Synergy of Activin and Ciliary Neurotrophic Factor Signaling Pathways in the Induction of Vasoactive Intestinal Peptide Gene Expression
Aviva J. Symes,
R. Lee Pitts,
Jill Conover,
Ksenija Kos and
James Coulombe
Departments of Pharmacology (A.J.S., R.L.P., J.C.) and Anatomy
and Cell Biology (K.K., J.C.) Uniformed Services University of the
Health Sciences Bethesda, Maryland 20814
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ABSTRACT
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Activin, a member of the transforming growth
factor-ß superfamily, can regulate neuropeptide gene expression in
the nervous system and in neuroblastoma cells. Among the neuropeptide
genes whose expression can be regulated by activin is the gene encoding
the neuropeptide vasoactive intestinal peptide (VIP). To investigate
the molecular mechanisms by which activin regulates neuronal gene
expression, we have examined activins regulation of VIP gene
expression in NBFL neuroblastoma cells. We report here that NBFL cells
respond to activin by increasing expression of VIP mRNA. Activin
regulates VIP gene transcription in NBFL cells through a 180-bp element
in the VIP promoter that was previously characterized to be necessary
and sufficient to mediate the induction of VIP by the neuropoietic
cytokines and termed the cytokine response element (CyRE). We find that
the VIP CyRE is necessary and sufficient to mediate the transcriptional
response to activin. In addition, ciliary neurotrophic factor (CNTF), a
neuropoietic cytokine, synergizes with activin to increase VIP mRNA
expression and transcription through the VIP CyRE. Mutations in either
the Stat (signal transducer and activator of transcription) or AP-1
sites within the CyRE that reduce the response to CNTF, also reduce the
response to activin. However, mutating both the Stat and AP-1 sites
within the wild-type CyRE, while reducing the separate responses to
either activin or CNTF, eliminates the synergy between them. These data
suggest that activin and CNTF, two factors that appear to signal though
distinct pathways, activate VIP gene transcription through a common
transcriptional element, the VIP CyRE.
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INTRODUCTION
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Activin was first identified because of its ability to stimulate
FSH release from cultured pituitary cells (1, 2). Because of the
presence of a conserved region containing seven cysteine residues,
activin is considered to be a member of the transforming growth factor
ß (TGF-ß) superfamily of polypeptide growth factors which includes
TGF-ß, mullerian inhibitory substance, Drosophila
decapentaplegic gene product, bone morphogenetic factors (BMPs), and
inhibin (3). Three separate genes encode the individual subunits that
comprise the activin molecule (4). These subunits dimerize in different
combinations to form the active activin molecule of approximately 30
kDa (2). While no functional differences between these different
isoforms have been described, the different activins appear to differ
in the locations of their expression (5, 6).
Activin signals through type II and type I receptors (7, 8), which both
have intrinsic serine threonine kinase activity (7, 8, 9, 10). While there is
significant homology among and between the type II and type I receptors
of the TGF-ß superfamily, the ligand specificity appears to derive
from the particular type II receptor expressed. The current model of
receptor activation proposes that ligand binding to type II receptors
causes recruitment of type I receptor to the ligand-type II receptor
complex, transphosphorylation of the glycine-serine rich domain in the
type I receptor by the type II receptor, and propagation of the signal
by the activated type I receptor to intracellular targets (reviewed in
Ref. 11).
Activin may be involved in several different regions of the nervous
system. Activin acts as a trophic factor to support the survival of
several neuronal cell lines (12), has trophic activity for cultured
midbrain dopaminergic neurons (13), supports the survival in culture of
hippocampal neurons (14), and influences the survival and/or
differentiation of retinal photoreceptors in culture (15). Activin
appears to act as a target-derived neurotransmitter differentiation
factor for parasympathetic neurons of the avian ciliary ganglion (16, 17). Activin immunoreactivity is present in neuronal nuclei scattered
throughout the central nervous system of the rat (18). Activin type II
receptor mRNAs have been detected by in situ hybridization
in embryonic chicken spinal cord (19), by Northern blot analysis in
human fetal spinal cord and cerebrum (20) and by RT-PCR in
neuroblastoma cells (21). Activins effects on neuronal cells in
culture, the expression of activin and its receptors within the nervous
system and within neuronal target tissues suggests that activin might
have several important roles in the development and function of the
nervous system.
In cultured rat sympathetic neurons activin A has been shown to induce
expression of mRNAs for choline acetyltransferase and for several
neuropeptides, including vasoactive intestinal peptide (VIP) (22, 23).
The VIP gene is a well characterized gene, which is regulated by a
variety of mediators, including cAMP, phorbol esters, calcium, ciliary
neurotrophic factor, and related neuropoietic cytokines (24). The
promoter elements that mediate these signals have been mapped either to
the 16-bp cAMP response element (CRE) located -70 bp upstream of the
transcription start site (25, 26, 27) or the 180-bp cytokine response
element (CyRE) located -1153bp upstream of the transcription start
site (28). However, the mechanism through which activin or other
members of the TGF-ß superfamily regulate VIP gene expression has not
been examined. To study the molecular mechanisms by which activin might
influence neuropeptide transmitter expression, we have turned to a
neuroblastoma cell line (NBFL) as a potential model system. We asked
whether and through which sequences activin influences the expression
of the VIP gene.
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RESULTS
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As a first step toward investigating the action of activin on
neuropeptide gene expression, we looked for a cell line that would
respond to activin to regulate specific neuropeptide genes. The human
neuroblastoma cell line, NBFL, has been well characterized and shown to
express and regulate a number of different neuropeptide genes in
response to other cytokines and growth factors (29). We therefore
decided to investigate whether or not activin altered expression of the
neuropeptide VIP gene in NBFL cells. VIP is expressed in NBFL cells,
and its expression is induced by another class of cytokine, the
neuropoietic or gp130 cytokines. Northern blots of RNA isolated from
NBFL cells treated for 66 h with either 25 ng/ml or 100 ng/ml
activin showed an approximately 2-fold induction of VIP mRNA (Fig. 1
). The neuropoietic cytokine, CNTF
(ciliary neurotrophic factor), induced VIP mRNA more robustly, leading
to a 14-fold induction in VIP mRNA after 66 h. To determine
whether activin increases VIP gene transcription, we examined the
effect of activin on the luciferase reporter gene fused to 5'-flanking
sequences from the human VIP gene (29). When NBFL cells were
transfected with a VIP-luciferase fusion gene containing 1929 p of
5'-flanking sequences, activin treatment of NBFL cells increased
luciferase activity 2-fold (Fig. 2A
). As
changes in luciferase activity reflect changes in the transcriptional
rate of the luciferase gene, this observation indicates that activin
acts to increase transcription from the VIP gene promoter. Thus,
activin induces both VIP mRNA and transcription driven by the VIP
promoter approximately 2-fold suggesting that the increase in VIP mRNA
expression is predominantly due to increased transcription.

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Figure 1. Activin Induces VIP mRNA Expression
A, Northern blot of RNA isolated from NBFL cells treated with CNTF (25
ng/ml) or activin A (25 ng/ml or 100 ng/ml) for 66 h. Total
cytoplasmic RNA (20 µg) was loaded in each lane. Two sizes of
VIP mRNA are detectable by Northern blotting. These transcripts are
thought to correspond to differential use of cleavage/polyadenylation
sites in VIP RNA (66 ). B, Quantification of data from triplicate lanes
of above experiment. Relative density of VIP mRNA was normalized to
that of cyclophilin mRNA for each lane. *, Data are significantly
different from control lane (P < 0.05). Data are
representative of three independent experiments.
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Figure 2. Activin Induces VIP Gene Transcription through the
Same DNA Element as CNTF
NBFL cells were transfected with the VIP promoter-luciferase plasmids
shown. Six hours later, transfected cells were untreated or treated
with either activin A (100 ng/ml) or CNTF (25 ng/ml) for 3640 h
before harvesting and analysis of resultant luciferase and
ß-galactosidase activity. Normalized luciferase data are presented,
with the value of VIP 2000 or Cy1luc within each experiment given as 1.
Data, expressed as mean ± SEM, are determined from
three independent experiments, each performed in duplicate.
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To localize more precisely the genomic sequences mediating activin
responsiveness of the VIP promoter, activin effects on a series of
transfected VIP promoter-luciferase fusion constructs were examined.
When NBFL cells were transfected with VIP-luciferase fusion genes
containing either 1929 or 1330 bp of 5'-flanking sequences, activin
treatment of the transfected cells increased luciferase activity 2- to
3-fold (Fig. 2A
). However, luciferase activity produced in cells
transfected with constructs containing 1151 or 340 bp of VIP
5'-flanking sequences was not induced by activin treatment. Therefore,
the 180-bp region between -1330 and -1151 upstream of the
transcription start site of the VIP gene contains cis-acting
sequences that are required to mediate the transcriptional response of
the VIP gene to activin. Interestingly, this is the same region of the
VIP promoter that we have previously shown to be necessary and
sufficient to mediate the effects of the neuropoietic cytokines,
including CNTF, on VIP gene transcription (28) and have named this
region the VIP cytokine response element (CyRE). To determine whether
the VIP CyRE could mediate the effects of activin on VIP gene
transcription, independent of surrounding sequences, we examined
activin regulation of the luciferase reporter Cy1luc. Cy1luc consists
of the VIP CyRE placed upstream of the heterologous basal promoter,
eRSV, driving expression of the luciferase reporter (28). Activin
treatment of NBFL cells transfected with Cy1luc led to a 5-fold
induction in luciferase activity (Fig. 2B
). The basal promoter
eRSV
was unable to mediate a transcriptional response to activin (data not
shown). Cotreatment of transfected cells with follistatin and activin
did not induce luciferase activity (data not shown), indicating that
activin is responsible for this induction. In NBFL cells transfected
with either VIP1929luc or Cy1luc, the induction in luciferase activity
was more robust with CNTF than activin. This correlates well with our
observation that CNTF induces VIP mRNA to a greater extent than activin
(Fig. 1
). Thus, the region of the VIP promoter from -1332 to -1153,
the VIP CyRE, is necessary and sufficient to mediate the effects of
either activin or CNTF on VIP gene transcription.
As CNTF and activin, members of very different cytokine families,
transduce their signal to the VIP gene through the same region of the
VIP promoter, we examined the effects of their joint action on VIP
mRNA. Treatment of NBFL cells with either activin or a low dose of CNTF
(0.5 ng/ml) led to a small induction in VIP mRNA (<2-fold); but
cotreatment with both these agents, gave a marked 8-fold induction
(Fig. 3
). Higher doses of CNTF (5 ng/ml)
induced VIP alone to a greater extent, yet this increase was also
considerably enhanced in the presence of activin (Fig. 3
). Thus,
activin and CNTF together induce VIP mRNA in a synergistic manner. To
ascertain whether or not this synergistic signaling was mediated
through the VIP CyRE, NBFL cells were transfected with Cy1luc and
treated with either activin, CNTF, or both cytokines together. To
exclude the possibility that activins effects were mediated through
stimulation of CNTF expression, we investigated the effects of
coadministration of activin with three different doses of CNTF. Activin
stimulated luciferase activity driven by Cy1luc to 5.8% of the
luciferase activity present when cells were maximally stimulated by
both cytokines (100 ng/ml activin; 25 ng/ml CNTF). CNTF treatment alone
stimulated Cy1luc-mediated luciferase activity to a greater extent than
activin alone, as we had noted previously. However, coadministration of
activin and CNTF led to a large increase in Cy1luc-directed luciferase
activity over that of either agent alone (Fig. 4
). Coadministration of activin together
with either 1 ng/ml, 5 ng/ml, or 25 ng/ml CNTF induced luciferase
activity 190%, 218%, or 200%, respectively, over that induced by
CNTF alone. As the luciferase activity induced by activin together with
1 ng/ml CNTF is substantially larger than that induced by either 5
ng/ml or 25 ng/ml CNTF alone, it is extremely unlikely that the
mechanism of activins action is to increase the amount of CNTF acting
on the CyRE. Thus, the synergistic signaling between CNTF and activin
is mediated by the sequences present in the VIP CyRE.

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Figure 3. Activin Enhances CNTF-Mediated Induction of VIP
mRNA Expression
Northern blot of RNA isolated from NBFL cells treated with CNTF and/or
activin (100 ng/ml) for 48 h. RNA (20 µg) is loaded onto each
lane. Data are representative of two independent experiments each
performed in duplicate.
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Figure 4. Activin Enhances CNTF-Mediated Induction of the VIP
CyRE
NBFL cells, transfected with Cy1luc, were treated with either activin
(100 ng/ml), CNTF, or activin and CNTF at the doses shown, for 36
h before harvesting and analysis of the resultant luciferase and
ß-galactosidase activity. Data are expressed as mean ±
SEM of three independent experiments performed in
duplicate.
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To determine whether another member of the TGF-ß superfamily of
cytokines could also induce VIP transcription through the CyRE and
synergize with CNTF, we assayed the ability of TGF-ß to activate
Cy1luc transcription. TGF-ß induced Cy1luc transcription
approximately 4-fold alone and more than doubled the transcriptional
activity of CNTF alone (Fig. 5A
).
Interestingly, TGF-ß and CNTF also synergistically activated
transcription from the TGF-ß reporter 3TPlux, suggesting that the
synergy between these two pathways is not restricted to the VIP CyRE
alone.

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Figure 5. CNTF and TGF-ß Synergistically Activate Gene
Transcription
A, NBFL cells, transfected with either Cy1luc or 3TPlux, were treated
with either CNTF (25 ng/ml), TGF-ß (2.5 ng/ml), or CNTF and TGF-ß
for 36 h before harvesting and analysis of the resultant
luciferase and ß-galactosidase activity. B, NBFL cells, transfected
with Cy1luc with or without the chimeric TrkC-gp130 receptor TGP, were
treated with either NT-3, TGF-ß, or NT-3 and TGF-ß for 36 h
before harvesting and analysis of the resultant luciferase and
ß-galactosidase activity. All data are expressed as mean ±
SEM, of three independent experiments performed in
duplicate. Numbers shown represent fold induction in luciferase
activity relative to Cy1luc in untreated cells.
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A possible explanation for this synergy between these cytokines is that
TGF-ß family cytokines may induce synthesis of a limiting component
of the CNTF receptor. To exclude this possibility we used a chimeric
gp130 receptor system, composed of a TrkC extracellular domain fused to
a gp130 cytoplasmic tail. Gp130 is the signal-transducing receptor
subunit shared by of all the neuropoietic cytokines (30). The chimeric
TrkC-gp130 receptor (TGP) has previously been characterized to
transduce gp130 signals in response to the TrkC ligand
neurotrophin-3 (NT-3) (31). In the presence of the TGP receptor, NT-3
induces Cy1luc transcription approximately 70-fold and is able to
synergize with TGF-ß to induce Cy1luc transcription more than
300-fold (Fig. 5B
). Without the transfected TGP chimeric receptor, NT-3
had no effect on Cy1luc transcription alone, nor on TGF-ß induced
Cy1luc transcription. These data suggest that signals initiating from
the gp130 receptor synergize with those from TGF-ß and imply that
this synergy will be common to all members of the neuropoietic cytokine
family.
We have previously shown that the VIP CyRE is a complex transcriptional
regulatory element composed of multiple domains, which act together to
mediate the effects of CNTF on VIP gene transcription. We have
described a Stat (signal transducer and activator of transcription)
site and a noncanonical AP-1 site within the CyRE which are both
important for the ability of CNTF to induce VIP gene transcription (28, 32). To determine whether the Stat or AP-1 sites are involved in
activin-mediated transcriptional induction of the VIP gene, we
introduced mutations into these sites, within the context of the VIP
CyRE. Treatment of NBFL cells transfected with Cy1mG3 luc, containing a
4-bp mutation within the Stat site (28), with either CNTF, activin, or
both cytokines, showed that levels of luciferase activity directed by
all these treatments was reduced relative to that of the wild type
Cy1luc (Fig. 6
). The mutation in the Stat
site reduced CNTF-mediated induction of luciferase activity to
approximately 20% of wild-type levels, whereas the activin induction
was reduced to 85% of wild-type levels, suggesting that the mutation
in the Stat site had a greater impact on CNTF signaling than activin
signaling. The induction by both cytokines together was 18% of the
wild-type Cy1luc. However, mutation in the AP-1 site with Cy1mG2
decreased both activin and CNTF stimulation of luciferase activity to
approximately 50% of wild type levels. Coadministration of CNTF and
activin to NBFL cells transfected with Cy1mG2luc, led to a 53-fold
induction in luciferase activity, 27% of that generated by Cy1luc
under similar treatment. However, mutation in either site did not
inhibit the synergistic signaling by CNTF and activin. Thus, both the
AP-1 site and the Stat site are important in mediating the
transcriptional induction by CNTF and activin, but neither alone
appears to be responsible for this signaling.

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Figure 6. Synergistic Signaling of CNTF and Activin Requires
the Stat and AP-1 Sites in the VIP CyRE
NBFL cells were transfected with the Cyluc plasmids shown and treated
with either CNTF (5 ng/ml), activin (100 ng/ml) or both, for 36 h
before harvesting and analysis of the resultant luciferase and
ß-galactosidase activity. Data are expressed as mean ±
SEM, of three independent experiments performed in
duplicate. Numbers shown represent fold induction in luciferase
activity relative to the activity of Cy1luc in untreated cells.
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To ascertain whether or not both the AP-1 and Stat sites might be
required for this signaling and regulation of gene expression, NBFL
cells were transfected with a CyRE luciferase reporter plasmid that
contained mutations in both the AP-1 and Stat sites. Activin induced
luciferase activity directed by this plasmid, approximately 2-fold, a
similar level to that directed by Cy1mG2luc. The induction mediated by
CNTF was much reduced from either of the single mutated plasmids, but
CNTF induced luciferase activity 5.7-fold or 7.9% of wild-type Cy1luc
levels. However, when NBFL cells transfected with this plasmid were
coadministered activin and CNTF, the luciferase activity was induced
7-fold, an induction not significantly different from the single CNTF
treatment. Thus the VIP CyRE with mutation in both Stat and AP-1 sites,
while still able to mediate transcriptional induction by either CNTF or
activin, is no longer able to transmit synergistic signaling by these
two classes of cytokine.
To determine whether TGF-ß can induce binding to either the AP-1 or
Stat site within the VIP CyRE, or alter the CNTF-mediated induction of
binding to these sites, DNA mobility shift assays were run with nuclear
extracts treated with CNTF and/or TGF-ß for various times. We
have previously shown that CNTF induces three complexes to bind to the
G3 Stat within 15 min of treatment (28). The slowest mobility and most
abundant Stat complex (A) is composed of a Stat3 homodimer, the fastest
mobility complex (C) is composed of a Stat1 homodimer, and the middle
complex (B) is a Stat3-Stat1 heterodimer (28). We have also
demonstrated that CNTF induces the AP-1 proteins c-Fos, JunB, and JunD
to bind to a modified noncanonical AP-1 site in NBFL cells (32). CNTF
induced AP-1 proteins to bind to the m3G2 site more slowly than Stat
proteins binding to the P3 site (Fig. 7
).
The different kinetics of nuclear protein binding to DNA may be due to
the requirement for protein synthesis for AP-1 induction, in contrast
to the posttranslational modification of Stat proteins by CNTF
signaling pathways (28, 32). TGF-ß treatment did not induce Stat
binding, nor did it alter the time or pattern of CNTF-induced
Stat proteins binding (Fig. 7A
). Surprisingly, similar results were
seen with binding to the modified noncanonical AP-1 site in the VIP
CyRE. TGF-ß did not induce binding to this site or to a consensus
AP-1 site (Fig. 7
) in NBFL cells, nor did it alter CNTF-induced binding
to this site. The apparent reduction in CNTF-induced nuclear protein
binding to m3G2 in the presence of TGF-ß is not reproducible. Thus,
TGF-ß does not alter CNTF-induced Stat or AP-1 binding to their
respective binding sites within the VIP CyRE.

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Figure 7. TGF-ß Does Not Activate Binding to the Stat or
AP-1 Sites of the VIP CyRE
DNA mobility shift assay with nuclear extracts prepared from NBFL cells
treated with CNTF (25 ng/ml), TGF-ß (2.5 ng/ml), or both CNTF and
TGF-ß for the times indicated. P3 is the VIP CyRE Stat binding site;
m3G2, a modified AP-1 binding site from the VIP CyRE that reduces
non-AP-1 proteins binding to the site (32 ), and AP-1 probe is a
canonical AP-1 site (Promega Corp.). B, The
arrow indicates the position of the CNTF-induced protein
complex binding to m3G2, that we have previously shown to consist of
the AP-1 proteins Jun-B, Jun-D, and c-Fos (32 ).
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DISCUSSION
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There is considerable evidence that growth factor and cytokine
production in the targets of the peripheral nervous system may serve to
regulate the expression of both classical and neuropeptide
transmitters. We have used the neuroblastoma cell line, NBFL, as a
model system to begin to examine the mechanisms by which activin might
regulate expression of the VIP neuropeptide gene. While NBFL cells
differ from primary neurons in many respects, like most neurons of the
peripheral nervous system, they are thought to originate from neural
crest cells (33). Moreover, like neurons, NBFL cells produce a number
of neuropeptides and regulate neuropeptide gene expression in response
to several growth factors and cytokines (29). We have found that
activin, a factor implicated in regulating neuropeptide expression,
stimulates transcription of the VIP gene and that this stimulation is
mediated through the CyRE, a region of the VIP gene that also mediates
this genes response to the neuropoietic cytokines. In addition, a
combination of activin and the neuropoietic cytokine CNTF produces a
synergistic stimulation of VIP gene transcription.
In our experiments, Northern blot analysis of NBFL cells showed a small
but significant 2-fold increase in VIP mRNA after activin treatment.
These data contrast with those of Fann and Patterson (34) who showed
that activin treatment of cultured rat sympathetic neurons did not
increase VIP mRNA expression unless depolarizing levels of potassium
were present. However, other members of the TGF-ß superfamily, BMP 2
and BMP 6, did induce VIP mRNA (34). These data may reflect either
differences in the cell type-specific response or the robustness of the
response to activin.
Our data indicate that members of very different cytokine families
regulate VIP gene expression through the same 180-bp cytokine response
element. One possible explanation is that activin/TGF-ß does not act
directly on the CyRE, but instead influences CNTF-mediated signaling
pathways to induce VIP gene transcription. Thus, activin/TGF-ß may
act by stimulating the secretion of CNTF resulting in an
autostimulation of VIP transcription. Or activin/TGF-ß may induce the
synthesis of additional functional CNTF receptors and thus increase the
potential stimulation of gp130 transduction pathways. Our data showing
that a chimeric TrkC-gp130 receptor can mediate synergy with TGF-ß in
response to NT-3 suggests that there is direct interaction between
gp130 and activin/TGF-ß signaling pathways. This conclusion is also
supported by the differential responses of mutant CyRE reporter
plasmids in mediating the response to activin and CNTF. Mutation of the
Stat site reduced both CNTF and activins induction of CyRE-driven
transcription but had a greater effect on the response to CNTF.
Conversely, mutation in the AP-1 site reduced both CNTF and activins
ability to stimulate transcription but with a larger reduction in the
effect of activin. Thus, if activin were acting only through gp130
signaling pathways, the effect of CNTF and activin on these CyRE
mutants would be identical. Our data showing a differential response of
mutant CyRE reporter plasmids in mediating the response to activin and
CNTF in addition to the synergy mediated by the activated chimeric
receptor with TGF-ß suggests that activin/TGF-ß and CNTF are acting
via different pathways to directly influence CyRE-mediated
transcription.
Activin induces transcription of the VIP gene through the 180-bp CyRE.
While rapid progress has been made in dissecting the molecular events
through which activin may regulate gene transcription, the mechanisms
of activin signal transduction have not been studied in cells of
nervous system origin. Thus, by establishing a model system in which to
study activin regulation of a neuronally restricted gene, we will be
able to investigate whether there exist any neuronal-specific
mechanisms of activin regulation of gene expression. Several different
DNA elements have now been reported that mediate transcriptional
induction by TGF-ß superfamily members. These include AP-1, Sp1, NF1,
and CarG elements (35, 36, 37, 38). This diversity of response elements
mediating somewhat similar signals was perplexing in comparison to the
conservation of response elements used by other cytokine signaling
pathways. However, the discovery of the Smad transcription
factors, which often form a transcriptional complex in combination with
various other transcription factors, gave a possible explanation for
this complexity (reviewed in Ref. 11). In a variety of cell types,
activin may regulate gene expression through phosphorylation of the
Smad transcription factors, Smad2 and Smad3 (39). TGF-ß also works
through these receptor-activated Smad proteins, Smad2 and Smad3 (40).
These proteins are usually localized in the cytoplasm in unstimulated
cells and are directly phosphorylated by a type I receptor after ligand
activation. Phosphorylated Smad2 and Smad3 hetero-oligomerize with the
pathway-independent Smad4 and the complex translocates to the nucleus
(41, 42). Smad3 and Smad4 may bind DNA directly, or complex, sometimes
together with Smad2, to other DNA binding proteins to induce gene
transcription of regulated genes (43, 44, 45, 46). We have found two Smad
binding sites within the VIP CyRE that are distinct from the Stat and
AP-1 sites and are characterizing their contribution to
activin-regulated VIP transcription and synergy with CNTF signaling
pathways (R. L. Pitts and A. Symes, paper in preparation).
It is clear that in vivo cytokines and growth factors do not
act in isolation to influence cellular differentiation and function.
When cells respond to two or more of these signaling pathways, the
responses may be synergistic or antagonistic. Smad proteins themselves
can mediate the cross-talk from different signaling pathways.
EGF-activated ERK activity phosphorylates Smad 1, inhibiting its
nuclear translocation (47) whereas Smad 2 can positively transduce
signals from both TGF-ß superfamily members, and hepatocyte growth
factor (48). Recently TGF-ß has been shown to synergize with glial
cell line-derived neurotrophic factor (GDNF) and CNTF to promote
neuronal survival (49, 50), suggesting that synergy between TGF-ß
family members and neurotrophic factors may be a more general
phenomenon in the development and maintenance of the mature nervous
system.
The synergy between CNTF and activin in regulating VIP gene
transcription through the VIP CyRE may be mediated at several different
levels. Activin may induce proteins important to CNTF signaling such as
receptors or kinases. Our data showing that the synergism of activin
and CNTF is notable even in the presence of saturating concentrations
of CNTF suggest that activin is acting, at least partially,
independently of the CNTF pathway. Activin and CNTF have both been
reported to induce members of the AP-1 transcription factor family
(51, 52, 53). Thus, the AP-1 site in the VIP CyRE is a potential target for
the synergistic action of CNTF and activin (32). However, the continued
presence of a synergistic response to CNTF and activin mediated by
Cy1mG2luc in which the AP1 site is mutated (Fig. 6
) argues against the
AP-1 site being a unique site through which this synergy is mediated.
Indeed, TGF-ß does not induce nor alter CNTF-induction of AP-1
proteins binding to either the VIP AP-1 site, or a canonical AP-1 site
(Fig. 7
). Thus, the AP-1 site in the VIP CyRE is not sufficient to
mediate the synergy between CNTF and activin/TGF-ß.
While the possibility exists of cooperativity of the activin and CNTF
signaling pathways at many points between the receptor and the nucleus,
our results suggest that both activin and CNTF stimulate VIP gene
transcription through the binding of transcriptional complexes to the
CyRE. Mutation analysis suggests that the Stat and AP-1 sites
contribute to both activin and CNTFs transcriptional induction of the
VIP CyRE, yet neither alone is responsible. Interestingly, mutations in
both the Stat and AP-1 sites abolish the synergistic effect of
coadministering activin and CNTF. These data suggest that several
different sites contribute to the synergistic interaction of activin
and CNTF. It is possible, therefore, that a different transcriptional
activation complex is formed when both signaling pathways are
stimulated than when either alone is activated. Coactivators, such as
CBP/p300, may be involved in mediating the synergistic response. Smads,
AP-1, and Stat transcription factors all interact directly with CBP
(54, 55, 56, 57, 58) potentially allowing CBP to act as a bridging molecule to
mediate the synergy between these pathways. Indeed, CBP/p300 is
implicated in the synergistic interaction between BMP2 and LIF
(leukemia inhibitory factor) induction of the GFAP (glial
fibrillary acidic protein) promoter in fetal neuroepithelial
cells (59) through bridging LIF-induced Stat3 with the BMP2-induced
Smad1. In the VIP CyRE, synergistic signaling between the
activin/TGF-ß pathway and that of CNTF will probably involve
interaction of Stat, Smad, and AP-1 proteins interacting with a
coactivator in a large multiprotein complex. Thus, eliminating the
contributions of DNA binding proteins activated by one signaling
pathway, e.g. by mutating the Stat and AP-1 binding sites
within the VIP CyRE, may be sufficient to destabilize the complex and
prevent the synergy. Thus, the synergistic effects of activin and CNTF
might result from cooperativity of CNTF- and activin-induced
transcriptional complexes.
Similar synergistic effects of cytokines and growth factors might be
important in bringing about a precise regulation of neurotransmitter
gene expression. If the availability of these factors is low enough to
allow the synergistic effects to contribute significantly toward
influencing gene expression, then target cells might be better able to
regulate more precisely the spatial and temporal availability of these
factors that serve to control neuronal expression of neurotransmitters
and neuropeptides.
 |
MATERIALS AND METHODS
|
---|
Materials
FBS and horse serum were obtained from Life Technologies (Gaithersburg, MD), other cell culture reagents
were purchased from Fisher Scientific (Pittsburgh, PA),
and culture plates were obtained from Becton Dickinson and Co. (Lincoln Park, NJ). Recombinant human CNTF was a gift from
Regeneron Pharmaceuticals, Inc. (Tarrytown, NY). TGF-ß
was purchased from R&D Systems (Minneapolis, MN).
Recombinant chicken activin A was produced using a baculovirus we
constructed to contain a complete chicken inhibin ß A (activin A)
cDNA (the kind gift of Patricia Johnson; Ref. 60). Suspension cultures
of Trichoplusia ni Hi 5 cells (Invitrogen, San Diego, CA)
in serum free medium were infected with the activin-encoding
baculovirus and the supernatant was collected. Activin, partially
purified by heparin affinity chromatography, was concentrated with a
15-kDa Centriprep filter (Amicon, Beverly, MA), and stored in 0.1
M acetic acid at 4 C. The presence of immunoreactive
activin A was confirmed by Western blot analysis and the biological
activity of the preparation was measured by determining the dose
required to obtain a half-maximal response in stimulating the
expression of somatostatin-like immunoreactivity in cultured CG neurons
(16).
Plasmids
Construction of the series of VIP promoter-luciferase plasmids
has been described (28). Cy1luc contains the entire 180-bp CyRE fused
to
eRSVluc (28). The sequence and construction of Cy1mG3 luc and
Cy1mG2 luc (m2CyBluc) have previously been described (32). 3TPlux is
composed of 3 AP-1 sites, linked to a 100-bp region of the plasminogen
activator inhibitor enhancer, upstream of a basal E4 promoter (61). The
chimeric receptor plasmid, TGP, is composed of the extracellular domain
of TrkC linked to the cytoplasmic tail of gp130 (31).
Cell Culture and Transfection
NBFL cells were maintained as previously described in DMEM (4.5
g glucose/liter) supplemented with 5% (vol/vol) FBS and 5% horse
serum (29). Cells were transfected by calcium phosphate precipitation.
Cells were plated at 1.5 x 105 cells per
35-mm well, 1 day before transfection. Each well received 3 µg
luciferase reporter plasmid and 1 µg of the cotransfection control,
RSVß-gal. Cells were left with precipitate overnight before washing
twice with DMEM and replacement of the media. Six hours later, activin
and/or CNTF were added for 3640 h. Cells were harvested and assayed
for luciferase activity as previously described (62) and
ß-galactosidase activity (Tropix, Bedford, MA). Luciferase activity
was normalized to ß-galactosidase activity to control for
transfection efficiency.
RNA Isolation and Analysis
Total cytoplasmic RNA was isolated from NBFL cells after lysis
with Nonidet P-40 and transferred to nylon membranes as previously
described (63). Northern blots were hybridized with a 580-bp
HindIII/EcoRI fragment of human VIP cDNA (64) and
rehybridized with a probe for the unregulated internal reference gene
cyclophilin (65). Blots were digitized using a Storm phosphorimager,
with Imagequant software (Molecular Dynamics, Inc.,
Sunnyvale, CA). The relative densitometric readings were normalized to
cyclophilin mRNA to account for loading differences between lanes.
DNA Mobility Shift Assays
NBFL cells were grown to confluency and serum starved overnight
before treatment with CNTF and/or TGF-ß for the times indicated.
Nuclear extracts were prepared and binding reactions performed as
previously described (28). Synthetic complementary oligonucleotides
with GGG overhangs were annealed and labeled with
[
-32P]-dCTP using M-MLV (moloney
murine leukemia virus) reverse transcriptase
(Promega Corp., Madison, WI). The sequences of the G3 and
m3G2 probes are described elsewhere (28, 32).
 |
ACKNOWLEDGMENTS
|
---|
We thank Regeneron Pharmaceuticals, Inc. for their
gift of CNTF, and Dr. J. Wrana for the 3TPlux plasmid. We are also
grateful to Dr. Elizabeth Jones, Dr. Robert Lechleider, and Dr. Fern
Murdoch for many helpful discussions.
This work was supported by NIH Grant R29 NS-35839 (A.J.S.) and by
Uniformed Services University of the Health Sciences (USUHS) intramural
support. The opinions and assertions contained herein are the private
opinions of the authors and are not to be construed as reflecting the
views of the Uniformed Services University of the Health Sciences or
the US Department of Defense.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Aviva Symes, Department of Pharmacology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814.
Received for publication March 25, 1999.
Revision received November 29, 1999.
Accepted for publication December 6, 1999.
 |
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