Bone 73 (2015) 242–248

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Original Full Length Article

CCN2 enhances RANKL-induced osteoclast differentiation via direct binding to RANK and OPG Eriko Aoyama a, Satoshi Kubota a,b, Hany Mohamed Khattab a, Takashi Nishida b, Masaharu Takigawa a,b,⁎ a b

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Okayama, Japan

a r t i c l e

i n f o

Article history: Received 6 August 2014 Revised 17 December 2014 Accepted 23 December 2014 Available online 30 December 2014 Edited by: Hong-Hee Kim Keywords: CCN2 family protein 2 (CCN2) Osteoclast Receptor activator of nuclear factor-κB ligand (RANK) RANK ligand (RANKL) Osteoprotegerin (OPG)

a b s t r a c t CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a multi-potent factor for mesenchymal cells such as chondrocytes, osteoblasts, osteoclasts, and endothelial cells. CCN2 is also known as a modulator of other cytokines and receptors via direct molecular interactions with them. We screened additional factors binding to CCN2 and found receptor activator of NF-kappa B (RANK) as one of them. RANK is also known as TNF-related activation-induced cytokine (TRANCE) receptor, and its signaling plays a critical role in osteoclastogenesis. Notable affinity between CCN2 and RANK was confirmed by using surface plasmon resonance (SPR) analysis. In fact, CCN2 enhanced the RANK-mediated signaling, such as occurs in NF-kappa B, p38 and JNK pathways, in pre-osteoclastic RAW264.7 cells; whereas CCN2 had no influence on RANK–RANK ligand (RANKL) binding. Moreover, CCN2 also significantly bound to osteoprotegerin (OPG), which is a decoy receptor of RANKL. Of note, OPG markedly inhibited the binding between CCN2 and RANK; and CCN2 canceled the inhibitory effect of OPG on osteoclast differentiation. These findings suggest CCN2 as a candidate of the fourth factor in the RANK/RANKL/OPG system for osteoclastogenesis, which regulates OPG and RANK via direct interaction. © 2014 Elsevier Inc. All rights reserved.

Introduction CCN is an acronym that stands for Cyr61 (cysteine-rich 61)/ CCN1, CTGF (connective tissue growth factor)/CCN2, and Nov (nephroblastoma overexpressed)/CCN3, which are the 3 founder members of this family. This family now consists of 6 distinctive members by the addition of 3 more members, named WISP (Wnt-induced secreted protein) 1–3/CCN4–6 [1–3]. They are all cysteine-rich secreted proteins and composed of 4 distinct modules connected in tandem, i. e., IGF binding protein-like, von Willebrand type C, thrombospondin type 1 repeat, and C-terminal modules, except for CCN5, which lacks the CT module [2,3]. CCN family member 2 (CCN2), also previously known as CTGF and hypertrophic chondrocyte specific gene product 24 (Hcs24) [1,2], has been shown to play important roles in skeletal formation, maintenance, and regeneration by acting

Abbreviations: CTGF, connective tissue growth factor; ELISA, enzyme-linked immuno assay; GST, glutathione transferase; M-CSF, macrophage colony-stimulating factor; NF-κB, nuclear factor-kappa B; OPG, osteoprotegerin; RANK, receptor activator of NF-κB; RANKL, receptor activator of NF-κB ligand; RANKL-GST, GST fusion RANKL; SPR, surface plasmon resonance; TACE, TNF-α-converting enzyme; TRAP, tartrate-acid resistant alkaline phosphatase; TRANCE, TNF-related activation-induced cytokine; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1. ⁎ Corresponding author at: Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan. Fax: +81 86 235 6842. E-mail address: [email protected] (M. Takigawa).

http://dx.doi.org/10.1016/j.bone.2014.12.058 8756-3282/© 2014 Elsevier Inc. All rights reserved.

on various types of skeletal cells [1–4]. CCN2 promotes the proliferation and differentiation of chondrocytes, osteoblasts, and vascular endothelial cells [1–4] and the fusion of osteoclast precursors [4,5] in vitro. Moreover, CCN2-deficient mice demonstrate multiple skeletal defects [6], whereas endochondral bone formation is promoted in transgenic mice overexpressing CCN2 in their cartilage and without skeletal disorder [4,7]. These findings suggest that multifunctional actions of CCN2 on various skeletal cells are orchestrated in the microenvironment of the skeletal tissues. One notable feature of CCN2 is that CCN2 binds to a variety of cytokines (e. g., FGF, BMP, and TGF-β) [8–11], membrane proteins (e.g., FGFR, TrkA, and LRP1) [12–15], extracellular matrix components (e.g., heparin, aggrecan and integrins) [16–18], and CCN proteins themselves with high affinities [18]. Therefore, it is critical to specify the binding partners in a given microenvironment for understanding the biological functions of CCN2. To explore other possible physiological molecular networks tied to CCN2, we screened for other CCN2-binding proteins. Previously we employed bacteriophage display screening in designing peptide aptamers binding to CCN2 [19]. Next, by utilizing the same screening system, we searched for peptides binding to human CCN2 and found some that are homologous to the amino acid sequence of receptor activator of nuclear factor kappa B (RANK). RANK, also known as TRANCE receptor, is a member of tumor necrosis factor (TNF) receptor family, and RANK signaling is essential for osteoclastogenesis [20–22]. RANK signaling is regulated by RANK ligand (RANKL) and osteoprotegerin (OPG), which is a decoy receptor

E. Aoyama et al. / Bone 73 (2015) 242–248

of RANK. In this present study we found that CCN2 bound directly to both RANK and OPG, regulating RANK/RANKL/OPG signaling. A few previous reports indicated that CCN2 is necessary for osteoclastogenesis induced by RANKL [5,23,24]. For example, we reported earlier that down-regulation of CCN2 represses osteoclastogenesis [25] and that recombinant CCN2 enhances RANKL-induced osteoclastogenesis [23]. In addition, we partly uncovered the molecular mechanism of its enhancing effect: CCN2 increases expression of dendritic cell-specific transmembrane protein (DC-STAMP) and fusion of mononuclear osteoclast precursor cells via interaction with DC-STAMP [5]. Moreover, Nozawa et al. suggested that CCN2 promotes the induction of osteoclastogenesis via integrin αvβ3 [24]. As a multiple regulator, CCN2 has a potential to modulate osteoclastogenesis at multiple stages in different signaling systems [5]. However, the precise mechanisms of CCN2 action in the RANK/RANKL/OPG system were not sufficiently elucidated though the system is known as crucial in osteoclast differentiation. To figure out the whole effect of CCN2 on osteoclastogenesis, we in the present study investigated the interaction of CCN2 with RANK and OPG. Here, this study presents data revealing a novel role of CCN2 in the RANK/RANKL/OPG system supporting osteoclastogenesis. Experimental procedures Reagents and antibodies Recombinant protein CCN2 from BioVendor Laboratory Medicine (Brno, Czech Republic), recombinant human RANK/TNFRSF11A Fc Chimera, recombinant OPG, and recombinant mouse M-CSF were purchased from R&D Systems (Minneapolis, MN, USA). GST-fused RANKL was prepared and purified as described in a previous report by T. Nishida et al. [5]. Anti-human IgG (Fc specific)-peroxidase conjugate was purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-OPG and anti-GST antibodies were from R&D Systems and GE Healthcare UK Ltd. (Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England), respectively. Anti-NF-κB p65 (A) antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-ACTIVE® MAPK pAb and anti-phospho-JNK antibody were supplied by Promega Corporation (Madison, Wl, USA); whereas anti-p44/42 MAPK antibody, anti-phospho-p38 MAPK antibody, anti-p38 MAPK antibody, and antiJNK antibody were purchased from Cell Signaling Technology (Danvers, MA, USA).

243

of OPG binding to CCN2 was measured by using a 1:2000 dilution of anti-OPG antibody. Similarly, RANKL binding to RANK was quantified by use of a 1:1000 dilution of anti-GST antibody.

Surface plasmon resonance measurements Human recombinant CCN2 was diluted to 25 μg/ml with 10 mM sodium acetate buffer (pH 4.0) and immobilized onto CM5 sensor chips (GE Healthcare UK Ltd.) according to standard amine coupling procedures. Recombinant RANK diluted with HBS-EP (GE Healthcare UK Ltd.) to concentrations of 1.6, 8, 40, 200, and 1000 nM was injected into the flow cells. For affinity measurements, binding and dissociation were monitored with Biacore X (GE Healthcare UK Ltd.). The data were fitted by using BIAevaluation software version 4.1 (GE Healthcare UK Ltd.) with the single-cycle kinetics support package (GE Healthcare UK Ltd.). Binding data were globally fit to the single-cycle kinetics 1:1 Langmuir binding model. Recombinant OPG diluted to 0. 63, 1.25, 2.5, 5, and 10 nM was used to determine the binding dissociation constant between OPG and CCN2.

Cell cultures and TRAP staining RAW264.7 cells were maintained in a humidified incubator (5% CO2 in air) at 37 °C with 10% heat-inactivated FCS/αΜΕΜ supplemented with 50 μg/ml streptomycin and 100 units/ml penicillin. Osteoclast was induced to differentiate from RAW264.7 cells with 100 ng/ml RANKL. Osteoclast formation from murine bone marrow cells was investigated as described previously [27]. Briefly, bone marrow cells from 8–12 w male Balb/cJ mice were plated at 5 × 105 cells/well in a 24-well plate and cultured with M-CSF for 3 days. After the floating cells had been washed out with PBS, the adherent cells were cultured with M-CSF and RANKL for 6 days. The medium was changed at day 3, and the cells were stained for tartrate-resistant acidic phosphatase (TRAP) activity after 6 days [5]. The staining was quantified as the ratio of the optical absorbance at wavelength 540 to that at 410 nm to correct the background derived from cell bodies, buffer and culture plates All animal experiments were performed in accordance with the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan and approved by the Animal Research Control Committee of Okayama University (Approval No.: OKU-2014284).

Screening of bacteriophage display library and homology search Screening of random dodecapeptides displayed on filamentous M13 bacteriophages was performed by a biopanning method, according to the manufacturer's instructions (Ph.D.-12 Library, New England Biolabs, Beverly, MA, USA) as described previously [26]. Thereafter, the sequences of these dodecapeptides were subjected to a BLAST homology search to specify proteins with the respective peptide sequences.

Evaluation of NF-κB nuclear translocation RAW264.7 cells stimulated with RANKL and/or CCN2 were stained with anti-p65 antibody. The cells were scanned to acquire images on an ArrayScan® HCS reader (Thermo Scientifics Arrayscan VTI, Cellomics, Inc., Pittsburgh, PA, USA), and the rate of nuclear translocation of NF-κB was analyzed according to the manufacturer's protocol.

Solid-phase binding assay Western blotting The binding assay was carried out as described previously [12]. Briefly, wells of an ELISA plate were coated with recombinant CCN2 diluted to 1 μg/ml with 50 mM NaHCO3 buffer (pH 9.6) at 4 °C overnight. After the wells had been masked with a blocking buffer (50 mM Tris– HCl [pH 7.4], 150 mM NaCl, 2% BSA, 0.05% Tween 20) for 2 h at 37 °C, 50 ml of recombinant RANK-Fc or recombinant OPG, which had been diluted with blocking buffer, was added to the wells; and incubation was continued for 2 h at 37 °C. The wells were then washed with wash buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 0.05% Tween 20) and subsequently incubated with 50 ml of a 1:2000 dilution of anti-human IgG-HRP. Bound IgG-HRP was measured by using TMB peroxidase substrate (Sigma-Aldrich, St Louis, MO, USA). The amount

The cells stimulated with RANKL and/or CCN2 were lysed with RIPA buffer [12]. Total protein (40–50 μg) was applied to SDS-PAGE and the separated proteins were then transferred to a polyvinyl difluoride (PVDF) membrane. After blocking, Western blotting was carried out with Anti-ACTIVE® MAPK pAb, p44/42 MAPK antibody, anti-phosphop38 MAPK antibody, anti-p38 MAPK antibody, anti-phospho-JNK antibody or anti-JNK antibody. Immunopositive signals were detected by use of a secondary antibody-HRP conjugate and chemiluminescent substrate. Quantitative densitometric analysis of blots was performed by using a ChemiDoc MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA) and ImageLab software version 5.1 (Bio-Rad Laboratories).

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CCN2 binding peptide: GRLPSAAHYLSS

Table. 1 Acceleratory effect of CCN2 on the nuclear translocation of NF-κB in RAW264.7 cells stimulated with RANKL.

- - - 499GRLPSSA505 - - -

- - - 55YMSS58 - - -

C

N : TNFR-like domain/Cysteine-Rich Domain (CRD) : Transmembrane site

Stimulator

None

GST

CCN2 + GST

CCN2 + RANKL-GST

RANKL-GST

Positive objects (%)

3.06

5.26

3.61

41.07

34.50

RAW264.7 cells (1 × 104/100 ml/well) were stimulated with 1 μg/ml RANKL-GST in the presence or absence of CCN2 for 15 min. GST was used as a negative control of RANKLGST. After fixation and permeabilization, the cells were stained with anti-p65 antibody and Alexa568-conjugated anti-rabbit antibody. The rate of translocation of NF-κB was calculated with ArrayScan® VTI HCS Reader as described in Section 2 “Experimental procedures”. A repeat experiment gave similar data.

: TRAF binding site

Fig. 1. Schematic diagram of RANK and its amino-acid sequence regions homologous to those of the CCN2-binding peptide. RANK protein has 2 sites homologous to the peptide binding to CCN2. The black box indicates the cysteine-rich domain (CRD); and the gray boxes, the TRAF-binding sites. The striped box shows the transmembrane domain.

Quantitative real-time PCR analysis Total RNA was isolated by using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and was reverse-transcribed to cDNA by using a Takara RNA PCR kit (AMV), version 3.0 (Takara Shuzo, Tokyo, Japan). Amplification reactions were performed with a SYBR® Green Real-time PCR Master Mix (Toyobo; Tokyo, Japan) by using StepOne™ software v2.1 (Applied Biosystems, Foster City, CA, USA). The nucleotide sequences of the primers used were as follows: 5′-GCCAAAAGGGTCATCATCTC-3′ (forward) and reverse 5′-GTCTTCTGGGTGGCAGTGAT-3′ (reverse) for Gapdh; 5′-ATGGGCTCTCCTGTCAACAC-3′ (forward) and reverse 5′GGCTGCCAAAATAAACTCA-3′ (reverse) for c-fos; 5′-GTGCAGCCAGAA AGCTCA-3′ (forward) and reverse 5′-TGAGGCTGGTCTTCCGAGTT-3′ (reverse) for Nfatc1.

Statistical analysis Unless otherwise specified, the experiments were repeated at least twice; and similar results were obtained in the repeated experiments. Statistical analysis was performed by performing Student's t-test, oneway ANOVA test or two-way ANOVA test, if necessary. Data were expressed as the means ± standard deviations (SD).

A

Results Identification of RANK as a CCN2-binding protein Earlier we had employed a bacteriophage display system to screen for peptides binding to each module of CCN2 [26]. As a result, three rounds of screening revealed that a peptide binding to the IGFBP-like module included sequences homologous to 2 separate parts of the amino-acid sequence of human receptor activator of NF-κB (RANK) (Fig. 1) [28,29]. One of them was located on the N-terminal TNF-R like domain/cysteine-rich domain (CRD) within the extracellular domain (amino acids 55–58), and the other was between TRAF-binding sites within the intracellular domain (amino acids 499–505). To confirm the binding of CCN2 to RANK, we carried out a solidphase binding assay. Briefly, the human IgG Fc-tagged RANK (RANKFc) diluted to several concentrations was incubated with recombinant CCN2 adsorbed on the ELISA plate. Fig. 2A shows that RANK bound specifically to CCN2. The same amounts of human IgG were used as a negative control, and it hardly bound to CCN2 at any of the doses tested. RANK binding to CCN2 increased in a dose-dependent manner, and statistical significance was demonstrated between RANK and IgG at 2 μg/ml and 4 μg/ml. Next, we used surface plasmon resonance methodology to kinetically analyze the interaction between RANK and CCN2. Recombinant CCN2 was absorbed on a CM5 chip and examined for its interaction with RANK-Fc (Fig. 2B). This analysis gave a dissociation constant value for the binding of CCN2 to RANK (95.1 nM) indicating the specificity of the binding [30].

B

0.08

300 250 200

0.06

RANK

*

0.04

IgG

Resonance (RU)

Abs450(sample-blank)

*

150 100 50 0

0.02

-50 -100

0 0

1

2

(µg/ml)

3

4

0

200

400

600

800

1000

1200

Second (s)

Fig. 2. Binding of RANK to CCN2 in vitro. (A) CCN2 (1 μg/ml) was added to and incubated on an ELISA plate overnight. After washing and blocking with BSA, recombinant RANK-Fc was added at the indicated doses to the recombinant CCN2-coated plate. After extensive washing with the wash buffer (described in Section 2 “Experimental procedures”) the amount of RANK-Fc bound to CCN2 was determined by using anti-human Fc antibody. The representative data from 3 independent experiments show the mean values from triple wells ± SD (* indicates p value b 0.01 compared with the value for the same dose of IgG). (B) The binding of recombinant CCN2 and RANK-Fc was analyzed by use of SPR. Recombinant CCN2 was coated on a CM5 chip, and the extracellular domain of RANK-Fc diluted to the indicated concentrations was injected. The Kd value was calculated from sensorgrams as described under Section 2 Experimental procedures.

E. Aoyama et al. / Bone 73 (2015) 242–248

RANKL +CCN2

time (min)

0

5 15 30 60 5 15 30 60

p-ERK ERK

p-ERK/ERK 4

Relative density

RANKL

245

3 2

CCN2(-)

1

CCN2(+)

0 RANKL(-)

RANKL +CCN2

RANKL

5 15 30 RANKL treatment (min)

60

p-JNK/JNK

time (min) 0

5 15 30 60

5

15 30 60

p-JNK JNK

Relative density

2.5 2 1.5 1

CCN2(-)

0.5

CCN2(+)

0 RANKL(-) RANKL

RANKL +CCN2

5 15 30 60

5 15 30 60

5 15 30 RANKL treatment (min)

60

0

p-p38 p38

Relative density

p-p38/p38 time (min)

5 4 3 2

CCN2(-)

1

CCN2(+)

0 RANKL(-)

5 15 30 RANKL treatment (min)

60

Fig. 3. Amplification of p38 and JNK activation induced by RANKL stimulation of RAW264.7 cells by CCN2. RAW264.7 cells (6.6 × 104/well) were plated overnight, and culture medium was changed to 1% FCS/αΜΕΜ. The next day, the cells were stimulated with RANKL-GST (25 ng/ml) in the presence or absence of CCN2 (100 ng/ml). After incubation for the indicated times, the cells were collected in RIPA buffer. The lysate was analyzed by Western blotting using anti-MAPK antibodies. Data are representative of those of 3 independent experiments.

Effect of CCN2 on RANK–RANKL signaling in osteoclast precursor RAW264.7 cells Subsequently, we investigated the effect of this interaction between RANK and CCN2 on the binding between RANK and RANKL by using the solid-phase binding assay. RANKL was adsorbed on the wells of an ELISA plate, and the mixture of RANK and CCN2 was then added to the wells. The results showed that CCN2 neither increased nor decreased the binding between RANK and RANKL adsorbed on the ELISA plate (data not shown). The amounts of RANK binding to RANKL were almost invariable in the presence of any concentration of CCN2, whereas we confirmed that OPG prevented the binding of RANK to RANKL in a

dose-dependent manner by acting as a decoy receptor of RANKL in the same assay system (data not shown). Murine macrophage cell line RAW264.7 cells differentiate into osteoclasts by RANKL stimulation mediated by NF-κB and MAPK [28]. In order to functionally characterize the effect of CCN2 on the signaling downstream of RANK, we investigated the effect of CCN2 on the RANKL-induced translocation of NF-κB from the cytosol to the nucleus of RAW264.7 cells. The cells were stimulated with RANKL in the presence or absence of CCN2; and after a 30-min incubation, NF-κB was stained with an anti-p65 antibody to assess the rate of NF-κΒ nuclear translocation by comparatively measuring the signal intensities in both the nucleus and cytosol of each cell. The values shown in Table 1

*

* 3.5

14

*

none CCN2

10 8 6 4

none

*

3 nfatc1/gapdh

c-fos/gapdh

12

CCN2

2.5 2 1.5 1 0.5

2 0

0

-

+ RANKL

-

+ RANKL

Fig. 4. Effect of CCN2 on the expressions of NFATc1 and c-Fos in RAW264.7 cells induced by RANKL. RAW264.7 cells (6.6 × 104/well) were plated and cultured overnight in 10% FCS/αΜΕΜ. The cells were then stimulated with RANKL-GST (25 ng/ml) in the presence or absence of CCN2 (100 ng/ml). After incubation for 48 h, the mRNA levels were quantified by real-time RTPCR analysis, as described in Section 2 Experimental procedures. The data are the mean values ± S.D. of values from triplicate samples from representative of three independent experiments. * p b 0.05 as compared with each value from the control wells.

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A

B

1.2

*

160 140

*

120

0.8 0.6

OPG IgG

*

0.4

100 80 60 40 20

*

0.2

Resonance (RU)

Abs450(sample-blank)

1

0 -20

0 0

0.2

0.4

0.6

0.8

1

0

500

1000

(µg/ml)

1500

Second (s)

Fig. 5. OPG bound to CCN2 specifically. (A) OPG diluted to the indicated concentrations was incubated on an ELISA plate bearing adsorbed CCN2. After washing of the plate with the wash buffer, anti-OPG antibody was added; and bound anti-OPG was measured with anti-rat antibody-HRP and TMB substrate. Representative data from 3 independent experiments are shown as the mean values from triple wells ± SD (* indicates p value b 0.01 compared with the value for same dose of IgG). (B) CCN2 was immobilized on a CM5 chip, and serially diluted OPG was injected onto the chip. The Kd value was calculated from sensorgrams, as described in under Section 2 Experimental procedures.

Direct interaction of CCN2 with OPG and its biological outcome It is well established that OPG binds to RANKL and disturbs the binding between RANK and RANKL [32,33]. Considering that both RANK and OPG bind to the same ligand, next we examined the binding of CCN2 to OPG. Surprisingly, the results of the solid-phase binding assay showed the binding of OPG to CCN2, which occurred in a dosedependent manner (Fig. 5A). SPR analysis showed the binding affinity of OPG for CCN2 to be 24.5 nM (Fig. 5B), which was almost as high as that of OPG to RANKL [34]. Then, the binding assay was conducted to test the influence of OPG on the binding of CCN2 to RANK. The results shown in Fig. 6 indicate that OPG remarkably prevented the binding between CCN2 and RANK in a dose-dependent manner. Indeed, OPG at the same concentration as that of RANK decreased the binding of RANK to CCN2 down to 25% of that without OPG. These data thus show that OPG is not only RANK–RANKL signal inhibitor, but also a potent inhibitor of the binding of CCN2 to RANK. Finally, to elucidate the effect of CCN2 on OPG function, we examined the inhibitory effect of OPG on RANKL-induced osteoclastogenesis in the presence or absence of CCN2. Murine bone marrow adherent cells

were cultured with M-CSF, RANKL, OPG and/or CCN2. Figs. 7A and B depict the results when the cells were stained for TRAP activity after 6 days in culture. Fifty nanograms/ml CCN2 slightly recovered the TRAP staining repressed by OPG but did not affect the polynuclear cells. However, the wells cultured with 100 ng/ml CCN2 had a significant number of polynuclear cells; and the wells with 200 ng/ml CCN2 contained as many polynuclear and TRAP-positive cells as the OPG-free wells. The optical quantification of each well shown in Fig. 7B indicates that TRAP staining of wells containing OPG was significantly reduced, in which reduction was prevented by 100 ng/ml and 200 ng/ml CCN2; and this staining was equivalent to that without OPG. CCN2 alone did not induce maturation of osteoclasts (data not shown). These data mean that CCN2 could disturb the inhibitory effect of OPG on RANK/ RANKL signaling.

Discussion In the present study, we found that CCN2 directly bound to RANK and enhanced RANK signaling induced by RANKL. SPR analysis revealed that the dissociation constant (Kd) of RANK and CCN2 was 95.1 nM. According to several previous studies, the dissociation constant of RANK–RANKL is reported to be between 0.109 and 752 nM [30,35,36]. Therefore, the Kd value for the dissociation between RANK and CCN2 in this study indicates that the binding of these proteins has affinity

0.25

Abs450(sample-blank)

indicate that the rate of NF-κB nuclear translocation was 34.1% for the cells stimulated with RANKL alone. However, the combination of RANKL and CCN2 enhanced the rate up to 41.07%. The rate for the cells incubated with CCN2 only was almost the same as that for the cells with GST (negative control). These data demonstrate that CCN2 amplified the NF-κB translocation induced by RANKL. Moreover, RANK signaling via MAPK in the presence or absence of CCN2 was investigated by using the Western blot assay. Fig. 3 shows that CCN2 enhanced the RANKL-induced activation of p38 and JNK and slightly that of ERK. CCN2 tested alone did not elicit phosphorylation of these MAPKs (data not shown). These results also supported the amplifying effect of CCN2 on RANK signaling. The expressions of NFATc1 and c-Fos are induced by RANKL and are known as osteoclast differentiation markers at early stage [31]. To analyze the effect of CCN2 on the expression of these markers, we quantified their mRNA levels by real-time RT-PCR. As shown in Fig. 4, CCN2 potentiated the expressions of NFATc1 and c-Fos stimulated by RANKL (25 ng/ml). No significant stimulatory effect of CCN2 was observed without RANKL. These results show that CCN2 has potentiating effect on the mRNA expression of osteoclastic markers under RANK/RANKL signaling.

0.2 0.15 0.1

* 0.05

*

*

0.5

1

0

OPG (µg/ml)

0

0.25

Fig. 6. OPG dose dependently decreased the binding of RANK-Fc to CCN2 coated on an ELISA plate. An ELISA plate was coated overnight with CCN2 (1 μg/ml). After the plate had been washed and blocked with BSA, recombinant RANK-Fc was pre-mixed with OPG at the indicated concentrations and applied to the CCN2-coated plate. After a wash with the wash buffer, the amount of RANK-Fc bound to CCN2 was measured by use of HRP-conjugated anti-human Fc antibody and TMB substrate. Representative data from 3 independent experiments are shown. * indicates p b 0.01, as compared with the value for the control wells incubated with RANK-Fc alone.

E. Aoyama et al. / Bone 73 (2015) 242–248

247

A

OPG (+) CCN2 (0)

B

Intensity of TRAP staining (Abs540/Abs410)

OPG (-) CCN2 (-)

2

OPG (+) CCN2 (50)

OPG (+) CCN2 (100)

OPG (+) CCN2 (200)

*

1.5 1

OPG(+) OPG(-)

0.5 0 0

50

100

200

CCN2 (ng/ml) Fig. 7. CCN2 nullified the inhibitory effect of OPG on RANKL-induced osteoclastogenesis in mouse bone marrow cultures. (A) Mouse bone marrow cells were cultured with RANKL and OPG as described under Section 2 Experimental procedures in the presence or absence of CCN2 diluted to the indicated concentrations. After 6 days the mature osteoclasts were stained for TRAP activity. (B) Quantitative evaluation of Fig. 7A as the ratio of the optical absorbance at wavelength 540 nm to that at 410 nm to correct the background derived from cell bodies. The representative data from 3 independent experiments are shown as the mean values from triple wells ± SD (* indicates p value b 0.01).

sufficient for exerting biological effects. In fact, CCN2 enhanced the translocation of NF-κB from the cytoplasm to the nucleus in RAW264. 7 cells stimulated by RANKL. As is widely recognized, NF-κB plays an essential role in osteoclast maturation induced by RANK signaling [37, 38]. Furthermore, the effect of CCN2 on activation of MAPKs such as p38, JNK, and ERK was tested, and we found that CCN2 amplified the RANK-induced phosphorylation of p38 and JNK. JNK and p38 are known to mediate RANK signaling and induce activation or expression of several important factors downstream, such as AP-1 or NFATc1, in osteoclastogenesis [39–41]. However, the role of ERK in osteoclastogenesis is controversial [31,42]. Previous reports described that the phosphorylation of ERK is elevated by stimulation with RANKL, whereas a MEK inhibitor increased the number of TRAP-positive cells. These data collectively indicate that CCN2 positively regulated osteoclastogenesis via enhancement of RANK signaling. Moreover CCN2 showed the potentiating effect on the mRNA expression of NFATc1 and c-Fos induced by RANKL. NFATc1 has a crucial transcriptional factor in the early stage of osteoclast differentiation and its expression is positively regulated by c-Fos [43,44]. These findings further confirm that CCN2 intensifies the osteoclast differentiation via MAPK under RANK signaling. In order to clarify the mechanism of RANK signal enhancement by CCN2, we examined the effect of CCN2 on the binding between RANK and RANKL in the solid-phase binding assay. Unexpectedly, CCN2 neither increased nor decreased the binding. This result indicates that CCN2 did not enhance the RANK signaling via modification of the binding between RANK and RANKL. In addition, no effect of CCN2 on the amount of RANK protein on pre-osteoclastic cells was observed (data not shown), suggesting that the signal enhancement by CCN2 may not be ascribed to an increase in RANK expression itself. Therefore, the mechanism of CCN2 action enhancing RANK signaling remains unclear; but a few hypotheses can be suggested. The binding of CCN2 to RANK might elicit a change in the tertiary structure of RANK, resulting perhaps in trimerization [45] or cleavage by TNF-α converting enzyme

(TACE) [46]. Alternatively, CCN2 might bind to the soluble RANK ectodomain cleaved by TACE and prevent its negative effect on RANK signaling. CCN2 also bound to OPG, and the binding revealed significant affinity (Kd value: 24.5 nM); and this Kd value was comparable to that of the binding of OPG to RANKL (8.4 nM at 20 °C or 23 nM at 37 °C) [34]. Furthermore, OPG inhibited the binding of CCN2 to RANK. This fact suggests that OPG uses an alternative inhibitory pathway in counteracting osteoclastogenesis. As is widely recognized, OPG acts as a negative regulator via inhibition of RANK–RANKL binding; but our findings indicate that it also may act via inhibition of CCN2–RANK binding. It should be also noted that this study uncovered a novel role of CCN2 as an OPG inhibitor. Hence, CCN2 is the negative regulator of OPG function; but the mechanism of its inhibitory effect on OPG remains unclear. CCN2 did not directly prevent the binding of OPG to RANKL (data not shown). These findings suggest that the site of binding between OPG and CCN2 is different from that between OPG and RANKL. These 3 proteins might form a tri-molecular complex that could interact with RANK to modulate its signaling pathway. In this study, CCN2 failed to induce the formation of TRAP-positive cells without RANKL (data not shown), which is consistent with several previous reports [5,24,25]. We previously showed that CCN2 enhances RANKL-induced osteoclastogenesis [5]. However, this result showed that CCN2 produced by RANKL-stimulated osteoclast precursors promotes the fusion of the mononuclear precursor cells via binding to DC-STAMP. This is a phenomenon of late-stage osteoclastogenesis. In contrast, in the present study, we revealed the molecular interaction of CCN2 with RANK, which should occur in the early stage of osteoclastogenesis and have a sufficient effect on RANKL-signaling leading to osteoclastogenesis. Further studies in vivo with mutant animals will provide an integrated view of these CCN2 functions in bone resorption and homeostasis.

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Conclusion Three new systems operative in osteoclastogenesis conducted by CCN2 were clarified. First, CCN2 significantly bound to RANK and enhanced RANK signaling. Second, CCN2 prevented the inhibitory effect of OPG. Third, OPG remarkably inhibited the binding of CCN2 to RANK by directly binding to CCN2, thereby attenuating the enhancing effect of CCN2 on RANK signaling. Therefore, we propose CCN2 as a candidate of the fourth key molecule working in RANK/RANKL/OPG signaling. Acknowledgments We thank Dr. Takako Hattori and Dr. Harumi Kawaki for their helpful suggestions and Ms. Eri Yashiro and Yoshiko Miyake for their secretarial assistance. This study was supported in part by the programs JSPS KAKENHI Grants-in-aid for Young Scientists (B), No. 25861755 (to EA) and JSPS KAKENHI Grants-in-aid for Scientific Research (S), No. 19109008 (to MT) and (B), No. 24390415 (to MT) and (C), No. 26462810 (to TN), and (C), No. 25462886 (to SK), and by JSPS KAKENHI Challenging Exploratory Research, No. 266708 (to MT). References [1] Perbal B, Takigawa M. CCN proteins: a new family of cell growth and differentiation regulators. In: Perbal B, Takigawa M, editors. London, UK: Imperial College Press; 2005. p. 1–311. [2] Takigawa M, Nakanishi T, Kubota S, Nishida T. Role of CTGF/HCS24/ecogenin in skeletal growth control. J Cell Physiol 2003;194(3):256–66. [3] Kubota S, Takigawa M. Role of CCN2/CTGF/Hcs24 in bone growth. Int Rev Cytol 2007;257:1–41. [4] Takigawa M. CCN2: a master regulator of the genesis of bone and cartilage. J Cell Commun Signal 2013;7(3):191–201. [5] Nishida T, Emura K, Kubota S, Lyons KM, Takigawa M. CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes osteoclastogenesis via induction of and interaction with dendritic cell-specific transmembrane protein (DC-STAMP). J Bone Miner Res 2011;26(2):351–63. [6] Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, et al. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 2003;130(12):2779–91. [7] Tomita N, Hattori T, Itoh S, Aoyama E, Yao M, Yamashiro T, et al. Cartilage-specific over-expression of CCN family member 2/connective tissue growth factor (CCN2/ CTGF) stimulates insulin-like growth factor expression and bone growth. PLoS One 2013;8(3):e59226. [8] Nishida T, Kubota S, Aoyama E, Janune D, Maeda A, Takigawa M. Effect of CCN2 on FGF2-induced proliferation and MMP9 and MMP13 productions by chondrocytes. Endocrinology 2011;152(11):4232–41. [9] Abd El Kader T, Kubota S, Nishida T, Hattori T, Aoyama E, Janune D, et al. The regenerative effects of CCN2 independent modules on chondrocytes in vitro and osteoarthritis models in vivo. Bone 2014;59:180–8. [10] Maeda A, Nishida T, Aoyama E, Kubota S, Lyons KM, Kuboki T, et al. CCN family 2/ connective tissue growth factor modulates BMP signalling as a signal conductor, which action regulates the proliferation and differentiation of chondrocytes. J Biochem 2009;145(2):207–16. [11] Abreu JG, Ketpura NI, Reversade B, De Robertis EM. Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol 2002;4(8): 599–604. [12] Aoyama E, Kubota S, Takigawa M. CCN2/CTGF binds to fibroblast growth factor receptor 2 and modulates its signaling. FEBS Lett 2012;586(24):4270–5. [13] Wahab NA, Weston BS, Mason RM. Connective tissue growth factor CCN2 interacts with and activates the tyrosine kinase receptor TrkA. J Am Soc Nephrol 2005; 16(2):340–51. [14] Kawata K, Kubota S, Eguchi T, Aoyama E, Moritani NH, Kondo S, et al. Role of LRP1 in transport of CCN2 protein in chondrocytes. J Cell Sci 2012;125(Pt 12):2965–72. [15] Segarini PR, Nesbitt JE, Li D, Hays LG, Yates III JR, Carmichael DF. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor. J Biol Chem 2001;276(44):40659–67. [16] Ball DK, Rachfal AW, Kemper SA, Brigstock DR. The heparin-binding 10 kDa fragment of connective tissue growth factor (CTGF) containing module 4 alone stimulates cell adhesion. J Endocrinol 2003;176(2):R1–7. [17] Aoyama E, Hattori T, Hoshijima M, Araki D, Nishida T, Kubota S, et al. N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan. Biochem J 2009;420(3):413–20. [18] Hoshijima M, Hattori T, Aoyama E, Nishida T, Yamashiro T, Takigawa M. Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes. FEBS J 2012;279(19):3584–97. [19] Kawasaki M, Fujishiro M, Yamaguchi A, Nozawa K, Kaneko H, Takasaki Y, et al. Possible role of the JAK/STAT pathways in the regulation of T cell-interferon related genes in systemic lupus erythematosus. Lupus 2011;20(12):1231–9.

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