the calcineurin/nfat signaling pathway regulates … · 2004-01-13 · 2 summary although best...

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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: [email protected]

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: [email protected]

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: [email protected].

## To whom correspondence may be addressed. Tel.: 312-503-8290; Fax: 312-503-

5349; e-mail: [email protected].

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

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RANKL (ng/ml)

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0

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*

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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

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FP-fluoresence Intensity P-fluoresence IntensityG GF

0

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0 3 10 30 100RANKL (ng/ml)

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GFP+RANKLGFP

0.62 )

Bar : 0.5mm

*** *

**

*

##

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E

B

0

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Pit a

rea

(mm

GFP+RANKL GFP

*

35

VIVIT+RANK
D
C

VIVIT+RANKL

VIVIT+RANKL

#

Bar : 0.5mm

http://www.jbc.org/

Fig. 3

C

cDNA

NFATc1

NFATc2

NFATc3

yclophilin

Control no cDNA

Dow

nloaded
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0

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GFP NFAT

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AP+

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Fig. 4

NFAT

Bar : 0.5mm

D

0

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Pit a

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E F NFAT

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Fig. 5

NFATc GFP

ca

P

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cy

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thepsin K

beta 3

alpha V

alcitonin eceptor

clophilin

no DNA

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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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