ayv1_/v....v1.'ul V fin.» .v . wig. “3331.....L5 I ‘ - . Q .. L < .a. . _ , , ... I 32.... 5%»... , ‘ . . ‘ . ‘ ‘ , ‘ .. ‘ «r uMwm 3v a, 4 . h .4. , V . ‘ . ,, ‘ . .35an . ‘ 155‘ ;. {$5.de . A, ‘ . r: .- r}? . s ... am vi : A , H A . mm gm & m... L: r \ 553. 2w... 6%.. g vb: L. J: 2M4... In any”! maqg-pnfl “3!:- ‘unCN-q . . Lit, . "1.5,... This is to certify that the dissertation entitled REGULATION OF INTERLEUKlN-8 EXPRESSION BY DEOXYNIVALENOL IN U937 HUMAN MONOCYTES presented by JENNIFER S. GRAY LIBRARY Michigan State University has been accepted towards fulfillment of the requirements for the Ph. D. degree in Microbiology and Environmental ToxicologL JWJWWW Major Professor’s Signature 5/30/2007 Date MSU is an aflinnative-action, equal-opportunity employer 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. DAIEDUE DAIEDUE DATEDUE 6/07 p:/ClRC/DaleDue.inddop.1 REGULATION OF INTERLEUKlN-8 EXPRESSION BY DEOXYNIVALENOL IN U937 MONOCYTES By Jennifer S. Gray A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Molecular Genetics 2007 ABSTRACT REGULATION OF INTERLEUKIN-8 EXPRESSION BY DEOXYNIVALENOL IN U937 MONOCYTES By Jennifer S. Gray Deoxynivalenol (DON, vomitoxin) is a worldwide contaminant of a variety of grain crops. Since humans and animals are exposed to DON through these contaminated grains, health agencies across the world have set maximal tolerable levels for exposure. In vitro models can provide insight into DON’s effects on human cells. Based on observations that monocytes are the primary responders in DON-treated human peripheral blood mononuclear cells, U937 monocytes were chosen here to investigate mechanisms responsible for DON-induced IL-8 expression. Elevated levels of IL-8, 3 neutrophil chemoattractant, have been linked to a variety of inflammatory conditions. DON was found to induce marked mitogen-activated protein kinase (MAPK) activation in the U937 cells. p38 inhibition completely suppressed IL-8 protein and mRNA expression whereas ERK and JNK inhibition partially inhibited IL-8 mRNA but had little effect on IL-8 protein levels. As DON has previously been found to increase expression of immune molecules through an activation of transcription, studies were conducted on the effect of this toxin on the expression of an lL-8 promoter-driven luciferase construct. Mutations in five transcription factors previously implicated in lL-8 expression, AP-l, C/EBPB, Oct-l, NF-KB, and NRF, revealed that only an intact NF-ICB site was necessary for DON-induced IL-8 promoter-driven luciferase. Examination of NF-KB binding, utilizing a transcription factor ELISA, indicated that only p65 binding was increased in the presence of DON. Two other NF -KB subunits, p50 and p52, were present in U937 nuclear protein. Levels of p50 were equivalent in untreated and DON- treated samples, however, DON treatment decreased p52 binding. These findings were further confirmed by the observation that an NF -KB inhibitor, caffenic acid phenyl ester (CAPE) could inhibit both DON-induced IL-8 protein and mRNA. When CAPE was further used to assess DON’s post-transcriptional effects, the toxin was not found to increase lL-8 mRNA stability. Double-stranded RNA protein kinase R (PKR) has been linked to DON—induced MAPK activation and cytokine expression. PKR can mediate MAPK activation as well as NF-KB activation. Investigation into the role of PKR in U937 cells revealed that this kinase was essential for DON-induced lL-8 protein expression. Since protein synthesis inhibitors other than DON are also capable of activating MAPKs and inducing IL-8 protein, two of these, Shiga toxin 1 (Stxl) and ricin, were investigated for a common reliance on PKR as an upstream activator of IL-8 expression. Both Stxl- and ricin- induced IL-8 protein production in U937 cells was found here to be PKR-dependent. In summary, DON-induced IL-8 expression in U937 monocytes was regulated primarily at the transcriptional level and was p38- and NF-KB-dependent. DON-induced IL-8 protein was PKR-dependent with similar effects being observed for the translational inhibitors, Stxl and ricin suggesting that these toxins might share a common mechanism for IL-8 expression in the U937 cell line. Increased knowledge of the mechanisms triggered by these three toxins might aid in dealing with accidental or deliberate intoxication by these agents. DEDICATION Without the patience and support of my family, especially my parents William and Susan Gray and my grandparents Edward and Ardis Drabik none of this would have been possible. Grandpa, I’m sure you’ll want to discuss this when you finish reading it — just let me know. iv ACKNOWLEDGMENTS I’d like to thank my advisor, Dr. James Pestka for his continued funding and support as well as my committee members, Dr. Norbert Kaminski, Dr. Linda Mansfield, and Dr. Richard Schwartz for their advice and technical assistance. I’d also like to thank present and past Pestka lab members for interesting conversations/discussions and assistance in the lab. A special shout out to Sarah Godbehere for her assistance in RNA isolations. TABLE OF CONTENTS DEDICATION .................................................................................................................. iv ACKNOWLEDGMENTS ................................................................................................ v TABLE OF CONTENTS ................................................................................................ vi LIST OF TABLES ............................................................................................................ x LIST OF FIGURES ......................................................................................................... xi LIST OF ABBREVIATIONS -- -_ -- -_ - - -- - ..... - ..xvii INTRODUCTION ............................................................................................................. 1 Overall hypothesis .......................................................................................................... 5 Chapter summaries and hypotheses ................................................................................ 5 Significance ..................................................................................................................... 9 CHAPTER 1: LITERATURE REVIEW..." -- - - -- _- -- _- - - 11 Deoxynivalenol ............................................................................................................. l I General immune system eflects of DON ................... . ................................................ I I Underlying mechanisms for DON ’s induction of immune molecules ....................... 13 Modulation of intracellular signaling by DON ........................................................ 14 Interleukin (IL)-8 .......................................................................................................... 15 Correlation of IL-8 levels with disease ..................................................................... I 6 Mechanisms of IL-8 induction in disease ................................................................. 20 Regulation of IL-8 expression ...................................................................................... 24 IL-8 expression due to multiple transcription factors ............................................... 25 NF -KB as the only transcription factor necessary for IL-8 expression ..................... 33 IL-8 expression is also regulated post-transcriptionally .......................................... 35 Multiple mechanisms trigger IL-8 expression .......................................................... 36 Involvement of chromosome remodeling or basal transcription machinery in the expression of IL-8 ..................................................................................................... 3 7 Mitogen activated protein kinases (MAPKs) ................................................................ 4O Linkage of MAPKs pathways to IL-8 expression ...................................................... 40 MAPKs activate transcription factors ...................................................................... 44 M4PKs also mediate post-transcriptional mechanisms ........................................... 4 7 MAPKs acts in tandem for both transcription and post-transcriptional mechanisms ................................................................................................................................... 48 Other kinases implicated in IL-8 expression ............................................................ 49 U937 cells ..................................................................................................................... 50 vi CHAPTER 2: ROLE OF MITOGEN-ACTIVATED PROTEIN KINASES IN DEOXYNIVALENOL-INDUCED INTERLEUKIN-8 EXPRESSION ..................... 52 Abstract ......................................................................................................................... 52 Introduction ................................................................................................................... 53 Materials and Methods .................................................................................................. 56 Cell culture ................................................................................................................ 56 Western Blot .............................................................................................................. 56 MTT viability assay ................................................................................................... 5 7 IL-8 protein determination ........................................................................................ 58 RNA isolation ............................................................................................................ 58 Reverse transcription real-time PC R ........................................................................ 58 Statistics .................................................................................................................... 59 Results ........................................................................................................................... 6O DON activated p38 and JNK in U93 7 cells .............................................................. 60 Cell viability was not aflected by the MAPKs inhibitors .......................................... 60 MAPK inhibitors were able to suppress DON-induced IL-8 protein levels in U93 7cells .................................................................................................................. 63 MAPK inhibitors were able to suppress DON-induced IL-8 mRNA levels in U93 7 cells ........................................................................................................................... 63 MAPK inhibitors were able to suppress DON-induced IL-8 hnRNA levels in U93 7 cells ........................................................................................................................... 66 Discussion ..................................................................................................................... 66 CHAPTER 3: DON-INDUCED IL-8 EXPRESSION IN U937 CELLS IS NF -KB DEPENDENT 72 Abstract ......................................................................................................................... 72 Introduction ................................................................................................................... 73 Materials and Methods .................................................................................................. 76 Cell culture ................................................................................................................ 76 Site-directed mutagenesis ......................................................................................... 77 Transient transfection of U93 7 cells ......................................................................... 79 Assessment of NF -KB binding ................................................................................... 79 IL-8 protein determination ........................................................................................ 80 RNA isolation and reverse transcription real-time PCR .......................................... 82 Results ........................................................................................................................... 83 DON-induced IL-8 transcription requires NF -KB .................................................... 83 DON increases binding activity of p65 ..................................................................... 91 DON-induced IL-8 is not due to increased mRNA stability in U93 7 cells ............... 93 DON-induced IL-8 requires PKR ............................................................................. 96 IL-8 induction by the translational inhibitors, Stxl and ricin, is also suppressed by a specific PKR inhibitor in U93 7 cells ........................................................................ 96 Discussion ..................................................................................................................... 99 CHAPTER 4: SUMMARY AND CONCLUSIONS 108 Overview ..................................................................................................................... 108 Summary of key findings ............................................................................................ 108 vii Proposed model for IL-8 induction ............................................................................. l 12 Significance and future studies ................................................................................... 115 APPENDIX A: IL-8 PRODUCTION IN MULTIPLE HUMAN CELL LINES ..... 117 Abstract ....................................................................................................................... 117 Introduction ................................................................................................................. 1 17 Materials and Methods ................................................................................................ 119 Cell culture .............................................................................................................. I I 9 IL-8 protein determination ...................................................................................... 120 MTT assay for cell viability .................................................................................... 12] RNA isolation .......................................................................................................... 121 Reverse transcription real-time PCR ...................................................................... 12] Statistics .................................................................................................................. 122 Results and Discussion ............................................................................................... 124 DON induces IL-8 protein in several human cell lines ........................................... I24 DON induces IL-8 RNA in undiflerentiated U93 7 cells .......................................... 129 Conclusions ................................................................................................................. 133 APPENDIX B: OPTIMIZATION OF TRANSFECTIONS USING THE IL-8 PROMOTER-DRIVEN LUCIFERASE CON STRUCT-- 134 Abstract ....................................................................................................................... 134 Introduction ................................................................................................................. 134 Materials and Methods ................................................................................................ 137 Transformation of E. coli and isolation of plasmids .............................................. 13 7 Cell culture .............................................................................................................. 138 Electroporation of cells and reporter assays .......................................................... I 3 8 Luciferase real-time PCR ....................................................................................... 140 Results and Discussion ............................................................................................... 141 Luciferase levels in U93 7 cells normalized with ,B—galactosidase .......................... I 41 Luciferase real-time PCR with U93 7 or RA W 264. 7 cells ...................................... I44 Luciferase levels in RA W 264. 7 cells (without transfection normalization) ........... 1 4 9 Constitutive expression of luciferase in U93 7 cells ................................................ 153 T ransfection normalization using a constitutively expressed Renilla luciferase gene in U93 7 cells ........................................................................................................... 155 Confirmation of a successful transfection system in U93 7 cells ............................. 158 Conclusions ................................................................................................................. 160 APPENDIX C: OPTIMIZATION OF mRNA STABILITY EXPERIMENTS ...... 161 Abstract ....................................................................................................................... 161 Introduction ................................................................................................................. 161 Materials and Methods ................................................................................................ 164 Cell culture .............................................................................................................. 164 RNA isolation .......................................................................................................... 164 Reverse transcription real-time PCR ...................................................................... I 65 IL-8 protein determination ...................................................................................... 166 Results and Discussion ............................................................................................... 166 viii IL-8 stability experiments using general transcription inhibitors .......................... 166 Assessment and optimization of NF -KB inhibitors .................................................. l 71 Conclusions ................................................................................................................. 180 Abstract ....................................................................................................................... 181 Introduction ................................................................................................................. l 8 1 Methods ....................................................................................................................... 182 Results and Discussion ............................................................................................... 183 Predicted transcription factor binding sites using T F SEARCH ............................. 183 Predicted transcription factor binding sites using PROMO ................................... 185 Conclusions ................................................................................................................. 188 REFERENCES 189 LIST OF TABLES Table 1.1: Conditions with lL-8 involvement ................................................................... 17 Table 1.2: Summary studies investigating regulation of IL-8 .......................................... 26 Table 1.3: Summary of studies investigating the role of signaling pathways in IL-8 expression. ................................................................................................................ 42 Table 3.]: Primers used for site-directed mutagenesis of the IL-8 promoter. Mutations are indicated in lower case bold font. ............................................................................. 78 Table 3.2: TransAM ELISA probes. Mutations are in lower case bold font. ................... 81 Table B.1: Plasmids used for optimization of U937 cell transfection. ........................... 139 Table C. 1: Summary of selected studies using NF-KB inhibitors ................................... 163 Table C. 2: Summary of IL-8 mRNA stability experiments. Numbers in parentheses indicates the number of experiments where IL-8 hnRN A was also measured. ...... 167 Table D. 1: Transcription factor binding site predictions using TF SEARCH. ................ 184 Table D2: Transcription factor binding site predictions using PROMO. ...................... I86 LIST OF FIGURES AN IMAGE IN THIS DISSERTATION IS PRESENTED IN COLOR Figure I. 1: General model for the mechanism of action of DON and other trichothecene. (9) ................................................................................................................................ 4 Figure 1.1: Structure of DON. The epoxide and double bond at C9-CIO are responsible for biological activity of this molecule. .................................................................... 12 Figure 1.2: Representation of the lL-8 promoter with major regulatory elements. .......... 28 Figure 1.3: General MAPK pathway.(This image is in color.) ......................................... 41 Figure 2.1: Exposure to DON increases phosphorylation of p38, JNK, and ERK in U937 cells. Western blot of U937 total protein samples isolated after a 30 minute exposure to 500 ng/ml DON. (A) Phospho-p38 and nonphospho-p38 (B) Phospho- JNK and nonphospho-JNK (C) Phospho-ERK and nonphospho-ERK. (n = 3) These data are representative of 2 independent experiments. ............................................. 61 Figure 2.2: Viability of U937 cells does not decrease with exposure to MAPK inhibitors. MTT with U937 cells after a 3 hour exposure to 500 ng/ml DON, 2.0 uM 88203580, 10 uM PD98059, or 1.0 uM SP600125. Data are the mean i SEM (n = 3). These data are representative of 2 independent experiments. ............................. 62 Figure 2.3: DON-induced IL-8 protein levels are suppressed by a p38 inhibitor. IL-8 protein levels in U937 culture supernatant after 3 hours exposure to 500 ng/ml DON, and/or 2.0 uM 83203580, 10 uM PD98059, or 1.0 uM SP600125. Bars without the same letter are statistically different (p < 0.05). Data are the mean i SEM (n = 5). These data are representative of 3 independent experiments .................................... 64 Figure 2.4: DON-induced IL-8 mRNA levels are suppressed by MAPKs inhibitors. IL-8 mRNA levels in U937 after a 3 hour exposure to 500 ng/ml DON, and/or 2.0 uM SBZO3580, 10 uM PD98059, or 1.0 uM SP600125. Bars without the same letter are statistically different (p < 0.05). Data are the mean :t SEM (n = l 1). These data were collected from 4 separate experiments. ............................................................ 65 Figure 2.5: DON-induced IL-8 hnRNA is suppressed by MAPKs inhibitors. IL-8 hnRNA levels in U937 after a 3 hour exposure to 500 ng/ml DON, and/or 2.0 uM SBZO3580, 10 uM PD98059, or 1.0 uM SP600125. Bars without the same letter are statistically different (p < 0.05). Data are the mean :I: SEM (n = 11). These data were collected from 4 separate experiments ...................................................................... 67 xi Figure 2.6: Comparison of relative levels of IL-8 protein, mRNA, and hnRNA expression in U937 cells exposed to 500 ng/ml DON, and/or 2.0 uM SB203580, 10 uM PD98059, or 1.0 uM SP600125. (see Figures 2.3, 2.4, and 2.5) ............................. 70 Figure 3.1: DON induces IL-8 promoter driven luciferase in U937 cells. U937 cells were transfected with a wild-type IL-8 promoter luciferase (-162/+44 IL-8 LUC) construct incubated for 1 h, then treated with 0, 1 ug/ml DON, or 1 Itng LPS for 11 h. Data are mean :l: SEM combined from two or more independent experiments. (n 2 12) ...................................................................................................................... 84 Figure 3.2: The NF-KB site is required for DON-induced IL-8 promoter driven luciferase in U937 cells. U937 cells were transfected with either a wild-type IL-8 promoter luciferase construct or NF-KB-mutated IL-8 promoter construct, incubated for 1 h, then treated with 0, 1 ug/ml DON, or 1 ug/ml LPS for 11 h. Data are mean i SEM combined from two or more independent experiments. (n 2 12) ........................... 86 Figure 3.3: The AP-l site is not required for DON-induced IL-8 promoter driven luciferase in U937 cells. U937 cells were transfected with either a wild-type IL-8 promoter luciferase construct or AP-l-mutated IL-8 promoter construct, incubated for l h, then treated with 0, l ug/ml DON, or 1 ptng LPS for 11 h. Data are mean i SEM combined from two or more independent experiments. (n 2 12) ................... 87 Figure 3.4: The C/EBPB site is not required for DON-induced IL-8 promoter driven luciferase in U937 cells. U937 cells were transfected with either a wild-type IL-8 promoter luciferase construct or C/EBPfi-mutated IL-8 promoter construct, incubated for 1 h, then treated with 0, 1 ug/ml DON, or l 1.1ng LPS for 11 h. Data are mean a: SEM combined from two or more independent experiments. (n 2 12) 88 Figure 3.5: The Oct-l site is not required for DON-induced IL-8 promoter driven luciferase in U937 cells. U937 cells were transfected with either a wild-type IL-8 promoter luciferase construct or Oct-l-mutated IL-8 promoter construct, incubated for l h, then treated with 0, 1 ug/ml DON, or 1 trng LPS for 11 h. Data are mean :1: SEM combined from two or more independent experiments. (n 2 12) ................... 89 Figure 3.6: The NRF site is not required for DON-induced lL-8 promoter driven luciferase in U937 cells. U937 cells were transfected with either a wild-type lL-8 promoter luciferase construct or NRF-mutated IL-8 promoter construct, incubated for 1 h, then treated with 0, l ug/ml DON, or 1 ug/ml LPS for 11 h. Data are mean :I: SEM combined from two or more independent experiments. (11 Z 12) ................... 90 Figure 3.7: DON alters the binding of three NF-kB subunits, p65 (A), p50 (B), and p52 (C), in U937 cells. U937 cells were treated with 0, 1 ug/ml DON, or LPS for 3 h, nuclear protein was isolated, and then NF-KB binding was measured (20 pg per binding reaction). Data are mean :t SEM and pooled from two independent experiments. (n = 6) ...... ‘ ............................................................................................ 92 xii Figure 3.8: CAPE inhibits DON-IL-8 protein in U937 cells. Cells were pretreated for 2 h with either 0 or 100 ug/ml CAPE then treated for 12 h with 0, 250, 500, or 1000 11ng DON. Data are mean :t SEM and representative of two independent experiments (n=3) ..................................................................................................... 94 Figure 3.9: CAPE inhibits IL-8 mRNA in U937 cells. Cells were pretreated for 2 h with either 0 or 100 ug/ml CAPE then treated for 3 h with 0, 500 ng/ml DON, or 5 ng/ml LPS. Data are mean :I: SEM and representative of two independent experiments. (11 = 3) ............................................................................................................................... 95 Figure 3.10: DON does not stabilize lL-8 mRNA. U937 cells were pretreated for 2 h with 100 ug/ml CAPE then treated with 0, 250, 500 ng/ml DON, or 5 ng/ml LPS at the timepoints indicated. Data are mean i: SEM pooled from two independent experiments. (n = 6) .................................................................................................. 97 Figure 3.11: A PKR inhibitor suppresses DON-induced IL-8 protein (~98%). U937 cells were pretreated with or without PKR inhibitor (2.5 uM) or a Negative control inhibitor (2.5 uM) for 45 minutes before addition of 0, 500, or 1000 ng/ml DON. Culture supernatant was collected 12 h afier addition of DON and IL-8 protein was assessed by ELISA. Data is mean 3: SEM. Representative of 3 independent experiments. (n = 3) .................................................................................................. 98 Figure 3.12: A PKR inhibitor suppresses Stxl-induced IL-8 protein (~80%). U937 cells were pretreated with or without PKR inhibitor (2.5 uM) or a Negative control inhibitor (2.5 uM) for 45 minutes before addition of O, 500, or 1000 ng/ml Stxl. Culture supernatant was collected 12 h after addition of Stxl and IL-8 protein was assessed by ELISA. Data is mean i SEM. Representative of two independent experiments. (n = 3) ................................................................................................ 100 Figure 3.13: A PKR inhibitor suppresses ricin-induced IL-8 protein (~30-60%). U937 cells were pretreated with or without PKR inhibitor (2.5 M) or a PKR inhibitor negative control (2.5 uM) for 45 minutes before addition of 0, 10, 50, 500, or 1000 ng/ml ricin. Culture supematent was collected 12 h afier addition of ricin and IL-8 protein was assessed by ELISA. Data is mean d: SEM. Representative of 2 independent experiments. (n = 3) ........................................................................... 101 Figure 4.1 ........................................................................................................................ 114 Figure A. 1: Diagram of hnRNA primers and probe based on the IL-8 DNA sequence (Genbank sequence NM_006216). ......................................................................... 123 Figure A.2: lL-8 protein production by macrophage differentiated U937 cells after a 12 h exposure to DON. U937 cells were differentiated with PMA and allowed to rest for 48 h after removal of the PMA. Data are mean i SEM (n = 3). This is xiii representative of 2 independent experiments. Bars without the same letter are statistically different (p < 0.05) ............................................................................... 125 Figure A.3: IL-8 protein production from undifferentiated U937 cells after a 12 h exposure to DON. Data are mean i SEM (n = 3). This is representative of 2 independent experiments. Bars without the same letter are statistically different (p < 0.05). ....................................................................................................................... 126 Figure A.4: IL-8 protein production from THP-1 cells after a 12 h exposure to DON. Data are mean 1 SEM (n = 3). This is representative of 2 independent experiments. Bars without the same letter are statistically different (p < 0.05). .......................... 127 Figure A.5: IL-8 protein production from HEK-293 cells after a 12 h exposure to DON. Data are mean :t SEM (n = 6). This is an average of 2 independent experiments. Bars without the same letter are statistically different (p < 0.05). .......................... 128 Figure A.6: MTT of U937 cells with a 3 h DON exposure. There is no statistical difference among the treatment groups. Data are mean a: SEM (n = 6). This is an average of 2 independent experiments .................................................................... 130 Figure A.7: IL-8 mRNA levels in undifferentiated U937 cells after 3 h exposure to DON. Data are mean :t SEM (n = 6). This is an average of 2 separate experiments. Bars without the same letter are statistically different (p < 0.05). .................................. 131 Figure A.8: IL-8 hnRNA levels in undifferentiated U937 cells after a 3 h exposure to DON. Data are mean i SEM (n = 6). This is an average of 2 separate experiments. Bars without the same letter are statistically different (p < 0.05). .......................... 132 Figure B. 1: Basic electroporation scheme for both RAW 264.7 and U937 cells. .......... 142 Figure 3.2: Unequal suppression of constitutive expression of luciferase (Luc) and B- galactosidase (B-gal) in U937 cells with 500 ng/ml DON. Cells were allowed to rest for 6 h after electroporation (15 ug pGL2 Control and 5 pg pSV40 Bgal) and were treated for 12 h with or without DON. (n = 8) ....................................................... 143 Figure B.3A: Summary of Cok and Morrison (2001) method for measurement of luciferase mRNA using real-time PCR and transfection normalization using using luciferase protein ..................................................................................................... 145 Figure B.3B: Final RNA and DNA isolation scheme for luciferase real-time PCR ....... 146 Figure B.4: Preliminary real-time PCR results for luciferase mRNA and DNA in RAW 264.7 cells are highly variable. RNA and DNA, isolated from RAW 264.7 cells electroporated with 20 pg of -1 62/+44 IL-8 LUC plasmid, were assessed for luciferase. Samples were normalized with either luciferase DNA alone or 18S RNA xiv levels and luciferase DNA. cDNA was made with either random hexamers (rh) or luciferase-specific (ls) primers. * = values that are 0.0002 or lower (n = 2) ......... 148 Figure B.5: Increasing the amount of plasmid increases the luciferase protein levels (non- normalized) in LPS-treated RAW 264.7 cells. Cells were electroporated with 5, 15, or 25 pg -162/+44 lL-8 LUC and after a 24 h recovery time were treated for 0.5, 2, or 4 h with or without 1 pg/ml LPS. (n = 2) ........................................................... 151 Figure B.6: Longer treatment times have higher luciferase protein levels (non- normalized) in RAW 264.7 cells. Cells were electroporated with 15 pg of -l62/+44 IL-8 Luc, allowed to recover for 24 h and treated for 0.5, 2, 4 h with the concentrations of DON and LPS indicated. ND = not determined (11 = 2) ............. 152 Figure B.7: Shorter recovery times have higher luciferase levels (non-normalized) in U937 cells. Cells were electroporated with 0, 5, or 10 pg of pGL2 control plasmid and allowed to recover for either 12 or 24 h. Luciferase levels were measured after either a 2 (14 h and 26 h) or 4 h (16 h or 28 h) treatment time. (n = 2) .................. 154 Figure 3.8: DON causes approximately equivalent suppression of firefly and Renilla luciferase levels in U937 cells. Cells were electroporated with 10 pg of pGL2 Control and 1 pg pRL SV40 and allowed to recover 1 h before treatment. Cells were treated with 0, 500 or 1000 ng/ml DON for 2, 4, 6, or 12 h. (n = 3) ............. 156 Figure 8.9: DON-induction of IL-8 promoter—driven luciferase levels (normalized with Renilla luciferase levels) in U937 cells. Cells were electroporated with 15 pg - l62/+44 lL-8 LUC and 1 pg pRL SV40, allowed to recover for 1 h and treated with 0, 500, or 1000 ng/ml DON for 2, 4, 6, or 12 h. (n = 3) ......................................... 157 Figure B. 10: DON induces IL-8 promoter driven luciferase levels in U937 cells. Cells were electroporated with 15 pg -162/+44 lL-8 LUC and 0.5 pg pRL SV40, allowed to recover for 1 h, and treated for 11 h with 0, 500, 1000 ng/ml DON, or 1000 ng/ml LPS. Representative of several independent experiments. (n = 20) ....................... 159 Figure C.1A: IL-8 mRNA in U937 cells after exposure to 100 pM of DRB alone or with 250, 500, or 1000 ng/ml DON. Cells were co-treated with various concentrations of DON and DRB for the specified times, then total RNA was isolated and assessed for IL-8 mRNA. Data are mean :t SEM (n = 3). .......................................................... 169 Figure C.le IL-8 hnRNA in U937 cells afier exposure to 100 pM of DRB alone or with 250, 500, or 1000 ng/ml DON. Cells were co-treated with various concentrations of DON and DRB for the specified times, then total RNA was isolated and assessed for IL-8 hnRNA. Data are mean 3: SEM (n = 3) ........................................................... 170 Figure C.2: BAY 11-7085 does not inhibit DON-induced IL-8 protein production. U937 cells were exposed to a 12 h co-treatment of BAY 11-7085 (0, 5, 20, or 40 pM) with DON (0, 250, or 500 ng/ml). Data are mean i: SEM (n = 3). ................................. 172 XV Figure C.3: CAPE inhibits DON-induced IL-8 protein production. U937 cells were co- exposed to CAPE (0, 25, 50, or 100 pg/ml) with DON (0, 250, or 500 ng/ml) for 12 h. Data are mean i: SEM (n = 3). ............................................................................ 173 Figure C.4: Gliotoxin does not inhibit IL-8 protein production. U937 cells were co- treated with gliotoxin (O, 1, or 2 pg/ml) with DON (O, 250, or 500 ng/ml) for 12 h. Data are mean :t SEM (n = 3). ................................................................................ 174 Figure C.5: BAY ”-7085 does not inhibit DON-induced lL-8 protein with a 2 h pretreatment. U937 cells were treated for 2 h with BAY 11-7085 (0, 5, 20, or 40 pM) then a 12 h treatment with DON (0, 250, or 500 ng/ml). Data are mean 3: SEM (n = 3) ...................................................................................................................... 176 Figure C.6: CAPE inhibits DON-induced IL-8 protein. U937 cells were treated with CAPE (0, 25, 50, or 100 pg/ml) for 2 h then treated for 12 h with DON (0, 250, or 500 ng/ml). Data are mean i SEM (n = 3). ............................................................ 177 Figure C.7: Gliotoxin does not inhibit DON-induced IL-8 protein. U937 cells were given a 2 h pretreatment of gliotoxin (0, 1, or 2 pg/ml) then a 12 h treatment with DON (0, 250, or 500 ng/ml). Data are mean :t SEM (n = 3). ................................................ 178 Figure C.8: CAPE inhibits IL-8 mRNA in U937 cells. Cells were pretreated for 2 h with either 0 or 100 pg/ml CAPE then treated for 3 h with 0 or 500 ng/ml DON. Representative of two independent experiments. Data are mean :1: SEM (n = 3)... 179 xvi 3 ’ UTR ActD Ad7 ANOVA AP-l ARE C/EBPB CAPE CAT CBP COX-2 CPPD CREB CYP DON DRB EHEC ELISA EMSA ERK LIST OF ABBREVIATIONS 3’ untranslated region actinomycin D Adenovius serotype 7 one way analysis of variance activator protein-1 AU rich region CCAAT/enhancer binding protein B caffenic acid phenyl ester chloramphenical acetyltransferase CREB binding protein cyclooxygenase-2 calcium pyrophosphate dihydrate cAMP response element binding protein cytochrome P450 deoxynivalenol dichlorobenzimidazole riboside enterohemorrhagic E. coli enzyme-linked immunosorbant assay electrophoretic mobility gel shift assay extracellular signal-regulated kinases xvii FDA FLS FMF I-lck HDAC HI FBS hnRNA HRP IgG IKK lL-8 IPF IVIG IKB JNK/SAPK LPS Luc MAPK MEK mIL-8Rh KO MIP-2 MKK mRNA Food and Drug Administration fibroblast-like synoviocytes familial Mediterranean fever hematopoietic cell kinase histone deacetylase Heat-inactivated fetal bovine serum heteronuclear RNA horseradish peroxidase immunoglobulin G IKB kinase interleukin-8 idiopathic pulmonary fibrosis intravenous immunoglobulin inhibitor of NF-KB c-Jun N-terrninal kinase/stress-activated protein kinases lipopolysaccharide luciferase mitogen-activated protein kinase mitogen-activated or ERK kinase mice that lacked the murine homologue of the lL-8 receptor macrophage-inflammatory protein-2 MAPK kinase messenger RNA xviii MSU MTT NAC NE NF-KB NIK NRF Oct- 1 PBMC PBS PDAR PGE2 PKA PKC PKR PMA PMN POU SDS leAM Stx 1 TAP TBP monosodium urate monohydrate 3-[4, 5—dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide N-acetyl-cysteine neutrophil elastase nuclear factor- KB NF-KB inducing kinase NF-KB repressing factor octamer-l peripheral blood mononuclear cell phosphate buffered saline Pre-Developed Assay Reagents prostaglandin E2 CAMP-dependent protein kinase protein kinase C double-stranded RNA protein kinase R phorbol- l 2-myristate- 1 3-acetate polymorphonuclear cells Pit-Oct-Unc sodium dodecyl sulfate soluble intercellular adhesion molecule Shiga toxin 1 TBP-associated factors TATA-binding protein xix TDI TGF—B TNF-a TSA VIP WHO B-gal Tolerable Daily Intake transforming growth factor-B tumor necrosis factor- a trichostatin A vasoactive intestinal peptide World Health Organization B-galactosidase XX INTRODUCTION Deoxynivalenol (DON; vomotoxin) is a member of the trichothecene mycotoxins, which are commonly found in grain species worldwide. DON is produced by F usarium graminearum, which is the causative agent of head blight affecting maize, wheat, barley, and other grain crops. DON exposure through the ingestion of contaminated grains has been seen worldwide, due in part to the toxin’s resistance to high temperatures, such as those used in food processing and cooking. This makes it a common threat to animal and human health. Additionally, DON contamination has been projected to potentially cause annual losses up to $637 million for food for human consumption, $18 million for feed and $2 million for livestock (1). During the past decade, there have been several surveys on the presence of naturally occurring foodbome contaminants in a variety of foods. One of these surveys collected 11,022 samples from 12 countries and found 57% of the samples were positive for DON (2). DON was found primarily in cereal grains and grain products, with wheat, wheat flour, and bread concluded to be primarily responsible for DON intake by humans. Two countries, France and Germany, were concluded to have human intake levels above the Tolerable Daily Intake (TDI; 1 pg/kg body weight) as set by the World Health Organization (WHO). In another survey published in 2004, Tritscher and Page (3) determined that of 5 regions tested, Africa, Europe, Latin America, the Middle East, and the Far East, 4 exceeded the TD] set by the WHO and estimated the DON intake for the USA to be approximately 51 pg/person/day. In a more recent survey of the French diet, Leblanc et al. (4) determined that 5% or less of adults, children, or vegetarians exceeded the TD] for DON with cereal products consumption responsible for DON exposure. Based on the persistence of DON in foods, it is not surprising that DON has been linked to numerous instances of human toxicosis in the last several decades. For example, gastroenteritis outbreaks that have occurred in the past several decades in China and India, have been linked to elevated levels of DON in food and feed. Reported symptoms in those outbreaks were similar to symptoms observed with acute toxicity in animal studies. In the United States, recent outbreaks (October 1997 to October 1998) that occurred in schools were linked to burrito consumption. Of note, DON contaminated wheat was found in the tortillas used for the burritos, although the levels of DON were below the Food and Drug Administration (FDA) advisory level (5). Along with potential effects from acute toxicity, there are also symptoms associated with chronic toxicity have been observed in various animal studies. These symptoms, including altered nutritional efficiency, anorexia, and decreased weight gain, are not as severe as those seen with one large dose of DON, can also be detrimental to food animal health (6). Due to the concern over human health, various regulatory agencies around the world have set limits on foods and feeds to reduce human and animal exposure to DON. Advisory levels in the USA, set by the FDA, are 1 ppm for finished wheat products, 10 ppm for grains used for cattle older than 4 months and chickens, and 5 ppm for feed for swine and all other animals (7). Health Canada has a slightly higher guideline of 2 ppm for uncleaned wheat and 1.2 ppm for wheat flour for adults, but a 0.6 ppm level for wheat flour for infants (7). The European Union has set an even lower level than Health Canada of 0.75 ppm for raw flour and 0.5 ppm for retail stage cereal products (7). The symptoms of DON intoxication could, in part, be caused by cytokine and chemokine dysregulation. One of the immune molecules that DON can induce is interleukin (IL)-8 (8). IL-8 is a chemokine involved in the inflammatory process. Its main function is to attract neutrophils to sites of inflammation. In addition, secondary roles include a number of other processes, including angiogenesis, obesity, cardiovascular disease, and contribute to the severity of autoimmune diseases. The overproduction of lL-8 as a result of DON exposure could contribute to chronic inflammation (for example, exacerbate arthritis) or exacerbate other preexisting conditions. Over the last fifty years, many aspects of trichothecenes’ effect on cells have been investigated ranging from interaction with the ribosome and inhibition of protein synthesis to increase the proinflammatory gene expression (9). Figure 1.1 provides an overview of trichothecene effects. The figure starts by depicting the toxin interacting with the ribosome and/or interaction with upstream kinases, potentially PKR and/or Hck, which causes inhibition of protein synthesis and an activation of signaling cascades upstream of mitogen-activated protein kinase (MAPK) pathways. Those upstream kinases can, in turn, activate MAPKs. By causing an increase in MAPKs, increases in transcription, mRNA stability, and apoptosis can occur in cells and lead to a variety of effects in both cell lines and animal models. l TRICi-IO'I'HECE’15:)..‘5- e ' e " "r RibosomaI/Receptor Binding ??? Hck PKR \ I MAPK Activation \ ........ p53 ...00 ooooo 0.000 f ..... .g ‘ ”l “235°45‘10““ 4‘ mRNA Stability t Leukocyte tva ton\ \ / apoptosis \ * 1~ Proinflammato . Anorexia. Reduced /\Genes ry !m_9‘P"QSFPPT¢$5I00 Weight Gain Immune stimulation! ; Autoimmune Effects ‘ Tissue Injury Figure 1.1: General model for the mechanism of action of DON and other trichothecene. (9) Overall hypothesis In light of the ubiquitous role of lL-8 in many human conditions and its increased levels in the presence of DON, the overall goal of this dissertation was to determine the mechanisms by which IL-8 is induced in U937 cells, a human monocyte line. The guiding hypothesis is that DON induces increases in both transcription and mRNA stability, through the activation of signaling cascades, which are responsible for the increase in IL-8 protein and IL-8 mRNA. The specific aims in this dissertation were (1) to determine if DON-induced IL-8 protein and IL-8 RNA in undifferentiated U937 cells was mediated through the mitogen-activated protein kinases (MAPKs), (2) to determine if transcriptional alteration occurs due to exposure to DON, and if so which transcription factors are involved, (3) to determine the contribution that post-transcriptional stability has on elevated mRNA levels, and (4) to determine the role of an upstream kinase in the elevation of IL-8 levels. Qh_apter summggi_es. and hypotheses Chapter 1 provides background information relevant to the research presented in this dissertation. This includes the effect that DON has on eukaryotic cells, primarily murine and human macrophages, IL-8, the regulation of IL-8, a brief overview of mitogen-activated protein kinases (MAPKs), and U937 cells. General characteristics of lL-8 are given and the relationship of IL-8 and various human diseases are also explored. lL-8 expression is elevated in many human inflammatory conditions and is a chemokine of importance because DON has been shown to elevate various inflammatory molecules in vivo and in vitro. The mechanisms of IL-8 expression are presented for both mRNA stability and transcription. Transcription is of particular importance because it involves a variety of proteins necessary for the process both before the actual start of transcription and during transcription. Eukaryotic transcription is a complex process involving individualized proteins specific to the molecule that will be expressed and general proteins ubiquitous to the process. The process of transcription occurs at the promoter, which is the region of the DNA that is 5’ to the coding sequence. This area contains many binding sites for proteins known as transcription factors along with binding sites for basal transcription machinery. RNA polymerase II, which is responsible for mRNA synthesis, starts as a preinitiation complex consisting of the polymerase along with a variety of TFII proteins, such as TFIID (TATA-binding protein (TBP) and TBP- associated factors (TAFs)). (10) After the polypeptide complex forms, of approximately 40 proteins, it binds to the promoter along with transcription factors specific to an individual promoter, which is collectively known as the initiation complex. After the actual transcription starts, preinitiation factors are replaced by elongation factors. (I 1) The elongation factors function to speed up the transcription. Once the transcript is produced the multi-protein complex dissociates from the DNA. The specific transcription factors for each promoter are subject to activation as a response to a variety of stimuli. The IL-8 promoter has binding sites for 5 main transcription factors (AP-1, C/EBPB, Oct-l, NF -KB, and NRF) that have been found to play a role in IL-8 expression under different conditions. Along with alterations in transcription factor activation, lL-8 expression has also been linked to histone alteration, which controls the accessibility of the promoter to the transcription machinery. Both transcription factor activation and alteration of the transcription machinery and histones can be due to multiple signaling cascades. The role of upstream signaling cascades, MAPKs in particular, will also be discussed in relation to IL-8 expression in this chapter. Upstream signaling cascades are important due to the involvement of MAPKs pathways in transmission of a response to a specific stimulus. In the last section, information regarding U937 cells will be presented. U937 cells are a human monocyte line that was first isolated in 1976 (12). They were chosen as the model for this research for several reasons. The most important is that DON targets actively dividing cells, such as immune cells (9). The second reason is that U937 cells, macrophage differentiated, have already been reported to have elevated IL-8 in response to DON exposure. Finally, U937 cells were chosen as a model for this research is because this cell line has been widely used for a variety of studies and has well characterized signaling pathways. Chapter 2 examines the role that MAPKs might play in the observed alteration of the normal expression of lL-8. The hypothesis is that one, if not more, of the MAPKs families are partly responsible for the increase in IL-8 expression resulting from DON exposure. MAPK phosphorylation was assessed using Western Blot and three MAPKs inhibitors, one each for the p38, ERK, and JNK pathways, to determine the relative contribution of each pathway for IL—8 expression in U937 cells. Data included in this chapter were published, the complete paper can be found at http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXH-4HVDY12- 2&__user=l l l l 158&'coverDate=06%2F l 5%2F2006&_rdoc= l &_fmt=&_orig=search&_ sort=d&view=c&iacct=C00005l676&_vcrsion=I&_urlVcrsion=0&_userid=l l l l 158& md5=78 l acZ66c645297d87ce1874a6l 71 38c , and provided in vitro support to ex vivo experiments with cells isolated from human blood. Chapter 3 explores the hypothesis that DON-induced lL-8 expression is due to increases at both the transcriptional and post-transcriptional levels. The transcriptional level is examined using an lL-8 promoter-driven luciferase construct with a wild-type sequence or containing mutations in the binding sites of five main transcription factors for lL-8 expression. Additionally, results obtained with the luciferase construct were confirmed with a transcription factor ELISA to measure the binding activity of the transcription factor found to be primarily responsible for IL-8 expression. Chapter 3 also investigates the role of increased mRNA stability in DON-induced IL-8 expression. Several immune molecules have been shown to have a DON-induced increase in mRNA stability. IL-8 mRNA was measured during a time course after U937 cells were exposed to a transcriptional inhibitor to determine if DON causes an increase in the stability of the IL-8 transcript. Chapter 4 is a summary chapter integrating how research presented in this dissertation fits into the model of DON response mechanisms. There are also five Appendices (A, B, C, D, and E). Appendix A presents additional IL-8 protein induction data in differentiated U937 cells as well as two additional cell lines, THP-l and HER-293. Differentiated U937 cells were originally considered as the primary cell line for this research. However, they were replaced with undifferentiated U937 cells after the differentiation process was determined to be inconsistent and subject to variability in excess of what can be reasonably expected. The two additional cell lines are both human and had been considered as replacement cell lines for the U937 cells. Appendix B contains data collected while optimizing the transfection experiments. Experiments were conducted in both RAW 264.7 cells and U937 cells with three different reporter genes, different plasmids concentrations, and different times for both recovery time after the electroporation and exposure time to DON or LPS. Appendix C contains data obtained while optimizing the mRNA stability experiments. Originally mRNA timecourses were conducted using general transcription inhibitors. However, during the course of those experiments inconsistent and inconclusive data were obtained. It was determined that the general transcription inhibitors were inappropriate for the cell line used (possibly because of metabolism) and utilization of more specific inhibitors were explored. Appendix C contains the data obtained while selecting an inhibitor and optimizing both dose and treatment conditions. Appendix D contains the sequences of the mutated IL-8 promoter used for the luciferase constructs and presents data obtained from using two different web-based transcription factor binding site recognition programs. Appendix E contains a complete journal article containing much of the data in Chapter 3 as originally published in Toxicolggy and Applied Pharmacology (2006). Significance Due to the fact that DON is a common contaminant of grains worldwide, and that it can adversely affect both animal and human immune systems at low concentrations, it is important to obtain further information on DON’s effects on the human immune system. DON exposure at low doses can induce expression of cytokines, including IL-8 in human cells. Because aberrant regulation of lL-8 and other cytokines can adversely impact human and animal health, it is necessary to understand if DON interferes with the regulation of lL-8. This dissertation investigates the mechanisms behind the DON- induced elevation of lL-8. 10 CHAPTER 1: LITERATURE REVIEW Deoxvnivglenol Deoxynivalenol (DON, vomitoxin) is a fungal secondary metabolite that is produced primarily by F usarium graminearum or F usarium culmorum and classified as a trichothecene. It is one of the most common mycotoxins found in cereal grains (6) and is a frequent contaminant of wheat, corn, and barley worldwide (13). Since DON is a very stable compound, it is capable of withstanding the high temperatures used in processing and cooking of foods (1 3). Because of this, DON is present in processed foods and be consumed by humans and animals. DON inhibits protein synthesis by binding to ribosomes and interfering with chain elongation (13). The unsaturated bond at C9-C10 and an epoxide at C12-C13 are responsible for biological activity (13). (Figure 1.1) Actively dividing cells, such as immune cells, are very susceptible to the effects of DON. Immune system effects in response to DON exposure include impairment of human and murine lymphocyte proliferation, decreased host resistance to bacterial infection in mice, increased total serum immunoglobulin A production in mice, and increased expression of cytokines in mice and in cell lines (14) (13). General immune system eflects of DON DON has been found to be immunosuppressive and immunostimulatory in experimental animals, depending on dose and duration of exposure (15). At high doses, 1.] H C 3 \MO I/(l) I V-n-OH 9 2 3 H2C—-(’) 8 6 r," W5 5 4 o I 'CH3 ‘ ""H (DH i CH3 H OH Figure 1.1: Structure of DON. The epoxide and double bond at C9-C10 are responsible for biological activity of this molecule. DON exhibits immunosuppressive effects. These effects could be mediated by several mechanisms including the inhibition of translation and increased apoptosis (13). Acute poisoning, from extremely high doses of DON, has also been observed in both humans and animals (6). The poisoning presents as a multi-system shock-like syndrome with symptoms of dermal irritation, nausea, vomiting, diarrhea, hemorrhage, and hematological lesions (13). The immunostimulatory effects of DON, seen at lower doses (16) include the production of cytokines in vitro and in vivo that apparently results from interference with normal gene regulation. Previous laboratory data have shown that interleukin (IL)-6 protein levels are superinduced in RAW 264.7 murine macrophage cells when activated with lipopolysaccharide (LPS) (17). Levels of both IL-6 protein and mRNA are elevated afier DON exposure in B6C3F1 mice (18). In EL-4 human thymoma cells, after exposure to DON, levels of IL-2 protein and mRNA are elevated (19). IL-2 protein is also induced by DON, along with the chemokine IL-8, in Jurkat human T cells (6). In the U937 cell line, a human monocyte cell line that can be differentiated into macrophage cells, DON induces IL-6 and lL-8 protein (8). Underlying mechanisms for DON ’s induction of immune molecules Increased transcription and/or post-transcriptional stability are likely to be responsible, in part, for the DON-stimulated cytokine expression. Previously, DON has been shown to alter transcription factor binding. NF-tcB (20) and AP-l (21) binding are increased in EL-4 cells after DON exposure. Similar effects are seen in RAW 264.7 murine macrophage cells after exposure to DON as evidenced by increased binding, to 13 consensus sequences, for NF-KB, AP-l, and C/EBPB (22). Increased binding of CREB and AP-l were also observed in murine peritoneal macrophages (23). Additionally, cyclooxygenase-2 (COX-2) also seems to be induced by DON at the transcriptional level. Moon et al. (24) observed that a COX-2 promoter-driven luciferase construct was inducible by DON in RAW 264.7 cells. DON has also been shown to increase post-transcriptional stability of certain mRNA transcripts. Reports have shown an increase of TNF-a and IL-6 mRNA stability in RAW 264.7 cells as a result of DON exposure (25, 26). Increased IL-6 mRNA stability was also identified in murine peritoneal macrophages treated with DON (23). IL-2, another cytokine increased in the presence of DON, was found to have increased mRNA stability in DON-exposed EL-4 thymoma cells (19, 27). Additionally, the stability of luciferase transcripts was increased in a construct including the COX-2 3’ untranslated region (UTR) in RAW 264.7 cells (24). Taken together, DON is capable of inducing a variety of immune molecules, both in cell culture and in whole animals. The induction has been attributed to alterations at both the transcriptional level, through increased activation of transcription factors, and the post-transcriptional level, by increasing mRNA stability. Modulation of intracellular signaling by DON DON modulates intracellular signaling cascades. Yang et al. (28) showed that mitogen-activated protein kinases (MAPKs) were activated by trichothecenes in RAW 14 264.7 cells. Additionally, Zhou et al. (29) determined that along with increased activation of MAPKs in U937 cells, double-stranded RNA-activated protein kinase R (PKR) was also involved in DON’s effect on cells. PKR is a kinase activated by extracellular stimuli, such as exposure to double-stranded RNA, which has been linked to activation of NF-KB through the ItcB kinase (IKK) complex (30, 31). PKR has also been found to be an upstream element in the “ribotoxic stress response”. The ribotoxic stress response is a pathway of signal transduction whereby damage to the 28S rRNA by a chemical triggers activation of a downstream signaling pathway, such as those mediated by MAPKs (32). In U937 cells transfected with a PKR antisense vector, reduced phosphorylation was detected for all three MAPK pathways as well as a decrease in apoptosis following DON exposure (29). Interleukin (IL)—8 Chemokines, a subgroup of cytokines, affect chemotaxis and other aspects of leukocyte behavior including the extravasation process important in the inflammatory cascade. Chemokines are broken down into 2 families based on the position of conserved cysteines. One family is the CC chemokines and the other is the CXC chemokines, which includes IL-8. Along with the distinctive CXC sequence, IL-8 protein has sequences specific for neutrophil chemotaxis (33) and for angiogenesis (34). IL-8 can be produced in almost all nucleated cells (35) under a variety of conditions. Cell types that produce IL-8 include monocytes (36-3 8), neutrophils (39, 40), T lymphocytes (41-43), natural killer cells (44), fibroblasts (45, 46), macrophages (8, 47), 15 endothelial cells (48-51) and epithelial cells (52, 53). Stimuli for IL-8 production can act on a variety of cells or may be highly selective (54). Stimuli include tumor necrosis factor (TNF)-a, IL-1 [3, H202, as well as viral and bacterial infections. IL-8 induces adherence of neutrophils to vascular endothelium and triggers extravasation into tissues (55). Other functions of IL-8 include initiation of the acute inflammatory response (56) and cell type specific functions such as chemotaxis and shape change in neutrophils, induction of histamine and leukotriene release in basophils (44). The chromosomal location for lL-8 gene in humans is at 4q12-21 and this gene consists of 4 exons with 3 introns (54, 57) and results in a precursor composed of 99 amino acids. After cleavage of a 20 amino acid leader sequence, the functional IL-8 is secreted (3 5). This protein is resistant to inactivation by plasma peptidases, heat, pH extremes, and other denaturing treatments. Activity of the IL-8 protein is lost, however, if the disulfide bonds holding the cysteines together are broken (35). Correlation of IL-8 levels with disease Elevated IL-8 levels have been seen in serum, urine, and at sites of inflammation in a variety of conditions (58-61). Due to its varied functions, IL-8 has been implicated in many diseases. Much damage can be done during disease states by the presence of IL- 8 drawing neutrophils to specific areas and triggering activation of an inflammatory response. Table 1.1 presents a list of the various conditions in which IL-8 plays a role. Several studies have focused on identifying gene products and mediators that might be responsible for symptoms observed in inflammatory conditions. Elevated levels of multiple proinflammatory molecules, including IL-8, are expressed in psoriatic skin 16 Table 1.1: Conditions with lL-8 involvement. Condition Reference Anti-IL-8 treatment can lessen disease acute immune complex-type glomerulonephritis (i.e. IgA nephropathy); LPS/IL-l induced (61) arthritis; LPS-induced dermatitis; lung perfusion injury Elevated IL-8 levels implicated in disease psoriasis (62, 63) poststreptococcal glomerulonephritis (64) Familial Mediterranean fever (65) ovarian hyperstimulation syndrome (66) pulmonary fibrosis (67) alcoholic hepatitis (68, 69) IgA nephropathy (70) joint inflammatory disease (rheumatoid . . (45, 46, 71) arthritis and gout) cystic fibrosis (72, 73) hyperoxia (74, 75) inflammatory bowel disease (Crohn's . (76, 77) disease) bacterial toxin exposure or bacterial . . (78-81) infections Kawasaki disease (82) l7 models when compared to normal controls (63). Besbas et al. (64) found elevated levels of IL-8 in urine and serum of children in the acute phase of poststreptococcal glomerulonephritis compared to the resolution phase and the controls. Familial Mediterranean fever (F MP) is another condition with elevated lL-8 (65). FMF has recurrent attacks of inflammation in the membranes that line the abdominal cavity and lungs, arthritis, and fever. Elevated IL-8 and soluble intercellular adhesion molecule (sICAM)-l was seen in patients with F MF, both during the active attacks and while in remission, compared to patients without F MF . The elevated IL-8 and sICAM-l levels lead the authors to suggest that neutrophil activation and adhesion is increased in FMF. While the abovementioned studies have not reported a statistical correlation between increased IL-8 to events leading to inflammation or severity of the condition, several studies have. Most commonly, elevated IL-8 levels have correlated with an increased presence of a cell type necessary for symptoms of the condition. Elevated levels of IL-6 and IL-8 significantly correlated with biological characteristics of ovarian hyperstimulation syndrome (66). A similar trend was seen with lL-8 and psoriasis. An increased density of mast cells, thought to have a role in inflammation in psoriasis, as well as increased density of IL-8 producing keratinocytes in the psoriatic skin with lesions when compared to the‘psoriatic skin without lesions and normal skin was observed (62). There was a correlation between the elevated IL-8 levels produced by the keratinocytes and increased density of mast cells. In idiopathic pulmonary fibrosis (IPF), Beeh et al. (67) presented data that correlated increased sputum lL-8 levels with increased sputum neutrophil levels. Accumulation of neutrophils and other inflammatory cells in the alveolar space is a characteristic seen in IPF. The increased sputum 18 neutrophil levels also correlated with impairment of lung function. The authors concluded that neutrophils were responsible for the inflammation observed with IPF . In patients with alcoholic hepatitis, Bird (68) found a correlation between increased plasma and liver IL-8 levels and increased infiltration of neutrophils into the liver. This study also reported that the highest IL-8 levels were seen in patients that died within four weeks of hospital admission. Huang et al. (69) also observed a correlation between patient death and the high IL-8 levels in alcoholic hepatitis. A correlation was also seen between elevated IL-8 levels and the severity of liver injury. A similar trend was seen in a study conducted by Huang et al. (70) on IgA nephropathy. IL-8 levels were measured in patients with IgA nephropathy, a type of glomerulonephritis. There was a significant correlation between elevated IL-8 levels and increases in disease activity as measured by urinary protein and urinary casts. The authors also found a significant correlation between increased IL-8 levels and increased tubular damage in the kidneys. Harada et al. (61) summarized data on the effects of a neutralizing antibody against IL-8 in LPS-induced dermatitis, LPS+IL-1-induced arthritis, lung reperfusion injury, and acute immune complex-type glomerulonephritis. The authors found that neutrophil infiltration is inhibited with anti-IL-8 injections in all models examined. Anti- IL-8 is able to prevent skin redness in the dermatitis, destruction of the pulmonary architecture in lung reperfusion, and brought the urinary levels of protein and albumin back to normal in the acute immune complex-type glomerulonephritis. These data led the authors to conclude that IL-8 is involved in the activation and infiltration of neutrophils in the model inflammatory conditions and that by preventing normal neutrophil function, tissue damage could be avoided in these conditions. 19 Mechanisms of IL-8 induction in disease In contrast to the above studies, which noted elevated IL-8 alone or correlated with disease markers, other studies have focused on discovering potential pathways that lead to IL-8 production in inflammatory conditions. Mechanisms of IL-8 production in rheumatoid arthritis were investigated by Georganas et al. (45). Fibroblast-like synoviocytes (FLS), obtained from patients with rheumatoid arthritis, produced IL-8 after exposure to IL-10, mediated by NF -icB activation. AP-l and C/EBPB were not involved in IL-8 expression by FLS. Suzuki et al. (46) investigated the role of p38 in IL-8 production in rheumatoid synovial fibroblasts (RSF) isolated from tissue biopsy samples of patients with active rheumatoid arthritis. A p38 inhibitor was able to suppress TNF-or- or IL-lfi-induced IL-8 protein, but was unable to inhibit IL-8 mRNA or block p65 translocation. The authors concluded that while the p38 pathway was not involved in the NF-KB cascade, an inhibitor of p38 may be useful in addition to conventional anti- rheumatic drugs that do act through the NF-KB pathway. In gout, another inflammatory condition that affects joints, IL-8 expression in the joint space is a necessary event in the pathogenesis of the disease. A model of gout was investigated with THP-l monocytes exposed to monosodium urate monohydrate (MSU) or calcium pyrophosphate dihydrate (CPPD) (71). MSU and CPPD triggered an increase in ERK, p38, and JN K activation. Additionally, both an ERK inhibitor and a NF-KB inhibitor were able to reduce IL-8 mRNA levels in THP-1 cells stimulated with either MSU or CPPD. A p38 inhibitor was able to suppress the level of CPPD-induced IL-8 20 mRNA but not MSU-induced IL-8 mRNA. Both MSU and CPPD increased the binding of AP-l and NF-KB to the IL-8 promoter. The ERK inhibitor was able to block the increased binding of AP-l and reduce the binding of NF -KB for both MSU and CPPD treatments, but the p38 inhibitor was only able to block the increased AP-l binding. Using an IL-8 promoter-driven luciferase construct, mutations in the NF-KB and AP-l sites reduced MSU- and CPPD-induced luciferase. Mutation in the C/EBPB site only partially reduced CPPD-induced luciferase but did not reduce MSU-induced levels. The authors concluded that these data suggested potential targets for treatment of gout (MSU deposition) and CPPD crystal deposition disease. In lungs of patients with cystic fibrosis, inflammation is primarily caused by neutrophils and their protein secretions, including neutrophil elastase (NE) (73). Devaney et al. (72) examined NE-induced IL-8 production in human bronchial epithelial cells. The authors found that an anti-Toll-like receptor (TLR) was able to inhibit NE- induced IL-8 production significantly. Identification of events involved in NE-induced inflammation provides potential targets for treatments that may be more effective than the standard anti-protease therapies. Deaton et al. (75) examined the effects of hyperoxia in U937 cells. Oxygen toxicity occurs in patients that require high oxygen concentrations. Infiltration of neutrophils and alveolar damage can be seen in these patients. Both alveolar macrophages and U937 cells had hyperoxia-induced IL—8 levels. The hyperoxia-induced IL-8 expression in U937 cells could be inhibited by treatment with dexamethasone, a corticosteroid. Chemotactic activity of the alveolar macrophages and U937 cells was reduced when an antibody to IL-8 was added. These results indicate that IL-8 is 21 produced after exposure to high levels of oxygen and that the lL-8 production may play a role in neutrophil infiltration. The authors concluded that agents that suppress IL-8, such as dexamethasone, may be useful treatments to help prevent lung injury induced by oxygen toxicity if additional evidence can link IL-8 to this condition. A later study by Allen et al. (74) indicated that IL-8 is important for high oxygen level-induced lung injury. Elevated levels of IL-8 and other chemokines occur in Crohn’s disease, a chronic inflammatory bowel disease. Banks et al. (77) correlated increased chemokine expression with increased activity of the disease, characterized by local inflammation and tissue damage. Alzoghaibi et al. (76) investigated the role of dietary fatty acids in stimulation of lL-8 in patients with Crohn’s disease. Intestinal smooth muscle cells from patients with Crohn’s disease exhibited a higher level of IL-8 production than cells from healthy patients. In the cells from patients with Crohn’s disease, linoleic acid, but not oleic acid, increased IL-8 production, leading the authors to suggest that a diet with oleic acid, rather than linoleic acid may help to decrease the inflammation that occurs with this disease. Several other studies have focused on IL-8 involvement in disease conditions triggered by bacteria or bacterial toxins. Hang et al. (80) examined a model urinary tract infection using mice that lacked the murine homologue of the IL-8 receptor (mIL-8Rh KO). Neutrophils in these mice could not respond to IL-8-like stimuli, but retained other normal functions. mIL-8Rh KO mice had elevated MlP-2 (a molecule in mice that has a similar role to lL-8 in humans) levels in response to an intravesical Escherichia coli infection that continued to increase in the mIL-8Rh KO mice after control mice returned 22 to normal. While neutrophils in mIL-8h KO mice were able to migrate to the site of the E. coli infection, they were unable to clear the infections and, rather, caused kidney damage due to aberrant, uncontrolled responses. In another study with a different bacterial toxin, Wamy et al. (81) treated THP-l monocytes with Clostridium diflicile toxin A, which is enterotoxic and causes acute inflammation with neutrophil infiltration. This toxin induced IL-8 production and this induction was significantly reduced by an inhibitor of ERK or p38. Using an IL-8 promoter-driven luciferase construct, it was observed that dominant negative inhibition of the upstream kinases of the p38 pathway blocked luciferase expression. While oral treatment with the p38 inhibitor did not prevent intestinal inflammation, direct injection of the chemical into the intestinal lumen significantly reduced the severity of the inflammation, including reduction of neutrophil infiltration. These data implicate p38 in the neutrophil recruitment seen in the enteritis as a result of toxin A exposure. In a later study of C. difiicile toxin A, Johal et al. (79) used T84 epithelial cells to determine the effect of different toxin concentrations on expression of IL-8 and TGF-B, a protective cytokine. Toxin A concentrations of 10 ng/ml or less increased TGF-B levels but did not alter IL-8 levels. Concentrations of toxin A greater than 10 ng/ml increased IL-8 but not TGF-B levels. The authors concluded that the development and severity of the inflammation as a result of C. diflicile infection may depend on the exposure of the epithelial cells to the toxin and the expression of proinflammatory or protective molecules. Dahan et al. (78) examined the mechanisms behind IL-8 production in T84 intestinal epithelial cells as a result of infection with enterohemorrhagic E. coli (EHEC). 23 EHEC causes gastroenteritis and hemorrhagic colitis. Activation of both AP-l and NF- KB resulted from EHEC infection in T84 cells. Also activated were p3 8, ERK, and JNK. Inhibitors for ERK, p38, and NF -KB significantly inhibited IL-8 levels, individually, and had an even greater inhibition when used in combination. These results may help develop treatments to lessen the severity of EHEC infections. Although several of the above-mentioned studies related the host response to IL—8 with the severity of the inflammatory condition, one study utilized IL-8’s Chemotactic ability to reduce potential complications from an inflammatory condition. Asano et al. (82) determined that mononuclear cells and neutrophils had elevated IL-8 protein and mRNA along with decreased Chemotactic activity of the neutrophils in patients with Kawasaki disease (acute multisystem vasculitis) when treated with intravenous immunoglobulin (IVIG). The authors concluded that the increased IL-8 in mononuclear cells, neutrophils, and plasma may help to prevent neutrophils from accumulating at inflammation sites, thereby reducing the risk of an aneurysm. Regulation of IL-8 expression Since IL-8 is a biologically potent molecule, it is very important that its production be strictly regulated by cells. Regulation of IL-8 can occur on both the transcriptional and post-transcriptional level. (Table 1.2) On the transcriptional level, silencing is done to suppress basal transcription (83, 84). Various transcription factors act in combination to suppress transcription of a gene. Genes, such as the cytokine genes, 24 require multiple steps in order to activate gene expression and there are numerous sites and levels where negative regulation could occur (19, 83). IL-8 expression due to multiple transcription factors Transcription factor binding sites located in the IL-8 promoter include those for activator protein-1 (AP-1), CCAAT/enhancer binding protein B (C/EBPB), octamer-l (Oct-1), nuclear factor- KB (NF -i Cell survival 3 mRNA stability " ctin reo canizatio ' P i ' ll n‘smplmr AU. .atm’l www.merckbiosciences.co. uk/popup/CBC/ip_mapk_si gnaling_pathway.htm Figure 1.3: General MAPK pathway. (This image is in color.) 41 owmmommm . o - . - 3 50:08 .. Gm: oz Em: an _ “2 8. z a _ at 58824833. $422.. 83:3 - 830258 owmmommm £83,233 Ann: 02 £88m 0585C. mu. 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(130) found that Mycoplasmafermentans lipid-associated membrane proteins (LAMPf) triggered production of IL-8 in primary human monocytes and polymorphonuclear cells (PMN). Using chemical inhibitors, protein tyrosine kinases were found to be necessary for both monocytes and PMN while ERK activation had a larger role in PMNs than monocytes. When THP-l monocyte cells were transfected with a NF-KB or AP-l luciferase constructs, LAMPf was able to increase expression of both constructs. A NF-KB inhibitor was able to prevent IL-8 production in both monocytes and PMNs. Albanyan et al. (39) examined the role of ERK and p38 in type III group B Streptococcus-induced lL-8 in primary human neutrophils. In contrast to the previous study linking ERK activation to IL-8 expression, an ERK inhibitor was only able to partially reduce lL-8 protein levels in Streptococcus-treated primary human neutrophils. Another MAPK family, p38, was found to be responsible for the induction of IL-8 protein as determined with a p38 inhibitor. This difference in the degree of importance of the ERK and p38 pathways could be due to the fact that the cells were isolated from different individuals or use of different stimuli. MAPKs activate transcription factors MAPKs can also play a role in both transcriptional induction and in post- transcriptional stability. Several studies have linked the three MAPKs families to activation of transcription factors involved in lL-8 expression. Alcom et al. (122) also found that the ERK pathway had a role in lL-8 production in A549 epithelial cells. 44 Adenovius serotype 7 (Ad7) increased IL-8 production through an activation of transcription. Ad7 was found to increase ERK activation, and with an inhibitor of ERK, IL-8 production was inhibited at the transcriptional level, though the specific transcription factors were not determined. Li et al. (121) used bronchial epithelial cells, 16HBE14o-, to examine the role of the MAPKs pathways in TNF-a-induced IL-8 expression. Increased NF -KB binding, composed of p65 and p50 subunits, occurred as a result of TNF-a exposure. With an IL- 8 promoter-driven luciferase construct, the authors determined that NF-KB was required for TNF-a-induced transcription. Inhibition of ERK or JNK prevented transcription, while inhibition of p38 and ERK significantly inhibited only IL-8 protein. JNK was found to be important for NF-KB activation, while ERK was found to be important for AP—l activation. The AP-l site also was critical for basal transcription as a mutation reduced basal transcription. Using U937 monocytes, Josse et al. (124) found that H202 causes an increase in IL-8 mRNA. Using IL-8 promoter-driven luciferase constructs, the authors determined that an increase in NF -KB, specifically p65, p50, c-Rel was responsible for transcriptional activation. AP-l was also found to contribute to activation of the IL-8 promoter, although a mutation in the AP-l binding site had only half the decrease in luciferase levels than that seen with a mutation in the NF-KB binding site. Examination of the p38 and ERK pathway revealed that p38 was necessary for increased IL-8 mRNA stability while ERK was not. Na et al. (126) determined that radicicol, an anti-fungal antibiotic, is able to inhibit both IL-8 protein and mRNA expression in PMA + LPS stimulated THP-l 45 monocytes. Both NF -KB and AP-l are necessary for PMA + LPS-induced IL-8, with increased binding observed after treatment. C/EBPB was not involved in either the PMA + LPS-induced lL-8 production or the inhibition by radicicol. The authors found, however, that THP-l cells had a strong constitutive binding of C/EBPB. PMA + LPS treatment also activated ERK, p38, and JN K, with inhibition of ERK and p38 occurring afier radicicol treatment. Additional inhibitors for ERK and p38 were also able to inhibit PMA + LPS-induced IL-8 mRNA levels. Further examination of the ERK pathway revealed that binding of both PMA + LPS-induced AP-I and NF-KB were inhibited after addition of an ERK inhibitor. Addition of a p38 inhibitor did not alter NF-KB binding and only partially inhibited AP-l binding. Bhattacharyya et al. (127) used both primary human monocytes and THP-l monocytes to determine the role of the MAPKs pathways in Helicobacter pylori-induced lL-8. H. pylori activated ERK, p38, and JNK in human monocytes and THP-1 cells. Both an ERK and p38 phosphorylation was prevented by kinase inhibitors. The upstream signaling molecules of ERK (MEKI), p38 (MKK6 and MKK3), and JNK (MKK7) were found to be necessary for H. pylori activation of the MAPKs pathways. NF-KB binding was increased, though not due to activation of the MAPKs pathways, while increased AP-l binding was attributed to the activation of the ERK and JNK pathways. The NF-KB binding site was also necessary for H. pylori-induced luciferase in THP-1 cells transfected with an IL-8 promoter-driven luciferase construct. However, although increased AP-I binding was seen, a mutation in the AP-l binding site only partially reduced luciferase levels. 46 Friedland et al. (128) examined IL-8 production afier phagocytosis of zymosan in THP-l monocyte cells. Zymosan treatment induced NF-KB binding. PKA, PKC, and tyrosine kinase inhibitors were unable to prevent both the increase in NF-KB binding and the IL-8 mRNA production. Significant inhibition of zymosan-induced IL-8 production was seen after inhibition of the p38 pathway while only partial inhibition was observed with an ERK inhibitor. An interesting aspect of their study was that the number of NF - KB binding complexes changed depending on the clone of TI-IP-l cell used for the experiment. The authors noted the reason for this difference was unknown, as all cells used behaved in a similar manner. MAPKs also mediate post-transcriptional mechanisms MAPKs pathways also play a role in increasing mRNA stability. A substrate of p38, MK2, is involved in stabilization of mRNAs containing AU rich elements (132), including TNF-a, in murine cells. In another study, Ridley et al. (133) found that blocking p38 increased the decay of COX-2 mRNA. An increase in p38 activation leads to increased stability of IL-8 mRNA in several cell types. Winzen et al. (119) found that, in HeLa epithelial cells, constitutive expression of MEKK] was capable of increasing mRNA stability of IL-8 mRNA from less than 15 minutes to around 110 minutes. Since MEKKI can be involved in multiple pathways, the effects of constitutive expression of MKK7, NIK, and MKK6 were also checked. It was determined that only MKK6, an upstream signaling molecule in the p38 pathway, was involved in stabilization of IL-8 mRNA. 47 Frevel et al. (123) similarly found involvement of the p38 pathway in increased IL-8 mRNA stability. Here, a 43.5 fold increase in IL-8 mRNA levels was observed in TNF-a-treated THP-l monocytes. With the addition of SB203580, a p38 inhibitor, the half-life of the lL-8 transcript decreased to 17 minutes; almost 3 times lower than in cells without the inhibitor thus implicating a role for p38 in the increased half-life. Another study (120), investigated the role both the ERK and p38 pathways have in stabilization of the lL-8 mRNA in HT-29 epithelial cells. The ERK and p38 pathways both were determined to have a role in stabilization of TNF-a-induced IL-8 expression. Both an ERK inhibitor, PD98059, and a p38 inhibitor, SB203580, significantly reduce TNF-a- induced IL-8 protein levels by greater than 40% and about 30%, respectively. The authors concluded that the inhibition of IL-8 seen with the two inhibitors was not due to a difference in AP-I or NF-KB binding, but to a destabilization of IL-8 mRNA. AMPKS acts in tandem for both transcription and post-transcriptional mechanisms MAPKs pathways have also been found to cause increases in IL-8 by stimulating a signaling cascade that can end in activation of transcription and an increase in mRNA stability. Holtmann et al. (118) used KB epithelial and HEK-293 cells to determine the kinases involved in lL-8 transcription and lL-8 mRNA stability. Cells were transfected with constitutive expression vectors and IL-8 protein was measured. The authors determined that MKK7, SAPK/JN K pathway, NIK, and MEKKI were important for IL-8 expression. Mutations of the AP-l and NF -KB site caused a decrease in basal activity and prevented induction by NIK or MKK7. Increased IL-8 mRNA stability was also seen. It was determined that MKK6 increased IL-8 mRNA stability. 48 Other kinases implicated in IL-8 expression Additional kinases have also been implicated in IL-8 expression. Muhl et al. (129) found that inhibition of protein tyrosine kinases or p38 was able to prevent nitric oxide-induced IL-8 protein in U937 cells. Ameixa and Friedland (125) also found that protein tyrosine kinases were important for Mycobacterium tuberculosis-induced IL-8 expression. They examined the role of ERK, p38, and protein tyrosine kinases in M. tuberculosis-induced IL-8 expression in primary human monocytes. Using inhibitors, the authors found that ERK was not involved in M. tuberculosis-induced IL-8, though necessary for LPS-induced IL-8. The p38 inhibitor did not reduce either M tuberculosis- or LPS-induced IL-8. A broad spectrum protein tyrosine inhibitor inhibited both the M. tuberculosis— and LPS-induced IL-8 protein and mRNA. PKR has also been linked to IL-8 expression via NF «B, either by activating NF - KB inducible kinase (NIK) (31) or by interacting with the IKK complex (30, 31). Rho proteins were also found to have a role in IL-8 expression. Hippenstiel et al. (51) determined that inhibition of small GTP-binding Rho proteins prevented LPS-induced IL- 8 expression in human umbilical vein endothelial cells (HUVECs). Additionally, inhibition of the p38 pathway also reduced IL-8 expression. Both the p38 and Rho inhibitors prevented LPS-induced activation of a NF -KB dependent luciferase reporter construct. The authors concluded that different pathways, one involving Rho proteins and the other consisting of the p38 pathway, both lead to NF -l(B activation necessary for IL-8 expression. Buss et al. (131) found that, in HeLa epithelial and HEK-293 cells phosphorylation of p65 was induced by lL—IB or TNF-a. The activation of p65 was not 49 altered by ERK, JN K, or p38 inhibitors; however a proteosome inhibitor, that prevented IKB degradation, increased basal activation but prevented inducible activation. As shown by these previous studies, stimuli can activate multiple signaling pathways, other than the MAPK pathways, that result in activation of downstream proteins and drive lL-8 expression. U937 cells U937 cells are promonocytes that were isolated from the pleural effusion of an individual with diffuse histiocytic lymphoma (12). These cells can be differentiated to form neutrophils, with exposure to retinoic acid, or macrophages, with exposure to PMA. U937 cells can produce a variety of cytokines and chemokines, including IL-8, both in the undifferentiated monocytic form and as a macrophage. Using cytogenetic methods four different sublines of U937 cells have been discovered: U937-1, U937-2, U937-3, and U937-4 (134, 135). This cell line has between 40 and 60 chromosomes, double to triple the normal complement in human cells (134, 136). Examination of U937-1 with whole chromosome painting and comparative genomic hybridization has provided evidence that chromosome 4, the location of the lL-8 gene, has losses of genomic material at 4q and an extra copy of 4p added to chromosome 16p (137). In a separate cytogenetic study of U937 cells, obtained directly from American Type Culture Collection, a translocation from 4p to 16p was observed. However, losses of genomic material on chromosome 4 were not observed with comparative genomic hybridization 50 (138). This difference could be a reflection of differences in the cells or could be due to the different sources of DNA used in the comparative genomic hybridization. Another alteration in U937 cells is the lack of production of p53, a tumor suppressor that can induce apoptosis and cell cycle arrest, protein or mRNA (139). In an additional study published in 1992, Sugimoto et al. (140) observed that 46 bases were missing from'an exon of the p53 mRNA as a result of an abnormal splicing event. In an interesting connection, mutations of the p53 gene have been linked to both constitutive NF-KB activation and high basal IL-8 protein production (141). The high basal expression was decreased in cells transfected with an expression vector for normal p53. Transfection with normal p53 also reduced NF-KB transcriptional activity, measured with a NF-KB-driven luciferase construct, but not NF-KB DNA binding as seen in an EMSA. Experiments conducted in an earlier study observed that mutations in either p65, a subunit of NF-KB, or p53 result in an increase in transcriptional activity of p53 or p65, respectively (142). U937 cells were chosen for the research in this dissertation for a variety of reasons. First, actively dividing cells, such as immune cells are very susceptible to the effects of DON (9, 13). Secondly, as DON can be found in plasma of mice, given an oral exposure, within 30 minutes it is likely that blood monocytes are in contact with DON (143). Third, U937 cells, in a macrophage differentiated form, have already been shown to produce lL-8 and cytokines as a result of DON exposure (8). Fourth, U937 cells have been utilized in numerous studies (75, 99, 101, 102, 144) and produce robust levels of IL- 8. 51 CHAPTER 2: ROLE OF MlTOGEN-ACTIVATED PROTEIN KINASES IN DEOXYNIVALENOL-INDUCED INTERLEUKlN-8 EXPRESSION Part of the data in this chapter has been published in Islam, 2., IS. Gray, and J .J. Pestka, p38 Mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes. Toxicol Appl Pharmacol, 2006. 213(3): p. 235-44. @2101 Deoxynivalenol (DON), a trichothecene mycotoxin, is a common contaminant of grain crops worldwide. At low concentrations, DON is capable of stimulating production of immune proteins in a variety of cells. Previous reports have determined that mitogen- activated protein kinase pathways (MAPKs) are also activated upon exposure to DON and these kinases might be upstream elements in the expression of immune mediators. The hypothesis tested in this study was that MAPKs play a central role in DON-induced lL-8 expression in the U937 human monocyte model. Activation of three major pathways, p38, ERK, and JNK, was involved in the DON-induced lL-8 expression. Of these, the p38 pathway contributed to the greatest extent as reflected by increased IL-8 protein, mRNA, and hnRNA levels and the extent of inhibition with selective MAPK inhibitors. 52 Introduction Deoxynivalenol (DON, vomitoxin) is a fungal secondary metabolite, classified as a trichothecene, which is produced by F usarium gramineamm or F usarium culmorum. It is one of the most common mycotoxins found in cereal grains (6) and is a frequent contaminant of wheat, corn, and barley worldwide (13). DON is a very stable compound that is capable of withstanding the high temperatures used in processing and cooking of foods (13). Because of this, DON that is present in the unprocessed grains can still be present in processed foods and be consumed by humans and animals. DON is both immunosuppressive and immunostimulatory in experimental animals, depending on dose (15). The immunostimulatory effects of DON, seen at lower doses (16), includes the induction of cytokines in vitro and in vivo. Interference with normal mechanisms of gene regulation appears to drive cytokine production. In macrophage-differentiated U937 cells, DON can induce secretion of IL-6 and IL-8 (8). As a prerequisite of the induction of cytokines, DON activates signaling cascades in cells. Yang et al. (28) showed that mitogen-activated protein kinases (MAPKs) were activated by trichothecenes in RAW 264.7 cells. Additionally, Zhou et al. (29) determined that, prior to inducing MAPK activation in U937 cells, DON activates double stranded RNA activated protein kinase (PKR). IL-8 is a biologically potent molecule that is produced by a wide variety of cell types, including monocytes (36-3 8), neutrophils (39, 40), T lymphocytes (41 , 42), natural killer cells (44), fibroblasts (44-46), macrophages (8, 47) and endothelial cells (49-51). IL-8 is classified as a CXC chemokine containing sequences specific for neutrophil chemotaxis (33) and for angiogenesis (34). Additional functions of IL-8 include 53 initiation of the acute inflammatory response (56) and cell specific functions such as neutrophil shape change as well as histamine induction and leukotriene release in basophils (44). IL-8’s varied actions have been implicated in several diseases. Extensive tissue damage can occur following lL-8-mediated recruitment of neutrophils to specific areas and subsequent inflammation. Elevated lL-8 levels have been seen in serum from patients with severe ovarian hyperstimulation (58) and respiratory syncytial virus bronchiolitis (59). Elevated urinary IL-8 is seen in patients with glomerulonephritis (61). IL-8 is found in inflammatory sites in patients with psoriatic scales, rheumatoid arthritis, cystic fibrosis (60), gouty arthritis, and adult respiratory distress syndrome (61). In support of lL-8’s role in disease, administration of an IL-8 antibody lessens disease severity in several animal models, including lung perfusion injury and lipopolysaccaride (LPS)/IL-1-induced arthritis (61). Regulation of IL-8 is critical and can occur at both the transcriptional and post- transcriptional levels. Transcription factor binding sites located in this promoter include AP-l, C/EBPB, nuclear factor- KB (NF-KB), Octamer-l (Oct-1), and NF-KB repressing factor (NRF). Combinations of two or three transcription factors, which varies according to cell type, are required to obtain the highest level of transcription. The three transcription factors that usually are present, in such complexes, are AP-l , C/EBPB, and NF-KB (54, 56, 145). One way that transcription factors are activated is via the MAPK signaling cascade. Once MAPKs are activated by phosphorylation, they translocate to the nucleus where they are capable of activating, primarily by phosphorylation, other proteins such as 54 transcription factors (1 16). The three main families of MAPKs are p38, extracellular signal-regulated kinases (ERKs), and c—Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKS) (115). p38 has been correlated to IL-8 transcription with cells stimulated by Clostridium dzflicile toxin A (81), Mycoplasma fermentans lipid-associated membrane protein (LAMPf ) (130), urate crystals (71), and phorbol-lZ-myristate-l3- acetate (PMA)/LPS (126). ERK (120, 121, 130) and JNK (121 , 127) have also been linked to IL—8 transcription. Besides transcriptional induction, MAPKs can also modulate in mRNA stability. A substrate of p38, MK2, has been found to be involved in stabilization of mRNA containing AU rich elements (132). Ridley et al. (133) found that blocking p38 increases the decay of COX-2 mRNA. p38 also increases the stability of TNF-induced IL—8 transcripts (120, 121), in cells transfected with constitutively active MEKI, which is upstream of p38, ERK, and JN K, and constitutively active MKK (MAPK kinase) 6, upstream of p38 (1 18). In the same study, constitutively active MKK7, a kinase capable of activating JNK, also could induce IL-8 expression. Our laboratory has previously shown that DON induces lL-8 protein production in macrophage-differentiated U937 cells (8). The purpose of this study was to test the hypothesis that MAPKs play a central role on DON-induced IL-8 expression in U937 cells, a human monocyte model. The results indicate that all three major MAPKs families were involved in the DON-induced expression of IL-8, although there was a difference in the relative contributions of p38, ERK, and JNK. Notably, p38 played the largest role in DON-induced lL-8 expression, with ERK and JNK having lesser roles. 55 Materials and Methods Cell culture U937 cells, obtained from American Type Culture Collection (ATCC; Manassas, VA), were isolated from the pleural effusion of an individual with diffuse histiocytic lymphoma (Sundstrom and Nilsson 1976). U937 cells were grown in RPMI-1640 supplemented with 10% FBS and 100 U/ml penicillin and 100 ug/ml streptomycin (Gibco BRL; Rockville, MD). Cells were incubated at 37°C with 6% CO2. Fresh grth media (RPMI-l640 supplemented with 10% FBS and 100 U/ml penicillin and 100 pg/ml streptomycin) was added as necessary to passage the cells. DON and/or the MAPKs inhibitors were incubated with U937 cells (1x106 cells/ml) for 3 h. The MAPK inhibitors SP600125 (JNK), PD98059 (ERK), and SB203580 (p38) were obtained from Calbiochem (San Diego, CA) and dissolved in dimethyl sulfoxide (DMSO). All other reagents are fi'om Sigma (St. Louis, MO) unless otherwise noted. Western Blot Total protein was isolated from U937 cells after a 30 minute exposure to 500 ng/ml DON. Briefly, cells were lysed with hot 1% (w/v) SDS buffer (1 mM sodium ortho-vanadate in 10 mM Tris, pH 7.4) collected, boiled for 5 minutes, sonicated for 30 sec, and centrifuged at 12,000 x g for 15 minutes. The supernatant was collected and the protein was quantitated with the DC Protein Quantitation kit (BioRad; Hercules, CA). Total protein (10 pg) was loaded on a 10% SDS-PAGE gel and run at 100 V. The gel was electrotransferred to a PDVF membrane (Amersham; Piscataway, NJ) overnight at 56 4°C. The membrane was then blocked in 5% (w/v) bovine serum albumin in TBST (20 mM Tris—HCI, pH 8 and 137 mM NaCl containing 0.1% Tween 20), washed and incubated with a primary antibody for phospho-p38, phospho-ERK, or phospho-JNK (rabbit IgG) (Cell Signaling; Beverly, MA) at room temperature for l h. The membrane was then washed and incubated with an anti-rabbit whole IgG (Cell Signaling) antibody conjugated with horseradish peroxidase for 1 h at room temperature. Bands were visualized with the ECL detection system (Amersham). Membranes were stripped, blocked, and incubated with nonphospho p38, ERK, or JN K (anti-rabbit IgG) (Cell Signaling) overnight at 4°C. The next day, the membrane was then washed and incubated with an anti-rabbit whole IgG (Cell Signaling) antibody conjugated with HRP for 1 h at room temperature. Bands were visualized with the ECL detection system (Amersham). MTT viability assay Cells were plated (200 pl/well) in 96 well plates at 1x106 cells/ml. DON, dissolved in RPMI-1640, and 25 pl of 5 mg/ml of MTT reagent (3-[4, 5-dimethylthiazol- 2-yl]-2, S-diphenyltetrazolium bromide) dissolved in Dulbecco’s phosphate buffered saline (PBS) were added and cells were incubated at 37°C with 6% CO2 for 3 h. Plates were centrifiiged at 300 x g for 10 minutes and the supernatant was removed. DMSO (125 pl) was added and the absorbance was read at 690-570 nm on a Vmax Kinetic Microplate Reader (Molecular Devices; Menlo Park, CA). Percent of the control was calculated for each well. 57 IL-8 protein determination After treatment, cell culture plates were centrifuged for 10 minutes at 300 x g and the supernatant collected and stored at -20°C. An OptELISA IL-8 kit (Pharmingen; San Diego, CA) was used 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 lOOpl. All samples were read at 450 nm in a Vmax Kinetic Microplate Reader (Molecular Devices). RNA isolation U937 cells were treated with DON for the 3 h and then the plates were centrifuged for 10 minutes at 300 x g, the supernatant removed, and the RNaqeous kit (Ambion; Austin, TX) was used to isolate the RNA. Samples were treated with DNA-free (Ambion) to remove gross DNA contamination. The manufacturer’s instructions were followed for both kits. Reverse transcription real-time PCR Reverse transcription real-time PCR was performed using One-Step PCR Master Mix (Applied Biosystems; Foster City, CA) and IL-8 Pre-Developed Assay Reagents (PDAR; Applied Biosystems) multiplexed with the 188 PDAR (Applied Biosystems). For the hnRNA, Primer Express software v 1.5 (Applied Biosystems) was used to make primer (base numbers 31 13351-31 13369 and 31 13431-31 13452) and probe (base 58 numbers 311341 1-3113429) selections from the NM_006216 (Genbank) sequence (IL-8 DNA). The primer/probe set was selected to include an exon-intron junction. Fold change was determined using the relative quantitation method. First, standard curves are created using dilutions of total RNA from LPS-treated U937 cell total RNA. An equation, for the trend line of the standard values, is used to convert the Ct values obtained in the assay to nanogram amounts of the target. The amounts were normalized by dividing the IL-8 value by the 18S (the endogenous control) value. Relative expression is obtained by dividing all normalized values by the average of the control normalized value. Reaction conditions and PCR program were all following the manufacturer’s instructions using an ABI 7700 (96 wells) or 7900HT (384 wells), both at the Michigan State University’s Genomics Technology and Support Facility, depending on the number of samples to be analyzed. Statistics Data were analyzed with SigmaStat v 1.0 (Jandel Scientific; San Rafael, CA). [L- 8 protein and RNA data were analyzed using one way analysis of variance (ANOVA) with Student-Newman-Keuls Method for pairwise comparisons. Treatment groups that have a p-value of < 0.05 were considered significant. 59 BM DON activated p38 and MK in U93 7 cells To confirm that MAPKs are activated by exposure to DON, U937 cells were treated with 500 ng/ml of DON for 30 minutes. Phospho-p38 levels were increased in the DON group as compared to untreated cells (Figure 2.1A). Nonphospho-p38 levels were approximately equivalent in all samples. A similar increase was observed for phospho- JNK samples even though the nonphospho-JNK bands was less abundent in DON-treated samples as compared to the control samples (Figure 2.1 B). There were no apparent differences in the phospho-ERK levels in the DON-treated and untreated cells (Figure 2.1 C). Basal ERK activation in control samples was apparent. This was possibly due to the fact that the U937 cells were in suspension and required centrifugation to isolate the total protein, which might result in artifactual activation. Phospho-ERK levels were also examined at a 3 h timepoint, with no difference between the control and DON-treated samples. However, the overall activation level was reduced compared to the 30 minute samples (data not shown). Cell viability was not affected by the MAPKs inhibitors An MTT assay was conducted on the U937 cells to determine if the MAPK inhibitors alter the viability of the treated cells using concentrations employed previously by other investigators (25, 39, 127, 128). If the inhibitors were toxic, any observed inhibition may be the caused by fewer cells producing less IL-8 rather than an actual inhibition of IL-8 expression. When cells were treated with 2 pM SB203580, 10 pM PD98059, 1 pM SP600125 or 500 ng/ml DON (Figure 2.2), none of the three MAPK 60 A. Control 500ng/ml DON Control 500ng/ml DON P-JNK (p54) ‘ ‘""‘”"""‘ P-JNK (p46) . _ JNK (p54) . JNK (“46) if... ' C. P-ERK (p44) P-ERK (p42) ERK (p44) _f ERK (p42) Figure 2.1: Exposure to DON increases phosphorylation of p38, JN K, and ERK in U937 cells. Western blot of U937 total protein samples isolated after a 30 minute exposure to 500 ng/ml DON. (A) Phospho-p38 and nonphospho-p38 (B) Phospho-JNK and nonphospho-JNK (C) Phospho-ERK and nonphospho-ERK. (n = 3) These data are representative of 2 independent experiments. 61 140 120 ‘ 100 - _T_ _L_ __.L.1 80 - I 60~ MTT Response 40- 20— VH DON SB PD SP Figure 2.2: Viability of U937 cells does not decrease with exposure to MAPK inhibitors. MTT with U937 cells after a 3 hour exposure to 500 ng/ml DON, 2.0 pM SB203580, 10 pM PD98059, or 1.0 pM SP600125. Data are the mean i SEM (n = 3). These data are representative of 2 independent experiments. 62 inhibitors significantly altered the viability of the cells. At 3 h, viabilities of the cells treated with 500 ng/ml DON was not significantly different than from non-treated cells. AMPK inhibitors were able to suppress DON-induced IL-8 protein levels in U93 7cells Since 500 ng/ml DON strongly induced lL-8 (Figure 2.3), it was used in all subsequent experiments. IL-8 concentration in culture media was measured afler 3 h exposure to 500 ng/ml DON and/or MAPKs inhibitors (2 pM 83203580, 1 pM SP600125, or 10 pM PD98059). IL-8 protein from the cells co-treated with DON and SB203580, the p38 inhibitor, was reduced by 97% (p < 0.05) (Figure 2.3). Neither the JNK inhibitor, SP600125, nor the MEK/ERK inhibitor, PD98059, significantly inhibited the DON-induced lL-8 protein levels. MAPK inhibitors were able to suppress DON-induced IL-8 mRNA levels in U93 7 cells 1L-8 mRNA levels were determined afier 3 h exposure to 500 ng/ml DON and/or 2 pM SB203580, 1 pM SP600125, or 10 pM PD98059 (Figure 2.4). All three of the inhibitors significantly inhibited IL-8 mRNA levels (p < 0.05). SB203580 inhibited approximately 90% of the DON-induced IL-8 mRNA levels while SP600125 and PD98059 caused approximately 50% inhibition. Marked SB203580 inhibition of the IL- 8 mRNA was similar to that seen at the protein level. 63 1200 b 1000 ~ i b E T b g: 800 - I .2; 0) § 600 ~ 0. °? =3 400 - 200 - a a a a a 0 F71 . "7 I“?! FT? . [—77 . Control DON SB DON SP DON PD DON +SB +SP +pD Figure 2.3: DON-induced 1L-8 protein levels are suppressed by a p38 inhibitor. IL-8 protein levels in U937 culture supernatant after 3 hours exposure to 500 ng/ml DON, and/or 2.0 pM 83203580, 10 pM PD98059, or 1.0 pM SP600125. Bars without the same letter are statistically different (p < 0.05). Data are the mean :1: SEM (n = 5). These data are representative of 3 independent experiments. 64 20 b g T 9 Q 15- < z D: E e co :2 10- fi' 0) .2 E m (I 5- a a a * a a O *I I If I :1 I ETj I Control DON SB DO SP DON PD 00 +SB +sp +PD Figure 2.4: DON-induced IL-8 mRNA levels are suppressed by MAPKs inhibitors. IL-8 mRNA levels in U937 after a 3 hour exposure to 500 ng/ml DON, and/or 2.0 pM SB203580, 10 pM PD98059, or 1.0 pM SP600125. Bars without the same letter are statistically different (p < 0.05). Data are the mean :t SEM (n = 11). These data were collected from 4 separate experiments. 65 MAPK inhibitors were able to suppress DON-induced IL-8 hnRNA levels in U93 7 cells Levels of IL-8 heteronuclear RNA (hnRN A) were determined after 3 h exposure to DON with or without SB203580, SP600125, or PD98059 (Figure 2.5). hnRNA levels can be used to measure transcription as hnRNA is pre-processed mRN A. The levels of the IL-8 hnRNA are equivalent to the levels of IL-8 mRNA in all treatment groups. All inhibition was statistically significant (p < 0.05) although, as with the mRNA levels, SP600125 and PD98059 only inhibit approximately half of the induction by DON while SB203580 provides an almost complete inhibition (~90%). Discussion The results presented here indicate that p38 and JNK were activated in U937 cells after exposure to DON and that this activation, primarily p38, contributed to the signaling cascades leading to IL-8 expression. SB203580, a p38 inhibitor, PD98059, a MEK/ERK inhibitor and SP600125, a JN K inhibitor, were used to determine if specific MAPK families are involved in the DON-induction of IL-8. Neither the MAPK inhibitors nor the DON concentration used (500 ng/ml) altered the cells’ viability, indicating that the differences seen in lL-8 protein and mRNA with the MAPKs inhibitors were not due to reduced cell viability. Increased phosphorylation of p38 and JNK was found in U937 cells after a 30 minute DON exposure. Similar results have been obtained in RAW 264.7 cells (28). 66 25 b % 20- ‘l' > .9 , <1: 2 DC 15* C .C w 2' § 1 5.. a a a a a 0 [I] I C.‘ [I [AT—l . [—77 . Control DON SB DON SP DON PD DON +SB +SP +PD Figure 2.5: DON-induced IL-8 hnRNA is suppressed by MAPKs inhibitors. IL-8 hnRNA levels in U937 after a 3 hour exposure to 500 ng/ml DON, and/or 2.0 pM SB203580, 10 pM PD98059, or 1.0 pM SP600125. Bars without the same letter are statistically different (p < 0.05). Data are the mean i SEM (n = 11). These data were collected from 4 separate experiments. 67 ERK phosphorylation was equivalent between the untreated and DON-treated U937 cells. Constitutive activation of ERK has been found in several cell lines, though has not been seen in U937 cells as reported by Ajenjo et al. (146). The possibility exists that there were alterations to the specific U937 cells utilized for these experiments that may have resulted in constitutive activation of ERK. The high levels of phosphorylation of ERK in the untreated U937 cell may also be an artifact of the sample preparation method. U937 cells are in suspension and require centrifugation and removal of the supernatant before lysis of the cells. ERK may have been activated during that procedure. SB203580, a p38 inhibitor, was able to completely inhibit DON-induced IL-8 protein and RNA levels. Similar inhibition, with a p38 inhibitor, was previously observed with DON-induced TNF-a (25) and COX-2 (24) with both of those studies found p38 responsible for increased stability of the transcripts investigated. Activation of p38 has been linked to both an increase in IL-8 transcription (78, 147, 148) and in IL-8 mRNA stability (120, 149). More specifically on the transcriptional level, inhibition of p38 has been shown to reduce NF-KB activity (51, 150, 151). There is a NF -KB binding site on the IL-8 promoter and increases in NF-KB activation have been shown to be responsible for increasing IL-8 expression in numerous models (38, 52, 95, 125). In agreement with results for U937 cells, p38 was also found to be important in human PBMC’s response to DON (152). DON induced IL-8 protein in PBMCs isolated from seven different individuals. In all cases, IL-8 protein production was found to be p38-dependent. Furthermore, in individuals with a greater number of CD14+ monocytes, there was an increased sensitivity to DON. This suggests that the monocyte population is 68 responding to DON exposure. These data provides additional corroboration for the relevance of U937 cells as a model of primary human cells. Both the JNK and ERK inhibitors were unable to inhibit IL-8 protein and only able to partially inhibit 1L-8 RNA. This difference can be seen clearly by comparing fold induction of IL-8 protein, mRNA, and hnRNA. (Figure 2.6) DON induced equivalent levels of all three of the IL-8 forms measured. Several possible mechanisms might explain the discrepancy between the IL-8 RNA and protein amounts in the presence of the ERK and JN K inhibitors. A feedback mechanism might be involved in these differences. With a large amount of transcript, translation could be slowed in response to a certain amount of IL-8 protein released into the culture supernatant. With the ERK and JNK inhibitors, the amount of transcript is reduced and accumulation of the IL-8 protein in the culture supernatant might not have reached the level necessary to trigger a reduction in protein production due to an autocrine effect. IL-8 protein is a fairly stable protein, being resistant to inactivation by plasma peptidases, heat, pH extremes, and other denaturing treatments (35), thus allowing for an accumulation in the media. IL-8 protein might need to be measured after a longer time of exposure (12 h to 24 h) to determine if the protein remains high or is reduced, with levels closer to the RNA levels. Other reasons for the difference between protein and mRNA expression might include decreased degradation of the lL-8 protein in the presence of the ERK or JNK inhibitors or the possibility that the ERK and JNK contribute in a reduced manner to IL-8 transcription, relative to the p38 contribution. Other studies have shown that ERK and/or JNK are necessary for lL-8 expression (12], 122, 126) 69 N 01 — mRNA average m :1 hnRNA average 2 — protein average Q 20 - = l .9 8 2 15 - Q. X (D °? 3 1O - d) .2 I a l a s- 0 v ' v v Control DON SB DON SP DON PD DON +SB +SP +PD Figure 2.6: Comparison of relative levels of IL-8 protein, mRNA, and hnRNA expression in U937 cells exposed to 500 ng/ml DON, and/or 2.0 pM SB203580, 10 pM PD98059, or 1.0 pM SP600125. (see Figures 2.3, 2.4, and 2.5) 70 To summarize, activation of the MAPK pathways was important for increased transcriptional expression of DON-induced IL-8. Of the three examined, p38 was the most important in this model. This might be due to interaction of the MAPKs with transcription factors or due to increased mRNA stability. Future studies should focus on downstream events resulting from the activation of the MAPK pathways that mediate IL- 8 expression. 71 CHAPTER 3: DON-INDUCED IL-8 EXPRESSION IN U937 CELLS IS NF -1 O A 2.0 - A Relative lL-8 promoter-driven luciferase —k 01 §¥% 00 >04 00 >04 00 >04 00 >04 00 %fi§ >04 0wr 00 >04 _ 00 . 00 >04 04 00 0 >04 04 00 00 >04 00 00 >04 04 00 00 >04 00 00 >04 — 00 . 00 >04 00 00 >04 04 00 00 >04 0 did 3&4 0 0 >04 ,00 . I 1 7 V I V Control DON LPS Figure 3.6: The NRF site is not required for DON-induced [L-8 promoter driven luciferase in U937 cells. U937 cells were transfected with either a wild- type IL-8 promoter luciferase construct or NRF-mutated IL-8 promoter construct, incubated for l h, then treated with O, l ug/ml DON, or 1 ug/ml LPS for 11 h. Data are mean :t SEM combined from two or more independent experiments. (n 2 12) 90 Based on the observation that the NF-KB site mutation specifically reduced DON- induced luciferase, further investigations into the effects of DON on NF-KB binding were carried out. DON increases binding activity ofp65 An increase in transcription ofien is due to increased DNA binding of transcription factors in promoter regions. The effects of DON on binding of five NF-KB subunits (p65/RelA, c-Rel, RelB, p50/NF-KBl, and p52/NF-KBZ) were assessed using an ELISA-based binding assay. Two of the five subunits, RelB and c-Rel, were undetectable in all U937 cell samples tested (data not shown) while levels of p65, p50, and p52 were measurable. Additionally, specificity of the IL—8 wild-type probe was assessed by incubating with unlabeled wild-type or mutant probe. Unlabeled mutant probe did not reduce the binding, while competition with the unlabeled wild-type probe reduced the binding measured by at least 80% (data not shown). Significantly (p<0.05) increased binding of p65 was observed for both the DON- and LPS-treated U937 cells (Figure 3.7A). Although, p50 binding was equivalent in untreated and DON-treated samples, there was a small, but significant (p<0.05) increase in p50 binding in LPS- treated U937 cells (Figure 3.78). The binding of p52 was quite different from the p65 and p50 binding. While in untreated and LPS-treated samples, binding of p52 was equivalent, there was a reduction (p=0.056) of p52 binding in the DON-treated samples (Figure 3.7C). Taken together DON treatment affected NF-KB binding by decreasing p52 and significantly increasing p65 binding. 91 60 p° o‘ .0 @. l. lL-8 expression Figure 4.1 114 release of NF-KB and p38-dependent increase in NF -KB transactivation as, in U937 cells DON-induced IL-8 expression is PKR-, p38- and NF -KB-dependent. Significance and future studies The findings of this dissertation indicate that DON-induced IL-8 expression in U937 cells is p38- and NF-KB-dependent and that DON-, Stxl-, and ricin-induced IL-8 protein production share a common reliance on PKR. IL-8 induction is solely regulated on the transcriptional level. This contrasts with previous findings that indicate both an increase of transcription and in mRNA stability are responsible for upregulation of several immune proteins after exposure to DON. It is unclear at this time whether that difference is specifically due to the target investigated, IL-8, or a characteristic of the cell line used, U937 monocytes. Additional investigation into DON-induced IL-8 in human primary cells or alternative monocyte lines might clarify this issue. The potential mechanism of DON-induced expression might provide insight into chronic inflammatory conditions with IL-8 dysregulation, such as rheumatoid arthritis, psoriasis, or inflammatory bowel disease. Additionally, both elevated serum IL-8 and endothelial damage due to increase neutrophil activation is observed in human patients with hemolytic uremic syndrome (HUS), which can lead to renal failure (192). Stx1 and ricin are able to cause HUS in a mouse model (193, I94). Insight into potential mechanisms leading to IL-8 expression might assist in developing additional therapeutics to minimize damage seen in HUS and increase patient survival. p38 inhibitors have already been suggested to minimize the damage seen in HUS (193) and research in this 115 dissertation potentially supports that finding if indeed DON, Stxl, and ricin share a common mechanism. Further investigation into DON-induction of IL-8 is warranted, if a common mechanism for IL-8 induction is shared with other translational inhibitors. For one, clarification of the mechanism leading to NF -KB activation needs to be examined. It is notable that, in these cells, NF-KB is extremely important compared to its relative unimportance in DON induced gene expression in a variety of cells. Several possible mechanisms leading to NF-KB activation are present in the U937 model system. Both PKR and p38 have been observed to activate NF-KB, but the mechanisms by which they do this are quite different. PKR is involved with phosphorylation and degradation of IKB while p38 increases the transactivation of NF -i Primer _ Probe Figure A. 1: Diagram of hnRNA primers and probe based on the IL-8 DNA sequence (Genbank sequence NM_006216). 123 Results and Discussion DON induces IL-8 protein in several human cell lines DON caused an increase in IL-8 protein in macrophage-differentiated U937 cells after an exposure time of 12 h. (Figure A.2) Undifferentiated U937 cells also responded to DON by producing IL-8 protein. (Figure A.3) IL-8 protein was reproducibly induced, in the undifferentiated U937 cells, as early as 3 h afier exposure to DON (data not shown). Although both macrophage-differentiated and undifferentiated U937 cells produced IL-8 protein as a result of exposure to DON, much higher levels of IL-8 protein were secreted from the differentiated cells. Two other human cell lines were also examined for DON-induced IL-8 protein production. THP-l, a monocyte cell line, displayed concentration-dependent DON- induced IL-8 protein production, though at lower levels than U937 cells. (Figure A.4) The difference in the IL-8 expression level is not surprising because, although THP-l and U937 cells are classed as monocytes, they are at different stages of differentiation (199). The difference in IL-8 protein levels seen could also be due to the random mutations that each cell line possesses simply by being cancer cells. The second cell line HEK-293, embryonic kidney cells, also demonstrated DON- induced IL-8 protein production. (Figure A5) The induction in the HEK-293 cells, however, was not in a concentration-dependent fashion nor was it as high as the IL-8 protein levels seen with the two monocytic lines. This difference could simply be a result of cell type differences. Based on robust IL-8 production and simplicity of experimental set-up, undifferentiated U937 cells were chosen for all further studies. An MTT assay was run to 124 0.8 - 06- ab lL—8 protein (pg/ml x 106) 0.0 a b a b 0.4 i a 0.2 - ’ 10 50 DON (ng/ml) Figure A.2: IL-8 protein production by macrophage differentiated U937 cells after a 12 h exposure to DON. U937 cells were differentiated with PMA and allowed to rest for 48 h afier removal of the PMA. Data are mean 1: SEM (n = 3). This is representative of 2 independent experiments. Bars without the same letter are statistically different (p < 0.05). 125 O a .. t 4. b r . .w ‘4 ‘0 if.- .' . " 1 ,— 39:.1.’ ‘4' Afltv ’ .4“ ~ - ""741“; a. ..l. o' .;-o.,. . .‘./ MM‘ 4 4. A‘. f ‘7“: $53 ‘1‘; i ‘ ’ . . a 4' .40 , ‘ “_ '4 N. " . . k l , 1-! .y‘ M- '0. me;;.:.l..jm,-f .‘ - (Li-I t." .w. ,4 . a... 40' q.“ v. .,- . g , .. . \ ,1 .‘, < . \- ‘I v. . ,1. “151- ,,A . , lL-8 protein (pg/ml x 104) -> N Wg’f’“ _. a a E .4” ‘04-‘flfi A1" -_,- _2 “(*8 v Ll ‘— ._ '1 k‘.‘ A, ".1“: kid-g 0" . 33'.) n'\ f ' , ' .231”, . o it I l I 0 1 10 72.5 125 250 DON (ng/ml) Figure A.3: IL-8 protein production from undifferentiated U937 cells after a 12 h exposure to DON. Data are mean i SEM (n = 3). This is representative of 2 independent experiments. Bars without the same letter are statistically different (p < 0.05). 126 400 d T c 300 - E O) 3 .s (D 5 200 - a co 3' c 100 . T a b 0 ND r—fi 0 250 500 1000 DON (ng/ml) Figure A.4: IL-8 protein production from THP-1 cells after a 12 h exposure to DON. Data are mean 1 SEM (n = 3). This is representative of 2 independent experiments. Bars without the same letter are statistically different (p < 0.05). 127 120 b 100 - b I b E 80 - T T U) 9, C '93, 60 - e O. a Z 40 ~ '1’ 20 - 0 I I l I 0 250 500 1000 DON (ng/ml) Figure A.5: IL-8 protein production from HEK-293 cells after a 12 h exposure to DON. Data are mean :1: SEM (n = 6). This is an average of 2 independent experiments. Bars without the same letter are statistically different (p < 0.05). 128 determine if cell viability was affected after a 3 h exposure to various concentrations of DON. (Figure A.6) There were no statistical differences among the treatment groups, indicating that 3 h exposure to DON did not reduce cell viability. A 3 h timepoint was chosen to examine cell viability because that was the timepoint that was selected in order to measure IL-8 RNA levels. DON induces IL-8 RNA in undifferentiated U93 7 cells DON produced an increase in IL-8 mRNA levels in U937 cells as early as 1 h afler exposure although the induction was not concentration dependent (data not shown). Concentration-dependent DON-induced IL-8 mRNA expression was seen after 3 h (Figure A.7). An increase in mRNA can indicate that there is an increase in transcription or an increase in mRNA stability. Either is possible with DON exposure. Increases in activity of a variety of transcription factors have been seen in various cells after exposure to DON (20, 21, 25). Similarly, increases in mRNA stability of several immune molecules have also been seen with exposure to DON (19, 25, 26). Data were obtained to provide circumstantial evidence for an increase in transcription is the heteronuclear RNA (hnRNA) levels. hnRNA, lacking the processing seen in mRNA, was used to observe whether transcription was affected. Concentration-dependent DON induction of IL-8 hnRNA levels was also seen in U937 cells (Figure A.8). IL-8 hnRNA and mRNA levels were approximately equivalent. Increased IL-8 hnRNA levels could also be seen as early as l h after exposure with DON (data not shown). 129 120 100 - T T :6 T 'T . E 80 4 ,’ o .- . o .;' '46 60 ‘J l i E .. 8 5 40 - ‘ r o. .2" 20 J ‘ l 0 l l I [1‘7 0 250 500 1000 DON (ng/ml) Figure A.6: MTT of U937 cells with a 3 h DON exposure. There is no statistical difference among the treatment groups. Data are mean :1: SEM (n = 6). This is an average of 2 independent experiments. 130 16 14 d g T 9 c D. _ 6 10 T ‘2‘ e- b g T «9 6- =' .3 4- E R 2- a 0 I l 1' Di I 0 250 500 1000 DON (ng/ml) Figure A.7: IL-8 mRNA levels in undifferentiated U937 cells after 3 h exposure to DON. Data are mean :l: SEM (n = 6). This is an average of 2 separate experiments. Bars without the same letter are statistically different (p < 0.05). 131 25 .5 20 1 C 3 l 0) E. - a" 15 - I '- < z “E g 10 - f _l 7.); a,b E 5 - T (D n: a O I T r 1 ‘ 0 250 500 1000 DON (ng/ml) Figure A.8: IL-8 hnRNA levels in undifferentiated U937 cells after a 3 h exposure to DON. Data are mean :I: SEM (n = 6). This is an average of 2 separate experiments. Bars without the same letter are statistically different (p < 0.05). 132 Conclusions DON was able to induce IL-8 protein in all three cell lines tested, differentiated and undifferentiated U937, THP-l , and HEK-293, though in differening amounts. The differences observed in the levels of IL-8 production may be due to cell type differences or even the maturity of the cells. Even though differentiated U937 cells produce higher levels of IL—8 protein, undifferentiated U937 cells have both DON-induced IL-8 protein and RNA. Because of its simplicity, this model was chosen for experiments in this dissertation. 133 APPENDIX B: OPTIMIZATION OF TRANSFECTIONS USING THE IL-8 PROMOTER-DRIVEN LUCIFERASE CONSTRUCT Am This appendix describes the optimization experiments for transfections with an lL-8 promoter construct in both U937 and RAW 264.7 cells. Initially, B—galactosidase was used as the reporter gene for transfection normalization. However, this reporter gene was found to be unsuitable. Real-time PCR was then used to measure luciferase for both expression (luciferase mRNA) and normalization (luciferase DNA, plasmid). This technique also was found to be problematic, possibly due to variability problems with U937 cells. Subsequent experiments were attempted in RAW 264.7 murine macrophage cells, which were also found to be unsuitable for studying DON stimulation of an IL-8 promoter-driven luciferase construct. Finally, U937 cell transfections were assessed using the Renilla luciferase reporter construct for normalization. This appears to be an accurate and reproducible way to normalize transfections with DON treatment. Introduction Many studies use reporter constructs to examine transcriptional regulation triggered by various stimuli in a variety of cell types. These experiments provide information purported to be more reflective of the cell’s actual function rather than protein binding differences obtained with Electrophoretic Mobility Gel Shift Assays (EMSA). EMSA data can vary depending on the conditions of the binding reactions and 134 to probe sequences. Additionally, EMSAs can only provide information about binding differences to a relatively short and specific oligonucleotide sequence. Reporter assays rely upon functioning transcription machinery within the cells along with revealing alterations in transcription factors. However, there are several concerns that arise from the use of reporter assays. One concern is that the method used for transfection of the reporter construct into specific cells can impact the results. There are several transfection methods that can be used, which include electroporation, lipid reagents, and calcium phosphate. Each method might have different efficiencies and consistency relative to moving the plasmid into the cells as well as inherent toxicity and activation of the target cell (203). A second concern for reporter assays is the choice of cell line. Different cell lines vary widely relative to transfection efficiency, with some cell lines being particularly problematic. U937 cells are found to be difficult to transfect, partly because of the transfection method (176) and partly a result of the response of the cells to foreign DNA (175). When undifferentiated U937 cells were compared to differentiated U937 cells and to other human monocyte lines (176) using either a lipid reagent or electroporation, it was found that the transfection efficiency for the undifferentiated cells lines was below 10% whereas the differentiated cells could not be transfected. The authors concluded that the lipid reagent had the advantage of using a lower amount of plasmid and that fewer cells were necessary to obtain similar results as to electroporation (176). As well as having low transfection efficiencies, U937 cells were also found to undergo apoptosis afier the foreign DNA was electroporated into the cells (175). Apoptosis was found to be unique to electroporation and did not occur with the 135 two lipid reagents tested. Another problem seen in U937 cells is poor expression of the reporter gene (178). An additional concern with reporter assays is normalization of the transfection. Efficiency of the transfection and consistency among the samples are typically assayed using a second plasmid that has constitutive expression of a reporter gene. Use of the second plasmid to normalize the transfection depends on the assumption that a reporter that is not altered by the conditions in the experiment. Several studies have reported conditions in which the internal control has been altered by the treatments (204-206) as well as ways to compensate for internal control alterations (207-209). Other variables that can influence the success of reporter assays are the concentration of plasmid utilized and recovery time. The latter item refers to the amount of time cells are allowed to recover fiom the transfection itself and the amount of time cells are allowed to express the reporter gene. Expression of the reporter gene is usually assessed by measuring the amount of protein present. In the case of protein synthesis inhibitors, such as DON, protein levels may be altered by exposure of the chemical of interest. The goal of this study was to optimize transfection conditions so that, when using an IL-8 promoter-driven luciferase construct, the luciferase levels would more accurately reflect IL-8 expression levels in the U937 cells. This appendix summarizes extensive experiments over several years that were undertaken to correct for alteration of the internal control, as a result of exposure to DON, in order to provide more consistent results. Variables assessed include the use of several reporter genes, B—galactosidase, firefly luciferase and Renilla luciferase in two different cell lines, RAW 264.7 and U937 136 cells. Conditions were optimized for the recovery time after electroporation, plasmid amount, treatment time, molecule measured (protein or mRNA), and DON concentration. Materials 3nd Methods Transformation of E. coli and isolation of plasmids Plasmids employed in these studies are summarized in Table B. I. Plasmids (50 ng) were incubated, for 10 minutes, with chemically competent JMIO9 E. coli (Promega, Madison, WI) on ice before incubation at 37°C for 5 minutes. The mixture was immediately placed back on ice and 1 ml of LB broth was added and incubated at 37°C for 1 h with shaking (250 rpm). After incubation, 75 pl of JM109 cells were plated on LB + ampicilin (100 pg/ml) and incubated overnight at 37°C. A single colony was picked for growth in a 3 ml LB + ampicillin culture and incubated overnight at 37°C with shaking (250 rpm). A 0.2 ml or 1 ml aliquot of bacterial culture was added to 100 or 500 ml LB + ampicillin culture, respectively, and incubated overnight at 37°C with shaking (250 rpm). Cells were pelleted in 50 ml tubes (Corning, Corning, NY) at 2000 x g for 15 minutes and pellet wet weight was determined. Pellets were stored at -20°C until isolation with either an EndoFree Plasmid Maxi or Mega kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. 137 Cell culture U937 cells, obtained from American Type Culture Collection (ATCC; Manassas, VA), were grown in RPMI-l640 supplemented with 10% heat-inactivated fetal bovine serum (HI F BS) and 100 U/ml penicillin and 100 pg/ml streptomycin (Gibco BRL; Rockville, MD). RAW 264.7 cells (ATCC) were grown in DMEM supplemented with 10% HI FBS and 100 U/ml penicillin and 100 pg/ml streptomycin. All cells were incubated at 37°C with 6% C02. Electroporation of cells and reporter assays U937 cells (107 cells/210 pl of RPMI-I640) were electroporated using a Bio-Rad (Hercules, CA) Gene Pulser at 280 V and 960 pF in a 0.4 cm gap cuvette (Bio-Rad) with various amounts of plasmids. See Table 3.1 for the plasmids utilized for these studies. RAW 264.7 cells (5 x 106 cells/210 p1 of RPMI-l640) were electroporated with a Bio- Rad (Hercules, CA) Gene Pulser at 300 V and 960 pF in a 0.4 cm gap cuvette (Bio-Rad) with various amounts of plasmids. After electroporation, cells were diluted in 12 ml growth media and allowed to rest for various times. Cells were then treated with 250, 500, or 1000 11ng DON or 1000 ng/ml LPS and plated at 1 ml/well in a 24 well plate. After the selected incubation times, the cells were lysed with 100 pl of Reporter Lysis Buffer (Promega) or 100 pl of Passive Lysis Buffer for the Dual-Luciferase Assay Kit (Promega). The Bright-Glo Luciferase assay kit (Promega) and the Beta-Glo Assay kit (Promega) were used to ascertain luciferase and B-galactosidase levels when cells were 138 Table B. l: Plasmids used for optimization of U937 cell transfection. Name of P1 a sm i d3 Promoter Reporter gene pGL2 Control SV40 (constitutive) Firefly luciferase pSV40 B-gal SV40 (constitutive) B—galactosidase -162/+44 IL-8 Luc IL-8 (inducible) Firefly luciferase pRL SV40 SV40 (constitutive) Renilla luciferase a = All plasmids except -l62/+44 IL-8 Luc were from Promega. The -l62/+44 IL-8 Luc plasmid was a gift from Dr. Antonella Casola, Division of Child Health Research Center, University of Texas; (103) 139 co-transfected with luciferase and B-galactosidase reporter constructs. Normalization of transfections was accomplished by dividing luciferase levels with B-galactosidase levels from the same cell lysate. For cells co-transfected with firefly luciferase and Renilla luciferase reporter constructs, luciferase levels were measured with the Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s instructions. Firefly luciferase levels were normalized against Renilla luciferase levels, in the same aliquot of cell lysate. A Turner 20e Luminometer (Turner Designs; Sunnyvale, CA) was used for all luminometer measurements. Luciferase real-time PCR Due to difficulties with the B-galactosidase reporter gene, the possibility of measuring both the luciferase mRNA (for expression) and luciferase DNA (plasmid for normalization) was explored. Cells (either U937 cells or RAW 264.7) were electroporated and treated with DON. RNA and DNA were originally isolated with TRI reagent (Ambion) following the manufacturer’s protocol. The optional linear acrylamide was used to aid recovery of DNA. However, because of poor RNA recovery, subsequent studies employed the RNAqueous Kit (Ambion) to isolate RNA from half the sample, while the TR] reagent DNA isolation protocol was used on the other half of the sample. Primers for luciferase (forward primer 5’-GCACATATCGAGGTGAACATCAC- 3’ and reverse primer 5’-TGCCAACCGAACGGACAT-3’) were selected using Primer Express software (Applied Biosystems). cDNA was synthesized from the RNA using the Taqman Reverse Transcription Kit (Applied Biosystems) using random hexamers provided with the kit according to the manufacturer’s instructions. SYBR Green Master 140 Mix (Applied Biosystems) was used according to the manufacturer’s instructions. Additionally, cDNA was synthesized using luciferase specific primers and the 188 PDAR (Applied Biosystems) was utilized to normalize the sample amount, both alterations were to reduce variability. Results and Discussion Luciferase levels in U93 7 cells normalized with ,B-galactosidase Figure 3.1 shows the basic experimental set-up for all transfection experiments conducted. Experiments were conducted using constitutively expressed reporter genes. B-galactosidase, a reporter that is commonly used to normalize transfection experiments, was chosen to use for normalization of transfection due to the ease of measurement and the compatibility of reagents with luciferase assays. Several lipid reagents were screened (Invitrogene’s Lipofectamine and DMRIE-C and Roche’s FuGENE 6) and were found to be ineffective for transfection of U937 cells (data not shown). Electroporation was chosen as a reproducible means of transfecting the U937 cells. U937 cells were electroporated with a total of 20 pg plasmid (15 pg of pGL2 Control and 5 pg of pSV4O B-galactosidase). Cells were allowed to recover from the electroporation for 6 h, and then were exposed to 0 or 500 ng/ml DON for 12 h. By comparing the pGL2 Control, which contains a luciferase reporter gene under the control of the constitutive SV40 promoter, to the pSV40 B—galactosidase plasmid, which has a SV40 promoter controlling expression of a B—galactosidase reporter, the effects of DON on both luciferase and B—galactosidase could be compared. As Figure B.2 shows, DON l4l Electroporate Recovery time ii Plate cells and treat with DON or LPS Exposure time lr Lyse cells and measure reporter genes Figure B]: Basic electroporation scheme for both RAW 264.7 and U937 cells. 142 120 _ 100- T T .9. C 8 1: 80— 2 8 i.— g 60- T S 3: O E 40- S o o. 20 - T 0 I ,i ., .. "a Luc B—gal Luc B-gal Control DON Figure B.2: Unequal suppression of constitutive expression of luciferase (Luc) and B-galactosidase (B-gal) in U937 cells with 500 ng/ml DON. Cells were allowed to rest for 6 h after electroporation (15 pg pGL2 Control and 5 pg pSV40 Bgal) and were treated for 12 h with or without DON. (n = 8) 143 w.-- ”a. suppressed both luciferase and B—galactosidase, a result not surprising because of DON’s capacity to inhibit of protein synthesis. However, B-galactosidase was suppressed approximately 40% while luciferase were suppressed approximately 80%. Thus, reporter expression data using 1.3-galactosidase normalization will not reflect the expression of IL- 8 in the U937 cells. Attempts to correct for the differences by measuring total protein in the cell lysate were not successful (data not shown). Since the differences in the protein levels could not be corrected, the endpoint of the experiments was changed to luciferase mRNA. It seemed reasonable that luciferase mRNA could be measured by real-time PCR and the transfections normalized with the plasmid levels in the cells. The next section discusses these experiments. Luciferase real-time PCR with U93 7 or RA W 264. 7 cells Over the course of optimizing the luciferase real-time PCR, concerns over luciferase expression levels in U937 cells prompted a switch to RAW 264.7 cells using a previously described method (210) for assessing luciferase mRNA (Figure B.3A). The method used for these experiments (Figure B.3B) included specific modifications of the Cok and Morrison (210) method (reagents used are detailed in the methods section above). The general strategy of this technique was to use one plasmid and then use the luciferase mRNA to measure expression and the luciferase DNA for normalization. This necessitated isolation of both the DNA and the RNA from a single sample. However, it is possible to have significant DNA contamination of the RNA sample. Based on the amount of plasmid transfected into the cells, approximately 2 pg of plasmid are present 144 Transfect cells using transfection reagent Rest 16 h v / Isolate RNA (Trizol-like reagent) Luciferase assay for protein 1 (normalize transfection) Re-isolate RNA (Trizol-like reagent) Treat RNA with DNase 1 Make cDNA with random hexamers Luc cDNA normalized Real-time PCR with luc and with GAPDH < GAPDH primers and SYBR Green cDNA/luciferase protein dye Figure B.3A: Summary of Cok and Morrison (2001) method for measurement of luciferase mRNA using real-time PCR and transfection normalization using using luciferase protein. 145 Electroporate cells Rest 24 h v Treat 4h Isolate DNA (TRI reagent) ISOlate RNA (RNAqueous kit) Treat RNA with DNase 1 Make cDNA (random hexamers or luciferase specific primers) it / Real-time PCR with luciferase primers and SYBR Green dye (Iuc cDNA amount normalized by luc DNA amount — RNA/DNA) Figure B.3B: Final RNA and DNA isolation scheme for luciferase real-time PCR. 146 per sample thus necessitating rigorous clean-up of the RNA. In fact, Cok and Morrison re-isolated the RNA in order to remove any gross DNA contamination before DNase treating the RNA sample. Since there is a potential DNA contamination and the low yield of DNA and RNA, several alterations were made to this single isolation technique yielding both DNA and RNA. First, a linear acrylamide carrier was used to increase the yield of DNA. Linear acrylamide was added to the sample and, during the ethanol precipitation step, pulls down a larger amount of DNA than would otherwise precipitate. This step solved the DNA yield difficulties. Next to be addressed was the RNA yield and DNA contamination issues. Although approximately 1 x 106 cells/ml were used, this number did not take into account cell death resulting from the electroporation and/or plasmid uptake. It has been observed that with lower cell number, such as those seen in cell culture experiments, reagents such as the TR] reagent (Ambion) or TRIzol (Invitrogen) are less efficient (unpublished observations). For this reason, the amount of cells in each sample was doubled and half of the sample was used for the DNA isolation and the other half was used for a RNA isolation using the RNAqueous kit (Ambion). This had an additional benefit of providing less DNA contamination of the RNA. RNA samples were treated with a DNase before reverse transcription. With nucleic acid isolation problems minimized, the reverse transcription was examined. Cok and Morrison (210) used random hexamers to synthesize the cDNA and measured luciferase cDNA, normalized the sample loading with GAPDH amounts, by real-time PCR then normalized the transfection itself with constitutive protein expression from a second plasmid. The experiment presented in Figure 3.4 included random I47 a) Q — luc cDNA/18$lluc DNA (rh) .93 20 :3 luc cDNA/luc DNA (rh) 'g — luc cDNA/188/luc DNA (Is) 2 [SS luc cDNA/luc DNA (Is) .2 I? 15 - In} 0 E 2 o. m 10 ‘1 _.u_ “6 c .9 a 5 . 2 a. x m 9 a; 0 a; Control LPS DON Figure B.4: Preliminary real-time PCR results for luciferase mRNA and DNA in RAW 264.7 cells are highly variable. RNA and DNA, isolated from RAW 264.7 cells electroporated with 20 pg of -l62/+44 IL-8 LUC plasmid, were assessed for luciferase. Samples were normalized with either luciferase DNA alone or 188 RNA levels and luciferase DNA. cDNA was made with either random hexamers (rh) or luciferase-specific (ls) primers. * = values that are 0.0002 or lower (n = 2) I48 ,2..- .., l hexamers and luciferase specific primers for cDNA synthesis and normalized the sample loading in the real-time PCR by measuring 188 levels. Transfections were normalized by measuring luciferase DNA (plasmid) with real- time PCR. Additionally, normalization of the real-time PCR was carried out in two different ways. One was calculated by dividing the luciferase cDNA amount by the ISS amount (to normalize for loading) then dividing by the luciferase DNA amount to normalize for the transfection efficiency. This normalization was represented with luc cDNA/ 1 8S/luc DON For the other normalization, 188 levels were not considered, represented by luc cDNA/luc DNA in the figure. Neither of the normalization methods reduced the variability seen in the samples. While results obtained using random hexamers were equivalent for both of the calculations, using luciferase specific primers increased the variability observed. Inclusion of 188 values for sample loading in the real- time PCR reaction produced a much larger value (around 10 fold) than the 50.0002 fold calculated without normalizing for sample loading with 18S. Additionally, all values obtained from DON-treated cells were equal or lower than values from untreated cells. Due to the extreme variability of these preliminary real-time data, it was concluded that optimization at the protein level must again be considered. This optimization was carried out in RAW 264.7 cells. Luciferase levels in RA W 264. 7 cells (without transfection normalization) Since there was extremely high variability found in the luciferase real-time PCR data, further optimization was deemed necessary using RAW 264.7 cells. Luciferase protein was used for optimization due to the ease of measurement. The maximum 149 .1. .¢-v--uo_nu 1 .,..-........., l 1 treatment time of 4 h was chosen based on a study performed by Thompson et al. (203), which determined that the optimal levels of luciferase, induced by a LPS responsive promoter, occurred using a 24 h recovery time followed by a 4 h treatment with 1000 ng/ml LPS. Additional, earlier, timepoints were also examined in order to aid in the selection of a timepoint suitable for RNA isolation to measure luciferase mRNA. Figure 85 presents luciferase expression in RAW 264.7 cells electroporated with various (5, 15, or 25 pg of -162/+44 IL-8 Luc plasmid, which contains the wild-type IL-8 promoter driving a firefly luciferase gene). After electroporation, the cells were allowed to recover for 24 h and then the cells were treated with or without 100 ng/ml LPS for 0.5, 2, or 4 h. Luciferase levels were found to be increased in the LPS-treated groups both with increasing plasmids amounts and with increased exposure time. These data indicate that the -l62/+44 IL-8 Luc plasmid was LPS responsive, confirming that the plasmid was functioning properly and that increasing amounts of plasmid produced higher luciferase levels. Based on Figure 8.5, 15 pg of plasmid was selected for subsequent experiments as that amount produced high levels of luciferase while limiting the amount of plasmid actually used. Figure 8.6 summarizes the different treatment times tested using the 15 pg plasmid amount. RAW 264.7 cells were electroporated and treated as before, with treatment groups of 0, 100, 250 ng/ml DON, 100 ng/ml LPS, or 100 ng/ml DON + 100 ng/ml LPS. The results from this figure indicate that, although the -l62/+44 IL-8 Luc plasmid was LPS responsive in RAW 264.7 cells, RAW 264.7 cells transfected with the - l62/+44 IL-8 Luc plasmid were not DON responsive. In. addition, neither the 100 nor 150 + 5 pg Control 500 —O— 15 pg Control + 25 pg Control 400 + 5 pg LPS + 15 pg LPS 300 g —0— 25 pg LPS 5 200 5 100 / .5; 50 // // .2 ‘ a 'l a i 0‘ 25 ~ 0 J / 4 a O _s N (a) A Hours after treatment Figure B.5: Increasing the amount of plasmid increases the luciferase protein levels (non-normalized) in LPS-treated RAW 264.7 cells. Cells were electroporated with 5, 15, or 25 pg -1 62/+44 IL-8 LUC and after a 24 h recovery time were treated for 0.5, 2, or 4 h with or without 1 pg/ml LPS. (n = 2) 151 35 _ 0.5 h — 2 h 30 A 1:: 4 h a, 25 7 (D S 0 2'5 20 - 2 ,2 15 ~ 3 0 0‘ 10 - 5 .. _ ND ND ND DON 0 100 250 100 (nglml) LPS 100 (nglml) 0 0 Figure 3.6: Longer treatment times have higher luciferase protein levels (non- normalized) in RAW 264.7 cells. Cells were electroporated with 15 pg of - 162/+44 IL-8 Luc, allowed to recover for 24 h and treated for 0.5, 2, 4 h with the concentrations of DON and LPS indicated. ND = not determined (n = 2) 152 {tflmfit'fiw‘ 250 ng/ml DON concentrations induced luciferase levels. Furthermore, even though the LPS group showed an exposure time dependent increase in luciferase, the co-treatment of DON + LPS did not increase the luciferase levels and in fact slightly reduced the levels in comparison to the LPS alone group. Other studies in our lab suggested that RAW 264.7 cells were unsuitable for this study. Dr. H-R Zhou determined that RAW 264.7 cells treated with LPS would induce an increase in phosphorylated p65 but DON-treated cells did not have an increase in p65 phosphorylation (data not shown). Thus, this cell line might be unsuitable as DON was unable to activate NF -KB. Experiments were therefore re-initiated in U937 cells. Constitutive expression of luciferase in U93 7 cells Figure 8.7 presents data indicate that the shorter the recovery time employed, the higher the luciferase response. U937 cells were electroporated with 0, 5, or 10 pg of the pGL2 Control plasmid. Cells were allowed to recover for either 12 or 24 h. After recovery, cells were incubated an additional 2 or 4 h in order to simulate a treatment time. After the simulated treatment time, cells were lysed and the lysate measured for luciferase. Results indicated that higher luciferase levels occur in cells with the shorter recovery time (12 h recovery versus 24 h). However, differences between the constitutive promoter in the pGL2 Control and the inducible promoter in the IL-8 promoter-driven luciferase construct must be kept in mind. The pGL2 Control plasmid is constantly producing luciferase in fairly large quantities while the IL-8 promoter-driven luciferase construct would produce differing levels depending on the stimulus. Thus, luciferase levels with the IL-8 promoter construct are likely to be dramatically lower. For 153 '1 T—fi‘fi—“T { f ' T .xns Htizféw \y l 60 - 14h [:3 16h 50 2 — 26 h 28h m 'E 3 40 ~ f.» / / _l 10;/ ;/ a) .2 E m CK in: 5 a . W 0 U in] 0 ug 5 ug 10 ug Amount of plasmid transfected Figure B.7: Shorter recovery times have higher luciferase levels (non- normalized) in U937 cells. Cells were electroporated with 0, 5, or 10 pg of pGL2 control plasmid and allowed to recover for either 12 or 24 h. Luciferase levels were measured after either a 2 (14 h and 26 h) or 4 h (16 h or 28 h) treatment time. (n = 2) 154 (3.15.” .' '. ' \i this reason, when continuing to optimize recovery and treatment times for eventual use with the IL-8 promoter construct, a substantially shorter recovery time of l h was tested with 2, 4, 6, and 12 h exposure times in order to determine the best luciferase expression time. T ransfizction normalization using a constitutively expressed Renilla luciferase gene in U93 7 cells The utility of a Renilla luciferase plasmid for normalization purposes was also examined. This was done by co-electroporating U937 cells with two SV40 promoter- driven luciferase plasmids, one the pGL2 Control (10 pg) that contained a firefly luciferase reporter and the other the pRL SV40 (1 pg) that contained a Renilla luciferase reporter. Cells were allowed to recover for l h, and then were treated with 0, 500, or 1000 ng/ml DON. Figure 88 presents all luciferase levels relative to the untreated, electroporated control luciferase levels. A dramatic decrease in luciferase was observed for both the firefly and Renilla luciferase. This decrease was seen at all exposure timepoints and was larger the longer the cells were exposed to DON. However, unlike the unequal suppression of luciferase and B-galactosidase, both firefly and Renilla luciferase was inhibited equivalently. Under the same recovery time (1 h) and exposure times to DON (2, 4, 6, or 12 h) the luciferase response to the -l62/+44 IL-8 Luc promoter luciferase construct was tested. For Figure 89, firefly luciferase (under control of the IL-8 promoter) was divided by the amount of Renilla luciferase (under control of a constitutive promoter) for transfection normalization. Figure 39 presents normalized data, indicating that the 155 110 \\ \\ — 2 h 1:] 4 h g — 6 h ‘g I [:1 12 h 0 100 - 3 i 8 ~37, / E 30 7 “5 E 20 ~ 0) 9 1 °’ 1 ‘1 10 . .l o _ 11 4 . Firefly Renilla Firefly Renilla Firefly Renilla DON 0 500 1000 (nglml) Figure 3.8: DON causes approximately equivalent suppression of firefly and Renilla luciferase levels in U937 cells. Cells were electroporated with 10 pg of pGL2 Control and 1 pg pRL SV40 and allowed to recover l h before treatment. Cells were treated with 0, 500 or 1000 ng/ml DON for 2, 4, 6, or 12 h. (n = 3) 156 2.5 - 2 h g ::1 4 h it") 6 h 1:; 2.0 - I 1:: 12 h 'E ."g’ I 1? 1.5 - .25. O E a 1.0 ‘ fl '— 3 I f .“z’ 0.5 . ‘- 16 L. 6‘2 1" 0.0 L -- '- 0 ng/ml 500 ng/ml 1000 ng/ml DON Figure B.9: DON-induction of IL-8 promoter-driven luciferase levels (normalized with Renilla luciferase levels) in U937 cells. Cells were electroporated with 15 pg -l62/+44 IL-8 LUC and 1 pg pRL SV40, allowed to recover for l h and treated with 0, 500, or 1000 ng/ml DON for 2, 4, 6, or 12 h. (n = 3) 157 ”‘7 luciferase levels for both 500 and 1000 ng/ml DON are equivalent in the earlier timepoints (2, 4, or 6 h), but the later (12 h) timepoint showed a clear increase over the control at both DON concentrations tested. Recovery (1 h) and exposure (12 h) times were selected for further testing. At this point, since there were high levels of Renilla luciferase in the control cells the amount of pRL SV40 plasmid was reduced from 1 pg to 0.5 pg. C onfirmation of a successfitl transfection system in U93 7 cells Figure 8.10 presents data from several independent experiments, showing the consistency among independent experiments. U937 cells were electroporated with 15 pg -l62/+44 IL-8 Luc promoter-driven luciferase construct and 0.5 pg of pRL SV40. Cells were allowed to recover for 1 h and then were treated with 0, 500, 1000 ng/ml DON or 1000 11ng LPS for l l h. Cells were lysed and assayed for luciferase according to the abovementioned methods. Figure 8.10 represents a sample size of 20 and there was found to be statistically significant differences between the control and DON or LPS treated groups. Thus, DON induced IL-8 promoter-driven transcription in the U937 cells. The observation that there was only a one-fold increase in luciferase levels was not surprising. In a study conducted by Roux et al. (211) the authors found an approximately 40-fold increase in IL-8 protein induced by LPS in U937 cells but only a 3-fold increase in luciferase levels driven by an IL-8 wild-type promoter also in U937 cells. This optimized experimental set-up was therefore selected to assay mutations in the IL-8 promoter in order to determine the transcription factors that are important in DON- induced IL-8 transcription. 158 3.0 2.5 4 2.0 4 0.5 ~ Relative lL-8 promoter-driven luciferase _L 01 l 0.0 Figure 3.10: DON induces IL-8 promoter driven luciferase levels in U937 cells. Cells were electroporated with 15 pg -l62/+44 IL-8 LUC and 0.5 pg pRL SV40, allowed to recover for l h, and treated for 11 h with 0, 500, 1000 ng/ml DON, or 1000 ng/ml LPS. Representative of several independent experiments. (n = 20) 159 Conclusions Since B—galactosidase was found to be unsuitable for normalizing transfection efficiencies afler DON exposure, additional methods for normalizing transfections were examined. Utilizing real-time PCR to measure both the inducible luciferase mRNA and the constant luciferase DNA levels had high levels of variation for several reasons. However, use a second plasmid containing a Renilla luciferase gene was assessed and was an acceptable method for transfection normalization in U937 cells treated with DON. 160 APPENDIX C: OPTIMIZATION OF mRNA STABILITY EXPERIMENTS Am Deoxynivalenol (DON) induces expression of a variety of immune proteins due to, in part, an increase in mRNA stability. The assessment of mRNA stability is generally carried out by using general transcriptional inhibitors to block transcription and then measuring the time the transcript of interest remains. However, assessment of IL-8 mRNA stability in U937 cells using general transcriptional inhibitors provided inconsistent results possibly due to the well-known drug resistance of this cell line. Additional inhibitors, those that are specific to IL-8, were therefore assessed for use in this model system. Caffeic acid phenethyl ester was found to be an effective inhibitor of both IL-8 protein and mRNA and was selected to use in the assessment of IL-8 mRNA stability. Introduction Deoxynivalenol (DON) has been found to increase the protein levels of various immune molecules (8, 17, 24-26, 163). One of the ways that DON induces an increase in immune molecules is to increase the stability of the mRNA (25, 26) (19). This allows a larger amount of protein to be translated than if the transcript were degraded quickly. Conventionally, measurement of mRNA stability is done using a general transcriptional inhibitor to stop transcription and then the amount of the mRNA of 161 l 1‘1‘3119L. ‘ -n interest is measured over specific time intervals. Two inhibitors that are commonly used are dichlorobenzimidazole riboside (DRB) and actinomycin D (ActD). DRB halts transcription by preventing elongation of the transcript and can also induce p53- dependent apoptosis (212). ActD also inhibits the function of RNA polymerase 11, thus preventing mRNA from being synthesized (213). However, there have been reports that ActD can interfere with mRNA degradation making assessment of mRNA stability problematic (119). Since using general transcription inhibitors did not provide clear results in DON— treated U937 cells, alternative inhibitors were considered. Alternative inhibitors were selected based on their ability to inhibit IL-8 transcription specifically. NF-KB inhibitors were the first group selected for consideration based on preliminary experiments showing the importance of this transcription factor for DON-induced IL-8 as well as previously published studies using a variety of these inhibitors. Three NF-KB inhibitors, BAY 11- 7085, caffeic acid phenethyl ester (CAPE), and gliotoxin, were selected to be tested in this model system. BAY 11-7085 was successfully used in U937 (214) and human retinal endothelial cells (215) and resulted in decreased NF-KB binding activity and inhibition of IL-8 protein, respectively. CAPE was used successfully in human retinal endothelial cells (215) to inhibit IL-8 protein and also in microglia (216) to inhibit IL-6 and nitric oxide as well as inhibiting NF-KB binding. Notably, two studies found CAPE to be effective in U937 cells. Nardini et al. (217) found that there was an inhibition of ceramide-induced NF-KB binding. In the other study, CAPE was able to selectively inhibit NF-KB binding in U937 after exposure to a variety of stimuli (187). Table 01 is a summary of selected studies using these three inhibitors. 162 VF—‘—-" "" ‘7 I Table 01: Summary of selected studies using NF -1— DON 250 ng/mI+DRB .5 —-— DON 500 ng/ml+DRB E 1'0 ‘ 1 —a—— DON 1000 ng/ml+DRB Q) . .§ 0.8 . , .. j 00 0.6 ‘3. .5 1 I E l .C c 09 0.4 - , =' (D :5 0.21 2 (D II 0.0 1 1 I I l I I 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Hours after addition of DRB Figure C.lB: IL-8 hnRNA in U937 cells after exposure to 100 pM of DRB alone or with 250, 500, or 1000 ng/ml DON. Cells were co-treated with various concentrations of DON and DRB for the specified times, then total RNA was isolated and assessed for IL-8 hnRNA. Data are mean :1: SEM (n = 3). 170 mechanisms of drug resistance suggest that these cells are capable of neutralizing various chemicals, possibly including those used to inhibit transcription such as DRB or ActD. Due to the inefficiency of the two general transcription inhibitors, additional inhibitors were evaluated in this model. Assessment and optimization of NF -KB inhibitors Since NF-KB is critical for DON-induced IL-8 expression (see Chapter 3) the NF - KB inhibitors chosen to screen for inhibition of DON-induced IL-8. Based on the ease of the assay as well as amount of inhibitor and cells required, assessment and optimization was carried out using an ELISA for IL-8 protein. For an ELISA, less than 200 pl of culture supernatant is necessary, which allows for a much smaller amount of both inhibitor and cells required for the assay then would be required for real-time PCR measurement of IL-8 mRNA. ’ U937 cells were either co-treated with an inhibitor and DON or pretreated for 2 h with each inhibitor before treatment with DON. Once DON was added, cells were incubated for 12 h before the supernatant was collected. Figure 02 presents the results from cotreating DON and BAY l 1-7085. BAY 11-7085, at all three concentrations tested, was unable to inhibit DON-induced IL-8 protein levels and in some samples seemed to increase the IL-8 protein produced by the U937 cells. Co-treatment of CAPE was far more successful (Figure 03). At 100 pg/ml CAPE, DON-induced IL-8 protein was completely inhibited. Gliotoxin co-treatment was also ineffective, at both concentrations tested, and seemed to also increase IL-8 protein levels in the DON-treated samples (Figure 04). Along with cotreating the cells with each inhibitor and DON cells 171 8 - Vehicle F I: 5 pM BAY 6 - _ 20 pM BAY E :3 40 pM BAY B» I 5 .5 4 _ ‘é Q 00 I =I' 2 a O _ j I 0 ng/ml 250 ng/ml 500 ng/ml DON Figure 02: BAY 1 1-7085 does not inhibit DON-induced IL-8 protein production. U937 cells were exposed to a 12 h co-treatment of BAY 11-7085 (0, 5, 20, or 40 pM) with DON (0, 250, or 500 ng/ml). Data are mean :1: SEM (n = 3). 172 5 — Vehhicle 4 _ CI! 25 pg/ml CAPE - 50 pg/ml CAPE E [:3 100 pg/ml CAPE a 3 _ E» 5 .E 9. 1.11 9 . o- 2 d i o? =1. 1 - 0 1.. I '— 0 ng/ml 250 ng/ml 500 ng/ml DON Figure 03: CAPE inhibits DON-induced lL-8 protein production. U937 cells were co-exposed to CAPE (0, 25, 50, or 100 pg/ml) with DON (0, 250, or 500 ng/ml) for 12 h. Data are mean d: SEM (n = 3). 173 30 25 _ _ Vehicle I: 1 pg/ml glio c _ 2 pg/ml glio g 20 - O) 5 1.5, 15 - ’6 a 00 ,—l_', 10 a 5 .. 0 — — - ‘ I 0 ng/ml 250 ngml 500 ng/ml DON Figure C.4: Gliotoxin does not inhibit lL-8 protein production. U937 cells were co-treated with gliotoxin (0, l, or 2 pg/ml) with DON (0, 250, or 500 ng/ml) for 12 h. Data are mean :t SEM (n = 3). 174 fi."-ii were also pretreated with the inhibitors before DON exposure. A 2 h timepoint was chosen based on the studies in Table 01 (187, 215, 223). Even with the 2 h pretreatment, BAY 11-7085 (Figure 05) was still unsuccessful in inhibiting DON- induced IL-8 protein. As Figure C.6 shows, CAPE pretreatment produced similar successful results to the CAPE co-treatment, with 100 pg/ml of CAPE completely inhibiting DON-induced IL-8 protein. Gliotoxin remained unsuccessful even with the 2 h pretreatment at both concentrations tested (Figure C.7). CAPE was the only effective inhibitor of DON-induced IL-8 protein with both the co-treatment and the pretreatment of 100 pg/ml CAPE causing complete inhibition of DON-induced IL-8 protein. The next step was evaluating the effect of CAPE on IL-8 mRNA. For measuring IL-8 mRNA from CAPE- and DON-treated cells, a 2 h pretreatment was used. U937 cells were pretreated with 0, 50, or 100 pg/ml of CAPE and then treated for 3 h with or without 500 ng/ml DON or 5 ng/ml LPS. Both the 50 and 100 pg/ml of CAPE were effective at inhibiting DON-induced IL-8 mRNA, though the 100 pg/ml concentration showed the most inhibition of DON-induced IL-8 mRNA (Figure C8). The 100 pg/ml CAPE concentration was chosen for the IL-8 mRNA stability experiments due to the complete inhibition seen of both DON-induced IL-8 protein and mRNA. 175 7 6 - — Vehicle I [2:1 5 pM BAY 2 5 - — 20 pM BAY I g 1:] 40 pM BAY 19 C 4 _ E’ a 3 ‘ E (I) .2. 2 . 1 a 0 .. I 0 ng/ml 250 ng/ml 500 ng/ml DON Figure C.5: BAY 11-7085 does not inhibit DON-induced IL-8 protein with a 2 h pretreatment. U937 cells were treated for 2 h with BAY 11-7085 (0, 5, 20, or 40 pM) then a 12 h treatment with DON (O, 250, or 500 ng/ml). Data are mean :1; SEM (n = 3). 176 truce—r1 i... - Vehicle 4 - III] 25 pglml CAPE A — 50 pglml CAPE :E [:1 100 pglml CAPE U) 3 - 5 .E .03. o '5. 2 - Q a 1 _ 0 0 ng/ml 250 ng/ml 500 ng/ml DON Figure 06: CAPE inhibits DON-induced IL-8 protein. U937 cells were treated with CAPE (0, 25, 50, or 100 pg/ml) for 2 h then treated for 12 h with DON (0, 250, or 500 ng/ml). Data are mean :t SEM (n = 3). 177 inf-- 6 - Vehicle 5 ‘ 1:: 1 pglmlglio A — 2 pg/ml glio s 4 . O) 5 '5 3 - it ’5 a w __.'_ 2 ‘ 1 .. O I' 0 ng/ml 250 ng/ml 500 ng/ml DON Figure C.7: Gliotoxin does not inhibit DON-induced IL-8 protein. U937 cells were given a 2 h pretreatment of gliotoxin (0, l, or 2 pg/ml) then a 12 h treatment with DON (0, 250, or 500 ng/ml). Data are mean :t SEM (n = 3). 178 20 _r - Vehicle III] 50 pglml CAPE '5 - 100 pglml CAPE g 151 a X 0) <2 E 101 E w 2 m .2 a 5 - (I) a: 0 0 nglml 500 nglml DON Figure 08: CAPE inhibits IL-8 mRNA in U937 cells. Cells were pretreated for 2 h with either 0 or 100 pg/ml CAPE then treated for 3 h with 0 or 500 ng/ml DON. Representative of two independent experiments. Data are mean i SEM (n = 3). 179 Conclusions U937 cells did not appear to have transcription suppressed with two general transcriptional inhibitors, DRB and ActD. NF-KB inhibitors were assessed for suppression of IL-8 transcription and one of the inhibitors, CAPE, was found to completely inhibit IL-8 expression. CAPE was selected for use in experiments to determine if DON increased the stability of IL-8 mRNA. I80 ‘i ' "" APPENDIX D: IL-8 PROMOTER CONSTRUCT MUTATIONS AND PREDICTIONS OF BINDING SITES USING TRANSCRIPTION FACTOR BINDING SITE RECOGINITION DATABASES Ahém Two different web-based transcription factor binding site recognition programs (TF SEARCH and PROMO) were used to determine if the site-directed mutations in transcription factor binding sites, specifically AP-I, C/EBPB, Oct-l, NF -KB, or NRF, of the IL-8 promoter-driven luciferase constructs would prevent binding of the transcription factors examined. Both web-based programs gave similar results and, in general, predicted that the mutations present in the luciferase constructs would prevent the specific transcription factors of interest from binding. Furthermore, the programs suggested that, except for one potential increase in a basal transcription factor in the construct with the AP-1 mutation, most other potential binding sites would not be altered. Introduction Many published studies have used mutations in either promoter constructs or EMSA probes to weigh the involvement of various transcription factors. In the past several decades, web-based programs have been available to determine potential binding sites and provide in silico verification that the mutations are actually preventing binding. An additional advantage of using these programs is that transcription factors other than the ones directly examined can be screened for differential binding. The wild-type sequence for the IL-8 promoter (Genbank Accession number NT_0062I6.14 base pairs 31 13125 to 3113282) contains 5 major transcription factor 181 binding sites, AP-l, C/EBPB, Oct-l, NF-KB, and NRF, that are important for transcription of IL-8 in multiple cell types under a wide variety of condition. In these studies, a wild-type IL-8 promoter-driven luciferase construct was employed and the site-directed mutagenesis was utilized to assess the role of specific transcription factors in DON-induced IL-8 (see Materials and Methods, Chapter 3). Two web-based programs were used to examine the wild-type IL-8 promoter and effects of mutations in the IL-8 promoter. MM Both TFSEARCH (http://www.cbrc.ip/research/db/TFSEARCH.html) (174) and PROMO (http://alggen.lsi.igaccs/cgi- bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) (172, 173) employ a version ofthe TRANSFAC database that compiles transcription factors and binding site sequences. All of the IL-8 promoter sequences, wild-type and all five mutant sequences, were entered individually into each of web-based programs and the results were assessed. For TFSEARCH, sequences were entered to the site and the vertebrate matrix was selected for comparison. The threshold score was left at the default of 85% similarity, indicating that sites with less than 85% similarity to sites in the IL-8 promoters would be disregarded. The sequence was submitted and the results were examined. The PROMO program had more complex options. For PROMO, only human factors were considered with binding sites used from eukaryota. The maximum matrix dissimilarity rate was left at the default of 15, 182 indicating that binding sites would only be predicted if the site similarity was equal to or greater than 85%. The sequences were then submitted and the output was examined. Results and Discussion Predicted transcription factor binding sites using T F SEARCH TFSEARCH was the first program used to assess the mutations of the IL-8 promoter. When the wild-type sequence was searched using the vertebrate sequences AP-l and NF-KB binding sites were found. NRF was not found at all on the promoter sequence and potential C/EBPB and Oct-1 binding sites were not found in their correct locations. Use of all possible sequences for these transcription factors did not alter these basic findings. Table D.1 contains a summary of this information. Mutations of the NRF, C/EBPB, and Oct-1 sites did not change the potential binding of AP-l or NF-KB and did not alter the basic score of these two transcription factors. Scores are the percent similarity to the consensus sequences and were approximately 90% for both AP-l and NF-KB. When the AP-l mutation IL-8 promoter was assessed, binding of AP-l was absent and there were no alterations to scores of other potential transcription factor binding sites in that area. Similar results were obtained with the NF-KB mutated IL-8 promoter. Potential binding of NF-KB was completely absent and other potential 183 o . LWWWM 8.2.3. 0: 8.0-2.3 2.3-2.3 2.3.2.3 Ximoécow .omo aw” o 9 .8352 8 .8352 9 .8352 3 $8.08 8.85 o: 83%...» miz maiz - - - - - . 090 2.3 09¢ 2.3 2.5 2.3 090 2.3 099 2.3 252 282 252 287. -80 28 9 .8352 2 .8352 8 .8352 8 .8352 9 .8352 ammO 28-2.3 28-28 88-2? 28-2.3 . . - - 8 .8352 8 .8352 8 .8352 2 .8352 8.2.... o: o\om 382% mm . m< . m< 88an 88:8ch 88:88 58an w 88:88 88:88 85 an: .326 3: maize ma: .808 ma: .3. ES: 28 See 8.8323 we .5 Emma... .28 3. .u . 3.303 102(32me 23 8 8.3808 28:85 3595; .Iufimmm... wEm: £830.58 37. 3383. 882 8.8.888... “.d 032. 184 q rim transcription factor binding was unaltered. Since TFSEARCH only assessed the binding of AP-I and NF-KB for the IL-8 promoter 3 second web-based program, PROMO, was used. Predicted transcription factor binding sites using PROMO PROMO was also used to assess potential transcription factor binding sites. As with TF SEARCH, NRF was absent from the assessment. Additionally, the specific factor that was Oct-1 was difficult to identify. This database contains several POU domain entries and different octamer family proteins. Using the wild-type IL-8 promoter, AP-l and various constituents (such as c-Jun and c-Fos), C/EBPB, a variety of Oct family factors, and NF -KB potential binding sites were identified in expected locations. All mutated promoters assessed had no alterations except in the area of the mutation. Table D2 contains a summary of this information. In the mutated NRF promoter, there was a decrease of the basic NF-KB score and potential binding sites of components of NF -KB (NF «B1 and RelA) were found. For the mutated NF-KB promoter, no binding was found for NF -KB or members of the NF-KB family. This matches the results obtained using the TFSEARCH program. With the Oct-l mutated promoter, several increases to three different potential binding sites occurred. Two of these were C/EBPB transcription factors and one was a member of the Oct family. There was no change to a majority of the Oct family and POU domain members. This is not alarming because the specific Oct-1 transcription factor that interacts with a binding site on the IL-8 promoter might not be in the databas 185 $3.3 35:5 3 35:5 3 <3. $3.33 3.35 3 35:5 3 8:2 3. a. 2. ..\ :3 ..\ .2 a. 8..-... {WNW 3....35 3 28.2.: 28-2.3 28-2.3 chasm 3.32 3.2 o . . 2 .8352 2 .8352 9 .8352 o 3.8 mziz $3.3 $3.3 $33.33 a. 33.. $3.3 $3.3 $33.33 5 33.. $3.3 $3.3 $3.33 330.. $33.33 3.35 3 $3.3 3339. $33 3.35 3 $3.3 .88 3.35-5588 $3.3 3.35 3 $3.33 23-8. 330.. $33.33 3.35 0: $3.33 330,. 28-2.3 28-2.3 <33 3.35 3 2.8.2.3 £33 830.. .30 3.. 2 .855... 9 33:3. .. . . o. .833. c 33.8 $3.33. 3.35 3 $3.33 3.3.8 $3.33 3.35 2. $3.33 9.3.8 $3.3 35:5 3 $33.3 8.8 $3.3 3.35 3 $3.... 3330.. $3.3 $3.33 3...: .8 8 3. 8 3.35 3 $3.33. .-..< o -33 u -23 o -33 o -33 . - o. .8352 o. .8352 8 .8352 o. .8382 8.2.3 o: .3: t. 05... . 8< 35:5 2. $3.33. 2. 8%... 35:5 3 $33. .53 88:88 88:88 88:88 88Eo8 88:88 w 88:88 8.3 .3. 82:. 3-.. -5. .80.: 3-.. 3-5. ....<5 2:. .98. 33:33; 3.38 3.32:. 388:. -33 3-5. . . 6.2023. 3.3: 33.8.83 2.88.. E. 38.8.88 23 3.383 88.3 8.8.888... .md 83“... 186 or the sequence on the lL-8 promoter may be different enough from the consensus sequences to be unrecognizable with the cut-off of 85% similarity. This percent similarity means that of sequences are more then 15% different they will not even be on the results from the program. The mutated AP-l promoter results were the most interesting. Along with no AP- 1 binding for the mutated promoter there was an increase in percent similarity of a basal transcription factor, TFIIB. Potential TFIIB binding increased from 89.48% in the wild- type IL-8 promoter to 95.36%. While it is unclear exactly what biological significance of this difference is, it provides a possible reason for the increased IL-8 promoter-driven luciferase expression observed for the mutant AP-l construct (see Chapter 3). TFIIB is responsible for stabilizing the interaction of TBP with the DNA as well as recruiting the RNA Polymerase II complex and selection of the transcriptional start site (224, 225). It should be mentioned that both of these web-based programs might not contain specific transcription factors important for IL-8 transcription or that if transcription factors are present in the databases there is some uncertainty about the form of the transcription factor. For example, neither of the programs have entries on NRF and some binding sites are just listed as AP-I or NF -l(B without any mention of the specific dimer composition of the factors. Additionally, experimental reports might show that binding is altered with certain mutations that the programs will not detect due to reliance on consensus Sequences. 187 Conclusions The wild-type IL-8 promoter and the five promoters with mutations in transcription factor binding sites (AP-1, C/EBPB, Oct-l, NF-KB, or NRF) were screened using two web-based transcription factor binding site recognition programs, TFSEARCH and PROMO. Overall, results from these two programs indicated that the wild-type IL-8 promoter had the transcription factor binding sites of interest and that the point mutations used should disrupt these binding sites. Additionally, a potential increase in the binding of a basal transcription factor, TFIIB, was found in the mutant AP-l promoter, which would provide a tentative explanation for the increased luciferase expression results presented in Chapter 3. 188 10. 11. REFERENCES Council for Agricultural Science and Technology. 2003. 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