The Zinc Finger-Containing Transcription Factor Gata-4 Is Expressed in the Developing Endocrine Pancreas and Activates Glucagon Gene Expression (original) (raw)

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1Diabetes Unit, University Hospital Geneva, 1211 Genève 14, Switzerland

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1Diabetes Unit, University Hospital Geneva, 1211 Genève 14, Switzerland

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1Diabetes Unit, University Hospital Geneva, 1211 Genève 14, Switzerland

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1Diabetes Unit, University Hospital Geneva, 1211 Genève 14, Switzerland

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1Diabetes Unit, University Hospital Geneva, 1211 Genève 14, Switzerland

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1Diabetes Unit, University Hospital Geneva, 1211 Genève 14, Switzerland

*Address all correspondence and requests for reprints to: Jacques Philippe, M.D., Diabetes Unit, University Hospital Geneva, 24, rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland.

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Received:

05 February 2004

Accepted:

03 November 2004

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Beate Ritz-Laser, Aline Mamin, Thierry Brun, Isabelle Avril, Valérie M. Schwitzgebel, Jacques Philippe, The Zinc Finger-Containing Transcription Factor Gata-4 Is Expressed in the Developing Endocrine Pancreas and Activates Glucagon Gene Expression, Molecular Endocrinology, Volume 19, Issue 3, 1 March 2005, Pages 759–770, https://doi.org/10.1210/me.2004-0051
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Abstract

Gene inactivation studies have shown that members of the Gata family of transcription factors are critical for endoderm development throughout evolution. We show here that Gata-4 and/or Gata-6 are not only expressed in the adult exocrine pancreas but also in glucagonoma and insulinoma cell lines, whereas Gata-5 is restricted to the exocrine pancreas. During pancreas development, Gata-4 is expressed already at embryonic d 10.5 and colocalizes with early glucagon+ cells at embryonic d 12.5. Gata-4 was able to transactivate the glucagon gene both in heterologous BHK-21 (nonislet Syrian baby hamster kidney) and in glucagon-producing InR1G9 cells. Using gel-mobility shift assays, we identified a complex formed with nuclear extracts from InR1G9 cells on the G5 control element (−140 to −169) of the glucagon gene promoter as Gata-4. Mutation of the GATA binding site on G5 abrogated the transcriptional activation mediated by Gata-4 and reduced basal glucagon gene promoter activity in glucagon-producing cells by 55%. Furthermore, Gata-4 acted more than additively with Forkhead box A (hepatic nuclear factor-3) to trans-activate the glucagon gene promoter. We conclude that, besides its role in endoderm differentiation, Gata-4 might be implicated in the regulation of glucagon gene expression in the fetal pancreas and that Gata activity itself may be modulated by interactions with different cofactors.

TRANSCRIPTION FACTORS OF the Gata family contain a zinc finger DNA binding domain and interact with the consensus binding site A/T (GATA) A/G. They have been found throughout eukaryotes and shown to play critical roles in cell fate specification and differentiation (1). In flies, worms, Xenopus, and mice, Gata factors are critical for endoderm development (2–10). In vertebrates, six family members have been identified so far that can be subdivided into two groups on the basis of sequence homology and expression pattern: Gata-1, Gata-2, and Gata-3 are expressed in the hematopoetic system in a superposed fashion, whereas Gata-4, Gata-5, and Gata-6 are critical for differentiation and cell-specific gene expression in different endoderm and mesoderm derived tissues (8).

Mammalian Gata-4 is first expressed in visceral and parietal endoderm, precardiatic mesoderm and later in the heart, gonads, liver, small intestine, and pancreas (11–13). Mice deficient in Gata-4 display absence of ventral foregut endoderm, visceral endoderm, and Cardia bifida and die at embyronic d (e) 7.5–9.5 because of defects of heart morphogenesis (in part secondary to endoderm defects) and ventral closure of the foregut (14–16). The essential role of Gata-4 for endoderm development is further demonstrated by the fact that Gata-4 null embryonic stem cells are partially defective in generation of proper visceral endoderm and definitive endoderm of the foregut (16, 17). In addition, Gata-4 is required for the development and function of murine gonads (18).

Mammalian Gata-5 is expressed in the developing heart, lung, vasculature, genitourinary system and the adult intestine, stomach, bladder, and lung. Gata-5 −/− mice are viable and present defects in female genitourinary tract development (19–21).

The expression pattern of Gata-6 is similar to Gata-4, with Gata-6 being detected during embryonic development in the primitive streak, allantois, visceral endoderm, heart, lung buds, urogenital ridge, vascular smooth muscle, epithelial layer of the stomach, small and large intestine whereas expression in the adult is found in the heart, aorta, stomach, small intestine, bladder, liver, lung, and pancreas (12, 22). Homozygous mutant mice for Gata-6 die at e5.5–7.7 due to defects in visceral extraembryonic endoderm function and subsequent lack of survival and proliferation in embryonic ectoderm (23, 24). Interestingly, Gata-4 and Gata-6 have a strong interplay inasmuch as Gata-6 induces Gata-4, absence of Gata-4 leads to up-regulation of Gata-6, and both factors interact functionally and physically in transcriptional activation (8, 15, 23, 25). These data indicate that Gata-4 and Gata-6 function in concert to direct tissue-specific gene expression in the developing heart and gastrointestinal tract, whereas Gata-4 and Gata-6 have unique functions in differentiation of the liver and of the arterial system, bladder, and lung, respectively (22, 26). As shown by the targeted disruption studies, both Gata-4 and Gata-6 are master control genes of visceral endoderm formation, and this function is underlined by their ability to induce the proper differentiation of embryonic stem cells toward extraembryonic endoderm (27).

Expression of Gata-4 and Gata-6 in the adult exocrine pancreas and transcriptional activation of the gene for the pancreatic exocrine enzyme elastase by Gata-4 has previously been described (12, 28, 29). Moreover, single-cell transcript analysis has recently detected coexpression of Gata-4 and Gata-6 with 70–100% of early glucagon- and insulin-producing cells at e10.5 of mouse embryonic development (30). In this report, we therefore analyzed the expression of Gata-4, Gata-5, and Gata-6 in the endocrine pancreas and their potential function in hormone gene expression. We found Gata-4 and Gata-6 transcripts in the pancreas as early as e10.5 and thereafter throughout pancreas development until adulthood. Gata-4 was coexpressed with early glucagon-, but not insulin-producing cells at e12.5. Furthermore, we identified Gata-4 as a transcriptional activator of the glucagon gene promoter by gel-mobility shift assays and transient transfections in heterologous and glucagon-producing cells. Taken together, our data indicate a potential role for Gata-4 as an early activator of glucagon gene expression.

RESULTS

Gata-4 and Gata-6 Are Present in the Pancreas and in Pancreatic Endocrine Cell Lines

To study the expression of transcription factors of the Gata family in the pancreas, RT-PCR was performed using primers specific for Gata-4, Gata-5, and Gata-6. Whereas expression of Gata-5 was restricted to the intestine and low levels in the whole pancreas, Gata-4 and Gata-6 transcripts were detected in the intestine, whole pancreas, islets, and mouse glucagon- and insulin producing endocrine cell lines (Fig. 1); we were unable to detect transcripts from the hamster cell lines, InR1G9 and HIT-15, suggesting that hamster Gata-4 and Gata-6 transcripts differ notably in sequence from that of mouse and rat. We next studied expression of Gata-4 and Gata-6 during pancreas development using pancreas at embryonic day (e) 10.5, 11.5, 12.5, 14.5, and 17.5 as well as from adult BL6xCBAJ mice. Transcripts of both factors were detected as early as e10.5 and persisted during pancreas development until adulthood (Fig. 2A).

Fig. 1.

Gata-4 and Gata-6 Are Expressed in the Islet and in Endocrine Cell Lines RT-PCRs (35 cycles) from mouse whole pancreas, rat islets and intestine and pancreatic endocrine cell lines using primers specific for Gata-4, Gata-5, Gata-6, or β-actin.

Fig. 2.

Gata-4 and Gata-6 Are Expressed in the Developing Pancreas A, RT-PCRs (37 cycles) from dorsal and ventral mouse developing pancreas, kidney, liver, testis, and intestine. Numbers on top indicate embryonic days. B, Immunofluorescence of cryosections of developing and adult mouse pancreas using antiglucagon, and anti-Gata-4 antibodies. Gata-4 colocalizes with about 40% of Glu+ cells at e12.5 (a–f), whereas at e14.5, Gata-4 expression is detected only rarely in Glu+ cells (g–i) and not any more at e17.5 (k–m). In adult pancreas (n–p), Gata-4 expression is restricted to the exocrine tissue. Images were obtained using an Axiocam microscope with magnification ×40 (a–c and n–p) or ×100 (d–f) or a confocal microscope with magnification ×100, zoom ×2.5 (d–f). Arrows indicate cells positive for both Gata-4 and glucagons.

To confirm Gata-4 and Gata-6 expression in the endocrine and exocrine pancreas at the protein level, we performed immunohistochemical analysis of adult and embryonic pancreas. We first analyzed the specificity of anti-Gata antisera using nonislet Syrian baby hamster kidney (BHK-21) cells overexpressing Gata-4 and Gata-6. All antibodies analyzed (anti-Gata-4 sc-1237, anti-Gata-6 sc-7244, and sc9055) were specific to their respective antigen (data not shown). Unfortunately, both anti-Gata-6 antisera did not elicit any signal on pancreas sections. During the embryonic development of the pancreas, Gata-4 was present in about 40% of glucagon-producing (Glu+) cells at e12.5 (Fig. 2B, a–i). Later in development at e14.5, only rare Glu+ cells coexpressed Gata-4 and none at e17.5 (Fig. 2), which was in contrast detected in most amylase+ cells (data not shown). We never observed cells coexpressing insulin and Gata-4 (data not shown). In the adult pancreas, Gata-4 was exclusively detected in the exocrine tissue (Fig. 2B), suggesting that detection of gata-4 transcripts in islets by RT-PCR might reflect either untranslated mRNA, insufficient levels of the respective proteins to be detected by immunofluorescence, or contamination by exocrine tissue.

Gata-4 Binds to the G5 Element of the Glucagon Gene Promoter

Because Gata-4 was coexpressed with glucagon-producing cells during embryonic development and because the glucagon gene promoter contains a putative Gata binding site on the G5 element (TGATAC), we analyzed by EMSA whether Gata-4 and Gata-6 interacted with this element. As shown in Fig. 3A, both Gata-4 and Gata-6 overexpressed in human embryonic kidney (HEK) 293T cells are able to bind to G5Gata, and the respective complexes that exhibited identical electrophoretic mobility, were recognized by anti-Gata-4 and anti-Gata-6 antibodies. In extracts from glucagon- or insulin-producing, exocrine and enteroendocrine cell lines, a complex comigrating with Gata-4 and Gata-6 was displaced by anti-Gata-4 but not by anti-Gata-6 antibodies indicating that only Gata-4 interacts with G5 in extracts from the analyzed cell lines. Of note, both Gata-4 and Gata-6 formed two complexes with different electrophoretic mobilities; the faster migrating complex was present in variable intensities and may correspond to a degradation product. Because both Gata-4 and Gata-6 transcripts were present in endocrine cell lines but only Gata-4 interacted with G5, we assessed the relative affinity of Gata-4 and Gata-6 for G5 in EMSA competition experiments. A similar molar excess of cold G5Gata oligonucleotides competed for the Gata-4 and Gata-6 complexes formed with G5Gata indicating that both transcription factors bind to this element with roughly the same affinity and that Gata-6 is probably not translated in the endocrine cell lines (Fig. 3B).

Fig. 3.

Gata-4 Binds to the G5 Element of the glucagon Gene Promoter A, EMSA using an oligonucleotide comprising a putative Gata binding site on the G5 element of the glucagon gene promoter and nuclear extracts from different pancreatic and enteroendocrine cell lines or HEK 293T cells transfected with Gata-4 or Gata-6. Although both Gata-4 and Gata-6 are able to bind to G5, only Gata-4 can be identified in extracts from glucagon- or insulin-producing, exocrine and enteroendocrine cell lines using specific antisera. A and B represent specific but as yet uncharacterized complexes. B, EMSA competition experiment analyzing the relative affinity of Gata-4 and Gata-6 for the G5 element. Protein-DNA complexes formed with nuclear extracts from HEK 293T cells overexpressing Gata-4 or Gata-6 and labeled G5Gata were competed for by the indicated molar excess of cold oligonucleotides.

The first 350 bp of the rat glucagon gene promoter that are sufficient for cell-specific and maximal expression (31), comprise three additional potential binding sites of Gata transcription factors, one within G1 (AGATAT) and two, less conserved sites, within G3 (AGATTG) and G4 (TGATTT) (Fig. 4A). We therefore assessed the relative affinity of Gata-4 for these elements as compared with a known Gata-4 binding site on the steroidogenic acute regulatory protein (StAR) (32) in EMSA. As shown in Fig. 4B, a similar molar excess of cold G5Gata, G1–50, and StAR oligonucleotides competed for the Gata-4 complex formed with G5Gata indicating that Gata-4 binds to these elements with roughly the same affinity. The less conserved potential Gata binding sites on G3 and G4 displayed at least 5-fold less affinity for Gata-4 and control oligonucleotides comprising a mutated G5 element (G5GataMut) or a sequence without Gata binding site did not compete at all for Gata-4. To confirm these results, we performed EMSA using nuclear extracts from HEK 293T cells overexpressing Gata-4 and Gata-6. Both proteins were able to bind to G1–50; however, Gata-4 only formed a very weak complex with nuclear extracts from glucagon-producing InR1G9 cells and Gata-6 was not detected (data not shown). Furthermore, neither Gata-4 nor Gata-6 formed complexes with G3 or G4, and binding factors from InR1G9 cell extracts were Pbx1/Prep1 and Pax-6 as described previously (Refs. 33 and 34 and data not shown). We therefore conclude that despite the ability of Gata-4 and Gata-6 to bind to the glucagon promoter elements in vitro, only Gata-4 is implicated in glucagon gene expression in glucagon-producing cell lines and that the major Gata-4 binding site is G5.

Fig. 4.

Potential Gata Binding Sites on the Rat Glucagon Gene Promoter A, Schematic diagram illustrating potential binding sites of Gata proteins on the glucagons gene promoter and known protein complexes formed on the glucagon gene control elements (G1 to G4). Oligonucleotides used for EMSA are indicated below. B, EMSA competition experiments analyzing the relative affinity of Gata-4 for its potential binding sites. Protein-DNA complexes formed with nuclear extracts from HEK 293T cells overexpressing Gata-4 and labeled G5Gata were competed for by the indicated molar excess of cold oligonucleotides.

Gata-4 Transactivates the Glucagon Gene Promoter

To test the effect of Gata-4 on the transcriptional activation of the glucagon gene promoter, we cotransfected the nonislet cell line BHK-21 with a chloramphenicol acetyltransferase (CAT) reporter plasmid driven by the full-length promoter (−292Glu) and increasing amounts of Gata-4 expression plasmids. Gata-4 dose-dependently increased basal CAT activity by up to 16-fold (Fig. 5). Similar results were obtained using the Gata-6 expression vector consistent with the ability of both proteins to interact with G5 and G1. In contrast, neither Gata-4 nor Gata-6 had any effect on the insulin I gene promoter in transient transfection studies of BHK-21 cells (data not shown).

Fig. 5.

Dose-Dependent Activation of the Glucagon Gene Promoter by Gata-4 and Gata-6 BHK-21 cells were cotransfected with 10 μg of −292GluCAT and increasing amounts of Gata-4 or Gata-6 expression vectors.

To further analyze the _cis_-acting elements conferring transcriptional activation of the glucagon gene promoter by Gata-4, we performed transient transfections of BHK-21 cells using CAT reporter constructs driven by successive 5′ and internal deletions of the rat glucagon gene promoter (Fig. 6A) and increasing amounts of Gata-4 expression plasmids. Gata-4 transactivated 5.4-fold −175GluCAT comprising Gata elements on G5 and G1; deletion or mutation of the G5 element (−138GluCAT and −175G5MGluCAT, respectively) reduced maximal activation to 2.7- and 2.6-fold, and further deletion of the two most proximal Gata elements on G4 and G1 (−31GluCAT) abolished transcriptional activation by Gata-4 (Fig. 6B). Because transactivation of −292GluCAT, containing the low-affinity binding site G3, by Gata-4 was much higher compared with −175GluCAT, 15.5-fold vs. 5.4-fold, we used a reporter construct containing G3 directly upstream of the first 138 bp of the glucagon gene promoter. Indeed, Gata-4 activated G3–138GluCAT by 6.1-fold vs. 2.7- and 2.9-fold for −138GluCAT and G2–138GluCAT, which only contain the G1–50 Gata binding site, respectively. Because the sequence upstream of the characterized glucagon gene promoter comprises additional potential Gata binding sites at −353 (GGATGG), −572 (TGATAG), −762 (AGATAC), −880 (TGATGA), −931 (TGATTT), −1000 (TGATCT), relative to the transcriptional start site, we tested two longer promoter fragments in transient trans-fection assays. However, neither −2500GluCAT nor −1100GluCAT conferred stronger activation by Gata-4 than −292GluCAT (Fig. 6). To test whether G5 was able to confer Gata-4 responsiveness, we placed three copies of this element upstream of the glucagon gene TATA box [3x(G5Gata)-31GluCAT]; this construct was activated 9.8-fold by Gata-4. Our data thus indicate that Gata-4 transactivates the glucagon promoter through G5 although other proximal elements are implicated at least in overexpression assays in heterologous BHK-21 cells.

Fig. 6.

Gata-4 Transactivates the Glucagon Gene Promoter Trough G5 A, Schematic representation of glucagon reporter gene constructs used in this study. CRE, cAMP response element. B, BHK-21 cells were cotransfected with 10 μg of the indicated reporter plasmids and increasing amounts of Gata-4 expression vector. Gata-4 induces activity of the glucagon gene promoter through G5 although other elements also mediate transcriptional activation in BHK-21 cells overexpressing Gata-4.

Mutation of the Gata Binding Site on G5 Abrogates Transcriptional Activation by Gata-4 and Reduces Basal Glucagon Gene Promoter Activity in Glucagon-Producing Cells

To analyze the effect of Gata-4 on the transcriptional activation of the glucagon gene promoter in glucagon-producing cells, we performed transient transfections in InR1G9 cells. As previously reported, basal activity of the enhancerless construct −175GluCAT was much lower than that of the full-length promoter construct −292GluCAT (31). Gata-4 transactivated −292GluCAT and −175GluCAT 1.7- and 4-fold, respectively (Fig. 7). Mutation of the Gata binding site on G5 abolished this transactivation in both reporter constructs indicating that in glucagon-producing cells, G5 is the major element interacting with Gata-4. Importantly, basal activity of G5M-292GluCAT was reduced by 55% as compared with the wild-type promoter construct −292GluCAT, suggesting that the Gata binding element on G5 is required for glucagon promoter activity in glucagon-producing cells.

Fig. 7.

Effect of a Gata Site Mutation on G5 in Glucagon-Producing Cells InR1G9 cells were cotransfected with 3 μg of either wild-type or G5Gata mutated −292GluCAT and −175GluCAT and Gata-4. Mutation of the Gata binding site on G5 abrogates transcriptional activation by Gata-4 and reduces basal glucagon gene promoter activity in glucagon-producing cells. * and **, Statistical significance vs. basal activation with P < 0.05 and P < 0.01, respectively; #, statistical significance of CAT activity of the mutated vs. wild-type promoter.

Gata-4 Activates the Glucagon Gene Promoter with Forkhead Box A (Foxa) [HNF3 (Hepatic Nuclear Factor-3)]

The glucagon gene promoter is well characterized and some critical _trans_-acting factors have been identified (see Fig. 4A). We therefore tested, whether Gata-4 interacts with any of these transcription factors. As shown in Fig. 8A, Gata-4 did not modify activation by the major glucagon gene transcription factor, Pax-6, which can interact as a monomer with G1 and G3 and as a heterodimer with Cdx-2/3 on G1. E47 and Beta2 that bind as a heterodimer to G4 had additive effects when combined with Gata-4. Heterodimers of Pbx proteins and Prep1 have been characterized on G3 and suggested to interact with G5 (34); however, no functional interaction was detected with Gata-4. Gata-4 acted more than additively with Foxa1 and Foxa2 (previously called HNF3α and HNF3β), which both can bind to the G2 and G1 elements of the glucagon gene promoter (Fig. 8, A and B). To further analyze the potential functional interaction of Foxa and Gata-4, we used promoter constructs comprising either G5 and both Foxa binding sites on G2 and G1 (−213GluCAT) or only G5 and G1 (−175GluCAT) for transfection assays in BHK-21 cells. Gata-4 transactivated −213GluCAT 6.3-fold and Foxa1 and Foxa2 3.9- and 4.5-fold, respectively. Together with Gata-4, activation by Foxa1 and Foxa2 reached 22.1- and 23.5-fold, respectively (Fig. 8B). This effect was lost when G2 was deleted (−175GluCAT) with a 12-fold activation, which was clearly less than additive, with both Gata-4 and Foxa1 indicating that potential interactions between Foxa and Gata-4 on the glucagon gene promoter depends on G2 and G5.

Fig. 8.

Interaction of Gata-4 with Known Glucagon Gene Transcription Factors A, BHK-21 cells were cotransfected with 10 μg of −292GluCAT and different glucagon gene transcription factors (0.25 μg each) alone or in combination with Gata-4 (0.25 μg). Gata-4 acts synergistically with Foxa1 and Foxa2, but has only additive effects when combined with other factors and no influence on activation by Pax-6 and Cdx-2/3. B, Transient cotransfection of Gata-4 and Foxa1 or Foxa2 (0.25 μg each) using reporter constructs comprising Gata-4 and Foxa binding sites on G5 and G1, respectively, but including (−213GluCAT) or excluding (−175GluCAT) a second Foxa binding site on G2. # and *, Statistical significance of transcriptional activation of a particular transcription factor cotransfected with Gata-4 vs. activation by Gata-4 and by the factor without Gata-4, respectively.

Expression of a Dominant-Negative Gata-4 Mutant [Double Fingers Wild Type (DF WT)] in Glucagon-Producing Cells Decreases Glucagon Gene Expression

To analyze the importance of endogenous Gata-4 in glucagon-producing cells, we expressed a dominant-negative Gata-4 mutant (Gata-4 DF WT) containing only the two zinc fingers and previously shown to bind GATA sites but to inhibit wild-type Gata-4-induced transactivation in InR1G9 cells (35). This Gata-4 mutant was previously shown to act as a dominant-negative competitor of wild-type Gata-4 both by direct GATA binding to DNA and protein-protein interactions involving Gata factors. We first assessed the ability of the mutant Gata-4 protein to inhibit the Gata-4-induced transactivation of −292GluCat by transient transfections in BHK cells. Cotransfection of increasing amounts of mutant Gata-4 cDNA, at 0.25, 0.5, and 1.0 μg along with 1.0 μg of wild-type Gata-4 cDNA led to a dose-dependent decrease in Gata-4-induced transactivation of −292GluCat reaching 50% at equal amounts of both cDNAs (data not shown).

Pools of clones of InR1G9 cells containing either the empty vector or expressing the mutant Gata-4 were first selected through the antibiotic geneticin. We then analyzed pools of clones for the presence of the mutant Gata-4 protein by gel retardation assays. We selected two pools of clones, A1 and A2, expressing the mutant Gata-4 protein at equivalent levels; these levels were similar to that of the endogenous wild-type Gata-4 (Fig. 9A). Glucagon mRNA levels were first quantified in both A1 and A2 pools and in control pools transfected with the vector. As seen on Fig. 9B, glucagon mRNA levels were decreased by 51 and 58%, respectively, in pools A1 and A2 compared with control cells.

Fig. 9.

Dominant-Negative Competitor Gata-4 DF WT Decreases Glucagons Gene Transcription InR1G9 cells were transfected with either 3 μg of pCDNA3 containing a gene coding for geneticin resistance (mock) with or without the Gata-4 DF WT cDNA (37 ). Pools of clones were then selected by geneticin treatment and expression of the Gata-4 mutant was assessed by gel retardation assays. A, EMSA using the labeled G5 oligonucleotide with nuclear extracts from two pools of InR1G9 cells expressing the mutant Gata-4 DF WT (A1 and A2), mock-transfected InR1G9 cells and HEK 293T cells transfected with the wild-type Gata-4 cDNA (WT in HEK) as well as with _in vitro_-translated Gata-4 mutant protein (DF WT) as positive controls. Arrows indicate Gata-4 WT or Gata-4 mutant (DF) proteins. A and B represent specific but as yet uncharacterized complexes. B, Northern blot from two pools of InR1G9 cells (A1 and A2) expressing the mutant Gata-4 DF WT compared with mock-transfected cells. C, Pools of mock-transfected and mutant Gata-4 DF WT-expressing InR1G9 cells (A1 and A2) were transiently transfected with 3 μg of −175GluCat or 3x(G5Gata)-31GluCat. * and **, Statistical significance of P < 0.05 and <0.005, respectively.

We then transfected the promoter constructs −175GluCat and 3x(G5Gata)-31GluCat in the different pools of both control and Gata-4 mutant-expressing cells (Fig. 9C). The transcriptional activity was decreased by 75 and 71% for −175GluCat and by 57 and 70% for 3x(G5Gata)-31GluCat in pools A1 and A2, respectively, compared with the activity obtained in control cells. These data suggest that, in InR1G9 cells, endogenous Gata-4 is important in glucagon gene expression.

DISCUSSION

In the present study, we have analyzed the expression pattern of Gata-4, -5, and -6 in the embryonic and adult endocrine pancreas and their potential implication in hormone gene expression. We found low Gata-5 expression restricted to the adult exocrine pancreas, whereas Gata-4 and Gata-6 transcripts were present in the endocrine pancreas and cell lines. In a recent single-cell transcript analysis at e10.5 of mouse embryonic development; however, Gata-5 transcripts had been described in 10/11 Glu+/Ins+ cells but only rarely in single hormone expressing cells (30), indicating that Gata-5 might possibly have a role in early pancreas development. Gata-4 and Gata-6 are expressed early during pancreas development, starting at least at e10.5. Using immunofluorescence, we found Gata-4 in about 40% of Glu+ cells at e12.5, whereas they are restricted to the exocrine pancreas in adult mice. Previously, coexpression of Gata-4 with 10/10 of Glu+/Ins− cells has been detected at e10.5 of mouse embryonic development (30). Taken together, these data indicate that most or all early glucagon-producing cells express Gata-4 (e10.5), and as development proceeds, the number of glucagon-producing cells expressing Gata-4 markedly decreases and none are detected in the adult pancreas. Already at e10.5, 65% of Gata-4+ cells do not coexpress an islet hormone (30) suggesting an additional role of Gata-4 in pancreatic precursors or nonislet cell lineages. Indeed, at e14.5, we observe coexpression of Gata-4 with amylase and in the adult pancreas (data not shown) and later Gata-4 is restricted to the exocrine tissue. Strikingly, we did not detect colocalization of insulin and Gata-4, although at e10.5, 8/11 Glu+/Ins+ cells had previously been found positive for Gata-4 transcripts (30). This divergence might be explained by developmental changes between e10.5 and e12.5 with the disappearance of cells positive for both hormones. By contrast, Gata-4 is found in insulin-producing cells in culture (at the mRNA protein levels), a finding that might reflect the dedifferentiated state of these cells. Because the early cells coexpressing glucagon and insulin are not precursor cells for mature β-cells (36), we suggest that Gata-4 is not implicated in the differentiation of the β-cell lineage.

We found only Gata-4 to be present in nuclear extracts from endocrine cell lines although both Gata-4 and Gata-6 are transcribed in these cells (Figs. 1 and 3). The discrepancy between Gata-6 RNA and protein expression might be due to the marked difference in detection levels of PCR and immunofluorescence/EMSA or alternatively to physiological regulation by translational control or protein-turnover rate. Indeed, nonequivalence of Gata-6 RNA and protein expression has been described in certain regions of the heart, which display highest levels of Gata-6 transcripts but contain no or very little Gata-6 protein (37). We detected Gata-4 proteins not only in pancreatic endocrine cells, but also in the enteroendocrine cell line GluTag; similarly, Gata-4 has been described in the intestinal K-cell line STC1 (38), both cell lines transcribing the preproglucagon gene. These data suggest that Gata-4 may have a role not only in pancreatic but also in enteroendocrine proglucagon-producing cells.

Gata-4 transactivates the glucagon gene promoter through its interaction with the G5 element in glucagon-producing and in heterologous cell lines. Consistent with most functional Gata binding elements described to date (39), the Gata interaction site on the glucagon gene promoter is located proximal to the transcriptional start site. The relative affinity of Gata-4 for G5 is similar to that for a known and well-studied Gata-binding site on the StAR promoter (32) and importantly, mutation of the Gata element on G5 drastically reduced basal transcriptional activity of the glucagon gene promoter indicating that this Gata binding element is required for glucagon promoter activity in glucagon-producing cells. Furthermore, the decrease in glucagon mRNA levels and in the activity of glucagons promoter DNA constructs containing G5 in cells expressing the dominant-negative competitor mutant Gata-4 indicate that the endogenous Gata-4 is important in the expression of the glucagons gene in InR1G9 cells. Interestingly, we found that Fog1 (Friend-of-Gata), a Gata-specific cofactor, is expressed in the endocrine pancreas and represses transcriptional activation by Gata-4 in cotransfections of BHK-21 cells (data not shown). Fog1 may thus regulate transcriptional activation of the glucagon gene promoter by Gata-4.

Gata-4 has previously been shown to functionally interact with a variety of transcription factors such as Nkx2–5 on cardiac, steroidogenic factor-1 and CCAAT enhancer binding protein β on gonadal, HNF1α and Cdx-2 on intestinal, and Foxa2 on hepatic gene promoters (32, 33, 39–44). When analyzing for potential interactions with known transcription factors of the glucagon gene, we found synergistic transcriptional activation of the glucagon gene promoter by Gata-4 and Foxa binding to the adjacent G5 and G2 elements, respectively. Similar to Gata proteins, members of the Fox family are critical for endoderm development in different phyla and importantly, Gata-4:Foxa2 interactions have been demonstrated to confer developmental competence of the gut endoderm. During liver formation, Gata-4 and Foxa2 interact to induce the early liver-enriched transcription factor Hex and both proteins have been shown to be the first transcription factors to bind to the albumin enhancer in precursor cells of the liver and to open compacted chromatin thereby facilitating access of other enhancer binding factors (44–46). Interestingly, the liver and ventral pancreas arise from a common region of the embryonic foregut; signals from the cardiac mesenchyme induce hepatic differentiation, thereby actively excluding the pancreatic fate, whereas the region extending away from the developing heart develops into the pancreas (47, 48). Analysis of the implication of Foxa2 and Gata-4 in pancreatic specification and lineage differentiation is hampered by the early embryonic lethality of both targeted mutations. However, studies using embryoid bodies lacking Foxa2 have suggested that Foxa2 may lie directly upstream of Pdx1, a critical pancreas transcription factor, in the cascade directing pancreas development (49). In addition, Gata-4 and Gata-6 have recently been shown to directly activate bone morphogenic protein-4, a member of the TGF-β superfamily, which in turn, has been implicated in pancreatic epithelial cell development and the formation of islet-like structures in vitro (29, 50). It will thus be interesting to directly analyze the respective role of Gata-4 and Gata-6 in pancreatic exocrine and endocrine differentiation using lineage-specific gene activations in mice. Given the role of Foxa in glucagon gene expression (51, 52), and our identification of Gata-4 as transcriptional activator of the glucagon gene promoter, we suggest that besides its role in endoderm differentiation, Gata-4 might be implicated in the regulation of glucagon gene expression in the fetal pancreas. Despite silencing of Gata-4 expression later in the α cell lineage, this may play a role in promoting transcriptional potency of the glucagon gene by facilitating access of other transcription factors.

MATERIALS AND METHODS

Cell Culture and DNA Transfection

The glucagon-producing hamster InRIG9 (53), insulin-producing hamster HIT-T15 and the BHK-21 and HEK 293T cell lines were grown in RPMI 1640 (Seromed, Basel, Switzerland) supplemented with 5% heat-inactivated fetal calf serum, 5% heat-inactivated newborn calf serum, 2 mm glutamine, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. The enteroendocrine GluTag cells were cultured as described (54). HEK 293T and BHK-21 cells were transfected by the calcium phosphate precipitation technique (55) using 10–15 μg of total plasmid DNA per 10-cm petri dish. One microgram of pSV2A placental alkaline phosphatase, a plasmid containing the human placental alkaline phosphatase gene, driven by the simian virus 40 early promoter was added to monitor transfection efficiency (56). Transfection of InR1G9 cells was done using the diethylaminoethyl-dextran method as described previously (57). Expression vectors contained cDNAs for the rat Gata-4 (M. Nema, Institut de Recherches Cliniques de Montréal, University of Montréal, Montréal, Canada), mouse Gata-5 and Gata-6 (E. Morrisey, University of Pennsylvania, Philadelphia, PA), human dominant-negative Gata-4 DF WT mutant (coding for a truncated Gata-4 protein containing only its two zinc fingers, J. J. Tremblay, Laval University, Quebec, Canada), rat Foxa1 and −2 (previously HNF-3α and −β, R. Costa, University of Illinois, Chicago, IL), human Pbx1a (M. Cleary, Stanford University School of Medicine, Stanford, CA), human Pbx1b and Prep-1 (F. Blasi, Department of Biological and Technological Research Istituto Scientifico Hospital San Raffaele, Milano, Italy), human E47 (Z.-S. Ye, Rockefeller University, New York, NY), hamster Beta2 (M. J. Tsai, Baylor College of Medicine, Houston, TX), hamster Cdx-2/3 and Pdx1 (M. S. German, University of California, San Francisco, CA), rat Isl-1 (D. Drucker, University of Toronto, Toronto, Canada), and quail Pax-6 (S. Saule, Institut Curie, Orsay, France). In experiments using variable quantities of expression vectors, coding for different transcription factors, total amount of DNA was kept constant by adding appropriate amounts of empty expression vector. Reporter plasmids consisted of the CAT reporter gene driven by different fragments of the rat glucagon gene promoter (−292GluCAT, −213GluCAT, −175GluCAT, −138GluCAT, G3–138GluCAT, G2–138GluCAT, −75GluCAT, −31GluCAT (31), three concatameritzed copies of the G5 element upstream of the glucagon gene TATA box 3x(G5Gata)-31GluCAT or the rat insulin I gene promoter (−410InsCAT) (58). Reporter plasmids mutated in the Gata binding site on G5 (−292 G5M GluCAT and −175 G5M GluCAT) were constructed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands) according to the manufacturer’s protocol and the following primers: forward 5′-gtctcaccccggttggagcgtgaggagc and reverse 5′-gctcctcacgctccaaccggggtgagac.

For the generation of InR1G9 cells expressing the Gata-4 mutant DF WT, cells were transfected as mentioned above with 3 μg of either pCDNA3 (mock) or Gata-4 DF WT plasmids. Forty-eight hours after transfection, 600 μg/ml of G418-sulfate (CLONTECH, Palo Alto, CA) were added for selection. Resistant cells were tripsinized, counted and plated in 24-wells to obtain one to five cells/well. Each pool of cells was then amplified, tested for Gata-4 mutant expression and used for transfection of glucagons gene promoter constructs.

Data are presented as fold stimulation of the CAT activity obtained with the reporter plasmid alone and are the mean ± sem of at least three experiments.

CAT and Protein Assays

Cell extracts were prepared 48 h after transfection and analyzed for CAT and alkaline phosphatase or luciferase activities as described previously (59). Quantification of acetylated and nonacetylated forms was done with a PhosphorImager (Molecular Dynamics). A minimum of three independent transfections was performed; each of them carried out in duplicate.

RT-PCR Analysis

Total RNA was isolated from mouse embryonic and adult pancreas and kidney, different rat tissues and αTC1 (mouse glucagon-producing; Ref. 60), Min6 (mouse insulin-producing; Ref. 61), AR42J (rat pancreatic acinar cell derived, ATCC, Manassas, VA), HIT-T15 (hamster insulin-producing), and InR1G9 (hamster glucagon-producing) cell lines using Trizol Reagent (Invitrogen Life Technologies, Gaithersburg, MD) according to the manufacturer’s specifications. First-strand cDNA synthesis was performed with random hexamer primers and SuperScript II Reverse Transcriptase (Invitrogen Life Technologies) as recommended by the supplier. One tenth of the resulting cDNA was used for semiquantitative PCR using HotStar (QIAGEN, Valencia, CA) or Goldstar (Eurogenetech, Brussels, Belgium) DNA polymerase as recommended by the provider. The following primers were used: Gata4 sense 5′-ccccaatctcgatatgtttg, antisense 5′-aggacctgctggtgtcttag; Gata5 sense 5′-agttccgacgtagccccttc, antisense 5′-ttgctgtggatcctgaggag; Gata6 sense 5′-cttctaattcagatgactgc, antisense 5′-gcacggaggatgtgacttc; Fog1 sense 5′-atccacatgcgcagccacag, antisense 5′-agtagatctcacccttggag; Fog2 sense 5′-gaaaatctgagctgcgaag, antisense 5′-tgc tgt aca caa tgc agt tg; β-actin sense 5′-cccagatcatgtttgagacc, antisense 5′-agg atcttcatgaggtagtc.

Immunofluorescence

Cryosections of adult or embryonic mouse pancreas fixed with 4% paraformaldehyde were treated with 10 mm NH4Cl and immunolabeled using monoclonal mouse antiinsulin (Sigma, St. Louis, MO; I-2018) and antiglucagon (Sigma G-2654) antisera followed by goat antimouse cyanin 3 antibodies (Molecular Probes, Eugene, OR; A-11031). To immunolabel Gata4 using goat polyclonal anti-Gata4 serum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; sc-1237), signals were amplified with donkey antigoat biotin (Jackson ImmunoResearch Laboratories, West Grove, PA; no. 705-065-147) and streptavidin-Alexa488 (Molecular Probes S-11223) antibodies after standard procedures. Gata-6 was stained with anti-Gata-6 antibodies (Santa Cruz sc-7244 and 9055) and signals amplified as for Gata-4.

Northern blot analysis were performed as previously described (31) and hybridized with a 32P-labeled rat glucagons cDNA probe.

EMSAs

Nuclear extracts from pancreatic exocrine, endocrine, enteroendocrine, or HEK 293T cells transfected with Gata-4 or Gata-6 expression plasmids or the empty vector pSG5 were prepared according to Schreiber et al. (62). EMSAs were performed as described previously (63) using 8 μg of nuclear extracts and oligonucleotides containing the rat glucagon gene. _In vitro_-translated proteins were made with a T3, T7 couple reticulocyte lysate system (Promega, Madison, WI). Antibodies specific for Gata-4 (sc-1237) and Gata-6 (sc-7244) used in EMSA were purchased at Santa Cruz.

Data analysis

Data are presented as mean ± sem, and statistical significance was tested by analysis of variance and Student’s t test where applicable. The threshold for statistical significance was P < 0.05. * and **, Statistical significance with P < 0.05 and P < 0.01, respectively.

Acknowledgments

We thank P. Meda, C. Mas, D. Caille, and M. Heikinheimo for valuable advice with immunohistochemistry, S. Charbon for excellent experimental assistance, and the bioimaging platform National Centres of Competence in Research in Geneva for confocal images. We are indebted to M. Nema, E. Morrisey, M. S. German, S. Saule, C. V. E. Wright, and M. D. Walker for generously providing molecular probes.

This work was supported by the Swiss National Found, and the Institute for Human Genetics and Biochemistry.

Abbreviations:

• BHK-21,
  Nonislet Syrian baby hamster kidney;
• CAT,
  chloramphenicol acetyltransferase;
• DF WT,
  double fingers wild type;
• e,
• Fog,
• Foxa,
• HEK,
• HNF3,
  hepatocyte nuclear factor 3;
• StAR,
  steroidogenic acute regulatory protein.

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