From the 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 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.
Reagents--
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
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
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
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- 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- 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.
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).
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).
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).
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.
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-
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- 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).
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.
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- 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- The role of lipid rafts as a signaling platform has been well
recognized for immune cell surface receptors such as TCR, BCR, and
Fc 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- 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.
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
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-
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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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-
-D-glucopyranoside,
methyl-
-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).
-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
-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.
8 M 1
,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).
-minimum essential
medium plus 10% FBS. Generation of osteoclasts was confirmed by the
tartrate-resistant acid phosphatase (TRAP) staining (28).
-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.
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[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.
View larger version (39K):
[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.
View larger version (28K):
[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.
View larger version (48K):
[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.
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-
B
(Fig. 5A, middle and bottom
sets). Instead, MCD treatment rather potentiated the JNK and
NF-
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 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 I 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.
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-
B. To our knowledge, this is
the first reported instance of a dissection of receptor-mediated
signaling requiring the differential involvement of lipid rafts.
View larger version (30K):
[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.
View larger version (56K):
[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.
, 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 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
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.
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.
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.
View larger version (30K):
[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.
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-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-
-cyclodextrin;
FBS, fetal bovine serum;
FITC, fluorescein
isothiocyanate;
Ctx, choleratoxin;
TRITC, tetramethylrhodamine
isothiocyanate;
PBS, phosphate-buffered saline;
GM1, monosialoganglioside 1;
GM3, monosialoganglioside 3.
![]() |
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 |
5. |
Ko, Y. G.,
Lee, J. S.,
Kang, Y. S.,
Ahn, J. H.,
and Seo, J. S.
(1999)
J. Immunol.
162,
7217-7223 |
6. |
Cottin, V.,
Doan, J. E.,
and Riches, D. W.
(2002)
J. Immunol.
168,
4095-4102 |
7. |
Vidalain, P. O.,
Azocar, O.,
Servet-Delprat, C.,
Rabourdin-Combe, C.,
Gerlier, D.,
and Manie, S.
(2000)
EMBO J.
19,
3304-3313 |
8. |
Hostager, B. S.,
Catlett, I. M.,
and Bishop, G. A.
(2000)
J. Biol. Chem.
275,
15392-15398 |
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 |
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 |
12. |
Teitelbaum, S. L.
(2000)
Science
289,
1504-1508 |
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 |
15. |
Galibert, L.,
Tometsko, M. E.,
Anderson, D. M.,
Cosman, D.,
and Dougall, W. C.
(1998)
J. Biol. Chem.
273,
34120-34127 |
16. |
Wong, B. R.,
Josien, R.,
Lee, S. Y.,
Vologodskaia, M.,
Steinman, R. M.,
and Choi, Y.
(1998)
J. Biol. Chem.
273,
28355-28359 |
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 |
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 |
19. |
Kobayashi, N.,
Kadono, Y.,
Naito, A.,
Matsumoto, K.,
Yamamoto, T.,
Tanaka, S.,
and Inoue, J.
(2001)
EMBO J.
20,
1271-1280 |
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 |
22. | Wong, B. R., Besser, D., Kim, N., Arron, J. R., Vologodskaia, M., Hanafusa, H., and Choi, Y. (1999) Mol. Cell 4, 1041-1049[Medline] [Order article via Infotrieve] |
23. | Soriano, P., Montgomery, C., Geske, R., and Bradley, A. (1991) Cell 64, 693-702[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] |
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 |
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 |
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 |
30. |
Martin, M.,
Schneider, H.,
Azouz, A.,
and Rudd, C. E.
(2001)
J. Exp. Med.
194,
1675-1681 |
31. | Brown, D. A., and Rose, J. K. (1992) Cell 68, 533-544[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 |
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 |
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 |
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 |
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 |
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 |
38. |
Arron, J. R.,
Pewzner-Jung, Y.,
Walsh, M. C.,
Kobayashi, T.,
and Choi, Y.
(2002)
J. Exp. Med.
196,
923-934 |
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 |
40. |
Datta, S. R.,
Brunet, A.,
and Greenberg, M. E.
(1999)
Genes Dev.
13,
2905-2927 |
41. | Pleiman, C. M., Hertz, W. M., and Cambier, J. C. (1994) Science 263, 1609-1612[Medline] [Order article via Infotrieve] |
42. |
Cantley, L. C.
(2002)
Science
296,
1655-1657 |
43. | Horne, W. C., Neff, L., Chatterjee, D., Lomri, A., Levy, J. B., and Baron, R. (1992) J. Cell Biol. 119, 1003-1013[Abstract] |
44. | Tansey, M. G., Baloh, R. H., Milbrandt, J., and Johnson, E. M., Jr. (2000) Neuron 25, 611-623[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 |
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 |
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 |
48. | Wu, H., and Parsons, J. T. (1993) J. Cell Biol. 120, 1417-1426[Abstract] |
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 |
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] |