Ectopic expression of the beta-cell specific transcription factor Pdx1 inhibits glucagon gene transcription
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- Ritz-Laser, B., Gauthier, B.R., Estreicher, A. et al. Diabetologia (2003) 46: 810. doi:10.1007/s00125-003-1115-7
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The transcription factor Pdx1 is required for the development and differentiation of all pancreatic cells. Beta-cell specific inactivation of Pdx1 in developing or adult mice leads to an increase in glucagon-expressing cells, suggesting that absence of Pdx1could favour glucagon gene expression by a default mechanism.
We investigated the inhibitory role of Pdx1 on glucagon gene expression in vitro. The glucagonoma cell line InR1G9 was transduced with a Pdx1-encoding lentiviral vector and insulin and glucagon mRNA levels were analysed by northern blot and real-time PCR. To understand the mechanism by which Pdx1 inhibits glucagon gene expression, we studied its effect on glucagon promoter activity in non-islet cells using transient transfections and gel-shift analysis.
In glucagonoma cells transduced with a Pdx1-encoding lentiviral vector, insulin gene expression was induced while glucagon mRNA levels were reduced by 50 to 60%. In the heterologous cell line BHK-21, Pdx1 inhibited by 60 to 80% the activation of the α-cell specific element G1 conferred by Pax-6 and/or Cdx-2/3. Although Pdx1 could bind three AT-rich motifs within G1, two of which are binding sites for Pax-6 and Cdx-2/3, the affinity of Pdx1 for G1 was much lower as compared to Pax-6. In addition, Pdx1 inhibited Pax-6 mediated activation through G3, to which Pdx1 was unable to bind. Moreover, a mutation impairing DNA binding of Pdx1 had no effect on its inhibition on Cdx-2/3. Since Pdx1 interacts directly with Pax-6 and Cdx-2/3 forming heterodimers, we suggest that Pdx1 inhibits glucagon gene transcription through protein to protein interactions with Pax-6 and Cdx-2/3.
Cell-specific expression of the glucagon gene can only occur when Pdx1 expression extinguishes from the early α cell precursor.
KeywordsEndocrine pancreasPdx1Pax-6Cdx-2/3glucagontranscriptional regulationα-cell
electrophoretic mobility shift assay
The mammalian pancreas arises as two evaginations, the dorsal and ventral pancreatic anlage, from the embryonic endoderm in the region of the future duodenum. Determination of the pancreatic fate of the dorsal anlage requires signals from the notochord repressing endodermal Sonic hedgehog expression, whereas the ventral anlage forms independently from the notochord [1, 2]. In the mouse, the first two transcription factors expressed in the prospective pancreatic endoderm are HB9 and Pdx1, which are detected at the 8 to 10 somite stage, respectively (embryonic day (E) 8–8.5) [3, 4, 5]; both factors are transiently expressed in the entire pancreatic anlage and are crucial for its development. Mice deficient in HB9 selectively lack the dorsal pancreas due to a defect in specification of the pancreatic epithelium, whereas the ventral pancreas develops and exhibits more subtle defects in beta-cell differentiation and islet organization [3, 4]. Inactivation of Pdx1 results in pancreatic agenesis through a failure in proliferation of the pancreatic epithelium as well as in the branching and differentiation of the pancreatic buds [5, 6]. The widespread expression of HB9 and Pdx1 in the embryonic pancreas declines at E9.5–10.5 (HB9) and E10.5 (Pdx1) and they are later restricted to beta cells (HB9) or beta and δ cells (Pdx1). Similarly, the homeobox gene Isl-1 is transiently expressed in the entire dorsal bud and later restricted to pancreatic endocrine cells; mice deficient in Isl-1 lack the dorsal exocrine pancreas and have no differentiated islet cells [7, 8, 9]. Another transcription factor expressed very early (E9) during development in discrete cells of the pancreatic epithelium is Pax-6, a member of the paired-homeobox family. Pax-6 is crucial for the differentiation of glucagon-producing α cells inasmuch as Pax-6 homozygous mutant mice have no or few α cells [10, 11]. In contrast, inactivation of Pax-4, another paired homeobox gene, results in the selective lack of beta and δ cells; mice lacking both Pax-6 and Pax-4 have no endocrine cells .
Several lines of evidence suggest that Pdx1 plays a key role not only in islet cell differentiation but also in maintaining the differentiated state and control of islet-hormone gene expression. Pdx1 is a major transactivator of the insulin and somatostatin genes through its synergistic interaction with E47/Beta2 and Pax6 or Pbx/Prep1, respectively [13, 14, 15, 16, 17, 18]. Beta-cell specific inactivation of Pdx1 in developing mice resulted in a decrease in beta-cells with a concomitant 2.5-fold increase in Glu+ cells and to coexpression of insulin and glucagon in 22% of cells suggesting that α-cell differentiation and glucagon gene expression are favored by the absence of Pdx1 in vivo . Furthermore, transient inhibition of Pdx1 in pancreatic beta cells of adult mice by antisense RNA expression using a Tet-On system led to reduction of Pdx1 dependent beta-cell specific gene expression and to a striking increase in the number of glucagon gene expressing cells that were homogeneously distributed within the islet . Similarly, we could recently show in a Tet-On system in insulinoma cells that functional inactivation of Pdx1 resulted in differentiation of insulin-producing beta cells into glucagon-producing α-cells .
In this work, we aimed to complement the studies of Pdx1 function in beta cells by analysing the effect of ectopic Pdx1 expression in α cells. Since α-cell specificity is mainly characterized by glucagon gene expression, we studied Pdx1 effects on glucagon gene transcription in glucagonoma (InR1G9) cells by Lentiviral transduction and on glucagon promoter activity in non-islet cells. We show a new potential function of Pdx1 as mediator of cell-specific expression of the glucagon gene through inhibition of transcription.
Materials and methods
Cell culture and DNA transfection
The glucagon-producing hamster InRIG9  and mouse αTC1 , the insulin-producing hamster HIT-T15 , the non-islet Syrian baby hamster kidney (BHK-21) and human embryonic kidney (HEK) cell lines were grown in RPMI 1640 (Seromed; Basel, Switzerland) supplemented with 5% heat-inactivated fetal calf serum and 5% heat-inactivated newborn calf serum, 2 mmol/l glutamine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin. BHK-21 and HEK cells were transfected by the calcium phosphate precipitation technique  using 10–15 µg of total plasmid DNA per 10-cm petri dish. One µg of pSV2A PAP, a plasmid containing the human placental alkaline phosphatase gene, driven by the simian virus 40 (SV40) early promoter was added to monitor transfection efficiency . Transfection of InR1G9 cells was done using the DEAE-dextran method as described previously . cDNAs for the hamster Cdx-2/3 and Pdx1 (M.S. German, University of California, San Francisco, Calif., USA), rat Isl-1 (D. Drucker, University of Toronto, Toronto, Canada), mouse Meis2 (N. Copeland, National Cancer Institute, Fredrick, USA), and quail Pax-6 (S. Saule, Institut Curie, Orsay, France), were cloned in the expression vector pSG5 (Stratagene). In experiments using variable quantities of expression vectors coding for Pdx1, Pax-6, or Cdx-2/3, total amount of DNA was kept constant by adding appropriate amounts of the empty vector pSG5. Reporter plasmids consisted of the CAT reporter gene driven by different fragments of the rat glucagon gene promoter (-292GluCAT, -138GluCAT, G3–138GluCAT, G3–31GluCAT  or the rat insulin I gene promoter (-410InsCAT, comprising 410 bp of the 5'flanking sequence and 49 bp of exon 1 and intron 1 of the rat insulin I gene) . Data are presented as fold stimulation of the CAT activity obtained with the reporter plasmid alone and are the means +/- SEM of at least three experiments.
Transduction of InR1G9 cells with a lentiviral vector encoding hPDX1
A Human PDX1 cDNA (hPDX1), kindly provided by V. Schwitzgebel (University Hospital Geneva, Geneva, Switzerland), was subcloned into pBluescript (Stratagene) and then inserted into an optimized HIV-based vector (pWPT, kindly provided by M. Wiznerowicz, Faculty of Medecine, Geneva, Switzerland), generating the pWPT-PDX transfer plasmid. Map and sequence are available at http://www.medecine.unige.ch/~salmon/pWPT-PDX.html. Stocks of pWPT-PDX lentiviral vectors were produced using transient cotransfection of 293T cells with pWPT-PDX, R8.91 (second generation HIV-packaging plasmid , and pMDG (VSV envelope-expression plasmid) as described previously . Vectors were concentrated by ultracentrifugation and resuspended in serum-free medium (CellGro SCGM, CellGenix, Freiburg, Germany) as described previously . Vector stocks were stored at −70°C and titres were determined by enzymatic assay of HIV reverse transcriptase. For hPDX1 transduction, 3×104 INR1G9 cells were transduced in 50 µl of RPMI 1640 10% FCS with 2.5×104 pPWT-PDX transducing units (as determined by RT assay). After 24 h, cells were washed twice with RPMI 10% FCS and cultured for further 72 h before testing for Pdx1 expression by immunocytochemistry using anti-Pdx1 antibodies kindly provided by C. V. E. Wright (Vanderbilt Medical Center, Nashville, Tenn., USA). RNA was isolated for northern blotting using Trizol (Invitrogen) and whole-cell protein extracts were prepared for EMSA. Northern blots were sequentially hybridized with random labelled complementary DNA (cDNA) probes for rat glucagon and proinsulin I and an 18S ribosomal (rRNA) oligonucleotide probe (5′-GCCGTCCCTCTTAATCATGGCCTCAGTTCC). Hybridization signals of the pWPT-PDX transduced InR1G9 cells were quantified using a PhosphoImager (Molecular Dynamics) and expressed as glucagon mRNA/18S rRNA ratio relative to the control cells.
Reverse transcription was done by using 2 µg of total RNA isolated from HIT-T15 and from non-infected or lentivirus-infected InR1G9 cells, random hexanucleotide primers and Superscript II reverse transcriptase (Invitrogen). One tenth of the resulting cDNA was used for real-time PCR in the LightCycler instrument (Roche) using the QuantiTect SYBR Green PCR kit (Qiagen) as recommended by the provider. The following primers were used: insulin sense 5′-tcttctacacacccaagacc, antisense 5′-gttccacaatgccacgcttc; glucagon sense 5′-gatcattcccagcttcccag, antisense 5′-ctggtaaaggtcccttcagc; 28S rRNA sense 5′-tagccaaatgcctcgtcatc, antisense 5′-acctctcatgtctcttcacc.
Chloramphenicol aetyltransferase (CAT) and protein assays
Cell extracts were prepared 48 h after transfection and analysed for CAT and alkaline phosphatase activities as described previously . Quantification of acetylated and non-acetylated forms was done with a PhosphorImager (Molecular Dynamics). A minimum of three independent transfections was done; each of them carried out in duplicate.
Electrophoretic Mobility Shift Assays (EMSAs)
Nuclear extracts from InR1G9, HIT-T15 or BHK-21 cells transfected with Pax-6 or Pdx1 expression plasmids or the empty vector pSG5 were prepared according to . EMSAs were carried out as described previously  adding first 8 to 28 µg (8 µg in standard reactions) of nuclear extracts and subsequently labelled oligonucleotides containing the rat glucagon gene G1 (G1–56, G1–52, G1–54, G1–50) or G3 (5′-GCTGAAGTAGTTTTTCACGCCTGACTGAGATTGAAGGGTGTATTTC) sequence [35, 36], or the A3/A4 element of the rat insulin I gene (InsA3/A4, 5′-GATCTTGTTAATAATCTAATTACCCTAGAACAATTATTAGATTAATGG) . In competition experiments, labelled and cold oligonucleotides were added simulaneously and when specific antibodies were used, they were added after half of the incubation time. Antisera were generously provided by H. Edlund (anti-Pdx1; University of Umea, Umea, Sweden), S. Saule (anti-Pax-6 homeodomain, serum no. 13; Institut Curie, Orsay, France), D. Melloul (anti full-length Pdx1; Hebrew University Hadassah Medical Center, Jerusalem, Israel), and M.S. German (anti-Cdx-2/3; University of California, San Francisco, Calif., USA).
Mutagenesis of Pdx1
In vitro mutagenesis was done by using the QuikChangeSite-Directed Mutagenesis Kit (Stratagene, The Netherlands) according to the manufacturer's protocol using the following primers for the I192Q mutation of Pdx1: forward 5′-gagacacatcaagcagtggttccaaaacc and reverse 5′-ggttttggaaccactgcttgatgtgtctc.
GST fusion proteins and GST-precipitation
Hamster Pax-6 GST fusion proteins have been described . GST fusion proteins were expressed in E. coli and purified according to the manufacturer recommendations (Pharmacia). L-[35S]methionine-labelled Pdx1 was generated in vitro using the TNT wheat germ extract system (Promega) and GST-precipitations were done as described previously .
Data are presented as means ± SE and statistical significance was tested by analysis of variance and Student's t test where applicable. The threshold for statistical significance was a p value of less than 0.05 (* and ** indicate statistical significance with p<0.05 and p<0.01, respectively).
Lentiviral transduction of Pdx1 in InR1G9 cells inhibits transcription of the endogenous glucagon gene
Pdx1 inhibits transcriptional activation of the glucagon gene promoter by Pax-6 and Cdx-2/3
Pdx1 binds to the G1 element
To assess the relative affinity of Pdx1 for G1 compared to the A3/A4 of the rat insulin I gene, a known Pdx1 binding site, we carried out EMSA competition experiments. A similar molar excess of cold G1–56 and InsA3/A4 oligonucleotides competed for the Pdx1 complex formed with InsA3/A4 indicating that Pdx1 binds to both elements with roughly the same affinity (Fig. 4C). These data are in agreement with the equivalent transactivation of the glucagon and insulin gene promoters by Pdx1. When the individual glucagon gene binding sites were used for EMSA, the most proximal motif G1–50 displayed a slightly higher affinity for Pdx1 compared to G1–52 and G1–54 located within G1–56 but lower compared to InsA3/A4 (Fig. 4D).
Pdx1 forms heterodimers with Pax-6 and Cdx-2/3 on G1
Alternatively to a mechanism of direct competition for the binding site, Pdx1 could interact with Pax-6 and/or Cdx-2/3 and either form a heterodimer or impair DNA binding by protein to protein interaction. We therefore analysed the effect of adding various amounts of Pdx1 on Pax-6 binding to G3 and G1. Pdx1 overexpressed in BHK-21 cells did not interact with G3, only heterodimers of the ubiquitous proteins Pbx and Prep1  were observed (Fig. 5C). Pax-6 formed a complex with G3 that was not affected by the addition of increasing amounts of Pdx1-containing extracts (Fig. 5C). In contrast to G3, Pdx1 bound as a monomer and a homodimer to G1 (Fig. 5D). In addition, a Pax6:Pdx1 heterodimer was formed that was recognized by both anti-Pdx1 and anti-Pax-6 antibodies, thus confirming the protein to protein interaction of both transcription factors (Fig. 5D). When Pdx1 and Cdx-2/3 containing extracts were mixed, both proteins bound as monomers and Pdx1 did not impair Cdx-2/3 binding. In addition, both proteins formed a heterodimer that was recognized by anti-Cdx-2/3 and anti-Pdx1 antibodies (Fig. 5E). Note that when anti-Pdx1 antibodies were used, the Cdx-2/3-Pdx1 heterodimer was displaced by a Cdx-2/3 homodimer with only slightly slower migration than Cdx-2/3-Pdx1. We therefore conclude that Pdx1 could exert its transcriptional inhibition on the glucagon gene promoter through protein to protein interaction with Pax-6 and Cdx-2/3 that could change their conformation and interfere with their transcriptional properties. Furthermore, inhibition of Pax-6 mediated activation could also in part be mediated by competition of binding to the G1 but not the G3 element.
Pdx1 interacts with the homeodomain of Pax-6
Inhibition of Pax-6 and Cdx-2/3 mediated activation is independent of Pdx1 DNA binding
In cotransfection assays, a quantitatively similar repression of Cdx-2/3 was observed with the Pdx1 wt and mutant I192Q protein (Fig. 7C) confirming that the inhibition of Cdx-2/3 mediated transcriptional activation is independent of Pdx1 DNA binding and rather occurs through protein to protein interactions. In contrast, repression of Pax-6 mediated activation was attenuated by the Pdx1 mutation (78% and 54% by wt and I192Q Pdx1, respectively). We therefore tested by GST pulldown whether the I192Q mutation affected interactions between Pdx1 and Pax-6. Protein to protein interactions of both transcription factors were reduced by approximately 65% indicating that the Pdx1 homeodomain is critical for protein interactions with Pax-6 (Fig. 7D). Since the reduced repressor activity of Pdx1 I192Q on Pax-6 corresponds quantitatively with the decreased protein to protein interaction of both factors, we suggest that Pdx1 could exert its repressor activity primarily through protein to protein interaction. Our data cannot, however, rule out a contribution of DNA binding competition to its inhibitory effect on Pax-6.
We show that Pdx1 is able to act as a repressor of the endogenous glucagon gene in InR1G9 glucagonoma cells while simultaneously inducing insulin gene transcription. Similarly, Pdx1-induction of insulin and islet-amyloid-polypeptide gene expression has been shown in stable clones of glucagon-producing AN697 and αTC1 cells, but in these studies, no effect on glucagon gene expression has been observed [46, 47]. Although we have no clear explanation for this discrepancy, our recent results showing de-repression of the glucagon gene through overexpression of a dominant-negative Pdx1 protein in insulinoma cells  and in vivo data showing de-repression of glucagon gene expression by functional inactivation of Pdx1 in beta cells of developing and adult mice [19, 20] support our findings. However, these studies do not discriminate between direct effects of Pdx1 on glucagon gene expression and indirect effects mediated by the transcriptional network of the beta cell. Overexpressing Pdx1 in glucagonoma cells allowed us to assess the role of Pdx1 on the relative expression of the insulin and glucagon genes in the absence of other beta-cell specific factors and demonstrates the critical direct role of Pdx1 in the cell's ability to transcribe these genes.
The inhibitory effect of Pdx1 on glucagon gene expression is mediated in vitro by impairing Pax-6 and Cdx-2/3 driven activation of the G3 and G1 promoter elements. Pdx1 did not interact directly with G3 and although it bound to three AT-rich elements within G1, two of which are binding sites for Pax-6 and Cdx-2/3, its relative affinity was lower as compared to Pax-6. Pdx1 is subject to post-translational modifications such as phosphorylation altering its subcellular localization . We cannot exclude that these modifications could enhance Pdx1 binding to the G1 element in vivo. However, the DNA binding deficient Pdx1 mutant I92Q repressed Cdx-2/3 mediated activation quantitatively similarly to the wild-type protein and although Pax-6 driven activation was reduced with Pdx1 I192Q, protein-to-protein interactions with Pax-6 were equally affected. Moreover, since Pdx1 repressed Pax-6 mediated activation also through G3, a paired box binding site, we suggest that the major molecular mechanism of transcriptional inhibition by Pdx1 could be through protein to protein interaction although we cannot exclude a contribution of DNA binding competition to its inhibitory effect on Pax-6.
Two mechanisms might thus explain the repression of transcriptional activation by Pdx1; (i) through direct interactions with Pax-6 and Cdx-2/3, Pdx1 could change their conformations thereby impairing contacts to coactivator proteins or favour contacts with corepressors. We show by GST precipitation assays protein to protein interactions between Pdx1 and Pax-6 that are mediated in part by the Pax-6 homeodomain and the capacity of Pdx1 to form a heterodimer with both Pax-6 and Cdx-2/3. Similarly, Pdx1 has also been shown to interact with Cdx-2/3 in solution  thus potentially neutralizing Cdx-2/3 function independent of DNA binding. In the same study, Pdx1 was able to inhibit the Cdx-2/3 mediated activation of the sucrose-isomaltase gene in heterologous cells. (ii) Alternatively, free Pdx1 might efficiently compete for common coactivators and impair their interaction with DNA-bound transcription factors thus blocking transcriptional activation. In this regard it is interesting that overexpression of Pdx1 in insulin-producing cells has recently been shown to repress insulin promoter activity . Similarly, an excess of Pdx1 impaired its synergy with E47 on the insulin mini-enhancer in non-islet cells . One common cofactor linking specific transcription factors to the basal transcription machinery, CBP/p300, is able to physically interact with Pax-6, Cdx-2/3, and Pdx1 [38, 51]. We therefore cotransfected BHK-21 cells with increasing amounts of CBP or p300; however, this complementation with transcriptional coactivators failed to compensate for the transcriptional repression by Pdx1. Pax-6 contains an unusually long transactivation domain that has been reported to directly interact with the TATA binding protein within TFIID (TBP) . Pdx1 could thus impair Pax-6 interactions with TBP or alternatively, act by disturbing a multifactorial transcription complex by altering the ratio of its free components.
Pdx1 has been implicated in the tissue-specific activation of different genes through cell type and promoter context dependent protein to protein interactions. In acinar cells, Pdx1 associates with Pbx1 and Meis2 on the B element of the elastase I gene  and endocrine-specific complexes consist of Pdx1/Pbx, Pdx1/Pax-6 or E47/Beta2/Pdx1 on the somatostatin and insulin gene promoter, respectively [13, 14, 15, 16, 17]. These protein contacts allow for cooperative DNA binding and synergistic transcriptional activation. We show a new potential function of Pdx1 as mediator of cell-specific expression of the glucagon gene through its inhibition of Pax-6 and Cdx-2/3 mediated transcription.
We thank V. Schwitzgebel, D. Drucker, H. Edlund, M.S. German, S. Saule, C.V.E. Wright, D. Melloul, N. Copeland, and M.D. Walker for generously providing molecular probes. This work was supported by the Swiss National Fund (J. Philippe, P.A. Halban), the Institute for Human Genetics and Biochemistry (J. Philippe), the Berger Foundation (J. Philippe), Juvenile Diabetes Research Foundation International (P.A. Halban) and the Carlos and Elsie de Reuters Foundation (J. Philippe).