©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
C/EBP-related Sites in Addition to a Stat Site Are Necessary for Ciliary Neurotrophic Factor-Leukemia Inhibitory Factor-dependent Transcriptional Activation by the Vasoactive Intestinal Peptide Cytokine Response Element (*)

(Received for publication, November 11, 1994; and in revised form, January 27, 1995)

Aviva J. Symes Prithi Rajan Lisa Corpus J. Stephen Fink (§)

From the Molecular Neurobiology Laboratory, Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 02114

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The family of neuropoietic cytokines, including ciliary neurotrophic factor (CNTF) (^1)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, LIFRbeta(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 beta 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.


EXPERIMENTAL PROCEDURES

Materials

Cell culture reagents were obtained from Life Technologies, Inc., fetal bovine serum from Sigma, and culture plates from Becton Dickinson Labware (Lincoln Park, NJ). Recombinant human CNTF was a gift from Regeneron Pharmaceuticals (Tarrytown, NY). Recombinant human LIF, IL-6, and sIL-6R were purchased from R & D Systems (Minneapolis, MN). Recombinant human oncostatin M was a gift from Bristol-Myers Squibb (Seattle, WA). Acrylamide was purchased from National Diagnostics (Atlanta, GA). Anti-C/EBPalpha and anti-C/EBPbeta antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-C/EBP was a gift from Dr. Steven McKnight (Tularik, CA). Bacteria expressing C/EBP or NF-IL6 DNA-binding domains and eukaryotic expression plasmids for C/EBP and LAP were a gift of Dr. David Ron (New York University, NY). Oligonucleotides were synthesized in our laboratory on an Applied Biosystems 380B DNA synthesizer. APRE-M6 oligonucleotide was a gift from Dr. Mario Vallejo (Massachusetts General Hospital, Boston). Cycloheximide was obtained from Sigma.

Cell Culture and Transfection

NBFL cells were maintained and transfected as described previously(22) . Cells were transfected by the calcium phosphate precipitation method with 20 µg of luciferase reporter plasmid and 2.5 µg of RSVCAT plasmid/10-cm plate. In cotransfection experiments, 10 µg of reporter plasmid was transfected together with a total of 6 µg of expression plasmid. Cytokine treatment began 24 h after transfection; 36 h later cells were harvested and assayed for luciferase (58) and CAT activity(59) . When cells were not treated with cytokines, cells were harvested 48 h after transfection. Luciferase activity was normalized to CAT activity to control for transfection efficiency.

DNase I Footprinting

Bacterially expressed C/EBPalpha- and NF-IL6 DNA-binding domains were isolated as described previously from Escherichia coli BL21 plys.S(60) . DNA probes were synthesized in a polymerase chain reaction with one oligonucleotide end-labeled with [P]ATP by T4 polynucleotide kinase. The oligonucleotides used were either (A1) 5`-CCGGGTACCTAAAAAAGATTTCCTGG3-` with (A4) 5`-CACCTGCAGCGTTCAAAGTCAGAC-3` or (A11) 5`-CCGAAGCTTGGGTGATGAAGAGCTAAGG-3` with (A12) 5`-CCGCTGCAGTCCTCTTTCTTCTAC-3`. VIP1929luc (22) was used as a template. Labeled DNA fragments were purified on a 10% non-denaturing polyacrylamide gel before footprinting as described previously(61) . Between 10 and 30 ng of bacterial extract was incubated with probe for 20 min before DNase I digestion (1:600 dilution) for 1 min. Reactions were stopped, phenol/chloroform extracted, ethanol precipitated, and analyzed on a 8% denaturing polyacrylamide gel using DNA sequencing reactions from the same probe as size markers.

Plasmids

Cy1luc contains the entire 180-bp CyRE fused to DeltaeRSVluc(24) . C/EBPalpha and C/EBPbeta were expressed in the pCDNA1 vector under the control of the cytomegalovirus promoter(62) . Plasmids containing mCyA, mCyB, and mCyC sites were constructed by polymerase chain reaction site-directed mutagenesis as described by Ho et al.(63) . The following pairs of oligonucleotides were used to create the mutant sites within Cy1luc: mCyA: 5`-ACAAATTTCCAGACATGGTTAGGCTTAATTCATTTAATTT-3` and 5`-AAATTAAATGAATTAAGCCTAACCATGTCTGGAAATTTGT-3`; mCyB 5`-GATTAGAAAATATGATGGATCGGAGCAGGATATTCTTTTA-3` and 5`-TAAAAGAATATCCTGCTCCGATCCATCATATTTTCTAATC-3`; and mCyC 5`-TCCCAGTTGACAGCTGAGAATTGTACCAGAGTTCCTGTGG-3` and 5`-CCACAGGAACTCTGGTACAATTCTCAGCTGTCAACTGGGA-3`. In each case these oligonucleotides were paired with oligonucleotide A1 or A4 with Cy1luc as a template, to create new fragments. These fragments were gel-purified and used as template in a subsequent polymerase chain reaction with oligonucleotides A1 and A4 as primers. The resultant 180-bp fragment was digested with KpnI and PstI, gel-purified, and ligated into KpnI-PstI-digested DeltaeRSVluc (24) to create plasmids containing the mutant site. All plasmids were sequenced to confirm their fidelity.

RNA Isolation and Analysis

Total cytoplasmic RNA was isolated from tissue culture cells after lysis with Nonidet P-40(64) . Each RNA sample was separated by electrophoresis on formaldehyde, 1.4% agarose gels and electrotransferred onto GeneScreen membranes (DuPont NEN). Northern blots were hybridized as described previously (22) with probes labeled (65) with [alpha-P]dCTP to a specific activity of 0.5-2 times 10^9 counts/min/µg. Blots were washed to 0.2 times SSC (1 times = 0.015 M NaCl, 0.0015 M sodium citrate pH 7.0) containing 0.1% SDS at 65 °C. VIP mRNA was detected with a 580-bp HindIII-EcoRI fragment of human VIP cDNA(66) . The membranes were hybridized with a probe for the unregulated internal reference gene cyclophilin(67) . Autoradiograms were digitized with a Microtek scanner and analyzed with the NIH Image 1.41 program. The relative densitometric readings were normalized to cyclophilin mRNA to account for loading differences between lanes.

DNA Mobility Shift Assays

Synthetic complementary oligonucleotides with GGG or GATC overhangs were annealed and labeled with [alpha-P]dCTP using Superscript reverse transcriptase (Life Technologies, Inc.) or Klenow fragment. Nuclear extracts were isolated and binding reactions were performed as described previously(24) . Nuclear extracts (approximately 15 µg of protein) were incubated with 0.5 ng of labeled probe (approximately 250,000 counts/min) for 20 min at room temperature before running on a 5% non-denaturing polyacrylamide gel (37.5:1) in 0.5 times TBE at 200 V. The following pairs of complementary oligonucleotides were used in DNA mobility shift assays (mutated residues are underlined): G3: 5`-GATTTCCTGGAATTAAGATC-3` and 3`-CTAGGCTAAAGGACCTTAATT-5`; CyA: 5`-TAAGTTTCAAAATGTCTGGAAATTTGGATC-3` and 3`-CTAGATTCAAAGTTTTACAGACCTTTAAAC-5`; NF-kappaB: 5`-AGTTGAGGGGACTTTCCCAGGC-3` and 3`-TCAACTCCCCTGAAAGGGTCCG5-`; CyB 5`GGGAAAATATGATTAAGCATAG-3` and 3`-TTTTATACTAATTCGTATCGGG-5`; CyC 5`-GGGCTCTGGCTTAAGTCAGAGCTGTCA-3` and 3`-GAGACCGAATTCAGTCTCGACAGTGGG; mCyA 5`-TAAGCCTAACCATGTCTGGAAATTTGGATC3` and 3`-CTAGATTCGGATTGGTACAGACCTTTAAAC; mCyB 5`-TGAAAATATGATGGATCGGAGGATC-3` and 3`-CTAGACTTTTATACTACCTAGCCTC-5`; mCyC 5`-CTCTGGTACAATTCTCAGCTGTCAGATC-3` and 3`-CTAGGAGACCATGTTAAGAGTCGACAGT-5`.


RESULTS

Soluble IL-6 Receptor Confers IL-6 Responsiveness to NBFL Neuroblastoma Cells

We have previously shown that treatment of NBFL cells with CNTF, LIF, and oncostatin M increases levels of VIP mRNA(22, 23) . However, the related cytokine IL-6 did not increase VIP mRNA(23) . The failure of IL-6 to increase VIP mRNA may be due to differences in the signal transduction pathways among the different cytokines or to the absence of functional IL-6 receptors in NBFL cells. To test these hypotheses, NBFL cells were treated with a soluble form of the ligand-binding subunit of the IL-6 receptor (sIL-6R). The sIL-6R has been shown to confer IL-6 responsiveness to IL-6-resistant cells (39, 68) . Together, IL-6 and sIL-6R induced VIP mRNA, but neither of these proteins alone did (Fig. 1A). VIP mRNA was induced 5-fold by IL-6 and sIL-6R, 8-fold by CNTF, 10-fold by LIF, and 11-fold by oncostatin M. This experiment suggests that the failure of IL-6 alone to induce VIP mRNA is due to the absence of functional IL-6 receptors in NBFL cells. Functional IL-6 receptors can be reconstituted by the addition of sIL-6R permitting induction of VIP mRNA in response to IL-6.


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 times 10^5 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 DeltaRSV 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.

C/EBP Transcription Factors Bind to and Transactivate the VIP CyRE

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) . Since our data suggest that IL-6 and the CNTF family of cytokines transmit signals to the nucleus by similar mechanisms, we sought to investigate the role of C/EBP proteins in mediating the CNTF induction of the VIP gene in NBFL cells. To examine whether C/EBP proteins are able to transactivate the CyRE, expression plasmids encoding full-length C/EBPalpha or C/EBPbeta proteins were cotransfected with the reporter plasmid Cy1luc into NBFL cells, and luciferase activity was measured. C/EBP alpha and C/EBPbeta transactivated Cy1luc (Fig. 2). C/EBP alpha was a stronger activator of transcription through the CyRE, inducing luciferase activity to a maximum of 7.8-fold compared to 4.7-fold for C/EBPbeta cotransfection. Thus, C/EBP transcription factors are able to transactivate the VIP CyRE.


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 alpha or C/EBP beta 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/EBPalpha and C/EBPbeta 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/EBPalpha DNA-binding domain, CyA, CyB, and CyC (Fig. 3). The C/EBPbeta DNA-binding domain protected the same region as C/EBPalpha (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/EBPalpha or C/EBPbeta, 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/EBPalpha and C/EBPbeta 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/EBPalpha. DNA probe was incubated either with extract from bacteria alone (lanes 1, 4, and 5) or with extract from bacteria expressing a truncated C/EBPalpha 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.



The Role of C/EBP in CNTF-mediated Induction of VIP Transcription

As C/EBP proteins are able to bind and transactivate the VIP CyRE, we sought to determine whether the possible C/EBP-binding sites were important to the CNTF-mediated regulation of the CyRE. Reporter plasmids were constructed which had either CyA, CyB, or CyC sites individually mutated within Cy1luc. The mutated C/EBP-binding sites did not compete for nuclear protein binding to the CyA, CyB, or CyC sites in DNA mobility shift assays (Fig. 6, B and C). Mutation of the C/EBP sites within the CyRE-luciferase plasmid Cy1luc attenuated CNTF-dependent transcriptional activation (Fig. 4). Mutation of the CyA, CyC, and CyB sites reduced the CNTF-dependent induction of luciferase by 30, 40, and more than 75%, respectively (Fig. 4). Therefore, the three possible C/EBP-binding sites in the CyRE are important to CNTF-mediated activation of VIP transcription.


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/EBPalpha or C/EBPbeta decreased CNTF-mediated transcriptional activation (Fig. 5). This inhibition of CNTF-dependent transcriptional activation was marked; 0.5 and 6 µg of C/EBPalpha expression plasmid reduced luciferase induction by 63 and 76%, respectively. C/EBPbeta was a more potent inhibitor of CNTF-dependent transcriptional activation: 0.5 and 6 µg of C/EBPbeta 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).



Inducible and Basal NBFL Nuclear Proteins Bind to C/EBP Sites in the VIP CyRE

DNA mobility shift assays were performed to examine whether nuclear proteins from NBFL cells bind to the putative C/EBP sites in the CyRE. Oligonucleotides corresponding to the putative C/EBP-binding sites were incubated with nuclear extracts prepared from NBFL cells treated for varying times with CNTF. CyA, CyB, and CyC probes bound nuclear proteins from untreated NBFL cells (Fig. 6A). Using the CyB probe with nuclear extracts from CNTF-treated NBFL cells, an additional DNAbulletprotein complex was detected (complex IV, Fig. 6A). This CyB-protein complex was induced by 1 h of CNTF treatment and was still detectable after 6 h (Fig. 6A). Further analysis showed that the inducible CyB-protein complex was not present 15 min after CNTF treatment and had disappeared by 24 h (data not shown). The CyA and CyC nuclear protein complexes were unaltered after CNTF treatment of NBFL cells.

Competition analysis of these DNAbulletprotein 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.

NBFL Nuclear Proteins Binding to the Putative C/EBP Sites in the VIP CyRE Are Distinct from C/EBPs

To obtain more direct evidence for C/EBP proteins in NBFL cell nuclear extracts binding to CyA, CyB, and CyC sites, DNA mobility shift assays were performed in the presence of C/EBP antisera. Neither the basal nor CNTF-inducible nuclear protein complexes binding to CyA, CyB or CyC probes were altered by incubation with C/EBPalpha, C/EBPbeta, or C/EBP antisera (Fig. 7). Furthermore, when the C/EBP antisera were used in Western blots of NBFL cell lysates, before and after CNTF treatment, C/EBPalpha, C/EBPbeta, or C/EBP proteins could not be detected (data not shown). The C/EBP antisera used in these experiments appear to be adequate for detecting C/EBP proteins. These antisera have been used previously to detect C/EBP proteins in DNA mobility shift assays (53, 60) and are able to detect C/EBP protein binding to the M6 site using nuclear extracts prepared from COS cells transfected with C/EBPalpha or C/EBPbeta (data not shown).


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/EBPalpha and C/EBPbeta, 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/EBPbeta-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 DNAbulletprotein complexes formed with NBFL nuclear extracts do not contain known C/EBP proteins.

Characterization of the CNTF-inducible CyB Nuclear Protein Binding

To determine whether de novo protein synthesis is required for induction of CyB complex IV binding, NBFL cells were pretreated with the protein synthesis inhibitor, cycloheximide, before CNTF treatment. Cycloheximide prevented the inducible complex IV formation (Fig. 8A), indicating that de novo protein synthesis is required for the formation of this complex.


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.


DISCUSSION

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 LIFRbeta(44) . As the cytoplasmic tail of the LIFRbeta 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 LIFRbeta 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. (^2)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/EBPalpha, C/EBPbeta, 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^2 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.


FOOTNOTES

*
This work was supported by the Dysautonomia Foundation Inc., Alzheimer's Association H. Houston Speck Research grant and National Institutes of Health Grant NS27514. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Molecular Neurobiology Lab., Dept. of Neurology, Massachusetts General Hospital, Boston MA 02114. Tel.: 617-726-5717; Fax: 617-726-5677.

(^1)
The abbreviations used are: CNTF, ciliary neurotrophic factor; LIF, leukemia inhibitory factor; IL, interleukin; VIP, vasoactive intestinal peptide; bp, base pair(s); CyRE, cytokine response element; GST, glutathione S-transferase; IFN, interferon.

(^2)
A. J. Symes, and J. S. Fink, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Susan Lewis, Michael Schwarzchild, Mahendra Rao, David Ron, and Mario Vallejo for many helpful discussions and technical advice. We acknowledge Regeneron Pharmaceuticals for their gift of CNTF, Bristol Myers Squibb for their gift of Oncostatin M, Dr. Steven McKnight for C/EBP antiserum, Dr. David Ron for bacteria expressing C/EBP DNA-binding domains and C/EBP and LAP expression plasmids, and Dr. Mario Vallejo for the M6 oligonucleotide.


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