Hydrogen peroxide enhances the expression of Giα proteins in aortic vascular smooth cells: role of growth factor receptor transactivation

Nathan Mbong, Madhu B. Anand-Srivastava

Abstract

Oxidative stress has been shown to increase the expression of Giα proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats. The present study was undertaken to examine if H2O2, which induces oxidative stress, could also enhance the expression of Giα proteins in VSMC and to further explore the underlying signaling pathways responsible for this response. Treatment of VSMC with H2O2 increased the expression of Giα proteins and not of Gsα protein in a concentration- and time-dependent manner. A maximal increase of ∼40–50% was observed at 100 μM and 1 h and was restored to control levels by AG1295 and AG1478, inhibitors of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R), respectively, and PD98059 and U126, inhibitors of extracellular signal-regulated kinase (ERK1/2), and wortmannin and AKT inhibitor VIII, inhibitors of PKB/AKT, respectively. In addition, H2O2 also increased the phosphorylation of EGF-R, PDGF-R, ERK1/2, and AKT, which was attenuated by the respective inhibitors, whereas the inhibitors of EGF-R and PDGE-R also inhibited the enhanced phosphorylation of ERK1/2 and AKT. Furthermore, transfection of cells with short interfering RNA of EGF-R and PDGF-R restored the H2O2-induced enhanced expression of Giα proteins to control levels. The increased expression of Giα proteins was reflected in enhanced Gi functions as demonstrated by enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by a low concentration of GTPγS, whereas Gsα-mediated stimulations of AC were significantly decreased. Furthermore, H2O2-induced enhanced proliferation of VSMC was attenuated by dibutyryl-cAMP. These results suggest that H2O2 increases the expression of Giα proteins in VSMC through the transactivation of EGF-R/PDGF-R and ERK1/2 and phosphatidylinositol-3 kinase signaling pathways.

  • hydrogen peroxide
  • extracellular-regulated kinase
  • phosphatidylinositol-3 kinase
  • adenylyl cyclase

guanine nucleotide proteins, known as G proteins, are a family of GTP-binding proteins that play a critical role in the regulation of a variety of signal transduction systems, including the adenylyl cyclase (AC)/cAMP system (31). The AC system is composed of three components: a receptor, a catalytic subunit, and a guanine nucleotide regulatory protein that transmits the signal from the hormone-occupied receptor to the catalytic subunit (30).Well-characterized members of G protein family include Gs, Gi, Gq, and Go. Gs and Gi are involved in the stimulation and inhibition of hormone-sensitive AC (14). Each of the G proteins is heterotrimeric, consisting of α-, β-, and γ-subunits (Gα, Gβ, and Gγ). The α-subunit binds and hydrolyzes GTP and confers specificity in receptor and effector interactions (14). Molecular cloning has revealed four different forms of Gsα, resulting from differential splicing of one gene, and three distinct forms of Giα, Giα-1, Giα-2, and Giα-3, encoded by three distinct genes (6, 14, 18).

Alterations in Giα proteins and associated AC signaling have been shown to be implicated in various pathological conditions, such as hypertension (2), diabetes (16), and heart failure (11). We previously have reported an increased expression of Giα-2 and Giα-3 proteins and mRNA in hearts and aortas from spontaneously hypertensive rats (SHR; Ref. 2), 1K1C (13), N-nitro-l-arginine methyl ester (l-NAME; Ref. 10), and DOCA-salt hypertensive rats (4) with established hypertension. The enhanced expression of Giα proteins was shown to be attributed to the enhanced levels of vasoactive peptides including angiotensin II (ANG II) because the treatment of hypertensive rats with captopril, an angiotensin-converting enzyme inhibitor, was shown to restore the enhanced expression of Gi proteins to control levels (26). The enhanced expression of Giα proteins occurs before the onset of hypertension in SHR and DOCA-salt (23, 24), suggesting the implication of increased expression of Giα protein in the pathogenesis of hypertension. Furthermore, we have shown that vascular smooth muscle cells (VSMC) from SHR also exhibit enhanced levels of Giα protein compared with Wistar-Kyoto rats, which were restored to control levels by antioxidants, suggesting a role of oxidative stress in increased expression of Giα protein in SHR (19). In addition, ANG II-induced enhanced expression of Giα proteins in A10 VSMC has also been attributed to the enhanced oxidative stress due to the enhanced activity of NADPH oxidase and enhanced production of the superoxide anion (O2; Ref. 21). ANG II has also been reported to increase the levels of H2O2, another reactive oxygen species (ROS) inducing oxidative stress (35).

G-protein-coupled receptor-induced transactivation of tyrosine kinase receptors has also been shown as a common pathway for transmission of ROS-sensitive signals (8). Furthermore, H2O2 has been shown to increase the tyrosine phosphorylation of tyrosine kinase receptors in the absence of growth factors in VSMC and induce the activation of downstream pathways such as mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3 kinase (PI3K/protein kinase B/AKT; Ref. 25). The activation of vascular MAPK induced by ANG II has also been demonstrated in VSMC (21, 32). Studies have shown that the antioxidants diphenyleneiodonium and N-acetyl cysteine inhibit extracellular-regulated kinase (ERK1/2) activation induced by ANG II in VSMC (12, 21) and that exogenous H2O2 stimulates protein tyrosine phosphorylation of ERK1/2 (25). We (19) have recently shown that enhanced oxidative stress exhibited by VSMC from SHR increased the expression of Gi proteins through the activation of MAPK signaling. However, it remained unexplored whether VSMC isolated from normal aorta will exhibit a similar response when subjected to increased oxidative stress. Therefore, the current study was undertaken to investigate if the increased oxidative stress induced by H2O2 in aortic VSMC could also increase the expression of Giα proteins and the associated AC signaling and to further explore the underlying signaling mechanisms responsible for this response.

We showed for the first time that H2O2 increased the expression of Giα proteins in aortic VSMC through the transactivation of growth factor receptors and MAPK/PI3K signaling.

MATERIALS AND METHODS

IBMX, glucagon, oxotremorine, isoproterenol, forskolin (FSK), guanosine 5′-[3-thio] triphosphate (GTPγS), and GTP were purchased from Sigma-Aldrich Chemical (St. Louis, MO). Adenosine triphosphate isotope [α-32P]ATP was purchased from PerkinElmer (Boston, MA). The PDGF-Rβ inhibitor AG1295, EGF-R inhibitor AG1478, and MEK1 inhibitor PD98059 were purchased from Calbiochem (Gibbstown, NJ). EGF-R small interfering (si)RNA(r), PDGF-Rβ siRNA(m), siRNA transfection medium, and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine 2000 was purchased from Life Technologies. Antibodies against Giα-2 (L5), Giα-3 (C-10), ERK1/2 (C-14), p-ERK1/2 (phosphospecific-tyrosine204), p-AKT (phosphospecific-serine473), AKT (H-136), p-PDGF-R (phosphospecific-tyrosine857), PDGF-R (958), p-EGF-R (phosphospecific-tyrosine1173), and EGF-R (1005) were from Santa-Cruz Biotechnologies. All other chemicals used in the experiments were purchased from Sigma-Aldrich.

Cell culture and incubation.

VSMC were cultured from explants of rat aorta as described previously (5). Colonies of VSMC grew out from some of the explants within 7–14 days. When sufficiently confluent, the colonies were trypsinized with 0.05% trypsin in PBS (Ca2+ and Mg2+ free, containing 0.02% EDTA). The resulting suspension of cells was plated in 75-cm2 flasks and incubated at 37°C in a 95% air-5% CO2 humidified atmosphere in DMEM (with glucose, l-glutamine, and sodium bicarbonate) containing antibiotics and 10% FBS. The cells were passaged upon reaching confluence with 0.5% trypsin and used between passages 5 and 20. Confluent cells were starved for 24 h in DMEM without FBS at 37°C. To study the effect of pharmacological inhibitors on Giα expression, the cells were incubated in the absence or presence of PD98059 (10 μM), U126 (10 μM), wortmannin (0.1 μM), AKT inhibitor VIII (0.5 μM), AG1478 (5 μM), and AG1295 (5 μM) before treatment with H2O2 (100 μM) for 1 h. The cells were scraped into ice-cold homogenization buffer containing 10 mM Tris·HCl buffer and 1 mM EDTA (pH 7.5). The homogenate was centrifuged at 1,000 g for 10 min. The supernatant was used for immunoblotting, and the pellet was resuspended in 10 mM Tris·HCl buffer containing 1 mM EDTA (pH 7.5) and used for AC assay. All the animal procedures used in the present studies were approved by the Comité de Déontologie de l′Expérimentation sur les Animaux of the University of Montreal (No. 99050). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

AC activity determination.

AC activity was determined by measuring [α-32P]cAMP formation from [α-32P]ATP as described previously(5). The assay medium contained 50 mmol/l of glycylglycine (pH 7.5), 0.5 mmol/l MgATP, [α-32P]ATP (1.5 ×106 count/min), 5 mmol/l MgCl2 (in excess of the ATP concentration), 100 mmol/l NaCl, 0.5 mmol/l cAMP, 1 mmol/l IBMX, 0.1 mmol/l EGTA, 10 μmol/l GTPγS, and an ATP-regenerating system consisting of 2 mmol/l creatine phosphate, 0.1 mg of creatine kinase/ml, and 0.1 mg of myokinase/ml in a final volume of 200 μl. Incubation was initiated by the addition of the membrane preparation (20–30 μg) to the reaction mixture, which had been thermally equilibrated for 2 min at 37°C. The reactions were conducted in triplicate and were terminated by the addition of 0.6 ml of 120 mM Zinc acetate. cAMP was purified by coprecipitation of other nucleotides with ZnCO3, an addition of 0.5 ml of 144 mM Na2CO3, and subsequent chromatography by the double column system as previously described

Transfection of small interfering RNA.

Confluents cells were starved for 5 h in siRNA transfection medium. Cells were transfected with 90 nM siRNA and 4 μg/ml of lipofectamine per 60-mm cell dishes. After 48 h of transfection, the cells were lysed and used for Western blotting.

Western blotting.

Western blotting of G proteins (Gsα, Giα-2, and Giα-3), AKT, ERK1/2, PDGFβ-R, and EGF-R was performed using specific antibodies as described previously (15, 19). After SDS-PAGE, the separated proteins were electrophoretically transferred to a nitrocellulose membrane (Bio-Rad) with a semidry transblot apparatus (Bio-Rad) at 15 V for 45 min. After transfer, the membranes were washed twice in PBS and were incubated in PBS containing 3% skim milk at room temperature for 2 h. The blots were then incubated with antibodies against Giα-2 (L5), Giα-3 (C-10), p-ERK1/2(phosphospecific-tyrosine204), ERK1/2 (C-14) p-AKT (phosphospecific-serine473), AKT (H-136) p-EGF-R (phosphospecific-tyrosine1173), EGF-R (1005), p-PDGF-R (phosphospecific-tyrosine857), and PDGF-R (958) in PBS containing 1.5% skim milk and 0.1% Tween-20 at room temperature overnight. The antigen-antibody complexes were detected by incubating the blots with goat anti-rabbit IgG (Bio-Rad) conjugated with horseradish peroxidase for 1 h at room temperature. The blots were then washed three times with PBS before reaction with enhanced chemiluminescence Western blotting detection reagents (Amersham). Quantitative analysis of the protein was performed by densitometric scanning of the autoradiographs employing the Enhanced Laser Densitometer LKB Ultroscan XL and quantified using the gel-scan XL evaluation software (version 2.1) from Pharmacia (Baie d'Urfé, Québec, Canada). The scanning was one dimensional and scanned the entire area of protein bands in the blot.

Determination of cell proliferation.

Cell proliferation was quantified by DNA synthesis that was evaluated by incorporation of [3H]thymidine into cells as described earlier (22). Subconfluent aortic VSMC were plated in six-well plates for 24 h and were serum deprived for 24 h to induce cell quiescence. VSMC were incubated in the absence or presence of H2O2 (100 μM) and dibutyryl cAMP (500 μM) alone or in combination for 24 h. [3H]thymidine (1 μCi) was added and further incubated for 4 h before the cells were harvested. The cells were rinsed twice with ice-cold PBS and incubated with 5% trichloroacetic acid for 1 h at 4°C. After being washed twice with ice-cold water, the cells were incubated with 0.4 N sodium hydroxide solution for 30 min at room temperature, and radioactivity was determined by liquid scintillation counter. Cell viability was checked by the trypan blue exclusion technique and indicated that >90∼95% cells were viable.

Statistical analysis.

Results are expressed as means ± SE and were analyzed by t-test and one-way ANOVA followed by Newman-Keul test. Results were considered statistically significant at a value of P < 0.05.

RESULTS

Effect of H2O2 on the expression of Giα-2, Giα-3, and Gsα proteins in aortic VSMC.

We (19) previously showed that oxidative stress contributes to the enhanced expression of Giα proteins in VSMC from SHR. To further establish the relationship between oxidative stress and Giα proteins levels, we examined the effect of various concentrations of H2O2 (50–250 μM) on the levels of Giα-2 and Giα-3 proteins in aortic VSMC. Results shown in Fig. 1, A and B, indicates that treatment of VSMC with H2O2 for 1 h increased the levels of both Giα-2 and Giα-3 proteins in a concentration-dependent manner. The maximal increase of ∼75% in Giα-2 and 60% in Giα-3 was observed at 100 μM. In addition, the increased expression of Giα-2 and Giα-3 proteins by H2O2 (100 μM) was also time dependent. As shown in Fig. 1, C and D, the levels of Giα-2 and Giα-3 were enhanced as early as 30 min and reached a maximum of ∼180% of control at 1–2 h and level after 3 h; however, the levels of Gsα were not affected by H2O2 treatment (Fig. 1, E and F).

Fig. 1.

Effect of various concentrations and time periods of H2O2 treatment on the levels of Giα-2, Giα-3, and Gsα proteins in aortic vascular smooth muscle cells (VSMC). Aortic VSMC were incubated in the absence (0 μM) or presence of H2O2 (50 to 250 μM; A and B) for different time periods (30 min to 4 h; C and D) as described in materials and methods. Cell lysates were prepared and subjected to Western blotting using specific antibodies against Giα-2 (A and C), Giα-3 (B and D), or Gsα (E and F). Dynein was used as a loading control. For time-course experiments (C and F), same membranes were used to probe Giα-2, Gsα, and dynein and therefore same dynein control was shown in C and F. Proteins were quantified by densitometric scanning and plotted as a percentage of control (CTL) taken as 100%. Data are means ± SE of 3 separate experiments. *P < 0.05, **P < 0.01 vs. CTL.

Effect of actinomycin D on H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins in aortic VSMC.

To investigate whether H2O2-induced enhanced levels of Giα-2 and Giα-3 proteins were due to increased RNA synthesis, the effect of actinomycin D, an inhibitor of RNA synthesis was examined on H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins. Results shown in Fig. 2 indicate that actinomycin D attenuated the H2O2-induced enhanced level of Giα-2 and Giα-3 proteins to control levels.

Fig. 2.

Effect of actinomycin D on H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins in aortic VSMC. Aortic VSMC were pretreated without or with actinomycin D (5 μM) for 24 h and were further incubated in the absence or presence of 100 μM of H2O2 for 1 h. Cell lysates were prepared and subjected to Western blotting using specific antibodies against Giα-2 and Giα-3 as described in materials and methods. Dynein was used as a loading control. Proteins were quantified by densitometric scanning and plotted as a percentage of CTL taken as 100%. Values are means ± SE of 3 separate experiments. **P < 0.01 vs. CTL. §P < 0.05 vs. H2O2-treated group.

Effect of H2O2 on Gi functions in aortic VSMC.

To investigate if the H2O2-induced enhanced expression of Giα is also reflected in enhanced Giα functions, the effect of H2O2 on receptor-dependent and -independent functions of Giα proteins was examined. The receptor-independent function of Giα was investigated by studying the effect of low concentrations of GTPγS (10−12 to 10−7 M) on FSK-stimulated AC activity. As illustrated in Fig. 3A, GTPγS inhibited FSK-stimulated AC activity in a concentration-dependent manner in both control and H2O2-treated cells; however, the inhibition was greater by about 25% in H2O2-treated cells.

Fig. 3.

A: effect of H2O2 on GTPγS-mediated inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity in aortic VSMC. Aortic VSMC were incubated in the absence or presence of 100 μM H2O2 for 1 h. Membranes were prepared as described in materials and methods. Adenylyl cyclase activity was determined in these membranes in the presence of 100 μM FSK alone, taken as 100%, and in the presence of various concentrations of GTPγS (10−12 to 10−7 M). Basal enzyme activity values in the absence of GTPγS in control or H2O2-treated cells were 102 ± 2.1 and 68 ± 1.5 cAMP·mg protein−1·min−1. Values are means ± SE of 3 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal. §§P < 0.01 vs. H2O2-treated group. B: effect of H2O2 on hormonal inhibition of adenylyl cyclase activity in aortic VSMC. Aortic VSMC were treated without or with H2O2 (100 μM) for 1 h. Membranes were prepared as described in materials and methods. Adenylyl cyclase activity was determined in the presence of 10 μM GTPγS alone, taken as 100% or in combination with 10 μM angiotensin II (ANG II), 0.1 μM C-ANP4–23, or 50 μM oxotremorine (Oxo). Basal enzyme activity values in the absence of GTPγS in control or H2O2-treated cells were 21 ± 0.45 and 10 ± 0.25 pmol cAMP·mg protein−1·min−1, respectively. Values are means ± SE of 3 separate experiments. **P < 0.01 vs. CTL. C and D: effect of H2O2 on agonist-mediated stimulation of adenylyl cyclase in aortic VSMC. Aortic VSMC were treated without or with H2O2 (100 μM) for 1 h. Membranes were prepared as described in materials and methods. Adenylyl cyclase activity was determined in the presence of 10 μM GTP alone, taken as 100% or in combination with 50 μM isoproterenol or 1 μM glucagon (C), in the absence or presence of 10 mM sodium fluoride (NaF) or 50 μM FSK (D). Basal adenylyl cyclase activity values in the absence of GTP in control or H2O2-treated cells were 24.6 ± 0.18 and 16 ± 0.71 cAMP·mg protein−1·min−1, respectively. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. CTL.

The receptor-dependent function of Giα proteins was also examined by studying the effects of H2O2 treatment on ANG II-, C-ANP4–23-, and oxotremorine-mediated inhibition of AC activity in aortic VSMC. The results in Fig. 3B indicate that ANG II, C-ANP4–23, and oxotremorine inhibited AC activity by ∼25–30% in control cells. However, these inhibitions were significantly augmented (∼55%) by H2O2 treatment.

Effect of H2O2 on Gs-mediated stimulation of AC activity in aortic VSMC.

The interaction of Giα and Gsα has been well established (7). Since H2O2 enhanced Giα protein expression without altering the levels of Gsα, we investigated whether increased level of Giα induced by H2O2 could affect Gsα-mediated stimulation of AC. To test this, the effects of isoproterenol, a β-adrenergic agonist, glucagon, sodium fluoride (NaF), and FSK on AC activity was examined. Figure 3C illustrates that isoproterenol and glucagon stimulated AC activity in both control cells and H2O2-treated cells to various degrees; however, the extent of stimulation was significantly decreased (∼40%) in H2O2-treated cell compared with control cells. In addition, FSK- and NaF-stimulated AC activities were also attenuated by about 35% by H2O2 treatment (Fig. 3D).

Role of ERK1/2 and PI3K in H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins in aortic VSMC.

The involvement of ERK1/2 and PI3K in enhanced expression of Giα proteins in VSMC from SHR has been demonstrated (19). To investigate whether ERK1/2 and PI3K is also implicated in H2O2-induced enhanced expression of Giα in VSMC, the effects of PD98050 (10 μM) and U126 (10 μM), inhibitors of ERK phosphorylation, and wortmannin (0.1 μM), an inhibitor of PI3K, and AKT inhibitor VIII (0.5 μM) were examined on H2O2-induced enhanced expression of Giα proteins. Results shown in Figs. 4 and Fig. 5, indicate that the increased expression of Giα-2 (A and C) and Giα-3 (B and D) proteins in H2O2-treated cells were restored to control levels by PD98059 ( Fig. 4, A and B) and U126 ( Fig. 4, C and D) as well as by wortmannin (Fig. 5, A and B) and AKT inhibitor VIII (Fig. 5, C and D) treatments. However, these inhibitors did not have any significant effect on the levels of Giα proteins in control cells.

Fig. 4.

Effect of ERK1/2 inhibitors on H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins in aortic VSMC. Cells were pretreated without or with PD98050 (10 μM; A and B) or U126 (10 μM; C and D) for 1 h and then stimulated with 100 μM of H2O2 for 1 h. Cell lysates were prepared and subjected to Western blotting using specific antibodies against Giα-2 (A and C) and Giα-3 (B and D) as described in materials and methods. Dynein was used as a loading control. Proteins were quantified by densitometric scanning and plotted as a percentage of CTL taken as 100%. Values are means ± SE of 8 separate experiments. **P < 0.01, ***P < 0.001 vs. CTL. #P < 0.05, ##P < 0.01 vs. H2O2-treated group.

Fig. 5.

Effect of phosphatidylinositol-3 kinase (PI3K)/AKT inhibitors on H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins in aortic VSMC. Cells were pretreated without or with wortmannin (Wort; 0.1 μM; A and B) or AKT inhibitor VIII (0.5 μM; C and D) for 1 h and then stimulated with 100 μM of H2O2 for 1 h. Cell lysates were prepared and subjected to Western blotting using specific antibodies against Giα-2 (A and C) and Giα-3 (B and D) as described in materials and methods. Dynein was used as a loading control. Proteins were quantified by densitometric scanning and plotted as a percentage of CTL taken as 100%. Values are means ± SE of 8 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CTL. #P < 0.05, ##P < 0.01 vs. H2O2-treated group.

Effect of H2O2 on the phosphorylation of ERK1/2 and AKT in aortic VSMC.

Figure 6A shows the effect of H2O2 treatment on ERK1/2 phosphorylation in aortic VSMC. Treatment of cells with H2O2 increased the phosphorylation of Tyr204 on ERK1/2 by ∼90% compared with control cells which was attenuated towards control level by PD98059 as well as by U126. In addition, these inhibitors also inhibited the ERK1/2 phosphorylation in control cells by ∼50%.

Fig. 6.

Effect of H2O2 on ERK1/2 and AKT phosphorylation in aortic VSMC. Confluent cells were incubated in the absence or presence of PD98059 (10 μM) or U126 (10 μM; A) or wortmannin (0.1 μM) or AKT inhibitor VIII (0.5 μM; B) for 1 h and then challenged with 100 μM of H2O2 for 1 h. Cell lysates were prepared and subjected to Western blotting using phospho-specific-Tyr204 ERK , phosphospecific-serine473 AKT antibodies (top), and ERK1/2 or AKT antibodies (bottom), as described in materials and methods. Proteins were quantified by densitometric scanning and plotted as a percentage of CTL taken as 100%. Values are means ± SE of 6 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CTL. #P < 0.05, ##P < 0.01 vs. H2O2-treated group.

Since H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins was also abolished by PI3K inhibitors, it was of interest to examine whether treatment of aortic VSMC with H2O2 would increase the phosphorylation of AKT. To test this, the effect of H2O2 on AKT phosphorylation was investigated in VSMC. As shown in Fig. 6B, H2O2 increased the phosphorylation of phosphospecific-serine473 on AKT by ∼60% compared with control cells and this increased phosphorylation was attenuated to control levels by wortmannin as well as by AKT inhibitor VIII. In addition, these inhibitors also inhibited the AKT phosphorylation in control cells by ∼50–60%.

Effect of growth factors receptor inhibitors on H2O2-induced enhanced expression of Giα proteins in aortic VSMC.

Since H2O2 has been shown to transactivate growth factor receptor such as EGF-R and PDGF-Rβ in A10 VSMC (25), it may be possible that the H2O2-induced enhanced expression of Giα-2 and Giα-3 could also be due to the enhanced activity of both EGF-R and PDGF-Rβ. To investigate this, the effect of AG1478, an inhibitor of EGF-R and AG1295, an inhibitor of PDGF-Rβ, on H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins in aortic VSMC was examined. The results shown in Fig. 7, indicate that the increased expression of Giα-2 and Giα-3 proteins (∼50% ) in H2O2-treated cells compared with control cells was restored to control levels by AG1478 (Fig. 7, A and B) and AG1295 (Fig. 7, C and D) . However, these inhibitors did not have any significant effect on the levels of Giα-2 and Giα-3 proteins in control cells.

Fig. 7.

Effect of EGF-R and PDGF-R inhibitors on H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins in aortic VSMC. Aortic VSMC were treated with either AG1478 (5 μM; A and B) or AG1295 (5 μM; C and D) for 1 h and then treated with 100 μM H2O2 for 1 h. Cell lysates were prepared and subjected to Western blotting using specific antibodies against Giα-2 (A and C) and Giα-3 (B and D) as described in materials and methods. Dynein was used as a loading control. Proteins were quantified by densitometric scanning and plotted as a percentage of CTL taken as 100%. Values are means ± SE of 4 separate experiments. *P < 0.05, **P < 0.01 vs. CTL. §P < 0.05, §§P < 0.01, §§§P < 0.001 vs. H2O2-treated group.

Effect of siRNA of growth factor receptors (EGF-R and PDGF-R) on H2O2-induced enhanced expression of Giα proteins in aortic VSMC.

To further confirm the implication of EGF-R and PDGF-R in H2O2-induced enhanced expression of Giα proteins, the effect of siRNA of EGF-R and PDGF-Rβ on the expression of Giα proteins was investigated in control and H2O2-treated cells. Results shown in Fig. 8, indicate that the increased expression of Giα-2 (A and C) and Giα-3 (B and D) proteins (by ∼60 and 50%, respectively) in H2O2-treated cells compared with control cells, was attenuated to control levels by EGF-R and PDGF-R siRNA. However, cells transfected with lipofectamine alone, scrambled siRNA, and siRNA of EGF-R or PDGF-R did not affect the expression of Giα-2 and Giα-3 proteins in control cells. In addition, the levels of total EGF-R (Fig. 7E) and PDGF-R (Fig. 7F) were also significantly decreased by 65 and 80%, respectively, in cells transfected with EGF-R siRNA and PDGF-Rβ siRNA.

Fig. 8.

Effect of EGF-R and PDGF-R silencing on H2O2-induced enhanced expression of Giα-2, Giα-3, EGF-R, and PDGF-R proteins in aortic VSMC. Aortic VSMC were silenced with either EGF-R (A, B, and E) or PDGF-R-β (C, D, and F) siRNA or control siRNA (scrambled) for 48 h and then treated with 100 μM H2O2 for 1 h. Cell lysates were prepared and subjected to Western blotting using specific antibodies against Giα-2 (A and C), Giα-3 (B and D), EGF-R (E), and PDGF-R (F) as described in materials and methods. Dynein was used as a loading control. Proteins were quantified by densitometric scanning and plotted as a percentage of CTL taken as 100%. Values are means ± SE of 4 separate experiments. **P < 0.01 vs. CTL. §P < 0.05, §§§P < 0.001 vs. H2O2-treated group.

Effect of H2O2 on the phosphorylation of growth factor receptors in aortic VSMC.

To investigate whether H2O2 could also transactivate growth factor receptors in aortic VSMC, the phosphorylation of EGF-R and PDGF-R in response to H2O2 was examined and the results are shown in Fig. 9. Treatment of aortic VSMC with H2O2 increased the phosphorylation of Tyr1173on EGF-R (Fig. 9A) and Tyr857on PDGF-R (Fig. 9B) by ∼40% compared with control cells. However, the increased phosphorylation of EGF-R and PDGF-Rβ induced by H2O2 was attenuated to control levels by AG1478 and AG1295, respectively.

Fig. 9.

A and B: effect of H2O2 on the phosphorylation of EGF-R and PDGF-R in aortic VSMC. Confluents aortic VSMC were incubated in the absence or presence of AG1478 (5 μM; A), or AG1295 (5 μM; B) for 1 h and then challenged with 100 μM of H2O2 for 1 h. Cell lysates were prepared and subjected to Western blotting using specific antibodies as described in materials and methods. Proteins were quantified by densitometric scanning and plotted as a percentage of CTL taken as 100%. Values are means ± SE of 4 separate experiments. *P < 0.05, **P < 0.01 vs. CTL. §§P < 0.01 vs. H2O2-treated group. C and D: effect of growth factor receptor inhibitors on H2O2-induced enhanced phosphorylation of ERK1/2 and AKT in aortic VSMC. Confluent aortic VSMC were incubated in the presence of 5 μM of AG1478 or 5 μM of AG1295 for 1 h and then stimulated with 100 μM H2O2 for 1 h. Cell lysates were prepared and subjected to Western blotting using specific antibodies as described in materials and methods. Proteins were quantified by densitometric scanning and plotted as a percentage of CTL taken as 100%. Values are means ± SE of 3 separate experiments. **P < 0.01, ***P < 0.001 vs. CTL. §§P < 0.01, §§§P < 0.001 vs. H2O2-treated group.

Implication of growth factor receptors in H2O2-induced enhanced activation of ERK1/2 and AKT signaling in aortic VSMC.

To investigate the implication of growth factor receptors in H2O2-induced enhanced activation of ERK1/2 and AKT, the effect of the EGF-R and PDGF-R inhibitors AG1478 and AG1295 on the phosphorylation of ERK1/2 and AKT was examined in control H2O2-treated cells. As illustrated in Fig. 9, H2O2 increased the phosphorylation of p-ERK1/2 (C) and AKT (D) by ∼60 and 50%, which was restored to control levels by AG1478 and AG1295, whereas these inhibitors did not have any effect on the levels of p-ERK1/2 and pAKT in control cells.

H2O2 increased the proliferation of aortic VSMC through the reduced levels of cAMP.

Since enhanced oxidative stress has been shown to contribute to hyperproliferation of VSMC from SHR (22), it was of interest to examine if H2O2 could also augment the proliferation of VSMC and whether this enhanced proliferation is due to enhanced levels of Giα proteins and resultant decreased levels of intracellular cAMP. To test this, we determined the effect of H2O2 on DNA synthesis and the results are shown in Fig. 10. Treatment of VSMC with H2O2 enhanced the DNA synthesis by ∼30% which was reversed to control levels by dibutyryl-cAMP. In addition, dibutyryl-cAMP was also able to decrease the DNA synthesis (∼25%) in control cells.

Fig. 10.

Effect of dibutyryl cAMP on H2O2-induced proliferation of aortic VSMC. Aortic VSMC were incubated in the absence (control) or presence of dibutyryl (db)-cAMP (0.5 mM) for 1 h and then stimulated with 100 μM H2O2 for 1 h. [3H]thymidine incorporation was determined as described in materials and methods. Results are expressed as a percentage of CTL, taken as 100%. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. CTL. §P < 0.05 vs. H2O2-treated group.

DISCUSSION

We earlier reported that the enhanced expression of Giα proteins in VSMC from SHR was attributed to the increased oxidative stress and increased MAP kinase activity (19). However, in the present study, we demonstrate that H2O2, a mimicker of oxidative stress, could also increase the levels of Giα proteins in aortic VSMC through the transactivation of growth factors receptors.

Although the role of oxidative stress in ANG II-mediated cell signaling has been well established (33), evidence for a direct role of oxidative stress in the increased expression of Giα proteins and associated AC has been lacking. We report that H2O2 increases the expression of Giα proteins in a time- and concentration-dependent manner; however, a slight decrease in the expression of Giα-2, and Giα-3 proteins that was observed beyond 150 μM H2O2 and at 3 and 4 h of treatment may not be due to apoptosis, because the total cell count by hemocytometer was not different between control and H2O2-treated cells. Furthermore, the H2O2-evoked enhanced expression of Giα-2 and Giα-3 in aortic VSMC may be at the transcriptional level because actinomycin D, an inhibitor of RNA synthesis, inhibited the H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins in these cells.

The enhanced expression of Giα-2 and Giα-3 by H2O2 treatment was also reflected in increased Giα functions as demonstrated by the enhanced inhibition of AC by inhibitory hormones such as ANG II, C-ANP4–23, and oxotremorine as well as the enhanced inhibition of FSK-stimulated AC activity by GTPγS in H2O2-treated cells compared with control cells resulting in decreased levels of cAMP. In this regard, the relationship between increased expression of Giα proteins and enhanced Giα function has previously been reported (2). In addition, the decreased responsiveness of AC to isoproterenol and glucagon stimulation in H2O2-pretreated cells and resultant diminished levels of cAMP may not be attributed to Gsα proteins, because the levels of Gsα proteins were not altered by H2O2 treatment and may be due to the overexpression of Giα proteins. In this regard, the interaction between Giα and Gsα is well established (7). For example, the overexpression of Giα proteins has been shown to attenuate the stimulatory responses of hormones on AC activity (2, 4, 10, 13), whereas the downregulation of Giα proteins augmented the hormonal stimulations of AC activity (1, 3).

We also showed that H2O2 enhanced the proliferation of VSMC, which may be attributed to the increased expression of Giα proteins and resultant decreased levels of intracellular cAMP. This notion was supported by the fact that the enhanced proliferation induced by H2O2 was reversed to control levels by dibutyryl-cAMP. The role of oxidative stress in enhanced proliferation of VSMC from SHR has been reported earlier (22). In addition, cAMP has also been shown to regulate the proliferation of VSMC (17). The decreased levels of cAMP have been reported to result in hyperproliferation of the cells (29).

Our results showing that blockade of PDGF-R and EGF-R by specific inhibitors restored the H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins to control levels without affecting the expression of Giα proteins in control cells suggest the implication of growth factor receptors in enhanced expression of Giα proteins induced by H2O2 in VSMC. This is further supported by the results showing that silencing of PDGF-R and EGF-R by siRNA attenuated H2O2-induced enhanced expression of Giα proteins. The underlying mechanism by which H2O2 enhances the expression of Giα-2 and Giα-3 proteins in VSMC appears to involve the transactivation of growth factor receptors, such as PDGF-R and EGF-R, because H2O2 enhanced the phosphorylation and not the levels of PDGF-R and EGF-R in VSMC. In addition, the fact that siRNA of PDGF-R and EGF-R decreased the H2O2-induced enhanced phosphorylation of these receptors further suggests the role of transactivation of growth factor receptor in enhanced expression of Giα proteins by H2O2. This notion is further supported by our results showing that siRNA of PDGF-R and EGF-R that decreased the levels of these receptors in control cells was without effect in altering the levels of Giα proteins. Taken together, it may be suggested that the transactivation of growth factor receptors by H2O2 contributes to the enhanced expression of Giα proteins in VSMC. In this regard, the implication of EGF-R in ANG II-induced enhanced expression of Giα proteins and proliferation in A10 VSMC has recently been shown (15).

The role of MAPK and PI3K in protein synthesis has been well established (20, 34). We (19) previously showed the implication of ERK1/2 in the enhanced expression of Giα-2 and Giα-3 protein in VSMC from SHR. In addition, ANG II has also been shown to enhance the expression of Giα proteins through ERK1/2 signaling pathways in A10 VSMC (21). However, in the present study, we demonstrate that the phosphorylation of ERK1/2 and AKT by H2O2 in aortic VSMC may be responsible for the H2O2-induced enhanced expression of Giα proteins because the inhibitors of MAP kinase and PI3K/AKT, PD98059, U126, wortmannin, and AKT inhibitorVIII, respectively, restored the H2O2-induced enhanced level of Giα-2 and Giα-3 proteins to control levels. Furthermore, our results showing that the inhibitors of PDGF-R and EGF-R attenuated the H2O2-induced enhanced phosphorylation of ERK1/2 to control levels suggest a role for PDGF-R and EGF-R in the enhanced activity of ERK1/2 induced by H2O2 in VSMC. These results are in agreement with the studies of other investigators (27) who have also reported the activation of MAPK by growth factor receptors. Thus, taken together, it may be suggested that the phosphorylation of PDGF-R and EGF-R by H2O2 activates downstream signaling pathways ERK1/2 and PI3K, which in turn may be responsible for the increased expression of Giα-2 and Giα-3 proteins in aortic VSMC. The precise mechanism by which H2O2 transactivates growth factor receptor-protein tyrosine kinases (PTKs) is still not clear. However, ROS-mediated inhibition of tyrosine phosphatases (9) that can shift the equilibrium of the phosphorylation-dephosphorylation cycle resulting in a net increase of tyrosine phosphorylation of R-PTKs and nonreceptor PTKs (28) may be a possible mechanism by which H2O2 induces transactivation of growth factor receptors.

In summary, we provide the first direct evidence that H2O2 that causes oxidative stress transactivates PDGF-R and EGF-R, which through the activation of downstream signaling pathways including ERK1/2 and PI3K contribute to the enhanced expression of Giα-2 and Giα-3 proteins and inhibition of AC activity in VSMC. It may be suggested that the increased expression of Giα proteins and resultant decreased levels of cAMP induced by oxidative stress may be one of the factors responsible for the vascular remodeling and thereby vascular complications observed in various pathological states including hypertension and atherosclerosis (Fig. 11).

Fig. 11.

Schematic diagram summarizing the possible mechanisms by which H2O2 increases the expression of Giα proteins in vascular smooth muscle cells.

GRANTS

This study was supported by the Canadian Institutes of Health Research Grant MOP-53074.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: N.M. performed experiments; N.M. and M.B.A.-S. analyzed data; N.M. prepared figures; N.M. and M.B.A.-S. drafted manuscript; M.B.A.-S. conception and design of research; M.B.A.-S. interpreted results of experiments; M.B.A.-S. edited and revised manuscript; M.B.A.-S. approved final version of manuscript.

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View Abstract