Platelet-derived Growth Factor Induces Interleukin-6 Transcription in Osteoblasts through the Activator Protein-1 Complex and Activating Transcription Factor-2
1999; Elsevier BV; Volume: 274; Issue: 10 Linguagem: Inglês
10.1074/jbc.274.10.6783
ISSN1083-351X
AutoresNathalie Franchimont, Deena Durant, Sheila Rydziel, Ernesto Canalis,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoPlatelet-derived growth factor (PDGF) BB, a mitogen that stimulates bone resorption, increases the expression of interleukin-6 (IL-6), a cytokine that induces osteoclast recruitment. The mechanisms involved in IL-6 induction by PDGF BB are poorly understood. We examined the effect of PDGF BB on IL-6 expression in cultures of osteoblasts from fetal rat calvariae (Ob cells). PDGF BB increased IL-6 mRNA and heterogeneous nuclear RNA levels, the rate of transcription, and the activity of base pairs (bp) −2906 to +20 IL-6 promoter fragments transiently transfected into Ob cells. Deletion analysis revealed two responsive regions, one containing an activator protein-1 (AP-1) site located between bp −276 and −257, and a second, less well defined, downstream of −257. Targeted mutations of a cyclic AMP-responsive element (CRE), and nuclear factor-IL-6 and nuclear factor-κB binding sites in a bp −257 to +20 IL-6 construct that was transfected into Ob cells, revealed that the CRE also contributed to IL-6 promoter induction by PDGF BB. Electrophoretic mobility shift assay revealed AP-1 and CRE nuclear protein complexes that were enhanced by PDGF BB. Supershift assays revealed binding of Jun and Fos to AP-1 and CRE sequences and binding of activating transcription factor-2 to CRE. In conclusion, PDGF BB induces IL-6 transcription in osteoblasts by regulating nuclear proteins of the AP-1 complex and activating transcription factor-2. Platelet-derived growth factor (PDGF) BB, a mitogen that stimulates bone resorption, increases the expression of interleukin-6 (IL-6), a cytokine that induces osteoclast recruitment. The mechanisms involved in IL-6 induction by PDGF BB are poorly understood. We examined the effect of PDGF BB on IL-6 expression in cultures of osteoblasts from fetal rat calvariae (Ob cells). PDGF BB increased IL-6 mRNA and heterogeneous nuclear RNA levels, the rate of transcription, and the activity of base pairs (bp) −2906 to +20 IL-6 promoter fragments transiently transfected into Ob cells. Deletion analysis revealed two responsive regions, one containing an activator protein-1 (AP-1) site located between bp −276 and −257, and a second, less well defined, downstream of −257. Targeted mutations of a cyclic AMP-responsive element (CRE), and nuclear factor-IL-6 and nuclear factor-κB binding sites in a bp −257 to +20 IL-6 construct that was transfected into Ob cells, revealed that the CRE also contributed to IL-6 promoter induction by PDGF BB. Electrophoretic mobility shift assay revealed AP-1 and CRE nuclear protein complexes that were enhanced by PDGF BB. Supershift assays revealed binding of Jun and Fos to AP-1 and CRE sequences and binding of activating transcription factor-2 to CRE. In conclusion, PDGF BB induces IL-6 transcription in osteoblasts by regulating nuclear proteins of the AP-1 complex and activating transcription factor-2. Skeletal cells synthesize growth factors with important effects on the replication and the differentiated function of cells of the osteoblast and osteoclast lineages. Platelet-derived growth factor (PDGF) 1The abbreviations used are: PDGF, platelet-derived growth factor; MRE, multiple response element; PKC, protein kinase C; PKA, protein kinase A; IL, interleukin; AP, activator protein; bp, base pair(s); DMEM, Dulbecco's modified Eagle's medium; CRE, cyclic AMP-responsive element; CREB, CRE-binding protein; NF, nuclear factor; ATF, activating transcription factor; hnRNA, heterogeneous nuclear RNA; PCR, polymerase chain reaction.1The abbreviations used are: PDGF, platelet-derived growth factor; MRE, multiple response element; PKC, protein kinase C; PKA, protein kinase A; IL, interleukin; AP, activator protein; bp, base pair(s); DMEM, Dulbecco's modified Eagle's medium; CRE, cyclic AMP-responsive element; CREB, CRE-binding protein; NF, nuclear factor; ATF, activating transcription factor; hnRNA, heterogeneous nuclear RNA; PCR, polymerase chain reaction. is released by platelets following aggregation and is synthesized by osteoblasts and osteosarcoma cells (1Hart C.E. 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Acta Oncol. 1989; 28: 331-334Crossref PubMed Scopus (25) Google Scholar). Cells of the osteoblast lineage express the PDGF A gene and, to a lesser extent, the PDGF B gene, although PDGF BB has the greatest activity and has been studied more extensively for its biological properties (2Rydziel S. Ladd C. McCarthy T.L. Centrella M. Canalis E. Endocrinology. 1992; 130: 1916-1922Crossref PubMed Scopus (37) Google Scholar, 3Rydziel S. Canalis E. Endocrinology. 1996; 137: 4115-4119Crossref PubMed Scopus (17) Google Scholar, 7Centrella M. McCarthy T.L. Kusmik W.F. Canalis E. J. Cell. Physiol. 1991; 147: 420-426Crossref PubMed Scopus (62) Google Scholar). In bone, PDGF stimulates the replication of cells of the osteoblastic lineage, and it does not acutely enhance the differentiated function of the osteoblast (8Canalis E. McCarthy T.L. Centrella M. J. Cell. Physiol. 1989; 140: 530-537Crossref PubMed Scopus (168) Google Scholar, 9Centrella M. McCarthy T.L. Canalis E. Endocrinology. 1989; 125: 13-19Crossref PubMed Scopus (86) Google Scholar). PDGF also increases bone resorption, most likely by increasing the number of osteoclasts, an effect associated with an increase in the bone eroded surface (10Cochran D.L. Rouse C.A. Lynch S.E. Graves D.T. Bone. 1993; 14: 53-58Crossref PubMed Scopus (35) Google Scholar, 11Hock J.M. Canalis E. Endocrinology. 1994; 134: 1423-1428Crossref PubMed Scopus (138) Google Scholar). Although PDGF may act directly on skeletal cells, some effects may be mediated by other cytokines present in the bone microenvironment. Interleukin-6 (IL-6) is a multifunctional cytokine that enhances bone resorption by increasing osteoclast formation and recruitment (12Roodman G.D. J. Bone Miner. Res. 1992; 7: 475-478Crossref PubMed Scopus (293) Google Scholar, 13Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1282) Google Scholar). Hormones that increase bone resorption, such as parathyroid hormone, 1,25-dihydroxyvitamin D3, and IL-1 enhance IL-6 production in skeletal cells, indicating that it may be an important intermediary in their effects on bone resorption (14Feyen J.H. Elford P. Di Padova F.E. Trechsel U. J. Bone Miner. Res. 1989; 4: 633-638Crossref PubMed Scopus (220) Google Scholar, 15Franchimont N. Vrindts Y. Gaspar S. Lopez M. Gathy R. DeGroote D. Reginster J.Y. Franchimont P. Christiansen C. Riis B. Proceedings 1993: Fourth International Symposium on Osteoporosis and Consensus Development Conference. Handelstrykkeriet Aalborg ApS/Aalborg, Denmark, 1993: 249-250Google Scholar, 16Helle M. Brakenhoff J.P.J. DeGroot E.R. Aarden L.A. Eur. J. Immunol. 1988; 18: 957-959Crossref PubMed Scopus (350) Google Scholar, 17Littlewood A.J. Russell J. Harvey G.R. Hughes D.E. Russell R.G.G. Gowen M. Endocrinology. 1991; 129: 1513-1520Crossref PubMed Scopus (160) Google Scholar). 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Recently, we demonstrated that PDGF BB increases IL-6 synthesis in osteoblast cultures by activating protein kinase C (PKC) and calcium-dependent pathways (22Franchimont N. Canalis E. Endocrinology. 1995; 136: 5469-5475Crossref PubMed Google Scholar). The mechanisms involved were not determined, and the induction of IL-6 by PDGF BB may play a central role in the effects of PDGF on bone remodeling. In the present study, we examined the mechanisms involved in the induction of IL-6 expression by PDGF BB in cultures of osteoblast enriched cells from 22-day fetal rat calvariae (Ob cells). We also determined the regulatory elements of the rat IL-6 gene promoter responsible for the stimulatory effect of PDGF BB on IL-6 expression. The culture method used was described in detail previously (23McCarthy T.L. Centrella M. Canalis E. J. Bone Miner. Res. 1988; 3: 401-408Crossref PubMed Scopus (186) Google Scholar). Parietal bones were obtained from 22-day old fetal rats immediately after the mothers were sacrificed by blunt trauma to the nuchal area. This project was approved by the Animal Care and Use Committee of Saint Francis Hospital and Medical Center. Cells were obtained by five sequential digestions of the parietal bone, using bacterial collagenase (CLS II, Worthington Biochemical Corp., Freehold, NJ). Cell populations harvested from the third to the fifth digestions were previously shown to express osteoblastic characteristics and were cultured as a pool (23McCarthy T.L. Centrella M. Canalis E. J. Bone Miner. Res. 1988; 3: 401-408Crossref PubMed Scopus (186) Google Scholar). Ob cells were plated at a density of 8000–12,000 cells/cm2 and cultured in a humidified 5% CO2 incubator at 37 °C, maintaining a pH of 7.5. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with nonessential amino acids (Life Technologies, Inc.), and 10% fetal bovine serum (Summit, Fort Collins, CO). For RNA and nuclear protein analyses, cells were grown to confluence (∼50,000 cells/cm2), transferred to serum-free medium for 20–24 h, and then exposed to test agents in serum-free medium for 30 min to 4 h as indicated in the text and figure legends. For the nuclear run-on experiment, subconfluent cultures of Ob cells were trypsinized, subcultured at a 1:8 dilution, and grown to confluence in DMEM supplemented with 10% fetal bovine serum. Cells were serum-deprived for 24 h and treated for 15 and 30 min in serum-free DMEM. For transient transfections, cells were grown to 70% confluence, transfected in 10% fetal bovine serum, serum-deprived, and treated. Recombinant human PDGF BB (Austral, San Ramon, CA) was added directly to the culture medium. Cycloheximide (Sigma) was dissolved in ethanol and diluted 1:1000 in culture medium; an equal amount of ethanol was added to control cultures. Total cellular RNA was extracted with the RNeasy Kit per the manufacturer's instructions (Qiagen, Chatsworth, CA). The RNA recovered was quantitated by spectrophotometry, and equal amounts of RNA from control or test samples were loaded on a formaldehyde agarose gel following denaturation. The gel was stained with ethidium bromide to visualize ribosomal RNA (rRNA), documenting equal RNA loading of the various experimental samples. RNA was then blotted onto Gene Screen Plus charged nylon membrane (DuPont), and the uniformity of transfer was documented by revisualization of rRNA. A 900-base pair (bp)BamHI-PstI restriction fragment of rat IL-6 cDNA, and a 700-bp BamHI-SphI fragment of a mouse 18 S rRNA cDNA clone (both from ATCC, Rockville, MD) were purified by agarose gel electrophoresis (24Northemann W. Braciak T.A. Hattori M. Lee F. Fey G.H. J. Biol. Chem. 1989; 264: 16072-16082Abstract Full Text PDF PubMed Google Scholar). IL-6 and 18 S rRNA cDNAs were labeled with [α-32P]dATP and [α-32P]dCTP (50 μCi each at a specific activity of 3000 Ci/mmol; DuPont) using the random hexanucleotide primed second strand synthesis method (25Feinberg A.P. Vogelstein B. Anal. Biochem. 1984; 137: 266-267Crossref PubMed Scopus (5193) Google Scholar). Hybridizations were carried out at 42 °C for 16–48 h, and posthybridization washes were performed at 65 °C in 1× SSC for IL-6 and 0.1× SSC for 18 S. The bound radioactive material was visualized by autoradiography on Kodak X-AR5 film (Eastman Kodak), employing Cronex Lightning Plus intensifying screens (DuPont). To examine changes in IL-6 hnRNA, a sense strand intron 2-specific amplimer, 5′-GAATTGGGAATTCTCTGCTG-3′ and an antisense strand intron 2 amplimer, 5′-GAAGGCCAAGAGATCTTACT-3′, were synthesized in accordance with published sequences (24Northemann W. Braciak T.A. Hattori M. Lee F. Fey G.H. J. Biol. Chem. 1989; 264: 16072-16082Abstract Full Text PDF PubMed Google Scholar). IL-6 hnRNA levels were determined by reverse transcription-PCR (26Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology: Preparation and Analysis of RNA. John Wiley and Sons Inc., New York1995: 4.10.5-4.10.9Google Scholar). For this purpose, total RNA from control and test samples was prepared as described for Northern analysis. One μg of RNA was treated with amplification grade DNase I and reverse-transcribed in the presence of the IL-6 intron 2 specific antisense primer at 42 °C for 30 min with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). The newly transcribed cDNA was amplified by 22 PCR cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min in the presence of the IL-6 intron 2-specific sense primer described, Taq DNA polymerase, and 5 μCi of [α-32P]dATP (3000 Ci/mmol, DuPont). The PCR products were fractionated by electrophoresis on a 6% polyacrylamide denaturing gel and visualized by autoradiography. An internal DNA standard at a concentration of 20 fg was included in the PCR to correct for variations in amplification. The standard was obtained by amplification of SV40 promoter sequences in the pGL2-Basic plasmid DNA (Promega, Madison, WI) using the composite sense primer 5′-GAATTGGGAATTCTCTGCTGattagtcagcaaccatagtc-3′ and the antisense primer 5′-GAAGGCCAAGAGATCTTACTggttccatcctctagaggat-3′. The uppercase letters indicate IL-6 sequences, and the lowercase letters represent pGL2-Basic plasmid sequences. To examine changes in the rate of transcription, nuclei were isolated by Dounce homogenization in a Tris buffer, pH 7.4, containing 0.5% Nonidet P-40 (27Greenberg M.E. Ziff E.B. Nature. 1984; 311: 433-438Crossref PubMed Scopus (2010) Google Scholar). Nascent transcripts were labeled by incubation of nuclei in a reaction buffer containing 500 μμ each adenosine, cytidine, and guanosine triphosphates, 150 units of RNasin (Promega), and 250 μCi [α-32P]UTP (800 Ci/mmol, DuPont) (27Greenberg M.E. Ziff E.B. Nature. 1984; 311: 433-438Crossref PubMed Scopus (2010) Google Scholar). RNA was isolated by treatment with DNase I and proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. Linearized plasmid DNA containing 1 μg of cDNA was immobilized onto GeneScreen Plus by slot blotting according to the manufacturer's directions (DuPont). The plasmid vector pGL2-Basic (Promega) was used as a control for nonspecific hybridization, and a mouse 18 S rRNA cDNA clone was used to estimate uniformity of the loading. Equal counts per min of [32P]RNA from each sample were hybridized to cDNAs at 42 °C for 72 h and washed in 1× SSC at 58 °C for 30 min. Hybridized cDNAs were visualized by autoradiography. To determine changes in promoter activity, chimeric constructs of the 5′ flanking region of the IL-6 promoter and a luciferase reporter gene (kindly provided by Dr. G. Fey, Erlanghen, Germany) were tested (28Baffet G. Braciak T.A. Fletcher R.G. Gauldie J. Fey G.H. Northemann W. Mol. Biol. Med. 1991; 8: 141-156PubMed Google Scholar, 29Franchimont N. Rydziel S. Canalis E. J. Clin. Invest. 1997; 100: 1797-1803Crossref PubMed Scopus (52) Google Scholar). Ob cells were cultured to approximately 70% confluence and transiently transfected by calcium phosphate-DNA co-precipitation as described (29Franchimont N. Rydziel S. Canalis E. J. Clin. Invest. 1997; 100: 1797-1803Crossref PubMed Scopus (52) Google Scholar). Cotransfection with a construct containing the cytomegalovirus promoter-driven β-galactosidase gene (CLONTECH, Palo Alto, CA) was used to control for transfection efficiency. After 4 h, cells were exposed for 3 min to 10% glycerol. Ob cells were allowed to recover in serum-containing DMEM for 24 h, serum-deprived for 20–24 h, and exposed to control or test medium for 1–4 h as described in the text and figure legends. Cells were washed with phosphate buffered saline and harvested in reporter lysis buffer (Promega). Luciferase activity was measured using a luciferase assay kit (Promega), and β-galactosidase activity was measured using Galacton reagent (Tropix, Bedford, MA), both in accordance with manufacturer's instructions. Luciferase activity was corrected for β-galactosidase activity, and data are expressed as treated:control ratios. Site-directed mutagenesis was performed by using the method of gene splicing by overlap extension or using the Morph Mutagenesis kit (5 Prime → 3 Prime, Inc., Boulder, CO) (30Horton R.M. White B.A. Methods in Molecular Biology. 15. Humana Press Inc., Totowa, NJ1993: 251-261Google Scholar). The Morph Mutagenesis kit was used in accordance with the manufacturer's instructions to create targeted mutations of the cyclic AMP-responsive element (CRE), within the multiple response element (MRE), and of the nuclear factor for IL-6 (NF-IL-6) binding site. Gene splicing by overlap extension was used to create mutations of the nuclear factor-κB (NF-κB) site. For this purpose, wild-type and mutant constructs were generated by PCR using a 5′ primer containing a sequence corresponding to bp −257 to −249 of the rat IL-6 promoter (5′-AGGCGAGCTCAAAGAAAGA-3′) and a 3′ primer that included bp +6 to +20 of the rat IL-6 gene (5′-CCGCTCGAGACAGAATGA-3′). A chimeric construct containing bp −257 to + 20 of the IL-6 promoter was used as a template. Mutant sense and antisense primers, used to synthesize the intermediate products, contained two or three altered bases, creating the desired targeted mutations (29Franchimont N. Rydziel S. Canalis E. J. Clin. Invest. 1997; 100: 1797-1803Crossref PubMed Scopus (52) Google Scholar). Newly synthesized wild-type and mutated IL-6 promoter fragments were cloned into the plasmid pGL3-Basic (Promega), containing the luciferase reporter gene. Sequences of the wild-type and mutated constructs generated either by the Morph Mutagenesis kit or by PCR were confirmed by DNA sequence analysis using the Sequenase Version 2.0 DNA sequencing kit (United States Biochemical Corp., Cleveland, OH). For gel shift assays, nuclear extracts from control and treated cultures were prepared as described (31Scott V. Clark A.R. Docherty K. Harwood A.J. Methods in Molecular Biology. 31. Humana Press Inc., Totowa, NJ1994: 339-347Google Scholar). Cells were washed with phosphate buffered saline, suspended in Hepes/KOH, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol buffer, allowed to swell on ice for 15 min, and lysed with 10% Nonidet P-40 (Sigma). Following centrifugation, the nuclear pellet was resuspended in a Hepes/KOH buffer in the presence of protease inhibitors at 4 °C, incubated for 30 min, and centrifuged, and the supernatant stored at −70 °C. Protein concentrations were determined by DC protein assay in accordance with the manufacturer's instructions (Bio-Rad). Synthetic oligonucleotides (Life Technologies, Inc., or National Biosciences Inc., Plymouth, MN) were labeled with [γ-32P]ATP using polynucleotide kinase. Nuclear extracts and labeled oligonucleotides were incubated for 20 min at room temperature in a 10 mm Tris buffer (pH 7.5), containing 1 μg of poly(dI-dC). The specificity of binding was determined by the addition of homologous or mutated unlabeled synthetic oligonucleotides in 100-fold excess (31Scott V. Clark A.R. Docherty K. Harwood A.J. Methods in Molecular Biology. 31. Humana Press Inc., Totowa, NJ1994: 339-347Google Scholar). DNA-protein complexes were resolved on nondenaturing, nonreducing 4% polyacrylamide gels, and the complexes were visualized by autoradiography. For gel supershift assays, rabbit affinity purified antibodies to Fos and Jun family of nuclear factors, activating transcription factor-2 (ATF-2), and other cyclic AMP response element-binding proteins (CREB-1, CREM-1, ATF-1, ATF-3, and ATF-4) (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated for 1 h with nuclear extracts at room temperature, prior to the addition of synthetic oligonucleotides and electrophoretic resolution. Data are presented as means ± S.E. Statistical differences were determined by analysis of variance and post hoc examination by the Ryan-Einot-Gabriel-Welch F test (32Wall F.J. Statistical Data Analysis Handbook. 1st Ed. McGraw-Hill, New York1986Google Scholar). Northern blot analysis of total RNA from serum-deprived confluent Ob cells revealed limited expression of IL-6 transcripts. Confirming our initial observation, PDGF BB at 3.3 nm caused a marked time-dependent stimulation of IL-6 mRNA levels, which was maximal after 60 min (Fig. 1). To determine whether the induction of IL-6 was dependent on protein synthesis, Ob cells were treated with PDGF BB in the presence or absence of the protein synthesis inhibitor, cycloheximide at 3.6 μm. Cycloheximide did not prevent the effect of PDGF BB (Fig. 2). To analyze the mechanisms involved in the regulation of IL-6, we examined the effects of PDGF BB on IL-6 hnRNA and on the rate of transcription of the IL-6 gene. PDGF BB markedly increased IL-6 hnRNA after 30 min, and the effect was sustained for 120 min (Fig. 3). PDGF BB at 3.3 nm increased the rate of IL-6 transcription, as determined by a nuclear run-on assay on nuclei from Ob cells, by 2–3-fold after 15 min (not shown) and by 6-fold after 30 min (Fig.4).Figure 2Effect of PDGF BB at 3.3 nm on IL-6 mRNA levels, in the presence or absence of cycloheximide (Cx) at 3.6 μm, in cultures of Ob cells treated for 1 h. Total RNA from control (C) or PDGF BB-treated (BB) cells was subjected to Northern blot analysis and hybridized with an α-32P-labeled IL-6 cDNA. IL-6 transcripts of 1.2 and 2.4 kilobase pairs were visualized by autoradiography. Blots were stripped and rehybridized with an α-32P-labeled 18 S rRNA cDNA. IL-6 transcripts are shown in the upper panel, and 18 S rRNA transcripts are shown in the lower panel.View Large Image Figure ViewerDownload (PPT)Figure 3Effect of PDGF BB at 3.3 nm on IL-6 hnRNA levels in cultures of Ob cells treated for 30–240 min.Total RNA from control (C) or PDGF BB-treated (BB) cells was extracted, and 1 μg was subjected to reverse transcription-PCR in the presence of IL-6 sense and antisense intron 2 primers and α-32P-dATP. Reverse transcription-PCR products were fractionated by polyacrylamide gel electrophoresis and visualized by autoradiography. Internal standard (std) is shown at the top of the blot and IL-6 hnRNA at the bottom.View Large Image Figure ViewerDownload (PPT)Figure 4Effect of PDGF BB at 3.3 nm on IL-6 transcription rates in cultures of Ob cells treated for 30 min. Nascent transcripts from control (C) or PDGF BB (BB)-treated cultures were labeled in vitro with [α-32P]UTP, and the labeled RNA was hybridized to immobilized cDNA for IL-6. 18 S rRNA cDNA was used to demonstrate loading, and pGL2-Basic (pGL2-B) vector DNA was used as a control for nonspecific hybridization.View Large Image Figure ViewerDownload (PPT) To define gene elements responsible for the PDGF BB effect, Ob cells were transiently transfected with chimeric constructs containing fragments of the IL-6 promoter linked to the luciferase reporter gene. The effects of PDGF BB at 3.3 nm were tested initially for 1–4 h on a bp −276 to +20 fragment of the rat IL-6 promoter. After 2 h, PDGF BB caused a 3.5-fold increase in promoter activity, an effect sustained for 4 h (Fig. 5). To characterize the regulatory elements involved, 5′ deletion constructs of the IL-6 promoter ranging from bp −2906 to +20 to bp −34 to +20 were tested in six independent experiments. 5′ Deletions from bp −2906 to bp −276 of the IL-6 promoter resulted in a decrease in basal activity (not shown) but did not preclude the response to PDGF BB, which induced the bp −276 to +20 promoter construct by about 3-fold (Fig. 6). Deletion to bp −257, with the consequent removal of an AP-1 site, resulted in a partial loss of the stimulatory effect by PDGF BB, so that it increased the activity of the bp −257 to +20 IL-6 construct 2-fold (Fig. 6). These results suggest that the AP-1 site between −276 and −257 is in part responsible for the effect of PDGF BB, and that additional sequences contained in the bp −257 to +20 region of the IL-6 promoter play a role in the response to PDGF BB. To define additional elements responsible for the regulation of IL-6 by PDGF BB, a bp −257 to +20 fragment of the IL-6 promoter was cloned into pGL3-Basic, and targeted mutations of the known consensus sequences of CRE, NF-IL-6, and NF-κB binding sites were made and tested. PDGF BB at 3.3 nmincreased the activity of the bp −257 to +20 wild-type IL-6 construct by 1.5–2-fold (Fig. 7). Targeted mutations of NF-IL-6 and NF-κB did not result in a change in the response to PDGF BB. In contrast, a mutation of the CRE resulted in a total loss of the IL-6 promoter response to PDGF BB (Fig. 7).Figure 6Effect of PDGF BB on IL-6 promoter activity in transiently transfected Ob cells. Ob cells were transfected by calcium phosphate-DNA co-precipitation with chimeric constructs containing fragments spanning the −2906 to +20 region of the rat IL-6 promoter sequence linked to a luciferase reporter gene. The 5′ deletion end points of the IL-6 promoter are indicated immediately under thecolumns, and a diagram with selected putativecis-regulatory elements present in the −276 to +20 region of the IL-6 promoter is depicted at the bottom. To control for transfection efficiency, a cytomegalovirus-β-galactosidase expression vector was co-transfected. 24 h after transfection, Ob cells were serum-deprived for 20–24 h and exposed to control medium (white columns) or PDGF BB at 3.3 nm(black columns) for 4 h. Cells were harvested, and luciferase activity was corrected for β-galactosidase activity and expressed as treated:control ratios for each construct, following normalization of control values to 100%. Values are means ± S.E. of 12–21 observations, pooled from six independent experiments. *, significantly different from the respective control (p< 0.05).View Large Image Figure ViewerDownload (PPT)Figure 7Effect of PDGF BB on IL-6 promoter activity in transiently transfected Ob cells. Ob cells were transfected by calcium phosphate-DNA co-precipitation, with a chimeric construct containing a bp −257 to +20 fragment of the IL-6 promoter, without (wild-type (WT)) or with mutations of CRE, NF-IL-6, and NF-κB as indicated under the columns. A diagram of the −257 to +20 region of the IL-6 promoter outlining the location and sequence of the three binding sites examined is depicted at thebottom. Arrows point to mutations used to alter CRE, NF-IL-6, and NF-κB consensus sequences. Constructs were linked to a luciferase reporter gene. To control for transfection efficiency, a cytomegalovirus-β-galactosidase expression vector was co-transfected. 24 h after transfection, Ob cells were serum-deprived for 20–24 h and exposed to control medium (white columns) or PDGF BB at 3.3 nm (black columns) for 4 h. Cells were harvested, and luciferase activity was corrected for β-galactosidase activity and expressed as treated:control ratios for each construct following normalization of control values to 100%. Values are means ± S.E. of 12 observations pooled from two experiments. *, significantly different from the respective control (p < 0.05).View Large Image Figure ViewerDownload (PPT) To confirm the functional data, nuclear extracts, obtained from control and PDGF BB-treated cells, were incubated with radiolabeled oligonucleotides containing AP-1 and CRE/MRE sequences in the context of the IL-6 promoter. Binding studies revealed increased protein binding to AP-1 sequences in nuclear extracts from PDGF BB-treated cultures (Fig. 8). Unlabeled homologous oligonucleotides prevented, whereas mutated oligonucleotides did not prevent, the binding of the 32P-labeled sequences to nuclear proteins.
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