From the Department of Molecular Pharmacology, University of Göttingen, D-37070 Göttingen, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
To maintain blood glucose levels within narrow
limits, the synthesis and secretion of pancreatic islet hormones is
controlled by a variety of extracellular signals.
Depolarization-induced calcium influx into islet cells has been shown
to stimulate glucagon gene transcription through the transcription
factor cAMP response element-binding protein that binds to the glucagon
cAMP response element. By transient transfection of glucagon-reporter
fusion genes into islet cell lines, this study identified a second
calcium response element in the glucagon gene (G2 element, from Activation of gene transcription allows cells to adapt to changes
in environmental conditions through a new pattern of expressed proteins. In cells that are electrically excitable, calcium is an
important intracellular second messenger that directs the genomic response of the cell. Transcription factors that have been shown to
mediate calcium-induced gene transcription include
CREB1 (1-4), serum response
factor (2, 5), and C/EBP Like neurons, endocrine cells of the pancreatic islets are electrically
excitable and express L-type voltage-dependent calcium channels (7, 8). By virtue of stimulation of glycogenolysis and
gluconeogenesis in the liver, the islet hormone glucagon is an
important regulator of blood glucose levels (9). Glucagon-producing islet cells show spontaneous electrical activity (10). Membrane electrical activity and calcium influx into glucagon-producing pancreatic islet cells is tightly controlled by extracellular messengers. Whereas L-arginine increases spike frequency
(10), Previous experiments have shown that membrane depolarization and
calcium influx stimulate glucagon gene transcription (3). A mechanism
involved has been characterized. Through a
calcium-/calmodulin-dependent protein kinase calcium
stimulates the phosphorylation of the transcription factor CREB on the
same serine residue that is also phosphorylated by
cAMP-dependent protein kinase A. CREB binds to a CRE in the 5'-flanking region of the glucagon gene and stimulates transcription (3, 13-15). The present study addressed the question whether there are
additional calcium-responsive elements in the glucagon gene. By the
results obtained, the G2 element is identified as a second calcium
response element. The further characterization suggests that calcium
responsiveness is conferred by the calcium/calcineurin-regulated transcription factor NFATp functionally synergizing with the
cell-specifically expressed transcription factor HNF-3 Plasmids--
The plasmids Cell Culture and Transfection of DNA--
The pancreatic islet
cell line HIT-T15 (27) was grown in RPMI 1640 medium supplemented with
10% fetal calf serum, 5% horse serum, 100 units/ml penicillin, and
100 µg/ml streptomycin. Cell Extracts--
Nuclear extracts were prepared from Electrophoretic Mobility Shift Assay--
Synthetic
complementary oligonucleotides with 5'-GATC overhangs were annealed and
labeled by a fill-in reaction with [ Oligonucleotides--
The sequences of the G2 oligonucleotides
(wild type and mutants 1, 3, and 5) were as described previously
(20) and read as follows (only one strand with the 5'-GATC overhang is
shown, and mutated bases are underlined): G2,
5'-GATCCAGGCACAAGAGTAAATAAAAAGTTTCCGGGCCTCTGA-3'; G2 m1,
5'-GATCCAGGCACAGTTCGAAATAAAAAGTTTCCGGGCCTCTGA-3'; G2 m3, 5'-GATCCAGGCACAAGAGTAAATAAGGGATTTCCGGGCCTCTGA-3'; G2 m5,
5'-GATCCAGGCACAAGAGTAAATAAAAAGTTTGGGGGCCTCTGA-3'. The
sequence of the oligonucleotide (NFATcons) containing a well characterized NFAT-binding site was as described (36) except that
BamHI (upstream) and BglII ends (downstream) with
5'-GATC overhangs were added.
RT-PCR--
Poly (A)+ RNA was extracted from Western Blot--
Nuclear extracts were resolved on a 5.5%
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
incubated for 2 h in 10% nonfat dry milk dissolved in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2% Tween
20), incubated for 1 h in 2% gelatin dissolved in TBST, and then
incubated with anti-NFATp antiserum (anti-67.1) (36), diluted 1:3,000
in TBST, overnight at 4 °C. Antibody-antigen complexes were detected
with ECL reagents (Amersham). Using the same samples, immunoblots were
performed with an anti-CREB antiserum as has been described previously
(15).
Materials--
A stock solution of TPA (1 mM) was
prepared in dimethyl sulfoxide and further diluted in cell culture
medium. FK506 (provided by Fujisawa) was dissolved in ethanol. Controls
received the solvent only.
Identification of a Second Calcium-responsive Element in the
Glucagon Gene--
To study the calcium responsiveness of the
glucagon gene in the absence of a functional CRE, the CRE in the
5'-flanking region of the rat glucagon gene was removed by either
5'-deletion to
Known elements within the enhancer region of the glucagon gene include
the G2 element (20, 40, 41), the G3 element (30, 40, 42-44), as well
as a binding site for C/EBP proteins (CS) (23) (Fig.
2). To study their role, four copies of
oligonucleotides containing the G2 element (from Characterization of Protein Binding to the G2 Element--
The G2
element has been shown before to confer cell-specific basal activity
(20, 40, 41) and Ras/protein kinase C responsiveness (20) to the
glucagon gene. The binding of two transcription factors is required for
both of these activities in
The binding specificity of the depolarization-induced protein complex
was studied using G2 mutants. The depolarization-induced nuclear
protein complex did bind to labeled mutant 1 (Fig. 3B, lanes
7 and 8) but not to labeled mutant 5 (Fig.
3B, lanes 11 and 12). In mutant 1 and
mutant 5, 5 and 2 bases, respectively, have been mutated that are
required for the binding of HNF-3
The binding specificity was further characterized in competition
experiments using G2 wild type, mutant 1, mutant 5, and also mutant 3. In mutant 3, 4 bases have been mutated (see top of Fig. 3A). As shown in Fig. 3C, the binding of HNF-3
The mutations in mutants 3 and 5 alter a sequence within G2,
5'-GGAAACTT-3', which shares extensive homology with a sequence in the
murine IL-2 gene that binds transcription factors of the NFAT family
(match of 7 bases out of 8) (18, 19). In T cells, NFAT transcription
factors have been shown to undergo a calcium- and
calcineurin-dependent translocation from the cytosol to the nucleus (18, 19, 46). Therefore, a possible relationship between the
depolarization-induced G2-binding protein complex and NFAT was studied.
First, the oligonucleotide NFATcons, which contains a well
characterized NFAT-binding site, was used as probe and cold competitor
in electrophoretic mobility shift assays. As shown in Fig.
3D, NFATcons specifically competed for the binding of the
depolarization-induced nuclear protein to labeled G2 (lane 4). When NFATcons was used as probe, membrane depolarization
induced the binding of a protein complex that comigrated with the
depolarization-induced nuclear protein binding to labeled G2 (Fig.
3D, compare lane 7 with lanes 5 and
2; the depolarization-induced protein complexes are
indicated by arrows); it also shown a binding specificity that was indistinguishable from that of the depolarization-induced nuclear binding to labeled G2, since the depolarization-induced binding
to labeled NFATcons was competed away by G2 and NFATcons but not by G2
mutant 5 (Fig. 3D). In addition to the
depolarization-induced nuclear protein, several additional complexes
were detected with labeled NFATcons; these were not further
characterized. The cross-competition between G2 and NFATcons supports
the notion that the depolarization-induced nuclear complex on G2 has a
binding specificity that is related to NFAT. Second, the effect of
FK506 was studied. FK506 is an inhibitor of the
calcium-/calmodulin-dependent protein phosphatase calcineurin (47). Previous studies have shown that FK506 and cyclosporin A effectively inhibit calcineurin activity also in pancreatic islet cells (14, 15). Whereas membrane depolarization induced the binding of a protein complex to the G2 element in controls
(Fig. 3E, lanes 5 and 6), treatment of
the cells with FK506 (167 nM) blocked this effect (Fig.
3E, lanes 7 and 8), suggesting that
the induction by membrane depolarization of nuclear protein binding to
G2 depends on calcineurin phosphatase activity. Third, the effect of an
antiserum directed specifically against NFATp was studied (donated by
A. Rao, Boston, MA) (36). Whereas the addition of a preimmune serum had
no effect, the addition of the anti-NFATp antiserum abolished
specifically the depolarization-induced G2-binding protein (Fig.
3F, compare lane 6 with lanes 4 and
2), indicating that this protein complex possesses
NFATp-like immunoreactivity. Finally, using another protocol, separate
nuclear and cytosolic extracts were prepared and used in
electrophoretic mobility shift assays. A G2-binding protein complex was
found in cytosolic extracts from unstimulated cells that comigrated
with the nuclear complex induced by membrane depolarization (Fig.
3G, compare lane 7 with lane 2).
Concomitant with the induction of protein binding in nuclear extracts,
membrane depolarization markedly decreased the binding of this complex
in cytosolic extracts (Fig. 3G, compare lane 7 with lane 8), consistent with a depolarization-induced translocation from the cytosol to the nucleus. The binding of both the
protein complex induced by membrane depolarization in nuclear extracts
as well as the binding of the comigrating protein complex in cytosolic
extracts from unstimulated cells was not affected by the addition of a
preimmune serum but was abolished by the addition of an anti-NFATp
antiserum (Fig. 3G). Taken together, these results indicate
that the depolarization-induced nuclear G2-binding protein is NFATp
which in pancreatic islet cells undergoes a calcium- and
calcineurin-dependent translocation from the cytosol to the
nucleus in response to membrane depolarization.
The results obtained in the electrophoretic mobility shift assay
indicate that NFATp is expressed in pancreatic islet Functional Analysis of NFATp and HNF-3 Role of the G2 Element and NFATp in the Regulation of the Intact
Glucagon Promoter by Membrane Depolarization--
To study the role of
the G2 element in depolarization-induced activation of the intact
glucagon promoter, glucagon-reporter fusion genes were used that carry
an internal deletion of the G2 element without (
To explore further the role of the G2-binding transcription factor
NFATp for glucagon promoter activity, FK506 and the expression vector
pBK-CMV-NFATpDBD were used. When nuclear translocation of NFATp was
blocked by FK506, the activation of glucagon promoter activity by
membrane depolarization and calcium influx was markedly reduced (Fig.
7B), suggesting that calcium-induced activation of the
glucagon promoter depends on NFATp binding. In these experiments 292 base pairs of the glucagon promoter were used which lack the CRE,
because the transactivation of serine 119-phosphorylated CREB is
inhibited by FK506 (13-15). Although the minimal DNA-binding domain of
NFATp is sufficient for cooperation with Fos and Jun proteins, it lacks
an intrinsic transactivation domain (25) and, in the absence of
interaction with AP-1 on composite NFAT-binding sites, is thus expected
to inhibit NFAT transactivation when overexpressed by blocking
NFAT-binding sites (25). As shown in Fig. 7C, overexpression of the NFATp DNA-binding domain inhibited the depolarization-induced activation of glucagon gene transcription by 40%. The inhibition of
depolarization-induced activation of the glucagon promoter by NFATpDBD
was less than that by G2 deletion or FK506, which may be explained by a
lower efficiency of NFATpDBD overexpression. Taken together, all these
data suggest a role of the G2 element and of NFATp in the regulation of
the intact glucagon promoter by membrane depolarization.
In this report we describe experiments that have led to the
identification and characterization of a second calcium response element within the glucagon promoter. Evidence is presented that the
transcription factor NFATp is expressed in glucagon-producing islet
cells and is directed to the nucleus by calcium/calcineurin in response
to membrane depolarization. NFATp is known to cooperate with AP-1
proteins in T cells (16-19). By contrast, this study shows a novel
pairing of NFATp with the cell-specific transcription factor HNF-3 Calcium influx through voltage-dependent calcium channels
stimulates both glucagon secretion (9) and biosynthesis through gene
transcription (3, 13, 15). The present study shows that, in addition to
the glucagon CRE described previously (3, 13, 15), the glucagon G2
element is a second calcium response element of the glucagon gene. As
is indicated by the effects of internal deletions of the CRE and G2,
both elements are required for full depolarization responsiveness of
the glucagon gene, similar to what has been shown for the calcium
regulation of the c-fos gene by the c-fos CRE and
the c-fos serum response element (5, 48). Using primary
hippocampal neurons, Bading et al. (49) demonstrated that
calcium, depending on its mode of entry into neurons, activates
distinct signaling pathways that lead to gene activation via different
cis-acting regulatory elements. Furthermore, differential
activation of transcription factors induced by calcium response
amplitude and duration has been shown in B lymphocytes (50). Thus, it
is possible that depending on the type of the calcium signal the G2
element and the glucagon CRE could subserve distinct functions in
calcium signaling to the glucagon gene. The ability of membrane
depolarization to potentiate cAMP-induced glucagon gene transcription
in a synergistic fashion (3) may be physiologically important. Through
synergistic interaction the calcium response of G2 and the CRE may
participate in a cross-talk between intracellular signaling systems
that in A cells integrates multiple stimuli to an appropriate
transcriptional response of the glucagon gene in order to maintain
glucose homeostasis.
NFATp was originally characterized as the preexisting cytoplasmic
component of a transcription factor in T cell hybridomas implicated in
the induction of several cytokine genes during the immune response
(19). NFATp has been molecularly cloned (19, 32) and is now known to be
a member of a family of transcription factors that contain at least
four members and share a 165
to
200). Membrane depolarization was found to induce the binding of a
nuclear complex with NFATp-like immunoreactivity to the G2 element.
Consistent with nuclear translocation, a comigrating complex was found
in cytosolic extracts of unstimulated cells, and the induction of
nuclear protein binding was blocked by inhibition of calcineurin
phosphatase activity by FK506. A mutational analysis of G2 function and
nuclear protein binding as well as the effect of FK506 indicate that
calcium responsiveness is conferred to the G2 element by NFATp
functionally interacting with HNF-3
binding to a closely associated
site. Transcription factors of the NFAT family are known to cooperate
with AP-1 proteins in T cells for calcium-dependent
activation of cytokine genes. This study shows a novel pairing of NFATp
with the cell lineage-specific transcription factor HNF-3
in islet
cells to form a novel calcium response element in the glucagon gene.
INTRODUCTION
Top
Abstract
Introduction
References
(6).
-adrenergic cell-surface receptor stimulation by
catecholamines through cAMP enhances the L-type calcium current,
increasing the influx of calcium associated with each action potential
(11). It is well known that membrane depolarization and calcium influx increase the cytosolic free calcium concentration, which stimulates hormone secretion by exocytosis (8, 12). The question is whether, and
if so, how, this calcium signal reaches also into the nucleus and
regulates gene transcription.
. NFAT family
proteins are known to cooperate with newly synthetized AP-1 proteins in
T cells (16-19). This study shows a novel pairing of NFATp with the
constitutively expressed cell-specific transcription factor HNF-3
forming a novel calcium response element in the glucagon gene.
EXPERIMENTAL PROCEDURES
350GluLuc (3),
350(
297/
292)GluLuc (3),
350
GluLuc (20),
292GluLuc (20),
pT81Luc (21), 4xG3T81Luc (22), 4xCST81Luc (23), 4xGluCRET81Luc (24),
4xG2T81Luc, 4xG2 m1T81Luc, 4xG2 m3T81Luc, and 4xG2 m5T81Luc (20) have
been described previously. The plasmid
350(2
)GluLuc was prepared by subcloning the SstI fragment of
350
GluLuc into the
SstI site of pT81
N (pT81Luc (21) with the
AatII site deleted); four bases in the CRE octamer (from
296 to
293) were then deleted with the restriction enzyme
AatII and T4 DNA polymerase; after religation the
SstI fragment was subcloned into the SstI site of
pXP2 (21). An expression vector encoding the DNA-binding domain of
NFATp (amino acids 398-584) (19, 25) (pBK-CMV-NFATpDBD) was prepared by PCR using pLGPmNFAT1-B (26) as template and the oligonucleotides 5'-GATTGAGCTCGCAGCTCCACGGCTACAT-3' and
5'-GCGCGAATTCTCCACCGTAGCTTCCATC-3' as upstream and downstream primers,
respectively; the PCR product was digested with SacI and
EcoRI and subcloned into the
SacI-EcoRI sites of pBK-CMV (Stratagene,
Heidelberg, Germany). All constructs were confirmed by sequencing.
TC2 cells (28, 29) were grown in
Dulbecco's modified Eagle's medium (4.5 g of glucose/liter)
supplemented with 2.5% fetal calf serum, 15% horse serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were
trypsinized and transfected in suspension by the DEAE-dextran method
(3) with 2 µg of indicator plasmid per 6-cm dish. Rous sarcoma
virus-chloramphenicol acetyltransferase plasmid (0.4 µg/6-cm dish)
was added as a second reporter to check for transfection efficiency.
When indicated, 0.7 µg of pBK-CMV-NFATpDBD was co-transfected per
6-cm dish; these co-transfections were done with a constant DNA
concentration, which was maintained by adding Bluescript (Stratagene, Heidelberg, Germany). Cells were stimulated with high KCl (45 mM final concentration) or TPA (300 nM) for
6 h before harvest. FK506 (167 nM, unless noted
otherwise) was added 1 h before stimulation. Cell extracts (3)
were prepared 48 h after transfection. A chromatographic
chloramphenicol acetyltransferase assay was performed as described
previously (30). Thin layer chromatography plates were analyzed with a
Fuji PhosphorImager. The luciferase assay was performed as described
previously (3).
TC2
cells by the method of Schreiber et al. (31), except for the
experiments shown in Fig. 3G. In the experiments shown in
Fig. 3G, separate nuclear and cytosolic extracts were
prepared as described previously (32-34).
-32P]dCTP and
Klenow enzyme. By using 15 µg of protein from cell extracts, the
electrophoretic mobility shift assay was performed as described by
Klemsz et al. (35). In some binding reactions, a specific
anti-NFATp antiserum (kindly provided by A. Rao, Harvard Medical
School, Boston) was used. This antiserum (anti-67.1) is directed
against the 67.1 peptide of murine NFATp; it does not cross-react with
NFATc, NFAT3, or NFATx (36). Cell extracts were incubated with 1 µl
of a 1:10 dilution of preimmune serum or anti-NFATp antiserum in the
reaction buffer with probe for 15 min at room temperature. Following a
15-min incubation on ice, the samples were loaded onto the gels and
electrophoresed as described above.
TC2
cells using a commercial kit (Fast Track 2.0TM,
Invitrogen). RT-PCR was performed using a commercial kit (Gene AmpTM Thermostable rTth Reverse Transcriptase RNA PCR Kit,
Roche Molecular Systems) with primers and PCR reaction conditions as
follows: upstream primer 5'-AGTCCCCAAGACGAGCT-3', downstream primer
5'-CGGGCTCAGAAAGTTCTGG-3'; 15 s at 95 °C, 30 s at
57 °C, 45 s at 72 °C, for 34 cycles; the expected product is
234 base pairs long. After agarose gel electrophoresis, the product
obtained was verified by extraction, subcloning (pCR© 2.1, Invitrogen), and cycle sequencing (Thermo Sequenase fluorescent labeled
primer cycle sequencing kit, Amersham, Braunschweig, Germany; M13
fluorescent primer).
RESULTS
292 or by internal deletion of four bases within the
CRE octamer motif, yielding the constructs
292GluLuc and
350(
297/
292)GluLuc, respectively (Fig.
1). The corresponding glucagon-reporter
fusion genes were transiently transfected into two pancreatic islet
cell lines, HIT and
TC2. These cell lines have been used previously to demonstrate that membrane depolarization and calcium influx induce
glucagon gene transcription through the CRE of the glucagon gene (3,
13-15). HIT cells (27) express glucagon, although only at a low level
(37, 38), and have the advantage to respond to second messenger
stimulation and to be well characterized with respect to electrical
activity, voltage-dependent calcium channels, cytosolic
calcium concentration, and secretion (see Ref. 3). Increases in
extracellular potassium concentration to 40 mM have been
shown to depolarize HIT cells with action potentials continuing at the
peak of the depolarizing phase of the spontaneous activity; at the same
time, high potassium initiates the influx of calcium through
dihydropyridine-sensitive, L-type Ca2+ channels
in the cell membrane and elevates the cytosolic calcium concentration
(39). Membrane depolarization was induced by elevating the potassium
chloride concentration in the incubation medium from 5 to 45 mM. As shown in Fig. 1B, the high
potassium-induced increase in glucagon gene transcription was decreased
in HIT cells by 77% by the 4-base deletion in the CRE octamer motif,
as has been reported previously (3). It was decreased by 46% when the
CRE was removed by 5'-deletion to
292 (Fig. 1B). Basal
reporter activity was not changed by these deletions (data not shown). Thus, although the depolarization-induced increase in glucagon gene
transcription was reduced using both constructs, glucagon gene
transcription did still respond to membrane depolarization, resulting
in a 2.0- and 3.3-fold stimulation of transcription in the absence of
the CRE, respectively (Fig. 1B). After transfection of
292GluLuc, the stimulation of transcription by high potassium was
abolished when extracellular calcium was bound by 1.5 mM
EGTA added to the medium (data not shown), suggesting that gene
induction by membrane depolarization depends on calcium influx
elevating intracellular calcium levels. The pancreatic
-like cell
line
TC2 has been established from a glucagonoma arising in
transgenic mice expressing the SV40 large T-antigen oncogene (28, 29). These cells express predominantly glucagon in a rather uniform pattern
(28). In
TC2 cells, the depolarization-induced stimulation of
glucagon gene transcription was only slightly reduced by 5'-deletion to
292 or by the internal deletion of four bases within the CRE octamer
(Fig. 1C). When compared with
350GluLuc, basal reporter activity of
350(
297/
292)GluLuc was not changed, and basal
reporter activity of
292GluLuc was reduced to 38 ± 7%
(n = 12). The results obtained in both pancreatic islet
cell lines thus indicate that besides the CRE the rat glucagon gene
5'-flanking region contains at least one more calcium-responsive
element within 292 bases in front of the transcription start site.
View larger version (15K):
[in a new window]
Fig. 1.
Stimulation of glucagon reporter gene
expression by high potassium-induced membrane depolarization in the
absence of a functional CRE. A, relative position of
the CRE octamer 5'-TGACGTCA-3' in the rat glucagon gene 5'-flanking
region. In the plasmid 350GluLuc, the rat glucagon gene from
350 to
+58 was fused to the coding region of the luciferase reporter gene
(LUC). The CRE was removed by either 5'-deletion to
292
(plasmid
292GluLuc) or by internal deletion of four bases within the
CRE octamer motif (plasmid
350(
297/
292)GluLuc). The plasmids
350GluLuc,
350(
297/
292)GluLuc, and
292GluLuc were
transiently transfected into the pancreatic islet cell lines HIT
(B) or
TC2 (C). The cells were stimulated by
high potassium-induced membrane depolarization (KCl, 45 mM). Luciferase activity is expressed as percentage of the
mean value in each experiment of the activity measured in the
respective controls (5 mM KCl). Values are mean ± S.E. of three independent experiments, each done with two
(B) or four (C) dishes.
200 to
165), the
G3 element (from
274 to
234), or the CS element (from
241 to
212) were placed in front of the truncated viral thymidine kinase
promoter of herpes simplex virus (from
81 to +52) fused to the
luciferase reporter gene (plasmid pT81Luc) (21). As shown in Fig. 2,
the promoter alone did not respond to membrane depolarization in
TC2 cells. The G3 element and the binding site for C/EBP proteins were also
inactive (Fig. 2). However, the G2 element did confer depolarization
responsiveness to the promoter (Fig. 2). The blocker of L-type,
voltage-dependent calcium channels, diltiazem, inhibited the high potassium-induced transcriptional activation of the G2 element
(data not shown). Depolarization responsiveness conferred by the G2
element to the nonresponsive thymidine kinase promoter was only
somewhat less than that conferred by the glucagon CRE. When in separate
experiments the plasmids 4xG2T81Luc and 4xGluCRET81Luc were transfected
in parallel into
TC2 cells, membrane depolarization stimulated
transcription to 182 ± 14% of controls (n = 6)
through the G2 element and to 225 ± 18% of controls
(n = 6) through the glucagon CRE. These data indicate
that the G2 element is a calcium-responsive element of the glucagon
gene.
View larger version (20K):
[in a new window]
Fig. 2.
The G2 element of the glucagon gene confers
depolarization responsiveness. Constructs with the luciferase
reporter gene under the control of four copies of the G3 element, the
G2 element, or a C/EBP-binding site in the glucagon gene (CS) linked to
a truncated viral thymidine kinase promoter were transiently
transfected into TC2 cells. The relative position of these elements
in the rat glucagon gene 5'-flanking region is shown at the
top of the figure. TK, promoter alone (pT81Luc);
292, plasmid
292GluLuc. The cells were stimulated by high
potassium-induced membrane depolarization (KCl, 45 mM).
Luciferase activity is expressed as percentage of the mean value in
each experiment of the activity measured in the respective controls (5 mM KCl). When compared with pT81Luc (100 ± 12%), the
basal activity of the constructs was 1,196 ± 193% (4xG3T81Luc),
77 ± 9% (4xCST81Luc), 184 ± 22% (4xG2T81Luc), and
6,020 ± 752% (
292GluLuc). Values are mean ± S.E. of
three independent experiments, each done in duplicate.
TC2 cells, HNF-3
and an Ets-like
protein (20). However, the precise role of HNF-3
in the
islet-specific, basal transcriptional activity of G2 remains to be
defined (20, 41). To study the effect of membrane depolarization and
calcium influx on nuclear protein binding to the G2 element,
electrophoretic mobility shift assays were performed. Nuclear extracts
were prepared from pancreatic islet
TC2 cells with or without
stimulation by membrane depolarization. It has been shown previously
that under basal nonstimulated conditions band h (Fig.
3A) represents the binding of
HNF-3
, whereas several weak bands collectively labeled e
(Fig. 3A) represent proteins with a binding specificity
related to the Ets family of transcription factors (20). Membrane
depolarization for 15 or 120 min induced the binding of a protein
complex that migrated among the bands e (Fig. 3A, marked by
an arrow). Whereas the intensities of some bands e varied
somewhat between the experiments, the depolarization-induced nuclear
protein binding was consistently observed (Fig. 3,
A-F).
View larger version (56K):
[in a new window]
Fig. 3.
Membrane depolarization induces protein
binding to the G2 element adjacent to the binding of
HNF-3 as revealed by the electrophoretic
mobility shift assay. A, depolarization-induced nuclear
protein binding.
TC2 cells were stimulated for 15 or 120 min with
high potassium-induced membrane depolarization (KCl, 45 mM)
or were left unstimulated. Nuclear extracts were prepared and incubated
with labeled G2 oligonucleotide (wild type). It has been shown
previously that under basal nonstimulated conditions band h
represents the binding of HNF-3
, whereas several weak bands
collectively labeled e represent proteins with a binding
specificity related to the Ets family of transcription factors (20).
f, free probe; ns, nonspecific bands. The
arrow indicates the depolarization-induced protein binding.
The G2 wild-type sequence is presented at the top of the
figure, and the bases mutated in mutants 1, 3, and 5 are indicated. The
boxed sequence motifs show the homology to the HNF-3
consensus binding site (20, 45) as well as the GGAA motif, an essential
purine-rich core of binding sites for members of the Ets family of
transcription factors. Also boxed is a sequence,
5'-GGAAACTT-3', which shares extensive homology with a sequence in the
murine IL-2 gene that binds transcription factors of the NFAT family (18). B, binding
specificity as analyzed by using wild-type and mutant probes. Labeled
G2, mutant 1 (m1) or mutant 5 (m5) were incubated
with nuclear extracts prepared from
TC2 cells that had been
stimulated for 120 min with high potassium-induced membrane
depolarization (KCl, 45 mM) or had been left unstimulated.
The arrow indicates the depolarization-induced protein
complex. f, free probe; ns, nonspecific bands.
C, binding specificity as analyzed in competition
experiments. Labeled G2 (wild type) was incubated with nuclear extracts
prepared from
TC2 cells that had been stimulated for 120 min with
high potassium-induced membrane depolarization (KCl, 45 mM)
or had been left unstimulated. Competitors were added at a 500-fold
(lanes 3-6) or 175-fold (lanes 8-11) molar
excess as indicated. wt, wild type; m1,
3, or 5, mutants 1, 3, or 5. The arrow
indicates the depolarization-induced protein binding. f,
free probe; ns, nonspecific bands. D,
cross-competition between G2 and an oligonucleotide containing a well
characterized NFAT-binding site, NFATcons, for binding of the
depolarization-induced nuclear protein. Labeled G2 or labeled NFATcons
were incubated with nuclear extracts prepared from
TC2 cells that
had been stimulated for 30 min with high potassium-induced membrane
depolarization (KCl, 45 mM) or had been left unstimulated.
Competitors were added at a 500-fold molar excess. m5, G2
mutant 5. The arrows indicate depolarization-induced protein
binding. f, free probe; ns, nonspecific bands.
The asterisk indicates HNF-3
binding to labeled G2.
E, effect of FK506 on the induction of nuclear protein
binding by membrane depolarization. Labeled G2 was incubated with
nuclear extracts from
TC2 cells that had been stimulated with high
potassium-induced membrane depolarization (KCl, 45 mM) for
120 min or had been left unstimulated. FK506 (167 nM) was
added to the cultures 1 h prior to KCl. The arrow
indicates the depolarization-induced protein binding. f,
free probe; ns, nonspecific bands. F, the
depolarization-induced nuclear G2-binding protein is recognized by a
specific anti-NFATp antiserum. Labeled G2 was incubated with nuclear
extracts prepared from
TC2 cells that had been stimulated for 60 min
with high potassium-induced membrane depolarization (KCl, 45 mM) or had been left unstimulated. Preimmune serum
(Pre) or anti-NFATp antiserum (Anti-NFATp) were
added to the binding reaction as indicated on top of the
lanes. The arrow indicates the
depolarization-induced protein binding. The asterisk
indicates a retarded band that appeared in the presence of anti-NFATp
antiserum when added to the binding reaction with nuclear extracts from
stimulated cells ("super-shifted band"). f, free probe;
ns, nonspecific bands; h, HNF-3
. G,
evidence for depolarization-induced translocation from the cytosol to
the nucleus of a protein with NFATp-like immunoreactivity. Separate
nuclear and cytosolic extracts were prepared from
TC2 cells
stimulated or not with high potassium-induced membrane depolarization
(KCl, 45 mM, for 120 min) and incubated with labeled G2
oligonucleotide. Preimmune serum (Pre) or anti-NFATp
antiserum (Anti) as indicated on top of the lanes
were added to the binding reaction. The arrow on the
left indicates the depolarization-induced nuclear protein
binding, whereas the arrow on the right indicates
a comigrating complex in cytosolic extracts from unstimulated cells.
Note that the depolarization-induced nuclear protein binding and the
comigrating complex in cytosolic extracts from unstimulated cells are
abolished by the anti-NFATp antiserum. The asterisk
indicates a retarded band that appeared in the presence of anti-NFATp
antiserum when added to the binding reaction with nuclear extracts from
stimulated cells (super-shifted band). f, free probe;
ns, nonspecific bands.
and the Ets-like proteins,
respectively (see Ref. 20; see also top of Fig.
3A). Consequently, band h was not formed when G2 mutant 1 was used as probe, and bands e were not detectable when G2 mutant 5 was
used as probe (Fig. 3B). This indicates that the
depolarization-induced nuclear complex binds to a site within G2 that
is distinct from the HNF-3
-binding site; in contrast, the two
guanine bases (noncoding strand) exchanged in mutant 5 are essential
for binding (see top of Fig. 3A).
(band h) was competed away by mutants 5 and 3 but not by mutant 1, which carries mutations within the HNF-3
-binding sequence. In
contrast, mutant 1 competed for the binding of the
depolarization-induced protein as well as did the wild-type sequence
(Fig. 3C, compare lane 4 with lane 3 and lane 9 with lane 8), whereas mutant 3 and,
even more so, mutant 5 were less effective competitors (Fig.
3C). This suggests that the depolarization-induced protein
complex has critical contacts with bases that are mutated in mutants 3 and 5.
TC2 cells. This
was further investigated using different approaches. In an RT-PCR
analysis, a primer pair that is specific for NFATp generated from
poly(A)+ RNA of
TC2 cells a product of the expected size
(Fig. 4A). Subcloning and
sequencing confirmed that this product is NFATp cDNA, indicating that NFATp is expressed at the RNA level. A more detailed analysis using primer pairs, which target the divergent C-terminal ends of NFATp
isoforms (19, 26), detected transcripts encoding the B and C isoforms
but not the A isoform (not shown). To confirm further the expression of
NFATp at the protein level Western blotting was used. NFAT proteins are
known to migrate on SDS gels with an apparent molecular mass of
120-140 kDa (19). As shown in Fig. 4B (upper
panel), a protein with NFATp-like immunoreactivity and an apparent
molecular mass of about 120 kDa was detected in nuclear extracts from
TC2 cells that had been stimulated by membrane depolarization. No
such band was detected in nuclear extracts from unstimulated cells
(Fig. 4B, lane 1). As a control for the quality
of the nuclear extracts, an antiserum directed against a constitutively
nuclear protein, CREB, was also used and yielded bands of about 43 kDa
with similar signal intensities in both extracts (Fig. 4B,
lower panel). Therefore, the results obtained in the
electrophoretic mobility shift assay, in the RT-PCR analysis, and in
Western blotting experiments concur that NFATp is expressed in these
cells. When taken together, our data suggest that membrane depolarization induces in pancreatic islet cells through activation of
calcineurin the nuclear translocation of NFATp, which binds to the G2
enhancer-like element of the glucagon gene.
View larger version (18K):
[in a new window]
Fig. 4.
Expression of NFATp in pancreatic islet
TC2 cells. A, RT-PCR of NFATp
transcripts. The relative position of the primers and the size of the
expected fragment are indicated. An RT-PCR product of the expected size
was obtained as shown by agarose gel electrophoresis (lane
2). Lane 1, size markers. The RT-PCR product was
verified by subcloning and sequencing. B, Western blot.
Nuclear extracts from
TC2 cells that had been stimulated for 60 min
with high potassium-induced membrane depolarization (KCl, 45 mM) or had been left unstimulated were subjected to
immunoblotting. Upper panel, anti-NFATp antiserum;
lower panel, anti-CREB antiserum.
Binding to the G2
Element--
To study the role of NFATp binding in calcium-induced
activation of G2 transcriptional activity, transfection experiments were performed. As shown in Fig. 5, when
nuclear translocation of NFATp was blocked by FK506 (167 nM), the activation of G2 transcriptional activity by
membrane depolarization and calcium influx was markedly reduced. This
suggests that calcium-induced activation of G2 transcriptional activity
depends on calcineurin phosphatase activity and NFATp binding. The
regulatory sequences within G2 that are required for depolarization
responsiveness were characterized. Four copies of G2 wild-type sequence
or mutants 1, 3, and 5 were placed in front of the heterologous
thymidine kinase promoter of herpes simplex virus (pT81Luc). In
contrast to the G2 wild-type sequence, mutants 1, 3, and 5 failed to
confer depolarization responsiveness (Fig.
6). Thus, the depolarization
responsiveness of G2 was abolished by mutant 1 as well as by mutants 3 and 5, which interfere with the binding of HNF-3
(mutant 1) as well
as with the depolarization-induced NFATp binding (mutants 3 and 5).
Membrane depolarization was unable to stimulate transcription through
four copies of a high affinity AP-1 site or a composite IL-2 NFAT:AP-1
site (data not shown). Taken together, the results of the present study
suggest that the depolarization responsiveness of G2 may depend on the
binding of both HNF-3
and NFATp. It is noteworthy that, in contrast
to membrane depolarization, the protein kinase C-activating phorbol ester TPA stimulated transcription through mutant 3 at least as well as
through G2 wild-type sequence (Fig. 6), consistent with published data
(20). This indicates that distinct proteins mediate protein kinase C
and depolarization responsiveness of the G2 element. Whereas HNF-3
and an Ets-like protein have been shown to confer Ras and protein
kinase C responsiveness (20), NFATp and HNF-3
are required for the
stimulation of G2 activity by membrane depolarization and calcium
influx.
View larger version (18K):
[in a new window]
Fig. 5.
Effect of FK506 (167 nM) on
depolarization-induced activation of G2 transcriptional activity.
The plasmid 4xG2T81Luc was transiently transfected into TC2 cells.
The cells were stimulated by high potassium-induced membrane
depolarization (KCl, 45 mM). Luciferase activity is
expressed as percentage of the mean value in each experiment of the
activity measured in the controls (5 mM KCl, no FK506).
Values are mean ± S.E. of four independent experiments, each done
in duplicate.
View larger version (31K):
[in a new window]
Fig. 6.
Mutational analysis of sequences within G2
required for depolarization responsiveness. Four copies of the G2
oligonucleotide with wild-type sequence (G2) or the G2 mutants 1, 3, or
5 (G2 m1, G2 m3, and G2 m5) were placed in front
of the truncated viral thymidine kinase promoter of herpes simplex
virus fused to the luciferase reporter gene. The plasmids were
transfected into TC2 cells. KCl, 45 mM; TPA, 300 nM. Luciferase activity is expressed as percentage of the
mean value, in each experiment, of the activity measured in the
respective control (no treatment). When compared with 4xG2T81Luc
(100 ± 6%), the basal activity was not changed by mutants 1 or 5 and was 677 ± 65% for 4xG2 m3T81Luc, as has been reported
previously (20). Values are mean ± S.E. of three independent
experiments, each done in duplicate.
350
GluLuc) or with
an additional internal deletion inside the CRE (
350(2
)GluLuc).
Deletion of the G2 element markedly reduced the depolarization-induced
increase in transcriptional activity of the glucagon promoter in
TC2
cells (Fig. 7A, left panel), suggesting that G2 is required for depolarization
responsiveness of the glucagon promoter. However, interpretation of the
data is complicated by the marked decrease in basal activity by the G2
deletion (Fig. 7A, left panel). Therefore,
similar transfection experiments were performed in HIT cells. In this
pancreatic islet
-cell line, the internal deletion of G2 caused only
a slight decrease in basal activity (Fig. 7A, right
panel). The internal deletion of G2 reduced the
depolarization-induced activation of the glucagon promoter by 76%
(Fig. 7A, right panel), suggesting again that G2
is required for depolarization responsiveness of the glucagon promoter.
Similar results were obtained with the double deletion mutant (Fig.
7A, right panel). Depolarization responsiveness
of the glucagon promoter was inhibited in HIT cells by the internal
deletion of G2 (Fig. 7A, right panel) to the same degree (by about 75%) as by the deletion inside the CRE (Fig. 1B), suggesting that both G2 and the CRE are required for
full activation of glucagon gene transcription by membrane
depolarization; furthermore, this suggests that the depolarization
responsiveness mediated by one element to the promoter depends in part
on the other element.
View larger version (15K):
[in a new window]
Fig. 7.
Role of the G2 element and NFATp in the
regulation of the glucagon promoter by membrane depolarization.
A, effect of deleting G2 ( G2) or G2 plus CRE
(
G2
CRE) on glucagon promoter activity. The plasmids
350GluLuc,
350
GluLuc, or
350(2
)GluLuc were transfected into
TC2 or HIT cells. The cells were stimulated by high
potassium-induced membrane depolarization (45 mM KCl,
black bars) or were left unstimulated (white
bars). Luciferase activity is expressed as percentage of the mean
value in each experiment of the activity measured in the controls
(unstimulated
350GluLuc). Values are mean ± S.E. of three
independent experiments, each done in duplicate. B, effect
of FK506 on depolarization-induced activation of glucagon gene
transcription. The plasmid
292GluLuc was transfected into
TC2
cells. The cells were stimulated by high potassium-induced membrane
depolarization (KCl, 45 mM). FK506, 16.7 nM.
Luciferase activity is expressed as percentage of the mean value in
each experiment of the activity measured in the controls (5 mM KCl, no FK506). Values are mean ± S.E. of three
independent experiments, each done in duplicate. C, effect of overexpression of the
DNA-binding domain of NFATp (NFATpDBD) on depolarization
responsiveness of the glucagon promoter.
TC2 cells were
co-transfected with
350GluLuc and the expression vector
pBK-CMV-NFATpDBD as indicated. The cells were stimulated by high
potassium-induced membrane depolarization (KCl, 45 mM).
Luciferase activity is expressed as percentage of the mean value in
each experiment of the activity measured in the controls (unstimulated
350GluLuc alone). Values are mean ± S.E. of three independent
experiments.
DISCUSSION
,
building a novel calcium response element.
300-amino acid DNA-binding domain distantly
related to the Rel homology region (18, 19, 51). The NFAT family of
transcription factors is a target for the clinically important
immunosuppressant drugs cyclosporin A and FK506, which inhibit the
calcium-/calmodulin-dependent serine/threonine phosphatase
calcineurin (47, 52, 53). In resting T cells, NFATp resides in the
cytoplasm and is fully phosphorylated; following stimulation, it
rapidly becomes dephosphorylated by calcineurin activated by an
increase in cytosolic calcium concentration, and NFATp then
translocates to the nucleus (19, 26, 46, 54-56). Although some cells
lack the requisite mechanisms for regulating NFATp subcellular
localization (26), our results suggest that NFATp is expressed and
regulated in glucagon-producing islet cells in a similar way as in T
cells and binds to the glucagon gene. The G2 element within the
glucagon promoter contains a typical NFAT-binding site
(5'-GGAAACTT-3'). Using an electrophoretic mobility shift assay, a
protein complex with NFATp-like immunoreactivity was found to bind to
this site and was detected in cytosolic extracts from unstimulated
cells and in nuclear extracts from cells stimulated by membrane
depolarization, consistent with depolarization-induced translocation of
NFATp from the cytosol to the nucleus. The mutations in G2 mutant 3 were less detrimental for NFATp binding than those of G2 mutant 5, consistent with the finding that the bases exchanged in G2 mutant 3 are
less conserved (72-78%) than those of G2 mutant 5 (100%) in aligned
sequences derived by PCR-based site selection of an optimal
NFAT-binding site (57). Furthermore, NFAT makes sequence-specific major
groove contacts in the 5'-half-site (58), which is altered in G2 mutant
5 but not in G2 mutant 3. The antiserum used is specific for NFATp (36)
and completely blocked the depolarization-induced nuclear protein
binding, providing no evidence that in addition to NFATp other members
of the NFAT family of transcription factors may be involved. NFATp has
been shown before to be expressed in a wide variety of tissues
including pancreas (36, 51, 59). The expression of NFATp in pancreatic
islet
TC2 cells was confirmed by RT-PCR of NFATp mRNA as well as
by Western blotting. Inhibition of calcineurin phosphatase activity by
FK506 blocked both the depolarization-caused induction of nuclear NFATp
binding and G2-mediated transcription. The latter was also prevented by
mutating the binding sites for NFATp or HNF-3
within G2. Taken
together, these data suggest that the molecular mechanism by which
glucagon gene transcription is induced by membrane depolarization and
calcium influx in pancreatic islet cells through the G2 element may be
consistent with a model in which calcium activates calcineurin
phosphatase activity which then, probably by direct dephosphorylation,
induces the translocation of NFATp from the cytosol to the nucleus
where it cooperates with HNF-3
at the G2 element to stimulate
transcription (Fig. 8).
View larger version (21K):
[in a new window]
Fig. 8.
Novel pairing of NFATp with the cell-specific
transcription factor HNF-3 in islet cells to
form a novel calcium response element in the glucagon gene. The
results of the present study are consistent with a model in which
membrane depolarization and calcium influx activate calcineurin
phosphatase activity which then, probably by direct dephosphorylation,
induces the translocation of NFATp from the cytosol to the nucleus
where it cooperates with HNF-3
to stimulate transcription.
The N-terminal transactivation domain of NFATp has been shown to
recruit the coactivators p300/CBP (60), which are also bound by CREB
after phosphorylation in response to calcium-induced signaling (61).
NFATp-CBP interactions may involve the CREB-binding domain and the
cysteine-histidine-rich region 3 of CBP (60). Thus, cooperative
recruitment of a common coactivator by CREB, binding to the CRE, and
NFATp, binding together with HNF-3 to G2, is a potential explanation
for the effects of the internal deletion of either the CRE or G2 on
glucagon promoter activity in HIT cells, which indicate that
depolarization responsiveness conferred to the glucagon promoter by G2
or the CRE depends in part on the other element.
Although NFATp appears to be regulated by calcium/calcineurin in both T
cells and islet cells, there are significant differences. First, in T
cells, NFATp is recruited by an inositol trisphosphate-induced release
of calcium from intracellular stores and capacitative calcium entry
(19, 55), whereas in this study in islet cells NFATp was mobilized by
membrane depolarization and calcium influx through
voltage-dependent, L-type calcium channels. This may be of
general significance also in neurons (36, 51). Second, this study shows
a novel pairing of NFATp. An important component of transactivation by
NFATp family proteins in T cells is their cooperation in the nucleus
with Fos and Jun AP-1 proteins (16-19). The interaction with AP-1 is
essential for NFAT transcriptional activity. Since AP-1 is newly
induced upon T cell receptor activation through separate signals, two
signaling pathways are needed to trigger the IL-2 NFAT site for a full
transcriptional response, Ras/protein kinase C for AP-1 induction and
calcium/calcineurin for NFAT activation (19, 47, 52, 53, 62). In the
IL-2 enhancer, a consensus recognition site for NFAT is closely
juxtaposed with a nonconsensus AP-1 site (18, 19). NFAT and AP-1
together form a highly stable cooperative complex (16-19, 58, 63). A structural model has been proposed (58, 63), wherein the so-called insert region of NFAT lies closest to the spacer region of c-Fos/c-Jun, allowing a single arginine residue in the c-Jun spacer (Arg-285) to
make an especially important contact. This study shows a novel pairing
of NFATp with HNF-3. Both proteins bind to closely associated sites
within the G2 element. HNF-3
binds immediately 3' to the NFAT site
and, thus, at a relative position occupied by AP-1 proteins in
composite NFAT:AP-1 sites (19). The binding of NFATp or HNF-3
was
not affected when the binding of HNF-3
(in labeled mutant 1) or
NFATp (in labeled mutant 5), respectively, was eliminated, providing no
evidence that, as with AP-1, the interaction between NFATp and HNF-3
involves cooperative binding; it appears to be based on a functional
synergism that may or may not include a direct protein-protein
interaction. HNF-3
is a member of the winged helix family of
transcription factors (64). It is expressed early in development
(64-67). In adults, it is expressed in the liver (68), lungs, small
intestine (68, 69), exocrine pancreas (70), and pancreatic islets (20,
41, 70, 71), being involved in cell-specific gene transcription in
these endoderm-derived tissues. Instead of integration into Ras/protein
kinase C pathways through AP-1 proteins (19, 47, 52, 53, 62), the
interaction between NFATp and HNF-3
may not confer the requirement
for an input from signaling pathways others than those regulating
NFATp. Thus, the present study shows a new combinatorial
usage of NFATp with HNF-3
, which ties the
calcium/calcineurin/NFAT pathway directly to cell-specific gene transcription.
The present findings expand the functions of the transcription factor
HNF-3 to include a role in calcium-regulated gene transcription. HNF-3
was first defined as a transcription factor involved in developmental and cell-specific gene transcription (see above). Subsequently, it has been suggested that HNF-3
synergizes with the
glucocorticoid receptor in the liver to confer glucocorticoid responsiveness to the tyrosine aminotransferase gene (72) and with
an Ets-like transcription factor in islet cells to confer Ras and
protein kinase C responsiveness to the glucagon gene (20). The results
presented here suggest that HNF-3
potentiates transcriptional activation by the calcium-regulated transcription factor NFATp. Thus,
HNF-3
is able to cooperate with several regulated transcription factors and in this way seems to function as a mediator of
environmental signals in endoderm-derived tissues in which
it is expressed.
There are several examples where multiple signaling pathways focus on a single DNA control element, although the mechanisms by which these signals are integrated or selected are different (1, 2, 73). The binding sites within G2 of calcium-regulated NFATp and of the Ets-like proteins, probably regulated by protein kinase C and Ras (20), overlap, suggesting that these proteins compete for binding to G2 and thereby determine which signal is mediated through G2.
Diverse calcium response elements have already been described,
including elements binding the transcription factors CREB (1-4), serum
response factor (2, 5), and C/EBP (6). The calcium-responsive glucagon G2 element differs from these elements by DNA sequence, interacting proteins, and the lack of evidence for an involvement of
calcium-/calmodulin-dependent protein kinases in signaling to this element. It also differs from composite NFAT-binding sites in
the IL-2 and other cytokine genes (18, 19) by a novel pairing of NFATp.
In this sense, the depolarization-induced combination of NFATp with the
cell lineage-specific transcription factor HNF-3
at the glucagon G2
element represents a novel calcium response element.
![]() |
ACKNOWLEDGEMENTS |
---|
We greatly appreciate the generous gifts of anti-NFATp antiserum from Dr. A. Rao, Harvard Medical School, Boston, and of anti-CREB antiserum from Dr. J. F. Habener, Harvard Medical School, Boston. We thank E. Oetjen for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft Grants SFB402/A3 and Kn 220/8-1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Neurology, University of Heidelberg,
69120 Heidelberg, Germany.
§ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, University of Göttingen, Robert-Koch-Str. 40, D-37070 Göttingen, Germany. Fax: 49-551-399652. E-mail: wknepel{at}med.unigoettingen.de.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CREB, CRE-binding protein; CRE, cAMP response element; IL-2, interleukin 2; NFAT, nuclear factor of activated T cells; RT-PCR, reverse transcription and PCR amplification; TPA, 12-O-tetradecanoylphorbol-13-acetate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|