is involved in RANKL-induced osteoclastogenesis ...

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Prepublished online April 28, 2005; doi:10.1182/blood-2004-12-4975

Transcriptional induction of cyclooxygenase-2 in osteoclast precursors is involved in RANKL-induced osteoclastogenesis Song Y Han, Na K Lee, Kyung H Kim, In W Jang, Mijung Yim, Jae H Kim, Won J Lee and Soo Y Lee

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Blood First Edition Paper, prepublished online April 28, 2005; DOI 10.1182/blood-2004-12-4975

Transcriptional induction of cyclooxygenase-2 in osteoclast precursors is involved in RANKL-induced osteoclastogenesis (Short title: COX-2 induction and osteoclastogenesis by RANKL) Song Yi Han,1* Na Kyung Lee,1* Kyung Hee Kim,1 In Whan Jang,1 Mijung Yim,2 Jae Hong Kim,3 Won Jae Lee,1 and Soo Young Lee1,4

1

Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha

Womans University, Seoul, 120-750, Korea; 2College of Pharmacy, Sookmyung Women’s University, Seoul 140-742, Korea; 3School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea

* These authors have contributed equally for this work. Supported in part by the Molecular and Cellular BioDiscovery Research Grant (M1-0401-000045) from the Ministry of Science and Technology (to S.Y.L), by the Korea Science and Engineering Foundation (KOSEF) through the Center for Cell Signaling Research at Ewha Womans University, and by grant No. R01-2005-000-10305 from the Basic Research Program of the KOSEF. S.H., N.L. and K.K. were supported by BK21 fellowship from the Ministry of Education.

4

To whom correspondence should be addressed

Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha Womans University, Seoul, 120-750, Korea Tel: 82-2-3277-3770 Fax: 82-23277-3760 E-mail: [email protected]

Word count: 3555 Scientific heading: Hematopoiesis

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Copyright © 2005 American Society of Hematology


Abstarct Regulation of osteoclast differentiation is key to understanding the pathogenesis and to developing treatments for bone diseases such as osteoporosis. To gain insight into the mechanism of the RANKL-specific induction of the osteoclast differentiation program, we took suppression-subtractive hybridization screening approach to identify genes specifically induced via RANKL-Rac1 signaling pathway. Among identified targets, we show that RANKL selectively induces cyclooxygenase (COX)-2 expression via Rac1 that results in turn in PGE2 production in RAW 264.7 cells. By using transient transfection assays, we found that the -233/206 region of the COX-2 promoter gene was critical for RANKL-induced promoter activity. This RANKL-responsive region contained a NF- B site that, when mutated, completely abolished the induction of NF- B DNA binding activity by RANKL. Blockade of COX-2 by celecoxib inhibits differentiation of bone marrow-derived monocyte/macrophage precursor cells (BMMs) into TRAP-positive osteoclastic cells. This inhibition can be rescued by the addition of exogenous PGE2, suggesting that COX-2-dependent PGE2 induction by RANKL in osteoclast precursors is required for osteoclast differentiation.

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Introduction Bone remodeling depends on a delicate balance between bone formation and bone resorption, wherein bone-forming osteoblasts and bone-resorbing osteoclasts play central roles.1 Tipping this balance in favor of osteoclasts leads to pathological bone resorption, as seen in bone diseases such as autoimmune arthritis and postmenopausal osteoporosis.2 Thus, the investigation of the regulatory mechanisms of osteoclast differentiation is critical in the understanding of physiology and pathology of the skeletal system. Receptor activator of NF- B ligand (RANKL; also called TRANCE, ODF, and OPGL), a member of the tumor necrosis factor (TNF) family,3-6 plays an essential role in osteoclastogenesis, and its signaling mechanism has been extensively studied.7 Briefly, binding of RANKL to its receptor, RANK, results in the recruitment TRAF family proteins such as TRAF6, which in turn activates the NF- B and JNK pathways. RANKL also induces c-Fos by an as yet unknown mechanism. The essential role of the TRAF6 and c-Fos pathways in osteoclastogenesis determined by gene targeting studies has been well documented.8,9 In addition, several negative regulators of RANKL signaling have been reported.10-12 A typical example is osteoprotegerin (OPG), which functions as a soluble, decoy receptor for RANKL, thereby attenuating excessive RANKL signaling. To gain insight into how osteoclasts differentiate and function to control bone metabolism,

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we took suppression-subtractive hybridization (SSH) screening approach to identify genes specifically induced via RANKL-Rac1 signaling pathway. In this screening, we found that cyclooxygenase (COX)-2 is one of the strongly induced genes following RANKL stimulation in osteoclast precursors. COX-2 is known to be expressed at very low levels and is strongly induced by pro-inflammatory stimuli, including LPS, as well as by several activated oncogenes.13,14 The significance of COX-2 in inflammation is highlighted by the observation that COX-2 inhibitors block the synthesis of prostaglandin (PG) E2 (PGE2) and, as a result, inhibit inflammation and confer analgesia.15 In bone, PGE2 is produced mainly by osteoblasts and acts as a potent stimulator of bone resorption.16 The production of PGE2 by osteoblasts is regulated by several cytokines, including IL-1 and IL-6. The addition of indomethacin, an inhibitor of PG synthesis, to mouse bone marrow cultures strikingly suppresses osteoclast formation induced by IL-1, indicating that PGE2 production by osteoblasts is involved in the mechanism of bone resorption induced by IL-1.17,18 In the inflammatory processes, in which the mononuclear phagocyte family plays a crucial part, PGs by themselves have little inflammatory capacity, but in the presence of other mediators they can synergistically amplify the local inflammatory responses.19,20 Because the rate-limiting step in PGE2 synthesis is catalyzed by COX-2,21 the fact that osteoclast precursors express COX-2 by RANKL stimulation led to us investigation of direct involvement of PGE2 in osteoclast differentiation induced by RANKL

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signaling. Here, we provide evidences that COX-2 expression is dependent on RANKL-regulated small GTPase Rac1 pathway. We also show that RANKL stimulation results in the production of PGE2, which contributes to the accelerated osteoclast differentiation. We further demonstrate that the ability of Rac1 to regulate COX-2 expression depends on NF- B, a key regulator of COX-2 transcription. Finally, we show that inhibition of COX-2 by celecoxib severely suppresses differentiation of BMMs into TRAP-positive osteoclasts. This inhibition can be rescued by the addition of exogenous PGE2. Collectively, our results show that RANKL-Rac1NF- B-dependent COX-2 expression plays a key role in the RANKL-induced osteoclast differentiation via PGE2 production.

Materials and methods Cell culture and reagents RAW264.7 monocyte/macrophage cell line was maintained at 37°C in 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). RAW264.7 cells stably transfected with a cDNA encoding a dominant negative mutant of Rac1, RacN17 (RAW-RacN17) were maintained in DMEM supplemented with 10% FBS and 400 µg/ml G418. Celecoxib, purchased from LKT Laboratories (Minneapolis, MN), was

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dissolved in DMSO as a 100 mM stock solution and stored at -20°C. Recombinant M-CSF was purchased from R & D Systems; RANKL was from Peprotech (London, United Kingdom); and all other chemicals were from Sigma (St. Louis, MO). Anti-COX-2 (C-20) was from Santa Cruz Biotechnology (Santa Cruz, CA). In vitro osteoclastogenesis Nonadherent bone marrow-derived monocytes/macrophages cells derived from C57BL/6 mice were seeded and cultured in -MEM (GIBCO-BRL) with 10% FBS containing 10 ng/ml MCSF. After 2 days, nonadherent cells including lymphocytes were washed out, and adherent cells were used as bone marrow-derived monocytes/macrophages (BMMs). These osteoclast precursor cells were further cultured in the presence of 50 ng/ml RANKL and 10 ng/ml M-CSF to generate osteoclasts. After 5 days of culture, cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). TRAP-positive multinucleated (>3 nuclei) cells were counted as osteoclast-like cells. Subtractive hybridization and differential screening Total RNA was prepared using TRIZOL reagent (GIBCO). 1 mg of total RNA was subjected into Oligotex poly A+ RNA column (QIAGEN) to purify poly A+ RNA. 2 µg of poly A+ RNA from RANKL-treated RAW264.7 and RAW-RacN17 cells was used to make tester and driver cDNAs. Subtracted PCR was performed using the PCR-select cDNA subtraction kit according

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to the manufacturer’s protocol (CLONTECH). Briefly, tester and driver cDNAs were digested with RsaI, and the tester was ligated to adapter DNA. After two hybridizations with the tester and driver cDNAs, the resulting mixture was diluted 1:1000 and amplified by PCR using flanking and nested primers to produce a subtracted and normalized PCR fragment library. The efficiency of subtraction was verified via Southern blot analysis of the unsubtracted and subtracted PCR products using a 32P-labeled GAPDH cDNA probe. Subtracted PCR products were cloned into pGEM-T easy vector (Promega) for DNA sequence analysis. Northern hybridization and immunoblotting analysis For the Northern analysis, 20 µg total RNAs extracted from RANKL-treated RAW264.7 and RAW-RacN17 cells were separated and transferred to nylon membranes as described.22 The membranes were hybridized with [ -32P]dCTP probe prepared using a Ready-to-go labeling kit and a ProbeQuant G-50 purification kit (Amersham Biosciences). For immunoblotting analysis, 106 cells of RAW264.7 and RAW-RacN17 cells were stimulated with RANKL (50 ng/ml) for the indicated times and then lysed in 20 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol and proteinase inhibitors mix for 30 minutes on ice. After centrifugation, supernatants were subjected to 10% SDS-PAGE and transfered to PVDF membranes (Amersham Biosciences). The proteins were detected with specific Abs against COX-2 and tubulin (Santa Cruz Biotechnology).

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PGE2 measurements RAW264.7 cells and RAW-RacN17 cells were cultured for 15 hours in 96-well plates. Indomethacin (10 µM) and celecoxib (100 ng/ml) were applied 30 minutes prior to RANKL (50 ng/ml) stimulation for 8 hours. PGE2 level was measured in cells plated 104 per well, using a commercially available PGE2 enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI).

Transient transfections and luciferase assay Luciferase reporter pGL3 plasmid including 5'-flanking region (from -1838 to +123 bp) of the human COX-2 gene was kindly provided by Dr. Prescott (University of Utah). DNA fragments of various lengths of the COX-2 promoter regions (-1838/+123, -518/+123, -361/+123, 200/+123, -96/+123, -78/+123 and -44/+123) were prepared by PCR using the above plasmid as the template and subsequently sub-cloned into pGL3 plasmid. For luciferase assay, RAW264.7 and RAW-RacN17 cells were (1 x 107) suspended in 400 µl of serum-free medium were mixed with 10 µg of pGL3-basic or pGL3-COX-2 promoter-luciferase reporter and 2 µg of pCMVgalactosidase plasmid and electroporated (BTX-co.) using the pre-set protocol for RAW264.7 cells (155V, 70 msec, 1 pulse). 30 hours after electroporation, RANKL (50 ng/ml) was applied for 6 hours. Cells were then harvested and lysed in a reporter lysis buffer (Promega). Luciferase value was normalized to -galactosidase value. The relative luciferase value (RLV) was defined as the ratio of the COX-2 luciferase activity divided by the activity of pGL3-basic luciferase in

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cell lysates. The RLV of RANKL-stimulated pGL3-basic transfected cultures was given the arbitrary value of 1. The fold induction was calculated by dividing the RLV of a given culture by the RLV of the pGL3-basic control. Electrophoretic mobility shift assay (EMSA) RANKL-stimulated or unstimulated cells were lysed and nuclear extracts were prepared as described previously.23 For EMSA, 1 x 105 cpm of a 32P-end-labeled probe was incubated with a mixture of nuclear extract (5 µg) in the binding buffer [10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and 2 µg of poly (dI-dC) plus 5% NP-40] for 15 minutes at room temperature, and the protein-DNA complexes were resolved in a 5% native poly-acrylamide gel and detected by autoradiography. We used the following double-stranded probes:

proximal

NF- B

site

of

wild

type

probe

(5’-

CAGGAGAGTGGGGACTACCCCCTCTGCT-3’) (-233/-206), distal NF- B site of wild type probe (5’-GGCGGGAGAGGGGATTCCCTGCGCCCCCG-3’) (-457/-429), proximal NF- B site of mutant probe (5’-CAGGAGAGTGGccACTACCCCCTCTGCT-3’), and distal NF- B site of mutant probe (5’-GGCGGGAGAGGGGATctCCTGCGCCCCCG-3’). A double-stranded, NF- B-specific oligonucleotide probe containing the two tandemly repeated NF- B sites derived from the human immunodeficiency virus (HIV) long terminal repeat (5’ATCAGGGACTTTCCGCTGGGGACTTTCCG-3’) was also used as controls.24 The underlined

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nucleotides indicate the NF- B binding sites, and the lowercase nucleotides indicate the mutations.

RESULTS Identification of Rac1-dependent genes highly expressed during osteoclastogenesis upon RANKL stimulation. We have recently reported that RANKL activates Rac1 in RAW264.7 cells thereby regulating osteoclast differentiation.25(Fig7) To identify genes selectively activated by RANKL through Rac1-dependent pathway, differentially expressed genes between RANKLstimulated RAW264.7 cells and RANKL-stimulated RAW-RacN17 cells (denoted as RacN17) expressing a dominant interfering mutant RacN17 were isolated using suppression subtractive hybridization, a cDNA subtraction technique based on suppression PCR that is sensitive to rare transcripts. The subtractive cDNA libraries were constructed at the two different RANKLstimulated time points (8 hours and 3 days) and were analyzed as described previously.26 The most abundant clones chosen from the subtraction were carbonic anhydrase II, osteopontin, and cathepsin K, all of which were previously known to play critical roles in osteoclast function.27,28 As shown in Figure 1, mRNAs for carbonic anhydrase II and cathepsin K both of which are known to be strongly expressed in osteoclasts were specifically induced by RANKL in RAW264.7 cells but not in RAW-RacN17 cells, confirming the validity of our screening

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protocol. Among genes that have not previously been functionally implicated in osteoclast differentiation were immune response gene (IRG) 1,29 interferon (IFN)-inducible gene (Ifi) 202,30 and COX-2 (Figure 1). In osteoblasts, COX-2 is known to play a critical role in synthesis of PGE221 thereby stimulating osteoclast differentiation indirectly.31 Since RANKL-stimulated COX-2 induction in osteoclast precursors has not been reported previously, COX-2 was chosen for further analysis in this study.

Requirement of the Rac1 pathways in RANKL-induced COX-2 expression and PGE2 induction. To confirm that Rac1 controls the induction of COX-2 by RANKL, we measured COX-2 expression by Northern blotting with RNA preparation from RAW264.7 and RAWRacN17 cells at various time points after RANKL stimulation. The results showed that COX-2 induction of RAW264.7 cells was seen from 2 hours after RANKL stimulation and decreased toward basal level after 24 hours (Figure 2A). In contrast, COX-2 induction by RANKL was significantly lower in RAW-RacN17 cells. Consistent with this result, immunoblotting analysis revealed that robust induction of COX-2 protein occurred in RAW264.7 cells but not in RAWRacN17 cells (Figure 2B).

To determine whether PGE2 secretion depends on RANKL-Rac1-dependent pathway, we measured the level of PGE2 in supernatants of RAW264.7 and RAW-RacN17 cell cultures

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harvested before and after 8 hours of stimulation with RANKL. As shown in Figure 3A, RANKL stimulation induced PGE2 production in RAW264.7 cells whereas RAW-RacN17 cells were defective in PGE2 induction by RANKL. Consistently, COX inhibitors, celecoxib and indomethacin, completely inhibited RANKL-induced PGE2 secretion (Figure 3B). These data showed that COX-2 expression by Rac1-transduced signals is required for PGE2 induction by RANKL.

Functional activity of COX-2 promoter in response to RANKL.

To explore the

transcriptional regulation of COX-2 gene by RANKL stimulation, we studied the COX-2 promoter-driven transcription in RAW264.7 cells transiently transfected with DNAs containing a region spanning from -1838 to +123 bp relative to the transcription start site of the human COX-2 gene in the reporter plasmid pGL3 Basic (P1-1838). As shown in Figure 4A, luciferase activity of this construct was strongly up-regulated upon activation by RANKL. To map the regions responsible for this induction, subsequent 5’-deletion constructs ranging from -518 down to -44 bp were generated (Figure 4A). An induction in promoter activity upon RANKL stimulation was observed only in the reporter construct, P-518, indicating that a critical regulatory region at -518/-362 bp was responsible for this induction. Sequence analysis revealed the existence of a potential distal NF- B-binding site at -457/-429 bp.32 In contrast, the promoter activity of a shorter construct, P-361, containing a proximal NF- B-binding site at -

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233/-206 bp could not be induced by RANKL stimulation, suggesting that the distal NF- Bbinding site but not the proximal one is important for RANKL-induced activation of COX-2 promoter. To determine whether Rac1 regulates COX-2 promoter activity, RAW264.7 and RAWRacN17 cells were electroporated with a reporter construct containing the luciferase gene under the control of the COX-2 promoter (P-1838) or reporter basic (pGL3-basic). After 30 hours, cells were untreated or treated with RANKL or LPS for 6 hours. Cells were then lysed and luciferase activity was measured in the lyastes. The results showed that LPS increased COX-2 promoter activity in both RAW264.7 and RAW-RacN17 cells. However, RANKL activated the COX-2 promoter in RAW264.7 cells but not RAW-RacN17 cells (Figure 4B). Taken together, these data indicate that the transcriptional induction of COX-2 in RAW264.7 cells upon RANKL stimulation occurs via Rac1-NF- B signaling cascades. To further analyze the functionality of these sequences as NF- B binding elements, we performed electrophoretic mobility shift assay (EMSA) with nuclear extracts of RAW264.7 cells using DNA probes of the two putative NF- B binding sites. A retarded complex was resolved in nuclear extracts from RAW264.7 cells stimulated by RANKL with the distal NF- B probe (-457/-429) but not with the proximal probe (-233/-206) (Figure 5A). This band was not seen with a distal NF- B probe (-457/-429M) containing a mutation in the NF- B binding

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element, confirming the specificity of the reaction. As expected, the retarded complex could not be seen in RAW-RacN17 cells upon RANKL stimulation (Figure 5B). We also performed EMSA with an oligonucleotide probe containing the two NF- B sites derived from the HIV long terminal repeat. Consistent with above, RANKL-induced NF- B activation was severely affected in RAW-RacN17 cells (Figure 5C). Inhibition of osteoclast differentiation by COX-2 inhibitors. In order to investigate the role of RANKL-stimulated COX-2 expression in osteoclast precursors, we examined the effect of a specific inhibitor of COX-2, celecoxib, on osteoclast differentiation of BMMs in the absence of stromal cells. Celecoxib inhibited RANKL-induced osteoclast differentiation from BMMs in a dose-dependent manner (Figure 6A; c, d, and e). The similar inhibitory effect was observed with indomethacin, COX-1 and -2 inhibitors in BMMs (data not shown). Since the expression of COX-2 is clearly seen within 15 hours after RANKL stimulation (Figure 2), we next examined whether celecoxib affects the early stage or the late stage of osteoclast differentiation by performing temporal treatment with celecoxib. Addition of celecoxib 2 days after RANKL stimulation had no effect on osteoclast formation (Figure 6A; f, g, and h), consistent with that the early time induction of PGE2 by RANKL stimulation is necessary for osteoclast differentiation. Exogenous PGE2 reversed the inhibitory effects of celecoxib. Celecoxib is well known to

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inhibit COX-2,33 the enzyme responsible for catalyzing the formation of PGE2 from arachidonic acid. Whether the inhibitory effects of celecoxib on osteoclastogenesis are mediated through the suppression of COX-2 was investigated. To determine this, BMMs were exposed to exogenous PGE2 together with celecoxib and then examined for RANKL-induced osteoclast differentiation. The results in Figure 7 show that suppressive effects of osteoclast differentiation by celecoxib could be reversed by PGE2. The latter alone had no effect on osteoclast differentiation. Thus, these results suggest that celecoxib inhibits osteoclast differentiation through the suppression of COX-2 activity, thereby resulting in the inhibition of PGE2 induction.

Discussion RANKL signaling is known to be essential for osteoclast differentiation, at the stage after the commitment of the bone marrow progenitor cells into the myelomonocytic lineage.7 Although our understanding of signaling pathways associated with osteoclast differentiation advanced considerably recently, the regulatory factor(s) of RANKL-mediated osteoclastogenesis, involved in transmission and modulation of RANKL signaling is still unknown. By employing PCRbased suppression subtractive hybridization, we have identified COX-2 gene whose expression is highly enriched during RANKL stimulation and whose induction is impaired in RAW264.7 cells expressing a dominant negative form of Rac1.

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Our study clearly demonstrates a pathway uniquely linked to RANKL signaling, culminating in the expression of COX-2 through RANK-Rac1-NF- B-dependent pathway. Osteoclast formation induced by RANKL was accompanied with PGE2 production, which was blocked by a selective COX-2 inhibitor celecoxib. We also showed that suppression of osteoclastogenesis by celecoxib could be reversed by PGE2. Moreover, simultaneous treatment with PGE2 in the presence of RANKL cooperatively induced osteoclast differentiation. Taken together, these data show RANKL-induced COX-2 is responsible for PGE2 production in osteoclast precursors for regulating osteoclast differentiation. PGE2 has been known to play a crucial role in regulation of bone resorption.34 Agonists reported to stimulate PGE2-dependent osteoclast formation include IL-1,35 TNF- ,36 and PTH.37 Most of these agonists induce PGE2 production in osteoblasts and stromal cells, and osteoclast formation induced by these agonists is dependent on PGE2 synthesis in mouse bone marrow cultures.38,39 It has also been reported that COX-2 and PGE2 play important roles in bone formation of rat long bones as shown in an experiment using systemic injection of PGE240 and are required for mesenchymal cell differentiation into osteoblasts.41 Because of the complexity of responses to PGE2 in bone cells including osteoclasts and osteoblasts, it is difficult to define precisely the net effect of PGE2 on bone resorption and formation. However, our data suggest that RANKL-induced PGE2 synthesis in osteoclast precursors is cell-autonomous and that PGE2

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acts directly on the precursors for osteoclast formation in a paracrine or an autocrine manner. This is consistent with the previous finding of a synergistic effect of PGE2 on the ability RANKL to induce osteoclast differentiation.42 In addition, Okada et al43 showed a reduction in RANKL-stimulated osteoclast formation in spleen cell cultures in the absence of osteoblasts from COX-2-/- mice, suggesting that PGE2 originates from multiple sources during osteoclastogenesis in vivo. PGE2 exerts its effects through interaction with specific cell surface receptors.44 The PGE receptors, divided into four subtypes (EP1, EP2, EP3, and EP4), are important in regulating PGE2 action in the target cells. Among them, EP4 and EP2 are coupled to activation of adenyl cyclase and are implicated in PGE2-induced osteoclastogenesis.45-47 It is tempting to speculate that the PGE2-EP signaling works in osteoclast precursors to induce a maximal osteoclast formation. Evidence for expression of both EP2 and EP4 receptors in the osteoclast precursors including RAW264.7 cells and BMMs48 supports this possibility. It remains to be clarified under what physiological or pathological contexts the PGE2-EP signaling is mobilized for bone resorption. Whatever the detailed mechanism underlying the above process, our results indicate that RANKL induces COX-2 expression in osteoclast precursors through Rac1-dependent NF- B activation pathway thereby resulting in PGE2 production. Although PGE2 itself is not sufficient

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for regulating osteoclast formation, PGE2 likely acts in an autocrine or a paracrine loop to enhance the early phase differentiation of osteoclasts. These findings suggest that signaling molecules involved in COX-2-PGE2 pathway in osteoclast precursors could potentially be novel targets for drugs to inhibit osteoclast function.

Acknowledgements We thank Jaesang Kim for critical reading of the manuscript and S. Prescott for providing the COX-2 promoter vector.

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References 1. Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev. 2000;21:115-137. 2. Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science. 2000;289:15081514. 3. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, Teepe MC, DuBose RF, Cosman D, Galibert L. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature. 1997;390:175-179. 4. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, Kalachikov S, Cayani E, Bartlett FS III, Frankel WN, Lee SY, Choi Y. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinases in T cells. J Biol Chem. 1997;272:25910-25914. 5. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998;95:3597-3602. 6. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX,

ዊዊ


Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998; 93:165-176. 7. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423:337-342. 8. Grigoriadis AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, Fleisch HA, Wagner EF. cFos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science. 1994;266:443-448. 9. Lomaga MA, Yeh WC, Sarosi I, Duncan GS, Furlonger C, Ho A, Morony S, Capparelli C, Van G, Kaufman S. et al., TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 1999;13:1015-1024. 10. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Boyle WJ, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 1997;89:309-319. 11. Takayanagi H, Ogasawara K, Hida S, Chiba T, Murata S, Sato K, Takaoka A, Yokochi T, Oda H, Tanaka K, Nakamura K, Taniguchi T. T-cell-mediated regulation of osteoclastogenesis

ዊዊ


by signalling cross-talk between RANKL and IFN- . Nature. 2000;408:600-605. 12. Takayanagi H, Kim S, Matsuo K, Suzuki H, Suzuki T, Sato K, Yokochi T, Oda H, Nakamura K, Ida N, Wagner EF, Taniguchi T. RANKL maintains bone homeostasis through c-Fosdependent induction of interferon- . Nature. 2002;416:744-749. 13. Caivano M, Cohen P. Role of mitogen-activated protein kinase cascades in mediating lipopolysaccharide-stimulated induction of cyclooxygenase-2 and IL-1

in RAW264

macrophages. J. Immunol. 2000;164:3018-3025. 14. Sheng H, Williams CS, Shao J, Liang P, DuBois RN, Beauchamp RD. Induction of cyclooxygenase-2 by activated Ha-ras oncogene in Rat-1 fibroblasts and the role of mitogenactivated protein kinase pathway. J Biol Chem. 1998;273:22120-22127. 15. Meade EA, Smith WL. DeWitt DL. Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs. J Biol Chem. 1993;268:6610-6614. 16. Suda T, Takahashi N, Martin TJ. Modulation of osteoclast differentiation. Endocr Rev. 1992;13:66-80. 17. Tai H, Miyaura C, Pilbeam CC, Tamura T, Ohsugi Y, Koishihara Y, Kubodera N, Kawaguchi H, Raisz LG, Suda T. Transcriptional induction of cyclooxygenase-2 in osteoblasts is involved in interleukin-6-induced osteoclast formation. Endocrinology. 1997;138:2372-2379.

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18. Chen QR, Miyaura C, Higashi S, Murakami M, Kudo I, Saito S, Hiraide T, Shibasaki Y, Suda T. Activation of cytosolic phospholipase A2 by platelet-derived growth factor is essential for cyclooxygenase-2-dependent prostaglandin E2 synthesis in mouse osteoblasts cultured with interleukin-1. J Biol Chem. 1997;272:5952-5958. 19. Williams TJ, Morley J. Prostaglandins as potentiators of increased vascular permeability in inflammation. Nature. 1973;246:215-217. 20. Moncada S, Ferreria SH, Vane JR. Prostaglandins, aspirin-like drugs and the oedema of inflammation. Nature. 1973;246:217-219. 21. Herschman HR. Prostaglandin synthase 2. Biochim Biophys Acta. 1996;1299:125-140. 22. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, Kalachikov S, Cayani E, Bartlett FS 3rd, Frankel WN, Lee SY, Choi Y. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem. 1997;272:25190-25194. 23. Lee SY, Reichlin A, Santana A, Sokol KA, Nussenzweig MC, Choi Y. TRAF2 is essential for JNK but not NF-kappaB activation and regulates lymphocyte proliferation and survival. Immunity. 1997;7:703-713. 24. Pfeffer K, Matsuyama T, Kundig TM, Wakeham A, Kishihara K, Shahinian A, Wiegmann K, Ohashi PS, Kronke M, Mak TW. Mice deficient for the 55 kd tumor necrosis factor receptor

ዊዊ


are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell. 1993;73:457-467. 25. Lee NK, Choi YG, Baik JY, Han SI, Jeong D, Bae YS, Kim N, Lee SY. A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood. Prepublished on April 7, 2005, as DOI 10.1182/blood-2004-09-3662. 26. Rho J, Altmann CR, Socci ND, Merkov L, Kim N, So H, Lee O, Takami M, Brivanlou AH, Choi Y. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis. DNA Cell Biol. 2002;21:541-549. 27. Zaidi M, Blair HC, Moonga BS, Abe E, Huang CL. Osteoclastogenesis, bone resorption, and osteoclast-based therapeutics. J Bone Miner Res. 2003;18:599-609. 28. Sodek J, Ganss B, McKee MD. Osteopontin. Crit Rev Oral Biol Med. 2000;11:279-303. 29. Cheon YP, Xu X, Bagchi MK, Bagchi IC. Immune-responsive gene 1 is a novel target of progesterone receptor and plays a critical role during implantation in the mouse. Endocrinology. 2003;144:5623-5630. 30. Rozzo SJ, Allard JD, Choubey D, Vyse TJ, Izui S, Peltz G, Kotzin BL. Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus. Immunity. 2001;15:435-443.

ዊዊ


31.Smith WL, Dewitt DL. Prostaglandin endoperoxide H synthases-1 and -2. Adv Immunol. 1996;62:167-215. 32. Tazawa R, Xu XM, Wu KK, Wang LH. Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene. Biochem Biophys Res Commun. 1994;203:190-199. 33. Kiefer W, Dannhardt G. COX-2 inhibition and the control of pain. Curr Opin Investig Drugs. 2002;3:1348-1358. 34. Kawaguchi H, Pilbeam CC, Harrison JR, Raisz LG. The role of prostaglandins in the regulation of bone metabolism. Clin Orthop. 1995;313:36-46. 35. Sato T. et al. Involvement of prostaglandin endoperoxide H synthase-2 in osteoclast-like cell formation induced by interleukin-1 . J Bone Miner Res. 1996;11:392-400. 36. Lader CS, Flanagan AM. Prostaglandin E2, interleukin 1 , and tumor necrosis factorincrease human osteoclast formation and bone resorption in vitro. Endocrinology. 1998;139:3157-3164. 37. Sato T, Morita I, Murota S. Prostaglandin E2 mediates parathyroid hormone induced osteoclast formation by cyclic AMP independent mechanism. Adv Exp Med Biol. 1997;407:383-386. 38. Raisz LG. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, San

ዊዊ


Diego, CA;1996: 661-670. 39. Miyaura C, Inada M, Matsumoto C, Ohshiba T, Uozumi N, Shimizu T, Ito A. An essential role of cytosolic phospholipase A2alpha in prostaglandin E2-mediated bone resorption associated with inflammation. J Exp Med. 2003;197:1303-1310. 40. Suponitzky I, Weinreb M. Differential effects of systemic prostaglandin E2 on bone mass in rat long bones and calvariae. J Endocrinol. 1998;156:51-57. 41. Zhang X, Schwarz EM, Young DA, Puzas J, Rosier RN, O’Keefe RJ. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest. 2002;109:1405-1415. 42. Wani MR, Fuller K, Kim NS, Choi Y, Chambers T. Prostaglandin E2 cooperates with TRANCE in osteoclast induction from hemopoietic precursors: synergistic activation of differentiation, cell spreading, and fusion. Endocrinology. 1999 ;140:1927-1935. 43. Okada Y, Lorenzo JA, Freeman AM, Tomita M, Morham SG, Raisz LG, Pilbeam CC. Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture. J Clin Invest. 2000;105:823-32. 44. Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and function. Physiol Rev. 1999;79:1193-1226. 45. Sakuma Y, Tanaka K, Suda M, Yasoda A, Natsui K, Tanaka I, Ushikubi F, Narumiya S, Segi

ዊዊ


E, Sugimoto Y, Ichikawa A, Nakao K. Crucial involvement of the EP4 subtype of prostaglandin E receptor in osteoclast formation by proinflammatory cytokines and lipopolysaccharide. J Bone Miner Res. 2000;15:218-227. 46. Miyaura C, Inada M, Suzawa T, Sugimoto Y, Ushikubi F, Ichikawa A, Narumiya S, Suda T. Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice. J Biol Chem. 2000;275:19819-19823. 47. Li X, Okada Y, Pilbeam CC, Lorenzo JA, Kennedy CR, Breyer RM, Raisz LG. Knockout of the murine prostaglandin EP2 receptor impairs osteoclastogenesis in vitro. Endocrinology. 2000;141:2054-2061. 48. Kobayashi Y, Mizoguchi T, Take I, Kurihara S, Udagawa N, Takahashi N. Prostaglandin E2 enhances osteoclastic differentiation of precursor cells through protein kinase A-dependent phosphorylation of TAK1. J Biol Chem. 2005;280:11395-11403.

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Figure legends Figure 1. Identification of specifically expressed genes in RANKL-stimulated RAW 264.7 cells. Northern analysis of genes expressed during osteoclast differentiation. Total RNA was extracted from RAW264.7 and RAW-RacN17 cells stimulated with RANKL for the indicated periods. 20 µg of total RNA was applied to each lane. cDNAs for cathepsin K, carbonic anhydrase II, osteopontin, Ifi202, IRG1, and COX-2 were used to prepare probes and hybridized to total RNA. GAPDH, glyceraldehydes-3-phosphate dehydroganase was used as a control for total RNA loading. Figure 2. COX-2 is rapidly induced by RANKL stimulation in a Rac1-dependent manner. (A) Expression of COX-2 mRNA in RAW264.7 and RAW-RacN17 cells stimulated with RANKL for the indicated periods was examined by Northern analysis. GAPDH was used as a control for total RNA loading. (B) Expression of COX-2 protein in RAW264.7 and RAW-RacN17 cells stimulated with RANKL for the indicated periods. Whole cell lysates (50 µg each) were immunoblotted with anti-COX-2 Ab and reprobed with anti-tubulin Ab. Figure 3. RANKL-stimulated PGE2 secretion is compromised in RAW-RacN17 cells. (A) RAW264.7 cells and RAW-RacN17 cells were stimulated with or without RANKL (50 ng/ml) for 8 hours. Culture supernatants were collected and PGE2 concentration was measured as described in Materials and methods. Unstimulated cultures from RAW264.7 and RAW-RacN17

ዊዊ


cells secreted similar levels of PGE2. PGE2 levels produced by unstimulated cells were given the arbitrary value of 1. Data are expressed as mean (+/-SD) of triplicates. Similar results were obtained in two additional experiments. (B) RAW264.7 cells were pretreated for 30 minutes with indomethacin (10 µM) and celecoxib (100 ng/ml) and stimulated with RANKL for 8 hours. PGE2 levels were measured as in (A). Data are expressed as mean (+/-SD) of triplicates. Figure 4. Analysis of the region responsible for the promoter activity of COX-2 gene in response to RANKL stimulation. (A) Putative consensus sequences in the 5’-flanking region of human COX-2 gene are illustrated in the upper left. Each deleted promoter fragment was inserted into a pGL3 basic luciferase vector. Numbers indicate distance in bp from the start site of transcription. RAW264.7 cells were transiently transfected with these plasmids along with galactosidase plasmid. The transfected cells were stimulated with RANKL (50 ng/ml) for 6 hours, and then the activities of luciferase and -galactosidase were measured. The normalized luciferase activities in the cells transfected with various reporter plasmids are presented as the fold induction calculated by dividing the relative luciferase value (RLV) of a given culture by the RLV of the pGL3-basic control. Data are expressed as mean (+/-SD) of triplicates. (B) As in (A), except that RAW264.7 cells and RAW-RacN17 cells were transfected with the reporter plasmid P-1838 and stimulated with RANKL (50 ng/ml) and LPS (1 µg/ml), respectively. PBS indicates phosphate buffered saline used for a control. Data are expressed as mean (+/-SD) of

ዊዊ


triplicates. Figure 5. Electrophoretic mobility shift assay targeting NF- B sites. (A) Nuclear extracts from RAW264.7 cells stimulated with RANKL (50 ng/ml) for 15 minutes were analyzed by electrophoretic mobility shift assay. Gel shift assays were performed using oligonucleotides corresponding to the COX-2 distal NF- B site (nucleotides -457/-429) and the COX-2 proximal site (nucleotides -233/-206). The COX-2 distal site (nucleotides -457/-429) probe containing a mutation in NF- B element abolished the complex formation. (B) As in (A), except that nuclear extracts were prepared from RAW-RacN17 cells. Gel shift assays were performed using oligonucleotides corresponding to the COX-2 distal NF- B site (nucleotides -457/-429). (C) Gel shift assays were performed using oligonucleotides containing the two tandemly repeated NFB sites derived from the HIV long terminal repeat. The arrowheads indicate specific RANKLinduced complexes. The NS indicates a nonspecific protein complex. The arrows point to free probes. Similar results were obtained in two additional experiments. Figure 6. Suppressive effects of COX-2 inhibitor, celecoxib on RANKL-induced osteoclastogenesis in BMMs. (A) Demonstration of TRAP-positive cells differentiating from BMMs unstimulated (a) or stimulated (b) with RANKL. On day 5 with RANKL stimulation, the cells were fixed and stained for TRAP. TRAP-positive cells appear as red cells. (c-h) Dosedependent effects of celecoxib on the formation of TRAP-positive multinucleated osteoclasts

ዊዊ


(MNC) with RANKL stimulation. BMMs were pretreated (c, 100 ng/ml; d, 50 ng/ml; e, 25 ng/ml) with different concentrations of celecoxib or treated (f, 100 ng/ml; g, 50 ng/ml; h, 25 ng/ml) 2 days after RANKL stimulation. On day 5 with RANKL stimulation, the cells were fixed and stained for TRAP (original magnification, x 100). (B) Total numbers of TRAPpositive MNCs are shown. Note that formation of TRAP-positive MNCs was inhibited by pretreatment of celecoxib in a dose-dependent manner. The open and filled arrowheads indicate pretreatment and post-treatment with celecoxib, respectively. Similar results were obtained in two additional experiments. Figure 7. Exogenous PGE2 reversed the inhibitory effects of celecoxib. BMMs were pretreated with celecoxib (50 ng/ml) and PGE2 either alone or in combination as indicated and then stimulated with RANKL. On day 5, the cells were fixed and stained for TRAP as described in Materials and methods. Similar results were obtained in two additional experiments.

ዊዊ


RANKL

8 hours

3 days

7 17 N1 W cN W W c a RA R RA RA Ra

Cathepsin K Carbornic anhydraseዊ Osteopontin Ifi202

IRG1 COX-2 GAPDH

ዊዊ


A

hours

RANKL – 2

5 8 15 1 2 3

days 5

7

COX-2 RAW264.7

GAPDH COX-2

RAW-RacN17 GAPDH

B

RAW264.7

RAW-RacN17

RANKL 0 2 5 8 15 24 0 2 5 8 15 24 (hours) COX-2

tubulin

ዊዊ


B

ዊዊዊ ዊዊዊ

PGE2 synthesis (fold induction)

RAW264.7 RAW-RacN17

ዊዊዊ ዊዊዊ ዊዊዊ

ዊዊዊ ዊዊዊ ዊዊዊ ዊዊዊ ዊዊዊ

control

Ce lec ox In ib do m et ha cin

ዊዊዊ

ዊዊዊ

co nt ro l RA NK L

PGE2 synthesis (fold induction)

A

RANKL

ዊዊ

pNFAT AP1 ATF/CRE E-box TATA

dNFAT

NF- B

A

NF- B


+1

Luc

P-1838

Luc

P-518

Luc

P-361

Luc

P-200

Luc

P-96 P-78 P-44

Luc

-RANKL +RANKL

Luc

pGL3 Basic Luc 0

5

10

15

20

25

30

Relative luciferase value

B P-1838

LPS RANKL PBS

pGL3 Basic

RAW264.7 RacN17

PBS 0

5

10

15

20

Relative luciferase value

ዊዊ

25

30


B

+

+

RAW264.7

-233/-206M

–

-457/-429

-233/-206

+

-233/-206

-457/-429 -457/-429M

-457/-429

RANKL – +

C RAW-RacN17

RAW264.7

RANKL – +

RANKL – +

NF- B

NF- B

NS

NS

ዊዊ

RAW-RacN17

A

– +

NF- B


a

c

f

b

d

g

e

h

TRAP-positive MNCs/well

B

A

160 120

80 40

0

M-CSF + RANKL Celecoxib

+ +

+ +

+ +

+ +

+ +

+ +

+ +

100 50 25 100 50 25 (ng/ml)

ዊዊ


TRAP-positive MNCs/well

300

200

100

0 M-CSF + RANKL Celecoxib PGE2

+

+ +

+

+ + +

ዊዊ

+ + +

+ + + +