Signals for death and differentiation: a two-step mechanism for in vitro transformation of adult islets of Langerhans to duct epithelial structures

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1 (23) 1, & 23 Nture Pulishing Group All rights reserved /3 $25. Signls for deth nd differentition: two-step mechnism for in vitro trnsformtion of dult islets of Lngerhns to duct epithelil structures A-M Jml 1, M Lipsett 1, A Hzrti 1, S Prskevs 1, D Agpitos 1, D Mysinger 2 nd L Rosenerg n,1 1 Deprtment of Surgery, Reserch Institute of the McGill University Helth Center, Montrel, Cnd 2 Deprtment of Phrmcology, Reserch Institute of the McGill University Helth Center, Montrel, Cnd Corresponding uthor: L Rosenerg, Montrel Generl Hospitl, 165 Cedr Avenue, Room L9-424, Montrel, Cnd H3G 1A4. Tel: +1 (514) ext ; Fx: +1 (514) ; E-mil: lwrence.rosenerg@mcgill.c Received ; revised ; ccepted Edited y CJ Thiele Astrct Phenotypic chnge of dult pncretic islets hs een implicted in the development of certin pncretic cncers nd in islet trnsplnt filure. The im of this study ws to chrcterize intrcellulr events tht medite chnges in dult islet phenotype. Using n in vitro islet-to-duct trnsformtion model, cnine islets were induced to undergo phenotypic trnsformtion to duct-like epithelil structures through two-stge process. Stge one ws chrcterized y widespred islet cell poptosis ssocited with the formtion of cvitry spces within the islets. During this stge, c-jun N- terminl regulted kinse (JNK) nd cspse-3 ctivities were elevted, while extrcellulr signl-regulted kinse (ERK) nd Akt ctivities were decresed. The second stge of the process ws chrcterized y n inversion in the lnce in ctivity etween these signl trnsduction pthwys nd y concomitnt decrese in poptosis. The trnsformed islets were no longer immunorective for islet cell hormones, ut expressed the duct epithelil cell mrker CK-AE1/AE3. In contrst to islet cells, these duct epithelil cells were highly prolifertive. To clrify the role of the identified chnges in signl trnsduction events, we performed dditionl studies using phrmcologicl inhiitors of enzyme ctivity nd demonstrted tht inhiition of JNK nd cspse-3 ctivity prevented cystic trnsformtion. Our results indicte tht the lnce in signling ctivity etween ERK/Akt nd JNK/ cspse-3 ppers to e n importnt regultor of islet cell deth nd differentition. (23) 1, doi:1.138/ sj.cdd Keywords: islets of Lngerhns; ductl epithelium; poptosis; differentition; intrcellulr signl trnsduction pthwys; dietes; pncretic cncer Arevitions: ECM, extrcellulr mtrix; MAPK, mitogenctivted protein kinse; ERK, extrcellulr signl-regulted kinse; JNK, c-jun N-terminl regulted kinse; PI3K, phosphtidylinositol 3-kinse; IHC, immunohistochemistry; ELISA, enzyme-linked immunosorent ssy; TUNEL, terminl deoxynucleotidyl trnsferse-medited dutp nick end leling; MTT, (3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrzolium romide); BrdU, 5-romo-2 deoxyuridine Introduction In the dult pncres, the endocrine cells tht comprise the islets of Lngerhns hve long een regrded s stle popultion of terminlly differentited cells. 1 In comprison, it is well known tht other pncretic cells, the cinr nd ductl cells, exhiit plsticity. 2 4 While recent studies hve egun to explore the potentil of dult islet plsticity, 5 7 the cellulr nd moleculr events tht regulte this process remin to e elucidted. 8 An pproprite model for the study of islet plsticity will provide n understnding of mechnisms underlying dult islet cell differentition. Such model would e useful for the investigtion of vriety of pncretic pthologies, including mny pncretic cncers, where the cell of origin remins to e identified. 7 In ddition, this model would llow the investigtion of chnges in islet cell phenotype following islet isoltion. These chnges in the differentited stte of isolted islets prior to nd following trnsplnttion re suggested to led to the filure of islets to restore glucohomeostsis in significnt popultion of trnsplnt recipients This model could, therefore, provide vlule insight into the control of dult islet cell differentition. We hve previously descried n in vitro model of the trnsformtion of freshly isolted dult humn islets into ductlike epithelil structures. 5,14 In this model, isolted islets re emedded into type-1 collgen mtrix nd supplemented with defined medi. This phenotypic chnge ppers to involve two-stge process entiling islet cell loss nd chnge in cell phenotype. Hence, our in vitro system offers n excellent opportunity to understnd the complex events tht re involved in mediting dult islet cell deth nd differentition. In mny cell types, phenotypic stility nd survivl is conferred y trophic fctors present in the microenvironment, including extrcellulr mtrix (ECM) proteins such s collgen-i nd lminin, 2,15,16 s well s endogenous growth fctors including FGF-2 nd TGF- nd -. 17,18 The isoltion nd purifiction of islets from the dult pncres leds to the destruction of the ECM nd disruption of potentilly importnt islet mtrix nd islet cinr cellulr reltions. 8,17 19 Intrcellulr signl trnsduction pthwys tht medite the

2 988 Signls for islet deth nd differentition effects of these trophic fctors hve een ssocited with the processes of cell deth nd differentition. 2,21 Of these, mong the etter chrcterized signling pthwys re the mitogen-ctivted protein kinses (MAPKs) extrcellulr signl-regulted kinse (ERK) nd c-jun N-terminl regulted kinse (JNK) s well s the serine/threonine kinse Akt Activtion of the ERK nd PI3-K/Akt signling pthwys hve een demonstrted to result in the survivl nd differentition of numer of cell types. 21,23,25,26 Alterntively, ctivtion of JNK nd cspse-3 hs een ssocited with the induction of progrmmed cell deth in severl cell types. 22,23,25,27 Moreover, it is the dynmic lnce etween ctivities in the ERK/Akt nd the JNK/cspse-3 signl trnsduction pthwys tht is recognized s criticl determinnt of cell differentition, prolifertion nd poptosis ,28 In this study, we demonstrte tht islet-to-duct trnsformtion is chrcterized y two-stge process involving islet cell poptosis nd chnge in the differentited stte of the islet. We report tht the reltive lnce etween ERK/Akt ctivities nd those of JNK/cspse-3 ppers to define nd medite these two stges in this model of islet cell deth nd differentition. c e d f Results Islet-to-duct-like trnsformtion Freshly isolted islets of Lngerhns (>95% purity) re solid spheroid structures s visulized under the inverted microscope (Figure 1). By immunohistochemistry (IHC), insulin immunorective cells comprised the mjority of the islet tissue (B85%) immeditely following islet isoltion. Glucgon immunorective cells nd somtosttin immunorective d cells surrounded the inner -cell mss of the islets, together ccounting for pproximtely 15% of the islet tissue (dt not shown). From Dy 3 onwrds, the islets underwent cystic trnsformtion to duct-like epithelil structures (Figures 1c nd e). This process ws first oserved in the mntle region of the islets nd progressed until solid islets hd een completely replced y cystic structures. This chnge from n endocrine to duct-like epithelil phenotype ws ssocited with significnt decline in the presence of islet cell hormones, s indicted y IHC. The re of insulin immunorective tissue, s determined y computerided morphometric nlysis, ws gretest on Dy, flling 83% y Dy 3 in culture (Po.1) (Figure 2). Similr decreses were lso oserved in glucgon (69% Po.1) nd somtosttin (8% Po.1) immunorective tissue res (Figures 2 nd c). Islet cell hormone immunorectivities continued to decline to rely detectle levels y Dy 12 in culture. Histologiclly, the cystic structures were comprised of cuoidl nd low-columnr duct-like epithelil cells (Figures 1d nd f). In order to etter chrcterize these cells, we nlyzed cytokertin (CK) immunorectivity s mrker of duct epithelil cell phenotype. CK-AE1/AE3 immunorective cells were not identified in islets t Dy (Figure 3). With the onset of cystic trnsformtion, the cells tht lined the cystic spces demonstrted CK-AE1/AE3 immunorectivity. By Dy Figure 1 Representtive photomicrogrphs of islet-to-duct trnsformtion tken fter islet isoltion (, ), t Dy 3 (c, d) nd t Dy 6 (e, f) ( 1). Morphologicl chnges re depicted y inverted microgrphs (, c, e) nd histologicl chnges re depicted y Hemtoxylin & eosin (H&E) stining (, d, f) ( 1). The formtion of cvitry spces within islet structures is pprent y Dy 3 nd continues until Dy 6 when cystic trnsformtion is complete 6, more thn 95% of islets hd trnsformed into cystic structures tht were comprised of cells of which over 9% were CK-AE1/AE3 immunorective. The pprent loss of cells during the cystic trnsformtion led us to investigte the occurrence of progrmmed cell deth. Islet to duct-like trnsformtion involves cell deth followed y prolifertion Apoptosis ws most pprent during the initil 3 dys in culture, s demonstrted y progrmmed cell deth-specific enzyme-linked immunosorent ssy (ELISA) (Figure 4). In comprison with islets ssessed t Dy, there ws 57% decrese in poptosis (3872 nm/mgdna versus 1671) on Dy 3 in culture (Po.1). On Dy 6, there ws further 42% decrese in poptosis (171 nm/mgdna) (Po.1) nd on Dy 12, this vlue hd decresed nother 2% (7.471 nm/ mgdna) (Po.5). To determine which cells undergo poptosis, coleling for insulin, glucgon or somtosttin nd for TUNEL (terminl

3 Signls for islet deth nd differentition 989 c Insulin-positive re reltive to Dy (%) Glucgon-positive re reltive to Dy (%) Somtosttin-positive re reltive to Dy (%) Dy Dy 12 deoxynucleotidyl trnsferse-medited dutp nick end leling) ws performed (Figure 4). Using this pproch, it ppered tht most of the cells undergoing poptosis were cells Dys in culture Figure 2 Decline in islet cell hormone immunorectivity during islet-to-duct trnsformtion, s visulized y immunohistochemistry. The rtio of insulin (), glucgon (), nd somtosttin (c) immunorective re s percentge of totl tissue re ws quntified y computer-ided morphometric nlysis t different times in culture. Significnt differences re shown etween ll consecutive time points (Po.5, Po.1, Po.1). Representtive photomicrogrphs demonstrting islet cell hormone immunorectivity t Dy nd Dy 12 re shown djcent to ech grph ( 1) CK-AE1/AE3 positive cells/totl cells (%) Dy Dy Dys in culture Figure 3 Increse in immunorectivity of the duct epithelil cell mrker CK- AE1/AE3 during islet-to-duct trnsformtion s visulized y immunohistochemistry. () Microgrphs representtive of CK-AE1/AE3 immunorectivity fter islet isoltion (Dy ) nd t Dy 12 ( 1). () The rtio of CK-AE1/AE3 immunorective cells to the totl numers of cell present ws determined t different times in culture. Significnt increses re shown etween vrious consecutive time points (Po.1, Po.1) As 5nm/µg DNA Insulin/TUNEL In keeping with this initil wve of poptosis, islet cell survivl ws found to e 62% higher (Po.5) on Dy 3 of culture, compred to Dy (Figure 5), s determined y (3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrzolium romide) (MTT) ssy. On Dy 6 of culture, there ws further 3% increse in cell survivl (Po.5). Expnsion of the emerging cystic structures ws noted from Dy 6 onwrds. This my hve occurred s result of incresed fluid secretion y the cells lining the cysts nd/or y cell prolifertion. Hence, we ssessed prolifertion using Bromo-deoxy-Uridine (BrdU) incorportion. At Dy, the incorportion of BrdU into islet cells ws elow detectle levels (Figure 6). On Dy 3 of culture, concurrent with the onset of cystic trnsformtion, BrdU incorportion ws detected in 23% of the nuclei of single-lyered cuoidl epithelil cells, nd not in ny of the remining islet cells (Po.1). By Dy 6, when >95% of the islets hd trnsformed into duct-like epithelil structures, this vlue incresed y 58% (Po.1) to 55%. By Dy 12, there ws further increse of 37% (Po.5), s 87% of ll cells in culture hd incorported BrdU into their nuclei s the duct-like epithelil structures continued to proliferte nd expnd rpidly Dys in culture Glucgon/TUNEL Somtosttin/TUNEL Figure 4 () Apoptosis of cells during islet-to-duct trnsformtion s quntified y progrmmed cell deth specific ELISA. Significnt decreses in cytoplsmic oligonucleosome enrichment re shown etween ll consecutive time points (Po.5, Po.1). () Photomicrogrphs demonstrting et cell poptosis during islet-to-duct trnsformtion s visulized y immunohistochemistry for islet cell hormones (red) nd TUNEL ssy for poptotic nuclei (rown) ( 1) As 595nm/1 IEQ Dys in culture Figure 5 Increse in cell survivl during islet-to-duct trnsformtion s quntified y MTT ssy. Significnt differences re shown t consecutive time points (Po.5)

4 99 Signls for islet deth nd differentition BrdU leling index (%) Dy Dy 12 Signl trnsduction events during islet-to-duct trnsformtion In order to etter understnd the underlying events mediting the phenotypic switch from endocrine to duct-like epithelil cells, we exmined signling events known to e ssocited with poptosis (JNK nd cspse-3). 22,27 nd survivl, prolifertion nd differentition (ERK nd PI3-K/Akt). 21,23,25,26 On Dy, JNK nd cspse-3 ctivities (ctivtion reltive to expression) were highest in contrst with susequent time points (Figure 7), s determined y Western lot nlysis. This ws in keeping with the initil high level of poptosis previously descried. JNK nd cspse-3 ctivities susequently declined y 61 (Po.1) nd 34% (Po.5) respectively, y Dy 3. There ws further 76 (Po.5) nd 64% (Po.1) decrese, respectively, y Dy 6. JNK nd cspse-3 ctivities persisted to Dy 12 in culture. ERK nd Akt ctivities were highest on Dy nd susequently decresed y 92 (Po.1) nd 69% (Po.1), respectively, y Dy 3 (Figure 8). Therefter, ERK nd Akt ctivities incresed significntly throughout the reminder of the culture period. Compred to Dy 3, ERK nd Akt ctivities incresed 5 (Po.1) nd 98% (Po.1), respectively, y Dy 6, with further increse of 16 (Po.5) nd 12% (Po.1), respectively, y Dy 12. To determine the contriution of specific signl trnsduction events to the cystic trnsformtion of islets, phrmcologicl inhiitors of propoptotic events (JNK phosphoryltion nd cspse-3 clevge) nd prosurvivl events (ERK nd Akt phosphoryltion) were exmined (Figures 9,1 respectively, compred to the untreted control. Following 6 or 12 dys of culture, there were no significnt differences in the level of poptosis with tretment, though poptosis ws lredy mrkedly decresed in comprison to Dy controls Dys in culture Figure 6 Increse in cell prolifertion during islet-to-duct trnsformtion, s visulized y BrdU immunohistochemistry. () Representtive photomicrogrphs of BrdU immunorectivity fter islet isoltion (Dy ) nd t Dy 12 ( 1). () The rtio of BrdU immunorective nuclei to the totl numer of nuclei present ws determined t different times in culture. Significnt increses were determined etween consecutive time points (Po.5, Po.1, Po.1) Reltive density Reltive density Inhiition of oth JNK nd cspse-3 ctivities together cused 96% decrese on Dy 3 ( control. Following 6 or 12 dys of culture, there were no significnt differences in the level of poptosis with tretment, though poptosis ws lredy mrkedly decresed in comprison to Dy controls. Inhiition of oth JNK nd cspse-3 ctivities together cused 96% decrese on Dy 3 (Po.1), reltive to the untreted control. Thus, the level of poptosis, which ppered to decrese spontneously etween Dy nd Dy 3, ws further diminished y the ddition of phrmcologic inhiitors of JNK ctivtion nd cspse-3 clevge. This dditionl reduction in poptosis, however, did not pper to reflect ny dditive or synergistic ction of these two inhiitors. Islet-to-cyst trnsformtion in the presence of these inhiitors ws severely impired. We oserved tht y Dy 3, fewer thn 2% of the islets treted with SB2358 formed cystic structures, wheres none of the islets treted with z- VAD-FMK formed cysts. Similrly, none of the islets trnsformed into cystic structures when oth inhiitors were dministered together Dys in culture P-JNK2 P-JNK1 JNK2 JNK1 Cleved Cspse-3 Cspse-3 Figure 7 () Inset: JNK1/2 phosphoryltion (ctivtion) nd expression s determined y Western lot nlysis. Blots re representtive of six independent experiments. Grph: Time course of ggregte JNK1/2 ctivity (ctivtion normlized to expression) during islet-to-duct trnsformtion s determined y computer-ided densitometric nlysis. Significnt decreses re shown t vrious consecutive time points (Po.1). () Inset: Cspse-3 clevge (ctivtion) nd expression s determined y Western lot nlysis. Blots re representtive of six independent experiments. Grph: Time course of cspse-3 ctivity (ctivtion normlized to expression) during islet-to-duct trnsformtion s determined y computer-ided densitometric nlysis. Significnt decreses re shown t vrious consecutive time points (Po.5, Po.1, Po.1)

5 Signls for islet deth nd differentition 991 Reltive Density Reltive Density When ERK or Akt ctivity ws inhiited y PD9859 or wortmnnin, respectively, the level of poptosis reched t Dy 6 incresed y 46 (Po.1) nd % (Po.1), respectively (Figure 1), in comprison to untreted controls, while t Dy 12, poptosis, reltive to the untreted control, ws incresed y 71 (Po.1) nd 51% (Po.1), respectively. Inhiition of oth ERK nd Akt ctivities together resulted in 59% increse in poptosis on Dy 6 (Po.1) nd 79% increse t Dy 12 (Po.1), reltive to control. An dditive or synergistic effect ws not oserved. With respect to cellulr prolifertion, ERK inhiition ws ssocited with n 8% decrese in BrdU leling t Dy 6 (Po.1) nd n 84% decrese t Dy 12 (Po.1) (Figure 1c), in comprison to the untreted control. Akt inhiition ws ssocited with decrese in BrdU leling y 41% t Dy 6 (Po.1) nd y 49% t Dy 12 (Po.1), reltive to the untreted control. Inhiition of oth ERK nd Akt ctivities together cused 9% decrese in BrdU leling t Dys in culture P-ERK2 P-ERK1 ERK2 ERK1 P-Akt (S473) Akt Figure 8 () Inset: ERK1/2 phosphoryltion (ctivtion) nd expression s determined y Western lot nlysis. Blots re representtive of six independent experiments. Grph: Time course of ggregte ERK1/2 ctivity (ctivtion normlized to expression) during islet-to-duct trnsformtion s determined y computer-ided densitometric nlysis. Significnt decreses re shown t vrious consecutive time points (Po.5, Po.1, Po.1). () Inset: Akt phosphoryltion (ctivtion) nd expression s determined y Western lot nlysis. Blots re representtive of six independent experiments. Grph: Time course of Akt ctivity (ctivtion normlized to expression) during islet-to-duct trnsformtion s determined y computer-ided densitometric nlysis. Significnt decreses re shown t vrious consecutive time points (Po.5, Po.1, Po.1) Dy Dy 6 (Po.1) nd 92% decrese t Dy 12 (Po.1), reltive to the uninhiited control, which is indictive of n dditive effect. Previously, it hd een reported tht Akt ctivity is elevted in humn endothelil cells cultured in type-1 collgen. 29 Hving lredy demonstrted tht type-1 collgen is necessry for islet-to-duct trnsformtion, 5,14 nd since this process ppers to e ssocited with n increse in Akt ctivity, we sought to exmine the independent effect of type-1 rt til collgen on Akt ctivtion in dult islets immeditely fter culture. Compred to freshly isolted islets mintined overnight in sl medi lone, the ctivity of Akt in islets cultured overnight in type-1 collgen with sl medi ws two-fold greter (Figure 11), suggesting tht some of the chnges in Akt ctivity during islet-to-duct trnsformtion my e due to the presence of type-1 collgen. Discussion P-JNK2 P-JNK1 JNK2 JNK1 Cleved Cspse-3 Cspse-3 P-ERK2 P-ERK1 ERK2 ERK1 P-Akt (S473) Akt Figure 9 The phrmcologic inhiitors SB2358, z-vad-fmk, PD9859 nd wortmnnin decresed JNK, ERK nd Akt phosphoryltion nd cspse-3 clevge, respectively, to undetectle levels. Blots re representtive of four independent experiments Phenotypic chnges in dult pncretic islets hve een implicted in the development nd progression of severl disese sttes. Pour et l., 3 for exmple, hve suggested tht islet-to-duct trnsformtion my ply role in the development of pncretic ductl-type denocrcinoms. 3 However, the specific cell of origin of these tumors remins to e identified. 7,31 In ddition, the loss of endocrine phenotype, function nd survivl following islet isoltion 6,9,12,13,32,33 nd trnsplnttion 9 11 hs een implicted in the filure of islet grfts to restore euglycemi in dietic trnsplnt recipients. In the present study, we extend our previous work on n in vitro model of islet-to-duct trnsformtion 5,8,14 to the chr-

6 992 Signls for islet deth nd differentition As 5nm/ug DNA As 5nm/ug DNA BrdU leling index (%) Control SB2358 z-vad-fmk SB z-vad-fmk Control PD9859 wortmnnin PD wortmnnin Control PD9859 wortmnnin PD wortmnnin Dy Dy 3 Dy 6 Dy 12 Figure 1 The effect of signl trnsduction event inhiition on poptotic cell deth, s determined y progrmmed cell deth ELISA. () SB2358 nd/or z- VAD-FMK tretment re ssocited with significnt decreses in the level of poptosis t Dy 3. () Significnt increses in the level of poptosis t Dy 6 nd Dy 12 were oserved fter PD9859 nd/or wortmnnin tretment. (c) PD9859 nd/or wortmnnin tretment ws ssocited with significnt decrese in cell prolifertion t Dy 6 nd Dy 12, s visulized y BrdU immunohistochemistry. Significnt differences re shown t vrious time points (Po.1, Po.1, Po.5) Reltive Density Immeditely fter isoltion Liquid medium overnight Collgen overnight P-Akt (S473) Akt Figure 11 Effects of type-1 collgen on Akt ctivity (ctivtion normlized to expression). Inset: Akt phosphoryltion (ctivtion) nd expression s determined y Western lot nlysis. Blots re representtive of four independent experiments. Grph: Akt ctivity (ctivtion normlized to expression) ws two-fold higher in islets cultured overnight in sl medi in comprison with islets cultured overnight in sl medi lone (Po.5, Po.1) cteriztion of the opposing signling events tht underlie this phenotypic switch. Until now, the mechnisms underlying phenotypic chnges in dult pncretic islets hve not een well understood. An in vitro model of islet cell plsticity would fcilitte the elucidtion of puttive regultory mechnisms of islet cell differentition nd the susequent development of strtegies to prevent chnges in islet cell phenotype. Hence, the purpose of this study ws to delinete the morphologicl chnges nd ssocited signling events tht occur during islet-to-duct differentition. We demonstrte tht this trnsformtion entils two-stge process medited y lnce in ctivity etween ERK/Akt nd JNK/cspse-3 signl trnsduction pthwys. The first stge of the trnsformtion process is chrcterized y islet cell loss through poptosis. This is followed y second stge in which there is phenotypic switch from solid islet to cystic duct-like structures lined y columnr nd cuoidl epithelium. This process is reminiscent of the cvittion process oserved in the erly mouse emryo in which cell loss y poptosis is followed y the trnsformtion of the solid emryonic ectoderm into cvity surrounded y columnr epithelium. 34 Coucouvnis et l. 34 hve suggested tht this cvittion process is comprised of two distinct stges, ech with seemingly opposing underlying signls nd outcomes. During the first stge of islet phenotypic trnsformtion, widespred cell loss occurs y poptosis, process proly initited y the islet isoltion procedure itself, 8,32,33 during which criticl islet mtrix interctions re disrupted. 8 Meredith et l. 14,15 nd Boudreu et l. 35 hve shown tht humn endothelil cells, nd gut nd mmmry epithelil cells undergo poptosis when the cell mtrix reltion is pertured. Moreover, given tht the ECM functions to instruct cellulr phenotype (reviewed in Streuli 36 ), it is quite possile tht the islet-to-duct phenotypic switch tht we descrie my e initited, t lest in prt, y the disruption of the islet ECM reltion. Interestingly, Hisok et l. 37 suggest tht the ECM protein lminin my provide guidnce for terminl cellulr differentition during pncretic morphogenesis in rts. Similrly, Menko et l. 38 found tht in the sence of the 3 1 integrin receptor for lminin, the ctivtion of the prosurvivl kinse ERK ws suppressed nd slivry epithelil cells exhiited n ltered phenotype. We oserved tht islet cell poptosis led to the formtion of cvitry spces within the islets. This morphologicl chnge correlted with the oserved ptterns of signl trnsduction ctivtion events such tht JNK nd cspse-3 ctivities were incresed reltive to ERK nd Akt, oth of which decresed nd remined low during this first stge of the trnsformtion process. This is keeping with results descried y Dvis in which the withdrwl of trophic support induces poptosis through n elevtion in JNK nd cspse-3 ctivities. 22 Furthermore, role for JNK nd cspse-3 involvement in the induction of poptosis hs previously een demonstrted in neuronl cells 22 s well s in islets immeditely fter isoltion. 27,32,33 The reciprocl reltion etween the propoptotic signl cspse-3 nd the prosurvivl signl ERK erly in the trnsformtion process my e explined, t lest in prt, y the ility of cspse-3 to cleve nd inctivte Rf-1, n

7 Signls for islet deth nd differentition 993 upstrem ctivtor of the ERK signl trnsduction pthwy. 39 The dt support the notion tht it is the suppression of the ERK pthwy together with the ctivtion of JNK nd cspse-3 tht is ssocited with the widespred occurrence of islet cell poptosis during the first stge of islet-to-duct trnsformtion. These oservtions lend further support to the notion tht the lnce in ctivity etween deth nd differentition/prolifertion signl trnsduction pthwys is criticl determinnt of cell fte. The imlnce in ctivity etween these opposing sets of signl trnsduction pthwys shrply delinetes the first stge of the islet-to-duct trnsformtion from the second. During this ltter stge, the oserved disppernce of islet hormones cn e explined y the preceding wve of islet cell poptosis nd phenotypic switch of the remining cells to duct-like epithelil cells, which no longer express islet cell hormones. In the second stge of islet-to-duct trnsformtion, ERK nd Akt ctivities re incresed, reltive to the first stge. This increse in signling through prosurvivl pthwys occurs coincident with decline in the ctivity of cspse-3, iomrker nd executioner of poptosis. Hence, it is not surprising tht during this second stge of the trnsformtion process, there is significnt reduction in poptosis. This result my e further explined y the ility of ctive Akt to inhiit poptosis y phosphoryltion nd inctivtion of the poptosis-inititing enzyme cspse-9, 41 the propoptotic Bcl-2 homolog Bd 42,43 nd the Forkhed fmily trnscription fctor FKHRL1, which medites the trnscription of propoptotic gene products. 44,45 Levresse et l. 46 hve lso documented Akt-medited inhiition of the JNK signl trnsduction pthwy, further highlighting Akt s ntipoptotic ctions. 46 Hong et l. 26 found tht incresed ctivtion of PI3-K/Akt nd ERK, nd inhiition of JNK ctivtion, were necessry nd sufficient to prevent poptosis in crdic myolsts, 26 while Yoon et l. 25 reched similr conclusions in dipocytes. 25 The shift in the reltive lnce in these signling ctivities hs lso een shown to regulte cell deth nd differentition in primitive neuroectoderml tumors. 24 Owing to the complexity of signl trnsduction networks nd the crosstlk tht is common etween different pthwys, we hve egun to gther informtion on the role of other signling events in this model. For exmple, elevtion of intrcellulr level of camp ppers to e necessry for isletto-duct trnsformtion to occur. 14 Additionlly, we hve determined in preliminry studies tht the onset of cystic trnsformtion is lso relted in time-dependent fshion to the upregultion of TGF-y-1 protein nd receptor during the first 48 h which is rpidly reversed, suggesting the involvement of TGFy-1-medited utocrine loop. 47 Thus, it ppers tht the initil stge of islet-to-duct trnsformtion is medited y network of signl trnsduction events tht re interrelted y crosstlk etween the respective pthwys ,46,48 Of the signling molecules we studied, Akt is the one tht ppers to ply mjor role in islet cell survivl nd differentition. 44,45,29 Hence, n understnding of the fctors tht regulte Akt ctivity in isolted islets my prove to e of use in controlling phenotypic stility. Recently, constitutively ctive Akt hs een implicted in the induction nd regultion of islet cell differentition nd survivl in trnsgenic mice. 49,5 This finding, coupled with our oservtions tht elevted Akt ctivity regultes chnges in cell phenotype, survivl nd prolifertion, suggest tht type-1 collgen-medited Akt ctivity my e criticl meditor of the differentition/ prolifertion signl tht chrcterizes the second stge of our in vitro islet-to-duct model. While cellulr trnsformtion is known to e importnt during emryonic development, 34,51,52 the significnce of its occurrence in dult islets, including involvement in the loss of islet phenotype following trnsplnttion or during pncretic crcinogenesis, remins to e fully elucidted. Our dt suggest tht t lest prt of the sis of stility of cell survivl nd phenotype in dult islets is regulted y the reltive lnce in ctivity etween the ERK/Akt nd JNK/cspse-3 signl trnsduction pthwys. Mterils nd Methods Islet isoltion nd purifiction Pncret from six mongrel dogs of oth sexes (ody weight 25 3 kg, ge 2 3 yers) were resected under generl nesthesi in ccordnce with the Cndin Council for Animl Cre guidelines. Prior to orgn removl, pncretic ducts were cnnulted to permit intrductl infusion with Lierse CI (1.25 mg/ml) (Boehringer Mnnheim, Indinpolis, IN, USA), ccording to estlished protocols. 53,54 Purifiction ws chieved with the use of continuous grdient of Ficoll-ditrizoic cid (Seromed- Biochrom KG) in n pheresis system (model 2991, COBE Lortories) (COBE BCT, Denver, CO, USA). 55 The purity of the finl preprtions ws greter thn 95%, s indicted y dithizone stining of tissues with dimeters rnging from 5 to 5 mm. Immunohistochemistry to detect the presence of mylse nd cytokertin ws negtive, consistent with the sence of ductl nd exocrine tissue (dt not shown). Islet cell culture Freshly isolted islets were emedded in type-1 rt til collgen gel. 5 nd cultured in Dulecco s modified Egle medium (DMEM/F12) (GIBCO, Burlington, ON, Cnd) supplemented with 1% fetl ovine serum (FBS), epithelil growth fctor (EGF) (1 ng/ml) (Sigm, St Louis, MO, USA) nd choler toxin (CT) (1 ng/ml) (Sigm, MO, USA). Approximtely 3 islets equivlents (IEQ) per group per time point were used. Islets were cultured in 95% ir, 5% CO 2 t 371C, nd the medium ws chnged on lternte dys. To determine whether the oserved chnges in intrcellulr kinse ctivity were directly implicted in cell deth nd differentition in this model, we employed phrmcologicl inhiitors of JNK (SB2358 t 5 mm) (Cliochem-Noviochem, Sn Diego, CA, USA), cspse-3 (z-vad-fmk t 1 mm) (Promeg Corp., Mdison, WI, USA), ERK (PD9859 t 5 mm) (New Englnd Biols, Beverly, MA, USA) nd Akt (wortmnnin t 1 nm) (Cliochem-Noviochem, Sn Diego, CA, USA). Dose response curves were estlished for ech inhiitor in order to determine optiml concentrtions (dt not shown). Representtive islets from ech group were exmined fter n overnight recovery following isoltion (Dy ) nd on Dys 3, 6 nd 12 in culture using the following investigtions. Islet cell lysis nd protein content Islet smples were spun t 9 rpm for 2 min t 41C. The pellet ws wshed twice with ice-cold phosphte-uffered solution (PBS) nd then dissolved in lysis uffer (5 mm Tris-HCl, ph 8., 1.37 mm NCl, 1% (v/v)

8 994 Signls for islet deth nd differentition nonidet P-, 1% (v/v) glycerol,.1 mm sodium orthovndte nd complete protese inhiitor cocktil tlet (Boehringer-Mnnheim)). Smples were sonicted nd the superntnt ws kept for Western lot nlysis. The protein content ws determined using BIO-RAD Protein Assy Dye Regent (BIO-RAD, Hercules, CA, USA). Lystes were diluted 6 : 1 with 6 Lemmli smple uffer (.375 M Tris-HCl ph 6.8, 12% w/v SDS, 3% w/v glycerol,.2% w/v romophenol lue, 12% -mercptoethnol in ddh 2 O) nd oiled for 3 min. Western lot nlysis An equl mount of protein (75 mg) ws loded for ech smple onto 12% polycrylmide gel run t 1 V for 9 min. Trnsfer onto nitrocellulose ws conducted t 25 ma for 9 min. Memrnes were locked with 2% ovine lumin in wshing uffer (25 mm Tris, 15 mm NCl,.5% Tween 2 in ddh 2 O). Blocked memrnes were then proed with primry ntiodies. Anti-Akt nd nti-ctive-akt (Ser-473) (New Englnd Bio-Ls, Beverly, MA, USA) were used t 1 : 1 dilutions. Anti-ctive-ERK (Snt Cruz Biotechnology, CA, USA), nti-erk (Promeg Corp., Mdison, WI, USA) nd nti-ctive-jnk (Promeg Corp., Mdison, WI, USA) were used t 1 : 5. Anti-cspse-3 (ctive nd expression) (New Englnd Bio-Ls, Beverly, MA, USA) nd nti-jnk (Snt Cruz Biotechnology, CA, USA) were used t 1 : 1. Following primry ntiody incution, lots were wshed for 1 h in wshing uffer, then incuted for 1 h in nti-rit horserdish peroxidse-linked secondry ntiody (1 : 2) (Amershm Life Sciences Inc., Buckinghmshire, UK). Following nother 1 h wshing, the lots were developed using the enhnced chemiluminescence system (ECL) (Amershm Life Sciences Inc., Buckinghmshire, UK) nd Kodk X- OMAT film (Kodk, Rochester, NY, USA). Memrnes were stripped y incuting t 651C for hlf n hour in stripping uffer (1 mm - mercptoethnol, 2% w/v SDS nd 62.5 mm Tris-HCl ph 6.7) nd reproed with primry ntiody. Developed lots were scnned on UMAX Astr 2P scnner-using Presto! PgeMnger version (NewSoft, Inc.). Morphologicl nlysis Immunohistochemistry Tissue ws fixed in 4% pr-formldehyde (PFA) nd emedded in 3% grose following stndrd protocol of dehydrtion nd prfin emedding. 8 Seril sections (thickness 4 mm) were cut from ech prffin lock. Consecutive sections were processed for routine histology nd immunostined for the pncretic hormones insulin, glucgon nd somtosttin (Biogenex, Sn Rmon, CA, USA) s well s for CK-AE1/ AE3 (Dko Dignostics Cnd, Mississug, ON, Cnd) t 1 : 1 dilution, using AB complex method (streptvidin iotin horserdish peroxidse; Dko), s descried previously. 55 The sections were incuted overnight t 41C with the pproprite primry ntiodies. Negtive controls involved the omission of the primry ntiodies. Cell deth nd prolifertion Collgen-emedded tissue cultured in DMEM/F12-CT ws hrvested using collgense XI (.25 mg/ml) (Sigm, Montrel, Que., Cnd) nd processed using progrmmed cell deth ELISA (Roche Moleculr, Montrel, Que., Cnd). 56 Cells were incuted in lysis uffer for 3 min, nd the sornce of the superntnt, which contined oligonucleosomes, ws mesured t 5 nm. Vritions in smple size were corrected for y mesuring totl smple DNA content nd stndrdizing etween smples. 8 To evlute cell prolifertion, cells cultured in DMEM/F12-CT were preincuted with 1 mm 5-romo-2 deoxyuridine (BrdU, Sigm, Montrel, Que., Cnd) for 3 h t 371C. Hrvested cells were fixed in 4% PFA s descried ove. Immunostining for BrdU ws performed using the AB complex method. The sections were pretreted with.1% trypsin nd 3 N HCl to denture the DNA. A monoclonl nti-brdu ntiody ws used t 1 : 5 dilution (Sigm, Montrel, Que., Cnd). To clculte BrdU leling index, t lest 5 cells were counted for ech time point. A BrdU leling index ws determined s the percentge of totl cells stined positive for BrdU incorportion. MTT ssy Aliquots contining 5 IEQ were plced in sterile Eppendorf tues nd 5 ml of stock MTT (5 mg/ml) (Sigm, Montrel, Que., Cnd) ws dded to ech smple. The smples were incuted t 371C for 2 h, wshed twice with cold PBS nd lysed with 2 ml of DMSO (Sigm, Montrel, Que., Cnd). Two 1 ml liquots from ech smple were loded onto 96-well plte nd the sornce ws mesured t 595 nm using Benchmrk Microplte Reder (BIO-RAD, Hercules, CA, USA). Dt from susequent time points were expressed s IEQ y clirting the ssy using the sornce of 5 IEQ immeditely following isoltion. Ech experiment ws performed in triplicte. Sttisticl nlysis The scnned Western lots were semiquntified using Gel-Pro Anlyzer version 3. (Medi Cyernetics, LP). All experiments were performed minimum of four times from seprte islet isoltions. Sttisticl nlysis ws performed using the Student s t-test with significnce defined s Po.5. 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