Departments of Laboratory Medicine and Pathobiology and Medicine, Banting and Best Diabetes Centre, The Toronto General Hospital, University of Toronto, Toronto, Canada M5G 1L5
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ABSTRACT |
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A single mammalian proglucagon gene is expressed in the brain, islets, and intestinal enteroendocrine cells, which gives rise to a unique profile of proglucagon-derived peptides (PGDPs) in each tissue. The biological importance of glucagon, glucagon-like peptide (GLP)-1, and GLP-2 has engendered considerable interest in the factors regulating the synthesis and secretion of the PGDPs in vivo. Although rat proglucagon gene transcription has been extensively studied, the factors important for control of human proglucagon gene expression have not been examined. We now report that, despite conservation of proximal promoter G1-G4 enhancer-like elements, human proglucagon reporter plasmids containing these elements are transcriptionally inactive in islet cell lines. Remarkably, larger human proglucagon promoter fragments, such as the 1604 hGLU-Luc, are expressed in GLUTag enteroendocrine cells but not in islet cell lines. A total of 5775 bases of human proglucagon promoter were required for expression in islet cell lines. Analysis of human proglucagon promoter expression in transgenic mice demonstrated that ~1.6 kb of human proglucagon gene sequences directs expression of a human growth hormone reporter gene to the brain and intestinal enteroendocrine cells but not islet cells in vivo. These findings provide the first evidence demonstrating divergence in the mechanisms utilized for tissue-specific regulation of the human and rodent proglucagon genes.
islets; glucagon-like peptide-1; glucagon-like peptide-2; intestine
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INTRODUCTION |
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THE MAMMALIAN PROGLUCAGON gene is expressed in the A cells of the pancreatic islets, the L cells of the small and large intestine, and selected neurons of the brain. In mammals, tissue-specific differences in the posttranslational processing of proglucagon result in the liberation of glucagon in the pancreas and two glucagon-like peptides, GLP-1 and GLP-2, in the intestine (7). The proglucagon-derived peptides (PGDPs) have diverse and essential roles in human physiology. Glucagon is an important regulator of carbohydrate, lipid, and amino acid metabolism and acts as a counterregulatory hormone to insulin in regulating levels of blood glucose levels. GLP-1 is an incretin hormone that potentiates insulin release from the pancreas and also plays a role in gastric emptying and feeding behavior (7). GLP-2 stimulates intestinal hexose transport and is trophic to the mucosal epithelium of the small and large intestine (6). In contrast, the biological activities and physiological relevance of oxyntomodulin and glicentin, PGDPs cosecreted with GLP-1 and GLP-2 from the gut, are less well established.
The finding that pancreatic and intestinal PGDPs have pleiotropic
biological effects has stimulated considerable interest in
understanding the regulation of PGDP biosynthesis. The majority of
these studies have utilized rodent islet cell lines to identify the
determinants regulating proglucagon gene expression in vitro. Transfection studies have identified three enhancer-like elements (designated G2, G3, and G4), and an islet-specific promoter element (G1), in the first 300 bp of the rat glucagon gene promoter (4, 32).
Glucagon gene expression and secretion of the PGDPs are regulated by a
cAMP-dependent pathway in both islets and intestinal cells, consistent
with the functional localization of a cAMP response element in the rat
proglucagon gene promoter (8, 24). Complementary studies using
transgenic mice demonstrated that 1252 nt of rat glucagon promoter
sequences are sufficient for targeting transgene expression to the A
cells of the pancreatic islets, and transfection experiments have
implicated a role for cdx-2/3,
isl-1,
brn4,
pax6, and both hepatocyte nuclear
factor (HNF)-3 and HNF-3
in the control of islet
proglucagon gene transcription (17, 22, 23, 33, 36, 40).
The interest in gut-derived PGDPs such as GLP-1 and GLP-2 has stimulated interest in understanding the regulation of intestinal proglucagon gene expression. Studies using rodent intestinal cell lines demonstrate that peptide hormones such as gastrin-releasing peptide activate proglucagon gene transcription (27). Complementary experiments in rats indicate that enteral nutrition stimulates intestinal proglucagon gene expression, predominantly in the jejunum (16), whereas diets enriched in fiber or fatty acids increase proglucagon mRNA transcripts in the small and large bowel (35, 38). Intriguingly, although 1252 bp of rat proglucagon promoter sequences are sufficient for correct targeting of transgene expression to islet A cells (12), additional 5'-flanking sequences are required for specifying rat intestinal proglucagon gene transcription in vivo (25).
Despite the importance of human PGDPs in normal physiology and metabolic disorders such as diabetes, paradoxically little is known about the regulation of human proglucagon gene transcription. The structural organization of the human and rodent proglucagon cDNAs and genes appears similar (3, 15, 41), with significant nucleotide identity extending to key regulatory sequences such as the G1-G4 elements in the human and rat proglucagon gene 5'-flanking regions. Although numerous studies have analyzed rat proglucagon gene transcription in both transgenic and transfection experiments, no reports have yet described the regulation of the human glucagon gene. In this study, we isolated human proglucagon gene regulatory sequences and analyzed human proglucagon gene transcription using transfected reporter genes and cell lines in vitro and transgenic mice in vivo. Our findings demonstrate that the human and rat proglucagon genes utilize different regulatory regions to achieve identical patterns of tissue-specific gene expression.
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MATERIALS AND METHODS |
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Reagents. Reagents and chemicals were purchased from Bioshop Canada (Burlington, ON), Caledon (Georgetown, ON), Difco (Detroit, MI), Pharmacia (Baie d'Urfe, PQ), Sigma Chemical (St. Louis, MO), and Canadian Life Technologies (Burlington, ON). Restriction endonucleases and DNA modifying enzymes were from New England Biolabs (Mississauga, ON). Taq DNA polymerase, 10× PCR buffer, and deoxynucleotide triphosphates were obtained from Boehringer Mannheim (Laval, PQ). Oligonucleotide primers were synthesized by ACGT (Toronto, ON). T7 sequencing kit was purchased from Pharmacia (Montreal, PQ). Radioactive deoxynucleotide labels were obtained from Amersham (Oakville, ON).
Plasmids. A human P1 DNA clone encoding glucagon gene sequences was isolated, using a PCR-generated probe, by Genome Systems (St. Louis, MO). Human proglucagon promoter sequences were isolated, characterized by restriction mapping and DNA sequencing, and subcloned into the promoterless plasmid SK-Luc (21) immediately adjacent to the coding sequences of the firefly luciferase reporter gene.
Cell culture and transfections. The hamster islet cell line InR1-G9 (11) and baby hamster kidney fibroblasts (BHK cells) were grown in DMEM (4.5 g glucose/l) supplemented with 5% calf serum and 10% penicillin and streptomycin. The mouse enteroendocrine cell line GLUTag (9) was grown in DMEM supplemented with 10% fetal bovine serum. InR1-G9 and BHK cells were transfected with 10 µg of plasmid DNA/5-cm plate by the calcium phosphate method. GLUTag cells were transfected with 10 µg of plasmid DNA/5-cm plate by electroporation, and cells were harvested 20-36 h after transfection. Each set of transfections was carried out on three separate occasions in triplicate for each plasmid. The promoterless plasmid SK-Luc and CMV-Luc were used as negative and positive controls in each transfection. Luciferase activities were analyzed using a Lumat LB 9501 (EG&G Berthold, Wellesley, MA).
A fragment of the human proglucagon gene 5'-flanking region from an Xba I site (Generation of transgenic mice. The purified 3.8-kb human proglucagon promoter/hGH chimeric gene was used to generate transgenic founders (Chrysalis DNX Transgenics, Princeton, NJ), and mice were characterized by Southern blot analysis of tail DNA as previously described (25). Nylon membranes were exposed on the phosphorimage screen overnight and analyzed using a STORM 840 (Molecular Dynamics, Sunnyvale, CA).
RNA isolation and analysis.
Total cellular RNA was isolated from various tissues using TRIzol
reagent (Life Technologies). cDNA was synthesized by reverse transcription using a cDNA synthesis kit (Amersham Pharmacia Biotech, Toronto, ON). Primers for the hGH coding sequence were
5'-GAAGAAGCCTATATCCCAAAG-3' and
5'-GAGTAGTGCGTCATCGTTGTG-3', generating a 378-bp cDNA. The PCR reaction was carried out for 35-40 cycles (94°C for 1 min and then annealing at 55°C for 1 min and extension at 72°C for 1 min), and amplification products were analyzed on 2% agarose gels
stained with ethidium bromide. mRNA was isolated from 400 µg of total
RNA using Oligotex mRNA Mini kit (Qiagen, Chatsworth, CA). RNA
hybridization and washing were carried out as described, using
[-32P]dATP randomly
labeled hGH and mouse glyceraldehyde-3-phosphate dehydrogenase cDNA
probes (8).
Immunohistochemical analysis. Tissues from 6- to 8-wk-old mice were fixed in buffered neutral formalin for 24 h. Tissues were processed for immunohistochemistry and/or intestinal morphometry as previously described (1, 2). Formalin-fixed, paraffin-embedded tissue was sectioned at 4 µm for imunohistochemistry using the streptavidin-biotin-peroxidase complex technique. Primary antisera and antibodies were directed against the following antigens and were used at the specified dilutions: GLP-1 (polyclonal antiserum prepared by D. J. Drucker) at 1:2,500 for 30 min and hGH at 1:1,000 (DAKO, Toronto, ON). The reactions were visualized using 3,3'-diaminobenzidine and hydrogen peroxide. Appropriate positive and negative controls were performed in each case.
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RESULTS |
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Regulation of human proglucagon gene expression in transfected cell
lines.
Because a 300-bp fragment of rat proglucagon promoter sequences is
sufficient for directing reporter gene expression in pancreatic islet
cell lines (10), we initially surmised that similarly sized fragments
of the proximal human proglucagon gene promoter would also activate
luciferase gene expression in islet cells. Three plasmids containing
254, 327, or 602 bp of human proglucagon gene 5'-flanking
sequences ligated upstream of the luciferase-coding sequence were
transfected into islet InR1-G9 cells, mouse GLUTag intestinal cells,
and BHK fibroblasts. Surprisingly, no significant luciferase activity
was detected after transfecting the hGLU-Luc fusion genes into hamster
islet InR1-G9 cells (Fig.
1A).
The 602-bp hGLU-Luc plasmid generated <5% of the luciferase activity
generated using 476 bp of the rat proglucagon promoter. Similarly, the
hGLU-Luc plasmids were significantly less transcriptionally active in
GLUTag enteroendocrine cells compared with 476 rat GLU-Luc (Fig.
1B). Neither the human nor rat
GLU-Luc plasmids induced significant luciferase activity in BHK
fibroblasts, with luciferase activities <1% of those obtained with
CMV-Luc.
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Regulation of human proglucagon gene expression transgenic mice. The results of the cell transfection studies suggested that transcription of the human proglucagon gene is differentially regulated in both islet and intestinal cell lines, since different promoter sequences are required for human vs. rat proglucagon gene transcription in islet and intestinal endocrine cells. To exclude the possibility that these results reflected species-specific differences in the ability of immortalized rodent cell lines to support reporter gene expression and not true biological differences in promoter regulation, we introduced a human proglucagon promoter-reporter transgene, 1.6hGLU-GH (Fig. 3) into mice. This transgene was predicted, on the basis of cell line experiments, to support transcriptional activation in the intestine, but not the pancreas, of transgenic mice. The transgene contains 1604 bases of human proglucagon promoter linked to the hGH gene. Six transgenic founders were obtained, and germ line transmission was observed in two founder lines as assessed by Southern blot analysis (data not shown).
Transgene expression was assessed using a combination of RT-PCR, Northern blot analyses, and immunocytochemistry. hGH mRNA transcripts were detected in the stomach, jejunum, ileum, colon, and brain but not in the pancreas or liver of two lines of 1.6hGLU-GH transgenic mice (Fig. 4A). Northern blot analysis demonstrated an ~1-kb hGH mRNA transcript in colon mRNA, consistent with the predicted size of the transgene transcript if expression initiated correctly from the human proglucagon promoter sequences. To determine if the 1.6-kb human proglucagon promoter contained the necessary elements required for directing transgene expression to enteroendocrine cells, hGH expression was localized by immunocytochemistry. hGH-immunoreactive cells were detected in the stomach, jejunum, ileum, and colon (Fig. 5). Furthermore, staining of adjacent sections for hGH and GLP-1 demonstrated that the same population of enteroendocrine cells contained both hGH and GLP-1 immunopositivity (data not shown). Similar colocalization of GLP-1 and hGH immunopositivity was observed in occassional brainstem neurons, consistent with the known localization of central nervous system (CNS) proglucagon gene expression. In contrast, no transgene expression was detected, as assessed by either RT-PCR or immunocytochemistry, in the pancreas of 1.6hGLU-GH transgenic mice.
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DISCUSSION |
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The rodent and human proglucagon genes share an identical exon/intron
organization, and studies of tissue-specific processing of the PGDPs
demonstrate an identical profile of peptide processing in human vs.
rodent tissues (29, 30). Remarkably, despite the strong conservation of
proximal promoter elements known to be important for islet
cell-specific rat proglucagon gene transcription, human proglucagon
reporter genes containing either 254, 327, or 602 bp of
5'-flanking sequences were transcriptionally inactive in
pancreatic islet InR1-G9 cells. Comparison of the rat and human proximal proglucagon promoter sequences (Fig.
6) show that,
although they are indeed highly related, several differences in
nucleotide sequence are apparent. The G1 promoter element is almost
perfectly conserved, as are the binding sites for
isl-1,
cdx-2/3, and
Brn-4 (17, 21, 40). In contrast to G1,
the upstream G2-G4 enhancer sequences are less well conserved
(Fig. 6). Nucleotide changes are observed between the rat and human
proglucagon promoters at the binding sites of four transcription
factors that interact with upstream promoter sequences, including cAMP
response element binding protein,
pax6, HNF-3 and HNF-3
(9, 23,
33, 36) (Fig. 6). Whether changes in the interaction of one or more of these factors with the human proglucagon promoter is sufficient to
extinguish expression in human islets seems unlikely, given our
findings that addition of more distal 5'-flanking sequences restores expression in islet cell lines.
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The results obtained from analysis of the hybrid HR and RH proximal
promoter plasmids demonstrate that, whereas the rat upstream G2-G4
enhancers can effectively activate expression of the more proximal
human promoter, the comparable human sequences homologous to the
G2-G4 enhancers are not sufficient to activate reporter expression
from the proximal rat promoter in islet and intestinal cells (Fig. 2).
These results strongly suggest that changes in sequence between
66 and
300 bp of human proglucagon promoter (i.e., G2,
G3, and G4) contribute to the lack of expression of the human
proglucagon promoter in islet cell lines. To compensate, it seems
likely that the human proglucagon gene has evolved one or more
additional upstream regions that direct expression in islet cells. The
available evidence from our transfection studies suggests that a
pancreatic islet enhancer is likely located between
3355 and
5775 in the human proglucagon gene promoter, a hypothesis that
should be further explored in future experiments.
The results of previous studies examining rat proglucagon gene
expression in cell lines and transgenic mice demonstrated that, whereas
1252 bp of rat proglucagon 5'-flanking sequences directed transgene expression to the brain and islets of transgenic mice, no
expression was observed in enteroendocrine cells of the small or large
intestine (12). Addition of upstream 5'-flanking sequences extending to 2252 was required for expression of the rat
proglucagon promoter in intestinal enteroendocrine cells (25). In
contrast, our results from both cell lines and transgenic mice clearly
demonstrate that, although ~1600 bp of human proglucagon gene
sequences are sufficient for expression in brain and intestine,
additional regulatory sequences are required for human islet cell expression.
These findings suggest that, although the mechanisms specifying CNS and
enteroendocrine proglucagon gene transcription are likely highly
conserved across species, the DNA sequences and transcription factors
regulating human and rat proglucagon expression in the islet have
clearly diverged. Previous studies of rat proglucagon promoter
expression using transgenic mice and cell transfections identified a
modular glucagon gene intestinal enhancer, designated GUE (glucagon
upstream enhancer), between 2292 and
1253 in the rat
proglucagon gene 5'-flanking region (20). A smaller 45-bp subdomain of the GUE was identified, between
1431 and
1387, that exhibited intestinal enhancer-like activity in
enteroendocrine cells (20). The results of our human proglucagon gene
transgenic and transfection studies demonstrate that intestine-specific
expression is mediated by sequences contained within the first ~1600
bp of the human proglucagon gene 5'-flanking region. Hence, it
seems reasonable to infer, based on the available rat and human data, that sequences comprising a functional GUE likely reside between
1252 and
1600 bp. Within this region, there are conserved
potential binding motifs for GATA, basic helix-loop-helix, and
cdx-2/3 transcription factors.
The reason(s) for utilization of different regulatory sequences in the
human and rat proglucagon genes is not apparent. Studies of the human
and rat insulin promoter in transgenic mice have not revealed major
differences in the sequences required for -cell-specific expression
in vivo (14). In humans, glucagon secretion appears regulated primarily
by nutrients, especially glucose, insulin, and the autonomic nervous
system. To date, the results of experiments in rats and mice suggest
that the regulation of rodent glucagon secretion is comparable to that
described in human studies (7). No studies have examined the regulation
of human proglucagon gene expression in human islet cells; hence,
whether additional differences exist in the regulation of human vs.
rodent proglucagon gene expression remains to be determined.
Although rodents are excellent models for studying gene expression and physiology, there are several instances in which significant differences exist between rodent and human physiology and regulation of gene expression. For example, the human albumin promoter has tissue-specific enhancers immediately adjacent (within 500 bases) to its promoter (13), whereas the rat albumin gene enhancers are located far upstream (8-10 kb to its promoter) (34). Similarly, the murine and human P-selectin gene promoters exhibit several structural nucleotide differences that correlate with differential transcription factor binding and species-specific differences in gene regulation (31). Moreover, species-specific responses to fibrate administration have been mapped to differences in promoter elements of the rat vs. human apolipoprotein A-I gene promoters (39). The results of our experiments provide an additional example of species-specific differences in the control of tissue-specific gene transcription. Previous studies have demonstrated that gene duplication, tissue-specific mRNA splicing, and tissue-specific posttranslational processing contribute to diversity of PGDP expression in different species (5, 18, 19, 28, 29). Our findings extend these results by demonstrating that diversity in tissue-specific proglucagon gene expression in mammals is also achieved via utilization of different promoter elements. Future studies directed at identifying the precise molecular basis for and physiological relevance of these findings are clearly warranted.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. Irwin, Univ. of Toronto, Dept. of Laboratory Medicine and Pathobiology, 100 College St., Toronto, Canada M5G 1L5 (E-mail: david.irwin{at}utoronto.ca).
Received 5 May 1999; accepted in final form 22 July 1999.
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