From the Department of Pediatrics, Cell and Molecular
Biology Unit, Washington University School of Medicine, St. Louis,
Missouri 63110 and the § Department of Oral and
Maxillofacial Surgery, Mie University School of Medicine, Tsu, Mie,
514-8507, Japan
Received for publication, September 11, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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PTEN (also known as MMAC-1 or TEP-1) is a
frequently mutated tumor suppressor gene in human cancer. PTEN
functions have been identified in the regulation of cell survival,
growth, adhesion, migration, and invasiveness. Here, we characterize
the diverse signaling networks modulated by PTEN in osteoclast
precursors stimulated by RANKL and osteopontin (OPN). RANKL
dose-dependently stimulated transient activation of Akt
before activation of PTEN, consistent with a role for PTEN in
decreasing Akt activity. PTEN overexpression blocked RANKL-activated
Akt stimulated survival and osteopontin-stimulated cell migration while
a dominant-negative PTEN increased the actions of RANKL and OPN. PTEN
overexpression suppressed RANKL-mediated osteoclast differentiation and
OPN-stimulated cell migration. The PTEN dominant-negative
constitutively induced osteoclast differentiation and cell
migration. Our data demonstrate multiple roles for PTEN in
RANKL-induced osteoclast differentiation and OPN-stimulated cell
migration in RAW 264.7 osteoclast precursors.
The tumor suppressor gene
PTEN1
(phosphatase and tensin homolog deleted from
chromosome 10), also known as MMAC1 (mutated in multiple advanced cancers) or TEP1
(TGF- RANKL is expressed by osteoblastic lineage cells and stimulates its
specific receptor, the receptor activator of
NF Osteopontin (OPN) is one of the major noncollagenous bone matrix
proteins produced by osteoblasts and osteoclasts (22-25). It also
stimulates PI3K activity (10-24, 26, 27), which is a target of PTEN
(28) and induces cell migration (26). In addition, a recent study
indicates that Akt is essential for cell migration (29).
The major function of PTEN appears to be down-regulation of the PI3K
product PtdIns(3,4,5)P3, which regulates Akt and complex downstream pathways affecting cell growth, survival, and migration. In
addition, PTEN has weak protein tyrosine phosphatase activity, which
may target focal adhesion kinase (FAK) and Shc, and thereby modulate
other complex pathways (1, 7). In this report, we show that PTEN
regulates the RANKL-activated Akt survival signaling pathway and the
OPN-stimulated cell migration in RAW 264.7 osteoclast precursors.
Moreover, we found that PTEN also regulates RANKL-induced osteoclast
differentiation from RAW 264.7 osteoclast precursors. In addition, we
suggest that RANKL may regulate balance of activated Akt and activated
PTEN and have influence osteoclast differentiation. Thus, it is likely
that PTEN plays multiple roles involving osteoclast formation,
survival, and migration.
Materials--
RAW 264.7 cells were obtained from
American Type Culture Collection (Manassas, VA). Polyclonal anti-Akt,
anti-phospho-Akt (Thr-308), anti-I Cell Cultures and Transient Transfection--
RAW 264.7 cells (a
murine macrophage line capable of RANKL-mediated osteoclastogenesis)
were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen)
supplemented with 10% heat-inactivated fetal bovine serum (FBS). After
24 h, GFP, GFP-PTEN (WT), GFP-PTEN (C124A; phosphatase dead
mutant), Myc (empty), or Myc-Akt (K179M) dominant-negative expression
vector were transiently cotransfected into the cells using the
LipofectAMINE reagent System (Invitrogen) according to the
manufacturer's instructions. Then, the GFP- or GFP-PTEN (WT)-
expressing cells treated with or without RANKL were fixed and stained
for tartrate-resistant acid phosphatase (TRAP) to observe cell morphology.
Mouse bone marrow macrophages (BMMs) were prepared from the femur and
tibia of 4-6-week-old C57BL/6 mice and incubated in tissue culture
dishes (100-mm dishes) in the presence of recombinant mouse
macrophage-colony-stimulating factor (20 ng/ml). After 24 h
in culture, the non-adherent cells were collected and layered on
Histopaque gradient, and the cells at the gradient interface were
collected. The cells were replated (60-mm dishes) at
65,000/cm2 in Preparation of Cell Lysates and Immunoblotting--
24-36 h
after transfection, medium was removed, and cells were washed two times
with phosphate-buffered saline (PBS) and then cultured in DMEM
serum-free medium for 24 h. For RANKL (100 ng/ml) or OPN (25 µg/ml) stimulation experiments, RANKL or OPN were added to the
culture medium and incubated for 5, 10, 15, 30, and 60 min. The cells
were then washed once with ice-cold PBS and lysed in a cell lysis
buffer (New England Biolabs) to prepare whole cell lysates. Lysates
were clarified by centrifugation at 14,000 × g for 10 min, and protein concentrations in the supernatants were measured using
the Bio-Rad protein assay reagent kit (Bio-Rad). Proteins were resolved
by SDS-PAGE, electroblotted to polyvinylidene difluoride membrane
(Millipore, Bedford, MA), blocked in 5% skim milk, 1× PBS, 0.05%
Tween 20, and probed with primary antibodies. For the detection of
NF Apoptosis Assay--
After cotransfected, cells were treated
with RANKL (100 ng/ml). Then, whole cell lysates were prepared as
above. Lysates were clarified by centrifugation at 14,000 × g for 10 min, and the supernatant fractions were harvested.
Caspase-3 activity assay of cell extracts were measured using a kit
(CaspACETM assay system; Promega) according to the
manufacturer's instructions.
Osteoclast Formation Assay--
Cells were cultured in a 60-mm
dish (40 × 104 cells/5-ml dish) in DMEM containing
10% FBS overnight. Cells were then transfected with five expression
vectors, respectively. After 24 h, media was removed, and cells
were washed two times with PBS and then cultured in the above-mentioned
medium with RANKL (100 ng/ml). After culturing for 2 days, cells were
added to fresh medium and RANKL. Then, after culturing for 2 days,
cells were fixed and stained for TRAP (30). TRAP-positive
multinucleated cells (MNCs) containing more than three nuclei were
counted as osteoclasts under microscopic examination (31).
Affinity Precipitation of Cellular Rho--
The cells (RAW cells
or RAW cells with GFP-PTEN·WT) were washed once with ice-cold PBS and
lysed in Rho-binding lysis buffer (Upstate Biotechnology) to prepare
whole cell lysates. Lysates were clarified by centrifugation at
14,000 × g for 10 min, and equal volumes of lysates
were incubated with the Rhotekin Rho binding domain (20 µg; Upstate
Biotechnology) beads at 4 °C for 45 min. The beads were washed three
times with wash buffer (Tris buffer containing 1% Triton X, 150 mM NaCl, 10 mM MgCl2, 10 µg/ml each of leupeptin and aprotinin, and 0.1 mM
phenylmethylsulfonyl fluoride). Bound Rho proteins were detected by
immunoblotting using polyclonal anti-Rho.
Protein Purification--
Constitutively active Rho (V14Rho),
Rac (L61Rac), and cdc42 (V12cdc42) were cloned in-frame into a
bacterial expression vector, pTAT-HA, to produce TAT fusion proteins.
The vector pTAT-HA has an N-terminal His6 leader followed
by the 11-amino acid TAT protein transduction domain flanked by glycine
residues, a hemagglutinin (HA) tag, and polylinker. The cDNAs
encoding V14Rho, L61Rac, and V12cdc42 were cloned into the Tat-HA
plasmid. High copy number plasmids were obtained by transformation of
the pTAT-HA with V14Rho, L61Rac, and V12cdc42 in BL21. The purification
protocol was adapted from the published procedure using a Ni-NTA column
(32, 33). Briefly, bacterial pellets were resuspended in a buffer
containing 100 mM NaCl, 20 mM Hepes (pH 8.0),
and 8 M urea and sonicated and centrifuged at 12,000 rpm
for 10 min at 4 °C. Imidazole was added to the supernatant to a
final concentration of 10-20 mM and purified in the Ni-NTA
column as described (32, 33). Addition of 8 M urea to the
sonication buffer allows for the isolation of insoluble protein in
bacterial inclusion bodies and efficient transduction into cells. Bound
proteins were eluted with stepwise addition of 5-10 ml each of 100, 250, and 500 mM and 1 M imidazole in the above
buffer. Urea was removed by rapid display by using the Slide-A-Lyzer
cassette (Pierce) or by the use of desalting PD-10 columns (Sephadex
G-25; Amersham Biosciences).
Migration Assay--
Cell migration assays were performed using
transwell migration chambers (Corning Inc., Corning, NY) (10).
Membranes with a pore size of 8 µm (Corning Inc.) were coated with
OPN (25 ng/ml) at 4 °C overnight (haptotaxis) and dried under air.
Approximately 5 × 104 cells (RAW cells with or
without GFP, GFP-PTEN WT, GFP-PTEN C124A, Myc, or Myc-Akt K179M) were
added to the upper chamber in DMEM containing 1% FBS and 2% bovine
serum albumin (100 µl) and allowed to adhere for 1-2 h. After cells
with GFP-PTEN (WT) attached to the membrane, TAT fusion protein was
added to a final concentration of 100 nM in the upper
chamber in the above medium (100 µl). Substrates such as OPN (25 µg/ml) were added to the lower chamber in DMEM containing 1% FBS and
2% bovine serum albumin (600 µl; chemotaxis). The cells were allowed
to migrate for 12-14 h at 37 °C in a tissue culture incubator with
5% CO2. After the incubation period, nonmigrated cells on
the upper side on the membrane were removed with a cotton swab. Wells
were fixed with an alcohol/formaldehyde/acetic acid mixture (20:2:1)
for 15 min. Filters were stained with hematoxylin stain (Sigma), rinsed
well with water, and dried. Dried filters were cut out and mounted with
permount solution (Thomas Scientific, Swedesboro, NJ) on a glass slide.
Cells were viewed under a ×40 objective in an inverted microscope and
counted (Zeiss microscope). Data are presented as the number of
cell-migrated fields (mean ± S.D.), and all assays were performed
in triplicate.
PTEN Activity Assay--
For the generation of whole cell
lysates prepared from cells with or without RANKL (100 ng/ml)
treatment, cells were washed once with ice-cold PBS and lysed in a cell
lysis buffer (New England Biolabs) to prepare whole cell lysates. The
lysates were centrifuged and supernatants analyzed using PTEN malachite
green assay kit (Upstate Biotechnology) according to the
manufacturer's instructions.
Expression of PTEN in RAW 264.7 Osteoclast Precursors Regulates
RANKL-stimulated Signal Transduction--
RANKL activates PI3K/Akt
survival signaling and activity of osteoclasts (17, 19-21). It also
prompts macrophages to develop the osteoclast phenotype. PTEN, a tumor
suppressor, is frequently mutated in human cancers and is a negative
regulator of PI3K/Akt survival signaling (1-7). However, the role of
PTEN in osteoclast precursors is unknown. Therefore, we examined
whether PTEN regulates Akt survival signaling pathway in RANKL-treated
RAW 264.7 osteoclast precursors. Activation of Akt and Bad has been
implicated in anti-apoptotic signaling pathways (8, 9). As shown in
Fig. 1, RANKL activated Akt and Bad in 5 min in GFP-expressing cells (peaks at 5 min and 10 min, respectively),
and the effects were blocked by GFP-PTEN (WT) expression (Fig. 1,
A and B). Moreover, RANKL altered cell morphology
in RAW 264.7 osteoclast precursors with and without GFP (Fig.
1C). RANKL-treated GFP-PTEN (WT)-expressing cells revealed no obvious morphological change (Fig. 1C). In most
cell types, mobilization of NF Expression of PTEN in RAW 264.7 Osteoclast Precursors Regulates
Osteoclast Differentiation in Vitro--
Osteoclasts are derived from
hemopoietic progenitors of the monocyte macrophage lineage (13-18).
Therefore, we examined whether GFP-PTEN (WT) suppresses osteoclast
differentiation from RAW 264.7 osteoclast precursors. Nuclear
translocation of NF Expression of Akt Dominant-negative in RAW 264.7 Osteoclast
Precursors Induces Apoptosis, but Does Not Influence Osteoclast
Differentiation--
Recent studies have demonstrated that Akt
regulates apoptosis at multiple sites and identified direct Akt targets
including Bad, caspase 9, the forkhead family of transcription factors, and the NF Expression of PTEN in RAW 264.7 Osteoclast Precursors Regulates the
OPN Signaling Pathway--
OPN stimulates monocyte/macrophage
migration, and it also stimulates osteoclasts migration via Rho
activation (10, 24). Rho and Rac, which are members of the Rho-GTPase
family, play an important role in the organization of the actin
cytoskeleton in osteoclasts (10, 41-46). Recent studies have reported
that Akt is essential for endothelial cell chemotaxis, whereas PTEN reconstitution or overexpression inhibits cell migration (29, 47, 48).
Therefore, we examined whether PTEN regulates OPN-activated the Akt,
Rho, and Rac signaling pathway in RAW 264.7 osteoclast precursors. OPN activated Akt (at 5 min; peak 10 min), Rho (at 5 min),
and Rac/cdc42 (at 5 min; peak 10 and 15 min) in GFP-expressing cells,
and the effects of OPN on Akt and Rac/cda42 were completely blocked by
GFP-PTEN (WT) expression (Fig. 4).
However, GFP-PTEN (WT) did not inhibit activated Rho expression (Fig.
4B). OPN also activated Rho (at 5 min) in GFP-PTEN (C124A)
mutant-expressing cells (Fig. 4B). Myc-Akt (K179M)
dominant-negative also blocked OPN-stimulated Rac/cdc42 phosphorylation
(Fig. 4C) compared with Myc-expressing cells. In contrast,
GFP-PTEN (C124A) mutant enhanced Akt (peak 10 min) and Rac/cdc42
phosphorylation (peak 10 min) compared with GFP-expressing cells, but
not Rho, and these effects were continued until the end of the assays
(60 min) (Fig. 4). These results demonstrate that PTEN regulates
OPN-activated cell survival and migration signaling pathways in RAW
264.7 osteoclast precursors.
Expression of PTEN and Akt in RAW 264.7 Osteoclast Precursors
Regulates OPN-stimulated Migration--
PTEN suppresses migration of a
variety of cell types, including primary human fibroblasts,
non-transformed mouse fibroblasts, and tumor cells (47, 49).
PTEN-null mouse fibroblasts also show enhanced rates of
migration, which are reduced by reintroduction of PTEN (48). Since the
role of PTEN in OPN-activated signaling pathway has not
been studied, we investigated whether PTEN and Akt regulate
OPN-stimulated migration in RAW 264.7 osteoclast precursors using
migration assays. We have previously demonstrated that OPN stimulates
osteoclast migration (10). RAW 264.7 osteoclast precursor migration was
also strongly stimulated by OPN (Fig. 5A). GFP-PTEN (WT) and Myc-Akt
(K189M) dominant-negative suppressed OPN-stimulated cell migration in
haptotaxis and chemotaxis assays (Fig. 5, B and
C). In contrast, GFP-PTEN (C124A) mutant stimulated OPN-stimulated cell migration in chemotaxis assays, but not
significantly in haptotaxis assays (Fig. 5, B and
C). These data support the hypothesis that OPN-activated Rac
and cell migration were suppressed by GFP-PTEN (WT) and Myc-Akt (K189M)
dominant-negative in RAW 264.7 osteoclast precursors.
Rac Rescues the Suppression of Migration by GFP-PTEN (WT) in RAW
264.7 Osteoclast Precursors--
Rho and Rac are essential for
osteoclast migration (10, 41, 44, 45). Rho acts upstream of PI3K in
osteoclasts (10). Rac also acts upstream of PI3K in breast carcinoma
epithelial cells (43), whereas Rac acts downstream of Akt in
endothelial cells (50). Therefore, the Rac signaling pathway in
osteoclast migration is not clear. We found that Rac is downstream of
Akt in RAW 264.7 osteoclast precursors since GFP-PTEN (WT) and Myc-Akt (K179M) dominant-negative blocked Rac phosphorylation (Fig.
4C). To further examine this issue, we performed migration
assays following transduction of constitutive active Rho, Rac, and
cdc42 in GFP-PTEN (WT)-transfected Raw cells. Constitutively active
Rac, but not Rho and cdc42, rescued the suppression of migration in
GFP-PTEN (WT)-expressing cells (Fig. 5D). We conclude that
Rac acts downstream of Akt, and PTEN has influence on cell migration by
Rac, not Rho, in RAW 264.7 osteoclast precursors.
RANKL May Regulate PTEN Activity in RAW 264.7 Osteoclast Precursors
and BMMs--
Little is known about modes of PTEN regulation. Recent
studies have reported that PTEN transcription is regulated by p53 in immortalized mouse embryonic fibroblasts (51). However, it is not clear
how PTEN activity is regulated in osteoclast precursors. To determine
whether RANKL regulates PTEN activity, we examined expression of PTEN
phosphorylation and its activity in RANKL-treated RAW 264.7 osteoclast
precursors and BMMs. RANKL activated Akt (RAW: 5 min, BMMs: 5 and 10 min) and suppressed PTEN activity (RAW: 5 min, BMMs: 5 and 10 min)
(Fig. 6). After activation by RANKL,
activated Akt gradually decreased (Fig. 6, A and
C). In contrast, PTEN activity was stimulated by RANKL at 15 and 30 min (Fig. 6, B and D). PTEN also activated
by RANKL at 10 min (peak 15 min) in RAW 264.7 osteoclast precursors and
30 min in BMMs (Fig. 6, A and C). These data
indicate that RANKL directly or indirectly may regulate PTEN activity
in RAW 264.7 osteoclast precursors and BMMs.
RANKL produced by osteoblastic lineage cells and activated T
lymphocytes is an essential factor for osteoclast differentiation, fusion, activation, and survival, thus resulting in bone resorption and
bone loss (13-18). We demonstrate that RANKL activates Akt, which is
essential for cell survival in osteoclasts in agreement with others
(17, 19-21). Although RANKL is known to stimulate phosphorylation of a
serine residue at position 473 in Akt (Ser-473), we have shown
stimulation of threonine phosphorylation at position 308 in Akt
(Thr-308) (data not shown) in agreement with Wong et al.
(19). However, RANKL did not stimulate Akt (Ser-473) phosphorylation (data not shown) in our hands except in GFP-PTEN (C124A)
mutant-expressing cells (data not shown). Akt requires phosphorylation
at positions Ser-473 and Thr-308 for full activation (8, 9, 39).
Therefore, the mechanism of RANKL-stimulated activation of Akt
demonstrated here is not yet clear.
Bad is a Bcl-2 family member regulated through phosphorylation at
Ser-136 by activated Akt resulting in its inactivation and cell
survival (9). We demonstrated that RANKL phosphorylates Bad, and the
GFP-PTEN (C124A) mutant enhances the effect of RANKL, whereas the RANKL
effect is negatively regulated by GFP-PTEN (WT). These results indicate
that PTEN activity down-regulates both RANKL-activated Akt and Bad
phosphorylation in RAW 264.7 osteoclast precursors, regulating
RANKL-stimulated survival signals.
We demonstrated that GFP-PTEN (WT) negatively regulates RANKL-activated
Akt survival signaling and suppresses osteoclast differentiation of RAW
264.7 osteoclast precursors. Mice deficient in NF
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-regulated and epithelial cell-enriched
phosphatase), is located on chromosome 10q23, a genomic
region that suffers loss-of-heterozygosity in many human cancers
(1-7). Recent studies have demonstrated that PTEN plays an essential
role in regulating signaling pathways involved in cell growth,
adhesion, migration, invasion, and apoptosis, and mutations in the
PTEN gene cause tumorigenesis in a number of human tissues
(1-5, 7). Germline mutations of PTEN cause Cowdend syndrome, a
multiple hamartoma condition associated with high incidence of breast,
brain, and thyroid neoplasia (1-7). Biochemical studies of the PTEN
phosphatase have revealed a molecular mechanism by which tumorigenesis
may be caused in individuals with PTEN mutations. The
protein product, PTEN, has homology to dual-specificity phosphatases,
and PTEN functions not only as a protein phosphatase, but also as a
lipid phosphatase (1-7). As a lipid phosphatase, PTEN functions
through dephosphorylation of the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), a product
of phosphatidylinositol 3-kinase (PI3K) activity, and as a protein
phosphatase by negatively regulating survival signaling mediated by
protein kinase B/Akt (PKB/Akt) (1-7). PTEN converts the biologically
active lipid PtdIns(3,4,5)P3 to PtdIns(4,5)P2 (1-7), and it dephosphorylates Akt phosphorylated at Thr-308 and
Ser-473. Since PI3K-stimulated PtdIns(3,4,5)P3 is an
important signaling molecule in cytoskeletal rearrangement,
differentiation, migration, proliferation, and survival (8-12), PTEN
may antagonize the actions of PI3K by decreasing
PtdIns(3,4,5)P3 levels (1-7). Mutations that impair PTEN
function result in a marked increase in cellular levels of
PtdIns(3,4,5)P3 and constitutive activation of Akt survival
signaling pathways, leading to inhibition of apoptosis, hyperplasia,
and tumor formation (1-5,7).
B (RANK), which located on osteoclasts, to promote
differentiation, survival, fusion, and activation of osteoclasts and to
prevent osteoclast apoptosis. The signaling cascade of RANK activation
involves stimulation of the c-Jun, NF
B, and serine/threonine kinase
Akt pathways (13-18). RANKL also activates the antiapoptotic
serine/threonine kinase Akt through a signaling complex involving c-Src
and TRAF 6 in primary osteoclasts (17, 19-21).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-
, anti-phospho-I
B-
(Ser-32), anti-Bad, anti-phospho-Bad (Ser-136), and
anti-phospho-Rac/cdc42 (Ser-71) were purchased from New England Biolabs
(Beverly, MA). Polyclonal anti-NF
B/p50 is obtained from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Polyclonal anti-Rho is purchased
from Upstate Biotechnology (Lake Placid, NY). Green fluorescent protein
(GFP), GFP-PTEN wild-type (WT), and GFP-PTEN point mutant C124A
(Cys-124
Ala) cDNA plasmid were kindly provided by Dr. Kenneth
M. Yamada (Craniofacial Developmental Biology and Regeneration Branch,
NIH, Bethesda, MD). Myc (empty) and Myc-Akt1 (K179M) dominant-negative (Lys-179
Met) cDNA plasmid were purchased from Upstate
Biotechnology. The cDNA encoding Rho (V14Rho), Rac (L61Rac), and
cdc42 (V12cdc42) were kindly provided by Dr. Alan Hall (MRC Laboratory
for Molecular Cell Biology, Department of Biochemistry, University
College of London, London, UK).
MEM, supplement with 10% heat-inactivated
FBS in the presence of M-CSF (100 ng/ml). After 3 days in culture,
cells were harvested for immunoblotting.
B/p50, nuclear extracts were used instead of whole cell lysates.
Following incubation with horseradish peroxidase-conjugated goat
anti-rabbit antibody (New England Biolabs), bound immunoglobulins were
detected using enhanced chemiluminescence (Pierce).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B involves phosphorylation I
B-
serine residues 32/36, resulting in ubiquitination and rapid
proteasomal degradation of the phosphorylated inhibitory protein
(34-36). RANKL stimulation also stimulated I
B-
phosphorylation
by 5 min (peak 5 min), leading to a decrease of total cellular
I
B-
beginning at 10 min in GFP-expressing cells, events required
for NF
B activation (Fig. 1D) (13-18). In contrast,
in GFP-PTEN (WT) transfectants, RANKL failed to activate I
B-
(Fig. 1D). NF
B is essential for osteoclast
differentiation and anti-apoptosis (13-18, 37, 38). RANKL stimulated
nuclear translocation of NF
B at 5 min (peak 5 and 10 min) in
GFP-expressing cells, and the effect was delayed (peak 15 min) by
GFP-PTEN (WT) expression (Fig. 1E). On the other hand, in
transfectants expressing the GFP-PTEN (C124A) mutant enhanced Akt, Bad,
and I
B-
phosphorylation, and nuclear translocation of NF
B in
the absence of RANKL compared with GFP-expressing cells. (Fig. 1,
0 min). To further confirm the above results, we measured
GFP-PTEN (WT) induced apoptosis and its suppression by GFP-PTEN (C124A)
(Fig. 2). After cotransfection, cells
were treated with RANKL for 5 min, because, RANKL activated Akt, Bad,
and I
B-
and induced nuclear translocation of NF
B at 5 min.
GFP-PTEN (WT), but not GFP-PTEN (C124A) mutant, markedly induced
apoptosis in RANKL-treated RAW 264.7 osteoclast precursors. Our results
indicate that PTEN regulates the RANKL-activated survival signaling
pathway and apoptosis in RAW 264.7 osteoclast precursors.
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Fig. 1.
PTEN regulates RANKL-activated Akt survival
signaling pathway in RAW 264.7 osteoclast precursors. RAW 264.7 osteoclast precursors were transiently cotransfected with GFP, GFP-PTEN
wild-type (WT), GFP-PTEN (C124A) mutant, Myc, or Myc-Akt (K179M)
dominant-negative. Cells were treated with RANKL (100 ng/ml) for the
indicated time, and whole cell extracts or nuclear extracts (for
NF B) were electrophoresed and analyzed by immunoblotting with
antibodies against Akt (A), phospho-Akt
(A), Bad (B), phospho-Bad (B),
I
B-
(D), phospho-I
B-
(D), and NF
B
(E). C, the GFP- or GFP-PTEN (WT)-expressing
cells treated with or without RANKL (5 min) were fixed and stained for
TRAP (reddish brown).
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Fig. 2.
GFP-PTEN wild-type and Myc-Akt (K179M)
dominant-negative induces apoptosis in RANKL-treated RAW 264.7 osteoclast precursors. RAW 264.7 osteoclast precursors were
transiently cotransfected with GFP, GFP-PTEN wild-type (WT), GFP-PTEN
(C124A) mutant, Myc, or Myc-Akt (K179M) dominate-negative. 24 h
after transfection, cells were treated with RANKL (100 ng/ml) for 5 min, and then caspase-3 activity of whole cell extracts were measured.
Data represent means ± S.D. of three experiments in duplicate. *,
p < 0.01 compared with control or GFP-, GFP-PTEN
(C124A) mutant-, or Myc-expressing cells.
B is essential for osteoclasts differentiation,
and it was delayed in GFP-PTEN (WT)-expressing cells (Fig. 1,
E). GFP-PTEN (WT) suppressed TRAP-positive MNCs number
compared with control and other vector-expressing cells. In contrast,
GFP-PTEN (C124A) stimulated TRAP-positive MNC number compared with
other conditions (Fig. 3).
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Fig. 3.
PTEN regulates osteoclast differentiation
from RAW 264.7 osteoclast precursors, but not Akt. RAW 264.7 osteoclast precursors were transiently cotransfected with GFP, GFP-PTEN
wild-type (WT), GFP-PTEN (C124A) mutant, Myc, or Myc-Akt (K179M)
dominant-negative, and cells were treated with RANKL (100 ng/ml). Cells
were then fixed and stained for TRAP, and the number of TRAP-positive
MNCs was scored. Values are expressed as the mean ± S.E. of
quadruplicate cultures. Similar findings were obtained in four
independent sets of experiments. *, p < 0.01 compared
with control or GFP-, Myc-, or Myc-Akt(K179M)
dominant-negative-expressing cells.
B regulator IKK, each of which plays a critical role in
mediating cell death (8, 9, 39, 40). We showed that Myc-Akt (K179M)
dominant-negative delayed nuclear translocation of NF
B (Fig.
1E) compared with Myc-expressing cells and induced apoptosis
(Fig. 2). In contrast, TRAP-positive MNC numbers were not influenced by
Myc-Akt (K179M) dominant-negative-expressing cells (Fig. 3). These data
indicate that the suppression of osteoclast differentiation may be
attributed to a decreased number of RAW 264.7 osteoclast precursors by
GFP-PTEN (WT)-induced apoptosis and that delayed nuclear translocation
of NF
B did not affect osteoclast production. Indeed, GFP-PTEN (WT)
more strongly induced apoptosis, compared with the Myc-Akt (K179M)
dominant-negative (Fig. 2).
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Fig. 4.
PTEN regulates OPN-activated Akt and
Rac/cdc42, but not Rho, in RAW 264.7 osteoclast precursors. RAW
264.7 osteoclast precursors were transiently cotransfected with GFP,
GFP-PTEN wild-type (WT), GFP-PTEN (C124A) mutant, Myc, or
Myc-Akt(K179M) dominant-negative, and cells were treated with OPN
(25 µg/ml) for the indicated time. Whole cell extracts were
electrophoresed and analyzed by immunoblotting with antibodies against
Akt (A), phospho-Akt (A), and phospho-Rac/cdc42
(C). B, lysates from transfected cells were
incubated with Rhotekin Rho binding domain beads, the beads washed, and
the bound protein analyzed by immunoblotting with a monoclonal antibody
against RhoA.
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Fig. 5.
PTEN and Akt regulate OPN-stimulated cell
migration in RAW 264.7 osteoclast precursors. A, RAW
264.7 osteoclast precursor migration was assessed in both haptotaxis
and chemotaxis assays. Data represent means ± S.D. of three
experiments in triplicate. B and C, transfected
RAW 264.7 osteoclast precursors were added to the upper chamber and
assessed in both haptotaxis and chemotaxis assays. Data represent
means ± S.D. of three experiments in triplicate. *,
p < 0.01 compared with control or GFP-, GFP-PTEN
(C124A) mutant-, or Myc-expressing cells in haptotaxis assay and
control, or GFP- or Myc-expressing cells in chemotaxis assay.
D, after GFP-PTEN wild-type (WT)-expressing RAW 264.7 osteoclast precursors attached to the membrane, TAT fusion protein
(V14Rho, L61Rac, V12cdc42) added to the upper chamber and assessed
chemotaxis assays. Data represent means ± S.D. of three
experiments in triplicate. *, p < 0.01 compared with
GFP-PTEN (WT)- and GFP-PTEN (WT) + HA-TAT-expressing cells.
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Fig. 6.
RANKL may regulate PTEN activity in RAW 264.7 osteoclast precursors and BMMs. A and C, RAW
264.7 osteoclast precursors and BMMs were treated with RANKL (100 ng/ml) for the indicated time, and whole cell extracts were
electrophoresed and analyzed by immunoblotting with antibodies against
phospho-Akt, phospho-PTEN, and PTEN. B and D,
whole cell extracts were prepared from RAW 264.7 osteoclast precursors
with or without RANKL (100 ng/ml)-treated, and PTEN activity was
measured using PTEN malachite green assay kit. Data represent
means ± S.D. of three experiments in duplicate. *,
p < 0.01 compared with control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B develop severe
osteoporosis because of failed osteoclastogenesis (37). Therefore, NF
B is essential for osteoclasts differentiation. GFP-PTEN (WT) and Myc-Akt (K179M) dominant-negative delayed nuclear translocation of NF
B compared with GFP-expressing cells. However, the Myc-Akt (K179M) dominant-negative failed to suppress osteoclast differentiation in RAW 264.7 osteoclast precursors. We suggest that the
number of RAW 264.7 osteoclast precursors were decreased by GFP-PTEN
(WT) because of its strong induction of apoptosis compared with Myc-Akt
(K179M) dominant-negative-expressing cells. Moreover, GFP-PTEN (WT) may
directly suppress RANKL-activated TRAF 6/NF
B signaling
pathway, but how it does this is unclear (Fig.
7). In contrast, the GFP-PTEN (C124A)
mutant enhanced RANKL-activated Akt survival signaling pathway and
markedly induced nuclear translocation of NF
B. It also stimulated
osteoclast differentiation. Our data suggest that PTEN regulates the
RANKL-activated Akt survival signaling pathway and RANKL-induced
osteoclast differentiation in RAW 264.7 osteoclast precursors (Fig.
7).
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[in a new window]
Fig. 7.
A model of a novel PTEN function for
osteoclast differentiation and migration in RAW 264.7 osteoclast
precursors. PTEN regulates RANKL-activated Akt survival signaling
pathway for cell survival. It also may regulate RANKL-activated
TRAF6/NF B signaling pathway for osteoclast differentiation.
Furthermore, it regulates the OPN-activated migration signaling
pathway. Moreover, in cell migration, Rac acts downstream of Akt and
Rho acts upstream of PI3K in RAW 264.7 osteoclast precursors.
OPN has been shown to stimulate macrophage and osteoclast migration
(10, 24). It is not known, however, whether OPN stimulates cell
migration via PI3K/Akt pathway activation. It has been previously reported in LNCap cells, that v
3 mediates
cell migration by the PI3K/Akt pathway activation. However, adhesion to
OPN did not support
v
3-mediated cell
migration or activate the PI3K/Akt pathway (11). We have demonstrated
that OPN stimulates production of phosphoinositides, including
phosphatidylinositol trisphosphate in osteoclasts and activates
gelsolin-associated PI3K (10, 26). Here, we have demonstrated that OPN
stimulates cell migration via the Akt pathway activation including Rac,
and the effect is regulated by PTEN in RAW 264.7 osteoclast precursors
(Fig. 7). Moreover, we demonstrated that Rac acts downstream of Akt.
Because, GFP-PTEN (WT) negatively regulated OPN-activated Rac, but not activated Rho. In addition, L61Rac, but not V14Rho, rescued cell migration in GFP-PTEN-expressing cells.
It has been reported that PTEN inhibits cell migration (1-5,7). One recently identified mechanism is through its effects on PtdIns (3, 4, 5) P3 levels, which have downstream effects on Rac and cdc42 signaling (48). However, tyrosine-phosphorylated FAK can bind to and activate PI3K (52), and a contributing pathway affecting PtdIns (3, 4, 5) P3 through FAK and PI3K has also been identified (53). This is a second mechanism by which PTEN may inhibit phosphotyrosine-based signaling pathways that has proven useful for dissecting the signaling pathways that regulate cell migration, although they do not prove that PTEN normally regulates these pathways. In the studies reported here, we found that PTEN inhibition of OPN-stimulated cell migration by PTEN through negative regulation of OPN-activated Akt and Rac, but not Rho.
As shown in Fig. 7, it is likely that PTEN plays multiple roles in RAW 264.7 osteoclast precursors. One question that remains is how PTEN activity is regulated. There is very little information on the regulation of PTEN expression, localization, or activity. In this study, we demonstrate that one possibility is RANKL regulation of PTEN activity. RANKL activates the PI3K/Akt survival signaling pathway and osteoclast differentiation, which PTEN also regulates (Fig. 7). Our data support that RANKL may regulate the balance between activated Akt and PTEN, which influences osteoclast differentiation.
In summary, we provide the first evidence that PTEN regulates the
RANKL- and OPN-activated signaling pathways. Furthermore, PTEN activity
influences osteoclast differentiation, survival, and migration in
osteoclast precursors. The molecular target of PTEN is
PtdIns(3,4,5)P3 (28), but whether PTEN directly or
indirectly regulates the RANKL-activated TRAF 6/NFB signaling
pathway is not yet clear (Fig. 7).
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AR41677 and grants from Pharmacia to Washington University (to K. A. H.).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 should be addressed: Dept. of Pediatrics, Medicine, and Cell and Molecular Biology, Washington University School of Medicine, 5th Floor McDonnell Pediatric Research Bldg. Campus Box 8208, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-286-2772; Fax: 314-286-2894; E-mail: hruska_k@kids.wustl.edu.
Published, JBC Papers in Press, November 30, 2002, DOI 10.1074/jbc.M209299200
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ABBREVIATIONS |
---|
The abbreviations used are:
PTEN, phosphatase
and tensin homolog deleted from chromosome 10;
GFP, green fluorescent
protein;
RANKL, receptor activator of nuclear factor-B ligand;
OPN, osteopontin;
NF
B, receptor activator of nuclear factor;
TRAF, tumor
necrosis factor receptor-associated factor;
HA, hemagglutinin;
PtdIns, phosphatidylinositol;
FBS, fetal bovine serum;
PBS, phosphate-buffered
saline;
OPN, osteopontin;
DMEM, Dulbecco's modified Eagle's medium;
TRAP, tartrate-resistant acid phosphatase;
NTA, nitrilotriacetic acid;
BMM, mouse bone marrow macrophage;
MNC, multinucleated cell.
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