(Received for publication, August 29, 1996, and in revised form, November 20, 1996)
From the Department of Pathology and the
§ Department of Molecular and Cellular Biochemistry, Stritch
School of Medicine, Loyola University Chicago,
Maywood, Illinois 60153
Invasive and metastatic cells require protease
expression for migration through the extracellular matrix. Metastatic
NIH 3T3 fibroblasts transformed by different activated ras
genes showed two different protease phenotypes,
rasuPA+/CL and
rasCL+/uPA
(Zhang, J-Y., and Schultz, R. M. (1992) Cancer Research 52, 6682-6689). Phenotype
rasuPA+/CL
is dependent on expression of the
serine-type protease urokinase plasminogen activator (uPA) and the
phenotype rasCL+/uPA
on the cystine-type
protease cathepsin L (CL) for lung colonization in experimental
metastasis. The existence of multiple invasive phenotypes on
ras-isoform transformation implied the activation of
alternative pathways downstream from Ras. We now show that c-Raf-1,
extracellular signal-regulated protein kinase (ERK)-1, and ERK-2 are
hyperphosphorylated, and the ERK activity is high in both the uPA- and
CL-dependent ras-transformed invasive
phenotypes. Levels of c-Jun and c-Jun NH2-terminal kinase
(JNK) activity are also high in the uPA-dependent
phenotype, but they are almost undetectable in the
CL-dependent phenotype. The uPA Ras-response element is a
PEA3/URTF element, and mobility shift assays show a strong PEA3/URTF
protein band in the uPA-dependent phenotype. This band is
competed by a consensus AP-1 DNA sequence and by antibodies to PEA3 and
c-Jun. Thus, the uPA-invasive phenotype appears to require the
activation of Ets/PEA3 and c-Jun transcription factors activated by the
ERK and JNK pathways, while the CL-invasive phenotype appears to
require ERK activity with suppression of JNK and c-Jun activities.
These postulates are supported by the introduction of a dominant
negative c-Jun, TAM67, into cells of phenotype
rasuPA+/CL
, which down-regulated the high uPA
mRNA levels characteristic of this phenotype to basal levels and
up-regulated basal levels of CL mRNA to levels similar to those
observed in cells of phenotype rasCL+/uPA
. We
conclude that the JNK pathway acts as a switch between two distinct
protease phenotypes that are redundant in their abilities to grow
tumors and metastasize.
Increased protease expressions are correlated with the invasive properties of migrating cells. The proteases are required for the degradation of the ECM1 through which cells must invade during the course of their invasions and migrations. Proteases that are expressed and secreted by invading cells include urokinase plasminogen activator, the cathepsins B, D, and L, and the matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1), the 72- and 92-kDa type IV collagenases (MMP-2 and MMP-9), stromelysins 1 and 3 (MMP-3, MMP-11), and matrilysin (MMP-7) (1-7). Since any one migrating cell only expresses or induces a limited spectrum of these proteases, redundant protease mechanisms must exist for cellular migration through ECM under both normal and pathological conditions. Furthermore, switching between redundant mechanisms may be a common event. For example, prior data have shown a dependence on plasminogen activator activity in the cellular invasions that occur during fertilization, embryogenesis, angiogenesis, neuronal development, and macrophage migration (1, 2, 4, 5, 8-10). However, only slight perturbations in these processes are observed in plasminogen activator-deficient mice (4, 8), suggesting that readily available compensatory protease mechanisms exist. The pathways that regulate these protease expression patterns and, therefore, the mechanisms responsible for the compensatory switching between protease-induction pathways are essentially unknown.
In a previous report, we showed two distinct ras-transformed
metastatic phenotypes in NIH 3T3 fibroblasts, based on differences in
expression of either uPA or CL and the corresponding ability of either
uPA or CL to facilitate lung colonization in nude mice (11). The NIH
3T3 cells transformed by the EJ/vHa-ras gene are an example
of phenotype rasuPA+/CL. These cells showed a
high constitutive expression of uPA, low constitutive expression of CL,
and uPA-dependent (CL-independent) lung colonization in
nude mice. In contrast, NIH 3T3 cells transformed by the
RAS1Leudel gene were of phenotype
rasCL+/uPA
. These cells showed high
constitutive expression of CL, low constitutive expression of uPA, and
CL-dependent (uPA-independent) lung colonization (11).
A role for uPA in invasion and metastasis has been demonstrated by experiments in which metastasis was inhibited by anti-uPA antibodies and by the expression of uPA sense or antisense cDNAs in transformed cells that either promoted or inhibited, respectively, tumor cell invasion and metastasis (12-14). Pro-uPA may be secreted by migrating or stromal cells in the environment of migrating cells and may be activated while bound to specific uPA receptors expressed on the cell surface of invading cells (15). uPA promotes the degradation of ECM proteins either acting alone or through activation of plasminogen to plasmin (6), which, in turn, may initiate an extracellular cascade leading to the activation of the pro-metalloproteinases (6, 16, 17). A possible role for CL in invasion and metastasis is supported by reports of a correlation between a high CL expression and high levels of invasion or metastasis in particular tumor types (reviewed by Gottesman and co-workers (7)). CL is secreted by transformed cells in the pro-form and is processed extracellularly to an enzymatically active form (18-20). CL has been shown to act on types I and IV collagens and other major components of the ECM (21-23).
Multiple pathways, in addition to the c-Raf ERK pathway, have
recently been shown to be activated by Ras (24, 25). Ras may thus be a
switching molecule that interprets multiple cellular physiological
signals to control such diverse physiological processes as
proliferation and differentiation (26) through selective activation of
downstream pathways. Different Ras isoforms may discriminate between
downstream pathways through structurally-dependent differences in affinities for downstream effector protein molecules (27), resulting in an affinity-dependent selection among
the possible downstream effectors and their corresponding pathways.
This paper will provide evidence that the Ras ERK and Ras
JNK
pathways are required for the rasuPA+/CL
phenotype; while in the rasCL+/uPA
phenotype,
the ERK pathway is activated, but the JNK pathway leading to the
activation of c-Jun is inhibited. These data support a hypothesis that
the JNK pathway regulates a switch between two different transformed
phenotypes that carry out metastasis by different protease
mechanisms.
NIH 3T3 cells and NIH 3T3 cells containing the EJ/vHa-ras and RAS1Leudel oncogene constructs were originally obtained from Merck Sharp & Dohme Research Laboratories (West Point, PA) and cultured at low passage number as described previously by Bradley et al. (28) The characteristics and metastatic potential of these transformed cells have been previously reported by Bradley et al. (28) and by Zhang and Schultz (11). Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a 5% CO2 atmosphere as described previously (11).
Electrophoretic Mobility Shift AssayNuclear extracts were
prepared by the method of Digman et al. (29) from NIH 3T3
(control), EJ/vHa-ras, and RAS1Leudel
cells. The cells (1 × 107) were washed with cold PBS
and suspended in hypotonic buffer (10 mM HEPES (pH 7.9),
1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.2 mM in freshly added
phenylmethylsulfonyl fluoride). The swollen cells were homogenized, and
the nuclei were pelleted and resuspended in extraction buffer (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM
MgCl2, 20 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM phenylmethylsulfonyl fluoride). A high salt buffer (with 2 M KCl in place of 20 mM KCl) was added dropwise to release soluble proteins. The
solution was centrifuged, the supernatant was dialyzed, and the protein concentrations in the extracts were determined by a Bio-Rad protein kit
with bovine serum albumin solutions as standards and normalized so that
each aliquot contained an equivalent concentration of protein. The
aliquots were stored at 80 °C until utilized.
Double-stranded DNA oligonucleotides were purified by agarose gel electrophoresis and labeled with [32P]ATP and T4 polynucleotide kinase for 30 min at 37 °C. The nuclear extracts (1-2 µg of protein) were preincubated at room temperature in a reaction mixture containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 10% glycerol (w/v), 2 µg of poly(dI-dC) and unlabeled competitor oligonucleotide or antibody (when utilized) at room temperature for 15 min. The labeled probe (1 ng) was added, and the incubation continued for another 20 min. The protein-DNA complexes were resolved on a 4% non-denaturing polyacrylamide gel at room temperature in 0.5 × TBE (1 × TBE was 89 mM in Tris-HCl, 2.5 mM EDTA, and 89 mM boric acid) at 100 V for 3 h, and the gels were dried and analyzed by autoradiography.
Western BlottingCells (2 × 105 cells/well) were plated onto a 6-well plate and grown overnight at 37 °C in a 5% CO2 incubator. The cells were washed with cold PBS and lysed with 400-500 µl of RIPA buffer (10 mM sodium phosphate (pH 7.5), 100 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 µg/ml aprotinin, and 100 µM sodium orthovanadate). Lysates were assayed for total cell protein (Bio-Rad protein kit) and normalized for protein concentrations. The samples were boiled at 95 °C for 5 min in SDS-PAGE buffer. About 25-50 µg of total protein was loaded per lane onto a 12% polyacrylamide gel (30% acrylamide/0.8% bisacrylamide) and electrophoresed in a Protean mini-gel apparatus at 150 V for 1.5 h. The separated proteins were transferred to a nitrocellulose membrane in a Bio-Rad transblot apparatus at 200 mA for 90 min in cold transfer buffer (20% methanol/0.05% SDS, 25 mM Tris, 192 mM glycine). The blots were blocked for 1 h in 5% (w/v) non-fat dried milk in Tris-buffered saline (TBS) (20 mM Tris, 137 mM NaCl). They were washed in 0.1% Tween-20 in TBS (TBST) and incubated in a 1:500 dilution of primary rabbit polyclonal antibody (10 µg/0.1 ml) for 1 h, followed by three washes in TBST. The membranes were then incubated in a 1:5000 dilution of goat anti-rabbit peroxidase (standard solution of 1 µg/ml) secondary antibody for 1 h. The membranes were washed in TBST, and immunoreactivity was detected by the enhanced chemiluminescence (ECL) protocol (Amersham, Life Science, Inc.) with the chemiluminescence recorded on Kodak X-Omat film. In some experiments, visualization of antigen was made with an alkaline phosphatase secondary antibody and BCIP (bromochloroindoyl phosphate) and NBT (nitro blue tetrazolium) as chromogenic substrates.
All antibodies for Western blot analyses and for immunoprecipitation were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The specific antibodies used in this work are identified in the respective figure legends.
[32P]Phosphate-labeling and ImmunoprecipitationCells were plated in 6-well plates as described above. Prior to [32P]phosphate-labeling, the cells were starved for phosphate for 1 h in phosphate-free DMEM (Life Technologies, Inc.) containing 10% FBS that had previously been dialyzed against phosphate-free DMEM. Orthophosphate (100 µCi 32P) was added to each well and incubated for 4 h. The radiolabeled cells were washed with cold PBS and lysed with RIPA buffer, and equal amounts of RIPA-buffered lysate from each cell line (normalized by their protein concentrations) were treated overnight for immunoprecipitation by addition of the indicated antiserum and protein A/G-Sepharose beads. The immune complexes were collected by centrifugation for 30 min at 2500 rpm in a microcentrifuge and washed 4 times with 1 ml of RIPA buffer. Samples were boiled in SDS-PAGE sample buffer and resolved on a 12% SDS-PAGE gel. The gels were transferred and analyzed by autoradiography with quantification by densitometry (Bio-Imaging analyzer).
MAPK In-gel AssayThe procedure was similar to that previously described by Gotoh et al. (30). Cells were plated as in the Western blot analysis procedure and lysed with RIPA buffer, the lysates were normalized for protein concentration, and the samples were boiled in the SDS-PAGE sample buffer. Approximately 50 µg of protein were loaded per lane in an SDS-PAGE 12% polyacrylamide gel in which myelin basic protein (Sigma) was incorporated at a concentration of 0.4 mg/ml. After electrophoresis, the gel was washed twice with 20% (v/v) 2-propanol in 50 mM Tris-HCl (pH 7.4) for 30 min each wash, followed by a wash with 5 mM mercaptoethanol in 50 mM Tris-HCl (pH 7.4). The gel was further washed twice (30 min each) with 6 M guanidine HCl in 50 mM Tris-HCl (pH 7.4). The proteins were renatured by incubating the gel overnight in 50 mM Tris-HCl (pH 7.4) containing 0.04% (v/v) Tween 40 and 5 mM mercaptoethanol at 4 °C. The gel was preincubated at 30 °C for 30 min in 50 mM HEPES, 100 µM sodium orthovanadate, 10 mM magnesium chloride, and 5 mM mercaptoethanol. The gel was then placed in the same buffer containing 50 µM cold ATP and 100 µCi [32P]ATP for 1 h at 30 °C. After extensive washing with 5% (w/v) trichloroacetic acid and 10 mM sodium pyrophosphate, the gels were dried and autoradiographed. The relative intensity of the 32P-bands were determined by densitometry (Bio-Imaging analyzer).
JNK AssayThe plasmid pGEX-GST-c-Jun-1-135 synthesized by
J. R. Woodgett (31) was obtained from J. M. Kyriakis (Massachusetts
General Hospital) and used to transform Escherichia coli.
The GST-c-Jun substrate for JNK was isolated as described by Frangioni
and Neel (32). GST-c-Jun was attached to hexylglutathione-agarose beads (Sigma) and used to assay JNK as described by Westwick and Brenner (33). The cellular lysates incubated with the GST-Jun-agarose were
obtained from NIH 3T3 cells and ras-transformed NIH 3T3
cells grown in 100 mm plates, were washed with ice-cold PBS, and were treated with cell lysis buffer (20 mM HEPES, 25% glycerol,
0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT and 0.3% Nonidet P-40).
After 30 min at 4 °C, the plates were scraped and the lysate clarified by centrifugation at 14,000 rpm for 10 min. The supernatant was assayed for protein concentration by the Bio-Rad protein kit, normalized for protein concentration, and stored in aliquots at 80 °C. For the kinase assay, 80 µg protein aliquots were
incubated with 1 µg of GST-c-Jun bound to glutathione-agarose beads
for 3 h at 4 °C. The kinase assay was carried out as described
by Westwick and Brenner (33), and the 32P-c-Jun-GST product
was analyzed in a 12% SDS-polyacrylamide gel. The gel was dried and
exposed overnight for autoradiography with quantification by
densitometry.
The mouse CL cDNA in pUC-19 was a gift from G. Gary Sahagian (Tufts University School of Medicine, MA), and the mouse uPA cDNA plasmid pDB4501 (a mouse uPA PstI-HindIII cDNA fragment in pSP64) was a gift of Dominique Belin (Center Medical Universitaire, Switzerland). The CL mRNA 32P-labeled hybridization probe was produced from the 766-base pair EcoRI fragment of the pUC19-CL by random priming (Ready-to-Go Kit, Pharmacia Biotech Inc.). The 32P-labeled uPA mRNA antisense hybridization probe was prepared using SP6 RNA polymerase (Life Technologies, Inc.) from the SP6 promoter site in the pDB4501 plasmid.
Generation and Characterization of EJ/vHa-ras-transformed Cells Containing a Dominant Negative c-Jun (TAM67)The plasmid pCMV-TAM67 containing the dominant negative c-Jun, TAM67, and the control plasmid pCMV (empty vector) were a generous gift from Michael J. Birrer (NCI, MD). TAM67 is a deletion mutant of the c-jun gene that lacks the N-terminal transactivation domain (amino acids 3-122) (34, 35). Cells were co-transfected with pcDNA3 (Invitrogen, CA) along with pCMV-TAM67 or pCMV (empty vector) using the calcium phosphate method (36). In brief, cells were grown to a concentration of 3 × 105 cells/100-mm dish, the media replaced with 5 ml of serum-free DMEM, and incubated 2 h. Cells were then exposed in DNA/calcium phosphate solution (2 µg of pCMV-TAM67 or of pCMV-empty vector along with 1 µg of pcDNA3 and made up to 10 µg of DNA with salmon sperm DNA) for 4 h, were rinsed with PBS, and were grown overnight in 10% FBS-DMEM. The following day, cells were divided into three 100-mm plates, and the media was changed to 10% FBS-DMEM containing 200 µg/ml G418. After 48 h, the concentration of G418 was increased to 600 µg/ml, and the cells were selected for 14 to 21 days. G418-resistant colonies were isolated for expansion using cloning cylinders, and cells were maintained in 10% FBS-DMEM containing 200 µg/ml G418 until analysis.
The presence of TAM67 mRNA, which migrates lower than indigenous c-Jun mRNA (2.7 or 3.0 kb), in the transfected cell colonies was shown by Northern analysis for TAM67 using a BamHI fragment from pCMV-TAM67 to hybridize with TAM67 mRNA (868 base pairs).
The persistence of the transformed phenotype in the TAM67 expressing cells was shown by the ability of the cells to form colonies in soft agar, utilizing the procedure of Cox and Der (37).
Several parallel pathways are known to exist downstream from Ras
(24, 25, 38). A well characterized downstream pathway is from Ras c-Raf-1
MEK
ERK-1/ERK-2 (Fig. 1). The
phosphorylation of transcription factors by the ERKs activate the
expression of genes that are believed to cause new DNA synthesis and
cell division (24, 38). A parallel kinase cascade pathway from Ras
utilizes the Ras-like p21 GTP binding proteins Rac and CDC42, leading
to the activation of JNK and p38 kinase. Activation of JNK leads to the
phosphorylation and activation of the transcription factors c-Jun and
ATF-2 (24, 39-43). This pathway is also activated in a Ras-independent
manner by cytokines and other stress factors (39-43). In this work, we
have compared the signal transduction pathway from Ras to ERK and Ras
to JNK in EJ/vHa-ras-transformed (phenotype
rasuPA+/CL
),
RAS1Leudel-transformed (phenotype
rasCL+/uPA
), and untransformed NIH 3T3
fibroblasts to determine the Ras downstream signals that may regulate
the switching between the uPA-dependent and
CL-dependent invasion mechanisms.
Assay of the Raf
The relative concentrations of c-Raf-1
and the MAP kinases ERK-1 and ERK-2 were determined by Western blot
analysis (Fig. 2). The levels of c-Raf-1, ERK1, and ERK2
were not significantly different between the untransformed NIH 3T3
cells and the EJ/vHa-ras- or
RAS1Leudel-transformed NIH 3T3 cells. Therefore,
differences between the untransformed and Ras-transformed phenotypes
are not due to underlying differences in the concentrations of these
downstream Ras signal transduction proteins.
Activation of protein molecules downstream from Ras in the Raf MEK
ERK kinase cascade occurs on their phosphorylation by the preceding
kinase in the cascade (44-46). To assay the phosphorylation state of
c-Raf-1 and c-ERK-1/-2, cells were grown in
[32P]phosphate for 4 h prior to lysis and
immunoprecipitation with c-Raf-1 or c-ERK-1 specific antibodies. The
polyclonal ERK-1 antibody cross-reacts with ERK-2, and both antigens
are immunoprecipitated. 32P-c-Raf-1 was at a 2- to 3-fold
higher concentration in both EJ/vHa-ras- and
RAS1Leudel-transformed cells than in
untransformed NIH 3T3 cells (Fig. 3A,
Table I). Levels of 32P-ERK-1 and
32P-ERK-2 were 9- to 13-fold higher in the
EJ/vHa-ras-transformed cells than in untransformed NIH 3T3
cells (Fig. 3B and Table I). In the
RAS1Leudel-transformed cells, the
32P-ERK-1 and 32P-ERK-2 levels were 6- to
7-fold higher than in NIH 3T3 cells. The difference in
32P-ERK levels between EJ/vHa-ras-transformed
cells and RAS1Leudel-transformed cells does not
appear functionally significant since we observed similar ERK
catalytic rates (see below) in both these
ras-transformed phenotypes.
|
ERK activity was measured directly by an in-gel substrate assay with
the ERK specific substrate myelin basic protein (33, 47).
32P incorporation into myelin basic protein was observed at
a position corresponding to the molecular weights of the ERKs (Fig.
3C). ERK activities were significantly higher in the
EJ/vHa-ras- and RAS1Leudel-transformed cell lines (lanes 2 and 3, 3.4 ± 0.3-fold and 3.0 ± 0.4-fold,
respectively) than for NIH 3T3 controls (Fig. 3C, lane
1). Thus, the high ERK catalytic activities and the
hyperphosphorylation of c-Raf-1 and ERK-1/ERK-2 in the EJ/vHa- and
RAS1Leudel-transformed cells show that the
c-Raf-1 ERK pathway is constitutively activated to approximately an
equal extent in both the uPA- and CL-dependent
ras-transformed phenotypes relative to NIH 3T3 controls.
Western blot analysis of cell
lysates for c-Fos levels showed that levels of c-Fos were equal in the
untransformed NIH 3T3 and EJ/vHa-ras- and
RAS1Leudel-transformed cells
(Fig. 4A). However, Western blot analysis of
c-Jun showed a relatively high concentration of c-Jun in the EJ/vHa-ras-transformed cells (phenotype
rasuPA+/CL), intermediate amounts of c-Jun in
untransformed NIH 3T3 control cells, and low concentrations of c-Jun
in RAS1Leu-del-transformed cells (phenotype
rasCL+/uPA
) (Fig. 4B).
Treatment of cells with [32P]phosphate followed by
immunoprecipitation of the cell lysates with c-Jun antibody,
SDS-polyacrylamide electrophoresis of the immunoprecipitate, and
autoradiography to visualize 32P-labeled proteins showed
two major bands of molecular weights characteristic of both
32P-c-Jun and 32P-c-Fos (Fig.
5B). Western blot analysis of the immunoprecipitate with
anti-Fos antibody showed the band at 61 kDa to be a Fos antigen (Fig.
5C). The 32P-c-Jun and 32P-c-Fos
bands were present at high relative concentrations in the
EJ/vHa-ras-transformed cells, intermediate levels in
untransformed NIH 3T3 cells, and near or below our limits of detection
in the RAS1Leudel-transformed cells (Fig.
5B and Table I). An assay of JNK was made directly against a
substrate GST-Jun-1-135, which specifically assays JNK but not ERK
(33). Fig. 5A shows a 3-fold higher level of
32P-GST-Jun in EJ/vHa-ras-transformed cells than
in untransformed NIH 3T3 cells, while in RAS1Leudel cells
(phenotype rasCL+/uPA), JNK activity is barely
observable (
20% of the level in NIH 3T3 controls).
The lower levels of JNK catalytic activity and 32P-c-Jun in the RAS1Leudel-transformed cells suggests that the JNK activity may be inhibited in the CL-dependent phenotype. Western blot analysis for JNK protein showed similar concentrations in RAS1Leudel-transformed cells and NIH 3T3 cells (data not shown), demonstrating that the low activity found for JNK in RAS1Leudel cells is not due to a decrease in JNK protein levels in these cells but to a lower enzymatic activity of the JNK protein.
Analysis by Mobility Shift Assays of the PEA3/AP-1-like uPA Ras-response ElementThe mouse and human uPA genes have been
sequenced and several functional enhancer elements in their promoter
regions identified (48-50). Lengyel et al. (51) showed that
the Ras response element of the uPA promoter is a PEA3/AP-1-like
element located upstream of the start of transcription between 1973
and
1960 in the human uPA gene. Nerlov et al. (52) had
previously shown this same sequence acts as an uPA enhancer in
transformed human HepG2 and HT 1080 cells. In the mouse uPA promoter,
the homologous element is located
2.4 kb upstream from the start of
transcription (50). The element is similar to the PEA3/AP-1 element
identified as a Ras-response element in the collagenase and stromelysin
protease genes by Wasylyk and co-workers (53) and Wasylyk et
al. (54). However, the consensus AP-1 sequence, TGACTCA, is a
heptamer, and the AP-1-like sequence of the PEA3/AP-1-like element of
the uPA promoter is an octomer sequence (TGAGGTCA in mouse (50) and
TGAaGTCA in human (48)). These octomer sequences of the uPA promoter
are more similar to a consensus octomer CRE enhancer element, TGACGTCA,
than to AP-1. Because of the similarity to both the consensus AP-1 and
CRE, this sequence has been designated URTF (
okinase
ranscription
actor binding element) rather
than AP-1-like by Rørth et al. (50). In agreement with the
nomenclature of Rørth, we refer to this AP-1/CRE-like element as the
URTF element.
Fig. 6 shows gel retardation assays with a
32P-double-stranded DNA sequence containing the PEA3/URTF
enhancer sequence treated with nuclear extracts from
EJ/vHa-ras, RAS1Leudel, and
untransformed NIH 3T3 cells. The gel shifts show two bands (bands
1 and 2) corresponding to protein-DNA complexes of faster and slower mobilities that are competed by unlabeled PEA3/URTF. The slower mobility complex (band 1) is present in high
concentrations in the EJ/vHa-ras-transformed cells,
intermediate concentrations in untransformed NIH 3T3 cells, and low
concentrations in RAS1Leudel-transformed cells
(Fig. 6). The variation in concentration of band 1 among the three cell
lines positively correlates with the previously reported levels of uPA
mRNA and uPA gene transcription rates among the three cell lines
(11).
Fig. 7A shows gel retardation experiments
with the double-stranded deoxyoligonucleotide sequences, given in
Table II, in EJ/vHa-ras- and
RAS1Leudel-transformed cells. The gel
retardation of the AP-1 consensus sequence gives an intense broad band
with the nuclear extract of EJ/vHa-ras-transformed cells and
a less broad and less intense band with
RAS1Leudel-transformed cells (Fig.
7A, lanes 1 and 6). A complex is not formed with the mutant AP-1 sequence (lanes 2 and
7). The broadness of the AP-1 complex in the gel retardation
experiments may be due to the presence of multiple members of the Jun,
Fos, and CREB/ATF families that can associate with the consensus AP-1
enhancer element. A similar broad AP-1 band has been previously
observed in other cell types and shown to be due to the binding of
multiple AP-1 proteins to the AP-1 enhancer element (55). The narrower
and less intense band observed for the AP-1 complex with the
RAS1Leudel-transformed cells indicates the
presence of lower concentrations and/or less diversity of AP-1 binding
proteins in these cells. In addition to the broad AP-1 band, the
RAS1Leudel-transformed cells also show a slower
migrating AP-1 DNA-protein complex band with identical mobility to band
2 observed with the PEA3/URTF sequence (lane 6). While the
consensus AP-1 forms a broad protein-DNA complex band, the mutant AP-1
sequence formed no strong protein-DNA complexes (lanes 2 and
7). The URTF element shows a complex with overlapping
mobility to the complexes observed with the consensus AP-1 in both
EJ/vHa-ras- and
RAS1Leudel-transformed cells (lanes 3 and 8). However, the URTF bands are not as intense or
broad as that seen for the consensus AP-1 sequence.
|
The mutant PEA3/URTF sequence showed no retarded complex with the nuclear extract of EJ/vHa-ras-transformed cells (Fig. 7A, lane 4). However in the RAS1Leudel-transformed cells, a complex with mobility identical to that of the band 2 complex is observed with the mutant PEA3/URTF sequence (Fig. 7A, lane 9). A similar mutant PEA3/URTF has been shown to be unresponsive to Ras stimulation in a chloramphenicol acetyltransferase enhancer assay system (51), suggesting that the band 2 complex formed between mutant-PEA3/URTF and nuclear proteins in RAS1Leudel-transformed cells is non-functional.
Fig. 7B shows the competition between 32P-labeled sequences and unlabeled sequences for proteins in gel retardation. In these experiments, only the nuclear extracts of the EJ/vHa-ras-transformed cells were utilized. The experiment shows the broad complex formed with the consensus 32P-AP-1 sequence is completely competed by a 100-fold excess unlabeled AP-1. The 32P-URTF sequence is competed by both the unlabeled URTF sequence and by the unlabeled consensus AP-1 sequence, indicating an overlap of binding specificity between the consensus AP-1 and URTF sequences. The 32P-PEA3/URTF complex (band 1) is efficiently competed by the unlabeled consensus AP-1 sequence. This competition further indicates an overlap of specificity between the consensus AP-1 sequence and the URTF sequence of the mouse uPA PEA3/URTF enhancer.
Fig. 8 shows the inhibition of the PEA3/URTF band 1 by
both a PEA3 antibody and an antibody against c-Jun, indicating the presence of both PEA3 and c-Jun specific antigens in the band 1 complex. The inhibition of complex formation by anti-PEA3 and anti-c-Jun specific antibodies is in agreement with previously reported
experiments showing that Ets transcription factors and c-Jun
trans-activate through the PEA3/AP-1-like element (48, 50-52, 56-58).
Conversion of the rasuPA+/CL
A dominant
negative c-Jun, TAM67, missing the c-Jun N-terminal
trans-activating domain (amino acids 3-122) was transfected into the EJ/vHa-ras-transformed,
RAS1Leudel-transformed, and NIH 3T3 cells. The
expression of the TAM67 gene in G418-resistant clones was shown by
Northern analysis to migrate at 868 base pairs, while native c-Jun
mRNA migrates at approximately 2.7-3.2 kilobase pairs. Of 11 clones analyzed, 5 were positive for TAM67 mRNA expression. The
levels of CL mRNA and uPA mRNA were then determined in all
TAM67 mRNA-expressing clones. Fig. 9 shows the
results for controls transfected with pCMV-empty vector and for all 5 of the EJ/vHa-ras-TAM67 clones isolated. In cells
transfected with pCMV-empty vector, all G418-resistant clones tested
maintained their expected phenotype. Also as expected, RAS1Leudel and NIH 3T3 cells, which normally
contain low constitutive levels of c-Jun (Fig. 4), show no changes in
protease phenotype on their transfection with TAM67 (data not shown).
However, TAM67-expressing EJ/vHa-ras cells showed a
conversion in levels of uPA and CL mRNAs from those of a
rasuPA+/CL phenotype to levels characteristic
of RAS1Leudel-transformed cells of
phenotype rasCL+/uPA
. These data suggest
that activation of c-Jun is necessary for the
rasuPA+/CL
phenotype and inhibition of c-Jun
activity generates a switch to the rasCL+/uPA
phenotype.
Previous data from our laboratory demonstrated that two distinct
metastatic phenotypes were generated in NIH 3T3 cells transformed by
EJ/vHa-ras and RAS1Leudel genes (11).
One phenotype, designated rasuPA+/CL and
represented by the EJ/vHa-ras-transformed NIH 3T3 cells, shows high constitutive levels of uPA mRNA and low constitutive CL
mRNA levels. Experiments utilizing uPA and CL antisense
oligonucleotides to selectively suppress uPA or CL gene expressions
demonstrated that lung colonization by
EJ/vHa-ras-transformed cells was uPA-dependent and CL-independent. In contrast, the second ras-transformed
phenotype, designated rasCL+/uPA
and
represented by RAS1Leudel-transformed NIH 3T3 cells, shows
high constitutive levels of CL mRNA and low constitutive levels of
uPA mRNA. Antisense oligonucleotide suppression experiments
demonstrated that lung colonization of
RAS1Leudel-transformed cells was
CL-dependent and uPA-independent. Both these cell lines
were found to give a similar large number of tumors in nude mice lung
colonization assays (11). This report characterizes the signal
transduction pathways downstream from mutant Ras for the two redundant
metastatic ras-transformed phenotypes that utilize either
uPA or CL to achieve experimental metastasis. Metastasis is an ECM
invasive process that requires proteases to degrade the proteins of the
ECM to allow the movement of migrating cells through the ECM (6).
Fig. 1 displays pathways downstream from Ras that lead to the
activation of gene transcription factors. The pathway through c-Raf-1
MAPK kinase/MEK
MAPK/ERK-1/ERK-2 leads to activation by
phosphorylation of transcription factors of the Ets family and Myc
(59-61). Early studies indicated that the ERK1 and/or the ERK2 may
also activate c-Jun and c-Fos (62, 63). While some evidence exists for
activation of c-Jun and c-Fos by ERK (40, 62, 63), phosphorylation of
c-Jun by ERK may also inhibit c-Jun mediated
trans-activations (40). Accordingly, whether activation of
c-Jun occurs by ERK phosphorylation is not clear. However, c-Jun and
c-Fos activation does occur on phosphorylation by the c-Jun and c-Fos
specific kinases, JNK and FRK, that are distinct from the MAPKs ERK-1
and ERK-2. The activation of JNK and FRK occur via a
Ras-dependent pathway not involving c-Raf-1, MEK, or ERK
(31, 38-43) (Fig. 1).
The concentrations of c-Raf-1, ERK-1, and ERK-2 in the Ras ERK
pathway were determined by Western blot analysis and shown to be at
approximately equal concentrations in the
EJ/vHa-ras-transformed cells, the
RAS1Leudel-transformed cells, and the
untransformed NIH 3T3 cells (Fig. 2). Accordingly, differences in the
transformed phenotypes are not due to underlying differences in the
cellular concentrations of these proteins. However, the
[32P]phosphate-labeled forms of c-Raf-1, ERK-1, and ERK-2
were present at high concentrations only in the
ras-transformed cells (Fig. 3, A and
B, and Table I). Direct kinase assays confirm the high activity of MAPK/ERK in both ras-transformed phenotypes
(Fig. 3C). Therefore, the c-Raf-1
ERK pathway may be
required for transformation by both mutant Ras isoforms.
This agrees with evidence from other laboratories in which transfection
of NIH 3T3 cells with constitutively active forms of Raf or MEK lead to
transformation. Conversely, experiments in which the Raf
ERK
pathway is inhibited by dominant negative c-Raf-1, ERK, or phosphatase
deactivators of ERK have been shown to lead to reversion of Ras-induced
transformation (26, 64-66).
In contrast to the observation of ERK activation in both
ras-transformed phenotypes, the c-Jun activating kinase,
JNK, shows high activity only in the EJ/vHa-ras transformed
cells while its activity is barely detectable in the
RAS1Leudel-transformed cells (Fig.
5A). Not surprisingly since c-Jun is known to autoregulate
its own expression (67), we found low levels of both c-Jun and
32P-c-Jun levels in
RAS1Leudel-transformed cells (Figs.
4B and 5B). The low levels of JNK activity and
c-Jun in RAS1Leudel-transformed cells relative to NIH 3T3
controls suggests that the rasCL+/uPA
phenotype is JNK- and c-Jun-independent. This result may be surprising, as inhibition of c-Jun expression has been shown to cause reversion of
the transformed phenotype in several ras-transformed cell
lines. For example, a dominant-negative mutant of c-Jun has been shown to partially revert Ki-ras-transformed
NIH 3T3 cells to a normal phenotype (68, 69). However, other data have
shown that rat embryo fibroblasts, unable to express c-Jun, could be transformed into a tumorigenic and metastatic phenotype by an activated
ras gene (70). Our data show in the
RAS1Leudel-transformed cells low concentrations
of c-Jun and phosphorylated-c-Jun, corresponding with an inhibition of
JNK activity and indicating that a c-Jun-independent transformation
pathway exists in the RAS1Leudel-transformed
cell line. In addition, the inhibition of c-Jun activity with TAM67 in
EJ/vHa-ras- and
RAS1Leudel-transformed cells results in the
maintenance of the transformed state as assayed by colony formation in
soft agar (data not shown), supporting the existence of
c-Jun-independent transformed phenotypes.
The Ras-response element in the uPA promoter has been shown in
ras-transformed human OVCAR cells to be the PEA3/URTF
sequence (51). The mouse uPA promoter contains an upstream PEA3/URTF enhancer sequence identical to the human sequence, except for a single
G A change in the URTF site (see Table II). A PEA3/URTF enhancer
has also been found in the porcine uPA gene promoter and has an
identical sequence to that of mouse (57). Mutational studies of the
PEA3/URTF sequence in chloramphenicol acetyltransferase expression
vectors with native and mutant human PEA3/URTF sequences have shown
that both the PEA3 and URTF elements are required for enhancer
stimulation by Ras (51). The PEA3 element binds members of the Ets
family of transcription factors which are activated upon stimulation of
the Raf/ERK pathway (61). The URTF element in the human PEA3/URTF
sequence appears to be trans-activated in
ras-transformed human OVCAR cells by c-Jun as a dominant
negative c-Jun, TAM67, inhibits PEA3/URTF enhancer stimulation (51). Further evidence that c-Jun activates the URTF enhancer comes from
experiments in porcine LCC-PK1 cells in which okadaic acid inhibition of the JNK phosphatase increased the activity of JNK, the
corresponding concentration of 32P-c-Jun, and the enhancer
activity of the URTF element in the PEA3/URTF sequence (57). The
immunological characterization of the URTF gel retardation complex with
human HepG2 cell nuclear extract by monospecific antibodies showed the
complex to be a heterodimer of c-Jun and ATF-2 (58). It has been
suggested that ATF-2 is activated by the same pathway as c-Jun (43)
(Fig. 1). Activation of the Ras-response element in the uPA promoter
thus appears to require the activation of an Ets family transcription factor through stimulation of the c-Raf-1
ERK-1/ERK-2 pathway (51,
61, 68, 71) and of c-Jun and perhaps ATF-2 through activation by the
JNK pathway (43, 51, 57, 58). In this work, we show by gel shift
analysis that the intensity of one of the retarded bands (band 1)
positively correlates with uPA mRNA levels and the rate of uPA
expression among the cell lines (Figs. 6 and 7). Other bands were shown
to be nonspecific since they appear in the presence of a mutant URTF
sequence and/or with the consensus AP-1 alone (Fig. 7). Further
analysis of band 1 supports the AP-1-like character of the octomer URTF
mouse sequence in the PEA3/URTF element. The URTF gel retardation band
was shown to be competed by a consensus AP-1, and the consensus AP-1
also competed for the band 1 proteins of the complete PEA3/URTF (Fig. 7). Furthermore, an antibody to the AP-1 transcription factor, c-Jun,
prevented formation of the PEA3/URTF gel retardation band 1 (Fig. 8).
An antibody to transcription factor PEA3, part of the PEA3 subfamily of
the Ets family of transcription factors, also inhibited the formation
of the PEA3/URTF band 1 complex (Fig. 8). PEA3 proteins are a subfamily
of the Ets family of transcription factors. Recently discovered members
of the Ets family of transcription factors that may regulate uPA
through the PEA3 element include ERM, a ubiquitous member of the PEA3
subfamily, and the Ets transcription factor E1A-F, which has been shown
to up-regulate the stromelysin, type I collagenase, and 92-kDa type IV
collagenase genes (72, 73).
Therefore, the simplest explanations for our data are that: (1) in the
untransformed NIH 3T3 cells, neither the ERK or JNK signaling pathways
are active, leading to low levels of active Ets and c-Jun/ATF-2 and low
constitutive expression of uPA and CL. (2) In the EJ/vHa-ras
cells, both the Ras ERK and the Ras
JNK pathways are active,
and their combined activities are required to activate the PEA3 and
c-Jun transactivating proteins on the uPA PEA3/URTF enhancer. (3) In
the RAS1Leudel-transformed cells, the activated
Ets transcription factors are present since the Ras
ERK pathway is
active, but JNK activity is absent and therefore insufficient levels of
active c-Jun exist to activate the uPA PEA3/URTF enhancer. We have
shown a switch in phenotype from rasuPA+/CL
to
rasCL+/uPA
by the selective inhibition of
c-Jun with TAM67 in EJ/vHa-ras cells. Thus, the presence of
active Ets and an absence of active c-Jun lead to increased expression
of CL by an unknown mechanism. We cannot at present eliminate the
possibility that other pathways downstream of Ras are active in the
EJ/vHa-ras- and
RAS1Leudel-transformed cells.
Only recently have the details of redundant pathways begun to be explored as greater knowledge of the similarities and differences of functionally homologous protein families become available. We have shown that in ras-transformed NIH 3T3 cells, redundant transformed phenotypes exist that differ in both signaling pathway activations and genes expressed. While we have studied only changes in the regulation of uPA and CL gene expressions, we expect that the differential activation of pathways we observe affect a whole spectrum of gene expressions.