Fluid Shear Stress-induced Cyclooxygenase-2 Expression Is Mediated by C/EBP β, cAMP-response Element-binding Protein, and AP-1 in Osteoblastic MC3T3-E1 Cells*

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 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 (1)(2)(3). On the other hand, an increase in mechanical loading causes a gain in bone density (4,5). 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 (6). Evidence obtained from in 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 (7,8). 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-␤, 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 (9 -15). 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 (16,17), 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-␤ in response to mechanical stress was at least in part mediated by the PG production (10,18). PGs have anabolic effects on proliferation and differentiation of bone-forming cells via diverse signal transduction systems dependent on their concentration and species (19 -21). PGs also regulate the differentiation and function of boneresorbing cells such as osteoclasts (22,23). 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 (24). Fluid shear stress has been reported to stimulate rapidly PGE 2 production in osteocytes through a cascade of sequential activation of cytoskeleton-associated Ca 2ϩ channel, phospholipase C, intracellular Ca 2ϩ , protein kinase C (PKC), and phospholipase A 2 (25). 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 (26). 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-2 transcription, whereas the cytokine-or growth factor-dependent factors and regulatory elements have been extensively reported (27)(28)(29)(30)(31).
In the present study, we attempted to identify transcription factors and transcriptional regulatory elements located in 5Јflanking 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 ␤, AP-1, and CREB, which bound to their respective sites on the cox-2 promoter gene in osteoblastic MC3T3-E1 cells.

EXPERIMENTAL PROCEDURES
Cell Culture-MC3T3-E1 cells (2500 cells/cm 2 ) were seeded on type 1 collagen-coated slide glasses (Matsunami, Tokyo, Japan; 25 ϫ 50 ϫ 1 mm) in 100-mm plastic dishes and were cultured in ␣-minimum essential medium (␣-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 CO 2 incubator (5% CO 2 , 95% air) as described previously (21). After 4 days, the culture medium was removed and replaced with fresh ␣-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.
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 ϫ 25 mm wide ϫ50 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 CO 2 incubator. The pH of the medium was kept constant by gassing with humidified 95% air and 5% CO 2 . 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/cm 2 , respectively.
Reverse Transcription-Polymerase Chain Reaction (PCR)-After MC3T3-E1 cells had been subjected to shear stress for the desired time, total RNA (1 g) 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 ␤-actin. Sequences of the primers used for PCR were as follows: cox-2 forward, 5Ј-GGG TTG CTG GGG GAA GAA ATG TG-3Ј; COX-2 reverse, 5Ј-GGT GGC TGT TTT GGT AGG CTG TG-3Ј; ␤-actin forward, TCA CCC ACA CTG TGC CCA TCT AC-3Ј; ␤-actin reverse, 5Ј-GAG TAC TTG CGC TCA GGA GGA GC-3Ј. 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-l reaction mixture containing 0.5 l 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 l 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 ␤-actin were 479 and 538 bp, respectively.
Western Blot Analysis-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 mM EDTA, 1 mM p-aminoethylben-zenesulfonyl fluoride (p-ABSF), 10 g/ml leupeptin, and 10 g/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 antirabbit IgG antibody. Immunoreactive proteins were visualized with Western blot chemiluminescence reagents (PerkinElmer Life Sciences) following the manufacturer's instructions.
Preparation of the 5Ј-flanking Region of Mouse cox-2 Gene and Construction of Luciferase Reporter Vectors-Luciferase reporter pGL2 plasmid including 5Ј-flanking region of the murine cox-2 gene from Ϫ3195 to ϩ39 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 Pyrobest TM DNA polymerase (Takara, Kyoto, Japan). Mutated fragments were prepared by two-stage bridge PCR, using mutated primers previously reported by Brunner et al. (32). These fragments were inserted into pGL2 basic vectors (Promega, Madison, WI), by using a Ligation kit version II® (Takara).
Transfection of MC3T3-E1 Cells with Plasmids and Luciferase Assay-MC3T3-E1 cells were cultured for 4 days on type 1 collagencoated slide glasses in 100-mm dishes containing ␣-MEM supplemented with 10% FBS. For transfection, the cells were treated for 24 h with plasmid DNA (0.7 g) containing cox-2 promoter and luciferase reporter gene, standard plasmid DNA (0.4 g) containing ␤-galactosidase gene, and 18 l of Effectene transfection reagent® (Qiagen) in 4 ml of ␣-MEM with 10% FBS. Then the medium was changed to 15 ml of ␣-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/cm 2 ) 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 l 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 ␤-galactosidase activity, the cell lysate (15 l) was incubated in a 335-l reaction buffer (0.1 M sodium phosphate (pH 7.5, 2 mM o-nitrophenyl-␤-galactopyranoside), 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X-100, 5 mM ␤-mercaptoethanol) at 37°C for 24 h. The reaction was then terminated with 150 l of 1 M sodium carbonate, and the absorbance was measured at 420 nm. The luciferase activities were normalized on the basis of ␤-galactosidase activities. A part of the cell lysate was used to fluorometrically determine DNA content by the method of Kissane and Robins (33).
Data Analysis of Luciferase Activity-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 Ϫ959 to ϩ39 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 the cox-2 promoter region from Ϫ959 to ϩ39 bp.
Preparation of Nuclear Extract-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 g/ml aprotinin, 2 g/ml pepstatin, 2 g/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 ϫ 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 mM MgCl 2 , 20% glycerol, 1 mM dithiothreitol, 1 mM p-ABSF, 2 g/ml aprotinin, 2 g/ml pepstatin, 2 g/ml leupeptin. The extracts then were centrifuged at 14,000 ϫ g for 30 min, and the supernatants were used for the electrophoretic mobility shift assay (EMSA).
EMSA-As shown in Table I, three oligonucleotides were synthesized on the basis of the sequence of putative binding sites of C/EBP-␤, AP-1, and CREB located in the promoter region of the murine cox-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 CREbinding 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 [␥-32 P]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 g of protein) were incubated in binding buffer (10 mM Tris-HCl (pH 7.5), 4% glycerol, 50 mM NaCl, 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM dithiothreitol containing 32 P-labeled C/EBP ␤, AP-1, and CREB probes) for 20 min at room temperature. The protein-DNA complexes were resolved by PAGE (5% gel) in 0.5ϫ TBE buffer and visualized by autoradiography. For supershift experiments, the nuclear extracts were incubated with anti-c-Jun, anti-c-Fos, anti-C/EBP-␤, anti-CREB, or anti-phospho-CREB antibody for 30 min on ice after binding to the oligonucleotides and then were subjected to PAGE.

Up-regulation of COX-2 Expression in Osteoblastic MC3T3-E1 Cells by Fluid Shear Stress-When osteoblastic
MC3T3-E1 cells were exposed to 2.88 dynes/cm 2 of fluid shear stress, expression of cox-2 mRNA was increased as early as 1 h after the start of exposure and reached a maximum at 3 h (Fig.  1A). The increase in the expression continued at least for 9 h. The up-regulation of cox-2 mRNA expression depended on the magnitude of the fluid shear stress, with a significant increase even at 0.18 dynes/cm 2 (Fig. 1B). In addition, the fluid shear stress also induced the expression of COX-2 protein in a timedependent manner, with a maximal effect at 6 h after the shear stress application (Fig. 1C).
Functional Activity of cox-2 Promoter in Response to Fluid Shear Stress-About 1,000 bp of the 5Ј-flanking region of the mouse cox-2 gene contained various putative transcription response elements as described previously (28). When MC3T3-E1 cells were transfected with luciferase-reporter plasmid including the 5Ј-flanking region of the cox-2 gene (Ϫ959 to ϩ39 bp), luciferase activities in the cells were time-dependently in-creased in response to the fluid shear stress (2.88 dynes/cm 2 , 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. 1C). 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 5Ј-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/cm 2 ). As shown in Fig. 3, in the cells transfected with a plasmid having a region of cox-2 gene promoter from Ϫ959 to ϩ 39 bp, an ϳ4-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 Ϫ959 to Ϫ172 bp. However, when the region from Ϫ172 to Ϫ100 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 Ϫ100 to Ϫ79 bp was the same as that in the cells transfected with the plasmid deleted from Ϫ172 to Ϫ100 bp. In addition, deletions from Ϫ79 to Ϫ46 bp further reduced the shear stress-induced luciferase activity. These deletion data suggested that two regions (Ϫ172 to Ϫ100 bp and Ϫ79 to Ϫ46 bp) represented possible shear stress-response elements. Based on analysis of consensus sequence, these regions contained presumed cis-elements for C/EBP ␤, 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-2 promoter (Ϫ959 to ϩ39 bp) gene, as shown in Fig. 4. Introduction of a mutation into CRE (1) (Ϫ447 to Ϫ440 bp), NF-B (Ϫ401 to Ϫ393 bp), C/EBP ␤ (2) (Ϫ93 to Ϫ85 bp), and E-box (Ϫ53 to Ϫ48 bp) did not affect the stimulation of luciferase activity by the fluid shear stress. On the other hand, a mutations in C/EBP ␤ (1) (Ϫ138 to Ϫ130 bp), AP-1 (Ϫ73 to Ϫ61 bp), or CRE (2) (Ϫ59 to Ϫ52 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.

FIG. 1. Effects of shear stress on COX-2 expression in MC3T3-E1 cells.
A, osteoblastic MC3T3-E1 cells were exposed (S) or not exposed (N) to fluid shear stress (2.88 dynes/cm 2 ) for the indicated times. Then total RNA was extracted, and expression of COX-2 and ␤-actin mRNA in the cells was analyzed by reverse transcription-PCR. Numbers in parentheses indicate the number of PCR cycles. B, the cells were subjected to various magnitudes of the fluid shear stress for 1 h. Numbers in parentheses indicate the number of PCR cycles. C, after the cells were exposed (S) or not exposed (N) to the shear stress (2.88 dynes/cm 2 ) for the indicated times, the cells were extracted. The cell extracts were subjected to Western blotting analysis with anti-COX-2 antibody.

FIG. 2. Fluid shear stress-induced COX-2 protein and luciferase activity of WT COX-2 reporter plasmids in osteoblastic MC3T3-E1 cells.
Osteoblastic MC3T3-E1 cells were transiently transfected with 0.7 g of pGL2 reporter plasmid including the COX-2 gene promoter from Ϫ959 to ϩ39 bp (WT [-959]) and 0.4 g of ␤-galactosidase plasmid. The transfected cells were subjected or not subjected to the shear stress (2.88 dynes/cm 2 ) for the indicated times. The cells were then extracted in a lysis buffer, and the extracts were used to determine the activities of luciferase and ␤-galactosidase. The luciferase activity in the transfectants was normalized to the ␤-galactosidase activity and then revised by the intracellular DNA content.
Furthermore, to confirm the functional elements responsive to shear stress, we performed double and triple mutation analyses (Fig. 5). When both C/EBP ␤ (1) (Ϫ138 to Ϫ130 bp) and CRE (2) (Ϫ59 to Ϫ52 bp) sites were mutated, the shear stressinduced 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-2 promoter gene (Ϫ959 to ϩ39 bp). In addition, by triple mutation in C/EBP ␤ (1) (Ϫ138 to Ϫ130 bp), AP-1 (Ϫ73 to Ϫ61 bp), and CRE (2) (Ϫ59 to Ϫ52 bp) sites, the stimulation of luciferase activity in response to

FIG. 3. Construction of luciferase reporter plasmids containing COX-2 gene promoter regions and deletion analysis of COX-2 gene promoters.
Putative consensus sequences in the 5Ј-flanking region of murine COX-2 gene are illustrated in the upper left. Each deleted promoter fragment was inserted into a pGL2 basic luciferase vector. Numbers indicate distance in bp from the start site of transcription. Osteoblastic MC3T3-E1 cells were transiently transfected with these plasmids along with the ␤-galactosidase plasmid. The transfected cells were exposed or not exposed to shear stress at 2.88 dynes/cm 2 for 6 h, and then the activities of luciferase and ␤-galactosidase were measured. The luciferase activities were normalized to the ␤-galactosidase activities. The normalized luciferase activities in the cells transfected with various reporter plasmids are 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 the COX-2 promoter region from Ϫ959 to ϩ39 bp (WT [Ϫ959]). Electrophoretic Mobility Shift Assay Targeting Possible Shear Stress-response Elements-To identify further the possible shear stress-response elements, we carried out an EMSA using the nuclear extracts from MC3T3-E1 subjected to fluid shear stress. As shown in Table I, we constructed three doublestranded oligonucleotide probes for C/EBP ␤, AP-1, and CREB based on the sequence of each binding site in the cox-2 promoter gene. When the C/EBP ␤ probe was incubated with nuclear extracts from shear stress-unloaded cells, the band indicating their complex was observed in the gel (Fig. 6A). The formation of the complex increased with fluid shear stress loading, the increase being significant as early as 1 h after the application of the shear stress. When the probe and the nuclear extracts were incubated with anti-C/EBP ␤ antibody, the complex was further shifted to the upper position in the gel, whereas other antibodies did not change the mobility of the complex. Likewise, the fluid shear stress increased the formation of the complex with the nuclear extracts and AP-1 probe, and the complex was supershifted by incubating with anti-AP-1 antibody but not with anti-CREB and anti-C/EBP ␤ antibodies (Fig. 6B). These results suggest that the shear stress increased the bindings of C/EBP ␤ and AP-1 to their respective sites in cox-2 promoter in MC3T3-E1 cells. On the other hand, the fluid shear stress did not affect the binding of the nuclear extracts to the CREB probe, whereas the shifted bands were recognized by anti-CREB antibody but not by anti-AP-1 antibody (Fig. 6C). However, it was reported that a translocation of CREB to the nucleus or its binding to DNA was independent of activation of transcription of target genes and that the phosphorylation of CREB positively regulated the activation (34). Therefore, by the supershift experiments with phospho-CREB antibody, we finally examined the effect of fluid shear stress on the phosphorylation of CREB in the complex of the nuclear extracts with CREB probe at 3 h. We found that the fluid shear stress at least in part induced the phosphorylation of CREB in their complex, suggesting the transactivation of CREB in the cells. DISCUSSION Mechanical loading on bone generates extracellular matrix deformation and fluid flow, and the mechanical stimuli are translated into mechanical signals such as mechanical strain and fluid shear stress, respectively. These mechanical signals could adaptively change functions of bone-forming cells (osteocytes and osteoblasts). The results of extensive in vivo and in vitro studies indicated that these mechanical signals induced the expression of COX-2, an inducible isoform of prostaglandin G/H synthase, via a complexity of signal transduction systems (15,26,(35)(36)(37). However, little is known about the mechanism that regulates cox-2 transcription in response to mechanical stress. In this study, we demonstrated that the cox-2 expression induced by fluid shear stress was mediated by C/EBP ␤, AP-1, and CREB, which bound to their respective elements on FIG. 5. Double and triple mutation analyses of luciferase activities in response to fluid shear stress. C/EBP ␤(Ϫ138 to Ϫ130 bp), AP-1 (Ϫ73 to Ϫ61 bp), and CRE (2) (Ϫ59 to Ϫ52 bp) sites were mutated (cross) by the two-stage Bridge PCR method. The double-and triple-mutated COX-2 gene promoters were ligated into pGL2 basic luciferase vectors. The MC3T3-E1 cells were transiently transfected with the wild-type and mutated constructs. The transfected cells were subjected or not subjected to shear stress for 6 h at 2.88 dynes/cm 2 and then the activities of luciferase and ␤-galactosidase were measured. The luciferase activities were normalized to the ␤-galactosidase activities. The normalized luciferase activities in the cells transfected with various reporter plasmids are 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 the COX-2 promoter region from Ϫ959 to ϩ39 bp (WT [Ϫ959]). for mouse cox-2 gene The constructed three double-strand oligonucleotide probes for C/EBP ␤, 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 ␤, 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 Ebox-binding sites, respectively, indicated by underlines.
FIG. 6. Electrophoretic mobility shift assay targeting C/EBP ␤, AP-1, and CRE sites. Osteoblastic MC3T3-E1 cells were exposed (S) or not exposed (N) to fluid shear stress (2.88 dynes/cm 2 ) for 1 or 3 h. After the exposure, nuclear extracts were prepared. The extracts (1.8 g) were incubated with 32 P-labeled oligonucleotide probes for C/EBP ␤ (A), AP-1 (B), or CRE (C) site in the presence or absence of normal IgG, anti-C/EBP ␤, anti-c-Jun/AP-1, anti-c-Fos, anti-CREB or anti-phospho-CREB antibody, and the mixtures were then subjected to PAGE (5% gel). Closed and open arrows indicate shifted and supershifted bands, respectively; and dotted arrow in C shows supershifted band with anti-phospho-CREB antibody.
the cox-2 gene promoter in osteoblastic MC3T3-E1 cells. To our knowledge, this is the first report defining the shear stressresponse elements in the cox-2 promoter in bone-forming osteoblasts.
The types of mechanical stimulus that produce the adaptive response of bone-forming cells are still under investigation in in vivo studies. Evidence obtained from in vitro study has, however, shown that bone-forming cells are more sensitive to fluid shear stress than to mechanical strain, suggesting that the fluid shear stress could be a dominant signal in mechanotransduction (38,39). When mechanical loading is applied to bone, the flow of extracellular fluid filled in the space between canaliculi and osteocyte process is increased by the local deformation. A magnitude of 8 -30 dynes/cm 2 of fluid shear stress on the osteocyte process is predicted during physiological mechanical loading (40). Although osteocytes are considered to be primary cells responsible for mechanosensing (8), the fluid shear stress even at a low magnitude of the shear stress (Ͻ1 dynes/cm 2 ) also increased the cox-2 mRNA expression in osteoblastic MC3T3-E1, as demonstrated in this study. These data suggest that osteoblasts as well as osteocytes are sensitive to mechanical loading beyond expectation.
In this study, we attempted to identify transcription response elements in the cox-2 promoter region that contribute to the induction of cox-2 mRNA expression in response to the fluid shear stress. In the 5Ј-flanking region from Ϫ959 to ϩ39 bp of COX-2 gene promoter, there are two CRE elements (CRE (1) (Ϫ447 to Ϫ440 bp), CRE (2) (Ϫ59 to Ϫ52 bp)), two C/EBP ␤ elements (C/EBP ␤ (1) (Ϫ138 to Ϫ130 bp), C/EBP ␤ (2) (Ϫ93 to Ϫ85 bp)), NF-B element (Ϫ401 to Ϫ393 bp), AP-1 element (Ϫ73 to Ϫ61 bp), E-box (Ϫ53 to Ϫ48 bp), and TATA-box (Ϫ30 to 25 bp). The deletion analysis indicated that the shear stressinduced luciferase responses were dependent on two regions, from Ϫ172 to Ϫ100 bp and from Ϫ79 to Ϫ46 bp, which include C/EBP ␤ (1), AP-1, or CRE (2) sites, and E-box. The single mutation of the each site of C/EBP ␤ (1), AP-1, and CRE (2), but not that of the E-box, decreased the shear stress-induced luciferase activities. In addition, the triple mutations of C/EBP ␤ (1), AP-1, and CRE sites abolished the shear stress-induced luciferase response. These results indicate that C/EBP ␤ (1), AP-1, or CRE (2) sites are required for the induction of COX-2 mRNA expression by the fluid shear stress.
The involvement of these sites in the shear stress-induced COX-2 mRNA expression was also confirmed by the mobility gel shift assays. The bindings of C/EBP ␤ and AP-1 to their respective oligonucleotides were increased by exposure to the fluid shear stress, suggesting a transactivation by these transcription factors. Such transactivation has been demonstrated to require phosphorylation of the factors (41)(42)(43)(44). Although the CREB binding to its CRE site was unaffected by the shear stress, phosphorylation of CREB was induced by the shear stress. The activated function of CREB is also modulated by phosphorylation by several kinases (32,44) and is mediated by coactivators such as CBP and p300 (45). Thus, shear stressinduced kinase activities could have induced the phosphorylation of the above transcription factors, by which the COX-2 transcription could be activated. Several signal transduction pathways for response to fluid shear stress have been proposed. In bone and endothelial cells, the fluid shear stress induced activation of a variety of protein kinases and phospholipases (PLs), suggesting that putative mediators for the mechanotransduction are Ca 2ϩ channels, phospholipase C, protein kinase (PK) A, PKC, phospholipase A 2 , protein tyrosine kinases, CaMK-II, or mitogen-activated protein kinases (15, 25, 26, 46 -48). Both C/EBP ␤ and CREB are known to be phosphorylated by PKA, PKC, CaMK-II or p38 mitogen-activated protein kinase (49 -51), and AP-1 is phosphorylated by c-Jun N-terminal kinase and p42/44 mitogen-activated protein kinase (52). It remains to be clarified which signal transduction pathways dominantly contribute to the induction of COX-2 expression during shear stress loading.
C/EBP ␤, AP-1, and CREB have a leucine zipper domain for dimerization. It was reported that c-Jun and CREB formed a heterocomplex, and this complex played an important role in the transcription of some genes (53)(54)(55). In addition, evidence concerning cross-talk in signal transduction demonstrated that AP-1 efficiently transactivates CRE sequences and that Fos and Jun efficiently bind and cooperate in activating CRE promoter elements (56). It is therefore possible that these transcription factors associate with each other and regulate the COX-2 transcription in combination in response to fluid shear stress. In this study, however, only antibody against a given factor could recognize the shifted band of this factor with its respective oligonucleotide. Thus, these transcription factors seem to individually regulate the COX-2 transcription.
Besides sites of C/EBP ␤ (Ϫ138 to Ϫ130 bp), AP-1 (Ϫ73 to Ϫ61 bp), and CRE (Ϫ59 to Ϫ52 bp), other putative response elements exist in the promoter region of COX-2 gene, another CRE site (Ϫ447 to Ϫ440 bp), NF-B site (Ϫ401 to Ϫ393 bp), and another C/EBP ␤ (Ϫ93 to Ϫ85 bp). However, the deletion and mutation of these elements had no effect on the fluid shear stress-induced increase in luciferase activity, suggesting that these sites are not associated with the shear stress-activated COX-2 transcription. In addition, the COX-2 promoter region also contains a sequence (GAGACC, Ϫ305 to Ϫ300 bp) of shear stress response element (SSRE), which was initially reported in the promoter of the PDGF-B gene (57). However, the deletion of the COX-2 promoter region from Ϫ366 to Ϫ172 bp including the SSRE site failed to decrease the shear stress-induced luciferase response, implying the independence of the response from SSRE. On the contrary, the deletion (Ϫ366 to Ϫ172 bp) rather enhanced the luciferase response, suggesting that there might be suppressor sites in this region.
Regarding NF-B, evidence has been accumulating that shear stress regulates gene expression of endothelial cells via activation of NF-B (58). Bhullar et al. (59) reported that shear stress-induced NF-B translocation into the nucleus was dependent on integrin associated with cytoskeleton in endothelial cells. Pavalko et al. (15) reported that an inhibition of the cytoskeleton organization reduced COX-2 or c-Fos expression induced by 12 dynes/cm 2 of shear stress in MC3T3-E1 cells; however, the magnitude of the shear stress was about 5 times higher than that in this study. Taken together, these results suggest that mechanotransduction requires cytoskeleton-integrin interactions and raise the possibility that NF-B may be involved in the activation of COX-2 transcription by strong shear stress. In addition, the activation of NF-B has been reported to trigger the COX-2 expression elicited by some cytokines such as IL-1␤ and TNF-␣ in a variety of cells (60,61). Yamamoto et al. (28) demonstrated that the same mutated sequences of NF-B site employed in this study inhibited both the NF-B DNA binding and the NF-B-dependent COX-2 transcription induced by TNF-␣ in MC3T3-E1 cells. In our present study, however, the mutation and deletion of the NF-B site did not affect the increase in luciferase activity induced by shear stress. Thus, transcription response elements for COX-2 gene seem to vary in the types of stimuli and cells, but it should be noted that NF-B might regulate the shear stress-induced COX-2 induction under a high magnitude of shear stress.
In conclusion, our present study demonstrates that C/EBP ␤, AP-1, and CREB sites located at Ϫ138 to Ϫ130 bp, Ϫ73 to Ϫ61 bp, and Ϫ59 to Ϫ52 bp, respectively, in a COX-2 promoter region were required for the COX-2 transcription induced by fluid shear stress and that the shear stress increased the DNA binding activity of C/EBP ␤ and AP-1 and enhanced CREB phosphorylation. These data suggest that C/EBP ␤, AP-1, and CREB via each DNA-binding site regulate the fluid shear stress-induced COX-2 expression.