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Volume 17, Number 16,
Issue of August 15, 1997
pp. 6189-6202
Copyright ©1997 Society for Neuroscience
Calcium Controls Gene Expression via Three Distinct Pathways That
Can Function Independently of the Ras/Mitogen-Activated Protein Kinases
(ERKs) Signaling Cascade
Claire M. Johnson1,
Caroline S. Hill2,
Sangeeta Chawla1,
Richard Treisman2, and
Hilmar Bading1
1 Division of Neurobiology, Medical Research Council
Laboratory of Molecular Biology, Cambridge CB2 2QH, England, and
2 Transcription Laboratory, Imperial Cancer Research Fund
Laboratories, London WC2A 3PX, England
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Calcium ions are the principal second messenger in the control of
gene expression by electrical activation of neurons. However, the full
complexity of calcium-signaling pathways leading to transcriptional activation and the cellular machinery involved are not known. Using the
c-fos gene as a model system, we show here that the activity of its complex promoter is controlled by three independently operating signaling mechanisms and that their functional significance is cell type-dependent. The serum response element (SRE), which is
composed of a ternary complex factor (TCF) and a serum response factor
(SRF) binding site, integrates two calcium-signaling pathways. In PC12
cells, calcium-regulated transcription mediated by the SRE requires the
TCF site and is not inhibited by expression of the dominant-negative
Ras mutant, RasN17, nor by the MAP kinase kinase 1 inhibitor PD 98059. In contrast, TCF-dependent transcriptional regulation by nerve growth
factor or epidermal growth factor is mediated by a Ras/MAP kinases
(ERKs) pathway targeting the TCF Elk-1. In AtT20 cells and hippocampal
neurons, calcium signals can stimulate transcription via a
TCF-independent mechanism that requires the SRF binding site. The
cyclic AMP response element (CRE), which cooperates with the TCF site
in growth factor-regulated transcription, is a target of a third
calcium-regulated pathway that is little affected by the expression of
RasN17 or by PD 98059. Thus, calcium can stimulate gene expression via
a TCF-, SRF-, and CRE-linked pathway that can operate independently of
the Ras/MAP kinases (ERKs) signaling cascade in a cell type-dependent
manner.
Key words:
gene regulation;
c-fos;
calcium signaling;
cyclic AMP response element;
serum response element;
ternary complex
factor
INTRODUCTION
Changes in gene expression are important
mechanisms by which neurons transform short-lasting electrical events
into long-lasting functional and morphological alterations that may be
responsible for plasticity-related events such as memory formation (for
review, see Sheng and Greenberg, 1990 ; Morgan and Curran, 1991).
Calcium entry into neurons through voltage or ligand-gated ion channels serves as the trigger for electrical activity-dependent transcriptional responses (for review, see Ghosh and Greenberg, 1995). Signal transduction cascades initiated by increases in intracellular calcium
concentrations control the transcription rate of inducible genes by
modifying transcription factors that interact with specific cis-acting
DNA regulatory elements in the promoter of the gene. However, the exact
signal transduction mechanisms involved in calcium-regulated
transcription are unknown. To analyze this, we have used the
c-fos gene, a strongly calcium-inducible gene, as a model
system. Previous work has identified two control regions in the
c-fos promoter, the cyclic AMP response element (CRE) and the serum response element (SRE), as calcium-responsive
promoter/enhancer elements (Sheng et al., 1988, 1990; Bading et al.,
1993; Misra et al., 1994; Miranti et al., 1995; Robertson et al.,
1995). Our recent study demonstrates that CRE-dependent transcriptional
activation is controlled by increases in nuclear calcium concentrations
(Hardingham et al., 1997), suggesting a model in which a nuclear
calcium-responsive enzyme, possibly calcium/calmodulin (CaM)-dependent
protein kinase IV (Jensen et al., 1991), mediates this response.
Consistent with this hypothesis is the finding that CaM kinases can
phosphorylate the CRE binding protein (CREB), which can mediate
calcium-activated transcription of c-fos (Sheng et al.,
1990, 1991). This phosphorylation occurs on residue serine 133 that is
critical for CREB to function as a transcriptional activator (Gonzales
and Montminy, 1989; Sheng et al., 1991; Matthews et al., 1994; Sun et
al., 1994).
Calcium signaling at the c-fos SRE is still an enigma.
Mechanisms of calcium-activated transcription by the SRE can function independently of increases in nuclear calcium concentrations
(Hardingham et al., 1997), suggesting that a signal transduction
machinery localized to the cytoplasm can control this response. A
candidate signaling cascade is the Ras/MAP kinases (ERKs) pathway,
which is activated in response to calcium signals (Bading and
Greenberg, 1991; Rosen et al., 1994; Rusanescu et al., 1995). Indeed, a
recent study suggested that, in rat cortical neurons, transcriptional induction of a c-fos-based model reporter gene that follows
calcium entry through the NMDA receptor occurs via a MAP
kinase-dependent pathway targeting the SRE-interacting protein ternary
complex factor (TCF) Elk-1 (Xia et al., 1996). However, analysis of
similar model reporter genes in the rat pheochromocytoma cell line,
PC12, indicates that calcium activates transcription by a
TCF-independent pathway involving the principal SRE binding protein,
the serum response factor (SRF), and CaM kinases (Misra et al., 1994;
Miranti et al., 1995).
In this study we have focused on the analysis of the role of the
Ras/MAP kinases (ERKs) signaling cascade in calcium-activated transcription via the SRE and the CRE. Because SRE and CRE function may
depend on their context within the c-fos promoter, we
constructed mutants of the human c-fos gene by altering
these regulatory sites without disturbing surrounding sequences and
relative spacing. These in-context mutations allow us, first, to
determine the contribution of an individual element to the overall
transcriptional response elicited by the intact c-fos
promoter and, second, to dissect the multitude of signaling pathways
targeting the c-fos promoter and to investigate their
nature. We used DNA transfection and microinjection techniques to
introduce these plasmids into cells, analyzed them for transcriptional
inducibility by calcium signals, and compared them with inducibility by
nerve growth factor (NGF) and epidermal growth factor (EGF), two
classical activators of the Ras/MAP kinases (ERKs) pathway (Thomas et
al., 1992; Wood et al., 1992) (for review, see Marshall, 1994, 1995).
Because the functional significance of signal transduction pathways
targeting the CRE or the SRE may be cell type-dependent, we have
studied calcium-activated transcription in PC12 cells, in the mouse
pituitary cell line AtT20, and in primary rat hippocampal neurons.
MATERIALS AND METHODS
Cell culture and transfection. PC12 cells were grown
on collagen-coated dishes in DMEM containing 10% heat-inactivated
horse serum, 5% fetal calf serum (FCS), 100 U/ml penicillin G, and 100 µg/ml streptomycin (PC12 media) at 37°C in an atmosphere consisting of 10% CO2/90% air. AtT20 cells were grown in DMEM
containing 10% FCS, 100 U/ml penicillin G, and 100 µg/ml
streptomycin at 37°C in 5% CO2/95% air. For DNA
transfection, cells were transferred to
poly-D-lysine-coated dishes 1-2 d before transfection.
Transfection of PC12 cells [20-30 µg of human c-fos
plasmid and 1.5 µg of  -globin plasmid (pSV1) (Sheng et al.,
1988) per 100 mm dish] was performed by the calcium phosphate method
(Sheng et al., 1988) with the following modifications: cells were kept
in DMEM containing 10% FCS at 37°C in 5%
CO2/95% air for 1 hr before transfection. After the
addition of calcium phosphate precipitates, cells were incubated in
DMEM containing 10% FCS at 37°C in 5% CO2/95%
air for 5-6 hr. Cells were shocked with 25% (v/v) glycerol in
HEPES-buffered saline, pH 7.05, for 2 min and washed three times with
PBS, followed by incubation in PC12 media at 37°C in 10%
CO2/90% air until stimulation. AtT20 cells and, in
some experiments, PC12 cells were transfected with Lipofectamine (Life
Technologies, Gaithersburg, MD).
Analysis of gene expression. At 40-48 hr after
transfection, cells were stimulated with NGF (100 ng/ml NGF-7S or 50 ng/ml NGF-2.5S; Sigma, Poole, UK; no difference was observed between the two NGF preparations) for 30-40 min or with 3 ng/ml EGF (Sigma) for 30-40 min or 10 µM forskolin (Calbiochem, Lucerne,
Switzerland) and 0.5 mM IBMX (3-isobutyl-1-methylxanthine;
Sigma) for 50 min or by adding to the medium 0.41 volume of KCl
depolarization solution [containing (in mM) 10 HEPES, pH
7.2, 170 KCl, 1 MgCl2, and 2 CaCl2] for 50 min. Calcium influx into AtT20 cells was induced by adding to the
medium 0.41 volume of KCl depolarization solution containing a 5 µM concentration of the L-type calcium channel agonist
FPL 64176 (Zheng et al., 1991). Extraction of total RNA and RNase
protection assay were as described (Treisman, 1985; Sheng et al., 1988;
Bading et al., 1993). In each reaction 10-20 µg of total RNA was
hybridized to the c-fos and -globin antisense probe. Data
were quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA)
or densitometer analysis and normalized for transfection efficiency by
reference to expression of the -globin gene. CAT and
 -galactosidase assays were performed as described (Ausubel et al.,
1987); pCH110 containing the -galactosidase gene under the control
of an SV40 enhancer was used as a reference plasmid.
Western blot analysis. Lysis of cells, separation of
proteins by SDS-PAGE (10% polyacrylamide), and blotting onto
polyvinylidene fluoride membranes was done as described (Bading and
Greenberg, 1991). Immunoblot analysis with phospho-MAP kinase/ERK or
MAP kinase/ERK-specific antibodies (New England Biolabs, Beverly, MA)
and signal detection by chemiluminescence were performed according to
the manufacturer's instruction (New England Biolabs).
Plasmids. The wild-type c-fos plasmid, pF711,
contains 711 base pairs (bp) of 5 flanking sequences; pF222 is
identical to pF711 except that it contains only 222 bp of 5 flanking
sequences (Treisman, 1985). All c-fos mutants are
derivatives of pF711 and were named according to the particular
regulatory element mutated. The following mutants have been
described previously: pFos TCF (Hill et al., 1994), pFosSRF
(Hill and Treisman, 1995), and pFosSIF (Hill and Treisman,
1995). Plasmids MLV128, NL.Elk, NL.Elk307, NL.Elk383/389,
RSV128, RSVrasN17, (Gal)2-TATA-CAT, Gal-ElkC, and
Gal-ElkC383/389 also have been described previously (Hill et al., 1993,
1995; Marais et al., 1993). Plasmid pFosCRE was made by cloning the
NotI fragment of pF711 containing the CRE sequence into
pGEM13Zf(+) (Promega, Madison, WI). The
NarI/BssH2 fragment containing the CRE was
replaced with annealed oligonucleotides 1 and 2 (see below) in which
the CRE sequence was replaced with a Gal4 binding site. The resulting
plasmid contained a point mutation that was repaired by PCR
mutagenesis. Then the NotI fragment containing the Gal4
binding site was cloned into pF711 to create pFosCRE. By swapping
the BssH2/NcoI fragment of pFosCRE into
plasmids containing mutations of the SRF binding site, TCF binding
site, or SIE, we generated plasmids pFosCRESRF, pFosCRETCF,
pFosCRESIF, pFosCRESRFSIF, and pFosCRETCFSIF.
The c-fos genomic constructs were tagged with the 9E10 myc
epitope (EQKLISEEDL, single letter code) by inserting annealed
oligonucleotides 3 and 4 (see below) into the NcoI site
located in the fourth exon of the c-fos gene. All DNA
manipulations were performed by standard techniques, and plasmids were
verified by sequencing.
Oligonucleotides. Oligonucleotides used included the
following: (1)
CG-CGCCACCCCTCTGGCGCCACCGTGGTTGACGGAGTACTGTCCT-CCGTCATTCATAAAACGCTTGTTATAAAAGCAGTGGCTGCGG (top strand), (2)
CGCCGCAGCCACTGCTTTTATAACAAGCGTTTT-ATGAATGACGGAGGACAGTACTCCGTCAACCACGGTGGCGCC-AGAGGGGTGG (bottom strand), (3),
CATGAAGCTTGAGCAGAAG-CTGATCAGCGAGGAAGATCTGGC (top strand),
and (4) CATGGC-CAGATCTTCCTCGCTGATCAGCTTCTGCTCAAGCTT (bottom strand).
Microinjection of primary hippocampal neurons and
immunofluorescence. Hippocampal neurons were isolated and cultured
as described (Bading and Greenberg, 1991). At 9 d after plating
the growth medium was replaced by transfection medium (Bading et al.,
1993). Neurons were injected 11-12 d after plating, using a Zeiss
Microinjection Workstation (Oberkochen, Germany). Plasmids were
injected into the nucleus at 100 µg/ml in half-strength PBS
containing 0.9% (w/v) Texas Red-conjugated 70 kDa dextran (Molecular
Probes, Eugene, OR) as an injection marker. At 24 hr after
microinjection, neurons were stimulated with 20 µM
glutamate. Glutamate stimulation was terminated after 10 min by adding
1 mM sodium kynurenate and 11 mM
MgCl2 to the medium, as described (Bading et al., 1993).
Cells were fixed 2 hr after stimulation in 3% paraformaldehyde in PBS containing 4% sucrose for 20 min, washed twice with 10 mM
glycine in PBS for 10 min, permeabilized in 0.5% NP-40 in PBS for 5 min, and then incubated in blocking solution (1.5% normal goat serum in PBS) for 20 min. All steps were at room temperature. Incubation with
the anti-myc monoclonal antibody 9E10 (Santa Cruz Biotechnology, Tebu,
France) (1:200 dilution in PBS) was overnight at 4°C. Incubations with biotinylated anti-mouse antibody (Jackson ImmunoResearch, West
Grove, PA) and with fluorescein-avidin (Vector Laboratories, Burlingame, CA), both diluted 1:200 in PBS, were for 1 hr each at room
temperature. Cells were washed with PBS and mounted in Vectashield
(Vector). Immunofluorescence was quantitated by an MRC 600 confocal
laser scanning microscope.
RESULTS
c-fos promoter in-context mutations
To study the function of the SRE and the CRE in the context of the
intact c-fos gene, we introduced specific mutations into the
promoter of the human c-fos gene by changing the key native regulatory sequences to heterologous DNA binding sites for the yeast
proteins Gal4 or MCM1 or to a half-site for the bacterial LexA protein
(see Fig. 1 for schematic representation). The parental plasmid pF711 contains the entire transcribed region of the
c-fos gene and 711 bp of upstream regulatory sequence
(Treisman, 1985). The SRE, centered on nucleotide  310 relative to the
transcription start site, was changed to an MCM1 site that abrogates
binding of SRF (plasmid pFosSRF; Hill and Treisman, 1995). SRF is
the principal transcription factor that interacts with the SRE (Prywes and Roeder, 1987; Schröter et al., 1987; Treisman, 1987; Norman et al., 1988). At the c-fos SRE, SRF can form a ternary
complex with Ets domain proteins such as Elk-1 or SAP-1 [also termed
ternary complex factors (TCFs)] that are transcription factor targets of the Ras/MAP kinases (ERKs) pathway and contain growth
factor-regulated transcriptional activation domains (Shaw et al., 1989;
Hipskind et al., 1991; Dalton and Treisman, 1992; Gille et al., 1992;
Hill et al., 1993; Janknecht et al., 1993; Marais et al., 1993) (for review, see Treisman, 1994). Recruitment of Elk-1 or SAP-1 to the
SRE/SRF complex requires the TCF site (Dalton and Treisman, 1992;
Treisman et al., 1992), which is an Ets motif located 5 to the SRF
binding site and critical for c-fos activation by certain stimuli (Shaw et al., 1989; Graham and Gilman, 1991; Hill and Treisman,
1995). To disrupt ternary complex formation at the c-fos SRE, we replaced the TCF site by a LexA half-site (plasmid pFosTCF; Hill et al., 1994). The CRE, located at nucleotide 60 relative to the
transcription start site, was changed to a Gal4 binding site (plasmid
pFosCRE; see Materials and Methods). The sis-inducible element
(SIE), which is located at nucleotide 345 and plays a role in growth
factor and cytokine-activated gene expression (Hayes et al., 1987;
Wagner et al., 1990; Fu and Zhang, 1993; Sadowski et al., 1993; Zhong
et al., 1994) (for review, see Darnell et al., 1994; Ihle et al.,
1994), also was changed to a Gal4 binding site (plasmid pFosSIF;
Hill and Treisman, 1995). Additional constructs contain combinations of
these mutations.
Fig. 1.
Schematic representation of the human
c-fos promoter illustrating the in-context mutations.
All c-fos gene constructs contain 711 bp of
c-fos upstream regulatory sequence and the entire
transcribed region. The start of transcription is indicated by an
arrow. The base changes used to create the promoter
mutants are shown in bold below the wild-type sequence.
The CRE and SIF mutations both
generate Gal4 binding sites and abrogate the binding of CREB/ATF and
sis-inducible factor (SIF)/STAT, respectively. The
TCF mutation generates a LexA half-site and blocks
ternary complex formation; the SRF mutation creates
an MCM1 site that cannot bind SRF. The consensus binding sites for STAT
factors, TCFs, SRF, and CREB/ATF are indicated by
asterisks.
[View Larger Version of this Image (19K GIF file)]
c-fos induction by NGF and EGF is mediated by a Ras/MAP
kinases (ERKs) signaling pathway targeting the TCF Elk-1
To establish our experimental system, we first tested in DNA
transfection experiments the wild-type and mutant c-fos gene constructs in PC12 cells for inducibility by NGF and EGF treatment, which is known to signal to the nucleus via the Ras/MAP kinases (ERKs)
signaling pathway (Thomas et al., 1992; Wood et al., 1992). Expression
of the endogenous rat c-fos (c-fosR) and
the transfected human c-fos (c-fosH) was
measured by RNase protection assay. A plasmid containing the human
-globin gene under the control of the SV40 enhancer was
cotransfected to normalize for transfection efficiency. The result of a
representative experiment is shown in Figure 2. The basal level of expression of the endogenous rat c-fos gene
and the transfected wild-type and mutant human c-fos gene
constructs was low (Fig. 2A, lanes 1, 4, 7, 10, 13, and 16). Stimulation of PC12 cells with NGF and
EGF resulted in robust transcriptional induction of the endogenous and
transfected c-fos genes (Fig. 2A, lanes 2 and 3). Mutation of the SRF binding site (plasmid pFosSRF) reduced inducibility by NGF and EGF treatment to 23 ± 1% (n = 3) and 18 ± 1% (n = 3)
(Fig. 2A, lanes 5 and 6),
respectively, demonstrating that the SRF binding site is critical for
transcriptional regulation by NGF and EGF. We next investigated whether
the SRF binding site or the adjacent TCF binding site (which is
nonfunctional in the absence of SRF binding) is the site of regulation
by NGF and EGF signaling pathways. We tested the c-fos gene
construct pFosTCF that contains an intact SRF binding site but no
longer allows ternary complex formation (Hill et al., 1993, 1994).
Mutation of the TCF site reduced the growth factor-induced
transcriptional responses to 27 ± 6% (NGF; n = 5) and 31 ± 4% (EGF; n = 5) (Fig. 2A, lanes 14 and 15). This demonstrates
that, in the context of the intact c-fos gene, the SRF
binding site is necessary, but not sufficient, for transcriptional
induction by NGF and EGF and that ternary complex formation is critical
for transcriptional regulation by NGF and EGF signaling pathways.
Mutation of the SIE (plasmid pFosSIF) did not reduce
c-fos inducibility after growth factor treatment (NGF:
91 ± 19%, n = 3; EGF: 86 ± 14%, n = 3) (Fig. 2A, lanes 8 and
9). Double mutations of the SRF binding site and the SIE
(plasmid pFosSRFSIF; Fig. 2A, lanes 11 and 12) or of the TCF site and the SIE (plasmid
pFosTCFSIF; Fig. 2A, lanes 17 and
18) resulted in levels of transcriptional induction similar
to those seen with the TCF site or SRE mutations alone. Therefore, the
SIE appears to play only a minor role in transcriptional induction by
NGF and EGF. Mutation of the CRE reduced the growth factor-stimulated
transcriptional responses to 45 ± 5% (NGF; n = 3) and 46 ± 8% (EGF; n = 5) of the induction
observed with the c-fos wild-type gene (Fig.
2B, lanes 5 and 6). Double mutation of the CRE and either the SRF binding site or the TCF site resulted in
a virtually complete loss of inducibility (pFosCRESRF: 6 ± 1%, n = 2, NGF; 5 ± 1%, n = 2, EGF; pFosCRETCF: 8 ± 1%, n = 2, NGF;
12 ± 2%, n = 3, EGF) (Fig. 2B,
lanes 8 and 9, 11 and 12). These
results indicate that the CRE is required for a maximal growth factor
response and suggest a mechanism by which TCFs cooperate with
CRE-interacting proteins in NGF and EGF-regulated c-fos
transcription.
Fig. 2.
The TCF site and the CRE control
c-fos transcriptional induction in PC12 cells after NGF
and EGF treatment. RNase protection analysis was used to measure
expression of the endogenous rat c-fos gene
(c-fosR), the transfected
human -globin gene (globin; to indicate
transfection efficiency), and human c-fos gene
(c-fosH) after
transfection of plasmid pF711 (wild-type c-fos gene;
A, lanes 1-3; B, lanes 1-3) or one
of the following plasmids containing in-context mutations:
pFosSRF (A, lanes 4-6), pFosSIF (A,
lanes 7-9), pFosSRFSIF (A, lanes
10-12), pFosTCF (A, lanes 13-15), pFosTCFSIF (A, lanes 16-18), pFosCRE
(B, lanes 4-6), pFosCRE-SRF (B,
lanes 7-9), or pFosCRETCF (B, lanes
10-12). RNA was isolated from unstimulated cells (lanes marked
) or cells stimulated with NGF (N) or EGF
(E).
[View Larger Version of this Image (59K GIF file)]
Having established that the TCF site is a critical target for NGF and
EGF signaling pathways, we next tested the function of TCF Elk-1 as an
NGF- and EGF-responsive transcription factor. Mutation of the TCF site
introduces a half-site for the bacterial LexA protein into the
c-fos promoter, which allowed us to determine whether Elk-1
can restore NGF and EGF inducibility by expressing a LexA-Elk-1 fusion
protein (NL.Elk) in PC12 cells. LexA-Elk-1 binds to the TCF mutation
in a SRF-dependent manner (Hill et al., 1993). As shown in Figure
3A, expression of NL.Elk restored inducibility of pFosTCF to 81 ± 14% (NGF; n = 3) and 60 ± 14% (EGF; n = 4), respectively, of
the induction observed with the c-fos wild-type pF711 (Fig.
3A, compare lanes 2 and 3 with
lanes 8 and 9; 3B). Expression of a
truncated form of Elk-1 that lacks C-terminal sequences (NL.Elk307),
including phosphorylation sites or the mutation of two MAP kinase/ERK
phosphorylation sites on Elk-1 (serine 383 and serine 389;
NL.Elk383/389) previously shown to be critical for Elk-1 activity (Hill
et al., 1993; Janknecht et al., 1993; Marais et al., 1993), renders
Elk-1 functionally inactive (Fig. 3A, lanes 11 and
12, 14 and 15; 3B). To
investigate whether Elk-1 is sufficient to mediate this transcriptional
response, we examined the ability of a Gal4-Elk-1 fusion protein
(Gal-ElkC) to transactivate a Gal4-CAT reporter construct in
NGF-treated PC12 cells. The basal level of expression of the Gal4-CAT
reporter gene in unstimulated cells was low (Fig. 3C, lanes 1, 3, and 5). NGF stimulation of PC12 cells cotransfected
with the Gal-ElkC expression plasmid, but not with the vector control,
resulted in a robust transactivation of the Gal4-CAT reporter gene
(Fig. 3C, lanes 2 and 4). Mutations of
serine 383 and serine 389 on Elk-1 (Gal-ElkC383/389) abolished its
ability to transactivate the reporter gene (Fig. 3C, lane
6). Similar results were obtained with other reporter gene
constructs, using EGF as the stimulus (data not shown). These results
demonstrate that Elk-1 is sufficient for mediating NGF- and EGF-induced
transcriptional responses in PC12 cells.
Fig. 3.
NGF and EGF treatment of PC12 cells activates
c-fos expression by a Ras/MAP kinases (ERKs) signaling
pathway targeting TCF Elk-1. A, RNase protection
analysis was performed as described in Figure 2. PC12 cells were
transfected either with 30 µg of plasmid pF711 (wild-type
c-fos gene, lanes 1-3) or with 30 µg of pFosTCF (lanes 4-15) plus 1 µg of vector
plasmid (MLV128, lanes 4-6), plasmid NL.Elk,
which expresses an altered binding specificity Elk-1 protein that can
bind the TCF mutation (lanes 7-9; Hill et al.,
1994), plasmid NL.Elk307 (lanes 10-12), or NL.Elk383/389 (lanes 13-15). Cells were stimulated with
NGF (N) or EGF (E).
B, Quantitation of RNase protection experiments by PhosphorImager and densitometer analysis. The levels of
c-fos mRNA transcribed from plasmid
pFosTCF, normalized for transfection efficiency to
that of the -globin gene, are expressed as a percentage of the level
of mRNA produced by the transfected wild-type c-fos gene
construct pF711, normalized to -globin expression, in response to
the same stimulus. The mean ± SEM of three independent
experiments is shown. C, NGF-regulated transcriptional
activation by Gal4-Elk-1 fusion proteins measured with a
(Gal)2-TATA-CAT reporter gene construct. PC12 cells were
transfected with 2 µg of plasmid (Gal)2-TATA-CAT and 1 µg of vector (pUC19; lanes 1 and 2),
Gal-ElkC expression plasmid (lanes 3 and
4), or Gal-ElkC383/389 expression plasmid (lanes 5 and 6). Transfections
also included 0.5 µg of a reference plasmid, pCH110, containing the
-galactosidase gene under the control of the SV40 enhancer. Extracts
were prepared from unstimulated cells (lanes marked ), and cells were
stimulated with 100 ng/ml NGF-7S (N) for 8 hr and
analyzed for CAT activity. Data were quantified by the PhosphorImager
and normalized for transfection efficiency to expression of the
-galactosidase reference gene. NGF-induced levels of CAT activities,
relative to that obtained with PC12 cells expressing GalElk
C, which was taken as 100%, were 6% (Vector) and 5% (Gal-ElkC383/389). Similar results were obtained
in other experiments. D, RNase protection analysis was
performed as described in Figure 2. PC12 cells (60 mm dish) were
transfected by using Lipofectamine with 4 µg of plasmid pF711
(wild-type c-fos gene) plus 1 µg of either vector
plasmid (RSV128, lanes 1-4) or plasmid RSVrasN17 (lanes 5-8; Hill et al., 1995).
Transfections also included 0.75 µg of a reference plasmid pSV1.
Cells were stimulated with NGF (N), EGF
(E), or forskolin/IBMX
(F/I) or were left unstimulated (lanes marked
). NGF-induced levels of c-fos mRNA transcribed from
pF711, normalized for transfection efficiency to the levels of
-globin mRNA, in cells expressing RasN17 are 17 + 1%
(n = 7) of that obtained in cells transfected with
the vector control. Similar results were obtained in EGF-treated cells.
cAMP-induced expression of the pF711, normalized to the levels of
-globin mRNA, in cells expressing RasN17 is 133 ± 15%
(n = 6) of that in cells transfected with the
vector control.
[View Larger Version of this Image (41K GIF file)]
We next directly tested the role of the Ras/MAP kinases (ERKs)
signaling pathway in NGF- and EGF-activated transcription. Expression
of a dominant interfering Ras mutant, RasN17 (Feig and Cooper, 1988),
blocked NGF- and EGF-induced expression of the wild-type
c-fos gene construct pF711 (Fig. 3D, compare
lanes 2 and 3 with lanes 6 and
7). In contrast, expression of RasN17 did not reduce
c-fos induction in response to increased intracellular levels of cAMP after treatment of the cells with a combination of
forskolin (stimulator of adenylate cyclase) and IBMX (inhibitor of cAMP
phosphodiesterase) (Fig. 3D, compare lanes 4 and
8).
This analysis of NGF- and EGF-induced gene expression, which
established a direct link between a growth factor receptor signal, a
particular signal transduction cascade, and a transcription factor
target, illustrates that the c-fos gene constructs when used
in conjunction with other genetic or pharmacological means are powerful
tools to investigate the mechanisms of signal-regulated transcription.
We next applied this experimental approach to the analysis of gene
expression controlled by calcium signals.
In PC12 cells, calcium-signaling pathways control transcription via
CRE- and TCF-dependent mechanisms
To activate calcium-signaling pathways in PC12 cells, we exposed
cells to elevated extracellular KCl, which causes membrane depolarization and the opening of L-type voltage-gated calcium channels. In contrast to NGF and EGF stimulation, induction of c-fos transcription by KCl treatment was not reduced by
mutations of either the TCF site or the SRF binding site (Fig.
4A, compare lane 2 with
lanes 6 and 8; 4B). Because
this is most likely caused by the presence of the CRE (at position 60
relative to the start site of transcription), which previously has been
shown to function as a calcium-responsive element (Sheng et al., 1988,
1990; Bading et al., 1993), we tested c-fos gene constructs
that lack this CRE. In-context mutation of the CRE reduced
c-fos induction after KCl treatment to 54 ± 3%
(n = 13) (Fig. 4A, lane 4;
4B), demonstrating the importance of the CRE in
calcium-activated transcription. However, significant induction still
occurred in the absence of the CRE, indicating the existence of at
least one other calcium-responsive element in the c-fos
promoter. To determine whether CRE-independent activation of the
c-fos promoter by calcium signals is mediated by the SRE, we
transfected the c-fos gene construct pFosCRESRF in
which both the CRE and the SRF binding site had been mutated. Compared
with construct pFosCRE, transcriptional induction of pFosCRESRF is reduced further (Fig. 4A, compare
lanes 4 and 12; 4B), indicating
that binding of SRF is required for CRE-independent calcium-activated
transcription. We next investigated whether this response is
TCF-dependent and tested the c-fos gene construct pFosCRETCF that contains an intact SRF binding site but does not
allow for ternary complex formation. Mutation of the TCF site resulted
in a reduction of the CRE-independent calcium response that was similar
to that seen with the mutation of the SRF binding site (Fig.
4A, compare lanes 12 and 14;
4B). This indicates that, in the absence of the CRE,
calcium-signaling pathways can stimulate transcription via a
TCF-dependent mechanism. To identify transcription factors involved in
this response, we used the same strategy that was used to investigate
NGF- and EGF-regulated transcription factors. Similar to the
experiments described in Figure 3A, we used a LexA-Elk-1 fusion protein to test whether Elk-1 can mediate the TCF-dependent calcium response. Expression of NL.Elk resulted in only a very small
increase in calcium-dependent inducibility of the c-fos gene
constructs pFosCRETCF and pFosCRE-TCFSIF (data not
shown). Although this may indicate that TCFs other than Elk-1 function as a calcium-activated transcription factor, it is also possible that,
in the absence of the CRE, TCF-LexA fusion proteins are unable to
reconstitute the complex required to activate transcription in response
to calcium-signaling pathways.
Fig. 4.
The TCF site and the CRE are targets of
calcium-signaling pathways in PC12 cells. A, RNase
protection analysis was performed as described in Figure 2. PC12 cells
were transfected with plasmid pF711 (wild-type c-fos
gene, lanes 1 and 2) or one of the
following plasmids containing in-context mutations: pFosCRE
(lanes 3 and 4), pFosSRF
(lanes 5 and 6), pFosTCF
(lanes 7 and 8), pFosSIF (lanes
9 and 10), pFosCRESRF (lanes
11 and 12), pFosCRETCF (lanes
13 and 14), pFosCRESRFSIF
(lanes 15 and 16), or
pFosCRETCFSIF (lanes 17 and 18).
RNA was isolated from unstimulated cells (lanes marked ) or cells
stimulated with KCl (K).
B, Quantitation of RNase protection experiments by
PhosphorImager analysis. The levels of c-fos mRNA
transcribed from the indicated plasmids and normalized for transfection
efficiency to the level of -globin mRNA are expressed as a
percentage of the amount of mRNA produced by the transfected wild-type
c-fos gene construct pF711, normalized to -globin
expression, in response to the same stimulus. The mean ± SEM is
shown. Compared with the wild-type construct pF711, mutation of the SRF
binding site consistently caused a small but significant (p < 0.001; paired t test)
enhancement of the KCl-induced transcriptional response.
[View Larger Version of this Image (58K GIF file)]
Double mutations of either the CRE and TCF site or the CRE and
SRE abolish most of the calcium-activated transcriptional response. However, a small portion of the induction, ~25-35%, still remains. Introduction of an SIE mutation further reduced this remaining response
to 15 ± 2% (pFosCRESRFSIF, n = 3) and
to 17 ± 3% (pFosCRE-TCFSIF, n = 6)
(Fig. 4A, lanes 16 and 18;
4B), This indicates that the SIE can, in the absence
of the CRE and SRE, make a small contribution to transcriptional
activation by calcium signal.
In AtT20 cells, calcium-signaling pathways control transcription
via CRE and TCF-independent SRF-linked mechanisms
AtT20 cells were stimulated by exposing them to elevated
extracellular KCl in the presence of a 5 µM concentration
of the L-type calcium channel agonist FPL 64176 (Zheng et al., 1991) (KCl/FPL stimulation). KCl treatment alone gives rise to only small
increases in intracellular calcium concentrations, insufficient to
activate gene transcription (Hardingham et al., 1997). Similar to the
results obtained with PC12 cells, mutation of either the SRF binding
site or the TCF site did not affect KCl/FPL-induced c-fos
expression (Fig. 5A, compare lane
2 with lanes 6 and 8; 5B), and
mutation of the CRE reduced the response to 45 ± 2%
(n = 13) (Fig. 5A, lane 4;
5B) of that obtained with pF711. However, in contrast to
PC12 cells, calcium-activated transcription via the SRE (plasmid
pFosCRE: 45 ± 2%, n = 13) was reduced by a
mutation of the SRF binding site (plasmid pFosCRESRF: 12 ± 1%, n = 8), but not by a mutation of the TCF site
(plasmid pFosCRETCF: 43 ± 8%, n = 3) (Fig.
5A, compare lane 4 with lanes 10 and
12; 5B). This indicates that, in AtT20 cells,
SRE-mediated calcium-activated transcription is controlled by a
TCF-independent SRF-linked pathway.
Fig. 5.
The SRF-binding site and the CRE are targets of
calcium-signaling pathways in AtT20 cells. A, RNase
protection analysis was performed as described in Figure 2. AtT20 cells
were transfected with plasmid pF711 (wild-type c-fos
gene, lanes 1 and 2) or one of the
following plasmids containing in-context mutations: pFosCRE (lanes 3 and 4), pFosSRF
(lanes 5 and 6), pFosTCF
(lanes 7 and 8), pFosCRESRF
(lanes 9 and 10), or pFosCRETCF
(lanes 11 and 12). RNA was isolated from
unstimulated cells (lanes marked ) or cells stimulated with KCl/FPL
64176 (K/F). B, Quantitation of
RNase protection experiments by the PhosphorImager. Analysis was done
as described in Figure 4B.
[View Larger Version of this Image (68K GIF file)]
NMDA receptor activation can mediate transcriptional induction by a
TCF-independent mechanism in hippocampal neurons
The NMDA receptor is a major site for calcium entry into
hippocampal neurons and is the principal ion channel responsible for
transcriptional regulation by the excitatory neurotransmitter glutamate
(Cole et al., 1989; Wisden et al., 1990; Lerea et al., 1992; Bading et
al., 1993, 1995; Lerea and McNamara, 1993). A previous study has shown
that the c-fos SRE can function as an NMDA
receptor-responsive element (Bading et al., 1993). However, it is
unknown whether NMDA receptor-induced transcription is mediated by the
SRF binding site or requires, in addition, ternary complex formation
(as is the case for calcium, NGF, and EGF induction of c-fos
expression in PC12 cells; see Figs. 2, 4). We initially addressed this
issue in DNA transfection and RNase protection experiments, similar to
those described above that used PC12 cells. However, the basal level of
expression of the transfected c-fos gene constructs pF711
and pFosCRE in unstimulated hippocampal neurons was very high and
only moderately induced on NMDA receptor activation (data not shown),
which made the interpretation of the data very difficult. In the same
experiments, the endogenous c-fos gene exhibited low basal
expression levels and was robustly induced after NMDA receptor
activation, suggesting that deregulation of the transfected
c-fos constructs may be a result of the transfection procedure. We, therefore, established a microinjection technique as an
alternative means of introducing DNA into hippocampal neurons. To allow
transcriptional induction of the injected c-fos gene constructs to be measured at the single cell level, we inserted an
oligonucleotide encoding the 9E10 myc epitope in frame into the
c-fos coding region. Expression of the myc-tagged
c-fos protein was detected by immunocytochemical methods,
using the 9E10 antibody, and quantified by the confocal laser scanning
microscope. Similar to the DNA transfection experiments, microinjection
into primary hippocampal neurons of the myc-tagged wild-type
c-fos gene, pF711myc, gave rise to high basal levels of
expression in many unstimulated cells (data not shown). However,
in-context mutation of the CRE reduced this basal level of expression,
providing an assay for analyzing mechanisms of NMDA receptor-regulated
gene expression at the single-cell level. To determine whether ternary
complex formation is critical for transcriptional regulation by the
NMDA receptor pathway, we tested the myc-tagged constructs
pFosCREmyc, pFosCRE-SRFmyc, and pFosCRETCFmyc (Fig.
6, Table 1). Expression of pFosCREmyc
was very low in the majority (91%) of unstimulated cells and in most
cells induced to moderate or high levels in response to glutamate
treatment, which was used to activate NMDA receptors. We observed,
however, a small number of unstimulated neurons that express
pFosCREmyc at moderate and even high levels. Although this may be
attributable to spontaneous electrical activation of the neurons, as
described previously for the endogenous c-fos gene (Bading
et al., 1995), it is also possible that the stress associated with
microinjection of DNA constructs triggers a transcriptional response in
some hippocampal neurons, giving rise to false-positive cells. In a
fraction of the neurons (28%) expression of pFosCREmyc was not
induced after glutamate stimulation, which resembles results obtained
previously with the endogenous c-fos gene (Bading et al.,
1995) and may reflect a lack of functional NMDA receptors. Glutamate
regulation of pFosCRETCFmyc was virtually identical to that of
pFosCREmyc, with most cells showing moderate or strong levels of
induction and a fraction of neurons (27%) with no detectable transcriptional response. In contrast, mutation of the SRE
(pFosCRESRFmyc) resulted in a loss of inducibility in most
neurons (74%). These results indicate that the NMDA receptor signaling
pathway can activate transcription by a TCF-independent mechanism that
requires the SRF binding site. Table 1 summarizes our microinjection
experiments; Figure 6 shows representative examples.
Fig. 6.
Analysis of NMDA receptor regulation of
c-fos expression in primary hippocampal neurons by
microinjection technique. The example shown illustrates neurons that
were injected with plasmid pFosCREmyc. Similar experiments were
performed with other c-fos gene constructs, the results
of which are summarized in Table 1. Injected neurons, left unstimulated
(A-C) or stimulated with 20 µM
glutamate (D-F), are identified by the
fluorescence of the Texas Red-conjugated dextran (A and
D) present in the injection solution and are indicated with arrows in the phase-contrast photographs
(B and E). Expression of myc-tagged
c-fos protein in unstimulated neurons (C;
positions of injected cells are marked by arrows) and
glutamate-stimulated neurons (F) was detected by
immunofluorescence technique with the 9E10 monoclonal antibody. Scale
bar, 50 µm.
[View Larger Version of this Image (74K GIF file)]
Role of Ras/MAP kinases (ERKs) pathway in
calcium-activated transcription
Expression of a dominant-negative form of Ras, RasN17, previously
has been shown to block calcium activation of MAP kinase kinase (MEK)
and MAP kinases/ERK (Rosen et al., 1994; Rusanescu et al., 1995). To
investigate the role of MAP kinases/ERK in the regulation of gene
expression by calcium signals, we tested the effect of expression of
RasN17 on CRE and TCF-mediated calcium-activated transcription in PC12
cells. Because expression of RasN17 may affect the function of L-type
voltage-gated calcium channels (Rosen et al., 1994), we activated
calcium-signaling pathways in PC12 cells by adding 10 µM
ionomycin to the medium, which, similar to KCl treatment, activated
c-fos expression via a CRE-dependent and TCF-dependent
mechanism (data not shown). Expression of RasN17 had very little effect
on calcium-induced transcription from the transfected wild-type gene
construct pF711, the CRE-dependent gene construct pFosSRF, and the
TCF-dependent gene construct pFosCRE (Fig.
7A, compare lanes 2 and
6, 10 and 12, 14 and
16; 7B). Calcium-activated transcription of
another CRE-dependent c-fos gene construct, pF222, which
contains a deletion of upstream regulatory sequence 5 to nucleotide
222 and therefore lacks the SRE, also was affected only very little
by expression of RasN17 (Fig. 7A, compare lanes
18 and 20; 7B). cAMP-induced transcription from pF711 after treatment of the cells with forskolin and IBMX also
was not inhibited by the expression of RasN17 (Fig. 7A,
compare lanes 4 and 8; 7B), whereas
NGF-induced transcription from pF711 was blocked by RasN17 (Fig.
7A, compare lanes 3 and 7;
7B). These results indicate that calcium can induce
CRE-mediated and TCF-mediated transcription independently of Ras
activation. Because Ras activation is critical for the stimulation of
the MAP kinases (ERKs) signaling cascade, our results suggest that
calcium-activated transcription operates independently of the Ras/MAP
kinases (ERKs) pathway. To test this hypothesis directly, we treated
PC12 cells with the MAP kinase kinase (MEK) 1 inhibitor, PD 98059. This
treatment blocked KCl-induced and ionomycin-induced MAP kinases/ERKs
activation as assessed by Western blot analysis, using antibodies
specific for the phosphorylation sites on MAP kinases/ERKs that are
indicative of stimulation of their enzymatic activity (Fig.
8A, compare lanes 2 and
3 with 5 and 6). In contrast,
similar to the results obtained in the RasN17 expression experiments,
PD 98059 treatment had little effect on calcium-induced transcriptional
activation of the transfected wild-type gene construct pF711, the
TCF-dependent construct pFosCRE, and the CRE-dependent constructs
pFosTCF and pF222 (Fig. 8B, compare lanes
2 and 3, 7 and 8, 10 and 11, 13 and 14; 8C). In contrast, NGF-induced transcription from pF711 was inhibited by PD
98059 (Fig. 8B, lanes 4, 5;
8C). This result provides further evidence against a
critical role of the Ras/MAP kinases (ERKs) signaling cascade in
calcium-regulated transcription.
Fig. 7.
Effect of expression of RasN17 on CRE-dependent
and TCF-dependent calcium-activated transcription in PC12 cells.
A, RNase protection analysis was performed as described
in Figure 2. PC12 cells were transfected with plasmid pF711 (wild-type
c-fos gene, lanes 1-8), pFos-CRE
(lanes 9-12), pFosTCF (lanes
13-16), or pF222 (lanes 17-20) plus
either vector plasmid (RSV128, lanes 1-4, 9 and
10, 13 and 14,
17 and 18) or plasmid RSVrasN17
(lanes 5-8, 11 and 12, 15
and 16, 19 and 20). Cells
were stimulated with ionomycin (Ion), NGF
(N), and forskolin/IBMX
(F/I) or were left unstimulated (lanes marked
). B, Quantitation of RNase protection experiments by
the PhosphorImager. The levels of c-fos mRNA transcribed from the indicated plasmids, normalized for transfection efficiency to
the level of -globin mRNA, in cells expressing RasN17 are shown as a
percentage of the amount of c-fos mRNA, normalized to
-globin expression, produced by cells transfected with the vector
control in response to the same stimulus. The mean ± SEM is
shown.
[View Larger Version of this Image (55K GIF file)]
Fig. 8.
Effect of the MAP kinase kinase 1 inhibitor PD
98059 on MAP kinases/ERKs activation (A) and
CRE-dependent and TCF-dependent calcium-activated transcription in PC12
cells (B, C). A,
Immunoblot analysis of lysates from PC12 cells before (lanes 1, 4, 7, 10) and 5 min after either KCl stimulation
(K; lanes 2, 5, 8, 11) or ionomycin
treatment (Ion; lanes 3, 6, 9, 12), using
antibodies specific for either phospho-MAP kinases/ERKs (lanes
1-6) or for MAP kinases/ERKs (lanes
7-12) to control for even protein loading. Treatment of the
cells with 50 µM PD 98059 (lanes 4-6 and
10-12) was done for 60 min before stimulation.
Mr, Protein molecular weight
standards (× 10 3). B, RNase
protection analysis was performed as described in Figure 2. PC12 cells
were transfected with plasmid pF711 (wild-type c-fos
gene, lanes 1-5), pFosCRE (lanes
6-8), pFosTCF (lanes 9-11), and pF222
(lanes 12-14). RNA was isolated from
unstimulated cells (lanes marked ) or cells stimulated with ionomycin
(Ion) or NGF (N). Treatment of the
cells with 50 µM PD 98059 (lanes 3, 5, 8, 11, 14) was done for 60 min before stimulation. Similar results were obtained with PD 98059 on KCl-induced TCF-dependent and
CRE-dependent transcriptional activation (data not shown). C, Quantitation of RNase protection experiments by the
PhosphorImager. The levels of c-fos mRNA transcribed
from the indicated plasmids and normalized for transfection efficiency
to the level of -globin mRNA in cells treated with PD 98059 are
expressed as a percentage of the amount of c-fos mRNA,
normalized to -globin expression, produced by untreated cells in
response to the same stimulus. The mean ± SEM is shown.
[View Larger Version of this Image (52K GIF file)]
We next investigated, using AtT20 cells, the involvement of MAP
kinases/ERKs in TCF-independent SRF-linked calcium-activated transcription. We were unable to perform experiments with the RasN17
expression vector because nontoxic concentrations of ionomycin, which
may be necessary to activate calcium entry into RasN17-expressing cells
(see above), only poorly induces c-fos expression (our
unpublished observation). However, we performed experiments with the
MEK 1 inhibitor PD 98059. Treatment of AtT20 cells with PD 98059 blocked KCl/FPL-induced activation of MAP kinases (ERKs) (Fig.
9A, compare lanes 2 and
4) but decreased inducibility of the transfected
constructs pF711, pFosCRE, and pFosSRF to only ~65-75% (Fig.
9B, compare lanes 2 and 3,
5 and 6, 8 and 9;
9C). A slightly larger effect of PD 98059 was seen on
KCl/FPL inducibility of transcription from pF222, which when compared
with cells not treated with PD 98059 was reduced to 44 ± 1%
(n = 3) (Fig. 9B, compare lanes
11 and 12; Fig. 9C).
Fig. 9.
Effect of the MAP kinase kinase 1 inhibitor PD
98059 on MAP kinases/ERKs activation (A) and on
CRE-dependent and SRF-linked calcium-activated transcription in AtT20
cells (B, C). A,
Immunoblot analysis of lysates from AtT20 cells before (lanes 1, 3, 5, 7) and 5 min after KCl/FPL stimulation
(K/F; lanes 2, 4, 6, 8), using antibodies
specific either for phospho-MAP kinases/ERKs (lanes 1-4) or for MAP kinases/ERKs (lanes
5-8) to control for even protein loading. Treatment of the
cells with 50 µM PD 98059 (lanes 3, 4, 7, 8) was done for 60 min before stimulation.
Mr, Protein molecular weight
standards (× 103). B, RNase
protection analysis was performed as described in Figure 5. AtT20 cells
were transfected with plasmid pF711 (wild-type c-fos
gene, lanes 1-3), pFosCRE (lanes
4-6), pFosSRF (lanes 7-9), and pF222
(lanes 10-12). RNA was isolated from unstimulated cells
(lanes marked ) or cells stimulated with KCl/FPL
(K/F). Treatment of the cells with 50 µM PD 98059 (lanes 3, 6, 9, 12) was done
for 60 min before stimulation. C, Quantitation of RNase protection experiments by the PhosphorImager. Analysis was done as
described in Figure 8C.
[View Larger Version of this Image (38K GIF file)]
These results indicate that the propagation of calcium signals to the
nucleus and calcium-dependent transcriptional activation can function
independently of the Ras/MAP kinases (ERKs) signaling cascade.
DISCUSSION
In this study we have investigated the complexity of
calcium-signaling mechanisms to the nucleus, using the c-fos
promoter as a model system. We demonstrate that calcium controls gene
expression by multiple mechanistically distinct pathways that operate
in a cell type-dependent manner. Calcium activates transcription via
the CRE, and, at the SRE, via a TCF-dependent and a TCF-independent SRF-linked mechanism. A model illustrating calcium signaling to the
c-fos promoter is shown in Figure 10.
Fig. 10.
Schematic model of calcium-signaling pathways to
the c-fos promoter.
[View Larger Version of this Image (18K GIF file)]
CRE-mediated calcium-activated transcription
The presence of an intact CRE in the c-fos promoter
can, in the absence of other regulatory sequences such as the SRE,
confer a calcium-induced transcriptional response that is similar in magnitude to that obtained with the wild-type c-fos gene.
This confirms previous observations that the CRE is a potent mediator of transcriptional activation by calcium-signaling pathways (Sheng et
al., 1988, 1990; Bading et al., 1993). However, we recently demonstrated that increases in cytoplasmic calcium concentrations are
not sufficient to activate a CRE and that increased nuclear calcium
concentrations are required (Hardingham et al., 1997). This finding,
together with our observation that CRE-mediated transcription is
largely independent of the Ras/MAP kinases (ERKs) cascade, suggests
that a nuclear localized mechanism directly activated by calcium
controls gene expression via the CRE. This has important implications
for gene expression by electrical activation of neurons. Calcium
signals, associated with electrical activity, per se will not
necessarily activate CRE-dependent gene expression, although they may
activate cytoplasmic signaling pathways such as the Ras/MAP kinases
(ERKs) cascade. Only stimuli that give rise to increases in nuclear
calcium will cause CRE-mediated transcriptional responses. This
provides a possible explanation for the finding that CRE-mediated gene
expression can be induced by multiple trains of high-frequency
electrical stimulation of neurons, but not by a single high-frequency
stimulus (Impey et al., 1996). The former, but not the latter, stimulus
may be associated with a sustained calcium influx into the cell, giving
rise to larger increases in nuclear calcium. In contrast, SRE-dependent
transcription (discussed below) can be stimulated independently of
increases in nuclear calcium by a rise in cytoplasmic calcium
(Hardingham et al., 1997). This may explain why the zif/268
gene, which contains several SREs in its promoter (Changelian et al.,
1989; Christy et al., 1989), is activated even with moderate
LTP-inducing stimuli that may not increase the nuclear calcium
concentration (Cole et al., 1989; Wisden et al., 1990; Worley et al.,
1993). The molecular mechanisms controlled by nuclear calcium may
involve CaM kinase IV. CaM kinase IV is localized to the nucleus of
neuronal cells (Jensen et al., 1991) and can phosphorylate CREB, a
mediator of calcium-activated transcription that interacts with the
CRE, on serine 133 (Sheng et al., 1991; Matthews et al., 1994; Sun et al., 1994). This residue is critical for CREB to function as a transcriptional activator (Gonzales and Montminy, 1989; Sheng et al.,
1991; Matthews et al., 1994; Sun et al., 1994). In addition, expression
of a constitutively active form of this CaM kinase IV is sufficient to
activate CRE/CREB-dependent transcription (Matthews et al., 1994; Sun
et al., 1994). Further studies are required to investigate the
expression, subcellular localization, and function of CaM kinase IV in
PC12 and AtT20 cells.
The SRE integrates two calcium-signaling pathways
The SRE is a second site of transcriptional regulation by
calcium-signaling pathways. The mechanisms by which calcium signals activate the SRE are cell type-dependent. In PC12 cells,
calcium-activated transcription via the c-fos SRE requires
the TCF site, whereas this response in AtT20 cells and primary
hippocampal neurons occurs via a TCF-independent SRF-linked mechanism.
These cell type differences may be also relevant for gene regulation in
the brain. A TCF-dependent pathway may be functional in one area of the
brain, whereas in a different area the SRF-linked pathway may be
operating. As a consequence, genes that contain SRF binding sites
without adjacent TCF binding motif in their promoter will respond to
calcium signals only in those cell types in which the SRF-linked
signaling pathway is functional. One example is the -actin gene that
contains in its promoter a binding site for SRF and an adjacent
inverted Ets motif, which when compared with the c-fos SRE,
less efficiently forms a ternary complex (Treisman et al., 1992).
Indeed, in PC12 cells in which the TCF-dependent mechanism targets the
c-fos SRE, -actin expression is not induced by
KCl-induced membrane depolarization (Bartel et al., 1989). In contrast,
in hippocampal neurons in which the c-fos SRE is activated
by an SRF-linked mechanism, transcription of -actin is stimulated by
calcium signals (Bading et al., 1995). Thus, the cell type is an
important determinant for the functional significance of a particular
calcium-signaling pathway and provides a means for differential gene
expression by calcium signals.
The nature of the calcium-signaling pathway targeting the TCF site
remains unclear. In cortical neurons, calcium signals generated by NMDA
receptor activation can stimulate transcription through a MAP kinase
signaling cascade targeting the TCF Elk-1 (Xia et al., 1996). However,
our analysis of the TCF-dependent pathway in PC12 cells demonstrated
that neither expression of RasN17 nor treatment of the cells with MAP
kinase kinase 1 inhibitor PD 98059 significantly reduced the
TCF-dependent calcium response, indicating that the Ras/MAP kinases
(ERKs) pathway is not critical. Moreover, we find that, in primary
hippocampal neurons, NMDA receptor-regulated gene expression is mainly
independent of the TCF site and requires the SRF binding site (see
below). In the experiments described by Xia et al. (1996), a
c-fos-based reporter gene was used in which the binding site
for TCF Elk-1 was inserted in close proximity to the c-fos
TATA box, whereas in our experiment the TCF site remained in its
natural position (nucleotide 320) in the intact c-fos
promoter. It is possible that MAP kinases/ERK, which are known to be
activated in response to NMDA receptor activation (Bading and
Greenberg, 1991), can stimulate transcription through TCF Elk-1 if the
TCF binding site is close to the start site of transcription. If,
however, the TCF site is several hundred base pairs upstream of the
TATA box (as in the constructs used in this study), transcriptional
activation through this site may be mediated by a mechanism that
functions independently of the Ras/MAP kinases (ERKs) signaling
cascade. Thus, differences in the promoter constructs used for the
transcriptional analysis and cell type differences may explain this
discrepancy.
The TCF-independent SRF-linked pathway of calcium-activated
transcription in AtT20 cells and primary hippocampal neurons resembles the mechanism by which serum stimulation or activation of
heterotrimeric G-proteins induces c-fos expression. Signals
generated by these stimuli are transduced to the nucleus by a mechanism
involving members of the rho family of GTPases (Hill et al., 1995).
This raises the possibility that calcium signals also stimulate
transcription by a rho-dependent mechanism. Our finding that the
TCF-independent SRF-linked pathway is insensitive to PD 98059 is
consistent with this hypothesis, because a rho-mediated mechanism of
transcriptional activation may function independently of MAP kinase
kinase 1 activation. Although our experiments demonstrate that, in the
context of the entire c-fos gene, the SRF binding site is
not sufficient to mediate c-fos induction in PC12 cells upon
KCl stimulation, two other studies indicate that a TCF-independent
SRF-linked pathway may be also functional in PC12 cells (Misra et al.,
1994; Miranti et al., 1995). In these studies it was shown that the SRF
binding site, when placed close to the c-fos TATA box, is
capable of mediating calcium-dependent transcriptional activation via a
mechanism that involves CaM kinases (Misra et al., 1994; Miranti et
al., 1995). These results also illustrate that the promoter context in
which a particular transcriptional control element is analyzed is an important, often unappreciated, determinant of the signaling mechanism that is functionally significant.
Conclusion
Calcium controls gene expression by multiple mechanistically
distinct pathways activating transcription via the CRE and the SRE. The
SRE is bifunctional and integrates two signaling mechanisms. The
significance of these pathways for the regulation of transcription depends on the spatial properties of the calcium signals (Hardingham et
al., 1997) and the cell type. Given the differences in the composition
and arrangements of calcium response elements in transcriptional control regions, these findings constitute the basic principles of
calcium-activated transcription that may underlie differential gene
expression by electrical activity in neurons.
FOOTNOTES
Received April 17, 1997; accepted May 28, 1997.
This work was supported by the Medical Research Council, Glaxo
Wellcome, Imperial Cancer Research Fund, and the Howard Hughes Medical
Institute. We thank Bill Wisden for helpful discussions and comments on
this manuscript.
Correspondence should be addressed to Dr. Hilmar Bading, Division of
Neurobiology, Medical Research Council Laboratory of Molecular Biology,
Hills Road, Cambridge CB2 2QH, England.
Dr. Hill's present address: Ludwig Institute for Cancer Research, 91 Riding House Street, London W1P 8BT, England.
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