Activation of activating transcription factor by p38 MAP kinase during apoptosis induced by human amylin in cultured pancreatic b-cells Shaoping Zhang1, Hong Liu1, Junxi Liu1, Cynthia A Tse1, Michael Dragunow2 and Garth J S Cooper1 The School of Biological Sciences, Faculty of Science, University of Auckland, New Zealand Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, New Zealand Keywords activating transcription factor 2; amylin; b-cell apoptosis; p38 kinase; type-2 diabetes Correspondence G J S Cooper, School of Biological Sciences, University of Auckland, Level 4, 3A Symonds Street, Private Bag 92019, Auckland, New Zealand Fax: +64 93737045 Tel: +64 93737599 ext 87239 E-mail: g.cooper@auckland.ac.nz (Received May 2006, accepted 16 June 2006) doi:10.1111/j.1742-4658.2006.05386.x Amylin-mediated islet b-cell death is implicated in diabetogenesis We previously reported that fibrillogenic human amylin (hA) evokes b-cell apoptosis through linked activation of Jun N-terminal kinase (JNK 1) and a caspase cascade Here we show that p38 kinase [p38 mitogen-activated protein (MAP) kinase] became activated by hA treatment of cultured b-cells whereas extracellular signal-regulated kinase (ERK) did not; by contrast, nonfibrillogenic rat amylin (rA) altered neither Pretreatment with the p38 kinase-inhibitor SB203580 decreased hA-induced apoptosis and caspase-3 activation by 30%; as did combined SB203580 and JNK inhibitor I, by about 70%; and the combination of SB203580, the JNK inhibitor I and a caspase-8 inhibitor, by 100% These findings demonstrate the requirement for concurrent activation of the p38 kinase, JNK and caspase-8 pathways We further showed that hA elicits time-dependent activation of activating transcription factor (ATF-2), which was largely suppressed by SB203580, indicating that this activation is catalyzed mainly by p38 kinase Furthermore, hA-induced apoptosis was suppressed by specific antisense ATF-2, and increased phospho-ATF-2 (p-ATF-2) was associated with increased CRE (cAMP-response element) DNA binding and CRE-mediated transcriptional activity, as well as enhancement of c-jun promoter activation We also detected changes in the phosphorylation status and composition of the CRE complex that may play important roles in regulation of distinct downstream target genes These studies establish p38 MAP kinase-mediated activation of ATF-2 as a significant mechanism in hA-evoked b-cell death, which may serve as a target for pharmaceutical intervention and effective suppression of b-cell failure in type-2 diabetes Progressive b-cell loss and defective insulin production and secretion accompanied by the presence of islet amyloid deposits are characteristic pathological features of type diabetes mellitus (T2DM) [1–3] Current studies have indicated that amyloid formation may contribute to the development of hyperglycemia by causing islet dysfunction [4,5] The major protein component of islet amyloid has been identified as a 37 amino acid peptide, called amylin (also known as islet amyloid polypeptide) [6–8] Human amylin (hA) can self-assemble to form b-sheet-containing aggregates that are cytotoxic to b-cells, as observed in vitro and Abbreviations AP-1, activator protein-1; AS-jnk1, antisense jnk1; ATF-2, activating transcription factor 2; CAT, chloramphenicol acetyltransferase; CRE, cAMP-response element; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GST, glutathione S-transferase; hA, human amylin; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; p38 kinase, p38 MAP kinase; p-ATF-2, phosphorylated activating transcription factor 2; rA, rat amylin; T2DM, type diabetes mellitus FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS 3779 ATF-2 activation mediates hA-evoked apoptosis S Zhang et al in vivo [9–13] In contrast, rat amylin (rA), whose sequence varies from the human at six residues, does not aggregate and exhibits random conformations in physiological solutions [9,14] Extracellular application of fibrillogenic hA, but not nonfibrillogenic rA, induces apoptosis in cultured human and rat b-cells [10,15,16] In addition, onset of diabetes, associated with islet amyloid formation and decreased b-cell mass, has been demonstrated in transgenic mice expressing hA in their b-cells [13,17] Formation of amylin aggregates in the pancreatic islets may thus play an important role in triggering islet b-cell death and dysfunction in T2DM Since all humans produce amylin with the propensity to self-assemble, but most not lose b-cell mass and develop diabetes, the mechanism by which amylin aggregates and causes cytotoxicity is attracting increasing attention as a potential molecular target for pharmacological intervention Several putative molecular mechanisms by which hA might lead to or cause b-cell toxicity have been identified One envisages that hA evokes b-cell toxicity through apoptosis (programmed cell death), wherein contact of protein aggregates with b-cell membranes is necessary for death induction [10,11,16] Another possibility is increased cellular pro-oxidant responses and low density lipoprotein uptake evoked by aggregate– cell interactions [18] Small or intermediate-sized hA aggregates ⁄ oligomers, rather than monomers or large, mature amylin fibrils, have been associated with b-cell membrane leakage, instability and apoptosis [19,20] More recent studies suggest that Ca2+ signaling disruptions may be the common mechanism for oligomermediated cytotoxicity in many amyloidogenic diseases including T2DM [21] Thiol reducing agents can prevent hA-induced b-cell cytotoxicity [22] In addition, it is now known that hA-induced b-cell apoptosis entails alterations in RNA and protein synthesis from genes such as p53, p21WAF1 ⁄ CIP1 and c-jun [10,15,16] We previously showed that hA elicited b-cell apoptosis via stimulated expression and activation of c-Jun accompanied by increased activator protein-1 (AP-1) DNA binding and c-Jun transcriptional activation [15] We also found that fibrillogenic amylin evoked b-cell apoptosis through linked activation of a caspase cascade and Jun N-terminal kinase (JNK) [23] However, non-b-sheet forming ⁄ nonfibrillogenic amylin variants such as rA or triprolyl-hA elicited neither apoptosis nor caspase ⁄ JNK activation [23] Together, these findings support the hypothesis that small hA aggregates or oligomers interact with b-cell membranes in a specific, conformation-dependent manner that in turn activates specific intracellular signal transduction pathways that elicit apoptosis 3780 The intracellular signaling pathways mediating hA-induced b-cell apoptosis are incompletely understood hA-evoked cytotoxicity has been associated with activation of JNK and p38 mitogen activating protein (MAP) kinase (p38 kinase) followed by caspase-3 activation [24] Mitogen-activated protein kinases (MAPKs) are a group of protein serine ⁄ threonine kinases that play central roles in cellular responses to various extracellular stimuli [25–27] In general, the extracellular signal-regulated kinase (ERK) pathway is required for cell proliferation and differentiation [27,28] Conversely, the JNK and p38 kinase pathways are preferentially activated by genotoxic agents and cytokines, and tend to mediate the stress response, growth arrest and apoptotic pathways [26,29,30] However, activation of ERK1 ⁄ was reported to contribute to cytokine-evoked apoptosis in primary rat pancreatic b-cells [31] Stressactivated JNK and p38 kinase were also reportedly associated with cell proliferation, anticytotoxicity and antiapoptotic activity [32–34] Thus, these MAPK pathways fulfill complex physiological roles in mediation of distinct cellular responses in different cell lineages MAPK may either contribute to or prevent cell death, depending on the duration of activation and the balance of activity between the MAP kinase, ERK, and the stress-activated kinases, JNK and p38 [26] Activation of MAPK-mediated signaling pathways could result in phosphorylation of several protein targets, including activating transcription factor (ATF2) This protein regulates gene expression by binding either to cAMP-response element (CRE) DNA response elements as a homodimer, or to both AP-1 and CRE sequences as a heterodimer, which it can form with other members of the ATF family or with Jun ⁄ Fos family members [35,36] The most common of these is the ATF-2 ⁄ c-Jun heterodimer that recognizes both AP-1 and CRE sites in the promoter regions of its target genes The c-jun gene is a major ATF-2 target, and both c-Jun and ATF-2 are influential regulators of its expression [37] ATF-2, together with c-Jun, has been implicated in a wide variety of biological processes, for example, neuronal apoptosis [38] ATF-2 activity is regulated by phosphorylation of Thr69 and Thr71 residues in its NH2-terminal region [39], and either JNK or p38 kinase can catalyze these phosphorylation events in vitro and in vivo [27,40,41] We have previously shown that human amylin elicits c-Jun activation in islet b-cells, through activation of the JNK pathway [15,23] In the current study, we planned to determine whether either of the other two MAP kinases, ERK and p38 kinase, and activation of their target transcription factors, such as ATF-2, could modulate this apoptotic pathway If they do, how might they co-operate to control FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS S Zhang et al ATF-2 activation mediates hA-evoked apoptosis CRE-mediated transcriptional activity of downstream genes? The results from the current study were expected to contribute to a better molecular understanding of nuclear events that occur in b-cells in response to hA treatment Results Increased p38 kinase activity, but not ERK activity, in hA-induced b-cell apoptosis Given the important role played by MAPK in the regulation of transcription factor activities and gene expression, we sought here to investigate whether hA treatment might increase the activity of ERK1 ⁄ or p38 kinase, and whether such activation by phosphorylation could contribute to subsequent hA-evoked b-cell death Two insulinoma b-cell lines, rat RINm5F and human CM, were cultured and exposed to hA for various periods The hA solutions employed were prepared in water as previously described [16] We have analyzed equivalent preparations and shown them to contain polymorphic fibrillar structures, composed predominantly of protofibrils, and also minute soluble oligomeric aggregates [14,42] We believe that the latter are likely to directly elicit hA-mediated cytotoxicity The amylin concentrations and time-points used in these experiments were based on previous studies in which hA-elicited activation of the caspase cascade and the JNK pathway were characterized [23] The calculated EC50-value for the concentration dependence of hA cytotoxicity was determined to be 10 lm [43] Studies of time dependence of cell killing by 10 lm hA indicated that cell death reached half-maximal after 24 h Figure shows that hA induced time-dependent activation of p38 kinase, which was detectable from h, peaked at h, then declined by h and had returned to the 1-h level by 16 h after treatment In contrast, hA rA 0h 1h 2h 4h 8h 16h 24h 1h 4h 8h 24h 1.0 2.9 4.8 3.3 1.6 0.6 ratio -p-p38 CM 1.0 2.1 3.7 1.9 1.2 0.5 RINm5F ratio -p-p38 Fig Western blot analysis of p-p38 kinase protein in RINm5F and CM cells Total cell extracts were prepared from cells treated with 10 lM hA or rA at the indicated time-points and subjected to western blot analysis using anti-p-p38 kinase IgG Fold induction of p-p38 kinase (shown as a ratios) was calculated based on levels at h, which were set at one All results shown are the average of three independent experiments hA did not activate ERK1 ⁄ over the 24-h time course studied (data not shown) Thus, hA treatment elicited activation of p38 kinase, but not pERK1 ⁄ 2, in both RINm5F and CM b-cells Furthermore, nonfibrillogenic rA activated neither ERK1 ⁄ nor p38 kinase activity, as shown in Fig 1, indicating that sequence differences between hA and rA and the fibrillogenic potential of the human peptide are required for hAinduced p38 kinase activation In contrast, the same effects are not observed when b-cells are exposed to solutions containing large mature hA fibrils, prepared by dissolution in NaCl ⁄ Pi and 7-day incubation prior (data not shown) This finding is consistent with the current view that the early aggregates rather than the mature fibrils, are the primary toxic species [20,44] Similar results regarding hA-elicited p38 kinase activation were obtained in studies wherein an immunocomplex kinase assay was employed (shown in Fig 2A) Here, p38 kinase immunoprecipitated from 4-h hAtreated cells catalyzed phosphorylation of glutathione S-transferase (GST)-ATF-2, whereas phosphorylation of GST-Elk-1 mediated by ERK1 ⁄ immunoprecipitation did not increase with 4-h treatment and did not differ from that in untreated controls These results are consistent with the observations obtained from direct western blot analysis described above In addition, a dose-dependence experiment showed that p38 kinase activity increased with hA concentrations (Fig 2B), demonstrating a dose-responsive effect of hA on activation of p38 kinase In parallel experiments, we studied the effects of inhibition of ERK1 ⁄ and p38 kinase on hA-induced caspase-3 activation and apoptosis Figure shows that pretreatment of RINm5F or CM cells with the selective p38 kinase-inhibitor SB203580 for h prior to hA exposure significantly inhibited apoptosis by 32% (Fig 3A) and caspase-3 activation by 30% (Fig 3B) In contrast, no pretreatment with either SB202474 (negative inhibitor-control) or PD98059 (inhibitor of the kinase upstream from ERK1 ⁄ 2) elicited increased apoptosis or caspase-3 activation when compared with non-pretreated controls Thus, activation of p38 kinase, but not the ERK pathway, contributes to the molecular mechanism through which hA induces b-cell apoptosis The inhibitory effect of SB203580 was incomplete, however, indicating that p38-kinase activation is not the only mechanism by which hA induces b-cell apoptosis We detected a further reduction in caspase-3 activation and apoptosis in cells pretreated with combined SB203580 and JNK inhibitor I (70% reduction in total), and full suppression with the combination of SB203580, JNK inhibitor I and caspase-8 inhibitor (Fig 3A,B) In addition, treatment of cells with hA and JNK inhibitor I can FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS 3781 ATF-2 activation mediates hA-evoked apoptosis S Zhang et al GST-ATF-2 (p38 immunoprecipitated) GST-Elk-1 (ERK immunoprecipitated) Relative phosphorylation A 4.5 3.5 2.5 1.5 0.5 * * co hA rA co B hA rA RINm5F hA rA Relative phosphorylation p38 immunoprecipitated CM * * * * * * 0 10 CM 20 40 10 20 40 μΜ RINm5F Fig Immunocomplex kinase assay for hA-induced ERK and p38 kinase activity (A) Total cell extracts were prepared from RINm5F and CM cells untreated (co) or treated with 10 lM of hA or rA for h Whole cell kinase activity assay was performed using ERK and p38 kinase immunoprecipitated with c-32P-ATP and GST-Elk-1 (for ERK assay) or GST-ATF-2 (for p38 kinase assay) as substrates The phosphorylation reactions were visualized by autoradiography after SDS ⁄ PAGE, quantified by PhosphorImager and presented relative to untreated control The results are mean ± SEM of three independent experiments, each performed in duplicate *P < 0.01 versus respective controls (B) RINm5F and CM cells were untreated (co) or treated with various concentrations of hA or rA for h as indicated p38 kinase activity was measured as described above using p38 kinase immunoprecipitated with c-32P-ATP and GSTATF-2 as substrates significantly but not fully suppress caspase-3 activity and apoptosis, whereas the inhibitors themselves did not prevent apoptosis in the absence of hA (data not shown) These findings support a mechanism in which multiple apoptotic pathways, including those mediated via JNK, p38 and initiator caspase-8, cooperate to mediate hA-evoked b-cell apoptosis Increased phosphorylation of ATF-2 in response to hA treatment is catalyzed mainly by p38 kinase We examined protein expression and phosphorylation of ATF-2 by p38 kinase during hA-evoked b-cell 3782 Fig Effects of MAPK inhibitors on hA-induced apoptosis and activation of caspase-3 Cultured RINm5F and CM cells were pre-incubated with specific MAPK inhibitor alone (SB203580, JNK inhibitor I or PD98059), or combinations of inhibitors (SB203580 + JNK inhibitor I) or (SB203580 + JNK inhibitor I + caspase-8 inhibitor) or inhibitor-negative control (SB202474) for h before exposure to hA (A) Apoptosis was assessed after 24-h exposure using a quantitative cell death detection ELISA Results shown represent enrichment of nucleosomes (fragmented DNA) (B) Caspase-3 activity was determined after 16-h hA-exposure using synthetic fluorogenic oligopeptide substrate z-DEVD-AFC The fluorescence was measured at excitation k ¼ 400 nm and emission ¼ 540 nm All data were presented relative to the untreated control (co) and calculated as mean ± SEM of four independent experiments, each performed in duplicate †P < 0.01 versus control; * P < 0.01 versus hA-treated cells FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS S Zhang et al 1.0 co hA hA +S B2 +J 358 N K in hA hi +A b I Sjn hA k +P D 98 05 indicating that increased activation of ATF-2 is correlated with induction of apoptosis and the ability of hA to form b-sheet-containing aggregates In contrast, we found that levels of nonphosphorylated ATF-2 were unaffected by hA treatment throughout the 24-h study (Fig 4B) In addition, hA-induced apoptosis was suppressed by specific antisense ATF-2, demonstrating the important role played by ATF-2 in cell death (Fig 4C) Effects of hA on ATF-2 mRNA expression were also measured using quantitative RT-PCR, which showed that tissue ATF-2 mRNA content remained unchanged throughout this period (data not shown) Thus, hA treatment had no measurable effect on ATF-2 mRNA or protein expression, and hA-stimulation of ATF-2 activity was not attributable to enhanced tissue ATF-2 content ATF-2 is a transcription factor whose activation can be catalyzed by either p38 or JNK, or by both [27,41] To further clarify the role of the MAPKs in hA-evoked activation of ATF-2, we used selective MAPK inhibitors to ascertain the major upstream kinase that activates ATF-2 Figure 5A shows that hA apoptosis Figure shows the result of a representative western blot analysis using specific antibodies for ATF-2 and phosphorylated activating transcription factor (p-ATF-2) Human amylin-induced apoptosis in CM cells was accompanied by time-dependent increases in phosphorylation (activation) of ATF-2 (Fig 4A) Phosphorylated ATF-2 had reached maximal levels by h after initiation of hA treatment (four- to five-fold increase), which coincided with the time at which the level of p-p38 kinase had increased Augmented phosphorylation of ATF-2 was also detected in RINm5F cells after hA treatment (data not shown) However, no increment in p-ATF-2 level was detected in cells treated with either vehicle alone or noncytotoxic rA in either RINm5F or CM cells, ATF-2 activation mediates hA-evoked apoptosis 0.19 0.81 0.79 1.1 A ratios - pATF-2 1.0 0.42 0.38 0.45 0.92 B ratios - p-c-JunSer63 C Fig Activation of ATF-2 is required for human amylin-induced b-cell apoptosis (A,B) Representative western blot analysis of time-dependent activation and expression of ATF-2 Total cell extracts were prepared from CM cells untreated (co) or treated with 10 lM hA or rA at the indicated time-points Western blots were performed using anti-p-ATF-2 IgG (A) or ATF-2 IgG (B) and specific protein bands were visualized using ECL chemiluminescence reagent Fold induction of p-ATF-2 (shown as ratios) was calculated based on levels at h, which were set at one Results shown are the average of three independent experiments (C) RINm5F and CM cells were transfected with antisense ATF-2 (ASATF-2) or sense ATF-2 (S-ATF-2) for 24 h before exposure to hA Apoptosis was assessed after 24-h exposure using a quantitative cell death detection ELISA All data were presented relative to the untreated control (co) and calculated as mean ± SEM of four independent experiments, each performed in duplicate †P < 0.01 versus control; *P < 0.01 versus hA-treated cells D - ATF-2 1.0 0.37 0.40 0.36 0.96 ratios - c-Jun Fig Effects of inhibition of MAPK on hA-evoked expression and activation of ATF-2 and c-Jun (A) CM cells were pre-incubated with specific MAPK inhibitors (SB203580, PD98059 or JNK inhibitor I) for h or transfected with AS-jnk1 for 24 h before exposure to hA Total cell extracts were prepared and subjected to western blot analysis using anti-p-ATF-2 IgG (B) The same western blot membrane as in (A) was stripped and re-probed with anti-p-c-Jun IgG (C) Cell treatments were performed as in (A) and western blot analyzed using anti-ATF-2 IgG (D) Cell treatments were performed as in (A) and western blot analyzed using anti-c-Jun IgG All changes of protein levels were calculated based on those in corresponding hA-treated cells, which were set at one Results shown are the average of three independent experiments FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS 3783 ATF-2 activation mediates hA-evoked apoptosis S Zhang et al pretreatment of CM cells with SB203580 caused a large decline (averaging about 80%) in hA-induced ATF-2 phosphorylation compared with non-pretreatment controls Pretreatment of CM cells with JNK inhibitor I or transfection with antisense jnk1 (ASjnk1) caused lesser inhibitory effects (20% decrements), and PD98059 failed to decrease hA-induced ATF-2 phosphorylation at all, indicating that ATF-2 phosphorylation is catalyzed mainly by p38 kinase and, to a lesser extent, by JNK1 Similar effects were observed following pretreatment with MAPK inhibitors of RINm5F cells, wherein we also found that SB203580 could largely inhibit hA-induced ATF-2 phosphorylation (data not shown) Therefore, p38 kinase rather than JNK is the primary upstream kinase for ATF-2 in hA-induced b-cell apoptosis It is known that, unlike JNK, p38 kinase does not directly phosphorylate c-Jun [27] However, we set out to investigate whether p38 activation has any ultimate indirect downstream effect on hA-induced activation of c-Jun The same western blot membrane, which had been used for analysis of p-ATF-2 above, was stripped and re-probed with the p-c-JunSer63-specific antibody The results shown in Fig 5B demonstrate that suppression of p38 kinase activation by SB203580, as well as suppression of JNK1 activation by JNK inhibitor I and AS-jnk1, caused equal inhibition of c-Jun phosphorylation ATF-2 and c-Jun protein levels were analyzed by western blot, as shown in Fig 5C,D ATF-2 protein level was unchanged and c-Jun protein levels were equivalently decreased by pretreatment with SB203580, JNK inhibitor I, or AS-jnk1 These data indicate that the decreased c-Jun phosphorylation evoked by SB203580 may be due to decreased c-jun transcription Furthermore, we found that inhibition of c-jun expression was more pronounced by simultaneous treatment with both SB203580 and JNK inhibitor I, indicating that p38 kinase and JNK1 act co-operatively to control c-Jun expression and activation In addition, pretreatment of PD98059 did not decrease activity of either p-ATF-2 or p-c-JunSer63 (Fig 5A,B), further indicating that hA-elicited activation of c-Jun and ATF-2 are independent of the ERK pathway Activation of ATF-2 is associated with increased CRE-DNA binding activity To determine whether increased ATF-2 activation following hA treatment is associated with a change in the DNA binding activity at the CRE site, nuclear proteins were extracted from 8-h hA-treated and untreated RINm5F and CM cells and subjected to electrophoretic mobility shift assay (Fig 6A) Two shifted bands 3784 corresponding to two different forms of CRE DNAbinding complexes were detected ATF-2-CRE DNA binding activity was markedly induced following 8-h hA treatment, as shown by increased intensities of both shifted bands in hA-treated cells In addition, the increased CRE binding activity was suppressed by JNK inhibitor I and SB 203580, implying that hA-evoked CRE binding is mediated by both the JNK and the p38 kinase pathways Additionally, supershift assays (antibody pre-incubations) were performed to determine which types of CRE-binding protein complexes were induced upon hA treatment We detected the appearance of two supershifted bands, corresponding to the CRE-antibody supershifted complexes, in hA-treated cells following pre-incubation of antibody against p-c-JunSer63, indicating that both of these shifted CRE complexes contain p-c-JunSer63 (Fig 6B) We also found that pre-incubation with antibodies against c-Jun, ATF-2 or p-ATF-2 enable competition of these antibodies on binding of labeled CRE to protein complexes (shown by weakening in the two shifted bands) However, pre-incubation of specific blocking peptide with these antibodies before incubation with nuclear extract, did not weaken the shift bands (data not shown) Thus c-Jun and p-c-JunSer63, ATF-2 and p-ATF-2 are all part of the two CRE-binding complexes in hA-treated cells Interestingly, the antibodies for p-c-JunSer63 and p-ATF-2 were more efficient than the antibodies for unphosphorylated ATF-2 and c-Jun in supershifting or competing with the CRE complexes, suggesting that these complexes are mainly composed of the active forms of c-Jun and ATF-2 In contrast, only antibodies for unphosphorylated ATF-2 and c-Jun competed with binding of labeled DNA to the CRE complex from untreated control cells, indicating that the unphosphorylated forms of ATF-2 and c-Jun are the major components of the complexes associated with CRE DNA sequences in untreated control cells Taken together, our results demonstrate changes in protein composition and phosphorylation state of the CRE-binding complexes, with the emergence of functionally significant p-ATF-2 and p-c-JunSer63 in hA-treated apoptotic cells Activation of ATF-2 increases transcriptional transactivation potential of ATF-2 The correlation of ATF-2 activation with CREmediated transcriptional activity after hA treatment was studied using a CRE-driven luciferase reporter construct (pCRE-luc) CM cells were transiently transfected with pCRE-luc and luciferase activity was FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS S Zhang et al ATF-2 activation mediates hA-evoked apoptosis B RINm5F p co+anti-c-Jun co+anti-ATF-2 co+anti-p-c-Jun co+anti-p-ATF-2 co hA hA+anti-p-c-Jun hA+anti-p-ATF-2 hA+anti-c-Jun hA+anti-ATF-2 p CM co hA hA+specific oligo hA+non-specific oligo hA+JNK inhib I hA+SB203580 co hA hA+specific oligo hA+non-specific oligo hA+JNK inhib I hA+SB203580 A p-c-Jun-CREcomplexes CREcomplexes _ _ _ _ _ _ _ _ _ _ _ CRE_ complexes Fig Representative electrophoretic mobility shift assays of CRE-DNA binding activated by hA (A) The binding reactions were performed using nuclear extracts prepared from RINm5F and CM cells that had been treated with hA or vehicle control (co) for h Nuclear extracts were also prepared from cells that had been pre-incubated with SB203580 or JNK inhibitor I (JNK inhib I) before treatment with hA For the assay of CRE binding specificity, nuclear extracts were incubated for h with unlabelled specific or nonspecific oligonucleotides, respectively, before addition of labeled CRE probe P denotes reaction containing only labeled CRE probe without nuclear extract (B) Supershift experiments were carried out by incubation of nuclear extracts with different antibodies for h, before addition of labeled CRE probe The binding reaction samples were then analyzed as described above measured to determine the effects of amylin on modulation of CRE-mediated transcriptional activity Results demonstrate that treatment of transfected cells with hA caused increased transactivation activity in comparison with untreated control samples, as measured by increased production of relative light units of luciferase activity (Fig 7) Luciferase activity reached maximum induction at h (about four-fold increase), which coincided with the observed elevation in CREbinding activity In contrast, noncytotoxic rA, which does not elicit ATF-2 activation, had no effect on CRE-mediated transcriptional activation Thus, hA activates CRE-driven gene transcription and the increased transactivation potential of ATF-2 is correlated with hA’s fibrillogenic and cytotoxic properties Also shown in Fig is evidence that suppression of p38 kinase by SB203580 inhibits ATF-2-mediated transcriptional activation Together, these data demonstrate a role for the p38 kinase-mediated signal transduction pathway in transcriptional responses mediated by ATF-2 in hA-treated b-cells The cooperative effect of JNK and p38 kinase on CRE-mediated transcriptional activation was also demonstrated by inhibition of luciferase expression using JNK inhibitor I, as well as its simultaneous use with SB203580 We showed that suppression of CRE-luciferase activity was more pronounced by combined inhibition of JNK and p38 kinase (Fig 7) Control treatment of transfected cells with inhibitors alone had no effect on luciferase activity (data not shown) To determine whether hA-stimulated activation of ATF-2 activates ATF-2-dependent transcription of c-jun, a time course experiment was performed wherein CM cells were transfected with a c-jun promoterchloramphenicol acetyltransferase (CAT) reporter construct CAT activity was measured at various timepoints in transfected-cells pretreated with SB203580, which selectively inhibits phosphorylation of ATF-2 but not of c-Jun, and hA stimulated ATF-2-mediated c-jun expression (Fig 8) The maximum induction of CAT activity was about three- to four-fold above control values following h of exposure, whereas in contrast, CAT activity remained consistently low in rA-treated b-cells The time at which the CAT activity FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS 3785 ATF-2 activation mediates hA-evoked apoptosis S Zhang et al Fig Analysis of CRE driven luciferase activity induced by hA treatment CM cells were transfected with a CRE-driven luciferase reporter construct (pCRE-luc) for 24 h before exposure to hA, rA or vehicle control (co) for various times as indicated Transfected CM cells were also pre-incubated with SB203580, JNK inhibitor I or with combination of SB203580 and JNK inhibitor I before exposure to hA Cell lysates were then prepared and analyzed using a luciferase reporter gene assay system Resulting values, shown as relative light units, are mean ± SEM of four independent experiments, each performed in duplicate †P < 0.01 versus control; *P < 0.01 versus hA-treated cells reached maximal coincided with the observed elevation in CRE binding and CRE-mediated transcriptional activity SB203580, a suppressor of p38 kinase-mediated ATF-2 activation, inhibited c-jun promoter transactivation Thus, activated ATF-2 and p38 kinase play critical roles in stimulation of c-jun transcription during hA-evoked b-cell apoptosis In addition, control treatment with inhibitors alone had no effect on CAT activity (data not shown); hA-induced c-jun promoter activation was partially suppressed by JNK inhibitor I and more completely suppressed by combined pretreatment with SB203580 and JNK inhibitor I (Fig 8), further indicating that both the JNK- and p38 kinasemediated pathways are necessary for hA-induced transactivation of c-jun gene expression Discussion We have previously shown that hA elicits islet b-cell apoptosis through activation of c-Jun and the JNK pathway [15,23] We have also shown that activated JNK1 interacts with a caspase cascade in controlling this apoptotic process [23] The objective of the current studies was to clarify possible roles of ERK and p38 kinase and their downstream target ATF-2, in hA-elicited b-cell apoptosis using the same b-cell lines, rat 3786 Fig Analysis of ATF-2 dependent c-jun promoter activation in hA treated CM cells Cells were transfected with a c-jun promoter-CAT reporting construct for 24 h before exposure to hA, rA or vehicle control (co) for various times as indicated Transfected CM cells were also pre-incubated with SB203580, JNK inhibitor I or with combination of SB203580 and JNK inhibitor I before exposure to hA Cell lysates were then prepared and analyzed using a CAT Elisa kit Results were presented as relative CAT activities based on untreated control levels, which were set at one All values are mean ± SEM of four independent experiments, each performed in duplicate †P < 0.01 versus control; *P < 0.01 versus hA-treated cells RINm5F and human CM that we previously employed in our studies of JNK activation The CM line was originally established from ascitic cells taken from a human subject with a malignant insulinoma [45] CM cells express genes typical of the islet b-cell lineage, such as insulin and certain of the GLUT genes, respond to glucose stimulation and posses a functional glucose-signaling pathway, thus representing a good model for studies of b-cell function and signaling [46] We show here that, in addition to the JNK pathway, the p38 kinase pathway is also required for hA-evoked b-cell apoptosis, whereas no role for the ERK pathway was apparent p38 kinase activation is related to the presence of fibrillogenic hA, and hA-induced activation of the p38 kinase pathway in b-cells is consistent with the general role of the p38 kinase pathway in cellular regulation of antiproliferation and apoptosis However, this pathway is only partially p38-dependent and targeting multiple pathways, including caspase-8, JNK and p38 kinase, is required for complete suppressed of hA-induced b-cell apoptosis Our results are supported by the report that hA, at nanomolar concentrations, FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS S Zhang et al induced strong and sustained phosphorylation of JNK and p38 kinase in RINm5F cells [24] Consistent with our current results, these data also indicate that ERK activation does not play a role in hA-induced RINm5F cell apoptosis, although therein an early ERK activation was detected at which the effect was not concomitant with JNK and ⁄ or p38 activation [24] We have shown here that hA elicits distinct and specific effects on phosphorylation of ATF-2 by p38 kinase-mediated signaling pathways, although a lesser effect of JNK was also detected Alterations in ATF-2 phosphorylation correspond closely with the previously observed pattern of changes in the levels of phosphorylated c-JunSer63 [15] p-ATF-2 has been identified previously as part of the AP-1 complex that regulates AP-1-mediated transcriptional activation evoked by hA in apoptotic b-cells [15] Collective results from previous and current studies indicate that ATF-2 could form homodimers with itself or heterodimers with c-Jun to bind to the specific AP-1 and CRE consensus sites in the promoter regions of target genes, including those of c-jun Inhibition of p38 kinase by SB203580, which decreases ATF-2 phosphorylation, could suppress induction of CRE binding and c-jun promoter activation in response to hA, consistent with the ability of activated-ATF-2 to transactivate the expression of target genes Although p38 kinase does not directly phosphorylate c-Jun, we detected a decrease in c-Jun activation as a result of pretreatment of b-cells with SB203580 This is likely because the suppression in p38 kinase activity caused by SB203580 can cause decreased p-ATF-2, which in turn lessens its binding to and transcriptional activation of the c-jun promoter The resulting decrease in c-jun expression would cause diminished amounts of c-Jun protein to be available for JNK1-mediated phosphorylation In addition, our studies with JNK and p38 kinase inhibitors showed that effects of direct and indirect inhibition of c-Jun phosphorylation were similar, indicating that there is no major competition between JNK and p38 kinase on direct and indirect activation of c-Jun Furthermore, JNK is responsible for increasing the activity of c-Jun during hA-evoked b-cell apoptosis, as shown by our previous study [15] However, although both JNK and p38 kinase elicited phosphorylation of ATF-2, our current data show that p38 is more important for the activation of ATF-2 evoked by hA in both our b-cell systems Thus, these parallel pathways may well converge at AP-1 and CRE sites, mediating hA-induced induction of expression of their target genes, including c-jun as demonstrated herein The composition of the ATF-2-associated transcription factor complexes may differ between various ATF-2 activation mediates hA-evoked apoptosis physiological and pathological states, so that even closely related members of the same protein family may contribute to quite distinct biological phenomena We have demonstrated changes in the protein composition and phosphorylation state of the CRE complex during hA-induced b-cell apoptosis The two shifted bands, corresponding to hA-induced CRE-binding complexes detected here, may represent different dimers formed from some of the identified components, including c-Jun, p-c-Jun, ATF-2 and p-ATF-2 This supports our idea that variation in CRE-complex composition and phosphorylation between hA-treated and untreated cells, can result in formation of different dimers that may have distinguishable CRE-binding specificity and activity Moreover, changes in the composition or phosphorylation state of CRE complexes can modulate their transcriptional activity and thereby alter target-gene specificity, leading to apoptosis in hA-treated b-cell systems Apoptosis is an important form of b-cell death in diabetes Formation of islet amyloid, rather than the presence of islet amyloid per se, was related to increased b-cell apoptosis in a mouse model of T2DM [5] We expect that our current investigations into the molecular mechanisms relating the structure of amylin aggregates ⁄ oligomers to their function and the associated b-cell apoptosis will ultimately lead to a better understanding of the causes of b-cell failure and islet dysfunction in T2DM These insights may allow the development of new approaches to preserve islet b-cell survival in vivo Moreover, the current findings may also be relevant to other forms of amyloid-associated cell death, such as occur in Alzheimer’s disease and the prion encephalopathies Experimental procedures Cell culture treatments For amylin treatment, peptide solutions were prepared by dissolving hA (Lot 524836; Bachem, Torrance, CA, USA) or rA (Lot ZM275; Bachem) in water and incubation at room temperature for 10 min, as previously described [15,16] Rat and human insulinoma cell lines, RINm5F and CM, were cultured and treated with hA or rA as previously described [15,16] Both cell lines were originally derived from transformed b-cells, and retain numerous differentiated features of their cell lineage (e.g insulin synthesis and secretion) For MAPK-inhibitor treatment, a selective p38 kinase inhibitor (SB203580), a selective ERK inhibitor (PD 98059) or negative inhibitor control (SB 202474) (Calbiochem, La Jolla, CA, USA) were prepared by dissolution in dimethyl sulfoxide The inhibitors were then added to RINm5F or FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS 3787 ATF-2 activation mediates hA-evoked apoptosis S Zhang et al CM cell cultures, to final concentrations of 10 lm or 100 lm, respectively, h before exposure to hA Alternatively, JNK inhibitor I or JNK inhibitor I-negative control peptides (Calbiochem) were dissolved in water and applied to cell cultures to final concentrations of lm, h before hA addition The doses selected have been tested and treatments with inhibitors alone shown to have no effect on b-cell proliferation and viability Quantitative cell death detection ELISA RINm5F and CM cells were cultured in 96-well plates in the presence or absence of specific MAPK inhibitors for h before exposure to hA for 24 h as described above For ATF-2 antisense and sense oligonucleotide transfection, cells were incubated with 0.2 lm phosphothiorate-modified antisense and sense ATF-2 (antisense: CACATGTAACTT GAATTTCAT and sense: ATGAAATTCAAGTTACAT GTG) using lipofectin reagent as previous described for transfection of antisense c-jun [15] Cells were then exposed to hA and apoptotic cell death measured using a cell deathdetection ELISA system (Roche Applied Science, Mannheim, Germany) as previously described [15,23] Caspase-3 activity assay RINm5F and CM cells were cultured on 24-well plates in the presence or absence of specific MAPK inhibitors for h before exposure to synthetic hA for 16 h, as described above Cells were then lyzed and caspase-3 activity assays performed as previously described [23] One hundred micrograms of each cell extract was incubated in reaction buffer containing 40 ngỈlL)1 of the specific fluorogenic caspase-3 substrate, Ac-DEVD-AFC (Bio-Rad Hercules, CA, USA), in the presence or absence of a caspase-3 inhibitor (z-DEVD-FMK), in a black 96-well plate at 37 °C for 3– h Caspase activity was determined by measuring the AFC released using a fluorescence MP reader (Spectra Max Gemini XS; Molecular Devices: excitation at 400 nm; emission at 540 nm) Western blot analysis RINm5F and CM cells were untreated, or treated with MAPK inhibitors as indicated, before exposure to hA or rA CM cells were also transfected with AS-jnk1 as previously described, before exposure to hA [23] Total cell lysates were then prepared and protein concentrations determined as previously described [15,23] Twenty-five micrograms of each whole-cell extract were separated by 12% SDS ⁄ PAGE, and Ponceau S staining was performed to confirm the equal loading Western blots were performed using either rabbit anti-p-p38 kinase (Cell Signaling, Beverly, MA, USA), rabbit anti-p-ERK1 ⁄ (Cell Signaling), 3788 rabbit anti-ATF-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-p-ATF-2 (Cell Signaling), mouse anti-p-c-JunSer63 (Santa Cruz Biotechnology) or rabbit anti-c-Jun (Oncogene Science, San Diego, CA, USA) Specific signals were detected using a horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse IgG (Jackson Immuno Research, Soham, UK) and an enhanced ECL reagent according to the manufacturer’s instructions (Roche Applied Science) Intensities of the reactive bands were determined by scanning autoradiography on an imaging densitometer (ScanMaker, Microtek) Immunocomplex kinase assay RINm5F and CM cells were cultured in six-well plates and exposed to hA as described above Cells were lyzed and kinase activities of p38 and ERK1 ⁄ determined by in vitro immunocomplex kinase assay, as described [47] Briefly, 100 lg of protein from each cell extract were incubated with lg of an antibody for ERK1 ⁄ or p38 kinase for h at °C, respectively, in the presence of protein A–Sepharose (Amersham Biosciences, Uppsala, Sweden) The immunocomplexes were then collected by centrifugation and resuspended in 30 lL of kinase reaction buffer (20 mm Hepes, pH 7.5, 20 mm b-glycerophosphate, 10 mm p-nitrophenol phosphate, mm MgCl2, mm 2-mercaptoethanol and 50 lm Na3VO4), containing 10 lCi of c-32P-ATP (Amersham Biosciences) Following incubation for 30 at 30 °C with lg of GSTATF-2 (for p38 kinase assay) or GST-Elk-1 (for ERK1 ⁄ assay), the reactions were terminated by addition of SDS ⁄ PAGE sample buffer and heating for at 95 °C The samples were then analyzed using 12% SDS ⁄ PAGE The protein bands (phosphorylated substrates) were analyzed using a phosphorImager (FLA 2040 Fuji, Japan) Electrophoretic mobility shift assay Cells were grown in T25 tissue culture flasks and either left untreated or treated with MAPK inhibitors before exposure to hA, as described above Cells were then harvested for preparation of nuclear extracts, as described [15] The double-stranded DNA-binding probe for the CRE complex was 5¢-end labeled with c-32P-ATP using T4 polynucleotide kinase (Invitrogen, Carlsbad, CA, USA) The top strand consensus sequence for complex binding was: 5¢-TCGATT GGCTGACGTCAGAGAGAG-3, where the CRE binding site is underlined CRE binding reaction and electrophoretic mobility shift assays were carried out as previously described [15] For competition experiments, unlabelled oligonucleotides, either specific (containing the CRE sequence), or nonspecific [containing the SP1 binding site (5¢-AT TCGATCGGGGCGGGGCGAGC-3¢) in 200-fold excess], were added to the reaction before addition of the labeled probe For supershift experiments, specific antibodies (anti- FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS S Zhang et al ATF-2, anti-p-ATF2, anti-c-Jun, anti-p-JunSer63) were mixed with nuclear protein extract for h prior to the addition of the labeled probe A total of 15 lL of the binding reaction mixture was then electrophoresed on 5% nondenaturing polyacrylamide gels and the DNA-protein binding signals were visualized by phosphor-imaging (FLA 2040, Fuji, Japan) Construction of c-jun promoter-reporting construct Total genomic DNA from human CM cells was isolated using DNAzol reagent (Invitrogen) as previously described [16] A 636 nt DNA fragment for the c-jun promoter was generated by PCR using primers: 5¢-CCCAAAACCACTG GCCTGGTTC-3¢ and 5¢-CACAGGCGCTAGATCTGGG CAG-3¢ This fragment was then cloned into a promoter activity assay vector pOPI3CAT (Stratagene, La Jolla, CA, USA) between BstX I and Bgl II restriction sites using standard molecular cloning techniques [48] The cloned c-jun promoter sequences were verified by DNA sequencing (DNA sequencing service, Centre for Genomics and Proteomics, the University of Auckland, New Zealand) Luciferase reporter gene assay CM cells were plated (12-well plates at a density of · 104 cellsỈwell)1) one day before transfection A total of 1.5 lg of either luciferase-reporter gene construct (pCRE-Luc; Stratagene) or vector DNA was transfected into cells using Fugene reagent (Roche) according to the manufacturer’s protocol Transfection efficiency was checked by cotransfection with 0.5 lg of pEGFP plasmid DNA (Clontech, Mountain View, CA, USA) and green fluorescent protein (GFP) expression measured (excitation at 395 nm; emission at 510 nm) Cells were pretreated with inhibitors and exposed to hA or rA for 1, 4, 8, 16 and 24 h, beginning 24 h after transfection Luciferase activity assays were performed using a highly sensitive luciferase reporter gene assay system (Roche) as previously described Luciferase activity was determined by measuring light emission at 562 nm using a luminescence MP reader (SpectraMAX Gemini XS; Molecular Devices), and the results were normalized relative to the levels of GFP expression CAT activity assay A total of 1.0 lg of either c-jun promoter-CAT reporter construct or vector DNA was transfected into cells using Fugene reagent (Roche) according to the manufacturer’s protocol Transfection efficiency was checked by cotransfection with 0.5 lg of pEGFP plasmid DNA (Clontech) as described above CM cells were subsequently pretreated with inhibitors and exposed to hA or rA for 1, 4, 8, 16 and ATF-2 activation mediates hA-evoked apoptosis 24 h, 24 h after transfection Cells were then harvested and CAT activity assays performed using a CAT Elisa kit (Roche) Briefly, cell extracts were prepared using the lysis buffer provided and 200 lg of each sample were incubated in the anti-CAT-coated MP modules (covered with foil) for h at 37 °C, followed by washing and addition of 200 lL of anti-CAT-DIG working solution The MP modules were further incubated for h at 37 °C and re-rinsed Two hundred microliters of anti-digoxigenin-peroxidase (anti-DIGPOD) was then added to each well and incubated a further h at 37 °C After washing, 200 lL of POD substrate 2,2¢azino-di-[3-ethylbenzthiazoline sulfonate (6)]diammonium salt (ABTS) with substrate enhancer was added to each well and incubated with shaking for about 30 at room temperature to enable photometric reaction Absorbance was measured at k ¼ 405 nm (MP reader; SPECTRA MAX 340, Molecular Devices) Results were normalized relative to the levels of GFP expression as described above Statistical analysis All results are presented as mean ± sem Differences between experimental groups were analyzed by paired Student’s t-tests or, in the case of multiple comparisons, by anova followed by Dunnett’s or Tukey’s post hoc multiple comparisons tests (to analyze more than two conditions) as appropriate Statistical significance was determined at P < 0.05 Acknowledgements We wish to thank P Pozzilli (Department of Diabetes and Metabolism, St Bartholomew’s Hospital, London, UK) for kindly providing the CM cells and H K Oie (NIH, Bethesda, MD, USA) for kindly providing the RINm5F cells We thank X Li for her enthusiastic assistance with statistical analyses This work was supported by the Endocore Research Trust, the Maurice and Phyllis Paykel Trust, and the New Zealand Lottery Grants Board, and by Programme Grants from the New Zealand Health Research Council to GC and 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c-Jun N-terminal kinase ⁄ p38 stress signaling in 1-b-darabinofuranosylcytosine-induced apoptosis Biochem Pharmacol 59, 407–418 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbour Laboratory, Cold Spring Harbor, NY FEBS Journal 273 (2006) 3779–3791 ª 2006 The Authors Journal compilation ª 2006 FEBS 3791 ... beta-cells J Mol Biol 324 , 27 1? ?28 5 16 Zhang SP, Liu JX, Saafi EL & Cooper GJS (1999) Induction of apoptosis by human amylin in RINm5F 3790 17 18 19 20 21 22 23 24 25 26 27 28 29 islet beta-cells... phosphorylation of ATF -2 in response to hA treatment is catalyzed mainly by p38 kinase We examined protein expression and phosphorylation of ATF -2 by p38 kinase during hA-evoked b-cell 37 82 Fig Effects of MAPK... ATF -2 and phosphorylated activating transcription factor (p-ATF -2) Human amylin -induced apoptosis in CM cells was accompanied by time-dependent increases in phosphorylation (activation) of ATF-2