Published online before print June 14, 2007, doi: 10.1161/ATVBAHA.107.146746
© 2007 American Heart Association, Inc.
Vascular Biology |
Endothelin-1, but not Ang II, Activates MAP Kinases Through c-Src–Independent Ras-Raf–Dependent Pathways in Vascular Smooth Muscle Cells
From the Kidney Research Centre (A.Y., G.E.C., A.C.I.M., A.B.A., R.M.T.), Ottawa Health Research Institute, University of Ottawa, Ontario, Canada; the Department of Pharmacology (A.Y., R.C.T.), Institute of Biomedical Sciences-USP, Sao Paulo, Brazil; and Lady Davis Institute for Medical Research (E.L.S.), Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Canada.
Correspondence to Rhian M. Touyz, MD, PhD, Kidney Research Centre, University of Ottawa/Ottawa Health Research Institute, 451 Smyth Rd, Ottawa, ON, KIH 8M5. E-mail rtouyz{at}uottawa.ca
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Abstract |
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Objective— Endothelin-1 (ET-1) and angiotensin II (Ang II) activate common signaling pathways to promote changes in vascular reactivity, remodeling, inflammation, and oxidative stress. Here we sought to determine whether upstream regulators of mitogen-activated protein kinases (MAPKs) are differentially regulated by ET-1 and Ang II focusing on the role of c-Src and the small GTPase Ras.
Methods and Results— Mesenteric vascular smooth muscle cells (VSMCs) from mice with different disruption levels in the c-Src gene (c-Src+/– and c-Src–/–) and wild-type (c-Src+/+) were used. ET-1 and Ang II induced extracellular signal-regulated kinase (ERK) 1/2, SAPK/JNK, and p38MAPK phosphorylation in c-Src+/+ VSMCs. In VSMCs from c-Src+/– and c-Src–/–, Ang II effects were blunted, whereas c-Src deficiency had no effect in ET-1–induced MAPK activation. Ang II but not ET-1 induced c-Src phosphorylation in c-Src+/+ VSMCs. Activation of c-Raf, an effector of Ras, was significantly increased by ET-1 and Ang II in c-Src+/+ VSMCs. Ang II but not ET-1–mediated c-Raf phosphorylation was inhibited by c-Src deficiency. Knockdown of Ras by siRNA inhibited both ET-1 and Ang II–induced MAPK phosphorylation.
Conclusions— Our data indicate differential regulation of MAPKs by distinct G protein–coupled receptors. Whereas Ang II has an obligatory need for c-Src, ET-1 mediates its actions through a c-Src–independent Ras-Raf–dependent pathway for MAPK activation. These findings suggest that Ang II and ET-1 can activate similar signaling pathways through unrelated mechanisms. MAP kinases are an important point of convergence for Ang II and ET-1.
c-Src and Ras involvement in MAPK activation by ET-1 and Ang II was examined. ET-1, but not Ang II, induced MAPK phosphorylation in c-Src–deficient VSMCs. Ras knockdown by siRNA inhibited both Ang II– and ET-1–induced effects. Our findings demonstrate that whereas MAPK regulation by Ang II is c-Src–sensitive, ET-1–mediated actions involve c-Src–independent Ras-Raf–dependent pathways.
Key Words: MAPK • Src tyrosine kinases • Ras • c-Raf • signal transduction • G protein–coupled receptors
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Introduction |
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Angiotensin II (Ang II) and endothelin-1 (ET-1) are vasoactive agents that mediate multiple vascular actions through activation of distinct G protein–coupled receptors (GPCR).1,2 Both peptides play an important role in hypertension and cardiovascular diseases by promoting changes in vascular reactivity and endothelial function, cardiovascular fibrosis, tissue remodeling, inflammation, and oxidative stress.2,3 These actions are mediated through a myriad of signaling pathways, including mitogen-activated protein kinases (MAPKs), small GTPases, and receptor and nonreceptor tyrosine kinases.4–12
MAPKs are a family of serine/threonine kinases which are classically associated with vascular smooth muscle cell (VSMC) contraction, migration, adhesion, collagen deposition, cell growth, differentiation, and survival.13 Of the major MAPKs, extracellular signal-regulated kinases (ERK1/2), p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNK) are the best characterized.13 The complex signaling networks that underlie MAPK activation typically require phosphorylation by a MAPK kinase also known as MEK. The ERK1/2 phosphorylation cascade involves MEK1/2 (MAP/ERK kinase) whereas the signaling processes leading to SAPK/JNK and p38 MAPK activation involve MEK4/7 and MEK3/6, respectively.13 Activation of MAPKs has been reported to be primarily dependent on the nonreceptor tyrosine kinase c-Src in different cell types.14–16 To date, at least 14 Src-related kinases have been identifed,17 of which the 60 kDa c-Src is the most abundantly expressed isoform in VSMCs and rapidly activated by GPCR.14,18–20 Other proximal regulators of MEK include the Ras-Raf pathway, which may not necessarily involve c-Src.21
Cellular mechanisms and signaling pathways that are involved in hypertensive vascular damage are currently subjects of intensive investigation. We highlighted the importance of c-Src in the molecular and cellular processes underlying the activation of ERK1/2-dependent growth signaling by Ang II in VSMCs from resistance arteries of essential hypertensive patients.15 In addition, c-Src, as a critical proximal regulator of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase-driven superoxide anion generation, once activated by Ang II, contributes to the amplification of oxidative stress–induced vascular redox–sensitive MAPK activation.22,23 Although it is now clearly established that c-Src is an important mediator of Ang II effects,16,24,25 evidence for a direct role of c-Src in ET-1–induced MAPK activation in VSMCs is still lacking.
Here we sought to determine whether upstream regulators of MAPK activation, in particular c-Src and the small GTPase Ras, are differentially regulated by ET-1 and Ang II in VSMCs. To address this issue we used VSMCs from c-Src–deficient mice with different levels of disruption of the c-Src gene. We also downregulated the Ras-Raf pathway with siRNA. Our findings indicate that ET-1 and Ang II, which both signal through GPCRs, modulate MAPK phosphorylation through distinct pathways. Whereas Ang II has an obligatory need for c-Src, ET-1 mediates its actions through a Ras-Raf–dependent c-Src–independent pathway for MAPK activation. Such differences may contribute to distinct functional responses of these agonists in VSMCs.
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Methods |
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Cell Culture
The study was carried out according to the recommendations of the Canadian Council for Animal Care. VSMCs from c-Src+/– and c-Src–/– mice, which are heterozygous and homozygous, respectively for a disruption in the c-Src gene, and wild-type c-Src+/+ mice were studied. The c-Src+/–- and c-Src–/– mice were generated from a c-Src+/– F1 cross26 and were genotyped by polymerase chain reaction (PCR). VSMCs derived from mice (8 to 10 weeks old) mesenteric arteries were isolated and characterized as described in detail previously.27 Briefly, mesenteric beds were cleaned of adipose and connective tissue; VSMCs were dissociated by enzymatic digestion of vascular arcades for 30 to 60 minutes at 37°C. Cell suspension was centrifuged and resuspended in Dulbecco modified Eagle medium containing 10% fetal calf serum, 2 mmol/L glutamine, 20 mmol/L HEPES (pH 7.4), and antibiotics. At subconfluence, the culture medium was replaced with serum-free medium for 24 hours to render the cells quiescent. Low-passage cells (passages 4 to 7) were studied.
Western Blotting
VSMCs from c-Src+/+, c-Src+/–, and c-Src–/– mice were stimulated with either Ang II (0.1 µmol/L) or ET-1 (0.1 µmol/L). After SDS-PAGE separation of proteins, samples were transferred to nitrocellulose membranes as previously described (available online at http://atvb.ahajournals.org).28 Membranes were then incubated with the following primary antibodies (1:1000): anti–c-Src (Tyr418, anti-p38MAPK (Thr180/Tyr182), anti-ERK1/2 (Thr202/Tyr204), anti-SAPK/JNK (Thr183/Tyr185), anti-c-Raf (Ser338), anti-Yes, anti-Fyn, anti-AT1 anti-AT2, anti-ETA and anti-ETB. Washed membranes were incubated with horseradish peroxidase–conjugated second antibody. Immunoreactive proteins were detected by chemiluminescence. For ERK1/2 and JNK, where 2 bands (isoforms) were visible, combined density of the 2 bands was evaluated. β-actin were used as loading control.
RNA Interference and Cell Transfection
Small interfering RNAs (siRNA) technique was used to downregulate Ras-Raf pathway in VSMCs (available online at http://atvb.ahajournals.org). After 24 hours transfection, cells were stimulated with ET-1 (0.1 µmol/L, 2 and 5 minutes) or Ang II (0.1 µmol/L, 1 and 2 minutes) and MAPKs phosphorylation was evaluated by Western blotting as described above.
Data Analysis
Ang II– and ET-1–stimulated effects were determined as the percent increase over control, with the control normalized to 100%. Results are presented as mean±SEM and compared by ANOVA. P<0.05 was considered significant.
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Results |
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Effects of ET-1 and Ang II on ERK 1/2 Phosphorylation in VSMCs
As shown in Figure 1A, ET-1 time-dependently increased ERK 1/2 phosphorylation. In VSMCs from c-Src+/+ mice, maximal responses were achieved within 2 minutes and the phosphorylation returned to basal levels after 30 minutes. Similar responses were observed in VSMCs from c-Src+/– and c-Src–/– mice. Ang II induced a biphasic increase in ERK 1/2 phosphorylation in VSMCs from c-Src+/+ mice with a peak response obtained within 2 minutes, followed by a second response within 10 minutes (Figure 1B). Unlike ET-1, phosphorylation levels of ERK 1/2 were not increased by Ang II in VSMC from c-Src+/– and c-Src–/– mice.
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Effects of ET-1 and Ang II on SAPK/JNK Phosphorylation in VSMCs
After ET-1 stimulation, SAPK/JNK was rapidly phosphorylated in VSMCs from c-Src+/+, c-Src+/–, and c-Src–/– mice (Figure 2A). Phosphorylation levels returned to basal after 30 minutes of stimulation. Ang II induced SAPK/JNK phosphorylation within 2 minutes. This effect was transient and phosphorylation levels returned to basal after 5 minutes. In VSMCs from c-Src+/– and c-Src–/– Ang II failed to induce SAPK/JNK phosphorylation (Figure 2B).
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Effects of ET-1 and Ang II on p38MAPK Phosphorylation in VSMCs
ET-1 induced a time-dependent increase in p38MAPK phosphorylation in c-Src+/+ VSMCs. Maximal responses were observed after 10 minutes and phosphorylation levels remained increased after 30 minutes of stimulation. In c-Src+/– and c-Src–/– cells the earlier ET-1 effect on p38MAPK phosphorylation was similar to that observed in c-Src+/+ cells (Figure 3A). However, in c-Src–deficient VSMCs, responses were attenuated at 10 minutes. Ang II also induced p38MAPK phosphorylation in cells from c-Src+/+. Peak responses were achieved within 2 minutes of stimulation and remained slightly increased after 10 minutes. Ang II did not induce p38MAPK phosphorylation in cells from c-Src+/– or c-Src–/– mice (Figure 3B).
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ET-1 and Ang II Receptors Expression in VSMCs from c-Src+/+, c-Src+/–, and c-Src–/– Mice
To verify whether altered ET-1 and Ang II signaling is attributable to changes in receptor status, we determined the expression of ETA, ETB, AT1, and AT2 receptors in unstimulated cells from c-Src+/+, c-Src+/–, and c-Src–/– mice. c-Src gene knockout had no effect in ET-1 or Ang II receptors expression (supplemental Figures I and II, available online at http://atvb.ahajournals.org).
ET-1 and Ang II Effects on c-Src Phosphorylation in VSMCs
We evaluated whether ET-1 influences activation of c-Src in VSMCs from c-Src+/+ mice. As we previously reported,25 Ang II stimulation rapidly increased phosphorylation of c-Src, with responses maintained for up to 5 minutes, as shown in Figure 4. However unlike Ang II, ET-1 did not significantly modify phosphorylation of c-Src (Figure 4).
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Effect of c-Src Gene Knockout on Src Family Members Fyn and Yes Expression
To rule out whether c-Src knockout might be compensated by overexpression of other members of the Src family, we analyzed the expression of Fyn and Yes, 2 major Src family kinases, in VSMCs from c-Src+/+, c-Src+/–, and c-Src–/– mice. c-Src deficiency had no effect on Fyn or Yes expression (supplemental Figure III).
ET-1 and Ang II Effects on c-Raf Phosphorylation in VSMCs
Ras-dependent pathways may be involved in ET-1 and Ang II–induced MAPK activation. To address this issue we investigated whether ET-1 and Ang II were able to induce phosphorylation of c-Raf, an effector of Ras, in VSMCs from c-Src+/+ mice. As shown in Figure 5A, ET-1 induced rapid phosphorylation of c-Raf. Phosphorylated c-Raf levels remained increased after 30 minutes of stimulation. c-Src gene knockout had no effect on ET-1–induced c-Raf phosphorylation (Figure 5A). Ang II also induced c-Raf phosphorylation in VSMCs from c-Src+/+ mice, this effect was rapid and transient and phosphorylation levels returned to basal within 5 minutes of stimulation (Figure 5B). In VSMCs from c-Src+/– or c-Src–/– mice Ang II failed to induce c-Raf phosphorylation (Figure 5B).
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Effects of Ras siRNA on ET-1 and Ang II–Induced MAPK Phosphorylation in VSMCs
To further investigate the putative role of c-Raf in MAPK regulation by ET-1 and Ang II, we investigated MAPK responses in VSMCs in which Ras, a proximal Raf regulator, was downregulated by siRNA. Ras protein abundance was markedly reduced in VSMCs from c-Src+/+ mice transfected with siRNA for 24 hours, but not in control cells or cells transfected with nonsilencing siRNA. After 48 hours Ras protein expression returned to basal levels (data not shown). As shown in Figure 6A, ET-1–induced ERK 1/2 phosphorylation is significantly blunted (P<0.01 versus control cells) after 2 and 5 minutes of stimulation in transfected cells. Similar results were observed for SAPK/JNK and p38MAPK (supplemental Figure IV and V). Ang II–mediated phosphorylation of ERK 1/2 (Figure 6B) SAPK/JNK and p38MAPK (supplemental Figure IVB and VB) was also inhibited in cells transfected with siRNA.
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Discussion |
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Major findings in the present study demonstrate that MAPKs are differentially regulated by Ang II and ET-1 in VSMCs. Whereas Ang II stimulates phosphorylation of ERK1/2, SAPK/JNK, and p38MAPK via c-Src–dependent pathways, ET-1 induces MAPK activation through c-Src–independent mechanisms. Ras-Raf pathway is involved in Ang II–induced c-Src–mediated MAPK phosphorylation. Here we also identify the Ras-Raf pathway as a putative proximal regulator of MAPKs by ET-1. This is supported by the findings that c-Raf is potently activated by ET-1 in a c-Src–insensitive manner and ET-1–mediated MAPK effects are attenuated in cells in which the Ras-Raf pathway is downregulated by siRNA. These data indicate the existence of 2 distinct signaling pathways leading to GPCR-mediated MAPK activation in VSMCs. Ang II and ET-1 elicit diverse cellular responses in vascular and nonvascular cells, such as contraction, growth, migration, and inflammation.2,3 Many of these processes are mediated through activation of MAPKs which in most cases are dependent on transactivation of the epidermal growth factor (EGF) receptor.29,30 Ang II– and ET-1–mediated transactivation of the EGF receptor is mediated by several intermediary signaling molecules including Ca2+, reactive oxygen species (ROS), metalloproteases that generate EGF-like ligands, and tyrosine kinases such as c-Src.29,31–35 c-Src is one of the first kinases to be activated by Ang II and it plays a key role in VSMC signaling events.14,18–20,24 Previous studies demonstrated that c-Src is an effector of cell signaling by ET-1 in nonvascular cells,30,36–42 but the role of this tyrosine kinase in VSMC signaling mediated by ET-1 and the role of EGFR transactivation remains to be determined.
We investigated in greater detail whether c-Src is in fact a target for ET-1 to activate MAPKs in VSMCs as it is for Ang II. From our study it is evident that c-Src does not contribute significantly to ET-1–mediated MAPK activation. This is supported by the observations that ET-1, but not Ang II, was able to induce MAPK phosphorylation in VSMC from c-Src–deficient mice. In addition, Ang II induces rapid phosphorylation of c-Src in VSMCs from c-Src+/+ mice whereas ET-1 has no effect on the kinase activation. These findings here support our earlier in vivo studies, in which we reported that Src tyrosine kinases do not contribute to vascular trophic signaling of ET-1.43 One may argue that c-Src knockout might be compensated by the expression and activity of other members of the Src family like Fyn and Yes. However, c-Src gene knockout had no effect on the expression of Fyn and Yes. Moreover, it has been demonstrated that in c-Src–/– mice cells neither Fyn or Yes are able to fully restore Ang II–induced activation of ERK 1/2,16 suggesting specificity for Src family kinases. The lack of Ang II–induced phosphorylation of MAPKs in c-Src gene knockout mice could also be attributable to decreased expression of its receptors. However no significant changes were observed regarding Ang II and ET-1 receptors expression in the mice studied.
There is a paucity of information on the signaling pathways leading to MAPK activation by ET-1 in VSMCs. G protein–coupled receptors can also signal by interacting with various small G proteins that are generally classified, by structural similarity, into 5 subfamilies: Ras, Rho, Arf, Rab, and Ran family GTPases.44–46 The biological effects of Ras proteins are exerted through the activation of several downstream effectors, including Raf, Rac, phosphatidylinositol 3-kinase (PI3K), and Ral.46 Ras stimulation of the serine/threonine kinase Raf is followed by activation of the downstream kinase MEK 1/2, which in turn phosphorylates ERK 1/2.46 These events may bypass c-Src. We found that ET-1 induces c-Raf phosphorylation, and this event is not affected by c-Src knockout. This suggests that ET-1–induced phosphorylation of MAPKs might be mediated by c-Src–independent and Ras-Raf–dependent mechanisms.
Ras can activate ERK1/2, SAPK/JNK, and p38MAPK47,48 via Cdc42 and Rac in different cell types. Ras-dependent signaling by ET-1 has been demonstrated in mesangial cells.49 We have also shown that the Ang II signaling cascade leading to MAPKs phosphorylation is also Ras-Raf dependent because Ras downregulation with siRNA completely blocks Ang II–induced MAPK activation. Furthermore, this event seems to be c-Src–dependent because c-Raf phosphorylation is inhibited in VSMCs from c-Src+/– and c-Src–/– mice. These findings suggest that Ras-Raf may be both upstream and downstream of c-Src in response to Ang II stimulation.
In view of our findings, Ang II and ET-1 induce phosphorylation of MAPKs through distinct pathways in VSMCs. Whereas Ang II–mediated activation of ERK 1/2, SAPK/JNK, and p38MAPK is dependent on the tyrosine kinase c-Src, ET-1 induces the same effect via c-Src–independent Ras-Raf–dependent mechanisms. Taken together our data suggest that different ligands to GPCR can activate similar signaling pathways through unrelated mechanisms. MAP kinases are an important point of convergence for Ang II and ET-1.
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Acknowledgments |
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Sources of Funding
This study was supported by grant 44018 from the Canadian Institutes of Health Research (CIHR). Dr Touyz is supported through a Canada Research Chair/Canadian Foundation for Innovation award. Dr Callera received a fellowship from the CIHR and Dr Montezano received a fellowship from Amgen.
Disclosures
None.
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Footnotes |
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Original received January 8, 2007; final version accepted May 23, 2007.
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