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Vascular Endothelial Growth Factor Induction by Prostaglandin E2 in Human Airway Smooth Muscle Cells Is Mediated by E Prostanoid EP2/EP4 Receptors and SP-1 Transcription Factor Binding Sites *

Vascular Endothelial Growth Factor Induction by Prostaglandin E2 in Human Airway Smooth Muscle... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 34, Issue of August 26, pp. 29993–30000, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Vascular Endothelial Growth Factor Induction by Prostaglandin E in Human Airway Smooth Muscle Cells Is Mediated by E Prostanoid EP /EP Receptors and SP-1 Transcription Factor Binding Sites* 2 4 Received for publication, December 23, 2004, and in revised form, May 19, 2005 Published, JBC Papers in Press, June 21, 2005, DOI 10.1074/jbc.M414530200 Dawn Bradbury‡, Deborah Clarke‡, Claire Seedhouse§, Lisa Corbett‡, Joanne Stocks‡, and Alan Knox‡ From the ‡Division of Respiratory Medicine, §Academic Haematology, University of Nottingham, City Hospital, Nottingham NG5 1PB, United Kingdom a critical role in physiological and pathological angiogenesis in Prostaglandin E (PGE ) can increase vascular endo- 2 2 thelial growth factor A (VEGF-A) production but the most biological systems (2). VEGF is implicated in tumor neo- mechanisms involved are unclear. Here we character- vascularization and in angiogenesis associated with a number ized the transcriptional mechanisms involved in human of chronic inflammatory diseases, such as asthma, chronic ob- airway smooth muscle cells (HASMC). PGE increased 2 structive pulmonary disease, inflammatory bowel disease, VEGF-A mRNA and protein but not mRNA stability. rheumatoid and osteoarthritis (3–7). VEGF is secreted by a PGE stimulated the activity of a transiently transfected variety of cell types, but not by endothelial cells themselves and 2068-bp (2018 to 50) VEGF-A promoter-driven lucifer- mesenchymal cells serve as an important source of VEGF in a ase construct. Functional 5 deletional analysis mapped number of inflammatory and neoplastic processes (8). There the PGE response element to the 135-bp sequence (85/ are at least five members of the VEGF family including pla- 50) of the human VEGF-A promoter. PGE -induced lu- cental growth factor, VEGF-A, VEGF-B, VEGF-C, and ciferase activity was reduced in cells transfected with a VEGF-D (9). The most potent angiogenic factor in vivo is 135-bp VEGF promoter fragment containing mutated VEGF-A, which has six splice variants: 121, 145, 165, 183, 189, Sp-1 binding sites but not in cells transfected with a and 204 amino acids (10). construct containing mutated EGR-1 binding sites. Elec- A number of stimuli are capable of increasing VEGF release trophoretic mobility shift assay and chromatin immuno- in different biological systems. Inflammatory cytokines such as precipitation assay confirmed binding of Sp-1 to the interleukin-1 and transforming growth factor- increased VEGF promoter. PGE increased phosphorylation of VEGF release in human cholangiocellular carcinoma cells, sy- Sp-1 and luciferase activity of a transfected Sp-1 re- porter construct. PGE receptor agonists EP (ONO-AE1 novial fibroblasts, cardiac myocytes, and airway smooth muscle 2 2 259) and EP (ONO-AE1 329) mimicked the effect of cells (11–14). We have previously shown that the pro-inflam- PGE , and reverse transcription-PCR, Western blotting, matory asthma mediator, bradykinin, increased VEGF produc- and flow cytometry confirmed the presence of EP and 2 tion in human airway smooth muscle cells (HASMC) (15). A EP receptors. VEGF protein release and Sp-1 reporter 4 number of studies have shown that the products of COX-2, the activity were increased by forskolin and isoproterenol, inducible form of cyclooxygenase, may mediate the effect of which increase cytosolic cAMP, and the cAMP analogue, cytokines and mediators on the release of chemokines and 8-bromoadenosine-3,5-cyclophosphoric acid. These cytokines in an autocrine manner through a mechanism involv- studies suggest that PGE increases VEGF transcrip- ing endogenous prostanoid production. Recent work suggests tionally and involves the Sp-1 binding site via a cAMP- that this is also true of VEGF. Autocrine PGE increases VEGF dependent mechanism involving EP and EP receptors. 2 4 release in response to interleukin-1 in synovial fibroblasts and in response to bradykinin in HASMC (12, 15). Further- more, exogenous PGE increases VEGF expression in fibro- Vascular endothelial growth factor (VEGF) is a 45-kDa he- blasts and osteoblasts (16–18). These studies are consistent parin-binding homodimeric glycoprotein that is an important with the known role of COX products in angiogenesis (19, 20): growth and survival factor for endothelial cells (1). VEGF plays COX-2-derived thromboxane A , prostacyclin, and PGE stim- 2 2 ulate endothelial cell migration and angiogenesis (21), whereas COX inhibitors have protective effects on angiogenesis in ex- * This work was supported by Asthma UK. The costs of publication of this article were defrayed in part by the payment of page charges. This perimental models (22, 23). In asthma both COX-2 and VEGF article must therefore be hereby marked “advertisement” in accordance are increased but the two have not been firmly linked (24, 25). with 18 U.S.C. Section 1734 solely to indicate this fact. Collectively these studies suggest that PGE can contribute To whom correspondence should be addressed: Division of Respira- to angiogenesis via increased VEGF production but the molec- tory Medicine, Clinical Science Bldg., City Hospital, Hucknall Rd., Nottingham NG5 1PB, United Kingdom. Tel.: 44-115-8404775; Fax: ular mechanisms involved have not been studied in detail, 44-115-8404771; E-mail: alan.knox@nottingham.ac.uk. particularly the balance between transcriptional and post-tran- The abbreviations used are: VEGF, vascular endothelial growth scriptional events. factor: HASMC, human airway smooth muscle cells; EMSA, electro- The VEGF promoter contains the hypoxia response element, phoretic mobility shift assay; ChIP, chromatin immunoprecipitation; 8-Br-cAMP, 8-bromoadenosine 3,5-cyclophosphoric acid; COX, cy- hypoxia inducible factor-1, p53/Von Hippel Lindau, NFB, clooxygenase: RT, reverse transcription; PGE , prostaglandin E ; EP, E 2 2 and AP-1 as well as several potential transcription factor bind- prostanoid; ELISA, enzyme-linked immunosorbent assay; PKA, protein ing sites for Sp-1 and AP-2 (26). PGE binds to G protein- kinase A; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3- coupled membrane receptors, the E prostanoid (EP) receptors. phosphate dehydrogenase; PKA, protein kinase A; AP, activator pro- tein; EGR, early growth response. Four subtypes of EP receptors have been described, EP ,EP , 1 2 This paper is available on line at http://www.jbc.org 29993 This is an Open Access article under the CC BY license. 29994 PGE Induces VEGF via Sp-1 72 °C for 1 min, and a final extension of 72 °C for 10 min. The following EP , and EP , encoded by different genes (27). Each subtype is 3 4 primers were used: EP -R sense 5-TCCAATGACTCCCAGTCTGAGG- tissue-specific and uses different intracellular signaling mech- A-3, antisense 5-TCAAAGGTCAGCCTGTTTAC-3;EP -R sense 5-T- anisms suggesting potentially different inflammatory re- CTGACCTCGGTGTCCAAAAATCG-3, antisense 5-TGGGTACTGCA- sponses depending on receptor subtype binding (28). The re- GCCGCGAGCTA-3 (33), and GAPDH sense 5-CCACCCATGGCAAA- ceptor used by PGE to increase VEGF is unknown. 2 TTCCATGGCA-3, GAPDH antisense 5-TCTAGACGGCAGGT- Here we determined the molecular mechanisms involved in CAGGTCCACC-3. The PCR products were visualized by electrophoresis on a 2% agar- the transcriptional regulation of the VEGF promoter by exog- ose gel in 0.5 TBE buffer after staining with 0.5 g/ml ethidium enous PGE in human airway smooth muscle cells. Mutational bromide. The ultraviolet (UV)-illuminated gels were photographed, and and deletional analysis of the VEGF promoter showed that the densitometry was analyzed using a GeneGenius gel documentation Sp-1 transcription factor binding was essential for the increase and analysis system (Syngene, Cambridge, UK). in VEGF promoter activity produced by PGE . PGE caused Quantitative Real-time RT-PCR—VEGF-A expression was deter- 2 2 phosphorylation of Sp-1 and electrophoretic mobility shift as- mined using primer sequences: sense 5-CTTGCCTTGCTGCTCTAC- C-3 and antisense 5-CACACAGGATGGCTTGAAG-3 (34).  -Micro- say (EMSA), and chromatin immunoprecipitation (ChIP) dem- 2 globulin was used as the housekeeping gene (35). 1 ng of reverse- onstrated that PGE increased Sp-1 binding to the VEGF pro- transcribed cDNA was subjected to real-time PCR using Excite Real- moter. Furthermore, studies with EP and EP receptor 2 4 time Mastermix with SYBR green (Biogene, Cambridge, UK) and the subtype agonists, the cAMP analogue 8-bromoadenosine-3,5- ABI Prism 7700 detection system (Applied Biosystems, Warrington, cyclophosphoric acid (8-Br-cAMP), forskolin, which increases Cheshire, UK). Each reaction consisted of 1 Excite mastermix, SYBR adenylyl cyclase activity, and the 2 receptor agonist isoprot- green (1:60000 final concentration), 40 nM of both sense and antisense primers, 1.6 l of DNA (or dH O), and H O to a final volume of 20 l. erenol, showed that PGE -induced activation of Sp-1 was me- 2 2 Thermal cycler conditions included incubation at 95 °C for 10 min diated by EP and EP receptors via cAMP. 2 4 followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Integration of the fluorescent SYBR green into the PCR product was monitored MATERIALS AND METHODS after each annealing step. Amplification of one specific product was Cell Culture—Human tracheas were obtained from three post-mor- confirmed by melting curve analysis, where a single melting peak tem individuals. Primary cultures of human ASM cells were prepared eliminated the possibility of primer-dimer association. For melting from explants of ASM according to methods previously reported (29, curve analysis to be performed the products were heated from 60 to 30). Cells at passage 6 were used for all experiments. We have previ- 95 °C over 20 min after the 40 cycles. ously shown that cells grown in this manner depict the immunohisto- To enable the levels of transcripts to be quantified, standard curves chemical and light microscopic characteristics of typical ASM cells (30). were generated using serial dilutions of KG1a cDNA. Negative controls Experimental Protocol—The cells were cultured to confluence in 24- consisting of no template were included, and all reactions were set up in well culture plates in a humidified, 5% CO , 37 °C incubator using 2 triplicate. VEGF-A expression was normalized to the housekeeping Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% gene by dividing the mean of the VEGF-A triplicate value by the mean fetal calf serum (Seralab, Crawly Down, Sussex, UK), 100 units/ml of the  -microglobulin triplicate value. This was then expressed as penicillin, 100 g/ml streptomycin, 4 mML-glutamine, and 2.5 g/ml -fold increase over unstimulated cells at each time point. amphotericin B (Sigma). The cells were growth-arrested in serum-free Flow Cytometric Analysis of EP Receptors—HASMC were detached medium for 24 h prior to experiments. Immediately before each exper- using a sterile scraper, washed, and incubated with polyclonal rabbit iment, fresh serum-free medium containing PGE or ethanol vehicle anti-human EP -R (Cayman Chemical, Ann Arbor, MI). The cells were (Sigma) was added. In time course experiments cells were incubated washed twice and incubated with fluorescein isothiocyanate-conjugated with 1 M PGE for 2–24 h. In the concentration response experiments goat anti-rabbit secondary antibody (Sigma). Preimmune rabbit serum cells were incubated for 24 h with 1 nM to 10 M PGE . In subsequent was used as the negative control (Sigma). Using the FACSCalibur flow experiments, 24-h incubation times were used. At the indicated times, cytometer (BD Biosciences) and logarithmic amplification of the green the culture media were harvested and stored at 20 °C. The highly fluorescence channel (FL-1), 10,000 events were acquired and analyzed selective EP and EP receptor subtype agonists ONO-AE1 259 and with CellQuest software (BD Biosciences). 2 4 ONO-AE1 329, which were a gift from ONO Pharmaceuticals, Osaka, Western Blotting—The nuclear protein fractions were prepared using Japan, were used in the PGE receptor studies (31, 32). Nu-Clear extraction kit (Sigma) following the manufacturer’s protocol. The cAMP analogue 8-Br-cAMP, the PKA inhibitor H-89, and fors- Western blotting was performed as described previously using a specific kolin and isoproterenol, which increase cytosolic cAMP, were purchased polyclonal rabbit anti-human EP receptor antibody (Cayman Chemi- from Sigma. Mithramycin was purchased from Tocris Cookson Ltd. cal) or mouse monoclonal anti-human Sp1 antibody (1C6; Santa Cruz (Avonmouth, Bristol, UK). The cells were preincubated for 1 h with Biotechnology, Santa Cruz, CA) and horseradish peroxidase-conjugated the inhibitors. secondary antibody (DakoCytomation, Ely, Cambridgeshire, UK) (33). VEGF-A Assay—The enzyme-linked immunosorbent assay (ELISA) The human histiocytic lymphoma cell line U937, which is known to was used to measure VEGF-A (R&D Systems, Abingdon, Oxon, UK) express EP receptors, was used as the positive control. The human T according to the manufacturer’s instructions and has been described by lymphoblastic leukemia cell line Jurkat was used as a positive Sp-1 us in detail elsewhere (15). control. RNA Isolation and Reverse Transcription-Polymerase Chain Reac- Cell Viability—The toxicity of all the chemicals and vehicles used in tion (RT-PCR)—Cells in 6-well plates were treated with PGE and this study was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- collected at time 0, 1, 2, 4, 8, and 24 h, respectively. Total RNA was tertrazolium bromide assay (29). At the end of the experiment the isolated by using the RNeasy mini kit (Qiagen, West Sussex, UK) culture media was removed and replaced with 250 l of media contain- following the manufacturer’s protocol with on-column DNase digestion. ing 1 mg/ml thiazolyl blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylter- 1 g of total RNA was reverse transcribed in a total volume of 20 l trazolium bromide (Sigma) and incubated for 20 min in 37 °C. This including 200 units of Moloney murine leukemia virus reverse tran- medium was removed and 250 lofMe SO was added to solubilize the scriptase (Promega, Madison, WI), 25 units of RNase inhibitor (Pro- blue-colored tetrazolium. The optical density was read at 550 nm in a mega), 0.5 g of oligo(dT) primer, 0.5 mM of each dNTPs, and 1 TECAN GENios (Tecan UK Limited, Theale, Reading, UK) microplate first-strand buffer provided by Promega. The reaction was incubated at reader. Viability was set as 100% in control cells. 42 °C for 90 min. Transfection with VEGF Promoter-driven Luciferase Constructs and Aliquots of the RT products were subsequently used for PCR ampli- Sp-1 Reporter Luciferase Construct—Cells were cultured in 24-well fication. 10 l of RT products was brought to a volume of 50 l contain- plates to confluence, growth arrested for 24 h, and transfected using 1 ing1mM MgCl , 0.12 mM of each dTNPs, 1 unit of Taq polymerase l of LF2000 (Lipofectamine LF2000, Invitrogen) and 0.8 g of DNA per (Promega), 0.5 M of both the upstream and downstream PCR primers, well according to the manufacturer’s instructions. The cells were co- and 1 PCR buffer, provided by Promega. transfected with 1 ng/well of the internal control plasmid pRL-SV40 Amplification was carried out with a PTC-100 programmable (Promega UK, Southampton, UK) containing the Renilla luciferase thermal controller (MJ Research Inc., Watertown, MA) after an initial gene. After 24 h incubation with or without 1 M PGE , the cells were denaturation at 94 °C for 3 min. This was followed by 35 cycles of PCR harvested and the firefly and Renilla luciferase activities were meas- using the following temperature and time profile: denaturation at 94 °C ured using the Dual Luciferase Assay System Kit (Promega) and Mi- for 1 min, primer annealing at 56 °C for 1 min, primer extension at crolumat Plus LB 96V luminometer (Berthold Technologies GmbH & PGE Induces VEGF via Sp-1 29995 FIG.1. A, VEGF production in response to 1 nM to 1 M PGE . The VEGF released into the culture medium was measured by ELISA. B, time course of VEGF production in HASMC treated with 1 M PGE for 0, 2, 4, 6, 8, 16, and 24 h compared with unstimulated control cells. Each point represents the mean  S.E. of quadruple determinations from FIG.2. A, time course of PGE -mediated VEGF mRNA induction. three independent experiments (*, p  0.05; **, p  0.01; and ***, p ASMC were incubated with and without 1 M PGE for 0, 1, 2, 4, 8, and 0.001 by ANOVA). 24 h. The housekeeping gene  -microglobulin and VEGF mRNA were measured by quantitative real-time RT-PCR. Preincubation with 5 Co. KG, Bad Wildbag, Germany). The VEGF promoter-driven luciferase g/ml actinomycin D (ACT D), an inhibitor of transcription, inhibits constructs were a kind gift from Professor Dieter Marme´, Institute of PGE -mediated VEGF mRNA at all time points. The VEGF results Molecular Oncology, Tumor Biology Center, Freiburg, Germany (36). were normalized by dividing the mean of the triplicate VEGF result by The Sp-1 reporter construct containing 6 Sp-1 binding sites was a kind the mean of the triplicate  -microglobulin result and are expressed as -fold increase over control. B, luciferase activity in ASMC transiently gift from Professor Jeffrey E. Kudlow, School of Medicine, The Univer- transfected for 24 h with either a 2068-bp fragment of the VEGF sity of Alabama at Birmingham (37). promoter (2018/50) or control vector pGL3 basic, ligated to a lucif- EMSA—The nuclear protein fractions for EMSA were prepared erase reporter construct. Cells were cultured to confluence, growth using the Nu-Clear extraction kit (Sigma) following the manufacturer’s arrested, and transfected with 1 l of LF2000 and 0.8 g of DNA per protocol. Protein concentrations were determined using the Bio-Rad well. There was a significant increase in promoter activity in cells protein assay. Consensus Sp-1, AP-2, and EGR-1 oligonucleotides were stimulated for 24 h with 1 nM up to 1 M PGE compared with unstimu- purchased from Santa Cruz Biotechnology. VEGF promoter-specific oli- lated control cells (**, p  0.01; ***, p  0.001). The figure represents gonucleotides that recognized the 85/50 binding region: sense 5- the mean  S.E. of three experiments performed in triplicate. CCCGGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCGGAG-3 and antisense 5-CTCCGCCCCGCCGGGACCCCGCCCCCGGCCCGCCCC- was performed using the GeneGenius gel documentation and analysis GGG-3 were purchased from Sigma. system (Syngene, Cambridge, Cambridgeshire, UK). All oligonucleotides were labeled using [- P]ATP (Amersham Bio- Statistical Analysis—VEGF ELISA and luciferase levels were ex- sciences) and T4 polynucleotide kinase (Promega). Fifteen micrograms pressed as the mean of triplicate or quadruplicate wells for that exper- of nuclear protein, P-labeled double-stranded probe (40,000 counts iment. The experiments were repeated at least three times and the per min/ng), and 2 lof5 binding buffer (20% glycerol, 5 mM MgCl , results shown represent the mean  S.E. Analysis of variance (ANOVA) 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 50 mM Tris-HCl (pH was used to determine significant differences. A p value of 0.05 7.5), 0.25 mg/ml poly(dI-dC)poly(dI-dC)) were mixed in a total volume (2-tailed) was regarded as statistically significant. of 10 l. In competition assays, 50 unlabeled competitors were added at the same time of probe addition. The mixture was incubated at room RESULTS temperature for 30 min, then loaded on a 5% polyacrylamide gel in 0.5 PGE Increases VEGF-A Protein Production—There was a TBE buffer, and subjected to electrophoresis for 60 min. The gel was 2 165 dried and exposed for autoradiography on Kodak XAR film at 70 °C significant increase in VEGF release above control in cells for 19–48 h. Supershift were performed using 4 g of specific goat cultured for 24 h with concentrations of PGE ranging from 1 polyclonal anti-human Sp-1 (PEP 2), AP-2 (C-18), or EGR-1 (588) anti- nM to 1 M (Fig. 1A). Cells treated with 1 M PGE for 2, 4, 8, body (Santa Cruz Biotechnology). 16, and 24 h also showed significantly increased VEGF levels ChIP Assay—HASM cells were cultured to confluence in 75-cm compared with unstimulated control cells (Fig. 1B). flasks, growth arrested, and incubated with ethanol vehicle or 1 M PGE Increased VEGF Is Transcriptional—Real-time RT- PGE for 30 min. The ChIP assay was performed using the ChIP-IT kit (Active Motif, Rixensart, Belgium) following the manufacturer’s proto- PCR showed that PGE increased VEGF-A mRNA levels with col and using 4 g of goat anti-human polyclonal Sp-1 antibody (PEP 2) time with a 6-fold increase at 60 min and a peak 12-fold (Santa Cruz Biotechnology) for each immunoprecipitation. increase by 4 h compared with controls at these times (Fig. 2A). The VEGF primers used yielded a 202-bp product corresponding to To confirm that this was because of VEGF-A gene transcription 199 to 3 of the VEGF gene promoter and were: forward 5-GGTC- rather than stabilization of mRNA, the cells were cultured for GAGCTTCCCCTTCA-3, and reverse 5-GATCCTCCCCGCTACCAG- 30 min with 5 g/ml actinomycin D, an inhibitor of RNA po- 3. Forty cycles of a two-step PCR program, 95 °C for 1 min and 60 °C for 1 min, in the presence of 6% Me SO and 1 M betaine using Red Taq lymerase II, followed by 1 M PGE . Pretreatment with actino- 2 2 and 2.5 mM magnesium chloride (Sigma, Poole, Dorset, UK) were used mycin D prevented the PGE -induced increase in VEGF mRNA (38). Potential problems with PCR resulting from high melting temper- (Fig. 2A). atures were reduced by addition of Me SO (39), and the amplification of The cells were transfected with a 2068-bp VEGF promoter GC-rich templates was enhanced by betaine (40). fragment (2018 to 50) ligated to firefly luciferase. There was The PCR products were visualized by electrophoresis on 2% agarose a 5.6  0.48-fold increase in luciferase activity in cells treated gel in 0.5 TBE buffer after staining with 0.5 g/ml ethidium bromide. The ultraviolet-illuminated gels were photographed, and densitometry with 1 M PGE compared with unstimulated cells (Fig. 2B). 2 29996 PGE Induces VEGF via Sp-1 FIG.4. A, position of the transcription factor binding sites present on the 135-bp VEGF promoter. B, the effect of binding site mutations on VEGF promoter activity in response to 1 M PGE . Three Sp-1 binding sites, GGGCGG mutated to GTTCGG, and both EGR-1 binding sites, GCGGGGGCG, mutated to GCTAGGGCG. The graph shows -fold in- crease of luciferase activity in cells treated with PGE compared un- stimulated cells. The figure represents the mean  S.E. of three exper- iments performed in triplicate and was analyzed by ANOVA (***, p  0.001). Transfection studies using the wild type construct and a construct containing mutations of the three Sp-1 (88/50) binding sites showed a significant reduction in luciferase ac- tivity (Fig. 4, A and B), suggesting that these factors were necessary for VEGF induction by PGE . PGE Increases Sp-1 Binding to the VEGF Promoter—We used EMSA to determine whether PGE treatment increased Sp-1 binding to the VEGF promoter. Incubation with 1 M FIG.3. A, representation of the VEGF promoter-driven luciferase constructs used in the transfection studies showing the positions of the PGE for 60 min induced binding activity with Sp-1 consensus transcription factor binding sites. B, increase in luciferase expression in and VEGF promoter oligonucleotides (Fig. 5, A and E). This ASMC transiently transfected with the deletion series of the VEGF was not seen with AP-1 and EGR-1 consensus oligonucleotides promoter luciferase constructs after 24 h incubation with 1 M PGE . (Fig. 5, B and C). Supershift studies using a monoclonal anti- Cells were cultured in 24-well plates to confluence, growth arrested, and transfected using 1 l of LF2000 and 0.8 g of DNA per well. The body to Sp-1 produced gel retardation with the consensus se- figure represents the mean  S.E. of three experiments performed in quence (Fig. 5A) and a reduction in binding with the VEGF triplicate (*, p  0.05; **, p  0.01; and ***, p  0.001 by ANOVA). promoter-specific primers (Fig. 5F). Competition with 50-fold excess unlabeled VEGF promoter oligonucleotides blocked transcription factor binding, whereas excess irrelevant AP-1 These results suggest that induction of VEGF by PGE is oligonucleotides did not block Sp-1 transcription factor binding, transcriptional and not mediated by post-transcriptional stabi- demonstrating that the binding was specific (Fig. 5E). There lization of PGE mRNA. was no transcription factor binding in experiments using mu- Mutations in the Sp-1 Binding Sites in the VEGF Promoter tated consensus Sp-1 oligonucleotides (Fig. 5D). Reduce PGE -stimulated Luciferase Activity—To determine ChIP—The PGE -mediated increase in Sp-1 binding to the which transcription factors are involved, the cells were trans- VEGF promoter demonstrated by EMSA was confirmed using fected with 2068 bp of the wild type VEGF promoter and a the ChIP assay. Protein-DNA complexes were immunoprecipi- series of deletion constructs ligated to a firefly luciferase re- tated with antibody to Sp-1 and the DNA isolated and purified. porter plasmid. A diagram representing the VEGF promoter An aliquot of non-immunoprecipitated chromatin was used as showing the key transcription factor binding sites and the the input control, and no antibody control was included to show positions where the restriction enzymes cleave the promoter to specificity. Input, control, and immunoprecipitated DNA were generate the series of deletions is shown in Fig. 3A. One mi- subjected to 40 cycles of a two-step PCR in the presence of 1 M cromolar PGE increased the luciferase levels 4.2  0.65 in 2 betaine and 6% Me SO using VEGF promoter-specific primers cells transfected with the 2068 construct. There was a signifi- spanning 199 to 3 bp (Fig. 6A). Densitometry showed a cant PGE -mediated increase in luciferase activity with all of significant increase in Sp-1 binding to the VEGF promoter the deletions series except the smallest 102-bp fragment of the following incubation with PGE . The results were normalized VEGF promoter (Fig. 3B). However, deleting the sequences to the input control (Fig. 6B). between 1286 and 789 bp resulted in a reduction in the EP and EP Receptor Agonists Mimic the Effect of PGE — 2 4 2 stimulatory effect of the PGE . This suggests that the up- Both EP and EP receptors, which positively couple to adeny- 2 2 4 stream AP-1, AP-2, or hypoxia-inducible factor-1 sequences lyl cyclase, are present on HASMC as demonstrated by flow may also be involved in PGE -mediated VEGF increase. cytometry and a specific antibody to the EP receptor (Fig. 7, A 2 4 PGE Induces VEGF via Sp-1 29997 FIG.5. A, PGE increases consensus Sp-1 binding and addition of anti-Sp-1 antibody to the nuclear extracts from cells treated with 1 M PGE 2 2 for 60 min resulted in a supershift. B,1 M PGE for 60 min did not increase the consensus AP-2 binding or C, EGR-1 binding. Addition of antibodies to AP-2 or EGR-1 to the nuclear extracts did not result in gel retardation. D, Sp-1 binding is inhibited when a mutated consensus Sp-1 construct is used. E, PGE increases transcription factor binding to the VEGF promoter (88/50). Binding was specific as shown by competitive binding. Nuclear extracts from PGE -treated cells were incubated with labeled VEGF promoter (hot VEGF) in the presence of a 50-fold excess of unlabeled VEGF promoter (cold VEGF) or unlabeled AP-1 (cold AP-1). F, antibody to Sp-1 diminishes Sp-1 binding to the VEGF promoter. The figures shown are representative of three experiments. and B), by Western blotting and a specific antibody to the EP creased VEGF and luciferase activity in cells transfected with receptor (Fig. 7C) and RT-PCR (Fig. 7D). the 2068 VEGF promoter construct (Fig. 8C). To determine which PGE receptors were important we Mithramycin Inhibits PGE -induced Activation of VEGF— 2 2 looked at the effect of PGE receptor agonists on VEGF pro- The anticancer antibiotic, mithramycin A selectively binds to duction together with luciferase activity in cells transfected GC-rich regions of DNA preventing Sp-1 binding. Preincubation with the 2068 VEGF promoter construct. We found that both for 1 h with 500 nM and 1 M mithramycin significantly reduced EP (ONO-AE1 259) and EP (ONO-AE1 329) receptor agonists PGE -stimulated VEGF protein release in a concentration-de- 2 4 2 increased VEGF production and luciferase activity (Figs. 7E pendent manner. Maximal inhibition was seen using 1 M mith- and 8C) in the same way as PGE . There was an additive effect ramycin (Fig. 9A). Basal levels of VEGF production were not when both agonists were used in suboptimal concentrations changed significantly by mithramycin treatment (data not (Fig. 7E). This suggests that PGE is acting via both EP and shown). 2 2 EP receptors. Inhibition of PKA Abrogates PGE -induced Activation of 4 2 Increasing Intracellular cAMP Mimics the Effect of PGE — VEGF—Preincubation for 1 h with 10 M H-89, an inhibitor of The cAMP analogue 8-Br-cAMP increased VEGF protein pro- PKA, prior to a 24-h culture with 1 M PGE , resulted in a duction in a concentration-dependent manner (Fig. 8A). Agents significant reduction in secreted VEGF as measured by ELISA that increase cAMP also similarly increased VEGF release. (Fig. 9A). Forskolin, a direct activator of adenylyl cyclase (Fig. 8A), and Nuclear Sp-1 Protein Is Phosphorylated by PGE —Western the -adrenoreceptor agonist isoproterenol (Fig. 8B), both in- blotting demonstrated that Sp-1 protein expression was con- 29998 PGE Induces VEGF via Sp-1 FIG.6. A, representative ChIP assay PCR showing PGE increases in Sp-1 binding to the VEGF promoter. Immunoprecipitation (IP) was carried out using antibody to Sp-1. The PCR primers were amplified in the 199 to 3 region of the VEGF promoter. B, densitometry of ChIP PCR normalized to the input. Duplicate experiments were repeated in triplicate (***, p  0.001 by ANOVA). fined to the nucleus and was phosphorylated by PGE . Previous studies have shown that the 106k Sp-1 band represents the phosphorylated protein (41). GAPDH and the nuclear-specific proteins lamin A and C were used as controls (Fig. 9B). Increasing Intracellular cAMP Increases Sp-1 Luciferase Re- porter Activity—Agents that increase cAMP also increased a 6-repeat Sp-1/luciferase reporter construct. The cAMP ana- logue 8-Br-cAMP, forskolin, a direct activator of adenylyl cy- clase, and the -adrenoreceptor agonist salbutamol all in- creased the activity of a transiently transfected Sp-1 reporter FIG.7. A, flow cytometry histogram of unstimulated ASMC stained with preimmune rabbit serum control and fluorescein isothiocyanate- luciferase construct. (Fig. 9C). conjugated secondary antibody. B, flow cytometry histogram of un- stimulated ASMC stained with polyclonal rabbit anti-human EP re- DISCUSSION ceptor and fluorescein isothiocyanate-conjugated secondary antibody. There are several key novel findings in this study. We found C, Western blotting of ASMC showing EP receptor protein. U937 cells that PGE increases VEGF-A expression through transcrip- were used as a positive control. D, RT-PCR demonstrating mRNA for both EP and EP receptors. E, agonists to EP and EP receptors also tional mechanisms involving the GC-rich Sp-1 transcription 2 4 2 4 increase VEGF protein production by ELISA over control. 1 M PGE is factor binding sites on the proximal (88/50) region of the also shown (***, p  0.001 by ANOVA). VEGF promoter. Furthermore, the effect was mediated by EP and EP recep- 2 4 tors via cAMP and PKA. These studies are the first in any a series of deletions of the VEGF promoter ranging from 2068 biological system to study the transcription factors involved in to 102 bp. We found that promoter activity was maintained VEGF production by PGE and also delineate the upstream down to the 135-bp construct. However, all luciferase activity signaling cascade components. was lost using the 102-bp construct, suggesting that the main We first determined whether PGE was acting via transcrip- regulatory sites were contained within the 102–135-bp region. tional or post-transcriptional mechanisms. Stimulation with This region contains one AP-2, two EGR-1, and three Sp-1 PGE resulted in increased VEGF-A protein release after2has transcription factor binding sites. To explore this further we measured by ELISA. Quantitative real-time RT-PCR also used constructs with mutations in the Sp-1 or EGR-1 binding showed that PGE increased VEGF-A mRNA after 1 h. Pre- sites. We found no reduction in luciferase levels using con- treatment of the cells with the RNA polymerase II inhibitor structs containing mutated EGR-1 sites, whereas a construct actinomycin D prevented this, suggesting that the increased with mutations in all three Sp-1 sites resulted in loss of lucif- VEGF mRNA was because of transcriptional rather than post- erase activity, suggesting that Sp-1 binding was crucial to transcriptional mechanisms. Furthermore, mRNA stability ex- VEGF induction by PGE . These observations were also sup- periments showed no alteration in mRNA half-life after PGE ported by EMSA results, which showed that PGE increased 2 2 treatment. Collectively these studies suggest that VEGF was Sp-1 but not AP-2 or EGR-1 binding. Specificity of binding was regulated transcriptionally by PGE and this was confirmed by demonstrated by experiments using excess unlabeled oligonu- studies using VEGF promoter luciferase constructs. A few pre- cleotides and supershift with Sp-1 antibody. The EMSA results vious studies have looked at whether PGE increases VEGF were confirmed by the ChIP assay and specific antibody to transcriptionally or post-transcriptionally. PGE -mediated Sp-1. Consistent with a role for Sp-1, VEGF production was VEGF up-regulation was transcriptional in osteoblasts (16) inhibited by the Sp-1 inhibitor mithramycin (43). Using West- and overexpression of myc in B cells increased the initiation of ern blotting we also showed that Sp-1 is a nuclear protein that VEGF mRNA translation (42). is phosphorylated by PGE . To determine key transcription factor binding sites we used Ours are the first studies to show that Sp-1 is involved in PGE Induces VEGF via Sp-1 29999 FIG.9. A, reduction of VEGF protein release by the Sp-1 inhibitor mithramycin A (MTR) and PKA inhibitor H-89. Preincubation with 500 nM and 1 M MTR or 10 M H-89 prior to 24 h culture with 1 M PGE significantly reduced PGE stimulated VEGF measured by ELISA (* and #, p  0.05; ** and ##, p  0.01; *** and ###, p  0.001 by ANOVA). NS, not significant. B, Western blotting showing the nuclear localiza- tion of Sp-1 and increased phosphorylation of 106k Sp-1 by 15 min FIG.8. Agents that increase intracellular cAMP levels mimic 1 M PGE and increase VEGF production. A, concentration re- incubation with 1 M PGE . Lanes 1 and 2 are cytosolic fractions: 2 2 sponse to forskolin (FSK), which directly activates adenylyl cyclase and control (1) and PGE (2). Lanes 3 and 4 are nuclear fractions: control (3) the cAMP analogue, 8-Br-cAMP, increases VEGF, the -adrenergic and PGE (4). GAPDH and nuclear lamin were used as housekeeping agonists. B, isoproterenol (ISO). C, agents that act via cAMP mimic controls. C, PGE , cAMP analogue, and salbutamol and forskolin, which PGE and stimulate VEGF promoter-driven luciferase activity (*, p  increase cellular cAMP, increased luciferase activity of a transfected Sp-1 luciferase reporter construct (**, p  0.01; and ***, p  0.001 by 0.05, **, p  0.01; and ***, p  0.001 by ANOVA). ANOVA). PGE -induced VEGF production, although Sp-1 is important in tory responses depending on receptor subtype binding (28). We the activation of genes involved in tumor proliferation and the focused on the two EP receptors, EP and EP , which activate 2 4 induction of VEGF in response to some other stimuli (11, 34, cAMP. We found that both EP and EP receptors were ex- 2 4 44, 45). For example, VEGF induction by interleukin-1 in pressed in HASMC. This contrasts to a previous study that cardiac myocytes and by tumor necrosis factor- in glioma cells reported EP but not EP receptor expression in HASM (48). 2 4 is mediated through Sp-1 sites (13, 46). In contrast, transform- We confirmed our findings using both RT-PCR and either ing growth factor- induced VEGF via AP-2 transcription fac- Western blotting or fluorescence-activated cell sorter for EP tor binding (47). and EP , respectively, using antibodies designed for these Having shown that VEGF production was transcriptionally methodologies. The fact that both mRNA and protein to EP mediated via Sp-1, we then went on to characterize the pros- receptors was present suggests that these cells do indeed ex- tanoid receptor involved. PGE binds to a family of 7 trans- press EP receptors. Furthermore, experiments with EP and 2 4 2 membrane G protein-coupled membrane receptors, the EP re- EP receptor agonists mirrored the effect of PGE on VEGF 4 2 ceptors. Four subtypes of EP receptors have been described protein production and reporter activity, suggesting that both EP ,EP ,EP , and EP encoded by different genes (27). Each of these receptors are implicated in this process. This is similar 1 2 3 4 subtype is tissue-specific and uses different intracellular sig- to Clarke et al. (49) who suggested that positive regulation of naling mechanisms, suggesting potentially different inflamma- granulocyte colony-stimulating factor by E-Ring 8-isoprostanes 30000 PGE Induces VEGF via Sp-1 1955–1967 in HASMC was mediated by EP and EP receptors. 2 4 14. Wen, F. Q., Liu, X., Manda, W., Terasaki, Y., Kobayashi, T., Abe, S., Fang, Q., EP and EP receptors couple to G protein, which stimu- 2 4 S Ertl, R., Manouilova, L., and Rennard, S. I. (2003) J. Allergy Clin. Immunol. 111, 1307–1318 lates adenylyl cyclase activity increasing the intracellular 15. Knox, A. J., Corbett, L., Stocks, J., Holland, E., Zhu, Y. M., and Pang, L. (2001) cAMP levels resulting in PKA signaling. We performed exper- FASEB J. 15, 2480–2488 iments using a variety of pharmacological tools to probe the 16. Harada, S., Nagy, J. A., Sullivan, K. A., Thomas, K. A., Endo, N., Rodan, G. A., and Rodan, S. B. (1994) J. Clin. Investig. 93, 2490–2496 role of different components of this pathway. Isoproterenol, 17. Harada, S., Rodan, S. B., and Rodan, G. A. (1995) Clin. Orthop. Relat. Res. 313, which elevates cAMP via -adrenoceptors and forskolin, a di- 76–80 18. Ben Av, P., Crofford, L. J., Wilder, R. L., and Hla, T. (1995) FEBS Lett. 372, rect activator of adenylyl cyclase, had similar effects to PGE 83–87 on VEGF protein production and VEGF promoter luciferase 19. Gately, S. (2000) Cancer Metastasis Rev. 19, 19–27 expression, suggesting that cAMP pathways regulate VEGF 20. Gately, S., and Li, W. W. (2004) Semin. Oncol. 31, 2–11 21. Daniel, T. O., Liu, H., Morrow, J. D., Crews, B. C., and Marnett, L. J. (1999) release. Further evidence in support of a role for cAMP was Cancer Res. 59, 4574–4577 obtained from studies using 8-Br-cAMP, a cell-permeable 22. Jones, M. K., Wang, H., Peskar, B. M., Levin, E., Itani, R. M., Sarfeh, I. J., and Tarnawski, A. S. (1999) Nat. Med. 5, 1418–1423 cAMP analogue. Increasing cAMP also increased the activity of 23. Peterson, H. I. (1986) Anticancer Res. 6, 251–253 a transfected Sp-1 luciferase reporter construct. To explore the 24. Pang, L., Pitt, A., Petkova, D., and Knox, A. J. (1998) Clin. Exp. Allergy 28, main downstream target of cAMP, PKA, we studied the effect 1050–1058 25. Hoshino, M., Takahashi, M., and Aoike, N. (2001) J. Allergy Clin. Immunol. of the PKA inhibitor H-89. We found that H-89 markedly in- 107, 295–301 hibited PGE -induced VEGF protein production, suggesting 26. Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D., Fiddes, J. C., and Abraham, J. A. (1991) J. Biol. Chem. 266, 11947–11954 that it was PKA mediated, although it is possible that other 27. Coleman, R. A., Smith, W. L., and Narumiya, S. (1994) Pharmacol. Rev. 46, kinases mediated this effect (50). 205–229 Our studies have relevance for asthma where several immu- 28. Narumiya, S., Sugimoto, Y., and Ushikubi, F. (1999) Physiol. Rev. 79, 1193–1226 nohistochemical studies have shown that COX-2 and VEGF are 29. Pang, L., and Knox, A. J. (1997) Am. J. Physiol. 273, L1132–L1140 both up-regulated (24, 25). They are also of relevance to a wide 30. Pang, L., and Knox, A. J. (1997) Br. J. Pharmacol. 121, 579–587 range of inflammatory and malignant diseases where increased 31. Maruyama, T., Asada, M., Shiraishi, T., Ishida, A., Egashira, H., Yoshida, H., Maruyama, T., Ohuchida, S., Nakai, H., Kondo, K., and Toda, M. (2001) prostanoid production has been implicated in angiogenic pro- Bioorg. Med. Chem. Lett. 11, 2029–2031 cesses mediated via VEGF release. Strategies targeting Sp-1- 32. Tani, K., Naganawa, A., Ishida, A., Egashira, H., Sagawa, K., Harada, H., Ogawa, M., Maruyama, T., Ohuchida, S., Nakai, H., Kondo, K., and Toda, mediated gene transcription may provide a new therapeutic M. (2001) Bioorg. Med. Chem. Lett. 11, 2025–2028 approach to influence remodeling processes. In conclusion, our 33. Clarke, D. L., Belvisi, M. G., Smith, S. J., Hardaker, E., Yacoub, M. H., Meja, K. K., Newton, R., Slater, D. M., and Giembycz, M. A. (2005) Am. J. Physiol. studies provide evidence that PGE induces VEGF via Sp-1 288, L238–L250 binding sites on the VEGF promoter via EP and EP receptors 2 4 34. Schafer, G., Cramer, T., Suske, G., Kemmner, W., Wiedenmann, B., and in a cAMP- and PKA-dependent mechanism involving phospho- Hocker, M. (2003) J. Biol. Chem. 278, 8190–8198 35. Pallisgaard, N., Clausen, N., Schroder, H., and Hokland, P. (1999) Genes rylation of Sp-1. Chromosomes Cancer 26, 355–365 36. Finkenzeller, G., Technau, A., and Marme, D. (1995) Biochem. Biophys. Res. Acknowledgments—We thank Dieter Marme´ for providing the VEGF Commun. 208, 432–439 luciferase promoter constructs, and Jeffrey E. Kudlow for the Sp-1 37. Biggs, J. R., Kudlow, J. E., and Kraft, A. S. (1996) J. Biol. Chem. 271, 901–906 reporter construct. 38. 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Vascular Endothelial Growth Factor Induction by Prostaglandin E2 in Human Airway Smooth Muscle Cells Is Mediated by E Prostanoid EP2/EP4 Receptors and SP-1 Transcription Factor Binding Sites *

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Publisher
American Society for Biochemistry and Molecular Biology
Copyright
Copyright © 2005 Elsevier Inc.
ISSN
0021-9258
eISSN
1083-351X
DOI
10.1074/jbc.m414530200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 34, Issue of August 26, pp. 29993–30000, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Vascular Endothelial Growth Factor Induction by Prostaglandin E in Human Airway Smooth Muscle Cells Is Mediated by E Prostanoid EP /EP Receptors and SP-1 Transcription Factor Binding Sites* 2 4 Received for publication, December 23, 2004, and in revised form, May 19, 2005 Published, JBC Papers in Press, June 21, 2005, DOI 10.1074/jbc.M414530200 Dawn Bradbury‡, Deborah Clarke‡, Claire Seedhouse§, Lisa Corbett‡, Joanne Stocks‡, and Alan Knox‡ From the ‡Division of Respiratory Medicine, §Academic Haematology, University of Nottingham, City Hospital, Nottingham NG5 1PB, United Kingdom a critical role in physiological and pathological angiogenesis in Prostaglandin E (PGE ) can increase vascular endo- 2 2 thelial growth factor A (VEGF-A) production but the most biological systems (2). VEGF is implicated in tumor neo- mechanisms involved are unclear. Here we character- vascularization and in angiogenesis associated with a number ized the transcriptional mechanisms involved in human of chronic inflammatory diseases, such as asthma, chronic ob- airway smooth muscle cells (HASMC). PGE increased 2 structive pulmonary disease, inflammatory bowel disease, VEGF-A mRNA and protein but not mRNA stability. rheumatoid and osteoarthritis (3–7). VEGF is secreted by a PGE stimulated the activity of a transiently transfected variety of cell types, but not by endothelial cells themselves and 2068-bp (2018 to 50) VEGF-A promoter-driven lucifer- mesenchymal cells serve as an important source of VEGF in a ase construct. Functional 5 deletional analysis mapped number of inflammatory and neoplastic processes (8). There the PGE response element to the 135-bp sequence (85/ are at least five members of the VEGF family including pla- 50) of the human VEGF-A promoter. PGE -induced lu- cental growth factor, VEGF-A, VEGF-B, VEGF-C, and ciferase activity was reduced in cells transfected with a VEGF-D (9). The most potent angiogenic factor in vivo is 135-bp VEGF promoter fragment containing mutated VEGF-A, which has six splice variants: 121, 145, 165, 183, 189, Sp-1 binding sites but not in cells transfected with a and 204 amino acids (10). construct containing mutated EGR-1 binding sites. Elec- A number of stimuli are capable of increasing VEGF release trophoretic mobility shift assay and chromatin immuno- in different biological systems. Inflammatory cytokines such as precipitation assay confirmed binding of Sp-1 to the interleukin-1 and transforming growth factor- increased VEGF promoter. PGE increased phosphorylation of VEGF release in human cholangiocellular carcinoma cells, sy- Sp-1 and luciferase activity of a transfected Sp-1 re- porter construct. PGE receptor agonists EP (ONO-AE1 novial fibroblasts, cardiac myocytes, and airway smooth muscle 2 2 259) and EP (ONO-AE1 329) mimicked the effect of cells (11–14). We have previously shown that the pro-inflam- PGE , and reverse transcription-PCR, Western blotting, matory asthma mediator, bradykinin, increased VEGF produc- and flow cytometry confirmed the presence of EP and 2 tion in human airway smooth muscle cells (HASMC) (15). A EP receptors. VEGF protein release and Sp-1 reporter 4 number of studies have shown that the products of COX-2, the activity were increased by forskolin and isoproterenol, inducible form of cyclooxygenase, may mediate the effect of which increase cytosolic cAMP, and the cAMP analogue, cytokines and mediators on the release of chemokines and 8-bromoadenosine-3,5-cyclophosphoric acid. These cytokines in an autocrine manner through a mechanism involv- studies suggest that PGE increases VEGF transcrip- ing endogenous prostanoid production. Recent work suggests tionally and involves the Sp-1 binding site via a cAMP- that this is also true of VEGF. Autocrine PGE increases VEGF dependent mechanism involving EP and EP receptors. 2 4 release in response to interleukin-1 in synovial fibroblasts and in response to bradykinin in HASMC (12, 15). Further- more, exogenous PGE increases VEGF expression in fibro- Vascular endothelial growth factor (VEGF) is a 45-kDa he- blasts and osteoblasts (16–18). These studies are consistent parin-binding homodimeric glycoprotein that is an important with the known role of COX products in angiogenesis (19, 20): growth and survival factor for endothelial cells (1). VEGF plays COX-2-derived thromboxane A , prostacyclin, and PGE stim- 2 2 ulate endothelial cell migration and angiogenesis (21), whereas COX inhibitors have protective effects on angiogenesis in ex- * This work was supported by Asthma UK. The costs of publication of this article were defrayed in part by the payment of page charges. This perimental models (22, 23). In asthma both COX-2 and VEGF article must therefore be hereby marked “advertisement” in accordance are increased but the two have not been firmly linked (24, 25). with 18 U.S.C. Section 1734 solely to indicate this fact. Collectively these studies suggest that PGE can contribute To whom correspondence should be addressed: Division of Respira- to angiogenesis via increased VEGF production but the molec- tory Medicine, Clinical Science Bldg., City Hospital, Hucknall Rd., Nottingham NG5 1PB, United Kingdom. Tel.: 44-115-8404775; Fax: ular mechanisms involved have not been studied in detail, 44-115-8404771; E-mail: alan.knox@nottingham.ac.uk. particularly the balance between transcriptional and post-tran- The abbreviations used are: VEGF, vascular endothelial growth scriptional events. factor: HASMC, human airway smooth muscle cells; EMSA, electro- The VEGF promoter contains the hypoxia response element, phoretic mobility shift assay; ChIP, chromatin immunoprecipitation; 8-Br-cAMP, 8-bromoadenosine 3,5-cyclophosphoric acid; COX, cy- hypoxia inducible factor-1, p53/Von Hippel Lindau, NFB, clooxygenase: RT, reverse transcription; PGE , prostaglandin E ; EP, E 2 2 and AP-1 as well as several potential transcription factor bind- prostanoid; ELISA, enzyme-linked immunosorbent assay; PKA, protein ing sites for Sp-1 and AP-2 (26). PGE binds to G protein- kinase A; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3- coupled membrane receptors, the E prostanoid (EP) receptors. phosphate dehydrogenase; PKA, protein kinase A; AP, activator pro- tein; EGR, early growth response. Four subtypes of EP receptors have been described, EP ,EP , 1 2 This paper is available on line at http://www.jbc.org 29993 This is an Open Access article under the CC BY license. 29994 PGE Induces VEGF via Sp-1 72 °C for 1 min, and a final extension of 72 °C for 10 min. The following EP , and EP , encoded by different genes (27). Each subtype is 3 4 primers were used: EP -R sense 5-TCCAATGACTCCCAGTCTGAGG- tissue-specific and uses different intracellular signaling mech- A-3, antisense 5-TCAAAGGTCAGCCTGTTTAC-3;EP -R sense 5-T- anisms suggesting potentially different inflammatory re- CTGACCTCGGTGTCCAAAAATCG-3, antisense 5-TGGGTACTGCA- sponses depending on receptor subtype binding (28). The re- GCCGCGAGCTA-3 (33), and GAPDH sense 5-CCACCCATGGCAAA- ceptor used by PGE to increase VEGF is unknown. 2 TTCCATGGCA-3, GAPDH antisense 5-TCTAGACGGCAGGT- Here we determined the molecular mechanisms involved in CAGGTCCACC-3. The PCR products were visualized by electrophoresis on a 2% agar- the transcriptional regulation of the VEGF promoter by exog- ose gel in 0.5 TBE buffer after staining with 0.5 g/ml ethidium enous PGE in human airway smooth muscle cells. Mutational bromide. The ultraviolet (UV)-illuminated gels were photographed, and and deletional analysis of the VEGF promoter showed that the densitometry was analyzed using a GeneGenius gel documentation Sp-1 transcription factor binding was essential for the increase and analysis system (Syngene, Cambridge, UK). in VEGF promoter activity produced by PGE . PGE caused Quantitative Real-time RT-PCR—VEGF-A expression was deter- 2 2 phosphorylation of Sp-1 and electrophoretic mobility shift as- mined using primer sequences: sense 5-CTTGCCTTGCTGCTCTAC- C-3 and antisense 5-CACACAGGATGGCTTGAAG-3 (34).  -Micro- say (EMSA), and chromatin immunoprecipitation (ChIP) dem- 2 globulin was used as the housekeeping gene (35). 1 ng of reverse- onstrated that PGE increased Sp-1 binding to the VEGF pro- transcribed cDNA was subjected to real-time PCR using Excite Real- moter. Furthermore, studies with EP and EP receptor 2 4 time Mastermix with SYBR green (Biogene, Cambridge, UK) and the subtype agonists, the cAMP analogue 8-bromoadenosine-3,5- ABI Prism 7700 detection system (Applied Biosystems, Warrington, cyclophosphoric acid (8-Br-cAMP), forskolin, which increases Cheshire, UK). Each reaction consisted of 1 Excite mastermix, SYBR adenylyl cyclase activity, and the 2 receptor agonist isoprot- green (1:60000 final concentration), 40 nM of both sense and antisense primers, 1.6 l of DNA (or dH O), and H O to a final volume of 20 l. erenol, showed that PGE -induced activation of Sp-1 was me- 2 2 Thermal cycler conditions included incubation at 95 °C for 10 min diated by EP and EP receptors via cAMP. 2 4 followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Integration of the fluorescent SYBR green into the PCR product was monitored MATERIALS AND METHODS after each annealing step. Amplification of one specific product was Cell Culture—Human tracheas were obtained from three post-mor- confirmed by melting curve analysis, where a single melting peak tem individuals. Primary cultures of human ASM cells were prepared eliminated the possibility of primer-dimer association. For melting from explants of ASM according to methods previously reported (29, curve analysis to be performed the products were heated from 60 to 30). Cells at passage 6 were used for all experiments. We have previ- 95 °C over 20 min after the 40 cycles. ously shown that cells grown in this manner depict the immunohisto- To enable the levels of transcripts to be quantified, standard curves chemical and light microscopic characteristics of typical ASM cells (30). were generated using serial dilutions of KG1a cDNA. Negative controls Experimental Protocol—The cells were cultured to confluence in 24- consisting of no template were included, and all reactions were set up in well culture plates in a humidified, 5% CO , 37 °C incubator using 2 triplicate. VEGF-A expression was normalized to the housekeeping Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% gene by dividing the mean of the VEGF-A triplicate value by the mean fetal calf serum (Seralab, Crawly Down, Sussex, UK), 100 units/ml of the  -microglobulin triplicate value. This was then expressed as penicillin, 100 g/ml streptomycin, 4 mML-glutamine, and 2.5 g/ml -fold increase over unstimulated cells at each time point. amphotericin B (Sigma). The cells were growth-arrested in serum-free Flow Cytometric Analysis of EP Receptors—HASMC were detached medium for 24 h prior to experiments. Immediately before each exper- using a sterile scraper, washed, and incubated with polyclonal rabbit iment, fresh serum-free medium containing PGE or ethanol vehicle anti-human EP -R (Cayman Chemical, Ann Arbor, MI). The cells were (Sigma) was added. In time course experiments cells were incubated washed twice and incubated with fluorescein isothiocyanate-conjugated with 1 M PGE for 2–24 h. In the concentration response experiments goat anti-rabbit secondary antibody (Sigma). Preimmune rabbit serum cells were incubated for 24 h with 1 nM to 10 M PGE . In subsequent was used as the negative control (Sigma). Using the FACSCalibur flow experiments, 24-h incubation times were used. At the indicated times, cytometer (BD Biosciences) and logarithmic amplification of the green the culture media were harvested and stored at 20 °C. The highly fluorescence channel (FL-1), 10,000 events were acquired and analyzed selective EP and EP receptor subtype agonists ONO-AE1 259 and with CellQuest software (BD Biosciences). 2 4 ONO-AE1 329, which were a gift from ONO Pharmaceuticals, Osaka, Western Blotting—The nuclear protein fractions were prepared using Japan, were used in the PGE receptor studies (31, 32). Nu-Clear extraction kit (Sigma) following the manufacturer’s protocol. The cAMP analogue 8-Br-cAMP, the PKA inhibitor H-89, and fors- Western blotting was performed as described previously using a specific kolin and isoproterenol, which increase cytosolic cAMP, were purchased polyclonal rabbit anti-human EP receptor antibody (Cayman Chemi- from Sigma. Mithramycin was purchased from Tocris Cookson Ltd. cal) or mouse monoclonal anti-human Sp1 antibody (1C6; Santa Cruz (Avonmouth, Bristol, UK). The cells were preincubated for 1 h with Biotechnology, Santa Cruz, CA) and horseradish peroxidase-conjugated the inhibitors. secondary antibody (DakoCytomation, Ely, Cambridgeshire, UK) (33). VEGF-A Assay—The enzyme-linked immunosorbent assay (ELISA) The human histiocytic lymphoma cell line U937, which is known to was used to measure VEGF-A (R&D Systems, Abingdon, Oxon, UK) express EP receptors, was used as the positive control. The human T according to the manufacturer’s instructions and has been described by lymphoblastic leukemia cell line Jurkat was used as a positive Sp-1 us in detail elsewhere (15). control. RNA Isolation and Reverse Transcription-Polymerase Chain Reac- Cell Viability—The toxicity of all the chemicals and vehicles used in tion (RT-PCR)—Cells in 6-well plates were treated with PGE and this study was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- collected at time 0, 1, 2, 4, 8, and 24 h, respectively. Total RNA was tertrazolium bromide assay (29). At the end of the experiment the isolated by using the RNeasy mini kit (Qiagen, West Sussex, UK) culture media was removed and replaced with 250 l of media contain- following the manufacturer’s protocol with on-column DNase digestion. ing 1 mg/ml thiazolyl blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylter- 1 g of total RNA was reverse transcribed in a total volume of 20 l trazolium bromide (Sigma) and incubated for 20 min in 37 °C. This including 200 units of Moloney murine leukemia virus reverse tran- medium was removed and 250 lofMe SO was added to solubilize the scriptase (Promega, Madison, WI), 25 units of RNase inhibitor (Pro- blue-colored tetrazolium. The optical density was read at 550 nm in a mega), 0.5 g of oligo(dT) primer, 0.5 mM of each dNTPs, and 1 TECAN GENios (Tecan UK Limited, Theale, Reading, UK) microplate first-strand buffer provided by Promega. The reaction was incubated at reader. Viability was set as 100% in control cells. 42 °C for 90 min. Transfection with VEGF Promoter-driven Luciferase Constructs and Aliquots of the RT products were subsequently used for PCR ampli- Sp-1 Reporter Luciferase Construct—Cells were cultured in 24-well fication. 10 l of RT products was brought to a volume of 50 l contain- plates to confluence, growth arrested for 24 h, and transfected using 1 ing1mM MgCl , 0.12 mM of each dTNPs, 1 unit of Taq polymerase l of LF2000 (Lipofectamine LF2000, Invitrogen) and 0.8 g of DNA per (Promega), 0.5 M of both the upstream and downstream PCR primers, well according to the manufacturer’s instructions. The cells were co- and 1 PCR buffer, provided by Promega. transfected with 1 ng/well of the internal control plasmid pRL-SV40 Amplification was carried out with a PTC-100 programmable (Promega UK, Southampton, UK) containing the Renilla luciferase thermal controller (MJ Research Inc., Watertown, MA) after an initial gene. After 24 h incubation with or without 1 M PGE , the cells were denaturation at 94 °C for 3 min. This was followed by 35 cycles of PCR harvested and the firefly and Renilla luciferase activities were meas- using the following temperature and time profile: denaturation at 94 °C ured using the Dual Luciferase Assay System Kit (Promega) and Mi- for 1 min, primer annealing at 56 °C for 1 min, primer extension at crolumat Plus LB 96V luminometer (Berthold Technologies GmbH & PGE Induces VEGF via Sp-1 29995 FIG.1. A, VEGF production in response to 1 nM to 1 M PGE . The VEGF released into the culture medium was measured by ELISA. B, time course of VEGF production in HASMC treated with 1 M PGE for 0, 2, 4, 6, 8, 16, and 24 h compared with unstimulated control cells. Each point represents the mean  S.E. of quadruple determinations from FIG.2. A, time course of PGE -mediated VEGF mRNA induction. three independent experiments (*, p  0.05; **, p  0.01; and ***, p ASMC were incubated with and without 1 M PGE for 0, 1, 2, 4, 8, and 0.001 by ANOVA). 24 h. The housekeeping gene  -microglobulin and VEGF mRNA were measured by quantitative real-time RT-PCR. Preincubation with 5 Co. KG, Bad Wildbag, Germany). The VEGF promoter-driven luciferase g/ml actinomycin D (ACT D), an inhibitor of transcription, inhibits constructs were a kind gift from Professor Dieter Marme´, Institute of PGE -mediated VEGF mRNA at all time points. The VEGF results Molecular Oncology, Tumor Biology Center, Freiburg, Germany (36). were normalized by dividing the mean of the triplicate VEGF result by The Sp-1 reporter construct containing 6 Sp-1 binding sites was a kind the mean of the triplicate  -microglobulin result and are expressed as -fold increase over control. B, luciferase activity in ASMC transiently gift from Professor Jeffrey E. Kudlow, School of Medicine, The Univer- transfected for 24 h with either a 2068-bp fragment of the VEGF sity of Alabama at Birmingham (37). promoter (2018/50) or control vector pGL3 basic, ligated to a lucif- EMSA—The nuclear protein fractions for EMSA were prepared erase reporter construct. Cells were cultured to confluence, growth using the Nu-Clear extraction kit (Sigma) following the manufacturer’s arrested, and transfected with 1 l of LF2000 and 0.8 g of DNA per protocol. Protein concentrations were determined using the Bio-Rad well. There was a significant increase in promoter activity in cells protein assay. Consensus Sp-1, AP-2, and EGR-1 oligonucleotides were stimulated for 24 h with 1 nM up to 1 M PGE compared with unstimu- purchased from Santa Cruz Biotechnology. VEGF promoter-specific oli- lated control cells (**, p  0.01; ***, p  0.001). The figure represents gonucleotides that recognized the 85/50 binding region: sense 5- the mean  S.E. of three experiments performed in triplicate. CCCGGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCGGAG-3 and antisense 5-CTCCGCCCCGCCGGGACCCCGCCCCCGGCCCGCCCC- was performed using the GeneGenius gel documentation and analysis GGG-3 were purchased from Sigma. system (Syngene, Cambridge, Cambridgeshire, UK). All oligonucleotides were labeled using [- P]ATP (Amersham Bio- Statistical Analysis—VEGF ELISA and luciferase levels were ex- sciences) and T4 polynucleotide kinase (Promega). Fifteen micrograms pressed as the mean of triplicate or quadruplicate wells for that exper- of nuclear protein, P-labeled double-stranded probe (40,000 counts iment. The experiments were repeated at least three times and the per min/ng), and 2 lof5 binding buffer (20% glycerol, 5 mM MgCl , results shown represent the mean  S.E. Analysis of variance (ANOVA) 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 50 mM Tris-HCl (pH was used to determine significant differences. A p value of 0.05 7.5), 0.25 mg/ml poly(dI-dC)poly(dI-dC)) were mixed in a total volume (2-tailed) was regarded as statistically significant. of 10 l. In competition assays, 50 unlabeled competitors were added at the same time of probe addition. The mixture was incubated at room RESULTS temperature for 30 min, then loaded on a 5% polyacrylamide gel in 0.5 PGE Increases VEGF-A Protein Production—There was a TBE buffer, and subjected to electrophoresis for 60 min. The gel was 2 165 dried and exposed for autoradiography on Kodak XAR film at 70 °C significant increase in VEGF release above control in cells for 19–48 h. Supershift were performed using 4 g of specific goat cultured for 24 h with concentrations of PGE ranging from 1 polyclonal anti-human Sp-1 (PEP 2), AP-2 (C-18), or EGR-1 (588) anti- nM to 1 M (Fig. 1A). Cells treated with 1 M PGE for 2, 4, 8, body (Santa Cruz Biotechnology). 16, and 24 h also showed significantly increased VEGF levels ChIP Assay—HASM cells were cultured to confluence in 75-cm compared with unstimulated control cells (Fig. 1B). flasks, growth arrested, and incubated with ethanol vehicle or 1 M PGE Increased VEGF Is Transcriptional—Real-time RT- PGE for 30 min. The ChIP assay was performed using the ChIP-IT kit (Active Motif, Rixensart, Belgium) following the manufacturer’s proto- PCR showed that PGE increased VEGF-A mRNA levels with col and using 4 g of goat anti-human polyclonal Sp-1 antibody (PEP 2) time with a 6-fold increase at 60 min and a peak 12-fold (Santa Cruz Biotechnology) for each immunoprecipitation. increase by 4 h compared with controls at these times (Fig. 2A). The VEGF primers used yielded a 202-bp product corresponding to To confirm that this was because of VEGF-A gene transcription 199 to 3 of the VEGF gene promoter and were: forward 5-GGTC- rather than stabilization of mRNA, the cells were cultured for GAGCTTCCCCTTCA-3, and reverse 5-GATCCTCCCCGCTACCAG- 30 min with 5 g/ml actinomycin D, an inhibitor of RNA po- 3. Forty cycles of a two-step PCR program, 95 °C for 1 min and 60 °C for 1 min, in the presence of 6% Me SO and 1 M betaine using Red Taq lymerase II, followed by 1 M PGE . Pretreatment with actino- 2 2 and 2.5 mM magnesium chloride (Sigma, Poole, Dorset, UK) were used mycin D prevented the PGE -induced increase in VEGF mRNA (38). Potential problems with PCR resulting from high melting temper- (Fig. 2A). atures were reduced by addition of Me SO (39), and the amplification of The cells were transfected with a 2068-bp VEGF promoter GC-rich templates was enhanced by betaine (40). fragment (2018 to 50) ligated to firefly luciferase. There was The PCR products were visualized by electrophoresis on 2% agarose a 5.6  0.48-fold increase in luciferase activity in cells treated gel in 0.5 TBE buffer after staining with 0.5 g/ml ethidium bromide. The ultraviolet-illuminated gels were photographed, and densitometry with 1 M PGE compared with unstimulated cells (Fig. 2B). 2 29996 PGE Induces VEGF via Sp-1 FIG.4. A, position of the transcription factor binding sites present on the 135-bp VEGF promoter. B, the effect of binding site mutations on VEGF promoter activity in response to 1 M PGE . Three Sp-1 binding sites, GGGCGG mutated to GTTCGG, and both EGR-1 binding sites, GCGGGGGCG, mutated to GCTAGGGCG. The graph shows -fold in- crease of luciferase activity in cells treated with PGE compared un- stimulated cells. The figure represents the mean  S.E. of three exper- iments performed in triplicate and was analyzed by ANOVA (***, p  0.001). Transfection studies using the wild type construct and a construct containing mutations of the three Sp-1 (88/50) binding sites showed a significant reduction in luciferase ac- tivity (Fig. 4, A and B), suggesting that these factors were necessary for VEGF induction by PGE . PGE Increases Sp-1 Binding to the VEGF Promoter—We used EMSA to determine whether PGE treatment increased Sp-1 binding to the VEGF promoter. Incubation with 1 M FIG.3. A, representation of the VEGF promoter-driven luciferase constructs used in the transfection studies showing the positions of the PGE for 60 min induced binding activity with Sp-1 consensus transcription factor binding sites. B, increase in luciferase expression in and VEGF promoter oligonucleotides (Fig. 5, A and E). This ASMC transiently transfected with the deletion series of the VEGF was not seen with AP-1 and EGR-1 consensus oligonucleotides promoter luciferase constructs after 24 h incubation with 1 M PGE . (Fig. 5, B and C). Supershift studies using a monoclonal anti- Cells were cultured in 24-well plates to confluence, growth arrested, and transfected using 1 l of LF2000 and 0.8 g of DNA per well. The body to Sp-1 produced gel retardation with the consensus se- figure represents the mean  S.E. of three experiments performed in quence (Fig. 5A) and a reduction in binding with the VEGF triplicate (*, p  0.05; **, p  0.01; and ***, p  0.001 by ANOVA). promoter-specific primers (Fig. 5F). Competition with 50-fold excess unlabeled VEGF promoter oligonucleotides blocked transcription factor binding, whereas excess irrelevant AP-1 These results suggest that induction of VEGF by PGE is oligonucleotides did not block Sp-1 transcription factor binding, transcriptional and not mediated by post-transcriptional stabi- demonstrating that the binding was specific (Fig. 5E). There lization of PGE mRNA. was no transcription factor binding in experiments using mu- Mutations in the Sp-1 Binding Sites in the VEGF Promoter tated consensus Sp-1 oligonucleotides (Fig. 5D). Reduce PGE -stimulated Luciferase Activity—To determine ChIP—The PGE -mediated increase in Sp-1 binding to the which transcription factors are involved, the cells were trans- VEGF promoter demonstrated by EMSA was confirmed using fected with 2068 bp of the wild type VEGF promoter and a the ChIP assay. Protein-DNA complexes were immunoprecipi- series of deletion constructs ligated to a firefly luciferase re- tated with antibody to Sp-1 and the DNA isolated and purified. porter plasmid. A diagram representing the VEGF promoter An aliquot of non-immunoprecipitated chromatin was used as showing the key transcription factor binding sites and the the input control, and no antibody control was included to show positions where the restriction enzymes cleave the promoter to specificity. Input, control, and immunoprecipitated DNA were generate the series of deletions is shown in Fig. 3A. One mi- subjected to 40 cycles of a two-step PCR in the presence of 1 M cromolar PGE increased the luciferase levels 4.2  0.65 in 2 betaine and 6% Me SO using VEGF promoter-specific primers cells transfected with the 2068 construct. There was a signifi- spanning 199 to 3 bp (Fig. 6A). Densitometry showed a cant PGE -mediated increase in luciferase activity with all of significant increase in Sp-1 binding to the VEGF promoter the deletions series except the smallest 102-bp fragment of the following incubation with PGE . The results were normalized VEGF promoter (Fig. 3B). However, deleting the sequences to the input control (Fig. 6B). between 1286 and 789 bp resulted in a reduction in the EP and EP Receptor Agonists Mimic the Effect of PGE — 2 4 2 stimulatory effect of the PGE . This suggests that the up- Both EP and EP receptors, which positively couple to adeny- 2 2 4 stream AP-1, AP-2, or hypoxia-inducible factor-1 sequences lyl cyclase, are present on HASMC as demonstrated by flow may also be involved in PGE -mediated VEGF increase. cytometry and a specific antibody to the EP receptor (Fig. 7, A 2 4 PGE Induces VEGF via Sp-1 29997 FIG.5. A, PGE increases consensus Sp-1 binding and addition of anti-Sp-1 antibody to the nuclear extracts from cells treated with 1 M PGE 2 2 for 60 min resulted in a supershift. B,1 M PGE for 60 min did not increase the consensus AP-2 binding or C, EGR-1 binding. Addition of antibodies to AP-2 or EGR-1 to the nuclear extracts did not result in gel retardation. D, Sp-1 binding is inhibited when a mutated consensus Sp-1 construct is used. E, PGE increases transcription factor binding to the VEGF promoter (88/50). Binding was specific as shown by competitive binding. Nuclear extracts from PGE -treated cells were incubated with labeled VEGF promoter (hot VEGF) in the presence of a 50-fold excess of unlabeled VEGF promoter (cold VEGF) or unlabeled AP-1 (cold AP-1). F, antibody to Sp-1 diminishes Sp-1 binding to the VEGF promoter. The figures shown are representative of three experiments. and B), by Western blotting and a specific antibody to the EP creased VEGF and luciferase activity in cells transfected with receptor (Fig. 7C) and RT-PCR (Fig. 7D). the 2068 VEGF promoter construct (Fig. 8C). To determine which PGE receptors were important we Mithramycin Inhibits PGE -induced Activation of VEGF— 2 2 looked at the effect of PGE receptor agonists on VEGF pro- The anticancer antibiotic, mithramycin A selectively binds to duction together with luciferase activity in cells transfected GC-rich regions of DNA preventing Sp-1 binding. Preincubation with the 2068 VEGF promoter construct. We found that both for 1 h with 500 nM and 1 M mithramycin significantly reduced EP (ONO-AE1 259) and EP (ONO-AE1 329) receptor agonists PGE -stimulated VEGF protein release in a concentration-de- 2 4 2 increased VEGF production and luciferase activity (Figs. 7E pendent manner. Maximal inhibition was seen using 1 M mith- and 8C) in the same way as PGE . There was an additive effect ramycin (Fig. 9A). Basal levels of VEGF production were not when both agonists were used in suboptimal concentrations changed significantly by mithramycin treatment (data not (Fig. 7E). This suggests that PGE is acting via both EP and shown). 2 2 EP receptors. Inhibition of PKA Abrogates PGE -induced Activation of 4 2 Increasing Intracellular cAMP Mimics the Effect of PGE — VEGF—Preincubation for 1 h with 10 M H-89, an inhibitor of The cAMP analogue 8-Br-cAMP increased VEGF protein pro- PKA, prior to a 24-h culture with 1 M PGE , resulted in a duction in a concentration-dependent manner (Fig. 8A). Agents significant reduction in secreted VEGF as measured by ELISA that increase cAMP also similarly increased VEGF release. (Fig. 9A). Forskolin, a direct activator of adenylyl cyclase (Fig. 8A), and Nuclear Sp-1 Protein Is Phosphorylated by PGE —Western the -adrenoreceptor agonist isoproterenol (Fig. 8B), both in- blotting demonstrated that Sp-1 protein expression was con- 29998 PGE Induces VEGF via Sp-1 FIG.6. A, representative ChIP assay PCR showing PGE increases in Sp-1 binding to the VEGF promoter. Immunoprecipitation (IP) was carried out using antibody to Sp-1. The PCR primers were amplified in the 199 to 3 region of the VEGF promoter. B, densitometry of ChIP PCR normalized to the input. Duplicate experiments were repeated in triplicate (***, p  0.001 by ANOVA). fined to the nucleus and was phosphorylated by PGE . Previous studies have shown that the 106k Sp-1 band represents the phosphorylated protein (41). GAPDH and the nuclear-specific proteins lamin A and C were used as controls (Fig. 9B). Increasing Intracellular cAMP Increases Sp-1 Luciferase Re- porter Activity—Agents that increase cAMP also increased a 6-repeat Sp-1/luciferase reporter construct. The cAMP ana- logue 8-Br-cAMP, forskolin, a direct activator of adenylyl cy- clase, and the -adrenoreceptor agonist salbutamol all in- creased the activity of a transiently transfected Sp-1 reporter FIG.7. A, flow cytometry histogram of unstimulated ASMC stained with preimmune rabbit serum control and fluorescein isothiocyanate- luciferase construct. (Fig. 9C). conjugated secondary antibody. B, flow cytometry histogram of un- stimulated ASMC stained with polyclonal rabbit anti-human EP re- DISCUSSION ceptor and fluorescein isothiocyanate-conjugated secondary antibody. There are several key novel findings in this study. We found C, Western blotting of ASMC showing EP receptor protein. U937 cells that PGE increases VEGF-A expression through transcrip- were used as a positive control. D, RT-PCR demonstrating mRNA for both EP and EP receptors. E, agonists to EP and EP receptors also tional mechanisms involving the GC-rich Sp-1 transcription 2 4 2 4 increase VEGF protein production by ELISA over control. 1 M PGE is factor binding sites on the proximal (88/50) region of the also shown (***, p  0.001 by ANOVA). VEGF promoter. Furthermore, the effect was mediated by EP and EP recep- 2 4 tors via cAMP and PKA. These studies are the first in any a series of deletions of the VEGF promoter ranging from 2068 biological system to study the transcription factors involved in to 102 bp. We found that promoter activity was maintained VEGF production by PGE and also delineate the upstream down to the 135-bp construct. However, all luciferase activity signaling cascade components. was lost using the 102-bp construct, suggesting that the main We first determined whether PGE was acting via transcrip- regulatory sites were contained within the 102–135-bp region. tional or post-transcriptional mechanisms. Stimulation with This region contains one AP-2, two EGR-1, and three Sp-1 PGE resulted in increased VEGF-A protein release after2has transcription factor binding sites. To explore this further we measured by ELISA. Quantitative real-time RT-PCR also used constructs with mutations in the Sp-1 or EGR-1 binding showed that PGE increased VEGF-A mRNA after 1 h. Pre- sites. We found no reduction in luciferase levels using con- treatment of the cells with the RNA polymerase II inhibitor structs containing mutated EGR-1 sites, whereas a construct actinomycin D prevented this, suggesting that the increased with mutations in all three Sp-1 sites resulted in loss of lucif- VEGF mRNA was because of transcriptional rather than post- erase activity, suggesting that Sp-1 binding was crucial to transcriptional mechanisms. Furthermore, mRNA stability ex- VEGF induction by PGE . These observations were also sup- periments showed no alteration in mRNA half-life after PGE ported by EMSA results, which showed that PGE increased 2 2 treatment. Collectively these studies suggest that VEGF was Sp-1 but not AP-2 or EGR-1 binding. Specificity of binding was regulated transcriptionally by PGE and this was confirmed by demonstrated by experiments using excess unlabeled oligonu- studies using VEGF promoter luciferase constructs. A few pre- cleotides and supershift with Sp-1 antibody. The EMSA results vious studies have looked at whether PGE increases VEGF were confirmed by the ChIP assay and specific antibody to transcriptionally or post-transcriptionally. PGE -mediated Sp-1. Consistent with a role for Sp-1, VEGF production was VEGF up-regulation was transcriptional in osteoblasts (16) inhibited by the Sp-1 inhibitor mithramycin (43). Using West- and overexpression of myc in B cells increased the initiation of ern blotting we also showed that Sp-1 is a nuclear protein that VEGF mRNA translation (42). is phosphorylated by PGE . To determine key transcription factor binding sites we used Ours are the first studies to show that Sp-1 is involved in PGE Induces VEGF via Sp-1 29999 FIG.9. A, reduction of VEGF protein release by the Sp-1 inhibitor mithramycin A (MTR) and PKA inhibitor H-89. Preincubation with 500 nM and 1 M MTR or 10 M H-89 prior to 24 h culture with 1 M PGE significantly reduced PGE stimulated VEGF measured by ELISA (* and #, p  0.05; ** and ##, p  0.01; *** and ###, p  0.001 by ANOVA). NS, not significant. B, Western blotting showing the nuclear localiza- tion of Sp-1 and increased phosphorylation of 106k Sp-1 by 15 min FIG.8. Agents that increase intracellular cAMP levels mimic 1 M PGE and increase VEGF production. A, concentration re- incubation with 1 M PGE . Lanes 1 and 2 are cytosolic fractions: 2 2 sponse to forskolin (FSK), which directly activates adenylyl cyclase and control (1) and PGE (2). Lanes 3 and 4 are nuclear fractions: control (3) the cAMP analogue, 8-Br-cAMP, increases VEGF, the -adrenergic and PGE (4). GAPDH and nuclear lamin were used as housekeeping agonists. B, isoproterenol (ISO). C, agents that act via cAMP mimic controls. C, PGE , cAMP analogue, and salbutamol and forskolin, which PGE and stimulate VEGF promoter-driven luciferase activity (*, p  increase cellular cAMP, increased luciferase activity of a transfected Sp-1 luciferase reporter construct (**, p  0.01; and ***, p  0.001 by 0.05, **, p  0.01; and ***, p  0.001 by ANOVA). ANOVA). PGE -induced VEGF production, although Sp-1 is important in tory responses depending on receptor subtype binding (28). We the activation of genes involved in tumor proliferation and the focused on the two EP receptors, EP and EP , which activate 2 4 induction of VEGF in response to some other stimuli (11, 34, cAMP. We found that both EP and EP receptors were ex- 2 4 44, 45). For example, VEGF induction by interleukin-1 in pressed in HASMC. This contrasts to a previous study that cardiac myocytes and by tumor necrosis factor- in glioma cells reported EP but not EP receptor expression in HASM (48). 2 4 is mediated through Sp-1 sites (13, 46). In contrast, transform- We confirmed our findings using both RT-PCR and either ing growth factor- induced VEGF via AP-2 transcription fac- Western blotting or fluorescence-activated cell sorter for EP tor binding (47). and EP , respectively, using antibodies designed for these Having shown that VEGF production was transcriptionally methodologies. The fact that both mRNA and protein to EP mediated via Sp-1, we then went on to characterize the pros- receptors was present suggests that these cells do indeed ex- tanoid receptor involved. PGE binds to a family of 7 trans- press EP receptors. Furthermore, experiments with EP and 2 4 2 membrane G protein-coupled membrane receptors, the EP re- EP receptor agonists mirrored the effect of PGE on VEGF 4 2 ceptors. Four subtypes of EP receptors have been described protein production and reporter activity, suggesting that both EP ,EP ,EP , and EP encoded by different genes (27). Each of these receptors are implicated in this process. This is similar 1 2 3 4 subtype is tissue-specific and uses different intracellular sig- to Clarke et al. (49) who suggested that positive regulation of naling mechanisms, suggesting potentially different inflamma- granulocyte colony-stimulating factor by E-Ring 8-isoprostanes 30000 PGE Induces VEGF via Sp-1 1955–1967 in HASMC was mediated by EP and EP receptors. 2 4 14. Wen, F. Q., Liu, X., Manda, W., Terasaki, Y., Kobayashi, T., Abe, S., Fang, Q., EP and EP receptors couple to G protein, which stimu- 2 4 S Ertl, R., Manouilova, L., and Rennard, S. I. (2003) J. Allergy Clin. Immunol. 111, 1307–1318 lates adenylyl cyclase activity increasing the intracellular 15. Knox, A. J., Corbett, L., Stocks, J., Holland, E., Zhu, Y. M., and Pang, L. (2001) cAMP levels resulting in PKA signaling. We performed exper- FASEB J. 15, 2480–2488 iments using a variety of pharmacological tools to probe the 16. Harada, S., Nagy, J. A., Sullivan, K. A., Thomas, K. A., Endo, N., Rodan, G. A., and Rodan, S. B. (1994) J. Clin. Investig. 93, 2490–2496 role of different components of this pathway. Isoproterenol, 17. Harada, S., Rodan, S. B., and Rodan, G. A. (1995) Clin. Orthop. Relat. Res. 313, which elevates cAMP via -adrenoceptors and forskolin, a di- 76–80 18. Ben Av, P., Crofford, L. J., Wilder, R. L., and Hla, T. (1995) FEBS Lett. 372, rect activator of adenylyl cyclase, had similar effects to PGE 83–87 on VEGF protein production and VEGF promoter luciferase 19. Gately, S. (2000) Cancer Metastasis Rev. 19, 19–27 expression, suggesting that cAMP pathways regulate VEGF 20. Gately, S., and Li, W. W. (2004) Semin. Oncol. 31, 2–11 21. Daniel, T. O., Liu, H., Morrow, J. D., Crews, B. C., and Marnett, L. J. (1999) release. Further evidence in support of a role for cAMP was Cancer Res. 59, 4574–4577 obtained from studies using 8-Br-cAMP, a cell-permeable 22. Jones, M. K., Wang, H., Peskar, B. M., Levin, E., Itani, R. M., Sarfeh, I. J., and Tarnawski, A. S. (1999) Nat. Med. 5, 1418–1423 cAMP analogue. Increasing cAMP also increased the activity of 23. Peterson, H. I. (1986) Anticancer Res. 6, 251–253 a transfected Sp-1 luciferase reporter construct. To explore the 24. Pang, L., Pitt, A., Petkova, D., and Knox, A. J. (1998) Clin. Exp. Allergy 28, main downstream target of cAMP, PKA, we studied the effect 1050–1058 25. Hoshino, M., Takahashi, M., and Aoike, N. (2001) J. Allergy Clin. Immunol. of the PKA inhibitor H-89. We found that H-89 markedly in- 107, 295–301 hibited PGE -induced VEGF protein production, suggesting 26. Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D., Fiddes, J. C., and Abraham, J. A. (1991) J. Biol. Chem. 266, 11947–11954 that it was PKA mediated, although it is possible that other 27. Coleman, R. A., Smith, W. L., and Narumiya, S. (1994) Pharmacol. Rev. 46, kinases mediated this effect (50). 205–229 Our studies have relevance for asthma where several immu- 28. Narumiya, S., Sugimoto, Y., and Ushikubi, F. (1999) Physiol. Rev. 79, 1193–1226 nohistochemical studies have shown that COX-2 and VEGF are 29. Pang, L., and Knox, A. J. (1997) Am. J. Physiol. 273, L1132–L1140 both up-regulated (24, 25). They are also of relevance to a wide 30. Pang, L., and Knox, A. J. (1997) Br. J. Pharmacol. 121, 579–587 range of inflammatory and malignant diseases where increased 31. Maruyama, T., Asada, M., Shiraishi, T., Ishida, A., Egashira, H., Yoshida, H., Maruyama, T., Ohuchida, S., Nakai, H., Kondo, K., and Toda, M. (2001) prostanoid production has been implicated in angiogenic pro- Bioorg. Med. Chem. Lett. 11, 2029–2031 cesses mediated via VEGF release. Strategies targeting Sp-1- 32. Tani, K., Naganawa, A., Ishida, A., Egashira, H., Sagawa, K., Harada, H., Ogawa, M., Maruyama, T., Ohuchida, S., Nakai, H., Kondo, K., and Toda, mediated gene transcription may provide a new therapeutic M. (2001) Bioorg. Med. Chem. Lett. 11, 2025–2028 approach to influence remodeling processes. In conclusion, our 33. 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Journal of Biological ChemistryAmerican Society for Biochemistry and Molecular Biology

Published: Aug 26, 2005

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