Isolation and Characterization of the Human gp130 Promoter
REGULATION BY STATS*

(Received for publication, January 6, 1997, and in revised form, March 14, 1997)

Charles A. O'Brien Dagger and Stavros C. Manolagas

From the Division of Endocrinology, University of Arkansas for Medical Sciences Center of Osteoporosis and Metabolic Bone Diseases, and GRECC, Veterans Administration Medical Center, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Glycoprotein 130 (gp130), a shared component of all the receptors for the interleukin-6 cytokine family, transduces cytokine signals in part by activating latent cytoplasmic signal transducers and activators of transcription (STATs). STATs subsequently translocate into the nucleus and stimulate gene expression. In the studies reported here, the 5'-flanking region of the human gp130 gene was isolated and the transcription initiation sites were mapped. To demonstrate that the isolated DNA fragment contained a functional promoter, a plasmid construct containing 2433 base pairs of the gp130 5'-flanking region, inserted upstream from the firefly luciferase gene, was transiently transfected into HepG2 hepatoma cells. The construct exhibited constitutive promoter activity. In addition, a 5-h treatment with interleukin-6 or oncostatin M stimulated the activity of this promoter severalfold. Localization of the cytokine response element by 5'-deletion analysis and site-directed mutagenesis revealed a cis-acting binding site for activated STAT complexes. Furthermore, DNA binding analysis demonstrated that this element binds activated STAT1 and STAT3 homo- and heterodimers. This STAT-binding element was sufficient to confer cytokine stimulation to a minimal herpesvirus thymidine kinase promoter. These results establish that the DNA fragment we have isolated contains the human gp130 promoter and that interleukin-6 type cytokines may influence the activity of this promoter via activated STATs.


INTRODUCTION

Members of the interleukin-6 (IL-6)1 cytokine family, which includes IL-6, interleukin-11 (IL-11), oncostatin M (OSM), ciliary neurotrophic factor, leukemia inhibitory factor, and Cardiotropin-1, have pleiotropic but functionally redundant effects on a wide variety of mammalian cells (1, 2). This redundancy can now be explained, at least in part, by the discovery that the receptors for each of these cytokines share a common signal transducing component known as gp130 (1, 3-5). Ligand binding to the specificity determining subunit of these receptors (alpha  subunits) causes tyrosine phosphorylation of gp130 (beta  subunit) by members of the JAK family of tyrosine kinases (6). This event results in tyrosine phosphorylation of several downstream signaling molecules including members of the STAT family of transcription factors (7, 8). Phosphorylated STATs in turn undergo homo- and heterodimerization and translocate to the nucleus where they activate transcription of cytokine responsive genes (9).

In mammals, the STAT family includes at least six members which are differentially activated by a variety of growth factors and cytokines (10). Ligand-activated gp130-JAK complexes predominantly phosphorylate STAT3 (also known as acute-phase response factor or APRF) and to a lesser extent STAT1 (11-13). The cis-acting DNA sequences recognized by STAT complexes, termed STAT-binding elements (SBEs) have the general structure TT(N)5AA (9, 14). The sequence, and in some cases the size, of the spacer region between the palindromic TT-AA motif confers specificity for different STATs (11, 14). An alternative sequence (CTGGGA), originally termed acute-phase response element (APRE), is required for IL-6 induction of many acute-phase response genes (3) and is found in the spacer region of some palindromic SBEs or in incomplete palindromic forms which may also bind STATs (11, 15).

Intrigued by the evidence that IL-6 increases gp130 mRNA in vitro in human monocytes, epithelial cells, or hepatoma cells (16-18); that administration of IL-6 to mice produces a striking up-regulation of gp130 mRNA levels in several tissues (19); and that several systemic hormones regulate gp130 expression (20-22), we have cloned and characterized the 5'-flanking region of the human gp130 gene. Using chimeric gp130 promoter/luciferase reporter constructs, we demonstrate that this region functions as a constitutively active transcriptional promoter and that its activity is stimulated by IL-6-type cytokines. In addition, we have localized a cis-acting sequence element responsible for induction by these cytokines and show that it conforms to the consensus binding site for activated STAT complexes. The STAT complexes which bind the gp130 cytokine response element are identified as STAT1 and STAT3 homo and heterodimers. Finally, we show that this SBE is sufficient to confer cytokine inducibility on a heterologous promoter to a level comparable to previously characterized SBEs.


EXPERIMENTAL PROCEDURES

Isolation and Mapping of a Genomic Fragment Containing the gp130 5'-Flanking Region

A human fibroblast genomic library in the Lambda FIX II vector (Stratagene) was screened with a probe consisting of the 5'-terminal 550 bp of the human gp130 cDNA (4) according to established methods (23). DNA was purified from positive plaques and mapped by partial restriction enzyme digestion combined with Southern blot analysis (24). A 2.7-kb EcoRI fragment which hybridized to the probe was isolated from a clone containing a 12.5-kb insert and was subcloned into pBluescript II KS+ (Stratagene) to yield the plasmid pAE3. Both strands of the 2.7-kb fragment were completely sequenced by the chain termination method (25) using sequence-derived primers (GenBank accession number U70617[GenBank]).

Primer Extension and S1 Nuclease Analysis

Primer extension was performed using an oligonucleotide primer complementary to bases +116 to +136 of the first exon shown in Fig. 1B. The 5'-32P-labeled primer was annealed to 20 µg of poly(A)+ RNA from the ARD cell line in 1 × first strand buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2) for 1 h at 60 °C and then placed on ice. Dithiothreitol and deoxynucleoside triphosphates were added to a final concentration of 10 and 500 mM, respectively, followed by 200 units of Superscript II reverse transcriptase (Life Technologies, Inc.). The reaction was incubated at 45 °C for 2 h followed by the addition of ribonuclease A to a final concentration of 0.25 mg/ml and a 10-min incubation at 37 °C. The reaction mixture was then precipitated with ethanol, resuspended in 95% formamide, heat denatured, and fractionated on a 5% polyacrylamide, M urea sequencing gel.


Fig. 1. Isolation of the human gp130 5'-flanking region. A, a partial restriction enzyme map of the 12.5-kb insert containing exon 1 of the gp130 gene is shown indicating sites for EcoRI (E) and PvuII (P). The location of exon 1 is denoted by the black box and an arrow below the box indicates the direction of transcription. B, a partial sequence of the gp130 5'-flanking region is presented with the nucleotides corresponding to the reported cDNA sequence double underlined. The sequence is numbered such that +1 corresponds most 3'-transcription start site. Positions of transcription start sites (see Fig. 2) are indicated by the vertical arrows and homologies to transcription factor consensus binding sites are underlined and labeled. The first 10 nucleotides of intron 1 are shown in lowercase letters.
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The S1 nuclease assay was performed using a 32P-labeled, single-stranded DNA probe (23) generated by extension of the 116-136 oligonucleotide, described above, after it was annealed to single-stranded DNA from the pAE3 plasmid. The probe was annealed to 50 µg of total RNA from HepG2, ARD, or Escherichia coli cells in 30 µl of hybridization buffer (40 mM 1,4-PIPES, pH 6.4, 1 mM EDTA, 400 mM NaCl, and 80% formamide) overnight at 30 °C. S1 nuclease digestion was carried out by the addition of 300 µl of S1 mapping buffer (280 mM NaCl, 50 mM sodium acetate, pH 4.5, 4.5 mM ZnCl2, 20 µg/ml single-stranded DNA, and 1000 units/ml S1 nuclease) and incubation at 42 °C for 1 h. The reaction was terminated with 80 µl of stop buffer (4 M ammonium acetate, 50 mM EDTA, and 50 µg/ml E. coli tRNA) and the products were ethanol precipitated and fractionated as described for the primer extension products.

Cell Culture and Transient Transfections

HepG2 hepatoma cells and HeLa epithelial carcinoma cells were obtained from the American Type Culture Collection (ATCC) and maintained in phenol red-free minimum essential medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum (Sigma). A human myeloma cell line (ARD) was kindly provided by Dr. Bart Barlogie (University of Arkansas for Medical Sciences) and was cultured in RPMI 1640 medium (Life Technologies, Inc.) containing 10% fetal bovine serum. A murine preadipocyte cell line, +/+LDA.11, was cultured in McCoy's 5A medium (Sigma) containing 10% fetal bovine serum. All cytokines were obtained from R & D Systems and were used at the following final concentrations: IL-6, 20 ng/ml; OSM, 20 ng/ml; IL-6sR, 40 ng/ml; IFN-gamma , 5 ng/ml.

Transient transfections of all cell types were carried out in 12-well tissue culture plates (2.2-cm diameter wells) using LipofectAMINE (Life Technologies, Inc.) as described by the manufacturer. Briefly, the day before transfection, cells were seeded at 2-4 × 104 cells/well in medium containing 10% fetal bovine serum. The next day, the cells were washed once with serum-free medium and each well was incubated with serum-free medium containing 200 ng of chimeric luciferase reporter plasmid, 200 ng of control plasmid (pSVbetagalactosidase Vector, Promega), and LipofectAMINE for 5 h. The medium was replaced with serum-containing medium and the cells were allowed to recover for 24 h before treatment with cytokines or hormones. The only exception was the experiment described in Fig. 3 in which the cells were cultured for 48 h following transfection. Lysate preparation and luciferase activity assays were performed using a kit (Promega) according to the manufacturer's instructions. Light intensity was measured with a Turner luminometer. The colorometric beta -galactosidase assay was performed using standard protocols (23) and luciferase activity was divided by the beta -galactosidase activity to normalize for transfection efficiency.


Fig. 3. Constitutive activity of the gp130 promoter. HepG2 cells were transiently transfected with plasmid constructs containing the gp130 5'-flanking region in the sense (p-2433) or antisense (p-2433-AS) orientation. The parental plasmid pGL3-Basic and the ptk-Luc construct were transfected as negative and positive controls, respectively. Following transfection, the cells were cultured for an additional 48 h before lysates were prepared. The values represent the mean normalized luciferase activity of three independent transfections and the error bars indicate the standard deviation. Similar results were obtained in three independent experiments.
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DNA Constructions

All of the chimeric luciferase reporter constructs in this study were prepared using the pGL3-Basic vector (Promega). A region spanning -2433 to +64 of gp130 5'-flanking region was prepared by partial digestion of the 2.7-kb EcoRI fragment of pAE3 with PvuII and purification of the 2.5-kb fragment. The 2.5-kb fragment was blunt-ended with the Klenow fragment of E. coli DNA polymerase I (Klenow) and ligated into the SmaI site of pGL3-Basic to produce constructs with the insert in either the sense (p-2433) or antisense (p-2433-AS) orientation. Each of the 5'-deletion constructs was prepared from p-2433 utilizing existing restriction enzyme sites (indicated in Fig. 5A). Constructs in which the APRE sequence (mt1) or the SBE sequence (mt2) were eliminated from the p-381 5'-deletion construct were prepared using the Chameleon site-directed mutagenesis kit (Stratagene) and the mutations were confirmed by sequencing (25). The herpesvirus thymidine kinase (tk) promoter/luciferase construct (ptk-LUC) was prepared by insertion of the BamHI/BglII fragment of pBLCAT2 (26), which spans positions -105 to +51 of the thymidine kinase gene, into the BglII site of pGL3-Basic. The heterologous constructs depicted in Fig. 7 were prepared by insertion of the following double-stranded oligonucleotides (only the top strands are shown) into the SmaI site of ptk-LUC: gp130, GATCGCGTTACGGGAATCG; SIE, GATCGATTGACGGGAACT; rat alpha 2-macroglobulin, GATCCTTCTGGGAATTC. Plasmids with single or double insertions were identified by restriction enzyme analysis and confirmed by sequencing.


Fig. 5. Localization of the cytokine response elements in the gp130 5'-flanking region. A, constructs containing successive 5' deletions of the gp130 5'-flanking region were transiently transfected into HepG2 cells and treated with either IL-6 or OSM for 5 h. The values represent the mean of three independent transfections with the error bars indicating the standard deviation. Similar results were obtained in at least three separate experiments. B, either the potential acute-phase response element (APRE) or the palindromic STAT-binding element (SBE) was eliminated by site-directed mutagenesis from the reporter construct containing the smallest region of 5'-flanking region which still possessed cytokine inducibility (p-381). These constructs were transfected into HepG2 cells and treated with either IL-6 or OSM for 5 h. The values represent the mean of three independent transfections and the error bars indicate the standard deviation. Similar results were obtained in three separate experiments.
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Fig. 7. The gp130 SBE confers cytokine inducibility to a heterologous promoter. SBEs from the gp130, c-fos, or rat alpha 2-macroglobulin promoters were inserted upstream from the herpesvirus thymidine kinase (tk) promoter driving the expression of the luciferase reporter gene. Reporters were constructed with either 1 or 2 copies of the SBE placed upstream of the tk promoter. HepG2 cells were transiently transfected with these constructs and treated with IL-6, OSM, or IFN-gamma for 5 h. The value shown is the mean fold induction (induced/uninduced) calculated from two different experiments in which the value for induced and uninduced were the means of three independent transfections. The error bars indicate the standard error of the mean from two experiments.
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Nuclear Extracts and DNA Mobility Shift Assays

Nuclear extracts were prepared from HepG2 cells using the method described by Sadowski and Gilman (27). Cells grown to approximately 70% confluence in 10-cm dishes were treated with cytokines for 15 min. After treatment, the cells were washed twice with ice-cold phosphate-buffered saline and once with hypotonic buffer (20 mM HEPES, pH 7.9, 20 mM sodium fluoride, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of leupeptin, aprotinin, and pepstatin). The cells on each plate were lysed with 0.3 ml of ice-cold lysis buffer (hypotonic buffer containing 0.2% Nonidet P-40), scraped into microcentrifuge tubes, vortexed for 5 s, and centrifuged for 30 s at 15,000 × g. Nuclear pellets were resuspended in 0.2 ml of high salt buffer (hypotonic buffer with NaCl and glycerol added to 420 mM and 20%, respectively) and incubated on ice for 30 min with occasional mixing. Nuclear debris were pelleted at 15,000 × g for 20 min at 4 °C, and the supernatants were quick-frozen on dry ice and stored at -80 °C.

Probes for the DNA mobility shift assay were prepared by filling in GATC overhangs of double-stranded oligonucleotides (described in the DNA construction section) using Klenow and [alpha -32P]dCTP. DNA binding reactions were performed by preincubating 15 µg of nuclear extract with 1 µg of poly(dI-dC)·poly(dI-dC) in 1 × binding buffer (10 mM Hepes, pH 7.9, 50 mM NaCl, 1 mM dithiothreitol, and 5% glycerol) for 15 min on ice followed by the addition of approximately 5 fmol of probe (20,000 cpm) and an additional 15-min incubation at room temperature. The reactions were resolved on 5% polyacrylamide gels (39:1 acrylamide:bis) containing 1% glycerol in 0.5 × TBE (Tris borate/EDTA) buffer. For supershift assays, binding reactions were preincubated with either anti-STAT1 or anti-STAT3 antibodies (both from Santa Cruz Biotechnology) or non-immune rabbit IgG for 15 min at room temperature.


RESULTS

Isolation of the Human gp130 Promoter

A human fibroblast genomic DNA library was screened with a probe consisting of the extreme 5'-end of the cloned human gp130 cDNA (4). Several clones were isolated and the hybridizing region from each was subcloned and partially sequenced. One of the clones contained a region which was identical to the first 152 bp of the reported gp130 cDNA sequence (Fig. 1, A and B). This clone was selected for further analysis and a partial restriction enzyme map of the 12.5-kb insert is shown in Fig. 1A. One of the remaining clones possessed approximately 3 kb of continuous homology to the gp130 cDNA but contained several mismatches, small insertions and deletions (not shown). Since the homologous region in this clone was uninterrupted by introns, it was concluded that this fragment represented a processed gp130 pseudogene. This conclusion is consistent with the previous observation that two distinct loci, one each on chromosomes 5 and 17, hybridized to gp130 probes (28) and that a pseudogene-like sequence was polymerase chain reaction amplified from chromosome 17 DNA (29).

A 2.7-kb EcoRI fragment containing the 152-bp identity with the gp130 cDNA was subcloned and completely sequenced. To identify the transcription start sites, primer extension analysis was performed using a primer complementary to bases 116- 136 of the exon 1 sequence (from Fig. 1B) and poly(A)+ RNA from ARD cells. ARD cells were chosen for these experiments because they produce relatively large amounts of gp130 mRNA compared with other cell lines examined (not shown). The primer extension products indicated that there are three groups of start sites, designated A, B, and C, which begin 17 bp upstream from the 5'-end of the known cDNA sequence (Fig. 2, left panel). S1 nuclease protection assays performed on total RNA from HepG2 and ARD cells revealed protected fragments corresponding to the same three sets of start sites as the primer extension analysis ( Fig. 2, right panel).


Fig. 2. Mapping of the transcription start sites on the human gp130 gene. The left panel shows a primer extension analysis using 10 µg of poly(A)+ RNA from ARD cells and a primer complementary to bases +116 to +136 of exon 1 of the gp130 gene. The same primer was used to generate the sequence shown at the right of the primer extension. Bands representing three sets of start sites are indicated by the brackets and letters to the left of the gel. The panel on the right shows fragments protected from S1 nuclease digestion by a single-stranded DNA probe that was annealed to 30 µg of total RNA from HepG2, ARD, or E. coli cells. The probe was prepared by annealing the 116-136 primer to single-stranded DNA containing exon 1 and 5'-flanking sequence followed by extension with Klenow enzyme. The same primer was used to generate the sequence to the left of the protected bands to allow a precise localization of the transcription start sites. Three sets of bands corresponding to the same start sites identified by the primer extension analysis are indicated by brackets and letters on the right.
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Sequence analysis of the region upstream of the transcription start sites revealed a G + C-rich region that did not contain a TATA box (Fig. 1B). However, a potential Sp1-binding site was present at -47 bp and two CCAAT motifs were located at -135 and -167 bp. Besides several additional Sp1 sites, the first 600 bp of 5'-flanking region also contained several sequences homologous to IL-6 type cytokine response elements including a palindromic SBE (TTACGGGAA), a non-palindromic SBE or APRE (CTGGGA), and two adjacent NFIL6/C-EBPbeta -binding sites (3, 30).

Constitutive and Stimulated Activity of the Human gp130 Promoter

To confirm that the isolated DNA fragment contained a functional promoter, we prepared reporter gene constructs by inserting the 5'-flanking region (spanning bases -2433 to +64 relative to the most 3'-transcription start site) upstream from the firefly luciferase gene in the pGL3-Basic vector. When a construct with this fragment in the sense orientation (p-2433) was transiently transfected into HepG2 cells, a significant level of transcriptional activity was observed (Fig. 3). The parental vector or the parental vector containing the fragment in the antisense orientation produced only trace amounts of transcriptional activity. Similar results were obtained when this set of constructs was transfected into HeLa or +/+LDA.11 cells (not shown). These results establish that the 5'-flanking region of gp130 contains a promoter that is constituitively active in epithelial and fibroblastoid cell types.

Transcriptional regulation of the gp130 promoter was subsequently investigated in HepG2 cells transiently transfected with the p-2433 construct. Consistent with earlier evidence suggesting that gp130 production is regulated, 5-h treatment with IL-6 in combination with the IL-6-soluble receptor (IL-6sR) or OSM stimulated the activity of the promoter approximately 4-fold, while IL-6 alone produced approximately a 2-fold increase (Fig. 4). Essentially the same results were obtained when the treatment time was extended to 24 h, with the only exception that the effects of IL-6 + IL-6sR and OSM were slightly lower and that for IL-6 was higher compared with the 5-h time point. In line with the evidence that interferon (IFN)-gamma and IL-6 activate overlapping sets of transcription factors and genes (31), 5-h treatment with IFN-gamma stimulated the reporter construct to a level similar to that produced by IL-6. However, after 24 h of treatment, the response to IFN-gamma was significantly decreased.


Fig. 4. Response of gp130 promoter activity to cytokines. HepG2 cells were transiently transfected with the p-2433 construct and treated with the indicated cytokine for 5 (left panel) or 24 h (right panel). The values represent the mean normalized luciferase activity of three independent transfections with the error bars indicating the standard deviation. Similar results were obtained in at least two experiments.
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Localization of the Cytokine Response Elements in the gp130 5'-Flanking Region

To identify the cis-acting sequence elements responsible for the induction of the gp130 promoter by IL-6 type cytokines, sequential 5'-deletion constructs of the plasmid p-2433 were prepared (depicted in Fig. 5A). Transfection of these constructs into HepG2 cells and treatment with either IL-6 or OSM for 5 h revealed that deletion beyond -191 bp abolished the response to either cytokine (Fig. 5A). Besides failing to exhibit cytokine responsiveness, the -191 construct also displayed a significant decrease in basal transcriptional activity. Taken together, these results indicate that the region between -381 and -191 bp contained sequences responsible for the majority of the cytokine responsiveness as well as sequences contributing to the basal transcriptional activity of the gp130 promoter.

The region between -381 and -191 bp contains two potential cytokine response elements identified by comparison to consensus sequences: a palindromic SBE (TTACGGGAA) and a non-palindromic SBE or APRE (CTGGGA). Based on this and evidence that IL-6-type cytokines utilize STAT proteins in their signal transduction pathway, we reasoned that one or both of these potential SBEs might be responsible for the cytokine induction of the gp130 promoter. To address this issue, we prepared reporter constructs in which either of these potential response elements were eliminated from the -381 promoter fragment by site-directed mutagenesis. The constructs were then transiently transfected into HepG2 cells and their activity was examined following 5 h of treatment with IL-6 or OSM. The wild-type -381 promoter fragment responded to IL-6 and OSM as expected from the 5'-deletion analysis and this response was unaffected in the construct containing the mutated APRE consensus sequence (mt1) (Fig. 5B). However, the construct containing the mutated palindromic SBE consensus sequence was unresponsive to either cytokine (mt2 in Fig. 5B). Thus, the SBE-like sequence located between -381 and -191 bp is required for stimulation of the gp130 promoter by IL-6 and OSM.

Characterization of the gp130 SBE

The sequence between the TT-AA motif of the gp130 cytokine response element is identical to the IL-6-response element from the human alpha 2-macroglobulin promoter (32) and to the c-fos SBE known as the sis-inducible element (SIE) (33). The SIE binds STAT1 and STAT3 homo- and heterodimers (12, 34) and is required in vivo for the induction of the c-fos gene by a variety of stimuli (35). A comparison of the gp130 SBE-like sequence with SBEs from various cytokine or growth factor-inducible promoters is shown in Table I. Both a mutant version of the SIE, denoted SIEm67, and a SBE from the rat alpha 2-macroglobulin promoter, which contains the APRE consensus (CTGGGA), have been used as classical IL-6 response elements (12, 31, 34, 36). In our studies we employed the latter for comparison purposes.

Table I. Comparison of the gp130 SBE with SBEs from different mammalian promoters

The following abbreviations were used: halpha 2M, human alpha 2-macroglobulin; ralpha 2M, rat alpha 2-macroglobulin. The underlined sequence in the ralpha 2M sequence corresponds to the previously described APRE (3). SIEm67 is the high affinity binding mutant of the SBE from the c-fos promoter.

Gene SBE Reference

gp130 GCGTTACGGGAATCG This report
Halpha 2M CTCTTACGGGAATGG 32
Ralpha 2M TCCTTCTGGGAATTC 43
c-fos(SIE) GATTGACGGGAACTG 33
SIEm67 GATTTACGGGAAATG 33
SBE motif    TTNNNNNAA

Previous studies have shown that in HepG2 cells, IL-6 and OSM induce DNA binding homodimers of STAT1 and STAT3 as well as heterodimers of STAT1 and STAT3; while IFN-gamma induces predominantly STAT1 homodimers (12, 13, 36). In electrophoretic mobility shift assays with the SIEm67 probe, these complexes migrate as a set of three bands with the slowest corresponding to homodimers of STAT3, the middle to heterodimers of STAT1 and STAT3, and the fastest to homodimers of STAT1.

To determine if the gp130 cytokine response element is in fact bound by activated STAT complexes, we performed electrophoretic mobility shift assays using an oligonucleotide probe corresponding to the gp130 cytokine response element and nuclear extracts from cytokine-treated HepG2 cells. IL-6 type cytokines induced the formation of the three characteristic DNA binding complexes designated A, B, and C, while IFN-gamma induced the formation of a single complex that co-migrated with complex C (Fig. 6A). Complexes of the same mobility and intensity were seen when the same extracts were incubated with a probe corresponding to the wild-type SIE from the c-fos promoter (compare lanes 2-5 with lanes 7-10). The specificity of the protein binding to the gp130 probe was demonstrated by competition with a 100-fold molar excess of either the wild-type gp130 or the SIE oligonucleotide but not a mutated gp130 oligonucleotide (Fig. 6B). The identity of the binding proteins in the OSM-treated extract was established by demonstrating that an anti-STAT1 antibody inhibited the formation of both the fastest and the intermediate migrating complexes, whereas an anti-STAT3 antibody supershifted the slowest and the intermediate complexes (Fig. 6C). As expected, the anti-STAT1 antibody inhibited the formation of the fastest migrating complex from the IFN-gamma -treated extract whereas anti-STAT3 had no effect. These results demonstrate that the cytokine response element in the gp130 promoter is bound by activated STAT1 and STAT3 proteins and that the gp130 SBE binds these proteins with an affinity similar to that of the c-fos SBE.


Fig. 6. A, the gp130 SBE-like sequence binds complexes induced by IL-6, OSM, and IFN-gamma . DNA mobility shift assays were performed with extracts made from HepG2 cells treated with the indicated cytokine for 15 min. Each extract was incubated with one of the indicated 32P-labeled oligonucleotide probes (sequences compared in Table I) and treated as described under "Experimental Procedures." The intense, uninduced complex formed with the SIE probe is due to the presence of a CCAAT-box binding protein site (34). B, specificity of the gp130 SBE. Competition binding reactions were performed by preincubating nuclear extracts, from OSM or IFN-gamma treated HepG2 cells, with a 100-fold molar excess of the indicated unlabeled double-stranded oligonucleotide (gp130SBE (wt); c-fos SBE (SIE); mutant gp130 SBE (mt)) followed by incubation with the 32P-labeled gp130 SBE probe. C, identification of STAT proteins associated with the gp130 SBE. Nuclear extracts from OSM or IFN-gamma treated HepG2 cells were preincubated with the following antibodies before addition of the 32P-labeled gp130 SBE probe: rabbit non-immune IgG (IgG), anti-STAT1 (S1), or anti-STAT3 (S3). The identities of the proteins associated with the SBE are indicated at the left of the panel.
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Finally, to determine if the gp130 SBE was sufficient to confer cytokine inducibility on a heterologous promoter, we inserted an oligonucleotide corresponding to a single copy of the gp130 SBE upstream from an herpes simplex virus-thymidine kinase promoter/luciferase reporter gene. To compare the stimulatory ability of the gp130 SBE to previously characterized SBEs, we also prepared similar constructs containing single copies of either the c-fos SBE (SIE) or the rat alpha 2-macroglobulin SBE. The gp130 SBE was sufficient to confer IL-6 and OSM inducibility to the promoter to an extent equal to or greater than that of either the c-fos or rat alpha 2-macroglobulin SBE (Fig. 7). Multimerization of the SBEs increased responsiveness to IL-6 and OSM, and conferred IFN-gamma induction as well.


DISCUSSION

In the work described in this report, we have isolated and characterized the human gp130 5'-flanking region. We determined that this DNA fragment is similar to the 5'-flanking regions of genes for other members of the cytokine receptor family in that it lacks a recognizable TATA box and is rich in G + C residues (37). These properties along with multiple transcription start sites are also found in many constitutively expressed genes (38). In addition, we found that IL-6-type cytokines stimulate the activity of this promoter. Although several potential sites for IL-6-inducible transcription factors were identified within the first 600 bp upstream from the transcription start sites, deletion and mutagenesis analysis revealed that, of these, only the consensus palindromic SBE was required for IL-6 stimulation under the conditions used in our experiments.

The observation that the gp130 promoter is stimulated by IL-6 and OSM suggests that the previously reported increases in gp130 mRNA in response to these cytokines might be the result of transcriptional regulation. Consistent with this suggestion, the 2-4-fold stimulation of the isolated promoter, in our studies, corresponded closely with the earlier described magnitude of gp130 mRNA induction by IL-6 in HepG2 cells (18). OSM exhibited reproducibly greater stimulation of the gp130 promoter, as compared with IL-6. This phenomenon may be due to a greater number of functional receptors at the cell surface and/or more effective activation of STAT complexes by the former cytokine. Several genes are induced by both IL-6 and IFN-gamma , in many cases through the same SBEs (31). Nonetheless, we found that IFN-gamma , although it stimulated promoter activity following a 5-h treatment, had no effect on the gp130 promoter in HepG2 cells at 24 h. The lack of a sustained effect of IFN-gamma is consistent with the results of studies by Schooltink et al. (18) demonstrating lack of an effect of this agent on gp130 expression in HepG2 cells.

Lamb and co-workers (13) have suggested that promoters containing a single SBE, like gp130, are stimulated preferentially by STAT1/STAT3 heterodimers as compared with homodimers of either STAT1 or STAT3. This suggestion is in agreement with our finding of a preferential stimulation of the gp130 promoter by IL-6 or OSM as compared with IFN-gamma , and can explain the apparent discrepancy between the amount of STAT-DNA binding activity and transactivation effect produced by these cytokines, which has also been demonstrated in other systems (14, 39). Recent experiments in STAT1-deficient mice demonstrated that STAT1 is not required for IL-6 induction of several IL-6-responsive genes (40). Therefore it remains possible that the STAT3 homodimer, and potentially other STAT proteins (41), may be responsible for the stimulation of the gp130 promoter by IL-6 type cytokines.

The level of IL-6-type cytokine stimulation of the gp130 promoter is slightly lower than that reported for some of the acute-phase response and immediate early gene promoters, which demonstrate 4-8-fold stimulation by IL-6 in hepatoma cell lines (15, 42-44). This is the case even though the gp130 SBE binds activated STAT dimers with a similar affinity as the c-fos SBE and is able to confer cytokine stimulation to a heterologous promoter to approximately the same extent as the c-fos and rat alpha 2-macroglobulin SBEs. A possible explanation for this difference comes from the observation that many acute-phase response gene promoters contain multiple SBEs or an SBE in close proximity to binding sites for other inducible transcription factors. However, we have identified only a single SBE in the gp130 promoter which appears to confer the majority of the cytokine responsiveness.

The biologic significance of the in vitro evidence for the stimulation of the gp130 promoter by IL-6 type cytokines, to the in vivo situation, is at this stage a matter of conjecture. Nonetheless, it is well established that following cytokine binding, gp130, as well as the alpha  subunit of the IL-6 receptor, is internalized and degraded (45-48). Considering this and the evidence that several cell types express receptors for more than one member of the IL-6 type cytokine family (49, 50), and that all of them utilize gp130, maintenance of responsiveness to other members of the family, following stimulation by one of them, may require gp130 replenishment. The STAT-mediated increase in gp130 promoter activity may therefore be part of a mechanism to achieve this replenishment. A similar mechanism may be operative for other receptors that activate STAT proteins as evidenced by the recent finding that STAT6 binds to and activates the IL-4 receptor promoter (51).

Currently, it is unknown whether, in at least some of the target cells of IL-6-type cytokines, the abundance of gp130 may be limiting so as to render regulation of gp130 expression consequential (47). Nonetheless, it has been shown that not only IL-6 type cytokines, but also IL-1, TNFalpha , and the systemic hormones estrogen, androgen, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), all-trans-retinoic acid, and parathyroid hormone regulate gp130 expression in vitro (17, 20-22). Specifically, in studies reported elsewhere, we have shown that estrogen and androgen decrease the expression of the gp130 transcript and its protein by cells of the bone marrow stromal/osteoblastic lineage in vitro (22). Conversely, ovariectomy in mice causes an increase in the expression of gp130 mRNA and protein in ex vivo bone marrow cell cultures.2 In contrast to sex steroids, we found that parathyroid hormone and 1,25-(OH)2D3 stimulate gp130 expression. A stimulatory effect of gp130 by parathyroid hormone and 1,25-(OH)2D3 has been also found in studies by Romas et al. (21). Furthermore, Sidell and co-workers (20) have observed that all-trans-retinoic acid decreases the cell surface expression of gp130 in freshly isolated myeloma cells. Moreover, it has been demonstrated that anti-gp130 antibodies dose dependently inhibit IL-6-type cytokine-induced osteoclast formation (21) and Kaposi's sarcoma cell proliferation in vitro (52); and that the decrease in gp130 induced by all-trans-retinoic acid is associated with inhibition of IL-6-dependent human myeloma cell growth (20). Finally, in studies reported elsewhere, we have shown that the inhibitory effect of estrogen on gp130 is accompanied by a decrease in the amount of activated STAT complexes induced in response to IL-6 type cytokines (53). These latter observations may bear relevance to the evidence that bone marrow cells from estrogen-deficient mice are more sensitive to osteoclastogenic signals mediated through gp130, than cells from estrogen replete animals (54).

In conclusion, our results establish that the DNA fragment we have isolated contains the human gp130 promoter and that IL-6 type cytokines can influence the activity of this promoter via activated STATs. In addition, the in vitro demonstration of a STAT mediated up-regulation of gp130 suggests a mechanism whereby activation of gp130 by its ligands may modulate the production of new gp130. Whether modulation of gp130 production by its cognate ligands and/or other agents may serve to replenish receptor consumed upon ligand activation and/or to modulate the sensitivity of cells to the actions of IL-6 type cytokines will of course require future studies, as will the mechanisms by which other cytokines and hormones regulate gp130 production.


FOOTNOTES

*   This work was supported by the National Institutes of Health Grants P01 AG13918-01 and AR43003, the Department of Veterans Affairs, and a University of Arkansas for Medical Sciences institutional grant (to C. A. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U70617[GenBank].


Dagger    To whom correspondence and reprint requests should be addressed: University of Arkansas for Medical Sciences, 4301 West Markham St., Mail Slot 587, Little Rock, AR 72205. Tel.: 501-686-5607; Fax: 501-686-8148; E-mail: cobrien{at}life.uams.edu.
1   The abbreviations used are: IL, interleukin; OSM, oncostatin M; STAT, signal transducers and activators of transcription; SBE, STAT binding element; APRE, acute-phase response element; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; bp, base pair(s); PIPES, 1,4-piperazinediethanesulfonic acid; tk, thymidine kinase; sR, soluble receptor; SIE, sis-inducible element; IFN, interferon; kb, kilobase pair(s).
2   S.-C. Lin, T. Yamate, Y. Taguchi, V. Borba, G. Girasole, C. A. O'Brien, T. Bellido, E. Abe, and S. C. Manolagas, submitted for publication.

ACKNOWLEDGEMENTS

We thank T. Bellido and R. Jilka for useful discussions, T. Kishimoto for reagents, and N. Stahl and J. Darnell for reviewing the manuscript.


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