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Department of Pharmacology, Fukushima Medical University School of Medicine, Fukushima Japan (S.M., I.M., J.K.), Department of Pharmacology, Fukuoka University School of Medicine, Fukuoka, Japan (T.I.); and Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Science, Kyushu University, Fukuoka, Japan (H.K.)
Received March 30, 2004; accepted May 6, 2005
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Abstract |
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). Several studies have shown that the expression of NCX1 mRNA is up-regulated in animal models of cardiac hypertrophy and heart failure (Pogwizd et al., 1999) and in human end-stage heart failure (Hasenfuss et al., 1999). On the other hand, NCX1 expression was down-regulated in some other heart failure models (Yao et al., 1998). However, the mechanism regulating NCX gene expression is still unknown.
HMG-CoA reductase inhibitors, known as statins, are widely used clinically to prevent coronary heart disease and systemic atherosclerosis. It is generally assumed that the beneficial effects of statins result from the inhibition of cholesterol synthesis (Goldstein and Brown, 1990; Levine et al., 1995). However, by inhibiting HMG-CoA reductase and thereby mevalonic acid (Mev) synthesis, statins also have other effects through prevention of the synthesis of various isoprenoids, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) (Grunler et al., 1994), both of which are important lipid attachments for the post-translational modification of a variety of small GTP-binding proteins, such as Ras and Rho GTPases (Casey, 1995). Ras and Rho family proteins coordinately regulate various cellular processes such as differentiation, proliferation, and apoptosis (Van Aelst and D'Souza-Schorey, 1997; Scita et al., 2000).
We postulated that fluvastatin (Flv), one of the lipophilic statins, may modulate NCX1 mRNA expression in cardiac myocytes by affecting small GTP-binding proteins. In the present study, we investigated the effects of Flv on NCX1 mRNA levels in H9c2 cardiomyoblasts.
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Materials and Methods |
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-lysophosphatidylcholine, palmitoyl (LPC), and 5,6-dichlorobenzimidazol riboside (DRB) were purchased from Sigma-Aldrich (St. Louis, MO). Cell Culture. H9c2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. Cells were plated onto 35-mm dishes at 104 cells/cm2.
RT-PCR. Total RNA was extracted from H9c2 cells by the acid-guanidine thiocyanate/phenol/chloroform method (Chomczynski and Sacchi, 1987). First-strand cDNA primed by random hexamers was prepared from total RNA (1 µg) using Moloney murine leukemia virus reverse transcriptase in a final reaction volume of 20 µl. The cDNA was diluted 5-fold with water and was used as a template for PCR analysis. Primers used to amplify NCX1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were designed on the basis of the published cDNA sequences as described previously (Watanabe et al., 2002). The primers for RhoA and RhoB were GTG GTA AGA CAT GCT TGC TC (sense, 47–66) and GAT GAT GGG CAC ATT TGG AC (antisense, 339–320), and ATG TGC TTC TCG GTA GAC AG (sense, 526–545) and AGA AAA GGA CGC TCA GGA AC (antisense, 1133–1114), respectively (GenBank accession numbers AY026068
[GenBank]
and NM022542). PCR was carried out with different amplification cycles as described previously (Ohkubo et al., 2000). The predicted lengths of PCR products for NCX1, GAPDH, RhoA, and RhoB were 302, 500, 293, and 608 base pairs, respectively. The PCR products were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. The images were analyzed using NIH Image software. To quantify mRNA levels, we first determined optimal PCR conditions with different amplification cycles. With our experimental conditions, PCR products of NCX1 and GAPDH with control H9c2 cells were increased during 22 to 28 and 16 to 22 cycles, respectively. Therefore we used 24 and 26 amplification cycles for NCX1 and 18 and 20 cycles for GAPDH. Under these conditions, the amounts of PCR products correlated well with the mRNA levels (0.1–5 µg) analyzed. In some experiments, mRNA levels were quantified using a real-time PCR system (LightCycler; Roche Diagnostics GmbH, Mannheim, Germany) with LightCycler FastStart DNA Master SYBR Green I kit. All results shown by semiquantitative RT-PCR analysis were confirmed by the real-time PCR system.
Adenovirus Infection. Recombinant adenovirus pAdTrack-CMV, pAdTrack-CMV containing cDNA encoding C3 toxin, constitutive active mutant of ras (RasV12), and constitutive active mutant of RhoA (RhoAV14) were prepared as described previously (Arai et al., 2003; Tashiro et al., 2003). Twenty-four hours before infection H9C2 cells were plated onto collagen-coated 50-ml (25-cm2) culture flask at 5 x 105 cells/cm2. Adenovirus infection was done for 2 h at a multiplicity of infection of 100 in 0.5 ml of culture medium. The cells were then cultured for 24 h in DMEM containing 10% fetal bovine serum. Under these conditions, almost 100% of the cells expressed recombinant proteins as determined by visualization of green fluorescent protein. Cells were then treated with Flv or LPC for an additional 24 h. RNA was extracted, and mRNA levels of NCX1 and GAPDH were determined by RT-PCR as described above.
Western Blotting. H9c2 cells were incubated with or without flv or LPC for 24 h and then washed twice with phosphate-buffered saline. Cells were then harvested with lysis buffer (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1 mM dithiothreitol, 10 µg/ml antipain, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Whole-cell lysates were sonicated and centrifuged at 500g for 5 min at 4°C. The supernatants (12.5 µg of protein) were subjected to Western blot analysis using anti-NCX1 rabbit polyclonal antibody as described previously (Iwamoto et al., 1998). For measurement of RhoB protein, whole-cell lysates after removing nuclei were centrifuged at 25,000g for 10 min at 4°C. The supernatant was collected and used as cytosolic fraction. The pellet was washed twice with the lysis buffer by centrifuging at 25,000g for 10 min at 4°C. The pellet was resuspended with the lysis buffer and used as crude membrane fraction. Membrane and cytosolic fractions were then subjected to Western blot analysis using anti-RhoB rabbit polyclonal antibody (sc-180; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The intensities of immunoreactive protein bands were analyzed using NIH Image software.
siRNA Reagents and Transfection. siRNA sequences were selected by siDirect (RNAi, Tokyo, Japan), which is a web-based online software system for selecting highly effective siRNA sequences (Naito et al., 2004). RNA oligonucleotides were synthesized by Proligo Biochemie GmbH (Hamburg, Germany). Sequence CUA UCG AUC GGA CGU CGU ACG was used as a control siRNA. siRNA against RhoA and RhoB corresponded to coding regions +255 to +270 and +1005 to +1026, respectively (GenBank accession numbers, AY026068
[GenBank]
and NM022542). H9c2 cells were plated in 6-cm culture dishes at a density of 4 x 104 cells/dish. Transfection of siRNA (100 pmol/well) was carried out using Polyfect (QIAGEN, Valencia, CA) according to the manufacturer's protocol. After transfection, cells were cultured for 24 to 72 h and subjected to RNA extraction and Western blot analysis. Changes in mRNA levels of NCX1, RhoA, RhoB, and GAPDH were determined by a real-time PCR system and semiquantitative RT-PCR analysis as described above. Changes in protein levels of NCX1 and RhoB were determined by Western blot analysis as described above.
Treatment of Rat with Flv In Vivo. Six-week-old male Wister rats weighing 107 to 123 g were divided into two groups (control and Flv groups). The rat was housed individually in one cage and fed a grained diet, and it was daily given approximately 10% of body weight. The Flv group was fed the same diet, but it contained 1 mg/g Flv. The Flv group reveived approximately 60 mg/kg Flv each day. After 1 week, rats were sacrificed, and total RNA was extracted from heart as described above. NCX1 mRNA levels were examined by RT-PCR using the real-time PCR system.
Data Analysis. Statistical analyses of the data were performed by the paired Student's t tests for two data comparison and one-way analysis of variance with Dunnett's two-tailed test for multiple data comparison. P values of less than 0.05 were considered to be statistically significant.
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Involvement of Small G-Protein Signaling Pathway in the Regulation of NCX1 mRNA Level. Because geranylgeranylation and farnesylation is required for activation of small G-proteins, we examined the effect of the expression of constitutive active mutant of RhoA, RhoAV14, and that of Ras, RasV12. Infection of recombinant adenovirus encoding RhoAV14 or RasV12 did not alter NCX1 mRNA. In contrast, expression of botulinum C3 exoenzyme, a specific inhibitor of Rho family protein (Sekine et al., 1989; Paterson et al., 1990; Ridley and Hall, 1992), significantly decreased NCX1 mRNA (Fig. 3). Infection with the control adenovirus did not affect NCX mRNA. These results suggest that a Rho family protein other than RhoA is involved in the regulation of NCX1 mRNA.
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Effect of LPC on NCX1-mRNA Level. We showed previously that in vascular endothelial cells (Yokoyama et al., 2002) and guinea pig cardiac ventricular cells (Li et al., 2002), LPC stimulates nonselective cation currents via activation of the Rho-signaling pathway. Therefore, we examined whether LPC-induced Rho activation causes the accumulation of NCX1 mRNA. Treatment of H9c2 cells with 5 µM LPC for 24 h significantly increased NCX1 mRNA. This effect of LPC was inhibited by the expression of C3 exoenzyme (Fig. 4). The LPC-induced increase in NCX1 mRNA was blocked by Flv (data not shown), which is consistent with our previous data obtained with cardiac ventricular myocytes and endothelial cells. These results support the view that NCX1 mRNA levels are regulated by a Rho signaling pathway.
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). Western blot analysis with RhoB-specific antibody revealed that H9c2 cells possess RhoB protein (Fig. 6A). Incubating cells with 5 µM Flv for 24 h resulted in a decrease to 44% of the control in membrane-associated RhoB protein level and in an increase by 432% in cytosolic RhoB protein level (Fig. 6B), suggesting that Flv blocks isoprenylation of RhoB and its function (Stamatakis et al., 2002). On the other hand, incubating cells with 5 µM LPC for 24 h resulted in an increase to 157% of the control in membrane-associated RhoB protein and in a decrease to 76% of the control in cytosolic RhoB protein (Fig. 6B). These results suggest that RhoB could be a candidate regulator of NCX1 mRNA.
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Effect of RhoB siRNA on NCX1 mRNA and Protein in H9c2 Cells. To directly examine the role of RhoB in NCX1 mRNA expression, we used RhoB-specific siRNA to inhibit endogenous RhoB function and compared with the effect of RhoA-specific siRNA in H9c2 cells. In preliminary experiments, we tested several different transfection methods to load siRNAs in H9c2 cells, and the highest transfection efficacy, which was still less than 30%, was obtained with the Polyfect transfection reagent. Even with such relatively low transfection efficacy, RT-PCR analyses revealed that siRNA for RhoA and RhoB selectively decreased their own target mRNA (Fig. 7A). Negative control siRNA had no effect on gene expressions of RhoA, RhoB, or GAPDH. Transfection with RhoB siRNA for 24 h decreased NCX1 mRNA significantly to 71.3 ± 0.02% of the control cells (n = 6, Fig. 7B). In contrast, RhoA siRNA did not have a significant effect on NCX1 mRNA. Western blot analysis revealed that siRNA for RhoB selectively decreased its own target protein (Fig. 8A). Transfection with RhoB siRNA for 72 h decreased NCX1 protein significantly to 50.2 ± 0.05% of the control cells (n = 3, Fig. 8B). In contrast, negative control and RhoA siRNAs did not have a significant effect on RhoB and NCX1 protein. These results suggest that RhoB, but not RhoA, is involved in the regulation of NCX1 mRNA and protein.
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Effect of Flv on Rat Cardiac NCX1 mRNA Level In Vivo. To examine whether Flv affects the cardiac NCX1 mRNA levels in vivo, rats were fed for 1 week with either control or Flv (1 mg/g)-containing diet. Before the experiment, the body weight did not differ between control and Flv groups. After 1 week, the body weight of the control group increased from 116 ± 2.7 to 128 ± 2.3 g (n = 5). In contrast, the body weight of the Flv group did not increase at all (from 112 ± 1.6 to 111 ± 2.2 g, n = 5). The total amount of diet taken by Flv group was slightly less than that in the control group (approximately 90% of control), and the average daily dose of Flv was approximately 60 mg/kg/day. As shown in Fig. 10, cardiac NCX1 mRNA levels in Flv-fed rats were significantly decreased to 65% of that in control rats.
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Discussion |
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; Levine et al., 1995). However, several recent studies demonstrated that, in addition to lowering cholesterol, statins prevent the synthesis of various isoprenoids, such as FPP and GGPP (Grunler et al., 1994). These isoprenoids are important lipid attachments for post-translational modification of a variety of small GTP-binding proteins, such as Ras and Rho small G-proteins (Casey, 1995). Flv down-regulation of NCX1 mRNA was prevented by GGPP and FPP, suggesting that small G-proteins are targets for the effect of Flv. This was supported by the observation that the down-regulation of NCX1 mRNA by Flv was mimicked by the expression of C3 toxin, a specific inhibitor of Rho family G-proteins. These results indicate that the level of NCX1 mRNA is regulated by the Rho-signaling pathway in H9c2 cells.
Geranylgeranylation is required for membrane association of activated Rho (Casey, 1995). Many studies demonstrated that unlike GGPP, FPP usually does not reverse statin inhibition of Rho-mediated responses (Laufs and Liao, 1998; Wassmann et al., 2001; Yokoyama et al., 2002). However, in this study, both GGPP and FPP prevented the down-regulation of NCX1 mRNA by Flv. FPP is required for isoprenylation of Ras. However, Ras-mediated signaling seems to have a minor role, if any, in the regulation of NCX1 mRNA levels because the expression of the constitutive active mutant of Ras did not modulate NCX1 mRNA levels. In addition, we could not observe a significant alteration of NCX1 mRNA by the expression of the constitutive active mutant of RhoA. These results, together with the effects of C3 exoenzyme, suggest that Rho family proteins other than RhoA may be responsible for the regulation of NCX1 mRNA levels.
The involvement of certain Rho family proteins in the regulation of NCX1 mRNA levels was also suggested by experiments with LPC. We showed previously that in vascular endothelial cells (Yokoyama et al., 2002) and guinea pig cardiac ventricular cells (Li et al., 2002), LPC induces nonselective cation currents via activation of the Rho-signaling pathway. In the present study, we found that LPC up-regulated the NCX1 mRNA in H9c2 cells. This effect of LPC was inhibited by the expression of C3 exoenzyme, suggesting that an activation of Rho family G-protein is involved in the LPC-induced increase in NCX1 mRNA.
Among several C3 exoenzyme substrates, RhoB is known to be isoprenylated not only by GGPP but also by FPP (Adamson et al., 1992). We identified RhoB protein in H9c2 cells (Fig. 6). In addition, membrane-associated RhoB was decreased by Flv treatment but was increased by LPC, reflecting that RhoB function is inhibited by the depletion of isoprenoids by Flv but is stimulated by LPC in H9c2 cells. These results may suggest that RhoB is a regulator of NCX1 mRNA level. However, recent studies demonstrated that differential isoprenoid modification of RhoB altered its function and cellular localization. Thus, farnesylated RhoB localizes to the plasma membrane (Lebowitz et al., 1995) and induces growth promotion (Lebowitz et al., 1997), whereas geranylgeranylated RhoB localizes to multivesicular late endosomes (Lebowitz et al., 1995) and has apoptotic and growth-inhibitory effects (Du and Prendergast, 1999). Nevertheless, possible involvement of RhoB in the regulation of NCX1 mRNA was further supported by experiments with RhoB siRNA. We found that NCX1 mRNA and protein in H9c2 cells were decreased by transfection of siRNA specific for RhoB but not for RhoA. When H9c2 cells were treated with Flv, we observed not only a decrease in membrane-associated RhoB but also a marked increase in cytosolic RhoB. This is consistent with the interpretation that isoprenylation is necessary for rapid turnover of RhoB (Stamatakis et al., 2002). The cytosolic RhoB accumulated by Flv in H9c2 cells must be the nonprenylated inactive form, which cannot be used for activation by GTP loading. Therefore, the regulation of NCX1 mRNA levels may be a downstream event of activated RhoB. Although the molecular mechanisms remain to be elucidated, our data suggest that RhoB plays an important role in the regulation of NCX1 mRNA levels.
Rho-mediated alterations of mRNA levels, based on statin effects, have been demonstrated with various proteins. For example, statins up-regulate the mRNA expressions of endothelial nitric-oxide synthase (Laufs and Liao, 1998) and tissue plasminogen activator (Essig et al., 1998) but down-regulate the expression of plasminogen activator inhibitor-1 (Ishibashi et al., 2002) and AT1 angiotensin II receptor (Wassmann et al., 2001). The present results show for the first time that NCX1 mRNA levels are regulated by Rho family G-protein RhoB. Furthermore, the results obtained with DRB-treated cells suggest that the down-regulation of NCX1 mRNA by Flv is caused by a decrease in NCX1 mRNA stability, although a direct effect of Flv on NCX1 transcription remains to be investigated. Increased mRNA stability by the Rho-signaling pathway was demonstrated with the angiotensin AT1 receptor (Wassmann et al., 2001). In addition, an opposite effect of Rho (decrease in mRNA stability) was described with endothelial nitric-oxide synthase (Laufs and Liao, 1998), suggesting that the Rho-signaling pathway may be involved in the regulation of mRNA stability. Further studies are necessary to clarify how the RhoB-signaling pathway regulates NCX1 mRNA stability.
A regulatory role of the Rho-signaling pathway in NCX1 expression provides a new perspective for understanding the mechanism underlying pathophysiological changes in cardiac NCX1 expression. In many animal models of cardiac hypertrophy and heart failure, both NCX expression and activity are increased (Hatem et al., 1994; Studer et al., 1994; Hasenfuss et al., 1999; Pogwizd et al., 1999; Gomez et al., 2002). The myocardial Rho-signaling pathway is activated by various extracellular signaling molecules, such as angiotensin II (Aoki et al., 1998) and endothelin (Kuwahara et al., 1999), which are also considered to be factors mediating cardiac hypertrophy and heart failure. In this study, we found that long-term treatment of rat with Flv in vivo significantly decreased cardiac NCX1 mRNA. Although the dose of Flv used in this study is higher than that of clinical medication, this result indicates that the effects of Flv on NCX1 mRNA observed in H9c2 cells occur in the cardiac tissue in vivo.
In conclusion, we have found that the inhibition of HMG-CoA reductase in H9c2 cells leads to a decrease in NCX1 mRNA stability through an inhibition of the RhoB signaling pathway. Our data also indicate that activation of Rho increases NCX1 expression. These observations show for the first time that Mev-derived metabolites play regulatory roles in the expression of NCX1. These results may have important implications for understanding the mechanism of altered NCX1 expression in cardiac hypertrophy and heart failure.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: NCX, Na+/Ca2+ exchanger; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; Mev, mevalonic acid; Flv, fluvastatin; LPC, lysophosphatidylcholine; Y27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; DRB, 5,6-dichlorobenzimidazol riboside; siRNA, short interfering RNA.
Address correspondence to: Dr. Junko Kimura, Department of Pharmacology, Fukushima Medical University School of Medicine, Fukushima, 960-1295, Japan. E-mail: jkimura{at}fmu.ac.jp
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