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Endocrinology Vol. 142, No. 3 1179-1187
Copyright © 2001 by The Endocrine Society

ARTICLES

Insulinotropic Hormone Glucagon-Like Peptide 1 (GLP-1) Activation of Insulin Gene Promoter Inhibited by p38 Mitogen-Activated Protein Kinase1

Daniel M. Kemp and Joel F. Habener2

Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts

Address all correspondence and requests for reprints to: Joel F. Habener, M.D., Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street–WEL320, Boston, Massachusetts 02114. E-mail: jhabener{at}partners.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The insulin gene promoter contains many transcriptional response elements that predispose the gene to a wide range of regulatory signals. Glucagon-like peptide 1 (GLP-1) stimulates insulin gene transcription by intracellular second messenger cascades leading to direct transcription factor activation or to the up-regulation of insulin promoter specific transcription factors. In these studies, we have identified a novel regulatory signaling mechanism acting on the rat insulin 1 promoter (rINS1) in the INS-1 {beta}-cell line. In the presence of stimulatory concentrations of GLP-1 (0.1–100 nM) on rINS1 activity, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) using SB 203580 resulted in a marked increase in promoter activity (maximum 3-fold) over GLP-1 alone, as determined by rINS1 promoter-luciferase reporter gene expression. This effect was revealed to be mediated via the cAMP response element (CRE) of rINS1, because site directed mutagenesis of the CRE motif in rINS1 abolished the increased response to SB 203580. Furthermore, inhibition of p38 MAPK uncovered a similar, more pronounced, response in the expression of a generic CRE promoter driven reporter gene. Time course dose-response studies indicate that the p38 MAPK induced inhibitory response may involve expression of immediate early genes (IEGs); maximum repression of rINS1 activity occurred after 4 h of treatment, comparable with regulatory responses by IEGs. In conclusion, these results demonstrate a novel signaling mechanism whereby p38 MAPK represses rINS1 promoter activity in response to GLP-1, suggesting the involvement of a robust regulatory control by p38 MAPK in insulin gene expression. The relevance of this mechanism may be most apparent during periods of cellular stress in which p38 MAPK activity is stimulated. In this regard, reduced insulin expression levels caused by chronic hyperglycemia (glucotoxicity) and/or hyperlipidemia (lipotoxicity) may be a direct consequence of this mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCAGON-LIKE PEPTIDE 1 (GLP-1) is secreted from the enteroendocrine L-cells in response to nutrient intake and acts on the pancreatic {beta}-cells to augment glucose-induced insulin secretion (1, 2). Apart from its role as a potent insulin secretagogue, GLP-1 also induces multiple intracellular mechanisms intrinsic to {beta}-cell function. Up-regulation of insulin gene expression is central to its function in these cells and may be achieved by a variety of mechanisms that influence transcriptional activity through a number of different cis-acting promoter sequence elements (3, 4). The {beta}-cell-specific transcription factor IDX-1 (which binds A-box elements in the insulin promoter) is up-regulated by GLP-1 coincident with increased insulin expression (5, 6, 7). GLP-1 also stimulates insulin promoter activity through its cAMP response element(s) (CREs) by multiple, incompletely understood signaling pathways (8, 9). Other transcription factors (either stimulators, inhibitors, or synergists) that regulate the activity of the insulin gene promoter include the helix-loop-helix proteins E47/Pan1, E12, and BETA2/NeuroD1, the bZIP proteins C/EBP{beta}, CREB, ATF-2, and the winged helix-like protein NF-Y (10, 11, 12, 13, 14). The complexity of the transcriptional machinery acting on the insulin gene promoter creates difficulty in dissecting specific GLP-1-induced effects on the promoter. Furthermore, other, as yet unidentified, transcriptional regulators may be involved in GLP-1-induced insulin gene expression. In addition to its effects on insulin gene expression, GLP-1 induces other distinct responses in {beta}-cells, which include up-regulation of several immediate early genes such as c-Jun, c-Fos, and JunB, proposed to be important for {beta}-cell adaptation to hyperglycemia (15).

Further challenges in characterizing GLP-1-mediated responses in {beta}-cells arise from the ability of the GLP-1 receptor to activate multiple signaling pathways. Early studies on the GLP-1 receptor uncovered a robust stimulation of cAMP levels, mediated by the activation of the G protein Gs and adenylyl cyclase (9, 16). Subsequent studies determined activation of several other second messengers including mitogen-activated protein kinase (17, 18), phospholipase C (19), Ca2+ mobilization (20, 21, 22) and phosphatidylinositol 3-kinase (6). The ability of the GLP-1 receptor to regulate such a diverse response suggests a prominent role of the receptor in intracellular cross-talk. Promiscuous G protein coupling of the GLP-1 receptor has recently been demonstrated (17), and this phenomenon has long been ascribed as the reason for the induction of multiple pathways by this receptor and other receptor family members including the calcitonin and PACAP receptors (19, 23, 24). Thus, the reported pleiotropic effects of GLP-1 receptor signaling prompts further investigations to characterize the functional responses of this receptor. This need for further investigation is further emphasized by the recent findings that secreted insulin feedback on {beta}-cells is strongly augmented by GLP-1 by its secretogogic activity (25, 26, 27, 28).

Recently, GLP-1-mediated activation of p38 MAP kinase (p38 MAPK) was identified in CHO cells and also in RIN-1046–38 insulinoma cells (17). p38 MAPK is characteristically induced by activation of cellular stress-mediated signaling pathways that modify inflammation, cell growth, cell differentiation, the cell cycle, and apoptosis (reviewed in Ref. 29). Substrates for p38 MAPK include several transcription factors including ATF-2 (30) and CHOP (31, 32), both of which regulate gene expression mediated by CREs in the promoters of target genes. Furthermore MAPKAP-K2 (a substrate for p38 MAPK), also activated by p38 MAPK, activates CREB, an important transcriptional activator of CRE-regulated gene expression (33, 34). These observations suggest a role for p38 MAPK in the regulation of genes under the transcriptional control of CREs, and moreover, the potential involvement of p38 MAPK in the regulation of insulin gene transcription by GLP-1. Notably, the expression of the insulin gene was recently shown to be regulated by p38 MAPK activity in response to glucose and insulin in human islets and MIN6 cells (27, 35, 36), further supporting the hypothesis that p38 MAPK is an important mediator of insulin gene transcription.

Here we describe the identification of a novel signaling mechanism stimulated by GLP-1 that involves the activation of p38 MAPK leading to a potent repression of rat insulin 1 promoter (rINS1) activity via a specific targeting of the CRE sequence in the promoter of the insulin gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All cell culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY) except FBS (Omega Scientific, Tarzana, CA). GLP-1 was obtained from BioNebraska Inc. (Lincoln, NE). Spruce SB 203580, H-89, forskolin, and adenosine-3',5'-cyclic monophosphorothioate, RP-isomer (Rp-cAMPS) were purchased from Calbiochem (San Diego, CA). Wortmannin was obtained from Sigma (St. Louis, MO). SB 203580, H-89, wortmannin, and forskolin were dissolved in DMSO-vehicle.

Cell culture
INS-1 cells (passage numbers 99–110) were cultured in RPMI 1640 containing 10 mmol/liter HEPES, 11.1 mmol/liter glucose, 10% FBS, 100 µU/ml penicillin G, 100 µg/ml streptomycin, 1 mmol/liter sodium pyruvate, and 50 µmol/l 2-mercaptoethanol (37). Cells were maintained at 37 C in a humidified incubator gassed with 5% CO2. Cell cultures were passaged by trypsinization and subcultured every 5 days.

Plasmid DNA constructs
The rat insulin promoter sequence, rINS1, was provided by Dr. M. German (University of California at San Francisco Medical School, San Francisco, CA). It was fused to the coding sequence of luciferase in the pxp2-basic vector to generate -410rINS1-LUC. The construct containing deletions (8) of the rINS1 CRE were prepared by site-directed mutagenesis (8). The pCRE-LUC expression plasmid was obtained from Stratagene (La Jolla, CA).

Transfection of INS-1 cells
Adherent INS-1 cells grown to 80–90% confluence in Falcon 100-mm tissue culture dishes were transfected using Lipofectamine 2000 (Life Technologies, Inc.). Cells were rinsed twice in serum-free culture medium before the addition of 1.0 ml transfection cocktail containing 1.0 µg plasmid DNA. Cells were incubated in this mixture for 4 h. Cells were then trypsinized and transferred in culture medium to 24-well plates (Costar Corning, Inc., Acton, MA) at 500 µl cell suspension per well and incubated for 48 h.

Luciferase assays
Test substances were dissolved in RPMI culture medium and added to 24-well plates at a final volume of 300 µl/well. Cells were exposed to test substances for 4 h unless stated otherwise at 37 C in a humidified incubator. Inhibitory test substances were added 30 min before addition of GLP-1. After 4 h, cells were lysed, and measurements of luciferase activity were performed using a luciferase assay kit (Promega Corp., Madison, WI) in conjunction with a dual injection port luminometer (Wallac, Inc., Gaithersburg, MD) with automated application of ATP and luciferin assay substrates. All experiments were carried out in triplicate.

Immunoblotting
Immunoblotting for p38 MAPK and dual phospho-p38 MAPK was carried out in accordance with the manufacturer’s instructions with modifications (Cell Signaling Technology, Beverly, MA). After treatment, cells were washed with PBS, and lysed in 70 µl lysis buffer (25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 1% Triton X-100, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM {beta}-glycerophosphate, 10% glycerol and 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid). Cell lysates were then collected into Eppendorf tubes, vortexed and frozen over night. After centrifugation at 14,000 x g for 5 min., the resultant supernatant was collected, followed by addition of SDS-PAGE sample buffer containing 50 mM Tris-Cl, 2% SDS, 10% glycerol, 5% {beta}-mercaptoethanol, and bromophenol blue (pH 6.8). Samples were boiled for 2 min. and centrifuged at 14,000 x g for 2 min, then analyzed on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 10% nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBS/T) for 1 h at 22 C. The blots were then treated with the primary antibody in TBS/T containing 5% BSA at 4 C overnight. After washing, immunolabeling was detected by LumiGLO chemiluminescent reagent according to the manufacturer’s instructions. Membranes were then stripped by incubation for 45 min at 65 C in a solution containing 10 mM Tris (pH 6.7), 100 mM {beta}-mercaptoethanol, and 2% SDS. After washing, efficacy of stripping was determined by reexposure of the membranes to LumiGLO reagent. Thereafter, blots were reblocked and immunolabeled as described above.

Statistical analysis
Concentration-response curves were analyzed by nonlinear regression with variable slope, using GraphPad Software, Inc. Prism. Other graphs were generated using Microsoft Corp. Excel. Data represents mean ± SEM. Statistical significance was determined using the Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GLP-1 induces CRE-luc promoter activity via cAMP/PKA signaling
To determine the specific signaling pathway by which GLP-1 mediates gene transcription via CREs, we used a reporter gene plasmid construct (pCRE-LUC) containing the luciferase gene under the transcriptional control of a consensus CRE rich promoter (4x CRE). Specific inhibitors of second messenger signaling pathways were then used to identify the pathway of the transduced signal determined by their effects on reporter gene expression induced by GLP-1. pCRE-LUC was transiently transfected into the pancreatic {beta}-cell line INS-1. Treatment of the INS-1 cells with increasing concentrations of GLP-1 resulted in a dose-dependent increase in luciferase gene expression with an EC50 value of 0.43 nM (Fig. 1AGo) corresponding with reported observations of cAMP accumulation in INS-1 cells in response to GLP-1 (16). The maximal response was 7-fold above basal level, which corresponded to only 26% of that attributed to forskolin (10 µM) treatment in the same experiment (Fig. 1BGo). To determine whether CRE-directed luciferase expression was mediated by cAMP/PKA, cells were treated with the specific cAMP pathway inhibitors, Rp-cAMPS (50 µM), and H-89 (10 µM). Both treatments blocked GLP-1 stimulated luciferase expression whereas the PI3-kinase specific inhibitor wortmannin (50 nM) had no effect on CRE-directed luciferase expression (Fig. 1CGo), suggesting that GLP-1 induction of the CRE luciferase reporter gene is mediated through cAMP and PKA signal transduction pathways.



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  		Figure 1. Glucagon-like peptide-1 activates a cAMP response element transcriptional reporter in INS-1 cells. INS-1 cells expressing the pCRE-LUC reporter plasmid were treated for 4 h at 37 C in a humidified atmosphere. A, Concentration-response curve of reporter gene expression in response to increasing concentrations of GLP-1. B, Stimulation of luciferase gene expression with GLP-1 (100 nM) or forskolin (10 µM). C, Luciferase expression response to 100 nM GLP-1 in the presence of H-89 (10 µM), Rp-CAMPS (50 µM) or wortmannin (50 nM). Cells were incubated for 30 min with respective inhibitor compounds before GLP-1 treatment. Expression levels represent fold increase over basal level (control). Data points are mean values (± SEM) of at least three independent experiments carried out in triplicate.

 
The rat insulin I promoter is activated by a PKA-independent pathway in response to GLP-1
A similar approach was taken to determine whether rINS1 was regulated by cAMP/PKA signaling. In cells transiently expressing the luciferase reporter gene under the transcriptional control of the rat insulin 1 promoter (rINS1-LUC), luciferase expression was again induced in a dose-dependent manner in response to GLP-1 treatment (EC50 value = 0.39 nM) and reached a maximum of 2-fold above the basal level (Fig. 2AGo). However, insulin promoter activity was not inhibited by H-89 suggesting that the activity was mediated by an alternate signaling pathway to that of the CRE promoter. Treatment with wortmannin appeared to partially attenuate the insulin promoter response to GLP-1, but was not statistically significant (Fig. 2BGo), suggesting that PI3-K may mediate GLP-1 stimulated rINS1 activity but that this signaling pathway is not sufficient for elicitation of the full response.



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  		Figure 2. Glucagon-like peptide-1 activates the rat insulin-1 gene promoter independently of cAMP signaling. A, Concentration-response curve of -410rINS1-LUC gene expression treated with increasing concentrations of GLP-1. B, Effects of treatment with 100 nM GLP-1 in the presence of H-89 (10 µM) or wortmannin (50 nM) on -410rINS1-LUC reporter plasmid expression. Cells were treated for 4 h at 37 C in a humidified atmosphere and incubated for 30 min with respective inhibitor compounds before GLP-1 treatment. Expression levels represent fold increase over basal level (control). Data points are mean values (± SEM) of at least three independent experiments carried out in triplicate.

 
GLP-1 activates p38 MAPK
Because ATF-2 and CREB transcription factors are phosphorylated by p38 MAPK and MAPKAP-K2 (a substrate for p38 MAPK), respectively, we investigated whether the induction of rINS1 by GLP-1 is mediated by p38 MAPK. To determine whether p38 MAPK was regulated by GLP-1 we measured levels of phosphorylated p38 MAPK by immunostaining cell lysates with a phospho-specific antibody. Treatment for 15 min with 10 nM GLP-1 increased the level of phosphorylated p38 MAPK by 2- to 3-fold (Fig. 3Go, A and B). In the presence of the p38 MAPK-specific inhibitor SB 203580, the response was attenuated. Treatment with 100 nM GLP-1 increased p38 MAPK phosphorylation to a lesser extent than 10 nM GLP-1. The immunoblot was stripped of antibody and reprobed using a p38 antibody to confirm equal protein levels in each lane. Anisomycin treated INS-1 cells (30 min) served as a positive control for p38 MAPK activity.



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  		Figure 3. Immunoblot analysis of p38 MAPK phosphorylation in response to GLP-1. A, Cells were treated for 15 min with GLP-1 following 30 min preincubation with SB 203580 (20 µM). Samples were electrophoresed and immunoblotted with the antibody specific for activated p38 MAPK (phospho-Thr 180/Tyr 182) then stripped and reprobed with antibody specific for total p38 MAPK. Immunoblots were quantitated by scanning densitometry, and the results were expressed as the fold stimulation over the control value. This data are representative of three independent experiments. B, Cells were treated for 30 min with Anisomycin (10 µg/ml) in the presence or absence of SB 203580 (20 µM). For comparison purposes the data in the left most lane in (A) and the right most lane in (B) are identical (duplicated). All data are derived from the same representative experiment.

 
p38 MAPK represses GLP-1 induced gene expression
INS-1 cells expressing the rINS1-LUC reporter plasmid were treated with increasing concentrations of GLP-1 in the presence or absence of the p38 MAPK specific inhibitor SB 203580 (20 µM). Remarkably, the dose response to GLP-1 was significantly increased in the presence of the inhibitor compared with that in its absence (maximum of 3-fold) (Fig. 4AGo). This finding indicated that p38 MAPK activity regulates rINS1 activity by a potent inhibitory mechanism in response to GLP-1. Notably, in the absence of GLP-1, there was no significant effect of SB 203580 treatment on basal activity of the promoter, suggesting that the p38 MAPK-mediated repression of rINS1 activity was a direct result of GLP-1 signaling. Thus, in INS-1 cells, GLP-1 appears to induce a bifurcating signal that both activates and represses rINS1 promoter activity.



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  		Figure 4. Enhanced stimulation of cAMP response element and rat insulin-1 gene promoter activities by inhibition of p38 kinase activity. Concentration-response curves of -410rINS1-LUC (A), or pCRE-LUC (B) gene expression in response to increasing concentrations of GLP-1 in the presence or absence of 20 µM SB 203580. Cells were incubated with SB 203580 (open circles) or vehicle (closed circles) for 30 min before GLP-1 treatment. Cells were treated for 4 h at 37 C in a humidified atmosphere. Expression levels represent fold increase over basal level (control). Data points are mean values (± SEM) of at least three independent experiments carried out in triplicate.

 
To determine whether p38 MAPK activity affects GLP-1 induced expression of the CRE promoter, we repeated the above experiment in cells expressing the pCRE-LUC reporter gene. Again, treatment with SB 203580 resulted in a marked increase in luciferase expression that reached 5-fold that in the absence of the inhibitor (Fig. 4BGo). In the experiments using either the pCRE-LUC or rINS1-LUC reporter gene constructs, luciferase expression was down regulated by GLP-1 at concentrations above 10 nM. We contribute this effect to a desensitization of the GLP-1 receptor by prolonged elevated concentrations of agonist, which has been demonstrated in earlier studies of {beta}-cell lines (38, 39). Furthermore, similar effects of GLP-1 occur at the level of cAMP accumulation in RIN-1046–38 cells, also suggesting that this observed down-regulation occurs at the receptor level (9).

Repression of rat insulin I promoter activity by p38 MAPK is mediated through the CRE motif
Because the inhibitory response by p38 MAPK was particularly strong in the expression of pCRE-LUC, we investigated whether repression of rINS1 activity was mediated through the CRE motif in the rINS1 promoter. Mutation of the rINS1 sequence by a base deletion within the CRE sequence generated a construct that displayed a nonfunctional CRE sequence (rINS1{Delta}CRE-LUC) (8). GLP-1 treatment in cells transiently expressing the rINS1{Delta}CRE-LUC plasmid induced a 40% increase in luciferase expression above basal level compared with a 114% increase with the similarly transiently transfected intact promoter (rINS1-LUC) (Fig. 5Go), presumably as a result of the inability of GLP-1 to induce CRE-mediated promoter activity. However, in the presence of SB 203580, luciferase expression of rINS1{Delta}CRE-LUC was not augmented, suggesting that the p38 MAPK induced effect in rINS1-LUC activity is mediated through the CRE of the rINS1 insulin promoter.



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  		Figure 5. Stimulation of rat insulin-1 gene promoter by GLP-1 activation of p38 MAPK is mediated by the cAMP response element in the insulin gene promoter. INS-1 cells expressing the -410rINS1-LUC reporter plasmid (solid bars) or the -410rINS1{Delta}CRE-LUC reporter plasmid (hatched bars) were treated with GLP-1 in the presence or absence of 20 µM SB 203580. Results are presented as % control, where basal expression equals 100%. Cells were treated for 4 h and then incubated for 30 min with SB 203580 before GLP-1 treatment. Data points are mean values (± SEM) of three independent experiments done in triplicate.

 
Repression of rINS1 activity by p38 MAPK is transient
To investigate the dynamic regulation of the effect on rINS1 activity, we performed time course studies of luciferase expression in response to treatment with GLP-1 +/- SB203580. Cells were incubated for various time periods with test reagents (GLP-1 +/- SB203580), followed by a 2 h further incubation in fresh media devoid of the test reagents. The second incubation period (2 h) was designed to allow the processes of transcription and translation to occur to reflect luciferase protein expression levels following short time courses (0.5–2 h). The media were changed to fresh media to prevent further signal activation. Furthermore, this postincubation period allowed for a more accurate determination of changes in transcriptional expression at longer time courses as a result of the degradation of residual luciferase due to its approximately 3 h half-life in vitro. The peak effect of p38 MAPK induced repression was observed at 4 h of incubation and was decreased at 6 h. Interestingly, GLP-1 induced expression in the absence of SB 203580 remained constant at approximately 2-fold above basal level at all time points (Fig. 6Go).



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  		Figure 6. Time course of enhanced GLP-1 mediated activation of the rat insulin-1 promoter in response to inhibition of p38 MAPK activity. INS-1 cells expressing the -410 rINS1-LUC reporter plasmid were treated with GLP-1 in the presence (hatched bars) or absence (filled bars) of 20 µM SB 203580. Following treatment with GLP-1 for the respective incubation times, the media were changed and cells were further incubated in fresh media devoid of GLP-1 and SB203580 for 2 h before measuring luciferase expression levels. Expression levels represent fold increase over basal level (control). Data points are mean values (± SEM) of at least three independent experiments carried out in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to better characterize the signaling mechanisms involved in GLP-1-induced insulin gene transcription in pancreatic {beta}-cells. We used luciferase reporter constructs to assess the functional activity of the insulin gene by employing various promoter sequences to direct luciferase expression. Using the pCRE-LUC reporter plasmid, the ability of GLP-1 to induce CRE-mediated gene transcription to only 26% of that observed with forskolin corresponds with observations at the level of cAMP accumulation in INS-1 cells (16). This proportionality suggests a direct relationship between cAMP levels and CRE-mediated gene expression. The relatively low response to GLP-1 maybe explained by several possibilities including the low GLP-1 receptor density in INS-1 cells (16), receptor desensitization (38, 39), or by the potent stimulatory effect of forskolin on adenylyl cyclase. Treatment with two specific inhibitors of PKA activity (H-89 and Rp-cAMPS) abolished the response to GLP-1, further evidence of the direct signaling cascade. Conversely, GLP-1 induced activation of the rat insulin 1 promoter (-410rINS1-LUC) was insensitive to H-89, suggesting that the insulin gene is regulated by a different signaling pathway in response to GLP-1. However, involvement of a CRE-mediated response in rINS1 activity was evident because mutation of the CRE in the rINS1 promoter construct led to a decreased fold-induction in reporter gene expression by GLP-1 (Fig. 4Go; Ref. 8). Therefore, these results suggest that although GLP-1 can induce CRE-mediated gene expression via orthodox signaling through cAMP, PKA, and presumably CREB, induction of the rat insulin 1 gene appears to be mediated by a distinct pathway, but which also displays partial dependence on a functional CRE. These observations are in agreement with the recent studies of Skoglund et al. (8).

Following treatment of INS-1 cells with the p38 MAPK specific inhibitor SB 203580, GLP-1 induced augmented responses in both CRE and rINS1 promoter activity. This effect was dose dependent, suggesting that p38 MAPK activity may be induced by GLP-1 (Fig. 4Go). Indeed this was shown by immunoblot analysis of phospho-p38 MAK activity in response to GLP-1 treatment (Fig. 3Go). The fact that reporter gene expression was enhanced by SB 203580 treatment indicated that p38 MAPK activity provokes a marked inhibition of GLP-1 mediated promoter activity. Also, the correlating response between the two promoter-reporter constructs implied that the p38 MAPK-induced inhibition of rINS1 promoter activity was directed by its CRE sequence. This conjecture was further evidenced by the finding that a mutation of the CRE in the rINS1 insulin promoter construct abrogated the CRE responsiveness and resulted in a complete loss of response to SB 203580 treatment. The reduced fold expression level in response to GLP-1 compared with the insulin promoter construct with an intact CRE was presumably due to the abolition of CRE-mediated activity (8). In conclusion, it appears that the CRE motif in the insulin promoter is the direct target of repression by p38 MAPK.

Interestingly, p38 MAPK phosphorylation was maximal at 10 nM GLP-1 and was reduced at 100 nM. This observation correlates well with the extent by which SB 203580 treatment augmented the transcriptional response of the CRE promoter in response to GLP-1 (Fig. 4BGo). This supports the evidence that the reporter activity in response to GLP-1 and SB 203580 directly reflects the activity of p38 MAPK.

The inhibitory effect of p38 MAPK peaked at 4 h and was reduced at 6 h, as shown in a time course experiment comparing reporter gene expression in response to GLP-1 in the presence and absence of SB 203580 (Fig. 6Go). This temporal pattern of promoter activity may indicate the involvement of immediate early gene (IEG) regulation as opposed to direct phospho-activation of transcription factors by p38 MAPK or MAP/KAP-K2, MAP/KAP-K3, or MSK1/2 (substrates for p38). p38 MAPK has been implicated in the regulation of several IEGs including c-jun, c-fos, and nur-77 (29).

c-jun is a particularly attractive candidate target for further investigation as previous studies provide strong evidence for the involvement of this protein in regulatory effects of p38 MAPK (29, 40, 41). Furthermore, up-regulation of c-jun expression was recently demonstrated in response to GLP-1 treatment in INS-1 cells (15) along with several other IEGs. The PACAP receptor, a member of the GLP-1 receptor family was also shown to stimulate c-jun transcription (42), which may signify a common feature of these receptors. Even more evidence can be derived from observations that activating protein-1 (AP-1) binding activity in INS-1 cells is significantly increased in response to GLP-1 (6), consistent with increased expression of the AP-1 subunits, Fos, Jun, and ATF proteins. Increased AP-1 activity was also observed in rat pancreatic carcinoma cells in response to PACAP (42). A recent study describes a complete signal transduction pathway that involves direct regulation of the c-jun promoter by GPCRs (e.g. the m1 muscarinic receptor) via p38 MAPK (41). Perhaps the most convincing existing evidence in support of the possible involvement of c-jun in the repression of promoter activity in our studies is the observation that c-jun represses human insulin promoter activity, and that the mechanism of repression may involve the inhibition of CRE-binding proteins (43). Interestingly, not only c-jun but also c-fos represses human insulin promoter activity and thus c-fos may be an equally attractive target for p38 MAPK.

The rat insulin 1 promoter CRE sequence (TGACGTCC) is positioned between -178 and -185 relative to the transcriptional start site and closely matches but is not identical with the CRE octamer consensus motif (TGACGTCA) (44). Studies of transcriptional activities of 5' deleted and point mutated promoter-reporter plasmids demonstrated that this CRE-like sequence is necessary for cAMP induction of rat insulin 1 gene transcription in HIT cells and INS-1 cells (8, 44). However, cellular CREB binds only weakly to this CRE and was not detected by elecrophoretic mobility shift assays using the insulin CRE sequence, although three protein complexes from HIT cell nuclear extracts were identified, none of them containing proteins with CREB like immunoreactivity (45). This circumstance contrasts with the binding profiles of the somatostatin and glucagon gene CRE motifs, which display characteristic CREB binding patterns. Notably, this situation may help explain the relatively weak stimulation of rINS1 activity through cAMP/PKA-mediated signaling in response to GLP-1 compared with that seen with the generic CRE promoter used in these studies. A possible reason for the unusual behavior of the insulin gene CRE could be the overlapping CCAAT sequence beginning two base pairs from the 3' end of the CRE motif. Cross-reactivity with CCAAT/enhancer binding proteins (C/EBPs) and/or NF-Y family member proteins at this site may cause steric-hindrance resulting in a competitive binding relationship between CRE and CCAAT binding proteins. Disruption of the 3' CCAAT motif overlapping the CRE of the rINS1 promoter (TGACGTCCAAT) markedly enhanced the cAMP-inducible activity of the promoter (14). Evidence was presented that NF-Y conferred basal activity to the CRE and decreases the ability of CREB to mediate cAMP-stimulated transcription (14). Another possible factor in the p38 MAPK regulated repression of insulin gene promoter activity may be the transcriptional repressors C/EBP{beta} or CHOP, both members of the C/EBP transcription factor family. C/EBPs have been shown to bind to and activate transcription of CREs in the promoters of genes (46). CHOP is activated by phosphorylation by p38 MAPK (47), although there is as yet no evidence of up-regulation of CHOP gene expression by p38 MAPK.

In a recent study to identify possible substrates for MAPKAP-K2 (a downstream substrate for p38 MAPK), the E2A gene product E47 was isolated (48). This transcription factor binds two distinct enhancer elements (E boxes) in the rat insulin 1 promoter and is required for transcriptional activation (49, 50). Interestingly, it was shown that MAPK-K2 repressed transcriptional activity by interaction with E47 linking the E47 function to the MAPK signaling network. However, the involvement of this mechanism in our studies is doubtful because repression of promoter activity was dependent on the CRE rather than the E boxes.

What is the mechanism by which GLP-1 activates p38 MAPK? An increasing amount of information suggests that in contrast to the initial lock and key concept of receptor signaling, G protein-coupled receptors can regulate multiple signaling pathways by binding and functionally activating several trimeric G proteins (reviewed in Ref. 51). This concept has lead to the identification of receptor cross-talk, in which supposedly distinct GPCRs can interregulate the functional response of each other’s signal (reviewed in Ref. 52). The GLP-1 receptor couples to multiple G proteins including Gs, Gq/11, and Gi1,2 (17). Although G protein promiscuity has not been directly demonstrated in {beta}-cells, the potential for such promiscuous signaling of various pathways and signaling cross-talk is evident. In accordance with this notion, activation of the GLP-1 receptor increases p38 MAPK activity by 2-fold in RIN-1046–38 insulinoma cells and transfected CHO cells. This response is dependent on Gs protein activation, although not by {alpha}-subunit signaling, suggested by the failure of 8-bromo-cAMP to activate p38 MAPK activity (17). The authors proposed that G{beta}{gamma} subunits associated with Gs protein could be responsible for GLP-1 induced p38 MAPK activity, and this hypothesis is supported by studies in which p38 MAPK activation by several GPCRs was identified, with the implication of G{beta}{gamma} subunits in the response (53).

What is the functional purpose of the repression by p38 MAPK in insulin promoter activity? In accord with our findings, p38 MAPK activity prevents a much greater expression potential of the insulin gene than is seen in response to GLP-1 treatment. Interestingly, although the time course study demonstrated a transient increase in the p38 MAPK-mediated repression in insulin promoter activity, the insulin- promoter luciferase reporter expression level at all time points was consistently similar, suggesting that the repression is a regulatory mechanism to retain insulin transcription levels at a precisely controlled level. If this is the case, it could be hypothesized that diverse p38 MAPK activating stimuli (e.g. ER stress, cytokines, growth factors, and UV irradiation) could lead to detrimental repression of insulin gene expression. This regulatory response may function as a conservation mechanism in response to a stressful environment or as an early response in the process of programmed cell death.

Glucose toxicity and lipotoxicity results in decreased levels of insulin biosynthesis along with other important functional components of {beta}-cells (54, 55), followed by the commitment to programmed cell death (56, 57). Although many reports detail a decrement of the {beta}-cell specific transcription factor IDX-1 levels in association with insulin gene transcription in response to sustained supraphysiological levels of glucose or lipid treatment (58, 59, 60), Kajimoto et al. (61) demonstrated that suppression of IDX-1 causes no decrease in insulin messenger RNA in MIN6 cells, thus it could be inferred that contrary to general opinion, other inhibitory factors cause the suppression of insulin gene expression, independently of IDX-1 activity, which may simply be down regulated coincident with the down-regulation of the insulin gene. Therefore, the mechanism described in our studies may play an important role in the stress related decrease in insulin gene expression.

Our findings also suggest that agents that inhibit p38 MAPK activity in vivo could lead to increased insulin transcription levels, signifying a potentially new target for the treatment of type 2 diabetes mellitus. In conclusion, our results highlight a novel mechanism whereby GLP-1 induces a bifurcating signal that imparts opposing transcriptional responses by affecting both stimulatory and inhibitory signals on the same insulin gene promoter.


   Acknowledgments
 
We thank M. Ubeda for helpful advice and R. Larraga and T. Budde for help in the preparation of the manuscript. rINS1{Delta}CRE-Luc plasmid was constructed by M. Hussain.


   Footnotes
 
1 The studies were supported in part by USPHS Grant DK-30834. Back

2 Investigator with the Howard Hughes Medical Institute. Back

Received September 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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