Mitogen-activated Protein Kinase Phosphatase-1 (MKP-1) Expression Is Induced by Low Oxygen Conditions Found in Solid Tumor Microenvironments
A CANDIDATE MKP FOR THE INACTIVATION OF HYPOXIA-INDUCIBLE STRESS-ACTIVATED PROTEIN KINASE/c-Jun N-TERMINAL PROTEIN KINASE ACTIVITY*

Keith R. LaderouteDagger §, Holly L. MendoncaDagger , Joy M. CalaoaganDagger , A. Merrill KnappDagger , Amato J. Giaccia, and Philip J. S. Storkparallel

From the Dagger  Pharmaceutical Discovery Division, SRI International, Menlo Park, California 94025, the  Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305, and the parallel  Vollum Institute and Department of Pathology, Oregon Health Sciences University, Portland, Oregon 97201

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pathophysiological hypoxia is an important modulator of gene expression in solid tumors and other pathologic conditions. We observed that transcriptional activation of the c-jun proto-oncogene in hypoxic tumor cells correlates with phosphorylation of the ATF2 transcription factor. This finding suggested that hypoxic signals transmitted to c-jun involve protein kinases that target AP-1 complexes (c-Jun and ATF2) that bind to its promoter region. Stress-inducible protein kinases capable of activating c-jun expression include stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 members of the mitogen-activated protein kinase (MAPK) superfamily of signaling molecules. To investigate the potential role of MAPKs in the regulation of c-jun by tumor hypoxia, we focused on the activation SAPK/JNKs in SiHa human squamous carcinoma cells. Here, we describe the transient activation of SAPK/JNKs by tumor-like hypoxia, and the concurrent transcriptional activation of MKP-1, a stress-inducible member of the MAPK phosphatase (MKP) family of dual specificity protein-tyrosine phosphatases. MKP-1 antagonizes SAPK/JNK activation in response to diverse environmental stresses. Together, these findings identify MKP-1 as a hypoxia-responsive gene and suggest a critical role in the regulation of SAPK/JNK activity in the tumor microenvironment.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa 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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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 SAPKgamma /JNK1 antibody (JNK1 (FL); immunizing antigen full-length recombinant human JNK1; cross-reactive with p54 SAPKalpha 2/JNK2 and SAPKbeta /JNK3), monoclonal anti-p46 SAPKgamma /JNK1 antibody (JNK1 (F-3); immunizing antigen full-length recombinant human JNK1), rabbit polyclonal anti-SAPKalpha /JNK2 (JNK2 (FL); immunizing antigen full-length recombinant human JNK2; cross-reactive with p46 SAPKgamma /JNK1 and SAPKbeta /JNK3), and monoclonal anti-phospho-SAPK/JNK antibody (p-JNK (G-7); immunizing antigen amino acids 183-191 of human SAPKgamma /JNK1 phosphorylated on Thr-183 and Tyr-185; identical with the corresponding region of phosphorylated human SAPKalpha /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 [gamma -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 beta -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 beta -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 [gamma -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 [alpha -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 [gamma -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 [alpha -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, beta -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 beta -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.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-SAPKgamma /JNK1 or -SAPKalpha /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 approx  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 SAPKgamma /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 SAPKgamma /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 SAPKgamma /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 SAPKgamma /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 SAPKgamma /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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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 SAPKgamma /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 SAPKgamma /JNK1 or p38 MAPK protein levels in SiHa cells. Photograph of a Western blot of total SiHa cell protein probed with the anti-SAPKgamma /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 SAPKgamma /JNK1 isoforms and to p38 MAPK are indicated by arrows.

Hypoxia-inducible SAPK/JNK Activation Involves Both SAPKalpha /JNK2 and SAPKgamma /JNK1-- The anti-SAPKgamma /JNK1 antibody used for the immunoprecipitations shown in Fig. 2A cross-reacts with both human SAPKalpha /JNK2 and SAPKbeta /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-SAPKgamma /JNK1 antibody. To confirm that SAPKgamma /JNK1 is activated by hypoxia, an identical immunoprecipitation study was performed involving a monoclonal antibody specific for p46 SAPKgamma /JNK1. Fig. 3A shows that hypoxia transiently stimulated p46 SAPKgamma /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 SAPKgamma /JNK1 activity detected by the monoclonal compared with the polyclonal SAPKgamma /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 SAPKgamma /JNK1 in SiHa cells and that the total basal level of this SAPKgamma /JNK1 isoform remained constant for up to 6 h of hypoxia. Fig. 3C shows that SAPKalpha /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 SAPKalpha /JNK2 protein levels remained constant for at least 6 h of hypoxia. Finally, using a specific cDNA probe for SAPKbeta /JNK3 (36), no signal was detected on a Northern blot of SiHa cell total RNA (data not shown) indicating that SAPKbeta /JNK3 is not significantly expressed in these cells. It has been reported that SAPKbeta /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 SAPKalpha /JNK2 and SAPKgamma /JNK1.


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Fig. 3.   Transient SAPK/JNK activation in hypoxic SiHa cells involves both SAPKgamma /JNK1 and SAPKalpha /JNK2. A, representative autoradiograph showing phosphorylation of the GST-ATF2-(1-94) substrate by p46 SAPKgamma /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 SAPKgamma /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 SAPKgamma /JNK1 isoform is indicated by an arrow. C, representative autoradiograph showing phosphorylation of the GST-ATF2-(1-94) substrate by SAPKalpha /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-SAPKalpha /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 SAPKalpha /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 SAPKalpha /JNK2 and SAPKgamma /JNK1; see "Experimental Procedures"). This finding parallels that of the immunocomplex kinase assay shown in Fig. 3 for the monoclonal antibody specific for p46 SAPKgamma /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 TNFalpha (37). Together, the findings shown in Figs. 1-4 provide strong evidence to support the hypothesis that pathophysiological hypoxia induces both SAPKalpha /JNK2 and SAPKgamma /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.

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).

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, beta -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).

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Right-arrow  MAPK kinase Right-arrow  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, SAPKgamma /JNK1, and p38 MAPK, whereas rat MKP-2 (related to human VH-2) preferentially recognizes ERK2 and SAPKalpha /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 SAPKgamma /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-alpha (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 SAPKalpha /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 Ikappa 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 SAPKgamma /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.

    FOOTNOTES

* This work was supported by Grants CA73807, CA20329, and CA67166 from the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Pharmaceutical Discovery Div., SRI International, 333 Ravenswood Ave., Menlo Park, CA 94025. Tel.: 650-859-3080; Fax: 650-859-5816; E-mail: keith.laderoute{at}sri.com.

2 K. R. Laderoute, H. L. Mendonca, J. M. Calaoagan, and A. M. Knapp, unpublished data.

    ABBREVIATIONS

The abbreviations used are: ATF2, activating transcription factor 2, SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal protein kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PBS-T, PBS with Tween 20; MKP, MAPK phosphatase; TPA, 12-O-tetradecanoylphorbol-13-acetate; PTK, protein-tyrosine kinase; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.

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TOP
ABSTRACT
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RESULTS
DISCUSSION
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