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The Transcription Factor PAX4 Acts As a Survival Gene in INS-1E Insulinoma Cells

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The Transcription Factor PAX4 Acts As a Survival Gene in INS-1E Insulinoma Cells Oncogene (2007) 26, 4261–4271 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc 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 domain nuclear factor Pax4 is essential for b-cell essential for islet b-cell generation during pancreas maturation. Indeed, Pax4 mRNA is detected in the development and their survival in adulthood. High Pax4 mouse pancreatic bud at E9.5, becoming maximal at expression was reported in human insulinomas indicating E13.5–15.5 during the period of massive b-cell expan- that deregulation of the gene may be associated with sion and thereafter declining. In mature islets, low levels tumorigenesis. We report that rat insulinoma INS-1E of Pax4 expression are detected in human, mouse and cells express 25-fold higher Pax4 mRNA levels than rat rat b-cells (Zhang et al., 2001; Heremans et al., 2002; islets. In contrast to primary b-cells, activin A but not Kojima et al., 2003; Zalzman et al., 2003; Brun et al., betacellulin or glucose induced Pax4 mRNA levels 2004, 2005; Theis et al., 2004; Gunton et al., 2005). indicating dissociation of Pax4 expression from insulino- Consistent with its tissue-specific expression pattern, ma cell proliferation. Short hairpin RNA adenoviral targeted disruption of the pax4 gene in mice results constructs targeted to the paired domain or homeodomain in the absence of mature pancreatic b- and d-cells with (viPax4PD and viPax4HD) were generated. Pax4 mRNA a commensurate increase in the glucagon-producing levels were lowered by 73 and 50% in cells expressing a-cells (Sosa-Pineda et al., 1997; Wang et al., 2004). This either viPax4PD or viPax4HD. Transcript levels of the increase was attributed to the a-cell-specific transcrip- Pax4 target gene bcl-xl were reduced by 53 and 47%, tion factor Arx, which is repressed by Pax4 during whereas Pax6 and Pdx1mRNA levels were unchanged. development (Collombat et al., 2003). Interestingly, viPax4PD-infected cells displayed a twofold increase simultaneous inactivation of the arx and pax4 gene in spontaneous apoptosis and were more susceptible in mice islets promotes a somatostatin-producing dÀ to cytokine-induced cell death. In contrast, proliferation cell fate specification to the detriment of the a-and was unaltered. RNA interference-mediated repression of b-cell lineages, indicating that Pax4 is redundant for d- insulin had no adverse effects on either Pax4 or Pdx1 cell formation under certain conditions (Collombat expression as well as on cell replication or apoptosis. et al., 2005). These results indicate that Pax4 is redundant for Several studies in the Japanese population and one in proliferation of insulinoma cells, whereas it is essential Afro-Americans have associated mutations in the pax4 for survival through upregulation of the antiapoptotic gene gene to type II diabetes, whereas polymorphisms have bcl-xl. been linked to type I diabetes in Scandinavian, German Oncogene (2007) 26, 4261–4271; doi:10.1038/sj.onc.1210205; and Swiss families (Shimajiri et al., 2001, 2003; published online 29 January 2007 Kanatsuka et al., 2002; Holm et al., 2004; Mauvais- Jarvis et al., 2004; Biason-Lauber et al., 2005; Tokuya- Keywords: RNAi; insulin; Pax4; proliferation; apoptosis; ma et al., 2006). These genetic studies suggest an survival 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 Introduction Pax4 in rat islets resulted in the induction of the c-myc/ Id2 proliferation pathway and of the antiapoptotic gene Normal islet b-cell mass is achieved through the inter- bcl-xl. The coordinated stimulation of these genes by play of numerous transcription factors during pancreas Pax4 prompted b-cell replication and conferred protec- organogenesis. In particular, the paired homeo- tion against cytokine-induced apoptosis in human islets (Brun et al., 2004). These results clearly show that Pax4 Correspondence: Dr BR Gauthier and T Brun, Department of Cell acts as a key regulator of b-cell plasticity in mature Physiology and Metabolism, University Medical Center, 1211 Geneva islets, substantiating the concept that terminally differen- 4, Switzerland. tiated b-cells retain a significant capacity to proliferate E-mails: [email protected] and thierry.brun@ medecine.unige.ch in response to physiological stimuli (Dor et al., 2004). Received 5 May 2006; revised 23 October 2006; accepted 9 November Human insulinomas were shown to contain aber- 2006; published online 29 January 2007 rantly high expression levels of Pax4 as compared to Pax4 and apoptosis T Brun et al 4262 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 mem- bers 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 down- regulation 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 Figure 1 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 (a) Higher levels of Pax4 and Bcl-xL transcripts are expressed in in human insulinomas and proposed to be a predisposi- the rat insulinoma INS-1E cell line as compared to adult rat islets. tion factor for tumour formation and/or progression QT-PCR using RNA purified from INS-1E cells and freshly (Miyamoto et al., 2001). Accordingly, we found that the isolated rat islets were performed on Pax4, Bcl-xL (left panel) and rat insulinoma INS-1E cell line contained 25-fold more Serca3 (right Panel). Data are presented as fold change mRNA levels as compared to primary cells and normalized to the Pax4 mRNA as compared to rat islets (Figure 1a). housekeeping gene transcript cyclophilin. Each value represents Interestingly, transcript levels for the anti-apoptotic the mean7s.e. of four independent experiments performed in gene bcl-xl, a downstream target of Pax4, were also duplicate. *Po0.05; **Po 0.01. (b) Pax4 mRNAs in INS-1E cells increased fivefold in INS-1E cells. In contrast, mRNA were treated with increasing doses of activin A and betacellulin as indicated on the graph. The results are normalized to cyclophilin levels for the unrelated serca3 gene were decreased (or Rsp291, see Supplementary Information) and are expressed as twofold as compared to islets (Figure 1a). To determine fold change of mRNA as compared to control. (c) INS-1E cells and whether a correlation could be established between Pax4 rat islets were incubated with increasing concentrations of glucose. expression levels and the capacity of INS-1E cells to Pax4 and c-myc transcript abundance levels were then estimated by proliferate, the cells were exposed to the mitogens QT-PCR. Statistical significances were carried out in between control INS-1E cells and islets incubated with the various growth activin A or betacellulin, which were shown to stimulate factors by Student’s t-test. *Po0.05; **Po0.01. 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 dose- dependent stimulation of Pax4 expression, reaching constant when normalized to a second housekeeping maximal induction of 11-fold at 0.5 nM. Surprisingly, gene (RpS29) (Theander-Carrillo et al., 2006) indicating betacellulin provoked only a small nonsignificant that cyclophilin transcription is refractory to the effect increase in Pax4 mRNA levels (Figure 1b). The of mitogens (Supplementary data, panel A). Consistent reference gene cyclophilin expression levels remained with this premise, similar activin A-induced increases in Oncogene Pax4 and apoptosis T Brun et al 4263 Pax4 transcript levels were observed when the expres- abrogated the shRNA-mediated repression of insulin sion profile was normalized with RpS29, whereas no mRNA levels. More importantly, downregulation of the alterations were detected in the presence of betacellulin insulin transcript had no effect on either Pdx1 or Pax4 (Supplementary data, panel B). The discrepancy ob- mRNA levels (Figure 3b).
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