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The calcineurin/NFAT signaling pathway regulatesosteoclastogenesis in RAW264.7 cells*
Hiroaki Hirotani, Nathaniel A. Tuohy, Je-Tae Woo¶, Paula H. Stern##and Neil A. Clipstone§#.
Departments of Molecular Pharmacology and Biological Chemistry,and §Microbiology-Immunology,
Feinberg School of Medicine,Northwestern University,
303 East Chicago Avenue,Chicago, IL 60611
Running title:The calcineurin/NFAT signaling pathway and osteoclastogenesis
Keywords:Calcineurin, NFAT, osteoclast, RANKL, CsA, FK506, osteoclast differentiation,RAW264.7
Corresponding authors:Neil A. ClipstoneDepartment of Microbiology-ImmunologyFeinberg School of Medicine,Northwestern University,303 East Chicago Avenue,Chicago, IL 60611Phone: (312) 503 8233FAX: (312) 503 1339e-mail: n-clipstone@northwestern.edu
Paula H. SternDepartment of Molecular Pharmacology and Biological Chemistry,Feinberg School of Medicine,Northwestern University,303 East Chicago Avenue,Chicago, IL 60611Phone: (312) 503-8290FAX: (312) 503-5349e-mail: p-stern@northwestern.edu
JBC Papers in Press. Published on January 13, 2004 as Manuscript M213067200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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SUMMARY
Although best known for its role in T lymphocyte activation, the calcineurin/nuclear factor
of activated T cells (NFAT) signaling pathway is also known to be involved in a wide
range of other biological responses in a variety of different cell types. Here we have
investigated the role of the calcineurin/NFAT signaling pathway in the regulation of
osteoclast differentiation. Osteoclasts are bone resorbing multinucleated cells that are
derived from the monocyte-macrophage cell lineage following stimulation with a member
of the TNF family of ligands known as receptor activator of NF-kB ligand (RANKL). We
now report that inhibition of calcineurin with either the immunosuppressant drugs
cyclosporin A (CsA) and FK506, or the retrovirally-mediated ectopic expression of a
specific calcineurin inhibitory peptide, all potently inhibit the RANKL-induced
differentiation of the RAW264.7 monocyte-macrophage cell line into mature
multinucleated osteoclasts. In addition, we find that NFAT family members are
expressed in RAW264.7 cells and that their expression is upregulated in response to
RANKL stimulation. Most importantly, we find that ectopic expression of a constitutively
active, calcineurin-independent NFATc1 mutant in RAW264.7 cells is sufficient to induce
these cells to express an osteoclast-specific pattern of gene expression and differentiate
into morphologically distinct, multinucleated osteoclasts capable of inducing the
resorption of a physiological mineralized matrix substrate. Taken together, these data
define calcineurin as an essential downstream effector of the RANKL-induced signal
transduction pathway leading towards the induction of osteoclast differentiation, and
furthermore, indicate that the activation of the NFATc1 transcription factor is sufficient to
initiate a genetic program that results in the specification of the mature functional
osteoclast cell phenotype.
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INTRODUCTION
Bone is a dynamic tissue that is under a constant state of remodeling or homeostasis.
This remodeling process is a delicate balance between the activities of osteoblasts, the
cells that deposit bone and osteoclasts, the cell type that is responsible for bone
resorption (1,2). Interference with this delicate balance can result in very serious human
pathologies that affect bone integrity, such as osteoporosis and osteopetrosis.
Accordingly, the molecular signaling pathways that regulate osteoblast and osteoclast
differentiation and function have come under intense scrutiny.
Osteoclasts, the cells that resorb bone, are hematopoietically-derived,
multinucleated cells that arise from the monocyte-macrophage lineage (3). It is now clear
that osteoclast differentiation is dependent upon the intimate cellular interaction of
myeloid pre-osteoclast precursors with either osteoblasts or stromal cells and is
influenced by a wide range of local factors (4,5). In fact, a wealth of data has indicated
that a member of the tumor necrosis factor (TNF) family of ligands known as receptor
activator of NF-kB ligand (RANKL; also known as ODF, OPGL and TRANCE), that is
expressed on both stromal cells and osteoblasts, plays an essential role in the regulation
of osteoclast differentiation (6-8). Thus, a soluble recombinant form of RANKL (sRANKL)
is sufficient to fully replace the requirement for osteoblast and stromal cell interactions in
the induction of osteoclast differentiation in in vitro cultures (6,7), while agents that are
known to stimulate osteoclastogenesis and bone resorption have been shown to
increase the expression of RANKL on osteoblast cells (9-12). RANKL interacts with its
specific receptor RANK, a member of the TNF receptor family, that is expressed on
osteoclast precursors (8,13). The importance of the RANKL/RANK receptor-ligand pair in
the regulation of osteoclastogenesis is underscored by the observation that mice that are
genetically deficient in either RANKL or RANK exhibit a severe osteopetrotic phenotype,
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due to a profound defect in osteoclast differentiation (14-16). RANK is known to interact
with a number of tumor necrosis factor receptor-associated factor (TRAF) signaling
adaptor proteins, including TRAF-2, TRAF-5 and TRAF-6 (17) and stimulation of RANK
activates a number of downstream signaling pathways, including mitogen-activated
protein kinases (MAPK), the src tyrosine kinase, phosphatidylinositol-3 kinase (PI-3K),
and the transcription factors NF-kB and AP-1 (17-23). In fact, genetic studies in knockout
mice have revealed that the p50 and p52 subunits of NF-kB, as well as c-fos, a
component of the AP-1 transcription factor complex are essential for efficient osteoclast
maturation in vivo (24-26), while c-src is required for mature osteoclasts to resorb bone
(27). Furthermore, in vitro studies with specific chemical inhibitors have suggested
important roles for the p38 MAPK and PI-3K in the regulation of RANKL-induced
osteoclast differentiation (20,22,23). However, while RANKL/RANK signaling is clearly
indispensable for osteoclast differentiation, our complete understanding of the contingent
series of signaling events involved in RANKL-induced osteoclastogenesis is far from
complete.
Previous studies have indicated that the immunosuppressant drugs cyclosporin A
(CsA) and FK506 have pronounced effects on bone. Thus, the addition of either CsA or
FK506 to in vitro bone organ or marrow cultures has been shown to inhibit both
osteoclastogenesis and bone resorption induced by a variety of different agents
including parathyroid hormone, calcitriol, prostaglandins and cytokines (28-32).
Furthermore, CsA has also been shown to inhibit bone resorption in isolated osteoclast
cultures (33). Although these results suggest that CsA and FK506 are likely to inhibit an
important step in the regulation of osteoclast differentiation and activation, this
interpretation is complicated by the fact that these cultures contain both osteoclast
precursors and RANKL-expressing osteoblasts and stromal cells. Hence, it is unclear
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from these latter studies whether the relevant cellular target for the inhibitory effects of
these drugs is the osteoclast per se, or alternatively, the osteoblast or stromal cell. This
is an important issue to resolve, as the inhibitory effects of CsA and FK506 on the
generation of mature functional osteoclasts may help define a novel step in the
regulation of osteoclastogenesis.
It is now well established that both CsA and FK506 are specific inhibitors of the
calcium/calmodulin-regulated serine/threonine phosphatase, calcineurin (34).
Calcineurin is best known for the role that it plays during T cell activation, where it acts to
regulate the activity of the nuclear factor of activated T cells (NFAT) family of
transcription factors, and thereby couples stimulation of the T cell antigen-receptor to
changes in the expression of cytokines and other important immunoregulatory genes
(35,36). In T cells, calcineurin is activated in response to the T cell receptor–induced
increase in the intracellular calcium concentration. Once activated, calcineurin directly
dephosphorylates NFAT proteins that are present in a hyperphosphorylated latent form
in the cytoplasm, and induces their rapid translocation into the nucleus, where in concert
with nuclear partner proteins such as the AP-1 transcription factor complex, they are
able to bind cooperatively to their target promoter elements and activate the transcription
of specific NFAT target genes (35,36). By potently inhibiting the in vivo activity of
calcineurin, both CsA and FK506 are able to block the calcineurin-dependent nuclear
translocation of NFAT proteins and thereby prevent the NFAT-dependent expression of
a large number cytokines and other genes known to play an essential role in the
initiation and regulation of the immune response. While the calcineurin/NFAT signaling
pathway is certainly best known for its role in the regulation of the immune response, it
has become increasingly apparent that this pathway is also active in a wide range of cell
types and tissues outside of the immune system (36,37) and therefore plays a much
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broader role in the regulation of cell growth and development than previously
appreciated.
In the current study we have investigated the potential role of the
calcineurin/NFAT signaling pathway in the regulation of osteoclast differentiation. For a
model system, we have used the RAW264.7 cell line, as RANKL-induced stimulation of
these monocyte-macrophage-like cells is known to be sufficient to induce osteoclast
differentiation in the complete absence of either stromal cells, osteoblasts or other
exogenously supplied differentiation-inducing factors (8). We now report that the
RANKL-induced differentiation of RAW264.7 cells is potently inhibited by both CsA and
FK506. Furthermore, we find that ectopic expression of VIVIT-GFP, a previously
characterized specific peptide inhibitor of calcineurin that is believed to selectively
prevent calcineurin from activating NFAT proteins, but not other calcineurin-dependent
signaling pathways (38), also potently inhibits RANKL-induced osteoclast differentiation.
In fact, we show that members of the NFAT family of transcription factors are expressed
in RAW264.7 cells and that their expression is upregulated in response to stimulation
with RANKL. Most importantly, we demonstrate that ectopic expression of a
constitutively active NFATc1 mutant is sufficient to induce RAW264.7 cells to express an
osteoclast-specific pattern of gene expression and undergo terminal differentiation into
mature functional multinucleated osteoclast-like cells. Taken together, these data identify
a novel role for the calcineurin/NFATc1 signaling pathway in the regulation of osteoclast
differentiation and demonstrate that the NFATc1 transcription factor is sufficient to
induce the transcriptional program leading to the acquisition of the mature osteoclast cell
phenotype.
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EXPERIMENTAL PROCEDURES.
Cell culture. RAW 264.7 cells (American Type Culture Collection) were maintained at
37°C, 5% CO2 in growth media: a-MEM (Invitrogen) supplemented with 10% (v/v) fetal
bovine serum and 1% penicillin/streptomycin (Invitrogen). For osteoclastogenesis
assays, cells were resuspended in fresh medium, and cultured for 5 - 8 days in 96-well
plates at cell a density of either 500 or 3000 cells per well. Soluble RANKL (Peprotech)
was added at 3 to 100 ng/ml as indicated, CsA (Calbiochem) was used at 2 mg/ml and
FK506 (Calbiochem) at 1 ng/ml.
Determination of osteoclast differentiation. Osteoclast differentiation was assessed
by measuring the cellular enzymatic activity of tartrate-resistant acid phosphatase
(TRAP), a marker enzyme of osteoclasts and by the direct enumeration of TRAP-
positive, morphologically distinct, multinucleated osteoclasts (39,40). For the
measurement of TRAP activity, cells were fixed in 10% (v/v) formalin for 10 min, washed
in 95% (v/v) ethanol and then incubated in 0.1 ml phosphatase substrate (3.7 mM p-
nitrophenyl phosphate in 50 mM citrate buffer, pH 4.6) in the presence of 10 mM sodium
tartrate at 25°C for 30 min. After the incubation, the solution was from each well was
transferred to a tube containing 0.1 ml 0.1 N NaOH and the absorbance at 410 nm was
determined with a Dynatech MR5000 microplate reader. After the assay for TRAP
activity, the cells were stained for TRAP with 0.1 mg/ml naphthol AS-MX phosphate and
0.6 mg/ml of fast red violet LB salt in 0.1 M sodium acetate buffer (pH 5.0) containing 50
mM sodium tartrate. All of the TRAP-positive cells with 3 or more nuclei were counted.
Cultures were photographed with a Nikon Coolpix 4500 digital camera and images were
transferred with NikonView 5 software.
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Recombinant retroviruses and infection of RAW264.7 cells. The retroviral
expression vectors pMSCV-GFP, MSCV-VIVIT-GFP and pMSCV-caNFATc1 have been
previously described (41,42). Recombinant retroviruses were produced by co-
transfecting either the pMSCV-GFP, pMSCV-VIVIT-GFP or pMSCV-caNFATc1 proviral
vectors together with pVSV-G (Clontech), encoding the vesicular stomatitis virus-
glycoprotein, into the GP293 pantrophic packaging cell line (Clontech) using
Lipofectamine Plus (Invitrogen). Media was replaced after 24 hrs and viral supernatants
were harvested 2 days post-transfection and stored at –80°C. For infections, 3000 RAW
264.7 cells were plated per well of a 96-well plate. The next day, media was replaced
with 100 ml of viral supernatant containing 8 mg/ml polybrene (Sigma) and plates were
centrifuged at 2000 rpm for 2 hr at room temperature. After removal of viral supernatant,
cells were cultured in growth media for subsequent analysis.
Determination of functional osteoclast activity by calcium phosphate resorption
assay and pit formation on dentine slices. Cells were seeded onto 16 well BD
BioCoat‚ Osteologic‚ calcium phosphate-coated quartz slides and incubated in aMEM
supplemented with 15% fetal bovine serum, 1% penicillin/ streptomycin, 50 mg/ml L-
ascorbic acid and 10 mM Na b-glycerophosphate. The cells were infected with the
appropriate recombinant retrovirus as described above and medium was changed after
24 hr and three times weekly thereafter. After 10 days, bleach solution (6% NaOCl,
5.2% NaCl) was added to each well. The dish was agitated for 5 minutes and the
medium/bleach mixture removed. Wells were washed 2x in de-ionized water, and then
in running de-ionized water and allowed to air dry. Cultures were photographed as
described above, and the transferred images quantified to determine the area of
resorbed matrix using Scion Image‚ software. For direct examination of bone resorbing
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activity, cells were seeded in 24 well plates directly onto 0.5mm thick dentine slices (a
kind gift of Professor Hisashi Shinoda, Tohoku University Dental School, Sendai, Japan)
and infected with either the MSCV-GFP or MSCV-caNFATc1 retrovirus exactly as
described above. After 10 days in culture, cells were removed by vigorous washing with
bleach solution, dentine slices were washed in de-ionized water and resorption pits were
visualized by staining with hematoxylin and light microscopy. For visualization of
resorption pits by scanning electron microscopy, dentine slices were first dehydrated in a
series of ascending ethanol washes, then critically point dried under CO2. Dentine slices
were then mounted on stubs, sputter coated with a 10 nm layer of gold and examined by
scanning electron microscopy (Hitachi S3500) using a Robinson backscattered electron
detector.
Semi-quantitative analysis of mRNA expression by reverse transcriptase-
polymerase chain reaction. Total RNA was isolated from cells using the RNeasy mini-
prep method (Qiagen) and first strand cDNA was synthesized in 20 ml reactions using
the Promega reverse transcription system following the manufacturer’s protocol. For
analysis of the expression of NFAT family members, serial two-fold dilutions of the first
strand cDNA synthesis reaction were used for PCR amplification using Platinum Taq
polymerase (Invitrogen) and the following gene specific primer pairs: NFATc1 sense 5’-
CAACGCCCTGACCACCGATAG-3’ and antisense 5’-GG CTGCCTTCCGTCTCATAGT-
3’; NFATc2 sense 5’-GGGCCATGTGAGCAGGAGGAGA- 3’ and antisense 5’-
GCGTTTCGGAGCTTCAGGATGC-3’; NFATc3 sense 5’-CTTTCAGTTCCTTCACCCTT
TACCT-3’ and antisense 5’-TGCCAATATCAGTTTCTCCTTTTC-3’; cyclophilin sense 5'-
TGGCACAGGAGGAAAGAGCATC-3' and antisense 5'-AAAGGGCTTCTCCACCTC
GATC-3'. Thermal cycling conditions were 32 cycles of 94°C for 30 sec, 57°C for 30 sec
and 72°C for 60 sec (NFATc1 and NFATc3) and 32 cycles of 94°C for 30 sec, 61°C for
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30 sec and 72°C for 60 sec (NFATc2), and 32 cycles of 94°C for 1 min, 60°C for 1 min,
72°C for 2 min (cyclophilin). For analysis of osteoclast-specific gene expression PCR
amplification was performed using the following sets of gene specific primers: TRAP
sense 5’-CACGATGCCAGCGACAAGAG-3’ and antisense 5’-TGACCCCGTATGTGGC
TAAC-3’; aV integrin sense 5’-GCCAGCCCATTGAGTTTGATT-3’ and antisense 5’-GCT
ACCAGGACCACCGAGAAG-3’; b3 integrin sense 5’-TTACCCCGTGGACATCTACTA-3’
and antisense 5’-AGTCTTCCATCCAGGGCAATA-3’; cathepsin K sense 5’-GGAGAA
GACTCACCAGAAGC-3’ and antisense 5’-GTCATATAGCCGCCTCCACAG-3’;
calcitonin receptor sense 5’-ACCGACGAGCAACGCCTACGC-3’ and antisense 5’-GCC
TCACAGCCTTCAGGTAC-3’. Thermal cycling conditions were 22-27 cycles of 94°C for
1 min, 58°C for 1 min and 72°C for 1 min (TRAP, aV and cathepsin K); 30 cycles of
94°C for 1 min, 50°C for 1 min and 72°C for 1 min (b3); and 33 cycles of 94°C for 1 min,
52°C for 1 min and 72°C for 1 min (calcitonin receptor). PCR products were resolved by
2% agarose gel electrophoresis and visualized by Vistra Green staining on a Storm
phosphoimager using ImageQuant software.
Statistics: Significance was determined by Scheffe’s F-test.
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RESULTS
CsA and FK506 inhibit the RANKL-induced osteoclast differentiation of RAW 264.7
cells
To initially determine the potential role of the calcineurin/NFAT signaling pathway in
osteoclastogenesis, we first evaluated the effects of the specific calcineurin inhibitors
CsA and FK506 on the ability of RANKL to induce the differentiation of RAW264.7 cells.
Thus, cultures of RAW264.7 cells were stimulated with a range of sRANKL
concentrations (3 - 100 ng/ml) in the presence and absence of either CsA or FK506 and
osteoclast differentiation was assessed after 8 days by the production of TRAP
enzymatic activity, a marker enzyme of osteoclast differentiation and by the direct
enumeration of multinucleated TRAP-positive cells. As indicated in Fig. 1, sRANKL-
stimulation induced a dose dependent increase in both TRAP activity and the number of
multinucleated TRAP-positive cells. Notably, both of these RANKL-induced parameters
of osteoclast differentiation were significantly inhibited in the presence of either CsA or
FK506 (Fig. 1). However, while the inhibitory effects of CsA and FK506 on sRANKL-
induced TRAP activity were quite modest, both of these immunosuppressant drugs
potently inhibited the appearance of morphologically distinct, TRAP-positive,
multinucleated osteoclasts. Hence, these results indicate that while both CsA and FK506
inhibit the RANKL-induced differentiation of RAW264.7 cells, it is the later steps in the
osteoclast differentiation pathway leading to fusion and multinucleation that appear to be
more sensitive to the drugs, rather than the early production of TRAP activity.
Ectopic expression of the specific calcineurin inhibitory peptide VIVIT-GFP blocks
RANKL-stimulated osteoclastogenesis.
The above results demonstrating that CsA and FK506 both inhibit the RANKL-induced
osteoclast differentiation of RAW264.7 cells suggest that calcineurin activity is required
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for RANKL-induced osteoclastogenesis. To investigate this further, we took advantage of
a 16 amino acid high affinity calcineurin-binding peptide identified by Rao and
colleagues (known as VIVIT), that when fused to the N-terminus of GFP has been shown
to selectively inhibit the ability of calcineurin to activate NFAT proteins (38). In order to
efficiently introduce this VIVIT-GFP fusion protein into RAW264.7 cells, we utilized our
previously described retroviral vector in which VIVIT-GFP is under the transcriptional
control of the MSCV-long terminal repeat (42). By pseudotyping this recombinant
retroviruses with the vesicular stomatitis virus glycoprotein-G we were able to generate
high titer virus capable of routinely infecting >95% of RAW264.7 cells (Fig 2A). This high
level of infection efficiency obviates the laborious process of isolating and expanding
clonal cell lines and means that we are able to analyze bulk populations of cells
immediately after infection. To determine the effects of VIVIT-GFP on RANKL-induced
osteoclast differentiation, RAW264.7 cells were infected with either MSCV-VIVIT-GFP or
a control MSCV-GFP retrovirus and then induced to differentiate in the presence of
sRANKL. As indicated in Fig. 2B, ectopic expression of VIVIT-GFP in RAW 264.7 cells
modestly inhibited the RANKL-induced increase in TRAP activity. However, like our
results with CsA and FK506, we found that ectopic expression of VIVIT-GFP potently
blocked the RANKL-induced formation of morphologically distinct, TRAP-positive,
multinucleated cells (Figs. 2C and 2D). To determine whether calcineurin was also
required for the RANKL-dependent induction of osteoclast functional activity, we tested
the effects of VIVIT-GFP on the ability of RANKL to induce the resorption of a synthetic
bone substrate. For this analysis we took advantage of BD BioCoat‚ Osteologic‚ slides
that are coated with calcium phosphate and which have been extensively used in the
analysis of osteoclast function (43-45). Thus, RAW264.7 cells infected with either the
control MSCV-GFP or the MSCV-VIVT-GFP retrovirus were plated on the BD Osteologic
slides, then treated with sRANKL and the extent of calcium phosphate resorption was
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determined after 10 days in culture. As shown in Fig. 2E, we found that RAW264.7 cells
infected with the control retrovirus readily resorbed the calcium phosphate matrix when
stimulated with RANKL, whereas the RANKL-induced resorption activity of cells infected
with MSCV-VIVT-GFP was markedly reduced. Taken together with the effects of CsA
and FK506, these data strongly suggest that calcineurin activity is required for the
RANKL-stimulated differentiation of RAW264.7 cells into morphologically distinct, TRAP-
positive, multinucleated functional osteoclasts. Moreover, since VIVIT-GFP has been
demonstrated to preferentially block the ability of calcineurin to dephosphorylate NFAT
proteins, but not other calcineurin substrates, these data raise the possibility that the
NFAT family of transcription factors might play a role in the regulation of RANKL-induced
osteoclast differentiation and function.
NFAT family members are expressed in RAW264.7 cells and their expression is
induced by stimulation with RANKL.
Although NFAT proteins were originally identified in cells of the immune system, it has
become increasingly apparent that these proteins are also expressed in a wide variety of
other cell types. In order to determine whether NFAT family members are expressed in
RAW264.7 cells we performed RT-PCR analysis on mRNA isolated from either
unstimulated or RANKL-induced RAW264.7 cells using oligonucleotide primers specific
for individual members of the NFAT family. As shown in Fig. 3, we find that NFATc1 and
NFATc3 mRNAs are indeed expressed at a low level in resting non-stimulated
RAW264.7 cells, and that that this level of expression is further increased in response to
stimulation with sRANKL. The mRNA for NFATc2 was barely detectable in resting
RAW264.7 cells, but like NFATc1 and NFATc3, its expression was also induced
following stimulation with sRANKL. NFATc4 mRNA was not detected in RAW264.7 cells
(data not shown).
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Ectopic expression of a constitutively active NFATc1 mutant in RAW264.7 cells is
sufficient to induce osteoclastogenesis.
Having established that NFAT family members are expressed in RAW264.7 cells and
that the RANKL-induced differentiation of RAW 264.7 cells into multinucleated TRAP-
positive bone resorbing osteoclasts is potently inhibited by VIVIT-GFP, which is believed
to selectively inhibit the ability of calcineurin to activate NFAT proteins (38), we next
wished to directly assess the potential contribution of the NFAT proteins themselves to
osteoclast differentiation. Towards this end we took advantage of our previously
characterized constitutively active NFATc1 mutant (caNFATc1), that is known to be
constitutively localized to the nucleus, bind DNA with high affinity and is capable of
activating endogenous chromatin-embedded NFAT target genes (41). We used this
NFATc1 mutant to determine whether activation of the NFATc1 signaling pathway by
itself in RAW264.7 cells was sufficient to induce osteoclast differentiation. Thus,
RAW264.7 cells were infected with either a control MSCV-GFP retrovirus or our
previously described MSCV-caNFATc1 virus, and the effects on osteoclast differentiation
were examined. As shown in Fig. 4, enforced expression of caNFATc1 in RAW264.7
cells was sufficient to induce TRAP activity and the appearance of large morphologically
distinct, TRAP-positive, multinucleated cells in the complete absence of RANKL
signaling. Conversely, in the absence of RANKL stimulation, cells infected with the
control virus never underwent a morphological change towards the osteoclast
phenotype. Notably, expression of caNFATc1 significantly increased the kinetics of
osteoclast differentiation, as multinucleated cells appeared as early as forty eight hours
after infection with the MSCV-caNFATc1 virus, as compared to the 4-6 days required for
the initial appearance of these cells following RANKL stimulation (data not shown).
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To determine whether these caNFATc1-induced TRAP-positive multinuclear cells
were capable of mediating mature osteoclast function, we examined their activity in two
independent functional osteoclast assays. First, we assessed the ability of either control
or caNFATc1-expressing RAW264.7 cells to resorb a calcium phosphate substrate.
Thus, RAW264.7 cells infected with either the control MSCV-GFP or MSCV-caNFATc1
retrovirus were plated on BD Osteologic slides and the extent of calcium phosphate
resorption was determined after 10 days in culture. As shown in Fig. 4D, we found that
RAW264.7 cells infected with the caNFATc1 virus, but not the control virus, were
capable of mediating significant resorption of the calcium phosphate matrix. Second, we
examined whether caNFATc1-expressing RAW264.7 cells were capable of directly
resorbing a physiological mineralized substrate, dentine. As shown in Fig. 4E, after 10
days in culture on dentine slices, cells infected with the caNFATc1 virus formed clearly
discernable resorption pits as visualized by hematoxylin-staining, whereas no such pits
were observed on dentine slices cultured with control MSCV-GFP virus-infected
RAW264.7 cells under identical conditions. In order to formally establish that these latter
hematoxylin-staining areas detected on the dentine slices cultured with caNFATc1-
expressing RAW264.7 cells truly represented the formation of caNFATc1-induced
resorption pits, and were not merely an artifact due perhaps to the staining of residual
cell debris, we performed scanning electron microscopy of the dentine slices. As clearly
indicated in Fig. 4F, this analysis revealed that the hematoxylin-staining areas detected
do indeed correspond to resorption pits formed on the surface of the dentine slice.
Taken together as a whole therefore, these data provide clear evidence that the ectopic
expression of caNFATc1 is sufficient to induce RAW264.7 cells to acquire mature
osteoclast functional activity.
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Finally, in order to gain independent molecular evidence that activation of the
NFATc1 transcription factor is capable of inducing RAW264.7 cells to undergo terminal
osteoclast differentiation, we determined the effects of caNFATc1 on the expression of a
panel of genes that are known to be selectively expressed in terminally differentiated
osteoclasts. As shown in Fig. 5, expression of caNFATc1 in RAW264.7 cells is sufficient
to induce the expression of TRAP, the aV and b3 integrin subunits, cathepsin K and the
calcitonin receptor, which are all known to be induced during RANKL-stimulated
osteoclastogenesis (1,46-49). This result indicates that activation of the NFATc1
transcription factor is able to mimic RANKL-induced signals and induce RAW264.7 cells
to acquire molecular markers associated with terminally differentiated osteoclasts. Taken
together, therefore, the collective data presented in Fig. 4 and Fig. 5 provides strong and
compelling evidence that the activation of the NFATc1 transcription factor is, in and of
itself, sufficient to induce the differentiation of RAW264.7 cells into cells with a
morphologically distinct, terminally differentiated functional osteoclast phenotype.
DISCUSSION.
The calcineurin/NFAT signaling pathway was originally identified in T cells, where it is
known to play a key role in the regulation of immunologically important cytokine genes
during the initiation of the T cell immune response (35,36). However, it has now become
increasingly clear that this signal transduction pathway is also active in a number of
other distinct cell and tissue types and plays a much broader role in the regulation of cell
growth and development than was initially appreciated. These additional roles include
the regulation of cardiac valve morphogenesis, patterning of the vasculature, cardiac
hypertrophy, angiogenesis, regulation of chondrocyte differentiation and multiple roles in
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the regulation of skeletal muscle, such as myogenesis, muscle fiber-type gene
expression and myocyte hypertrophy (36,37,50-54). Our current data, along with other
recent results (55,56), now extends to osteoclasts, the list of cell types that take
advantage of the calcineurin/NFAT signaling pathway as a means of regulating cell fate
choices and cellular functions.
In our initial studies we used specific inhibitors of the calcium/calmodulin-
regulated serine/threonine phosphatase, calcineurin, together with the well established
RAW264.7 monocyte-macrophage-like/pre-osteoclast cell line to investigate the potential
role of calcineurin in the regulation of RANKL-induced osteoclastogenesis. As a result of
these studies, we found that the structurally distinct immunosuppressant drugs CsA and
FK506, which are each known to specifically inhibit calcineurin’s calcium-induced
enzymatic activity (34), both potently blocked the RANKL-induced differentiation of
RAW264.7 cells into mature TRAP-positive, multinucleated osteoclasts. Similar inhibitory
effects of CsA and FK506 on the RANKL-induced osteoclastogenesis of bone marrow-
derived monocyte/macrophage precursors have also recently been reported (55,56). In
addition to the effects of the immunosuppressant drugs, we found that ectopic
expression of VIVIT-GFP, a previously characterized specific peptide inhibitor of
calcineurin (38), also potently inhibited the ability of RAW264.7 cells to undergo terminal
osteoclast differentiation in response to treatment with RANKL. Taken together, the
observation that three structurally distinct inhibitors of calcineurin all act to independently
block RANKL-induced osteoclast differentiation, provides strong and compelling
evidence to implicate calcineurin as an essential downstream effector of the RANKL-
induced signal transduction pathway leading towards the induction of terminal osteoclast
differentiation, and thereby helps define a novel role for calcineurin in the regulation of
osteoclastogenesis.
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Interestingly, the VIVIT-GFP calcineurin inhibitory peptide used in our studies,
was initially reported by Rao and colleagues to selectively inhibit the ability of calcineurin
to activate NFAT proteins, but not other downstream targets such as NF-kB (38). Hence,
our observation that VIVIT-GFP potently blocked RANKL-induced osteoclastogenesis
suggested a potential role for NFAT family members in the regulation of RANKL-induced
osteoclast differentiation. This was an initially surprising notion, as NFAT family
members were not previously known to be expressed in cells of the
monocyte/macrophage lineage. However, as revealed by our RT-PCR analysis, NFAT
family members NFATc1, NFATc2 and NFATc3 are indeed expressed in RAW264.7
cells. Moreover, we found that their expression is upregulated in response to stimulation
of the RANKL/RANK signaling pathway, thereby establishing a direct link between a
signal known to be pivotal in the induction of osteoclast differentiation and the increased
expression of members of the NFAT family of transcription factors. Similar RANKL-
induced increases in the expression of NFATc1 mRNA in bone marrow-derived
monocyte/macrophage precursors have also recently been reported (55,56). In fact,
these later studies also revealed that RANKL stimulation leads not only to an increase in
NFATc1 gene expression, but also to the translocation of NFATc1 proteins from the
cyctoplasm into the nucleus.
A significant question that is raised by these studies, is how does RANKL-
induced stimulation of RANK lead to the activation of the calcium/calmodulin-dependent
phosphatase, calcineurin and in turn the NFAT family of transcription factors? In this
regard, while RANK-mediated signal transduction has been extensively studied for a
number of years, much of the focus has been on the mechanisms by which RANK
stimulation couples to the activation of the MAPK, AP-1 and NF-kB signaling pathways
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(57). In fact, it is only very recently that RANKL treatment has also been shown to lead
directly to an increase in the intracellular calcium concentration (55,56). However,
potential insights into the molecular mechanism underlying RANKL-induced changes in
intracellular calcium are provided by the analysis of RANKL/RANK signaling in the
regulation of angiogenesis in endothelial cells, which has revealed that RANK
stimulation can activate calcium-dependent signaling events via the effects of the Src
tyrosine kinase on phospholipase C (58). The Src tyrosine kinase is coupled to RANK
via its interaction with the cytoplasmic adaptor protein, TRAF6, which is known to play an
essential role in the regulation of RANKL-induced osteoclast differentiation and the
stimulation of bone resorbing activity (59). In fact, in addition to playing a role in the
regulation of RANK-induced phospholipase C activity, the Src tyrosine kinase has also
been shown to play a role in the RANK-induced regulation of protein kinase B (18). This
is of interest, as protein kinase B is known to phosphorylate and inhibit the activity of
glycogen synthase kinase-3 (60), which is known to be a potent inhibitor of NFATc1
activity (61,62). Accordingly, we believe that the RANK-induced activation of Src is likely
to affect the activity of the calcineurin/NFATc1 signaling pathway in two distinct ways.
First, the TRAF6/Src-dependent activation of phospholipase C and the subsequent
production of the calcium mobilizing agent inositol trisphosphate leads to an increase in
the intracellular calcium concentration that in turn presumably activates calcineurin,
thereby inducing the dephosphorylation of NFATc1 and its rapid transport into the
nucleus. Second, the TRAF6/Src-dependent activation of protein kinase B-dependent
likely results in the phosphorylation and subsequent inhibition of the principal NFATc1
inhibitory kinase, glycogen synthase kinase-3, thereby preventing this kinase from
directly phosphorylating active NFATc1, which is known to both inhibit its DNA binding
activity and promote its export back into the cytoplasm (61,62). In fact, support for this
proposed role of Src in the regulation of the calcineurin/NFAT signaling pathway is
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provided by the observation that activation of Src family members has previously been
shown to be sufficient to fully induce the calcineurin-dependent activation of NFAT
proteins in T cells (63). Hence, we believe that RANK is likely to affect the activity of the
calcineurin/NFATc1 signaling pathway via the TRAF6-dependent activation of the Src
tyrosine kinase. Interestingly, several other members of the TNFR family, such as CD40
and TACI, that are also known to regulate the calcineurin/NFAT signaling pathway have
also been shown to interact with TRAF6 (64,65), raising the possibility that potentially
other TRAF6-associated receptors, such as the interleukin-1 and the toll-like family of
receptors, may also couple to the stimulation of the calcineurin/NFATc1 signaling
pathway.
Clearly, the most striking result in our current study, is the observation that
enforced expression of an activated NFATc1 allele in RAW264.7 cells is sufficient to
mimic the effects of RANKL stimulation and induce these cells to acquire a
morphologically distinct, TRAP-positive, multinucleated osteoclast-like bone resorbing
phenotype. Importantly, in addition to these morphological and functional criteria, we
also observe that constitutively active NFATc1 is able to induce the expression of a
panel of genes including TRAP, cathepsin K, the calcitonin receptor and the aV and b3
integrin subunits, that together are considered to be molecular markers of fully
differentiated osteoclasts (1,46-49). The induction of the calcitonin receptor is particularly
significant, as amongst cells of the myelomonocytic lineage, the expression of this gene
is restricted to osteoclasts (47-49). Accordingly, this result indicates that the caNFATc1-
induced cells do indeed represent bona fide terminally differentiated osteoclasts and not
merely macrophage polykaryons that are known to be generated under certain
conditions. Thus, based upon these collective morphological, functional and molecular
criteria, our data strongly indicates that the activation of the NFATc1 transcription factor
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is alone sufficient to induce the program of transcriptional events leading to the
acquisition of the mature osteoclast phenotype. The importance of the NFATc1
transcription factor to the regulation of osteoclastogenesis is further underscored by two
other recent sets of studies. First, Ishida et al, found that the inhibition of NFATc1
expression in RAW264.7 cells with an NFATc1 antisense construct was able to
attenuate RANKL-induced osteoclast differentiation (55). Second, in an article published
concommitantly with the original submission of our current manuscript, Takayanagi et al
(56) have demonstrated that NFATc1-deficient monocyte/macrophage percursor cells
derived from cytokine treatment of NFATc1-null ES cells are unable to undergo
osteoclast differentiation in response to RANKL stimulation, indicating that NFATc1 plays
an essential non-redundant role in the regulation of RANKL-induced osteoclastogenesis.
Furthermore, in agreement with our current findings, this later group also demonstrated
that ectopic expression of NFATc1 in bone marrow-derived monocyte/macrophage
percursor cells was sufficient to promote the formation of TRAP-positive multinuclear
osteoclast-like cells in the absence of RANKL signaling. Taken as a whole, these
combined results strongly suggest that the activation of NFATc1 is both necessary and
sufficient for the induction of osteoclastogenesis and the acquisition of the mature
functional osteoclast phenotype.
The observation that the activation of the NFATc1 transcription factor in
osteoclast precursor cells, is, by itself, sufficient to initiate a genetic program that results
in the specification of the mature functional osteoclast cell fate, serves to operationally
define NFATc1 as a potential master regulatory transcription factor for osteoclast
differentiation. However, to what extent NFATc1 is directly involved in regulating the
expression of the terminal class of genes responsible for specifying the mature
osteoclast phenotype, remains to be seen, as it is also possible that NFATc1 may merely
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induce the expression of other transcription factors that are in turn responsible for the
expression of osteoclast-specific genes. Resolution of these alternatives will require the
identification of the specific NFATc1 target genes in osteoclast precursor cells. Although
in this regard, it is interesting to note that NFATc1 has already been shown to regulate
the activity of the TRAP promoter (56) and that putative NFAT binding sites have also
been identified in the osteoclast-specific P3 promoter of the calcitonin receptor (66).
Finally, while it appears that the activation of the NFATc1 transcription factor is
both necessary and sufficient for osteoclastogenesis, it is important to note that several
other transcription factors that affect various aspects of osteoclast development and
function have been previously identified. Thus, animals that are genetically deficient in
either the ETS family member PU.1, c-Fos, both the p50 and p52 subunits of NF-kB, or
both of the helix-loop-helix transcription factors microphthalmia-associated transcription
factor and TFE3, have been shown to exhibit an osteopetrotic phenotype (24-26,67,68).
However, unlike our results with NFATc1, none of these factors alone has been shown
to be sufficient to induce osteoclastogenesis independently of other osteoclast-inducing
conditions, although overexpression of the c-Fos family member Fra-1 in osteoclast
precursor cells has been reported to significantly enhance osteoclastogenesis in the
presence of stromal cells, prostaglandin E2 and 1, 25(OH)2 vitamin D3 (69). In fact,
members of the c-Fos family of proteins are of particular interest, as they are subunits of
the AP-1 transcription factor complex that is known to be a nuclear partner for NFAT
proteins (35). Together, NFAT and AP-1 proteins are known to bind co-operatively to a
number of functionally important sites in the promoters of numerous cytokine genes (35).
Given the essential roles of both NFATc1 and c-Fos in the regulation of
osteoclastogenesis, it is tempting to speculate that they will directly co-operate in the
regulation of osteoclast-specific genes. In fact, the interaction between NFATc1 and c-
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Fos has been shown to be necessary in the regulation of the TRAP promoter (56),
although whether NFATc1 interactions with AP-1 are absolutely required for the
induction of other osteoclast-specific genes remain to be determined. While NFATc1 and
c-Fos, together with other AP-1 family members, may co-operate in the regulation of
certain osteoclast-specific genes, it appears that the functional relationship between
NFATc1 and c-Fos in the regulation of osteoclast differentiation is likely to be quite
complex, as recent studies have also revealed an essential role for c-Fos in the initial
RANKL-induced upregulation of NFATc1 mRNA in osteoclast precursors (56). This
observation highlights what will be a major challenge for the future, namely attempting to
understand the molecular road map by which each of the transcription factors known to
be important for osteoclast differentiation collaborate with each other to ensure the
successful completion of the genetic program leading to the acquisition of the mature
functional osteoclast cell fate. Nonetheless, whatever the relative functional relationship
between these various transcription factors, it is clear from our current data that the
calcineurin/NFATc1 signaling pathway plays a prominent role in the regulation of
osteoclast differentiation and function, with NFATc1 likely to serve a pivotal role as a
critical molecular switch in the regulation of the mature functional osteoclast cell
phenotype.
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FOOTNOTES
* This work was supported in part by National Institutes of Health grant R29 GM55292
(to N.A.C) and a grant from the Chicago Chapter of the National Arthritis Foundation and
pilot project on P60 AR30692, National Insitutes of Health Multipurpose Arthritis Center,
Northwestern University (to P.H.S).
¶ Present address: Department of Bioengineering, Chubu University, College of
Bioscience and Biotechnology, Matsumoto 1200, Kasugai-shi, Aichi-ken, 487-8501,
Japan.
# To whom correspondence may be addressed. Tel.: 312-503-8233; Fax: 312-503-1339;
e-mail: n-clipstone@northwestern.edu.
## To whom correspondence may be addressed. Tel.: 312-503-8290; Fax: 312-503-
5349; e-mail: p-stern@northwestern.edu.
ABBREVIATIONS
The abbreviations used in this paper are: NFAT, nuclear activated factor of T cells; CsA,
cyclosporin A; RANKL, receptor activator of NF-kB ligand; sRANKL, soluble RANKL;
TNF, tumor necrosis factor, TRAF, tumor necrosis factor receptor-associated factor;
MAPK, mitogen-activated protein kinase; PI-3K, phosphatidylinositol-3 kinase; GFP,
green fluorescent protein; TRAP, tartrate-resistant acid phosphatase; MSCV, murine
stem cell virus; PCR, polymerase chain reaction; RT, reverse transcribed; MNC,
multinucleated cells.
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FIGURE LEGENDS
Figure 1. Effects of cyclosporin and FK506 on RANKL-stimulated TRAP activity
and formation of TRAP-positive multinucleated cells in cultures of RAW264.7
cells. Cells were seeded at 500 cells per well in a 96 well plate and cultured for 8 days
with a range of sRANKL concentrations (3-100 ng/ml) in the presence and absence of
either CsA or FK506. A, Effect of CsA, 2 mg/ml, on RANKL-stimulated TRAP activity. B,
Effect of 2 mg/ml CsA on RANKL-stimulated formation of TRAP-positive multinucleated
cells. C, Effect of 1 ng/ml FK506 on RANKL-stimulated TRAP activity. D, Effect of 1
ng/ml FK506 on RANKL-stimulated formation of TRAP-positive multinucleated cells.
Results represent the means +/- SEM of responses from 6 determinations. * p<0.05 vs.
untreated control; # p<0.05 vs. RANKL alone; MNC, multinucleated cells.
Figure 2. Effects of VIVIT on RANKL-stimulated TRAP activity, formation of TRAP-
positive multinucleated cells and resorption of a calcium phosphate substrate by
RAW264.7 cells. RAW264.7 cells infected with either the control MSCV-GFP or the
MSCV-VIVIT-GFP retrovirus were cultured for 8 days in the presence of a range of
sRANKL concentrations (3-100 ng/ml) and then analyzed for parameters of osteoclast
differentiation. A, Flow cytometric analysis of virally infected RAW264.7 cells
demonstrating efficient infection with the MSCV-VIVIT-GFP retrovirus. (Left panel)
uninfected RAW264.7 cells, (right panel) RAW264.7 cells infected with the MSCV-VIVIT-
GFP retrovirus. B, Effect of VIVIT-GFP expression on RANKL-stimulated TRAP activity.
C, Effect of VIVIT-GFP expression on RANKL-stimulated formation of TRAP-positive
multinucleated cells. D, Effect of VIVIT-GFP expression on the morphological
appearance of RANKL-treated RAW264.7 cells. (Left panel) control MSCV-GFP virus-
infected cells left unstimulated, (middle panel) RANKL-treated control MSCV-GFP virus-
infected cells, and (right panel) RANKL-treated MSCV-VIVIT-GFP-infected cells. E,
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Effect of VIVIT-GFP expression on RANKL-stimulated resorption of a calcium phosphate
substrate after 10 days of culture. RAW264.7 cells grown on BioCoat‚ Osteologic‚
calcium phosphate-coated quartz slides were infected with either control MSCV-GFP or
the MSCV-VIVIT-GFP virus and either left unstimulated or treated with 100 ng/ml
sRANKL. After 10 days, the area of resorption was determined as described in
Experimental Procedures and is presented as mean area of resorption (mm2). Results
represent the means +/- SEM of the responses from 6 cultures. * p<0.05 vs. cells
infected with control retrovirus only; # p<0.05 vs. cells infected with control retrovirus and
treated with RANKL. Representative images showing the appearance of the substrate
after removal of untreated GFP-expressing cells (left panel), RANKL-treated GFP-
expressing cells (middle panel), and RANKL-treated VIVIT-GFP-expressing cells (right
panel) are presented.
Figure 3. Effect of RANKL stimulation on the expression of NFAT family members
in RAW 264.7 cells. Cells were either left unstimulated or treated with RANKL (100
ng/ml) for 24 hr at which point RNA was isolated for RT-PCR analysis. Serial fold
dilutions of cDNA samples from each cell population were analyzed by PCR using gene
specific primers for NFATc1, NFATc2, NFATc3 and cyclophilin followed by agarose-gel
electrophoresis.
Figure 4. Ectopic expression of a constitutively active NFATc1 mutant is sufficient
to induce RAW264.7 cells to acquire a mature functional osteoclast-like
phenotype. RAW264.7 cells infected with either control MSCV-GFP or MSCV-
caNFATc1 virus encoding a constitutively active form of NFATc1 (caNFATc1) were left
unstimulated and analyzed for parameters of osteoclast differentiation after 8 days in
culture. A, Effect of ectopic expression of caNFATc1 on TRAP activity. B, Effect of
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ectopic expression of caNFATc1 on the number of multinucleated TRAP-positive cells.
ND, none detected. C, Morphological appearance of cultures infected with either the
control MSCV-GFP retrovirus (left panel) or the MSCV-caNFATc1 retrovirus (right
panel). D, Effect of ectopic expression of caNFATc1 on the ability of RAW264.7 cells to
resorb a calcium phosphate substrate. RAW264.7 cells grown on BioCoat‚ Osteologic‚
calcium phosphate-coated quartz slides were infected with either the control MSCV-GFP
(left panel) or the MSCV-caNFATc1 virus (middle panel) and representative images
showing the appearance of the matrix after 10 days of culture are presented.
Quantitation of the area of resorption induced by either control or MSCV-caNFATc1
virally-infected cells was determined as described in “Experimental Procedures” and is
presented as mean area of resorption (mm2) (right panel). Results are means +/- SEM
of responses from 6 cultures. * p<0.05 vs. cells infected with control retrovirus. E, Effect
of ectopic caNFATc1 expression on the ability of RAW264.7 cells to form resorption pits
on dentine slices. RAW264.7 cells infected with either control MSCV-GFP (left panel) or
the MSCV-caNFATc1 (middle panel) retrovirus were incubated on dentine slices for 10
days, at which point cells were removed and resorption pits were visualized by
hematoxylin staining. Resorption pits appear as dark spots stained with hematoxylin.
Quantitation of the data showing mean area of resorption (mm2) +/- SEM is presented
(right panel). ND, none detected. F, Representative scanning electron microscope
images illustrating the formation of resorption pits in the surface of dentine slices
cultured in the presence of caNFATc1-expressing cells. Original magnification 150X and
100X, respectively.
Figure 5. Effect of constitutively active NFATc1 on gene expression in RAW264.7
cells. RAW264.7 cells infected with either the control MSCV-GFP or the MSCV-
caNFATc1 virus were cultured for 6 days, at which point RNA was isolated for RT-PCR
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analysis using gene specific primers for TRAP, alpha V, beta 3, cathepsin K, calcitonin
receptor and cyclophilin. PCR products were resolved by 2% agarose-gel
electrophoresis and visualized by Vistra Green staining using a Storm phosphoimager.
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Fig. 1
0
0.1
0.2
0.3
0.4
0.5
0 3 10 30 100
RANKL (ng/ml)
TR
AP
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ity (A
410) Control
CsA
A
0
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100
150
200
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RANKL (ng/ml)
TR
AP+
MN
C /
wel
l ControlCsA
B
#
*
#
*
#
***
***
*
##
#
0
0.1
0.2
0.3
0.4
0.5
0 3 10 30 100
RANKL (ng/ml)
TR
AP
activ
ity (A
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FK506
C
0
50
100
150
200
250
0 3 10 30 100
RANKL (ng/ml)
TR
AP+
MN
C /
wel
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D
#
*#
*
#
*#
*
**
*
**#
*
34
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Uninfected MSCV-VIVIT-GFPUninfected Fig. 2
A
100 101 102 103 104
0.68%
GFP- fluoresence Intensity
Cel
l num
ber
100 101 102 103 104
95.23%
Cel
l num
ber
GFP- fluoresence Intensity100 101 102 103 104
0.68%
GFP- fluoresence Intensity
Cel
l num
ber
Cel
l num
ber
FP-fluoresence Intensity P-fluoresence IntensityG GF
0
0.5
1
1.5
2
0 3 10 30 100RANKL (ng/ml)
TR
AP
activ
ity (A
410) GFP
VIVIT
0
50
100
150
200
0 3 10 30 100RANKL (ng/ml)
TR
AP+
MN
C /
wel
l
GFP
VIVIT
#
*
#
*
L
GFP+RANKLGFP
0.62 )
Bar : 0.5mm
*** *
**
*
##
*
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E
B
0
0.2
0.4
GFP GFP+RANKL
Pit a
rea
(mm
GFP+RANKL GFP
*
35
VIVIT+RANK
DC
VIVIT+RANKL
VIVIT+RANKL
#
Bar : 0.5mm
Fig. 3
C
cDNA
NFATc1
NFATc2
NFATc3
yclophilin
Control no cDNA
Dow
nloaded
+RANKL36
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0
100
200
300
400
500
GFP NFAT
TR
AP+
MN
C /
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0
0.05
0.1
0.15
0.2
GFP NFAT
TR
AP
activ
ity (A
410)
B
GFP C
A
* *
ND
Fig. 4
NFAT
Bar : 0.5mm
D
0
0.2
0.4
0.6
0.8
GFP NFAT
Pit a
rea
(mm
2 )
NFAT
Bar : 0.25mm
NFAT GFP
0
0.2
0.4
0.6
0.8
1
1.2
GFP NFAT
Pit a
rea
(mm
2 )
Bar : 0.5mm
*
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NFAT GFP
E F NFAT
Bar : 0.1mmBar : 0.1mm
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Fig. 5
NFATc GFP
ca
P
cr
cy
TRA
thepsin K
beta 3
alpha V
alcitonin eceptor
clophilin
no DNA
38
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Hiroaki Hirotani, Nathaniel A. Tuohy, Je-Tae Woo, Paula H. Stern and Neil A. Clipstonecells
The calcineurin/NFAT signaling pathway regulates osteoclastogenesis in RAW264.7
published online January 13, 2004J. Biol. Chem.
10.1074/jbc.M213067200Access the most updated version of this article at doi:
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