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* Fondation pour Recherches Médicales, University of Geneva, 1211 Geneva, Switzerland; and
Department of Nutrition and Institut du Cancer, University of Montreal, Centre de Recherche L. C. Simard, Montreal H2L 4M1, Canada
1Correspondence: Fondation pour Recherches Médicales, University of Geneva, 64 avenue de la Roseraie, 1211 Geneva, Switzerland. E-mail: vanhaast{at}cmu.unige.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: immediate-early response genes • proto-oncogenes • intragenic response element • glucagon-like peptide 1 • intracellular Ca2+
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and translation
(2)
. In the long term, glucose also controls the
expression of a variety of glycolytic (3)
and lipogenic
(4)
genes implicated in the fuel sensing process and
promotes ß-cell growth (5)
, differentiation
(6)
, and apoptosis (7)
at elevated levels of
sugar, notably in pathophysiological situations such as obesity and
type II diabetes. If much knowledge has accumulated recently on the
short-term actions of glucose on ß-cell signaling, the mechanisms
implicated in the ß-cell adaptation to hyperglycemia are poorly
understood. Since these are long-term processes they likely
implicate changes in the expression of transcription factors,
which in turn might regulate ‘late’ genes such as cell cycle and
metabolic genes.
To link glucose signaling to the late phenotypic actions of the
sugar, we recently investigated the effect of glucose on the expression
of immediate-early genes (IEGs) coding for known transcription factors
implicated in the regulation of cell differentiation, proliferation,
and apoptosis (8
, 9)
. Glucose and other fuel secretagogues
were shown to increase the expression of IEGs (c-fos,
c-jun, junB, zif268, and
nur-77) in INS-1 ß-cell (10)
and
zif-268 in primary rat islet cells (11)
.
Physiologically, this costimulation would be expected to involve
neurohormonal agonists such as glucagon-like peptide 1 (GLP-1),
glucose-dependent insulinotropic polypeptide (GIP), and pituitary
adenylyl cyclase-activating peptide 38 (PACAP-38). GLP-1 and GIP are
released from duodenal cells after oral absorption of glucose
(12)
and PACAP-38 is detected in pancreatic nerve fibers
and rat islets (13)
. Glucoincretins enhance glucose
sensitivity of ß-cells acutely and enable an insulin secretory
response in previously glucose-unresponsive cells (14)
.
GLP-1 is also an insulinotropic agent by virtue of its stimulation of
insulin gene expression and proinsulin biosynthesis (15)
.
Our previous work in INS-1 cells demonstrated that the same
glucoincretins enable ß-cells to respond to glucose in terms of
induction of the IEGs listed above (10)
.
Glucose stimulation generates intracellular signals in
ß-cells—initially a rise in the reduction state of
pyridine nucleotides, followed by an elevation in the ATP/ADP ratio
(16
, 17)
. These changes lead to plasma membrane
depolarization and an enhancement of Ca2+ influx
(16)
. Furthermore, glucose leads to activation of the ERK
mitogen-activated protein kinase (MAPK) cascade (18)
,
phosphatidylinositide 3-kinase (PI3-K) (19
, 20)
, and
protein kinase C (PKC) (21)
, the latter as a result of
enhanced phospholipid synthesis and elevated cytosolic free
Ca2+ (16)
.
What are the signal transduction pathways and DNA elements implicated
in the induction of IEG by the combined presence of glucose and
glucoincretins? To answer this question, we have used the
c-fos gene as a paradigm of IEG induction in response to
various neurohormonal agonists. The transcription of c-fos
by a variety of neurohormonal agonists is controlled by both the
promoter and an intragenic site localized in intron 1
(22)
. The promoter regulating sequences include the
cAMP/Ca2+ (CRE) response element
(23)
and a serum response element (SRE) (24)
.
These regulatory sequences bind proteins that are differentially
sensitive to phosphorylation by
Ca2+/calmodulin-dependent protein kinase (CaM
kinase), protein kinase A (PKA), and MAPK (25)
. An
additional mechanism controlling the abundance of c-fos mRNA
is a block to transcriptional elongation situated in the proximal part
of the first intron (26)
, which can be relieved by
Ca2+ signals (27
, 28)
.
To better understand the molecular mechanism whereby glucose promotes c-fos induction, we have studied the relative contributions of various promoter and intragenic (intron 1) elements of c-fos. The results indicate that the first intron contains regulatory sequences beyond the elongation block site that play an essential and permissive role for the transcriptional activation of this IEG by glucose in the presence of glucoincretins. The CRE, but not the SRE, is an additional element implicated in the action of both glucose and glucoincretins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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32P-dCTP (3000
Ci/mmol) and nylon hybridization membranes were obtained from Amersham
(Zürich, Switzerland). Random priming kits were from Qiagen
(Basel, Switzerland). Dual-luciferase reporter assay sytem was from
Promega (Luzern Switzerland) and measured with an EG&G Berthold Lumat
LB 9507 luminometer (Bad Wildbad, Germany).
Cell culture and incubation
INS-1 cells were grown in monolayer cultures with regular RPMI
medium (3 mM glucose) supplemented with 10 mM HEPES, 10%
heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/ml
penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 50 µM
ß-mercaptoethanol at 37°C in a humidified (5%
CO2, 95% air) atmosphere. 3 x
106 cells were seeded in 24-well plates (day 0);
48 h later, cells were used for transfection (day 2).
Transfection
Transfections were performed with the DOSPER liposomal
transfection reagent from Boehringer Mannheim (Rotkreuz, Switzerland).
In each well containing 200 µl of RPMI culture medium without fetal
calf serum; 30 µl of the transfection solution was added; 800 µl of
the transfection solution HEPES-buffered saline (20 mM HEPES, 150 mM
NaCl, pH 7.4) contained 40 µl of DOSPER, 11 µg of the selected
c-fos plasmid containing the firefly luciferase reporter
gene, and 2.75 µg of Renilla luciferase for normalization. After
6 h (afternoon of day 2), cells were washed and incubated in
regular RPMI medium containing serum for the next 24 h (until
afternoon of day 3). Cells were deprived of serum, L-glutamine, and
pyruvate overnight (until morning of day 4) in modified RPMI.
Experiments were performed on day 4 (morning) in the same modified RPMI
medium deprived of serum, L-glutamine, and pyruvate.
mRNA measurements
Total RNA was extracted from cells by the guanidinium
isothiocyanate method. RNA samples (12 µg) were denatured by
incubation in glyoxal, subjected to electrophoresis in 1.2% agarose
gels, and transferred to a nylon membrane by capillarity. The mRNAs
were detected by Northern blot hybridization with
32P-labeled cDNA probes obtained by random
priming. The insert used were a c-fos EcoRI
1–2116 EcoRI fragment from rat subcloned in plasmid pSP65,
and a human alpha-actin PstI 1–720 PvuII
fragment was used to assess RNA loading on the gel. All membranes were
Coomassie blue stained as another means of controlling the amount of
total RNA loaded in each lane.
Plasmids
The PGL3 enhancer vector (Promega, Catalys AG, Luzern,
Switzerland) containing fragments of the c-fos promoter and
gene linked to the luciferase reporter gene was used for quantitative
analysis of the effect of glucose, cpt-cAMP, and glucoincretins on
c-fos reporter gene expression. Mutations and polymerase
chain reaction (PCR) products were verified by ABI PRISM dye terminator
cycle sequencing (Perkin Elmer, Rotkreuz, Switzerland). Numbers for
base positions are cited relative to the transcriptional start site of
the murine c-fos gene sequence (EMBL database access number:
V00727).
Plasmid c-fos/intron was generated by cloning the region of
the mouse c-fos proto-oncogene extending from positions
-379 to +1073 into the HindIII restriction site of the pGL3
enhancer vector. Plasmid c-fos/basic was obtained by
subcloning the -379 to +119 fragment of the mouse c-fos
gene into the HindIII site of the pGL3 enhancer vector. For
plasmid c-fos/intron(
119), the sequence between +298 to
+416 (119 bp) was deleted as follows. Using plasmid
c-fos/intron as a template, two sets of primers were
designed to amplify fragment -379 to +298 (primer definition: 5'-CCT
ACG CGT TGC ACC CTC AGA GTT GG-3' and 5'-AAC ATA TGC TCA CCT GTG TGT
TGA CA-3') and fragment +416 to +1073 (primer definition: 5'-TGC ATA
TGT CAG AGC AGC CTT AGC CT-3' and 5'-CCT AAG CTT CGG ACA GAT CTG CGC
AA-3'). The PCR reactions to amplify different parts of plasmid
c-fos/intron were performed in a final volume of 50 µl
containing 10 pg of c-fos/intron, 100–150 pmol of each
primer, 200 mM of the four deoxyribonucleotides, 1.5 mM
MgCl2, 5 µl of 10x PCR buffer, and 2.5 units
of Taq DNA polymerase. The PCR was run for 25 cycles (94°C
for 1 min, 52°C for 2 min, 72°C for 2 min), followed by a final
extension step at 72°C for 5 min. Twenty microliters of the PCR
products were run on 1% agarose gel and bands were isolated according
to the QIAEX II agarose gel extraction protocol (QIAGEN Gmbh, Ilden,
Germany). After purification, the two fragments were ligated via an
inserted NdeI restriction site. The Mlu I and
HindIII restriction sites of the pGL3 polylinker, which had
also been introduced into the external primers, were used to insert
this fragment in the pGL3 enhancer vector. To prepare plasmid
c-fos/intron(534), the sequence between +337 and +870
(534 bp) was deleted. Using plasmid c-fos/intron as
template, a set of primers was designed to amplify the -379 to +337
region (primer definition: 5'-CCT ACG CGT TGC ACC CTC AGA GTT GG-3' and
5'-GCG AAT TCG GCG GCT ACA CAA AGC CAA-3'). The +416 to +1073 fragment
was also amplified using the same set of primers used for the
preparation of plasmid c-fos/intron(119). Conditions and
purification procedures of the fragments identical to those described
above were used. The resulting +416 to +1073 fragment was then cut with
EcoRI at position +870. The +870 to +1073 fragment was then
inserted into the pGL3 enhancer vector, using the XhoI and
HindIII polylinker restriction sites, which were also
present in the external site of the primers used for amplification.
Inactivating mutations in the CRE and the SRE
The two major response elements in the c-fos promoter
were mutated following the example of Hardingham et al.
(29)
. Thus, the SRE sequence GTC CAT ATT
AGG AC (-255 to -242) was changed to GTC CCA
ATC TAG AC; the CRE sequence CCG CCC AGT
GAC GTA GGA AGT (-487 to -479) was modified to
CCC GGA AGT ACT GTC CTC CGT.
Mutagenesis was performed using the Quick-Change mutagenesis kit from
Stratagene (San Diego, Calif.). The sequences of the mutated clones
were checked using the ABI dye terminator method (Perkin Elmer
Corporation). Inactivation of SRE was demonstrated in transfection
experiments using the CHO and GH4C1 cell lines in which stimulation by
serum and PMA were abolished (data not shown).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
We thus proceeded to further study transcriptional activation of
c-fos by glucose with a reporter gene approach. Two
constructs were initially used (see Fig. 1B
). Both constructs link the c-fos promoter to the
expression of the luciferase reporter gene that was inserted in-frame
into either the first or second exon; the former (termed
c-fos/basic) includes the SRE and CRE elements from the
c-fos promoter, whereas the latter construct
(c-fos/intron) also includes the first intron as further
potential control element.
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As shown in Fig. 1B
(upper panel), cpt-cAMP stimulated the
expression of the c-fos/basic reporter gene twofold at 3 mM
glucose and threefold when combined with high (11 mM) glucose. However,
the effects were modest in comparison to the stimulation of endogenous
c-fos mRNA accumulation by the same stimuli (Fig. 1A
). A robust stimulation of luciferase activity was
observed for the c-fos/intron construct (Fig. 1B
,
lower panel): 7-fold and 16-fold for cpt-cAMP alone or in conjunction
with 11 mM glucose, respectively. The degrees of stimulation for the
c-fos/intron reporter gene construct were similar to that of
the c-fos mRNA transcript. Thus, the first intron contains
essential element(s) for the control of c-fos by glucose and
cAMP.
Glucose and glucoincretins rely on intragenic element(s) to
stimulate any c-fos reporter gene construct
We determined whether the essentiality of the first intron
was also applicable to the physiologically relevant synergistic
stimulation of c-fos transcription by elevated glucose and
glucoincretin hormones. Figure 2A
shows the Northern blot analysis of the stimulation of
endogenous c-fos transcription by elevated glucose in the
presence of three glucoincretins used at their maximally effective
concentrations (13
, 30)
: 10 nM GLP-1, 1 nM PACAP-38, and
10 nM GIP. At low glucose these hormones did not modify
c-fos mRNA level; in conjunction with high glucose, they
caused a six- to sevenfold increased c-fos mRNA expression.
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The corresponding reporter gene analysis is shown in Fig. 2B
(upper panel) for the c-fos/basic and in Fig. 2B
(lower panel) for the c-fos/intron constructs. Expression of
the c-fos/basic construct was not significantly modified by
the presence of the three glucoincretins alone or in combination with
high glucose. In contrast, the stimulation of INS-1 cells by elevated
glucose in conjunction with the three glucoincretin hormones GLP-1,
PACAP-38, and GIP resulted in a strong stimulation of the expression of
the c-fos/intron reporter gene. The data underscore the
regulatory importance of the first intron of c-fos that is
essential to obtain any c-fos expression by physiological
stimuli.
A large part of the first intron that is unrelated to the
elongation block site contributes to the control of
c-fos transcription by glucose
The only c-fos intragenic control element described to
date is present in the first intron of c-fos. It is a
transcriptional elongation block site that is derepressed by agents
raising cytosolic free Ca2+ such as the
Ca2+ ionophore A23189 (27)
. Since
glucose signals via cytosolic Ca2+ changes
(17)
, we hypothesized that dual control by promoter
elements and the Ca2+-sensitive block of
elongation are the keys to glucose control of c-fos
expression. To test this hypothesis, a 119 bp deletion variant of the
c-fos/intron reporter construct,
c-fos/intron(119), was prepared that lacked region +298
to +416 around the block site. A larger part (534 bp) of intron 1 was
also deleted from position +337 to +870, yielding the construction
c-fos/intron(534). The reporter gene expression from the
four constructs described so far were compared with respect to
stimulation by cpt-cAMP alone (white bars), cpt-cAMP with 11 mM Glucose
(gray bars), and GLP-1 with 11 mM glucose (black bars) (Fig. 3
). For all three conditions, the results were qualitatively the same. A
small 119 bp deletion in intron 1 eliminating the transcription block
site was insufficient to suppress the intragenic contribution to
expression control. The stimulation of reporter gene activity based on
the c-fos/intron(119) construct (Fig. 3B
) was
in all cases significantly better than with the c-fos/basic
construct (Fig. 3D
) but, surprisingly, was not significantly
lower than the c-fos/intron construct (Fig. 3A
).
In contrast, a larger deletion in intron 1 (534) abolished the
intron contribution to expression control. Thus, stimulation of
reporter gene activity based on the c-fos/intron(534)
construct (Fig. 3C
) was no longer different from that seen
with the c-fos/basic construct. In conclusion, the first
intron of c-fos plays an essential role in the control of
c-fos gene expression by glucose and glucoincretins based on
more sequence elements than those in the immediate vicinity of the site
where transcriptional elongation blocking occurs.
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Transcriptional activation of c-fos by glucose
involving the first intron is based on the Ca2+/cAMP
response element of the c-fos promoter
Two major candidate response elements mediating glucose action on
c-fos are CRE and SRE. Proteins interacting with CRE are
phosphorylated and activated by PKA and CaM kinases (31)
and will reflect Ca2+ and cAMP signaling by
glucose and glucoincretins, respectively. The SRE binds proteins that
are targets for MAP-kinases, which are activated by elevated glucose in
INS-1 cells (18)
. To directly assess the role of these
elements, we mutated both SRE and CRE (alone and in combination) in the
c-fos/intron reporter gene. The effects of an inactivating
mutagenesis on the stimulation of transcription by cpt-cAMP, elevated
glucose, and cpt-cAMP as well as high glucose in conjunction with GLP-1
are shown in Fig. 4
. When normalized to the stimulation by the nonmutated
c-fos/intron construct, the results were identical in all
cases. Inactivating SRE had no significant effect. In contrast,
inactivating CRE caused a more than 60% reduction of stimulation.
Inactivating SRE in addition to CRE did not cause a significant further
reduction in stimulation. This establishes CRE as an essential element
in the control of c-fos transcription by glucose and
glucoincretin hormones acting via cAMP.
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Transcriptional activation of c-fos reporter gene by
glucose and glucoincretins involves the PKA and CaM kinase signaling
pathways
To delineate the glucose signaling systems implicated in
c-fos induction, we used specific inhibitors of the major
candidate pathways. The actions of the tested inhibitors on the
transcriptional activation of the c-fos/intron reporter gene
are presented in Fig. 5
. The costimulation of 11 mM glucose and 0.5 mM cpt-cAMP (upper panel)
served as a reference (white bar) for the effects of the various
inhibitors tested singly (dotted bars) or in combination (striped
bars). Separately, the L-type Ca2+ channel
blocker nifedipine and the PKA inhibitor H89 reduced by half of the
glucose/glucoincretin stimulation. The CaM kinase II inhibitor KN-93
reduced the inductive process to an extent similar to nifedipine,
suggesting that Ca2+ acts via
Ca2+/calmodulin kinase II. This was further
confirmed by the use of combinations of inhibitors: nifedipine and
KN-93 had no additive effects. In contrast, nifedipine or KN-93 used in
conjunction with the PKA inhibitor H-89 exerted a 90% inhibition on
c-fos/intron reporter gene activation. Moreover, the PI3-K
inhibitor wortmannin (50 nM), the MEK1 inhibitor PD98059 (10 µM), the
p38 MAPK inhibitor SB203580 (5 µM), and the
Ca2+-dependent PKC inhibitor Gö6976 (50 nM)
had no inhibitory effect (data not shown). Taken together, these
results show that the PKA and CaM kinase II pathways are two major
determinants for c-fos/intron activation.
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In view of the results obtained with cpt-cAMP, we tested whether the same conclusions can be drawn for the physiologically relevant synergistic stimulation of c-fos transcription by elevated glucose and glucoincretin hormones (lower panel), since the latter could possibly stimulate c-fos/intron reporter gene through other pathways. The costimulation of 11 mM glucose and 10 nM GLP-1 was used as reference (white bar) for the effects of nifedipine, PKA, and CaM kinase inhibitors, singly (dotted bars) or combined (striped bars). Wortmannin, PD98059, SB203580, and Gö6976 (nM) had no inhibitory effects (data not shown). The results are more similar qualitatively and quantitatively than with cpt-cAMP (upper panel), and demonstrate that PKA and CaM kinase II pathways are also the major determinants in c-fos/intron activation by glucose and GLP-1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 32)
, SRE (24
, 33)
, and AP-1 binding site (34)
. Our initial
attempts to stimulate INS-1 ß-cells with a combination of glucose and
glucoincretin hormones and then to measure c-fos reporter
gene activation with a construct containing only the c-fos
promoter failed. This led us to include in the c-fos
reporter gene construct parts of transcribed regions considered so far
as nonessential in gene activation control, notably the first intron.
They proved to be crucial. We observed with the c-fos/intron
constructs considerable levels of induction that match the fold
induction of c-fos mRNA by glucose and glucoincretins.
How important is this finding in general terms? So far, the promoter
has been considered the decisive element in most studies of signaling
and gene transcription, and those studies have overlooked the possible
contribution of intragenic control elements. Furthermore, many previous
studies have addressed transcriptional control elements with a reporter
gene approach using extremely nonphysiological stimulation, e.g., with
Ca2+ ionophores, phorbol esters, and forskolin.
We show here that cpt-cAMP is able to cause c-fos reporter
gene activation in the absence of glucose, whereas GLP-1 and the other
glucoincretins could provoke these changes only in the presence of
elevated glucose and with the inclusion of intronic elements. From our
study it can thus be hypothesized that intronic elements are of
particular importance in the stimulus-transcription coupling via
moderate intracellular physiological signals. Those signals are
generated not only by glucose in the ß-cells, but by many other
modulators of neuroendocrine secretion (35)
.
Physiological signaling toward gene expression is usually accomplished
via several pathways. In our study, glucose effects based mainly on
Ca2+ signaling are dependent on the activation of
the cAMP signaling cascade by glucoincretins and the intronic control
of transcription is dependent on the CRE in the promoter. None of these
elements can independently trigger transcriptional activation, and
therefore c-fos expression is tightly controlled.
Interactive control implicating promoter and intragenic elements is a
new concept for c-fos transcription, although a few studies
of other genes (36
, 37)
suggested the general importance
of intragenic elements (38)
. We have preliminary evidence
in pituitary cells stimulated by TRH or EGF that transcription of
another IEG, mitogen-activated protein kinase phosphatase, is
controlled by intronic elements (39
; S. Ryser, and W.
Schlegel, unpublished results). This suggests that intragenic elements
are also important in the physiological transcriptional control of
other genes.
Transcription is controlled both by stimulating the assembly of the
polymerase II complex and by providing factor(s) allowing the complex
to pass negative control sequences, causing arrest or slow progression
of transcription. An early observation that hinted at the importance of
the first intron of c-fos described here was a
transcriptional block site (26)
. Relief of the block
allows rapid completion of transcript synthesis and thus reduces the
time for given stimuli to generate the requested response. It was shown
that this block could be relieved with a rise in
Ca2+ (27
, 28)
. This ion is an
essential signal for glucose-induced c-fos expression, which
is strongly blocked by inhibitors of voltage gated
Ca2+ channels (10)
. Since the CRE
mediates both Ca2+ and cAMP signaling
(32)
, the synergy between glucose and glucoincretins as
well as these two second messenger molecules could not be explained
considering only the promoter CRE because the 534 bp deletion almost
completely abolished c-fos gene activation. Using T cells,
Lee and Gilman (40)
have speculated that the synergy
arises because these stimuli affect different steps in c-fos
transcription, cAMP inducing initiation of transcription and
Ca2+ promoting elongation. Thus, the simplest
interpretation for the synergy between Ca2+
(glucose) and cAMP (GLP-1) signals was to assume that cAMP would
address the CRE and that Ca2+ would relieve the
block. We built a construct in which the region mapped in previous
studies (27
, 28)
as responsible for the transcriptional
arrest was deleted (c-fos/intron(119). This construct was
compared to the induction of the normal c-fos/intron
construct. Surprisingly, the results contradicted this view, since the
synergistic activation was maintained. We therefore had to further
define the intronic region implicated in c-fos regulation.
This was achieved using a construct in which a large region of 534 bp
after the elongation block was deleted:
c-fos/intron(534). The resultant reduction of 75% in
reporter gene induction by glucose and glucoincretins clearly indicated
that one or more control elements are present between position +337 and
+870 and confirmed the essentiality of the intron in c-fos
regulation.
A systematic search for the intronic response element(s) is a
substantial endeavor that we have begun to undertake. It should reveal
whether intronic elements are similar or identical to elements found in
promoters or whether they are novel sequences. Among the candidate
novel elements is downstream regulatory elements (DRE), an
orientation-independent derepressive element also present in the
nontranslated region of intron 1 (38)
. It is similar to an
asymmetric CRE-like sequence and shares homology with the AP-1 binding
site (34)
. As DRE interact with a
Ca2+ binding protein named DRE antagonist
modulator (DREAM) (41)
, this response element is
particularly interesting in our system. However, the site containing
the DRE was removed in the c-fos/intron(119) construct
without major consequences on the synergistic stimulation of the
reporter gene by glucose and glucoincretins. Therefore, the DRE and the
elongation block site are not the sole answers to c-fos
control by intragenic elements. Moreover, the consensus sequence for
DRE is still uncertain and DREAM homologues may exist that target other
consensus sequences. It can be speculated that the first intron
contains several response elements, each contributing to the overall
control. Indeed, elements such as Sp1-like binding sites, CRE-like
sequences, AP-1-like sequences, or Hairpin structures are located
within the 534 bp deletion of the c-fos/intron(534).
Further work will be necessary to delineate the existing candidate
response elements and to appreciate the contribution of each to
glucose-induced c-fos gene expression.
Being a target for intracellular signaling, the promoter is essential
to activate the initiation of transcription. To delineate the
c-fos promoter contribution to transcriptional activation,
we tested the consequences of inactivating mutations in the two major
promoter elements of the c-fos reporter gene construct
c-fos/intron: the CRE and the SRE. The SRE is responsible
for serum and growth factors c-fos induction (23
, 32)
through binding of the serum response factor (SRF) and is an
important target for the MAP kinase cascade (25)
. The ERK
group of MAPK phosphorylate Elk, a protein associated with SRF in order
to activate it (42)
. Moreover, it has been reported that
Ca2+ can also enhance transcription through
phosphorylation of SRF by CaM kinase. The inactivating mutations
performed on SRE did not alter c-fos reporter gene
activation, indicating that the SRE is not a relevant target of glucose
and glucoincretins signaling cascades in INS-1 cells. We confirmed this
conclusion with the use of MAPK inhibitors: ERK is known to be
implicated in c-fos gene activation by several growth
factors (25)
, but the inhibition of the MAP kinase kinase
MEK with PD98059 did not alter the inductive process of glucose and
glucoincretins. Similarly, SB203580, an inhibitor of p38, the
stress-activated protein kinase had no effect. Finally, a potential
target of glucose and GLP-1 signaling cascades described in INS-1 cells
is PI3 kinase, an enzyme implicated in ß-cell growth
(19)
. However, wortmannin, a PI3 kinase inhibitor, did not
affect c-fos reporter gene activation by glucose and
glucoincretins.
The results with the inactivated SRE are in contrast with the marked
(60%) decrease of induction measured with the c-fos
construct with an inactivated CRE. CRE binds
Ca2+/cAMP response element binding protein
(CREB), a factor known to integrate independent
Ca2+ and cAMP signaling cascades
(43)
. Glucose only slightly elevates cAMP in INS-1
ß-cell and the action of glucose and GLP-1 on the cellular cAMP
content are not additive (10)
. Thus,
Ca2+ signaling based on
Ca2+ influx likely plays the main role in the
action of glucose since inhibiting voltage gated
Ca2+ channels with nifedipine markedly reduced
c-fos reporter gene activation and c-fos mRNA
accumulation in response to the sugar (10)
. We show in the
present study that Ca2+ acts through the
activation of CaM kinase II, and that the inhibition of the enzyme by
KN-93 concomitantly with PKA inhibition by H-89 abolished 90% of the
c-fos reporter gene activation. The data demonstrate that
the regulation of the c-fos reporter gene promoter by
glucose and glucoincretins is based almost exclusively on two distinct
pathways, PKA and Ca2+, the latter promoting CaM
kinase II activation. The results also indicate that glucose-regulated
gene expression in mammalian cells occurs by diverse mechanisms. Thus,
the sugar << directly >> modulates the expression of a number of
genes such as L-type pyruvate kinase (44)
through a CACGTG
motif (45)
whereas it << indirectly >> regulates the
expression of at least another i.e., c-fos via
Ca2+ and cAMP signaling converging to the
promoter CRE and intronic elements.
In conclusion, the results underscore a synergistic interaction between glucose and three glucoincretins, GLP-1, GIP, and PACAP-38 in terms of transcriptional activation of c-fos, which is dependent on the presence of the first intron. This is of potential interest to ß-cell pathophysiology since c-fos gene activation is one of the earliest event in ß-cell activation and a candidate for the long-term phenotypic changes induced by hyperglycemia and related pathological situations, like obesity and type II diabetes. In contrast to the usual transcription analysis aimed at the gene promoter only, our study emphasizes the importance of downstream elements for the control of gene transcription. It will be of interest to determine whether the essentiality of intronic elements in gene control is a general phenomenon in higher eukaryotic cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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