1Vascular Biology Program, Departments of Pathology and Surgery, Children's Hospital and Harvard Medical School; and 2Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115
Submitted 3 July 2003 ; accepted in final form 12 October 2003
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ABSTRACT |
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fibronectin; cell mechanics; hypertension
Cell contractility results from the action of tensional forces that are generated within the actin cytoskeleton. Tension generation results from binding interactions between actin and myosin filaments that activate a myosin ATPase that drives filament sliding. These actin-myosin interactions are promoted by phosphorylation of the regulatory myosin light chain (MLC) (45) by MLC kinase (MLCK) (14). MLCK activity is controlled by intracellular Ca2+ and calmodulin levels (45); however, this stimulatory influence is balanced by MLC phosphatase, which can dephosphorylate MLC (16). Various signaling molecules contribute to control of MLC phosphatase activity, including the Rho effector, Rho-associated kinase (ROCK) (16), and protein kinase C (46). Interestingly, binding and clustering of integrin receptors can activate both the Rho (38) and protein kinase C pathways (2, 6, 50, 55).
Cell interactions with ECM differ from those with soluble regulators, because cells also apply traction to their integrin receptors that mediate matrix adhesion (1). Tensional forces generated within the cytoskeleton and resisted by the ECM feed back to alter cell shape and thereby influence various cellular responses, including growth, differentiation, and motility. Importantly, cultured VSM cells exhibit different levels of contractility on ECM substrates that differ in their ability to support cell spreading (26, 47). The Rho/ROCK pathway and associated focal adhesion formation can also be stimulated by increasing the mechanical stiffness of the ECM substrate (13) or by direct distortion of cell surface integrins (1, 31, 39). However, these local events at the site of integrin binding still remain under the control of global biochemical events that are linked to cell distortion: spread VSM cells respond to vasoagonists that work through the Rho pathway (e.g., lysophosphatidic acid), whereas retracted cells do not (47).
Cell shape stability requires the establishment of a mechanical force balance within the cytoskeleton (20). Tensional forces that are generated within contractile microfilaments are partially balanced by the cell's external tethers to the ECM substrate and partly by internal microtubules that resist compression inside the cell. By balancing these forces between microfilaments, microtubules, and ECM, the cell generates a "prestress" or state of isometric tension in the cytoskeleton that mechanically stabilizes cell shape. Moreover, these internal forces can be shifted between these different load-bearing elements. For example, chemical disruption of microtubules increases the level of traction exerted on the ECM as compressive forces normally borne by these cytoskeletal elements are transferred to complementary ECM adhesion sites (51). Conversely, modifying ECM mechanics can also alter the level of forces carried by microtubules and microfilaments (33, 36, 37). The point is that both the level of prestress in the cytoskeleton and the net contractile force cells exert on their ECM substrate depend on a cellular mechanical force balance between the cytoskeleton and the ECM. The purpose of the present study was therefore to gain further insight into the fundamental mechanism by which cell adhesion and spreading on ECM regulate pulmonary VSM cell contractility by exploring whether ECM-dependent changes in this cellular force balance feed back to modulate the contractility set-point.
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MATERIALS AND METHODS |
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Various densities (0-1 µg/cm2) of FN (Collaborative Research) were immobilized on nonadhesive, bacteriological, plastic dishes, as described (24). Projected cell areas were determined from phase contrast images using IPLab image analysis software (Scanalytics); at least 20 cells from at least 4 randomly selected fields of each culture dish were analyzed for each condition. For bead-binding experiments, tosyl-activated 4.5 µm beads (Polysciences) were coated with a saturating FN density (50 µg/ml) as described (43). Coated beads were added to suspended cells (20 beads/cell) and rocked at 37°C for 30 min; bead attachment was verified by microscopy. Microfabricated micrometer-sized adhesive islands were produced on glass slides and coated with a saturating density of FN (50 µg/ml) as described (8, 9). Flexible polyacrylamide gel culture substrates were prepared according to published methods (33, 51) and coated with high-density FN (1 µg/cm2). The substrate flexibility was manipulated by varying both the total acrylamide concentration (2-4%) and the bis-acrylamide component (0.1-0.5%); the Young's modulus (stiffness) for each substrate was determined as described previously (52).
MLC phosphorylation was assayed using glycerol-PAGE in combination with immunoblotting (12, 51). MLC bands were detected with an anti-MLC monoclonal antibody (MY-21; Sigma). The stoichiometry of MLC phosphorylation (mol PO4/mol MLC) was determined by densitometry using Scion Image software (Scion). MLC phosphorylation was calculated from the following formula: (P1 + 2 x P2)/(P0 + P1 + P2) where P0 = unphosphorylated MLC band, P1 = singly phosphorylated MLC band, and P2 = doubly phosphorylated MLC band. Microfluorimetric quantitation of intracellular calcium levels was carried out using fura 2 in conjunction with ratio imaging, as described (51).
The tractional stresses exerted by individual cultured VSM cells and cellular prestresses were quantitated using a modified form of traction force microscopy, as previously described (48). Briefly, VSM cells were cultured on flexible polyacrylamide gels substrates (Young's modulus = 3,750 Pa) coated with various densities of fibronectin (0.02 to 1 µg/cm2) that contained green fluorescent nanobeads (500-nm diameter; Molecular Probes) as fiducial markers. Nanobead positions were measured before and after addition of warm SDS (1% final concentration) to remove the cells. Isometric tension forces (prestress) exerted by each of the adherent cells were determined by comparing bead position before and after cell detachment and calculating relative bead displacements. Active traction forces produced by stimulation with the vasoagonist, ET-1, were calculated by subtracting the total traction force exerted minus the prestress calculated for cells under similar conditions without ET-1. Statistical analysis of total MLC phosphorylation and prestress was carried out using means calculated from all experiments of a defined condition, and significance was determined by the Student's t-test for unpaired samples. The Student's t-test for paired samples was used for statistical analysis of changes in MLC phosphorylation and tractional forces induced by various agents.
To confirm changes in the organization of microfilaments and microtubules after drug treatment, cells cultured on glass coverslips (Lab-Tek, Nalge Nunc, Naperville, IL) coated with FN (1 µg/cm2) were fixed with 2% paraformaldehye in microtubule stabilization buffer (0.1 M PIPES, pH 6.75, 1 mM EGTA, 1 mM MgSO4, 2 M glycerol, and protease inhibitors) (5, 30). Microtubules were visualized using a mouse monoclonal anti-tubulin primary antibody (Sigma) and a FITC-conjugated anti-mouse IgG secondary antibody (Amersham). Microfilaments were visualized using rhodamine-phalloidin (300 ng/ml; Sigma). Fluorescent images were captured using a Nikon Diaphot 300 inverted microscope and a Photometrics Sensys KAF 1400 charge-coupled device digital camera.
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RESULTS |
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To directly determine whether these FN-dependent changes in MLC phosphorylation resulted in actual changes in cell prestress (level of isometric tension in the cell before vasoagonist addition) and active contraction in the presence of ET-1, we measured interfacial traction on the adhesive substrate and cellular prestress within pulmonary VSM cells cultured on flexible, FN-coated, polyacrylamide substrates using traction force microscopy (34, 48). In this technique, cell-generated forces are quantitated by analyzing stress-dependent changes in the movement of small (0.2 µm) fluorescent beads that are contained within the flexible substrate, both before and after removal of the cells from the substrate. Increasing FN coating densities on these flexible substrates resulted in a FN density-dependent increase in cell prestress (Fig. 4A). ET-1 induced significant increases in traction at each FN density (Fig. 4B). Unlike MLC phosphorylation, the mean net change in traction in response to ET-1 was greatest in cells cultured on moderate FN density (Fig. 4B). This result correlated with the fact that a higher percentage of cells demonstrated a statistically significant increase in traction on moderate FN density compared with low and high densities (Fig. 4C). However, analysis of ET-1-induced changes in traction in this responsive (contractile) subset of cells again revealed a direct correlation between FN density and the magnitude of ET-1-induced traction forces (Fig. 4D). Analysis of the contractile response to ET-1 over time in this same subset of cells (Fig. 4E) showed that traction forces closely paralleled MLC phosphorylation (Fig. 2D), reaching maximal levels by about 3 min and remaining high through 8 min. Micrographs of responsive cells carried out during traction force microscopy also revealed increases in the number and size of regions where highest traction stresses were exerted beneath individual cells in response to both increasing FN densities and adding ET-1 (Fig. 5).
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These initial findings are consistent with the concept that different densities of FN modulate MLC phosphorylation, cell prestress, and the contractile response to ET-1 based on their ability to promote cell extension. However, varying the ECM coating density also alters local integrin receptor clustering and activates associated integrin signaling cascades (24, 28, 43). To discriminate between these two mechanisms, suspended cells were allowed to bind to FN-coated microbeads (4.5-µm diameter), which induce integrin clustering and signaling without promoting VSM cell spreading (28). Cell binding to FN-coated microbeads for 30 min was not sufficient to induce an increase in MLC phosphorylation, either alone or after stimulation with ET-1 (Fig. 6, A and B).
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A potential caveat in these latter experiments is that cells may engulf attached microbeads after periods greater than 30 min (8), thereby limiting the duration of integrin-FN engagement. To address this limitation, cells were cultured for 24 h on microfabricated substrates containing 30 x 30 µm2 adhesive islands coated with high FN that limit cell spreading to the size and shape of the islands because they are surrounded by nonadhesive regions (8). Spreading of cells on these small adhesive islands has been shown previously to optimally activate integrin signaling pathways (56). With the use of this method, cell spreading was restricted to 30% of that observed for cells on control (unpatterned) FN (Fig. 6C), and MLC levels were significantly reduced (Fig. 6, D and E).
The mechanism by which ECM density controls cell extension appears to rest, in part, on the ability of the ECM to physically resist cell tractional forces (23). To independently determine whether the physical properties of the ECM and associated changes in the cellular mechanical force balance are critical for controlling MLC phosphorylation, VSM cells were cultured on flexible substrates that were coated with a constant (saturating) FN density but varied in their mechanical compliance (33). Cells dissipate more stress when adherent to flexible substrates than on rigid dishes that more effectively resist cell-generated tractional forces, and thus the level of prestress in the cytoskeleton is reduced. Cell spreading was suppressed when substrate rigidity (Young's modulus) was decreased (Fig. 7, A, C-F), as previously observed when FN densities were lowered (Fig. 1). MLC phosphorylation levels in VSM cells scaled directly with the rigidity of these substrates (Fig. 7B) and, hence, with their ability to both maintain isometric tension in the cytoskeleton and support cell spreading.
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Because cell spreading decreases as substrate compliance is increased, it could be argued that cell spreading itself is the critical determinant of MLC phosphorylation. To address this possibility, we employed various pharmacological agents that alter cytoskeletal mechanics (and, hence, prestress) in fully spread cells on rigid FN-coated dishes. First, cells were treated with cytochalasin D (1 µg/ml), which disrupts microfilaments (Fig. 8, A and B) and thus dissipates tensional stress. MLC phosphorylation was greatly reduced in these cells, both before and after stimulation with ET-1 (Fig. 9, A and C). However, cytochalasin D also has a dramatic effect on cell morphology, inducing cell retraction and rounding (Fig. 8B). To dissipate cell prestress without disrupting cytoskeletal integrity or changing cell shape, we used BDM, which inhibits myosin ATPase downstream of MLC phosphorylation (Fig. 8C). Computerized image analysis confirmed that BDM did not significantly alter VSM cell spreading under these conditions (average projected cell areas for cells in the presence or absence of BDM were, respectively, 3,241 ± 228 and 3,209 ± 171 µm2). In spread cells treated with BDM, MLC phosphorylation was significantly reduced (Fig. 9, A and C) to levels similar to those exhibited by cells on low FN (Fig. 2), on highly flexible substrates (Fig. 7), or by retracted cells treated with cytochalasin D (Fig. 9, A and C). Although BDM had a potent inhibitory effect in cells on moderate and high FN, it did not produce a significant change in MLC phosphorylation in cells on low FN (Fig. 9D). One potential complication of BDM treatment is that it can inhibit calcium transport (4, 32). However, no significant changes in intracellular calcium were detected in BDM-treated VSM cells using the Ca2+-sensitive dye fura 2 and microfluorimetry (data not shown). Treatment of cells with BDM and cytochalasin D also decreased the total level of MLC phosphorylation in ET-1-stimulated cells; however, ET-1 produced a similar net increase in MLC phosphorylation (0.25 to 0.3 mol PO4/mol MLC) even under conditions in which prestress was dissipated and the cytoskeleton was disrupted (Fig. 9, A and C).
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In contrast to agents that dissipate cellular prestress, treatment of VSM cells with nocodazole at a dose (10 µM) that completely depolymerizes microtubules (Fig. 8, D and E) elevated MLC phosphorylation to levels comparable to those observed in spread cells maximally stimulated with ET-1 (Fig. 9, A-C). Similar induction of MLC phosphorylation by microtubule-disrupting agents has been observed in other cell types (25). Microtubule disruption also was able to increase MLC phosphorylation in cells that were pretreated with either BDM or cytochalasin D to first dissipate cytoskeletal prestress (Fig. 9, B and C); however, the maximal level of MLC phosphorylation was significantly decreased in the drug-treated cells. This ability of depolymerized microtubules to increase VSM cell contractility required cell adhesion to FN as treatment of suspended cells with the same dose of nocodazole failed to produce any effect on MLC phosphorylation (Fig. 9E).
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DISCUSSION |
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Past studies have shown that biochemical signals are initiated upon integrin engagement and clustering, even in the absence of changes in cell shape or external mechanical loading of integrin adhesion sites (28, 35, 43). But in the present study, integrin engagement alone was not sufficient to induce MLC phosphorylation in the absence of cell spreading. Moreover, flexible FN substrates that similarly did not support cell extension also failed to increase MLC phosphorylation. These observations are consistent with the findings of past studies that similarly demonstrated that alterations in the cell that are secondary to integrin binding, specifically, changes in mechanical loading of focal adhesion sites and associated changes in cell shape, are also critical for an integrated contractile response in VSM cells (26, 47). For example, when cells were prevented from spreading using microfabricated elastomeric substrates, they display greatly reduced basal tractional forces (prestress) and failed to contract in response to soluble contractile agonists (serum or lysophosphatidic acid), whereas expression of constitutively active RhoA rescued the contractile response (47).
The present study extends these findings by showing that ECM-dependent changes in cell spreading modulate signal transduction at the level of MLC phosphorylation, both at steady state and in response to another vasoconstrictor, ET-1. In light of these observations, it is interesting that VSM cells are more responsive to ET-1 on the moderate FN density compared with high FN in terms of increasing the tractional forces they exert on the substrate. A similar relationship between contraction induced by ET-1 and FN density was demonstrated in these same cells using magnetic twisting cytometry (26). This could be explained, in part, if the nonresponsive cells on high FN were already maximally contracted before ET-1 addition. In fact, the mean prestress of the responsive (ET-1-sensitive) cells on high FN was significantly lower than that in the unresponsive cells on the same substrate: 189 ± 29 and 336 ± 21 (Pa, respectively). In contrast, on low FN, both prestress and MLC phosphorylation were low, supporting our argument that contractility is sensitive to cell spreading in these cells.
Nevertheless, it remains unclear how cell spreading impacts biochemical signaling pathways that ultimately modulate MLC phosphorylation and contractility. One potential clue to this mechanism stems from our finding that cell spreading can be dissociated from MLC phosphorylation by altering cell-generated tensile forces independently of cytoskeletal integrity and cell shape. This behavior was observed when actomyosin tension generation was inhibited by BDM, which acts downstream of MLC phosphorylation (17, 27) and does not significantly affect cell shape. Because of BDM's known phosphatase activity (53, 54) and effects on intracellular Ca2+ (4, 32), we cannot totally exclude the possibility that BDM modulates MLC phosphorylation by pathways separate from its effect on prestress. However, our finding that BDM had only a minimal effect on MLC phosphorylation in cells with low prestress on low FN suggests that its inherent phosphatase activity did not contribute significantly to the results observed under our assay conditions. Likewise, no diminution of intracellular Ca2+ was observed in cells treated with BDM in the present study. Thus changes in cytoskeletal prestress that accompany cell spreading may be a key determinant of MLC phosphorylation.
We also found that disruption of microtubules elevated MLC phosphorylation levels in adherent VSM cells, even in cells treated with BDM and cytochalasin D, as previously described (25). These findings indicate that microtubule depolymerization or release of tubule monomers can activate the contractile machinery independently of intact actin filaments or cytoskeletal prestress. However, the total increase in MLC phosphorylation was significantly decreased in cells with lower prestress due to these drug treatments, and the effects of microtubule disruption were completely inhibited in suspended cells that lacked ECM adhesions. Combined with recent findings that demonstrate a direct shift of forces from microtubules to sites of ECM adhesion after microtubule disruption (51), it is possible that part of the observed increases in MLC phosphorylation in adherent cells may be due to changes in tensional forces exerted on ECM adhesions. ET-1 also failed to increase MLC phosphorylation in round cells in suspension, whereas it induced a significant increase in cells adherent to FN that were treated with cytoskeletal disruptor drugs. These findings again suggest that some signaling structure that is formed during the initial process of cell spreading, and is sufficient to support vasoagonist signaling in the absence of prestress, may remain intact under these disruptive conditions. Interestingly, the focal adhesion exhibits this property at least for the short times (minutes) examined in this study.
How might cell spreading and prestress influence biochemical signaling leading to MLC phosphorylation? Increases in cell spreading, cytoskeletal prestress, and microtubule disassembly are all accompanied by increases in traction forces exerted on the ECM (52), which, in turn, promote focal adhesion formation (7, 11, 39). Thus changes in cell shape and prestress may modulate focal adhesion signaling pathways that control MLC phosphorylation. A likely candidate for mediating this type of signaling is RhoA acting through ROCK. Alternatively, prestress may alter the conformation and/or kinetics of other biochemical regulators that are tethered to the cytoskeleton (22).
Whatever the mechanism, the results of this study raise the intriguing possibility that prestress may feed back to regulate contractile signaling, much as it regulates growth signaling (19, 40) and ET-1 gene expression (10). Specifically, our findings suggest that if tension is dissipated in the cytoskeleton (i.e., prestress is decreased) because adhesion sites are of insuffi-cient strength or the compliance of ECM increases, then the cell will remain in a low state of contractility, regardless of the presence of soluble vasoagonists. This contractile set-point is reflected by the level of MLC phosphorylation in the cell before stimulation by soluble agonists, and it is governed by the level of prestress in the cytoskeleton independently of integrin binding or cell spreading. In this way, ECM mechanics may feed back via a mechanochemical mechanism to modulate further tension generation so that an appropriate cellular force balance is achieved to match the physical characteristics of the microenvironment, thereby ensuring "compliance matching" within the vascular wall. These data also suggest that chemical agents that alter ECM mechanics, cytoskeletal structure, or basal prestress may be useful for treatment of hypertensive diseases that are characterized by abnormal VSM cell contractility.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-67669 (to D. E. Ingber) and National Aeronautics and Space Administration Grant NAG2-1509 (to N. Wang).
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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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