JBC Focus on PI3-Kinase with Echelon

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M212626200 on March 11, 2003

J. Biol. Chem., Vol. 278, Issue 20, 18573-18580, May 16, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/20/18573    most recent
M212626200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ha, H.
Right arrow Articles by Kim, H.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ha, H.
Right arrow Articles by Kim, H.-H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Membrane Rafts Play a Crucial Role in Receptor Activator of Nuclear Factor kappa B Signaling and Osteoclast Function*

Hyunil HaDagger §, Han Bok KwakDagger §, Seung Ku LeeDagger §, Doe Sun Na||, Christopher E. Rudd**, Zang Hee LeeDagger §DaggerDagger, and Hong-Hee KimDagger §§§

From the Dagger  National Research Laboratory for Bone Metabolism,  Research Center for Proteineous Materials, and § School of Dentistry, Chosun University, Gwangju 501-759, Korea, the || Department of Biochemistry, University of Ulsan College of Medicine, Seoul 138-736, Korea, and the ** Department of Haematology, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, London W12 ONN, United Kingdom

Received for publication, December 11, 2002, and in revised form, February 24, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Membrane lipid rafts play a key role in immune cell activation by recruiting and excluding specific signaling components of immune cell surface receptors upon the receptor engagement. Despite this, the role of these microdomains in the regulation of osteoclasts as controlled by receptor activator of nuclear factor kappa B (RANK) has yet to be established. In this study, we demonstrate that the raft microdomain expression plays an essential role in osteoclast function and differentiation. Expression of raft component flotillin greatly increased during osteoclast differentiation, whereas engagement of RANK induced the translocation of tumor necrosis factor receptor-associated factor 6 to rafts where Src was constitutively resident. Disruption of rafts blocked TRAF6 translocation and Akt activation by RANK ligand in osteoclasts and further reduced the survival of osteoclasts. Actin ring formation and bone resorption by osteoclasts were also found to require the integrity of rafts. Our observations demonstrate for the first time that RANK-mediated signaling and osteoclast function are critically dependent on the expression and integrity of raft membrane microdomains.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rafts are specialized membrane microdomains enriched in glycosphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)1-anchored proteins (1, 2). Raft microdomains are most abundant at the plasma membrane but may also be present in endocytic and secretory pathways. Proteins modified with saturated acyl chain groups, such as GPI-anchored proteins and double acylated proteins, have been found to be preferentially targeted to rafts. However, certain transmembrane proteins can also be enriched in rafts through a mechanism that is still unclear. The involvement of rafts has been implicated in many important cellular processes, which include generation and maintenance of cellular polarity, chemotactic migration, and cell surface receptor signaling. For T cell and B cell antigen receptors, raft domains function as signaling platforms where selective signaling molecules are recruited or segregated away (3). Antigen- or antibody-mediated cross-linking of the immune cell receptor facilitates its translocation into raft microdomains containing myristate- and palmitate-modified Src family kinases, which initiate signaling cascades by phosphorylating tyrosine residues on the nonenzymatic receptor complexes.

Recently, the association with rafts of some members of the tumor necrosis factor receptor (TNFR) family, including CD40, has been reported (4-8). The tumor necrosis factor receptor-associated factor (TRAF) proteins are key signaling adaptor molecules utilized by many TNFR family receptors. Among the six mammalian TRAF family proteins, TRAF2 and TRAF3 were shown to be recruited to raft microdomains during CD40 signaling (7, 8). Similarly, the association of TRAF2 with caveolin-1, a component that along with rafts constitutes caveolae, has been reported (9). The association of CD40 and TRAF in raft microdomains raises the possibility that rafts may function as a signaling platform for the TNFR group of transmembrane proteins as observed for the immune cell antigen receptors.

Osteoclasts are multinucleated cells specialized for bone resorption (10). These cells are formed by fusion of committed mononuclear cells of the monocyte/macrophage lineage hematopoietic cells. In this context, many studies have documented the importance of the TNF family member receptor activator of nuclear factor kappa B ligand (RANKL; also known as ODF, OPGL, and TRANCE) in the regulation of osteoclast differentiation, activation, and survival of osteoclasts (11, 12). Its receptor RANK can directly bind several TRAF proteins, which in turn trigger downstream signaling molecules for the activation of NF-kappa B and mitogen-activated protein kinases (MAPKs) (13-16). That mice deficient in RANKL, RANK, or TRAF6 commonly show osteopetrotic phenotype due to defective osteoclastic bone resorption points out the particular importance of TRAF6 for RANK signaling in osteoclasts (17-21). The association of TRAF6 with Src family tyrosine kinases and subsequent stimulation of the Src kinase activity has been suggested to mediate phosphoinositide 3-kinase (PI3K)/Akt activation (22). The biological significance of the TRAF6-Src signaling pathway is consistent with the osteopetrotic phenotype of Src-deficient mice that displayed osteopetrosis due to a defect in resorption function of osteoclasts (23-25).

Given the crucial role of TRAF6 and Src in osteoclast function and RANK signaling and the fact that Src family kinases preferentially segregate to raft microdomains, we sought to address the potential role of membrane rafts for signaling by RANK/TRAF6 in bone resorption function of osteoclasts. Our findings demonstrate that raft expression increases during the osteoclastogenesis and that TRAF6 is recruited to rafts by RANKL stimulation in osteoclasts. Further, we show that disruption of rafts interfere with a variety of parameters required for osteoclast function and differentiation. These include impeded RANK signaling to Akt, defective actin ring formation, and resorption activity of osteoclasts and the reduced survival of osteoclasts. Overall, our findings demonstrate for the first time a crucial role for membrane lipid rafts in the function, and potentially differentiation, of osteoclasts.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- alpha -Minimum essential medium and fetal bovine serum (FBS) were purchased from Invitrogen. Recombinant human soluble RANKL and macrophage colony-stimulating factor (M-CSF) were from PeproTech EC (London, UK). n-Octyl-beta -D-glucopyranoside, methyl-beta -cyclodextrin (MCD), filipin, leukocyte acid phosphatase assay kit, FITC-conjugated choleratoxin (Ctx) B subunit, and anti-Ctx were obtained from Sigma. Glutathione S-transferase-RANKL (extracellular domain, amino acids 158-316) was prepared in our laboratory and conjugated to TRITC by Advanced Biochemicals, Inc. (Chonju, Korea).

Anti-TRAF2 (H-249), anti-TRAF6 (H-274), and anti-actin (I-19) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Src was from Upstate Biotechnology, Inc. (Waltham, MA). Anti-flotillin and anti-caveolin-1 were obtained from BD Biosciences (Lexington, KY). The rabbit antiserum raised against the cytoplasmic domain of human RANK was previously described (13). All other antibodies were purchased from Cell Signaling (Beverly, MA).

Osteoclast Culture-- Osteoclast differentiation from bone marrow cells was achieved as previously described with a slight modification (26). Bone marrow cells from 6-7-week-old ICR mice were cultured for 24 h at 37 °C in a humidified atmosphere of 5% CO2 in alpha -minimum essential medium containing 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10 ng/ml M-CSF. The nonadherent cells were collected and separated on the Histopaque (Sigma) gradient. Cells at the interface were harvested, resuspended at 1 × 106 cells/ml in alpha -minimum essential medium plus 10% FBS, and cultured in the presence of 30 ng/ml M-CSF and 50 ng/ml RANKL for 6 days. The complete medium was changed on the third day.

Osteoclasts were also generated by cocultures of mouse bone marrow cells and osteoblasts. 1 × 107 bone marrow cells and 1 × 106 calvarial osteoblasts were seeded on a collagen gel-coated 90-mm dish and cultured for 6-7 days in the presence of 10-8 M 1alpha ,25-dihydroxyvitamin D3 and 10-6 M prostaglandin E2. Cells were detached by treating with 0.2% collagenase (Invitrogen) at 37 °C for 10 min, replated on 60-mm culture dishes, and incubated for another day. Osteoblasts were removed by treating with 0.1% collagenase at 37 °C for 30 min followed by an intensive pipetting. The remaining cells were considered enriched mature osteoclasts. Calvarial osteoblasts were prepared as previously described (27).

The osteoclastogenic differentiation of the murine monocyte/macrophage cell line Raw264.7 cells was achieved by seeding the cells at the density of 1 × 104/well in 48-well plates and culturing for 4 days with 100 ng/ml RANKL in alpha -minimum essential medium plus 10% FBS. Generation of osteoclasts was confirmed by the tartrate-resistant acid phosphatase (TRAP) staining (28).

Human osteoclasts were obtained by culturing peripheral blood mononuclear cells separated on the Histopaque gradient in the presence of 50 ng/ml M-CSF and 100 ng/ml RANKL. Cells were plated at 1.5 × 106/well in six-well plates and cultured for 14 days with the medium changed every 3 days. For the sucrose density gradient experiment, cells from 36 wells were pooled and lysed as below.

Isolation of Rafts-- Rafts were isolated by a discontinuous sucrose density gradient ultracentrifugation. Cells were washed with ice-cold PBS and lysed in 2 ml of ice-cold TNE buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1 mM EDTA with protease and phosphatase inhibitors) containing 0.5% Brij 58. The lysate was incubated on ice for 30 min and mixed with an equal volume of 80% (w/v) sucrose in TNE. The mixture was overlaid with 4 ml of 35% sucrose, which in turn was topped with 4 ml of 5% sucrose. The gradient was subjected to ultracentrifugation at 38,000 rpm in an SW41 rotor (Beckman Instruments) for 18 h at 4 °C. After centrifugation, 1-ml fractions were collected from the top of the gradient. Fractions were analyzed for the raft marker protein flotillin.

Cell Fractionation-- Cells were washed with ice-cold PBS and lysed in ice-cold TNE buffer containing 0.5% Brij 58 followed by incubation on ice for 30 min. Insoluble fractions were pelleted by microcentrifugation at 14,000 rpm for 20 min. The supernatant was removed and considered the soluble (S) fraction. The insoluble pellet was resuspended in the lysis buffer supplemented with 60 mM n-octyl-beta -D-glucopyranoside and 0.3% deoxycholic acid, incubated for 1 h on ice, and microcentrifuged for 20 min at 14,000 rpm. The supernatant from this step was referred to as the insoluble (I) fraction. The whole process was performed below 4 °C.

Actin Ring Formation Assay-- Cells were seeded on glass coverslips and incubated in medium containing 0.5% FBS for 1-4 h in the absence of RANKL. Cells were pretreated with MCD or filipin and stimulated with RANKL. Cells were fixed in 3.7% formaldehyde for 10 min, washed, and stained with rhodamin-conjugated phalloidin for 10 min. The actin ring formation was observed under the Zeiss Axiolab fluorescence microscope.

Resorption Assay-- Cocultured osteoclasts were replated on dentine slices and allowed to settle for 2 h. Cells on dentine slices were incubated with MCD or filipin. After 30 min, dentine slices were washed with medium to remove MCD, and incubation was continued for 12 h. After the incubation period, cells were completely removed from the plate by abrasion with a cotton tip, and dentine slices were stained with hematoxylin solution. Photographs were taken under a light microscope at ×40 magnification, and total areas of resorption pits were analyzed by the Image Pro-Plus program version 4.0 (Media Cybernetics).

Western Blotting Analysis-- Total cell lysates were prepared by lysing cells in TNE plus 0.5% Brij 58 buffer supplemented with 60 mM n-octyl-beta -D-glucopyranoside and 0.3% deoxycholic acid for 1 h on ice and obtaining the supernatants by microcentrifugation at 14,000 rpm for 20 min. Total cell lysates or the fractionated cellular proteins described above were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was probed with a primary antibody followed by incubation with an appropriate secondary antibody conjugated to horseradish peroxidase. The immune complexes were detected with an enhanced chemiluminescence system.

Immunofluorescence Microscopy-- RNAK was transfected to 293 cells using SuperFect reagent (Qiagen, Valencia, CA). Cells were incubated with TRITC-conjugated glutathione S-transferase-RANKL for 30 min on ice, washed twice with PBS containing 0.5% bovine serum albumin and fixed with 3.7% formaldehyde. After washing with PBS/bovine serum albumin, cells were incubated for 30 min at 4 °C with FITC-conjugated Ctx for GM1 staining. In experiments where membrane raft patches were induced, the labeling with TRITC-RANKL was followed by incubation with FITC-Ctx. Bound Ctx was cross-linked by incubation with anti-Ctx (diluted to 1:250) for 20 min at 37 °C. Cells were fixed, and fluorescence microscopy was performed with the Zeiss Axiolab microscope.

Osteoclast Survival and Apoptosis Assays-- For survival assays, purified mature osteoclasts prepared as above were pretreated with MCD for 30 min, washed in medium, and incubated in the presence of RANKL for 24 h. After washing, cells remaining attached were stained for TRAP. TRAP-positive viable cells were scored. For apoptosis assays, cells were pretreated with MCD for 30 min, washed, and incubated with RANKL for 9 h. Cells were fixed with 10% formaldehyde and stained with 1 µg/ml 4',6-diamidino-2-phenylindole for 20 min. Cells displaying condensed chromatin or fragmented nuclei were considered apoptotic.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression Levels of Flotillin, a Raft Marker Protein, and TRAF6 Increase during Osteoclast Differentiation-- Despite the growing appreciation of the importance of lipid raft microdomains in signaling by membrane receptors, the role played by these microdomains in signaling events needed for osteoclast function has not been addressed. The possibility of raft involvement has been suggested by an increase in glycosphingolipids GM1 and GM3 (i.e. gangliosides enriched in rafts) during osteoclast generation from bone marrow cells (BMC) (29). In this context, RANKL binding to RANK provides essential signals for osteoclast differentiation as well as for the activation and survival of mature osteoclasts (11). We therefore investigated whether lipid rafts were important in RANK signaling and for the bone resorption function of osteoclasts. We first examined the expression profile of the raft marker protein flotillin in osteoclasts relative to the caveolae marker protein caveolin and other molecules implicated in RANK signaling. Osteoclast differentiation was induced by culturing mouse BMC with M-CSF plus RANKL or Raw264.7 cells with RANKL only. Under these conditions, flotillin expression was found to increase during osteoclastogenesis from BMC (Fig. 1A, top panel, bands indicated by an arrow). Expression levels in human endothelial cells served as control. A slightly lower band reactive to the flotillin antibody was also present at a high level in unstimulated bone marrow cells and gradually decreased as osteoclastogenesis progressed (indicated by an asterisk). Although we do not presently know the identify this band, it may be flotillin from cell types other than osteoclast lineage, such as lymphocytes, present at the beginning but which gradually disappear during the bone marrow cell culture, and flotillin in these cell types may have slightly different mobility. This may be supported by the appearance of only a single band in blots of Raw264.7 cell lysates with the same antibody (Fig. 1B, top panel). Caveolin-1 was hardly detected in BMC, which was in striking contrast to the high expression level in endothelial cells (Fig. 1A, second panel). During the bone marrow differentiation into osteoclasts, the expression level of TRAF6 increased, whereas that of TRAF2 rather slightly decreased (Fig. 1A). The Src protein level was found to be greatly elevated during the osteoclast differentiation from BMC (Fig. 1A).


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 1.   The raft protein flotillin and TRAF6 increase during osteoclastogenesis. A, mouse bone marrow cells were cultured in an osteoclastogenic medium (30 ng/ml M-CSF plus 50 ng/ml RANKL) for 6 days, and total cell lysates were prepared as described under "Experimental Procedures." 30 µg of lysates from cells cultured for the indicated days and 10 µg of human endothelial cell lysate were resolved and subjected to Western blotting. B, Raw264.7 cells were cultured for 4 days with RANKL (100 ng/ml). Cells were lysed, and 30 µg of total cell lysates were analyzed by Western blotting.

In Raw264.7 cells induced to differentiate to osteoclasts in the presence of RANKL, similar patterns of increase were observed in the expression of flotillin, TRAF6, and Src (Fig. 1B). Caveolin-1 was not detected in these cells (Fig. 1B). Different from BMC, the TRAF2 expression increased during osteoclast formation from Raw264.7 cells (Fig. 1B).

RANK Is Localized in Rafts in Osteoclasts-- The increase in the raft component flotillin and the two important molecules for osteoclast function TRAF6 and Src during osteoclast formation (Fig. 1) suggested that rafts may function in RANK signaling. To explore this possibility, we first examined the potential of RANK associating with rafts in cells that overexpress RANK. The human embryonic kidney cell line 293 was transfected with a RANK expression plasmid. RANK then was detected with TRITC-conjugated RANKL. At the same time, surface membrane rafts were detected using FITC-conjugated cholera toxin B subunit that binds to the marker GM1, as described (30). Under this regime, immunofluorescence microscopy revealed the colocalization of RANK and GM1 throughout the plasma membrane (Fig. 2A, top panel). When GM1 was cross-linked to induce formation of raft patches, concentrated double staining of RANK and GM1 was observed at these places (Fig. 2A, bottom panel). Next, we sought to obtain evidence for the raft localization of RANK endogenously expressed in osteoclasts. Osteoclasts were generated from human blood mononuclear cells, and raft fractions were separated by sucrose density gradient centrifugation as described (31). A substantial portion of endogenous RANK was detected in the low density raft fractions, where the raft marker protein flotillin was exclusively present (Fig. 2B).


View larger version (39K):
[in this window]
[in a new window]
  		Fig. 2.   RANK is associated with membrane rafts. A, 293 cells were transfected with RANK and cultured for 40 h. Cells were incubated with TRITC-RANKL to label RANK, fixed, and stained with FITC-Ctx to label GM1 as described under "Experimental Procedures" (top). Alternatively, cells were stained with TRITC-RANKL and FITC-Ctx, and GM1 was cross-linked with anti-Ctx before fixation of cells (bottom). Fluorescence microscopic images of red and green channels and the merged pictures are shown. B, osteoclasts generated from human peripheral blood mononuclear cells were lysed in a buffer containing 0.5% Brij 58, and the lysates were fractionated on sucrose density gradient as described under "Experimental Procedures." Fractions were collected from the top of the gradients and subjected to Western blotting with an antiserum against human RANK. The same membrane was stripped and reprobed with anti-flotillin antibody.

TRAF6 but Not TRAF2 Is Localized in Rafts in Osteoclasts Differentiated from Raw264.7 Cells-- We next examined whether the localization of TRAF6, TRAF2, and Src could be regulated by RANKL in the Raw264.7 cell line. These cells have been shown to differentiate to cells that display features unique to osteoclasts including the bone resorption activity in response to RANKL stimulation (32). Raft complexes are resistant to solubilization in nonionic detergents at low temperatures. When cells were lysed in 0.5% Brij 58 (a mild nonionic detergent) below 4 °C, the raft protein flotillin was exclusively localized in the insoluble fraction (Fig. 3A, bottom panel, lanes 4-6), confirming that the raft separation was successful. In the undifferentiated Raw264.7 cells, a negligible amount TRAF6 was detected in the detergent-resistant fraction (Fig. 3A, top panel, lane 4). However, when these cells were induced to differentiate by RANKL, a significant amount of detergent-insoluble TRAF6 was observed (Fig. 3A, top panel, lane 6). In contrast, TRAF2 remained detergent-soluble during osteoclastogenesis of these cells (Fig. 3A, second panel). A substantial amount of Src, which is known to be associated with rafts (33), was also detected in the insoluble fraction (Fig. 3A, third panel). The increase in the amount of TRAF6 and Src in the detergent-insoluble fraction during differentiation of these cells could be due to either simple induction of the protein expression level and nonselective subcellular allocation or a response regulated by the RANKL cytokine added to drive the differentiation. To investigate the latter possibility, subcellular distribution of TRAF6 and Src in response to short exposure to RANKL was examined in the differentiated cells. The osteoclasts differentiated from Raw264.7 in the presence of RANKL were depleted of the cytokine to restore the receptor to an inactive state. Cells were then restimulated with RANKL for 15 min, and the protein distribution was assessed. RANKL stimulation caused the translocation of TRAF6 from 5 min (Fig. 3B, top panel). Again, translocation of TRAF2 could not be observed (Fig. 3B, second panel). A substantial amount of Src remained in the detergent-insoluble fraction in the RANKL-deprived cells (Fig. 3B, third panel, lane 4), and this level did not change by the restimulation with RANKL (lanes 5 and 6).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 3.   RANKL induces TRAF6 translocation into the detergent insoluble fraction. A, Raw264.7 cells were driven to differentiate to osteoclasts by culturing in the presence of RANKL (100 ng/ml) for 4 days. Cells on day 0, 2, and 4 were lysed in a buffer containing 0.5% Brij 58 as described under "Experimental Procedures." 30 µg of lysates from the soluble and insoluble fractions were subjected to Western blotting with the indicated antibodies. B, osteoclasts generated by culturing Raw264.7 cells with RANKL for 4 days were incubated in medium containing 0.5% FBS and no RANKL for 4 h. Cells were restimulated with RANKL (1 µg/ml) for the indicated times and lysed. The Brij 58-soluble and -insoluble lysates were immunoblotted.

Raft-disrupting Agents Block TRAF6 Translocation and Akt Activation Induced by RANKL-- To evaluate the significance of RANK-induced TRAF6 translocation to rafts (Fig. 3), we next assessed the effects of MCD and filipin on TRAF6 distribution and RANK signaling. MCD and filipin have been reported to extract and sequester, respectively, cholesterol and thereby disrupt raft microdomains (34, 35). In osteoclasts derived from Raw264.7 cells, MCD treatment abolished the effect of RANKL on TRAF6 translocation (Fig. 4, top panel). The amount of Src in the insoluble fraction, which was not affected by RANKL stimulation, was reduced in MCD-treated cells (Fig. 4, second panel). Again, TRAF2 was hardly detected in the insoluble fraction of these cells (data not shown). These observations demonstrate that the integrity of rafts is needed for RANK/RANKL-induced translocation of TRAF6 to this structure.


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 4.   MCD treatments block TRAF6 translocation. Osteoclasts generated from Raw264.7 cells were incubated in medium containing 0.5% FBS for 4 h, pretreated with or without MCD (15 mM) for 30 min, and then stimulated with RANKL (1 µg/ml) for the indicated time. Cells were lysed in a buffer containing 0.5% Brij 58, and the insoluble fractions were subjected to Western blotting with antibodies for the indicated proteins.

Given this effect, we next examined effects of raft disruption on RANK signaling. RANKL-induced signaling in osteoclasts has been shown to activate Akt/protein kinase B, MAPKs, and NF-kappa B (22, 26, 36). RANKL-induced Akt/PKB activation was confirmed in osteoclasts generated from Raw264.7 (Fig. 5A, top panel set, lanes 1-3). By contrast, MCD treatment blocked the Akt activation by RANKL (lanes 4-6). On the other hand, the same treatment did not inhibit the RANKL activation of JNK and NF-kappa B (Fig. 5A, middle and bottom sets). Instead, MCD treatment rather potentiated the JNK and NF-kappa B activation by RANKL. The effect of MCD on extracellular signal-regulated kinase activation was similar to JNK, whereas RANKL activation of p38 was hardly detected in these cells (data not shown). When filipin was used, similar results were obtained as with MCD (Fig. 5B).


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 5.   Raft disruption inhibits RANKL-induced Akt activation. A, osteoclasts derived from Raw264.7 cells were serum-starved in medium containing 0.5% FBS for 4 h, pretreated with or without MCD (10 mM) for 30 min, and stimulated with RANKL (500 ng/ml) for the indicated time. The activated forms of Akt, JNK, and Ikappa B in whole-cell extracts were detected with phospho-specific antibodies. Membranes were stripped and reprobed with the control antibodies. B, Raw264.7 cell-derived osteoclasts were treated as in A, except that filipin (5 µg/ml) was used to disrupt rafts. C, osteoclasts were derived from bone marrow cells and purified as described under "Experimental Procedures." Cells were then treated as in A, and cell lysates were subjected to Western blotting analyses.

In addition to these observations of raft involvement in the transformed model cell line of osteoclasts, it was also important to assess the role of rafts in osteoclasts derived from bone marrow cells. Osteoclasts were generated from hematopoietic cells by coculturing bone marrow cells with calvarial osteoblasts as described under "Experimental Procedures." Also in these cells, RANKL activation of Akt was blocked by MCD (Fig. 5C, top), whereas that of NF-kappa B was not affected (Fig. 5C, bottom). JNK activation was not observed in these cells in most cases (Fig. 5C, middle). Activation of extracellular signal-regulated kinase and p38 was rarely detectable, and, when detected, MCD did not inhibit the activation (data not shown). Overall, our findings demonstrate that raft expression on osteoclasts plays a key role in ensuring optimal downstream signaling via RANK/RANKL ligation. Further, it shows a specific requirement for raft integrity in the activation of the Akt/PKB pathway without affecting the activation of JNK and NF-kappa B. To our knowledge, this is the first reported instance of a dissection of receptor-mediated signaling requiring the differential involvement of lipid rafts.

Raft Disruption Blocks Actin Ring Formation in and Bone Resorption by Osteoclasts-- The perturbations of RANK signaling by compounds that disrupt rafts (Fig. 5) suggest that rafts are likely to play a crucial role for osteoclast function. To address this issue, we next investigated whether the raft-disrupting agents affect formation of actin ring, a unique cytoskeletal structure required for bone resorption by osteoclasts (10). Staining F-actin with rhodamin-phalloidin showed a ring structure in osteoclasts generated from Raw264.7 cells, which became more dense and smooth with the addition of RANKL stimulation (Fig. 6A, middle). Significantly, MCD treatment destroyed the ring structure in the presence and absence of RANKL, showing a pattern where the F-actin was dispersed toward the inside (Fig. 6A, right). Further, the effect of raft disruption on the actin ring was more dramatic in osteoclasts derived from bone marrow cells (Fig. 6B). Filipin treatment had a similar effect on the actin ring staining (data not shown).


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 6.   Rafts are required for actin ring formation induced by RANKL. A, osteoclasts generated from Raw264.7 cells were incubated in medium containing 0.5% FBS for 4 h. Cells were incubated with or without MCD (10 mM) for 30 min, washed with medium, and stimulated with RANKL (500 ng/ml) for 30 min. Cells were fixed and stained for F-actin with rhodamin-phalloidin. B, purified osteoclasts derived from bone marrow cells were incubated in medium containing 0.5% FBS for 1 h. Cells were treated with MCD (10 mM) for 15 min and then stimulated with RANKL (100 ng/ml) for 30 min. Cells were fixed and stained for F-actin.

We next examined the effect of raft disruption on the bone resorption activity of osteoclasts. Osteoclasts derived from bone marrow cells were placed on dentine slices and exposed to MCD for a short time (30 min) before the resorption period or to filipin for the whole incubation period (12 h). Consistent with the result of actin ring experiments, raft disruption by MCD or filipin led to the inhibition of bone resorption by osteoclasts (Fig. 7A). Both the area and the number of resorption pits were reduced, suggesting that both resorption activity per se and migration of osteoclasts were affected by the MCD treatment (Fig. 7, B and C). These results underlie the importance of raft microdomains for proper cytoskeletal organization, which is necessary for the resorption function of osteoclasts.


View larger version (56K):
[in this window]
[in a new window]
  		Fig. 7.   Raft disruption inhibits the bone resorption function of osteoclasts. Mature osteoclasts generated by co-culturing bone marrow cells with osteoblasts as described under "Experimental Procedures" were plated on dentine slices. After incubation for 2 h to allow cell attachment to dentine, MCD (10 mM for A and indicated doses for B and C) or filipin (2.5 µg/ml) was added. After 30 min, MCD-treated dentine slices were washed with medium, and incubation was continued for 12 h. Cells were removed from dentine slices, and resorbed pits were visualized by staining with hematoxylin before taking photographs. Total areas and the number of resorption pits were determined with an image analysis program.

Rafts Are Required for Survival of Osteoclasts-- Given the importance of rafts in the RANKL-induced Akt/PKB activation in osteoclasts (Fig. 5) and the importance of Akt in preventing the induction of apoptosis in cells (22, 27), we next examined whether raft disruption would affect the survival of osteoclasts. In this context, mature osteoclasts undergo apoptosis in the absence of a survival factor such as RANKL, TNF-alpha , and M-CSF (27, 36, 37). As previously reported, RANKL stimulated osteoclast survival (Fig. 8A). Treatment with MCD reduced the RANKL stimulation of osteoclast survival in a dose-dependent manner (Fig. 8A, right panel). Also, the effect of MCD on osteoclast apoptosis was assessed by counting cells displaying nuclear fragmentation and chromatin condensation, visualized by staining with 4',6-diamidino-2-phenylindole (Fig. 8B, indicated by arrows). RANKL reduced the portion of apoptotic cells. Prior exposure to MCD abrogated the antiapoptotic effect of RANKL (Fig. 8B). Overall, our findings show that membrane rafts are required for RANKL/RANK regulation of osteoclast survival, a finding that is consistent with the importance of rafts in the activation of antiapoptotic regulator Akt/PKB by RANK (Fig. 5).


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 8.   Effects of MCD on the survival of purified osteoclasts. A, purified osteoclasts were prepared from the co-culture of bone marrow cells and osteoblasts. After pretreatment with MCD at indicated doses for 30 min, cells were washed and incubated in the presence (100 ng/ml) or absence of RANKL for 24 h. Detached cells were washed away, and remained cells were stained for TRAP. Multinucleated TRAP-positive cells were counted. B, purified osteoclasts were pretreated with MCD for 30 min. After washing, cells were treated with RANKL (100 ng/ml) for 9 h. Cells were fixed and stained with 4',6-diamidino-2-phenylindole. The proportion of cells with apoptotic nuclei was assessed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we provided important evidence for the role of membrane lipid rafts in responses of osteoclasts to RANK stimulation. Activation of RANK provoked the translocation of TRAF6 into the detergent-insoluble raft fraction where Src was constitutively present (Figs. 3 and 4). Raft disruption with cholesterol sequestering agents selectively impaired the RANKL signaling pathways of Akt but not those of MAPK and NF-kappa B in osteoclasts (Fig. 5). Furthermore, raft-disrupting agents destroyed the integrity of actin ring structure and hampered the RANKL stimulation of bone-resorbing activity and cell survival of osteoclasts (Figs. 6-8). Overall, we presented data demonstrating that the raft microdomain is essential for the proper signaling by RANK and the cellular function of osteoclasts. To our knowledge, our report is the first to implicate rafts in RANK signaling and osteoclast activation.

The role of lipid rafts as a signaling platform has been well recognized for immune cell surface receptors such as TCR, BCR, and Fcepsilon R (3). More recently, the raft association and its requirement for signaling have been demonstrated for the TNFR family members CD40 and TNFR1 (5-8). Accordingly, TRAF2 and TRAF3 were shown to translocate into rafts in response to CD40 ligation (7, 8). In addition, TRAF1 was suggested to play a role in regulating the raft localization of TRAF2 for sustained CD40 signaling (38). On the other hand, TRAF6 was found to remain unassociated with rafts in CD40-stimulated B cells (8). TRAF6 has been shown to be more essential for osteoclast activation than other TRAF proteins (19, 39). In this study, we detected the raft association of endogenous TRAF6 in conditions under which RANK is engaged. Stimulation with RANKL induced TRAF6 recruitment to rafts in osteoclasts differentiated from Raw264.7 cells (Figs. 3B and 4). An increase in the amount of TRAF6 protein in the detergent-insoluble fraction also occurred during the RANKL-driven osteoclastogenesis of Raw264.7 cells (Fig. 3A). To the contrary, raft association of TRAF2 was hardly detected in osteoclasts (Fig. 3). The selective recruitment of TRAF6 to rafts in osteoclasts may be in part accountable for the more pronounced role of TRAF6 than that of other TRAFs for RANK signaling in osteoclasts.

One intriguing observation of our study was selective dependence of RANK-mediated pathways but not others on the integrity of rafts. Raft disruption impaired the RANKL signaling pathways of Akt/PKB but not those of MAPK and NF-kappa B in osteoclasts (Fig. 5). The selective involvement of Akt was consistent with the requirement for rafts in protection against apoptosis. Akt promotes cell survival by phosphorylating and inactivating proapoptotic molecules and by modulating the transcription of survival and death genes (40). Given that Src family kinases can stimulate PI3K activity (41), that PI3K activity is critical for Akt activation (42), and that pharmacological inhibitors of PI3K or Src family kinases decrease the Akt activation in and the survival of osteoclasts (22, 27), it has been postulated that Src mediates, through PI3K, the activation of Akt by RANKL and thereby increases osteoclast survival. Our findings that raft disruption blocked RANKL-induced Akt activation (Fig. 5) and cell survival (Fig. 8) underscore the importance of raft microdomains for the RANKL-induced activation of Akt and subsequent antiapoptotic signaling cascades in osteoclasts.

Another protein of key importance to osteoclast function is the nonreceptor protein-tyrosine kinase pp60src (23-25). The expression of Src greatly increases during osteoclast differentiation (see Ref. 43 and Fig. 1). As it has been reported in other cell types (33, 44), the raft association of Src was evidently observed in osteoclasts (Fig. 3). RANKL stimulation of the catalytic activity of Src was reported, and the recruitment of TRAF6 to form a trimolecular complex with RANK and Src was suggested to result in the Src activation (22). In our study, the constitutive level of Src in the detergent-insoluble raft fraction did not significantly change in response to RANKL (Figs. 3 and 4). Given that RANKL induces TRAF6 translocation into the detergent-insoluble fraction (Figs. 3 and 4), it is possible that Src, resident in rafts, functions as a docking site for the RANK signaling complexes containing TRAF6. Consistent with this notion, the expression of Src251, a truncated Src mutant that functions in a dominant negative manner and reduced the level of endogenous wild-type Src in the detergent-insoluble fraction, was implied to alter the detergent solubility of TRAF6 (45). Whereas the question of whether the recruitment of TRAF6 to Src in raft microdomains is a prerequisite for Src activation by RANKL and, if it is, exactly how TRAF6 activates Src remains to be addressed, our preliminary data support a role of lipid rafts for RANKL activation of Src by showing the detrimental effect of raft disruption on the Src activation by RANKL in osteoclasts (data not shown).

Osteoclasts activated to resorb bone display a distinct organization of F-actin, a ring or belt-like cytoskeletal architecture called actin ring (10). The actin ring structure is required for the formation of the sealing zone and the bone resorption function of osteoclasts. Accumulating evidence indicates that TRAF6 is pivotal for the resorption function of osteoclasts. Mice deficient in TRAF6 displayed an osteopetrotic phenotype resulting from the defective resorption activity of osteoclasts. Osteoclasts in these mice lacked the sealing zone and the ruffled border (18). Recently, it was shown that the retroviral gene transfer of RANK lacking the TRAF6 binding site into hematopoietic progenitor cells resulted in the generation of osteoclasts defective in the actin ring formation and resorption capability (39). Our finding that disruption of rafts impaired the resorption activity of osteoclasts with the concomitant loss of the actin ring integrity (Figs. 6 and 7) indicates that rafts are required for osteoclast function. However, we cannot completely exclude the possibility that the cholesterol extraction had effects on other aspects of cellular integrity and affected osteoclast function in a way independent of rafts.

Rafts in osteoclasts may function as the platform for a network of signaling molecules that start to assemble in response to external stimuli such as RANKL. In the assembly process, RANKL-induced recruitment of TRAF6 to RANK and to rafts may be an early event (Fig. 9). The raft recruitment may stimulate the interaction of Src, resident constitutively in rafts in osteoclasts, with the RANK-TRAF6 complexes (22), leading to the activation of Src and PI3K. On one hand, the lipid product of PI3K activity, phosphatidylinositol 3,4,5-trisphosphate, mediates recruitment of pleckstrin homology domain proteins PDK1 and Akt and consequent activation of Akt. Activated Akt then operates antiapoptotic machinery for osteoclast survival. On the other hand, Src may provoke tyrosine phosphorylation of other protein-tyrosine kinases and adaptor proteins, such as Pyk2 and p130cas (46, 47), and perhaps actin-binding proteins like cortactin (48). These molecules may work in concert with PI3K downstream targets known to regulate cytoskeletal organization, such as Rho, Rac, and ARF6 (42, 49, 50), to construct and stabilize the actin ring structure. Overall, the requirement for rafts in the stability of the actin ring, which may be crucial for the maintenance of polarity of osteoclasts, and in the increased cell survival mechanism via Akt/PKB would be critical for proper bone resorption function.


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 9.   A model of raft involvement in RANK signaling for osteoclast function. Upon RANKL stimulation, the raft microdomains recruit and concentrate the RANK-TRAF6 complex and facilitate its interaction with Src. Consequently, Src- and PI3K-dependent signaling pathways are triggered. The recruitment of PDK1 and Akt to the plasma membrane through the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3) results in Akt activation, which turns on antiapoptotic machinery. Src provokes cascades of coordinated interactions of proteins including tyrosine kinases, adaptors, and actin-binding proteins, some of which are also downstream of PI3K, for organization of the actin ring, the structure required for resorption function of osteoclasts.

Detailed analyses of the components recruited to rafts in response to stimuli of osteoclasts may provide an insight into the mechanism by which osteoclasts are activated. In addition, such analyses may reveal new molecular targets for the development of antiresorptive drugs.

    ACKNOWLEDGEMENTS

We thank Drs. N. Takahashi and N. Udagawa (Matsumoto Dental University, Japan) for advice on osteoclast cultures.

    FOOTNOTES

* This work was supported by grants from the Ministry of Science and Technology, Korea and the Korea Science and Engineering Foundation through the Research Center for Proteineous Materials, the National Research Laboratory, and 21C Frontier Functional Proteomics Project Grant FPR02A3-5-110.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence may be addressed: Chosun University School of Dentistry, 375 Seosuk-Dong, Dong-Gu, Gwangju 501-759, Korea. Tel.: 82-62-230-6853; Fax: 82-62-227-6589; E-mail: jhblee@chosun.ac.kr.

§§ To whom correspondence may be addressed: College of Dentistry, Seoul National University, Seoul 110-749, Korea. Tel.: 82-2-740-8686; Fax: 82-2-765-8656; E-mail: hhbkim@snu.ac.kr or hhbkim{at}yahoo.com.

Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M212626200

    ABBREVIATIONS

The abbreviations used are: GPI, glycosylphosphatidylinositol; TNF, tumor necrosis factor; TNFR, TNF receptor; TRAF, TNF receptor-associated factor; RANK, receptor activator of NF-kappa B; RANKL, RANK ligand; M-CSF, macrophage colony-stimulating factor; TRAP, tartrate-resistant acid phosphatase; BMC, bone marrow cell; JNK, c-Jun N-terminal kinase; MAPK, mitogen activated protein kinase; PI3K, phosphoinositide 3-kinase; MCD, methyl-beta -cyclodextrin; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; Ctx, choleratoxin; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; GM1, monosialoganglioside 1; GM3, monosialoganglioside 3.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Simons, K., and Toomre, D. (2000) Nat. Rev. Mol. Cell. Biol. 1, 31-39[CrossRef][Medline] [Order article via Infotrieve]
2. Brown, D. A., and London, E. (1998) Annu. Rev. Cell Dev. Biol. 14, 111-136[CrossRef][Medline] [Order article via Infotrieve]
3. Cherukuri, A., Dykstra, M., and Pierce, S. K. (2001) Immunity 14, 657-660[CrossRef][Medline] [Order article via Infotrieve]
4. Bilderback, T. R., Grigsby, R. J., and Dobrowsky, R. T. (1997) J. Biol. Chem. 272, 10922-10927[Abstract/Free Full Text]
5. Ko, Y. G., Lee, J. S., Kang, Y. S., Ahn, J. H., and Seo, J. S. (1999) J. Immunol. 162, 7217-7223[Abstract/Free Full Text]
6. Cottin, V., Doan, J. E., and Riches, D. W. (2002) J. Immunol. 168, 4095-4102[Abstract/Free Full Text]
7. Vidalain, P. O., Azocar, O., Servet-Delprat, C., Rabourdin-Combe, C., Gerlier, D., and Manie, S. (2000) EMBO J. 19, 3304-3313[CrossRef][Medline] [Order article via Infotrieve]
8. Hostager, B. S., Catlett, I. M., and Bishop, G. A. (2000) J. Biol. Chem. 275, 15392-15398[Abstract/Free Full Text]
9. Feng, X., Gaeta, M. L., Madge, L. A., Yang, J. H., Bradley, J. R., and Pober, J. S. (2001) J. Biol. Chem. 276, 8341-8349[Abstract/Free Full Text]
10. Bilezikian, J. P., Raisz, L. G., and Rodan, G. A. (2002) Principles of Bone Biology , 2nd Ed. , Academic Press, Inc., San Diego, CA
11. Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M. T., and Martin, T. J. (1999) Endocr. Rev. 20, 345-357[Abstract/Free Full Text]
12. Teitelbaum, S. L. (2000) Science 289, 1504-1508[Abstract/Free Full Text]
13. Kim, H.-H., Lee, D. E., Shin, J. N., Lee, Y. S., Jeon, Y. M., Chung, C. H., Ni, J., Kwon, B. S., and Lee, Z. H. (1999) FEBS Lett. 443, 297-302[CrossRef][Medline] [Order article via Infotrieve]
14. Darnay, B. G., Ni, V. J., Moore, P. A., and Aggarwal, B. B. (1998) J. Biol. Chem. 273, 20551-20555[Abstract/Free Full Text]
15. Galibert, L., Tometsko, M. E., Anderson, D. M., Cosman, D., and Dougall, W. C. (1998) J. Biol. Chem. 273, 34120-34127[Abstract/Free Full Text]
16. Wong, B. R., Josien, R., Lee, S. Y., Vologodskaia, M., Steinman, R. M., and Choi, Y. (1998) J. Biol. Chem. 273, 28355-28359[Abstract/Free Full Text]
17. Naito, A., Azuma, S., Tanaka, S., Miyazaki, T., Takaki, S., Takatsu, K., Nakao, K., Nakamura, K., Katsuki, M., Yamamoto, T., and Inoue, J. (1999) Genes Cells 4, 353-362[Abstract]
18. Lomaga, M. A., Yeh, W. C., Sarosi, I., Duncan, G. S., Furlonger, C., Ho, A., Morony, S., Capparelli, C., Van, G., Kaufman, S., van der Heiden, A., Itie, A., Wakeham, A., Khoo, W., Sasaki, T., Cao, Z., Penninger, J. M., Paige, C. J., Lacey, D. L., Dunstan, C. R., Boyle, W. J., Goeddel, D. V., and Mak, T. W. (1999) Genes Dev. 13, 1015-1024[Abstract/Free Full Text]
19. Kobayashi, N., Kadono, Y., Naito, A., Matsumoto, K., Yamamoto, T., Tanaka, S., and Inoue, J. (2001) EMBO J. 20, 1271-1280[CrossRef][Medline] [Order article via Infotrieve]
20. Kong, Y. Y., Yoshida, H., Sarosi, I., Tan, H. L., Timms, E., Capparelli, C., Morony, S., Oliveira-dos-Santos, A. J., Van, G., Itie, A., Khoo, W., Wakeham, A., Dunstan, C. R., Lacey, D. L., Mak, T. W., Boyle, W. J., and Penninger, J. M. (1999) Nature 397, 315-523[CrossRef][Medline] [Order article via Infotrieve]
21. Dougall, W. C., Glaccum, M., Charrier, K., Rohrbach, K., Brasel, K., De Smedt, T., Daro, E., Smith, J., Tometsko, M. E., Maliszewski, C. R., Armstrong, A., Shen, V., Bain, S., Cosman, D., Anderson, D., Morrissey, P. J., Peschon, J. J., and Schuh, J. (1999) Genes Dev. 13, 2412-2424[Abstract/Free Full Text]
22. Wong, B. R., Besser, D., Kim, N., Arron, J. R., Vologodskaia, M., Hanafusa, H., and Choi, Y. (1999) Mol. Cell 4, 1041-1049[CrossRef][Medline] [Order article via Infotrieve]
23. Soriano, P., Montgomery, C., Geske, R., and Bradley, A. (1991) Cell 64, 693-702[CrossRef][Medline] [Order article via Infotrieve]
24. Boyce, B. F., Yoneda, T., Lowe, C., Soriano, P., and Mundy, G. R. (1992) J. Clin. Invest. 90, 1622-1627[Medline] [Order article via Infotrieve]
25. Lowe, C., Yoneda, T., Boyce, B. F., Chen, H., Mundy, G. R., and Soriano, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4485-4489[Abstract/Free Full Text]
26. Lee, S. E., Woo, K. M., Kim, S. Y., Kim, H.-M., Kwack, K., Lee, Z. H., and Kim, H.-H. (2002) Bone 30, 71-77[Medline] [Order article via Infotrieve]
27. Lee, S. E., Chung, W. J., Kwak, H. B., Chung, C. H., Kwack, K. B., Lee, Z. H., and Kim, H.-H. (2001) J. Biol. Chem. 276, 49343-49349[Abstract/Free Full Text]
28. Shin, J. N., Kim, I., Lee, J. S., Koh, G. Y., Lee, Z. H., and Kim, H. -H. (2002) J. Biol. Chem. 277, 8346-8353[Abstract/Free Full Text]
29. Iwamoto, T., Fukumoto, S., Kanaoka, K., Sakai, E., Shibata, M., Fukumoto, E., Inokuchi, J., Takamiya, K., Furukawa, K., Furukawa, K., Kato, Y., and Mizuno, A. (2001) J. Biol. Chem. 276, 46031-46038[Abstract/Free Full Text]
30. Martin, M., Schneider, H., Azouz, A., and Rudd, C. E. (2001) J. Exp. Med. 194, 1675-1681[Abstract/Free Full Text]
31. Brown, D. A., and Rose, J. K. (1992) Cell 68, 533-544[CrossRef][Medline] [Order article via Infotrieve]
32. Hsu, H., Lacey, D. L., Dunstan, C. R., Solovyev, I., Colombero, A., Timms, E., Tan, H. L., Elliott, G., Kelley, M. J., Sarosi, I., Wang, L., Xia, X. Z., Elliott, R., Chiu, L., Black, T., Scully, S., Capparelli, C., Morony, S., Shimamoto, G., Bass, M. B., and Boyle, W. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3540-3545[Abstract/Free Full Text]
33. Waheed, A. A., Shimada, Y., Heijnen, H. F., Nakamura, M., Inomata, M., Hayashi, M., Iwashita, S., Slot, J. W., and Ohno-Iwashita, Y. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4926-4931[Abstract/Free Full Text]
34. Kilsdonk, E. P., Yancey, P. G., Stoudt, G. W., Bangerter, F. W., Johnson, W. J., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 17250-17256[Abstract/Free Full Text]
35. Vereb, G., Matko, J., Vamosi, G., Ibrahim, S. M., Magyar, E., Varga, S., Szollosi, J., Jenei, A., Gaspar, R., Jr., Waldmann, T. A., and Damjanovich, S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6013-6018[Abstract/Free Full Text]
36. Jimi, E., Akiyama, S., Tsurukai, T., Okahashi, N., Kobayashi, K., Udagawa, N., Nishihara, T., Takahashi, N., and Suda, T. (1999) J. Immunol. 163, 434-442[Abstract/Free Full Text]
37. Lacey, D. L., Tan, H. L., Lu, J., Kaufman, S., Van, G., Qiu, W., Rattan, A., Scully, S., Fletcher, F., Juan, T., Kelley, M., Burgess, T. L., Boyle, W. J., and Polverino, A. J. (2000) Am. J. Pathol. 157, 435-448[Abstract/Free Full Text]
38. Arron, J. R., Pewzner-Jung, Y., Walsh, M. C., Kobayashi, T., and Choi, Y. (2002) J. Exp. Med. 196, 923-934[Abstract/Free Full Text]
39. Armstrong, A. P., Tometsko, M. E., Glaccum, M., Sutherland, C. L., Cosman, D., and Dougall, W. C. (2002) J. Biol. Chem. 277, 44347-44356[Abstract/Free Full Text]
40. Datta, S. R., Brunet, A., and Greenberg, M. E. (1999) Genes Dev. 13, 2905-2927[Free Full Text]
41. Pleiman, C. M., Hertz, W. M., and Cambier, J. C. (1994) Science 263, 1609-1612[Abstract/Free Full Text]
42. Cantley, L. C. (2002) Science 296, 1655-1657[Abstract/Free Full Text]
43. Horne, W. C., Neff, L., Chatterjee, D., Lomri, A., Levy, J. B., and Baron, R. (1992) J. Cell Biol. 119, 1003-1013[Abstract/Free Full Text]
44. Tansey, M. G., Baloh, R. H., Milbrandt, J., and Johnson, E. M., Jr. (2000) Neuron 25, 611-623[CrossRef][Medline] [Order article via Infotrieve]
45. Xing, L., Venegas, A. M., Chen, A., Garrett-Beal, L. B., Boyce, F., Varmus, H. E., and Schwartzberg, P. L. (2001) Genes Dev. 15, 241-253[Abstract/Free Full Text]
46. Duong, L. T., Lakkakorpi, P. T., Nakamura, I., Machwate, M., Nagy, R. M., and Rodan, G. A. (1998) J. Clin. Invest. 102, 881-892[Medline] [Order article via Infotrieve]
47. Lakkakorpi, P. T., Nakamura, I., Nagy, R. M., Parsons, J. T., Rodan, G. A., and Duong, L. T. (1999) J. Biol. Chem. 274, 4900-4907[Abstract/Free Full Text]
48. Wu, H., and Parsons, J. T. (1993) J. Cell Biol. 120, 1417-1426[Abstract/Free Full Text]
49. Zhang, D., Udagawa, N., Nakamura, I., Murakami, H., Saito, S., Yamasaki, K., Shibasaki, Y., Morii, N., Narumiya, S., Takahashi, N., and Suda, T. (1995) J. Cell Sci. 108, 2285-2292[Abstract]
50. Lakkakorpi, P. T., Wesolowski, G., Zimolo, Z., Rodan, G. A., and Rodan, S. B. (1997) Exp. Cell Res. 237, 296-306[CrossRef][Medline] [Order article via Infotrieve]

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
H. J. Kim, Y. Lee, E.-J. Chang, H.-M. Kim, S.-P. Hong, Z. H. Lee, J. Ryu, and H.-H. Kim
Suppression of Osteoclastogenesis by N,N-Dimethyl-D-erythro-sphingosine: A Sphingosine Kinase Inhibition-Independent Action
Mol. Pharmacol., August 1, 2007; 72(2): 418 - 428.
[Abstract] [Full Text] [PDF]