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Tumor Biology |
/ERK/AP-1 Signaling Cascade1
Center for Molecular Medicine, Samsung Biomedical Research Institute and Molecular Therapy Research Center, Sungkyunkwan University, Seoul, 135-230 [M. S. S., J-H. L., K. P.], and Graduate School of Biotechnology, Korea University, Seoul, 136-701 [Y. K. P.], Korea
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ABSTRACT |
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expression level and kinase activity were increased by hypoxia. Transfection of MKN28 cells with a dominant-negative PKC- blocked the induction of GRP78 through ERK by hypoxia, indicating that PKC- directly participated in GRP78 induction under hypoxia. Taken together, this study shows that a PKC--Raf-1-MEK-ERK-AP1 signaling cascade acts on a 12-O-tetradecanoylphorbol-13-acetate response element-like element to mediate hypoxia-induced GRP78 expression in human gastric cancer cells. We also confirmed in vivo the overexpression of GRP78 in surgical specimens of human primary gastric tumors. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MAPKs are serine/threonine kinases present in all of the cell types. They are the central mediators that transduce diverse extracellular signals, including mitogens, growth factor, cytokines, and hypoxia, from the cell membrane to the nucleus (6
, 7)
. Activation of ERK via a Raf-1
MEKERK signaling cascade mediates biological responses by phosphorylating a large number of substrates including transcription factors such as Elk-1, c-Myc, NF-IL6, ATF-2, and AP-1 (8, 9, 10)
. The AP-1 transcription factor complex is a major target of MAPK signaling pathways and consists of dimers of c-Jun/c-Jun, c-Jun/c-Fos, or c-Jun/ATF-2. The AP-1 complex binds to TREs with the consensus nucleotide sequence TGA(C/G)TCA present in the promoters of genes that regulate cell differentiation and proliferation (10
, 11)
. Activation of MAPK is regulated by phosphorylation of several signaling protein kinases including Ras, Raf-1, and PKC. Activation of Raf-1 can be achieved in a Ras-dependent or -independent pathway (12)
. PKC is a ubiquitous calcium- and phospholipid-dependent serine/threonine protein kinase and can be another upstream activator of Raf-1 (13
, 14)
. At present, 12 isoenzymes of PKC have been identified and can be divided into the following three groups: (a) conventional PKCs or cPKCs (
, ßI, ßII, and 
), which are activated by calcium or diacylglycerol; (b) novel PKCs or nPKCs (
, , 
, 
, and µ), which are independent of calcium for activity but responsive to diacylglycerol; and (c) atypical PKCs or aPKCs (
, 
, and 
), which do not require either calcium or diacylglycerol for activation (15)
. Although these isoforms of PKC share highly conserved domains, they differ in substrate specificity, tissue expression, and cellular distribution, indicating that they play different roles in the regulation of important cellular processes (16)
.
Hypoxia is a common feature of solid tumors and has the potential to promote malignant progression by altering gene expression. These alterations promote the expression of tumorigenic proteins, including cell-cycle-regulatory proteins, angiogenesis-regulatory proteins, metastasis-promoting proteins, metabolic enzymes, and transcription factors (17) . These changes in expression can occur at the transcriptional level through changes in transcriptional rates or at the post-transcriptional level through changes in mRNA stability (17) . Tumor hypoxia can also act as a trigger for enhanced growth, metastasis, radiation resistance, and chemotherapy resistance. Thus, understanding the induction of chronic hypoxia-inducible gene expression and the associated signal transduction pathways could provide clues to selectively preventing stress protein induction and selectively killing hypoxic cells in solid tumors.
Although adenocarcinoma of the stomach is the second most common cancer worldwide, little is known about stress responses in stomach cancers. In this study, we investigated the mechanism and signaling pathway involved in the induction of the GRP78 gene by chronic hypoxia in human gastric tumor cells. We demonstrate that GRP78 is induced in vivo in primary gastric tumors, and we show that transcriptional activation rather than mRNA stabilization contributes to the induction of GRP78 by chronic hypoxia. Furthermore, we demonstrate, for the first time, that a PKC-/ERK/AP-1 signaling cascade mediates the induction of GRP78 by chronic hypoxia.
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MATERIALS AND METHODS |
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Materials.
We obtained RPMI 1640, FBS, and penicillin/streptomycin from Life Technologies, Inc. (Gaithersburg, MD); actinomycin D, PD98059, GF-109203X, Gö6976, manumycin A, BAPTA/AM, GST-MEK1, and histone H1 from Calbiochem (La Jolla, CA); ß-actin monoclonal antibody, MBP, dexamethasone, and PMA from Sigma Chemical Co. (St. Louis, MO); anti-GRP78, anti-c-Jun, anti-c-Fos antibodies, anti-ERK2, anti-MEK1, and anti-Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); antiphosphospecific ERK1/2 antibody, GST-c-Jun, GST-ATF2, and T4 polynucleotide kinase from New England Biolabs (Beverly, MA); PKC sampler kit from Transduction Labs (Lexington, KY); poly(dI·dC), protein G-Sepharose, [-32P]ATP (specific activity, >5000 Ci/mmol), [-32P]UTP (specific activity, 3000 Ci/mmol), and [-32P]dCTP (specific activity, 3000 Ci/mmol) from Amersham Pharmacia (Uppsala, Sweden).
Cell Culture and Hypoxia.
Human gastric-tumor cell line MKN28, established from moderately differentiated adenocarcinoma (18)
, was provided by the Korean Cell Line Bank (KCLB number 80102) and maintained in RPMI 1640 with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2 humidified air. For all of the experiments, subconfluent cells were incubated in 0.5% FBS-containing RPMI 1640 overnight before exposure to hypoxia. Hypoxic conditions were generated by placing the cells in a Gas-pak pouch (Becton Dickinson, Cockeysville, MD). Oxygen deprivation is almost complete after 1 h of incubation.
Northern Blot Analysis.
Total cellular RNA was isolated from cells or tissues using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Total cellular RNA (15 µg) was separated on 1.2% agarose gels containing 2.2 M formaldehyde. The cDNA probe for human GRP78 was amplified by PCR using the following primers: 5'-GCCACGGGATGGTTCCTTGCC-3' and 5'-GCGGATCCAGGTCGACGCCGGCCA-3'. After purification, this 1.38-kb fragment was labeled with [-32P]dCTP using a PrimeIt II labeling kit (Stratagene, Cedar Creek, TX) according to the manufacturer’s instructions. Probe was hybridized to the membrane at 42°C overnight in 10 ml of hybridization buffer (50% formamide, 5 x SSC, 1 x Denhardt’s solution, 1% SDS, 9% dextran sulfate, and 0.1 mg/ml salmon sperm DNA). The blots were washed twice in 2 x SSC, 0.1% SDS for 20 min at 42°C and once in 0.2 x SSC, 0.1% SDS for 30 min at 65°C. The radioactive bands corresponding to the mRNA signals for GRP78 were quantified using a PhosphorImager (Molecular Dynamics). The blots were stripped of probes by washing in 0.5% SDS at 90°C for 20 min and then rehybridized with a cDNA probe to ß-actin. Signal intensities were calculated relative to ß-actin signals.
Nuclear Run-On Transcription Analysis.
Nuclei were isolated from MKN28 cells grown under normoxia or hypoxia for 24 h. Run-on transcription analysis was carried out as described by Greenberg and Ziff (19)
except that radioactively labeled RNAs were extracted with phenol-chloroform and purified on a QIAshredder spin column (Qiagen, Hilden, Germany). RNAs were hybridized with GRP78 or ß-actin cDNA and blotted onto a nylon membrane (Hybond-XL; Amersham) with a slot blot apparatus (Bio-Rad). Blots were washed, treated with RNase to digest nonhybridized RNA, and autoradiographed.
Immune Complex Kinase Assays.
Immune complex kinase assays were performed as described by Derijard et al. (20)
except that cells were washed twice with ice-cold PBS and lysed in Triton X-100 lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 50 mM NaF, 10 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 7 µg/ml pepstatin, and 5 µg/ml leupeptin] for 30 min at 4°C. Insoluble material was removed by centrifugation at 12,000 x g for 10 min at 4°C. Protein from whole cell lysates (400 µg) was incubated with specific antibody to JNK, p38, ERK2, Raf-1, or PKCs, for 2 h at 4°C. After addition of Protein G-Sepharose, immunoprecipitation was continued for 1 h. Immune complexes were washed twice with Triton X-100 lysis buffer and three times with 20 mM HEPES (pH 7.4) and resuspended in 20 µl of kinase assay buffer containing 10 mM MgCl2, 2 mM DTT, 0.2 mM sodium orthovanadate, and 1 µg of substrate. GST-c-Jun, GST-ATF2, MBP, GST-MEK1, or histone H1 was used as a substrate for JNK, p38, ERK, Raf-1, or PKCs. The MAPK, Raf-1, or PKC activity assays were carried out in the presence of 20 µM of unlabeled ATP and 2 µCi of [-32P]ATP for 30 min at 30°C, for 20 min at room temperature, or for 5 min at 30°C, respectively, and stopped by addition of 5 µl of 5 x SDS sample buffer [312.5 mM Tris-Cl (pH 6.8), 10% 2-mercaptoethanol, 10% (w/v) SDS, 0.5% bromophenol blue, 10% (v/v) glycerol] and boiling for 5 min. The reaction mixtures were separated on 10 or 15% SDS-polyacrylamide gels. Phosphorylated substrates were visualized by autoradiography.
Western Blot Analysis.
Cells were lysed in radioimmunoprecipitation assay buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 7 µg/ml pepstatin, 5 µg/ml aprotinin, and 5 µg/ml leupeptin] for 20 min at 4°C. Insoluble material was removed by centrifugation at 12,000 x g for 10 min at 4°C. Protein from whole cell lysates (25 µg) was separated by SDS-polyacrylamide gel and electrophoretically transferred onto an enhanced chemiluminescence nitrocellulose membrane (Amersham Pharmacia). Antisera used for Western blot analysis were anti-GRP78 monoclonal (1:1,000), anti-ß-actin monoclonal (1:5,000), anti-c-Jun and anti-c-Fos antibodies (1: 1,000), anti-p-ERK1/2 monoclonal, anti-ERK2 polyclonal, anti-MEK1 polyclonal (1:2,000), or anti-PKC antibodies (1:500). Horseradish peroxidase-conjugated IgGs were used as secondary antibodies. Rabbit antigoat IgG (Zymed, San Francisco, CA) was used against anti-GRP78 antibody (1:5,000), goat antimouse IgG (Zymed) was used against anti-ß-actin antibody (1:10,000), and anti-PKC antibodies (1:2,000) or goat antirabbit IgG (Amersham Pharmacia) was used against anti-ERK2 and anti-MEK1 antibody (1:5,000). Western blots were developed using the enhanced chemiluminescence detection kit (Amersham Pharmacia) according to the manufacturer’s instructions.
Transient Transfection and Luciferase Assays.
Cells were transiently transfected with reporter constructs (designed as pGRP78-Luc or pAP-13-Luc). pGRP78-Luc contains the 374-bp stretch of human GRP78 promoter (21)
generated by PCR amplification (primers: 5'-CCCGGGGTCACTCCTGCTGGACCTA-3' and 5'-CCTCACCGTCGCCTACTCGGCTAT-3') and inserted into pGL3-basic vector (Promega, Madison, WI). The pAP13-Luc construct containing three TRE-like sites from GRP78 promoter corresponding to the region from nucleotides -180 to -163 was amplified by PCR (primers: 5'-TCGAAATGAATCAGCTGGGGGG-3' and 5'-TCGACCCCCCAGCTGATTCATT-3', with linker sequences shown in italics) and then inserted into pGL3 promoter vector (Promega). MKN28 cells in 60-mm dishes were transfected at 40–50% confluency with 1 µg of pGRP78-Luc or pAP-13-Luc using Effectene transfection reagent (Qiagen) according to manufacturer’s instructions. As an internal control to correct for variations in transfection efficiency, 50 ng of pRL-SV40 (Promega) was cotransfected. After 12 h of incubation, cells were given fresh medium containing 0.5% FBS and then additionally incubated for 12 h before exposure to normoxia or hypoxia. After 24 h, the cells were harvested, and luciferase activity was measured using a dual Luciferase Reporter Assay system (Promega) according to the manufacturer’s instructions. The assay was normalized for Renilla luciferase activity to correct for variations in transfection efficiency. To examine the functional role of ERK, MAPK, and specific PKC isoforms in MKN28 cells exposed to hypoxia, cells were transfected with either 0.5 µg of a DN-MEK1 (22)
or 1 µg of a DN-PKC- or DN-PKC- (23)
.
Preparation of Nuclear Extracts, EMSAs, and Coimmunoprecipitation.
Nuclear extracts were prepared from cultured MKN28 cells by the method of Schreiber et al. (24)
. EMSAs were performed with the oligonucleotides derived from the TRE-containing region of the human GRP78 promoter (nucleotides -180 to -163). Sense (5'-AATGAATCAGCTGGGGGG-3') and antisense (5'-CCCCCCAGCTGATTCATT-3') strands of oligonucleotides were annealed into double-stranded oligonucleotides and were 5' end-labeled with [-32P]ATP (5000 Ci/mmol) using T4 polynucleotide kinase and then purified through 15% nondenaturing polyacrylamide gel. Dialyzed nuclear extracts (5 µg) were preincubated on ice for 15 min with reaction buffer containing 15 mM HEPES (pH 7.9), 100 mM KCl, 3 mM MgCl2, 1 mM EDTA, 10% glycerol, 0.5 mM DTT, and 2 µg of poly(dI·dC). A 32P-labeled oligonucleotide probe (0.5 ng) was added to the reaction mixture and then incubated at room temperature for 20 min. For binding competition experiments, the unlabeled AP-1 oligonucleotide competitor was added at 100-fold molar excess 15 min before an addition of 32P-labeled probe. Antibody interference assays were carried out by incubation with anti-cJun (sc-44X) or anti-cFos (sc-413X) for 20 min before an addition of 32P-labeled probe. DNA-protein complexes were resolved on a 5% nondenaturing polyacrylamide gel in 0.5 x Tris-borate EDTA buffer [45 mM Tris-borate, 1 mM EDTA]. AP-1 complexes in the nuclear extracts (200 µg) were coimmunoprecipitated with anti-c-Jun antibody (Santa Cruz Biotechnology) and protein G-Sepharose (Amersham Pharmacia). Proteins in the immune complex were separated by 10% SDS-PAGE and then analyzed by Western blot analysis with anti-c-Fos antibody.
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RESULTS |
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. The amount of GRP78 mRNA additionally increased after 36 h (data not shown). Increases in gene expression can occur at the transcriptional level by increasing the transcriptional rate or at the post-transcriptional level by stabilizing mRNA (17)
. To distinguish between these possibilities, first, nuclear run-on assays were performed with MKN28 nuclei to determine whether hypoxia leads to an increase in transcription. 32P-labeled RNAs transcribed from MKN28 nuclei were hybridized to filter-immobilized human GRP78 cDNA and ß-actin cDNA (standard for quantitation). The transcriptional rate of the GRP78 gene was 7-fold greater after 24 h of hypoxic incubation, whereas the transcription of the ß-actin gene was only slightly greater (Fig. 1B)
. Second, to determine whether hypoxia leads to stabilization of GRP78 mRNA, actinomycin D was used to block transcription. Treatment of hypoxic MKN28 cells with actinomycin D (7.5 µg/ml) made no significant difference in GRP78 mRNA (Fig. 1C)
. These results demonstrate that transcriptional activation, not mRNA stabilization, contributes to the induction of GRP78 by chronic hypoxia.
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3-fold greater than in the control (Fig. 2A)
. This activation of ERK was time-dependent and resulted in maximal activation at 16 h (Fig. 2B)
. The enhancement of the activity of ERK by hypoxia was additionally confirmed by Western blotting with a phosphospecific ERK antibody. A highly specific inhibitor of MEK1, PD98059 (50 µM), completely blocked the phosphorylation of ERK by hypoxia (Fig. 2C)
. When cells were pretreated with PD98059 (50 µM) for 1 h and incubated under normoxia and hypoxia, PD98059 completely abolished the induction of GRP78 mRNA and protein under hypoxia (Fig. 2D)
. Taken together, these results demonstrate that ERK is necessary for the induction of GRP78 by hypoxia in MKN28 cells.
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, Lanes 1 and 3). The role of ERK on the activation of the GRP78 promoter under chronic hypoxia was additionally evaluated with MEK1 inhibitor PD98059. Pretreatment of the transfected cells with PD98059 (50 µM) inhibited the transcriptional activation of the GRP78 promoter by hypoxia (Fig. 3A
, Lane 4). To confirm the significance of ERK activation in GRP78 induction, we examined the effect of a DN-MEK1. The overexpression of DN-MEK1 was confirmed by Western blot analysis (Fig. 3B)
. The increase in the transcriptional activity of GRP78 promoter by hypoxia was also inhibited by DN-MEK1 (Fig. 3A
, Lane 5). Taken together, these results demonstrate that activation of ERK is necessary for the transcriptional activation of the GRP78 promoter by chronic hypoxia.
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, Lanes 1–4). This induction of AP-1 binding activity precedes GRP78 induction. Because ERK activity is required for the activation of the GRP78 promoter by hypoxia, we examined the role of ERK on AP-1 DNA binding activity. Enhancement of AP-1 DNA binding activity by hypoxia was inhibited by pretreatment with PD98059 (50 µM; Fig. 4A
, Lanes 5–7). To confirm the binding specificity of the DNA-protein complex, a competition DNA binding assay was performed with unlabeled TRE-like oligonucleotide. The formation of the AP-1 complex was completely inhibited by the addition of 100-fold excess of unlabeled competitor (Fig. 4A
, Lane 8). Thus, the DNA-protein interactions were sequence-specific.
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, Lanes 9–12). These results demonstrate that hypoxia-enhanced DNA binding activity of AP-1 is probably composed of c-Jun/c-Fos dimer, and ERK mediates enhancement of AP-1 binding activity. Next, the induction of AP-1 components was examined by Western blot analysis. A dramatic induction of AP-1 components c-Fos and c-Jun was evident in the nuclear extract prepared from cells grown under hypoxia (Fig. 4B)
. This induction was inhibited by PD98059. To demonstrate the interaction between c-Jun and c-Fos as a functional AP-1 complex, coimmunoprecipitation was performed. Direct interaction between c-Jun and c-Fos was evident (Fig. 4C)
.
Because changes in AP-1 DNA binding activity do not mirror the transcriptional activity of the complex, we next tested whether hypoxia-induced AP-1 was capable of transcriptionally activating an AP-1-dependent reporter gene. Transient transfection assays in MKN28 cells with a reporter construct, pAP13-Luc, which contains three TRE-like sequences from the GRP78 promoter, demonstrated that hypoxia increased the luciferase activity of transfected cell extracts by 3-fold (Fig. 4D
, Lanes 1 and 3). To additionally evaluate the role of ERK on the activation of AP-1 transcriptional activity, we again used PD98059 and DN-MEK1. Both PD98059 and DN-MEK1 completely prevented the increase in the AP-1-dependent transcriptional activity by hypoxia (Fig. 4D
, Lanes 4 and 5). These data prompted us to hypothesize that the overall transcriptional activity of the GRP78 promoter may be regulated by AP-1 during chronic hypoxia in MKN28 cells. The importance of AP-1 activity in hypoxia-induced expression of the GRP78 gene was confirmed by using dexamethasone, an inhibitor of AP-1 activity. Induction of GRP78 mRNA by hypoxia was completely blocked by pretreatment with dexamethasone (0.5 µM; Fig. 4E
).
ERK-mediated GRP78 Induction by Chronic Hypoxia Involves Activation of Raf-1 and PKC.
We next examined the upstream signaling molecules that may activate ERK during chronic hypoxia. Because the activation of ERK by growth factors leads to the activation of Ras followed by recruitment of Raf-1 to the plasma membrane and then subsequent activation of MEK1 (12)
, we explored whether Raf-1 and Ras are involved in ERK-mediated GRP78 induction. Chronic hypoxia induced Raf-1 activity >3-fold when measured by immune complex kinase assays using GST-MEK as a substrate (Fig. 5
, Lanes 1 and 2). In contrast, Ras did not act as an activator of Raf-1, because activation of Raf-1 and ERK was not affected by manumycin A (30 µM), a potent inhibitor of Ras (Fig. 5
, Lane 5).
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, ßI, ßII, , , and isoforms (25)
, whereas Gö6976 selectively inhibits Ca2+-dependent PKC and ß, but does not affect Ca2+-independent PKC, , and isoforms, even at micromolar levels (26)
. Pretreatment with GF-109203X (10 µM) inhibited kinase activities of both Raf-1 and ERK as well as the induction of GRP78 transcripts during chronic hypoxia (Fig. 5
, Lane 3). In contrast, pretreatment with Gö6976 (2 µM) had little effect on ERK activity and GRP78 mRNA (Fig. 5
, Lane 4). These results demonstrate that ERK-mediated GRP78 induction by chronic hypoxia is dependent on specific PKC isoforms.
PKC- Is Necessary for ERK-mediated GRP78 Induction by Chronic Hypoxia.
To additionally identify the specific PKC isoforms responsible for the ERK-mediated GRP78 induction by hypoxia, the induction of ERK activity and GRP78 mRNA by hypoxia were examined during treatment with PMA or BAPTA. Prolonged treatment with PMA completely inhibited both hypoxia-induced GRP78 induction and the activity of ERK (Fig. 6A
, Lane 3). In contrast, pretreatment of MKN28 cells with BAPTA/AM (50 µM for 30 min), a membrane permeable calcium-chelator, did not inhibit the induction of ERK activity or GRP78 mRNA by hypoxia (Fig. 6A
, Lane 4). Taken together, these results indicate that PMA-sensitive and Ca2+-independent nPKCs (PKC and/or PKC) are involved in the ERK-mediated GRP78 induction by hypoxia. To additionally identify the PKC isoform responsible, we used immune kinase assays to measure the enzymatic activities of six PKC isoforms expressed in MKN28 cells. Hypoxia dramatically enhanced the activity of PKC- (Fig. 6B)
. This enhanced PKC- kinase activity was time-dependent and peaked at 4 h (Fig. 6C)
. On the other hand, PKC kinase activity was inhibited (Fig. 6B)
.
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Is Necessary for ERK-mediated GRP78 Induction by Chronic Hypoxia in MKN28 Cells. in the ERK-mediated GRP78 induction, we transfected cells with a DN-PKC- or a DN-PKC-. The expression of each transfected DN-PKC gene was verified by Western blotting (Fig. 7A)
. In both cases, kinase activity was dramatically lower than in transfected cells, as expected from its dominant-negative property (data not shown). The induction of ERK activity, GRP78 mRNA, and AP-1-dependent transcriptional activity by chronic hypoxia was significantly blocked by DN-PKC- but not by DN-PKC- (Fig. 7B–D)
. Taken together, our results demonstrate that PKC- is necessary for ERK-mediated GRP78 induction by chronic hypoxia.
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. On the other hand, little or no GRP78 mRNA was detected in normal stomach tissues. After normalization with 28S rRNA, quantitation of mRNA bands revealed a 3–10-fold increase in GRP78 mRNA in gastric tumors compared with normal tissues of the same patient. In addition, paraffin sections of advanced gastric tumor specimens were processed for the induction of GRP78 protein by immunohistochemistry using a polyclonal GRP78 antibody. Strong GRP78 immunoreactivity was detected in tumor tissues, whereas little or no immunoreactivity was detected in normal gastric tissues (Fig. 8B)
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DISCUSSION |
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Previous studies by other investigators have shown rapid and transient activation of ERK, JNK, and p38 MAPK during hypoxia (28
, 29)
. In this study, we investigated which MAPK is involved in the signaling cascade of GRP78 induction by chronic hypoxia, because it occurs in gastric tumors. We showed that ERK activation is necessary for the induction of GRP78 in MKN28 cells by using both PD98059, a highly specific inhibitor of MEK1 and a DN-MEK1. Pretreatment of cells with PD98059 prevented the increase in the activity of ERK, the level of GRP78 mRNA, the level of GRP78 protein, and the transcriptional activity of the GRP78 promoter. Whereas pretreatment of MKN28 cells with BAPTA did not inhibit either the induction of ERK activity or GRP78 mRNA by hypoxia (Fig. 6A)
, treatment of MKN28 cells with Tg, an ER Ca2+-ATPase inhibitor, blocked the activation of ERK, and PD98059 could not block the Tg-induced accumulation of GRP78 mRNA and protein, even at a high concentration of PD98059 (150 µM).4
A recent report also demonstrated that GRP78 induction by Tg treatment was inhibited by chelation of intracellular calcium with BAPTA (30)
. Taken together, these results support the notion that different signaling pathways mediate induction of GRP78 by Tg and hypoxia.
We hypothesized that AP-1 might be involved in the regulation of GRP78. Induction of AP-1 activity can occur either by an increase in the abundance of AP-1 components or by an increase in their transactivating potential without affecting their binding activities (11)
. Our data revealed dramatic increases in the levels of AP-1 components of the c-Fos and c-Jun family during hypoxia and showed that these increases were strongly dependent on ERK activity (Fig. 4B)
. In addition, our transfection studies in human gastric cancer cells demonstrated that enhancement of AP-1 DNA binding activity also involves transcriptional induction of the GRP78 gene through a TRE-like motif (Fig. 4D)
. Inhibition of GRP78 induction by dexamethasone, an inhibitor of AP-1 activity, additionally supported the requirement of AP-1 activity for the induction of GRP78 by hypoxia (Fig. 4E)
. However, we did not address whether or not induction of AP-1 activity by itself, in the absence of hypoxia, was sufficient to mediate the induction of GRP78 transcription. The abundance of AP-1 protein is usually regulated by transcriptional regulation of the genes encoding AP-1 components (11)
or by modulation of the stability of these components through ubiquitination (31)
.
Chronic hypoxia leading to ERK activation can enhance the activities of other transcription factors. So we looked for other factors that might bind to the GRP78 promoter. One of the important motifs involved in responses to hypoxia is the HIF-1 binding motif (17) . However, HIF-1 does not appear to play a role in the induction of GRP78 under chronic hypoxia, because no recognizable HIF-1 binding motif is present in the sequence of the GRP78 promoter. Using the CRE, ER stress response element, or the core region of GRP78 as an EMSA probe, no specific protein-DNA complexes were induced by chronic hypoxia in MKN28 cells.4
Other investigators have found that several signaling pathways, including PKC, PKA, and p38 MAPK, are involved in the induction of GRP78 in a variety of cell culture systems (32, 33, 34)
. The GRP78 gene was reported to be rapidly activated after treatment with okadaic acid followed by heat shock in rat brain tumor cells; a CRE element upstream of rat GRP78 was necessary for this rapid induction, and this activation involved the PKA signaling pathway and p38 MAPK signaling pathways (33
, 34)
. However, the mechanisms for immediate response to hypoxia and chronic hypoxia can be quite different. Depending on cell type and stimulus, ERK activation may occur by either PKC-dependent or -independent pathways (12
, 35)
and duration of ERK activation can be critical for determining signaling pathways (36)
. In this study, we also showed that hypoxia specifically increases both the level of expression and the enzymatic activity of PKC- of six PKC isoforms expressed in MKN28 cells (Fig. 6, B and C)
. We used PKC inhibitors and DN-PKC isoforms to demonstrate that ERK-mediated GRP78 induction by hypoxia in MKN28 cells depends on the PKC- isoform. Catalytically inactive dominant-negative molecules are known to act by competing with the corresponding endogenous isoforms (37)
. The essential role of PKC- in the ERK-mediated GRP78 induction as a delayed and sustained response to hypoxia in MKN28 cells was additionally confirmed by showing its inhibition by DN-PKC- but not by DN-PKC- (Fig. 7)
.
This study did not examine whether hypoxia directly modulates the function of other signal transducing molecules (e.g., receptors, G proteins, and phospholipase) or leads to the release of soluble factors (e.g., angiotensin II and endothelin) that act on receptors to stimulate intracellular signal transduction pathways (38, 39, 40)
. Earlier reports showed that phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate generated by PI3k activity are potent and selective activators of nPKC isoforms and have little effect on cPKCs or aPKCs (41)
. Furthermore, Moriya et al. (42)
reported that both the PI3k and phospholipase C pathways can activate PKC- in a cell-specific and stimulus-specific manner. The specificity of the PI 3k pathway for PKC- suggests its involvement in the signaling pathway leading to the induction of GRP78 by chronic hypoxia. Our preliminary findings with PI3k inhibitors suggest the involvement of PI3k in GRP78 induction in MKN28 cells (data not shown).
In this study, we did not address why cancer cells induced GRP78 mRNA and protein under chronic hypoxia. However, studies by other investigators have reported evidence suggesting that endoplasmic reticulum resident chaperones, including GRP78, are involved in cellular survival during chronic hypoxia (43) . In addition, the induction of GRP78 was reported to protect cells by suppressing oxidative damage and stabilizing calcium homeostasis (44 , 45) . Furthermore, the degree of GRP78 induction appears to regulate Tg-induced apoptosis (46) . Usually, normal mammalian cells use the ERK signaling pathway to transduce survival signals for cell growth and development by hormones and growth factors. But cancer cells might modify the ERK signaling pathway to transduce signals that prevent hypoxia-induced apoptosis. Future studies will address the roles of GRP78 in the survival of cancer cells in a hypoxic microenvironment.
In summary, we demonstrated that GRP78 mRNA and protein are overexpressed in human gastric cancer cells exposed to chronic hypoxia as well as in surgical specimens of primary gastric tumors. Our results suggest that GRP78 induction has clinical importance in human gastric tumors. In addition, we also elucidated that sustained induction of GRP78 by chronic hypoxia is mainly attributable to transcriptional activation rather than mRNA stability, and we uncovered a novel signaling pathway involving a PKC-/ERK/AP-1 signaling cascade mediating the induction of GRP78 in human gastric tumor cells. To our knowledge, this is the first report to link chronic hypoxia to the induction of the GRP78 gene in human gastric tumors.
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ACKNOWLEDGMENTS |
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and PKC- expression plasmids. We are grateful to General Director Jung-Don Seo at Samsung Biomedical Research Institute, Seoul, Korea for his support and encouragement throughout this work. |
FOOTNOTES |
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1 Supported by Samsung Grant SBRI B-98-024 and SRC Grant from Korea Science and Engineering Foundation. 
2 To whom requests for reprints should be addressed, at Molecular Therapy Research Center, Sungkyunkwan University, SMC Annex B252, 50 Ilwon-dong, Kangnam-ku, Seoul, 135-230, Korea. Phone: 82-2-3410-3629; Fax: 82-2-3410-3649; E-mail: hannah05{at}dreamwiz.com
3 The abbreviations used are: GRP78, Mr 78,000 glucose-regulated protein; DN, dominant-negative mutant; ERK, extracelluar signal-regulated protein kinase; FBS, fetal bovine serum; MBP, myelin basic protein; JNK, c-Jun-NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAP/ERK kinase; AP-1, activator protein-1; Tg, thapsigargin; TRE, 12-O-tetradecanoylphorbol-13-acetate response element; PKC, protein kinase C; CRE, cAMP response element; EMSA, electrophoretic mobility shift assay; PMA, phorbol 12-myristate 13-acetate; PI3k, phosphatidylinositol 3'-kinase; BAPTA/AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester; GST, glutathione S-transferase; HIF, hypoxia inducing factor 1.
4 M. S. Song and K. Park, unpublished observations.
Received 4/17/01. Accepted 9/16/01.
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B. J. Biol. Chem., 274: 27981-27988, 1999. or phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. USA, 93: 151-155, 1996.This article has been cited by other articles:
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