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VASCULAR BIOLOGY

Rho GTPase signaling modulates cell shape and contractile phenotype in an isoactin-specific manner

Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Submitted 2 May 2003 ; accepted in final form 27 June 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Rho family small GTPases (Rho, Rac, and Cdc42) play an important role in cell motility, adhesion, and cell division by signaling reorganization of the actin cytoskeleton. Here, we report an isoactin-specific, Rho GTPase-dependent signaling cascade in cells simultaneously expressing smooth muscle and nonmuscle actin isoforms. We transfected primary cultures of microvascular pericytes, cells related to vascular smooth muscle cells, with various Rho-related and Rho-specific expression plasmids. Overexpression of dominant positive Rho resulted in the formation of nonmuscle actin-containing stress fibers. At the same time, -vascular smooth muscle actin (VSMactin) containing stress fibers were disassembled, resulting in a dramatic reduction in cell size. Rho activation also yielded a disassembly of smooth muscle myosin and nonmuscle myosin from stress fibers. Overexpression of wild-type Rho had similar but less dramatic effects. In contrast, dominant negative Rho and C3 exotransferase or dominant positive Rac and Cdc42 expression failed to alter the actin cytoskeleton in an isoform-specific manner. The loss of smooth muscle contractile protein isoforms in pericyte stress fibers, together with a concomitant decrease in cell size, suggests that Rho activation influences "contractile" phenotype in an isoactin-specific manner. This, in turn, should yield significant alteration in microvascular remodeling during developmental and pathologic angiogenesis.

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.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell culture and transfection. Pericytes were isolated from bovine retinas as previously described (13, 20) and used through passage 10 in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Gaithersburg, MD) containing 10% bovine calf serum (BCS; Hyclone, Logan, UT) supplemented with penicillin, streptomycin, fungizone, and L-glutamine (GIBCO). Cells from three separate primary preparations were used. Cells were plated on 22-mm square glass coverslips (Corning, Big Flats, NY) in Costar six-well plates at a density of 150,000 cells per well to reach 60-80% confluence 72 h after being plated. Pericytes were transfected as previously described (29) with 0.5 µg DNA per coverslip by using Effect-ene transfection reagent (Qiagen, Valencia, CA) in Optimem media (GIBCO) with 10% BCS. Cultures were incubated with transfection mixture for 16 h and then washed with PBS and incubated in fresh DMEM with 10% of BCS for an additional 24 h. For each plasmid, at least 10 separate transfection experiments were performed.

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.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To study the function of the small GTPases in isoactin array regulation, we specifically blocked or increased their activity by transiently transfecting cells with dominant negative or dominant positive mutants. We used expression plasmids with the dominant positive mutant of RhoA, RhoL63, which mimics the constitutively GTP-bound state of the GTPase, the dominant negative mutant RhoN19, which mimics the GDP-bound state of GTPase (9, 10), and wild-type RhoA (RhoWT) for transient transfection of pericytes. To mark transfectants, the GFP reporter was used.

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).

View larger version (100K): [in this window] [in a new window]   Fig. 1. Overexpression of wild-type and dominant positive RhoA alters isoactin arrays in stress fibers. A-D: cells were transfected with RhoA-expressing plasmid, pExvRhoWT. After 48 h, cells were fixed and stained against Myc-epitope as a marker for transfected cells (B), antibodies against -vascular smooth muscle actin (VSMactin) (C), and phalloidin-Alexa 633 (D). E-H: cells were cotransfected with pExvRhoL63 plasmid and pEGFP-N3 plasmids. After 48 h, cells were fixed and stained with an antibody against GFP as a marker for transfected cells (F), an antibody against VSMactin (G), and phalloidin-Alexa 350 (H). I-L: pericytes were cotransfected with pEF-pLinkC3 and pEGFP-N3 plasmids. After 48 h, cells were fixed and stained for GFP as a marker for transfected cells (I), with an antibody against VSMactin (K), and phalloidin-Alexa 350 (L). Scale bar = 50 µm.

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).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Quantitation of fluorescence intensity in cells transfected with RhoL63 expression plasmid and untransfected cells stained with VSMactin antibody (VSMA) or phalloidin (Phall). Cells were transfected with RhoL63 expression plasmid. After 48 h, cells were stained with VSMactin antibodies or phalloidin-Alexa 546. Results are normalized to the fluorescence level of control cells (100%) and expressed as means ± SE (n = 10 cells; *P < 0.01). Cells examined were from 2 separate transfection experiments, 5 from each.

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).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Overexpression of RhoA, RhoA mutants, and C3 exotransferase affect pericyte cell size. Cells were cotransfected with GFP-expressing plasmid and either an empty vector, RhoWT, RhoL63, RhoN19, or C3 exotransferase. After 48 h, cells were stained with anti-GFP antibody and cell size was measured with Photoshop 5.5 software. The number of cells in each group is indicated at bottom. Cells from 3-5 separate transfection experiments were examined for each group. NS, nonsignificant; *P < 0.05; **P < 0.01, compared with control.

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).

View larger version (163K): [in this window] [in a new window]   Fig. 4. Effect of RhoA mutants overexpression on pericyte myosin isoform organization. A-I: cells were cotransfected with pEGFP-N3 and either wild-type RhoA, dominant positive mutant RhoL63, or dominant negative mutant RhoN19 as designated on the figure. After 48 h, cells were fixed and stained with polyclonal antibodies against GFP (B, E, H) and nonmuscle myosin (C, F, I). J-L: cells were transfected with RhoL63 plasmid. After 48 h, the cells were fixed and stained with polyclonal antibodies against smooth muscle myosin (SMmyosin) (K) and phalloidin-Alexa 488 (L). Scale bar = 50 µm.

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.

View larger version (92K): [in this window] [in a new window]   Fig. 5. Overexpression of dominant positive mutants of different members of Rho/Ras family small GTPases does not alter isoactin arrays in microvascular pericytes. A-L: cells were cotransfected with pEGFP-N3 and constitutively active Cdc42L61, RacL61, or RasL61 as designated. After 48 h, cells were fixed and stained with antibody against GFP, VSMactin, or phalloidin-Alexa 546 as designated. Scale bar = 50 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we report a Rho GTPase-dependent signaling cascade that alters pericyte shape and phenotype in a contractile protein isoform-specific manner. Rho activation in pericytes leads to selective disassembly of VSMactin-containing stress fibers, but not -actin-containing stress fibers, and also causes NMmyosin and SMmyosin disassembly from stress fibers. Such modification of the isoactin array is Rho specific because the activation of other small GTPases, such as Rac, Cdc42, or Ras, does not produce similarly specific effects.

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.

	DISCLOSURES

 
This work was supported by National Institutes of Health Grants NIH-GM-55110 and EY-09033 (to I. M. Herman).

	ACKNOWLEDGMENTS

 
We thank Dr. Deniz Toksoz (Tufts University School of Medicine, Boston) for helpful discussions.

	FOOTNOTES

 

Address for reprint requests and other correspondence: I. M. Herman, Dept. of Physiology, Tufts Univ. School of Medicine, 136 Harrison Ave., Boston, MA 02111 (E-mail: ira.herman{at}tufts.edu); and A. Y. Kolyada, Dept. of Physiology, Tufts Univ. School of Medicine, 136 Harrison Ave., Boston, MA 02111 (E-mail: alexey.kolyada{at}tufts.edu).

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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