Integration of Jak-Stat and AP-1 signaling pathways at the vasoactive intestinal peptide cytokine response element regulates ciliary neurotrophic factor-dependent transcription.

Ciliary neurotrophic factor (CNTF)-dependent induction of expression of the neuropeptide vasoactive intestinal peptide (VIP) gene is mediated by a 180-base pair cytokine response element (CyRE) in the VIP promoter. To elucidate the molecular mechanisms mediating the transcriptional activation by CNTF, intracellular signaling to the CyRE has been studied in a neuroblastoma cell line. It has been shown previously that CNTF induces Stat proteins to bind to a site within the CyRE. CNTF also induces a second protein to bind to a C/EBP-like site within the CyRE. In this report, we show that this inducible CyRE binding protein is composed of the AP-1 proteins c-Fos, JunB, and JunD. These proteins bind to a non-canonical AP-1 site located near the previously characterized C/EBP site. The serine/threonine kinase inhibitor H7 prevents CNTF-dependent induction of AP-1 binding and CyRE-mediated transcription, suggesting that an H7-sensitive kinase is important to mediating CNTF effects on VIP transcription. The integration at the VIP CyRE of the Jak-Stat and AP-1 signaling pathways with other pre-existing proteins provides a cellular mechanism for cell- and cytokine-specific signaling.


Ciliary neurotrophic factor (CNTF)-dependent induction of expression of the neuropeptide vasoactive intestinal peptide (VIP) gene is mediated by a 180-base pair cytokine response element (CyRE) in the VIP promoter.
To elucidate the molecular mechanisms mediating the transcriptional activation by CNTF, intracellular signaling to the CyRE has been studied in a neuroblastoma cell line. It has been shown previously that CNTF induces Stat proteins to bind to a site within the CyRE. CNTF also induces a second protein to bind to a C/EBPlike site within the CyRE. In this report, we show that this inducible CyRE binding protein is composed of the AP-1 proteins c-Fos, JunB, and JunD. These proteins bind to a non-canonical AP-1 site located near the previously characterized C/EBP site. The serine/threonine kinase inhibitor H7 prevents CNTF-dependent induction of AP-1 binding and CyRE-mediated transcription, suggesting that an H7-sensitive kinase is important to mediating CNTF effects on VIP transcription. The integration at the VIP CyRE of the Jak-Stat and AP-1 signaling pathways with other pre-existing proteins provides a cellular mechanism for cell-and cytokine-specific signaling.
CNTF and LIF induce expression of the neuropeptide vasoactive intestinal peptide (VIP) in sympathetic neurons in culture (28,29) and in a human neuroblastoma cell line, NBFL (30,31). To gain insight into the mechanisms underlying cytokine-mediated activation of neuronal gene expression, we have investigated the genomic regulatory elements mediating the induction of the VIP gene by CNTF and related cytokines. We previously identified a large, 180-base pair cytokine responsive element (CyRE) within the 5Ј-flanking region of the VIP gene, which is located 1330 bp from the transcription start site (32). The CyRE is necessary and sufficient to mediate transcriptional activation by CNTF, LIF, IL-6, and oncostatin M (32,33). CNTF treatment induces binding of Stat1␣ and Stat3 proteins to a region of the 5Ј end of the CyRE (32). This STAT site is important to transcriptional activation mediated by the CyRE as a mutation in this site reduces the cytokine-dependent induction of a CyRE-linked luciferase reporter by 80% (32). As mutation or deletion of regions distinct from the STAT site also attenuate transcription mediated by the CyRE (33,34), other regions within the CyRE appear to be important to CNTFmediated induction of VIP gene expression. Serine-threonine kinases are important to CNTF-dependent increases in VIP transcription as induction of VIP mRNA by CNTF in NBFL cells is prevented by the serine-threonine kinase inhibitor H7 (24). It is not known whether the H7-sensitive regulation of VIP gene expression by CNTF in NBFL cells is mediated by the CyRE.
We have previously shown that three regions within the VIP CyRE have sequence homology to C/EBP binding sites and bind purified C/EBP proteins (33). While these C/EBP-related sites are important to the CNTF-mediated induction of CyRE transcriptional activity, no evidence was found that C/EBP proteins interact with these sites in NBFL cells. CNTF induces a nuclear protein complex to bind to one of these C/EBP-related sites within the CyRE. CNTF-dependent induction of this DNA-protein complex was protein-synthesis dependent, in contrast to the protein synthesis-independent induction of STAT proteins. In this report, we show that the CNTF-induced nuclear protein complex that binds to the C/EBP-related site in the CyRE is composed of AP-1 proteins and that AP-1 activation represents an H7-sensitive nuclear signaling pathway required for CyRE-mediated transcriptional activation by CNTF.

EXPERIMENTAL PROCEDURES
Materials-Cell culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD), fetal bovine serum was from Sigma, and culture plates were from Becton Dickinson Labware (Lincoln Park, NJ). Recombinant human CNTF was a gift from Regeneron Pharmaceuticals (Tarrytown, NY). Oligonucleotides encoding the consensus sites for the transcription factors AP-1, NF-B, and CREB were purchased from Promega (Madison, WI). The remaining oligonucleotides were synthesized in our laboratory on an Applied Biosystems 380B DNA synthesizer. Antisera to c-Jun, c-Fos, fosB, and an epitope common to all Jun family members were from Santa Cruz Biotechnology (Santa Cruz, CA). Antisera to JunB and JunD were a gift of Dr. Rodrigo Bravo (Bristol Myers Squibb, NJ). H7 was purchased from Calbiochem (La Jolla, CA).

12-O-Tetradecanoylphorbol-13-acetate was obtained from Sigma
Cell Culture and Transfection-NBFL cells were maintained and transfected as described previously (31). Cells were transfected by the calcium phosphate co-precipitation method. Each 10-cm plate received 20 g of luciferase reporter plasmid and 3 g of RSVCAT plasmid. Cytokine and/or inhibitors were added 24 h after transfection; cells were harvested 6 or 36 h later and assayed for luciferase (35) and CAT (36) activities. Luciferase activity was normalized to CAT activity to control for transfection efficiency.
Plasmids-Cy1luc contains the entire 180-bp CyRE fused to ⌬eRS-Vluc (32). m2CyBluc was constructed by PCR site-directed mutagenesis as described by Ho et al. (37) with the oligonucleotides 5Ј-GGTAACT-GGATTAGAAAATACACTTAAGCATAGCAGG-3Ј and 5Ј-CCTGCTAT-GCTTAAGTGTATTTTCTAATCCAGTTACC3-Ј. These oligonucleotides were paired with oligonucleotide A1 or A4 (32), with Cy1luc as a template, to create new fragments. The fragments were gel-purified and used as template in a subsequent PCR, with oligonucleotides A1 and A4 as primers, to create m2CyBluc. Cy1mG3luc was constructed by PCR amplification of Cy1luc with the primers 5Ј-CCGGGTACCTAAAAAA-GATGTACTGGTATTAAGCCACAGGAACTCTGG3-Ј and A4. The resultant 180-bp fragments were digested with KpnI and PstI, gel purified, and ligated into KpnI/PstI-digested ⌬eRSVluc (32) to create plasmids containing the mutant sites. Both plasmids were sequenced to confirm their fidelity.
RNA Isolation and Analysis-Total cytoplasmic RNA was isolated from NBFL cells and transfered to nylon membranes as described previously (33). Northern blots were hybridized with either a 1.2-kilobase NaeI fragment of human c-fos or a 0.39-kilobase BamHI fragment of jun-B and rehybridized with a probe for the unregulated internal reference gene cyclophilin to correct for loading differences.

RESULTS
We have previously shown that the CyB site, one of three C/EBP-like sites within the VIP CyRE, is functionally important to CNTF-dependent activation of transcription mediated by the CyRE (33). In EMSAs, we previously showed that the CyB site binds two protein complexes from NBFL cells. The more slowly migrating DNA-protein complex (referred to previously as complex III) is present in nuclear extract from untreated NBFL cells. The faster migrating complex (complex IV) is only present in nuclear extracts prepared from NBFL cells that have been treated with CNTF, LIF, or OM. Neither of these nuclear protein complexes is recognized by antibodies against known C/EBP proteins. Complex IV is induced to bind to the CyB probe within 1 h of CNTF treatment. This induction is inhibited by the protein synthesis inhibitor cycloheximide. The rapid induction and protein synthesis dependence of the induction suggested that the protein complex may be composed of immediate early gene products such as AP-1 proteins.
To test the hypothesis that AP-1 proteins are present in CyB complex III, the CyB DNA-protein complexes were competed with excess unlabeled consensus AP-1 binding sites. Complex IV was competed by excess unlabeled AP-1, CRE, and CyB oligonucleotides ( Fig. 1). In contrast, the uninduced complex III is competed only by excess cold CyB oligonucleotide (Fig. 1). These results suggest that complex III and complex IV are composed of distinct nuclear proteins and that proteins that bind to AP-1 and CRE oligonucleotides also bind to the CyB site.
Inspection of the sequence of the CyB oligonucleotide revealed an imperfect homology with a consensus AP-1 site (see Fig. 4A). Within the CyB site, the AP-1 sequence is located 5Ј to the previously identified homology to a C/EBP site (33). To ascertain whether complexes III and IV had different DNA recognition requirements, a series of mutations were made in the CyB region. EMSAs performed with these CyB mutations and nuclear extracts prepared from CNTF-treated NBFL cells showed that the two CyB binding complexes were distinguishable (Fig. 2). Mutation of three base pairs in the AP-1 homology region abolished binding of the inducible complex IV (m2CyB). In contrast, mutation of three base pairs immediately 3Ј to this site (m3CyB) reduced the binding of complex III but did not affect complex IV (Fig. 2B). Mutation of three base pairs 5Ј to the AP-1 site (m4CyB) did not affect the binding of complexes III or IV. Thus, complexes III and IV bind to different regions of the CyB probe, and the CNTF-inducible complex IV binds to the DNA sequence most closely resembling the AP-1 site.
To determine whether complex IV is composed of AP-1 proteins, antisera to members of the Fos and Jun families of proteins were incubated, prior to running an EMSA, with nuclear extract prepared from CNTF-treated NBFL cells. Binding to the m3CyB probe was examined to allow easier visualization of the inducible complex IV. Complex IV, but not complex III, binding was altered by several Fos and Jun antisera (Fig. 3). Antisera that recognize an epitope common to Jun proteins inhibited the binding of complex IV (Fig. 4). When antisera DNA mobility shift assay with nuclear extracts prepared from NBFL cells treated for 1 h with CNTF (25 ng/ml). Arrows indicate complex III, whose binding to the CyB probe is unaffected by CNTF treatment, and complex IV, which is induced to bind to the CyB probe by CNTF treatment (33). Competing unlabeled oligonucleotides were present at 10 and 100-fold molar excess. Complex IV is competed by CyB, CRE, and AP-1 oligonucleotides, but the uninduced complex III is only competed by CyB.

AP-1 Proteins and CNTF Regulation of VIP Gene Expression
specific to JunB and JunD were added to the AP-1 binding reaction, the more slowly migrating complex ("supershift") was formed (Fig. 4). The addition of c-Fos antisera to the m3CyB EMSA inhibited complex IV binding. Antisera to c-Jun and fos-B did not affect complex IV binding to the m3CyB probe. Similar results were obtained when the native CyB site was used as a probe (data not shown). Thus, CNTF induces an AP-1 protein complex to bind to the CyB site within the VIP CyRE. This AP-1 complex contains c-Fos, JunB, and JunD.
To characterize the differences between m3CyB and a consensus AP-1 site, the binding of CNTF-induced complex IV to both probes was examined. Complex IV binding to m3CyB was competed with equal efficiency by itself, AP-1, or CRE-unlabeled oligonucleotide. In contrast, CNTF-induced AP-1 binding to the consensus AP-1 site was most strongly competed by unlabeled AP-1 and most weakly by m3CyB (Fig. 4B). These data suggest that AP-1 proteins have a lower affinity for the m3CyB site than for a typical AP-1 site. The mobilities of the CNTF-induced AP-1 complex to the AP-1 consensus site, the CyB, and the m3CyB probe were identical (data not shown). Complex III did not bind to the consensus AP-1 site, nor did excess AP-1 and CRE sites interfere with complex III binding to the CyB probe (Figs. 2 and 4B), thus demonstrating that complexes III and IV have different DNA binding specificities.
We next sought to determine whether the AP-1 site within the CyRE was important to the CNTF-dependent activation of transcription mediated by the 180-base pair CyRE linked to the luciferase reporter. A luciferase reporter plasmid was constructed that had three base pairs of the CyB AP-1 site mutated to produce the m2CyB site (Fig. 2) within the context of the CyRE (m2CyBluc). The m2CyB mutation did not bind the CNTF-induced AP-1 proteins (Fig. 2) or compete for binding of these proteins to the CyB probe in competition assays (data not shown). However, the m2CyB mutation did not alter the binding of the non-inducible complex III proteins (Fig. 2). Therefore, the m2CyB mutation was used to assess the importance of the AP-1 complex to CNTF-mediated transcriptional activation through the CyRE. Mutation of the CyB AP-1 sequence within the CyRE reduced CNTF-mediated induction of luciferase by 50% compared with the native CyRE contained in the Cy1luc reporter (Fig. 5). These data demonstrate that the AP-1 site within the CyB region of the CyRE is required for CNTF-dependent transcriptional activation mediated by the CyRE and further suggest that AP-1 protein binding to this site is important to this activation. However, a luciferase reporter containing three multimerized copies of the CyB site directing transcription from a basal promoter was not induced by CNTF (data not shown). Therefore, the CyB AP-1 site appears to act in concert with other sites within the CyRE to mediate the full CNTF-dependent transcriptional activation mediated by the CyRE.
It has been previously shown that CNTF-mediated induction of VIP mRNA in NBFL cells is sensitive to the serine/threonine protein kinase inhibitor H7 (24). However, binding of STAT proteins to the STAT site within the CyRE was not inhibited by H7 treatment (32), indicating that the H7 sensitivity of VIP mRNA induction was not due to alteration in STAT protein binding to the CyRE (32, 38, 39). To determine whether the AP-1-like CyB site was a location at which the H7 sensitivity of CNTF-dependent VIP transcriptional activation was mediated, we determined whether AP-1 induction and binding to the CyRE was inhibited by H7 treatment. We first examined the kinetics and sensitivity of CNTF-mediated induction of mRNA encoding the immediate early genes c-fos and jun-B, which comprise the AP-1 proteins binding to the CyB site. H7 treatment abolished CNTF-mediated c-fos mRNA induction and significantly attenuated jun-B mRNA induction, also delaying its induction (Fig. 6A). H7 pretreatment of NBFL cells also attenuated CNTF-dependent AP-1 binding (Fig. 6). H7 treatment alone did not induce binding to the AP-1 site. Similar results were obtained using the CyB site as a probe (data not shown). Thus, H7 inhibits the CNTF-mediated induction of c-fos and jun-B mRNA and formation of the AP-1 complex, suggesting that the CyB AP-1 site is a possible location through which H7 inhibits the CNTF-mediated induction of VIP mRNA.
Multiple sites within the 180-bp CyRE or sites outside the CyRE in the VIP promoter are potential sites of H7 inhibition. To determine whether H7 inhibited the CNTF-mediated induction of transcription through the VIP CyRE, NBFL cells transfected with the CyRE-luciferase reporter Cy1luc were treated with H7, and the effect on CNTF induction was assessed. H7 completely inhibited CNTF induction of luciferase activity mediated by Cy1luc (Fig. 7) without significantly reducing luciferase activity driven by RSVluc (data not shown). To ascertain whether H7-mediated repression of CNTF-dependent transcriptional activation was mediated by the CyB AP-1 site, NBFL cells were transfected with the m2CyBluc reporter, and the effect of H7 on CNTF-induced transcription was assessed. If H7 acted through the CyB AP-1 site, then mutation of this site should abrogate the H7 inhibition. Similar to its effect on the native CyRE, however, H7 completely inhibited CNTF induction of transcription mediated by a CyRE containing a mutation at the CyB AP-1 site (m2CyBluc; Fig. 7). This indicates that the ability of H7 to inhibit CNTF-dependent transcriptional activation depends on other sites within the CyRE.
As serine-threonine phosphorylation has been shown to affect the ability of STAT proteins to activate transcription (40), H7 could influence transcriptional activation by CNTF by altering serine-threonine phosphorylation of STAT proteins. We have shown that STAT proteins contribute to the CNTF-de-pendent activation of transcription by interacting with a site within the 5Ј region of the CyRE (32). Therefore, we investigated the importance of the CyRE STAT binding site to H7mediated repression of CNTF-dependent transcription activation. H7 inhibited CNTF-dependent transcription mediated by a CyRE reporter (Cy1mG3luc) containing a mutation in the STAT binding site in NBFL cells (Fig. 7). The similar inhibitory effects of H7 on the native CyRE and the CyRE STAT site mutant indicates that H7 repression of CNTF-dependent transcription depends on other sites within the CyRE. DISCUSSION These experiments demonstrate that CNTF induces an AP-1 complex to bind to a region within the VIP CyRE, the CyB site (33). AP-1 activation is necessary for the full CNTF-dependent activation of transcription mediated by the CyRE. These data present the first functional evidence for the role of AP-1 proteins in CNTF-dependent regulation of transcription. Although CNTF stimulation of AP-1 protein binding and activation contributes to CNTF-dependent transcriptional activation (Figs. 5 and 7), interaction of AP-1 proteins with multimerized CyB or canonical AP-1 sites is not able to mediate transcriptional activation in response to CNTF. 2

FIG. 6. CNTF-mediated AP-1 induction is inhibited by H7. A,
NBFL cells were pretreated with either no inhibitor or the serine/ threonine kinase inhibitor H7 (50 M) and then treated with CNTF (25 ng/ml) for the times indicated, after which cells were harvested and cytoplasmic RNA was isolated. Blots were probed with a cDNA probe to either c-fos (69) or jun-B (70) followed by a cyclophilin probe (41) to normalize for loading differences. B, DNA mobility shift assay of nuclear extracts from NBFL cells pretreated with either no inhibitor or H7 (50 M) and then treated for 1 h with CNTF (25 ng/ml). H7 inhibits the CNTF-induced binding to the AP-1 probe. a non-canonical AP-1 site. The two nucleotides that distinguish the CyB AP-1 and canonical AP-1 sites have been shown by mutational analysis to be critical for AP-1 binding (42) although AP-1 proteins can bind to this sequence in other genes (43). The sequence difference between the CyB AP-1 site and the canonical AP-1 site may be reflected in the differences in affinity of AP-1 proteins for these sites (Fig. 4).
There are two general cellular mechanisms by which CNTF may activate AP-1. Expression of the Fos-Jun components of the AP-1 complex may be induced, or phosphorylation of Fos and Jun proteins by CNTF-activated kinases may lead to a more transcriptionally active AP-1 complex. In NBFL cells, we have shown that CNTF induces c-fos and JunB mRNA (Fig. 6A and Ref. 30) and that CNTF induction of AP-1 binding is protein synthesis-dependent (33). The pathways that lead to induction of IEG transcription are complex and involve multiple pre-existing transcription factors such as STATs, serum response factor, and CREB (44 -48). STAT proteins themselves appear to interact with the c-fos promoter to contribute to c-fos induction by CNTF (19). Similar pathways may activate transcription of the Jun-B gene (49) in response to CNTF or related cytokines.
The intracellular signaling pathways that lead to regulation of AP-1 activity have been partially characterized (50). The MAP kinases, JNK, ERK, and FRK increase c-Fos and c-Jun synthesis and phosphorylation, thereby increasing AP-1 binding and activation (51)(52)(53). These kinases are, therefore, candidate molecules for mediating the CNTF-induced activation of AP-1 in NBFL cells. While it has been shown that neuropoietic cytokines activate ERK 1 and 2 in several different cell types (10,25,26), it is not known whether other MAPKs are activated by these cytokines and act to regulate gene expression. Consistent with the involvement of MAPKs in CNTF gene regulation, we have previously shown that CNTF activates Ras, an upstream regulator of MAPKs, in NBFL cells (24). CNTFmediated induction of AP-1 binding is also partially sensitive to H7, consistent with the role of MAPK or other serine-threonine kinases in AP-1 activation in response to CNTF.
Inhibition of serine-threonine kinases by H7 may not only inhibit AP-1 activation but may regulate the Jak-Stat pathway directly. Serine-threonine phosphorylation of STATs can increase the ability of these proteins to activate transcription and bind DNA (54). STAT proteins contain a MAPK phosphorylation site and demonstrate ligand-dependent association with MAPK that is required for interferon ␤-dependent activation of STATs (55). Therefore, H7 may attenuate CNTF-dependent transcription by inhibiting MAPK phosphorylation of Stat proteins and limiting their ability to activate transcription. In NBFL cells, H7 does not affect CNTF-induced Stat binding detected by EMSA (32), but serine-threonine phosphorylation of Stat3 may enhance its ability to activate transcription without a change in the amount of STAT binding (54).
The sensitivity of CNTF-induction of VIP mRNA to the serine/threonine kinase inhibitor H7 (24) supports a role of kinases, such as MAPKs or protein kinase C, in CNTF-mediated induction of VIP gene expression. We show here that this H7 sensitivity of CNTF-dependent induction of VIP mRNA in NBFL cells is mediated, at least in part, through the CyRE (Fig. 7). H7 does not appear to inhibit transcription one of the CyRE sites (STAT and CyB AP-1 sites) that we have shown to bind inducible proteins (Stat1, Stat3, and AP-1) in response to CNTF. Instead, H7 may be acting at a point further upstream in CNTF-mediated nuclear signaling, inhibiting a kinase or kinases which have effects on multiple regions of the CyRE. If H7 sensitive kinases are acting through both the AP-1 and Stat sites, then the transcriptional activity remaining when one site CNTF binding to its receptor activates the receptor-associated tyrosine kinases of the Jak/Tyk family to phosphorylate a number of intracellular substrates including the cytoplasmic tails of the receptor complex. These phosphotyrosines provide docking ports for SH2 domain proteins, bringing them into close proximity with the kinases. The SH2 domain Stat proteins are tyrosine phosphorylated and subsequently serine/ threonine phosphorylated by an H7-sensitive kinase. They translocate to the nucleus and bind to the STAT site in the VIP CyRE where they contribute to the CNTF-mediated transcriptional activation. CNTF transcriptionally activates some immediate early genes and potentially also post-translationally modifies their protein products. As there are stat sites in the promoters of many immediate early genes, Stat proteins may contribute to the transcriptional induction of immediate early genes. Fos, JunB and JunD are induced to bind to the AP-1 site in the CyRE. AP-1 mRNA and protein induction is H7 sensitive, suggesting that one or more H7-sensitive kinases are involved in the induction and post-translational modification of these proteins. The induced proteins combine with other constitutive and cell-specific proteins to activate transcription in a cell-and cytokine-specific fashion. VIP transcriptional induction is blocked by H7.

AP-1 Proteins and CNTF Regulation of VIP Gene Expression
is mutated would be abolished by H7 effects at the other. If these effects are mediated by two separate H7-sensitive kinases, then use of more specific kinase inhibitors than H7 may assist in clarifying this issue. Alternatively, H7-sensitive kinases may affect proteins binding to regions of the CyRE other than the AP-1 and STAT sites, affecting activity of a transcription factor we have not yet identified.
The mechanism by which Jak-Stat and AP-1 intracellular signals integrate to regulate CNTF transcription through the CyRE is not known. AP-1 and STAT transcription factors, which appear to be required for full CNTF-dependent transcription by the CyRE, may directly interact to activate transcription (56). A direct interaction between c-Jun and a STAT protein (Stat3␤) binding to adjacent AP-1 and STAT sites in the IL6-responsive element of the ␣2 macroglobulin gene promoter has been postulated to result in synergistic transcriptional activation (56). Direct protein-protein interaction between STATs and AP-1 proteins could stabilize protein-DNA binding complex formation and contribute to CNTF-mediated transcriptional activation in NBFL cells. However, there is approximately 100 bp separating the Stat and AP-1 sites with many other proteins binding to intermediate sites (33,41). Therefore, the integration of these two signaling pathways may be through complex formation with other basal or cell-specific factors.
The composition of the AP-1 complex with different members of the Fos/Jun family may have important biological consequences (57,58). Jun family members have similar DNA binding and dimerization domains but differ in their activation domains (59,60). JunB and JunD are weaker transcriptional activators than c-Jun and may antagonize its function (59,61,62). The JunB gene is often activated during differentiation, in contrast to the c-Jun gene that is induced during proliferation and cell death (57,58,63). Fos and JunB genes are often co-induced in the nervous system in response to a variety of extracellular signals (64,65). It has been previously shown that neuropoietic cytokines induce the c-Fos and JunB genes in several types of transformed and primary cells (7,23,27,30,66). In NBFL cells, cytokine treatment induced an AP-1 complex that contained c-Fos, JunB, and JunD. CNTF does not produce a proliferative response in NBFL cells but induces several neuropeptide genes that mimic the cytokine-mediated differentiation of sympathetic neurons (31,67). Thus, induction of AP-1 proteins (JunB, in particular) in NBFL cells by CNTF is another example of the participation of this transcriptional activator in differentiation-related process.
A model summarizing CNTF-induced signaling to the VIP CyRE is shown in Fig. 8. AP-1 and Stat proteins are induced by CNTF treatment to bind to sites in the VIP CyRE. Stat proteins also bind to the promoters of immediate early genes and may thereby participate in the transcriptional induction of AP-1 proteins. AP-1 and Stat-mediated transcriptional activation is sensitive to the protein kinase inhibitor H7, indicating possible sites where H7-sensitive kinases may act during CNTF-mediated induction of CyRE transcription. The AP-1 complex contributes to CNTF-mediated transcriptional activation of the VIP CyRE, acting in a combinatorial manner with other proteins binding to the CyRE. Many diverse extracellular stimuli activate AP-1 proteins (68). Integration of the AP-1 and Jak-Stat signaling pathways at the CyRE with other DNA binding proteins provides a mechanism whereby the VIP CyRE mediates transcriptional regulation by neuropoietic cytokines in a cytokine-and cell-specific manner (34). Integration of multiple signaling pathways with pre-existing proteins at the CyRE may provide biological specificity of cytokine action leading to selective effects of neuropoietic cytokines on differentiation, proliferation, and survival in susceptible cells in the nervous system.