©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (*)

(Received for publication, May 3, 1995; and in revised form, May 31, 1995)

Kazuo Obara (1) Gordana Nikcevic (2) Lidija Pestic (2)(§),   Grzegorz Nowak (2) Donald D. Lorimer (2)(§),   Vince Guerriero , Jr. (3) Elliot L. Elson (4) Richard J. Paul (1) Primal de Lanerolle (2)(¶)

From the (1)Department of Physiology and Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45221, the (2)Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago, Illinois 60612, the (3)Department of Animal Sciences, University of Arizona, Tucson, Arizona 85721, and the (4)Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63130

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We investigated the role of myosin light chain (MLC) phosphorylation (MLC-P) in non-muscle contractility by comparing MLC-P and the contractile properties of wild type 3T3 fibroblasts and 3T3 fibroblasts expressing the catalytic domain of myosin light chain kinase (tMK). MLC-P is 0.96 mol of PO(4)/mol of MLC in cells expressing tMK compared to 0.20 mol of PO(4)/mol of MLC in control cells. Expressing tMK also results in a 2-fold increase in cortical stiffness compared to control cells. Contractile properties were quantified by growing wild type and transfected fibroblasts in collagen and attaching the ensuing fibers to an apparatus for performing mechanical measurements. Serum stimulation resulted in a dose-dependent increase in force with maximal force generated in the presence of 30% (v/v) serum. Surprisingly, MLC-P did not increase in wild type cells following stimulation with 30% serum, and tMK expression did not affect the contractile properties of fibers made from these cells. Moreover, the dose responses to serum, maximal force, force-velocity relationships, and dynamic stiffness were similar in the wild type cells and fibroblasts expressing tMK. These data demonstrate that non-muscle cells can generate force without an increase in MLC-P, and that an increase in MLC-P does not affect the contractile properties of fibroblast fibers.


INTRODUCTION

Cellular force generation requires energy transduction. ATP hydrolysis due to the cyclic interaction of actin and filamentous myosin (i.e. myosin II) is a major pathway for converting chemical energy into mechanical work(1) . This reaction is regulated in mammalian smooth muscle (2) and non-muscle (3) cells by the phosphorylation of the 20-kDa light chain of these myosins (MLC) (^1)by the calcium-calmodulin-dependent enzyme myosin light chain kinase. MLC phosphorylation (MLC-P) is essential for force generation by smooth muscles, and this reaction has also been implicated in modulating the rate of cross-bridge cycling and the speed of contraction(2) . Increases in MLC-P have also been correlated with force generation (4, 5) and filament dynamics (6) in fibroblasts following serum stimulation. However, the role of MLC-P in fibroblast contractility is not known with certainty.

In order to better understand the mechanisms that regulate non-muscle contractility, we compared the contractile properties and MLC-P in wild type 3T3 fibroblasts and fibroblasts expressing the catalytic domain of myosin light chain kinase (tMK). tMK was expressed using a retroviral vector based on the murine Moloney leukemia virus(7) . The contractile properties of the fibroblasts were quantified by growing cells in collagen and performing mechanical studies on the ensuing fibers in myobaths(8) . Although tMK expression substantially increases MLC-P and cortical stiffness, MLC-P did not change with stimulation and force generation. In addition, the contractile properties are similar in the wild type cells and cells expressing tMK following serum stimulation. These data suggest that an increase in MLC-P is not obligatory for force generation by non-muscle cells.


MATERIALS AND METHODS

Cell Culture

Wild type Swiss or NIH 3T3 fibroblasts and NIH 3T3 fibroblasts expressing tMK were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% calf serum and antibiotics at 37 °C in an atmosphere containing 5% CO(2).

Construction of Expression Vectors

The pLNCX murine Moloney leukemia virus-based vector (7) was used to express tMK in NIH 3T3 cells. In this vector, the neomycin resistance gene is driven from the viral long terminal repeat, and the cloned genes are driven from the cytomegalovirus promoter. The tMK gene was constructed by inserting a stop codon following lysine 793 of the chicken gizzard myosin light chain kinase gene(9) . The 1995-base pair tMK gene was then cloned into the XbaI site of pBluescript II SK, cut with NotI, and the ends were filled in with Klenow fragment. The product was cut with HindIII, and the resulting 2005-base pair, HindIII-blunt end fragment was cloned between HindIII-HpaI sites of the pLNCX vector to form the tMK vector. The resulting construct (pLNC-tMK) was verified by restriction digestion.

Retroviral Expression of Proteins in 3T3 Cells

The pLNCX and pLNC-tMK vectors were first passaged through the GP+envAM12 amphotropic packaging cell line(10) . In order to do so, the packaging cells were transfected using 15 µg of either DNA using CaPO(4) precipitation(11) . Following transfection, cells were selected for 8 days in 0.8 mg/ml G418 (effective concentration). Selected cells were expanded in culture, and 4 10^5 cells were plated in a 100-mm dish. Twenty four hours later, viral media were collected, passed through a 0.45-µm filter, and used to infect 3T3 cells in log phase growth. Infected cells were selected for 8 days in 0.9 mg/ml G418 (effective concentration). Cells surviving G418 treatment were expanded and used in subsequent studies. Cells infected with the pLNCX and pLNC-tMK constructs are referred to as neo and tMK cells, respectively.

MLCPhosphorylation

To quantitate the stoichiometry of MLC-P, control or tMK cells grown in 100-mm dishes were washed in PO(4)-free DMEM and incubated with 300 µCi of P(i) in 3 ml of PO(4)-free DMEM for 6 h. The cells were washed in ice-cold PO(4)-free Gey's solution and removed by trypsinization. The trypsin was neutralized with 10% serum, and equal aliquots of the cells were placed in two Eppendorf tubes and washed extensively in ice-cold PO(4)-free Gey's solution. One aliquot of the cells was extracted, and the myosin was immunoprecipitated as described previously(12) . The immunoprecipitated myosin, along with known amounts of macrophage myosin II, were separated by SDS-PAGE, stained with Coomassie Blue, and the MLC bands were identified by autoradiography. The MLC bands were then excised and subjected to scintillation counting. The staining of the myosin heavy chains was quantitated densitometrically, and the data from the scans of the purified myosins were used to generate a standard curve. The amount of myosin heavy chain in each immunoprecipitate was then calculated from the standard curve, and the level of MLC-P was expressed as counts/min/pmol of myosin. Simultaneously, the other aliquot was extracted in perchloric acid and used to isolate creatine phosphate and inorganic phosphate as described previously(13) . Aliquots of the purified creatine phosphate and inorganic phosphate were subjected to liquid scintillation counting, and the specific activity of the creatine phosphate, corrected for inorganic phosphate, was expressed as counts/min/pmol of creatine phosphate. The stoichiometry of MLC-P was then calculated from the counts/min/pmol of myosin, following correction for the presence of 2 mol of MLC/mol of myosin, and the corrected specific activity of the creatine phosphate, which is in equilibrium with ATP(13) . This experiment involved substantial handling of a large amount of radioactivity and was performed once in duplicate.

Relative changes in MLC-P were quantitated in the following manner. Monolayers of wild type or infected cells grown on 60-mm dishes or cells grown into fibers in silicone rubber ``molds'' (see below) were washed in PO(4)-free DMEM and incubated in 1 ml of PO(4)-free DMEM containing 50 µCi of P(i) for 6 h at 37 °C. Cells grown in dishes or fibers were stimulated by adding fetal bovine serum (30%, v/v) to the media containing P(i) for 5 or 20 min. The cells or fibers were washed twice at the appropriate times with ice-cold Gey's buffer and extracted, and the myosin immunoprecipitated as described previously(12) . The immunoprecipitates were then separated by SDS-PAGE (5%-20% gradient). Following Coomassie staining, the gels were air-dried and exposed to x-ray film, and the bands on the autoradiogram representing MLC were quantified densitometrically. Coomassie Blue staining of the myosin heavy chain in each of the immunoprecipitates following SDS-PAGE was also quantified densitometrically. The areas of scans of the light chains were then divided by the areas of the corresponding scans of the heavy chain. The MLC-P data were normalized by dividing the ratios at 5 and 20 min following serum stimulation in control and tMK cells by their respective ratios at time 0.

Phosphopeptide Mapping of MLCfrom tMK Cells

Myosin was immunoprecipitated from tMK cells that were grown in two 100-mm dishes and labeled with 300 µCi of P(i) in 3 ml of PO(4)-free DMEM, each, for 6 h. Myosin was also immunoprecipitated from unlabeled control (neo) cells grown in one 100-mm dish and, along with myosins purified from tracheal smooth muscle and from macrophages, were phosphorylated in vitro using -labeled [P]ATP and purified smooth muscle myosin light chain kinase as described previously(14) . The immunoprecipitated and purified myosins were separated by SDS-PAGE, and the MLC bands were identified by autoradiography. The MLC bands were excised and digested with trypsin, and the phosphopeptides were analyzed by one-dimensional isoelectric focussing PAGE and autoradiography as described(15) .

Quantitation of Cortical Stiffness

Cortical stiffness was measured on cells grown on glass coverslips coated with 15 µg/ml poly(2-hydroxyethyl methacrylate)(16) . Plated cells were allowed to attach for 4-6 h, and the coverslips were then mounted in the chamber of the cell poker in the inverted position. Stiffness measurements were performed in degassed media containing fetal bovine serum at 37 °C. The surface of the cells was indented near their center (depth of indentation < 2.6 µm and velocity of indentation = 5.1 µm/s) with a glass microprobe (tip diameter = 2 µm) attached to a flexible glass beam of known bending constant. The degree of bending of the glass beam is used to calculate cellular deformability (i.e. stiffness), which is the force resisting indentation (in millidynes) per unit area of indentation (in micrometers), as described previously (16, 17) .

Construction of Fibroblast Fibers

These were formed by growing cells in collagen as described by Kolodney and Wysolmerski (4) and modified by Obara et al.(8) . Briefly, dispersed cells were suspended in an ice-cold collagen solution, which contained 2 10^6 cells/ml, 0.5 mg/ml rat tail collagen in DMEM. An aliquot (2 ml) of the collagen/cell suspension was poured into a trough (0.8 5 cm 0.5 cm deep) cut in a layer of silicone rubber in 100-mm glass Petri dishes and placed in a CO(2) incubator at 37 °C. After 8 h, an additional 1 ml of DMEM was added to each well. The preparations were incubated for 3-5 days or until the cells shrank the gel and formed a fiber.

Contractility Measurements

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 connected to an AME 801 silicon strain gauge (SensoNor) force transducer. The fibers were bathed in MOPS-PSS buffer, which contained (mM): NaCl, 140; KCl, 4.7; NaH(2)PO(4), 1.2; EDTA, 0.02; MgSO(4), 1.2; CaCl(2), 2.5; dextrose, 5.5; and MOPS, 20, pH 7.4 at 37 °C. Contractile studies were performed by adding calf serum to the final concentration desired.

The length of the fiber was controlled with a Cambridge Technologies ergometer. Force-velocity relations were measured by imposing constant shortening velocities on the fiber and measuring the resulting force. Stiffness was measured by imposing rapid (<1 ms) shortening and stretching steps (-2.6 to +0.6% L(0)). A series of 8 step changes at 60-s intervals constituted an experimental set for the measurement of stiffness; the slope of the peak force versus length curve was taken as stiffness.

Data Analysis

All data are presented as mean ± S.E. Standard analysis of variance and unpaired t tests were used to assess differences which were accepted as statistically significant for p < 0.05.


RESULTS

Expression of tMK in active form was demonstrated by immunoprecipitating myosin from cells grown in tissue culture dishes and labeled with P(i). SDS-PAGE followed by autoradiography demonstrated a substantial increase in MLC-P in cells infected with tMK compared to wild type cells or cells infected with the empty plasmid (Fig.1). Quantitation of the stoichiometry of MLC-P demonstrated a 4.8-fold increase in MLC-P in tMK cells compared to control cells (Table1). Phosphopeptide maps of trypsin digests demonstrated the presence of a phosphopeptide in myosin immunoprecipitated from tMK cells that had a pI of 4.5, which is the pI reported for the MLC phosphopeptide containing the Ser residue phosphorylated by myosin light chain kinase(15) . This peptide also comigrated with a peptide present in purified smooth muscle and macrophage myosins and myosin immunoprecipitated from neo cells that were phosphorylated in vitro (Fig.2).


Figure 1: MLC phosphorylation in NIH 3T3 fibroblasts. Wild type cells (WT), neo (NEO), and tMK cells (tMK CELLS) grown in tissue culture dishes and wild type cells grown into fibers (WILD TYPE FIBERS) were labeled with P(i). Wild type cells grown in fibers and the tMK cells were stimulated with serum for 5 or 20 min and extracted, and the myosin immunoprecipitated as described under ``Materials and Methods.'' The immunoprecipitates were separated by SDS-PAGE and exposed to x-ray film. Note the increased level of MLC-P (MLC) in tMK cells compared to the wild type and neo cells and the absence of an increase in MLC-P following serum stimulation in wild type fibers or tMK cells. The amount of myosin heavy chain (MHC) in each of the immunoprecipitates was similar based on Coomassie Blue staining.






Figure 2: Phosphopeptide map analysis of the site of MLC-P in tMK cells. Myosin was immunoprecipitated from tMK cells (tMK) labeled with P. Myosin immunoprecipitated from unlabeled control cells (NEO) and myosin purified from tracheal smooth muscle (TSM) and macrophages (MØ) were phosphorylated with purified chicken gizzard myosin light chain kinase, in vitro, and the MLC in each sample was digested and analyzed as described under ``Materials and Methods.'' This is a picture of an autoradiogram of a single isoelectric focussing gel showing the region around pH 4.5. The lane marked NEO was exposed for a longer period of time because the immunoprecipitated myosin incorporated phosphate poorly.



Experiments were then performed to investigate the effect of tMK expression and the resulting increase in MLC-P on the stiffness of the cortical cytoskeleton. Cortical stiffness was quantified using the ``cell poker,'' which measures the force required to indent the free surface of adherent cells(16, 17) . Analysis of histograms of stiffness values obtained by poking individual neo or tMK cells demonstrated a shift to higher stiffness values in tMK cells, and that the mean stiffness in tMK cells was almost twice that in neo cells (Table1).

We then compared the contractile properties of wild type Swiss or NIH 3T3 cells and cells expressing tMK. Fibers composed of these three cell types were mounted under isometric conditions in MOPS-PSS, and the length was adjusted to match the original fiber length in the mold. After stress relaxation, force measurements indicated a steady level of baseline force that was virtually identical in wild type Swiss and NIH 3T3 cells and tMK cells (Table2). Addition of serum increased isometric force (Fig. 3). Force fell below the initial baseline level upon the addition of 1 µM cytochalasin D (Table2), and the extent and time courses for relaxation were very similar in wild type and tMK cells ( Fig.3and Table 2). The latter finding suggests that the intrinsic mechanical activity in all types of fibers was similar. Contracted fibers also relaxed when the serum was replaced with MOPS-PSS, and these contraction/relaxation cycles could be repeated for at least 8 h(8) . Serum induced virtually identical dose-dependent contractions in wild type Swiss or NIH 3T3 cells and tMK cells (Fig.4). Maximal isometric forces, which were produced in response to 30% serum, developed by fibers made from wild type Swiss 3T3 cells, NIH 3T3 cells, and tMK cells were also very similar (Table2).




Figure 3: Contraction-relaxation cycle of wild type (WT) and tMK (tMK) cells. Fibers made from wild type NIH 3T3 cells and tMK cells were mounted in myobaths in MOPS-PSS buffer as described under ``Materials and Methods.'' Addition of calf serum (CS) to a final concentration of 30% resulted in force generation. These contractions could be reversed by washing out the serum or by adding cytochalasin D (Cyto D) to a final concentration of 1 µM. The time courses for contraction and relaxation are virtually identical in the two types of fibers.




Figure 4: Dose-response relationship for serum and force. Fibers made from wild type Swiss (SW) or NIH (NIH) 3T3 cells or NIH 3T3 cells expressing tMK (tMK) were mounted in myobaths in MOPS-PSS buffer (see ``Materials and Methods''). Cumulative dose-response curves were performed by serially replacing the buffer with new buffer containing increasing concentrations of serum. A total number of 6 fibers were analyzed in each group, and there were no statistically significant differences in the data at any serum concentration. The basal and maximal levels of force generated by the three types of fibers are given in Table2, and these values were not significantly different.



The contractile properties of the two types of fibers were characterized further by imposing constant shortening velocities and measuring the consequent force responses as described previously(8) . The velocity-force relations were fitted with the Hill equation, (P + a) (V + b) = b(P(0) + a), using a nonlinear least squares routine (Origin) and taking P(0) as the initial force. For contractions induced by 30% serum (n = 24), the velocities at zero force (V(max)) were virtually identical, as was the dimensionless curvature parameter a/P(0), for wild type and tMK fibers (Table2). Fiber stiffness was measured by imposing rapid (<1 ms) step changes in length. A plot of the peak force responses against the imposed step length change showed that the peak force responses of fibroblast fibers were linear in the region where stretches were imposed, and this linear range extended to short step decreases(8) . Stiffness values were not significantly different in fibers composed of wild type and tMK cells (Table2).

Finally, MLC-P following serum stimulation was quantified by immunoprecipitating myosin from cells grown in monolayer or from cells grown into collagen fibers. Most studies were performed on cells grown on dishes because it was much more difficult to handle the P(i)-labeled fibers. Similar phosphorylation results were obtained with either cells grown on dishes or into fibers. As shown in Fig.1, there was no detectable increase in MLC-P following serum stimulation of wild type cells grown into fibers. Statistical analysis of the normalized phosphorylation data (Table1) at times 0, 5, and 20 min (force generation plateaued at 20 min, Fig.3) did not demonstrate statistically significant differences in these data (p > 0.05, n = 4). Moreover, MLC-P was initially high and did not increase further following serum stimulation of tMK cells grown in culture ( Fig.1and Table 1).


DISCUSSION

We investigated the role of MLC-P in regulating the contractile properties of non-muscle cells by comparing the contractile properties and time courses of MLC-P in fibers composed of wild type and fibroblasts expressing the catalytic domain of myosin light chain kinase. To do so, we used a retroviral system to express the catalytic domain of myosin light chain kinase in 3T3 fibroblasts. Proteolytic digestion of purified myosin light chain kinase (18) or insertion of a stop codon following lysine 793 (9) results in a constitutively active, Ca-calmodulin independent form of the enzyme that retains its substrate specificity for MLC. Fig.1demonstrates a high level of MLC phosphorylation in unstimulated cells expressing tMK, thereby verifying that tMK is expressed in active form in the infected cells.

Based on the well-characterized relationship between MLC-P and smooth muscle contraction (2) and previous studies on non-muscle cells(4, 5) , we had anticipated (a) an increase in MLC-P during force generation by fibroblasts and (b) that a constitutive increase in MLC-P would enhance the mechanical properties (i.e. maximal force, time to peak force, V(max), stiffness). Our data do not support either of these predictions. Both types of fibers contract and relax at the same rate (Fig.2), they generate similar levels of isometric force in response to increasing levels of serum stimulation (Fig.3), and the contractile parameters (Table 2) are virtually identical despite substantial differences in the levels of MLC-P. Moreover, there is no increase in MLC-P following serum stimulation (Fig.1) in fibers composed of either cell type. These data demonstrate that MLC-P is neither required for force generation nor does increasing MLC-P affect the contractile properties of fibroblast fibers.

In contrast, increasing MLC-P increases cortical stiffness. The cell poker quantifies the force required to indent the free surface of adherent cells, which is determined mainly by the cortical cytoskeleton (16, 17) . Myosin II is an important contributor to cortical stiffness in Dictyostelium discoideum(17) , and MLC-P has been shown to stabilize myosin filaments in mammalian cells(19) . Thus, the observation that myosin hyperphosphorylation increases cortical stiffness establishes two important points. First, it demonstrates for the first time that cortical stiffness is determined, at least in part, by the level of MLC-P. Second, it establishes that an increase in MLC-P has a local cellular effect. Surprisingly, this local effect does not appear to be transmitted to the fiber as a whole.

Perhaps the most striking aspect of this study is that the apparent dissociation between contractile parameters, which reflect an actomyosin-dependent system(8) , and MLC-P. Most of the contractile parameters measured in these fibers are very similar to those measured in smooth muscle, a tissue whose contractile properties have previously been shown to be regulated by MLC-P(2) . With the exception of maximal isometric force, which is considerably lower than that reported for smooth muscles (0.2-1.0 mN/mm^2versus 10-200 mN/mm^2, (8) ), V(max), the curvature parameter a/F(0) and stiffness values are comparable in the fibroblast fibers and smooth muscle fibers(8) . Nevertheless, there is no increase in MLC-P under conditions that result in force generation in these fibers, nor does increasing the level of MLC-P by expressing tMK affect the contractile properties. These data suggest that the molecular mechanisms involved in regulating energy transduction and determining the contractile responses of fibroblast fibers and smooth muscles are fundamentally different. Additional studies are required to determine the basis for these differences.


FOOTNOTES

*
This work was supported, in part, by National Institutes of Health Grants HL 23240 (to R. J. P.), HL 43651 (to V. G., Jr.), GM 38838 (to E. E.), and HL 35808 and HL 02411 (to P. de L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Training Grant HL 076922.

Florence and Arthur Brock Established Investigator of the Chicago Lung Association. To whom correspondence and reprint requests should be addressed: Dept. of Physiology and Biophysics, University of Illinois at Chicago (M/C 901), 901 S. Wolcott, Chicago, IL 60612. Tel.: 312-996-6430; Fax: 312-996-1414.

^1
The abbreviations used are: MLC, the 20-kDa light chain of myosin II; MLC-P, MLC phosphorylation; tMK, the catalytic domain of myosin light chain kinase; neo cells, NIH 3T3 fibroblasts expressing the neomycin resistance gene; tMK cells, NIH 3T3 fibroblasts expressing tMK and the neomycin resistance genes; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Rob Wysolmerski for assistance with the phosphopeptide mapping.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.