1 Division of Experimental Pathology, Department of Laboratory Medicine, Lund
University, University Hospital Malmö, SE-205 02 Malmö
2 Department of Cell and Molecular Biology, University of Umeå, SE-901 87
Umeå, Sweden
3 Division of Molecular Medicine, Department of Laboratory Medicine, Lund
University, University Hospital Malmö, SE-205 02 Malmö
* Author for correspondence (e-mail: anita.sjolander{at}exppat.mas.lu.se )
Accepted 30 January 2002
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Summary |
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Key words: Leukotriene D4, Ras, Protein kinase C, Raf-1, MAPK, Intestinal epithelial cell proliferation
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Introduction |
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Furthermore, ulcerative colitis is associated with an increased incidence
of neoplastic transformation (Ekbom et
al., 1990), and several studies have shown that colon cancer is
under-represented in populations treated with non-steroidal anti-inflammatory
drugs (Smalley and DuBois,
1997
). A possible link between inflammation and the occurrence of
cancer has been suggested (Sheng et al.,
1997
). To determine whether LTD4 is involved in the
coupling between inflammatory bowel conditions and an increased risk of
cancer, we have previously exposed non-transformed intestinal epithelial cells
to LTD4 for prolonged periods of time
(Öhd et al., 2000
). Such
exposure caused an upregulation of the cancer-associated proteins COX-2 and
ß-catenin, as well as the anti-apoptotic protein Bcl-2. Furthermore,
LTD4 also caused a PKC-dependent upregulation of active ß1
integrins and an enhanced ß1-integrin-dependent adhesion of intestinal
epithelial cells (Massoumi and
Sjölander, 2001
). Taken together, these results suggest that
LTD4 signal a switch from cell death to cell survival.
MAPKs belong to a group of serine threonine kinases, and the MAPK family in
mammalian cells includes extracellular signal-regulated kinase-1 and -2
(Erk-1/2), the c-Jun NH2-terminal kinases (JNK) and p38 MAPK
(Garrington and Johnson,
1999). These MAPKs integrate multiple signals from various
receptors and second messengers and are involved in the regulation of cellular
proliferation and differentiation
(Garrington and Johnson,
1999
). Once activated, a MAPK can translocate to the nucleus,
where it presumably regulates the expression of different transcription
factors (Garrington and Johnson,
1999
; Velarde et al.,
1999
). It has been shown that Erk-1/2 is activated by a variety of
receptor tyrosine kinases and G-protein-coupled receptors. The mechanism
underlying activation of Erk-1/2 seems to be highly receptor and cell specific
(Daulhac et al., 1999
), and,
for many different types of receptors and cells, such activation is induced by
a PKC- and/or a Ras-dependent signaling pathway
(Hawes et al., 1995
). It has
been shown that different PKC isoforms stimulate Erk-1/2 along Ras-dependent
or Ras-independent signaling pathways (Li
et al., 1998
), suggesting that PKC acts upstream of Ras and Raf-1
(Miranti et al., 1999
) or
directly upstream of Raf-1 (Kolch et al.,
1993
; Cheng et al.,
2001
). Activation of the serine/threonine kinase Raf-1 is a
complicated and not fully elucidated event that includes association with the
active GTP-bound form of Ras at the membrane, Ras-dependent phosphorylation of
Ser338 and most probably an additional phosphorylation of Tyr341
(Mason et al., 1999
).
Different second messengers converge at Raf for subsequent downstream
activation of MAPKs.
It has also been proposed that activation of Erk-1/2 by G-protein-coupled
receptors occurs through transactivation of receptor tyrosine kinases
(Daub et al., 1996;
Rao et al., 1995
). In Rat-1
fibroblasts and COS-7 cells, inhibition of EGF receptor function abrogates
tyrosine phosphorylation of Shc and the subsequent activation of Erk-1/2 in
response to LPA, endothelin-1 and thrombin, all of which bind to
G-protein-coupled receptors (Daub et al.,
1996
; Daub et al.,
1997
). Such crosstalk between EGF and LTD4 receptors has been
demonstrated in experiments showing that EGF-induced modulations of the
cytoskeleton in fibroblasts are mediated by the CysLT1 receptor
(Peppelenbosch et al.,
1995
).
In light of our previous study of intestinal epithelial cells, showing that LTD4 can reduce apoptosis and upregulate distinct proteins such as COX, we conducted the present investigation to determine if and how LTD4 affects proliferation in these cells.
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Materials and Methods |
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Cell culture
Human embryonic intestinal epithelial cells [Intestine 407
(Henle and Dienhardt, 1957)],
which exhibit typical epithelial morphology and growth, were cultured as a
monolayer to approximately 80% confluence for 5 days. Cell cultures were kept
at 37°C in a humidified atmosphere of 5% CO2 and 95% air in
Eagle's basal medium supplemented with 15% new-born calf serum, 55 IU/ml
penicillin and 55 µg/ml streptomycin. The cells were regularly tested to
ensure the absence of mycoplasma contamination.
MTS assay
The cells were cultured on Nunclon (Nalge Nunc International, Denmark)
96-well plates (5-10x103 cells per well) for three days. They
were subsequently pre-incubated in the absence or presence of the MEK
inhibitor PD98059 (50 µM for 30 minutes), the PKC inhibitor GF109203X (30
µM for 30 minutes) or the farnesyltransferase inhibitor FTI-277 [20 µM
for 48 hours (Lerner et al.,
1995)]. The cells were then allowed to grow in fresh media for
another two days in the absence or presence of 80 nM LTD4 or 100
ng/ml EGF and the above inhibitors. The control cells were allowed to grow in
the absence of LTD4, EGF and any inhibitor for the same period of
time as the treated cells. The MTS assay (Promega, Madison, WI) was carried
out according to the protocol provided by the manufacturer. Briefly, the cells
were incubated in 20 µl of MTS/PMS solution for 2 hours, after which
soluble formazan (reduced MTS tetrazolium) was measured at 490 nm in a BMG
plate reader (Offenburg, Germany).
Cell counting
The cells were cultured for three days in 35x10 mm Petri dishes. The
cells were subsequently pre-incubated with or without pertussis toxin, PTX
(500 ng/ml for 2 hours) or FTI-277 (20 µM for 48 hours). Then the cells
were allowed to grow in fresh media for 2 days in the absence or presence of
80 nM LTD4 or 100 ng/ml EGF and the above inhibitors. The effects
of 80 nM LTD4 or 100 ng/ml EGF were also tested in cells
transfected with N17 Ras or the empty vector. To determine the number of
viable cells, all cell counts were performed in the presence of 0.2% trypan
blue.
Expression of N17 Ras, K-PKC, K-Raf-1 and
RD-PKC in Int 407 cells
Cells were transfected for 24 or 48 hours with a full-length human
HA-tagged N17 Ras construct (Odajima et
al., 2000), GFP-tagged RD-PKC
, RD-PKC
(Zeidman et al., 1999
) or a
W437 kinase-dead PKC
construct (K-PKC
), which was
generously provided by Arthur Mercurio (Beth Israel Deaconess Medical Center,
Boston, MA, USA) or HA-tagged kinase inactive c-Raf construct
(K-Raf-1), generously provided by Larry Karnitz (Mayo Clinic,
Rochester, MN, USA). Control cells were transfected with empty pEGFP-N1 vector
from Clontech. Transient transfections of the cells were achieved using 3.5
µl of Lipofectamine (Gibco) and 1.8 µg of plasmid DNA/ml and were
performed in serum-free medium, essentially according to the protocol provided
by the supplier.
Cell lysis
Cells were serum-starved for 2 hours, pre-incubated with inhibitors for the
indicated periods of time and stimulations were terminated by adding ice-cold
lysis buffer (50 mM Tris [pH 7.5], 1 mM EDTA, 1 mM EGTA, 1 mM
Na3VO4, 1% Triton X-100, 50 mM NaF, 5 mM sodium
pyrophosphate, 10 mM sodium glycerophosphate, 4 µg/ml Leupeptin and 30
µg/ml phenylmethanesulfonyl-fluoride, PMSF). Thereafter, the cells were
kept on ice for 30 minutes in the lysis buffer, and the remaining cell debris
was scraped loose into the buffer. The lysates were homogenized 10 times on
ice in a glass tissue grinder (Dounce) and then centrifuged at 10,000
g for 15 minutes. The protein content of each supernatant was
measured and compensated for prior to electrophoresis.
Cell fractionation
Cell stimulations were 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 loose into the cold buffer, homogenized 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.
GST fusion proteins and binding assays
The cDNA clone encoding the GST fusion protein of the Raf minimal binding
domain (RBD) of Ras in pGEX vector was transformed into Escherichia
coli and cultured at 30°C
(Hallberg et al., 1994).
Expression of the GST fusion proteins was induced with 1 mM
isopropyl-l-thio-D-galactopyranoside, and the E. coli were
subsequently collected by centrifugation at 3500 g for 15 minutes
followed by sonication in phosphate-buffered saline. Triton X-100 was added to
the lysate (final concentration 1%), and particulate matter was removed by
centrifuging at 5000 g for 15 minutes. The cleared lysate was
incubated with glutathione-agarose beads (Sigma) for 1 hour at 4°C, and
the beads were subsequently washed three times with ice-cold PBS. Lysates of
unstimulated or stimulated Int 407 cells were prepared in 1.0 ml of the lysis
buffer supplemented with 10 mM MgCl2. GST fusion protein (5-10
µg) or GST alone was pre-bound to agarose beads and incubated with 1.0 ml
of one of the cell lysates (1 mg/ml total cell protein) for 2 hours at
4°C. Thereafter, the beads were washed once with ice-cold lysis buffer
supplemented with 0.5 M NaCl and twice with buffer A.
Gel electrophoresis
Cell lysates, membrane fractions or precipitated proteins were solubilized
by boiling at 100°C for 5 minutes in a sample buffer [62 mM Tris (pH 6.8),
1.0% SDS, 10% glycerol, 15 mg/ml dithiothreitol, and 0.05% bromophenol blue].
The solubilized proteins were subjected to electrophoresis on 10-12%
homogeneous polyacrylamide gels in the presence of SDS.
Immunoblotting
The separated proteins were electrophoretically transferred to a PVDF
membrane. All membranes were blocked for 1 hour with 5% non-fat dried milk at
room temperature and then incubated with a primary antibody for 1 hour at room
temperature or overnight at 4°C. A 1:500 dilution was used for the
anti-Ras (RO2120) antibody, whereas 1:1000 dilutions were used for all other
antibodies. Subsequently all membranes were washed extensively and incubated
with a horseradish-peroxidase-linked goat anti-rabbit, anti-sheep or
anti-mouse antibody (1:5000) for 1 hour at room temperature. Thereafter, the
membrane was again washed extensively, incubated with ECL western blot
detection reagents and finally exposed to hyperfilm-ECL to visualize
immunoreactive proteins. The phospho-MAPK blots were stripped and reprobed to
detect total MAPK.
MEK-1, Raf-1 and B-Raf kinase assays
MEK-1, Raf-1 and B-Raf kinase were assayed using commercial kits from
Upstate Biotechnology. The cells were first pre-incubated in the absence or
presence of the MEK inhibitor PD98059 (50 µM for 30 minutes), the Src
tyrosine kinase family inhibitor PP1 (10 µM for 15 minutes), PTX (500 ng/ml
for 2 hours) or the PKC inhibitor GF109203X (30 µM for 30 minutes).
Alternatively, the cells were depleted of PKC by incubation with 1 µM TPA
(12-O-Tetradecanoylphorbol 13 acetate) for 24 hours. Thereafter, the cells
were incubated in the absence or presence of 80 nM LTD4 for 3
minutes and lysed. The cell lysates (1 mg aliquots) were then used for
immunoprecipitation (1 hour at 4°C) with either 2 µg of an anti-MEK-1,
2 µg of an anti-Raf-1 or 4 µg of an anti-B-Raf antibody. Thereafter, 30
µl of a 3 mg/ml solution of protein G-agarose beads was added, and the
mixture was allowed to stand for an additional hour at 4°C. The
immunoprecipitates were washed twice with lysis buffer, after which kinase
activities were measured with a coupled-enzyme assay. In short, the immune
complexes were incubated for 30 minutes at 30°C with 10 µl of cold
Mg-ATP buffer (Upstate Biotechnology) and the specified substrates (0.4 µg
of inactive MEK-1 and 1 µg of inactive Erk-2 for Raf assays and MEK
assays). This mixture (4 µl) was incubated for 10 minutes at 30°C with
10 µl of 2 mg/ml MBP substrate and 10 µl of a 1:10 dilution of
[-32P]ATP (1 mCi/100 µl) diluted with the cold Mg-ATP
buffer. The reaction mixture (25 µl) was spotted onto the center of a P81
phosphocellulose paper square and washed thoroughly several times with 0.75%
phosphoric acid and then once with acetone and thereafter subjected to liquid
scintillation counting.
Immunofluorescence
The cells were seeded onto glass coverslips and grown for 5 days, during
the last 24 hours they were contransfected with N17 Ras and EGFP (empty
vector). Thereafter, the cells were serum-starved for 2 hours and stimulated
with 80 nM LTD4 for 3 minutes or 100 ng/ml EGF for 5 minutes at
37°C. The stimulations were terminated by fixation of the cells for 10
minutes at room temperature in a 3.7% paraformaldehyde/PBS solution, after
which the cells were permeabilised in a 0.5% Triton X-100/PBS solution for 5
minutes. The coverslips were subsequently washed twice in PBS and incubated at
room temperature in a 3% BSA/PBS solution for 15 minutes. The cells were
stained for 1 hour with a phospho-specific antibody against Erk-1/2.
Thereafter, the coverslips were washed six times in PBS and incubated with a
1:200 dilution (in blocking buffer) of Alexa Fluor 568 goat anti-rabbit
secondary antibody. The coverslips were finally washed six times in PBS and
mounted in fluorescent mounting medium (DAKO A/S). Samples were examined and
photographed in a Nikon Eclipse 800 microscope, using a 60x objective.
Images were recorded with a scientific-grade, charge-coupled device (CCD)
camera (Hamamatsu, Japan) and subsequently analysed with HazeBuster
deconvolution software (VayTek, Inc., Fairland, CT, USA).
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Results |
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LTD4 activates Erk-1/2 in intestinal epithelial cells
We observed a significant activation of Erk-1/2 in cells stimulated with
LTD4 (Fig. 2),
although this was not as pronounced as the response induced by EGF
(Fig. 3C). A concentration of
0.8 nM LTD4 was sufficient to induce activation of Erk-1/2, and the
response at that level was half of that noted at 80 nM LTD4
(Fig. 2A). We refrained from
using higher and non-physiological concentrations. The specific
LTD4 receptor CysLT1 antagonist ZM198,615 (ICI-198,615,
50 µM for 15 minutes) abolished Erk-1/2 activation induced by 80 nM
LTD4, indicating that the effect is mediated by the
CysLT1 receptor (Fig.
2A). The response to LTD4 was rapid, reached a peak
after 3 minutes and returned to basal level after about 30 minutes
(Fig. 2B).
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LTD4-induced Erk-1/2 activation is mediated via a pathway
sensitive to PTX, GF109203X, PP1 and PD98059
As mentioned above, G-protein-coupled receptors are known to activate
Erk-1/2 through different second messenger pathways or by transactivation of
EGF receptors. To identify the signals involved in LTD4-induced
activation of Erk-1/2 and to compare them with those involved in
LTD4-effected cell proliferation
(Fig. 1), we initially used
compounds that inhibit various signaling molecules. We found that
pre-incubation with 500 ng/ml PTX for 2 hours, 30 µM GF109203X for 30
minutes, 10 µM PP1 for 15 minutes (a Src family kinase inhibitor) and 50
µM PD98059 for 30 minutes blocked LTD4-induced activation of
Erk-1/2 (Fig. 2C). Neither the
protein kinase A inhibitor Rp-cAMPS (50 µM for 30 minutes) nor the PI
3-kinase inhibitors wortmannin (100 nM for 10 minutes) and LY294002 (50 µM
for 30 minutes) had any inhibitory effect on the LTD4-induced
activation of Erk-1/2 (Fig.
2D). These results suggest that a heterotrimeric G-protein, PKC,
and MEK are involved in LTD4-mediated activation of Erk-1/2.
Inhibition of LTD4-induced Erk-1/2 activation by the PP1 can
readily be explained by the involvement of a Src-like kinase(s), although it
could also imply crosstalk between the LTD4 receptor and the EGF
receptor.
Transactivation of the EGF receptor is not involved in
LTD4-mediated activation of Erk-1/2
To determine whether crosstalk occurs between LTD4 and EGF
receptors, we first investigated possible signals involved in EGF-induced
Erk-1/2 activation. As expected, EGF-induced activation of Erk-1/2 was
inhibited by genistein (a protein tyrosine kinase inhibitor; 50 µg/ml for
30 minutes), PD153035 (an EGF-receptor inhibitor; 2 µM for 30 minutes),
PD98059 (50 µM for 30 minutes) and to a lesser extent by PP1 (10 µM for
15 minutes), but not by 500 ng/ml PTX (for 2 hours), GF109203X (30 µM for
30 minutes) or PKC depletion by 1 µM TPA for 24 hours
(Fig. 3A,C). The lack of effect
of PTX indicates that EGF-mediated Erk-1/2 activation in intestinal epithelial
cells does not occur via the LTD4 receptor, which has been
suggested for the effects of EGF on the cytoskeleton in fibroblasts
(Peppelenbosch et al., 1995).
We also used an antibody that recognizes the phosphorylated form of the EGF
receptor to determine whether LTD4 participates in activation of
the EGF receptor. As shown in Fig.
3B, stimulation with LTD4 did not lead to any
detectable phosphorylation of the EGF receptor, whereas treatment with EGF
caused a 40-fold increase in EGF receptor phosphorylation. Furthermore, the
LTD4-induced activation of Erk-1/2 was not affected by the
EGF-receptor inhibitor PD153035 (Fig.
3C). Nor did pretreatment with the FGF-1-receptor inhibitor SU5402
or the more general receptor-tyrosine kinase inhibitor SU4984 (blocking FGF-,
PDGF- and insulin-receptors) affect the LTD4-induced activation of
Erk-1/2 (data not shown). These results suggest that EGF-induced activation of
Erk-1/2 is not mediated through the CysLT1 receptor and that
LTD4 is not involved in stimulation of the EGF receptor.
Identification of a specific PKC isoform(s) involved in the
LTD4-induced activation of Erk-1/2
We have previously shown that LTD4 induces translocations (i.e.
activation) of ,
and
PKC isoforms but not of
PKCßII, PKCµ or PKC
(no other novel PKC isoforms is expressed)
in intestinal epithelial cells (Thodeti et
al., 2001
). Furthermore when these cells were subjected to
prolonged (24 hours) treatment with TPA they exhibited total downregulation of
PKC
and PKC
but only partial downregulation of PKC
(Thodeti et al., 2001
). In the
leukemia cell line THP-1, LTD4 has been suggested to activate
Erk-1/2 through a PKC
-dependent pathway
(Hoshino et al., 1998
).
However, LTD4-induced activation of Erk-1/2 in intestinal
epithelial cells was only abolished by a high concentration (30 µM) of the
PKC inhibitor GF109203X (Fig.
2C). A lower concentration of GF109203X (2-10 µM) impaired the
TPA- but not the LTD4-induced activation of Erk-1/2 (data not
shown), suggesting that a novel PKC isoform(s) is involved in producing the
effects of LTD4 on Erk-1/2. In order to identify the PKC isoform
involved in Erk-1/2 activation in intestinal cells, we first investigated the
effect of PTX, which blocks the LTD4-mediated activation of
Erk-1/2, on the LTD4-induced translocation of PKC
,
and
. LTD4 induced a rapid PTX-dependent translocation of PKC
,
and
to a membrane fraction and a subsequent reduction
of these isoforms in a cytosolic fraction from these cells
(Fig. 4A). We then investigated
a possible involvement of calcium-dependent PKCs. Cells were preincubated with
the calcium chelator MAPTAM (10 µM for 1 hour) before stimulation with
LTD4. Such a chelation of cytosolic free calcium did not reduce the
LTD4-induced activation Erk-1/2
(Fig. 4B). Furthermore,
addition of the calcium ionophore ionomycin (1 µM for 5 minutes) did not
stimulate Erk-1/2 nor did it affect the LTD4-induced activation of
ERK-1/2 (Fig. 4B). These data
make participation of the calcium-dependent PKC
isoform unlikely. We
also noted that the PKC
inhibitor rottlerin (10 or 30 µM was added
for 30 minutes) had no effect on the LTD4-induced activation of
Erk-1/2 (Fig. 4C). These data
argue against an involvement of PKC
in the LTD4-induced
activation of Erk-1/2. To obtain more direct evidence for a role of PKC
in the LTD4-induced Erk-1/2 activation, we performed the following
three experiments. Firstly, we studied the time course of TPA-induced
downregulation of the different PKC isoforms, and this revealed that PKC
was downregulated much earlier (4 hours) than PKC
and PKC
(Fig. 4D). Parallel experiments
showed that such a pretreatment with TPA for only 4 hours abolished the
LTD4-induced activation of Erk-1/2
(Fig. 4D), suggesting that
PKC
is involved in such activation. Secondly, we examined the effect of
LTD4 on Erk-1/2 activation in cells transfected with either the
regulatory domain of PKC
(RD-PKC
) or the regulatory domain of
PKC
(RD-PKC
). The isolated regulatory domains have been
suggested to work as isoform-specific dominant-negative inhibitors of PKC
(Jaken, 1996
), and inhibition
of specific isoforms with these domains has been successfully utilized in
several studies (Cai et al.,
1997
; Kiley et al.,
1999
; Massoumi and
Sjölander, 2001
). We noted that expression of RD-PKC
blocked the LTD4-induced activation of Erk-1/2, whereas expression
of RD-PKC
did not (Fig.
4E). These results were obtained even though the expression level
of the GFP-tagged RD-PKC
is less than that of the GFP-tagged
RD-PKC
, which was revealed by reprobing the western blot with an
anti-GFP antibody (Fig. 4E).
Thirdly, we transfected cells with kinase-dead PKC
(K-PKC
) or the corresponding empty vector and examined their
effects on LTD4-induced Erk-1/2 activation. We noted that
expression of K-PKC
totally inhibited the
LTD4-induced activation of Erk-1/2, whereas transfection of the
empty vector had no effect (Fig.
4F). These results clearly show that PKC
is the isoform
involved in the LTD4-induced activation of Erk-1/2 in intestinal
cells.
|
LTD4 induces activation of Raf-1 and MEK via a
PKC-dependent signaling pathway
Employing in vitro kinase assays, we found that LTD4 induced
rapid activation of Raf-1 (Fig.
5A), but not B-Raf (Fig.
5B), in intestinal epithelial cells. The stimulation of Raf-1
peaked approximately 2 minutes after addition of the leulotriene
(Fig. 5A). G-protein- and
PKC-dependent activation of Erk has also been found to be mediated by MEKK1
rather than Raf-1 (Vuong et al.,
2000). To investigate whether the LTD4-induced
activation of Erk-1/2 is mediated by Raf-1, we transfected cells with either a
HA-tagged kinase-dead Raf-1 expressing vector [K-Raf-1
(Sutor et al., 1999
)] or an
empty vector and examined their effect on LTD4-induced Erk-1/2
activation in these cells. The results clearly show that expression of
K-Raf-1 inhibited the LTD4-induced activation of
Erk-1/2, whereas the empty vector had no such effect
(Fig. 5C). In subsequent
experiments, LTD4-mediated activation of Raf-1 was demonstrated to
be abolished by pre-incubation with PTX (500 ng/ml for 2 hours) or GF109203X
(30 µM for 30 minutes) or by TPA-induced downregulation of PKC
(Fig. 5D). These data indicate
that Raf-1 is located downstream of PKC in LTD4-induced activation
of Erk-1/2 (Fig. 5D). On the
basis of these results, we performed immunoprecipitations to examine the
possibility of an LTD4-mediated association between PKC
and
Raf-1, but we found no such association (data not shown). It is quite likely
that Raf-1 is activated by PKC
, even if there is no physical connection
between the two proteins; alternatively PKC
could stimulate Raf-1
indirectly via activation of Ras. It has previously been demonstrated that PKC
can play a role in the activation of Ras in lymphocytes
(Downward et al., 1990
). To
test the ability of PD98059 to inhibit the LTD4-induced activation
of MEK, we employed an in vitro kinase assay
(Fig. 5E). We found that
pre-incubation with PD98059 (50 µM for 30 minutes), PTX (500 ng/ml for 2
hours) or PP1 (10 µM for 15 minutes) inhibited the LTD4-induced
activation of MEK.
|
LTD4 activates Erk-1/2 via a Ras-independent
mechanism
In the present study we clearly show that LTD4 causes a rapid
and transient activation of Ras (Fig.
6A). To explore a possible role for active Ras in the
LTD4-induced activation of Erk-1/2, we either incubated the cells
with the Ras farnesyltransferase inhibitor FTI-277 (20 µM for 48 hours) or
transfected them with HA-tagged N17 Ras. The latter is an Asn-17 mutant of
Ha-Ras, which blocks multiple downstream signals such as activation of Raf-1
and phosphorylation of MAPK (Odajima et
al., 2000). In cells pre-incubated with FTI-277, we detected
almost no increase in GTP-Ras in the pull-down assay of cells stimulated with
LTD4 for 1 minute (Fig.
6B); however the LTD4-induced activation of Erk-1/2 was
not affected at all. Under identical conditions we noted reductions of
EGF-induced Ras activation and simultaneous and similar reductions in
EGF-induced Erk-1/2 activation. To gain further support for
LTD4-induced Ras-independent activation of Erk-1/2, we
cotransfected cells with N17 Ras and empty EGFP vector, and following
stimulation with LTD4 or EGF the cells were immunostained with a
phospho-Erk antibody (Fig. 7).
In unstimulated cells, the staining with the phospho-Erk antibody was weak in
both N17-Ras-transfected and non-transfected cells. In contrast, cells
stimulated with LTD4 stained brightly with the phospho-Erk antibody
regardless, of whether the cells were transfected with N17 Ras or not
(Fig. 7), thus indicating that
LTD4-induced Erk-1/2 activation is Ras-independent. As a positive
control, cells transfected with or without N17 Ras were stimulated with EGF
(Fig. 7). Cells stimulated with
EGF stained brightly with the phospho-Erk antibody, provided that they were
not transfected with N17 Ras (Fig.
7). These latter control data are in agreement with the previously
reported Rasdependency of EGF-induced Erk-1/2 activation
(Daub et al., 1996
).
Furthermore, parallel experiments revealed that neither preincubation with PTX
or GF109203X nor TPA downregulation of PKC impaired the
LTD4-induced activation of Ras (data not shown). These findings
clearly indicate that LTD4-mediated activation of Ras is separate
from the Erk-1/2 signaling pathway.
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Discussion |
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|
Our finding that LTD4 causes a transient, time- and
concentration-dependent activation of Erk-1/2 in intestinal epithelial cells
is compatible with the effect of PD98059, a specific MEK inhibitor, on the
LTD4-induced proliferative response in these cells. Also in
accordance with effects on LTD4-mediated cell proliferation, we
found that activation of Erk-1/2 by LTD4 involves stimulation of a
PTX-sensitive G-protein, a Src-like protein and MEK. Other types of
Gi-protein-coupled receptors in other kinds of cells have been
shown to initiate the MAPK signaling cascade through release of Gß
subunits and activation of a Src-like kinase(s)
(van Biesen et al., 1996
). We
found both similarities and discrepancies between the signaling pathways
leading to activation of Erk-1/2. These observations agree with the conclusion
drawn by Luttrell and coworkers that the mechanisms involved in activation of
MAPK are heterogeneous and appear to depend not only on the nature of the
G-protein-coupled receptor but also on the cell type
(Luttrell et al., 1996
). In
addition to the signals discussed above, our findings that PKC inhibitors and
downregulation of PKC isoforms impair the LTD4-induced activation
of Erk-1/2 in intestinal epithelial cells suggest that PKC plays an important
role in this signaling cascade.
Hoshino and colleagues (Hoshino et al.,
1998) have demonstrated that LTD4 activates Erk-1/2 via
a PTX-insensitive but PKC
- and Raf-1-dependent pathway in the monocytic
leukemia cell line THP-1. However, we found that LTD4 activates
Erk-1/2 via a PTX-sensitive G-protein/PKC-dependent pathway in Int 407
intestinal epithelial cells. We also obtained evidence that it is the
isoform of PKC that is involved in LTD4-mediated stimulation of
Erk-1/2. First, a high concentration of GF109203X was required to impair
LTD4-induced activation of Erk-1/2, and this compound is known to
be a more potent inhibitor of classical PKC than of novel PKC isoforms
(Chen et al., 1999
), implying
involvement of a novel isoform, such as PKC
. Second, we have earlier
shown that LTD4 not only resulted in translocation/activation of
PKC
but also of the
and
isoforms
(Thodeti et al., 2001
). Third,
exposing the cells to TPA for 4 hours, a sufficient amount of time for such a
TPA treatment to abolish the LTD4-induced activation of Erk-1/2,
caused a significant downregulation only of PKC
. Fourth, by transfecting
and using the regulatory domains of PKC
and PKC
as
isoform-specific dominant-negative inhibitors of PKC
(Jaken, 1996
), we conclude
that PKC
, but not PKC
, is involved in the LTD4-induced
activation of Erk-1/2. Finally, the LTD4-induced activation of
Erk-1/2 was totally inhibited in cells transfected with K-PKC
.
The exact location at which PKC takes part in the activation of Erk-1/2
most probably depends on the stimulus and the cell type examined. It has been
shown that the signaling pathway triggered by activation of the T-cell
receptor on T-lymphocytes involves a PKC upstream of Ras
(Downward et al., 1990).
Nonetheless, we found that LTD4-induced activation of Ras was
insensitive to both PKC inhibitors and downregulation of PKC. In addition,
despite our demonstration that LTD4 can activate Ras, this
activation does not seem to be involved in the LTD4-effected
stimulation of Erk-1/2. This conclusion was formed on the basis of the
observation that inhibition of Ras, by either the Ras inhibitor FTI-277 or
transfection with N17 Ras, had no effect on the LTD4-induced
activation of Erk-1/2. Our results instead agree with data showing that
activation of Erk-1/2 by the M1 receptor involves PKC at a point that is
upstream of Raf-1 activation (Marais et
al., 1998
).
We performed in vitro assays for Raf activities and observed that
LTD4 caused activation of Raf-1 with a peak around 2 minutes, which
is in line with the LTD4-induced activation of Erk-1/2.
LTD4-mediated activation of Raf-1 is sensitive to PTX, PKC
inhibitors and downregulation of PKC by TPA. In accordance with our data
indicating involvement of PKC in LTD4-induced activation of
Erk-1/2, several investigators have shown that, once activated, PKC
can
stimulate Raf-1 via phosphorylation of serine
(Kolch et al., 1993
;
Khalil and Morgan, 1993
;
Cai et al., 1997
). In support
of our results, Velarde (Velarde et al.,
1999
) studied vascular smooth muscle cells and demonstrated that
activation of PKC
is required for bradykinin-induced stimulation of
Erk-1/2.
In conclusion, our results demonstrate that LTD4 can promote
proliferation of intestinal epithelial cells by a traditional Ras-dependent
pathway but, more interestingly, even in the absence of Ras activity a
G-protein/PKC/Raf-1/MEK signaling pathway can induce proliferation via
activation of Erk-1/2 in these cells. Activation of such signaling pathways
and the subsequent increase in proliferation indicate that this inflammatory
mediator can contribute to growth of intestinal cells during pathological
inflammatory conditions.
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Acknowledgments |
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