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q interaction and protein kinase C-/CaMKII activation in H9c2 cardiomyoblast cells
Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan, ROC1 Division of Medical Technology, Department of Internal Medicine, Armed-Force, Taichung General Hospital, Taichung 402, Taiwan, ROC2 Laboratory of Exercise Biochemistry, TPEC, Taipei 105, Taiwan, ROC3 Emergency Department, China Medical University Hospital, Taichung 413, Taiwan, ROC4 Department of Pediatrics, Medical Research and Medical Genetics, China Medical University, Taichung 413, Taiwan, ROC5 Department of Healthcare Administration, Asia University, Taichung 413, Taiwan, ROC6 Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan, ROC7 Graduate Institute of Chinese Medical Science, China Medical University, Taichung 404, Taiwan, ROC8 Graduate Institute of Basic Medical Science, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan, ROC9 Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan, ROC
(Correspondence should be addressed to C-Y Huang; Email: cyhuang{at}mail.cmu.edu.tw)
* (W-W Kuo and C-Y Huang contributed equally to this work) 
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Abstract |
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and calcium/calmodulin-dependent protein kinase II (CaMKII) in a Gq-dependent manner. By the inhibition of PKC-/CaMKII activity, we found that IGF-II and Leu27IGF-II-induced cell hypertrophy and upregulation of ANP and BNP were significantly suppressed. Taken together, this study provides a new insight into the effects of the IGF2R and its downstream signaling in cardiac hypertrophy. The suppression of IGF2R signaling pathways may be a good strategy to prevent the progression of pathological hypertrophy. |
Introduction |
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have been reported to be involved in the development of cardiac hypertrophy (Frey et al. 2004, Heineke & Molkentin 2006). After selectively binding to receptors on the membrane, these hormones trigger intracellular signaling cascades into the cell nuclei that cause an increase in the expression of the hypertrophic response genes. IGF-I and IGF-II are members of the IGF family of peptide growth factors. They can bind to two types of cell surface receptors, IGF1R and IGF2R, and have been shown to play a critical role in the development and growth of cells (Jones & Clemmons 1995). IGF1R acts as a receptor tyrosine kinase to trigger a series of mitotic signaling cascades after binding with IGFs (McMullen et al. 2004). In contrast, the IGF2R, also known as the ‘clearance receptor’, stabilizes local IGF concentration through internalization and lysosomal degradation (Boker et al. 1997). It has been shown that the binding of IGF-I and IGF-II to the IGF1R induces cell hypertrophy in neonatal ventricular cardiomyocytes (Adachi et al. 1994, Miyashita et al. 2001). Several investigations further observed that the activation of IGF1R signaling cascade can improve heart contractions and attenuate pathological hypertrophy and fibrosis. However, the increased expression of IGF-II in several animal models with pathological cardiac hypertrophy raised doubts about the role of IGF-II in mediating stressful responses in the heart (Kluge et al. 1995, Lee et al. 2006).
A few studies have indicated that after binding with IGF-II, IGF2R not only functions in the degradation of IGF-II, but also triggers an intracellular signaling pathway that contributes to the regulation of a variety of physiological functions, such as calcium influx, acetylcholine (ACh) release, and cell migration (Nishimoto et al. 1987, McKinnon et al. 2001, Hawkes et al. 2006). Moreover, the existence of a putative G-protein-binding site within the cytoplasmic domain of the IGF2R suggests that IGF-II may regulate small G proteins that activate signaling pathways through IGF2R (Nishimoto et al. 1989, Murayama et al. 1990, Okamoto & Nishimoto 1991, Ikezu et al. 1995). Based on these findings, we propose that the binding of IGF-II to IGF2R may trigger an intracellular signaling cascade response to cardiac hypertrophy and that the role of this signaling is completely different from that of IGF1R-derived physiological hypertrophy.
In this study, we investigated whether the IGF2R signaling pathway may induce myocardial hypertrophy pathologically by the activation of small G protein and its downstream signaling pathway. We found that the IGF2R was aberrantly expressed in the myocardial infarction tissue. We further found that treatment with IGF-II induced cell hypertrophy in a time-dependent manner and an increase in the protein level of pathological hypertrophy markers ANP and BNP in the H9c2 cardiomyoblast cell. We also found that using Leu27IGF-II, an analog of IGF-II that interacts selectively with the IGF2R (Beukers et al. 1991), to specifically activate IGF2R signaling cascades led to an increase in the phosphorylation of protein kinase C (PKC)- and calcium/calmodulin-dependent protein kinase II (CaMKII) in a Gq-dependent manner. By inhibiting the activity of PKC-/CaMKII, the IGF-II-induced cell hypertrophy and upregulation of protein expression of ANP and BNP significantly decreased. Our findings indicated that IGF2R may function as a G-protein-coupled receptor to induce the downstream modulation of PKC- and CaMKII via the Gq signaling pathway, thereby contributing to the progression of pathological hypertrophy. The suppression of the IGF2R signal may help to prevent the transition from physiological hypertrophy to pathological hypertrophy.
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Materials and Methods |
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The human cardiovascular tissue array (Provitro, Berlin, Germany) was immunostained with an anti-IGF2R antibody (SantaCruz Biotechnology, SantaCruz, CA, USA) using the Ultra Vision LP Detection System (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. The tissue array section was dried at 58 °C overnight. Then, this section was deparaffinized in xylene and sequentially hydrated using a graded series of ethanol. The endogenous peroxidase activity was blocked with hydrogen peroxide blocking buffer for 13 min. After rinsing in water for 15 min, the microarray slide was microwave-treated in citrate buffer for 15 min, cooled down to room temperature (RT) for 30 min, and blocked with an UV blocking buffer for 5 min. The primary antibody directed against the peptides 1030–1209 of the rat IGF2R (1:100) was incubated for 30 min. The slide was incubated with the primary antibody enhancing buffer at RT for 20 min. HRP Polymer was added and incubated at RT for 20 min. The IGF2R antibody was located by a universal secondary antibody formulation conjugated to an enzyme-labeled HRP Polymer. After staining with an appropriate substrate/chromogen for 5 min, the slide was counterstained with Harris hematoxylin, dehydrated through a graded series of ethanol to xylene washes, and cover slipped with a permanent mounting medium (Sigma Chemical). The polymer complex was then detected by microscopy (magnification 200x).
Cell culture
H9c2 cardiomyoblast cells were obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM pyruvate in humidified air (5% CO2) at 37 °C. H9c2 cells were cultured in serum-free medium for 12 h and then treated with or without IGF-I (10–8 M; Sigma Chemical), IGF-II (10–8 M; Sigma Chemical), and Leu27IGF-II (10–8 M; GroPep, Adelaide, Australia). After further incubation for 12 or 24 h, the cells were harvested and extracted for analysis.
Measurement of cell surface area
To measure the cell surface area, cardiomyocytes were stained with rhodamine phalloidin (1:50 dilution) for 20 min to visualize F-actin. Cell images from at least ten randomly chosen fields (x40 objective) of 60 cardiomyocytes were measured in three separate experiments using NIH image software. Only myocytes that were completely in the field were measured. The effect of IGF-II-mediated increase in cell size was determined.
Protein extraction and western blot analysis
Cultured H9c2 cells were scraped and washed once with PBS. The cell suspension was then spun down, and the cell pellets were lysed for 30 min in lysis buffer (50 mM Tris (pH 7.5), 0.5 M NaCl, 1.0 mM EDTA (pH 7.5), 10% glycerol, 1 mM basal medium Eagle, 1% Igepal-630, and proteinase inhibitor cocktail tablet (Roche)) and spun down at 12 000 g for 10 min. Then, the supernatants were removed and placed into new Eppendorf tubes for western blot analysis. Proteins from the H9c2 cell line were separated on 12% gradient SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific protein binding was blocked in blocking buffer at RT for 1 h (5% milk, 20 mM Tris–HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20). The membranes were blotted with specific antibodies as indicated for each experiment in the blocking buffer at RT for 1 h. Chemiluminescent detection was accomplished with Western Blotting Luminol Reagent (SantaCruz Biotechnology). The densitometric analysis of immunoblots was performed using the AlphaImager 2200 Digital Imaging System (Digital Imaging System, CA, USA).
Antibodies and reagents
The following antibodies were used in this study: anti-ANP (SantaCruz Biotechnology), anti-BNP (SantaCruz Biotechnology), anti-ERK1/2 (SantaCruz Biotechnology), anti-phospho-ERK1/2 (SantaCruz Biotechnology), anti-phospho-PKC- (Cell Signaling, MA, USA), anti-PKC- (Cell Signaling), anti-phospho-CaMKII (Cell Signaling), anti-CaMKII (Cell Signaling), anti-Gq (SantaCruz Biotechnology), and anti--tubulin (SantaCruz Biotechnology). All of the secondary antibodies (anti-rabbit-HRP, anti-mouse-HRP, and anti-goat-HRP) and the enhanced chemiluminescence kit were purchased from Santa Cruz Biotechnology. IGF-I and IGF-II were purchased from Sigma, and Leu27IGF-II was obtained from GroPep. The calcineurin inhibitor FK506 and cyclosporin A were purchased from Calbiochem (CA, USA) and Sigma respectively. PKC-/CaMKII inhibitor cocktail was obtained from Upstate. SB203580 (p38 MAP kinase inhibitor; Promega), U0126 (MEK1 and MEK2 inhibitor) and SP600125 (JNK inhibitor) were purchased from Promega.
siRNA and transfection
Double-stranded small interfering RNA (siRNA) sequences targeting guanine nucleotide-binding protein, q polypeptide (Gq; GenBank accession number NM_031036), mRNAs were obtained from Dharmacon (Lafayette, CO, USA). A nonspecific duplex (5'-CAGUGGAGAUCAACGUGCAAGUU-3'; Dharmacon), which did not significantly affect Gq mRNA and protein levels relative to the untransfected controls, was used as a control. The concentration of siRNA and the time of incubation were tested. H9c2 cardiomyoblast cells were plated in 100 mm well plates in DMEM without fetal bovine serum and transfected with double-stranded siRNA using the DharmaFECT Duo Transfection Reagent (Dharmacon) according to the manufacturer's instructions. To assess gene silencing, Gq protein levels were detected by immunoblotting.
Statistical analysis
The relative intensities of protein were analyzed using the Digital Sciences 1D program from Kodak Scientific Imaging Systems. All of the results were expressed as means±S.D., or as the means and the coefficient of variation of three to five separate experiments, as indicated. The transfection experiments were performed in triplicate. Standard curves were run, and the data that were obtained fell within the linear range of the curve. In addition, all values were normalized to their respective lane loading controls. The densitometric analysis of immunoblots in bar figures (Figs 3f, 4b, d, 5
b and d) was performed using one-way ANOVA with preplanned contrast comparisons against the control group (serum-free) or against the Leu27IGF-II group. Results in Figs 2b, d, 3b and d were analyzed by unpaired Student's t-test. In all cases, P<0.05 was considered significant.
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Results |
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To examine the correlation between the expression levels of IGF2R protein and myocardial infarction, we performed immunohistochemical analysis of human cardiovascular tissue array containing 10 normal heart and 28 infarcted myocardium tissues. Representative images demonstrating positive or negative infarcted myocardium staining compared with normal human heart tissue are shown in Fig. 1. A total of 25 (65.7%) showed positive staining for IGF2R. Thus, 13 (34.3%) could be categorized as absent or minimal expression for IGF2R. Out of the infarcted myocardium samples, 18 (64%) showed a strong expression of IGF2R and 7 (25%) showed moderate expression. The remainder appeared to have no more staining than the normal heart tissue. None of the ten normal tissue samples on the slide showed any IGF2R overexpression. Only 3 out of the 28 infarcted myocardium samples showed staining beyond background levels and none had strong staining. We found a significant association between IGF2R overexpression (scored as moderate/strong) and myocardial infarction. Overall, then, 25 out of the 28 infarcted myocardium samples (89%) examined by immunohistochemistry showed a significant overexpression of IGF2R.
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We investigated whether the treatment with IGF-II would directly induce cell hypertrophy and reactivate the expression of hypertrophy markers ANP and BNP in H9c2 cardiomyoblast cells, and compared its effect with IGF-I. The rhodamine phalloidin stain revealed, when compared with untreated controls, that there was 1.8- and 2.3-fold increase in cell surface size in the cells treated with IGF-I and IGF-II respectively (Fig. 2a and b) for 24 h. However, the treatment with IGF-II, but not with IGF-I, for 12 h significantly elevated the cell surface size (Fig. 2a and b) suggested that IGF-I and IGF-II induced cell hypertrophy probably by triggering a distinct signal transduction. Furthermore, western blot revealed that there was a sixfold increase in the protein levels of ANP and BNP in cells treated with IGF-II (Fig. 2c and d) but not in cells treated with IGF-I. Although IGF-I and IGF-II both induced cell hypertrophy (Fig. 2a and b), the increased ANP and BNP levels were detected only in the cells treated with IGF-II, indicating that IGF2R plays a crucial role in the induction of ANP and BNP by activating the intracellular signaling pathway involved in pathological hypertrophy (Dietz et al. 1989, Tissandier et al. 1995).
Leu27IGF-II activation of the PKC-/CaMKII signaling through Gq
Using Leu27IGF-II to exclude other effects derived from insulin and IGF1Rs in H9c2 cardiomyoblasts (Beukers et al. 1991), we attempted to clarify whether the IGF-II-induced small G-protein-sensitive signaling pathway is mediated by IGF2R. Western blots revealed that treatment with IGF-I and IGF-II, but not with Leu27IGF-II, increased the level of ERKI/2 phosphorylation at 30 min, (Fig. 3a and b), suggesting that Leu27IGF-II did not activate the IGF1R downstream effectors. We also observed that treatment with IGF-II and Leu27IGF-II both resulted in a significant increase in the phosphorylation of PKC- and CaMKII (Fig. 3c and d), suggesting that the effects might occur through IGF2R. When compared with IGF-II, Leu27IGF-II had stronger and faster effects on the phosphorylation of PKC- and CaMKII (Fig. 3c and d), which might be due to the fact that Leu27IGF-II has a higher affinity to bind with IGF2R than with IGF-II. We further investigated whether Gq might be involved in the Leu27IGF-II-induced phosphorylation of PKC- and CaMKII, used guanine nucleotide-binding protein, alpha q polypeptide (Gq), siRNA to disrupt the expression of Gq protein in H9c2 cardiomyoblast cells. As shown in Fig. 3e and f, we found a significantly greater reduction in the phosphorylation of PKC- and CaMKII in cells transfected with Gq siRNA than in cells treated with Leu27IGF-II alone, implying that the siRNA-mediated knockdown of Gq inhibits Leu27IGF-II-induced PKC- and CaMKII activation. Taken together, these findings indicate that, in response to the ligand, IGF2R may act as a GPCR to modulate Gq and activate its downstream effectors.
Inhibition of PKC-/CaMKII signaling blocks IGF-II and Leu27IGF-II-induced cell hypertrophy and upregulating ANP/BNP
In order to find out whether the activation of PKC- and CaMKII is required for IGF-II and Leu27IGF-II to induce cell hypertrophy and upregulate ANP and BNP, we used a variety of protein kinase inhibitors to suppress intracellular signaling cascades in H9c2 cardiomyoblast cells exposed to IGF-II or Leu27IGF-II. We found that the inhibition of either PKC-/CaMKII or ERK1/2 activities significantly prevented IGF-II-induced cell hypertrophy (Fig. 4a and b). The results suggest that IGF-II induced cell hypertrophy via two distinct pathways. However, the Leu27IGF-II-induced cell hypertrophy was rescued by inhibiting the PKC-/CaMKII activity (Fig. 5a and b), indicating that the binding of Leu27IGF-II to IGF2R induced cell hypertrophy by activating the PKC-/CaMKII signaling pathway, but not the ERK1/2 pathway. We further found that once PKC-/CaMKII activity was inhibited, both IGF-II and Leu27IGF-II induction of ANP and BNP protein expression could be reduced (Figs 4c and d, 5c and d). Taken together, these findings demonstrate that IGF2R signaling induced cell hypertrophy and upregulated ANP and BNP by activating PKC-/CaMKII, whereas IGF1R signaling only induced cell hypertrophy, but did not regulate ANP and BNP, via ERK1/2 activation.
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Discussion |
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q and the activation of its downstream effectors PKC-/CaMKII, the IGF2R signaling pathway may contribute to the progression of pathological hypertrophy. A depiction of this process is shown in Fig. 6.
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The physiological functions of IGFs mediated by three plasma membrane receptors (Jones & Clemmons 1995), including the IGF-I, IGF-II, and insulin receptors, have made it difficult to identify the specific role that IGF2R plays in the mediation of a given biological response. We used the Leu27IGF-II analog, which interacts selectively with the IGF2R (Beukers et al. 1991), to overcome the cross talk among these receptors, providing us an opportunity to reexamine the role of IGF2R in cell signaling. We found that treatment with either IGF-II or Leu27IGF-II led to an increase in the phosphorylation of PKC- and CaMKII (Fig. 3). Studies have found that the cross-talk between IGF2R signals and the small G-protein Gi regulates cell behavior by activating specific intracellular signaling cascades in a pertussis toxin-sensitive manner (Nishimoto et al. 1987, McKinnon et al. 2001, Hawkes et al. 2006). Our data indicated that Leu27IGF-II-induced the increment of phospho-PKC- and phospho-CaMKII is Gq required (Fig. 3). It would be interesting to further investigate how IGF2R cooperates with Gq to activate its downstream signaling cascades.
In this study, we demonstrated that there are two signaling pathways, ERK1/2 and PKC-/CaMKII, involved in IGF-II-induced cell hypertrophy, and that only PKC-/CaMKII activation is needed for IGF-II-increased expression of ANP and BNP (Fig. 4a–d). Our findings showed that Leu27IGF-II enhanced the phosphorylation of PKC- and CaMKII, but did not have the same effect on ERK1/2 phosphorylation (Fig. 3), suggesting that binding of IGF-II to IGF2R could induce cell hypertrophy and induction of ANP and BNP. Moreover, specifically activated IGF2R signaling by Leu27IGF-II triggered the PKC-/CaMKII signaling, in order to induce cell hypertrophy, and upregulated ANP and BNP (Fig. 5), which confirm the results in Figs 3 and 4 respectively. For further research, it would be interesting to investigate whether IGF2R, in addition to serving as ‘clearance receptor’ for IGF-II, is involved in the regulation of various physiological functions including cell metabolism, development and growth, all of which are modulated by IGF-II.
Numerous studies have implicated that the activation of PKC- and CaMKII signaling in response to calcium influx (Molkentin 2006, Ferrero et al. 2007) might play a critical role in the Gq-induced pathological cardiac hypertrophy, cardiac contractile failure, and apoptosis of cardiomyocytes (D'Angelo et al. 1997, Adams et al. 1998, Mende et al. 1998, Wettschureck et al. 2001, Braz et al. 2004). It is possible that by activating PKC- and CaMKII, IGF2R signaling may be involved in the regulation of pathological cardiac remodeling and the progression from adaptive cardiac hypertrophy to cardiac failure. The study of BALB/c 3T3 cells shows that IGF-II stimulates the calcium influx involved with GTP-binding protein (Nishimoto et al. 1987). Based on our results, we propose that after IGF-II-binding, the IGF2R changes conformation, allowing Gq to bind and activate phospholipase C (PLC)-β in the cardiomyocytes (Rockman et al. 2002). This results in a change in the influx of intracellular calcium via inositol trisphosphate and contributes to the activation of calcium-dependent protein kinase PKC- and CaMKII (Rockman et al. 2002).
In conclusion, the results of this study suggest that an overactivation of the IGF2R pathway might cause pathological hypertrophy by modulating Gq and the activity of its downstream effectors PKC-/CaMKII to induce ANP and BNP expression. These new insights might be used to prevent the transition from physiological hypertrophy to the pathological hypertrophy in the heart.
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Acknowledgements |
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References |
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Received in final form 1 February 2008
Accepted 14 February 2008
Made available online as an Accepted Preprint 14 February 2008
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2.1- and 3-fold in cell surface size respectively, when compared with untreated control. The cell surface area of at least 50 myocytes from 10 randomly selected fields in three separate experiments (the number of replications (n value) is three to five for each groups) was measured by a computed image analyzer; data are presented as a percentage compared with untreated control (mean±S.E.M). Bars indicate averages. Statistical significance: *P<0.05; #P<0.01, IGF-I or IGF-II versus untreated controls. (c and d) Western blot of protein lysates from H9c2 cardiomyoblasts treated with IGF-I (10–8 M) and IGF-II (10–8 M) at 12 and 24 h using anti-ANP antibody and anti-BNP antibody respectively as the cardiac hypertrophy marker. Protein levels of ANP and BNP increased in the presence of IGF-II, but not in cells treated with IGF-I. The blots were measured by densitometry. Data are presented as means±S.E.M. Bars indicate averages, *P<0.05. n=three independent experiments for each data point.
