Published online before print July 5, 2007, doi: 10.1161/CIRCRESAHA.107.156463
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© 2007 American Heart Association, Inc.
Integrative Physiology |
Abl Silencing Inhibits CAS-Mediated Process and Constriction in Resistance Arteries
From the Center for Cardiovascular Sciences, Albany Medical College, NY.
Correspondence to Dale D. Tang, The Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue, MC-8, Albany, NY 12208. E-mail tangd{at}mail.amc.edu
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Abstract |
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The tyrosine phosphorylated protein Crk-associated substrate (CAS) has previously been shown to participate in the cellular processes regulating dynamic changes in the actin architecture and arterial constriction. In the present study, treatment of rat mesenteric arteries with phenylephrine (PE) led to the increase in CAS tyrosine phosphorylation and the association of CAS with the adapter protein CrkII. CAS phosphorylation was catalyzed by Abl in an in vitro study. To determine the role of Abl tyrosine kinase in arterial vessels, plasmids encoding Abl short hairpin RNA (shRNA) were transduced into mesenteric arteries by chemical loading plus liposomes. Abl silencing diminished increases in CAS phosphorylation on PE stimulation. Previous studies have shown that assembly of the multiprotein compound containing CrkII, neuronal Wiskott–Aldrich Syndrome Protein (N-WASP) and the Arp2/3 (Actin Related Protein) complex triggers actin polymerization in smooth muscle as well as in nonmuscle cells. In this study, Abl silencing attenuated the assembly of the multiprotein compound in resistance arteries on contractile stimulation. Furthermore, the increase in F/G-actin ratios (an index of actin assembly) and constriction on contractile stimulation were reduced in Abl-deficient arterial segments compared with control arteries. However, myosin regulatory light chain phosphorylation (MRLCP) elicited by contractile activation was not inhibited in Abl-deficient arteries. These results suggest that Abl may play a pivotal role in mediating CAS phosphorylation, the assembly of the multiprotein complex, actin assembly, and constriction in resistance arteries. Abl does not participate in the regulation of myosin activation in arterial vessels during contractile stimulation.
Key Words: tyrosine kinase • actin cytoskeleton • contraction • vascular smooth muscle • adapter protein
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Introduction |
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Actin cytoskeleton remodeling has recently emerged as an important cellular process mediating smooth muscle contraction.1–7 A pool of globular actin (G-actin) is stimulated onto filamentous actin (F-actin) in a variety of smooth muscle cells and tissues in response to agonist stimulation. Blockage of actin polymerization by the inhibitors cytochalasin and latrunculin attenuates active force on activation with contractile stimuli whereas myosin regulatory light chain phosphorylation (MRLCP) is not disrupted.5,8–10 These studies suggest that actin dynamics and MRLCP are independently regulated, and that both dynamic changes in the actin cytoskeleton and myosin activation are required for force development during contractile stimulation of smooth muscle.4,8–11 However, the mechanisms that regulate the actin cytoskeleton in smooth muscle are not completely understood.
The tyrosine phosphorylated protein Crk-associated substrate (CAS) has been implicated in the modulation of the actin cytoskeleton in smooth muscle cells as well as in nonmuscle cells including COS-7 cells and NIH3T3 cells.9,12–14 Downregulation of CAS by antisense dramatically attenuates force development and actin polymerization in carotid arteries in response to contractile stimulation.9 CAS undergoes tyrosine phosphorylation in vascular smooth muscle cells and tissues on stimulation with angiotensin II or serotonin12,15 as well as in nonmuscle cells in response to growth factors and cell adhesion.13,16 CAS phosphorylation has been proposed to be a key event regulating the actin cytoskeleton; phosphorylated CAS may enhance its binding to the adapter protein CrkII, which may facilitate the formation of the multiprotein compound including CrkII, neuronal Wiskott–Aldrich Syndrome Protein (N-WASP) and the Arp2/3 (Actin Related Protein) complex and initiating actin polymerization and branching mediated by the Arp2/3 complex.1–3,17,18
The upstream regulatory molecules of CAS in smooth muscle are not completely elucidated. The nonreceptor tyrosine kinase Abl (Abelson tyrosine kinase, c-Abl) is ubiquitously expressed and has been implicated to function in a range of cellular processes including the regulation of the actin cytoskeleton that mediates cell migration and spreading, membrane ruffling, cell adhesion, neurite extension, and cell growth and survival in nonmuscle cells including NIH3T3 fibroblasts, mouse embryonic fibroblasts, neurons, epithelial cells, and COS-7 cells.19,20 Abl is able to stimulate the formation of actin microspikes in fibroblasts spreading on fibronectin; this function of Abl is dependent on kinase activity and is not shared by c-Src tyrosine kinase.21 In addition, platelet-derived growth factor (PDGF) enhances the activity of Abl and increases plasma membrane ruffling. Mouse embryonic fibroblasts lacking Abl exhibit 4- to 5-fold fewer dorsal/circular membrane ruffles when stimulated by PDGF. Expression of wild-type Abl in these cells rescues the effects of PDGF.22,23
The objective of this study was to test the hypothesis that Abl may be a pivotal upstream molecule of CAS in the resistance artery, a well-recognized arterial smooth muscle model.6,24 Our results demonstrate that silencing of Abl by small interfering RNA (siRNA) attenuates CAS phosphorylation, the formation of the protein complex containing CrkII and dynamic changes in the actin cytoskeleton without affecting myosin activation during stimulation with the 
1-adrenoceptor agonist phenylephrine (PE).
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Materials and Methods |
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Materials, tissue preparation, immunoblot analysis, in vitro kinase assay, cDNA constructs, chemical loading and organ culture, fluorescence microscopy, coimmunoprecipitation, analysis of F/G-actin ratios, analysis of myosin regulatory light chain phosphorylation (MRLCP), and statistical analysis are described in the Materials and Methods section of the supplemental materials available online at http://circres.ahajournals.org.
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Results |
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Increases in CAS Phosphorylation and the Association of CAS with CrkII Are Dose- and Time-Dependent in Mesenteric Arteries on PE Stimulation
Our previous studies suggest that CAS participates in the cellular processes that mediate vascular smooth muscle contraction.8,9 Recent report has shown that serotonin stimulation elicits the tyrosine phosphorylation (Tyr-410) of CAS in rat aorta (elastic artery).12 To evaluate whether CAS phosphorylation occurs in resistance arteries, rat mesenteric rings were treated with different concentrations of PE for different time points, or they were not stimulated. CAS tyrosine phosphorylation was assessed by immunoblot analysis using phospho-CAS (Tyr-410) antibody. Stimulation with PE (5 minutes) resulted in the dose-dependent increase in CAS phosphorylation in mesenteric arteries (Figure 1A). CAS phosphorylation was increased as early as 1 minute after stimulation with PE (10 µmol/L) and maintained for the 30-minute period (Figure 1B).
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CAS phosphorylation enhances its ability to interact with the SH2-containing adapter protein CrkII in nonmuscle cells in response to integrin activation.25,26 To determine whether contractile stimulation leads to the increase in the association of CAS with CrkII, blots of CrkII immunoprecipitates from unstimulated and PE-stimulated arteries were detected for CAS and CrkII. Activation with PE led to the increase in the amount of CAS coimmunoprecipitated with CrkII; CAS/CrkII ratios on PE stimulation were obviously augmented in a dose- and time-dependent manner (Figure 1C and 1D).
Activation With PE Induces Abl Phosphorylation in Resistance Arteries
We also evaluated Abl phosphorylation in response to contractile stimulation. In nonmuscle cells, Abl has been implicated in regulating actin cytoskeleton remodeling.19,20 Immunoblot analysis was used to assess Abl phosphorylation (Tyr-412) in mesenteric arteries treated with PE. The phosphorylation level of Abl in resistance arteries was augmented on contractile activation, which was dose- and time-dependent (Figure 1E and 1F). The increase in Abl phosphorylation in response to PE stimulation was slightly earlier than the enhancement of CAS phosphorylation and the interaction of CAS with CrkII.
CAS Phosphorylation Is Catalyzed by Abl In Vitro
Src has been implicated in the regulation of CAS phosphorylation in vascular smooth muscle tissues and nonmuscle cells.12,25 Abl phosphorylation is attenuated by a Src inhibitor in cultured smooth muscle cells.27 Thus, Abl kinase may be able to catalyze CAS phosphorylation in smooth muscle.
To assess whether Abl mediates CAS phosphorylation directly, Abl (enzyme) was immunoprecipitated from rat resistance arteries followed by the addition of purified CAS (substrate). In vitro kinase assay was initiated by the addition of ATP, and CAS phosphorylation was evaluated by immunoblot analysis. CAS tyrosine phosphorylation increased by 2.5-fold in the presence of Abl (Figure 2).
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Abl Silencing Attenuates CAS Phosphorylation on Contractile Stimulation
To elucidate the role of Abl in regulating CAS in smooth muscle, we used plasmid-based short hairpin RNA (shRNA) to silence Abl in resistance arteries and then determine the effects of Abl silencing on CAS phosphorylation. Green fluorescence protein (GFP)-plasmids encoding Abl-shRNA or luciferase-shRNA (control RNA) were introduced into mesenteric arteries using a modified method of chemical loading (also known as reversible permeabilization).1,2,28 Protein expression in these tissues was assessed by immunoblot analysis. Fluorescence microscopy was used to assess whether GFP fluorescence (an indication of transfection) is present in the smooth muscle layer of the arterial wall.
As shown in Figure 3A, the level of Abl protein was reduced in arteries producing Abl shRNA compared with untreated tissues or arteries generating luciferase shRNA. However, smooth muscle–specific -actin content was not different in these tissues. Ratios of Abl/actin were significantly lower in Abl-deficient tissues than in untreated arteries or tissues producing luciferase shRNA (Figure 3B). Moreover, GFP fluorescence was widely detected in the medial smooth muscle layer of arteries treated with plasmids, indicating efficient transfection in the tissues (Figure 3C).
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CAS phosphorylation in Abl-depleted arteries and control tissues was evaluated by immunoblot analysis. Basal CAS phosphorylation (Tyr-410) was similar in untreated arteries or in segments producing luceferase shRNA or Abl shRNA. However, CAS phosphorylation on stimulation with PE (10 µmol/L, 5 minutes) was significantly lower in arterial segments generating Abl shRNA compared with untreated tissues or arteries producing luciferase shRNA (Figure 4A; n=5, P<0.01).
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We also observed that stimulation with KCl resulted in the increase in phosphorylation of Abl and CAS (Figure 4B and 4C). Therefore, we determined whether Abl silencing affects KCl-induced phosphorylation of CAS in resistance arteries. CAS phosphorylation in response to stimulation with KCl (80 mmol/L, 5 minutes) was significantly reduced in segments producing Abl shRNA compared with untreated arteries or tissues generating luciferase shRNA (Figure 4D; n=5, P<0.01).
Abl Silencing Inhibits the Association of CAS With CrkII and Formation of the Protein Complex Containing CrkII During PE Stimulation
We evaluated the effects of Abl silencing on CAS/CrkII coupling and formation of the complex containing CrkII. Untreated arteries or arteries that had been treated with plasmids encoding luciferase shRNA, or Abl shRNA were stimulated with PE, or left unstimulated. Blots of CAS or CrkII immunoprecipitates from these arteries were probed with antibodies against CAS, CrkII, N-WASP, or Arp2.
In untreated arteries and arteries producing luciferase shRNA, the amount of CrkII in CAS immunoprecipitates was increased during PE stimulation. In contrast, the CrkII amount coimmunoprecipitated with CAS in response to PE stimulation was attenuated in tissues producing Abl shRNA (Figure 5A). Similarly, the amounts of N-WASP and Arp2 coimmunoprecipitated with CrkII were increased on contractile stimulation in untreated arteries and arteries producing luciferase shRNA; however, the increase in the amounts of N-WASP and Arp2 in CrkII immunoprecipitates elicited by PE stimulation was reduced in tissues generating Abl shRNA (Figure 5B). Treatment with shRNAs did not affect CAS/CrkII coupling and the complex formation in unstimulated arteries (Figure 5A and 5B). Increases in protein ratios of CrkII/CAS, N-WASP/CrkII, and Arp2/CrkII in response to PE activation were significantly reduced in arteries producing Abl shRNA compared with untreated tissues and tissues generating luciferase shRNA (Figure 5C, n=3, P<0.05).
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Increases in F/G-Actin Ratios Elicited by PE Are Attenuated in Abl-Depleted Arteries
Contractile stimulation increases ratios of F/G-actin in smooth muscle tissues and cells.1–3,8,9,29 Abl has been implicated in regulating the actin cytoskeleton in nonmuscle cells such as fibroblasts and COS cells.19,20 We evaluated the effects of Abl downregulation on F/G-actin ratios in resistance arteries on contractile stimulation. Untreated arteries or arteries that had been treated with plasmids encoding luciferase shRNA or Abl shRNA were stimulated with PE or they were not stimulated. Ratios of F/G-actin in these tissues were evaluated using a fractionation assay.1,3,8,9,29
Stimulation with PE led to the increase in F/G-actin ratios in untreated arteries and segments producing luciferase shRNA. However, the increase in F/G-actin ratios on PE stimulation was dramatically depressed in Abl-deficient resistance arteries. Treatment with shRNAs did not affect the F/G-actin ratios in unstimulated arteries (Figure 6).
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Constriction in Response to Contractile Stimulation Is Depressed in Abl-Deficient Arteries
We assessed the effects of Abl silencing on arterial constriction in response to contractile stimulation. The contractile responses of rat mesenteric arteries to PE were determined, after which plasmids encoding luciferase shRNA (Luc shRNA) or Abl shRNA were transduced into these arteries, and were incubated for 2 days. Constriction of these arteries was then determined. Dose-response curves on stimulation with PE were compared between 3 groups. In untreated segments or in arteries treated with plasmids producing luciferase shRNA, contractile responses to PE were dose-dependent; maximal constriction was 70% of the corresponding maximal value before preincubation. However, the dose-response curve in Abl-deficient tissues was significantly right shifted. Maximal contraction was reduced to 30% of the preincubation value (Figure 7A).
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We also assessed the effects of Abl silencing on KCl-induced vasoconstriction. KCl-induced dose-response curve in Abl-deficient segments was right shifted with a reduced maximal response compared with untreated arteries or segments producing luciferase shRNA (Figure 7B). There were no significant differences in tension among the 3 experimental groups before contractile stimulation.
Abl Silencing Does Not Inhibit the Increase in MRLCP on Contractile Stimulation
MRLCP has been thought to initiate crossbridge cycling and force generation in smooth muscle.30,31 To determine whether the depressed contractile response in Abl-depleted arteries stem from the inhibition of MRLCP, a subset of mesenteric segments that had been treated with no plasmids, or plasmids encoding luciferase shRNA or Abl shRNA were stimulated with 10–5 mol/L PE for 5 minutes, or they were not stimulated. These arterial vessels were then frozen for the determination of MRLCP.
In spite of reduced constriction, PE stimulation was able to lead to a significant increase in MRLCP in Abl-deficient arteries. The average increases in MRLCP in segments not treated with shRNA, or luciferase shRNA-treated and Abl shRNA-treated tissues were not significantly different 5 minutes after PE stimulation (Figure 8).
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Discussion |
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CAS is a 130-kDa tyrosine phosphorylated protein participating in the cellular processes that transduce external signals onto the actin cytoskeleton in various cell types including smooth muscle cells. In this report, Abl is able to catalyze CAS phosphorylation in the in vitro study. Moreover, Abl silencing attenuates CAS phosphorylation, the formation of the multiprotein complex containing CrkII, actin polymerization, and arterial constriction during contractile activation. These results suggest that Abl plays an essential role in regulating CAS-mediated signaling and constriction in resistance arteries.
CAS is required for actin filament assembly and active force development in arterial smooth muscle.9 In the present report, activation with the adrenoceptor agonist led to the enhancement of CAS phosphorylation and the association of CAS with CrkII in resistance arteries. These observations are supported by previous studies on elastic artery and in cultured cells.12,25 More importantly, our results showed that Abl underwent phosphorylation at Tyr-412 in resistance arteries on contractile stimulation, implying its role in modulating arterial constriction. Tyr-412 is located at the activation loop of Abl kinase domain. When unstimulated, the activation loop of the Abl kinase domain folds into the active site, thereby preventing binding of both the substrate and ATP. Phosphorylation at Tyr-412 induces conformation changes; the activation loop no longer blocks the active site, which leads the increase in kinase activity.32
In the present study, arterial segments were transfected with plasmids encoding Abl shRNA using the method of chemical loading plus liposomes.1,2,28,33,34 Plasmid-based shRNA has been shown to inhibit gene expression in mammalian cells; shRNA can be cleaved by intracellular RNase III (dicers) to generate siRNA with 21 to 22 bases, which initiates the formation of RNA-induced silencing complex (RISC) and induces gene silencing.35 Abl silencing by shRNA depressed the expression of Abl and attenuated CAS tyrosine phosphorylation in response to contractile activation (Figures 3 and 4
). Moreover, our in vitro study suggests a direct role for Abl in catalyzing CAS tyrosine phosphorylation. Because Src may participate in the regulation of CAS phosphorylation in vascular smooth muscle tissues and nonmuscle cells,12,36 and Abl phosphorylation is attenuated by a Src inhibitor in cultured vascular smooth muscle cells,27 it is possible that contractile stimulation may activate Abl tyrosine kinase via Src; the activated Abl may directly catalyze CAS phosphorylation in resistance arteries.
CAS phosphorylation has been shown to occur in its substrate domain, which contains 15 YXXP tyrosine residues.13 Using site-directed mutagenesis combined with tryptic phosphopeptide mapping, Hanks et al. have documented that Tyr-410 is 1 of the major phosphorylation sites on CAS; phosphorylation on the tyrosine residues creates docking sites for the SH2-containing signaling effector CrkII.13,25 Previous studies have shown that CrkII is able to regulate the activity of N-WASP. When unstimulated, the C-terminal portion of N-WASP binds to its GTP-binding domain, masking its binding motif for the Arp2/3 complex and inhibiting the activity of N-WASP. When bound to CrkII, N-WASP undergoes conformational changes, exposing the binding motif for the Arp2/3 complex and initiating actin polymerization and branching mediated by the Arp2/3 complex.1–3,17,18
In this report, Abl silencing inhibited the association of CAS with CrkII, the formation of protein complex containing CrkII, N-WASP, and the Arp2/3 complex, and actin filament assembly in arterial segments during contractile activation. Because Abl mediates CAS phosphorylation in the in vitro study and in arteries in response to contractile stimulation, we propose that Abl may regulate CAS tyrosine phosphorylation, which may sequentially facilitate the interaction of CAS with CrkII and the activation of N-WASP, promoting the Arp2/3 complex-mediated actin polymerization in arterial smooth muscle in response to contractile activation. In addition, Abl silencing did not attenuate increases in MRLCP during contractile stimulation, suggesting that Abl is not involved in the regulation of myosin activation in resistance arteries.
Actin polymerization may enhance force development by the following mechanisms. First, the actin filaments of smooth muscle cells connect to extracellular matrix via transmembranous β integrins and linker proteins such as vinculin and talin, facilitating force transmission between actin filaments to matrix.1,2,37,38 Actin polymerization is initiated by the Arp2/3 complex in nonmuscle and smooth muscle cells, promoting nascent actin polymerization at cell cortex. Cortical actin assembly may reinforce the linkage of actin filaments to integrins strengthening the transduction of mechanical force.1–3,17,18 Second, actin assembly has been shown to increase the numbers of contractile units and the length of actin filaments, providing more and efficient contractile elements for force development.5,39 Third, actin polymerization may also be a part of the reorganization process that allows for the rapid adjustment of cytoskeletal organization elicited by external stimulation.1,3,40–43
In the present study, both PE stimulation and KCl depolarization induced the tyrosine phosphorylation of Abl and CAS. Furthermore, Abl silencing attenuated CAS phosphorylation and force development in response to activation with PE and KCl. These results suggest that Abl tyrosine kinase is an essential convergent molecule that regulates constriction in resistance arteries on the activation with -receptor and membrane depolarization. An interpretation for this is that KCl depolarization increases intracellular Ca2+ (well demonstrated), which may activate Abl and CAS via a Ca2+-dependent mechanism.31,44 Therefore, Abl deficiency is able to disrupt the KCl-induced contraction. Because PE is also able to activate L-type channel and increase intracellular Ca2+,44 it is possible that PE-mediated Abl/CAS activation and constriction may be mediated in part by Ca2+-dependent mechanisms. As some other protein kinases associated with smooth muscle contraction, Abl may also be activated by other signaling molecules such as small GTPases.31,44
Summary
Although the role of CAS in regulating smooth muscle constriction has been recognized, the upstream regulator of CAS has not been well identified. In this study, we demonstrate a critical role of Abl in modulating CAS tyrosine phosphorylation and constriction in resistance arteries. Abl-mediated arterial constriction may be regulated by the formation of the multiprotein complex and actin filament assembly.
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Acknowledgments |
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Sources of Funding
This work was supported by The National Heart, Lung, and Blood Institute Grant HL-75388 and an American Heart Association Scientist Development Grant (to D.D.T.).
Disclosures
None.
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Footnotes |
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Original received February 8, 2007; resubmission received May 21, 2007; revised resubmission received June 19, 2007; accepted June 26, 2007.
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