Important role for Rac1 in regulating reactive oxygen species generation and pulmonary arterial smooth muscle cell growth
Sandip Patil,1,*
Melisa Bunderson,2,*
Jason Wilham,2 and
Stephen M. Black2,3
1Department of Pediatrics, Northwestern University, Chicago, Illinois 60611; and 2Department of Biomedical and Pharmaceutical Sciences and 3International Heart Institute of Montana, University of Montana, Missoula, Montana 59802
Submitted 10 November 2003
; accepted in final form 4 August 2004
 |
ABSTRACT
|
---|
Vascular NADPH oxidases have been shown to be a major source of reactive oxygen species (ROS). Recent studies have also implicated ROS in the proliferation of vascular smooth muscle cells. However, the components required for activation of the NADPH oxidase complex have not been clearly elucidated. Here we demonstrate that ROS generation in ovine pulmonary arterial smooth muscle cells (PASMCs) requires the activation of Rac1, implicating this protein as an important subunit of the NADPH oxidase complex. Our results, using a geranylgeranyl transferase inhibitor (GGTI-287), demonstrated a dose-dependent inhibition of Rac1 activity and ROS production. This was associated with an inhibition of PASMC proliferation with an arrest at G2/M. The inhibition of Rac1 by GGTI-287 led us to more specifically target Rac1 to investigate its role in the generation of ROS and cellular proliferation. To accomplish this, we utilized a dominant negative Rac1 (N17Rac1) and a constitutively active Rac1 (V12Rac1). These two forms of Rac1 were transiently expressed in PASMCs using adenovirus-mediated gene transfer. N17Rac1 expression resulted in decreased cellular Rac1 activity, whereas V12Rac1 infection showed increased activity. Compared with controls, the V12Rac1-expressing cells had higher levels of ROS production and increased proliferation, whereas the N17Rac1-expressing cells had decreased ROS generation and proliferation and cell cycle arrest at G2/M. However, the inhibition of cell growth produced by N17Rac1 overexpression could be overcome if cells were co-incubated with the Cu,Zn superoxide dismutase inhibitor DETC. These results indicate the importance of Rac1 in ROS generation and proliferation of vascular smooth muscle cells.
vascular reduced nicotinamide adenine dinucleotide phosphate oxidase
THE NADPH OXIDASE COMPLEX is a multisubunit enzyme that has been widely studied with respect to its role in the innate immune response by phagocytic cells (4). Activation of NADPH oxidase results in generation of reactive oxygen species (ROS), which are important in the host defense against infection (4). More recently, ROS have been demonstrated to be important modulators of vascular cell function. ROS have been shown to be generated in response to a number of ligands such as angiotensin II (10), epidermal growth factor (37), platelet-derived growth factor (21), basic fibroblast growth factor (16), phenylephrine (38), endothelin-1 (5, 34, 41), and serotonin (18, 19). In addition, the proliferation of smooth muscle cells (SMCs) has been strongly linked to the generation of ROS such as superoxide and H2O2 (5, 34, 41). It has been demonstrated that the major source of ROS in vascular cells is the NADPH oxidase complex (3, 23, 26, 28).
The Rac1 enzyme has been shown to play a critical role in the assembly and activation of NADPH oxidase in the neutrophil form of the enzyme complex (1, 13). However, the role of Rac1 in the vascular NADPH oxidase system remains unresolved. Rac1 is a 21-kDa GTP binding protein belonging to the Ras superfamily. Rac1 cycles between an inactive GDP bound and, upon stimulation, an active GTP bound form. This cycle is regulated by GDP exchange factors and GTPase activating factors. Although much of the work on the regulation of NADPH oxidase signaling has been in the phagocytic context (4), recent strides have been made in the understanding of its role in cardiac and vascular cells (11). However, key differences exist between the neutrophil and the cardiovascular NADPH oxidases such as the location and the rate of generation of ROS. Thus it will be important to evaluate the role of potential subunit components in the vascular NADPH oxidase complex and contrast and compare with the phagocytic form. To begin to examine the components required for formation of an active vascular NADPH oxidase complex we chose Rac1. Amongst the structural features involved in its activation, the Rac1 protein contains a cysteine residue in its COOH terminus to which a geranylgeranyl group is attached by a thioether linkage (36). Thus geranylgeranylation is a necessary posttranslational modification that allows Rac1 to participate in the formation and activation of the NADPH oxidase complex. Proteins such as Rac1, Rho1, and Rap1A are geranylgeranylated by enzymes known as geranylgeranyl transferase I (GGTase I). GGTase I recognizes proteins that end with the motif CAAX, where X is leucine or isoleucine (6). Recently, CAAX peptidomimetics have been made that selectively inhibit the geranylgeranylation by GGTase I (31).
Here we show that pharmacological inhibition of geranylgeranylation blocks the production of ROS and cell growth in vascular SMCs. By more specifically targeting Rac1 using dominant negative and constitutively active Rac1 proteins expressed from adenoviral constructs, we have shown that the modulation of ROS generation can alter SMC proliferation, indicating the critical requirement for Rac1 in regulating both NADPH oxidase activity and subsequently SMC growth. The effect of the overexpression of the dominant negative Rac1 mutation was the inhibition of SMC proliferation at the G2/M phase of the cell cycle. This inhibition could be overcome if ROS levels were increased in the SMCs. These data suggest that the targeting of Rac1 may have therapeutic potential for the treatment of cardiovascular disease in which increased oxidative stress is thought to play an important role.
 |
MATERIALS AND METHODS
|
---|
Cell culture and adenoviral infections.
Pulmonary arterial smooth muscle cells (PASMCs) from 4-wk-old lambs were isolated by techniques previously described (41, 42). PASMC identity was confirmed as being positive for SMC actin, caldesmon, and calponin. Cells were maintained in Dulbecco's modified Eagle's medium (Mediatech) containing 10% fetal bovine serum (FBS, Hyclone) and antibiotics. Cells were maintained in a 5% CO2 atmosphere at 37°C and split 1:4 at 80% confluence.
Adenoviral constructs for constitutively active (AdV12Rac1) and dominant negative (AdN17Rac1) Rac were a gift from Dr. Toren Finkel (National Institutes of Health). The viruses were amplified in HEK-293 cells and titered. AdV12Rac1 was titered at 5 x 109 plaque-forming units (pfu)/ml, whereas AdN17Rac1 was 4 x 109 pfu/ml. For infections PASMCs were plated at 500,000 cells/well in a six-well plate and allowed to attach. Cells were then infected with the viruses at a multiplicity of infection (MOI) of 200:1 in 2% FBS. For proliferation assays, cells were split into 96-well plates (2,000 cells/well) 12 h postinfection in the presence or absence of FBS (10%).
Antibodies and reagents.
Rac1 expression was determined using an anti-Rac1 antibody (Upstate Biotechnologies, Lake Placid, NY). Rac1 activation assays were performed with the Rac1 activation assay kit (Upstate Biotechnologies). The geranylgeranyl transferase-1 inhibitor 287 (GGTI-287) was obtained from Calbiochem (La Jolla, CA). The SMC actin antibody was purchased from Sigma.
Western blotting and Rac activation assays.
For Western blotting, cells grown to 80% confluence were washed in PBS and harvested in lysis buffer (20 mmol/l Tris pH 7.5, 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 1% Triton, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l
-glycerophosphate, 1 mmol/l sodium vanadate, and 1 mg/ml leupeptin). The lysates were pelleted (10,000 g for 15 min at 4°C), and the supernatant was assayed for protein content. Lysates (100 µg) were then separated on a 12% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane (Hybond ECL, Amersham Biosciences). The membrane was blocked for 1 h at 25°C in a solution of 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). It was incubated with the appropriate antibody overnight at 4°C or according to the manufacturer's protocols. The blot was then incubated with the recommended horseradish peroxidase-conjugated secondary antibody for 1 h and was washed several times in TBS-T solution. Bands were detected by chemiluminescence (Pierce).
Rac1 activation assays were performed as described in the manufacturer's protocol. Briefly, cell lysates (200 µg) were incubated with p21-activated kinase-1 (PAK-1) agarose (5 µl/sample) for 1 h at 4°C. The beads were then washed three times with the wash buffer, resuspended in 2x Laemmli buffer, boiled, and run on a 12% SDS polyacrylamide gel. Electroblotting was performed, and the bands were detected as described above using an anti-Rac1 antibody.
Proliferation assays.
Proliferation assays were performed using the Titer 96 AQueous One Solution kit (Promega, Madison, WI), the basis of which has been shown to be a reliable alternative to [3H]thymidine incorporation (7). PASMCs were seeded at 2,000 cells/well in 96-well plates. The cells were starved for 12 h postattachment in serum-free media. Cells were then grown for a further 72 h in 100 µl of media in the presence or absence of 10% FBS or media containing 10% FBS in the presence of increasing concentrations of GGTI-287 or after adenoviral vector expressing only green fluorescence protein (AdGFP), V12Rac1, or N17Rac1 infection. After 24, 48, and 72 h, 20 µl of the assay reagent were added to each well, and the absorbance was read at 492 nm after 4 h of incubation at 37°C. In some experiments PASMCs were plated on 96-well plates at a density of 1,000 cells/well and allowed to attach overnight. Cells were then infected with the AdN17Rac1 virus at an MOI of 200:1 in 50 µl and incubated for 2 h. Then, an additional 50 µl of fresh media were added. At 24 h postinfection, the Cu-Zn superoxide dismutase (SOD1) inhibitor diethyldithiocarbamate (DETC) was added in serum-free medium for 4 h and removed, and untreated medium containing 10% FBS was reapplied. We then allowed cells to proliferate for 48 h before assaying for cell growth again using the CellTiter 96 Aqueous One Solution Proliferation Assay (Promega).
Fluorescence analysis.
PASMCs were plated at 2,000 cells/well in 96-well plates and treated as described above. Treatment was carried out for 72 h. Dihydroethidium (DHE, 20 µM; Molecular Probes) was added to the media 15 min before the end of the experiment. Cells were washed with PBS and imaged under a Nikon Eclipse TE-300 fluorescent microscope. DHE-stained cells were observed after excitation at 518 nm and emission at 605 nm. Fluorescent images were captured with a CoolSnap digital camera, and the average fluorescent intensities (to correct for differences in cell number) were quantified using Metamorph imaging software (Fryer) as we have previously described (41). Statistical analysis was carried out as detailed below (see Statistical analysis).
Terminal deoxynucleotidyltransferase dUTP nick end labeling assays.
Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assays were performed using the Dead End Fluorometric TUNEL System Kit (Promega) according to the manufacturer's protocol. Briefly, cells grown in 96-well plates were fixed in 4% formaldehyde at 4°C for 25 min. The cells were washed, treated with 0.2% Triton X, and equilibrated in equilibration buffer followed by further incubation in assay buffer containing fluorescein-12 dUTP for 60 min at 37°C. The reaction was terminated with 2x saline-sodium citrate buffer incubated at room temperature with 4',6-diamidino-2-phenylindole (DAPI, 5 µmol/l) stain in PBS for 15 min and viewed under a Nikon TE300 fluorescent microscope. DAPI is a blue fluorescent nuclear stain and was used to confirm the presence of cells that were negative for TUNEL staining. Again, fluorescent images were captured with a CoolSnap digital camera.
Flow cytometry.
Cell cycle analysis was performed with propidium iodide-stained nuclei according to the manufacturer's protocol (Becton-Dickinson). Briefly, cells were washed and reconstituted at 106 cells/ml. Cells were stained with propidium iodide stain solution for 20 min at 37°C. To this, an equal volume of 4 M NaCl salt solution was added. This was stored for 6 h before flow cytometry analysis. The distribution was determined using ModFit software.
Statistical analysis.
All values are expressed means ± SE. Band intensities from Western blot analyses were determined densitometrically using Kodak 1D software version 3.5. Comparisons were made by ANOVA. A value of P < 0.05 was considered statistically significant.
 |
RESULTS
|
---|
Initially we used GGTI-287 (05 µmol/l) to examine its effects on Rac1 expression and activity in PASMCs. Results obtained indicated that GGTI-287 had no effect on Rac1 expression at any concentration used (Fig. 1, A and B). However, treatment with GGTI-287 produced a dose-dependent inhibition of Rac1 activity with activity being reduced by 62% at 25 µmol/l (P < 0.05 vs. untreated, Fig. 1, C and D). Next, using the oxidation of DHE to ethidium as an estimate of cellular ROS generation, we examined the dose-dependent effect of GGTI-287 on cellular ROS levels. The results obtained indicate that GGTI-287 dose dependently inhibited ethidium fluorescence, indicating a key role for Rac1 in the production of ROS in PASMCs (Fig. 2).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1. Geranylgeranyl transferase inhibitor (GGTI)-287 dose dependently reduces Rac1 activity in pulmonary arterial smooth muscle cells (PASMCs). Cells were grown in DMEM containing 10% FBS in the presence and absence of GGTI-287 (025 µmol/l) for 72 h and then analyzed for Rac1 expression and activity. A: total cell lysates (100 µg) were separated on a 12% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against Rac1. In addition, blots were reprobed with smooth muscle cell (SMC) actin to ensure equal loading. Blot is representative of 3 independent experiments. B: GGTI-287 has no effect on Rac1 expression. Densitometric values for Rac1 were normalized to those obtained with SMC actin and then expressed as means ± SE; n = 3. C: total lysates (100 µg) prepared from GGTI-287-treated cells were immunoprecipitated with p21-activated kinase binding domain (PBD) agarose to isolate active Rac1. The isolated protein was then separated on a 12% denaturing polyacrylamide gel, electrophoretically transferred to Hybond, and analyzed using a specific antiserum raised against Rac1. Blot is representative for 3 independent experiments. D: densitometric values for Rac1 activation expressed as percentage of untreated cells (100%). Values are means ± SE; n = 3. *P < 0.05 compared with untreated. GGTI-287 dose dependently decreases Rac1 activity. P < 0.05 vs. previous dose.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2. Reactive oxygen species (ROS) production in PASMCs is decreased by GGTI-287. Cells were grown in DMEM containing 10% FBS in the presence and absence of GGTI-287 (025 µmol/l) for 72 h. Oxidation of dihydroethidium (DHE) to ethidium was then quantified as an estimate of cellular ROS levels using Metamorph imaging software. Ethidium fluorescence was dose dependently decreased by GGTI-287. Values are means ± SE; n = 8. *P < 0.05 compared with untreated.
|
|
Because we have previously demonstrated the importance of ROS in regulating SMC growth (41), the inhibition of ROS generation led us to determine whether there were alterations in PASMC growth. As in the previous experiments, PASMCs were exposed to GGTI-287 (050 µmol/l), and the effect on proliferation to 10% FBS was determined at 72 h (Fig. 3). At concentrations of up to 5 µmol/l, GGTI-287 had no effect on the inhibition of cell growth compared with control. However, at 25 µmol/l, GGTI-287 inhibited the proliferation of PASMCs (Fig. 3A). Correlating with the effect of GGTI-287 on Rac1 activity (Fig. 1D), this result suggests that inhibition of PASMC growth requires that Rac1 activity be less than a threshold level (
40% of untreated cells, Fig. 1D). Using GGTI-287 at 25 µmol/l, we next performed cell cycle analysis using flow cytometry to compare the cellular distribution in various phases of the cell cycle (Fig. 3B). The results obtained indicate that there was a twofold increase in the number of cells in the G2/M phase compared with untreated cells (P < 0.05 vs. untreated, Fig. 3C).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3. GGTI-287 inhibits the proliferation of PASMCs. A: PASMCs were plated in 96-well plates (2,000/well) and allowed to attach for 12 h. Cells were treated with increasing doses of GGTI-287 (025 µmol/l) in the presence of 10% FBS as a stimulus for proliferation. The number of viable cells was determined at 0 and 72 h. Values are means ± SE relative to viable cells at t = 0 h (100%); n = 8. *P < 0.05 compared with no GGTI-287. B: representative Mod-Fit data from untreated PASMCs and cells treated with 25 µmol/l GGTI-287 for 72 h. A minimum of 5,000 events were collected and analyzed. C: changes in PASMC cell cycle distribution in response to treatment with 25 µmol/l GGTI-287. GGTI-287 induces a significant accumulation in PASMCs in G2/M. Values are means ± SE; n = 3 samples. *P < 0.05 vs. untreated cells.
|
|
Our previous studies have detected an increase in programmed cell death associated with a decrease in cellular ROS (41). Thus using the TUNEL assay, we determined whether the decrease in ROS induced by GGTI-287 exposure was associated with an increase in PASMC apoptosis. However, cells treated with GGTI-287 were TUNEL negative for all doses tested (Fig. 4), although the addition of 4-(2-aminoethyl)-benzenesulfonyl fluoride, which inhibits NADPH oxidase assembly, (8) induced SMC apoptosis as we have previously described (41).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4. GGTI-287 does not induce apoptosis in PASMCs. Cells were plated in 96-well plates, allowed to attach, and then were treated or not with GGTI-287 (5 and 25 µmol/l) for 72 h in the presence of 10% FBS. Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay was performed as described in MATERIALS AND METHODS. Cells were costained with 4',6-diamidino-2-phenylindole (DAPI) to show identify all cells in each field. GGTI-287 did not induce a marked increase in TUNEL-positive nuclei relative to untreated cells. However, the presence of 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, 1 mmol/l) to prevent NADPH oxidase assembly did induce an increase in TUNEL-positive nuclei. This experiment was repeated 3 times with the same result.
|
|
GGTI-287 inhibits the activation of a number of small GTP binding proteins. Thus we next wanted to determine whether the effects we observed with this agent were specifically due to its ability to inhibit Rac1. To examine this, adenoviruses containing Rac1 mutants were used to infect PASMCs. The overexpression of the Rac1 mutants was initially confirmed by Western blot analysis (Fig. 5). Results obtained demonstrate that Rac1 levels were increased by twofold in cells infected with the dominant negative (N17Rac1) or constitutively active (V12Rac1) mutant proteins compared with uninfected cells or with cells infected with AdGFP (P < 0.05, Fig. 5, A and B). The lack of significant differences in Rac1 expression between the uninfected and AdGFP-infected cells indicates that adenoviral infection by itself did not alter Rac1 expression (Fig. 5, A and B). We next determined Rac activity. No significant differences were observed between the uninfected PASMCs and the GFP-infected cells, indicating that adenoviral infection by itself did not alter Rac activity. However, the results obtained indicate that Rac1 activity was altered by the presence or absence of serum. The levels of Rac1 activity in the uninfected PASMCs and the GFP-infected cells was significantly decreased in the absence of 10% FBS (Fig. 5, C and D). Cells infected with AdV12Rac1 (constitutively active) showed a twofold increase in Rac1 activity compared with controls in the absence of 10% FBS (Fig. 5, C and D). In the presence of 10% FBS, the AdV12Rac1-infected cells maintained the twofold increase in Rac1 activity compared with the controls in the presence of 10% FBS (P < 0.05, Fig. 5, C and D). PASMCs infected with the AdN17Rac1 (dominant negative) had similar levels of active Rac1 compared with controls in the absence of 10% FBS. However, Rac1 activity was not increased by the presence of 10% FBS (P < 0.05; Fig. 5, C and D).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5. Constitutively active and dominant negative Rac1 infections increase Rac1 expression and modulate cellular Rac1 activity. Cells were infected with V12Rac1 (AdV12Rac1) and N17Rac1 (AdN17Rac1) and harvested after 72 h. Cells were also infected with a green fluorescence protein-expressing adenoviral construct (AdGFP). AdGFP and uninfected cells were used as controls. A: total cell lysates (100 µg) were separated on a 12% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against Rac1. In addition, blots were reprobed with SMC actin to ensure equal loading. Blot is representative of 3 independent experiments. B: infection with either V12Rac1 and N17Rac1 increases cellular Rac1 expression. Densitometric values for Rac1 were normalized to those obtained with SMC actin and then expressed as means ± SE; n = 4. Infection with either AdV12Rac1 or AdN17Rac1 increases cellular Rac1 expression, but infection with AdGFP does not. *P < 0.05 compared with uninfected cells. C: cells infected with V12 Rac1, N17Rac1, or GFP along with uninfected controls were grown in the presence (+) or absence () of 10% FBS (serum). After 72 h total lysates (100 µg) were immunoprecipitated with PBD agarose to isolate active Rac1. The isolated proteins were separated on a 12% denaturing polyacrylamide gel, electrophoretically transferred to Hybond, and analyzed using a specific antiserum raised against Rac1. Blot is representative of 3 independent experiments. D: densitometric values for Rac1 activation expressed as percentage of uninfected cells in the absence of FBS (100%). Values are means ± SE; n = 3. *P < 0.05 compared with no FBS. P < 0.05 compared with uninfected cells in the presence of 10% FBS.
|
|
To determine whether the modulation of Rac1 activity altered ROS generation in PASMCs, changes in ethidium fluorescence were determined in the presence and absence of 10% FBS. Confirming the importance of ROS in regulating SMC growth, the presence of 10% FBS significantly increased ethidium fluorescence in uninfected cells and cells infected with either AdGFP or AdV12Rac1, whereas no such increase was observed in cells infected with AdN17Rac1 (Fig. 6). Further comparisons of the ethidium fluorescence intensities demonstrate that the constitutively active Rac1-infected cells (AdV12Rac1) had significantly higher levels of ethidium fluorescence compared with either uninfected cells or cells infected with AdGFP (P < 0.05, Fig. 6). In addition, no significant differences were observed between uninfected cells or cells infected with AdGFP, indicating that adenoviral infection by itself did not alter cellular ROS levels (Fig. 6).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6. Detection of ROS generation in Rac1-infected SMCs. Cells were infected with V12Rac1 (AdV12Rac1) and N17Rac1 (AdN17Rac1) and plated in a 96-well plate and grown for 72 h either in the presence (+) or absence () of 10% FBS (serum). Oxidation of DHE to ethidium was then quantified as an estimate of cellular ROS levels using Metamorph imaging software. Ethidium fluorescence was increased in the presence of 10% FBS except in N17Rac1-infected cells. V12Rac1 infection increased ethidium fluorescence in both the presence and absence of 10% FBS. GFP infection did not significantly alter cellular ROS levels compared with uninfected cells. Values are means ± SE; n = 8. *P < 0.05 compared with no FBS. P < 0.05 compared with uninfected cells in the presence of 10% FBS.
|
|
Next, we determined the effect of modulating Rac activity on PASMC proliferation. PASMCs were again infected with the Rac1 mutants or the adenovirus containing GFP, in the presence and absence of 10% FBS for 72 h. The results obtained indicated that 10% FBS increased the number of PASMCs by 75% over a 72-h time period (Fig. 7A). This growth was attenuated in serum-free conditions (Fig. 7A). Cells expressing constitutively active Rac1 (V12Rac1) showed a significantly greater increase in cell number compared with the GFP-infected cells, in both the presence and absence of 10% FBS (P < 0.05, Fig. 7A). Conversely, the PASMCs expressing the dominant negative Rac1 had significantly decreased proliferation in the presence of 10% FBS (P < 0.05, Fig. 7). Cell cycle analysis of these cells showed that the dominant negative N17Rac1 cells were accumulated in the G2/M phase of the cell cycle (Fig. 7, B and C). This is in contrast to GFP- and V12Rac1-infected cells that showed no such accumulation (Fig. 7, B and C). Again GFP infection did not alter the percentage of cells in G2/M, indicating that infection alone did not alter PASMC cell cycle (data not shown).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 7. Modulation of Rac1 activity alters PASMC growth. A: PASMCs infected or not with AdV12Rac1, AdN17Rac1, or AdGFP were plated in 96-well plates (2,000/well) and grown in the presence or absence of 10% FBS (serum). Cell proliferation was determined at 0, 24, 48, and 72 h. FBS induces a significant increase in cell number in AdGFP- and AdV12Rac1-infected cells but not in AdN17Rac1-infected cells. The t = 0 time point indicates the time at which the cells were plated in 96-well plates. Values are means ± SE; n = 8. *P < 0.05 compared with no FBS. P < 0.05 compared with AdGFP in the presence of FBS. B: representative Mod-Fit data from cells infected with AdGFP, AdV12Rac1, or AdN17 and exposed to 10% FBS for 72 h. A minimum of 5,000 events were collected and analyzed. C: changes in PASMC cell cycle distribution in response to treatment with 25 µmol/l GGTI-287. Infection with AdN17Rac1 produces a significant accumulation of cells in G2/M. Values are means ± SE; n = 3 samples. *P < 0.05 vs. AdGFP-infected cells.
|
|
Finally, we determined whether the reduction in ROS levels associated with N17Rac1 overexpression was solely responsible for the reduction in SMC growth. To accomplish this we again infected SMCs with N17Rac1. After 24 h, the cells were exposed to increasing concentrations of the SOD1 inhibitor DETC (01 µM) for 4 h. The medium was then replaced with fresh DMEM containing 10% FBS. After a further 48 h, cell viability was determined. The results obtained indicate that N17Rac1 overexpression (Fig. 8A) reduced SMC growth by 48% (Fig. 8B). However, the presence of DETC was able to overcome the inhibitory effect of N17Rac1, indicating that increasing ROS levels (Fig. 8C) is sufficient to restore SMC cell growth.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 8. Increasing cellular ROS levels overcomes the inhibitory effect of N17Rac1 on PASMC proliferation. A: PASMCs were infected or not with AdN17Rac1, and then 72 h later total cell lysates (100 µg) were separated on a 12% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against Rac1. In addition, blots were reprobed with SMC actin to ensure equal loading. Blot is representative of 3 independent experiments. B: PASMCs were plated overnight on 96-well plates at a density of 1,000 cells/well and then infected with the AdN17Rac1 virus. At 24 h postinfection, the Cu,Zn superoxide dismutase (SOD1) inhibitor diethyldithiocarbamate (DETC, 01 µM) was added in serum-free medium for 4 h, removed, and then replaced with regular medium supplemented with 10% FBS. Cell proliferation was then determined after 48 h. The presence of the N17Rac1 protein significantly reduces PASMC growth, and this effect is overcome in the presence of DETC. Values are means ± SD; n = 8. P < 0.05 vs. AdN17Rac1 in the absence of DETC. *P < 0.05 vs. uninfected in the absence of DETC. C: ethidium fluorescence was decreased in N17Rac1-infected cells compared with uninfected cells. However, ethidium fluorescence was increased in the presence of the SOD1 inhibitor DETC. Values are means ± SD; n = 8. *P < 0.05 compared uninfected. P < 0.05 compared with AdN17Rac1 in the absence of DETC.
|
|
 |
DISCUSSION
|
---|
Vascular SMC proliferation is a significant contributing factor in the onset and progression of pulmonary hypertension as well as other cardiovascular pathological states, such as atherosclerosis, restenosis, and stent placement (30). Recently ROS have been implicated as important mediators of SMC growth, and increased ROS generation has been implicated in conditions involving vascular remodeling such as hypercholesterolemia, hypertension, diabetes, and balloon injury (12, 25). Given the importance of ROS in vascular remodeling, a better understanding of how their generation is regulated will be important if better therapies to treat these pathologic conditions are to be developed. In this study, we have addressed the role of Rac1 in the generation of ROS and its role in regulating SMC proliferation. The results we have obtained indicate the critical importance of Rac1 in regulating SMC ROS generation and cell proliferation.
The exact mechanism by which Rac1 is involved in the activation of the vascular NADPH oxidase complex remains unclear. This is in contrast to phagocytotic cells, where the role of Rac1 has been elucidated. In the phagocytic context it has been shown that one of the oxidase complex subunit p67phox is sequestered by p40phox. Rac1 binding to p67phox results in the disruption of p67phox/p40phox association allowing NADPH oxidase activation (29). However, whether this is also true of vascular oxidases has not been elucidated. Indeed, until this study, it has not been clear whether Rac1 is involved in regulating ROS generation in SMCs. To begin to elucidate the role of Rac1 in regulating the vascular NADPH oxidase complex, we have used various strategies to modulate the cellular activity of Rac1. The use of GGTIs, which block a broad range of small GTP binding proteins, inhibited Rac 1 activity in PASMCs. This is in agreement with previous studies in which another GGTI, GGTI-298, was shown to inhibit Rac1 rat pulmonary aortic microvascular SMCs stimulated by FBS (31). However, this study found that GGTI treatment also resulted in a reduction in cell growth due to an increase in apoptosis. In our study, though, PASMC growth was inhibited and cells were not apoptotic, as determined by TUNEL analysis. However, in the previous study, only cells cultured in the absence of serum became apoptotic (31). As all our treatments were carried out in the presence of serum, this may explain why our studies did not induce PASMC apoptosis. However, a more likely explanation is that the GGTI-287 failed to reduce Rac activity and ROS levels below the threshold required to trigger apoptosis in our cells. We have previously shown, using SMCs isolated from the pulmonary vessels of fetal lambs, that ROS levels need to fall to <20% to induce apoptosis (39). This may also explain why GGTI-287 at 5 µmol/l does not inhibit PASMC proliferation. At this concentration GGTI-287 inhibits Rac activity and ROS levels by <50% (see Figs. 1D and 2), and we have previously shown, using SMCs isolated from the pulmonary vessels of fetal lambs, that ROS levels need to fall to <50% of control levels to produce an inhibition of cell growth (40).
Our next strategy involved the use of both constitutively active and dominant negative mutants of Rac1 to more specifically target this protein and better delineate its role in the regulation of ROS induction and SMC proliferation. We found that the dominant negative Rac1 phenotype inhibited the proliferation of SMCs. This inhibition resulted in a lower cell number compared with uninfected cells grown in the presence of 10% FBS. This correlated with N17Rac1-mediated inhibition of cellular ROS generation. In addition, increasing cellular ROS levels by inhibiting SOD1 was able to overcome the inhibitory effect of N17Rac1 overexpression, supporting our conclusion that ROS levels play an important role in regulating PASMC growth. We also found that the constitutively active V12Rac1 stimulated PASMC proliferation even in the absence of 10% FBS. This suggests that increasing ROS generation alone is sufficient to stimulate SMC growth. This is in agreement with our previous studies, in which the addition of H2O2 in the absence of serum stimulated SMC growth in cells isolated from late-gestation fetal lambs (41). V12Rac1-infected cells also exhibited higher levels of proliferation in the presence of 10% FBS than under serum-free conditions. However, our data also demonstrate that 10% FBS stimulates Rac1 activity and ROS generation. Thus the result that FBS increased proliferation in V12Rac1-infected cells may be explained by an increase in the activation of the NADPH oxidase complex by the growth factors present in FBS. Indeed several studies have indicated that growth factors can activate NADPH oxidase activity and increase ROS levels in SMCs (16, 21, 37).
Our studies have focused on the role of Rac1 in NADPH oxidase activity. However, a previous study showed that Rac2 is present in SMCs isolated from human aorta (27). This study found that Rac2 protein expression was induced by thrombin in a biphasic fashion with expression peaking at 1 h and again at 6 h after treatment (27). This study also found that in addition to changes in expression, thrombin also induced Rac2 translocation to the membrane fraction of human aortic smooth muscle cells. Accumulation of ROS was also found to occur shortly after translocation of Rac2 to the membrane fraction (27). Together our data and this previous study suggest that either Rac1 or Rac2 can be utilized to form the active NADPH oxidase complex. The Rac isoforms share 92% homology (20), and Rac1 or Rac2 overexpression has been shown to increase ROS generation in cell culture models suggestive of overlapping function (32, 35). In addition, Rac2 appears to be the principal GTPase interacting with components of the NADPH oxidase, at least in some systems (9, 14, 15). However, our results utilizing the dominant negative Rac1 completely abrogated the proliferative response of our PASMCs to 10% FBS, suggesting that Rac1 may be the dominant Rac isoform, at least in PASMCs. It is not clear whether all SMCs express both Rac1 and Rac2 or only one or the other, and further studies will be needed to address the relative expression and activity of Rac2 compared with Rac1 in SMCs.
Our cell cycle analyses using the Rac1 inhibitor GGTI-287 and the N17Rac1 mutant showed a significant accumulation of cells in the G2/M phase. There was a twofold increase in the accumulation of cells in the G2/M phase upon GGTI-287 treatment and a fivefold greater number of cells in the G2/M phase compared with the G1/S phase. Our results are in agreement with other studies investigating the effect of Rac1 inhibition in cell proliferation. For example the overexpression of N17Rac1 in fibroblasts also inhibited proliferation and G2/M progression (24). Similarly, a novel Rac protein, racE, regulates cytokinesis in Dictyostelium (17). It should be noted that several studies of the inhibition of cell proliferation by GGTIs have suggested this inhibition occurs at the G1/S phase of the cell cycle (22, 31, 33). These were found to induce the hypophosphorylation of the retinoblastoma protein and also regulated the expression of cell cycle proteins such as cyclin A, p21, and the activation of cyclin-dependent kinase (CDK) 2 and CDK4 (2). However, these studies were carried out in tumor models and not in primary cells. As tumor cells usually demonstrate aberrant regulation of one or more cellular functions, this may account, at least in part, for the differences in the phase in which the cell cycle arrest occurred. Also, as mentioned above, GGTIs can alter proteins other than Rac1, and this may also produce effects on cell cycle progression. It should also be noted that a majority of our cells were still in the G1/S phase. It is unclear why the N17Rac1 results in a greater accumulation of cells in the G2/M phase than GGTI-287 treatment. However, this may be related to their respective reduction in cellular ROS levels. We have previously shown, using SMCs isolated from the pulmonary vessels of fetal lambs, that ROS levels need to fall to <50% of control levels to produce an inhibition of cell growth and to <10% to induce apoptosis (39, 40). As N17Rac reduces ROS levels by
65% (Fig. 6) whereas GGTI-287 reduces ROS by
50%, this may account for the relative increase in cells in the G2/M phase.
In summary, this study demonstrates an important role for Rac1 in the signal transduction pathway leading to the proliferation of SMCs via the generation of ROS. Inhibition of Rac1 results in a decrease in ROS generation and a reduction in cell growth. This inhibition of proliferation appears to be at the G2/M transition. Thus our data strongly suggest an important role for Rac1 in the activation of the NADPH oxidase complex and in the ROS generation required to stimulate the proliferation of SMCs. We speculate that the directed targeting of the Rac1 protein may have potential therapeutic efficacy in the treatment of cardiovascular disease where increased SMC proliferation is involved. However, further studies will need to be undertaken using animal models to better define this possibility.
 |
GRANTS
|
---|
This research was supported in part by National Institutes of Health Grants HL-67841, HL-60190, HD-398110, HL-072123, and HL-070061 (to S. M. Black).
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: S. M. Black, International Heart Inst. of Montana, Rm. 3300, St. Patrick Hospital, 554 W. Broadway, Missoula, MT 59802 (E-mail: Stephen.black{at}umontana.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* S. Patil and M. Bunderson contributed equally to this work. 
 |
REFERENCES
|
---|
- Abo A, Pick E, Hall A, Totty N, Teahan CG, and Segal AW. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature 353: 668670, 1991.[CrossRef][ISI][Medline]
- Adnane J, Bizouarn FA, Qian Y, Hamilton AD, and Sebti SM. p21(WAF1/CIP1) is upregulated by the geranylgeranyltransferase I inhibitor GGTI-298 through a transforming growth factor beta- and Sp1-responsive element: involvement of the small GTPase rhoA. Mol Cell Biol 18: 69626970, 1998.[Abstract/Free Full Text]
- Berry C, Hamilton CA, Brosnan MJ, Magill FG, Berg GA, McMurray JJ, and Dominiczak AF. Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries. Circulation 101: 22062212, 2000.[Abstract/Free Full Text]
- Bokoch GM and Diebold BA. Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood 100: 26922696, 2002.[Abstract/Free Full Text]
- Brennan L, Steinhorn RH, Wedgwood S, Mata-Greenwood E, Roark EA, Russell JA, and Black SM. Increased superoxide generation is associated with pulmonary hypertension in fetal lambs: a role for NADPH oxidase. Circ Res 92: 683691, 2003.[Abstract/Free Full Text]
- Casey PJ. Biochemistry of protein prenylation. J Lipid Res 33: 17311740, 1992.[ISI][Medline]
- Crouch S, Kozlowski R, Slater K, and Fletcher J. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J Immunol Methods 160: 8188, 1993.[CrossRef][ISI][Medline]
- Diatchuk V, Lotan O, Koshkin V, Wikstroem P, and Pick E. Inhibition of NADPH oxidase activation by 4-(2-aminoethyl)-benzenesulfonyl fluoride and related compounds. J Biol Chem 272: 1329213301, 1997.[Abstract/Free Full Text]
- Dorseuil O, Reibel L, Bokoch GM, Camonis J, and Gacon G. The Rac target NADPH oxidase p67 interacts preferentially with Rac2 rather than Rac1. J Biol Chem 271: 8388, 1996.[Abstract/Free Full Text]
- Griendling KK, Minieri CA, Ollerenshaw JD, and Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 11411148, 1994.[Abstract]
- Griendling KK, Sorescu D, and Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 86: 494501, 2000.[Abstract/Free Full Text]
- Grunfeld S, Hamilton CA, Mesaros S, McClain SW, Dominiczak AF, Bohr DF, and Malinski T. Role of superoxide in the depressed nitric oxide production by the endothelium of genetically hypertensive rats. Hypertension 26: 854857, 1995.[Abstract/Free Full Text]
- Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, Sundaresan M, Finkel T, and Goldschmidt-Clermont PJ. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 275: 16491652, 1997.[Abstract/Free Full Text]
- Knaus U, Heyworth PG, Evans T, Curnutte JT, and Bokoch GM. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science 254: 15121515, 1991.[ISI][Medline]
- Knaus UG, Heyworth PG, Kinsella BT, Curnutte JT, and Bokoch GM. Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase. J Biol Chem 267: 2357523582, 1992.[Abstract/Free Full Text]
- Krieger-Brauer HI, Medda P, and Kather H. Basic fibroblast growth factor utilizes both types of component subunits of Gs for dual signaling in human adipocytes. Stimulation of adenylyl cyclase via Galph(s) and inhibition of NADPH oxidase by Gbeta gamma(s). J Biol Chem 275: 3592035925, 2000.[Abstract/Free Full Text]
- Larochelle DA, Vithalani KK, and De Lozanne A. A novel member of the rho family of small GTP-binding proteins is specifically required for cytokinesis. J Cell Biol 133: 13211329, 1996.[Abstract]
- Lee SL, Simon AR, Wang WW, and Fanburg BL. H2O2 signals 5-HT-induced ERK MAP kinase activation and mitogenesis of smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 281: L646L652, 2001.[Abstract/Free Full Text]
- Lee SL, Wang WW, and Fanburg BL. Superoxide as an intermediate signal for serotonin-induced mitogenesis. Free Radic Biol Med 24: 855858, 1998.[CrossRef][ISI][Medline]
- Leusen J, Verhoeven AJ, and Roos D. Interactions between the components of the human NADPH oxidase: intrigues in the phox family. J Lab Clin Med 128: 461476, 1996.[ISI][Medline]
- Marumo T, Schini-Kerth VB, Fisslthaler B, and Busse R. Platelet-derived growth factor-stimulated superoxide anion production modulates activation of transcription factor NF-kappaB and expression of monocyte chemoattractant protein 1 in human aortic smooth muscle cells. Circulation 96: 23612367, 1997.[Abstract/Free Full Text]
- Mazet JL, Padieu M, Osman H, Maume G, Mailliet P, Dereu N, Hamilton AD, Lavelle F, Sebti SM, and Maume BF. Combination of the novel farnesyltransferase inhibitor RPR130401 and the geranylgeranyltransferase-1 inhibitor GGTI-298 disrupts MAP kinase activation and G(1)-S transition in Ki-Ras-overexpressing transformed adrenocortical cells. FEBS Lett 460: 235240, 1999.[CrossRef][ISI][Medline]
- Mohazzab KM and Wolin MS. Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle. Am J Physiol Lung Cell Mol Physiol 267: L815L822, 1994.[Abstract/Free Full Text]
- Moore KA, Sethi R, Doanes AM, Johnson TM, Pracyk JB, Kirby M, Irani K, Goldschmidt-Clermont PJ, and Finkel T. Rac1 is required for cell proliferation and G2/M progression. Biochem J 326: 1720, 1997.[ISI][Medline]
- Ohara Y, Peterson T, and Harrison D. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 91: 25462551, 1993.[ISI][Medline]
- Pagano PJ, Ito Y, Tornheim K, Gallop PM, Tauber AI, and Cohen RA. An NADPH oxidase superoxide-generating system in the rabbit aorta. Am J Physiol Heart Circ Physiol 268: H2274H2280, 1995.[Abstract/Free Full Text]
- Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C, Ballinger CA, Brasier AR, Bode C, and Runge MS. Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo. J Biol Chem 274: 1981419822, 1999.[Abstract/Free Full Text]
- Rajagopalan S, Kurz S, Münzel T, Tarpey M, Freeman BA, Griendling KK, and Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest 97: 19161923, 1996.[Abstract/Free Full Text]
- Rinckel LA, Faris SL, Hitt ND, and Kleinberg ME. Rac1 disrupts p67phox/p40phox binding: a novel role for Rac in NADPH oxidase activation. Biochem Biophys Res Commun 263: 118122, 1999.[CrossRef][ISI][Medline]
- Ross R. A pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362: 801809, 1993.[CrossRef][ISI][Medline]
- Stark WW Jr, Blaskovich MA, Johnson BA, Qian Y, Vasudevan A, Pitt B, Hamilton AD, Sebti SM, and Davies P. Inhibiting geranylgeranylation blocks growth and promotes apoptosis in pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 275: L55L63, 1998.[Abstract/Free Full Text]
- Sulciner DJ, Irani K, Yu ZX, Ferrans VJ, Goldschmidt-Clermont P, and Finkel T. Rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation. Mol Cell Biol 12: 71157121, 1996.
- Sun J, Qian Y, Chen Z, Marfurt J, Hamilton AD, and Sebti SM. The geranylgeranyltransferase I inhibitor GGTI-298 induces hypophosphorylation of retinoblastoma and partner switching of cyclin-dependent kinase inhibitors. A potential mechanism for GGTI-298 antitumor activity. J Biol Chem 274: 69306934, 1999.[Abstract/Free Full Text]
- Sundaresan M, Yu ZX, Ferrans VJ, Irani K, and Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270: 296299, 1995.[Abstract]
- Sundaresan M, Yu ZX, Ferrans VJ, Sulciner DJ, Gutkind JS, Irani K, Goldschmidt-Clermont PJ, and Finkel T. Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem J 318: 379382, 1996.[ISI][Medline]
- Vignais PV. The superoxide-generating NADPH oxidase: structural aspects and activation mechanism. Cell Mol Life Sci 59: 14281459, 2002.[CrossRef][ISI][Medline]
- Wassmann S, Laufs U, Muller K, Konkol C, Ahlbory K, Baumer AT, Linz W, Bohm M, and Nickenig G. Cellular antioxidant effects of atorvastatin in vitro and in vivo. Arterioscler Thromb Vasc Biol 22: 300305, 2002.[Abstract/Free Full Text]
- Wassmann S, Laufs U, Stamenkovic D, Linz W, Stasch JP, Ahlbory K, Rosen R, Bohm M, and Nickenig G. Raloxifene improves endothelial dysfunction in hypertension by reduced oxidative stress and enhanced nitric oxide production. Circulation 105: 20832091, 2002.[Abstract/Free Full Text]
- Wedgwood S and Black SM. Combined superoxide dismutase/catalase mimetics alter fetal pulmonary arterial smooth muscle cell growth. Antioxid Redox Signal 6: 191197, 2004.[CrossRef][ISI][Medline]
- Wedgwood S and Black SM. Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic. Am J Physiol Lung Cell Mol Physiol 285: L305L312, 2003.[Abstract/Free Full Text]
- Wedgwood S, Dettman R, and Black S. ET-1 stimulates pulmonary arterial smooth muscle cell proliferation via induction of reactive oxygen species. Am J Physiol Lung Cell Mol Physiol 281: L1058L1067, 2001.[Abstract/Free Full Text]
- Wedgwood S, McMullan DM, Bekker JM, Fineman JR, and Black SM. Role for endothelin-1-induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res 89: 357364, 2001.[Abstract/Free Full Text]