Departments of 1 Physiology, 3 Internal Medicine, and 2 Anatomy and Cell Biology, University of Michigan, Ann Arbor, Michigan 48109-0622
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ABSTRACT |
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We investigated how heat shock protein 27 (HSP27) and its phosphorylation are involved in the action of cholecystokinin (CCK) on the actin cytoskeleton by genetic manipulation of Chinese hamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. This effect could be blocked with the p38 MAP kinase inhibitor SB-203580. Examination by confocal microscopy of cells stained with rhodamine phalloidin showed that CCK dose-dependently induced changes of the actin cytoskeleton, including cell shape changes, which were coincident with actin cytoskeleton fragmentation and formation of actin filament patches in the cells. To further evaluate the role of HSP27, CHO-CCK-A cells were transfected with expression vectors for either wild-type (wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27 and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen after high-dose CCK stimulation. In contrast, overexpression of nonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition of phosphorylation of HSP27 by preincubation of wt-HSP27 transfected cells with SB-203580 did not protect the actin cytoskeleton. These results suggest that phosphorylation of HSP27 is required to stabilize the actin cytoskeleton and to protect the cells from the effects of high concentrations of CCK.
heat shock protein; cholecystokinin; p38 mitogen-activated protein kinase; microfilaments
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INTRODUCTION |
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IT IS WELL ESTABLISHED that induction of heat shock proteins (HSPs) in response to various stresses correlates with increased resistance to subsequent cellular damage. However, HSPs are also present in significant amounts in unstressed cells, where they are believed to accomplish essential cellular functions. For HSP60, HSP70, and HSP90, molecular chaperone activities involved in protein translocation, protein folding, and signal transduction have been documented (for reviews see Ref. 5). For HSP27 there are several proposed activities. It has been shown that HSP27 in vitro acts as a molecular chaperone, facilitating the refolding of partially denatured proteins into active conformations (13, 14) and that it can also act as a phosphorylation-regulated F-actin barbed end capping protein capable of inhibiting actin polymerization (2, 26, 27). The physiological significance of the accumulation of HSP27 after stress has been demonstrated in gene transfection studies, in which overexpression of HSP27 was shown to confer resistance against heat shock and oxidative stress (11, 12, 15, 17, 24). Inhibition of protein kinase activity that results in reduced HSP27 phosphorylation (8) or expression of a nonphosphorylatable mutant HSP27 in fibroblasts inhibits actin filament stabilization (18), suggesting that this function of HSP27 is phosphorylation dependent. It has become apparent that the function and role of HSP27 not only is dependent on the total amount of HSP27 and its ability to become phosphorylated but also is cell type specific. For example, it appears that the effects on the actin cytoskeleton in human umbilical vein endothelial cells (HUVEC), which contain a relatively high amount of endogenous HSP27 (5-7 µg HSP27/mg total protein compared with 1-2 µg in CCL39 cells), are different from that in fibroblasts (10). In HUVEC cells treatment with H2O2 induced accumulation of stress fibers, recruitment of vinculin to focal adhesions, and the loss of membrane ruffles, whereas in fibroblasts severe fragmentation of F-actin was observed (10). In addition, data showed that a prior activation of the p38 mitogen-activated protein (MAP) kinase cascade, leading to the phosphorylation of HSP27, increased the stability of actin microfilaments in cells exposed to cytochalasin D (8). This effect was dependent on the expression of HSP27, and the effect was totally inhibited by blocking p38 activity with SB-203580 (8). These results support the concept that activation of p38 MAP kinase during adverse environmental conditions serves a homeostatic function of regulating microfilament dynamics that would otherwise be destabilized during stress. P38 MAP kinase activation during normal agonist stimulation may constitute an additional actin-signaling pathway, the importance of which depends on the level of expression of HSP27 (8). Biochemical analyses have revealed that HSP27 is phosphorylated on the same serine residues and by the same protein kinase regardless of the triggering agents or treatments (for detailed reviews see Ref. 16). The protein kinase that phosphorylates HSP27 has been identified as MAP kinase-activated protein (MAPKAP) kinase 2/3, which itself is activated by p38 MAP kinase (35). So far, there are no reports showing that members of the extracellularly regulated kinase (ERK) or c-jun NH2-terminal kinase (JNK) pathways are involved in phosphorylation of HSP27 in vivo.
Previously, we have shown that cholecystokinin (CCK) activates p38 MAP kinase and MAPKAP kinase 2, leading in vitro and in vivo to phosphorylation of HSP27 in isolated pancreatic acini (7, 34). Furthermore, we showed that CCK induced changes in the actin cytoskeleton. Changes in the apical cytoskeleton of intact and permeabilized pancreatic acini after treatment with CCK have been reported earlier (28, 30), and there is evidence that an intact actin cytoskeleton is necessary for regulated secretion (28). In the present study, we investigated the effects of CCK on the actin cytoskeleton in CHO-CCK-A cells and the role of HSP27 in modulating these effects after transfection of human wild type (wt)-HSP27 or of various mutants of HSP27. In addition to previously reported transfection studies, where oxidative stress induced changes of the actin cytoskeleton (10, 11, 24), we now show that CCK, which acts via a Gq protein-coupled receptor signaling pathway, activates p38 MAP kinase in a dose-dependent manner and that the effects seen on the actin cytoskeleton are dependent not only on the concentration of CCK but also on the amount of phosphorylatable HSP27. Together with our previously reported study, these results suggest that the p38 MAP kinase/HSP27 plays an important role in regulating microfilaments after CCK treatment. These findings may also help to further understand the pathophysiology of a model of acute pancreatitis, in which injections of high concentrations of caerulein disrupt the actin cytoskeleton in exocrine pancreatic acinar cells.
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EXPERIMENTAL PROCEDURES |
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Materials. CCK octapeptide (CCK-8) was
from Research Plus (Bayonne, NJ). Rhodamine phalloidin, Alexa 488 phalloidin, and ProLong Anti-fade kit were purchased from Molecular
Probes (Eugene, OR). Rabbit polyclonal anti-p38 (C-20) antibody was
from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal
anti-HSP27 antibody (SPA-801) and mouse monoclonal anti-human HSP27
antibody (SPA-800) were from Stressgen Biotechnologies (Victoria, BC,
Canada). The mouse monoclonal anti-vinculin antibody was from Sigma
(St. Louis, MO). Fibronectin-coated coverslips were obtained from
Becton Dickinson (Bedford, MA). Ampholytes for isoelectric focusing
(IEF) gel electrophoresis and protein assay reagents were from Bio-Rad
(Hercules, CA), nitrocellulose membranes were from Schleicher & Schuell
(Keene, NH).
[-32P]ATP (3,000 Ci/mmol) was from DuPont NEN Life Science Products (Boston, MA). The
pcDNA 3.1(+), version A, was from Invitrogen (San Diego, CA). The
enhanced chemiluminescence (ECL) detection system, protein A conjugated
to horseradish peroxidase, and X-ray film were from Amersham (Arlington
Heights, IL). Protein A-agarose was from Pierce (Rockford, IL). All
other reagents were obtained from Sigma.
Culture and transfection of CHO-CCK-A cells. CHO-K1 cells stably transfected with the cloned rat CCK-A receptor as described earlier (37) were used. Cells were routinely cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 0.25 mg/ml G418, penicillin, streptomycin, and amphotericin B at 37°C in a humidified atmosphere containing 5% CO2. For transient transfection, CHO-CCK-A cells were grown to ~60% confluence in six-well plates and transfected with 1 µg of plasmid using FuGENE transfection reagent (Roche, Indianapolis, IN) following the instructions of the manufacturer. Transfection efficiency was ~40%. The cells were transfected with pcDNA 3.1 plasmids containing a cDNA insert for human HSP27 (9) or with mutagenized HSP27 cDNAs in which the codons for Ser15,78,82 were altered to encode for either glycine (3G), alanine (3A), or aspartic acid (3D). cDNAs coding for human HSP27 were cut out from the vector pBluescript II KS using restriction enzymes ECL 136 II and Xho I and ligated into eukaryotic expression vector pcDNA 3.1(+) (version A) using the restriction enzymes EcoR V and Xho I. For immunocytochemistry, cells were plated on fibronectin-coated glass coverslips 24 h before transfection.
P38 MAP kinase assays. Cells were
washed once with 1 ml of ice-cold PBS containing 1 mM
Na3VO4,
pH 7.4, and were scraped and sonicated for 5 s in 0.1 ml of ice-cold
lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton
X-100, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM dithiothreitol,
0.2 mM
Na3VO4,
25 mM NaF, 10 mM sodium pyrophosphate, 25 mM -glycerophosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. The lysates were then centrifuged in a microcentrifuge at
4°C for 15 min and assayed for p38 MAP kinase activity as described earlier (34). Briefly, immunoprecipitated p38 MAP kinase from 200 µg
of cell lysate protein was used to phosphorylate 5 µg of GST-ATF-2
(1-109) in 20 µl of kinase buffer (18 mM HEPES, pH 7.4, 10 mM
magnesium acetate, 50 µM ATP, 2.5 µCi
[
-32P]ATP per
sample). The reaction mixture was incubated at 30°C for 20 min with
shaking. Reactions were terminated by addition of four times SDS sample
buffer, GST-ATF-2 was resolved by SDS gel electrophoresis, and
incorporated 32P was visualized
and quantitated using a phosphoimager system (Bio-Rad GS-505) with
Molecular Analyst software.
IEF, SDS-PAGE, and Western analysis. Cells were lysed in urea buffer containing 9.0 M urea, 4% Nonidet P-40, and 1% 2-mercaptoethanol. Twenty micrograms of total lysate protein per sample were subjected to IEF using the Bio-Rad 111 mini cell system. A monomer solution containing 5% ampholytes (vol/vol), pH 3-10 was used. After samples were absorbed into the gel, IEF was performed using 15 min at 100 V, 15 min at 200 V, and at least 1 h at 450 V followed by electrophoretic transfer to nitrocellulose membrane. HSP27 and its isoforms were detected using rabbit polyclonal anti-HSP27 antibodies and monoclonal anti-HSP27 antibodies followed by labeling with anti-rabbit IgG and anti-mouse IgG-peroxidase conjugates, respectively. Images of the antigen bands were revealed with the ECL kit from Amersham and recorded onto Hyperfilm from Amersham (34). To dephosphorylate HSP27 in vitro, CHO-CCK-A cells were lysed in dephosphorylation buffer containing 5 mM Tris, pH 8.5, and 0.1 mM EDTA. Three hundred micrograms of total cell lysate were incubated with 200 units of calf intestinal alkaline phosphatase (Roche, Indianapolis, IN). The reaction was performed at 37°C for 30 min with constant shaking and terminated by the addition of IEF urea buffer. For SDS-polyacrylamide gel electrophoresis, confluent CHO cells in 100-mm dishes were lysed with 0.25 ml of ice-cold buffer containing 20 mM Tris, pH 7.8, 2 mM EDTA, 50 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 0.1% (wt/vol) Triton X-100. Samples were adjusted to a final concentration of 62.5 mM Tris · HCl, pH 6.8, 2% SDS (wt/vol), 10% glycerol, 0.05% bromphenol blue, and 0.5% 2-mercaptoethanol (vol/vol), boiled for 5 min, and the proteins (30 µg/lane) were separated by 10% SDS-PAGE followed by a transfer onto a nitrocellulose membrane. HSP27 was detected with anti-HSP27 antibodies and the ECL reagent as described above. All Images were scanned using an Agfa Arcus II Scanner. Quantification and computerized densitometry of the specific bands on all films was performed using Molecular Analyst software. The analysis was performed within the range of proportionality of the film, as assessed in earlier experiments using recombinant purified mouse HSP27 protein in the range of 1-100 ng as a standard.
Cell fractionation with Triton X-100. Triton X-100-soluble and -insoluble fractions were prepared as described previously (21, 33) with the use of a slightly modified protocol. Briefly, CHO-CCK-A cells were grown in 100-mm plastic dishes until ~80% confluent and lysed as described above. The cell lysates were sonicated and promptly centrifuged at 100,000 g for 30 min at 4°C, and the pellets were washed with the lysis buffer and resedimented. The first supernatants (Triton-soluble cellular fraction) and the washed pellets (Triton-insoluble cellular fraction) were prepared as samples for SDS-PAGE or IEF after determination of the protein concentration using Bio-Rad protein assay reagent.
Fluorescence staining and confocal microscopy analysis. Cells were grown on fibronectin-coated glass coverslips and transfected with the indicated plasmid. Twenty-four hours after transfection the cells were serum starved for 24 h and received treatment as indicated. The cells were then washed once with ice-cold PBS and fixed in 3.7% formaldehyde in PBS for 10 min as described earlier (31). For phalloidin staining, 5 U/ml of rhodamine phalloidin was used. For antibody staining, cells on coverslips were incubated with anti-HSP27 monoclonal antibody (SPA-800) at 1:800 dilution for 1 h, washed three times, and then incubated with a 1:200 dilution of TRITC-conjugated anti-mouse secondary antibody (Sigma T2408) with 5 U/ml of Alexa 488-conjugated phalloidin. After a 1-h incubation, the coverslips were washed three times and then mounted onto glass slides with anti-fade mounting medium. Confocal microscopy was performed with a Noran Oz microscope. Image-acquisition settings (laser intensity, iris size, and amplifier gain) were strictly maintained constant for all images taken on the same day to allow quantitative, paired comparisons. Images were stored on rewritable 4.2-GB disks and further processed using Noran Intervision deconvolution software and Adobe Photoshop 4.0. All images were processed identically.
Statistical analysis. The presented results were obtained from several independent experiments on CHO-CCK-A cultures. Quantitative data are given as means ± SE and are analyzed using ANOVA and the Dunnett multiple comparison test. Differences with P < 0.05 were considered to be significant.
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RESULTS |
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Activation of p38 MAP kinase by CCK.
Stimulation of serum-starved CHO-CCK-A cells with various
concentrations of CCK for 10 min induced a dose-dependent increase in
p38 MAP kinase activity (Fig. 1). P38 MAP
kinase activity was increased after stimulation with 1 nM CCK by 100%
(P < 0.05) and maximally increased
with 10 nM and 100 nM CCK, each by 200%
(P < 0.01), which was similar to the
activation seen after stimulation with 10% FBS.
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CCK-induced phosphorylation of HSP27.
We next examined the effects of CCK on the phosphorylation of HSP27 by
IEF followed by Western blotting using a specific antibody. In
serum-starved CHO-CCK-A cells, HSP27 appeared as two bands representing
the unphosphorylated and monophosphorylated isoforms, with the major portion being monophosphorylated (Fig. 2).
Pretreatment for 60 min with 20 µM SB-203580, a specific p38 MAP
kinase inhibitor, had no effect on the phosphorylation pattern.
Treatment with 10 nM or 100 nM CCK led to the appearance of the
diphosphorylated isoform, which then constituted 38 ± 2.6% and
41.7 ± 0.8% of the total HSP27, respectively. The phosphorylation
of HSP27 seen after treatment with CCK for 10 min could be blocked by
pretreatment with SB-203580, indicating that p38 MAP kinase was
responsible for the observed effects. Treatment with alkaline
phosphatase as an internal control to identify the unphosphorylated
form of HSP27 completely dephosphorylated HSP27 (Fig. 2,
lane 1).
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Effects of CCK on the actin
cytoskeleton. To investigate whether CCK affects the
actin cytoskeleton, CHO-CCK-A cells were treated for 30 min with
various concentrations of CCK and were fixed, stained with rhodamine
phalloidin, and examined by confocal fluorescence microscopy. Cells
that were serum starved for 24 h showed a typical fibroblast polygonal
cell shape with predominant actin staining in the cortical membrane
region and a few stress fibers (Fig.
3A).
Addition of 100 pM CCK significantly increased the formation of stress
fibers and filopodia, with maximal effect occurring between 30 and 60 min (Fig. 3B). In contrast, addition of 10 nM CCK for 30 min induced in the majority of the cells a change
of their cell shape, resulting in a more rounded form with very intense
staining of actin under the plasma membrane and fewer stress fibers
(Fig. 3C). Treatment with 100 nM CCK
resulted in formation of clumps or patches of actin filaments (Fig.
3D). These effects were even more
pronounced after 60-120 min.
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Appearance of vinculin. Vinculin is
one of the major components of focal contacts and is thought to be
involved in linking actin filaments to integral membrane proteins (3,
20). In serum-starved CHO-CCK-A cells, vinculin could be localized
predominantly to the cortical membrane and colocalized to the end of
the few stress fibers seen in these cells (Fig.
4, A-C).
Treatment with 100 pM CCK, which induces the formation of stress
fibers, significantly increased the amount of vinculin seen in punctate
spots at the end of the stress fibers (Fig. 4,
D-F).
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Analysis of phosphorylation of human HSP27 expressed
in CHO-CCK-A cells. To further investigate the
potential role of HSP27 and its phosphorylation in modulating the actin
cytoskeleton, human wt-HSP27 or various mutant forms of human HSP27
were transfected into CHO-CCK-A cells. Two different
nonphosphorylatable mutants, 3A and 3G, were used, because it has been
previously shown that they might behave differently in their effect on
cells, with 3A forming large oligomers and 3G not forming them (23).
The 3D mutant was used to imitate the phosphorylated form of HSP27 by inserting the three negatively charged aspartate residues in the serine
phosphorylation sites. With the use of IEF followed by Western blotting
using a specific anti-human HSP27 antibody, the effects of the
different forms of human HSP27 were tested. Figure 5 shows that cells transfected with human
wt-HSP27 contained nearly equal amounts of unphosphorylated and
monophosphorylated isoforms of the human protein (lane
1) and that stimulation with 10 nM CCK resulted in
increased amounts of the diphosphorylated form (lane
2). Cells transfected with the mutant forms 3A and 3G
contained only unphosphorylated HSP27 (lanes
3 and 7), and
treatment with 10 nM CCK for 10 min did not cause any increase in
phosphorylation of the human HSP27 (lanes
4 and 8). Cells
transfected with the 3D form of human HSP27 expressed a protein that
migrated on IEF gels to a position close to the most phosphorylated
isoform of wt-HSP27, and treatment with 10 nM CCK did not change this
pattern (lanes 5 and
6). There was no HSP27 recognized by
the antibody when the vector lacking any human HSP27 cDNA insert was
used for transfection of the CHO-CCK-A cells, indicating the
specificity of the anti-human HSP27 antibody (lane
9).
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Immunocytochemical analysis of HSP27 transfected
CHO-CCK-A cells. CHO-CCK-A cells transfected with the
different human HSP27 constructs were examined using confocal
fluorescence microscopy. In serum-starved CHO-CCK-A cells, phalloidin
staining of transfected and untransfected cells was similar.
Transfection of the cells did not affect their cell shape, and both
untransfected and transfected cells showed few stress fibers and the
typical polygonal cell shape (Fig.
6A).
Figure 6B represents the same optical
field as seen in Fig. 6A, showing the
transfected cells stained with the human HSP27-specific antibody. This
specificity was further proved when cells were labeled only with the
secondary antibody, resulting in no detectable staining signal (not
shown). Figure 6C shows a superimposed
image of Fig. 6, A and
B. Treatment of untransfected cells
with 10 nM CCK for 60 min induced the same changes of the actin
cytoskeleton as described above, with actin fragmentation and cell
rounding (Fig. 6, D-F). Changes of
the cell shape and actin fragmentation was seen in 93% of the cells
examined (Table 1). These
effects were not seen in the cells transfected with the human wt-HSP27
identified by immunohistochemistry with a specific antibody against
human HSP27 (Fig. 6E). None of 20 transfected and examined cells showed signs of actin fragmentation
(Table 1). These data indicate a protective role of wt-HSP27.
Transfection with the mutant forms 3A, 3G, or 3D of human HSP27 did not
affect the actin cytoskeleton in untreated serum-starved CHO-CCK-A
cells (data not shown). Treatment of human 3G-HSP27 transfected cells with 10 nM CCK resulted in the same disruption of the actin
cytoskeleton as seen in untransfected cells (Fig. 6,
G-I and Table 1). Figure 6H shows that 3G-HSP27 transfected
cells round up after CCK treatment. Human 3A-HSP27 transfected cells
behaved in the same manner as the 3G-HSP27 mutant transfected cells,
with no protective effect having been observed (data not shown). In
contrast, cells transfected with the human 3D-HSP27 mutant did not show
signs of actin fragmentation or changes of cell shape after 10 nM CCK
treatment (Fig. 6, J-L and Table 1).
In all cells shown in Fig. 6, I and
L, although HSP27 colocalizes with the
actin cytoskeleton, indicating that HSP27 is partially associated with
the cytoskeleton, only the cells expressing the phosphorylatable 3D
mutant did not show signs of actin fragmentation. These results
indicate that not only the amount of HSP27 is important for stabilizing
the actin cytoskeleton but also this protective effect is
dependent on HSP27 phosphorylation.
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HSP27 distribution in Triton X-100-soluble and
-insoluble fractions. Figure
7, A and
B, shows the results from immunoblots of Triton X-100-soluble and -insoluble fractions of CHO-CCK-A cells
after treatment with 10 nM CCK compared with control. Each experiment
was performed three times. Figure 7C
shows the corresponding densitometric data of Triton X-100-soluble and
-insoluble diphosphorylated HSP27 as percent of total HSP27. Under
control conditions, the distribution of HSP27 between the Triton
X-100-soluble and -insoluble fraction was ~70% and 30%,
respectively. After 30 min of treatment with 10 nM CCK, HSP27
redistributed into the insoluble fraction, mounting to ~55% of total
HSP27, with some 45% left in the soluble fraction. This was
accompanied by an increase in diphosphorylated HSP27 in the insoluble
fraction (Fig. 7, A-C). The
percentage of diphosphorylated HSP27 compared with the total amount of
HSP27 in the detergent-soluble fraction was not significantly higher than in the detergent-insoluble fraction
(P > 0.05).
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Phosphorylation of HSP27 in cells transfected with
human wt-HSP27. To demonstrate that the p38 MAP kinase
pathway mediates phosphorylation of human wt-HSP27, transfected cells
were stimulated with 10 nM CCK for 30 min, with or without SB-203580
pretreatment, and phosphorylation of the human HSP27 was analyzed by
IEF and Western blotting. When serum-starved CHO-CCK-A cells were left untreated or were pretreated with SB-203580, human wt-HSP27 was predominantly in the unphosphorylated form, and little was in the mono-
or diphosphorylated form (Fig.
8A,
lanes 1-2). Stimulation with
CCK resulted in an increase in the diphosphorylated isoform (lane 3), and this was inhibited by
pretreatment with SB-203580 (Fig. 8, lane
4). Data from at least three independent experiments were quantified by densitometry (Fig.
8B).
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Effect of SB-203580 on the actin cytoskeleton of cells
overexpressing wt-HSP27 or 3D-HSP27. To further
demonstrate that not only the amount of HSP27 present in cells but
also phosphorylation of HSP27 is important for regulating the
organization of the actin cytoskeleton, CHO-CCK-A cells overexpressing
human wt-HSP27 or 3D-HSP27 were pretreated with SB-203580 and analyzed
after stimulation with or without CCK. When the cells were left
untreated or treated with SB-203580, the actin cytoskeleton could be
observed as shown in Figs. 3A,
4A,
6A, and
9A. The
predominant cytoplasmic localization of HSP27 is shown in Fig.
9B. The protective effect of
overexpressing human wt-HSP27 against damage caused by stimulation with
CCK was totally nullified when these cells were pretreated with the p38 inhibitor SB-203580 (Fig. 9, C and
D). In contrast, when 3D-HSP27 was
overexpressed and cells were stimulated with CCK, SB-203580 had no
inhibitory function of the protective effect seen in Fig. 9C indicating that phosphorylation of
HSP27 stabilizes the actin cytoskeleton (Fig. 9,
E and F).
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DISCUSSION |
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Several modes of action for the small HSPs, including HSP27, have been proposed during recent years. These actions include chaperone activity, protection against apoptosis, protease inhibition, and cross-linkage to other proteins (1, 25). However, much evidence suggests that HSP27 plays a major role in regulating the organization of the actin cytoskeleton under both control and stress conditions (1, 2, 11, 16-18).
We recently reported that treatment of isolated rat pancreatic acini with CCK activates the p38 MAP kinase cascade, including MAPKAP kinase 2, which results in an increase in HSP27 phosphorylation (7, 34). Furthermore, we showed that CCK at supraphysiological concentrations induces changes of the actin cytoskeleton, which could be partially blocked by inhibiting p38 MAP kinase activity with SB-203580 (34). We now have evaluated the effects of CCK on the actin cytoskeleton in CHO-CCK-A cells, which overexpress human wt-HSP27 or various mutant forms of human HSP27. First, we demonstrate that CCK activates p38 MAP kinase dose dependently in CHO-CCK-A cells. Second, treatment with 10 nM CCK increased the phosphorylation of HSP27. Preincubation with SB-203580 blocked the increase of the diphosphorylated isoform of HSP27 seen after CCK treatment, indicating that the p38 MAP kinase pathway is responsible for phosphorylation of HSP27. The lack of effect of SB-203580 on the monophosphorylated isoform could be due to a longer half-life of the monophosphorylated isoform after its phosphorylation relative to the diphosphorylated isoform. This is consistent with our data obtained in rat pancreatic acini, where SB-203580 had no effect on MAPKAP kinase 1 (RSK-90) or p70 S6 kinase (34).
Next, we examined the effects of CCK on the actin cytoskeleton as seen by labeling with rhodamine phalloidin and analyzed by confocal fluorescence microscopy. CCK, in both physiological and supraphysiological concentrations, induced, in as little as 1-3 min, changes in actin cytoskeleton, seen as membrane ruffles (data not shown here). At later time points (30 min), the effects seen after treatment with physiological concentrations of CCK were different from the effects seen with a high dose of CCK. After treatment with high concentrations of CCK, there were clear signs of disruption of the actin cytoskeleton (Fig. 3, B-D).
It has been reported that growth hormone induces reorganization of the actin cytoskeleton, including a rapid actin depolymerization seen after 30 s in CHO cells expressing growth hormone receptors followed by a slow repolymerization of actin stress fibers (6). Another study reported stress fiber formation after treatment with CCK-8 in mouse Swiss 3T3 and NIH/3T3 fibroblasts expressing the human CCK-B receptor (36). In this study the effects were seen as early as 1 min with maximal stress fiber formation after 30 min, similar to our observations. The concentration of CCK used in that report was 10 nM, which is in contrast to our data, which show that 10 nM CCK causes disruption of the actin cytoskeleton. It is possible that CCK acts differently in binding to the CCK-B receptor, whereas our data were obtained in CHO cells transfected with the CCK-A receptor.
To further demonstrate that CCK affects the cytoskeleton, we examined the effects of CCK on vinculin. Vinculin and talin are major components of focal contacts in the cytoskeleton of animal cells (3, 20). Our data clearly show that treatment with 100 pM CCK for 30 min not only induces stress fiber formation but also induces appearance of vinculin in focal contacts (Fig. 4, A-F).
It has been shown before that, in various cell types, including fibroblasts, oxidative stress produces a severe disruption of the microfilament cytoskeleton characterized by the fragmentation and patching of F-actin (11). We now report that CCK, a gastrointestinal hormone, which acts via a Gq-coupled receptor, in high concentration shows similar effects on the actin cytoskeleton. These effects are different from the effects seen in HUVECs, which contain a high amount of endogenous HSP27, where oxidative stress induced accumulation of stress fibers, recruitment of vinculin to focal adhesions, and the loss of membrane ruffles (10). To further evaluate whether amount and phosphorylation of HSP27 play an important role in regulating the organization of the actin cytoskeleton, we transfected CHO-CCK-A cells to express either wt-human HSP27 or mutant forms of HSP27. Expression of wt-HSP27 clearly showed a protective effect. No signs of actin fragmentation, patching of F-actin, or change in cell shape as observed in untransfected CHO-CCK-A cells were seen (Fig. 6, D-F). The same protective effects were seen when the 3D mutant of human HSP27, which mimics the phosphorylated state, was used for transfection (Fig. 6, J-L, and Fig. 9, E and F). In contrast, when cells were transfected to express nonphosphorylatable mutants (3A or 3G) of human HSP27, there was no protective effect seen (data for 3G shown in Fig. 6, G-I; data not shown for 3A). Recently, it was demonstrated in vitro that HSP27 has an actin-barbed end-capping activity (27), which depends on the phosphorylation status of HSP27 (16), with the unphosphorylated monomeric or small oligomeric form being the active fraction (2). It was demonstrated that heat shock in CHO cells increased the amount of endogenous HSP27, human wt-HSP27, and a nonphosphorylatable mutant of human HSP27 recovered in the detergent-insoluble fraction by 30-40% (19). The authors concluded that HSP27 insolubilization following heat shock is not mediated by phosphorylation. Another study showed that ~30% of cellular endogenous and transfected HSP27 fractionates with plasma membrane components and that a nonphosphorylatable HSP27 mutant partitioned with that fraction to the same extent as transfected wt-HSP27 and endogenous HSP27 after phorbol ester treatment, indicating that phosphorylation is not necessary for the association with membrane components (32). In contrast to the first study (19), where heat shock increased the amount of HSP27 found in the detergent-insoluble pool, these authors did not observe such an effect after treatment of bovine arterial endothelial cells with phorbol ester, suggesting that these results depend on the cell type and amount of HSP27. In our study, we demonstrate that HSP27 redistributes from the Triton X-100-soluble to the -insoluble fraction after CCK treatment, which is accompanied by increased amounts of diphosphorylated HSP27 in the insoluble fraction. The fact that HSP27 and its diphosphorylated isoform can be detected in nearly equal amounts in the detergent-soluble and -insoluble pool after stimulation with CCK may indicate that diphosphorylated HSP27 is not specifically recruited to the cytoskeleton and that the protective functions of HSP27 are multiple. Nevertheless, CCK induces redistribution of HSP27, and further studies are needed to clarify the role of amount and phosphorylation of HSP27 associated with the detergent-insoluble pool. Because of the relatively low amounts of endogenous HSP27 in CHO cells, the endogenous phosphorylated HSP27 probably is not sufficient to stabilize the actin cytoskeleton when stimulated with high concentrations of CCK. The results shown in Fig. 6 support the hypothesis that not only the amount of HSP27 but also phosphorylation of HSP27 is important in regulating the organization of the actin cytoskeleton. Consistent with this hypothesis were our results showing that preincubation with SB-203580 inhibited the increased phosphorylation of HSP27 seen after CCK treatment of human wt-HSP27-expressing cells (Fig. 8) and blocked the protective effect of overexpression of human wt-HSP27 (Fig. 9, C and D) but not the protective effect of overexpression of human 3D-HSP27 (Fig. 9, E and F ). These data are also consistent with other reports on regulation of actin by the p38 MAP kinase/HSP27 pathway (8, 10, 11). To ensure that these effects are not part of activation of the ERKs, cells were pretreated with PD-098059 and with or without 10 nM CCK. PD-098059 alone or the combination with CCK did not affect the actin cytoskeleton (data not shown).
Although CCK activates p38 MAP kinase in physiological concentrations in both CHO-CCK-A cells and isolated rat pancreatic acinar cells (34), the importance of that signaling pathway might depend on the level of expression of HSP27. It is believed that an intact cytoskeleton is necessary for regulated secretion in pancreatic acini as well as in other secretory cell types (28). As polarized cells, pancreatic acini possess a dense net of filamentous actin near the lumen at the apical site where secretion of the zymogen granules occurs. We and others have previously shown that high doses of CCK led to remarkable changes of this actin cytoskeleton in pancreatic acini in vitro (30, 34). Additionally, there is further evidence that HSP27 may play an important role in protecting the actin cytoskeleton using a new rat model of acute necrotizing pancreatitis (unpublished data).
It should be mentioned that, besides HSP27, further members of the family of stress proteins apparently interact with the microfilaments and even may protect them, which complicates our understanding of how the organization of these structures is regulated. For example, B-crystallin (a protein sharing sequence similarity to HSP27) was found to be associated with microfilaments in cardiomyocytes (22) and cultured eye lens cells (4). In human colonic epithelial Caco2/bbe cells, HSP72 can protect the barrier function against an oxidant-induced injury, largely by protecting the integrity of the actin cytoskeleton (29). However, so far it is unknown if and how stress proteins other than HSP27 are involved in CCK-mediated rearrangements of microfilaments as described in this study.
In conclusion, we have shown that CCK treatment of CHO-CCK-A cells induces major changes in microfilament organization, from increased formation of stress fibers to breakdown of the actin cytoskeleton. Our results provide evidence that HSP27 is an effector downstream of p38 MAP kinase and that this signaling cascade regulates actin filament dynamics. These insights into pathways regulating microfilament dynamics should contribute to investigations designed to better understand different models of acute pancreatitis and to help to identify new treatment strategies. As a first step, we are currently in the process of doing studies with transgenic animals, overexpressing wt-HSP27 or mutant HSP27 in pancreas.
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ACKNOWLEDGEMENTS |
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We thank T. Komorowski for assistance with confocal microscopy and Lee A. Weber (University of Nevada) for human HSP27 cDNA pBluescript plasmids.
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FOOTNOTES |
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This work was supported by Deutsche Forschungsgemeinschaft Grant SCHA 7661/-1 (C. Schäfer) and by National Institutes of Health Grants DK-52860 (J. A. Williams), ES-06265 and ES-07006 (M. J. Welsh), DK-34933 (to the Michigan Gastrointestinal Peptide Center), and DK-20572 (to the Michigan Diabetes Research and Training Center).
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. §1734 solely to indicate this fact.
Address for reprint requests: J. A. Williams, 7744 Medical Science II, Dept. of Physiology, Univ. of Michigan 48109-0622.
Address for correspondence: C. Schäfer, Dept. of Gastroenterology, Philipps-Universität Marburg, 35033 Marburg, Germany.
Received 26 April 1999; accepted in final form 9 August 1999.
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REFERENCES |
---|
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---|
1.
Arrigo, A.-P.,
and
J. Landry.
Expression and function of the low-molecular-weight heat shock proteins.
In: The Biology of Heat Shock Proteins and Molecular Chaperones, edited by R. I. Morimoto,
A. Tissieres,
and C. Georgopoulos. New York: Cold Spring Harbor Laboratory, 1994, p. 335-374.
2.
Benndorf, R.,
K. Hayess,
S. Ryazantsev,
M. Wieske,
J. Behlke,
and
G. Lusch.
Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity.
J. Biol. Chem.
269:
20780-20784,
1994
3.
Burridge, K.,
K. Fath,
T. Kelly,
G. Nuckolls,
and
C. Turner.
Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton.
Annu. Rev. Cell Biol.
4:
487-525,
1988.
4.
Del Vecchio, P. J.,
K. S. MacElroy,
M. P. Rosser,
and
R. L. Church.
Association of -crystallin with actin in cultured lens cells.
Curr. Eye Res.
3:
1213-1219,
1984[Medline].
5.
Gething, M. J.,
and
J. Sambrook.
Protein folding in the cell.
Nature
355:
33-45,
1992[Medline].
6.
Goh, E. L. K.,
T. J. Pircher,
T. J. J. Wood,
G. Norstedt,
R. Graichen,
and
P. E. Lobie.
Growth hormone-induced reorganization of the actin cytoskeleton is not required for STAT5 (signal transducer and activator of transcription-5)-mediated transcription.
Endocrinology
138:
3207-3215,
1997
7.
Groblewski, G. E.,
T. Grady,
N. Mehta,
H. Lambert,
C. D. Logsdon,
J. Landry,
and
J. A. Williams.
Cholecystokinin stimulates heat shock protein 27 phosphorylation in rat pancreas both in vivo and in vitro.
Gastroenterology
112:
1354-1361,
1997[Medline].
8.
Guay, J.,
H. Lambert,
G. Gingras-Breton,
J. N. Lavoie,
J. Huot,
and
J. Landry.
Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.
J. Cell Sci.
110:
357-368,
1997
9.
Hickey, E.,
S. E. Brandon,
R. Potter,
G. Stein,
J. Stein,
and
L. A. Weber.
Sequence and organization of genes encoding the human 27 kDa heat shock protein.
Nucleic Acids Res.
14:
4127-4145,
1986[Abstract].
10.
Huot, J.,
F. Houle,
F. Marceau,
and
J. Landry.
Oxidative stress-induced reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells.
Circ. Res.
80:
383-392,
1997
11.
Huot, J.,
F. Houle,
D. R. Spitz,
and
J. Landry.
HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress.
Cancer Res.
56:
273-279,
1996[Abstract].
12.
Huot, J.,
G. Roy,
H. Lambert,
P. Chretien,
and
J. Landry.
Increased survival after treatments with anticancer agents of Chinese hamster cells expressing the human Mr 27,000 heat-shock protein.
Cancer Res.
51:
5245-5252,
1991[Abstract].
13.
Jakob, U.,
M. Gaestel,
K. Engel,
and
J. Buchner.
Small heat shock proteins are molecular chaperones.
J. Biol. Chem.
268:
1517-1520,
1993
14.
Knauf, U.,
U. Jakob,
K. Engel,
J. Buchner,
and
M. Gaestel.
Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance.
EMBO J.
13:
54-60,
1994[Abstract].
15.
Landry, J.,
P. Chretien,
H. Lambert,
E. Hickey,
and
L. A. Weber.
Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells.
J. Cell Biol.
109:
7-15,
1989[Abstract].
16.
Landry, J.,
and
J. Huot.
Modulation of actin dynamics during stress and physiological stimulation by a signaling pathway involving p38 MAP kinase and heat-shock protein 27.
Biochem. Cell Biol.
73:
703-707,
1995[Medline].
17.
Lavoie, J. N.,
G. Gingras-Breton,
R. M. Tanguay,
and
J. Landry.
Induction of Chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock. HSP27 stabilization of the microfilament organization.
J. Biol. Chem.
268:
3420-3429,
1993
18.
Lavoie, J. N.,
H. Lambert,
E. Hickey,
L. A. Weber,
and
J. Landry.
Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27.
J. Biol. Chem.
268:
24210-24214,
1993
19.
Lavoie, J. N.,
H. Lambert,
E. Hickey,
L. A. Weber,
and
J. Landry.
Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27.
Mol. Cell. Biol.
15:
505-516,
1995[Abstract].
20.
Lee, S.-W.,
and
J. J. Otto.
Vinculin and talin: kinetics of entry and exit from the cytoskeletal pool.
Cell Motil. Cytoskeleton
36:
101-111,
1997[Medline].
21.
Loktionova, S. A.,
and
A. E. Kabakov.
Protein phosphatase inhibitors and heat preconditioning prevent Hsp27 dephosphorylation, F-actin disruption and deterioration of morphology in ATP-depleted endothelial cells.
FEBS Lett.
433:
294-300,
1998[Medline].
22.
Lutsch, G.,
R. Vetter,
U. Offhaus,
M. Wieske,
H. J. Grone,
R. Klemenz,
I. Schimke,
J. Stahl,
and
R. Benndorf.
Abundance and location of the small heat shock proteins HSP25 and B-crystallin in rat and human heart.
Circulation
96:
3466-3476,
1997
23.
Mehlen, P.,
E. Hickey,
L. A. Weber,
and
A. P. Arrigo.
Large unphosphorylated aggregates as the active forms of hsp27 which controls intracellular reactive oxygen species and glutathione levels and generates a protection against TNF in NIH-3T3-ras cells.
Biochem. Biophys. Res. Commun.
241:
187-192,
1997[Medline].
24.
Mehlen, P.,
X. Preville,
P. Chareyron,
J. Briolay,
R. Klemenz,
and
A. P. Arrigo.
Constitutive expression of human hsp27, Drosophila hsp27, or human B-crystallin confers resistance to TNF- and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts.
J. Immunol.
154:
363-374,
1995
25.
Mehlen, P.,
K. Schulze-Osthoff,
and
A. P. Arrigo.
Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1 and staurosporine-induced cell death.
J. Biol. Chem.
271:
16510-16514,
1996
26.
Miron, T.,
K. Vancompernolle,
J. Vandekerckhove,
M. Wilchek,
and
B. Geiger.
A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein.
J. Cell Biol.
114:
255-261,
1991[Abstract].
27.
Miron, T.,
M. Wilchek,
and
B. Geiger.
Characterization of an inhibitor of actin polymerization in vinculin-rich fraction of turkey gizzard smooth muscle.
Eur. J. Biochem.
178:
543-553,
1988[Abstract].
28.
Muallem, S.,
K. Kwiatkowska,
X. Xu,
and
H. L. Yin.
Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells.
J. Cell Biol.
128:
589-598,
1995[Abstract].
29.
Musch, M. W.,
K. Sugi,
D. Strauss,
and
E. B. Chang.
Heat-shock protein 72 protects against oxidant-induced injury of barrier function of human colonic epithelial Caco2/bbe cells.
Gastroenterology
117:
115-122,
1999[Medline].
30.
O'Konski, M. S.,
and
S. J. Pandol.
Cholecystokinin JMV-180 and caerulein effects on the pancreatic acinar cell cytoskeleton.
Pancreas
8:
638-646,
1993[Medline].
31.
Piotrowicz, R. S.,
E. Hickey,
and
E. G. Levin.
Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration.
FASEB J.
12:
1481-1490,
1998
32.
Piotrowicz, R. S.,
and
E. G. Levin.
Basolateral membrane-associated 27-kDa heat shock protein and microfilament polymerization.
J. Biol. Chem.
272:
25920-25927,
1998
33.
Sakamoto, K.,
T. Urushidani,
and
T. Nagao.
Translocation of Hsp27 to cytoskeleton by repetitive hypoxia-reoxygenation in the rat myoblast cell line, H9c2.
Biochem. Biophys. Res. Commun.
251:
576-579,
1998[Medline].
34.
Schäfer, C.,
S. E. Ross,
M. J. Bragado,
G. E. Groblewski,
S. A. Ernst,
and
J. A. Williams.
A role for the p38 mitogen-activated protein kinase/Hsp27 pathway in cholecystokinin-induced changes in the actin cytoskeleton in rat pancreatic acini.
J. Biol. Chem.
273:
24173-24180,
1998
35.
Stokoe, D.,
K. Engel,
D. G. Campbell,
P. Cohen,
and
M. Gaestel.
Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small heat shock proteins.
FEBS Lett.
313:
307-313,
1992[Medline].
36.
Taniguchi, T.,
K. Takaishi,
T. Murayam,
M. Ito,
N. Iwata,
K. Chihara,
T. Sasaki,
Y. Takai,
and
T. Matsui.
Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers.
Oncogene
12:
1357-1360,
1996[Medline].
37.
Yule, D. I.,
M.-J. Tseng,
J. A. Williams,
and
C. D. Logsdon.
A cloned CCK-A receptor transduces multiple signals in response to full and partial agonists.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G999-G1004,
1993