* Department of Food Science and Human Nutrition, Department of Microbiology and Molecular Genetics, and
Center for Integrated Toxicology, Michigan State University, East Lansing, Michigan 488241224
1 To whom correspondence should be addressed at 234 G.M. Trout Building, Michigan State University, East Lansing, MI 48824. Fax: (517) 3538963. E-mail: pestka{at}msu.edu.
Received December 9, 2004; accepted March 11, 2005
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ABSTRACT |
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Key Words: macrophages; apoptosis; protein kinases/phosphatases; cytokines; cell activation.
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INTRODUCTION |
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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., 1997; Laskin et al., 2002
; Shifrin and Anderson, 1999
; Yang et al., 2000
). A host can potentially encounter such ribotoxins as a result of food/environmental contamination, infection, or chemical terrorism (Henghold, 2004
; Madsen, 2001
). 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., 2003
; Hassoun and Wang, 1999
, 2000
; Higuchi et al., 2003
; Mengeling et al., 2001
; Pestka et al., 2004
; Rosser et al., 2004
).
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, 2005). 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, 2003
). 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., 1997). More recently, DON has been found to induce cyclooxygenase 2 (COX-2) in mice (Moon and Pestka, 2003
) and many chemokines (Chung et al., 2003a
; Kinser et al., 2004
) in vivo. Upregulation of TNF-
, IL-1ß, IL-6, IL-8, and COX-2 also occurs in macrophage cultures treated with DON (Moon and Pestka, 2003
; Sugita-Kinoshi and Pestka, 2001
; Wong et al., 1998
). DON-induced gene expression has been linked to both transactivation (Li et al., 2000
; Ouyang et al., 1996
; Wong et al., 2002
; Yang and Pestka, 2002
) and increased mRNA stability (Chung et al., 2003b
; Li et al., 1997
; Moon et al., 2003
; Wong et al., 2001
). 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., 2003a
). DON and other trichothecenes similarly activate MAPKs in vitro (Shifrin and Anderson, 1999
; Yang et al., 2000
; Zhou et al., 2003b
). Involvement of ERK and p38 has been conclusively demonstrated in expression of TNF-
(Chung et al., 2003b
) and COX-2 (Moon and Pestka, 2003
; Moon et al., 2003
) 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., 2004). These apoptotic effects have been corroborated in vitro using leukocyte cultures (Pestka et al., 1994
; Poapolathep et al., 2004
; Uzarski and Pestka, 2003
). Trichothecene-induced apoptosis in vitro is dependent on p38 and JNK activation, but can be downregulated by ERK (Shifrin and Anderson, 1999
; Uzarski and Pestka, 2003
; Yang et al., 2000
).
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., 2003b). 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., 2003
; Konakova et al., 1998
; Schorey and Cooper, 2003
). The results suggest that Src family kinases, notably hematopoietic cell kinase (Hck), were critical upstream signal-transducing components to the ribotoxic stress response.
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MATERIALS AND METHODS |
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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., 2003b). 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) SDSPAGE 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), 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, 1970). 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
, C/EBPß, c/EBP
), and NF
B (NF
B p65, NF
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 NFB transcription factors in nuclear extracts (Zhou et al., 2003a
). Double-stranded AP-1, C/EBP, and NF-
B consensus probes (Santa Cruz) were radiolabeled with [
-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-.
Cell supernatants were analyzed for TNF- by enzyme-linked immunosorbent assay (ELISA) using a mouse TNF-
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). 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., 2004).
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.
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RESULTS |
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DISCUSSION |
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The observation that DON activated all three MAPK families confirms previous findings for this and other trichothecenes (Iordanov et al., 1997; Shifrin and Anderson, 1999
; Yang et al., 2000
; Zhou, 2003a
,b
). PP1 and PP2 are highly selective inhibitors of Src family tyrosine kinases (Bain et al., 2003
; Hanke et al., 1996
; Liu et al., 1999
;
u
a et al., 2000
), 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., 1999
). Khadaroo et al. (2003)
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., 1998
). Using PP1 as well as mutant macrophages deficient in expression of Src- family kinases (Hck-/Fgr-/Lyn-), Mocsai et al. (2000)
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., 2004
). 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-B, AP-1, and C/EBP (Li et al., 2000
; Ouyang et al., 1996
; Wong et al., 2002
; Zhou et al., 2003a
). Both p38 and ERK have been shown to mediate DON-induced transactivation TNF-
(Chung et al., 2003b
) and cycloxygenase-2 (Moon and Pestka, 2003
; Moon et al., 2003
). Congruent with the postulated role for Hck in TNF-
production, LPS and IFN-
are also known to activate Hck and Lyn, as well as Src-dependent induction of IL-6 and TNF-
production in human monocytes (Beaty et al., 1994
; Uptain et al., 1997
; Yeung et al., 1996
). Ceramide-mediated stimulation of inducible nitric oxide synthase and TNF accumulation in murine macrophages requires Src activity (Knapp and English, 2000
). Ernst et al. (2002)
demonstrated that constitutive expression of Hck in "knock-in" mice renders them highly sensitive to LPS, with aberrantly elevated TNF-
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, 2004
). However, Hck has recently been shown to mediate caspase-driven apoptosis in human myelomonocytic THP-1 cells (Shivakrupa et al., 2003
) 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., 1997; Laskin et al., 2002
; Shifrin and Anderson, 1999
; Yang et al., 2000
). 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., 2002
), 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, 2004
). 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, 2004
). 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., 2003b). The ribosome can specifically bind to PKR, rendering the kinase inactive, whereas dissociation results in PKR activation (Erickson et al., 2001
; Fernandez et al., 2002
; Raine et al., 1998
; Vattem et al., 2001
; Wu et al., 1998
; Zhu et al., 1997
). 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- 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.
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ACKNOWLEDGMENTS |
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