ARTHRITIS & RHEUMATISM Vol. 50, No. 7, July 2004, pp 2309–2316 DOI 10.1002/art.20352 © 2004, American College of Rheumatology Expression and Function of RANK in Human Monocyte Chemotaxis Birgit A. Mosheimer, Nicole C. Kaneider, Clemes Feistritzer, Daniel H. Sturn, and Christian J. Wiedermann sequential process of bone resorption by osteoclasts followed by deposition of new bone by osteoblasts. Bone loss is a common feature of various inflammatory arthritides (1). Osteoclasts, which can be generated from peripheral monocytes (2,3), are known to contribute to focal bone erosion in rheumatoid arthritis (RA) (4–6) and in animal models of arthritis (7,8). RANK, which is expressed on osteoclasts and osteoclast precursor cells, T cells, and dendritic cells (9–11), mediates intracellular signals obligatory for osteoclastogenesis as well as osteoclast activation and survival in vivo (11,12). The effects are mediated through RANKL, a member of the tumor necrosis factor superfamily that is expressed on osteoblast/stromal cells and activated T cells (13,14). RANKL exists as a membrane-bound peptide, a soluble fragment cleaved from the cell surface, and a secreted protein (15). Osteoprotegerin (OPG) is a secreted decoy receptor that binds and thereby inactivates RANKL (16). OPG messenger RNA (mRNA) is expressed in a number of tissues (16,17). It was previously shown that OPG expression on macrophage-type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis (18). OPG administration reduced osteoclast numbers and bone erosions in collagen-induced arthritis (15). In this study, we investigated the effects of recombinant human RANKL on the chemotaxis of monocytes and the possible involvement of phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases in RANKL-induced monocyte migration. Furthermore, we report the expression of RANK mRNA and surface protein in monocytes. Objective. RANKL, a member of the tumor necrosis factor superfamily, is a central regulator of osteoclast recruitment and activation. Whether RANKL affects monocyte locomotion in vitro via RANK and a possible signaling pathway were investigated. Methods. Monocytes were obtained from venous blood of healthy donors. Cell migration was studied by micropore filter assays. The signaling mechanisms required for RANKL-dependent migration were tested using signaling enzyme blockers and Western blot analyses. Expression of RANK messenger RNA (mRNA) in monocytes was demonstrated by reverse transcriptase– polymerase chain reaction, and receptor expression on cell surface was investigated by fluorescence-activated cell sorting analyses. Results. RANKL significantly stimulated monocyte chemotaxis via activation of phosphatidylinositol 3-kinase, phosphodiesterase, and Src kinase. The effect on migration was inhibited by osteoprotegerin, which is the decoy receptor for RANKL. Expression of RANK receptor mRNA was shown, and synthesis of RANK in monocytes was suggested by the detection of RANK immunoreactivity on the cell surface. Conclusion. These data suggest that RANK is expressed by monocytes whose activation by RANKL stimulates directed migration involving phosphatidylinositol 3-kinase, phosphodiesterase, and Src kinases. Normal skeletal maintenance occurs by a tightly coupled process of bone remodeling. It consists of a Supported by the Verein zur Förderung von Forschung und Fortbildung in klinischer Kardiologie und Intensivmedizin–Innsbruck. Birgit A. Mosheimer, MD, Nicole C. Kaneider, MD, Clemes Feistritzer, MD, Daniel H. Sturn, MD, Christian J. Wiedermann, MD: University of Innsbruck, Innsbruck, Austria. Address correspondence and reprint requests to Christian J. Wiedermann, MD, Department of Internal Medicine, University of Innsbruck, Anichstrasse 35, A-6020, Innsbruck, Austria. E-mail: email@example.com. Submitted for publication November 3, 2003; accepted in revised form March 22, 2004. MATERIALS AND METHODS Reagents and materials. All stock solutions were stored at ⫺20°C before use. RPMI 1640 with phenol red was purchased from Biological Industries (Kibbutz Beit Haemek, 2309 2310 Israel). Bovine serum albumin (BSA) was from Dade-Behring (Marburg, Germany). Dextran, staurosporine, 3-isobutyl-1methyl xanthine (IBMX), wortmannin, tyrphostin 23, Triton X-100, and recombinant human interleukin-8 (IL-8) were from Sigma (St. Louis, MO). Bisindolylmaleimide I (GF 109203X) was obtained from Boehringer Ingelheim (Ingelheim, Germany). Lymphoprep was from Nycomed Pharma (Oslo, Norway). Dulbecco’s phosphate buffered saline (PBS) and fetal calf serum were from PAA Laboratories (Linz, Austria). Hanks’ balanced salt solution (HBSS) without phenol red was obtained from Invitrogen (Carlsbad, CA). The biotinylated mouse anti-mouse antibody, the IgG isotype control, and anti-human RANK antibody were from eBioscience (San Diego, CA). Streptavidin–phycoerythrin was from Becton Dickinson (San Jose, 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). RNA-Bee was from Tel-Test (Friendswood, TX). Reverse transcriptase (RT) was from Gibco BRL (Vienna, Austria). HotStarTaq polymerase was purchased from Qiagen (Valencia, CA). Primers were from MWG Biotech (Ebersberg, Germany). Certified polymerase chain reaction (PCR) agarose was from Bio-Rad (Hercules, CA). RANKL was from Serotec (Oxford, UK). PP1, PP2, PP3, and Tween 20 were from Calbiochem (San Diego, CA). Hybond P membrane was from Amersham Biosciences (Little Chalfont, UK). Recombinant human procalcitonin (PCT) was from Brahms Diagnostica (Berlin, Germany). Monocyte chemotactic protein 1 (MCP-1) was from R&D Systems (Minneapolis, MN). PAGEr Duramide gels were from Cambrex (Rockland, ME). Phospho-Src Tyr527 antibody, phospho-Src Tyr416 antibody, and anti-rabbit IgG horseradish peroxidase–linked antibody were from Cell Signaling Technology (Beverly, MA). 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 (PBMCs) were collected and washed twice with HBSS. Positive selection of CD14⫹ monocytes was performed by adding MACS colloidal superparamagnetic microbeads conjugated with monoclonal anti-human CD14 antibodies to cooled, freshly prepared PBMC preparations in MACS buffer (PBS with 5 mM EDTA and 0.5% BSA) according to manufacturer’s instructions. Cells and microbeads were incubated for 15 minutes 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 withdrawn, 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 ⬃98% (19). Monocyte migration assay. Migration assays were performed by using a modified 48-well Boyden microchemotaxis chamber (Neuroprobe) in which a 5-m–pore-size cellulose nitrate filter separated the upper and the lower chambers. Monocytes were resuspended in RPMI 1640/0.5% BSA (1 ⫻ 106 cells/ml). Fifty microliters of the cell suspension was placed MOSHEIMER ET AL into the upper chamber and allowed to migrate toward various concentrations of RANKL (1 aM to 10 nM) placed in the lower chamber for 90 minutes at 37°C in a humidified atmosphere (5% CO2). After the migration period, the nitrocellulose filters were dehydrated, fixed, and stained with hematoxylin. The migration depth of the cells into the filters was quantified by means of microscopy, measuring the distance (in micrometers) from the surface of the filter to the leading front of 3 cells. Data are expressed as a chemotaxis index, which is the ratio between the distance of directed migration and random migration of monocytes into the nitrocellulose filters. Intracellular signaling of RANKL on monocytes was tested by preincubation of the cells with the intracellular enzyme blockers staurosporine (from Streptomyces species) (10 ng/ml), GF 109203X (500 nmoles/liter), wortmannin (from Penicillium fumiculosum) (10 nmoles/liter), IBMX (100 M), and tyrphostin 23 (10 ng/ml) for 30 minutes at 37°C in a humidified atmosphere (5% CO2). In further experiments, monocytes were preincubated with different concentrations of PP1, which is a selective inhibitor of Src kinases, and PP2, which acts in a manner similar to that of PP1 and specifically inhibits the tyrosine kinases lck, fyn, and hck, for 30 minutes at 37°C. Incubation of monocytes with PP3 served as a negative control. The cells were then washed twice, resuspended in RPMI/0.5% BSA, and tested in the migration assay toward RANKL. 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 RANKL just before they were transferred to the upper chamber. The same concentrations of RANKL remained beneath the filter of the Boyden chamber; thus, distinct concentration gradients could be formed. Data are expressed as a chemotaxis index within a matrix. RT-PCR. Total RNA was isolated from 107 cells by an acid guanidinium thiocyanate–phenol–chloroform mixture. An RT reaction was performed on 1 g of RNA using random hexamers RT. One microgram of the resulting complementary DNA was then subjected to 35 cycles of PCR in a 50-l reaction mixture containing 1 pmole of sense and antisense primer pairs in a Biometra thermocycler, as follows: 95°C for 60 seconds (denaturation), 62°C for 60 seconds (annealing), and 72°C for 60 seconds (extension). Primers were designed to amplify a 400-bp coding sequence of human RANK receptor. The sense primer sequence was 5⬘-GCA-AAC-TTT-GGTCAG-CAG-GGA-G-3⬘. The antisense primer sequence was 5⬘-GCT-CAG-TGC-AGT-TGC-AGC-TTT-C-3⬘. The PCR products were subjected to agarose gel analysis. CD3⫹ lymphocytes were used as a positive control, because RANK expression on T cells has previously been described (9). PCR without template was used as a negative control. Fluorescence-activated cell sorting (FACS) analysis. Fluorometric analysis for RANK expression on the cell surface of monocytes was performed. A total of 5 ⫻ 105 cells were washed twice in Dulbecco’s PBS containing 0.5% BSA and incubated with 150 g/ml of human IgG for 20 minutes at 4°C. After pelleting, cells were incubated with 10 g/ml of mouse anti-human RANK (eBioscience) or the respective isotypematched control mouse IgG (eBioscience) for 30 minutes at 4°C. After washing, 10 g/ml of biotinylated mouse anti-mouse RANK AND MONOCYTES 2311 according to manufacturer’s instructions, and blots were incubated overnight at room temperature. Immunoreactivity was determined using peroxidase-conjugated goat anti-rabbit IgG and SuperSignal chemiluminescent substrate (Pierce, Rockford, IL). The intensity of the Western blot bands was quantified using the Fluor-S MultiImager system and Quantity One software (Bio-Rad). Statistical analysis. Data are expressed as the mean ⫾ SEM. Means were compared by the Mann-Whitney U test, the paired t-test, and Kruskal-Wallis analysis of variance. P values less than 0.05 were considered significant. Statistical analyses were performed using the StatView software package (Abacus Concepts, Berkeley, CA). RESULTS Figure 1. Effects of RANKL on migration of human monocytes. The direct chemotactic effects of different concentrations of RANKL on human monocytes were investigated. Monocyte chemoattractant protein 1 (MCP-1) and procalcitonin (PCT) served as positive controls, and interleukin-8 (IL-8) served as a negative control. Chemotaxis experiments were performed in modified Boyden chambers. Data shown are the mean ⫾ SEM of the chemotaxis index, which is the ratio of the distance of migration (in micrometers) toward attractant and the distance toward medium. The mean distance of random migration was 51.9 ⫾ 2.18 m. ⴱ ⫽ P ⬍ 0.05 versus medium, by Mann-Whitney U test, after multiple group comparison using the Kruskal-Wallis test (n ⫽ 6). IgG (eBioscience) was incubated for another 30 minutes. Cells were washed twice, and the monocytes were subsequently incubated with a 1:25 dilution of streptavidin–phycoerythrin for 30 minutes, washed twice, then immediately analyzed on a FACScan with CellQuest software (Becton Dickinson). Western blot analysis. Cells were incubated with RANKL at various concentrations (0.1 fM to 100 pM) for 120 minutes. Cells were lysed in lysis buffer containing 1% Triton X-100. Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and blotted onto polyvinylidene difluoride membranes, which were blocked with 2% phosphatase-free milk powder in Tris buffered saline with 0.1% Tween. The antibody was then diluted in 0.2% milk Table 1. Effects of RANKL on monocyte migration. To explore for chemotactic properties of RANKL in the absence of other chemoattractants, freshly prepared monocytes were allowed to migrate toward different concentrations of RANKL (1 aM to 10 nM). The same concentrations of MCP-1 and PCT were used as a positive control (20). IL-8 (1 aM to 10 nM) served as a negative control. Concentrations of RANKL ranging from 10 fM to 10 nM significantly increased migration in a dose-dependent manner (Figure 1). RANKL stimulated migration maximally at a concentration of 1 pM. In checkerboard analysis, the migratory response was confirmed as true chemotaxis. Maximal induction of migration occurred in the presence of a positive concentration gradient between the 2 compartments of the Boyden chamber (higher concentration below the filter). In the presence of either equal concentrations of RANKL above and below the filter or a negative gradient (higher concentration above the filter), no significant enhancement of migration occurred. These results indicate that RANKL is able to activate a chemotactic response in human monocytes with no appreciable chemokinetic activity (Table 1). Effect of concentration gradients of RANKL on monocyte migration* Upper chamber Medium RANKL, lower chamber Medium 10⫺16M 10⫺14M 10⫺12M 1.00 ⫾ 0.00 1.29 ⫾ 0.08 1.62 ⫾ 0.09† 1.81 ⫾ 0.12† ⫺16 10⫺14M 10⫺12M 1.06 ⫾ 0.03 1.21 ⫾ 0.08 1.43 ⫾ 0.05 1.70 ⫾ 0.08† 1.00 ⫾ 0.02 1.08 ⫾ 0.03 1.25 ⫾ 0.11 1.49 ⫾ 0.10 1.05 ⫾ 0.02 1.16 ⫾ 0.05 1.20 ⫾ 0.07 1.15 ⫾ 0.10 10 M * Values are the mean ⫾ SEM of the chemotaxis index. Different concentrations of RANKL were supplied to the upper and/or lower compartment of the chemotaxis chamber. The mean distance of random migration was 36.8 ⫾ 3.8 m (n ⫽ 6). † P ⬍ 0.05 versus medium, by Mann-Whitney U test, after multiple group comparison using the Kruskal-Wallis test (n ⫽ 6). 2312 MOSHEIMER ET AL Table 2. Effect of blocking of signaling enzymes on monocyte migration toward RANKL* Figure 2. Effects of osteoprotegerin (OPG) on RANKL-induced monocyte migration. Chemotaxis of freshly prepared monocytes toward the optimal concentrations of RANKL (1 pM), MCP-1 (10 nM), and PCT (0.1 nM) in the presence of different concentrations of OPG (100 aM to 1 M) was monitored. Data shown are the mean ⫾ SEM of the chemotaxis index. The mean distance of random migration was 55.2 ⫾ 5.72 m. ⴱ ⫽ P ⬍ 0.05 versus RANKL stimulation, by Mann-Whitney U test, after multiple group comparison using the Kruskal-Wallis test (n ⫽ 4) (see Figure 1 for other definitions). Effects of OPG on RANKL-induced monocyte migration. Because we observed a chemotactic effect of RANKL on monocytes, we were interested to determine whether this effect could be inhibited by OPG, the soluble decoy receptor for RANKL. Untreated monocytes were allowed to migrate toward the optimal concentration of RANKL (1 pM) in the presence of different concentrations of OPG (100 aM to 1 M). Nearly equimolar concentrations of OPG significantly inhibited monocyte migration toward RANKL in a dosedependent manner. An excess of OPG over RANKL, however, abrogated the inhibitory effect. The addition of OPG to the lower chamber had no effect on monocyte migration toward MCP-1 and PCT (Figure 2). Blocking of intracellular signaling enzymes in RANKL-induced chemotaxis of monocytes. To elucidate signaling pathways involved in transmitting RANKL effects in monocyte migration, different intracellular enzyme blockers were tested. Monocytes were freshly isolated, incubated with the enzyme blockers for 30 minutes, and washed twice. The blockers staurosporine, GF 109203X, IBMX, wortmannin, and tyrphostin 23 were used at established signal-blocking concentrations. Staurosporine, which is a nonspecific inhibitor of protein kinase C (PKC) that also affects protein kinase A signaling, and the specific PKC inhibitor GF 109203X did not affect RANKL-induced monocyte migration. Wortmannin, a specific inhibitor of PI 3-kinase, the phosphodiesterase inhibitor IBMX, and tyrphostin 23, a Treatment Monocyte migration Medium Wortmannin, 10 nmoles/ml Tyrphostin 23, 10 ng/ml GF 109203X, 500 nmoles/liter Staurosporine, 10 ng/ml IBMX, 100 M 1.70 ⫾ 0.05 1.14 ⫾ 0.06† 1.26 ⫾ 0.06† 1.47 ⫾ 0.06 1.62 ⫾ 0.05 1.23 ⫾ 0.06† * Values are the mean ⫾ SEM of the chemotaxis index. Cells incubated with enzyme blockers for 30 minutes were washed twice, resuspended in RPMI 1640/0.5% bovine serum albumin, and chemotaxis toward RANKL (1 pM) was monitored. The mean distance of random migration was 48.19 ⫾ 0.95 m. GF 109203X ⫽ bisindolylmaleimide I; IBMX ⫽ 3-isobutyl-1-methyl xanthine. † P ⬍ 0.05 versus medium incubation toward RANKL, by MannWhitney U test, after multiple group comparison by the Kruskal-Wallis test (n ⫽ 4). tyrosine kinase inhibitor, significantly decreased RANKL-induced chemotaxis in monocytes (Table 2). In further experiments, we investigated whether Src tyrosine kinases are involved in RANK-mediated monocyte migration in response to RANKL. Monocytes were preincubated with different concentrations of PP1 and PP2, which are selective inhibitors of Src kinases, Figure 3. Effect of PP1, PP2, and PP3 pretreatment on monocyte chemotaxis in response to RANKL. Monocytes were incubated with different concentrations of PP1, PP2, and PP3 for 30 minutes. Cells were washed twice, resuspended in RPMI 1640/0.5% bovine serum albumin, and chemotaxis toward RANKL (1 pM) was monitored. Data shown are the mean ⫾ SEM of the chemotaxis index. The mean distance of random migration was 47.1 ⫾ 4.78 m. ⴱ ⫽ P ⬍ 0.05 versus medium incubation toward RANKL, by Mann-Whitney U test, after multiple group comparison using the Kruskal-Wallis test (n ⫽ 4) (see Figure 1 for the definition of chemotaxis index). RANK AND MONOCYTES 2313 kinases could be detected in response to RANKL, whereas the inactive, Tyr527-phosphorylated form was not significantly affected. Time course experiments with an optimal concentration of RANKL (1 pM) revealed an Figure 4. Phosphorylation status of Src kinases in RANKLstimulated monocytes. Freshly prepared monocytes were incubated with RANKL. Cells then were washed twice and lysed. Equal protein concentrations of lysates were loaded onto lanes. The phosphorylation status of Src kinase was visualized after incubation with different concentrations of RANKL (100 aM to 100 pM) for 120 minutes using a monoclonal antibody against phospho-Src Tyr416 (A) and phosphoSrc Tyr527 (B). Time-dependent phosphorylation of Tyr416 was detected after stimulation of cells with RANKL (100 pM) for different time spans (C). Src kinase protein is represented by the 60-kd product. and PP3, which served as a negative control. Chemotaxis experiments revealed a significant dose-dependent inhibition of monocyte migration after incubation with PP1 and PP2 toward RANKL. PP3 did not affect RANKLinduced monocyte migration (Figure 3). Activation of Src kinase in monocytes. In order to confirm involvement of Src kinases in RANKLdependent signaling in monocytes, Western blot analysis was performed. To investigate the phosphorylation status of Src kinases after incubation with RANKL, monocytes were incubated with different concentrations of RANKL (100 aM to 10 nM) for 2 hours. Using phosphoSrc antibodies against the 2 major phosphorylation sites Tyr416 and Tyr527, a concentration-dependent increase of the active Tyr416 phosphorylation status of Src Figure 5. Reverse transcriptase–polymerase chain reaction (PCR) and FACScan analysis of RANKL in monocytes. A, RANK mRNA in monocytes and T cells. One microgram of total RNA from each sample was reverse transcribed into cDNA and amplified for the RANK gene using PCR. RANK is represented by a 400-bp product. B, Fluorescence-activated cell sorting analysis of anti-RANK monoclonal antibody (mAb) binding to monocytes. A FACScan flow cytometer was used for fluorescence analysis, and a histogram of phycoerythrin fluorescence is shown. Cells were incubated with either isotypematched control IgG or anti-RANK mAb and stained with phycoerythrin-conjugated streptavidin. 2314 MOSHEIMER ET AL increase of the active, Tyr416-phosphorylated form in a time-dependent manner (Figure 4). Expression of RANK in monocytes. Because RANKL-induced effects on chemotaxis of monocytes may be mediated by its binding to and activation of RANK, the surface expression of RANK on these cells and the mRNA content of monocytes were tested. To determine whether RANK mRNA is expressed in monocytes, RT-PCR analysis was performed. Expression of RANK mRNA in T cells served as a positive control, and PCR without any template served as a negative control. Data confirmed that RANK mRNA could be found in human peripheral blood monocytes obtained from healthy donors. In FACS analysis, a significant shift of fluorescence in monocytes by anti-RANK antibody was observed. An IgG antibody was used as a negative control (Figure 5). DISCUSSION Arthritis in humans is characterized by synovial inflammation, erosion of bone and cartilage, severe joint pain, and finally loss of function (21). In RA synovial membrane, recently immigrated monocytes differentiate into mature macrophages (22). Macrophages possess broad proinflammatory, destructive, and remodeling capacities and contribute considerably to inflammation and joint destruction in both the acute and chronic phases of RA (23). Osteoclasts, which can derive from monocytes (2,3), are known to contribute to focal bone erosion in RA and in animal models of arthritis (7,8). A recent study by Breuil et al (24) demonstrated that RANKL, which is expressed on osteoblast/stromal cells in cultured synovial fibroblasts from patients with RA (25,26) and in CD4⫹ and CD8⫹ T lymphocyte subsets in RA synovium (27,28), has chemotactic properties on monocytes. This effect can be abrogated by the addition of OPG, the decoy receptor of RANKL (24). In our study, we confirm these results and reveal the involvement of PI 3-kinase, phosphodiesterase, and Src kinases in the signaling pathway. Moreover, we demonstrate the expression of RANK by means of identifying the expression of mRNA and detecting cell surface RANK by FACS analysis. RANKL has been shown to be chemotactic on various cell types, including osteoclasts and human umbilical vein endothelial cells (29,30). Confirming recent work, in our experiments RANKL stimulated monocyte migration in a dose-dependent manner. Breuil et al reported maximum RANKL-induced migration at a concentration of 100 ng/ml, using MonoMac-6 cells (24). This dose is equivalent to 3 nM. In our setting, maximal response could be seen at RANKL concentrations of 100 pM for freshly prepared human monocytes from peripheral blood, which is in good correlation with physiologic RANKL levels in the picomolar range measured in serum from healthy subjects (31) and with RANKL levels in synovial fluid from patients with RA (3). Differences in maximal responses may be attributable to different cell types used. In our study, checkerboard analysis confirmed the activity of RANKL on monocytes as chemotactic. Checkerboard experiments clearly showed that monocyte migration depends on the presence of RANKL as a concentration gradient. Results of the checkerboard analysis were internally consistent with those of migration assays and demonstrated gradientdependent effects of RANKL on monocytes, with significant responses at picomolar concentrations. Moreover, the effect of RANKL was significantly inhibited by the addition of equimolar concentrations of its decoy receptor, OPG, whereas migration of monocytes toward MCP-1 and PCT was not affected by OPG. The decoy receptor OPG binds RANKL with high affinity, thereby preventing RANKL from interacting with RANK (16,17). This strengthens the idea that RANKL-induced monocyte migration is specific and may depend on RANKL binding to its receptor, RANK. Nevertheless, an excess of OPG over RANKL abrogated the inhibitory effect of OPG on RANKL-induced chemotaxis. Data suggest that OPG has chemotactic properties on monocytes that may serve as an explanation for this observation (Mosheimer BA, et al: unpublished observations). How OPG may induce migration remains to be studied. RANKL mediates osteoclastogenesis as well as osteoclast activation and survival in vivo via its receptor RANK (10,11). RANK mRNA is expressed in many tissues, including skeletal muscle, liver, small intestine and colon, thymus, and adrenal gland (9), yet expression of RANK protein appears to be restricted to osteoclasts and osteoclast precursor cells, T cells, dendritic cells, and endothelial cells (9–11,32). This study is the first to show that freshly isolated CD14⫹ PBMCs express not only RANK mRNA but also RANK immunoreactivity on their surface, as shown by FACS analysis. Various effects of RANKL, including bone resorption by osteoclasts and inhibition of cell death, are mediated through PI 3-kinase, PKC, and phospholipase C pathways (33–35). Previous studies have shown that Src kinases and phospholipase C are implicated in RANKL-induced endothelial cell migration, whereas RANK AND MONOCYTES osteoclast chemotaxis toward RANKL is ERK-1/2 dependent (29,30). To analyze which of these pathways mediates the chemotactic response of monocytes toward RANKL, cells were incubated with different specific enzyme blockers. The specific inhibitor of PI 3-kinase wortmannin, as well as the phosphodiesterase inhibitor IBMX and the tyrosine kinase inhibitor tyrphostin 23, significantly inhibited RANKL-induced chemotaxis in monocytes, while the PKC inhibitors staurosporine and GF 109203X did not affect RANKL-induced monocyte migration. These results indicate that PI 3-kinase, phosphodiesterase, and tyrosine kinases are also involved in RANKL-induced monocyte migration. Because of the effects by the tyrosine kinase inhibitor tyrphostin 23, we tested the influence of specific inhibitors of the Src tyrosine kinase family members on cell migration toward RANKL. Src kinases have been implicated in multiple signaling pathways that regulate migration, cellular growth, and cell survival (36,37). The specific inhibitors of the Src family tyrosine kinases PP1 and PP2, but not the inactive analog PP3, which inhibits epidermal growth factor receptor tyrosine kinase, abolished RANKL-induced chemotaxis. Thus, by combining these data with those from studies demonstrating that RANKL stimulates Src activation (38,39), we performed Western blot analyses to further investigate the phosphorylation status of Src kinases in monocytes after stimulation with RANKL. Src activity is regulated by tyrosine phosphorylation at 2 sides with opposing effects: phosphorylation of Tyr416 in the activation loop of the kinase domain up-regulates the enzyme activity, while phosphorylation of Tyr527 renders the enzyme less active (40). In our experiments, incubation of monocytes with RANKL led to a clear time- and dose-dependent effect on Src kinase phosphorylation status: the active Tyr416-phosphorylated form of Src kinase increased in a dose-dependent manner, whereas no positive effect could be seen for the inactive Tyr527-phosphorylated form of Src kinases on Western blot. Time course experiments revealed that the increase of Tyr416-phosphorylated Src kinases in monocytes after RANKL stimulation is also time dependent. Taken together, these results suggest that RANKL enhances monocyte migration via binding to its receptor RANK and consequent activation of PI 3-kinase, phosphodiesterase, and Src kinases. Because inflammatory macrophages and osteoclasts can derive from peripheral blood monocytes, RANKL-induced monocyte migration may contribute to bone loss in arthritides. 2315 REFERENCES 1. Rehman Q, Lane NE. Bone loss: therapeutic approaches for preventing bone loss in inflammatory arthritis. 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