1 Experimental Pathology, Department of Laboratory Medicine, Lund University,
Malmö University Hospital, SE-205 02 Malmö, Sweden
2 Molecular Medicine, Department of Laboratory Medicine, Lund University,
Malmö University Hospital, SE-205 02 Malmö, Sweden
* Author for correspondence (e-mail: anita.sjolander{at}exppat.mas.lu.se)
Accepted 27 June 2002
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Summary |
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Key words: LTD4, CNF-1, Stress fibres, RhoA, PKC-
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Introduction |
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Activation of the LTD4 receptor CysLT1 triggers a
cascade of intracellular signalling events, including activation of the
serine/threonine protein kinase C (PKC)
(Thodeti et al., 2001;
Vegesna et al., 1988
). PKC can
be classified into three major subgroups: the classical PKCs (PKC
,
ßI, ßII and
), which are Ca2+/diacylglycerol
dependent; the novel PKCs (PKC
,
,
and
). which are
Ca2+ independent but require diacylglycerol for activation; and the
atypical PKCs
and
, which are neither Ca2+ nor
diacylglycerol dependent (Ron and
Kazanietz, 1999
). PKC isoforms are expressed to varying degrees in
all mammalian cells (Martiny-Baron et al.,
1993
; Mellor and Parker,
1998
; Nishizuka,
1992
) and have been linked to changes in the cytoskeleton in many
different cell types (for a review, see
Keenan and Kelleher, 1998
). If
a PKC isoform(s) is involved in the LTD4-induced changes of the
actin cytoskeleton in intestinal cells, it has to be one of the
,
ßII,
or
isoforms, since these are the ones activated by
this LT (Thodeti et al.,
2001
).
RhoA belongs to the family of Rho GTPases, which are key regulators of the
actin cytoskeleton in many types of cells
(Hall, 1998) and function as
molecular switches by alternating between a GDP-bound inactive form and a
GTP-bound active form (Hall,
1998
). There are at least 14 Rho GTPases, which share more than
50% sequence identity, and the most extensively studied members of this family
are RhoA, Cdc42 and Rac1 (Hall,
1998
). RhoA regulates the formation of stress fibres and focal
adhesions, whereas Cdc42 and Rac1 are believed to stimulate the formation of
filopodia and lamellipodia, respectively
(Ridley and Hall, 1992
). The
C3 exoenzyme from Clostridium botulinum and cytotoxic necrotising
factor-1 (CNF-1) from Escherichia coli are important tools for
studying the regulation of the actin cytoskeleton by their ability to regulate
the Rho protein. Treatment with C3 exoenzyme, which is an
ADP-ribosyltransferase that catalyses mono-ADP ribosylation of RhoA at Asp41,
results in inhibition of RhoA and selective disorganisation of actin stress
fibres (Chardin et al., 1989
);
exposure to CNF-1 induces the opposite effects. CNF-1 exhibits catalytic
deamidase activity specific to Rho Gln63. Modification of Gln63 to Asp63 in
Rho blocks the intrinsic and GAP-stimulated hydrolysis of GTP, resulting in
permanent activation of the GTP-binding protein and thereby provoking
prominent bundling of actin stress fibres
(Flatau et al., 1997
;
Schmidt et al., 1997
).
In the present study, we show that LTD4 causes stress-fibre
production via activation of PKC and a subsequent downstream activation
of RhoA. To our knowledge, this is the first study to show that RhoA is
involved in rearrangement of the actin cytoskeleton through a downstream
effect on PKC
.
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Materials and Methods |
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Cell culture
The intestinal epithelial cell line Intestine 407
(Henle and Deinhardt, 1957)
was cultured as a monolayer in 75 cm2 flasks in Eagle's basal
medium supplemented with 15% new-born calf serum, 55 µg/ml streptomycin and
55 IU/ml penicillin. Cell cultures were kept at 37°C in a humidified
environment of 5% CO2 and 95% air. The cells, which exhibit typical
epithelial morphology and growth, were regularly tested to ensure the absence
of mycoplasma contamination.
Cell fractionation, gel electrophoresis and immunoblotting
The cells were serum-starved for 12 hours and then stimulated with 40 nM
LTD4 in the absence or presence of a mixture of 5 µg/ml
lipofectamine and 5 µg/ml C3 exoenzyme for 10 hours as described elsewhere
(Borbiev et al., 2000). The
stimulation was terminated by adding ice-cold buffer A containing 20 mM
NaHepes (pH 8), 2 mM MgCl2 1 mM EDTA, 2 mM
Na3VO4, 4 µg/ml leupeptin and 30 µg/ml PMSF.
Thereafter, the cells were scraped into the cold buffer, homogenised 10 times
on ice in a glass tissue grinder (Dounce) and then centrifuged at 200
g for 10 minutes. The protein content of the supernatant was
measured and compensated for, and the supernatant was subsequently centrifuged
at 1000 g for 5 minutes. The supernatant of the 1000
g fraction was further centrifuged at 200,000
g for 30 minutes. The resulting membrane-rich pellet was
suspended in 150 µl of buffer A, and boiled in SDS-sample buffer for 10
minutes and loaded onto 8% polyacrylamide gels. The separated proteins were
transferred to PVDF membranes, which were blocked in 3% BSA/PBS and incubated
for 1 hour at 25°C in a 1:500 dilution of PKC
,
or
antibodies in PBS with 3% BSA. The membranes were probed with a 1:5000
dilution of a secondary antibody coupled to horseradish peroxidase for 1 hour
at 25°C and thereafter developed with an ECL kit (Amersham).
Confocal microscopy
The cells were seeded onto glass coverslips and grown for five days. These
cells were serum starved for 12 hours and then stimulated with either 40 nM
LTD4 for 5 minutes or 100 nM TPA for 15 minutes in a tissue culture
incubator at 37°C; this was done with or without pre-incubation with 2
µM GF109203X or 2 µM Gö6976 for 15 minutes, 300 ng/ml CNF-1 for 16
hours or a mixture of 5 µg/ml lipofectamine and 5 µg/ml C3 exoenzyme for
10 hours. The stimulations were terminated by fixing the cells for 10 minutes
at room temperature in 3.7% paraformaldehyde/PBS solution, after which the
cells were permeabilised in 0.5% Triton X-100/PBS solution for 5 minutes. The
coverslips were subsequently washed twice in PBS and incubated at room
temperature in 3% BSA/PBS solution for 15 minutes. The cells were then stained
for 30 minutes with Alexa 488 phalloidin or Alexa 546 phalloidin (5 µg/ml).
Thereafter, the coverslips were washed six times in PBS and mounted in a
fluorescent mounting medium (DAKO A/S). Confocal images were recorded using a
Bio-Rad Radiance 2000 confocal laser scanning system with a Nikon microscope
(model TE300) equipped with a 60x1.4 Plan-APOCHROMAT oil immersion
objective.
Construction and transfection of EGFP-RhoA and EGFP-RD-PKC in Int 407
cells
L63-RhoA and N19-RhoA cDNAs were originally obtained from Alan Hall (MRC
Laboratory, London) and cloned into the pEGFP-N1 vector (Clontech
Laboratories, Inc.). Expression vectors encoded the regulatory domain or
PKC, ßII,
,
or full-length PKC
EGFP-fusion
proteins (Zeidman et al.,
1999
). Transient transfections of the cells were performed using
5.0 µg/ml Lipofectin (GIBCO) and 1.8 µg of plasmid DNA/ml in serum-free
medium, essentially according to the protocol provided by the supplier.
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Results |
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|
|
The PKC inhibitor GF109203X blocks LTD4- and TPA-induced
stress-fibre formation
Phorbol esters such as 12-O tetradecanoylphorbol 13 acetate (TPA) can
directly stimulate classic and novel PKCs, and they stimulate microfilament
remodelling in most cells. Fig.
3 shows generation of stress fibres induced in intestinal
epithelial cells by 100 nM TPA (15 minutes), and the pattern in this case is
similar to that seen in such cells treated with 40 nM LTD4 for 5
minutes. We have previously established that LTD4 activates
PKC, ßII,
and
in these cells
(Thodeti et al., 2001
). In the
present experiments, we used the PKC inhibitors bisindoylmaleimide GF109203X
and Gö6976 to evaluate the contribution of different PKC isoforms to
LTD4-induced reorganisation of F-actin in these cells. GF109203X is
an inhibitor of both classic and novel PKC isoforms, whereas Gö6976 is a
potent inhibitor of classic PKC isoforms
(Davies et al., 2000
;
Martiny-Baron et al., 1993
).
Pretreatment of the cells with 2 µM GF109203X for 15 minutes, before
stimulation with either 40 nM LTD4 for 5 minutes or 100 nM TPA for
15 minutes, inhibited stressfibre formation
(Fig. 3). By contrast,
identical pretreatment with 2 µM Gö6976 did not cause such inhibition
(Fig. 3). These results suggest
that a novel PKC isoform is involved in the LTD4-induced
rearrangement of the cytoskeleton in epithelial cells.
|
PKC is involved in the stress-fibre formation induced by
LTD4
To further investigate the identity of the PKC isoform(s) involved in
LTD4-induced stress-fibre formation, we transfected cells with
vectors encoding the regulatory domain (RD) of PKC, ßII,
or
fused to EGFP. These RDs have previously been documented to act as
specific dominant-negative inhibitors of their respective PKC isoform
(Cai et al., 1997
;
Jaken, 1996
;
Kiley et al., 1999
;
Paruchuri et al., 2002
). The
results obtained with cells transfected with vectors encoding these RDs
revealed that the LTD4-induced formation of stress fibres was
inhibited by the RD of PKC
but was not affected by the RD of
PKC
, ßII or
(Fig.
4).
|
The activation of PKC occurs downstream of RhoA
Considering the above results, we addressed the question of whether the
LTD4-induced activation of PKC- occurs upstream or
downstream of RhoA. We treated cells with LTD4 in the presence or
absence of the Rho inhibitor C3 exoenzyme and then measured activation of
PKC
,
, and
by measuring their translocation to the
membrane fraction. As outlined in Fig.
5, these three PKC isoforms were rapidly translocated to the
membrane fraction upon exposure to 40 nM LTD4, and the maximum
effect of the translocation was seen after 90-300 seconds. However, in the
presence of C3 exoenzyme, the translocation to the plasma membrane induced by
LTD4 treatment was reduced for PKC
and
and was
totally inhibited for PKC
(Fig.
5). In additional experiments, we transfected the cells with
N19-RhoA (the dominant-negative of RhoA) or the RD of PKC
and directly
stimulated either PKC with TPA or RhoA with CNF-1
(Fig. 6A,B). Cells transfected
with EGFP-N19-RhoA produced stress fibres when exposed to 100 nM TPA for 15
minutes. By contrast, when cells were transfected with the RD of PKC
,
no tendency towards generation of stress fibres was seen after treatment with
CNF-1 (Fig. 6B). To confirm
these findings, we used another approach to inhibit PKC
. The cells were
stimulated with CNF-1 for 16 hours in the absence or presence of 2 µM
GF109203X, and, in agreement, the formation of stress fibres was completely
inhibited by GF109203X (Fig.
6C). Together, these results show that activation of PKC
is
needed for stress-fibre formation and that it occurs downstream of RhoA.
|
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Discussion |
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LTD4 can activate different PKC isoforms in Int 407 cells,
including PKC, ßII,
and
(Thodeti et al., 2001
). In
addition, we have previously shown that activation of PKC
in
LTD4-treated cells is involved in the formation of focal adhesions
and activation of ß1 integrins
(Massoumi and Sjölander,
2001
). In our present investigation, we used two different PKC
inhibitors, one for classic isoforms (Gö6976) that does not inhibit the
LTD4- or TPA-induced production of stress fibres and one for
classic and novel isoforms (GF109203X) that completely inhibits the effects of
both LTD4 and TPA. In addition, we also transfected the cells with
vectors encoding the RD of different PKC isoforms to specifically inhibit them
(Jaken, 1996
) and thereby
study the involvement of these enzymes in the LTD4-induced
production of stress fibres. Expression of RD-PKC
in intestinal cells
provided compelling evidence that activation of PKC
is required for
LTD4-induced stress-fibre formation. It has previously been shown
that PKC activation induces or enhances the formation of stress fibres in
Chinese hamster ovary cells and fibroblasts
(Defilippi et al., 1997
;
Woods and Couchman, 1992
) but
not in Swiss 3T3 fibroblasts (Ridley and
Hall, 1994
). These results and our present observations indicate
that PKC activation has cell-type-specific effects on the generation of stress
fibres. These specific effects may be caused by altered PKC expression or
dissimilarities in PKC-mediated signalling pathways among different cell
types. Taken together, our findings suggest that the novel PKC isoform
PKC
is important for actin reorganisation into stress fibres in
LTD4-stimulated Int 407 cells. In contrast to our data, Brandt and
co-workers have recently shown that prolonged TPA-induced activation of PKC
leads to a disassembly of actin stress fibres after 2 hours by decreasing the
activity of RhoA in the rat aortic smooth muscle cell line A7r5 cells
(Brandt et al., 2002
). It
should be pointed out that the cell type used in this study had a well
developed actin stress-fibre network that crossed the whole cell body in the
resting state, that is, prior to stimulation TPA. The difference between our
findings could therefore be ascribed to the cell types used and/or the time of
agonist exposure.
We performed several different kinds of analysis to explore a possible
relationship between the roles of RhoA and PKC in
LTD4-induced stress-fibre formation. First, we could clearly
document that the LTD4-induced translocation of PKC
to a
membrane fraction was blocked by pretreating the cells with the RhoA inhibitor
C3 exoenzyme. Thereafter, we examined whether direct activation of PKC
was sufficient to produce stress fibres when RhoA is inhibited. The results in
this case showed that such activation induced by TPA did indeed cause
stress-fibre formation in intestinal epithelial cells, even after transfection
with dominant-negative RhoA (N19-RhoA). In another attempt to understand the
interrelationship between RhoA and PKC
, we activated RhoA directly by
using CNF-1 and inhibited PKC
by either transfecting the cells with the
regulatory domain of PKC
or pre-incubating the cells with the PKC
inhibitor GF109203X. The results revealed that cells with CNF-1-activated RhoA
could not produce stress fibres when PKC-
was inhibited. Together,
these data indicate that PKC
and RhoA are located on the same
LTD4-triggered signalling pathway and that involvement of
PKC
in the present production of stress fibres occurs downstream of
RhoA activation. This idea is compatible with our previous observation that
inhibition of RhoA impairs the LTD4-induced and PLC-dependent
mobilisation of intracellular Ca2+
(Grönroos et al., 1996
),
since activation of PLC also leads to the formation of diacylglycerol, an
endogenous activator of PKC. In contrast to our data, Strassheim and
co-workers showed that RhoA and the classic PKCs are involved in separate
signalling pathways that regulate the organisation of myosin and stress
fibres, and, according to their model of M3 receptor activation,
PKC-induced actin reorganisation does not depend on RhoA activation in a
CHO-K1 cell line (Strassheim et al.,
1999
). However, several studies have demonstrated a convergence
between PKC- and Rho GTPase-regulated signalling pathways
(Coghlan et al., 2000
;
Hippenstiel et al., 1998
;
Nozu et al., 1999
). More
specifically, Slater and co-workers found that RhoA acts in close association
with PKC
in an in vitro assay
(Slater, 2001
). A direct
interaction between Rho and PKC has also been observed in a study of yeast
extracts in vitro, in which Pkc1 co-immunoprecipitated with Rho1 in a
GTP-dependent manner (Kamada et al.,
1996
), perhaps even indicating that Rho1 is located upstream of
Pkc1. Interestingly, LTD4 causes cytoskeleton rearrangements in
intestinal epithelial cells by activation of at least two different PKC
isoforms. Previous results have shown that LTD4-induced activation
of PKC
affects the formation of focal adhesions and regulation of
ß1 integrins in intestinal epithelial cells
(Massoumi and Sjölander,
2001
), whereas the present data show that PKC
has a
specific role in the formation of stress fibres in these cells. Apart from
this difference in the dependency on specific PKC isoforms, the formation of
stress fibres is a more rapid event, seen already after 5 minutes, in
comparison with the formation of focal adhesions, which require 15 minutes
exposure to LTD4. This difference could be explained by the present
observation (Fig. 5) that
LTD4 causes a more rapid activation of PKC
than PKC
does.
We cannot yet define how PKC participates in the cytoskeletal
rearrangements caused by LTD4, but interestingly enough Lopez-Lluch
and colleaguesrecently reported that the C2-like module, which is located in
the N-terminal of PKC-
, interacts with G-actin in migrating neutrophils
and co-localises with F-actin in TPA-stimulated cells
(Lopez-Lluch et al., 2001
). In
order to clarify whether such an event is part of the mechanism whereby
PKC-
participates in LTD4-induced stress-fibre formation or
if PKC-induced phosphorylation of myosin light chain II is involved, as shown
for TPA (Masuo et al., 1994
),
additional experiments have to be performed.
Our results show that activation of the LTD4 receptor
CysLT1 alters the organisation of the actin network by an extensive
production of stress fibres in intestinal epithelial cells. This effect is
mediated by activation of RhoA and a subsequent downstream activation of the
novel PKC isoform PKC, both of which are essential for this formation
of stress fibres.
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Acknowledgments |
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