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Endocrinology Vol. 147, No. 8 3727-3736
Copyright © 2006 by The Endocrine Society

Role of the Exchange Protein Directly Activated by Cyclic Adenosine 5'-Monophosphate (Epac) Pathway in Regulating Proglucagon Gene Expression in Intestinal Endocrine L Cells

Shamim Lotfi, Zhihong Li, Jane Sun, Yang Zuo, Patrick P. L. Lam, Youhou Kang, Mehdi Rahimi, Diana Islam, Peixiang Wang, Herbert Y. Gaisano and Tianru Jin

Division of Cell and Molecular Biology (S.L., Z.L., J.S., Y.Z., M.R., D.I., P.W., H.Y.G., T.J.), Toronto General Research Institute, University Health Network; Institute of Medical Science (M.R., T.J.); and Departments of Medicine (P.P.L.L., Y.K., H.Y.G., T.J.), Laboratory Medicine and Pathobiology (S.L., J.S., D.I., T.J.), and Physiology (P.P.L.L., Y.K., H.Y.G., T.J.), University of Toronto, Toronto, Ontario, Canada M5G 2M1

Address all correspondence and requests for reprints to: T. Jin, Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network, 67 College Street, Toronto, Ontario, Canada M5G 2M1. E-mail tianru.jin{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although proglucagon gene expression and the synthesis of proglucagon encoded peptide hormones could be activated by protein kinase A (PKA) activators such as forskolin/3-isobutyl-1-methylxanthine (IBMX) and cholera toxin, whether the activation is entirely attributed to PKA has not been previously examined. We found that forskolin/IBMX also activate ERK1/2 phosphorylation in intestinal and pancreatic proglucagon-producing cell lines. The MEK inhibitors PD98059 and U0126 were found to repress the expression of proglucagon promoter as well as endogenous proglucagon mRNA in two intestinal proglucagon-producing cell lines and to block the stimulatory effect of forskolin/IBMX on proglucagon mRNA expression. The repressive effect of the PKA-specific inhibitors H-89 and KT-5720, however, was either not observable or much less potent. Forskolin could activate ERK1/2 phosphorylation and proglucagon gene transcription on its own, whereas forskolin plus IBMX are required to effectively activate the PKA pathway in the proglucagon-producing cells. Exchange protein directly activated by cyclic AMP 2 (Epac2, or cAMP-binding guanine nucleotide exchange factor-2) was found to be expressed in gut and pancreatic proglucagon-producing cell lines, whereas the Epac-pathway-specific cAMP analog, 8-pMeOPT-2'O-Me-cAMP, effectively stimulated ERK1/2 phosphorylation as well as proglucagon mRNA expression. We therefore suggest that cAMP at least partially regulates proglucagon gene expression via the Epac-Ras/Rap-Raf-MEK-ERK signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GENE THAT ENCODES proglucagon is expressed in pancreatic islet A cells and intestinal endocrine L cells as well as in certain endocrine neurons in the brain. A single proglucagon gene gives rise to an identical mRNA transcript and an identical prehormone but different active hormonal products in these three types of cells/tissues (1, 2, 3). Of the hormones encoded by the proglucagon gene, glucagon, glucagon-like peptide (GLP)-1, and GLP-2 have received much of the attention and been studied intensively (4, 5). These hormones play opposite or overlapping roles in blood glucose homeostasis as well as in controlling satiety (1, 2, 3, 4, 5, 6, 7).

Intensive investigations have been conducted during the past two decades on transcription factors and signaling molecules that regulate proglucagon expression (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). These investigations have revealed the function of several enhancer-like elements and a cAMP response element (CRE) within the first 300 bp of the 5' flanking sequence of the proglucagon gene (8, 9). A number of independent studies have also indicated that the expression of proglucagon promoter and endogenous proglucagon mRNA and the synthesis and secretion of glucagon and GLP-1 could be activated by forskolin/3-isobutyl-1-methylxanthine (IBMX) or by other agents that increase the intracellular levels of cAMP (10, 11, 12). These observations would suggest that proglucagon gene expression could be activated by protein kinase A (PKA). In fact, PKA signaling via the CRE-binding protein and CRE has been shown to stimulate proglucagon transcription and glucagon and GLP-1 synthesis in both primary and transformed intestinal endocrine cells as well as in rat primary pancreatic islet cultures (3, 11, 12, 13). However, deleting or mutating the CRE motif within the proglucagon promoter only partially attenuates forskolin/IBMX-mediated activation (3, 10, 12, 14), indicating that PKA may activate proglucagon expression via mechanisms other than using its CRE. We have suggested recently that PKA may cross-talk with the canonical Wnt pathway in regulating proglucagon expression (14, 15). Furthermore, although PKA is a major effector of cAMP, we have learned from many recent studies in other cell lineages that it is not the sole target of this second messenger. For example, cAMP has been shown to activate ion-gated channels that are involved in olfactory and visual signaling as well as the cAMP-binding guanine nucleotide exchange factors [cAMP-GEFs, or exchange protein directly activated by cyclic AMP (Epac)]. Recently, the role of the Epac pathway in regulating insulin expression, as well as the synthesis and secretion of insulin, has been studied by a few laboratories. However, no study has been conducted on the role of Epac in proglucagon-producing cells.

In the present study, we demonstrate that in the gut proglucagon-producing cells, PKA inhibition could not effectively block the stimulation by forskolin/IMBX treatment on the expression of proglucagon promoter as well as endogenous proglucagon mRNA; that forskolin or forskolin plus IBMX treatment stimulated ERK1/2 phosphorylation, whereas the MAPK kinase (MEK) inhibitors repressed both the basal and forskolin/IBMX-stimulated proglucagon expression. Furthermore, we found that Epac2 is expressed in gut proglucagon-producing cells, and the Epac-pathway-specific cAMP analog 8-pMeOPT-2'O-Me-cAMP activated ERK1/2 phosphorylation and proglucagon expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture medium and fetal bovine serum were purchased from Invitrogen Life Technology Inc. (Burlington, Ontario, Canada). Radioisotopes were obtained from Amersham Pharmacia Biotech (Baie d’Urfe, Quebec, Canada). Forskolin, dideoxyforskolin, IBMX, 8-Br-cAMP, 8-Br-cGMP, and PKA inhibitors H-89 and KT-5720 were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). The MEK inhibitor PD98059 was purchased from Calbiochem (EMD Biosciences, Inc., San Diego, CA), and U1206 from Promega (Madison, WI). The Epac-pathway-specific cAMP analog 8-pMeOPT-2'O-Me-cAMP was purchased from BIOLOG Life Science Institute (Bremen, Germany).

Plasmids, cell culturing, transfection, and luciferase (LUC) reporter gene analysis
Construction of the 2.4-kb proglucagon/luciferase (GLU/LUC) reporter gene plasmid has been previously described (16). The pCRE-LUC reporter gene plasmid was purchased from BD Biosciences (Mountain View, CA). The PKA-deficient pancreatic InR1-G9 and PKA-active intestinal GLUTag and STC-1 cell lines were maintained as previously described (16, 17, 18). For LUC reporter gene analysis, STC-1 and GLUTag cell lines were transfected using the electroporation method (16).

Northern blotting and RT-PCR
RNA from cultivated cell lines was extracted using the TRIzol reagent (Invitrogen Life Technology) (15). The method for Northern blotting analysis was described previously, using the rat proglucagon cDNA as the probe for detecting proglucagon mRNA expression (11, 17, 18, 19). Epac2 mRNA expression in the cultivated cell lines was examined by RT-PCR using a pair of primers as follows: forward, GCCTATTCGTGGCTCTG; reverse, CAGACCTCAGTGACAACC. These primers were designed based on the mouse Epac2 mRNA sequence (GenBank accession no. NM_019688, 36).

Western blotting analysis and confocal microscopy
For Western blot analysis, whole-cell lysate (~20 µg protein) of each sample was size-fractionated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Protran; Schleicher & Schuell, Keene, NH). The separated proteins were identified by the indicated primary antibodies, which were then detected with an ECL Western blot analysis system (Amersham Pharmacia Biotech), per the manufacturer’s instruction, with corresponding secondary antisera, including peroxidase-linked antirabbit, antimouse, or antigoat Ig (19). Primary antibodies include antiactin antibody from Sigma-Aldrich, antibodies against phosphorylated ERK1/2 (sc-7383, 1:3000) and total ERK1/2 (sc-94, 1:3000), and anti-Epac2 (sc-9383, 1:300), which were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

For confocal microscopy, each cell line plated on a glass coverslip was fixed in 2% formaldehyde for 1 h at room temperature, washed with PBS, and blocked with 5% normal goat serum and 0.1% saponin overnight at 4 C. Cells were next immunolabeled with a goat polyclonal anti-cAMP-GEFII (Epac2) (sc 9383; Santa Cruz Biotechnology) at 1:50 dilution overnight at 4 C; and for specificity, the antibody was preadsorbed with its immunizing peptide (sc-9383 P; Santa Cruz Biotechnology) at 10x concentration of primary antibody. After rinsing with 0.1% saponin and PBS, the coverslips were incubated with the fluorescent-labeled appropriate secondary antibodies for 1 h at room temperature, mounted on slides in a fading retarder (0.1% p-phenylenediamine in glycerol), and examined using a laser scanning confocal imaging system (LSM 510; Carl Zeiss, Oberkochen, Germany).

Data analysis
LUC reporter gene analysis results are expressed as mean ± SD (n ≥ 3). Statistical differences between samples were assessed using Student’s t test. Significance was assumed when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PKA is not the sole effector of cAMP in activating proglucagon promoter by cAMP
Forskolin plus IBMX has been used as the major tool in examining the effect of cAMP or PKA on proglucagon expression (3, 10, 12, 15). To determine the existence of pathways other than PKA, we used the potent PKA inhibitors H-89 and KT-5720 in our LUC reporter gene analysis. As shown in Fig. 1AGo, when the STC-1 cell line was examined, forskolin/IBMX stimulated the 2.4-kb GLU/LUC fusion gene activity approximately 8-fold. The GLU/LUC fusion gene activity remained at the basal level when the cells were pretreated with 0.1, 1.0, or 10 µM H-89. If PKA were the only target of the cAMP signal in activating proglucagon expression in this cell line, it would be expected that pretreating the cells with H-89 would prevent the stimulation by forskolin/IBMX. Interestingly, GLU/LUC fusion gene activity was further stimulated instead of repressed when the STC-1 cells were pretreated with 0.1 or 1.0 µM H-89, followed by forskolin/IBMX treatment. When the concentration of H-89 was increased to 10 µM, forskolin/IBMX-stimulated GLU/LUC activity was attenuated but was still significantly higher than that of the untreated cells (Fig. 1AGo). When the concentration of H-89 was increased to 30 µM, cells were dying and the LUC activity was approximately 10 times lower than that in the untreated cells (data not shown). Similar results were obtained for the GLUTag cell line (Fig. 1BGo). To verify whether H-89 is able to inhibit PKA in the proglucagon-producing cells, we conducted additional LUC reporter gene analysis using the PKA-responsive reporter pCRE-LUC. As expected, forskolin/IBMX stimulated pCRE-LUC activity more than 17-fold in the STC-1 cell line (Fig. 1CGo), whereas pretreating the cells with 1.0 and 10 µM H-89 generated 45 and 85% attenuation, respectively. The Epac-specific cAMP analog 8-pMeOPT-2'O-Me-cAMP, however, did not stimulate pCRE-LUC activity (Fig. 1CGo). Furthermore, forskolin/IBMX-stimulated GLU/LUC activity was not significantly attenuated by pretreating the STC-1 cell line with 10 or 30 µM KT-5720 (data not shown).


Figure 1
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  		FIG. 1. PKA inhibition cannot effectively attenuate the activation on 2.4-kb GLU/LUC by forskolin/IBMX. A and B, The 2.4-kb GLU/LUC (3 µg) was transfected into STC-1 (A) or GLUTag (B) cells. Forskolin plus IBMX (F/I, 10 µM each) or dimethylsulfoxide (as vehicle for forskolin/IBMX) was added 14 h after the transfection. After another 4 h, cells were harvested for LUC reporter gene analysis. The indicated amount of PKA inhibitor H-89 was added to the indicated samples 45 min before the addition of forskolin/IBMX. C, pCRE-LUC reporter (3 µg) was transfected into the STC-1 cell line. Forskolin plus IBMX (F/I, 10 µM each) or dimethylsulfoxide (as vehicle for forskolin/IBMX) or 8-pMeOPT-2'O-Me-cAMP (Epac) (10 mM) was added 14 h after the transfection. After growing for another 4 h, cells were harvested for LUC reporter gene analysis. The PKA inhibitor H-89 was added to cells 45 min before the addition of forskolin/IBMX or 8-pMeOPT-2'O-Me-cAMP. Relative LUC activity was calculated as the fold increase with the activity in the vehicle-treated cells defined as 1-fold (mean ± SE; n = 3). * or #, P < 0.05. ND, No difference.

 
The ERK signaling pathway is involved in cAMP-activated proglucagon gene transcription
If Epac is a component in activating proglucagon expression by cAMP, one may expect that the activation would be via the Ras/Rap-Raf-MEK-ERK signaling pathway. To initiate the investigation on this hypothesis, we assayed whether forskolin, IBMX, or forskolin plus IBMX would stimulate ERK1/2 phosphorylation. When STC-1 and GLUTag cell lines were examined, forskolin plus IBMX stimulated ERK1/2 phosphorylation within 5 min to 4 h of the experimental period. Our representative results are shown in Fig. 2AGo for the STC-1 cell line and in Fig. 2BGo for the GLUTag cell line. In addition, we found that although the phosphodiesterase (PDE) inhibitor IBMX on its own could not consistently stimulate ERK1/2 phosphorylation (data not shown), forskolin on its own could (Fig. 2Go, A and B, bottom panels). In conducting another line of research, we observed that forskolin/IBMX also stimulated ERK1/2 phosphorylation in the PKA-deficient cell line InR1-G9 (19). We present here that in this cell line, forskolin, IBMX, or forskolin plus IBMX stimulated ERK1/2 phosphorylation (Fig. 2CGo). Interestingly, the activation was not observed in the absence of serum (data not shown). Therefore, ERK activation by cAMP could occur in the absence of PKA. Figure 2DGo shows that forskolin/IBMX-stimulated ERK activation cannot be blocked by H-89 or KT-5720 pretreatment. H-89 or KT-5720 alone had no appreciable effect on ERK activation in the STC-1 cell line (data not shown). Figure 2EGo shows that although forskolin on its own stimulated pCRE activity close to 4-fold, the addition of IBMX led to more than 20-fold activation. IBMX on its own, however, has no stimulatory effect.


Figure 2
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  		FIG. 2. Forskolin/IBMX stimulates ERK activation PKA independently. A–C, The PKA-active STC-1 (A) and GLUTag (B) cell lines and the PKA-deficient InR1-G9 (C) cell line were grown in the presence of vehicle (dimethylsulfoxide, V), forskolin (10 µM, F), IBMX (10 µM, I), or forskolin plus IBMX (10 µM each, F/I) for the indicated period before the cells were harvested for the examination of ERK1/2 phosphorylation. The same membranes were stripped and followed by hybridization with anti-total ERK1/2 antibody (loading control). D, STC-1 cells were grown in the presence of vehicle (V) or forskolin plus IBMX (10 µM each) for 2 h before the cells were harvested for the examination of ERK1/2 phosphorylation. H-89 or KT-5720 was added 45 min before the addition of F/I. E, pCRE/LUC (3 µg) was transfected into the STC-1 cells. Forskolin (10 µM, F), IBMX (10 µM, I), forskolin plus IBMX (F/I, 10 µM each), or dimethylsulfoxide (as vehicle, V) were added 14 h after the transfection. After another 4 h, cells were harvested for LUC reporter analysis. E, Relative LUC activity was calculated as the fold increase with the activity in the vehicle-treated cells defined as 1-fold (mean ± SE; n = 3). *, P < 0.05.

 
To investigate whether ERK activation plays a role in stimulating proglucagon expression, we have, first of all, examined the effect of forkolin, IBMX, or forskolin plus IBMX on proglucagon mRNA expression in the two intestinal endocrine cell lines. As shown in Fig. 3Go, A and B, forskolin plus IBMX substantially stimulated endogenous proglucagon mRNA expression in the GLUTag and STC-1 cell lines. Forskolin on its own stimulated proglucagon mRNA expression nearly as potently as forskolin plus IBMX, consistent with its potent capability in stimulating ERK activation (Fig. 2Go, A and B). IBMX on its own also moderately activated proglucagon mRNA expression, although it cannot stimulate ERK activation and PKA on its own (Fig. 2EGo and data not shown). Figure 3Go, C and D, show that the PKA inhibitors H-89 and KT-5720 generated no appreciable repression on forskolin/IBMX-stimulated proglucagon mRNA expression in STC-1 and GLUTag cell lines, respectively.


Figure 3
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  		FIG. 3. PKA inhibition cannot effectively attenuate the activation on proglucagon mRNA expression by forskolin/IBMX. A and B, GLUTag (A) and STC-1 (B) cell lines were grown in the presence of vehicle (dimethylsulfoxide, V), forskolin (10 µM, F), IBMX (10 µM, I), or forskolin plus IBMX (10 µM each, F/I) for 4 h before being harvested for total RNA extraction. Ten micrograms of RNA were used for Northern blotting, using rat proglucagon cDNA as the probe. The same membranes were stripped and rehybridized with the mouse tubulin cDNA probe. C and D, STC-1 and GLUTag cells were treated with forskolin plus IBMX (10 µM each, F/I) in the presence or absence of the PKA inhibitors H-89 (10 µM) or KT-5720 (30 µM) for 4 h before being harvested for total RNA extraction and Northern blotting analysis.

 
To directly investigate the role of ERK activation, two MEK inhibitors were then used. As shown in Fig. 4Go, forskolin/IBMX stimulated GLU/LUC activity around 5- and 8.5-fold when transfected into the GLUTag (Fig. 4AGo) and STC-1 (Fig. 4BGo) cell lines, respectively. Similar activation was generated by forskolin alone but not by IBMX alone (data not shown). The MEK inhibitors PD98059 and U0126 moderately repressed the basal activity of GLU/LUC. When these two cell lines were pretreated with either one of these two inhibitors, forskolin/IBMX-stimulated GLU/LUC reporter gene activity was attenuated 40–65%. The effect of PD98059 on endogenous proglucagon mRNA expression was then examined. Figure 4CGo shows that treating GLUTag cells with either 50 or 100 µM PD98059 substantially repressed the basal proglucagon mRNA expression. Figure 4DGo shows that forskolin/IBMX-stimulated proglucagon mRNA expression also could be substantially repressed by 50–100 µM PD98059.


Figure 4
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  		FIG. 4. The MEK inhibitors repress proglucagon promoter and endogenous proglucagon mRNA expression. A and B, The 2.4-kb GLU/LUC reporter plasmid (3 µg) was transfected into GLUTag (A) or STC-1 (B) cell lines. Forskolin plus IBMX (10 µM each, F/I) was added 14 h after the transfection. After another 4 h, cells were harvested for LUC reporter gene analysis. For indicated samples, the MEK inhibitor PD98059 (100 µM) or U0126 (25 µM) was added 45 min before the addition of forskolin/IBMX. Relative LUC activity was calculated as the fold increase with the activity in the vehicle-treated cells defined as 1-fold (mean ± SE; n = 3). *, P < 0.05. C and D, GLUTag cells were grown in the presence of vehicle (dimethylsulfoxide, V), the MEK inhibitor PD98059, and/or forskolin/IBMX (10 µM each, F/I) for 4 h before being harvested for total RNA extraction and Northern blotting, using rat proglucagon cDNA as the probe. The same membranes were stripped and rehybridized with mouse tubulin cDNA probe.

 
8-pMeOPT-2'O-Me-cAMP stimulates ERK1/2 activation and proglucagon expression
We then examined Epac2 expression in the proglucagon-producing cell lines, including STC-1, GLUTag, and the PKA-deficient InR1-G9. As expected, Epac2 expression in pancreatic B cell line INS-1 was abundant when examined by immunocytochemistry (Fig. 5AGoGo). As shown in the middle panel, Epac2 was detected to be abundant in the cytosol but was also present in the plasma membrane. For specificity, the top panel shows the lack of fluorescence signal in the absence of the primary antibody, and the bottom panel shows a substantially reduced signal, particularly the cytosolic signal, when the primary antibody was first absorbed with the immunizing peptide. Like INS-1, Epac2 expression is abundant in the gut intestinal STC-1 cell line (Fig. 5BGoGo) and also in the GLUTag and PKA-deficient pancreatic InR1-G9 cell lines (data not shown). No signal was detected in the STC-1 cells in the absence of primary antibody (top panel), and the Epac2 signal was substantially reduced when the antibody was blocked by the immunizing peptide (bottom panel). Figure 5CGoGo shows Western blots of Epac2 protein (130 kDa) expression in INS-1 and in the three proglucagon-producing cell lines, with the rat brain lysate as the positive control (panel i). For specificity, the 130-kDa Epac2 signal could be completely blocked by the immunizing peptide, without affecting the actin signals (panel ii). These results taken together indicate that Epac2 is indeed expressed in intestinal proglucagon-producing cells. Furthermore, we examined Epac2 mRNA expression in INS-1 and two intestinal proglucagon-producing cell lines by RT-PCR. A single DNA fragment of anticipated size (509 bp) was obtained for each of the three cell lines (Fig. 5DGoGo), and the identity of the fragment obtained from the GLUTag cell line was confirmed to be Epac2 by DNA sequencing analysis.


Figure 5
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  		FIG. 5. Detection of Epac expression in proglucagon-producing cell lines. A and B, Confocal microscopy shows the location of Epac2 in the cytosol (indicated by arrows) and plasma membrane (indicated by arrowheads) in the pancreatic B cell line INS-1 (A) and in the intestinal proglucagon-producing cell line STC-1 (B). Top, Absence of primary antibody; middle, with primary antibody; bottom, primary antibody adsorbed with the immunizing peptide. C, Western blotting shows the expression levels of Epac2 proteins (~20 µg protein) in the indicated cell lines, in the absence (i) or presence (ii) of the immunizing peptide, which blocked the specific Epac2 signal. The same blots were probed for actin, which serves as protein loading control and whose signals were not blocked by the Epac2 immunizing peptide (ii). D, RT-PCR show the expression of Epac2 mRNA in INS-1 as well as two intestinal proglucagon-producing cell lines. –, No cDNA.

 

Figure 5
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  		FIG. 5A. Continued

 
The Epac-specific cAMP analog 8-pMeOPT-2'O-Me-cAMP was then used to verify the implication of the Epac pathway in regulating proglucagon gene expression in the intestinal endocrine cells. As shown in Fig. 6Go, A and B, 50 µM 8-pMeOPT-2'O-Me-cAMP (Epac) effectively stimulated ERK1/2 phosphorylation in both GLUTag (A) and STC-1 (B) cell lines, starting from 5 min. In the GLUTag cells, stimulated ERK1/2 phosphorylation was also observed with 10 µM 8-pMeOPT-2'O-Me-cAMP (data not shown) and 1 mM 8-Br-cAMP (cAMP, Fig. 6AGo) but not with 1 mm 8-Br-cGMP (cGMP, Fig. 6AGo). Figure 6Go, C and D, show that 10 and 50 µM 8-pMeOPT-2'O-Me-cAMP substantially stimulated proglucagon mRNA expression in the GLUTag and STC-1 cell lines, respectively.


Figure 6
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  		FIG. 6. 8-pMeOPT-2'O-Me-cAMP stimulates ERK activation and proglucagon mRNA expression. A and B, GLUTag and STC-1 cell lines were grown in the presence of 8-Br-cAMP (1 mM, cAMP), 8-Br-cGMP (1 mm, cGMP), or the Epac-pathway-specific cAMP analog 8-pMeOPT-2'O-Me-cAMP (Epac, 50 µM) for the indicated period before the cells were harvested for the examination of ERK1/2 phosphorylation. The same membranes were stripped and followed by hybridization with anti-total ERK1/2 antibody. C and D, GLUTag and STC-1 cells were grown in the presence of vehicle (dimethylsulfoxide, V), forskolin plus IBMX (10 µM each, F/I), or the indicated amount of 8-pMeOPT-2'O-Me-cAMP (Epac) for 4 h before being harvested for total RNA extraction and Northern blotting, using rat proglucagon cDNA as the probe. The same membranes were stripped and rehybridized with mouse tubulin cDNA probe.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mechanisms underlying proglucagon expression have been extensively studied during the past two decades (8, 20, 21, 22, 23, 24). The control of proglucagon expression was initially examined using the pancreatic islet cell lines, and the studies have identified four enhancer or enhancer-like elements, namely G2, G3, G4, and G5, and an islet-specific promoter element G1 (8, 9, 22, 25, 26, 27). After the recognition of the biological functions of GLP-1, extensive examinations have been conducted using the gut endocrine L cell lines (10, 28) and the primary cell cultures (3, 10, 11, 12, 14, 29, 30). Because the activation by cAMP was previous attributed to PKA, the main focus was the CRE motif, located at –291 to –298 bp, upstream of the G3 enhancer element (22). However, a number of studies have shown that deleting or mutating the CRE motif generated only a moderate attenuation on cAMP-stimulated proglucagon promoter activity (10, 11, 12, 14), although the interaction between CRE-binding protein and CRE has been demonstrated (31). In addition, the CRE may mediate the activation via other transcription factors, such as activated transcription factor (ATF) (32). Furthermore, Furstenau et al. (27) have identified an additional CRE within the G2 element, and our group has demonstrated the role of cross-talk between PKA and the canonical Wnt pathways in proglucagon expression, using a T-cell factor (TCF)-binding site within the G2 element (14, 15). More recently, in examining the role of protein hydrolysates (peptones) in regulating proglucagon expression, Gevrey et al. (33) identified an adjacent putative CRE site (CRE-like1) upstream of the canonical CRE. These observations clearly indicate that multiple potential cAMP-responsive cis-elements and signaling pathways are involved in mediating the effect of the second messenger cAMP in regulating proglucagon gene expression.

Although cAMP has been initially shown to mediate its intracellular events through PKA, more recent observations have revealed additional mechanisms. For example, cAMP was shown to activate ion-gated channels (34, 35, 36). In addition, the identification of Epac molecules, as novel cAMP-binding proteins, has opened a new research direction for understanding the function of cAMP (35, 36, 37). The role of Epac has been extensively examined in other endocrine cell lineages. For example, Bremier et al. (38) have assessed the role of cAMP-Epac-Rap1 pathway in TSH-cAMP-mediated effects in thyroid cells. Gonzalez-Robayna et al. (39) have demonstrated that FSH stimulates phosphorylation and activation of PKB/Akt and serum and glucocorticoid-induced kinase (Sgk) in a PKA-independent manner. Using a series of kinase inhibitors in their studies, Gonzalez-Robayna et al. (39) came to the conclusion that the activation involves the cAMP-Epac-Rap/Ras-Raf-MEK pathway. Via binding to a Gs-coupled receptor on pancreatic B cells, GLP-1 is able to potentiate glucose-induced insulin secretion, insulin gene expression, and B cell proliferation. These stimulatory effects have been attributed to the second messenger cAMP. To investigate the existence and the nature of PKA-independent pathways that may mediate these functions, Leech et al. (40) examined and demonstrated the expression of both Epac1 and Epac2 in the pancreatic B cells. They also reported that the Epac pathway was activated by cAMP in the pancreatic B cells and proposed that the Ras/MAPK pathway plays an important role in a PKA-independent, GLP-1-mediated pathway in regulating pancreatic B cell growth and gene expression (40). However, no examination has been conducted on the role of this pathway in the proglucagon-producing endocrine cells.

We found that PKA inhibition does not effectively repress forskolin/IBMX-stimulated proglucagon promoter and endogenous proglucagon mRNA expression, that forskolin or forskolin plus IBMX stimulated ERK activation in both PKA-active and -deficient cell lines (19), and that MEK inhibitors repressed proglucagon promoter and endogenous proglucagon mRNA expression. These observations collectively suggest that cAMP could stimulate proglucagon expression via ERK activation in a PKA-independent manner. Using 8-pMeOPT-2'O-Me-cAMP, we then clearly demonstrated the capability of this Epac-specific cAMP analog in stimulating ERK activation and proglucagon expression and therefore suggest that Epac-Ras/Rap-Raf-MEK-ERK is an alternative pathway in activating proglucagon expression by cAMP.

Synthetic cAMP analogs are not as efficient in stimulating cAMP pathways as endogenous cAMP in the long term, because of their half-life, potency, and membrane permeability. For most of the studies, forskolin plus IBMX has been used. However, to differentiate PKA and Epac pathways, we had to use the Epac-specific cAMP analog. It appears that maximum PKA activation would require both forskolin and IBMX, although forskolin could stimulate ERK activation and proglucagon expression on its own, nearly as potently as forskolin plus IBMX. This observation is of interest. In the test tube, the binding affinity of Epac to cAMP is lower than that of PKA (41, 42). Forskolin stimulates adenocyclase for producing endogenous cAMP, whereas IBMX is an inhibitor of PDE for stabilizing cAMP. One may expect that to use both of them would achieve high levels of intracellular cAMP to activate both PKA and Epac. A question is why Epac, which possesses lower affinity to cAMP in the test tube, could be activated by forskolin alone (indicated by ERK activation), although maximum PKA activation could not be reached by forskolin alone. As suggested by Bos (43), Epac may have higher sensitivity for cAMP compared with PKA in intact cells. This could be because of the differences in cellular compartmentation of PKA and Epac and the presence of additional accessory components. Finally, although IBMX on its own cannot stimulate GLU/LUC activity and ERK activation, it is able to activate endogenous proglucagon mRNA expression, especially in the STC-1 cell line (Fig. 3AGo). Whether this indicates the existence of a yet to be identified mechanism needs to be investigated.

We suggest that Epac mediates the effect of cAMP in regulating proglucagon expression through the Ras/Rap-Raf-MEK-ERK pathway. It is necessary to examine in detail the involvement of each of the components. We have recently reported that cotransfecting a dominant-negative Ras into the proglucagon-producing cells generated no substantial repressive effect on proglucagon promoter (19). Whether a Rap molecule or other members of the Ras-like protein family are involved in regulating proglucagon expression by cAMP deserves more examination. Furthermore, it is also desirable to identify the involvement of cis-elements and transcription factors that carry the ERK signal to proglucagon gene transcription. In a previous study, we reported that H-89 (0.1 and 1.0 µM) could partially repress forskolin/IBMX-stimulated –476 GLU/GLU activity (19), in contrast with no repression at all at these two concentrations for the 2.4-kb GLU/LUC presented in this study (Fig. 1Go). One possible explanation would be the existence of a yet to be defined CRE between the –2.4-kb to –476-bp region. Finally, Epac-mediated insulin secretion has recently attracted our attention (44, 45, 46, 47, 48). 8-pMeOPT-2'O-Me-cAMP was demonstrated to stimulate Ca-induced Ca release and exocytosis in pancreatic B cells (45, 46, 47, 48). The secretion of GLP-1 from intestinal endocrine L cells is also evidently mediated by cAMP-elevating chemicals (11). Whether the Epac pathway is also involved in mediating the cAMP messenger in regulating GLP-1 secretion, and the participation of other molecules for the formation of the secretory granules, deserves a detailed investigation.


   Footnotes
 
This work was supported by the operating grants from the Canadian Institute of Health Research (CIHR 68991-G to T.J. and MOP-64465 to H.Y.G.) and the Canadian Diabetes Association (CDA, 1198 to T.J.). J.S. and D.I. are recipients of Banting and Best Diabetes Center (BBDC) Novartis Graduate Award, P.P.L.L. is a recipient of a BBDC Tamarack Graduate Studentship Award, and S.L. is supported by a University of Toronto Open Scholarship.

Author Disclosure Summary: All authors have nothing to declare

First Published Online April 27, 2006

Abbreviations: CRE, cAMP response element; Epac, exchange protein directly activated by cAMP; GLP, glucagon-like peptide; IBMX, 3-isobutyl-1-methylxanthine; LUC, luciferase; MEK, MAPK kinase; PKA, protein kinase A.

Received January 17, 2006.

Accepted for publication April 18, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mojsov S, Heinrich G, Wilson IB, Ravazzola M, Orci L, Habener JF 1986 Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing. J Biol Chem 261:11880–11889[Abstract/Free Full Text]
  2. Drucker DJ, Asa S 1988 Glucagon gene expression in vertebrate brain. J Biol Chem 263:13475–13478[Abstract/Free Full Text]
  3. Drucker DJ, Brubaker PL 1989 Proglucagon gene expression is regulated by a cyclic AMP-dependent pathway in rat intestine. Proc Natl Acad Sci USA 86:3953–3957[Abstract/Free Full Text]
  4. Drucker DJ 2003 Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis. Mol Endocrinol 17:161–171[Abstract/Free Full Text]
  5. Kieffer TJ 2004 Gastro-intestinal hormones GIP and GLP-1. Ann Endocrinol (Paris) 65:13–21[Medline]
  6. Drucker DJ, Erlich P, Asa SL, Brubaker PL 1996 Induction of intestinal epithelial proliferation by glucagon-like peptide 2. Proc Natl Acad Sci USA 93:7911–7916[Abstract/Free Full Text]
  7. Brubaker PL, Drucker DJ 2004 Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system. Endocrinology 145:2653–2659[Abstract/Free Full Text]
  8. Philippe J, Drucker DJ, Habener JF 1987 Glucagon gene transcription in an islet cell line is regulated via a protein kinase C-activated pathway. J Biol Chem 262:1823–1828[Abstract/Free Full Text]
  9. Herzig S, Fuzesi L, Knepel W 2000 Heterodimeric Pbx-Prep1 homeodomain protein binding to the glucagon gene restricting transcription in a cell type-dependent manner. J Biol Chem 275:27989–27999[Abstract/Free Full Text]
  10. Gajic D, Drucker DJ 1993 Multiple cis-acting domains mediate basal and adenosine 3',5'-monophosphate-dependent glucagon gene transcription in a mouse neuroendocrine cell line. Endocrinology 132:1055–1062[Abstract]
  11. Drucker DJ, Jin T, Asa SL, Young TA, Brubaker PL 1994 Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line. Mol Endocrinol 8:1646–1655[Abstract]
  12. Lu F, Jin T, Drucker DJ 1996 Proglucagon gene expression is induced by gastrin-releasing peptide in a mouse enteroendocrine cell line. Endocrinology 137:3710–3716[Abstract]
  13. Drucker DJ, Campos R, Reynolds R, Stobie K, Brubaker PL 1991 The rat glucagon gene is regulated by a protein kinase A-dependent pathway in pancreatic islet cells. Endocrinology 128:394–400[Abstract]
  14. Ni Z, Anini Y, Fang X, Mills G, Brubaker PL, Jin T 2003 Transcriptional activation of the proglucagon gene by lithium and ß-catenin in intestinal endocrine L cells. J Biol Chem 278:1380–1387[Abstract/Free Full Text]
  15. Yi F, Brubaker PL, Jin T 2005 TCF-4 mediates cell type-specific regulation of proglucagon gene expression by ß-catenin and glycogen synthase kinase-3ß. J Biol Chem 280:1457–1464[Abstract/Free Full Text]
  16. Jin T, Drucker DJ 1995 The proglucagon gene upstream enhancer contains positive and negative domains important for tissue-specific proglucagon gene transcription. Mol Endocrinol 9:1306–1320[Abstract]
  17. Jin T, Drucker DJ 1996 Activation of proglucagon gene transcription through a novel promoter element by the caudal-related homeodomain protein cdx-2/3. Mol Cell Biol 16:19–28[Abstract]
  18. De Leon DD, Deng S, Madani R, Ahima RS, Drucker DJ, Stoffers DA 2003 Role of endogenous glucagon-like peptide-1 in islet regeneration after partial pancreatectomy. Diabetes 52:365–371[Abstract/Free Full Text]
  19. Chen L, Wang P, Andrade CF, Zhao IY, Dube PE, Brubaker PL, Liu M, Jin T 2005 PKA independent and cell type specific activation of the expression of caudal homeobox gene Cdx-2 by cyclic AMP. FEBS J 272:2746–2759[CrossRef][Medline]
  20. Lee YC, Asa SL, Drucker DJ 1992 Glucagon gene 5'-flanking sequences direct expression of simian virus 40 large T antigen to the intestine, producing carcinoma of the large bowel in transgenic mice. J Biol Chem 267:10705–10708[Abstract/Free Full Text]
  21. Nian M, Gu J, Irwin DM, Drucker DJ 2002 Human glucagon gene promoter sequences regulating tissue-specific versus nutrient-regulated gene expression. Am J Physiol Regul Integr Comp Physiol 282:R173–R183
  22. Philippe J, Drucker DJ, Knepel W, Jepeal L, Misulovin Z, Habener JF 1988 {alpha}-Cell-specific expression of the glucagon gene is conferred to the glucagon promoter element by the interactions of DNA-binding proteins. Mol Cell Biol 8:4877–4888[Abstract/Free Full Text]
  23. Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, Zhang C, Laurent V, Lindberg I, Ugleholdt R, Holst JJ, Steiner DF 2002 Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects. Proc Natl Acad Sci USA 99:10293–10298[Abstract/Free Full Text]
  24. Efrat S, Teitelman G, Anwar M, Ruggiero D, Hanahan D 1988 Glucagon gene regulatory region directs oncoprotein expression to neurons and pancreatic {alpha} cells. Neuron 1:605–613[CrossRef][Medline]
  25. Drucker DJ, Philippe J, Jepeal L, Habener JF 1987 Glucagon gene 5'-flanking sequences promote islet cell-specific gene transcription. J Biol Chem 262:15659–15665[Abstract/Free Full Text]
  26. Drucker DJ, Philippe J, Mojsov S 1988 Proglucagon gene expression and posttranslational processing in a hamster islet cell line. Endocrinology 123:1861–1867[Abstract]
  27. Furstenau U, Schwaninger M, Blume R, Jendrusch EM, Knepel W 1999 Characterization of a novel calcium response element in the glucagon gene. J Biol Chem 274:5851–5860[Abstract/Free Full Text]
  28. Gomes BP, Drucker DB, Lilley JD 1994 Associations of specific bacteria with some endodontic signs and symptoms. Int Endod J 27:291–298[Medline]
  29. Zhao Y, Liu T, Zhang N, Yi F, Wang Q, Fantus IG, Jin T 2005 Role of Cdx-2 in insulin and proglucagon gene expression: a study using the RIN-1056A cell line with an inducible gene expression system. J Endocrinol 186:179–192[Abstract/Free Full Text]
  30. Knepel W, Chafitz J, Habener JF 1990 Transcriptional activation of the rat glucagon gene by the cyclic AMP-responsive element in pancreatic islet cells. Mol Cell Biol 10:6799–6804[Abstract/Free Full Text]
  31. Diedrich T, Knepel W 1995 Interaction of the transcription factor CREB with pancreatic islet cell-specific enhancer elements. Biol Chem Hoppe Seyler 376:39–44[Medline]
  32. Wang J, Cao Y, Steiner DF 2003 Regulation of proglucagon transcription by activated transcription factor (ATF) 3 and a novel isoform, ATF3b, through the cAMP-response element/ATF site of the proglucagon gene promoter. J Biol Chem 278:32899–32904[Abstract/Free Full Text]
  33. Gevrey JC, Malapel M, Philippe J, Mithieux G, Chayvialle JA, Abello J, Cordier-Bussat M 2004 Protein hydrolysates stimulate proglucagon gene transcription in intestinal endocrine cells via two elements related to cyclic AMP response element. Diabetologia 47:926–936[CrossRef][Medline]
  34. Schwede F, Maronde E, Genieser H, Jastorff B 2000 Cyclic nucleotide analogs as biochemical tools and prospective drugs. Pharmacol Ther 87:199–226[CrossRef][Medline]
  35. Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM 1998 A family of cAMP-binding proteins that directly activate Rap1. Science 282:2275–2279[Abstract/Free Full Text]
  36. de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL 1998 Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396:474–477[CrossRef][Medline]
  37. Richards JS 2001 New signaling pathways for hormones and cyclic adenosine 3',5'-monophosphate action in endocrine cells. Mol Endocrinol 15:209–218[Abstract/Free Full Text]
  38. Dremier S, Vandeput F, Zwartkruis FJ, Bos JL, Dumont JE, Maenhaut C 2000 Activation of the small G protein Rap1 in dog thyroid cells by both cAMP-dependent and -independent pathways. Biochem Biophys Res Commun 267:7–11[CrossRef][Medline]
  39. Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL, Richards JS 2000 Follicle-stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-induced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells. Mol Endocrinol 14:1283–1300[Abstract/Free Full Text]
  40. Leech CA, Holz GG, Chepurny O, Habener JF 2000 Expression of cAMP-regulated guanine nucleotide exchange factors in pancreatic ß-cells. Biochem Biophys Res Commun 278:44–47[CrossRef][Medline]
  41. Rehmann H, Prakash B, Wolf E, Rueppel A, De Rooij J, Bos JL, Wittinghofer 2003 A Structure and regulation of the cAMP-binding domains of Epac2. Nat Struct Biol 10:26–32[CrossRef][Medline]
  42. de Rooij J, Rehmann H, van Triest M, Cool RH, Wittinghofer A, Bos JL 2000 Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs. J Biol Chem 275:20829–20836[Abstract/Free Full Text]
  43. Bos JL 2003 Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol 4:733–738[CrossRef][Medline]
  44. Kashima Y, Miki T, Shibasaki T, Ozaki N, Miyazaki M, Yano H, Seino S 2001 Critical role of cAMP-GEFII–Rim2 complex in incretin-potentiated insulin secretion. J Biol Chem 276:46046–46053[Abstract/Free Full Text]
  45. Holz GG 2004 Epac: a new cAMP-binding protein in support of glucagon-like peptide-1 receptor-mediated signal transduction in the pancreatic ß-cell. Diabetes 53:5–13[Abstract/Free Full Text]
  46. Holz GG 2004 New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic ß-cells. Horm Metab Res 36:787–794[CrossRef][Medline]
  47. Kang G, Chepurny OG, Rindler MJ, Collis L, Chepurny Z, Li WH, Harbeck M, Roe MW, Holz GG 2005 A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic ß cells. J Physiol. 566:173–188
  48. Kang G, Joseph JW, Chepurny OG, Monaco M, Wheeler MB, Bos JL, Schwede F, Genieser HG, Holz GG 2003 Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exocytosis in pancreatic ß-cells. J Biol Chem 278:8279–8285[Abstract/Free Full Text]



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