© 2001 American Heart Association, Inc.
Vascular Biology |
Myosin Light Chain Kinase Regulates Capacitative Ca2+ Entry in Human Monocytes/Macrophages
From the Departments of Internal Medicine III (Q.-K.T., L.P., K.T.) and Clinical Pharmacology and Therapeutics (H.W., H.-Y.L., K.O.), Hamamatsu University School of Medicine, Hamamatsu, Japan, and Life Science Center (M.S.), Asahi Chemical Industries, Co Ltd, Fuji City, Shizuoka, Japan.
Correspondence to Hiroshi Watanabe, MD, PhD, Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan. E-mail hwat{at}hama-med.ac.jp
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Abstract |
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Abstract—Monocytes/macrophages are present in all stages of atherosclerosis. Although many of their activities depend to various extents on changes in intracellular Ca2+ concentration ([Ca2+]i), mechanisms regulating [Ca2+]i in these cells remain unclear. We aimed to explore the role of myosin light chain kinase (MLCK) in Ca2+ signaling in freshly isolated human monocytes/macrophages. Large capacitative Ca2+ entry (CCE) was observed under fura 2 fluoroscopy in human monocytes/macrophages treated with thapsigargin and cyclopiazonic acid. ML-9 and wortmannin, 2 structurally different inhibitors of MLCK, dose-dependently (1 to 100 µmol/L) prevented CCE and completely did so at 100 µmol/L, whereas inhibitors of tyrosine kinase and protein kinase C had only partial effects. Western blotting showed that thapsigargin significantly caused myosin light chain phosphorylation, which was almost completely blocked by ML-9 (100 µmol/L) and wortmannin (100 µmol/L). ML-9 also dose-dependently (1 to 100 µmol/L) inhibited this phosphorylation, which was well correlated with its inhibition of CCE. Transfection with MLCK antisense completely prevented CCE in response to thapsigargin and cyclopiazonic acid, whereas MLCK sense had no effect. These data strongly indicate that MLCK regulates CCE in human monocytes/macrophages. The study suggests a possible involvement of MLCK in many Ca2+-dependent activities of monocytes/macrophages.
Key Words: monocytes/macrophages • capacitative Ca2+ entry • myosin light chain kinase
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Introduction |
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Monocytes/macrophages are present in all stages of atherosclerosis.1 2 3 Many of their functions depend on changes in intracellular Ca2+ concentration ([Ca2+]i). For example, leukocytes adhering to endothelial cells demonstrate multiple Ca2+ transients4 ; the binding of oxidatively damaged red blood cells by macrophages is almost totally Ca2+ dependent5 ; and oxidized LDL–induced activation of protein kinase C (PKC) leading to macrophage growth also involves a rise in [Ca2+]i.6 In addition, optimal oxidative modification of LDL by monocytes/macrophages requires Ca2+ release from intracellular stores and Ca2+ entry,7 and LDL receptor–mediated lipoprotein degradation is also Ca2+ dependent.8
In many cells, intracellular Ca2+ stores are mobilized after either the production of inositol trisphosphate or blockade of the endoplasmic reticulum (ER) Ca2+ pump, and it has been postulated that Ca2+ store depletion can trigger transmembranous Ca2+ entry, the capacitative Ca2+ entry (CCE) model.9 Such a mechanism has not been demonstrated in human monocytes/macrophages. Furthermore, although more human homologues of the Drosophila gene products transient receptor potential and transient receptor potential-like are being cloned and have proven to be candidate genes for CCE channels,10 further information is required to determine how intracellular Ca2+ store depletion can activate CCE. Numerous second messengers have been implicated in CCE activation, including inositol trisphosphate,11 cGMP,12 a Ca2+ influx factor,13 a product of Cytochrome P 450 activity,14 and tyrosine phosphorylation.15 16 We have recently reported that myosin light chain (MLC) kinase (MLCK) inhibitors prevent agonist-stimulated, fluid flow–stimulated, and chloride-sensitive Ca2+ entry in endothelial cells.17 18 In monocytes/macrophages, MLCK is implicated in several activities important in atherosclerosis. Thus, MLCK is important for macrophage motility19 and regulates neutrophil migration across the endothelium.20 MLCK activation was recently proven to be critical in cytoskeletal changes resulting in pseudopod formation during phagocytosis.21 Migration and recruitment of macrophages and macrophage-derived foam cells are, in turn, stimulated by increased Ca2+ concentration.22 Although these observations, taken together, indicate a likelihood of an involvement of MLCK in Ca2+ signaling and thus in many Ca2+-dependent activities of these cells, the role of MLCK in monocyte/macrophage Ca2+ signaling has never been investigated.
In the present study, we have investigated the effects of various inhibitors of MLCK and other protein kinases on CCE and MLC phosphorylation caused by thapsigargin (TG) and cyclopiazonic acid (CPA) in human monocytes/macrophages, and we have tested the effects of MLCK sense and antisense oligonucleotides on the CCE. Our results indicate that MLCK is an important regulator of CCE in human monocytes/macrophages and suggest the possible involvement of the kinase in many Ca2+-dependent activities of these cells.
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Methods |
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Cell Isolation
The investigation was approved by the institutional ethics committee. Informed consent was obtained from healthy volunteers, and heparinized blood was collected, diluted in PBS containing 2% FCS, and subjected to gravity centrifugation (400g) in Ficoll-Paque isolating solution (Pharma Biotech) for 30 minutes at 18°C. The upper plasma layer was removed, leaving the leukocyte layer undisturbed; this layer was then gently collected and washed several times with PBS containing 2% FCS at 4°C. The final pellets were suspended in RPMI 1640 medium containing 2% FCS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µmol/L streptomycin at a density of 2x106 cells/mL, aliquoted onto polybiphenyl dishes fixed on 10x10-mm glass coverslips, and incubated at 37°C under 5% CO2 for several hours. Before the experiments, samples were washed several times with the same culture medium to remove nonadhering leukocytes, leaving only adherent monocytes/macrophages on glass coverslips. Differential cell counts under Wright’s Giemsa staining showed that >90% of the isolated cells were mononucleocytes. Cell viability was >95%, as determined by trypan blue dye exclusion test.
[Ca2+]i
Measurement
[Ca2+]i
was measured as previously
described.18 TG, CPA, ML-9,
wortmannin, genistein, herbimycin A, staurosporine,
bisindolylmaleimide I, MLCK sense, MLCK antisense, and Superfect
reagent (QIAGEN Inc) at the concentrations used in the present
study did not affect fura 2 fluorescence or
autofluorescence of unloaded cells.
Western Blotting
MLC phosphorylation in
monocytes/macrophages was measured by separation of
nonphosphorylated and phosphorylated
forms by glycerol-PAGE according to their respective charges of
phosphate molecules, followed by electrophoretic transfer of the
proteins to a nitrocellulose membrane. The relative amounts of each
form were quantified by immunoblotting with an anti-MLC
antibody, as described in a previous study with
[
-32P]ATP incorporation into separated
MLC bands and phosphomyosin-specific
phosphatase.23 It has been
demonstrated that electrophoresed protein bands represent
nonphosphorylated and phosphorylated
MLC. Briefly, after stimulation with TG in the presence or absence of
wortmannin or ML-9, cells were exposed to 5% trichloracetic acid
containing 2 mmol/L dithiothreitol. After
centrifugation at
2500g for 3 minutes, the pellet
was washed with 10 mmol/L dithiothreitol/acetone, resuspended in
urea sample buffer, and processed for urea/glycerol/PAGE and
immunoblotting by a modification of the method by
Persechini et al.24 The urea
extracts before and after stimulation with TG contained
nonphosphorylated and phosphorylated
MLC. Relative quantification of nonphosphorylated and
phosphorylated MLC was made by
densitometry.
MLCK Sense and Antisense
Transfection
MLCK antisense oligonucleotides (5'
GGGCTTTCATCTCCG 3') were produced according to the mRNA of porcine
smooth muscle MLCK (DDBJ access identification No. D89497). The
target sequence started at the 170-base position. Respective sense
oligonucleotides were produced for use as controls. The
oligonucleotides were dissolved in Tris-HCl plus
EDTA (pH 7.4) to 0.1 µg/µL. Then 10 µL of this mixture was
diluted in RPMI medium containing no serum, proteins, or antibiotics to
300 µL. The solution was mixed for a few seconds before Superfect
transfection reagent (30 µL, QIAGEN Inc) was added and mixed, and the
mixture was left at room temperature for 10 minutes to allow complex
formation. Meanwhile, the cells were washed with PBS several times,
leaving the adherent ones on glass coverslips. Then, 2 mL of RPMI
solution containing 10% FCS and antibiotics was added to the reaction
tube containing the transfection complexes. The solution was mixed,
immediately transferred to the cell wells at 500 µL each, and
incubated for 3 hours at 37°C and 5% CO2. The
medium was then removed, the cells were washed once with PBS, and the
culture medium containing serum and antibiotics was added. The samples
were incubated for 20 hours. Over 95% of the cells remained viable
under the trypan blue exclusion test before the experiments. To assess
whether the oligonucleotides were successfully
transfected, the 5' ends were conjugated with fluorescein
isothiocyanate (FITC) and transfected, and then the cells were observed
under a fluorescence microscope (Axiophot,
Zeiss).
Statistical Analysis
Data are expressed as mean±SD from at least 3
separate experiments. Statistical analysis was performed with
the Student t test for unpaired
data. Correlation analysis was performed by using GraphPad
Prism (GraphPad Software, Inc). A value of
P<0.05 was considered
significant.
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Results |
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CCE Exists in Human Monocytes/Macrophages
To investigate Ca2+ signaling in human monocytes/macrophages, we used TG, an irreversible inhibitor of the ER Ca2+-ATPase,25 and CPA, a reversible inhibitor of the enzyme.26 Under Ca2+-free conditions, TG (1 µmol/L) and CPA (100 µmol/L) increased the fluorescence ratios at 340 and 380 nm (F340/380 ratios) very slightly, from 0.88±0.10 and 0.92±0.02 to 1.07±0.06 and 1.10±0.11 after 90 seconds of TG and CPA treatment, respectively (open circles, Figures 1A
and 1B). In
Ca2+-containing medium, TG (1 µmol/L)
significantly increased the F340/380 ratio from 0.87±0.17 to
4.28±0.39 and 4.00±0.30 after 2 and 8 minutes, respectively (closed
circles,
Figure 1A). Likewise, CPA (100 µmol/L) largely increased
the F340/380 ratio from 0.92±0.12 to 4.10±0.41 and 3.15±0.32 after
3.5 and 8 minutes, respectively (closed circles,
Figure 1B). To determine whether CCE was at work, the
reversible ER Ca2+-ATPase
inhibitor CPA (100 µmol/L) was first applied in
Ca2+-free medium, which slightly increased
the F340/380 ratio. CPA was then washed out, and the cells were kept in
Ca2+-free medium for 5 minutes. When 1
mmol/L Ca2+ was introduced without CPA, the
F340/380 ratio was largely increased
(Figure 1C). These results clearly implicate CCE in human
monocytesmacrophages.
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MLCK Inhibitors Prevented
Human Monocyte/Macrophage CCE
To investigate the role of MLCK in
Ca2+ signaling in human
monocytes/macrophages, we first examined the effects of ML-9, a
strong MLCK
inhibitor,27 on
TG-induced Ca2+ response. Five-minute ML-9
pretreatment did not increase the basal F340/380 ratio but
dose-dependently (1 to 100 µmol/L) prevented the TG-induced
Ca2+ response, with almost complete
inhibition at 100 µmol/L
(Figure 2A). In the same manner as used with ML-9, a
30-minute pretreatment with wortmannin, a different MLCK
inhibitor, did not increase the basal F340/380 ratio but
dose-dependently (1 to 100 µmol/L) inhibited the TG-induced
Ca2+ response, with complete inhibition at
100 µmol/L
(Figure 2B).
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To confirm that MLCK inhibition could block CCE, CPA was
applied for 5 minutes under Ca2+-free
conditions, which slightly raised the F340/380 ratio. CPA was then
removed, and ML-9 (100 µmol/L) was applied 5 minutes before 1
mmol/L Ca2+ was added. Even after the
addition of Ca2+, the F340/380 ratio
did not increase in the presence of ML-9. When ML-9 was removed 7.5
minutes later in Ca2+-containing medium, the
F340/380 ratio increased
(Figure 2C
).
To test whether MLCK inhibition could affect
intracellular Ca2+ store depletion,
monocytes/macrophages pretreated with 100 µmol/L ML-9
(Figure 3A and 3C) or 100 µmol/L wortmannin
(Figure 3B and 3D) were stimulated with TG (1 µmol/L) or
CPA (100 µmol/L) in Ca2+-free medium. TG
and CPA still transiently increased the F340/380 ratios as they did in
controls. All in all, these data demonstrate that different MLCK
inhibitors prevent CCE without affecting ER
Ca2+ release in response to TG and CPA in
human monocytes/macrophages.
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MLCK Inhibitors Blocked
TG-Stimulated MLC Phosphorylation
Because a key function of MLCK is to
phosphorylate MLC, we investigated the effects of the MLCK
inhibitors on TG-induced MLC
phosphorylation in monocytes/macrophages
(Figure 4). In control conditions, MLC was not
phosphorylated (the total extracted MLC being in the
nonphosphorylated form); TG (1 µmol/L) increased
phosphorylated MLC to as much as 77% of the total
extracted MLC. Wortmannin (100 µmol/L) pretreatment for 30 minutes or
ML-9 (100 µmol/L) pretreatment for 5 minutes completely inhibited the
formation of phosphorylated MLC bodies, which were now
only 6% and 0%, respectively, of the total extracted MLC
(Figure 4A). ML-9 dose-dependently (1 to 100 µmol/L)
inhibited this MLC phosphorylation
(Figure 4B), and the effects of ML-9 to inhibit CCE and MLC
phosphorylation were closely correlated
(P<0.05,
r=0.95197;
Figure 4C). These findings substantiate MLCK involvement in
the effects shown by both inhibitors on
CCE.
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Effects of Other Kinase Inhibitors
on CCE
Various protein kinases have been implicated in CCE.
Therefore, we compared the effects of inhibitors of MLCK,
protein tyrosine kinase (PTK), and PKC on TG- and CPA-induced
Ca2+ responses.
Figure 5 compares the effects on the peak and sustained
phases of the Ca2+ responses to 1 µmol/L
TG
(Figure 5A) and 100 µmol/L CPA
(Figure 5B) by the MLCK inhibitors ML-9 (100
µmol/L) and wortmannin (100 µmol/L), the PTK inhibitors
genistein (100 µmol/L) and herbimycin A (100 µmol/L), and the PKC
inhibitors bisindolylmaleimide I (10 µmol/L) and
staurosporine (0.3 µmol/L). Pretreatment for 5 minutes
with either the PTK or PKC inhibitors significantly, but
only partially, inhibited TG- and CPA-induced
Ca2+ influxes, whereas the MLCK
inhibitors almost abolished these influxes. Five minutes
was sufficient for the inhibitors to exert their maximal
effects, except for wortmannin, which required 30
minutes.
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P<0.05 vs respective values by ML-9.
Effects of MLCK Sense and Antisense on TG- and
CPA-Induced Ca2+ Responses
To provide further independent evidence that MLCK
regulates CCE, we transfected human monocytes/macrophages with
MLCK sense or antisense oligonucleotides and tested
Ca2+ responses to TG and CPA in these cells.
To confirm that the oligonucleotides were transfected
into cells, the 5' ends were conjugated with FITC, the complexes were
transfected, and the cells were observed under a fluorescence
microscope. All the cells displayed bright FITC fluorescence,
indicating that they all contained MLCK sense and antisense
oligonucleotides (please see
http://atvb.ahajournals.org).
Figure 6 shows the Ca2+ responses
of control, sense, and antisense cells to 1 µmol/L TG
(Figure 6A) and 100 µmol/L CPA
(Figure 6C). In antisense cells,
Ca2+ responses to TG and CPA were completely
prevented; there were only small transient rises similar to those
observed under Ca2+-free conditions.
However, MLCK sense oligonucleotides had no effects on
TG- and CPA-induced Ca2+ responses. In
Ca2+-free medium, small and transient rises
in [Ca2+]i were
still observed after treating MLCK antisense cells with 1 µmol/L TG
(Figures 6B) or 100 µmol/L CPA
(Figure 6D). A similar transfection protocol with only
Superfect Reagent showed no effect on TG- and CPA-induced CCE (data not
shown).
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Discussion |
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The ER Ca2+-ATPase inhibitors TG and CPA largely increased [Ca2+]i in human monocytes/macrophages in Ca2+-containing medium but did so only very slightly under Ca2+-free conditions. This indicates that the bulk of [Ca2+]i rise was due to Ca2+ entry. In Figure 1C
, Ca2+ entry was
observed on the restoration of extracellular
Ca2+ after a short incubation in
Ca2+-free conditions after CPA removal.
Because the store depletion stimulated by CPA was apparently the
only mechanism to trigger the Ca2+ entry
under this condition, this observation clearly indicates CCE in human
monocytes/macrophages. This is similar to
Ca2+ entry mechanisms in many other cell
types.28
The first clue to the involvement of MLCK in the regulation
of CCE in human monocytes/macrophages was that ML-9 and
wortmannin, structurally different MLCK inhibitors,
completely prevented this entry at 100 µmol/L. ML-9 is a potent MLCK
inhibitor
(Ki 3.8
µmol/L) that competes with ATP for binding to the
kinase.29 Because ML-9 at
high doses can also inhibit PKC
(Ki 54
µmol/L) and PKA
(Ki 32
µmol/L), its dose-dependent inhibition of the
Ca2+ response was not enough to implicate
MLCK in CCE. However, similar effects were observed with structurally
unrelated wortmannin, an inhibitor of
phosphatidylinositol-3 kinase and
MLCK.30 It was used in the
present study as an MLCK inhibitor, because TG and CPA
mobilize ER Ca2+ not through the activation
of phosphatidylinositol-3 kinase. This similarity suggests specificity
to MLCK of the effects by both agents to inhibit CCE. The peaks of the
Ca2+ responses in
Figure 2B seemed delayed, but this cannot be interpreted as
an inhibitory effect of wortmannin on store depletion.
Ca2+ rises due to store release constitute a
very small portion of the rising phases of the
Ca2+ response curves. Furthermore, as shown
in
Figure 3, wortmannin did not affect store mobilization. This
delay was not seen with ML-9. In
Figure 2C, Ca2+ entry was
observed only after the removal of ML-9. This observation and a
comparison with
Figure 1C clearly show that ML-9 was able to inhibit CCE in
monocytes/macrophages.
Further clues to the involvement of MLCK in CCE came from
the findings that wortmannin and ML-9 were able to completely inhibit
TG-induced MLC phosphorylation at concentrations that
completely inhibited TG-induced CCE. IC50 values
for ML-9 to block MLC phosphorylation and CCE were also
similar, according to
Figure 4C. The correlation between the effects of ML-9 to
inhibit CCE and MLC phosphorylation further indicates
that MLCK was involved in the effect of the compound to inhibit CCE.
MLC phosphorylation by MLCK is linked to cellular
contractile activity; therefore, it could be that MLCK activation after
Ca2+ store release might rearrange the
cytoskeleton, which would open the gate for
Ca2+ entry. Nevertheless, MLCK-regulated CCE
and MLC phosphorylation could just as likely be
independent events; thus, the extent of MLC
phosphorylation would be considered in this regard only
as a measure of MLCK activity. In fact, in another series of
experiments, calyculin A, a phosphatase inhibitor that
should increase MLC-phosphorylated forms by preventing
them from being degraded, neither caused any
Ca2+ entry nor enhanced the TG-induced
Ca2+ responses (authors’ unpublished data,
2000).
Various kinases have been implicated in CCE. Particularly,
the PTK inhibitor genistein was found to inhibit CCE in
various cells, including fibroblasts, 15
lymphocytes,31
platelets,32 and
endothelial
cells.16 33 PKC
involvement in CCE has been controversial. In
Xenopus oocytes, activation of
PKC was found to inhibit
CCE,34 whereas in
endothelial cells, we showed that
inhibitors of PKC and PKA did not affect agonist-induced
Ca2+
entry.35 In the present
study, ML-9 and wortmannin abolished TG- and CPA-induced CCE, whereas
inhibitors of PTK and PKC had only partial effects. The
observations that bisindolylmaleimide I, a strong PKC
inhibitor
(Ki 0.01
µmol/L), PKA
(Ki 2.0
µmol/L),36 and
staurosporine only partially inhibited TG- and CPA-induced
CCE make it more likely that the effects seen with ML-9 shown in
Figure 2A were due to MLCK inhibition. These data suggest
that MLCK may play a more important role than do PTK and PKC in CCE in
human monocytes/macrophages. MLCK has several potential
phosphorylatable sites that can be phosphorylated by
other protein kinases, including PKC and
PTK.37 In fact, the PTK
inhibitor genistein was shown to reduce MLCK activity in
MLCK immunoprecipitates, and it has been suggested that
genistein-sensitive tyrosine kinase activities are involved in
thrombin-mediated MLCK activation and MLC
phosphorylation.38
It is possible, then, that MLCK, partially influenced by PKC and PTK,
lies downstream from these 2 kinases in the CCE signaling cascade and
plays a more decisive role in the regulation of this cascade in human
monocytes/macrophages. This hypothesis seems to explain the
proportional contribution of PTK, PKC, and MLCK to the coupling of
internal store depletion and transmembranous
Ca2+ entry in these cells.
The experiments above suggest that MLCK may be important in CCE activation in human monocytes/macrophages and that MLCK may be more important than PTK and PKC in this regard. Nevertheless, because all the inhibitors used significantly inhibited CCE, they could be accomplishing this by some nonspecific effects on the cells. The observations that transfection with MLCK antisense completely prevented CCE but that MLCK sense had no effect provide solid independent evidence that MLCK plays a crucial role in the regulation of CCE in human monocytes/macrophages. The coupling role is clear because neither the MLCK inhibitors nor MLCK antisense affected the emptying of the Ca2+ store by TG and CPA.
In conclusion, we have provided solid evidence that MLCK has a crucial role in the regulation of CCE in human monocytes/macrophages. Together with our previous findings in endothelial cells, the present study suggests a possible involvement of MLCK in the pathogenesis of atherosclerosis, the initiation of which involves both of these cell types. The present study suggests the involvement of MLCK in many Ca2+-dependent activities of these cells. Further investigations are under way to test these hypotheses.
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Acknowledgments |
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This study was supported by a grant-in-aid for scientific research (No.10670642) from the Ministry of Education, Science, Sports, and Culture of Japan.
Received August 21, 2000; accepted December 12, 2000.
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