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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departament de Oncològia Molecular,
Institut de Recerca Oncològica (IRO), Barcelona, Spain; Facultat
de Farmacia, Universitat de Barcelona, Barcelona, Spain.
The monofunctional alkylating agent
N-methyl-N-nitro-N-nitrosoguanidine (MNNG) is
a widespread environmental carcinogen that causes DNA lesions, leading
to cell death. However, MNNG can also trigger a cell-protective
response by inducing the expression of DNA repair/transcription-related
genes. We demonstrate that the urokinase-type plasminogen activator
(uPA) gene product, a broad spectrum extracellular protease
to which no DNA repair function has been assigned, is transcriptionally
induced by MNNG in C2C12 and NIH3T3 cells. This induction required an
AP1-enhancer element located at Urokinase-type plasminogen activator (uPA)
is a secreted serine protease that converts the zymogen plasminogen to
plasmin, a trypsin-like serine protease capable of degrading
extracellular matrix components and of activating other proteinases
(for revision see Irigoyen et al1). Analysis of the
consequences of loss in uPA function of uPA-deficient mice has
confirmed the participation of this protease in physiologic processes
such as fibrinolysis and angiogenesis, as well as in pathologic events
such as wound healing, inflammation, and tumor
invasiveness.2-9 In addition to these proteolytic
functions, recent studies have demonstrated mitogenic and chemotactic
properties of uPA through interaction with its cell surface
high-affinity receptor.10,11 Reflecting its wide spectrum
of functions, uPA expression is regulated by numerous extracellular
stimuli depending on the cell type. uPA gene transcription
can be induced by growth factors, phorbol esters, cytokines,
cytoskeletal reorganization, and several
oncoproteins.12-22
We have recently shown that ultraviolet (UV) light irradiation induces
the expression of urokinase-type plasminogen activator (uPA)
gene in NIH3T3 fibroblasts.23 uPA was the first protein shown to be inducible in xeroderma pigmentosum cells at much lower UV
doses than in parental heterozygotic cells,24 suggesting that DNA damage might be responsible for this induction. Damage to DNA
can be inflicted by a broad spectrum of agents, including UV light,
ionizing radiation (IR), and alkylating agents. Recent studies have
demonstrated that extracellular uPA activity is inversely related to
the cell capacity to repair the DNA lesions induced by alkylation
agents such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG).25 Monofunctional alkylating agents like MNNG and
methyl-methanesulfonate (MMS) are widely distributed environmental
mutagens and carcinogens that, on activation, react with DNA and
proteins generating aducts.26-28 Among the aducts,
O6-alkyl guanine (generated by N-alkylation of the DNA base) is the
predominant cytotoxic and mutagenic lesion, because of its mispairing
properties, which leads eventually to chromosomal aberrations, point
mutations, and cell killing.29,30 This lesion also appears
to be involved in tumor induction, in particular gastric
carcinogenesis.31-34 However, monofunctional alkylating
agents not only cause cell destruction, but also induce the
transcription of many genes, including genes coding for transcriptions factors such as c-fos, c-jun, junB, and junD,35 for cell
cycle regulatory proteins such as p53 and p21,36-38 for
growth arrest and DNA damage (GADD) proteins39,40 and for
DNA repair proteins such as O6-methylguanine-DNA-methyltransferase
(MGMT) and DNA polymerase In this study, we show that the monofunctional alkylating agent MNNG is
a potent inducer of uPA gene expression in 2 different murine cell lines. Specifically, we have examined the mechanism(s) of
MNNG-induced uPA transcription. This induction required an AP1-enhancer element, which is located at Cell culture
RNA analysis
Plasmids The p-8.2Luc, a murine uPA-promoter luciferase reporter plasmid (kindly provided by Dr Y. Nagamine), contains 8.2 kb of murine uPA promoter region, as described.13 The p-2.0Luc ( 2 kb of murine uPA promoter), p-4.9Luc (4.9 kb of uPA promoter), and p-6.6Luc (6.6 kb of uPA promoter) were derived from
p-8.2Luc.13,23,72 the p-8.2( AP1)Luc, p-6.6(AP1)Luc,
and p-4.9(AP1)Luc, lacking the AP1 enhancer element, are described
elsewhere.13,23,72 Expression vectors pSR -MEKK1(K432M),
pcDNAIII-MKK6b(A), pcDNAIII-JIP-1, pcDNAIII-SEK1/MKK4(KR),
pcDNAIII-MKK7(A) were kindly provided by Drs M. Karin, J. Han, R. Davis, and E. Nishida.57,70,73-77
Western blotting Cells were cultured in 0.5% FBS and, at the indicated time points after treatment, whole cell extracts (WCE) were prepared as described in Miralles et al.23 Total JNK1 and ERK2 were detected in 30 µg WCE by immunoblotting using specific antibodies at 1:1000 dilution. JNK1 and ERK antibodies are from Santa Cruz Biotechnology (sc-474 and sc-154, respectively). Alternatively, phosphorylated p38 was detected using an antiphospho p38 antibody (New England Biolabs No 9211S). Immunoblots were developed using ECL detection system (Amersham).Protein kinase assays JNK and ERK were immunoprecipitated from WCE with anti-JNK1 and anti-ERK2 antibodies, respectively, and immunocomplexes were recovered with protein A-Sepharose and washed, as described previously.23 Phosphorylation reactions were performed in a 30-µL volume containing kinase buffer supplemented with 20 µmol/L ATP, 0.0185 MBq (0.5 µCi) -[32P]ATP and 1 µg GST-cJun1-79 or myelin basic protein (MBP) as
substrates for JNK and ERK assays, respectively, at 30°C for 30 minutes. Reactions were stopped by the addition of 4 × Laemmli sample
buffer and resolved by 10% or 12% SDS-PAGE for JNK or ERK assays, respectively.
Transfections assays 2.5 × 104 cells were cotransfected using the transfection reagent DOTAP (Boehringer Mannheim) with 300 ng of uPA-luciferase plasmid and 50 ng of RSV- Gal, as internal control.
After transfection, cells were cultured in DMEM containing 0.5% FBS
for 16 hours before MNNG stimulation (70 µmol/L), and reporter
activities were analyzed after 8 hours. When indicated, cells were
cotransfected with 150 ng of reporter plasmid and 150 ng of expression
plasmids or empty vector alone, together with 50 ng of internal
control. Inhibition of MEK and p38 kinase was performed by pretreating
transfected cells with 50 µmol/L PD98059 and 10 µmol/L SB203580,
respectively, for 30 minutes before MNNG treatment. Alternatively,
transfected cells were pretreated with 30 µmol/L curcumin for 30 minutes before an 8-hour period of incubation with MNNG.
Firefly luciferase activities were standardized for
-galactosidase activity, used as internal control. All
transfection/reporter assays were repeated at least 3 times, showing
less than 25% variability. A Student t test was used to
validate the results.
C2C12 cell lines containing stables transfected the different uPA promoter-luciferase plasmids have recently been characterized.72 Electrophoretic mobility shift assays Nuclear extracts were obtained from C2C12 cells before and after MNNG treatment. The extraction of nuclear proteins was performed as described by Miralles et al.23 For electrophoretic mobolity shift assays (EMSAs), 5 µg of nuclear extracts was incubated in 50 mmol/L Tris-HCl pH 7.9, 12.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L DTT, 20% glycerol, 0.5 mmol/L PMSF, and 2 µg of poly dI-dC for 10 minutes at room temperature to titrate out nonspecific binding before the addition of 15 000-20 000 cpm-labeled oligonucleotide and the reaction was further incubated for 20 minutes. When unlabeled competing olinucleotides were added, nuclear extracts were preincubated for 30 minutes at room temperature before the addition of the labeled probe. Samples were loaded on a prerun 5% polyacrylamide gel (29:1 in 0.25 × TBE) and electrophoresed at 200 V. Gels were dried and autoradiographed at80° C. The sequences of the
sense strands of the oligonucleotides used in EMSAs are as
follows:
AP1A, 5'-GAGGAAATGAGGTCATCTTGCTCTG-3'; AP1B, 5'-GGCCATGTGAATCACGACAGCCTG-3'; IgkB, 5'-CAGAGGGGACTTTCCGAG-3'.
MNNG induces murine uPA messenger RNA (mRNA) expression.
We have previously shown that uPA expression is induced during the UV
response.23 To extend this observation, we analyzed whether other environmental mutagenic agents, such as the
monofunctional alkylating agent MNNG, modulate the expression of the
uPA gene. As shown in Figure
1A, treatment of C2C12 and NIH3T3 murine
cell lines with MNNG increase the expression of a 2.7-kb transcript corresponding to murine uPA mRNA, with respect to untreated cells. uPA
mRNA induction was not due to an unspecific up-regulation of RNA
synthesis, because MNNG did not significantly modify the levels of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in either cell
line. The increase in uPA gene induction was time-dependent, being first observed after 30 minutes after stimulation in C2C12 cells
and reaching its maximum 2 hours after MNNG treatment; uPA mRNA
induction in these cells decreased 4 hours after treatment, returning
to basal levels after 8 hours (Figure 1A, top). Induction of uPA mRNA
in NIH3T3 cells was maximal after 4 hours stimulation with MNNG, and
was undetectable by 8 hours after treatment (Figure 1A, bottom). To
gain an insight into the mechanisms leading to increased uPA mRNA
expression in MNNG-treated cells, we studied the effects of RNA and
protein synthesis inhibitors on the uPA transcript level in cells that
were stimulated with the alkylating agent. The protein synthesis
inhibitor cycloheximide did not inhibit uPA mRNA induction by MNNG in
either cell type (Figure 1B, and data not shown); moreover, cells
treated only with cycloheximide expressed higher levels of uPA
transcripts, suggesting that MNNG stimulation of uPA mRNA did not
require de novo protein synthesis. In contrast, uPA mRNA induction by
MNNG was abrogated in cells treated with the RNA synthesis inhibitor
actinomycin D (Figure 1B). These data suggested that increased uPA
transcription, rather than message stabilization, was the mechanism
responsible for the MNNG-induced effect.
An AP1-enhancer element is involved in uPA promoter induction by MNNG We next examined the effect of MNNG on the activity of the murine uPA gene promoter. A murine uPA genomic fragment (8.2 kb to +398 base pairs [bp]), ligated upstream the luciferase
reporter gene, p-8.2Luc,13 was assessed for
luciferase activity after transient transfection in C2C12 and NIH3T3
cells. Comparison of luciferase activities generated by
p-8.2Luc, between unstimulated and MNNG-stimulated cells,
showed that the uPA promoter activity increased an average
4-fold after MNNG treatment in both cell types, whereas activity of
p-0.035Luc (a plasmid including only 35-bp of the uPA minimal promoter
sequence) was unaffected (Figure 2A), indicating that
the murine uPA promoter contains MNNG-responsive sequences that might
account, at least in part, for the MNNG-mediated induction of uPA in
these cells. To begin identifying the main regions involved in the uPA
transcriptional response to MNNG, different murine uPA
promoter-deletion luciferase constructs, generated from the full-length
8.2-kb promoter plasmid, were stably trasfected into C2C12
cells,72 and the corresponding uPA promoter-luciferase
(uPA-Luc)-containing cell lines were analyzed for luciferase
inducibility after treatment with MNNG. As shown in Figure 2B, deletion
of 1.6 and 3.3 kb from the 8.2-kb construct (generating plasmids
p-6.6Luc and p-4.9Luc, respectively) did not alter the luciferase
induction of the full-length 8.2-kb plasmid (P > .05),
suggesting that these upstream promoter regions were irrelevant for uPA
transcriptional induction by MNNG in the uPA-Luc-containing cell
lines. However, although C2C12 cells containing p-4.9Luc retained full
MNNG-induced luciferase activity, C2C12 cells transfected with
p-2.0Luc, a plasmid containing 2.0 kb of the murine uPA minimal
promoter, showed no luciferase inducibility (P < .01)
(Figure 2B), clearly indicating the location within this region of
cis-element(s) relevant for MNNG-induced uPA transcription. As expected
(according to transient transfection results shown in Figure 2A), cells
stably transfected with p-0.035Luc showed no luciferase induction after
stimulation with MNNG (Figure 2B).
The uPA promoter contains an AP1-enhancer element, located at Induction of MAP kinases by MNNG in C2C12 and NIH3T3 cells The alkylating agent MMS has been shown to promote the induction of JNK and p38 activities in human 293 cells.48 MNNG could also induce JNK activity in these cells. However, JNK activation is not a general response to alkylating drugs, because alkylating agents such as ENU cannot exert this effect. To determine the potential activation of the 3 main classes of MAPKs (JNK, ERK and p38) by MNNG in C2C12 and NIH3T3 murine cell lines, we measured the activities of JNK, ERK and p38, respectively, in response to MNNG treatment (Figure 3). Accordingly, immune complex kinase assays with C2C12 and NIH3T3 cell extracts, using an antibody against JNK1 and GST-cJun as substrate, were performed to determine JNK activation by MNNG in these cells. MNNG induced a very strong activation of JNK activity, peaking 1 hour after stimulation, remaining high after 2 hours, and returning to basal levels 4 hours after treatment in C2C12 cells (Figure 3A, left). MNNG was also a very potent inducer of JNK activity in NIH3T3 cells, reaching maximal levels between 30 minutes and 1 hour after stimulation, decreasing after 2 hours, and returning to basal levels 4 hours after treatment (Figure 3A, right). Similarly, we measured the activation of ERK using an antibody against ERK2 and myelin basic protein (MBP) as a substrate. As shown in Figure 3B, no activation of ERK2 was observed after MNNG treatment (up to 2 hours) in C2C12 cells, whereas ERK2 was potently activated in these cells within 6 minutes (0.1 hour) after serum (20% FBS) treatment. As in C2C12 cells, no ERK activation was observed after MNNG treatment of NIH3T3 cells (data not shown). Finally, activation of p38 by MNNG in C2C12 cells was assessed by Western blotting using an antibody specific for phospho-p38. As shown in Figure 3C, phosphorylation of p38 by MNNG was detected within 30 minutes after treatment of C2C12 cells, and still apparent after 4 hours. However, the signal corresponding to phosphorylated p38 in response to MNNG was weaker than that obtained in response to UV-C irradiation in C2C12 cells (Figure 3C, last lane). Furthermore, the activation of p38 induced by MNNG (Figure 3C) is almost negligible in comparison with the potent activation of JNK induced by the same alkylating agent (Figure 3A). Similar differences in the activation of JNK and p38 by MNNG were obtained on transient transfection of epitope-tagged HA-JNK1 and HA-p38, followed by immunoprecipitation with anti-HA antibody and measurement of kinase activities using GST-cJun and MBP substrates, respectively (data not shown). As in C2C12 cells, MNNG induced a very weak phosphorylation of p38 in NIH3T3 cells (data not shown). Altogether, these results suggested that JNK is the most potently activated MAPK in response to MNNG in C2C12 and NIH3T3 cells, whereas p38, but not ERK, was weakly activated by MNNG in these cells.
Curcumin blocks JNK activation by MNNG PD98059 and SB203580 are specific inhibitors of ERK and p38 activation, respectively. However, no synthetic inhibitor of JNK activation has yet been identified. The dietary pigment curcumin is a potent inhibitor of JNK activation by various agonists, including PMA plus ionomycin, anysomycin, UV-C, and TNF.78 To test
whether curcumin could also inhibit JNK activation in response to MNNG, C2C12 cells were pretreated with curcumin before their stimulation with
MNNG, and cell extracts were immunoprecipitated with an antibody against JNK1, and the enzymatic activity assessed using GST-cJun as the
substrate. As shown in Figure 4,
activation of JNK activity after a 1-hour treatment with MNNG was
completely abrogated by curcumin pretreatment, thus providing a useful
tool for blocking JNK activation by this alkylating agent. As expected,
neither 10 µmol/L SB203580 nor 50 µmol/L PD98059 affected JNK
activation by MNNG, whereas these compounds inhibited activation of p38
by UV-C irradiation and of ERK2 by 100 nmol/L TPA, respectively, in
NIH3T3 and C2C12 cells (results not shown).
Curcumin abrogates induction of AP1 binding, uPA transcription, and uPA mRNA expression by MNNG Once we had shown that both JNK and p38 MAPKs are induced in response to MNNG in C2C12 cells, we analyzed whether any of these MAPK signaling pathways were responsible for the induction of uPA gene expression by MNNG. We studied the effect of JNK and p38 inhibitors on uPA expression at different levels (Figure 5). As shown by Northern blotting, uPA mRNA expression was induced in C2C12 cells after a 2-hour treatment with MNNG. Curcumin pretreatment of C2C12 cells resulted in a complete inhibition of uPA mRNA induction by MNNG (Figure 5A); in contrast, pretreatment of cells with p38 inhibitor SB203580 did not alter this induction and, as expected, ERK inhibitor PD98059 had no effect on uPA induction by MNNG (Figure 5A, right). These data suggested that uPA gene induction by MNNG most likely occurred via the JNK signaling pathway. We next examined whether these MAPK inhibitors modify the MNNGtranscriptional response of uPA-Luc-containing C2C12 cell lines. As shown in Figure 5B, MNNG induced luciferase activity from the p-8.2Luc-C2C12 cell line an average of 4-fold. Although neither PD98059 nor SB203580 had any significant effect on uPA promoter induction by MNNG, curcumin fully abrogated uPA transcriptional induction in response to MNNG. Moreover, pretreatment of C2C12 cells with curcumin decreased AP1-binding activity to the uPA 3'-TRE (AP1B) after stimulation with MNNG (Figure 5C). In summary, curcumin specifically inhibits uPA induction by MNNG at 3 different levels: (1) AP1-binding to the uPA enhancer element, (2) uPA transcriptional activity, and (3) uPA mRNA expression.
Involvement of the JNK signaling pathway in uPA transcriptional induction by MNNG Because only curcumin seemed to abrogate uPA induction by MNNG, we hypothesized that the JNK signaling cascade was the intracellular mediator of this effect. To determine the specific involvement of the JNK pathway in uPA gene induction by MNNG, we determined uPA transcriptional activation by this agent in the absence or presence of specific MAPK inhibitors. MEKK1(K432M),79 a dominant negative form of MEKK1, was overexpressed in transient cotransfection experiments with p-6.6Luc in C2C12 cells, with or without MNNG. As shown in Figure 6, the uPA promoter activity induced by MNNG was strongly reduced by the catalytically inactive mutant form of MEKK1. Furthermore, overexpression of JIP-1, a cytoplasmic protein known to cause retention of JNK in the cytoplasm and subsequent inhibition of the JNK pathway,57 and to act as a scaffold protein for JNK signaling,58 also down-regulated MNNG-induced uPA promoter activity in these cells. In addition, the expression of MKK7(A), dominant negative form of MKK7, a specific activator of JNK, but not of p38, in response to TNF and
UV,77 abrogated uPA transcriptional induction by MNNG. A
similar inhibitory effect on uPA promoter induction by MNNG was caused
by overexpression of SEK1(KR), a dominant negative form of SEK1 (JNK
kinase, also known as MKK4) (Figure 6). These results, together with
those showing the inhibitory effect of curcumin on uPA
promoter-luciferase stimulation (Figure 5B), indicated that the
transcriptional induction of the uPA gene by MNNG was
mediated, at least in part, by the MEKK1/JNK pathway. In agreement with
the results shown in Figure 5B, SB203580, a specific inhibitor of 2 p38
isoforms (p38 and p38),68,80 did not block the
MNNG-induced activation of the 8.2-kb promoter-containing C2C12 cell
line. However, because 2 additional p38 isoforms (p38 and
p38 )64-67,69 are activated by stresses such as UV but
insensitive to SB203580,68,81 the potential involvement of
these latter kinases in uPA induction by MNNG was assessed.
Accordingly, MKK6b(A), a dominant negative form of MKK6b, which
diminishes the activation of all p38 isoforms70,71 was
cotransfected together with p-6.6Luc in C2C12 cells. As shown in Figure
6, the uPA promoter activity induced by MNNG was not suppressed by the
catalytically inactive mutant form of MKK6b. Taken together, these
results indicated that the JNK pathway was directly involved in the
transcriptional activation of the murine uPA gene by MNNG in
C2C12 cells, via its AP1-enhancer element.
Genotoxic agents like UV light irradiation and monofunctional
alkylating carcinogens trigger a rapid, highly regulated adaptative response, known as the cellular stress response, which involves coordinate control of multiple signal transduction pathways, leading to
the induction of many genes. The gene inductive response to UV has been
analyzed extensively, and it is known to promote transcription of genes
coding for transcription factors, growth factors, viral proteins, and
proteases (reviewed in Bender et al50). However, less is
known about the inductive response to alkylating agents such as MMS and
MNNG. These agents induce the early expression of several
proto-oncogens including c-fos, c-jun, junB, and junD, although to a
different extent.35 They also induce the level of cell
cycle regulatory/tumor suppressor proteins such as p53, p21, and
adenomatous polyposis coli (APC),36-38,82 and of DNA repair proteins such as 06-methylguanine-DNA methyltransferase (MGMT)
and The cellular response to genotoxic agents is complex. UV irradiation
activates different MAPKs (ERK, JNK, and p38) as well as NF Promoter deletion analysis revealed that the murine AP1-enhancer
element located at Reports from over a decade ago indicate that the alkylating agents
mechlorethamine and MNNG could induce the production of plasminogen
activator in U-87MG cells, an alkylation repair-deficient (Mer
We are grateful to Drs Y. Nagamine, M. Karin, J. Han, R. Davis, and E. Nishida for generously providing us with various plasmids. We also thank A. Martin, G. Aniorte, and D. Fernandez for assistance.
Submitted December 13, 1999; accepted April 3, 2000.
Supported by DGES (PM97-0088) and Fundació La Marató-TV3.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Pura Muñoz-Cánoves, Institut de Recerca Oncològica (IRO), Aut. Castelldefels, km 2.7, E-08907-L'Hospitalet Ll. (Barcelona), Spain; e-mail: pmunoz{at}iro.es.
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Abstract
Introduction
B (
