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INTRODUCTION |
Tumor hypoxia contributes directly to processes involved in
malignant progression, such as angiogenesis (1, 2), the elimination of
p53 tumor suppressor activity (3), and genetic instability (4).
Recognition that hypoxic tumor microenvironments are important for the
development of these phenotypes has stimulated interest in how
transformed cells respond to hypoxic signals (2, 5, 6). Several
hypoxia-sensitive mammalian transcription factors have been described,
including heat shock transcription factor-1 (HSF-1) (7), c-Fos and
c-Jun (5, 8, 9), NF-
B (10), a retrotransposon-like VL30 element
(11), p53 (3), and hypoxia-inducible factor-1 (HIF-1) (12).
We reported that the c-jun proto-oncogene is induced at the
message and protein levels in hypoxic SiHa human squamous carcinoma cells (5). Further investigation of the mechanism of this induction demonstrated that activation of the c-jun promoter by
hypoxia correlates with phosphorylation of the transactivation domain of the ATF21 transcription
factor (13). Since c-Jun and ATF2 dimers are AP-1 complexes that bind
to the c-jun promoter region (14), this finding suggested
that hypoxic signals transmitted to the promoter are mediated in part
by protein kinases that target both ATF2 and c-Jun. Stress-inducible
protein kinases capable of activating the c-jun promoter
include the SAPK/JNK and p38 MAPK families of the MAPK superfamily of
serine/threonine kinases (15, 16). Since both SAPK/JNKs and p38 MAPK
are sensitive to redox stresses, such as those associated with
ischemia-reperfusion events (17-20), we investigated the effect of
tumor-like hypoxia on their induction in transformed cells. In these
studies, which are described in detail below, we observed that both
SAPK/JNK and p38 MAPK activities are induced by hypoxia, but the
inductions are transient. Because activated SAPK/JNKs and p38 MAPK can
be deactivated by members of a family of dual-specificity phosphatases,
called MAPK phosphatases (MKPs) (21-23), we hypothesized that the
induction of these MAPKs in hypoxic cells is antagonized by
redox-responsive members of the MKP family. In particular, we evaluated
MKP-1 and -2 as possible contributors to this inhibitory activity, as
they are widely expressed immediate-early gene products that are
induced by a variety of stimuli (23-26).
Here, we report that hypoxia transiently induces SAPK/JNK as well as
p38 MAPK activity in SiHa cells, and concurrently induces a SAPK/JNK
phosphatase activity. This transient induction of SAPK/JNK activity
correlates with both the transcriptional activation of the gene for the
MKP family member MKP-1 and the enhanced expression of MKP-1 mRNA.
The hypoxia-inducible expression of MKP-1 mRNA is reversible,
returning to the aerobic level on reoxygenation. Together, these
findings show that MKP-1 is a hypoxia-responsive phosphatase and imply
that it contributes to the attenuation of SAPK/JNK activity stimulated
in hypoxic cells. In the context of tumor biology, the poised and
reversible responses of these MKP and MAPK pathways to hypoxic signals
suggest that they are tightly regulated within the tumor microenvironment.
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EXPERIMENTAL PROCEDURES |
Materials--
The GST-ATF2-(1-94) fusion protein was expressed
from a pGEX-KG plasmid (obtained from Dr. John Kyriakis, Massachusetts
General Hospital, Charlestown, MA), and the GST-c-Jun-(1-141) fusion
protein was expressed from a pGEX-2T plasmid (obtained from Dr. James Woodgett, Ontario Cancer Institute, Toronto, Ontario). Mammalian expression vectors (pcDNAIII) for full-length mouse MKP-1 and rat
MKP-2 cDNAs are described elsewhere (26). The following antibodies
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): rabbit
polyclonal anti-mouse p38 MAPK antibody (p38 (C-20); cross-reactive
with human p38 MAPK), rabbit polyclonal SAPK
/JNK1 antibody (JNK1
(FL); immunizing antigen full-length recombinant human JNK1;
cross-reactive with p54 SAPK
2/JNK2 and SAPK
/JNK3), monoclonal
anti-p46 SAPK
/JNK1 antibody (JNK1 (F-3); immunizing antigen
full-length recombinant human JNK1), rabbit polyclonal
anti-SAPK
/JNK2 (JNK2 (FL); immunizing antigen full-length recombinant human JNK2; cross-reactive with p46 SAPK
/JNK1 and SAPK
/JNK3), and monoclonal anti-phospho-SAPK/JNK antibody (p-JNK (G-7); immunizing antigen amino acids 183-191 of human SAPK
/JNK1 phosphorylated on Thr-183 and Tyr-185; identical with the corresponding region of phosphorylated human SAPK
/JNK2).
Cell Culture and Hypoxic Treatments--
The SiHa human cervical
carcinoma cell line was acquired from the American Type Culture
Collection (Rockville, MD). Details of the preparation and treatment of
SiHa cultures have been described elsewhere (5, 13). Because our system
for exposing cell cultures to hypoxia creates defined atmospheric
oxygen partial pressure (pO2) values within the range of
approximately 1% to
0.01% (relative to air at approximately 21%),
we define the low oxygen conditions used for these studies as hypoxia,
not anoxia. These conditions simulate those detectable in hypoxic
regions of solid tumors and in solid tumor models (3, 27, 28). The
hypoxia experiments described in this study were all performed at
pO2
0.01%. SiHa cells were plated at 106
cells/60-mm diameter glass culture dish in Eagle's basal medium containing 10% bovine calf serum (JRH Biosciences, Lenexa, KN) and 25 mM HEPES buffer (pH 7.4), and incubated in a 5%
CO2/air atmosphere at 37 °C. Cells were incubated for 3 days after plating before exposure to hypoxia. Briefly, aluminum
gas-exchange chambers containing the cells were placed in a 37 °C
circulating water bath and the original atmosphere was repeatedly
exchanged with 5% CO2/95% N2 by using a
manifold equipped with a vacuum pump and a gas cylinder (5).
Atmospheric oxygen levels in this apparatus were calibrated by using a
polarographic oxygen electrode (Oxygen Sensors, Inc., Norristown, PA)
in an attached test chamber. At the completion of various hypoxic
exposure times, the chambers were opened in an anaerobic box (Bactron
X, Sheldon Manufacturing Inc., Cornelius, OR) maintained at 5%
CO2/balance N2 to prepare cell lysates without
significant reoxygenation. For reoxygenation experiments, hypoxic cells
were incubated in 5% CO2/air. Based on the criterion of
trypan blue exclusion, these hypoxic exposures had no acute toxicity
for the cells used in these studies. In addition, clonogenic assays
have shown that prolonged hypoxia (pO2
0.01% for up to
16 h) is not significantly toxic to SiHa cells (29). The hypoxic
exposures used for these studies also do not deplete total ATP levels
in SiHa cells.2
ATF2 and c-Jun Kinase Assays--
ATF2 and c-Jun kinase
activities in cell lysates were assayed by using appropriate GST fusion
proteins of ATF2 or c-Jun transactivation domains as substrates
(GST-ATF2-(1-94) or GST-c-Jun-(1-141)) (13, 30). Briefly, the fusion
protein was expressed in Escherichia coli
BL21(DE3)pLysS cells (Stratagene Cloning Systems, La Jolla, CA), and
the cells were suspended in PBS-T (20 mM sodium phosphate (pH 7), 150 mM NaCl, 1% Triton X-100, 0.1 mM
PMSF, 20 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM
benzamidine hydrochloride, 100 µM
Na3VO4, 50 mM NaF). The bacterial
cells were lysed by subjecting them to three freeze/thaw cycles
followed by sonication. The fusion protein was extracted by adding
suspensions of glutathione-Sepharose 4B beads (Amersham Pharmacia
Biotech, Uppsala, Sweden) to samples of the cleared bacterial lysates
and then tumbling the mixtures overnight at 4 °C. The beads were
washed four times with ice-cold PBS-T.
Following a hypoxic treatment, plates of cells were immediately placed
on ice in air or on Super Ice® cold packs in the anaerobic
box and the medium was removed. Each dish was washed with 2 ml of
ice-cold PBS before adding 1 ml of ice-cold lysis solution (0.1 mM PMSF, 20 µg/ml aprotinin, 0.5 µg/ml leupeptin, 1 mM benzamidine hydrochloride, 100 µM
Na3VO4, 50 mM NaF). These solutions
were degassed before harvesting protein from hypoxic cells. The plates
were scraped, and the resultant cell suspensions were disrupted in
ice-cold Dounce homogenizers. The disrupted cell mixtures were
transferred to gasket-cap microcentrifuge tubes for spinning at
15,000 × g for 15 min at 4 °C. The protein concentrations of the supernatants were determined by a bicinchoninic acid (BCA) assay (Pierce), and the concentrations were normalized with
lysis buffer. The kinase assays were performed by first adding suspensions of Sepharose beads with adducted GST fusion protein to
tubes of supernatant fractions containing 100 µg of cell protein. The
tubes were gently rotated at 4 °C for 1 h, spun at 11,000 × g for 1 min at 4 °C, and washed four times with 1 ml
of ice-cold PBS-T. The PBS-T was removed from the beads, and 20 µl of
kinase buffer (50 mM Tris-HCl (pH 7.5), 10 mM
MgCl2, 100 mM NaF, 1 mM Na3VO4, 0.4 mM ATP) and 4 µCi of
[
-32P]ATP (6,000 Ci/mmol; Amersham Pharmacia Biotech)
was added to initiate each reaction. The samples were incubated for 30 min at 30 °C, and the reactions were stopped by adding 40 µl of a 2× SDS sample buffer (125 mM Tris-HCl (pH 6.8), 4.6% SDS,
10% mercaptoethanol, 20% glycerol) and boiling for 5 min. Samples were resolved in 12% discontinuous SDS-polyacrylamide gels and the
gels were stained with colloidal Coomassie Brilliant Blue R-250 to
confirm equal protein loading. The gels were dried and exposed to Kodak
XAR film to prepare autoradiographs. Densitometry was performed by
using a Lynx video densitometer (Applied Imaging Corp., Santa Clara, CA).
Immunocomplex Kinase Assays--
To perform these assays the
medium was removed from the cells in air or in the anaerobic box, as
described above. After washing with ice-cold PBS, each dish of cells
received 500 µl of ice-cold detergent lysis buffer (20 mM
Tris-HCl (pH 7.5), 1% Triton X-100, 10% glycerol, 137 mM
NaCl, 2 mM EDTA, 25 mM sodium
-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM PMSF,
20 µg/ml aprotinin, 10 µg/ml leupeptin). The cells were lysed by
scraping and the suspensions were transferred to gasket-cap
microcentrifuge tubes for spinning at 14,000 × g at
4 °C for 15 min. The protein concentrations of the supernatants were
determined by a BCA assay, and the concentrations were normalized with
the lysis buffer. Each lysate was precleared by adding 60 µl of
Protein A/G Plus-agarose beads (Santa Cruz Biotechnology) and tumbling
for at least 1 h at 4 °C. The lysates were spun at 600 × g at 4 °C for 10 min, and each lysate was normalized to
100 or 200 µg of total protein and divided into two equal samples. One sample received an amount of a specific antibody (3 µg of anti-p38 MAPK antibody; 1 µg of anti-SAPK/JNK antibody), and the other sample was used as a control for nonspecific protein binding to
the Protein A/G Plus-agarose beads. Each sample then received 20 µl
of the agarose beads and was gently tumbled for at least 1 h at
4 °C. The beads were spun at 600 × g at 4 °C for
10 min and washed twice with ice-cold lysis buffer and three times with ice-cold kinase buffer (25 mM HEPES (pH 7.4), 25 mM MgCl2, 2 mM DTT, 25 mM sodium
-glycerophosphate, 0.1 mM
Na3VO4). The kinase buffer was removed, and 5 µg of substrate was added to each sample followed by 20 µl of
kinase buffer containing 20 µM ATP and 3-5 µCi of
[
-32P]ATP (6,000 Ci/mmol; Amersham Pharmacia Biotech).
The samples were incubated for 30 min at 30 °C, and the reactions
were stopped by adding 40 µl of 2× SDS sample buffer and boiling for
5 min. Samples were resolved in 10% or 12% discontinuous
SDS-polyacrylamide gels, and relative kinase activities were determined
as described above.
Immunoblotting Procedure--
To obtain total cellular lysates
for detecting SAPK/JNK or p38 MAPK protein, cells were washed once with
ice-cold PBS and lysed in the ice-cold detergent buffer used for the
immunocomplex kinase assays. The lysates were frozen in dry ice and
stored at
80 °C. Frozen lysates were thawed on ice and centrifuged
at 10,000 × g for 5 min at 4 °C. The protein
concentrations of the supernatants were measured by a BCA assay. Equal
protein samples (5-10 µg) for gel electrophoresis were diluted with
equal volumes of the 2× SDS sample buffer and boiled for 5 min.
Proteins were resolved in discontinuous 11% SDS-polyacrylamide gels
and electroblotted in a buffer containing 24 mM Tris-HCl
(pH 8.3), 192 mM glycine, and 15% methanol onto Immobilon
P membranes (Millipore, Marlborough, MA) by using a TR 70 Semiphor
semidry transfer unit (Hoefer Scientific Instruments, San Francisco,
CA). Blots were incubated in 1% nonfat dried milk at 4 °C overnight
and then incubated at room temperature for 2 h with the
anti-SAPK/JNK or the anti-p38 MAPK antibodies used for the
immunocomplex kinase assays diluted 1:100 in PBS containing 5% horse
serum. Antibody binding was detected by using a biotin-labeled
anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA),
streptavidin alkaline phosphatase (Vector), and the substrates nitro
blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Life
Technologies, Inc.). Alternatively, binding was detected by using an
IgG antibody conjugated with horseradish peroxidase (IgG-HRP; Santa
Cruz Biotechnology) diluted 1:10,000 in PBS/0.1% Tween 20, and the ECL
Plus Western blotting detection system (Amersham Pharmacia Biotech).
Assay for SAPK/JNK (Thr-183+Tyr-185)
Phosphorylation--
Aerobic and hypoxic SiHa cells were placed on ice
in air or on Super Ice® cold packs in the anaerobic box,
and the medium was removed. Each dish was washed twice with 1 ml of
ice-cold PBS containing 1 mM Na3VO4
and 50 mM NaF, and then 200 µl of ice-cold lysis solution (10 mM HEPES (pH 7.9), 1 mM EDTA, 60 mM KCl, 1 mM DTT, 0.5% Nonidet P-40, 1 mM Na3VO4, 50 mM NaF,
0.5 µM okadaic acid, 1 mM PMSF) were added.
Degassed solutions were used with hypoxic cells. The plates were
scraped, and the resultant cell suspensions were transferred to
gasket-cap microcentrifuge tubes for spinning at 15,000 × g for 10 min at 4 °C. Samples of the lysates were treated
with 10 mM iodoacetamide for 15 min to remove DTT before
measuring protein concentrations by a BCA assay. The protein
concentrations were normalized by dilution with the lysis buffer, and
samples containing 500 µg of total protein in a total volume of 600 µl were prepared by adding the lysis buffer used for the ATF2 and
c-Jun kinase assays. Suspensions (40 µl) of Sepharose beads with
adducted GST-ATF2-(1-94) fusion protein were added to the lysates, and
they were gently rotated at 4 °C for 1 h. Then the lysates were
spun at 13,000 × g for 1 min at 4 °C, washed four
times with 1 ml of ice-cold PBS-T, diluted with an equal volume of 2×
SDS sample buffer, and boiled for 5 min. Proteins were resolved in
discontinuous 11% SDS-polyacrylamide gels and electroblotted as
described above. Blots were blocked in 4% nonfat dried milk in PBS
containing 0.1% Tween 20 at 4 °C for 1 h and then incubated at
room temperature for 1 h with anti-phospho-SAPK/JNK antibody
diluted 1:1,000 in PBS/0.1% Tween 20. Antibody binding was detected by
using the ECL Plus Western blotting detection system, as described
above (the anti-mouse IgG antibody conjugated with horseradish
peroxidase was diluted 1:10,000 in PBS/0.1% Tween 20).
Northern Analysis--
Purification of total cellular RNA for
Northern analysis was performed by using the Trizol®
reagent (Life Technologies, Inc.) or the RNeasy method (Qiagen Inc.,
Santa Clarita, CA) according to the manufacturers' instructions. RNA
was resolved in 1% denaturing agarose gels and blotted onto Magna NT
nylon membranes (MSI, Westboro, MA), as described elsewhere (29). A
1.9-kilobase pair MKP-1 cDNA probe was prepared by digestion of
pcDNAIII/MKP-1 with HindIII and BamHI,
and a 2.4-kilobase pair MKP-2 cDNA probe was prepared by digestion
of pcDNAIII/MKP-2 with EcoRV. The probes were labeled
with [
-32P]dCTP (Amersham Pharmacia Biotech) by the
random primer method. To provide a normalization standard for RNA
loading, ethidium bromide fluorescence from the 28 S rRNA band of total
RNA was photographed or blots were stripped and probed with a DNA
oligomer corresponding to a human 28 S rRNA sequence
(CLONTECH) end-labeled with
[
-32P]ATP (Amersham Pharmacia Biotech). Exposure of
control cells to UV radiation involved using a 254-nm wavelength source
(UV-C) at a calibrated fluence of 40 J/m2 (31). UV-treated
cells were harvested for RNA 1 h after exposure.
Message Stability--
The half-life of MKP-1 mRNA was
determined according to a protocol described elsewhere (5). Briefly,
SiHa cells were exposed to hypoxia for 4 h or to UV, as described
above. Hypoxic cells were removed from the aluminum hypoxia chambers in
the anaerobic box held at 37 °C. Both hypoxic and UV-treated cells
were given 5 µg/ml actinomycin D (Sigma) for 10 min (time zero), and
then cells were harvested for total RNA at various times afterward. Total RNA was processed for Northern analysis, and MKP-1 mRNA signals on the blots were measured by using a phosphorimager (Storm 840, Molecular Dynamics, Santa Clara, CA). The half-lives of MKP-1 mRNA were calculated from plots of the natural log (intensity) against the time of actinomycin D exposure starting at time zero.
Nuclear Runoff Transcription Assay--
This assay is a
modification of a protocol described elsewhere (5). For each
experimental condition, eight dishes of SiHa cells were plated at
2 × 106 cells/100-mm diameter plastic culture dish 4 days before treatment. To harvest nuclei, dishes were placed on ice,
the medium was removed, and the cells were washed twice with ice-cold
PBS followed by scraping in 800 µl of ice-cold lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40). After removal of
the original lysate, each dish was washed with another 800 µl of
ice-cold lysis buffer and the combined suspensions were kept on ice.
The lysates were spun at 500 × g at 4 °C for 5 min, and the nuclear pellets were resuspended in 5 ml of ice-cold lysis buffer. After another spin at 500 × g, each pellet was
resuspended in 200 µl of storage buffer (50 mM Tris-HCl
(pH 8.3), 5 mM MgCl2, 0.1 mM EDTA,
40% glycerol) and frozen in liquid N2. To perform nuclear
runoff transcription reactions, each sample of frozen nuclei was thawed
at room temperature and added to 200 µl of 2× reaction buffer (10 mM Tris-HCl (pH 8.0), 5 mM MgCl2,
300 mM KCl, 2 mM DTT; 1 mM each of
ATP, CTP, and GTP; 25 units/ml RNase inhibitor; Life Technologies,
Inc.). Then 10 µl of 10 mCi/ml [
-32P]UTP (Amersham
Pharmacia Biotech) were added to each sample, and the samples were
incubated at 30 °C for 30 min. The reactions were stopped by
digestion with 10 µg of RNase-free DNase I (Life Technologies, Inc.)
at 30 °C for 5 min. Nascent RNA samples were harvested and purified
by the RNeasy® method (Midi Kit, Qiagen).
Nylon membrane (MSI) slot blots of 250 ng each of MKP-1,
-actin, and
pBluescript II KS+ (pBSK; Stratagene, La Jolla, CA)
cDNA were prepared by using a Hoefer PR 648 slot blot filtration
manifold according to the manufacturer's instructions. The MKP-1 and
-actin cDNAs were inserts in pBSK, and the plasmids were
linearized before blotting. Membranes were prehybridized at 42 °C
for at least 4 h in 4× Denhardt's solution containing 1 µg/ml
Saccharomyces cerevisiae tRNA, and hybridized at 42 °C
for 36-48 h with 1-5 × 106 cpm of nascent RNA in
50% formamide hybridization solution (10 mM TES (pH 7.4),
500 mM NaCl, 2 mM EDTA, 0.4% SDS, 2 units/ml RNase inhibitor). After hybridization, the membranes were washed twice
at 42 °C for 1 h with buffer A (10 mM Tris-HCl (pH
7.4), 300 mM NaCl, 2 mM EDTA), and then once at
42 °C for 30 min in buffer B (5 mM Tris-HCl (pH 7.4), 10 mM NaCl, 2 mM EDTA, 0.4% SDS). The blots were
then washed twice in buffer A and incubated at 37 °C in buffer A
containing 10 mg/ml RNase A. After washing the membranes in buffer A
twice at 42 °C for 1 h, they were exposed to Kodak BioMax x-ray
film for autoradiography.
 |
RESULTS |
Hypoxia without Reoxygenation Transiently Induces Phosphorylation
of the Transactivation Domains of the ATF2 and c-Jun Transcription
Factors by SAPK/JNKs and p38 MAPK in SiHa Cells--
Previously, we
reported that exposure of SiHa cells to a range of low oxygen
conditions (pO2
0.1%) without reoxygenation caused
transcriptional activation of c-jun (5) and phosphorylation of the ATF2 transactivation domain (13). As mentioned above, the
c-jun promoter is sensitive to activation by both SAPK/JNK and p38 MAPK members of the MAPK superfamily (15, 16). In the present
study, we investigated the activation of these MAPKs by hypoxia
(pO2
0.01%) by using aerobic and hypoxic SiHa cell lysates in the following assays: 1) kinase assays involving the GST-ATF2-(1-94) and GST-c-Jun-(1-141) fusion proteins as substrates; and 2) immunocomplex kinase assays involving anti-p38 MAPK and anti-SAPK
/JNK1 or -SAPK
/JNK2 antibodies, and the GST-ATF2-(1-94) fusion protein as a substrate. To avoid possible effects of
reoxygenation on SAPK/JNK and p38 MAPK activation, hypoxic cells were
harvested for these assays exclusively under anaerobic conditions. It
is important to note that, although time zero for hypoxia is defined as
the start of the protocol described under "Experimental
Procedures," the time required to attain a pO2
0.01%
by this method is 2 h. Thus, the earliest observations reported
here are 2 h following the onset of hypoxic conditions.
Fig. 1 shows that both ATF2 and c-Jun
kinase activities were stimulated in SiHa cells under low oxygen
conditions, and that these activities peaked within 2-4 h of hypoxia.
Both the degree of hypoxia (i.e. pO2 and
duration of exposure) and the cell type may be important determinants
of the onset of SAPK/JNK and p38 MAPK activity. For example, in a
previous study involving NIH 3T3 cells, c-Jun kinase activity was not
detected following hypoxic exposures of less than 1 h at
pO2
0.1% (32). Fig.
2A shows that p38 MAPK
activity was transiently stimulated in hypoxic SiHa cells under the
same conditions as those that induced ATF2 kinase activity, and that
this response persisted for at least 4 h of hypoxia. This
induction of p38 MAPK activity was approximately 3-fold greater than
that of the aerobic control (e.g. 3.2 ± 1.1 at 4 h, sample S.D., n = 4). For comparison, sorbitol (300 mM for 1 h) induced p38 MAPK activity by approximately
8-fold relative to the control (8.4 ± 3.1, n = 3, data not shown). Thus, as reported by others for heart (33, 34), p38
MAPK can be activated in a human carcinoma cell line by hypoxia. Fig.
2A also shows that these hypoxic conditions strongly and
transiently induced SAPK
/JNK1 activity relative to the aerobic
control, giving a maximum value within the interval of 2-4 h of
hypoxia (e.g. 31.8 ± 3.3 at 4 h,
n = 3). This activation of SAPK
/JNK1 is consistent
with the finding shown in Fig. 1 of enhanced c-Jun kinase activity from SiHa cells exposed to identical hypoxic conditions. The Western blots
shown in Fig. 2B demonstrate that total basal SAPK
/JNK1 and p38 MAPK protein levels in SiHa cells did not change during hypoxic
exposures of up to 6 h. These findings indicate that the transient
induction of SAPK
/JNK1 and p38 MAPK activities in hypoxic SiHa cells
cannot be attributed to stress-induced MAPK protein synthesis and
degradation. While ischemia-inducible p38 MAPK activity has been
reported (33, 34), to our knowledge SAPK/JNK activation by hypoxia
per se has not been established. Hypoxia was also found to
induce both transient p38 MAPK and SAPK
/JNK1 activities in identical
experiments using immortalized mouse embryo fibroblasts (T-MEFs,
obtained from Dr. Randall Johnson, University of California, San Diego;
data not shown). This finding suggests that the activation of these
stress-inducible MAPKs by pathophysiological hypoxia can occur in a
variety of mammalian cell types.

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Fig. 1.
Hypoxia transiently induces both ATF2 and
c-Jun kinase activities in SiHa cells. Autoradiographs showing
phosphorylation of the GST-ATF2-(1-94) and GST-c-Jun-(1-141)
substrates in kinase assays using SiHa cell lysates. Cells were
incubated at 37 °C in 5% CO2/air (lanes
1 and 5) or under hypoxia (pO2 0.01%; lanes 2-4 and 6-8) for the indicated
times. In this experiment and in all others, hypoxic cells were
harvested for protein kinase assays under anaerobic conditions. For
details, see "Results."
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|

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Fig. 2.
Hypoxia transiently induces SAPK/JNK and p38
MAPK activities in SiHa cells. A, representative
autoradiograph showing phosphorylation of the GST-ATF2-(1-94)
substrate by p38 MAPK and SAPK /JNK1 immunoprecipitated from lysates
of aerobic SiHa cells (5% CO2/air; lanes
5 and 9), and from lysates of SiHa cells exposed
to hypoxia (pO2 0.01%; lanes 6-8
and 10-12) for the indicated times. The histograms show
inductions of protein kinase activities in hypoxic cells normalized to
those in aerobic controls (error bars represent
sample standard deviations or S.D., n 3).
I.P., immunoprecipitations. B, hypoxia does not
change SAPK /JNK1 or p38 MAPK protein levels in SiHa cells.
Photograph of a Western blot of total SiHa cell protein probed with the
anti-SAPK /JNK1 antibody (top panels) or the
anti-p38 MAPK antibody (bottom panels) used for
the immunoprecipitations described in A. Cells were exposed
to hypoxia (pO2 0.01%) for 2, 4, or 6 h before
harvesting protein. The protein bands corresponding to the p46 and p54
SAPK /JNK1 isoforms and to p38 MAPK are indicated by
arrows.
|
|
Hypoxia-inducible SAPK/JNK Activation Involves Both SAPK
/JNK2
and SAPK
/JNK1--
The anti-SAPK
/JNK1 antibody used for the
immunoprecipitations shown in Fig. 2A cross-reacts with both
human SAPK
/JNK2 and SAPK
/JNK3 (see "Experimental
Procedures"). Thus, it is possible that other members of the SAPK/JNK
family (16, 35) can contribute to the activity immunoprecipitated by
the anti-SAPK
/JNK1 antibody. To confirm that SAPK
/JNK1 is
activated by hypoxia, an identical immunoprecipitation study was
performed involving a monoclonal antibody specific for p46
SAPK
/JNK1. Fig. 3A shows
that hypoxia transiently stimulated p46 SAPK
/JNK1 activity relative
to the aerobic control, giving a maximum induction within 2-4 h of
stress (e.g. 2.8 ± 1.0 at 2 h,
n = 3). The difference in the -fold induction of
SAPK
/JNK1 activity detected by the monoclonal compared with the
polyclonal SAPK
/JNK1 antibody can be attributed in part to the lower
aerobic background signal consistently found with the polyclonal
antibody. In addition, the larger -fold induction in Fig. 2A
may reflect the contribution of more than one SAPK/JNK to the signal.
The Western blot shown in Fig. 3B confirms that the
monoclonal antibody detected p46 SAPK
/JNK1 in SiHa cells and that
the total basal level of this SAPK
/JNK1 isoform remained constant
for up to 6 h of hypoxia. Fig. 3C shows that
SAPK
/JNK2 was also transiently activated in hypoxic SiHa cells
within 2-4 h of stress (e.g. 3.3 ± 1.2 at 4 h,
n = 3), and Fig. 3D indicates that total
basal SAPK
/JNK2 protein levels remained constant for at least 6 h of hypoxia. Finally, using a specific cDNA probe for SAPK
/JNK3
(36), no signal was detected on a Northern blot of SiHa cell total RNA
(data not shown) indicating that SAPK
/JNK3 is not significantly
expressed in these cells. It has been reported that SAPK
/JNK3 is
primarily expressed in neuronal tissue (16, 36) whereas SiHa cells are
of cervical origin. Together, these studies demonstrate that the
transient SAPK/JNK activity induced in hypoxic SiHa cells consists of
contributions from both SAPK
/JNK2 and SAPK
/JNK1.

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Fig. 3.
Transient SAPK/JNK activation in hypoxic SiHa
cells involves both SAPK /JNK1 and
SAPK /JNK2. A, representative
autoradiograph showing phosphorylation of the GST-ATF2-(1-94)
substrate by p46 SAPK /JNK1 immunoprecipitated from lysates of
aerobic SiHa cells (5% CO2/air; lane
5), and from lysates of SiHa cells exposed to hypoxia
(pO2 0.01%; lanes 6-8) for the
indicated times. The histogram shows inductions of protein kinase
activities in hypoxic cells normalized to those in aerobic controls
(error bars represent S.D., n = 3). B, photograph of a Western blot of total SiHa cell
protein probed with the same anti-p46 SAPK /JNK1 antibody as that
used for the immunoprecipitations described in A. Cells were
exposed to hypoxia (pO2 0.01%) for 2, 4, or 6 h
before harvesting protein. The protein band corresponding to the p46
SAPK /JNK1 isoform is indicated by an arrow. C,
representative autoradiograph showing phosphorylation of the
GST-ATF2-(1-94) substrate by SAPK /JNK2 immunoprecipitated from
lysates of aerobic and hypoxic SiHa cells, as described above. The
histogram shows inductions of protein kinase activities in hypoxic
cells normalized to those in aerobic controls (S.D., n = 3). D, photograph of a Western blot of total SiHa cell
protein probed with the anti-SAPK /JNK2 antibody used for the
immunoprecipitations described in C. Cells were exposed to
hypoxia as described above. The protein bands corresponding to the p46
and p54 SAPK /JNK2 isoforms are indicated by arrows.
|
|
Hypoxia Induces a Phosphatase Activity in SiHa Cells That
Dephosphorylates the TPY Signature Motif of SAPK/JNKs--
The finding
of a transient activation of both SAPK/JNKs and p38 MAPK in hypoxic
cells suggested a hypoxia-inducible negative regulatory mechanism for
these MAPKs. To investigate this possibility, we focused on the
attenuation of hypoxia-inducible SAPK/JNK activity because it has a
strong response in SiHa cells. Fig. 4
shows that endogenous SAPK/JNKs in hypoxic SiHa cells are transiently
phosphorylated during 2-4 h of stress on Thr-183 and Tyr-185 in the
activating TPY signature motif (15, 16). In addition, unlike
anisomycin, hypoxia seems to preferentially phosphorylate/activate p46
isoforms of SAPK/JNKs in SiHa cells (the antibody recognizes the
phosphorylated TPY motif in both SAPK
/JNK2 and SAPK
/JNK1; see
"Experimental Procedures"). This finding parallels that of the
immunocomplex kinase assay shown in Fig. 3 for the monoclonal antibody
specific for p46 SAPK
/JNK1, in which transient SAPK/JNK activation
occurred within 2-4 h of hypoxia. Although an adducted
GST-ATF2-(1-94) fusion protein rather than an immunoprecipitating
antibody was used to isolate activated SAPK/JNKs for the
anti-phospho-SAPK/JNK Western blot, both p46 and p54 SAPK/JNK isoforms
bind to this adducted protein (Fig. 4, anisomycin
lane). Interestingly, using a similar assay, others have
reported preferential activation of a p46 SAPK/JNK isoform in cells
stimulated by TNF
(37). Together, the findings shown in Figs. 1-4
provide strong evidence to support the hypothesis that
pathophysiological hypoxia induces both SAPK
/JNK2 and SAPK
/JNK1
activity by phosphorylation on the TPY signature motif, with a possible
preference for activation of the p46 isoforms. In addition, the decline
in (Thr-183+Tyr-185)-phosphorylated SAPK/JNK protein in SiHa cells by
4 h of hypoxia (Fig. 4) is consistent with the stimulation of a
specific phosphatase activity capable of antagonizing concurrent
SAPK/JNK activation.

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Fig. 4.
Hypoxia induces a SAPK/JNK phosphatase
activity in SiHa cells. Autoradiograph of a Western blot of
SAPK/JNK protein isolated from lysates of SiHa cells on agarose beads
adducted with GST-ATF2-(1-94). The blot was probed with an antibody
that detects SAPK/JNKs phosphorylated on Thr-183 and Tyr-185 in the TPY
signature motif. Cells were exposed to hypoxia (pO2 0.01%) for 2, 4, or 6 h, or to anisomycin (10 µg/ml) for 1 h before lysis and extraction of activated SAPK/JNK protein. Hypoxic
cells were harvested under anaerobic conditions. Protein bands are
shown corresponding to the p46 and p54 isoforms of the endogenous
(Thr-183+Tyr-185)-phosphorylated SAPK/JNKs.
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|
Hypoxia Stimulates Expression of the MAPK Phosphatase MKP-1 in SiHa
Cells--
Activation of SAPK/JNKs can be antagonized by members of
the MKP family of dual-specificity phosphatases (22, 23, 31, 38).
Prompted by evidence that the MKP gene family members
MKP-1/CL100 and MKP-2 are stress-inducible (23,
26, 31), we investigated whether the SAPK/JNK phosphatase activity
induced in hypoxic SiHa cells could be associated with the accumulation
of the mRNAs for these MKPs. Although originally identified as
specific for ERK1/2 dephosphorylation (24, 25), recent reports provide
evidence that MKP-1 and MKP-2 can also recognize SAPK/JNKs (22, 23, 38,
39). Fig. 5A shows that MKP-1
mRNA accumulated in hypoxic SiHa cells as early as 2 h and
remained elevated for up to 24 h of stress. This mRNA
accumulation ranged from 2- to 4-fold relative to that in aerobic cells
(e.g. 1.8 ± 0.2 at 2 h of hypoxia,
n = 3), and returned to the aerobic level by 2 h
of reoxygenation (Fig. 5B). In contrast, MKP-2 mRNA
accumulation did not change appreciably in response to hypoxia (Fig.
5A). For comparison, UV radiation, a strong inducer of MKP-1
expression in some cells (31), caused a 3-fold accumulation relative to
the control (3.5 ± 0.8, n = 3) of MKP-1 mRNA
in SiHa cells (data not shown). These findings indicate that MKP-1 is a
candidate for a hypoxia-inducible SAPK/JNK phosphatase activity in SiHa
cells.

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Fig. 5.
MKP-1/CL100 mRNA is induced in hypoxic
SiHa cells and returns to the aerobic level on reoxygenation.
A, autoradiographs of a Northern blot of total RNA from
aerobic SiHa cells (5% CO2/air; lanes
1, 3, 5, 7, and
9) and SiHa cells exposed to hypoxia (pO2 0.01%; lanes 2, 4, 6,
8, and 10) for the indicated times. The blot was
probed sequentially for the mRNAs for MKP-1 (top
panel) and MKP-2 (middle panel). The
bottom panel shows ethidium bromide (EtBr)
fluorescence from the 28 S rRNA band of the total RNA samples used for
this Northern analysis. B, autoradiograph of a Northern blot
of total RNA from aerobic SiHa cells (lane 1) and
SiHa cells exposed to hypoxia (Hx; lanes
2 and 3) or reoxygenation following 2 h of
hypoxia (Reox; lanes 4-6).
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|
The Induction of MKP-1 Expression by Hypoxia Is the Result of
Transcriptional Activation--
Hypoxia can influence gene expression
at the transcriptional level through the activity of specific
transcription factors and at the post-transcriptional level by
stabilizing mRNA (5, 40, 41). To determine whether mRNA
stabilization contributes to MKP-1 mRNA accumulation in hypoxic
cells, we used actinomycin D to block transcription in hypoxic SiHa
cells or in aerobic SiHa cells exposed to positive controls for MKP-1
expression (i.e. UV radiation, TPA). Fig.
6A shows a Northern blot of
total RNA obtained from hypoxic and UV-treated SiHa cells at 0, 15, 30, 45, 60, and 75 min after a 10-min incubation time with actinomycin D. Analysis of plots of the natural log (signal intensity)
versus time from two independent Northern blotting
experiments gave a value of 20.9 ± 2.9 min for the half-life of
MKP-1 mRNA in hypoxic SiHa cells. For comparison, the half-life of
MKP-1 mRNA in UV- or TPA-treated SiHa cells was found to be 20.1 and 22.1 min, respectively. Thus, the half-life of MKP-1 mRNA in
SiHa cells is essentially the same following induction by such
disparate stimuli as hypoxia, UV radiation, and TPA. This finding
indicates that transcriptional activation rather than mRNA
stabilization is primarily responsible for the hypoxia-inducible
accumulation of MKP-1 mRNA.

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Fig. 6.
The induction of MKP-1/CL100
expression by hypoxia involves transcriptional activation and not
mRNA stabilization. A, autoradiograph of a Northern
blot of total RNA from hypoxic SiHa cells (pO2 0.01%;
lanes 1-6) and UV-treated aerobic SiHa cells
(5% CO2/air; 40 J/m2; lanes
7-12) harvested at the indicated times after exposure to
the transcriptional inhibitor actinomycin D (5 µg/ml). Time zero was
defined as 10 min after the addition of actinomycin D at 37 °C.
Hypoxic cells were incubated with actinomycin D and harvested under
anaerobic conditions at 37 °C. B, autoradiograph of nylon
slot blots of linear MKP-1 cDNA, -actin cDNA, and
pBluescript II KS+ (pBSK) hybridized with nascent RNA from
SiHa cell nuclei labeled with [32P]UTP, as described
under "Experimental Procedures." RNA was obtained from aerobic SiHa
cells, TPA-treated aerobic SiHa cells (100 ng/ml for 1 h), and
hypoxic SiHa cells (pO2 0.01% by 2 h). Each strip
contained triplicate slot blots of each plasmid DNA (250 ng/slot
blot).
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|
To investigate the contribution of transcriptional activation to the
induction of MKP-1 expression by hypoxia, we performed nuclear runoff
transcription assays to measure MKP-1 promoter activity
directly (Fig. 6B). This transcriptional analysis
demonstrated that the endogenous MKP-1 promoter in SiHa
cells exposed to 2 h of hypoxia was activated 6-fold (6.1 ± 1.1, n = 3) relative to the aerobic control. For
comparison, exposure of SiHa cells to TPA, a known stimulus for
MKP-1 transcription (42), activated the promoter by 2-fold
(2.3 ± 0.4, n = 3) relative to the control (Fig.
6B). Taken with the finding from the actinomycin D study that hypoxia does not stabilize MKP-1 mRNA, the nuclear runoff analysis confirms that hypoxia induces the accumulation of MKP-1 mRNA primarily by transcriptional activation, and that this
induction can occur by 2 h of the onset of the stress.
 |
DISCUSSION |
The MAPK superfamily of proline-directed Ser/Thr kinases includes
the mitogen-responsive members ERK1/2, the SAPK/JNKs, p38 MAPKs,
Fos-regulating kinase or FRK, and ERK3/BMK (15, 16, 43-45). Signaling
pathways for the MAPKs involve the general sequence MAPK kinase kinase
MAPK kinase
MAPK. The MAPK kinases are dual-specificity protein
kinases that phosphorylate their substrates on threonine and tyrosine
within the conserved signature motif TXY (X is E
for ERK1/2, P for SAPK/JNKs, and G for p38 MAPKs) (15, 16).
Deactivation of these signaling cascades at the MAPK level is
critically dependent on dephosphorylation of the TXY motif
by members of the MKP family of dual-specificity phosphatases (21, 23).
Currently, the known MKP family includes nine members (22, 23, 26, 46,
47). Although various MKPs can dephosphorylate different members of the
MAPKs, individual members of the family possess some substrate
specificity (23, 38, 46-48). For example, mouse MKP-1 (3CH134/ERP;
related to human CL100) preferentially recognizes ERK1/2, SAPK
/JNK1,
and p38 MAPK, whereas rat MKP-2 (related to human VH-2) preferentially
recognizes ERK2 and SAPK
/JNK2, but not p38 MAPK (23-25, 38). While
some MKPs have tissue-specific patterns of expression (47), MKP-1 and
MKP-2 are widely distributed. Moreover, like other immediate-early gene
products (e.g. c-Jun), their expression is responsive to
various environmental stimuli, including mitogens, oxidative stress, UV
radiation, heat shock, and alkylating agents (22, 23, 31, 49).
The major finding of this study is that exposure of human carcinoma
cells to tumor-like low oxygen conditions (3, 28, 50) stimulates
transient SAPK/JNK activity while simultaneously activating
transcription of the MKP-1 gene. Thus, MKP-1 is a
hypoxia-responsive gene. In terms of functional expression, we
demonstrated that MKP-1 inhibits hypoxia-inducible SAPK
/JNK1
activity in co-transfected SiHa cells.2 Taken with our
observation that hypoxic SiHa cells contain a (Thr-183+Tyr-185)-phosphorylated SAPK/JNK phosphatase activity (Fig.
4), these findings suggest that MKP-1 contributes to the attenuation of
SAPK/JNK activation in transformed cells exposed to pathophysiological
hypoxia. In support of this idea, others have reported that an MKP such
as MKP-1 antagonizes transient SAPK/JNK activation in
mitogen-stimulated Jurkat human T-cells (51) and in rat mesangial cells
treated with TNF-
(52, 53). To establish the contribution of
endogenous MKP-1 to hypoxia-inducible SAPK/JNK dephosphorylation, it
will be necessary to obtain effective anti-MKP-1 antibodies for
immunodepletion and immunoprecipitation studies, as commercially
available antibodies are either nonspecific or cross-react with
multiple MKPs (23, 26).
MKP-1 is regarded as an immediate-early gene (31, 39, 42,
54), but little is known concerning the transcriptional and
post-transcriptional controls on its expression and/or activity. At the
protein level, MKP-1/CL100 has a short half-life (54), suggesting that
it is targeted for rapid proteolysis like other immediate-early genes.
At the transcriptional level, a model for stress-inducible MKP-1
expression has been proposed in which SAPK/JNKs transcriptionally
activate the MKP-1 gene in a negative feedback loop (22,
39). Consistent with this model, the promoter region for the human
MKP-1 gene (i.e. MKP-1/CL100) contains
cis-acting elements for AP-1 and ATF/CREB transcription
factors (42) both of which are physiological targets of SAPK/JNKs
and/or p38 MAPK (16, 55). However, we observed that activation of the
MKP-1/CL100 promoter in SiHa cells occurs by 2 h of the
initiation of hypoxia (Fig. 6), overlapping with the onset of transient
SAPK/JNK activity (i.e. 2-4 h of hypoxia, Figs. 2-4). We
also determined that hypoxia-inducible expression of the mouse
MKP-1 gene (i.e. 3CH134/ERP) does not require c-Jun, using c-jun null T-MEFs (56).2
Interestingly, it has been reported that the activation of MAPKs including SAPK/JNKs is not sufficient for the induction of MKP-1 expression in rodent fibroblasts (57-59). Although these findings do
not necessarily exclude a model of hypoxia-inducible
MKP-1/CL100 expression involving a SAPK/JNK feedback loop,
they suggest that other models are also possible. For example, the
hypoxic response of the MKP-1/CL100 promoter may be mediated
by its Sp1 and/or CRE sites, shown to be hypoxia-responsive elements in
some systems (60, 61). Alternatively, hypoxia-responsive elements may
be present at distant sites in the regulatory regions of the
MKP-1/CL100 gene, as has been demonstrated for the human
erythropoietin and mouse heme oxygenase-1 genes (62, 63). Given this
potential complexity, it is likely that identifying the
hypoxia-responsive elements in the MKP-1/CL100 gene will
require detailed knowledge of both the 5'- and 3'-regulatory regions.
Although not established in vivo, an epitope of MKP-1 can be
phosphorylated by SAPK
/JNK2 in vitro (64), raising the
possibility that stress-inducible MKP-1 activity could be regulated at
the post-translational level by phosphorylation as well as by
proteolysis. Hypoxia can modulate the activities of protein
phosphatases (65, 66) and activate protein kinases (8-10, 32, 33, 60,
66, 67). If MKP-1 and SAPK/JNK activation in hypoxic SiHa cells are
interrelated, it is conceivable that early signals for their induction
share upstream activators. We observed that genistein (50 µM), a broadly active PTK inhibitor (68), inhibited
hypoxia-inducible SAPK/JNK and p38 MAPK activity in SiHa cells, while
suramin (0.3 mM), which disrupts receptor PTK
oligomerization (69), had no effect.2 These findings are
consistent with a role for non-receptor PTK activity in the activation
of SAPK/JNK and p38 MAPK pathways by hypoxia. Members of the Src family
of PTKs have been implicated in hypoxia-responsive signaling pathways
(10, 66, 67). Finally, a report demonstrating that both an MPK protein
and the upstream SAPK/JNK activator MEKK-1 are components of the I
B
kinase complex (64) suggests an integrating mechanism for the upstream
regulation of redox-responsive MKP and MAPK pathways. The potential
role of multi-protein complexes in the transmission of signals
generated by hypoxia and reoxygenation is an important area for further research (70).
Up-regulation of normal MKP-1 mRNA and protein has been detected in
clinical specimens of a group of early stage carcinomas and in various
stages of breast and prostate carcinoma (71-74). The biological
function of MKP-1 activity in tumors is not clear, but it is reasonable
to hypothesize that the induction of MKP-1 expression in hypoxic or
reoxygenated tumor microenvironments is associated with
stress-inducible MAPK activation. Evidence has been presented showing
that both SAPK/JNKs and p38 MAPK can promote apoptosis in cells exposed
to toxic stimuli (reviewed in Refs. 75 and 76). If MKP-1 inhibits
SAPK/JNK- or p38 MAPK-dependent apoptosis by preventing
prolonged MAPK activation (51-53), then stress-inducible MKP-1
expression may contribute to the net growth of a solid tumor. In
support of an anti-apoptotic function for MKP-1 in tumors, patterns of
MKP-1 mRNA expression in early stage prostate carcinoma specimens
were found to be inversely correlated both with apoptosis as determined
by a TUNEL assay and with SAPK
/JNK1 protein expression (73, 74). As
opposed to stress-inducible apoptosis mediated by prolonged SAPK/JNK
and/or p38 MAPK activity, in some cell types MKP-1 may actually promote
apoptosis in response to a transient receptor-dependent
signal (77). Because overexpressed MKP-1 can down-regulate
ras-dependent mitogenic signals (21, 78), the
suggestion has also been made that it could act as a tumor suppressor
(72). Regardless of the potential role of MKP-1 in oncogenesis,
observations of heterogeneous, up-regulated MKP-1 expression in human
tumor specimens provide evidence of an important contribution of this
MKP to tumor pathophysiology, and suggest that it may be protective for
hypoxic cells.