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Cardiovascular Research 2003 57(1):139-146; doi:10.1016/S0008-6363(02)00610-7
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Copyright © 2003, European Society of Cardiology

Protein kinase C βII activation induces angiotensin converting enzyme expression in neonatal rat cardiomyocytes

Yuke Zhang, Laura J. Bloem, Lan Yu, Thomas B. Estridge, Philip W. Iversen, Christine E. McDonald, James P. Schrementi, XuShan Wang, Chris J. Vlahos and Jian Wang*

Cardiovascular Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA

* Corresponding author. Tel.: +1-317-277-6767; fax: +1-317-433-2815. jyw{at}lilly.com

Received 6 February 2002; accepted 5 August 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Members of the protein kinase C (PKC) family are important mediators of cell signaling underlying multiple aspects of myocardial function. Activation of the βII isoform of PKC is thought to be involved in the development of congestive heart failure. To investigate the biological effect of PKC-βII, we measured gene expression of angiotensin converting enzyme (ACE) and angiotensin II (AngII) receptors AT1A and AT1B in cardiomyocytes overexpressing PKC-βII. Methods: An adenovirus construct expressing PKC-βII was introduced into cultured neonatal rat ventricular myocytes (NRVMs). Western blot and in situ kinase assay was used to measure PKC-βII level and activity in NRVMs. Real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to measure the mRNA levels of several genes following PMA stimulation of either un-infected or ad-PKC-βII infected cells. Results: Our data show that activation of PKC-βII in cardiomyocytes leads to elevated expression of angiotensin-converting enzyme (ACE) gene. Treatment of adeno-PKC-βII infected cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) resulted in an 8-fold increase of ACE mRNA expression, whereas ACE mRNA levels only increased around 2-fold in uninfected or adeno-GFP (green fluorescent protein) infected cardiomyocytes with similar PMA treatment. The induction of ACE mRNA was blocked by the PKC-β-specific antagonist LY379196. No significant change of angiotensin II receptors AT1a and AT1b could be detected in the cardiomyocytes expressing PKC-βII. Conclusion: These data indicate that ACE is a transcription target of PKC-βII activation in cardiomyocytes, and also suggest a mechanism for the involvement of PKC in cardiac hypertrophy and fibrosis through increased activity of angiotensin converting enzyme in the myocardium.

KEYWORDS Gene expression; Heart failure; Protein kinases; Renin angiotensin system


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Protein kinase C (PKC) is a family of serine–threonine kinases that function as important mediators of cell signaling [1,2]. To date, 12 isoforms of PKC have been identified that can be divided into three groups. Classical PKCs (isoforms {alpha}, βI, βII, and {gamma}) are activated by phosphatidylserine, Ca2+, and diacylglycerol (or PMA). Novel PKCs ({delta}, {epsilon}, {eta}, {theta}, and µ) are not activated by Ca2+, but are activated by PMA and diacylglycerol. The atypical PKCs ({zeta}, {iota}, and {lambda}) are not activated by Ca2+, PMA, or diacylglycerol. In cultured cardiomyocytes, PKC activation is involved in the signaling of a diverse set of cytokines and stimuli, such as angiotensin II (AngII), endothelin-1, vascular endothelial growth factor (VEGF) and the {alpha}-adrenergic receptor agonists. For example, activation of {alpha}-adrenergic receptor by phenylephrine (PE) has been shown to activate PKC and induce expression of c-fos [3], atrial natriuretic peptide (ANP) and cardiac myosin light chain (MLC)-2 genes [4]. Similarly, angiotensin II (AngII) also induced ANF and MLC expression in neonatal rat cardiomyocytes [3].

Expression of the different PKC isoforms is tissue-specific. Studies using rat ventricular cardiomyocyte preparations have shown that Ca2+-dependent PKC isoforms {alpha} and β are expressed in fetal and neonatal heart [4,5], while PKC-β is only sparsely detected in adult cardiac tissue [6]. Increased expression of these PKC isoforms in adult heart is associated with pathologies such as diabetes and heart failure. Specifically, elevated expression and activation of PKC β has been observed in failing human myocardial tissue [7] and in an animal model of heart failure [8]. Transgenic mice with targeted cardiac overexpression of PKCβII exhibit gross cardiac hypertrophy and diminished ventricular function [9,10].

To investigate the biological effect of PKC-βII activation in cardiomyocytes, we introduced an adenovirus construct encoding full-length PKC-βII into cultured neonatal rat ventricular myocytes. We found that PKC-βII activation in these cells stimulates the expression of angiotensin converting enzyme (ACE). Our data suggest that PKC-βII activation during CHF may lead to elevated ACE activity in the myocardium.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Adenovirus PKC construct
Adenoviral construct containing human PKC-βII cDNA was generated with the AdEasy kit (Quantum Biotechnologies, Rockville, MD). Briefly, pLYPKCβII was digested with XhoI followed by generation of blunt ends using T4 DNA polymerase. The PKC-βII cDNA was released with BamHI and ligated to EcoRV–BamHI site of RB11174A1 (a modified version of pQBI-AdCMV5 from Q-BIOgene, Carlsbad, CA). The recombinant adenovirus was generated by homologous recombination in QBI293A cells (Q-BIOgene) co-transfected with the desired transfer vectors along with linearized viral DNA according to the manufacturer suggested protocol (Q-BIOgene). Plaques were selected, amplified, and analyzed for protein expression by Western blot. Appropriate plaques were grown to high titer in QBI293A cells and purified over cesium chloride gradients. Tissue culture infectious dose (TCID) was used to titer the amplified viral stock. Adenoviral construct containing green fluorescent protein (GFP) cDNA was from Quantum Biotechnologies.

2.2 Cell cultures
NRVMs were prepared from 1- to 2-day-old Sprague–Dawley rats as described previously [11]. Contamination of non-myocytes was reduced through pre-plating for 2 h in regular culture dishes and further reduced by incubation in medium containing 5% horse serum and 0.1 mM bromodeoxyuridine (BrdU). After 24 h, cells were changed to medium without serum and BrdU. The investigation conforms with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Adenovirus containing either GFP or PKC-βII cDNA stock solutions were diluted in cardiomyocyte culture medium and applied to NRVM cultures on the third day of cell culture. Cells were incubated with medium containing adenovirus for 24 h, after which the medium was aspirated and cells were incubated with medium containing phorbol 12-myristate 13-acetate (PMA) in the presence or absence of PKC inhibitors. Protein kinase C inhibitor Go6976 (Sigma, St. Louis, MO) or LY379196 [12] were dissolved in DMSO and diluted into serum-free medium before use. Control cells were treated with the DMSO vehicle alone. Cells were treated with these compounds for 24 h before RNA isolation.

2.3 Immunoblotting
Polyclonal antibodies against PKC-βII and PKC{alpha} were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA). Cell lysates were prepared with the 1x sodium dodecyl sulfate (SDS) sample buffer (Novex, San Diego, CA) according to the manufacturer's suggested protocol. Equal amount of cell lysates were separated on pre-cast SDS–12% polyacrylamide gel (Novex) and transferred to nylon membranes (Novex). Membranes were probed with indicated primary antibodies and horseradish peroxidase (HRP) conjugated secondary antibody (Santa Cruz). Membranes were developed with the enhanced chemiluminescent reagent (Amersham Biosciences, Piscataway, NJ) and exposed to X-ray film.

2.4 In situ PKC activity assay
NRVMs were cultured at 2.5x104 cells/well in 96-well plates in the serum-free medium (SFM) described above. On day 3, cells were changed to medium containing either adeno-GFP or adeno-PKC-βII. On day 5, the medium was removed and the cells were incubated with or without LY379196 in SFM (90 µl) for 1 h at 37 °C in a humidified chamber. PMA (100 nM final) was then added to appropriate wells and the cells were incubated for an additional 10 min. Medium was then removed and PKC activity was determined with a modification of published procedure [13] using the EGF-R peptide (651–658, RKRTLRRL, Biomol, Plymouth Meeting, PA) as substrate. Briefly, 40 µl of the enzyme mix (137 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM K2HPO4, 20 mM HEPES, 10 mM MgCl2, 1 mg/ml glucose, 25 mM β-glycerophosphate, 5 mM EGTA, 50 µg/ml digitonin, 2.5 mM CaCl, pH 7.2, 100 µM [{gamma}-33P]ATP, and 200 µM EGF-R peptide) was added to each well. After 15 min incubation, 10 µl 30% TCA was added to stop the reaction, and then 40 µl from each well was transferred to individual wells of phosphocellulose MultiScreen-PH 96-well plates (Millipore, Bedford, MA) and counted. Endogenous activity was determined in the absence of peptide and subtracted from each sample to determine the PMA-activated PKC activity.

2.5 RNA isolation and quantitative RT-PCR (TaqMan)
Total cellular RNA from neonatal rat cardiac myocytes was isolated using the Trizol reagent (Life Technologies, MD) according to the manufacturer's protocol. RNA samples were treated with DNase I and extracted with phenol/chloroform to remove any contaminating genomic DNA. First strand cDNA was prepared by reverse transcription with 3 µg of RNA in a 20-µl reaction volume using the Superscript II kit (Life Technologies).

The relative mRNA levels were measured by real-time quantitative PCR analysis utilizing 5' nuclease assays (TaqMan, Applied Biosystems, Foster City, CA). Oligonucleotide probes were labeled with FAM (6-carboxy fluorescent, reporter dye) at the 5' end and TAMRA (6-carboxy-tetramethylrhodomine, quencher dye) at the 3' end. Probes and primers were designed using Primer Express version 1.0 (Applied Biosystems) and were synthesized by Biosource International (Camarillo, CA). Primer and probe sequences are listed in Table 1. PCR reactions included 2x Universal Master Mix, a specific probe and primer set, and a cDNA aliquot from each RT reaction. PCR reactions were performed in triplicate in an ABI 7700 instrument (Applied Biosystems) using standard conditions. Relative expression levels of a specific gene in each sample were calculated from a standard curve made from a dilution series of one of the samples. RNA levels of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and 18S RNA was also measured using pre-developed reagents (part 4308313 and 4308329, Applied Biosystems), simultaneously with the gene of interest.


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  		Table 1 Primers and probes used for TaqMan analysis

 
Two different genes, GAPDH and 18S RNA, were used as endogenous controls [14]. Similar data were obtained by normalizing with either GAPDH or 18S RNA. It has been reported that the GAPDH mRNA level can be modified by PMA treatment [14]. Under our experiment conditions, no significant difference in GAPDH level was detected among different RNA samples that were prepared from a similar number of cells treated with or without PMA.

2.6 Angiotensin-converting enzyme activity assay
NRVMs cultured in 6-well plates were washed with phosphate-buffered saline (PBS) and scraped into centrifuge tubes. The cell pellets were resuspended in lysis buffer containing 50 mM Tris, pH 7.4, 1% NaCl and 8 mM CHAPS, and then sonicated and centrifuged to recover the clear cell lysate. ACE activity was measured by using a method adapted from that described by Corvol et al. [15]. Cell lysates were used directly or diluted with assay buffer consisting of 50 mM HEPES, 300 mM NaCl and 0.01% Brij detergent (Pierce, IL) at pH 7.5. The reaction was started by the addition of 40 µl of synthetic ACE substrate (125 µM), Abz-Gly-p-nitro-Phe-Pro-OH (Bachem, Bubendorf, Switzerland), solution to 60 µl of sample in each well. After the addition of substrates, each plate was briefly mixed and incubated in the dark at room temperature for periods ranging up to 6 h. The plates were read on a BMG Fluostar Galaxy fluorometer (BMG Labtechnologies, Durham, NC) at excitation and emission wavelengths of 340 and 405 nm, respectively.

2.7 Statistical methods
Unpaired Student's t-test was used for determining the significance of differences (Fig. 5); values of P<0.01 were considered statistically significant. Results are expressed as mean±standard error of the mean. Mean values were calculated from data of triplicate experiments.


Figure 5
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  		Fig. 5 PKC-βII activation induced ACE activity. NRVMs were infected with adenovirus PKCβII (lane 2 through 4) and treated with 10 nM PMA for 24 h in the presence (lane 4) or absence (lane 3) of 500 nM LY379196. Cell lysates were prepared and ACE activity was measured as described in Section 2. The ACE activity in untreated NRVM (lane 1) was set as 100%. Mean values are calculated from data of triplicate experiments (** P<0.01 versus untreated).

 
For Figs. 2–4GoGo, the data were analyzed by analysis of variance (ANOVA) with variance components for between- and within-experiment variability. Statistical comparisons were based on the between-experiment variability. The data were transformed to the log scale to equalize variances across groups. The Hochberg procedure for multiple comparisons was used to control the false positive rate.


Figure 2
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  		Fig. 2 PKC-βII activation leads to increased ACE mRNA expression in cardiomyocytes. NRVMs were left untreated, or infected with adeno-GFP (ad-GFP) or adeno-PKCβII (ad-PKC) construct as indicated. After 24 h of virus infection, the indicated concentration of PMA was added for 24 h and total cellular RNA isolated. TaqMan analysis was performed to determine the relative amount of ACE (A), AT1A (B), or AT1B (C) mRNA level in these cells. Relative mRNA abundance was normalized against GAPDH level in each sample and expressed as arbitrary units in the bar graph. ACE/GAPDH ratio in the uninfected and untreated cells (baseline) was set as 1. Mean values were calculated from data of triplicate experiments (** P<0.01 versus baseline).

 

Figure 3
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  		Fig. 3 Effect of PKC-βII expression level on induction of ACE mRNA level in NRVMs. (A) NRVMs were infected with increasing amounts of PKCβII adenovirus as indicated. The cells were stimulated with 10 nM PMA for 24 h and relative ACE mRNA level was measured by TaqMan. The ACE/GAPDH ratio in uninfected and untreated cells (baseline) was set as 1 on the bar graph (** P<0.01 versus baseline). (B) NRVMs infected with adeno-PKCβII (at an MOI of 40) were treated with 10 nM PMA and RNA was collected at the indicated time points. ACE mRNA levels were determined using TaqMan. ACE/GAPDH ratio in PMA untreated cells was set as 1 on the bar graph (** P<0.01 versus untreated).

 

Figure 4
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  		Fig. 4 A PKC-β-specific inhibitor blocked ACE mRNA induction in cardiomyocytes. NRVMs infected with adeno-PKC-βII or uninfected cells (no virus) were pretreated with the cPKC-specific inhibitor Go6976 (Go) or the PKCβ-specific inhibitor LY379196 (LY) at 500 nM for 1 h before PMA stimulation. Twenty-four hours later, total cellular RNA was isolated and relative ACE mRNA level was determined by TaqMan. ACE/GAPDH ratio in uninfected and unstimulated NRVMs was set as 1 on the bar graph. Mean values were calculated from data of triplicate experiments. The letters, a, b and c, define groups of data that are not statistically different within a group, but are statistically different between groups (P<0.01).

 

   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Expression of protein kinase C-βII in adeno-PKC-βII infected cardiomyocytes
NRVM cultures were infected with different dilutions of the PKC-βII adenovirus construct in order to determine the optimal virus to cardiomyocyte ratio for PKC-βII expression. After 24 h of infection, cell lysates were collected and subjected to Western blot analysis using a PKC-βII specific antibody. As shown in Fig. 1A, increasing amounts of PKC-βII protein in the cell lysates correlates with an increasing multiplicity of infection (MOI) of the PKC-βII adenovirus. Since substantial elevated expression of PKC-βII was detected at MOI of 40, this MOI was used for all subsequent studies.


Figure 1
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  		Fig. 1 (A) Adeno-PKC-βII induced PKC-βII over-expression in cardiomyocytes. Cell lysates were prepared from uninfected NRVMs (lane 2), NRVMs infected with adeno-GFP construct (lane 3) or infected with adeno-PKC-βII at MOI of 10 (lane 4), 20 (lane 5), 40 (lane 6) and 100 (lane 7). Recombinant PKC-βII protein (5 pg) was loaded as a positive control (lane 1). Samples were separated using SDS–PAGE followed by Western blot analysis with a PKC-βII specific antibody (Santa Cruz Biotechnology). A representative blot from three different experiments is shown. An identical blot was also probed with a PKC{alpha} specific antibody to verify equal loading. (B) Inhibition of PKC-βII activity by LY379196 in adenovirus infected cardiomyocytes. NRVMs infected with adeno-PKCβII were pretreated for 60 min with LY379196 (0.5 nM–1 µM). Following treatment with 100 nM PMA, PKC activity was then determined by measuring 33P incorporation on the peptide RKRTLRRL as described in Section 2. Results are presented as mean±S.E.M., n=3 for each condition. Unstimulated: basal PKC activity in unstimulated adeno-PKCβII-infected NRVMs; +PMA: maximal PKC activity in adeno-PKCβII infected NRVMs treated with 100 nM PMA; +PMA+LY: PMA-induced PKC activity in adeno-PKCβII infected NRVMs in the presence of increasing concentrations of LY379196.

 
To confirm that the cells infected with PKC-βII adenovirus express active kinase, PKC activity was determined using an in situ kinase assay in intact NRVMs infected with either PKC-βII adenovirus or GFP adenovirus. PKC activity was determined by phosphorylation of a substrate peptide in permeabilized cells in the presence of 33P-ATP. NRVMs expressing PKC-βII show an approximately 12-fold higher PMA-stimulated PKC activity over the PMA-stimulated GFP-expressing myocytes (23829±1640 counts per minute (cpm) versus 1945±66 cpm, data not shown). In Fig. 1B, preincubation of the adeno-PKC-βII cells with an increasing concentration of LY379196 prior to PMA stimulation shows a complete inhibition of substrate peptide phosphorylation (IC50=0.01 µM), a value consistent with the previously reported IC50 against PKC-βII in cell-free systems (IC50=0.03 µM) [12]. This also demonstrates that the compound is able to cross the cell membrane and act to specifically inhibit PKC-βII in intact cells.

3.2 PKC-βII augments PMA induced ACE transcription in cardiomyocytes
Using real-time quantitative RT-PCR, the relative mRNA level of ACE was measured following PMA treatment of uninfected, as well as adeno-PKC-βII or adeno-GFP infected, NRVMs. As shown in Fig. 2A, 10 nM PMA induced an 8-fold increase in ACE mRNA level in adeno-PKC βII infected cardiomyocytes, while a 2-fold increase in ACE mRNA expression in adeno-GFP or uninfected cardiomyocytes was observed. A maximal ACE mRNA induction of 12-fold was achieved by 50 nM PMA in adeno-PKC βII infected NRVMs, compared to a 3-fold induction in adeno-GFP and uninfected cardiomyocytes. Moreover, there is no difference in ACE mRNA expression in the adeno-GFP infected cells compared to uninfected cells at any PMA concentration used. The mRNA levels of the angiotensin II receptors AT1A and AT1B were also measured from these same treated cells. As shown in Fig. 2B and C, mRNA level of neither AT1A nor AT1B changed significantly in cardiomyocytes with PKC-βII over-expression.

To determine the effect of PKC-βII expression, the dose–response curve of ACE induction with increasing concentrations of PKC-βII activation was measured with Taqman. As shown in Fig. 3A, increasing concentrations of PKC-βII adenovirus resulted in a corresponding increase in ACE mRNA level. ACE induction reached maximal levels at an MOI of 40. The time-course of ACE mRNA induction was also measured. As shown in Fig. 3B, ACE induction was initially present after 14 h of treatment with 10 nM PMA and reached a maximum at 24 h.

3.3 Inhibition of ACE induction with the PKCβ-selective inhibitor, LY379196
To further verify that the induction of ACE expression is mediated by PMA-stimulation of the βII isoform of protein kinase C, the effect of PKC inhibitors on ACE expression was evaluated. As shown in Fig. 4, both Go6976, a specific inhibitor for the classical PKC isoforms [12], and LY379196, a selective PKC-β inhibitor [12], blocked PMA-induced ACE transcription in NRVMs infected with PKC-βII adenovirus. The slight increase in PMA-induced ACE expression in cells expressing only the endogenous PKCs was also blocked by both compounds.

3.4 PKC-β-mediated induction of ACE enzyme activity
To determine if increased ACE mRNA level resulted in a corresponding increased ACE protein and activity in PKC-βII over-expressing NRVMs, ACE activity was measured in cell lysates using a peptide substrate for ACE. As shown in Fig. 5, PMA stimulation induced a three-fold increase in angiotensin conversion. Moreover, the PKCβ-selective inhibitor, LY379196, inhibited the PMA-induced ACE activity in adeno-PKC-βII infected cells. Captopril (1 µM), a selective inhibitor of ACE, was able to block this peptidase activity in the cardiomyocyte lysate (data not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The renin–angiotensin system plays a major role in the physiology of the cardiovascular system, as well as in the pathogenesis of cardiovascular diseases [16]. AngII has been shown to stimulate cardiomyocyte hypertrophy [17], as well as to induce the proliferation of cardiac fibroblasts and the expression of a number of pro-fibrotic mediators [18]. In experimental animal models, infusion of AngII promoted increased intramyocardial fibrosis that was attenuated by treatment with the AT-1 antagonist Losartan [19]. Similarly, ACE inhibitors and AT1 receptor antagonists prevented cardiac fibrosis in response to myocardial infarction [20,21], as well as improved cardiac function and survival in ischemic heart failure [22]. Mice deficient in AT-1 showed reduced cardiac fibrosis and left ventricular remodeling, as well as improved survival following myocardial infarction compared to wild type mice [23]. Most importantly, in addition to the benefits of lowering systemic blood pressure, treatment of heart failure patients with ACE inhibitors or AT1 receptor antagonists have shown clinical benefit in the form of reduction of both mortality and morbidity [24], which may be the result of reducing vascular and arrhythmic events, as well as benefits in ventricular remodeling and in modulating interstitial fibrosis [25].

Classically, the renin–angiotensin system is considered a circulating hormone system. However, recent experimental evidence suggests the existence of a local angiotensin generating system in several tissues, including the heart. Increased ACE mRNA level and activity have been detected in the myocardium of heart failure patients [26,27]. Similarly, expression of angiotensinogen, the precursor for angiotensin, was also upregulated in ischemic rat heart [28]. Increased local availability of angiotensin II in the myocardium is thought to be one of the mechanisms responsible for cardiac hypertrophy and fibrosis, which are typical pathological changes in the failing heart. However, the molecular mechanisms underlining increased myocardium ACE expression in heart failure patients have not been delineated.

While the ability of AngII to stimulate PKC has been well established in cardiomyocytes, activation of PKC may also affect activity of the renin–angiotensin system. Previous investigators have demonstrated that cultured neonatal or adult cardiomyocytes will release AngII when exposed to mechanical or chemical stress. For example, mechanical stretch applied to in vitro cultures of NRVMs caused a release of AngII, as well as other hypertrophic growth factors [17]. More recently, it was reported that treatment of isolated adult rat cardiomyocytes with high levels of glucose (5 mmol/l) also stimulated the release of AngII [29]. In each of these examples, however, the AngII secretion was caused by release from intracellular stores rather than by new protein synthesis. Whereas others have speculated that exposure to high levels of glucose can activate PKC-induced gene transcription [30], there is no reported evidence that PKC activation can induce expression of mediators of the renin–angiotensin pathway in cardiomyocytes.

Here we report that PKC activation by PMA induces ACE transcription in cultured neonatal rat ventricular myocytes. Moreover, by using an adenovirus construct of PKC-βII, we demonstrated PKC-βII overexpression in NRVMs greatly augmented PMA-induced ACE transcription (Fig. 2A), but had no effect on the mRNA expression of AngII receptor (Fig. 2B and C). The level of ACE mRNA expression correlated to amount of PKC-βII present (Fig. 3). Furthermore, a PKC-β-selective inhibitor, LY379196, blocked PMA induction of ACE expression in both untreated and adeno-PKC-βII infected NRVMs (Fig. 4). Finally, an increase in ACE enzymatic activity was demonstrated in PMA-treated cells infected with adeno-PKC-βII, but not in uninfected or untreated cells. However, PMA also induced a two to three fold induction of ACE mRNA level in NRVMs; therefore, the contribution of other PKC isoforms, such as PKC-{alpha}, cannot be excluded. Taken together, these results suggest that PKC activation could result in increased expression of ACE, and thereby account for local production of angiotensin II in the myocardium.

Since residual non-myocytes (mostly cardiac fibroblasts) are present in our NRVM preparation, it is possible that cardiac fibroblasts may have also contributed to the robust ACE induction by PMA. However, under our experimental conditions, we found that PMA was not able to stimulate significant elevation in ACE mRNA level (<2-fold) in neonatal rat fibroblasts, either untreated or infected with adeno-PKC-βII construct (data not shown). This may be due to the fact that ACE mRNA level is very low in neonatal rat cardiac fibroblasts compared to that in cardiac myocytes [31].

PKC activation-induced ACE transcription in NRVMs was not detectable at 1, 4 and 6 h after PMA treatment, became evident at 14 h and remained at elevated levels up to 48 h (Fig. 3B), suggesting that an intermediate step is involved. The signaling pathway mediating PKC activation-induced ACE expression remains to be delineated. There are several possible mechanisms worth exploring. Several reports have shown that the M-CAT (myocyte-specific CAT) cis element is required for {alpha}1-adrenergic and PKC induction of cardiac hypertrophy related genes, such as ANP, brain natriuretic peptide (BNP), {alpha}-skeletal actin ({alpha}SKA) and β-myosin heavy chain (β-MHC), in NRVMs [32,33]. The genomic sequences 5' to rat ACE are not known, but the human and mouse ACE gene promoter region sequences are available in the NCBI (National Center for Biotechnology Information) database. Sequence search revealed two stretches containing tandem M-CAT elements in the human genomic sequence 5' to the ACE gene, at position –788 to –814 and at –187 to –247, respectively. Similarly, tandem M-CAT elements are also present in 5' flanking sequences of the mouse ACE gene, at position –216 to –224 and between –552 to –780. The early growth response factor 1 (egr-1) might also be involved. Previously, Villard et al. [34] reported that PMA treatment increased ACE expression and activity in cultured human endothelial cells. In human endothelial cells, induction of egr-1 transcription by PMA precedes that of ACE. Multiple egr-1 response elements are present in both human and mouse ACE promoter region. Further study of the ACE promoter/enhancer region could help to understand the signaling pathway by which PKC-β stimulates ACE expression.

Saijonmaa et al. [35] showed that, in cultured human vascular endothelial cells (HUVECs), ACE expression is upregulated by VEGF, whereas treatment with a PKC inhibitor prevents the induction of ACE. Williams et al. [36] have shown that activation of the PKC-βII isoform has been linked to increased expression of VEGF. It is possible that PKC-βII activation by PMA induces release of VEGF, which in turn leads to increased expression of ACE. Future studies, beyond the scope of this manuscript, will examine VEGF production by NRVMs in response to PMA stimulation.

Both PMA and AngII have been shown to stimulate cardiomyocyte hypertrophy [17]. However, protein synthesis determination in NRVMs indicates that the PMA-induced hypertrophy response in cultured NRVMs can be blocked by the PKC-βII inhibitor LY379196, but not by either ACE-inhibitor captopril or AT1-receptor blocker Losartan (data not shown). Thus, AngII is not likely the mediator of PKC stimulated cardiomyocyte hypertrophy. Nevertheless, it will be interesting to investigate if PKC activation could stimulate AngII synthesis and release by cultured NRVMs.

Our findings are the first to demonstrate that PMA can also induce ACE expression in the cardiomyocyte, and that the PKC-β isoform plays a key role in ACE expression in these cells. In addition, elevated PKC-β isoform expression and activation is observed in hearts of CHF patients [1,7]. These data suggest that PKC-βII induced ACE expression may, at least in part, contribute to the development of CHF.

Time for primary review 28 days.


   Acknowledgements
 
The authors wish to thank Najia Jin for technical assistance and Robin J. Bowman for her assistance with the manuscript.


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

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