From the Department of Molecular and Cellular
Biochemistry and the ¶ Department of Pathology, Stritch School of
Medicine, Loyola University Chicago, Maywood, Illinois 60153 and the
Molecular Signaling Unit, Laboratory of Cellular Development and
Oncology, NIDR, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
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Ras activates a multitude of
downstream activities with roles in cellular proliferation, invasion
and metastasis, differentiation, and programmed cell death. In this
work we have evaluated the requirement of extracellular
signal-regulated protein kinase (ERK), c-Jun
NH2-terminal kinase kinase (JNKK), and c-Jun/AP-1
activities in transformation and extracellular matrix invasion of
ras oncogene expressing NIH 3T3 fibroblasts by expressing
stable mutant genes that constitutively inhibit these activities.
Whereas the inhibition of ERK activity reverts the transformed and
invasive phenotype, the inhibition of the JNK pathway and AP-1
trans-activating activities by JNKK[K129R] and
c-Jun(TAM67) had no effect on the ability of the ras
oncogene-expressing cells to grow in soft agar or invade Matrigel
basement membrane. Thus an elevated JNK activity and/or c-Jun/AP-1
trans-activating activity are not absolute requirements for
ras transformation or invasion through basement membrane, and the dependence on AP-1 activity for transformation is
cell-specific. However, inhibition of JNK kinase (JNKK) in
ras-transformed cells with normally elevated JNK activity
switches the protease-dependent invasive phenotype from a
urokinase plasminogen activator (uPA)-dependent to a
cathepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-transformed cells of low constitutive JNK
activity with the JNK stimulator, anisomycin, converts the protease
mRNA levels from those characteristic of a CL-dependent
to a uPA-dependent phenotype. These protease phenotypes can
be duplicated in untransformed NIH 3T3 cells that express
platelet-derived growth factor receptors and m1 muscarinic receptors
that selectively stimulate the ERK or JNK pathways, respectively. It is
concluded that high ERK activity is required for both protease
phenotypes, whereas the JNK pathway and c-Jun/AP-1 activity are not
required for transformation but regulate a switch between uPA and CL
protease phenotypes in both transformed and untransformed cells. In
ras-transformed NIH 3T3 fibroblasts, the uPA- and
CL-dependent protease phenotypes are redundant in their
ability to invade through basement membrane.
The Ras protein is at the center of a web of multiple downstream
pathways that are differentially regulated to produce signals that
control processes as disparate as cell division, cell differentiation, and programmed cell death (reviewed in Refs. 1-3). Two of the better
characterized pathways downstream from Ras are from Ras to
ERK-11 and -2 through the
sequential activation of Raf and MEK1, and the pathway from Ras leading
to the activation of JNK1 through the activation of Cdc42 and Rac 1 which then sequentially activate PAK, MEK kinase-2 and/or -3, JNKK/SEK/MKK4, and JNK1. The ERKs and JNK1 are mitogen-activated
protein kinases that translocate to the nucleus and activate
transcription factors involved in Ras-regulated processes. The protein
c-Jun is a nuclear substrate of JNK1 (4-7), and c-Jun is a prominent
component of many AP-1 transcription factors that bind to AP-1/TRE
enhancer elements in gene regulatory sites (8, 9). Such an AP-1
enhancer element is a part of the ras response element
present in many protease genes with roles in cellular migration and
invasion. These protease gene products are secreted to degrade the
proteins of the extracellular matrix through which the cells invade
during cell migration (10). In addition, the secreted proteolytic
activities activate growth and migratory factors that regulate cellular
invasion (10). Proteases implicated in invasion processes include the
serine-type protease uPA, the cysteine-type proteases cathepsins B and
L, the aspartate-type protease cathepsin D, and the matrix metallo-type proteases (MMPs) (10-12). Specific protease genes up-regulated by AP-1
induction include the type I collagenases (MMP-1,-13), type IV
collagenase (MMP-9), stromelysin-1 (MMP-3), matrilysin (MMP-7), and
urokinase plasminogen activator (13-21). Other genes up-regulated by a
ras-responsive AP-1 regulatory element with possible roles
in generating the properties exhibited by transformed cells are cyclin
D1 and uPA-receptor (22, 23).
The introduction of an activated ras gene into NIH 3T3
fibroblasts results in transformed cells capable of metastasizing to the nude mouse lung following tail vein injection (24, 25). We have
previously characterized protease gene expressions in ras-transformed NIH 3T3 cells and have shown that at least
two distinct protease phenotypes are generated, apparently dependent on
the isoform of ras oncogene expressed by the cell (25).
Cells transformed by the EJ/vHa-ras oncogene had elevated
levels of uPA mRNA and secreted uPA protease activity but basal
expression of CL. Inhibition of uPA and CL protease gene expressions
with antisense oligonucleotides showed that the
EJ/vHa-ras-transformed cells metastasized to the nude mouse
lung after tail vein injection in a uPA-dependent and
CL-independent manner. In contrast to the EJ/vHa-ras cells,
NIH 3T3 cells transformed by the RAS1Leudel
oncogene showed elevated levels of CL mRNA and secreted CL protease
activity but low basal expression of uPA. Selective inhibition of CL
and uPA gene expressions with antisense oligonucleotides showed the
RAS1Leudel-transformed cells colonized the lung
in a CL-dependent and uPA-independent manner. The
EJ/vHa-ras-transformed cells were designated a
rasuPA+/CL Based on the observation of the two different
protease-dependent metastatic phenotypes
(rasuPA+/CL In this paper we show that expression of TAM67 inhibits both wild-type
c-Jun expression and total AP-1 trans-activating activity in
the ras-transformed NIH 3T3 cells. However, the cells remain transformed and invasive, as shown by growth in soft agar and invasion
through Matrigel basement membrane. This result is surprising, since
prior reports in other cell lines showed a requirement for AP-1
activity to maintain the transformed state induced by a ras oncogene (26-31). We also show that inhibition of JNK1 in
ras-transformed cells by expression of a dominant negative
JNKK/SEK similarly has no effect on the ability of the cell to grow in
soft agar or invade basement membrane. However, as with the inhibition
of c-Jun/AP-1 through TAM67, expression of the mutant JNKK[K129R] in
EJ/vHa-ras-transformed cells switches the protease phenotype for invasion from rasuPA+/CL Plasmids and Cell Lines--
NIH 3T3 cells and cells transformed
with EJ/vHa-ras and RAS1Leudel were
gifts from Merck (West Point, PA) and cultured at low passage number as
described (24, 25). These cells have previously been characterized with
regard to Ras activity and metastatic potential (24, 25). The cells
expressed either the chimeric EJ/vHa-ras gene constructed by
Gibbs et al. (32) or the RAS1Leudel
gene constructed by DeFoe-Jones et al. (33).
The c-Jun(TAM67) genes 297-CMV-c-TAM and 296-CMV-vector (empty vector)
were a gift from Michael Birrer (National Cancer Institute, Biomarkers
and Prevention Research Branch, Rockville, MD). c-Jun(TAM67) codes for
a c-Jun protein lacking the trans-activation domain (residues 2-122) (30, 34). The plasmids pCEP4 ERK1-K71R and pCEP4-ERK2-K52R (5, 35) containing an NH2-terminal 6×His motif were gifts of Melanie Cobb (University of Texas Southwestern Medical Center, Dallas). The dominant negative JNKK/SEK1 plasmid pEBG-SEK1(K129R) (6) was a gift from Leonard Zon (Dana Farber Cancer
Institute, Boston). The pGEX-c-Jun-(1-135) plasmid containing an
NH2-terminal GST utilized in the JNK assay was synthesized by Woodgett and co-workers (7) and was obtained from John Kyriakis (Massachusetts General Hospital, Boston). The plasmid pBL-CAT2 (36)
containing a 3×AP1 consensus sequence from human collagenase upstream
of the thymidine kinase minimal promoter and the chloramphenicol acetyltransferase (CAT) gene was a gift from Michael Karin (University of California, San Diego). The pRSVZ plasmid containing the
Introduction of Stably Expressing Genes into Cells by Calcium
Phosphate Transfection--
Genes were introduced into NIH 3T3 cells
by the calcium phosphate precipitation method of Wigler et
al. (37). The experimental plasmid DNA (1-2 µg) was transfected
along with 1 µg of pcDNA3 (neomycin resistance expression
plasmid, Invitrogen, San Diego, CA) and made up to 10 µg of total DNA
with puc19 DNA. Cells were exposed to the DNA/calcium phosphate (10 µg DNA, 125 mM CaCl2, 0.75 mM
Na2HPO4, 5 mM KCl, 140 mM NaCl, 6 mM glucose, 25 mM HEPES (pH 7.0) solution) for 4 h, rinsed with phosphate-buffered saline, and allowed to grow overnight in complete media. On the following day
cells were divided into three 100-mm plates, and media were changed to
DMEM, 10% FBS containing 200 µg/ml G418. Twenty-four hours later the
media were changed to complete media containing 600 µg/ml G418, and
the cells were selected for 14-21 days. G418-resistant colonies were
isolated for expansion using cloning cylinders, and cells were
maintained in DMEM, 10% FBS containing 200 µg/ml G418 until analysis.
Preparation of Cellular Extracts--
Cells were grown to
confluence in 100-mm plates, washed 2 times with 5 ml of
phosphate-buffered saline, and lysed in 500 µl/dish RIPA buffer (0.15 M NaCl, 50 mM Tris (pH 7.2), 1% (w/v)
deoxycholate, 1% (v/v) Triton X-100, 0.1% SDS) containing protease
inhibitors (2 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml
pepstatin A) and phosphatase inhibitors (20 mM
Electrophoresis and Western Blot Analysis--
Protein
concentrations were determined with the Bio-Rad Protein Assay Kit, and
the samples normalized, the samples boiled for 2 min in 6× PAGE
buffer, and subjected to PAGE electrophoresis. Silver staining of PAGE
gels was performed with the Bio-Rad Silver Stain Kit (Bio-Rad).
For Western blotting, cells were grown to confluence in 100-mm dishes;
the cell lysate was isolated as above, and equal amounts of protein
were electrophoresed through a 12% SDS-PAGE gel at 150 V for 90 min;
the gels washed for 15 min in 100 ml of transfer buffer (20% methanol,
0.05% SDS, 25 mM Tris, 192 mM glycine) and transferred to nitrocellulose membranes (Nytran) in a Bio-Rad transblot
apparatus at 200 mA for 90 min or overnight at 20 mA in ice-cold
transfer buffer. Blots were blocked for at least 2 h in 5% (w/v)
non-fat dried milk in TBST buffer (20 mM Tris-HCl (pH 7.0),
137 mM NaCl and containing 0.1% Tween 20); primary
antibody was added to the blocking solution at a concentration
recommended by the manufacturer and allowed to incubate overnight.
Blots were rinsed with 10 ml of TBST and incubated with a 1:2000
dilution of secondary antibody (goat anti-rabbit peroxidase conjugate) for 2 h. The blots were washed 3 times with 10 ml of TBST, blotted dry, immersed for 30 s in 5 ml of LumiGLO chemifluorescence
substrate (Kirkegaard & Perry, Gaithersburg, MD), briefly allowed to
drip dry, placed between plastic sheets, and exposed to x-ray film (Sterling x-ray Film, Dublin, OH).
Verification of c-Jun(TAM67) Expression--
c-Jun(TAM67)
mRNA was verified by Northern blot analysis using the 820-base pair
BamHI fragment of the pCMV-TAM67 plasmid which binds to both
the native c-Jun message (2.7-3.0 kilobase pairs) and the truncated
c-Jun(TAM67) message (866 base pairs). Cell lines that showed
c-Jun(TAM67) mRNA were further analyzed by Western blot analysis
using a c-Jun antibody (sc-44-G, Santa Cruz Biotechnology Inc., Santa
Cruz, CA) which recognizes the COOH-terminal DNA-binding region
(residues 247-263) of c-Jun. Clones expressing TAM67 showed a primary
band at 29 kDa and the absence of a native c-Jun band at 39 kDa.
Verification of Dominant Negative ERK Expression--
Dominant
negative plasmids pCEP4-ERK1[K71R] and pCEP4-ERK2[K52R] express
catalytically inert ERKs with an NH2-terminal
His6-tag (5, 35). Transfected cells showing G418 resistance
were grown to confluence in 100-mm plates and treated with denaturing
lysis buffer (DLB) (8 M urea, 0.1 M sodium
phosphate, 0.01 M Tris-HCl (pH 8.0)), and the clarified
cell lysates (see above) were purified over Ni2+ spin
columns (Quiagen, Chatsworth, CA) that had been pre-equilibrated with
600 µl of DLB (pH 6.3). Loaded columns were washed twice with 600 µl of DLB containing 20 mM imidazole to remove
non-binding proteins. Bound proteins were then eluted in 2 washes of 75 µl each of DLB (pH 4.5) containing 200 mM imidazole. The
Ni2+ column eluents were analyzed by SDS-PAGE and Western
blotting to show the expression of mutant ERK1 and ERK2 in transfected cells. Western analyses utilized anti-ERK antibody SC-94 (Santa Cruz
Biotechnology, Santa Cruz, CA) which recognizes both ERK1 and ERK2. In
addition, ras-transformed cells expressing the mutant ERKs
genes showed an inhibition of the normally elevated ERK kinase activity
to basal levels, identical to the levels of ERK activity present in
untransformed NIH 3T3 cells.
Kinase Assays--
The JNK kinase assay was carried out as
described previously (21).
The ERK assays were performed on immunoprecipitated ERK1/2 from cell
lysates normalized to a concentration of 100 µg of total protein
using 1 µl of anti-ERK1 antibody (SC-94-G, Santa Cruz Biotechnology,
Santa Cruz, CA) which recognizes both ERK1 and ERK2. The
immunoprecipitate was washed 3 times with 500 µl of kinase buffer (50 mM HEPES (pH 7.4), 10 mM MgCl2, 5 mM mercaptoethanol, 2 µg/ml aprotinin, 0.5 µg/ml
leupeptin, 0.7 µg/ml pepstatin A, 20 mM
Measurement of AP-1 Activity with 3×AP1-pBL2-CAT
Vector--
Cells lines were plated in triplicate in six-well plates
at 25 × 103 cells/well in DMEM containing 10% FBS 1 day prior to transfection. Cells were transfected by treatment with a
DNA/FuGENE solution containing 1 µg of 3×AP-1 pBL2-CAT DNA, 1 µg
of Growth in Soft Agar--
The procedure utilized was similar to
Clark et al. (39). Cells were grown in 6-well plates in
complete media, lifted by incubation in trypsin/EDTA (Sigma), counted
in a hemocytometer, and mixed at a concentration of 10,000 cells/2 ml
of DMEM, 0.33% agar (Difco). The cells in 0.33% agar were plated over
2 ml of DMEM, 0.5% agar that had been allowed to harden in a 6-well
dish. Cells were fed 0.5 ml of DMEM, 0.33% agar twice a week and
allowed to grow for 14 days at which time colony formation was
determined under low magnification (× 10) in an inverted microscope.
Matrigel Invasion Assays--
The Matrigel invasion chambers
were prepared at a 1:20 dilution of Matrigel (Becton Dickinson) with
cold serum-free DMEM by the procedure described by Repesh (40).
Conditioned media were prepared from untransformed NIH 3T3 cells and
stored in aliquots at
Invasions in the presence of antibody were carried out as follows.
Polyclonal anti-CL antibody (Athens Research and Technology, Athens,
GA) was diluted to a stock concentration of 80 µg/ml, and polyclonal
anti-uPA (mouse) antibody (American Diagnostica, Greenwich, CT) was
diluted to a stock concentration of 37.5 µg/µl. Antibody solution
(15 µl) was added to a Matrigel insert (CoStar, Cambridge, MA), and
the treated insert was incubated for 15 min. Cells (50,000 in 200 µl
of DMEM/BSA) were added and the inserts incubated for a further 15 min.
The inserts were placed into an invasion chamber over conditioned media
(750 µl) prepared from untransformed NIH 3T3 fibroblasts. The
chambers were incubated for 24 h at 37 °C in 5%
CO2.
Treatment of Cells with JNK and ERK Pathway
Stimulators--
M1-3T3 cells expressing the acetylcholine
Gq-linked m1 muscarinic receptor have been previously
characterized by Coso et al. (41). The m1-3T3 cells were
grown to confluency in 100-mm culture plates in complete media
containing 10% FBS. Sixteen hours prior to treatment, the media were
changed to DMEM containing 0.1% FBS. Immediately prior to treatment
the media were changed to serum-free DMEM. The stimulators utilized
were PDGF (type B, B), obtained from Upstate Biotechnology (Lake
Placid, NY), and carbachol and anisomycin (Sigma). The concentrations
utilized and the time course of treatment are described in the figure legends.
Northern Blot Analyses for uPA and CL--
The mouse CL cDNA
plasmid was a gift from Dr. G. Gary Sahagian (Tufts University School
of Medicine, Boston), and mouse uPA cDNA was from Dominique Belin
(Center Medical Universitaire, University of Geneva). The Northern
blots were standardized for equal application of RNA by comparing the
UV fluorescence of the 18 S and 28 S rRNA bands in the agarose gels
according to the method of Bonini and Hofmann (42). Several blots were
reprobed with glyceraldehyde-3-phosphate dehydrogenase, according to
methods previously described (21), to verify the equal loading of RNA
in the Northern analyses.
Quantification of Autoradiograms--
Autoradiograms were
scanned into Photoshop 3.05 for the Macintosh, and the relative
intensity of the bands was analyzed using the public domain NIH image
software.2 Statistical
analyses were carried out utilizing analysis of variance, and the
reported values represent the mean ± S.D.
Characterization of Cells Expressing Dominant Negative Mutant
Genes--
Fig. 1 shows that in
agreement with a previous report (21), parental
EJ/vHa-ras-transformed cells have a 6-fold higher JNK activity than untransformed NIH 3T3 cells, while in
RAS1Leudel-transformed cells JNK activity is
inhibited to approximately 30% that observed in NIH 3T3 cell controls
(lanes 1-3). Fig. 1 then shows the JNK activity of two
typical EJ/vHa-ras clones stably transformed with the
dominant negative JNKK[K129R] plasmid (lanes 4 and
5) showing the inhibition of JNK activity to a level similar to the RAS1Leudel-transformed cells. While other
EJ/vHa-ras[K129R] clones tested showed inhibition of JNK
activity (data not shown), clone 12 was neomycin-resistant but showed
only partial inhibition of its JNK activity (Fig. 1, lane
6). This cell line was retained in subsequent experiments as a
negative control. RAS1Leudel-transformed cells
were similarly transfected with the JNKK[K129R] and the neomycin
resistance plasmids, and clones were selected for neomycin resistance.
The RAS1Leudel cells have an intrinsically low
JNK activity (Fig. 1), and therefore assays of the JNK activity in
RAS1Leudel cells expressing
JNKK[K129R] are not informative.
TAM67 lacks the NH2-terminal activation domain (amino
acid residues 3-122) of c-Jun and acts to down-regulate c-Jun/AP-1
trans-activating enhancer activity (30).
EJ/vHa-ras-transformed cells expressing the dominant
negative c-Jun, TAM67, have been previously reported (21). However, the
evidence for the expression of TAM67 in the EJ/vHa-ras-TAM
clones has not been presented. In addition, we have overexpressed TAM67
in RAS1Leudel-transformed cells, even though
c-Jun levels are normally low in these cells (21), to prevent the
possible induction of c-Jun by environmental stimuli. Expression of
TAM67 was verified in transfected cells by the appearance of the
truncated message (866 nucleotides) in Northern analyses. In addition,
Western blotting showed the appearance of a 29-kDa protein due to the
expression of TAM67 and the disappearance of the band due to native
c-Jun of 39 kDa in c-Jun-expressing cells (Fig.
2). Levels of native wild-type c-Jun are
decreased in TAM67-expressing cells as c-Jun gene expression is
auto-regulated by a c-Jun/AP-1 enhancer site (43). The observation of
c-Jun down-regulation supports the ability of TAM67 to act as a
dominant negative gene in our stably transfected cells. Clone
EJ/vHa-ras-TAM5 was neomycin-resistant but showed no TAM67
expression (Fig. 2, lane 12). This clone was incorporated
into subsequent experiments as a negative control to test for possible
biases introduced by the selection process.
It has been previously reported that TAM67 not only represses c-Jun
activity but also quenches total AP-1 trans-activating activity through its association with other Jun and Fos family members
to form inactive complexes at AP-1 gene enhancer sites (34).
Accordingly, the expression of TAM67 and similar c-Jun mutants lacking
a functional activation domain have been previously used to inhibit
total AP-1 trans-activating activity in designated cells
(26, 27, 34). In order to determine if AP-1 trans-activating activity was repressed in EJ/vHa-ras-TAM cells, parental
cells and cells stably expressing TAM67 were transiently transfected with a 3×AP-1-tk-CAT reporter plasmid (pBL-CAT2) to assay AP-1 trans-activation activity (Fig.
3). Parental
EJ/vHa-ras-transformed cells showed a 3.5-fold higher AP-1
trans-activating activity than untransformed NIH 3T3 cells
and a 6-fold higher activity than
RAS1Leudel-transformed cells.
EJ/vHa-ras-TAM4 cells showed a 5-fold decrease in AP-1
activity from that of parental EJ/vHa-ras cells (significant at p < 0.001) to levels identical to the activity of
RAS1Leudel cells (not significant at
p > 0.05). EJ/vHa-ras-TAM5 cells, which
were previously shown not to express observable TAM67 (Fig. 2) and were
incorporated as a negative control, showed an elevated AP-1 activity
(2-fold greater than NIH 3T3 controls and 3-fold greater than
EJ/vHa-ras-TAM4 cells).
EJ/vHa-ras- and
RAS1Leudel-transformed cells were prepared
expressing the catalytically inert mutant ERK genes,
(His)6-ERK1[K71R] and (His)6-ERK2[K52R],
which act as dominant negatives genes for ERK-1 and ERK-2,
respectively, and also express an NH2-terminal 6×His tag
(5, 35). Expression of mutant ERK1 and ERK2 genes was verified by passing the cell lysates from transfected cells over a
Ni2+ affinity column which binds the mutant proteins
through their 6×His motif and then eluting the mutant proteins in
imidazole buffer. The eluted mutant ERK-1 and ERK-2 protein bands were
observed in SDS-PAGE and identified by Western analysis with anti-ERK
antibody. Control cells transfected with mock plasmid showed no protein bands in the same analysis. In addition, measurement of ERK activity in
mutant ERK-expressing cells against the ERK-specific substrate myelin
basic protein showed levels of ERK activity at or below that present in
untransformed NIH 3T3 control cells, whereas the parental
EJ/vHa-ras- and
RAS1Leudel-transformed cells had an elevated ERK
activity (3-fold higher than untransformed NIH 3T3 cells). The level of
activated JNK1 was measured in EJ/vHa-ras clones expressing
dominant negative ERK1/2 and found to be identical to the level in the
parental EJ/vHa-ras-transformed cells (data not shown),
demonstrating that the inhibition of ERK1/2 activities had no effect on
the high activity of JNK1 in EJ/vHa-ras cells.
Neither JNK Activity nor c-Jun/AP-1 Are Required for the
Maintenance of the Transformed and Invasive Phenotype of EJ/vHa-ras-
and RAS1Leudel-transformed
Cells--
EJ/vHa-ras-JNKK clones 7-12 and
RAS1Leudel-JNKK clones 1-3 expressing dominant
negative JNKK were tested for growth in soft agar. Similarly,
EJ/vHa-ras-TAM clones 1-6 and
RAS1Leudel-TAM clones 1-3 expressing TAM67 were
also tested for growth in soft agar. All clones expressing
JNKK[K129R] or TAM67 formed greater than 100 non-adhering colonies
(foci) visible under low magnification. The number and types of
non-adhering colonies were identical to the number of colonies formed
in soft agar by the parental EJ/vHa-ras-transformed and
RAS1Leudel-transformed cells. The untransformed
NIH 3T3 cells formed no foci in soft agar in the same assay.
EJ/vHa-ras (5 clones tested) cells and
RAS1Leudel (4 clones tested) cells expressing
dominant negative ERK1 and ERK2 also did not form foci in the soft agar
assay. The results showed that elevated JNK or c-Jun/AP-1 activities
are not required for growth in soft agar, even by the
EJ/vHa-ras cells which normally have a constitutively
elevated JNK and c-Jun/AP-1 activities. However, elevated ERK activity
is required for growth in soft agar.
Selected clones of EJ/vHa-ras- and
RAS1Leudel-transformed cells expressing dominant
negative JNKK[K129R] or TAM67 were tested in a Matrigel basement
membrane invasion assay. In this assay cells are placed on top of a
Matrigel basement membrane in an invasion chamber, and conditioned
media were placed below the membrane in the chamber. The chamber is
incubated for 18 h at 37 °C and 5% CO2, and the
number of cells that invade through to the bottom side of the basement
membrane are counted. Matrigel invasion has been found to generally
correlate with the metastatic potential of the tumor cell (40, 44). In
support of its applicability as an assay of the invasive phenotype,
inhibitors of in vivo invasion and metastasis are found to
similarly inhibit invasion of transformed cells through Matrigel (45,
46). In this assay, both the EJ/vHa-ras and
RAS1Leudel cells expressing JNKK[K129R] or
TAM67 showed an identical number of invading cells as did parental
EJ/vHa-ras-transformed cells and
RAS1Leudel-transformed cells, whereas
untransformed NIH 3T3 cells showed a low level of invasion (3-20-fold
lower, depending on the experiment) (Fig.
4). Cells expressing the dominant
negative ERK1 and ERK2 also showed a low invasion cell number,
identical to that observed for untransformed NIH 3T3 cells (Fig.
4C). Thus, elevated JNK activity or c-Jun and AP-1
trans-activating activity are not required for invasion
through the Matrigel basement membrane by the EJ/vHa-ras- and RAS1Leudel-transformed cells, whereas
elevated ERK activity is required.
Switch of the rasuPA+/CL
RAS1Leudel-transformed cells were also
transfected with the JNKK[K129R] plasmid, and isolated
neomycin-resistant cells showed no change in protease phenotype from
parental RAS1Leudel-transformed cells of
phenotype rasCL+/uPA Expression of the Dominant Negative JNKK[K129R] in
EJ/vHa-ras-transformed Cells Converts Its Protease Dependence for
Basement Membrane Invasion from uPA-dependent to
CL-dependent--
Fig. 6
shows the number of EJ/vHa-ras-transformed cells and
JNKK[K129R]-expressing EJ/vHa-ras cells that invaded the
Matrigel membrane in the presence of inhibiting antibodies against uPA or CL. Invasion of EJ/vHa-ras-transformed cells through
Matrigel was inhibited by 59% (significant at p = 0.0049) with anti-uPA antibody and was not significantly inhibited by
anti-CL antibody, as expected for cells of phenotype
rasuPA+/CL Decrease of Elevated uPA and CL mRNA Levels by Expression of
Dominant Negative ERK1[K71R] and ERK2[K52R] in Ras-transformed
Cells--
Fig. 7 shows the expression
of the dominant negative ERK-1 and -2 decreased the concentration of CL
mRNA in RAS1Leudel cells and of uPA mRNA
in EJ/vHa-ras cells to levels identical to those in
untransformed NIH 3T3 cells. EJ/vHa-dnERK-clone 5 cells have been shown
to express neomycin resistance but not to express dominant negative ERK
activity. It was included as a control cell line and shows the
selection procedure did not preferentially select for a low protease
phenotype. Accordingly, a stimulated ERK activity is required for the
elevated expression of uPA or CL mRNA characteristic of the
EJ/vHa-ras- or RAS1Leudel-transformed
phenotypes, respectively.
Switch of a rasCL+/uPA Treatment of Non-transformed m1-NIH 3T3 Cells with PDGF Stimulates
ERK Activity Alone and Leads to an Elevation of CL mRNA
Levels--
We determined whether the mRNA levels characteristic
of the rasuPA+/CL Treatment with Both PDGF and Carbachol or PDGF and Anisomycin
Stimulates both ERK and JNK Activity and Increases uPA mRNA
Levels--
Fig. 10A shows
that the combined treatment of cells with PDGF and carbachol increased
both ERK and JNK activity 2-3-fold by 6 and 8 h. Anisomycin
activates c-Jun by a complementary pathway not utilizing the
Gq protein (47), and the combined treatment of m1-3T3
cells with PDGF and anisomycin similarly increased both ERK and JNK
activities 2-3-fold above basal levels at 4-8 h (Fig. 10B). Treatment of the m1-3T3 cells with both the PDGF and
m1 agonist carbachol or PDGF and anisomycin increased by 2-3-fold the
uPA mRNA levels at 6-8 h after initiation of treatment but not the CL mRNA levels (Fig. 10, C and D). These
stimulated levels are similar to the uPA and CL mRNA levels
observed in the EJ/vHa-ras-transformed cells (Fig. 5).
The EJ/vHa-ras- and RAS1Leudel Oncogene- transformed
Cells Do Not Require Elevated JNK or AP-1 Activities to Maintain the
Transformed State or for Invasion through Basement Membrane--
The
expression of JNKK[K129R] and c-Jun(TAM67) activity did not inhibit
the ability of cells containing the EJ/vHa-ras or RAS1Leudel oncogenes to form colonies in soft
agar or to invade Matrigel. These results indicate a lack of the
requirement of JNK activity and Jun/AP-1 activity in transformation,
even in the EJ/vHa-ras-transformed cells which normally have
a high JNK activity and a corresponding high activity of c-Jun/AP-1.
This is the first report showing (i) constitutive inhibition of the JNK
pathway is compatible with ras transformation, and (ii) that
expression of a dominant negative c-Jun which acts to quench AP-1
activity is compatible with ras transformation.
Previous experiments indicated that c-Jun activity is required for the
transformed state induced by activated ras (9, 26, 27,
48-52). Furthermore, the lack of reversion of the transformed state on
expression of dominant negative c-Jun(TAM67) is in conflict with
previous reports of reversion of transformation in other cell types
(28-31, 53). However, there are two reports in conflict with the
concept that c-Jun expression is essential for the initiation and/or
maintenance of the ras-transformed state. Whereas Johnson et al. (26) showed that mutant Ha-ras could not
initially transform c-jun
The apparent conflict between our observations and previous reports may
possibly be explained by a difference in genetic background among cell
lines. The requirement of multiple mutational events involving the
activation of oncogenes and loss of tumor suppressor genes leading to
the transformation of a primary cell to a neoplastic cell is a
generally accepted principle of carcinogenesis (54). As a hypothetical
example we consider the opposing effects of c-Jun and tumor suppressor
genes in the regulation of cyclin D1/Cdk4 activity. c-Jun/AP-1 activity
increases the expression of the cyclin D1 gene (22), which when
complexed with Cdk4 has a kinase activity required for the progression
of cells through the critical control point between the G1
and S phases in the cell cycle (55, 56). However, counteracting cyclin
D1 activity are tumor suppressor proteins that act to inhibit the
activity of the cyclin D1·Cdk4 complex (55, 56). If the tumor
suppressor activity is lost, as occurs in many tumor cells, this may
reduce the requirement for AP-1 stimulation of cyclin D1 expression. In
carcinogenesis, the progression of genetic changes leading to a highly
malignant cell can involve the loss of a requirement for AP-1 activity.
Several AP-1-responsive genes with roles in facilitating the invasion
of malignant cells have been described including uPA, the uPA receptor,
as well as the metalloproteinases MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9
(13-21). Thus, when AP-1 activity is inhibited and the invasive
malignant phenotype is maintained, new genes may be expressed to
compensate for the loss of these AP-1-dependent genes. An
example of such a compensatory change, shown in this work, is the
substitution of CL protease gene expression for uPA protease gene
expression in the context of suppressed c-Jun/AP-1 activity.
ERK activities are constitutively elevated in both EJ/vHa- and
RAS1Leudel-transformed NIH 3T3 cells. This is in
agreement with prior reports indicating that ERK activity is required
for cell proliferation and the transformed phenotype (57-59). It was
therefore not surprising that the expression of dominant negative
ERK1[K71R] and ERK2[K52R] in the ras-transformed cells
inhibited the ability of these cells to grow in soft agar and to invade
Matrigel basement membrane. We were unable to isolate a single clone
that expressed detectable levels of dominant negative ERK1 and ERK2 and
was still transformed.
The JNK Pathway Regulates a Switch between uPA- and
CL-dependent Protease Phenotypes That Are Redundant in
Their Ability to Invade Basement Membrane--
In this paper we show
the JNK pathway can act as a switch between uPA- and
CL-dependent protease-invasive phenotypes that are
redundant in their ability to invade basement membrane. The EJ/vHa-ras-transformed cells have an intrinsically high
activity of JNK and high levels of c-Jun and phosphorylated c-Jun (21). The inhibition of JNKK activity in EJ/vHa-ras-transformed
cells decreased its JNK activity to basal levels. Correlated with a decrease in JNK activity was a decreased uPA mRNA concentration and
increased CL mRNA concentration, resulting in an apparent switch of
protease mRNA levels from those characteristic of the EJ/vHa-ras of phenotype rasuPA+/CL
In agreement with the hypothesis that JNK activity acts as a switch
between transformed protease phenotypes, the stimulation of JNK
activity by anisomycin in RAS1Leudel-transformed
cells, which normally contain low JNK and AP-1 activities and low basal
levels of uPA mRNA, was sufficient to transiently increase uPA
mRNA levels to the levels observed in EJ/vHa-ras-transformed cells (Fig. 8). Corresponding with
the increase in JNK activity and the increase in uPA mRNA levels
was a decrease in CL mRNA levels. This represents an apparent
switch of phenotype from rasCL+/uPA
Whereas the JNK pathway acts as a switch between protease phenotypes,
ERK pathway activity is required for the elevated levels of either uPA
or CL mRNA. Expression of dominant negative ERK1[K71R] and
ERK2[K52R] both in EJ/vHa-ras cells and in
RAS1Leudel cells resulted in a decrease in
elevated uPA mRNA and CL mRNA levels, respectively, to basal
levels (Fig. 7). The requirement for ERK activity for uPA expression
found in these experiments is in agreement with prior data which showed
ERK pathway activity was required for the enhanced expression of uPA in
a squamous cell carcinoma cell line (UM-SCC-1) and in an ovarian
carcinoma cell line (OVCAR-3) (20, 60, 61).
The uPA gene contains the PEA3-AP-1-like ras-responsive
enhancer element (20, 21, 62). The PEA3 portion of the element binds
transcription factors of the Ets family that have been shown to be
activated by the ERK pathway, whereas the AP-1-like portion of the
enhancer binds c-Jun activated by the JNK pathway (20, 21, 63).
Accordingly, it is not surprising that the activation of both the ERK
and JNK pathways are required to stimulate uPA gene expression, and
thus the inhibition of JNK activity and/or c-Jun/AP-1 activity in
EJ/vHa-ras-transformed cells is sufficient to inhibit
ras-stimulated uPA gene expression. Although the loss of
elevated uPA expression on inhibition of JNK and/or c-Jun/AP-1 is
understood, it is not known why the inhibition of JNK activity leads to
elevated CL mRNA levels or the expression of high JNK activity
decreases elevated CL mRNA levels in the ras-transformed cells.
Cells of the different protease mRNA phenotypes were tested for
their ability to invade through Matrigel basement membrane in the
presence of inactivating antibodies directed against secreted uPA or
CL. Invasion of the EJ/vHa-ras-transformed cells with uPA and CL mRNA levels characteristic of the
rasuPA+/CL Regulation of CL and uPA mRNA Profiles in Untransformed Cells
by Stimulation of ERK Activity and by Stimulation of ERK Plus JNK
Activities--
The activated ras oncogene can activate
additional pathways to those that lead to the activation of ERK and JNK
(1-3). To determine whether constitutively activated ras is
necessary for the generation of the CL+/uPA
The various ways that the rasuPA+/CL
In summary, we have shown that JNK and c-Jun/AP-1 activities are
not necessarily required to maintain a transformed and invasive phenotype. Rather in our cells, they regulate a switch between uPA- and
CL-dependent protease invasive phenotypes. In
ras-transformed NIH 3T3 cells these phenotypes are redundant
in their ability to facilitate invasion through basement membrane.
Although we have analyzed the expression of only two genes, other genes
that contribute to the phenotypic properties of these cells most likely depend on whether the JNK pathway is activated or inhibited. Future work will be required to identify these accompanying genes.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
phenotype and the
RAS1Leudel-transformed cells a reciprocal
rasCL+/uPA
phenotype.
and
rasCL+/uPA
) generated by the
EJ/vHa-ras and RAS1Leudel oncogenes,
it was hypothesized that the different ras isoforms activated different combinations of Ras downstream activities. Analysis
of the ERK and JNK pathway activities downstream from Ras showed that
in the EJ/vHa-ras-transformed cells of phenotype rasuPA+/CL
both ERK and JNK activities were
elevated (21). Correlated with the elevated JNK activity, the
EJ/vHa-ras-transformed cells showed high levels of c-Jun and
hyperphosphorylated c-Jun. The RAS1Leudel-transformed cells had a
constitutively elevated ERK activity, identical in magnitude to the ERK
activity in the EJ/vHa-ras-transformed cells. However, in
the RAS1Leudel-transformed cells of phenotype
rasCL+/uPA
, the JNK activity and c-Jun
levels appeared inhibited (JNK activity was ~30% of the activity in
untransformed NIH 3T3 cells) (21). When a dominant negative c-Jun,
TAM67, was expressed in EJ/vHa-ras-transformed cells of
phenotype rasuPA+/CL
, the uPA
mRNA levels were decreased to basal, and the CL mRNA levels
were elevated to that identical to the level of CL mRNA in
RAS1Leudel of phenotype
rasCL+/uPA
(21). This indicated that c-Jun
levels regulate a switch between the uPA- and CL-dependent
protease phenotypes, with high levels of c-Jun generating a uPA+/CL
protease phenotype and low levels of c-Jun a CL+/uPA
protease
phenotype in the ras-transformed NIH 3T3 cells.
to
rasCL+/uPA
. The protease phenotype could be
switched in reverse, from rasCL+/uPA
to
rasuPA+/CL
, by stimulating the
intrinsically low JNK activity in
RAS1Leudel-transformed cells with the JNK
stimulator anisomycin. Finally, the mRNA levels
characteristic of the rasCL+/uPA
or
rasuPA+/CL
phenotypes can be generated in
non-transformed NIH 3T3 fibroblasts with selective inducers that
activate either ERK alone or both ERK and JNK activities, respectively,
indicating that a ras oncogene is not required to regulate
the switch between CL and uPA expression. A switch in protease
expressions in cellular invasions through regulation of JNK or c-Jun
activities allows an invasive cell to overcome inhibitors of one class
of protease (i.e. serine- or cysteine-type proteases) by
switching to the expression of another class of protease.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-galactosidase gene under control of a Rous sarcoma virus long
terminal repeat promoter was obtained from the American Tissue Culture
Collection (ATCC).
-glycerophosphate, 50 µM
Na3VO4, 10 mM
p-nitrophenol phosphate). Cell lysates were clarified by centrifugation at 12,000 rpm for 10 min at 4 °C. Protein levels were
determined on the clarified supernatants using the Bio-Rad Protein
Assay Kit (Bio-Rad).
-glycerophosphate, 50 µM
Na3VO4, and 10 mM
p-nitrophenol phosphate) followed by incubation for 30 min
at room temperature in 30 µl of kinase buffer containing 0.25 mg/ml
myelin basic protein and 50 µM ATP of 10 µCi/ml
[32P]ATP. The reaction was terminated by addition of 10 µl of 6× PAGE loading buffer, boiled 2 min, PAGE electrophoresed
through a 15% acrylamide gel, and the gel dried and autoradiographed. To quantitate the incorporation of 32P into myelin basic
protein, the bands determined by autoradiography were cut out, put into
1 ml of scintillation fluid (Bio-Scint II, Research Products
International, Mount Prospect, IL), and counted in a scintillation counter.
-galactosidase pRSVZ DNA, and 3 µl of FuGENE reagent
(Boehringer Mannheim) mixed according to the manufacturer's
instructions. The FuGENE DNA reagent was added dropwise to cells and
the plates returned to the incubator for 48 h. The media were
removed, and the cells were washed with phosphate-buffered saline, and
350 µl of TEN buffer (40 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 7.8), and 150 mM NaCl) was added,
and the cells were scraped with a rubber policeman. Following
centrifugation in the cold, the pellet was resuspended in 100 µl of
0.25 M Tris-Cl (pH 7.5), and subjected to three freeze-thaw
cycles. The cell lysate was centrifuged and the supernatant frozen at
80 °C. A 20-µl aliquot of each cell lysate was assayed for
-galactosidase activity against the substrate
2-nitrophenyl-
-galactopyranoside as described by Hauser et
al. (38). A 5-µl aliquot of each cell lysate was analyzed for
CAT activity by the addition to the aliquot 2 µl of 100 µCi/ml
[14C]chloramphenicol (40-60 mCi/mmol), 20 µl of 4 mM acetyl-CoA, 25 µl of 1 M Tris-Cl (pH 7.5)
and H2O to a final volume of 150 µl, and incubated at
37 °C for 1.5 h. The reaction was extracted with 1 ml of ethyl
acetate and the ethyl acetate evaporated in a Speedvac evaporator.
Samples were resuspended in 30 µl of ethyl acetate and spotted 2 cm
above the edge of a plastic-backed TLC sheet. Thin layer chromatography
was run in 19:1 chloroform/methanol, and the sheet air-dried and placed
on film for autoradiography. The film was aligned with the TLC sheet,
and the radioactive spots were cut out and added to scintillation fluid
and counted in a scintillation counter. CAT activity was calculated as
percent acetylated product that was normalized by the
-galactosidase values for each sample to correct for transcription efficiency.
70 °C prior to use. The conditioned media
were allowed to thaw, and 750 µl was added to the bottom of each well
of a 24-well plate. Cells (50,000 cells in 0.5 ml of DMEM/BSA (0.1%
BSA)) were added to the top of Matrigel inserts in invasion chambers
(Corning Costar, Kennebunk, ME), and the invasion chambers were
incubated for 18-20 h at 37 °C and 5% CO2.
Non-invading cells were removed from the upper surface of the membrane
by "scrubbing" with a cotton-tipped swab moved gently over the
membrane surface. The membrane was fixed in methanol (1-2 min) and
stained with Diff-Quik (Scientific Products, McGaw Park, IL). Dyed
inserts were dried and the membranes removed, placed on top of a drop
of immersion oil on a slide, and covered with a coverslip. Invading
cells were counted under the microscope at × 100, with at least
15 fields counted and the average number of cells per field calculated.
In some experiments the total number of invading cells was determined
with similar results.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
JNK activity is inhibited by expression of
JNKK[K129R] in EJ/vHa-ras oncogene-containing
cells. Top, solid phase JNK activity assay performed on
100 µg of cell extract. Lane 1, parental
RAS1Leudel-transformed cells transfected with
empty vector plasmid and showing low constitutive JNK activity, as
previously reported for RAS1Leudel-transformed
cells (21); lane 2, untransformed NIH 3T3 cells transfected
with empty vector plasmid and showing basal JNK activity; lane
3, parental EJ/vHa-ras-transformed cells transfected
with empty vector plasmid and showing high JNK activity, as previously
reported (21); lanes 4 and 5,
EJ/vHa-ras clones expressing the dominant negative
JNKK[K129R] gene; lane 6, clone 12 of
EJ/vHa-ras cells transfected with JNKK[K129R] plasmid but
showing no inhibition of JNK activity and included in subsequent
experiments as a control. Bottom, bar graph of
data for cell clones described on top, showing mean ± S.D. of repeated measurements (n = 3).
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Fig. 2.
Western blot showing c-Jun and c-Jun(TAM67)
expressions in cells. The upper band is due to
wild-type c-Jun of 39 kDa, and multiple lower
band(s) are due to c-Jun(TAM67) of apparent molecular
mass 29 kDa. The multiplicity of c-Jun(TAM67) bands in
c-Jun(TAM67)-expressing cells has been previously observed by others
(30). In most cells expressing c-Jun(TAM67), endogenous wild-type c-Jun
levels are no longer observable.
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Fig. 3.
AP-1 trans-activating enhancer
activity assayed with a 3×AP1-tk-CAT reporter plasmid in untransformed
NIH 3T3, RAS1Leudel-transformed,
EJ/vHa-ras-transformed, and EJ/vHa-ras cells
expressing c-Jun(TAM67). Cells were co-transfected with the
pBL-CAT2 (3×AP1-tk-CAT) and pRSVZ ( -galactosidase) plasmids, and
the CAT activity was assayed after 48 h. Results were normalized
to
-galactosidase activity to control for transfection efficiency.
Three independent transfections were performed for each data point,
with the mean transcriptional activity plotted. The error
bars show the standard deviation from the mean. The experiment was
carried out two additional times with similar results.
Asterisk shows cell lines significantly different from
EJ/vHa cells at p < 0.001. Thus,
EJ/vHa-ras-transformed cells are significantly different
from RAS1Leudel-transformed
(RAS1del)-transformed, EJ/vHa-ras-TAM4-transformed, and NIH
3T3 cells at p < 0.001, whereas NIH 3T3,
RAS1Leudel-transformed, and
EJ/vHa-ras-TAM4-transformed cells showed no significant
difference in their AP-1 activity (not significant at p > 0.05).
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Fig. 4.
Assay of Matrigel invasion for
ras-transformed cells expressing dominant negative
JNKK[K129R], c-Jun(TAM67), or ERK1[K71R] and ERK2[K52R].
Numbers represent the -fold change in the average number of
cells invading ± S.D. relative to untransformed NIH 3T3 controls.
Top panel, clones EJ/vHa-ras-TAM4,
EJ/vHa-ras-TAM5, and RAS1Leudel-TAM2
expressing c-Jun(TAM67) were not statistically different at
p > 0.05 from parental EJ/vHa-ras- or
RAS1Leudel-transformed cells. Middle
panel, clones EJ/vHa-ras-JNKK[K129R]-JNKK7,
EJ/vHa-ras-JNKK[K129R]-JNKK8, and
RAS1Leudel- JNKK were not significantly different at p > 0.05 from parental EJ/vHa-ras- or
RAS1Leudel-transformed cells. Bottom
panel, EJ/vHa-ras and RAS1Leudel
cells expressing ERK1[K71R] and ERK2[K52R] were not
significantly different at p > 0.05 from untransformed
NIH 3T3 cells.
Phenotype to a
rasCL+/uPA
Phenotype by Expression of the Dominant
Negative JNKK[K129R] in EJ/vHa-ras-transformed Cells--
Fig.
5 shows the uPA and CL mRNA levels
characteristic of the parental NIH 3T3, EJ/vHa-ras- and
RAS1Leudel-transformed cell lines transfected
with the empty expression vector pEBG and with the corresponding
plasmid encoding a dominant negative JNKK, JNKK[K129R]. The empty
vector transfected EJ/vHa-ras-transformed cells showed a
3-4-fold elevation of uPA mRNA over untransformed NIH 3T3 cells
and a basal level of CL mRNA as expected of cells of phenotype
rasuPA+/CL
(lane 2 versus lane 1), whereas the parental
RAS1Leudel-transformed cells of phenotype
rasCL+/uPA
expressed a 4-fold elevated level
of CL mRNA and a low basal concentration of uPA mRNA
(lane 3 versus lane 1). These levels of uPA and CL mRNA are similar to those previously reported by EJ/vHa-ras- and
RAS1Leudel-transformed cells of phenotype
rasuPA+/CL
and
rasCL+/uPA
, respectively (21, 25). Fig. 5 then
shows that the expression of dominant negative JNKK in the
EJ/vHa-ras cells causes a switch in the protease mRNA
levels from those characteristic of the parental EJ/vHa-ras
cells (phenotype rasuPA+/CL
) to the phenotype
rasCL+/uPA
characteristic of the
RAS1Leudel-transformed cells. This switch
involves a 2.5-3.5-fold increase in the level of CL mRNA and the
3-fold decrease in the level of uPA mRNA. The negative control
Clone 12 cells, which we have previously shown to express neomycin
resistance but lack detectable expression of JNKK[K129R], showed uPA
and CL mRNA levels identical to that of the parental
EJ/vHa-ras-transformed cells and indicate that the selection
procedure did not preferentially select for a CL+/uPA- phenotype. Other
EJ/vHa-JNKK1 clones showed a similar switch in protease mRNA levels
from rasuPA+/CL
to
rasCL+/uPA
as shown for clone 7 and clone
8.
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Fig. 5.
Expression of dominant negative JNKK[K129R]
leads to increased CL and decreased uPA mRNA levels in
EJ/vHa-ras-transformed NIH 3T3 cells. Cells were
co-transfected with empty vector plasmid pEBG or with plasmid
containing JNKK[K129R] and with the plasmid pcDNA3(neo) and
selected for neomycin resistance. Top, untransformed NIH 3T3
(lane 1); parental EJ/vHa-ras-transformed cells
(phenotype rasuPA+/CL ) (lane 2);
parental RAS1Leudel-transformed cells (phenotype
rasCL+/uPA
) (lane 3);
EJ/vHa-ras oncogene clones expressing JNKK[K129R]
(lanes 4 and 5); EJ/vHa-ras oncogene
clone 12 (lane 6) was shown to express neomycin resistance
but not JNKK[K129R], and it was incorporated in experiments as a
negative control. Lanes were shown to contain equal applications of RNA
(see "Experimental Procedures"). Bottom, summary of
multiple independent determinations of the relative uPA and CL mRNA
concentrations in experiments like that shown in the top
panel, giving the mean ± S.D. The Northern blots were
standardized for equal application of RNA by comparing the UV
fluorescence of the 18 S and 28 S rRNA bands in the agarose gels (see
"Experimental Procedures"). The uPA and CL mRNA levels of
EJ/vHa-JNKK clones 7 and 8 are significantly different from
EJ/vHa-ras parental cells at p < 0.05.
(data not shown).
Similarly, transfection of untransformed NIH 3T3 cells with
JNKK[K129R] yielded neomycin-resistant clones with low basal levels
of uPA and CL mRNA, similar to the level of parental NIH 3T3 cells.
These results were expected, as the
RAS1Leudel-transformed cells and untransformed
NIH 3T3 cells normally show low JNK activity, and the expression of
JNKK[K129R] would not be predicted to change the protease phenotype
of the cells.
. In contrast, invasion of the
RAS1Leudel-transformed cells was inhibited by
anti-CL antibody by 41% (significant at p = 0.01) and
was not inhibited by anti-uPA antibody, as expected for cells of
phenotype rasCL+/uPA
. The invasion of
the EJ/vHa-ras-transformed cells expressing JNKK[K129R]
was not inhibited by anti-uPA antibody, but its invasion was inhibited
by anti-CL antibody by 34% (significant at p = 0.0006), demonstrating a switch from a
rasuPA+/CL
-invasive phenotype to a
rasCL+/uPA
-invasive phenotype upon expression
of JNKK[K129R].
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Fig. 6.
Invasion through Matrigel
membranes of EJ/vHa-ras-transformed cells
and EJ/vHa-ras expressing dominant negative JNKK[K129R]
in the absence and presence of inhibiting antibodies to uPA and
CL. Values are mean ± S.D. of units of relative invasion
cell numbers with respect to NIH 3T3 cells (set to 1.0). * or + show
experiments where the presence of antibody significantly inhibited
invasion with respect to the invasion in the absence of the antibody.
Top, graph of invasion of cell lines in the presence and
absence of anti-CL antibody. EJ/vHa-ras cells showed
identical invasion numbers in the presence and absence of anti-CL
antibody (p = not significant, n = 3).
However, EJ/vHa-ras cells expressing dominant negative JNKK
showed a significant inhibition in numbers of invading cells in the
presence of the anti-CL antibody (34% decrease, p = 0.0006, n = 3). The RAS1Leudel
cells similarly showed a significant decrease in the number of invading
cells in the presence of CL antibody (41% decrease, p < 0.01 (n = 3)), as expected for cells of phenotype
rasCL+/uPA . Bottom, graph of
invasion of cell lines in the presence and absence of anti-uPA
antibody. EJ/vHa-ras cells showed a significant decrease in
the number of invading cells in the presence of anti-uPA antibody
(59% decrease, p < 0.005, n = 3), as expected for cells of phenotype
rasuPA+/CL
. However, the EJ/vHa-ras
cells expressing dominant negative JNKK showed no decrease in invasion
numbers in the presence of the anti-uPA antibody nor did the
RAS1Leudel-transformed cells of phenotype
rasCL+/uPA
.
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Fig. 7.
Expression of dominant negative ERK1[K71R]
and ERK2[K52R] in ras-transformed NIH 3T3 cells leads to
the decrease of elevated uPA and CL mRNA concentrations to basal
levels. Cells were co-transfected with empty vector plasmid pCEP4
or with pCEP4 plasmids containing ERK1[K71R] and ERK2[K52R] and
pcDNA3(neo), and selected for neomycin resistance. Top,
CL and uPA mRNA levels in NIH 3T3 cells and parental
EJ/vHa-ras- and
RAS1Leudel-transformed cells co-transfected with
empty and neomycin-expressing vectors (first three cell lines) followed
by selected clones of EJ/vHa-ras-transformed cells
co-transfected with the dominant negative ERK1[K71R] and ERK2[K52R]
and the neomycin-resistant vector. All cells were selected for neomycin
resistance, and the relative levels of CL and uPA mRNA were
determined with respect to NIH 3T3 controls. The Northern blots were
standardized for equal application of RNA by comparing the UV
fluorescence of the 18 S and 28 S rRNA bands in the agarose gels (see
"Experimental Procedures"). Error bars represent the
S.D. of multiple determinations. Clone EJ/vHa-ERK5 was
neomycin-resistant but did not express dominant negative ERK activity.
Bottom, CL and uPA mRNA levels in NIH 3T3 cells, and
parental EJ/vHa-ras- and
RAS1Leudel-transformed cells co-transfected with
empty and neomycin-expressing vectors (first three cell lines) followed
by selected clones of RAS1Leudel-transformed
cells co-transfected with the dominant negative ERK1[K71R] and
ERK2[K52R] and the neomycin-resistant vector. All cells were selected
for neomycin resistance and the relative levels of CL and uPA mRNA
determined relative to NIH 3T3 control. The Northern blots were
standardized for equal application of RNA by comparing the UV
fluorescence of the 18 S and 28 S rRNA bands in the agarose gels (see
"Experimental Procedures"). Error bars represent the
S.D. of multiple determinations.
to a rasuPA+/CL
Phenotype by Treatment of RAS1Leudel-Transformed Cells with
the JNK Pathway Stimulator Anisomycin--
Treatment of cells with low
concentrations of anisomycin has been shown to selectively stimulate
c-Jun phosphorylation in the absence of ERK activation (47). The
RAS1Leudel-transformed cells of phenotype
rasCL+/uPA
have an intrinsically low JNK
activity (21). Accordingly, we determined whether treatment of
RAS1Leudel cells with anisomycin would be
sufficient to switch its phenotype (rasCL+/uPA
) to the
rasuPA+/CL
phenotype. Fig.
8 shows that treatment of
RAS1Leudel-transformed cells with anisomycin
increased JNK activity by 2.5-4-fold from 1 to 8 h after
initiation of treatment. Parallel with the rise in JNK activity is a
2.5-4-fold increase in uPA mRNA levels at 2 and 4 h (Fig. 8).
Corresponding to the increase in uPA mRNA levels, we observed a
decrease in CL mRNA to levels in untransformed NIH 3T3 cells (data
not shown).
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Fig. 8.
Time course of change in JNK activity leads
to an increase in uPA mRNA levels on treatment of
RAS1Leudel-transformed cells with
anisomycin. Cells were treated with 25 ng/ml anisomycin, and the
cell lysate was analyzed at the times indicated. Top, solid
phase JNK assay against the substrate GST-c-Jun-(1-135) at various
time points after treatment with anisomycin. Levels of JNK activity
were significantly increased at all time points measured. Repeated
measurements gave a p < 0.05 (n = 3)
at 1-8 h. Middle, Northern blot for uPA mRNA at various
time points after treatment with anisomycin. Lanes were shown to have
equal application of RNA by the method described (see "Experimental
Procedures"). Bottom, graph of mean ± S.D. for
repeated measurement of time course of JNK activity (white
box) and uPA mRNA levels (shaded box) after
treatment of RAS1Leudel-transformed cells with
anisomycin. Northern blots were normalized for equal application of RNA
by the method described (see "Experimental Procedures"). Levels of
uPA mRNA are significantly increased at all time points following
treatment (n = 3).
and
rasCL+/uPA
phenotypes could be reproduced in
non-transformed cells by stimulating ERK activity or ERK and JNK
activities in the absence of constitutively activated ras.
m1-3T3 cells contain the Gq trimeric protein-linked acetylcholine receptors (AChRs) as well as PDGF receptors (41). Cells
treated with PDGF showed a sustained 2-3-fold stimulation of ERK
activity at 4, 6, and 8 h after PDGF treatment (Fig.
9B). PDGF treatment had no
effect on the low basal activity of JNK observed in these cells. If the
same cells were treated with the m1-AChR receptor agonist, carbachol,
ERK activity was stimulated only briefly (at 5-10 min, see Ref. 41 and
data not shown) and otherwise remained at low basal levels at 2-8 h
after initiation of carbachol treatment (Fig. 9A). However,
carbachol treatment increased JNK activity by 4-fold in a sustained
manner at 4-8 h (Fig. 9A). Similar results were previously
reported by Coso et al. (41). Fig. 9D shows that
the PDGF-induced increase in ERK but not JNK activity correlated with a
3-fold increase in the levels of CL mRNA, whereas the levels of uPA
mRNA remained at basal levels. This mRNA profile is similar to
the uPA and CL mRNA levels observed in the
RAS1Leudel-transformed cells (Fig. 5). The
elevation of JNK activity alone had no effect on the level of either
uPA mRNA or CL mRNA (Fig. 9C). This is in agreement
with the findings that high levels of ERK are required for elevated
expression of CL or uPA mRNA in RAS1Leudel
and EJ/vHa-ras oncogene-containing cells (Fig. 7).
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Fig. 9.
Time course of changes in JNK and ERK
activities and the corresponding CL and uPA mRNA levels on
treatment of m1-3T3 cells with carbachol or PDGF. m1-3T3 cells
were treated with PDGF or carbachol for the times indicated, and their
JNK and ERK activities were determined using GST-c-Jun-(1-135) and
myelin basic protein as substrates. The same cells were analyzed for
uPA and CL mRNA levels by Northern blotting (see "Experimental
Procedures"). Experiments were repeated at least three times, and the
means ± S.D. for each value are shown. A, m1-3T3
cells were treated with 100 µM carbachol, and the JNK
activity was significantly increased (p < 0.001, n = 3) at 4, 6, and 8 h after treatment, whereas
ERK activity showed no significant change (p = not significant, n = 3). B,
m1-3T3 cells were treated with 10 ng/ml PDGF(B,B), and the JNK
activity showed no significant increase (p = not
significant, n = 6), but ERK activity was significantly
increased (p < 0.05, n = 4) at 4, 6, and 8 h following treatment. C, treatment with 100 µM carbachol led to no change in levels of CL and uPA
mRNA (p = not significant, n = 3).
D, treatment with 10 ng/ml PDGF led to increased levels of
CL mRNA at 4,6 and 8 h (p < 0.01, n = 3), and no significant change in the level of uPA
mRNA (p = not significant, n = 3).
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Fig. 10.
Time course of change in JNK and ERK
activities and the corresponding changes in the levels of CL or uPA
mRNA on treatment of m1-3T3 cells with PDGF and carbachol or PDGF
and anisomycin. M1-3T3 cells were treated with PDGF plus
carbachol or PDGF plus anisomycin, and their JNK and ERK activities
were determined using GST-c-Jun-(1-135) and myelin basic protein as
substrates. The same cells were analyzed for uPA and CL mRNA levels
by Northern blotting (see "Experimental Procedures"). Experiments
were carried out at least 3 times, and the means ± S.D. for each
value are shown. A, treatment of m1-3T3 cells with 10 ng/ml
PDGF and 100 µM carbachol increased both JNK and ERK
activities significantly (p < 0.05, n = 4) at 4, 6, and 8 h. B, treatment of m1-3T3 cells
with 10 ng/ml PDGF and 25 ng/ml anisomycin increased both JNK and ERK
activities significantly (p < 0.05, n = 3) at 4, 6, and 8 h. C, m1-3T3 cells treated with 10 ng/ml PDGF and 100 µM carbachol led to a significant
increase in uPA mRNA levels (p < 0.05, n = 3) at 8 h but not CL mRNA levels
(p = not significant, n = 3).
D, m1-3T3 cells treated with 10 ng/ml of PDGF and 25 ng/ml
anisomycin led to a significant increase in uPA mRNA levels at 6 and 8 h (p < 0.05, n = 3) but not
CL mRNA levels (p = not significant,
n = 3).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/
mouse embryo
fibroblasts unless a c-Jun plasmid was also co-expressed, after a
period of time c-jun null fibroblasts containing a
ras oncogene spontaneously developed an ability to form
tumors and acquired other properties of the transformed phenotype.
Similarly, Marshall-Heyman et al. (27) reported two
ras oncogene-transformed rat fibrosarcoma cells lines had
spontaneously acquired a deficiency in c-jun expression,
suggesting that c-Jun is not essential for the maintenance of
transformation. However, both authors showed that in the
c-jun-deficient ras-transformed cells other Jun
family AP-1 proteins had increased their activity, presumably
compensating for the loss of c-Jun in the AP-1 complex. The requirement
of AP-1 activity in these c-jun-deficient cells was
demonstrated by the expression of dominant negative c-jun
genes, such as TAM67, that act as general quenchers of AP-1
trans-activation (34). For both cases, expression of the
mutant c-Jun down-regulated AP-1 activity and reverted the transformed
cells to a normal phenotype (26, 27). Thus although c-Jun was not
specifically required, AP-1 activity appeared to be required for
transformation in these c-Jun-deficient cells. However in our cells,
AP-1 activity is not required for the maintenance of transformation.
to those characteristic of
RAS1Leudel-transformed cells of phenotype
rasCL+/uPA
(Fig. 5). This switch in phenotype
is similar to that observed previously on introduction of the dominant
negative c-Jun(TAM67) into EJ/vHa-ras-transformed cells
(21). As the expression of both dominant negative JNKK and c-Jun change
the protease phenotype from rasuPA+/CL
to
rasCL+/uPA
in
EJ/vHa-ras-transformed cells, it is suggested that the
JNK pathway in these cells is required for production of the
rasuPA+/CL
protease phenotype through
activation of c-Jun.
to
rasuPA+/CL
in
RAS1Leudel-transformed cells.
phenotype was significantly
inhibited by anti-uPA antibody and not by anti-CL antibody. Expression
of JNKK[K129R] in the EJ/vHa-ras-transformed cells
reversed its protease dependence for invasion through Matrigel from
uPA-dependent to CL-dependent, as shown by
inhibition of Matrigel invasion by anti-CL antibody instead of anti-uPA
antibody. Accordingly, the switch in the relative levels of CL and uPA
mRNA correlated with a switch in protease dependence for basement
membrane invasion.
and uPA+/CL
protease
phenotypes, we investigated the role of ERK and JNK activities in
inducing uPA and CL mRNA levels in untransformed m1-3T3 cells.
These cells contain the Gq-coupled m1 muscarinic
acetylcholine receptor (AChR) which is stimulated by carbachol to
induce a sustained high JNK activity with only a brief stimulation of
ERK activity (Fig. 9 and Ref. (41). Anisomycin also stimulates the
activation of c-Jun by promotion of the phosphorylation of its
NH2-terminal trans-activation domain but by a
different pathway than does the carbachol-stimulated AChR (47).
Treatment of untransformed m1-3T3 cells with PDGF alone was found to
stimulate ERK and CL mRNA levels, whereas treatment with PDGF and
carbachol or with PDGF and anisomycin increased both ERK and JNK
activities and increased uPA mRNA levels but not CL mRNA levels
(Figs. 9 and 10). Thus stimulation of ERK activity in the absence of
elevated JNK activation generates a protease phenotype in untransformed
m1-3T3 cells similar to the rasCL+/uPA
phenotype formed by RAS1Leudel-transformed
cells, whereas the combined stimulation of ERK and JNK activities
generate a protease mRNA profile similar to that observed in the
rasuPA+/CL
phenotype of
EJ/vHa-ras-transformed cells. An activated ras is not required to generate these protease phenotypes.
or
rasCL+/uPA
mRNA phenotypes were generated
in this work are summarized in Fig. 11.
The rasuPA+/CL
phenotype was formed in NIH 3T3
cells by (a) transformation with the EJ/vHa-ras
oncogene, (b) treatment of
RAS1Leudel-transformed cells with anisomycin,
(c) treatment of untransformed m1-3T3 cells with both PDGF
and carbachol, or (d) treatment of untransformed m1-3T3
cells with PDGF and anisomycin. The rasCL+/uPA
phenotype was generated by (a) expression of the
RAS1Leudel oncogene, (b) expression
of the dominant negative JNKK[K129R] gene in
EJ/vHa-ras-transformed cells, (c) expression of
the dominant negative c-Jun(TAM67) in EJ/vHa-ras-transformed
cells, or (d) treatment of untransformed m1-3T3 cells with
PDGF. In the rasuPA+/CL
phenotype both the ERK
and JNK activities are elevated, whereas in the
rasCL+/uPA
phenotype the ERK activity is
elevated but the JNK activity and/or c-Jun and AP-1 activity is
inhibited.
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Fig. 11.
Conditions for generation of the
rasuPA+/CL or
rasCL+/uPA
protease phenotype. All
uPA+/CL
phenotypes showed elevated ERK and JNK and/or AP-1
activities, whereas all CL+/uPA
phenotypes show only the elevation of
ERK activity, with inhibition of basal activities of JNK and/or AP-1.
In boxes, first line describes cell type and second line the
treatment or dominant negative gene expression. AChR refers
to the presence of acetylcholine m1 receptor.
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FOOTNOTES |
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* 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. Section 1734 solely to indicate this fact.
§ Portions of this work submitted in partial fulfillment of the requirements for a Ph.D. degree in the Dept. of Molecular and Cellular Biochemistry, Loyola University of Chicago.
** To whom correspondence and reprint requests should be addressed: Dept. of Molecular and Cellular Biochemistry, Loyola University Stritch School of Medicine, 2160 S. First Ave., Maywood, IL 60153. E-mail: rschult{at}luc.edu.
The abbreviations used are: ERK, extracellular signal-regulated protein kinase; JNK, c-Jun NH2-terminal kinase; JNKK, JNK kinase; BSA, bovine serum albumin; CL, cathepsin L; FBS, fetal bovine serum; GST, glutathione S-transferase; MMP, matrix metallo-proteinase; PDGF, platelet-derived growth factor; uPA, urokinase plasminogen activator; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; AChR, acetylcholine receptor; MEK, MAP kinase kinase; PAK, p21-activated kinase; TRE, 12-O-tetradecanoylphorbol-13-acetate response element.
2 Available at the following on-line address: http://rsb.info.nih.gov/ nih-image.
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