AJP - Endo Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 290: E1287-E1295, 2006. First published January 10, 2006; doi:10.1152/ajpendo.00535.2005
0193-1849/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/E1287    most recent
00535.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boylan, M. O.
Right arrow Articles by Wolfe, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boylan, M. O.
Right arrow Articles by Wolfe, M. M.

Sp1/Sp3 binding is associated with cell-specific expression of the glucose-dependent insulinotropic polypeptide receptor gene

Michael O. Boylan, Lisa I. Jepeal, and M. Michael Wolfe

Section of Gastroenterology, Boston University School of Medicine and Boston Medical Center, Boston, Massachusetts

Submitted 3 November 2005 ; accepted in final form 3 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor (GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between –1 and –100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5'-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between –85 and –40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions –77, –60, and –50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at –50 and –60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at –70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between –100 and –40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter.

gastric inhibitory polypeptide; transcription factors; RIN38 cells; insulinoma; rat-2 cells; chromatin immunoprecipitation assay

GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE (GIP) is a member of the secretin-VIP family of gastrointestinal regulatory peptides and was originally named gastric inhibitory polypeptide (6). Targets for the hormone include fundic cells of the stomach, where GIP has been shown to inhibit acid secretion (49), and beta-cells of the pancreatic islets, where GIP potentiates insulin release (15, 39). Because of its insulin-releasing properties, GIP has been designated a physiological incretin, the proposed substance that mediates the enteroinsular axis and appears to play an important role in the maintenance of glucose homeostasis. Studies with GIP-specific antagonists demonstrated that GIP contributes up to 72% of the postprandial insulin release (46).

GIP exerts its biological properties through a specific G protein-coupled receptor possessing seven transmembrane domains (48). The binding of GIP to its receptor (GIPR) is thought to trigger the activation of a heterotrimeric Gs protein that, in turn, stimulates adenylate cyclase. The accumulation of intracellular cAMP leads to an increase in Ca2+ influx through voltage-gated Ca2+ channels. These events, along with the activation of a wortmannin-sensitive pathway, are thought to be responsible for the stimulation of insulin release by GIP (44).

In addition to the pancreatic islets and the stomach, RNA encoding the GIPR has been detected in the adrenal cortex, heart, adipose tissue, and several regions in the brain, including the cerebral cortex, hippocampus, and the olfactory bulb (48). However, the precise physiological and pathological significance of GIP in these other tissues remains to be elucidated. Initial studies have demonstrated that GIP inhibits glucagon-stimulated lipolysis in adipose tissue (14), and the intraventricular injection of GIP results in a decrease in plasma follicle-stimulating hormone and an increase in plasma growth hormone levels (38).

Previous studies have suggested that aberrant expression of the GIPR may play a role in the etiology of food-induced Cushing's syndrome (FICS), type 2 diabetes mellitus, and obesity. In the case of FICS, ectopic expression of GIPR on adrenal cells has been associated with increased postprandial cortisol secretion (25, 41). In type 2 diabetes mellitus, the loss of GIPR expression on islet beta-cells has been hypothesized to contribute to an altered enteroinsular axis (24, 33, 37); consistent with this hypothesis, Lynn et al. (32) reported suppressed levels of GIPR expression in islet beta-cells isolated from a rat model of type 2 diabetes mellitus. Recent studies have also indicated that GIP and GIPR may contribute to the development of obesity. Miyawaki et al. (35) generated a GIPR-deficient mouse that, unlike its wild-type littermates, did not develop diet-induced obesity and its associated complications, including abnormal glucose tolerance, diabetes mellitus, and fatty liver, when fed a high-fat diet (35).

To characterize the regulation of GIPR gene expression, specifically at the level of gene transcription, we cloned the rat GIPR gene and identified the promoter region. Preliminary analysis indicated that GIPR transcription is initiated from a promoter containing neither a CAAT motif nor a TATA box. Transient transfection analyses demonstrated that the first 100 bp of the 5'-flanking region of the GIPR gene are sufficient to direct transcription in a rat insulinoma cell line 2 (RIN38), a cell line that expresses the endogenous gene. In addition, analysis of GIPR promoter activity in a cell line that does not express GIPR (rat-2) suggested that the cell-specific expression of the GIPR gene is controlled by distal negative regulatory sequences (5). In this report, we have used transient transfection analyses of mutant derivatives of the GIPR promoter combined with electromobility shift assays to demonstrate that binding of the transcription factors Sp1 and Sp3 is critical for high levels of GIPR transcription in RIN38 cells. In addition, chromatin immunoprecipitation (ChIP) analysis indicates that binding of Sp1 and Sp3 to the GIPR promoter in intact cells correlates with cell-specific expression of the GIPR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue culture. RIN38 cells (rat insulinoma) were a gift from Dr. A. Lernmark (Seattle, WA), whereas rat-2 fibroblast cells and Drosophila Schneider S2 cells were purchased from American Type Culture Collection (Manassas, VA). RIN38 and rat-2 cells were grown in DMEM containing 10% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C in an atmosphere of 5% CO2. Schneider S2 cells were maintained in Schneider medium containing 10% heat-inactivated serum (Sigma) and grown at 20°C.

Rat and human GIPR sequences. The sequence of the rat GIPR promoter has been previously described, and its GenBank accession number is AF050667. The corresponding Human GIPR sequence was identified with the nucleotide-nucleotide BLAST (blastn) tool provided by the National Center for Biological Information. The first 20 bp of the human cDNA sequence (GenBank no. NM 000164) were used as the query.

GIP luciferase plasmids and Sp1/Sp3 expression vectors. The wild-type plasmid pGL-660 has been described previously (5). Constructs pGL-40, PGL-52, and pGl-85 were prepared using the PCR with the GIPR-specific forward primers 5'-gaagatctcctcccaacaccccagg-3', 5'-gaagatctggccccgcctcctccca-3', and 5'-tttagatctccagagctcccccct-3', respectively, as previously described (5). The mutant constructs pGL-660 (mutA), pGL-660 (mutB), and pGL-660 (mutC) were designed with mismatched primers in a two-step PCR-based method for introducing point mutations (20), as previously described (21). The plasmid pGL-660 was used as the template in the initial PCRs. The following GIPR specific primers were used: for pGL-660(mutA), 5'-cgccctggccccgAAtcctcccaa-3' and 5'-ttgggaggaTTcggggccagggcg-3'; for pGl-660(mutB), 5'-ctgttctctgttccgAAAtggccc-3' and 5'-gggccaTTTcggaacagagagaacag-3'; and for pGL-660(mutC), 5'-tccccagagctccccAAtcctgttc-3' and 5'-gaacaggaTTggggagctctgggga-3'.

The plasmids pPAC, pPACSp1, pPACSp3, and pPACUSp3 were a kind donation from Dr. Guntram Suske (Institute of Molecular Biology and Tumor Research, Philipps-University, Marburg, Germany).

Transient transfection assays. One day before transfection, RIN38 cells were plated at a density of ~3–6 x 105 cells per 60-mm dish in the appropriate growth media. A mixture containing 2.5 µg pGL2 reporter plasmid, 14 µl lipofectamine (Invitrogen, Carlsbad, CA), 0.5 µg pCMVbeta-galactosidase DNA (a control for transfection efficiency), and 600 µl of serum-free medium was incubated at room temperature. After 15 min, 2.4 ml of media were added, and the resultant DNA mixture was then added to cells previously washed twice with serum-free medium. After 5 h, 3 ml of media containing twice the normal concentration of serum were added, and the incubation was continued for 48 h, after which the cells were harvested. For enzymatic analysis, the cells were first washed twice with PBS and then were lysed in 500 µl of lysis buffer following the manufacturer's instructions (Analytical Luminescence, San Diego, CA). Schneider S2 cells were transfected by calcium phosphate precipitation with 10 µg of luciferase reporter DNA, 1 µg transfection control plasmid (CMV-driven beta-galactosidase), and 100 ng of either pPAC, pPACSp1, pPACSp3, or pPACUSp3 per 100-mm dish. Each experimental condition was tested in triplicate. Cells were harvested 48 h after transfection by first washing twice with PBS and then adding 1.5 ml of lysis buffer.

Luciferase and beta-galactosidase measurements. To assay luciferase activity, 100 µl of the cell lysate were mixed with 100 µl of luciferase substrate solution A (Analytical Luminescence). With a luminometer with automatic injection, 100 µl of substrate solution B (Analytical Luminescence) were then added, and luciferase activity was measured as light emission over a 30-s period. beta-Galactosidase activity in 40 µl of the cell lysate was determined after a 5- to 30-min incubation at 37°C with 2 mM chlorophenol red beta-galactopyranoside (Boehringer Mannheim, Indianapolis, IN) in 2 mM MgCl2, 0.1 mM MnCl2, 45 mM 2-mercaptoethanol, and 100 mM NaHPO4, pH 8. The reactions were stopped by adding 500 µl of 0.5 M EDTA, pH 8.0, and absorbance at 570 nm was measured with a spectrophotometer. Within each experiment, luciferase activity was determined in duplicate and normalized to beta-galactosidase activity for each dish. Each plasmid was tested at least six times between two separate experiments.

EMSAs. Nuclear extracts were prepared according to the method of Dignam et al. (12). The protein concentration was subsequently estimated with a commercial Bio-Rad protein assay (Bio-Rad, Hercules, CA). EMSAs were performed using 4% polyacrylamide gels (44:1 acrylamide/bisacrylamide) in 40 mM Tris·HCl and 195 mM glycine (pH 8.5) at 4°C. The following probe sequences were used: site Sp1-A, (start position –45): 5'-CGCCCTGGCCCCGCCTCCTCCCAA-3'; site Sp1-B (start position –57): 5'-ctgttctctgttccgccctggcc-3'; and site Sp1-C (start position –75): 5'-TCCCCAGAGCTCCCCCCTCCTGTTC-3' (presumptive binding sites are underlined). Each reaction contained 10,000 cpm (~7 fmol) of double-stranded oligonucleotide (ds-oligo) probe, end-labeled by T4 kinase in the presence of [{gamma}-32P]ATP and purified on a Quik Spin G25 column (Boehringer Mannheim). The reactions were performed in 20 µl of a mixture containing 20 mM HEPES (pH 7.3), 50 mM KCl, 5 mM beta-mercaptoethanol, 20% glycerol, 1 µg poly(dI-dC), 1 µg BSA, and 2.5 µg RIN38 nuclear extract. The reaction mixtures were incubated for 30 min at room temperature, followed by 5 min on ice before loading. The samples were separated by electrophoresis for 1.5–2.5 h at 250 V. For competition studies, the samples were preincubated with unlabeled ds-oligos with wild-type (same as above) or mutant sequences: mut-A: 5'-CGCCCTGGCCCCGAATCCTCCCAA-3'; mutB: 5'-ctgttctctgttccgAAAtggccc-3'; and mut-c: 5'-TCCCCAGAGCTCCCCAATCCTGTTC-3', prior to the addition of the probe (substituted bases are in bold type). A ds-oligo with the sequence 5'-ATTCGATCGGGGCGGGGCGAGC-3' was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used as a positive control. For supershift experiments, specific antibodies were preincubated with the nuclear extracts for 10 min at room temperature before the addition of probe. The antisera to Sp1 (H-225), Sp2 (K-20), Sp3 (H-225), and Sp4 (V-20) were purchased from Santa Cruz Biotechnology.

Western analysis. Cells grown on 10-cm plates to 80% confluence were washed once with 1x PBS and then suspended by scraping in 1 ml of RIPA buffer (Boston Bioproducts, Ashland, MA) containing Complete, a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) followed by incubation on ice for 15 min. Cell lysates were centrifuged at 14,000 rpm at 4°C for 5 min, and the supernatant was aspirated. Protein concentrations were measured using the BCA protein assay kit (Pierce, Rockford, IL), and protein (50 µg) was resolved on a 12% SDS-polyacrylamide gel and transferred to Protran nitrocellulose membranes (PerkinElmer Life Sciences, Boston, MA). The membranes were blocked in 5% nonfat milk, 1x PBS, and 0.5% Tween 20 at room temperature for 2 h and then incubated with rabbit anti-pituitary tumor-transforming gene 1 (PTTG1; H-160) that was purchased from Santa Cruz Biotechnology and diluted 1:2,000 in 1x PBS containing 5% nonfat milk. After a 4-h incubation, the membranes were washed three times for 5 min at room temperature with wash buffer (1x PBS, 0.5% Tween 20) and incubated with the appropriate horseradish peroxidase-conjugated secondary antiserum diluted in 5% nonfat milk in 1x PBS for an additional 1 h. The membranes were then rinsed three times for 5 min in wash buffer, soaked in chemiluminescence reagent, as instructed by the manufacturer (Boston Bioproducts), and exposed to X-ray film for 0.1–5 min.

CHiP analysis. CHiP assays were performed by following the protocol of Cissell and Gerrish and colleagues (8, 17). RIN38 and rat-2 cells grown on 10-cm plates to 90% confluence were exposed to 1% formaldehyde in DMEM for 5 min at 23°C. Glycine was added to 0.125 M to quench the formaldehyde, and the cultures were incubated for 2 min. The cells were collected in ice-cold PBS, pelleted by centrifugation, and incubated for 10 min on ice in 0.6 ml of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris·HCl, pH 8.1, 1 mM PMSF). Lysed samples were transferred to prechilled microcentrifuge tubes and subjected to sonication consisting of twelve 10-s pulses. The reactions were centrifuged for 10 min at 4°C and stored at –70°C. For each condition, a 100-µl aliquot was diluted with 0.9 ml of buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris·HCl, pH 8.1, 167 mM NaCl, containing 0.1% protease inhibitor mixture for mammalian cells) (Sigma, St. Louis, MO) and precleared with 60 µl of BSA-blocked protein A/G-agarose (Santa Cruz Biotechnology) for 1 h at 4°C. After removal of the agarose beads by centrifugation, either 2 µl of specific antiserum or 10 µl of normal rabbit IgG (Santa Cruz Biotechnology) and the mixture was incubated overnight at 4°C. Specific rabbit polyclonal antibodies directed against either Sp1 (H-225), Sp3 (H-225), or GATA-4 (H-112) were used in this study (Santa Cruz Biotechnology). Antibody-protein-DNA complexes were isolated by incubation with 60 µl of blocked protein A/G agarose for 1 h at 4°C. After an extensive washing period, bound DNA fragments were eluted with 300 µl of elution buffer (50 mM NaHCO3, 1% SDS) and analyzed by PCR using PCR Master mix (Roche, Mannheim, Germany), 15 pmol of each primer, and 10 µl of the immunoprecipitated DNA/reaction. Cycling parameters were as follows: one cycle at 95°C for 2 min and 28 cycles at 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s. The primers used for amplification of the GIPR promoter fragment bound by Sp1/Sp3 were 5'-ggactggctctctcagcatg-3' and 5'-ctgccagagagctctggaag-3'. The primers used for amplification of the PTTG1 promoter fragment bound by Sp1/Sp3 were 5'-ggctgacccaaaccatctca-3' and 5'-gagtgaagcacaagtcccga-3'. Amplified products were electrophoresed through a 1.4% agarose gel in Tris acetate-EDTA buffer and visualized after ethidium bromide staining.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The first 100 bp of the GIPR promoter is highly conserved between rat and human and contains potential transcription factor binding sites. As previously demonstrated, the first 100 bp upstream of the transcription start site of the rat GIPR gene are sufficient to direct high levels of transcriptional activity in a number of established cell lines (5). The corresponding Human GIPR sequence was identified by using the first 20 bp of the human cDNA sequence (GenBank no. NM 000164) as a query for a nucleotide-nucleotide BLAST (blastn). The human GIPR 5'-flanking region was contained within the Homo sapiens chromosome 19, cosmid R28204 [GenBank] , with the complete sequence having GenBank accession no. AC006132. A comparison of the 5'-flanking region of the rat and human GIPR gene demonstrated 88% identity within the first 92 bp of this region (Fig. 1). Analysis of the next 2,000 upstream base pairs did not show any additional regions of shared homology. Neither the rat nor the human GIPR promoter contains a TATA box, and, like many TATA-less promoters, the sequence is GC-rich (~68% and ~73%, respectively, for human and rat in the first 92 bp). As an initial step in the identification of important regulatory elements in the GIPR promoter, we analyzed the rat and human sequences using the Web-based programs rVISTA 2.0 and TRANSFAC 6.0. rVISTA 2.0 combines database searches with a comparative sequence analysis to identify potential transcription factor binding motifs that are conserved between species (30). TRANSFAC compares a sequence with a database containing eukaryotic transcription factors, their genomic binding sites, and DNA-binding profiles (52). Using these programs, we identified three binding motifs that are present in both the human and rat sequences. With rVISTA 2.0, putative binding sites for Sp1 family of transcription factors were found at positions –45 and –57, whereas a consensus site for transcription factors of the zinc finger family, such as myeloid zinc finger gene (MZF1), was identified at position –75. The TRANSFAC program also identified the Sp1 binding motifs at positions –45 and –57, as well as an additional Sp1 site in the rat gene starting around –77. The TRANSFAC program did not recognize an Sp1 site at or near position –77 in the human gene.


Figure 1
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 1. Comparison of the 5'-flanking region of the human and rat glucose-dependent insulinotropic polypeptide receptor (GIPR) genes identifies 2 highly conserved elements. Sequence –1 to –92 of the human GIPR gene (hGIPR) shares 88% identity with the analogous region of the rat GIPR gene (rGIPR). The location of a myeloid zinc finger gene (MZF1) and/or Sp1 motif(s) and an Sp1 motif identified using the rVISTA 2.0 and TRANSFAC programs and the transcription start sites are indicated. The human sequence was obtained from the National Center for Biological Information by using the first 20 bp of the human cDNA sequence (GenBank no. NM 000164) as a query for a nucleotide-nucleotide BLAST (blastn). The human GIPR 5'-flanking region was contained within the Homo sapiens chromosome 19, cosmid R28204 [GenBank] , with the complete sequence having GenBank accession no. AC006132. The rat sequence was generated by Sanger dideoxy sequence analysis of a rat genomic clone (GenBank accession no. AF050667).

 
Deletion of a region containing putative transcription factor binding sites abolishes GIPR promoter activity. To further characterize the functional properties of the first 100 bp of the rat GIPR 5'-flanking sequence and in particular the regions containing potential transcription factor binding motifs identified above, we made deletion mutants of our GIPR promoter region in the luciferase reporter vector pGL2-basic. These mutants were analyzed in transient transfection assays using RIN38 cells. As shown in Fig. 2, deletion of the sequence between –660 and –85 did not affect transcriptional activity. However, removal of the sequence between –85 and –50 decreased promoter activity by ~65% (Fig. 2). Further deletion of sequences down to position –40 led to an additional ~23% loss of promoter activity, for a total loss of ~88% (Fig. 2). This loss of promoter activity following the deletion of the sequence between –85 and –40 suggests of the presence of at least 2 positive regulatory elements in the region.


Figure 2
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2. Deletion analysis of the GIPR promoter in RIN38 cells. Top: schematic representation of the upstream region of the GIPR gene, depicting the approximate location of the Sp1 motifs at positions –45 (A), –57 (B), and –75 (C). Bottom: schematic representation of the GIPR-luciferase (LUC) chimeras (left) is shown adjacent to the relative transcriptional activity of the chimeric genes (right). All constructs are derivatives of pGL2-basic, and all include the same 3' end (+30) fused to the firefly luciferase gene. The number to the left of each construct indicates the 5' extent of the GIPR-specific insert. Chimeric constructs were cotransfected with pCMV-beta-galactosidase, and the data represent mean activity ± SE of 6 separate transfections normalized to the activity of the promoterless construct, pGL2-basic, after correcting for differences in transfection efficiencies by the measurement of beta-galactosidase activity. Data are expressed relative to the activity measured for the parental vector pGL-660, which was set to 100%.

 
EMSAs demonstrate specific binding of Sp1 and Sp3 to sequences in the GIPR promoter. Because the removal of DNA sequences containing potential binding motifs led to a reduction in promoter activity, we next characterized the binding properties of these sequences. EMSAs were performed using nuclear extracts from RIN38 cells and ds-oligos representing each of the three potential regulatory regions of the GIPR gene. A commercially available ds-oligo that included a well-characterized Sp1 binding site was used as a control. Figure 3, left, demonstrates competitive binding analysis using a radiolabeled synthetic ds-oligo (probe) that represents the region surrounding the putative Sp1 binding motif at position –45 (Sp1-A in Fig. 1). Three DNA-protein complexes (a, b, and c in Fig. 3), consistent with Sp1/Sp3 binding, were identified (4). The binding of the nuclear proteins to the probe was specific because an excess of unlabeled wild-type (wt), but not mutant ds-oligo, inhibited the formation of bands produced by the probe in a concentration-dependent manner. The control ds-oligo was also able to displace binding in a concentration-dependent manner, further implicating the presence of an Sp1/Sp3 binding site. Figure 3, middle, demonstrates a competitive binding analysis using a probe representing the region around the Sp1 binding motif at position –57 (Sp1-B in Fig. 1). Again, three DNA-protein complexes (a, b, and c in Fig. 3), consistent with Sp1/Sp3 binding, were identified (4). The binding of nuclear proteins to the probe was specific because an excess of unlabeled wild-type, but not mutant ds-oligo, inhibited the formation of bands produced by the probe in a concentration-dependent manner. The control ds-oligo was also able to displace binding in a concentration-dependent manner, further implicating the presence of an Sp1/Sp3 binding site. Figure 3, right, demonstrates a competitive binding analysis using a probe representing the region around the MZF1/Sp1 binding motif at position –77 (Sp1-C in Fig. 1). As with the ds-oligo representing the putative Sp1 sites at positions –45 and –57, three specific complexes were identified that could be competed with an excess of either unlabeled wild-type ds-oligo or the control ds-oligo, but not with an excess of a mutant ds-oligo. These observations are consistent with the presence of an Sp1/Sp3 binding site in the corresponding region of the GIPR promoter. The binding affinity of nuclear proteins to the probe representing a region now referred to as the Sp1-C site appears to be lower than that for the probe representing the Sp1-A or Sp-1 B sites. Whereas as little as a 10-fold excess of control ds-oligo displaced binding to site Sp1-C, even a 100-fold excess of control could not completely displace binding to sites Sp1-A or Sp1-B.


Figure 3
View larger version (66K):
[in this window]
[in a new window]
  		Fig. 3. EMSAs demonstrate specific binding of RIN38-derived nuclear protein to 3 elements in the 5'-flanking sequences of the GIPR gene. Wild-type (wt) double-stranded (ds) oligonucleotides, radiolabeled using T4 polynucleotide kinase, were examined in EMSAs with nuclear extract derived from RIN38 cells. The binding reaction mixtures were preincubated with increasing amounts of either unlabeled wt ds-oligonucleotide, the corresponding mutant (mut) ds-oligonucleotide, or an Sp1-containing commercial (com) ds-oligonucleotide. The complexes were resolved on a 4% acrylamide-0.5x TBE. The sequences of the various ds-oligonucleotides are given in MATERIALS AND METHODS. Left: analysis of the sequences between –56 and –33 of the GIPR gene (Sp1-A). Middle: analysis of the sequences between –62 and –47 of the GIPR gene (Sp1-B). Right: analysis of the sequences between –88 and –64 of the GIPR gene (Sp1-C). Arrowheads (left) indicate position of the DNA-protein complexes (designated a, b, c).

 
EMSAs were performed to confirm the binding of Sp1 and Sp3 nuclear protein to probes representing the Sp1-A, Sp1-B, and Sp1-C sites (Fig. 4). Incubation of probes representing either the Sp1-A, Sp1-B, or the Sp1-C site with nuclear proteins and rabbit antisera specific for Sp1 resulted in a loss of the slowest migrating complex in the EMSA (band a) and the appearance of an even slower complex (lane 1). The loss of the band is indicative of polyclonal antibodies reacting with epitopes on Sp1 and inhibiting interaction with the probe. The appearance of the slower migrating or "supershifted" band is consistent with the binding of antibodies to an Sp1 complex. When the probes representing either the Sp1-A, Sp1-B, or the Sp1-C site were incubated with nuclear proteins and rabbit antisera specific for Sp3 (lane 3), both faster-moving complexes in the EMSAs were lost (bands b and c), and two additional slower migrating complexes appeared. The presence of two bands representing Sp3 immunoreactivity is the result of the interaction of two differently sized isoforms of Sp3 with the radiolabeled ds-oligo. Although these isoforms are encoded by the same mRNA, they are derived from protein translation initiation at different start codons, resulting in a long form and a short form that are both recognized by the Sp3 antisera. Rabbit antisera directed against Sp2 (lane 2) and Sp4 (lane 4) were used as negative controls and failed to alter the appearance or migration of the DNA-protein complexes.


Figure 4
View larger version (66K):
[in this window]
[in a new window]
  		Fig. 4. EMSAs identifying Sp1 and Sp3 immunoreactivity in DNA-protein complexes. RIN38 nuclear extract was incubated with either Sp1, Sp2, Sp3, or Sp4 antisera before the addition of radiolabeled wild-type probe. The complexes were resolved on a 4% acrylamide-0.5x TBE gel. Left: region of the GIPR gene between –56 and –33 (Sp1-A). Middle: region of the GIPR gene between –62 and –47 (Sp1-B). Right: region of the GIPR gene between –88 and –64 (Sp1-C). Arrowheads (left) indicate the position of the DNA-protein-antibody complexes. The specific antisera added to each sample are indicated above the corresponding lanes.

 
Mutation of Sp1/Sp3 binding sites leads to a loss of promoter activity. To evaluate the contribution of the three Sp1/Sp3 binding sites in the regulation of the GIPR promoter, we introduced point mutations into each of the Sp1 binding sites described above in the context of our GIPR-luciferase reporter construct, pGL-660, and then evaluated the effects of the mutations in transient transfection assays using RIN38 cells (Fig. 5). The point mutations introduced were the same changes that abolished Sp1 and Sp3 binding in the above EMSAs. Analysis of the construct containing a mutation in the Sp1 motif located at –45 (Sp1-A) showed an ~64% reduction in promoter activity [compare pGL-660 (mutA) to pGL-660]. Similarly, analysis of the construct containing a mutation in the Sp1 motif located at –57 (Sp1-B) showed an ~74% reduction in promoter activity [compare pGL-660 (mutB) with pGL-660]. In contrast, a mutation of the Sp1 motif located at position –75 (Sp1-C) had little effect on promoter function [see pGL-660 (mutC)]. Therefore, although all three sites are capable of binding protein in vitro, only the Sp1 sites located at position –45 and –57 act as a transcription enhancer element in transient transfection experiments.


Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 5. Mutation analysis of the GIPR promoter constructs containing specific mutations in the Sp1 binding motifs. Top: schematic representation of the upstream region of the GIPR gene depicting the approximate location of the Sp1 motifs at positions –45 (A), –57 (B), and –75 (C). Bottom: schematic representation of the GIPR-LUC chimeras (left) is shown adjacent to the relative activity of the chimeric genes (right). The constructs pGL-660 (mutA), pGL-660 (mutB), and pGL-660 (mutC) are derivatives of pGL-660, with base pair substitutions that are predicted to render inactive the putative Sp1 sites at positions –45 (A), –57 (B), and –75 (C), respectively. Chimeric constructs were cotransfected with pCMV-beta-galactosidase, and data represent mean activity ± SE of 6 separate transfections that are normalized to the activity of pGL2-basic (with beta-galactosidase activity used to correct for differences in transfection efficiencies). Data are expressed relative to the activity measured for the parental vector pGL-660, which was set to 100%.

 
Sp1 and Sp3 activate the GIPR promoter in an Sp1/Sp3 null cell line. Drosophila Schneider S2 cells lack endogenous Sp1 and Sp3 expression and consequently are useful for demonstrating the specific activation of a promoter on exogenous expression of these factors (11). Using these cells, we cotransfected expression vectors containing cDNA for either Sp1 (pPACSp1), the long-form of Sp3 (pPACUSp3), the short-form Sp3 (pPACSp3), or empty vector (pPAC) with our GIPR reporter constructs. We expressed the data as relative light units after correcting for differences in transfection efficiencies by dividing by the associated beta-galactosidase activity. As shown in Fig. 6, expression of either Sp1 or the long form of Sp3 enhanced the activity of all the GIPR reporter constructs, whereas expression of the short form of Sp3 had no effect. The inclusion of pPACSp1 or pPACUSP3 with the reporter constructs pGL-660, which contains both Sp1-A and Sp1-B sites, resulted in an ~58- and 29.3-fold increase in promoter activity, respectively, over the combination of PGL-660 and the empty vector, pPAC (Fig. 6). The inclusion of pPACSp1 or pPACUSP3 with pGL-40, which lacks both the Sp1-A and Sp1-B sites, resulted in only 6.3- and 2-fold increases in promoter activity, respectively (Fig. 6). These results are consistent with both Sp1 and the long form of Sp3 being able to activate transcription on binding to the Sp1 sites in the GIPR promoter.


Figure 6
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 6. Effect of exogenous expression of Sp1 and Sp3 on the GIPR promoter. Drosophila Schneider S2 cells were transfected with 10 µg of reporter DNA and 1 µg of a lacZ containing control vector in the presence of either 100 ng Sp1, Sp3, (short form) or USp3 (long form) expressing vectors or empty vector. Top: schematic representation of the upstream region of the GIPR gene showing the approximate location of the Sp1 binding motifs at positions –45 (A), –57 (B), and –75 (C). Bottom: schematic representation of the GIPR-LUC chimeras (left) is shown adjacent to the relative activity of each chimeric gene resulting from cotransfection with the various pPAC expression vectors (right). Data represent mean activity ± SE of 6 separate transfections that are normalized to the activity of the pGL2-basic cotransfected with the empty vector (pPAC), after using beta-galactosidase activity to correct for differences in transfection efficiencies. Data are expressed relative to the activity measured for the parental vector pGL-660 in the presence of pPACSp1, which was set to 100%.

 
Sp1 and Sp3 associate with the GIPR promoter in living cells. To demonstrate Sp1/Sp3 binding to the endogenous GIPR gene, ChIP analyses were performed with formaldehyde cross-linked chromatin isolated from RIN38 and rat-2 cells. Rat-2 embryonic fibroblast cells do not express the GIPR and thus were included as a negative control for Sp1/Sp3 binding to the GIPR promoter. After precipitation of chromatin in the presence of anti-Sp1, anti-Sp3, or control sera, the GIPR 5'-flanking sequence spanning nucleotides –349 to +16 was selectively amplified by PCR. As shown in Fig. 7, RIN38 samples treated with an Sp1- or Sp3-specific antiserum yielded a PCR product, whereas the corresponding rat-2 samples were negative. Rabbit IgG and anti-GATA-4 were used as negative controls for the ChIP assay, and, as expected, the PCRs for these samples did not yield a product. As a positive control for the ChIP analyses, sequences from the rat pituitary tumor-transforming gene 1 (PTTG1) were also amplified (9, 40). As expected, PTTG1-specific sequences were identified in samples prepared from both RIN38 and rat-2 cells. Expression of PTTG1 in RIN38 and rat-2 cells was confirmed by Western analysis (data not shown). These results demonstrate that Sp1/Sp3 bind to the GIPR promoter region in RIN38 cells and the binding is cell specific.


Figure 7
View larger version (50K):
[in this window]
[in a new window]
  		Fig. 7. Sp1 and Sp3 bind to the GIPR gene control sequences in intact RIN38 cells. Formaldehyde cross-linked chromatin from RIN38 or Rat-2 cells was incubated with either Sp1, Sp3, or control antisera. Immunoprecipitated DNA was analyzed by PCR with primers to transcriptional regulatory sequences of the rat GIPR gene (top) or the rat pituitary tumor-transforming gene 1 (PTTG1) (bottom). Total input DNA at a 1:100 dilution was used as a positive control for the PCR reactions. DNA prepared from chromatin that was immunoprecipitated with either normal rabbit immune sera or GATA-4 antisera was used as negative controls for assays. The lanes are designated either according to the antisera used for the chromatin immunoprecipitation assay or by "input," which indicates total chromatin was used in the PCR. The gel shown is representative of 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It has been well established that the incretins GIP and glucagon-like peptide-1 (GLP-1) are important regulators of postprandial insulin release (47, 50, 51), and it is becoming increasingly apparent that these peptides play a major role in regulating body weight (28, 35). Initially, because of its potent insulinotropic actions, GLP-1 and its analogs were proposed as pharmacological agents for the treatment of type 2 diabetes mellitus. However, recent clinical studies indicate that the inhibition of gastric emptying by GLP-1 analogs may account for the beneficial effects of improved glucose homeostasis and weight loss (28). In contrast to GLP-1, antagonizing the effects of GIP may have beneficial consequences. It was demonstrated that GIPR-null mice are protected from diet-induced obesity and its associated complications, including type 2 diabetes mellitus and fatty liver. Although these mice exhibit transient mild elevations in postprandial glucose levels, it does not appear to affect the overall health of the animals (35).

Antagonism of GIP may also be beneficial for patients with FICS, which results from ectopic expression of GIPRs on cortisol-producing adrenal hyperplasia (26). Normally, cortisol is released from the adrenal cortex in response to ACTH activation of its specific G protein-coupled receptor (43). Apparently, in patients with FICS, binding of GIP to the ectopically expressed GIPRs produces the same signals that lead to cortisol release (26). Therefore, antagonizing GIP could in theory relieve the symptoms associated with FICS, thereby controlling patients’ cortisol levels before surgical excision of the adrenal gland or by obviating the need for surgery in those for whom operations are risky.

GIP may also play a role in the etiology of type 2 diabetes mellitus. It has been shown that patients with type 2 diabetes mellitus have both a diminished incretin effect and a reduced response to exogenous GIP. It has been speculated that this loss of the incretin effect and the reduced insulinotropic action of GIP may occur as a result of either a chronic desensitization of the GIPR (45) or a reduction in the expression of GIPRs in pancreatic beta-cells (18). Evidence from the study of an animal model of obesity-related type 2 diabetes appears to support the latter hypothesis. Lynn et al. (32) showed that, in pancreatic islets of VDF rats, a strain of the fatty Zucker rat containing a leptin receptor missense mutation, the expressions of both GIP receptor mRNA and protein are decreased.

Because GIPR expression has been implicated in the etiology of FICS and possibly type 2 diabetes mellitus and obesity, we initiated this project to identify and characterize the factors regulating GIPR gene expression. We previously reported that the rat GIPR gene contained a TATA-less promoter and that the first 100 bp of this promoter directed high levels of gene expression in both GIPR expressing and nonexpressing cells (5). In addition, sequences distal to the transcription start site were found to be required for directing cell-specific expression of the GIPR, and we hypothesized that upstream elements negatively regulate the basal promoter (5). In the present report, we describe the characterization of this basal promoter. A comparison of the 5'-flanking sequences between the rat and human GIPR genes demonstrated 88% identity within the first 92 bp, after which the sequences diverged abruptly. This high degree of conservation suggests an important role for this region in the regulation of GIPR. In addition, the GC content of this conserved region is high (67 and 72% for human and rat, respectively). Next, we used two Web-based programs to identify transcription factor binding motifs to analyze this region and identified two Sp1 motifs and an MZF1/Sp1 binding motif. Sp1 is just one member of a family of transcription factors that include Sp2, Sp3, and Sp4. Sp1 family members all possess highly conserved DNA binding domains consisting of three zinc fingers and regulate gene expression upon binding to GC-rich sequences (10). Two members, Sp1 and Sp4, are transcription activators, whereas Sp2 possesses repressor activity. As a result of the initiation of translation at two separate start codons, two isoforms of Sp3 are synthesized, a long form that activates transcription and a short form that represses transcription. MZF1 is a member of the Krüppel family of zinc finger containing transcription factors and activates transcription primarily in myeloid progenitor cells (19).

The observations that the GIPR promoter possesses a high GC content and multiple Sp1 binding motifs are consistent with reports from analysis of other TATA-less promoters (3, 22). The 5'-flanking sequences of other closely related G protein-coupled receptors, such as GLP-1, glucagon, VIP, PACAP, PTH, and PTHRH, also contain GC-rich regions and possess multiple Sp1 motifs, suggesting evolutionary conservation of not only the structural gene but also the regulatory portion of the gene (1, 2, 7, 27, 34, 42). When the regulatory regions of these other G protein-coupled receptor genes were analyzed, they were shown to bind the transcription factors Sp1 and the two isoforms of Sp3. In addition, the mutation of all but one of these Sp1 sites led to a decrease in promoter activity, suggesting that the majority of these sites act as transcription enhancers. The mutation of one Sp1 site within the GLP-1 promoter led to an increase in promoter activity (16), which the authors speculated may have resulted from the loss of a "silencing" element. In the case of the GIPR gene, we found that removal of part of the GIPR promoter containing the two Sp1 motifs and the MZF1/Sp1 binding motif at position –45, –57, and –75, respectively, led to an ~88% reduction in activity, suggesting that the regulatory elements in this region bind activators of transcription. EMSAs employing nuclear extracts from RIN38 cells demonstrated specific binding of Sp1 and the two isoforms of Sp3 to sequences representing the two Sp1 motifs and the MZF1/Sp1 motif at positions –45, –57, and –75, respectively (Fig. 1).

The inability to demonstrate specific binding of other members of the Sp1 family to the Sp1-binding motifs at positions –45 and –57 or specific binding of MZF1 and these other Sp1 family members to the MZF1/Sp1 binding motif at position –75 may be because of lack of expression of these factors in RIN38 cells rather than to their binding properties. Sp1, Sp2, and Sp3 are ubiquitously expressed, whereas the expressions of Sp4 and MZF1 are limited to specific cell types (10, 23, 29). The inability to detect binding of either Sp4 or MZF1 to the GIPR-specific sequences may be a consequence of the lack of expression of these proteins in RIN38 cells. In contrast, the lack of Sp2 binding may reflect the low binding properties of Sp2 to GC-rich sequences bound by Sp1 and Sp3 (36).

Mutational analysis of these putative transcription factor binding motifs in transient transfection assays suggests that binding of Sp1 and Sp3 to the Sp1 binding motifs at positions –45 and –57 are important for regulation of the GIPR in RIN38 cells, whereas the binding of these factors to the MZF1/Sp1 site at position –77 is not essential. In transient transfection assays using RIN38 cells, mutation of the Sp1 motif at position –45 (Sp1-A) led to an ~64% reduction in promoter activity, whereas mutation of the Sp1 motif at position –57 (Sp1-B) led to an ~74% reduction in promoter activity. In contrast, mutation of the MZF1/Sp1 motif at position –75 did not affect promoter activity in the same assay. Although we have identified two important regulatory elements in the GIPR promoter, our results do not exclude the possibility of additional regulatory elements downstream of this site that are dependent on these upstream regulatory sequences.

Further evidence to support the notion that the Sp1/Sp3 binding to the GIPR promoter positively regulates gene transcription was obtained using the Schneider S2 cell model. Because these cells lack endogenous expression of Sp1/Sp3, they can be used to examine specifically the effect of exogenous expression of Sp1 or Sp3 on transcriptional activity without interference from endogenous protein (31). Consistent with our finding using the RIN38 cell model, the segment of the GIPR promoter containing the Sp1 binding motifs at positions –45 and –57 was required for maximal promoter activity as seen for both Sp1 and the long form of Sp3.

The EMSAs and transient transfection analysis provide additional evidence for the involvement of Sp1 and Sp3 in the regulation of GIPR. However, what occurs in these artificial in vitro systems may not necessarily reflect what is occurring for the endogenous gene. In the nucleus of living cells, the initiation of transcription is affected by closed chromatin structure or competition from other nuclear factors that may exclude Sp1 and/or Sp3 from binding to the GIPR promoter. In this study, we were able to corroborate the EMSA and transient transfection data using ChIP analysis. Data from these experiments demonstrated that both Sp1 and Sp3 specifically associated with the GIPR promoter in RIN38 cells but not rat-2 cells. As previously described, RIN38 cells express endogenous GIPR, whereas rat-2 cells do not. In an earlier study, we demonstrated that transcription initiated at the GIPR promoter in rat-2 cells was repressed by sequences located upstream of the basal promoter (5). Our results are similar to those observed with the rat luteinizing hormone receptor, in which a DNA domain located between –1266 and –1307 bp repressed, in a cell-dependent manner, transcription of a promoter containing multiple Sp1 binding motifs within the first 200 bp (13). Based on data derived from our CHiP analysis, the upstream negative regulatory elements in the GIPR promoter presumably bind factors that prevent the interaction of ubiquitously expressed Sp1 and Sp3 with the GIPR promoter in nonexpressing cells.

Our results support the involvement of both the transcription factors Sp1 and Sp3 in the regulation of GIPR. Both of these factors can bind to the GIPR promoter, leading to an enhancement of transcriptional activity. The loss of Sp1 and Sp3 binding to the promoter is associated with an absence of GIPR expression, and the prevention of binding of these factors may represent an important factor determining cell-specific expression. Further studies will be necessary to determine whether dysregulation of GIPR expression, such as seen in FICS and type 2 diabetes mellitus, might involve factors that alter the binding of Sp1 and Sp3 to important regulatory elements within the GIPR promoter.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK53158 (M. M. Wolfe).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Michael Wolfe, Section of Gastroenterology, Boston Medical Center, 650 Albany St., Boston, MA 02118 (e-mail: michael.wolfe{at}bmc.org)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aino H, Hashimoto H, Ogawa N, Nishino A, Yamamoto K, Nogi H, Nagata S, and Baba A. Structure of the gene encoding the mouse pituitary adenylate cyclase-activating polypeptide receptor. Gene 164: 301–304, 1995.[CrossRef][ISI][Medline]
  2. Alimov AP, Langub MC, Malluche HH, and Koszewski NJ. Sp3/Sp1 in the parathyroid gland: identification of an Sp1 deoxyribonucleic acid element in the parathyroid hormone promoter. Endocrinology 144: 3138–3147, 2003.[Abstract/Free Full Text]
  3. Azizkhan JC, Jensen DE, Pierce AJ, and Wade M. Transcription from TATA-less promoters: dihydrofolate reductase as a model. Crit Rev Eukaryot Gene Expr 3: 229–254, 1993.[Medline]
  4. Bilsland AE, Anderson CJ, Fletcher-Monaghan AJ, McGregor F, Evans TR, Ganly I, Knox RJ, Plumb JA, and Keith WN. Selective ablation of human cancer cells by telomerase-specific adenoviral suicide gene therapy vectors expressing bacterial nitroreductase. Oncogene 22: 370–380, 2003.[CrossRef][ISI][Medline]
  5. Boylan MO, Jepeal LI, and Wolfe MM. Structure of the rat glucose-dependent insulinotropic polypeptide receptor gene. Peptides 20: 219–228, 1999.[CrossRef][ISI][Medline]
  6. Brown JC and Dryburgh JR. A gastric inhibitory polypeptide. II. The complete amino acid sequence. Can J Biochem 49: 867–872, 1971.[CrossRef][ISI][Medline]
  7. Buggy J, Hull J, and Yoo-Warren H. Isolation and structural analysis of the 5' flanking region of the gene encoding the human glucagon receptor. Biochem Biophys Res Commun 208: 339–344, 1995.[CrossRef][ISI][Medline]
  8. Cissell MA, Zhao L, Sussel L, Henderson E, and Stein R. Transcription factor occupancy of the insulin gene in vivo. Evidence for direct regulation by Nkx22. J Biol Chem 278: 751–756, 2003.[Abstract/Free Full Text]
  9. Clem AL, Hamid T, and Kakar SS. Characterization of the role of Sp1 and NF-Y in differential regulation of PTTG/securin expression in tumor cells. Gene 322: 113–121, 2003.[CrossRef][ISI][Medline]
  10. Cook T, Gebelein B, and Urrutia R. Sp1 and its likes: biochemical and functional predictions for a growing family of zinc finger transcription factors. Ann NY Acad Sci 880: 94–102, 1999.[Abstract/Free Full Text]
  11. Courey AJ and Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55: 887–898, 1988.[CrossRef][ISI][Medline]
  12. Dignam JD, Lebovitz RM, and Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475–1489, 1983.[Abstract/Free Full Text]
  13. Dufau ML, Tsai-Morris CH, Hu ZZ, and Buczko E. Structure and regulation of the luteinizing hormone receptor gene. J Steroid Biochem Mol Biol 53: 283–291, 1995.[CrossRef][ISI][Medline]
  14. Dupre J, Greenidge N, McDonald TJ, Ross SA, and Rubinstein D. Inhibition of actions of glucagon in adipocytes by gastric inhibitory polypeptide. Metabolism 25: 1197–1199, 1976.[ISI][Medline]
  15. Dupre J, Ross SA, Watson D, and Brown JC. Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J Clin Endocrinol Metab 37: 826–828, 1973.[ISI][Medline]
  16. Galehshahi FS, Goke B, and Lankat-Buttgereit B. A novel silencer element repressing expression of the GLP-1 receptor gene in fibroblasts and pancreatic A-cells, but not in pancreatic B- and D-cells. Peptides 21: 1169–1176, 2000.[CrossRef][ISI][Medline]
  17. Gerrish K, Cissell MA, and Stein R. The role of hepatic nuclear factor 1{alpha} and PDX-1 in transcriptional regulation of the pdx-1 gene. J Biol Chem 276: 47775–47784, 2001.[Abstract/Free Full Text]
  18. Holst JJ, Gromada J, and Nauck MA. The pathogenesis of NIDDM involves a defective expression of the GIP receptor. Diabetologia 40: 984–986, 1997.[CrossRef][ISI][Medline]
  19. Hromas R, Collins SJ, Hickstein D, Raskind W, Deaven LL, O'Hara P, Hagen FS, and Kaushansky K. A retinoic acid-responsive human zinc finger gene, MZF-1, preferentially expressed in myeloid cells. J Biol Chem 266: 14183–14187, 1991.[Abstract/Free Full Text]
  20. Innis MA and Gelfand DH. Optimization of PCRs. In: PCR Protocols: A Guide to Methods and Application. San Diego, CA: Academic, 1990.
  21. Jepeal LI, Boylan MO, and Wolfe MM. Cell-specific expression of the glucose-dependent insulinotropic polypeptide gene functions through a GATA and an ISL-1 motif in a mouse neuroendocrine tumor cell line. Regul Pept 113: 139–147, 2003.[CrossRef][ISI][Medline]
  22. Jones KA, Yamamoto KR, and Tjian R. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell 42: 559–572, 1985.[CrossRef][ISI][Medline]
  23. Kingsley C and Winoto A. Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. Mol Cell Biol 12: 4251–4261, 1992.[Abstract/Free Full Text]
  24. Krarup T, Madsbad S, Moody AJ, Regeur L, Faber OK, Holst JJ, and Sestoft L. Diminished immunoreactive gastric inhibitory polypeptide response to a meal in newly diagnosed type I (insulin-dependent) diabetics. J Clin Endocrinol Metab 56: 1306–1312, 1983.[Abstract]
  25. Lacroix A, Bolte E, Tremblay J, Dupre J, Poitras P, Fournier H, Garon J, Garrel D, Bayard F, Taillefer R, and et al. Gastric inhibitory polypeptide-dependent cortisol hypersecretion—a new cause of Cushing's syndrome. N Engl J Med 327: 974–980, 1992.[Abstract]
  26. Lacroix A, Ndiaye N, Tremblay J, and Hamet P. Ectopic and abnormal hormone receptors in adrenal Cushing's syndrome. Endocr Rev 22: 75–110, 2001.[Abstract/Free Full Text]
  27. Lankat-Buttgereit B and Goke B. Cloning and characterization of the 5' flanking sequences (promoter region) of the human GLP-1 receptor gene. Peptides 18: 617–624, 1997.[CrossRef][ISI][Medline]
  28. Larsen PJ, Fledelius C, Knudsen LB, and Tang-Christensen M. Systemic administration of the long-acting GLP-1 derivative NN2211 induces lasting and reversible weight loss in both normal and obese rats. Diabetes 50: 2530–2539, 2001.[Abstract/Free Full Text]
  29. Le Mee S, Fromigue O, and Marie PJ. Sp1/Sp3 and the myeloid zinc finger gene MZF1 regulate the human N-cadherin promoter in osteoblasts. Exp Cell Res 302: 129–142, 2005.[CrossRef][ISI][Medline]
  30. Loots GG and Ovcharenko I. rVISTA 2.0: evolutionary analysis of transcription factor binding sites. Nucleic Acids Res 32: W217–W221, 2004.[Abstract/Free Full Text]
  31. Lyko F, Ramsahoye BH, and Jaenisch R. DNA methylation in Drosophila melanogaster. Nature 408: 538–540, 2000.[CrossRef][Medline]
  32. Lynn FC, Pamir N, Ng EH, McIntosh CH, Kieffer TJ, and Pederson RA. Defective glucose-dependent insulinotropic polypeptide receptor expression in diabetic fatty Zucker rats. Diabetes 50: 1004–1011, 2001.[Abstract/Free Full Text]
  33. Mazzaferri EL, Starich GH, Lardinois CK, and Bowen GD. Gastric inhibitory polypeptide responses to nutrients in Caucasians and American Indians with obesity and noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 61: 313–321, 1985.[Abstract]
  34. McCuaig KA, Lee HS, Clarke JC, Assar H, Horsford J, and White JH. Parathyroid hormone/parathyroid hormone related peptide receptor gene transcripts are expressed from tissue-specific and ubiquitous promoters. Nucleic Acids Res 23: 1948–1955, 1995.[Abstract/Free Full Text]
  35. Miyawaki K, Yamada Y, Ban N, Ihara Y, Tsukiyama K, Zhou H, Fujimoto S, Oku A, Tsuda K, Toyokuni S, Hiai H, Mizunoya W, Fushiki T, Holst JJ, Makino M, Tashita A, Kobara Y, Tsubamoto Y, Jinnouchi T, Jomori T, and Seino Y. Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nat Med 8: 738–742, 2002.[CrossRef][ISI][Medline]
  36. Moorefield KS, Fry SJ, and Horowitz JM. Sp2 DNA binding activity and trans-activation are negatively regulated in mammalian cells. J Biol Chem 279: 13911–13924, 2004.[Abstract/Free Full Text]
  37. Osei K, Falko JM, O'Dorisio TM, Fields PG, and Bossetti B. Gastric inhibitory polypeptide responses and glucose turnover rates after natural meals in type II diabetic patients. J Clin Endocrinol Metab 62: 325–330, 1986.[Abstract]
  38. Ottlecz A, Samson WK, and McCann SM. The effects of gastric inhibitory polypeptide (GIP) on the release of anterior pituitary hormones. Peptides 6: 115–119, 1985.[ISI][Medline]
  39. Pederson RA, Schubert HE, and Brown JC. The insulinotropic action of gastric inhibitory polypeptide. Can J Physiol Pharmacol 53: 217–223, 1975.[ISI][Medline]
  40. Pei L. Genomic organization and identification of an enhancer element containing binding sites for multiple proteins in rat pituitary tumor-transforming gene. J Biol Chem 273: 5219–5225, 1998.[Abstract/Free Full Text]
  41. Reznik Y, Allali-Zerah V, Chayvialle JA, Leroyer R, Leymarie P, Travert G, Lebrethon MC, Budi I, Balliere AM, and Mahoudeau J. Food-dependent Cushing's syndrome mediated by aberrant adrenal sensitivity to gastric inhibitory polypeptide. N Engl J Med 327: 981–986, 1992.[Abstract]
  42. Sreedharan SP, Huang JX, Cheung MC, and Goetzl EJ. Structure, expression, and chromosomal localization of the type I human vasoactive intestinal peptide receptor gene. Proc Natl Acad Sci USA 92: 2939–2943, 1995.[Abstract/Free Full Text]
  43. Stocco DM and Clark BJ. Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17: 221–244, 1996.[CrossRef][ISI][Medline]
  44. Straub SG and Sharp GW. Glucose-dependent insulinotropic polypeptide stimulates insulin secretion via increased cyclic AMP and [Ca2+]1 and a wortmannin-sensitive signalling pathway. Biochem Biophys Res Commun 224: 369–374, 1996.[CrossRef][ISI][Medline]
  45. Tseng CC, Boylan MO, Jarboe LA, Usdin TB, and Wolfe MM. Chronic desensitization of the glucose-dependent insulinotropic polypeptide receptor in diabetic rats. Am J Physiol Endocrinol Metab 270: E661–E666, 1996.[Abstract/Free Full Text]
  46. Tseng CC, Kieffer TJ, Jarboe LA, Usdin TB, and Wolfe MM. Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat. J Clin Invest 98: 2440–2445, 1996.[ISI][Medline]
  47. Tseng CC, Zhang XY, and Wolfe MM. Effect of GIP and GLP-1 antagonists on insulin release in the rat. Am J Physiol Endocrinol Metab 276: E1049–E1054, 1999.[Abstract/Free Full Text]
  48. Usdin TB, Mezey E, Button DC, Brownstein MJ, and Bonner TI. Gastric inhibitory polypeptide receptor, a member of the secretin-vasoactive intestinal peptide receptor family, is widely distributed in peripheral organs and the brain. Endocrinology 133: 2861–2870, 1993.[Abstract]
  49. Villar HV, Fender HR, Rayford PL, Bloom SR, Ramus NI, and Thompson JC. Suppression of gastrin release and gastric secretion by gastric inhibitory polypeptide (GIP) and vasoactive intestinal polypeptide (VIP). Ann Surg 184: 97–102, 1976.[ISI][Medline]
  50. Wang Z, Wang RM, Owji AA, Smith DM, Ghatei MA, and Bloom SR. Glucagon-like peptide-1 is a physiological incretin in rat. J Clin Invest 95: 417–421, 1995.[ISI][Medline]
  51. Weir GC. Glucagon-like peptide-1 (GLP-1): a piece of the incretin puzzle. J Clin Invest 95: 1, 1995.[ISI][Medline]
  52. Wingender E. Recognition of regulatory regions in genomic sequences. J Biotechnol 35: 273–280, 1994.[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/E1287    most recent
00535.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boylan, M. O.
Right arrow Articles by Wolfe, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation