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The Journal of Immunology, 2006, 177: 2384-2390.
Copyright © 2006 by The American Association of Immunologists

Activation of NFAT Signal In Vivo Leads to Osteopenia Associated with Increased Osteoclastogenesis and Bone-Resorbing Activity1

Fumiyo Ikeda2,*, Riko Nishimura3,*, Takuma Matsubara*, Kenji Hata*, Sakamuri V. Reddy{dagger} and Toshiyuki Yoneda*

* Department of Molecular and Cellular Biochemistry, Osaka University Graduate School of Dentistry, Osaka, Japan; and {dagger} Children’s Research Institute, Medical University of South Carolina, Charleston, SC


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The transcription factor family member NFAT plays an important role in the regulation of osteoclast differentiation. However, the role of NFAT in osteoclasts in vivo is still not fully understood. Thus, we generated transgenic mice in which constitutively active-NFAT1/NFATc2 (CA-NFAT1) is specifically expressed in the osteoclast lineage, using the tartrate-resistant acid phosphatase gene promoter. Both x-ray and histological analyses demonstrated an osteopenic bone phenotype in the CA-NFAT1 transgenic mice, whereas the number of tartrate-resistant acid phosphatase-positive osteoclasts was markedly higher in the long bones of these mice. Furthermore, the bone-resorbing activity of mature osteoclasts derived from the transgenic mice was much higher than that of wild-type mice. Interestingly, the introduction of CA-NFAT1 into osteoclasts or RAW264 cells increased the expression and activity of c-Src and stimulated actin ring formation. In contrast, CA-NFAT1 or GFP-tagged VIVIT peptide, a specific inhibitor of NFAT, did not affect the survival of mature osteoclasts. Collectively, our data indicate that NFAT controls bone resorption in vivo by stimulating the differentiation and functioning of osteoclasts but not their survival.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Osteoclasts, which are derived from hemopoietic stem cells under the control of M-CSF and the receptor activator of the NF-{kappa}B ligand (RANKL)4 play a central role in bone resorption (1, 2, 3). RANKL, a membrane-bound molecule expressed in osteoblasts and stromal cells, binds to its receptor, receptor activator of the NF-{kappa}B (RANK) and activates intracellular signaling cascades in osteoclasts and their precursors (4). Consistent with in vitro studies, mice deficient in either the RANKL or the RANK gene show severe osteopetrosis due to a complete lack of osteoclast development, indicating that the RANKL/RANK signal is indispensable for osteoclastogenesis (2, 3, 4). RANKL has also been shown to use TNFR-associated factor 6 (TRAF6), an essential adaptor molecule in RANKL signaling and osetoclast development, to activate the IKK/NF-{kappa}B, JNK/c-Jun, and p38/ATF2 pathways (5, 6, 7). These signaling pathways are essential for osteoclastogenesis (8, 9, 10, 11).

NFAT2/NFATc1 is a target transcription factor of RANKL and regulates osteoclastogenesis (12, 13). Furthermore, overexpression of NFAT2 in spleen cells promotes osteoclast differentiation even in the absence of RANKL, and mouse ES cells isolated from NFAT2-deficient mice fail to form osteoclasts in vitro (13). NFAT1/NFATc2 activated by RANKL, and in cooperation with the AP-1 complex, controls the expression of NFAT2 (14). Consistent with these findings, NFAT2 expression is diminished in spleen cells from c-Fos- or TRAF6-deficient mice (13, 14). Moreover, the induction of NFAT2 expression during osteoclast differentiation is critically dependent on NF-{kappa}B, as demonstrated in a recent study using an inhibitor of NF-{kappa}B (15). These findings suggest an important role for the NFAT family in the regulation of osteoclast differentiation.

Although the importance of the NFAT family in osteoclastogenesis has been well demonstrated in vitro, the relationship between the NFAT family and osteoclasts in vivo remains unclear. NFAT2-deficient mice are embryonic lethal at the early stage (16, 17), and mice deficient in other NFAT family member genes do not show a distinctive skeletal phenotype, presumably because compensation is supplied by each family member (18, 19, 20). In a very recent study, mouse ES cells isolated from normal mice but not from NFAT2-deficient mice rescued the osteopenic phenotype of c-Fos-deficient mice (21), illustrating the importance of NFAT2 in osteoclastogenesis in the c-Fos-deficient condition. Nonetheless, it remains unclear how NFAT contributes to the regulation of differentiation and functioning of osteoclasts in the presence of the c-Fos gene, which is an essential transcription factor for osteoclast development (22) and critical for NFAT functioning as a transcriptional partner (10, 13, 14).

To investigate the functional role of the NFAT family in the osteoclast story, we generated transgenic (Tg) mice in which the active form of NFAT1 was specifically expressed in the osteoclast lineage, under the control of the tartrate-resistant acid phosphatase (TRAP) gene promoter. This transgenic model showed a clear osteopenic phenotype with a marked increase in osteoclasts. In addition, the activation of NFAT signaling in vivo led to increased bone resorption with consequent induction of the expression and activation of c-Src. Thus, our study demonstrates that NFAT is an important transcription factor for osteoclast differentiation and functioning.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

A monocytic cell line, RAW264, was purchased from the RIKEN cell bank (Tsukuba) and cultured in {alpha}-MEM containing 10% FCS (Valley Biomedical). Soluble RANKL (sRANKL) and M-CSF were purchased from Pepro-Tech EC and Green Cross, respectively. Anti-c-Src and anti-active-c-Src Abs were purchased from Cell Signaling. 4,6-diamidino-2-phenylindole was purchased from Molecular Probes, and rhodamine-labeled phalloidin was from Fluka.

Constructs and transfection

The constitutively active NFAT1/NFATc2 (CA-NFAT1) construct (23), GFP-VIVIT construct (24), TRAP gene promoter (25), and TRAF6 cDNA (7) have been described previously (10).

Generation and analysis of Tg mice

CA-NFAT1 cDNA tagged with an HA epitope was fused to 1.8 kb of the mouse TRAP gene promoter (10, 25). Subsequently, Tg mice were generated using pronuclear injection of TRAP-CA-NFAT1 cDNA into C57BL/6 x DBA/2 F1 mice (10). Genomic DNA isolated from the tails of these mice was then analyzed by PCR and Southern blot analysis using specific primers or the probes for the transgene. Expression of the transgene was also confirmed by RT-PCR analysis. An x-ray analysis of long bones and spines was performed with an analyzer from SOFTEX. Three-dimensional microcomputed tomography (3D-micro CT) was performed, and the trabecular thickness was measured using a composite x-ray analysis system (SMX-100CT-SV; Shimadzu). Spleen cells or bone marrow cells isolated from control or TRAP-CA-NFAT1 Tg mice were analyzed by the in vitro osteoclast differentiation assay or by the pit formation assay. All experiments were performed with sex- and age (2- to 4-wk-old)-matched mice under protocols approved by the Osaka University Graduate School of Dentistry Animal Care Committee.

Histological and histomorphometric analyses of long bones

Bones isolated from Tg or control mice were fixed in 3.8% buffered formalin, decalcified in 4% EDTA, and then histologically analyzed by H&E, TRAP, or Alcian blue staining (26). Undecalcified sections of bones were also analyzed for mineral content using von Kossa staining as previously described (27). Bones isolated from the transgenic or control mice were fixed and embedded for frozen section. Sections were cut using tungsten blades and left for 1 h in 5% silver solution in sunlight. Thereafter, the sections were washed with distilled water for 5 min and incubated in 5% sodium thiosulfate for 5 min in preparation for the histomorphometric analysis. The growth plate width, as stained with Alcian blue, and the area in square meters of the tissue stained by the von Kossa method were analyzed by Scion Image analysis software (Scion Cooperation). Three sites from independent sections were analyzed. Bone volume/total tissue volume (BV/TV) and osteoclast number/perimeter of bone surface were determined using the sagittal section of the middle part of each bone as previously described (28), using the image analysis system Image-Pro Plus (Silver Spring, MD). To determine BV/TV, the bone volume of three independent sections was measured and divided by the total tissue volume. To determine osteoclast number/perimeter of bone surface, in three independent TRAP-stained sections the number of TRAP-positive osteoclasts on the trabecular bone in each section was counted and divided by the length of the bone surface.

RT-PCR

Total RNA was isolated from brain, kidney, liver, lung, and spleen cells that had been cultured in the presence or absence of M-CSF and sRANKL or both. The RNA was then treated with RNase-free DNase (Wako Pure Chemical Industries), and after denaturation of the total RNA at 70°C for 10 min, cDNAs were synthesized using an oligodeoxythymidylate primer and reverse transcriptase (Qiagen). PCR amplification was performed using specific primers for CA-NFAT1 tagged with an HA epitope (forward primer, 5'-ttgcattcattttatgtttcagg-3'; reverse primer, 5'-ggccgcgactctagatcat-3'). The PCR products were then electrophoresed through a 2% agarose gel and visualized by ethidium bromide staining.

Osteoclast differentiation in vitro

Bone marrow cells and spleen cells were isolated from the Tg mice, the littermate control mice, or C57BL/6 mice (Nihon SLC; Hamamatsu) and incubated with M-CSF (30 ng/ml) and sRANKL (10–100 ng/ml) for 4–6 days (7). The cells were then analyzed by TRAP staining, and TRAP-positive multinucleated cells were counted as osteoclasts (7). RAW264 cells were incubated in the presence of sRANKL (20 ng/ml) for 4 days (10).

Nonspecific esterase (NSE) staining

Spleen cells isolated from the Tg or control mice were cultured with M-CSF (10 ng/ml) for 6 days and stained using an NSE staining kit (Sigma-Aldrich) according to the manufacturer’s protocol.

Pit formation assay

To examine the function of TRAP-positive cells derived from spleen cells or bone marrow cells, we induced osteoclast formation on dentin slices or Osteologic Plates (calcium phosphate-covered plates; BD Biosciences). After 6 days of culture, cells were first stained for TRAP activity, and the number of TRAP-positive cells was counted. Cells were then removed from the dentin slices or Osteologic Plates, and the resorption pits formed on the dentine slices were stained with toluidine blue (29). The area encompassed by the pits was quantified using the image analysis system Image-Pro Plus.

Analysis of survival rates of osteoclasts in vitro

Osteoclasts prepared as above were further incubated in the presence or absence of sRANKL for 20 h, after which the cells were washed with PBS and stained for TRAP activity. Multinucleated TRAP-positive cells were counted. Osteoclasts at the beginning (t = 0) of incubation were also analyzed. The survival rate of the osteoclasts was then calculated as the percentage of TRAP-positive multinuclear cells remaining after the 20 h of treatment divided by the TRAP-positive cell number at t = 0.

Immunoblotting analysis

Protein was isolated from RAW264 cells, and the concentration was determined as previously described (30). Equivalent amounts of protein were loaded for SDS-PAGE, and immunoblotting then performed using Abs specific for c-Src and active c-Src. In all immunoblotting analyses, we confirmed that equal amounts of protein had been loaded by staining the transferred membrane with Ponceau S. Densiometric analysis was performed using the image analysis system Scion Image.

Generation of adenovirus

The recombinant adenoviruses carrying CA-NFAT1, TRAF6, GFP, and GFP-VIVIT were constructed by recombination between the expression cosmid cassette and the parental virus genome in 293 cells, as previously described (31). The viruses were confirmed to retain no proliferative activity in cells other than 293 cells. Virus titers were determined using the modified point assay (32).

Immunocytochemistry

RAW264 cells were washed 3 times with ice-cold PBS, fixed in 3.8% paraformaldehyde-PBS, and permeabilized by incubation for 15 min in 0.1% Triton-PBS. The cells were then blocked with 1% BSA-PBS and incubated with rhodamine-labeled phalloidin and 4,6-diamidino-2-phenylindole for 30 min. The cells were extensively washed with PBS and visualized using a fluorescent microscope (Carl Zeiss Vision).

Statistical analysis

Data were analyzed by ANOVA. The number of experiments performed or the number of samples analyzed is indicated in each figure legend.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Using RT-PCR, we confirmed the specific expression of CA-NFAT1 in osteoclasts from transgenic mice overexpressing CA-NFAT1 under the control of the TRAP gene promoter (Fig. 1A). The Tg mice (TRAP-CA-NFAT1 Tg mice) were born alive and fertile but were smaller in overall body size than the control mice. Furthermore, the TRAP-CA-NFAT1 Tg mice weighed <30% of the weight of the control mice at 3 wk after birth, a significant difference (Fig. 1B).


Figure 1
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  		FIGURE 1. Osteopenic phenotype of TRAP-CA-NFAT1 Tg mice. A, Total RNA was extracted from brain, kidney, liver, spleen, and osteoclast-like cells (OCL) derived from bone marrow cells induced by M-CSF and sRANKL and assessed by RT-PCR to examine specific expression of CA-NFAT1 mRNA in osteoclasts. Expression of beta-actin was examined as the control (Cont). TG, TRAP-CA-NFAT1 Tg mice. B, Appearance and body weight of control and TRAP-CA-NFAT1 Tg littermate mice at 3 wk after birth. The body weight of the TRAP-CA-NFAT1 Tg mice was significantly lower than that of the control mice (four control mice and six TRAP-CA-NFAT1 mice were analyzed). C, Soft x-ray analysis of long bones and spines in control and TRAP-CA-NFAT1 Tg mice. The pictures were scanned from a representative x-ray picture, in which control and Tg mice x-ray images were on the same film. D, 3D-micro CT analysis of femurs in control and TRAP-CA-NFAT1 Tg mice. The trabecular thickness (Tr.Th) in TRAP-CA-NFAT1 Tg mice was significantly smaller than in the control mice. Femurs from three mice were analyzed.

 
To examine the phenotype of these mice bones, we analyzed the long bones of TRAP-CA-NFAT1 Tg and control mice by soft x-ray analysis. As shown in Fig. 1C, the radiodensities in the long bones and spines of the CA-NFAT1 Tg mice were lower than those of the control mice. These findings were confirmed by 3D-micro CT analysis, which showed that the trabecular thickness of femurs of the TRAP-CA-NFAT1 Tg mice were clearly reduced compared with those of the control mice (Fig. 1D). These results suggested that TRAP-CA-NFAT1Tg mice have an osteopenic phenotype.

To further analyze this osteopenic phenotype, we histologically examined the femur of TRAP-CA-NFAT1 Tg mice. As shown in Fig. 2A, the bone volume of the trabecular bones was markedly reduced in theTRAP-CA-NFAT1 Tg mice. Moreover, histomorphometric analysis showed a statistically significant reduction in the bone volume of the TRAP-CA-NFAT1 Tg mice (Fig. 2A). These data confirm the osteopenic phenotype of TRAP-CA-NFAT1 Tg mice.


Figure 2
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  		FIGURE 2. Stimulated osteoclastogenesis and bone destruction in TRAP-CA-NFAT1 Tg mice (TG). A, H&E staining of long bones from control (Cont) and TRAP-CA-NFAT1 Tg littermate mice (TG). Original magnification, x50. Histomorphometric analysis demonstrated a significant decrease in BV/TV in TRAP-CA-NFAT1 Tg mice. B, TRAP staining of long bones of control and TRAP-CA-NFAT1 Tg littermate mice. Original magnification: top, x50; bottom, x100. The number of TRAP-positive osteoclasts/perimeter of bone surface (OC no./BS mm) was higher in the femurs of TRAP-CA-NFAT1 Tg mice. Alcian blue staining (C) and von Kossa staining (D) of long bones of control and TRAP-CA-NFAT1 Tg littermate mice. Original magnification: top, x100; bottom, x250). The width of the growth plate (GP) of TRAP-CA-NFAT1 Tg mice was smaller than that of the control mice. The Von Kossa-stained area of the CA-NFAT Tg mice was lower than that of the control mice. Each staining procedure (A–D) was performed on three sections each from three mice.

 
To clarify whether the osteopenic phenotype observed in the Tg mice was the result of enhanced osteoclastogenesis, we evaluated osteoclast formation in TRAP-CA-NFAT1 Tg mice by TRAP staining of the femurs of these mice. The number of TRAP-positive osteoclasts was markedly higher in the TRAP-CA-NFAT1 Tg mice than in the control mice (Fig. 2B), suggesting that the osteopenic phenotype of TRAP-CA-NFAT1 Tg mice is due to an increase in TRAP-positive osteoclasts. We also observed that the growth plate of TRAP-CA-NFAT1 Tg mice was significantly thinner than that of the control mice (Fig. 2C). Moreover, von Kossa staining of the femurs indicated a reduction in mineralized areas in TRAP-CA-NFAT1 Tg mice (Fig. 2D).

We next performed ex vivo experiments using bone marrow cells isolated from the transgenic and control mice to confirm the stimulatory effect of CA-NFAT1 on osteoclast formation in CA-NFAT1 Tg mice. The bone marrow cells were cultured in the presence of M-CSF and sRANKL, and the osteoclastogenic activity of these cells then examined. TRAP-positive osteoclast-like cell formation was significantly higher in the bone marrow cell culture of TRAP-CA-NFAT1 Tg mice compared with that of control mice, at both a low (30 ng/ml) and high (100 ng/ml) concentration of sRANKL (Fig. 3A). We also found that NFAT2 expression was markedly up-regulated in the osteoclasts formed from CA-NFAT1-Tg mice (Fig. 3B). In addition, spleen cells that had been isolated from control and CA-NFAT1 Tg mice and then treated with M-CSF (10 ng/ml) were stained for NSE activity to examine whether macrophage differentiation was intact in the Tg mice. We confirmed that macrophage differentiation was not affected in the CA-NFAT1 Tg mice (Fig. 3C). These findings demonstrate that activation of NFAT1 specifically stimulates osteoclastogenesis in these transgenic mice.


Figure 3
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  		FIGURE 3. Increased number of TRAP-positive osteoclasts and activation of mature osteoclasts derived from TRAP-CA-NFAT1 Tg cells. A, An increased number of TRAP-positive osteoclasts induced by sRANKL in TRAP-CA-NFAT1 Tg mice. Bone marrow cells isolated from either control (Cont) or TRAP-CA-NFAT1 Tg littermate mice were cultured with M-CSF (30 ng/ml) and different concentrations of sRANKL (0, 10, 30, or 100 ng/ml). After 4 days of culture, TRAP staining was performed, and the number of TRAP-positive multinucleated osteoclast-like cells (MNC) in each group was then counted. Four independent views were analyzed to determine the number of TRAP-positive cells. B, RANKL-induced expression of NFAT2 and c-Src was increased in osteoclast-like cells (ocl) of TRAP-CA-NFAT1 Tg bone marrow cells. Bone marrow cells derived from control or TRAP-CA-NFAT Tg mice were cultured under the control of M-CSF with or without sRANKL (100 ng/ml) for 6 days. The expression of NFAT2 and c-Src and the activation of c-Src were examined using the same amount of cell lysate for each. Expression of beta-actin was examined to confirm equal loading of protein. Activation of c-Src was also detected in TRAP-CA-NFAT1 Tg cells by anti-c-Src527 Ab. The numbers indicate the fold increase in NFAT2 and c-Src expression as determined by densitometric analysis. C, Macrophage differentiation was examined by NSE staining. Spleen cells isolated from control and TRAP-CA-NFAT1 Tg mice were cultured in M-CSF (10 ng/ml)-supplemented medium for 6 days, and NSE staining was then performed. NSE activity was not affected in M-CSF-treated bone marrow cells derived from TRAP-CA-NFAT1 Tg mice. D, Increased bone-resorbing activity of TRAP-CA-NFAT1 Tg littermate mice compared with control mice. Bone marrow cells isolated from control or TRAP-CA-NFAT1 Tg littermate mice were cultured with M-CSF (30 ng/ml) and sRANKL (100 ng/ml) on dentin slices or osteologic plates for 6 days and then stained for TRAP activity. Cells were then removed and stained with toluidine blue solution. The pit formation area on each osteologic plate was then quantified by dividing the pit formation area in square meters by the number of TRAP-positive cells that had formed on the same plates. The pit formation area per single TRAP-positive cell (mm2/ocl, in which mm2 is square millimeter and ocl is osteoclast-like cell) derived from TRAP-CA-NFAT1 Tg mice was significantly higher than that of control mice. Three osteologic plates were analyzed to measure the pit area per osteoclast. Original magnification, x250 for A, C, and D. Similar results were obtained from three independent experiments.

 
On the basis of the severe osteopenic phenotype observed in the CA-NFAT1 Tg mice, we examined the possibility that the activation of NFAT1 stimulates osteoclastic bone resorption in addition to osteoclastogenesis. To address this possibility, we performed a pit assay to determine the bone-resorbing activity of bone marrow cells isolated from CA-NFAT1 Tg or control mice (Fig. 3D). The pit formation area formed on the calcium phosphate-covered plates per osteoclast was markedly greater in the TRAP-CA-NFAT1 Tg mice than in the control mice (Fig. 3D). These data demonstrate that the osteopenic phenotype in TRAP-CA-NFAT1 Tg mice is due to increases in osteoclastogenesis and bone-resorbing activities by mature osteoclasts.

c-Src plays an essential role in ruffled border formation and osteoclastic bone resorption (33, 34). Thus, we examined the expression and activity of c-Src in osteoclast-like cells from CA-NFAT1 Tg or control mice. As shown in Fig. 3B, both c-Src expression and activity was clearly up-regulated in the cells from CA-NFAT1 Tg mice. We next evaluated the effect of activation of NFAT1 on formation of the actin ring, a distinctive cytoskeleton structure for osteoclasts, using RAW264 cells, which rarely form the actin ring even in the presence of sRANKL (Fig. 4A, middle). We introduced CA-NFAT1 into RAW264 cells using an adenovirus system and then cultured the cells in the presence of sRANKL. This resulted in actin ring formation (Fig. 4A, right). Western blot analysis demonstrated that the introduction of CA-NFAT1 induced the expression and activation of c-Src even in the absence of sRANKL (Fig. 4B). These data collectively suggest that the activation of NFAT signal up-regulates c-Src expression and function, and consequently promotes actin ring formation.


Figure 4
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  		FIGURE 4. Actin ring formation and c-Src expression by induction of CA-NFAT1 in osteoclast precursor cells. A, Actin ring formation induced by CA-NFAT1 in RAW264 cells. TRAP-positive multinucleated osteoclast-like cells, which do not contain an actin ring, were induced by sRANKL. Additional introduction of CA-NFAT1 by adenovirus with sRANKL induced actin ring formation, as determined by immunofluorescence using rhodamine-labeled phalloidin. Original magnification, x400 for all panels. B, Induction of expression and activation of c-Src by CA-NFAT1 in RAW264 cells. The introduction of CA-NFAT1 into RAW264 cells using an adenovirus system induced the expression and activation of c-Src in the absence of sRANKL, as determined by immunoblotting with anti-c-Src and anti-active-c-Src. Similar results were obtained from three independent experiments. Cont, Control.

 
NFAT family members are involved in the regulation of survival in T cells (35, 36). Because the life span of osteolcasts is controlled by RANKL, which prevents apoptosis of osteoclasts, we next investigated whether the activation of NFAT1 affected the survival of osteoclasts. After the induction of mature osteoclast formation from bone marrow cells isolated from CA-NFAT1 Tg or control mice, we cultured these cells in the absence of sRANKL for an additional 20 h. As shown previously (37), mature osteoclasts derived from the control mice disappeared within 20 h of sRANKL starvation (Fig. 5A, top). Mature osteoclasts derived from CA-NFAT1 Tg mice also disappeared in the absence of sRANKL, and the survival rate of TRAP-CA-NFAT1 Tg osteoclasts was very similar to that of the control osteoclasts (Fig. 5A, bottom). To confirm the effect of NFAT1 on the survival of osteoclasts, we performed in vitro assay using primary osteoclast-like cells derived from bone marrow cells of wild-type mice (Fig. 5, B and C). Overexpression of TRAF6 in the osteoclast-like cells supported the survival of the cells even in the absence of sRANKL (Fig. 5B, middle). On the other hand, overexpression of CA-NFAT1 did not support the survival of osteoclast-like cells (Fig. 5B, bottom). Moreover, VIVIT peptide, a specific inhibitor of the NFAT family, had little effect on the survival of the osteoclast-like cells (Fig. 5C, right). These data collectively indicate that the activation of NFAT is not involved in the programmed cell death of osteoclasts.


Figure 5
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  		FIGURE 5. Effects of CA-NFAT1 on survival in mature osteoclasts. A, CA-NFAT1 did not affect the survival of mature osteoclasts. Bone marrow cells isolated from control (Cont) and TRAP-CA-NFAT1 Tg littermate mice were cultured with M-CSF (30 ng/ml) and sRANKL (100 ng/ml) for 6 days and for a further 20 h with or without sRANKL, after which the number of TRAP-positive cells was counted. sRANKL starvation had little effect on the survival rate of mature osteoclasts derived from TRAP-CA-NFAT1 Tg mice compared with control mice. B, Effects of CA-NFAT1 overexpression on mature osteoclasts. Bone marrow cells were isolated from wild-type mice and then treated with M-CSF (30 ng/ml) and sRANKL (100 ng/ml) for 6 days and infected with control, TRAF6, or CA-NFAT1 adenovirus. After 20 h of culture in the presence or absence of sRANKL, TRAP staining was performed to determine the survival of mature osteoclasts. TRAF6 infection rescued the survival of TRAP-positive cells in the absence of sRANKL. On the other hand, the introduction of CA-NFAT1 had little effect on the survival of mature osteoclasts. C, Effects of an NFAT-specific inhibitor, VIVIT, on survival of mature osteoclasts. The introduction of GFP-conjugated VIVIT, via an adenovirus system, into wild-type bone marrow cells treated with M-CSF (30 ng/ml) and sRANKL (100 ng/ml) for 6 days had no significant effect on the survival rate of mature osteoclasts. GFP adenovirus was used as the control. Four independent wells were analyzed to calculate the survival rate of the osteoclasts for A and C. Original magnification, x400 for all panels. Similar results were obtained from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although the importance of the NFAT family in the regulation of osteoclastogenesis has been previously demonstrated (10, 13, 14, 21), the role of this family in the osteoclast lineage has not been fully established. To this end, we examined the effects of activation of the NFAT signal on osteoclast differentiation and function by generating Tg mice overexpressing CA-NFAT1, specifically in the osteoclast lineage. We previously showed that a CA-NFAT1 mutant protein, which predominantly localizes in the nucleus (10), promotes the differentiation of mouse spleen cells into TRAP-positive osteoclasts and induces NFAT2 expression even in the absence of RANKL (10). Asagiri et al. (21) also showed that overexpression of NFAT1 promotes osteoclast differentiation even in the absence of the NFAT2 gene. As expected, we found that the Tg mice manifested an osteopenic phenotype associated with an increase in osteoclast formation and bone resorption. In addition, ex vivo experiments demonstrated high osteoclastogenic activity of bone marrow cells isolated from the Tg mice. Thus, activation of the NFAT family leads to osteoclast differentiation in vivo.

NFAT1 and NFAT2 are expressed in mature osteoclasts (10, 13), which suggests that the NFAT family is involved in control of osteoclast function. In fact, we found that osteoclastic bone-resorbing activity was markedly increased in TRAP-CA-NFAT1 Tg mice. To confirm whether the stimulated osteoclastic bone resorption was due not only to activated osteoclastogenesis but also to higher bone-resorbing activity, we examined the bone-resorbing activity per osteoclast using bone marrow cells isolated from TRAP-CA-NFAT1 Tg mice. Our ex vivo experiments indicated that activation of NFAT1 resulted in high osteoclastic bone-resorbing activity per osteoclast. Consistent with this, treatment with cyclosporin A, which inhibits the NFAT signal, suppresses bone resorption by osteoclasts (38). Furthermore, the expression and activity of c-Src, which is essential for osteoclastic bone resorption and ruffled border formation (33, 34), were substantially elevated in the osteoclasts from TRAP-CA-NFAT1 Tg mice compared with those from the control mice. In addition, we found that CA-NFAT1 stimulated actin ring formation in RAW264 cells. In contrast, we did not detect any effects of CA-NFAT1 on proton pump expression (data not shown). However, RANKL/NFAT signaling may actually be involved in the regulation of proton pump function, because the cellular localization of the vacuolar proton-ATPase is controlled during osteoclast differentiation (39). Taken together, our results strongly suggest that CA-NFAT1 up-regulates c-Src function and ruffled border formation, thereby stimulating osteoclastic bone resorption. Thus, it is highly likely that the NFAT family controls osteoclast function in addition to osteoclast development.

Interestingly, the TRAP-CA-NFAT1 Tg mice were significantly smaller in overall size than the control mice, and the growth plate of the TRAP-CA-NFAT1 Tg mice was much thinner than that of the control mice. Although NFAT1 and NFAT4 are known to be involved in the regulation of chondrogenesis (18, 40), it is unlikely that CA-NFAT1 directly affected chondrocyte differentiation in the present study because the transgene was not induced in the chondrocytes of the transgenic mice. In contrast, we observed numerous TRAP-positive osteoclasts around the growth plate of the TRAP-CA-NFAT1 Tg mice. Consistent with this, endochondral ossification was substantially impaired in the TRAP-CA-NFAT1 Tg mice. These data support the important role of osteoclasts, or chondroclasts, in regulation of growth plate development and raise the possibility that aggressive and excessive osteoclast function affects endochondral bone formation during the developmental stage.

RANKL is critical for the survival of osteoclasts, given that it inhibits apoptosis. We showed that the overexpression of TRAF6 prevents the apoptosis of mature osteoclasts, as does treatment with sRANKL. Therefore, we were curious as to whether RANKL/TRAF6 signaling supports the survival of osteoclasts through the NFAT family. However, osteoclasts formed from the bone marrow cells of TRAP-CA-NFAT1 Tg mice showed a survival rate similar to those of the control mice. Furthermore, the introduction of CA-NFAT1 did not prevent the apoptosis of the osteoclasts. Moreover, a specific inhibitor of NFAT function, the VIVIT peptide, also did not affect the survival of the osteoclasts. In contrast to other systems (35, 36, 41, 42), the NFAT family does not appear to be involved in the survival of osteoclasts. Further analysis of RANKL/TRAF6 signaling would contribute to the understanding of the molecular mechanisms by which RANKL regulates osteoclast apoptosis.

The NFAT 1/2pick;3657f5a;0;1family members interact with partner transcription factors to regulate the transcription of their target genes (43, 44, 45). The interaction of NFAT with AP-1 or NF-{kappa}B, both of which play a critical role in the regulation of osteoclastogenesis, has been associated with the regulation of the transcriptional activity of target genes in T cells (46). Recent studies have determined the molecular interaction between NFAT and AP-1 in the control of osteoclastogenesis (10, 13, 14). On the other hand, the mechanism of the regulation of NFAT and NF-{kappa}B in RANKL/RANK signaling remains unclear. Moreover, it is not yet clear why the RANKL/RANK/TRAF6 system shows unique osteoclastogenic action, whereas other stimulatory cytokines including TNF-{alpha} or IL-1, which also activate AP-1 or NF-{kappa}B, do not. Thus, the precise molecular mechanism that regulates NFAT activity through the RANKL/RANK/TRAF6 system in osteoclasts should be clarified. Our previous data indicate that the transcriptional activity of NFAT is enhanced by TRAF6 (10) and is induced not only by wild-type TRAF6 but also by a deletion mutant of TRAF6 that lacks the ability to mediate NF-{kappa}B or AP-1 signaling (data not shown). Nevertheless, further analysis of the molecular mechanism that regulates bone metabolism via osteoclastic activity is required for better understanding of the unique function of the RANKL/RANK system together with NFAT activation.

In conclusion, we demonstrated that the NFAT family plays a critical role in the regulation of osteoclast development and bone resorption. Thus, an appropriate dissection of clinical application of VIVIT peptide and NFAT-calcineurin association compounds (47, 48) or development of more selective NFAT inhibitors would contribute to the establishment of treatment for bone destructive diseases such as osteoporosis, rheumatoid arthritis, bone metastases, and Paget’s disease.


   Acknowledgments
 
We thank Dr. G. David Roodman for providing the TRAP gene promoter construct and Dr. Anjana Rao for providing the CA-NFAT1 and GFP-VIVIT constructs. We also thank Drs. Kazuya Oshima and Hideki Yoshikawa for technical support in the 3D-micro CT analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Ministry of Education, Science, Sports and Culture Grant-in-Aid for Scientific Research A 11307041(to T.Y.), B 15390560 (to R.N.), and C 10671739 (to R.N.); Grant-in-Aid for Scientific Research on Priority Areas B 12137205 (to T.Y.); Naito Foundation (to R.N.); and The 21st Century COE Program (to T.Y. and R.N.). Back

2 Current address: Institute for Biochemistry II, Goethe University Medical School, Frankfurt 60590, Germany. Back

3 Address correspondence and reprint requests to Dr. Riko Nishimura, Department of Molecular and Cellular Biochemistry, Osaka University Graduate School of Dentistry, Osaka, Japan 565-0871. E-mail address: rikonishi{at}dent.osaka-u.ac.jp Back

4 Abbreviations used in this paper: RANKL, receptor activator of the NF-{kappa}B ligand; RANK, receptor activator of the NF-{kappa}B; sRANKL, soluble RANKL; TRAF6, TNFR-associated factor 6; Tg, transgenic; CA-NFAT1/NFATc2, constitutively active-NFAT1/NFATc2; 3D-micro CT, three-dimensional microcomputed tomography; TRAP, tartrate-resistant acid phosphatase; BV/TV, bone volume/total tissue volume; NSE, nonspecific esterase. Back

Received for publication February 17, 2006. Accepted for publication May 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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