Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111
Submitted 2 May 2003 ; accepted in final form 27 June 2003
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ABSTRACT |
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vascular smooth muscle actin; Rho GTPase; pericyte; myosin; cytoskeleton
In the arterial wall, Rho activation leads to a dramatic alteration of vascular smooth muscle cell contractile function. The regulation occurs principally through the actin-based cytoskeleton and the Rho GTPase effector, Rho kinase (2-4, 18, 28). Presumably, Rho kinase-dependent alterations in contractility may be directly linked to the role that Rho GTPase plays in signaling actin-based cytoskeletal reorganization (6).
In the microvasculature, pericytes regulate vessel integrity through their interactions with the capillary and postcapillary venular endothelial cells (7, 13, 14, 20). Pericyte control of endothelial cell growth and microvascular blood flow appears to occur through a cytoskeleton-dependent mechanism analogous to that seen in the arterial circulation. Indeed, the contractile phenotype of pericytes is marked by the expression of the specific actin isoform, -vascular smooth muscle actin (
VSMactin) (21), that participates in stress fiber formation and drives contractility. At the same time, pericytes express the ubiquitous
-actin isoform, which also participates in the formation of stress fibers, and
-actin, another nonmuscle actin family member, which mediates cell spreading and motility (16). Stress fiber formation is regulated by Rho GTPase, but whether that regulation applies equally to the stress fibers containing
-actin and those containing specialized contractile
VSMactin is not known. Herein, we report the influence of Rho signaling on isoactin array, contractile phenotype, and cell shape by using pericytes as a model for contractile cells. On the basis of the important roles that the Rho GTPase signaling pathways play in regulating vascular cytoskeletal reorganization and because of the complexity and functional diversity of the actin isoforms themselves, we set out to determine whether Rho GTPase-dependant signal transduction affects the cytoskeleton in an isoactin-dependent manner. Here, we demonstrate that activation of Rho leads to the selective disassembly of
VSMactin containing stress fibers, yielding a significant decrease in cell size. This observation lends strong support to the hypothesis that Rho GTPase signaling is isoactin-specific and points to the possibility that regulation of vascular cell tone, contractile potential, and growth can be targeted through this developmentally important and disease-associated pathway.
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MATERIALS AND METHODS |
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Plasmids. Green fluorescent protein (GFP)-expressing plasmid, pEGFP-N3, was purchased from Clontech (Palo Alto, CA) and has a cytomegalovirus (CMV) immediate early promoter. RhoA-expressing plasmids pExvRhoWT, dominant positive RhoA mutant pExvRhoL63, dominant negative RhoA mutant, pZipNeoRhoN19, and dominant positive mutant of Ras, pZipNeoRasL61, and expression plasmid for C3 exotransferase, pEFpLinkC3, were the generous gift of Dr. Deniz Toksoz (Tufts University School of Medicine, Boston, MA). pExv plasmids have an SV40 promoter, pZipNeo plasmids have Moloney murine leukemia virus (M-MuLV) long terminal repeats (LTRs), and pEFpLinkC3 has the elongation factor-1 (EF-1
) gene promoter. Dominant positive Rac1, pMT3RacL61, and dominant positive Cdc42, pMT3Cdc42L61, were contributed by Dr. Larry Feig (Tufts University School of Medicine, Boston, MA) and have the adenovirus major late promoter (AdMLP).
Antibodies. Anti-VSMactin and anti-nonmuscle myosin (NMmyosin) antibodies were prepared as described (13, 15). Anti-smooth muscle myosin (SMmyosin) rabbit polyclonal antibodies were purchased from Biomedical Technologies (Stoughton, MA). Monoclonal antibodies against Myc-epitope and GFP were purchased from Santa Cruz (Santa Cruz, CA). Goat secondary antibodies conjugated with Alexa-488 or Alexa-546 and phalloidins conjugated with Alexa-350, Alexa-488, Alexa-546, and Alexa-633 were purchased from Molecular Probes (Eugene, OR).
Immunofluorescence analysis. Cells on glass coverslips were fixed with 4% formaldehyde in DMEM and permeablized with 0.1% Triton in 40 mM HEPES, 50 mM PIPES, 75 mM KCl, 1 mM MgCl2, and 0.1 mM EGTA for 90 s at room temperature (16). Cells were incubated in 20 µg/ml of primary antibody for 1 h at room temperature. Cells were incubated with secondary antibodies diluted 1:100 for 45 min at room temperature. Images were captured on a Zeiss Axiovert fluorescence microscope digital imaging workstation with cooled charge-coupled device (Hamamatsu, Orca II) camera, x40 (NA = 0.75) oil-immersion objective, by using Metamorph software (Universal Imaging). For the comparison of the levels of fluorescence of transfected and untransfected cells, three representative square regions within one transfected cell were selected in Adobe Photoshop 5.5, and the levels of fluorescence in those regions were measured. Within the same image, three regions of the same size were selected in an untransfected cell and the levels of fluorescence were measured.
Measurement of cell size. Cells were transfected with the GFP-expressing plasmid and with one of the RhoA mutants, C3 exotransferase, or an empty vector. Images were captured as described above. Cell area measurements were made by selecting GFP-positive transfected cells in Adobe Photoshop 5.5 and calculating the area in square microns.
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RESULTS |
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Pericytes overexpressing RhoWT demonstrate a more robust stress fiber network than control cells (Fig. 1, A-D). Staining with phalloidin, which binds F-actin regardless of isoform, reveals a strong stress fiber network in the cells transfected with RhoWT expression plasmid (Fig. 1D), but VSMactin stress fiber staining is greatly reduced compared with untransfected control cells. Instead, transfected cells possess
VSMactin that is uniformly dispersed throughout the cytoplasm (Fig. 1C).
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Pericytes overexpressing the dominant positive mutant RhoL63 have an even more pronounced isoactinspecific phenotype (Fig. 1, E-H) than RhoWT overexpressing cells. Whereas phalloidin staining reveals a pronounced stress fiber network in cells transfected with RhoL63 compared with untransfected cells or RhoWT expressers (Fig. 1G), anti-VSMactin staining demonstrates selective
VSMactin disassembly from stress fibers (Fig. 1H). This is similar to the effect seen in fibroblasts, in which Rho activation leads to increased formation of stress fibers, predominantly composed of
-actin (16). Fluorescent phalloidin and
VSMactin antibody localization studies reveal an increase in fluorescence in RhoL63-transfected cells, averaging 406 and 215%, respectively, compared with untransfected cells (P < 0.001) (Fig. 2). The four-fold increase in
-actin is associated with the accumulation of
-actin in stress fibers as seen in Fig. 1H, whereas the two-fold increase in
VSMactin is associated with the loss of coaxially aligned
VSMactin-containing stress fibers and
VSMactin homogenous redistribution throughout the cytoplasm (Fig. 1G).
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Expression of C3 exotransferase, which irreversibly inactivates RhoGTPase, or dominant negative mutant RhoN19 (data not shown) fails to cause any significant changes in pericyte isoactin array. In contrast to control cells, in cells expressing C3 exotransferase, we observed strong actin staining at the edges of the cell and an atypical radial scaffold of actin fibers (Fig. 1, I-L), as well as an increase in overall cell size. These changes are observed with both phalloidin (Fig. 1L) and VSMactin staining (Fig. 1K), indicating that the effect of Rho inactivation by C3 exotransferase is not isoform specific.
Because our data reveal that activation of Rho causes pericytes to become smaller and suppression of Rho results in larger pericytes, we quantified the changes in cell size in response to mutant Rho overexpression. RhoWT causes a slight reduction in cell size (77% of control), RhoL63 significantly reduces (P < 0.05) the size of the cell (44% of control), Rho N19 expression leads to a slight increase in cell size (108% of control), and C3 transferase significantly increases (P < 0.01) cell size (298%) (Fig. 3). It is believed that cell size after Rho activation depends on the ability of the cell to form integrin-adhesion complexes and stress fibers. If cells are capable of forming stress fibers but not contacts, they will be small and round, as has been demonstrated for macrophages (1).
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To explore whether other vascular smooth muscle-associated cytoskeletal proteins were similarly disassembled from pericyte stress fibers, we examined the localization of myosin isoforms by using immunofluorescence microscopy. We found that RhoWT and RhoL63 have a dramatic effect on NMmyosin distribution (Fig. 4, C and F), causing disassembly and dispersion similar to that seen in VSMactin, and expression of dominant positive RhoL63 leads to a similar redistribution of SMmyosin (Fig. 4, J-L). Expression of RhoN19 does not alter pericyte NMmyosin localization (Fig. 4I).
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We next studied the effect of other members of the Rho family on VSMactin presence in stress fibers. We transfected cultured pericytes with constitutively active Rac and Cdc42. In addition, we tested Ras as a control. Constitutively active Ras (RasL61) has no effect on cell shape,
VSMactin localization, or stress fiber arrays (Fig. 5, A-D). Expression of constitutively active Rac (RacL61) leads to lamellipodia formation and stress fiber redistribution, but phalloidin and
VSMactin staining both reveal a normal stress fiber network (Fig. 5, E-H). Expression of constitutively active Cdc42, Cdc42L61, leads to filopodia formation, cytoskeletal redistribution, and some reduction of the
VSMactin levels in stress fibers (Fig. 5, I-L). However, Cdc42L61 does not lead to disassembly and uniform dispersion of
VSMactin in the cell cytoplasm, as seen with Rho overexpression.
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DISCUSSION |
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To date, no information has appeared in the scientific literature regarding the isoform-specific effect of Rho activation on the actin cytoskeletal array. Vascular smooth muscle cells are closely related to pericytes and demonstrate "contractile" and "synthetic" phenotypes. In the synthetic stage, vascular smooth muscle cells are able to proliferate in response to injury or in the process of normal angiogenesis, but they down-regulate vascular smooth muscle-specific proteins, such as VSMactin and SMmyosin. Smooth muscle contractility is modulated by Rho and its target, Rho kinase.
There are reports that Rho activation in vascular smooth muscle cells leads to differentiation and a switch from the synthetic to the contractile phenotype, but the effect of Rho activation on VSMactin has not been studied (19, 30). In our work, we observe that pericytes in the contractile stage, expressing
VSMactin and SMmyosin, respond to Rho activation by disassembling
VSMactin- and SMmyosin-containing stress fibers. Whereas the bundled arrays of actin filaments are noticeably altered, fluorescent phalloidin localization suggests that specific populations of isoactin filaments persist. Clearly, the dynamic reorganization of the actin cytoskeleton, shifting from a stress fiber-rich array to one comprised of orthogonally oriented interconnected filaments, is under Rho GTPase control. However, the precise mechanism leading to this isoactinspecific reorganization is not completely understood.
The results of our study suggest that an optimal steady-state Rho GTPase expression level is needed to maintain the delicate balance required to sustain cell adhesion, growth, and proliferation, or contractile potential via an isoactin-specific cytoskeletal signaling mechanism. Interestingly, Rho kinase functions as one of the major downstream effectors of Rho GTPase signal transduction. Through its counteracting effects on myosin light-chain kinase and phosphatase activities, calcium- and calmodulin-dependent actomyosin-based contraction could be reversibly regulated (5, 8, 18, 25). Also, the Rho kinase inhibitor, Y-27632, selectively suppresses smooth muscle contractility by inhibiting calcium sensitization. This, in turn, suppresses stress fiber formation and dramatically augments hypertension in rat models (28). There are numerous other pivotal actin-associated effectors that are downstream of Rho GTPase signaling, including myristolated ala-nine-rich C kinase substrate (MARCKS), the LIM kinases, and their downstream phosphoeffector capable of promoting actin filament disassembly, cofilin (11, 26). These observable pathways that converge on actin-based cytoskeletal remodeling represent a limited molecular understanding of Rho GTPase function and cytoskeletal signal transduction. Our work adds a new level of complexity to our understanding the manner in which Rho GTPase and Rho GTPase effectors function to alter cell shape and contractile potential in an isoactin-specific manner. Clearly, more work will be needed before we fully appreciate how these complex signaling cascades orchestrate isoactin dynamics and cell behavior during development or in association with disease.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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