Regulation of endothelial cell barrier function by
calcium/calmodulin-dependent protein kinase II
Talaibek
Borbiev,
Alexander D.
Verin,
Shu
Shi,
Feng
Liu, and
Joe G. N.
Garcia
Division of Pulmonary and Critical Care Medicine, Johns Hopkins
University School of Medicine, Baltimore, Maryland 21224
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ABSTRACT |
Thrombin-induced
endothelial cell barrier dysfunction is tightly linked to
Ca2+-dependent cytoskeletal protein reorganization. In this
study, we found that thrombin increased
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II)
activities in a Ca2+- and time-dependent manner in bovine
pulmonary endothelium with maximal activity at 5 min. Pretreatment with
KN-93, a specific CaM kinase II inhibitor, attenuated both
thrombin-induced increases in monolayer permeability to albumin and
decreases in transendothelial electrical resistance (TER). We next
explored potential thrombin-induced CaM kinase II cytoskeletal targets
and found that thrombin causes translocation and significant
phosphorylation of nonmuscle filamin (ABP-280), which was attenuated by
KN-93, whereas thrombin-induced myosin light chain phosphorylation was
unaffected. Furthermore, a cell-permeable N-myristoylated synthetic
filamin peptide (containing the COOH-terminal CaM kinase II
phosphorylation site) attenuated both thrombin-induced filamin
phosphorylation and decreases in TER. Together, these studies indicate
that CaM kinase II activation and filamin phosphorylation may
participate in thrombin-induced cytoskeletal reorganization and
endothelial barrier dysfunction.
thrombin; filamin; myosin light chain phosphorylation; transendothelial electrical resistance
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INTRODUCTION |
THROMBIN IS A
MULTIFUNCTIONAL serine protease that plays a central role in
hemostasis by regulating platelet aggregation and blood coagulation,
with profound effects on vascular endothelial cell function. Thrombin
promotes endothelial cell activation and increases vascular
permeability, producing an elevation in intracellular Ca2+
as a direct result of phosphatidylinositol-specific
phospholipase C activation as well as increases in phospholipase
A2, phospholipase D, protein kinase C, and myosin light
chain kinase (MLCK) activities (6-9, 16, 17).
Potential mechanisms of endothelial cell barrier dysfunction induced by
thrombin include increases in contractile forces, decreases in
intercellular junctional connections, and reductions in endothelial
cell-extracellular matrix adhesive forces (10).
Furthermore, thrombin challenge produces dramatic cytoskeletal rearrangements concomitant with phosphorylation of myosin light chains
(MLC) (6, 26) and caldesmon (30), an
actin-binding protein that mediates smooth muscle contraction and may
be involved in the contractile mechanism of endothelial cell barrier
regulation. Another actin-binding protein that may also be important
for cell shape regulation, cell locomotion, and potentially endothelial cell barrier regulation is the ubiquitously distributed nonmuscle filamin, a 280-kDa homodimer present in the cortical cytoplasm and
responsible for three-dimensional actin network formation (12). Filamin provides attachment of filamentous actin to
plasma membrane glycoproteins (GP1b
in platelets, Fc
R1 in
leukocytes), binds directly to cytoplasmic domains of
1-
and
2-integrins, and associates with furin, presenilin,
and SEK-1, indicating its important regulatory role in actin
cytoskeleton organization and multifunctional properties (4, 18,
19, 21, 25, 29, 38, 39). It has been suggested that the
translocation of filamin from the endothelial cell membrane to the
cytosol may be actually involved in the disassembly of the
membrane-cytoskeleton network and subsequent increases in vascular
permeability, whereas association of filamin with the
membrane-cytoskeleton interface may contribute to the establishment or
maintenance of barrier function (13). Filamin function is
modulated by cAMP-dependent protein kinase A-mediated
phosphorylation, which increases the resistance of filamin to calpain
proteolysis (1), whereas Ca2+-dependent CaM
kinase II-induced filamin phosphorylation decreases its actin
filament cross-linking activity (24). Furthermore, filamin
phosphorylation induced by bradykinin and H2O2
via protein kinase A and CaM kinase II pathways is associated with
redistribution of filamin between cytosol and membrane compartments
(13, 35). In this study, we examined whether CaM kinase II
activities are involved in thrombin-induced endothelial cell barrier
dysfunction and explored filamin as a potential cytoskeletal target for
CaM kinase II. Our data indicate that both CaM kinase II and filamin are important participants in vascular barrier regulation.
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MATERIALS AND METHODS |
Reagents.
Bovine thrombin was obtained from Sigma (St. Louis, MO), KN-93 and
ionomycin were purchased from Calbiochem (La Jolla, CA), fura 2-AM was
obtained from Molecular Probes (Junction City, OR), antibody to MLC was
produced in rabbit against purified baculovirus-expressed and purified
smooth muscle MLC by Biodesign International (Kennebunk, ME), antibody
to CaM kinase II was purchased from Transduction Laboratories
(Lexington, KY), phospho-CaM kinase II antibody was obtained from ABR
(Golden, CO), antibody to smooth muscle MLCK was obtained from Sigma,
and antibody to human filamin was purchased from Chemicon (Temecula,
CA). Synthesized myristoylated and purified peptides were obtained from
the Biochemistry and Biotechnology Facility, Indiana University
(Indianapolis, IN) and were based on the deduced amino acid sequence of
human umbilical vein endothelial cell filamin (11). The
CaM site containing filamin peptide contains amino acid residues
2517-2529 of filamin: NH2-TGPRLVS*NHSLHE-COOH, with
Ser* corresponding to the CaM kinase II phosphorylation site. Control
peptide has the sequence of NH2-SPFEVKVGTECGNQK-COOH
representing 564-579 amino acid residues, which is not
phosphorylated by CaM kinase II. Both peptides were successfully
reported in the investigation of endothelial cell permeability
(37).
Bovine pulmonary artery endothelial cell culture.
Bovine pulmonary artery endothelial cells were obtained frozen at 16 passages from American Tissue Type Culture Collection (CCL 209;
Manassas, VA) and were utilized at passages 19-24
(6). Endothelial cells were cultured in complete medium
and maintained at 37°C in a humidified atmosphere of 5%
CO2-95% air and grew to contact-inhibited monolayers with
the typical cobblestone morphology. Cells from each primary flask were
detached with 0.05% trypsin and resuspended in fresh culture medium
and passaged to appropriate size flasks or dishes.
CaM kinase II activity assay.
Endothelial cell monolayers grown in 60-mm dishes were incubated with
thrombin for indicated times and lysed in 500 ml of lysis buffer
containing 50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM
Na3VO4, 0.1% 2-mercaptoethanol, 1% Triton
X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium
glycerophosphate, and 1:500 diluted protease inhibitory cocktail
(Calbiochem) for 30 min. Cell debris was removed by 10 min
centrifugation at 16,000 g, and the supernatants were
incubated with 2.5 mg of CaM kinase II monoclonal antibodies
(Transduction Laboratories) at 4°C for 1 h, followed by
incubation with 100 µl of protein G Sepharose slurry (containing 30%
protein G Sepharose 4 fast flow; Amersham Pharmacia Biotech,
Piscataway, NJ) for 1 h at 4°C with gentle agitation.
Immunoprecipitates were washed twice with lysis buffer, and the CaM
kinase II activity was determined by the incorporation of
[32P]orthophosphate into its specific substrate peptide
using the CaM kinase II assay kit (Upstate Biotechnology, Lake Placid, NY).
MLC phosphorylation in endothelium.
Endothelial cell monolayers were analyzed for MLC phosphorylation by
urea- PAGE as we have previously described (6), followed by Western immunoblotting with specific anti-MLC antibodies.
Intracellular Ca2+ determinations
assessed by fura 2 fluorescence.
Intracellular Ca2+ concentration
([Ca2+]i) was measured by fluorescence in
bovine pulmonary artery endothelial cells loaded with fura 2-AM.
Confluent endothelial cell monolayers were removed from
75-cm2 tissue flasks and seeded onto glass coverslips. When
confluent, the endothelial cells were exposed to loading buffer
(Hanks' buffer supplemented with 0.1% pluronic acid, 1 mM
CaCl2 and 5 µM fura 2-AM) for 20 min at 37°C. After
fura 2-AM loading, endothelial cells were washed twice in buffer
supplemented with 1 mM Ca2+ and then maintained in the same
buffer. Continuous monitoring of the cellular fluorescence was
performed at 37°C with constant stirring in a Perkin Elmer LS-50
fluorometer by ratio of the 340/380-nm excitation signal.
Transendothelial electrical resistance.
Endothelial cells were seeded onto evaporated gold microelectrodes and
grown to confluence as previously described (34). The
endothelial cell monolayer-covered microelectrodes were then connected
to an electrical cell substrate impedance system (Applied Biophysics,
Troy, NY) and rinsed with medium 199 (GIBCO BRL), and the
transendothelial electrical impedance was monitored for ~1 h to
establish a baseline resistance. Resistance values from each
microelectrode (measured in ohms) were normalized as the ratio of
measured resistance to baseline resistance and plotted vs. time.
Albumin clearance measurement of endothelial cell permeability.
Macromolecule permeability of cultured endothelial cell monolayers was
performed as previously described (5, 6). Gelatinized polycarbonate micropore membranes (Nucleopore, Pleasanton, CA) were
mounted on the base of plastic cylinders and endothelial cells and then
were seeded on the membranes and grown to confluence. The system
consists of two compartments, the upper compartment (luminal) and the
lower compartment (abluminal), which are separated by a polycarbonate
filter on which the endothelial cell monolayer is grown. The lower
compartment was stirred continuously and kept at a constant temperature
of 37°C by use of a thermally regulated water bath. BSA (4%)
complexed to Evans blue dye was added to the luminal compartment for
1 h to establish the basal albumin clearance rate, and then
samples were taken from the abluminal compartment every 10 min for
measurements. Transendothelial cell albumin transport was determined by
measuring the absorbance of Evans blue dye present in the abluminal
chamber at 620 nm in a spectrophotometer (Vmax Multiplate Reader;
Molecular Devices, Menlo Park, CA).
CaM kinase II, MLCK, and filamin immunoprecipitation and Western
immunoblotting.
Confluent endothelial cells were rinsed with PBS, then lysed with the
addition of either boiling 1% SDS, 1 mM sodium vanadate, and 10 mM
Tris · HCl, pH 7.4, or cold immunoprecipitation buffer (1%
Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA,
0.5% NP-40, and protease inhibitors). To a microcentrifuge tube, 50 µl of Omnisorb (Calbiochem), 400 µl of H2O, 400 µl of immunoprecipitation buffer, and 100 µl of total endothelial cell lysate were added and incubated for 30 min at 4°C, followed by centrifugation for 15 min. To supernatants, 10-100 µg of
polyclonal or monoclonal antibody were added and incubated for 1 h
at 4°C. Omnisorb (50-200 µl; Calbiochem) was added to each
tube and incubated for an additional 30 min followed by centrifugation
for 1 min. Pellets were washed three times with immunoprecipitation
buffer and resuspended in electrophoresis sample buffer, boiled for 5 min, and centrifuged for 5 min, and supernatants were loaded onto SDS-PAGE, transferred to nitrocellulose (30 V for 18 h or 90 V for
2 h) and reacted with antibody of interest. Immunoreactive proteins were detected using enhanced chemiluminescent detection system
according to the manufacturer's directions (Amersham, Little Chalfront, UK). The relative intensities of the protein bands were
quantified by scanning densitometry.
Preparation of subcellular fractions.
Confluent endothelial cells (80-100%) were fractionated into
cytosolic, membrane, and nuclear/cytoskeletal fractions as previously described (27). Cells were rinsed with PBS and incubated
in ice-cold cytosolic buffer (0.01% digitonin, 10 mM PIPES, pH 6.8, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 5 mM EDTA, and 5 µM phallicidin) and protease inhibitory cocktail (1: 500 diluted;
Calbiochem) with agitation for 10 min at 4°C. The soluble (cytosolic)
fraction was collected, the dishes were rinsed with cytosolic buffer
without protease inhibitors, and the residual material was extracted
with membrane buffer (0.5% Triton X-100, 10 mM PIPES, pH 7.4, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 3 mM EDTA, 5 µM
phallicidin, and protease inhibitory cocktail) with agitation for 20 min at 4°C. The soluble (membrane) fraction was collected, and
protein material remaining on the dish was scraped into SDS buffer
(0.5% Triton X-100, 0.5% SDS, 10 mM Tris · HCl, pH 6.8, and
protease inhibitory cocktail), sonicated, heated at 100°C for 5 min,
and centrifuged, and the supernatant (cytoskeletal fraction) together with other obtained fractions was used for SDS-PAGE and subsequent Western blotting analysis with human nonmuscle filamin-specific antibodies.
Statistics.
ANOVAs with a Student-Newman-Keuls test were used to compare means of
clearance rates, CaM kinase II activities, and ratios of un-, mono-,
and diphosphorylated MLC of two or more different treatment groups. An
independent Student's t-test was performed to analyze the
differences between means of electrical resistance (ohms) in
transendothelial electrical resistance (TER) experiments. The
comparisons of two means from different subcellular fractions were
performed using Welch's approximate t-test. Results are
expressed as means ± SD. Differences in two groups are considered
statistically significant when P < 0.05.
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RESULTS |
Thrombin-induced CaM kinase II activation in bovine pulmonary
artery endothelial cells.
We first confirmed that CaM kinase II activities in endothelial cells
are dependent on Ca2+/calmodulin availability
(2) using confluent intact endothelial cell monolayers
treated with the Ca2+ ionophore ionomycin (5 µM, 10 min)
in the absence and presence of the Ca2+ chelator EGTA (2 mM, 30 min; Fig. 1). CaM kinase II
activity was determined by the phosphorylation of exogenous CaM
kinase II peptide substrate and is expressed as picomoles per minute per 106 cells. In the presence of Ca2+,
ionomycin (5 µM, 10 min) caused a three- to fourfold increase in CaM
kinase II activity compared with control cells. When extracellular Ca2+ was eliminated by chelation with EGTA,
ionomycin-induced CaM kinase II activity was significantly reduced,
consistent with the Ca2+ dependence of CaM kinase II
activity (Fig. 1A). Pretreatment with the CaM kinase II
inhibitor KN-93 (4 µM) abolished ionomycin-induced CaM kinase II
activation, confirming complete inhibition of CaM kinase II activity
under our experimental conditions (Fig. 1B). We next
examined changes in CaM kinase II activity in intact thrombin-activated endothelial cells. Consistent with the well-known observation that
thrombin produces Ca2+ mobilization in endothelial cells
(9), we found that thrombin-induced CaM kinase II
activation is dependent on increases in
[Ca2+]i and occurs in a time-dependent manner
(0-30 min), with maximal effect at 5 min (Fig. 1C)
(2). Moreover, thrombin induced significant CaM kinase II
autophosphorylation, a reflection of kinase activation, in CaM kinase
II immunoprecipitates as detected by Western immunoblotting with
anti-phospho-CaM kinase II antibodies (Fig.
2, inset). Taken together,
these results demonstrate that thrombin and ionomycin induce
significant and rapid Ca2+-dependent CaM kinase II
activation in endothelium.

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Fig. 1.
Ionomycin- and thrombin-induced
Ca2+/calmodulin (CaM)-dependent kinase II activity in
bovine endothelium. CaM-dependent protein kinase activity in
endothelial cell homogenates was determined by the phosphorylation of
exogenous substrate peptide and is expressed as
pmol · min 1 · 106
cells 1. A: ionomycin (5 µM, 10 min)
increased CaM kinase II activity in the presence (+) of
Ca2+, whereas in the absence ( ) of Ca2+
(chelation with EGTA, 2 mM, 30 min), ionomycin-induced CaM kinase II
activity was reduced, suggesting that CaM kinase II activity is
Ca2+ dependent. Error bars are means ± SD.
*P < 0.05 compared with vehicle. B:
ionomycin-induced CaM kinase II activity was completely abolished with
KN-93 (5 µM, 30 min), a specific inhibitor of CaM kinase II. Error
bars are means ± SD. *P < 0.05 compared with
control. ** P < 0.05 compared with ionomycin.
C: effect of Ca2+ chelation on thrombin-induced
CaM kinase II activity in endothelial cells. Confluent endothelial
cells were pretreated with EGTA (2 mM, 30 min) or vehicle control
followed by thrombin challenge (100 nM, 15 min). Thrombin-induced CaM
kinase II activity was blocked by EGTA pretreatment indicating that CaM
kinase II activity is Ca2+ dependent. Error bars are
means ± SD. *P < 0.05 compared with control.
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Fig. 2.
Temporal analysis of thrombin-induced endothelial cell
activation. Thrombin increases CaM kinase II activity in a
time-dependent manner with maximal effect at 5 min. Ca2+
and myosin light chain (MLC) phosphorylation measurements were made as
described in MATERIALS AND METHODS. Inset:
thrombin-induced CaM kinase II autophosphorylation as evidenced by
Western immunoblotting with anti-phospho-CaM kinase II antibodies.
Endothelial cell monolayers were stimulated by thrombin (100 nM) for 15 min followed by immunoprecipitation of CaM kinase II as described in
MATERIALS AND METHODS. [Ca2+]i,
intracellular Ca2+ concentration.
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Effect of CaM kinase II inhibition on thrombin-induced endothelial
cell barrier dysfunction and MLC phosphorylation.
Thrombin is a potent bioactive agonist that produces increased vascular
permeability (5, 30). Because thrombin-induced CaM kinase
II activation in endothelial cells was completely abolished by the
specific CaM kinase II inhibitor KN-93, we next examined the role of
CaM kinase II inhibition in thrombin-induced endothelial cell
permeability and barrier dysfunction as assessed by alterations in
albumin clearance and TER. CaM kinase II inhibition with KN-93 significantly attenuated both thrombin-induced clearance of Evans blue
dye-albumin across confluent endothelial cell monolayers grown on
polycarbonate filters (Fig. 3) and
thrombin-induced declines in TER (Table
1). Together, these data suggest an
important role of CaM kinase II in the regulation of thrombin-induced
endothelial cell barrier dysfunction.

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Fig. 3.
Effect of CaM kinase II inhibition on thrombin-induced
endothelial cell permeability. Endothelial cell monolayers grown on
polycarbonate filters were pretreated with vehicle or KN-93 (5 µM)
followed by the addition of 100 nM thrombin or vehicle at 60 min.
Clearance of Evans blue dye-albumin across endothelial cell monolayers
was determined during each hour from 0 to 180 min. KN-93 significantly
attenuated the marked increase in thrombin-induced albumin clearance.
Error bars are means ± SD. *P < 0.05 compared
with control. ** P < 0.05 compared with thrombin.
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We have previously shown that thrombin-induced contractile events, gap
formation, and barrier dysfunction occur via MLCK-dependent mechanisms,
indicating an important role of MLC phosphorylation in this model of
vascular permeability (6). We next examined the
involvement of CaM kinase II in the MLCK-dependent pathway of
thrombin-induced endothelial cell barrier dysfunction. Confluent endothelial cell monolayers pretreated with KN-93 and challenged with
thrombin were used for assessing the levels of nonphosphorylated, monophosphorylated, and diphosphorylated MLC in confluent
endothelium. CaM kinase II inhibition did not alter the stoichiometry
of thrombin-induced MLC-phosphorylated species (Fig.
4A), suggesting that CaM
kinase II activity is more likely to be involved in MLCK-independent regulatory pathways that contribute to a thrombin-induced endothelial cell barrier dysfunction. Consistent with these data,
immunoprecipitation of MLCK from 32P-labeled endothelial
cell homogenates after KN-93 pretreatment failed to demonstrate
significant alterations in thrombin-induced MLCK phosphorylation (Fig.
4B).

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Fig. 4.
Effect of CaM kinase II inhibition on thrombin-induced
MLC and MLCK phosphorylation. MLC phosphorylation was detected by using
urea gel electrophoresis, which separates non-, mono-, and
diphosphorylated MLC. A: thrombin-induced MLC
phosphorylation (100 nM) was not altered by pretreatment with KN-93 (5 µM, for 30 min). Error bars are means ± SD. *P < 0.05 compared with vehicle. B: endothelial cell
monolayers were pretreated with KN-93 (5 µM for 30 min) and
challenged with thrombin (100 nM for 15 min) followed by MLC kinase
(MLCK) immunoprecipitation as described in MATERIALS AND
METHODS. Thrombin-induced MLCK phosphorylation (100 nM) was not
changed after KN-93 pretreatment (5 µM, for 30 min).
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Effect of thrombin-induced filamin phosphorylation and
translocation on TER.
We next addressed whether filamin, an actin-binding protein known to be
phosphorylated by CaM kinase II in vitro (24), may represent a relevant CaM kinase II target in thrombin-stimulated endothelium. Differential detergent subcellular fractionation revealed bovine endothelial cell filamin to be distributed in both cytosolic (~58%) and membrane (~42%) endothelial
cell fractions. Thrombin stimulation of confluent bovine endothelium
resulted in rapid but transient translocation of filamin from the
cytosolic to the membranous fraction after 1 min, quickly returning to
the cytosolic fraction by 5 min (Fig. 5).
We pretreated endothelial cells with either CaM peptide (5 µM, 30 min), a synthetic myristoylated peptide that contains a previously
described serine phosphorylation site (11) for CaM kinase
II within the human endothelial cell filamin sequence
(NH2-T2517GPRLVS*NHSLHE2529-COOH)
or with a synthetic myristoylated control filamin peptide (NH2-S564PFEVKVGTECGNQK579-COOH),
which does not contain a CaM kinase II consensus site. We found that
the CaM peptide but not the control peptide significantly attenuated
thrombin-stimulated filamin translocation (100 nM, 1 min; Fig.
6A). To explore whether
thrombin induces filamin phosphorylation, endothelial cell monolayers
were labeled with [32P]orthophosphate for 2.5 h,
pretreated with KN-93, and challenged with thrombin (100 nM, 10 min).
Bovine endothelial cell filamin was then immunoprecipitated and
detected in homogenates as a single ~250-kDa band. Thrombin induced
significant filamin phosphorylation that was partially attenuated by
KN-93 pretreatment, suggesting that CaM kinase II phosphorylates
nonmuscle filamin in vivo (Fig. 6B). Together, these results
indicate that thrombin-induced CaM kinase II activation results in
nonmuscle filamin phosphorylation and subcellular translocation.

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Fig. 5.
Thrombin-induced filamin translocation. Time course of
thrombin-induced filamin redistribution between cytosolic and membrane
fractions. Cells were exposed to 100 nM thrombin for 1, 2, and 5 min.
Error bars are means ± SD. *P < 0.05 compared
with control (0 min). Inset: representative
(n = 4) immunoblot of filamin from cytosolic and
membrane fractions of endothelial cells treated with thrombin (100 nM)
for the indicated times.
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Fig. 6.
Inhibition of thrombin-induced filamin translocation and
phosphorylation by KN-93 and CaM peptide. A: effect of
myristoylated peptides on thrombin-induced subcellular filamin
distribution. Endothelial cells were pretreated with either control or
CaM peptide (5 µM, 30 min) followed by thrombin challenge (100 nM, 1 min). CaM peptide, but not control peptide, prevented thrombin-induced
filamin subcellular redistribution. B: endothelial cells
were labeled with [32P]orthophosphate for 2.5 h in
phosphate-free medium and then treated with 100 nM thrombin in either
the presence or absence of KN-93 pretreatment (5 µM for 30 min).
Filamin immunoprecipitates were obtained as described in
MATERIALS AND METHODS. Thrombin induced filamin
phosphorylation, which was attenuated by KN-93 pretreatment.
C: filamin immunoprecipitates were obtained from endothelial
cell monolayers as described in MATERIALS AND METHODS.
Cells were pretreated with either 5 µM CaM peptide or 5 µM control
peptide for 30 min followed by thrombin (100 nM) treatment for 15 min.
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Our next experiments utilized 32P-labeled endothelial cell
monolayers previously pretreated with the myristoylated peptides (5 µM; CaM peptide or control) added for 30 min before thrombin stimulation (100 nM, 10 min) and filamin immunoprecipitation. Thrombin-induced filamin phosphorylation was significantly attenuated by the CaM peptide but not by the control peptide (Fig. 6C),
thereby confirming the observed inhibition of filamin phosphorylation by KN-93 (Fig. 6B) and identifying a potentially important
and physiologically relevant site of filamin phosphorylation by CaM kinase II in situ. To explore the physiological consequences
of these events, TER measurements were obtained in endothelial cell monolayers pretreated with either CaM peptide or control peptide (5 µM) followed by thrombin (100 nM) stimulation. The CaM kinase II
inhibitory peptide, but not the control peptide, significantly attenuated thrombin-induced decreases in TER (Table
2), consistent with an important role for
CaM kinase II and filamin phosphorylation in thrombin-induced
endothelial cell barrier dysfunction.
 |
DISCUSSION |
Our data indicate that thrombin-induced Ca2+
mobilization in bovine pulmonary artery endothelial cells leads to CaM
kinase II activation and autophosphorylation. Inhibition of CaM kinase
II activity with the specific inhibitor KN-93 attenuated both
thrombin-induced increases in albumin clearance and decreases in TER,
suggesting an important role of CaM kinase II activities in
thrombin-induced endothelial cell barrier dysfunction. We have
previously shown that thrombin-induced endothelial cell barrier
dysfunction is tightly linked to cytoskeletal rearrangement and stress
fiber formation and involves, at least in part, increases in the level of phosphorylated regulatory MLCs catalyzed by MLCK with Rho kinase involvement (6, 10). However, pretreatment with KN-93
before thrombin challenge on endothelial cells does not alter the level of MLC phosphorylation in endothelial cells, indicating that CaM kinase
II involvement in endothelial cell barrier regulation is not likely via
modifications in thrombin-induced MLCK of Rho kinase activation.
Because neither thrombin-induced MLCK nor MLC phosphorylation was
inhibited by KN-93 pretreatment, CaM kinase II does not appear to
phosphorylate the endothelial cell MLCK isoform (8), as demonstrated in smooth muscle cells (32, 33). Smooth
muscle MLCK phosphorylation by CaM kinase II leads to decreases in
Ca2+ sensitivity and attenuation of kinase activity
(32, 33), although other reports suggest a lack of
correlation between CaM kinase II activation and parameters of MLC
phosphorylation and force in histamine- and KCl-stimulated arterial
smooth muscle (28). Because inhibition of CaM kinase II
attenuates thrombin-induced endothelial cell barrier dysfunction
without altering either MLCK or MLC phosphorylation status, we
speculated the existence of additional CaM kinase II-dependent targets
affecting EC barrier function.
Assuming an important role for the actin cytoskeleton in maintenance of
endothelial cell barrier function, potential substrates for CaM kinase
II include an abundant number of actin-binding proteins that may affect
endothelial cell permeability. For example, caldesmon is a regulatory
contractile protein that is phosphorylated by several protein kinases
(including CaM kinase II), and thereby promotes actomyosin interaction
in smooth muscle (22, 23). Our unpublished data indicate
that CaM kinase II is consistently found in caldesmon
immunoprecipitates from endothelial cells, suggesting that caldesmon
phosphorylation by CaM kinase II after thrombin may participate in
modulation of endothelial cell barrier function. In this study, we have
focused on another important CaM kinase II target potentially involved
in endothelial cell barrier regulation, the actin-binding and
cross-linking protein filamin (ABP-280). Filamin is located at the
interface between the cytoskeleton and the plasma membrane, where it
anchors filamentous actin to plasma membrane proteins. In this spatial
locale, filamin is uniquely qualified to control cell shape and cell
locomotion, stabilize attachments to other cells and to the substratum,
and regulate cellular responses to external stimuli (20).
The importance of filamin was demonstrated in human malignant melanoma
cell lines that lack ABP-280 (3) and display impaired
locomotion and circumferential blebbing of the plasma membrane, whereas
reserved expression of ABP-280 restores motility and reduces membrane
blebbing. Actin cross-linking properties of filamin are affected by CaM
kinase II-dependent phosphorylation, which occurs with a maximal
stoichiometry of 1 mol of phosphate per mole of filamin dimer and leads
to a decrease in filamin-regulated actin filament cross-linking
activity (24). We speculate that filamin phosphorylation
causes a shift in the cortical actin filament sol-gel transitions that
are critical for cell movement (31) and thus leads to
changes in endothelial cell barrier function. Thus regulation
of the mechanical properties of the cytoplasmic actin network by
filamin phosphorylation may alter endothelial cell permeability. Our
TER experiments using a CaM peptide, which functions as a specific
competitive inhibitor of nonmuscle filamin phosphorylation by CaM
kinase II, strongly suggest that filamin phosphorylation is involved in
endothelial cell barrier regulation, although the exact mechanism is
not clear and consequent signaling events are not well understood. The
subcellular redistribution of filamin appears to be involved in the
disassembly of the membrane-cytoskeleton network, loss of junctional
integrity between cells, and an increase in vascular permeability
(13-15, 35-37). In this case, observed direct
association of filamin with integrins (19, 29), which play
major roles in cell-cell and cell-extracellular matrix interactions,
may be important in endothelial cell barrier regulation.
Physiologically relevant filamin subcellular redistribution may also
affect protein interactions within the complexes of focal contacts and
adherens junctions, triggering correspondent signal transduction
processes. Further work is necessary to link changes in filamin
phosphorylation and subcellular distribution to endothelial cell
barrier regulation.
In conclusion, we have shown that CaM kinase II activation is involved
in thrombin-induced endothelial cell barrier dysfunction. CaM kinase II
activation does not alter the level of MLC phosphorylation but leads to
phosphorylation and translocation of the cytoskeletal actin-binding
protein nonmuscle filamin, with subsequent modulation of endothelial
cell barrier function, presumably via changes in the mechanical
properties of the cytoplasmic actin network. Further examination of
filamin-mediated cytoskeletal rearrangement as well as other CaM kinase
II cytoskeletal targets may reveal new insights into nonmuscle
cytoarchitectural regulation.
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ACKNOWLEDGEMENTS |
We thank Steve Durbin and Lakshmi Natarajan for technical
assistance and Ellen Reather for assistance with manuscript preparation.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-50533, HL-58064, and HL-57402 and by the Dr. David Marine Endowment.
Address for reprint requests and other correspondence: J. G. N. Garcia, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, 4B.77, Baltimore, MD 21224-6801 (E-mail: drgarcia{at}welch.jhu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
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