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Myosin light chain kinase transference induces myosin light chain activation and endothelial hyperpermeability

John H. Tinsley1, Primal De Lanerolle2, Emily Wilson1, Weiya Ma1, and Sarah Y. Yuan1

1 Departments of Surgery and Medical Physiology, Texas A&M University System Health Science Center, Temple, Texas 76504; and 2 Department of Physiology, University of Illinois at Chicago, Chicago, Illinois 60612


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The actomyosin complex is the major cytoskeletal component that controls cell contraction. In this study, we investigated the effects of actomyosin interaction on endothelial barrier function and gap formation. Activated myosin light chain kinase (MLCK) protein was transferred into coronary venular endothelial cell (CVEC) monolayers. Uptake of the activated protein resulted in a significant shift in myosin light chain (MLC) from an unphosphorylated to a diphosphorylated form. In addition, MLCK induced a hyperpermeability response of the monolayer as measured by albumin transendothelial flux. Microscopic examination of MLCK-treated CVECs revealed widespread gap formation in the monolayer, loss of peripheral beta -catenin, and increases in actin stress fibers. Inhibition of all of the above responses by a specific MLCK inhibitor suggests they are the direct result of exogenously added MLCK. These data suggest that activation of MLCK in CVECs causes phosphorylation of MLC and contraction of CVECs, resulting in gap formation and concomitant increases in permeability. This study uses a novel technique to measure the effects of an activated kinase on both its substrate and cellular morphology and function through direct transference into endothelial cells.

endothelial permeability; actin cytoskeleton; phosphorylation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE MICROVASCULAR ENDOTHELIUM consists of a layer of closely opposed cells that serves as a barrier to control the transvascular passage of solutes, fluid, and blood cells. On one hand, endothelial adherens junctions, which are formed by transmembrane adhesive proteins called cadherins, appear to be the main complex guarding against macromolecular leakage through the interendothelial pathway. On the other hand, the contractile forces generated by actomyosin interaction tend to pull the tightly connected endothelial cells apart, facilitating macromolecular efflux. A balance between the adhesive and contractile forces maintains the endothelial monolayer in a semipermeable status. Disruption of the equilibrium can lead to barrier dysfunction and microvascular leakage, resulting in the development of tissue edema.

Phosphorylation of myosin light chain (MLC) by activated myosin light chain kinase (MLCK) plays a critical role in the development and regulation of contractile forces within cells (2, 3, 10). Studies have shown that alterations in MLC phosphorylation affect the contractile properties of smooth muscle (4-6, 15, 18). MLCK phosphorylates MLC at Ser-19 and secondarily at Thr-18, resulting in increases in myosin activity (11, 12). In terms of endothelial cells, recent studies have shown that the phosphorylation condition of MLC affects the permeability of cultured cells and intact venules (7, 17, 22). Inhibition of serine phosphorylation attenuates thrombin-induced MLC phosphorylation and decreases permeability across endothelial cells derived from large arteries (7). Others have shown that on inflammatory stimulation, myosin associates with actin stress fibers to produce cell contraction (9). In this process, serine/threonine phosphorylation of MLC is required to increase the ATPase activity of myosin.

Although work on endothelial cell myosin and myosin-associated activities has increased over the past few years, many questions remain regarding precise controls and mechanisms. Even less is known about the role of myosin in controlling contraction of microvascular endothelial cells. The purpose of this study was to examine the effects of activated MLCK on MLC phosphorylation and the permeability property of coronary venular endothelial cells (CVECs). To accomplish this, the activated MLCK protein was directly transferred to the CVECs using a previously published protocol (19) in which MLCK is taken in by the cells as a polyamine complex. The phosphorylation of MLC, hyperpermeability response, loss of peripheral beta -catenin, and actin stress fiber formation we observed can all be directly attributed to activated MLCK. Whereas pharmacological agents exist to inhibit MLCK, no agents are currently available to specifically upregulate MLCK activity. Our results indicate that it may be possible to study the functions of other cellular proteins through direct transference of the protein into the cells.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell culture. Bovine CVECs were isolated from postcapillary venules (~15 µm in diameter) as previously described (16). CVECs were routinely maintained on gelatin-coated dishes that contained 10% fetal bovine serum in complete DMEM (DMEM with 1 mM sodium pyruvate, 2 mM L-glutamine, 15 mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 25 U/ml heparin). The cells exhibited properties characteristic of the endothelial cell, such as typical cobblestone morphology, positive immunofluorescent staining for factor VIII antigen, uptake of diacetylated low-density lipoprotein, and the ability to form tubes (16). Cells were used at passages 4-14.

MLCK transference into CVECs. Smooth muscle MLCK was isolated from chicken gizzards and purified by affinity chromatography using calmodulin coupled to Sepharose 4B as previously described (1). The kinase was 98-99% pure, as judged by SDS-PAGE. MLCK was transferred into the CVECs at a concentration of 0.1 mg/ml using a previously described protocol (19). Briefly, MLCK was activated by digesting with trypsin (1 µg/20 µg MLCK) for 15 min. This proteolysis of MLCK generates a protein fragment that is active in the absence of Ca2+/calmodulin (14). Next, the MLCK was added to TransIT-LT1 polyamine reagent (24 µl/ml; PanVera, Madison, WI) and incubated at room temperature for 10 min. Soybean trypsin inhibitor (50 µg/1 µg trypsin) was then added for 5 min, and the mixture was applied to the cells for incubation at 37°C for 6 h. In previous experiments, we were able to introduce the beta -galactosidase protein to ~88% of the cells in a monolayer (19). Additionally, we were able to introduce a protein kinase C (PKC)-inhibitor peptide to cells and show an ~55% decrease in PKC activity (19).

MLC phosphorylation. Cells were lysed 6 h post MLCK delivery and analyzed for MLC phosphorylation by urea PAGE as previously described (7, 21). In some cells, the selective MLCK inhibitor ML-9 (10-5 M) and the serine phosphatase inhibitor calyculin A (10-8 M) were applied for 1 h before lysis. Mono- and diphosphorylated forms of MLC migrate more rapidly through the gel than the unphosphorylated form. A monoclonal anti-MLC antibody, clone no. MY-21 (Sigma, St. Louis, MO), was used for immunoblotting. Protein bands were detected using LumiGLO chemiluminescent substrate (New England BioLabs, Beverly, MA). Images of the immunoreactive bands were scanned by reflectance scanning densitometry, and the intensity of the bands was quantified using NIH Image software. The percentage of MLC at each of the three levels of phosphorylation was calculated by dividing by the total MLC present in all three forms.

Measurement of endothelial permeability. CVEC monolayer permeability was determined with the use of FITC-labeled bovine serum albumin (Sigma) as previously described (20). Cells were grown on gelatin-coated Costar transwell membranes (VWR, Houston, TX) and exposed to MLCK (6 h), ML-9 (1 h), and calyculin A (10-8 M; 1 h). The tracer protein FITC-albumin was added to the luminal chamber for 45 min, and samples were taken from both the luminal and abluminal chambers for fluorometry analysis. The readings were converted with the use of a standard curve to albumin concentration. These concentrations were then used in the following equation to determine the permeability coefficient of albumin (Pa)
P<SUB>a</SUB><IT>=</IT><FR><NU>[A]</NU><DE><IT>t</IT></DE></FR><IT>×</IT><FR><NU><IT>1</IT></NU><DE><IT>A</IT></DE></FR><IT>×</IT><FR><NU>V</NU><DE>[L]</DE></FR>
where [A] is abluminal concentration; t is time in seconds; A is area of membrane in cm2; V is volume of abluminal chamber; and [L] is luminal concentration.

beta -catenin and actin localization. For microscopy, CVECs were grown to confluence on coverslips and treated by protein transference and/or drugs in the same time frames as those used for permeability measurements. After fixation and permeabilization, primary antibody to beta -catenin (Santa Cruz Biotechnology, Santa Cruz, CA) was applied for 1.5 h followed by secondary antibody conjugated to fluorescein isothiocyanate for 45 min. Actin was stained using rhodamine phalloidin (Molecular Probes, Eugene, OR) after permeabilization. Coverslips were then mounted on slides, and cells were examined under a Zeiss Axiovert 135 inverted microscope equipped with fluorescence filter sets and photographed using Kodak GoldMax 400 film.

Data analysis. In the immunoblot studies, a representative image of Western blots was selected to present. Analysis of variance was used to evaluate the significance of intergroup differences in the immunoblot analyses and permeability studies. A value of P < 0.05 was considered significant for the comparisons.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activated MLCK induces hyperphosphorylation of MLC in CVECs. Activated MLCK was introduced to CVEC monolayers and internalized by the cells through a polyamine/MLCK conjugate. Figure 1 shows that the activated MLCK increased phosphorylation of MLC. In treated cells, ~61% of the MLC was phosphorylated, compared with ~38% in control cells (compare lanes a and c). Furthermore, the increase appears to be exclusive to the diphosphorylated form of MLC; ~39% in MLCK-treated cells vs. ~12% in control cells. It is important to note that the polyamine reagent TransIT-LT1 did not have an effect on phosphorylation when applied in the absence of MLCK (Fig. 1, lane b). The serine phosphatase inhibitor calyculin A drove MLC to a virtually 100% diphosphorylated state (Fig. 1, lane f). A specific MLCK inhibitor, ML-9, when applied to the cells, completely abolished phosphorylation of MLC, and, in tandem with MLCK, held phosphorylation levels well below that of control cells (Fig. 1, lanes d and e).


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Fig. 1.   Myosin light chain kinase (MLCK)-induced myosin light chain (MLC) phosphorylation. Coronary venular endothelial cells (CVECs) were treated with MLCK for 6 h and calyculin A and ML-9 for 1 h. A: total cell lysate was used for Western blotting, and MLC was separated into unphosphorylated (U), monophosphorylated (M), and diphosphorylated (D) forms. B: the bands from A were quantitated by scanning densitometry, and the amounts of each of the 3 forms of MLC were expressed as a percentage of total MLC. From left to right, the order in B is unphosphorylated, monophosphorylated, and diphosphorylated. For both A and B, results are shown with control (a); TransIT-LT1 (b); TransIT-LT1 and MLCK (c); TransIT-LT1, MLCK, and ML-9 (d); ML-9 (e); and calyculin A (f). This experiment was repeated 3 times. A is a representative Western blot. Error bars are shown in B. *P < 0.05 vs. control unphosphorylated; omega P < 0.05 vs. control diphosphorylated; and epsilon P < 0.05 vs. control monophosphorylated.

Hyperpermeability of CVEC monolayers in response to activated MLCK. Increases in MLC phosphorylation have been implicated in the breakdown of endothelial monolayer integrity. To determine the effect of activated MLCK on monolayer integrity, we monitored the clearance of albumin across CVEC monolayers. Figure 2 shows that activated MLCK significantly increased permeability of treated cells to ~128% of control cell levels (compare bar a vs. bar c). This increase was attenuated by the MLCK inhibitor ML-9, and, in fact, permeability seems to have decreased below control levels in response to ML-9 (Fig. 2, bar d). Not surprisingly, calyculin A induced a large hyperpermeability response, ~174% of control levels (Fig. 2, bar f), corresponding to its large effect on MLC phosphorylation.


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Fig. 2.   Albumin permeability of CVEC monolayers in response to MLCK, ML-9, and calyculin A. CVECs were grown to confluence on Transwell membranes and treated with control (a); TransIT-LT1 (b); TransIT-LT1 and MLCK (c); TransIT-LT1, MLCK, and ML-9 (d); ML-9 (e); and calyculin A (f). FITC-albumin was then added to the luminal chamber, and permeability of albumin (Pa) was calculated after 45 min. Pa values were expressed as percentage of control cells. Data are shown with SE bars. For each treatment, n = 12. *P < 0.05.

CVEC beta -catenin and actin localization in response to activated MLCK. We examined endothelial cell morphology by staining for beta -catenin, a junctional protein that we had previously shown to disappear from the cell periphery under conditions of hyperpermeability (20). In addition, because cellular contraction and gap formation are linked to formation of stress fibers, we looked at actin distribution in response to activated MLCK. Control cells exhibit a tight monolayer with closely opposed neighboring cells, as well as uniform beta -catenin and actin staining at the cell periphery (Fig. 3, A and B). However, when activated MLCK is introduced to the monolayer, intercellular gaps form concomitantly with the disappearance of beta -catenin at the cell periphery and formation of actin stress fibers (Fig. 3, C and D). The same effects are seen with calyculin A treatment (Fig. 3, G and H), and in both cases, it is presumable that increases in albumin clearance are due to this cellular separation. When the MLCK inhibitor ML-9 is applied to the cells in tandem with activated MLCK, cells remain closely apposed, with beta -catenin and actin localization confined primarily to the cell periphery (Fig. 3, E vs. F).


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Fig. 3.   CVEC beta -catenin and actin localization. CVECs were grown to confluence and treated with control (A and B), MLCK/TransIT-LT1 (C and D), MLCK/ML-9/TransIT-LT1 (E and F), and calyculin A (G and H). After treatments, cells were fixed and stained for beta -catenin and actin as described in EXPERIMENTAL PROCEDURES. Note the increase in gap formation, loss of beta -catenin staining at cell periphery, and actin stress fiber formation with MLCK (C and D) and calyculin (G and H) treatments. These 3 phenomena are not seen with the addition of ML-9 (E and F). Images are representative of 50 fields observed for each treatment.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The role of MLC phosphorylation in smooth muscle contraction has been extensively studied over the last 15-20 yr. However, the study of the contractile apparatus in endothelial cells, and more directly, microvascular endothelial cells, is in its infancy. We must understand the mechanisms that regulate endothelial cell contraction to develop therapeutic agents to combat endothelial hyperpermeability and macromolecular leakage, processes that have been implicated in the pathogenesis of various inflammatory diseases and ischemia-reperfusion injury. The signaling events that lead to intercellular gap formation and increases in endothelial permeability are not completely understood. Evidence is accumulating that actomyosin interaction-induced cell contraction plays an important role in eliciting endothelial hyperpermeability responses to inflammatory mediators. We have previously shown that MLC phosphorylation results in increased albumin permeability in intact coronary venules (22). We are now able to study the regulatory mechanisms at the molecular level using a monolayer permeability system. We have the ability to transfer proteins directly into cells, alleviating the need to wait for expression when transfecting DNA. Additionally, this technique allows us to transfer protein into a monolayer of cells, which makes permeability and biochemical analyses possible. This is an obvious advantage over microinjection, which cannot presently be used on the large numbers of cells necessary for these studies.

To study MLC phosphorylation, we used an urea PAGE protocol that takes advantage of the fact that mono- and diphosphorylated forms of MLC migrate through the gel faster than unphosphorylated MLC (7, 8). Our results showed that the amount of diphosphorylated MLC increased significantly above control cell levels, whereas there was no apparent increase in monophosphorylated MLC when activated MLCK was introduced to the CVECs. This is important because evidence suggests that diphosphorylation involving Thr-18 and Ser-19 brings about a larger increase in the ATPase activity of myosin than does monophosphorylation of Ser-19 alone (13). Therefore, the increase we saw in diphosphorylated MLC might be capable of producing enough intracellular contractile force to cause intercellular gap formation and hyperpermeability. In fact, we did observe significant increases in permeability associated with the introduction of MLCK and MLC phosphorylation. Additionally, exposure to MLCK evoked beta -catenin loss at the cell periphery as well as increases in actin stress fiber formation, events we had previously associated with hyperpermeability. All of the above responses can be attributed to an increase in the amount of activated MLCK. This is supported by the fact that a specific MLCK inhibitor produced a concomitant attenuation of these responses.

In summary, this study uses a novel approach to directly examine the effects of activated MLCK on MLC and morphology in microvascular endothelial cells. We previously reported that phosphorylation of adherens junction and focal adhesion proteins in CVECs modulates the permeability of the same type of cells (20, 23). Further studies will be needed to correlate these events and their roles in the regulation of endothelial barrier function. This study provides a technical basis for further transference of MLCK and other proteins directly into cultured cells as well as intact microvessels to determine their physiological functions.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-61507 (to S. Y. Yuan) and HL-59618 and an American Heart Association Grant (to P. de Lanerolle). S. Y. Yuan is a recipient of National Institutes of Health Research Career Award K02 HL-03606.


    FOOTNOTES

Address for reprint requests and other correspondence: J. H. Tinsley, Depts. of Surgery and Medical Physiology, Texas A&M Univ. System Health Science Center, 1901 Veterans Memorial Dr., Bldg. 4, Temple, TX 76504 (E-mail: jht{at}tamu.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.

Received 21 March 2000; accepted in final form 20 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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