(Received for publication, April 21, 1995; and in revised form, July 31, 1995)
From the
The expression of the urokinase-type plasminogen activator, which plays a crucial role in tissue remodeling by controlling the synthesis of the broadly acting plasmin serine protease, is regulated by several tyrosine kinases. Since the actions of these tyrosine kinases is dependent on the activation of ras proteins, we undertook a study to identify signaling events downstream of ras responsible for the stimulation of urokinase promoter activity. Transient expression of an activated c-Ha-ras in OVCAR-3 cells, which do not harbor the mutated oncogene, led to a dose-dependent trans-activation of the urokinase promoter. A sequence residing between -2109 and -1964 was critical for the stimulation of the urokinase promoter by c-Ha-ras. Mutation of an AP-1 and a PEA3 site at -1967 and -1973, respectively, or the co-expression of a transactivation domain-lacking c-jun substantially impaired the ability of c-Ha-ras to stimulate urokinase promoter activity. The induction of the urokinase promoter by ras was completely blocked by expression of a dominant negative c-raf expression vector and substantially reduced in cells made to co-express a catalytically inactive mitogen-activated protein kinase kinase. Further, the expression of an ERK1/ERK2-inactivating phosphatase (CL100) abrogated the stimulation of the urokinase promoter by c-Ha-ras. These data argue for a role of a mitogen-activated protein kinase-dependent signaling pathway in the regulation of urokinase promoter activity by ras.
The urokinase-type plasminogen activator has been implicated in
a variety of physiological and pathological processes including
prostatic involution, cytotrophoblast implantation, and tumor cell
invasion, all of which require extensive extracellular matrix
proteolysis(1, 2, 3) . Urokinase facilitates
this process by mediating the conversion of the abundant zymogen,
plasminogen, into the widely acting serine protease plasmin, the latter
which degrades several extracellular matrix components including
laminin, fibronectin, and possibly type IV
collagen(4, 5) . Transcription of the urokinase gene
gives rise to a 2.5-kilobase mRNA(6) , which is subsequently
translated into a single chain 50-kDa proenzyme (7, 8) and secreted into the extracellular space. The
steady state level of urokinase mRNA is determined at two levels.
Firstly, the expression of the gene is regulated by 2.1 kilobases of
the 5`-flanking sequence(9) . Secondly, recent studies have
indicated that 3`-untranslated sequences play a role in determining the
stability of the urokinase mRNA(10) .
Several studies have
indicated that urokinase expression is regulated by growth factors
(HGF/SF, EGF), ()which bind to transmembrane receptor
protein tyrosine kinases, and by non-receptor protein tyrosine kinases
including v-src and
v-yes(11, 12, 13) . One of the early
events in this pathway involves the coupling of the ligand-activated
receptor protein tyrosine kinase to ras through Grb2.sos or
Shc Grb2.sos, this increasing the exchange of GTP for GDP and
culminating in an activated ras(14, 15) . The
pivotal role of ras in mediating the signal generated by
tyrosine kinases has been deduced from several experimental
observations. First and foremost, inhibitory ras mutant
proteins (16) block the stimulation of growth and
transformation of NIH 3T3 cells by oncogenic tyrosine kinases.
Secondly, revertant clones isolated from activated ras-transformed NIH 3T3 cells are resistant to
retransformation by protein tyrosine kinase-encoding
oncogenes(17) . Thirdly, ras, like protein tyrosine
kinases, elevates urokinase mRNA levels in chick embryo
fibroblasts(13) .
However, the events connecting the ras signal to altered gene expression are still not entirely clear.
Certainly, the involvement of c-raf, mitogen-activated protein
kinase kinase (MAPKK also referred to as MEK) and mitogen-activated
protein kinases (MAPK), which form successive tiers in a ras-dependent signal transduction pathway, has been suggested
from several reports. c-raf is a protein serine/threonine
kinase which acts as a cytoplasmic signal transducer downstream of ras(18) and is an immediate upstream activator of
MAPKK (MEK1)(19, 20) . MEK1, in turn, activates the
extracellular signal-regulated kinase (ERK) group of MAPK (21) which regulates AP-1 activity via increased transcription
of the c-fos gene (22) as a consequence of
p62/elk phosphorylation. Alternatively,
modulation of gene expression by ras can also be achieved in a
c-raf-independent pathway involving the c-jun amino-terminal kinase (JNK)(23, 24) .
Although ras increases the steady state level of urokinase mRNA(13) , the mechanism by which this is accomplished has not been elucidated. We therefore undertook a study to determine the role of a MAPK pathway in the regulation of urokinase expression by c-Ha-ras. We report, herein, that the stimulation of urokinase promoter activity by ras, is dependent on one, or multiple, MAPKs and is mediated via the stimulation of an AP-1 and PEA3 sequence located at -1967 and -1973, respectively, as well as a region residing between -1956 and -1905 in the urokinase promoter.
Figure 1: OVCAR-3 cells are low expressors of urokinase. Conditioned medium collected from OVCAR-3 cells and from the high urokinase-producing OC-7 cells (Positive Control) was harvested, clarified by centrifugation, and the cells were enumerated. Conditioned medium, normalized to cell number, and authentic, recombinant, urokinase (rSC-uPA) was denatured, in the absence of reducing agent, and electrophoresed in a 12.5% acrylamide gel. The resolved proteins were transferred to a nitrocellulose filter. The filter was blocked with a solution containing 3% bovine serum albumin and incubated with a polyclonal antibody to human urokinase. Reactive proteins were visualized by ECL.
Figure 2:
Transient expression of c-Ha-ras increases urokinase promoter activity in a dose-dependent manner.
OVCAR-3 cells were transiently transfected at 50% confluency with 10
µg of a CAT reporter driven by the wild type (2109 bp) urokinase
promoter in the absence, or in the presence, of varying amounts of a
vector bearing the c-Ha-ras sequence or 4 µg of the vector
alone (pSV2neo). All transfections were performed in the
presence of 5 µg of a -galactosidase-expressing vector. After
5 h, the medium was changed and cells were cultured for an additional
36 h. The cells were harvested and assayed for
-galactosidase
activity. Cell extracts, corrected for differences in transfection
efficiency, were incubated with
[
C]chloramphenicol for 8 h. The mixture was
extracted with ethyl acetate and subjected to thin layer
chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope.
Figure 3:
Activation of the urokinase promoter by
c-Ha-ras requires a sequence residing between -2109 and
-1964 in the 5`-flanking sequence of the urokinase gene. OVCAR-3
cells were transiently co-transfected, as described in the legend to Fig. 2, with 4 µg of the c-Ha-ras expression vector
or an equimolar amount of the pSV2neo vector, 10 µg of a CAT
reporter driven by the indicated 5`-deleted fragment of the urokinase
promoter, and 5 µg of a vector encoding the -galactosidase
gene. After 48 h, the cells were harvested and transfection
efficiencies were determined by
-galactosidase activity. Cell
extract, normalized for transfection efficiency, was incubated with
[
C]chloramphenicol for 8 h, and acetylated
products were extracted with ethyl acetate and subjected to thin layer
chromatography. The conversion of
[
C]chloramphenicol to acetylated derivatives was
determined with a 603 Betascope.
Figure 4:
The stimulation of urokinase promoter
activity by c-Ha-ras requires AP-1 and PEA3 binding sites in
the 5`-flanking sequence of the urokinase gene. OVCAR-3 cells were
transiently co-transfected, as described in the legend to Fig. 2, with 8 µg of a CAT reporter driven by the wild type (WT) or the point-mutated (A, B, I, F, G) urokinase promoter with (+) or
without(-) 4 µg of c-Ha-ras expression vector or the
empty expression vector alone (pSV2neo). CAT activity is
indicated numerically as the percent of
[C]chloramphenicol converted. The schematic to
the right shows a representation of the mutated region (solid boxes) of the urokinase promoter. The numbers above
the arrows refer to the position relative to the urokinase
transcription start site. Mutated urokinase promoter constructs are
described in more detail
elsewhere(32) .
Figure 5:
The stimulation of the urokinase promoter
by c-Ha-ras is attenuated by the expression of a
transactivation domain-lacking c-jun mutant. OVCAR-3 cells
were transiently transfected with 4 µg of a CAT reporter driven by
the wild type urokinase promoter and, where indicated, 4 µg of the
c-Ha-ras expression vector and the indicated amount of an
expression vector encoding a transactivation domain-lacking c-jun protein (TAM 67) or an amount of the empty cytomegalovirus vector
equimolar to 10 µg of TAM 67. Transfection efficiencies were
determined by assaying for -galactosidase activity. After an 8-h
incubation with [
C]chloramphenicol, the cell
lysate was extracted with ethyl acetate, and the acetylated products
were resolved by thin layer chromatography. The conversion of
[
C]chloramphenicol was determined with a 603
Betascope.
Figure 6:
Abrogation of the
c-Ha-ras-dependent induction of urokinase promoter activity by
an ERK1/ERK2-inactivating phosphatase. OVCAR-3 cells were transiently
transfected and assayed for CAT activity as described in the legend to Fig. 2. Cells were transfected with a CAT reporter driven by
either the wild type urokinase promoter (7 µg) (A) or 3
AP-1 tandem repeats (3 AP-1 pBLCAT) upstream of a thymidine
kinase minimal promoter (pBLCAT) (1 µg) (B) with (+),
or without(-), 3 µg of the c-Ha-ras expression
vector (c-Ha-ras) or a vector control (pSV2neo) in the
presence of the indicated amount of the CL100-encoding expression
vector (CL100 SG5) or the empty vector (pSG5 vector equimolar with 10
µg of CL100 SG5). Cell extract, normalized for differences in
transfection efficiency, was incubated with
[
C]chloramphenicol, and the reaction products
were extracted with ethyl acetate and subjected to thin layer
chromatography. The conversion of
[
C]chloramphenicol was determined with a 603
Betascope.
Since AP-1-binding transcription factors
are required for the stimulation of the urokinase promoter by ras, we determined whether the expression of CL100 could also
repress the induction of an AP-1-driven promoter by ras.
OVCAR-3 cells were transiently transfected with a CAT reporter driven
by 3 AP-1 tandem repeats (3 AP-1 pBLCAT) upstream of a
thymidine kinase minimal promoter (pBLCAT) and the ras expression vector with, or without, the CL100-encoding expression
vector. Expression of ras caused a marked stimulation of the
AP-1-driven CAT reporter (Fig. 6B) in OVCAR-3 cells,
whereas the activity of the thymidine kinase minimal promoter-CAT
reporter (pBLCAT), which was negligible, was unaffected by the
oncogene. Co-expression of the CL100-encoding vector (CL100 SG5)
countered the ras-dependent induction of the AP-1-driven CAT
reporter. The highest level of the CL100-encoding vector (10 µg),
but not the empty vector (pSG5), completely abolished the stimulation
of the AP-1-driven CAT reporter by ras. Although the
CL100-encoding vector also repressed the basal activity of the 3
AP-1 pBLCAT vector, the amount of this repression was
insufficient to account for the MAPK phosphatase-mediated suppression
of the ras response. These data indicate that the induction of
AP-1 activity by ras, which is critical for the stimulation of
the urokinase promoter by the oncogene, is effectively countered by
co-expression of a MAPK-inactivating phosphatase.
Figure 7:
Expression of a catalytically inactive
MAPKK attenuates the ability of ras to stimulate the urokinase
promoter. OVCAR-3 cells were transiently transfected and assayed for
CAT activity as described in the legend to Fig. 2. Cells were
co-transfected with 7 µg of a CAT reporter driven by the wild type
urokinase promoter (u-PA) with (+) or without(-) 4
µg of the c-Ha-ras expression vector (c-Ha-ras)
in the presence of the indicated amount of an expression vector
encoding either the wild type (MEK wt) or point-mutated (MEK K97M) MEK1 sequence(29) . Cell extract,
normalized for differences in transfection efficiency, was incubated
with [C]chloramphenicol, and the reaction
products were extracted with ethyl acetate and subjected to thin layer
chromatography. The amount of acetylated
[
C]chloramphenicol was determined on a 603
Betascope.
Figure 8:
Abrogation of the
c-Ha-ras-dependent induction of urokinase promoter activity by
expression of a dominant negative c-raf. OVCAR-3 cells were
transiently transfected and assayed for CAT activity as described in
the legend to Fig. 2. Briefly, cells were transfected with 7
µg of a CAT reporter driven by the wild type urokinase promoter
with (+) or without(-) 4 µg of the c-Ha-ras expression vector (c-Ha-ras) in the presence of the
indicated amount of an expression vector encoding a kinase
domain-lacking c-raf (raf C4) or an amount of the
empty expression vector (Vector) equimolar with 4 µg of
the raf C4. Cell extract, normalized for differences in
transfection efficiency, was incubated with
[C]chloramphenicol, and the reaction products
were extracted with ethyl acetate and subjected to thin layer
chromatography. The amount of acetylated
[
C]chloramphenicol was determined on a 603
Betascope.
Urokinase gene expression is regulated by a diverse set of
growth factors which interact with transmembrane receptor tyrosine
kinases including HGF/SF and EGF as well as by non-membrane tyrosine
kinases including
v-src(11, 12, 13) . Although there
is ample evidence that these tyrosine kinases mediate their effects via ras-dependent pathways(16) , the downstream event(s)
by which ras stimulates urokinase gene expression has not been
determined, and it now appears that ras can stimulate at least
2 distinct pathways, one which is c-raf-dependent, the other
c-raf-independent(24) . Stimulation of the
c-raf-dependent pathway by ras leads to the
sequential activation of MAPKK and the ERK group of the MAPKs (19, 20, 21) . The ERKs phosphorylate several
transcription factors including p62/elk, this
leading to increased AP-1 activity as a consequence of enhanced fos gene transcription (22) . Several observations in this,
and other, studies suggest that the induction of urokinase promoter
activity by ras occurs through this pathway. Firstly,
expression of the ERK1/ERK2-inactivating CL100 phosphatase efficiently
blocked the ras signal in OVCAR-3 cells. Secondly, an
expression construct encoding a catalytically inactive MEK1, which is
specific for the ERKs, substantially reduced the ability of ras to
stimulate the urokinase promoter. Thirdly, the block of c-raf,
which is an upstream effector of MEK1(19, 44) , with a
dominant negative expression vector, completely abrogated the ras-dependent stimulation of the urokinase promoter. Thus,
together these data would suggest a pivotal role for one, or multiple,
ERKs in the stimulation of the urokinase promoter by ras and
are consistent with the hypothesis that this is achieved by the
c-raf-dependent pathway described by Minden et
al.(24) . Additionally, since dominant negative mutants to
ERK1 and ERK2 also block AP-1 activation (an event necessary for the
induction of the urokinase promoter by ras) as well as the
proliferation response to growth factors including
EGF(40, 46) , it may very well be that the ability of
these stimuli to elevate urokinase expression (47) is mediated
through this pathway.
Further evidence that the regulation of urokinase expression by ras is mediated through a c-raf-dependent pathway comes from studies with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. TPA, via protein kinase C, is an activator of c-raf(48) , and treatment of cells with this agent leads to a strong activation of urokinase gene transcription(49) . The transcriptional requirements for urokinase promoter stimulation by TPA (32) and ras are similar in that mutation of the PEA3 and AP-1 motifs (-1973 and -1967, respectively), as well as a region shown to be required for the efficient induction of urokinase gene expression by the phorbol ester (32) , substantially impaired the ability of the oncogene as well as TPA to stimulate the promoter. Presumably, these findings for ras reflect the well characterized observations of increased AP-1 and PEA3 activity brought about by the oncogene as a consequence of increased synthesis, or activation by phosphorylation, of c-jun, c-fos, and c-ets family members (50, 51) .
While these data suggest the involvement of ERKs in the regulation of urokinase expression by ras, we cannot currently exclude a role for the JNKs in this process. ras, via MEKK, is an inducer of JNK(24) , and this leads to increased AP-1 activity subsequent to c-jun phosphorylation(23) . However, any speculation as to the role of JNKs in the regulation of urokinase expression by ras must take into account our observations that interfering with the ERK pathway with a MEK1 mutant, or with a dominant negative c-raf expression vector, effectively suppresses the ras stimulus. Thus, either the JNKs play only a minor role in the regulation of urokinase expression by the oncogene or alternatively both ERKs and JNKs are concurrently required for the stimulation of urokinase promoter activity by ras. Notwithstanding these considerations, our findings favor a key role for the ERKs in the stimulation of urokinase promoter by ras.
In conclusion, we
have shown that the induction of urokinase promoter activity by an
activated c-Ha-ras requires a PEA3/AP-1 sequence in the
urokinase promoter and is mediated through a c-raf and
MAPK-dependent signal transduction pathway. Since several tyrosine
kinases including the receptors for EGF (c-erbB1), and HGF/SF
(p190), as well as the non-receptor tyrosine kinase
v-src, mediate their effects through ras, it is
interesting to speculate that c-raf and/or the MAPKs may
represent novel therapeutic targets for repressing the elevated
synthesis of the plasminogen activator in invasive cancer which is
driven by both autocrine and paracrine mechanisms.