Dermatan sulfate inhibits osteoclast formation by binding to receptor activator of NF-κB ligand

Biochemical and Biophysical Research Communications 354 (2007) 447–452 www.elsevier.com/locate/ybbrc

Dermatan sulfate inhibits osteoclast formation by binding to receptor activator of NF-jB ligand Kouhei Shinmyouzu a, Tetsu Takahashi a, Wataru Ariyoshi a, Hisashi Ichimiya a, Shin Kanzaki a, Tatsuji Nishihara b,* a

b

Division of Oral and Maxillofacial Reconstructive Surgery, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580, Japan Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan Received 14 December 2006 Available online 10 January 2007

Abstract Dermatan sulfate (DS) is a major component of extracellular matrices in mammalian tissues. In the present study, DS demonstrated a high level of binding activity to receptor activator of NF-jB ligand (RANKL) and obstructed the binding of RANK to RANKL, determined using a quartz-crystal microbalance (QCM) technique. Further, when mouse bone marrow cells were cultured with RANKL and macrophage colony-stimulating factor, DS suppressed tartrate-resistant acid phosphatase-positive multinucleated cell formation in a dose-dependent manner. In addition, immunoblot analyses revealed that DS reduced the levels of phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase protein in mouse osteoclast progenitor cells stimulated with RANKL. Together, these results indicate that DS regulates osteoclast formation through binding to RANKL and inhibition of signal transduction in osteoclast progenitor cells, suggesting that it has an important role in bone metabolism in pathological conditions.  2007 Elsevier Inc. All rights reserved. Keywords: Dermatan sulfate; Glycosaminoglycan; Osteoclast; Receptor activator of NF-jB ligand; Macrophage colony-stimulating factor; Quartz-crystal microbalance technique

Glycosaminoglycans (GAGs), acidic polysaccharide complexes involved in a variety of physiological conditions, are long-chain compounds composed of repeating disaccharide units with a carboxyl group and one or more sulfates, in which one sugar is N-acetylgalactosamine or N-acetylglucosamine. Recently, the potential roles of GAGs in some biological processes [1–4], including angiogenesis [5], viral invasion [6–8], tumor growth [9–11], and bone metabolism [12–14], have been reported. Dermatan sulfate (DS) is an endogenous GAG, with repeating disaccharide units of N-acetylglucosamine and glucuronic acid. It is abundant in mammalian tissues and present in high concentrations in connective tissues, such as the vitre*

Corresponding author. Fax: +81 93 581 4984. E-mail address: [email protected] (T. Nishihara).

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.12.221

ous humor of the eye and joint cartilage [15]. It has also been reported that DS has biological effects on bone metabolism [16], cell adhesion [17,18], homing of lymphocytes [19,20], and development of the central nervous system [21]. The receptor activator of NF-jB ligand (RANKL), a cognate ligand for receptor activator of NF-jB (RANK), is expressed on osteoblastic cells and plays an important role in osteoclast differentiation [22]. RANK is also expressed on osteoclasts, and its progenitors regulate bone mass and calcium metabolism by resorbing bone. It is important to note that some sulfated GAGs have been shown to prolong the half-life of RANKL through its inhibition of osteoprotegerin binding to the RANKL–RANK complex and mediate the functions of osteoclasts [23]. Although DS is known to bind to proteins, as well as play important roles with cell to cell interactions and

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subsequent biological reactions [17–20], there are no known reports regarding the effects of DS on bone resorption. In the present study, we examined the effects of DS on osteoclast formation and attempted to elucidate the precise mechanism by which DS regulates osteoclast differentiation in vitro.

deviation of three examinations, with similar results obtained in each experiment.

Materials and methods

To determine the interaction of DS and RANKL, we examined the affinity between DS and RANKL using a QCM technique. When DS (300 lg/ml; volume 100 ll) was injected into the equilibrated solution containing a RANKL-immobilized sensor chip at 25 C, the frequency decreased by 60 Hz, while the frequency decreased by 150 Hz, when RANK (100 lg/ml; volume 30 ll) was injected (Fig. 1A). In contrast no frequency decrease was observed when DS (300 lg/ml; volume 100 ll) was injected into the equilibrated solution containing an M-CSF-immobilized sensor chip at 25 C. For these experiments, we used c-fms, which is known to bind to M-CSF as a specific ligand. When c-fms (10 lg/ml; volume 100 ll) was injected, the frequency decreased by 160 Hz (Fig. 1B). Next, we examined the competitive ability of DS with RANK

Reagents. DS was kindly supplied by Seikagaku Corporation (Tokyo, Japan). Human recombinant RANKL was purchased from Pepro Tech EC, Ltd. (London, UK). Human recombinant RANK was purchased from R&D Systems Inc. (Minneapolis, MN, USA). Human M-CSF was purchased from Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan), and human c-fms from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p38 mitogen-activated protein kinase (MAPK), anti-phosphorylated p38 MAPK, anti-extracellular signal-regulated kinase (ERK), and antiphosphorylated ERK polyclonal antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, CA, USA). Cell culture. Mouse bone marrow cells were prepared by flushing the marrow cavities of femora and tibiae from 6-week-old female ddY mice (Kyudo Co., Ltd., Fukuoka, Japan). The cells were centrifuged and resuspended in a-minimal essential medium (a-MEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) with penicillin G (100 U/ml) and streptomycin (100 lg/ml). Mouse monocytic RAW 264.7 cells (ATCC TIB71) were cultured in a-MEM supplemented with 10% FBS and antibiotics. Kinetic analysis using quartz-crystal microbalance (QCM). A 27-MHz QCM (AfinixQ; Initium Inc., Tokyo, Japan) was employed to analyze the affinity of RANKL and DS. RANKL (2 ll; 10 11 M) was immobilized directly on the gold electrode surface of the QCM ceramic sensor chip, after which the sensor chip was soaked in a chamber containing 10 ml of phosphate-buffered saline (PBS, pH 7.2) at 25 C until frequency equilibrium was attained. DS (300 lg/ml; volume 100 ll) was applied into the equilibrated solution containing the RANKL-immobilized sensor chip. The binding of DS to RANKL was determined by monitoring the alterations in frequency resulting from changes in mass on the electrode surface. In some experiments, the affinity of M-CSF (2 ll; 10 11 M) and DS were examined in the manner described above. Western blot analysis. RAW 264.7 cells (1 · 105 cells/well) were cultured in 6-well plates in a-MEM containing 10% FBS in the presence or absence of RANKL (40 ng/ml) and DS (300 lg/ml). The cells were washed twice with PBS and lysed in lysis buffer (75 mM Tris–HCl containing 2% SDS and 10% glycerol, pH 6.8). Protein contents were measured using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). The samples were then subjected to 10% SDS–PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). Non-specific binding sites were blocked by immersing the membrane in 10% skimmilk in PBS for 1 h at room temperature, after which the membrane was washed four times with PBS, followed by incubation with the diluted primary antibody for 2 h at room temperature. After washing the membrane, chemiluminescence was produced using ECL reagent (Amersham Pharmacia Biotech, Uppsala, Sweden) and detected with Hyperfilm-ECL (Amersham Pharmacia Biotech). To confirm that equal loading had occurred, the membranes were stained with Coomassie brilliant blue G-250 after exposure to film. Differentiation of osteoclasts. Mouse bone marrow cells were cultured in 24-well plates (5 · 106 cells/well) with RANKL (40 ng/ml) and M-CSF (50 ng/ml) in the presence of DS (300 lg/ml). The cells were fixed and stained with tartrate-resistant acid phosphatase (TRAP) (Sigma Chemical Co., St. Louis, MO, USA) [24]. TRAP-positive multinucleated cells containing three or more nuclei were considered to be osteoclasts and counted under a microscope. Statistical analysis. Statistical differences were determined using an unpaired Student’s t-test. All data are expressed as means ± standard

Results DS binds to RANKL and blocks binding of RANK to RANKL

Fig. 1. RANKL (2 ll; 10 11 M) and M-CSF (2 ll; 10 11 M) were immobilized directly on a QCM ceramic sensor chip soaked in PBS solution at 25 C, as described in Materials and methods. DS (300 lg/ml; volume 100 ll), RANK (100 lg/ml; volume 30 ll), and c-fms (10 lg/ml; volume 100 ll) were applied separately to the equilibrated solution. (A) Binding ability between RANKL on the sensor chip and PBS (a), DS (b), and RANK (c). (B) Binding ability between M-CSF on the sensor chip and PBS (a), DS (b), and c-fms (c).

K. Shinmyouzu et al. / Biochemical and Biophysical Research Communications 354 (2007) 447–452

Fig. 2. To examine the competitive ability of DS against RANKL– RANK binding, RANKL (2 ll; 10 11 M) was immobilized on a QCM ceramic sensor chip soaked in a PBS solution at 25 C as described in Materials and methods, after which DS (300 lg/ml; volume 100 ll) or RANK (100 lg/ml; volume 30 ll) was injected into the solution. (A) DS was injected into the solution ( ), then RANK was injected after the solution was equilibrated ( ). (B) RANK was injected into the solution ( ), and then DS was injected after the solution was equilibrated ( ).

through binding to RANKL. Following the binding of DS to RANKL, RANK was unable to bind to RANKL (Fig. 2A). Further, after RANK was bound to RANKL, DS did not bind to RANKL (Fig. 2B). Effect of DS on phosphorylation of signaling molecules involved osteoclast differentiation To examine the effect of DS on MAPK signal transduction in the process of osteoclast differentiation, we used a homogenous clonal population of murine monocytic RAW 264.7 cells. When cultured with RANKL, this cell line is known to express RANK and differentiate into TRAP-positive cells. In the present study, immunoblot analysis detected phosphorylated p38 MAPK within 15 min after the addition of RANKL (40 ng/ml) and the expression reached a plateau by 30 min. When the cells were incubated with both RANKL and DS (300 lg/ml), the level of p38 MAPK phosphorylation was lower than that in cells treated with RANKL alone. In contrast, the total amounts of p38 MAPK were not affected by treatment with RANKL and DS (Fig. 3A). We also examined the effects of DS on the phosphorylation of ERK in osteo-

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Fig. 3. DS suppresses the phosphorylation of p38 MAPK and ERK in RAW 264.7 cells stimulated with RANKL. RAW 264.7 cells (1 · 105 cells/ well) were stimulated with RANKL (40 ng/ml) in the presence or absence of DS (300 lg/ml) for the indicated times, after which whole cell lysates were subjected to immunoblotting analyses. (A) Expression of p38 MAPK and phosphorylated p38 MAPK. (B) Expression of ERK and phosphorylated ERK. Lane 1, RANKL (0 ng/ml). Lane 2, RANKL (40 ng/ml). Lane 3, RANKL (40 ng/ml) and DS (300 lg/ml).

clast progenitors. Fig. 3B shows the time course of changes in phosphorylation levels of ERK in RAW 264.7 cells. ERK was phosphorylated within 30 min in response to RANKL, and phosphorylation reached a maximum level within 60 min, while DS (300 lg/ml) reduced the level of RANKL-induced phosphorylation of ERK. However, DS had no effect on the total amount of ERK protein in RAW 264.7 cells stimulated with RANKL. DS inhibits osteoclast formation in mouse bone marrow cultures We also investigated the effects of DS on osteoclast differentiation in mouse bone marrow cultures. When mouse bone marrow cells were cultured with RANKL (40 ng/ml) and M-CSF (50 ng/ml) for 8 days, DS (300 lg/ml) remarkably reduced the number of osteoclasts formed (Fig. 4). Discussion Bone remodeling is regulated by numerous parameters including cytokines, growth factors, and extracellular matrix components, which are associated with the cell membrane. Recently, GAGs were found to have several kinds of biological activities by kinetic analyses

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Fig. 4. DS inhibits osteoclast formation in bone marrow cultures. Mouse bone marrow cells (5 · 106 cells/well) were stimulated with RANKL (40 ng/ml) and M-CSF (50 ng/ml) in the presence or absence of DS (300 lg/ml) for 8 days. (A) Images showing osteoclast formation (magnification, 20·). (B) The number of osteoclasts was counted after TRAP staining. Data are expressed as means ± standard deviation of triplicate cultures. The experiment was performed three times, with similar results obtained.

[17,18,25], while other reports have shown that numerous proteins containing a heparin-binding domain interact specifically with GAGs [26–30]. The interactions of these parameters with GAGs cause a modification of their biological activities and properties. It has also been reported that, heparin bound to bone morphogenetic protein to regulate bone formation [31], and that low molecular weight hyaluronic acid (HA) enhanced both osteoclast formation and function [32]. Although, DS is rich in bone and the cartilage matrix, and clinically used to prevent cartilage resorption in joint lesions, there are few reports regarding the binding of DS to bone metabolism-related cytokines. Further, the exact mechanisms by which DS causes such profound effects and its interactions with each protein remain unknown.

It is important to note that GAGs, such as heparin, HA, and DS, are abundant in joint synovial fluids, suggesting a critical role of DS in bone metabolism in joint lesions [33]. Recently, Theoleyre et al. reported that heparin binds to osteoprotegerin (OPG), a decoy receptor for RANKL, with a high-affinity and that preincubation of OPG with heparin inhibited the binding of OPG to the RANKL– RANK complex [23]. Further, our research group found that high sulfated GAGs such as heparin and chondroitin sulfate E inhibited osteoclast formation and function (data not shown). In the present study, we showed that DS strongly bound to RANKL and completely inhibited RANK binding to RANKL, and subsequently suppressed osteoclast formation. These findings suggest that the sulfated portion of GAGs, such as heparin, heparan sulfate,

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chondroitin sulfate E, and DS, may recognize and bind to osteoclastogenesis-related proteins, as previously described by Gange et al. [25]. Studies are under way to clarify the mechanisms of binding between GAGs and RANKL. MAPK family members are proline-directed serine/threonine kinases that play important roles in cell growth, differentiation, and apoptosis [34–37]. Some external stimuli activate the phosphorylation of threonine and tyrosine [38,39]. MAPK family members are classified into the ERK and p38 MAPK groups, and it is widely accepted that peptide growth factors and phorbol esters preferentially activate ERK [38–40]. Further, there are some studies that found that RANKL–RANK binding causes the phosphorylation of p38 MAPK and ERK, and that such phosphorylation leads to osteoclast differentiation [32,41]. In the present study, we used a homogenous clonal population of murine monocytic RAW 264.7 cells to clarify the effects of DS on the signaling pathway in osteoclast progenitor cells, and found that DS strongly inhibited the phosphorylation of p38 MAPK and ERK in RAW 264.7 cells stimulated with RANKL (Fig. 3). These findings suggest that the inhibitory effect of DS on osteoclast differentiation into mature osteoclasts is also responsible for the regulation of phosphorylation of p38 MAPK and ERK. Further, when mouse bone marrow cells were cultured for 8 days in the presence of both RANKL and M-CSF, DS significantly reduced osteoclast formation (Fig. 4). Based on our findings, we speculated that this phenomenon was caused by the inhibitory effect of DS on RANK binding to RANKL and subsequent phosphorylation of MAPK signal transduction. In summary, the present results demonstrated that DS inhibits the binding of RANK to RANKL and the subsequent phosphorylation of MAPK signal transduction. In addition, DS suppressed osteoclast formation induced by RANKL and M-CSF in mouse bone marrow cultures. These findings suggest that DS plays an important role in bone and cartilage tissues in both physiological and pathological conditions. Acknowledgments The authors thank Seikagaku Corporation, Japan, for their generous gift of DS for use in this study. This work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] U. Lindahl, ‘Heparin’—from anticoagulant drug into the new biology, Glycoconj. J. 17 (2000) 597–605. [2] R. Sasisekharan, G. Venkataraman, Heparin and heparan sulfate: biosynthesis, structure and function, Curr. Opin. Chem. Biol. 4 (2000) 626–631. [3] B. Casu, U. Lindahl, Structure and biological interactions of heparin and heparan sulfate, Adv. Carbohydr. Chem. Biochem. 57 (2001) 159–206.

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