1 Department of Pharmacology and Toxicology, University of Ulm, 89069 Ulm,
Germany
2 MRC Laboratory for Molecular Cell Biology, University College London, London
WC1E 6BT, UK
* Author for correspondence (e-mail: klaudia.giehl{at}medizin.uni-ulm.de)
Accepted 22 May 2003
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Summary |
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Key words: LPA, Migration, Ras, Rho, ERK, Pancreatic carcinoma
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Introduction |
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Lysophosphatidic acid (LPA; 1-acyl-sn-glycerol-3-phosphate) is one
of the simplest natural phospholipids. The estimated concentrations of active,
albumin-bound LPA in serum are in the range of 1-5 µM
(Eichholtz et al., 1993). LPA
acts on different cell types and tissues as an intercellular lipid messenger
and has growth factor-like properties
(Moolenaar, 2000
). It is
mainly produced and released by activated platelets
(Eichholtz et al., 1993
), but
under pathological conditions, LPA can also be produced extracellularly by
secreted phospholipase D, which is also referred to as autotaxin
(Fourcade et al., 1995
;
van Dijk et al., 1998
;
Umezu-Goto et al., 2002
). LPA
exerts its biological functions by binding to at least three different
high-affinity receptors, LPA1/Edg2, LPA2/Edg4 and
LPA3/Edg7 (Contos et al.,
2000
), which interact with both pertussis toxin (PTX)-sensitive
and PTX-insensitive G proteins. Agonist-activated LPA receptors promote
diverse signaling responses, including inhibition of adenylyl cyclase,
Ca2+-mobilization, activation of mitogen-activated protein kinases
(MAPKs), DNA synthesis and Rho GTPase-dependent cytoskeletal changes. In
agreement with this diversity in signal transduction, LPA mediates numerous
cellular responses, including platelet aggregation, smooth muscle contraction,
cell proliferation and differentiation, protection from apoptosis, stress
fiber formation and tumor cell invasion (reviewed by
An et al., 1998b
;
Imamura et al., 1993
;
Goetzl et al., 1999
;
Moolenaar, 1999
;
Swarthout and Walling, 2000
;
Fishman et al., 2001
).
Moreover, an accumulation of LPA has been described in ascites of cancer
patients, implying a possible role for LPA in the peritoneal spread of some
malignancies (Xu et al., 1995
;
Westermann et al., 1998
).
Thus, the characterization of the relevant signal transduction pathways in
cancer cells should facilitate an understanding of the role of LPA as a
regulator of tumor cell function.
To determine the effects of LPA on the metastatic behavior of pancreatic carcinoma cells, we have examined the expression pattern of LPA receptors, the LPA-induced signal transduction pathways and the biological responses to LPA in the pancreatic carcinoma cell lines PANC-1, BxPC-3 and MiaPaCa-2. Our findings provide evidence that LPA-mediated signal transduction via pertussis toxin-sensitive Gi/o, Ras- and Rho-GTPases, and especially the MAP kinase ERK play a critical role in the LPA-induced migration of human pancreatic cancer cells.
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Materials and Methods |
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Cell lines and culture conditions
Human pancreatic carcinoma cell lines PANC-1 (ATCC CRL 1469), BxPC-3 (ATCC
CRL 1687) and MiaPaCa-2 (ATCC CRL 1420) were obtained from American Type
Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in
Dulbecco's modified Eagle medium (DMEM, high glucose, without sodium pyruvate)
supplemented with 10% (v/v) fetal calf serum (FCS), L-glutamine (2 mM) and
penicillin-streptomycin (100 IU/ml-0.1 mg/ml). Cell culture media and
supplements were from Invitrogen (Groningen, The Netherlands). Cells were
incubated at 37°C in a humidified atmosphere with 10% CO2. For
PTX treatment, the culture medium was supplemented with 25-100 ng/ml PTX and
cells were incubated overnight. Serum starvation was performed in DMEM without
supplements or DMEM supplemented with 0.1% (v/v) FCS or 0.1% (w/v) bovine
serum albumin (BSA) (fraction V, cell culture tested, Sigma).
RT-PCR analysis of LPA receptor mRNA expression
Total mRNA was extracted from cells grown to confluence using the RNeasy
Midi Kit from Qiagen (Hilden, Germany) according to the manufacturer's
instructions (1-2x107 cells/column). For RT-PCR, 10 µg of
total RNA were reverse transcribed to first strand cDNA using oligo-(dT)
primers and the SuperScript preamplification system (Invitrogen). LPA receptor
cDNAs were amplified from single-stranded cDNA by PCR using 0.5 U SAWADY Taq
DNA Polymerase (PeqLab, Erlangen, Germany) and 0.4 µM of each primer. The
amounts of single-stranded cDNAs used as templates were adjusted to similar
levels according to the amount of single-stranded -actin cDNA present
in the sample. The amplification protocols for the LPA receptors were: 35
cycles: 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 2
minutes, followed by a final extension time of 10 minutes at 72°C. The PCR
primers were designed according to the published sequences
(Moller et al., 2001
):
LPA1/Edg2: nucleotides 623-642, exon 3 (sense), nucleotides
952-971, exon 4 (antisense); LPA2/Edg4: nucleotides 251-270, exon 2
(sense), nucleotides 1036-1055, exon 3 (antisense); LPA3/Edg7:
nucleotides 500-520, exon 2 (sense), nucleotides 863-881, exon 3 (antisense).
The
-actin cDNA fragment was amplified using the following
amplification protocol: 25 cycles: 95°C for 45 seconds, 60°C for 1
minute, 72°C for 1 minute, followed by a final extension time of 10
minutes at 72°C. The PCR primers were:
5'-GCGTACCTCATGAAGATCCT-3' (sense) and
5'-GCGGATGTCCACGTCACACT-3' (antisense). The PCR products contained
in 25 µl of the reaction volume were fractionated by agarose [2% (w/v)] gel
electrophoresis and visualized by ethidium bromide staining. The sequences of
the amplified cDNAs were verified by direct sequencing using the PCR
primers.
Membrane preparation
Confluent cells from a 150 cm2 flask were washed once with
ice-cold PBS (140 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5
mM KH2PO4, pH 7.2) and collected by scraping into 500
µl of ice-cold lysis buffer [10 mM PIPES/KOH, pH 7.3, 100 mM KCl, 3.5 mM
MgCl2, 3 mM NaCl, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 10
µg/ml aprotinin, 10 µg/ml benzamidine, 10 µg/ml leupeptin and 5
µg/ml pepstatin A]. Cells from 4 flasks were homogenized by nitrogen
cavitation at 25 bar for 30 minutes at 4°C in a Kontes Mini-Bomb (Kontes,
Vineland, NJ, USA). The homogenate was applied to an equal volume (2 ml) of
lysis buffer containing 0.43 g/ml sucrose. The gradient was centrifuged at
200,000 g in a swinging bucket rotor for 30 minutes at
4°C. The membranes were recovered from the interphase by aspiration,
diluted with an equal volume of lysis buffer, and collected by centrifugation
at 100,000 g for 30 minutes at 4°C. The pellet was
resuspended in lysis buffer to a final concentration of 1 mg protein/ml, snap
frozen and stored at -80°C.
[35S]GTP[S] binding
Binding of [35S]GTP[S] to membranes was assayed as described in
Moepps et al. (Moepps et al.,
1997) with some modifications. Membranes (6 µg protein/sample)
were incubated at 30°C in a mixture (100 µl) containing 50 mM
triethanolamine/HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10
µM GDP and 0.4 nM [35S]GTP[S] (1250 Ci/mmol). The reaction was
started by addition of the receptor ligand and [35S]GTP[S] and
terminated after 2 hours by rapid filtration through 0.45 µm-pore size
nitrocellulose membranes (Advanced Microdevices, Ambala Cantonment, India).
Membranes were washed four times with 4 ml each of ice-cold buffer (50 mM
Tris/HCl, pH 7.5, 5 mM MgCl2), dried, and the retained
radioactivity was determined by liquid scintillation counting. Non-specific
binding was defined as the binding not competed for by 10 µM unlabeled
GTP[S].
GTPase activity assays
Expression and purification of glutathione S-transferase
(GST)-fusion proteins: The GTP-bound form of Ras was determined by using a
GST-fusion protein of the Ras-binding domain (RBD) of Raf-1 (amino acids
51-131) as an activation-specific probe for endogenous Ras-GTP
(de Rooij and Bos, 1997). The
preparation of recombinant GST-RBD was performed as described in Giehl et al.
(Giehl et al., 2000
). The
recombinant GST-PAK-CD fusion protein, encompassing amino acids 56-141 of the
CRIB-domain of PAK1B, was used as a probe for GTP-bound Rac1
(Sander et al., 1998
) and the
recombinant GST-TRBD fusion protein encoding the Rho-binding domain of
Rhotekin, amino acids 7-89, was used as an activation-specific probe for
RhoA-GTP (Ren and Schwartz,
2000
). GST-PAK-CD (encoded by pGEX2TK/PAK-CD) and GST-TRBD
(encoded by pGEX2T/TRBD) were expressed in E. coli BL21(DE3) for 2
hours at 30°C or 6 hours at 20°C, respectively, after induction with
0.5 mM isopropyl-
-D-thiogalactopyranoside (IPTG). Bacteria (500 ml)
were harvested and lysed in 10 ml ice-cold lysis buffer [GST-PAK-CD: 50 mM
Tris/HCl, pH 7.5, 150 mM NaCl, 1% (w/v) Triton X-100, 5 mM MgCl2, 1
mM dithiothreitol (DTT), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM
PMSF; GST-TRBD: 50 mM Tris/HCl, pH 8.0, 20% (w/v) sucrose, 10% (v/v) glycerol,
2 mM MgCl2, 0.2 mM Na2S2O5, 2 mM
DTT, 0.2 mM PMSF, 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml
leupeptin] by sonication. Clarified lysates were mixed with 0.5 ml of lysis
buffer-equilibrated Glutathione Sepharose 4B [75% slurry (Amersham
Biosciences, Freiburg, Germany)] and incubated by end-over-end rotation for 60
minutes at 4°C. The beads were collected by centrifugation at 1000
g for 1 minute at 4°C in a benchtop centrifuge, washed
four times with 5 ml each of wash buffer [GSTPAK-CD: 50 mM Tris/HCl, pH 7.5,
0.5% (v/v) Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1
µg/ml aprotinin, 1 µg/ml leupeptin, 0.1 mM PMSF; GST-TRBD: 50 mM
Tris/HCl, pH 7.6, 1% (v/v) Triton X-100, 150 mM NaCl, 10 mM MgCl2,
10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.2 mM PMSF], and finally
resuspended in 2 ml of wash buffer containing 10% (v/v) glycerol. Aliquots
were snap frozen and stored at -80°C.
GTPase activity assays: For detection of GTP-bound Ras, Rac or RhoA, confluent cells were incubated in medium without supplements for 16-20 hours and treated with 10 µM LPA for the indicated periods of time at 37°C and 10% CO2. Cells (1-2 107 cells/10 cm dish) were washed with 10 ml of ice-cold TBS (50 mM Tris/HCl, pH 7.5, 150 mM NaCl) and lysed for Ras-/Rac-GTP pull-down experiments in 500 µl Ras/Rac-RIPA buffer [50 mM Tris/HCl, pH 7.2, 150 mM NaCl, 10 mM MgCl2, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.5% (w/v) SDS, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 mM PMSF] or for Rho-GTP pull-down assays in Rho-RIPA buffer [50 mM Tris/HCl, pH 7.2, 500 mM NaCl, 10 mM MgCl2, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF]. The cell lysates were cleared by centrifugation in a benchtop centrifuge at 15,800 g for 15 minutes at 4°C and the protein concentration was determined by the bicinchoninic acid assay (Pierce, Sankt Augustin, Germany) using BSA fraction V (2 mg/ml) as a standard. Aliquots of the cell lysates (0.5-2 mg of protein) in 500 µl of Ras/Rac- or Rho-RIPA buffer were mixed with 20-40 µg GST-fusion protein immobilized to Glutathione Sepharose 4B beads and the mixture was rotated for 45 minutes (Rho assay) or 90 minutes (Ras and Rac assay) at 4°C. The beads were washed four times with 300 µl each of Ras/Rac-RIPA buffer (Ras assay) or wash buffer [Rac/Rho assay: 50 mM Tris/HCl, pH 7.2, 150 mM NaCl, 10 mM MgCl2, 1% (v/v) Triton X-100, 10 µg/ml aprotinin, 10 mg/ml leupeptin, 0.1 mM PMSF]. Beads were dried with a Hamilton syringe, resuspended in 30 µl of SDS-gel loading buffer, and the suspension was boiled for 10 minutes at 95°C. Bound proteins were separated on 12.5% SDS-polyacrylamide gels (5x8 cm) and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Equal loading of GST-fusion proteins and protein transfer were controlled by Ponceau S staining (Sigma). Precipitated Ras, Rac and RhoA proteins were detected by using the appropriate antibodies and ECL Western Blotting Detection System (Amersham Biosciences).
ERK2 activity assay
Confluent cells were incubated overnight in DMEM containing 0.1% FCS and
treated with PTX (100 ng/ml), PD98059 (25 µM), or growth factors as
indicated. Cells were washed twice with 10 ml PBS and scraped into 500 µl
of MAPK-RIPA buffer per 10 cm dish [MAPKRIPA buffer: 50 mM Tris/HCl, pH 7.2,
150 mM NaCl, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v)
SDS, 2 mM sodium orthovanadate, 25 mM -glycerophosphate, 10 mM sodium
pyrophosphate, 400 µM aprotinin]. Cells were homogenized by forcing the
suspension ten times through a 0.5x25 mm needle attached to a disposable
syringe. ERK2 activity was determined after immunoprecipitation of the kinase
in in vitro phosphorylation assays using a synthetic MBP peptide (APRTPGGRR)
as substrate as described in Giehl et al.
(Giehl et al., 2000
).
Determination of HA-ERK2 activity in transfected cells
The cDNAs of human H-Ras(S17N), K-Ras(S17N), Rac1(T17N) and RhoA(T19N) were
cloned into pEGFP-C expression vectors (Clontech, Heidelberg, Germany) to
produce N-terminally tagged EGFP-GTPase fusion proteins. PANC-1 cells
(3x106 cells/10 cm dish) were cotransfected using 50 µl
DMRIE-C reagent (Life Technologies, Karlsruhe, Germany), 8 µg of
pcDNA3/HA-ERK2 and 8 µg of the vector encoding the EGFP-GTPase fusion
protein. Plasmids and DMRIE-C reagent were preincubated in 500 µl each of
DMEM without supplements for 15 minutes at room temperature, pooled and
incubated for another 15 minutes for complex formation. Cells were washed once
and covered with 4 ml each of DMEM without supplements. The transfection
mixture (1 ml) was added and cells were incubated for 4 hours at 37°C and
10% CO2. The transfection medium was replaced by DMEM with 10% FCS.
After additional 24 hours, cells were starved for 4 hours in DMEM containing
0.1% FCS. Treatment with growth factors was performed in the same medium.
Cells were lysed as described for ERK2-assays and HA-epitope-tagged proteins
were immunoprecipitated from the cleared cell lysate (1 mg of protein per
sample) using 2.5 µg of anti-HA antibody (12CA5) and 30 µl of protein
G-agarose [50% slurry (Roche Diagnostics), washed and equilibrated with
MAPK-RIPA buffer]. The activity of the immunoprecipitated HA-tagged ERK2
kinase activity was determined as described for endogenous ERK2.
Cell migration assays
For ligand-induced cell migration, 5x104 cells were seeded
in 500 µl DMEM containing 10% FCS on top of non-coated polyethylene
terephthalate (PET) membranes of transwell cell culture inserts (12-well
inserts, 8 µm pore size; BD Biosciences, Heidelberg, Germany). The bottom
chamber was filled with 1.2 ml of DMEM with 10% FCS. After 1 hour, mitomycin C
(10 µg/ml) was added for 2 hours to the cells to inhibit cell
proliferation. Thereafter, cells in the upper chamber were washed with DMEM
and incubated in 400 µl DMEM with or without inhibitor. Medium in the
bottom chamber was replaced by 1.2 ml of DMEM containing 0.1% (w/v) BSA with
or without growth factors and/or inhibitors. After 24 to 40 hours, the cells
were fixed in 4% (w/v) paraformaldehyde in PBS, stained for 15 minutes with
Harris' hematoxylin solution (Merck, Darmstadt, Germany), and washed three
times for 15 minutes each in 2 ml each of tap water. Cells that had remained
on top of the membrane were wiped away. Cells that had migrated to the bottom
side of the membrane were visualized under the microscope and quantified by
counting the number of cells in three randomly chosen visual fields.
For wound healing assays, PANC-1 and BxPC-3 cells were seeded at high density [2x105 cells on cover slips (diametre 13 mm) or 1.5x106 cells on 35 mm tissue culture dishes with 2x2 mm grids on the bottom (Nunc, Wiesbaden, Germany)], and incubated for 1 to 2 days until a confluent monolayer was formed. Cells were incubated overnight in DMEM supplemented with 0.1% (w/v) BSA. Wounding was performed by scraping through the cell monolayer with a pipette tip. Afterwards, cells were washed twice with PBS and migration was induced by addition of 10 µM LPA. For kinetic analysis, two individual fields of the grid of the 35 mm dish were photographed every 6-12 hours after wounding.
Microinjection
For microinjection, PANC-1 cells were seeded on cover slips and incubated
for 1 to 2 days until a confluent monolayer was formed. After wounding, cells
were incubated for 2 hours in DMEM without supplements to allow respreading of
the cells that had rounded up. Expression vectors encoding EGFP-GTPase fusion
proteins were injected into the nuclei of the cells in the first 3 to 5 rows
at the wound edge at a concentration of 0.1 µg DNA per µl PBS using an
Eppendorf Microinjection Unit (Microinjector model 5242; Micromanipulator
model 5170; CO2-Controller model 3700 and Heat Controller model
3700). Microinjection was performed in DMEM without supplements at 37°C
and 10% CO2. Cell migration was induced 3 hours after injection by
addition of 10 µM LPA to the culture medium. The cells were incubated for
24 hours, fixed with 4% (w/v) paraformaldehyde in PBS at RT and well-spread,
green-fluorescent cells were counted. Cells that had migrated into the
woundspace were calculated as per cent of injected cells.
Immunofluorescence staining
Cells used for immunostaining were fixed for 15 minutes at room temperature
in 4% (w/v) paraformaldehyde in PBS and then permeabilized for 15 minutes in
0.1% (v/v) Triton X-100 in PBS. Unspecific binding of antibodies or phalloidin
was blocked by incubating the cells for 60 minutes at room temperature with 3%
(w/v) BSA in PBS. The distribution of vinculin was determined using a
monoclonal anti-vinculin antibody (1:100) and AlexaFluor488- or Cy3-conjugated
anti-mouse antibody (1:1000). Filamentous actin was visualized by staining
with TRITC-conjugated phalloidin (50 nM). Dually phosphorylated ERK1/2 was
detected by a polyclonal phospho-p42/44 MAP kinase (Thr202/Tyr204) antiserum
(1:100), and an AlexaFluor488-conjugated anti-rabbit antibody (1:1000).
Antibody incubation (40 µl per cover slip) was performed for 1 hour at room
temperature in a moist chamber. The cover slips were finally mounted by
inverting them into 10 µl Mowiol [2.8 g Mowiol 4-88 (Hoechst, Frankfurt am
Main, Germany), 6 g glycerol, 6 ml H2O, 12 ml of 200 mM Tris/HCl,
pH 8.5]. Cells were examined with an inverse IX70 fluorescence microscope
(Olympus, Hamburg, Germany) and images were recorded using a CCD camera and
analySIS 3.1 imaging system (Soft Imaging System, Münster, Germany).
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Results |
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LPA receptor mRNA expression and activation of PTX-sensitive
heterotrimeric G proteins
To characterize the signal transduction components involved in LPA-mediated
cell migration, we investigated the mRNA expression of the three known
high-affinity LPA receptors by RT-PCR analysis in PANC-1, BxPC-3 and MiaPaCa-2
pancreatic carcinoma cells. Primers were designed according to the published
sequences of human LPA1/Edg2, LPA2/Edg4 and
LPA3/Edg7 receptors (Moller et
al., 2001). Analysis of the amplified DNA by gel electrophoresis
(Fig. 2A) revealed that the
LPA1/Edg2 and LPA2/Edg4 receptor mRNAs were
well-expressed in PANC-1 and BxPC-3 cells. The LPA3/Edg7 mRNA was
clearly expressed in PANC-1 and to a lesser extent in BxPC-3 cells. MiaPaCa-2
cells showed no LPA receptor mRNA expression. To investigate whether LPA
interacts with these LPA receptors and activates heterotrimeric G proteins in
the human pancreatic carcinoma cell lines, the effect of LPA on
[35S]GTP[S] binding to isolated cell membranes was determined. With
this method, receptor-mediated activation of heterotrimeric G proteins can be
analyzed in membrane preparations
(Gierschik et al., 1991
).
Fig. 2B shows that LPA
concentration-dependently stimulated [35S]GTP[S] binding to both
PANC-1 and BxPC-3 cell membranes. Maximal stimulation, which was 1.8-fold for
PANC-1 membranes and 2.7-fold for BxPC-3 membranes was observed at 30 µM
LPA. Because LPA concentrations well above 10 µM tend to precipitate
(Moolenaar, 1999
), 10 µM
LPA was used in most of the experiments shown in this paper. To determine
which heterotrimeric G proteins are activated by LPA in human pancreatic
carcinoma cells, intact cells were incubated with 50 ng/ml PTX for 12 to 16
hours prior to membrane preparation. As shown in
Fig. 2B, this treatment
completely abolished LPA-induced [35S]GTP[S] binding in PANC-1 and
BxPC-3 cell membranes. Taken together, these results show that PANC-1 and
BxPC-3 cells express mRNAs of the three known high-affinity LPA receptors,
whereas MiaPaCa-2 cells express little, if any, of these mRNAs. Furthermore,
LPA activates receptors coupled to PTX-sensitive Gi/o-type
proteins.
|
Activation of monomeric GTPases by LPA
The biological responses of LPA are known to be mediated by multiple signal
transduction pathways involving the monomeric GTPases Ras and Rho
(van Corven et al., 1993;
Kumagai et al., 1993
).
Fig. 3 represents the GTPase
activation pattern for PANC-1 cells after treatment of serum-starved cells
with LPA, as determined by in vitro pull-down experiments using specific
GST-fusion proteins as activation-specific probes for Ras, Rac and RhoA. The
left panel shows the precipitated GTP-bound GTPases, whereas the right panel
represents the amounts of the GTPases in the analyzed lysates. LPA induced a
rapid activation of Ras proteins with a maximum around 1 minute, which
gradually declines within 5 minutes. After approximately 10 to 15 minutes, Ras
activation was no longer apparent (data not shown). We have previously shown
that signal transduction via the Ras-Raf-MEK-ERK cascade is maintained in
PANC-1 cells harboring a mutationally activated K-ras allele and is
required for growth factor-induced cell proliferation and migration. Addition
of EGF or FCS does not result in further activation of the constitutively
activated K-Ras, but markedly activates wild-type N-Ras proteins. HRas is only
marginally expressed in PANC-1 cells
(Giehl et al., 2000
). To
determine whether treatment of the cells with LPA also leads to activation of
N-Ras, isoform-specific analysis of Ras activation was performed. As shown in
Fig. 3, N-Ras was activated 1
minute after addition of LPA and this activation gradually declined within the
next 5 minutes. In contrast, K-Ras was not activated by LPA. Thus,
Ras-dependent signal transduction by LPA is because of N-Ras activation. For
RhoA (Fig. 3), we observed a
pronounced, rapid and short-lasting activation after treatment with LPA, again
with a maximum around 1 minute. Interestingly, Rac-GTPases were also activated
by LPA, but here maximal activation was observed at 3 minutes.
|
Activation of the mitogen-activated protein kinase ERK2 by LPA
Our previous findings indicated that growth factor-mediated signal
transduction via the Ras-Raf-MEK-ERK pathway is required for directed cell
migration of PANC-1 cells (Giehl et al.,
2000). Addition of LPA to serum-starved PANC-1 cells also resulted
in activation of ERK2 as analyzed in in vitro phosphorylation assays with
immunoprecipitated ERK2 and a MBP-peptide as kinase substrate
(Fig. 4). Time course analysis
after addition of LPA (10 µM) to PANC-1 cells
(Fig. 4A) revealed that ERK2
activation was maximal after 10 to 15 minutes and returned to basal levels
after approximately 2 hours. In additional experiments (results not shown), we
found that ERK2 activity was stimulated by LPA in a concentration-dependent
manner, starting at approximately 10 nM LPA and reaching a maximum at 1 µM
LPA. To examine the involvement of Gi/o-type G proteins and
activated MEK1, the upstream activator of ERK1/2, for LPA-induced ERK
activation, PTX and the MEK-1 inhibitor PD98059, respectively, were used. The
effects of these inhibitors on EGF-induced ERK activation were examined for
comparison (Weisz et al.,
1999
; Giehl et al.,
2000
). Fig. 4B
shows that pretreatment of serum-starved PANC-1 cells with PD98059 (25 µM)
prior to addition of LPA resulted in complete inhibition of LPA-induced ERK2
activation, and in an approximately 70% reduction of EGF-induced kinase
activation. PTX (100 ng/ml) markedly, but not completely reduced LPA-mediated
activation of ERK2, yet did not affect EGF-induced kinase stimulation.
Comparable results were obtained for serum-starved BxPC-3 cells, demonstrating
that a Gi/o-MEK1-ERK2 pathway mediates LPA-induced signal
transduction in human pancreatic carcinoma cells. Given that the monomeric
GTPases Ras, Rac and RhoA can influence the activity of ERK
(Tang et al., 1999
;
Li et al., 2001
), transient
cotransfection experiments with expression vectors encoding either enhanced
green fluorescent protein (EGFP), dominant negative mutants of EGFP-H-Ras,
-K-Ras, -Rac1 and -RhoA fusion proteins and HA-tagged ERK2 were performed.
Cytotoxic effects of these GTPases were not detectable under the conditions
used here, and it was even possible to establish stably transfected PANC-1
cells overexpressing dominant negative GTPases (K. Giehl et al., unpublished).
The kinase activity of the cotransfected HAERK2 was determined after treatment
of starved cells for 10 minutes with LPA. These experiments revealed that
overexpression of dominant negative K-Ras and Rac1 led to a striking (>80%)
inhibition of LPA-induced ERK activation. EGFP-RhoA(T19N) and EGFP-H-Ras(S17N)
inhibited LPA-induced ERK activation by approximately 70% and 60%,
respectively. Forced expression of activated mutants of K-Ras
(Giehl et al., 2000
), Rac1 and
RhoA combined with HA-tagged ERK2 caused activation of the cotransfected
kinase in PANC-1 cells (K. Giehl et al., unpublished). These results show,
that LPA-induced activation of ERK requires the activity of the three GTPases,
Ras, Rac1 and RhoA.
|
Inhibition of LPA-induced cell migration
To investigate whether the activation of Ras, Rac1 and/or RhoA proteins are
necessary for LPA-induced cell migration, wound healing assays were performed.
Confluent, serum-starved PANC-1 cells were wounded and allowed to reattach at
the wound edge. Expression plasmids encoding either EGFP or dominant negative
EGFP-H-Ras(S17N), -Rac1(T17N) or -RhoA(T19N) fusion proteins were injected
into the nucleus of cells at the wound edge. The inset in
Fig. 5A shows that expression
of EGFP proteins in injected cells was evident 3 hours after injection.
Migration of serum-starved PANC-1 cells was induced by addition of LPA, after
EGFP protein expression was observed, and cells were incubated for another 24
hours. Cell migration was quantified by calculating the ratio of
EGFP-expressing cells, which had migrated into the cell-free space, to all
EGFP-expressing cells by fluorescent microscopy. Only well-attached, outspread
cells were taken into account for quantification. The diagram in
Fig. 5A shows that
approximately 60% of EGFP-injected control cells had moved into the free
space, but only 15% of EGFP-H-Ras(S17N)-expressing and 13% of
EGFP-Rac1(T17N)-expressing PANC-1 cells had migrated. The most obvious
inhibitory effect was caused by EGFP-RhoA(T19N), because only 4% of the
injected cells showed LPA-induced cell migration.
|
Next, the chemotactic response of PANC-1 cells to LPA was studied using
trans-well cell culture inserts. Fig.
5B shows the bottom sides of the porous membranes with PANC-1 and
BxPC-3 cells that had migrated through the filter. Addition of LPA resulted in
a marked enhancement of directed cell migration of PANC-1 and BxPC-3 cells. To
elucidate whether activation of PTX-sensitive Gi/o-type proteins
and ERK are required for migration in response to LPA or hepatocyte growth
factor (HGF), chemotaxis was measured in the presence of PTX or PD98059,
respectively. HGF activates the receptor tyrosine kinase c-Met
(Birchmeier et al., 1997).
Fig. 5C demonstrates that
chemotaxis in response to LPA was completely inhibited by PTX in both cell
lines, whereas HGF-induced chemotaxis was not affected by PTX. PD98059 had no
effect on LPA- or HGF-mediated cell motility of NIH/3T3 cells (data not
shown), but, very interestingly, resulted in a 50-60% reduction of LPA- as
well as HGF-induced chemotaxis of the pancreatic carcinoma cell lines. The
importance of activated MEK-ERK for LPA-induced cell migration was verified by
using a second MEK inhibitor, U0126
(Favata et al., 1998
). As
shown in Fig. 5C, 10 µM of
U0126 was as effective as 25 µM of PD98059 in inhibiting LPA-induced
chemotaxis of PANC-1 and BxPC-3 cells and also inhibited LPA-induced ERK
phosphorylation (data not shown). LPA also induced activation of the
mitogen-activated kinase JNK in PANC-1 and BxPC-3 cells (data not shown).
Inhibition of JNK by treatment of these cells with SP600125, an anthrapyrazole
inhibitor of JNK (Bennett et al.,
2001
), resulted in an approximately 50% inhibition of cell
migration (Fig. 5C). Therefore,
activation of Gi/o-type proteins, of Ras, Rac1 and RhoA as well as
of MEK, ERK and JNK are required for LPA-mediated migration of pancreatic
carcinoma cells.
LPA-induced rearrangement of actin cytoskeleton and focal adhesion
structures
To examine whether the Gi/o-MEK-ERK pathway is required for
LPA-induced changes of the actin cytoskeleton and of the distribution of focal
complexes during migration, wound-healing assays were performed. LPA-treated
PANC-1 cells were fixed 3 to 8 hours after wounding and LPA addition.
Afterwards, the cells were stained with phalloidin for filamentous actin and
for vinculin, a component of focal adhesions.
Fig. 6a illustrates that
serum-starved PANC-1 cells at the wound edge exhibited only few, if any, actin
stress fibers. Distinct vinculin-containing structures were also not apparent
(Fig. 6b). Treatment of PANC-1
cells with LPA led to spreading of the cells into the cell-free space, to
formation of actin stress fibers and to formation of an actin-rich
lamellipodia at the leading edge (arrows in
Fig. 6c). The stress fibers
were oriented towards the wound edge and radially across the cell
(Fig. 6c). Furthermore, fine,
linear vinculin-containing focal contacts were apparent directly at the
leading edge (arrows in Fig.
6d). Both structures, actin-rich protrusions and focal contacts,
have been suggested to support the protrusive activity during cell locomotion
(Small et al., 1999).
Pretreatment of cells with PTX led to a complete inhibition of LPA-induced
lamellipodia and focal complex formation
(Fig. 6e,f). Interestingly,
inhibition of ERK activity by PD98059 resulted in marked alterations of the
actin cytoskeleton organization (see Fig.
6g and Fig. 6c).
Thus, treatment of cells with the inhibitor resulted in a disruption of the
dense actin network underneath the leading lamellae, which is normally seen
after LPA treatment. Furthermore, the radially organized actin stress fibers
(Fig. 6c) were now found
unorganized in the center of the cell. Notably, the tips of these actin
filaments now colocalized with large, vinculin-containing focal adhesions, and
adhesion sites were no longer apparent at the leading edge, but distributed
over the entire substrate-attached cell surface
(Fig. 6h). This colocalization
of actin and vinculin becomes clearly evident in the merged image shown in
Fig. 6i. Similar results were
obtained in BxPC-3 cells (data not shown).
|
To investigate the role of activated ERK in LPA-induced cell motility and focal contact assembly in more detail, we used phospho-specific ERK1/2 antibodies to detect the localization of the activated kinase. Fig. 7a-a" illustrates that neither phosphorylated ERK1/2 (pT/pY-ERK1/2) nor vinculin exhibited a specific, distinct staining in serum-starved PANC-1 cells. After treatment of wounded PANC-1 cells with LPA, phosphorylated ERK1/2 was localized in distinct structures at the leading lamellae (Fig. 7B). Vinculin was localized in focal contacts at the leading edge of migrating cells (Fig. 7b'). Colocalization analysis (merged image in Fig. 7b") demonstrated that active ERK1/2 is part of the vinculin-containing, newly formed focal contact sites. As shown in Fig. 7c-c", treatment of PANC-1 cells with PD98059 inhibited localization of phosphorylated ERK into the remaining focal contacts. Further studies of the localization of phosphorylated ERK1/2 showed that the kinase was found in focal contact sites and in the cytoplasm within the first 3 hours after addition of LPA [Fig. 7B (a-c)]. Two hours later, the cytoplasmic and focal contact staining diminished, whereas the nuclear staining clearly increased. Eight hours after addition of LPA, phosphorylated ERK1/2 was nearly completely localized in the nucleus. These results demonstrate that inhibition of LPA-induced ERK activation results in disintegration of the actin cytoskeleton and in a loss of focal contact formation at the leading edge. Furthermore, localization of active ERK to focal contacts is an early event during LPA-induced cell migration, followed by translocation of the kinase into the nucleus.
|
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Discussion |
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---|
LPA-induced ERK activation has been shown to be mediated by both
Gi/o-proteins and the Gq-PLC-Ca2+-PKC pathway
(Kranenburg and Moolenaar,
2001; van Leeuwen et al.,
2003
). LPA-induced ERK activation in PANC-1 cells was markedly,
but not completely inhibited by PTX, indicating that there might be other G
proteins involved. Our analysis on Gq-mediated ERK activation in
PANC-1 cells, using transiently coexpressed Gq-coupled mouse
muscarinic acetylcholine receptor M1 and ERK2, revealed that
activation of this receptor with carbachol resulted in a more than fivefold
activation of HA-ERK2 (K. Giehl et al., unpublished). These results indicate
that the residual, PTX-insensitive ERK2 activation after LPA-treatment (cf.
Fig. 4) may be because of
Gq activation.
The results presented in this paper show that LPA is an efficacious
chemoattractant for pancreatic carcinoma cells and, moreover, that activation
of ERK and JNK is crucial for LPA-induced pancreatic carcinoma cell migration.
LPA-induced JNK activation was first described by Sasaki et al. in Swiss 3T3
fibroblasts (Sasaki et al.,
1998). The recent analyses of LPA-receptor knockout mice revealed
that JNK was activated by LPA in wild-type
lpA1-/- and
lpA2-/- mouse embryonic fibroblasts, but not in
cells derived from double knockout lpA1-/-
lpA2-/- mice
(Contos et al., 2002
).
Although JNK was initially characterized as a cytokine- and stress-activated
kinase involved in regulation of apoptosis, its activity relates to a wider
spectrum of cellular activities, such as tumor development, cytoskeletal
rearrangement or cell motility (Davies,
2000
; Shin et al.,
2001
). Although the role of JNK activity in pancreatic carcinoma
cells is currently unclear, the results of this study implicate a function of
LPA-induced JNK-activation in tumor cell migration. Furthermore, initial
experimentation shows that the GTPase Rac1 is involved in JNK activation in
PANC-1 cells (data not shown). Studies on other G protein-coupled receptors
showed that G
and Gß
subunits, as well as Ras and Rho
family members are involved in JNK activation in a cell-type-specific manner
(reviewed by Lowes et al.,
2002
). The significant role of active ERK in directed epitheloid
carcinoma cell migration was also evident for HGF-induced (cf.
Fig. 5) or EGF-induced
(Giehl et al., 2000
)
migration. Here, we extended our investigation and demonstrate, for the early
phase of migration, that active ERK colocalized with vinculin at newly formed
cell adhesion sites. Formation of these attachment sites was dependent on
active Gi/o-MEK-ERK. Inhibition of LPA-induced ERK activation by
either PTX or PD98059 resulted in loss of contact formation, disintegration of
the actin cytoskeleton and impaired cell motility. The localization of active
ERK to focal contacts was temporally restricted and preceded the translocation
of the kinase into the nucleus (cf. Fig.
7B). Thus, activation as well as appropriate subcellular
localization of the kinase is crucial for the migratory response of these
epitheloid carcinoma cells to LPA. The relevance of active MEK and ERK in cell
migration is controversial with regard to cell type and chemoattractant
(Klemke et al., 1997
;
Nobes and Hall, 1999
;
Sliva et al., 2000
) (reviewed
by Stupack et al., 2000
).
However, there is growing evidence for a synergistic interaction between
integrin-mediated cell adhesion and ERK activation. For fibroblasts it has
been shown that activation of ERK by mitogens depends on an intact
cytoskeleton and cell adhesion and is associated with the formation of actin
structures (Renshaw et al.,
1997
; Klemke et al.,
1997
; Howe et al.,
1998
; Renshaw et al.,
1999
; Aplin and Juliano,
2001
). Fincham and coworkers showed a spatial and temporal
association of active ERK to newly forming integrin-containing focal contact
sites in cell-matrix adhesion assembly during cell spreading in rat embryo
fibroblasts and colon carcinoma cells
(Fincham et al., 2000
;
Brunton et al., 2001
).
According to their data, translocation of ERK to the cell periphery is
initiated by integrin-clustering or activation of c-src and activation of MEK.
Regulation of the localization of ERK and other MAP kinases is mediated by
changes in their interaction with several scaffold proteins achieved through
specific MAPK-docking sites (Tanoue et
al., 2000
; Cyert,
2001
). In a recent study by Ahmed and coworkers a direct
interaction of ERK2 with the cytoplasmic domains of
6- and
5-integrin subunits was shown (Ahmed
et al., 2002
). This direct link might explain how ERK is retained
at integrin-containing focal contact sites. Although the exact function of ERK
at focal adhesions remains to be clarified, a potential role of ERK might be
the phosphorylation of paxillin, a scaffold protein in focal adhesion
complexes. This was demonstrated for HGF-induced cell adhesion and migration
of mIMCD-3 renal epithelial cells (Liu et
al., 2002
). According to these data, activated ERK associates with
paxillin, which leads to paxillin phosphorylation at specific serine residues
at the amino terminus and subsequent interaction of paxillin with focal
adhesion kinase (FAK). These phosphorylation events are essential for cell
spreading and adhesion (Liu et al.,
2002
). These results, together with our findings on the spatial
distribution of phosphorylated ERK in PANC-1 cells, highlight the importance
of this kinase in integrating cell adhesion- and receptor-mediated signal
transduction processes in the control of cell locomotion and gene
transcription.
Collectively, our results show that LPA is an efficacious chemoattractant for human pancreatic carcinoma cells. LPA-directed cell motility is mediated by activation of PTX-sensitive heterotrimeric G-proteins and dependent on the LPA-induced activation of Ras, Rac1 and RhoA. Moreover, activation of ERK and translocation of the active kinase to newly forming focal contact sites at the leading lamellae of migrating cells is crucial for the accurate organization of the actin cytoskeleton and for the migratory response of pancreatic carcinoma cells.
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Acknowledgments |
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