(Received for publication, November 11, 1994; and in revised form, January 27, 1995)
From the
The neuropoietic cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) regulate VIP gene expression through a cytokine response element (CyRE) which interacts with members of the STAT transcription factor family. The CyRE STAT site is, however, insufficient to mediate full transcriptional activation by CNTF/LIF, suggesting that other sequences and nuclear proteins are also important. As C/EBP proteins participate in the transcriptional effects of the related cytokine, interleukin-6, we investigated the role of possible C/EBP-binding sites in the response of the VIP CyRE to CNTF/LIF. Using DNase I footprinting, transactivation studies, DNA mobility shift assays, and mutational analysis, three sites within the VIP CyRE were identified as C/EBP-related binding sites and shown to be important to CNTF/LIF-mediated transcriptional activation. The CyRE C/EBP-related sites interact with nuclear proteins from the human neuroblastoma cell line, NBFL, including a novel, protein synthesis-dependent, nuclear protein complex, induced by CNTF treatment. These nuclear proteins are not, however, recognized by antisera to known C/EBP proteins. Therefore, other nuclear proteins regulated by independent pathways act in concert with the JAK-STAT pathway to mediate CNTF/LIF regulation of VIP gene expression through the CyRE.
The family of neuropoietic cytokines, including ciliary
neurotrophic factor (CNTF) ()and leukemia inhibitory factor
(LIF), function as differentiation and survival
factors(1, 2, 3, 4) . These proteins
are most closely related to two other cytokines, oncostatin M, and
interleukin-6 (IL-6)(5, 6) . LIF, oncostatin M, and
IL-6 have a broad spectrum of effects on diverse cell
types(7, 8, 9) . In contrast, the biological
actions of CNTF are largely confined to the nervous
system(10) . The signaling mechanisms utilized by these
cytokines are beginning to be
understood(11, 12, 13, 14) . Much
less is known about the nuclear mechanisms by which the neuropoietic
cytokines regulate neuronal gene expression.
Members of the CNTF family of cytokines are capable of altering the neurotransmitter phenotype of post-mitotic neurons. Treatment of sympathetic neurons with CNTF or LIF results in coordinate regulation of expression of genes encoding neurotransmitter synthesizing enzymes, neurotransmitter receptors, and neuropeptides(15, 16, 17, 18, 19, 20) . Vasoactive intestinal peptide (VIP) is one of the neuropeptides whose synthesis is increased in sympathetic neurons by CNTF and LIF(15, 16, 19, 21) . Expression of the VIP gene is also increased by CNTF, LIF, and oncostatin M in the human neuroblastoma cell line, NBFL(22, 23) . The increase in VIP gene expression by CNTF in NBFL cells is rapid and prolonged(22) .
To gain insight into the nuclear events underlying cytokine-mediated activation of neuronal gene expression, we have investigated the mechanisms of transcriptional activation of the VIP gene by the CNTF family of cytokines. Recently, we characterized a genomic regulatory region and nuclear proteins that are important to the transcriptional activation of the VIP gene by CNTF in NBFL cells(22, 24) . CNTF-dependent increases in VIP gene expression are mediated by a 180-bp cytokine-responsive element (CyRE) located 1.3 kilobases from the transcription initiation site in the VIP gene(24) . This CyRE also mediates transcriptional activation by LIF and oncostatin M(24) . To one region within the CyRE, CNTF treatment induces binding of a protein complex composed of Stat1 and Stat3/APRF. Stat1 and Stat3/APRF are members of the recently described STAT transcription factor family and are activated in response to numerous extracellular activators (25, 26, 27, 28, 29) . As in other cell types, activation of STAT proteins in NBFL cells by CNTF is independent of de novo protein synthesis (24) .
Several lines of evidence suggest the existence of additional nuclear signaling pathways activated by CNTF in NBFL cells. Deletion analysis of the CyRE indicated that additional regions, distinct from the STAT-binding site, are important for CNTF-dependent transcriptional activation mediated by this element(24) . The biologic effects of CNTF, including its effect on VIP mRNA, are often prolonged(19, 22) , but STAT transcription factors are rapidly activated and inactivated (30, 31, 32) . CNTF increases ras-GTPase activity, a pathway which is independent of STAT activation(33) . The differential effects of kinase inhibitors on VIP mRNA induction by CNTF also suggest the existence of multiple CNTF signaling pathways in NBFL cells(33) . However, the precise identification of important sequences within the CyRE, distinct from the STAT site, and the nuclear proteins with which they interact have not been determined.
IL-6 and LIF activation of gene
transcription has been studied in both hemeatopoetic and hepatic
cells(34, 35, 36, 37, 38) ,
but it is not known whether IL-6 and the CNTF family of cytokines
utilize similar nuclear mechanisms to regulate neuronal gene
expression. A signal receptor subunit, gp130, is a common component of
the CNTF, LIF, oncostatin M, and IL-6 receptor
complexes(14, 39, 40) . In the presence of
IL-6, homodimerization of gp130 results in transduction of a receptor
signal (41, 42, 43) . CNTF and LIF signaling
depends on formation of a heterodimer of gp130 and a gp130-like
receptor subunit, LIFR(44) . Transduction of receptor
signals by IL-6 and the CNTF family of cytokines activates
receptor-associated tyrosine kinases of the JAK-TYK
family(12, 13, 44) . The similarities in the
receptor signaling mechanisms utilized by IL-6 and the CNTF family of
cytokines suggests that these cytokines might utilize common nuclear
mechanisms for regulating gene expression.
Members of the C/EBP
transcription factor family are involved in mediating the effect of
IL-6 on hepatic acute-phase
genes(46, 47, 48) . C/EBP proteins are
members of the b-Zip family of transcription factors and regulate the
transcription of a diverse array of
genes(49, 50, 51, 52) . Several
members of this family have been cloned and all bind similar
sequences(47, 53, 54) . Expression of C/EBP
and C/EBP
is elevated in several tissues during the
acute-phase response(46, 55, 56) , and there
is increased binding of these proteins to their DNA-binding sites in
regulated genes(46, 47, 56, 57) .
In this study we sought to identify regions of the CyRE, distinct from the STAT-binding site, which are important to CNTF/LIF-dependent transcriptional activation mediated by the CyRE in NBFL cells, and considered the C/EBP proteins as candidate transcription factors which may interact with these regions. We find evidence that IL-6, similar to the effects of the CNTF family of cytokines, activates transcription through the CyRE in NBFL cells. We identify sites within the CyRE which bind purified C/EBP proteins and demonstrate that these sites are important for activity of the CyRE. These C/EBP-related binding sites interact with nuclear proteins from NBFL cells, one of which is activated in a protein synthesis-dependent manner by CNTF/LIF. We find no evidence that C/EBP proteins from NBFL cells interact with these C/EBP-related sites. Thus, CNTF/LIF regulation of VIP gene expression depends on activation of protein synthesis-dependent and -independent nuclear signaling pathways interacting with multiple regions of the CyRE.
Figure 1:
sIL-6R and
IL-6 induce VIP mRNA in NBFL cells. A, NBFL cells were treated
with IL-6 (40 ng/ml), sIL-6R (25 ng/ml), IL-6 (40 ng/ml), and sIL-6R
(25 ng/ml), CNTF (25 ng/ml), LIF (10 ng/ml), or Oncostatin M (10
ng/ml). After 24 h cells were harvested and cytoplasmic RNA isolated.
Northern analysis of NBFL RNA with 20 µg loaded in each lane. Blots
were probed with a human VIP cDNA probe followed by a cyclophilin probe
to normalize for loading differences. B, NBFL cells were
plated at 5 10
cells/35-mm plate 1 day before
transfection. Cells were transfected as described under
``Experimental Procedures'' with 6 µg of Cy1luc and 2
µg of RSVCAT/35-mm plate. Twenty-four h after transfection cells
were treated with 10, 20, or 40 ng/ml IL-6 and harvested 36 h later for
luciferase and CAT activity determination.
We have previously mapped the CNTF, LIF, and oncostatin
M-responsive region of the VIP promoter to a 180-bp CyRE(24) .
To establish whether IL-6 induction of VIP mRNA was mediated through
the same region as that utilized by CNTF, LIF, and oncostatin M, NBFL
cells were transfected with the CyRE-luciferase reporter construct,
Cy1luc(24) . Cy1luc contains the 180-bp VIP CyRE upstream of a
heterologous promoter RSV and linked to the luciferase reporter
gene. In the absence of sIL-6R, treatment of Cy1luc-transfected NBFL
cells with IL-6 (10, 20, and 40 ng/ml) did not increase luciferase
activity (Fig. 1B). However, the addition of 25 ng/ml
sIL-6R together with IL-6 led to an dose-dependent induction of
luciferase activity (Fig. 1B). Thus, the VIP CyRE is
sufficient to mediate transcriptional activation to IL-6 as well as to
CNTF, LIF, and oncostatin M. This suggests that IL-6 transduces a
receptor signal to the nucleus by mechanisms similar to that utilized
by CNTF, LIF, and oncostatin M.
Figure 2:
C/EBP transcription factors transactivate
the VIP CyRE. NBFL cells were cotransfected with 10 µg of Cy1luc
and expression vectors for either C/EBP or C/EBP
under the
control of the cytomegalovirus promoter(62) . The total amount
of cytomegalovirus promoter was kept constant by the addition of the
parent vector pCDNA1. Cells were harvested 48 h after transfection, and
luciferase and CAT activity were
determined.
To determine whether transactivation of
the CyRE by C/EBP and C/EBP
could be a result of C/EBP
proteins binding to sequences within the CyRE, DNase I footprinting was
performed using bacterially expressed C/EBP DNA-binding domains and a
probe containing the entire CyRE. Three regions within the CyRE were
protected by the C/EBP
DNA-binding domain, CyA, CyB, and CyC (Fig. 3). The C/EBP
DNA-binding domain protected the same
region as C/EBP
(data not shown). Identically prepared bacterial
extracts without C/EBP expression plasmids did not protect the CyRE DNA (Fig. 3). The sequences of the CyA-, CyB-, and CyC-binding sites
conformed loosely to the C/EBP consensus binding site,
TT/GNNGNAAG/T(50) . Additionally, in DNA mobility shift assays,
nuclear extracts prepared from COS cells transfected with C/EBP
or
C/EBP
, but not from untransfected COS cells, bound to
oligonucleotides composed of the CyA or CyC sites (data not shown). We
were unable to detect C/EBP proteins binding to the CyB site because
the CyB probe bound proteins of similar size as C/EBP proteins
expressed in untransfected COS cells (data not shown). The presence of
three possible binding sites for C/EBP proteins within the CyRE
indicates that transactivation of the VIP CyRE by C/EBP
and
C/EBP
may result from direct interaction of C/EBP proteins with
nucleotide sequences within the CyRE.
Figure 3:
C/EBP proteins bind to the VIP CyRE. DNase
I footprinting analysis of the VIP CyRE (-1330 to -1150)
with bacterially expressed DNA binding domain of C/EBP. DNA probe
was incubated either with extract from bacteria alone (lanes
1, 4, and 5) or with extract from bacteria
expressing a truncated C/EBP
DNA-binding domain (lanes 2 and 3). The sites protected from DNase I digestion are
indicated by boxes (CyA, CyB, and CyC). The nucleotide
sequences of the protected regions is shown at the base of the
gel.
Figure 6: Binding of NBFL nuclear extracts to the Cy sites within the VIP CyRE. A, DNA mobility shift assay with nuclear extracts prepared from CNTF-treated (25 ng/ml) NBFL cells, harvested at the indicated times, binding to CyA, CyB, or CyC sites. The arrows indicate specific complexes binding to the CyB and CyC probes. Complex IV is induced to bind by CNTF. B, DNA mobility shift assay with nuclear extracts prepared from untreated NBFL cells with the CyA or CyC probe. Competing unlabeled oligonucleotides were present at 100-fold molar excess. Sequence of the oligonucleotides is given under ``Experimental Procedures.'' C, DNA mobility shift assay using the CyB probe and nuclear extracts prepared from NBFL cells treated for 2 h with CNTF (25 ng/ml). Competing unlabeled oligonucleotides were added at 100-fold molar excess.
Figure 4: Effect of mutations in putative C/EBP sites on activity of the CyRE. A, NBFL cells transfected with 20 µg of luciferase reporter plasmids were treated with CNTF (25 ng/ml) for 36-40 h before harvesting and analysis of luciferase and CAT activity. The fold induction of luciferase activity by Cy1luc is given the value of 100% and that of the mutant Cy1luc plasmids are represented as a percentage of Cy1luc activity (±S.E. n = 3). B, sequence of mutations made in putative C/EBP sites. The bases mutated are shown in bold.
We sought to assess
whether C/EBP proteins are involved in the CNTF-dependent
transcriptional activation through the VIP CyRE. Surprisingly,
transfection of NBFL cells with increasing amounts of expression
plasmids encoding full-length C/EBP or C/EBP
decreased
CNTF-mediated transcriptional activation (Fig. 5). This
inhibition of CNTF-dependent transcriptional activation was marked; 0.5
and 6 µg of C/EBP
expression plasmid reduced luciferase
induction by 63 and 76%, respectively. C/EBP
was a more potent
inhibitor of CNTF-dependent transcriptional activation: 0.5 and 6
µg of C/EBP
expression plasmid reduced CNTF-dependent
transcriptional activation by 83 and 91%, respectively (Fig. 5).
Thus, C/EBP proteins inhibit CNTF-dependent activation of transcription
mediated by the CyRE, in contrast to their effect in untreated NBFL
cells.
Figure 5: C/EBP transcription factors inhibit the induction of the CyRE by CNTF. NBFL cells transfected as in Fig. 2were treated with CNTF (25 ng/ml) for 36-40 h before harvesting and analysis of luciferase and CAT activity. The fold induction of luciferase activity by Cy1luc in the presence of pCDNA1 plasmid alone is given the value of 100%, and the fold induction of Cy1luc in the presence of varying amounts of C/EBP expression plasmid are represented as a percentage of Cy1luc activity (±S.E., n = 5).
Competition analysis of these DNAprotein
complexes using unlabeled oligonucleotides confirmed that nuclear
protein binding to CyA, CyB, and CyC probes was sequence-specific (Fig. 6, B and C). Furthermore, each putative
C/EBP site binds different proteins (Fig. 6, B and C). CyA protein binding is competed by a 100-fold molar excess
of itself, but not by any other oligonucleotide tested, including CyB,
CyC, M6 (a high affinity C/EBP-binding site(69) ), mCyA, or
G3-STAT (the CyRE STAT-binding site (24) ). The CyC probe binds
two predominant complexes, I and II (Fig. 6A).
CyC-protein complexes I and II are competed by a 100-fold molar excess
of unlabeled CyC, but neither is competed by CyA, mCyC, G3-STAT, or M6.
Complex I, but not complex II, is specifically competed by 100-fold
molar excess of CyB, suggesting that complexes I and II are composed of
different proteins and that CyB binds those proteins contained in
complex I.
The basal and inducible CyB-containing complexes III and IV, respectively, are competed by 100-fold molar excess of CyB. The oligonucleotides CyA and G3-STAT did not compete for basal or inducible CyB binding. However, mCyB, CyC, and the high affinity C/EBP-binding site, M6, competed weakly for the inducible complex IV binding. Thus, nuclear proteins bind specifically to all three putative C/EBP sites within the CyRE. Most of these proteins appear to be unrelated. The exceptions are the CyC complex I which is recognized by the CyB site, and the CyB complex IV which is weakly recognized by the CyC site. Additionally, most of these protein complexes do not appear to be C/EBP proteins because they are not competed by a C/EBP consensus oligonucleotide (M6), or by a different C/EBP consensus oligonucleotide (data not shown). However, the weak competition of M6 for CyB complex IV suggests that this protein may be related to the C/EBP transcription factor family.
Figure 7: NBFL nuclear proteins are not recognized by anti-C/EBP specific antisera. DNA mobility supershift assay using the CyA, CyB, or CyC probes. Nuclear extracts from NBFL cells either untreated (CyA and CyC probes), or treated for 2 h with CNTF (25 ng/ml) (CyB probe) were incubated in the presence of anti-C/EBP antisera. No change in binding was detectable.
C/EBP
proteins are able to form heterodimers with other C/EBP proteins
through their leucine zipper
regions(47, 54, 62) . Additionally, other
members of the C/EBP family, distinct from C/EBP and C/EBP
,
have been
identified(53, 54, 56, 62) .
Therefore, it is possible that NBFL nuclear proteins binding to the
CyA, CyB, and CyC sites are previously uncharacterized C/EBP proteins
binding as hetero- or homodimers. To establish whether the NBFL
proteins which bind to CyA, CyB, or CyC are able to heterodimerize with
other C/EBP members, a DNA mobility shift assay was performed in the
presence of CHOP-10 fused to glutathione S-transferase (GST).
CHOP-10 is a member of the C/EBP family which does not bind DNA but is
able to form heterodimers with other C/EBP members and thereby inhibit
their binding to DNA(62) .
In a DNA mobility shift assay
GST-CHOP-10 protein inhibited the binding of nuclear proteins from
C/EBP-transfected COS cells to the high affinity C/EBP site, M6
(data not shown). This observation is consistent with the ability of
CHOP-10 to form heterodimers with C/EBP proteins which are incapable of
binding DNA. However, incubation of NBFL nuclear proteins with
bacterially expressed GST-CHOP-10 protein did not perturb the nuclear
protein complexes formed with CyA, CyB, or CyC probes. This suggests
that proteins interacting with the CyA, CyB, and CyC sites are not
C/EBP proteins capable of heterodimer formation with CHOP-10.
Additionally, CyA, CyB, or CyC NBFL protein complexes were not heat
stable (data not shown) which is not characteristic of C/EBP
proteins(51, 70) . Taken together, these data strongly
suggest that the CyA, CyB, and CyC sites are C/EBP-binding sites, but
the DNA
protein complexes formed with NBFL nuclear extracts do not
contain known C/EBP proteins.
Figure 8:
Characterization of inducible binding to
the CyB probe. A, DNA mobility shift assay with nuclear
extracts prepared from NBFL cells, pretreated for 30 min with either no
inhibitor or the protein synthesis inhibitor cycloheximide (CHX 100 µM) and then treated for 1 or 3 h with CNTF (25
ng/ml). Cycloheximide prevents the CNTF-induced binding to the CyB
probe. B, DNA mobility shift assay with nuclear extracts
prepared from NBFL cells treated for 1 h with either CNTF (25 ng/ml, C), LIF (10 ng/ml, L), Oncostatin M (10 ng/ml, O), or IFN- (10 ng/ml, I). The inducible protein
complex IV (indicated by the arrow) is induced by CNTF, LIF,
and Oncostatin M, but not by IFN-
.
To establish whether other members of the CNTF
family of cytokines also induced CyB complex IV formation, NBFL cells
were treated with either LIF or oncostatin M. These CNTF-related
cytokines induced CyB complex IV formation, supporting the similarity
of nuclear signaling by these cytokines (Fig. 8B).
Interferon- (IFN-
), which activates similar kinases as the
CNTF family of cytokines(12) , and induces binding to the VIP
CyRE STAT site(24) , did not induce CyB complex IV formation.
This study identifies regions of the VIP CyRE, distinct from the previously described STAT transcription factor binding site, which are important for transcriptional activation of the VIP gene by the CNTF family of cytokines. Using DNase I footprinting, transactivation studies, DNA mobility shift assays, and mutation analysis, three C/EBP-related binding sites (CyA, CyB, and CyC) were demonstrated to be important to the CNTF-mediated activation of VIP transcription. The existence of additional functional domains within the CyRE, and separate from the STAT-binding site, were predicted on the basis of deletion analysis of the CyRE(24) . Integrity of the C/EBP-related binding sites and the STAT-binding site are required for CNTF-dependent transcriptional activation mediated by the CyRE. Thus, the CyA, CyB, and CyC sites represent new functional domains within the 180-bp VIP CyRE. The CyRE C/EBP-related sites interact with nuclear proteins from NBFL cells including a novel, protein synthesis-dependent, nuclear protein complex which is induced by CNTF to bind to the CyB site. Although these C/EBP-related sites interact with purified C/EBP proteins, they do not interact with known C/EBP proteins from NBFL cells.
Soluble IL-6R and sCNTFR are among the
only known soluble receptors which are able to promote, rather than
inhibit, the effects of their
ligands(39, 68, 71) . This property allowed
us to examine the similarities between CNTF and IL-6 signaling to the
VIP gene in a neuronal cell line. It is thought that in the presence of
IL-6, sIL-6R forms a functional IL-6 receptor with
gp130(41, 42, 43) . CNTF and LIF signaling,
in contrast, are dependent on a heterodimer of gp130 and
LIFR(44) . As the cytoplasmic tail of the LIFR
is
able to transmit signals without gp130(72) , it is notable that
the signaling pathways initiated by cytokine receptors of different
subunit composition (the heterodimer of gp130 and LIFR
for the
CNTF-related cytokines and the homodimer of gp130 for IL-6) converge on
the VIP CyRE to regulate gene expression.
The importance of the C/EBP-related binding sites CyA, CyB, and CyC to transcriptional activation mediated by the CyRE is supported by the ability of mutations at these sites and of cotransfected expression plasmids for C/EBP proteins to substantially reduce transcriptional activation of Cy1luc by CNTF ( Fig. 4and Fig. 5). The inhibitory effect of C/EBP proteins on CNTF-dependent transcriptional activation contrasts with experiments showing that transfection of C/EBP expression plasmids increases the response to IL-6 in hepatic cell lines through IL-6 response elements in acute-phase genes(47) . Inhibition of CNTF inducibility in NBFL cells may result from binding of the transfected C/EBP proteins to the C/EBP-related binding sites and displacement of the endogenous NBFL proteins. Alternatively, the transfected C/EBP proteins may form heterodimers, or interact in other ways, with NBFL proteins and prevent them from binding to the CyRE.
Despite evidence in support of CyA, CyB, and CyC as C/EBP-related
binding sites, we are unable to find evidence that the NBFL proteins
interacting with the C/EBP-related sites are known C/EBP proteins.
Using extracts from NBFL cells, DNA mobility shift assays with C/EBP
antisera and GST-CHOP-10 fusion proteins indicate that C/EBP proteins
are not binding to these sites. Furthermore, Western blots with
anti-C/EBP antisera demonstrate that C/EBP proteins are not present in
NBFL cells, before or after treatment with CNTF. ()Although
CyA, CyB, and CyC sites are functionally important to the CNTF-mediated
induction of Cy1luc in NBFL cells, these sites do not appear to be
acting as C/EBP-binding sites to activate transcription in response to
cytokines.
The inability of antisera to C/EBP, C/EBP
, and
C/EBP
to recognize the NBFL proteins binding to CyA, CyB, and CyC
sites strongly suggests that known C/EBP proteins do not participate in
CyRE-mediated transcriptional activation in NBFL cells. All C/EBP
antisera were raised to specific, non-shared epitopes of individual
C/EBP family members, permitting discrimination among them. However,
none of the known C/EBP cDNAs were isolated from neuronal tissue.
C/EBPs in brain may be different from those in the liver and
kidney(51) . As NBFL cells are of neuronal origin it is
possible that novel C/EBP-like proteins may be present in these cells
and would not be recognized by the C/EBP antisera used in this study.
Therefore, it remains a possibility that C/EBP-like proteins are
involved in mediating the CNTF response in NBFL cells. Recently,
evidence has been presented for the existence of neuronal C/EBP
proteins which function in the nervous system of Aplysia (74) .
These Aplysia C/EBP proteins may have mammalian homologues which are
expressed in NBFL cells.
One hour after CNTF/LIF/oncostatin M treatment of NBFL cells, a protein complex (IV) is induced to bind to the CyB site. Mutation of this site reduces CNTF-mediated transcriptional activation of Cy1luc by 75% demonstrating that the CyB site is important to full CNTF-mediated transcriptional activation through the CyRE. However, activation of CyB-complex IV is not the earliest detectable nuclear effect of CNTF signaling in NBFL cells. Within 15 min of CNTF treatment of NBFL cells, STAT transcription factors are induced to bind to the Stat site within the CyRE(24) . The slower time course of CyB-complex IV activation is reflected in its dependence on de novo protein synthesis in contrast to the more rapid, protein synthesis-independent activation of STAT protein binding(24) . Thus, the nuclear signaling mechanisms by which the CNTF family of cytokines regulates VIP gene expression in NBFL cells involve multiple basal and inducible transcription factors with different time courses and requirements for protein synthesis.
CNTF, LIF, oncostatin M, and IFN- activate
STAT proteins in NBFL cells(24) . However, the CNTF family of
cytokines, but not IFN-
, induce the CyB-complex IV protein. The
inability of IFN-
to induce VIP mRNA
and CyB-complex
IV protein in NBFL cells is consistent with the importance of the CyB
site in the activation of VIP gene expression. The identification of
proteins which bind to the CyB site will lead to further understanding
of the mechanisms through which the CNTF family of cytokines regulate
neuropeptide gene expression.
The reduction of CNTF-dependent transcriptional activation by mutations in CyA and CyC sites indicates that these C/EBP-related binding sites which interact with constitutive proteins are also required for a full CNTF response. The VIP CyRE, therefore, is similar to many IL-6-inducible hepatic acute-phase genes in which several interacting regulatory sequences are required for full IL-6-dependent transcriptional activation(73, 75, 76) . The juxtaposition of C/EBP and putative STAT sites is a structural feature of some IL-6-inducible gene promoters(38) . The VIP CyRE is similarly organized with STAT and C/EBP-related binding sites required for full activity. This organization suggests that combinatorial interaction among nuclear proteins is required for transcriptional regulation of the VIP gene by the CNTF family of cytokines and may be a general feature of nuclear signaling by the neuropoietic cytokines.