Published online before print March 3, 2003, doi: 10.1161/01.HYP.0000060821.62417.35
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(Hypertension. 2003;41:882.)
© 2003 American Heart Association, Inc.
Scientific Contributions |
Akt Is a Major Downstream Target of PI3-Kinase Involved in Angiotensin II–Induced Proliferation
From INSERM U36, Collège de France, Paris, France.
Correspondence to Catherine Monnot, PhD, INSERM U36–Collège de France, 11, place Marcelin Berthelot, 75005 Paris, France. E-mail catherine.monnot{at}college-de-france.fr
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Abstract |
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Different signal transduction cascades have been implicated in angiotensin II (Ang II)–mediated cell growth, such as the extracellular signal-regulated kinase 1/2 (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K) pathways. To identify the downstream targets of PI3K involved in Ang II–induced proliferation, we used both rat aortic smooth muscle (RASM) cells and a CHO cell line stably expressing the rat AT1A receptor. The ERK1/2 and PI3K pathways are independently activated and implicated in Ang II–mediated DNA synthesis and cell number increase in these 2 cell lines. In addition, a specific inhibitor of Akt inhibited Ang II–induced Akt phosphorylation, DNA synthesis and proliferation in CHO-AT1A or RASM cells. A dominant-negative mutant of Akt was also found to selectively block Ang II–induced proliferation of CHO-AT1A cells. To further elucidate the signaling events leading to Akt activation, we used an AT1 receptor mutant (AT1AD74E), deficient for Gq protein coupling, and the intracellular calcium chelator BAPTA-AM. Although altered Akt and ERK1/2 activation was observed in the CHO-AT1AD74E cell line, blockade of intracellular calcium elevation did not affect phosphorylation of these kinases. These results provide the first evidence of a specific and necessary role of Akt in Ang II–induced proliferation through a Gq protein–dependent calcium-independent pathway.
Key Words: angiotensin II • muscle, smooth, vascular • kinase • hyperplasia • signal transduction
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Introduction |
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Besides its key regulatory role in cardiovascular and renal homeostasis, the octapeptide hormone angiotensin II (Ang II) plays a central role in the progression of cardiovascular diseases involving growth-stimulating activity such as hypertension, atherosclerosis, and restenosis after balloon angioplasty. Vascular smooth muscle cells (VSMC) are the critical targets of hypertrophic and hyperplasic responses to Ang II. The proliferative response of Ang II in VSMC involves the Ang II type 1 receptor (AT1) and both tyrosine and serine/threonine kinases. AT1 is a heterotrimeric G protein–coupled receptor (GPCR) containing 7 transmembrane helixes that is mainly coupled to a G protein from the Gq subfamily. Many components of the AT1 receptor signaling pathways have been investigated for their involvement in the mitogenic response. A direct role of the extracellular signal-regulated kinase 1/2 (ERK1/2) and of the JAK/STAT pathway has been demonstrated in Ang II–induced DNA synthesis and proliferation of cultured rat aortic smooth muscle (RASM) cells.1
Also, Ang II has been shown to activate phosphatidylinositol 3-kinases (PI3K) in cultured porcine carotid artery VSMC.2 PI3K are lipid kinases that phosphorylate the 3'-OH group of the inositol ring in inositol phospholipids after activation by a variety of extracellular stimuli. Several downstream targets of PI3K have been identified such as the phosphoinositide-dependent kinase-1 (PDK-1), the serine/threonine kinase Akt, also known as protein kinase B (PKB), and the p70 ribosomal protein S6 kinase (p70S6K). PI3K play a major role in a wide range of cellular processes, including motility and cell cycle progression.3 Recent evidence suggests that the PI3K pathway is implicated in VSMC mitogenesis. Shigematsu et al4 have shown that activation of PI3K is essential for the initiation of medial VSMC replication after balloon injury in rats. In addition, a direct role of PI3K in Ang II–induced DNA synthesis and proliferation has been described in cultured porcine carotid artery VSMC.2 However, the involvement of the downstream targets of PI3K in the Ang II–induced proliferative response is still unknown.
One of the major downstream targets of PI3K is Akt. Three isoforms of Akt have been identified in mammalian cells: Akt1/PKB
, Akt2/PKBß and Akt3/PKB
. The amino acid sequence analysis of the 3 Akt proteins reveals an N-terminal region with homology to a modular domain termed the pleckstrin homology (PH) domain. Binding of PI3K phospholipid products to the PH domain of Akt results in translocation of the Akt to the plasma membrane, where it is activated by phosphorylation through upstream kinases such as PDK-1. Once phosphorylated, Akt activates various proteins involved in many cellular responses, including cell survival and growth promotion.5 It has recently been shown that Akt is phosphorylated and activated by the AT1 receptor in a PI3K-dependent manner in various cell types.6–10 Regarding its functional role in Ang II–induced cellular responses, Akt has been shown to regulate neuritogenesis in brain neurons,11 to prevent apoptosis in HEK 293 cells,12 and to promote hypertrophy and protein synthesis in primary culture of VSMC13 and in glomerular mesangial cells.14 However, the potential implication of Akt in the cell proliferation induced by Ang II has not been established.
Therefore, the aim of our study was to examine the specific role of Akt in the proliferative effect of Ang II in RASM cells and Chinese hamster ovary (CHO) cells, stably expressing the rat AT1A receptor (CHO-AT1A). Our results show that PI3K and mitogen-activated protein kinase/ERK kinase (MEK) pathways are independently stimulated and required for the Ang II–induced proliferation in both cell lines. In addition, we demonstrated that incubation of RASM or CHO-AT1A cells with a phosphatidylinositol analog reduces both Akt phosphorylation and cell proliferation. The direct implication of Akt in Ang II–stimulated mitogenesis is further emphasized because DNA synthesis and increase in cell number of CHO-AT1A cells are abolished by the expression of an inducible Akt dominant-negative mutant. To understand the upstream pathway leading to Ang II–induced Akt activation, Akt stimulation was examined in a CHO cell line expressing the D74E mutant of the AT1A receptor, which is unable to stimulate the Gq-dependent signaling pathways. In those cells, Akt activation is completely abolished, suggesting that Gq-dependent signaling events of the AT1 receptor could be necessary for Akt phosphorylation. In conclusion, our data demonstrate a correlation between Akt activity and the cellular proliferative response to Ang II, a finding that has implications for understanding the many pathological processes involving Ang II.
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Materials
Ang II, wortmannin, rapamycin, and bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) were obtained from Sigma. LY294002 and inhibitor of Akt were purchased from Calbiochem. U0126 was obtained from Promega. Irbesartan was from Sanofi Synthelabo Recherche. 3H-thymidine was from Amersham Pharmacia Biotech.
Cell Culture
CHO-AT1A and CHO-AT1A D74E cell lines, stably expressing the rat AT1A receptor or its D74E mutant, were previously described.15,16 Wild-type CHO cells, CHO-AT1A, and CHO-AT1A D74E cell lines were grown in Ham’s F12 medium supplemented with 7.5% fetal calf serum. RASM were isolated from the aortas of male Sprague-Dawley rat as described previously.17 RASM cells were grown in DMEM supplemented with 7.5% fetal calf serum and used between passages 8 and 22. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
Transfection
The pcDNA3.1-Akt mutant mammalian expression plasmid encoding the dominant-negative Akt mutant was kindly provided by Dr Stéphanie Dimmeler, Frankfurt, Germany.18 This sequence, consisting of a truncated form of Akt (PH domain, amino acids 1 to 147), was subcloned in a mifepristone-regulated expression system (GeneSwitch System, Invitrogen) and verified by DNA sequencing. For the generation of a stable cell line expressing dominant-negative (DN) Akt, the CHO-AT1A cell line was transfected with the use of Fugene 6 (Roche).
Immunoblotting
Before Ang II stimulation (100 nmol/L), cell growth was arrested by 16 hours of serum starvation. Cell lysates were prepared with ice-cold lysis buffer, subjected to SDS-PAGE, and transferred to PVDF membranes. The membranes were probed with polyclonal anti-Akt (New England Biolabs), polyclonal phospho-Ser473 specific anti-Akt (New England Biolabs), polyclonal anti-Akt1 PH domain (Upstate Biotechnology), polyclonal anti-ERK1/2 (Upstate Biotechnology), and monoclonal phosphospecific anti-ERK1/2 (Upstate Biotechnology) overnight at 4°C. After incubation with peroxidase- or alkaline phosphatase-linked secondary antibodies, immunoreactive proteins were visualized by ECL reagent or NBT/BCIP substrates, respectively. Quantification of the relative amounts of phosphorylated proteins was performed with the National Institutes of Health Image 1.61 software and normalized for the amount of total proteins in each experiment.
DNA Synthesis
Before analysis, cell growth was arrested by 48 hours of serum starvation. Cells were then stimulated with Ang II (100 nmol/L) for 16 hours and labeled with 1 µCi/mL of 3H-thymidine for 2 hours. After washing with trichloroacetic acid, radioactivity was measured by liquid scintillation counting.
Cell Proliferation Assay
Cell proliferation was measured with the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega). Before analysis, cell growth was arrested by 48 hours of serum starvation. CHO-AT1A or RASM cells were then stimulated with Ang II (100 nmol/L) for 24 or 48 hours, respectively. After 1 hour of incubation with a phenazine methosulfate/MTS (3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium salt) mix, the absorbance was measured at 490 nm.
Statistical Analysis
The Student t test was used for statistical analysis, and data are represented as mean±SEM, where n=3 or more. A value of P<0.05 was considered statistically significant.
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Results |
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Recombinant and Endogenous AT1 Receptor–Mediated Akt Phosphorylation Requires PI3K and Is Independent of the MEK/ERK1/2 Pathway
In the CHO-AT1A cell line, Kd and Bmax values of the AT1 receptor are similar to those measured in rat smooth muscle cells in vivo. In addition, these cells express mRNAs encoding Akt 1, 2 and 3, as detected by RT-PCR, using specific primers for each isoform (data not shown), and contain detectable amounts of endogenous ERK1/2 and Akt proteins. In response to Ang II, Akt was rapidly phosphorylated in CHO-AT1A cells, with a maximal stimulation 12.5 minutes after addition of 100 nmol/L Ang II (Figure 1A, left panel), whereas the amount of total Akt remained unchanged. Akt phosphorylation was abolished by the AT1 antagonist irbesartan (88% inhibition, P<0.05; Figure 1A, left panel) indicating that Ang II–induced Ser473 phosphorylation of Akt was mediated by the AT1 receptor. Although the downstream effectors of PI3K and MEK have been thought to lie on separate pathways in response to Ang II,7,12 the presence of signaling cross-talk between PI3K and ERK1/2 has been reported.19 In CHO-AT1A cells, the concomitant phosphorylation of ERK 1 and 2 observed in response to Ang II was significantly reduced by irbesartan, (62% inhibition, P<0.05; Figure 1A, right panel).
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To investigate the role of PI3K in Ang II–induced Akt phosphorylation, the effect of 2 structurally unrelated PI3K inhibitors, LY294002 and wortmannin, was tested. Incubation of the CHO-AT1A cells with increasing concentrations of LY294002 reduced the levels of active Akt with a maximum inhibition at 10 µmol/L (91±6% of inhibition, P<0.001) and an IC50 of 2.4±0.9 µmol/L (Figure 1B), which is in agreement with the previously reported IC50 for LY294002 inhibition of PI3K activity (1.4 µmol/L).20 In contrast, doses up to 10 µmol/L LY294002 had no effect on Ang II–induced ERK1/2 phosphorylation (Figure 1B). Similarly, incubation of the cells with 100 nmol/L wortmannin resulted in a complete inhibition of Ang II–induced Akt phosphorylation without affecting ERK1/2 activation (data not shown).
U0126, the selective inhibitor of the upstream kinase of ERK1/2, strongly inhibited Ang II–induced phosphorylation of ERK1/2 at 20 µmol/L (98±1% of inhibition, P<0.001) and displayed an IC50 of 2.4±0.4 µmol/L (Figure 1C) in CHO-AT1A cells. On the contrary, U0126 had no effect on Ang II–induced Akt phosphorylation (Figure 1C).
We also investigated phosphorylation of Akt and of ERK1/2 in RASM cells. Again, Ang II–induced Akt and ERK1/2 phosphorylation were selectively inhibited in presence of LY294002 or U0126, respectively (Figure 1D). Thus, our results indicate that Ang II–induced phosphorylation of Akt is downstream of PI3K and that activation of the PI3K/Akt and MEK/ERK1/2 pathways by Ang II is independent in CHO-AT1A and RASM cells.
PI3K and MEK Pathways Are Independently Required for Recombinant and Endogenous AT1 Receptor–Mediated Proliferation
The proliferative effect of Ang II was investigated in CHO-AT1A cells using 2 approaches. First, treatment of the cells with graded concentrations of Ang II for 16 hours induced a dose-dependent stimulation of 3H-thymidine incorporation. The EC50 was 0.12±0.02 nmol/L, with a maximal stimulation observed with 100 nmol/L Ang II (data not shown). This increase in DNA synthesis was markedly reduced in the presence of 1 µmol/L irbesartan (122±10% stimulation versus 573±58%, P<0.01). To assess the importance of the PI3K/Akt pathway on Ang II–induced DNA synthesis, thymidine incorporation was measured in the presence of graded concentrations of LY294002; 10 µmol/L LY294002 resulted in 97±1% of inhibition of thymidine uptake (P<0.001; Figure 2A). Interestingly, U0126 was almost as effective as the PI3K inhibitor at blocking Ang II–stimulated DNA synthesis in these cells. Specifically, 20 µmol/L U0126 reduced DNA synthesis by over 80% (P<0.01; Figure 2A). Second, the proliferative effect of Ang II was assessed by measuring cell number by MTS assay. After 24 hours, incubation with 100 nmol/L Ang II, a sharp cell number increase was observed in CHO-AT1A cells (Figure 2B, inset). This effect was markedly reduced in presence of 1 µmol/L irbesartan (0.043±0.001 versus 0.248±0.044 increase over control in presence or absence of irbesartan, respectively, P<0.001). A smaller but significant increase in the number of RASM cells was observed after Ang II stimulation (0.180±0.006 increase over control; Figure 2B, inset). Thus, these results showed that Ang II stimulates DNA synthesis and cell proliferation, through the AT1A receptor, in CHO-AT1A cells. These cells are a valid and sensitive cellular system since their Ang II–induced proliferative response mimics the cell number increase observed in RASM cells.
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In addition, the cell number increase of CHO-AT1A or RASM was inhibited by increasing concentrations of LY294002 and U0126 (Figure 2B). The strong correlation of these data with LY294002 and U0126 inhibition of Akt and ERK1/2 phosphorylation suggests that phosphorylation of these kinases is intimately linked to the Ang II–mediated proliferative response. Furthermore, our data emphasize that although independent, both these signaling pathways must function for Ang II–induced proliferation in CHO-AT1A and RASM cells.
Ang II–Induced Endogenous Akt Activation Through PI3K Is Required for Cellular Proliferation
The role of Akt was directly examined with the use of a phosphatidylinositol analog. This compound is a pharmacological inhibitor of Akt (Akti), displaying an IC50 of 5.0±1.7 µmol/L for Akt.21 Incubation of CHO-AT1A cells with increasing concentrations of Akti inhibited Ang II–induced Akt phosphorylation (62±5% of inhibition, P<0.01), without affecting Ang II–stimulated ERK1/2 activation (Figure 3A). Similar results were obtained in RASM cells (data not shown). In addition, inhibition of Akt with Akti blocked Ang II–induced DNA synthesis in CHO-AT1A cells in a dose-dependent manner (Figure 3B). Thus, 10 µmol/L Akti eliminated 95±4% of Ang II–mediated thymidine uptake (P<0.01). Cell number increase was similarly reduced by incubation with the inhibitor of Akt in both CHO-AT1A and RASM cells (Figure 3C), demonstrating for the first time the role of Akt in the proliferative response induced by Ang II in 2 different cell types.
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In addition, CHO-AT1A cells were transfected with a dominant-negative (DN) mutant of Akt.18 This truncated form of Akt (amino acids 1 to 147) encompasses the Akt PH domain. Polyclonal antibody against Akt PH domain was used to monitor expression of the DN Akt mutant by an inducible promoter system. Treatment of the CHO-AT1A DN Akt cell line with the inducing agent mifepristone led to the appearance of a 20-kDa band (Figure 4A). The DN Akt mutant expression was induced without modification of the endogenous Akt content (60-kDa band) when compared with cells without mifepristone treatment (Figure 4A). Mifepristone concentration of 10-10 mol/L significantly reduced Ang II–induced Akt phosphorylation in CHO-AT1A DN Akt cells as compared with CHO-AT1A cells (46% inhibition, P<0.05; Figure 4B), whereas it did not affect Ang II–induced ERK1/2 phosphorylation (data not shown). Interestingly, mifepristone induction significantly decreased Ang II–induced 3H-thymidine incorporation (54±8% inhibition in CHO-AT1A DN Akt cells versus 6±4% in CHO-AT1A, P<0.05; Figure 4C). Cell number increase was strongly inhibited in presence of mifepristone in CHO-AT1A DN Akt cells (Figure 4D). Taken together, these results show that Akt inhibition achieved either with a pharmacological inhibitor or with a dominant-negative mutant of Akt correlates with a decrease of Ang II–induced cell proliferation. In summary, in response to Ang II, Akt is activated by PI3K and is involved in the cellular proliferation mediated by the AT1 receptor both in a CHO-AT1A cellular model and in the physiologically relevant RASM cell line.
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Among the diverse PI3K targets, p70S6K was also a good candidate to investigate since Ang II can stimulate it through AT1 receptor.7,22 Rapamycin, a potent inhibitor of p70S6K, did not have any discernible effect on Ang II–induced Akt or ERK1/2 activation in the CHO-AT1A cells (102±16% and 94±14% of stimulation in 100 nmol/L rapamycin, respectively). Moreover, 100 nmol/L rapamycin failed to inhibit Ang II–induced 3H-thymidine uptake in CHO-AT1A cells (105±10% of stimulation) and increase in CHO-AT1A and RASM cell number (data not shown). These data show that p70S6K is not involved in Ang II–induced proliferation of the CHO-AT1A and RASM cell lines.
Expression of an AT1A Mutant, Uncoupled From Gq Protein Activation, Leads to the Alteration of Akt and ERK1/2 Phosphorylation in Response to Ang II Through a Calcium-Independent Pathway
To gain further insight into the upstream mechanisms responsible for Akt activation, an AT1 receptor mutant was investigated for its ability to induce Akt or ERK1/2 phosphorylation in response to Ang II. The AT1A D74E mutant contains a glutamate at position 74 in the second transmembrane domain instead of the natural aspartate. Previous analysis showed the inability of this mutant in mediating agonist binding–induced G-protein activation and in stimulating Ang II–induced proliferation.16 In wild-type CHO-AT1A cells, Ang II induced Akt phosphorylation within 5 minutes. In contrast, no Akt phosphorylation was detected in CHO-AT1A D74E cells (Figure 5A). Moreover, although Ang II–induced ERK1/2 phosphorylation in wild-type CHO-AT1A cells was biphasic (first peak reached after 5 minutes followed by a sustained level maintained for 30 minutes after ligand addition), only the initial peak of ERK1/2 phosphorylation was retained in the CHO-AT1A D74E cells (Figure 5B). These results suggest that Gq-dependent signaling events are necessary for Ang II–induced Akt phosphorylation as well as for sustained ERK1/2 activation.
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In this study, we showed that the activation of Akt was dependent on aspartate 74 of the AT1 receptor. This residue is implicated in several aspects of AT1 receptor signaling, including mobilization of intracellular calcium.16 To directly elucidate the role of intracellular calcium elevation, Ang II–mediated signaling was investigated in the presence of the calcium chelator BAPTA-AM. The blockade of calcium mobilization by BAPTA-AM did not affect Ang II–induced Akt and ERK1/2 phosphorylation (Figure 5C), demonstrating that activation of these proteins is not secondary to intracellular calcium elevation.
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Discussion |
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Ang II–dependent altered VSMC growth is associated with cardiovascular diseases such as hypertension and atherosclerosis. In VSMC, Ang II leads to hyperplasic growth characterized by an increase in cell number or hypertrophic growth in the absence of cell division. To elucidate the role of the serine/threonine kinase Akt in mediating Ang II mitogenic properties, we used RASM cells expressing endogenous AT1 receptors and CHO cells stably transfected with rat AT1A receptor. The CHO-AT1A cell line exhibits a marked cell proliferation in response to Ang II when compared with more physiological models.15,19 In addition, CHO cells express little, if any, endogenous AT1 receptor and are easy to transfect, allowing us to explore the effect of Akt or AT1 receptor mutants on the signaling pathways of Ang II.
Stimulation of the MEK/ERK1/2 cascade1 and activation of PI3K2 are implicated in Ang II–induced proliferation of cultured VSMC. Previous studies have shown that Ang II–induced ERK1/2 activity and phosphorylation could be modulated by PI3K in CHO-AT1A cells19 or in RASM cells.23 However, some authors have reported an independent activation of these 2 signaling pathways in VSMC, in which inhibitors of PI3K did not block Ang II–induced ERK1/2 activation24 and inhibitors of MEK signaling had no effect on Ang II–induced activation of Akt.7 Similarly, in our study, MEK or PI3K inhibitors did not show any significant effect on Ang II–induced phosphorylation of Akt or ERK1/2, respectively, demonstrating that activation of the 2 pathways is independent in RASM and CHO-AT1A cell lines. This discrepancy most probably results from differences in the experimental procedures, including higher inhibitors concentrations, leading to blockade of unrelated pathways. Using measurement of 3H-thymidine incorporation and MTS cell proliferation assay, the present data demonstrate that the hyperplasic effect of Ang II is mediated by the PI3K/Akt pathway and the ERK1/2 cascade in CHO-AT1A and RASM cells. Indeed, both DNA synthesis and increase in cell number are completely inhibited by PI3K and MEK inhibitors. These data establish a requirement for these 2 independent parallel signaling pathways in the Ang II–induced proliferation. Similarly, independent activation of the PI3K and MEK cascades has been reported as necessary for Ang II–induced p70S6K activation in RASM cells7 and Ang II–mediated apoptosis inhibition in HEK 293 cells.12
PI3K involvement in the Ang II–induced growth-stimulating activity has been reported for protein synthesis in RASM cells25 and for proliferation in porcine VSMC.2 In the present study, the role of downstream effectors of PI3K, such as Akt and p70S6K, in Ang II proliferative response was investigated. p70S6K has been implicated in growth factor–induced cell cycle progression in VSMC26 and in Ang II–induced VSMC hypertrophy,22 but its specific role in Ang II–induced proliferation is still a matter of debate. In our study, rapamycin had no effect on Ang II–induced 3H-thymidine incorporation or increase in cell number of CHO-AT1A and RASM cells, demonstrating that p70S6K is not the downstream target of PI3K responsible for Ang II–induced proliferation.
We show the first evidence for a direct role of Akt in Ang II–mediated cell proliferation. A consistent correlation between the level of PI3K-mediated Akt phosphorylation and the efficacy of Ang II–mediated cell proliferation was observed. In addition, we found that the inhibition of Akt phosphorylation by a phosphatidylinositol analog completely blocked Ang II–mediated increase in cell number in RASM and CHO-AT1A cell lines. Finally, the use of an inducible dominant-negative Akt further emphasized the correlation between inhibition of Akt phosphorylation and inhibition of Ang II–mediated cell proliferation in CHO-AT1A cells.
The pivotal role of Akt in Ang II–mediated cell proliferation is consistent with previously described functions of Akt. Indeed, Akt is directly required downstream of PI3K for PDGF-induced DNA synthesis and c-fos gene transcription in glomerular mesangial cells.27 Conversely, Hixon et al28 reported that exclusive activation of Akt eliminates the activity of mitotic spindle cell cycle checkpoint, resulting in polyploidization and hypertrophy of RASM cells. However, as suggested by the authors, factors that promote both Akt and MEK/ERK1/2 signaling would restore a normal cell cycle progression. In the present study, stimulation of the 2 endogenous PI3K/Akt and MEK/ERK1/2 pathways leads to proliferation of RASM and CHO-AT1A cells and is consistent with this hypothesis. Therefore, the balance between the PI3K/Akt and ERK1/2 pathways activation appears to be responsible for different cellular responses.
The mechanism of Akt activation by GPCR is still unclear. In the present study, Ang II–induced Akt phosphorylation is both wortmannin and LY294002-sensitive, suggesting that Akt stimulation by the AT1 receptor takes place downstream of PI3K activation. In COS-7 cells, activation of the PI3K/Akt pathway is achieved by either the -subunit or the ß-subunits of the heterotrimeric proteins Gi and Gq.29 In contrast, Bommakanti et al30 obtained a similar ß-mediated activation of this pathway but no activation with the -subunit of heterotrimeric G proteins in HEK-293 cells. Although controversial, these results indicate a possible activation of the PI3K/Akt pathway by heterotrimeric G proteins. In VSMC, Ang II–induced Akt activity is pertussis toxin–insensitive, indicating that a Gq subfamily member rather than Gi is involved in Ang II–mediated Akt activation.6 Similarly, our experiments suggest that activation of Akt is dependent on Gq-mediated signaling events, as Ang II failed to activate Akt through the Gq protein uncoupled mutant AT1AD74E. The rapid stimulation of ERK1/2 was unchanged, whereas the late phase of ERK1/2 activation was eliminated. Furthermore, the blockade of calcium mobilization by BAPTA-AM did not affect Ang II–induced Akt and ERK1/2 phosphorylation in CHO-AT1A cells, demonstrating that activation of these pathways by the AT1 receptor is calcium-independent. This is in agreement with previous results in HEK 293 cells in which mutation of the AT1 receptor resulted in a significant inhibition of Akt and ERK1/2, through blockade of a calcium-independent pathway.12 We have previously shown that the AT1A D74E receptor is unable to stimulate Ang II–induced DNA synthesis.16 Seta et al31 also described an AT1 receptor mutant, lacking heterotrimeric G-protein coupling, still able to stimulate the early phase of ERK1/2 stimulation but unable to induce increase in cell number. In addition, Doan et al32 described another AT1 receptor–mutant deficient for intracellular calcium increase but still able to induce increase in cell number in CHO cells. Therefore, G protein–mediated calcium signaling is not necessary for the proliferation initiated by Ang II. Taken together, these results suggest that in CHO cells the onset of proliferation observed with Ang II can be attributed to G protein–dependent calcium-independent pathways such as the PI3K/Akt and the MEK/ERK1/2 pathways.
Perspective
Ang II plays a pivotal role in VSMC hyperplasia associated with cardiovascular diseases such as atherosclerosis and restenosis. Both the PI3K/Akt pathway and the ERK1/2 cascade are necessary for Ang II–induced VSMC proliferation. The current results show that the serine/threonine kinase Akt is essential for this response. Future studies can be directed toward understanding how the balance and cross-regulation between these 2 signaling pathways may influence the cellular response.
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Acknowledgments |
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This work was supported by Grant 98126 from Hoechst Marion Roussel. Céline Dugourd was awarded a PhD fellowship from Ministère de l’Education Nationale, de la Recherche et de la Technologie. We are grateful to Corinne Ardidie and Véronique Neiveyans for methodological assistance and to Kristen Frenzel for helpful discussions.
Received November 22, 2002; first decision December 27, 2002; accepted January 28, 2003.
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