From the Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School, Worcester, Massachusetts 01613
Received for publication, November 28, 2000, and in revised form, March 21, 2001
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ABSTRACT |
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During neuronal differentiation, an exquisitely
controlled program of signal transduction events takes place, leading
to the temporally and spatially regulated expression of genes
associated with the differentiated phenotype. A critical class of genes
involved in this phenomenon is that made up of genes encoding
neurotransmitter-gated ion channels that play a central role in signal
generation and propagation within the nervous system. We used the well
established PC12 cell line to investigate the molecular details
underlying the expression of the neuronal nicotinic acetylcholine
receptor class of ion channels. Neuronal differentiation of PC12 cells can be induced by nerve growth factor, leading to an increase in
neuronal nicotinic acetylcholine receptor gene expression. Nerve growth
factor initiates several signal transduction cascades. Here, we show
that the Ras-dependent mitogen-activated protein kinase and phosphoinositide 3-kinase pathways are critical for the
nerve growth factor-mediated increase in the transcriptional activity
of a neuronal nicotinic acetylcholine receptor gene promoter. In
addition, we show that a component of the
Ras-dependent mitogen-activated protein kinase
pathway, nerve growth factor-inducible c-Jun, exerts its effects
on receptor gene promoter activity most likely through protein-protein
interactions with Sp1. Finally, we demonstrate that the target for
nerve growth factor signaling is an Sp1-binding site within the
neuronal nicotinic acetylcholine receptor gene promoter.
Nerve growth factor
(NGF)1 is critical for the
survival and differentiation of sensory and sympathetic neurons in the
peripheral nervous system (1) and of basal forebrain and hippocampal
cholinergic neurons in the central nervous system (2). NGF signals
through binding to its receptors, a high affinity tyrosine kinase
receptor, TrkA, and a low affinity receptor, p75 (3). Binding of NGF to
its receptors initiates several signal transduction cascades, including, among others, ras-dependent mitogen-activated
protein kinase (MAPK), phosphoinositide 3-kinase (PI3-K), and the
cAMP-dependent protein kinase pathways (3). Some of
the downstream targets of the activated protein kinase pathways are
transcription factors such as AP-1, cAMP responsive element-binding
protein, and NF- Rat pheochromocytoma PC12 cells have been extensively used to study the
molecular mechanisms involved in NGF signaling. PC12 cells are
chromaffin-like cells that in response to NGF treatment withdraw from
the cell cycle and differentiate into sympathetic-like neurons, a
process accompanied by neurite outgrowth, increased electrical
excitability, and changes in neurotransmitter synthesis (4-6). Among
the signal transduction cascades activated by NGF in PC12 cells, the
MAPK and the PI3-K pathways are thought to play a central role,
although other pathways are clearly important (7-10). Activated TrkA
promotes conversion of the membrane-anchored Ras-GDP into Ras-GTP. Ras
then cooperates in activation of a serine/threonine kinase, Raf-1,
which phosphorylates the dual specificity MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK), which in turn
phosphorylates and activates the ERKs, ERK1 and ERK2. Recently, it was
demonstrated that activation of ERK in response to NGF in both primary
sensory neurons and in PC12 cells also requires activation of the PI3-K pathway (11, 12). PI3-K is required for internalization of TrkA and for
its signaling to ERK via a Ras family member, Rap 1 (11, 12). Rapid
activation of ERK in response to NGF is mediated through Ras, whereas
the sustained activation is Rap 1-dependent (11).
Activation of ERK is thought to be required for PC12 responsiveness to
NGF, because NGF-induced differentiation can be blocked by specific
kinase inhibitors, by antibodies against Ras and MEK, or by expression
of dominant negative mutants of Ras and MEK (13-16). In addition,
constitutively active forms of Ras, Raf-1, and MEK1 can induce neuronal
differentiation of PC12 cells (15, 17-19). Activated ERKs translocate
to the nucleus, where they phosphorylate and activate the existing
transcription factor c-Jun, as well as induce expression of c-Jun (20).
Interestingly, overexpression of activated c-Jun in PC12 cells induces
neurite outgrowth, suggesting that c-Jun plays an important role in
promoting PC12 neural differentiation (20).
Among the neuronal genes whose expression is up-regulated in response
to NGF in PC12 cells are the genes encoding several subunits ( Cell Culture and Transfections--
SN17 cells (26) and
Drosophila melanogaster Schneider SL2 cells were maintained
as described (27, 28). PC12 cells (29) were cultured and differentiated
with NGF (Upstate Biotechnology, Lake Placid, NY) as previously
described (25). Twenty-four h prior to transfections, SN17 cells were
plated at a density of 250,000 cells per 35-mm dish. Transfections were
performed by a calcium phosphate method using a commercially available
kit (Eppendorf-5 Prime, Inc., Boulder, CO). The wild type rat
Immunoprecipitations and Western
Blotting--
Immunoprecipitations and Western blotting were performed
as previously described (36) using 250 µg of nuclear extracts
prepared from NGF-treated PC12 cells. Anti-c-Jun rabbit polyclonal
antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Nuclear extracts were prepared by the method of Dignam et
al. (37) as previously described (38), except that nuclear
extracts were dialyzed against RIPA buffer (50 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 1% nonidet P-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate).
PI3-K and MEK Activation Are Necessary for the Induction of the
c-Jun Transcativates the
Jun proteins bind to the DNA sequence 5'-TGACTCA-3', referred to as an
AP-1 site, as homodimers or as heterodimers with Fos proteins (39).
Interestingly, sequence analysis of the c-Jun and Sp1 Physically Interact in PC12 Cells--
c-Jun has
been shown to functionally interact with a number of transcription
factors, including members of the Sp family, Sp1 and Sp3 (35, 40-42).
Moreover, c-Jun can directly interact with Sp1 in vitro
(41). This is particularly relevant to the c-Jun Transcativates the c-Jun and Sp1 Synergistically Activate the
To investigate the importance of the Sp1-binding site for the ability
of Sp1 and c-Jun to synergistically activate the The CA Box Is Important for the NGF Responsiveness of the To date, twelve genes ( The MAPK pathway, through a series of phosphorylation events (that
include phosphorylation of ERK), is thought to relay signals received
by NGF receptors in the plasma membrane to specific transcription factors in the nucleus (51). These activated transcription factors are
required for expression of the delayed early and late genes, some of
which, such as neuronal nAChR, contribute to the neuronal phenotype
developed by PC12 cells in response to NGF. c-Jun is believed to be one
of the key factors activated by the MAPK signaling cascade (39). c-Jun
is an inducible transcription factor that is activated in response to
multiple extracellular stimuli, including growth factors, cytokines,
neurotransmitters, T cell activators, various forms of stress, and UV
radiation (39). Regulation of c-Jun activity occurs at the level of
transcription, resulting in an increase in c-Jun mRNA, as well as
post-transcriptionally, resulting in an enhancement of its
transactivation potential through phosphorylation (39). Leppa et
al. (20) recently demonstrated that in PC12 cells, activation of
the MAPK pathway, more specifically ERK, results in stimulation of both
c-Jun synthesis and phosphorylation. Moreover, they showed that an
exogenous activated form of c-Jun induced marked neurite outgrowth and
that overexpression of wild type c-Jun potentiated differentiation
induced by MEK1, whereas dominant-negative mutants of c-Jun inhibited
it (20). Thus, proper c-Jun activation appears to be necessary for
neuronal differentiation of PC12 cells. We therefore investigated
whether c-Jun is capable of regulating the promoter of the c-Jun has been shown to directly interact with a number of
transcription factors, for example, members of the cAMP responsive element-binding protein family, glucocorticoid receptor (53), Smad
proteins (54, 55), and members of the Sp family, Sp1 and Sp3 (35,
40-42). Interestingly, we have previously demonstrated that both Sp1
and Sp3 can strongly transactivate the It should be noted that the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B.
3,
5,
7,
2, and
4) of neuronal nicotinic acetylcholine receptors (nAChRs) (21-23). nAChRs are pentameric ligand-gated ion
channels important for synaptic transmission in the nervous system
(24). Previously, we demonstrated that the NGF-mediated increase in
nAChR gene expression is independent of cAMP-dependent protein kinase signaling (21) and that NGF treatment of PC12 cells
increases the transcriptional activity of the
4 gene promoter (25).
In this study we investigated the involvement of the other NGF-signaling pathways, namely MAPK, PI3-K, protein kinase C, and phospholipase C, in the regulation of nAChR gene expression. We
show that in PC12 cells, activation of PI3-K and one of the components
of the MAPK pathway, MEK, is important for up-regulation of
4
subunit gene promoter activity in response to NGF. We also show that
NGF-inducible c-Jun can transactivate the promoter of the
4
gene through cooperation with Sp1 protein bound to the
4 promoter at
a previously identified Sp1-binding site, a CA box. We further
demonstrate that this Sp1-binding site is necessary for the
responsiveness of the
4 promoter to NGF.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4-luciferase expression plasmid, pX1B4FH, containing a 226-bp
FokI/HindIII fragment spanning nucleotides
89
to +137, relative to the
4 transcription initiation site, and the
construct pX1B4FHmut4, containing a mutation in the CA box of the
4
promoter, were described previously (25, 30). Cells were transfected
with 2.5 µg of test DNA (pX1B4FH or pX1B4FHmut4), 2.5 µg of
effector DNA (the empty pCMV5 vector (Invitrogen, Carlsbad, CA) or
pCMV-jun constructs), and 2.5 µg of a
-galactosidase expression
vector, RSV-
gal. In some cases, no effector DNA was included in the
transfections. To ensure that the calcium phosphate/DNA precipitates
had equal amounts of DNA, appropriate quantities of pBluescript II SK
DNA (Stratagene, La Jolla, CA) were added to each sample. PC12 cells were transfected in 60-mm dishes at a density of 106
cells/ml using 30 µl of LipofectAMINE (2 mg/ml; Life Technologies, Inc.) and 2.5 µg of each DNA. After a 5-h incubation in the
lipid/DNA mix, cells were washed twice with Dulbecco's modified
Eagle's medium and fed with growth medium. For experiments involving
the pharmacological kinase inhibitors, growth media were
supplemented with or without 100 ng/ml NGF and with or without
inhibitors, as indicated. All inhibitors were purchased from Calbiochem
(San Diego, CA) and were used at the following concentrations: MEK inhibitor PD98059 at 100 µM (31), PI3-K inhibitor
LY294002 at 50 µM (12, 32), phospholipase C inhibitor
U-73122 at 1 µM (33), and protein kinase C inhibitors
calphostin C (33) and bisinodylmalemide (BIM) (34) at 400 nM and 1 µM, respectively. SL2 cells
were transfected as previously described (28), except that 100 ng of
the effector DNAs (pActSp1 or pPacJun) were used. The pPacJun plasmid
was a kind gift of Dr. J. Noti and is described elsewhere (35).
Forty-eight h following transfection, cells were harvested and assayed
for luciferase activity using a commercially available kit (Promega
Corp., Madison, WI) and an Autolumat LB953 luminometer (EG&G Berthold,
Gaithersburg, MD). All transfections were done a minimum of two times
with two different preparations of plasmid DNAs. To correct for
differences in transfection efficiencies between dishes, the luciferase
activity in each sample was normalized to the
-galactosidase
activity in the same sample, which was measured using a commercially
available kit (Galacto-Light; Tropix, Inc., Bedford, MA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 Promoter in Response to NGF--
NGF-dependent
up-regulation of neuronal nAChR gene expression in PC12 cells is
thought to occur partly at the level of transcription (25). To
investigate which of the NGF-activated pathways is involved in the
transcriptional regulation of neuronal nAChR gene expression in PC12
cells, we examined the effects of blocking several of these pathways on
the transcriptional activity of the
4 gene promoter. We previously
identified a 226-bp promoter fragment of the
4 gene that can confer
neuron-specific expression to a reporter gene in transient transfection
assays (27). We have also demonstrated that treatment of PC12 cells
with NGF results in a significant increase in
4 promoter activity
and that the 226-bp region is sufficient to mediate this response (25,
27). We therefore investigated whether MEK, PI3-K, protein kinase C, or
phospholipase C is involved in the regulation of transcription of the
4 gene through elements in the 5' regulatory region of the gene. The
226-bp
4 fragment fused upstream of the luciferase gene (pX1B4FH)
was transiently transfected into PC12 cells. Following transfection
cells were treated with NGF for 36 h. A parallel set of
transfected cells was left untreated and served as a control. Consistent with our previous results, 36 h of NGF treatment
stimulated
4 promoter activity and resulted in induction of reporter
gene activity (Fig. 1). When NGF
treatment of the transfected cells was carried out in the presence of
PD98059, a selective pharmacological agent that blocks activation of
MEK, or LY294002, a specific pharmacological inhibitor of PI3-K,
stimulation of the
4 promoter was significantly reduced, as judged
by reporter gene activity (Fig. 1). In contrast, inhibitors of
phospholipase C, U-73122, and protein kinase C, calphostin C, and
bisinodylmalemide, had little effect on the activity of the
4
promoter in transfected cells in response to NGF (Fig. 1). These
results suggest that activation of the
4 promoter in response to NGF
is mediated in part via the MAPK- and PI3-K-dependent
pathways in PC12 cells. This is consistent with a model suggesting that
these two pathways share overlapping functions in neuronal cells (12).
In addition, these data indicate that the 226-bp fragment of the
4
promoter contains the necessary elements to mediate the response to ERK
activation, a common downstream target of MEK and PI3-K (12).
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Fig. 1.
Pharmacological blockade of MEK and PI3-K
prevents NGF activation of the 4
promoter. PC12 cells were transiently transfected with pX1B4FH as
described under "Experimental Procedures." Following transfection,
cells were treated with 100 ng/ml NGF in the presence or absence of
specific pharmacological kinase inhibitors for 36 h, as indicated.
Luciferase values were normalized to
-galactosidase expression as
driven by the CMV promoter (see "Experimental Procedures"). Fold
induction was calculated relative to the normalized luciferase activity
obtained from transfected cells that were not treated with NGF. Error
bars represent standard deviations of the means. PD,
PD98059; LY, LY294002; U, U-73122;
CalC, calphostin C.
4 Promoter in PC12 and SN17
Cells--
It is well established that in PC12 cells, activation of
the ERK cascade by NGF induces c-Jun expression and phosphorylation of
its gene product (20). c-Jun is a transcription factor that has been
implicated in regulation of gene expression in response to multiple
extracellular stimuli (39). To address the question whether c-Jun is
capable of regulating the activity of the
4 promoter, PC12 cells
were transiently transfected with the 226-bp
4
promoter-luciferase construct, pX1B4FH, alone or in combination with an expression construct for c-Jun, pCMV-jun, in which the c-Jun
gene is under the control of the CMV promoter, or with an empty
parental vector, pCMV5. As shown in Fig.
2A (left side), transfections of the empty expression vector pCMV5 had no effect on
reporter gene activity. However, when the
4-luciferase construct was
cotransfected with c-Jun, a dramatic increase in reporter gene activity
was observed (Fig. 2A). Similar results were observed in the
cholinergic cell line SN17 (Fig. 2B). Together, these data indicate that, indeed, c-Jun is capable of transactivating the
4
promoter and suggest that c-Jun-responsive elements are present within
this 226-bp fragment of the
4 promoter.
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Fig. 2.
c-Jun transactivates the
4 promoter through an Sp1-binding site. PC12
cells (A) or SN17 cells (B) were transfected with
either the wild type
4 promoter/luciferase construct (WT)
(pX1B4FH) or the mutant construct (mut) (pX1B4FHmut4) alone
or with pCMV5 or pCMV-jun (c-Jun). Luciferase values were normalized to
-galactosidase expression as driven by the CMV promoter. Error bars
represent standard deviations of the means.
4 promoter fragment used in
these experiments indicates that there are no AP-1-binding sites within
this region. It is therefore possible that c-Jun transactivates the
4 promoter through protein-protein interactions with a factor that
can bind to
4 regulatory elements.
4 promoter, because we
previously reported that both Sp1 and Sp3 can efficiently transactivate
this promoter (28, 30, 36). Therefore, it is possible that c-Jun
activates the
4 promoter through protein-protein interactions with
Sp1 and/or Sp3. To test this hypothesis, we first wanted to address the
question of whether c-Jun and Sp1 are physically associated in PC12
cells. To that end, we performed immunoprecipitation experiments
followed by Western blot analysis. Anti-Sp1 or anti-c-Jun antiserum was
used to immunoprecipitate proteins from nuclear extracts prepared from NGF-treated PC12 cells. The precipitated proteins were separated via
SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane that was subsequently incubated with anti-Sp1 or anti-c-Jun antiserum. As shown in Fig.
3, preimmune serum did not precipitate
either Sp1 or c-Jun in this experiment. However, complexes containing
Sp1 and c-Jun were detected by immunoprecipitations using antibodies
directed toward one or the other protein (Fig. 3). It should be noted
that the levels of Sp1 found in c-Jun immunoprecipitates were somewhat
lower when compared with those precipitated with anti-Sp1 antibody
(Fig. 3), suggesting that not all cellular Sp1 is engaged in a complex
with c-Jun. The same was true with c-Jun as well (Fig. 3). These data
provide compelling evidence for direct physical interactions between
Sp1 and c-Jun in PC12 cells and support the idea that c-Jun may
regulate the
4 promoter through its association with Sp1.
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Fig. 3.
Immunoprecipitations of c-Jun-Sp1 complexes
from NGF-treated PC12 cell lysates. Anti-c-Jun, anti-Sp1, or
preimmune rabbit serum (PIRS) was used to immunoprecipitate
(IP) proteins from a PC12 cell extract. Western analysis of
the immunoprecipitated material was carried out using anti-Sp1 or
anti-c-Jun antiserum as indicated.
4 Promoter through an Sp1-binding
Site--
Previously, we showed that Sp1 can specifically bind to a CA
box in the 226-bp region of the
4 promoter (30, 36) and that this
interaction is critical for Sp-mediated transactivation of the
4
promoter (36). If c-Jun activates the
4 promoter through its
association with Sp1, then the Sp1-binding site within this promoter,
the CA box, should be important for the ability of c-Jun to regulate
the
4 promoter. To test this hypothesis, PC12 cells were
cotransfected with a wild type
4 promoter-luciferase expression
construct, pX1B4FH, or with a construct in which the CA box is mutated
at three nucleotide positions, pX1B4FHmut4, and with an expression
construct for c-Jun, pCMV-jun. As mentioned earlier, c-Jun was able to
strongly transactivate the wild type
4 promoter, pX1B4FH (Fig.
2A). However, when the
4 promoter-luciferase reporter
containing mutations in the CA box, pX1B4FHmut4, was used in this
experiment, c-Jun activation of the promoter was marginal (Fig.
2A). Similar results were obtained from transfections performed in SN17 cells (Fig. 2B). Thus, the CA box appears
to be critical for c-Jun-mediated transactivation of the
4 promoter.
4 Promoter in
Drosophila SL2 Cells--
To further investigate the importance of
c-Jun-Sp1 interactions for the transactivation of the
4
promoter, we performed transfection experiments using the
Drosophila SL2 cell line. These cells lack endogenous Sp1
activity (43-46), as well as endogenous c-Jun activity (35), making
them a useful system to study the functional consequences of the
protein-protein interactions mentioned above. We previously reported
that Sp1 can strongly transactivate the
4 promoter when transfected
into SL2 cells (28, 30, 36). To determine whether c-Jun activates the
4 promoter through its interactions with Sp1, SL2 cells were
cotransfected with a wild type
4 promoter-luciferase expression
construct (pX1B4FH) and pActSp1 or pPacJun or both. To confirm that Sp1
and c-Jun expression was involved in transactivation, the reporter DNA
was also transfected with the parental vectors devoid of Sp1 and c-Jun
coding sequences, pAct and pPacO, alone. As expected, Sp1 was capable
of transactivating the wild type
4 promoter-luciferase construct
~20-fold (Fig. 4). Consistent with the
observation that the
4 promoter fragment used in these experiments
does not contain an AP-1-binding site, c-Jun by itself did not have any
effect on reporter gene activity (Fig. 4). However, when Sp1 and c-Jun
were cotransfected with the
4 promoter-luciferase construct, an
~120-fold increase in reporter gene activity was observed, indicating
a synergistic effect of the two regulatory factors (Fig. 4). These
results are consistent with the hypothesis that c-Jun activates the
4 promoter through direct interactions with Sp1.
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Fig. 4.
Sp1 and c-Jun synergistically transactivate
the 4 promoter in Drosophila
cells. Drosophila SL2 cells were transfected with
pX1B4FH or pX1B4FHmut4 (WT or mut, respectively)
alone, with "empty" expression vectors (pAct, pPacO), or with
expression constructs for c-Jun and Sp1, individually and together.
Fold induction was calculated as above.
4 promoter, similar
transfection experiments were performed using a
4
promoter-luciferase construct in which the CA box is mutated at three
nucleotide positions (pX1B4FHmut4). Mutation in this site reduces the
ability of Sp1 to transactivate the
4 promoter in SL2 cells (Fig. 4)
(36). The residual activation seen with the mutated promoter is most
likely a result of Sp1 binding to downstream low affinity Sp-binding
sites (30, 36). As in the case of the wild type
4 promoter, c-Jun
was unable to transactivate the mutated promoter (Fig. 4). Synergistic
transactivation of the wild type
4 promoter by Sp1 and c-Jun was
dramatically reduced when the mutated promoter was used. The observed
reporter gene activity was comparable with that of Sp1 alone on the
wild type promoter (Fig. 4). Together, these results indicate that Sp1
and c-Jun can cooperate to activate the
4 promoter in
Drosophila cells and that an intact Sp1-binding site is
important for this cooperativity.
4
Promoter in PC12 Cells--
Collectively, the data presented above
suggest that the CA box of the
4 promoter might be a target for NGF
signaling in PC12 cells. To address this question, we transfected PC12
cells with a wild type
4 promoter-luciferase expression construct,
pX1B4FH, or with the construct in which the CA box is mutated,
pX1B4FHmut4. Consistent with our previous findings (30), the basal
activity of the mutant promoter was significantly lower than that of
the wild type in untreated PC12 cells (Fig.
5). Thirty-six h of NGF treatment of the
transfected cells resulted in a dramatic increase in the activity of
the wild type
4 promoter, as judged by reporter gene activity (Fig.
5). However, activity of the mutated promoter was increased only
slightly, resulting in reporter gene activity similar to that produced
by the wild type
4 promoter in unstimulated PC12 cells (Fig. 5).
These data suggest that the CA box is necessary, but probably not
sufficient, to mediate the response of the
4 promoter to NGF in PC12
cells.
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Fig. 5.
Mutation of the Sp1-binding site prevents NGF
activation of the 4 promoter. PC12 cells
were transfected with pX1B4FH (WT) or pX1B4FHmut4
(mut). Following transfection, cells were treated with 100 ng/ml NGF for 36 h. A parallel set of cells was left untreated.
Luciferase values were normalized to
-galactosidase expression as
driven by the CMV promoter. Error bars represent standard deviations of
the means.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-
10,
2-
4) encoding subunits of
neuronal nAChRs have been identified. Assembly of the different subunits into pentameric homo- or heteromeric receptors results in
formation of ion channels with distinct physiological and
pharmacological profiles (24). This diversity is thought to be a
consequence of differential expression of the subunit genes and
subsequent incorporation of their respective products into mature
receptors (47). Whereas the implications of the functional diversity of nAChR are beginning to be understood, relatively little is known regarding regulation of their gene expression in general and in response to neurotrophin signaling in particular. NGF-driven
differentiation of PC12 cells has been extensively used as a model
system to study the physiological, biochemical, and molecular
mechanisms of neurotrophin signaling. In regard to neuronal nAChR, it
has been demonstrated that in PC12 cells, NGF increases ACh-induced
channel activity and ACh-induced macroscopic current density (4, 21,
48), phenomena that are thought to be a consequence of an increase in
the number of functional nAChR (21). This hypothesis is consistent with
observed increases in the binding of [3H]nicotine (49)
and of anti-nAChR antibodies (50) that occur in PC12 cells in response
to NGF. Whereas the exact mechanisms of the increase in the number of
nAChRs are unknown, NGF-driven induction of nAChR gene expression is
clearly important (21). Previously, we showed that the NGF-induced
up-regulation of nAChR expression and increases in ACh-induced current
density are independent of cAMP-dependent protein kinase
activity (21). In this study, using specific chemical inhibitors, we
demonstrate that the MAPK and PI3-K signal transduction cascades, but
not the phospholipase C or protein kinase C pathways, are important for
NGF-mediated induction of the promoter of the
4 subunit gene in PC12
cells. Significantly, positive stimulation of
4 promoter activity in response to NGF was considerably reduced by the addition of a pharmacological inhibitor of MEK, PD98059, and was reduced to a similar
extent by addition of a PI3-K inhibitor, LY294002. These data suggest
that activation of the MAPK and PI3-K pathways is clearly necessary for
the induction of nAChR gene expression. Moreover, they indicate that
MAPK and PI3-K pathways might have common downstream targets in PC12
cells. Originally, PI3-K and MAPK were thought to initiate two distinct
signal transduction cascades in neuronal cells. However, in a recent
report York et al. (12) convincingly demonstrated that
inhibitors of PI3-K block well known targets of the MAPK pathway,
namely ERK and B-Raf, therefore indicating that the MAPK and PI3-K
pathways are indeed interconnected.
4 subunit
gene. Using the same fragment of the
4 promoter that confers
neuron-specific expression to a reporter gene (24) and that is
sufficient to mediate responses to NGF in PC12 cells (25), we showed
that c-Jun can strongly transactivate the
4 promoter in both PC12
and SN17 cells. Surprisingly, this 226-bp region of the
4 promoter
does not contain "classic" Jun-binding sites, AP-1 elements, to
which c-Jun normally binds as a dimer with Jun-Fos proteins (52). Thus,
we hypothesized that c-Jun transactivates the
4 promoter through
protein-protein interactions with a nuclear factor bound to
4
regulatory elements.
4 promoter (28, 30, 36). Sp1
and Sp3 are zinc finger DNA-binding proteins that bind a CA box element
in the 226-bp region of the
4 promoter (30, 36). Originally, Sp
family members were thought to be ubiquitous factors contributing to
core promoter activities (56). However, more recently, they have been
shown to participate in regulated and cell type-specific gene
expression, including regulation of transcription of a number of
neuron-specific genes (30, 36, 57-62). Moreover, in PC12 cells, NGF
treatment has been shown to stimulate Sp1-dependent
transcription (63). Although the exact mechanism of this stimulation is
unknown, it is thought that post-translational modifications and/or
association with other factors may play a role (63). Hence, NGF-induced
activation of
4 gene expression may occur through functional
interactions between c-Jun and Sp1. Consistent with this idea are
results of our immunoprecipitation/Western blot analysis demonstrating
that c-Jun and Sp1 can physically associate in NGF-treated PC12 cells.
Furthermore, the Sp1-binding site in the 226-bp region of the
4
promoter, a CA box, is important for the ability of c-Jun to
transactivate this promoter in both PC12 and SN17 cells. In contrast to
PC12 and SN17 cells, c-Jun by itself had no effect on reporter gene
activity in Drosophila SL2 cells that were devoid of
endogenous Sp proteins. c-Jun was able to transactivate the
4
promoter in SL2 cells only when cotransfected with Sp1, providing
additional support for the hypothesis that it does so through direct
interactions with the latter protein. Interestingly, transactivation by
c-Jun and Sp1 occurred in a synergistic manner and did so in a CA
box-dependent manner. Together, these data suggest that Sp1
and c-Jun can cooperate to activate the
4 promoter and that an
intact Sp1-binding site is important for this cooperativity. Moreover,
the CA box of the
4 promoter appears to be critical for NGF
signaling, because the responsiveness of the
4 promoter to NGF is
dramatically reduced when the CA box is mutated. Previous studies
demonstrated that c-Jun and Sp1 functionally interact on both synthetic
and natural promoters (40, 41); however, to our knowledge, this study
is the first demonstration of cooperativity of these proteins in
neuronal cells. Whether, similar to Sp1, Sp3 can functionally interact
with c-Jun to regulate nAChR gene expression remains to be elucidated.
4 subunit, together with the
3 and
5 subunits, forms the predominant neuronal nAChR subtype expressed
in the peripheral nervous system (64, 65). These three genes are
tightly clustered in mammalian genomes, raising the possibility that
they are coordinately expressed via a common regulatory mechanism.
Indeed, it has been demonstrated that Sp1 is important for the
regulation of expression of all three genes (30, 36, 61, 62).
Therefore, it is possible that through interactions with Sp1, c-Jun
might regulate the promoters of the
3 and
5 subunit genes as well.
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ACKNOWLEDGEMENTS |
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We thank John Noti, Mangkey Bounpheng, Barbara Christy, Ed Seto, and Haley Melikian for expression constructs and useful discussions. A portion of the work described was performed at the University of Texas Health Science Center at San Antonio.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institutes of Health and the Smokeless Tobacco Research Council, Inc. (to P. D. G.).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.
Supported by a POWRE grant from The National Science Foundation.
§ To whom correspondence should be addressed: Brudnick Neuropsychiatric Research Inst., Dept. of Psychiatry, University of Massachusetts Medical School, 55 Lake Ave. North, P.O. Box 2795, Worcester, MA 01613. Tel.: 508-856-4035; Fax: 508-856-4130; E-mail: paul.gardner@umassmed.edu.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M010735200
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ABBREVIATIONS |
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The abbreviations used are: NGF, nerve growth factor; MAPK, ras-dependent mitogen-activated protein kinase; PI3-K, phosphoinositide 3-kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; ACh, acetylcholine; nAChR, neuronal nicotinic acetylcholine receptor; bp, base pair.
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