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Inactivation of Protein Tyrosine Phosphatases Enhances Interferon Signaling in Pancreatic Islets

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Inactivation of Protein Tyrosine Phosphatases Enhances Interferon Signaling in Pancreatic Islets Diabetes Volume 64, July 2015 2489 William J. Stanley,1,2 Sara A. Litwak,1 Hong Sheng Quah,1,2 Sih Min Tan,3 Thomas W.H. Kay,1,2 Tony Tiganis,4 Judy B. de Haan,3 Helen E. Thomas,1,2 and Esteban N. Gurzov1,2 Inactivation of Protein Tyrosine Phosphatases Enhances Interferon Signaling in Pancreatic Islets Diabetes 2015;64:2489–2496 | DOI: 10.2337/db14-1575 Type 1 diabetes (T1D) is the result of an autoimmune that phosphorylate STAT1. These then dimerize, translo- assault against the insulin-producing pancreatic b-cells, cate to the nucleus, and bind g-activated sites of a diverse where chronic local inflammation (insulitis) leads to array of genes. Blocking activation of this transcriptional b-cell destruction. T cells and macrophages infiltrate into pathway protects islets from immune destruction (3–5). islets early in T1D pathogenesis. These immune cells se- These data indicate that excessive activation of JAK/STAT crete cytokines that lead to the production of reactive signaling in islets during the inflammatory process con- oxygen species (ROS) and T-cell invasion and activation. tributes to b-cell dysfunction and death. Cytokine signal- Cytokine-signaling pathways are very tightly regulated by ing results in the upregulation and release of factors by ISLET STUDIES protein tyrosine phosphatases (PTPs) to prevent exces- b-cells, such as chemokines, that attract immune cells and sive activation. Here, we demonstrate that pancreata amplify the inflammatory process. from NOD mice with islet infiltration have enhanced Protein tyrosine phosphatases (PTPs) are a large oxidation/inactivation of PTPs and STAT1 signaling com- superfamily of enzymes that dephosphorylate tyrosine pared with NOD mice that do not have insulitis. Inactiva- tion of PTPs with sodium orthovanadate in human and phosphorylated proteins to oppose the actions of protein rodent islets and b-cells leads to increased activation tyrosine kinases (6,7). PTPs play an important role in the of interferon signaling and chemokine production medi- development of both forms of diabetes (8). The architec- ated by STAT1 phosphorylation. Furthermore, this exac- ture and low thiol pKa of the Cys residue in the active erbated STAT1 activation–induced cell death in islets was site of PTPs renders these proteins highly susceptible to prevented by overexpression of the suppressor of cyto- oxidation by reactive oxygen species (ROS) (6,8). ROS- kine signaling-1 or inactivation of the BH3-only protein mediated oxidation of the PTP active site Cys inhibits PTP Bim. Together our data provide a mechanism by which activity and prevents substrate binding. Recent studies PTP inactivation induces signaling in pancreatic islets have established that PTP oxidation occurs in vivo under that results in increased expression of inflammatory physiological and pathological conditions such as inflam- genes and exacerbated insulitis. mation (6,8,9). The total serum antioxidant levels of patients with prediabetes and patients with T1D are reduced compared Type 1 diabetes (T1D) is caused by progressive loss of with age-matched controls (10,11). Furthermore, ROS pancreatic b-cells due to an autoimmune assault. Local and oxidative stress have been linked to b-cell cytotoxicity inflammation and proinflammatory cytokines, particularly and are believed to be involved in pathology (12). The interferons (IFNs), play an important role in b-cell loss mechanism, however, remains unclear. Here, we show (1,2). IFN-g signal transduction involves activation of that oxidative stress in pancreatic islets during insulitis the tyrosine kinases Janus kinase (JAK) 1 and JAK2 results in the enhanced oxidation of PTPs and that 1St Vincent’s Institute of Medical Research, Melbourne, Victoria, Australia Received 14 October 2014 and accepted 22 February 2015. 2 ’ Department of Medicine, St Vincent s Hospital, The University of Melbourne, This article contains Supplementary Data online at http://diabetes Melbourne, Victoria, Australia .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1575/-/DC1. 3Diabetic Complications Division, Baker IDI Heart and Diabetes Institute, Mel- © 2015 by the American Diabetes Association. Readers may use this article as bourne, Victoria, Australia long as the work is properly cited, the use is educational and not for profit, and 4Department of Biochemistry and Molecular Biology, Monash University, Mel- the work is not altered. bourne, Victoria, Australia Corresponding author: Esteban N. Gurzov, [email protected]. 2490 PTP Oxidation Promotes Inflammation Diabetes Volume 64, July 2015 inactivation of PTPs in human and rodent islets and to sulfonic acid (PTP-SO3H) using pervanadate (Sigma- b-cells leads to increased INF signaling, chemokine pro- Aldrich). Then, “hyperoxidized” PTPs were detected by duction, and cell death. immunoblotting with the PTPox antibody (R&D Systems, Minneapolis, MN). Immunoprecipitation was performed RESEARCH DESIGN AND METHODS using the PTPox antibody (R&D Systems) and radioimmu- Mice noprecipitation assay buffer. Antibody/protein complexes Mice were maintained at St Vincent’s Institute, and were collected with Protein G PLUS-Agarose (Santa Cruz experiments were approved by the institutional animal Biotechnology, Santa Cruz, CA), washed, and then boiled ethics committee. The list of mice is provided in Supple- in sample buffer (Santa Cruz Biotechnology) to remove mentary Table 1. the antibody/protein complex. Equal amounts of proteins Immunohistochemistry and Immunofluorescence were resolved by 10% SDS-PAGE and immunoblotted Mouse pancreata were frozen in optimal cutting temper- with the antibodies indicated in Supplementary Table 3. ature embedding medium (Sakura Finetek, Tokyo, Japan) All blots shown are representative for two to four inde- or fixed with formalin and embedded in paraffin. Sections pendent experiments. were incubated with mouse anti-phosphorated (p)STAT1 Cell Viability (BD Biosciences, San Jose, CA) and guinea pig anti-insulin Human islet preparations were assessed for viability by (DAKO, Glostrup, Denmark) or rabbit anti-nitrotyrosine simultaneously staining monodispersed islet cells with 5 (Millipore, Billerica, MA) primary antibodies. The sections mg/mL 7-aminoactinomycin D (7-AAD) for islet viability, were then incubated with secondary Alexa Fluor 488 anti- 10 mmol/L Newport green (NG) for b-cells, and 1 mmol/L mouse and Alexa Fluor 568 anti-guinea pig (both from tetramethylrhodamine-ethyl-ester (TMRE) for apoptosis Molecular Probes, Life Technologies, Carlsbad, CA) and (Molecular Probes, Invitrogen, Grand Island, NY), and counterstained with DAPI for immunofluorescence or analyzing by flow cytometry following the method of Ichii secondary biotinylated anti-rabbit (Vector Laboratories, et al. (16). Mouse islets were dispersed into single cells Burlingame, CA) for immunohistochemistry. with trypsin. DNA fragmentation was analyzed by staining with propidium iodide, as previously described (17). The Cell Culture and Treatments percentage cell death of MIN6 cells was determined in at Human pancreata were obtained, with informed consent least 600 cells per experimental condition by inverted from next-of-kin, from heart-beating, brain-dead donors by fluorescence microscopy after staining with the DNA the Australian Islet Transplant Consortium, and approved dyes Hoechst-33342 (10 mg/mL) and propidium iodide by the human ethics committees of the hospitals involved (5 mg/mL). and the Australian Red Cross. Human islets were isolated as described previously (13). Islets were used from donors Statistical Analysis without diabetes who were an average age of 64 6 3years. Data are means 6 SEM of three to five independent Mouse islets were isolated as described previously (14). The experiments. Comparisons between groups were made by insulin-producing MIN6 and NIT-1 cell lines were cultured paired t test or by ANOVA, followed by t test with the in DMEM (Life Technologies) supplemented with 10% fetal Bonferroni correction. calf serum. Cytokine concentrations were selected based on previous time course and dose-response studies (15). RESULTS PTPs Are Oxidized in Pancreata From NOD Mice Real-Time PCR To directly evaluate the role of insulitis-induced PTP Analyses were performed with the ddCT method using oxidation, we used nontransgenic NOD mice and trans- b -actin as internal controls. Probes are provided in Sup- genic NOD mice expressing proinsulin (PI) in MHC class II plementary Table 2. bearing cells (NOD PI mice) (18). NOD PI mice do not PTP Oxidation, Immunoprecipitation, and Western develop insulitis due to immune tolerance to PI, but other- Blotting wise have a normal, functional immune system. At 12 Total (reversible and irreversible) PTP oxidation was weeks of age, NOD mice developed insulitis and oxidative assessed essentially as described previously (9). Briefly, stress that correlated with increased nitrotyrosine-positive PTP oxidation results in two pools of PTPs: oxidized islets in the pancreas (Fig. 1A–C), as previously reported 2 (PTP-SOH; inactive) and reduced (PTP-S ; active). To de- (19). Insulitis and nitrotyrosine staining were markedly tect oxidized PTPs in NOD mice, pancreas samples were reduced in NOD PI transgenic mice at 12 weeks of age alkylated with N-ethylmaleimide (NEM, Sigma-Aldrich), (Fig. 1A–C). Enhanced nitrotyrosine staining is indicative rendering active PTPs resistant to further modification, of oxidative stress and results from the increased produc- whereas oxidized PTPs remain unaffected. Excess NEM tion of superoxide and nitric oxide and the generation
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