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Annals of Clinical & Laboratory Science 39:351-360 (2009)
© 2009 Association of Clinical Scientists

A Peptide from a ras Effector-Domain Blocks ras-Dependent Cardiac Hypertrophy in Myocytes

Lili Deng1,5, Mohamed Boujdir2,6, Anthony Tremontano3,7, Matthew R Pincus4,8 and Nabil El-Sherif3,7
1 Molecular Cardiology Program, 2 Research and Development Service, 3 Department of Cardiology, and 4 Department of Pathology & Laboratory Medicine, NY Harbor VA Medical Center, Brooklyn, New York; 5 Molecular and Cell Biology Program, 6 Department of Pharmacology and Physiology, 7 Department of Cardiology, and 8 Department of Pathology, SUNY Downstate Medical Center, Brooklyn, New York

Address correspondence to Nabil El-Sherif, M.D., Cardiology Service, NY Harbor VA Healthcare System, 800 Poly Place, Brooklyn, NY 11209, USA; tel 718 630 3740; fax 718 630 3740; e-mail nelsherif{at}aol.com; or to Matthew R. Pincus, M.D., Ph.D., Department of Pathology & Laboratory Medicine, NY Harbor VA Medical Center, 800 Poly Place, Brooklyn, NY 11209, USA; tel 718 630 3688; fax 718 630 2960; e-mail matthew.pincus2{at}med.va.gov.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
PNC-2 is a peptide corresponding to an effector domain (residues 96–110) of ras-p21 that strongly and specifically blocks mitogenic signal transduction by oncogenic but not activated, normally-expressed wild-type ras-p21 protein. Since myocardial hypertrophy can be induced both by oncogenic and overexpressed wild-type ras-p21, we investigated whether PNC-2 can block norepinephrine (NE)-induced, ras-dependent myocardial hypertrophy in cardiac myocytes. Since PNC-2 blocks oncogenic ras-p21-induced activation of JNK and ERK, we further determined whether this peptide blocks activation of these kinases in NE-treated myocytes. Using cultured neonatal rat ventricular myocytes (NRVM), we found that NE alone significantly increased NRVM surface area, 3H-leucine uptake, protein/DNA ratio, and atrial nartiuretic factor (ANF) mRNA levels in these cells. However, pretreatment of the NRVM with PNC-2 linked on its carboxyl terminal end to a transmembrane-penetrating leader sequence (PNC-2-leader) resulted in strong inhibition of NE-mediated cell growth and 3H-leucine uptake and in significantly lower protein/DNA ratios. Induction of ANF mRNA levels was likewise inhibited by PNC-2-leader. In contrast, no inhibition of any of these NE-induced events was observed with a negative control peptide, X13-leader. Western blot analysis showed that JNK and ERK1/2 activity, but not p38 activity, was increased in NRVM within 5 min of exposure to NE (2 µM). Pretreatment with PNC-2-leader decreased ERK1/2 and JNK activity to basal levels. We conclude that a synthetic peptide designed to block oncogenic ras can also counter the effects of NE-induced hypertrophy associated with overexpression of ras p21 by blocking JNK/ jun and ERK activation. PNC-2 may provide a prototype for novel therapy in cardiac conditions associated with activation of NE.

Keywords: myocardial hypertrophy, ras-p21, jun-N-terminal kinase (JNK), ERK, epinephrine-induced hypertrophy, atrial natriuretic factor (ANP), RNase protection assay

Abbreviations: ANF = atrial natriuretic factor; Ang II = angiotensin II; EP = epinephrine; ERK = extracellular growth factor-responsive kinase also called mitogen-activated protein kinase; ET-1 = endothelin-1; GPCR = G-protein-coupled receptors; JNK = jun N-terminal kinase; NE = norepinephrine; NRVM = cultured neonatal rat ventricular myocytes; p38 = p38 MAP kinase (MAPK); PE = phenylephrine; PNC-2-leader = ras peptide containing amino acids 96-110 attached at its carboxyl terminus to transmembrane-penetrating leader sequence; RPA = ribonuclease protection assay; X13-leader, control peptide from cytochrome P450 attached at carboxyl terminus to transmembrane-penetrating leader sequence


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Acknowledgements
 References
 
Ras gene-encoded p21 proteins are a family of small guanine nucleotide-binding proteins (21 kDa). Ras-p21 activity is switched on by stimulation with the transition of ras proteins from GDP-bound to GTP-bound. A single amino acid substitution for glycine at position 12 (oncogenic p21) results in malignant transformation of cells [1,2]. Using computer-based molecular dynamics calculations, we previously reported that the average 3-dimensional structures of activated normal (GTP-bound) and oncogenic forms of p21 all exhibit similar regional conformational changes at residues 6–15, 35–47, 55–71, 81–93, 96–110, and 115–126, when superimposed on the average structure of normal p21 bound to GDP [1,2]. PNC-2 is a peptide corresponding to one of those effector domains, ie, residues 96–110. We found that this peptide completely blocks oncogenic ras-p21-induced oocyte maturation by inhibiting phosphorylation of JNK and ERK [2] while having no effect on insulin-induced maturation that requires activation of endogenous wild-type ras-p21. Recently, we found that PNC-2, attached on its carboxyl terminal end to a transmembrane-penetrating peptide that enables transport of the peptide across cell membranes, called PNC-2-leader, induces either tumor cell necrosis of cancer cells or reversion of cancer cells to the untransformed phenotype, but has no effect on the viability or growth of untransformed cells [3]. In cancer cells treated with PNC-2-leader, there was marked diminution in phosphorylated JNK and ERK although expression of total levels of both kinases remained unaffected [3].

Ras p21 proteins are universally important in regulating intracellular signaling events in mammalian cells and controling their growth, differentiation, and survival. Recently, involvement of ras p21 proteins in cardiac hypertrophy has become an area of significant interest. Previous work in cultured cardiac myocyte systems and in transgenic mice demonstrated that oncogenic ras induces a hypertrophic response in the myocardium [4]. Ras is rapidly activated in myocytes exposed to Ang II, ET-1, PE, and NE [5–7]. On the other hand, transfection with dominant negative ras inhibits agonist-stimulated ANF expression [8]. These in vitro and in vivo studies suggest that ras may play a major role in the regulation of cardiac hypertrophy. The signal transduction pathway that transmits signals from membrane-bound ras to the nucleus includes three mitogen-activated kinase cascades (MAPKs), JNK, ERK, and p38.

In the present study, we determined if PNC-2-leader peptide blocks ras-dependent myocardial hypertrophy with concurrent inhibition of JNK/ jun and ERK activation. In these experiments, we used NE, a potent growth factor for cardiac myocytes [9], as the agent to induce hypertrophy via a ras-dependent pathway. NE has been postulated to have a hypertrophic effect on cardiac myocytes through {alpha}1-, and β-adrenoceptors, which are G-protein-coupled receptors (GPCR). GPCR agonists activate ras-MAPK pathways [7,10,11].


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Acknowledgements
 References
 
Preparation of peptides.  PNC-2 peptide, containing residues 96–110 of ras-p21, YREQIKRVKDSDDVP, was synthesized with an antennapedia leader sequence on its carboxyl terminal end to enable the peptide to traverse cell membranes [3]. The leader peptide sequence was KKWKMRRNQFVKVQRG. Since a growing number of studies suggest that scrambled peptides may behave similarly to the original peptide [12,13], to exclude the nonspecific effect of peptide or the effect from leader sequence, X13, an unrelated peptide from cytochrome P450, called X13, whose sequence is MPFSTGKRIMLGE, attached on its carboxyl terminal end to the antennapedia leader sequence as above, was used as a control. This peptide is referred to as X13-leader. Both peptides were synthesized by solid phase methods and purified by HPLC to >95% purity as evaluated by mass spectroscopy.

Neonatal ventricular cardiac myocytes culture.  Ventricles from 1- to 3-day-old Sprague-Dawley rats were minced and subjected to consecutive digestion with trypsin (Sigma, St Louis, MO). The supernatants collected from each digestion were centrifuged at 700 g for 10 min, and subsequently resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% calf serum (Gibco BRL, Grand Island, NY), 0.1mM bromodeoxyuridine (BrdU, Sigma), 50 units/ml penicillin, and 50 µg/ml streptomycin (Sigma), then centrifuged at 700 g for 5 min. The pellet was resuspended in the same medium and filtered through a cell strainer. Cells were preplated for 30 min at 37°C to decrease contamination of non-muscle cells. The non-adherent cardiac myocytes were then plated on 1% gelatin-coated Petri dishes. Myocytes were incubated at 37°C in humidified air with 5% CO2 overnight. The medium was then changed to serum-free medium (DMEM with 10 µg/ml insulin, 10 µg/ml transferrin).

Study protocol.  In all cases, cardiac myocytes were incubated in serum-free medium 24 hr before peptide (PNC-2-leader, or X13-leader) addition. Five study groups were investigated: 1). Control group (cardiac myocytes with no norepinephrine [NE, Sigma] and no peptide incubation); 2). NE group (cardiac myocytes incubated with NE alone); 3). PNC-2-leader group (cardiac myoctyes incubated with PNC-2-leader peptide alone); 4). NE+PNC-2-leader group (cardiac myocytes pretreated 48 hr with PNC-2-leader peptide, then incubated with NE in the presence of PNC-2-leader peptide); and 5). NE+X13-leader (cardiac myocytes pretreated 48 hr with control peptide X13-leader, then incubated with NE in the presence of X13-leader peptide). A concentration of NE (2 µM) was used that is maximal for myocyte growth and gene transcription [14]. For each group, the medium was changed daily.

Comparison of cell size.  Cardiac myoctyes were plated at a density of 200 cells/mm2. Fields were randomly selected from the dishes. The photographs were taken from the same fields 24 hr before and 72 hr following NE stimulation. Cell area was compared by using the NIH UTHSCSA Image Tool.

Comparison of protein synthesis.  Cardiac myocytes were plated at a density of 500 cells/ mm2. Protein synthesis was evaluated by 3H-leucine uptake. Briefly, after 48 hr peptide pretreatment, NE (2 µM, Sigma) was added for a period of 24 hr in the presence of 2 µCi/ml 3H-leucine (Amersham, Piscataway, NJ). Following the 24 hr period of agonist treatment, cardiac myoyctes were washed with cold PBS (pH 7.4), and cold 5% TCA was added for 30 min to precipitate proteins. The precipitates were dried in air, resuspended in 0.4M NaOH, and aliquots were counted in a scintillation counter.

Determination of protein/DNA ratio.  Total cellular protein was normalized to DNA as an index of cell hypertrophy. At the completion of 48 hr NE stimulation, the cells were rinsed 3 times with PBS (triplicate dishes for each group), scraped into 1 ml of sodium citrate buffer containing 0.25% SDS, and immediately frozen and stored at –20°C [15]. Protein was measured by the Bradford assay [16], using bovine serum albumin as the standard. DNA concentration was determined by dsDNA Quantitation Kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. Briefly, 10 µl samples and 190 µl TE were added into the PicoGreen predispensed wells of the microplate. PicoGreen shows bright green fluorescence upon binding to double-stranded DNA (dsDNA). The fluorescence of samples was determined by a fluorescence-based microplate reader; the amount of DNA was calibrated with known concentrations of DNA standards (included in the kit).

Western blots.  Cardiac myocytes were plated at a density of 1,000 cells/mm2. Following 5 min exposure to NE, myocytes were washed twice with cold PBS and then scraped into extraction buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.25% Triton X-100, 10% glycerol, 1 mM PMSF, 1 mM DTT, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM NaF, 2 mM Na3VO4). Samples were transferred to an Eppendorf tube and placed on ice for 30 min. Cell debris was removed by centrifugation. The supernatants containing equal amounts of protein were subjected to 10% polyacrylamide-SDS gel. The expression levels of phospho-JNK (using anti-phospho JNK [JNK-P)] phosphorylated at positions Thr 183 and Tyr 185 [Promega, Madison, WI], diluted 1:800); total-JNK (using anti-JNK polyclonal antibody [Sigma] which recognizes both JNK-1 and JNK-2, diluted 1:1000); phospho-ERK (phosphorylated at Thr 183 and Tyr 185 [Promega], diluted 1:2500); and total-ERK (New England Biolabs, Beverly, MA, diluted 1:500) were detected by western blotting. All incubations were performed for 12 hr at 4°C, after which the membranes were washed three times with TBS-T and incubated with anti-rabbit secondary antibody (Amersham) at 1:4000 dilution. Detection was performed by the ECL chemiluminescence detection kit (Amersham) [3].

RNase protection assay (RPA).  Cardiac myocytes were plated at a density of 1,000 cells/mm2. Following a 24 hr period of NE stimulation, total RNA was isolated by a modification of the technique of Chromczynski and Sacchi [17]. ANF mRNA levels in different experimental groups were determined by RPA [18]. Briefly, RPA’s were performed with concomitant measurements of cyclophilin mRNA (internal standard). The cDNA fragment of Kv4.2 expressing ANF was kindly provided by Dr. Tamkun of Vanderbilt University. DNA template was prepared by ligating the cDNA fragment into pCRtm II vector (Invitrogen, Grand Prairie, TX). The Kv4.2 cDNA template was used to prepare 32P-UTP radiolabeled antisense cRNA probe (MAXIscript, Ambion, Austin, TX). cRNA probe was purified before use over 5% polyacrylamide/8 M/L urea gel. Concomitant hybridization of the two probes (1 x 104 cpm Kv4.2 cRNA and 1 x 104 cpm cyclophilin cRNA per 10 ug RNA sample) was carried out at 48°C for 18 hr followed by digestion with RNases A and T1 (Ambion), 37°C for 30 min. The reaction was terminated by the adding SDS and proteinase K, followed by phenol/chloroform extraction and ethanol precipitation. The protected fragments were visualized by autoradiography after electrophoresis on a 5% polyacrylamide/8 M/L urea gel. Yeast RNA (10 ug) was used as a negative control to test for the presence of probe self-complementation bands. Quantitative evaluation was carried out using scanning densitometric analysis. For comparisons among different groups, the arbitrary densitometric units were normalized to the value of the cyclophilin gene.

Statistics.  All data are expressed as mean ± SE. Comparisons between two groups were made using the unpaired Student’s t test. Multiple comparisons among groups were determined by ANOVA. When the F ratio exceeded the critical value (p <0.05), Tukey’s post-hoc test was performed for group-to-group differences; p <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Acknowledgements
 References
 
PNC-2-leader peptide inhibits NE-induced neonatal rat ventricular myocyte hypertrophy.  Fig. 1Go shows the % change of myocyte surface area at day 8 among different experimental groups. Exposure to NE (2 µM) significantly increased the myocyte size compared to control group without NE stimulation (n ≥ 15, p <0.05). Pretreatment with PNC-2-leader peptide (10 µg/ml) totally inhibited NE-mediated cell growth. X13-leader (10 µg/ml) did not affect NE-mediated cell growth. Treatment with PNC-2-leader had no effect (not shown).


Figure 1
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  		Fig. 1. The percentage change of myocyte surface area at day 8 in different study groups. Each condition is described on the X-axis. Concentrations of PNC-2-leader and X13-leader were each 10 ug/ml. The control consisted of untreated cells; N = 4 for each group; * indicates p <0.05.

 
Consistent with these observations, as shown in Fig. 2Go, exposure of cardiac myocytes to NE (2 µM) for 24 hr caused 74% ± 6% (p <0.01, N = 4) increase in 3H-leucine uptake compared to control. The 48 hr pretreatment of cardiac myocytes with PNC-2-leader peptide (10 µg/ml) prior to addition of NE resulted in a significant decrease (94% ± 7%, N = 4, p <0.01) of NE-mediated 3H-leucine uptake. X13-leader peptide pretreatment did not affect 3H-leucine uptake compared to the NE group (Fig. 2Go). Treatment with PNC-2-leader alone had no effect on 3H-leucine uptake compared to untreated controls (not shown).


Figure 2
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  		Fig. 2. Rate of protein synthesis, assessed by 3H-leucine incorporation, induced by NE (2 uM, 24 hr) in different conditions as listed on the X-axis; N ≥4 for each group; * indicates p <0.01. The control consisted of untreated cells. Incubation with PNC-2-leader and X13-leader, each present at 10 ug/ml, was for 48 hr prior to NE treatment and during NE treatment.

 
Fig. 3Go shows that myocardial response to PNC-2-leader, as measured by 3H-leucine uptake, is dose-dependent. Myocytes were stimulated by NE (2 µM) and treated with different concentrations of PNC-2-leader peptide (0, 0.5, 1, 2.5, 5, 10, 25, and 50 µg/ml). Scintillation readings were normalized to control (no NE stimulation). PNC-2-leader peptide, at a concentration of 5 µg/ml or higher, completely inhibited NE-induced 3H-leucine uptake (N = 4 for each concentration).


Figure 3
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  		Fig. 3. PNC-2-leader dose-response curve for inhibition of NE-stimulated hypertrophy. 3H-leucine incorporation into protein was used as an index of hypertrophy; experimental conditions are the same as for Fig. 2Go. Scintillation readings were normalized to the control (value was set as 1.0); 5 µg/ml or higher concentration of PNC-2 peptide completely inhibited NE-stimulated 3H-leucine incorporation. Values are means ± SE; N = 4 for each group.

 
Table 1Go shows the total cellular protein and DNA content of cardiomyocytes. Protein accumulation was normalized to the DNA content per dish for minor variations in cell number. There was no significant change for DNA content among all 5 study groups. However, protein content and protein/DNA ratios were significantly increased in NE-stimulated or X13-leader + NE-stimulated myocytes compared with those for myocytes treated with NE + PNC-2-leader-peptide whose protein and protein/DNA ratios were almost identical to the values for the control (no NE, no peptide).


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  		Table 1. Protein and DNA contents and protein/DNA ratios for cardiac myocytes in 5 different conditions.
 
The induction of ANF expression is a conserved feature of cardiac hypertrophy. In NE-stimulated cardiac hypertrophy, the increase of ANF mRNA level is at least partially ras-dependent [7]. Fig. 4Go shows that 24 hr treatment with NE (2 µM) caused a significant increase in ANF mRNA expression over the control value. In contrast, pretreatment with PNC-2-leader peptide strongly inhibited NE-induced ANF mRNA (Fig. 4Go). X13-leader peptide pretreatment had no effect on NE-stimulated ANF expression. PNC-2-leader pretreatment did not change steady-state ANF mRNA levels. These results suggest that PNC-2-leader strongly and specifically blocks ANF expression in NE-treated myocytes, with no toxicity to myocytes, consistent with our findings that this peptide blocks myocyte hypertrophy (Figs. 1Go–3GoGo).


Figure 4
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  		Fig. 4. Panel A: Blots of gene expression levels of ANF assessed by RPA. Samples contained 10 µg of total RNA, and the negative control sample contained 10 µg of yeast RNA. The conditions for each result are given above each lane. Panel B: Quantitative analysis of the blots of ANF mRNA levels in the 5 study groups from Panel A. Results were normalized to cyclophilin mRNA. Values are means ± SE, N = 4 for each group; * indicates p <0.05.

 
PNC-2-leader peptide blocks activation of JNK and ERK on the oncogenic ras-p21 pathway.  In previous studies, we found that PNC-2 blocked oncogenic ras-p21-induced phosphorylation of JNK and ERK in oocytes that were co-injected with oncogenic (Val 12-containing) ras-p21 and PNC-2 and that PNC-2-leader blocked these phosphorylations in 3 different oncogenic ras-transformed cancer cell lines [1–3]. In oocytes, we found that PNC-2 blocked the direct interaction of oncogenic ras-p21 with JNK [1]. Since PNC-2 blockade of phosphorylation of JNK and ERK correlated with inhibition of oncogenic ras-p21-induced oocyte maturation and since PNC-2-leader-induced blockade of phosphorylation of JNK and ERK correlated with tumor cell necrosis or phenotypic reversion of ras-transformed tumor cells, we explored the effects of PNC-2-leader on JNK and ERK phosphorylation in NE-treated myocytes. As shown in Fig. 5Go, we studied phosphorylation of JNK and ERK at time points up to 6 hr following NE stimulation of myocytes (5 min, 30 min, 2 hr, 6 hr). JNK and ERK phosphorylation strongly increased after 5 min of NE stimulation, was sustained for at least 30 min, and then returned to basal levels (Fig. 5Go).


Figure 5
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  		Fig. 5. Phosphorylations of JNK (Panel A) and ERK (Panel B) at 5 min, 30 min, 2 hr, and 6 hr after NE stimulation of myocytes.

 
Fig. 6Go shows that 5 min stimulation of cardiac myocytes with NE (2 µM) increased JNK activity by 2.1 fold (p <0.01, N = 4). NE-stimulated JNK activation was totally inhibited by 10 µg/ml PNC-2-leader peptide pretreatment while X13-leader peptide pretreatment had no effect.


Figure 6
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  		Fig. 6. PNC-2-leader peptide completely inhibited NE-stimulated JNK phosphorylation. Representative comparison of phosphorylated and total JNK protein levels in control (untreated myocytes), NE, PNC-2-leader + NE, and X13-leader + NE groups. Each condition is labeled above the appropriate lane. Proteins were collected 5 min after NE addition. Topmost panel: blots for phosphorylated JNK (labeled as phospho-JNK). Middle panel: blots for total JNK. Lowermost panel: ratio of phosphorylated JNK to total JNK for each condition from the above two blots. JNK phosphorylation level in the control (untreated) group was set equal to 1.0. Values are means ± SE; N = 4 for each group; ** indicates p <0.01.

 
Fig. 7Go shows that 5 min stimulation of cardiac myocytes with NE (2 µM) likewise increased ERK phosphorylation by 2.3 fold (p <0.01, N = 4). The NE-stimulated ERK activation was totally inhibited by 10 µg/ml PNC-2-leader peptide pretreatment while X13-leader peptide pretreatment had no effect (Fig. 7Go). In contrast, as shown in Fig. 8Go, when we blotted for phosphorylated and total p38, we found that NE induced no significant increase in the levels of phosphorylated p38.


Figure 7
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  		Fig. 7. PNC-2-leader peptide completely inhibited NE-stimulated ERK phosphorylation. Representative comparison of phosphorylated and total ERK protein levels in control, NE, PNC-2-leader + NE, and X13-leader + NE groups. Each condition is labeled above the appropriate lane. Proteins were collected 5 min after NE addition. Topmost panel: blots for phosphorylated ERK (labeled as phospho-p44/42). Middle panel: blots for total ERK (labeled as total-p44/42). Lowermost panel: ratio of phosphorylated ERK to total ERK for each condition from the above two blots. ERK phosphorylation level in control (untreated) group was set equal to 1.0. Values are means ± SE; N = 4 for each group; ** indicates p <0.01.

 

Figure 8
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  		Fig. 8, Upper panel: Blots for phosphorylation of p38 in myocytes under different conditions, as labeled on top of the figure, at 5 min, 30 min, 2 hr, and 6 hr after NE stimulation of myocytes. Lower panel: Blots for total p38. The conditions are the same as for the blots shown in the upper panel. Con = control (untreated cells).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Acknowledgements
 References
 
Our results suggest that the anti-oncogenic ras-p21 peptide, PNC-2-leader, is effective in blocking norepinephrine-induced myocardial hypertrophy. This conclusion is based on our findings that PNC-2-leader, but not the control peptide, X13-leader, blocks NE-induced increase in cell size, a direct measure of myocyte hypertrophy. In addition, PNC-2-leader, but not the control peptide, blocks new protein synthesis, as measured by 3H-leucine uptake, which occurs in myocardial hypertrophy, but has no effect on DNA levels in myocytes. Importantly, a hallmark event of myocardial hypertrophy in myocytes is enhanced expression of ANP as we found occurs in myocytes induced with NE to undergo hypertrophy. Expression of ANP in NE-treated myocytes, however, is completely blocked by PNC-2-leader but not by the control peptide. Thus PNC-2-leader strongly and specifically blocks NE-induced myocardial hypertrophy. PNC-2-leader-induced blockade of hypertrophy occurs without any resulting toxicity to myocytes, since cell survival is the same for myocytes treated with PNC-2-leader alone or combined with NE. In addition, PNC-2-leader has no effect on myocyte DNA levels (Table 1Go) and PNC-2-leader pretreatment of myocytes does not change steady-state ANF mRNA levels throughout the course of the experiment as shown in Fig. 4Go.

As shown in Fig. 5Go, treatment of myocytes with NE results in elevated intracellular levels of phosphorylated forms of JNK and ERK that, as shown in Figs. 6Go and 7Go, are strongly downregulated by PNC-2-leader. These findings are similar to previous results in which we found that Val 12-ras-p21 induced high levels of phosphorylated JNK and ERK in oocytes [1,2]. This induction was blocked by PNC-2 [1,2]. High levels of the phosphorylated forms of these kinases also occurred in ras-transformed cancer cells [1–3], but were strongly downregulated by PNC-2-leader. In contrast, insulin-induced oocyte maturation, which occurs via activation of endogenous wild-type ras-p21 [1,2], is not blocked by PNC-2 and is not associated with significant elevations of phosphorylated forms of JNK and ERK. Thus, in these cells, oncogenic and activated wild-type ras-p21 utilize differing signal transduction pathways [1–3]. In our present study, it appears that NE, which is known to induce ras-p21 overexpression in myocytes, causes activation of ras pathways that appear to be similar to those induced by oncogenic ras-p21, thereby making its effects susceptible to inhibition by PNC-2-leader.

However, one difference between our current and previous results concerns the time-course of activation of JNK and ERK. NE-induced phosphorylations of these kinases in myocytes occurs within 30 min, following which the phosphorylated forms are reduced to their baseline levels. In our previous studies, we found that oncogenic ras-p21 induced high and increasing levels of phosphorylated JNK and ERK in oocytes and sustained high levels of these kinases in ras-transformed cancer cells [1–3]. This difference may imply that brief activation of these kinases in myocytes is sufficient to induce sustained hypertrophy. It may also point to a difference in downstream signaling pathways induced by oncogenic and overexpressed wild-type ras-p21.

While several in vitro and in vivo studies have implicated ras activation in cardiac hypertrophy [7, 19–22], as we have observed in this study, the downstream signaling events that mediate hypertrophy are incompletely understood. Ras activity is known to result in activation of the 3 MAPK branches (ERK, JNK, and p38). Although all 3 MAPK pathways have been shown to be sufficient, if constitutively activated, to induce hypertrophic responses in neonatal cardiomyocytes [19,23,24], there has been disagreement over which, if any, of the pathways is necessary for hypertrophy to physiologically relevant stimuli. The role of p38 MAP kinase in regulation of cardiac hypertrophy has primarily been investigated in culture-based systems. p38 MAPK has been shown to be activated by hypertrophic agonists such as phenylephrine (PE) or endothelin-1 (ET-1), and pharmacologic inhibition of p38 activity blocked agonist-stimulated cardiomyocyte hypertrophy in vitro [25–27]. In vivo, p38 activity is elevated by pressure overload hypertrophy in aortic banded mice [23]. These data suggest a role of p38 activation in the regulation of cardiomyocyte hypertrophy. In contrast, other groups have reported that pharmacologic inhibition of p38 activity does not affect agonist-induced myocyte hypertrophy [28,29]. In the present study, 2 µM NE treatment successfully induced hypertrophy without p38 activation (Fig. 8Go) suggesting that p38 activation is not a requisite event in NE-induced hypertrophy. Since we found that oncogenic ras-p21 induces high levels of phosphorylated p38 in oocytes [30], NE-induced overexpression of endogenous ras-p21 may induce hypertrophy utilizing pathways downstream of JNK and ERK that are different from those induced by oncogenic ras-p21.

Similar to p38, the role of ERK in hypertrophy is controversial. In response to agonist stimulation or mechanical stretching, ERK1/2 has been shown to be activated in cultured cardiac myocytes [29,32,33] and in acute pressure overload heart in Throdents [34,35]. These observations suggested that ERK1/2 might regulate the hypertrophic response. This notion has been strongly supported by multiple pharmacologic inhibition or transfection studies in vitro [36–41]. However, a number of studies have reported contradictory conclusions (28,42–44). Some studies have proposed that ras-mediated activation of the JNK cascade is more relevant to hypertrophy compared to ERK [6]. Choukroun et al [28] reported that adenovirus-mediated gene transfer of SEK-1 (KR), a dominant inhibitory mutant of the immediate upstream activator of JNK, blocked ET-1-induced hypertrophy in vitro. In addition, gene transfer of SEK-1(KR) to the adult rat heart inhibited pressure overload-induced cardiac hypertrophy in vivo.

Our results suggest that PNC-2-leader peptide prevents NE-mediated hypertrophic response by blocking both ras-dependent ERK and JNK activation. In this regard we have recently shown that there is a positive feedback interaction between the raf-MEK-ERK and JNK-jun pathways [45] explaining how inhibition of one pathway can result in inhibition of the other pathway.

In summary, our results strongly support the hypothesis that, besides blocking oncogenic ras signal transduction in oocytes and transformed cell lines, PNC-2 peptide also blocks overexpression of normal cellular p21-induced cell growth by blocking JNK/jun and ERK activation in cardiac myocytes. NE plays a critical role in the development of hypertrophy in vivo [34]. Our data showing that a synthetic peptide designed to selectively block oncogenic ras can also counter the effects of NE-induced hypertrophy associated with overexpresison of ras p21 by blocking JNK/jun and ERK activation clearly warrants further studies. These studies have the potential of introducing novel therapeutic approaches to clinical management of the negative sequelae of cardiac remodeling.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
References
 
The work was supported by MERIT and REAP grants to Dr. El-Sherif from the Veterans Administration Central Research Department, by NIH RO1 Grant CA42500, and by a VA MERIT grant to Dr. Pincus.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Pincus MR. Development of new anti-cancer peptides from conformational energy analysis of the oncogenic ras-p21 protein and its complexes with target proteins. Frontiers in Bioscience 2004;9:3486–3509.[Medline]
  2. Pincus MR, Michl J, Bowne W, Zenilman M. Anti-cancer peptides from the ras-p21 and p53 proteins. In: Research Advances in Cancer (Mohan RM, Ed), Global Research Network Pub, Kerala, India, 2007; pp 65–90.
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