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Published as doi: 10.1096/fj.06-7798com.
 (The FASEB Journal. 2007;21:3240-3249.)
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Sequential actions of ERK1/2 on the AP-1 transcription factor allow temporal integration of metabolic signals in pancreatic ß cells

Dominique A. Glauser and Werner Schlegel1

Fondation pour Recherches Médicales, University of Geneva, Switzerland

1Correspondence: Fondation pour Recherches Médicales, Av. de la Roseraie 64, 1211 Geneva, Switzerland. E-mail: werner.schlegel{at}medecine.unige.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The AP-1 transcription factor composed of fos and jun gene products mediates transcriptional responses to hormonal and metabolic stimulations of pancreatic beta cells. Here, we investigated the mechanisms that dynamically control expression of AP-1 subunit proteins. In MIN6 cells, glucose and GLP-1 raised c-FOS protein with biphasic kinetics, an initial peak being followed by a plateau that persisted as long as stimuli were maintained. ERK1/2 activation paralleled c-FOS expression. Whereas initial induction of c-FOS protein required ERK1/2-dependent activation of c-fos transcription and de novo protein synthesis, persistent accumulation of c-FOS under sustained stimulation did not. Indeed, dependent on ERK1/2 activation, c-FOS accumulated in its hyperphosphorylated form protected from degradation through the proteasome pathway. The implication of ERK1/2 in the accumulation of c-FOS protein was confirmed in rat primary ß cells, and the functional consequences of this mechanism were demonstrated with DNA-binding and reporter assays. Altogether these findings reveal a sequential regulation of AP-1 by ERK1/2, which initially increases transcription of c-fos and, if stimulation persists, stabilizes freshly synthesized c-FOS protein to efficiently activate the transcription of AP-1-regulated genes. This ERK1/2-AP-1 module can function as a temporal integrator converting metabolic stimuli of different durations into differential transcriptional outputs.—Glauser, D. A., Schlegel, W. Sequential actions of ERK1/2 on the AP-1 transcription factor allow temporal integration of metabolic signals in pancreatic ß cells.


Key Words: Langerhans islets • c-fos • protein stability • proteolysis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN MAMMALS, THE ENDOCRINE PANCREAS COORDINATES metabolism of various organs to optimize uptake, storage, and use of nutrients for the whole body. To this end, pancreatic ß cells continuously adjust their insulin secretion according to circulating nutrient levels and other signals like hormones, growth factors, or neurotransmitters. In addition, all of these factors also control gene expression in ß cells, allowing their long-term adaptation to ensure glucose homeostasis throughout changing metabolic status and insulin demand (1 2 3 4) . For instance, glucose regulates many genes involved in ß cell metabolism, in its secretory pathway, or in the control of ß cell mass (5 6 7) .

These pleiotropic effects of glucose are initially mediated via intracellular signaling pathways. Metabolism of glucose leads to increased ATP/ADP ratio and subsequent closure of ATP-dependent K+ channels. The resulting depolarization causes Ca2+ entry through voltage-gated Ca2+ channels. Apart from eliciting exocytosis, this Ca2+ influx activates calmodulin kinases II and IV, protein kinase A (via cAMP), protein kinase C, phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated kinases 1/2 [ERK1/2, also known as p44/p42 mitogen-activated protein (MAP) kinases; ref. 8 ]. Some of these pathways are also activated by incretin hormones, like glucagon-like peptide-1 (GLP-1), which activates PI3K and protein kinase A through cAMP production (9) . As a result, glucose and incretins synergize not only to promote insulin exocytosis but also to regulate gene expression (7 , 10) . Indeed, through these signaling cascades, transcription factors will be activated or inhibited resulting in changed gene expression.

The first genes to respond to intracellular signals are immediate-early genes, which are induced independently of de novo protein synthesis (7 , 11 , 12) . Immediate-early gene levels in ß cells react rapidly to changing metabolic state. Many of these immediate-early genes encode transcription factors and regulate, in turn, transcription of downstream target genes. The importance of such indirect mechanism have recently been highlighted in genome-scaled studies (13 ; Glauser, D. A., Brun, T., Gauthier, B. R., and Schlegel, W., unpublished observations). An important actor in this system is the AP-1 transcription factor. AP-1 binds DNA as a dimer composed of fos and jun gene products. AP-1 subunits are subjected to post-translationnal modifications (phosphorylation, redox regulation of disulfide bridges) that can alter their function by modulating their trans-activation activity or their stability (14 , 15) . In ß cells, AP-1 composition is regulated by glucose and GLP-1 in a coordinated manner. Indeed, these two factors modulate the expression pattern of fos and jun genes, their most important effect being a strong induction of c-fos, an immediate-early gene (7) . Consequently, newly synthesized c-FOS is recruited to the AP-1 complex, thus enhancing AP-1 target gene trans-activation. Importantly, c-FOS expression levels respond rapidly to both an increase and a drop of glucose concentrations and remain elevated as long as the metabolic stimulation is maintained (13) . Hence, expression of c-FOS is tightly coupled to ß cell metabolic states.

The aim of the present study was to elucidate the molecular mechanisms through which expression of c-FOS is continuously maintained at a level reflecting the metabolic state of the pancreatic ß cell.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Glucagon-like peptide-1(7–37) (human), actinomycin D (act D), cycloheximide (CHX), UO126, MG132, and chlorophenylthio-cyclicAMP (cpt-cAMP) were purchased from Sigma (Buchs, Switzerland). Stock solutions of actinomycin D (100 mg/ml, 2000x) and UO126 (10 mM, 2000x) were prepared in DMSO. When these inhibitors were used, control treatments included vehicle (1:2000 v/v of DMSO). Note that DMSO vehicle had no detectable effect on its own. Rabbit polyclonal antibodies were as follows: anti-c-FOS (sc-52), anti-TFIIB (sc-225), anti-ERK1/2 (p44/p42 MAPK, sc-94), anti-p38 (sc-728; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-specific ERK1/2 (p44/p42 MAPK #9101), and anti-phospho-specific p38 (#9211; Cell Signaling, Danvers, MA, USA). Monoclonal anti-insulin was from Sigma (St. Louis, MO, USA; I-2018). The secondary antibodies were as follows: HRP-conjugated anti-rabbit-IgG (Cell Signaling), alexa-488 labeled anti-rabbit-IgG, and alexa-568 labeled anti-mouse-IgG (both from Molecular Probes, Eugene, OR, USA). The two p38 antibodies were a kind gift of Dr. Eric Féraille (Service de Nephrologie, Fondation pour Recherches Medicales, Geneva, Switzerland).

Cell culture and stimulation
MIN6 B1 cells were generously provided by Dr. Philippe Halban (Dept. of Development and Medical Genetics, Medical Faculty of Geneva University) and maintained as described earlier (16) . Twenty-four hours before experiments, cells were transferred to low glucose medium (DMEM supplemented with 1 mM glucose, 1% fetal calf serum, 71 µM 2-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 µg/ml gentamicin). This preincubation period did not alter cell morphology nor the ability of cells to secrete insulin in response to stimulation with glucose and cpt-cAMP (data not shown). Furthermore, FOXO transcription factor activity in the MIN6 cell nucleus was not enhanced by the preincubation (Supplemental Fig. 1 ). FOXO activation is generally associated with stress situations and a deficit in Akt activation (17) . Similarly, the stress p38 MAPK was not activated during the low glucose preincubation period (Supplemental Fig. 2 ). There is thus no evidence that the preincubation produces any serious deleterious effect on cell viability and physiology. Sustained stimulations were maintained all along the experiments. Transient stimulation was achieved by replacing the medium with low glucose medium after 1 h of stimulation.


Figure 1
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  		Figure 1. Induction of c-fos gene expression by glucose and GLP-1 is biphasic. MIN6 cells were stimulated with 10 mM glucose, 10 nM GLP-1, or costimulated with glucose and GLP-1 for the indicated period of time, before analysis of c-fos mRNA and c-FOS protein expression. A) c-fos mRNA quantification by real-time RT-PCR; normalized with 18S rRNA and expressed as mean results with SD as error bars (n≥3). B, C) c-FOS expression assessed by Western blot analysis of nuclear extracts. Pretreatments were made with act D (50 mg/l), cycloheximide (CHX, 100 mg/l), and UO126 (5 µM) 1 h before the stimulation.


Figure 2
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  		Figure 2. Sustained expression of c-FOS does not depend on long-term c-fos mRNA accumulation. MIN6 cells were costimulated with 10 mM glucose and 10 nM GLP-1 at t = 0. Additional treatments were performed at t = 1 h: stimuli removal (for the transient stimulation), addition of 50 mg/l act D, or addition of 5 µM UO126. A) c-fos mRNA quantification by real-time RT-PCR; normalized with 18S rRNA and expressed as mean results with SD as error bars (n≥3). B) c-FOS expression assessed by Western blot analysis of nuclear extracts. NS: no stimulation. Representative of 3 different experiments.

Reverse transcriptase-polymerase chain reaction
Total mRNA was extracted with acid phenol-guanidinium reagent (TRI-Reagent, Molecular Research Center, Cincinnati, OH, USA) following the manufacturer’s instructions. For each experimental condition, reverse transcription was realized in triplicate and cDNA was analyzed by quantitative real-time polymerase chain reaction as described previously (13) . Relative cDNA quantity for each reaction sample was normalized with the corresponding 18S rRNA quantity. As a supplemental control, the constitutively expressed gene junD was analyzed in parallel to c-fos.

Protein extracts and Western blotting
Nuclear protein extracts were prepared according to Schreiber et al. (18) , except that phosphatase inhibitors (50 mM NaF and 1 mM sodium orthovanadate) were added throughout the procedure. Total protein extraction was made according to Reffas et al. (19) . Nuclear protein extracts (15 µg) or total protein extracts (30 µg) were resolved on SDS-PAGE followed by Western blot analysis as described previously (19) . Primary antibody dilutions were as follows: anti-c-FOS (1:1000), anti-TFIIB (1:10,000), anti-ERK1/2 (p44/p42 MAPK, 1:5000), anti-p38 (1:500), anti-phospho-specific ERK1/2 (p44/p42 MAPK, 1:1000), and anti-phospho-specific p38 (1:1000).

CIP treatment
Nuclear extracts (15 µg in 15 µl) were incubated 30 min at 37°C in presence of 20 U of calf intestine phosphatase [CIP (alkaline phosphatase), Roche Diagnostics GmbH, Mannheim, Germany] before SDS-PAGE analysis (20) .

DNA binding and Luciferase reporter
FOS and JUND specific binding to AP-1 consensus site was detected with the ELISA-like TransFactor Kit Inflammation II (BD Biosciences AG, Alschwil, Switzerland), and FOXO DNA-binding activity was assessed with the TransAM FKHR (FOXO1; active motif) as detailed in the manufacturer’s instructions with minor modifications described previously (13) . For reporter experiments, PathDetect cis-Reporting System pAP-1-Luc or pCIS CK (control) plasmids (Stratagene Europe, Amsterdam Zuidoost, The Netherlands) were cotransfected with Renilla luciferase plasmid (for normalization; Promega, Luzern, Switzerland) with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) as described previously (13) .

Islet isolation and immunocytochemistry
Seven-week-old male Wistar rats (~250 g) were purchased from Elevage Janvier (Le Genest-St-Isle, France). Pancreatic islets were isolated by collagenase digestion, handpicked, and maintained in 11.1 mM glucose/RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum (Amimed, BioConcept Allschwil, Switzerland), 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamicin (Sigma). For immunocytochemistry, partially trypsin dispersed rat islets were cultivated and preincubated 20 h in low glucose RPMI 1640 medium (1 mM glucose RPMI 1640, 0.1% BSA, and same antibiotics as above). After stimulation, cells were subjected to cytospin on SuperFrostPlus slides (Menzel GmbH and Co KG, Braunschweig, Germany) and fixed in 4% paraformaldehyde pH 7.0 for 30 min at room temperature. After three PBS washes and two incubations with boiling 10 mM citrate pH 6.0 for 2 min, cells were permeabilized with 0.2% Triton PBS for 15 min. Primary antibody for c-FOS (1:200) and mouse anti-insulin (1:1000) were diluted in 0.05% triton PBS and used for an overnight incubation. After three washes, cells were incubated 1 h with secondary antibodies (alexa-488 labeled anti-rabbit-IgG and alexa-568 labeled anti-mouse-IgG; both 1:300). After washings, cells were incubated three minutes in 5 mg/ml 4',6-diamidino-2-phenylindol (DAPI), washed three times, and mounted in Dakocytomation fluorescent mounting medium (DakoCytomation AG, Untermüli, Switzerland). Images were acquired with a Zeiss Axiocam Imaging System (Bioimaging Core Facility, Medical Faculty, Geneva University, Geneva, Switzerland).

Statistical analyses
Student’s t tests were used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Elevated glucose and GLP-1 induce c-fos immediate-early gene expression in primary ß cells and different insulinoma cell lines (7 , 12 and Glauser et al.,unpublished observations). The mechanisms underlying changes in c-fos transcription and c-FOS protein levels were investigated here in the MIN6 ß cell line. MIN6 cells are mice insulinoma cells that have been obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice (21) .

Induction of c-fos mRNA by GLP-1 and glucose is biphasic
At low glucose, GLP-1 produced a modest and transient rise in c-fos mRNA levels (Fig. 1 A). Elevated glucose produced a similarly modest accumulation that, however, persisted over at least 4 h. Combination of glucose and GLP-1 produced a strong initial increase in c-fos mRNA levels (~8-fold increase after 30 min), followed by a persistent plateau (3- to 4-fold increase; Fig. 1A ). Thus, kinetics of c-fos mRNA exhibit two phases: first, a strong initial peak dependent on the synergistic action of GLP-1 and glucose; second, a period of persistent elevation that depends mainly on glucose.

Initial accumulation of c-FOS protein requires ERK1/2 activation, c-fos mRNA, and protein de novo synthesis
To test whether the initial induction of c-fos mRNA by GLP-1 and glucose was translated into protein accumulation, we assessed c-FOS protein levels by Western blotting. In nuclear extracts from nonstimulated cells, basal level of c-FOS protein was almost undetectable (Fig. 1B ). Within 1 h of stimulation with elevated glucose and GLP-1, c-FOS becomes prominent. As expected, this massive rise was totally abolished by pretreatment with actinomycin D (transcription inhibitor) or cycloheximide (protein synthesis inhibitor), indicating that it relies on de novo RNA and protein synthesis (Fig. 1C ). Of note, the pretreatment with actinomycin D completely abolished c-fos mRNA induction (Supplemental Fig. 3). Interestingly, pretreatment with UO126 (ERK1/2 inhibitor) also efficiently inhibited c-FOS induction, showing that ERK1/2 activation is necessary for the initial accumulation of c-FOS. This latter effect may partially depend on initial activation of c-fos transcription, since pretreatment with UO126 significantly inhibited by ~35% (n=3; P<0.05 by Student’s t test) the c-fos mRNA peak at 30 min. However, the drastic reduction by ERK1/2 inhibition of c-FOS protein levels (Fig. 1C ) suggested the implication of further mechanisms.

Prolonged accumulation of c-fos transcripts requires persistent stimulation by glucose and ERK1/2 activation
We then evaluated the mechanism underlying the second phase of c-fos induction, consisting of a persistent elevation of c-fos mRNA (from 1 to 4 h). This persistent accumulation can be rapidly reversed upon removal of the stimuli (i.e., replacement of the medium with low glucose medium), confirming previous observations (13 ; Fig. 2 A). In this study, this type of stimulation protocol, with stimuli removal after 1 h, will be referred to as "transient stimulation" and compared to "sustained stimulation," for which the stimulation is maintained all along the experiment. When stimulation is sustained, delayed addition of actinomycin D (1 h after the beginning of the stimulation) strongly decreased c-fos mRNA to basal levels, indicating that long-term maintenance of elevated c-fos transcript levels relies on mRNA synthesis and not on mRNA stabilization. Similar to actinomycin D, UO126 abolished the sustained elevation of c-fos mRNA. Thus, in addition to contribute to the initial c-fos induction, ERK1/2 activation is required for prolonged c-fos transcription.

Prolonged accumulation of c-FOS protein after sustained glucose and GLP-1 stimulation is independent of prolonged mRNA accumulation
To determine how the sustained elevation of c-fos mRNA was translated to the protein level, the kinetics of c-FOS protein induction were analyzed by Western blotting. A peak and a plateau were also observed for c-FOS protein, following c-fos transcript levels with a slight delay (Figs. 1B and 2B , top). c-FOS level remained high only upon sustained stimulation, but decreased to basal level after transient stimulation (Fig. 2B ), strictly parallel to what was observed for c-fos mRNA (Fig. 2A ). This correlation suggested a tight coupling between transcript and protein levels. To test this, sustained gene transcription was inhibited with actinomycin D. Surprisingly, despite the drastic reduction in c-fos mRNA level produced by actinomycin D (Fig. 2A ), the sustained elevation of c-FOS protein was completely maintained (Fig. 2B ). Noteworthy, the plateau of c-FOS protein observed in presence of actinomycin D was still dependent on sustained stimulation, since c-FOS protein levels returned to basal levels after transient stimulation. Thus, persistent expression of c-FOS protein does not require the sustained transcription of the c-fos gene, indicating that post-transcriptional mechanisms are involved.

c-FOS is stabilized by sustained stimulation with elevated glucose and GLP-1
c-FOS prolonged expression may rely on the up-regulation of its mRNA translation. To test this possibility, the protein synthesis inhibitor cycloheximide was used in a concentration that had been shown to completely abolish c-FOS initial induction (Fig. 1C ). When added after 1 h of stimulation, cycloheximide was not able to reduce the extended c-FOS accumulation upon sustained stimulation (Fig. 3 A). Thus, prolonged up-regulation of c-FOS protein levels is not dependent on its de novo synthesis.


Figure 3
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  		Figure 3. Multiple phosphorylations of c-FOS and protection against proteolysis. A) MIN6 cells were cultured and c-FOS expression was assessed as in Fig. 2B . MG132, 10 µM. TFIIB as loading control. Representative of 3 different experiments. B) Nuclear extracts from MIN6 cells submitted to a sustained stimulation (4 h) with glucose plus GLP-1 were treated with CIP and analyzed by gel electrophoresis followed by Western blotting. Representative of 2 different experiments. C) Cells were stimulated as in A (3 h) and nuclear extracts analyzed by gel electrophoresis followed by Western blotting.

The latter observation suggests that sustained stimulation stabilizes c-FOS protein. As shown in other systems, c-FOS turns over rapidly due to its degradation via the proteasome pathway (22 , 23) . We tested the role of this pathway using the proteasome inhibitor MG132 (Fig. 3A ). MG132 added 1 h after stimulation stabilized c-FOS protein such that a transient stimulation produced a persistently elevated c-FOS level, indistinguishable from the level seen with sustained stimuli in the presence of MG132. Note that some levels of proteasome-dependent degradation occur even with sustained stimuli, since the c-FOS levels seen after 4 h in the presence (for the last 3 h) of MG132 are higher than the corresponding levels with uninhibited proteasomes. Our results show, however, clearly that the removal of stimuli after transient stimulation increases c-FOS degradation. Importantly, MG132 acted identically when used conjointly with cycloheximide, ruling out that a MG132 side effect could have compensated for c-FOS degradation by promoting somehow its de novo synthesis.

Altogether, these results show that sustained stimulation with glucose and GLP-1 stabilizes c-FOS through a mechanism relying, at least partially, on its protection from degradation by the proteasome.

Stabilization of c-FOS is linked to multiple phosphorylation
Western blotting experiments suggested a slight increase in the apparent molecular weight of c-FOS extracted from cells exposed for 3 h or more to high glucose (Fig. 2B ). Furthermore, multiple bands could be detected (Fig. 3B ). These features likely reflect post-translational modifications such as phosphorylation. In other systems, phosphorylation can stabilize c-FOS (24) . To test whether c-FOS was phosphorylated in cells submitted to a sustained stimulation, their nuclear extracts were treated with CIP and analyzed by SDS-PAGE followed by Western blotting (Fig. 3B ). Similar to an earlier study (20) , and as controlled with ERK1/2 phosphorylation, CIP efficiently hydrolyzes phosphate groups from proteins. CIP treatment produced a reduced apparent molecular size of c-FOS on SDS gels, with the disappearance of the multiple bands of higher apparent molecular weight. These findings are consistent with multiple phosphorylation of c-FOS in cells under sustained stimulation.

Further experiments aimed to determine whether c-FOS dephosphorylation after transient stimulation may cause its destabilization. It was, however, difficult to detect a clear reduction of c-FOS phosphorylation, as a shift back to lower apparent size, since the Western blot signals for c-FOS become very small (Fig. 3A ). This reflects the rapid degradation of non- or hypophosphorylated c-FOS forms. To circumvent this problem, cells were treated with MG132, which blocks c-FOS degradation by inhibiting the proteasome pathway (see Fig. 3A ). As depicted in Fig. 3C , rescued c-FOS protein has an enhanced mobility (i.e., a smaller apparent molecular size) in SDS-PAGE analysis. Thus, shortening the stimulation reduces c-FOS phosphorylation level. This strongly suggests that c-FOS phosphorylation contributes to its stabilization under sustained elevated glucose levels.

Sustained elevated glucose promotes long-term ERK1/2 activation
In other systems, ERK1/2 is implicated in c-FOS phosphorylation, both directly and indirectly through activation of ribosomal S6 kinase (RSK; ref. 25 ). This prompted us to examine the activation of ERK1/2, and we performed a kinetic analysis over 4 h after stimulation by elevated glucose and/or GLP-1 (Fig. 4 A). Upon glucose elevation, a slight early ERK1/2 activation (observed from 5 min) was followed by a more massive activation after 2 h. In contrast GLP-1 produced an important early effect, which was reduced after 15 min. Combined stimulation by GLP-1 and elevated glucose resulted in a biphasic response, with an early peak of activation followed later by a prolonged activation of ERK1/2. The strong prolonged activation of ERK1/2 is coherent with a model in which this activation leads to c-FOS phosphorylation and consequently its stabilization.


Figure 4
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  		Figure 4. ERK1/2 prolonged activation by glucose is essential to c-FOS stabilization. MIN6 cells were cultured and treated as in Fig. 2B . A, B) Phosphorylation of ERK1/2 was assessed by Western blot analysis of whole cell extracts, using antibodies recognizing phosphorylated forms of ERK1/2 (phospho-ERK1/2) and total ERK1/2, respectively. B) 1 of 5 blots used for the densitometric quantification presented below for ERK1 (p44) and ERK2 (p42) (top). *P < 0.01 (n=5). C, D) Western blot analysis of nuclear extracts. 5 µM UO126 and 10 µM MG132 were added after 1 h of stimulation. Representative of 3 different experiments.

Sustained ERK1/2 activation by glucose is essential for c-FOS stabilization
If ERK1/2 activation is responsible for c-FOS stabilization by phosphorylation, one would expect that transient stimulation would be insufficient to cause sustained ERK1/2 activation. This is precisely what was observed (Fig. 4B ), with a >50% reduction in ERK1/2 activation 2 h after stimuli removal (P<0.01).

To further establish the implication of ERK1/2 in c-FOS stabilization, we used pharmacological inhibitors. UO126 drastically accelerated the decline c-FOS level (Fig. 4C ) seen under sustained stimulation. As this effect was abolished by concomitant use of MG132 inhibitor (Fig. 4D ), it is clear that ERK1/2 activation affects c-FOS level through a stabilization mechanism.

An ERK1/2-dependent stabilization mechanism regulates JUNB expression similarly to c-FOS
The ERK1/2-dependent mechanism of stabilization may not be restricted to c-FOS. Hence, we tested if this mechanism would affect other transcription factors. c-FOS contain DEF domain, i.e., a specific binding site for ERK1/2 that could be involved in its stabilization. We therefore evaluated regulation of JUNB, another transcription factor that harbors a DEF domain (26) .

We stimulated MIN6 cells with elevated glucose in combination with either GLP-1 or chlorophenylthio-cyclicAMP (cpt-cAMP), a membrane-permeant cAMP analog that mimics the activation of the cAMP pathway by GLP-1. Similarly to c-FOS, long-term expression of JUNB for up to 4 h depended on the maintenance of the stimuli (Fig. 5 A). When UO126 and MG132 were used, JUNB behaved like c-FOS. Indeed, JUNB degradation was blocked by MG13 and its stabilization was inhibited by UO126 (Fig. 5B, C ). Thus, sustained stimulation by glucose and cAMP agonists protects JUNB from degradation via the proteasome pathways in an ERK1/2-dependent manner.


Figure 5
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  		Figure 5. An ERK1/2-dependent stabilization mechanism regulates JUNB sustained expression. MIN6 cells were cultured and treated as in Fig. 3A . 0.2 mM cpt-cAMP were used. JUNB expression was assessed by Western blot analysis in parallel to c-FOS and TFIIB (exposure times vary between A–C). A) Kinetics of JUNB and c-FOS expression after sustained or transient stimulation by cpt-cAMP plus glucose. B, C) Effects of the MEK-1 inhibitor UO126 and/or the preteasome inhibitor MG132 on JUNB and c-FOS expression induced by glucose plus cpt-cAMP (B) or GLP-1 (C).

Functional consequences of ERK1/2-dependent c-FOS stabilization
c-FOS activates transcription when binding to DNA as part of the AP-1 complex. We thus investigated the impact of ERK1/2-dependent c-FOS stabilization on the amount of c-FOS protein recruited to the DNA-binding AP-1 complex. To that purpose, we used an ELISA quantification of FOS and JUN proteins binding to a surface-attached dsDNA with an AP-1 consensus sequence (TransFactor Assay). After stimulation, newly synthesized c-FOS protein was recruited to AP-1 complexes, as indicated by a significant increase in c-FOS DNA-binding activity (Fig. 6 A). Reduced ERK1/2 activation upon transient rather than sustained stimulation as well as inhibition of ERK1/2 by UO126 resulted in reduced c-FOS content of the AP-1 DNA binding complexes (Fig. 6A ). In contrast, the level of JUND, a constitutively expressed component of the AP-1 complex, remained unchanged on stimulation or inhibition of ERK1/2. Thus, ERK1/2 activation is essential for sustaining changes in AP-1 composition by allowing prolonged recruitment of c-FOS.


Figure 6
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  		Figure 6. Recruitment of c-FOS to AP-1 DNA binding complex and trans-activation enhancement require ERK1/2 activation. A) DNA binding activities of c-FOS and JUND were measured in nuclear extracts from MIN6 cells stimulated with 10 mM GLP-1 plus 10 mM glucose (3 h). Results as means (+/– SD, n=4). B) MIN6 cells were transfected with pAP-1-luc (luciferase reporter vector harboring repeated AP-1 consensus enhancers) or corresponding control vector, and stimulated with 10 nM glucose plus 0.2 mM cpt-cAMP. Luciferase activity was measured after 6 h and expressed in arbitrary units (+/– SD, n=3). *P < 0.05, #P < 0.01, **P < 0.001 vs. sustained stimulation. UO126: 5 µM added after 1 h stimulation.

Changes in AP-1 composition by recruitment of c-FOS enhance transactivation of AP-1 target genes (13) . Further investigations were undertaken to establish whether ERK1/2 inhibition (and its consequences on AP-1 composition) could attenuate transcription of AP-1-regulated genes. To this end, we assessed the transcriptional activation of an AP-1 luciferase reporter (driven by multiple AP-1 enhancer sites). In transfected MIN6 cells, stimulation with high glucose and cpt-cAMP induced transcription of the reporter (Fig. 6B ). When added after the 1 h of stimulation, UO126 significantly attenuated this up-regulation by >50%. Thus duration of ERK1/2 activation is important for the regulation of AP-1 target gene expression.

ERK-1/2-dependent stabilization of c-FOS occurs in ß cells of isolated rat islets
It was important to verify the key finding of the present study in normal nontransformed pancreatic ß cells. Using immunocytochemistry, we have previously shown a prominent accumulation of c-FOS in the nuclei of ß cells, after 1 h of stimulation by GLP-1 and glucose (Glauser et al., unpublished observations). Pursuing these studies, we report here that nuclear c-FOS elevation is still detectable after 3 h (Fig. 7 ). In contrast, when UO126 is added after an initial 1 h period of stimulation, c-FOS level is drastically reduced within the subsequent 2 h. The results obtained with the immunocytochemistry approach in primary ß cells are completely consistent with those obtained in MIN6 cells by Western blotting (see Fig. 4C ). This demonstrates that delayed ERK1/2 activation is crucial for the prolonged expression of c-FOS also in primary ß cells.


Figure 7
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  		Figure 7. c-FOS accumulation relies on ERK1/2 activation in ß cells from rat dispersed islets. Primary islet cells were stimulated with 15 mM glucose plus 10 nM GLP-1 for 3 h, fixed, and analyzed by immunofluorescent costaining for c-FOS and insulin (INS) and nuclear staining (DAPI). Individual fluorescence channels and merged images presented here are representative of 3 different experiments. Bar = 50 µm. UO126 (5 µM) was added after 1 h of stimulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our findings show that ERK1/2 acts sequentially to regulate c-FOS expression in ß cells stimulated with glucoincretins and glucose. Indeed, ERK1/2 promotes c-fos gene transcription and post-translational modifications on c-FOS, which lead to c-FOS stabilization, its long-term recruitment to the AP-1 complex and the transcriptional activation of AP-1 regulated genes. This multistep mechanism results in differential gene regulation by AP-1 in response to different durations of metabolic stimulation. Fluctuations in nutrients sensed by the endocrine pancreas may thus be integrated over time to produce a gene expression output in the pancreatic ß cell, which is governed by long-term changes in nutrient supply.

Stimulation by GLP-1 and glucose activates c-fos transcription and its mRNA reaches an elevated level within 30 min. This strong initial induction relies on the activation of CaMkinase II and PKA, which converge to regulate cis-elements in the c-fos promoter (12) . In addition, our results show a moderate implication of ERK1/2 in this early stimulatory effect, which probably reflects a contribution of GLP-1 signaling. In beta cells, GLP-1 activates ERK1/2 via activation of PKA and Ca2+ signaling (27 , 28) . ERK1/2 can activate Elk-1 transcription factor, which binds a serum response element (SRE) in the c-fos promoter and promotes initiation of transcription (29) . Another possibility by which c-fos gene transcription can be increased is through ERK1/2-dependent modulation of transcriptional elongation. This type of stimuli-dependent mechanisms targeting elongation has been recently described in pituitary neuroendocrine cells (30 , 31) .

The mRNA peak is subsequently reduced to a more moderate level, which persists over hours provided that high glucose levels are maintained. This prolonged steady-state accumulation is not linked to c-fos messenger stabilization (32) but due to a somewhat elevated continuous transcriptional rate balanced by constant c-fos mRNA degradation. In contrast to the initial induction, this long-lasting effect on transcription is fully dependent on ERK1/2 activation. Thus, the c-fos mRNA down-regulation observed when stimulation is transient would be due to the ERK1/2 inactivation and the ensuing decrease in c-fos transcription.

c-FOS protein expression kinetics followed that of its mRNA with a small delay. This was nicely evident after transient stimulation where mRNA down-regulation was followed closely by the reduction of c-FOS protein levels. This correlation suggested a tight coupling between c-fos mRNA and c-FOS protein assuming rapid turnover of both protein and mRNA. Therefore, it came as a surprise when we found that drastically reducing c-fos mRNA level with actinomycin D had no detectable effect on the prolonged c-FOS protein expression. Even if we cannot exclude a small contribution of the prolonged mRNA accumulation, our data clearly reveal that the major event leading to prolonged c-FOS expression does not rely on continuous c-FOS de novo synthesis but on protein stabilization.

c-FOS degradation after transient stimulation was abolished by the use of MG132 proteasome inhibitor. The proteasome pathway is known to be the main route for c-FOS degradation (see ref. 22 for a review). Interestingly, it does not obligatorily require ubiquitination and is regulated by c-FOS post-translational modifications, such as the phosphorylation of Ser-362 and of Ser-374 providing efficient stabilization (24) . Phosphorylation at these two residues is dependent on ERK1/2 activation as it directly phophosylates Ser-374 and activates RSK which in turn will phosphorylate Ser-362 (25) . Our data are in line with this, providing evidence that ERK1/2 dependent phosphorylation has a stabilizing effect on c-FOS in ß cells exposed to high glucose. In addition, ERK1/2-dependent phosphorylation has been shown to prime c-FOS for further phosphorylation events and control its transactivation potential (26 , 33) . This suggests that the function of ERK1/2 action on c-FOS may extend beyond its stabilization.

Glucose and GLP-1 activate ERK1/2 through different signaling pathways converging on MEK (27 , 28 , 34 35 36) . Our kinetic analysis of ERK1/2 activation has revealed that GLP-1 and glucose elicit different temporal responses. GLP-1 was more efficient than glucose in early ERK1/2 phosphorylation, but this effect was only transient. In contrast, glucose produced a delayed and long-lasting pronounced effect. Among the possible explanations for this prolonged activation is an insulin autocrine feedback, maintaining a high cytoplasmic Ca2+ level by inhibiting its uptake in endoplasmic reticulum (37) . Down-regulation of some phosphatases (such as MAPK phosphatase-1, MKP-1) represents an alternative mechanism by which ERK1/2 could achieve long-term activation (38 39 40) . Further investigations will be required to define these upstream mechanisms.

The way ß cells temporally interpret metabolic intracellular signaling to control long-term gene expression is a largely unanswered question. Recently, we have provided evidence that the induction of immediate-early genes acts as a temporal filter such that transient inductions do not induce the expression of downstream target genes (13) . The new insights into the mechanism by which signaling duration can be integrated at the expression level of a single gene are summarized in Fig. 8 . Initial induction of c-fos transcription, which is rather massive, can be viewed as a priming step allowing the synthesis and accumulation of c-FOS protein. Subsequently, the limiting factor determining c-FOS expression becomes protein stability, which is controlled by the ERK1/2 signaling pathway. If metabolic stimulation persists, ERK1/2 activation will last sufficiently to stabilize c-FOS and affect target gene transcription. Inversely, if metabolic stimulation is only transient, ERK1/2 activation will be too short and c-FOS will be rapidly degraded through the proteasome pathway with little impact on target genes.


Figure 8
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  		Figure 8. Schematic model for ERK1/2 multiples roles in regulation of c-fos expression and function. Dotted-line arrows = weak effects; solid-line arrows = strong effects.

A similar mechanism has been proposed to contribute to the control of cell cycle progression in fibroblast in response to different mitogenic growth factors (26 , 41 , 42) . In the fibroblast model, EGF or PGDF, which produce distinct ERK1/2 activation kinetics, have a differential impact on AP-1 controlled gene expression. Our results demonstrate that the ERK1/2-AP-1 module can also function to sense the dynamic input of a single stimulus, namely the metabolic activation of pancreatic ß cells caused by continuously fluctuating glucose concentrations. Duration of glucose stimulation and ensuing ERK1/2 activation are determinant for ß cell mitogenesis (43) and insulin production (44) . Such a mechanism would be of particular interest in the context of type-2 diabetes emergence and progression, during which temporal deregulation of circulating glucose and hormone levels are marked.


   ACKNOWLEDGMENTS
 
We would like to acknowledge I. Piuz, A. Massiha, M. Steidel, and A. Gjinovci, for technical assistance, as well as P. Halban and E. Féraille for the gift of materials. We thank C. B. Wollheim, B. Gauthier, and T. Brun for fruitful discussion and for help with the islet experiments. Financial support was from Swiss National Foundation, Grants No. 3100A0–102147/1 to W. Schlegel, and from the Fondation pour Recherches Médicales, Geneva.

Received for publication December 1, 2006. Accepted for publication April 19, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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