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Role of RhoA Inactivation in Reduced Cell Proliferation of Human Airway Smooth Muscle by Simvastatin

Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan

Correspondence and requests for reprints should be addressed to Hiroaki Kume, M.D., Ph.D., Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: hkume{at}med.nagoya-u.ac.jp

	Abstract

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Enhanced proliferation of smooth muscle cells contributes to airway remodeling of bronchial asthma. Recently, statins, inhibitors of 3-hydroxy-3-methylglutaryl–coenzyme A reductase, have been shown to inhibit proliferation of both vascular and airway smooth muscle cells independently of lowering cholesterol. However, the mechanisms remain to be elucidated. The purpose of this study was to determine molecular processes by which statins inhibit proliferation of human bronchial smooth muscle cells. Simvastatin (0.1–1.0 µM) significantly inhibited cell proliferation and DNA synthesis induced by FBS in a concentration-dependent manner. The inhibitory effects of simvastatin were antagonized by mevalonate and geranylgeranylpyrophosphate, whereas the effects were not affected by squalene and farnesylpyrophosphate. The antiproliferative effects of simvastatin were mimicked by GGTI-286, a geranylgeranyltransferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho, and Y-27632, an inhibitor of Rho-kinase, a target protein of RhoA. Western blot analysis showed that the level of membrane localization of RhoA (active Rho) was markedly increased by FBS, and that the level of active RhoA increased by FBS was reduced by simvastatin. Moreover, the inhibitory effect of simvastatin on FBS-induced RhoA activation was also antagonized by geranylgeranylpyrophosphate, but not by farnesylpyrophosphate. Because these isoprenoids are required for prenylation of small G proteins RhoA and Ras, respectively, the present results demonstrate that an inhibition in airway smooth muscle cell proliferation by simvastatin is due to prevention of geranylgeranylation of RhoA, not farnesylation of Ras. Therefore, statins may have therapeutic potential for prohibiting airway remodeling in bronchial asthma.

Key Words: asthma • airway remodeling • 3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitors (statins) • Rho-kinase • isoprenoids

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   Cell proliferation of airway smooth muscle is suppressed by RhoA/Rho-kinase inactivation regulated by the mevalonate pathways in cholesterol synthesis. Statins may be a novel therapeutic agent for prohibiting airway remodeling via these processes.

 
Remodeling in the airway walls leads to an augmentation in airway hyperresponsiveness and to a reduction in reversibility of airflow limitation, and is responsible for the severity of bronchial asthma (1, 2). The increased airway smooth muscle (ASM) mass, which is due to the enhanced proliferation (hyperplasia) and hypertrophy of ASM cells, may be implicated in the processes concerning the reconstruction of airway structure (3–8). ASM cells proliferate in response to three groups of mitogens; for example: (1) polypeptide growth factors (platelet-derived growth factor [PDGF], epidermal growth factor [EGF], etc.); (2) contractile agents (thromboxanes, leukotriene D4, etc.); and (3) proinflammatory cytokines (IL-1, TNF-, etc.). To regulate cell cycle entry and DNA synthesis, these mitogens activate Ras, a 21-kD guanosine triphosphatase (GTPase), which interacts with the major downstream effectors, such as extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) (7, 9–11). Moreover, as another signal transduction pathway, PI3K activates Rho family GTPase via Akt.

It has been revealed that statins, inhibitors of 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase, have pleiotropic effects, and cause an inhibition in cell growth in vascular smooth muscle cells, independent of lowering the concentration of plasma cholesterol (12, 13). The antiproliferative activity of statins may be mediated by suppressing the synthesis of isoprenoids, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) (14), which are involved in membrane localization and activation of small GTPases Ras and Rho families, respectively (13, 15, 16). Therefore, these small GTPases may play a key role in the signal transduction pathways for regulation of cell proliferation induced by mitogens (17). A previous report has suggested that isoprenoids are related to cell proliferation in ASM cells (18). However, little is currently known about intracellular mechanisms underlying the anti-proliferative effects of statins.

Although various mitogens are proposed as major contributors (19), it is still unclear which mitogen is related to the hyperplasia involved in the pathophysiology of bronchial asthma. Serum may cause proliferation of ASM cells (6, 20, 21), because it contains various growth factors, such as PDGF, EGF, and thrombin. Because plasma leakage from the microvasculature occurs in the airway walls in response to inflammation of bronchial asthma (22), exudated serum may play an important role as a mitogen in the airway remodeling in this disease. Therefore, an investigation in to the relationship between excessive exposure to serum and cell proliferation in ASM has lead to the development of new concepts about airway remodeling.

This study was designed to determine molecular mechanisms underlying the inhibitory effects of simvastatin, a commonly prescribed statin for therapy of hypercholesterolemia, on cell proliferation induced by serum using cultured human ASM cells. Moreover, we examined, in detail, the involvement of isoprenoid/small GTPase processes in the functional antagonism between simvastatin and serum as it relates to regulation of ASM cell proliferation.

	MATERIALS AND METHODS

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Cell Culture
Primary cultures of normal human bronchial smooth muscle (BSM) cells from multiple donors were obtained from Cambrex (Walkersville, MD). The cells were maintained in culture medium (SmBM; Cambrex), containing 5% FBS, human recombinant EGF (1 ng/ml), insulin (10 mg/ml), human recombinant fibroblast growth factor (2 ng/ml), gentamicin (50 mg/ml), and amphotericin B (0.05 mg/ml) (SmGM-2 Bulletkit; Cambrex) in an atmosphere of 5% CO₂ and 95% air at 37°C. Cells at the eighth–ninth passage were used for subsequent experiments. Cell viability was determined by morphology, trypan blue exclusion, and Annexin V-Biotin Apoptosis Detection kit (Medical and Biological Laboratories, Nagoya, Japan), according to the manufacturer's instructions.

Cell Proliferation Assay
BSM cells were subcultured at a density of 1.0 x 10⁴ cells/well in 12-well plates, and were incubated in 1 ml Dulbecco's modified Eagle's medium/F-12 culture medium (Invitrogen, Carlsbad, CA), containing 10% FBS or 0.1% FBS with growth factors (10 ng/ml PDGF, 10 ng/ml EGF, and 1 U/ml thrombin) and antibiotics–antimycotic (100 U/ml penicillin, 100 µl/ml streptomycin, 250 ng/ml amphotericin B; Invitrogen). Culture medium and reagents were replaced every day. Cell counts were evaluated after 24 h, 3 d, 5 d, and 7 d by using the mitochondria-dependent reduction of 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (WST-1) to formazan (WST-1 assay). WST-1 assay was performed by using cell proliferation reagent WST-1 (Roche, Basel, Switzerland). WST-1 (10%/well) was added to each well, and cells were incubated for 1 h in an atmosphere of 5% CO₂ and 95% air at 37°C. The amount of formazan in the obtained solution was estimated by measuring with a multiwell spectrophotometer (Wallac 1420 ARVOsx; PerkinElmer Japan, Tokyo, Japan) at a wavelength of 450 nm with a reference at 650 nm.

DNA Synthesis Assay
BSM cells in a subconfluent state were starved in serum-free medium for 24 h, and cells were exposed to each agent for 24 h. After the supernatant was removed, cells were washed twice with PBS and then stimulated with the medium containing 10% FBS or 0.1% FBS with growth factors (10 ng/ml PDGF, 10 ng/ml EGF, and 1 U/ml thrombin) and antibiotics. For experiments using simvastatin, isoprenoids, and inhibitors, simvastatin, isoprenoids, and C3 exoenzyme were added 24 h before stimulation with mitogen; Y-27632 and GGTI-286 were added 30 min before. After 24 h stimulation, 5-Bromo-2'-deoxy-uridine (BrdU) solution (BrdU Labeling and Detection kit 3; Roche) in serum-free medium was added to each well for 4 h. After the serum-free medium containing BrdU solution was removed, cells were fixed and incubated with anti-BrdU antibody conjugated with peroxidase for 30 min. After washing three times with PBS, peroxidase substrate solution was added to each well and incubated for 30 min. Optical density was measured by a scanning multiwell spectrophotometer at a wavelength of 405 nm with a reference at 490 nm.

Membrane Association Assay—Preparation of Membrane Fraction
BSM cells were grown to confluence in 100-mm dishes and then placed in serum-free medium for 24 h. Cells were stimulated with 10% FBS for 15 min. Simvastatin and FPP or GGPP were added 24 h before stimulation. After stimulation by FBS, cells were washed with ice-cold PBS. The membrane-enriched fractions for the assay of RhoA, Rac1, and Cdc42 membrane localization were prepared using the ProteoExtract subcellular proteome extraction kit (EMD Biosciences, San Diego, CA), according to the manufacturer's instructions.

Rho Family Activation Assay—Rhotekin and Pak1 Pull-Down Assay
RhoA and Rac1/Cdc42 activation were determined by Rhotekin and Pak1 pull-down assay, respectively. BSM cells were grown to confluence in 100-mm dishes and then placed in serum-free medium for 24 h. Cells were stimulated with 10% FBS for 15 min. Simvastatin and FPP or GGPP were added 24 h before stimulation. After stimulation by FBS, cells were washed with ice-cold PBS. The GTP-RhoA-, GTP-Rac1-, and GTP-Cdc42-enriched lysates were prepared using the EZ-Detect Rho, EZ-Detect Rac1, and EZ-Detect Cdc42 Activation kits (Pierce Biotechnology, Rockford, IL), respectively, according to the manufacturer's instructions.

Phosphorylation of ERK1/2 and Akt/Protein Kinase B Assay
Phosphorylation of ERK1/2 and Akt/protein kinase B (PKB) were evaluated by using specific antibodies for each protein. BSM cells were grown to confluence in 60-mm dishes and then placed in serum-free medium for 24 h. Cells were stimulated with 10% FBS for 15 min. Simvastatin was added 24 h before stimulation. After stimulation by FBS, cells were washed with ice-cold PBS. Whole cellular lysates were prepared with lysis buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 5% 2-mercaptoethanol).

Western Blot Analysis
Protein contents of cellular lysates were measured by using a Bio-Rad protein assay reagent kit (Bio-Rad, Hercules, CA). Equal amounts of lysates, adjusted to protein content, were resolved by SDS-PAGE using a 4–20% linear gradient running gel (Daiichi Pure Chemicals, Tokyo, Japan). Proteins were transferred to nitrocellulose membrane, and the membrane was incubated at room temperature in PBS for 1 h. Immunoblotting was performed using antibodies against RhoA (RhoA [26C4]: sc-418; Santa Cruz Biotechnology, Santa Cruz, CA), phospho-ERK1/2 (Anti-ACTIVE MAPK pAb; Promega, Madison, WI), ERK1/2 (Anti-ERK1/2 pAb; Promega), phospho-Akt/PKB (Anti-pS⁴⁷³ Akt pAb; Promega), and Akt/PKB (Akt antibody; Cell Signaling Technology, Beverly, MA). Immunodetection was accomplished using a sheep anti-mouse secondary antibody or donkey anti-rabbit secondary antibody and the Enhanced Chemiluminescence kit (Amersham Biosciences, Piscataway, NJ). The intensity was quantified by using Scion image software (Scion Corporation, Frederick, MD).

Cell Permeabilization
Cell permeabilization with streptolysin O was done according to previous reports (23). BSM cells were washed twice with permeabilization buffer (120 mM KCl, 30 mM NaCl, 10 mM Hepes [pH 7.2], 10 mM EGTA, 10 mM MgCl₂). C3 exoenzyme (10–30 ng/ml), 5 mM dithiothreitol, 1 mM ATP, and 0.5 U/ml streptolysin O were added to the buffer for permeabilization and incubated for 2 min at 37°C. Resealing was achieved by the addition of Dulbecco's modified Eagle's medium/F-12 medium containing 10% FBS. Control cells were treated with permeabilization buffer in the absence of C3 exoenzyme.

Reagents
Simvastatin and Y-27632 were obtained from Wako (Osaka, Japan). Mevalonate, FPP, GGPP, squalene, and thrombin were obtained from Sigma (St. Louis, MO). Human recombinant PDGF-BB and human recombinant EGF were from Invitrogen. GGTI-286 was from EMD Biosciences. C3 exoenzyme was from List Biological Laboratories, Incorporated (Campbell, CA).

Statistical Analysis
The effects of agents on cell proliferation and DNA synthesis were expressed taking response to medium in the presence of 10% FBS (control condition) as 100% in the WST-1 assay and BrdU uptake assay. Data are presented as mean ± SD, and compared using ANOVA followed by Bonferroni-Dunn post hoc test. P < 0.05 was considered significant.

	RESULTS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Proliferation of BSM Cells Induced by Mitogens
When human BSM cells were exposed to 10 ng/ml PDGF, 10 ng/ml EGF, and 1 U/ml thrombin with 0.1% FBS for 7 d, these growth factors caused cell proliferation in a time-dependent manner. However, cell numbers augmented by these growth factors were much lower than those augmented by 10% FBS (P < 0.01). After exposure to 10 ng/ml PDGF, 10 ng/ml EGF, 1 U/ml thrombin with 0.1% FBS, and 10% FBS for an equivalent period, the absorbance values were increased by 1.7- (± 0.1), 1.5- (± 0.1), 1.7- (± 0.1), and 3.7- (± 0.3) fold compared with that value after exposure to 0.1% FBS alone for an equivalent period (Figure 1A).

View larger version (17K): [in this window] [in a new window]   Figure 1. Cell proliferation induced by various mitogens. The effects of 0.1% FBS (open circles), 0.1% FBS with growth factors (closed triangles, PDGF; closed inverted triangles, EGF; closed squares, thrombin), and 10% FBS (closed circles) on cell number (A) and DNA synthesis (B). Data are expressed as means ± SD from three experiments. Each experimental condition was tested in triplicate. Responses to these mitogens were expressed taking cell number and DNA synthesis by 0.1% FBS as 100%. **P < 0.01 versus the condition containing 0.1% FBS.

Inhibitory Effects of Simvastatin on Proliferation and DNA Synthesis
When the human BSM cells were exposed to 10% FBS with simvastatin for 7 d, application of simvastatin (0.1–1 µM) caused a significant inhibition in augmented cell number induced by 10% FBS in a concentration-dependent manner (Figure 2A). The values of percent absorbance for 10% FBS with 0.1, 0.3, and 1.0 µM simvastatin for 7 d were 91.0 (± 3.6%; n = 3; P < 0.01), 56.0 (± 2.1%; n = 3; P < 0.01), and 34.6 (± 2.0%; n = 3; P < 0.01), respectively (Figure 2A). Simvastatin had no effect on cell viability, as determined by trypan blue exclusion and apoptosis assay (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 2. The inhibitory effects of simvastatin (closed circles, 0.1 µM; closed triangles, 0.3 µM; closed inverted triangle, 1.0 µM) against augmented cell number (A) and DNA synthesis (B) induced by 10% FBS (open circles). The antiproliferative effects against augmented cell number induced by growth factors (C). Data are expressed as means ± SD from three experiments. Each experimental condition was tested in triplicate. Response to simvastatin was expressed taking cell number and DNA synthesis induced by 10% FBS as 100%. *P < 0.05 and **P < 0.01 versus control.

Simvastatin tended to inhibit augmented cell number induced by growth factors; however, the inhibitory effects on growth factors were not dramatic compared with the effect on FBS (n = 3; not significant; Figure 2C).

Regulation of Antiproliferative Effects of Simvastatin by Intermediate Isoprenoids
Human BSM cells were exposed to 10% FBS with 1 µM simvastatin in the presence of 300 µM mevalonate and 100 µM squalene for 7 d. When mevalonate was applied, the inhibitory effect of simvastatin was markedly attenuated. The value of percent absorbance for FBS with simvastatin was increased to 90.1 ± 6.6% (n = 3) in the presence of 300 µM mevalonate (P < 0.01, Figure 3A); the effect of mevalonate was concentration-dependent (data not shown). In contrast, when 100 µM squalene was applied, the inhibitory effect of simvastatin was not affected. That value for FBS with simvastatin was 44.7 ± 4.1% (n = 3) in the presence of 100 µM squalene (not significant; Figure 3A). The effect was not affected by squalene in higher concentrations (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 3. Regulation of simvastatin-induced antiproliferative effects by isoprenoid intermediates. The effects of mevalonate and squalene (A) on inhibiting the FBS-augmented cell number. The effects of FPP and GGPP on inhibiting the augmented cell number (B) and DNA synthesis (C) by FBS. Data are expressed as means ± SD from three experiments. Each experimental condition was tested in triplicate. Responses to these isoprenoid intermediates are expressed as shown in Figure 2. **P < 0.01 versus the value for 10% FBS; P < 0.01 versus the value for 10% FBS with 1 µM simvastatin.

The inhibitory effect of simvastatin against DNA synthesis induced by exposure to FBS for 24 h was also reversed by an equimolar concentration of GGPP. The value of percent BrdU uptake for 10% FBS with 1 µM simvastatin was increased to 89.6 ± 5.0% (n = 3) in the presence of GGPP (P < 0.01; Figure 3C). In contrast, the inhibitory effect of simvastatin was not affected by an equimolar concentration of FPP. That value for FBS with simvastatin was 67.2 ± 4.9% (n = 3) in the presence of FPP (not significant; Figure 3C).

The Inhibitory Effects of Rho/Rho-Kinase Inhibitors on Cell Proliferation and DNA Synthesis
Human BSM cells were exposed to 10% FBS with GGTI-286 (1–10 µM), Y-27632 (1–30 µM), and C3 exoenzyme (10–30 ng/ml) for 7 d. Augmented cell number by FBS was markedly attenuated in a concentration-dependent manner by addition to these agents. The values of percent absorbance for FBS with 1, 3, and 10 µM GGTI-286 were 100.3 ± 0.8 (n = 3; not significant), 81.4 ± 3.1 (n = 3; P < 0.05), and 62.0 ± 1.3% (n = 3; P < 0.01), respectively (Figure 4A). The values for FBS with 1, 10, and 30 µM Y-27632 were 95.8 ± 6.1 (n = 3; not significant), 89.0 ± 4.3 (n = 3; P < 0.05), and 65.3 ± 1.5% (n = 3; P < 0.01), respectively (Figure 4B). The values for FBS with 10 and 30 nM C3 exoenzyme were 85.0 ± 8.3 (n = 3; not significant) and 65.2 ± 1.9% (n = 3; P < 0.01), respectively (Figure 4C).

View larger version (19K): [in this window] [in a new window]   Figure 4. The inhibitory effects of GGTI-286 (A), Y-27632 (B), and C3 exoenzyme (C) on the cell number augmented by 10% FBS. The effects of GGTI-286, Y-27632, and C3 exoenzyme on the augmented DNA synthesis by 10% FBS (D). Data are expressed as means ± SD from three experiments. Each experimental condition was tested in triplicate. Responses to these agents are expressed as shown in Figure 2. *P < 0.05; **P < 0.01.

Regulation of Membrane Localization of RhoA by Simvastatin
After 15-min exposure of human BSM cells to 10% FBS, percent association of RhoA with plasma membrane was increased by 3-fold compared with that after exposure to FBS-free medium (Figure 5A). Application of 1 µM simvastatin caused a marked reduction in the membrane-associated RhoA induced by FBS. The value of percent RhoA associated with membrane for FBS was decreased to 57.3 ± 5.4% by simvastatin (n = 3; P < 0.05; Figure 5A). However, in the presence of 30 µM GGPP, the inhibitory effect of simvastatin against membrane-associated RhoA for FBS was markedly suppressed. The value of percent membrane-associated RhoA for FBS with simvastatin was increased to 75.2 ± 1.6% by GGPP (n = 3; P < 0.05; Figure 5A). In contrast, in the presence of 30 µM FPP, the inhibitory effect of simvastatin against FBS-induced RhoA association with membrane was not affected. The value of percent RhoA associated with membrane was 58.1 ± 2.6% (n = 3) under this experimental condition (not significant; Figure 5A).

View larger version (31K): [in this window] [in a new window]   Figure 5. (A) Regulation of membrane-associated RhoA by 10% FBS in the absence and presence of simvastatin with and without FPP and GGPP. Membrane localization of RhoA was determined by immunoblotting membrane fractions with RhoA antibody. Response to these agents was expressed taking membrane-associated RhoA for 10% FBS as 100%. (B) RhoA activation by 10% FBS in the absence and presence of simvastatin with and without FPP and GGPP. RhoA activation was evaluated by rate of GTP-Rho (active form) and total RhoA. Response to these agents was expressed taking membrane-associated RhoA for 10% FBS as 100%. (C) Effect of simvastatin on phosphorylation of ERK1/2 and Akt/PKB. Phosphorylation of ERK1/2 and Akt/PKB were determined by immunoblotting with phospho-ERK1/2 and phospho-Akt/PKB antibodies, respectively. Response to these agents was expressed taking phosphorylated ERK1/2 and Akt/PKB for 10% FBS as 100%. Data are expressed as mean ± SD from three experiments. **P < 0.01; *P < 0.05 versus control. P < 0.05 versus the value for 10% FBS with 1 µM simvastatin.

After a 15-min exposure of human BSM cells to 10% FBS, the percentage of the rate of GTP-RhoA and total RhoA was increased by 5-fold compared with that after exposure to FBS-free medium (Figure 5B). Application of 1 µM simvastatin markedly reduced RhoA activation induced by FBS. The value of percent GTP-RhoA for FBS was decreased to 29.5 ± 12.2% by simvastatin (n = 3; P < 0.05; Figure 5B). However, in the presence of 30 µM GGPP, the inhibitory effect of simvastatin against activated RhoA by FBS was markedly suppressed. The value of percent GTP- RhoA for FBS with simvastatin was increased to 94.6 ± 13.2% by GGPP (n = 3; P < 0.05; Figure 5B). In contrast, in the presence of 30 µM FPP, the inhibitory effect of simvastatin against FBS-induced RhoA activation was not affected. The value of percent GTP-RhoA was 35.0 ± 17.7% (n = 3) under this experimental condition (not significant; Figure 5B).

Effects of Simvastatin on Phosphorylation of ERK1/2 and Akt/PKB
Exposure to 10% FBS caused a marked increase in the level of phosphorylation of ERK1/2 and Akt/PKB. However, in the presence of 1 µM simvastatin, FBS-induced phosphorylation of ERK1/2 and Akt/PKB was not affected. The values of percent phosphorylated ERK1/2 and Akt/PKB for FBS with simvastatin were 102.3 ± 2.1% and 97.5 ± 7.2%, respectively (n = 3; not significant; Figure 5C).

Effects of Simvastatin on Membrane Localization and Activation of Rac1 and Cdc42
After a 15-min exposure of human BSM cells to 10% FBS, there were no significant increases in percent association of Rac1 or Cdc42 with plasma membrane (n = 3; not significant; Figure 6A). Moreover, application of 1 µM simvastatin did not affect Rac1 and Cdc42 membrane localization (n = 3; not significant; Figure 6A).

View larger version (41K): [in this window] [in a new window]   Figure 6. Effects of simvastatin on membrane localization (A) and activation (B) of Rac1 and Cdc42. Membrane localization of Rac1 and Cdc42 were determined by immunoblotting on membrane fractions with their specific antibody. Rac1 and Cdc42 activation were evaluated by rate of GTP formation (active form) and total of these proteins. Response to these agents was expressed taking the values of membrane-associated Rac1 and Cdc42 for 10% FBS as 100%. Data are expressed as mean ± SD from three experiments.

	DISCUSSION

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Our results have demonstrated that simvastatin inhibits cell proliferation and DNA synthesis induced by FBS in human BSM cells independently of inhibiting synthesis of cholesterol, and that these inhibitory effects are mediated by geranylgeranylation of Rho, but not by farnesylation of Ras. When human BSM cells were exposed to PDGF, EGF, and thrombin with a lower concentration of FBS for 7 d, cell proliferation, as measured by cell number mediated by these growth factors, was not so dramatically increased (Figure 1A). The numbers of cells associated with these growth factors were increased by 1.5-fold compared with that of the control, whereas the value for 10% FBS was increased by fourfold. However, in DNA synthesis assay, after 24-h exposure to these growth factors and 10% FBS, BrdU uptake by these growth factors was not so markedly different from that value for 10% FBS (Figure 1B). It is unknown whether there was a discrepancy between the WST-1 cell proliferation assay and DNA synthesis in terms of growth factors such as PDGF, EGF, and thrombin. Incubation with a single agent of growth factors may be insufficient to maintain cell proliferation fully over an extended period (7 d). This phenomenon may be mediated by involvement of cell death, as a previous report has shown that serum prevents apoptosis, whereas these growth factors do not (24). As shown here, a high concentration (10%) of FBS was more potent than these growth factors in the enhancement of cell proliferation of human BSM cells (Figure 1A). Therefore, we believe that serum is suitable for a long-term cell culture, such as a mitogen culture.

As shown in Figure 2A, simvastatin inhibited cell proliferation induced by 10% FBS in a concentration-dependent manner, similar to previously reported results (18). To confirm this antiproliferative effect of simvastatin, DNA synthesis assay was performed. Increased BrdU uptake by 10% FBS was also reduced by simvastatin in a concentration-dependent manner (Figure 2B). Moreover, simvastatin, GGTI-286, Y-27632, and C3 exoenzyme did not effect cell viability, as determined by trypan blue exclusion and apoptosis assay (data not shown). These data indicate that simvastatin inhibits cell proliferation by reducing DNA synthesis, not by promoting apoptosis. Previously, it has been suggested that statins inhibit the proliferation of ASM cells mediated by inhibiting synthesis of isoprenoids, such as mevalonate and geranylgeraniol (18). However the key isoprenoids and molecule concerning this phenomenon were not clarified in detail. In the present study, involvement of FPP and GGPP was examined to determine molecular mechanisms underlying the antiproliferative action of statins. As shown in Figures 3A–3C, inhibitory effects of simvastatin on cell proliferation and DNA synthesis were reversed by mevalonate and GGPP, but were not affected by squalene and FPP. The antiproliferative action of statins is related to GGPP, and independent of reducing cholesterol synthesis (Figure 7). These results are consistent with those of previous reports using vascular smooth muscle cells and atrial fibroblasts (25, 26).

View larger version (13K): [in this window] [in a new window]   Figure 7. Schematic representation of the antiproliferative effect of simvastatin via regulation of the mevalonate pathway and Rho/Rho-kinase activation on human ASM cells.

As shown in Figures 3B and 3C, FPP, which is needed for farnesylation of Ras, is not related to the functional antagonism between FBS and simvastatin in the cell proliferation of the human BSM. However, it is generally considered that ERK and PI3K/Akt, which are regulated by Ras, are involved in the postreceptor processes of mitogenic signals from receptors with intrinsic protein tyrosine kinase and G protein–coupled receptors related to ASM cell proliferation (7, 27–30). To confirm involvement of farnesylation of Ras in the antiproliferative action by statins, phosphorylation of ERK1/2 and Akt/PKB were examined (Figure 5C). Exposure to 10% FBS caused an increase in activity of ERK1/2 and Akt/PKB; however, application of simvastatin did not reduce the augmented activity of these kinases by FBS. These results support the idea that the antiproliferative action of statins is not due to inhibition of Ras farnesylation processes in ASM cells.

However, the lack of inhibition of Akt/PKB phosphorylation may not rule out a role for Rac1 and Cdc42, downstream effectors of Akt/PKB (7, 29). Rac1 and Cdc42 are known to have multiple downstream effectors including PAK, JNK, p38, SRF, and NF-B (7, 31). Moreover, previous reports have shown that bovine ASM cell growth induced by PDGF is not reduced by Rho-kinase inhibitor, and is predominantly regulated by Rac1 and Cdc42, not by RhoA (32, 33). Rac1 and Cdc42 are also geranylgeranylated small GTPases; therefore, they may be inhibited by simvastatin directly, not via reducing Ras activation. Next, we examined whether Rac1 and Cdc42 are related to FBS-induced ASM cell proliferation. Although these proteins are generally considered to play an important role in cell proliferation, membrane association and activated forms of Rac1 and Cdc42 were not increased by FBS (Figures 6A and 6B). Moreover, they were not affected by simvastatin (Figures 6A and 6B). In contrast, as shown in this study, RhoA was activated by FBS, and activated RhoA was attenuated by simvastatin (Figure 5B). A previous report indicated that an inhibition in RhoA, Rac, or Cdc42 leads to an activation of the other two proteins (34). In the present experiments, RhoA activated by FBS may cause an inhibition in activity of Rac1 and Cdc42. Moreover, recent reports have shown that the relationship between simvastatin and Rac1 is complicated. Activation of Rac1 is attenuated by simvastatin in heart muscle (35), whereas Rac1 is activated by simvastatin in endothelium (36). Further studies are needed to clarify the involvement of Rac1 and Cdc42 in the inhibitory effects of simvastatin on cell proliferation by FBS.

In ASM, the signal pathway of RhoA/Rho-kinase plays functional roles in responsiveness to muscarinic receptor agonists (37), reduced responsiveness to -adrenergic receptor agonists after exposure to a lipid mediator (38), cytoskeletal reorganization (39), and migration (40), which are implicated in the pathophysiology of bronchial asthma. In addition to these effects, it has been revealed that the RhoA/Rho-kinase pathways are essential for cell proliferation by serum, and that statins suppress ASM cell proliferation mediated by inhibiting geranylgeranylation of Rho. RhoA may be a target molecule for therapy of airway hyperreactivity, -adrenergic desensitization, and airway remodeling. Our results provide evidence that statins may have therapeutic potential for prohibiting airway remodeling of bronchial asthma via inactivation of Rho.

	Footnotes

 
This work was supported by Grant-in-Aid 17590785 (H.K.) from the Ministry of Education, Science, Sports, and Culture of Japan.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0034OC on July 20, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 26, 2006

Accepted in final form July 12, 2006

	References

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 

1. Dunnill MS, Massarella GR, Anderson JA. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis, and in emphysema. Thorax 1969;24:176–179.[Abstract/Free Full Text]
2. Elias JA. Airway remodeling in asthma. J Clin Invest 1999;104:1001–1006.[Medline]
3. James AL, Pare PD, Hogg JC. The mechanics of airway narrowing in asthma. Am Rev Respir Dis 1989;139:242–246.[Medline]
4. Ebina M, Takahashi T, Chiba T, Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma: a 3-D morphometric study. Am Rev Respir Dis 1993;148:720–726.[Medline]
5. Bousquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM. Asthma: from bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med 2000;161:1720–1745.[Free Full Text]
6. Johnson PR, Roth M, Tamm M, Hughes M, Ge Q, King G, Burgess JK, Black J. Airway smooth muscle cell proliferation is increased in asthma. Am J Respir Crit Care Med 2001;164:474–477.[Abstract/Free Full Text]
7. Hirst SJ, Martin JG, Bonacci JV, Chan V, Fixman ED, Hamid QA, Herszberg B, Lavoie JP, McVicker CG, Moir LM, et al. Proliferative aspects of airway smooth muscle. J Allergy Clin Immunol 2004;114:S2–S17.[CrossRef][Medline]
8. Woodruff PG, Dolganov GM, Ferrando RE, Donnelly S, Hays SR, Solberg OD, Carter R, Wong HH, Cadbury PS, Fahy JV. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med 2004;169:1001–1006.[Abstract/Free Full Text]
9. Panettieri RA. Cellular and molecular mechanisms regulating airway smooth muscle proliferation and cell adhesion molecule expression. Am J Respir Crit Care Med 1998;158:S133–S140.[Medline]
10. Ammit AJ, Panettieri RA Jr. Invited review: the circle of life: cell cycle regulation in airway smooth muscle. J Appl Physiol 2001;91:1431–1437.[Abstract/Free Full Text]
11. Citro S, Ravasi S, Rovati GE, Capra V. Thromboxane prostanoid receptor signals through Gi protein to rapidly activate extracellular signal-regulated kinase in human airways. Am J Respir Cell Mol Biol 2005; 32:326–333.[Abstract/Free Full Text]
12. Sparrow CP, Burton CA, Hernandez M, Mundt S, Hassing H, Patel S, Rosa R, Hermanowski-Vosatka A, Wang PR, Zhang D, et al. Simvastatin has anti-inflammatory and antiatherosclerotic activities independent of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol 2001;21:115–121.[Abstract/Free Full Text]
13. Liao JK, Laufs U. Pleiotropic effects of statins. Annu Rev Pharmacol Toxicol 2005;45:89–118.[CrossRef][Medline]
14. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990;343:425–430.[CrossRef][Medline]
15. Casey PJ. Protein lipidation in cell signaling. Science 1995;268:221–225.[Abstract/Free Full Text]
16. Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev 2001;81:153–208.[Abstract/Free Full Text]
17. Tomlinson PR, Wilson JW, Stewart AG. Salbutamol inhibits the proliferation of human airway smooth muscle cells grown in culture: relationship to elevated cAMP levels. Biochem Pharmacol 1995;49:1809–1819.[CrossRef][Medline]
18. Vigano T, Hernandez A, Corsini A, Granata A, Belloni P, Fumagalli R, Paoletti R, Folco G. Mevalonate pathway and isoprenoids regulate human bronchial myocyte proliferation. Eur J Pharmacol 1995;291: 201–203.[CrossRef][Medline]
19. Hershenson MB, Abe MK. Mitogen-activated signaling in airway smooth muscle: a central role for Ras. Am J Respir Cell Mol Biol 1999;21:651–654.[Free Full Text]
20. Johnson PR, Armour CL, Carey D, Black JL. Heparin and PGE2 inhibit DNA synthesis in human airway smooth muscle cells in culture. Am J Physiol 1995;269:L514–L519.
21. Lanner MC, Raper M, Pratt WM, Rhoades RA. Heterotrimeric G proteins and the platelet-derived growth factor receptor- contribute to hypoxic proliferation of smooth muscle cells. Am J Respir Cell Mol Biol 2005;33:412–419.[Abstract/Free Full Text]
22. Shiels IA, Bowler SD, Taylor SM. Airway smooth muscle proliferation in asthma: the potential of vascular leakage to contribute to pathogenesis. Med Hypotheses 1995;45:37–40.[CrossRef][Medline]
23. Chellaiah MA, Soga N, Swanson S, McAllister S, Alvarez U, Wang D, Dowdy SF, Hruska KA. Rho-A is critical for osteoclast podosome organization, motility, and bone resorption. J Biol Chem 2000;275: 11993–12002.[Abstract/Free Full Text]
24. Freyer AM, Johnson SR, Hall IP. Effects of growth factors and extracellular matrix on survival of human airway smooth muscle cells. Am J Respir Cell Mol Biol 2001;25:569–576.[Abstract/Free Full Text]
25. Laufs U, Marra D, Node K, Liao JK. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1). J Biol Chem 1999;274:21926–21931.[Abstract/Free Full Text]
26. Porter KE, Turner NA, O'Regan DJ, Balmforth AJ, Ball SG. Simvastatin reduces human atrial myofibroblast proliferation independently of cholesterol lowering via inhibition of RhoA. Cardiovasc Res 2004; 61:745–755.[Abstract/Free Full Text]
27. Page K, Li J, Hershenson MB. Platelet-derived growth factor stimulation of mitogen-activated protein kinases and cyclin D1 promoter activity in cultured airway smooth-muscle cells: role of Ras. Am J Respir Cell Mol Biol 1999;20:1294–1302.[Abstract/Free Full Text]
28. Krymskaya VP, Penn RB, Orsini MJ, Scott PH, Plevin RJ, Walker TR, Eszterhas AJ, Amrani Y, Chilvers ER, Panettieri RA Jr. Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation. Am J Physiol Lung Cell Mol Physiol 1999; 277:L65–L78.[Abstract/Free Full Text]
29. Hirst SJ, Walker TR, Chilvers ER. Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma. Eur Respir J 2000;16:159–177.[Abstract]
30. Zhou L, Hershenson MB. Mitogenic signaling pathways in airway smooth muscle. Respir Physiol Neurobiol 2003;137:295–308.[CrossRef][Medline]
31. Westwick JK, Lambert QE, Clark GJ, Symons M, Van Aelst L, Pestell RG, Der CJ. Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways. Mol Cell Biol 1997;17:1324–1335.[Abstract]
32. Bauerfeld CP, Hershenson MB, Page K. Cdc42, but not RhoA, regulates cyclin D1 expression in bovine tracheal myocytes. Am J Physiol Lung Cell Mol Physiol 2001;280:L974–L982.[Abstract/Free Full Text]
33. Gosens R, Schaafsma D, Meurs H, Zaagsma J, Nelemans SA. Role of Rho-kinase in maintaining airway smooth muscle contractile phenotype. Eur J Pharmacol 2004;483:71–78.[CrossRef][Medline]
34. Moorman JP, Luu D, Wickham J, Bobak DA, Hahn CS. A balance of signaling by Rho family small GTPases RhoA, Rac1 and Cdc42 coordinates cytoskeletal morphology but not cell survival. Oncogene 1999;18:47–57.[CrossRef][Medline]
35. Laufs U, Kilter H, Konkol C, Wassmann S, Bohm M, Nickenig G. Impact of HMG CoA reductase inhibition on small GTPases in the heart. Cardiovasc Res 2002;53:911–920.[Abstract/Free Full Text]
36. Jacobson JR, Dudek SM, Birukov KG, Ye SQ, Grigoryev DN, Girgis RE, Garcia JG. Cytoskeletal activation and altered gene expression in endothelial barrier regulation by simvastatin. Am J Respir Cell Mol Biol 2004;30:662–670.[Abstract/Free Full Text]
37. Ito S, Kume H, Honjo H, Katoh H, Kodama I, Yamaki K, Hayashi H. Possible involvement of Rho kinase in Ca²⁺ sensitization and mobilization by MCh in tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol 2001;280:L1218–L1224.[Abstract/Free Full Text]
38. Kume H, Ito S, Ito Y, Yamaki K. Role of lysophosphatidylcholine in the desensitization of -adrenergic receptors by Ca²⁺ sensitization in tracheal smooth muscle. Am J Respir Cell Mol Biol 2001;25:291–298.[Abstract/Free Full Text]
39. Hirshman CA, Emala CW. Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho. Am J Physiol 1999;277: L653–L661.
40. Parameswaran K, Cox G, Radford K, Janssen LJ, Sehmi R, O'Byrne PM. Cysteinyl leukotrienes promote human airway smooth muscle migration. Am J Respir Crit Care Med 2002;166:738–742.[Abstract/Free Full Text]

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