inflammatory -cell inhibition and apoptosis are increasingly accepted as key contributors to failing insulin secretion leading to type 1 and type 2 diabetes [ 1 , 2 ] .
interleukin-1 , a 17  kda proinflammatory cytokine and mediator of inflammation , fever , and acute - phase responses [ 1 , 3 ] , inhibits -cell function in vitro and in vivo by signaling via nfb and mitogen activated protein kinases ( mapk ) to activate endoplasmic reticulum and mitochondrial death pathways [ 47 ] .
the mapk family encompasses the extracellular signal - regulated kinase ( erk ) , c - jun n - terminal kinase ( jnk ) , and p38 cascades .
the precise contribution of the jnk pathway to stress - induced -cell failure is not fully understood , especially with regard to the differential roles of the jnk subtypes , jnk1 , jnk2 , and jnk3 .
the jnk proteins control the expression of immediate early genes such as ap-1 transcription factor family members and are important regulators of both apoptosis and survival , depending on the biological context [ 8 , 9 ] .
three mammalian genes encoding for jnk have been identified : jnk1 , jnk2 , and jnk3 , located on different chromosomes [ 10 , 11 ] .
jnk1 and jnk2 are expressed in a large variety of tissues regulating cell proliferation , differentiation , inflammation , autoimmunity , obesity , and tumorigenesis .
jnk3 expression was thought to be restricted to the brain , heart , and testes [ 13 , 14 ] ; however , more recently , jnk3 was found to be expressed also in human and mouse pancreatic -cells .
mice deficient in a single jnk allele survive , as do jnk1  jnk3   and jnk2  jnk3   mice , whereas combined genetic disruption of jnk1 and jnk2 alleles causes early embryonic death due to severe dysregulation of apoptosis in the brain . additionally , jnk1  /jnk2  
mouse embryonic fibroblasts ( mefs ) are protected against uv - induced apoptosis , indicating that jnk1 and jnk2 are required for a normal apoptotic response to uv exposure .
furthermore , jnk3   mice are protected from stroke , neuronal death , and oxidative stress .
supporting these direct observations of the importance of jnk in proapoptotic signalling , cell - permeable jnk inhibitory protein-1 ( jip-1 ) derived jnk inhibitory peptides or the jnk inhibitory small molecule sp600125 decrease intracellular jnk signalling and improve cell survival in vitro and in vivo [ 1822 ] .
splicing of the jnk genes gives rise to more than twelve different transcript variants [ 13 , 23 ] translating into proteins with and without a cooh - terminal extension to generate both 46  kda and 54  kda isoform proteins with a high level of homology .
initially , the different jnk subtypes were thought to have largely redundant functions , but different tissue distribution , substrate preferences , and expression patterns support that the jnk subtypes also have nonredundant functions and are involved in distinct cellular processes [ 10 , 12 , 13 , 23 , 25 ] .
however , little is known about how the individual jnk isoforms and subtypes mediate apoptosis .
apart from phosphorylating and activating members of the activating protein-1 ( ap-1 ) transcription factor family , jnk proteins regulate other proteins involved in cell proliferation and apoptosis , including p53 , myc , and members of the bcl-2 family of proteins [ 6 , 26 , 27 ] .
the precise contribution of the individual jnk subtypes in mediating il-1-induced -cell apoptosis is largely unknown , although a protective role of jnk3 in il-1 , tnf- , and ifn--induced -cell apoptosis via akt2 signaling has been suggested [ 15 , 28 ] . in these studies
, no differentiation was made between the roles of the jnk subtypes in the signaling pathways of the three individual proinflammatory cytokines . since jnk subtype activation is stimulus dependent and as il-1 is indispensable in the proapoptotic combination of inflammatory cytokines , the action of tnf- and inf- being mainly to synergize with il-1 , it is important to investigate the differential role of the jnk subtypes in stimulus - specific pancreatic -cell signalling . here , we investigated the individual roles of jnk1 , jnk2 , and jnk3 in il-1-induced apoptosis in insulin - producing ins-1 cells and provide evidence for a critical role of jnk1 in mediating il-1-induced -cell apoptosis , whereas jnk2 but not jnk3 conferred protection .
the clonal rat -cell line ins-1   ( gift from wollheim , geneva , switzerland ) and ins-1 cell lines stably expressing shrna   were grown in rpmi-1640 medium with 11  mmol / l glucose ( invitrogen , naerum , denmark ) supplemented with 50  mol / l -mercaptoethanol , 100  u / ml penicillin , 100  g / ml streptomycin , and 10% heat - inactivated fetal bovine serum ( fbs ) ( invitrogen ) .
recombinant mouse il-1 was from bd bioscience pharmingen ( san diego , ca , usa ) .
jnk1(f-3 ) mouse monoclonal antibody raised against amino acid 1 - 384 of full length jnk1 p46 human origin was purchased from santa cruz technologies ( santa cruz , ca , usa , catalog number : sc-1648 , used at 1  :  1000 dilution ) .
jnk2 rabbit polyclonal antibody raised against a synthetic peptide for human jnk2 ( catalog number : # 4672 , used at 1  :  1000 dilution ) , jnk3 rabbit monoclonal antibody raised against a synthetic peptide for human jnk3 ( catalog number : # 55a8 , used at 1  :  1000 dilution ) , p - jnk ( thr183/tyr185 ) polyclonal rabbit antibody raised against a synthetic phosphopeptide corresponding to residues surrounding thr183/tyr185 of human sapk / jnk ( catalog number : # 9251 , used at 1  :  1000 dilution ) , t - jnk polyclonal rabbit antibody raised against a recombinant human jnk2 fusion protein ( catalog number : # 9252 , used at 1  :  1000 dilution ) , cleaved caspase 3 ( asp175 ) rabbit polyclonal antibody raised against amino terminal residues adjacent to asp175 in human caspase 3 ( catalog number : # 9661 , used at 1  :  500 dilution ) , myc ( d84c12 ) rabbit monoclonal antibody raised against synthetic peptide corresponding to amino - terminal residues of c - myc ( catalog number : # 5605 , used at 1  :  1000 dilution ) , and -tubulin ( 9f3 ) rabbit monoclonal antibody raised against human -tubulin ( catalog number : # 2128 , used at 1  :  1000 dilution ) were all from cell signalling ( beverly , ma , usa ) .
the mouse monoclonal -actin antibody raised against -cytoplasmic actin n - terminal peptide , ac - asp - asp - asp - ile - ala - ala - leu - val - ile - asp - asn - gly - ser - gly - lys , conjugated to keyhole limpet haemocyanin ( klh ) was obtained from abcam ( cambridge , uk , catalog number : ab6276 , used at 1  :  10000 dilution ) .
the specificity of the jnk antibodies was previously verified against recombinant jnk1 , jnk2 , and jnk3 protein using western blot analysis .
ft cells , used to produce lentivirus , were cultured in d - mem medium ( invitrogen ) supplemented with 100  u / ml penicillin , 10  nm mem nonessential amino acids , 1  mm sodium pyruvate , and 10% fbs ( invitrogen ) ( complete d - mem ) .
ht1080 cells , used for virus titration , were cultured in d - mem medium ( invitrogen ) supplemented with 100  u / ml penicillin , 100  g / ml streptomycin , and 10% fbs ( invitrogen ) .
ins-1 cell lines , stably expressing jnk1 , jnk2 , or jnk3 shrnas , nonsense shrna , or empty vector , were created as described in . in brief
, ins-1 cells were transduced with an moi of 5 with virus generated by transfection of hek293 ft cells with the lentiviral vectors plko.1 jnk1 ( trcn0000055115 ) , plko.1 jnk2 ( trcn0000012590 ) , plko.1 jnk3 ( trcn0000012634 ) , or empty vector ( all from open biosystems , thermo scientific , st .
leon - rot , germany ) or plko.1 nonsense ( shc002 ) ( sigma , brondby , denmark ) .
all constructs contained a puromycin - resistance gene as mammalian selection marker , and stably transduced ins-1 cells were selected using 1  g / ml puromycin ( sigma ) .
ins-1 cell lines were passaged a minimum of three times and tested for knockdown efficiency and specificity compared to empty vector and nonsense shrna expressing ins-1 cell lines .
total rna was isolated using trizol ( sigma ) followed by rneasy mini elute kit ( qiagen , hilden , germany ) purification .
1  g of total rna was used to prepare targets by one - cycle target labeling kit ( affymetrix , santa clara , ca , usa ) following the instructions of the manufacturer .
hybridization cocktails were hybridized onto rat genome 230 2.0 genechips , containing 31,099 rat gene probes , at 45c for 17  h ( 60  rpm ) in a hybridization oven 640 ( affymetrix ) .
genechips were rinsed and stained in a genechip fluidics station 450 using the fluidics protocol  eukge - ws2v5_450  ( affymetrix ) .
for all conditions , three separate experiments were analyzed on separate arrays ; that is , a total of 36 arrays were used .
array data is available at arrayexpress   ( https://www.ebi.ac.uk/arrayexpress ) , accession number e - mtab-3146 . to adjust for nonspecific hybridization , optical effects , and comparability ,
significant differential expression was assessed using the moderated t - statistics for pairwise comparison between conditions implemented in the limma package .
the comparisons performed were 45  min il-1 exposed jnk1 knockdown ( kd ) ins-1 cells adjusted for nonexposed jnk1 kd versus 45  min il-1 exposed ns control ins-1 cells adjusted for nonexposed ns control ins-1 cells ; 45  min il-1 exposed jnk2 kd ins-1 cells adjusted for nonexposed jnk2 kd versus 45  min il-1 exposed ns control ins-1 cells adjusted for nonexposed ns control ins-1 cells ; and 45  min il-1 exposed jnk3 kd ins-1 cells adjusted for nonexposed jnk3 kd versus 45  min il-1 exposed ns control ins-1 cells adjusted for nonexposed ns control ins-1 cells .
genes were considered to be significantly regulated if the log2 fold change was > 1 or < 1 and the p value was < 0.05 . only probes that could be mapped to gene identifiers using the  rat2302.db  probe annotation package were considered for further analysis . for clustering analysis , we used hierarchical clustering as implemented in the heatmap.2 function in the gplots r package .
briefly , the mean log2 expression value for three replicates of the 12 conditions was calculated for each probe .
overrepresented biological processes among groups of regulated genes were identified by hypergeometric testing of gene ontology ( go ) terms using david [ 32 , 33 ] . only go terms reaching a benjamini corrected p value < 0.05 were considered significantly overrepresented .
ins-1 cells were exposed to 150  pg / ml il-1 or vehicle over either a 12  h ( stable shrna cell line experiments ) or 24  h ( ins-1 cell line experiments ) time course and total rna was isolated using the rneasy kit ( qiagen ) and quantified on a nanodrop 1000 microvolume spectrophotometer .
the rna was treated with recombinant shrimp dnase ( affymetrix ) , and first strand cdna was synthesized from 2  g of rna using the high capacity cdna reverse transcription kit ( applied biosystems , carlsbad , ca , usa ) following the manufacturer 's protocol .
quantitative rt - pcr was performed in a 10  l volume using 0.5  l of premixed taqman primer / probes ( applied biosystems ) , 5  l 2x taqman universal master mix ( applied biosystems ) , and 4.5  l template ( 20  ng ) and run on a 384-well plate in triplicate on the applied biosystems prism 7900ht real - time pcr machine for 40 cycles .
the following primer / probes were purchased from applied biosystems : hprt1 ( rn01527840_m1 ) , rn18s1 ( hs03928990_g1 ) , jnk1 ( rn01453358_m1 ) , jnk2 ( rn00569058_m1 ) , jnk3 ( rn00563035_m1 ) , jun ( rn00572991_s1 ) , trp53 ( rn00755717_m1 ) , junb ( rn00572994_s1 ) , jund ( rn00824678_s1 ) , and myc ( rn00561507_m1 ) .
the primer / probe sequences for the jnk isoforms are listed in table 1 and synthesized by integrated dna technologies ( idt , berchem , belgium ) .
we utilized the human isoform - specific primer and probe sequences determined by dreskin et al .   and modifying them based upon a cross species homology comparison between human and mouse , as the rat jnk isoform sequences were not published .
relative transcript quantities were calculated by the standard curve method and normalized to the average of the housekeeping genes 18s and hprt1 .
the appropriate housekeeping genes were selected by analyzing the mrna expression of four common housekeeping genes , gapdh , ppia , 18s , and hprt1 , in ins-1 cells after 12 and 24  h exposures to il-1. the two genes used were the least variable following il-1 treatment .
ins-1 cells ( 0.8  10  cells / well for western blotting and 0.075  10  cells / well for cell death detection assay ) were transfected with sirna directed against rat myc ( sigma ) , negative control sirna ( mission sirna universal negative control , sigma ) , or vehicle .
the cells were transfected using dharmafect4 ( thermo scientific , denmark ) according to the manufacturer 's protocol , with a final concentration of sirna of 50  nmol / l .
the cells were incubated for 22  h with sirna or vehicle and then exposed to il-1 ( 150  pg / ml ) or left nonexposed for 24  h. protein was isolated and analyzed by western blotting and apoptotic cell death was measured by the cell death detection elisa ( roche , hvidovre , denmark ) .
ins-1 cells or ins-1 cell lines stably expressing shrna were grown to 8090% confluence , exposed to il-1 ( 150  pg / ml ) or vehicle for up to 24  h , washed in ice - cold pbs , and lysed for 15  min on ice using 1x lysis buffer ( cell signaling ) .
the protein concentration was measured by the bradford method ( bio - rad , copenhagen , denmark ) .
3040 micrograms of total protein were mixed with 4x lds sample buffer ( invitrogen ) , heated for 5  min at 70c , and loaded on 10% nupagebis - tris gel ( invitrogen ) .
the blots were blocked for 1  h in 1x tris - buffered saline ( ph 7.6 ) containing 0.1% tween 20 and 5% bsa and afterwards incubated overnight at 4c with primary antibody .
blots were rinsed and incubated with a rabbit secondary horseradish peroxidase - conjugated antibody for 1  h. immune complexes were detected by chemiluminescence using super signal west dura extended duration substrate ( thermo scientific ) , and images were captured digitally by use of the fuji las3000 platform ( fujifilm , tokyo , japan ) or the alpha innotech flourchem q imaging platform ( kem - en - tec , taastrup , denmark ) .
apoptotic cell death was measured by the detection of dna - histone complexes released from the nucleus to the cytosol of cells by using cell death detection elisa ( roche ) as described by the manufacturer . in brief , 0.075  10 ins-1 cells stably expressing jnk1 , jnk2 , jnk3 , nonsense shrna , or empty vector were cultured 24  h prior to exposure to il-1 ( 150  pg / ml ) or vehicle .
after an additional 24  h , the culture medium was removed and cells lysed in 200  l 1x lysis buffer for 30  min at room temperature .
the lysate was then centrifuged for 10  min at 200  g , incubated with anti - dna peroxidase and anti - histone - biotin , and added to streptavidin - coated wells for 2  h at room temperature .
absorbance was measured after addition of peroxidase substrate abts ( 2.2-azino - bis-3-ethylbenzthiazoline-6-sulfonate ) at 405 and 490  nm .
all statistical analyses were performed at raw data using graphpad prism ( graphpad software inc . , san diego , ca , usa ) .
where the figures show normalized data , the mean of the controls of the individual experiment was set to 1 and the error bars show the standard error calculated from the raw data .
statistical significance was determined using one- or two - way anova with post hoc tests , or student 's t - test as appropriate .
to determine which isoforms are expressed in the ins-1 cell model , we created rat - specific primers and probes ( table 1 ) , utilizing the human isoform - specific primer and probe sequences determined by dreskin et al .   and modifying them based upon a cross species homology comparison between human and mouse , as the rat jnk isoform sequences have not been published . utilizing quantitative rt - pcr , we found that the ins-1 cells expressed jnk11 , jnk11 , jnk21 , jnk22 , jnk31 , and jnk32 .
jnk12 and jnk12 were expressed at very low levels and jnk21 and jnk22 were not expressed in ins-1 cells ( data not shown ) .
we chose to examine il-1-induced regulation of the six isoforms with the higher level of expression in the ins-1 cell line .
we exposed ins-1 cells to 150  pg / ml of il-1 or vehicle for 2 to 24  h. we used 150  pg / ml il-1 to induce apoptosis in the ins-1 cells as titration experiments showed that this was the lowest concentration sufficient to induce maximal apoptosis ( data not shown ) . utilizing rt - pcr , we found that jnk11 , jnk11 , jnk21 , and jnk22 were not regulated by il-1 , whereas jnk31 was significantly upregulated at 8  h and jnk32 was upregulated from 8 to 24  h ( figures 1(a)1(f ) ) .
comparison of the data obtained with jnk isoform - specific primers with data obtained with nonisoform - specific taqman primers confirmed that jnk1 and jnk2 were not regulated by il-1 , whereas jnk3 was upregulated from 6 to 12  h ( supplementary figures 1a1c , in supplementary material available online at http://dx.doi.org/10.1155/2016/1312705 ) .
as there are no available commercial jnk isoform - specific antibodies , we used jnk1 , jnk2 , or jnk3 subtype specific antibodies .
we exposed ins-1 cells to 150  pg / ml il-1 for 4 to 24  h to measure the expression and regulation of the jnk subtype proteins by western blot analysis .
the jnk1 subtype encompasses jnk11 and jnk11 isoforms translating into 46  kda and jnk12 and jnk12 isoforms translating into 54  kda isoform proteins , respectively .
we found that only jnk1 46 and 54  kda isoform proteins expressions were significantly regulated in that they were decreased by il-1 in a time - dependent manner ( figure 2(a ) ) .
the jnk2 subtype includes jnk21 and jnk22 translating into 46  kda and 54  kda isoform proteins , respectively .
the jnk2 antibody reacts only with the 54  kda isoform protein , compatible with jnk22 being the predominating jnk2 isoform expressed in ins-1 cells ( unpublished data ) .
the jnk3 subtype specific antibody reacts only with the 54  kda protein isoform corresponding to jnk32 ; however , jnk31 that translates into a 46  kda protein isoform and jnk32 are expressed at equally high levels in ins-1 cells ( unpublished data ) .
however , the observed il-1-induced jnk3 mrna expression did not translate into increased jnk3 54  kda isoform protein expression ( figure 2(a ) ) . of note ,
detection of il-1-regulated jnk31 isoform protein expression was not possible with this jnk3 subtype antibody .
finally , we exposed ins-1 cells to 150  pg / ml il-1 or vehicle for 0.5 to 24  h to measure jnk phosphorylation as a measure of jnk activity .
jnk 46 and 54  kda isoforms were both significantly phosphorylated after 0.5 to 6  h of il-1 exposure , with maximum peak phosphorylation of the isoforms after 0.5 to 1  h of il-1 exposure ( figure 2(b ) ) . in summary ,
il-1 increased jnk31 and jnk32 mrna expression and decreased jnk1 subtype protein expression in ins-1 cells . furthermore , il-1-induced jnk phosphorylation peaks after 0.5 to 6  h of stimulation . to investigate the differential roles of the jnk subtypes in il-1-induced cell death , we produced stable ins-1 knockdown cell lines expressing shrnas directed against jnk1 , jnk2 , or jnk3 , as well as nontarget ( nonsense , ns ) shrna and empty vector ( ev ) controls .
the jnk knockdown cell lines were tested for knockdown specificity and efficiency by western blot analysis using jnk subtype specific antibodies ( figure 3(a ) ) .
we achieved specific knockdown of the intended jnk subtypes by approximately ~80% jnk1 , ~75% jnk2 , and ~45% jnk3 knockdown compared to control ns ( ns shrna ) ins-1 cells ( figure 3(a ) ) .
we next wished to measure early response jnk activity in the stable jnk knockdown ins-1 cell lines to investigate how knockdown of the individual jnk subtype affected total phosphorylation status of the different jnk isoforms ( 46 or 54  kda ) .
we exposed the stable jnk knockdown ins-1 cells to 150  pg / ml il-1 or vehicle for 0.5  h ( figure 3(b ) ) as we detected peak jnk phosphorylation at this time point in nontransduced ins-1 cells ( figure 2(b ) ) . though the jnk knockdown ins-1 cell lines showed specific jnk1 , jnk2 , or jnk3 knockdown ( figure 3(b ) ) , this did not affect the total level of jnk ( all jnk subtypes ) ( figure 3(b ) ) .
interestingly , jnk1 knockdown ( jnk1 shrna ) ins-1 cells showed significantly reduced il-1-induced phosphorylation of the 46  kda isoforms compared to control ns expressing ins-1 cells .
phosphorylations of the 46  kda isoforms in jnk2 knockdown ( jnk2 shrna ) and jnk3 knockdown ( jnk3 shrna ) ins-1 cells were not different compared to the control ns ins-1 cells ( figure 3(b ) ) .
there were no differences in phosphorylation of the 54  kda jnk isoforms between 0.5  h il-1 exposed ns and jnk1 , jnk2 , or jnk3
knockdown ins-1 cell lines ( figure 3(b ) ) . to summarize , only jnk1 knockdown reduced early il-1-mediated activity of the 46  kda jnk isoforms as determined by phosphorylation in ins-1 cells .
next , to investigate the functional impact of knockdown of the individual jnk subtypes , we exposed the stable jnk knockdown and control cell lines to 150  pg / ml of il-1 or vehicle for 24  h , after which apoptosis markers were measured by elisa and western blotting .
in contrast , knockdown of jnk1 prevented il-1-induced apoptosis , measured as decreased levels of cytoplasmic nucleosomes ( figure 4(a ) ) . jnk1
interestingly , knockdown of jnk2 significantly potentiated il-1-induced apoptosis and caspase 3 activation , while jnk3 knockdown did not significantly affect il-1-induced apoptosis or cleavage of caspase 3 compared to ns cells .
since the jnk members had differential effects on il-1-induced apoptosis , we next determined if there were unique early response gene clusters regulated by each jnk member .
ins-1 cells stably expressing shrnas directed against jnk1 , jnk2 , jnk3 , or ns were exposed to 150  pg / ml of il-1 or vehicle for 45  min .
mrna expression was analyzed by microarray analysis . when we compared the normalized gene expression profile of the 45  min il-1 exposed jnk1 , jnk2 , or jnk3
knockdown ins-1 cells with the gene expression profile of 45  min il-1 exposed ns ins-1 cells , adjusted for their il-1 nonexposed control conditions , 74 , 144 , and 134 genes , respectively , were found to be differentially regulated ( supplementary tables 13 ) .
the jnk1 knockdown ins-1 cell line gene expression pattern was significantly different from that of jnk2 or jnk3 knockdown and ns ins-1 cells ( supplementary figure 2 ) .
however , overall , there were no significant clusters or pathway associations to cast light on the potential pathways or early il-1-induced mechanisms underlying the unique actions of the jnk subtypes , and we therefore decided to proceed with a candidate gene approach to understand the mechanisms of action of the jnk subtypes .
we therefore next analyzed in the jnk knockdown cells specific jnk - related signalling molecules known to be regulated by the jnk proteins to determine if the regulation of these genes by il-1 is mediated specifically by jnk1 .
ins-1 cells stably expressing shrna directed against jnk1 , jnk2 , or jnk3 , or the ns or ev controls , were exposed to 150  pg / ml of il-1 for 2 , 6 , and 12  h. il-1 upregulated jun mrna from 2 to 12  h , trp53 mrna at 6 and 12  h , junb mrna from 2 to 12  h , jund mrna at 2  h , and myc mrna at 12  h ( figures 5(a)5(e ) ) .
these findings indicate that many of the common jnk - related signalling proteins are regulated by il-1. interestingly , there was a temporal difference in the regulation of the mrna transcripts , as jun and junb were upregulated at all time points , whereas myc was upregulated only at 12  h and jund was upregulated significantly only at 2  h. knockdown of jnk1 , jnk2 , or jnk3 did not affect the regulation of trp53 or junb mrna expression by il-1. this indicates that these mrna transcripts are not regulated by jnk1 , jnk2 , or jnk3 specifically , either due to redundancy in the actions of the jnk members or due to the fact that they are not regulated by the jnk proteins in ins-1 cells .
jnk1 knockdown increased basal jun mrna expression at 2  h ( figure 5(a ) ) and attenuated the upregulation of myc by il-1 at 12  h ( figure 5(e ) ) , indicating that jnk1 may be involved in the basal regulation of jun and that it specifically mediates the regulation of myc mrna expression by il-1. next , we wished to determine if il-1 regulates the levels of myc protein and if jnk1 mediates this regulation in response to il-1. ins-1 cells were exposed to 150  pg / ml of il-1 or vehicle for 4 to 24  h , and protein was analyzed by western blot analysis .
il-1 increased the total amount of myc protein from 12 to 24  h ( figure 6(a ) ) .
the increase in total myc protein by il-1 corresponded to the timing of the upregulation of myc mrna by il-1 , which we noted at 12  h ( figure 5(e ) ) .
next , we exposed the ins-1 cells stably expressing shrna directed against jnk1 , jnk2 , or jnk3 , or the ns or ev controls to 150  pg / ml of il-1 or vehicle for 16  h. knockdown of jnk1 prevented il-1 induction of total myc protein expression ( figure 6(b ) ) .
these results , taken together with the data in figure 5(e ) , indicate that jnk1 mediates inflammatory regulation of myc transcription and translation in ins-1 cells .
the myc protein has been shown previously to be involved in jnk - mediated apoptosis triggered by uv irradiation or antineoplastic drugs [ 26 , 35 ] .
we therefore wished to determine if myc mediates il-1-induced apoptosis in the ins-1 -cell model .
ins-1 cells were transiently transfected with either sirna directed against myc or a control ns oligonucleotide sequence and then exposed to 150  pg / ml of il-1 or vehicle for 24  h ( figure 7 ) .
transfection with sirna directed against myc decreased the total level of myc by ~2.8-fold detected by western blot analysis ( figure 7(a ) ) and , interestingly , attenuated the il-1-induced caspase 3 activation by ~3.2-fold ( figure 7(b ) ) .
in addition , transfection of ins-1 cells with myc sirna attenuated il-1-induced apoptosis by ~1.6-fold ( figure 7(c ) ) . this indicates that myc may mediate il-1-induced apoptosis in the ins-1-cell line and , taken together with the previous results , that the jnk1-myc pathway is an important mediator of il-1-induced apoptosis .
few studies have investigated the differential roles of the jnk subtypes in cellular apoptosis , or if there are specific downstream signaling proteins utilized by a specific jnk subtype to mediate its actions .
it is known that il-1 does not induce apoptosis in most cells but the pancreatic -cells belong to the exceptions .
many studies have shown that jnk plays an important role in cellular apoptosis and specifically in cytokine and il-1-induced -cell apoptosis   most using the nonspecific jnk inhibitor sp600125 .
we show here that jnk11 , jnk11 , jnk21 , jnk22 , jnk31 , and jnk32 isoforms are expressed in ins-1 cells whereas only the jnk3 isoforms mrna is regulated by il-1. when we analyzed jnk1 , jnk2 , and jnk3 subtype protein expression , only jnk1 subtype was significantly downregulated by il-1 after 24  h of exposure .
we believe that this downregulation of jnk1 subtype is not mediated by proteases after cell lysis since we use a broad protease inhibitor cocktail in our lysis buffer . the reduced jnk1 subtype expression may though be a side effect of apoptosis due to caspase 3-mediated cleavage ; however , in other stress - induced apoptotic cell lines , jnk protein expression is shown to be caspase - independent [ 37 , 38 ] .
gene expression profiling studies followed by functional characterization of candidate genes have demonstrated that the exposure of insulin - producing cell lines , fluorescence - activated cell - sorted primary rodent -cells , intact rodent islets , and human islets to proinflammatory cytokines induces not only upregulation of proapoptotic / downregulation of antiapoptotic genes , but also a protective response including downregulation of proapoptotic / upregulation of antiapoptotic genes , with a high degree of concordance between the different model systems [ 39 , 40 ] .
these findings suggest that inflammatory attack on the pancreatic -cell induces a race between deleterious and protective responses , of which the deleterious pathways eventually prevail .
we believe that the observed decrease in jnk1 content following 16  h of exposure to il-1 is a late compensatory negative feedback mechanism serving to suppress signaling via jnk1 ; however , this late suppression in jnk1 content is insufficient to prevent apoptosis .
knockdown of jnk1 completely prevented il-1-induced ins-1 cell apoptosis , while jnk2 knockdown potentiated cytokine - induced ins-1 cell apoptosis .
this is an interesting observation as jnk2 has been shown to inhibit jnk1 signaling , and thus knockdown of jnk2 may act to increase jnk1 activity .
knockdown of jnk3 did not significantly affect il-1-induced apoptosis compared to ns cells but an increase could be observed , but not in levels of cleaved caspase 3 .
both jnk1 and jnk2 knockdown protected ins-1e cells against apoptosis by 40 and 60% , respectively , in that study .
we found complete protection of apoptosis by jnk1 knockdown and found that jnk2 knockdown potentiated the effect of il-1. our study and   differed in the following respects : we utilized ins-1 cells stably expressing shrna , whereas abdelli et al .
used a cytokine combination , containing 10  ng / ml il-1 , 25  ng / ml tnf- , and 150  ng / ml ifn- , while we exposed ins-1 cells to only il-1 at 150  pg / ml .
thus , using a cytokine combination , confounding pathways and downstream signaling molecules are activated compared to testing il-1 alone .
however , as we only achieved ~50% knockdown of jnk3 in this study , new studies with more efficient jnk3 knockdown might acknowledge jnk3 as a protective protein .
we also investigated early il-1-induced jnk phosphorylation as a surrogate for jnk activity in the jnk knockdown ins-1 cells . here , we only observed significant reduction of phosphorylation of the 46  kda isoform in 0.5  h il-1 exposed jnk1 knockdown ins-1 cells when compared to ns ins-1 cells .
this was not surprising to us as the major jnk1 isoforms expressed in ins-1 cells translate into 46  kda proteins and as we achieved a high jnk1 knockdown efficiency in the jnk1 knockdown ins-1 cell line .
despite high specific knockdown of the individual jnk subtypes in the shrna expressing ins-1 cell lines , we did not see any changes in total levels of jnk .
the total jnk antibody recognizes all three jnk subtypes and therefore a reduction in only one subtype is unlikely to affect the total amount of jnk .
the reduced 46  kda phosphorylation might explain the decreased il-1-induced apoptosis in jnk1 knockdown ins-1 cells as inhibition of total jnk activity decreases intracellular jnk signalling and improves rodent -cell survival in vitro in response to cytokines [ 5 , 18 , 19 , 43 ] .
we did not observe altered 54  kda isoform phosphorylation between 0.5  h il-1 exposed ns and jnk1 , jnk2 , or jnk3 perhaps indicating that il-1-induced phosphorylation in the individual jnk knockdown subtype ins-1 cells is covered by the remaining jnk isoforms .
there are many potential downstream targets of jnk1 that activate apoptosis , such as the mitochondrial stress pathway and caspase activation , but there are few studies analyzing if there are specific signaling proteins or transcription factors that are uniquely regulated by each jnk subtype .
we performed microarray mrna expression profiling to investigate the genes regulated by the jnk subtypes .
there were no differences in basal gene expression levels in jnk1 , jnk2 , or jnk3 shrna expressing ins-1 cells when compared to ns shrna expressing ins-1 cells , indicating that individual jnk subtype knockdown does not initiate differential basal gene regulation .
exposure of the cells to il-1 for 45  min initiated differential gene regulation ; however , no significant clusters were identified .
this indicates that the jnk subtypes might mediate their major differential effect by modulating activity of proteins rather than affecting early il-1-induced gene transcription in -cells . using a transcription factor candidate gene expression analysis
, we found that myc was specifically regulated by jnk1 after 12  h of il-1 exposure .
we found that myc mrna and total protein levels were specifically downregulated in jnk1 knockdown ins-1 cells and that knockdown of myc decreased il-1-induced apoptosis and cleaved caspase 3 .
in addition to activating myc expression , jnk proteins are known to increase the stability of the myc protein   that normally has a short half - life of approximately 30 minutes .
our data also suggests that jnk1 increases the stability of total myc , as myc protein levels were increased in wild - type ins-1 cells by il-1 from 16  h to 24  h ; additionally , we found that myc mrna expression was increased at 12  h. this increase could be due to an increased stability of the mrna transcript , as opposed to transcriptional activation , given that there is a delayed increase in myc mrna expression relative to other jnk - regulated genes , such as jun , junb , and jund , which were all increased at 2  h. myc is involved in mediating numerous cellular functions , including cell proliferation , growth , differentiation , and apoptosis .
there is much debate as to how myc regulates these opposing cellular processes , but it seems to be through a complex and intricate balance between myc and its protein interaction partner max , as well as other signaling pathways .
myc induces the expression of the tumor suppressor protein cyclin - dependent kinase inhibitor 2a , which stabilizes p53 , an important regulator of apoptosis , by sequestering the e3 ubiquitin - protein ligase , mouse double minute 2 homolog , in turn degrading p53 .
myc also induces the proapoptotic bh3-only protein , bim , and blocks the expression of the antiapoptotic proteins bcl-2 and bcl - xl .
myc has been shown to promote intrinsic cell death by destabilizing via the bax protein .
thus , myc knockdown in cisplatin - induced apoptosis in a549 human lung carcinoma cells blocked cytochrome c release and prevented bax oligomerization .
p53 induction of p21cip1 drives the cell into senescence , and blocking p21 upregulation redirects the actions of p53 to activate apoptotic pathways .
p53 was significantly upregulated in ins-1 cells following il-1 treatment ; however , when we analyzed p21cip1 mrna expression in the ins-1 cells stably expressing jnk1 shrna , we did not observe a decrease in expression , and thus we do not favor this mechanism of action . in nonstressed islets , myc expression is low   and -cell specific myc overexpression markedly increases -cell apoptosis . here
, we suggest that myc is a key mediator of il-1-induced apoptosis in the ins-1 cell model .
we additionally propose that jnk1 specifically regulates myc , implicating myc as a potential downstream relay of the apoptotic effect of jnk1 .
further experiments are likewise required to verify the direct involvement of myc in mediating the apoptotic actions of jnk1 , such as overexpression of jnk1 combined with myc knockdown .
in summary , this study suggests that jnk1 is a key mediator of il-1-induced ins-1 cell apoptosis , that the transcription factor myc is regulated specifically by jnk1 , and that myc may mediate the apoptotic actions of jnk1 .
future studies are required to investigate if the jnk1-myc pathway is involved in cytokine - induced apoptosis in other cell types to determine if this is a pancreatic -cell specific effect or a more general cellular effect .
we propose that the jnk1-myc pathway may be a target for protecting pancreatic -cells from inflammatory stress , and thus a potential treatment target in diabetes .
 the relative contributions of the jnk subtypes in inflammatory -cell failure and apoptosis are unclear . 
 the jnk protein family consists of jnk1 , jnk2 , and jnk3 subtypes , encompassing many different isoforms . 
 ins-1 cells express jnk11 , jnk12 , jnk11 , jnk12 , jnk21 , jnk22 , jnk31 , and jnk32 mrna isoform transcripts translating into 46 and 54  kda isoform jnk proteins . utilizing lentiviral mediated expression of shrnas against jnk1 , jnk2 , or jnk3 in insulin - producing ins-1 cells , we investigated the role of individual jnk subtypes in il-1-induced -cell apoptosis . 
 jnk1 knockdown prevented il-1-induced ins-1 cell apoptosis associated with decreased 46  kda isoform jnk protein phosphorylation and attenuated myc expression . 
 transient knockdown of myc also prevented il-1-induced apoptosis as well as caspase 3 cleavage . 
 jnk2 shrna potentiated il-1-induced apoptosis and caspase 3 cleavage , whereas jnk3 shrna did not affect il-1-induced -cell death compared to nonsense shrna expressing ins-1 cells . in conclusion , jnk1 mediates ins-1 cell death associated with increased myc expression . 
 these findings underline the importance of differentiated targeting of jnk subtypes in the development of inflammatory -cell failure and destruction . 