Regulation of na+ h+ exchanger 1 (NHE 1) gene expression by mild oxidative stress

+ + REGULATION OF Na -H EXCHANGER (NHE-1) GENE EXPRESSION BY MILD OXIDATIVE STRESS CHANG KER XING BSc (Honours) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009  LIST OF FIGURES .6 LIST OF TABLE. 11 ACKNOWLEDGEMENTS .13  ABBREVIATIONS USED 14  SUMMARY OF THIS STUDY .16  PUBLICATIONS AND PRESENTATIONS 18  CHAPTER 1: INTRODUCTION 20  1.1  FREE RADICALS AND REACTIVE SPECIES .20  1.1.1  Overview of free radicals and their derivative reactive species . 20  1.1.2  Reactive Oxygen Species 21  1.1.2 A  Major types of free radicals and their derivatives .21  1.1.2 B  Redox signaling .23  1.1.3  Reactive Nitrogen Species 24  1.1.3 A  The production of NO from nitric oxide synthase 24  1.1.3 B  NO and its derivatives .25  1.1.3 C  Peroxynitrite 25  1.1.4  The antioxidant system 26  1.1.5  Oxidative stress 27  1.1.6  The NOX family NADPH oxidases 28  1.1.7  Hydrogen peroxide as a signaling molecule 32  1.2  REDOX REGULATION OF GENE EXPRESSION 35  1.2.1    Binding of transcription factors to DNA is influenced by redox balance 35  1.2.2  Transcription factors that are responsible for gene induction mediated  by ROS  36  1.2.2 A  NF-κB 36  1.2.2 B  AP-1 .37  Pg|1 1.2.2 C  HSF1 38  1.2.2 D  Nrf2 .40  1.2.3  Gene repression mediated by ROS 42  1.3  ROLES OF SUPEROXIDE AND HYDROGEN PEROXIDE IN CELL SURVIVAL AND TUMORIGENESIS .43  1.4  SODIUM-HYDROGEN EXCHANGER (NHE-1) .46  1.4.1  NHE and intracellular pH regulation . 46  1.4.2  The mammalian NHE family . 47  1.4.3  Physiological functions of NHE‐1 48  1.4.3. A  NHE-1 and myocardial diseases 48  1.4.3. B  NHE-1 in tumor cells .48  1.4.3. C  Regulation of cells’ volume during hypertonic stress .53  1.4.3. D  NHE-1 as a cytoskeleton anchoring protein and signalplex 53  1.4.3. E  NHE-1 and cell differentiation 53  1.4.4  1.5  Regulation of activity and expression of NHE‐1 54  1.4.4. A  Regulation of NHE-1 activity 54  1.4.4. B  Transcriptional regulation of NHE-1 expression .58  AIM OF STUDY 61  CHAPTER 2: MATERIALS AND METHODS .62  2.1  MATERIALS 62  2.1.1  Chemicals and reagents 62  2.1.2  Antibodies 63  2.1.3  Plasmids . 64  2.1.4  Cell lines and cell culture 65  Pg|2 2.2  METHODS 66  2.2.1  Treatment of cells with Hydrogen peroxide (H2O2) and Other  Compounds 66  2.2.2  Mammalian Cell Expression by Transient Transfection . 66  2.2.3  Luciferase Gene Reporter Assay 67  2.2.4  Chloramphenicol Acetyl Transferase (CAT) assay 68  2.2.5  Caspase Activity Assay 69  2.2.6  Cell viability estimation by Crystal Violet Assay 69  2.2.7  DNA Fragmentation Assay 70  2.2.8  SDS‐PAGE and Immunoblotting 71  2.2.9  RNA interference (RNAi) . 73  2.2.10  Nuclear‐Cytoplasmic Fractionation . 74  2.2.11  Intracellular pH (pHi) Measurement and NHE activity Assay . 74  2.2.12  RNA Isolation and Measurement of mRNA levels by Real‐time PCR 76  2.2.13  Immunofluorescence Assay using Confocal Microscopy 77  2.2.14  Extracellular H2O2 measurement using Amplex Red Assay 78  2.2.15  Intracellular ROS Measurement by CM‐DCFDA . 78  2.2.16  Intracellular Nitric Oxide (NO) Measurement by DAF‐FM . 79  2.2.17  Morphology Studies . 80  2.2.18  Protein Determination 80  2.2.19  Statistical Analysis 80  CHAPTER 3: RESULTS .81  3.1  MILD OXIDATIVE STRESS INDUCED BY H2O2 INHIBITS GENE EXPRESSION OF NHE-1 INVOLVED AN EARLY OXIDATION PHASE 81  3.1.1  To determine a non‐toxic dose of H2O2 in L6 rat muscle cell 81  3.1.2  H2O2 down‐regulates NHE‐1 gene expression . 87  3.1.3  H2O2 initiates the signal for NHE‐1 promoter repression . 94  3.1.4  Oxidation is involved in the early phase of NHE‐1 promoter inhibition  mediated by H2O2 . 103  Pg|3 3.2  ACTIVATION OF CASPASE AND IS REQUIRED FOR MILD OXIDATIVE STRESS-INDUCED DECREASE IN NHE-1 GENE EXPRESSION.115  3.2.1  Caspases are involved in the sustained inhibition of NHE‐1 gene  expression mediated by H2O2 . 115  3.2.2  Caspases 3 and 6 are involved in the H2O2‐mediated inhibition of NHE‐ 1 promoter activity . 120  3.2.3  Caspase 3 activity found in the nucleus is important for NHE‐1 gene  regulation induced by H2O2 126  3.2.4  Down‐regulation of NHE‐1 protein expression induced by H2O2 is  mainly attributed to the inhibition of NHE‐1 gene transcription . 135  NHE-1 0  β-actin 0  3.3  ACTIVATION OF CASPASES AND MEDIATED BY MILD OXIDATIVE STRESS INVOLVES IRON .140  3.3.1  Sustained repression of NHE‐1 mediated by H2O2 is iron‐dependent140  3.4  DOWN-REGULATION OF NHE-1 PROMOTER ACTIVITY IS DEPENDENT ON THE PRODUCTION OF PEROXYNITRITE .155  3.4.1  An initial stimulus mediated by H2O2 induces a transient increase of  ONOO‐ at a later phase that is responsible for NHE‐1 gene regulation . 155  3.4.2    ONOO‐ participates in the down‐regulation of NHE‐1 gene expression . . 166  3.5  PRODUCTION OF ROS AT THE LATE PHASE IS DEPENDENT ON CASPASE AND MAY BE GENERATED IN THE NUCLEUS OF L6 CELL 178  3.5.1  The production of the second ROS/ONOO‐ is caspase 3 dependent. 178  3.5.2  The production of ONOO‐ at 12 hour following L6 cells exposure to  H2O2 may be from the cell’s nucleus . 180  3.5.3  3.6  NOX2 and n‐NOS are found in L61.1 cells 182  INCREASED HO-1 EXPRESSION AND ACTIVATION OF p38MAPK .185  Pg|4 3.6.1  Induction of HO‐1 by H2O2 may be responsible for the sustained NHE‐1  gene repression 185  3.6.2  Activation of p38MAPK pathway is important for the down‐regulation  of NHE‐1 promoter activity . 194  3.7  LOCALIZATION OF THE H2O2 RESPONSE ELEMENT 206  3.7.1  An AP‐2 binding site found in the NHE‐1 promoter region is  responsible to induce the inhibition of NHE‐1 promoter by H2O2 207  3.8  PHYSIOLOGICAL IMPORTANCE OF NHE-1 GENE REGULATION .212  3.8.1  Effect of mild oxidative stress on NHE‐1: The regulation of  intracellular pH and cell cycle . 212  CHAPTER 4: DISCUSSION .220  4.1  NHE-1 GENE EXPRESSION IS REDOX-REGULATED .221  4.1.1  Down-regulation of NHE-1 gene expression by H2O2 .221  4.1.2  Thiol oxidation of an AP-2 or AP-2-like transcription factor could be responsible for the initial inhibition of NHE-1 gene expression mediated by H2O2 222  4.2  ROLE OF CASPASES AND IN THE INHIBITION OF NHE-1 EXPRESSION BY MILD OXIDATIVE STRESS .225  4.3  IRON AND THE ACTIVATION OF CASPASES AND BY MILD OXIDATIVE STRESS .232  4.3.1  Iron is required for the activation of caspases and by non-toxic doses of H2O2 .232  4.3.2  Activation of HO-1: A possible mechanism involved in the increase of labile iron pool (LIP) .234  4.4  INTRACELLULAR PRODUCTION OF ROS/ONOO- AT THE LATE PHASE IS CRUCIAL FOR A SUSTAINED REPRESSION OF NHE-1 GENE EXPRESSION .240  4.5  NUCLEAR LOCALIZATION OF CASPASE PROTEINS: FOR EFFICIENT GENE REGULATION OF NHE-1 DURING MILD OXIDATIVE STRESS .243  Pg|5 4.6  ACTIVATION OF P38MAPK PATHWAY INVOLVES IN THE DOWNREGULATION OF NHE-1 PROMOTER ACTIVITY .248  4.7  NHE-1 EXPRESSION AS AN IMPORTANT DETERMINANT FOR CELL TRANSFORMATION: A POSSIBLE INITIATOR FOR TUMORIGENESIS? .251  4.8  CONCLUSION 255  REFERENCES 256  Pg|6 LIST OF FIGURES Figure A: Pathways of ROS production and clearance 22  Figure B: Structural organization of NOX protein family members .29  Figure C: Roles of H2O2 in a mammalian cell .34  Figure D: Schematic illustration of the steps in transcription factors NF-kB, AP-1, HSF1 and p53 activation that may be influenced by ROS and thiols-containing molecules .39  Figure E: Redox-mediated activation of transcription factor Nrf2 41  Figure F: Schematic representation of the role of ROS in oncogenesis 45  Figure G: Topology of NHE-1 and its regulatory elements .57  Figure H: DNA sequence of the promoter/enhancer region of the human NHE .60  Figure 1: Establish non-toxic concentrations of H2O2 in L61.1 cells 86  Figure 2: Illustration of a L61.1 cell stably expressing full length 1.1kb proximal fragment of the mouse NHE-1 gene promoter inserted 5’ to the luciferase reporter gene 87  Figure 3: Down-regulation of NHE-1 promoter activity by H2O2 is dose-dependent .89  Figure 4: NHE-1 promoter repression by H2O2 is truly a regulatory process and is not due to the degradation of the luciferase proteins .91  Figure 5: Down-regulation of NHE-1 mRNA and protein expression by H2O2 is dosedependent .93  Figure 6: Consumption of extracellular H2O2 by L61.1 cells results in the inhibitory effect of H2O2 seen on NHE-1 promoter activity 96  Figure 7: NHE-1 promoter down-regulation was due to H2O2 and not due to other products present in the extracellular medium 99  Figure 8: Recovery of NHE-1 promoter activity and cell proliferation ability when serum was re-introduced in L6 cells 102  Figure 9: βME dose response effect on H2O2-mediated NHE-1 promoter activity repression .104  Pg|7 Figure 10: Reducing agents, DTT and ME inhibited H2O2-mediated repression of NHE-1 gene expression .106  Figure 11: Cellular reducing agents NAC and GSH rescued the inhibitory effect of H2O2 on NHE-1 promoter activity .108  Figure 12: Diamide dose response repression of NHE-1 expression .111  Figure 13: Thiol oxidizing agent diamide mimicked the effect of H2O2 in the downregulation of NHE-1 gene expression 113  Figure 14: ME inhibited H2O2-mediated repression of NHE-1 gene expression if it was added prior to H2O2 incubation or maximally hours post-H2O2 treatment 114  Figure 15: Inhibition of NHE-1 gene expression by H2O2 is caspases dependent 117  Figure 16: Reducing agent βME and pan-caspases inhibitor z-VAD-fmk rescue the inhibition of NHE-1 gene expression by H2O2 at different time points .118  Figure 17: Reducing agent βME but not pan-caspases inhibitor z-VAD rescues the inhibition of NHE-1 gene expression by thiol-oxidant diamide 119  Figure 18: Caspases and 10 are not activated by H2O2 in L61.1 cells 121  Figure 19: H2O2 induced activation of caspases and independent of the initiator caspases 122  Figure 20: H2O2 induced inhibition of NHE-1 promoter activity involves the activation of caspases and 124  Figure 21: siRNA gene silencing of caspases and abolished the inhibitory effect of H2O2 on NHE-1 promoter activity .125  Figure 22: H2O2-mediated increased in caspase activity is more pronounced in the nucleus than in the cytosol .127  Figure 23: Increase in cleaved caspase activity and expression induced by H2O2 is more pronounced in the nucleus than cytosol of L61.1 cells .131  Figure 24: Increase in active caspase in the nucleus post-H2O2 treatment detected by immunofluorescence 134  Figure 25: DNA transcriptional inhibitor actinomycin D increases caspase activities in L61.1 cells 136  Figure 26: Decrease of NHE-1 mRNA level induced by H2O2 is of similar degree to transcriptional inhibition by actinomycin D 137  Pg|8 Figure 27: Down-regulation of NHE-1 protein expression by H2O2 is mainly due to the transcriptional repression of NHE-1 promoter .138  Figure 28: Summary diagram illustrating the contribution of transcription and posttranscriptional event that result in the total decrease of NHE-1 protein expression mediated by non-toxic doses of H2O2 in L61.1 cells .139  Figure 29: Scavenging of HO• by HCOONa does not rescue the repression of NHE-1 promoter mediated by H2O2 .142  Figure 30: Chelating of iron by DFO inhibits the repression of NHE-1 promoter mediated by H2O2 145  Figure 31: Chelating of iron by DFO prevent the increase of caspases and activities mediated by H2O2 .146  Figure 32: Chelating of iron by phenanthroline rescue the decrease in NHE-1 promoter activity mediated by H2O2 148  Figure 33: Chelating of iron by phenanthroline prevents the increase of caspases and activities mediated by H2O2 149  Figure 34: Chelating of iron by DFP inhibits the repression of NHE-1 promoter mediated by H2O2 150  Figure 35: Activities of caspases and are required for FeCl3-induced NHE-1 promoter inhibition 154  Figure 36: Increased level of ROS production is detected at hour and 12 hour postH2O2 treatment .156  Figure 37: ONOO- is detected at hour by DCFDA fluorophore after H2O2 treatment in L61.1 cells 158  Figure 38: Increase in DAF fluorescence is detected at hour after H2O2 treatment in L61.1 cells 159  Figure 39: ONOO- at the concentrations of 150µM to 200µM generate similar level of DCF fluorescence as 50µM H2O2 at 14 hour in L61.1 cells 161  Figure 40: DCF fluorescence produced at 14 hour post-H2O2 was abolished when L61.1 cells were pre-treated with ONOO- decomposition catalyst FeTPPS .163  Figure 41: DAF fluorescence produced at 14 hour post-H2O2 treatment was abolished when L61.1 cells were pre-treated with ONOO- decomposition catalyst FeTPPS 165  Pg|9 Lim, S., and Clement, M.V. 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P g | 287 [...]... induced by H2O2 and ONOO- in L 61. 1 cells 18 8  Figure 57: H2O2 increases HO -1 protein expression in both the cytosol and nucleus of L 61. 1 cell 18 9  Figure 58: Silencing of HO -1 expression reduces the activation of caspases 3 and 6 induced by H2O2 19 1  P g | 10 Figure 59: Silencing of HO -1 expression reduces the inhibitory effect of NHE -1 promoter activity by H2O2... Breakdown of ONOO- and chelating of iron blocked the inhibition of NHE -1 promoter activity mediated by H2O2 .16 7  Figure 43: Breakdown of ONOO- by FeTPPS prevents the decrease of NHE -1 promoter activity by different doses of H2O2 16 8  Figure 44: Breakdown of ONOO- by FeTPPS prevents the inhibitory effects of H2O2 on NHE -1 promoter activity from early part of the time kinetics 16 9  Figure... MV Repression of the Na+/ H+ exchanger 1 expression by PPARγ activation is a potential new approach for specific inhibition of tumor cells’ growth in vitro and in vivo Cancer Research (in press) Poster presentations: Chang MK, Kumar AP, Pervaiz S and Clement MV Biphasic Effects of H2O2 on Na+/ H+ exchanger 1 (NHE -1) Gene Expression: Reminiscent for a Pro-apototic Cellular Course Mediated by Redox Controlled... University of Singapore (2008) Chang MK, Kumar AP, Pervaiz S and Clement MV Down -regulation of Na+/ H+ Exchanger 1 gene expression by hydrogen peroxide via activation of caspases: A new pathway involved in the redox inhibition of gene transcription Presented at 1st Biochemistry Student Symposium Clinical Research Centre, National University of Singapore (2008) P g | 19 CHAPTER 1: INTRODUCTION 1. 1 FREE... of NHE -1 promoter activity mediated by ONOO- 17 4  Figure 49: Exogenously added ONOO- does not activate caspases 3 and 6 in L 61. 1 cells 17 6  Figure 50: ONOO- donor, SIN -1 activates caspases 3 .17 7  Figure 51: Inhibiting the activities of caspase 3 by specific inhibitor and siRNA gene silencing prevent the increase of DCF fluorescence at 14 hour following exposure of L 61. 1... inhibition of NHE -1 promoter by H2O2 209  Figure 67: Over -expression of a dominant negative AP-2 (AP2) protein inhibited NHE -1 gene expression . 211   Figure 68: H2O2 at 50µM decreases NHE -1 set-point pH but not the rate of H+ extrusion 215   Figure 69: Pan-caspases inhibitor z-VAD prevents the drop of set-point pH induced by 50µM H2O2 218   Figure 70: Percentage of. .. mechanism involved in the inhibition of NHE -1 gene expression by H2O2 2 31 Figure 74: Classical endocytosis pathway of transferrin-receptor (TfR) 237  P g | 11 Figure 75: Hypothetical pathway showing the involvement of ONOO- in the inhibition of NHE -1 gene expression by an initial H2O2 stimulus 242  Figure 76: Postulated pathway describing the generation of ONOO- in the nucleus upon an initial... of proteins and gene expression (Sies, 19 97) Oxidative stress can up-regulate or down-regulate gene expression depending on the transcription factor and the mechanism of activation (Arrigo, 19 99) In eukaryotes, to induce the expression of specific genes, transcription factors must bind to the promoter regions of the target genes to initiate the transcription by RNA polymerase II (Zahradka et al., 19 89;... 17 9  Figure 52: Increase in the level of DCF fluorescence is detected in the cell nucleus with H2O2 treatment 18 1  Figure 53: NOX 2 is expressed in the nuclei of L 61. 1 cells 18 3  Figure 54: n-NOS is the predominant nitric oxide synthase found in L6 cells 18 4  Figure 55: H2O2 induces the expression of HO -1 18 7  Figure 56: Time kinetic studies of HO -1 protein expression. .. activation of caspase 3 and 6: a new pathway involved in the redox inhibition of gene transcription Presented at SFRBM 14 th Annual Meeting Renaissance Washington DC Hotel Washington, D.C USA (2007) *Poster won a travel award P g | 18 Oral presentations: Chang MK, Kumar AP, Pervaiz S and Clement MV Role of Transcription Factor, AP-2 in H2O2-mediated Repression of the Na+/ H+ exchanger 1 (NHE1 ) Gene Expression . 53 1. 4.3. E NHE -1 and cell differentiation 53 1. 4.4 Regulation of activityand expression of NHE 1 54 1. 4.4. A Regulation of NHE -1 activity 54 1. 4.4. B Transcriptional regulation of NHE -1. and nucleus of L 61. 1 cell 18 9  Figure 58: Silencing of HO -1 expression reduces the activation of caspases 3 and 6 induced by H 2 O 2 19 1 Pg| 11 Figure 59: Silencing of HO -1 expression. REGULATION 212  3.8 .1 Effect of mild oxidative stress onNHE 1: The regulation of intracellularpHandcellcycle 212  CHAPTER 4: DISCUSSION 220 4 .1 NHE -1 GENE EXPRESSION IS REDOX-REGULATED 2 21 4 .1. 1