Research Article 1463 The scaffold protein IB1/JIP-1 is a critical mediator of cytokine-induced apoptosis in pancreatic β cells Jacques-Antoine Haefliger1,*,‡, Thomas Tawadros1,*, Laure Meylan1, Sabine Le Gurun1, Marc-Estienne Roehrich1, David Martin1, Bernard Thorens2 and Gérard Waeber1 1Department of Internal Medicine, CHUV-1011 Lausanne, Switzerland 2Institute of Pharmacology and Toxicology, University Hospital, CHUV-1011 Lausanne, Switzerland *These authors contributed equally to this work ‡Author for correspondence (e-mail: jhaefl[email protected]) Accepted 7 January 2003 Journal of Cell Science 116, 1463-1469 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00356 Summary In insulin-secreting cells, cytokines activate the c-Jun N- cells by overproducing IB1/JIP-1 and this effect was terminal kinase (JNK), which contributes to a cell signaling associated with inhibition of caspase-3 cleavage. towards apoptosis. The JNK activation requires the Conversely, reducing IB1/JIP-1 content in INS-1 cells and presence of the murine scaffold protein JNK-interacting isolated pancreatic islets induced a robust increase in basal protein 1 (JIP-1) or human Islet-brain 1(IB1), which and cytokine-stimulated apoptosis. In heterozygous mice organizes MLK3, MKK7 and JNK for proper signaling carrying a selective disruption of the IB1/JIP-1 gene, the specificity. Here, we used adenovirus-mediated gene reduction in IB1/JIP-1 content in happloinsufficient transfer to modulate IB1/JIP-1 cellular content in order to isolated pancreatic islets was associated with an increased investigate the contribution of IB1/JIP-1 to β-cell survival. JNK activity and basal apoptosis. These data demonstrate Exposure of the insulin-producing cell line INS-1 or that modulation of the IB1-JIP-1 content in β cells is a isolated rat pancreatic islets to cytokines (interferon-γ, crucial regulator of JNK signaling pathway and of tumor necrosis factor-α and interleukin-1β) induced a cytokine-induced apoptosis. marked reduction of IB1/JIP-1 content and a concomitant increase in JNK activity and apoptosis rate. This JNK- Key words: Type-I diabetes, β-Cell line, Islet-brain-1, IB1/JIP-1, induced pro-apoptotic program was prevented in INS-1 INS-1, Pancreatic islets, Apoptosis, Adenovirus, JNK activity Introduction which is in turn associated with an increase phosphorylation The c-Jun N-terminal kinase (JNK) is a mitogen-activated of c-Jun and apoptosis rate (Bonny et al., 2000). In transient protein (MAP) kinase (MAPK) that is activated by cytokines transfection studies of β-TC3, overproduction of IB1/JIP-1 and exposure to environmental stress (Davis, 2000; Dickens was shown to prevent JNK-mediated activation of the et al., 1997; Whitmarsh et al., 1998; Yang et al., 1997a; Yang transcription factors c-Jun, Elk1 and ATF2 (Bonny et al., et al., 1997b). Phosphorylation of the AP-1 transcription 2000). The IB/JIP proteins contain several interacting factor is a key target of JNK, which in turn regulates various domains including an SH3 domain, a JNK domain and a cellular functions such as proliferation, differentiation and phosphotyrosine-interacting domain (Davis, 2000). Small- apoptosis (Davis, 2000). In pancreatic β cells, JNK molecule inhibitors such as SP600125 or small interacting activation might play a crucial role in the pro-apoptotic peptides (Barr et al., 2002; Bonny et al., 2001) were program observed during the onset of type-1 diabetes described to interfere with specific domains of IB1/JIP-1. In (Waeber et al., 2000). For proper JNK activation, the vitro, these molecules and peptides can inhibit JNK activity presence of the scaffold protein JNK-interacting protein 1 and could represent an useful tool for studying the JNK (JIP-1) or Islet-brain 1 (IB1) (Bonny et al., 1998; Dickens et signaling pathway (Barr et al., 2002; Bonny et al., 2001). al., 1997; Mooser et al., 1999) is required. This protein The molecular basis of JNK activation and the functional interacts with mixed-lineage protein kinases (MLKs), the role of IB1/JIP-1 were mostly assessed in the transformed MAPK kinase MKK7, JNK, the cytoskeleton-interacting mouse insulin-secreting cell line βTC3. Accordingly, we protein p190RhoGEF, kinesin, the amyloid precursor protein investigated the contribution of IB1/JIP-1 to rat β-cell survival and the low-density-lipoprotein-receptor-related family in freshly isolated pancreatic islets and in the differentiated members to allow proper signaling specificity (Davis, 2000; insulin-secreting β-cell line INS-1. Exposure of primary Gotthardt et al., 2000; Meyer et al., 1999; Scheinfeld et al., pancreatic islets and INS-1 cells to cytokines drastically 2002; Stockinger et al., 2000; Verhey et al., 2001; Verhey reduced the IB1/JIP-1 content. Using adenovirus-mediated and Rapoport, 2001). In pancreatic β cells, IB1/JIP-1 was gene transfer of IB1/JIP-1 to increase or reduce the IB1/JIP-1 found to be highly produced and to modulate JNK activity content, we could modulate the content of the scaffold protein (Bonny et al., 1998; Bonny et al., 2000). Exposure of insulin- in primary pancreatic islets and in INS-1 cells. Decreasing secreting cell lines to cytokines reduces the IB1 content, IB1/JIP-1 content induced an increase in JNK-activity and a 1464 Journal of Cell Science 116 (8) concomitant increase in cytokine-mediated apoptosis rate, separate experiments was counted for each condition (Bonny et al., whereas cells with higher levels of IB1/JIP-1 are protected 2000; Hoorens et al., 2001). Mouse islets were cultured for 48 hours against the cytokine-induced apoptosis. Last, we assessed the and then fixed in 1% paraformaldehyde, stained with Hoechst 33342 sensitivity of isolated pancreatic islets obtained from and mounted in mowiol (Calbiochem) before counting the apoptotic haploinsufficient mice (IB1/JIP-1+/–) and found that, compared nuclei. with wild-type pancreatic islets, the reduction of IB1/JIP-1 content is associated with an increase JNK activity and basal Western blotting apoptosis. Polyclonal IB1/JIP-1 antiserum was described previously (Bonny et al., 1998). These antibodies were affinity purified from crude serum using a Hitrap NHS-activated affinity Sepharose column (Amersham Materials and Methods Pharmacia Biotech, Switzerand) coupled to the immunogenic protein. Rodent islet isolation and cell lines Polyclonal antibodies against cleaved caspase-3 (Asp175, CST) and Our institutional review committee for animal experiments approved monoclonal antibodies against α-tubulin (T5168, Sigma, Switzerland) all the procedures for rat and mouse care, surgery and euthanasia. were diluted 1:100 and 1:10,000, respectively. The cells were lysed in Adult rats weighing 250-350 g were anesthetized using Halothane 5% SDS. Protein content was determined using the DC protein assay (Arovet AG, Switzerland), sacrificed and immediately used for reagent kit (Bio-Rad Laboratories, Switzerland). Aliquots were pancreas sampling. Rat islets were isolated from male Wistar rats by fractionated by electrophoresis in a 10% polyacrylamide gel and collagenase (NJ, USA) digestion. The pancreas were excised and immunoblotted onto Immobilon PVDF membranes (Millipore, MA) placed in a 2 mg ml–1 collagenase solution (collagenase type IV; overnight, at a constant voltage of 30 V. The membranes were blocked Worthington) in Hank’s balanced salt solution containing 10 mmol l–1 for 3 hours at room temperature in PBS containing 3% bovine serum Hepes, pH 7.4. Tissue was minced and incubated for 40 minutes at albumin and 0.1% Tween 20, and then incubated for 24 hours with 37°C. After purification on a Ficoll gradient (Sigma, Switzerland), the antibodies diluted in blocking buffer. After repeated rinsing in PBS and isolated islets were washed twice in a phosphate-buffered saline (PBS) PBS + 0.1% Tween20, immunoblots were incubated overnight at 4°C and the islets were cultured in RPMI-1640 supplemented with 10% with an anti-mouse antibody coupled to alkaline phosphatase (Dako fetal calf serum, 10 mmol l–1 Hepes, 1 mmol l–1 sodium pyruvate and Diagnostic, Zug, Switzerland), diluted 1:5000. Specific antigen- 50 µmol l–1 β-mercaptoethanol. The isolated islets were then exposed antibody complexes were detected using the alkaline phosphatase for 6-7 minutes at 37°C to the same medium supplemented with 0.16 development reagent kit (BCIP-NBT) method (AP development mg ml–1 trypsin (1:250) (Gibco, Grand Island, NY). The incubation reagent, BioRad Laboratories, Glattburg, Switzerland). For the was stopped by the addition of 10 ml ice-cold Krebs-Ringer- Phototope-HRP western blot detection system, membranes were bicarbonate buffer, supplemented with 0.5% bovine serum albumin incubated for 1 hour at room temperature in PBS containing 5% milk (BSA), 2.8 mM glucose and 10 mM Hepes, pH 7.4 (KRB+ buffer). and 0.1% Tween 20 (blocking buffer) and then incubated for 2 hours at The resulting suspension, which comprised mostly single cells, was room temperature with antibodies directed against IB1/JIP-1. Specific centrifuged for 5 minutes at 130 g. The pellet was resuspended in antigen-antibody complexes were detected with the Phototope-HRP KRB+ buffer to obtain a final concentration of 3×106 cells ml–1. western blot detection system (Amersham, Switzerland). For mouse islet isolation, pancreases were excised and placed in a 2 mg ml–1 collagenase solution (collagenase type IV; Worthington) in Hank’s balanced salt solution containing 10 mmol l–1 Hepes, pH 7.4. Protein-kinase assays Tissue was minced and incubated for 38 minutes at 37°C. After JNK activity was measured using glutathione-S-transferase/c-Jun(1-79) washing of the digested tissue, islets were hand picked under a bound to glutathione/Sepharose-4B (Bonny et al., 1998; Tawadros et stereomicroscope. Rodent islets were cultured in RPMI-1640 al., 2002). INS-1 cells or rodent islets were lysed in 0.5% Nonidet P- supplemented with 10% fetal calf serum, 10 mmol l–1 Hepes, 40, 20 mM Tris-HCl, pH 7.6, 0.25 mM NaCl, 3 mM EDTA, 3 mM 1 mmol l–1 sodium pyruvate and 50 µmol l–1 β-mercaptoethanol EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (Guillam et al., 2000).