Ste20-Related Kinase SLK Phosphorylates Ser188 of RhoA to Induce Vasodilation in Response to Angiotensin II Type 2 Receptor Activation
The small G protein Rho signaling pathways are recognized as major regulators of cardiovascular functions, and activation of Rho proteins appears to be a common component for the pathogenesis of hypertension and vascular proliferative disorders. Recent evidence suggests that modulation of Rho protein signaling by phosphorylation of Rho proteins provides an additional simple mechanism for coordinating Rho protein functions. Phosphorylation of RhoA by cAMP- or cGMP-activated kinase on Ser188 induces cytosolic sequestration of RhoA through increased interaction with guanine dissociation inhibitor, thereby resulting in inhibition of RhoA-dependent functions. Here we show that stimulation of angiotensin II (Ang II) type 2 receptor (AT2R) in vascular smooth muscle cells induces Ser188 phosphorylation of RhoA independently of cAMP- or cGMP-activated kinase. We identify the Ser/Thr kinase Ste20-related kinase SLK as a new kinase phosphorylating RhoA on Ser188. Activation of the signaling cascade involving Src homology 2 domain–containing protein-tyrosine phosphatase 1, casein kinase II and SLK is responsible for RhoA phosphorylation and inhibition of RhoA-mediated arterial contraction induced by AT2R activation. These results thus identify the molecular mechanism linking AT2R to RhoA inhibition and vasodilation.
RhoA is a member of the Rho protein family that has been identified as an essential regulator of vascular smooth muscle cell functions. Through the activation of its target Rho kinase, RhoA is the major regulator of the tonic component of vascular smooth muscle cell contraction and plays a critical role in the control of vascular smooth muscle differentiation, proliferation, and migration.1 Subsequent studies have demonstrated the participation of the RhoA/Rho kinase signaling pathway in several vascular pathologies, including hypertension, coronary artery spasm, effort angina, atherosclerosis, and restenosis.1,2 Indeed, although basal RhoA activity is required for homeostatic functions in physiological conditions, its sustained overactivation has pathological consequences in the vascular system, particularly in vascular smooth muscle cells. Activation of RhoA-dependent pathways is involved in excessive contraction, and thereby increases blood pressure but also in excessive cell growth and migration that participate in pathological cardiovascular remodeling.1
RhoA acts as a molecular switch. In the inactive GDP-bound form, RhoA is locked in the cytosol by guanine dissociation inhibitors (GDIs). In the active GTP-bound form released from GDI, RhoA translocates to plasma membrane where it interacts with effectors to transduce the signal downstream. GTPase-activating proteins then turn off activation. In addition to this regulation, we and others have demonstrated that phosphorylation/dephosphorylation cycle also controls RhoA activity.3 Cyclic GMP-dependent protein kinase (PKG) or cAMP-dependent protein kinase A (PKA) phosphorylate Ser188 of RhoA.4,5 Both in vitro and in vivo experiments indicated that Ser188 phosphorylation of RhoA induces increased association to GDI, leading to cytosolic accumulation of RhoA,6 and inhibition of RhoA-mediated functions.5 RhoA phosphorylation thus appears as a simple mechanism that could be used to control the dynamics of RhoA protein actions and to permit specific termination of RhoA protein signals.
It has been recently shown that inactivation of RhoA/Rho kinase signaling pathway may play a role in the vasodilation induced by angiotensin II (Ang II) type 2 receptor (AT2R) activation.7 Although most physiological responses triggered by Ang II such as vascular smooth muscle cell contraction, growth and inflammation have been ascribed to Ang II type 1 receptor (AT1R) activation, accumulating evidence indicates that AT2R antagonizes the effects of the AT1R, especially by inducing vasodilation, antigrowth and antiinflammatory actions.8 Coupling of AT2R to intracellular signaling pathways is less well understood than that of AT1R and seems to depend on the cell type. Three major cascades can be activated following AT2R stimulation including: (1) activation of protein phosphatases, in particular Src homology 2 domain–containing protein-tyrosine phosphatase 1 (SHP-1); (2) regulation of the nitric oxide/cGMP system; and (3) stimulation of PLA2 and release of arachidonic acid.9
Here we directly analyze the effect of Ang II on serine phosphorylation of RhoA both in vitro in vascular smooth muscle cells and in/ex vivo in artery samples. We describe the SHP-1/caseine kinase II (CK2)/Ste20-related kinase (SLK) pathway as a new signaling cascade activated following AT2R stimulation and identify SLK as a novel kinase phosphorylating RhoA on Ser188. SHP-1–dependent SLK-mediated RhoA phosphorylation is responsible for AT2R-induced vasodilation and may contribute to the antihypertensive effects of AT1R inhibitors.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Cell Culture and Transfection
Rat aortic smooth muscle cells were isolated by enzymatic dissociation. The different constructions used and small interfering (si)RNA were transfected by electroporation (Nucleofector, Amaxa). Protein extracts form cell cultures were used for coimmunoprecipitation and Western blot analysis.
Recombinant Protein Expression and Kinase Assays
GST-WT-RhoA and GST-S188A-RhoA were expressed in Escherichia coli and purified as previously described.5 Phosphorylation of recombinant RhoA was determined using immunoprecipitated SLK and SLK mutants. SLK activity was assessed by measuring the extent of SLK autophosphorylation.
Measurement of RhoA/Rho Kinase Activity
Activation of the RhoA/Rho kinase pathway was monitored by Western blot analysis of the phosphorylation level of the Rho kinase target myosin phosphatase target subunit 1 (MYPT).
Thoracic aorta rings were transfected with siRNA for 48 hours, then suspended under isometric conditions and connected to a force transducer (Pioden Controls Ltd).
Male WKY rats and SHR (250 g) were treated for 2 weeks with placebo or AT1R antagonist (candesartan; 2 mg/kg per day) in their drinking water. All experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals.
All results are expressed as the means±SD of sample size n. Significance was tested by ANOVA or Student t test.
AT2R Stimulation Induces Serine Phosphorylation of RhoA
RhoA phosphorylation, association to GDI, activation, and expression were analyzed in rat aortic smooth muscle cells stimulated with Ang II (0.1 μmol/L) for 1 to 360 minutes. Figure 1A shows that serine-phosphorylated RhoA was detected under control condition. Ang II induced a gradual increase in the amount of serine-phosphorylated RhoA, associated with an increased association to GDI (7.3±0.2 fold over control at 360 minutes, n=3), indicating cytosolic relocalization of RhoA. Analysis of MYPT phosphorylation indicates that Ang II also induced activation of RhoA/Rho kinase, with 2 peaks of activation at 5 (2.8±0.1 fold over control, n=3) and 60 to 120 minutes (3.0±0.1 fold over control, n=3).
In the presence of the AT1R inhibitor losartan, Ang II–induced increase in MYPT phosphorylation was totally inhibited, but the Ang II–induced phosphorylation of RhoA and increased GDI association were still present and even more pronounced for short Ang II applications (Figure 1A and 1B). The increase in RhoA phosphorylation occurred in a concentration-dependent manner (additional data, Figure I). Ang II–mediated increase in RhoA phosphorylation and association to GDI was abolished in the presence of the AT2R antagonist PD123319 (Figure 1C) and was mimicked by stimulation with the AT2R agonist CGP42112A (Figure 1D). In cells stimulated with CGP42112A for 360 minutes, the amount of serine-phosphorylated was increased by 7.6±0.2-fold over control (n=3), and the association to GDI enhanced by 7.2±0.2-fold over control (n=3). In contrast, CGP42112A did not stimulate MYPT phosphorylation and Ang II–mediated increase in MYPT phosphorylation was still observed in the presence of PD123319 (Figure 1C and 1D). This pharmacological analysis thus suggests that whereas Ang II activates the RhoA/Rho kinase pathway through its interaction with AT1R, stimulation of AT2R increases RhoA phosphorylation and its interaction with cytosolic GDI. This conclusion is supported by Western blot analysis and quantitative RT-PCR experiments, demonstrating the expression of both AT1R and AT2R in cultured smooth muscle cells (Figure 1E).
To further confirm this result, we used specific siRNA to selectively knockdown AT1R and AT2R. In cells treated with AT1R siRNA, a strong increase in RhoA phosphorylation was still produced by Ang II (Figure 1F). In contrast, siRNA-mediated AT2R silencing completely prevented Ang II–induced RhoA phosphorylation (Figure 1F). Therefore, in addition to pharmacological data, specific genetic manipulation of AT1R and AT2R demonstrates that Ang II–induced RhoA phosphorylation is mediated by AT2R. This role of AT2R was also confirmed in CHO cells that overexpressed AT1R but did not express AT2R (additional data, Figure II).
Ang II Signaling Regulates RhoA Phosphorylation In Vivo
Chronic pharmacological inhibition of AT1R is associated with Ang II and AT2R upregulation.10 If AT2R-mediated serine phosphorylation of RhoA is a physiological process regulating RhoA-dependent function in vivo, it is thus expected that the amount of phosphorylated RhoA is increased in animals treated with AT1R inhibitor. To assess this hypothesis, we analyzed RhoA phosphorylation and RhoA binding to GDI in pulmonary artery and aorta of candesartan-treated normotensive (WKY) and spontaneously hypertensive rats (SHR). Serine-phosphorylated RhoA is detected under control condition both in WKY and SHR (Figure 2A). The amount of serine-phosphorylated RhoA immunoprecipitated from pulmonary artery and aorta of candesartan-treated SHR and WKY rats and its association to GDI was strongly increased compared to their controls (Figure 2A). These results thus provide evidence that phosphorylation and cytosolic localization of RhoA in arteries are regulated by Ang II signaling pathway in vivo.
AT2R Stimulation Induces Phosphorylation of RhoA on Ser188
The only serine residue shown to be subjected to phosphorylation in the RhoA sequence is the Ser188.4,5 We thus analyzed whether the Ser188 was the site for AT2R-mediated RhoA phosphorylation by expressing hemagglutinin (HA)-tagged wild-type RhoA (WT-RhoA) or Ser188 phospho-resistant (S188A-RhoA) or Ser188 phospho-mimetic (S188E-RhoA) RhoA mutants in aortic smooth muscle cells. As expected, WT-RhoA was phosphorylated on serine by Ang II, in association with its increased interaction with cytosolic GDI (Figure 2B). Under basal conditions, S188E-RhoA showed an enhanced association to GDI, but neither its binding to GDI nor its serine phosphorylation was increased by Ang II stimulation (Figure 2B). In agreement with this observation, the level of serine phosphorylation and interaction with GDI of S188A-RhoA was not changed in presence of Ang II. These data indicate that AT2R stimulation induces phosphorylation of RhoA on Ser188.
AT2R-Induced Ser188 Phosphorylation of RhoA Does Not Need NO Synthase, PKA, or PKG Activity
The involvement of NO synthase (NOS) and PKG activity in Ang II–mediated Ser188 RhoA phosphorylation has been assessed by the use of their respective inhibitors l-NG-nitro arginine (L-NNA) and Rp-8-Br-PET-cGMPS. The increase in RhoA phosphorylation induced by Ang II for was not affected by NOS or PKG inhibition (5.0±0.3-fold over control versus 5.4±0.2 in control, n=4, P>0.5; Figure 3A). Western blot using an anti-PKG antibody on immunoprecipitated RhoA showed that PKG, associated with RhoA under basal condition, was released by AT2R stimulation (Figure 3B). In similar experiments, the use of H-89 revealed that inhibition of PKA did not modify AT2R stimulation–induced RhoA phosphorylation (Figure 3C), and the same observation was made in the presence of both PKG and PKA inhibitors (data not shown). Examination of PKA/RhoA interaction indicated that PKA interacted with RhoA under control condition but AT2R stimulation abolished this interaction (Figure 3D). The concentration of PKG and PKA inhibitors used efficiently inhibited PKG and PKA activity (additional data, Figure III). These results thus provide evidence that AT2R-induced increase in RhoA phosphorylation is not mediated through the activation of NOS, PKG, or PKA. The involvement of other kinases such as phosphatidylinositol 3-kinase, MAPK/ERK kinase (MEK1/2), and protein kinase C has also been ruled out by the use of LY 294006, PD98059, and U0126, and GF109203X, respectively (Figure 3E). In our experimental conditions, the efficiency of LY294006 has been checked by measuring Akt phosphorylation, PD98059 and U0126 by measuring ERK1/2 phosphorylation and GF109203X, by measuring CPI-17 phosphorylation (data not shown).
The involvement of serine phosphatase activity in AT2R-induced Ser188 phosphorylation of RhoA revealed that AT2R-induced serine phosphorylation of RhoA could not result from reduction of phosphatase activities which, in fact, are increased by AT2R stimulation (Figure IV in the online data supplement).
SLK Associates to RhoA
SLK is a serine/threonine kinase ubiquitously expressed in adult tissues. Expression of active SLK induced loss of actin stress fibers and, therefore, mimicked the effects of RhoA phosphorylation.5,11 This observation thus prompted us to select SLK as a potential RhoA-phosphorylating kinase. Western blot analysis indicated that SLK was present in vascular smooth muscle cells (Figure 4A). Specificity of the immunoreactive band has been checked (supplemental Figure V). Coimmunoprecipitation experiments further revealed that SLK interacted with RhoA in vascular smooth muscle cells, and the amount of SLK that coimmunoprecipitated with RhoA was not significantly modified by AT2R stimulation (Figure 4B and supplemental data Figure VI).
SLK Phosphorylates RhoA on Ser188
An in vitro kinase assay was performed using recombinant RhoA as substrate. WT-SLK and the active 1-373-SLK but not the kinase-dead mutant K63R-SLK induced phosphorylation of RhoA, indicating that RhoA was indeed a substrate for SLK (Figure 4C). The use of S188A-RhoA mutant prevented phosphorylation of RhoA by SLK and 1-373-SLK, indicating that SLK-mediated RhoA phosphorylation occurred on Ser188 (Figure 4C).
To investigate whether the phosphorylation of RhoA following AT2R stimulation was mediated by SLK, phosphorylation of endogenous RhoA has been analyzed in smooth muscle cells expressing WT-SLK and SLK mutants (Figure 4D). AT2R stimulation induced RhoA phosphorylation in cells expressing WT-SLK but not in cells expressing the inactive K63R-SLK mutant. On the opposite, expression of the active 1-373-SLK strongly increased the basal level of RhoA phosphorylation, which was not further increased on AT2R stimulation. A similar result was obtained with the SS/AA-SLK mutant. Serine phosphorylation of SLK at position 347/348 downregulates its kinase activity and the phosphorylation-resistant SS/AA-SLK mutant obtained by point mutation of these serine residues displayed a high kinase activity.12 These in vitro and in-cell results thus suggest that AT2R-induced Ser188 phosphorylation of RhoA is mediated by SLK.
AT2R Stimulation Induces Upregulation of SLK Kinase Activity
To further confirm the role of SLK in AT2R-induced RhoA phosphorylation, we directly measured the effect of AT2R stimulation on SLK activity by performing SLK kinase assays. SLK kinase activity was increased by 1.5- to 3-fold in cells stimulated with Ang II (Figure 4E). This effect was inhibited in the presence of PD123319, indicating that the stimulatory action of Ang II on SLK activity is mediated by AT2R stimulation.
AT2R Stimulation Induces Decrease of SLK Phosphorylation
The observation that the phosphorylation resistant mutant SS/AA-SLK increased RhoA phosphorylation and prevented further action of AT2R stimulation on RhoA phosphorylation prompted us to hypothesize that AT2R-induced stimulation of SLK activity could result from its dephosphorylation. To address this hypothesis, we examined the level of serine phosphorylation of immunoprecipitated SLK and SLK mutants. Under basal conditions, SLK and K63R-SLK mutant, but not the SS/AA-SLK mutant, are phosphorylated on serine residues, indicating that the phosphorylated serine residues recognized by the anti-phosphoserine antibody are the serine residues at position 347/348 and that these residues are basally phosphorylated (Figure 5A). Stimulation of AT2R produced a 5- to 8-fold decrease in the level of SLK and K63R-SLK phosphorylation (Figure 5A). No change was observed for the SS/AA-SLK mutant (Figure 5A). These results indicate that SLK is basally phosphorylated on serine at position 347/348 in vascular smooth muscle cells and that stimulation of AT2R decreased phosphorylation of these serine residues, thus suggesting that AT2R-induced stimulation of SLK kinase activity results from the release of the inhibitory action of the phosphoserine at position 347/348.
AT2R Stimulation–Induced RhoA Phosphorylation Involves CK2
CK2 has been identified as the kinase that phosphorylates SLK on serine residues 347 and 348.12 If CK2 is the kinase responsible for the basal phosphorylation of SLK in vascular smooth muscle cells, it is expected that inhibition of CK2 would lead to increase SLK kinase activity and, in turn, increase RhoA phosphorylation. The CK2 inhibitor 1 induced a 4- to 7-fold increase in RhoA phosphorylation in cells expressing SLK (Figure 5B). Under this condition, AT2R stimulation was no longer able to induce further increase in RhoA phosphorylation (data not shown). This effect was not observed in cells expressing the inactive K63R-SLK mutant, indicating that the increased RhoA phosphorylation induced by CK2 inhibition was attributable to stimulatory effect on SLK activity. This result has been confirmed by the use of siRNA targeting CK2 that produced a 4-fold increase in RhoA phosphorylation in cells expressing SLK but not in cells expressing the K63R-SLK mutant (supplemental Figure VI).
AT2R Stimulation–Induced SHP-1 Activation Mediates Dephosphorylation of CK2
As CK2 inhibition mimicked the effect of AT2R stimulation on RhoA phosphorylation, it could be envisaged that decreased phosphorylation and activation of SLK induced by AT2R indirectly resulted from AT2R-mediated inhibition of CK2. Tyrosine phosphorylation of CK2 by the Src family tyrosine kinase activates its kinase activity, whereas both tyrosine phosphorylation and activity of CK2 are inhibited by specific Src blockers.13 To investigate the potential role of CK2 in AT2R-induced SLK mediated RhoA phosphorylation, we thus examined the tyrosine phosphorylation of CK2. Figure 5C shows that CK2 is basally phosphorylated and that stimulation of AT2R decreased CK2 phosphorylation (Figure 5C).
The SH2 domain containing cytosolic tyrosine phosphatase, SHP-1, was identified as a major signal transducer of the AT2R.14 As shown in Figure 5C, siRNA-mediated SHP-1 silencing completely prevented the dephosphorylation of CK2 induced by AT2R stimulation. The role of SHP-1 was further confirmed by coimmunoprecipitation experiments. In cells expressing SHP-1, AT2R stimulation increased the amount of CK2 that coimmunoprecipitates with SHP-1 (Figure 5D). This AT2R-induced increase in CK2/SHP-1 interaction was even stronger in cell expressing the catalytically inactive substrate trapping SHP-1 mutant C453S-SHP-1 (Figure 5D). These results thus suggest that AT2R-induced dephosphorylation of CK2 through SHP-1.
AT2R Stimulation–Induced RhoA Phosphorylation Depends on SHP-1 Activity
We then directly analyzed the involvement of SHP-1 in the mechanism coupling AT2R stimulation to SLK-mediated RhoA phosphorylation. As expected, if SLK activity were indirectly upregulated by AT2R activation through SHP-1–mediated CK2 dephosphorylation, siRNA-mediated SHP-1 silencing abolished AT2R stimulation-induced RhoA phosphorylation (Figure 5E). This result was further confirmed by expression of catalytically inactive C453S-SHP-1 and D419A-SHP-1 mutants that completely inhibited AT2R stimulation–induced RhoA phosphorylation (Figure 5F). These results thus indicate that SLK-mediated AT2R stimulation–induced RhoA phosphorylation depends on SHP-1 phosphatase activity.
Essential Role of RhoA in AT2R Stimulation-Induced Vasodilation
To assess the functional consequence of the SHP-1–dependent SLK-mediated RhoA phosphorylation induced by AT2R stimulation, we performed contraction measurements in aorta rings from candesartan-treated SHR rats (Figure 6). As previously reported, increasing concentrations of Ang II–induced relaxation of aorta rings precontracted by noradrenaline (NA) (Figure 6A). This effect was abolished in the presence of the AT2R inhibitor PD123319 (Figure 6A) and in rings pretreated with AT2R siRNA (supplemental Figure VIII). NA-induced contraction resulted of both a calcium-dependent component, activated by a rise in intracellular calcium concentration, and a RhoA-dependent component, corresponding to the calcium sensitization of contractile protein. To identify which component was inhibited by AT2R stimulation, we selectively suppressed the RhoA-dependent component of the NA-induced contraction by a 48-hour treatment of aorta rings with siRNA targeting RhoA. Western blot analysis confirmed that siRNA efficiently produced 80% to 100% inhibition of RhoA expression (Figure 6B). Under these conditions, the amplitude of the NA-induced contraction was reduced by 40% to 50%, and the remaining contraction was not modified in the presence of the Rho kinase inhibitor Y-27632 (10 μmol/L, not shown). This calcium-dependent NA-induced contraction was not relaxed by AT2R stimulation, indicating that AT2R stimulation-induced relaxation completely depended on inhibition of the RhoA-dependent component of the contraction (Figure 6B). To further assess the involvement of the SHP-1/SLK signaling pathway, similar experiments were performed after treatment of aortic rings with siRNA targeting SHP-1 and SLK. Treatment with SHP-1 siRNA and SLK siRNA did not modify the amplitude of the NA-induced contraction but completely prevented the relaxing effect of AT2R stimulation (Figure 6C and 6D). These results thus support the essential role of SHP-1–dependent SLK-mediated RhoA phosphorylation in the relaxing effect of AT2R stimulation.
Our work identifies SLK as a new serine-threonine kinase that phosphorylates Ser188 of RhoA and establishes a novel signaling cascade downstream to AT2R stimulation. In vascular smooth muscle cells, AT2R stimulation by Ang II activates SLK through a coupling mechanism involving SHP-1 and CK2. This signaling pathway is responsible for AT2R stimulation–mediated vasodilation (Figure 7).
Although it is widely accepted that AT1R accounts for a large part of the cardiovascular effects induced by Ang II, an increasing body of evidence indicates that AT2R also contributes to the regulation of blood pressure and renal function.9 Stimulation of AT2R undoubtedly induces relaxation in several vascular territories.15 Studies in animal models of hypertension have revealed that AT2R is upregulated and mediates vasodilation only when AT1Rs are blocked, suggesting that AT2R participates in the mechanisms, whereby Ang II receptor antagonism lowers blood pressure.7 Recent study confirms these data in human by showing that AT2Rs are upregulated and contribute to Ang II–induced vasodilation in resistance arteries of hypertensive diabetic patients treated with AT1R blockers.16 Although the intracellular signaling pathways downstream to AT2R are now fully defined, several mechanisms have been proposed to participate to AT2R-mediated relaxation/vasodilation.15 Both endothelium-independent and endothelium-dependent AT2R-mediated vasodilations have been described, the latter being associated with stimulation of NO/cGMP pathway, either directly or indirectly through the increased release of bradykinin.15,17 It has been proposed that AT2R stimulation blocked the Na+–H+ exchanger, promoting intracellular acidosis, which, in turn, activates kininogenases in endothelial and smooth muscle cells to cleave bradykinin from intracellularly stored kininogens.18 Recently, the inhibition of the RhoA/Rho kinase signaling pathway has been suggested to play a role in AT2R-induced vasodilation.7 In conditions associated with AT2R upregulation (valsartan treated-hypertensive rats), Ang II–induced vasorelaxation is associated with reduced RhoA/Rho kinase activation that could result from phosphorylation of RhoA on Ser188.19 Stimulation of NOS activity and activation of PKG, the expression of which is increased in hypertensive rats treated with AT1R blockers, have been proposed to mediate AT2R-induced downregulation of RhoA/Rho kinase pathway.20 However, neither the level of RhoA phosphorylation nor the involvement of PKG have been directly assessed in these studies.
By using arteries from a similar experimental model, our results obtained by siRNA-mediated RhoA silencing confirm that AT2R-induced relaxation is attributable to inhibition of the RhoA-dependent component of the contraction.7 Our results show that AT2R stimulation indeed induced RhoA phosphorylation in vascular smooth muscle cells. Through this mechanism, AT2Rs counteract the activation of RhoA/Rho kinase mediated by vasoconstrictors, in particular by AT1R stimulation. On the other hand, AT1R seems to oppose to AT2R-mediated RhoA phosphorylation because RhoA phosphorylation induced by Ang II in the presence of losartan, or by the AT2 agonist CGP42112, is increased and occurred earlier. The effect of Ang II stimulation on RhoA phosphorylation and RhoA/Rho kinase activation is therefore likely to reflect the balance of AT1R/AT2R expression in given artery and condition.
In this study, we show that RhoA phosphorylation is responsible for the AT2R-induced relaxation. However, as the RhoA/Rho kinase pathways is also involved in a wide range of cellular processes,1 it could be supposed that AT2R-mediated inhibition of the RhoA/Rho kinase pathway also participates to other AT2R-mediated actions. Interestingly, among the numerous targets of the RhoA/Rho kinase pathway is the Na+–H+ exchanger NHE1, which is activated by Rho kinase.21 The inhibition of RhoA/Rho kinase, resulting from AT2R-mediated RhoA phosphorylation may therefore participate to the inhibition of the Na+-H+ exchanger observed in response to AT2R stimulation.18
Our analysis of the molecular mechanisms linking AT2R to RhoA in vascular smooth muscle cells did not confirm the role of NO and PKG previously suggested in AT2R-induced RhoA phosphorylation. Here, we demonstrate that inhibition of the RhoA-dependent contraction through SHP-1– and SLK-dependent mechanism is responsible for the relaxing effect of AT2R stimulation. This observation thus supports the major role of SHP-1–dependent SLK-mediated RhoA phosphorylation and consequent RhoA/Rho kinase inhibition in the vasodilator action of AT2R.
SHP-1 has been identified as an early transducer in AT2R signaling. SHP-1 is involved in AT2R-mediated MAPK cascades inhibition; however, its contribution to AT2-dependent processes that are independent of MAPK pathways remained questioned.14 By showing that SHP-1 is a key mediator of AT2R-mediated RhoA phosphorylation and relaxation, our results reinforce the large importance of SHP-1 as general signal transducer of AT2R and suggest that other upstream signals that lead to SHP-1 activation may also modulate RhoA phosphorylation. Stimulation of AT2R promotes apoptosis of cultured vascular smooth muscle cells and SHP-1 has been shown to play pivotal role in the proapoptotic effect of AT2R stimulation.22,23 SLK was also found to induce apoptosis in fibroblasts.24 Our observation that AT2R stimulation–mediated SLK activation depended on SHP-1 may suggests that SLK could also be involved in the proapoptotic effect of AT2R activation in vascular smooth muscle cells.
In conclusion, we identify SLK as a new kinase that regulates RhoA signaling and vascular smooth muscle contraction and describe a novel signaling pathway that negatively controls RhoA-dependent functions by phosphorylation of RhoA on Ser188. This signaling pathway involving SHP-1, CK2, and SLK is responsible for AT2R-induced vasodilation by inhibiting RhoA/Rho kinase-dependent contraction. Because blockade of AT1R increases both the plasma level of Ang II and the expression of AT2R, treatment with AT1R inhibitors presently used in clinical practice may lead to AT2R stimulation. By showing that AT2R activation induces Rho kinase inhibition, our results contribute to a better understanding of mechanisms whereby AT2Rs add to the antihypertensive effects of AT1R inhibitors. Our results also suggest that directly targeting the AT2R in cardiovascular disease would be useful.
We thank Dr R. Siraganian (National Institute of Dental and Craniofacial Research, NIH, Bethesda, Md) and Dr C. Monot (Collège de France, Paris, France) for providing us with SHP-1 constructs and AT1R-overexpressing CHO cells, respectively; N. Vaillant and C. Schleder for excellent technical assistance.
Sources of Funding
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Agence Nationale de la Recherche and the Fondation pour la Recherche Médicale. C.G. and M.R.-D. were supported by the Centre National de la Recherche Scientifique and the Société Française d’Hypertension Artérielle, respectively.
Original received September 24, 2007; revision received March 26, 2008; accepted April 3, 2008.
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