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Division of General Internal Medicine, Department of Internal Medicine, Medical University of Innsbruck, A-6020 Innsbruck, Austria
Address all correspondence and requests for reprints to: Professor Dr. Christian J. Wiedermann, Department of Internal Medicine, Medical University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail: christian.wiedermann{at}uibk.ac.at.
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Abstract |
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Data suggest that osteoprotegerin affects monocyte mi-gration and protein kinase C and phosphatidylinositol 3-kinase/Akt activation via syndecan-1. Osteoprotegerin-induced deactivation of monocyte chemotaxis toward different chemokines is due to interaction of osteoprotegerin with heparan sulfate and chondroitin sulfate.
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Introduction |
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B ligand (RANKL), its receptor RANK, and osteoprotegerin (OPG) (2, 3, 4, 5). Serum levels of OPG are elevated in patients with chronic inflammatory diseases (6, 7, 8, 9). OPG, a member of the TNF receptor superfamily, is a soluble decoy receptor that binds RANKL and thereby prevents interaction of RANKL with its receptor RANK (10). In addition to bone metabolism, RANKL and OPG play a role in the immune and vascular system (3, 10, 11, 12). Moreover OPG-deficient mice not only suffer from osteoporosis but also show arterial calcification (13). In men, OPG could be detected in the regions of Möncksberg’s sclerosis and in atherosclerotic tissue samples but not in control arteries (14). Recent data suggest that OPG is not only a modulator of RANKL but also may have RANKL-independent effects on leukocytes (15, 16, 17). Yet mechanisms involved in this direct cellular activity of OPG have not been elucidated.
Heparan sulfate proteoglycans (HSPGs) are important participants in cell-surface signaling and critical in controlling cell behavior. They are involved in actin cytoskeleton regulation, cell adhesion and migration, and modulation of specific receptor interactions (18). At the cell surface, proteoglycans of the syndecan family are the major source of heparan sulfates (19). OPG has a highly basic heparin-binding domain (20), making interactions with heparin and heparan sulfates possible.
In this study, we investigated the effects of recombinant human OPG on the chemotaxis of human monocytes and the possible involvement of the heparan sulfate proteoglycans in OPG-induced monocyte migration. Furthermore, we report the activation of phosphatidylinositol 3-kinase (PI3K)/Akt, protein kinase C (PKC), and tyrosine kinases by OPG in these cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All stock solutions were stored at –20 C before use. RPMI 1640 with phenol red was purchased from Biological Industries (Kibbutz Beit Hemeek, Israel). BSA was from Dade Behring (Marburg, Germany). Dextran, staurosporine, isobutylmethylxanthine (IBMX), wortmannin, tyrphostin 23, heparinase I, chondroitinase ABC, and Triton X-100 were from Sigma Chemical (St. Louis, MO). Bisindolylmaleimide I GF 109203X (GFX) was from Boehringer Ingelheim KG (Ingelheim am Rhein, Germany). Lymphoprep was from Nycomed Pharma AS (Oslo, Norway) and Hanks’ balanced salt solution (HBSS) without phenol red from Invitrogen (Carlsbad, CA). Magnetic-activated cell sorting (MACS) separation columns and microbeads were from Miltenyi Biotech (Auburn, CA). The microchemotaxis chambers were from Neuroprobe (Bethesda, MD), cellulose nitrate filters were from Sartorius (Göttingen, Germany). Syndecan antibodies (5G9 and DL-101) were from Santa Cruz Inc. (Wiltshire, UK). Phospho-PKC (pan) (Thr514), phospho-Akt (Ser473), phospho-Src (Tyr416), and phospho-Src (Tyr527) antibodies were from Cell Signaling (Beverly, CA). Biotinylated mouse antimouse IgG and IgG1 were from eBioscience (San Diego, CA). Streptavidin-PE was from Becton Dickinson (San Jose, CA). Hot Star Taq polymerase was purchased from QIAGEN Inc. (Valencia, CA). Primers were from MWG Biotech (Ebersdorf, Germany). Certified PCR agarose was from Bio-Rad Laboratories (Hercules, CA). PP1, PP2, PP3, and Tween 20 were from Calbiochem (San Diego, CA). Hybond-P membrane was from Amersham Biosciences (Little Chalfont, UK). Regulated upon activation normal T cell expressed and secreted (RANTES) was from PeproTech EC Ltd. (London, UK). Recombinant human procalcitonin (PCT) was from Brahms Diagnostica (Berlin, Germany). Monocyte chemotactic protein-1 (MCP-1) and recombinant human OPG were from R&D Systems (Minneapolis, MN). PAGEr duramide gels were from Cambrex (Rockland, ME).
Preparation of human monocytes
Monocytes were obtained from the peripheral blood of healthy donors (anticoagulated with EDTA). After Lymphoprep density gradient centrifugation, peripheral blood mononuclear cells were collected and washed twice with HBSS. Positive selection of CD14+ monocytes was performed by adding MACS colloidal superparamagnetic microbeads conjugated with monoclonal antihuman CD14 antibodies to cooled, freshly prepared peripheral blood mononuclear cell preparations in MACS buffer (PBS with 5 mM of EDTA and 0.5% BSA) according to the manufacturer’s instructions. Cells and microbeads were incubated for 15 min at 4–6 C. In the meantime the separation column was flushed with MACS buffer at room temperature. The cells were washed with MACS buffer, resuspended, and loaded at the top of the separation column. The eluent containing CD14– cells was withdraw, and after removal of the column from the magnet, trapped CD14+ monocytes were eluted with the 6-fold amount of cold MACS buffer, centrifuged, and resuspended in medium containing 0.5% BSA. By immunocytochemistry, preparations yielded a purity of approximately 98% (21).
Monocyte migration assay
Migration assays were performed by using a modified 48-well Boyden microchemotaxis chamber (Neuroprobe) in which a 5-µm-pore-size cellulose nitrate filter separated the upper and the lower chambers. Monocytes were resuspended in RPMI 1640/0.5% BSA (1 x 106 cells/ml). Fifty microliters of the cell suspension was placed into the upper chamber, and monocytes were allowed to migrate toward various soluble chemoattractants placed in the lower chamber for 90 min at 37 C in a humidified atmosphere (5% CO2). After the migration period, the nitrocellulose filters were dehydrated, fixed, and stained with hematoxylin. Migration depth of the cells into the filters was quantified by means of microscopy, measuring the distance (micrometers) from the surface of the filter to the leading front of three cells. Data are expressed as a chemotaxis index, which is the ratio between the distance of directed and random migration of monocytes into the nitrocellulose filters.
To test whether OPG itself has chemotactic properties on monocytes, different concentrations of OPG ranging from 1 aM up to 10 nM were added to the lower chamber as chemoattractant. For deactivation experiments, cells were incubated for 30 min with different concentrations of OPG (10 fM to 10 nM), washed twice with HBSS before testing chemotaxis toward RANTES (1.3 nM), MCP-1 (10 nM), or PCT (0.1 nM).
In other experiments, cells were pretreated with either heparinase I, an enzyme that cleaves highly sulfated regions of heparan-sulfate-like glycosaminoglycans (GAGs) at 2–0 sulfated uronic acids (22), or chondroitinase ABC, which cleaves chondroitin sulfate side chains of cell surface GAGs, for 30 min. Furthermore, monocytes were incubated with antibodies to syndecan-1 (DL-101) and -4 ectodomains for 15 min. After washing direct chemotaxis and deactivation experiments were performed.
Intracellular signaling of OPG on monocytes was tested by preincubation of the cells with the intracellular enzyme blockers staurosporine (10 ng/ml) (from streptomyces sp.); GFX (500 nmol/liter); wortmannin (10 nmol/liter) (from penicillium fumiculosum); IBMX (100 µM); tyrphostin 23 (10ng/ml); PP1 (1 µM), which is a selective inhibitor of Src kinases; PP2 (1 µM), which acts similarly to PP1 and specifically inhibits the tyrosine kinases lck, fyn, and hck; and PP3 (1 µM, which served as a negative control for 30 min at 37 C in a humidified atmosphere (5% CO2). The cells were then washed twice, resuspended in RPMI 1640/0.5% BSA and tested in the migration assay toward OPG (100 pM).
Checkerboard analysis
To ensure that the effect observed was true chemotaxis, checkerboard analyses were performed. Monocytes were resuspended in RPMI 1640/0.5% BSA containing various concentrations of OPG just before they were transferred to the upper chamber. The same concentrations of OPG remained beneath the filter of the Boyden chamber; thus, distinct concentration gradients could be formed. Data are expressed as chemotaxis index within a matrix.
RT-PCR
Total RNA was isolated from 107 cells by an acid guanidinium thiocyanate-phenol-chloroform mixture. A reverse transcriptase reaction was performed on 1 µg of RNA using random hexamers reverse transcriptase. One microgram of the resulting cDNA was then subjected to 35 cycles of PCR in a 50-µl reaction mixture containing 1 pmol of sense and antisense primer pairs in a thermocycler (Biometra, Göttingen, Germany): 95 C for 60 sec (denaturation), 56.4 C for 60 sec (annealing), and 72 C for 60 sec (extension). Primers were designed to amplify a 342-bp coding sequence of human syndecan-1. The sense primer sequence was 5'-CCC CAT CTT GCT TCC CTA ATC-3'. The antisense primer sequence was 5'-TCC CAG CAT TCA CTT CTC AC-3'. The PCR products were subjected to agarose gel analysis.
FACS analysis
Fluorometric analysis for syndecan expression on the cell surface of monocytes was performed. A total of 5 x 105 cells were washed twice in Dulbecco’s PBS containing 0.5% BSA and incubated with 150 µg/ml human IgG for 20 min at 4 C. After pelleting, cells were incubated with 10 µg/ml of mouse antihuman syndecan-1 or the respective isotype matched control mouse IgG (eBioscience) for 30 min at 4 C. After washing, 10 µg/ml were incubated for another 30 min. Cells were washed twice and the monocytes were subsequently incubated with a 1:25 dilution of streptavidin-PE for 30 min, washed twice, then immediately analyzed on a FACScan (Becton Dickinson) with CellQuest software (BD Biosciences, San Diego, CA).
Western blot analysis
Cells were either pretreated with OPG at various concentrations (0.1 fM to 10 nM) or coincubated with anti-syndecan-1 (2 µg/ml) and OPG (100 pM) for 15 min. Cells were lysed in lysis buffer containing 1% Triton X-100. Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gels and blotted onto polyvinyl difluoride membranes, which were blocked with 2% phophatase-free milk powder in Tris-buffered saline with 0.1% Tween 20. The antibodies were then diluted according to the manufacturer’s instructions, and blots were incubated overnight at room temperature. Immunoreactivity was determined using peroxidase-conjugated goat antirabbit, respectively, goat antimouse IgG and Super Signal chemiluminescent substrate (Pierce, Rockford, IL). Intensity of the Western blot bands was quantified using the Fluor-S MultiImager system and the Quantity One software (Bio-Rad Laboratories).
Statistical methods
Data are expressed as mean ± SEM. Means were compared by the Mann-Whitney U test and Kruskal-Wallis ANOVA. A difference with P < 0.05 was considered to be significant. Statistical analyses were performed using the StatView software package (Abacus Concepts, Berkeley, CA).
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Results |
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To explore for chemotactic properties of OPG in the absence of other chemoattractants, freshly prepared monocytes were allowed to migrate toward different concentrations of OPG (1 aM to 10 nM). Concentrations ranging from 10 fM to 10 nM of OPG significantly increased in vitro migration in a dose-dependent manner (Fig. 1
). OPG at a concentration of 100 pM was maximally stimulating.
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To investigate the role of intact HSPGs on the cell surface of monocytes for OPG-induced chemotaxis, monocytes were pretreated with heparinase I (50 nU/ml to 50 mU/ml) or chondroitinase (50 nU/ml to 50 mU/ml). Because glycipans carry heparan sulfate but not chondroitin sulfate side chains, whereas syndecans carry both (23), experiments were performed with heparinase I and chondroitinase to differentiate between the two HSPGs. Chemotactic effects of OPG (100 pM) were found to be significantly decreased in a dose-dependent manner by pretreatment with both heparinase I and chondroitinase (Fig. 2), thus suggesting syndecan involvement. Deactivation of migration also occurred when experiments were performed at 4 C (on ice). It is known that both, syndecan-1 and -4 are involved in cell migration (18) and expression of syndecan-4 in human monocytes has been recently reported (24). RT-PCR and FACS analyses revealed that monocytes have the ability to express syndecan-1 (Fig. 3). Assessment of syndecan surface expression by FACS showed that unstimulated monocytes have higher specific immunofluorescence activity for syndecan-1 than syndecan-4. To elucidate which of these syndecans are involved in the OPG-mediated cell migration, chemotaxis experiments toward OPG were performed using antibodies to the ectodomains of syndecan-1 or -4. Cells were pretreated with the two antibodies or isotype-matched IgG (2 ng/ml to 2 µg/ml) and then allowed to migrate toward OPG (100 pM). A specific antibody binding to the syndecan-1 but not to the syndecan-4 ectodomain inhibited directed migration of monocytes toward OPG in a dose-dependent manner (Fig. 4A). To rule out that syndecan-1 antibodies indirectly inhibit monocyte motility by inducing a signal that generally inhibits cell migration, we tested whether syndecan-1 antibodies (2 ng/ml to 2 µg/ml) influence monocyte migration toward RANTES (1.3 nM) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (10 nM). RANTES- as well as fMLP-induced chemotaxis was not significantly influenced by pretreatment with syndecan-1 antibodies (Fig. 4B).
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Blocking of intracellular signaling enzymes in OPG-induced chemotaxis of monocytes
To elucidate signaling pathways involved in transmitting OPG effects in monocyte migration, different intracellular enzyme blockers staurosporine, GFX, IBMX, wortmannin, tyrphostin 23, PP1, PP2, and PP3 were used at established signaling blocking concentrations. Staurosporine, which is a nonspecific inhibitor of PKC that also affects protein kinase A signaling, the specific PKC inhibitor GFX, wortmannin, a specific inhibitor of PI3K, and tyrphostin 23, a tyrosine kinase inhibitor, significantly decreased OPG-induced chemotaxis in monocytes. The phosphodiesterase inhibitor IBMX and the inhibitors of Src tyrosine kinases PP1 and PP2 as well as PP3, which served as negative control, had no effect on OPG-induced monocyte migration (Table 2).
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To investigate whether treatment of monocytes with OPG affects Akt, PKC, and Src-kinase activity, Western blot analyses were performed. Monocytes were incubated with different concentrations of OPG (0.1 fM to 10 nM) for 15 min. Using antibodies against phospho-Akt, phospho-PKC, and phospho-Src, a concentration-dependent increase of phospho-Akt and phospho-PKC could be detected in response to OPG, whereas OPG stimulation influenced neither Tyr416 nor Tyr527 phosphorylation of Src-kinases. To confirm syndecan involvement also in OPG-induced signaling, monocytes were incubated with the optimal concentration of OPG (100 pM) in the presence of antibodies to the ectodomain of syndecan-1 (2 µg/ml). Western blot analyses showed that antibody to the syndecan-1 diminished OPG-induced activation of Akt and PKC but had no effect on phosphorylation status of Src kinases (Fig. 5).
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Because syndecan ligands have previously been shown to interfere with chemokine-induced cell migration (24, 26), monocytes were incubated with various concentrations of OPG and then chemotaxis toward the monocyte chemoattractants MCP-1, RANTES, and PCT was investigated (27). Monocyte chemotaxis toward MCP-1 (10 nM), RANTES (1.3 nM), and PCT (0.1 nM) was significantly decreased by pretreatment with OPG at concentrations ranging from 1 pM to 10 nM (Fig. 6).
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Discussion |
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Monocyte migration and extravasation play a key role in chronic inflammation. Once the monocytes leave the circulation, they differentiate into macrophages, which contribute to chronic inflammation (1). In osteoclasts, OPG was shown to inhibit bone-resorbing activity and induce proteases and protease inhibitors expression (16, 17). Phosphorylation of ERK1/2 and p38 was seen after OPG stimulation in these cells (17). It is known that OPG has the ability to diminish RANKL-induced monocyte migration (29). However, preliminary data from our laboratory suggested that OPG may also have chemotactic properties on monocytes (30). In our present experiments, OPG stimulates monocyte migration in a dose-dependent manner in the absence of other chemoattractants. Maximal response for human monocytes could be seen at OPG concentrations of 100 pM, which is well within the range of OPG levels found in human plasma. Circulating OPG may prevent blood monocytes from premature activation due to its deactivating effect. However, because levels in synovial fluid are similar to plasma levels (31), a definitive biological role of our observation remains elusive. Checkerboard analyses confirmed the activity of OPG on monocytes as chemotactic because monocyte migration depends on the presence of an OPG concentration gradient. Results of the checkerboard analyses were internally consistent with that of migration assays. These observations suggest that OPG may have effects on functional responses of the cells, which may serve as bioassay for further biochemical analyses.
It is known that OPG can bind to heparin and heparan sulfates (20). A recent study shows that OPG is bound to syndecan-1 expressed on multiple myeloma cells and that this binding is due to interaction of OPG with heparan sulfates (32). Theoleyre et al. (17) show the involvement of OPG-RANKL-RANK complexes in direct OPG activity on osteoclasts. However, their results indicate the existence of another binding mechanism, and they suggest syndecans as possible candidates. Syndecans are type I integral membrane proteoglycans that contain both heparan sulfate and chondroitin sulfate groups. Besides their role in cell-extracellular matrix adhesion and growth factor binding (33), syndecans have also been implicated in cell migration (18, 24, 26). Interestingly, it was previously shown that both syndecan-1 and OPG are located on the uropods of the polarized myeloma cell (34). To study the possible binding of OPG to HSPG on monocytes, we employed the specificity of heparinase I to highly sulfated polysaccharide chains containing linkages to 2-O-sulfated 
-L-idopyranosyluronic acid residues (35). Heparinase I degrades the synthetic heparin pentasaccharide to a disaccharide and trisaccharide product (36). Pretreatment of monocytes with heparinase I abrogated the migratory response of monocytes toward OPG.
Membrane-bound HSPGs consist of two families, the syndecans and the glycipans. In contrast to syndecans, the glycipans do not posses chondroitin sulfate chains (33, 37). To rule out involvement of glycipans in OPG-induced monocyte migration, monocytes were treated with chondroitinase before testing cell migration toward OPG. Data demonstrating that OPG effects on monocyte migration are sensitive to both heparinase I and chondroitinase confirm that syndecans are involved in direct cellular actions of OPG. Other proteins with heparin-binding domains, including fibroblast growth factor-2 and VEGF, were tested and did not show activity like OPG. This indicates that OPG-induced monocyte migration via syndecan may be specific and is not commonly shared by other proteins with heparin-binding domains.
To investigate which type of syndecan is involved in OPG-induced monocyte chemotaxis, cells were incubated with antibodies against either syndecan-1 or -4 ectodomain. Incubation of monocytes with antibodies against syndecan-1 but not against syndecan-4 significantly inhibited cell migration, suggesting that OPG binding to syndecan-1 is involved in monocyte chemotaxis toward OPG. From our FACS data, syndecan-1 appears to be expressed more prominently on the surface of unstimulated monocytes than syndecan-4. Therefore, it is also possible that different blocking abilities of syndecan-1 and syndecan-4 antibodies derive from different surface expression patterns of these proteoglycans on monocytes. RANTES- and fMLP-induced monocyte migration was not influenced by syndecan-1 antibodies, thus ruling out that syndecan-1 antibody generally inhibits cell motility.
To investigate which signaling pathways are involved in the chemotactic response of monocytes toward OPG, cells were incubated with different specific enzyme blockers. The specific inhibitor of PI3K wortmannin, the PKC inhibitors staurosporine and GFX, and the tyrosine kinase inhibitor tyrphostin 23 significantly inhibited OPG-induced chemotaxis in monocytes, whereas the phosphodiesterase inhibitor IBMX and the specific inhibitors of the Src family tyrosine kinases PP1 and PP2 did not affect OPG-induced monocyte migration.
Previous studies revealed the involvement of PI3K, PKC, and Src-kinases in syndecan-dependent cell signaling (38, 39, 40). Thus, by combining these data with the results from our enzyme blocker studies, we performed Western blot analyses to further investigate OPG signaling pathways in monocytes and the possible involvement of syndecan-1 in the OPG-monocyte interaction. Akt is activated by phosphorylation (41). The phosphorylation of Akt requires PI3K-dependent generation of PI(3,4,5)P3 and therefore also serves as indicator of PI3K activity (42). The activity of PKC is under control of three distinct phosphorylation events. Specifically, the activation loop, the autophosphorylation site, and the hydrophobic site at the carboxy terminus of PKCßII are phosphorylated in vivo (43). However, the first step in activation is the phosphorylation at the activation loop, which occurs in all PKC isoforms. Src activity is regulated by tyrosine-phosphorylation at two sides with opposing effects. Phosphorylation of Tyr416 in the activation loop of the kinase domain up-regulates the enzyme activity, whereas phosphorylation of Tyr527 renders the enzyme less active (44). In our experiments, incubation of monocytes with OPG led to a clear dose-dependent increase of activated Akt and PKC phosphorylated at the activation loop. The maximal effect was seen at concentrations of 100 pM, which is in good correlation with chemotaxis experiments. Preincubation of cells with antibodies against the syndecan-1 ectodomain significantly reduced signal intensity, compared with the maximal effect. OPG stimulation of monocytes and preincubation of cells with syndecan-1 antibody did not affect phosphorylation status of Src kinases. Taken together, Western blot analyses further support the role of PI3K and PKC for OPG-dependent signal transduction and confirm the involvement of syndecan-1 in OPG-induced cell signaling.
Chemokines mediate their biological activity through activation of G protein-coupled receptors, but most chemokines are also able to bind to GAGs (45, 46, 47). This interaction is important for the formation of immobilized gradients of chemokines and facilitates the receptor binding process. Binding of chemokines to GAGs on leukocytes was shown to modify their chemotactic effect (47, 48). To investigate whether OPG interferes with chemokine function, we chose the monocyte chemoattractants RANTES, MCP-1, and PCT. Monocyte migration toward all, RANTES, MCP-1 and PCT, could be decreased in a dose-dependent manner by preincubation of the cells with OPG. This effect could be reversed by addition of heparinase I and chondroitinase to the preincubated cells, thus implicating that reduction of chemokine-induced migration by OPG is also due to interaction of OPG with monocyte cell surface HSPGs.
Our results provide evidence for the interaction of OPG with heparan sulfate and chondroitin sulfate chains attached to proteoglycans on the surface of monocytes leading to a previously unknown specific action of OPG on monocytes. OPG was shown to induce as well as inhibit monocyte migration, depending on the activation status of the cell. This dual role of OPG may be relevant in the light of changing levels and ratios of OPG and proinflammatory chemoattractants in physiological and pathological conditions. For example, the osteoclast precursor is believed to be a myeloid lineage cell. Hence, it may respond to the chemoattractant action of OPG. Because in the resting state, osteoblasts produce more OPG than RANKL (49), this may prevent activation of osteoclastogenesis but, based on the results from this study, may also attract osteoclast precursor cells. Production of OPG by osteoblasts may prevent the migration of osteoclast precursor cells to other areas of the bone marrow because OPG is able to overcome the chemoattractive activity of other factors. When osteoblasts are activated to stimulate resorption, RANKL production is increased and OPG production decreases (50). This stimulates mature osteoclast formation on the surface of bone and, based on the results of this study, may inhibit the recruitment of new osteoclast precursors to sites of active resorption. Such a scenario might explain the limited resorption that is seen in the remodeling cycle and permit the initiation of bone formation at sites of previous resorption.
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Footnotes |
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First Published Online February 22, 2005
Abbreviations: fMLP, N-Formyl-L-methionyl-L-leucyl-L-phenyl-alanine; GAG, glycosaminoglycan; GFX, bisindolylmaleimide I GF 109203X; HBSS, Hanks’ balanced salt solution; HSPG, heparan sulfate proteoglycan; IBMX, isobutylmethylxanthine; MACS, magnetic-activated cell sorting; MCP-1, monocyte chemotactic protein-1; OPG, osteoprotegerin; PCT, procalcitonin; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; RANKL, receptor activator of nuclear factor-B ligand; RANTES, regulated upon activation normal T cell expressed and secreted; VEGF, vascular endothelial growth factor.
Received September 24, 2004.
Accepted February 15, 2005.
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B ligand in serum of rheumatoid arthritis patients and their normalization after anti-tumor necrosis factor treatment. Arthritis Rheum 46:1744–1753[CrossRef][Medline]
vß 3-induced, NF-B-dependent survival factor for endothelial cells. J Biol Chem 275:20959–20962B ligand in Monckeberg’s sclerosis and atherosclerosis. J Clin Endocrinol Metab 89:4104–4112B ligand and MAPK. Biochim Biophys Acta 1644:1–7[Medline]
B ligand (RANKL) is a new chemotactic factor for human monocytes. FASEB J 17:1751–1753This article has been cited by other articles:
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 F. Lamoureux, G. Picarda, L. Garrigue-Antar, M. Baud'huin, V. Trichet, A. Vidal, E. Miot-Noirault, B. Pitard, D. Heymann, and F. Redini Glycosaminoglycans as Potential Regulators of Osteoprotegerin Therapeutic Activity in Osteosarcoma Cancer Res., January 15, 2009; 69(2): 526 - 536. [Abstract] [Full Text] [PDF]
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 R. Pakula, A. Melchior, A. Denys, C. Vanpouille, J. Mazurier, and F. Allain Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis Glycobiology, May 1, 2007; 17(5): 492 - 503. [Abstract] [Full Text] [PDF]
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