RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation

E XP ER I ME NT A L C EL L RE S EA R CH 3 13 ( 20 0 7 ) 1 6 8 –17 8

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation Kumaran Sundaram a , Riko Nishimura b , Joseph Senn a , Rimon F. Youssef a , Steven D. London c , Sakamuri V. Reddy a,⁎ a

Charles P. Darby Children's Research Institute, 173 Ashley Avenue, Charleston, SC 29425, USA Department of Biochemistry, Osaka University Graduate School of Dentistry, Osaka, Japan c College of Dental Medicine, Medical University of South Carolina, Charleston, SC 29425, USA b

ARTICLE

INFORMATION

ABS T R AC T

Article Chronology:

Osteoclast differentiation is tightly regulated by receptor activator of NF-κB ligand (RANKL)

Received 9 June 2006

signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in

Revised version received

osteoclast cells and plays an important role in degradation of extracellular matrix; however,

22 September 2006

the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study,

Accepted 2 October 2006

we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor

Available online 6 October 2006

cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580

Keywords:

increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-

RANK ligand (RANKL)

9 activity in RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9

Osteoclast

expression through p38 and ERK signaling pathways during osteoclast differentiation.

Matrix metalloproteinase-9 (MMP-9)

Transient expression of MMP-9 gene (+1 to −1174 bp relative to ATG start codon) promoter-

NFAT

luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant

TRAF6

increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to +1 bp to −446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from −446 bp to −1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (−1123 bp to −1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity in the absence of RANKL. Taken together, our results suggest that RANKL signals through TRAF6 and that NFATc1 is a downstream effector of RANKL signaling to modulate MMP-9 gene expression during osteoclast differentiation. © 2006 Elsevier Inc. All rights reserved.

⁎ Corresponding author. Fax: +1 843 792 7927. E-mail address: [email protected] (S.V. Reddy). 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.10.001

E XP E RI ME N TA L CE LL RE S EA RCH 3 13 ( 20 0 7 ) 1 6 8 –17 8

Introduction Bone remodeling is a physiological process that involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. Matrix metalloproteinases (MMPs) are a family of structurally conserved zinc-dependent proteolytic enzymes involved in the degradation of the extracellular matrix of various tissues including bone. Matrix metalloproteinase 9 (MMP-9) also termed gelatinase B/type IV collagenase is highly expressed at early stages of osteoclast development and in mature osteoclasts which resorb bone [1]. Increased MMP-9 activity has been detected in human osteoclastomas and osteoclasts in Paget's disease [2]. It has also been shown that upregulation of MMP-9 plays an important role in the pathogenesis of dental pulp inflammation and destruction [3]. Cytokines such as IL-1 markedly induce osteoclast formation and have been shown to upregulate MMP-9 expression responsible for bone matrix degradation [4]. Similarly, tumor necrosis factor-alpha (TNF-α) and granulocyte macrophage colony-stimulating factor (GM-CSF) have been shown to enhance MMP-9 activity and that interferon (IFN)-gamma inhibited TNF-α induced MMP-9 expression through a caspase-8dependent pathway [5]. It has also been shown that stromal cell-derived factor-1 (SDF-1) and RANKL increased MMP-9 expression which is essential for osteoclast precursor recruitment into bone [6]. MMPs may play a role in osteoclast migration, attachment/detachment and in regulating osteoclast behavior by cleaving the extracellular domains of regulatory proteins such as ICAM, IL-1β and RANKL [7]. Homozygous mice with a null mutation in the MMP-9 demonstrated an abnormal pattern of skeletal growth plate vascularization and ossification [8]. Recently it has been demonstrated that MMP-9 mediates vascular invasion of the hypertrophic cartilage callus, and that MMP-9 (−/−) mice have non-unions and delayed unions of their fractures caused by persistent cartilage at the injury site indicating that MMP-9 regulates crucial events during adult fracture repair [9]. MMP-9 deficiency results in accumulation of late hypertrophic chondrocytes. Galectin-3, an in vitro substrate of MMP-9 is a downstream regulator of MMP-9 function during endochondral bone formation [10]. RANK ligand (RANKL) is a member of the TNF family that is produced by osteoblasts and stromal cells in the bone microenvironment. Receptor activator of NF-κB (RANK) is expressed on committed osteoclast precursors. RANKL in combination with M-CSF induce differentiation of osteoclast precursors/spleen cells to form multinucleated osteoclasts in the absence of stromal/osteoblast cells. RANKL binding to RANK receptor signals through TRAF6 adapter protein resulting in activation of NF-κB and c-Jun N-terminal kinase (JNK) in osteoclast precursor cells, which then fuse to form multinucleated osteoclasts [11]. It has been suggested that macrophage fusion receptor (MFR) interaction with CD47 and CD44 mediate the adhesion/fusion to form multinucleated osteoclasts [12]. Furthermore, hyaluronan binding to CD44 resulted in down-regulation of MMP-9 expression affecting the motility of osteoclast-like cells on the permissive matrix substrates [13]. In addition, osteoprotegerin (OPG), a decoy receptor of RANKL which inhibits osteoclastogenesis has

169

been shown to induce promatrix MMP-9 activity and phosphorylation of p38 and ERK1/2 in osteoclast precursor cells [14]. Although TRAF6 is essential for both RANKL and TNF-α induced osteoclast differentiation, it has been reported that these cytokines did not effectively induce osteoclast precursor cells derived from TRAF5-deficient mice, however, JNK and NF-κB activation occurred in these cells [15]. These data suggest that other members of TRAF family may also be associated with RANK signaling in osteoclastogenesis. In vitro and in vivo molecular genetic approaches have provided evidence indicating several transcription factors play an important role in osteoclast differentiation and bone resorption activity [16]. Recently, it has been reported that calcineurin/NFAT (nuclear factor of activated T cells) signaling pathway regulates osteoclast differentiation. NFATc1-deficient embryonic stem cells fail to differentiate into bone resorbing osteoclasts in response to RANKL stimulation [17]. Furthermore, RANKL has been shown to upregulate NFATc1 expression during osteoclast differentiation. These results also indicated that calcineurin as an essential downstream effector of the RANKL signaling pathway. In addition, constitutively active calcineurin-independent NFATc1 mutant expression is sufficient to induce osteoclast differentiation of RAW264.7 cells in vitro [18]. Therefore, NFATc1 plays an important role in regulating osteoclast differentiation and function in response to RANKL stimulation. Although high levels of MMP-9 expression are detected in osteoclast precursors and bone resorbing osteoclasts, the molecular mechanisms that control MMP-9 gene expression during osteoclast differentiation are not well characterized. We and others have previously isolated the 5′-flanking region of the murine MMP-9 gene and characterized the promoter activity [19,20]. In this study, we show RANKL signals through TRAF6 and that NFATc1 is a downstream effector of RANKL signaling to modulate MMP-9 gene expression during osteoclast differentiation.

Materials and methods Reagents and antibodies The cell culture and DNA transfection reagents were purchased from Invitrogen (Carlsbad, CA). The ERK1/2, p38 MAPK inhibitors PD98059 and SB203580 were obtained from Calbiochem (La Jolla, CA). Rabbit anti-mouse MMP-9, anti-NFATc1, siRNAs and peroxidase conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human RANK ligand (RANKL) was purchased from R&D (Minneapolis, MN). Zymogram gels were obtained from Invitrogen (Carlsbad, CA).

Cell cultures RAW264.7 mouse macrophage cells (American Type Culture Collection) were cultured in α-minimum essential medium (αMEM) supplemented with 10% (v/v) fetal bovine serum and 1% penicillin and streptomycin at 37°C with 5% CO2 (Invitrogen, Carlsbad, CA). C57BL/6 mouse bone marrow cells from long

170

E XP ER I ME NT A L C EL L RE S EA R CH 3 13 ( 20 0 7 ) 1 6 8 –17 8

bones were suspended in culture medium (α-MEM containing L-glutamine, streptomycin and 10% fetal bovine serum) supplemented with M-CSF (10 ng/ml) and cultured overnight. Non-adherent cells obtained were cultured at a density of 5 × 104 cells/cm2 for 48 h in the presence of M-CSF. The resultant preosteoclast cells were used for RANKL stimulation experiments.

Gelatin zymography RAW264.7 and mouse bone-marrow-derived cell-conditioned medium was analyzed for MMP-9 activity by gelatin substrate gel electrophoresis as described [21]. Serum-starved cell cultured media samples were applied without reduction to a 10% polyacrylamide gel containing 0.1% gelatin. After electrophoresis, the gels were washed in washing buffer (50 mM Tris– HCl, pH 7.5, 5 mM CaCl2, 1 μM ZnCl2 and 2.5% Triton X-100) for 30 min at room temperature, and then incubated overnight at 37°C under shaking in the same buffer without TritonX-100. The gels were stained with a solution containing 0.1% Coomassie Brilliant Blue R-250. Formation of clear zone against the blue background on the polyacrylamide gels indicated the gelatinolytic activity, which was quantified by densitometric analysis using the NIH Image Program.

MMP-9 gene promoter analysis RAW264.7 cells were transfected with MMP-9 gene promoterluciferase reporter constructs, pGBcolSS3 (+1 to − 1174 bp) and pGBcol ΔNFATc#2 (+1 to − 446 bp relative to ATG start codon) using LipofectAmine-Plus transfection reagent (Invitrogen, Carlsbad, CA). Prior to transfection, the cells were cultured for 24 h in a six well plate at a density of 3 × 105 cells per well in α-MEM with 10% fetal bovine serum at 37°C. Fifteen microgram of Qiagen column (Qiagen, Chatsworth, CA) purified mouse MMP-9 gene promoter-luciferase reporter constructs and empty vector were co-transfected into RAW264.7 cells with 5 μg of pRSV β-galactosidase plasmid to normalize transfection efficiency [19]. The cells were cultured in the presence or absence of RANKL (100 ng/ml) for 48 h. The cell monolayer was washed twice with phosphate-buffered saline and incubated at room temperature for 15 min with 0.3 ml cell lysis reagent. The monolayer was scraped and spun briefly in a microfuge to pellet the debris. A 20 μl aliquot of each sample was mixed with 100 μl of the luciferase assay reagent. The light emission was measured for 10 s of integrated time using Sirius Luminometer following the manufacturer's instructions (Promega, Madison, WI, USA).

Quantitative real-time RT-PCR MMP-9 mRNA expression levels were determined by real-time reverse transcription-PCR as described previously [22]. Briefly, total RNA was isolated from RAW264.7 cells using RNAzol reagent (Invitrogen Inc., Carlsbad, CA) according to the manufacturer's protocol. Reverse transcription reaction was performed using poly-dT primer and Moloney murine leukemia virus reverse transcriptase (Applied Biosystem) in 25 μl reaction volume containing total RNA (5 μg), 1 x PCR buffer and 2 mM MgCl2, at 42°C for 15 min followed by 95°C for 5 min. The

quantitative real-time PCR was performed using IQTM SYBR Green Supermix in an iCycler (iCycler iQ Single–color Real Time PCR detection system; Bio-Rad, Hercules, CA). The primer sequences used to amplify glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA were 5′ CCTACCCCCAATGTATCCGTTGTG-3′ (sense) and 5′-GGAGGAATGGGAGTTGCTGTTGAA-3′ (anti-sense) and primers for mouse MMP-9 mRNA were 5′GTTTTTGATGCTATTGCTGAGATCCA-3′ (sense) and 5′-CCCACATTTGACGTCCAGAGAAGAA-3′ (anti-sense). Thermal cycling parameters were 94°C for 3 min, followed by 40 cycles of amplifications at 94°C for 30 s, 66°C for 1 min, 72°C for 1 min, and 72°C for 5 min as the final elongation step. Relative levels of MMP-9 mRNA expression were normalized in all the samples with respect to the levels of GAPDH amplification.

Western blot analysis RAW264.7 macrophage cells or mouse bone-marrow-derived mononuclear cells were seeded in 10-cm plates at a density of 106 cells in 10 ml of α-MEM medium containing 10% fetal calf serum and cultured for 24 h in the presence or absence of RANKL (100 ng /ml). The cells were lysed in a buffer containing 20 mM Tris–HCl at pH 7.4, 1% Triton X-100, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 150 mM NaCl, 0.1 mM Na3VO4 and 1× protease inhibitor cocktail (Sigma, St. Louis). The protein content of the samples was measured using the BCA protein assay reagent (Pierce). Protein (20 μg) samples were then subjected to SDSPAGE using 12% Tris–HCl gels and blot transferred on to a nitrocellulose membrane. Blocking was performed with 5% BSA in TBS-T (50 mM Tris, pH 7.2, 150 mM NaCl; 0.1% Tween 20). The membrane was incubated with a primary antibody against rabbit anti-mouse MMP-9 at 1:1000 dilution (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The blots were incubated for 1 h with horseradish peroxidase conjugated goat anti-rabbit antibody diluted 1:5000 in TBS-T buffer, and developed using ECL system (Amersham Biosciences).

siRNA interference RAW264.7 cells were seeded (5 × 105 cells/well) in 6-well plates and supplemented with α-MEM containing 10% fetal calf serum. A day after seeding, cells were transfected with double stranded NFATc1 siRNA (10 μM) containing 21 nucleotides (Santa Cruz Biotechnology, CA). The cells were stimulated with or without RANKL (100 ng/ml) for 48-h period. Suppression of NFATc1 expression by siRNA was confirmed by Western blot analysis of total cell lysates using anti-NFATc1 antibody (Santa Cruz biotechnology Inc., CA). Similarly, RAW264.7 cells stimulated with RANKL were transfected with siRNA against TRAF2 and TRAF6 and suppression of protein expression was confirmed by immunoblot analysis of total cell lysates using antibodies specific to TRAF2 and TRAF6. Total RNA isolated from the transfected cells was subjected to real-time PCR analysis for MMP-9 mRNA expression as described above.

Oligodeoxynucleotide pull-down assay We have previously reported the mouse MMP-9 gene 5′flanking nucleotide sequence [19]. Oligonucleotides corre-

E XP E RI ME N TA L CE LL RE S EA RCH 3 13 ( 20 0 7 ) 1 6 8 –17 8

171

sponding to the NFATc1 site (underlined) from MMP-9 gene promoter sequence (−1123 bp to −1152 bp relative to start codon) were synthesized as follows: sense 5′-biotin-TAAGAGAAGCTTGGGAGAACACCCAGCTCT-3′ and anti-sense 5′AGAGCTGGGTGTTCTCCCAAGCTTCTCTTA-3′. Doublestranded probes were made by annealing a 50 μM mixture of complementary oligonucleotides in TNE (10 mM Tris–HCl, 50 mM NaCl and 1 mM EDTA) buffer, heating to 95°C for 5 min, and then slowly cooling to room temperature. Nuclear extracts were prepared from mouse bone-marrow-derived mononuclear cells stimulated with RANKL (100 ng/ml) for 24 h. For pull-down assays, 30 μg of nuclear extracts was incubated in a 25 μl reaction mixture consisting of 10 μM probe and 1× binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris–HCl/pH 7.5, and 0.05 mg/ ml polydI–dC). After incubation for 2 h at room temperature, the reaction volume was increased to 0.5 ml with modified binding buffer, which does not contain poly(dI–dC). The complexes were captured by incubation with Dyna Beads M-280 streptavidin (Invitrogen) for 1 h at 4°C and washed three times with binding buffer. Proteins eluted were analyzed by immunoblotting with anti-NFATc1 antibody as described above.

Statistical analysis Results are presented as mean ± SD for three independent experiments and were compared by Student's t-test. Results were considered significantly different for p < 0.05.

Results RANKL induce MMP-9 expression in RAW264.7 cells MMP-9 is highly expressed in osteoclast lineage cells [2]. We have recently reported the capacity of RANKL stimulation and cytokine inhibition of osteoclast differentiation of RAW264.7 macrophage cells in vitro [23]. We therefore, examined the regulation of MMP-9 gene expression in RANKL stimulated RAW264.7 cells. As shown in Fig. 1A, gelatin zymogram analysis of conditioned media collected from RANKL (0– 100 ng/ml) stimulated RAW264.7 cells demonstrated a dosedependent increase (6.3-fold) in the levels of MMP-9 activity in a 24-h period. We further examined the time-dependent increase in the levels of MMP-9 activity upon RANKL stimulation of these cells. As shown in Fig. 1B, conditioned media obtained from RAW264.7 cells stimulated with RANKL (100 ng/ ml) for 0- to 48-h period demonstrated a significant increase (5-fold) in the levels of MMP-9 activity. Furthermore, real-time PCR analysis of total RNA isolated from these cells indicated that RANKL stimulation results in a significant increase (17fold) in the levels of MMP-9 mRNA expression in a time (0– 48 h)-dependent manner (Fig. 1C). These results suggest that RANKL signaling stimulates MMP-9 expression in preosteoclast cells.

RANKL regulates MMP-9 expression through TRAF6 signaling RANKL interaction with RANK receptor expressed on osteoclast precursors is critical for osteoclastogenesis. However,

Fig. 1 – RANKL induces MMP-9 expression in RAW264.7 cells. (A) RAW cells were cultured in serum free α-MEM and stimulated with RANKL (0–100 ng/ml) for 24 h and (B) different treatment period (0–48 h) as indicated. The conditioned media were collected from the cell cultures and MMP-9 activity was analyzed by gelatin zymography as described in Materials and methods. (C) Quantitative real-time RT-PCR analysis of MMP-9 mRNA expression in RAW264.7 cells. Reverse transcription was performed on total RNA isolated from RAW264.7 cells stimulated with RANKL (100 ng/ml) for the indicated period (0–48 h). Relative levels of MMP-9 mRNA expression were normalized with respect to the levels of GAPDH amplification. The data are shown as mean ± SD for three independent experiments (*p < 0.05).

RANK is known to interact with a number of tumor necrosis factor receptor-associated factor (TRAF) signaling adaptor proteins, including TRAF2 and TRAF6. Therefore, to determine the participation of TRAFs in RANKL signaling to induce MMP9 expression, we performed siRNA interference to suppress TRAF2 and TRAF6 expression during osteoclast differentiation. RAW264.7 cells were transfected with siRNA for TRAF2 and TRAF6 and stimulated with and without RANKL (100 ng/ ml) for 24 h. siRNA suppression of TRAF2 and TRAF6 protein expression was confirmed by immunoblot analysis (data not shown). Gelatin zymogram analysis of conditioned obtained from these cells demonstrated a significant increase (4.2-fold) in the levels of MMP-9 activity in RANKL stimulated RAW264.7

172

E XP ER I ME NT A L C EL L RE S EA R CH 3 13 ( 20 0 7 ) 1 6 8 –17 8

cells. Interestingly, MMP-9 activity was significantly decreased (2.8-fold) in RANKL stimulated RAW264.7 cells transfected with TRAF6 siRNA, however, no change in MMP-9 activity levels were detected in cells transfected with TRAF2 siRNA (Fig. 2A). In addition, total RNA isolated from these cells was subjected to real-time PCR analysis for MMP-9 mRNA expression. As shown in Fig. 2B, MMP-9 mRNA levels were significantly decreased (3.0-fold) in RANKL stimulated RAW264.7 cells transfected with TRAF6 siRNA, however, no change in MMP-9 mRNA expression levels was detected in cells transfected with TRAF2 siRNA compared to untransfected RANKL stimulated cells. These results indicate that RANKL-RANK interaction signals through TRAF6 to modulate MMP-9 expression in preosteoclast cells.

ERK1/2 and p38 MAPKs differentially regulates MMP-9 expression Recent studies have demonstrated that MMP-9 expression can be regulated by ERK1/2 and p38 MAPK signaling [24]. Since

RANKL signaling involves both ERK and MAPK, we examined whether RANKL induced MMP-9 expression was mediated through ERK1/2 and p38 MAPK-dependent signaling pathway. RAW264.7 cells were treated with ERK activation inhibitor PD98059 (2.5 mM) and p38 MAPK inhibitor SB203580 (35 nM) prior to 30 min of RANKL stimulation for 24 h. Gelatin zymogram analysis of conditioned media obtained from these cells demonstrated a significant decrease (3.0-fold) in MMP-9 activity in RANKL stimulated RAW264.7 cells treated with ERK inhibitor (Fig. 3A). In addition, total RNA isolated from these cells was subjected to real-time PCR analysis of MMP-9 mRNA expression. RANKL stimulated MMP-9 mRNA expression was significantly decreased (3.2-fold) in cells treated with ERK inhibitor. In contrast, MMP-9 activity and mRNA expression levels were increased (1.8-fold) in cells treated with p38 MAPK inhibitor alone (Figs. 3A and B). These results suggest a potential role for ERK signaling to enhance MMP-9 expression; however, p38 MAPK differentially regulates RANKL induced MMP-9 expression in preosteoclast cells.

Fig. 2 – RANKL signals through TRAF6 to enhance MMP-9 expression during osteoclast differentiation. RAW264.7 cells were transfected with TRAF2 and TRAF6 siRNA by Lipofectamine method. After 24-h incubation, the cells were stimulated with or without RANKL (100 ng/ml) for a further 24 h. (A) The conditioned media were collected and MMP-9 activity was analyzed by gelatin zymography. The relative band intensity of MMP-9 activity was quantified by densitometry as described in Materials and methods. (B) Total RNA isolated from these cells was subjected to real-time PCR analysis of MMP-9 mRNA expression as described in Materials and methods. The relative levels of MMP-9 mRNA expression were normalized with respect to the levels of GAPDH amplification. The data are shown as mean ± SD for three independent experiments (*p < 0.05).

E XP E RI ME N TA L CE LL RE S EA RCH 3 13 ( 20 0 7 ) 1 6 8 –17 8

RANKL stimulates MMP-9 gene promoter activity We have previously cloned the mouse MMP-9 gene promoter region and demonstrated promoter activity [19]. Recent evidence indicates that NFATc1/NFAT2 is a critical transcription factor involved in RANKL stimulated osteoclast differentiation [17]. Interestingly, nucleotide sequence analysis identified the presence of NFATc1 element at − 1123 to −1153 bp region relative to ATG start codon. To further define a functional role for NFAT element present in the MMP-9 gene promoter region, we developed plasmid constructs pGBcolSS3 (+1 to −1174 bp) and pGBcol ΔNFATc#2 (+1 to − 446 bp) with and without the NFAT element respectively driving luciferase reporter gene expression. To confirm the capacity of RANKL to stimulate MMP-9 gene promoter activity, MMP-9 gene promoter-luciferase reporter constructs, pGBcolSS3 and pGBcol ΔNFATc#2 were transiently transfected into RAW264.7 cells using the LipofectAMINE method and the cells cultured in the presence and absence of RANKL (100 ng/ml) for 48 h. As shown in Fig. 4 total cell lysates obtained from the RANKL stimulated RAW264.7 cells transfected with pGBcolSS3 (+1 to

173

−1174) showed 20-fold increase in luciferase activity compared with untreated cells. However, RANKL stimulation did not affect luciferase activity in cells that were transfected with the pGBcol ΔNFATc1#2 (+1 to −446 bp) plasmid and the empty vector. Transfection efficiency was normalized by co-expression of pRSV β-galactosidase plasmid and measuring the βgalactosidase activity in these cells. Taken together, these results suggest that MMP-9 gene promoter, −446 to − 1174 bp region containing NFATc1 transcription factor binding motif play a critical role in RANKL stimulation of MMP-9 gene expression.

Functional role for NFATc1 in RANKL regulation of MMP-9 gene expression To further confirm the participation of NFATc1 in RANKL induction of MMP-9 gene expression, we performed siRNA interference to suppress NFATc1 expression during osteoclast differentiation. Mouse bone marrow non-adherent cells were transfected with NFATc1 siRNA (Santa Cruz, USA) and stimulated with RANKL (100 ng/ml) for 48 h as described in

Fig. 3 – ERK and p38 signaling differentially regulate MMP-9 expression in RAW264.7 cells. Cells were pretreated with p38 MAPK inhibitor, SB203580 (35 nM) and ERK activation inhibitor PD98059 (2.5 mM) for 30 min prior to stimulation with RANKL (100 ng/ml) for 24 h. (A) The conditioned media were collected and MMP-9 activity was analyzed by gelatin zymography. The relative band intensity of MMP-9 activity was quantified by densitometry. (B) Total RNA was isolated and the MMP-9 mRNA expression was quantified by real-time PCR. The relative levels of MMP-9 mRNA expression were normalized with respect to the levels of GAPDH amplification. The data are shown as mean ± SD for three independent experiments (*p < 0.05).

174

E XP ER I ME NT A L C EL L RE S EA R CH 3 13 ( 20 0 7 ) 1 6 8 –17 8

Fig. 4 – RANKL enhances the MMP-9 gene promoter activity. RAW264.7 cells were transfected with MMP-9 gene promoter-luciferase reporter constructs, pGBcolSS3 (+1 to 1174 bp), pGBcol ΔNFATc1#2 (+1 to −446 bp) and empty vector (EV). The cells were cultured with and without 100 ng/ml of RANKL for 48 h. Total cell lysates prepared were assayed for Luciferase activity. Transfection efficiency was normalized by measuring β-galactosidase activity co-expressed in these cells. The data are shown as mean ± SD for three independent experiments (*p < 0.05).

Materials and methods. Serum-free conditioned media and total cell lysates obtained from these cultures were subjected to gelatin zymography and Western blot analysis respectively. As shown in Fig. 5A, zymogram analysis indicated that RANKL stimulation resulted in a 6-fold increase in MMP-9 activity in the conditioned media obtained from these cells compared to unstimulated control cells. Interestingly, MMP-9 activity was significantly decreased (3.8-fold) in NFATc1 siRNA transfected cells. Western blot analysis of total cell lysates demonstrated a significant decrease (4.5-fold) in the levels of MMP-9 protein

Fig. 6 – RANKL stimulation induces NFATc1 binding to MMP-9 gene promoter sequence. Nuclear extract (NE) was prepared from mouse bone marrow mononuclear cells stimulated with and without RANKL (100 ng/ml) for 24 h. The NE (30 μg protein) was incubated for 2 h with biotinylated oligonucleotides containing the NFATc binding sequence present in the MMP-9 gene promoter. The resulting DNA–protein complexes were isolated by streptavidin conjugated magnetic beads and analyzed by immunoblotting with anti-NFATc1 antibody as described in Materials and methods.

expression in these cells (Fig. 5B). In contrast, a control nonspecific siRNA did not affect MMP-9 and NFAT expression levels in these cells (data not shown). We have further confirmed RANKL enhanced NFATc1 binding to MMP-9 gene promoter sequence by oligonucleotide pull-down assay as described in Materials and methods. As shown in Fig. 6 nuclear extracts (NE) were prepared from the mouse bonemarrow-derived preosteoclast cells stimulated with RANKL for 24 h demonstrated high affinity binding of NFATc1 to MMP-

Fig. 5 – RANKL enhances MMP-9 expression through activation of NFATc1. Non-adherent mouse bone marrow cells stimulated with M-CSF (10 ng/ml) for 48 h. The resultant preosteoclast cells were transfected with NFATc1 siRNA. After 24 h, the cells were incubated with or without RANKL (100 ng/ml) for 24 h. (A) The conditioned media were collected and analyzed for MMP-9 activity by gelatin zymogram and (B) total cell lysates (20 μg of protein) obtained were subjected to Western blot analysis for NFATc1 and MMP-9 expression as described in Materials and methods.

E XP E RI ME N TA L CE LL RE S EA RCH 3 13 ( 20 0 7 ) 1 6 8 –17 8

9 promoter element compared to NE prepared from unstimulated cells. These data confirm the specificity of NFATc1 binding to NFAT recognition site present in the mouse MMP-9 gene promoter sequence. RANKL signaling activates calcineurin, which plays an essential role in dephosphorylation and nuclear translocation of NFATc1. Therefore, we further examined if calcineurin is involved in RANKL activation of NFATc1 to induce MMP-9 gene expression. Using a 16-amino-acid high affinity calcineurin binding peptide (VIVIT), we selectively inhibited the ability of calcineurin to activate NFAT proteins. RAW264.7 cells were stimulated with RANKL in the presence or absence of VIVIT peptide (1 ng/ml) for 24 h. As shown in Fig. 7, Western blot analysis of total cell lysates obtained from these cultures demonstrated that VIVIT peptide treatment resulted in a 4-fold and 3.7-fold decrease in the levels of RANKL induced MMP-9 and NFAT expression respectively compared to cells stimulated with RANKL alone. Gelatin zymography of serum-free conditioned media collected from these cultures further confirms that VIVIT peptide treatment significantly inhibited (3.8-fold) RANKL induced MMP-9 activity in RAW264.7 cells. VIVIT peptide treatment alone had no significant effect on MMP-9 expression (data not shown). These results suggest that calcineurin activated NFATc1 play an essential role in RANKL induced MMP-9 expression in preosteoclast cells.

Constitutively activated NFAT (caNFAT) modulate the MMP-9 gene promoter activity To further assess the potential of NFATc1 to modulate MMP-9 gene expression directly, we have co-expressed the caNFAT, which mimics NFATc1 [25], with MMP-9 gene promoterluciferase reporter constructs as described above. MMP-9 gene

175

Fig. 8 – Constitutive active NFAT (caNFAT) enhances the MMP-9 gene promoter activity. RAW264.7 cells were co-transfected with MMP-9 gene promoter-luciferase reporter constructs, pGBcolSS3 (+ 1 to −1174 bp), pGBcol ΔNFATc1#2 (+1 to − 446 bp) and empty vector (EV) with and without caNFAT. After, 48 h, total cell lysates obtained were assayed for luciferase activity and normalized for transfection efficiency by measuring β-galactosidase activity co-expressed in these cells. The data are shown as mean ± SD for three independent experiments (*p < 0.05).

promoter-luciferase reporter constructs, pGBcolSS3 (+ 1 to −1174 bp) and pGBcol ΔNFATc1#2 (+1 to −446 bp) were cotransfected with and without caNFAT into RAW264.7 cells using the LipofectAMINE method and the cells cultured for 48 h without RANKL stimulation. As shown in Fig. 8 total cell lysates obtained from the RAW264.7 cells co-transfected with caNFAT and pGBcolSS3 (+1 to −1174 bp) showed significant increase (5-fold) in luciferase activity compared to pGBcolSS3 MMP-9 promoterluciferase reporter construct alone. However, caNFAT co-expression did not affect luciferase activity in cells that were transfected with the pGBcol ΔNFATc1#2 (+1 to −446 bp) plasmid and the empty vector which lacks NFAT recognition site. Transfection efficiency was normalized by co-expression of pRSV β-galactosidase plasmid and measuring the β-galactosidase activity in these cells. These data further confirm that NFATc1 modulates MMP-9 gene expression in preosteoclast cells.

Discussion

Fig. 7 – Effect of calcineurin peptide inhibitor, VIVIT peptide on RANKL stimulated MMP-9 activity in RAW264.7 cells. RAW264.7 cells were stimulated with or without RANKL (100 ng/ml) and VIVIT (1 ng/ml) for 24 h. (A) Total cell lysates were subjected to Western blot analysis for MMP-9 and NFATc1 expression and (B) the conditioned media were analyzed for MMP-9 activity by gelatin zymogram as described in Materials and methods.

Osteoclast formation and bone resorption activity are regulated by the local factors produced in the bone marrow microenvironment. Tumor necrosis factor (TNF) gene family member, receptor activator of NF-κB (RANK) expressed on OCL precursors and RANK ligand (RANKL) expressed on osteoblast/stromal cells interaction is critical for OCL differentiation and bone resorption [26]. The RANKL-RANK signaling promotes the binding of TNF receptor associated factor (TRAF) family proteins such as TRAF6 to RANK receptor resulting in activation of NF-κB and Jun N-terminal kinase (JNK) pathways [27]. Although TRAF2 is essential for TNF-α induced osteoclastogenesis and TRAF5 functions in both RANKL and TNF-α induced osteoclastogenesis [15,28], our results with siRNA interference

176

E XP ER I ME NT A L C EL L RE S EA R CH 3 13 ( 20 0 7 ) 1 6 8 –17 8

show that RANKL signals through TRAF6, but not TRAF2, to enhance MMP-9 gene expression. Low level expression of NFATc1 in bone-marrow-derived osteoclast progenitors/ macrophages indicates the basal level expression of MMP-9 activity in these cultures. Previously, the MMP-9 promoter has been shown to contain nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) binding sites indicating that MMP-9 gene expression can be upregulated by stimuli such as IL-1 and TNF-α [29]. Therefore, it is possible that these cytokines synergize with RANKL to further enhance MMP-9 mRNA expression and increase the bone resorption capacity of the osteoclasts in the bone microenvironment and in pathologic conditions. In this study, we identified the functional NFATc1 recognition site and specific binding to the MMP-9 gene promoter sequence in response to RANKL stimuli. Since calcium differentially regulates p38 MAPK and ERK1/2 and induces MMP-9 gene expression [24], it is possible that calcium may modulate the constitutive levels of MMP-9 gene expression in the preosteoclasts. However, RANKL signaling induces the elevation of cytosolic Ca2+, which results in activation of calcineurin. Activated calcineurin dephosphorylates cytosolic NFAT followed by nuclear translocation and interaction with AP-1 transcription factor complex to modulate target gene expression in osteoclasts [30]. Recently, it has been reported that RANKL induced cathepsin K gene expression, during osteoclastogenesis, is cooperatively regulated by the complex of PU1 and NFATc1 transcription factors and p38 MAP kinase [31]. Additionally, phosphorylation of NFATc by glycogen synthase kinase-3 inhibits the ability of NFATc to bind DNA [32]. RANKL stimulates p38 activity and phosphorylation of Akt, a downstream target of PI3K and that of ERK suggests that these molecules may play important roles in osteoclast differentiation [33]. It has also been shown that TNF induces osteoclast differentiation and that p38 MAP kinase-deficient mice bone marrow cultures form reduced numbers of osteoclasts compared to control mice [34]. RANKL and TNFα induce phosphorylation of p38 MAPK in osteoclast precursors but not in mature osteoclasts [35]. It has also been demonstrated that differentiation of mouse bone marrow macrophages into osteoclasts in response to RANKL or TNF-α was strongly inhibited by a p38 MAPK inhibitor [36]. These data suggest that p38 MAP kinase pathway plays an important role in RANKL signaling of osteoclast differentiation but not for osteoclast function [37]. Recent evidence also indicates that decoy receptor 3 (DcR3) of the TNF receptor superfamily via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-α synthesis induces osteoclast formation from monocyte/macrophage lineage precursor cells [38]. In this study, we show that RANKL stimulation differentially regulates MMP-9 expression through p38 and ERK signaling pathways in RAW264.7 cells. Based on these results, it can be hypothesized that RANKL activation of p38 and ERK signaling pathways differentially regulate NFAT activation and its ability to interact with AP-1 transcription factor complex in modulating MMP-9 gene expression. In support of this, recent evidence suggests that ascochlorin, an anti-tumor compound, specifically inhibits MMP-9 activity through suppression of AP-1 transcription factor complex via the ERK signaling pathway [39]. Similarly, it has also been

shown that p38 kinase negatively regulates NFAT activation and nuclear translocation [40,41]. These studies further support our results indicating that inhibition of p38 signaling enhanced MMP-9 activity. It has been previously reported that RANKL enhances MMP9 expression in purified rabbit osteoclasts [42]. However, the present study provides molecular insights into the regulatory mechanisms operative in RANKL stimulated preosteoclast cells. Recently, it has been shown that NFATc1 regulates the calcitonin receptor, mouse tartrate-resistant acid phosphatase (TRAP) and human beta-3 integrin gene promoters during osteoclast differentiation [25,43,44]. Furthermore, NFATc1 expression is auto-regulated during osteoclastogenesis [45]. Therefore, in the present study, identification of NFATc1 transcription factor as an effecter of RANKL signaling to enhance MMP-9 expression provides a novel therapeutic target to control excess bone resorption activity and extracellular matrix degradation associated with pathologic conditions such as rheumatoid arthritis and Paget's disease of bone.

Acknowledgments We thank Rebecca Truesdell for her assistance with the preparation of manuscript. This work was supported by the National Institute of Health grants DE 12603 and C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources.

REFERENCES

[1] P. Reponen, C. Sahlberg, C. Munaut, I. Thesleff, K. Tryggvason, High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development, J. Cell Biol. 124 (1994) 1091–1102. [2] A.L. Wucherpfennig, Y.P. Li, W.G. Stetler-Stevenson, A.E. Rosenberg, P. Stashenko, Expression of 92 kD type IV collagenase/gelatinase B in human osteoclasts, J. Bone Miner. Res. 9 (1994) 549–556. [3] C.H. Tsai, Y.J. Chen, F.M. Huang, Y.F. Su, Y.C. Chang, The upregulation of matrix metalloproteinase-9 in inflamed human dental pulps, J. Endod. 31 (2005) 860–862. [4] K. Kusano, C. Miyaura, M. Inada, T. Tamura, A. Ito, H. Nagase, K. Kamoi, T. Suda, Regulation of matrix metalloproteinases (MMP-2, - 3, - 9, and - 13) by interleukin-1 and interleukin-6 in mouse calvaria: association of MMP induction with bone resorption, Endocrinology 139 (1998) 1338–1345. [5] M. Zhou, Y. Zhang, J.A. Ardans, L.M. Wahl, Interferon-gamma differentially regulates monocyte matrix metalloproteinase-1 and -9 through tumor necrosis factor-alpha and caspase 8, J. Biol. Chem. 278 (2003) 45406–45413. [6] X. Yu, Y. Huang, P. Collin-Osdoby, P. Osdoby, Stromal cell-derived factor-1 (SDF-1) recruits osteoclast precursors by inducing chemotaxis, matrix metalloproteinase-9 (MMP-9) activity, and collagen transmigration, J. Bone Miner. Res. 18 (2003) 1404–1418. [7] J.M. Delaisse, T.L. Andersen, M.T. Engsig, K. Henriksen, T. Troen, L. Blavier, Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities, Microsc. Res. Tech. 61 (2003) 504–513. [8] T.H. Vu, J.M. Shipley, G. Bergers, J.E. Berger, J.A. Helms, D. Hanahan, S.D. Shapiro, R.M. Senior, Z. Werb,

E XP E RI ME N TA L CE LL RE S EA RCH 3 13 ( 20 0 7 ) 1 6 8 –17 8

[9]

[10]

[11] [12]

[13]

[14]

[15]

[16] [17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes, Cell 93 (1998) 411–422. C. Colnot, Z. Thompson, T. Miclau, Z. Werb, J.A. Helms, Altered fracture repair in the absence of MMP9, Development 130 (2003) 4123–4133. N. Ortega, D.J. Behonick, C. Colnot, D.N. Cooper, Z. Werb, Galectin-3 is a downstream regulator of matrix metalloproteinase-9 function during endochondral bone formation, Mol. Biol. Cell 16 (2005) 3028–3039. W.J. Boyle, W.S. Simonet, D.L. Lacey, Osteoclast differentiation and activation, Nature 423 (2003) 337–342. A. Vignery, Osteoclasts and giant cells: macrophage-macrophage fusion mechanism, Int. J. Exp. Pathol. 81 (2000) 291–304. P. Spessotto, F.M. Rossi, M. Degan, R. Di Francia, R. Perris, A. Colombatti, V. Gattei, Hyaluronan-CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9, J. Cell Biol. 158 (2002) 1133–1144. S. Theoleyre, Y. Wittrant, S. Couillaud, P. Vusio, M. Berreur, C. Dunstan, F. Blanchard, F. Redini, D. Heymann, Cellular activity and signaling induced by osteoprotegerin in osteoclasts: involvement of receptor activator of nuclear factor kappaB ligand and MAPK, Biochim. Biophys. Acta 1644 (2004) 1–7. K. Kanazawa, Y. Azuma, H. Nakano, A. Kudo, TRAF5 functions in both RANKL- and TNFalpha-induced osteoclastogenesis, J. Bone Miner. Res. 18 (2003) 443–450. S.L. Teitelbaum, F.P. Ross, Genetic regulation of osteoclast development and function, Nat. Rev., Genet. 4 (2003) 638–649. H. Takayanagi, S. Kim, T. Koga, H. Nishina, M. Isshiki, H. Yoshida, A. Saiura, M. Isobe, T. Yokochi, J. Inoue, E.F. Wagner, T.W. Mak, T. Kodama, T. Taniguchi, Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts, Dev. Cell 3 (2002) 889–901. H. Hirotani, N.A. Tuohy, J.T. Woo, P.H. Stern, N.A. Clipstone, The calcineurin/nuclear factor of activated T cells signaling pathway regulates osteoclastogenesis in RAW264.7 cells, J. Biol. Chem. 279 (2004) 13984–13992. J. Roach, S.J. Choi, R.L. Schaub, R.J. Leach, G.D. Roodman, S.V. Reddy, Further characterization of the murine collagenase (type IVB) gene promoter and analysis of mRNA expression in murine tissues, Gene 208 (1998) 117–122. C. Munaut, T. Salonurmi, S. Kontusaari, P. Reponen, T. Morita, J.M. Foidart, K. Tryggvason, Murine matrix metalloproteinase 9 gene. 5′-upstream region contains cis-acting elements for expression in osteoclasts and migrating keratinocytes in transgenic mice, J. Biol. Chem. 274 (1999) 5588–5596. A.R. Quesada, M.M. Barbacid, E. Mira, P. Fernandez-Resa, G. Marquez, M. Aracil, Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases, Clin. Exp. Metastasis 15 (1997) 26–32. S. Srinivasan, M. Ito, H. Kajiya, L.L. Key Jr., T.L. Johnson-Pais, S.V. Reddy, Functional characterization of human osteoclast inhibitory peptide-1 (OIP-1/hSca) gene promoter, Gene 371 (2006) 16–24. M. Koide, H. Maeda, J.L. Roccisana, N. Kawanabe, S.V. Reddy, Cytokine regulation and the signaling mechanism of osteoclast inhibitory peptide-1 (OIP-1/hSca) to inhibit osteoclast formation, J. Bone Miner. Res. 18 (2003) 458–465. S. Mukhopadhyay, H.G. Munshi, S. Kambhampati, A. Sassano, L.C. Platanias, M.S. Stack, Calcium-induced matrix metalloproteinase 9 gene expression is differentially regulated by ERK1/2 and p38 MAPK in oral keratinocytes and oral squamous cell carcinoma, J. Biol. Chem. 279 (2004) 33139–43316. F. Ikeda, R. Nishimura, T. Matsubara, S. Tanaka, J. Inoue, S.V. Reddy, K. Hata, K. Yamashita, T. Hiraga, T. Watanabe,

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

177

T. Kukita, K. Yoshioka, A. Rao, T. Yoneda, Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation, J. Clin. Invest. 114 (2004) 475–484. T. Suda, N. Takahashi, N. Udagawa, E. Jimi, M.T. Gillespie, T.J. Martin, Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families, Endocr. Rev. 20 (1999) 345–357. H. Hsu, D.L. Lacey, C.R. Dunstan, I. Solovyev, A. Colombero, E. Timms, H.L. Tan, G. Elliott, M.J. Kelley, I. Sarosi, L. Wang, X.Z. Xia, R. Elliott, L. Chiu, T. Black, S. Scully, C. Capparelli, S. Morony, G. Shimamoto, M.B. Bass, W.J. Boyle, Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 3540–3545. K. Kanazawa, A. Kudo, TRAF2 is essential for TNF-alpha-induced osteoclastogenesis, J. Bone Miner. Res. 20 (2005) 840–847. Y. Kato, C.A. Lambert, A.C. Colige, P. Mineur, A. Noel, F. Frankenne, J.M. Foidart, M. Baba, R. Hata, K. Miyazaki, M. Tsukuda, Acidic extracellular pH induces matrix metalloproteinase-9 expression in mouse metastatic melanoma cells through the phospholipase D-mitogen-activated protein kinase signaling, J. Biol. Chem. 280 (2005) 10938–10944. A. Rao, C. Luo, P.G. Hogan, Transcription factors of the NFAT family: regulation and function, Annu. Rev. Immunol. 15 (1997) 707–747. M. Matsumoto, M. Kogawa, S. Wada, H. Takayanagi, M. Tsujimoto, S. Katayama, K. Hisatake, Y. Nogi, Essential role of p38 mitogen-activated protein kinase in cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1, J. Biol. Chem. 279 (2004) 45969–45979. P.G. Hogan, L. Chen, J. Nardone, A. Rao, Transcriptional regulation by calcium, calcineurin, and NFAT, Genes Dev. 17 (2003) 2205–2232. S.E. Lee, K.M. Woo, S.Y. Kim, H.M. Kim, K. Kwack, Z.H. Lee, H.H. Kim, The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation, Bone 30 (2002) 71–77. M. Matsumoto, T. Sudo, T. Saito, H. Osada, M. Tsujimoto, Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL), J. Biol. Chem. 275 (2000) 31155–31161. X. Li, N. Udagawa, K. Itoh, K. Suda, Y. Murase, T. Nishihara, T. Suda, N. Takahashi, p38 MAPK-mediated signals are required for inducing osteoclast differentiation but not for osteoclast function, Endocrinology 143 (2002) 3105–3113. X. Li, N. Udagawa, M. Takami, N. Sato, Y. Kobayashi, N. Takahashi, p38 Mitogen-activated protein kinase is crucially involved in osteoclast differentiation but not in cytokine production, phagocytosis, or dendritic cell differentiation of bone marrow macrophages, Endocrinology 144 (2003) 4999–5005. Z.H. Lee, H.H. Kim, Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts, Biochem. Biophys. Res. Commun. 305 (2003) 211–214. C.R. Yang, J.H. Wang, S.L. Hsieh, S.M. Wang, T.L. Hsu, W.W. Lin, Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells, Cell Death Differ. 11 (Suppl 1) (2004) S97–S107. S. Hong, K.K. Park, J. Magae, K. Ando, T.S. Lee, T.K. Kwon, J.Y. Kwak, C.H. Kim, Y.C. Chang, Ascochlorin inhibits matrix

178

E XP ER I ME NT A L C EL L RE S EA R CH 3 13 ( 20 0 7 ) 1 6 8 –17 8

metalloproteinase-9 expression by suppressing activator protein-1-mediated gene expression through the ERK1/2 signaling pathway: inhibitory effects of ascochlorin on the invasion of renal carcinoma cells, J. Biol. Chem. 280 (2005) 25202–25209. [40] C.M. Porter, M.A. Havens, N.A. Clipstone, Identification of amino acid residues and protein kinases involved in the regulation of NFATc subcellular localization, J. Biol. Chem. 275 (2000) 3543–3551. [41] S.V. Komarova, A. Pereverzev, J.W. Shum, S.M. Sims, S.J. Dixon, Convergent signaling by acidosis and receptor activator of NF-kappaB ligand (RANKL) on the calcium/calcineurin/NFAT pathway in osteoclasts, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 2643–2648. [42] Y. Wittrant, S. Theoleyre, S. Couillaud, C. Dunstan, D. Heymann, F. Redini, Regulation of osteoclast protease

expression by RANKL, Biochem. Biophys. Res. Commun. 310 (2003) 774–778. [43] K. Matsuo, D.L. Galson, C. Zhao, L. Peng, C. Laplace, K.Z. Wang, M.A. Bachler, H. Amano, H. Aburatani, H. Ishikawa, E.F. Wagner, Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos, J. Biol. Chem. 279 (2004) 26475–26480. [44] T.N. Crotti, M. Flannery, N.C. Walsh, J.D. Fleming, S.R. Goldring, K.P. McHugh, NFATc1 regulation of the human beta3 integrin promoter in osteoclast differentiation, Gene 372 (2006) 92–102. [45] M. Asagiri, K. Sato, T. Usami, S. Ochi, H. Nishina, H. Yoshida, I. Morita, E.F. Wagner, T.W. Mak, E. Serfling, H. Takayanagi, Autoamplification of NFATc1 expression determines its essential role in bone homeostasis, J. Exp. Med. 202 (2005) 1261–1269.