NOX enzymes and ROS generation in human microglia, rodent inner ear, and pancreatic islets. LI, Bin. Abstract

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1 Thesis NOX enzymes and ROS generation in human microglia, rodent inner ear, and pancreatic islets LI, Bin Abstract Les radicaux libres sont impliqués dans de nombreux processus physiologiques et pathologiques. Les protéines membranaires NOX sont une des principales sources de radicaux libres. Les radicaux libres induits par NOX peuvent influencer le développement, le métabolisme, la prolifération et la survie cellulaire. Mon travail de thèse étudie l'expression et la fonction de NOX dans différent type cellulaire et tissus: les cellules microgliales humaines (HMC3, ligne cellulaire humain microglia clone 3), la cochlée du rat nouveau-né, les îlots pancréatiques de souris. Reference LI, Bin. NOX enzymes and ROS generation in human microglia, rodent inner ear, and pancreatic islets. Thèse de doctorat : Univ. Genève, 2010, no. Méd.1 URN : urn:nbn:ch:unige DOI : /archive-ouverte/unige:5088 Available at: Disclaimer: layout of this document may differ from the published version.

2 Section de Fondamentale Département de Pathologie et Immunologie Thèse préparée sous la direction du Professeur Karl-Heinz KRAUSE NOX Enzymes and ROS Generation in Human Microglia, Rodent Inner Ear, and Pancreatic Islets Thèse présentée à la Faculté de Médecine de l'université de Genève pour obtenir le grade de Docteur en Sciences Médicales (MD-PhD) par Bin LI de Beijing, Chine Thèse n 1 GENÈVE 2010

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4 ACKNOWLEDGEMENTS This thesis I inscribe specifically to: My wife, Ms. Ping GUO, for her supporting my study aboard whole-heartedly & My parents, Mr. Yan-Qin LI and Ms. Hui-Zhen CHEN, for supporting my study all the time

5 Acknowledgements I thank my mentor, Prof. Karl-Heinz KRAUSE, for accepting me as his MD-PhD student. For years of my study, he has been giving me a correct direction in my thesis work, and encouraging me to learn more and to be ambitious in scientific research. I thank Prof. Pierre MAECHLER for giving me advices in my thesis work concerning NOX expression in pancreatic islets. I appreciate the kind help from Prof. Jean-Luc PUEL and Jing WANG (Institut National de la Sante et de la Recherche Médicale-UMR 583 and Université de Montpellier 1, Physiopathologie et thérapie des déficits sensoriels et moteurs, Montpellier, France.) who trained me in cochlea microdissection. I thank Dr. Michel DUBOIS-DAUPHIN, Jérôme BONNEFONT, Karen BEDARD, Olivier BASSET, and Vincent JAQURET for discussing about the project. I thank all the other members in Prof. KRAUSE s lab for their kind helps. They are: Hélène GFELLER, Christine DEFFERT, Stéphanie JULIEN, Olivier PLASTRE, Silvia SORCE, Diderik TIREFORT, Stefania SCHIAVONE, Yannick MARTINEZ, Olivier PREYNAT-SEAUVE and Michela SCHAEPPI. I remember to thank all the professors and teachers who taught me in degree courses. They helped me to strengthen my ability in scientific research.

6 CONTENTS ABBREVIATIONS & ACRONYMES RÉSUMÉ (French) THESIS ABSTRACT (English) RESEARCH BACKGROUND: 1. General overview: NOX enzymes Reactive oxygen species (ROS) General function of NOX NOX isoforms and their conserved structure NOX regulatory subunits Physiological and pathological roles of NOX Tools for NOX research NOX in microglia --- NOX expression / function 2.1. Origin, activation, and function of microglia NOX isoforms in rodent and human microglia NOX in cochlea --- cisplatin / ROS / apoptosis 3.1. Cochlea anatomy The organ of Corti and hearing function Cisplatin-induced hearing loss Cisplatin-induced ROS generation and NOX Cisplatin induces apoptosis Cisplatin-induced apoptosis vs. ROS Study of ROS (O 2 - ) generation in cochlea explant NOX in pancreatic islets --- ROS / insulin secretion / Diabetes 4.1. Pancreas anatomy Species difference in pancreatic islets

7 4.3. β cells and α cells Insulin vs. glucagon Insulin secretion and diabetes NOX dependent ROS generation and insulin secretion References RESEARCH ARTICLE 1: NOX4 expression in human microglia leads to constitutive generation of reactive oxygen species and to constitutive IL-6 expression Bin Li a, Karen Bedard a, Silvia Sorce a, Boris Hinz b, Michel Dubois-Dauphin a, Karl-Heinz Krause a* (J Innate Immun 2009; 1: ) Abstract Introduction Materials and methods Results Discussion Acknowledgement References Supplementary information RESEARCH ARTICLE 2: The relationship between NOX dependent ROS generation and cisplatininduced apoptosis in cochlea explants Bin Li a, Jing Wang b, Vincent Jaquet a, Michel Dubois-Dauphin a, Jean-Luc Puel b, Karl-Heinz Krause a* Abstract Introduction Materials and methods Results

8 Discussion Acknowledgement References Figures and legends RESEARCH ARTICLE 3: NOX2 deficiency increases glucose-stimulated insulin secretion Ning LI a#, Bin LI b#, Thierry Brun a, Olivier Basset b, Pierre Maechler a*, Karl Heinz Krause b* Abstract Introduction Research design and methods Results Discussion Acknowledgement Figures and legends References Supplementary information Global conclusions and perspectives

9 Abbreviations & Acronymes 8-iso 8-isoprostane Aβ amyloid-aβ ABC avidin biotin complex AD Alzheimer's disease AICA anterior inferior cerebellar artery ALS amyotrophic lateral sclerosis APCs antigen presenting cells Ara-C cytosine 1 β-arabinofuranoside ATP adenosine-5'-triphosphate b.p. base pair BBB blood brain barrier BDNF brain-derived neurotrophic factor BSA bovine serum albumin Caco-2 human colonic carcinoma cell line caspase cysteine-dependent aspartate-specific proteases CD11b cluster of differentiation 11b CD68 cluster of differentiation 68 CGD chronic granulomatous disease ClC-3 Cl-channel-3 CNS central nerve system CX3CL1 chemokine (C-X3-C motif) ligand 1 (C: cystine) CYBB cytochrome b558 subunit β DAB 3, 3 -diaminobenzidine (tetrahydrochloride dihydrate) DAPI 4,6-diamidino-2-phenylindole DHE dihydroethidium DMSO dimethyl sulphoxide DPI diphenylene iodonium eatp extracellular Adenosine-5'-triphosphate ECs endothelia cells ELISA enzyme-linked immunosorbent assay ER endoplasmic reticulum ESR electron spin resonance FCS fetal calf serum FPG fasting plasma glucose FPI fasting plasma insulin FRO formes réactives de l oxygène GAPDH glyceraldehyde-3-phosphate dehydrogenase GFAP glial fibrillary acidic protein GROα growth regulated oncogene α GSH reduced glutathione GSH-Px glutathione peroxidase 4

10 GSIS glucose-stimulated insulin secretion GSRNR glucose-stimulated ROS NBT reduction HBSS Hank's buffered salt solution HD Huntington s disease HE high energy (food) HEK human embryonic kidney (a cell line) HMC3 human microglia cell line clone 3 HSV-1 herpes simplex virus Iba ionized calcium-binding adapter molecule 1 IDF International Diabetes Federation IHC inner hair cell IL-1β interleukin-1β IL-6 interlukin-6 inos inducible nitride oxidase species IP-10 (gamma interferon) inducible protein-10 IpGTT intraperitoneal glucose tolerance test ITS insulin transferrin selenium LPS lipopolysaccharide LSD least significant difference MAC1 macrophage antigen complex 1 MARCO macrophage receptor with collagenous domain (scavenger receptor) MCP-1 monocyte chemoattractant protein-1 M-CSF (macrophage) colony stimulating factor MDC macrophage derived chemokine MHCII major histocompatibility complex MIP-1α/MIP-1β macrophage inflammatory protein-1α/-1β MIP-2 macrophage inflammatory protein-2 MIP-3β macrophage inflammatory protein-3β NAD(H) nicotinamide adenine dinucleotide (reduced form) NADP(H) nicotinamide adenine dinucleotide phosphate (reduced form) NBT nitroblue tetrazolium ncf1 neutrophil cytosolic factor 1 NO nitric oxide NOX NAD(P)H oxidase NT nitrotyrosine OHC outer hair cell PGE2 prostaglandin E2 PMA phobol myristate acetate PMN polymorphonuclear neutrophils PRRs pattern recognition receptors PVDF polyvinylidene fluoride RAGE receptor for advanced glycation end products RANTES regulated on activation, normal T cell expressed and secreted GSRNR glucose-stimulated ROS NBT reduction 5

11 RNS reactive nitrogen species ROS reactive oxygen species R-PIA R-phenylisopropyladenosine RPMI "Roswell Park Memorial Institute ; a cell culture medium SEM standard error of means SM scala media SOD superoxide dismutase SPF specific pathogen free SPSS statistics package for social sciences SR-A scavenger receptor class A ST scala tympani SV scala vestibuli T1DM type 1 diabetes mellitus T2DM type 2 diabetes mellitus TCA tricarboxylic acid TGFβ transforming growth factor β TLR2 Toll-like receptor 2 TNF-α, tumor necrosis factor-α VBM vessel of basilar membrane VEGF vascular endothelia growth factor VTL vessel of tympanic lip WHO World Health Organization 6

12 RÉSUMÉ Les radicaux libres sont impliqués dans de nombreux processus physiologiques et pathologiques. Les protéines membranaires NOX sont une des principales sources de radicaux libres. Les radicaux libres induits par NOX peuvent influencer le développement, le métabolisme, la prolifération et la survie cellulaire. Mon travail de thèse étudie l expression et la fonction de NOX dans différent type cellulaire et tissus: les cellules microgliales humaines (HMC3, ligne cellulaire humain microglia clone 3), la cochlée du rat nouveau-né, les îlots pancréatiques de souris. Première partie L expression constitutive de NOX4 dans les cellules HMC3 induit la production de ROS et l expression des IL-6 mrna Les ROS produits par la microglie jouent un rôle dans la neuroinflammation, la neuro-toxicité, le relâchement d acide aminé excitateurs, ainsi que dans la défense immunitaire et la prolifération cellulaire. Des études récentes montrent que les cellules microgliales primaires expriment non seulement NOX2, mais également les isoformes NOX1 et NOX4. Nous avons étudié la relation entre l expression des isoformes de NOX et la neuroinflammation dans la lignée cellulaire microgliale HMC3. De manière typique aux cellules microgliales, cette lignée exprime constitutivement les protéines Iba1 et CD14, et l interféron-gamma y induit l expression de CD11b, CD68 et MHCII. Cependant, de manière inattendue, et contrairement à ce qui est observé dans la microglie primaire où le mrna de NOX2 7

13 est prédominant, c est le mrna de l isoforme NOX4 qui est fortement exprimé au détriment de celui de l isoforme NOX2 virtuellement absent de la lignée HMC3. En conséquence, la production de ROS était constitutive et permanente, et détectée dans les vésicules intracellulaires. H 2 O 2, capable de traverser la membrane cellulaire, était détectée dans l espace extracellulaire. Deux constructions sirna dirigées contre NOX4 supprimaient spécifiquement la production de ROS par les cellules HMC3. Cette inhibition de NOX4 ne modifiait pas l expression des gènes MHCII, CD68, CD11b ni celle de inos, VEGF ou TGFβ. Cependant, l expression du mrna de IL-6 était notablement diminuée. Ces résultats suggèrent que l expression constitutive de NOX4 dans les cellules HMC3 conduit à l expression du mrna d IL-6. Il est donc possible que le passage d une activité NOX2 dominante dans la production des ROS vers une activité NOX4 dominante, et permanente, dans la production des ROS permette d expliquer le passage d une activité neuro-protective vers une activité neuro-inflammatoire par les cellules microgliales. Deuxième partie Corrélation entre les formes réactives de l oxygène produites par NOX et l apoptose induite par la cisplatine dans la cochlée But: L objectif de cette étude est d analyser la source des formes réactives de l oxygène (FRO) dans des explants cochléaires de rats et de comprendre leur rôle dans l apoptose induite par la cisplatine. Résultats: Plusieurs isoformes de NOX (NOX2, NOX3 et NOX4) sont détectées par RT-PCR dans les explants cochléaires. Le test de réduction du Nitroblue tetrazolium (NBT) a permis de mesurer un niveau 8

14 basal élevé de superoxide dans les cellules de toutes les structures des explants cochléaires (stria vascularis, organe de Corti et ganglion spiral). Après traitement à la cisplatine, une augmentation des FRO et de l apoptose est observée. Un traitement préventif au DPI (10 µm, un inhibiteur des flavoprotéines très efficace sur les enzymes NOX mais également sur d autres sources potentielle de FRO) bloque totalement la production de superoxide tant au niveau basal qu après traitement à la cisplatine. Toutefois, des résultats préliminaires montrent qu un traitement au DPI ou à l apocynine, une molécule antioxidante, ne protège pas les explants cochléaires de l apoptose révélée par l immuno-marquage de la caspase 3 active. Conclusions: Bien que les FRO soient produites par NOX dans toutes les régions de la cochlée, qu elles soient à l origine de l apoptose induites par la cisplatine reste à démontrer. Troisième partie La formation de ROS induite par le glucose est mediée par NOX2 et régule négativement la sécrétion d insuline Objectif : Etudier l expression des NOX dans les ilots de pancréas de souris et comprendre le rôle des NOX dans la formation des ROS et la sécrétion d insuline induites par le glucose. Méthodes : Après avoir établi le profil d expression des NOX dans les ilots de pancréas de souris sauvage (RT-PCR), la formation de ROS ainsi que la sécrétion d insuline ont été mesurées dans des ilots isolés de souris déficientes en NOX1, 2 et 4. Nous avons mesuré in vitro la sécrétion d insuline induite par le glucose. In vivo, 9

15 nous avons effectué des mesures en condition de jeûne de glucose, de sécrétion d insuline ainsi que l IpGTT. Résultats : Au niveau de l ARNm, les isoformes 1, 2 et 4 de NOX on été détectés dans les ilots provenant de souris sauvage et l isoforme NOX2 qui semble être le plus exprimé. La formation de ROS induite par le glucose est totalement supprimée dans les ilots provenant de souris déficientes en NOX2. Ces derniers ont une morphologie et un nombre de cellules normaux. Cependant, la sécrétion d insuline est fortement augmentée dans ces ilots. Ce résultat est nouveau et inattendu car des études précédentes utilisant le DPI, un inhibiteur non spécifique des NOX, ont suggéré que les ROS dérivées des NOX auraient un effet positif sur la régulation de la sécrétion d insuline. Les expériences in vivo chez les souris déficientes en NOX2 en condition de jeûne ont montré que le glucose est normal mais que le taux d insuline est augmenté. De même, le test IpGTT a suggéré une légère intolérance au glucose ainsi qu une faible augmentation de l insuline plasmatique, ce qui indiquerait une résistance modérée à l insuline. Conclusions : Après stimulation au glucose, NOX2 est la principale source de ROS dans les ilots du pancréas de souris. Ces ROS dérivés de NOX2 donnent un signal négatif pour la sécrétion d insuline et sont donc potentiellement important pour la régulation de la sécrétion d insuline et la disparition de la résistance à l insuline. 10

16 ABSTRACT Reactive oxygen species (ROS) take part in many physiological and pathological processes. NOX enzymes are important source of ROS, which can affect cell development, metabolism, proliferation, and death. My thesis work concerns the NOX expression and their function in different cells / tissue: human microglia cell line (HMC3, human microglia clone 3), rat cochlea, and mouse pancreatic islets. Part 1 NOX4 expression in human microglia leads to constitutive generation of reactive oxygen species and to constitutive IL-6 expression Bin Li a, Karen Bedard a, Silvia Sorce a, Boris Hinz b, Michel Dubois-Dauphin a, Karl-Heinz Krause a* (J Innate Immun 2009; 1: ) Reactive-oxygen-species generation by microglia is implicated in neuroinflammation, neuro-toxicity as well as in host defense, cell proliferation, and excitatory amino acid release. Recent studies demonstrate that primary microglia preparations express not only the phagocyte NADPH oxidase NOX2, but also the NOX1 and NOX4 isoforms. Here we investigated the relationship between neuroinflammation and NOX isoform expression in human microglia cell line clone 3 (HMC3). HMC3 cells are typical microglia, as suggested by the constitutive expression of Iba-1 and CD14, and IFNγ-induced expression of CD11b, CD68, and MHCII. However, the characteristics of NOX isoform expression and ROS generation 11

17 by HMC3 cells were unexpected. RT-PCR demonstrated abundant expression of NOX4, but almost not NOX2. ROS generation was constitutive and appeared predominantly intracellular, as superoxide was detected within intracellular vesicles, while the cell permeable H 2 O 2 was found in the extracellular space. ROS generation by HMC3 was efficiently suppressed by sirna directed against NOX4, but not by control sirna. NOX4 suppression did not alter expression of the microglia-typical genes MHCII, CD68, CD11b, nor did it affect the expression of inos, VEGF, or TGFβ. However, there was a marked decrease in IL-6 mrna. Taken together, we suggest that a constitutive NOX4-dependent ROS generation in microglia cell line leads to expression of IL-6 mrna. The possibility that microglia could switch from tightly regulated NOX2-dependent ROS generation to constitutive NOX4-dependent ROS generation is of interest for the understanding of the role of microglia in maintaining the balance between neuro-protection and neuroinflammatory damage. Part 2 The relationship between NOX dependent ROS generation and cisplatin- induced apoptosis in cochlea explants Aims: To study the source of reactive oxygen species (ROS) in rat cochlea explants and their role in cisplatin-induced apoptosis. Results: Several NOX isoforms, namely NOX2, NOX3, and NOX4 were detected by RT-PCR in cochlear explants. The Nitroblue tetrazolium (NBT) assay was used to detect superoxide in cochlear explants. A spontaneous superoxide generation was observed in the cell bodies of different structures of the cochlear explants i.e., stria vascularis, the organ of Corti, 12

18 and spiral ganglion. After cisplatin treatment, enhanced ROS generation and induced apoptosis were observed. A preventive treatment (10 µm) with the flavoprotein inhibitor diphenylene iodonium (DPI), a potent electron transporter inhibitor, completely blunted the spontaneous and cisplatin-induced superoxide generation. However, using immuno blotting assay, primary data demonstrated that DPI and apocynin did not reduce apoptosis by measuring caspase 3 activity. Conclusions: ROS generation distributes in all the structures with cell bodies of the cochlea explant. Cisplatin enhances spontaneous ROS generation and induces apoptosis. However, whether NOX-dependent ROS generation is correlated to cisplatin-induced apoptosis needs more research. Part 3 Glucose-dependent ROS generation is mediated by NOX2 and negatively regulates insulin secretion Objective: To study NOX expression in mouse pancreatic islets and understand its role in glucose-stimulated ROS generation and insulin secretion. Research design and methods: After establishing the NOX expression profile in islets of wild type mice (RT-PCR), ROS generation and insulin secretion were investigated in isolated NOX1-, NOX2- and NOX4-deficient pancreatic islets. In vitro, glucosestimulated insulin secretion was tested. In vivo, fasting glucose and insulin were measured and IpGTT test was performed. Results: In wild type islets, NOX1, NOX2, and NOX4 mrna were detected, with NOX2 appearing to be most heavily expressed. Glucose stimulated ROS generation was completely abolished in islets from NOX2-13

19 deficient mice. Islets from NOX2-deficient mice appeared morphologically normal and contained a normal number of β cells. However, insulin secretion from NOX2- deficient islets was markedly increased. This is unexpected and novel, as previous studies using the non-specific inhibitor DPI had suggested a positive regulation of insulin secretion by NOX-derived ROS. In vivo studies showed a normal fasting glucose, but increase fasting insulin levels in NOX2-deficient mice. The IpGTT test suggested the presence of mild glucose intolerance accompanied by a slight increase in plasma insulin levels, indicative of a moderate insulin resistance. Conclusions: NOX2 is the main source of glucose-stimulated ROS generation in mouse islets. NOX2-derived ROS provide a negative signal for insulin secretion, potentially important for insulin homeostasis and avoidance of insulin resistance. 14

20 RESEARCH BACKGROUND 1. General overview: NOX enzymes --- professional ROS producers 1.1. Reactive oxygen species (ROS) ROS are oxygen-derived small molecules, which easily react with each other and / or with other compounds including large organic molecules, such as proteins, lipids, and nucleic acids. Through redox reaction, they change molecule composition in the local environment, affecting cellular processes physiologically and/or pathologically. The following reactions summarize the redox reactions leading to ROS formation: 1. O 2 +e - O 2-2. O H + H 2 O 2 (non-radical ROS) 3. O H + HO - + HO (hydroxide ion and hydroxyl radical; the latter is highly reactive.) 4. H 2 O 2 + Fe 2+ HO - + HO + Fe 3+ (Fenton reaction) 5. Fe 3+ + H 2 O 2 Fe 2+ +H + + OOH (hydroperoxide; Fenton reaction) 6. O HO HO O 2 * (singlet oxygen; the natural form of oxygen; highly reactive) 7. HO + R HOR (R, organic substance; HOR, hydroxylated adduct; radical) 8. HO + R H + + OR (alkoxyl radical) 9. RO + O 2 ROO (peroxyl radical) In addition, cross-talks exist between ROS and reactive nitrogen species (RNS). For example: O NO OONO - (peroxynitrate). ROS affect cell functions in a complex way. Figure 1 shows an example as a 15

21 general overview of ROS participating in stress-induced cell responses [1]. Figure 1. Interaction between ROS generation and the cellular stress response. Acute stresses elicit stress-specific and general nonspecific responses. The cell may respond to acute stress in several ways. With persistence of stress, cells can adapt or even develop hormetic compensations --- becoming tolerable to a repeated stress. Secondary cell programs may ensue including cell differentiation (generally a result of relatively mild stresses) or phenotypic modulation usually seen in vascular smooth muscle cells. With persistent or un-adapted stress, cells may develop strategies to limit their propagation and/or survival, namely apoptosis, senescence, or autophagy. These programs can also be adaptive to some extent. Overwhelming stresses may induce necrosis. All such processes can be influenced by and, at the same time, feed-back on ROS production. The definition of whether ROS production is specific to a certain type of stress is therefore subjected to several biases. From: Santos CX, et al. Antioxid Redox Signal Oct;11(10): General function of NOX One important source of cellular ROS is the family of NOX NADPH oxidase enzymes. All NOX family members are transmembrane proteins that transport electrons across biological membranes to reduce oxygen into superoxide. The electron from cytoplasmic NADPH is transferred and bound to oxygen in the extracellular 16

22 space or in the lumen of intracellular organelles, via intermediate flavin adenine dinucleotide (FAD) and heme prosthetic groups [2] NOX isoforms and their conserved structure Tissue distribution of NOX isoforms To date, seven different NOX isoforms have been described: NOX1 through NOX5, DUOX1 and DUOX2 as shown in the figure 2. The family of NOX enzymes is widely distributed in a variety of tissues, but very high expression levels can be found in specific organs or cell types. NOX1 is abundant in colon epithelium. NOX2 (cytochrome b558 large subunit or heavy/β chain, gp91 phox, CYBB) is mainly expressed in phagocytes. NOX3 is mainly present in inner ear hair cells. NOX4 is highly detected in kidney and blood vessels. NOX5, which is absent in rodents, is predominantly expressed in lymph tissue and testis. DUOX1 and DUOX2 are mainly located in thyroid Conserved structure in NOX isoforms In accordance to their similar function, there are conserved structural properties of NOX enzymes that are common to all family members. Starting from the COOH terminus, these conserved structural features include 1) an NADPH-binding site at the COOH terminus, 2) a FAD-binding region in proximity of the most COOH-terminal transmembrane domain, 3) six conserved transmembrane domains, and 4) four highly conserved heme-binding histidines, two in the third and two in the fifth transmembrane domains (figure 3) [2]. 17

23 a b c d e f Figure 2. NOX isoform functioning groups The seven NOX family members need their own activation factors in superoxide generation. a: NOX1 activity requires p22 phox, NOXO1 (or possibly p47 phox in some cases) and NOXA1, and the small GTPase Rac. b: NOX2 requires p22 phox, p47 phox, p67 phox, and Rac; p47 phox phosphorylation is required for NOX2 activation. Although not absolutely required, p40 phox also associates with this complex and may contribute to activation. c: NOX3 requires p22 phox and NOXO1; the requirement for NOXA1 may be species dependent, and the requirement of Rac is still debated. d: NOX4 requires p22 phox, but in reconstitute systems it is constitutively active without the requirement for other subunits. However, in native NOX4-expressing cells, activation, possibly including Rac, has been described. e and f: NOX5, DUOX1, and DUOX2 are activated by Ca 2+ and do not appear to require subunits. (Bedard, K., and Krause, K. H. (2007) Physiol Rev 87, ) 18

24 Figure 3. Proposed structure of the core region of NADPH oxidase (NOX) enzymes. All NOX family members share six highly conserved transmembrane domains (I to VI). Transmembrane domains III and V each contain two histidines, spanning two asymmetrical hemes. The cytoplasmic carboxyl terminus contains conserved flavin adenine dinucleotide (FAD) and NADPH binding domains. NOX enzymes are thought to be single electron transporters, passing electrons from NADPH to FAD, to the first heme, to the second heme, and finally to oxygen. Enlarged circles represent amino acids that are conserved through human NOX1, NOX2, NOX3, and NOX4. (Bedard, K., and Krause, K. H. (2007) Physiol Rev 87, ) 1.4. NOX regulatory subunits To fulfill their function, NOX isoforms need the interaction with related subunits and regulatory proteins. p22 phox is needed for NOX1 to NOX4. NOXO1 and NOXA1 are for NOX1--- whether they are also needed for NOX3 remains to be investigated. p47 phox acts as an organizer for NOX2 function, while p67 phox acts as an activator. NOX5 and DUOX1/2 are calcium dependent. A small GTPase Rac is needed as regulator for NOX1 and NOX2 besides their ROS-generation regulatory function in mitochondria [3,4], thus it is not a strict regulatory subunit of NOX. 19

25 p22 phox NOX1, NOX2, NOX3, and NOX4 isoforms require the presence of the membrane subunit p22 phox for ROS generation. These NOX isoforms and p22 phox are thought to mutually stabilize each other in the membrane. Therefore, p22 phox is indissociable of NOX1-NOX4 expression. p22 phox contains several domains: a hydrophilic cytosolic N-terminus, a hydrophobic region interacting with the membrane, a NOX maturation region through which the protein interacts with the NOX enzymes, two transmembrane domains connected by an extracellular loop and a cytosolic tail containing a proline rich region that interacts with p47 phox, or NOXO1 tandem SH3 domains [5]. The membrane topology of p22 phox is difficult to predict, models are proposed with two [6,7], three [8,9], and four transmembrane domains [10]. In both fetal and adult tissues or cell lines [11], the mrna for p22 phox is widely expressed [12]. In response to angiotensin II, streptozotocin-induced diabetes [13], and hypertension [14], the expression of p22 phox increases [15] NOXO1 and p47 phox ---organizer subunits There are up to 25% sequence identity of proteins between NOXO1 and p47 phox with a high degree of similarity in their functional domains. p47 phox has an autoinhibitory region (AIR) that prevents this interaction until the protein is phosphorylated and undergoes a conformational change. AIR is absent in NOXO1. The molecular weights of p47 phox and NOXO1 are 47 kda and 41 kda respectively. They both are cytosolic proteins and not glycosylated. NOXO1 is highly expressed in colon [16-19], but also found in other tissues, including inner ear [16,19,20]. 20

26 p47 phox is highly expressed in myeloid cells [21,22], it has also been detected in other tissues, including inner ear [20,23], neurons[24], endothelial cells [25]etc. Upon phosphorylation of the autoinhibitory domain, p47 phox translocates to the plasma membrane and binds p22 phox. The lack of an autoinhibitory domain in NOXO1 might suggest that it is constitutively active. Indeed, NOXO1 is found localized at the cell membrane with NOX1 and p22 phox in transfected HEK293 cells [12], while p47 phox translocates to the membrane only after its autoinhibition has been released by phosphorylation [24,26,27] NOXA1 and p67 phox --- activator subunits p67 phox and NOXA1 share up to 28% amino acid identity with similar domain structures. Their molecular weights are 67 kda and 51 kda respectively. They are both cytoplasmic proteins and are not glycosylated. [2]. p67 phox is expressed in phagocytes [28], B lymphocytes [29], endothelial cells [25], neurons[30], astrocytes[30], kidney [31], and hepatic stellate cells [32]. NOXA1 is expressed in the spleen, inner ear, colon, small intestine [18,23], basilar arterial epithelial cells [33], airway-like normal human bronchial epithelial cells[34], vascular smooth muscle cells [35] etc. p67 phox and NOXA1 interact with p47 phox and NOXO1 [8,19,36]. Both interact with Rac [19,37-40] Physiological and pathological roles of NOX NOX family members are widely distributed in many types of cells, tissues, and organs, where they fulfill different functions. Physiological concentrations of ROS are necessary for the proper function of several biological processes, such as host defence, 21

27 biosynthesis or cellular signalling cascades [41-44]. NOX1 stimulates cell proliferation in actively dividing cells [45], and facilitates neointima formation [43]. Absence of NOX-dependent ROS production can be the cause of pathological conditions, such as the chronic granulomatous disease (CGD). In this case, mutations in NOX2 protein complex prevent the correct functionality of the enzyme and the production of ROS in phagocytes, which have a diminished bactericidal activity. Recently, a study has demonstrated that NOX2-derived ROS promote hypoxiainduced mobilization of endothelial progenitor cells and vascular repair by these cells [46]. In endothelial cells, NOX2 maintains the cytoskeleton and prevents apoptosis to support cell survival [47]. NOX4 promotes proliferation of endothelial cells [47]. In contrast, an excessive activation of NOX enzymes, associated with increased production of ROS, contributes to the progression of several diseases. For example, microglial NOX2 over expression might contribute to Alzheimer s disease and Parkinson s disease [48,49]; elevated NOX3 expression might be involved in various kinds of hearing loss including aging and noise [50,51]; increased NOX2 and/or NOX4 expression are thought to cause β-cell injury and diabetes [13,52]; NOX1 and NOX4 over expression are considered to be involved in hypertension [53] Tools for NOX research NOX inhibitors Pharmacological inhibitors Most of the chemical inhibitors currently used in pharmacological studies 22

28 targeting NOX enzymes are not NOX-specific [54], since they also block other targets. However, in a very controlled way, they are suitable for detecting NOX activity [55]. The most commonly used non specific NOX inhibitors are apocynin and DPI. Apocynin, ketone 4-hydroxy-3-methoxyacetophenone, is a medicinal plant extract. It has been described as a specific NOX2 inhibitor. However, it does not seem to interfere with neutrophils phagocytosis or intracellular killing [56,57]. The inhibition of NOX is reversible and due to the impeding assembly of the p47 phox subunit to the membrane complex [58]. Apocynin is thought to act as a prodrug and to form a dimer after peroxidase-mediated oxidation, the dimer being a more efficient NOX inhibitor than apocynin itself [59]. Since apocynin can be oxidized, it hence can act as an antioxidant and it has been suggested that apocynin is principally a ROS scavenger instead of NOX inhibitor in their experimental conditions [60-62], and that apocynin scavenges hydrogen peroxide instead of superoxide [56]. In in vitro studies, high concentration of apocynin have been used (<100 µm-1.2 mm [63]). In contrast, in animal models apocynin appears to be efficacious with a narrow therapeutic window from 2.5 mg/kg to 5 mg/kg [64]. Diphenylene iodonium (DPI) DPI is the most commonly used non specific NOX inhibitor. DPI acts by abstracting an electron from an electron transporter and forming a radical, which then inhibits the respective electron transporter through a covalent binding step [65]. Thus, it acts as an irreversible non-specific inhibitor in electron transporting systems. In the case of NOX enzymes, it is not clear whether the iodonium radical formation occurs through interaction with the flavin group [65] or the heme group [66]. Submicromolar concentrations of DPI inhibits not only all NOX isoforms, but also nitric oxide synthase [67], xanthine oxidase [68], mitochondrial complex I [69], 23

29 and cytochrome P-450 reductase [70] Genetic inhibition Since NOX specific inhibitors are not available, genetic engineering techniques appear as more valuable tools to study NOX activity. It is possible either to transduce or to transfect cells, which naturally do not express the specific NOX protein [44,47,71-77]; or knock down specific NOX, which is naturally expressed in the cells using mrna interference. In contrast, animal models of disease are better suited to understand the role of NOX enzyme in pathological conditions by comparing the effect of NOX-deficient with wild-type mice [46,78]. To our knowledge, at least the following NOX-specific knock-out animals or mutants with loss of related function have greatly helped to understand specific functions of related NOX and /or the subunit: (i) Knock-out animals: NOX1-deficient mice show decreased blood pressure upon angiotensin II infusion, but show no other abnormal phenotypes [43,79-81]. NOX2- [82-86], and p47 phox -deficient [87,88] mice are also available. Both NOX2-deficient and p47 phox - deficient mice show decreased bactericidal capacity of the neutrophils and represent a model of chronic granulomatous disease (CGD). Generally speaking, compared with p47 phox -deficiency, mice with NOX2-deficient mice are more susceptible to infection. Their courses of infection are usually shorter and end in death. That might be the main reason for the lacking of development in granulomatous inflammation at the site of infection as described by Sharon H. Jackson et al [88]. NOX4-deficient mice are available (Dr. KRAUSE, personal communication). (ii) Mutant animals: ncf1 (neutrophil cytosolic factor 1) mutant mice, which have lost the function of 24

30 p47 phox, have impaired bacteria-killing function [89]. p22 phox -, NOXO1- (head slant) [90], and NOX3- (head tilt) [91] mutants all show deficit in otoconia formation, and thus with balance problems. Interestingly, p22 phox -mutant rats show not only impaired bacteria-killing function and balance problems, but also increased eosinophilia [92]. Another rat lineage containing a polymorphism in p47 phox leading to a deceased oxidative burst has also been described [89] NOX antibodies Up to date, we are still looking for ideal NOX antibodies for all the NOX isoforms and their subunits. Specificity and sensitivity of antibodies are tested by using specific NOX-transduced cell lines through immuno blotting assay and/or immunofluorescence assay. In our studies, we specifically validated one NOX2 antibody (for human) [71] and one NOX4 antibody (in rat) by using NOX2 and NOX4 deficient mice showing [93] one clear specific band, while negative control showed no bands ROS measurement approaches There are several approaches in ROS measurement, but in our studies, we mainly used the first three of following mentioned assays: 1. Luminol enhanced chemiluminescence is widely used to detect extracellular ROS of different types [94-106]. 2. Amplex red assay is used to detects extracellular hydrogen peroxide; it is not sensitive to low level intracellular ROS generation [42,94,107]. 3. Nitro blue tetrazolium (NBT) assay is suitable to detect intracellular superoxide [41, ]. 4. Other techniques exist, such as dihydroethidium (DHE) and Electron spin 25

31 resonance (ESR), which are used to detect intracellular superoxide [42,94]. These techniques are reviewed by Dikalov S et al [114]. 2. NOX enzymes in microglia --- NOX expression / function Origin, activation, and function of microglia Besides the cells of the vasculature, the brain comprises two general cell types: neurons and glial cells. Glial cells provide physiological support to neurons and repair neuronal damage due to injury or disease [115]. Microglial cells account for approximately 5-20% of the total glial population in the central nervous system [ ]. They are resident macrophages of the central nerve system (CNS) involved in host defense. They are distributed with no significant local differences in the white and grey matters. In contrast to astrocytes they cover non-overlapping territories [120]. Astrocytes and oligodendrocytes are derived from neuroectoderm. In contrast, the origin of microglia is still debatable. It can derivate from: 1, neuroepithelial cells [ ], 2, hematopoietic cells (i.e. monocytes) [ ], and 3, mesodermal progenitors, distinct from monocytes, which colonize the nervous system via extravascular routes [127]. In the adult, healthy brain microglia appears as cells with a small cell body with ramified thin processes. At resting state, the expression of its surface antigens is low. When stimulated by injury, ischemia, and inflammatory states and neurotoxic and pro-inflammatory ligands microglial cells are activated (figure 4 and figure 5) [128,129]. Following stimulation or during neurodegenerative processes, they proliferate, migrate to the site of injury, and clear cell debris by phagocytosis [130]. In addition, they generate both neurotoxic and neurotrophic factors [116,131]. Thus, they 26

32 are highly reactive, mobile and multifunctional immune cells of the CNS, and play a universal role in the defence of the neural parenchyma [120]. In the last two decades, microglia became a research focus in many acute and chronic CNS diseases including stroke, Alzheimer s disease (AD), or multiple sclerosis [ ]. Figure 4. Microglia respond to several types of immunological signals. Endogenous factors include cytokines, material from apoptotic cells and aggregated proteins such as prions. Exogenous factors include viral envelope glycoproteins. In response, microglia can undergo several different levels of activation, finally resulting in a fully functioning phagocytic cell. Activated microglia can be friends or foes to neighboring neurons. As friends, they can clear toxic material (apoptotic neurons, protein aggregates), secrete neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and protective factors such as glutathione and increase clearance of excitotoxic glutamate by astrocytes. Microglia can also secrete potentially neurotoxic molecules such as proinflammatory cytokines (TNF-α, IL-1β), glutamate, free radical species and nitric oxide. (From: Peter N Monk & Pamela J Shaw; Nature Medicine; 2006; 12: ) 27

33 Figure 5. Microglia PRRs identify neurotoxic and pro-inflammatory ligands PRRs, pattern recognition receptors. Their responsible cytophatic functions include: 1, identify pathogens; 2, remove and clear cytotoxic materials through internalization and phagocytosis; 3, produce extracellular superoxide; and 4, release proinflammatory compounds. Ab, amyloid-ab; H 2 O 2, hydrogen peroxide; IL-1b, interleukin 1b; LPS, lipopolysaccharide; MAC1, macrophage antigen complex 1; MARCO, macrophage receptor with collagenous domain (scavenger receptor); NO, nitric oxide; O 2 -, superoxide; PGE2, prostaglandin E2; RAGE, receptor for advanced glycation end products; ROS, reactive oxygen species; SR-A, scavenger receptor class A; TLR2, Tolllike receptor 2; TNF-a, Tumour necrosis factor-a. (From: Michelle L. Block, Luigi Zecca and Jau-Shyong Hong; Nature Neuroscienc; 2007; 8: ) 2.2. NOX isoforms in rodent and human microglia Like monocyte-derived phagocytes, microglial cells produce ROS through NADPH oxidases. This ROS generation contributes not only to the host defence and the removal of cell debris, but also to neuroinflammation and neurodegeneration [49, ]. Microglia express NOX2 [141,142], and more recently it has been reported that other NOX isoforms, namely NOX1 and NOX4 [138,143], are also 28

34 expressed. NOX1 is suggested to mediate microglia neurotoxicity [144], while NOX4 is considered to involve in excitatory amino acid release via volume-regulated anion channels [143]. In mouse microglia, NOX2 and related cytosolic subunits p47 phox, p40 phox, and Rac proteins are detected [ ]. It has not been documented exactly what kind of NOX isoform expressed in human microglia or related cell line. In addition, species difference has been reported for ROS generation, especially between human and rodents [147]. Thus, it is not clear what kind of NOX isoforms are expressed in human microglia. 3. NOX enzymes in cochlea --- cisplatin, ROS and apoptosis 3.1. Cochlea anatomy The snail-shaped cochlea consists of multi-canal structures inside the bony labyrinth. These canals are named scala vestibule, scala media (or ductus cochlearis) and scala tympani respectively as shown in figure 6a [148]. The vasculature is similar in most mammalian. The blood supply of scala vestibule is mainly arterial; that of the scala media is capillary or by-pass, and that of the scala tympani is venous as shown in the figure 6b [149]. 29

35 a b Figure 6. Cochlea blood supply Panel a, 1. Vessel of the basilar membrane (VSM) 2. Vessel of the tympanic lip (VTL) --- These vessels belong to the spiral portion 3. Capillaries of the stria vascularis 4. Vessel of the spiral ligament. These vessels are belonging to the lateral portion. Arrows indicate the blood flow direction. SV, scala vestibuli; SM, scala media; ST, scala tympani. The arrows in the plots marked the direction of blood circulation. Panel b, 1. anterior inferior cerebellar artery (AICA) 2. labyrinthine artery (Inner artery) 3. common cochlear artery 4. anterior vestibular artery 5. cochlear artery (Spiral modiolar artery) 6. vestibulocochlear artery 7. cochlear branch 8. cochlear branch. Arrows indicate the blood flow direction. Capillaries of stria vascularis was shown in small plot. (From: Axelsson A. Am J Otolaryngol,1988.9: ) Corti s organ (figure 7 [150]) in scala media, contains several layers of sensory epithelial cells called hair cells, which are mechanotransducers of the inner ear. They are essential for both hearing and balance [151], and are supported in a rigid framework of supporting cells. The main structural support for the organ of Corti is provided by the pillar cells (cell's forming the outer and inner walls of the tunnel in the organ of Corti) and Deiter s cells (also called phalangeal cells) [152].There are two kinds of hair cells in the organ of Corti, and their names reflect their position with respect to the axis of the coiled cochlea: inner hair cells (IHCs) and outer hair cells (OHCs). IHCs are arranged in a single row, whereas outer hair cells are arranged in three or four rows. 30

36 There are fewer IHCs than OHCs (in human, approximately 3,500 versus 12,000; and in rat: 1,200 versus 4,000) [148, ]. These cells are innervated by the distal processes of bipolar primary sensory neurons located in the spiral ganglion. The central processes of the bipolar neurons are from the cochlear division of the vestibule-cochlear (eighth cranial) nerve. The IHCs are the actual sensory receptors, and 95% of the fibers of the auditory nerve that project to the brain arise from this subpopulation. The terminations on the project to the OHCs are almost all from efferent axons that arise from cells in the superior olivary complex [156,157]. In rat cochlea, the density of hair cells is not uniform [ ]. The lowest density for the OHCs is in the base of the cochlea with a trend of gradually increasing density toward the apex. The maximum density for the IHCs is found at about 25% of the basilar membrane length from the base. Both the OHCs and IHCs do not rest on the basilar membrane itself but on supporting cells as mentioned above. 31

37 a b c Figure 7. Cochlea structure and hearing a, The cochlea. Sound enters the outer ear canal, vibrates the eardrum and is transmitted to the cochlea by the middle ear bones. b, A transverse section through the organ of Corti. Inside the cochlea, the organ of Corti sits upon the basilar membrane. Its surface is covered by tectorial membrane. Vibration of the basilar membrane leads to lateral displacements of the mechanosensory bundles on the hair cells (colored orange). Inner hair cells form a row to one side (the left side here) of two rows of specialized supporting cells (pillar cells); outer hair cells form several rows on the other side. Inner hair cells are connected mainly to afferent nerve fibers and so are responsible for sending sensory information to the brain. Outer hair cells are connected to relatively few afferent fibers and are located above the most flexible region of the basilar membrane, where they can more easily influence its mechanical responses. c, The plasma membrane of outer hair cells contains a high density of membrane proteins embedded within the lipid layer. It is connected to relatively stiff, circumferential filaments that are cross-linked by thinner, more compliant filaments. This sub-membrane skeleton helps to convert changes of membrane surface area into changes in cell length. (from: Matthew Holley, Nature 405, (11 May 2000) ) 32

38 3.2. The organ of Corti and hearing function Sensory transduction in the cochlea has been investigated for many years. The initial step is to transform vibrations into action potentials. In Mammals, stereocilia of hair cells are sensitive to bending. The number of neurons in spiral ganglion of rat (depending on the strain and age) is about 15,800 to 19,900 [154, ]. The density of neurons varies along the cochlear spiral [154,162]. The number of nerve fibers synapse onto each IHC is uneven Cisplatin-induced hearing loss Injuries to any of these structures, i.e. stria vascularis, sensory epithelia and spiral ganglion, will damage the auditory function and cause hearing loss General information of cisplatin-induced ototoxicity Cisplatin is one of the most commonly used cytotoxic agents and concomitantly administered with radiotherapy and is increasingly used in locally advanced head and neck squamous cell carcinoma [163]. Ototoxicity is an important and dose-limiting side effect in cisplatin therapy. Audiometry shows cisplatin-induced ototoxicity in 75 to 100% of patients, which may be associated with tinnitus and hearing loss [164]. In cases of younger age, large cumulative doses, pre-existing hearing loss, renal disease, and irradiation of the brain or skull, patients are more vulnerable [ ] Injured cochlea structures caused by cisplatin Cisplatin ototoxicity occurs in at least three major tissues in cochlea: organ of Corti, spiral ganglion cells and lateral wall (stria vascularis and spiral ganglion) [165]. Cochlear damage was studied in the guinea pig model by daily administration of high cumulative doses of cisplatin, and was found to be dose-dependent and 33

39 selectively restricted to the OHCs of the inner ear and corresponding nerve fibers. It is found that hair cell degeneration was most severe in the basal turn of the cochlea, and progressed in an apical direction so that the cells in the first row were the most affected [169]. The time-sequence of damage to spiral ganglion cells and the outer hair cells in guinea pig follows a similar time course, suggesting that injury to both cochlear areas occurred in parallel, rather than sequentially [170]. TUNEL (terminal nuleotidyl transferase-mediated dutp-biotin nick end-labeling) staining in the stria vascularis of guinea pigs treated with marginally ototoxic dose of cisplatin showed positive in the stria vascularis, but no TUNEL staining in hair cells [171] ROS is involved in inner ear damage after cisplatin-treatment. ROS play important roles in the regulation of a number of normal physiological processes, including cell proliferation, survival, senescence and apoptotic cell death [172]. It is established that ROS are generated in hair cells exposed to cisplatin [51,173], aminoglycosides [ ], and noise [178,179]. Oxidative stress markers [antibodies to 8-isoprostane (8-iso) and nitrotyrosine (NT)] were found in spiral ganglion cells, in the blood vessels and fibrocytes of the lateral wall, as well as in supporting cells of the organ of Corti during the process of apoptosis [180]. ROS affect the OHCs in the organ of Corti [181]. Analysis of cochlea lesions in cisplatin-treated rats, guinea pigs, and gerbils indicates that OHC lesions are more severe than IHC lesions. Moreover, OHC lesions progress along a base to apex gradient as seen with other ototoxic drugs [ ]. Cisplatin has also been applied locally to the round window membrane of the chinchilla to avoid systemic toxicities 34

40 and mortalities; with this approach, OHC lesions are also more severe than IHC lesions [187].There are some studies reporting that enhancing antioxidant levels, either through drug application or genetic manipulation, promotes the survival of hair cell, thus preserves their function [ ]. Recent studies in the central nervous system have shown that mitochondria-associated oxidants are involved in pathways regulating cytochrome c translocation and caspase activation [191]. In addition, cisplatin administration has been shown to result in depletion of antioxidant enzymes and reduced glutathione (GSH), as well as increased malondialdehyde levels in the cochlea [192]. It is believed that cisplatin suppresses the formation of endogenous anti-oxidants which normally prevent the inner ear against ROS [181], and thus causes inner ear injury Cisplatin-induced ROS generation and NOX Since ROS play an important role in drug-, noise-, and age-dependent hearing loss, and NOX3 is found as a relevant source of ROS generation in the cochlear and vestibular systems; thus, it is hypothesized that NOX3-dependent ROS generation is contributing to hearing loss and balance problems in response to ototoxic drugs [23,90] Cisplatin induces apoptosis Drug-induced hearing loss is mostly, if not all, through apoptosis in particular through caspase activation. Caspases constitute a family of proteases normally existing as inactive enzymes. They are cysteine-dependent aspartate-specific proteases that function to mediate apoptotic destruction of the cells. Fourteen members of the caspase family have been 35

41 identified in mammals [193,194]. Intrinsic pathway is also found responsible for cisplatin-related apoptosis in cochlear hair cells [195,196]. Independent studies have shown that general caspase inhibitors are able to promote hair cell survival after treatment with cisplatin [197,198] and aminoglycosides [ ] Cisplatin-induced apoptosis and ROS Although most research to date reported that cisplatin induce ROS generation and is the causative factor in inner ear apoptosis. However, it is also possible that ROS generation is a secondary event in cisplatin ototoxicity [204]. So, this issue is matter of remained debate. For this reason, it is necessary to make it clear whether ROS generation will initiate and / or accelerate the process of apoptosis in cochlea Study of ROS (O 2 - ) generation in cochlea explants Intact cochlea samples are necessary for cochlea investigation In rat cochlea, the density of hair cells is not uniformly distributed along the basilar membrane in Corti s organ [ ]; therefore the extraction of intact cochlea was essential for our study. Extracted cochleae used in our study are shown in figure Reduced NBT as a method to detect superoxide generation in cochlea Applying NBT assay to intact cochlea uncovers a universal distribution of ROS (superoxide). Reduced NBT locates at all the structures with cell bodies seen in figure 9. 36

42 a Apical turn Middle turn Stria Vascularis VBM & VTL Modiolus b Hair cell layers Basal turn p c d Figure 8. Structure of cochlea explant from Wistar rat pup a. A cochlea explant maintains apical, middle and basal turns. Stria vascularis, vessel of basilar membrane (VBM) and vessel of tympanic lip (VTL) can be seen. b. Immunohistochemistry was applied to the intact cochlea. Myosin VIIa antibody specifically labels hair cell layers (lines with deep brown color). p marked the pealed down part of stria vascularis to show hair cells at basal turn. c. More samples available to choose qualified explants. d. Cochlea extracted from mouse pup on the 8 th post-natal day. Red circles mark the otolith in the broken semicircular tube. Scale bar=0.5 mm. a 1 b Figure 9. Superoxide distribution in cochlea explant can be revealed by NBT assay. Cochlea explants were incubated with 500 µg/ml nitroblue tetrazolium (NBT) for 2.5 hours. a. and b. show a different view of the same cochlea explant. Numbers in red circles designate respectively: 1. stria vascularis, 2. region of Corti s organ, 3. venous drainage net works in the surface of modiolar nerve fiber bundle, and 4. internal nerve fiber bundle of modiolus. 37

43 4. NOX enzymes in pancreatic islets --- ROS / insulin secretion / diabetes 4.1. Pancreas anatomy Pancreas is an endocrine and exocrine gland of the endocrine and digestive systems of vertebrates. The endocrine gland produces hormones including insulin, glucagon, and somatostatin; while the exocrine gland secrets pancreatic juice containing digestive enzymes into the small intestine. These enzymes help in the further breakdown of the carbohydrates, protein, and fat. Thus, pancreas is a gland organ with very complex physiological functions. In this part, we will focus mainly on its endocrine functions. The part of the pancreas, which is involved in the endocrine function, is composed by cell clusters called islets of Langerhans or pancreatic islets. Four types of cells are found in rat Langerhans islets [205]. They are distributed as shown in table 1 (based on mean volume density per islet by immunohistochemistry). The regions mentioned in table 1 are described in the figure

44 Table 1. Distribution of endocrine cells in rat pancreas The whole area of rat pancreas is divided into four regions shown in figure 10: lower duodenal, upper duodenal, gastric and splenic. Four kinds of cells in the pancreatic islets were counted after labeling with specific antibodies respectively. The values represent the percentage of the cell in the islet. A, α cells; B, β cells; D, δ cells; PP, pancreatic polypeptide producing. (From: AHMED A. et.al J Anat (Pt 3): ) Figure 10. Diagrammatic presentation of rat pancreas Rat pancreatic area is divided into four regions for specific cell counting as mentioned in table 1. Four regions: LD, lower duodenal; UD, upper duodenal; G, gastric; and SP, Splenic (from: AHMED A. et.al J Anat (Pt 3): ) Cell composition of rat pancreatic islet (percentage to the total islet) is the following: α cells, 15-20%; β cells, 65-80%; δ cells, 3-10%; PP cells, 3-5%; and ε- 39

45 cells, <1%. In contrast to the pancreas of mouse and/or rat, which looks like a piece of adipose tissue, loosely spreading behind the stomach and in the area between duodenum and spleen as shown in Figure 11 [206], the shape of human pancreas appears much more regular and compacted like some other organs (spleen, liver etc) in the body as shown by the model in Figure 12. stomach duodenum spleen transverse colon Figure 11. Rat pancreas and pancreatic ducts. Rat pancreas is irregular distributed in the area confined by stomach, spleen, transverse colon and duodenum as the region marked in red-dashed line. (from: Benedict J Page, et al. Journal of the Pancreas 2000; 1: ) 40

46 Figure 12. Human pancreas model Pancreas is a both endocrine and exocrine gland. It consists of three parts: head, body and tail. Acinar cells are involved in exocrine function. Pancreatic islet consisting of α,β, and δ cell is involved in endocrine function. (Figure from Species differences in pancreatic islets By using laser scanning confocal microscopy in human islets, Marcela Brissova et al found that islet composition in higher Mammals (human, non-human primate, and canine) is more diverse than in rodent islets [207]. Over Cabrera et al get similar conclusion. The structures of islets from different specie sources are shown in figure 13 [208]. 41

47 Figure 13. Interspecies difference in pancreatic islets Confocal micrographs (1-µm optical sections) demonstrated representative immunostained pancreatic sections containing islets of Langerhans from human(a), monkey (B), mouse (C), and pig (D). Insulin-immunoreactive (red), glucagonimmunoreactive (green), and somatostatinimmunoreactive (blue); (From: Cabrera O, et al, 2006) 4.3. β cells and α cells The most important cells controlling nutrient metabolism, especially in carbohydrates, are β cells and α cells β cells and insulin secretion Normally, β cell is the only source of insulin synthesis, which facilitates the usage of blood glucose and decrease blood glucose level; α cell produce glucagon which helps to elevate blood glucose level. Insulin comes from proinsulin. Proinsulin comes from preproinsulin which is a single chain precursor and the translation product of insulin mrna. During insertion into endoplasmic reticulum (ER), the signal peptide of preproinsulin is removed, thus forming proinsulin. There are three domains in proinsulin: B chain (an aminoterminal), A chain (a carboxyl-terminal) and C peptide (a connecting peptide). Within ER, C peptide is excised by several specific endopeptidases, thus forming insulin. 42

48 Both insulin and free C peptide are packaged in the Golgi complex into secretary granules in the cytoplasm before release (secretion). Primary stimulation to insulin secretion is elevated blood glucose concentration. However, the signal for insulin secretion must come from β cell when glucose is metabolized in it [209]. Glucose within β cell undergoes glycolysis to form pyruvate which then enters mitochondria where more ATP is generated through tricarboxylic acid (TCA) cycle in respiratory chain reactions. Thus, more ATP is released from mitochondria into the cytosol causing ATP/ADP ratio increase accordingly. This triggers the depolarization of membrane by closure of ATP sensitive K + channels and successive opening of Ca 2+ channels which are voltage-sensitive [210,211]. As a result, Ca 2+ concentration increased in the cytosol as the direct signal stimulating exocytosis of insulin granules [212,213]. Insulin controls glucose fundamentally by decreasing blood glucose through following mechanisms: 1, promoting cells in liver, muscle, and adipose tissue to take up glucose from the blood and increasing its usage there; or to store it as glycogen in the liver and muscle, and 2, stopping use of fat (lipid mobilization) as an energy source, thus decreasing gluconeogenesis in liver and kidney α cells and glucagon secretion Functionally opposite to insulin, glucagon secreted by α cells elevates the blood glucose level when it is too low by stimulating the liver to convert stored glycogen into glucose and release it into the bloodstream. However, glucagon also stimulates the release of insulin, so that newly-available glucose in the bloodstream can be taken up and used by insulin-dependent tissues. 43

49 4.4. Insulin vs. glucagon Concerning the functional contribution of the two hormones in regulating blood glucose, it is well-established that the first defence against falling plasma glucose concentrations is a decrease in insulin secretion, the second defence is an increase in glucagon secretion [214]. In diabetes, loss of the glucagon response to falling plasma glucose concentration is probably the result of β cell failure [215,216]. Plasma glucagon levels have been found to be elevated in patients with diabetes in some studies but not others [217]. Therefore the general concept is that a relative hyperglucagonaemia occurs in diabetes, since glucagon levels might be expected to be reduced in the setting of hyperglycaemia [215]. However, compelling evidence that glucagon contributes to the pathogenesis of hyperglycaemia in diabetes awaits longterm selective reduction of glucagon secretion or action in humans [215] Insulin secretion and diabetes Abnormality in insulin secretion and function causes one of the most common metabolic diseases --- diabetes mellitus. In human, diabetes mellitus has two forms: 1, juvenile-onset or Type 1 diabetes mellitus (T1DM) which arises from autoimmune destruction in pancreatic β-cells thus leading insulin deficiency, accounts for approximately 5 10% of individuals with diabetes; and 2, adult-onset or type 2 diabetes mellitus (T2DM), which arises from defects in insulin sensitivity (insulin resistance) and relative insulin deficiency, accounts for 90 95% of those with diabetes [218]. We already have a global accepted criteria in diabetes (and related disorder) diagnosis as shown in table 2. 44

50 Diabetes: Fasting plasma glucose 2-h plasma glucose* 7.0 mmol/l (126 mg/dl) or 11.1 mmol/l (200 mg/dl) Impaired Glucose Tolerance (IGT): Fasting plasma glucose 2-h plasma glucose* <7.0 mmol/l (126 mg/dl) and 7.8 mmol/l (140 mg/dl) and <11.1 mmol/l (200 mg/dl) Impaired Fasting Glucose (IFG): Fasting plasma glucose 2-h plasma glucose* 6.1 to 6.9 mmol/l (110 mg/dl to 125 mg/dl) and < 7.8 mmol/l (140 mg/dl) * Venous plasma glucose 2-h after ingestion of 75 g oral glucose load. If 2-hour plasma glucose is not measured, status is uncertain as diabetes or IGT cannot be excluded. Table 2. The 2006 WHO recommendations for the diagnostic criteria for diabetes and intermediate hyperglycaemia. There are three kinds of states in glucose metabolism disorder: diabetes, IGT and IFG. Both fasting plasma glucose and 2-h glucose are considered equally in diagnosis. (From: As the majority of glucose metabolism disorder cases, T2DM draws extensive attention in both clinical and fundamental research. T2DM patients without proper treatment will develop severe complications in long run. Most complications are rooted on vascular injuries, including blindness, end-stage renal failure, and atherosclerosis [219,220]. Hyperglycemia, resulting from insulin resistance and/or secretion, is the characteristic sign of the disease [218], and its chronic form is associated with endothelial dysfunction [221,222]. An increasing body of evidence suggests that elevated reactive oxygen species (ROS) produced by NADPH oxidase contribute to various diseases including endothelial dysfunction, atherosclerosis, hypertension, and tumor genesis [223]. 45

51 Pancreatic islets have a dense network of sinusoidal capillaries with a morphological resemblance to the renal glomerulus [224], thus dysfunction of ECs might associate with T2DM. The NAD(P)H oxidase isoforms of ECs are expressed in the endoplasmic reticulum (ER), perinuclear membranes generating ROS as modulators of redox-sensitive signaling pathways [ ]. In addition, anion channels [Cl-channel-3 (ClC-3)] expressed in the plasma membrane of ECs may - facilitate diffusion of O 2 between intracellular and/or extracellular compartments [228,229] NOX dependent ROS generation and insulin secretion ROS at physiological level are involved in signaling normal β-cell glucose response [ ] as reviewed in figure 14 [233], and figure 15 [252]. Pancreatic β- cell is particularly sensitive to oxidative stress due to the presence of low levels of antioxidant enzymes compared to other cell types [234,235]. Thus, over expressed ROS will affect β-cell function and cause injury in it. Increasing body of evidence demonstrates that oxidative stress occurs in obesity [ ] and diabetes [241, ]. The mechanisms involved in diabetes are reviewed in the figure 14, 15 from [252]. Concerning the source of ROS in pancreatic islet, mitochondria are thought to be the most important source by most investigators documented [ ]. However, a novel hypothesis is emerging, involving NOX enzymes [248]. Further research using NOX deficient mice should help deciphering this topic. 46

52 Figure 14. ROS generation and antioxidant systems in a generic cell type ROS can be generated through glucose metabolism in mitochondria by electron transport chain (ETC) activity and in the plasma membrane through NADPH oxidase NOX. The main antioxidant enzymes are superoxide dismutase (SOD), glutathione reductase, glutathione peroxidase and catalase. Figure from P. NewsholmeJ. et al. Physiol Aug 15;583(Pt 1):

53 Figure 15. NOX family and β-cell dysfunction in Type 2 diabetes Under normal conditions, low glucose induces a release of insulin by β-cells. Under chronic highglucose and NEFA conditions, several modifications lead to decreased β-cell mass and Type 2 diabetes. (I) Disturbances in the ER related to development of altered glucose homoeostasis are an emerging field of research. Impairment of normal protein folding in the ER during ER stress can redirect β-cells to apoptotic pathways via NOX4. (II) Hyperglycaemia is involved in cell death through the ROS inhibition of phosphatases of the JNK pathway, NF-κB activation and caspase activation. It also results in increased production of ROS by NOX1 and/or NOX2 and generation of AGEs, which in turn are associated with reduced transcription of genes involved in insulin production. (III) The endocrine pancreas is exposed not only to systemic, but also to locally produced components of the renin angiotensin system (RAS); in Type 2 diabetes, RAS activity is inappropriately up-regulated in β-cells and in endothelial cells of the pancreatic vasculature. The physiological role of the pancreatic RAS appears to involve islet blood flow regulation, an effect capable of affecting insulin secretion that may be triggered in part by NOX1. (IV) Pancreatic fibrosis seems to involve the increased production of TGF- β1, a classic profibrotic cytokine, and PSC activation, which may involve NOX2 and/or NOX4. (V) Finally, NOX2 in macrophages and polymorphonuclear neutrophils (yellow) contribute to inflammation by producing pro-inflammatory cytokines that lead to decreased β-cell mass and Type 2 diabetes. From Guichard C, et al. Biochem Soc Trans Oct;36(Pt 5):

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88 Supplementary information (protocol in detail) RNA interference targeting on NOX4 in HMC3 cells The protocol is used for experiments in 24-well plate, 500 ml medium (per well) containing sirna and HiPerfect transfection reagents. One day before transfection: HMC3 cells were seeded at 40,000/well in 24-well plate supplied with 1 ml medium with serum. On transfection day: 1. Remove old medium from the prepared cells. Add 400 µl new serum-free medium. 2. Prepare transfection mixture in to meet the following requests (use one well as example): sirna oligo: 300 ng/well and HiPerfect transfection reagent: 3 µl/well. 3. Mix sirna oligo and HiPerfect transfection reagent with additional serum-free medium to final volume of 30 µl. 4. Incubate mixture at room temperature for 7 minutes µl serum-medium was added into the mixture and mix well. 6. Add the mixture containing sirna oligo(s) to the prepared cells. 7. Incubate the cells at routine cell incubator for 6 hours. 8. At the end of incubation, remove old medium, re-fill the well with new routine culture medium (containing serum) hours later *, transfected HMC3 cells are ready for various kinds of detection --- by Q-PCR to detect target gene expression and / or ROS measurement by NBT assay. (*According to instruction of HiPerfect transfection reagent, 6 to 8 hours 83

89 after transfection transfected cells are ready for detection. The incubation time in the thesis part 1 was 24 hours as mentioned in the published paper.) a b Figure S-part 1. HMC3 cells transfected with fluorescein-tagged sirna. HMC3 cells were transfected with fluorescein-tagged non-sense sirna (300 ng/well, 24-well plate; 40,000 cells/ well at seeding one day before transfection) and HiPerfect transfection reagent (3 µl/well). At 18 hours post-transfection, old medium was removed. Cells were rinsed with new medium. Images were taken for the same sample. a, phase-contrast. b, under FITC filter to show fluorescein, which appeared in green-tiny particles. 84

90 Research article 2 The relationship between NOX dependent ROS generation and cisplatin- induced apoptosis in cochlea explants Bin Li a, Jing Wang b, Vincent Jaquet a, Michel Dubois-Dauphin a, Jean-Luc Puel b, Karl-Heinz Krause a* a Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Rue Michel-Servet 1, 1211 Geneva-4, Switzerland b INSERM - UMR 583, Institut des Neurosciences de Montpellier and Université Montpellier 1, Montpellier, France. * Correspondence to K.-H. Krause, M.D, Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Rue Michel-Servet 1, 1211 Geneva-4, Switzerland. Phone: ; Fax: ; Keys Words (3-10): Rat cochlea explants, cisplatin, reactive oxygen species, apoptosis, hearing loss, DPI, ototoxicity Word Count:

91 Abstract Aims: To study the source of reactive oxygen species (ROS) in rat cochlea explants and their role in cisplatin-induced apoptosis. Results: Several NOX isoforms, namely NOX2, NOX3, and NOX4 were detected by RT-PCR in cochlear explants. The Nitroblue tetrazolium (NBT) assay was used to detect superoxide in cochlear explants. A spontaneous superoxide generation was observed in the cell bodies of different structures of the cochlear explants i.e., stria vascularis, the organ of Corti, and spiral ganglion. After cisplatin treatment, enhanced ROS generation and induced apoptosis were observed. A preventive treatment (10 µm) with the flavoprotein inhibitor diphenylene iodonium (DPI), a potent electron transporter inhibitor, completely blunted the spontaneous and cisplatin-induced superoxide generation. However, using immuno blotting assay, primary data demonstrated that DPI and apocynin did not reduce apoptosis by measuring caspase 3 activity. Conclusions: ROS generation distributes in all the structures with cell bodies of the cochlea explant. Cisplatin enhances spontaneous ROS generation and induces apoptosis. However, whether NOX-dependent ROS generation is correlated to cisplatin-induced apoptosis needs more research. 86

92 Introduction The cochlea is a spiral shaped bony structure resembling a snail, which contains different canal structures filled with perilymph. These canals are the scala vestibule, the scala media (or ductus cochlearis) and the scala tympani [1]. The organ of Corti lies in scala media and contains several layers of sensory epithelial cells called hair cells, which play the role of mechanotransducers of the inner ear. They are essential for both hearing and balance [2], and are supported in a rigid framework of supporting cells [1]. Hair cells are innervated by the distal processes of bipolar primary sensory neurons located in the spiral ganglion. Vascularization of the cochlea is similar among most mammals. The blood supply of the scala vestibule is mainly arterial; that of the scala media is capillary or by-pass, and that of the scala tympani is venous [3]. Injuries to any of these structures will damage the auditory function and cause hearing loss. Cisplatin is a highly effective and widely used anticancer agent, concomitantly administered with radiotherapy to treat advanced head and neck squamous cell carcinoma [4,5]. However, it can cause the impairment of inner ear functions, including hearing and balance [6]. Cisplatin ototoxicity is clinically characterized by a dose-related, cumulative, and usually permanent sensorineural hearing loss, starting at higher frequencies and progressively extending to lower frequencies [7]. The variability in reported ototoxicity incidence may be related to several factors, including mode of drug administration, total dose per treatment and cumulative dose [8]. The risk of ototoxic (and nephrotoxic) side effects commonly hinder the use of higher doses that could maximize its anti-neoplastic effects [9]. The cytotoxic effects of cisplatin may occur via several putative pathways [10], including formation of DNA adducts, production of ROS, and depletion of 87

93 antioxidant enzymes in the cochlea [11-13]. Cisplatin-induced hearing loss has been linked to increased ROS formation in cochlear tissue [14,15]. Over production of ROS can lead to protein and DNA damage, cause cell membrane breakdown through lipid peroxidation [16], and act as a putative trigger for apoptotic cell death [17,18]. Enhancing antioxidant levels, either through drug application or genetic manipulation, promotes the survival of hair cells [19-21]. Local inner ear administration of cytoprotective antioxidant agents such as D- or L- methionine [22], trolox [23], and thiourea [24], or systemic administration of L- methionine [25] or salicylate effectively attenuates cisplatin ototoxicity [26]. Nevertheless, the source of ROS generation in the cochlea following cisplatin treatment is unclear. Recently, NOX3, an isoform of the ROS generating family of NADPH oxidases has been shown to be a relevant source of ROS generation in the cochlear and vestibular systems. The study of mouse mutants with a defect in vestibular function showed that NOX3 gene was affected and that its function is crucial for otoconia formation, a phenomenon that occurs at very early stage of embryonic development [27,28]. However, after birth, the function of NOX3 is unclear, but recent data support the hypothesis that cisplatin ototoxicity might be mediated by ROS generation by NOX3 because NOX3 mrna is up regulated in the cochlea of rats after cisplatin treatment [29,30]. In the present study, we used cochlea explants to investigate the expression profile of NOX enzymes by RT-PCR and found that, besides NOX3, NOX2 and NOX4 mrna were also detected. We demonstrated that cisplatin induced ROS generation and caspase 3 activation in cochlear explants. Our preliminary data showed that a preventive treatment with the non-specific electron transporter inhibitor 88

94 diphenyleneiodonium (DPI) did suppress ROS generation, but could not protect the cochlea explants from cisplatin-induced apoptosis. Apocynin, a natural compound acting principally as a ROS scavenger, but also a NOX inhibitor [31-33], was also unable to prevent cisplatin-induced apoptosis. Thus, whether the apoptotic cell death occurring in cochlea explants following cisplatin exposure is induced by increased oxidative stress needs further study. Materials and methods In accordance with Swiss legal requirements, all animal experiments were approved by an academic committee and supervised by the local veterinary agency. Cochlea extraction and ex vivo culture Cochleae were extracted from Wistar rat pups at the age of 2 to 5 post-natal days by micro-dissection. After extraction, cochlea explants were cultured overnight in routine cell culture incubator (37 C, 5% CO2, and 95% humidity) in DMEM-F12 (Gibco) supplemented with 2 mm L-glutamine (Gibco), 1 X N2 (Gibco), 1 X Insulin Transferrin Selenium (Gibco), 3 µg/ml cytosine 1 β-arabinofuranoside (Ara-C), (Sigma), 8.25 mm D-glucose (Invitrogen), and 62 µg/ml augmentin (GlaxoSmithKline). RT-PCR Total RNA was isolated using RNeasy mini kit (Qiagen) according to the manufacturer s instructions. Total RNA was reverse transcribed by Sensiscript reverse transcriptase (Qiagen). 89

95 PCR was performed using AccuPrime TM Taq DNA Polymerase (Invitrogen). Primers used for PCR detection are documented in Table 1. The primers were purchased from Invitrogen and have been documented before [34]. All the positive control cdna was synthesized from total RNA isolated from specific organs or cell lines expressing the target genes. Nitro Blue Tetrazolium chloride (NBT) assay NBT (Sigma-Aldrich) is reduced by superoxide into the water insoluble darkblue formazan [35-37]. In the present study, NBT was added in the medium at final concentration of 250 µg / ml and incubated the samples for 30 minutes. At the end of the incubation, the medium was removed, and samples were fixed with 4 % paraformaldehyde (PFA). Images were taken using Leica M165C binocular system. Immunoblotting Two cochleae were used per condition. Protein concentration was evaluated using the BioRad assay (BioRad, Hercules, CA, USA). The same amount of protein was loaded for sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE, 15%). After separation, the proteins were transferred onto polyvinylidene fluoride (PVDF) membrane. PVDF membranes were stained with FastGreen (Sigma) and were scanned for image by office scanner (hp scanjet 5530 photosmart scanner). After blocking with 0.05% Tween-20-PBS buffer containing 5 % (g/v) milk powder, the membranes were incubated at 4 C overnight with primary caspase 3 antibodies (1: 500) recognizing either cleaved caspase 3 (Cell Signaling #9661) or both cleaved and uncleaved forms of caspase 3 (Cell Signaling #9662). After washing, membranes were incubated with secondary antibody (horse radish peroxidase, HRP, conjugated 90

96 Goat-IgG to rabbit, Cappel; 1:10,000) for 60 minutes. Target proteins were revealed by using ECL kit (GE healthcare UK limited) according to instructions. Film was scanned for image. Scanned images were switched into inversed gray-scale mode for ImageJ (1.37V) analysis. Signals of target protein were normalized with the total protein signal. Statistical analysis All the experiments were done at least three times independently (detailed number of experiments are mentioned in figure legends). Data were analysed using the GraphPad Prism 4 software. For all tests, means were compared and analysed using a t-test. A p value 0.05 was considered statistically significant. 91

97 Results NOX2, NOX3, and NOX4 were detected by RT-PCR in cochlea explants Cochlear explants used for RNA extraction contained different tissues, such as the stria vascularis, the vessels of basilar membrane (VBM) and of tympanic lip (VTL) adjacent to the Corti s organ [38]. RT-PCR demonstrated the expression of NOX2, NOX3, and NOX4 in cochlea explants, but NOX1 was not detected (fig. 1a). NOX subunits p22 phox, p47 phox, and NOXA1 were detected. NOXO1 expression was low and p67 phox was not detected (fig. 1b). DPI prevented spontaneous superoxide generation and cisplatin-induced enhancement of superoxide in cochlea explants NBT reduction assay was adopted to visualize the distribution of superoxide generation (fig.2b to 2d). Cell bodies in all structures including in vascular system, Corti s organ, and spiral ganglion, were stained with reduced NBT (formazan). Formazan staining appeared stronger in groups treated with cisplatin (fig. 2c and fig. 2d) as compared with control (fig. 2b); the higher the concentration of cisplatin, the stronger was the formazan staining. No staining appeared on cochleae without NBT (fig. 2a), neither for cochleae pre-incubated with 10 µm DPI for one hour before 0.5 mm cisplatin incubation (fig. 2e and 2f). Thus, these results suggested that the non-specific NOX inhibitor DPI prevent the production of ROS after cisplatin treatment. explants Cisplatin induced apoptosis and generation of Caspase 3 protein in cochlea 92

98 Cochleae were treated with / without 0.5 mm cisplatin for 6 hours. Protein samples were analyzed by immunoblotting (fig.3). The analysis of band intensity showed that cleaved caspase 3 was stronger in 0.5 mm cisplatin-treated group (fig.3b; p<0.01) than in control groups. However, there was no difference in uncleaved caspase 3 between the two groups (fig. 3b; p>0.05). Thus, cisplatin induces apoptosis in cochlea explants, and generation of caspase 3. DPI or apocynin did not protect cochlea from cisplatin-induced apoptosis Cochlea explants were pre-incubated with / without 10 µm DPI for one hour and subsequently incubated with 0.5 mm cisplatin for six hours. For DPI groups, 10 µm DPI was used continuously with cisplatin to the end of the experiment. Proteins were extracted for analysis of caspase 3. In three independent experiments, DPI (10 µm) did not prevent the increase of cleaved caspase 3 in cisplatin-treated cochlea explants. Similar experiments were done by using apocynin, an antioxidant and NOX inhibitor at 2.5 mm concentration instead of 10 µm DPI. Data showed that there was no protection effect (p>0.05; fig. 4a and 4b). In addition, 10 µm DPI or 2.5 mm apocynin alone in treating the cochlea explants for seven hours did not cause change in either cleaved caspase 3 or uncleaved caspase 3 (p>0.05; data not shown). 93

99 Discussion In the present study, we used rat cochlea explants to detect the presence of NOX isoforms and to assess the effect of cisplatin in ROS generation and apoptosis. We showed that different NOX isoforms mrnas were expressed in the cochlea explants. Both cisplatin-enhanced ROS generation and cisplatin-induced apoptosis are present in this experiment model allowing the study of the relationship between cisplatin induced oxidative stress and apoptosis. Interestingly, we found that, in this system, the increased ROS generation induced by cisplatin toxicity appeared not the cause of apoptosis. Ototoxicity and nephrotoxicity are the two major adverse effects of cisplatin chemotherapy. Increased oxidative stress and apoptosis are the major features of this organ-specific toxicity. Sources of oxidants include mitochondria, xanthine oxidase, uncoupled inos and leukocyte infiltration. However, the phenomenon that cisplatininduced nephrotoxicity and ototoxicity share numerous pathological features suggested the presence of specific ROS generating systems in these organs. Interestingly, the kidneys have by far the highest expression level of NOX4 isoform, while NOX3 isoform is specifically expressed in inner ear [27,28]. Moreover, a recent report showed a marked superoxide generation as well as enhanced expression of NOX4 (and to a lesser extent NOX1) isoforms in the kidneys of mice treated with cisplatin [39]. Similarly, increased NOX3 mrna expression was demonstrated in the inner ear of rats treated with cisplatin [30]. In addition, since the recent discovery of all NOX isoforms, increasing evidence shows that up regulation or activation of NOX enzymes associated with oxidative stress are the key players causing cell damage in hypertension, atherosclerosis and neurodegenerative disease [40]. 94

100 RT-PCR detection of NOX expression demonstrated that NOX2, NOX3, and NOX4 were expressed in cochlea explants. The presence of NOX2 and NOX4 might come from stria vascularis and / or blood vessels within the cochlea explants, but NOX3 was probably from the organ of Corti and / or ganglion cells [30,41,42]. It has been documented that NOX3 requires p22 phox [43,44], NOXO1 [43,44] and NOXA1 [44] subunits for superoxide generation. Therefore the absence of NOXO1 in the cochlear explants raises the question of a functioning NOX3 isoform since NOXO1 mutant (head-slant mice) present a very similar phenotype as NOX3 mutant mice (head tilt) [28,45]. However, in HEK293 cells transfected with different combination of cytosolic subunits, Banfi et al. (2004) showed that the presence of NOXA1 and p47phox induced a strong superoxide generation [27]. Therefore all subunits required for NOX3 activity are present, but, as p67phox is absent, it is unlikely that NOX2 can be functional [27]. NOX4 is known to reduce NBT and to function without the presence of cytosolic subunits [46], and is therefore another candidate for the observed superoxide generation in the cochlea explants. The increased superoxide generation appeared to be uniformly distributed in all cytosolic structures of the cochlear explants, suggesting that increased ROS generation occurs in all cell types. The fact that superoxide generation was totally blunted by 10 µm DPI is interesting per se, but did not give favor to a particular source one over another. DPI inhibits inos, xanthine oxidase and NOX in the submicromolar range, and mitochondria at higher concentrations (10 µm), all possible sources of superoxide generation as reviewed in [47]. DNA damage and apoptosis via caspase 3 activation are well described in cisplatin ototoxicity [30,48,49]. In our experimental model, cleaved caspase increased in cisplatin treatment. However, while a concomitant decrease of uncleaved caspase 3 95

101 was expected, immunoblotting showed an unchanged level in uncleaved caspase 3. It was therefore possible that cisplatin induced apoptosis in specific cell types e.g. hair cells, while other tissues in the explants responded by an increased expression of caspase 3 gene. Another possibility is that, at the chosen time point, caspase 3 was increased, but not yet cleaved. Nevertheless, the complete inhibition of ROS generating by DPI had no effect to cisplatin-induced caspase 3 activation, suggested that ROS generation was not the cause of cisplatin-induced apoptosis in our model. Similar ineffectiveness in preventing apoptosis by apocynin supported this observation. Based on this model, it appeared that the protective effect of antioxidant therapies is not directly related to inhibition of cisplatin-enhanced ROS generation in cochlea itself, but rather through affecting other sources of oxidative stress such as leukocyte infiltration or the development of inflammation. Acknowledgement The authors would like to thank Karen Bedard for helpful discussion on NOX topics. We are also grateful to Olivier Plastre and Stéphanie Julien for technical support. 96

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107 Figure 1 A cdna sources cochlea colon spleen inner ear kidney NOX1 NOX2 NOX3 NOX4 NOX1 NOX2 NOX3 NOX4 marker (b.p.) Cycle# B cdna sources cochlea colon spleen colon spleen spleen NOXO1 p47 phox NOXA1 p67 pho p22 phox NOXO1 p47 phox NOXA1 p67 phox p22 phox

108 FIG. 1. NOX isoforms and subunits expressed in rat cochlea explants at mrna level. RT-PCR was performed using total RNA extracted from four cochleae at 35 and 40 cycles amplification. (A) NOX2, NOX3 and NOX4 isoforms were detected. The lengths of PCR products from designed primers for NOX isoforms were: 219 b.p.(nox1), 336 b.p.(nox2), 607 b.p.(nox3), and 454 b.p.(nox4). (B) NOX cytosolic subunits p22 phox, p47 phox, NOXA1 were detected. A molecular weight marker was used for size evaluation. The lengths of PCR products from designed primers for NOX subunits were: 266 b.p.(noxo1), 180 b.p.(p47 phox ), 266 b.p. (NOXA1), 260 b.p.(p67 phox ), and 435 b.p.(p22 phox ). b.p. = base pair. 103

109 A B Figure 2 C no NBT D + NBT E + cis 0.5 mm + NBT F + cis 1mM + NBT + DPI 10µM +NBT + DPI 10µM + cis 0.5mM + NBT 104

110 FIG.2. DPI inhibited ROS generation, and prevented ROS generation induced by cisplatin in cochlea explants. NBT assay was adopted to reveal superoxide generation. Cochlea samples were incubated with / without NBT (250 mg / ml) for 30 minutes to reveal reduced NBT in cochleae. Cisplatin (0.5 mm or 1 mm) incubation time was one hours. DPI (10 µμ) preincubation (before cisplatin treatment) was one hour. When combined with cisplatin treatment, DPI (10 µm) was used continuously to the end of experiment. (A). Cochleae without any treatment were used as negative controls for NBT-incubation groups. (B). Cochleae were treated only with NBT incubation. (C). and (D). Cochleae were treated with 0.5 mm or 1 mm cisplatin for one hour before NBT incubation. (E). Cochleae were treated with 10 µm DPI for two hours before incubation with NBT. (F). Cochleae were pretreated one hour with 10 µm DPI followed by additional one hour incubation with 0.5 mm cisplatin incubation. 105

111 Figure 3 A cisplatin 0.5 mm caspase 3 uncleaved (35 kda) Cleaved (19 KDa) B ratio to total protein cisplatin (0.5 mm) cleaved caspase3 uncleaved caspase3 p<0.01 n.s n=10 n=10 n=10 n=10 106

112 FIG.3. Cisplatin induced apoptosis in cochlea explants, but did not cause concomitant decrease in uncleaved caspase 3. Cochleae were treated with / without 0.5 mm cisplatin for six hours. Proteins were extracted for immunoblotting assay. Caspase 3 proteins, both cleaved and uncleaved, were analyzed semi-quantitatively. Data were from ImageJ readouts. (A) Representative image showed the total protein with Fast Green staining on PVDF membranes (left part) and probed with specific antibodies recognising caspase 3 (for both cleaved and uncleaved, right part). (B) Dot graph represented normalised values of cleaved and uncleaved caspase 3 following cisplatin treatment. Data presented the mean and SEM for each group. P values were calculated through t-test. Each group consisted of 10 samples from eight independent experiments. GraphPad Prism 4 (t-test) was used in analysis. n.s. = no significance. 107

113 A cleaved caspase 3 uncleaved caspase 3 Figure ratio to total protein n.s. n.s. B 0.0 cisplatin 0.5mM DPI 10µM n=3 n=3 n=3 n=3 cleaved caspase 3 uncleaved caspase 3 n.s. ratio to total protein n.s. 0.0 cisplatin 0.5mM apocynin 2.5mM n=5 n=5 n=5 n=5 108

114 FIG.4. Inhibition of ROS generation did not prevent cisplatin-induced apoptosis. Cochlea samples were incubated with / without ROS inhibitor for one hour followed by additional incubation with 0.5 mm cisplatin for six hours. At the end of treatment, proteins were extracted from each sample, and used for immunoblotting assay. Caspase 3 proteins, both cleaved and uncleaved, were analyzed semiquantitatively. Data were from ImageJ readouts. (A). DPI (10 µm) was used as the inhibitor in the experiments. (B). Apocynin (2.5 mm) was used as the inhibitor. GraphPad Prism 4 (t-test) was used in analysis. n.s. = no significance. The number of independent experiments was given as n in the plots. 109

115 Table 1 Primers used for RT-PCR detection of related genes expressed in rat cochlea explants mrna Primer sequence Product Tm size NOX1 F : 5 CTT CCT CAC TGG CTG GGA TA bp 60 C R: 5 CGA CAG CAT TTG CGC AGG CT 3 NOX2 F: 5 TGA CTC GGT TGG CTG GCA TC bp 60 C R: 5 CGC AAA GGT ACA GGA ACA TGG G 3 NOX3 F: 5 CGT GCC CTG TAC CTC AAT TT bp 60 C R: 5 AAC AGA TCG GCA AAC CAC TC 3 NOX4 F: 5 GTT AAA CAC CTC TGT CTG CTT G bp 60 C R: 5 CAC CTG TCA GGC CCG GAA CA 3 p22 phox F: 5 GCT CAT CTG TCT GCT GGA GTA bp 59 C R: 5 ACG ACC TCA TCT GTA ACT GGA 3 p47 phox F: 5 TCA CCG AGA TCT ACG AGT TC bp, 62 C R: 5 ATC CCA TGA GGC TGT TGA AGT 3 p67 phox F: 5 GAA AGC ATG AAG GAT GCC TGG 3 R: 5 ATA GCA CCA AGA TCA CAT CTC C bp 60 C NOXO1 F: 5 TTG ACA CGT CGG GGA CAT AC bp 62 C R: 5 CTG AGC CTC CAG GCT ATG AAT 3 NOXA1 F: 5 GAC TCA CTG CAG AGT GTA TGG 3 R: 5 TAC AAA GCA CTT GGG GAA AAT ACC bp 60 C 110

116 Research article 3 Glucose-dependent ROS generation is NOX2-dependent and negatively regulates insulin secretion Ning LI a#, Bin LI b#, Thierry BRUN a, Olivier BASSET b, Pierre MAECHLER a*, Karl-Heinz KRAUSE b* a Department of Cell Physiology and Metabolism, University Medical Centre, rue Michel-Servet 1, 1211 Geneva 4, Switzerland. b Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Rue Michel-Servet 1, 1211 Geneva-4, Switzerland #. The two authors contributed equally in the research. * Correspondent authors: Pierre Maechler, Ph.D., Department of Cell Physiology and Metabolism, University Medical Centre, rue Michel-Servet 1, 1211 Geneva 4, Switzerland. Phone: ; Fax: ; K.-H. Krause, M.D., Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Rue Michel-Servet 1, 1211 Geneva-4, Switzerland. Phone: ; Fax: ; Keys Words: NADPH oxidase, NOX, ROS, pancreatic islets, insulin resistance, diabetes, NOX deficiency, mouse, human Word Count: 225 Figures: 5 111

117 Abstract Objective: To study NOX expression in mouse pancreatic islets and understand its role in glucose-stimulated ROS generation and insulin secretion. Research design and methods: After establishing the NOX expression profile in islets of wild type mice (RT-PCR), ROS generation and insulin secretion were investigated in isolated NOX1-, NOX2- and NOX4-deficient pancreatic islets. In vitro, glucose-stimulated insulin secretion was tested. In vivo, fasting glucose and insulin were measured and IpGTT test was performed. Results: In wild type islets, NOX1, NOX2, and NOX4 mrna were detected, with NOX2 appearing to be most heavily expressed. Glucose stimulated ROS generation was completely abolished in islets from NOX2-deficient mice. Islets from NOX2-deficient mice appeared morphologically normal and contained a normal number of β cells. However, insulin secretion from NOX2-deficient islets was markedly increased. This is unexpected and novel, as previous studies using the non-specific inhibitor DPI had suggested a positive regulation of insulin secretion by NOX-derived ROS. In vivo studies showed a normal fasting glucose, but increase fasting insulin levels in NOX2-deficient mice. The IpGTT test suggested the presence of mild glucose intolerance accompanied by a slight increase in plasma insulin levels, indicative of a moderate insulin resistance. Conclusions: NOX2 is the main source of glucose-stimulated ROS generation in mouse islets. NOX2-derived ROS provide a negative signal for insulin secretion, potentially important for insulin homeostasis and avoidance of insulin resistance. 112

118 Introduction Diabetes is one of the most serious challenges to human health. Juvenile-onset or Type 1 diabetes mellitus (T1DM) accounts for approximately 5 10% of individuals with diabetes. It is arising from autoimmune destruction of pancreatic β-cells and leads to insulin deficiency. Adult-onset or type 2 diabetes mellitus (T2DM), arising from defects in insulin sensitivity (insulin resistance) and relative insulin deficiency, accounts for 90 95% of those with diabetes [1]. T2DM patients have an increased risk in developing cardiovascular complications. With progress of the disease, severe complications occur, including blindness, end-stage renal failure, and atherosclerosis [2,3]. Hyperglycemia, resulting from insulin resistance and/or secretion, is the characteristic sign of the disease[1], and its chronic form is associated with endothelial dysfunction [4,5]. An increasing body of evidence suggests that elevated reactive oxygen species (ROS) produced by NADPH oxidase (NOX) contribute to various diseases including endothelial dysfunction, atherosclerosis, hypertension, tumor genesis [6] and diabetes as reviewed in [7]. To date, seven NOX isoforms are known, namely NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Different species have their own profile of NOX isoforms. In human, all the NOX isoforms are detected respectively with tissue specificity. In rodents, NOX5 does not exist. NOX expression is tissue specific. NOX1 is abundantly found in colon. NOX2 is found in for example in phagocytes and smooth muscles cells. NOX3 exists predominantly in inner ear and was hardly detected in other tissue. NOX4 is the most widely expressed isoform. It is abundant in kidney. DUOX1 and DUOX2 mainly exist in thyroid gland. NADPH oxidase (NOX) 113

119 family interferes with life process in many aspects physiologically and pathologically, mainly through producing various reactive oxygen species [8]. In the pancreas, oxidative stress causes micro- / macro-vascular complications, β-cell damage and impairment of insulin secretion [9-12]. However the source of the ROS in β-cells might comes from at least two proposed candidates, i) leakage from the mitochondrial electron transport chain [13,14], and ii) superoxide-producing enzymes including NOX [9,15]. To date, little is known about NOX expression profile in mouse and human pancreatic islets. At function level, NADPH oxidase has been suggested as an important factor in affecting insulin secretion [16-18]. Glucose-stimulated insulin secretion was suppressed by non-specific NOX inhibitor DPI [19]. Therefore, it is considered valuable to investigate further [18]. In the current study, we show that NOX1, NOX2, and NOX4 were detected in isolated pancreatic islets derived from mouse. In human pancreatic islets, NOX2, NOX4, and NOX5 were detected. Our data tend to demonstrate that NOX2 deficiency affected ROS production and glucose-stimulated insulin secretion (GSIS). Data also revealed a negative correlation between ROS generation and GSIS. Immunofluorescence and in vivo experiments including fasting plasma insulin (FPI), fasting plasma glucose (FPG) measurement, and intra-peritoneal glucose tolerance test (IpGTT), demonstrated that NOX2-deficient mice developed insulin resistance. Research design and Methods In accordance with Swiss legal requirements, all animal experiments were approved by an academic committee and supervised by the local veterinary agency. Animals 114

120 Female C57BL/6 mice (Charles River, L Arbresle, France), NOX1-deficient, NOX2-deficient and NOX4-deficient (in C57BL/6 background) mice were kept in specific pathogen free (SPF) environment. All the mice used in the experiment were 2 to 6 months of age. Pancreatic islets isolation Pancreatic islets isolation approach in brief was as follows: in anesthetized animal, in situ digestion was performed with collagenase (0.5 mg/ml). Pancreas was transferred into a falcon tube for external digestion with collagenase (0.6 mg/ml). Digested pancreas was centrifuged several times, the supernatant was removed, and the isolated pancreatic islets were picked up under binocular. Isolated pancreatic islets were cultured in RPMI1640 medium (Sigma, St. Louis, MO) containing 11 mm glucose, 10% fetal bovine serum (FBS) (Gibco BRL, Life Technologies, Inc., Grand island, NY), 100 U/ml penicillin, and 100 µg /ml streptomycin. RT-PCR Total RNA was isolated by RNeasy mini kit (Qiagen) according to the manufacturer s instruction. Reverse transcription of RNA was performed using the superscript II kit (Invitrogen) according to manufacturer s instruction. PCR was performed using AccuPrime TM Taq DNA Polymerase (Invitrogen). Amplification of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control. Water was used for negative control; for primers see table 1. In mice, colon was used as positive control for NOX1, NOXO1, NOXA1 and p22 phox ; Spleen was used for NOX2, p47 phox, p67 phox and p40 phox. Inner ear was used for NOX3 and kidney was used for NOX4. For human tissue, Caco2 cells (a cell line 115

121 from human carcinoma in colon) were used as positive control for NOX1; blood for NOX2; HEK cells transduced with human NOX3 gene for NOX3; HMC3 (human microglia cell line) for NOX4 [20]; and HEK cells transduced with human NOX5 gene for NOX5 [21]. Combination measurements of glucose stimulated ROS generation detected by NBT reduction (GSRNR) and GSIS: After overnight recovery in culture medium containing 11 mm glucose, explanted pancreatic islets were used in GSRNR and GSIS measurements. Pancreatic islets samples (15 islets per sample) were incubated with HBSS containing 0.5 mg / ml NBT (Sigma-Aldrich) with 2.8 / 22.8 mm glucose for two hours. At the end of incubation, supernatant from each sample was used for insulin measurement (ELISA) according to the manufacturer s instruction of the kit (Mercodia AB, Uppsala, Sweden). For GSRNR measurement, each sample was homogenized in 42 µl 2M NaOH solution followed by addition of 49 µl 100% DMSO to dissolve reduced NBT. Mixture was transferred into 96-well-plate and absorption was measured by WALLAC Victor 1520 (λ= 630 nm). Immunofluorescence Pancreases from wild type C57BL/6 and NOX2-deficient mice were processed successively with fixation (4% paraformaldehyde), dehydration, and paraffin embedding. Embedded pancreas was sliced into sections of 7µm thickness and with 50µm interval between adjacent two. Sections were incubated for one hour with both antibodies, i.e., anti-insulin (Sigma, mouse monoclonal; dilution: 1/500) and anti-glucagon (Dako, rabbit 116

122 polyclonal, dilution 1/100), Then, sections were rinsed to clear redundant first antibodies. Alexa 488- (donkey anti-mouse, Molecular Probes) and Alexa 555- (donkey anti-rabbit, Molecular Probes) conjugated antibodies (dilution 1/2000) were used to reveal specific staining. DAPI (300 nm) was used to stain the nuclei. Sections were mounted in Dako mounting medium. Image analysis: Images of pancreatic sections were taken under the same condition. Insulin (labeled with Alexa 488, green) and glucagon (labeled with Alexa 555, red) signals were calculated respectively by ImageJ software. The area of target signals was used for analysis. Fasting plasma insulin (FPI), fasting plasma glucose (FPG) measurements and intraperitoneal glucose tolerance test (IpGTT): Food was withdrawn overnight before experiment. Sterilized glucose solution (22.5%; g/ml) was injected intra-peritoneally into the mouse at 3 g glucose / kg body weight. Blood was sampled from mouse tail. Blood glucose was detected at time points 0, 15, 30, 60 and 120 minutes after glucose injection. The number of independent tests and the number of mice used were mentioned in related figure legends. Statistical analysis All the experiments were done at least three times independently as indicated in the figure legends. Analysis was processed by SPSS (ver 13.0). In all tests, a p value 117

123 inferior to 0.05 was taken as statistically significant. Results are shown in the figures as means ± standard error of means (SEM). T-test was used to compare the means between groups. Boxplot model was used to demonstrate the median (the heavy black line), the 25 th and 75 th percentiles (the lower and higher box boundaries, mark the 25 th and 75 th percentiles of each distribution, respectively.), the minimum and maximum observed values that are not statistically outlying. The mean insulin (area) percentages were mentioned only in legends. Outliers were marked as tiny circles. Extremes were marked as stars. All the data were included in statistic analysis. Results NOX2 appeared to be the dominant NADPH oxidase isoform in mouse pancreatic islet; similarly in human. RT-PCR demonstrated that, in mouse pancreatic islets, NOX1, NOX2, and NOX4 existed at mrna level (Fig. 1a upper panel). NOX subunits NOXO1, p47 phox, NOXA1, p67 phox, p22 phox, and p40 phox were all detected (Fig. 1b). NOX3 was not detected. DUOX 1 and DUOX 2 were hardly detected (Fig.1a lower panel). NOX2 expression appeared dominant, since the strengths of NOX2 band (PCR products) were comparable between from pancreatic islets and from spleen cdna, which was used as NOX2 positive control. NOX2, NOX4, and NOX5 were detected in human pancreatic islets. Regardless of NOX5, which is absent in rodents, the shared profile of NOX isoform expressed in pancreatic islet of human and mouse were NOX2 and NOX4. 118

124 Pancreatic islets from NOX2-deficient mice did not exhibit ROS generation when stimulated with high (22.8 mm) glucose. However, under the same condition, insulin secretion was increased. The amplitude of the glucose (22.8 mm)-induced ROS generation in NOX1-deficient and NOX4-deficient mouse pancreatic islets (Fig. 2a) was the same than the one observed in wild type cells (Fig. 2a). However, 22.8 mm glucose did not stimulate ROS generation in the islets from NOX2-deficient mice (Fig. 2a). Compared with wild type mouse, insulin secretion in NOX2-deficient mice was elevated in the presence of 22.8 mm glucose (Fig. 2b). Multiple group comparisons among wild type, NOX1-deficient, NOX2-deficient, and NOX4-deficient mice demonstrated that insulin secretion activity in NOX2-deficient mouse pancreatic islets was enhanced in high glucose condition (Fig. 2b). Under 22.8 mm glucose stimulation, insulin secretion and ROS generation data showed a negative correlation between the two factors (Fig. 2c; R= ; p<0.05). Combination of GSRNR and GSIS measurements in the same set of samples revealed the negative correlation between ROS generation and insulin secretion under glucose stimulation. GSIS in NOX2-deficient islets was enhanced --- confirmed through analyzation by using either folds or absolute value, and in both conditions: with or without NBT. 1. By using insulin absolute values directly in analysis as shown in Fig. 3a: without NBT incubation: insulin level was higher in NOX2-deficient 22.8 mm group than in wild type 22.8mM (p<0.05); with NBT incubation: insulin secretion level was higher in NBT NOX2-deficient 22.8 mm group than in NBT wild type 119

125 22.8mM (p<0.005). By using insulin relative ratio, the folds of insulin level at 22.8 mm glucose condition to that at 2.8 mm glucose condition as shown in Fig. 3b: without NBT incubation: the folds of insulin secretion was greater in group NOX2-deficient 22.8 mm than in group wild type 22.8mM (p<0.001); with NBT incubation: insulin secretion fold did not show increase in NBT NOX2-deficient 22.8 mm group compared with NBT wild type 22.8mM (p=0.057, analyzed by t-test). 2. Introduction of NBT into the measurement did not change the main message in comparison between NOX2-deficient and wild type under 22.8 mm glucose stimulation condition, even though it decreased all the measured values in general no matter using absolute vale or folds in analyzing as shown in Fig. 3a and 3b. The glucagon signal (area) of pancreatic islets decreased in NOX2-deficient compared with wild type mice. Wild type and NOX2-deficient pancreatic islets were analyzed in immunofluorescence images (Fig. 4a to 4d). The parameter used in analysis was the percentage of target signal area in one islet to the total area of the islet. The mean value of glucagon was lower in NOX2-deficient (9.7%) than in wild type (14.8%) (Fig. 4e). Statistic analysis of insulin mean value showed no difference between wild type (75.0%) and NOX2-deficient (77.3%) (Fig. 4e). Statistic analysis showed no difference between the two genotypes in islet size and their variances (data not shown), neither in insulin signals. Glucagon was a bit lower in NOX2-/- than in wild type mice. NOX2-deficient mice showed higher FPI, normal FPG, and impaired glucose tolerance. 120

126 Data demonstrated that FPI was higher in NOX2-deficient than in wild type mice (Fig. 5a; p<0.05). There was not a difference in FPG between NOX2-deficient (Fig. 5a legend; 4.96 mm) and wild type (Fig.5a legend; 4.13 mm). Analysis of the data from ipgtt in six wild type and five NOX2-deficient mice, demonstrated an impaired glucose tolerance in NOX2-deficient mice compared with wild type. In NOX2-deficient mice, the glucose level at all the post-injection checkpoints were elevated (Fig. 5b). DPI inhibition effect on glucose-stimulated insulin secretion is probably through a pathway other than NOX. The non-specific electron transporter inhibitor DPI (10 µm) inhibited high glucose-induced insulin secretion in wild type mouse islets and all the three kinds of NOX deficiency mice with no difference (Fig.6). Discussion Our study demonstrates a high-level expression of NOX2 in mouse (and human) pancreatic islets. Islets from NOX2-deficient mice do not release ROS in response to glucose stimulation demonstrating that NOX2 is a predominant source of glucose-stimulated ROS. We demonstrate that NOX2-derived ROS provide a negative signal for insulin secretion, both in vitro and in vivo. Thus, NOX2 appears to be a novel player in insulin homeostasis, providing a physiologically relevant feed-back inhibition. NOX2 is also called the phagocyte NADPH oxidase and classically thought to be expressed in phagocytes, such as neutrophils, macrophages, and microglia. The robust expression of NOX2 in pancreatic islets raises the question whether this is due to 121

127 blood cells contamination or reflects expression of NOX2 in resident cells of the pancreas, such as endocrine pancreatic cells or pancreatic stellate cells. Given the relatively low abundance of blood cells and stellate cells in the purified preparations of pancreatic islets used in our study, we think that endocrine pancreatic cells, in particular β cells are most likely the cells expressing NOX2. The functional observation that the presence of NOX2 dampens insulin release from β cells also argues in favour of this proposed localization. In addition to NOX2, there is also NOX1 and NOX4 expression in mouse islets. NOX1-deficient islets show a trend towards decreased ROS generation and increased insulin release, but this is not statistically significant. NOX4-deficient islets do not behave differently from wild-type. Thus it is tempting to speculate that the cellular and/or the subcellular localization of NOX2 favoring inhibition of insulin secretion. Indeed, NOX4 is highly expressed in the vascular system and hence its presence in islets preparations might reflect simply their vascularization. Classically it has been suggested that glucose-induced ROS generation is due to metabolic activation of mitochondria and ROS generation by the mitochondrial oxidase. Our data provides a strong challenge to this view. Indeed, in the absence of NOX2, no glucose-stimulated ROS generation could be observed. Thus, NOX2 appears to be the primary source of glucose-stimulated ROS generation in pancreatic islets. It is however possible that mitochondrial damage through NOX2-derived ROS leads to a secondary ROS generation by mitochondria. What are the mechanisms of glucose-dependent NOX2 activation? A key step in the activation of NOX2 is the phosphorylation of the NOX2 regulatory subunit p47 phox by protein kinase C [22]. As glucose has been demonstrated to activate protein 122

128 kinase C [23,24], it is tempting to speculate that this is the underlying mechanism, however further studies will be necessary to prove the point. Our observation that NOX2 inhibits insulin secretion comes as a surprise. Indeed, based on the inhibition of insulin secretion by the non-selective electron transport inhibitors DPI, there are several studies that suggest that NOX enzymes stimulate insulin secretion. To better understand whether DPI inhibits insulin secretion through inhibition of NOX enzymes, we have added DPI to NOX-deficient islets. The compound is still a powerful inhibitor of insulin secretion, demonstrating that a DPI target unrelated to NOX enzymes mediates the inhibitor effects. For example, DPI inhibition of the mitochondrial oxidase [19] might account for the DPI effects. The inhibition of insulin secretion by NOX2 suggests a redox-sensitive negative feed-back mechanism in insulin homeostasis. The best studied redox-sensitive signaling pathways are MAP kinase activation, tyrosine phosphatase inhibition, PTEN inhibition leading to increased PI3-kinase activity, as well as alteration of ion channels (in particular Ca 2+ and K + ). Inhibition of Ca 2+ channels or activation of K + channels might provide such a negative feed-back. Another interesting possibility is NOX2-dependent inhibition of insulin secretion through metabolic coupling. Indeed, NOX2, being an NADPH oxidase, may lead to a significant decrease in cellular NADPH levels, while NADPH is an important metabolic factor required for insulin secretion. To which extent, can our results be extrapolated to humans? To our knowledge, insulin levels and glucose tolerance has not been in studies in human CGD patients (i.e. human NOX2-deficiency). Thus, such in-vivo studies will be necessary. However the expression profile of NOX enzymes in the human pancreatic islets (Fig. 1) suggests that NOX2 is also a predominant NOX isoforms. There is however an 123

129 interesting difference concerning NOX5. Indeed, the NOX5 gene is absent in rodents. However we observed expression of NOX5 in human islets. Thus, redox-sensitivity of insulin secretion might potentially be more complex in humans than in mice. In summary, we demonstrate a novel role of the ROS-generating enzyme NOX2, namely participation in insulin homeostasis through a negative feed-back mechanism. This negative feed-back might be due to redox-sensitive inhibitory signaling or through NADPH-dependent metabolic coupling. NOX2-dependent inhibition of insulin secretion provides a potentially interesting target for therapies directed towards enhancing insulin secretion. Acknowledgements: The authors would like to thank Dr. Michel Dubois-Dauphin for his helpful discussion about the project and Mr. Asllan GJINOVCI (Department of cell physiology and metabolism, C.M.U. Geneva University.) for his kind help in animal dissection and pancreas extraction. We also thank Christine Deffert and Olivier Plastre for their kind help in arrangement for the usage in NOX deficiency mice. 124

130 a Figure 1 cdna mouse pancreatic islets colon spleen inner ear kidney NOX1 NOX2 NOX3 NOX4 GAPDH NOX1 NOX2 NOX3 NOX4 cycle cdna m. islets thyroid DUOX1 DUOX2 D1 D2 marker (bp) cycle b cdna mouse pancreatic islets positive controls NOXA1 p67 phox NOXO1 p47 phox p40 phox p22 phox A1 67 O cycle c cdna human pancreatic islets positive controls NOX1 NOX2 NOX3 NOX4 NOX5 GAPDH N1 N2 N3 N4 N5 cycle

131 FIG. 1. RT-PCR detection of NOX enzymes in wild type mouse and human pancreatic islets. A: NOX isoforms in mouse pancreatic islets. The designed PCR product lengths were as following (in parentheses): mnox1 (300bp), mnox2 (652bp), mnox3 (769bp), mnox4 (311bp), mduox1 (558bp), mduox2 (586bp) and GAPDH (300bp). The same amount of cdna was used to detect NOX isoforms (upper panel): NOX1, NOX2, NOX3, and NOX4. GAPDH was used as a common control. Specific positive controls were displayed at right side of the panel. DUOX1 and DUOX2 were tested (lower panel). D1 and D2 designated DUOX1 and DUOX2 respectively. B: Related NOX subunits in mouse pancreatic islet. The designed PCR product lengths were as following: mnoxa1 (393bp), mp67 phox (698bp), mnoxo1 (336bp), mp47 phox (438bp), mp22 phox (430bp), and mp40 phox (793bp). The letters A1, 67, O1, 47, 22, and 40 (under positive controls in the panel) designated NOXA1, p67 phox, NOXO1, p47 phox, p22 phox and p40 phox respectively. C: NOX isoform expression in human pancreatic islets. The designed PCR product lengths were in parentheses as following: human NOX1 (353bp), NOX2 (475bp), NOX3 (432 bp), NOX4 (417 bp), NOX5 (535 bp) and GAPDH (302 bp). The positive controls: N1 to N5 designated as NOX1 to NOX5. These control cdna samples were from Caco 2 cells (for NOX1), human blood (for NOX2), HEK-NOX3 expression cell line, and human microglia cell line clone 3 (HMC3) and HEK-NOX5 transgenic cell line. The length of Marker molecular reference was noted at the end of the gel image shown in A (lower panel). All PCR were performed (for islets samples) at 35 and 40 cycles. H 2 O 2 instead of cdna used as negative control (not shown). The letter m before each NOX and subunits designated mouse. bp = base pair. 126

132 Figure 2 a 2.8 mm glucose 22.8 mm glucose b c NBT reduction (folds to 2.8mM glucose condition) Insulin secretion (folds to 2.8mMglucose condition) p<0.001 wild type p<0.05 p<0.05 NOX1-/- p<0.01 p<0.05 n.s. NOX2-/- p<0.01 p<0.005 NOX4-/- wild type NOX1-/- NOX2-/- NOX4-/- Insulin secretion (fodes to 2.8 mm glucose condition) NOX2-deficient NOX1-deficient wild type NOX4-deficient R = p < ROS generation (folds to 2.8 mm glucose condition) 127

133 FIG. 2. Combination measurements of ROS generation and insulin secretion Mouse pancreatic islets were incubated in HBSS with 2.8 or 22.8 mm glucose for two hours. Islets were used for ROS detection by NBT reduction assay and the supernatant was used to measure insulin secretion. A: 22.8 mm glucose-stimulated ROS generation. Data were normalized with the NBT signals of 2.8 mm glucose samples. B: Insulin secretion under 22.8 mm glucose stimulation. Data normalization was the same way as in NBT assay. The p values were calculated by SPSS, using one way ANOVA multiple group comparison (Post Hoc. LSD). C: Correlation analysis between ROS generation and insulin secretion. Data were from five independent experiments, except for the NOX2-deficient group in which four instead of five. The samples numbers for each condition (2.8 mm, 22.8 mm) in NBT reduction: wild (32, 32), NOX1-/- (37, 37), NOX2-/- (29, 29) and NOX4-/- (30, 30); in insulin secretion: wild (32, 32), NOX1-/- (36, 36), NOX2-/- (29, 29) and NOX4-/- (30, 29). Error bar = SEM. n.s. = not significant. 128

134 a Figure3 p<0.01 Insulin ng/islet p<0.05 p<0.05 p<0.005 p<0.05 n.s. b (n=30) (n=30) (n=28) (n=28) (n=32) (n=32) (n=29) (n=29) Insulin secretion (folds to 2.8mM glucose condition) p<0.001 p<0.001 p<0.001 p=0.057 (n=30) (n=30) (n=28) (n=28) (n=32) (n=32) (n=29) (n=29) 129

135 FIG.3. Insulin secretion in response to 22.8 mm glucose stimulation: comparisons between using insulin absolute values (ng/islet) and normalization by means at 2.8mM glucose state (folds); between without NBT and with NBT. Mouse islets after overnight culture in medium with 11 mm glucose were incubated with HBSS with 2.8 or 22.8 mm glucose for two hours. Supernatant was used to measure insulin level. Readouts (insulin, ng/islet) were used directly in analysis (A) or through normalization into folds to the mean values of each sample at 2.8 mm glucose within group (B). NBT was marked before the group name for NBT-treated samples. A: Insulin level (ng / islet) comparisons between groups. The mean values (ng /islet) of each group (not marked in the plot) were as following (the name of group was in parenthesis): 0.11 (wild type 2.8mM), 0.48 (wild type 22.8 mm), 0.06 (NOX2-deficient 2.8 mm), 1.06 (NOX2-deficient 22.8mM), 0.12 (NBT wild type 2.8 mm), 0.32 (NBT wild type 22.8 mm), 0.12 (NBT NOX2-deficient 2.8 mm), and 0.50 (NBT NOX2-deficient 22.8 mm). The data of NBT groups were those from former experiments. B: Insulin secretion (folds to 2.8 mm glucose condition) comparisons between groups. The means of folds for each group were as following (the name of group was in parenthesis): 6.22 (wild type 22.8 mm), (NOX2-deficient 22.8mM), 3.47 (NBT wild type 22.8 mm), and 4.52 (NBT NOX2-deficient 22.8 mm). T-test was used in all the comparisons. Data for groups without NBT were from three independent experiments; those with NBT: five for wild type; and four for NOX2-deficient. The number of n was the total sample number from each group. Boxplot model demonstrated the median, the 25 th and 75 th percentiles, the minimum and maximum observed values that are not statistically outlying. Outliers were marked as tiny circles. Extremes were marked as stars. -/-, deficient; n.s. = not significant. 130

136 Figure 4 a wild b wild c NOX2-/- d NOX2-/- e n.s. % of islet (area) p< wild type NOX2-deficient wild type NOX2-deficient (n=180) (n=105) (n=180) (n=105) insulin glucagon 131

137 FIG. 4. Insulin and glucagon immunolabeling in pancreas sections of wild type and NOX2-deficient mice. Pancreas samples were obtained from wild type and NOX2-deficient mice after food withdrawing overnight. A to D: Representative pictures of pancreatic islet from wild type (A, B) and NOX2-deficient (C,D) animals. Insulin was stained in green and glucagon in red. DAPI was used to stain the nuclei. B and D are higher magnification of A and C respectively. bar = 50 µm. E: ImageJ was used to quantify target signals (area of insulin and glucagon respectively) in the islet. SPSS (t-test) was used to compare the means between wild type and NOX2-deficient groups. Boxplot demonstrated the results. The mean insulin percentages were 75.0% in wild type and 77.3% in NOX2-deficient genotype, and both means were lower than their medians 77.2% and 80.9% respectively (not shown by Boxplot). The mean glucagon percentages were 14.8% in wild type, and 9.7% in NOX2-deficient genotype, and both means were higher than their medians 13.7% and 8.7% respectively (not marked either in Boxplot). Outliers were marked as tiny circles. Extremes were marked as stars. n.s. = not significant. 132

138 a FPI (µu/ml) Figure 5 values only (units marked in plot) FPG (mm) (n.s.) (p<0.05) n=7 n=5 n=7 n=5 wild type NOX2-deficient wild type NOX2-deficient b wild type mice (n=6) NOX2-deficient mice (n=5) * * * p< 0.05 glucose concentration (mm) * * min. 133

139 FIG.5. Insulin resistance analysis: fasting insulin, fasting glucose measurements and IpGTT in mice. A: After overnight food-withdraw, mice were used to detect fasting plasma glucose (FPG) and fasting insulin level (FPI). Four independent experiments were performed. Means were compared. p values were calculated through t-test. The mean values for the groups (not shown by the Boxplot) were the following: 4.13 mm (wild type, FPG), 4.96 mm (NOX2-deficient, FPG), 8.01 µu/ml (wild type, FPI) and µu/ml (NOX2-deficient, FPI). The number of n marked the number of mice used in each group from five independent experiment in wild type mice and four independent experiments in NOX2-deficient mice. B: IpGTT in wild type and NOX2-deficient mice. Glucose (peritoneal) injection amount was 3 g glucose per kilogram body weight. Blood glucose was measured at 0, 15, 30, 60, and 120 minutes after injection. Data were the collection of six wild type mice and five NOX2-deficient mice from five independent experiments. T-test was used in comparison of means for each time point. n.s. = not significant. 134

140 Figure 6 S.E. n.s. n.s. n.s. wild type NOX1-deficient NOX2-deficient NOX4-deficient (n=25) (n=24) (n=17) (n=22) 135

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