Mitogen-activated Protein Kinase Phosphatase-1 (MKP-1) Expression Is Induced by Low Oxygen Conditions Found in Solid Tumor Microenvironments

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.

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 ATF2 1 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)(18)(19)(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)(22)(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)(24)(25)(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.
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 (pO 2 ) values within the range of approximately 1% to Յ0.01% (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 pO 2 Յ 0.01%. SiHa cells were plated at 10 6 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% CO 2 /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% CO 2 /95% N 2 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% CO 2 /balance N 2 to prepare cell lysates without significant reoxygenation. For reoxygenation experiments, hypoxic cells were incubated in 5% CO 2 /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 (pO 2 Յ 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 g/ml aprotinin, 5 g/ml leupeptin, 1 mM benzamidine hydrochloride, 100 M Na 3 VO 4 , 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 g/ml aprotinin, 0.5 g/ml leupeptin, 1 mM benzamidine hydrochloride, 100 M Na 3 VO 4 , 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 ϫ 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 g of cell protein.
The tubes were gently rotated at 4°C for 1 h, spun at 11,000 ϫ 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 l of kinase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 100 mM NaF, 1 mM Na 3 VO 4 , 0.4 mM ATP) and 4 Ci of [␥-32 P]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 l of a 2ϫ 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 l 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 Na 3 VO 4 , 1 mM PMSF, 20 g/ml aprotinin, 10 g/ml leupeptin). The cells were lysed by scraping and the suspensions were transferred to gasketcap microcentrifuge tubes for spinning at 14,000 ϫ 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 l 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 ϫ g at 4°C for 10 min, and each lysate was normalized to 100 or 200 g of total protein and divided into two equal samples. One sample received an amount of a specific antibody (3 g of anti-p38 MAPK antibody; 1 g 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 l of the agarose beads and was gently tumbled for at least 1 h at 4°C. The beads were spun at 600 ϫ 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 MgCl 2 , 2 mM DTT, 25 mM sodium ␤-glycerophosphate, 0.1 mM Na 3 VO 4 ). The kinase buffer was removed, and 5 g of substrate was added to each sample followed by 20 l of kinase buffer containing 20 M ATP and 3-5 Ci of [␥-32 P]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 l of 2ϫ 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 Ϫ80°C. Frozen lysates were thawed on ice and centrifuged at 10,000 ϫ g for 5 min at 4°C. The protein concentrations of the supernatants were measured by a BCA assay. Equal protein samples (5-10 g) for gel electrophoresis were diluted with equal volumes of the 2ϫ 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 biotinlabeled 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-183ϩTyr-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 Na 3 VO 4 and 50 mM NaF, and then 200 l 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 Na 3 VO 4 , 50 mM NaF, 0.5 M 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 ϫ 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 g of total protein in a total volume of 600 l were prepared by adding the lysis buffer used for the ATF2 and c-Jun kinase assays. Suspensions (40 l) 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 ϫ g for 1 min at 4°C, washed four times with 1 ml of ice-cold PBS-T, diluted with an equal volume of 2ϫ 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 [␣-32 P]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 (CLON-TECH) end-labeled with [␥-32 P]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/m 2 (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 g/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 ϫ 10 6 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 l of ice-cold lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl 2 , 0.5% Nonidet P-40). After removal of the original lysate, each dish was washed with another 800 l of ice-cold lysis buffer and the combined suspensions were kept on ice. The lysates were spun at 500 ϫ 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 ϫ g, each pellet was resuspended Nylon membrane (MSI) slot blots of 250 ng each of MKP-1, ␤-actin, and pBluescript II KS ϩ (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 4ϫ Denhardt's solution containing 1 g/ml Saccharomyces cerevisiae tRNA, and hybridized at 42°C for 36 -48 h with 1-5 ϫ 10 6 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.

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 (pO 2 Յ 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 (pO 2 Յ 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 pO 2 Յ 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  1 and 5) or under hypoxia (pO 2 Յ 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." 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. pO 2 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 pO 2 Ϸ 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 Ϯ 1.1 at 4 h, sample S.D., n ϭ 4). For comparison, sorbitol (300 mM for 1 h) induced p38 MAPK activity by approximately 8-fold relative to the control (8.4 Ϯ 3.1, n ϭ 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 Ϯ 3.3 at 4 h, n ϭ 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 stressinduced 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.
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 Ϯ 1.0 at 2 h, n ϭ 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 Ϯ 1.2 at 4 h, n ϭ 3), and Fig.  3D indicates that total basal SAPK␣/JNK2 protein levels re-mained 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  5 and 9), and from lysates of SiHa cells exposed to hypoxia (pO 2 Յ 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 Ն 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 (pO 2 Յ 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.
that the transient SAPK/JNK activity induced in hypoxic SiHa cells consists of contributions from both SAPK␣/JNK2 and SAPK␥/JNK1.

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 tran-sient 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  5), and from lysates of SiHa cells exposed to hypoxia (pO 2 Յ 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 ϭ 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 (pO 2 Յ 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 ϭ 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.
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-183ϩTyr-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.
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 stressinducible (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 Ϯ 0.2 at 2 h of hypoxia, n ϭ 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 Ϯ 0.8, n ϭ 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.

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 Ϯ 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 hypoxiainducible accumulation of MKP-1 mRNA.
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 Ϯ 1.1, n ϭ 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 Ϯ 0.4, n ϭ 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.
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-183ϩTyr-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)(58)(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 5Ј-and 3Ј-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 M), 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 IB 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)(72)(73)(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)(52)(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 receptordependent 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.