The transcription factor PAX4 acts as a survival gene in INS ... - Nature

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Jan 29, 2007 - Department of Cell Physiology and Metabolism, University Medical Center, Geneva 4, Switzerland. The paired/homeodomain transcription ...
Oncogene (2007) 26, 4261–4271

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ORIGINAL ARTICLE

The transcription factor PAX4 acts as a survival gene in INS-1E insulinoma cells T Brun, DL Duhamel, KH Hu He, CB Wollheim and BR Gauthier Department of Cell Physiology and Metabolism, University Medical Center, Geneva 4, Switzerland

The paired/homeodomain transcription factor Pax4 is essential for islet b-cell generation during pancreas development and their survival in adulthood. High Pax4 expression was reported in human insulinomas indicating that deregulation of the gene may be associated with tumorigenesis. We report that rat insulinoma INS-1E cells express 25-fold higher Pax4 mRNA levels than rat islets. In contrast to primary b-cells, activin A but not betacellulin or glucose induced Pax4 mRNA levels indicating dissociation of Pax4 expression from insulinoma cell proliferation. Short hairpin RNA adenoviral constructs targeted to the paired domain or homeodomain (viPax4PD and viPax4HD) were generated. Pax4 mRNA levels were lowered by 73 and 50% in cells expressing either viPax4PD or viPax4HD. Transcript levels of the Pax4 target gene bcl-xl were reduced by 53 and 47%, whereas Pax6 and Pdx1 mRNA levels were unchanged. viPax4PD-infected cells displayed a twofold increase in spontaneous apoptosis and were more susceptible to cytokine-induced cell death. In contrast, proliferation was unaltered. RNA interference-mediated repression of insulin had no adverse effects on either Pax4 or Pdx1 expression as well as on cell replication or apoptosis. These results indicate that Pax4 is redundant for proliferation of insulinoma cells, whereas it is essential for survival through upregulation of the antiapoptotic gene bcl-xl. Oncogene (2007) 26, 4261–4271; doi:10.1038/sj.onc.1210205; published online 29 January 2007 Keywords: RNAi; insulin; Pax4; proliferation; apoptosis; survival

Introduction Normal islet b-cell mass is achieved through the interplay of numerous transcription factors during pancreas organogenesis. In particular, the paired homeoCorrespondence: Dr BR Gauthier and T Brun, Department of Cell Physiology and Metabolism, University Medical Center, 1211 Geneva 4, Switzerland. E-mails: [email protected] and thierry.brun@ medecine.unige.ch Received 5 May 2006; revised 23 October 2006; accepted 9 November 2006; published online 29 January 2007

domain nuclear factor Pax4 is essential for b-cell maturation. Indeed, Pax4 mRNA is detected in the mouse pancreatic bud at E9.5, becoming maximal at E13.5–15.5 during the period of massive b-cell expansion and thereafter declining. In mature islets, low levels of Pax4 expression are detected in human, mouse and rat b-cells (Zhang et al., 2001; Heremans et al., 2002; Kojima et al., 2003; Zalzman et al., 2003; Brun et al., 2004, 2005; Theis et al., 2004; Gunton et al., 2005). Consistent with its tissue-specific expression pattern, targeted disruption of the pax4 gene in mice results in the absence of mature pancreatic b- and d-cells with a commensurate increase in the glucagon-producing a-cells (Sosa-Pineda et al., 1997; Wang et al., 2004). This increase was attributed to the a-cell-specific transcription factor Arx, which is repressed by Pax4 during development (Collombat et al., 2003). Interestingly, simultaneous inactivation of the arx and pax4 gene in mice islets promotes a somatostatin-producing d cell fate specification to the detriment of the a- and b-cell lineages, indicating that Pax4 is redundant for dcell formation under certain conditions (Collombat et al., 2005). Several studies in the Japanese population and one in Afro-Americans have associated mutations in the pax4 gene to type II diabetes, whereas polymorphisms have been linked to type I diabetes in Scandinavian, German and Swiss families (Shimajiri et al., 2001, 2003; Kanatsuka et al., 2002; Holm et al., 2004; MauvaisJarvis et al., 2004; Biason-Lauber et al., 2005; Tokuyama et al., 2006). These genetic studies suggest an important role of this factor in regulating b-cell function and/or mass in adult islets. Accordingly, we have demonstrated that increased Pax4 mRNA levels in rat islets coincide with b-cell proliferation induced by the mitogens activin A and betacellulin. Overexpression of Pax4 in rat islets resulted in the induction of the c-myc/ Id2 proliferation pathway and of the antiapoptotic gene bcl-xl. The coordinated stimulation of these genes by Pax4 prompted b-cell replication and conferred protection against cytokine-induced apoptosis in human islets (Brun et al., 2004). These results clearly show that Pax4 acts as a key regulator of b-cell plasticity in mature islets, substantiating the concept that terminally differentiated b-cells retain a significant capacity to proliferate in response to physiological stimuli (Dor et al., 2004). Human insulinomas were shown to contain aberrantly high expression levels of Pax4 as compared to

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normal islets (Miyamoto et al., 2001). Furthermore, aberrant DNA demethylation in the promoter region of the pax4 gene was recently associated with induced expression of the transcript and development of haematologic malignancies (Li et al., 2006). These results clearly implicate the transcription factor in tumorigenesis. Consistent with the latter, several members of the PAX family members have been associated with cancer; increased levels of Pax3 and Pax7 are observed in sarcomas and neural crest-derived human tumours whereas uncontrolled expression of Pax2 and Pax8 is associated with kidney, prostate, breast and ovary carcinomas. Pax5 was also identified as a key factor for the maintenance of the neuroblastoma phenotype (for review, see Robson et al., 2006). The aim of the current study was to determine whether sustained deregulated expression of Pax4 is permissive for insulinoma growth or is facilitating tumour cell survival. To address these questions, we took advantage of the well-characterized rat insulinoma cell line INS-1E (Merglen et al., 2004). This stable clone was derived from the parental INS-1 cell line and exhibits a high proliferation index (Asfari et al., 1992). Thus, INS-1E provides a suitable cell model to investigate the impact of Pax4 on cell proliferation and/or survival. INS-1E cells were found to contain 25-fold higher levels of Pax4 mRNA as compared to rat islets. Targeted suppression of Pax4 using RNA interference resulted in downregulation of the bcl-xl gene with a concomitant increase in cell death. In contrast, cell proliferation was unaltered, suggesting that Pax4 is crucial for survival but not replication of the insulinoma. Results Pax4 is highly expressed in INS-1E cells but does not correlate with the proliferative capacity of the insulinoma High levels of Pax4 mRNA have been previously shown in human insulinomas and proposed to be a predisposition factor for tumour formation and/or progression (Miyamoto et al., 2001). Accordingly, we found that the rat insulinoma INS-1E cell line contained 25-fold more Pax4 mRNA as compared to rat islets (Figure 1a). Interestingly, transcript levels for the anti-apoptotic gene bcl-xl, a downstream target of Pax4, were also increased fivefold in INS-1E cells. In contrast, mRNA levels for the unrelated serca3 gene were decreased twofold as compared to islets (Figure 1a). To determine whether a correlation could be established between Pax4 expression levels and the capacity of INS-1E cells to proliferate, the cells were exposed to the mitogens activin A or betacellulin, which were shown to stimulate Pax4 transcription as well as replication in primary rat b-cells (Brun et al., 2004). Cells treated with increasing concentrations of activin A for 24 h exhibited a dosedependent stimulation of Pax4 expression, reaching maximal induction of 11-fold at 0.5 nM. Surprisingly, betacellulin provoked only a small nonsignificant increase in Pax4 mRNA levels (Figure 1b). The reference gene cyclophilin expression levels remained Oncogene

Figure 1 Pax4 is highly expressed in INS-1E cells but does not correlate with the proliferative capacity of the insulinoma. (a) Higher levels of Pax4 and Bcl-xL transcripts are expressed in the rat insulinoma INS-1E cell line as compared to adult rat islets. QT-PCR using RNA purified from INS-1E cells and freshly isolated rat islets were performed on Pax4, Bcl-xL (left panel) and Serca3 (right Panel). Data are presented as fold change mRNA levels as compared to primary cells and normalized to the housekeeping gene transcript cyclophilin. Each value represents the mean7s.e. of four independent experiments performed in duplicate. *Po0.05; **Po 0.01. (b) Pax4 mRNAs in INS-1E cells were treated with increasing doses of activin A and betacellulin as indicated on the graph. The results are normalized to cyclophilin (or Rsp291, see Supplementary Information) and are expressed as fold change of mRNA as compared to control. (c) INS-1E cells and rat islets were incubated with increasing concentrations of glucose. Pax4 and c-myc transcript abundance levels were then estimated by QT-PCR. Statistical significances were carried out in between control INS-1E cells and islets incubated with the various growth factors by Student’s t-test. *Po0.05; **Po0.01.

constant when normalized to a second housekeeping gene (RpS29) (Theander-Carrillo et al., 2006) indicating that cyclophilin transcription is refractory to the effect of mitogens (Supplementary data, panel A). Consistent with this premise, similar activin A-induced increases in

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Pax4 transcript levels were observed when the expression profile was normalized with RpS29, whereas no alterations were detected in the presence of betacellulin (Supplementary data, panel B). The discrepancy observed between the two mitogens prompted us to investigate whether glucose, a potent activator of INS1 proliferation (Hugl et al., 1998), could induce Pax4 expression. Similar to betacellulin, glucose failed to increase Pax4 mRNA levels in INS-1E, whereas a robust 10-fold induction was detected in rat islets (Figure 1c). The latter was also accompanied by a fourfold increase in c-myc mRNA at 30 mM glucose, correlating with stimulation of cell replication (Brun et al., 2004). Taken together, although high levels of Pax4 are detected in INS-1E cells, further induction is not observed with betacellulin and glucose, suggesting that mitogenmediated increases in INS-1E proliferation do not correlate with modulation in Pax4 expression levels. RNA interference-mediated suppression of Pax4 specifically inhibits mRNA levels of the antiapoptotic gene bcl-xl To determine the specific contribution of Pax4 to the insulinoma phenotype, we inhibited expression of the transcription factor by RNA interference. To this end, two regions of Pax4, the paired (PD) and the homeo (HD) domains, were individually targeted to circumvent potential nonspecific effects of a single short hairpin RNA (shRNA). As no satisfactory Pax4 antibodies are commercially available to detect the protein by immunohistochemistry or by Western blotting, repression was monitored by quantitative real-time polymerase chain reaction (QT-PCR). Pax4 steady-state mRNA levels were reduced by 54 and 40% in INS-1E cells coexpressing GFP (used to enrich the transfected cell population) and either siPax4PD or siPax4HD, whereas no inhibition was detected with either empty vector (U6) or a scrambled shRNA (siScr) (Figure 2a). Repression was specific to Pax4, as mRNA levels for the highly homologous Pax family member Pax6, also expressed in INS-1E cells, as well as the homeodomain containing transcription factor Pdx1 were unaltered (Figure 2b and c). Furthermore, STAT1 mRNA levels were refractory to siPax4PD and siPax4HD, indicating that the interferon system implicated in double-stranded RNAmediated cell apoptosis was not activated by the levels of shRNAs being transfected (Figure 2d) (Sledz et al., 2003). In contrast, the transcript of the Pax4 target gene bcl-xl (Brun et al., 2004) was inhibited by 55 and 30% in siPax4PD- and siPax4HD-expressing cells, respectively. Surprisingly, Id2 mRNA levels were significantly increased by approximately 75 and 50% in the presence of siPax4PD and siPax4HD, respectively, as compared to cells transfected with U6 (Figure 2f). To further substantiate the specificity of siPax4PD- and siPax4HDmediated repression of Pax4, we generated an shRNA targeted to insulin (siINS6) and evaluated its impact on insulin, Pdx1 and Pax4 transcripts (Figure 3). Insulin steady-state mRNA levels were inhibited by 75% in INS-IE cells coexpressing GFP and siINS6 (Figure 3a). A single point mutation in siINS6 (siMut1) completely

abrogated the shRNA-mediated repression of insulin mRNA levels. More importantly, downregulation of the insulin transcript had no effect on either Pdx1 or Pax4 mRNA levels (Figure 3b). Consistent with QT-PCR results, immunofluorescence experiments revealed that GFP-negative cells stained for insulin, whereas no staining was detected in GFP þ /siINS6 cells. Cells expressing siMut1 retained insulin staining (Figure 3c). Taken together, these results clearly demonstrate that specific repression can be achieved through RNA interference and that inhibition of Pax4 by siPax4PD or siPax4HD but not by siINS6 resulted predominantly in the suppression of Bcl-xL mRNA levels. Repression of Pax4 selectively induces apoptosis without altering proliferation We next sought to determine the physiological consequences of shRNA-mediated repression of Pax4 in INS-1E cells. This was initially studied by transient transfection assays in which cells coexpressing a phogrin-GFP recombinant protein and either siPax4PD or siPax4HD were analysed for cell apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay (Figure 4a) or proliferation by 5-bromo-20 -deoxy-uridine (BrdU) incorporation (Figure 4b). Increased colocalization of phogrin-GFP and TUNEL was observed by immunofluorescence in cells expressing either siPax4PD or siPax4HD as compared to U6- or siScr-transfected cells. In contrast, repression of insulin did not induce apoptosis in INS-1E cells (Figure 4a). Consistent with unaltered Id2 mRNA levels, cells retained their capacity to proliferate in the presence of siPax4PD and siPax4HD (Figure 4b). In order to measure accurately apoptosis and proliferation, INS-1E cells were infected with adenoviral constructs vU6, viScr, viPax4PD, viPax4HD or viINS6. The latter approach ensures delivery of the shRNAs in approximately 80% of cells (Brun et al., 2004). Gene profiling revealed that Pax4 mRNA levels were dose dependently inhibited by either viPax4PD or viPax4HD (Figure 5a). Consistent with transfection studies, viPax4PD was more effective than viPax4HD in suppressing Pax4, reaching 73710% inhibition as compared to 5075%. A similar dose- and siRNAdependent inhibition was observed for Bcl-xL mRNA levels (5376% for viPax4PD versus 4775% for viPax4HD; Figure 5d). In contrast, transcript levels of Pdx1 and Pax6 were unchanged (Figure 5b and c). Concomitantly, TUNEL immunostaining increased in viPax4PD- and viPax4HD-infected cells as compared to viScr-, vU6- and viINS6-transduced cells (Figure 6a). Quantification revealed that the highest concentration of viPax4PD and viPax4HD induced apoptosis in approximately 25.672 and 18.773% of INS-1E cells, respectively, whereas control viScr-infected cells exhibited on average 1271% cell death (Figure 6b). Interestingly, increasing the amount of either viPax4PD or viPax4HD resulted in massive apoptosis, indicating that further repression of Pax4 was lethal. Consistent with the lack of viINS6 effect on Pax4 expression, no significant Oncogene

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Figure 2 Repression of Pax4 by RNAi specifically inhibits mRNA levels of Bcl-xL. Attempts to inhibit Pax4 mRNA were made by creating shRNAs targeted to either the paired domain (siPax4PD) or to the homeodomain (siPax4HD) of the transcription factor. The empty vector U6 and U6 containing a scrambled shRNA (siScr) served as controls. Subsequent to co-transfection with the appropriate shRNA- and GFP-expressing vectors, INS-1E cells were FACS purified into GFP- (white bars) and GFP þ (black bars) subgroups, RNA extracted and transcript levels for (a) Pax4, (b) Pdx1, (c) Pax6, (d) Stat1, (e) Bcl-xL and (f) Id2 were determined by QT-PCR and normalized to cyclophilin. Results are the mean of 4–9 independent experiments performed in duplicate and are expressed as % change of mRNA as compared to GFP cells. *Po0.05 and **Po0.01.

difference was observed in cells expressing this shRNA as compared to viScr-transduced cells (Figure 6b). Addition of cytokines to the media provoked a further 1.7-fold increase in apoptosis, suggesting that INS-1E cells were more susceptible to apoptosis subsequent to Pax4 repression (Figure 6c). In contrast, quantification of BrdU staining confirmed the ability of INS-1E cells to sustain replication independent of the adenoviral construct applied (Figure 7a and b). To ensure that uniformity of labelling was not due to prolonged exposure to BrdU (72 h), the analogue was added 4 or 16 h before processing cells for immunofluorescence. The latter revealed a partial BrdU labelling, which increased with time (Figure 7c). However, no differences in proliferation were detected in viPax4PD-expressing cells as compared to non-infected or viScr-transduced cells (Figure 7d). Oncogene

Discussion The present study establishes a causal association between Pax4 mRNA levels and susceptibility of INS1E cells to apoptosis, suggesting an important physiological function of the transcription factor in cell survival. Accordingly, suppression of Pax2, Pax7 or Pax3 in tumour cell lines has been shown to increase cell death (Margue et al., 2000; Muratovska et al., 2003; He et al., 2005), indicating that high expression levels of pax genes are most likely essential for evading the apoptotic programme, a hallmark of cancer (Hanahan and Weinberg, 2000). Interestingly, deletion of two Pax2/5/ 8-related genes (egl-38 and pax2) in Caenorhabditis elegans was shown to induce apoptosis in somatic and germline cells via the antiapoptotic gene bcl-2 (Park et al., 2006). Similarly, we have previously demonstrated

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Figure 3 Specific downregulation of insulin mRNA levels in INS-1E cells expressing an insulin-targeted shRNA. (a) INS-1E cells were co-transfected with the empty vector (U6, control), the shRNA targeted to insulin (siINS6) or the siINS6 containing a single point mutation (siMut1) along with the reporter GFP-expressing vector and subsequently processed as in Figure 2. Transcript levels for (a) insulin, (b) Pdx1 and Pax4 were determined by QT-PCR and normalized to cyclophilin. Results are the mean of least three independent experiments performed in duplicate and are expressed as % change of mRNA as compared to GFP cells. **Po0.01. (c) Immunofluorescent detection of GFP (green) and insulin (red) in INS-1E cells 72 h after transfection with the indicated plasmids. No staining was detected in GFP þ /siINS6 cells (arrows) whereas cells expressing the control vectors U6 or siMut1 retained insulin staining.

that increased Pax4 expression not only protected against cytokine-induced apoptosis but also promoted proliferation of primary rat and human islet b-cells. Taken together, these observations indicate that INS-1E cells, in contrast to mature b-cells, have selectively exploited high expression levels of Pax4 to confer a strong advantage to survival while potentially shortcircuiting the impact of the transcription factor on cell proliferation. This premise is supported by our findings that repression of Pax4 in INS-1E by RNA interference did not inhibit proliferation. Furthermore, Id2, a downstream target of the proto-oncogene c-myc regulated by Pax4 in primary b-cells, was increased rather than decreased in Pax4-repressed INS-1E cells. Moreover, rat islets and INS-1E cells have similar Id2 mRNA levels (data not shown), suggesting that Id2 regulation is not important for proliferation in these transformed

cells. Interestingly, Pax6 is also expressed in endocrine cells as well as in INS-1E and was previously shown to induce pancreatic cystic adenoma in transgenic animals bearing the transcription factor under the Pdx1 promoter (Yamaoka et al., 2000). Pax6 could therefore potentially be involved in INS-1E cell proliferation by overriding the effect of Pax4. However, we have previously demonstrated that in contrast to Pax4, overexpression of Pax6 in islets did not promote proliferation (Brun et al., 2004). Consistent with the latter, several studies have also shown that increased Pax6 expression correlates with tumour suppression rather than stimulation of cell replication in human cancers (Zhou et al., 2003, 2005). Therefore, these studies do not support a fundamental role of Pax6 in b-cell replication. Most likely, other adaptive mechanisms such as upregulation of the telomerase gene prevail Oncogene

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Figure 4 Repression of Pax4 induces apoptosis without altering proliferation. INS-1E cells were co-transfected with the indicated vectors along with the reporter phogrin-GFP-expressing vector for 72 h as described in Materials and methods. Cell death (a) and cell proliferation (b) were measured in INS-1E cells by the TUNEL assay and by BrdU incorporation, respectively. For proliferation, cells were cultured in the presence of BrdU (10 mM) for 72 h. Representative composite images of INS-1E cells positive for TUNEL (a) or immunostained for BrdU (b) (red), GFP (green) and DAPI (nuclei, blue) are shown. Arrows depict INS1-E cells expressing siPax4PD or siPax4HD. Bars, 50 mM.

in the insulinoma to convey self sustained cell replication (Blasco, 2005). Consistent with the potential dissociation of Pax4 expression from proliferation, treatment of INS-1E cells with betacellulin or glucose did not stimulate Pax4 expression, whereas the sugar evoked an 11-fold induction of Pax4 mRNA with a concomitant increase in c-myc gene expression in rat islets. Accordingly, we have also demonstrated that betacellulin induced Pax4 expression in rat b-cells (Brun et al., 2004). Furthermore, we found that Pax4 was stimulated in human primary b-cells by both betacellulin and glucose (Brun et al., 2005). These growth factors are known to mediate their mitogenic effects on INS-1 cells through the PI3kinase signalling pathway (Barker et al., 2002; Buteau et al., 2003). Paradoxically, betacellulin was originally isolated from the conditioned medium of cell lines derived from mouse pancreatic b-cell tumours (Shing et al., 1993). Self-sufficiency in growth is a hallmark of cancer in which tumour cells escape the requirement of exogenous derived signals in order to survive (Hanahan and Weinberg, 2000). It is thus conceivable that deregulated high Pax4 expression in INS-1E cells is a consequence of elevated endogenous betacellulin levels and that further activation by the PI3-kinase pathway is inefficient. However, this does not preclude the potential action of betacellulin on other target genes involved in proliferation, as the mitogen stimulates thymidine Oncogene

incorporation in INS-1 cells (Huotari et al., 1998; Buteau et al., 2003). In agreement with a previous study (Ueda, 2000), we found that activin A stimulated Pax4 expression in INS-1E cells. This growth factor mediates its effect through the TGF-b signalling pathway independent of the PI3-kinase (Feng and Derynck, 2005), which may explain its ability to induce Pax4 expression in INS-1E cells. Thus, regulation of the pax4 gene in INS-1E cells appears to be governed by the action of several converging signal-transduction pathways but that intrinsic high levels of the transcription factor do not correlate with cell proliferation. Consistent with this premise, we found that inhibition of Pax4 expression using RNA interference induced apoptosis in the insulinoma cells without any impact on proliferation. We also observed a concomitant decrease in the antiapoptotic gene bcl-xl, which was previously demonstrated to be transcriptionally regulated by Pax4 (Brun et al., 2004). Accordingly, both Pax4 and Bcl-xL mRNA levels were dose- dependently inhibited by increasing amounts of viPax4PD and viPax4HD, resulting in a graded increase of apoptosis. Interestingly, Henderson et al. (2005) showed that the moderate reduction in Bcl-xL expression observed in bcl-xl þ / transgenic mice conferred protection against tumorigenesis. Conversely, high expression levels of Bcl-xL were detected in rhabdomyosarcoma cell lines and conveyed resistance to cell death (Margue et al., 2000). Remarkably, the

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Figure 5 Dose-dependent decrease in both Pax4 and Bcl-xL transcripts in INS-1E cells transduced with viral constructs harbouring either shPax4PD or shPax4HD. INS-1E cells were exposed to either 1  107 PFU/ml (white bars) or 2  107 PFU/ml (black bars) of the indicated adenoviruses for 72 h and transcript levels were subsequently measured for (a) Pax4, (b) Pdx1 (c) Pax6 and (d) Bcl-xL. Expression levels were normalized to the reference gene cyclophilin. Results are the mean of four independent experiments performed in duplicate and are expressed as % change of mRNA as compared to vU6-transduced INS-1E cells. *Po0.05 and **Po0.01.

latter anti-apoptotic effect was mediated through the direct transcriptional activation of Bcl-xL by Pax3. Likewise, Bcl-xL mRNA levels were found to be fivefold higher in INS-1E cells as compared to islets corroborating the increased Pax4 expression. Suppression of Pax4, and consequently Bcl-xl, leads to increased apoptosis, a phenomenon observed with other pax genes (Bernasconi et al., 1996; Margue et al., 2000; Ostrom et al., 2000; Henderson et al., 2005). Taken together, these findings strongly suggest that increased expression of Pax4 and the activation of its downstream target gene bcl-xl are critical determinants in sustaining the insulinoma phenotype. In support of this survival function, we find that subsequent to Pax4 inhibition, INS-1E cells are more susceptible to cytokine-induced apoptosis. In conclusion, we show that Pax4 acts as a survival gene in INS-1E cells, a process requiring upregulation of the antiapoptotic gene bcl-xl. Materials and methods Cell culture INS-1E cells (Merglen et al., 2004) were cultured in RPMI1640 (Invitrogen, Basel, CA, USA) supplemented with 10% fetal calf serum (FCS; Brunschwig AG, Basel, Switzerland) and other additions as described previously (Asfari et al., 1992). Pancreatic islets were isolated from Wistar rats as previously described (Gauthier et al., 2004). In several

instances, INS-1E cells were exposed to 0.1, 0.5 and 2 nM of activin A and betacellulin (Sigma, Basel, Switzerland) for 24 h. In parallel, islets as well as INS-1E cells were also cultured in the presence of 2.5, 10 and 30 mM glucose for 24 h. RNA interference RNAi experiments were performed using a novel expression vector that was created using the Adeno-X Tet-On expression system from BD Clontech (Basel, Switzerland). Briefly, a PmeI primer was introduced into the NheI site of the pTRE-Shuttle2 vector. Subsequently, restriction digest of the vector was performed using MluI and PmeI to release a fragment containing the Tet-responsive element as well as the CMV promoter. The MluI/PmeI vector backbone was then purified and ligated to a mouse U6 promoter MluI/PmeI fragment amplified by PCR from a construct kindly provided by Dr Altman (Yale University, CN, USA). The resulting plasmid, denoted as pDLDU6 (U6), contains a novel PmeI site that allows for the insertion of hairpin siRNA sequences at the þ 1 position of the endogenous U6 transcript. Two 21-nucleotide Pax4 shRNA structures with a 6-nucleotide loop were synthesized as two DNA oligonucleotides and annealed and ligated to the PmeI and XbaI sites of U6. The targeted sequences were; (1) siPax4PD, aimed at the sequence 50 -GGC TCG AAT TGC CCA GCT AAA-30 located in the paired domain and (2) siPax4HD, targeted to the sequence 50 -GCA GAC AAG AGA AGT TGA AAT-30 found in the homeodomain of Pax4. To monitor specificity of Pax4 repression and to control against physiological artefacts induced by RNAi, an shRNA directed against insulin (siINS6; 50 -CAG GCT TTT Oncogene

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Figure 6 Repression of Pax4 using adenoviral vectors induces cell death and increased susceptibility to cytokine-induced apoptosis. INS-1E cells were infected with the indicated adenoviral vectors and apoptosis was measured by the TUNEL assay 72 h posttransduction. (a) Representative immunofluorescent composite images of INS-1E cells revealing insulin- (green), DAPI- (nuclei, blue) and TUNEL (red)-positive cells. Arrows depict apoptotic INS1-E cells expressing viPax4PD or viPax4HD. (b) TUNEL-positive INS-1E cells were counted under a fluorescent microscope in five separate fields (each containing approximately 50 cells) of five independent experiments and graphical results are depicted as a percentage of TUNEL/insulin positive cells over the total amount of insulin positive cells. (c) Infected INS-1E cells were exposed for the last 24 h to IFN-g, IL-1b and TNF-a (2 ng/ml each) to induce apoptosis. Similar to (b), TUNEL-positive cells were counted and results are depicted as a percentage of TUNEL/insulin-positive cells over the total amount of insulin-positive cells. *Po0.05 and **Po0.01. Bars, 50 mM.

GTC AAA CAG CAC-30 ) and also a scrambled shRNA (siScr; 50 -CGG CGT TAG CGA TTA GAT GAT-30 ) were generated. Subsequently, the various U6/shRNAs cassettes were transferred into the promoter-deficient Adeno-X viral DNA to generate recombinant adenoviruses (viPax4HD, viPAx4PD, viINS6 and viScr).

monitored by real-time PCR. In parallel, cell proliferation as well as cell death assays were performed on GFP-phogrinco-transfected cells. The latter recombinant protein, targeted to insulin granules, was used for these experiments as GFP was found to interfere with the immunohistochemical detection of apoptosis detected by the TUNEL assay (data not shown).

Cell transfection Constructs were transfected into INS-1E cells along with a reporter GFP-expressing vector (ClonTech) using the Lipofectamine 2000 reagent according to the manufacturer’s guidelines (Invitrogen). The ratio of shRNA plasmid to reporter GFP vector was maintained at 1:2. Subsequent to cell sorting using GFP (72 h post-transfection), the effect of siPax4PD, siPax4HD, siINS6 and siScr on selected genes was

Adenoviral infection of INS-1E cells and cytokine treatment Before infection (24 h), INS-1E cells were seeded at 3  105 cells/ml in Falcon 24-well plates. Cells were then exposed to 1 or 2  107 PFU/ml of recombinant adenoviruses for 3 h, washed, replenished with fresh media and cultured for an additional 48–72 h. In some instances, infected cells were incubated in the presence of IFN-g, IL-1b and TNF-a (2 ng/ ml) for 24 h to induce apoptosis.

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Figure 7 Proliferation is sustained in Pax4-repressed INS-1E cells. INS-1E cells were transduced with the indicated concentrations of adenoviruses for 72 h. Cell proliferation was then assessed by BrdU incorporation. Representative composite images of INS-1E cells immunostained for BrdU (red), insulin (green) and DAPI (blue) are shown subsequent to a 72 h (a), as well as a 4 and 16 h (c) BrdU treatment (BrdU, green; and insulin, red). BrdU-positive cells were counted under a fluorescent microscope as described in Figure 6 and graphical results are depicted as a percentage of positive cells over the total amount of insulin-positive cells (b and d). Note that only a fraction of cells are labelled in (c) and that, similar to (b), no alterations in proliferation are detected among the various experimental groups. Data represent the mean7s.e. of at least three independent experiments.

Quantitative real-time PCR (QT-PCR) Total RNA was extracted from INS-1E cells using the Qiagen RNeasy mini kit and 2 mg was reversed transcribed into cDNA as described previously (Gauthier et al., 1999). Primers for cyclophilin, Serca3, insulin, Pdx1, Id2, Bcl-xL, Pax4, Pax6, Stat1 and RpS29 were designed using the Primer Express Software (Applera Europe, Rotkreuz, Switzerland) and sequences are shown in Table 1. QT-PCR was performed using an ABI 7000 Sequence Detection System (Applera Europe) and PCR products were quantified using the SYBR Green Core Reagent kit (Gauthier et al., 2004). Three distinct amplifications derived from 4–6 independent experiments were performed in duplicate for each transcript and mean values were normalized to the mean value of the reference mRNA cyclophilin or alternatively to the RpS29 transcript. Authenticity of each amplicon was verified by DNA sequencing.

Immunohistochemistry INS-1E cells were cultured on polyornithine-treated glass coverslips for 3 days, washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. Immunochemical detection of insulin was performed as described previously (Ishihara et al., 2003). Nuclei were stained with 40 ,6-diamino-2-phenylindole (DAPI) (10 mg/ml; Sigma). Coverslips were mounted using DAKO fluorescent mounting medium and visualized using a Zeiss Axiophot I. Cell proliferation and TUNEL assays For proliferation, transfected and/or infected INS-1E cells were labelled with 10 mM BrdU for 4, 16 or 72 h. Proliferation was estimated using an immunohistochemical assay kit as described by the manufacturer (BrdU labelling and detection Oncogene

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Primers used for QT-PCR analysis

Sequence

S: 50 -GGA TGC GCG GGA GGT AA-30 A: 50 -TCA TCG CCA GCC TCT CTC A-30 Cyclophilin S: 50 -TCA CCA TCT CCG ACT GTG GA-30 A: 50 -AAA TGC CCG CAA GTC AAA GA-30 Id2 S: 50 -CCG ACT GTA GAA AGG GCA TTG-30 A: 50 -GAT CAT CTC CCC CAG GTG TTC-30 Pax6 S: 50 -CCA GCT TCA CCA TGG CAA A-30 A: 50 -GCA GGA GTA CGA GGA GGT CTG A-30 Pax4 S: 50 -TGG ACA CCC GAC AGC AGA T-30 A: 50 -CTT AAG GCT CCG TGA GAT GTC A-30 Pdx1 S: 50 -CCG CGT TCA TCT CCC TTT C-30 A: 50 -CTC CTG CCC ACT GGC TTT T-30 RpS29 S: 50 -GCT GAAA CAT GTG CCG ACA GT-30 A: 50 -GGT CGC TTA GTC CAA CTT AAT GAA G-30 Serca3 S: 50 -ACC CTG TTG CTC CTT TG-30 A: 50 -TCA CAC TGA CAG GCG CTT TC-30 Stat1 S: 50 -CCT GCT TTG CCT CTG GAA TG-30 A: 50 -CCT TGA GCA GAG CAC GTT CTC-30 Bcl-xL

Kit, Roche Diagnostics, Rotkreuz, Switzerland). Cell death was measured by the TUNEL assay (In Situ Cell Death Detection Kit, Roche). Results are expressed as a percentage of BrdU or TMR-red-labelled nuclei (TUNEL-positive

cells) over the total amount of INS-1E cells (nuclei staining by DAPI). Statistical analysis Results are expressed as mean7s.e.m. Where indicated, the statistical significance of the differences between groups was estimated by Student’s unpaired t-test. * and ** indicate statistical significance with Po0.05 and Po0.01, respectively.

Abbreviations BrdU, 5-bromo-20 -deoxy-uridine; BSA, bovine serum albumin; DAPI, 40 , 6-diamidino-2-phenylindole; FCS, fetal calf serum; shRNA, short hairpin RNA; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick end labelling. Acknowledgements We are grateful to Elodie Husi, Mathurin Baquie´ and Delphine Chesnel for their expert technical assistance. This work was supported by grants from the Swiss National Science Foundation (#3100A0-107682/1 to BRG) and from the Juvenile Diabetes Research Foundation (#7-2005-1158 to CBW).

References Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB. (1992). Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology 130: 167–178. Barker CJ, Leibiger IB, Leibiger B, Berggren PO. (2002). Phosphorylated inositol compounds in beta-cell stimulus– response coupling. Am J Physiol Endocrinol Metab 283: E1113–E1122. Bernasconi M, Remppis A, Fredericks WJ, Rauscher III FJ, Schafer BW. (1996). Induction of apoptosis in rhabdomyosarcoma cells through down-regulation of PAX proteins. Proc Natl Acad Sci USA 93: 13164–13169. Biason-Lauber A, Boehm B, Lang-Muritano M, Gauthier BR, Brun T, Wollheim CB et al. (2005). Association of childhood diabetes mellitus with a genomic variant of Pax4: possible link to beta cell regenerative capacity. Diabetologia 48: 900–905. Blasco MA. (2005). Telomeres and human disease: ageing, cancer and beyond. Nat Rev Genet 6: 611–622. Brun T, Duhamel D, Sarret EJ, Bosco D, Wollheim CB, Gauthier BR. (2005). The diabetes-linked transcription factor Pax4 is expressed in human pancreatic islets and is activated by glucose activin A and betacellulin. Diabetologia 48(Suppl 1): A66. Brun T, Franklin I, St-Onge L, Biason-Lauber A, Schoenle E, Wollheim CB et al. (2004). The diabetes-linked transcription factor Pax4 promotes beta-cell proliferation and survival in rat and human islets. J Cell Biol 167: 1123–1135. Buteau J, Foisy S, Joly E, Prentki M. (2003). Glucagon-like peptide 1 induces pancreatic beta-cell proliferation via transactivation of the epidermal growth factor receptor. Diabetes 52: 124–132. Collombat P, Hecksher-Sorensen J, Broccoli V, Krull J, Ponte I, Mundiger T et al. (2005). The simultaneous loss of Arx and Pax4 genes promotes a somatostatin-producing cell fate specification at the expense of the alpha- and beta-cell Oncogene

lineages in the mouse endocrine pancreas. Development 132: 2969–2980. Collombat P, Mansouri A, Hecksher-Sorensen J, Serup P, Krull J, Gradwohl G et al. (2003). Opposing actions of Arx and Pax4 in endocrine pancreas development. Genes Dev 17: 2591–2603. Dor Y, Brown J, Martinez OI, Melton DA. (2004). Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429: 41–46. Feng XH, Derynck R. (2005). Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol 21: 659–693. Gauthier B, Robb M, McPherson R. (1999). Cholesteryl ester transfer protein gene expression during differentiation of human preadipocytes to adipocytes in primary culture. Atherosclerosis 142: 301–307. Gauthier BR, Brun T, Sarret EJ, Ishihara H, Schaad O, Descombes P et al. (2004). Oligonucleotide microarray analysis reveals PDX1 as an essential regulator of mitochondrial metabolism in rat islets. J Biol Chem 279: 31121–31130. Gunton JE, Kulkarni RN, Yim S, Okada T, Hawthorne WJ, Tseng YH et al. (2005). Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes. Cell 122: 337–349. Hanahan D, Weinberg RA. (2000). The hallmarks of cancer. Cell 100: 57–70. He SJ, Stevens G, Braithwaite AW, Eccles MR. (2005). Transfection of melanoma cells with antisense PAX3 oligonucleotides additively complements cisplatin-induced cytotoxicity. Mol Cancer Ther 4: 996–1003. Henderson CC, Zhang Z, Manson SR, Riehm JJ, Kataoka M, Flye MW et al. (2005). A moderate reduction of Bcl-x(L) expression protects against tumorigenesis; however, it also increases susceptibility to tissue injury. Oncogene 24: 7120–7124. Heremans Y, Van De Casteele M, in’t Veld P, Gradwohl G, Serup P, Madsen O et al. (2002). Recapitulation of

Pax4 and apoptosis T Brun et al

4271 embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3. J Cell Biol 159: 303–312. Holm P, Rydlander B, Luthman H, Kockum I. (2004). Interaction and association analysis of a type 1 diabetes susceptibility locus on chromosome 5q11–q13 and the 7q32 chromosomal region in Scandinavian families. Diabetes 53: 1584–1591. Hugl SR, White MF, Rhodes CJ. (1998). Insulin-like growth factor I (IGF-I)-stimulated pancreatic beta-cell growth is glucose-dependent. Synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-I in INS-1 cells. J Biol Chem 273: 17771–17779. Huotari MA, Palgi J, Otonkoski T. (1998). Growth factormediated proliferation and differentiation of insulin-producing INS-1 and RINm5F cells: identification of betacellulin as a novel beta-cell mitogen. Endocrinology 139: 1494–1499. Ishihara H, Maechler P, Gjinovci A, Herrera PL, Wollheim CB. (2003). Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells. Nat Cell Biol 5: 330–335. Kanatsuka A, Tokuyama Y, Nozaki O, Matsui K, Egashira T. (2002). Beta-cell dysfunction in late-onset diabetic subjects carrying homozygous mutation in transcription factors NeuroD1 and Pax4. Metabolism 51: 1161–1165. Kojima H, Fujimiya M, Matsumura K, Younan P, Imaeda H, Maeda M et al. (2003). NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and reverses diabetes in mice. Nat Med 9: 596–603. Li Y, Nagai H, Ohno T, Ohashi H, Murohara T, Saito H et al. (2006). Aberrant DNA demethylation in promoter region and aberrant expression of mRNA of PAX4 gene in hematologic malignancies. Leuk Res 9: 1547–1553. Margue CM, Bernasconi M, Barr FG, Schafer BW. (2000). Transcriptional modulation of the anti-apoptotic protein BCL-XL by the paired box transcription factors PAX3 and PAX3/FKHR. Oncogene 19: 2921–2929. Mauvais-Jarvis F, Smith SB, Le May C, Leal SM, Gautier JF, Molokhia M et al. (2004). PAX4 gene variations predispose to ketosis-prone diabetes. Hum Mol Genet 13: 3151–3159. Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB, Maechler P. (2004). Glucose sensitivity and metabolismsecretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology 145: 667–678. Miyamoto T, Kakizawa T, Ichikawa K, Nishio S, Kajikawa S, Hashizume K. (2001). Expression of dominant negative form of PAX4 in human insulinoma. Biochem Biophys Res Commun 282: 34–40. Muratovska A, Zhou C, He S, Goodyer P, Eccles MR. (2003). Paired-Box genes are frequently expressed in cancer and often required for cancer cell survival. Oncogene 22: 7989–7997. Ostrom L, Tang MJ, Gruss P, Dressler GR. (2000). Reduced Pax2 gene dosage increases apoptosis and slows the progression of renal cystic disease. Dev Biol 219: 250–258. Park D, Jia H, Rajakumar V, Chamberlin HM. (2006). Pax2/ 5/8 proteins promote cell survival in C. elegans. Development 133: 4193–4202.

Robson EJ, He SJ, Eccles MR. (2006). A PANorama of PAX genes in cancer and development. Nat Rev Cancer 6: 52–62. Shimajiri Y, Sanke T, Furuta H, Hanabusa T, Nakagawa T, Fujitani Y et al. (2001). A missense mutation of Pax4 gene (R121W) is associated with type 2 diabetes in Japanese. Diabetes 50: 2864–2869. Shimajiri Y, Shimabukuro M, Tomoyose T, Yogi H, Komiya I, Takasu N. (2003). PAX4 mutation (R121W) as a prodiabetic variant in Okinawans. Biochem Biophys Res Commun 302: 342–344. Shing Y, Christofori G, Hanahan D, Ono Y, Sasada R, Igarashi K et al. (1993). Betacellulin: a mitogen from pancreatic beta cell tumors. Science 259: 1604–1607. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. (2003). Activation of the interferon system by shortinterfering RNAs. Nat Cell Biol 5: 834–839. Sosa-Pineda B, Chowdhury K, Torres M, Oliver G, Gruss P. (1997). The Pax4 gene is essential for differentiation of insulin-producing beta cells in the mammalian pancreas. Nature 386: 399–402. Theander-Carrillo C, Wiedmer P, Cettour-Rose P, Nogueiras R, Perez-Tilve D, Pfluger P et al. (2006). Ghrelin action in the brain controls adipocyte metabolism. J Clin Invest 116: 1983–1993. Theis M, Mas C, Doring B, Degen J, Brink C, Caille D et al. (2004). Replacement by a lacZ reporter gene assigns mouse connexin36, 45 and 43 to distinct cell types in pancreatic islets. Exp Cell Res 294: 18–29. Tokuyama Y, Matsui K, Ishizuka T, Egashira T, Kanatsuka A. (2006). The Arg121Trp variant in PAX4 gene is associated with beta-cell dysfunction in Japanese subjects with type 2 diabetes mellitus. Metabolism 55: 213–216. Ueda Y. (2000). Activin A increases Pax4 gene expression in pancreatic beta cell lines. FEBS Lett 480: 101–105. Wang J, Elghazi L, Parker SE, Kizilocak H, Asano M, Sussel L et al. (2004). The concerted activities of Pax4 and Nkx2.2 are essential to initiate pancreatic beta-cell differentiation. Dev Biol 266: 178–189. Yamaoka T, Yano M, Yamada T, Matsushita T, Moritani M, Ii S et al. (2000). Diabetes and pancreatic tumours in transgenic mice expressing Pa6. Diabetologia 43: 332–339. Zalzman M, Gupta S, Giri RK, Berkovich I, Sappal BS, Karnieli O et al. (2003). Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc Natl Acad Sci USA 100: 2426–2431. Zhang YQ, Mashima H, Kojima I. (2001). Changes in the expression of transcription factors in pancreatic AR42J cells during differentiation into insulin-producing cells. Diabetes 50(Suppl 1): S10–14. Zhou YH, Tan F, Hess KR, Yung WK. (2003). The expression of PAX6, PTEN, vascular endothelial growth factor, and epidermal growth factor receptor in gliomas: relationship to tumor grade and survival. Clin Cancer Res 9: 3369–3375. Zhou YH, Wu X, Tan F, Shi YX, Glass T, Liu TJ et al. (2005). PAX6 suppresses growth of human glioblastoma cells. J Neurooncol 71: 223–229.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene