Further-more, 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
Trang 1kinase during apoptosis induced by human amylin in
cultured pancreatic b-cells
Shaoping Zhang1, Hong Liu1, Junxi Liu1, Cynthia A Tse1, Michael Dragunow2and
Garth J S Cooper1
1 The School of Biological Sciences, Faculty of Science, University of Auckland, New Zealand
2 Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, New Zealand
Progressive b-cell loss and defective insulin production
and secretion accompanied by the presence of islet
amyloid deposits are characteristic pathological
fea-tures of type 2 diabetes mellitus (T2DM) [1–3]
Cur-rent 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
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 3 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 pre-viously reported that fibrillogenic human amylin (hA) evokes b-cell apopto-sis through linked activation of Jun N-terminal kinase 1 (JNK 1) and a caspase cascade Here we show that p38 kinase [p38 mitogen-activated pro-tein (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 2 (ATF-2), which was largely suppressed by SB203580, indicating that this activation is catalyzed mainly by p38 kinase Further-more, 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 tran-scriptional 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
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 2 diabetes mellitus.
Trang 2in 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 do not lose b-cell mass and
develop diabetes, the mechanism by which amylin
aggregates and causes cytotoxicity is attracting
increas-ing attention as a potential molecular target for
phar-macological intervention
Several putative molecular mechanisms by which hA
might lead to or cause b-cell toxicity have been
identi-fied 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
pos-sibility 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
dis-ruptions may be the common mechanism for
oligomer-mediated cytotoxicity in many amyloidogenic diseases
including T2DM [21] Thiol reducing agents can
pre-vent 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
accom-panied 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) 1 [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
The intracellular signaling pathways mediating hA-induced b-cell apoptosis are incompletely under-stood hA-evoked cytotoxicity has been associated with activation of JNK and p38 mitogen activating protein (MAP) kinase (p38 kinase) followed by caspase-3 acti-vation [24] Mitogen-activated protein kinases (MAPKs) are a group of protein serine⁄ threonine kinases that play central roles in cellular responses to various extra-cellular stimuli [25–27] In general, the extraextra-cellular sig-nal-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 apop-totic pathways [26,29,30] However, activation of ERK1⁄ 2 was reported to contribute to cytokine-evoked apoptosis in primary rat pancreatic b-cells [31] Stress-activated JNK and p38 kinase were also reportedly associated with cell proliferation, anticytotoxicity and antiapoptotic activity [32–34] Thus, these MAPK path-ways 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 bal-ance 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 tar-gets, including activating transcription factor 2 (ATF-2) 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 fam-ily 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 expres-sion [37] ATF-2, together with c-Jun, has been implica-ted in a wide variety of biological processes, for example, neuronal apoptosis [38] ATF-2 activity is regulated by phosphorylation of Thr69 and Thr71 resi-dues in its NH2-terminal region [39], and either JNK or p38 kinase can catalyze these phosphorylation events
in vitroand 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
Trang 3CRE-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⁄ 2 or
p38 kinase, and whether such activation by
phosphory-lation 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
var-ious periods The hA solutions employed were
pre-pared in water as previously described [16] We have
analyzed equivalent preparations and shown them to
contain polymorphic fibrillar structures, composed
pre-dominantly 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
calcula-ted 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
indica-ted that cell death reached half-maximal after 24 h
Figure 1 shows that hA induced time-dependent
activa-tion of p38 kinase, which was detectable from 1 h,
peaked at 4 h, then declined by 8 h and had returned
to the 1-h level by 16 h after treatment In contrast,
hA did not activate ERK1⁄ 2 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,
nonfibrillogen-ic rA activated neither ERK1⁄ 2 nor p38 kinase activ-ity, as shown in Fig 1, indicating that sequence differences between hA and rA and the fibrillogenic potential of the human peptide are required for hA-induced 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 acti-vation were obtained in studies wherein an immunocom-plex kinase assay was employed (shown in Fig 2A) Here, p38 kinase immunoprecipitated from 4-h hA-treated cells catalyzed phosphorylation of glutathione S-transferase (GST)-ATF-2, whereas phosphorylation
of GST-Elk-1 mediated by ERK1⁄ 2 immunoprecipita-tion 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 activa-tion of p38 kinase
In parallel experiments, we studied the effects of inhi-bition of ERK1⁄ 2 and p38 kinase on hA-induced caspase-3 activation and apoptosis Figure 3 shows that pretreatment of RINm5F or CM cells with the selective p38 kinase-inhibitor SB203580 for 1 h prior to hA expo-sure significantly inhibited apoptosis by 32% (Fig 3A) and caspase-3 activation by 30% (Fig 3B) In contrast,
no pretreatment with either SB202474 (negative inhib-itor-control) or PD98059 (inhibitor of the kinase upstream from ERK1⁄ 2) elicited increased apoptosis or caspase-3 activation when compared with
non-pretreat-ed controls Thus, activation of p38 kinase, but not the ERK pathway, contributes to the molecular mechanism through which hA induces b-cell apoptosis The inhibi-tory effect of SB203580 was incomplete, however, indi-cating 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 addi-tion, treatment of cells with hA and JNK inhibitor I can
ratio 1.0 2.9 4.8 3.3 1.6 0.6
-p-p38
CM
0h 1h 2h 4h 8h 16h 24h 1h 4h 8h 24h
-p-p38
ratio 1.0 2.1 3.7 1.9 1.2 0.5
RINm5F
Fig 1 Western blot analysis of p-p38 kinase protein in RINm5F
and CM cells Total cell extracts were prepared from cells treated
with 10 l M 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
1 h, which were set at one All results shown are the average of
three independent experiments.
Trang 4significantly 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
0
1
2
3
4
5
6
7
8
0 5 10 20 40 0 5 10 20 40
hA rA
*
*
*
*
*
*
μΜ
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
co hA rA co hA rA
*
*
GST-ATF-2 (p38 immunoprecipitated)
GST-Elk-1 (ERK immunoprecipitated)
A
B
Fig 2 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 l M of hA or rA for
4 h Whole cell kinase activity assay was performed using ERK and
p38 kinase immunoprecipitated with c- 32 P-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
inde-pendent experiments, each performed in duplicate *P < 0.01
ver-sus respective controls (B) RINm5F and CM cells were untreated
(co) or treated with various concentrations of hA or rA for 4 h as
indicated p38 kinase activity was measured as described above
using p38 kinase immunoprecipitated with c-32P-ATP and
GST-ATF-2 as substrates.
Fig 3 Effects of MAPK inhibitors on hA-induced apoptosis and acti-vation of caspase-3 Cultured RINm5F and CM cells were pre-incuba-ted 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-neg-ative control (SB202474) for 1 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 nucleo-somes (fragmented DNA) (B) Caspase-3 activity was determined after 16-h hA-exposure using synthetic fluorogenic oligopeptide sub-strate z-DEVD-AFC The fluorescence was measured at excitation
k ¼ 400 nm and emission ¼ 540 nm All data were presented relat-ive 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.
Trang 5apoptosis Figure 4 shows the result of a representative
western blot analysis using specific antibodies for
ATF-2 and phosphorylated activating transcription
factor 2 (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
max-imal levels by 4 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
detec-ted 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,
indicating that increased activation of ATF-2 is corre-lated 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
- pATF-2
ratios 1.0 0.19 0.81 0.79 1.1
o hA
0 3 B S A h
I b i h i K J + A h
1 n j-S A + A h
9 8 D P A h
A
- p-c-JunSer63
ratios 1.0 0.42 0.38 0.45 0.92
B
- c-Jun 1.0 0.37 0.40 0.36 0.96 ratios
- ATF-2
C
D
Fig 5 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 1 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 mem-brane 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 ana-lyzed 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.
Fig 4 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 l M 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
chemilumines-cence reagent Fold induction of p-ATF-2 (shown as ratios) was
cal-culated based on levels at 1 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
(AS-ATF-2) or sense ATF-2 (S-(AS-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
inde-pendent experiments, each performed in duplicate †P < 0.01
versus control; *P < 0.01 versus hA-treated cells.
Trang 6pretreatment of CM cells with SB203580 caused a
large decline (averaging about 80%) in hA-induced
ATF-2 phosphorylation compared with
non-pretreat-ment controls Pretreatnon-pretreat-ment of CM cells with JNK
inhibitor I or transfection with antisense jnk1
(AS-jnk1) caused lesser inhibitory effects (20%
decre-ments), 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
inhibi-tors of RINm5F cells, wherein we also found that
SB203580 could largely inhibit hA-induced ATF-2
phosphorylation (data not shown) Therefore, p38
kin-ase rather than JNK is the primary upstream kinkin-ase
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
suppres-sion 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
phos-phorylation ATF-2 and c-Jun protein levels were
ana-lyzed 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-junexpression 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
fol-lowing 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
electropho-retic mobility shift assay (Fig 6A) Two shifted bands
corresponding to two different forms of CRE DNA-binding 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-incuba-tions) 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-anti-body supershifted complexes, in hA-treated cells following pre-incubation of antibody against p-c-Jun-Ser63, indicating that both of these shifted CRE com-plexes 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 anti-bodies on binding of labeled CRE to protein com-plexes (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 com-plexes 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, indica-ting that the unphosphorylated forms of ATF-2 and c-Jun are the major components of the complexes associated with CRE DNA sequences in untreated con-trol cells Taken together, our results demonstrate changes in protein composition and phosphorylation state of the CRE-binding complexes, with the emer-gence of functionally significant p-ATF-2 and p-c-Jun-Ser63 in hA-treated apoptotic cells
Activation of ATF-2 increases transcriptional transactivation potential of ATF-2
The correlation of ATF-2 activation with CRE-mediated transcriptional activity after hA treatment was studied using a CRE-driven luciferase reporter construct (pCRE-luc) CM cells were transiently trans-fected with pCRE-luc and luciferase activity was
Trang 7measured 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
meas-ured by increased production of relative light units of
luciferase activity (Fig 7) Luciferase activity reached
maximum induction at 8 h (about four-fold increase),
which coincided with the observed elevation in
CRE-binding 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
correla-ted with hA’s fibrillogenic and cytotoxic properties
Also shown in Fig 7 is evidence that suppression of
p38 kinase by SB203580 inhibits ATF-2-mediated
tran-scriptional activation Together, these data
demon-strate a role for the p38 kinase-mediated signal
transduction pathway in transcriptional responses
mediated by ATF-2 in hA-treated b-cells The
cooper-ative effect of JNK and p38 kinase on CRE-mediated
transcriptional activation was also demonstrated by inhibition of luciferase expression using JNK inhib-itor 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 trans-fected cells with inhibitors alone had no effect on lucif-erase 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 promoter-chloramphenicol acetyltransferase (CAT) reporter construct CAT activity was measured at various time-points 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 con-trol values following 8 h of exposure, whereas in contrast, CAT activity remained consistently low in rA-treated b-cells The time at which the CAT activity
p oc hA
h oc Ah
RINm5F CM
_ _CRE-complexes
_ _
A
p
_ _ CRE-complexes _ _ p-c-Jun-CRE-complexes
oc Ah
_
_ _ _
B
Fig 6 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 8 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 1 h with unlabelled specific or nonspecific oligonucleotides, respect-ively, 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 1 h, before addition of labeled CRE probe The binding reaction samples were then analyzed as described above.
Trang 8reached maximal coincided with the observed elevation
in CRE binding and CRE-mediated transcriptional
activity SB203580, a suppressor of p38
kinase-medi-ated ATF-2 activation, inhibited c-jun promoter
trans-activation Thus, activated ATF-2 and p38 kinase play
critical roles in stimulation of c-jun transcription
dur-ing 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
pretreat-ment with SB203580 and JNK inhibitor I (Fig 8),
further indicating that both the JNK- and p38
kinase-mediated 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-elici-ted b-cell apoptosis using the same b-cell lines, rat
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 tar-geting 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,
Fig 7 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
lucif-erase reporter gene assay system Resulting values, shown as
relative light units, are mean ± SEM of four independent
experi-ments, each performed in duplicate †P < 0.01 versus control;
*P < 0.01 versus hA-treated cells.
Fig 8 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.
Trang 9induced 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
activa-tion was detected at which the effect was not
concom-itant with JNK and⁄ or p38 activation [24]
We have shown here that hA elicits distinct and
spe-cific effects on phosphorylation of ATF-2 by p38
kin-ase-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
phosphor-ylated 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
sup-press 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
cur-rent data show that p38 is more important for the
acti-vation of ATF-2 evoked by hA in both our b-cell
systems Thus, these parallel pathways may well
con-verge at AP-1 and CRE sites, mediating hA-induced
induction of expression of their target genes, including
c-junas demonstrated herein
The composition of the ATF-2-associated
transcrip-tion factor complexes may differ between various
physiological and pathological states, so that even clo-sely 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 com-plexes can modulate their transcriptional activity and thereby alter target-gene specificity, leading to apopto-sis 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 associ-ated 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 trans-formed 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
Trang 10CM cell cultures, to final concentrations of 10 lm or
100 lm, respectively, 1 h before exposure to hA
Alternat-ively, JNK inhibitor I or JNK inhibitor I-negative control
peptides (Calbiochem) were dissolved in water and applied
to cell cultures to final concentrations of 1 lm, 1 h before
hA addition The doses selected have been tested and
treat-ments 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
1 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
death-detection ELISA system (Roche Applied Science,
Man-nheim, 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
1 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
micro-grams of each cell extract was incubated in reaction buffer
containing 40 ngÆlL)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 37C for 3–
4 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;
emis-sion 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
previ-ously 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,
Bev-erly, MA, USA), rabbit anti-p-ERK1⁄ 2 (Cell Signaling),
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 peroxi-dase-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 ima-ging 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 kin-ase activities of p38 and ERK1⁄ 2 determined by in vitro im-munocomplex kinase assay, as described [47] Briefly, 100 lg
of protein from each cell extract were incubated with 2 lg of
an antibody for ERK1⁄ 2 or p38 kinase for 2 h at 4 C, respectively, in the presence of protein A–Sepharose (Amer-sham 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,
5 mm MgCl2, 1 mm 2-mercaptoethanol and 50 lm Na3VO4), containing 10 lCi of c-32P-ATP (Amersham Biosciences) Following incubation for 30 min at 30C with 5 lg of GST-ATF-2 (for p38 kinase assay) or GST-Elk-1 (for ERK1⁄ 2 assay), the reactions were terminated by addition of SDS⁄ PAGE sample buffer and heating for 5 min at 95 C The samples were then analyzed using 12% SDS⁄ PAGE The protein bands (phosphorylated substrates) were ana-lyzed 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 dou-ble-stranded DNA-binding probe for the CRE complex was 5¢-end labeled with c-32P-ATP using T4 polynucleotide kin-ase (Invitrogen, Carlsbad, CA, USA) The top strand con-sensus 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 des-cribed [15] For competition experiments, unlabelled oligo-nucleotides, 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