Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation

Hiromi Nobe1, Koji Nobe1, Fabeha Fazal2, Primal de Lanerolle2, and Richard J. Paul1

1 Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio, 45267-0576; and 2 Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fibroblasts form fibers when grown in culture medium containing native type 1 collagen. The contractile forces generated can be precisely quantified and used to analyze the signal transduction pathways regulating fibroblast contraction. Calf serum (30%) induces a sustained contraction that is accompanied by a transient increase in intracellular calcium ([Ca2+]i). W-7, a calmodulin inhibitor, KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase, and ML-7, a myosin light-chain kinase inhibitor, had no effects on either the contraction or the [Ca2+]i responses. Neither genistein, a tyrosine kinase inhibitor, nor calphostin C, a protein kinase C inhibitor, had major effects on force or [Ca2+]i. In contrast, the Rho kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and HA1077 depressed the contraction in a dose-dependent manner without affecting the [Ca2+]i response. Stress fiber formation was also suppressed by Y-27632. Surprisingly, calf serum, Y-27632, and calf serum plus Y-27632 did not alter mono- or diphosphorylation of the myosin regulatory light chain (MRLC) compared with control untreated fibers. These results suggest that the sustained contraction of NIH 3T3 fibroblast fibers induced by calf serum is mediated by Rho kinase but is independent of a sustained increase in [Ca2+]i, calcium/calmodulin- or protein kinase C-dependent pathways, or increases in MRLC phosphorylation.

nonmuscle contractility; wound repair; calcium; stress fibers


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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FIBROBLASTS GENERATE a contractile force that is likely the basis for motility (4). Both motility and contractility are essential for their role in wound repair. The mechanism(s) of regulation of fibroblast contraction is thus of importance, both at the basic science and clinical levels. We have developed a model system that permits quantitative assessment of fibroblast contractility using NIH 3T3 fibroblast cells cultured in a three-dimensional collagen matrix (16). These collagen fiber preparations provide a powerful method for quantitative mechanical measurements (17) that cannot be easily achieved with monolayer culture and substrate wrinkling or gel shrinkage assays.

We previously reported that calf serum (CS) increased isometric force in these fibers in time- and dose-dependent manners (16, 17). The CS-induced force was abolished by cytochalasin D, indicating that the contraction in the reconstituted fiber is ascribable to the fibroblasts and not a property of the collagen matrix. Our observations are consistent with an earlier report by Kolodney and Wysolmerski (12) showing that the actin cytoskeleton is essential for force generation. Further, using fura 2 we found that 30% CS elicited a transient increase in intracellular calcium ([Ca2+]i), which was dependent on sarcoplasmic reticulum (SR) function. Inhibition of SR function led to a decrease in both force and [Ca2+]i in response to CS. This suggested that at least a transient [Ca2+]i increase was necessary to activate contraction but not essential for maintenance of force (15).

Increases in [Ca2+]i are known to regulate a variety of intracellular enzymes via calcium/calmodulin-dependent pathways. Phosphorylation of the 20-kDa regulatory light chain of myosin (LC20) by the calcium/calmodulin-dependent enzyme myosin light-chain kinase is necessary for smooth muscle contraction (3). A similar myosin light-chain kinase-based mechanism has also been postulated for nonmuscle contraction (12), but this hypothesis remains controversial (16). Modulation of the phosphorylation state of LC20 by myosin light-chain phosphatase also seems to play a major role in regulation of the level of force at constant [Ca2+]i (21). Rho-associated kinase, which is activated by the GTP-bound active form of Rho (7), phosphorylates the myosin-binding subunit of myosin light-chain phosphatase and inhibits its activity, thereby increasing myosin light-chain phosphorylation (8). Thus there is growing evidence that Rho-associated kinase plays a major role in regulating smooth muscle through a mechanism that involves an increase in myosin light-chain phosphorylation (21).

However, for nonmuscle tissues, the mechanism of regulation of actin-myosin interaction and contractility is not known with certainty. The present study was designed to use inhibitors of known regulatory pathways to probe the regulatory mechanism(s) of fibers made from NIH 3T3 fibroblasts. Our findings, based on measurements of isometric force, [Ca2+]i, and myosin light-chain phosphorylation, indicate that the sustained contraction in fibroblast fibers in response to CS is specifically mediated by Rho-associated kinase but is independent of myosin light-chain phosphorylation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and fibroblast fiber preparation. NIH 3T3 fibroblast fibers were subcultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% CS, 100 units/ml penicillin, and 100 µg/ml streptomycin. The cells were grown on 100-mm dishes in 5% CO2-95% air incubation at 37°C. The cells were propagated using 0.04% trypsin and 0.02% EDTA in phosphate-buffered saline, pH 7.4, at a split ratio of 1:3.

Fibroblast fibers were prepared according to Obara et al. (16). Rat tail collagen solution was neutralized with 0.1 M NaOH in an ice bath. Dispersed cells were suspended in a solution that contained 2 × 106 cells/ml and 0.5 mg/ml collagen in DMEM. A cell suspension of 2 ml was poured into a specially designed mold (0.8 cm × 5 cm × 0.5 cm deep) that was cut into a layer of silicone rubber in a 100-mm dish and placed in a CO2 incubator at 37°C. After 2 h, an additional 0.5 ml of DMEM was added to each well. The fiber preparations were incubated for 2-4 days.

Measurement of isometric force development. NIH 3T3 fibroblast fibers prepared as described above were cut into 5-mm pieces and mounted between glass posts with a cyanoacrylate glue. One post was fixed, and the other side was connected to a silicon strain gauge force transducer (SensoNor, AME801). The fibers were bathed in a MOPS-buffered physiological salt solution (MOPS-PSS) that contained (in mM) 140 NaCl, 4.7 KCl, 1.2 NaH2PO4, 0.02 EDTA, 1.2 MgSO4, 2.5 CaCl2, 11.1 glucose, and 20 MOPS, pH 7.4, at 37°C.

Measurement of [Ca2+]i. Measurements of [Ca2+]i levels were obtained from NIH 3T3 cells in fibers loaded with the Ca2+-sensitive fluorescent dye fura 2 based on the techniques of Grynkiewicz et al. (5). Fura 2-AM was prepared as a stock solution of 1 mM dye in DMSO. Our fura 2 loading solution contained 3 µM fura 2-AM, 0.015% pluronic F127, and 0.5% DMSO in MOPS-PSS buffer and was sonicated for at least 5 min to facilitate dispersion of the fura 2-AM. Fibroblast fibers were incubated in this solution at room temperature for 3 h. Fibroblasts were rinsed in MOPS-PSS buffer for 15 min to remove extracellular and nonhydrolyzed fura 2-AM.

A segment (~2 mm) of the fura 2-loaded fibroblast fiber was placed in a glass-bottomed culture dish and covered with nylon mesh, which kept the fiber isometric. The fiber was placed in a chamber with a total volume of 500 µl and perfused (5 ml/min) with MOPS-PSS, with a temperature of 37°C maintained. [Ca2+]i was measured with an intracellular imaging (INCA, Cincinnati, OH) microscope-based system. The chamber with the fiber was placed in a Nikon Diaphot inverted microscope with fluorphase objectives, permitting illumination at 340 nm. Fluorescent images of cells excited at 340 and 380 nm and emitting at 510 nm were obtained with a Dage silicon-intensified target camera. After subtraction of the background fluorescence, the 340- and 380-nm images were ratioed on a pixel-by-pixel basis at a frequency of 1 Hz. The ratios (R340/380) were converted to [Ca2+]i using a previously generated standard curve as described below. Quantitative analysis of the average [Ca2+]i was achieved by defining the outline of the cell, summing the signal in all the pixels within the defined area, and dividing by the number of pixels.

Solutions containing known concentrations of free-Ca2+ for standard curves were obtained from Molecular Probes (Eugene, OR). Fluorescence intensity was measured in 150 µl of each standard solution (0, 0.065, 0.100, 0.225, 0.351, and 0.602 µM free Ca2+ concentration) containing 13.3 µg/ml fura 2 pentapotassium salt. The R340/380 values obtained were used by the INCA system software for Ca2+ calibration of experimental data.

Measurement of myosin light-chain phosphorylation. After an experimental protocol was carried out, reactions in the isometrically mounted fibers were terminated by immersion in TCA in a dry ice-acetone slurry (10% TCA:90% acetone, vol/vol). Fibers were frozen in the steady state, generally 15 min after the test conditions were applied. The frozen fibers were homogenized by boiling in SDS sample buffer (1 ml) containing 50 mM Tris · Cl (pH 6.9), 5% SDS, 1% beta -mecaptoethanol, and 10% glycerol for 5 min and then centrifuged at 14,000 rpm for 10 min. Samples thus prepared from individual experiments were separated by SDS-PAGE on two 5-20% gradient gels and transferred to 0.2-µm pore size nitrocellulose membranes at 50 mA and 4°C for 16 h. After transfer, the blots were blocked with milk and probed with rabbit antibodies that recognize either the mono- or diphosphorylated forms of LC20 (provided by Fumio Matsumura, Rutgers University, and Peter Vincent, Albany Medical Center, respectively). The blots were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (Jackson ImmunoChemical) at 1:3,000 dilution. The blots were then washed extensively, and the signal was detected by using ECL reagent. To verify that there were similar amounts of LC20 in each lane, the blots were stripped and reprobed with a pan LC20 antibody that recognizes the un-, mono-, and diphosphorylated forms of LC20. Densitometry of protein bands was performed using a phosphorimager and analyzed using the Gel Pro Analyzer version 3.0 software. The data were normalized by dividing the areas of the bands for mono- and diphosphorylation by the corresponding areas from the pan LC20 antibody blot. To determine the relative changes in mono- and diphosphorylation in a given experiment, these normalized values in the experimental groups were divided by the ratio obtained from the control fibers in the same experiment. This procedure gives the fold increase in mono- or diphosphorylation of LC20 in the experimental groups compared with control as normalized to the total amount of LC20 in each sample. Data from 3-5 separate experiments were used to calculate the means ± SE.

Stress fiber measurement. The reconstituted NIH 3T3 fibroblast fibers were mounted isometrically with 50 µN resting tension at 37°C. After the experimental protocol fibers were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, samples were rinsed three times in PBS and then permeabilized with 0.2% Triton X-100 for 10 min. After being rinsed, samples were stained with rhodamine-phalloidin (60 min). The samples were then rinsed and intracellular actin levels were detected using confocal microscopy.

Materials. NIH 3T3 fibroblast cells were purchased from American Type Culture Collection (Manassas, VA). DMEM and CS were purchased from Life Technologies (Grand Island, NY). Rat tail collagen type-1 was purchased from Upstate Biotechnology (Lake Placid, NY). Fura 2 and fura 2-AM were obtained from Molecular Probes. Calphostin C, calyculin A, genistein, ML-7, and W-7 were purchased from Sigma Chemical (St. Louis, MO). HA1077 and KN-62 were purchased from Calbiochem (San Diego, CA). (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) was kindly provided by Welfide (Osaka, Japan). Calphostin C, calyculin A, genistein, KN-62, and ML-7 were dissolved in dimethyl sulphoxide. The final dimethyl sulphoxide concentration in the bathing medium was lower than 0.1% and had no effect on mechanical responses. The other agents were dissolved in deionized water.

Data analysis. Data are presented as means ± SE. Group data were compared using a one-way analysis of variance; the Bonferroni method was used to determine the level of significance of differences between groups. P < 0.05 was taken as indicative of statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of kinase inhibitors W-7, KN-62, and ML-7 on the contractile response and [Ca2+]i changes in NIH 3T3 fibroblast fibers. Figure 1 depicts a typical record of isometric force and changes in [Ca2+]i developed in NIH 3T3 fibroblast fiber. Resting levels of force and [Ca2+]i were 53.3 ± 4.4 µN and 61.3 ± 3.0 nM. Thirty percent CS induced the maximum increase in force, which reached a plateau within 15 min, averaging 168.8 ± 29.3 µN (n = 4). [Ca2+]i only transiently increased attaining peak levels of 119.3 ± 7.3 nM (n = 6) within 2 min before returning to baseline. Details of this behavior were reported in a previous study (15).


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Fig. 1.   Effects of calf serum (CS) and W-7 pretreatment in a typical NIH 3T3 fibroblast fiber. A: isometric force. Spikes in the record are due to surface tension changes and indicate when the solution was changed. B: intracellular calcium ([Ca2+]i) for 3 cells in the fiber under the conditions indicated. After relaxation in physiological salt solution (PSS), 10 µM W-7 was added to the PSS 10 min before the second CS stimulation.

To investigate the role of calmodulin-dependent pathways, we pretreated the fibroblast fiber with the inhibitor W-7 (10 µM) (23) for 10 min. As seen in Fig. 1, W-7 did not affect either the CS-induced contraction (Fig. 1A) or [Ca2+]i (Fig. 1B). Data from these types of experiments are summarized in Fig. 2A and show that W-7 at concentrations up to 100 µM did not affect either force or [Ca2+]i. Similarly, we investigated the role of calcium/calmodulin-dependent kinases using KN-62 (24). Fibroblast fibers were pretreated with various concentrations of KN-62 for 10 min before stimulation with CS. This inhibitor did not affect either the CS-induced contraction or the increase in [Ca2+]i (Fig. 2B). The role of myosin light-chain kinase was investigated using the inhibitor ML-7 (19). As shown in Fig. 2C, ML-7 up to 100 µM did not affect the CS-induced increased in [Ca2+]i or force. Another structurally distinct myosin light-chain kinase inhibitor, wortmannin (1 µM) (14), also did not inhibit the responses to CS (data not shown). Although pretreatment with these inhibitors did not affect baseline force or [Ca2+]i or block the responses to CS, it is possible that they might elicit a relaxation. Neither W-7 (10 µM), KN-62 (10 µM), nor ML-7 (30 µM) could relax the CS contraction when added after a sustained force was achieved (data not shown).


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Fig. 2.   Effects of W-7 (A), KN-62 (B), and ML-7 (C) on the CS-induced increases in isometric force (bars) and [Ca2+]i (circles) in NIH 3T3 fibroblast fibers. Fibroblast fibers were first challenged with 30% CS, serving as the control response. After relaxation in PSS, the indicated concentration of an inhibitor was added 10 min before CS stimulation/PSS relaxation cycle. Results are summarized as % of CS-induced maximal responses. Each value represents means ± SE of 3-6 independent determinations.

Effects of protein kinase inhibitors on CS-induced responses in fibroblast fibers. We further investigated tyrosine kinase and protein kinase C pathways using genistein (1) and calphostin C (9). Neither compound affected the basal levels of force or [Ca2+]i. As summarized in Table 1, 30 µM genistein did not affect the CS-induced contraction or increase in [Ca2+]i. Calphostin C (1 µM) also had no effect on [Ca2+]i but did cause a moderate (~20%) decrease in force.

                              
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Table 1.   Effects of protein kinase inhibitors on CS-induced isometric force and [Ca2+]i

Effects of Rho-associated kinase inhibition on the contractile response and changes in [Ca2+]i by CS. Because these kinase inhibitors were ineffective, we investigated whether Rho-associated kinase is involved in the fibroblast fiber contraction using Y-27632, a Rho-associated kinase inhibitor (25). Preincubation with 10 µM Y-27632 for 15 min slightly decreased the resting level of isometric force, but [Ca2+]i was not affected (Fig. 3). Y-27632 (10 µM) nearly abolished the CS-induced contraction, averaging 21.8 ± 5.5% of the control CS response (Fig. 4). Importantly, the CS-induced increase in [Ca2+]i was not affected (~90% of normal CS response; Fig. 3B). We also studied the effects of another, chemically distinct Rho-associated kinase inhibitor, HA1077 (25). Again, preincubation with 10 µM HA1077 blocked the force but not the CS-induced increase in [Ca2+]i (87.3 ± 14.6%). The results with HA1077 were qualitatively similar to the results with Y-27632, with the exception that addition of HA1077 did not decrease the resting level of isometric force. Y-27632 and HA1077 inhibited the CS-induced contraction in a dose-dependent manner with the half-maximum inhibition at ~3.5 and 7.0 µM, respectively (Fig. 4). Moreover, either Y-27632 (10 µM) or HA1077 (10 µM) relaxed force when added to the sustained phase of a CS-induced contraction (data not shown).


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Fig. 3.   Effects of the Rho-associated kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) on CS-induced responses in a typical NIH 3T3 fibroblast fiber. A: isometric force. B: [Ca2+]i responses for 3 cells in the fiber under the conditions indicated.



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Fig. 4.   Effects of Rho-associated kinase inhibitors on CS-induced increases in isometric force in NIH 3T3 fibroblast fibers. x-Axis shows the concentration of inhibitor added 15 min before CS stimulation. Each value represents means ± SE of 3-6 independent determination. * Significant (P < 0.05) differences from CS-induced maximal response as determined by one-way analysis of variance.

The Rho signaling pathway is thought to be involved in the phosphorylation and activation of myosin light-chain phosphatase by Rho-associated kinase. Inhibition of this phosphatase leads to an increase in myosin light-chain phosphorylation and force. Myosin light-chain phosphatase is a member of the family of phosphatase that is reported to be inhibited by the phosphatase inhibitor calyculin A (6). We confirmed that calyculin A itself could elicit a contraction, as well as enhance the response to CS (Fig. 5), in the fibroblast fibers. Treatment with 100 nM calyculin A alone increased fibroblast force to 53.1 ± 2.3% of the normal CS response. We also found that 100 nM of calyculin A did not increase [Ca2+]i (data not shown).


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Fig. 5.   Effects of the phosphatase inhibitor calyculin A on isometric force in NIH 3T3 fibroblast fibers. A cumulative dose-force record was generated by exposing the fiber to each dose of calyculin A (Cal-A) for 15 min; 30% CS was then added, which further increased force. Results are summarized as % of CS-induced maximal responses. Each value represents means ± SE of 5-8 independent determinations.

Myosin regulatory light-chain phosphorylation. Our evidence to this point suggests that inhibition of myosin light-chain kinase had little effect on force but contraction could be elicited by phosphatase inhibition. One interpretation of these observations is that a phosphorylation site other than the myosin regulatory light chain may be involved in activation of contraction. To test this hypothesis, we measured the LC20 phosphorylation (LC20-Pi) under a variety of experimental conditions. Measurements were made in the steady state, generally 15 min after the test conditions were applied. This is technically challenging due to the small number of cells in each fiber and the relatively low level of myosin in fibroblasts. We used an immunoblotting technique with antibodies to the mono- and diphosphorylated forms of LC20. The data were first normalized to the total LC20 as determined by striping and probing individual blots with a pan-LC20 antibody. The LC20-Pi states were then expressed relative to the basal level in a segment of the same fiber. This normalization significantly reduced the variability. As shown in Table 2, for all the experimental conditions studied, changes in LC20-Pi were modest. Thirty percent CS, Y-27632 alone, or the combination of 30% CS and Y-27632 did not result in LC20-Pi values that were significantly different from control. The exception was the positive control, i.e., LC20 phosphorylation measured in the presence of the phosphatase inhibitor calyculin A, which showed statistically significant increases in both mono- and diphosphorylated forms (Table 2). Under unstimulated conditions, reported values for control mono LC20-Pi are on the order of 5-10% of the total LC20 (10, 11, 16). Thus, even in the presence of calyculin-A, in which the highest phosphorylation levels were found, LC20-Pi would be <20% of the total LC20.

                              
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Table 2.   Phosphorylation state of the regulatory myosin light chain in fibroblast fibers

Effects of Rho-associated kinase inhibition on stress fiber formation. An alternate pathway for modulation of contractility by Rho-associated kinase might involve stress fiber formation and a coordinated change of cell shape. As shown by the confocal micrographs in Fig. 6, CS-induced force was associated with stress fiber formation. Inhibition of Rho-associated kinase by pretreatment with Y-27632 blocked stress fiber formation.


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Fig. 6.   Confocal micrographs showing the effects of the Rho-associated kinase inhibitor Y-27632 on actin stress fiber formation in NIH 3T3 fibroblast fibers. The reconstituted NIH 3T3 fibroblast fibers were incubated in the absence (A) or presence (B) of 30% CS for 15 min. C: fiber was pretreated for 10 min with the Rho kinase inhibitor Y-27632 (10 µM) before 15-min exposure to CS. Scale bars are 100 µm for the panels at left and 50 µm for the panels at right. Increased density of phalloidin staining in B indicates the CS-elicited stress fiber formation, which is blocked by Y-27632 shown in C.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study provides evidence indicating that the major mechanism regulating contraction in NIH 3T3 fibroblast fibers in response to 30% CS involves a Rho-associated kinase. Surprisingly, this sustained contraction is associated with only a transient increase in [Ca2+]i and is independent of an increase in LC20-Pi. Fibroblast contraction does not appear to involve pathways involving myosin light-chain kinase or tyrosine kinase, and inhibition of protein kinase C elicited only a minor (~20%) decrease in force with no changes in the [Ca2+]i signal. The latter characterizations are based on pharmacological evidence and are thus subject to its limitations. To this end, we used multiple agents, for example, to inhibit activation of the myosin light-chain kinase or Rho kinases pathways, to bolster our data. Similar approaches have been invaluable to our understanding of the regulation of smooth muscle contractility (21).

Fibroblast contraction is an important aspect of both normal wound healing and fibrosis, but the regulatory mechanism(s) is not yet established with certainty. We have previously demonstrated that CS induces a sustained, dose-dependent contraction (16). Further studies (15) indicated that the transient increase in [Ca2+]i in response to CS arises from intracellular stores. The transient increase in [Ca2+]i may play a role in initiation of the contraction induced by CS. However, the signaling system for the sustained contraction after [Ca2+]i returns to baseline is not known.

It is widely accepted that intracellular Ca2+ regulates cellular responses, often in association with calcium/ calmodulin-dependent pathways. Inhibition of calcium/ calmodulin interaction with W-7 (23) or the calcium/calmodulin kinase inhibitor KN-62 (24) did not affect the CS-induced contraction, suggesting that they do not play a major role in mediating this contractile response. Moreover, myosin light-chain kinase is known to mediate smooth muscle contraction (3, 22) and is suggested to regulate nonmuscle contraction as well (10, 21). However, the myosin light-chain kinase inhibitors ML-7 and wortmannin did not significantly reduce the contraction induced by CS in the fibroblast fibers.

Therefore, we investigated alternative signaling pathways that could modulate the actin-myosin interaction. It has been suggested that tyrosine kinases, protein kinase C, and Rho kinase may play a role in Ca2+ sensitization (22, 26) in smooth muscle. These kinases could modify the sensitivity of the contractile apparatus, thus modulating the contractile response and resulting in force maintenance in the presence of low [Ca2+]i. However, the tyrosine kinase inhibitor genistein did not block or attenuate the contraction induced by CS. The protein kinase C inhibitor calphostin C did not affect [Ca2+]i and only had a moderate (~20%) inhibitory effect on force.

In contrast, our data suggest a role for Rho-associated kinase in regulating fibroblast contraction. Rho-associated kinase has previously been implicated in increasing the calcium sensitivity of smooth muscles via inhibition of myosin phosphatase and/or direct phosphorylation of the 20-kDa myosin light chain (2). Both pathways increase LC20-Pi without activating myosin light-chain kinase (8). Similarly, myosin phosphatase is inhibited through the activation of Rho-associated kinase in NIH 3T3 cells (8). Recently, Y-27632 (25) and HA1077 (20) have been reported to inhibit Rho-associated kinase activity and to relax smooth muscles (21). Our results show that Y-27632 and HA1077 have a similar effect in fibroblasts, dramatically reducing the CS-induced contraction. Importantly, the [Ca2+]i response was not blocked by Y-27632 and HA1077, suggesting that an increase in [Ca2+]i per se is not sufficient to activate contraction. This is in agreement with our previous work and that of others (11, 15, 18), showing that an increase in [Ca2+]i arising from extracellular Ca2+ does not elicit a contraction. Based on these data, we conclude that the initial increase in [Ca2+]i is independent of Rho kinase activation but that the activation of pathways involving Rho-associated kinase are necessary for maintaining the sustained Ca2+-independent contraction elicited by CS.

The weight of our evidence suggests that LC20 phosphorylation is not part of this Rho kinase-mediated contraction of fibroblast fibers. This conclusion is based on multiple results. First, wortmannin and ML-7, inhibitors of myosin light-chain kinase, had no effect on isometric force generated in response to CS. Second, calyculin A, a protein phosphatase inhibitor, contracts fibroblast fibers. This result is consistent with the hypothesis that phosphatase inhibition, either by Rho-associated kinase or another mechanism, is sufficient to contract fibroblasts or smooth muscles. Interestingly, calyculin A treatment results in significant increases in mono- and diphosphorylation of LC20 but a relatively small increase in force (~50% of maximum) compared with fibers treated with CS. This increased MRLC-Pi may also be a consequence of inhibition of myosin phosphatase and just a correlated phenomenon, rather than causal, for force development. These data are consistent with our previous demonstration that large increases in LC20 phosphorylation are insufficient to support force generation by fibroblasts (16). Third, direct measurement of LC20-Pi (Table 2) did not reveal statistically significant changes in LC20 phosphorylation except in the presence of calyculin A. At first glance, the nonsignificant increases in LC20-Pi (Table 2, the largest being to 1.34 of control) might appear to be physiologically significant, although below our detectability. It is worth emphasizing that our LC20-Pi values are expressed relative to control. Because the reported control values unstimulated values of LC20-Pi are ~0.10 (10, 16), our largest "nonsignificant" increase in LC20-Pi would be from 0.10 to 0.13 of the total LC20. It is difficult to envision how this small change could lead to 100% activation of force and be a workable control mechanism.

On the other hand, the increase in LC20 monophosphorylation after calyculin A treatment (1.9-fold) is comparable to the increases in LC20-Pi (2-2.5-fold) observed in activated smooth muscles (21). This means that the method we have used is capable of detecting changes in LC20 phosphorylation of the magnitude that are associated with smooth muscle contraction. It is plausible that LC20-Pi might initiate the contraction and then decay to baseline before our steady-state measurements were made (15 min poststimulation). We have previously studied the time course of LC20-Pi (16), and as early as 5 min there was no change in LC20-Pi. However, given the measured initial [Ca2+]i transient, a potential signal for myosin light-chain kinase activation, a very rapid LC20-Pi transient cannot be unequivocally ruled out. We view this as unlikely given the lack of any effects on force of the inhibitors of myosin light-chain kinase activation. Moreover, Y-27632 did not affect the Ca2+ transient, but instead blocked force. Thus, if an initial MRLC-Pi transient is necessary for contraction, it certainly is not sufficient for generation or maintenance of force. Taken as a whole, our current data suggest a dissociation between LC20 phosphorylation and force generation by fibroblast fibers stimulated with CS.

These results are surprising in light of current convention that suggests the fibroblast contractility is mediated by LC20-Pi, based on the smooth muscle paradigm. The basis for these differences is not known but may be due to differences in the sources of fibroblasts (11, 18) or in the assessment of contractility (18). In some cases, LC20-Pi measurements were not made in the fibers themselves but in parallel studies on cells in culture (11), possibly providing another variable. Our current data are consistent with our previous work (16) in which the expression of a catalytically active myosin light-chain kinase in fibroblasts did not affect contractility despite complete phosphorylation of LC20-Pi. It is worth noting that in this previous study LC20-Pi was measured by an entirely different technique (16). Moreover, LC20 phosphorylation was found not to increase with force in response to CS in control fibers and the mechanical parameters (e.g., Vmax, stiffness, Hill coefficient, etc.) were identical in the two groups.

If not modulation of contraction via LC20, what is the mechanism affected by Rho kinase inhibition? Our data indicate that Rho kinase inhibition also prevents stress fiber formation (Fig. 6), suggesting a potential role in terms of cell shape changes underlying the CS increased force. It is possible that stress fiber formation, polymerization of the contractile proteins, and the reorganization of the complex of proteins at focal adhesions per se may not require light-chain phosphorylation, whereas it may be necessary for subsequent cell migration. There is also a growing smooth muscle literature that suggests that phosphorylation of cytoskeleton proteins, such as paxillin may modulate contractility (13). Unraveling the mechanism for modulation of fibroblast contractility by Rho kinase inhibition remains a formidable task but an important area of investigation.

In conclusion, our data show that activation of Rho-associated kinase is essential for the CS-induced contraction of NIH 3T3 fibroblast fibers. The data also demonstrate that this contraction is independent of calmodulin, calcium/calmodulin-dependent protein kinases, and myosin light-chain kinase but dependent on the activation of Rho-associated kinase. Moreover, our data suggest that LC20 phosphorylation is not an important part of the signaling pathway that results in fibroblast contraction after serum stimulation.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-59618 and HL-64702 (to P. de Lanerolle) and HL-54829 and HL-66044 (to R. J. Paul). F. Fazal was supported by National Institute of Diabetes and Digestive and Kidney Diseases Training Grant T32-DK-07739.


    FOOTNOTES

Address for reprint requests and other correspondence: R. J. Paul, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati, College of Medicine, Cincinnati, OH 45267-0576 (E-mail: Richard.Paul{at}uc.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.

First published October 3, 2002;10.1152/ajpcell.00188.2002

Received 20 September 2002; accepted in final form 24 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akiyama, T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, and Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262: 5592-5595, 1987[Abstract/Free Full Text].

2.   Amano, M, Mukai H, Ono Y, Chihara K, Matsui T, Hamajima Y, Okawa K, Iwamatsu A, and Kaibuchi K. Identification of a putative target for Rho as the serine-threonine kinase protein kinase N. Science 271: 648-650, 1996[Abstract].

3.   De Lanerolle, P, and Paul RJ. Myosin phosphorylation/dephosphorylation and regulation of airway smooth muscle contractility. Am J Physiol Lung Cell Mol Physiol 261: L1-L14, 1991[Abstract/Free Full Text].

4.   Ehrlich, HP, and Rajaratnam JB. Cell locomotion forces vs. cell contraction forces for collagen lattice contraction: an in vitro model of wound contraction. Tissue Cell 22: 407-417, 1990[ISI][Medline].

5.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

6.   Ishihara, H, Martin BL, Brautigan DL, Karaki H, Ozaki H, Kato Y, Fusetani N, Watabe S, Hashimoto K, Uemura D, and Hartshorne DJ. Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun 159: 871-877, 1989[ISI][Medline].

7.   Ishizaki, T, Maekawa M, Fujisawa K, Okawa K, Iwamatsu A, Fujita A, Watanabe N, Saito Y, Kakizuka A, Morii N, and Narumiya S. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J 15: 1885-1893, 1996[Abstract].

8.   Kimura, K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, and Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245-248, 1996[Abstract].

9.   Kobayashi, E, Nakano H, Morimoto M, and Tamaoki T. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 159: 548-553, 1989[ISI][Medline].

10.   Kolodney, MS, and Elson EL. Correlation of myosin light chain phosphorylation with isometric contraction of fibroblasts. J Biol Chem 268: 23850-23855, 1993[Abstract/Free Full Text].

11.   Kolodney, MS, Thimgan MS, Honda HM, Tsai G, and Yee HF, Jr. Ca2+-independent myosin II phosphorylation and contraction in chicken embryo fibroblasts. J Physiol 515: 87-92, 1999[Abstract/Free Full Text].

12.   Kolodney, MS, and Wysolmerski RB. Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study. J Cell Biol 117: 73-82, 1992[Abstract].

13.   Mehta, D, Tang DD, Wu MF, Atkinson S, and Gunst SJ. Role of Rho in Ca2+-insensitive contraction and paxillin tyrosine phosphorylation in smooth muscle. Am J Physiol Cell Physiol 279: C308-C318, 2000[Abstract/Free Full Text].

14.   Nakanishi, S, Kakita S, Takahashi I, Kawahara K, Tsukuda E, Sano T, Yamada K, Yoshida M, Kase H, Matsuda Y, Yoshiaki H, and Nonomura Y. Wortmannin, a microbial product inhibitor of myosin light chain kinase. J Biol Chem 267: 2157-2163, 1992[Abstract/Free Full Text].

15.   Nobe, K, Nobe H, Obara K, and Paul RJ. Preferential role of intracellular Ca2+ stores in regulation of isometric force in NIH 3T3 fibroblast fibres. J Physiol 529: 669-679, 2000[Abstract/Free Full Text].

16.   Obara, K, Nikcevic G, Pestic L, Nowak G, Lorimer DD, Guerriero V, Jr, Elson EL, Paul RJ, and de Lanerolle P. Fibroblast contractility without an increase in basal myosin light chain phosphorylation in wild type cells and cells expressing the catalytic domain of myosin light chain kinase. J Biol Chem 270: 18734-18737, 1995[Abstract/Free Full Text].

17.   Obara, K, Nobe K, Nobe H, Kolodney MS, de Lanerolle P, and Paul RJ. Effects of microtubules and microfilaments on [Ca2+]i and contractility in a reconstituted fibroblast fiber. Am J Physiol Cell Physiol 279: C785-C796, 2000[Abstract/Free Full Text].

18.   Parizi, M, Howard EW, and Tomasek JJ. Regulation of LPA-promoted myofibroblast contraction: role of Rho, myosin light chain kinase, and myosin light chain phosphatase. Exp Cell Res 254: 210-220, 2000[ISI][Medline].

19.   Saitoh, M, Ishikawa T, Matsushima S, Naka M, and Hidaka H. Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. J Biol Chem 262: 7796-7801, 1987[Abstract/Free Full Text].

20.   Seto, M, Sasaki Y, and Hidaka H. Effects of HA1077, a protein kinase inhibitor, on myosin phosphorylation and tension in smooth muscle. Eur J Pharmacol 195: 267-272, 1991[ISI][Medline].

21.   Somlyo, AP, and Somlyo AV. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522: 177-185, 2000[Abstract/Free Full Text].

22.   Somlyo, AP, and Somlyo AV. Smooth muscle: excitation-contraction coupling, contractile regulation, and the cross-bridge cycle. Alcohol Clin Exp Res 18: 138-143, 1994[ISI][Medline].

23.   Tanaka, T, Ohmura T, Yamakado T, and Hidaka H. Two types of calcium-dependent protein phosphorylations modulated by calmodulin antagonists. Naphthalenesulfonamide derivatives. Mol Pharmacol 22: 408-412, 1982[Abstract].

24.   Tokumitsu, H, Chijiwa T, Hagiwara M, Mizutani A, Terasawa M, and Hidaka H. KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 265: 4315-4320, 1990[Abstract/Free Full Text].

25.   Uehata, M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990-994, 1997[ISI][Medline].

26.   Walsh, MP, Horowitz A, Clement-Chomienne O, Andrea JE, Allen BG, and Morgan KG. Protein kinase C mediation of Ca2+-independent contractions of vascular smooth muscle. Biochem Cell Biol 74: 485-502, 1996[ISI][Medline].


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