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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 activin’s 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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). Activin’s 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 2AGo). 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 36–40 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.

 
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. 2AGo). 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, {Delta}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. 2BGo). The basal promoter {Delta}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. 1Go). 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. 3Go). 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. 3Go). 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 activin’s 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. 4Go). 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 activin’s 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.

 
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. 5AGo). 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.

 
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. 5BGo). 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. 6Go). 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.

 
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. 7Go). 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. 7AGo). 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. 7Go) 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 ).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 gene’s 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 activin’s 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 activin’s 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. 6Go) 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. 7Go). 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 CNTF’s 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {Delta}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 36–40 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 [{alpha}-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.


    REFERENCES
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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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