Ribotoxic Stress Response to the Trichothecene Deoxynivalenol in the Macrophage Involves the Src Family Kinase Hck

Hui-Ren Zhou*, Qunshan Jia* and James J. Pestka*,{dagger},{ddagger},1

* Department of Food Science and Human Nutrition, {dagger} Department of Microbiology and Molecular Genetics, and {ddagger} Center for Integrated Toxicology, Michigan State University, East Lansing, Michigan 48824–1224

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.

Received December 9, 2004; accepted March 11, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trichothecene mycotoxins and other translational inhibitors activate mitogen-activated protein kinase (MAPKs) by a mechanism called the "ribotoxic stress response," which drives both cytokine gene expression and apoptosis in macrophages. The purpose of this study was to identify upstream kinases involved in the ribotoxic stress response using the trichothecene deoxynivalenol (DON) and the RAW 264.7 macrophage as models. DON (100 to 1000 ng/ml) dose-dependently induced phosphorylation of c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPKs. MAPK phosphorylation in response to DON exposure occurred as early as 5 min, was maximal from 15 to 30 min, and lasted up to 8 h. Preincubation with inhibitors of protein kinase C, protein kinase A, or phospholipase C had no effect on DON-induced MAPK phosphorylation. In contrast, the Src family tyrosine kinase inhibitors, PP1 (4-amino-5-[4-methylphenyl)]-7-[t-butyl]pyrazolo[3,4-d]-pyrimidine) and, PP2 (4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]-pyrimidine) concentration-dependently impaired phosphorylation of all three MAPK families. PP1 suppressed DON-induced phosphorylation of the MAPK substrates c-jun, ATF-2, and p90Rsk. MAPK phosphorylation by two other translational inhibitors, anisomycin and emetine, were similarly Src-dependent. PP1 reduced DON-induced increases in nuclear levels and binding activities of several transcription factors (NF-{kappa}B, AP-1, and C/EBP), which corresponded to decreases in TNF-{alpha} production, caspase-3 activation, and apoptosis. Tyrosine phosphorylation of hematopoeitic cell kinase (Hck), a Src found in macrophages, was detectable within 1 to 5 min after DON addition, and this was suppressed by PP1. Knockdown of Hck expression with siRNAs confirmed involvement of this Src in DON-induced TNF-{alpha} production and caspase activation. Taken together, activation of Hck and possibly other Src family tyrosine kinases are likely to be critical signals that precede both MAPK activation and induction of resultant downstream sequelae by DON and other ribotoxic stressors.

Key Words: macrophages; apoptosis; protein kinases/phosphatases; cytokines; cell activation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitogen-activated protein kinases (MAPKs) mediate numerous physiological processes including proliferation, differentiation, and apoptosis in leukocytes (Rao, 2001Go). These kinases are composed of three major subfamilies. Extracellular signal-regulated kinases (ERK) are predominantly activated by mitogenic stimuli, whereas p38 MAPK and c-Jun N-terminal protein kinase (JNK) are activated by stress signals (Widmann et al., 1999Go). Activation of all three families occurs in macrophages in response to diverse stimuli that include LPS, lipoprotein, TNF-{alpha}, IFN-{gamma}, UV, and oxidative stress.

Many natural toxins that inhibit translation such as the trichothecenes, anisomycin, shiga toxin, and ricin are also effective activators of MAPKs via an incompletely characterized mechanism known as the "ribotoxic stress response" (Iordanov et al., 1997Go; Laskin et al., 2002Go; Shifrin and Anderson, 1999Go; Yang et al., 2000Go). A host can potentially encounter such ribotoxins as a result of food/environmental contamination, infection, or chemical terrorism (Henghold, 2004Go; Madsen, 2001Go). Activation of MAPKs by ribotoxins drives both inflammatory gene expression and apoptosis in the macrophage, making the innate immune system a critical target for these agents (Cameron et al., 2003Go; Hassoun and Wang, 1999Go, 2000Go; Higuchi et al., 2003Go; Mengeling et al., 2001Go; Pestka et al., 2004Go; Rosser et al., 2004Go).

The ribotoxin deoxynivalenol (DON or vomitoxin), a trichothecene mycotoxin that commonly contaminates cereal-based foods, has been linked to human and animal illnesses worldwide (Pestka and Smolinski, 2005Go). Depending on exposure frequency and timing of administration, DON and other trichothecenes stimulate or suppress leukocyte proliferation, cell-mediated immunity, immunoglobulin production, and host resistance in experimental animals (Pestka, 2003Go). Immune stimulation and suppression are likely to be mediated by upregulation of cytokine expression and apoptosis, respectively, in lymphoid tissue.

Acute oral exposure of mice to DON induces expression of proinflammatory cytokines as well as T-cell cytokines in spleen and Peyer's patches within 2 h (Zhou et al., 1997Go). More recently, DON has been found to induce cyclooxygenase 2 (COX-2) in mice (Moon and Pestka, 2003Go) and many chemokines (Chung et al., 2003aGo; Kinser et al., 2004Go) in vivo. Upregulation of TNF-{alpha}, IL-1ß, IL-6, IL-8, and COX-2 also occurs in macrophage cultures treated with DON (Moon and Pestka, 2003Go; Sugita-Kinoshi and Pestka, 2001Go; Wong et al., 1998Go). DON-induced gene expression has been linked to both transactivation (Li et al., 2000Go; Ouyang et al., 1996Go; Wong et al., 2002Go; Yang and Pestka, 2002Go) and increased mRNA stability (Chung et al., 2003bGo; Li et al., 1997Go; Moon et al., 2003Go; Wong et al., 2001Go). In the mouse, DON activates splenic JNK, ERK, and p38 MAPKs within 15 min, which is followed sequentially by activation of transcription factors and proinflammatory cytokine expression (Zhou et al., 2003aGo). DON and other trichothecenes similarly activate MAPKs in vitro (Shifrin and Anderson, 1999Go; Yang et al., 2000Go; Zhou et al., 2003bGo). Involvement of ERK and p38 has been conclusively demonstrated in expression of TNF-{alpha} (Chung et al., 2003bGo) and COX-2 (Moon and Pestka, 2003Go; Moon et al., 2003Go) in the RAW 264.7 macrophage model.

DON and other trichothecenes also induce apoptosis in spleen, Peyer's patches, thymus, and bone marrow of the mouse (Pestka et al., 2004Go). These apoptotic effects have been corroborated in vitro using leukocyte cultures (Pestka et al., 1994Go; Poapolathep et al., 2004Go; Uzarski and Pestka, 2003Go). Trichothecene-induced apoptosis in vitro is dependent on p38 and JNK activation, but can be downregulated by ERK (Shifrin and Anderson, 1999Go; Uzarski and Pestka, 2003Go; Yang et al., 2000Go).

Although differential upregulation of MAPKs clearly contributes to the characteristic duality of trichothecene immune effects, little is known about the upstream signaling molecules that mediate MAPK activation by DON and other ribotoxins. Recently, we have observed the double-stranded RNA (dsRNA) -activated protein kinase R (PKR) contributes, in part, to DON-induced MAPK activation (Zhou et al., 2003bGo). However, activation of this kinase alone does not appear to be sufficient to explain all downstream effects of DON. The purpose of this research was to identify other potential upstream kinases that contribute to the ribotoxic stress response using DON and the RAW 264.7 macrophage cell line as models. Candidate signaling proteins in the macrophage included PKA, PRC, phospholipase C, and the Src family tyrosine kinases (Khadaroo et al., 2003Go; Konakova et al., 1998Go; Schorey and Cooper, 2003Go). The results suggest that Src family kinases, notably hematopoietic cell kinase (Hck), were critical upstream signal-transducing components to the ribotoxic stress response.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture.
All chemicals were of cell culture grade and purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Inhibitors of protein kinase A, protein kinase C, phospholipase C, and Src tyrosine kinases were purchased from Calbiochem (San Diego, CA). RAW 264.7 murine macrophage cells (American Type Tissue Collection, Rockville, MD) were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma St. Louis, MO) supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml) and 10% (v/v) heat-inactivated fetal bovine serum (Gibco, Rockville, MD) in 6% CO2 at 37°C. Cells (5 x 106) were seeded in 10 ml in 100-mm2 sterile tissue culture dishes overnight to reach 80% confluency, preincubated with or without selected kinase inhibitors, and then incubated with DON or other ribotoxins for various time intervals. The concentrations of DON employed (100–1000 ng/ml) reflect concentrations found in spleen and other tissues in mice several h after given DON orally at 5 and 25 mg/kg (Azcona-Olivera et al., 1995Go). These doses are sufficient to activate MAPKs in spleens of mice within 30 min (Zhou et al., 2003aGo). Cultures were analyzed for MAPK phosphorylation and associated downstream activities. All inhibitors were used at concentrations which failed to alter viability as determined by the MTT assay (Uzarski and Pestka, 2003Go).

Phosphorylation of MAPKs and MAPK substrates.
Phosphorylation of MAPKs was assayed by Western analysis using rabbit IgGs specific for phospho-SAPK/JNK, phospho-p44/p42 ERK, and phospho-p38 (Cell Signaling, Beverly, MA) (Zhou et al., 2003bGo). Briefly, medium from culture dishes of treated adherent cells was decanted, and plates rinsed with 10 mM phosphate buffered saline (pH 7.4, PBS). Cells were scraped from dish into lysis buffer (1% [w/v] SDS, 1 mM sodium ortho-vanadate, 10 mM Tris [pH 7.4]), transferred to a microcentrifuge tube, boiled for 5 min, and sonicated briefly to reduce viscosity. Lysate was centrifuged at 12,000 x g for 15 min at 4°C, and supernatants analyzed with a Bio-Rad DC protein assay kit (Bio-Rad Laboratories Inc, Melville, NY). Lysate protein (40 µg) was resolved by 8% (w/v) SDS–PAGE and transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham, Arlington Heights, IL). After preincubating with 3% (w/v) bovine serum albumin (BSA) for 1 h at 25°C, membranes were incubated overnight with phospho-p38, ERK 1/2, and JNK 1/2 specific antibodies (Cell Signaling [1 µg/ml]) in 3% (w/v) BSA at 4°C, and then with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies (Amersham) (1 µg/ml) diluted in 3% (w/v) BSA for 1 h at 25°C. Bound HRP was determined with an ECL Chemiluminescence Detection kit (Amersham, Piscataway, NJ). To verify total MAPK protein concentrations, membranes were stripped and reprobed with specific antibodies that recognize both phosphorylated and unphosphorylated forms of each MAPK (Cell Signaling).

For detecting of the phosphorylation of c-jun (substrate of JNK), p90Rsk (substrate of p38 and ERK), and ATF-2 (substrate of p38 and JNK), PVDF membranes were incubated with rabbit polyclonal antibodies specific for phospho-c-jun, phospho-p90Rsk, and phospho-ATF-2 (Cell Signaling), followed by incubation of HRP-conjugated secondary antibodies and ECL detection as described above.

Preparation of nuclear extracts.
Cells were collected from cultures by centrifugation at 450 x g for 5 min. washed once with cold PBS. Nuclear extracts were prepared based on the method of Olnes and Kurl (1994)Go, with modifications to prevent protein modification or degradation. Briefly, cells were lysed in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mM dithiothreitol) with phosphatase inhibitors (1 mM sodium orthovanadate and 10 mM sodium fluoride), protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 mg/ml pepstatin A, 2 mg/ml leupeptin, and 2 mg/ml aprotinin), and 0.6% (w/v) Nonidet P-40. Nuclei were pelleted by centrifugation and suspended in hypertonic buffer containing 20 mM HEPES, pH 7.9, 0.4 M KCl, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 10% (w/v) glycerol, and the phosphatase and protease inhibitors indicated above. After 30 min at 4°C, soluble proteins released were collected by centrifugation at 12,000 x g for 10 min. Resultant extracts were analyzed for protein, aliquotted, and stored at –80°C until analysis.

Transcription factor detection and EMSA.
To detect nuclear transcription factor levels, electrophoresis of nuclear proteins was performed with 8% (w/v) polyacrylamide gel in the presence of 0.1% (w/v) SDS in the discontinuous buffer system (Laemmli, 1970Go). Proteins were transferred to a PVDF membrane (Amersham, Piscataway, NJ). Membranes were incubated with polyclonal antibodies (Santa Cruz Biotech, Santa Cruz, CA) for the transcription factor components AP-1 (phospho-c-Jun, Jun B, Jun D, c-Fos, Fos B, Fra-1, Fra-2), C/EBP (c/EBP{alpha}, C/EBPß, c/EBP{delta}), and NF{kappa}B (NF{kappa}B p65, NF{kappa}B p50, c-Rel, RelB), washed, and then incubated with HRP-conjugated donkey anti-rabbit IgG antibody (Amersham). HRP activity in bands was determined as described above.

Electrophoretic mobility shift assay [EMSA] was used to characterize the binding activity of AP-1, C/EBP, and NF{kappa}B transcription factors in nuclear extracts (Zhou et al., 2003aGo). Double-stranded AP-1, C/EBP, and NF-{kappa}B consensus probes (Santa Cruz) were radiolabeled with [{gamma}-32P-ATP] using Ready to GoTM Polynucleotide Kinase Kit (Pharmacia Biotech Inc. Piscataway, NJ). Nuclear extracts containing 10 µg protein were added to DNA-binding reaction buffer consisting of 20 mM HEPES pH 7.9, 60 mM KCl, 1 mM EDTA, 0.5 mM DTT, 1 µg poly dIdC in a total volume of 20 µl. These were preincubated on ice for 15 min to block the nonspecific binding. Following the addition of 1 µl 32P-labeled probe (30,000 cpm), the incubation was continued for 30 min at room temperature to promote the formation of nucleoprotein complexes. Resultant nucleoprotein complexes were separated on 4% (w/v) native polyacrylamide gels, dried, and visualized by autoradiography.

TNF-{alpha}.
Cell supernatants were analyzed for TNF-{alpha} by enzyme-linked immunosorbent assay (ELISA) using a mouse TNF-{alpha} kit (BD-PharMingen, San Diego, CA).

Caspase-3 measurement.
Cells were lysed in 300 µl of CHAPS buffer (100 mM HEPES [pH 7.5], 10% [w/v] sucrose, 0.5% [w/v] CHAPS, 1 mM EDTA, 10 mM DTT) containing 1:100 Protease Inhibitor Cocktail (Sigma), held on ice for 30 min, sonicated briefly, and then centrifuged 10,000 x g for 10 min. Following protein determination, 50 µg of lysate protein was incubated in 200 µl CHAPs buffer containing 12.5 µM DEVD-AMC (Calbiochem) substrate 30 min at 37°C. Substrate cleavage was detected using a CytoFluor II Microplate Fluorescence Reader (Biosearch, Bedford, MA) at 360 nm excitation and 460 nm emission.

DNA fragmentation analysis.
DNA fragmentation was determined by the method of Sellins and Cohen (1987)Go. Briefly, cells (1 x 107) were suspended in phosphate buffered saline (pH 7.4), centrifuged for 5 min (500 x g) at 4°C, and pellet suspended in 0.1 ml hypotonic lysing buffer (10 mM Tris, 10 mM EDTA, 0.5% [v/v] Triton X-100, pH 8.00) at 4°C for 10 min. Resultant lysate was centrifuged for 30 min (13,000 x g) at 4°C. The 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 13,000 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 60 V for 2 h in 2% (w/v) agarose gel (2 x 106 cells 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 a UV transilluminator. A 100-bp DNA ladder (GIBCO-BRL, Rockville, MD) was used as a molecular size marker.

Detection of Hck phosphorylation and activation.
Hck phosphorylation was detected by immunoprecipitation in conjunction with Western analysis. Adherent cells were scraped into RIPA lysis buffer (50 mM Tris [pH 7.4], 1% NP-40, 0.5% [w/v] sodium deoxycholate, 0.1% [w/v] SDS, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA) containing protease inhibitor cocktail (Roche Molecular Biochemicals) and phosphatase inhibitors (2 mM sodium vanadate and 2 mM sodium fluoride). Lysates were placed on ice for 30 min, sonicated briefly, precleared by adding 20 µl of nonimmune rabbit serum prebound to goat anti-rabbit IgG-agarose (50% [w/v]) (Sigma), and then incubated at 4°C for 30 min. Beads were centrifuged at 1000 x g for 5 min at 4°C, and protein concentration determined. Cell lysate (100 µg) was diluted into 300 µl in PBS, and 3 µg of anti-Hck rabbit polyclonal antibodies (Upstate Biotechnology, Lake Placid, NY) was added. Following overnight incubation at 4°C, lysates were further incubated with 30 µl of goat anti-rabbit IgG-agarose beads (50% [w/v]) for 2 h at 4°C. Beads were washed three times with RIPA for 10 min, once with PBS, eluted in 30 µl of boiling SDS sample buffer for 5 min, and then centrifuged at 3000 x g for 5 min. Supernatants were subjected to Western analysis as described for MAPKs using anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology).

For measuring Hck autophosphorylation activity, immunoprecipitation was carried out, beads washed three times with RIPA lysis buffer, and then once with kinase buffer containing 25 mM HEPES [pH 7.5], 0.1% Triton-X100, 100 mM NaCl, 10 mM MgCl2, 3 mM MnCl2, and 200 mM sodium vanadate. Tyrosine kinase reaction was carried out by incubation with 15 mM of ATP and incubated for 15 min at 37°C. The reaction was terminated by washing beads with RIPA lysis buffer three times and eluting bound Hck by addition of boiling SDS-sample buffer. Phosphorylated Hck was detected by Western analysis as described above.

Hck siRNA knockdown.
Hck siRNAs were prepared using a Silencer siRNA Cocktail Kit (Rnase III) (Ambion). Total RNA was extracted from RAW 264.7 cells and reverse transcribed to produce cDNA using RETRO script Kit (Ambion). PCR primers containing T7 RNA polymerase promoters were designed using Ambion software to amplify a 303-bp fragment of murine macrophage Hck gene (forward, 5'-TAATACGACTCACTATAGGGCAGAAGGGCCCTGTGTATGTG-3'; reverse, 5'-TAATACGA CTCACTATAGGGGCCACATAGTTGCTTGGGATG-3'). Resultant templates were used in an in vitro transcription reaction to generate dsRNA. Following column purification, dsRNA was digested with Rnase III and purified to remove any undigested dsRNA. siRNA was quantitated and transfections carried out using Silencer siRNA Transfection Kit (Ambion). SiRNA targeting Hck or Silencer Negative control SiRNA (Ambion) were transfected using siPORT lipid (Ambion) and siRNA (50 nM) in a 24-well tissue culture plate. The negative control sequence bears no significant homology to any known gene sequences in mouse, rat, or human. Hck knockdown efficacy was verified by real-time SYBR Green PCR using Hck-specific primers (forward 5'-CAAGCTGGGACCAAACAACA; reverse, 5'-GTGCGACCACAATGGTATCCT) using the CT method (Kinser et al., 2004Go).

Statistics.
Data were analyzed using Sigma Stat for Windows (Jandel Scientific, San Rafael, CA). Data were subjected to one-way ANOVA and pairwise comparisons made by Bonferroni or Student-Newman-Keuls methods. Differences were considered significant at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DON Induces p38, JNK, and ERK Phosphorylation
The capacity of the ribotoxic stressor DON to induce MAPK phosphorylation in the RAW 264.7 model was verified in RAW 264.7 cells. DON at concentrations as low as 100 ng/ml induced the phosphorylation of JNK 1/2, ERK 1/2 and p38 MAPK in a concentration-dependent manner after 15 min (Fig. 1A). Phosphorylation of all three MAPKs occurred within 5 min and was detectable up to 240 min for JNK and 360 min for ERK and p38 (Fig. 1B). A DON concentration of 250 ng/ml and incubation time of 30 min were selected as optimal for subsequent inhibitor studies.



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FIG. 1. DON induces JNK, ERK, and p38 phosphorylation. Cells (5 x 105/ml) were incubated with various DON concentrations for 15 min in dose response study (A) or with 250 ng/ml DON over time intervals (B). Lysate proteins (40 µg) were resolved by SDS–PAGE followed by Western analysis with antibodies to phosphorylated JNK 1/2, ERK 1/2 and p38. Membranes were stripped afterwards and probed with specific antibodies that recognized both phosphorylated and unphosphorylated forms of each MAPK. Results are representative of four independent experiments.

 
MAPK Activation by DON and Other Ribotoxins Involves Src Family Tyrosine Kinases
Many upstream signaling cascades have been reported for MAPKs (Widmann et al., 1999Go). We therefore screened for potential involvement of specific upstream kinases in DON-induced MAPK activation using pharmacological inhibitors. Inhibitors of protein kinase C, protein kinase A, and phospholipase C (Fig. 2) did not affect DON-induced MAPK phosphorylation in RAW 264.7 cells. In contrast, the Src family-selective tyrosine kinase inhibitors PP1 (Fig. 3A) and PP2 (Fig. 3B) abrogated DON-induced phosphorylation of MAPKs in a concentration-dependent manner. Consistent with these findings, PP1 inhibited DON-induced phosphorylation of the MAPK substrates (c-jun, ATF-2, and p90RSK) (Fig. 3C). PP1 also interfered with activation of JNK 1/2, ERK 1/2, and p38 by the ribotoxins anisomycin and emetine (Fig. 4A) but not protein levels of these MAPKs (Fig. 4B). These data suggest that induction of the ribotoxic stress response in macrophages by DON and other translational inhibitors might involve Src family tyrosine kinases. Accordingly, all further experiments focused on this family.



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FIG. 2. Protein kinase C, protein kinase A, and phospholipase C inhibitors do not affect the DON-induced MAPK phosphorylation. Cells (5 x 105/ml) were pretreated with inhibitors of PKC (staurosporine [Sta], 100 nM, or bisindolylmaleimide [Bis], 10 µM), PKA (H7 dihydrocholoride [H7], 50 µM, or HA-1004 [Ha], 50 µM), or PLC (U73122 [U], 10 µM, or D609 [D],10 µM) for 1 h, and then stimulated with 250 ng/ml DON for 30 min. Cell lysates (40 µg) were subjected to Western analysis. Results are representative of two independent experiments.

 


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FIG. 3. Src family-selective tyrosine kinase inhibitors PP1 and PP2 block DON-induced MAPK phosphorylation and activity. Cells (5 x 105/ml) were incubated with various PP1 (A) or PP2 (B) at various concentrations for 1 h and then with DON (250 ng/ml) for 15 min. Cell lysate protein (40 µg) was subjected to Western analysis to detect phosphorylation of (A,B) MAPKs or (C) MAPK substrates. Results are representative of three independent experiments.

 


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FIG. 4. Anisomycin- and emetine-induced phosphorylation of JNK, ERK, and p38 is Src-dependent. Cells (5 x 105/ml) were pretreated with or without PP1 (25 µM) for 1 h and then with anisomycin (AN, 15 ng/ml) or emetine (EM, 50 ng/ml) for 15 min, respectively. These concentrations inhibit translation by 50%, which equates to effects of 250 ng/ml DON. Cell lysate protein (40 µg) was subjected to Western analysis using specific antibodies for both phosphorylated (A) and unphosphorylated (B) forms of each MAPK. Results are representative of two independent experiments.

 
DON-Induced Transcription Factor Activation and TNF-{alpha} Production Are Src-Dependent
We have previously shown that short-term DON exposure induces MAPK-dependent transcriptional activation of inflammation-associated genes (Chung et al., 2003bGo; Moon and Pestka 2003Go). The effects of Src inhibition on transcription factors associated with inflammation in DON-treated cells were therefore assessed. PP1 inhibited DON-induced increases in nuclear levels of phospho-c-jun, Jun B, JunD, C/EBPß, c-Rel, and p50 (Fig. 5A). These effects corresponded with PP1 inhibition of DON-mediated increases in binding activity of the transcription factors AP-1, C/EBP, and NF-{kappa}B, which affect inflammatory gene transcription (Fig. 5B). TNF-{alpha} is one example of an inflammatory gene induced by DON (Chung et al., 2003bGo; Wong et al., 1998Go, 2001Go; Zhou et al., 1997Go). In RAW 264.7 cells, DON-induced TNF-{alpha} mRNA transcription and stability are regulated by ERK and p38, and this was reflected in secreted proteins (Chung et al., 2003bGo). Induction of this cytokine by DON was inhibited by PP1 (Fig. 5C). These results suggest that a Src might be involved in DON-induced inflammatory gene expression and is consistent with a sac being upstream of the MAPKs.



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FIG. 5. DON-induced nuclear transcription factor levels and binding activities as well as TNF-{alpha} production are Src-dependent. Cells (5 x 105/ml) were incubated with or without PP1 followed by DON (250 ng/ml). For nuclear transcription factor levels (A), cells were exposed to 25 µM PP1 for 1 h and then DON for 2 h, and nuclear extracts were analyzed by Western analysis using specific antibodies. For EMSA (B), cells were exposed to 25 µM PP1 for 1 h and then DON for 1 h, and binding activities of nuclear extracts (10 µg) were analyzed by using 32P-labeled double-stranded AP-1, C/EBP, or NF-{kappa}B consensus probes. Specificity of these bands has been verified previously (Wong et al., 2002Go). For TNF-{alpha} production (C), cells were incubated with PP1 for 1 h and then DON for 24 h, and supernatant analyzed by ELISA. Data are mean ± SEM (n = 4). Bars marked without the same letter differ significantly (p < 0.05). Results are representative of three independent experiments.

 
DON-Induced Caspase-3 Activation and DNA Fragmentation Are Src-Dependent
Prolonged or high concentration exposure to DON and other trichothecenes effectively induce apoptosis in leukocytes, and this is regulated by MAPK activation (Shifrin and Anderson, 1999Go; Uzarski et al., 2003Go; Yang et al., 2000Go). PP1 impaired both DON-induced caspase-3 activity (p < 0.05) (Fig. 6A) and DNA fragmentation (Fig. 6B). These findings suggest that a Src tyrosine kinase is critical to induction caspase-3 and apoptosis by DON and is further congruent with this kinase family being upstream of the MAPKs.



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FIG. 6. DON-induced caspase activation and DNA fragmentation are Src-dependent. Cells (5 x 105/ml) were incubated with or without PP1 (25 µM) followed by DON (250 ng/ml). For caspase activity (A), cells were incubated with DON for 3 or 6 h and analyzed using caspase 3 substrate DFVD-AMS, which emits fluorescence upon cleavage. Data are mean ± SEM (n = 4) and are expressed as fluorescence intensity relative to DON-exposed cells. Bars marked without the same letter differ significantly (p < 0.05). Internucleosomal DNA fragmentation (B) was assessed by agarose gel electrophoresis after incubation with DON for 3 and 6 h. Results are representative of three independent experiments.

 
DON Induces Phosphorylation and Activation of the of Hck, a Src Family Member
Hck is a Src family member that has been previously established to be critical for macrophage function (Ernst et al., 2002Go; Lowell, 2004Go; Lowell and Berton, 1998Go), thus making it a potential target for DON in RAW 264.7 cells. The toxin was found to be an effective inducer of Hck phosphorylation, which was inhibitable by PP1 (Fig. 7A). DON-induced Hck autophosphorylation was also observed and confirmed to be impaired by PP1 (Fig. 7B).



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FIG. 7. DON induction of Hck phosphorylation and activation. Cells (5 x 105/ml) were pretreated with or without PP1 (25 µM) for 1 h and then with DON (250 ng/ml) for 0 to 5 min. Hck phosphorylation (A) was detected by immunoprecipitation and by Western analysis. Hck activation (B) was detected by Hck immunoprecipitation in conjunction with tyrosine kinase assay using Western analysis. To potentiate effects on Hck protein levels, the membranes were stripped, then reprobed with anti-Hck polyclonal antibodies. Results are representative of two independent experiments.

 
Inhibition of Hck Expression Suppresses TNF-{alpha} Expression and Apoptosis
SiRNA inhibition was used to verify Hck involvement in DON-induced TNF-{alpha} expression and apoptosis in RAW 264.7cells. Treatment of cells with Hck SiRNA reduced Hck mRNA expression by 40% (p < 0.05) compared to cells treated with negative control (Fig. 8A). Both DON-induced TNF-{alpha} production (Fig. 8B) and caspase-3 activation (Fig. 8C) were significantly inhibited compared to negative control (p < 0.05) by Hck SiRNA knockdown. These results suggest that this Src family tyrosine kinase plays a key role in DON-induced gene expression and apoptosis in the macrophage.



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FIG. 8. Hck SiRNA inhibits DON-induced TNF-{alpha} production and caspase-3 activation. Cells transfected with Hck siRNA were verified to express significantly less Hck mRNA by real time PCR than cells treated with lipid translocation agent vehicle (Vh) (A). TNF-{alpha} ELISA (B) and fluorogenic caspase-3 assay (C) were used to measure effects of Hck SiRNAs on DON-treated cells. Results are mean ± SEM (n = 3). Bars without same letter differ significantly (p < 0.05). Results are representative of two independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Src proteins are a distinct family of protein tyrosine kinases that contain similar structural features and significant amino acid sequence homology (Lowell, 2004Go). Src family members include Hck, Fyn, Yes, Yrk, Blk, Fgr, Lck, Lyn, and Frk (Boggon and Eck, 2004Go). Src tyrosine kinases mediate innate immune signaling, responses to cytokines, apoptosis, antigen signaling, and responses to adhesive stimulation (Korade-Mirnics and Corey, 2000Go; Thomas and Brugge et al., 1997Go). The results presented here show, for the first time, that a Src family tyrosine kinase is likely to play a critical role in mediating the ribotoxic stress response. Srcs are expressed ubiquitously with different family members being found in specific cell types and subcellular locations. Hck is known to function in activated macrophages and appeared here to be an important Src target in macrophages exposed to the ribotoxin DON.

The observation that DON activated all three MAPK families confirms previous findings for this and other trichothecenes (Iordanov et al., 1997Go; Shifrin and Anderson, 1999Go; Yang et al., 2000Go; Zhou, 2003aGo,bGo). PP1 and PP2 are highly selective inhibitors of Src family tyrosine kinases (Bain et al., 2003Go; Hanke et al., 1996Go; Liu et al., 1999Go; Susa et al., 2000Go), and both concentration-dependently suppressed DON-induced phosphophorylation of MAPKs. The efficacy of PP1 in inhibiting Hck autophosphorylation verified that this compound inhibited Src activity in the RAW 264.7 macrophage cells. Several lines of evidence suggest linkages indeed exist between Srcs and MAPKS. In murine RAW 264.7 and human THP-1 monocyte-macrophage cell lines, expression of the HIV-1 protein Nef induces both ERK-mediated AP-1 binding and activation through Hck (Biggs et al., 1999Go). Khadaroo et al. (2003)Go observed that oxidant priming of RAW 264.7 macrophages involves activation of p38 through a Src-dependent pathway. Treatment of human fibrosarcoma cell line HT1080 with urokinase-type plasminogen activator induces Hck activation, leading to the subsequent activation of AP-1 via p38 and ERK signaling modules (Konakova et al., 1998Go). Using PP1 as well as mutant macrophages deficient in expression of Src- family kinases (Hck-/Fgr-/Lyn-), Mocsai et al. (2000)Go found that PP1 attenuates release of primary and secondary granules of neutrophils as well as the activation of p38 MAPK induced by chemoattractant fMLP. Mice deficient of Hck, Fgr, and Lyn are also defective in secondary granule release and p38 MAPK activation. Mechanical pressure-induced phosphorylation of p38 in epithelial cells involves a Src (Hofmann et al., 2004Go). Thus, the possibility proposed here that Hck and Srcs mediate ribotoxin-induced MAPK activation is reasonable and consistent with existing models for other Src stimuli.

DON has been previously shown to upregulate expression of inflammatory genes, both at the transcriptional and post transcriptional level. The toxin induces activation of NF-{kappa}B, AP-1, and C/EBP (Li et al., 2000Go; Ouyang et al., 1996Go; Wong et al., 2002Go; Zhou et al., 2003aGo). Both p38 and ERK have been shown to mediate DON-induced transactivation TNF-{alpha} (Chung et al., 2003bGo) and cycloxygenase-2 (Moon and Pestka, 2003Go; Moon et al., 2003Go). Congruent with the postulated role for Hck in TNF-{alpha} production, LPS and IFN-{gamma} are also known to activate Hck and Lyn, as well as Src-dependent induction of IL-6 and TNF-{alpha} production in human monocytes (Beaty et al., 1994Go; Uptain et al., 1997Go; Yeung et al., 1996Go). Ceramide-mediated stimulation of inducible nitric oxide synthase and TNF accumulation in murine macrophages requires Src activity (Knapp and English, 2000Go). Ernst et al. (2002)Go demonstrated that constitutive expression of Hck in "knock-in" mice renders them highly sensitive to LPS, with aberrantly elevated TNF-{alpha} expression being a key observation. Another key finding here was that DON-induced apoptosis was Src dependent. In general, Src activation is associated with downregulation of apoptosis (Playford and Schaller, 2004Go). However, Hck has recently been shown to mediate caspase-driven apoptosis in human myelomonocytic THP-1 cells (Shivakrupa et al., 2003Go) which are consistent with our results.

Trichothecenes and other translational inhibitors bind to the eukaryotic 60S ribosomal subunit, block peptidyl transferase, and inhibit translation, but concurrently can induce MAPK activation by via the "ribotoxic stress response" (Iordanov et al., 1997Go; Laskin et al., 2002Go; Shifrin and Anderson, 1999Go; Yang et al., 2000Go). However, it is not known how the ribosome transduces a signal following binding with these agents. Since both DON and two other translational inhibitors activated MAPKs in a Src-dependent fashion here, it is possible that this kinase family is important in mediating the ribotoxic stress response and, furthermore, that a linkage between the ribosome and Srcs might exist. Nck-1, an adaptor protein with Src deteminants, reportedly associates with ribosomes and affects their function (Kebache et al., 2002Go), suggesting the feasibility of Src interaction with ribosomal proteins. The SH3 and SH2 domains play a central role in regulating Src tyrosine kinase catalytic activity (Boggon and Eck, 2004Go). In inactivated Srcs, the SH3 domain represses kinase activity by interacting with amino acids within the catalytic domain as well as with linker region sequence lying between the SH2 and catalytic domains. Binding of the SH3 domain to a specific PXXP motif-containing cellular protein is one way to activate Src family kinases (Boggon and Eck, 2004Go). Since many ribosome proteins contain the PXXP motif (http://ribosome.miyazaki-med.ac.jp/; last accessed 3/17/2005), it is possible that DON alters ribosome confirmation sufficiently to reorient PXXP motifs on ribosomal proteins, thus resulting in immediate activation of adjacent Hcks. Further study is needed on how ribosomes interact with Src family kinases and MAPK signaling modules and to verify that ribosomal binding is indeed a prerequisite for trichothecene-induced stress effects.

Other intracellular signaling pathways are likely to function in parallel with Src. We have previously reported that PKR contributes partially to DON-induced MAPK activation (Zhou et al., 2003bGo). The ribosome can specifically bind to PKR, rendering the kinase inactive, whereas dissociation results in PKR activation (Erickson et al., 2001Go; Fernandez et al., 2002Go; Raine et al., 1998Go; Vattem et al., 2001Go; Wu et al., 1998Go; Zhu et al., 1997Go). Thus it is possible that, under normal conditions, a proliferating cell contains ribosome-PKR complexes. Binding of a trichothecene to a ribosome could cause a conformational change, releasing an activated PKR. Alternatively, trichothecene binding could damage rRNA, making it accessible for binding/activating PKR and evoking its release from the ribosome. Interaction between Src family proteins and PKR have not been reported to date. It will therefore be necessary to determine if Hck functions in parallel with PKR in signal transduction or whether these kinases are linked in some manner.

Taken together, the data presented herein support the contention that, in the macrophage, the ribotoxic stress response to DON and other translational inhibitors is, in part, Src-dependent. Subsequent downstream sequelae, including TNF-{alpha} expression and apoptosis, are similarly Src-dependent. Hck is a one likely candidate for mediating the ribotoxic pathway in the macrophage, but it is likely other Src family kinases contribute. Both the magnitude and duration of activation of individual MAPK families might preferentially direct a DON-exposed macrophage to either express inflammation-related genes or undergo apoptosis. In the future, it will be necessary to ascertain whether specific linkages exist between Hck and the ribosome and to understand how interaction with different ribotoxins might affect activation status of Src family tyrosine kinases. Additional insight is needed into how the activated Hck and other Srcs coordinate activity of MAPK signaling modules and how these subsequently modulate downstream gene expression and apoptosis.


    ACKNOWLEDGMENTS
 
This work was supported by Public Health Service Grants ES003358 (JJP) from the National Institute for Environmental Health Sciences and DK58833 (JJP) from the National Institute for Diabetes, Digestive and Kidney Diseases. We thank Theresa Bahns for technical assistance and Mary Rosner for manuscript preparation.


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 MATERIALS AND METHODS
 RESULTS
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