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Endocrinology Vol. 148, No. 12 5761-5768
Copyright © 2007 by The Endocrine Society

The Matricellular Protein CYR61 Inhibits Osteoclastogenesis by a Mechanism Independent of {alpha}vß3 and {alpha}vß5

Julie C. Crockett1, Norbert Schütze1, Denise Tosh, Susanne Jatzke, Angela Duthie, Franz Jakob and Michael J. Rogers

Bone and Musculoskeletal Research Programme (J.C.C., D.T., A.D., M.J.R.), Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom; and Orthopaedic Institute (N.S., S.J., F.J.), Molecular Orthopaedics, 97074 Wuerzburg, Germany

Address all correspondence and requests for reprints to: Professor Michael J. Rogers, Bone Research Group, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. E-mail: m.j.rogers{at}abdn.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cysteine-rich protein 61 (CYR61/CCN1) belongs to the family of CCN matricellular proteins. Most of the known effects of CCN proteins appear to be due to binding to extracellular growth factors or integrins, including {alpha}vß3 and {alpha}vß5. Although CYR61 can stimulate osteoblast differentiation, until now the effect of CYR61 on osteoclasts was unknown. We demonstrate that recombinant human CYR61 inhibits the formation of multinucleated, {alpha}vß3-positive, or tartrate-resistant acid phosphatase-positive human, mouse, and rabbit osteoclasts in vitro. CYR61 markedly reduced the expression of the osteoclast phenotypic markers tartrate-resistant acid phosphatase, matrix metalloproteinase-9, calcitonin receptor, and cathepsin K. However, CYR61 did not affect the formation of multinucleated osteoclasts when added to osteoclast precursors prior to fusion or affect the number or resorptive activity of osteoclasts cultured on dentine discs, indicating that CYR61 affects early osteoclast precursors but not mature osteoclasts. CYR61 did not affect receptor activator of nuclear factor-{kappa}B (RANK) ligand-induced phosphorylation of p38 or ERK1/2 in human macrophages and did not affect RANK ligand-induced activation of nuclear factor-{kappa}B, indicating that CYR61 does not appear to inhibit osteoclastogenesis by affecting RANK signaling. Furthermore, a mutant form of CYR61 defective in binding to {alpha}vß3 also inhibited osteoclastogenesis, and CYR61 inhibited osteoclastogenesis similarly in cultures of mouse wild-type or ß5–/– macrophages. Thus, CYR61 does not appear to inhibit osteoclast formation by interacting with {alpha}vß3 or {alpha}vß5. These observations demonstrate that CYR61 is a hitherto unrecognized inhibitor of osteoclast formation, although the exact mechanism of inhibition remains to be determined. Given that CYR61 also stimulates osteoblasts, CYR61 could represent an important bifunctional local regulator of bone remodeling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOCLASTS ARE THE specialized, multinucleated cells responsible for bone resorption and are derived from circulating hematopoietic precursors of the monocyte/macrophage lineage. Differentiation of these precursors into mature osteoclasts occurs within the bone microenvironment and is tightly controlled by a variety of growth factors and cytokines, including macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-{kappa}ß (NF{kappa}B) ligand (RANKL), which are critical for osteoclast formation (1). Via their receptors c-Fms and receptor activator of NF{kappa}B (RANK), respectively, M-CSF and RANKL activate a cascade of intracellular signaling pathways including p38, ERK1/2, and inhibitory-{kappa}B (2), which ultimately result in activation of the transcription factors NF{kappa}B, c-Fos, and nuclear factor of activated T cells-c1 (NFATc1), essential for osteoclastogenesis (3, 4, 5). These induce the expression of genes required for osteoclast differentiation and function and characteristic of the osteoclast phenotype, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, calcitonin receptor (CTR), matrix metalloproteinase (MMP)-9, and {alpha}vß3 integrin. The latter is critical for the fusion of osteoclast precursors to form mature, multinucleated osteoclasts as well as for osteoclast adhesion and bone resorption (6, 7, 8, 9, 10, 11, 12). {alpha}vß5 is also highly abundant on early osteoclast precursors and down-regulated during osteoclast differentiation (13).

Although it has become clear over recent years that RANKL and M-CSF are essential for osteoclast differentiation, a wide variety of other extracellular factors also play an important role in regulating osteoclast formation. Cysteine-rich protein 61 (CYR61/CCN1) is a member of the connective tissue growth factor (CCN) family of proteins that consists of CYR61, connective tissue growth factor (CTGF), nephroblastoma overexpressed (NOV), and Wnt-induced secreted proteins (WISP-1/CCN4, WISP-2/CCN5, and WISP-3/CCN6). All of these proteins have a similar structural organization consisting of four conserved domains: an IGF binding protein domain, a von Willebrand type C domain, a thrombospondin type 1 domain, and a C-terminal domain containing a putative cystine knot (see Ref. 14 for a review). The CCN proteins have been described as matricellular proteins (15) because they appear to be multifunctional, modular proteins that act as extracellular, matrix-associated molecules that affect a variety of processes including angiogenesis, inflammation, remodeling of extracellular matrix, and cell-matrix interactions (14). CYR61 (a 381 amino acid protein, molecular mass of 42 kDa) is encoded by an immediate-early gene on chromosome 1p22–31 and is expressed rapidly and transiently in response to growth and stress stimuli (16). It is highly conserved across species (more than 88% at the DNA level and more than 90% at the protein level in human vs. mouse). Many of the effects of CYR61 appear to be due to its ability to bind to heparan sulfate proteoglycans and cell surface integrins, including {alpha}vß3 and {alpha}vß5 (15, 17, 18, 19, 20). The role of CYR61 in the skeleton is poorly understood, although it appears to play a role in chondrogenesis and skeletogenesis during embryonic development (21, 22). CYR61 is expressed and secreted by osteoblasts and up-regulated in response to serum, 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3] and growth factors such as TNF{alpha} (23, 24, 25) and is a transcriptional target of canonical Wnt/ß-catenin signaling (26). The expression of CYR61 is also up-regulated in fracture callous during fracture repair (27, 28) and appears to stimulate osteoblast proliferation and differentiation (26). However, the effect of CYR61 on osteoclasts is unknown. In this study we examined the effect of CYR61 on osteoclast differentiation and bone resorption in vitro and show that CYR61 is a novel inhibitor of osteoclast formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of recombinant human CYR61
Human CYR61 was expressed in baculovirus infected Sf21 cells using the BakPak baculovirus expression system (BD Biosciences, Heidelberg, Germany), as previously described (29). The open reading frame of the human CYR61 gene (NM_001554) was amplified from a previously subcloned cDNA (23) and subcloned into the baculovirus transfer vector pBakPak8, which was modified to contain a C-terminal human IgG-Fc-domain. The expressed Fc-fusion protein (from hereon referred to just as CYR61) was purified from supernatants of infected Sf21 cells using protein G Sepharose columns (Amersham Biosciences, Freiburg, Germany) and subsequently desalted using a PD10 column (Amersham Biosciences). A purity of greater than 95% was determined by gel electrophoresis, silver staining, and Western blotting. A mutant form of CYR61, defective in the ability to bind {alpha}vß3, was generated by site-directed mutagenesis to cause substitution of Asp125 for Ala in domain II, as previously described (30).

Osteoclast formation assays
Human osteoclasts were generated from peripheral blood mononuclear cells obtained from healthy volunteers. Peripheral blood mononuclear cells were isolated by centrifugation over Lymphoprep and then seeded into 75-cm2 flasks (12–15 x 106 per flask) in {alpha}MEM containing 10% (vol/vol) fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml, and 0.1 mg/ml penicillin/streptomycin, respectively, and 20 ng/ml recombinant human M-CSF (R&D Systems, Abingdon, UK). After approximately 6 d, nonadherent cells were removed by washing and the adherent cells (highly enriched M-CSF dependent macrophages) were removed using trypsin and distributed into 96- or 48-well plates at a density of 2 x 104 or 5 x 104 cells/well in {alpha}MEM (Life Technologies, Paisley, UK) supplemented with 10% FCS, 50 ng/ml human soluble RANKL (Peprotech, Rocky Hill, NJ) and 20 ng/ml M-CSF, with or without 25–1000 ng/ml CYR61 (six replicate wells per treatment). After 6 d, cells were fixed with 4% formaldehyde and stained for TRAP (31) or stained for {alpha}vß3 using 23c6 mouse monoclonal antibody (kindly provided by Dr. S. Nesbitt, University College, London, UK) and Alexa-Fluor 488-conjugated antimouse secondary antibody (Molecular Probes, Eugene, OR), after which cells were counterstained with 4',6'-diamino-2-phenylindole (DAPI). Multinucleated (three or more nuclei), {alpha}vß3-positive cells were counted on a Axiovert microscope (Carl Zeiss Ltd., Welwyn Garden City, UK).

Mouse osteoclasts were generated in vitro from M-CSF-dependent murine bone marrow macrophages (32) from adult male C57BL/6 mice and adult wild-type and ß5–/– mice (129 SvJ strain) that were generously provided by Dr. Silvia Finneman (Cornell University, New York, NY) with kind permission from Professor Dean Sheppard (University of California, San Francisco, San Francisco, CA). These mice, lacking the ß5 integrin, had been generated as previously described (33). Bone marrow cells were flushed into 10-cm petri dishes (Falcon, Oxnard, CA) from the tibiae and femorae of all mice and were cultured in {alpha}MEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM glutamine, 10% (vol/vol) FCS, and 100 ng/ml M-CSF. After 3 d, nonadherent cells were removed and the adherent cells were reseeded into 96-well plates (Costar, Cambridge, MA) at a density of 5 x 103 cells/well in the medium described above containing 25 ng/ml M-CSF and 100 ng/ml murine soluble RANKL (both from R&D Systems), with or without CYR61 (at least four wells per treatment). Media were changed on the third day, and after 5 d the adherent cells were fixed and stained for TRAP (31). Multinucleated (three or more nuclei), TRAP-positive osteoclast-like cells were counted on a Zeiss Axiovert microscope. In some experiments, TRAP enzyme activity was also measured by lysing the cells in 20 µl PBS/0.1% (vol/vol) Triton X-100, followed by addition of 200 µl 0.1 M sodium acetate buffer (pH 5.2) containing 30 mM sodium tartrate and 8 mM paranitrophenylphosphate. The absorbance at 415 nm was then measured over 30 min at 37 C using a FL600 plate reader (Bio-Tek, Houston, TX).

Rabbit osteoclasts were generated from cultures of bone marrow (34). Bone marrow was flushed from the long bones of 3-d-old rabbits and cultured in 96-well plates at a density of 1.75 x 105/well, in {alpha}MEM supplemented with 10% (vol/vol) FCS and 1 x 10–8 M 1,25(OH)2D3 [without which few or no multinucleated cells are formed (35)]. Cells were cultured with or without 500 or 1000 ng/ml CYR61 (quadruplicate wells per treatment). Media were replaced every 4 d, and after 10 d, the adherent cells were fixed and stained for TRAP (34). Multinucleated, TRAP-positive osteoclasts were counted as described above. In some experiments, TRAP activity was measured as described above, but in the presence of 50 mM sodium tartrate.

RT-PCR analysis of osteoclast phenotypic markers
RT-PCR was used to assess the expression of cathepsin K, MMP-9, TRAP, and CTR during osteoclast differentiation in murine bone marrow macrophage cultures. M-CSF-dependent bone marrow macrophages were generated as described above, and total RNA was isolated using Trizol (Invitrogen, Paisley, UK) on d 0 and d 3 of culture with M-CSF + RANKL ± 1000 ng/ml CYR61. cDNA was prepared from 2 µg total RNA using Superscript II reverse transcriptase (Invitrogen). The following sets of primers were used for PCR analysis: cathepsin K, 5'-ggaagaagactcaccagaagc-3' (forward) and 5'-gtcatatagccgcctccacag-3' (reverse); MMP-9, 5'-cctgtgtgttcccgttcatct-3' (forward) and 5'-cgctggaatgatctaagccca-3' (reverse); TRAP, 5'-tacagcccccactcccaccct-3' (forward) and 5'-tcagggtctgggtctccttgg-3' (reverse); CTR, 5'-ccattcctgtacttggttggc-3' (forward) and 5'-agcaatcgacaaggagtgac-3' (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-actttgtcaagctcatttcc-3' (forward) and 5'-tgcagcgaactttattgatg-3' (reverse). For analysis of TRAP expression, the PCR was carried out using Pfx turbo polymerase (Invitrogen) and the cycling conditions were: 2 min at 94 C, then 25 cycles of 15 sec at 94 C, 30 sec at 61 C, and 1 min at 68 C followed by 5 min at 68 C. For all other osteoclast markers and GAPDH, the PCR was carried out using Taq polymerase (QIAGEN Ltd., Crawley, UK) and the cycling conditions were: 4 min at 94 C, 50 sec at 60 C, 90 sec at 72 C, then 15 (for MMP-9 and cathepsin K) or 25 (for CTR) cycles of 50 sec at 94 C, 50 sec at 60 C, 90 sec at 72 C followed by 50 sec at 94 C, 50 sec at 60 C, and 15 min at 72 C. All products were electrophoresed on a 1.5% agarose gel containing 0.5 µg/ml ethidium bromide.

Bone resorption assays
Mature osteoclasts were isolated from 2-d-old New Zealand White rabbits (34). Briefly, long bones were minced in {alpha}-MEM containing 100 U/ml penicillin and 100 µg/ml streptomycin, vigorously vortexed three times, and then the cell suspension was seeded onto 200-µm-thick discs of elephant ivory in 96-well plates (200 µl of cell suspension per well). After several hours the discs were washed gently with PBS and then cultured in fresh {alpha}-MEM containing 10% (vol/vol) FCS, 1 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, with or without 500 ng/ml or 1000 ng/ml CYR61 (six discs per treatment). After 48 h, the cultures were fixed with 4% formaldehyde and stained for TRAP. Resorption pits in the ivory surface were quantified by reflected light microscopy (31, 34). The resorptive activity of human osteoclasts was also assessed by culturing M-CSF-dependent human macrophages on ivory discs in the presence of RANKL (as described above). Then 100 ng/ml CYR61 was added to the cultures on d 6 (after the appearance of multinucleated osteoclasts); after a further 4 d the discs were analyzed for resorption area and number of TRAP-stained osteoclasts.

Analysis of RANK signaling
Cultures of highly enriched M-CSF-dependent macrophages, isolated as described above, were treated with 100 ng/ml RANKL ± 100 ng/ml CYR61 for 10–30 min and then lysed in ice-cold radioimmunoprecipitation assay buffer [10 mM potassium phosphate (pH 7.4), 137 mM NaCl, 0.1% (wt/vol) sodium dodecyl sulfate, 0.5% (wt/vol) sodium deoxycholate, 1% (vol/vol) Triton X-100, 1 mM sodium orthovanadate, 1 mM EDTA, protease inhibitors, and phosphatase inhibitor cocktail 1 (Sigma, Poole, UK)]. Lysates were cleared by centrifugation and assayed for protein content using the bicinchoninic acid assay (Pierce, Rockford, IL). Fifty micrograms of protein from each sample were then resolved by SDS-PAGE on 12% polyacrylamide-sodium dodecyl sulfate gels followed by transfer onto polyvinyl difluoride membranes. The blots were simultaneously hybridized with anti-dual-phosphorylated p38 (no. 9215; Cell Signaling Technology, Danvers, MA) and anti-p38 (no. 9217; Cell Signaling Technology) or with anti-phospho ERK1/2 (no. 9101; Cell Signaling Technology) and anti-ERK1/2 (no. 4696; Cell Signaling Technology), followed by AlexaFluor688-labeled antirabbit (A21076; Invitrogen) and IR800-labeled antimouse (LiCor Biosciences UK, Cambridge, UK) antibodies. Bands of phospho/total p38 and phospho/total ERK1/2 were visualized simultaneously using a LiCor Odyssey infrared imager and quantified using the Odyssey imaging software.

To measure RANKL-induced activation of the transcription factor NF{kappa}B, triplicate cultures of human M-CSF-dependent macrophages were treated for 30 min with 100 ng/ml RANKL in the absence or presence of 100 ng/ml CYR61. The level of NF{kappa}B (p65 subunit) in nuclear extracts, prepared using a commercial kit (Active Motif, Rixensort, Belgium), was then measured using a TransAM assay (Active Motif), according to the manufacturer’s instructions.

Detection of CYR61 expression
RT-PCR was used to assess the expression of CYR61 in TE85 osteoblast-like cells, human M-CSF-dependent macrophages, and human osteoclasts generated after culturing the macrophages for 6 d in M-CSF and RANKL. Total RNA was isolated using Trizol and cDNA was prepared from 2 µg total RNA using Superscript II reverse transcriptase (Invitrogen). For PCR analysis of CYR61 expression, the primers were 5'-caaccctttacaaggccaga-3' (forward) and 5'-tggtcttgctgcatttcttg-3' (reverse). GAPDH expression in each sample was assessed as a control using the primers 5'-gagtcaacggatttggtcgta-3' (forward) and 5'-gcagagatgatgacccttttg-3' (reverse). All PCRs were carried out using Taq polymerase (QIAGEN), and the cycling conditions were: 3 min at 94 C, 32 cycles of 30 sec at 94 C, 30 sec at 55 C, 30 sec at 72 C, and 5 min at 72 C. All products were electrophoresed on a 2% agarose gel containing 0.5 µg/ml ethidium bromide.

Statistical analysis
Data were analyzed using one-way ANOVA with Dunnett’s post hoc test. All analyses were performed on data pooled from at least three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CYR61 inhibits the formation of human, mouse, and rabbit osteoclasts in vitro
Cultures of M-CSF-dependent human macrophages formed large, multinucleated, TRAP-positive osteoclasts after 4 d or more in the presence of RANKL + M-CSF (Fig. 1AGo). These multinucleated cells did not form in the absence of exogenous RANKL. Addition of 25 ng/ml or more CYR61 at the start of the culture with RANKL caused a dramatic and concentration-dependent reduction in the number of multinucleated cells (MNCs) (Fig. 1Go, B and E). Because TRAP is a poor phenotypic marker of human osteoclasts generated in vitro, we also stained cells for {alpha}vß3. In control cultures (i.e. with RANKL + M-CSF), most cells were large, multinucleated, {alpha}vß3-positive osteoclasts (Fig. 1CGo). However, the addition of CYR61 to these cultures caused a significant, concentration-dependent decrease in the number of multinucleated, {alpha}vß3-positive osteoclasts (Fig. 1EGo, P < 0.001 for ≥ 25 ng/ml CYR61, three independent experiments), with only a few small, mononuclear {alpha}vß3-positive cells present (Fig. 1DGo) (these few {alpha}vß3-positive mononuclear cells were also present when human macrophages were cultured with M-CSF alone, i.e. in the absence of RANKL). Inhibition of osteoclast formation by CYR61 was not associated with increased morphological features of cell death (e.g. nuclear fragmentation) or any effect on the total cell viability of human monocytes using an Alamar Blue assay (data not shown). Furthermore, the purified IgG-Fc-domain alone had no effect on osteoclastogenesis (Fig. 1FGo); therefore, the inhibitory effect of the CYR61-Fc fusion protein was not due to the Fc tag.


Figure 1
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  		FIG. 1. CYR61 inhibits the formation of human osteoclasts in vitro. M-CSF-dependent human macrophages were cultured in 96-well plates (A–E) or 48-well plates (F) with M-CSF + RANKL in the absence (A and C) or presence (B and D) of 100 ng/ml CYR61 for 6 d and then fixed and stained for TRAP (A and B) or {alpha}vß3 (C and D). In cultures stained for {alpha}vß3, nuclei were counterstained with DAPI. CYR61 causes a concentration-dependent decrease in the number of multinucleated, {alpha}vß3-positive osteoclasts formed in the cultures (E), whereas the Fc tag alone has no effect (F). Data are the mean ± SD (n = 6 replicate wells) and are representative of three independent experiments.

 
Similar to the cultures of human monocytes described above, cultures of M-CSF-dependent, mouse bone marrow macrophages consisted entirely of numerous, multinucleated, strongly TRAP-positive osteoclasts and TRAP-positive mononuclear cells after 5 d treatment with RANKL + M-CSF (osteoclast formation did not occur in the absence of exogenous RANKL). Addition of 500 or 1000 ng/ml CYR61 at the start of the culture with RANKL caused a concentration-dependent decrease in the number of osteoclast-like, MNCs (Fig. 2Go, A and B). This decrease on MNC number was statistically significant in the presence of both 500 and 1000 ng/ml CYR61 (P < 0.001, three independent experiments). As with the cultures of human macrophages, mouse MNCs that were formed in the presence of CYR61 were smaller and less multinucleated than in control cultures. Notably, the presence of CYR61 also caused a very marked decrease in histochemical staining for TRAP in the entire culture (i.e. in both mononuclear cells and MNCs) (Fig. 2AGo). The latter effect was confirmed by measuring the level of TRAP activity in lysed cultures, because CYR61 caused a concentration-dependent decrease in total TRAP activity in the cultures after 5 d treatment (data not shown). Decreased expression of TRAP was confirmed by RT-PCR analysis of the bone marrow macrophage cultures after 3 d of CYR61 treatment (Fig. 2CGo). CYR61 also caused a decrease in the RANKL-induced expression of calcitonin receptor, cathepsin K, and MMP-9 (Fig. 2CGo). However, addition of CYR61 to the cultures immediately before the onset of cell fusion (usually the fourth day of incubation with RANKL + M-CSF) had little effect on the formation of osteoclast-like MNCs or on TRAP activity (Fig. 2BGo). Therefore, CYR61 did not appear to inhibit the fusion of differentiated, mononuclear osteoclast precursors.


Figure 2
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  		FIG. 2. CYR61 inhibits the formation of mouse osteoclasts in vitro. A, M-CSF-dependent mouse bone marrow macrophages were cultured with M-CSF + RANKL in the absence or presence of 500 or 1000 ng/ml CYR61 for 5 d and then fixed and stained for TRAP (arrows). B, CYR61 causes a concentration-dependent decrease in the number of multinucleated, TRAP-positive osteoclasts when added at the start of culture (d 0) but not when added to perfusion osteoclasts (d 4). Data are the mean ± SD (n = 6 replicate wells) and are representative of three independent experiments. C, CYR61 decreased expression of TRAP, MMP-9, CTR, and cathepsin K (Cat K) after 3 d culture with CYR61, determined by RT-PCR.

 
CYR61 also significantly inhibited (P < 0.001 for 500 and 1000 ng/ml CYR61, three independent experiments) the 1,25(OH)2D3-induced formation of osteoclast-like MNCs in cultures of rabbit bone marrow (Fig. 3Go, A and B). CYR61 also caused a marked decrease in the size and multinuclearity of the few rabbit MNCs that were able to form, and a marked decrease in histochemical TRAP staining of MNCs (Fig. 3AGo, arrows) and total TRAP enzyme activity (data not shown). Unlike in the cultures of human monocytes or murine bone marrow macrophages, the rabbit bone marrow cultures also contain a high proportion of stromal cells. Consistent with previous studies (14, 29), the presence of CYR61 appeared to stimulate the proliferation and/or survival of these cells, which were more numerous than in controls after 10 d culture (Fig. 3AGo).


Figure 3
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  		FIG. 3. CYR61 inhibits the formation, but not function, of rabbit osteoclasts in vitro. A, Rabbit bone marrow was cultured for 10 d with 1,25(OH)2D3 in the absence or presence of 500 or 1000 ng/ml CYR61 and then fixed and stained for TRAP (arrows). B, CYR61 causes a concentration-dependent decrease in the number of multinucleated, TRAP-positive osteoclasts formed in the cultures. C and D, Addition of CYR61 to cultures of mature osteoclasts does not decrease osteoclast number or inhibit bone resorption. Data are the mean ± SD (n = 6 replicate wells) and are representative of three independent experiments. CTL, Control.

 
CYR61 does not inhibit bone resorption by mature osteoclasts
When added to cultures of mature osteoclasts isolated from rabbit long bones, concentrations of 500 or 1000 ng/ml CYR61 had little effect on the number of TRAP-positive osteoclasts (Fig. 3CGo) or bone resorption (Fig. 3DGo). Similarly, addition of 100 ng/ml CYR61 to human osteoclasts (on d 6 of RANKL treatment) did not affect the number of osteoclasts or resorption area after a further 4 d culture (data not shown). Therefore, CYR61 does not appear to directly affect the resorptive activity or survival of mature osteoclasts.

CYR61 does not inhibit RANKL-induced activation of p38, ERK1/2, or NF{kappa}B in osteoclast precursors
As expected, addition of RANKL to cultures of M-CSF-dependent human macrophages caused the rapid phosphorylation of p38 and ERK1/2 and activation (i.e. nuclear accumulation) of NF{kappa}B (Fig. 4Go, A and B). Cotreatment of cells with 100 ng/ml CYR61 did not affect RANKL-induced activation of p38 or ERK1/2 (Fig. 4AGo) and did not inhibit RANKL-induced accumulation of nuclear NF{kappa}B (Fig. 4BGo). Furthermore, CYR61 alone had no effect on p38 or ERK1/2 phosphorylation or on NF{kappa}B activation. The same results were obtained using M-CSF-dependent mouse bone marrow macrophages (data not shown).


Figure 4
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  		FIG. 4. CYR61 does not inhibit RANK signaling. A, M-CSF-dependent human macrophages were treated with 100 ng/ml RANKL for 10 and 15 min and then analyzed by Western blotting for phosphorylated and total forms of p38 and ERK1/2. Data shown are representative of three independent experiments. B, M-CSF-dependent human macrophages were treated with 100 ng/ml RANKL alone or in the presence of 100 ng/ml CYR61 for 30 min and then NF{kappa}B was quantified in nuclear extracts. Values are mean ± SD (n = 3 replicate wells) and are representative of three independent experiments. CTL, Control.

 
CYR61 does not inhibit osteoclastogenesis via {alpha}vß3 or {alpha}vß5
A mutant form of CYR61 that lacks the ability to bind to {alpha}vß3 (30) (Asp125Ala substitution) was also tested for its ability to inhibit osteoclast formation in cultures of human M-CSF-dependent macrophages. The mutant form significantly (P < 0.001 for ≥ 25 ng/ml CYR61, three independent experiments) and concentration-dependently inhibited the formation of {alpha}vß3-positive cells (Fig. 5Go, A and B) and did not differ from wild-type CYR61 (Fig. 1Go), indicating that CYR61 does not appear to inhibit osteoclastogenesis by interfering with the function of {alpha}vß3. CYR61 also had a similar inhibitory effect on osteoclastogenesis in cultures of bone marrow macrophages from wild-type or ß5–/– mice (Fig. 5CGo), indicating that CYR61 does not appear to act via interaction with {alpha}vß5.


Figure 5
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  		FIG. 5. CYR61 does not inhibit osteoclastogenesis via {alpha}vß3 or {alpha}vß5. A, M-CSF-dependent human macrophages were cultured with M-CSF + RANKL in the absence or presence of 25–1000 ng/ml CYR61 or a mutant form (Asp125Ala, which lacks {alpha}vß3 binding) for 6 d and then fixed and stained for {alpha}vß3. Nuclei were counterstained with DAPI. B, The Asp125Ala mutant form of CYR61 causes a concentration-dependent decrease in the number of multinucleated, {alpha}vß33-positive osteoclasts formed in the cultures. Data are the mean ± SD (n = 6 replicate wells) and are representative of three independent experiments. CTL, Control. C, M-CSF-dependent bone marrow macrophages generated from ß5–/– mice were cultured with M-CSF + RANKL in the absence or presence of 50–1000 ng/ml CYR61 for 6 d and then fixed and stained for TRAP. CYR61 causes a similar decrease in the number of multinucleated, TRAP-positive osteoclasts formed in cultures of wild-type (solid line) or ß5 –/– (dashed line) cells. Data are the mean ± SD (n = 6 replicate wells) and are representative of two independent experiments.

 
CYR61 is not expressed in osteoclasts
When expression of CYR61 was examined by RT-PCR analysis of mRNA from human TE85 osteoblasts and human osteoclasts generated from MCSF-dependent macrophages, strong expression of CYR61 was detected in the osteoblasts (as expected) but was not detectable in the osteoclasts or macrophages (Fig. 6Go).


Figure 6
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  		FIG. 6. CYR61 is expressed by human osteoblasts but not osteoclasts. Total mRNA was extracted from human M-CSF-dependent monocyte/macrophages (Monocy), osteoclasts (Ocs), and human TE85 osteoblast-like cells (Obs) and then analyzed by RT-PCR for expression of CYR61. Data are representative of three independent experiments. CTL, Control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although several members of the CCN family (such as CTGF/CCN2 and CYR61/CCN1) appear to play a role in the proliferation and differentiation of osteoblasts (23, 29) and other mesenchymal cells (14), the effect of this family of proteins on osteoclasts has not been reported previously. Given that CYR61 can interact with the {alpha}vß3 integrin (14, 36), which is highly expressed on committed osteoclast precursors and mature osteoclasts, and that CYR61 can bind to a variety of growth factors and cytokines (14), we examined whether CYR61 could influence osteoclast formation or function. In this study we demonstrate that CYR61 inhibits the differentiation of hematopoietic osteoclast precursors in vitro and thereby prevents the formation of multinucleated osteoclasts.

In the three culture models examined (human M-CSF-dependent macrophages, mouse M-CSF-dependent bone marrow macrophages, and rabbit bone marrow), the formation of multinucleated osteoclast-like cells is dependent on the presence of the osteoclastogenic cytokine RANKL. This is added exogenously to human or mouse M-CSF-dependent osteoclast precursors, to form a relatively homogenous culture of {alpha}vß3-positive and TRAP-positive mononuclear and multinucleated cells. In cultures of rabbit bone marrow, osteoclast-like MNC formation is also dependent on RANKL, which is expressed on stromal cells in the culture and up-regulated by the addition of 1,25(OH)2D3 (37). The addition of CYR61 at the same time as addition of RANKL caused a striking inhibition of MNC formation and TRAP activity in all three culture models. When added to mouse bone marrow macrophage-derived osteoclast precursors at the terminal stages of differentiation, but before cell fusion, CYR61 did not prevent the formation of TRAP-positive, osteoclast-like MNCs. CYR61 therefore appears to inhibit the early stages of differentiation of osteoclast precursors into TRAP-positive cells. This is supported by the finding that CYR61 markedly decreased the RANKL-induced expression of phenotypic markers of osteoclasts (TRAP, cathepsin K, MMP-9, and calcitonin receptor) in mouse M-CSF-dependent bone marrow macrophages and dramatically decreased the RANKL-induced expression of {alpha}vß3 by human M-CSF-dependent macrophages.

CYR61 is known to bind to {alpha}vß3 integrin (14, 17, 36), which is highly expressed on osteoclast precursors and mature osteoclasts and is required for cell fusion and bone resorption (10, 38). Binding of {alpha}vß3 to osteopontin causes robust activation of ERK1/2 and cytoskeletal rearrangement in osteoclasts that promote osteoclast cell attachment and bone resorption (11, 39). Furthermore, {alpha}vß3 appears to cooperate with the M-CSF receptor for activation of ERK signaling required for osteoclastogenesis, and the defect in osteoclast formation from ß3-null bone marrow macrophages in vitro can be overcome by addition of high concentrations of M-CSF (12). Inhibition of {alpha}vß3 function might therefore account for the effects of CYR61 on osteoclastogenesis. However, the fact that CYR61 did not directly prevent the fusion of mouse osteoclast precursors and did not affect resorption by rabbit or human osteoclasts suggests that simple interference with the ligand-binding function of {alpha}vß3 integrin is unlikely. Also, addition of high concentrations (up to 500 ng/ml) of M-CSF could not overcome the inhibitory effect of CYR61 on osteoclastogenesis in cultures of mouse bone marrow macrophages or human macrophages (data not shown). Importantly, a mutant form of soluble CYR61, defective in the ability to bind {alpha}vß3 (30) (due to substitution of Asp125 for Ala in domain II of CYR61), still inhibited the formation of human osteoclasts just as effectively as wild-type CYR61. CYR61 is also known to interact with {alpha}vß5 (19, 20), which is highly abundant on osteoclast precursors (13). However, CYR61 had a similar inhibitory effect on osteoclast formation in cultures of bone marrow macrophages from wild-type and ß5–/– mice. Together, these observations demonstrate that CYR61 appears to inhibit osteoclastogenesis by a mechanism independent of {alpha}vß3 or {alpha}vß5.

To determine whether CYR61 prevents osteoclastogenesis by interfering with RANKL/RANK signaling, we examined the effect of CYR61 on signaling pathways downstream of RANK. CYR61 did not affect RANKL-induced activation of p38 or ERK1/2, and did not affect the nuclear translocation of the transcription factor NF{kappa}B, which is known to be critical for osteoclast formation (3). This suggests that CYR61, although capable of binding some growth factors, does not inhibit osteoclastogenesis by preventing the binding of RANKL to its cognate receptor, RANK, or by preventing RANK signaling but may activate signaling pathways that have a dominant-negative effect on osteoclastogenesis. Alternatively, CYR61 may inhibit costimulatory pathways required for efficient osteoclast differentiation (40, 41). The exact mechanism by which CYR61 inhibits osteoclast differentiation therefore remains to be determined.

CYR61 appears therefore to be a newly identified regulator of osteoclast formation. Because CYR61 is expressed and secreted by osteoblasts and mesenchymal stem cells (23, 24, 26), it may be involved in cross-talk between osteoblasts and osteoclasts in the local regulation of bone remodeling. This may be particularly relevant in conditions favoring enhanced bone formation such as fracture-healing (28) because CYR61 inhibits the formation of bone-resorbing osteoclasts but stimulates the proliferation and differentiation of bone-forming osteoblasts (23, 29). Furthermore, we show that CYR61 is not expressed by human osteoclasts or their monocytic precursors; therefore, CYR61 probably plays a paracrine role in the bone microenvironment as a negative regulator of osteoclastogenesis that is produced predominantly by osteoblasts and other mesenchymal cells.

Because CYR61 is expressed, and presumably secreted, by a variety of tumor cell types that metastasize to bone [including breast and prostate cancer cells (42, 43, 44, 45)], CYR61 could play a role in the alteration of bone cell activity that occurs in tumor-induced bone disease (46). In particular, given that CYR61 inhibits osteoclast formation but is up-regulated in osteoblasts in response to growth factors and cytokines and promotes osteoblast differentiation and function (23, 26, 29), it is tempting to speculate that CYR61 may be an important cause of the enhanced bone formation and predominantly osteosclerotic lesions that are characteristic of prostate cancer metastasis to the skeleton (47).


   Acknowledgments
 
We are very grateful to Dr. Silvia Finneman (Cornell University, New York, NY) and Professor Dean Sheppard (University of California, San Francisco, San Francisco, CA) for providing bones from ß5–/– mice and their wild-type littermates.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online September 6, 2007

1 J.C.C. and N.S. contributed equally to the study. Back

Abbreviations: CCN, CYR61/CTGF/NOV; CTR, calcitonin receptor; CYR61, cysteine-rich protein 61; DAPI, 4',6'-diamino-2-phenylindole; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M-CSF, macrophage-colony stimulating factor; MMP, matrix metalloproteinase; MNC, multinucleated cell; NF{kappa}B, nuclear factor-{kappa}B; 1,25-(OH)2D3, 1{alpha},25-dihydroxyvitamin D3; RANK, receptor activator of NF{kappa}B; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; WISP, Wnt-induced secreted protein.

Received April 12, 2007.

Accepted for publication August 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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