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Transcriptional Roles of Nuclear Factor κB and Nuclear Factor-Interleukin-6 in the Tumor Necrosis Factor α-Dependent Induction of Cyclooxygenase-2 in MC3T3-E1 Cells

Transcriptional Roles of Nuclear Factor κB and Nuclear Factor-Interleukin-6 in the Tumor Necrosis... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 52, Issue of December 29, pp. 31315–31320, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Transcriptional Roles of Nuclear Factor kB and Nuclear Factor- Interleukin-6 in the Tumor Necrosis Factor a-Dependent Induction of Cyclooxygenase-2 in MC3T3-E1 Cells* (Received for publication, August 21, 1995, and in revised form, October 6, 1995) Kei Yamamoto‡, Toshiya Arakawa§, Natsuo Ueda, and Shozo Yamamoto From the Department of Biochemistry, The University of Tokushima, School of Medicine, Tokushima 770, Japan When a mouse osteoblastic cell line MC3T3-E1 was boxanes from arachidonic acid. The enzyme is a bifunctional cultured in the presence of tumor necrosis factor a enzyme, and has the cyclooxygenase activity (from arachidonic (TNFa), the release of prostaglandin E and the cy- acid to PGG ) and the hydroperoxidase activity (from PGG to 2 2 clooxygenase activity increased in a dose- and time-de- PGH ). Two isozymes referred to as cyclooxygenase-1 and -2 in pendent manner. The increase of the enzyme activity this paper are found in the mammalian tissues (1, 2). Cyclooxy- was attributed mostly to the induction of cyclooxygen- genase-1 is generally considered as a constitutive enzyme, ase-2 rather than cyclooxygenase-1 as judged by the in- while cyclooxygenase-2 is rapidly and transiently induced by hibitory effect of NS398, Western blotting, and Northern various cytokines, hormones, and tumor promoters (1, 2). In blotting. In this system we attempted to elucidate the view of such an inducible nature the cyclooxygenase-2 has transcriptional regulation of the cyclooxygenase-2 gene. recently been a subject of active molecular biological investiga- As examined by the luciferase assay, two positive regu- tions. Promoter regions of the cyclooxygenase-2 genes of mouse latory regions (2186 to 2131 and 2512 to 2385 base (3), rat (4), and human (5) have been cloned and sequenced. pairs) were found in the 5*-flanking promoter region of Regardless of the animal species these promoter regions con- the mouse cyclooxygenase-2 gene in the TNFa-stimu- tained a canonical TATA box and various putative transcrip- lated cells. The former included putative NF-IL6 (C/ tional regulatory elements such as CRE, NF-IL6 (C/EBPb), EBPb) and AP2 elements, and the latter contained the AP2, SP1, NFkB, and GATA box. Among these elements, CRE NFkB motif. A DNA probe including the NF-IL6 and AP2 (6, 7) and C/EBPb (8) were shown to act as positive regulatory sites gave positive bands upon electrophoretic mobility elements for the cyclooxygenase-2 transcription. shift assay using the nuclear extracts of MC3T3-E1 cells. An osteogenic MC3T3-E1 cell line was established from new- The bands were supershifted by the addition of anti-NF- born mouse calvaria. The cells differentiate into osteoblasts, IL6 antibody but not by anti-AP2 antibody. A probe in- and show calcification in vitro (9, 10). With this cell line we cluding the NFkB site also gave positive bands, which were supershifted by anti-NFkB p50 and p65 antibodies. have been investigating the cyclooxygenase induction by epi- Furthermore, when the motif of NF-IL6 or NFkB or both dermal growth factor (11, 12), transforming growth factor b was subjected to point mutation, the luciferase activity (12), epinephrine (13), and PGs (14). More recently the cy- was markedly reduced. These data suggested a potential clooxygenase-2 induction was demonstrated with the role of both NF-IL6 and NFkB in the induction of cy- MC3T3-E1 cells by PGs (15, 16) and by transforming growth clooxygenase-2 by TNFa. factor b (16). In the present work, we attempted to find out the transcriptional regulatory factors involved in the induction of cyclooxygenase-2 by TNFa in the MC3T3-E1 cells. Prostaglandin (PG) endoperoxide synthase (EC 1.14.99.1) is EXPERIMENTAL PROCEDURES the key enzyme in the biosynthetic pathway of PGs and throm- Materials—The materials used in this work were purchased as fol- 14 35 lows: [1- C]arachidonic acid (2.1 GBq/mmol) and S-labeled anti-rab- bit IgG whole antibody (74 TBq/mmol) from Amersham International * This work was supported by grants-in-aid for scientific research (Bucks, United Kingdom); Immobilon-P transfer membrane from Mil- from the Ministry of Education, Science and Culture of Japan, the lipore (Bedford, MA); [g- P]ATP (220 TBq/mmol) from DuPont NEN; Japanese Foundation of Metabolism and Disease, the Japan Founda- fetal bovine serum and newborn bovine serum from Irvine Scientific tion for Applied Enzymology, Ono Pharmaceutical Company, Kissei Pharmaceutical Company, Sankyo Company, Takeda Pharmaceutical (Santa Ana, CA); penicillin G and phenylmethylsulfonyl fluoride from Industry, and the Japan Research Foundation for Clinical Pharmacol- Sigma; a-modified Eagle’s minimum essential medium (a-MEM), re- ogy. The costs of publication of this article were defrayed in part by the combinant mouse TNFa, and lipofectAMINE from Life Technologies, payment of page charges. This article must therefore be hereby marked Inc. (Gaithersburg, MD); ISOGEN from Nippon Gene (Tokyo, Japan); “advertisement” in accordance with 18 U.S.C. Section 1734 solely to EMBL3 mouse genomic library from Clonetech (Palo Alto, CA); Pica indicate this fact. Gene luciferase assay system from Toyo Ink (Tokyo, Japan); poly(dI- ‡ On leave from the Dept. of Biological Science and Technology, dC)-poly(dI-dC) from Pharmacia (Uppsala, Sweden); anti-AP2, anti-C/ Faculty of Engineering, The University of Tokushima, Minami Josan- EBPb (NF-IL6), and anti-NFkB p50 antibodies from Santa Cruz Bio- jima-cho, Tokushima, 770. technology, Inc. (Santa Cruz, CA); anti-NFkB p65 antibody from § Present address: Dept. of Biochemistry, Michigan State University, Serotec (Oxford, United Kingdom); and purified NFkB p50 protein from East Lansing, MI 48824. Promega (Madison, WI). MC3T3-E1 cell line was kindly provided by Dr. ¶ To whom correspondence should be addressed: Dept. of Biochemis- H. Kodama of Ohu University, luciferase plasmid pXP-1 by Dr. T. Sakai try, The University of Tokushima, School of Medicine, Kuramoto-cho, of Kyoto Prefectural University, and expression vectors containing the Tokushima 770, Japan. Tel.: 81-886-31-3111 (ext. 2220); Fax: genes of NF-IL6 and NFkB p50 by Dr. S. Akira of Osaka University. 81-886-33-6409. NS398 was donated by Taisho Pharmaceutical Co. (Saitama, Japan). The abbreviations used are: PG, prostaglandin; CRE, cyclic AMP Cell Culture—MC3T3-E1 cells (7.5 3 10 cells/dish) were plated in response element; NF-IL6, nuclear factor IL-6; C/EBP, CCAAT en- 150-mm plastic dishes with 30 ml of a-MEM containing 10% fetal hancer binding protein; NFkB, nuclear factor kB; a-MEM, a-modified bovine serum and 100 units/ml of penicillin G as described previously Eagle’s minimum essential medium; TNFa, tumor necrosis factor a; bp, base pairs; IL, interleukin. (11). The dishes were placed in a humidified 7% CO , 93% air incubator This is an Open Access article under the CC BY license. 31316 Transcriptional Regulation of Cyclooxygenase-2 at 37 °C. The cells were subcultured every 3 days. Determination of PGE Synthesis—MC3T3-E1 cells (6 3 10 cells/ dish) were plated in 35-mm dishes with 2 ml of a-MEM containing 10% fetal bovine serum. Confluent cultures were usually obtained on the 4th day. Then, the medium was changed to a-MEM supplemented with 2% newborn bovine serum, and TNFa was added. At indicated time inter- vals, the culture medium was removed and subjected to radioimmuno- assay for PGE (11). Cyclooxygenase Assay—MC3T3-E1 cells (9 3 10 cells/dish) were plated in 150-mm dishes with 30 ml of a-MEM containing 10% fetal bovine serum, and treated with TNFa. The cells were scraped from the dishes at various time intervals, suspended in 300 mlof20mM Tris-HCl (pH 7.4) containing 5 mM tryptophan, and sonicated twice each for 3 s at 20 kHz. The sonicates were incubated with 10 mM [1- C]arachidonic acid (50,000 cpm in 5 ml of ethanol) for 2 min at 24 °C in a 100-ml assay mixture containing 100 mM Tris-HCl (pH 8.0), 2 mM hematin, and 5 mM tryptophan. The reaction products were separated by TLC (15), and detected by a BAS2000 imaging analyzer (Fujix, Tokyo, Japan). Protein concentration was determined by the method of Lowry et al. (17) with bovine serum albumin as standard. FIG.1. Effects of TNFa on the PGE synthesis and the cy- Western Blotting—A polyclonal anti-cyclooxygenase-2 was prepared clooxygenase activity in MC3T3-E1 cells. The cells were incubated with a peptide corresponding amino acids 579–594 of murine enzyme, with TNFa at various concentrations for 12 h (A and C) or at a concen- and provided by Dr. Yoko Hayashi of this laboratory. The sonicates of tration of 20 ng/ml for the indicated time intervals (B and D). PGE MC3T3-E1 cells were subjected to 10% polyacrylamide gel electrophore- content in the culture medium was determined by radioimmunoassay sis in the presence of 0.1% SDS (10 mg of protein/lane). The protein (A and B). The cells were incubated in the absence (closed column or circle) or presence (open column or circle)of15 mM NS398, and the bands were transferred to an Immobilon-P membrane, which was in- cyclooxygenase activity of the sonicated cells was determined using cubated with the polyclonal antibody against cyclooxygenase-2 and [1- C]arachidonic acid as substrate (C and D). Data are means 6 S.E. then with S-labeled anti-rabbit IgG whole antibody as the second of triplicate determinations (A and B). C and D were repeated three antibody. Radioactivity of the immunocomplex was quantified by a times, and similar results were obtained. N.D., below detection limit. BAS2000 imaging analyzer. Northern Blotting—Total RNA was extracted from MC3T3-E1 cells using ISOGEN (a mixture of guanidium isothiocyanate and phenol) according to the manufacturer’s instruction. Northern blotting was 4% polyacrylamide gel electrophoresis gels at a constant 150 V for 2 h. performed as described previously (15, 18). The relative radioactivity of Distribution of the radioactivity on the dried gel was analyzed by a each band was estimated using a BAS2000. BAS2000 imaging analyzer. Preparation of the 59-Flanking Region of Mouse Cyclooxygenase-2 RESULTS Gene and Construction of Luciferase Reporter Vectors—We screened a mouse genomic library constructed in EMBL3 to clone the 59-flanking Induction of Cyclooxygenase-2 by TNFa—When MC3T3-E1 region of the mouse cyclooxygenase-2 gene by the method as described cells were incubated with various concentrations of TNFa for previously for 12-lipoxygenase (19). DNA fragments of mouse cyclooxy- 12 h, there was a dose-dependent increase in the amount of genase-2 promoter regions of various lengths or their point mutants PGE released into the medium (Fig. 1A). After the addition of were prepared from the clone by polymerase chain reaction, and were inserted into the plasmid pXP-1 as described previously for 12-lipoxy- 20 ng/ml TNFa, the PGE release increased with a lag time of genase (20). about 1 h, and reached a maximum at 9 h (Fig. 1B). As shown Transfection of Plasmids to MC3T3-E1 Cells and Luciferase Assay— in Fig. 1, C and D, TNFa increased the cyclooxygenase activity MC3T3-E1 cells (1.4 3 10 cells/dish) were cultured for 2 days in 60-mm dose and time dependently, and the enzyme activity continued dishes with 5 ml of a-MEM containing 10% fetal bovine serum. For to increase for 24 h to a specific activity of 0.8–2 nmol/2 min/mg transfection the subconfluent cells were treated with plasmid DNA (2.3 of protein. mg) containing cyclooxygenase-2 promoter and luciferase reporter gene, standard plasmid DNA (1.2 mg) containing the b-galactosidase gene, NS398, a specific inhibitor of cyclooxygenase-2 (22), inhibited and 9.2 ml of lipofectAMINE in 2.3 ml of serum-free a-MEM for4hat the enzyme activity almost completely at 15 mM concentration 37 °C according to the manufacturer’s instructions. The medium was (Fig. 1, C and D), suggesting that most of the increased enzyme changed to 5 ml of a-MEM with 10% fetal bovine serum, and the activity was attributable to cyclooxygenase-2 rather than cy- transfected cells were further incubated for 3 days. Then the cells were clooxygenase-1. To confirm this finding we carried out Western stimulated by 20 ng/ml TNFa in the presence of a-MEM supplemented blotting with an antibody specific for cyclooxygenase-2 (Fig. with 2% newborn bovine serum. After 12 h the cells were scraped from the dishes, and the luciferase activity was measured by a Lumat 2A). Without TNFa the cyclooxygenase-2 protein was undetect- LB9501 luminometer (Berthold, Germany) using a Pica Gene luciferase able, and the addition of 20 ng/ml TNFa increased the enzyme assay system according to the manufacturer’s instructions. The lucifer- amount time dependently up to 9 h. There was a slight but ase activities were normalized on the basis of b-galactosidase activities reproducible decrease around 12 h, followed by an increase which were assayed as described previously (20). 5 again. Preparation of Nuclear Extracts—MC3T3-E1 cells (9 3 10 cells/dish) Furthermore, as shown in Fig. 2B, the change in mRNA level were plated in 10 150-mm dishes with 30 ml of a-MEM containing 10% fetal bovine serum, and stimulated with various concentrations of was followed by Northern blot analysis. The addition of TNFa TNFa. After 1 h the cells were harvested, and nuclear extracts were brought about a biphasic increase in the cyclooxygenase-2 prepared as described previously (20). mRNA. First, there was a rapid 10-fold increase reaching a Electrophoretic Mobility Shift Assay—We synthesized the five oligo- maximum at 2 h, followed by a decrease at 3 h. Then, the nucleotides as shown in Fig. 4. The complementary oligonucleotides mRNA increased again at 6–12 h. However, after the first were annealed to the corresponding oligonucleotides as described by transient peak the presence of 15 mM NS398 reduced the second Berger and Kimmel (21), and the double-stranded oligonucleotides were purified electrophoretically on 15% polyacrylamide gel, end-labeled peak. The cyclooxygenase-1 mRNA was not detectable under with [g- P]ATP, and used as probes. The binding of the probes (10,000 these experimental conditions. cpm, about 10 fmol) to the nuclear extracts (2.3 mg protein) was per- These results presented in Figs. 1 and 2 demonstrated a formed in a 20-ml mixture containing 5 mg of poly(dI-dC)-poly(dI-dC), 15 rapid and marked induction of cyclooxygenase-2 by TNFa. The mM Tris-HCl at pH 7.5, 1 mM EDTA, 100 mM KCl, 5 mM MgCl , 12% second peak of the cyclooxygenase-2 may be attributable to the glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothre- enzyme which was induced by accumulating PGE produced by itol. For the supershift experiment, each antibody was added to the 2 mixture. The mixture was incubated at 25 °C for 30 min, and applied to the cyclooxygenase-2 at the first peak. Transcriptional Regulation of Cyclooxygenase-2 31317 (lanes 3–6). The complex a was hardly detectable before the addition of TNFa (lane 2). Considering the subunit structure of NFkB (p50 and p65), the g1 probe was incubated with the purified NFkB p50, and a band appeared at the position of complex a (lane 7). The complex a was supershifted to complex b by the addition of anti-p50 antibody (lane 10), and was supershifted to complex g by the addition of anti-p65 antibody (lane 11). The binding was not observed using the probe g2 with a mutation in NFkB motif (lanes 13 and 14). When gA probe was incubated with the nuclear extracts, three complexes (a, b, and c) appeared with increasing density depending on the amount of TNFa (Fig. 6, lanes 3–6). These complexes were supershifted to bands d, f, and g by the addi- tion of antibody against NF-IL6 (lane 8). The binding was scarcely observed by the use of the probe gC with a mutation in NF-IL6 (lanes 15–19). Moreover, the complex a was super- shifted to complex e by the addition of anti-NFkB p50 (lane 9). The bands a, b, and c were not supershifted by anti-AP2 anti- FIG.2. Western and Northern blot analyses of cyclooxygen- body (lane 7), and the binding profile was not affected by the ase-2 protein in MC3T3-E1 cells. A, the cells were incubated with 20 use of the probe gB with a mutation in AP2 (lanes 10–14). ng/ml TNFa. At the indicated time intervals, the cells were harvested These data suggested that NFkB and NF-IL6 were increased and subjected to Western blotting for cyclooxygenase-2 as described by the addition of TNFa in a dose-dependent manner. under “Experimental Procedures.” The immunocomplex was visualized (inset) and quantified (closed circles) utilizing S-labeled second anti- Possible Interaction of Both NFkB and NF-IL6 Binding body by a BAS2000 imaging analyzer. The experiment was repeated Sites—For mutation analysis of the promoter region, we con- three times, and similar results were obtained. PSL, photo-stimulated structed luciferase vectors including mouse cyclooxygenase-2 luminescence. B, the cells were incubated with 20 ng/ml TNFa in the promoter region (2512 to 1123 bp) with site-specific mutations absence (closed circle) or presence (open circle)of15 mM NS398 for indicated time periods. Total RNA (10 mg) was applied to Northern blot (Fig. 7). Each vector was transfected to MC3T3-E1 cells, and analysis. The relative amount of cyclooxygenase-2 mRNA was corrected the luciferase activity was measured as described above. As for b-actin mRNA. shown in Fig. 7, the luciferase activity decreased by 63% in a mutant of NFkB, 82% in a mutant of NF-IL6, and only 19% in Functional Activity of Cyclooxygenase-2 Promoters—As illus- a mutant of AP2 site. By mutation of both NFkB and NF-IL6, trated at the top of Fig. 3, about 600 bp of the 59-flanking region the luciferase activity was lost to the level of pXP1. These data of mouse cyclooxygenase-2 gene contained various putative suggested that both NFkB and NF-IL6 in combination acted as response elements; MEF-2 (a muscle-specific DNA binding pro- transcription factors for the induction of cyclooxygenase-2. tein, 2490 to 2481 bp), NFkB(2401 to 2393 bp), SP1 (2239 to Furthermore, expression vectors containing the genes of NF- 2234 bp), AP2 (2150 to 2142 bp), NF-IL6 (2138 to 2130 bp), IL6 and NFkB p50, respectively, were cotransfected to CRE (256 to 251 bp), and TATA box (230 to 225 bp). The MC3T3-E1 cells together with a luciferase plasmid containing sequences of 59-GGGATTCCC-39 (2401 to 2393 bp) and 59- the promoter region of cyclooxygenase-2 (2512 to 1123 bp). TTGCGCAAC-39 (2138 to 2130 bp) were identified as NFkB The luciferase activity increased depending on the amount of and NF-IL6 elements on the basis of their concensus sequences NF-IL6 cDNA (data not shown). A similar experiment using of 59-GGGA(A/C)TN(T/C)CC-39 and 59-T(T/G)NNGNAA(T/G)- the expression vector for NFkB p50 has so far been 39, respectively, as described previously (23). unsuccessful. For deletion analysis of the promoter region, we constructed DISCUSSION luciferase vectors of various lengths covering the region from 2621 to 241 bp (Fig. 3). Each vector was transfected to It was reported previously that TNFa increased the PGE MC3T3-E1 cells by the lipofection method, and the luciferase production and the cyclooxygenase activity in mouse osteoblas- activity of the cell lysate was measured at 12 h after the tic cell line MC3T3-E1 (24–26). We confirmed these findings addition of 20 ng/ml TNFa. The addition of TNFa markedly (Fig. 1), and demonstrated that the increased enzyme activity stimulated the luciferase activity (closed column versus slashed was attributed mostly to the induction of cyclooxygenase-2 column). The luciferase activity decreased when two regions according to the inhibitory effect of NS398, Western blotting, (2512 to 2385 bp, 2186 to 2131 bp) were deleted, and these and Northern blotting (Figs. 1 and 2). The mechanism of the regions were presumed to have positive response elements. We biphasic increase of cyclooxygenase-2 protein and mRNA (Fig. noted NFkB consensus element (2401 to 2393 bp), AP2 ele- 2) may be discussed as follows. First, TNFa activates phospho- ment (2150 to 2142 bp), and NF-IL6 element (2138 to 2130 lipase A (as reported in Ref. 27) and induces cyclooxygenase-2, bp). resulting in an increased production of PGE . Then, the pro- Electrophoretic Mobility Shift Assay Targeting Positive Reg- duced PGE binds to a PGE receptor, and increases the cy- ulatory Elements—For further identification of these positive clooxygenase-2 protein and mRNA again. Previously we re- regulatory elements, we prepared five double-stranded oligo- ported the cyclooxygenase-2 induction by various PGs, nucleotide probes (g1, g2, gA, gB, and gC). As shown in Fig. 4, including PGE which was a major arachidonate metabolite in g1 (2409 to 2385 bp) contained NFkB and their vicinity. gA this cell line (15). (2155 to 2121 bp) contained AP2 and NF-IL6. g2, gB, and gC Since the addition of TNFa brought about such a typical and were mutant probes for NFkB, AP2, and NF-IL6, respectively. prominent induction of cyclooxygenase-2 in MC3T3-E1 cells, We carried out electrophoretic mobility shift assay using the we attempted to elucidate the transcriptional regulation of the nuclear extracts of MC3T3-E1 cells preincubated with TNFa cyclooxygenase-2 gene in this system. The luciferase assay for 1 h. As shown in Fig. 5, when g1 probe was incubated with (Figs. 3 and 7) and electrophoretic mobility shift assay (Figs. 5 the nuclear extracts, a broad band of complex a was observed and 6) revealed two positive regulatory elements on the 59- with increasing density depending on the amount of TNFa flanking region of mouse cyclooxygenase-2 gene: NFkB motif 31318 Transcriptional Regulation of Cyclooxygenase-2 FIG.3. Construction of luciferase expression vectors and transfection analysis of cyclooxygenase-2 gene promoters. Putative consensus sequences in the 59-upstream region of mouse cyclooxygenase-2 gene are illustrated in the upper left. Each deleted promoter fragment was ligated into a luciferase plasmid. Numbers indicate distance in base pairs from the start of transcription. MC3T3-E1 cells (1.4 3 10 cells) were transfected with various luciferase plasmids (2.3 mg) including different promoter regions of mouse cyclooxygenase-2 gene and b-galactosidase plasmid (1.2 mg) in combination using the lipofection method as described under “Experimental Procedures.” The cells were cultured to confluency for 3 days, and incubated with (closed column) or without (slashed column) TNFa (20 ng/ml) for 12 h. The luciferase activity was assayed, and the results were normalized with the b-galactosidase activity. Data are means 6 S.E. of triplicate determinations. FIG.4. Syntheses of double stranded oligonucleotides as probes for mouse cyclooxygenase-2 gene. g1, 2409 to 2385 bp including the putative NFkB binding site indicated by a solid box; g2, GA of the NFkB was changed to CC; gA, 2155 to 2121 bp including AP2 and NF-IL6; gB, GC of AP2 was changed to TT; gC, TTGCG of the NF-IL6 was changed to CCGCT. FIG.5. Electrophoretic mobility shift assay targeting the (59-GGGATTCCC-39) located at nucleotides 2401 to 2393 bp NFkB site. Probe g1 was incubated for 0.5 h with the nuclear extracts (2.3 mg protein) of MC3T3-E1 cells treated with 0 ng/ml (lane 3), 0.2 and NF-IL6 motif (also referred to as C/EBPb,59-TTGCG- ng/ml (lane 4), 2 ng/ml (lane 5), and 20 ng/ml (lanes 6 and 9) TNFa. The CAAC-39)at 2138 to 2130 bp. NF-IL6 was originally identified nuclear extracts before the addition of TNFa were also incubated with as a nuclear factor binding to the IL-1 response element of the g1 (lane 2). Anti-NFkB p50 (lane 10) or anti-NFkB p65 (lane 11) anti- human IL-6 gene (28). By cDNA cloning NF-IL6 was found to body was incubated together with the nuclear extracts from the cells be a member of the CCAAT enhancer-binding protein (C/EBP) stimulated with 20 ng/ml TNFa. Purified NFkB p50 protein was incu- bated with probe g1 (lane 7). Probe g2 with a mutation for NFkB was family of basic-leucine zipper (bZIP) transcription factors (29). incubated with the nuclear extracts from the cells stimulated with 20 Subsequent studies suggested a regulatory role of NF-IL6 for ng/ml TNFa (lanes 13 and 14). Anti-NFkB p50 antibody was also the genes encoding many acute-phase proteins and cytokines present (lane 14). Lanes 1, 8, and 12 were control runs without the (30). The NF-IL6 motif on the rat cyclooxygenase-2 gene (59- nuclear extracts. Shifted bands indicated by a, b, and g are described in the text. TTATGCAAT-39 located at 2140 to 2132 bp) was previously reported to contribute to the induction of cyclooxygenase-2 by forskolin, follicle-stimulating hormone, and luteinizing hor- mone in granulosa cells (4, 8). Recently it was reported that the the involvement of the CRE motif in the TNFa-dependent cyclooxygenase-2 induction was mediated by an activating cyclooxygenase-2 induction in MC3T3-E1 cells. The AP2 ele- transcription factor/CRE element (59-CGTCACGTG-39 at 256 ment (59-CCGCTGCGG-39, 2150 to 2142 bp) close to the to 248 bp) on the mouse cyclooxygenase-2 gene promoter (7) or NF-IL6 was shown to be inactive (Figs. 3, 6, and 7). by CRE element (59-TTCGTCA-39 at 259 to 253) in the human NFkB was originally found as a binding protein to immuno- cyclooxygenase-2 gene (6). However, we could not demonstrate globulin k light-chain enhancer (31), and it is now considered as Transcriptional Regulation of Cyclooxygenase-2 31319 Individual binding sites for NF-IL6 and NFkB are present in the promoter of the IL-6 gene, and the cooperation of these two factors plays an important role in transcription of IL-6 (37). A similar activation was reported for the IL-8 promoter, which also contains both NF-IL6 and NFkB binding sites (37). The NFkB p50 and NF-IL6 proteins directly interact in vitro, and the Rel homology domain and leucine-zipper motif, respec- tively, are important for this interaction (38). TNFa enhanced the promoter activity of the cyclooxygen- ase-2 gene of MC3T3-E1 cells (Fig. 3), and increased the amounts of NFkB and NF-IL6 bound to the DNA probes in- cluding the corresponding cis-elements (Figs. 5 and 6). The results were in parallel with the marked induction of cyclooxy- genase-2 in the TNFa-stimulated cells (Figs. 1 and 2). It was shown that TNFa activated an endosomal acidic sphingomyeli- nase through the 55-kDa TNF receptor and then the produced ceramide triggered the activation of NFkB (27). TNFa also induced NF-IL6 in the mouse lung, liver, and kidney (30). By analogy we presume that TNFa triggers the activation or in- FIG.6. Electrophoretic mobility shift assay targeting AP2 and duction of both NFkB and NF-IL6, resulting in the induction of NF-IL6 sites. MC3T3-E1 cells were incubated for 1 h with various concentrations of TNFa. The nuclear extracts (2.3 mg of protein) from cyclooxygenase-2 in MC3T3-E1 cells. We should note that there the cells stimulated with 0 ng/ml (lane 3), 0.2 ng/ml (lane 4), 2 ng/ml are NFkB and NF-IL6 sites in human (5) and rat (4) cyclooxy- (lane 5), and 20 ng/ml (lanes 6–9, 11–14, and 16–19) TNFa were genase-2 genes: human, 59-GGGATTCCC-39 (2447 to 2439 bp, incubated with probe gA (wild type, lanes 3–9), gB (mutation for AP2, NFkB element) and 59-TTACGCAAT-39 (2132 to 2124 bp, lanes 11–14), or gC (mutation for NF-IL6, lanes 16–19). The nuclear extracts before the addition of TNFa was also incubated with gA (lane NF-IL6 element); rat, 59-GGGATTCCC-39 (2403 to 2395 bp, 2). Anti-AP2 (lanes 7, 12 and 17), anti-NF-IL6 (lanes 8, 13, and 18), or NFkB element) and 59-TTATGCAAT-39 (2140 to 2132 bp, NF- anti-NFkB p50 (lanes 9, 14, and 19) antibody was incubated together IL6 element). with the nuclear extracts. 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A. 89, 8145–8149 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Transcriptional Roles of Nuclear Factor κB and Nuclear Factor-Interleukin-6 in the Tumor Necrosis Factor α-Dependent Induction of Cyclooxygenase-2 in MC3T3-E1 Cells

Yamamoto, Kei; Arakawa, Toshiya; Ueda, Natsuo; Yamamoto, Shozo
Journal of Biological Chemistry – Dec 1, 1995
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 52, Issue of December 29, pp. 31315–31320, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Transcriptional Roles of Nuclear Factor kB and Nuclear Factor- Interleukin-6 in the Tumor Necrosis Factor a-Dependent Induction of Cyclooxygenase-2 in MC3T3-E1 Cells* (Received for publication, August 21, 1995, and in revised form, October 6, 1995) Kei Yamamoto‡, Toshiya Arakawa§, Natsuo Ueda, and Shozo Yamamoto From the Department of Biochemistry, The University of Tokushima, School of Medicine, Tokushima 770, Japan When a mouse osteoblastic cell line MC3T3-E1 was boxanes from arachidonic acid. The enzyme is a bifunctional cultured in the presence of tumor necrosis factor a enzyme, and has the cyclooxygenase activity (from arachidonic (TNFa), the release of prostaglandin E and the cy- acid to PGG ) and the hydroperoxidase activity (from PGG to 2 2 clooxygenase activity increased in a dose- and time-de- PGH ). Two isozymes referred to as cyclooxygenase-1 and -2 in pendent manner. The increase of the enzyme activity this paper are found in the mammalian tissues (1, 2). Cyclooxy- was attributed mostly to the induction of cyclooxygen- genase-1 is generally considered as a constitutive enzyme, ase-2 rather than cyclooxygenase-1 as judged by the in- while cyclooxygenase-2 is rapidly and transiently induced by hibitory effect of NS398, Western blotting, and Northern various cytokines, hormones, and tumor promoters (1, 2). In blotting. In this system we attempted to elucidate the view of such an inducible nature the cyclooxygenase-2 has transcriptional regulation of the cyclooxygenase-2 gene. recently been a subject of active molecular biological investiga- As examined by the luciferase assay, two positive regu- tions. Promoter regions of the cyclooxygenase-2 genes of mouse latory regions (2186 to 2131 and 2512 to 2385 base (3), rat (4), and human (5) have been cloned and sequenced. pairs) were found in the 5*-flanking promoter region of Regardless of the animal species these promoter regions con- the mouse cyclooxygenase-2 gene in the TNFa-stimu- tained a canonical TATA box and various putative transcrip- lated cells. The former included putative NF-IL6 (C/ tional regulatory elements such as CRE, NF-IL6 (C/EBPb), EBPb) and AP2 elements, and the latter contained the AP2, SP1, NFkB, and GATA box. Among these elements, CRE NFkB motif. A DNA probe including the NF-IL6 and AP2 (6, 7) and C/EBPb (8) were shown to act as positive regulatory sites gave positive bands upon electrophoretic mobility elements for the cyclooxygenase-2 transcription. shift assay using the nuclear extracts of MC3T3-E1 cells. An osteogenic MC3T3-E1 cell line was established from new- The bands were supershifted by the addition of anti-NF- born mouse calvaria. The cells differentiate into osteoblasts, IL6 antibody but not by anti-AP2 antibody. A probe in- and show calcification in vitro (9, 10). With this cell line we cluding the NFkB site also gave positive bands, which were supershifted by anti-NFkB p50 and p65 antibodies. have been investigating the cyclooxygenase induction by epi- Furthermore, when the motif of NF-IL6 or NFkB or both dermal growth factor (11, 12), transforming growth factor b was subjected to point mutation, the luciferase activity (12), epinephrine (13), and PGs (14). More recently the cy- was markedly reduced. These data suggested a potential clooxygenase-2 induction was demonstrated with the role of both NF-IL6 and NFkB in the induction of cy- MC3T3-E1 cells by PGs (15, 16) and by transforming growth clooxygenase-2 by TNFa. factor b (16). In the present work, we attempted to find out the transcriptional regulatory factors involved in the induction of cyclooxygenase-2 by TNFa in the MC3T3-E1 cells. Prostaglandin (PG) endoperoxide synthase (EC 1.14.99.1) is EXPERIMENTAL PROCEDURES the key enzyme in the biosynthetic pathway of PGs and throm- Materials—The materials used in this work were purchased as fol- 14 35 lows: [1- C]arachidonic acid (2.1 GBq/mmol) and S-labeled anti-rab- bit IgG whole antibody (74 TBq/mmol) from Amersham International * This work was supported by grants-in-aid for scientific research (Bucks, United Kingdom); Immobilon-P transfer membrane from Mil- from the Ministry of Education, Science and Culture of Japan, the lipore (Bedford, MA); [g- P]ATP (220 TBq/mmol) from DuPont NEN; Japanese Foundation of Metabolism and Disease, the Japan Founda- fetal bovine serum and newborn bovine serum from Irvine Scientific tion for Applied Enzymology, Ono Pharmaceutical Company, Kissei Pharmaceutical Company, Sankyo Company, Takeda Pharmaceutical (Santa Ana, CA); penicillin G and phenylmethylsulfonyl fluoride from Industry, and the Japan Research Foundation for Clinical Pharmacol- Sigma; a-modified Eagle’s minimum essential medium (a-MEM), re- ogy. The costs of publication of this article were defrayed in part by the combinant mouse TNFa, and lipofectAMINE from Life Technologies, payment of page charges. This article must therefore be hereby marked Inc. (Gaithersburg, MD); ISOGEN from Nippon Gene (Tokyo, Japan); “advertisement” in accordance with 18 U.S.C. Section 1734 solely to EMBL3 mouse genomic library from Clonetech (Palo Alto, CA); Pica indicate this fact. Gene luciferase assay system from Toyo Ink (Tokyo, Japan); poly(dI- ‡ On leave from the Dept. of Biological Science and Technology, dC)-poly(dI-dC) from Pharmacia (Uppsala, Sweden); anti-AP2, anti-C/ Faculty of Engineering, The University of Tokushima, Minami Josan- EBPb (NF-IL6), and anti-NFkB p50 antibodies from Santa Cruz Bio- jima-cho, Tokushima, 770. technology, Inc. (Santa Cruz, CA); anti-NFkB p65 antibody from § Present address: Dept. of Biochemistry, Michigan State University, Serotec (Oxford, United Kingdom); and purified NFkB p50 protein from East Lansing, MI 48824. Promega (Madison, WI). MC3T3-E1 cell line was kindly provided by Dr. ¶ To whom correspondence should be addressed: Dept. of Biochemis- H. Kodama of Ohu University, luciferase plasmid pXP-1 by Dr. T. Sakai try, The University of Tokushima, School of Medicine, Kuramoto-cho, of Kyoto Prefectural University, and expression vectors containing the Tokushima 770, Japan. Tel.: 81-886-31-3111 (ext. 2220); Fax: genes of NF-IL6 and NFkB p50 by Dr. S. Akira of Osaka University. 81-886-33-6409. NS398 was donated by Taisho Pharmaceutical Co. (Saitama, Japan). The abbreviations used are: PG, prostaglandin; CRE, cyclic AMP Cell Culture—MC3T3-E1 cells (7.5 3 10 cells/dish) were plated in response element; NF-IL6, nuclear factor IL-6; C/EBP, CCAAT en- 150-mm plastic dishes with 30 ml of a-MEM containing 10% fetal hancer binding protein; NFkB, nuclear factor kB; a-MEM, a-modified bovine serum and 100 units/ml of penicillin G as described previously Eagle’s minimum essential medium; TNFa, tumor necrosis factor a; bp, base pairs; IL, interleukin. (11). The dishes were placed in a humidified 7% CO , 93% air incubator This is an Open Access article under the CC BY license. 31316 Transcriptional Regulation of Cyclooxygenase-2 at 37 °C. The cells were subcultured every 3 days. Determination of PGE Synthesis—MC3T3-E1 cells (6 3 10 cells/ dish) were plated in 35-mm dishes with 2 ml of a-MEM containing 10% fetal bovine serum. Confluent cultures were usually obtained on the 4th day. Then, the medium was changed to a-MEM supplemented with 2% newborn bovine serum, and TNFa was added. At indicated time inter- vals, the culture medium was removed and subjected to radioimmuno- assay for PGE (11). Cyclooxygenase Assay—MC3T3-E1 cells (9 3 10 cells/dish) were plated in 150-mm dishes with 30 ml of a-MEM containing 10% fetal bovine serum, and treated with TNFa. The cells were scraped from the dishes at various time intervals, suspended in 300 mlof20mM Tris-HCl (pH 7.4) containing 5 mM tryptophan, and sonicated twice each for 3 s at 20 kHz. The sonicates were incubated with 10 mM [1- C]arachidonic acid (50,000 cpm in 5 ml of ethanol) for 2 min at 24 °C in a 100-ml assay mixture containing 100 mM Tris-HCl (pH 8.0), 2 mM hematin, and 5 mM tryptophan. The reaction products were separated by TLC (15), and detected by a BAS2000 imaging analyzer (Fujix, Tokyo, Japan). Protein concentration was determined by the method of Lowry et al. (17) with bovine serum albumin as standard. FIG.1. Effects of TNFa on the PGE synthesis and the cy- Western Blotting—A polyclonal anti-cyclooxygenase-2 was prepared clooxygenase activity in MC3T3-E1 cells. The cells were incubated with a peptide corresponding amino acids 579–594 of murine enzyme, with TNFa at various concentrations for 12 h (A and C) or at a concen- and provided by Dr. Yoko Hayashi of this laboratory. The sonicates of tration of 20 ng/ml for the indicated time intervals (B and D). PGE MC3T3-E1 cells were subjected to 10% polyacrylamide gel electrophore- content in the culture medium was determined by radioimmunoassay sis in the presence of 0.1% SDS (10 mg of protein/lane). The protein (A and B). The cells were incubated in the absence (closed column or circle) or presence (open column or circle)of15 mM NS398, and the bands were transferred to an Immobilon-P membrane, which was in- cyclooxygenase activity of the sonicated cells was determined using cubated with the polyclonal antibody against cyclooxygenase-2 and [1- C]arachidonic acid as substrate (C and D). Data are means 6 S.E. then with S-labeled anti-rabbit IgG whole antibody as the second of triplicate determinations (A and B). C and D were repeated three antibody. Radioactivity of the immunocomplex was quantified by a times, and similar results were obtained. N.D., below detection limit. BAS2000 imaging analyzer. Northern Blotting—Total RNA was extracted from MC3T3-E1 cells using ISOGEN (a mixture of guanidium isothiocyanate and phenol) according to the manufacturer’s instruction. Northern blotting was 4% polyacrylamide gel electrophoresis gels at a constant 150 V for 2 h. performed as described previously (15, 18). The relative radioactivity of Distribution of the radioactivity on the dried gel was analyzed by a each band was estimated using a BAS2000. BAS2000 imaging analyzer. Preparation of the 59-Flanking Region of Mouse Cyclooxygenase-2 RESULTS Gene and Construction of Luciferase Reporter Vectors—We screened a mouse genomic library constructed in EMBL3 to clone the 59-flanking Induction of Cyclooxygenase-2 by TNFa—When MC3T3-E1 region of the mouse cyclooxygenase-2 gene by the method as described cells were incubated with various concentrations of TNFa for previously for 12-lipoxygenase (19). DNA fragments of mouse cyclooxy- 12 h, there was a dose-dependent increase in the amount of genase-2 promoter regions of various lengths or their point mutants PGE released into the medium (Fig. 1A). After the addition of were prepared from the clone by polymerase chain reaction, and were inserted into the plasmid pXP-1 as described previously for 12-lipoxy- 20 ng/ml TNFa, the PGE release increased with a lag time of genase (20). about 1 h, and reached a maximum at 9 h (Fig. 1B). As shown Transfection of Plasmids to MC3T3-E1 Cells and Luciferase Assay— in Fig. 1, C and D, TNFa increased the cyclooxygenase activity MC3T3-E1 cells (1.4 3 10 cells/dish) were cultured for 2 days in 60-mm dose and time dependently, and the enzyme activity continued dishes with 5 ml of a-MEM containing 10% fetal bovine serum. For to increase for 24 h to a specific activity of 0.8–2 nmol/2 min/mg transfection the subconfluent cells were treated with plasmid DNA (2.3 of protein. mg) containing cyclooxygenase-2 promoter and luciferase reporter gene, standard plasmid DNA (1.2 mg) containing the b-galactosidase gene, NS398, a specific inhibitor of cyclooxygenase-2 (22), inhibited and 9.2 ml of lipofectAMINE in 2.3 ml of serum-free a-MEM for4hat the enzyme activity almost completely at 15 mM concentration 37 °C according to the manufacturer’s instructions. The medium was (Fig. 1, C and D), suggesting that most of the increased enzyme changed to 5 ml of a-MEM with 10% fetal bovine serum, and the activity was attributable to cyclooxygenase-2 rather than cy- transfected cells were further incubated for 3 days. Then the cells were clooxygenase-1. To confirm this finding we carried out Western stimulated by 20 ng/ml TNFa in the presence of a-MEM supplemented blotting with an antibody specific for cyclooxygenase-2 (Fig. with 2% newborn bovine serum. After 12 h the cells were scraped from the dishes, and the luciferase activity was measured by a Lumat 2A). Without TNFa the cyclooxygenase-2 protein was undetect- LB9501 luminometer (Berthold, Germany) using a Pica Gene luciferase able, and the addition of 20 ng/ml TNFa increased the enzyme assay system according to the manufacturer’s instructions. The lucifer- amount time dependently up to 9 h. There was a slight but ase activities were normalized on the basis of b-galactosidase activities reproducible decrease around 12 h, followed by an increase which were assayed as described previously (20). 5 again. Preparation of Nuclear Extracts—MC3T3-E1 cells (9 3 10 cells/dish) Furthermore, as shown in Fig. 2B, the change in mRNA level were plated in 10 150-mm dishes with 30 ml of a-MEM containing 10% fetal bovine serum, and stimulated with various concentrations of was followed by Northern blot analysis. The addition of TNFa TNFa. After 1 h the cells were harvested, and nuclear extracts were brought about a biphasic increase in the cyclooxygenase-2 prepared as described previously (20). mRNA. First, there was a rapid 10-fold increase reaching a Electrophoretic Mobility Shift Assay—We synthesized the five oligo- maximum at 2 h, followed by a decrease at 3 h. Then, the nucleotides as shown in Fig. 4. The complementary oligonucleotides mRNA increased again at 6–12 h. However, after the first were annealed to the corresponding oligonucleotides as described by transient peak the presence of 15 mM NS398 reduced the second Berger and Kimmel (21), and the double-stranded oligonucleotides were purified electrophoretically on 15% polyacrylamide gel, end-labeled peak. The cyclooxygenase-1 mRNA was not detectable under with [g- P]ATP, and used as probes. The binding of the probes (10,000 these experimental conditions. cpm, about 10 fmol) to the nuclear extracts (2.3 mg protein) was per- These results presented in Figs. 1 and 2 demonstrated a formed in a 20-ml mixture containing 5 mg of poly(dI-dC)-poly(dI-dC), 15 rapid and marked induction of cyclooxygenase-2 by TNFa. The mM Tris-HCl at pH 7.5, 1 mM EDTA, 100 mM KCl, 5 mM MgCl , 12% second peak of the cyclooxygenase-2 may be attributable to the glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothre- enzyme which was induced by accumulating PGE produced by itol. For the supershift experiment, each antibody was added to the 2 mixture. The mixture was incubated at 25 °C for 30 min, and applied to the cyclooxygenase-2 at the first peak. Transcriptional Regulation of Cyclooxygenase-2 31317 (lanes 3–6). The complex a was hardly detectable before the addition of TNFa (lane 2). Considering the subunit structure of NFkB (p50 and p65), the g1 probe was incubated with the purified NFkB p50, and a band appeared at the position of complex a (lane 7). The complex a was supershifted to complex b by the addition of anti-p50 antibody (lane 10), and was supershifted to complex g by the addition of anti-p65 antibody (lane 11). The binding was not observed using the probe g2 with a mutation in NFkB motif (lanes 13 and 14). When gA probe was incubated with the nuclear extracts, three complexes (a, b, and c) appeared with increasing density depending on the amount of TNFa (Fig. 6, lanes 3–6). These complexes were supershifted to bands d, f, and g by the addi- tion of antibody against NF-IL6 (lane 8). The binding was scarcely observed by the use of the probe gC with a mutation in NF-IL6 (lanes 15–19). Moreover, the complex a was super- shifted to complex e by the addition of anti-NFkB p50 (lane 9). The bands a, b, and c were not supershifted by anti-AP2 anti- FIG.2. Western and Northern blot analyses of cyclooxygen- body (lane 7), and the binding profile was not affected by the ase-2 protein in MC3T3-E1 cells. A, the cells were incubated with 20 use of the probe gB with a mutation in AP2 (lanes 10–14). ng/ml TNFa. At the indicated time intervals, the cells were harvested These data suggested that NFkB and NF-IL6 were increased and subjected to Western blotting for cyclooxygenase-2 as described by the addition of TNFa in a dose-dependent manner. under “Experimental Procedures.” The immunocomplex was visualized (inset) and quantified (closed circles) utilizing S-labeled second anti- Possible Interaction of Both NFkB and NF-IL6 Binding body by a BAS2000 imaging analyzer. The experiment was repeated Sites—For mutation analysis of the promoter region, we con- three times, and similar results were obtained. PSL, photo-stimulated structed luciferase vectors including mouse cyclooxygenase-2 luminescence. B, the cells were incubated with 20 ng/ml TNFa in the promoter region (2512 to 1123 bp) with site-specific mutations absence (closed circle) or presence (open circle)of15 mM NS398 for indicated time periods. Total RNA (10 mg) was applied to Northern blot (Fig. 7). Each vector was transfected to MC3T3-E1 cells, and analysis. The relative amount of cyclooxygenase-2 mRNA was corrected the luciferase activity was measured as described above. As for b-actin mRNA. shown in Fig. 7, the luciferase activity decreased by 63% in a mutant of NFkB, 82% in a mutant of NF-IL6, and only 19% in Functional Activity of Cyclooxygenase-2 Promoters—As illus- a mutant of AP2 site. By mutation of both NFkB and NF-IL6, trated at the top of Fig. 3, about 600 bp of the 59-flanking region the luciferase activity was lost to the level of pXP1. These data of mouse cyclooxygenase-2 gene contained various putative suggested that both NFkB and NF-IL6 in combination acted as response elements; MEF-2 (a muscle-specific DNA binding pro- transcription factors for the induction of cyclooxygenase-2. tein, 2490 to 2481 bp), NFkB(2401 to 2393 bp), SP1 (2239 to Furthermore, expression vectors containing the genes of NF- 2234 bp), AP2 (2150 to 2142 bp), NF-IL6 (2138 to 2130 bp), IL6 and NFkB p50, respectively, were cotransfected to CRE (256 to 251 bp), and TATA box (230 to 225 bp). The MC3T3-E1 cells together with a luciferase plasmid containing sequences of 59-GGGATTCCC-39 (2401 to 2393 bp) and 59- the promoter region of cyclooxygenase-2 (2512 to 1123 bp). TTGCGCAAC-39 (2138 to 2130 bp) were identified as NFkB The luciferase activity increased depending on the amount of and NF-IL6 elements on the basis of their concensus sequences NF-IL6 cDNA (data not shown). A similar experiment using of 59-GGGA(A/C)TN(T/C)CC-39 and 59-T(T/G)NNGNAA(T/G)- the expression vector for NFkB p50 has so far been 39, respectively, as described previously (23). unsuccessful. For deletion analysis of the promoter region, we constructed DISCUSSION luciferase vectors of various lengths covering the region from 2621 to 241 bp (Fig. 3). Each vector was transfected to It was reported previously that TNFa increased the PGE MC3T3-E1 cells by the lipofection method, and the luciferase production and the cyclooxygenase activity in mouse osteoblas- activity of the cell lysate was measured at 12 h after the tic cell line MC3T3-E1 (24–26). We confirmed these findings addition of 20 ng/ml TNFa. The addition of TNFa markedly (Fig. 1), and demonstrated that the increased enzyme activity stimulated the luciferase activity (closed column versus slashed was attributed mostly to the induction of cyclooxygenase-2 column). The luciferase activity decreased when two regions according to the inhibitory effect of NS398, Western blotting, (2512 to 2385 bp, 2186 to 2131 bp) were deleted, and these and Northern blotting (Figs. 1 and 2). The mechanism of the regions were presumed to have positive response elements. We biphasic increase of cyclooxygenase-2 protein and mRNA (Fig. noted NFkB consensus element (2401 to 2393 bp), AP2 ele- 2) may be discussed as follows. First, TNFa activates phospho- ment (2150 to 2142 bp), and NF-IL6 element (2138 to 2130 lipase A (as reported in Ref. 27) and induces cyclooxygenase-2, bp). resulting in an increased production of PGE . Then, the pro- Electrophoretic Mobility Shift Assay Targeting Positive Reg- duced PGE binds to a PGE receptor, and increases the cy- ulatory Elements—For further identification of these positive clooxygenase-2 protein and mRNA again. Previously we re- regulatory elements, we prepared five double-stranded oligo- ported the cyclooxygenase-2 induction by various PGs, nucleotide probes (g1, g2, gA, gB, and gC). As shown in Fig. 4, including PGE which was a major arachidonate metabolite in g1 (2409 to 2385 bp) contained NFkB and their vicinity. gA this cell line (15). (2155 to 2121 bp) contained AP2 and NF-IL6. g2, gB, and gC Since the addition of TNFa brought about such a typical and were mutant probes for NFkB, AP2, and NF-IL6, respectively. prominent induction of cyclooxygenase-2 in MC3T3-E1 cells, We carried out electrophoretic mobility shift assay using the we attempted to elucidate the transcriptional regulation of the nuclear extracts of MC3T3-E1 cells preincubated with TNFa cyclooxygenase-2 gene in this system. The luciferase assay for 1 h. As shown in Fig. 5, when g1 probe was incubated with (Figs. 3 and 7) and electrophoretic mobility shift assay (Figs. 5 the nuclear extracts, a broad band of complex a was observed and 6) revealed two positive regulatory elements on the 59- with increasing density depending on the amount of TNFa flanking region of mouse cyclooxygenase-2 gene: NFkB motif 31318 Transcriptional Regulation of Cyclooxygenase-2 FIG.3. Construction of luciferase expression vectors and transfection analysis of cyclooxygenase-2 gene promoters. Putative consensus sequences in the 59-upstream region of mouse cyclooxygenase-2 gene are illustrated in the upper left. Each deleted promoter fragment was ligated into a luciferase plasmid. Numbers indicate distance in base pairs from the start of transcription. MC3T3-E1 cells (1.4 3 10 cells) were transfected with various luciferase plasmids (2.3 mg) including different promoter regions of mouse cyclooxygenase-2 gene and b-galactosidase plasmid (1.2 mg) in combination using the lipofection method as described under “Experimental Procedures.” The cells were cultured to confluency for 3 days, and incubated with (closed column) or without (slashed column) TNFa (20 ng/ml) for 12 h. The luciferase activity was assayed, and the results were normalized with the b-galactosidase activity. Data are means 6 S.E. of triplicate determinations. FIG.4. Syntheses of double stranded oligonucleotides as probes for mouse cyclooxygenase-2 gene. g1, 2409 to 2385 bp including the putative NFkB binding site indicated by a solid box; g2, GA of the NFkB was changed to CC; gA, 2155 to 2121 bp including AP2 and NF-IL6; gB, GC of AP2 was changed to TT; gC, TTGCG of the NF-IL6 was changed to CCGCT. FIG.5. Electrophoretic mobility shift assay targeting the (59-GGGATTCCC-39) located at nucleotides 2401 to 2393 bp NFkB site. Probe g1 was incubated for 0.5 h with the nuclear extracts (2.3 mg protein) of MC3T3-E1 cells treated with 0 ng/ml (lane 3), 0.2 and NF-IL6 motif (also referred to as C/EBPb,59-TTGCG- ng/ml (lane 4), 2 ng/ml (lane 5), and 20 ng/ml (lanes 6 and 9) TNFa. The CAAC-39)at 2138 to 2130 bp. NF-IL6 was originally identified nuclear extracts before the addition of TNFa were also incubated with as a nuclear factor binding to the IL-1 response element of the g1 (lane 2). Anti-NFkB p50 (lane 10) or anti-NFkB p65 (lane 11) anti- human IL-6 gene (28). By cDNA cloning NF-IL6 was found to body was incubated together with the nuclear extracts from the cells be a member of the CCAAT enhancer-binding protein (C/EBP) stimulated with 20 ng/ml TNFa. Purified NFkB p50 protein was incu- bated with probe g1 (lane 7). Probe g2 with a mutation for NFkB was family of basic-leucine zipper (bZIP) transcription factors (29). incubated with the nuclear extracts from the cells stimulated with 20 Subsequent studies suggested a regulatory role of NF-IL6 for ng/ml TNFa (lanes 13 and 14). Anti-NFkB p50 antibody was also the genes encoding many acute-phase proteins and cytokines present (lane 14). Lanes 1, 8, and 12 were control runs without the (30). The NF-IL6 motif on the rat cyclooxygenase-2 gene (59- nuclear extracts. Shifted bands indicated by a, b, and g are described in the text. TTATGCAAT-39 located at 2140 to 2132 bp) was previously reported to contribute to the induction of cyclooxygenase-2 by forskolin, follicle-stimulating hormone, and luteinizing hor- mone in granulosa cells (4, 8). Recently it was reported that the the involvement of the CRE motif in the TNFa-dependent cyclooxygenase-2 induction was mediated by an activating cyclooxygenase-2 induction in MC3T3-E1 cells. The AP2 ele- transcription factor/CRE element (59-CGTCACGTG-39 at 256 ment (59-CCGCTGCGG-39, 2150 to 2142 bp) close to the to 248 bp) on the mouse cyclooxygenase-2 gene promoter (7) or NF-IL6 was shown to be inactive (Figs. 3, 6, and 7). by CRE element (59-TTCGTCA-39 at 259 to 253) in the human NFkB was originally found as a binding protein to immuno- cyclooxygenase-2 gene (6). However, we could not demonstrate globulin k light-chain enhancer (31), and it is now considered as Transcriptional Regulation of Cyclooxygenase-2 31319 Individual binding sites for NF-IL6 and NFkB are present in the promoter of the IL-6 gene, and the cooperation of these two factors plays an important role in transcription of IL-6 (37). A similar activation was reported for the IL-8 promoter, which also contains both NF-IL6 and NFkB binding sites (37). The NFkB p50 and NF-IL6 proteins directly interact in vitro, and the Rel homology domain and leucine-zipper motif, respec- tively, are important for this interaction (38). TNFa enhanced the promoter activity of the cyclooxygen- ase-2 gene of MC3T3-E1 cells (Fig. 3), and increased the amounts of NFkB and NF-IL6 bound to the DNA probes in- cluding the corresponding cis-elements (Figs. 5 and 6). The results were in parallel with the marked induction of cyclooxy- genase-2 in the TNFa-stimulated cells (Figs. 1 and 2). It was shown that TNFa activated an endosomal acidic sphingomyeli- nase through the 55-kDa TNF receptor and then the produced ceramide triggered the activation of NFkB (27). TNFa also induced NF-IL6 in the mouse lung, liver, and kidney (30). By analogy we presume that TNFa triggers the activation or in- FIG.6. Electrophoretic mobility shift assay targeting AP2 and duction of both NFkB and NF-IL6, resulting in the induction of NF-IL6 sites. MC3T3-E1 cells were incubated for 1 h with various concentrations of TNFa. The nuclear extracts (2.3 mg of protein) from cyclooxygenase-2 in MC3T3-E1 cells. We should note that there the cells stimulated with 0 ng/ml (lane 3), 0.2 ng/ml (lane 4), 2 ng/ml are NFkB and NF-IL6 sites in human (5) and rat (4) cyclooxy- (lane 5), and 20 ng/ml (lanes 6–9, 11–14, and 16–19) TNFa were genase-2 genes: human, 59-GGGATTCCC-39 (2447 to 2439 bp, incubated with probe gA (wild type, lanes 3–9), gB (mutation for AP2, NFkB element) and 59-TTACGCAAT-39 (2132 to 2124 bp, lanes 11–14), or gC (mutation for NF-IL6, lanes 16–19). The nuclear extracts before the addition of TNFa was also incubated with gA (lane NF-IL6 element); rat, 59-GGGATTCCC-39 (2403 to 2395 bp, 2). Anti-AP2 (lanes 7, 12 and 17), anti-NF-IL6 (lanes 8, 13, and 18), or NFkB element) and 59-TTATGCAAT-39 (2140 to 2132 bp, NF- anti-NFkB p50 (lanes 9, 14, and 19) antibody was incubated together IL6 element). with the nuclear extracts. 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Published: Dec 1, 1995

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