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• W2162214575 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. 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. Members of the interleukin-6 (IL-6) 1The abbreviations used are:ILinterleukinOSMoncostatin MSTATsignal transducers and activators of transcriptionSBESTAT binding elementAPREacute-phase response element1,25-(OH)2D31,25-dihydroxyvitamin D3bpbase pair(s)PIPES1,4-piperazinediethanesulfonic acidtkthymidine kinasesRsoluble receptorSIEsis-inducible elementIFNinterferonkbkilobase pair(s). 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 (1Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Google Scholar, 2Sehgal P.B. Wang L. Rayanade R. Pan H. Margulies L. Ann. N. Y. Acad. Sci. 1995; 762: 1-14Google Scholar). 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 (1Kishimoto T. Akira S. Narazaki M. Taga T. Blood. 1995; 86: 1243-1254Google Scholar, 3Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Google Scholar, 4Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Google Scholar, 5Taga T. Narazaki M. Yasukawa K. Saito T. Miki D. Hamaguchi M. Davis S. Shoyab M. Yancopoulos G.D. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10998-11001Google Scholar). Ligand binding to the specificity determining subunit of these receptors (α subunits) causes tyrosine phosphorylation of gp130 (β subunit) by members of the JAK family of tyrosine kinases (6Stahl N. Yancopoulos G.D. Cell. 1993; 74: 587-590Google Scholar). This event results in tyrosine phosphorylation of several downstream signaling molecules including members of the STAT family of transcription factors (7Boulton T.G. Stahl N. Yancopoulos G.D. J. Biol. Chem. 1994; 269: 11648-11655Google Scholar, 8Lutticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Kishimoto T. Barbieri G. Pellegrini S. Sendtner M. Heinrich P.C. Horn F. Science. 1994; 263: 89-92Google Scholar). Phosphorylated STATs in turn undergo homo- and heterodimerization and translocate to the nucleus where they activate transcription of cytokine responsive genes (9Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Google Scholar). interleukin oncostatin M signal transducers and activators of transcription STAT binding element acute-phase response element 1,25-dihydroxyvitamin D3 base pair(s) 1,4-piperazinediethanesulfonic acid thymidine kinase soluble receptor sis-inducible element interferon kilobase pair(s). In mammals, the STAT family includes at least six members which are differentially activated by a variety of growth factors and cytokines (10Ihle J.N. Cell. 1996; 84: 331-334Google Scholar). Ligand-activated gp130-JAK complexes predominantly phosphorylate STAT3 (also known as acute-phase response factor or APRF) and to a lesser extent STAT1 (11Wegenka U.M. Buschmann J. Lutticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Google Scholar, 12Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Google Scholar, 13Lamb P. Seidel H.M. Haslam J. Milocco L. Kessler L.V. Stein R.B. Rosen J. Nucleic Acids Res. 1995; 23: 3283-3289Google Scholar). The cis-acting DNA sequences recognized by STAT complexes, termed STAT-binding elements (SBEs) have the general structure TT(N)5AA (9Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Google Scholar, 14Seidel H.M. Milocco L.H. Lamb P. Darnell Jr., J.E. Stein R.B. Rosen J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3041-3045Google Scholar). The sequence, and in some cases the size, of the spacer region between the palindromic TT-AA motif confers specificity for different STATs (11Wegenka U.M. Buschmann J. Lutticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Google Scholar, 14Seidel H.M. Milocco L.H. Lamb P. Darnell Jr., J.E. Stein R.B. Rosen J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3041-3045Google Scholar). An alternative sequence (CTGGGA), originally termed acute-phase response element (APRE), is required for IL-6 induction of many acute-phase response genes (3Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Google Scholar) and is found in the spacer region of some palindromic SBEs or in incomplete palindromic forms which may also bind STATs (11Wegenka U.M. Buschmann J. Lutticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Google Scholar, 15Zhang Z. Fuentes N.L. Fuller G.M. J. Biol. Chem. 1995; 270: 24287-24291Google Scholar). Intrigued by the evidence that IL-6 increases gp130 mRNA in vitro in human monocytes, epithelial cells, or hepatoma cells (16Schoester M. Heinrich P.C. Graeve L. FEBS Lett. 1994; 345: 131-134Google Scholar, 17Snyers L. Content J. Biochem. Biophys. Res. Commun. 1992; 185: 902-908Google Scholar, 18Schooltink H. Schmitz-Van de Leur H. Heinrich P.C. Rose-John S. FEBS Lett. 1992; 297: 263-265Google Scholar); that administration of IL-6 to mice produces a striking up-regulation of gp130 mRNA levels in several tissues (19Saito M. Yoshida K. Hibi M. Taga T. Kishimoto T. J. Immunol. 1992; 148: 4066-4071Google Scholar); and that several systemic hormones regulate gp130 expression (20Ogata A. Nishimoto N. Shima Y. Yoshizaki K. Kishimoto T. Blood. 1994; 84: 3040-3046Google Scholar, 21Romas E. Udagawa N. Zhou H. Tamura T. Saito M. Taga T. Hilton D.J. Suda T. Ng K.W. Martin T.J. J. Exp. Med. 1996; 183: 2581-2591Google Scholar, 22Bellido T. Girasole G. Passeri G. Jilka R.L. Manolagas S.C. J. Bone Miner. Res. 1994; 9 (abstr.): S123Google Scholar), 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. 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 (4Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Google Scholar) according to established methods (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar). DNA was purified from positive plaques and mapped by partial restriction enzyme digestion combined with Southern blot analysis (24Southern E.M. J. Mol. Biol. 1975; 98: 503-517Google Scholar). 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 (25Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Google Scholar) using sequence-derived primers (GenBank accession number U70617). Primer extension was performed using an oligonucleotide primer complementary to bases +116 to +136 of the first exon shown in Fig. 1 B. 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 mmKCl, 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, 8 m urea sequencing gel. The S1 nuclease assay was performed using a 32P-labeled, single-stranded DNA probe (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar) 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 mmNaCl, 50 mm sodium acetate, pH 4.5, 4.5 mmZnCl2, 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. colitRNA) and the products were ethanol precipitated and fractionated as described for the primer extension products. 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-γ, 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 × 104cells/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 β-galactosidase assay was performed using standard protocols (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar) and luciferase activity was divided by the β-galactosidase activity to normalize for transfection efficiency. 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 theSmaI 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.5 A). 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 (25Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Google Scholar). The herpesvirus thymidine kinase (tk) promoter/luciferase construct (ptk-LUC) was prepared by insertion of theBamHI/BglII fragment of pBLCAT2 (26Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Google Scholar), which spans positions −105 to +51 of the thymidine kinase gene, into theBglII 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 α2-macroglobulin, GATCCTTCTGGGAATTC. Plasmids with single or double insertions were identified by restriction enzyme analysis and confirmed by sequencing.Figure 7The gp130 SBE confers cytokine inducibility to a heterologous promoter. SBEs from the gp130, c-fos, or rat α2-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-γ 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. Theerror bars indicate the standard error of the mean from two experiments.View Large Image Figure ViewerDownload (PPT) Nuclear extracts were prepared from HepG2 cells using the method described by Sadowski and Gilman (27Sadowski H.B. Gilman M.Z. Nature. 1993; 362: 79-83Google Scholar). 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 mmNa4P2O7, 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 mmand 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 [α-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 mmNaCl, 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. A human fibroblast genomic DNA library was screened with a probe consisting of the extreme 5′-end of the cloned human gp130 cDNA (4Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Google Scholar). 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. 1 A. 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 (28Kidd V.J. Nesbitt J.E. Fuller G.M. Somatic Cell Mol. Genet. 1992; 18: 477-483Google Scholar) and that a pseudogene-like sequence was polymerase chain reaction amplified from chromosome 17 DNA (29Rodriguez C. Grosgeorge J. Nguyen V.C. Gaudray P. Theillet C. Cytogenet. Cell Genet. 1995; 70: 64-67Google Scholar). 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. 1 B) 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). 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. 1 B). 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-EBPβ-binding sites (3Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Google Scholar, 30Lasfar A. Wietzerbin J. Billard C. Eur. J. Immunol. 1994; 24: 124-130Google Scholar). 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)-γ and IL-6 activate overlapping sets of transcription factors and genes (31Yuan J. Wegenka U.M. Lutticken C. Buschmann J. Decker T. Schindler C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1994; 14: 1657-1668Google Scholar), 5-h treatment with IFN-γ stimulated the reporter construct to a level similar to that produced by IL-6. However, after 24 h of treatment, the response to IFN-γ was significantly decreased. 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. 5 A). 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. 5 A). 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. 5 B). However, the construct containing the mutated palindromic SBE consensus sequence was unresponsive to either cytokine (mt2 in Fig.5 B). Thus, the SBE-like sequence located between −381 and −191 bp is required for stimulation of the gp130 promoter by IL-6 and OSM. The sequence between the TT-AA motif of the gp130 cytokine response element is identical to the IL-6-response element from the human α2-macroglobulin promoter (32Krause E. Wegenka U. Moller C. Horn F. Heinrich P.C. Biol. Chem. Hoppe-Seyler. 1992; 373: 509-515Google Scholar) and to the c-fos SBE known as thesis-inducible element (SIE) (33Wagner B.J. Hayes T.E. Hoban C.J. Cochran B.H. EMBO J. 1990; 9: 4477-4484Google Scholar). The SIE binds STAT1 and STAT3 homo- and heterodimers (12Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Google Scholar, 34Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Google Scholar) and is required in vivo for the induction of the c-fos gene by a variety of stimuli (35Robertson L.M. Kerppola T.K. Vendrell M. Luk D. Smeyne R.J. Bocchiaro C. Morgan J.I. Curran T. Neuron. 1995; 14: 241-252Google Scholar). 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 α2-macroglobulin promoter, which contains the APRE consensus (CTGGGA), have been used as classical IL-6 response elements (12Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Google Scholar, 31Yuan J. Wegenka U.M. Lutticken C. Buschmann J. Decker T. Schindler C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1994; 14: 1657-1668Google Scholar, 34Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Google Scholar, 36Raz R. Durbin J.E. Levy D.E. J. Biol. Chem. 1994; 269: 24391-24395Google Scholar). In our studies we employed the latter for comparison purposes.Table IComparison of the gp130 SBE with SBEs from different mammalian promotersGeneSBEReferencegp130GCGTTACGGGAATCGThis reportHα2MCTCTTACGGGAATGG32Krause E. Wegenka U. Moller C. Horn F. Heinrich P.C. Biol. Chem. Hoppe-Seyler. 1992; 373: 509-515Google ScholarRα2MTCCTT CTGGGA ATTC43Kunz D. Zimmermann R. Heisig M. Heinrich P.C. Nucleic Acids Res. 1989; 17: 1121-1138Google Scholarc-fos(SIE)GATTGACGGGAACTG33Wagner B.J. Hayes T.E. Hoban C.J. Cochran B.H. EMBO J. 1990; 9: 4477-4484Google ScholarSIEm67GATTTACGGGAAATG33Wagner B.J. Hayes T.E. Hoban C.J. Cochran B.H. EMBO J. 1990; 9: 4477-4484Google ScholarSBE motifTTNNNNNAAThe following abbreviations were used: hα2M, human α2-macroglobulin; rα2M, rat α2-macroglobulin. The underlined sequence in the rα2M sequence corresponds to the previously described APRE (3Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Google Scholar). SIEm67 is the high affinity binding mutant of the SBE from the c-fospromoter. Open table in a new tab The following abbreviations were used: hα2M, human α2-macroglobulin; rα2M, rat α2-macroglobulin. The underlined sequence in the rα2M sequence corresponds to the previously described APRE (3Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Google Scholar). SIEm67 is the high affinity binding mutant of the SBE from the c-fospromoter. 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-γ induces predominantly STAT1 homodimers (12Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Google Scholar, 13Lamb P. Seidel H.M. Haslam J. Milocco L. Kessler L.V. Stein R.B. Rosen J. Nucleic Acids Res. 1995; 23: 3283-3289Google Scholar, 36Raz R. Durbin J.E. Levy D.E. J. Biol. Chem. 1994; 269: 24391-24395Google Scholar). In" @default.
• W2162214575 created "2016-06-24" @default.
• W2162214575 creator A5027634434 @default.
• W2162214575 creator A5032280425 @default.
• W2162214575 date "1997-06-01" @default.
• W2162214575 modified "2023-09-27" @default.
• W2162214575 title "Isolation and Characterization of the Human gp130 Promoter" @default.
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