Published online before print January 8, 2009, doi: 10.1161/CIRCRESAHA.108.188524
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© 2009 American Heart Association, Inc.
Cellular Biology |
ROCK Isoform Regulation of Myosin Phosphatase and Contractility in Vascular Smooth Muscle Cells
From the Molecular Cardiology Research Institute and the Department of Medicine, Division of Cardiology (Y.W., N.R., M.B., W.B., H.K.S.), Tufts Medical Center, Boston; and the Laboratory for Microsystems Technology (X.R.Z., X.Z.), Department of Manufacturing Engineering, Boston University, Mass.
Correspondence to Howard K. Surks, Molecular Cardiology Research Institute, Tufts Medical Center, Box 80, 800 Washington St, Boston, MA 02111. E-mail hsurks{at}tuftsmedicalcenter.org
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Abstract |
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Abnormal vascular smooth muscle cell (VSMC) contraction plays an important role in vascular diseases. The RhoA/ROCK signaling pathway is now well recognized to mediate vascular smooth muscle contraction in response to vasoconstrictors by inhibiting myosin phosphatase (MLCP) activity and increasing myosin light chain phosphorylation. Two ROCK isoforms, ROCK1 and ROCK2, are expressed in many tissues, yet the isoform-specific roles of ROCK1 and ROCK2 in vascular smooth muscle and the mechanism of ROCK-mediated regulation of MLCP are not well understood. In this study, ROCK2, but not ROCK1, bound directly to the myosin binding subunit of MLCP, yet both ROCK isoforms regulated MLCP and myosin light chain phosphorylation. Despite that both ROCK1 and ROCK2 regulated MLCP, the ROCK isoforms had distinct and opposing effects on VSMC morphology and ROCK2, but not ROCK1, had a predominant role in VSMC contractility. These data support that although the ROCK isoforms both regulate MLCP and myosin light chain phosphorylation through different mechanisms, they have distinct roles in VSMC function.
Key Words: ROCK • myosin phosphatase • myosin light chain • myosin binding subunit
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Introduction |
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Increased vascular tone plays an important role in the pathophysiology of vascular diseases including hypertension, atherosclerosis, and myocardial infarction.1–3 Vascular tone is regulated by the contraction of vascular smooth muscle cells (VSMC) in the blood vessel wall. Vascular smooth muscle contraction is tightly coupled to the phosphorylation of the regulatory myosin light chain,4 which is regulated by the opposing activities of myosin light chain (MLC) kinase (MLCK) and myosin light chain phosphatase (MLCP) (reviewed elsewhere5). MLCP dephosphorylates MLC, leading to vascular smooth muscle relaxation (reviewed elsewhere6,7). MLCP activity is highly regulated by both vasoconstrictor and vasodilator signaling pathways. Nitrovasodilators stimulate cGMP-dependent protein kinase 1
, which activates MLCP to cause MLC dephosphorylation and smooth muscle relaxation.8–10 Vasoconstrictors, conversely, inhibit MLCP, leading to MLC phosphorylation and smooth muscle contraction (reviewed elsewhere6). Vasoconstrictor-mediated MLCP inhibition occurs by either phosphorylation of the MLCP inhibitory protein CPI-1711 or by phosphorylation of the myosin binding subunit (MBS) of MLCP at inhibitory sites T696 and T850.12–14 The RhoA/ROCK pathway is the most extensively studied mechanism of MLCP inhibition. Vasoconstrictor G protein–coupled receptor agonists lead to activation of RhoA guanine nucleotide exchange factors and GTP loading of the monomeric GTPase RhoA.15 GTP-bound RhoA then binds and activates its downstream effector ROCK,16–18 which in turn phosphorylates MBS19 at the 2 phosphorylation sites,12,13 leading to inhibition of MLCP activity.14 Phosphorylation at T850 also has been shown to cause dissociation of MBS from myosin.20 More recently, T850 has been implicated as the major ROCK phosphorylation site, whereas T696 is thought to be phosphorylated by other kinases.14 Many studies support that RhoA/ROCK signaling plays a role in the regulation of vascular tone and in the pathogenesis of vascular diseases,21–25 yet the precise mechanisms by which ROCK is targeted to and interacts with MLCP are not well understood.
We and others have recently characterized a new member of the MLCP complex, myosin phosphatase–Rho interacting protein (MP-RIP, also M-RIP, p116RIP).26–28 MP-RIP is a cytoskeletal scaffold that binds directly to both RhoA and MBS26 and targets MLCP to the contractile apparatus to dephosphorylate MLC.28,29 MP-RIP is also required to colocalize RhoA and MBS to regulate MLCP.30 MP-RIP, however, does not bind ROCK,28 leaving the issue of how ROCK is targeted to MLCP unresolved.
There are 2 isoforms of ROCK, ROCK1 and -2, that share overall 65% homology at the amino acid level.31 The tissue distribution of ROCK1 and -2 is similar, and relatively few studies have delineated the isoform specific roles of ROCK. Smooth muscle cells have been traditionally thought to express ROCK2 because it was purified from gizzard smooth muscle, although the expression of ROCK1 was not excluded.32 In this study, the specific roles of the ROCK isoforms in MLCP regulation and VSMC contractility were explored.
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Materials and Methods |
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Cell Culture
A7r5 cells were purchased from ATCC. Primary rat aortic smooth muscle cells were derived from rat aortas by the explant method and were identified by the expression of smooth muscle
-actin.
Smooth Muscle Cell Contractility Assay
Primary rat aortic smooth muscle cells were plated on a polymer substrate consisting of microfabricated posts made by replica molding of polydimethylsiloxane (PDMS). After incubation in serum-free media for 48 hours, the cells were stimulated with contractile agonist and imaged every minute for 30 minutes using a Cool SNAP EZ CCD camera (Photometrics) with NIS Elements software. Cell length change following agonist stimulation was measured using Image Pro Plus 6.0 software.
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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The MBS of MLCP Interacts With ROCK2 in VSMCs
To investigate the mechanism whereby ROCK interacts with MLCP, ROCK1 and -2 were individually immunoprecipitated from A7r5 VSMCs without contamination by the other isoform (Figure 1A).33 When the ROCK1 and -2 immunopellets were probed for MBS binding, ROCK2, but not ROCK1, was found to coimmunoprecipitate MBS (Figure 1B). In primary rat aortic smooth muscle cells, where the immunoreactivity of ROCK1 and -2 was similar, MBS was also found selectively in the ROCK2 immunopellet (Figure 1C). In a separate approach, ROCK2, but not ROCK1, was found in a myc-MBS immunopellet from transfected HEK293 cells (Figure 1D). Together, these experiments support that the MBS of MLCP interacts specifically with ROCK2.
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Cell Stimulation Augments the ROCK2-MBS Interaction
The ROCK2-MBS coimmunoprecipitation in resting VSMCs was augmented by exposure to the contractile agonist lysophosphatidic acid (LPA) or to serum (Figure 2A). These stimuli would be expected to increase ROCK activity, and the data therefore suggest that ROCK activity may modulate its interaction with MBS. When the time course of ROCK2-MBS binding in response to serum was examined, the interaction was significantly increased within 5 minutes of stimulation (P=0.008; n=4), was diminished by 40 minutes, and reached a second peak at 1 hour (P=0.04; n=4) (Figure 2B). When phosphorylation of MBS by ROCK was studied, a similar time course was observed (P=0.04 at 5 and 60 minutes; n=4), suggesting that the ROCK2-MBS interaction correlates with phospho-regulation of MLCP by ROCK (Figure 2C).
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Mechanism of ROCK-MBS Binding
To localize the ROCK2 binding domains within MBS, glutathione S-transferase (GST) fusion proteins were created that together encompassed the entire MBS molecule. These were individually tested for binding to ROCK from VSMC lysates. MBS peptides corresponding to amino acids 540 to 858 (with and without the central insert splice variant) and 683 to 866 both bound ROCK2 (Figure 3A). The minimal MBS domain tested that bound ROCK included amino acids 683 to 866, which contain the 2 major inhibitory phosphorylation sites, T696 and T850 (Figure 3C), and a predicted coiled coil structure (amino acids 704 to 764). Interestingly, MBS 683 to 866 was able to bind both ROCK2 and ROCK1 in vitro. When binding of mycROCK1 and mycROCK2 to MBS 683 to 866 was compared, 2-fold more ROCK2 bound MBS than ROCK1 (P=0.01, n=3; Figure 3B). MP-RIP targets RhoA to regulate MLCP26,28,30; however, Koga and Ikebe recently found that MP-RIP does not interact with ROCK.28 To confirm this observation in our binding studies, the same MBS domains were probed for MP-RIP binding. MP-RIP bound MBS 850 to 1030, confirming our previous findings26 and demonstrating that MP-RIP and ROCK bind to distinct domains of MBS (Figure 3A). Furthermore, when GST-MP-RIP domains, together encompassing the entire molecule, were compared with GST-MBS 683 to 866 for ROCK binding, only GST-MBS bound ROCK (Figure I in the online data supplement).
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To localize the MBS binding domain of ROCK, and to test direct binding between MBS and ROCK, 4 domains each of ROCK1 and -2, that together encompassed both molecules in their entirety, were separately tested for binding to GST-MBS 683 to 866. Of the ROCK2 domains, a coiled coil domain of ROCK2, 354 to 775, and to a much lesser extent the kinase domain of ROCK2, 1 to 360, bound MBS (Figure 3D). No domains of ROCK1 specifically bound MBS (Figure 3D and data not shown). The homology between ROCK2 354 to 775 and ROCK1 338 to 750 is only 58% (supplemental Figure II), compared to 86% homology between the catalytic domains of the 2 isoforms (data not shown). These studies suggest that ROCK2 amino acids 354 to 775 can directly bind MBS amino acids 683 to 866 (Figure 4).
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ROCK Isoform Regulation of MLCP and MLC Phosphorylation
When overexpressed in VSMCs, ROCK1 and -2 were localized diffusely in the cytoplasm (Figure 5A, right images). Immunofluorescent labeling with phospho-MLC antibodies showed that overexpression of either ROCK1 or ROCK2 dramatically increased MLC phosphorylation (Figure 5A, left images), whereas GFP did not affect MLC phosphorylation, and a dominant negative ROCK2 peptide reduced MLC phosphorylation in this assay (supplemental Figure III).
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When ROCK1 and -2 were individually silenced using RNA interference, upregulation of the other isoform was noted (Figure 5B). Combined ROCK isoform silencing was not as effective as individual silencing. MBS phosphorylation at the 2 inhibitory phosphorylation sites, T696 and T850, was probed in the setting of ROCK silencing. Silencing of either ROCK isoform caused a significant reduction in phosphorylation at T850 (P<0.05 for ROCK1 and ROCK2a; n=4) (Figure 5C). Silencing of both ROCK isoforms combined caused a significantly greater reduction in MBS T850 phosphorylation than individual silencing of either ROCK isoform (P=0.04 versus ROCK1 alone; n=4). ROCK silencing did not affect phosphorylation of MBS at T696 (supplemental Figure IV).
Phosphorylation of the MLCP downstream substrate MLC, the biochemical determinant of smooth muscle contraction, was also examined in the setting of ROCK isoform silencing. ROCK1 and -2 silencing both reduced MLC phosphorylation, although only ROCK2 silencing reached statistical significance (P<0.002; n=4) (Figure 5D). Combined ROCK silencing caused greater inhibition of MLC phosphorylation than silencing of individual ROCK isoforms (P<0.05 for R1, R2a, or R2b versus R1+R2; n=4) (Figure 5D). When examined by immunofluorescence microscopy, silencing of either ROCK isoform reduced phosphorylation of MLC, consistent with the biochemical data above (Figure 5E). These studies support that the ROCK isoforms both regulate MLCP activity and MLC phosphorylation and have an additive effect when silenced in combination.
ROCK Isoform Regulation of VSMC Morphology
The ROCK isoforms were found to have distinct morphological effects on primary rat aortic smooth muscle cells (Figure 6A and supplemental Figures V and VI). ROCK1-silenced cells had a smaller cell area (47% reduction, P<0.001 versus scrambled, 1-way ANOVA on ranks; Figure 6A and 6B), fewer stress fibers (39% reduction, P<0.001 versus scrambled, 1-way ANOVA on ranks; Figure 6A and 6C), and increased numbers of focal adhesions (40% increase, P<0.05, ANOVA Holm–Sidak test; Figure 6A and 6D) than scrambled control transfected cells. ROCK2-silenced cells had a larger cell area (65% increase, P<0.001 versus scrambled, 1-way ANOVA on ranks; Figure 6A and 6B) and greater number of stress fibers (49% increase, P<0.001 versus scrambled, 1-way ANOVA on ranks; Figure 6A and 6C) and fewer focal adhesions (63% reduction, P<0.001, ANOVA Holm–Sidak test; Figure 6A and 6D) compared to control VSMCs. These experiments support that whereas both ROCK isoforms regulate MBS and MLC phosphorylation, they have opposing effects on cell morphology. Treatment with the contractile agonist LPA modestly increased the number of stress fibers and reduced the number of focal adhesions under all silencing conditions (supplemental Figure V). Treatment with the ROCK inhibitor Y27632 dramatically reduced the numbers of stress fibers and focal adhesions under all silencing conditions, indicating that ROCK activity was high even in the resting cells in which 1 ROCK isoform had been silenced (supplemental Figure VI).
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ROCK Isoform Regulation of VSMC Contractility
The role of the ROCK isoforms in VSMC contractility was explored using an assay in which VSMCs are cultured on microfabricated posts which do not hinder cell contraction and whose movement can be used to measure cellular force production.34–39 Cells transfected with scrambled and ROCK2 small interfering (si)RNA were elongated, whereas cells transfected with ROCK1 siRNA appeared smaller than scrambled control and ROCK2-silenced cells, consistent with the immunofluorescence labeling in Figure 6 (also Figure 7A and supplemental videos). When the contractile agonist LPA was applied, control cells underwent a gradual contraction, primarily along the long axis of the cells, over a period of 15 to 20 minutes (see supplemental Video 1). LPA-mediated contractions were abolished by pretreatment with the ROCK1 and ROCK2 inhibitor Y27632, supporting that the contractility was mediated by ROCK (supplemental Figure VII) and by pretreatment with blebbistatin, indicating that the contraction required actin–myosin interactions (supplemental Figure VIII). ROCK1-silenced VSMCs also underwent a vigorous contraction (Figure 7A and supplemental Video 2). ROCK2-silenced cells contracted significantly less than scrambled control and ROCK1-silenced cells (Figure 7A and 7B and supplemental Video 3; P<0.001 versus scrambled siRNA cells, 1-way ANOVA).
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Intracellular force generation was calculated from the displacement of microfabricated posts. Force maps of control and ROCK1- and ROCK2-silenced cells (Figure 7A) confirmed that ROCK1 silencing did not significantly affect force generation, whereas ROCK2 silencing significantly reduced force (Figure 7C; scrambled versus ROCK1, P=NS; scrambled versus ROCK2, P<0.05; ANOVA on ranks). Interestingly, the difference in force between ROCK2 and controls cells was more pronounced than the difference in cell contraction in Figure 7B. When examined in more detail, control and ROCK1-silenced cells had a similar relationship between force production and the degree of contraction. However, ROCK2-silenced cells exhibited a steeper slope for this relationship, indicating that force production yielded a greater degree of contraction in these cells (Figure 7D).
The differences in force production and contraction between ROCK1- and ROCK2-silenced cells were explored further using immunofluorescence microscopy to image actin–myosin fibers and phosphorylation of MLC after treatment with LPA or Y27632 (Figure 7E and supplemental Figures IX and X). The stress fibers in control silenced VSMCs were aligned with and extended across the long axis of the cell and were diffusely localized throughout the cell body. The stress fibers displayed abundant MLC phosphorylation despite lack of agonist stimulation (Figure 7E). ROCK1-silenced cells had fewer stress fibers than control cells, yet these stress fibers extended across the long axis of the cell and were frequently localized at the cell periphery. MLC phosphorylation colocalized with the peripheral actin–myosin stress fibers (Figure 7E). The ROCK2-silenced cells displayed abundant stress fibers, but in many cells the stress fibers were short and haphazardly arranged. MLC phosphorylation also colocalized with stress fibers in the ROCK2-silenced cells (Figure 7E). For all silencing conditions, stimulation with LPA increased both stress fiber number and MLC phosphorylation slightly (supplemental Figure IX), whereas Y27632 dramatically reduced both stress fiber number and MLC phosphorylation (supplemental Figure X). The differences in stress fiber distribution and orientation between ROCK1- and ROCK2-silenced VSMCs raised the possibility that the axis of force generation may be altered when ROCK isoform expression is silenced. The direction of movement of the microfabricated posts was used to determine the average longitudinal and transverse components of force, which were plotted for each silencing condition (Figure 7F). For both scrambled and ROCK1-silenced cells, the longitudinal and transverse forces were similar and the force vectors pointed toward the center of the cell. However, in ROCK2-silenced cells, the force vectors were random and small, possibly reflecting the haphazard distribution of stress fibers as seen in Figures 5E, 6A, and 7E
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Discussion |
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There is little known about the ROCK isoform specificity of MLCP regulation or the mechanism whereby ROCK interacts with MLCP. We have found that ROCK2 specifically is found in a complex with MBS in the cell. Although MP-RIP targets RhoA to MLCP,30 MP-RIP does not bind ROCK.28 Our data confirm that ROCK and MP-RIP bind separate domains of MBS. The data presented here support a model where RhoA bound to MP-RIP and ROCK bound to MBS are brought into proximity by MP-RIP/MBS binding.
In the first demonstration of ROCK2-MLCP binding, we found that amino acids 683 to 866 bind directly to amino acids 354 to 775 of ROCK2. The ROCK2 binding domain on MBS is predicted to contain a coiled coil structure between amino acids 704 and 764. The MBS binding domain of ROCK2, amino acids 354 to 775, is also predicted to be a coiled coil structure, suggesting that coiled-coil binding mediates their interaction. Future studies will further characterize the specific residues involved in their interaction. Although ROCK1 338 to 750 is also predicted to form a coiled coil, the amino acid homology between ROCK1 and -2 in this domain is only 58%, suggesting that the specific residues that mediate the ROCK2-MBS interaction differ between the 2 ROCK isoforms. In fact, this domain of ROCK1, but not ROCK2, has been found to bind to RhoE and PDK1, further supporting that this region of ROCK1 and ROCK2 mediates isoform-specific interactions.40,41 Interestingly, in GST fusion protein interaction assays, both ROCK1 and -2 from cell lysates could interact with MBS 683 to 866. However, twice as much ROCK2 bound MBS. These data suggest that there may be an intermediary protein(s) involved in ROCK1-MBS binding and that this interaction may become apparent when relatively high concentrations of GST-MBS peptide are used for binding studies. This potential indirect interaction between ROCK1 and MBS will require further investigation.
When the roles of the ROCK isoforms in MBS and MLC phosphorylation were tested, overexpression of both isoforms increased MLC phosphorylation. A previous study has shown that the ROCK isoforms lose their specificity when overexpressed,33 and therefore silencing studies were also undertaken. Interestingly, silencing of each ROCK isoform led to upregulation of the other isoform, suggesting that the expression level of the ROCK isoforms is tightly controlled and inter-related in VSMCs. Silencing of either ROCK isoform lead to reduced MBS and MLC phosphorylation. Furthermore, combined ROCK isoform silencing, despite that downregulation of the ROCK isoforms was less efficient, lead to a significantly greater reduction in MBS and MLC phosphorylation than either isoform alone. These data support that both ROCK isoforms regulate MBS and MLC phosphorylation in VSMCs and, further, taken together with the binding data showing different mechanisms of interaction with MBS, suggest that their respective mechanisms are potentially distinct.
ROCK is known to regulate cell morphology, including the formation of actin–myosin stress fibers and focal adhesion complexes. Recently, 2 studies examined the roles of ROCK1 and -2 on fibroblast morphology with different results,33,42 and thus the effects of the ROCK isoforms on actin cytoskeletal architecture remains unresolved. In VSMCs, ROCK1 and ROCK2 had opposing effects, suggesting a cooperative role for the 2 ROCK isoforms where both are required to regulate cell morphology.
The measurement of contractility in single VSMCs is complicated by the strong focal adhesion contacts between the VSMC and the underlying substrate, limiting movement of the cell. This limitation has been addressed previously by plating VSMCs on flexible polymer substrates that can be pulled by the contracting cell. Force can be estimated by counting wrinkles in the polymer substrate or by measuring the movement of beads embedded in the polymer. In this study, a recently developed technology to precisely calculate intracellular forces from single cells was adapted to measure contractility in VSMCs in which ROCK1 or ROCK2 expression was silenced.
The present study is the first to demonstrate that ROCK2 is the isoform that regulates VSMC force production and contractility. Furthermore, the specificity of ROCK2 for direct MLCP binding suggests that this interaction may be critical for contractile regulation and is thus a potential target for the treatment of cardiovascular diseases. The regulation of actin cytoskeletal organization may also contribute to the differential roles of the ROCK isoforms in contractility. The plot of force direction versus contraction axis suggests that force remains aligned with the major axes of the cell in ROCK1 but not in ROCK2-silenced cells. This is corroborated by immunofluorescence microscopy showing preserved phospho-MLC along the long axis of the cell in ROCK1-silenced cells, but in fragmented multidirectional fibers in ROCK2-silenced cells. These data suggest that the abnormal distribution of actin–myosin fibers may cause a disorganized orientation of force production and thus contribute to the reduced contraction in ROCK2-silenced VSMCs. These data further suggest that both force production and the orientation of force producing fibers are important in VSMC contractility. Interestingly, the reduction in force production in ROCK2-silenced cells was more pronounced than the reduction in contractility. This discrepancy may be related to the fewer focal adhesions in ROCK2-silenced cells, causing reduced tethering of the cell to the microfabricated posts and allowing more length change per unit of force.
The role of the RhoA/ROCK signaling pathway in the regulation of vascular smooth muscle contractility and vascular tone is well established, although the mechanisms are incompletely understood. ROCK inhibitors have been shown to ameliorate vascular disease in animal models and are promising future therapies for human cardiovascular disease (reviewed elsewhere43). Although relatively specific for ROCK, the inhibitors may be active against other kinases, and do not exhibit specificity for the individual ROCK isoforms.44 A careful dissection of the specific roles of the 2 ROCK isoforms in vascular function may provide the opportunity to create therapies that target specific ROCK-mediated functions, while leaving other essential ROCK functions intact. This study indicates that whereas a balance of ROCK1 and ROCK2 activities is required to regulate VSMC actin cytoskeletal structure, ROCK2 is the predominant isoform that regulates VSMC contractility.
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Acknowledgments |
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The authors wish to thank Dr Shuh Narumiya for the ROCK1 and -2 cDNA, Dr Masumi Eto for the MYPT1 cDNA, and Dr Kozo Kaibuchi for the ROCK-Rho–binding domain cDNA. We also thank Drs Richard Karas and Michael E. Mendelsohn for helpful discussions.
Sources of Funding
Supported by NIH grants HL074069 and HL077378 to (to H.K.S.). X.Z. has grant support from the National Science Foundation.
Disclosures
None.
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Footnotes |
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Original received March 4, 2008; resubmission received October 2, 2008; revised resubmission received December 23, 2008; accepted December 30, 2008.
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References |
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