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
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ABSTRACT |
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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 -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
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INTRODUCTION |
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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 -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.
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EXPERIMENTAL PROCEDURES |
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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
-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
(105 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
(108 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)
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-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
-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.
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RESULTS |
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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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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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CVEC -catenin and actin localization in response to activated
MLCK.
We examined endothelial cell morphology by staining for
-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
-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
-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
-catenin and actin localization confined
primarily to the cell periphery (Fig. 3, E vs.
F).
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DISCUSSION |
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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 -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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adelstein, RS,
and
Klee CB.
Purification and characterization of smooth muscle myosin light chain kinase.
J Biol Chem
256:
7501-7509,
1981
2.
Cai, S,
Pestic-Dragovich L,
O'Donnell ME,
Wang N,
Ingber D,
Elson E,
and
de Lanerolle P.
Regulation of cytoskeletal dynamics and cell growth by myosin light chain phosphorylation.
Am J Physiol Cell Physiol
275:
C1349-C1356,
1998
3.
Chrzanowska-Wodnicka, M,
and
Burridge K.
Rho-stimulated contractility drives the formation of stress fibers and focal adhesions.
J Cell Biol
133:
1403-1415,
1996[Abstract].
4.
Colburn, JC,
Michnoff CH,
Hsu LC,
Slaughter CA,
Kamm KE,
and
Stull JT.
Sites phosphorylated in myosin light chain in contracting smooth muscle.
J Biol Chem
263:
19166-19173,
1988
5.
De Lanerolle, P,
Condit JR, Jr,
Tanenbaum M,
and
Adelstein RS.
Myosin phosphorylation, agonist concentration and contraction of tracheal smooth muscle.
Nature
298:
871-872,
1982[ISI][Medline].
6.
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
7.
Garcia, JGN,
Davis H,
and
Patterson CE.
Regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation.
J Cell Physiol
163:
510-522,
1995[ISI][Medline].
8.
Garcia, JGN,
Verin AD,
Schaphorst K,
Siddiqui R,
Patterson CE,
Csortos C,
and
Natarajan V.
Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60src.
Am J Physiol Lung Cell Mol Physiol
276:
L989-L998,
1999
9.
Goeckeler, ZM,
and
Wysolmerski RB.
Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation.
J Cell Biol
130:
613-627,
1995[Abstract].
10.
Hecht, G,
Pestic L,
Nikcevic G,
Koutsouris A,
Tripuraneni J,
Lorimer DD,
Nowak G,
Guerriero V, Jr,
Elson EL,
and
de Lanerolle P.
Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability.
Am J Physiol Cell Physiol
271:
C1678-C1684,
1996
11.
Ikebe, M,
and
Hartshorne DJ.
Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase.
J Biol Chem
260:
10027-10031,
1985
12.
Ikebe, M,
Hartshorne DJ,
and
Elzinga M.
Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin.
J Biol Chem
261:
36-39,
1986
13.
Ikebe, M,
Koretz J,
and
Hartshorne DJ.
Effects of phosphorylation of light chain residues threonine 18 and serine 19 on the properties and conformation of smooth muscle myosin.
J Biol Chem
263:
6432-6437,
1988
14.
Ikebe, M,
Stepinska M,
Kemp BE,
Means AR,
and
Hartshorne DJ.
Proteolysis of smooth muscle myosin light chain kinase. Formation of inactive and calmodulin-independent fragments.
J Biol Chem
262:
13828-13834,
1987
15.
Kamm, KE,
Hsu LC,
Kubota Y,
and
Stull JT.
Phosphorylation of smooth muscle myosin heavy and light chains.
J Biol Chem
264:
21223-21229,
1989
16.
Schelling, ME,
Meininger CJ,
Hawker JR,
and
Granger HJ.
Venular endothelial cells from bovine heart.
Am J Physiol Heart Circ Physiol
254:
H1211-H1217,
1988
17.
Sheldon, R,
Moy A,
Lindsley K,
Shasby S,
and
Shasby DM.
Role of myosin light chain phosphorylation in endothelial cell retraction.
Am J Physiol Lung Cell Mol Physiol
265:
L606-L612,
1993
18.
Strauss, JD,
de Lanerolle P,
and
Paul RJ.
Effects of myosin kinase inhibiting peptide on contractility and LC20 phosphorylation in skinned smooth muscle.
Am J Physiol Cell Physiol
262:
C1437-C1445,
1992
19.
Tinsley, JH,
Hawker J,
and
Yuan Y.
Efficient protein transfection of cultured coronary venular endothelial cells.
Am J Physiol Heart Circ Physiol
275:
H1873-H1878,
1998
20.
Tinsley, JH,
Wu MH,
Ma W,
Taulman AC,
and
Yuan SY.
Activated neutrophils induce hyperpermeability and phosphorylation of adherens junction proteins in coronary venular endothelial cells.
J Biol Chem
274:
24930-24934,
1999
21.
Verin, AD,
Patterson CE,
Day MA,
and
Garcia JGN
Regulation of endothelial cell gap formation and barrier function by myosin-associated phosphatase activities.
Am J Physiol Lung Cell Mol Physiol
269:
L99-L108,
1995
22.
Yuan, Y,
Huang Q,
and
Wu HM.
Myosin light chain phosphorylation: modulation of basal and agonist-stimulated venular permeability.
Am J Physiol Heart Circ Physiol
272:
H1437-H1443,
1997
23.
Yuan, Y,
Meng FY,
Huang Q,
Hawker J,
and
Wu HM.
Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function.
Am J Physiol Heart Circ Physiol
275:
H84-H93,
1998