we examined the ability of PKC-{alpha} and PKC-{delta} to translocate in response to PMA. Figure 2C shows that translocation of both isoforms occurred as indicated by decreases in the amount of each respective enzyme in the soluble fraction. Thus endogenous PKC isoforms remained capable of activation, which indicates that function was not impaired.

Treatment of neonatal rat cardiocytes with PMA leads to the activation of the p42 and p44 isoforms of ERK. To determine whether PKC-{epsilon} is coupled to ERK1/2, we measured the effects of PKC-{epsilon}/WT and PKC-{epsilon}/DN on the ability of PMA to increase ERK1/2 phosphorylation using an anti-phospho-Thr202/Tyr204 ERK1/2 antibody (Fig. 3). In cardiocytes overexpressing {beta}-Gal, PMA activated both isoforms of ERK as indicated by marked increases in phosphorylation compared with nontreated controls. This response to PMA was blunted in cardiocytes infected with PKC-{epsilon}/DN adenovirus relative to {beta}-Gal: a MOI of 2 decreased phosphorylation of p42 ERK and p44 ERK by 36 ± 23% and 41 ± 32% and a MOI of 10 decreased phosphorylation of p42 ERK and p44 ERK by 55 ± 28% and 55 ± 27% (means ± range of 2 experiments). Because the higher MOI of 10 did not completely block ERK1/2 phosphorylation, these results provide indirect evidence that endogenous isoforms of PKC remained active and that the inhibitory effects of PKC-{epsilon}/DN were isoform specific. Figure 3 also shows that ERK1/2 phosphorylation was not hindered by overexpression of PKC-{epsilon}/WT.



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  		Fig. 3. Effects of PKC-{epsilon}/WT and PKC-{epsilon}/DN on ERK1/2 activity. Cardiocytes were infected with each adenovirus at the indicated MOI. After a 48-h incubation, cardiocytes were either treated for 30 min with 200 nmol/l PMA (+) or not treated (–). Equivalent amounts of protein were used for Western blot analysis with anti-phospho-ERK1/2 (p-ERK) antibody. The signals for p42 and p44 ERK phosphorylation were quantified by digital image analysis and normalized to the corresponding amount of ERK1/2 in each lane. Summary data are provided in the text.

 
Regulation of cardiocyte growth by PKC-{epsilon}. To determine whether growth was dependent on activation of PKC-{epsilon}, cardiocytes were infected with either PKC-{epsilon}/WT or PKC-{epsilon}/DN adenovirus at a MOI of 2 and treated for 48 h with the {alpha}1-adrenergic agonist PE. Figure 4 shows that PE caused significant increases in protein-to-DNA and RNA-to-DNA ratios in the {beta}-Gal control, which indicates that growth occurred. By comparison, overexpression of PKC-{epsilon}/DN blocked cardiocyte growth induced by PE but did not alter protein-to-DNA or RNA-to-DNA ratios in nontreated cardiocytes. PKC-{epsilon}/WT alone produced a modest increase in protein-to-DNA and RNA-to-DNA ratios that did not achieve statistical significance. However, if overexpression of PKC-{epsilon}/WT was combined with PE treatment, then a statistically significant amount of growth occurred.



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  		Fig. 4. Effects of modifying PKC-{epsilon} activity on cardiocyte growth induced by phenylephrine (PE). Cardiocytes were infected with adenoviruses at a MOI of 2 and treated with PE for 40–44 h. Data are means + SE of 3 experiments. *P < 0.05 vs. the nontreated {beta}-Gal adenovirus as determined by ANOVA followed by the Student-Newman-Keuls test. A: protein-to-DNA ratio; B: RNA-to-DNA ratio.

 
To examine the effects of PKC-{epsilon} on morphology, a similar set of experiments was performed and the cardiocytes were fixed and stained with rhodamine phalloidin for fluorescence microscopy (Fig. 5). The surface area of cardiocytes overexpressing {beta}-Gal appeared greater after 2 days of PE treatment, which is consistent with the well-established growth response using this model. Myofibrils were well organized and abundant. In cardiocytes overexpressing PKC-{epsilon}/DN, the major difference in morphology was the appearance of slender, elongated processes that were devoid of myofibrils. The same morphology occurred in the presence or absence of PE. In contrast, the morphology of cardiocytes overexpressing PKC-{epsilon}/WT was indistinguishable from the {beta}-Gal controls. These results indicate that the effects of PKC-{epsilon}/DN on morphology were due to loss of function rather than nonspecific effects caused by overexpression of PKC-{epsilon}.



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  		Fig. 5. Effects of modifying PKC-{epsilon} activity on cardiocyte morphology. Cardiocytes were infected with {beta}-Gal adenovirus (A and B), PKC-{epsilon} /DN adenovirus (C and D), or PKC-{epsilon}/WT adenovirus (E and F) at a MOI of 2. The cardiocytes were maintained for an additional 40–44 h in the absence (A, C, and E) or presence (B, D, and F) of PE. Shown are immunofluorecent images of cardiocytes stained with rhodamine phalloidin (x400 magnification).

 
Regulation of ERK1/2 activity by PKC-{epsilon}. Recent evidence suggests that distinct PKC isoforms are involved in activation of ERK1/2. Given that {alpha}1-adrenergic stimulation of cardiocytes has been shown to cause the translocation of PKC-{epsilon} and PKC-{delta} (9), we used PKC-{epsilon}/DN adenovirus in addition to PKC inhibitors to investigate the possible contributions of specific PKC isoforms to ERK1/2 activation. First, a time course after PE treatment was done to establish the kinetics of ERK1/2 activation as determined by phosphorylation of p42 and p44 isoforms. Figure 6A shows that maximal phosphorylation occurred at 5 min and that ERK1/2 phosphorylation remained elevated for 15 min before returning to basal levels. The transient activation of ERK1/2 by PE has been reported previously (9). Next, the cardiocytes were infected with either PKC-{epsilon}/DN or the {beta}-Gal adenovirus and challenged after 2 days with PE for 15 min. Figure 6B shows that PE increased the phosphorylation of ERK1/2 in cardiocytes overexpressing {beta}-Gal. The increase in phosphorylation was blocked when cardiocytes were pretreated with the PKC inhibitor BIM, demonstrating that PE-induced ERK1/2 activation was dependent on PKC. In contrast, pretreatment with the PKC-{delta}-specific inhibitor rottlerin enhanced the effects of PE on ERK1/2 phosphorylation, although rottlerin treatment alone had no effect on ERK1/2 phosphorylation (data not shown). The finding that rottlerin potentiated the effects of PE on ERK1/2 phosphorylation suggests that PKC-{delta} has an inhibitory function in regulating ERK activity.



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  		Fig. 6. Activation of ERK1/2 in response to acute treatment with PE is dependent on PKC-{epsilon}. A: cardiocytes were treated with 100 µmol/l PE and whole cell lysates were prepared at the indicated time points. Equivalent amounts of protein were analyzed by Western blotting with anti-p-ERK1/2 antibody. The amount of ERK in each lane was analyzed with anti-ERK1/2 antibody. B: cardiocytes were infected with either {beta}-Gal or PKC-{epsilon}/DN adenovirus at the indicated MOI. After 48 h, cardiocytes were either treated with PE for 15 min or not treated. Two groups consisted of cardiocytes that were pretreated for 30 min with either 5 µmol/l bisindolylmaleimide (BIM) or10 µmol/l rottlerin (ROT). Whole cell lysates were prepared and equivalent amounts of protein were analyzed by Western blotting for phospho-ERK1/2 and total ERK. C: summary data for ERK1/2 phosphorylation were quantified by digital image analysis and normalized to nontreated cardiocytes infected with {beta}-Gal adenovirus. Data are means + SE of 3 experiments. *P < 0.05 vs. the nontreated {beta}-Gal group as determined by ANOVA followed by the Student-Newman-Keuls test.

 
The data shown in Fig. 6B further show that PKC-{epsilon}/DN adenovirus at a MOI of 2 did not alter ERK1/2 phosphorylation. An inhibitory effect was obtained by raising the MOI to 10, which indicates that the acute activation by PE is dependent on PKC-{epsilon}. The summary data shown in Fig. 6C confirm that ERK1/2 phosphorylation was significantly blocked by raising the MOI of PKC-{epsilon}/DN adenovirus and that basal phosphorylation of ERK1/2 in nontreated controls was not significantly altered.

PE has been shown to activate the p38 branch of the MAPK family in neonatal rat cardiocytes (11). Therefore, we examined whether PKC-{epsilon} was involved in activating p38 MAPK as measured by phosphorylation using an anti-phospho-Thr180/Tyr182 p38 MAPK antibody. Figure 7A demonstrates that phosphorylation of p38 MAPK increased after 5 min of PE treatment and that phosphorylation declined after 15 min toward baseline levels. Pretreatment with BIM did not block the ability of PE to increase phosphorylation of p38 MAPK, which indicates that PKC was not required for activation (Fig. 7B). Furthermore, the results shown in Fig. 7C show that PE was able to generate an increase in phosphorylation of p38 MAPK in cardiocytes overexpressing PKC-{epsilon}/DN. In three separate experiments, p38 phosphorylation was quantified by digital image analysis and normalized to total p38 MAPK. None of these experiments showed any differences in p38 phosphorylation in cardiocytes overexpressing PKC-{epsilon}/DN versus the {beta}-Gal control. These data indicate that, in contrast to ERK family members, p38 MAPK is not a direct effector of PKC-{epsilon}. However, when cardiocytes were pretreated with the PKC-{delta}-specific inhibitor rottlerin, the effect of PE on p38 MAPK phosphorylation was enhanced. Thus inhibiting PKC-{delta} potentiated the stimulatory effects of PE on both ERK and p38 MAPK family members.



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  		Fig. 7. Acute activation of p38 MAPK by PE does not require PKC-{epsilon}. A: cardiocytes were treated with PE and whole cell lysates were prepared at the indicated time points. Equivalent amounts of protein were analyzed by Western blotting with antibodies specific for phospho-p38 MAPK (p-p38) or total p38 MAPK. B: cardiocytes were infected with {beta}-Gal adenovirus. After 48 h, cardiocytes were treated for 15 min with PE. Pretreatment with 5 µmol/l BIM proceeded for 30 min before the addition of PE. C: cardiocytes were infected with either {beta}-Gal or PKC-{epsilon}/DN adenovirus. After 48 h, cardiocytes were treated for 15 min with PE. Pretreatment with 10 µmol/l ROT proceeded for 30 before the addition of PE. Total cell lysates were analyzed by Western blotting with a total p38-MAPK antibody and phospho-p38 MAPK antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In cardiac muscle, it has been shown that hypertrophic growth is regulated by activation of PKC isoforms such as PKC-{alpha} and PKC-{epsilon} (5, 30). The signaling mechanisms relevant to growth probably involve the MAPK superfamily because individual members are selectively regulated by specific PKC isoforms. For example, PKC-{epsilon} and PKC-{alpha} have been linked to ERK1/2 activation in cardiocytes, whereas PKC-{delta} has been linked to p38 and JNK activation (19, 23). The present study has extended these findings by establishing a cause-and-effect linkage between activity of PKC-{epsilon} and increased phosphorylation of ERK1/2. Furthermore, growth of neonatal rat cardiocytes produced in response to the {alpha}1-adrenergic agonist PE is dependent on activity of the PKC-{epsilon} isoform.

In PE-treated cardiocytes, overexpression of PKC-{epsilon}/DN was sufficient to block growth as measured using two endpoints of the hypertrophic response, namely, total protein and total RNA content. Using a similar strategy, Strait et al. (37) concluded that PKC-{epsilon} was not required to increase either cardiocyte protein content or surface area in response to ET-1. Given that both PE and ET-1 are Gq{alpha}-coupled receptor agonists that can activate PKC-{epsilon}, there are several possibilities to account for this apparent difference in the dependency on PKC-{epsilon} for growth. First, PE and ET-1 utilize distinct pathways to modulate cardiocyte growth, although both remained capable of stimulating the ERK pathway. In support of this possibility, it has been shown that an increase in cardiocyte protein synthesis induced by ET-1 was dependent on JNK activation but independent of ERK activation (8). Second, ET-1 produced a smaller growth response than PE, as indicated by 14% increase in protein content (37). Consequently, this relatively modest increase in cardiocyte growth could have limited the ability to detect the inhibitory effects of PKC-{epsilon}/DN. Third, ET-1 utilizes PKC-{epsilon} to activate an alternative signaling pathway in cardiocytes that proceeds through Rho, Rho-kinase, and cofilin (20). These findings have established that ET-1 can regulate focal adhesion kinase (FAK) and sarcomere assembly independent of ERK1/2 activation.

Adenoviruses were used at a MOI that was sufficient to achieve high-efficiency gene transfer into cardiocytes while avoiding levels of overexpression that promote nonspecific protein interactions. Our results show that overexpression of PKC-{epsilon}/DN was sufficient to block PKC-{epsilon} activation as determined by comparing the acute responsiveness to PMA. Furthermore, PKC-{epsilon}/DN had minimal effects on the functionality of other PKC isoforms expressed in the cardiocytes. This conclusion is based on the finding that endogenous PKC-{alpha} and PKC-{delta} were able to translocate in response to PMA treatment. In support this conclusion, ERK1/2 phosphorylation was reduced, but not completely inhibited, in cardiocytes infected with PKC-{epsilon}/DN adenovirus at a MOI of 10. Although this evidence is indirect, it suggests that other endogenous PKC isoforms were still capable of activation.

PE elicited an acute increase in ERK1/2 phosphorylation that was inhibited by PKC-{epsilon}/DN adenovirus, but a higher MOI was needed than that required to block cardiocyte growth (10 vs. 2, respectively). It is important to note that acute responses to either PMA or PE were used mainly because activation of PKC-{epsilon} was maximal. Although this approach is useful for defining the possible effectors of PKC-{epsilon}, acute ERK1/2 activation is likely to have distinct functions that are not directly relevant to sustained hypertrophic growth. For example, Barron et al. (2) demonstrated that the acute, transient activation of ERK1/2 in response to PE treatment was not responsible for the hypertrophic phenotype. The hypertrophic response was attributable a smaller, secondary rise in ERK activity that was maintained chronically. These findings suggest that infection of PKC-{epsilon}/DN adenovirus at a MOI of 2 is able to block cardiocyte growth by inhibiting this second peak of ERK1/2 activity. Our results also show that blockade of cardiocyte growth by PKC-{epsilon}/DN was accompanied by changes in morphology, which could reflect inhibition of signaling pathways not dependent on ERK1/2 such as the aforementioned Rho/ROCK pathway leading to FAK activation (20).

Besides PKC-{epsilon} and PKC-{delta}, recent studies have shown that PKC-{alpha} functions as a regulator of MAPK signaling in cardiocytes (5, 23). For example, overexpression of dominant negative PKC-{alpha} blocked PMA stimulated ERK1/2 phosphorylation in neonatal cardiocytes maintained in serum-supplemented medium (5). In contrast, we observed that PKC-{alpha} did not appear to activate ERK1/2 because the conventional PKC inhibitor Go6976 had no effect on ERK1/2 phosphorylation produced by PE treatment (data not shown). As shown in previous studies, it is likely that PKC-{alpha} activation did not occur because the cardiocytes were maintained in serum-free media (9, 10, 14). Serum is a potent anabolic stimulus in neonatal cardiocytes that exerts a marked effect on steady-state protein metabolism (26, 27). Furthermore, growth factors contained in serum-supplemented media are capable of activating PKC-{alpha} (25).

Our results support the conclusion that ERK1/2 activation is dependent on PKC-{epsilon} in neonatal rat cardiocytes. On the basis of evidence obtained using adult cardiocytes, the mechanism probably involves a direct link between PKC-{epsilon} and the c-Raf/MEK/ERK pathway (21). This notion is supported by proteome analyses, which have revealed that PKC-{epsilon} exists as a component of a larger protein complex that coimmunoprecipitates with ERK in several subcellular compartments of the cardiocyte (1, 33). Thus by directly regulating ERK1/2 activity, PKC-{epsilon} could modify the activity of multiple signaling pathways that control cardiocyte growth.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant PO1 HL-48788 and the Research Service of the Department of Veterans Affairs. V. U. Rao was supported in part by the Medical Scientist Training Program of the Medical University of South Carolina.


   ACKNOWLEDGMENTS
 
We thank Daisy Dominick, Christina DeRienzo, Aryan Namboodiri, Atif Saghir, and Jennifer MacDonald for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. J. McDermott, 303 Thurmond Bldg., 114 Doughty St., Charleston, SC 29403 (E-mail: mcdermp{at}musc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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 REFERENCES
 

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