AJP - Endo Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 292: E215-E222, 2007. First published August 22, 2006; doi:10.1152/ajpendo.00181.2006
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/E215    most recent
00181.2006v1
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 ISI Web of Science
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 ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kang, J.-H.
Right arrow Articles by Jo, Y.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kang, J.-H.
Right arrow Articles by Jo, Y.-H.

Proximal cyclic AMP response element is essential for exendin-4 induction of rat EGR-1 gene

Jung-Hoon Kang,1 Myung-Jun Kim,1 Hwa-In Jang,1 Kyung-Hee Koh,2 Keun-Sang Yum,3 Duck-Joo Rhie,1 Shin Hee Yoon,1 Sang June Hahn,1 Myung-Suk Kim,1 and Yang-Hyeok Jo1

1Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul; 2Department of Food Science and Nutrition, The Catholic University of Korea, Puchon; and 3Department of Family Medicine, Uijeongbu St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Uijeongbu, Korea

Submitted 14 April 2006 ; accepted in final form 14 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Glucagon-like peptide-1 and its potent agonist exendin-4 induce several immediate early response genes (IEGs) that code for transcription factors implicated in cell proliferation, differentiation, and apoptosis. We recently observed that early growth response factor-1 (EGR-1), an IEG product, was required for transcriptional activation of Ccnd1 (cyclin D1) gene by exendin-4. Herein, the regulatory mechanism whereby exendin-4 activates the transcription of EGR-1 gene was investigated in the pancreatic beta-cell line INS-1. Deletion analysis of rat EGR-1 promoter identified a critical region between –73 and –46 for the activation of EGR-1 in response to exendin-4. Mutation of the proximal putative cAMP response element (CRE, 5'-GTACGTCA-3') located at –69 resulted in a significant decrease in the EGR-1 transcription, whereas the mutation of the distal putative CRE at –139 was without such an effect. In immune supershift assays using exendin-4-treated cells, binding of cAMP response element-binding protein (CREB) phosphorylated on Ser133 to the proximal CRE was increased. Employment of a CREB mutant containing Ala substitution at Ser133 or a dominant negative CREB mutant that inhibits the binding of endogenous CREB to DNA significantly decreased the exendin-4-induced EGR-1 transcription. In experiments using specific protein kinase inhibitors, the effect of H-89 was more prominent than PD-98059, indicating the predominance of the PKA signaling over the MEK/ERK in induction of EGR-1. Therefore, it appears that the proximal CRE site is critical and the binding with CREB phosphorylated on Ser133 is necessary for induction of the EGR-1 transcription by exendin-4.

early growth response factor-1; cyclic adenosine monophosphate response element-binding protein; protein kinase A

GLUCAGON-LIKE PEPTIDE-1-(7–36) AMIDE (GLP-1), along with its analog exendin-4, is a potent insulinotropic hormone that enhances insulin secretion and whole body insulin sensitivity (8, 25). They also act as a growth factor that stimulates beta-cell proliferation and islet neogenesis in diabetic animal models (4, 15, 21). Activation of the GLP-1 receptor, which is G protein coupled, increases intracellular cAMP level and activates PKA. The cAMP/PKA signaling pathway has been shown to play a pivotal role in maintaining and promoting growth of the beta-cells (12). GLP-1 also activates other signaling pathways, such as phosphatidylinositol 3-kinase and mitogen-activated protein kinase, in these cells (15, 21). In addition, GLP-1 induces immediate early response genes, such as c-FOS, c-JUN, and early growth response factor-1 (EGR-1), that are implicated in beta-cell growth, differentiation, and apoptosis (21, 22).

Recently, we (13) reported that exendin-4 induced binding of EGR-1 protein to a cis-regulatory element between –153 and –134 on rat Ccnd1 (cyclin D1) promoter leading to activate cyclin D1 transcription. Other studies using small RNA interference demonstrated that reduced EGR-1 gene expression contributed to the decreased beta-cell proliferation and the consequent beta-cell failure, supporting the essential role of EGR-1 in normal pancreatic beta-cell function (7). EGR-1, also known as Zif268, NHFI-A, Tis8, and Krox24, is a 3-zinc-finger protein of a member of transcription factors, and its expression is rapidly and transiently induced by various extracellular stimuli (1–3, 23, 24). The induction of EGR-1 has been primarily known to be regulated at the transcriptional level through cis-regulatory elements of the 5'-regulatory region of the gene, which contains the binding sites for serum response factor, cAMP response element-binding protein (CREB), activating protein-1 (AP1), E26 (ETS), and EGR-1 itself (2, 3, 18–20).

Although the roles of GLP-1 in the proliferation of pancreatic beta-cells have been widely studied, the molecular mechanisms by which the signaling cascades lead to the regulation of their target gene such as EGR-1 have been less well known. Therefore, we investigated the cis-acting elements and the trans-acting factor involved in regulating EGR-1 gene expression in response to exendin-4 by use of the INS-1 pancreatic beta-cell line.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. All culture media, exendin-4, and poly(dI-dC) were purchased from Sigma-Aldrich (St. Louis, MO). Wild-type CREB, mutant-type CREB (Ser133Ala), and dominant negative Killer (CREB) were kindly provided by Dr. R. H. Goodman (Volum Institute, Oregon Health and Science University, Portland, OR). [32P]dCTP and [32P]ATP were obtained from PerkinElmer Life Sciences (Boston, MA). LipofectAMINE-PLUS reagent was from Life Technologies (Grand Island, NY). Antibodies to CREB and phosphorylated CREB were from Cell Signaling Technology (Beverly, MA). Anti-EGR-1, -c-JUN, and -Sp1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). QuikChange Site-Directed Mutagenesis Kit was from Stratagene (La Jolla, CA). Luciferase assay kit, rabbit anti-(mouse IgG)-peroxidase conjugate, and goat anti-(rabbit IgG)-peroxidase conjugate were from Promega (Madison, WI). PD-98059 and H-89 were from Calbiochem (San Diego, CA).

Cell culture and treatment. INS-1 cells (between passages 15 and 27) were cultured in RPMI 1640 medium (11.5 mM glucose) supplemented with 10% FBS, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 1 mM pyruvate, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37°C under an atmosphere of 95% air-5% CO2. For exendin-4 and inhibitor treatment, cells at an initial density of 1 x 105 cells/ml were cultured for 48 h and serum starved with RPMI 1640 (5 mM glucose) for 24 h. After starvation, the cells were treated with exendin-4 or pretreated with inhibitors, as described in the figure legends.

Preparation of nuclear extracts. Cells were washed twice with ice-cold PBS and were pelleted by centrifugation. The pellet was resuspended and incubated for 15 min on ice in a hypotonic solution consisting of 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.4% Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 2.5 mM dithiothreitol, 0.5 mM PMSF, and 1 µg/ml leupeptin. Nuclei were collected with centrifugation and incubated for 30 min on ice in a hypertonic buffer consisting of 20 mM HEPES, pH 7.9, 25% glycerol, 0.5 M NaCl, 1.5 mM MgCl2, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 5 mM dithiothreitol, 0.1 mM PMSF, and 1 µg/ml leupeptin. The nuclear extract was prepared by centrifugation and stored at –80°C prior to use. Protein concentrations were measured with the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).

Western blotting. Twenty micrograms from each sample were separated on an SDS-polyacrylamide gel. Following electrophoresis, proteins were transferred to PVDF membrane (Amersham Biosciences, Piscataway, NJ). Blocking was performed in 5% nonfat dried milk in PBS containing 0.2% Tween-20. Antibodies used to probe the blots were EGR-1, CREB, phosphorylated CREB, and beta-tubulin. The detection was performed by chemiluminescence system (Amersham Biosciences).

Northern blotting. Total cellular RNA was extracted using TRIzol reagent (Life Technologies) based on the manufacturer’s instructions. Ten micrograms of each sample were fractionated in a 1% formaldehyde agarose gel and transferred onto a Hybond-N nylon membrane (Amersham Biosciences). The membrane was hybridized in a rapid hybridization buffer (Amersham Biosciences) with a 0.7-kilobase rat EGR-1 cDNA labeled with [32P]dCTP (3,000 Ci/mmol) and washed under high-stringency conditions: once with 2x SSC and 0.1% SDS at room temperature for 15 min, once with 2x SSC and 0.1% SDS at 65°C for 15 min, and once with 0.1x SSC and 0.1% SDS at 65°C for 15 min. The membrane was subjected to autoradiography.

Plasmid constructions. The rat EGR-1 promoter fragment spanning from –462 to +27 was generated by a standard PCR method using genomic DNA from INS-1 cells as a template. The forward primer was 5'-TCAGGTACCAGGCTCCGGGTTGGGAAC-3' (KpnI site is underlined) and the reverse was 5'-TCACTCGAGCCAAGTTCTGCGCGCTGG-3' (XhoI site is underlined). The PCR product digested with KpnI and XhoI was cloned into the promoterless vector pGL3 basic (Promega), designated as p-462. The constructs with serially 5'-truncated fragments were synthesized using the p-462 as a template and cloned in the same manner. The forward primers with the KpnI site at the 5' end (underlined) were as follows: for p-324, 5'-TCAGGTACCTGCGCTTCCGGCTCTG-3'; for p-243, 5'-TCAGGTACCGCCACTGCCGCTGTTCC-3'; for p-159, 5'-TCAGGTACCCCGCTCTTGGATGGGAGGTC-3'; for p-124, 5'-TCAGGTACCGTCCTCCCGGTCGGTCC-3'; for p-73, 5'-TCAGGTACCCCATGTACGTCACGGCGGAG-3'; for p-46, 5'-TCAGGTACCCGTGCTGTTTCAGACCCTTG-3'. The reverse primer was the same as that used in p-462.

Site-directed mutagenesis. The mutant CRE constructs were prepared with a QuikChange Site-Directed Mutagenesis Kit according to the manufacturer’s protocol. The top strand of the primer set was as follows: for mpCRE-462 and mpCRE-73, 5'-ATATATGGCCATGTTGGTCACGGCGGAGGC-3', and for mdCRE-462, 5'-GATGGGAGGTCTTCTGGTCACTCCGGGTCC-3'. The two underlined nucleotides in the putative CRE sites were changed from AG (wild type) to TG. The mutation was verified by DNA sequence analysis.

Transient transfection and luciferase activity assay. Constructs of EGR-1 promoter-luciferase reporter gene were transiently transfected into INS-1 cells (0.5 µg/2 x 105 cells) without or with wild- or mutant-type CREB vector (1 µg) using LipofectAMINE-PLUS reagent. To control transfection efficiency and to normalize luciferase values in all experiments, pSV-beta-galactosidase (Promega) was used as an internal control. The fold induction was determined by dividing the luciferase activity of the exendin-4-treated cells by that of the untreated cells.

Electrophoretic mobility shift assay. Binding reaction to allow to form DNA/protein complexes was performed in 20 µl of 10 mM HEPES, pH 8, 0.1 mM EDTA, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 10% glycerol, 1 µg of poly(dI-dC), 32P-labeled oligonucleotide probe (30,000 cpm), and 5 µg of nuclear extract. The reaction was allowed to continue for 15 min at 4°C. In supershift studies, 2 µg of the appropriate antibody were preincubated with the nuclear extract for 15 min at 4°C before addition of the labeled probe. In competition experiments, the nuclear extract was incubated with a 100-fold molar excess of the appropriate unlabeled competitor oligonucleotides. Electrophoresis was carried out on 6% nondenatured polyacrylamide gels with 0.5x TBE (15 V/cm for ~2 h). The gel was dried under vacuum and subjected to autoradiography. Oligonucleotides used were as follows: CRE, 5'-CCATGTACGTCACGGCGGAG-3'; CRE mutant, 5'-CCATGTtgGTCACGGCGGAG-3'; CRE consensus, 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'; AP1 consensus, 5'-CGCTTGATGACTCAGCCGGAA-3'; and Sp1 consensus, 5'-ATTCGATCGGGGCGGGGCGAGC-3'.

Chromatin immunoprecipitation assays. Chromatin immunoprecipitation (ChIP) assays were performed with a ChIP assay kit (Upstate Biotechnology, Grand Island, NY) according to the manufacturer’s instructions. The conditions for PCR were as follows: 30 s at 95°C, 30 s at 62°C, and 30 s at 72°C for 25 cycles, with a final extension for 10 min at 72°C. Primers for ChIP were forward 5'-GTCCTCCCGGTCGGTCCTTC-3' and reverse 5'-GATCCCAGCGCGCAGAACTTGG-3'.

Statistical analysis. Data are expressed as means ± SD. Statistical analysis was performed using one-way ANOVA and Student’s t-test. P < 0.05 was accepted as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Activation of rat EGR-1 gene in response to exendin-4 in INS-1 cells. INS-1 cells transiently transfected with p-462 construct containing full-length EGR-1 promoter (from –462 to +27) were serum-starved and treated with varying concentrations of exendin-4 (10–10, 10–9, 10–8, and 10–7 M). As shown in Fig. 1A, exendin-4 induced the activity of the reporter gene in a dose-dependent manner, starting to induce at 10–9 M and peaking at 10–8 M without a further increase at 10–7 M. In examination of the kinetics of EGR-1 expression, exendin-4 (10–8 M) rapidly and transiently induced the expression of EGR-1 mRNA. The mRNA was detectable within 15 min after exendin-4 treatment, reaching maximum level at 30 min and sharply declining thereafter (Fig. 1B). The time course of the mRNA expression was well correlated with the kinetics of EGR-1 protein induction. The protein level started to increase within 30 min, peaked at 1 h, and returned to basal level by 2 h of exendin-4 treatment (Fig. 1C). In another pancreatic beta-cell line, RINm5F, exendin-4 treatment showed a similar response pattern of EGR-1 induction observed in INS-1 cells (data not shown).


Figure 1
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 1. Transcriptional activation of early growth response factor-1 gene (EGR-1) by exendin-4 in INS-1 cells. A: INS-1 cells transfected with 0.5 µg of full-length EGR-1 promoter construct (p-462) were serum starved for 24 h and treated with exendin-4 at the indicated concentrations for 6 h. After cell harvesting, luciferase activities were measured and normalized to beta-galactosidase activities. Data are expressed as fold induction of luciferase activity relative to the mean of the untreated control. Values are mean fold induction ± SD of 3 experiments with 2 replicate wells/experiments. *P < 0.05 vs. untreated control. B: Northern blot analysis of EGR-1 mRNA. C: Western blot of EGR-1 protein. Each lane contains 10 µg of total cellular RNA for Northern blot and 20 µg of nuclear extract for Western blot from cells treated with exendin-4 during the indicated time periods. Blots are representative of 3 independent experiments.

 
Determination of exendin-4 response elements of the rat EGR-1 promoter. The 5'-regulatory region of rat EGR-1 promoter contains several putative binding sites for transcription factors, which are known to mediate transcriptional activation of human EGR-1 gene (18–20). These regulatory sites include five sterol response elements (SREs), two ETSs, and two CREs. To determine the regulatory elements involved in inducing the rat EGR-1 gene in response to exendin-4, a series of 5' deletion constructs of the rat EGR-1 promoter were transiently transfected, and then the activities of the reporter gene were analyzed. As shown in Fig. 2, two regions were responsive to exendin-4, i.e., between –462 and –324 (designated as 5'-responsive region) and between –73 and –46 (designated as 3'-responsive region). The p-462, a full-length construct, showed an ~2.1-fold increase in promoter activity by exendin-4. Further serial deletion to –73, which eliminated five SREs and a distal CRE, maintained relatively constant responses of 1.6- to 1.7-fold induction. However, the deletion to –46, which eliminated the remaining proximal CRE site, resulted in a complete loss of the exendin-4 response. Thus, the 3' responsive region between –73 and –46 proved to be a principal site for induction of the EGR-1 gene activity in response to exendin-4. On the other hand, the 5'-responsive region between –462 and –324 may be required for the full activation of the EGR-1 promoter.


Figure 2
View larger version (6K):
[in this window]
[in a new window]

  		Fig. 2. Deletion analysis of rat EGR-1 promoter. Schematic description of constructs used in transient transfection assays is shown on the left. Putative regulatory elements [sterol response element (SRE), ETS, and cAMP response element (CRE)] are indicated. INS-1 cells transfected with the constructs (0.5 µg) were serum starved for 24 h and treated with exendin-4 for 6 h. After cell harvesting, luciferase activities were measured and normalized to beta-galactosidase activities. Numbers on the right are mean fold induction of luciferase activity induced by exendin-4 divided by the corresponding untreated control. Filled bars represent means ± SD of 5 experiments with 2 replicate wells/experiments. *P < 0.05 vs. p-462 and p-46.

 
Significant inhibition of exendin-4-induced EGR-1 transcription by mutation of the proximal CRE. To evaluate the functional significance of the proximal CRE site for the activation of the EGR-1 promoter by exendin-4, the proximal CRE sequences (5'-GTACGTCA-3') of the p-462 or p-73 construct were mutated to 5'-GTtgGTCA-3'. The distal CRE site located at –139 (5'-TCACGTCA-3') was also mutated to 5'-TCtcGTCA-3', as this distal CRE site was also shown to be involved in the regulation of the human EGR-1 gene by growth factors (5, 6, 18). As shown in Fig. 3, the introduction of the mutated proximal CRE into the p-462 construct (mpCRE-462) reduced the fold induction by ~28% compared with wild p-462 construct (1.5 ± 0.1 vs. 2.1 ± 0.2, P < 0.05), whereas the introduction of the same mutant CRE into the p-73 construct (mpCRE1–73) abolished the exendin-4 response compared with wild p-73 construct (1.0 ± 0.1 vs. 1.7 ± 0.1, P < 0.05). On the other hand, mutation of the distal CRE (mdCRE-462) decreased the fold induction by only 12% compared with the wild p-462 construct (1.8 ± 0.1 vs. 2.1 ± 0.2, P < 0.05). These results confirmed the critical role of the 3'-responsive region and the importance of the proximal CRE in activating the EGR-1 gene in response to exendin-4.


Figure 3
View larger version (6K):
[in this window]
[in a new window]

  		Fig. 3. Functional significance of proximal and distal CRE in inducing EGR-1 by exendin-4. Wild (wild p-462 and wild p-73) or mutant [mutation of proximal CRE site (mp)CRE-462, mutation of distal CRE site (md)CRE-462, and mpCRE-73] CRE constructs (0.5 µg) were transiently transfected into INS-1 cells. After transfection for 18 h, cells were serum starved for 24 h and treated with exendin-4 for 6 h. After cell harvesting, luciferase activities were measured and normalized to beta-galactosidase activities. Numbers on the right are mean fold induction of luciferase activity induced by exendin-4 divided by the corresponding untreated control. Filled bars represent means ± SD of 3 experiments with 2 replicate wells/experiments. *P < 0.05 vs. wild p-462, #P < 0.01 vs. wild p-73.

 
Induction of CREB phosphorylated on Ser133 associated with the proximal CRE site by exendin-4. To determine the nuclear factors bound to the proximal CRE site, electrophoretic mobility shift assays (EMSAs) were performed using nuclear extracts from exendin-4-treated INS-1 cells and a radiolabeled probe containing the proximal CRE sequence. As shown in Fig. 4A, the relative intensity of each band was irrelevant to the duration of exendin-4 treatment. The binding specificities of these complexes were determined using unlabeled oligonucleotides. The complexes were completely eliminated by 100-fold excess of unlabeled consensus or wild CRE oligonucleotides but not affected by mutant CRE oligonucleotide (Fig. 4B). Because the proximal CRE sequence of the radiolabeled probe used in this experiment also holds potential binding sites for c-JUN, one of the AP1 subunits, and Sp1 as shown in Fig. 4B, bottom, consensus AP1 and Sp1 oligonucleotides were also tested and proved to be without effect. Next, nuclear factors binding to the proximal CRE site were identified using relevant antibodies. As shown in Fig. 4C, incubation with an antibody against CREB resulted in a supershifted complex formation with similar intensity regardless of exendin-4 treatment. However, from the exendin-4-treated nuclear extract, an antibody (anti-pCREB) that specifically recognizes the CREB phosphorylated on Ser133, picked up a clear, supershifted band that was not detectable from the untreated nuclear extracts. The presence of phosphorylated CREB observed in the supershift assays was confirmed by Western blot analysis using the same nuclear extracts and the anti-phospho-CREB antibody. Exendin-4 treatment substantially induced the phosphorylation of CREB without affecting the levels of CREB proteins (Fig. 4D). Next, the in vivo effect of exendin-4 on the induction of the phosphorylation at Ser133 of CREB bound to rat EGR-1 promoter was examined by ChIP assay. The immunoprecipitated DNAs were amplified by standard PCR using a pair of primers flanking the proximal CREB-binding region (–124 to +27) of the EGR-1 promoter. As shown in Fig. 4E, in vivo binding of the phosphorylated CREB at Ser133 to the region of EGR-1 promoter was clearly detected after exendin-4 treatment, whereas the binding of CREB was not significantly changed. Thus the phosphorylation of CREB on Ser133 appears to be involved in the exendin-4-stimulated EGR-1 transcription.


Figure 4
View larger version (50K):
[in this window]
[in a new window]

  		Fig. 4. Activation of the proximal CRE site through binding of phosphorylated CREB on Ser133 by exendin-4. Nuclear extracts from INS-1 cells treated with exendin-4 for the indicated times were used in EMSA with a probe containing the putative CRE sequence. A: proximal CRE binding to nuclear factors at various time points. B: competition assays with unlabeled oligonucleotides. Unlabeled consensus CRE, wild, or mutant CRE and consensus activating protein-1 (AP1) or Sp1 were added at 100-fold molar excess. C: supershift assays with antibodies to CREB (anti-CREB), phospho-CREB on Ser133 (anti-pCREB), c-JUN (anti-c-JUN), or Sp1 (anti-Sp1). Nuclear extracts with exendin-4 treatment for 15 min were preincubated with antibodies for 15 min before addition of radiolabeled probe. D: Western blot analysis of phospho-CREB (p-CREB) and CREB using the same nuclear extracts used for supershift assays. E: ChIP assay was performed to amplify the region from –124 to +27. Input samples represent 0.001, 0.005, and 0.01% of total DNA, whereas PCRs of the immunoprecipitations include 5% of the resuspended DNA. IgG antibody was used as a negative control. Similar results were obtained from 3 independent experiments.

 
Requirement of CREB phosphorylation on Ser133 for transcriptional activation of EGR-1 gene. In activating EGR-1 transcription by exendin-4, whether phosphorylation on Ser133 of CREB is required needs to be confirmed. Hence, cotransfection with p-462 construct was performed using a wild-type and two mutant-type CREB-expressing vectors. Two mutant CREB vectors were Ser133Ala and KCREB. Ser133Ala expresses mutant CREB on which Ser133 was substituted with Ala to prevent phosphorylation of endogenous CREB. KCREB expresses an inhibitory protein, which binds to endogenous CREB to prevent the binding of CREB to the CRE site of gene promoters. Although wild-type CREB vector per se increased the luciferase activity, exendin-4 augmented the activity significantly. In contrast, mutant CREB vectors (Ser133Ala and KCREB) showed significantly less increased exendin-4 responses compared with the vector (Table 1). These results suggest that exendin-4 induction of CREB phosphorylation on Ser133 was directly involved in the transcriptional activation of EGR-1 promoter in response to exendin-4.


View this table:
[in this window]
[in a new window]

  		Table 1. Requirement of CREB phosphorylation on Ser133 for exendin-4 induction of EGR-1 promoter

 
Exendin-4 induction of EGR-1 expression via PKA and ERK signaling pathways. To investigate the signaling pathway for the exendin-4 induction of EGR-1 expression, specific kinase inhibitors (H-89 for PKA and PD-90059 for MEK) were used. As shown in Fig. 5A, at concentrations commonly used for the inhibition of PKA and MEK activities, pretreatment with H-89 (10 µM) completely inhibited the exendin-4 response, whereas PD-98059 (50 µM) decreased the response by 36%. In parallel with the transfection results, Western blot analysis showed that the exendin-4 induction of phosphorylation of CREB on Ser133 and of EGR-1 protein were completely blocked by H-89 but partly by PD-98059 (Fig. 5B). These results indicate that the PKA signaling pathway is more important for the exendin-4 activation of EGR-1 gene and for the phosphorylation of CREB than the MEK/ERK pathway.


Figure 5
View larger version (33K):
[in this window]
[in a new window]

  		Fig. 5. Involvement of PKA and ERK signaling pathways in exendin-4 induction of EGR-1 expression. A: INS-1 cells transfected with p-462 construct were serum starved for 24 h and then incubated for 1 h in the presence of H-89 (10 µM) or PD-98059 (PD, 50 µM). Cells were then treated with exendin-4 for 1 h. After cell harvesting, luciferase activities were measured and normalized to beta-galactosidase activities. Data are expressed as fold induction of luciferase activity relative to the untreated wild p-462. *P < 0.05 vs. untreated control or DMSO; {dagger}P < 0.05 vs. exendin-4 only. B: INS-1 cells serum starved for 24 h were incubated for 1 h in the presence of H-89 (10 µM) or PD-98059 (50 µM) and treated with exendin-4 for 10 min and for 1 h to determine levels of phospho-CREB and EGR-1, respectively. Similar results were obtained from 4 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Accumulating evidence strongly indicates that GLP-1 and its potent analog exendin-4 possess properties of enhancing pancreatic beta-cell growth and differentiation and of suppressing beta-cell apoptosis in both in vivo and in vitro models (4, 15, 21). In an effort to clarify the molecular mechanism of GLP-1 in regulating beta-cell proliferation, we (13) recently found that exendin-4 induced EGR-1 to bind the putative EGR-1 binding site of rat cyclin D1 promoter, leading to the transactivation of cyclin D1 gene. In this study, we further showed that the proximal CRE site of rat EGR-1 promoter in association with CREB phosphorylated on Ser133 contributed substantially to the transcriptional activation of EGR-1 gene in response to exendin-4 in INS-1 cells.

In humans, the rapid and transient activation of EGR-1 gene expression by many extracellular stimuli is mediated exclusively through the cis-regulatory elements within the 5'-regulatory region of the gene without involving posttranscriptional regulation such as mRNA stability. The rapid induction of EGR-1 gene is highly dependent on the binding sites for serum response factor, ternary complex factors, and CREB on the EGR-1 promoter. However, the molecular mechanism for the transient induction of EGR-1 gene is not well known. A negative feedback loop is suggested to downregulate the transcription, in which the EGR-1 protein can bind to the EGR-1 binding site of its own gene and allows only a transient activation of EGR-1 gene. In this study, our determination of the kinetics for induction of EGR-1 mRNA following exendin-4 treatment was well consistent with most other studies in that EGR-1 mRNA was induced within 15 min and significantly reduced after 30 min (2, 5, 22). The rapid and transient induction of EGR-1 mRNA, together with the activation of EGR-1 promoter, indicates the transcriptional regulation as a fundamental mechanism for the exendin-4 induction of EGR-1 gene.

Through serial-deletion analysis of rat EGR-1 promoter, we mapped the 5'- and 3'-responsive regions, which were located between –462 and –324 and between –73 and –46, respectively. Although the 5'-responsive region contains the SRE cluster and ETS sites important for EGR-1 transcription in human and mouse (3, 5, 18), and the region hence appears to be required for the full activation of the EGR-1 promoter in response to exendin-4, we focused our interest on the 3'-responsive region between –73 and –46. The reasons are as follows. First, deletion of this particular region eliminated almost completely the exendin-4 response, providing this region as a minimally essential promoter. Second, this region contains a proximal putative CRE site that could be the final target of the classic cAMP/PKA signaling pathway for GLP or exendin-4 (15).

Our site-directed mutation of the proximal CRE within the 3'-responsive region resulted in a complete loss of the responsiveness to exendin-4, confirming that the CRE is crucial in inducing the activation of the 3'-responsive region. The importance of the equivalent CRE in activating the gene was also reported with the human EGR-1 promoter (14, 19). The 3'-responsive regions of rat and human hold 100% identically conserved proximal CRE site (5'-GTACGTCA-3') extended from –69 to –62. However, the introduction of the mutated proximal CRE into p-462 construct resulted in only a one-third loss of the exendin-4 response instead of abolition of the response. This result might be attributed to the action of intact SRE cluster and ETS sites located within the 5'-responsive region between –462 and –324.

On the other hand, the distal CRE of human EGR-1 promoter has been shown to be involved in inducing EGR-1 genes (2, 5, 6, 18). In support of these studies, we also observed that mutation of the distal CRE of rat EGR-1 promoter resulted in a significant decrease in the exendin-4 induction of EGR-1 gene, although less than that of the proximal CRE (mdCRE-462 vs. mpCRE-462, Fig. 3). Interestingly, the deletion effect of the distal CRE at –139 on induction of EGR-1 promoter activation was negligible in our deletion analysis study (p-159 vs. p-124, Fig. 2). The discrepancy between the two studies is not clear; however, the strong stimulatory effect of the proximal CRE might have masked the effect of the distal CRE. Likewise, in human EGR-1 promoter, the importance of distal CRE was revealed only by mutation assay but not by deletion analysis (6).

CREB, a ubiquitously expressed transcription factor, plays a critical role in mediating many cellular responses to various physiological stimuli (16, 17). CREB activation is generally mediated through phosphorylation on the critical residue Ser133 within its kinase-inducible domain (16). The phosphorylation of CREB facilitates its association with the transcriptional coactivators such as CREB-binding protein, leading to promote the expression of CREB target genes. In our supershift and ChIP assays using an antibody against a phosphorylated CREB on Ser133, the binding activity of phospho-CREB was remarkably increased following treatment with exendin-4 in vitro and in vivo. In addition, cotransfection data also showed that overexpression of mutant CREB vectors resulted in a significant reduction of the activity of EGR-1 promoter. The involvement of CREB phosphorylation in our study is very well consistent with human EGR-1 studies showing that rapid phosphorylation of CREB mediated the activation of the promoter via binding to the proximal CRE site (14). These and our findings strongly indicate that exendin-4-induced phosphorylation of CREB is an important step in the transcriptional activation of EGR-1 gene.

Among multiple signaling pathways, PKA and ERK pathways have been well known to mediate EGR-1 expression in response to extracellular mitogens (1–3, 10, 24). In pancreatic beta-cells, cAMP/PKA/CREB pathway induced by GLP-1 or exendin-4 is believed to be a critical signaling pathway for cell survival and growth (12). In addition, the PKA signaling pathway was shown to mediate the induction of EGR-1 in insulinoma cell line MIN6 under high glucose concentration (2). In other cell types, the cAMP/PKA pathway has been suggested to cross-talk with Rap1/Raf/MEK to activate ERK (9). According to our observation employing pharmacological inhibitors, H-89 was more potent in suppressing the exendin-4-activated EGR-1 gene expression as well as pCREB induction than PD-98059, suggesting that the PKA pathway is mainly involved in exendin-4-induced EGR-1 expression. However, further experiments are required to clarify the exact signaling pathway for EGR-1 transcriptional activation by exendin-4 in beta-cells.

In conclusion, this study represents an important mechanism by which exendin-4 modulates the transcription of EGR-1 gene, the regulation of which is necessary for pancreatic beta-cell growth.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by a grant from the Korea Science and Engineering Foundation (R-2004-000-10127-0), Republic of Korea.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y.-H. Jo, Dept. of Physiology, The Catholic University of Korea, Seoul 137-701, Korea (e-mail: yhjo{at}catholic.ac.kr)

J.-H. Kang and M.-J. Kim contributed equally to this research.

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. Bauer I, Hohl M, Al-Sarraj A, Vinson C, Thiel G. Transcriptional activation of the EGR-1 gene mediated by tetradecanoylphorbol acetate and extracellular signal-regulated protein kinase. Arch Biochem Biophys 438: 36–52, 2005.[ISI][Medline]
  2. Bernal-Mizrachi E, Wice B, Inoue H, Permutt MA. Activation of serum response factor in the depolarization induction of EGR-1 transcription in pancreatic islet beta-cells. J Biol Chem 275: 25681–25689, 2000.[Abstract/Free Full Text]
  3. Thiel G, Cibelli G. Regulation of life and death by zinc finger transcription factor Egr-1. J Cell Physiol 193: 287–292, 2002.[CrossRef][ISI][Medline]
  4. Buteau J, Roduit R, Susini S, Prentki M. Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells. Diabetologia 42: 856–864, 1999.[CrossRef][ISI][Medline]
  5. Cui MZ, Laag E, Sun L, Tan M, Zhao G, Xu X. Lysophosphatidic acid induces early growth response gene 1 expression in vascular smooth muscle cells: CRE and SRE mediate the transcription. Arterioscler Thromb Vasc Biol 26: 1029–1035, 2006.[Abstract/Free Full Text]
  6. Duan WR, Ito M, Park Y, Maizels ET, Hunzicker-Dunn M, Jameson JL. GnRH regulates early growth response protein 1 transcription through multiple promoter elements. Mol Endocrinol 16: 221–233, 2002.[Abstract/Free Full Text]
  7. Garnett KE, Chapman P, Chambers JA, Waddell ID, Boam DS. Differential gene expression between Zucker Fatty rats and Zucker Diabetic Fatty rats: a potential role for the immediate-early gene Egr-1 in regulation of beta cell proliferation. J Mol Endocrinol 35: 13–25, 2005.[Abstract/Free Full Text]
  8. Gedulin BR, Nikoulina SE, Smith PA, Gedulin G, Nielsen LL, Baron AD, Parkes DG, Young AA. Exenatide (exendin-4) improves insulin sensitivity and (beta)-cell mass in insulin-resistant obese fa/fa Zucker rats independent of glycemia and body weight. Endocrinology 146: 2069–2076, 2005.[Abstract/Free Full Text]
  9. De Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396: 474–477, 1998.[CrossRef][Medline]
  10. Guha M, O’Connell MA, Pawlinski R, Hollis A, McGovern P, Yan SF, Stern D, Mackman N. Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression. Blood 98: 1429–1439, 2001.[Abstract/Free Full Text]
  11. Hokari R, Lee H, Crawley SC, Yang SC, Gum JR Jr, Miura S, Kim YS. Vasoactive intestinal peptide upregulates MUC2 intestinal mucin via CREB/ATF1. Am J Physiol Gastrointest Liver Physiol 289: G949–G959, 2005.[Abstract/Free Full Text]
  12. Jhala US, Canettieri G, Screaton RA, Kulkarni RN, Krajewski S, Reed J, Walker J, Lin X, White M, Montminy M. cAMP promotes pancreatic beta-cell survival via CREB-mediated induction of IRS2. Genes Dev 17: 1575–1580, 2003.[Abstract/Free Full Text]
  13. Kang JH, Kim MJ, Ko SH, Jeong IK, Koh KH, Rhie DJ, Yoon SH, Hahn SJ, Kim MS, Jo YH. Upregulation of rat Ccnd1 gene by exendin-4 in pancreatic beta cell line INS-1: interaction of early growth response-1 with cis-regulatory element. Diabetologia 49: 969–979, 2006.[CrossRef][ISI][Medline]
  14. Lee HJ, Mignacca RC, Sakamoto KM. Transcriptional activation of egr-1 by granulocyte-macrophage colony-stimulating factor but not interleukin 3 requires phosphorylation of cAMP response element-binding protein (CREB) on serine 133. J Biol Chem 270: 15979–15983, 1995.[Abstract/Free Full Text]
  15. List JF, Habener JF. Glucagon-like peptide 1 agonists and the development and growth of pancreatic beta-cells. Am J Physiol Endocrinol Metab 286: E875–E881, 2004.[Abstract/Free Full Text]
  16. Lonze BE, Ginty DD. Function and regulation of CREB family transcription factors in the nervous system. Neuron 35: 605–623, 2002.[CrossRef][ISI][Medline]
  17. Mayr B, Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2: 599–609, 2001.[CrossRef][ISI][Medline]
  18. Russell DL, Doyle KM, Gonzales-Robayna I, Pipaon C, Richards JS. Egr-1 induction in rat granulosa cells by follicle-stimulating hormone and luteinizing hormone: combinatorial regulation by transcription factors cyclic adenosine 3', 5'-monophosphate regulatory element binding protein, serum response factor, sp1, and early growth response factor-1. Mol Endocrinol 17: 520–533, 2003.[Abstract/Free Full Text]
  19. Sakamoto KM, Fraser JK, Lee HJ, Lehman E, Gasson JC. Granulocyte-macrophage colony-stimulating factor and interleukin-3 signaling pathways converge on the CREB-binding site in the human egr-1 promoter. Mol Cell Biol 14: 5975–5985, 1994.[Abstract/Free Full Text]
  20. Schwachtgen JL, Houston P, Campbell C, Sukhatme V, Braddock M. Fluid shear stress activation of egr-1 transcription in cultured human endothelial and epithelial cells is mediated via the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway. J Clin Invest 101: 2540–2549, 1998.[ISI][Medline]
  21. Stoffers DA. The development of beta-cell mass: recent progress and potential role of GLP-1. Horm Metab Res 36: 811–821, 2004.[CrossRef][ISI][Medline]
  22. Susini S, Roche E, Prentki M, Schlegel W. Glucose and glucoincretin peptides synergize to induce c-fos, c-jun, junB, zif-268, and nur-77 gene expression in pancreatic beta (INS-1) cells. FASEB J 12: 1173–1182, 1998.[Abstract/Free Full Text]
  23. Tsai JC, Liu L, Guan J, Aird WC. The Egr-1 gene is induced by epidermal growth factor in ECV304 cells and primary endothelial cells. Am J Physiol Cell Physiol 279: C1414–C1424, 2000.[Abstract/Free Full Text]
  24. Xiao D, Chinnappan D, Pestell R, Albanese C, Weber HC. Bombesin regulates cyclin D1 expression through the early growth response protein Egr-1 in prostate cancer cells. Cancer Res 65: 9934–9942, 2005.[Abstract/Free Full Text]
  25. Young AA, Gedulin BR, Bhavsar S, Bodkin N, Jodka C, Hansen B, Denaro M. Glucose-lowering and insulin-sensitizing actions of exendin-4: studies in obese diabetic (ob/ob, db/db) mice, diabetic fatty Zucker rats, and diabetic rhesus monkeys (Macaca mulatta). Diabetes 48: 1026–1034, 1999.[Abstract]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/E215    most recent
00181.2006v1
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 ISI Web of Science
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 ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kang, J.-H.
Right arrow Articles by Jo, Y.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kang, J.-H.
Right arrow Articles by Jo, Y.-H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.