Pancreatic Islet Cell Death

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 360 MEKK-1 and NF-κB Signaling in Pancreatic Islet Cell Death DARIUSH MOKHTARI ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2008 ISSN 1651-6206 ISBN 978-91-554-7222-1 urn:nbn:se:uu:diva-8896

Till Manfred

List of Papers This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I Hägerkvist R*, Mokhtari D*, Myers JW, Tengholm A, Welsh N. sirna produced by recombinant Dicer mediates efficient gene silencing in islet cells. Ann NY Acad Sci 2005;1040:114-22 II Mokhtari D, Myers JW, Welsh N. The MAPK kinase kinase-1 is essential for stress-induced pancreatic islet cell death. Endocrinology. In Press III Mokhtari D, Myers JW, Welsh N. The MAPK kinase kinase-1 is essential for cytokine-induced JNK and NF- B activation in human pancreatic islet cells. Diabetes. In Press IV Mokhtari D, Barbu A, Mehmeti I, Vercamer C, Welsh N. Overexpression of the NF- B subunit c-rel protects against islet cell death in vitro. Manuscript *Shared contribution as first author Reprints were made with permission from the publishers.

Contents Introduction...11 Background...13 Diabetes Mellitus...13 The islets of Langerhans...13 Type 1 diabetes: a combination of genes and environment...14 Pathology and pathogenesis of Type 1 diabetes...15 Mediators of -cell death in Type 1 diabetes...16 Pro-inflammatory cytokines...16 NOS and NO...17 NF- B...17 What is the role of NF- B in pancreatic islets?...19 MAPKs...19 The ERKs...20 The JNKs...21 The p38s...21 MAPKs in islet stress signaling...21 MEKK-1...22 RNA interference and dicer-generated sirna...24 Methodology...25 Cell culture...25 Islet isolation and culture...25 Plasmids...25 Liposome-mediated transfection of cell lines...25 Cell sorting...26 Liposome-mediated transfection of free islet cells with d-sirna...26 RNA isolation and cdna synthesis...26 Real time PCR...26 Evaluation of cell viability...27 Immunoblotting...27 Immunoprecipitation...27 Preparation of cytoplasmic and nuclear fractions....27 Confocal microscopy...28

Adenoviruses...28 In vitro transduction procedure...28 Extraction of nuclear proteins and electrophoretic mobility shift assay (EMSA)...29 Pancreas perfusion transduction...29 Results and discussion...30 Paper I...30 Paper II...31 Paper III...32 Paper IV...34 Conclusions...36 Paper I...36 Paper II...36 Paper III...36 Paper IV...36 Concluding remarks...37 Acknowledgements...39 References...40

Abbreviations ASK1 Apoptosis signal-regulating kinase ATF2 Activating transcription factor 2 ATP Adenosine triphosphate Bcl-2 B cell lymphoma associated protein-2 BSA Bovine serum albumin c-abl Cellular Abelson tyrosine kinase Cd42 Cell division cycle 42 cdna Complementary DNA c-iap-2 Cellular inhibitor of apoptosis protein-2 CMV Cytomegalovirus DETA/NO DETA/NONOate DMEM Dulbeccos modified eagles medium d-sirna Dicer-generated sirna dsrna Double stranded RNA EMSA Electrophoretic mobility shift assay ER Endoplasmic reticulum ERK Extracellular signal regulated kinase FACS Fluorescent activated cell sorter FCS Fetal calf serum FITC Fluorescein isothiocyanate GAPDH Glyceraldehyde 3-phosphate dehydrogenase GFP Green fluorescent protein IFN- IFN-gamma I B Inhibitory kappa B I K Inhibitory kappa kinase IL-1 Interleukin-1beta inos Inducible nitric oxide synthase JNK c-jun NH 2 -terminal kinase KRBH Krebs ringer bicarbonate HEPES MAPK Mitogen activated protein kinase MAP2K Mitogen activated protein kinase kinase MAP3K Mitogen activated protein kinase kinase kinase MEK MAP/ERK kinase MEKK MAP/ERK kinase kinase MEKK-1 MAP/ERK kinase kinase-1 MHC Major histocompatibility complex

MKK MAPK kinase MKKK MAPK kinase kinase MLDSTZ Multiple low dose streptozotocin MLK Mixed lineage kinase mrna Messenger RNA NF- B Nuclear factor kappa B NO Nitric oxide NOD Non-obese diabetic PBS Phosphate buffered saline PCR Polymerase chain reaction PFU Plaque forming units Rac Ras-related c3 botulinum toxin substrate Raf Rapidly accelerated fibrosarcoma RIP Receptor interacting protein RISC RNA induced silencing complex RNAi RNA interference ROS Reactive oxygen species RPMI Roswell park memorial institute rps3 Ribosomal protein S3 SDS Sodium dodecyl sulfate sirna Small interfering RNA STZ Streptozotocin TAK1 Transforming growth factor- -activated kinase 1 TNF- Tumor necrosis factor-alpha Tpl Tumor progression locus TRAF TNF receptor associated factor wt Wild type

Introduction Type 1 diabetes is a metabolic disease resulting from the destruction of the insulin-producing pancreatic -cells, located within the islets of Langerhans [1]. Although efforts to clarify the underlying mechanisms for -cell death have generated valuable understanding, the knowledge of the exact processes involved in the initiation and progression of -cell destruction still remain incomplete. Strong evidence points towards the early islet infiltration of immune cells as being important in the disease process. Upon activation, the immune cells produce and release toxic mediators such as proinflammatory cytokines and reactive oxygen species (ROS), believed to facilitate the dysfunction and death of the -cells [2]. Stimulation of insulin-producing cell lines and primary islets with proinflammatory cytokines in vitro leads to -cell death [3]. This response is in part, at least in rodent islets, mediated by the nuclear factor kappa B (NF- B)-inducible nitric oxide synthase (inos)-nitric oxide (NO) signaling pathway [4]. In human islets however, which seem to be less sensitive to the deleterious effects of NO [5], the NF- B-iNOS-NO signaling cascade may be of lesser importance. This discrepancy has led to a controversy over the role of NF- B in islets. Indeed there have been reports favoring both proand anti-apoptotic roles for NF- B in islets [6-8]. Therefore, the question about the exact role of NF- B in -cells remains unanswered. In addition to NF- B, the intracellular signaling pathways activated in islets by pro-inflammatory cytokines and ROS, have been shown to involve the mitogen activated protein kinases (MAPKs). In islets, MAPKs are rapidly activated by pro-inflammatory cytokines, NO-donors and hydrogen peroxide (H 2 O 2 ) [9-11]. It has been reported that activation of the MAPKs c- Jun N terminal kinase (JNK) and p38 leads to -cell death in vitro, in response to pro-inflammatory cytokines while inhibition of the same kinases protects -cells [10,12-15]. However, the upstream signaling events leading to MAPK activation in -cells are not well known. The work presented in this thesis therefore aims at characterizing the role of stress-induced MAPK and NF- B signaling in islets, with emphasis 11

on the role of the MAPK activator MAP/ERK kinase kinase-1 (MEKK-1) in islet cell death. Hopefully, the information gained in the present studies will increase the understanding of the signaling pathways important in the pathogenesis of Type 1 diabetes. The specific aims of this thesis were: to evaluate the suitability of dicer-generated sirna to silence endogenous gene expression in primary pancreatic islet cells. to characterize the role of MEKK-1 signaling in DETA/NONOate-, streptozotocin-, H 2 O 2 - or cytokine-induced human islet cell death. to study the expression and cytokine-induced translocation of NF- B subunits in human islet cells, and whether a gain of NF- B function by gene transfer affects islet cell survival in response to the toxins H 2 O 2, DETA /NONOate or streptozotocin. 12

Background Diabetes Mellitus The term Diabetes Mellitus is used to describe a variety of metabolic disorders characterized by elevated blood glucose levels. The hormone insulin, which is produced in the pancreatic -cells, plays a central role in Diabetes Mellitus. Insulin is a peptide hormone and the main regulator of glucose uptake in muscle, liver and fat cells. An insufficient production and/or response to insulin will therefore lead to hyperglycemia. The early symptoms of diabetes include increased urine production and thirst. However, even when treated, the disease often leads to more serious long time complications such as renal failure, nerve damage, cardiovascular disease and blindness. Diabetes can broadly be classified into two main types. Type 1 diabetes, which represents approximately 5-10 % of all cases of diabetes, is an autoimmune disease resulting in destruction of the insulin-producing -cells located in the pancreatic islets of Langerhans [1], and Type 2 which is estimated to represent 90-95 % of all cases, is due to -cell failure or various degrees of insulin resistance [16]. Type 1 diabetes usually has its onset before adulthood, whereas Type 2 most often develops in the elderly. Today, more than 230 million people have diabetes worldwide and by 2025 numbers are believed to reach 350 million [17]. Type 1 diabetes is today mostly treated by insulin injections. In severe cases, when insulin treatment fails to restore normo-glycemia, transplantation of islets into the liver has been shown to give promising results [18]. However, the shortage of donor islets today limits the use of islet transplantation as a treatment for Type 1 diabetes. The islets of Langerhans The islets of Langerhans are cell clusters scattered throughout the pancreas. The human pancreas contains about 1-2 million islets, each composed of about 2-3000 endocrine cells [19]. The islets consist of 5 major endocrine cell types [20]: 1) -cells, constituting 60-80 % of the islet cells. 2) -cells, constitute 10-20 % of the islet cells and produce glucagon, which promotes glycogen breakdown in the liver and thereby increases glucose concentra- 13

tions. 3) -cells, constitute 3-10 % of the islet cells and secrete somatostatin, which inhibits the release of other hormones including insulin and glucagon. 4) PP-cells, constituting 0,5-1% of the islets cells and secreting pancreatic polypeptide, which has been shown to stimulate gastric secretion and 5) ghrelin cells, constituting 0,5-1 % of the islets cells and producing the hormone ghrelin, regulating appetite and promoting the release of growth hormone. In addition of endocrine cells, the islets contain macrophages/dendritic cells, neurons, fibroblasts and endothelial cells. Histological sections of pancreata from deceased patients diagnosed with Type 1 diabetes, show a striking decrease in islet -cell number, whereas the number of, and PP-cells seem to be unaffected [21,22]. Type 1 diabetes: a combination of genes and environment Monozygotic twins show about a 50% concordance rate in developing Type 1 diabetes and the risk to a first degree relative is approximately 5 % [23]. The genetics of Type 1 diabetes, except for a few rare forms, cannot be classified according to a specific model of dominant or recessive inheritance of a specific set of genes [24]. Genes encoded by the major histocompatibility complex (MHC) have been shown to be the most important genes for the disease [25]. MHC functions as an antigen presenter for the T-lymphocytes and is thereby contributing to the activation of the immune response. The MHC locus accounts for approximately 50% of the familial aggregation and both susceptible and protective haplotypes have been defined [25]. In addition, a number of non-mhc genes, which contribute to the disease have been identified. However, the function of most of them are today unknown [26]. Since genetic factors are clearly insufficient in explaining the disease by themselves, there must also be environmental factors involved. The search for environmental factors has identified viral infections, toxins and early infant diet as possible events that could contribute to the precipitation of Type 1 diabetes [27,28]. The fact that the incidence of Type 1 diabetes has increased the last 50 years indicates that environmental factors are of importance for developing the disease. 14

Pathology and pathogenesis of Type 1 diabetes Patients diagnosed with Type 1 diabetes usually have lost the majority of their -cells and therefore their ability to maintain sufficient insulin production. However, the disease process seems to start years before the actual clinical outcome. During this long pre-clinical period, the patients usually do not display any prominent symptoms, although presence of autoantibodies to -cells and their antigens can be detected at an early stage [29]. The pathogenesis of Type 1 diabetes has been studied intensively, but there are still questions to answer about the exact mechanisms involved in the initiation and progression of -cell destruction. It is generally accepted that local inflammation, caused by infiltrating immune cells (insulitis), is a key event in the destruction of -cells. Histological stainings of pancreata from non-obese-diabetic (NOD) mice, an animal model for Type 1 diabetes, identified macrophages as being the first immune cells to invade the islets, followed by T-lymphocytes (CD4 + and CD8 + ) and B-lymphocytes [30]. Moreover, the immune cell infiltration preceded -cell destruction [31]. Dysfunction and damage of -cells in the early phase is thought to result from a direct contact with islet infiltrating cells (macrophages and CD4 + /CD8 + T-cells) and/or exposure to cytotoxic mediators (for example pro-inflammatory cytokines, ROS and Fas ligand), produced by these cells [32]. The main mode of -cell destruction is believed to be apoptosis [3]. However, necrosis is also observed and may be of importance in the early stage of the disease, as necrotic cells can activate an inflammatory response [33]. 15

Mediators of -cell death in Type 1 diabetes Pro-inflammatory cytokines Cytokines are small signaling proteins important in the regulation of the immune system and are mostly produced by immune cells, epithelial cells and stroma cells [34]. Pro-inflammatory cytokines such as Interleukin-1- beta (IL-1 ), Tumor necrosis factor-alpha (TNF- ) and Interferon-gamma (IFN- ) have been proposed as possible mediators of pancreatic -cell destruction [3]. This notion is based on the findings that soluble mediators secreted by activated immune cells from peripheral blood, induced cell death in human and rodent islets in vitro [35,36]. Subsequent studies revealed IL- as the main component responsible for the islet toxic effects [37,38]. Today, several cytokines produced by islet infiltrating immune cells have been identified. Most of these (e.g. IL-1, TNF- and IL-12) are produced by immune cells of the Th1 (T-helper cells-1) subtype [39,40], and it has been reported that the destructive insulitis within the islets, is connected to a Th1 cytokine profile, while a more non-destructive insulitis and disease protection is associated with a Th2 (T-helper cells-2) (e.g. IL-4 and IL-10) profile [41-44]. Cytokines released from Th1 and Th2 immune cells often have counteracting effects [40,45], and it might be that the balance of the different types may contribute to the outcome of the inflammatory process. Exposure of islets to pro-inflammatory cytokines in vitro induces apoptosis and necrosis. In rodent islets, the toxic effect has been shown to be caused by increased NO production, mediated by the NF- B inos NO pathway [46]. Human islets are less sensitive than rodent islets to the deleterious effects of cytokines, however, exposure for several days to proinflammatory cytokines induces functional impairment and apoptosis [5,47]. The intracellular signaling of pro-inflammatory cytokines starts with binding and activation of specific receptors. Following receptor stimulation, activation of the MAPKs c-jun NH 2 -terminal kinase (JNK), extracellular signal regulated kinase (ERK), p38 and various transcription factors such as NF- B, activating transcription factor 2 (ATF2), c-jun and signal transducer and activator of transcription-1 have been demonstrated [48-50]. 16

NOS and NO Nitric oxide (NO) is a small molecule with a broad range of biological effects, including involvement in neurotransmission, vascular homeostasis and cellular defense [51]. The radical is generated by NOS that converts L- arginine to citrulline and NO [52]. At low concentrations NO has been shown to have protective effects against pro-apoptotic stimuli. However, when produced in higher concentrations, NO induces cell death [53]. Out of three NOSs described, two are constitutively expressed and Ca 2+ - dependent (neuronal NOS (nnos) and endothelial NOS (enos)), while the third is inducible (inos) [51]. The nnos and enos forms are responsible for NO generation in nervous and endothelial tissues, respectively. Due to their dependency on intracellular Ca 2+ levels, they are usually active under short time periods and thereby generate only small amounts of NO. The inos is expressed in various cell types in response to pro-inflammatory cytokines and bacterial infections, generating larger amounts of NO. Therefore, toxic levels of NO are mostly associated with inos activation [54,55]. The concept that NO contributes to -cell destruction, is strengthened by the observations made in rodent islets, that inos inhibition blocks cytokineinduced NO formation and protects cells from cytokine-induced cell death in vitro and in vivo [56-59]. Also, inos knockout mice are protected from multiple low dose streptozotocin (MLDSTZ)-induced diabetes (a animal model for autoimmune-mediated diabetes) [60]. It should be emphasized, that in human islets, cytokine-induced cell death seems to be NOindependent [61,62], opening up alternative pathways for cytokine-induced cell death in human islets. The difference in NO-sensitivity between rodent and human islets has lead to the questioning of NO s role in -cell death. However, the higher scavenging capacity of free radicals and higher content of heat shock protein 70, observed in human islets, could in part explain the decreased sensitivity to NO seen in human islets [63,64]. Multiple signaling pathways have been proposed to mediate NO-induced cell death. It has been shown that NO activates both p53 and caspases and down-regulates the anti-apoptotic Bcl-2 protein [65,66]. In addition, inos induction leads to inhibition of aconitase, a decrease in mitochondrial membrane potential, inhibition of mitochondrial ATP production and endoplasmic reticulum (ER) stress [67-70]. NF- B NF- B was initially described as a regulator of the kappab light chain in B cells [71]. Since it s discovery, over 20 years ago, increasing interest in the NF- B family has given new insights in regulation and signaling of NF- B proteins. Today it is known that NF- B acts as a transcription factor 17

Figure 1. NF- B activation pathways. In the classical pathway, NF- B dimers such as p50/p65 are maintained in the cytoplasm by binding to I B. Receptor activation (TNF-rec, IL-1-rec) leads to recruitment of adaptor proteins and the activation of the I K complex consisting of and subunits and the scaffold NEMO. Phosphorylation of I B by I K results in ubiquitination followed by degradation of I B by the proteasome, facilitating NF- B dimer translocation to the nucleus. The alternative pathway usually mediates p52/rel-b translocation and involves activation of the NF- B inducing kinase (NIK), which phosphorylates a I K consisting only of subunits. I K phosphorylation of p100 leads to its partial degradation into p52 and translocation of p52/rel-b dimers. regulating a multitude of genes. The NF- B family consists of 5 subunit members, p50/p105, p52/p100, p65, c-rel and Rel-B [72]. These members share an N-terminal Rel homology domain responsible for DNA binding and subunit dimerization. NF- B is activated by different mitogenic stimuli, stress signals or inflammatory cytokines in a broad range of cell types [73]. The two main activation pathways are the classical (canonical) and the alternative (non-canonical) pathways (Fig.1) [73]. Activation of the classical pathway involves release of p50/p65 from inhibitor B (I B), as a result of phosphorylation by I B kinase (I K) and degradation of the I B by the proteasome. The alternative pathway involves p100 cleavage and the dimerization of the mature p52 product with Rel-B. In both cases, the ma- 18

ture dimeric NF- B proteins translocate to the nucleus and activate genes involved apoptotic signaling, immune and inflammatory response, cell proliferation, adhesion and angiogenesis [72]. Although NF- B mostly is associated with anti-apoptotic responses, pro-apoptotic functions have also been reported [74]. What is the role of NF- B in pancreatic islets? Even though the first study of NF- B in islets was published over 14 years ago, its role in islets is still a subject of controversy. It is known that the transcription factor is rapidly translocated from the cytosol to the nucleus upon treatment with IL-1, and in human islets, the activation of NF- B is necessary for cytokine-induced nitric oxide production [4]. In -cells from NOD mice, a protective effect of NF- B against TNF- -induced cell death has been reported [75]. It has also been demonstrated that NF- B activation in -cells leads to protection against cell death by induction of the antiapoptotic gene A20 [6] and that islet cell spreading, actin cytoskeletal organization and glucose stimulated insulin release was stimulated by a modest NF- B activation in response to signals from extracellular matrix components (76). Moreover, it was recently reported that NF- B maintains the highly differentiated -cell phenotype with insulin production capacity (7) and mediates the protective effects of Gleevec [77]. On the contrary, purified rat cells and intact human islets expressing a non-degradable form of I B were protected against cytokine-induced cell death [78]. In addition, a conditional knockout of NF- B protected mice against MLDSTZ-induced diabetes, suggesting a pro-apoptotic role of NF- B in -cells [8]. Therefore, the exact role of NF- B in pancreatic islets remains uncertain, warranting further studies to determine the consequences of NF- B activation for islet-cell survival and death. MAPKs MAPKs are expressed in all eukaryotic cells and transduce a large variety of external signals, leading to a wide range of cellular responses, including growth, differentiation, proliferation and apoptosis [79,80]. All MAPKs share a Thr-X-Tyr (TXY) motif within their activation loop and dual phosphorylation of these conserved threonine and tyrosine residues activates the MAPK, leading to downstream phosphorylation of other kinases and transcription factors [80]. MAPKs are organized in signaling cascades in which a MAPK kinase kinase (MAP3K, MKKK or MEKK) phosphorylates a MAPK kinase (MAP2K, MKK or MEK), which in turn activates a MAPK 19

(Fig.2) [80]. In human cells, the MAP3Ks are the largest group encoded by over 20 genes, while the MAP2Ks and MAPKs are encoded by 7 and 11 genes, respectively. Furthermore, different splice variants of MAPK cascade members have been observed [81]. The signaling cascade model of the MAPKs is straightforward, however the outcome of activation of a specific MAPK is complex. Some MAP3Ks can activate different MAPK pathways and the cellular outcome is dependent on the stimuli, signal duration and subcellular localization of the proteins involved [82]. For example, a transient activation of a MAPK, might have a protective effect in response to a stress stimuli, whereas a prolonged activation of the same MAPK might lead to apoptosis. The regulation of MAPK cascades has been shown to be dependent on docking motifs within the MAPKs and their substrates, assembly of MAPK cascade components by scaffolding proteins and phosphorylation/dephophorylation of MAPKs and substrates by protein kinases/phosphatases [83]. In concert with other signal transduction pathways, MAPKs transduce different signals leading to alterations in gene expression, thereby regulating cellular functions. In vertebrates, three major conserved MAPKs families have been described. These are the ERK, the p38 and the JNK family of MAPKs [80]. In general, the ERKs mediate proliferating and protective signals, whereas activation of the p38 and JNK families, more often is coupled to a cellular stress response. The ERKs The first members of the ERK family, the ubiquitously expressed 44 and 42 kda ERK1 and 2 proteins, were first identified over 20 years ago. Today, the family has grown to include a subgroup termed the large MAPKs with members such as ERK3, 5, 7 and 8. ERK1/2 and the large MAPKs share the TEY (Thr-Glu-Tyr) activation sequence within their kinase domain. However, the large MAPKs also have a large C-terminal domain ranging in size from 60-100 kda [84]. ERKs are strongly activated by proliferating stimuli, including growth factors and hormones and to lesser extent by proinflammatory cytokines, microtubule disruption and osmotic stress [80]. Upstream signaling of ERKs has been shown to involve the MAP3Ks Raf, Mos, MEKK-1 and Tpl and the MAP2Ks MKK1/MEK1, MKK2/MEK2 and MKK4/MEK4 (Fig.2) [79,84]. ERK activation leads to phosphorylation of numerous substrates including membrane proteins, transcription factors and cytoskeletal proteins [85]. 20

The JNKs The JNK family of MAPK consists of JNK1, 2 and 3 proteins. JNK1 and 2 are ubiquitously expressed whereas JNK3 mostly is found in the brain [80,83]. JNK proteins contain TPY (Thr-Pro-Tyr) in their activation motif. They are strongly activated in response to pro-inflammatory cytokines, UVirradiation, DNA damage, osmotic stress and growth factor deprivation [80,83]. Numerous MAP3Ks, such as MEKK-1-MEKK-4, MLKs, ASK1 and TAK1, have been described to be involved in mediating various upstream signals in the JNK pathway. However, only two MAP2K isoforms, MKK4/MEK4 and MKK7/MEK7, seem to be responsible for direct JNK phosphorylation (Fig.2) [80,83]. JNK activation leads to phosphorylation of additional non-nuclear substrates and activation of nuclear transcription factors [86]. The p38s The p38 family of MAPKs consists of the p38,, and isoforms, all sharing a common TGY (Thr-Gly-Tyr) motif [79,83]. The 38 kda protein family share about 40% sequence identity with other MAPKs, but only about 60 % identity within the family itself, suggesting different functions for the isoforms [87]. The p38 and isoforms are expressed in most tissues whereas p38 is expressed in skeletal muscle and p38 in intestine, lung, pancreas, kidney and testis [88]. p38 MAPKs respond to a wide range of stress stimuli including UV-irradiation, osmotic shock, pro-inflammatory cytokines and to a lesser extent growth factors [84,88]. Signals leading to p38 activation are mediated by the MAP3Ks TAK1, ASK1, MLK3, MEKK-3, and the MAP2Ks MKK3/MEK3 MKK4/MEK4 as well as MKK6/MEK6 (Fig.2) [79,84]. Downstream targets of p38 include both cytoplasmic proteins and transcription factors [79]. MAPKs in islet stress signaling In -cells, MAPKs are rapidly activated in response to IL-1, TNF- and the NO-donor DETA/NONOate (DETA/NO) [9-11]. It has been shown that a sustained JNK activation, in response to for example islet isolation or proinflammatory cytokines, leads to -cell apoptosis [12,13]. Moreover, inhibition of JNK or p38 has been reported to protect against cytokine-induced cell death [10,14,15,89]. Therefore, gaining new insights in the MAPK signaling pathways in pancreatic islets will improve our understanding of the molecular processes that may contribute to the destruction of -cells. 21

Figure 2. Simplified view over the MAPK signaling cascade, leading to ERK, JNK and p38 activation. The three-kinase module is shown, in where a MAP3K activates a MAP2K, in turn activating a MAPK. For simplicity not all known interactions are shown. MEKK-1 MEKK-1 is a 196-kDa serine/threonine MAP3K that has been shown to play an important role in both apoptosis and cell survival in various cell types. The MEKK-1 protein consists of a kinase domain in its C-terminal and an N-terminal domain containing various protein interaction motifs [90]. Recent findings also show that MEKK-1 can act as an E3 ubiquitin ligase and mediate ubiquitination and degradation of proteins through its plant homeobox domain [91]. In addition, it contains a caspase cleavage site [92]. In various cell types it has been reported that MEKK-1 promotes apoptosis in response to genotoxic stimuli, such as cisplatin, UV-irradiation and etoposid [92], and non-genotoxic stimuli, including anoikis and Fas stimulation [93,94]. When challenged by a pro-apoptotic stimulus, the full length MEKK-1 protein can be cleaved by DEVD caspases into an 80-95 kda proapoptotic fragment [90-94]. On the contrary, it has been reported that the full length MEKK-1 has anti-apoptotic effects in cardiomyocytes [95]. MEKK-1 preferentially activates the JNK pathway through phosphorylation of MKK4/MEK4 [96-99], although MEKK-1-mediated ERK and p38 activation has been demonstrated [100,101]. Moreover, MEKK-1 has been implicated in the regulation of NF- B and p53 [102,103]. The upstream 22

events of MEKK-1 signaling are today poorly understood. However, proteins of the Rho family of GTPases (Rac1, RhoA and Cdc42), the receptorinteracting protein (RIP) family of kinases and the TNF receptor-associated factor (TRAF) family have been suggested to promote MEKK-1 activation [104-106]. The role of MEKK-1 in -cells is not well characterized. It has been reported that expression of the constitutively active kinase domain of MEKK- 1 promotes JNK activation [13] and results in a lowered insulin gene transcription (107). 23

RNA interference and dicer-generated sirna RNA interference (RNAi) is a conserved natural mechanism of posttranscriptional gene silencing. RNAi has been shown to be important for the cellular defense against viral infections and cell fate specification [108]. Since the discovery made by Fire and Mello in 1998, that double stranded RNA (dsrna) could silence genes [109], a whole new area of research has developed, and today RNAi is a commonly used method to study gene function both in vitro and in vivo [110]. The pathway of RNAi can be broken down into two main components [108]. Firstly, when dsrna is detected within the cell, it will be recognized and cleaved into 21-23 nucleotide long fragments (small interfering RNA or sirna) by the RNase III enzyme Dicer. Secondly, the sirna is recognized by and binds to a protein complex named RNA induced silencing complex (RISC). Following RISC binding, one of the RNA strands is eliminated but the other remains bound to the RISC and is used as a probe to detect mrna sequences complementary to the sirna. If an mrna sequence binds to the sirna, an RNAase termed Slicer will degrade the mrna at sites not bound to the sirna, and the gene encoded by the mrna will be silenced. The most commonly used approach for gene silencing in mammalian cells is to introduce pre-designed synthetic sirnas, complementary to the targeted mrna, into cells. By doing this, the phase involving Dicermediated cleavage is bypassed and the risk of activating the interferon response is lowered [110]. The design of effective sirnas has been an obstacle in sirna-mediated gene silencing, since there is great variability between different sirnas in mediating effective gene silencing. Usually, 3-4 different sirna sequences need to be evaluated for efficiency, making the process both costly and time consuming. However the problems associated with designing sirna can be circumvented using a recombinant form of the Dicer enzyme. In this approach, the enzymatic activity of the recombinant Dicer is utilized for in vitro cleavage of dsrna, generating a pool of Dicergenerated sirna (d-sirna) sequences suitable for gene silencing [111]. 24

Methodology Cell culture Rat insulinoma cells (RIN-5AH), were maintained in RPMI 1640 medium supplemented with 10 % fetal calf serum (FCS), 2 mm L-glutamine and antibiotics (WS). TC-6 cells, COS, RAW 264.7 and human embryonic kidney (HEK) 293 cells, were maintained in DMEM supplemented as above. All cells were grown at 37 C in a humidified air incubator with 5 % CO 2. Islet isolation and culture Islets from Naval medical research institute-established mice, were isolated by collagenase digestion and incubated free floating in WS. For human islet culture, the glucose concentration of the WS was lowered to 5.6 mm. All islets were grown at 37 C in a humidified air incubator with 5 % CO 2. Plasmids Empty or PcDNA3 plasmids either containing the wild type (wt) mouse MEKK-1 or the mutant MEKK-1 cdna were used for MEKK-1 overexpression. For c-abl overexpression the psgtc-abl plasmid was used and for green fluorescent protein (GFP) expression, the pd2egfp-n1 vector was used. Liposome-mediated transfection of cell lines During transfection, cells were maintained in serum free RPMI 1640. The cells were transfected by adding a transfection mixture of 10 l Lipofectamine 2000 to 1 g DNA according to the instructions of the supplier. The DNA was pd2egfp-n1 or a combination of pdegfp-n1 and wt or mutant MEKK-1. For c-abl transfection, the cells were either transfected 25

with empty or psgtc-abl plasmid. Cells were centrifuged for 10 min at 550 x g and incubated for 1.5 h at 37 C. After the incubation, the serum free RPMI 1640 + transfection mix was replaced with WS. Cell sorting For RIN-5AH and TC-6 cells, GFP-positive cells were sorted out using a FACSCalibur flow cytometer. After sorting, the cells were pelleted by centrifugation for 2 min at 600 x g. The cells were then washed in phosphate buffered saline (PBS) and plated onto culture dishes as described above. Liposome-mediated transfection of free islet cells with d-sirna Free islet cells were prepared by treating islets with trypsin. Trypsination was terminated by addition of culture medium, followed by DNase treatment. Free islet cells were transfected with d-sirna and Lipofectamine 2000 in serum free media. RNA isolation and cdna synthesis Total RNA was isolated from cells using the Ultraspec RNA Isolation System reagent. Two g of RNA was used for cdna synthesis. cdna was synthesized using the M-MuLV reverse transcriptase and oligo-dt primers according to the manufacturer s instructions. Real time PCR PCR amplification was performed using the Lightcycler instrument and the Lightcycler FastStart DNA Master SYBR Green I kit. Specific primers for GAPDH (human), -actin (mouse) MEKK-1 (mouse and human) and c-abl (mouse), were used. Semi-quantitative data for MEKK-1 and c-abl expression were calculated using the formula: 2 MEKK-1/c-Abl. The different groups were expressed as % of GL3 d-sirna crossing point GAPDH or -actin - crossing point transfected cells. PCR products were analyzed by agarose gel electrophoresis and SYBR green staining to verify that the correct PCR products were obtained. 26

Evaluation of cell viability Cell viability for RIN-5AH and TC-6 cells was determined by staining the cells with propidium iodide for 10 min at 37 C. After careful washing, cells were trypsinized and analyzed for red fluorescence (FL-3) using flow cytometry. Islet cells were stained with propidium iodide and bisbenzimide for 10 min at 37 C and analyzed by fluorescence microscopy using Openlab 3.0.4 software. Total number of cells, as well as propidium iodide positive cells, were counted using the NIH Image 1.63 software by investigators not aware of sample identity. In paper IV, propidium iodide and bisbenzimide uptake was quantified using Adobe Photoshop software and the ratio red over blue was calculated as a relative measure of cell viability. Immunoblotting Cells were washed with ice cold PBS and suspended in sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min and separated by SDSpolyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to Hybond-P filters. Filters were blocked in milk or bovine serum albumin (BSA) and incubated with specific antibodies. Following a brief wash with PBS-Tween, membranes were probed with horseradish peroxidase-linked secondary antibodies. Unbound antibodies were washed away and the bound antibodies were visualized using enhanced chemoluminiscence and quantified by densitometry. Immunoprecipitation Cells were washed twice in ice-cold PBS and resuspended in lysis buffer on ice for 20 min. The lysed cells were cleared by centrifugation and remaining extracts were incubated with 5 g MEKK-1 rabbit polyclonal antibody or 5 g of purified rabbit IgG for 2.5 h on ice. Immune complexes were purified by binding to 50 l Protein A Sepharose for 1h at 4 C and thereafter washed 3 times with lysis buffer and once with H 2 O. The sepharose beads were resuspended in SDS sample buffer and immunoprecipitates were resolved by electrophoresis. Preparation of cytoplasmic and nuclear fractions. Human islets were either left untreated or treated with the cytokines IL-1ß (50U/ml) and TNF- (1000 U/ml) for 30 min. Following cytokine treatment, the islets were washed three times in cold PBS and resuspended in 27

solution A (10 mm HEPES ph 7.9, 1,5 mm MgCl 2, 10 mm KCL, 0,5 mm Dithiothreitol, protein inhibitor cocktail) and incubated on ice for 20 min. Following a brief centrifugation, the islets were again resuspended in solution A and lysed with a homogenizer. The lysates were then centrifuged for 5 min at 3200 rpm at 4 C and proteins in the supernatant fraction were precipitated using acetone for 10 min on ice followed by a 5 min centrifugation at 13000 rpm at 4 C. After removal of all acetone, the precipitated proteins were resuspended in SDS-sample buffer. The pelleted fractions of the lysates were resuspended in solution A and centrifuged a second time for 5 min at 3200 rpm at 4 C and resuspended in SDS-sample buffer. Confocal microscopy The imaging and analysis of intact isolated islets were performed by confocal laser scanning microscopy. Intact islets were cytospinned (1200 rpm for 3 min) to polylysine coated slides and fixed in 4% paraformaldehyde for 5 min. The islets were permeabilized in 0.1% Triton X-100 and then incubated for 60 min at 37C with guinea pig insulin antibodies, rabbit or mouse p65, Rel-B, p50 or p52 antibodies. The Alexa Fluor 488 goat anti-guinea pig, the Alexa Fluor 568 goat anti-mouse and the Cy3 donkey anti-rabbit IgGs were used as secondary antibodies. Samples were subjected to optical sectioning using a Nikon D-Eclipse C1 confocal laser scanner connected to a Nikon Eclipse C1-2000U inverted microscope. Fluorescence was excited at 488 and 543 nm and emitted light was collected between 535 and 650 nm. Adenoviruses We used serotype 5, E1-deleted/E3-deficient adenoviral vectors derived from the widely used AdEasy system, which express either nothing (control virus), GFP under the control of the cytomegalovirus (CMV) promoter (GFP virus) and c-rel under control of the CMV promoter (c-rel virus). Virus stocks were purified by caesium chloride density-gradient centrifugation and plaque titred by serial dilution and agar overlay on the HEK 293 cells. Typical titres were 10 10 plaque forming units (PFU)/ml or higher, representing 1 to 5% of the total viral particles as determined by readings of the optical densities. In vitro transduction procedure For the adenoviral-mediated transduction, rat or human islet cells, which had been dispersed into free islet cell suspension by trypsin treatment, were 28

incubated at 37 for 1 h in a volume of 0.4 ml RPMI-1640 supplemented with 2% FCS and containing 1 or 5 PFU/cell of the adenoviral vector. Transduced islets were washed and further cultured in complete RPMI-1640 medium. Extraction of nuclear proteins and electrophoretic mobility shift assay (EMSA) Cells were treated with IL-1 (50 U/ml) for 30 min, after which proteins were extracted for EMSA, which was performed as previously described (49). p65 antibody (0.2 mg) was used for supershift and c-rel antibody (0.2 mg) was used for blocking c-rel binding to the kappab-probe. Pancreas perfusion transduction Sprague-Dawley rats were anaesthetized with an intraperitoneal injection of thiobutabarbital sodium. A catheter connected to a peristaltic pump was placed in the abdominal aorta so that the perfusion medium could flow freely into the pancreas. The gland was removed from the animals and placed in a funnel at a constant temperature (30 C). The gland was perfused for 45 to 60 min at a flow rate of 1 ml/min with a continuously gassed (95% O2 5% CO2) Krebs Ringer bicarbonate HEPES (KRBH) buffer, supplemented with 20-mg/ml BSA, 20-mg/ml dextran T70 and 0.3-mg/ml glucose, and containing 0.5 x 10 9 PFUs per pancreas of the GFP-expressing adenoviral vector and 1 x 10 9 PFUs per pancreas of the c-rel adenoviral vector, which corresponds to 20 to 50 PFUs/islet cell. The capillary endothelium was disrupted before administering the viral vector by pre-perfusion for 40 s with medium containing 0.1% Triton X-100, followed by a 10-min wash with KRBH buffer only. Following transduction, islets were isolated and cultured for two days. After the culture period, GFP-positive islets were identified and separated from GFP-negative islets under a fluorescence stereomicroscope. 29

Results and discussion Paper I The use of sirna to silence genes has emerged as a powerful tool for studying gene function in different cell types. In the present study, we investigated whether d-sirna mediates efficient gene silencing in islet cells. In order to optimize liposomal delivery of sirna into islet cells, we first evaluated the efficiency of different commercially available liposome formulations to mediate uptake of fluorescein isothiocyanate (FITC)-labeled sirna into the -cell line TC-6. We observed that Lipofectamine was the most efficient formulation, mediating a transfection efficiency of 96,2 0,5 %. Oligofectamine transfected less than 40 % of the cells. None of the other formulations tested (Geneporter, GeneJuice or Genejammer) reached higher transfection rates than 5%. Secondly, we wanted to determine if Lipofectamine could be used to transfect primary islet cells. As expected, transfection was much more efficient in dispersed islet cells (90%) than in intact islets (10%). We also evaluated the potentially less toxic Lipofectamine 2000 on dispersed human islet cells, which promoted sirna uptake into 76% of the cells. Having established that Lipofectamine and Lipofectamine 2000 mediate efficient delivery of sirna into dispersed islet cells, we next attempted to silence the gene coding for the non-receptor tyrosine kinase cellular Abelson tyrosine kinase (c-abl). Since previous attempts from our group to silence c- Abl using pre-designed synthetic sirna have been inconsistent, we evaluated the use of in vitro generated d-sirna. By this approach, an in vitro transcribed dsrna corresponding to the c-abl gene will be cleaved by a recombinant form of the Dicer enzyme, yielding a pool of d-sirna molecules directed against the entire c-abl mrna instead of to a single target sequence when using synthetic sirna [111]. Following Lipofectamine 2000-mediated transfection of c-abl d-sirna into dispersed mouse islet cells, we found that c-abl mrna expression was decreased to 24,8 9,4% and 35,6 15,8% at 1 and 3 days post transfection, when compared to islet cells transfected with GL3 (control) d-sirna, using real time PCR. Not surprisingly, no silencing was observed 7 days post transfection, probably due to degradation of d-sirna. To confirm successful knockdown of the c-abl protein, we overexpressed c-abl in RAW 264.7 cells, a cell line easily lipofected. 24 hours post transfection, c-abl immuno- 30

reactivity was reduced by over 50 % in response to c-abl specific d-sirna when compared to cells treated with GL3 d-sirna. No toxic effect of the d- sirna treatment could be observed, using flow cytometry. In conclusion, we describe a non-toxic technique that mediates efficient delivery and gene silencing of d-sirna in primary islet cells. This technique can be used to study gene function and promote understanding of the intracellular pathways involved in the destruction of pancreatic -cells. Paper II MEKK-1 has been shown to activate JNK, ERK and p38 MAPKs, and implicated in both pro-apoptotic and anti-apoptotic signaling in various cell types [92-101]. The aim of the present study was therefore to characterize the role of MEKK-1 in stress-induced pancreatic islet cell death. First, we investigated whether overexpression of the wt MEKK-1 protein affected rates of stress-induced cell death. In two -cell lines, RIN-5AH and TC-6, MEKK-1 overexpression increased DETA/NO-, STZ- and H 2 O 2 - induced cell death. However, MEKK-1 overexpression alone did not increase cell death, indicating that an increase in MEKK-1 levels by itself, is not a sufficient event in promoting -cell apoptosis. Next, we observed that d-sirna-mediated down regulation of MEKK-1 gene expression in primary human islet cells, protected against DETA/NO-, STZ- and tunicamycin-induced cell death, supporting a contributing role for MEKK-1 in islet cell death. Moreover, MEKK-1 was activated in response to DETA/NO in TC-6 cells, as revealed by an increased threonine phosphorylation. Several studies have reported caspase-mediated cleavage of the MEKK-1 protein into an 80-95 kda pro-apoptotic fragment and that this leads to subcellular re-localization of the fragment from membrane bound to the cytosolic compartment [92-94]. However, we were not able to detect any cleavage of MEKK-1 in TC-6 cells in response to the different treatments. This strengthens the previously proposed possibility that MEKK-1 cleavage is not a prerequisite for its ability to activate down-stream MAPKs and participate in pro-apoptotic signaling [112]. Having established that MEKK-1 is activated in response to DETA/NO, and a necessary event in primary human islet stress-induced cell death, we next investigated the downstream effects of MEKK-1 in human islet cells. D-siRNA-mediated down-regulation of MEKK-1 resulted in decreased activation of JNK, but not of p38 and ERK, in DETA/NO- treated cells. These results are in line with previous reports showing that expression of the kinase domain of MEKK-1 promotes JNK activation in insulin-producing cells [13] and that MEKK-1 activation preferentially leads to the activation of JNK over p38 and ERK in non- -cells [113]. Interestingly, it is also in concert with prior reports stating that cell-permeable inhibitors of JNK pro- 31

tect against cytokine-induced cell death in insulin-producing cells [14,89]. To verify that the MEKK-1/JNK pathway actually promotes -cell death in response to DETA/NO or STZ, we treated TC-6 cells with the JNKinhibitor SP600125 prior to (30 min) and during exposure to DETA/NO or STZ. We observed a significantly lower cell death rate in SP600125 treated cells when challenged with DETA/NO or STZ. The exact down-stream events to MEKK-1/JNK activation are not well understood, but may involve phosphorylation of transcription factors c-jun, Ets-like transcription factor (Elk) and ATF2 [13], induction of p53 and ROS [13], release of Smac/Diablo from mitochondria [115] or release of the Bcl- 2 inhibitor nuclear receptor 77 from nuclei [116]. It is also possible that MEKK-1 interacts with the ER-stress pathway. Indeed, down-regulation of MEKK-1 presently counteracted islet cell death evoked by the ER-stress inducer tunicamycin. In line with this notion it has been observed that apoptosis signal-regulating kinase 1 (ASK1), which is also a known activator of JNK, is involved in ER-stress signaling [117]. Furthermore, results from paper III in this thesis indicate that MEKK-1 participates in NF- B activation. In summary, we propose a key role for MEKK-1 as a JNK activating MAP3K and in stress-induced -cell death. New insights in MEKK-1 signaling will hopefully increase our understanding of cellular processes contributing to Type 1 diabetes. Paper III The transcription factor NF- B and the MAP kinases JNK1/2 are known to play decisive roles in cytokine-induced damage of rodent -cells [4,6-8,10, 12,13,89]. The upstream events by which these factors are activated in response to cytokines are, however, uncharacterized. The aim of the present investigation was to elucidate a putative role of MEKK-1 in cytokineinduced signaling. First, to establish a functional role of MEKK-1, TC-6 cells were transiently transfected with wt or a kinase dead mutant MEKK-1, and subjected to a mix a cytokines (IL-, TNF-, IFN- ). Immunoprecipitation of the MEKK-1 protein followed by immunoblotting with phospho-mekk-1 (T1383) antibody, revealed MEKK-1 activation in response to 30 and 60 min cytokine stimulation in TC-6-cells. This is in line with previous studies identifying the amino acids positions T1383 and T1395 as activation sites in MEKK-1 (118,119). Overexpression of wt, but not the mutant MEKK-1, resulted in increased cytokine-induced JNK phosphorylation and I B degradation whereas no effect of MEKK-1 was seen in unstimulated cells. There was no effect of MEKK-1 on ERK and p38 phosphorylation. 32

Indeed, MEKK-1 preferentially activates the JNK pathway in other cell types [113]. Furthermore, cytokine-induced cell death was augmented in cells overexpressing wt MEKK-1 when compared to control and mutant transfected cells, whereas MEKK-1 had no effect on rates of cell death in untreated cells. This is in concert with the results from paper II, that elevated MEKK- 1 levels per se, is insufficient to induce apoptosis in TC-6-cells, however, when cells are subjected to stress, MEKK-1 signaling promotes cell death. Next, we addressed the role of MEKK-1 in primary human islets. MEKK-1 d-sirna transfection mediated successful knockdown of MEKK- 1 protein levels in dispersed human islet cells. Moreover, cytokine-induced JNK activation was abolished in islet cells treated with MEKK-1 d-sirna, when compared to control cells whereas no effect of MEKK-1 d-sirna could be seen on p38 and ERK activation. This strengthens the idea that MEKK-1 is a significant MAPK3, responsible for cytokine-induced JNK activation in human islets. Since direct JNK activation, has been reported to be mediated by the MAP2K MKK4, we next investigated if MEKK-1-JNK signaling, in response to IL1- in human islet cells, is mediated by MKK4. Indeed, a less pronounced IL1- -induced MKK4 activation was observed in MEKK-1 d- sirna treated human islet cells, when compared to control cells, indicating that IL-1 -induced MKK4 and JNK activation requires MEKK-1 activity in human islet cells. Furthermore, IL1- -induced I B degradation and NF- B translocation were attenuated in MEKK-1 d-sirna treated cells, signifying that MEKK-1 is required for NF- B activation in human islet cells. The toxic effect of pro-inflammatory cytokines in rodents have been shown to in part be mediated by the NF- B inos NO pathway [4]. We therefore addressed whether MEKK-1 knockdown had any effect on cytokine-induced cell death and inos induction. MEKK-1 knockdown in mouse islets and TC-6-cells, protected against cytokine-induced cell death. In TC-6-cells, the protection was accompanied by a markedly reduced IL1- induced inos expression in MEKK-1 d-sirna treated cells. We currently report that MEKK-1 is a key component in cytokineinduced JNK and NF- B activation, mediating inos induction and cell death. Prolonged and pronounced activation of JNK, which occurs in response to not only pro-inflammatory cytokines, but also to islet isolation and amyloid formation, leads to -cell death [12,120]. Activation of NF- B is also a pro-apoptotic event in rodent -cells as it participates in inos induction leading to the generation of toxic levels of nitric oxide. Since human islets are less sensitive to the deleterious effects of NO [61,62], it raises the possibility that NF- B could exert an anti-apoptotic effect in human islet cells, as it does in many other cell types [74], and that the pro-apoptotic effects of JNK-activation are in part neutralized by NF- B, thereby explaining the weaker apoptotic response of human -cells as compared to rodent 33