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Originally published In Press as doi:10.1074/jbc.M611780200 on May 28, 2007

J. Biol. Chem., Vol. 282, Issue 32, 23117-23128, August 10, 2007
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G{alpha}12/13-mediated Up-regulation of TRPC6 Negatively Regulates Endothelin-1-induced Cardiac Myofibroblast Formation and Collagen Synthesis through Nuclear Factor of Activated T Cells Activation*Formula

Motohiro Nishida{ddagger}, Naoya Onohara{ddagger}, Yoji Sato§, Reiko Suda{ddagger}, Mariko Ogushi{ddagger}, Shihori Tanabe§, Ryuji Inoue, Yasuo Mori||, and Hitoshi Kurose{ddagger}1

From the {ddagger}Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, §National Institute of Health Sciences, Setagaya, Tokyo 158-8501, the Department of Physiology, School of Medicine, Fukuoka University, Jonan-ku, Fukuoka 814-0180, and the ||Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan

Received for publication, December 22, 2007 , and in revised form, May 25, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sustained elevation of [Ca2+]i has been implicated in many cellular events. We previously reported that {alpha} subunits of G12 family G proteins (G{alpha}12/13) participate in sustained Ca2+ influx required for the activation of nuclear factor of activated T cells (NFAT), a Ca2+-responsive transcriptional factor, in rat neonatal cardiac fibroblasts. Here, we demonstrate that G{alpha}12/13-mediated up-regulation of canonical transient receptor potential 6 (TRPC6) channels participates in sustained Ca2+ influx and NFAT activation by endothelin (ET)-1 treatment. Expression of constitutively active G{alpha}12 or G{alpha}13 increased the expression of TRPC6 proteins and basal Ca2+ influx activity. The treatment with ET-1 increased TRPC6 protein levels through G{alpha}12/13, reactive oxygen species, and c-Jun N-terminal kinase (JNK)-dependent pathways. NFAT is activated by sustained increase in [Ca2+]i through up-regulated TRPC6. A G{alpha}12/13-inhibitory polypeptide derived from the regulator of the G-protein signaling domain of p115-Rho guanine nucleotide exchange factor and a JNK inhibitor, SP600125, suppressed the ET-1-induced increase in expression of marker proteins of myofibroblast formation through a G{alpha}12/13-reactive oxygen species-JNK pathway. The ET-1-induced myofibroblast formation was suppressed by overexpression of TRPC6 and CA NFAT, whereas it was enhanced by TRPC6 small interfering RNAs and cyclosporine A. These results suggest two opposite roles of G{alpha}12/13 in cardiac fibroblasts. First, G{alpha}12/13 mediate ET-1-induced myofibroblast formation. Second, G{alpha}12/13 mediate TRPC6 up-regulation and NFAT activation that negatively regulates ET-1-induced myofibroblast formation. Furthermore, TRPC6 mediates hypertrophic responses in cardiac myocytes but suppresses fibrotic responses in cardiac fibroblasts. Thus, TRPC6 mediates opposite responses in cardiac myocytes and fibroblasts.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Structural remodeling of the heart is a key determinant of clinical outcome in heart disease (1–3). Such remodeling involves the overproduction of ECM2 proteins, predominantly collagen types I and III, into the interstitial and perivascular space. Excessive collagen deposition increases myocardial stiffness and leads to diastolic dysfunction (2–4). Cardiac fibroblasts, constituting 60–70% of total cell numbers in the heart, are responsible for collagen deposition and create the scaffold for cardiac myocytes. Transformation of cardiac fibroblasts to myofibroblasts, characterized by the expression of {alpha}-SMA, has been implicated in diseases with increased ECM deposition and resultant cardiac fibrosis (4–6). Myofibroblast formation is controlled by various profibrotic stimuli, such as growth factors, cytokines, and mechanical stretch (5–8). Although several hormones, such as Ang II and ET-1, that stimulate G protein-coupled receptors also induce myofibroblast formation (9, 10), the molecular mechanism is not fully understood.

Sustained elevation of [Ca2+]i, induced by increased Ca2+ influx, is important for the regulation of diverse cellular processes, such as gene expression, cell proliferation, and differentiation. In the heart, a Ca2+-responsive serine/threonine phosphatase calcineurin is activated by sustained [Ca2+]i increase, and calcineurin activation has been implicated in hypertrophic growth of cardiomyocytes (11, 12). Activated calcineurin dephosphorylates the transcriptional factor NFAT, facilitating translocation of NFAT from cytosol to the nucleus, where it acts synergistically with other partners to mediate the expression of prohypertrophic genes. However, the physiological and pathological roles of calcineurin-NFAT signaling in cardiac fibroblasts are still controversial when the roles of the signaling pathway are analyzed by CysA, a calcineurin inhibitor. It has been reported that treatment with CysA inhibits pressure overload-induced cardiac hypertrophy and fibrosis (13) and enhances cardiac dysfunction during postinfarction failure (14). Other reports have demonstrated that myocardial fibrosis is promoted in transplanted hearts (15) or chronically aortic banded hearts (16) treated with CysA. Cardiac fibrosis in vivo often follows the development of cardiac hypertrophy. To understand fibrotic pathways, it is essential to examine mechanisms of cardiac fibrosis and hypertrophy individually. Although the role of NFAT in cardiac hypertrophy is established, the role of NFAT in cardiac fibrosis is still unknown.

We previously reported that G{alpha}12/13 mediate Ang II-induced NFAT activation in cardiac fibroblasts (17). We also demonstrated that activation of G{alpha}13 increases basal Ca2+ influx activity, which is completely suppressed by SK&F96365, an inhibitor of RACCs (18). Members of the TRPC family channels are thought to be molecular candidates for RACCs (19). Recent reports have implicated the involvement of TRPC up-regulation in diseases with abnormal Ca2+ handling and resultant heart failure (20, 21). We recently reported that TRPC3 and TRPC6 are involved in Ang II-induced hypertrophic responses of rat neonatal cardiomyocytes (12). However, it is still unknown whether TRPC channels participate in G{alpha}12/13-mediated NFAT activation and agonist-induced myofibroblast formation in cardiac fibroblasts.

In this study, we investigate how the expression of TRPC channels is regulated and whether TRPC channels are involved in the G{alpha}12/13-mediated increase in basal Ca2+ influx required for NFAT activation. We also examine the roles of TRPC channels and NFAT in transformation of cardiac fibroblasts to myofibroblasts.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Plasmid Construction—JNK inhibitor II (SP600125), cyclosporine A, and PP2 were purchased from Calbiochem. Ang II, ET-1, DPI, U73122 [GenBank] , Igepal CA-630, BQ123, BQ788, and anti-{alpha}-SMA antibody (clone 1A4) were purchased from Sigma. Fura-2/AM was from Dojindo (Kumamoto, Japan). Collagenase and Fugene 6 were from Roche Applied Science. Dual luciferase reagents was from Promega. pNFAT-Luc and pRL-SV40 vectors were from Stratagene. Anti-TRPC3 and anti-TRPC6 antibodies were from Alomone Laboratories. Anti-TRPC7 antibody was prepared by Y. Mori. Anti-phos-pho-ERK, anti-ERK, anti-phospho-Src (Tyr416), and anti-Src antibodies were from Cell Signaling. Anti-glyceraldehyde-3-phosphate dehydrogenase, anti-JNK1, horseradish peroxidase-conjugated anti-rabbit IgG, and anti-mouse IgG antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Rac antibody was from Transduction Laboratories. CA NFAT was first inserted into the pEGFP-C1 vector (Clontech) and transferred into pShuttle-cytomegalovirus vector to produce recombinant adenovirus. A 913-bp fragment containing the upstream region and the 5'-untranslated region of the rat TRPC6 gene3 was isolated by PCR using rat genomic DNA as a template, and the fragment was inserted upstream of a luciferase gene in the pGL3-Basic vector using KpnI/BglII sites.

Cell Culture—Cardiac fibroblasts and myocytes were prepared from ventricles of 1–2-day-old Sprague-Dawley rats (17, 22). Briefly, after digestion of ventricles with 0.1% collagenase, isolated fibroblasts were plated on a noncoated dish in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 50 units/ml penicillin/streptomycin. Subconfluent cells were serum-starved for 48 h and used for the experiments. Considering the possibility that cardiac fibroblasts may lose the original characteristics after prolonged culture, cells were used within two passages in all experiments.

Production of Adenoviruses, Infection, and Transfection—Recombinant adenoviruses used in this study, including WT TRPC6, DN TRPC6 (C6-3A and C6-{Delta}N), GFP-fused N-terminal region of NFAT4 (GFP-NFAT4), and CA NFAT, were produced as described previously (12, 23–25). Cells were infected with adenovirus(es) at a multiplicity of infection of 100 for 48 h. Small interference RNAs (100 nM) were transfected with Lipofectamine 2000 for 72 h.

Measurement of Intracellular Ca2+ Measurement and NFAT Activity—The intracellular Ca2+ concentration ([Ca2+]i)of cardiac fibroblasts was determined by a method described previously (17). Fluorescence images of GFP-positive cells were recorded and analyzed with a video image analysis system (Aquacosmos, Hamamatsu Photonics). Measurement of NFAT activity was performed as described previously (17). For measuring the translocation of GFP-NFAT4, cells (1.5 x 105) plated on glass bottom 35-mm dishes were infected for 48 h with adenovirus coding GFP-NFAT4 at a multiplicity of infection of 30. After ET-1 stimulation (100 nM) for 48 h, the localization of GFP-NFAT4 was determined with a laser-scanning confocal imaging system (LSM510; Carl Zeiss).

Measurement of ROS Production—Measurement of intracellular ROS concentration was performed in 2 mM Ca2+-containing Hepes-buffered saline solution (107 mM NaCl, 6 mM KCl, 11.5 mM glucose, 20 mM HEPES, pH 7.4, 1.2 mM MgSO4,2mM CaCl2) with a fluorescent dye, 2',7'-dichlorofluorescein diacetate as described previously (17). Fluorescence images were recorded and analyzed with a video image analysis system (Aquacosmos, Hamamatsu Photonics). The peak changes ({Delta}F/F0) of DCF fluorescence intensity were identified as values obtained by subtracting the basal fluorescence intensity (F0) from the maximal intensity during 5-min ET-1 treatment.

Expression Analysis of TRPC mRNAs—The RT-PCR protocol used for the expression analysis and the PCR primers used in this study were as described previously (26). Real time RT-PCR for quantitative measurement was performed as described previously (18). Briefly, total RNA (150 ng) was subjected to real time RT-PCR. Oligonucleotide primers and fluorescence-labeled Taq-Man® probes were designed with Primer Express software (Applied Biosystems, Foster City, CA). Primers and probe were as follows: for rat TRPC6 mRNA, forward primer (5'-AGCAGCAGCTCCTCTCCATATG-3'), reverse primer (5'-GAGGACCACGAGGAATTTCACT-3'), and TaqMan® probe (5'-TATGAGAACCTCTCTGGTTTACGGCAGCA-3'); for collagen type I, forward primer (5'-GGAGAGTACTGGATCGACCCTAAC-3'), reverse primer (5'-CTGACCTGTCTCCATGTTGCA-3'), and TaqMan® probe (5'-AAGGCTGCAACCTGGATGCCATCAA-3'); for collagen type III, forward primer (5'-CCTGCTTCACCCCTCTCTTATTT-3'), reverse primer (5'-TCCCGAGTCGCAGACACATAT-3'), and TaqMan® probe (5'-TAGAGATGTCTGGAAGCCAGAACCATGTCA-3'). All reactions were performed in TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems) and the Applied Biosystems 7500 real time PCR system. The rat glyceraldehyde-3-phosphate dehydrogenase mRNA (Applied Biosystems (catalog number 4308313)) was used as an internal control to normalize the differences in the amount of total RNA in each sample.


Figure 1
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  		FIGURE 1.
Up-regulation of TRPC6 by G{alpha}13 activation. A, RT-PCR analysis of RNA expression of TRPC channels in LacZ- and CA G{alpha}13-expressing cardiac fibroblasts. Bottom, real time RT-PCR analysis of RNA expression of TRPC6. B, expression of TRPC6 protein in LacZ-expressing and the respective G{alpha}-overexpressing cells. Cells were treated with forskolin (50 µM) for 24 h. C, effects of TRPC6 siRNAs (C6-1609 and C6-1786) on TRPC6 protein expression. D, effects of TRPC6 siRNAs on the basal Ca2+ influx activity in CA G{alpha}13-expressing cells. Four min after Ca2+ measurement in Ca2+-free solution, basal Ca2+ influx was determined by the addition of 2 mM Ca2+. E, the amplitude of maximum [Ca2+]i rises ({Delta}Ratio) induced by the addition of 2 mM Ca2+ was calculated. F, effects of TRPC6 siRNAs on CA G{alpha}13-induced increase in NFAT activity. *, p < 0.05; **, p < 0.01 versus control of LacZ-expressing cells. #, p < 0.05; ##, p < 0.01 versus control of CA G{alpha}13-expressing cells.

 
Western Blot Analysis—Cardiac fibroblasts (3 x 105 cells) plated on 6-well dishes were directly harvested with 200 µlof2x SDS sample buffer. The protein samples (20 µl) were fractionated by 8% SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride membrane. The expression of endogenous TRPC proteins was assessed by Western blotting using anti-TRPC antibodies (12, 27). The Src and ERK activities were assessed by using anti-phos-pho-specific antibodies. The JNK activity was assessed as described previously (23–25). For {alpha}-SMA expression, cells were harvested with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, and protease inhibitor mixture (6). The protein samples were fractionated by 10% SDS-PAGE.

Measurement of Rac Activity—Activation of small G proteins was determined by the method of Nishida et al. (25). Activated Rac was pulled down with 10 µg of glutathione S-transferase-fused Racinteracting domain of p21-activated kinase (PAK-CRIB). Pulled down small G proteins were detected with anti-Rac1 antibody.

Measurement of Cardiac Myofibroblast Formation—Transformation of cardiac fibroblasts to myofibroblasts was assessed by the expression of {alpha}-SMA and production of ECM components (6). Briefly, 24 h after infection, cardiomyocytes were stimulated with ET-1 (100 nM) for 48 h. The cells were washed, fixed, incubated with anti-{alpha}-SMA antibody, and then stained with Alexa Fluor 546 goat antimouse IgG. Morphological changes and {alpha}-SMA expression of cardiac fibroblasts were visualized with confocal microscopy. For the measurement of collagen synthesis, cells were harvested with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, and protease inhibitor mixture (Nacalai, Japan). Production of ECM components was assessed by [3H]proline incorporation. After cells were stimulated with ET-1 (100 nM)for 2 h,[3H]proline (1 µCi/ml) was added to the culture medium and further incubated for 6 h. The incorporated [3H]proline was extracted by 1 N NaOH overnight and measured using a liquid scintillation counter.

Measurement of Hypertrophic Responses of Cardiomyocytes—Preparation of rat neonatal cardiomyocytes and measurement of hypertrophic responses were performed as described (12). Briefly, 48 h after siRNA treatment, cardiomyocytes were stimulated with ET-1 (100 nM) for 24 h. The cells were stained with Alexa Fluor 594-phalloidin to visualize actin filament. Protein synthesis was measured by [3H]leucine incorporation. After cells were stimulated with ET-1 (100 nM)for 2 h,[3H]leucine (1 µCi/ml) was added and further incubated for 6 h.


Figure 2
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  		FIGURE 2.
Involvement of tyrosine kinase in CA G{alpha}13 -induced increase in basal Ca2+ influx activity in cardiac fibroblasts. A, expression of wild type TRPC6 (WT) and DN TRPC6 (C6-3A and C6-{Delta}N) proteins in cardiac fibroblasts. One-fourth volume of protein samples (5 µl) was subjected to 7% SDS-PAGE. The expression level of endogenous TRPC6 was low, and expression levels of wild type and mutated TRPC6 were very high. Therefore, the band intensity of endogenous TRPC6 is weak under these conditions. B and C, effects of DN TRPC6 proteins on the CA G{alpha}13 -induced increase in basal Ca2+ influx activity. B, average time courses of Ca2+ responses induced by the addition of 2 mM Ca2+. C, the amplitude of sustained [Ca2+]i increase 4 min after the addition of 2 mM Ca2+. D and E, effects of PP2, SP600125 (SP), and U73122 on the CA G{alpha}13-induced increase in basal Ca2+ influx activity. Cells were treated with PP2 (1 µM) or U73122 (1 µM) for 20 min and SP600125 (1 µM) for 48 h prior to [Ca2+]i measurement. D, average time courses of Ca2+ responses. E, peak changes in [Ca2+]i induced by the addition of 2 mM Ca2+. F, effects of SP600125, PP2, and AG1478 on CA G{alpha}13-induced increase in TRPC6 protein expression and Src phosphorylation. Cells were treated with SP600125 (1 µM) for 48 h and with PP2 (1 µM) or AG1478 (1 µM) for 30 min prior to cell harvest. *, p < 0.05 versus LacZ-expressing cells. #, p < 0.05 versus control (LacZ or Me2SO (DMSO)) of CA G{alpha}13-expressing cells.

 
Statistical Analysis—The results are presented as mean ± S.E. The data were accumulated under each condition from at least three independent experiments. For the measurements of [Ca2+]i, representative data of time course experiments were plotted from one of three similar experiments that were performed with more than 30 cells. Mean values were compared with control by Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Up-regulation of TRPC6 Proteins by G{alpha}13 Activation—We previously demonstrated that the expression of GTPase-defective mutants of G{alpha}12 (Q229L; CA G{alpha}12) and G{alpha}13 (Q226L; CA G{alpha}13) increases [Ca2+]i and NFAT activity through an SK&F96365-sensitve Ca2+ influx pathway (17, 18). TRPC channels are thought to be molecular candidates for RACCs. Thus, we examined which TRPC channels are expressed in rat cardiac fibroblasts. Among TRPC1 to -7 channels, TRPC1, TRPC3, TRPC6, and TRPC7 mRNAs were detected (Fig. 1A). Interestingly, only TRPC6 mRNA expression was increased 2.5-fold by the expression of CA G{alpha}13. The expression of TRPC6 proteins was also increased by CA G{alpha}12 (Fig. 1B). Expression of CA G{alpha}q (R183C) and WT G{alpha}i2 did not increase TRPC6 protein levels, although both mutants significantly increased ERK activity (supplemental Fig. 1). Treatment with forskolin (50 µM) rather decreased basal expression of TRPC6 proteins. These results indicate that G{alpha}12/13 specifically up-regulate TRPC6 proteins in cardiac fibroblasts. In order to examine whether up-regulation of TRPC6 participates in CA G{alpha}12/13-induced enhancement of Ca2+ responses, knockdown experiments of TRPC6 proteins were performed by using TRPC6 siRNAs (12). Since the extent of NFAT activation induced by CA G{alpha}13 was larger than that by CA G{alpha}12,CAG{alpha}13-expressing cells were used to examine the involvement of TRPC6 in G{alpha}12/13-mediated Ca2+ responses. Treatment with TRPC6 siRNAs completely suppressed CA G{alpha}13-induced up-regulation of TRPC6 protein expression (Fig. 1C). We confirmed that TRPC6 siRNAs did not affect the expression levels of TRPC3 and TRPC7 proteins (data not shown). The basal Ca2+ influx activity was determined by the increase in [Ca2+]i induced by the addition of 2 mM extracellular Ca2+ to cells in Ca2+-free Tyrode's solution (18). This basal Ca2+ influx activity was enhanced by the expression of CA G{alpha}13 and CA G{alpha}12 (Fig. 1D). The CA G{alpha}13-induced increase in [Ca2+]i was completely suppressed by the treatment with TRPC6 siRNAs (Fig. 1, C–E). The sustained increase in [Ca2+]i activated NFAT activity, and the treatment with TRPC6 siRNAs inhibited CA G{alpha}13-induced NFAT activation (Fig. 1F). These results suggest that up-regulation of TRPC6 by G{alpha}13 activation enhances Ca2+ influx and NFAT activation in cardiac fibroblasts.

Involvement of Tyrosine Kinase in TRPC6-mediated Ca2+ Influx by G{alpha}12/13 Activation—We next examined whether the channel activity of TRPC6 proteins is actually enhanced by G{alpha}13 activation. WT TRPC6 or two DN mutants of TRPC6 (C6-3A and C6-{Delta}N) were overexpressed in cardiac fibroblasts (Fig. 2A). The expression of DN TRPC6 significantly suppressed the sustained [Ca2+]i increase of CA G{alpha}13-expressing cells induced by the addition of 2 mM extracellular Ca2+ (Fig. 2, B and C), indicating that the increase in [Ca2+]i of CA G{alpha}13-expressing cells is enhanced by Ca2+ influx through TRPC6 channels. It has been reported that TRPC6 activity is regulated by DAG (28) and tyrosine phosphorylation (29), and activated G{alpha}13 induces activation of tyrosine kinase (30, 31) and PLC (32). We examined the involvement of TRPC6 in CA G{alpha}13-induced [Ca2+]i increase by determining the requirement of tyrosine phosphorylation or PLC for TRPC6 activation. Treatment with PP2, a Src tyrosine kinase inhibitor, and SP600125, a JNK inhibitor, but not U73122 [GenBank] , a PLC inhibitor, significantly suppressed the increase in [Ca2+]i of CA G{alpha}13-expressing cells induced by the addition of 2 mM Ca2+ (Fig. 2, D and E). Consistent with the involvement of Src, the expression of CA G{alpha}12 and CA G{alpha}13 increased Src activity about 2.5-fold (Fig. 2F). CA G{alpha}13-induced Src activation was suppressed by treatment with AG1478, an EGFR kinase inhibitor (Fig. 2F), suggesting that EGFR kinase mediates the CA G{alpha}13-induced Src activation. Treatment with SP600125, but not PP2 and AG1478, suppressed CA G{alpha}13-induced up-regulation of TRPC6 protein, whereas treatment with PP2 and AG1478, but not SP600125, suppressed CA G{alpha}13-induced Src activation (Fig. 2F). These results suggest that EGFR- and PP2-sensitive tyrosine kinase(s) participate in G{alpha}13-mediated enhancement of Ca2+ influx independently of TRPC6 up-regulation. These findings are consistent with the report that EGFR participates in G{alpha}13-mediated responses (33).


Figure 3
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  		FIGURE 3.
ET-1 up-regulates TRPC6 through the G{alpha}13-ROS-JNK-dependent pathway. A, changes in expression levels of TRPC3, TRPC6, and TRPC7 proteins by the treatment with Ang II (100 nM) and ET-1 (100 nM) for 48 h. B, concentration-dependent increase in TRPC6 protein expression by ET-1 treatment. C, effects of ET receptor antagonists on ET-1-induced increase in TRPC6 expression. Cells were treated with BQ123 (3 µM) or BQ788 (3 µM) for 5 min prior to ET-1 stimulation. D, involvement of G{alpha}12/13, ROS, and JNK in the ET-1-induced increase in TRPC6 expression. Twenty-four h after infection with LacZ or p115-RGS, cells were treated with ET-1 (100 nM) for 48 h. Cells were treated with DPI (1 µM), U73122 (1 µM), CysA (0.5 µg/ml), and SP600125 (SP;1 µM) for 20 min prior to ET-1 stimulation. E, effects of CysA, DPI, and SP600125 on ET-1-induced increase in the TRPC6-luciferase activity. Cells were treated with ET-1 (100 nM) for 6 h. F, effects of TRPC6 siRNAs on TRPC3 and TRPC6 protein expressions. *, p < 0.05 versus nontreatment (no ET-1) of control or LacZ-expressing cells. #, p < 0.05 versus ET-1 treatment of control or LacZ-expressing cells.

 
Up-regulation of TRPC6 Proteins by ET-1 and Ang II Stimulation—We next examined whether up-regulation of TRPC6 is actually induced by G13-coupled receptor stimulation. We have previously demonstrated that G{alpha}12/13 mediate Ang II- and ET-1-induced activation of JNK and hypertrophic responses in rat neonatal cardiomyocytes (24, 25). The TRPC6 protein expression levels were significantly increased by Ang II treatment and more potently by ET-1 treatment, in a concentration-dependent manner (Fig. 3, A and B). Treatment with ET-1 actually increased TRPC6 mRNA levels (supplemental Fig. 2A). The up-regulation of TRPC6 protein was detected at 6 h after ET-1 stimulation and the expression of TRPC6 proteins reached a peak at 48 h (data not shown). In contrast to TRPC6, Ang II and ET-1 treatment did not affect the expression levels of TRPC3 and TRPC7 proteins (Fig. 3A). These results indicate that ET-1 treatment selectively up-regulates TRPC6 proteins in cardiac fibroblasts. Since the ET-1-induced increase in TRPC6 protein expression levels was completely suppressed by BQ123, a selective ETA receptor antagonist, but not by BQ788, a selective ETB receptor antagonist (Fig. 3C), the ETA receptor predominantly mediates ET-1-induced TRPC6 up-regulation. It has been reported that the activation of JNK and STAT3 is required for up-regulation of TRPC6 proteins induced by ET-1 and PDGF stimulation in pulmonary vascular smooth muscle cells (34, 35). We previously demonstrated that G{alpha}12/13 mediate Ang II-induced Rac activation, ROS production, and JNK activation in cardiac myocytes and fibroblasts (17, 25). Thus, we examined whether ROS and JNK are involved in ET-1-induced TRPC6 up-regulation. Expression of p115-RGS, a selective inhibitory polypeptide of G{alpha}12/13, completely suppressed ET-1-induced increase in TRPC6 proteins, as expected (Fig. 3D). The ET-1-induced TRPC6 up-regulation was also suppressed by DPI and SP600125 but not by a PLC inhibitor, U73122 [GenBank] , and CysA. Since DPI is a selective inhibitor of ROS production by NADPH oxidase complex and SP600125 selectively inhibits JNK activation, these results suggest that the G{alpha}12/13-ROS-JNK pathway participates in ET-1-induced TRPC6 up-regulation. NFAT is activated through its dephosphorylation by calcineurin that is inhibited by CysA. Since the treatment with CysA increased TRPC6 mRNA levels and the expression of CA NFAT decreased TRPC6 mRNA levels (supplemental Fig. 2B), NFAT may negatively regulate ET-1-induced TRPC6 expression in cardiac fibroblasts. Furthermore, the ET-1-induced increase in TRPC6 promoter-dependent luciferase activity was completely suppressed by DPI and SP600125 but not by CysA (Fig. 3E). These results suggest that the G{alpha}12/13-ROS-JNK pathway participates in the transcriptional activation of the TRPC6 gene. ET-1-stimulated up-regulation of TRPC6 protein was completely suppressed by siRNAs against TRPC6 (Fig. 3F), suggesting the utility of these siRNAs for future analysis.

Up-regulation of TRPC6 Enhances Receptor-activated Ca2+ Responses but Not Store-operated Ca2+ Responses—TRPC proteins have been emerged as a candidate subunit of RACCs and SOCs (19). To examine whether up-regulation of TRPC6 enhances receptor-activated or store-operated Ca2+ entry, we have individually determined store-operated and receptor-activated Ca2+ entry. The increase in [Ca2+]i through receptoractivated Ca2+ entry is determined by the addition of Ca2+ after receptor stimulation in the absence of extracellular Ca2+. The increase in [Ca2+] through store-operated Ca2+i entry is determined by the addition of Ca2+ after the treatment of cells with Ca2+ ionophore ionomycin in the absence of extracellular Ca2+. Since the cells were treated with ET-1 for long period of time, we used Ang II as a receptor stimulant to avoid the under-estimation of ET-1-stimulated increase in [Ca2+]i through receptor-activated Ca2+ entry due to desensitization. The expression of TRPC6 did not affect the Ang II-induced increase in [Ca2+]i in the absence of extracellular Ca2+ but enhanced the increase in [Ca2+]i by the addition of extracellular Ca2+ (Fig. 4, A and B). The treatment with TRPC6 siRNA (C6-1609) did not affect the initial increase in [Ca2+]i of ET-1-treated cells (Fig. 4, A and B). However, TRPC6 siRNA (C6-1609) completely inhibited the increase in [Ca2+]i induced by the addition of Ca2+. These results are consistent with the findings that the prolonged treatment of cells with ET-1 increases TRPC6 expression. It has been known that TRPC6-mediated Ca2+ influx is enhanced by DAG derivative OAG (19). We examined whether OAG-stimulated increases in [Ca2+]i are suppressed by TRPC6 siRNA. Stimulation with OAG enhanced the increase in [Ca2+]i induced by the addition of extracellular Ca2+ of WT TRPC6-expressing or ET-1-treated cells (Fig. 4, C and D). TRPC6 siRNA (C6-1609) completely suppressed the OAG-induced increase in [Ca2+]i of WT TRPC6-expressing or ET-1-treated cells. Although compensatory up-regulation of TRPC3 mRNA expression is reported in TRPC6-deficient mice (36), TRPC6 siRNAs did not affect TRPC3 protein expression (Fig. 3F). These results suggest that up-regulation of TRPC6 enhances receptor-activated Ca2+ entry through a DAG-dependent mechanism. In contrast to the involvement of TRPC6 in receptor-activated Ca2+ entry, overexpression of TRPC6 did not affect Ca2+ entry-dependent increase in [Ca2+]i induced by ionomycin treatment that completely depletes intracellular Ca2+ stores (27) (Fig. 4, E and F). Up-regulation of TRPC6 by ET-1 treatment did not affect the increase in [Ca2+]i induced by Ca2+ entry after ionomycin treatment. These results suggest that the increased expression of TRPC6 does not contribute to the increase in [Ca2+] through SOC-mediated Ca2+i entry in cardiac fibroblasts. Since the Ang II-induced Ca2+ release and ionomycin-induced Ca2+ release were not affected by ET-1 treatment or TRPC6 siRNA treatment, the changes in TRPC6 proteins may not influence Ca2+ content of intracellular Ca2+ stores or IP3-mediated functions.


Figure 4
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  		FIGURE 4.
Up-regulation of TRPC6 enhances Ca2+ responses induced by Ca2+ entry upon receptor stimulation. A, average time courses of Ca2+ responses upon Ang receptor stimulation with Ang II (100 nM). Ca2+ release was first evoked in Ca2+-free solution, and Ca2+ entry-mediated Ca2+ responses were induced by the addition of 2 mM Ca2+. Forty-eight h after the transfection with siRNA (control or C6-1609), cells were treated with ET-1 (100 nM) for 48 h, and then [Ca2+]i levels were measured. B, peak Ang II-induced increase in [Ca2+]i in Ca2+ -free solution and after the addition of Ca2+. C, average time courses of Ca2+ responses induced by OAG (25 µM). D, peak OAG-induced [Ca2+]i increase observed after the addition of Ca2+ to Ca2+-free solution. E, average time courses of Ca2+ responses induced by ionomycin (1 µM). F, peak ionomycin-induced [Ca2+]i increase observed after the addition of Ca2+ to Ca2+-free solution. *, p < 0.05 versus nontreatment (no ET-1) of control cells.

 


Figure 5
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  		FIGURE 5.
Involvement of TRPC6 up-regulation in ET-1-induced sustained NFAT activation. A, effects of TRPC6 siRNA and DN TRPC6 (C6-{Delta}N) on the basal Ca2+ influx activity in ET-1-treated cells. Twenty-four h after the infection with adenovirus coding C6-{Delta}N or the transfection with siRNA (C6-1609), cells were treated with ET-1 (100 nM) for 48 h, and then basal Ca2+ influx activity was determined. B, peak amplitudes of [Ca2+]i increase, induced by the addition of 2 mM Ca2+. Cells were first treated with ET-1 for 48 h and then treated with SP600125 (SP;1 µM) or PP2 (1 µM) for 20 min prior to [Ca2+]i measurement. C, effects of p115-RGS, PP2, and SP600125 on ET-1-induced Src phosphorylation. Cells were first treated with PP2 and SP600125 for 20 min and then treated with ET-1 for 10 min. D, effects of p115-RGS and SP600125 on ET-1-induced nuclear localization of GFP-NFAT4 proteins. E, effects of TRPC6 siRNAs on ET-1-induced NFAT translocation. Twenty-four h after the transfection with siRNAs, cells were treated with ET-1 for 48 h, and the localization of NFAT was determined with confocal microscopy. F, quantification of nuclear predominant fluorescence of GFP-NFAT4. G, effects of TRPC6 WT, C6-{Delta}N, and C6-3A on the ET-1-induced increase in NFAT-luciferase activity. Twenty-four h after infection, cells were treated with ET-1 for 48 h. *, p < 0.05 versus nontreatment (no ET-1) of control or LacZ-expressing cells. #, p < 0.05 versus ET-1 treatment of control or LacZ-expressing cells.

 
Requirement of TRPC6 for ET-1-induced NFAT Activation—Since CA G{alpha}13-induced TRPC6 up-regulation enhances basal Ca2+ influx activity, we examined whether ET-1 treatment enhances basal Ca2+ influx activity through G{alpha}12/13-mediated TRPC6 up-regulation. Treatment with ET-1 for 48 h enhanced the increase in [Ca2+]i induced by the addition of 2 mM Ca2+ to ET-1-containing Ca2+-free solution, which was completely suppressed by TRPC6 siRNA (C6-1609) and DN TRPC6 (C6-{Delta}N) (Fig. 5, A and B). The ET-1-induced enhancement of Ca2+ influx was suppressed by p115-RGS and PP2. Treatment with ET-1 increased Src activity, which was completely suppressed by p115-RGS and PP2 but not by SP600125 (Fig. 5C). We confirmed that ET-1-induced TRPC6 up-regulation was not affected by PP2 (supplemental Fig. 2C). Src activation by ET-1 stimulation was mediated by ETA receptor, since ETA receptor-selective (BQ123) but not ETB receptor-selective (BQ788) blockers inhibited ET-1-stimulated Src activation (supplemental Fig. 2D). These results suggest that G{alpha}12/13-mediated activation of Src tyrosine kinase participate in ET-1-induced increase in basal Ca2+ influx activity apart from TRPC6 up-regulation. On the other hand, treatment with ET-1 for 48 h induced nuclear localization of GFP-NFAT4 proteins, which were inhibited by p115-RGS and SP600125 (Fig. 5, D and F). Treatment with SP600125 (1 µM) significantly inhibited the ET-1 treatment-induced increase in basal Ca2+ influx activity ({Delta}Ratio = 0.70 ± 0.05, n = 67 cells) and TRPC6 protein levels (Fig. 3D), suggesting that G{alpha}12/13-mediated activation of JNK participates in ET-1-induced NFAT translocation through TRPC6 up-regulation. The ET-1 treatment-induced NFAT translocation was also suppressed by TRPC6 siRNAs (Fig. 5, E and F). The expression of WT TRPC6 increased basal NFAT activity 2.5-fold, and it also enhanced NFAT activity induced by ET-1 treatment (Fig. 5G). In contrast, the expression of DN TRPC6 mutants completely suppressed the ET-1 treatment-induced increase in NFAT activity. These results suggest that G{alpha}12/13-mediated up-regulation and activation of TRPC6 channels participate in sustained activation of NFAT induced by ET-1 treatment in cardiac fibroblasts.

Inhibition of Cardiac Myofibroblast Formation by TRPC6 Activation—Transformation of cardiac fibroblasts to myofibroblasts is characterized by expression of {alpha}-SMA and production of ECM components that are a key event in connective tissue remodeling (6). Since NFAT is activated by the sustained increase in [Ca2+]i and the calcineurin-NFAT pathway plays a critical role in the pathogenesis of heart failure, we examined whether TRPC6-mediated NFAT activation participates in ET-1 treatment-induced transformation of cardiac fibroblasts. Treatment with ET-1 increased {alpha}-SMA expression in a concentration-dependent manner (Fig. 6, A and B). Treatment with ET-1 (1 nM) caused maximal {alpha}-SMA expression, whereas ET-1 treatment (>10 nM) slightly decreased {alpha}-SMA expression. Knockdown of TRPC6 did not affect basal {alpha}-SMA protein expression levels. However, it weakly but significantly enhanced ET-1-induced {alpha}-SMA expression (Fig. 6, A and C). Furthermore, TRPC6 siRNAs significantly enhanced the basal incorporation of [3H]proline, an index of synthesis of ECM proteins (Fig. 6D). TRPC6 siRNAs also enhanced ET-1-induced [3H]proline incorporation. Collagen expression is another index of transformation of cardiac fibroblasts to myofibroblasts. TRPC6 siRNA increased mRNAs of collagen type I and III, whereas overexpression of WT TRPC6 significantly suppressed the basal expression of collagen mRNAs (Fig. 6E). These results suggest that TRPC6 negatively regulates ET-1-induced transformation of cardiac fibroblasts to myofibroblasts and synthesis of ECM components.


Figure 6
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  		FIGURE 6.
Knockdown of TRPC6 enhances cardiac myofibroblast formation. A–C, effects of TRPC6 siRNAs on the ET-1-induced increase in {alpha}-SMA expression. Forty-eight h after transfection with siRNAs, cells were treated with ET-1 (100 nM) for 24 h. A, immunohistochemistry of cardiac fibroblasts stained with anti-{alpha}-SMA antibody. Scale bar,20 µm. B and C, concentration-dependent increase in {alpha}-SMA expression by ET-1 treatment (B) and changes in {alpha}-SMA expression by TRPC6 knockdown (C) were determined by Western blotting. D, effects of TRPC6 siRNAs on [3H]proline incorporation in cardiac fibroblasts with or without ET-1 treatment. E, effects of TRPC6 siRNA and WT TRPC6 on the mRNA expression of collagen type I and III. *, p < 0.05 versus nontreatment (no ET-1) of control cells. #, p < 0.05 versus ET-1 treatment of control cells.

 
Inhibition of Cardiac Myofibroblast Formation by NFAT Activation—The expression of TRPC6 WT inhibited the collagen mRNA expression and enhanced NFAT activation by ET-1 treatment (Figs. 5G and 6E). In addition to cardiac fibroblasts, we have previously demonstrated in cardiac myocytes that TRPC6 regulates NFAT activation (12). Then we examined whether TRPC6-mediated NFAT activation negatively regulates ET-1-induced transformation of cardiac fibroblasts to myofibroblasts. The expression of CA NFAT increased basal NFAT activity (8.3 ± 0.9-fold) and suppressed the increase in {alpha}-SMA expression by ET-1 treatment (Fig. 7, A and B). Expression of {alpha}-SMA by ET-1 treatment was also suppressed by expression of TRPC6 WT. In contrast, treatment with CysA increased the basal {alpha}-SMA expression levels and [3H]proline incorporation, which was further enhanced by ET-1 treatment (Fig. 7, B and D). These results suggest that TRPC6-mediated NFAT activation inhibits ET1-induced {alpha}-SMA expression in cardiac fibroblasts. Furthermore, the ET-1-induced increase in {alpha}-SMA expression and [3H]proline incorporation was suppressed by the expression of p115-RGS and DN Rac and treatment with SP600125 (Fig. 7, C and D). These results suggest that the G{alpha}12/13-mediated signaling pathway (G{alpha}12/13-Rac-JNK) mediates ET-1-induced myofibroblast formation in cardiac fibroblasts. However, G{alpha}12/13-mediated NFAT activation negatively regulates ET-1-induced cardiac myofibroblast formation.

Inhibition of ET-1-induced JNK Activation by TRPC6-mediated NFAT Activation—In order to examine how NFAT attenuates myofibroblast formation by ET-1 treatment, we next examined the effects of NFAT on ET-1-induced mitogen-activated protein kinase activation. Treatment with ET-1 increased JNK activity, which was suppressed by the expression of CA NFAT and WT TRPC6 but was potentiated by CysA (Fig. 8A). However, the ET-1-induced ERK activation was not affected by CA NFAT, TRPC6 WT, and CysA (Fig. 8B). This is consistent with the result that ERK activation is not involved in the increased expression of TRPC6 by ET-1 treatment (supplemental Fig. 2C). These results suggest that NFAT activation through TRPC6 up-regulation specifically inhibits ET-1-induced JNK activation in cardiac fibroblasts. The ET-1-induced ROS production and Rac activation, both of which work upstream of JNK, were suppressed by TRPC6 overexpression and NFAT activation (Fig. 8, C and D). These results suggest that NFAT inhibits JNK activation through inhibition of Rac activation and ROS production.

Requirement of TRPC6 Up-regulation in ET-1-induced Hypertrophic Responses of Cardiomyocytes—It has been reported that up-regulation of TRPC6 amplifies calcineurin signaling, leading to pathologic cardiac remodeling in mice (37). We have previously reported that G{alpha}12/13-mediated JNK activation participates in ET-1-induced hypertrophic responses of cardiomyocytes (24). We examined whether TRPC6 up-regulation is involved in ET-1-induced hypertrophic growth of rat neonatal cardiomyocytes. Treatment with ET-1 increased TRPC6 protein levels in a concentration-dependent manner, and the EC50 value was about 3 nM (Fig. 9A). The ET-1-induced increase in TRPC6 protein expression was completely suppressed by p115-RGS (Fig. 9B), suggesting that G{alpha}12/13 mediate ET-1-induced TRPC6 up-regulation. Treatment with ET-1 increased BNP-luciferase activity, a marker for cardiac hypertrophy, and the EC50 value was about 5 nM (Fig. 9C). ET-1-stimulated BNP-luciferase activity was blocked by expression of p115-RGS, an inhibitory polypeptide of G{alpha}12/13. This result suggests that G{alpha}12/13 mediate ET-1-stimulated hypertrophic response. Although we could not reliably determine the effects of TRPC6 siRNAs on ET-1-stimulated BNP-luciferase activity, the ET-1-induced NFAT activation and hypertrophic responses (actin reorganization and protein synthesis) were significantly suppressed by TRPC6 siRNAs (Fig. 9, D–F). These results suggest that G{alpha}12/13 also play a role in ET-1-induced increase in TRPC6 protein expression in cardiomyocytes. However, in the case of cardiomyocytes, the up-regulated TRPC6 may positively regulate hypertrophic responses induced by ET-1.


Figure 7
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  		FIGURE 7.
Inhibition of cardiac myofibroblast formation by NFAT activation. A and B, effects of TRPC6 WT, CA NFAT, CysA, and p115-RGS on the ET-1-induced increase in {alpha}-SMA expression. Cells were infected with adenovirus coding one of TRPC6 WT, p115-RGS, and CA NFAT at a multiplicity of infection of 100 for 24 h and then treated with ET-1 (100 nM) for 48 h. Cells were treated with CysA (0.5 µg/ml) for 20 min prior to ET-1 treatment. A, immunohistochemistry of cardiac fibroblasts stained with anti-{alpha}-SMA antibody. Scale bar,20 µm. B, changes in {alpha}-SMA expression determined by Western blotting. C, effects of p115-RGS, DN Rac, and SP600125 on the ET-1-induced increase in {alpha}-SMA expression. Twenty-four h after infection with adenovirus coding one of LacZ, p115-RGS, and DN Rac, cells were treated with ET-1 for 48 h. Cells were treated with SP600125 (SP;1 µM) for 20 min prior to ET-1 treatment. D, effects of CA NFAT, TRPC6 WT, CysA, and p115-RGS on [3H]proline incorporation in cardiac fibroblasts with or without ET-1 treatment. *, p < 0.05 versus nontreatment (no ET-1) of LacZ-expressing cells. #, p < 0.05 versus ET-1 treatment of LacZ-expressing cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that TRPC6 proteins are up-regulated by ET-1 treatment in cardiac fibroblasts. Furthermore, we demonstrated that the ET-1-induced increase in TRPC6 expression is critical for NFAT activation, which negatively regulates myofibroblast formation. Previous reports have shown that TRPC6 proteins are up-regulated by ET-1 and platelet-derived growth factor in pulmonary vascular smooth muscle cells (34, 35). These reports are consistent with the present results. We first demonstrated that G{alpha}12/13 mediate ET-1-induced up-regulation of TRPC6 mRNAs and proteins in cardiac fibroblasts. We previously reported that G{alpha}12/13 mediate Ang II-induced JNK activation through Rac-dependent ROS production (17, 25). Since it was reported that c-Jun, one of the target molecules of JNK, is required for agonist-induced up-regulation of TRPC6 proteins in pulmonary vascular smooth muscle cells (36), G{alpha}12/13-mediated JNK activation may participate in the increased expression of TRPC6 in cardiac fibroblasts. Furthermore, we clearly demonstrated that G{alpha}12/13 mediate ET-1-induced myofibroblast formation, since p115-RGS completely suppressed the increase in {alpha}-SMA expression and [3H]proline incorporation by ET-1 treatment (Fig. 6). Since ET-1-induced myofibroblast formation was also suppressed by DN Rac and SP600125 (Fig. 6C), the G{alpha}12/13-Rac-JNK signaling pathway may be involved in ET-1-induced myofibroblast formation. However, ET-1-induced fibrotic responses were inhibited by up-regulation of TRPC6 expression and resultant NFAT activation through the G{alpha}12/13-mediated pathway (Fig. 6). Therefore, ET-1 treatment caused fibrotic responses through the Rac-JNK pathway, and at the same time ET-1 treatment negatively regulated fibrotic responses through NFAT activation by the increased expression of TRPC (Fig. 10). The concentration of ET-1 to induce maximal {alpha}-SMA expression was about 1 nM, which was about 10 times lower than that of ET-1 to induce maximal expression of TRPC6 proteins (Figs. 3B and 6B). Thus, cardiac fibroblasts may turn on their own protective system against excess progression of fibrotic responses when the cells are exposed to a high concentration of ET-1.

Although we cannot explain in detail the mechanism of how NFAT inhibits JNK activation (Fig. 8A) and TRPC6 expression (supplemental Fig. 2) induced by ET-1 stimulation, there are a couple of possibilities. Since DPI and SP600125 suppressed TRPC6 promoter activity by ET-1 treatment, the ROS-JNK pathway plays an important role in TRPC6 expression (Fig. 3E). Expression of CA NFAT suppressed ET-1-stimulated Rac activation, and we previously reported that Rac mediates JNK activation (17, 25). Therefore, NFAT inhibits TRPC6 expression through inhibition of the Rac-JNK pathway. We can exclude down-regulation of ET receptors as a mechanism of NFAT-mediated inhibition of TRPC6 expression, since the ET-1-induced ERK activation was not affected by CA NFAT and TRPC6 WT (Fig. 8B). Since NFAT acts as a transcriptional factor, one possibility is that some factor(s) produced by NFAT activation negatively regulates JNK activity through inhibition of Rac activation in cardiac fibroblasts. For example, NFAT is reported to regulate the expression levels of BMP2 (bone morphogenetic protein 2), an antifibrotic factor (38). Further studies are required for understanding the mechanism of NFAT-dependent inhibition of JNK activation.


Figure 8
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  		FIGURE 8.
Inhibition of ET-1-induced Rac activation, ROS production, and JNK activation by TRPC6-mediated NFAT activation. A and B, effects of CA NFAT, CysA, and TRPC6 WT on ET-induced JNK activation (A) and ERK activation (B). Forty-eight h after infection with adenovirus coding one of LacZ, CA NFAT, and TRPC6 WT or 24 h after treatment with CysA (0.5 µg/ml), cells were treated with ET-1 (100 nM) for 20 min. C, effects of TRPC6 WT and TRPC6 siRNA (1609) on ET-1-induced ROS production. D, effects of CA NFAT and TRPC6 WT on ET-1-induced Rac activation. Cells were treated with ET-1 (100 nM) for 2 min. *, p < 0.05 versus nontreatment (no ET-1) of control or LacZ-expressing cells. #, p < 0.05 versus ET-1 treatment of control or LacZ-expressing cells.

 
It has been reported that TRPC6 channel activity is positively regulated by various stimuli, such as DAG (28), tyrosine phosphorylation (29), and mechanical stress (39). We found that the increase in [Ca2+]i of CA G{alpha}13-expressing cells was completely inhibited by PP2 but not by U73122 [GenBank] (Fig. 2D), suggesting that Src tyrosine kinase but not PLC-generated DAG is involved in CA G{alpha}13 -mediated Ca2+ influx. It also suggests that CA G{alpha}13 does not activate PLC in cardiac fibroblasts. We also found that AG1478 inhibits CA G{alpha}13-induced Src activation (Fig. 2F), suggesting the involvement of EGFR kinase in G{alpha}13-mediated Src activation. Our results suggest that CA G{alpha}13-mediated Src activation is involved in CA G{alpha}13 -induced Ca2+ influx.


Figure 9
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  		FIGURE 9.
Involvement of TRPC6 up-regulation in ET-1-induced hypertrophic responses of cardiomyocytes. A, concentration-dependent increase in TRPC6 protein expression by ET-1 stimulation. Cells were treated with ET-1 for 48 h. TRPC6 proteins were detected with Western blot. B, effects of p115-RGS on ET-1-induced increase in TRPC6 protein expression. Twenty-four h after infection, cells were treated with ET-1 (100 nM) for 48 h. C, concentration-dependent increase in BNP-luciferase activity by ET-1 stimulation. Cells were treated with ET-1 for 24 h, and luciferase activity was determined. D–F, effects of TRPC6 siRNAs on ET-1-induced NFAT activation (D), actin reorganization (E), and protein synthesis (F). Forty-eight h after transfection with siRNAs, cells were treated with ET-1 for 24 h (B and C) or 8 h (D). *, p < 0.05 versus ET-1 treatment of control or LacZ-expressing cells.

 
We also demonstrated that Ca2+ influx-induced increase in [Ca2+]i evoked by ionomycin is not enhanced by TRPC6 over-expression (Fig. 4C). This result suggests that TRPC6 does not participate in store-operated Ca2+ entry in cardiac fibroblasts. TRPC1 is often referred to as a SOC, but it is unknown whether TRPC1 works as a true SOC, because this can also be activated by DAG and membrane stretch (19, 40). In addition to TRPC1, it has been recently reported that STIM1, located predominantly in the endoplasmic reticulum, acts as a Ca2+ sensor for store-operated Ca2+ entry (41, 42) and that Orai1/CRACM1 is identified as the Ca2+ release-activated Ca2+ channel gene (43,44). Since TRPC1, STIM1, and Orai1/CRACM1 are ubiquitously expressed, these molecules may participate in store-operated Ca2+ entry in cardiac fibroblasts.


Figure 10
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  		FIGURE 10.
Scheme for two opposite roles of G{alpha}12/13 in cardiac fibroblasts. Stimulation of ETAR by ET-1 treatment activates G{alpha}12/13, which mediate ET-1-induced TRPC6 up-regulation and myofibroblast formation through Rac-dependent ROS production and JNK activation. On the other hand, G{alpha}12/13-mediated Ca2+ influx through Src-dependent activation of up-regulated TRPC6 induces NFAT activation. The activated NFAT inhibits ET-1-induced Rac activation, ROS production, and JNK activation through unknown mechanism(s). NFAT may work as a negative feedback regulator against ET-1-induced myofibroblast formation in cardiac fibroblasts.

 
A variety of evidence has implicated that the sustained increase in [Ca2+]i is involved in the pathogenesis of heart failure (45). For example, up-regulation of TRPC proteins are associated with the reduction of sarcoplasmic reticulum Ca2+-ATPase (20), suggesting the importance of TRPC in remodeling of the Ca2+ signaling mechanism in cardiac myocytes. Furthermore, it has been recently reported that diverse signals for cardiac hypertrophy stimulate the expression of TRPC channels and increase NFAT activity (21). Other groups demonstrated that the cardiac myocytes-specific expression of TRPC6 or CA calcineurin (upstream activator of NFAT) induces cardiac hypertrophy, and NFAT is activated in pathological but not physiological hypertrophy (46–48). We found in this study that G{alpha}12/13-mediated up-regulation of TRPC6 protein participates in ET-1-induced cardiomyocyte hypertrophy (Fig. 9). Thus, the TRPC-NFAT signaling pathway in myocytes plays an essential role in cardiac hypertrophy. In contrast to cardiac myocytes, we demonstrated in cardiac fibroblasts that up-regulation of TRPC6 expression and resultant activation of NFAT inhibit myofibroblast formation and synthesis of ECM components induced by ET-1 treatment. Therefore, we suggest that TRPC6 and TRPC6-mediated NFAT activation in cardiac fibroblasts work as negative regulators against cardiac fibrosis. Many lines of evidence have indicated that TRPC channels represent novel pharmacologic targets, since NFAT is activated by Ca2+ influx though TRPC channels in pathological hypertrophy. However, the role of TRPC channels in cardiac fibroblasts and cardiac fibrosis does not support this idea, since the TRPC6-NFAT signaling pathway works as a negative regulator of fibrotic responses. Inhibitor of TRPC channels may worsen the fibrotic process accompanied with hypertrophy. Since cardiac fibrosis leads to myocardial stiffness and diastolic dysfunction, future in vivo work will be required for understanding the role of TRPC proteins in the cardiac function and development of cardiac fibrosis. The present findings also indicate that activators of TRPC channel-NFAT signaling or downstream signaling components are novel anti-fibrotic drugs.

In summary, we have demonstrated a signal transduction pathway of ET-1-induced up-regulation of TRPC6 expression and NFAT activation. As the sustained increase in [Ca2+]i through TRPC6 activates NFAT, the G{alpha}12/13-mediated TRPC6 expression and NFAT activation may act as a negative feedback regulator against ET-1-mediated fibrotic responses.


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M. N. and H. K.); from the Ministry of Health, Labour, and Welfare of Japan and the National Institute of Biomedical Innovation (MF-16) (to Y. S.); from the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), the Naito Foundation, and Takeda Science Foundation (to M. N.); and from the Astellas Foundation for Research on Metabolic Disorders and the Kimura Memorial Heart Foundation Research Grant for 2006 (to H. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. Back

1 To whom correspondence should be addressed. Tel./Fax: 81-92-642-6884; E-mail: kurose{at}phar.kyushu-u.ac.jp.

2 The abbreviations used are: ECM, extracellular matrix; Ang, angiotensin; CA, constitutively active; CysA, cyclosporine A; DAG, diacylglycerol; DN, dominant negative; DPI, diphenyleneiodonium; EGFR, epidermal growth factor receptor; ET-1, endothelin-1; GFP, green fluorescent protein; JNK, c-Jun NH2-terminal kinase; NFAT, nuclear factor of activated T cells; p115-RGS, RGS domain of p115Rho guanine nucleotide exchange factor; PLC, phospholipase C; RACC, receptor-activated Ca2+ channel; RGS, regulator of G-protein signaling; ROS, reactive oxygen species; RT, reverse transcription; siRNA, small interfering RNA; {alpha}-SMA, {alpha}-smooth muscle actin; SOCs, store-operated Ca2+ channels; TRPC, canonical transient receptor potential; WT, wild type; ERK, extracellular signal-regulated kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; OAG, a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol. Back

3 The sequence was obtained from Ref. 37 (GenBankTM accession number NW_047798). Back


   ACKNOWLEDGMENTS
 
We thank Yuichi Nagamatsu for real time RT-PCR measurement during the early stage of this study and Yusuke Narita for production of CA NFAT adenovirus.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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