Role of Double-Stranded RNA-Activated Protein Kinase R (PKR) in Deoxynivalenol-Induced Ribotoxic Stress Response

Hui-Ren Zhou*,{dagger},{ddagger}, Allan S. Lau§ and James J. Pestka*,{dagger},{ddagger},1

* Departments of Microbiology and Molecular Genetics and {dagger} Food Science and Human Nutrition, and {ddagger} Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan, 48824-1224; and § Department of Pediatrics, Hong Kong University, Hong Kong, China

Received January 13, 2003; accepted May 5, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trichothecene mycotoxins and other protein synthesis inhibitors activate mitogen-activated protein kinase (MAPKs) via a mechanism that has been termed the "ribotoxic stress response." MAPKs are believed to mediate the leukocyte apoptosis that is observed following experimental exposure to these chemical agents in vitro and in vivo. The purpose of this research was to test the hypothesis that double-stranded, RNA-activated protein kinase R (PKR) is a critical upstream mediator of the ribotoxic stress response induced by the trichothecene deoxynivalenol (DON) and other translational inhibitors. DON was found to readily induce phosphorylation of JNK 1/2, ERK 1/2, and p38 in the murine macrophage RAW 264.7 cell line, within 5 min of culture addition, in a concentration-dependent fashion. Effects were maximal from 15 to 30 min and lasted up to 6 h. The translational inhibitors anisomycin and emetine also had similar effects when added to cultures at equipotent concentrations to DON. DON rapidly activated PKR within 1 to 5 min, as evidenced by autophosphorylation and by phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}). Interestingly, the latter effect was associated with rapid degradation of eIF2{alpha}. Pretreatment of RAW 264.7 cells with two inhibitors of PKR, 2-aminopurine (2-AP) or adenine (Ad), markedly impaired MAPK phosphorylation in RAW 264.7 cells according to the following rank order JNK > p38 > ERK. The capacity of DON to induce MAPK phosphorylation was also markedly suppressed in a stable transformant of the human promonocytic U-937 cell line containing an antisense PKR expression vector. This suppression followed a rank order of JNK > p38 > ERK in this PKR-deficient cell line when compared to control cells transfected with vector only. Apoptosis induction by DON and two other translational inhibitors, anisomycin and emetine, was almost completely abrogated in PKR-deficient cells. Together, the results indicate that PKR plays a critical upstream role in the ribotoxic stress response inducible by translational inhibitors.

Key Words: trichothecene; mycotoxin; mitogen-activated protein kinase; apoptosis; macrophage.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trichothecenes are sesquiterpenoid mycotoxins that contaminate agricultural commodities (Scott, 1990Go) and indoor air environments (Cooley et al., 1998Go). These toxins are of concern because of their documented adverse effects on human and animal health (Bhat, 1989; Etzel et al., 1998Go; Li et al., 1999Go, 2002Go). Trichothecenes can be immunostimulatory and immunosuppressive, depending on dose, exposure frequency, and timing of functional immune assay (Bondy and Pestka, 2000Go; Laskin et al., 2002Go). Macrophages, T cells, and B cells are all highly sensitive to trichothecenes and it is believed that induction of apoptosis by these toxins mediates immune suppression whereas induction of cytokines contributes to immune stimulation. Deoxynivalenol (DON, or vomitoxin) is the most frequently encountered trichothecene in grain-based foods worldwide, and thus its capacity to impact immune function is of particular interest (Rotter et al., 1996Go).

The molecular target of trichothecenes in leukocytes and other actively proliferating eukaryotic cells is the 60s ribosomal subunit (Middlebrook and Leatherman, 1989aGo,bGo; Witt and Pestka, 1990Go). Translational inhibitors that bind to ribosomes rapidly activate mitogen-activated protein kinases (MAPKs) and apoptosis in a process that has been termed the "ribotoxic stress response" (Iordanov et al., 1997Go). MAPKs impact many physiological processes, including cell growth, differentiation, and apoptosis (Cobb, 1999Go), and are important transducers of the immune response (Dong et al., 2002Go). The primary MAPK subfamilies include: (1) p44 and p42 MAPKs, also known as extracellular signal-regulated protein kinase 1 and 2 (ERK1 and 2), (2) p54 and p46 c-Jun N-terminal kinase 1 and 2 (JNK1/2), also referred to as stress-activated protein kinases (SAPK 1/2); and (3) p38 MAPK. Both our laboratory and others have demonstrated that DON and other trichothecenes activate JNK, ERK, and p38 in vitro (Moon et al., 2002; Shifrin and Anderson, 1999Go; Yang et al., 2000aGo) and in vivo (Zhou et al., 2003Go), suggesting that the ribotoxic stress response might be a critical transduction step during trichothecene toxicity. A major unresolved question regarding the ribotoxic stress response relates to the molecular linkage between ribosome interaction and MAPK activation (Laskin et al., 2002Go).

Double-stranded RNA-(dsRNA)-activated protein kinase (PKR) is a widely-expressed serine/theonine protein kinase that can be activated by dsRNA, interferon, and other agents (Williams et al., 2001Go). The earliest described role of PKR was translational inhibition via phosphorylation of eukaryotic initiation factor 2 {alpha}-subunit (eIF2{alpha}), which is an evolutionarily conserved antiviral response. Besides eIF2{alpha} phosphorylation and auto-phosphorylation activities, PKR appears to have a wide serine-theonine kinase substrate specificity. This kinase might also act through protein–protein interactions, which do not require catalytic activity. PKR reportedly functions as a signal integrator for ligand-activated, stress-activated protein kinase pathways leading to stimulation of JNK and p38 (Goh et al., 2000Go; Takizawa et al., 2002Go; Williams, 2001Go). The kinase is also believed to play a key role in mediating apoptosis induced by dsRNA, LPS, and TNF-{alpha} (Der et al., 1997Go; Gil and Esteben, 2000; Yeung and Lau, 1998Go; Yeung et al, 1996Go). PKR potentially modulates induction of cytokines including TNF-{alpha} (Meusel et al., 2002Go), IL-6, and IL-12 (Goh et al., 2000Go). Our laboratory has previously observed that DON downregulates expression of P58Ipk, which is the 58-{kappa}Da cellular inhibitor of PKR (Yang et al., 2000bGo). Thus, the potential exists for PKR to mediate early events leading to immunotoxicity associated with leukocyte exposure to DON and other trichothecenes and, additionally, to be an early step in the ribotoxic stress response.

The purpose of this study was to test the hypothesis that PKR is a critical upstream mediator of MAPK activation and apoptosis during the ribotoxic stress response. The results suggest that PKR can mediate activation of JNK, ERK, and p38 by DON and two other translational inhibitors, anisomycin and emetine. Furthermore, PKR contributes to the subsequent induction of apoptosis by these chemical agents.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell cultures.
RAW 264.7 murine macrophage cells (American Type Culture Collection, Rockville, MD) (2.5 x 105 per ml) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Chemical Co., St. Louis, MO) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (HI-FBS, Atlanta Biologicals, Inc, Norcross, GA), 100 U/ml penicillin (Sigma), and 100 µg/ml streptomycin (Sigma, St. Louis, MO) in a 5% CO2 humidified incubator at 37°C. Macrophage cell number and viability were assessed by trypan blue (Sigma) dye exclusion using a hematocytometer. For DON-exposure studies, cells (5 x 105/ml) were seeded in 10 ml of medium in 100 cm2 sterile tissue culture dishes overnight to achieve 80% confluence.

U-937 human monocyte-like cells were stably transformed with plasmids containing a parental control expression vector pRC-CMV (Invitrogen, Carlsbad, CA) or a vector constructed with anti-sense PKR and designated as U9K-C2 and U9K-A1, respectively, as described previously (Der and Lau, 1995Go). Cells were maintained in RPMI 1640 medium supplemented with 5% (v/v) FBS, and 400 µg/ml Geneticin (Gibco, Rockville, MD) in 6% CO2 at 37°C.

Cells were treated with DON, anisomycin, or emetine (Sigma) over specified time intervals in the absence or presence of various pharmacological inhibitors, and then were analyzed for MAPK phosphorylation, PKR activation, DNA fragmentation, and/or caspase activity.

Detection of MAPK phosphorylation.
Cells were washed with ice-cold phosphate buffer, lysed in boiling lysis buffer (1% [w/v] sodium dodecylsulfate (SDS), 1.0 mM sodium ortho-vanadate, and 10 mM Tris pH 7.4), and sonicated for 5 s. Lysate was centrifuged at 12, 000 x g for 15 min at 4EC. Protein was measured in the resultant supernatant by a Bio-Rad DC protein assay kit (Bio-Rad Laboratories Inc, Melville, NY). Extracts (40 µg) were mixed with protein sample loading buffer (Amersham, Arlington Heights, IL), and boiled for 5 min before resolving on an 8% (w/v) acrylamide gel and transfer to a Hybond-P polyvinylidene difluoride (PVDF) membrane (Amersham). After blocking with TRIS buffered (pH 7.5, 0.01 M) saline containing 0.1% (v/v) Tween 20 and 3% (w/v) bovine serum albumin (TBST-BSA), the membrane was incubated for 1 h with MAPK antibodies (rabbit IgG, Cell Signaling, Beverly, MA) at 1:1500 dilution in TBST-BSA, and then were washed three times with TBST. These antibodies reacted with both mouse and human MAPKs. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ) at 1:3000 dilution in TBST-BSA for MAPK detection. After washing three times with TBST, bound HRP-conjugated antibody was detected with an Enhanced Chemiluminescence (ECL) kit (Amersham) according to manufacturer’s instructions. Membranes were then stripped and reprobed with specific antibodies that recognize both phosphorylated and unphosphorylated forms of each MAPK as described above.

Detection of PKR activity.
For detection of the PKR phosphorylation and the phosphorylation of its substrate eIF2{alpha}, extracts were subjected to electrophoresis and transferred to PVDF membranes as described above. Some cell cultures for eIF2{alpha} studies were preincubated for 1 h with Phosphatase Inhibitor Cocktail (1 µl/ml medium; Sigma) or with Complete Mini protease inhibitor (1 tablet/10 ml medium, Roche) prior to the addition of DON. These were incubated with phospho-PKR(thr451)-specific rabbit polyclonal antibodies (Calbiochem, Lajolla, CA) or phospho-eIF2{alpha}(Ser51)-specific rabbit polyclonal antibodies (Cell Signaling), followed by incubation with secondary antibodies and ECL detection as described above. The total protein levels of PKR or eIF2{alpha} loaded were assessed, after stripping and incubating with the antibodies, recognizing both phosphorylated and unphosphorylated forms of the proteins (Santa Cruz, Santa Cruz, CA).

Measurement of protein synthesis.
Inhibition of protein synthesis was measured by 3H leucine incorporation using a modification of the method of Yang et al. (2000a)Go. Briefly, 2 x 105 in 50 µl of medium were added to each well of a 96-well tissue culture plate and cultured for 1 h. Various concentrations of DON anisomycin (AN) or emetine (EM) in 50 µl of medium along with 1 µCi of L-[3,4,5–3H(N)] leucine (NEN Life Science Products, Inc., Boston, MA; 5.6 Tbq/mmol) were then added. Cells were cultured for 3 h and supernatant transferred to a matching set of wells. Cells were suspended in 100 µl of 53 mM EDTA containing 0.5% (w/v) trypsin (GIBCO), then 25 µl of 50% (w/v) trichloroacetic acid (TCA) were added, absorbed to a glass filter paper using a Titertek cell harvester (Skatron Instruments, Inc., Sterling, VA), and filters were washed twice with cold 5% TCA and with 95% ethanol. The cell supernatant was also precipitated with TCA and harvested onto filters containing the corresponding precipitated cell protein. Thus, both total cellular and extracellular proteins containing radioactivity were measured. Radioactivity was measured in 5 ml of scintillation cocktail (Research Products International Col, Mount Prospect, IL) using a liquid scintillation counter (Packard Instrument Co., Downers Grove, IL).

DNA fragmentation assay.
DNA was extracted and electrophoresed as described by Sellins and Cohen (1987)Go. Briefly, cells (1 x 107) in phosphate-buffered saline (pH 7.4) were centrifuged for 5 min (500 x g) at 4°C, and the pellet was suspended in 0.1 ml hypotonic lysing buffer (10 mM Tris, 10 mM EDTA, 0.5% [v/v] Triton X-100, pH 8.0). Cells were incubated at 4°C for 10 min. The resultant lysate was centrifuged for 30 min (20,000 x g) at 4°C. Supernatant containing fragmented DNA was digested for 1 h at 37°C with Rnase A (0.4 µg/µl) and then incubated for 1 h at 37°C with proteinase K (0.4 µg/µl). DNA was precipitated in 50% isopropanol and 0.5 M NaCl overnight at -20°C. The precipitate was centrifuged at 20,000 x g for 30 min at 4°C. The resultant pellet was air dried, resuspended in 10 mM Tris, 1 mM EDTA, pH 8.0, then electrophoresed at 75 V for 2 h in 2% agarose gel (5 x 106 cells equivalent per lane) in 90 mM Tris-borate buffer (pH 8.0) containing 2 mM EDTA. After electrophoresis, the gel was stained with ethidium bromide (0.5 µg/ml), and the nucleic acids were visualized with UV transilluminator. A 100-bp DNA ladder (GIBCO) was used as a molecular size marker.

Caspase-3 assay.
Cells were suspended in 200 µl of CHAPS buffer (100 mM HEPES [pH 7.5] containing 10% (w/v) sucrose, 0.5% (w/v) CHAPS, 1mM EDTA, 10 mM DTT, and 100 µl protease inhibitor cocktail (1:100) (Sigma), placed on ice for 30 min, sonicated briefly, and then centrifuged 10,000 x g for 10 min. Following protein determination, lysates (50 µg in 100 µl CHAPS buffer) were incubated with an equal volume (100 µl) of fluorogenic substrate consisting of 25 µM DEVD-AMC (Calbiochem, San Diego, CA) dissolved in CHAPS buffer at 37°C for 30 min. Substrate cleavage was detected by a CytoFluor II Microplate fluorescence reader (Biosearch, Bedford, MA) at excitation 360 nm and emission 460 nm.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DON and Other Translational Inhibitors Induce JNK 1/2, ERK 1/2, and p38 Phosphorylation in RAW 264.7 Cells
Exposure to DON at concentrations as low as 100 ng/ml for 15 min induced phosphorylation of JNK 1/2, ERK 1/2, and p38 in RAW 264.7 cells (Fig. 1Go). Phosphorylation of the three MAPKs increased with increasing DON concentrations. Maximum phosphorylation was observed at 500 ng/ml DON or higher. When the kinetics of MAPK phosphorylation was assessed over a 4-h time span using 250 ng/ml of DON, JNK 1/2, ERK 1/2, and p38, phosphorylation was first observed at 5 min (Fig. 2Go). JNK 1/2 phosphorylation was maximal from 10 to 30 min and last detectable at 360 min. ERK 1/2 phosphorylation was maximal at 30 to 240 min and last observed at 360 min. Finally, maximal p38 phosphorylation occurred between 15 and 240 min.



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FIG. 1. DON induces concentration-dependent JNK, ERK, and p38 MAPK phosphorylation. RAW 264.7 cells (5 x 105/ml) were treated with DON for 15 min. Cell lysates (40 µg) were resolved on SDS–PAGE and subjected to Western blot analysis with antibodies specific for phosphorylated JNK, ERK, and p38. Bands were detected using the ECL system. Afterwards, blots were stripped and reprobed with specific antibodies that recognized both phosphorylated and unphosphorylated forms of each MAPK for assessment of protein loading.

 


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FIG. 2. Kinetics of DON-induced phosphorylation of JNK, ERK, and p38 MAPK. RAW 264.7 cells (5 x 105/ml) were cultured with 250 ng/ml of DON for various time intervals. Cells were analyzed as described in the Figure 1Go legend.

 
The capacity of DON to inhibit leucine incorporation in RAW 264.7 cells was compared to that for the translational inhibitors anisomycin and emetine. The concentrations required to inhibit activity by 50% (IC50) were 26, 85, and 24 ng/ml for DON, emetine, and anisomycin, respectively (Fig. 3AGo). Since the IC50 for DON was effective at activating MAPKs, the effect of equipotent concentrations of anisomycin and emetine on MAPK phosphorylation was measured. As found for DON, markedly increased JNK, ERK, and p38 phosphorylation was observed in anisomycin-treated cultures at 15, 30, and 60 min (Fig. 3BGo). Emetine also induced phosphorylation of the three MAPKs, but to a much lesser extent.



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FIG. 3. Effect of translational inhibitors on MAPK phosphorylation. (A) Inhibition of translation by DON, anisomycin (AN), and emetine (EM). RAW 264.7 cells (1 x 106/ml) were incubated with various concentrations of DON, AN, or EM concurrently with 1 µCi of [3H] leucine for 3 h. Leucine incorporation in the trichloroacetic acid protein precipitate was measured as a percentage of control cells treated with vehicle only. DON, EM, and AN concentrations required for 50% inhibition IC50 were 268, 85, or 24 ng/ml, respectively. (B) Anisomycin and emetine induce MAPK phosphorylation. RAW 264.7 cells were treated with AN (25 ng/ml) and EM (80 ng/ml) for 15 to 60 min and cytolysates were assessed for MAPK phosphorylation as described in the Figure 1Go legend. CONT indicates vehicle control.

 
DON Induces PKR Activity in RAW 264.7 Cells
The effects of DON on PKR activity were evaluated. PKR has been characterized by its capacities for autophosphorylation and to catalyze phosphorylation of eIF2{alpha} (Williams 2001Go). DON at 250 ng/ml induced PKR phosphorylation from 1 to 5 min (Fig. 4Go). DON, at the same concentration, also rapidly (1–2.5 min) induced eIF2{alpha} phosphorylation, although the end product rapidly decreased to levels below that for control (Fig. 5Go). The simultaneous disappearance of total eIF2{alpha} suggested that this protein was being degraded as a result of DON exposure. Inclusion of a phosphatase inhibitor prolonged the eIF2{alpha} phosphorylation response to DON as well as preventing protein degradation. Incorporation of a protease inhibitor prolonged DON-induced eIF2{alpha} phosphorylation and also prevented eIF2{alpha} degradation. Overall, these results suggest that DON might rapidly induce PKR activation in RAW 264.7 cells as evidenced by its own phosphorylation, as well as eIF2{alpha} phosphorylation. DON subsequently mediated a phosphatase-dependent proteolytic degradation of this latter important translation component. Importantly, the observation that the timing of PKR activation preceded phosphorylation of MAPKs is consistent with an upstream role for PKR.



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FIG. 4. Kinetics of DON-induced PKR phosphorylation. RAW 264.7 cells (5 x 105/ml) were incubated with DON (250 ng/ml) at intervals and resultant lysates (40 µg protein) were subjected to Western-blot analysis using anti-PKR (Thr451) phospho-specific antibodies.

 


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FIG. 5. Kinetics of DON-induced eIF2{alpha} phosphorylation. RAW 264.7 cells (5 x 105/ml) were incubated with 250 ng/ml DON with or without 1 h of preincubation with phosphatase or protease inhibitor cocktails. Cell lysates were collected at intervals and total cellular proteins were resolved by 8% SDS–PAGE, transferred to membrane, and analyzed for phosphorylated and nonphosphorylated eIF2{alpha} by Western blot.

 
PKR Mediates DON-Induced MAPK Phosphorylation in RAW 264.7 Cells
To assess the potential role of PKR in DON-induced MAPK phosphorylation, the effect of pretreating cells with adenine (Ad) or 2-aminopurine (2-AP), two previously described PKR inhibitors (Wong and Yen, 1998Go), were assessed. The two compounds inhibited DON-induced JNK 1/2 phosphorylation in a concentration-dependent fashion (Fig. 6Go). ERK and p38 phosphorylation were slightly inhibited by 2-AP and a similar weaker trend was observed for Ad. These data suggest that PKR activation might contribute, in part, to DON-induced activation of JNK and, to a much lesser extent, the other two MAPK families.



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FIG. 6. PKR inhibitors adenine (Ad) and 2-aminopurine (2-AP) inhibit DON-induced MAPK activation in a concentration-dependent manner. RAW 264.7. cells (5 x 105/ml) were treated with (A) Ad or (B) 2-AP for 1 h before exposure to DON (100 ng/ml). Cell lysates were prepared 15 min after DON treatment and analyzed for phosphorylated JNK, ERK, and p38 by Western blot.

 
PKR Mediates Induction of MAPK Phosphorylation by DON and Anisomycin in U-937 Cells
Fundamental limitations with inhibitor studies are the potential for the pharmacologic agents to be metabolized, to exert toxicity, or to be promiscuous with regard to their action. Therefore, we assessed the role of PKR by employing human U-937 monocyte cell lines that were stably transfected with either PKR antisense vector (U9K-A1) or control vector (U9K-C2) and that were thoroughly described previously (Der and Lau, 1995Go; Yeung and Lau, 1998Go; Yeung et al., 1996Go, 1999Go). As found in the RAW 264.7 cells, JNK 1/2 and p38 phosphorylation were effectively induced in U9K-C2 cells at DON concentrations of 100 to 1000 ng/ml, whereas ERK 2 was selectively activated (Fig. 7Go). However, JNK 1/2 phosphorylation was nearly completely abrogated in the PKR-deficient U9K-A1 cells with only a trace amount of phosphorylated JNK 1/2 being detectable at the 500 and 1000 ng/ml DON concentrations. To a lesser extent, PKR deficiency also partially impaired ERK 2 and p38 phosphorylation effects that were most apparent at low DON concentrations (100–250 ng/ml).



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FIG. 7. DON-induced MAPK phosphorylation is suppressed by PKR deficiency. U937 cells containing parental control expression vectors (U9K-C2) or anti-sense PKR expression vectors (U9K-A1) were cultured (5 x 105/ml) and were treated with various DON concentrations as indicated for 15 min. Cell lysates (40 µg) were analyzed by Western-blot analysis as described in the Figure 1Go legend.

 
When the kinetics of DON-induced MAPK phosphorylation was assessed in U9K-C2 cells, effects were detectable from 15–240 min (Fig. 8Go). In U9K-A1 cells, both DON-induced JNK 1/2 and ERK 1/2 phosphorylation were detectable only from 30 to 120 min and the magnitude of this response was reduced. The time window for DON-induced p38 phosphorylation in PKR-deficient cells was not decreased; however, the magnitude of the phosphorylation was depressed. Similar MAPK phosphorylation kinetics were observed for anisomycin (Fig. 9Go). Overall, these results suggested that PKR was involved upstream, in part, with both DON- and anisomycin-induced JNK 1/2 phosphorylation and to a much lesser extent, with p38 and ERK 1/2 phosphorylation.



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FIG. 8. DON-induced MAPK phosphorylation is delayed and inhibited in PKR-deficient cells. U9K-C2 and U9K-A1 cells were incubated with 250 ng/ml of DON for various times intervals. Cell lysates (40 µg) were analyzed by Western blot using antibodies as described in the Figure 1Go legend.

 


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FIG. 9. Anisomycin-induced MAPK phosphorylation is depressed in PKR-deficient cells. U9K-C2 and U9K-A1 cells were incubated with 25 ng/ml of anisomycin and MAPK phosphorylation measured as described in Figure 8Go.

 
PKR Mediates Induction of Apoptosis by DON and Other Translational Inhibitors in U-937 Cells
The capacity of trichothecenes and other translational inhibitors to induce MAPKs has been previously linked to apoptosis (Shifrin and Anderson, 1999Go; Yang et al., 2000aGo). Here, DON was found to induce DNA fragmentation in U9K-C2 cells, whereas the PKR deficient U9K-A1 cells were remarkably recalcitrant to these effects (Fig. 10Go). PKR-deficient cells were also resistant to apoptosis induction by anisomycin and emetine. Consistent with fragmentation data, DON, anisomycin, and emetine readily activated caspase-3 in U9K-C2 cells, whereas PKR-deficient cells did not appreciably activate this enzyme (Fig. 11Go). Thus, in the U937 model, PKR appeared to be required for apoptosis induction by translational inhibitors.



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FIG. 10. PKR deficiency impairs induction of DNA fragmentation by translational inhibitors. DNA fragmentation agarose gel electrophoresis was conducted on DNA isolated from lysates control (U9K-C2) and PKR-deficient (U9K-A1) cells after 6 h of exposure to DON: 0 (lanes 1, 5), 100 (lanes 2, 6), 250 (lanes 3, 7) and 500 (lanes 4, 8) ng/ml; anisomycin 0 (lanes 1, 5), 20 (lanes 2, 6), 50(lanes 3, 7) and 100 (lanes 4, 8) ng/ml; and emetine, 0 (lanes 1, 5), 40 (lanes 2, 6), 100 (lanes 3, 7), and 200 (lanes 4, 8) ng/ml. Gel was stained with ethidium bromide and photographed under UV light. Lane M is a 100-bp DNA ladder used for molecular size.

 


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FIG. 11. PKR deficiency impairs caspase induction by translational inhibitors. U9K-C2 and U9K-A1 cells (5 x 105 ml) were exposed to DON (250 ng/ml), AN (25 ng/ml), or EM (80 ng/ml) for various time intervals. Cell lysates were prepared and caspase-3 activity was measured.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The data presented here provide evidence, for the first time, that activation of PKR by DON contributes to downstream activation of MAPK cascades and, ultimately, to apoptosis. The capacity of DON and other translational inhibitors to activate PKR suggests that this class of chemicals can be added to a growing list of agents which activate this kinase and already includes dsRNA, interferon, LPS, cytokines, growth factors, and stress signals (Williams et al., 1999Go). The mechanisms by which DON activates PKR are unclear but, as suggested by kinetic data, appear to precede MAPK phosphorylation. Trichothecenes are not known to bind to a membrane-specific receptor but, rather, diffuse freely into the cell and bind to eukaryotic ribosomes (Middlebrook and Leatherman, 1989aGo,bGo; Witt and Pestka, 1990Go). PKR is found in the endoplasmic reticulum at high concentrations and associates with ribosomes via specific recognition sites (Wu et al., 1998Go). Thus, one putative model would be for a DON or other translation inhibitor to first bind ribosomes and then, in turn, transduce a signal via structural modification or kinase event to a closely associated PKR molecule (Fig. 12Go).



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FIG. 12. Depiction of PKR as transducer of ribotoxic stress response. Putative downstream effects include induction of proinflammatory and apoptotic genes. ER refers to endoplasmic reticulum.

 
An alternate explanation for PKR activation is that translational inhibition, per se, results in production of RNA polymerase III-(Pol III) directed transcripts of short, highly repetitive retro-transposed sequences (SINEs) that are analogous to human Alu sequences and can potentially drive PKR activation. In support of this possibility, Liu et al. (1995)Go found that cycloheximide induces SINE transcripts in mouse 3R3 cells. Furthermore, cellular exposure to different stresses, including translation inhibition, increases the abundance of human Alu RNAs that can form stable complexes with PKR (Chu et al., 1998Go). Williams et al. (1999)Go reported that, at low concentrations, Alu RNAs are efficient activators of PKR.

Recently, convincing evidence for at least one clear link between PKR and MAPK activation has been established (Takizawa et al., 2002Go). Specifically, a novel interaction was observed between PKR and apoptosis signal-regulating kinase (ASK1), a member of the MAPK kinase family. These conclusions were based on (1) colocalization and comimunoprecipitation of PKR and ASK1; (2) ASK1-mediated p38 phosphorylation in PKR deficient cells; and (3) reduced ASK-1 phosphorylation in PKR-deficient cells treated with poly (I)-poly (c), the classic activator of PKR. Further studies using these approaches could be used to better define the transduction mechanisms between PKR and MAPK during the ribotoxic stress response as well as the potential for cross-talk.

Trichothecene-induced apoptosis has been previously suggested to involve activation of JNK 1/2 and p38 (Yang et al., 2000aGo). Notably, DON-dependent phosphorylation of JNK might almost entirely be mediated through PKR, whereas DON-dependent phosphorylation of ERK and p38 were only partially mediated through PKR. The data presented here suggest that PKR activation may be upstream of this process. PKR-mediated apoptosis is driven by a large number of stimuli including virus infection, dsRNA, LPS, TNF-{alpha}, serum depletion, and cytokine withdrawal (Gil and Esteban, 2000Go). Furthermore, fibroblasts containing homozygous deletions in the PKR gene are resistant to apoptotic cell death in response to ds-RNA, TNF-{alpha} or LPS (Der et al., 1997Go). Although, PKR upregulates several apoptosis-related genes including Fas, Bax, TNF-{alpha}, and p53, and is known to involve the FADD-caspase 8 pathway, the precise mechanisms are not yet fully understood (Balachandran et al., 2000Go; Yeung et al., 1996Go).

PKR-mediated activation of JNK, p38, and ERK may also contribute to DON-induced upregulation of cytokines, chemokines, and cyclooxygenase-2 that have been observed previously in RAW 264.7 and U-937 cells (Ji et al., 1998Go; Moon and Pestka, 2002Go; Sugita-Konishi and Pestka, 2001Go; Wong et al., 1998Go; Zhou et al., 1997Go). A major consequence of MAPK phosphorylation is the activation of transcription factors (Cobb, 1999Go), which serve as immediate or downstream substrates of these kinases. For example, JNK 1/2 phosphorylates c-Jun, which is a component of AP-1 homodimer or heterodimer complexes (Dong et al., 2002Go). Also, p38 (Bhat et al., 2002Go) and ERK 1/2 (Hungness et al., 2002Go) drive activation of C/EBP. Phosphorylation/activation of CREB/ATF members is mediated by JNK (Dong et al., 2002Go), p38 (Bhat et al., 2002Go), and ERK (Belmonte et al., 2001Go). Finally, all three MAPK signaling pathways have been implicated in nuclear factor {kappa}B (NF-{kappa}B) activation through phosphorylation of its inhibitor I{kappa}B{alpha} (Alpert et al., 1999Go; Lee et al., 1998Go; Zhao and Lee, 1999Go). PKR-driven MAPK activation and subsequent activation of the aforementioned transcription factors, as observed in vitro and in vivo following DON treatment (Wong, 2002; Zhou et al., 2003Go), might likely play roles in these processes. Thus, it will be of interest to further define how PKR contributes to DON induction of proinflammatory genes.

Taken together, the data presented herein indicate that PKR is a transducer of the ribotoxic stress response that is initiated by trichothecenes and other translational inhibitors, and that evokes MAPK activation and culminates in apoptosis. Additional research is needed to discern the linkage between ribosomal binding by translational inhibitors and PKR activation as well as to understand how PKR mediates phosphorylation of the major MAPK families. In addition, there is need to examine whether these compounds can induce parallel signaling pathways that converge with PKR-directed pathways and, ultimately, generate diverse immunotoxic effects.


    ACKNOWLEDGMENTS
 
This work was supported by Public Health Service Grant ES-03358 (JJP) from the National Institute for Environmental Health Sciences and by USDA-ARS Grant 59-070-9-060. We thank Mary Rosner for assistance in preparing the manuscript and Theresa Bahns for technical assistance.


    NOTES
 
1 To whom correspondence should be addressed at 234 G.M. Trout Building, Michigan State University, East Lansing, MI 48824. Fax: (517) 353-8963. E-mail: pestka{at}msu.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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