+ + 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. (2007). Phosphorylation of the survival kinase Akt by superoxide is dependent on an ascorbate-reversible oxidation of PTEN. Free Radic Biol Med 42, 1178-1192. Lim, S.D., Sun, C., Lambeth, J.D., Marshall, F., Amin, M., Chung, L., Petros, J.A., and Arnold, R.S. (2005). Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate 62, 200-207. Lin, F., and Girotti, A.W. (1993). Photodynamic action of merocyanine 540 on leukemia cells: iron-stimulated lipid peroxidation and cell killing. Arch Biochem Biophys 300, 714-723. Lin, K.T., Xue, J.Y., Lin, M.C., Spokas, E.G., Sun, F.F., and Wong, P.Y. (1998). Peroxynitrite induces apoptosis of HL-60 cells by activation of a caspase-3 family protease. Am J Physiol 274, C855-860. Lin, Q., Weis, S., Yang, G., Weng, Y.H., Helston, R., Rish, K., Smith, A., Bordner, J., Polte, T., Gaunitz, F., et al. (2007). Heme oxygenase-1 protein localizes to the nucleus and activates transcription factors important in oxidative stress. J Biol Chem 282, 20621-20633. Liu, Y., Borchert, G.L., Surazynski, A., Hu, C.A., and Phang, J.M. (2006). Proline oxidase activates both intrinsic and extrinsic pathways for apoptosis: the role of ROS/superoxides, NFAT and MEK/ERK signaling. Oncogene 25, 5640-5647. Lu, M., and Gong, X. (2009). Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress-induced mitochondrial dysfunction. Cell Biol Int 33, 658664. Lu, S.C. (2009). Regulation of glutathione synthesis. Mol Aspects Med 30, 42-59. Lupertz, R., Chovolou, Y., Kampkotter, A., Watjen, W., and Kahl, R. (2008). Catalase overexpression impairs TNF-alpha induced NF-kappaB activation and sensitizes MCF-7 cells against TNF-alpha. J Cell Biochem 103, 1497-1511. Maccaglia, A., Mallozzi, C., and Minetti, M. (2003). Differential effects of quercetin and resveratrol on Band tyrosine phosphorylation signalling of red blood cells. Biochem Biophys Res Commun 305, 541-547. Maelfait, J., and Beyaert, R. (2008). Non-apoptotic functions of caspase-8. Biochem Pharmacol 76, 1365-1373. Maguire, J.J., Kellogg, E.W., 3rd, and Packer, L. (1982). Protection against free radical formation by protein bound iron. Toxicol Lett 14, 27-34. Malhotra, J.D., and Kaufman, R.J. (2007). Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxid Redox Signal 9, 2277-2293. Malik, F., Kumar, A., Bhushan, S., Khan, S., Bhatia, A., Suri, K.A., Qazi, G.N., and Singh, J. (2007). Reactive oxygen species generation and mitochondrial dysfunction P g | 272 in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by N-acetyl cysteine. Apoptosis 12, 2115-2133. Mallozzi, C., De Franceschi, L., Brugnara, C., and Di Stasi, A.M. (2005). Protein phosphatase 1alpha is tyrosine-phosphorylated and inactivated by peroxynitrite in erythrocytes through the src family kinase fgr. Free Radic Biol Med 38, 1625-1636. Mallozzi, C., Di Stasi, A.M., and Minetti, M. (1997). Peroxynitrite modulates tyrosine-dependent signal transduction pathway of human erythrocyte band 3. FASEB J 11, 1281-1290. Malo, M.E., and Fliegel, L. (2006). Physiological role and regulation of the Na+/H+ exchanger. Can J Physiol Pharmacol 84, 1081-1095. Malo, M.E., Li, L., and Fliegel, L. (2007). Mitogen-activated protein kinasedependent activation of the Na+/H+ exchanger is mediated through phosphorylation of amino acids Ser770 and Ser771. The Journal of biological chemistry 282, 62926299. Mandal, M.N., Patlolla, J.M., Zheng, L., Agbaga, M.P., Tran, J.T., Wicker, L., KasusJacobi, A., Elliott, M.H., Rao, C.V., and Anderson, R.E. (2009). Curcumin protects retinal cells from light-and oxidant stress-induced cell death. Free Radic Biol Med 46, 672-679. Manly, S.P., and Matthews, K.S. (1979). Activity changes in lac repressor with cysteine oxidation. J Biol Chem 254, 3341-3347. Mannick, J.B., Schonhoff, C., Papeta, N., Ghafourifar, P., Szibor, M., Fang, K., and Gaston, B. (2001). S-Nitrosylation of mitochondrial caspases. J Cell Biol 154, 11111116. Manucha, W., Carrizo, L., Ruete, C., and Valles, P.G. (2007). Apoptosis induction is associated with decreased NHE1 expression in neonatal unilateral ureteric obstruction. BJU Int 100, 191-198. Mao, Z., and Wiedmann, M. (1999). Calcineurin enhances MEF2 DNA binding activity in calcium-dependent survival of cerebellar granule neurons. J Biol Chem 274, 31102-31107. Marches, R., Vitetta, E.S., and Uhr, J.W. (2001). A role for intracellular pH in membrane IgM-mediated cell death of human B lymphomas. Proc Natl Acad Sci U S A 98, 3434-3439. Margariti, A., Xiao, Q., Zampetaki, A., Zhang, Z., Li, H., Martin, D., Hu, Y., Zeng, L., and Xu, Q. (2009). Splicing of HDAC7 modulates the SRF-myocardin complex during stem-cell differentiation towards smooth muscle cells. J Cell Sci 122, 460-470. Margolin, Y., and Behrman, H.R. (1992). Xanthine oxidase and dehydrogenase activities in rat ovarian tissues. Am J Physiol 262, E173-178. P g | 273 Martyn, K.D., Frederick, L.M., von Loehneysen, K., Dinauer, M.C., and Knaus, U.G. (2006). Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases. Cell Signal 18, 69-82. Matsuoka, Y., Okazaki, M., and Kitamura, Y. (1999). Induction of inducible heme oxygenase (HO-1) in the central nervous system: is HO-1 helpful or harmful? Neurotox Res 1, 113-117. McBride, A.A., Klausner, R.D., and Howley, P.M. (1992). Conserved cysteine residue in the DNA-binding domain of the bovine papillomavirus type E2 protein confers redox regulation of the DNA-binding activity in vitro. Proc Natl Acad Sci U S A 89, 7531-7535. McCord, J.M. (1998). Iron, free radicals, and oxidative injury. Semin Hematol 35, 512. McKallip, R.J., Jia, W., Schlomer, J., Warren, J.W., Nagarkatti, P.S., and Nagarkatti, M. (2006). Cannabidiol-induced apoptosis in human leukemia cells: A novel role of cannabidiol in the regulation of p22phox and Nox4 expression. Mol Pharmacol 70, 897-908. McKie, A.T., Marciani, P., Rolfs, A., Brennan, K., Wehr, K., Barrow, D., Miret, S., Bomford, A., Peters, T.J., Farzaneh, F., et al. (2000). A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell 5, 299-309. Meima, M.E., Mackley, J.R., and Barber, D.L. (2007). Beyond ion translocation: structural functions of the sodium-hydrogen exchanger isoform-1. Curr Opin Nephrol Hypertens 16, 365-372. Meischl, C., Krijnen, P.A., Sipkens, J.A., Cillessen, S.A., Munoz, I.G., Okroj, M., Ramska, M., Muller, A., Visser, C.A., Musters, R.J., et al. (2006). Ischemia induces nuclear NOX2 expression in cardiomyocytes and subsequently activates apoptosis. Apoptosis 11, 913-921. Miller, R.T., Counillon, L., Pages, G., Lifton, R.P., Sardet, C., and Pouyssegur, J. (1991). Structure of the 5'-flanking regulatory region and gene for the human growth factor-activatable Na/H exchanger NHE-1. J Biol Chem 266, 10813-10819. Min, K.S., Lee, H.J., Kim, S.H., Lee, S.K., Kim, H.R., Pae, H.O., Chung, H.T., Shin, H.I., and Kim, E.C. (2008). Hydrogen peroxide induces heme oxygenase-1 and dentin sialophosphoprotein mRNA in human pulp cells. J Endod 34, 983-989. Mishima, M., Wakabayashi, S., and Kojima, C. (2007). Solution structure of the cytoplasmic region of Na+/H+ exchanger complexed with essential cofactor calcineurin B homologous protein 1. The Journal of biological chemistry 282, 27412751. Misik, A.J., Perreault, K., Holmes, C.F., and Fliegel, L. (2005). Protein phosphatase regulation of Na+/H+ exchanger isoform I. Biochemistry 44, 5842-5852. P g | 274 Misko, T.P., Highkin, M.K., Veenhuizen, A.W., Manning, P.T., Stern, M.K., Currie, M.G., and Salvemini, D. (1998). Characterization of the cytoprotective action of peroxynitrite decomposition catalysts. J Biol Chem 273, 15646-15653. Miyano, K., Koga, H., Minakami, R., and Sumimoto, H. (2009). The insert region of the Rac GTPases is dispensable for activation of superoxide-producing NADPH oxidases. Biochem J. Montcourrier, P., Silver, I., Farnoud, R., Bird, I., and Rochefort, H. (1997). Breast cancer cells have a high capacity to acidify extracellular milieu by a dual mechanism. Clinical & experimental metastasis 15, 382-392. Moos, T., and Rosengren Nielsen, T. (2006). Ferroportin in the postnatal rat brain: implications for axonal transport and neuronal export of iron. Semin Pediatr Neurol 13, 149-157. Morel, Y., and Barouki, R. (1998). Down-regulation of cytochrome P450 1A1 gene promoter by oxidative stress. Critical contribution of nuclear factor 1. J Biol Chem 273, 26969-26976. Morel, Y., and Barouki, R. (2000). The repression of nuclear factor I/CCAAT transcription factor (NFI/CTF) transactivating domain by oxidative stress is mediated by a critical cysteine (Cys-427). Biochem J 348 Pt 1, 235-240. Myhre, O., Andersen, J.M., Aarnes, H., and Fonnum, F. (2003). Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol 65, 1575-1582. Nakagawa, H., Hasumi, K., Woo, J.T., Nagai, K., and Wachi, M. (2004). Generation of hydrogen peroxide primarily contributes to the induction of Fe(II)-dependent apoptosis in Jurkat cells by (-)-epigallocatechin gallate. Carcinogenesis 25, 1567-1574. Nakano, Y., Banfi, B., Jesaitis, A.J., Dinauer, M.C., Allen, L.A., and Nauseef, W.M. (2007). Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3. Biochem J 403, 97-108. Nakatsubo, N., Kojima, H., Sakurai, K., Kikuchi, K., Nagoshi, H., Hirata, Y., Akaike, T., Maeda, H., Urano, Y., Higuchi, T., et al. (1998). Improved nitric oxide detection using 2,3-diaminonaphthalene and its application to the evaluation of novel nitric oxide synthase inhibitors. Biol Pharm Bull 21, 1247-1250. Nathan, C., and Xie, Q.W. (1994). Nitric oxide synthases: roles, tolls, and controls. Cell 78, 915-918. Nauseef, W.M. (2008). Biological roles for the NOX family NADPH oxidases. J Biol Chem 283, 16961-16965. Nguyen, H.X., and Tidball, J.G. (2003). Interactions between neutrophils and macrophages promote macrophage killing of rat muscle cells in vitro. J Physiol 547, 125-132. P g | 275 Noel, J., and Pouyssegur, J. (1995). Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. The American journal of physiology 268, C283-296. Nyormoi, O., Wang, Z., Doan, D., Ruiz, M., McConkey, D., and Bar-Eli, M. (2001). Transcription factor AP-2alpha is preferentially cleaved by caspase and degraded by proteasome during tumor necrosis factor alpha-induced apoptosis in breast cancer cells. Mol Cell Biol 21, 4856-4867. Olson, C.E., Soll, A.H., and Kaplowitz, N. (1985). Modulating effect of thioldisulfide status on [14C]aminopyrine accumulation in the isolated parietal cell. J Biol Chem 260, 8020-8025. Orlowski, J., and Grinstein, S. (2004). Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflugers Arch 447, 549-565. Ozben, T. (2007). Oxidative stress and apoptosis: impact on cancer therapy. J Pharm Sci 96, 2181-2196. Ozkan, P., and Mutharasan, R. (2002). A rapid method for measuring intracellular pH using BCECF-AM. Biochim Biophys Acta 1572, 143-148. Pacher, P., Beckman, J.S., and Liaudet, L. (2007). Nitric oxide and peroxynitrite in health and disease. Physiol Rev 87, 315-424. Palmer, R.M., Rees, D.D., Ashton, D.S., and Moncada, S. (1988). L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun 153, 1251-1256. Pang, T., Su, X., Wakabayashi, S., and Shigekawa, M. (2001). Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers. The Journal of biological chemistry 276, 17367-17372. Paradiso, A., Cardone, R.A., Bellizzi, A., Bagorda, A., Guerra, L., Tommasino, M., Casavola, V., and Reshkin, S.J. (2004). The Na+-H+ exchanger-1 induces cytoskeletal changes involving reciprocal RhoA and Rac1 signaling, resulting in motility and invasion in MDA-MB-435 cells. Breast Cancer Res 6, R616-628. Park, C.I., Park, Y.N., and Jung, W.H. (1995). Transferrin receptor expression of the hyperplastic lesions of hepatocyte in experimental hepatocarcinogenesis. J Korean Med Sci 10, 183-188. Paroo, Z., Meredith, M.J., Locke, M., Haist, J.V., Karmazyn, M., and Noble, E.G. (2002). Redox signaling of cardiac HSF1 DNA binding. Am J Physiol Cell Physiol 283, C404-411. Pedersen, S.F. (2006). The Na+/H+ exchanger NHE1 in stress-induced signal transduction: implications for cell proliferation and cell death. Pflugers Arch 452, 249-259. P g | 276 Pedersen, S.F., Darborg, B.V., Rentsch, M.L., and Rasmussen, M. (2007a). Regulation of mitogen-activated protein kinase pathways by the plasma membrane Na+/H+ exchanger, NHE1. Arch Biochem Biophys 462, 195-201. Pedersen, S.F., King, S.A., Nygaard, E.B., Rigor, R.R., and Cala, P.M. (2007b). NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity. The Journal of biological chemistry 282, 1971619727. Pendergrass, K.D., Gwathmey, T.M., Michalek, R.D., Grayson, J.M., and Chappell, M.C. (2009). The angiotensin II-AT1 receptor stimulates reactive oxygen species within the cell nucleus. Biochem Biophys Res Commun 384, 149-154. Pennypacker, K.R., Hong, J.S., Douglass, J., and McMillian, M.K. (1992). Constitutive expression of AP-1 transcription factors in the rat adrenal. Effects of nicotine. J Biol Chem 267, 20148-20152. Pervaiz, S., Cao, J., Chao, O.S., Chin, Y.Y., and Clement, M.V. (2001). Activation of the RacGTPase inhibits apoptosis in human tumor cells. Oncogene 20, 6263-6268. Pervaiz, S., and Clement, M.V. (2004). Tumor intracellular redox status and drug resistance--serendipity or a causal relationship? Curr Pharm Des 10, 1969-1977. Pervaiz, S., and Clement, M.V. (2007). Superoxide anion: oncogenic reactive oxygen species? Int J Biochem Cell Biol 39, 1297-1304. Pesse, B., Levrand, S., Feihl, F., Waeber, B., Gavillet, B., Pacher, P., and Liaudet, L. (2005). Peroxynitrite activates ERK via Raf-1 and MEK, independently from EGF receptor and p21Ras in H9C2 cardiomyocytes. J Mol Cell Cardiol 38, 765-775. Petrecca, K., Atanasiu, R., Grinstein, S., Orlowski, J., and Shrier, A. (1999). Subcellular localization of the Na+/H+ exchanger NHE1 in rat myocardium. The American journal of physiology 276, H709-717. Piedrafita, F.J., and Pfahl, M. (1997). Retinoid-induced apoptosis and Sp1 cleavage occur independently of transcription and require caspase activation. Mol Cell Biol 17, 6348-6358. Pigeolet, E., Corbisier, P., Houbion, A., Lambert, D., Michiels, C., Raes, M., Zachary, M.D., and Remacle, J. (1990). Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals. Mech Ageing Dev 51, 283-297. Porreca, E., Del Boccio, G., Lapenna, D., Di Febbo, C., Pennelli, A., Cipollone, F., Di Ilio, C., and Cuccurullo, F. (1994). Myocardial antioxidant defense mechanisms: time related changes after reperfusion of the ischemic rat heart. Free Radic Res 20, 171179. P g | 277 Prus, E., and Fibach, E. (2008). Flow cytometry measurement of the labile iron pool in human hematopoietic cells. Cytometry A 73, 22-27. Putney, L.K., and Barber, D.L. (2003). Na-H exchange-dependent increase in intracellular pH times G2/M entry and transition. J Biol Chem 278, 44645-44649. Putney, L.K., Denker, S.P., and Barber, D.L. (2002). The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions. Annu Rev Pharmacol Toxicol 42, 527-552. Raman, M., Earnest, S., Zhang, K., Zhao, Y., and Cobb, M.H. (2007). TAO kinases mediate activation of p38 in response to DNA damage. EMBO J 26, 2005-2014. Ranawat, P., and Bansal, M.P. (2009). Apoptosis induced by modulation in selenium status involves p38 MAPK and ROS: implications in spermatogenesis. Mol Cell Biochem. Rao, G.N., Sardet, C., Pouyssegur, J., and Berk, B.C. (1993). Phosphorylation of Na(+)-H+ antiporter is not stimulated by phorbol ester and acidification in granulocytic HL-60 cells. The American journal of physiology 264, C1278-1284. Rebillard, A., Tekpli, X., Meurette, O., Sergent, O., LeMoigne-Muller, G., Vernhet, L., Gorria, M., Chevanne, M., Christmann, M., Kaina, B., et al. (2007a). Cisplatininduced apoptosis involves membrane fluidification via inhibition of NHE1 in human colon cancer cells. Cancer Res 67, 7865-7874. Rebillard, A., Tekpli, X., Meurette, O., Sergent, O., Lemoigne-Muller, G., Vernhet, L., Gorria, M., Chevanne, M., Christmann, M., Kaina, B., et al. (2007b). CisplatinInduced Apoptosis Involves Membrane Fluidification via Inhibition of NHE1 in Human Colon Cancer Cells. Haematologica 67, 7865-7874. Reinhardt, H.C., Aslanian, A.S., Lees, J.A., and Yaffe, M.B. (2007). p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 11, 175-189. Reshkin, S.J., Bellizzi, A., Albarani, V., Guerra, L., Tommasino, M., Paradiso, A., and Casavola, V. (2000a). Phosphoinositide 3-kinase is involved in the tumor-specific activation of human breast cancer cell Na(+)/H(+) exchange, motility, and invasion induced by serum deprivation. The Journal of biological chemistry 275, 5361-5369. Reshkin, S.J., Bellizzi, A., Caldeira, S., Albarani, V., Malanchi, I., Poignee, M., Alunni-Fabbroni, M., Casavola, V., and Tommasino, M. (2000b). Na+/H+ exchangerdependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformationassociated phenotypes. Faseb J 14, 2185-2197. Rhee, S.G., Chang, T.S., Bae, Y.S., Lee, S.R., and Kang, S.W. (2003). Cellular regulation by hydrogen peroxide. J Am Soc Nephrol 14, S211-215. P g | 278 Rich, I.N., Worthington-White, D., Garden, O.A., and Musk, P. (2000). Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na(+)/H(+) exchanger. Blood 95, 1427-1434. Robinson, K.M., and Beckman, J.S. (2005). Synthesis of peroxynitrite from nitrite and hydrogen peroxide. Methods Enzymol 396, 207-214. Rokutan, K., Kawahara, T., Kuwano, Y., Tominaga, K., Sekiyama, A., and TeshimaKondo, S. (2006). NADPH oxidases in the gastrointestinal tract: a potential role of Nox1 in innate immune response and carcinogenesis. Antioxid Redox Signal 8, 15731582. Roos, A., and Boron, W.F. (1981). Intracellular pH. Physiol Rev 61, 296-434. Rothe, F., Possel, H., and Wolf, G. (2002). Nitric oxide affects the phosphorylation state of microtubule-associated protein (MAP-2) and neurofilament: an immunocytochemical study in the brain of rats and neuronal nitric oxide synthase (nNOS)-knockouts. Nitric Oxide 6, 9-17. Roy, S., Sen, C.K., and Packer, L. (1999). Determination of cell-cell adhesion in response to oxidants and antioxidants. Methods Enzymol 300, 395-401. Ruiz-Ramos, R., Cebrian, M.E., and Garrido, E. (2005). Benzoquinone activates the ERK/MAPK signaling pathway via ROS production in HL-60 cells. Toxicology 209, 279-287. Russ, U., Balser, C., Scholz, W., Albus, U., Lang, H.J., Weichert, A., Scholkens, B.A., and Gogelein, H. (1996). Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes. Pflugers Arch 433, 26-34. Ryter, S.W., Xi, S., Hartsfield, C.L., and Choi, A.M. (2002). Mitogen activated protein kinase (MAPK) pathway regulates heme oxygenase-1 gene expression by hypoxia in vascular cells. Antioxid Redox Signal 4, 587-592. Sacca, P., Meiss, R., Casas, G., Mazza, O., Calvo, J.C., Navone, N., and Vazquez, E. (2007). Nuclear translocation of haeme oxygenase-1 is associated to prostate cancer. Br J Cancer 97, 1683-1689. Salerno, L., Sorrenti, V., Di Giacomo, C., Romeo, G., and Siracusa, M.A. (2002). Progress in the development of selective nitric oxide synthase (NOS) inhibitors. Curr Pharm Des 8, 177-200. Sardet, C., Fafournoux, P., and Pouyssegur, J. (1991). Alpha-thrombin, epidermal growth factor, and okadaic acid activate the Na+/H+ exchanger, NHE-1, by phosphorylating a set of common sites. The Journal of biological chemistry 266, 19166-19171. Sarma, B.K., and Mugesh, G. (2008). Thiol cofactors for selenoenzymes and their synthetic mimics. Org Biomol Chem 6, 965-974. P g | 279 Sauer, H., Wartenberg, M., and Hescheler, J. (2001). Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 11, 173-186. Schmid, E., Hotz-Wagenblatt, A., Hacj, V., and Droge, W. (1999). Phosphorylation of the insulin receptor kinase by phosphocreatine in combination with hydrogen peroxide: the structural basis of redox priming. FASEB J 13, 1491-1500. Scott, F.L., Fuchs, G.J., Boyd, S.E., Denault, J.B., Hawkins, C.J., Dequiedt, F., and Salvesen, G.S. (2008). Caspase-8 cleaves histone deacetylase and abolishes its transcription repressor function. J Biol Chem 283, 19499-19510. Serrander, L., Cartier, L., Bedard, K., Banfi, B., Lardy, B., Plastre, O., Sienkiewicz, A., Forro, L., Schlegel, W., and Krause, K.H. (2007a). NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation. Biochem J 406, 105-114. Serrander, L., Jaquet, V., Bedard, K., Plastre, O., Hartley, O., Arnaudeau, S., Demaurex, N., Schlegel, W., and Krause, K.H. (2007b). NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation. Biochimie 89, 1159-1167. Sharma, S.S., Dhar, A., and Kaundal, R.K. (2007). FeTPPS protects against global cerebral ischemic-reperfusion injury in gerbils. Pharmacol Res 55, 335-342. Sheng, J.Z., Wang, D., and Braun, A.P. (2005). DAF-FM (4-amino-5-methylamino2',7'-difluorofluorescein) diacetate detects impairment of agonist-stimulated nitric oxide synthesis by elevated glucose in human vascular endothelial cells: reversal by vitamin C and L-sepiapterin. J Pharmacol Exp Ther 315, 931-940. Shi, H., Hudson, L.G., Ding, W., Wang, S., Cooper, K.L., Liu, S., Chen, Y., Shi, X., and Liu, K.J. (2004). Arsenite causes DNA damage in keratinocytes via generation of hydroxyl radicals. Chem Res Toxicol 17, 871-878. Shono, T., Yokoyama, N., Uesaka, T., Kuroda, J., Takeya, R., Yamasaki, T., Amano, T., Mizoguchi, M., Suzuki, S.O., Niiro, H., et al. (2008). Enhanced expression of NADPH oxidase Nox4 in human gliomas and its roles in cell proliferation and survival. Int J Cancer 123, 787-792. Sies, H. (1997). Oxidative stress: oxidants and antioxidants. Exp Physiol 82, 291-295. Simmons, C.P., Goncalves, N.S., Ghaem-Maghami, M., Bajaj-Elliott, M., Clare, S., Neves, B., Frankel, G., Dougan, G., and MacDonald, T.T. (2002). Impaired resistance and enhanced pathology during infection with a noninvasive, attaching-effacing enteric bacterial pathogen, Citrobacter rodentium, in mice lacking IL-12 or IFNgamma. J Immunol 168, 1804-1812. Simon, H.U., Haj-Yehia, A., and Levi-Schaffer, F. (2000). Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5, 415-418. P g | 280 Slepkov, E.R., Rainey, J.K., Sykes, B.D., and Fliegel, L. (2007). Structural and functional analysis of the Na+/H+ exchanger. Biochem J 401, 623-633. Snabaitis, A.K., Cuello, F., and Avkiran, M. (2008). Protein kinase B/Akt phosphorylates and inhibits the cardiac Na+/H+ exchanger NHE1. Circ Res 103, 881890. Snabaitis, A.K., D'Mello, R., Dashnyam, S., and Avkiran, M. (2006). A novel role for protein phosphatase 2A in receptor-mediated regulation of the cardiac sarcolemmal Na+/H+ exchanger NHE1. J Biol Chem 281, 20252-20262. Soares, M.P., and Bach, F.H. (2009). Heme oxygenase-1: from biology to therapeutic potential. Trends Mol Med 15, 50-58. Sorensen, V., Zhen, Y., Zakrzewska, M., Haugsten, E.M., Walchli, S., Nilsen, T., Olsnes, S., and Wiedlocha, A. (2008). Phosphorylation of fibroblast growth factor (FGF) receptor at Ser777 by p38 mitogen-activated protein kinase regulates translocation of exogenous FGF1 to the cytosol and nucleus. Mol Cell Biol 28, 41294141. Soum, E., Brazzolotto, X., Goussias, C., Bouton, C., Moulis, J.M., Mattioli, T.A., and Drapier, J.C. (2003). Peroxynitrite and nitric oxide differently target the iron-sulfur cluster and amino acid residues of human iron regulatory protein 1. Biochemistry 42, 7648-7654. Stamler, J.S., and Meissner, G. (2001). Physiology of nitric oxide in skeletal muscle. Physiol Rev 81, 209-237. Stamler, J.S., Singel, D.J., and Loscalzo, J. (1992). Biochemistry of nitric oxide and its redox-activated forms. Science 258, 1898-1902. Stephenson, D., Yin, T., Smalstig, E.B., Hsu, M.A., Panetta, J., Little, S., and Clemens, J. (2000). Transcription factor nuclear factor-kappa B is activated in neurons after focal cerebral ischemia. J Cereb Blood Flow Metab 20, 592-603. Stock, C., and Schwab, A. (2006). Role of the Na/H exchanger NHE1 in cell migration. Acta Physiol (Oxf) 187, 149-157. Stone, J.R., and Yang, S. (2006). Hydrogen peroxide: a signaling messenger. Antioxid Redox Signal 8, 243-270. Sturrock, A., Huecksteadt, T.P., Norman, K., Sanders, K., Murphy, T.M., Chitano, P., Wilson, K., Hoidal, J.R., and Kennedy, T.P. (2007). Nox4 mediates TGF-beta1induced retinoblastoma protein phosphorylation, proliferation, and hypertrophy in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 292, L15431555. Su, Y.R., Linton, M.F., and Fazio, S. (2002). Rapid quantification of murine ABC mRNAs by real time reverse transcriptase-polymerase chain reaction. J Lipid Res 43, 2180-2187. P g | 281 Sugio, T., Mizunashi, W., Inagaki, K., and Tano, T. (1987). Purification and some properties of sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans. J Bacteriol 169, 4916-4922. Sumimoto, H., Ueno, N., Yamasaki, T., Taura, M., and Takeya, R. (2004). Molecular mechanism underlying activation of superoxide-producing NADPH oxidases: roles for their regulatory proteins. Jpn J Infect Dis 57, S24-25. Surguladze, N., Patton, S., Cozzi, A., Fried, M.G., and Connor, J.R. (2005). Characterization of nuclear ferritin and mechanism of translocation. Biochem J 388, 731-740. Szanto, I., Rubbia-Brandt, L., Kiss, P., Steger, K., Banfi, B., Kovari, E., Herrmann, F., Hadengue, A., and Krause, K.H. (2005). Expression of NOX1, a superoxidegenerating NADPH oxidase, in colon cancer and inflammatory bowel disease. J Pathol 207, 164-176. Taddei, M.L., Parri, M., Mello, T., Catalano, A., Levine, A.D., Raugei, G., Ramponi, G., and Chiarugi, P. (2007). Integrin-mediated cell adhesion and spreading engage different sources of reactive oxygen species. Antioxid Redox Signal 9, 469-481. Takano, H., Zou, Y., Hasegawa, H., Akazawa, H., Nagai, T., and Komuro, I. (2003). Oxidative stress-induced signal transduction pathways in cardiac myocytes: involvement of ROS in heart diseases. Antioxid Redox Signal 5, 789-794. Tanito, M., Agbaga, M.P., and Anderson, R.E. (2007). Upregulation of thioredoxin system via Nrf2-antioxidant responsive element pathway in adaptive-retinal neuroprotection in vivo and in vitro. Free Radic Biol Med 42, 1838-1850. Thomas, C., Mackey, M.M., Diaz, A.A., and Cox, D.P. (2009). Hydroxyl radical is produced via the Fenton reaction in submitochondrial particles under oxidative stress: implications for diseases associated with iron accumulation. Redox Rep 14, 102-108. Thome, U., Lazrak, A., Chen, L., Kirk, M.C., Thomas, M.J., Forman, H.J., and Matalon, S. (2003). Novel SIN-1 reactive intermediates modulate chloride secretion across murine airway cells. Free Radic Biol Med 35, 662-675. Thornton, T.M., and Rincon, M. (2009). Non-classical p38 map kinase functions: cell cycle checkpoints and survival. Int J Biol Sci 5, 44-51. Tominaga, K., Kawahara, T., Sano, T., Toida, K., Kuwano, Y., Sasaki, H., Kawai, T., Teshima-Kondo, S., and Rokutan, K. (2007). Evidence for cancer-associated expression of NADPH oxidase (Nox1)-based oxidase system in the human stomach. Free Radic Biol Med 43, 1627-1638. Topham, R., Goger, M., Pearce, K., and Schultz, P. (1989). The mobilization of ferritin iron by liver cytosol. A comparison of xanthine and NADH as reducing substrates. Biochem J 261, 137-143. P g | 282 Tortora, V., Quijano, C., Freeman, B., Radi, R., and Castro, L. (2007). Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation. Free Radic Biol Med 42, 1075-1088. Toualbi, K., Guller, M.C., Mauriz, J.L., Labalette, C., Buendia, M.A., Mauviel, A., and Bernuau, D. (2007). Physical and functional cooperation between AP-1 and betacatenin for the regulation of TCF-dependent genes. Oncogene 26, 3492-3502. Toyokuni, S. (1996). Iron-induced carcinogenesis: the role of redox regulation. Free Radic Biol Med 20, 553-566. Trackey, J.L., Uliasz, T.F., and Hewett, S.J. (2001). SIN-1-induced cytotoxicity in mixed cortical cell culture: peroxynitrite-dependent and -independent induction of excitotoxic cell death. J Neurochem 79, 445-455. Tsuruta, L., Lee, H.J., Masuda, E.S., Yokota, T., Arai, N., and Arai, K. (1995). Regulation of expression of the IL-2 and IL-5 genes and the role of proteins related to nuclear factor of activated T cells. J Allergy Clin Immunol 96, 1126-1135. Turturro, F., Driscoll, M., Friday, E., and Welbourne, T. (2007). ALK-mediated Na+/H+ exchanger-dependent intracellular alkalinization: does it matter for oncogenesis? Haematologica 92, 706-707. Ueno, N., Takeya, R., Miyano, K., Kikuchi, H., and Sumimoto, H. (2005). The NADPH oxidase Nox3 constitutively produces superoxide in a p22phox-dependent manner: its regulation by oxidase organizers and activators. J Biol Chem 280, 2332823339. Urban, T., Hurbain, I., Urban, M., Clement, A., and Housset, B. (1995). [Oxidants and antioxidants. Biological effects and therapeutic perspectives]. Ann Chir 49, 427-434. Ushio-Fukai, M. (2006). Localizing NADPH oxidase-derived ROS. Sci STKE 2006, re8. Verdin, E., Dequiedt, F., and Kasler, H. (2004). HDAC7 regulates apoptosis in developing thymocytes. Novartis Found Symp 259, 115-129; discussion 129-131, 163-119. Verdone, L., Caserta, M., and Di Mauro, E. (2005). Role of histone acetylation in the control of gene expression. Biochem Cell Biol 83, 344-353. Virag, L., Scott, G.S., Cuzzocrea, S., Marmer, D., Salzman, A.L., and Szabo, C. (1998). Peroxynitrite-induced thymocyte apoptosis: the role of caspases and poly (ADP-ribose) synthetase (PARS) activation. Immunology 94, 345-355. Vollgraf, U., Wegner, M., and Richter-Landsberg, C. (1999). Activation of AP-1 and nuclear factor-kappaB transcription factors is involved in hydrogen peroxide-induced apoptotic cell death of oligodendrocytes. J Neurochem 73, 2501-2509. P g | 283 Wakabayashi, S., Hisamitsu, T., Pang, T., and Shigekawa, M. (2003). Kinetic dissection of two distinct proton binding sites in Na+/H+ exchangers by measurement of reverse mode reaction. The Journal of biological chemistry 278, 43580-43585. Wakabayashi, S., Pang, T., Su, X., and Shigekawa, M. (2000). A novel topology model of the human Na(+)/H(+) exchanger isoform 1. J Biol Chem 275, 7942-7949. Wang, C., Fu, M., D'Amico, M., Albanese, C., Zhou, J.N., Brownlee, M., Lisanti, M.P., Chatterjee, V.K., Lazar, M.A., and Pestell, R.G. (2001). Inhibition of cellular proliferation through IkappaB kinase-independent and peroxisome proliferatoractivated receptor gamma-dependent repression of cyclin D1. Mol Cell Biol 21, 30573070. Wang, G., Anrather, J., Glass, M.J., Tarsitano, M.J., Zhou, P., Frys, K.A., Pickel, V.M., and Iadecola, C. (2006a). Nox2, Ca2+, and protein kinase C play a role in angiotensin II-induced free radical production in nucleus tractus solitarius. Hypertension 48, 482-489. Wang, X., Khaleque, M.A., Zhao, M.J., Zhong, R., Gaestel, M., and Calderwood, S.K. (2006b). Phosphorylation of HSF1 by MAPK-activated protein kinase on serine 121, inhibits transcriptional activity and promotes HSP90 binding. J Biol Chem 281, 782791. Webster, K.A., Prentice, H., and Bishopric, N.H. (2001). Oxidation of zinc finger transcription factors: physiological consequences. Antioxid Redox Signal 3, 535-548. Wei, C.C., Wang, Z.Q., Durra, D., Hemann, C., Hille, R., Garcin, E.D., Getzoff, E.D., and Stuehr, D.J. (2005). The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation. J Biol Chem 280, 8929-8935. Weiss, S.J., and Sagone, A.L., Jr. (1979). The effect of oxidant stress on diamidetreated human granulocytes. Biochim Biophys Acta 585, 620-629. Widera, A., Norouziyan, F., and Shen, W.C. (2003). Mechanisms of TfR-mediated transcytosis and sorting in epithelial cells and applications toward drug delivery. Adv Drug Deliv Rev 55, 1439-1466. Wiese, A.G., Pacifici, R.E., and Davies, K.J. (1995). Transient adaptation of oxidative stress in mammalian cells. Arch Biochem Biophys 318, 231-240. Williams, I.A., Xiao, X.H., Ju, Y.K., and Allen, D.G. (2007). The rise of [Na(+)] (i) during ischemia and reperfusion in the rat heart-underlying mechanisms. Pflugers Arch 454, 903-912. Williamson, J.A., Bosher, J.M., Skinner, A., Sheer, D., Williams, T., and Hurst, H.C. (1996). Chromosomal mapping of the human and mouse homologues of two new members of the AP-2 family of transcription factors. Genomics 35, 262-264. P g | 284 Witt, O., Deubzer, H.E., Milde, T., and Oehme, I. (2009). HDAC family: What are the cancer relevant targets? Cancer Lett 277, 8-21. Woenckhaus, C., Giebel, J., Failing, K., Fenic, I., Dittberner, T., and Poetsch, M. (2003). Expression of AP-2alpha, c-kit, and cleaved caspase-6 and -3 in naevi and malignant melanomas of the skin. A possible role for caspases in melanoma progression? J Pathol 201, 278-287. Wu, K.L., Khan, S., Lakhe-Reddy, S., Jarad, G., Mukherjee, A., Obejero-Paz, C.A., Konieczkowski, M., Sedor, J.R., and Schelling, J.R. (2004). The NHE1 Na+/H+ exchanger recruits ezrin/radixin/moesin proteins to regulate Akt-dependent cell survival. J Biol Chem 279, 26280-26286. Wu, K.L., Khan, S., Lakhe-Reddy, S., Wang, L., Jarad, G., Miller, R.T., Konieczkowski, M., Brown, A.M., Sedor, J.R., and Schelling, J.R. (2003). Renal tubular epithelial cell apoptosis is associated with caspase cleavage of the NHE1 Na+/H+ exchanger. Am J Physiol Renal Physiol 284, F829-839. Wu, W.S. (2006). The signaling mechanism of ROS in tumor progression. Cancer Metastasis Rev 25, 695-705. Wu, X., Bishopric, N.H., Discher, D.J., Murphy, B.J., and Webster, K.A. (1996). Physical and functional sensitivity of zinc finger transcription factors to redox change. Mol Cell Biol 16, 1035-1046. Xiong, S., She, H., Zhang, A.S., Wang, J., Mkrtchyan, H., Dynnyk, A., Gordeuk, V.R., French, S.W., Enns, C.A., and Tsukamoto, H. (2008). Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis. Am J Physiol Gastrointest Liver Physiol 295, G512-521. Xue, C., Pollock, J., Schmidt, H.H., Ward, S.M., and Sanders, K.M. (1994). Expression of nitric oxide synthase immunoreactivity by interstitial cells of the canine proximal colon. J Auton Nerv Syst 49, 1-14. Xue, J., Zhou, D., Yao, H., Gavrialov, O., McConnell, M.J., Gelb, B.D., and Haddad, G.G. (2007). Novel functional interaction between Na+/H+ exchanger and tyrosine phosphatase SHP-2. Am J Physiol Regul Integr Comp Physiol 292, R2406-2416. Yamaguchi, K., Mandai, M., Toyokuni, S., Hamanishi, J., Higuchi, T., Takakura, K., and Fujii, S. (2008). Contents of endometriotic cysts, especially the high concentration of free iron, are a possible cause of carcinogenesis in the cysts through the iron-induced persistent oxidative stress. Clin Cancer Res 14, 32-40. Yamakawa, H., Ito, Y., Naganawa, T., Banno, Y., Nakashima, S., Yoshimura, S., Sawada, M., Nishimura, Y., Nozawa, Y., and Sakai, N. (2000). Activation of caspase9 and -3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation. Neurol Res 22, 556-564. P g | 285 Yan, L., and Spallholz, J.E. (1993). Generation of reactive oxygen species from the reaction of selenium compounds with thiols and mammary tumor cells. Biochem Pharmacol 45, 429-437. Yang, C., Lai, H., Zhan, C., Xiao, Y., and Zheng, W. (2002). nNOS expression of hippocampal neurons in aged rats after brain ischemia/reperfusion and its role in DND development. Chin J Traumatol 5, 232-236. Yang, W., Dyck, J.R., and Fliegel, L. (1996a). Regulation of NHE1 expression in L6 muscle cells. Biochim Biophys Acta 1306, 107-113. Yang, W., Dyck, J.R., Wang, H., and Fliegel, L. (1996b). Regulation of NHE-1 promoter in mammalian myocardium. Am J Physiol 270, H259-266. Ye, J. (2008). Regulation of PPARgamma function by TNF-alpha. Biochem Biophys Res Commun 374, 405-408. Yu, H., Freedman, B.I., Rich, S.S., and Bowden, D.W. (2000). Human Na+/H+ exchanger genes : identification of polymorphisms by radiation hybrid mapping and analysis of linkage in end-stage renal disease. Hypertension 35, 135-143. Zachos, N.C., Tse, M., and Donowitz, M. (2005). Molecular physiology of intestinal Na+/H+ exchange. Annu Rev Physiol 67, 411-443. Zahradka, P., Larson, D.E., and Sells, B.H. (1989). RNA polymerase II-directed gene transcription by rat skeletal muscle nuclear extracts. Exp Cell Res 185, 8-20. Zechner, D., Craig, R., Hanford, D.S., McDonough, P.M., Sabbadini, R.A., and Glembotski, C.C. (1998). MKK6 activates myocardial cell NF-kappaB and inhibits apoptosis in a p38 mitogen-activated protein kinase-dependent manner. J Biol Chem 273, 8232-8239. Zhang, A.S., and Enns, C.A. (2009). Iron homeostasis: recently identified proteins provide insight into novel control mechanisms. J Biol Chem 284, 711-715. Zhang, B., Berger, J., Hu, E., Szalkowski, D., White-Carrington, S., Spiegelman, B.M., and Moller, D.E. (1996). Negative regulation of peroxisome proliferatoractivated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha. Mol Endocrinol 10, 1457-1466. Zhang, J., Bobulescu, I.A., Goyal, S., Aronson, P.S., Baum, M.G., and Moe, O.W. (2007). Characterization of Na+/H+ exchanger NHE8 in cultured renal epithelial cells. Am J Physiol Renal Physiol 293, F761-766. Zhang, M., Kho, A.L., Anilkumar, N., Chibber, R., Pagano, P.J., Shah, A.M., and Cave, A.C. (2006). Glycated proteins stimulate reactive oxygen species production in cardiac myocytes: involvement of Nox2 (gp91phox)-containing NADPH oxidase. Circulation 113, 1235-1243. P g | 286 Zhang, P., Wang, Y.Z., Kagan, E., and Bonner, J.C. (2000). Peroxynitrite targets the epidermal growth factor receptor, Raf-1, and MEK independently to activate MAPK. J Biol Chem 275, 22479-22486. Zhang, X., Bedard, E.L., Potter, R., Zhong, R., Alam, J., Choi, A.M., and Lee, P.J. (2002). Mitogen-activated protein kinases regulate HO-1 gene transcription after ischemia-reperfusion lung injury. Am J Physiol Lung Cell Mol Physiol 283, L815829. Zhao, R., Oxley, D., Smith, T.S., Follows, G.A., Green, A.R., and Alexander, D.R. (2007). DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH. PLoS Biol 5, e1. Zhu, X., Zeng, X., Huang, B., and Hao, S. (2004). Actin is closely associated with RNA polymerase II and involved in activation of gene transcription. Biochem Biophys Res Commun 321, 623-630. Zhuang, S., and Simon, G. (2000). Peroxynitrite-induced apoptosis involves activation of multiple caspases in HL-60 cells. Am J Physiol Cell Physiol 279, C341351. Zieleniak, A., Wojcik, M., and Wozniak, L.A. (2008). Structure and physiological functions of the human peroxisome proliferator-activated receptor gamma. Arch Immunol Ther Exp (Warsz) 56, 331-345. Zukor, H., Song, W., Liberman, A., Mui, J., Vali, H., Fillebeen, C., Pantopoulos, K., Wu, T.D., Guerquin-Kern, J.L., and Schipper, H.M. (2009). HO-1-mediated macroautophagy: a mechanism for unregulated iron deposition in aging and degenerating neural tissues. J Neurochem 109, 776-791. 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