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• W2090941846 abstract "Mechanical loading is crucial for maintenance of bone integrity and architecture, and prostaglandins are an important mediator of mechanosensing. Cyclooxygenase-2 (COX-2), an inducible isoform of prostaglandin G/H synthase, is induced by mechanical loading-derived fluid shear stress in bone-forming cells such as osteoblasts and osteocytes. In this study, we investigated transcription factor and transcriptional regulatory elements responsible for the shear stress-induced COX-2 expression in osteoblastic MC3T3-E1 cells. When the cells were transfected with luciferase-reporter plasmids including the 5′-flanking region of the murine cox-2 gene, the fluid shear stress increased the luciferase activities, consistent with the induction of COX-2 mRNA and protein expression. Deletion analysis of the promoter region revealed that the shear stress-induced luciferase responses were regulated by two regions, −172 to −100 base pair (bp) and −79 to −46 bp, of the cox-2 promoter, in which putativecis-elements of C/EBP β, AP-1, cAMP-response element-binding protein (CREB), and E box are included. Mutation of sites of C/EBP β, AP-1, and/or cAMP-response element decreased the shear stress-induced luciferase activities, whereas mutation of the E box did not affect the responses. In an electrophoretic mobility shift assay, shear stress enhanced nuclear extract binding to double-stranded oligonucleotide probes containing C/EBP β and AP-1-binding motifs, and the bands of the complexes were supershifted by the addition of antibody specific for each regulator. Although the binding activity of CREB toward its probe was unaffected by shear stress, the phosphorylation of CREB was enhanced by the stress. These data suggest that C/EBP β, AP-1, and CREB play crucial roles in the shear stress-induced cox-2 expression in osteoblasts. Mechanical loading is crucial for maintenance of bone integrity and architecture, and prostaglandins are an important mediator of mechanosensing. Cyclooxygenase-2 (COX-2), an inducible isoform of prostaglandin G/H synthase, is induced by mechanical loading-derived fluid shear stress in bone-forming cells such as osteoblasts and osteocytes. In this study, we investigated transcription factor and transcriptional regulatory elements responsible for the shear stress-induced COX-2 expression in osteoblastic MC3T3-E1 cells. When the cells were transfected with luciferase-reporter plasmids including the 5′-flanking region of the murine cox-2 gene, the fluid shear stress increased the luciferase activities, consistent with the induction of COX-2 mRNA and protein expression. Deletion analysis of the promoter region revealed that the shear stress-induced luciferase responses were regulated by two regions, −172 to −100 base pair (bp) and −79 to −46 bp, of the cox-2 promoter, in which putativecis-elements of C/EBP β, AP-1, cAMP-response element-binding protein (CREB), and E box are included. Mutation of sites of C/EBP β, AP-1, and/or cAMP-response element decreased the shear stress-induced luciferase activities, whereas mutation of the E box did not affect the responses. In an electrophoretic mobility shift assay, shear stress enhanced nuclear extract binding to double-stranded oligonucleotide probes containing C/EBP β and AP-1-binding motifs, and the bands of the complexes were supershifted by the addition of antibody specific for each regulator. Although the binding activity of CREB toward its probe was unaffected by shear stress, the phosphorylation of CREB was enhanced by the stress. These data suggest that C/EBP β, AP-1, and CREB play crucial roles in the shear stress-induced cox-2 expression in osteoblasts. cyclooxygenase-2 prostaglandins protein kinase cAMP-response element-binding protein cAMP-response element fetal bovine serum α-minimum essential medium polymerase chain reaction wild type p-aminoethylbenzenesulfonyl fluoride polyacrylamide gel electrophoresis base pair electrophoretic mobility shift assay Mechanical loading applied to the skeleton is crucial to the development and maintenance of bone integrity and architecture. A decrease in the mechanical loading due to prolonged immobilization or weightlessness in space reduces the bone formation rate, resulting in bone loss (1Jadvar H. Aviat. Space Environ. Med. 2000; 71: 640-646PubMed Google Scholar, 2Kannus P. Jarvinen T.L. Sievanen H. Kvist M. Rauhaniemi J. Maunu V.M. Hurme T. Jozsa L. Jarvinen M. J. Bone Miner. Res. 1996; 11: 1339-1346Crossref PubMed Scopus (47) Google Scholar, 3Jee W.S. Ma Y. Morphologie. 1999; 83: 25-34PubMed Google Scholar). On the other hand, an increase in mechanical loading causes a gain in bone density (4Turner C.H. Pavalko F.M. J. Orthop. Sci. 1998; 3: 346-355Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 5Daly R.M. Rich P.A. Klein R. Bass S. J. Bone Miner. Res. 1999; 14: 1222-1230Crossref PubMed Scopus (127) Google Scholar). Thus, bone tissue is sensitive to mechanical stimulation. Mechanical loading on bone generates extracellular matrix deformation and fluid flow, and the mechanical stimuli are translated to mechanical signals such as mechanical strain and fluid shear stress, respectively (6Cowin S.C. Bone ( NY ). 1998; 22 Suppl. 5: 119S-125SCrossref Scopus (48) Google Scholar). Evidence obtained fromin vitro studies indicates that osteocytes embedded in the lacunae/canaliculi system and osteoblasts and bone cells lining the bone surface are mechanosensors that detect load-derived mechanical stimuli (7Duncan R.L. Turner C.H. Calcif. Tissue Int. 1995; 57: 344-358Crossref PubMed Scopus (807) Google Scholar, 8Burger E.H. Klein-Nulend J. FASEB J. 1999; 13: 101-112Crossref PubMed Scopus (751) Google Scholar). By these bone-forming cells, the mechanical stimuli are translated into cellular signaling factors. Mechanical stress induces the expressions of several kinds of proteins in bone-forming cells such as insulin-like growth factor-I and -II, transforming growth factor-β, osteocalcin, osteopontin, c-Fos, nitric-oxide synthase, and cyclooxygenase-2 (COX-2,1 an isoform of prostaglandin G/H synthase), as reported in previous studies (9Perrone C.E. Fenwick-Smith D. Vandenburgh H.H. J. Biol. Chem. 1995; 270: 2099-2106Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 10Klein-Nulend J. Semeins C.M. Burger E.H. J. Cell. Physiol. 1996; 168: 1-7Crossref PubMed Scopus (49) Google Scholar, 11Akhouayri O. Lafage-Proust M.H. Rattner A. Laroche N. CaillotAugusseau A. Alexandre C. Vico L. J. Cell. Biochem. 1999; 76: 217-230Crossref PubMed Scopus (59) Google Scholar, 12Sodek J. Chen J. Nagata T. Kasugai S. Todescan Jr., R. Li I.W. Kim R.H. Ann. N. Y. Acad. Sci. 1995; 760: 223-241Crossref PubMed Scopus (156) Google Scholar, 13Raab-Cullen D.M. Thiede M.A. Petersen D.N. Kimmel D.B. Recker R.R. Calcif. Tissue Int. 1994; 55: 473-478Crossref PubMed Scopus (182) Google Scholar, 14Pitsillides A.A. Rawlinson S.C. Suswillo R.F. Bourrin S. Zaman G. Lanyon L.E. FASEB J. 1995; 9: 1614-1622Crossref PubMed Scopus (305) Google Scholar, 15Pavalko F.M. Chen N.X. Turner C.H. Burr D.B. Atkinson S. Hsieh Y.F. Qiu J. Duncan R.L. Am. J. Physiol. 1998; 275: C1591-C1601Crossref PubMed Google Scholar). In particular, the administration of NS-398, a selective inhibitor of COX-2, and indomethacin to rats in vivo inhibited mechanical loading-induced bone formation (16Forwood M.R. J. Bone Miner. Res. 1996; 11: 1688-1693Crossref PubMed Scopus (306) Google Scholar, 17Chow J.W.M. Jagger C.J. Chambers T.J. Calcif. Tissue Int. 1996; 59: 117-120Crossref PubMed Scopus (5) Google Scholar), suggesting that prostaglandins (PGs) are important mediators of the mechanical loading-induced bone response. In addition, up-regulation of expression of some skeletal growth factors including insulin-like growth factor-I and transforming growth factor-β in response to mechanical stress was at least in part mediated by the PG production (10Klein-Nulend J. Semeins C.M. Burger E.H. J. Cell. Physiol. 1996; 168: 1-7Crossref PubMed Scopus (49) Google Scholar, 18Chambers T.J. Fox S. Jagger C.J. Lean J.M. Chow J.W. Osteoarthritis Cartilage. 1999; 7: 422-423Abstract Full Text PDF PubMed Scopus (51) Google Scholar). PGs have anabolic effects on proliferation and differentiation of bone-forming cells via diverse signal transduction systems dependent on their concentration and species (19Raisz L.G. Osteoarthritis Cartilage. 1999; 7: 419-421Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 20Hakeda Y. Yoshino T. Natakani Y. Kurihara N. Maeda N. Kumegawa M. J. Cell. Physiol. 1986; 128: 155-161Crossref PubMed Scopus (115) Google Scholar, 21Hakeda Y. Hotta T. Kurihara N. Ikeda E. Maeda N. Yagyu Y. Kumegawa M. Endocrinology. 1987; 121: 1966-1974Crossref PubMed Scopus (100) Google Scholar). PGs also regulate the differentiation and function of bone-resorbing cells such as osteoclasts (22Kawaguchi H. Pilbeam C.C. Harrison J.R. Raisz L.G. Clin. Orthop. 1995; 313: 36-46PubMed Google Scholar, 23Suzawa T. Miyaura C. Inada M. Maruyama T. Sugimoto Y. Ushikubi F. Ichikawa A. Narumiya S. Suda T. Endocrinology. 2000; 141: 1554-1559Crossref PubMed Scopus (318) Google Scholar). Therefore, the mechanical stress-induced PG production by bone forming-cells may modulate the overall process of bone metabolism to adapt the skeleton to the mechanical environment. Production of PGs is kinetically controlled mainly by the release of arachidonic acid and expression of COX-2 in response to a variety of stimuli (24Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1860) Google Scholar). Fluid shear stress has been reported to stimulate rapidly PGE2 production in osteocytes through a cascade of sequential activation of cytoskeleton-associated Ca2+channel, phospholipase C, intracellular Ca2+, protein kinase C (PKC), and phospholipase A2 (25Ajubi N.E. Klein-Nulend J. Alblas M.J. Burger E.H. Nijweide P.J. Am. J. Physiol. 1999; 276: E171-E178PubMed Google Scholar). Regarding COX-2 induction, it has been reported that cox-2 expression induced by fluid shear stress could be dependent on cytoskeleton-integrin interactions and intracellular calcium release mediated by inositol trisphosphate in osteoblastic MC3T3-E1 cells (26Chen N.X. Ryder K.D. Pavalko F.M. Turner C.H. Burr D.B. Qiu J. Duncan R.L. Am. J. Physiol. 2000; 278: C989-C997Crossref PubMed Google Scholar). However, there has been no report indicating transcription factors or transcriptional regulatory elements in the cox-2 promoter region responsible for the shear stress-induced cox-2transcription, whereas the cytokine- or growth factor-dependent factors and regulatory elements have been extensively reported (27Crofford L.J. Tan B. McCarthy C.J. Hla T. Arthritis & Rheum. 1997; 40: 226-236Crossref PubMed Scopus (249) Google Scholar, 28Yamamoto K. Arakawa T. Ueda N. Yamamoto S. J. Biol. Chem. 1995; 270: 31315-31320Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar, 29Wadleigh D.J. Reddy S.T. Kopp E. Ghosh S. Herschman H.R. J. Biol. Chem. 2000; 275: 6259-6266Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 30Xie W. Fletcher B.S. Andersen R.D. Herschman H.R. Mol. Cell. Biol. 1994; 14: 6531-6539Crossref PubMed Scopus (189) Google Scholar, 31Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). In the present study, we attempted to identify transcription factors and transcriptional regulatory elements located in 5′-flanking region of the cox-2 promoter gene that contribute to the shear stress-induced cox-2 expression. Here, we report our findings indicating that the cox-2 expression induced by fluid shear stress was mediated by C/EBP β, AP-1, and CREB, which bound to their respective sites on the cox-2 promoter gene in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells (2500 cells/cm2) were seeded on type 1 collagen-coated slide glasses (Matsunami, Tokyo, Japan; 25 × 50 × 1 mm) in 100-mm plastic dishes and were cultured in α-minimum essential medium (α-MEM, ICN Biomedicals Inc., Aurora, OH) containing 10% fetal bovine serum (FBS, Intergen, NY) and 100 units/ml of penicillin G at 37 °C in a humidified CO2 incubator (5% CO2, 95% air) as described previously (21Hakeda Y. Hotta T. Kurihara N. Ikeda E. Maeda N. Yagyu Y. Kumegawa M. Endocrinology. 1987; 121: 1966-1974Crossref PubMed Scopus (100) Google Scholar). After 4 days, the culture medium was removed and replaced with fresh α-MEM containing 10% FBS. The removed medium was stored at 4 °C to be used as conditioned medium. The cells were cultured further for 3 more days prior to use in fluid shear stress experiments. A single-pass flow-through system was used. After MC3T3-E1 cells had been cultured on a slide glass (1-mm thick), the slide glass was carefully removed from and mounted on a parallel plate flow chamber that had been constructed by sandwiching silicone gaskets (1.5, 2, or 3 mm thick) between two acrylic plates, creating a flow channel (0.5, 1, or 2 mm deep × 25 mm wide ×50 mm long). Then the cells in the chamber were exposed to the fluid shear stress, which was generated by circulating the conditioned medium (0.36 ml/s) through a hydrostatic pump connected to the reservoirs at 37 °C in a CO2 incubator. The pH of the medium was kept constant by gassing with humidified 95% air and 5% CO2. As a control, the cells in the flow chamber were incubated for the same duration without having been exposed to the shear stress. When the rate of fluid flow is constant in the chamber, the magnitude of the shear stress is inversely proportional to the square of the depth of the flow channel. When the flow rate of the conditioned medium was 0.36 ml/s, the shear stress at 0.5-, 1.0-, and 2.0-mm depths of the flow channel was calculated to be 2.88, 0.72, and 0.18 dynes/cm2, respectively. After MC3T3-E1 cells had been subjected to shear stress for the desired time, total RNA (1 μg) extracted from the cells was used as a template for cDNA synthesis. cDNA was prepared by use of a Superscript II preamplification system (Life Technologies, Inc.). Primers were synthesized on the basis of the reported mouse cDNA sequences for COX-2 and β-actin. Sequences of the primers used for PCR were as follows: cox-2 forward, 5′-GGG TTG CTG GGG GAA GAA ATG TG-3′; COX-2 reverse, 5′-GGT GGC TGT TTT GGT AGG CTG TG-3′; β-actin forward, TCA CCC ACA CTG TGC CCA TCT AC-3′; β-actin reverse, 5′-GAG TAC TTG CGC TCA GGA GGA GC-3′. Amplification was carried out for 22–27 cycles under saturation, each at 94 °C, 45 s; 60 °C, 45 s; 72 °C, 1 min in a 50-μl reaction mixture containing 0.5 μl of each cDNA, 50 pmol of each primer, 0.2 mm dNTP, and 1.25 units of Taq DNA polymerase (Qiagen, Inc., Valencia, CA). After amplification, 10 μl of each reaction mixture was analyzed by 1.5% agarose gel electrophoresis, and the bands were then visualized by ethidium bromide staining. The PCR products for cox-2 and β-actin were 479 and 538 bp, respectively. Anti-COX-2, anti-c-Jun (sc-44x), anti-c-Fos (sc-253x), and anti-C/EBP β (sc-746x) antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA). Anti-CREB and anti-phospho-CREB antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). After exposure to shear stress, MC3T3-E1 was washed with PBS, scraped into a solution consisting of 10 mm sodium phosphate (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmEDTA, 1 mm p-aminoethylbenzenesulfonyl fluoride (p-ABSF), 10 μg/ml leupeptin, and 10 μg/ml aprotinin, and sonicated for 15 s. The protein concentration in the cell lysate was measured with a bicinchoninic acid protein assay kit (Pierce). Samples containing equal amounts of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE), and the proteins separated in the gel were subsequently electrotransferred onto a polyvinylidene difluoride membrane. After having been blocked with 5% skim milk, the membrane was incubated with anti-COX-2 antibody or nonimmune rabbit IgG and subsequently with peroxidase-conjugated anti-rabbit IgG antibody. Immunoreactive proteins were visualized with Western blot chemiluminescence reagents (PerkinElmer Life Sciences) following the manufacturer's instructions. Luciferase reporter pGL2 plasmid including 5′-flanking region of the murinecox-2 gene from −3195 to +39 bp was kindly provided by Dr. David L. DeWitt (Michigan State University). DNA fragments of various lengths of the cox-2 promoter regions were prepared by PCR using the above plasmid as a template and PyrobestTM DNA polymerase (Takara, Kyoto, Japan). Mutated fragments were prepared by two-stage bridge PCR, using mutated primers previously reported by Brunner et al. (32Brunner C. Kraft H.G. Utermann G. Muller H.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11643-11647Crossref PubMed Scopus (148) Google Scholar). These fragments were inserted into pGL2 basic vectors (Promega, Madison, WI), by using a Ligation kit version II® (Takara). MC3T3-E1 cells were cultured for 4 days on type 1 collagen-coated slide glasses in 100-mm dishes containing α-MEM supplemented with 10% FBS. For transfection, the cells were treated for 24 h with plasmid DNA (0.7 μg) containing cox-2promoter and luciferase reporter gene, standard plasmid DNA (0.4 μg) containing β-galactosidase gene, and 18 μl of Effectene transfection reagent® (Qiagen) in 4 ml of α-MEM with 10% FBS. Then the medium was changed to 15 ml of α-MEM with 10% FBS, and the transfected cells were further cultured for 3 days. Thereafter, the transfected cells were placed in the flow shear stress chamber and subjected to fluid shear stress (2.88 dynes/cm2) for 6 h at 37 °C. Control cells were also placed in other chambers without fluid flow for the same period. The cells were then washed twice with cold PBS and were scraped in 200 μl of Reporter lysis buffer (Promega). Luciferase activities in the cell lysate were measured by using a microplate luminometer (MicroLumat LB96P, EG & G Berthold, Aliquippa, PA) and a luciferase assay system (Promega), according to the manufacturer's instruction. For determination of β-galactosidase activity, the cell lysate (15 μl) was incubated in a 335-μl reaction buffer (0.1 m sodium phosphate (pH 7.5, 2 mm o-nitrophenyl-β-galactopyranoside), 10 mm KCl, 1 mm MgCl2, 0.1% Triton X-100, 5 mm β-mercaptoethanol) at 37 °C for 24 h. The reaction was then terminated with 150 μl of 1 msodium carbonate, and the absorbance was measured at 420 nm. The luciferase activities were normalized on the basis of β-galactosidase activities. A part of the cell lysate was used to fluorometrically determine DNA content by the method of Kissane and Robins (33Kissane J.M. Robins E. J. Biol. Chem. 1958; 233: 184-190Abstract Full Text PDF PubMed Google Scholar). In individual luciferase assays for deletion or mutation analysis, we always provided control cells that had been transfected with a reporter plasmid containing the cox-2 promoter region from −959 to +39 bp. The control cells were also subjected to shear stress together with cells transfected with other deleted or mutated reporter plasmids. The normalized luciferase activities in the cells transfected with various reporter plasmids were presented as percentage values, compared with the value of luciferase activities of the shear stress-loaded cells that had been transfected with the reporter plasmid containing thecox-2 promoter region from −959 to +39 bp. After MC3T3-E1 cells had been subjected to fluid shear stress for times indicated in the legends, the cells were washed twice with ice-cold PBS, incubated for 10 min on ice in 1 ml of ice-cold buffer A (10 mm Hepes, 10 mm KCl, 0.1 mm EDTA, 0.1% Nonidet P-40, 1 mm dithiothreitol, 1 mm p-ABSF, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 2 μg/ml leupeptin), and scraped. The cell lysates were incubated further for 10 min on ice and then transferred to tubes. The nuclei obtained by centrifugation for 1 min at 5,000 × g were extracted by a 30-min incubation in ice-cold buffer C, consisting of 50 mm Hepes (pH 7.5), 420 mm KCl, 0.1 mm EDTA, 5 mmMgCl2, 20% glycerol, 1 mm dithiothreitol, 1 mm p-ABSF, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 2 μg/ml leupeptin. The extracts then were centrifuged at 14,000 × g for 30 min, and the supernatants were used for the electrophoretic mobility shift assay (EMSA). As shown in Table I, three oligonucleotides were synthesized on the basis of the sequence of putative binding sites of C/EBP-β, AP-1, and CREB located in the promoter region of the murinecox-2 gene. The AP-1 probes and CREB probes were partially mutated to avoid the cross-bindings of other transcription factors, because the sequences of probes for AP-1 and CREB overlapped in part with CRE-binding site and AP-1- and E-box-binding sites, respectively. The oligonucleotides were annealed with their complementary oligonucleotides. The double-stranded oligonucleotides were end-labeled with [γ-32P]ATP by using T4 polynucleotide kinase (Promega) according to the manufacturer's instructions and were used as probes for EMSA. The nuclear extracts (1.8 μg of protein) were incubated in binding buffer (10 mm Tris-HCl (pH 7.5), 4% glycerol, 50 mm NaCl, 1 mm MgCl2, 0.5 mm EDTA, 0.5 mm dithiothreitol containing32P-labeled C/EBP β, AP-1, and CREB probes) for 20 min at room temperature. The protein-DNA complexes were resolved by PAGE (5% gel) in 0.5× TBE buffer and visualized by autoradiography. For supershift experiments, the nuclear extracts were incubated with anti-c-Jun, anti-c-Fos, anti-C/EBP-β, anti-CREB, or anti-phospho-CREB antibody for 30 min on ice after binding to the oligonucleotides and then were subjected to PAGE.Table ISyntheses of double-stranded oligonucleotides as probes for mouse cox-2 geneThe constructed three double-strand oligonucleotide probes for C/EBP β, AP-1, and CREB were synthesized based on the sequence of each binding site in mouse cox-2 promoter gene. Sequences in boxes indicate putative binding site for C/EBP β, AP-1, or CREB, respectively. Lowercase letters show mutated bases. The AP-1 probes and CREB probes were partially mutated to avoid the cross-bindings of other transcription factors, because the sequences of probes for AP-1 and CREB overlapped in part with CRE-binding site and AP-1- and E- box-binding sites, respectively, indicated by underlines. Open table in a new tab The constructed three double-strand oligonucleotide probes for C/EBP β, AP-1, and CREB were synthesized based on the sequence of each binding site in mouse cox-2 promoter gene. Sequences in boxes indicate putative binding site for C/EBP β, AP-1, or CREB, respectively. Lowercase letters show mutated bases. The AP-1 probes and CREB probes were partially mutated to avoid the cross-bindings of other transcription factors, because the sequences of probes for AP-1 and CREB overlapped in part with CRE-binding site and AP-1- and E- box-binding sites, respectively, indicated by underlines. When osteoblastic MC3T3-E1 cells were exposed to 2.88 dynes/cm2 of fluid shear stress, expression ofcox-2 mRNA was increased as early as 1 h after the start of exposure and reached a maximum at 3 h (Fig.1 A). The increase in the expression continued at least for 9 h. The up-regulation ofcox-2 mRNA expression depended on the magnitude of the fluid shear stress, with a significant increase even at 0.18 dynes/cm2 (Fig. 1 B). In addition, the fluid shear stress also induced the expression of COX-2 protein in a time-dependent manner, with a maximal effect at 6 h after the shear stress application (Fig. 1 C). About 1,000 bp of the 5′-flanking region of the mousecox-2 gene contained various putative transcription response elements as described previously (28Yamamoto K. Arakawa T. Ueda N. Yamamoto S. J. Biol. Chem. 1995; 270: 31315-31320Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). When MC3T3-E1 cells were transfected with luciferase-reporter plasmid including the 5′-flanking region of the cox-2 gene (−959 to +39 bp), luciferase activities in the cells were time-dependently increased in response to the fluid shear stress (2.88 dynes/cm2, Fig.2). A maximal increase was observed at 6 h after the shear stress application, consistent with the induction of COX-2 protein determined by the Western blotting analysis (Fig. 1 C). The increase in the luciferase activity was maintained up to 9 h. To identify what regions of the cox-2 promoter contributed to the shear stress-induced cox-2 transcription, we next constructed eight luciferase reporter plasmids containing various lengths of 5′-flanking region of the murine cox-2 gene as shown in Fig. 3. After each plasmid vector was introduced to MC3T3-E1 cells, the luciferase activities were measured at 6 h after application of fluid shear stress (2.88 dynes/cm2). As shown in Fig. 3, in the cells transfected with a plasmid having a region of cox-2 gene promoter from −959 to + 39 bp, an ∼4-fold increase in the activity was induced by the fluid shear stress. We defined this luciferase activity as 100% for comparison to the activities in the shear stress-loaded or -unloaded cells transfected with plasmids containing various deleted regions of the cox-2 gene promoter. The shear stress-induced luciferase activities were not decreased but rather increased by transfection with plasmids deleted from −959 to −172 bp. However, when the region from −172 to −100 bp in the promoter was deleted, the stimulatory effect of the shear stress on luciferase activity decreased markedly (Fig. 3). The shear stress-induced luciferase activity in the cells transfected with the plasmid deleted from −100 to −79 bp was the same as that in the cells transfected with the plasmid deleted from −172 to −100 bp. In addition, deletions from −79 to −46 bp further reduced the shear stress-induced luciferase activity. These deletion data suggested that two regions (−172 to −100 bp and −79 to −46 bp) represented possible shear stress-response elements. Based on analysis of consensus sequence, these regions contained presumedcis-elements for C/EBP β, AP-1, CRE, and E-box. To eliminate the possibility that the decreases in shear stress-induced luciferase activity were attributable to the difference in the length of the promoter region in the plasmids used, we introduced mutations in the presumed response elements in the same length of cox-2promoter (−959 to +39 bp) gene, as shown in Fig.4. Introduction of a mutation into CRE (1Jadvar H. Aviat. Space Environ. Med. 2000; 71: 640-646PubMed Google Scholar) (−447 to −440 bp), NF-κB (−401 to −393 bp), C/EBP β (2Kannus P. Jarvinen T.L. Sievanen H. Kvist M. Rauhaniemi J. Maunu V.M. Hurme T. Jozsa L. Jarvinen M. J. Bone Miner. Res. 1996; 11: 1339-1346Crossref PubMed Scopus (47) Google Scholar) (−93 to −85 bp), and E-box (−53 to −48 bp) did not affect the stimulation of luciferase activity by the fluid shear stress. On the other hand, a mutations in C/EBP β (1Jadvar H. Aviat. Space Environ. Med. 2000; 71: 640-646PubMed Google Scholar) (−138 to −130 bp), AP-1 (−73 to −61 bp), or CRE (2Kannus P. Jarvinen T.L. Sievanen H. Kvist M. Rauhaniemi J. Maunu V.M. Hurme T. Jozsa L. Jarvinen M. J. Bone Miner. Res. 1996; 11: 1339-1346Crossref PubMed Scopus (47) Google Scholar) (−59 to −52 bp) reduced the shear stress-induced luciferase activities to 41, 52, and 34%, respectively, of the value for the wild-type reporter, suggesting these sites to be shear stress-response elements. Furthermore, to confirm the functional elements responsive to shear stress, we performed double and triple mutation analyses (Fig. 5). When both C/EBP β (1Jadvar H. Aviat. Space Environ. Med. 2000; 71: 640-646PubMed Google Scholar) (−138 to −130 bp) and CRE (2Kannus P. Jarvinen T.L. Sievanen H. Kvist M. Rauhaniemi J. Maunu V.M. Hurme T. Jozsa L. Jarvinen M. J. Bone Miner. Res. 1996; 11: 1339-1346Crossref PubMed Scopus (47) Google Scholar) (−59 to −52 bp) sites were mutated, the shear stress-induced luciferase activity was further decreased to 14% of that in the shear stress-loaded cells transfected with the wild-type reporter plasmid containing the cox-2promoter gene (−959 to +39 bp). In addition, by triple mutation in C/EBP β (1Jadvar H. Aviat. Space Environ. Med. 2000; 71: 640-646PubMed Google Scholar) (−138 to −130 bp), AP-1 (−73 to −61 bp), and CRE (2Kannus P. Jarvinen T.L. Sievanen H. Kvist M. Rauhaniemi J. Maunu V.M. Hurme T. Jozsa L. Jarvinen M. J. Bone Miner. Res. 1996; 11: 1339-1346Crossref PubMed Scopus (47) Google Scholar) (−59 to −52 bp) sites, the stimulation of luciferase activity in response to the fluid shear stress fell to the level of the pGL2 basic vector. These results suggested that C/EBP β, AP-1, and CREB acted as functional transcription factors for up-regulation of cox-2transcript expression in response to the fluid shear stress.Figure 4Site mutation analysis of luciferase activities in response to fluid shear stress. The COX-2 gene promoter (−959 to +39 bp) was mutated at each putative transcriptional regulatory element by the two-stage Bridge PCR method. The mutated COX-2 gene promoters were ligated into pGL2 basic luciferase vectors.Lowercase letters in the upper sequence of each promoter indicate mutated bases, and the lower sequenceshows wild-type bases. Osteoblastic MC3T3-E1 cells were transiently transfected with the wild-type (−959 to +39 bp) and mutated construct along with the β-galactosidase plasmid. The transfected cells were subjected or not subjected to shear stress at 2.88 dynes/cm2 for 6 h and then assayed for luciferase and β-galactosidase activities. The luciferase activities were normalized to the β-galactosidase activities. The normalized luciferase activities in the cells transfected with various reporter plasmids are presented as percentage values, compared with the valu" @default.
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