v a: 4.. flit“: "f... .vmwh._m~fium.w%umh E. .hihuar? kflr v. ‘f. a A? a? a. .h. n a. ”la I: thus :2. - .. .i 55.]... ‘ 22.2.. 5. 1:11: run anilzx... 3:13.! 1.. . 152.11.21- iii-55rd. ”cl: .m- a a: .flfli‘la if: EL... . .n . 53.5... 1;. . $1.3! .chalsifiani..4 Stifiz.‘ t.Jfl»1..fa.awx .2. n!!! (in! 3.... 132.... .1... ,.,...:e.:u¥..tu LlUl’lnn l Michigan State University This is to certify that the dissertation entitled SIGNAL TRANSDUCTION MECHANISMS FOR DEOXYNIVALENOL-INDUCED p38 ACTIVATION presented by Heekyong Bae has been accepted towards fulfillment of the requirements for the PhD. degree in Food Science Major Professor’s Signature WM 979/ 2007 Date MSU is an Affirmative Action/Equal Opportunity Employer .-n-0-0-0-.-u-n---n-o--o-o-o-o----a-.--o-o-o----u-----———--.-------o--—-------—u-----—--u---._.-.-.- PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 KtlproleccaPrelelRClDateDue.indd __.--—._ . t_‘__._ __..._ “—u ng ..t__ SIGNAL TRANSDUCTION MECHANISMS FOR DEOXYNIVALENOL—INDUCED p38 ACTIVATION By Heekyong Bae A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Food Science 2009 ABSTRACT SIGNAL TRANSDUCTION MECHANISMS FOR DEOXYNIVALENOL- INDUCED p38 ACTIVATION BY Heekyong Bae Trichothecenes, mycotoxins prevalent in cereal foods, cause several toxic responses, and adversely affect human and animal health. Trichothecenes are ribosomal binding agents that inhibit translation and can induce activation of mitogen- activated protein kinases (MAPK) involved in cell survival or death. Mechanisms responsible for this “ribotoxic stress response”, however, are not well understood. The objective of this thesis was to characterize the ribotoxic stress response in macrophages stimulated by deoxynivalenol (DON). The central hypothesis was that DON induces p38 MAPK activation through ribosomal-mediated activation of double stranded RNA-activated protein kinase (PKR) and hematopoietic cell kinase (Hck). This hypothesis was tested using isolated ribosomes and murine macrophages and human monocyte cell line models. DON induced p38 binding to the ribosome within 5 min, and phosphorylated p38 was detectable in association with the ribosome shortly thereafter. Further fractionation of ribosomal subunits showed that p38 interacted with the 408 subunit initially and subsequently with the 608 subunit and monosome. Using an inhibitor of p38 phosphorylation, it was determined that phosphorylation was not required for p38 binding to the ribosome afier DON stimulus, suggesting that p38 might be activated via interaction with the ribosome. Two putative protein kinases, PKR and Hck, similarly regulate p38 phosphorylation and its downstream IL-8 mRNA and protein expression. PKR and Hck are localized in the 408 ribosomal subunit and DON induces PKR and Hck phosphorylation at this site. It was further determined that Hck directly interacts with ribosomal protein S3, which regulates translation initiation via a PKR substrate, alpha subunit of eukaryotic initiation factor 2a (eIFZa). Competitive binding studies with satratoxin G, another trichothecene, showed that DON rapidly bound to the ribosome after uptake into cells and targeted both free 408 and 608 ribosomal subunits. Taken together, these data suggest that free 408 and 608 ribosomal subunits might function as scaffolds for DON-induced p38 activation and downstream inflammatory gene expression, and that PKR and Hck likely play critical roles in this response. TABLE OF CONTENTS Page LIST OF TABLES .................................................................................. vi LIST OF FIGURES ................................................................................. vii ABBREVIATION ................................................................................... x INTRODUCTION ................................................................................. 1 CHAPTER 1. LITERATURE REVIEW ......................................................... 5 A. Trichothecenes ................................................................. 6 B. Deoxynivalenol (DON) ...................................................... 9 C. Mitogen-activated protein kinases (MAPK) .............................. 12 D. Ribotoxic stress response ................................................... 14 E. Double-stranded RNA activated protein kinase (PKR) ................. 20 F. Hematopoietic cell kinase (Hck) ............................................ 24 CHAPTER 2. ROLE OF THE RIBOSOME IN DON-INDUCED p38 ACTIVATION. . .28 ABSTRACT ................................................................................. 29 INTRODUCTION ........................................................................... 30 MATERIALS AND METHODS .......................................................... 32 RESULTS. ................................................................................... 36 DISCUSSION ............................................................................... 45 CHAPTER 3. ROLE OF PKR AND HCK IN DON-INDUCED p38 ACTIVATION. . ...51 ABSTRACT ................................................................................. 52 INTRODUCTION ........................................................................... 53 MATERIALS AND METHODS .......................................................... 56 RESULTS .................................................................................... 61 DISCUSSION ................................................................................ 78 iv CHAPTER 4. TRICHOTHECENES TARGET 408 AND 608 RIBOSOMAL SUBUNTTS .......................................................................................... 84 ABSTRACT ................................................................................. 85 INTRODUCTION ........................................................................... 85 MATERIALS AND METHODS .......................................................... 88 RESULTS .................................................................................... 92 DISCUSSION ............................................................................ 106 CHAPTER 5. SUNIMARY AND CONCLUSIONS ......................................... 111 BIBLIOGRAPHY ................................................................................. 1 15 LIST OF TABLES Page Table 1. Potential binding sites of ribosomal proteins to HCK ............................... 71 vi LIST OF FIGURES Egg Figure 1. Basic structure of trichothecenes ..................................................... 7 Figure 2. DON induces p38 phosphorylation in U937 monocytes ............................ 37 Figure 3. DON induces p38 association with the ribosome in U937 monocytes ........... 38 Figure 4. DON induces sequential interaction of p38 with 40S and 60S ribosomal subunits in U937 monocytes ....................................................................... 39 Figure 5. DON-induced p38 interaction with the ribosome does not require p38 phosphorylation in U937 monocytes ............................................................. 41 Figure 6. Rapid [3H]DON uptake in RAW 264.7 macrophages corresponds with p38 phosphorylation and ribosome interaction ....................................................... 42 Figure 7. DON-induced p38 interaction with the ribosome does not require p38 phosphorylation in RAW 264.7 macrophages ................................................... 43 Figure 8. DON induces JNK and ERK ribosomal interaction and phosphorylation in RAW 264.7 macrophages .......................................................................... 44 Figure 9. Proposed scaffolding role of ribosome in DON-induced ribotoxic stress response .............................................................................................. 46 Figure 10. PKR inhibition suppresses DON-induced IL-8 mRNA and protein expression in U937 cells. ...................................................................................... 62 Figure 11. Hck inhibition suppresses DON-induced IL-8 mRNA expression in U937 cells. ......................................................................................................... 63 Figure 12. p38 inhibitor blocks DON-induced IL-8 protein expression in U937 cells .................................................................................................... 64 Figure 13. PKR and Hck inhibition suppresses DON-induced p38 phosphorylation in U937 cells. .......................................................................................... 65 Figure 14. PKR antisense expression and Hck inhibition suppress DON-induced p38 phosphorylation and IL-8 production in U937 cells. .......................................... 67 Figure 15. DON induces phosphorylation of ribosome-associated PKR and Hck .........68 vii Figure 16. PKR is required for Hck interaction with the ribosome in U937 cells. ........70 Figure 17. Hck interacts with the ribosomal protein S3 (RPS3) .............................. 72 Figure 18. DON-induced MAPK interaction with the ribosome is suppressed in the peritoneal macrophages from PKR-deficient mice. ........................................... 73 Figure 19. PKR inhibition suppresses DON-induced Hck phosphorylation in ribosomal fractions of RAW 264.7 cells. .................................................................... 75 Figure 20. PKR and Hck phosphorylation precedes p38 interaction with the ribosome in RAW 264.7 cells. .................................................................................. 76 Figure 21. PKR and Hck inhibition suppresses DON-induced phosphorylation of ASK1, MKK3/6 and p38 in RAW 264.7 cells. .......................................................... 77 Figure 22. Ribosome functions as scaffold for PKR, Hck and p38 in DON-induced ribotoxic stress response. ......................................................................... 79 Figure 23. Putative role of ASK1 and MKK3/6 —in DON-induced p38 activation. . .....82 Figure 24. SG induces apoptosis in RAW 264.7 cells. ....................................... 94 Figure 25. UV absorption scan for SG. ........................................................ 95 Figure 26. SG binding to ribosomal subunits is concentration-dependent in RAW 264.7 cells. ................................................................................................ 96 Figure 27. Kinetics of SG binding to ribosomal in RAW 264.7 cells. ................... 97 Figure 28. High SG concentration induces rapid, saturable binding to 40S and 60S ribosomal subunits in RAW 264.7 cells. ...................................................... 99 Figure 29. DON competitively inhibits SG binding to 40S and 608 ribosomal subunits in RAW 264.7 cells. ............................................................................... 100 Figure 30. SG binding to the ribosome is reversible in RAW 264.7 cells. .............. 102 Figure 31. SG induces modest p38 and JNK phosphorylation in RAW 264.7 cells....103 Figure 32. SG and DON induce MAPK interaction with the ribosome. ............... 104 Figure 33. SG binds to 40S and 60S ribosomal protein fractions in PC-12 neuronal cells. ......................................................................................................... 105 viii LIST OF ABBREVIATIONS ANOVA 2-AP AP- 1 ASKl COX-2 DON eIF2a ELISA ERK Hck IgG IL-8 JNK/SAPK LPS MAPK PAP PAT one way analysis of variance 2-aminopurine activator protein-1 apoptosis signal kinase 1 cyclooxygenase-2 deoxynivalenol alpha subunit of eukaryotic initiation factor 2 enzyme-linked immunosorbant assay extracellular signal-regulated kinases hematopoietic cell kinase horseradish peroxidase irnmuno globulin G interleukin-8 c-Jun N-terminal kinase/stress-activated protein kinases lipopolysaccharide mitogen-activated protein kinase mitogen—activated or ERK kinase macrophage-inflammatory protein-2 MAPK kinase 3 and 6 3-[4, 5-dimethy1thiazol-2-yl]-2, 5-diphenyltetrazolium bromide Pokeweed antiviral protein patulin ix PBS PEB PKR PPl PP2 RIPS rRNA RPL3 RPL7 RPS3 RPS6 RS+M SDS SG SH phosphate buffered saline Polysome extraction buffer double-stranded RNA protein kinase R Src family kinase inhibitor, 4-Amino-5-(4-methylpheny1)-7-(t- butyl)pyrazolo[3,4—d]- pyrimidine Src family kinase inhibitor, 4-Amino-5-(4-chlorophenyl)-7-(t- butyl)pyrazolo[3,4-d]pyrimidine ribosome-inactivating proteins ribosomal RNA ribosomal protein large subunit L3 ribosomal protein large subunit L7 ribosomal protein smaill subunit S3 ribosomal protein smaill subtmit S6 ribosomal subunits and monosomes sodium dodecyl sulfate Satratoxin G Src homology Shiga toxin 1 Tolerable Daily Intake transforming grth factor-B tumor necrosis factor- a ultraviolet-B light INTRODUCTION Trichothecene mycotoxins, produced by F usarium, Myrotecium, T richoderma, Cephalosporium, Trichothecium, Verticimonosporium and Stachybotrys, are food and environmental (Hodgson et a1. 1998; Ueno 1985). Over 200 trichothecenes have been identified and all have a common 12,13-epoxide ring related to toxicity (Grove 2007). At high doses, trichothecenes are directly cytotoxic to many cells and can induce multiorgan damage. Early signs of trichothecene poisoning are irritation of the skin and mucous membranes with gastroenteritis. Trichothecenes rapidly diffuse through the cell membrane, bind to eukaryotic ribosomes and inhibit translation (Cundliffe and Davies 1977). Peptidyl transferase activity is thought to be a critical target in trichothecene inhibition of protein synthesis (Liao et a1. 1976). Deoxynivalenol (DON) is the most common trichothecene found in food. DON is referred as vomitoxin because it induces excessive salivation and vomiting when it is fed to animals. It is also thought to be similarly toxic to humans. Trichothecenes and various other xenobiotics inhibit protein synthesis and activate MAP kinase signaling via process known as “ribotoxic stress response.” This concept, initially proposed by Iordanov (1997), is thought to involve disruption of the 3’-end of large 28S ribosomal RNA, which typically functions in aminoacyl-tRNA binding, peptidyltransferase activity and ribosomal translocation. Ribotoxins, such as anisomycin and Shiga toxin, induce disruption of this area leading to JNK/SAPK and p38 activation (Bunyard et al. 2003; Smith et a1. 2003). Interestingly, ultraviolet radiation also triggers the ribotoxic stress response in mammalian cells via damage of the 3’-end of large 28S ribosomal RNA and activation of INK/p38 MAPK (Iordanov et al. 1998). A trichothecene-resistant yeast has been reported, that has a mutation of the ribosomal protein L3 conferring resistance to both trichothecenes and anisomycin (Fried and Warner 1981). If that same protein, L3, is transgenically modified in tobacco plants, it confers resistance to DON (Di and Turner 2005). Thus, ribosomes might mediate ribotoxin-induced MAP kinase activation through a conformational change of ribosome (Laskin et al. 2002). MAPK pathways are important intercellular signaling cascades that initiate cellular processes such proliferation, differentiation and apoptosis. Three MAPK cascades have been identified: extracellular signal-regulated kinases (ERKs), c-Jun N- terminal kinases (INKS), and p38-MAPK (Wada and Penninger 2004). ERK is important for cell survival, while the JNK and p38 pathways are involved in cell apoptosis. Trichothecenes induce all three MAPK in macrophages, T cells and B cells (Pestka et al. 2005; Shiftin and Anderson 1999). In trichothecene-treated macrophages, inhibition of ERK by chemical inhibitors potentiates p38- and p53- dependent apoptosis, whereas upon p38 inhibition, ERK and AKT survival pathways are activated (Zhou et al. 2005a). Thus, survival and apoptotic pathways compete with each other in trichothecene-stimulated macrophages, depending on dosage and timing (Pestka et al. 2004). Double-stranded RNA-(dsRNA)-activated protein kinase (PKR) is a widely- expressed serine/theonine protein kinase that has been shown to be induced not only by dsRNA, but by other agents including interferon (Williams 2001). This kinase is thought to play a critical role in trichothecene-induced inflammatory gene expression and apoptosis via the JNK and p38 MAPK pathways (Zhou et al. 2003c). However, the precise relationship of PKR to JNK and p38 MAPK has not yet been defined. PKR is known to interact with apoptosis signal-regulating kinase 1 (ASKI), one of the members of the MAPK kinase kinase family, which in turn activates JNK and p38 via MAPK kinases 4/7 or 3/6, respectively (Matsukawa et al. 2004; Takizawa et al. 2002) Src protein kinases also play critical roles in intracellular signaling cascades induced by external stimuli (Thomas and Brugge 1997) and share three conserved domains that include SH2, SH3 and tyrosine kinase domains (Boggon and Eck 2004). Hematopoietic cell kinase (Hck), one of the Src nonreceptor tyrosine kinases, is expressed primarily in myeloid cells (Quintrell et al. 1987) and the activation is regulated by SH2 and SH3 domains (Arold et al. 2001; Moarefi et al. 1997; Pellicena et al. 1998). Zhou et al. (2005b) reported that Hck is upstream of the p38 pathway in trichothecene-stimulated macrophages. However, it is not known how Hck activates p38 via interaction with the ribosome. Although Iordanov (1997) observed that the ribosome is important for MAPK activation in the ribotoxic stress response, the nature of the interactions between ribosomal proteins and MAPK is not well understood. To investigate the mechanism of the ribotoxic stress response in DON-stimulated macrophages, there were three specific aims in this dissertation; 1) to identify the role of PKR in DON-induced p38 activation, 2) to identify the role of Hck in DON-induced p38 activation, and 3) to assess the role of the ribosome in DON-induced p38 activation. Three chapters in this dissertation address these specific aims. Chaper I is a literature review of trichothecenes, deoxynivalenol (DON), ribotoxic stress response, MAPK and two upstream kinases, PKR and Hck. Chaper II is an investigation of the role of the ribosome inducing p38 activation in DON-stimulated murine macrophages and human monocytes. Chapter 111 includes studies of the roles of PKR and Hck in inducing p38 activation, and their interaction with the ribosome. Chapter IV contains competitive binding studies of DON and another trichothecene satratoxin G to the ribosome. Chapter V is a summary and conclusion for this dissertation. CHAPTER I LITERATURE REVIEW CHAPTER 1 LITERATURE REVIEW A. Trichothecenes A. 1. Introduction Trichothecene mycotoxins are secondary metabolites produced by various species of fungi, including Fusarium, Myrothecium, Trichoderma, Cephalosporium, Verticimonosporium, and Stachybotrys (Ueno 1985). There are over 200 members of this group and they share common structural features of a double bond between carbon 9 and 10 and an epoxide ring from carbon 12 to 13 in a sesquiterpenoid (Grove 2007). The double bond and epoxide ring are important for translation inhibition and primarily responsible for toxicity. Based on chemical structures and producing fungi, trichothecenes can be classified into four groups, Types A, B, C, and D (Betina 1989; Ueno 1985; Ueno et a1. 1973). Type A has a hydroxyl or acyl moiety at the R group locations and Type B has an additional carbonyl group at C-8. Type C has epoxide groups at C-7 and 8 or 09 and 10. Type D contains a diester or triester ring structure between C—4 and C-5. T-2 toxin and diacetoxyscirpenol are representative of Type A, and deoxynivalenol (DON) and nivalenol of Type B. Type C induces crotocin, and Type D includes satratoxin G, roridin A and verrucarin A (U eno 1985). Trichothecenes are also classified by the mode of translation inhibitory action, namely initiation inhibition (I-type), elongation inhibition (E-type) and termination inhibition (T-type). Protein synthesis inhibition is affected by the location of substitution of residue on C3, 4, 8 and 15 and dose (Carter and Cannon 1977). For example, 4-DON is classified as an E-type while 7-DON is classified as I-type (Ehrlich and Daigle 1987). Trichodermin completely stabilizes polysome profile at a high dose, while low doses of trichoderrnin partially break down polysomes as a characteristic of initiation inhibitors (Carter and Cannon 1977). H30 0. .,,R. .1 01 2°.-.- \ CH R3 14 3 Figure 1. Basic structure of trichothecenes A.2. Intracellular targets of trichothecenes Trichothecenes bind to ribosome and inhibit translation. Several reports have been shown that the ribosomal interaction with these toxins is critical for toxicity induction (Di and Tumer 2005; Femandez—Lobato et al. 1990; Fried and Warner 1981; Petrov et al. 2004). One of the in vivo and in vitro intracellular targets for trichothecenes is the 28S ribosomal RNA (rRNA). 28S rRNA is the site of peptidyl- transferase activity in the ribosome, and exposure to trichothecenes can prevent this part of protein synthesis (Shifiin and Anderson 1999). However, the protein synthesis inhibition in vitro is not always correlated with the toxicosis in vivo (Thompson and Wannemacher 1986). A. 3. T richothecenes induce immunotoxicity Acute toxicities of trichothecenes include gastroenteritis, destructed lymph nodes and spleens, cellular damage in bone marrow, hemorrhage and edema (Bhat et al. 1989; Luo et al. 1990; Ueno 1984, 1985). Chronic or subchronic exposure to trichothecenes causes anorexia, growth retardation and alimentary toxic aleukia (ATA) in livestock and humans (Bondy and Pestka 2000; Forsyth et al. 1977; Pestka et al. 1987; Ueno 1984). Trichothecenes modulate immunostimulatory or immunosuppressive response depending on the nature of substituent groups, dose and duration (Pestka et al. 2004). They target the immune cells such as mononuclearphagocyts, B- and T- cells, which induce proinflammatory gene expression in lower dosage and apoptosis in higher dosage in in vitro or ex vivo (Friend et al. 1983; Hughes et al. 1989; Hughes et a1. 1990; Hughes et al. 1988; Tomar et al. 1988; Tomar et al. 1987). B. Deoxynivalenol (DON) B. 1. Introduction Deoxynivalenol, a type B trichothecene, is produced by F usarium graminearun and F usarium culmarum, which is commonly found in corn and grain crops and is resistant to food processing. The World Health Organization (WHO) set the Tolerable Daily Intake (TDI) of DON to lug/kg body weight, however the actual exposure to DON can be higher in many countries (Leblanc et al. 2005; Schothorst and van Egmond 2004; Tritscher and Page 2004). Outbreaks in China, Japan, Korea and India have shown that the symptoms such as vomiting, nausea and gastroenteritis were associated with F usarium-contaminated crops (Bhat et al. 1989; Luo et al. 1990). In a recent survey of food from 12 countries in Europe, 57% of tested grain-based products had DON contamination (Schothorst and van Egmond 2004). Similarly, 237 commercial food products were tested for DON in southwest Germany and 71% of products found to have DON contamination (Schollenberger et al. 1999). Many countries have regulatory limits to DON, however, it has been hard to harmonize these regulations (van Egmond 2002). Exposure to DON induces a variety of toxicological and immunological effects. In the B6C3F1 female mouse, LDsos are 78 mg/kg orally and 49 mg/kg (i.p.) (Forsell et al. 1987). A high acute dose is capable of causing tremendous necrosis of the gastrointestinal tract, bone marrow, lymphoid tissues, heart and kidney. DON at 40 ppm induces 90% reduced feed consumption and weight loss in swine, the most susceptible species (Forsyth et al. 1977; Pestka et al. 1994). Subchronic exposure to DON (10 ppm) in mice reduces glucose transfer from the intestine and causes accumulation of S-methyltetrahydrofolic acid in tissues (Hunder et al. 1991). Dietary exposure to DON can also alter mitogen-induced proliferation of lymphocytes, host resistance, immunoglobulin production and cytokine expression (Rotter et al. 1996). DON triggers anorexia at low doses and at higher doses can cause emesis in animals (Forsyth et al. 1977; Pestka et al. 1987; Prelusky and Trenholm 1993). B. 2. DON-induced immunosuppression and immunostim ulation DON targets actively dividing cells, such as those in spleen, thymus and Peyer’s patches (Azcona-Olivera et al. 1992; Yan et al. 1998; Zhou et al. 2000) and can therefore alter immune function. Immune responses can either be suppressed or stimulated depending on the exposure time and dosage (Bondy and Pestka 2000). High doses of DON induce an immunosuppressive response by inhibiting immune cell proliferation and also induce apoptosis (F orsell and Pestka 1985; Robbana-Barnat et al. 1988; Tryphonas et al. 1986). In in vitro models, high concentrations of DON rapidly trigger apoptosis of immune cells, including macophages, monocytes, T cells, B cells and IgA secreting plasma cells in mice (Pestka et al. 1994; Yang et al. 2000; Zhou et al. 2003b). Low doses of DON stimulate the immune system inducing gene expression related to irnmunoglobulin secretion and proinflammatory responses. Proinflammatory proteins, such as tumor necrosis factor-alpha (TNFa), interleukin(IL)-l B and IL-6, are highly induced in the spleen and plasma of B6C3F1 10 mice (Gray and Pestka 2007; Zhou et al. 2003a). In murine splenic CD4+ cells, IL-2, IL-4, IL-5 and IL-6 mRNA expression was increased after stimulation by DON (Shi and Pestka 2006). DON increased macrophage inflammatory protein-2 (MIP-2) and complement 3A receptor in RAW 264.7 cells. COX-2 expression also increases in RAW 264.7 cells and in the spleen and Peyer’s patches from orally gavaged mice with DON (Moon and Pestka 2003). In vivo experiments show that mice orally exposed to DON (12.5 mg/kg) had significantly increased MIP-2 in the spleen and plasma, which was synergistically upregulated with LPS co-treatment (Chung et al. 2003b). In human Jurkat T cells, MAPK activation was critical for DON induced IL-2 and IL-8 expression (Pestka et al. 2005). In human U937 monocytes, DON induced IL-8 mRNA and protein expression was thought to be regulated by p38-mediated transcription, but not through mRNA stabilization (Gray and Pestka 2007; Islam et al. 2006) DON induces competing apoptotic and survival signaling pathways in murine macrophages via MAPK activation (Pestka et al. 2004; Zhou et al. 2005a). Alterations in 28S rRNA, due to protein synthesis inhibition by DON, are thought to be an initiation signal for activation of mitogen-activated protein kinases (MAPKs), such as JNK1/2 and p38 (Rocha et al. 2005). These two MAPKs have been observed to be associated with apoptotic cell death (Shifrin and Anderson 1999; Yang et al. 2000). Interestingly, ERK1/2, another MAPK, is considered an anti-apoptotic signal during DON-mediated apoptosis (Yang et al. 2000). Pharmaceutical inhibitors of p3 8, SB203580, and ERK, PD98059, have shown that p38 is critical for the apoptotic pathway, by inducing a tumor suppressor p53, pro-apoptotic protein Bax (Bel-2— 11 associated X protein), caspase-3 and cytochrome C release from mitochondria (Zhou et al. 2005a). Conversely, ERK is involved in a survival pathway via AKT (protein kinase B), p9ORSK (ribosomal S6 kinase) activation and pro-apoptotic protein Bad (Bcl-Z-associated death promoter) inactivation. C. Mitogen-activated protein kinases (MAPK) C.1. Introduction MAPK pathways are important intercellular signaling cascades that initiate cellular processes such proliferation, differentiation and apoptosis to extracellular stimuli (Avruch 2007). The basic cascades in the MAPK pathways are composed of three types of protein kinases. MAPK kinase kinase (MAPKKK or MEKK) stimulates the MAPK kinase (MAPK or MEK), which in turn stimulates MAP kinases (MAPK) (Enslen and Davis 2001; Tibbles and Woodgett 1999). Three distinct MAPKs have been characterized in mammals. There are extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (MS), and p3 8-MAPK (Wada and Penninger 2004). All three families are known to coordinately mediate diverse biological functions, such as cell proliferation, differentiation, translation, gene expression and apoptosis (Raingeaud et al. 1995; Wong et al. 1998). Although p38 and JNK are activated by environmental stress, including UV, heat stress, osmotic stress and endotoxin, ERK is typically activated by extracellular signals such as growth hormone and proliferative stimuli (Tibbles and Woodgett 1999). Typically, p38 mediates apoptosis and proinflammatory gene expression, whereas JNK and ERK are involved in regulation of both cell survival and death 12 depending on the cell type, stimuli, and the latency of the activated MAPKs (Himes et al. 2006; Kim et al. 2003; Shaul and Seger 2007; Wada and Penninger 2004). MAPK share various substrates localized in cytosol, nucleus as well as other cellular organelles, and MAPK relocalization in cells is important for interacting with discrete substrates and evoking diverse cellular regulatory response (Raman et al. 2007; Yoon and Seger 2006). C. 2. Role of MAPK in translation MAPK can regulate translation in response to environmental stimuli. For example, dsRNA-induced p38 and JNK activation can modulate inhibition of protein translation in murine fibroblasts (Iordanov et al. 2000; Iordanov et al. 2005). ERK competes with JNK to positively regulate protein translation via e1F2a dephosphorylation and prevents cell death by environmental stress in human alveolar macrophages (Monick et al. 2006). Shiga toxin-induced p38 and JNK suppress cap- dependent translation pathway in intestinal epithelial cells (IECs). Pharmaceutical inhibitors of p38 and ERK moderately inhibit eIF4E, which interacts with 4E-BP1 to inhibit translation phosphorylation (Colpoys et al. 2005). C3. WPKS and trichothecenes Trichothecenes induce phosphorylation of all three MAPK families in macrophages, T cells and B cells (Pestka et al. 2005; Shifiin and Anderson 1999). Interestingly, immunostimulatory or irnmunosuppressive responses are dose- and time-dependent, with p38 having a pivotal role in both outcomes. In trichothecene- 13 treated macrophages, inhibition of ERK by chemical inhibitors potentiates p3 8- and p53-dependent apoptosis, whereas upon p38 inhibition, ERK and AKT survival pathways are activated (Zhou et al. 2005a). ERK negatively regulates trichothecene- induced apoptosis in RAW264.7 murine macrophages, while p38 positively control apoptosis. ERK inhibitor PD98059 amplified SG and DON-induced apoptosis, while p38 inhibitor SB203580 moderately inhibited DON-induced apoptosis, not SG- induced apoptosis (Yang et al. 2000). Thus, survival and apoptotic pathways compete with each other in trichothecene-stimulated macrophages depending on dosage and timing (Pestka et al. 2004). D. Ribotoxic stress response D. I. Introduction Trichothecenes and other xenobiotics can inhibit protein synthesis and activate MAPK signaling via the process lmown as the “ribotoxic stress response.” This concept, initially proposed by Iordanov (1997), was thought to involve disruption of the 3’-end of the large 28S ribosomal RNA (rRNA). This area of the 28S rRNA typically functions in aminoacyl-tRN A binding, peptidyltransferase activity, and ribosomal translocation. Some toxins inducing ribotoxic stress response, called “ribotoxins”, are anisomycin, trichothecenes and ribosome-inactivating proteins (RIPS) such as ricin and Shiga toxin (Bender et al. 1997; Bunyard et al. 2003; Chung et al. 2003a; Iordanov et al. 1998; Pestka et al. 2004; Smith et al. 2003; Zinck et a1. 1995). Anisomycin, an antibiotic from Streptomyces griseolus, is the most well known 14 ribotoxin that induces MAPK activation via 28S rRNA damage (Iordanov et a1. 1997). Trichothecene mycotoxins target the peptyidyltransferase ring in the 60S ribosomal subunit and induce JNK and p38 MAPK activation. Trichothecenes inhibit anisomycin binding to the ribosome, and the trichothecene T-2 toxin inhibited anisomycin-induced JNK/SAPK activation, suggesting that trichothecenes also target 288 rRNA (Shifrin and Anderson 1999). RIPS are known to induce the ribotoxic stress response by depurinating 28S rRNA and inducing MAPK activation. They include ricin, from castor beans, abrin, from the seeds of jequirity plant, Shiga toxins, from E. coli, and pokeweed antiviral protein (PAP) (Bolognesi and Polito 2004; Stirpe 2004). RIPS are composed of A and B subunits, with the A subunit responsible for enzymatic cleavage of the rRN A. This action leads to MAPK activation and downstream proinflammatory gene expression and/or apoptosis. The B subunit of RIPS contains binding sites for cells surface receptors, allowing the protein to enter the cell via endocytosis (Bolognesi and Polito 2004; Smith et al. 2003; Stirpe 2004). The B subunit of ricin binds to both glycoproteins and glycolipids with a terminal galactose, while the B subunit of cholera toxin and Shiga toxin bind to structurally restricted receptors, such as glycolipid GM] and glycolipid receptor Gb3. After endocytosis, endosomes are transported to the Golgi apparatus, where the RIPS are released into the cell. (Sandvig and van Deurs 2002). Other potential ribotoxins are palytoxin, onnamide A, theopederin B, aplidin, 13-deoxytedanolid and patulin. Palytoxin induces the potassium influx into Ratl fibroblast cells, which leads to the ribotoxic stress response (Iordanov et al. 1998). 15 Onnamide A and theopederin B inhibit translation and induce MAPK activation, leading to a ribotoxic stress like response (Lee et al. 2005). Aplidin, produced from Aplidium albicans, has Shown a ribotoxin stress like response, inhibiting protein synthesis and inducing p3 8, JNK and ERK activation (Cuadrado et al. 2003). Patulin (PAT) is predominantly produced from Penicullium and Aspergillus. PAT inhibits protein translation and induces p38, ERK and JNK activation in HEK293 cells (Liu et al. 2006), l3-Deoxytedanolide, a macrolide produced by a marine Sponge, interacts with the 60S ribosomal subunit and potentially inhibits protein synthesis. 13- Deoxytedanolide is the first macrolide shown to inhibit eukaryotic ribosomes, which induce MAPK activation similarly to anisomycin (N ishimura et al. 2005). Along with proteins and chemicals, ultraviolet radiation also triggers the ribotoxic stress response in mammalian cells through damage to the 3’-end of large 28S ribosomal RNA and activation of JNK/p38 MAPK (Iordanov et al. 1998). 0.2. The role of ribosomes in the ribotoxic stress response Eukaryotic cells have 40S and 60S subunits, which combine to form an 80S monosome. The molecular weight of the 608 large subunit is 2,800,000 Daltons. It is composed of 5S, 5.88 and 28S rRNA and approximately 49 proteins of varying sizes. The molecular weight of 408 subunit is 1,400,000 Daltons. This subunit is composed of 18S rRNA and approximately 33 proteins of varying sizes. Translation is initiated in free 40S and 60S ribosomal subunits in the cytoplasm. Cytoplasmic proteins are mostly synthesized in the cytosol, whereas secreted proteins are synthesized in the endoplasmic reticulum (ER). In translation of secreted proteins, signal peptides which 16 are initial synthesized polypeptides, induce ribosome-translocation to the ER and discriminate between translation of ER-bound or cytoplasmic ribosomes (The Molecular Biology of the Cell, 2002) The ribosome is thought to have a critical role in the ribotoxic stress response. In eukaryotes, the peptydyl transferase ring in 28S rRNA is a known target for ribotoxins. The effect of the ribotoxins is Similar to the function of macrolide antibiotics in prokaryotes, which target 23S rRNA to inhibit translation. Macrolide antibiotics, such as erythromycin and clarithromycin are composed of a lactone ring with multiple side groups. Lactone rings are important for rRNA binding due to Van der Waals interaction, and side groups provide more specificity to binding in this region. Single nucleotide changes in the 23 rRNA in E. coli confer resistance to macrolide antibiotics (Xiong et al. 2005). RIPS, such as PAP and ricin, depurinate 288 rRNA, by cleaving a purine base (adenine or guanine) from a specific binding site, the alpha—sarcin/ricin loop of 28S rRNA. Mutation of catalytic activity of a subunit of Stxl blocked depurination of 28S rRNA and decreased p38 and JNK activation, Showing that ribotoxin binding to the ribosome induces MAPK activation, which leads to toxicity (Smith et al. 2003). Some trichothecene-resistant yeasts have a mutation in the ribosomal protein L3 gene. The ribosomal protein L3 (RPL3) is known to stabilize the peptydyl transferase ring in the 28S rRNA in yeast (F emandez-Lobato et al. 1990). Overexpressed RPL3 confers resistance to DON in tobacco plant (Di and Tumer 2005). RPL3 mutation changes the binding affinity of peptidyl-tRNA to the ribosome and even a few amino acid changes in the L3 protein leads to considerable rRNA 17 conformational change, suggesting that alteration of the peptydyl transferase ring is important for ribotoxin-induced toxicity (Petrov et al. 2004). Other evidence exists, showing how the ribosomal structure affects toxin binding to the peptydyl transferase ring. The trichothecene T—2 toxin is thought to target the peptydyl transferase ring. Emetine inhibits T—2 binding to cell free ribosomes isolated from CHO cells, showing that T-2 and emetine target similar binding Sites. The inhibitory effect of emetine on T-2 binding to the ribosome was different when the experiment was conducted at 4°C or 37°C, suggesting ribosomal structure is altered by temperature (Leatherman and Middlebrook 1993). Recent reports have shown that not only toxin binding to the ribosome but ribosomal structural alteration might be important for ribotoxic stress response. Emetine pretreatment inhibits ricin-induced JNK activation, while the ricin still depurinated the 28S rRNA, indicating that emetine-induced ribosomal structural alteration blocks MAPK activation induced by ricin-toxin interaction (Iordanov et al. 1997). These results suggest that the structural alteration to the ribosome by interaction of the toxin is necessary to induce MAPK activation. It has been also proposed that actively translating ribosomes are targets for ribotoxins, and run-ofl‘ ribosomes do not induce ribotoxin-induced toxicity, indicating that the structural alteration to the ribosome is important for toxicity (Iordanov et al. 1997; Ueno et al. 1969). Therefore, resistance to ribotoxins can not only be explained by binding inhibition but also, conformational change of the peptydyl transferase ring, which affects the efficiency of peptide bond formation (Jimenez and Vazquez 1975). Although Iordanov (1997) observed that the ribosome is important for MAPK 18 activation, the nature of the interaction between ribosomal proteins and MAPKs is not well understood. Recent reports have found that MAPK can directly interact with the ribosome. ERK interacts with ribosomal protein S3, which has a potential ERK binding motif, FXF P. Interestingly, only inactivated ERK can bind to the ribosome, while activated ERK has no interaction with the ribosome (Kim et al. 2005). These data suggest that inactivated ERK can bind to the ribosome and be phosphorylated under certain conditions. A proteomics study has shown that p38 MAPK co- irnmunoprecipitated with ribosomal proteins, as analyzed by LC-MS/MS, however, the ribosomal proteins were not specified in this report (Lin et al. 2003). D. 3. Ribotoxic stress response and oxidative stress response The oxidative stress response, triggered by the imbalance in production and elimination of reactive oxygen species, activates MAPK and leads to cellular toxicity (Scandalios 2002). The ribotoxic stress response, distinctive from the oxidative stress response, activates MAPKs via ribosomal interaction following the damage to 28S rRNA. One study reported that the mechanism of JNKl activation in Rat-1 cells is different when induced by the ribotoxic stress or oxidative stress. The JNK activation kinetics was slow in Rat-1 cells when stimulated with several pro-oxidants, but rapid when stimulated by ultraviolet-B light (UV-B). An antioxidant, N-acetyl cysteine, eliminated all pro-oxidant—induced INKl activation, but did not reduce UV-B induced JNKl activation. The ribotoxic stress response inhibitor, emetine, did not inhibit either the pro-oxidant or UV-B induced JNKl activation (Iordanov and Magun 1999). UV-B activated JNKl has different kinetics as compared to activation by pro-oxidants 19 such as sodium arsenite, cadmium chloride and hydrogen peroxide. Depending on the magnitude of the response to the ribotoxin, it is thought that ribotoxic stress response can also induce an oxidative stress response. Pulmonary toxicity of both ricin and abrin in alveolar macrophages was inhibited by antioxidant treatment. One proposed mechanism is the production of TNF-a (Lin et al. 2003). Ricin strongly induced TNF-o. expression, via p38/JNK MAPK activation, and reactive oxygen species are induced by the TNF-a-induced Signaling pathway. Therefore, it should be also considered that toxicity might be induced by the oxidative stress response triggered by the ribotoxic stress response. E. Double-stranded RNA-(dsRNA)—actlvated protein kinase (PKR) E. 1. Introduction PKR is a ubiquitously-expressed serine/theonine protein kinase that has been shown to be induced by dsRNA, LPS, interferon and TNF-o. (Williams 2001; Zhou et al. 2003c). PKR plays important roles in protein translation, transcription, cell growth control, cell differentiation, antiviral response and regulates expression of cytokines, such as T'NF-a, IL-6 and IL-12 (Der and Lau 1995; Der et al. 1997; Iordanov et al. 2000; Iordanov et al. 2005; Williams 2001; Zhou et al. 2003c). PKR has pro- or anti- apoptotic effects depending on cell types and duration of activation (Donze et al. 2004). PKR consists of two functional domains, one is a dsRNA binding domain (DRBD) and another is a catalytic domain (Zhu et al. 1997). DRBD binding to dsRNA leads to PKR activation and a catalytic domain phosphorylates eIF-Za on 20 serine residue 51 in order to regulate translation initiation (Kim et al. 2005; Samuel 1993). Although the DRBD is critical for the ribosomal protein interaction (Kumar et al. 1999), the kinase domain is also important for PKR localization in the ribosome. PKR is primarily localized in free ribosomal subunits, whereas deletion of kinase domain of PKR Shifts the PKR interaction with free ribosomal subunits to polysome (Zhu et al. 1997). Similary, K296D-PKR (dominant negative mutant-PKR) and K296D-KD (dominant negative mutant-PKR kinase domain) specifically bind to the polysome in COS-1 cells (Inokuchi et al. 1976). Thus, both DRBD and KD domain are important for PKR localization in the ribosome. PKR activation mechanisms are still controversial and several models have been proposed. One of prominent models is autoinhibition of PKR activation. Structurally, PKR is autoinhibited by DRBD, which inhibits PKR kinase activity by interaction with a catalytic domain. When dsRNA binds to DRBD, it leads to structural alteration of PKR and its activation (Wu and Kaufman 1997). However, a recent report has shown that binding affinity of nucleotide substrates to PKR was independent of PKR structural alteration by dsRNA binding, which conflicts with the previously proposed autoinhibitory model (Lemaire et al. 2006). Another model is autophosphorylation of PKR dimerization. Phosphorylation of Thr 446/451 in PKR is critical for its activation (Wu and Kaufman 2004). When dsRNA binds to DRBD, it induces PKR dimerization and subsequently leads to autophosphorylation of Thr446/451, which shows more preferential interaction with elF20. (Dey et al. 2005; Taylor et al. 2005; Wu and Kaufman 2004). However, PKR dimerization is not required for PKR activation. A PKR monomer can be activated by dsRNA binding 21 without dimerization (Wu and Kaufman 1996). The latter study’s investigators proposed that DRBD of PKR binds to ribosomal protein L18 and excess dsRNA detaches PKR from the ribosome and activates the kinase (Kumar et al. 1999). Although many mechanisms of PKR activation have been studied with dsRNA stimuli, dsRNA binding to DRBD is not necessary for PKR activation. PKR can be activated by non-dsRNA stimuli such as interferons, TNF-a, platelet-derived growth factor (PDGF) and LPS (Deb et al. 2001; Goh et al. 2000; Kumar et al. 1997; Yang et al. 1995). A possible mechanism is that PKR has a self-inhibitory region and can be structurally altered by multiple stimuli, which induces PKR activation. PKR localization and dimerization increase intermolecular autophosphorylation, enhancing eIF2a phosphorylation (Vattem et al. 2001). The proximity of PKR monomers in a concentration greater than 0.5 M can induce PKR activation, which is independent of dsRNA binding (Lemaire et al. 2005). PKR cleavage by caspases also can induce PKR activation. Caspase 8 and 9 induce alteration of PKR autoinhibitory structure and phosphorylation and DRBD cleavage, leading to elF2a phosphorylation (Saelens et al. 2001). E. 2. PKR role in the ribotoxic stress response PKR might play a critical role in trichothecene-induced inflammatory gene expression and apoptosis via the JNK and p38 MAPK pathways (Zhou et al. 2003c). PKR is closely localized to the peptidyl transferase ring (Kumar et al. 1999; Zhu et al. 1997). It is thought to function as a signal tranducer following 288 rRNA damage. However, the precise relationship of PKR to JNK and p38 MAPK has not yet been 22 defined. One mechanism is that PKR activates p38 via MAPK kinase kinase ASKl and MAP kinase 3 and 6. PKR is known to interact with apoptosis signal-regulating kinase 1 (ASKI), one of the members of the MAPK kinase kinase family, which in turn activates JNK and p38 via MAPK kinases 4/7 or 3/6, respectively (Kumar et al. 1999; Matsukawa et al. 2004; Takizawa et al. 2002; Zhu et al. 1997). Dominant negative PKR expressing cells exhibit impaired ASKl-induced p38 activation and apoptosis in COS-1 cells (Takizawa et al. 2002). PKR induces p38, JNK and ERK activation and apoptosis, via caspase-3, in the ribotoxic stress response (Zhou et al. 2003c). DON-induced p3 8, JNK and ERK activation is inhibited by the PKR inhibitors, adenine and aminopurine (2-AP). PKR deficient monocytes exhibit suppression of DON-induced p3 8, JNK and ERK activation. Anisomycin and emetine, as well as DON, induced caspase-3 activity and DNA fragmentation, which were suppressed in PKR deficient monocytes. PKR inhibitors, 2-AP and C16 strongly suppressed IL-8 expression by the ribotoxins ricin, Shiga toxin 1 and DON. Similarly, the cell line U9KM which is stably transfected and constitutively expressing dominant negative PKR, has significantly reduced IL-8 expression induced by ribotoxins (Gray et al. 2008). Our laboratory has recently reported that PKR is critical for apoptosis induction in PC-12 rat neuronal cell by another trichothecene, satratoxin G. The PKR inhibitor, C16, inhibited apoptotic gene expression and apoptosis-inducing factor (AIF) translocation to the cytoplasm, and also PKR siRNA suppressed SG-induced apoptosis. (Islam et al. 2008) 23 F. Hematopoietic cell kinase (Hck) E1. Introduction Src family kinases are a group of non-receptor protein tyrosine kinases (PTKs), which play critical roles in cell differentiation, morphology, motility, proliferation and survival by environmental stimuli (Boggon and Eek 2004; Roskoski 2004; Thomas and Brugge 1997). Most cells express mutiple isoforms of Src family kinases. However, distinct cell types have difierent expression levels of the Src family kinases and distinctive subcellular localization of these kinases (Thomas and Brugge 1997). In humans, eleven subfamily Src PT Ks have been identified, which include Fyn, Fgr, Frk, Blk, Brk, Lek, Lyn, Hck, Src, Srrn and Yes. They have different cellular locations and are present in specific cell types (Roskoski 2004). Hematopoietic cell kinase (Hck), one of the Src family kinases, is expressed primarily in myeloid cells. Hck functions in cytoskeletal rearrangement, gene transcription, phagocytosis, survival pathways, cell proliferation and apoptosis (Podar et a1. 2004; Quintrell et al. 1987; Schmidt et al. 2007; Sub et al. 2005). Hck has two isoforms, p59 and p61. These Hck isoforms result from alternative translation of one mRNA. Compared to p59 Hck, p61 Hck has an additional 21 amino acids at the N- terminal end. The two isoforms have different cellular functions with distinct subcellular localizations (Guiet et al. 2008; Schmidt et al. 2007). p59 Hck is primarily located in the plasma membrane, whereas p61 is found in the cytosol and intracellular vesicles such as lysosomes in human neutrophils (Guiet et al. 2008). In undifferentiated or differentiated U937 cells, the majority of the p59 Hck was detectable in separated membrane fractions, while p61 Hck was higher in the 24 cytosolic fraction (Robbins et al. 1995). Src family kinases including Hck share conserved domains that include a N- myristoyl group, Src homology (SH) 4, SH3, SH2, SH2 linkers and tyrosine kinase domains along with a C-terminal tail (Guiet et al. 2008; Thomas and Brugge 1997). The N-myristoryl group is important, but not required, for membrane localization, while a docking protein in N-terminus of Src family kinases is important for the membrane association (Roskoski 2004). The tyrosine kinase domain has an activation loop with tyrosines that are autophosphorylated. Src family kinases can be activated through disruption of either one or two inhibitory interactions, and autophosphorylation of a tyrosine kinase domain leads to fully functional Src (Guiet et al. 2008). One inhibitory interaction is the intramolecular interaction between the SH2 and SH3 linker domains. Another inhibitory interaction is the binding of SH2 domain and the phosphorylated tyrosine in a C-terminal tail (Guiet et al. 2008; Thomas and Brugge 1997). However, there could be multiple active conformational changes of Src family kinases that generate unique downstream signals (Lerner et al. 2005). As with other Src family members, the activation of Hck is regulated by SH2 and SH3 domains, and SH3 domain has an autoinhibitory effect when it interacts with SH2 (Yadav and Miller 2007). The SH3 domain of Hck is critical for protein-protein interaction. This region of Hck interacts with several cellular proteins by PXXP motifs, proline rich regions, in the HL-60 human monocytes (Gouri and Swarup 1997). For example, the SH3 domain is critical for Bruton’s tyrosine kinase (Btk) interaction with Src kinases such as Fyn, Lyn and Hck (Cheng et al. 1994). Another study 25 reported that the ORF3 protein of hepatitis E virus (HEV) interacts with SH3 domains, leading to ERK activation (Korkaya et al. 2001). E 2. Role of Hck in the ribotoxic stress response Zhou et al. (2005) reported that Hck is upstream of the p38 pathway in trichothecene-stimulated macrophages. Hck inhibitors, PP] and PP2, suppressed DON-induced p3 8, JNK and ERK activation and the downstream proinflammatory cytokine expression in RAW 264.7 cells. Phosphorylation of p38, JNK and ERK substrates, such as ATFz, c-jun, and p90RSK were also inhibited by PPl. Anisomycin and emetine induced p38, JNK and ERK phosphorylation within 30 min, Similar to results observed with DON treatment, and their phosphorylation was inhibited by PP]. In addition, the Hck inhibitor PPl significantly inhibited DON-induced caspase activation and apoptosis (Zhou et al. 2005b). G Research rationale Trichothecenes and other ribotoxins have etiologically been associated with human gastroenteritis. However, the mechanism that leads to gastroenteritis is not well identified. Several studies show that trichothecenes target gut immune tissue and induce inflammatory gene expression or apoptosis via p38 MAPK Thus, the identification of trichothecene-induced p38 mechanisms in macrophages will lead to a greater understanding of trichothecene-induced human gastroenteritis and yield strategies for treatment. Many known ribotoxins, such as ricin and Shiga toxin, induce Similar ribotoxic stress response mechanisms as trichothecenes including inhibition of 26 translation and induction of MPAK pathways as trichothecenes. This research will aid in understanding of how other ribotoxins affect human health. Additionally, further research of ribotoxic stress response mechanism could be used to make a predictive model for identification of adverse or therapeutic effects of unknown potential ribotoxins. 27 CHAPTER II ROLE OF THE RIBOSOME IN DON-INDUCED p38 ACTIVATION This chapter was published in Toxicological Sciences (2008). Bae H.K., Pestka J.J. Deoxynivalenol induces p38 Interaction with the ribosome in monocytes and macrophages. Toxicological Sciences, 2008, 105(1):59-66 28 ABSTRACT Trichothecene mycotoxins rapidly induce p3 8-mediated gene expression and apoptosis in mononuclear phagocytes via a process known as the ribotoxic stress response. We hypothesized that the trichothecene deoxynivalenol (DON) induces interaction of p38 with the ribosome. Two models, U937 human monocytes and RAW264.7 murine macrophages, were used to test this hypothesis based on their capacity to evoke rapid and robust p38 phosphorylation responses to DON. Following DON treatment of U937 cells, lysates were subjected to sucrose gradient fractionation and the resultant ribosomal fractions probed for p38 by Western blotting. p38 content in fractions containing ribosomal subunits and monosomes (RS + M) increased within 5 min of DON treatment and continued to increase up to 30 min. p38 appeared to be initially interact with the 40S subunit fiaction and then subsequently with the 60S unit and monosome fractions. Although p38 phosphorylation was blocked by the inhibitor SB203580, interaction of the kinase with the ribosome was unaffected, suggesting that ribosomal binding and phosphorylation were dissociable events. In RAW 264.7 cells, radiolabeled DON uptake occurred within 15 min and this corresponded to sequential increases nonphosphorylated p38 and phosphorylated p38 in the RS + M fraction. As observed for p3 8, DON similarly induced both ribosomal interaction with two mitogen—activated protein kinases, c-Jun N-terrninal kinase, and extracellular signal—regulated kinase, and their subsequent phosphorylation in RAW 264.7 cells. Taken together, these data suggest that, in mononuclear phagocytes, DON induced p38 mobilization to the ribosome and its subsequent phosphorylation. The ribosome mi t thus play a central role as a scaffold in the ribotoxic stress response. 29 INTRODUCTION Trichothecene mycotoxins, are a family of toxic sesquiterpenoids produced by foodbome and environmental fungi that are of concern to human and animal health. Over 200 trichothecenes have been identified and all have a common 12,13-epoxide ring that is related to their toxicity (Grove 2007). Trichothecenes rapidly diffuse through the cell membrane, bind to the eukaryotic ribosomes and inhibit translation by interacting with peptidyl transferase (Liao et al. 1976; Ueno 1985). Deoxynivalenol (DON), a trichothecene that frequently contaminates cereal-based foods, is a particular food safety concern because this toxin causes immune dysregulation and impairs growth in experimental animals (Pestka and Smolinski 2005). Widespread human exposure to DON has been recently demonstrated using a glucuronide metabolite as a urinary biomarker (Turner et al. 200 8). In addition to their translational effects, trichothecenes can activate mitogen- activated protein kinase (MAPK)—mediated signaling cascades that drive critical cellular processes such as proliferation, differentiation and apoptosis. Specifically, DON and other trichothecenes induce phosphorylation of p3 8, c-Jun N-terminal kinases (JNKs) and extracellular signal—regulated kinases (ERKs) in lymphoid tissues of experimental animals (Zhou et al. 2003b) as well as in mononuclear phagocytes and other leukocytes (Moon and Pestka 2002; Pestka et al. 2005; Shifiin and Anderson 1999). p38 is widely recognized to be a central mediator of many stress- activated Signaling pathways (Chen-Chih Wu et al. 2007). Notably, scaffolding proteins such as osmosensing scaffold for MEKK3 and TAK-l binding protein 1 enable this kinase to phosphorylate substrates in a selective fashion and thus 30 differentially modulate a variety of cellular functions (Kang et al. 2006; Raman et al. 2007). p38 activation is of particular importance relative to DON-induced gene transcription, messenger RNA (mRNA) stabilization and apoptosis (Chung et al. 2003b; Moon and Pestka 2002; Zhou et al. 2003a, b). The general mechanism by which trichothecenes and other translational inhibitors such as ricin, Shiga toxin, and anisomycin induce phosphorylation of p38 and other MAPK has been termed the ribotoxic stress response (Iordanov et al. 1997; Laskin et al. 2002). This response is believed to involve disruption of the 3’-end of large 28S ribosomal RNA (rRNA), which typically functions in aminoacyl-transfer RNA binding, peptidyl transferase activity and ribosomal translocation. Although the ribosome undoubtedly plays some role in MAPK activation by ribotoxins, the nature of interactions occurring among ribosomal proteins rRNA and MAPK is not well understood. In this study, we employed human monocyte and mouse macrophage cell lines to test the hypothesis that DON induces interaction of p38 with the ribosome. The large size of the ribosome and masking of critical epitopes makes it difficult to apply conventional irnmunoprecipitation method to identify potential kinase interactions with it. Therefore, we used sucrose density gradient fractionation in conjunction with ribosomal protein subunit markers to track interaction with p38 following DON stimulation. The results indicate that DON sequentially induced mobilization of p38 and other MAPKS to the ribosome and their phosphorylation. 31 MATERIALS AND METHODS Chemicals. DON and other chemicals were purchased from Sigma Chemical Co. (St Louis, MO) unless otherwise noted. DON was dissolved in distilled, filter- sterilized water for addition to cell cultures. The p38 kinase inhibitor SB203580 was obtained from Calbiochem (San Diego, CA). Cell cultures. All cultures were maintained at 37°C in a humidified 6% C02 incubator. U937 cells (ATCC, Rockville, MD) were grown in RPMI-1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (F BS) (Gibco BRL, Gaithersburg, MD), and 100 U/ml penicillin and 100 ug/ml streptomycin (Gibco BRL). RAW 264.7 cells (TTB 77, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% (v/v) heat-inactivated FBS, 1mM sodium pyruvate (Gibco BRL), 100 U/ml penicillin (Sigma), and 100 rig/ml streptomycin (Sigma). In typical experiments, U937 cells (1 x 105/ml) in 225-cm2 cell culture flasks (Corning, Lowell, MA) or RAW 264.7 cells (5 x 105/ml) in a lOO-mm cell culture dishes (Corning) were cultured overnight, and then treated with DON for various time intervals prior to analysis. DON concentrations of 500 ng/ml (1.68 uM) and 250 ng/ml (0.84 uM) were used for U937 and RAW 264.7 cells, respectively, based on optimal effects observed in previous studies (Moon and Pestka 2002; Zhou et al. 2003b). Sucrose density fractionation. Cytoplasmic fractions were prepared and ribosome fractions isolated as previously described (Galban et al., 2003). Briefly, 32 cells were washed with ice-cold phosphate buffered saline (PBS) twice and lysed in ice-cold polysome extraction buffer lysis buffer consisting of 0.3M NaCl, 15mM MgClz, 15mM Tris-HCl (pH 7.6), 1% (w/v) Triton X-100, 0.1 mg/ml cyclohexamide, 1 mg/ml heparin, and 0.01% (v/v) phosphatase inhibitor cocktails A and B (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Cell lysate was centrifuged at 10,000 x g for 15 min to remove nuclei, mitochondria and other cell debris. The protein concentration of the cell lysate supernatant was determined using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Cell lysate (1 mg in 1 ml) was layered over 9 m1 of linear sucrose gradient solution (5—45%) in a 11.5-m1 Sorvall centrifuge tube (Kendro Laboratories, Asheville, NC) and centrifuged at 35,000 x g for 3 h at 4°C in Sorvall TH-641 rotor. The gradient was fractionated at a rate of 1 mein by upward displacement with an ISCO system (Teledyne ISCO, Lincoln, NE) comprised of a syringe pump and needle-piercing system connected to an EM-l ultraviolet monitor for continuous measurement of absorbance at 254 nm. The protein from each fraction was precipitated by slow addition of trichloroacetic acid to a final concentration of 10% (w/v) and then overnight incubation at 4°C. Pellets were recovered by centrifugation (10,000 x g for 15 min), washed with cold acetone twice, and air dried. Pellets were suspended in sodium dodecyl sulfate lysis buffer, sonicated, and then subjected to Western analysis as described below. Western analyses. For conventional Western analysis, proteins were separated on 4% (w/v) polyacrylamide gels and transferred to a polyvinylidene 33 difluoride (PVDF) membrane (Millipore, Billerica, MA). After incubating with blocking buffer (Li-Cor, Lincoln, NE), membranes containing immobilized proteins were incubated with murine antibodies to phosphorylated p3 8, JNK, or ERK concurrently with corresponding rabbit antibodies to nonphosphorylated p3 8, JNK, or ERK (Cell Signaling Technology, Danvers, MA) overnight at 4°C. After washing, blots were incubated with secondary IRDye 680 goat anti-rabbit and IRDye 800CW goat anti-mouse IgG antibodies (Li-Cor) for 1 h at 25°C. Infrared fluorescence of bound secondary antibodies containing the two dyes were simultaneously measured using a Li-Cor Odyssey Infrared Imaging System, and relative phosphorylation determined using Odyssey Analysis Software. For identification of ribosomal fractions, PVDF membranes were incubated with mouse and rabbit antibodies to ribosomal protein S6 (RPS6) (cell Signaling) and ribosomal protein L7 (RPL7) (Bethyl Laboratories, Inc., Montgomery, TX), respectively, followed by IRDye 680 goat anti-species conjugates. For in-cell Western analysis, RAW 264.7 cells (1 x 10‘ cells/well) were incubated overnight in 96-well plates (Coming) and then subjected to DON treatments. Media were then removed and cells immediately fixed in 4% (v/v) formaldehyde in PBS at room temperature for 20 min. Wells were washed for 5 min with 0.1% (v/v) Triton X-100 in PBS five times and then incubated with blocking buffer (Li-Cor) with moderate agitation for 90 min at 25°C. Blocking buffer was removed and the cells were incubated with 25 u] of antibodies to phosphorylated and nonphosphorylated MAPK in blocking buffer as described above with gentle shaking at 4°C. Wells were washed for 5 min with 0.1% in PBS-Tween with gentle Shaking 34 . five times and then incubated with fluorescent-labeled secondary antibodies (IRDye 680CW goat anti-rabbit and IRDye 800 goat anti-mouse) in blocking buffer for 1 h at 25°C in the dark. Wells were washed for 5 min with 0.1% (v/v) PBS-Tween five times and the plate then scanned at 700 and 800 nm on a Li-Cor Odyssey Infrared Imaging System. Uptake of [3H] DON. [3H] DON was previously provided by Dr F. S. Chu (University of Wisconsin, Madison, WI) and repurified by preparative thin layer chromatography using waterzacetonitrilezisopropanol (1:2:3). Purified DON was quantified using a Veratox 5/5 Quantitative DON test (Neogen Corp., Lansing, MI) and specific activity calculated to be 800 Ci/M. RAW 264.7 cells were cultured in a 12-well plates (5 x lo‘ cells/mllwell) overnight. Cells were treated with DON (250 ng/ml) containing 675 uCi [3H] DON and uptake of [3H] DON was measured after 0, 2.5, 5, 15, and 30 min. Following washing in ice-cold PBS, cells were lysed in 100 pl of 1 N NaOH, and the extract was neutralized with 100 pl of 1 N HCl. The lysate was added to 5 m1 of Safety Solve Scintillant (Research Products International, Mt. Prospect, IL) and measured on a scintillation counter. Statistics. Data were analyzed by Student’s t-test or ANOVA with SigrnaStat v 3.1 (Jandel Scientific, San Rafael, CA) with the criterion for significance set at p < 0.05. 35 RESULTS Induction of p38 phosphorylation in U937 monocytes by DON was confirmed by Western analysis of whole cell extracts (Fig. 2). Incubation with DON at 500 ng/ml induced marked phosphorylation of p38 after 15 min and this was further increased after 30 min. To determine whether p38 interacts with the ribosome during DON treatment, ribosomal subunits (40S and 60S), monosomes (80S), and polysomes were separated by sucrose gradient fractionation (Fig. 3A). The identity of the fractions was verified using anti-RPS6 and anti-RPL7 as markers for 40S and 60S ribosomal subunits, respectively. Fractions were pooled into a ribosomal subunit plus monosome (RS + M) fiaction and a polysome (P) fiaction. p38 was detectable in RS + M fraction after 5 min and increased in a time-dependent manner (Fig. 3B). p38 was also detectable in the P fraction after 15 and 30 min, but to a lesser extent than in the RS + M fraction. Western blotting was conducted on individual fractions to identify the specific ribosomal components with which p38 interacts. Upon treatment of U937 cells with DON, p38 initially associated with the 40S ribosomal subunit fraction at 5 min (Fig. 4). However, after 15 min, p38 was predominantly found in the 60S and 80S fractions. Thus DON appeared to induce sequential p38 association with the 40S ribosomal subunit followed by the 60S subunit and 80S ribosome. To ascertain whether p38 required prior phosphorylation to bind the ribosome, cells were preincubated with the p38 kinase inhibitor SB203580 for 45 min before DON treatment. Although the inhibitor effectively suppressed phosphorylation of this kinase, p38 interaction with the ribosome was unaffected in DON-stimulated U937 36 15 min 30 min N .h 22- 201 18- 16~ 14~ 12~ 10- Relative Intensity co Vehicle DON Vehicle DON 15 min 30 min Figure 2. DON induces p38 phosphorylation in U937 monocytes. Cells were stimulated with 500 ng/ml of DON for 15 or 30 min. (A) Western analysis was conducted using antibodies Specific for total p38 and phospho-p38. (B) Infiared fluorescence intensities of phospho-p38 band normalized against intensity of corresponding total p38 band. Data are representative of three separate experiments. 37 608 A254 408 803 ‘. RPSG a. 4.. RPL7 4 ..... 4} p Non-ribosomal Ribosomal subunits and Polysome (P) protein monosome (RS+M) B Cont 5 min 15 min 30 min DON ' + "' '" RS+M P RS+M P RS+M P RS+M P p38 a.- 1......- w. Figure 3. DON induces p38 association with the ribosome in U937 monocytes. (A) Representative sucrose gradient separation of ribosomal subunit, monosome and polysome fractions of U937 cells. Fractions containing small (40S) and large (60S) ribosomal subunits were detected by Western blotting with RPS6 and RPL7 antibodies, respectively. (B) Fractions were pooled and designated as RS+M or P and analyzed for p38 by Western blotting. Data are representative of three separate experiments. 38 some EPoly Untreated . ..... «433 M3.3 333.333 ..... -...-.... 2 . , 1 . . s... F RPSG RPL7 ....L.Hflflhflflpum DON 5 min DON 15 min \ 8 3 p ribosomal subunits in U937 monocytes. Cells were incubated with 500 ng/ml of DON for 5 or 15 min and p38 interactions within the ribosomal fiactions compared with those for untreated cells. Data are representative 39 Figure 4. DON induces sequential interaction of p38 with 40S and 60S of three independent experiments. cells (Fig. 5). The kinetics of DON uptake was related to that for p38 activation in RAW 264.7 macrophages. [3H] DON uptake was rapid with 50 and 75% of maximum binding being observed after 2.5 and 5 min, respectively (Fig. 6A). Cells were saturated with [3 H] DON after 15 min. Rapid DON uptake closely corresponded with p38 phosphorylation which also peaked after 15 min (Figs. 6B and 6C). Consistent with U937 cells, DON induced p38 association with the pooled RS + M fractions of RAW 264.7 cells, with marked increases in total p38 being observed in this fraction from 5 to 30 min (Fig. 6D). p38 phosphorylation was also detectable from 5 to 30 min, with peak phospho-p38 levels being observed after 15 min. To determine cultures whether p38 requires prior phosphorylation to bind the ribosome in RAW 264.7, cells were preincubated with the p38 kinase inhibitor SBZO3580 for 45 min prior to DON treatment. As seen with U937 monocytes, the p38 kinase inhibitor blocked p38 phosphorylation but this did not affect p38 interaction with the ribosome (Fig. 7). The potential for other MAPKs to similarly bind to the ribosome following DON exposure was also addressed. AS observed for p3 8, DON induced phosphorylation of JNK and ERK from 15 to 30 min in RAW 264.7 cells (Figs. 8A and 8B). Also consistent with p38, DON-induced JNK 1/2 and ERK 1/2 association with the RS + M fraction from 5 to 30 min, whereas phosphorylation of JNK 1/2 and ERK 1 was observed only from 15 to 30 min (Fig. 8C). These data suggest that DON sequentially induced ribosomal interaction with all three major MAPK families and their subsequent phosphorylation. 4O DON - - + + P38 - + - + inhibitor Cell Lysate R8 + M Fraction Figure 5. DON-induced p38 interaction with the ribosome does not require p38 phosphorylation in U937 monocytes. Cells were preincubated with the p38 kinase inhibitor SBZO3580 (2 uM) for 45 min and treated with 500 ng/ml of DON for 30 min. Cell lysates and pooled RS+M were analyzed by Western blotting. Data are representative of two separate experiments. 41 8605.530 80.6900 0050 Mo 0303:0858 0.3 San $5303 E0303 .3 wma cBEbonmmonn use :33 com 008205 803 2+9“ 330m Ae Amodvnc St? 032 08.8 5055 80m .wmn :39 00330:: umfimwa Suzanne: 80.3 and Baflbofimonq .«o 0033005 00533 E00003 :02: 0.520% ADV $080 50303 200.5 .3 0050008 00083 E noun—boaqmofi wmq 0:0 SE on .8 m _ .8.“ GEE: 0va ZOO 5MB 0000—5830 0003 0:00 Amv 00.80608 0033: Ea £9,085 08: 93.5, com 98%: 38 ZOQEL 55 0003305 0.63 £00 33 £038.02: 088.2% 050 :ousbonnmona mad 5%? muaoqfiboo Swanaocoma 5ch Beam E 003% 2093.5 Sand .0 0.33m E25 0.5... 3:5 0:5 on 9. m o o 8‘ ‘ 1 wmaa . . Aw...” Nam... mma new. - ...... in}. O «my... n51 QM“ m 2.5 we: 2:5 95 on 900 . 8882200 ..lmu . mo H. . t . . . . . B H, W. e .3 0. mm m. Ne mic? n WA... U .mtw .3 s. e n - , n. aw H T o N 0 < 42 DON p38 _ + inhibitor Cell Lysate “a | , p38 R8 + M Fraction Figure 7. DON-induced p38 interaction with the ribosome does not require p38 phosphorylation in RAW 264.7 macrophages. Cells were preincubated with p38 kinase inhibitor (2 uM) for 45 min and treated with 250 ng/ml of DON for 30 min. Cells lysates and pooled RS+M were analyzed by Western blotting. Data are representative of two separate experiments. 43 .Bgfitomxo 88800 00:: .«o 3:85.332 03 8an .mch3 E0603 3 gm wed ME Bum—boammoam can :38 Ho.“ 005205 803 2+2 uBoom g .Amod v 3 00am. 00:2 083 0:233 mam Jam 05 M7: 33 00 0036085 0050mm 00338.5: 803 vam 0:0 ME vofibonmmozm 00 035803 00.8th 8000.03 :03: 0302316 238 E0603 :03: 3 350008 00009: a aofiEfieofi vim 05 ME 05 £505 85 “8.53 80 can: end zoo as, Basia 23 230 $5 .m0mmamoboafi Scam BE 5 noun—bonamona 05 8308003 RS809: Mmm Ea MZH 000.63 ZOO .w 0§mE A:_EV0E_._.o 3:): 95... oo cm 3 AEEV 0E_.r on m_. m 0 man: :11: oo on 9 o 0; LI. mvEm. xmwd 3W ... . . . . - I.’ all. N. ...... II! it xmm Va 9 .. I“ N._ 9 xzfia u .i .... ...m 9 O O .II'IIVAIU i... XZw . om Q . . 0 x23 ...N 0 mm 44 DISCUSSION MAPKs function as molecular rheostats in the regulation of DON-induced immune gene expression and apoptosis in leukocytes (Pestka and Smolinski 2005). p38 activation is particularly critical for not only DON-induced expression of proinflammatory cytokines such as interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-u or chemokines such as IL-8 and macrophage inflammatory protein-2 (Islam et al. 2006; Wong et al. 1998), but for DON-induced apoptosis as well (Zhou et al. 2005a). Other translational inhibitors such as anisomycin, ricin and Shiga toxin can also target innate immune system and activate MAPK-driven gene expression and apoptosis (Bunyard et al. 2003; Cherla et al. 2006; Korcheva et al. 2005). Although it has been postulated that ricin and other translational inhibitors induce MAPK activation following cleavage of the 28S rRNA (Iordanov et al. 1997), the underlying mechanisms of ribotoxic stress response remain incompletely understood. The results presented here indicate that, in clonal human monocyte and murine macrophage models, DON rapidly evokes both the mobilization of p38 to the ribosome and its subsequent phosphorylation (Fig. 9). These findings suggest the ribosome or associated proteins might play a scaffolding role during the ribotoxic stress response. DON and other trichothecene target actively dividing cells and are known to associate with 608 ribosomal subunits of ribosomes (Ueno et al. 1969; Wei et al. 1974). Here it was observed that, concurrent with DON uptake, p38 associated first with the 408 subunit fraction and then with fractions containing the 608 subunit and intact monosome. Using irnmunoprecipitation and peptide sequencing, Lin et al. (2003) demonstrated the capacity of p38 to interact with ribosomal proteins, however, 45 / ° Gene. mRNA mRNA Transcriptlon Stabilization Translation phosphorylated Figure 9. Proposed scaffolding role of ribosome in DON-induced ribotoxic stress response. DON-induced ribotoxic stress response in mononuclear phagocytes is pr0posed to involve the following sequential steps: (1) rapid DON uptake and binding to ribosome, (2) interaction of p38 with the ribosome, (3) p38 phosphorylation, and (4) putative induction of p3 8- mediated sequelae including mRNA transcriptioantabilization, protein translation and activation of apoptotic effectors. A similar sequence of events is possible for JNK and ERK. 46 stimuli capable of evoking such an interaction were not identified. As shown here, in the absence of a stimulus, p38 was minimally associated with the ribosomal components, whereas following treatment with DON, this kinase was readily detectable in ribosome fiactions. This is the first report to our knowledge that links a specific stimulus to induction of p38 binding to the ribosome. That observation that nonphosphorylated p38 was maximally associated with ribosomal fractions from 5 to 30 min, whereas the levels of phosphorylated form peaked at 15 min, indicated that p38 might be phosphorylated after binding to the ribosome. The p38 kinase inhibitor, SB203580, inhibits p38 phosphorylation by interacting with ATP binding site of this kinase. Our findings are consistent with those of Frantz et al. (1998) who observed that SB203580 at 0.1 and 1.0Lm suppressed p38 phosphorylation in THP-l (human monocyte) cells that were treated for 20 min with LPS or T'NF-a that are prototypical MAPK activators. These authors provided substantial evidence that pyridinylimidazoles block the p38 kinase biological activity by binding the inactive form of p38 and reducing its rate of activation. It should be noted that 83203580 sometimes yields disparate results. For example, Kong et al. (2008) observed that the inhibitor caused an increase in phosphorylation of p38 that was attributed to inhibition of a regulatory loop. However, that study differed extensively from both ours and that of Frantz et al. (1998). The former used BV2 microglial cells, 2-h SB203580 preincubation and a 20-h incubation with poly IC stimulus. A further issue is that Kong et al. (2008) did not show data for SB203580 alone making it difficult to know if this caused nonspecific stimulation of p38 phosphorylation. We have observed that prolonged (i.e., many hours) incubation 47 with 83203580 alone can actually induce p38 phosphorylation in absence of additional stimulus. The ability of inhibition with SB203580 to prevent DON-induced p38 phosphorylation at the Thr-Gly-Thr activation motif in our study but inability to block p38 association with the ribosome further suggest that p38 phosphorylation per se might not be required for the interaction. Accordingly, DON induced p38 binding to the ribosome appears to be dissociable from its phosphorylation. A critical question arising from these observations relates to the identity of the binding partner(s) for p3 8. Further studies are needed to determine whether p38 interacts with specific ribosomal proteins, rRNA or additional intermediary proteins that bind to ribosome. It is not yet clear how p38 is phosphorylated following interaction with the ribosome. Previous studies have shown that double-stranded RNA (dsRNA)—activated protein kinase (PKR) mediates DON-induced MAPK phosphorylation. PKR is a serine/threonine kinase known to localize with the ribosome (Wu et al. 1998; Zhu et al. 1997). PKR contains a specific dsRNA-binding motif that facilitates its activation (Williams 2001). This kinase is known to interact with apoptosis signaling kinase 1 (ASKl) (Takizawa et al. 2002) and mitogen-activated kinase kinase 6 (MKK6) (Silva et al., 2004), both of which can drive p38 phosphorylation. Recently, PKR has been shown to form a functional complex with p38 that contributes to muscle differentiation of committed myogenic cells (Alisi et al. 2008). PKR—deficient U937 monocytes exhibit reduced induction of p3 8, JNK, and ERK phosphorylation by DON (Zhou et al. 2003c). It is tempting to speculate that DON alters conformation of or damages 28 rRNA in a manner that enables it to bind and activate ribosomal-bound 48 PKR and that this subsequently drives p38 phosphorylation. An alternative explanation for p38 phosphorylation is that, upon binding to the ribosome, the kinase undergoes a conformational change that mediates its activation (Wilson et al. 1996; Zhang et al. 1994). Unphosphorylated MAPKs contain an “activation lip,” which is a loop located near the Thr-X-Tyr motif that might block their activation (Wilson et al. 1996). It might be speculated that DON-induced p38 binding to the ribosome disrupts this inhibitory structure thereby leading to phosphorylation of this kinase. The kinetics of JNK and ERK association with the ribosome and activation in DON-treated murine macrophages remarkably paralleled that observed for p38. All three of the kinase families are lmown to coordinately mediate diverse biological functions (Raingeaud et al. 1995; Wong et al. 1998). Although p38 and JNK are activated in many stress signaling pathways, ERK is typically activated in response to growth hormone and proliferative stimuli. p38 can promote apoptosis and proinflammatory gene expression, whereas JNK and ERK are involved in regulation of both cell survival and death depending on cell types and stimulus. Competing apoptotic and survival signaling pathways are induced by p38 and ERK, respectively, in DON-stimulated RAW 264.7 cells (Zhou et al. 2005a). The demonstration here that the ribosome functions as a scaffold for all three MAPKs suggests a unifying mechanism for coordinating these complex and seemingly disparate activities. MAPKs play significant roles in protein translation. Both ERK and JNK are involved in the initiation of protein synthesis by phosphorylation of eIF2a in human alveolar macrophages (Monick et al. 2006). p38 and ERK modulates translation 49 initiation via eIF-4E phosphorylation, also critical to translation (Sheikh and Fomace 1999). Pharmacologic inhibition of p38 and ERK impairs AKT-dependent cyclin D1 and c-myc mRN A translation by reducing entry into the internal ribosomal entry site (Shi et al. 2005). ERK can modulate translation initiation by binding to and phosphorylating ribosomal protein S3 (Kim et al. 2005). It thus seems reasonable to suggest that DONinduced mobilization of MAPK to ribosomes would impact translation in some manner. Taken together, the results presented herein demonstrate that following rapid uptake of DON by mononuclear phagocytes, p3 8, as well as JNK and ERK associate with the ribosome and are then phosphorylated. We propose that the ribosome functions first as an intracellular receptor for DON and then as scaffold to facilitate MAPK phosphorylation (Fig. 9). Ribosome-associated MAPKs might affect phosphorylation of protein substrates required for mRN A transcription, mRN A stabilization and translation. In the future it will be necessary to verify that ribosome- bound phosphorylated MAPKs are indeed activated. In addition, further study is needed to (1) identify critical ribosomal protein binding partners for p38 and other MAPK, (2) elucidate mechanisms by which these kinases are phosphoryated, (3) determine roles of ribosome-bound MAPKs in downstream events related to mRNA transcription, mRNA stability, protein translation and apoptosis, and (4) ascertain whether other translational inhibitors similarly induce MAPK binding to ribosomes. 50 CHAPTER III ROLE OF PKR AND HCK IN DON-INDUCED p38 ACTIVATION This chapter will be submited in Toxicological Sciences (2009). Bae H.K., Gray J., Li M., Lau A.S., Smithgall T., Kim J., Pestka J.J. Double-stranded activated kinase and Hematopoietic cell kinase associate with the 40S ribosomal subunit and mediate the ribotoxic stress response to deoxynivalenol in mononuclear phagocytes. 51 ABSTRACT The trichothecene deoxynivalenol (DON) binds to ribosomes and triggers a stress response which involves p3 8-driven proinflammatory gene expression and is dependent on both double-stranded RNA-activated protein kinase (PKR) and hematopoietic cell kinase (Hck), a Src family member. The purpose of this research was to characterize critical linkages that exist among these three kinases following stimulation of mononuclear phagocytes with DON. Two PKR inhibitors, 2-AP or C16, and the Hck inhibitor, PP2, suppressed p38 activation and p38-driven IL-8 expression in the U937 human monocyte cell line. U937 cells stably transfected with a PKR antisense vector (U 9K-Al) displayed marked reduction of DON-induced p38 activation and IL- 8 expression as compared to cells translocated with empty vector (U9K-C2), with both responses being completely ablated upon inclusion of PP2. Western analysis of sucrose density ultracentrifuge fractions revealed that PKR and Hck interacted with the 40S subunit in U9K—C2 but not U9K—A1 cells. Subsequent transfection and irnmunoprecipitation studies with HeLa cells indicated that Hck interacted with ribosomal protein S3 (RPSB). Consistent with U937 findings, DON-induced p38 interaction with the ribosome and phosphorylation were suppressed in peritoneal macrophages from PKR lorock-out mice. 2-AP also suppressed DON-induced phosphorylation of ribosome-associated Hck in RAW 264.7 murine macrophages. Furthermore 2-AP and PP2 inhibited DON- induced phosphorylation of p38 and two kinases, ASKl and MKK3/6, which are known to be upstream of p38 in RAW 264.7 cells. Taken together, 408- 52 bound PKR and Hck appear to be critical for DON-induced recruitment of p38 the ribosome, its subsequent phosphorylation, and p3 8-driven proinflammatory cytokine expression. INTRODUCTION Trichothecenes and other translational inhibitors are known to activate mitogen—activated protein kinase (MAPK) signaling pathways that are essential for cell survival and apoptosis via a poorly understood mechanism known as the ribotoxic stress response (Iordanov et al. 1997; Shifrin and Anderson 1999; Yang et al. 2000). Deoxynivalenol (DON) is a common trichothecene produced by F usarium graminearum in wheat, barley and com worldwide that results in contamination of human food (Pestka and Smolinski 2005). Upon binding to the ribosome in mononuclear phagocytes, DON inhibits translation and concurrently induces activation of p3 8, extracellular signal-regulated kinase (ERK) and c-Jun N-terrninal kinase (JNK) (Moon and Pestka 2002; Zhou et al. 2003). Studies with pharmacological inhibitors have shown that p38 plays a particularly critical role in DON-induced proinflammatory gene expression as well as apoptosis (Islam et al. 2006; Moon et al. 2003; Moon and Pestka 2002). Two kinases, double-stranded (ds) RNA-activated protein kinase (PKR) (Zhou et al. 2003) and hematopoietic cell kinase (Hck) (Zhou et al. 2005b), have been reported to be upstream of p38 in the DON-induced ribotoxic stress response. PKR is a serine/theonine protein kinase, induced by dsRNA, interferon, LPS, TNF-a and PACT, an intracellular activator of PKR. (Balachandran and 53 Barber 2007; Sadler and Williams 2007). PKR is present in many cell types and involved in regulation of antiviral responses, protein translation, transcription, and cellular fate. This enzyme has a functional dsRNA binding domain (DRBD) and a catalytic domain. The DRBD interacts with dsRNA, induces autophosphorylation and facilitates PKR association with the ribosome. The catalytic domain mediates phosphorylation of substrates including elongation initiation factor 2a (eIF2a) and regulates translation (Kim et al. 2005; Samuel 1993). The DRBD is believed to exert an autoinhibitory effect on the catalytic domain such that dsRNA binding leads to structural alteration of PKR and its activationWVu and Kaufman 1997). The autoinhibitory region of PKR can also be structurally altered by non-dsRNA stimuli (Iemaire et al. 2006; Vattem et al. 2001). PKR mediates DON-induced MAPK activation in mononuclear phagocytes as well as downstream effects including proinflammatory gene expression or apoptosis(Zhou et al. 2003). Induction of MAPK phosphorylation and apoptosis by DON and the translational inhibitor anisomycin are suppressed by the PKR inhibitor 2-AP in RAW 264.7 murine macrophages. Similarly, U937 human monocytes treated with pharmacologic inhibitors of PKR or those lacking functional PKR exhibit depressed induction of p38 phosphorylation and downstream IL-8 expression by DON and by the ribosome-inactivating proteins ricin and Shiga toxin (Gray et al. 2008; Islam et al. 2006). Lastly, PKR-deficient U937 cells exhibit suppressed p38 phosphorylation and apOptosis in response to DON or anisomycin (Zhou et al. 54 2003) Hck, a member of the Src kinase family, is a non-receptor protein tyrosine kinase expressed primarily in myeloid cells. Hck functions in cytoskeletal rearrangement, phagocytosis, gene transcription, cell proliferation and apoptosis (Guiet et al. 2008; Quintrell et al. 1987). Hck contains a SH2 domain, SH2 linker, SH3 domain and tyrosine kinase domain (Arold et al. 2001; Moarefi et al. 1997; Pellicena et al. 1998). The SH3 domain of Hck plays an important role in protein-protein interaction and interacts with PXXP motifs of several cellular proteins (Gouri and Swamp 1997). Hck activation is regulated by intracellular autoinhibitory interaction between SH2 and SH3 domains (Yadav and Miller 2007). Hck also appears to mediate ribotoxin-induced MAPK activation and related downstream effects (Zhou et al. 2005a). DON-induced MAPK activation and the downstream proinflammatory cytokine expression are suppressed by Src inhibitors PPl and PP2 or by Hck siRNA in RAW 264.7 macrophages. In addition, anisomycin- and emetine-induced p3 8, JNK and ERK phosphorylation are similarly inhibited by PPl. Furthermore, Hck inhibition significantly inhibits DON-induced caspase activation and apoptosis. Recently, we have observed in mononuclear phagocytes that DON induces the recruitment of p38 and other MAPKs to the ribosome and their subsequent phosphorylation (Bae and Pestka 2008). Despite the apparent importance of PKR and Hck in mediating the p38 activation in mononuclear phagocytes, relatively little is known about their relationship to p38 activation, 55 or how the ribosome might facilitate interaction among these three kinases. The purpose of this study was to identify in mononuclear phagocytes the linkages that exist between the ribosome and PKR, Hck and p38 following stimulation with DON. MATERIALS AND METHODS Materials. DON and other chemicals, unless otherwise noted, were purchased from Sigma-Aldrich, Inc. (St. Louis, MO) unless otherwise noted. Inhibitors were obtained from Calbiochem (La J olla, CA). Human- and mouse- specific PKR antibodies, mouse-specific Hck antibodies and phospho-PKR antibodies were supplied by Santa Cruz Biotech (Santa Cruz, CA). Human- specific Hck was purchased fiorn BD (Franklin Lakes, NJ). Human-specific phospho-PKR antibodies were from EMD Chemicals, Inc. (Gibbstown, NJ). Cell culture. All cultures were maintained at 37°C in a humidified 6% C02 incubator. U937 human monocyte cultures (ATCC, Rockville, MD) were grown in RPMI-l640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 ug/ml streptomycin (Gibco BRL, Gaithersburg, MD). Some U937 cultures were stably transfected with a constitutively expressed anti-sense PKR expression plasmid (U 9K-A1) or with an empty plasmid (U9K-C2) as previously described ((Cheung et al. 2005). U9K-Al and U9K-C2 cells were maintained in supplemented RPMI-1640 containing 0.5 mg/ml geneticin (Gibco BRL). RAW 264.7 murine macrophage cultures (TIB 77, ATCC) were cultured 56 in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% (v/v) heat-inactivated F BS, 1mM sodium pyruvate, 100 U/ml penicillin and 100 rig/ml streptomycin (Gibco BRL). Peritoneal macrophage cultures were elicited by i.p. injecting PKR knockout and wild-type control (C57Bl/6J) mice with 1.5 ml of thioglycollate (3%, w/v). PKR knockout mice (Hsu et al. 2004; Scheuner et al. 2003) were obtained from Dr. Randal Kaufman (U. Michigan, Ann Arbor) . After 72 h, macrophages were obtained by peritoneal lavage with cold Hank's buffer (Invitrogen) and collected by centrifirgation at 450 x g for 5 min. Cells were resuspended in supplemented DMEM and cultured overnight prior to experiment initiation. Experimental design. In typical experiments, U937 cells (1 x 105/ml) in 225-cm2 cell culture flasks (Corning, Lowell, MA) and peritoneal macrophages or RAW 264.7 cells (5 x 105/ml) in lOO-mm cell culture dishes (Corning) were cultured overnight, and then treated with DON for various time intervals prior to analysis. DON concentrations for U93 7, peritoneal macrophages and RAW 264.7 cells were 500-1000, 500 and 250 ng/ml respectively, and were based on optimal effects observed in previous studies (Gray et a1. 2008; Gray and Pestka 2007; Islam et al. 2006; Moon and Pestka 2002; Zhou et al. 2003; Zhou et al. 2005b). For some experiments, cells were treated with inhibitors of PKR (5.0 mM 2-AP or 2.5 uM C16), Hck (0.25-25 uM PP2) or p38 (2.0 uM SBZO3580) for l h before DON stimulation. A non-functional chemical analogue of C16, 57 the negative control inhibitor, was used to confirm that changes observed with C16 was due to the inhibition of PKR rather than a non-specific effect. There was no cytotoxicity at the concentrations of inhibitors used, as confirmed by the MTT viability assay. Quantitative real-time PCR. RNaqeous kits (Ambion, Austin, TX) were used to isolate RNA from cell pellets. Briefly, cells were lysed, nucleic acids precipitated with ethanol and RNA trapped in a glass fiber filter. The RNA was eluted and stored at -80°C. Reverse transcription real-time PCR was performed using One-Step PCR Master Mix and IL-8 Taqman Gene Expression Assay (NCBI NM 0005842) (NCBI BZM-NM 0040482) or the B- 2 microglobulin (housekeeping control) Taqman Gene Expression Assay (Applied Biosystems, Foster City, CA). Reaction conditions and PCR program followed manufacturer’s instructions using an ABI 7900HT (384 wells) at the Michigan State University’s Research Teclmology and Support Facility. Fold change was determined using a relative quantitation method (Smolinski and Pestka 2005). Enzyme-linked immunosorbent assay. After treatment, cell cultures were centrifuged for 10 min at 300 x g and the supernatant fraction collected. OptELISA kits (Phanningen, San Diego, CA) were used for IL-8 protein measurement according to manufacturer’s instructions with two modifications. First, the highest standard utilized was 1600 pg/ml, instead of 400 pg/ml. Second, to economize on reagents 50 pl of antibody dilutions and samples were used per well instead of 100 pl. All samples were read at 450 nm in a 58 Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA). Polysome separation. Cytoplasmic fractions were prepared by sucrose density gradient ultracentrifugation (Galban et al. 2003). Briefly, cells were washed with ice-cold PBS twice and lysed in ice-cold polysome extraction buffer (PEB) (0.3 M NaCl, 15 mM MgC12, 15 mM Tris-HCl [pH 7.6], 1% [w/v] Triton X-100, 0.1 mg/ml cyclohexirnide and 1 mg/ml heparin, 0.01% [v/v] and phosphatase inhibitors A and B [Santa Cruz Biotech, Santa Cruz, CA]. Cell lysate was centrifirged at 10,000 x g for 15 min to clear the resultant supernatant of nuclei, mitochondria and debris. Protein was measured using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Lysate (1 mg) was layered over 9 ml linear sucrose gradient solution (10-50 %) in a 11.5 ml Sorvall centrifuge tube and centrifuged at 35,000 x g for 3 h at 4°C in Sorvall TH-641 rotor. The gradient was fiactionated at a rate of 0.5 to 1.0 ml per min by upward displacement using an ISCO system (Teledyne ISCO, Lincoln, NE) comprised of a syringe pump, EM-l UV monitor for continuous measurement of absorbance at 254 nm and a fraction collector. Western analysis. Proteins were separated on 4% (w/v)polyacry1amide gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). After incubating with blocking buffer (Li-Cor, Lincoln, NE), membranes were incubated with murine antibodies or rabbit antibodies to immobilized proteins of interest overnight at 4°C. After washing, blots were incubated with secondary IRDye 680 goat anti-rabbit and/or IRDye 800CW goat anti-mouse IgG antibodies (Li-Cor) for 1 h at 25°C. Infiared 59 fluorescence of bound secondary antibodies containing the two dyes were simultaneously measured using a Li-Cor Odyssey Infrared Imaging System, and relative phosphorylation determined using Odyssey Analysis Software. Ribosomal fractions were identified with mouse and rabbit antibodies to ribosomal protein S6 (RPS6) (Cell Signaling Technology) and ribosomal protein L7 (RPL7) (Bethyl Laboratories, Inc., Montgomery, TX), respectively, followed by IRDye 680 goat anti-species conjugates (Bae and Pestka 2008). Plasmids, transfection and immunoprecipitation. Plasmids containing DNAs for full length human ribosomal protein S3 (RPS3) in-frame with a sequence coding for EGFP (pEGFPcl, Clontech) or human Hck were prepared as previously described (Kim et al. 2005; Trible et al. 2006) and reconstructed in pTRE-Tight Expression Plasmids. HeLa cells (Tet-Off, Clontech) were maintained in DMEM (Sigma) supplemented with 10% (v/v) PBS, 4 mM L-glutamine, 100 ug/ml G418, and 100 U/ml penicillin and 100 rig/ml streptomycin (Gibco BRL). Cells were tansiently transfected with the plasmids using Lipofectamine (Invitrogen), cultured for an additional 16 h. and then lysed in 1 ml non-denaturing lysis buffer with l rig/ml protease inhibitors (pepstatin, aprotinin, leupeptin) (Cell Signaling). After preclearing with mouse IgG and protein-A coupled sepharose beads for 10 min at 25°C, cell lysates were immunoprecipitated with anti-GFP mAb (Abcam, Cambridge, MA) or anti-Hck and then analyzed by Western analysis with Hck and RPS3 antibodies. Statistics. Data were analyzed with SigmaStat v 3.1 (Jandel Scientific, San Rafael, CA). IL-8 protein and RNA data were analyzed using one-way 60 analysis of variance with Student-Newman-Keuls Method for pairwise comparisons unless otherwise noted. p < 0.05 was considered significant. RESULTS U937 cells treated with DON expressed elevated levels of IL-8 mRNA and protein (Fig. 10). Pretreatment with 2-AP (5 mM) suppressed (~95%) DON-induced IL-8 mRNA (Fig. 10A) and protein (Fig. 10B) expression. Pretreatment with a second PKR inhibitor, C16 (2.5 uM), had similar effects (Fig 10C, D). Use of a non-functional chemical analogue of C16, the negative control inhibitor, confirmed that the effect observed with C16 was due to the inhibition of PKR rather than a non-specific chemical effect. To ascertain if Hck also played a role in DON-induced IL-8 mRNA expression, U937 cells were treated with PP2, a Src family inhibitor. DON-induced IL-8 mRNA was significantly inhibited by pretreatment of PP2 (2.5 uM) (Fig. 11). DON-induced IL-8 protein expression was suppressed in U937 cells co- treated with the p38 inhibitor SB203580 (Fig. 12). Pretreatment with either PKR (2-AP) (Fig. 13A) or Hck (PP2) (Fig. 13B) inhibitors markedly suppressed phosphorylation of p38 induced by DON. PKR inhibitor studies were confirmed using U937 cells stably transfected with either an expression plasmid constitutively expressing antisense PKR (U9K-A1) or an empty expression plasmid (U9K-C2). U9K-Al cells exhibited reduced DON-induced p38 phosphorylation as compared to the U9K-C2 control cells (Fig. 14A). Pretreatment with PP2 (0.25-25 uM) ablated 61 on a) ‘: -A~ .flaoEtonxo afiwgmovfi 685 we o>u3=omeaom .Amuev Sam H 568 0.8 Sea .983me ZOQ n c a Hot“ Mom czar—«8 .3 3.5308 mm? nommmoaxo SAME mam a .5 .” Im>l mr u r C l l o a a m < m... .8 .8 W ..I 8 OFUIrCV Ql Im>l W oo 62 30 < —VEH E PP2 E 20 - Q d o g. 1o~ E d) O! 0 DON - + Figure 11. Hck inhibition suppresses DON-induced IL-8 mRNA expression in U937 cells. Cells were pretreated with PP2 (2.5 1.1M) in DMSO or vehicle for 45 min before addition of 0 or 1000 ng/ml DON. IL-8 mRNA expression was measured by real-time PCR after a 6 h DON exposru'e. Data are mean :1: SEM (n=3). Representative of three independent experiments. 63 — VEH 2 ~ 83203580 E U) 5 09 1 ~ =' 0 Figure 12. p38 inhibitor blocks DON-induced IL-8 protein expression in U937 cells. Cells were co-treated with SBZO3580 (2.0 uM) in DMSO or vehicle and 0 or 1000 ng/ml DON. Culture supernatant was collected after 3 h, and IL-8 protein was assessed by ELISA. Data are mean :l: SEM (n=3). Representative of three independent experiments. 64 ADON - - + + 2AP - + - + p-p38 U m- DON - - + + PP2 - + - + p-p38 Figure 13. PKR and Hck inhibition suppresses DON-induced p38 phosphorylation in U937 cells. Cells were incubated for 45 min with (A) 2-AP (5.0 mM) or water vehicle or with (3) PP2 (0 or 2.5 uM) in DMSO or with vehicle before treating with 0 or 500 ng/ml DON for 15 min. Protein in cell lysate was analyzed by Western blotting for p38 and phospho-p3 8. Data are mean :1: SEM (n=3). Representative of three independent experiments. 65 both basal and residual DON-induced p38 phosphorylation in U9K—C2 and U9K-Al cells, respectively. Similarly, U9K-Al cells had significantly reduced levels of DON-induced IL-8 protein, as compared to the U9K—C2 control cells (Fig. 143). Levels of IL-8 protein, induced by DON, decreased further in U9K- Al cells upon treatment with PP2 (0.25-2.5 uM). This inhibitor also blocked basal DON-induced IL-8 production in U9K—C2 cells in a concentration- dependent manner. DON has previously been shown to induce p38 mobilization to the 40S subunit where it is phosphorylated (Bae et al. 2008). While PKR is known to associate with the 40S ribosomal subunit (Zhu et al. 1997), the capacity of Hck to interact with specific ribosomal subunits is unknown. Cell lysates from U937 cells treated with DON or vehicle for 1 min were therefore fractionated on a sucrose density gradient and fractions subjected to Western blotting. Fractions containing small (40S) and large (60S) ribosomal subunits were confirmed using antibodies against ribosomal proteins (RP) S6 and L7, respectively (Fig. 15). PKR and Hck were constitutively found in the 40S ribosomal fraction, and both were phosphorylated within 1 min after DON treatment. Comparatively little PKR or Hck was detectable in the 60S ribosomal unit fractions. U9K-C2 control and U9K-Al PKR antisense-containing cells were used to determine whether PKR affected Hck interaction with the ribosome. While Hck was found in lysates of both cell lines, PKR was only detectable lysates of U9K—C2 (Fig. 16A). Following separation on a sucrose gradient, 66 ADON --++++ PP2(IJM) - - - 0.25 2.5 25 U9K-CZ M p-p38 U9K-A1 ...... v p-p38 B — U9K-CZ b U9K-A1 800‘ E E “P 400 - :‘ oi . _ . DON - + + + + PP2 (uM) o o 0.25 2.5 25 Figure 14. PKR antisense expression and Hck inhibition suppress DON-induced p38 phosphorylation and IL-8 production in U937 cells. U937 cells expressing control (U 9K-C2) or PKR antisense vector (U 9K-Al) were pretreated with PP2 (0.25-25 MIT) in DMSO or vehicle for 45 min before treating with 500 ng/ml DON. (A) Cells were lysed with SDS after 30 min DON treatment protein analyzed by Western blotting for phospho-p38. (3) Cultm‘e supernatant was collected after a 6 h DON treatment, and IL-8 protein was assessed by ELISA. Data are mean :1: SEM (n=3). Representative of three independent experiments. 67 608 608 408 40s 30$ Os .. p-PKR - .. -- PKR "“ - p-Hck ‘ a: Hck 0.9- -'-- .. -— '-— RPS6 "'9‘" "‘ ‘- ...- RPL7 DON - + Figure 15. DON induces phosphorylation of ribosome-associated PKR and Hck. U937 cells were treated with vehicle of DON (500 ng/ml) for 1 min and subjected to sucrose density gradient fiactionation. Fractions were analyzed by Western blotting. Data are representative of three separate experiments. 68 PKR and Hck co-migrated with the 40S ribosomal fraction in U9K—C2 control cells (Fig. 163) but were nearly undetectable in the U9K-Al 40S fraction (Fig. 16C). The PXXP motif is critical for interaction of proteins with Src family kinases such as Hck. Bioinforrnatic analysis was used to determine numbers and location of PXXP motifs contained in over 70 known ribosomal proteins (supplementary data). Of these, ribosomal protein S3 (RPS3) was found to contain the largest number PXXP motifs (5) all of which were located in close proximity to one another, suggesting it to be a strong candidate for mediating Hck interaction with the 40S subunit. To ascertain whether RPS3 could directly interact with Hck, green fluorescent protein (GFP)-labeled RPS3 and Hck were expressed in HeLa cells (which do not express native Hck) (Fig. 17). Direct interaction of RPS3 and Hck was suggested upon irnmunoblotting of anti-GFP or anti-Hck immunoprecipitates with anti-RPS3 or anti-Hck. Peritoneal macrOphages from wild-type (WT) and PKR knock-out (KO) mice were treated with DON and relative p38 phosphorylation compared in cytoplasmic fractions from the two cultures (Fig. 18). Macrophages from PKR KO mice exhibited less p38 phosphorylation flran did macrophages from WT mice. p38 association with the pooled ribosomal subunits and monosome (RS+M fraction) was increased in peritoneal macrophages from WT mice following DON treatment, whereas p38 interaction with the ribosome was reduced in macrophages from PKR KO mice. Similarly, DON-induced p38 phosphorylation increased in the pooled RS+M fraction of macrophages from 69 A254 A U9K-c2 U9K-A1 5 —> ————§ % Sucrose 35 5 % Sucrose 35 §"""‘“ ‘ PKR PKR i'mw“ Hck Hck §-""""‘““" apss g...—--—--- ""’ RPse .....--._.__ RPL7 -..-...... - , RPL7 Figure 16. PKR is required for Hck interaction with the ribosome in U937 cells. (A) U9K-C2 control and U9K-Al PKR-antisense expressing were lysed with PEB and cytoplasmic fi‘actions were collected afier centrifugation at 10,000 x g for 15 min. Protein was analde by Western blotting with PKR and Hck antibodies. (3) U9K- C2 or (C) U9K-A1 cells were treated with 0 or 500 ng/ml DON for 1 min. Ribosomal fiactions were separated by sucrose gradient and protein was analyzed by Western blotting. Data are representative of three separate experiments. 70 Ribosomal protein 82 PXXP motifs 89 S10 $13 $20 826 L3 L4 L7 . 2 5f 1 1 1 1 2 1 2 2 4 1 1 1 1 2 1 1 Table 1. Potential binding sites of ribosomal proteins to HCK. PXXP sites were examined using Ribosome Protein Data Base (http://ribosomemedmiyazaki- u.ac.jp/). The numbers of PXXP binding sites are presented. 71 RPS3-GF P - + - + Hck - - + + "l-'- ...—...... . RPS3 Lysate -..—— Hck IP : GFP ~~~ «an-‘3 Hck III-'- I.” Hck IP I HCk ' - ...—..- “ GFP Figure 17. Hck interacts with the ribosomal protein S3 (RPS3). RPS3-GFP and Hck were overexpressed in Hela cells and lysed with non-denaturating buffer. Lysate and anti-GFP immunoprecipitate were analyzed by Western blotting with antibodies specific for Hck or RPS3. Results are representative of two separate experiments. 72 A. Cytoplasm MH' KO B. RS+M M" ’ .PKR swarwncr m '-~:. all-V1.2 .~ . . . .- Wuw- ‘tfiw" Q ~ Figure 18. DON-induced MAPK interaction with the ribosome is suppressed in the peritoneal macrophages fiom PKR-deficient mice. Peritoneal macrophages from wild-type (WT) and PKR knockout (KO) mice were cultured with O or 500 ng/ml DON for 15 min (A) Cells were lysed with PEB lysis buffer and analyzed by Western blotting. (3) Ribosomal proteins were separated using a sucrose density gradient fractionation (SDGF). Pooled RS + M fractions were analyzed by Western blotting. Data are representative of three separate experiments. 73 WT but not in those from PKR KO mice. The role of PKR in phosphorylation of ribosome-associated Hck was further confirmed in RAW 264.7 macrophages that were pretreated with either PKR or Hck inhibitor and then stimulated with DON. Following sucrose density gradient separation, and the pooled RS+M fraction was subjected to Western blotting. Phosphorylated Hck was not detectable in pooled RS+M fractions from RAW 264.7 cells treated with 2-AP or PP2 (Fig. 19). RPL7 again confirmed the presence of equal amounts of sample in each lane for the immunoblot. Phosphorylated PKR and Hck were detected in the pooled RS+M fraction within 5 min of DON treatment in RAW 264.7 cells, but disappeared within 15 min (Fig. 20). p38 associated with the RS+M fractions at 5, 15 and 30 rrrin afier DON exposure times. Protein loading in the RS+M combined fractions was again confirmed using RPL7 antibody. Apoptosis signal-regulating kinase 1 (ASKl) and mitogen-activated protein kinase 3 and 6 (MKK3/6) are kinases widely recognized to be upstream of p38. DON was found to induce ASKl and MKK3/6 phosphorylation in RAW 264.7 cells (Fig. 21A,3). Both the PKR inhibitor 2—AP (Fig. 21A) and the Hck inhibitor, PP2 (Fig. 213) suppressed DON-induced ASKl and MKK3/6 phosphorylation. ASKl and MKK 3/6 might thus serve as signaling cascade components that link PKR and/or Hck with p3 8. 74 - + + + DON - - + - PP2 - - - + 2AP - ~- - p-Hck v—I- RPL7 Figure 19. PKR inhibition suppresses DON-induced Hck phosphorylation in ribosomal fractions of RAW 264.7 cells. Cells were treated with PKR inhibitor, 2AP (5 mM) or HCK inhibitor, PP2 (2.5 uM) for 45 min before adding 250 ng/ml DON for 5 min. Ribosomes were fractionated on sucrose gradient, and the protein in RS+M was analyzed by Western blotting. Data are representative of three separate experiments. 75 min 0 5 15 30 ...... ... p-PKR 3:3; '1 * ' “F p-Hck _ ..-—~~ p38 ____.......... w _— RPL7 Figure 20. PKR and Hck phosphorylation precedes p38 interaction with the ribosome in RAW 264.7 cells. Cells were treated with 0 or 250 ng/ml DON for 0, 5, 15 or 30 min. Ribosomes were fiactionated on sucrose gradient, and the protein in RS+M was analyzed by Western blotting with antibodies against phospho-PKR, phospho-Hck, p38 or RPL7. Data are representative of three separate experiments. 76 DON - - - - + + + + 2-Ap - - + + - - + + -..... -- .. .... .— .. -. p-ASK1(967) -—- p-P38 DON - - + + PP2 - + - + ------ 3 --—-~ ---~— p-ASK1(967) ----3 p-MKK3/6 .- 3- p-p38 B --...._...—----— p38 Figure 21. PKR and Hck inhibition suppresses DON-induced phosphorylation of ASK1, MKK3/6 and p38 in RAW 264.7 cells. Cells were incubated for 45 min with (A) ZAP (5 mM) in DMSO or vehicle or with (3) PP2 (2.5 uM) or DMSO prior to treating with 0 or 250 ng/ml DON for 15 min. Cells were washed with cold PBS and lysed with hot SDS buffer then centrifuged at 10,000 x g for 15 min. Cell lysate was analyzed using Western blotting with specific antibodies. Data are representative of three separate experiments 77 DISCUSSION While PKR is known to bind to the ribosome, this is the first report to demonstrating that Hck similarly associates with the 40S ribosomal subunit in a PKR-dependent fashion. The results presented here further suggest that the 408 ribosomal subunit serves as a scaffold for Hck and PKR. Notably, both of the latter proteins appeared to be required for DON-induced p38 activation and p3 8-driven expression of proinflammatory genes such as IL-8. Furthermore, “ upon DON treatment, PKR and Hck were rapidly and transiently phosphorylated while in association with the ribosome, which was consistent with previous findings in whole cell lysates in RAW 264.7 and U937 cells (Zhou et al. 2005b; Zhou et al. 2003c). These mechanisms associated with the proposed scaffolding role of the ribosome are summarized in Fig. 22. The co-localization of PKR and Hck to the 40S subunit suggests that these kinases might associate in close proximity. This possibility is supported by the observation that Hck was not detectable in the pooled ribosomal fraction of U9K-Al cells even though cytoplasmic Hck expression per se was unaffected by PKR deficiency. The observation that the PKR inhibitor 2-AP suppressed Hck phosphorylation in the ribosome in RAW 264.7 cells, suggests that PKR activation might be required for Hck phosphorylation. It is of interest to understand how PKR and Hck associate with the ribosome. PKR is primarily localized in the 40S ribosome when human PKR is overexpressed in yeast, whereas, a DRBD PKR deletion mutant fail to interact with the ribosome (Zhu et al. 1997). This suggests that PKR might interact with the ribosome via a 78 DON DON binds n'bosome PKR and Hck phosphorylated p38 binds ribosome p38 is phosphorylated Gene Expression Figure 22. Ribosome frmctions as scaffold for PKR, Hck and p38 in DON-induced ribotoxic stress response. DON-induced ribotoxic is proposed to involve: (1) rapid DON uptake and binding to ribosome, (2)activation of ribosomal-associated PKR and Hck (3) interaction of p38 with the ribosome, (4) p38 phosphorylation and (5) induction of proinflammatory genes such as IL-8. 79 double stranded RNA binding domain (DRBD). In addition, several ribosomal proteins have been suggested to be binding partners for PKR. While association of Hck or other Srcs with the ribosome has not been described, the SH3 domain of Hck is known to be important for interaction with several cellular proteins containing PXXP binding motifs (Gouri and Swarup 1997). For example, Bruton’s tyrosine kinase (Btk), ORF3 protein of hepatitis E virus (HEV), and Nef protein of HIV interact with SH3 domains of Hck. (Cheng et al. 1994; Collette et al. 2000; Korkaya et a1. 2001; Stangler et al. 2007). Prototypical Hck binding sites contain proline- rich regions with PXXP motifs being particularly critical (Stangler et al. 2007). Upon characterizing potential binding sites of Hck to over 70 ribosomal proteins, we found that RPS3 to contain 5 PXXP motifs, all of which were localized in proline-rich region thus enhancing its potential to bind to the SH3 domain. The capacity for Hck to interact with RPS3 was strongly suggested following overexpression of these proteins in HeLa cells and immunoprecipitation with specific antibodies to these proteins. It is particularly notable that the RPS3 interacts with well-known PKR substrate eIF2a which regulates translation initiation (Samuel 1993). It might thus be speculated that PKR and Hck bind to the 40S ribosomal subunit in close proximity. We have previously demonstrated that p38 initially interacts with the the 408 ribosomal subunit in DON-stimulated macrophages (Bae and Pestka 2008). Consistent with the observation that peritoneal macrophages from PKR KO mice exhibit impaired p38 association with ribosome following DON 80 treatment, and that overall p38 phosphorylation was reduced in cell lysates from these cells. This suggests that PKR is critical for p38 ribosomal mobilization and phosphorylation in DON-induced macrophages. These findings correlated with the observation that PKR and Hck are transiently activated in the 408 ribosomal subunit and that both concurrent and subsequent p38 association was detectable in this fraction. Overall, these data suggest that the 408 ribosomal subunit might serve as a scaffold for signal transduction during the DON-induced ribotoxic stress response thus facilitating activation of p38 by PKR and Hck. A critical question arising from this work relates to the nature of the signaling cascade that links PKR/Hck and p3 8. PKR is known to interact with ASKl, a member of the MAPK kinase family, which can activate p38 via MKK3/6. Co-immunoprecipitation studies have demonstrated that PKR interacts with ASKl (Takizawa et al. 2002). Dominant negative PKR- expressing COS-1 monkey kidney fibroblast cells exhibit suppressed ASKI- induced p38 activation and apoptosis (Matsukawa et al. 2004; Takizawa et al. 2002). As shown here, DON-induced ASKl and MKK3/6 phosphorylation in RAW 264.7 cells was suppressed by the PKR inhibitor, 2AP. Analogous to the observations for PKR, inhibition of Hck found here was found to suppress ASKl and MKK3/6 phosphophorylation. Thus, PKR and Hck appear to share a signaling cascade involving ASK] and MKK3/6 that mediates p38 activation . Further studies are needed to determine if whether ASK] and MKK3/6 also interact with the ribosome. 81 DON l PKR l Hck l ASK1 l MKK3/6 p38 Figure 23. Putative role of ASK1 and MKK3/6 —in DON-induced p38 activation. It is not clear how PKR activation is triggered upon DON binding to the ribosome. Recently we observed that both DON and another trichothecene satratoxin G associate with both 40S and 60S ribosomal subunits (Bae et al, submitted). The peptidyl transferase ring has been suggested to be a critical and induction of binding site for toxins ribotoxic stress response (Foster and Tesh 2002; Iordanov et al. 1997). Ribotoxins, such as trichothecenes, anisomycin, ricin, Shiga toxin, promote damage to the peptidyl transferase ring in the 60S ribosomal subunit thus in playing a role for this site. However, in the present studies PKR, Hck and p38 localize to the 40S ribosomal subunit. Further studies should focus on determining how DON binding to the 40S ribosomal subunit induces PKR and Hck activation via the ribosomal interaction. 83 CHAPTER IV TRICHOTHECENES TARGET 408 AND 608 RIBOSOMAL SUBUNITS This chapter was published in Toxicology Applied Pharmacology. (2009). Bae H.K., Shinozuka J, Islam Z, Pestka J .J . Satratoxin G interaction with 40 S and 60 S ribosomal subunits precedes apoptosis in the macrophage. Toxicol Appl Pharmacol. 2009 Mar 20. [Epub ahead of print] PMID: 19306889 84 ABSTRACT Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially irnmunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultant fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min. and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model, which is similarly susceptible to ap0ptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis. INTRODUCTION 85 The trichothecene mycotoxins, toxic sesquiterpenoids produced by molds, are encountered in environment and food and are therefore of public health concern (Pestka 2008; Pestka et al. 2008). While trichothecenes share a common structure of a 12, 13 epoxide ring and a 9, 10 double bond, their toxicity varies widely based on differing R groups (Bamburg 1983). Three major types of trichothecene have been characterized. The most toxic of these are the macrocyclic trichothecenes (eg. satratoxins and roridins) (Pestka and Forsell 1988), which possess a cyclic diester or triester ring at C4 and C5 (Grove 1993) as compared to the less toxic Type A and Type 3 groups which contain simpler acyl substituents (Grove 1988; Grove 2000). Satratoxin G (SG) is a macrocyclic trichothecene that is produced by the black mold Stachybotrys chartarum, a fungus that often grows on cellulosic building materials in water-damaged buildings. Both S. chartarum and its mycotoxins have been suggested to contribute to a spectrum of adverse immune, respiratory and nervous system effects often referred to as damp-building related illness (Pestka et al. 2008). SG and other trichothecenes cause activation of the mitogen-activated protein kinases (MAPKs) p3 8, JNK and ERK as well as apoptosis in mononuclear phagocytes (Yang et al. 2000), which could potentially contribute to immunotoxicity. Recently, intranasal instillation of SG has been found to specifically induce apoptosis in olfactory neuronal cells, supporting a possible role in neurotoxicity within the respiratory tracts (Islam et al. 2006; Islam et al. 2007). Trichothecenes are widely considered to exert their toxicity by binding to the peptidyl transferase center of the 60S ribosomal subunit (Bamburg 1983; Middlebrook and Leatherman 1989a; Middlebrook and Leatherman 1989b). One 86 outcome of this binding is translational arrest, which can be differentially classified as initiation inhibition (I-type), elongation inhibition (E-type) and termination inhibition (T-type) (Iordanov et al. 1997). In I-type, there is a rapid conversion of polysomes into monosomes, whereas polysomes are stable in both the E- and T-type. The inhibition class can vary depending on toxin structure, concentration and exposure time. The ribosome also appears to play a pivotal role in the orchestration of trichothecene-induced toxic responses ranging from elevated gene expression to apoptosis. For example, induction of proinflammatory gene expression and apoptosis by the Type 3 trichothecene deoxynivalenol (DON) is mediated by mitogen-activated protein kinases (MAPK) (Pestka 2008). Recently, we have demonstrated that DON induces mobilization of p3 8, JNK and ERK to the ribosome and their subsequent phosphorylation (Bae and Pestka 2008). Interestingly, these effects correlate with cleavage of 18S and 28S rRNA at the peptidyl transferase center of the 60S ribosomal subunit (Li and Pestka 2008). Many critical questions remain relative to the interactions of trichothecenes with the ribosome and the ensuing ribotoxic stress response. First, the relationship between ribosomal binding and manifestations of cellular toxicity are not fully understood. Second, despite the potential importance of SG and other macrocyclic trichothecenes as etiologic agents in damp building-related illness, their capacities to interact with the ribosome have not been well-characterized. Third, SG has also been reported to covalently bind to proteins in vitro and in vivo (Yike et al. 2006), raising the possibility that this and other macrocyclic trichothecenes might form adducts with 87 the ribosome. Finally, while some trichothecenes have been shown to bind to the 60S ribosomal subunit under cell-free conditions, their potential to bind to the 40S subunit have not been fully addressed. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with the binding of this toxin to the ribosome. A major limitation to studying interaction of SG with the ribosome is the lack of either commercially available radiolabeled macrocyclic trichothecenes or suitable methods to carry out such labeling. Our laboratory has developed a specific antibody to SG and applied it to a highly sensitive ELISA (Chung et al. 2003a). This assay was used here to characterize the interaction of SG with the ribosome in the macrophage relative to ldnetic s, concentration-dependence, specificity, interacting subunits, and reversibility; these findings were further related to MAPK activation and apoptosis induction. MATERIALS AND METHODS Chemicals. SG (kindly provided by B. Jarvis, University of Maryland, College Park, MD) was purified from S. chartarum cultures as previously described (Hinkley and Jarvis 2001) and identity confirmed by electrospray ionization/collision-induced dissociation tandem mass spectroscopy (Tuomi et al. 1998). Purified SG was diluted in Tris-HCl buffer (pH 7.0) and the relative absorbance scanned over 220 to 340 nM using a NanoDrop spectrophotometer ND-1000 (Therrno Scientific, DE). DON and other chemicals were purchased from Sigma Chemical Co. (St Louis, MO) unless otherwise noted. 88 Cell cultures. RAW 264.7 cells American Type Culture Collection [ATCC] Manassas, VA) were cultured at 37°C in a humidified 6% C02 incubator in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% (vol/vol) heat- inactivated FBS, 100 U/ml penicillin (Sigma), and 100 ug/ml streptomycin (Sigma) (Zhou et al. 2005a). In a typical experiment, RAW 264.7 cells (5 x 105/ml) were cultured overnight to achieve 80% confluence in 100-mm cell culture dishes (Corning, NY), and then treated with SG at various concentrations and time intervals prior to analysis. PC-12 neuronal cells (ATCC) were grown to approximately 80% confluency on collagen-coated 6—we11 plates (BD Biosciences Phanningen, San Diego, CA) containing 2 ml of F-12K medium (ATCC) supplemented with 2.5% (v/v) fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 15% (v/v) horse serum (Atlanta Biologicals), 100 U/ml penicillin and 100 ug/ml streptomycin (Gibco-BRL, Rockville, MD)(Islam et al. 2008). DNA fragmentation ELISA. DNA fragmentation was measured with a Cell Death Detection ELISA (Boehringer Mannheim GmbH, Germany). Briefly, RAW 264.7 cells (1 x 105/ml) in a 12 well cell culture plate were cultured overnight, and then treated with SG at 10, 20, 40 and 80 ng/ml. After 1, 2, 4 and 6 h, cells were lysed and lysates were incubated in anti-histone-coated microtiter plate wells. Wells were washed and incubated with anti-DNA-peroxidase conjugates and unbound peroxidase conjugate removed by washing. Bound peroxidase retained in the irnmunocomplex was determined spectrophotometrically following addition of ABTS (2, 2’-azino-di- [3 -ethylbenzthiazoline sulfonate]) substrate. Relative increases in mono- and oligo- 89 nucleosomes in cytoplasm indicative of DNA fragmentation were determined by measuring absorbance at 405 nm on a Vmax Microplate Reader (Molecular Devices, Menlo Park, CA). Confirmation of DNA fragmentation by agarose gel electrophoresis. RAW 264.7 cells (2 x 105/ml) were incubated overnight in a 6 well cell culture plate, and then cultured with SG for 4 and 6 h. Cultures were centrifirged for 10 min (200 X g) at 4°C and the pellet suspended in 0.1 ml cell lysing buffer (10 mM Tris, pH 7.4, 10 mM EDTA, pH 8.0, 0.5% [v/v] Triton X-100)(Islam et al. 2002). Cells were incubated for 10 min at 4°C and the resultant lysate was centrifiiged for 30 min (12,000 x g) at 4°C. The supernatant, which contained fragmented DNA, was digested for 1 h at 37°C with 0.4 ug/ml of RNase A (Roche, Indianapolis, IN) and then incubated l h at 37°C with 0.4 rig/ml of proteinase K (Roche, Indianapolis, IN). DNA was precipitated with 50% (v/v) isopropanol in 0.5 M NaCl at 4°C overnight. The precipitate was centrifuged at 12,000 x g for 30 min at 4°C. The resultant pellet was air dried and resuspended in 20 pl of 10 mM Tris (pH 7.4), 1 mM EDTA (pH 8.0). DNA samples (10 Ill/well) were done electrophoresis at 50 V for about 3 h in 2% (w/v) agarose gel in 90 mM Tris— borate buffer containing 2 mM EDTA (pH 8.0). After electrophoresis, the gel was stained with ethidium bromide (0.5 ug/ml), and the nucleic acids were visualized with a UV transilluminator. A 100-bp DNA ladder (GIBCO-BRL, Rockville, MD) was used for molecular sizing. Sucrose density gradient fi'actionation. To prepare cytoplasmic extract for ribosomal fractionation, cells were washed with ice-cold PBS twice and lysed in ice- cold polysome extraction buffer(PEB) (0.3 M NaCl, 15 mM MgC12, 15 mM Tris-HCl 90 [pH 7.6], 1% (w/v) Triton X-100, 0.1 mg/ml cycloheximide and 1 mg/ml heparin) (Galban et al. 2003). This was centrifuged at 10,000 x g for 15 min to clear the resultant supernatant of nuclei, mitochondria and debris. Protein was measured using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Lysate protein (1 mg) was layered over 9 ml linear sucrose gradient solution (IO-50%) in a 11.5 ml Sorvall centrifuge tube and centrifuged at 35,000 x g for 3 h at 4°C in Sorvall TH-641 rotor. The gradient was fractioned at a rate of 0.25 to 1 ml per min by upward displacement using an ISCO system consisting of a fraction collector, a needle-piercing device and a syringe pump connected to an EM-l UV monitor for continuous measurement of the absorbance at 254 nm (Teledyne ISCO, Lincoln, NE). SG ELISA. SG was quantified by a competitive ELISA (Chung et al. 2003a). SG polyclonal antibodies (100 pl) diluted (0.5 pg/ml) in phosphate buffered saline (PBS) (pH 7.2, 10 mM) were incubated in 96-well ELISA plates (Corning) overnight at 4°C. Plates were aspirated, blocked with 300 pl of 3% (w/v) non-fat dried milk in PBS (NFDM-PBS), covered with parafihn and then incubated 60 min at 37°C. After washing four times with PBS containing 0.05 %(v/v) Tween 20 (PBS-Tween), 50 pl of standard or samples with 50 pl SG-horseradish peroxidase conjugate diluted (0.5 pg/ml) in NFDM-PBS at RT for 60 min. Plate was washed seven times with PBS- Tween, bound peroxidase was determined after incubation for 30 min at 25°C with 100 pl/well of K-Blue Substrate (Neogen, Lansing). The reaction was terminated with 100 pl/well of 6 N sulfuric acid stopping reagent and the plate at 450 nm by Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA). Western analysis. Proteins were separated on 4% (w/v) polyacrylamide gels 91 and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). After incubating with blocking buffer (Li-Cor, Lincoln, NE), membranes containing irrunobilized proteins were incubated with murine antibodies to phosphorylated p38 and JNK concurrently with corresponding rabbit antibodies to nonphosphorylated p38 or JNK (Cell Signaling Technology, Danvers, MA) overnight at 4°C. After washing, blots were incubated with IRDye 680 goat anti-rabbit IgG conjugates and IRDye 800CW goat anti-mouse IgG conjugates (Li-Cor) for 1 hr at 25°C. Infrared fluorescence of bound secondary antibodies containing the two dyes was simultaneously measured using a Li-Cor Odyssey Infrared Imaging System, and relative phosphorylation determined using Odyssey Analysis Software. Ribosomal fractions were identified with mouse and rabbit antibodies to ribosomal protein S6 (RPS6) (Cell Signaling Technology) and ribosomal protein L7 (RPL7) (Bethyl Laboratories, Inc., Montgomery, TX), respectively, followed by IRDye 680 goat anti- species conjugates (Bae and Pestka 2008). Statistics Data were analyzed by Student’s t-test or AN OVA with SigmaStat v 3.1 (Jandel Scientific, San Rafael, CA) with the criterion for significance set at p < 0.05. RESULTS The effect of SG concentration on apoptosis induction in RAW 264.7 cells was determined by measuring DNA fragmentation with a specific ELISA. SG- induced apoptosis was evident after 2 h at the two highest concentrations (40 and 80 ng/ml) and after 4 h at all concentrations (10-80 ng/ml) (Fig. 24A). Concentration- 92 dependent fragmentation was confirmed by gel electrophoresis (Figure 243). To relate apoptosis to ribosomal binding, RAW 264.7 cells were incubated with SG at 0, 20, 40 and 80 ng/ml for l h and binding of the toxin to ribosomal fractions was assessed. Since SG absorbs at 254 nm (Fig. 25), its binding to the ribosomes could be qualitatively followed by monitoring changes in absorbance in eluted sucrose density gradient fractions. Both the 40S and 60S absorbance values increased as SG in incubation mixture was elevated whereas the polysome level decreased (Fig. 26A). The relative contributions of SG and protein to absorbance at 254 were assessed by ELISA and protein assay, respectively. Concentration-dependent increases in SG binding to the 40S and 60 S subunits were confirmed by ELISA (Fig. 263). Likewise, protein in the 40S and 608 fiactions increased proportionally with SG in a manner that was suggestive of polysome disaggregation. Since SG binding (1 h) to the ribosome appeared to precede apoptosis induction (2-4 h), this early interaction was characterized further relative to kinetics, specificity, reversibility and relation to MAPK activation. The kinetics of SG binding to the ribosome were assessed in RAW 264.7 cells treated with low (10 nyml) and high (100 ng/ml) SG concentrations. As compared to untreated cells, cells treated with 10 ng/ml SG exhibited increases in A254 peaks corresponding with the 40S and 60S subunits within 30 min and these increased over time (Fig. 27A-F). ELISA confirmed that SG binding to specific ribosomal fractions increased from 30 to 240 min (Fig. 27C-F). No increases in colorimetrically- detemrined protein were observed in cells exposed to 10 ngml SG as were seen at higher SG concentrations (Fig. 263). 93 :1 Vehicle u 86 10 ng/ml I 86 20 ng/ml g I so 40 I 36 so ’a £9 5 E U) E u. < z D a: E. g g 1 2 4 6 Time (h) B SG (ng/ml), 4h SG (ng/ml), 6h M 010204080 010204080 Figure 24. SG induces apoptosis in RAW 264.7 cells. Cells were treated with different SG concentrations for various time intervals and then assessed for DNA fragmentation by (A) cell-death ELISA or (3) agarose gel electrophoresis. M retors to molecular marker. 94 1T)- (15 I Absorbance OI) . . 1+ +fi . 220 240 260 280 300 320 340 Wavelength (nm) Figure 25. UV absorption scan for SG. Purified SG was diluted in phosphate buffer (pH 7.0) and absorbance measured over the UV spectrum. 95 A Sucrose (%) 5 E45 5 “>45 Untreated 20 ng/ml 608 V K w LO <1? 40 ng/ml V 80 ng/ml * x x B 300 c ’g‘ E a, 200 3 5 .5 Q) 8 -100 E -o O 20 4O 80 SG added (ng/ml) Figure 26. SG binding to ribosomal subunits is concentration-dependent in RAW 264.7 cells. Cells were treated with SG (0, 20, 40, 80 nyml) for 1 h and then lysed with polysome extraction buffer (PEB). (A) Ribosomal fractions were separated on a sucrose gradient and the A254 monitored. (3) Individual fractions (0.5 ml) were analyzed for SG by competitive ELISA and for total protein colorimetrically. 96 Sucrose (%) B 5 ———> 45 E B) E, E 0) 4—5 0 S— n. D V In <3 5 10 15 5 10 15 I 15 2 15 2 E \ 9 or E E, O) .s 5 .‘1’. (D 9 a) ct Fraction number Figure 27. Kinetics of SG binding to ribosomal in RAW 264.7 cells. Cells were untreated or treated (A) with water or SG (10 ng/ml) for 15 (B), 30 (C), 60 (D), 120 (E), and 240 (F) min and then lysed with PEB. Ribosomal fractions were separated on a sucrose gradient A254 and monitored. Individual fiactions (0.5 ml) were analyzed for SG and total protein. 97 In cells treated with the high SG concentration(100 ng/ml), the toxin was detectable in the 408 and 608 ribosomal fiactions as early as 5 min with binding being nearly maximal after 15 min (Fig. 28A). To confirm that SG associated with both subunits, small volume ribosomal fractions (0.25 ml) were collected and analyzed by ELISA to increase resolution of the assay. Increased absorbances in the 40S and 60S peaks closely corresponded with increased SG concentrations in these fractions (Fig. 283). Unlike SG, DON neither absorbs at 254 nm, nor cross-reacts in the SG ELISA (Chung et al. 2003a). The specificity of SG binding to the ribosome fraction in RAW 264.7 cells was therefore ascertained by co-incubation with the Type 3 trichothecene DON. ELISA revealed that DON did not alter cytoplasmic SG concentrations (Fig. 29A). As expected, treatment with SG alone increased A254 in 408 and 608 ribosomal subunits (Fig. 293). However, SG and DON co-treatment generated A254 Peaks at 408 and 608 comparable to the untreated and DON-treated groups. Thus, DON appeared to compete with SG for binding to the ribosome, suggesting that the two trichothecenes bound to an identical site. SG has been previously reported to covalently bind to proteins (Yike et al. 2006) raising the possibility that adducts might form in the ribosome. The capacity of DON to compete bound SG from free ribosomal subunits was evaluated. RAW 264.7 cells were treated with SG (20 ng/ml) for l h and resultant cell lysates subjected to sucrose density gradient fiactionation. Pooled ribosome subunits and monosomes (RS+M) containing bound SG, were incubated with additional SG (20 ng/ml), DON (500 ng/ml) or both toxins for 1 h. Following these treatments the mixtures were 98 A Untreated 5min 60$ ' r 408 —-—> Sucrose (%) 45 B A20 ' 608 E 15 - 2’ (‘9’ 1° ‘ 403 U) 5 . o ' . . . - 5 10 15 20 25 30 Fraction number Figure 28. High SG concentration induces rapid, saturable binding to 408 and 60S ribosomal subunits in RAW 264.7 cells. Cells were untreated or treated with SG (100 nyml) for 5, 15, 30 min and then lysed with PEB. (A)Ribosomal fractions were separated on a sucrose gradient system and A254 monitored. (3) Cells were treated with water or SG (100 ng/ml) for 15 min.PEB lysates were separated at high resolution (0.25 ml/fraction) on a sucrose gradient and then fractions analyzed by SG ELISA. 99 A 5 2‘ U) 5 (D (013 0 .3:5:;:;E;;-.::;.~ 58‘ 00“ 90 ,e" 0% O B Untreated SG V f v LO <8” DON SG+DON 5 ——> 45 5 —> 45 Sucrose (%) Figure 29. DON competitively inhibits SG binding to 408 and 60S ribosomal subunits in RAW 264.7 cells. Cells were treated with vehicle, SG (100 ng/ml), DON (500 ng/ml) or both toxins for 15 min and then lysed with PEB. (A) SG content of cell lysate was analyzed by ELISA. (B) Ribosomal fractions were separated on a sucrose gradient and A254 monitored. 100 repeatedly washed and concentrated to remove unbound SG and then subjected to SG ELISA. Supplemental SG treatment did not affect SG interaction with the ribosome, whereas DON treatment dissociated bound SG from the ribosome (Fig. 30). These data suggested that SG association with the ribosome was reversible and therefore non-covalent. In addition, SG (20 ng/ml) and DON (500 ng/ml) co-treatment still resulted in maximum SG binding to the ribosome, suggesting that SG interacts more strongly with the ribosome than DON. While incubation with DON at 100 ng/ml for 15 min resulted in marked p38 and JNK phosphorylation being detectable in SDS extracts of whole cells, high SG concentrations (100-500 ng/ml) caused only modest kinase phosphorylation (Fig. 31). The effects of SO and DON on MAPK activation and mobilization to the ribosome were assessed in RAW 264.7 cells. DON-induced p38 and JNK phosphorylation was reduced by co-treatment with SG (Fig. 32A). Both SG (100 ng/ml) and DON induced p38 and JNK association with the pooled RS+M fractions (Fig. 32B). Phosphorylated p38 and JNK were detectable in the pooled RS+M fractions from DON-treated cells, but phosphorylation was not observed in these fractions from SG-treated cells. These data suggest that while both DON and SG could promote MAPK mobilization to the ribosome, only DON could induce MAPK phosphorylation at this site. Since SG has been recently shown to induce apoptosis in the PC-12 neuronal model (Islam et al. 2008), its capacity to bind ribosomes in this cell line was therefore also assessed. SG treatment of P012 cells increased the absorbance in both 408 and 608 peaks while the polysome profile decreased (Fig. 33). As observed for the RAW 264.7 cells, ELISA confirmed that SG preferentially bound to the fractions containing 101 O10) SG(ng/ml) N O.) A 0 J SG — + - DON _ _ + Figure 30. SG binding to the ribosome is reversible in RAW 264.7 cells. Cells were incubated with SG (20 ng/ml) for 60 min and ribosomal fractions were separated on sucrose gradient. Pooled RS+M fractions were incubated with additional SG (20 ng/ml) and/or (DON 500 ng/ml) for 1 h at 37°C. RS+M were repeatedly concentrated and washed to remove free SG and then analyzed by competitive SG ELISA. 102 ng/ml SG - 100 250 500 - DON - - - - 100 p-p38 ‘ ...-una- p38 W 4' ...... «- w w JNK mm,“ W Figure 31. SG induces modest p38 and JNK phosphorylation in RAW 264.7 cells. Cells were treated with SG (lOO-SOO ngml) or DON (100 ng/ml) for 30 min and then lysed with SDS buffer. Total protein was analyzed by Western blotting with specific antibodies to non- phosphorylated p38 and JNK and their phosphorylated forms. 103 A SG DON p-p38 p38 p-JNK JNK Cell Lysate - - +_+ - + - + . -“ ~‘- -.«, a. RS+M - - + + - + — + ...... -‘. a. sure-.4 Figure 32. SG and DON induce MAPK interaction with the ribosome. Cells were treated with vehicle, SG (100 ng/ml), DON (500 ng/ml) or both toxins for 15 min, and then lysed with PEB. (A) whole cell lysates were analyzed by Western blotting using specific antibodies to phosphorylated p38 and JNK and their non-phosphorylated forms. (B) Lysates were fractionated on sucrose gradient and pooled ribosomal subunits and monosomes (RS+M) analyzed by Western blotting. 104 Sucrose (%) A 5 ’45 5 > 45 l ll Control SG i ‘t 5 W N < 5 1o 15 5 1o 15 E O) E, .5 .9 2 o— “L 0 5 Fraction number Figure 33. SG binds to 408 and 608 ribosomal protein fi'actions in PC-12 neuronal cells. Cells were treated with water or SO (10 ng/ml) for 60 min and lysed with PEB. Ribosomal fractions were separated on a sucrose gradient system and A254 monitored. Individual fractions were analyzed for SG‘ and protein. 105 ribosomal subunits. Thus, 86 treatment evoked similar changes in toxin concentration in 408 and 608 ribosomal fractions of PC-12 cells to that seen for RAW 264.7 cells. DISCUSSION Iordanov et al. (1997) proposed that the ribosome mediate activation of protein kinases afier ribotoxic agents bind to or damage 28S rRNA in the 608 ribosomal subunit. Although several studies have investigated for the interaction of trichothecenes with the ribosome (Bamburg 1983; Carter and Cannon 1977; Middlebrook and Leatherman 1989a; Middlebrook and Leatherman 1989b), ribosomal interaction within cultured cells has not been as well-studied. Here we provide evidence for the first time that 86 rapidly associated with both the small and large ribosomal subunits prior to the induction of DNA fragmentation, a hallmark of apoptosis. SG binding to the ribosome was qualitatively evident based on increased A254 and this correlated closely with the ELISA quantitative data. 86 was not found in early fractions of the sucrose gradient, which contained cytoplasmic proteins, but rather, preferentially bound to the free 408 and 608 ribosomal subunits. The concurrent decreases in polysomes observed after treatment with SG at high concentrations or for prolonged time periods were consistent with previous reports that trichothecenes promote disaggregation of the polysome into 808 or free 408 and 608 subunits (Bamburg 1983). Polysome disassembly likely contributes to translational arrest by inhibiting the elongation process. Although trichothecenes are known to bind to the 608 ribosomal subunit 106 (Ueno 1985), 40S binding has been heretofore unreported. It was therefore particularly notable that the free 408 ribosomal subunit peak increased after SG treatment in both a concentration— and time-dependent manner. This observation might have biological significance relative to trichothecene effects because dsRNA- activated protein kinase (PKR), a kinase which is capable of driving apoptosis, is primarily localized in free 40S ribosomal subunit (Wu et al., 1998). PKR has been associated with trichothecene-induced MAPK activation and apoptosis in RAW 264.7 (Gray et a1. 2008; Zhou et al. 2003b) and PC-12 (Islam et al. 2008) cells. The possibility exists that SG binding to 40$ might mediate PKR activation. Relatedly, we recently observed that DON induces migration of p3 8, JNK and ERK to the 40S ribosomal subunit as well as their phosphorylation (Bae and Pestka 2008). It is possible that the 405 ribosomal subunit might act in concert with PKR as both a sensor and signal transducer for trichothecene-induced toxicity. The observation that DON competed for SO binding provides additional insight into the mechanisms of this Type B trichothecene. Produced by Fusarz’um graminearum and commonly found in cereal-based food, DON has been intensively studied relative to its toxicity in vitro and in vivo. The toxin has both immunostimulatory and immunosuppressive effects, depending on dose (Pestka 2008). Regarding the former, DON is rapidly taken up in mononuclear phagocytes where it induces immediate and robust MAPK activation (Bae and Pestka 2008). These kinases, notably p3 8, drive proinflammatory gene expression, mRNA stability and apoptosis (Chung et al. 2003b; Moon and Pestka 2002; Zhou et al. 2003a; Zhou et al. 20035; Zhou et al. 2005b). 107 The observation that DON did not competitively inhibit SG uptake into cells suggests that both 86 and DON might freely diffuse into the cell without requiring membrane receptors. Our finding that DON inhibited SG interaction with 408 and 608 ribosomal subunits indicates that DON binds to the same sites as SG These data filrther correlate with our recent reports that DON-induced MAPK interaction is mediated through both the 408 and 608 ribosomal subunits (Bae and Pestka 2008). Concentrations of 10 ng/ml SG induce apoptosis and inhibit over 90% of translation, whereas similar effects require 50 to 100 times more DON (Yang et al. 2000) confirming that this macrocyclic trichothecene is much more cytotoxic than the simpler Type B trichothecene. A critical question that arises relates to what is responsible for the different toxicities of SG and DON. Ueno et a1. (1968) first proposed that different binding affinities might affect toxicity among trichothecenes. It was notable that while treatment of the SG-saturated ribosomes with DON alone under cell-free conditions resulted in a reduction of SO binding, co-treatrnent with both toxins was insufficient to reduce levels of SG bound to the ribosomes. DON was not detectable in ribosomes by a DON-specific ELISA regardless of the concentration employed (data not shown), however, SG association with the ribosome was quiet stable during the fractionation. The lower potency of DON for apoptosis induction compared to SG might relate to DON’s lower affinity for the ribosome. Differences in relative toxicity between SG and DON might also result from differential MAPK activation. Importantly, DON-induced p38 activation mediates apoptosis in RAW 264.7 cells, whereas SG-induced apoptosis is p3 8-independent (Yang et al. 2000). SG did not appear to be as strong of an inducer of MAPK 108 activation as DON. Even though SG induced p38 and JNK migration to the RS+M fraction in this study, subsequent and robust p38 and JNK phosphorylation was not evident suggesting that binding to the ribosome per se is not sufficient to evoke MAPK activation. DON-induced MAPK phosphorylation was inhibited by SG co- treatment. The observation that DON but not SG induced MAPK phosphorylation following ribosomal interaction with these kinases might explain the different toxic effects of these two trichothecenes. The capacity of SO and other trichothecenes to induce apoptotic cell death in mononuclear phagocytes can potentially contribute to aberrant immune function. In addition to potential immunotoxic effects, these compounds are also selectively neurotoxic. Islam et a1 (2006, 2007) demonstrated that intranasal instillation of SG and other macrocyclic trichothecenes specifically induces apoptosis in olfactory sensory neurons in the nose and brain. More recently, PC-12 neuronal cells were used to investigate mechanisms of SG-induced death in neurons (Islam et al., 2008). Exposure to SG at 10 ng/ml or higher for 48 h was found to induce DNA fragmentation, which is a characteristic of apoptosis in PC-12 cells. SG-induced apoptosis was confirmed by microscopic morphology, hypodiploid fluorescence and annexin V-FITC uptake. Furthermore, mRNA expression of the proapoptotic genes p53, double stranded RNA-activated protein kinase (PKR), BAX and caspase- activated DNAse (CAD) was significantly elevated from 6 to 48 h after SG treatment. The results presented here suggest, as observed for RAW 264.7, that SG binding to the 408 and 60S subunits in PC-2 cells would be likely to occur prior to the onset of apoptosis. 109 Taken together, the results presented herein strongly suggest that SG binding to intracellular ribosomes proceeds rapidly, is both concentration- and time-dependent, and precedes apoptosis. In fiiture studies it will be important to determine the specific ribosomal binding sites for SG and other trichothecenes as well as identify the exact linkages between the ribosome and intracellular kinase signaling. It will be further critical to understand how cells respond to different trichothecenes with anti-apoptotic or pro-apoptotic mechanisms. Finally, these in vitro mechanisms must be applied to understanding the pathophysiologic effects of SO and other trichothecenes in vivo. 110 CHAPTER V SUMIVIARY AND CONCLUSIONS 111 Although Iordanov (1997) suggested that the ribosome is critical for MAPK activation during ribotoxic stress, the links between the ribosome and MAPK interaction are not understood. There are three major findings in this thesis. First, the ribosome can mediate activation of p38 via interaction with ribosomes in DON- stimulated murine macrophages and human monocytes (See Chapter H). When ribosomes were separated using a sucrose gradient, p38 was found to preferentially interact with the ribosomal subunits. Subsequently, p38 interacted with the 40S subunit initially and with the 608 subunit and monosome. Although both p38 and phosphorylated p38 were detectable in the ribosome, a pharmacological p38 inhibitor did not inhibit p38 interaction with the ribosome, indicating that p38 is activated following ribosomal interaction. Our data thus support the possibility that MAPK activation is dependent on ribosomal interaction. Second, PKR and Hck kinases upstream of p38, activate p38 via ribosomal interaction (See Chapter III). pharmacological inhibitors of PKR and Hck suppressed p38 activation and IL-8 mRNA and protein expression. PKR is known to bind to the 40S ribosomal subunit, but Hck binding to the ribosome has not been previously reported. Our results showed that Hck can directly interact with the ribosome via the ribosomal protein S3, and PKR is required for Hck interaction with the ribosome. Peritoneal macrOphages from PKR knock-out mice exhibit reduced DON-induced p38 interaction with the ribosome with concurrent inhibition of p38 phosphorylation. This is the first report to show that Hck interacts with the ribosome. Our data suggest that both PKR and Hck share a similar pathway to regulate p38 activation and PKR is necessary, through ribosomal interaction, for HCK activation 112 Third, although DON and other trichothecenes are known to target 28$ rRNA in the 60S ribosomal subunit, our findings suggest that DON binds to the 408 ribosomal subunit. DON binding to the 40S ribosomal subunit might induce a ribosomal conformational change, allowing kinase activation. Based on above results, PKR and Hck initially localized to the 408 ribosomal subunit. After DON stimulation, PKR and Hck were phosphorylated, and p38 initially interacted with the 408 ribosomal subunit and was subsequently phosphorylated. Taken together, the 40S ribosomal subunit might function as a signal transducer for DON-induced p38 activation, which is mediated by PKR and Hck via the ribosomal interaction. Future studies will focuse on how DON binding to the ribosome induces MAPK activation via the structural alteration. Firstly, it needs to be determined how DON activates PKR upon ribosomal interaction. One possibility is that DON induces cleavage of ribosomal RNA, leading to PKR activation (Li and Pestka 2008). A cell- free model such as rabbit reticulocytes could be used to study DON activation of PKR. Such a model could help to identify the mechanisms for MAPK activation following DON interaction with the ribosome. Secondly, the binding site for p38 in the ribosome needs to be characterized. Photoaffinity labeling of p38 with the ribosome in a cell-free system could be useful to analyze the binding site. When the binding site is identified, DON-induced p38 mobilization to the ribosome and its activation can be confirmed by live cell fluorescence microscopy (Hoppe et al. 2009) after mutation or blocking of p38 binding site to the ribosome. Finally, it needs to be confirmed in vivo whether p38 interaction to the ribosome can be induced after DON treatment As observed in vitro, DON is rapidly uptaken into tissues of the mouse within 15 min 113 after oral gavage and this results MAPK activation (Zhou et al. 2003b). Thus, DON- induced p38 interaction with the ribosome could be characterized in organs such as spleen and liver in dose and time-dependent manner. DON-related mechanisms could be explored using other ribotoxins such as anisomycin, ricin and Shiga toxin to identify common ribotoxic stress response mechanisms. It is possible that these ribotoxins have a similar mechanism to activate MAPK, leading to proinflammatory gene expression or cell apoptosis. Ultimally, understanding the mechanisms of ribotoxic stress response can be useful for a predictive or therapeutic model to ribotoxin-induced human toxicity. 114 BIBLIOGRAPHY 115 REFERENCES Alisi, A., Spaziani, A., Anticoli, S., Ghidinelli, M., and Balsano, C. (2008). 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