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(Hypertension. 2008;51:704.)
© 2008 American Heart Association, Inc.

Original Articles

Angiotensin II-Induced Extracellular Signal-Regulated Kinase 1/2 Activation Is Mediated by Protein Kinase C and Intracellular Calcium in Adult Rat Cardiac Fibroblasts

Erik R. Olson; Patricia E. Shamhart; Jennifer E. Naugle; J. Gary Meszaros

From the Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown; and the Graduate Program, School of Biomedical Sciences, Kent State University, Kent, Ohio.

Correspondence to J. Gary Meszaros, Northeastern Ohio Universities College of Medicine, Department of Physiology and Pharmacology, 4209 State Rt 44, Rootstown, OH 44272-0095. E-mail jgmeszar{at}neoucom.edu

	Abstract

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Angiotensin II (Ang II)-induced proliferation of cardiac fibroblasts is a major contributing factor to the pathogenesis of cardiac fibrosis. Ang II activates extracellular signal-regulated kinase (ERK) 1/2 to induce cardiac fibroblast proliferation, but the signaling pathways leading to ERK 1/2 activation have not been elucidated in these cells. The goal of the current study was to identify the intracellular mediators of Ang II-induced ERK 1/2 activation in adult rat cardiac fibroblasts. We determined that 100 nmol/L of Ang II-induced ERK 1/2 phosphorylation is inhibited by simultaneous chelation of cytosolic calcium and downregulation of protein kinase C (PKC) by phorbol ester or by the specific PKC inhibitor rottlerin, as well as PKC small interfering RNA, but not by inhibition of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate, phorbol ester, rottlerin, or PKC small interfering RNA alone. We also found that Ang II does not transactivate the epidermal growth factor receptor in adult cardiac fibroblasts, because pretreatment with 1 µmol/L of AG 1478 did not significantly inhibit [³H]-thymidine incorporation or ERK 1/2 activation. In addition, immunoprecipitation of the epidermal growth factor receptor demonstrated no significant Ang II-induced phosphorylation of tyrosine residues. Inhibition of phosphatidylinositide 3-kinase, PKC, and src tyrosine kinase had no effect on Ang II-induced ERK 1/2 activation. Collectively, these data demonstrate that Ang II does not transactivate the epidermal growth factor receptor in adult rat cardiac fibroblasts to activate ERK 1/2, a common pathway described in vascular smooth muscle and other cell types, but rather occurs via activation of distinct parallel signaling pathways mechanistically controlled by intracellular Ca²⁺ and PKC.

Key Words: cellular proliferation • mitogen-activated protein kinase • transactivation • heart failure • fibrosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cardiac remodeling is a dynamic process that is controlled by changes in both the cellular and extracellular compositions of the myocardium. The regulation of extracellular matrix remodeling is primarily mediated by cardiac fibroblasts (CFs), the major nonmuscle cell type of the heart.^1–3 CFs control matrix turnover in nonpathological states, and their activity is greatly enhanced after an acute cardiac event or during chronic cardiovascular disease states (ie, myocardial infarction or hypertension, respectively, see References ^4–7). Active CFs migrate to sites of damage, proliferate, and secrete large amounts of extracellular matrix proteins to repair the myocardium.⁸ Aberrant remodeling will cause cardiac fibrosis, a condition characterized by reduced contractility, which ultimately contributes to heart failure. Furthermore, the excess extracellular matrix disrupts the electrical circuitry of the myocardium, predisposing the heart to arrhythmias and potentially resulting in sudden cardiac death (reviewed in References ^9,10). Proliferation is a key early step in CF activation and a major contributing factor to the collective fibrotic potential of these cells.

A common occurrence during cardiovascular disease is the systemic elevation of angiotensin II (Ang II). Ang II has been shown to be a potent activator of CFs, and in cultured CFs, activation of the Ang II type 1 receptor stimulates both cellular proliferation and de novo collagen synthesis.^11–15 Our laboratory and others have demonstrated the mitogenic properties of Ang II on CFs, but the exact signaling mechanisms by which Ang II induces proliferation of CFs are not fully understood.^11,12,16 Ang II-induced proliferation of CFs has been shown to depend on activation of the extracellular signal-regulated kinase (ERK) 1/2 cascade^12,17; however, the signaling intermediates leading to ERK 1/2 activation are perhaps unique in the adult CFs.

A major effect of Ang II stimulation in CFs is the liberation of calcium from intracellular stores and the production of diacylglycerol, which induces protein kinase C (PKC) activation. However, calcium has additional cytosolic targets that have also been implicated in ERK 1/2 signaling. A key mechanism by which G protein-coupled receptors induce ERK 1/2 activation is by transactivation of the epidermal growth factor (EGF) receptor (EGFR). In vascular smooth muscle cells (VSMCs), Ang II type 1 receptor-induced ERK 1/2 activation occurs via Ca²⁺ and Src-dependent transactivation of the EGFR.^18–20 In neonatal CFs, phorbol ester-sensitive PKCs and intracellular Ca²⁺ have been shown to mediate mitogen-activated protein kinase signaling in response to Ang II.²¹ The EGFR also mediates β₂-adrenergic receptor-induced ERK 1/2 activation in adult CFs.^22,23 An alternative Ca²⁺-independent mechanism by which the Ang II type 1 receptor induces ERK 1/2 activation is through phosphatidylinositide 3-kinase (PI3K) and subsequent activation of atypical PKC.^24–26 In VSMCs and in MCF-7 cells, PKC is critical in mediating Ang II-induced ERK 1/2 activation and cellular proliferation, because direct inhibition of PKC resulted in a loss of Ang II-induced ERK 1/2 phosphorylation.^25,26 In addition, PKC inhibition reduced both basal and transforming growth factor-β-induced [³H]-thymidine incorporation in neonatal CFs.²⁷ Thus, the mechanism of ERK 1/2 activation by Ang II potentially involves multiple signaling mechanisms, including EGFR transactivation, PKCs, and intracellular Ca²⁺.

The goal of the current study was to identify the intracellular mediators of Ang II-induced proliferation and ERK 1/2 activation specifically in adult rat CFs. Our initial hypothesis was that Ang II activates ERK 1/2 and stimulates proliferation in a manner that is mechanistically dependent on PKC and either EGFR transactivation or an alternative mitogenic pathway involving PKC. However, contrary to our initial hypothesis, our findings demonstrate that concurrent inhibition of calcium signaling and PKC is required to prevent ERK 1/2 activation by Ang II. Surprisingly, transactivation of the EGFR by Ang II does not occur in adult CFs, a finding that differentiates Ang II and other G protein-coupled receptor signaling in adult CFs from other cells such as VSMCs and neonatal CFs. These data suggest, in contrast to several other cell types, that Ang II-induced ERK 1/2 activation in adult CFs proceeds through parallel Ca²⁺- and PKC-dependent pathways rather than through EGFR transactivation.

	Materials and Methods

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Materials
DMEM, penicillin/streptomycin, Fungizone, FBS, and Lipofectamine RNAiMax were all purchased from Invitrogen/GIBCO. [³H]-Thymidine was from ICN Biomedicals. Anti-phospho-tyrosine, anti-phospho-PKC, anti-PKC, and anti-EGFR antibodies were obtained from Cell Signaling Technology. Anti-phospho-ERK 1/2, anti-ERK 1/2, and anti-PKC antibodies were purchased from Santa Cruz Biotechnology. AG 1478 and anti-PKC antibodies were from Sigma-Aldrich. Fura-2/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA)/AM, LY 294002, PP2, rottlerin, hispidin, Gö6976, and the myristoylated PKC pseudosubstrate were from Calbiochem. PKC small interfering RNA (siRNA) was from Upstate/Millipore.

Isolation of Adult Rat Ventricular CFs
CFs were isolated as described previously^16,28,29 in accordance with the Institutional Animal Care and Use Committee guidelines. Briefly, left ventricles were excised from the hearts of anesthetized adult male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) and digested with collagenase type 2 (100 U/mL) and trypsin (0.6 mg/mL; Worthington Biochemical Corporation) for 90 minutes. The CFs were pelleted, resuspended, and plated in DMEM with 10% FBS. CFs were used only at early passages (2 or 3), where their morphology, expression markers, and growth properties remain consistent. The purity of first-passage cultures was >95% CFs as measured by vimentin and collagen (types I and III) expression, as described previously.^17,28,30 In all of the cases, CFs were placed in serum-free DMEM overnight and inhibitors added 30 minutes before agonist stimulation.

[³H]-Thymidine Incorporation Assay
Proliferation assays were performed as described previously.¹⁶ CFs were plated on 12-well tissue culture plates and grown to 40% to 50% confluence. Cells were treated in triplicate for a period of 48 hours, with [³H]-thymidine (2 µCi/mL) being added to the medium during the final 4 hours. The medium containing the label was removed, and the cells were washed twice with PBS and incubated in cold 5% trichloroacetic acid for 30 minutes. Cells were washed and solubilized in 1 mL of 0.5 mol/L of sodium hydroxide for 30 minutes at room temperature, and the amount of incorporated label was determined by liquid scintillation counting.

Pretreatment of CFs Using Pharmacological Inhibitors and PKC siRNA
CFs were pretreated with the specific PKC isoform inhibitors, 10 µmol/L of rottlerin for PKC, 10 µmol/L of hispidin for PKCβ, and 1 µmol/L of Gö6976 for PKC, in either the absence or presence of BAPTA for 30 minutes before 5 minutes of stimulation by 100 nmol/L of Ang II. CFs were incubated with Lipofectamine RNAiMax, 1.25 µg/mL of PKC siRNA, and serum/antibiotic free DMEM or DMEM alone for 24 hours, followed by pretreatment for 30 minutes with BAPTA and BAPTA with rottlerin before 5 minutes of stimulation by 100 nmol/L of Ang II. Equal protein amounts (10 µg) were loaded on SDS-PAGE gels and subjected to Western analysis as described below.

Protein Isolation and Western Analysis
Protein isolation and analysis were performed as described previously.^16,31 Briefly, whole-cell lysates were collected in lysis buffer (62.5 mmol/L of Tris-HCl, 2 mmol/L of EDTA, 2.3% SDS, and 10% glycerol [pH 6.8], with protease inhibitor mixture), and total protein content was determined by the bicinchoninic acid method (Pierce). Equal amounts of protein samples were separated by SDS-PAGE and transferred to nitrocellulose using standard techniques. Signals were detected by chemiluminescence, and densitometric data from Western blots were obtained and quantified using a Kodak 1D Digital Science Imaging System.

Immunoprecipitation of the EGFR
CFs were treated as indicated, washed with ice-cold PBS, and then scraped on ice in lysis buffer containing (in mmol/L): 20 Tris, 150 NaCl, 1 EDTA, 1% Triton X-100, 1 Na₃VO₄, and protease inhibitor mixture (pH 7.5). Cell lysates were sonicated 3 times for 5 seconds, centrifuged at 10 000g for 10 minutes at 4°C, and supernatants collected. Equal amounts of protein (500 µg) were immunoprecipitated with either the anti-EGFR or anti-phospho-tyrosine antibodies overnight at 4°C. Protein A-agarose beads were then added to the lysates and rotated for an additional 2 hours at 4°C. Antibody complexes were centrifuged and dissociated by incubating with 3x sample buffer (187.5 mmol/L of Tris-HCl, 6% SDS, 30% glycerol, 150 mmol/L of dithiothreitol, and 0.03% bromophenol blue [pH 6.8]) and boiling for 5 minutes. Equal protein amounts were loaded on SDS-PAGE gels and subjected to Western analysis as described above.

Measurement of Intracellular Calcium
CFs were loaded with 1 µmol/L of Fura-2/AM at 37°C for 30 minutes in HEPES buffered saline (in mmol/L: 130 NaCl, 5 KCl, 10 glucose, 1 MgCl₂, 1.0 CaCl₂, and 25 HEPES [pH 7.4]), and groups of 5 to 8 cells were monitored using an inverted Olympus IX-70 microscope. Spectrofluorometric measurements were collected using the Delta Scan System spectrofluorometer (Photon Technology), where the field was alternately excited at 380 and 340 nm and the emission ratio was collected at 511 nm.

Plasma Membrane Translocation of PKC
CFs were treated, washed with ice-cold PBS, and collected on ice in Buffer A (20 mmol/L of Tris, 250 mmol/L of sucrose, 1 mmol/L of EDTA, protease inhibitor mixture, and phosphatase inhibitor mixture). Cells were sonicated 3x5 seconds followed by centrifugation at 1000g for 10 minutes at 4°C. Cell lysates were then spun at 100 000g for 1 hour at 4°C, and the supernatants containing the cytosolic fraction were saved, whereas the pellets containing the membranes were resuspended in Buffer B (Buffer A containing 0.8% Triton-X 100) and kept on ice for 45 minutes. Cytosolic and membrane fractions were assessed by Western analysis as described previously.

Data Analysis/Statistics
Data analysis was performed using GraphPad Prism 4.0 statistical analysis software (GraphPad Software). Statistical significance between groups was determined by ANOVA with Tukey’s multiple comparison test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mechanism of Ang II-Induced ERK 1/2 Activation Is Controlled by Parallel Calcium and Phorbol Ester-Sensitive PKC Signaling Pathways
The release of calcium from intracellular stores contributes to PKC activation and potentially to ERK 1/2 phosphorylation. Incubating CFs with BAPTA/AM is an effective means in which to buffer intracellular Ca²⁺ and prevent downstream signaling (Figure 1A). Sequestering calcium with 30 µmol/L of BAPTA/AM inhibited intracellular Ca²⁺ elevation by Ang II but did not have a significant effect on Ang II-induced ERK 1/2 activation (Figure 1B). We attempted to use BAPTA/AM in longer-term proliferation assays in this study; however, overnight treatments of this chelator proved to be cytotoxic to the cells, hence a minor limitation of the study.

View larger version (17K): [in this window] [in a new window]   Figure 1. Chelation of intracellular Ca2+ does not block Ang II-induced ERK 1/2 activation. A, Cardiac fibroblasts were loaded with Fura-2/AM (1 µmol/L) and pretreated for 30 minutes with the calcium chelator BAPTA/AM. As anticipated, BAPTA/AM reduced the Ang II-induced intracellular Ca2+ elevation in a concentration-dependent manner with full blockade at 30 µmol/L. Triton X-100 was added after treatment with Ang II for normalization of the Ang II response. The fluorescence ratios are representative data of 3 similar experiments. B, Representative Western blots demonstrating that pretreatment for 30 minutes with BAPTA/AM had no effect on Ang II-induced ERK 1/2 phosphorylation after 5 minutes.

Short-term exposure to 12-myristate 13-acetate (PMA) stimulated translocation of phorbol ester-sensitive classic and novel PKC to the plasma membrane (Figure 2A) and induced rapid activation of ERK 1/2 (Figure 2B). When CFs are exposed to PMA for longer time periods, phorbol ester/diacylglycerol-sensitive PKCs are downregulated. Figure 2C demonstrates that CFs normally express PKC, but after treatment with 100 nmol/L of PMA for 18 hours, there is no apparent PKC expression. The downregulation of phorbol ester/diacylglycerol-sensitive PKCs by chronic PMA treatment did not have a significant effect on Ang II-induced activation of ERK 1/2 (Figure 2D). However, when PKC was downregulated and intracellular Ca²⁺ was chelated simultaneously, Ang II-induced ERK 1/2 phosphorylation was significantly inhibited (Figure 3), indicating that Ang II activates ERK 1/2 through parallel signaling of PMA-sensitive PKCs (phorbol ester-sensitive isoforms) and intracellular Ca²⁺.

View larger version (45K): [in this window] [in a new window]   Figure 2. Downregulation of PKC with PMA does not block Ang II-induced ERK 1/2 activation. A, Representative Western blot demonstrating PMA-induced activation of a classic isozyme PKC. Cytosolic and membrane fractions were separated as described in Materials and Methods and subjected to SDS-PAGE. Cytosolic PKC was reduced after treatment with 100 nmol/L of PMA for 15 minutes, whereas membrane-associated PKC was increased over basal. B, Representative Western demonstrating the rapid activation of ERK 1/2 in CFs in response to PMA (5 minutes). C, Representative Western blot displaying the effects of 18-hour treatment with PMA (100 nmol/L) on PKC expression. D, Representative Western blot demonstrating that downregulation of PKC with PMA treatment (18 hours) had no effect on Ang II-induced ERK 1/2 phosphorylation (5 minutes).

View larger version (29K): [in this window] [in a new window]   Figure 3. Inhibition of Ang II-induced ERK 1/2 activation by concurrent blockade of Ca2+ and PKC. A, Representative Western blot demonstrating that downregulation of PKC with 18 hours of 100 nmol/L of PMA and concomitant chelation of intracellular Ca2+ with 30 µmol/L of BAPTA/AM reduces Ang II-induced ERK 1/2 phosphorylation. B, Summary graph of all of the experiments measuring Ang II-induced ERK 1/2 activation in the presence of BAPTA/AM and/or PMA. All of the blots are representative of 3 separate experiments in cells cultured from 3 separate animals. Data are expressed as means±SEMs. *P<0.05, statistically significant vs basal; P<0.05, statistically significant vs Ang II. Significant differences between conditions were determined by 1-way ANOVA and Tukey’s multiple comparison test.

Pharmacological and siRNA-Mediated Inhibition of PKC Attenuate Ang II-Induced ERK 1/2 Phosphorylation
Because Ang II activates ERK 1/2 through both intracellular Ca²⁺ and phorbol ester-sensitive PKC pathways in isolated adult CFs, our next goal was to determine the specific PKC isoform involved in this pathway. Pretreatment of CFs with rottlerin, a PKC inhibitor, did not inhibit Ang II-mediated ERK 1/2 activation, but the combination of rottlerin and BAPTA blocked this activation (Figure 4A). In addition, Ang II-induced ERK 1/2 activation was not attenuated in cells pretreated with BAPTA and either Gö6976 (PKC) or hispidin (PKCβ), indicating that Ang II signals through PKC and not through PKC or PKCβ. PKC silencing by siRNA partially attenuated the Ang II-induced ERK 1/2 phosphorylation, whereas concurrent treatment with the siRNA and BAPTA led to a complete inhibition (Figure 4B). PKC siRNA treatment reduced PKC protein levels and had no effect on PKC protein levels (Figure 4C). Thus, pharmacological and molecular evidence demonstrate that Ang II uses PKC as a key signaling intermediate to activate ERK 1/2 in adult CFs.

View larger version (60K): [in this window] [in a new window]   Figure 4. Inhibition of PKC and intracellular Ca2+ attenuates ERK1/2 activation. A, Cardiac fibroblasts were pretreated for 30 minutes with the following isoform-specific PKC inhibitors: 10 µmol/L of rottlerin for PKC; 10 µmol/L of hispidin for PKCβ; 1 µmol/L of Gö6976 for PKC before 5 minutes of stimulation by 100 nmol/L of Ang II. Cell lysates were collected and subjected to SDS-PAGE and Western blot analysis as described above. The representative image was taken from 1 of 3 Western blot experiments performed in cells isolated from 3 separate animals and demonstrates that pharmacological inhibition of PKC, but not PKC or PKCβ, attenuates Ang II-induced ERK 1/2 activation. B, CFs were incubated with PKC siRNA for 24 hours and then pretreated for 30 minutes with BAPTA alone or BAPTA with rottlerin before 5 minutes of stimulation by 100 nmol/L of Ang II. Cell lysates were collected and subjected to SDS-PAGE and Western blot analysis as described above. The representative Western blot validates the pharmacological data displaying that Ang II activates ERK 1/2 via PKC. C, Western blot confirmation of PKC siRNA efficiency demonstrating that PKC protein levels decreased and PKC protein levels were unaffected on PKC siRNA treatment.

Ang II Induces CF Proliferation and ERK 1/2 Activation in an EGFR-Independent Manner
EGFR transactivation is a common mechanism for ERK activation and proliferation by Ang II and a number of other G protein-coupled receptor ligands in several cell types. Stimulation of CFs with Ang II resulted in a 44.6±2.6% increase in [³H]-thymidine incorporation over basal (Figure 5A). In CFs pretreated with AG 1478 (EGFR kinase inhibitor) 30 minutes before Ang II stimulation, the measured increase in [³H]-thymidine incorporation was 30.2±6.2% over basal and was not significantly different from Ang II alone. However, EGF-induced proliferation (75.3±19.6% over basal) was completely inhibited by pretreatment with AG 1478, as expected.

View larger version (39K): [in this window] [in a new window]   Figure 5. Ang II-induced cardiac fibroblast proliferation and ERK 1/2 activation are EGFR independent. A, [3H]-Thymidine incorporation was used to measure Ang II- (100 nmol/L) or 10 nmol/L of EGF-induced proliferation of serum-starved CFs over 48 hours. Each independent experiment was performed in triplicate. Data were pooled and expressed as the percentage of basal ±SEM. *P<0.05, statistically significant vs basal; P<0.05, statistically significant vs EGF. B, Pretreatment of CFs with 1 µmol/L of AG 1478 for 30 minutes was effective in blocking the activation of ERK 1/2 induced by stimulation with 10 nmol/L of EGF for 5 minutes. However, AG pretreatment had no significant effect on 100 nmol/L of Ang II-induced ERK 1/2 activation. Western blot is representative of 3 separate experiments (n=3). Data were pooled and expressed as percentage of the Ang II response ±SEM. *P<0.05, statistically significant vs basal; P<0.05, statistically significant vs EGF. C, Stimulation with 10 nmol/L of EGF for 5 minutes induced EGFR phosphorylation as determined by immunoprecipitiation. There was no significant tyrosine phosphorylation induced by 100 nmol/L of Ang II after 5 minutes. D through F, Pretreatment of cultured cardiac fibroblasts with the PP2 (src inhibitor), LY294002 (PI3K inhibitor), or -PS (PKC inhibitor) did not alter ERK 1/2 activation by Ang II.

Because ERK 1/2 is a common mediator of both Ang II- and EGF-induced CF proliferation, we determined whether ERK 1/2 phosphorylation was affected by AG 1478 pretreatment. Figure 5B demonstrates that AG 1478 blocked the EGF-induced increase in phosphorylated ERK 1/2 (107.2% reduction) but had no effect on phosphorylation induced by Ang II (21.2% reduction, not significant versus Ang II alone). This finding suggests that Ang II activates ERK 1/2 through a pathway that does not involve transactivation of the EGFR. To confirm this hypothesis, immunoprecipitation of phospho-tyrosine-containing proteins in cell lysates was performed followed by Western blotting for the EGFR. We determined that EGFR phosphorylation was not induced by Ang II, because densitometric analysis indicated that the levels of tyrosine phosphorylation were not significantly different from basal (Figure 5C). Src tyrosine kinase is a common intermediate of Ang II-induced mitogenesis and ERK 1/2 activation through transactivation of the EGFR.¹⁸ PI3K has been identified as a potential upstream mediator of the Ang II pathway as well in various cell types. Inhibition of c-src signaling with PP2 or PI3K inhibition by LY29004 had no effect on Ang II-induced ERK 1/2 activation (Figure 5D and 5E). Lastly, because PKC has been shown previously to be activated by Ang II in vascular smooth muscle and MCF-7 cells,^25,26 we treated adult CFs with the myristoylated inhibitory PKC pseudosubstrate (Figure 5F) and found that Ang II-induced ERK 1/2 activation was unaffected by this inhibitor, contrary to the signaling in these other cells. The multiple signaling pathways examined in this study and the proposed mechanism of Ang II-induced ERK 1/2 activation in adult CFs are summarized in Figure 6.

View larger version (17K): [in this window] [in a new window]   Figure 6. Model for Ang II-induced ERK 1/2 activation in adult rat cardiac fibroblasts. Ang II induces ERK 1/2 activation through independent parallel pathways originating at Ca2+ and PKC. Inhibition of one pathway does not block Ang II-induced ERK 1/2 phosphorylation, whereas simultaneous chelation of intracellular Ca2+ and downregulation of phorbol ester-sensitive PKCs and, more specifically, PKC suppresses Ang II-induced ERK 1/2 signaling. The EGFR, c-Src, PI3K, and the atypical PKC do not have roles in Ang II-induced ERK 1/2 activation in adult rat cardiac fibroblasts.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is well established that Ang II-induced proliferation of CFs depends on ERK 1/2 phosphorylation, because it has been shown that selective inhibition of this pathway will attenuate this process.¹⁷ However, several of the intermediates between the Ang II type 1 receptor and ERK 1/2 in CFs remain to be elucidated. The current study, therefore, was designed to identify the signaling mediators of Ang II-induced proliferation and ERK 1/2 activation specifically in adult rat CFs.

Intracellular calcium and PKC are common intermediates between ERK 1/2 and Gq-coupled receptors. Classic and novel PKCs are activated by diacylglycerol and phorbol esters and can directly phosphorylate Raf-1 to stimulate mitogen-activated protein kinase activation.³² Ca²⁺ has several intracellular targets, including multiple cytosolic tyrosine kinases, like the Src family and the focal adhesion kinase/PYK2 family, which have been implicated in ERK 1/2 activation.³³ We report here that concurrent cytosolic Ca²⁺ chelation and PKC downregulation were necessary to fully attenuate ERK 1/2 activation in our cells. This result is similar to one report that focused on Ang II and platelet-derived growth factor effects on mitogen-activated protein kinase signaling in neonatal CFs²¹; we have extended this to include the identification of the PKC isoform as a specific mediator of ERK 1/2 signaling in adult CFs. Our current findings indicate 2 separate and parallel pathways involved in ERK 1/2 activation; one through PKC (and/or phorbol ester-sensitive PKCs) and another that is mediated by intracellular Ca²⁺.

In addition to signaling through PKC, ERK 1/2 phosphorylation resulting from G protein-coupled receptor stimulation has been attributed to multiple alternative intracellular signaling pathways. Ang II has been shown to induce ERK 1/2 activation by both Ca²⁺/c-Src-dependent and -independent EGFR transactivation.^18,34,35 Our findings indicate that, in adult CFs, neither the EGFR nor c-Src is involved in signal transduction between the Ang II receptor and ERK 1/2, which distinguishes adult CFs from both neonatal CFs and VSMCs, which use EGFR transactivation.

Another important mediator of Ang II-induced mitogenesis is PI3K, which has been found to mediate Ang II-induced growth and proliferation by activation of ERK 1/2³⁶ or through ERK 1/2-independent pathways.^37,38 PI3K-induced ERK 1/2 activation and proliferation are commonly found in cell types where Ang II induces EGFR transactivation.^18,38 Through pharmacological inhibition of PI3K with LY 294002, we demonstrate that ERK 1/2 activation by Ang II is not dependent on this pathway in adult CFs. An alternative pathway from PI3K to ERK 1/2 involves activation of protein kinase C, which is also a proposed mediator of Ang II-induced signaling. ERK 1/2 activation and cellular proliferation caused by agonist stimulation depend on PKC in VSMCs, MCF-7 cells, and neonatal rat CFs.^25–27 In mouse embryonic fibroblasts expressing the Ang II type 1 receptor, Ang II-induced ERK 1/2 activation and cell proliferation have been shown to be mediated by PKC and c-src.³⁹ Collectively, our data suggest that PI3K and PKC are not signaling intermediates of Ang II-induced mitogenesis in adult CFs, which points out another major difference in the signaling pathways used in adult CFs versus those in neonatal CFs, VSMCs, and other cell types. Moreover, this result is consistent with our previous finding that Ang II did not stimulate Akt phosphorylation, because both Akt activation and PKC translocation require PI3K.¹⁶

Our findings demonstrate that Ang II-induced ERK 1/2 activation in adult CFs occurs through parallel Ca²⁺-dependent and PKC-dependent mechanisms, neither of which require the activity of the EGFR or c-Src. Selective inhibition of PI3K and PKC also had no effect on ERK 1/2 activation. These findings indicate that there are significant differences between the signaling pathways mediating Ang II-induced ERK 1/2 activation in adult CFs as compared with VSMCs and neonatal CFs. In conclusion, both calcium and PKC independently mediate the activation of ERK 1/2 by Ang II through parallel signaling pathways to control proliferation of adult CFs.

Perspectives
Overall, this study highlights the complexity of signaling involved in Ang II-induced ERK 1/2 activation and describes significant differences between adult CFs and other Ang II-responsive cell types. Differentiation of cell type-specific signaling pathways that cause Ang II-induced proliferation, hyperplasia, and hypertrophy of cardiovascular cells will uncover new molecular targets for the treatment of hypertension and heart failure. Our study indicates that CFs and vascular smooth muscle cells, in particular, use very distinct intracellular signaling pathways to induce Ang II-dependent growth and proliferation. EGFR transactivation in smooth muscle cells is a major mediator of Ang II signaling, and the lack of this signaling mechanism in fibroblasts, along with the dependence on intracellular calcium and PKC, suggests that pharmacological interventions targeting these distinct cardiovascular cells can be developed to prevent the aberrant growth and remodeling that occur during hypertension and other cardiovascular diseases.

	Acknowledgments

 
We appreciate the helpful critical review of this article by Dr Hans G. Folkesson, Department of Integrative Medical Sciences, Northeastern Ohio Universities College of Medicine.

Sources of Funding

This work was supported by an American Heart Association Ohio Valley Affiliate Beginning Grant-in-Aid (0160162B) and an Ohio Board of Regents Research Challenge Award (34177).

Disclosures

None.

	Footnotes

 
The first 2 authors contributed equally to this article.

Received July 23, 2007; first decision August 17, 2007; accepted December 18, 2007.

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