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Fos plays an essential role in the upregulation ofRANK expression in osteoclast precursors within thebone microenvironment

Atsushi Arai1,2,*, Toshihide Mizoguchi1,*, Suguru Harada1, Yasuhiro Kobayashi1, Yuko Nakamichi1,Hisataka Yasuda3, Josef M. Penninger4, Kazuhiro Yamada2, Nobuyuki Udagawa5 and Naoyuki Takahashi1,`

1Institute for Oral Science, Matsumoto Dental University, Nagano 399-0781, Japan2Department of Orthodontics, Matsumoto Dental University, Nagano 399-0781, Japan3Bioindustry Division, Oriental Yeast Company Limited, Tokyo 174-8505, Japan4Institute of Molecular Biotechnology of the Austrian Academy of Sciences, A-1030 Vienna, Austria5Department of Biochemistry, Matsumoto Dental University, Nagano 399-0781, Japan

*These authors contributed equally to this work`Author for correspondence (takahashinao@po.mdu.ac.jp)

Accepted 15 February 2012Journal of Cell Science 125, 2910–2917� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.099986

SummaryFos plays essential roles in the osteoclastic differentiation of precursor cells generated by colony-stimulating factor 1 (CSF-1) andreceptor activator of NF-kB ligand (RANKL; also known as tumor necrosis factor ligand superfamily member 11, Tnsf11). RANKL-deficient (RANKL2/2) mice and Fos2/2 mice exhibit osteopetrosis due to an osteoclast deficiency. We previously reported that RANK-

positive osteoclast precursors are present in bone of RANKL2/2 mice but not Fos2/2 mice. Here we report the role of Fos in RANKexpression in osteoclast precursors. Medullary thymic epithelial cells and intestinal antigen-sampling microfold cells have been shownto express RANK. High expression of RANK was observed in some epithelial cells in the thymic medulla and intestine but not inosteoclast precursors in Fos2/2 mice. RANK mRNA and protein levels in bone were lower in Fos2/2 mice than RANKL2/2 mice,

suggesting that Fos-regulated RANK expression is tissue specific. When wild-type bone marrow cells were inoculated into Fos2/2 mice,RANK-positive cells appeared along bones. RANK expression in wild-type macrophages was upregulated by coculturing withRANKL2/2 osteoblasts as well as wild-type osteoblasts, suggesting that cytokines other than RANKL expressed by osteoblasts

upregulate RANK expression in osteoclast precursors. CSF-1 receptor-positive cells were detected near CSF-1-expressing osteoblasticcells in bone in Fos2/2 mice. CSF-1 upregulated RANK expression in wild-type macrophages but not Fos2/2 macrophages.Overexpression of Fos in Fos2/2 macrophages resulted in the upregulation of RANK expression. Overexpression of RANK in Fos2/2

macrophages caused RANKL-induced signals, but failed to recover the RANKL-induced osteoclastogenesis. These results suggest thatFos plays essential roles in the upregulation of RANK expression in osteoclast precursors within the bone environment.

Key words: Osteoclasts, Osteoclast precursors, RANK, CSF-1, Fos

IntroductionOsteoclasts, multinucleated giant cells responsible for bone

resorption, exist along the bone surface (Chambers, 2000; Martin

et al., 1998; Roodman, 1999). Osteoclasts form from monocyte–

macrophage lineage precursors under the tight regulation of

bone-forming osteoblasts. Osteoblasts express two cytokines,

colony-stimulating factor 1 (CSF-1, also called macrophage

colony-stimulating factor) and receptor activator of NF-kBligand (RANKL; also known as tumor necrosis factor ligand

superfamily member 11, Tnsf11), both of which are essential to

osteoclastic differentiation (Arron and Choi, 2000; Boyle et al.,

2003; Hofbauer et al., 2000; Kong et al., 1999; Lacey et al., 1998;

Suda et al., 1999; Yasuda et al., 1998; Yoshida et al., 1990).

Osteoclastic differentiation is severely depressed in the bone of

CSF-1-mutated osteopetrotic op/op mice and RANKL-deficient

(RANKL2/2; Tnsf112/2) mice (Boyle et al., 2003; Kong et al.,

1999; Wiktor-Jedrzejczak et al., 1990; Yoshida et al., 1990). CSF-

1 is constitutively expressed, whereas RANKL is expressed

inducibly by osteoblasts in response to bone-resorbing stimuli,

such as parathyroid hormone and 1a, 25-dihydroxyvitamin D3(Suda et al., 1999; Yoshida et al., 1990). In the presence of tumor

necrosis factor a (TNF-a) and transforming growth factor b (TGF-b), hematopoietic precursors from RANKL2/2 or RANK2/2 micecan differentiate into osteoclasts in vitro, suggesting the existence

of alternative routes for osteoclastic differentiation (Kim et al.,

2005; Kobayashi et al., 2000).

After the discovery of the RANKL signaling in

osteoclastogenesis, RANKL–RANK signaling was shown to be

involved in other crucial biological processes such as the

development of lymph nodes, the development of medullary

thymic epithelial cells and intestinal antigen-sampling M

(microfold) cells, lactation, breast cancer metastasis to bone and

the central fever response in inflammation (Akiyama et al., 2008;

Cao et al., 2001; Fata et al., 2000; Hanada et al., 2009; Hikosaka

et al., 2008; Jones et al., 2006; Knoop et al., 2009). Medullary

thymic epithelial cells necessary for maintaining self-tolerance and

2910 Research Article

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intestinal M cells necessary for intestinal immunity have been

shown to highly express RANK (Akiyama et al., 2008; Hikosaka

et al., 2008; Knoop et al., 2009). Thus, RANKL–RANK

interaction is now recognized to participate in biological

responses beyond those in bone.

Fos, a component of activator protein 1 (AP-1), is a

transcription factor essential for osteoclastogenesis (Grigoriadis

et al., 1994; Wang et al., 1992). Fos-deficient (Fos2/2) mice

exhibit severe osteopetrosis because of a lack of osteoclasts. In

striking contrast, F4/80-positive macrophages have been detected

in bone of Fos2/2 mice (Grigoriadis et al., 1994). These results

suggest that hematopoietic progenitor cells can differentiate into

macrophages but not into osteoclasts in the absence of Fos,

because if the F4/80-positive macrophages in bone were

osteoclast precursors then Fos would be required for the

differentiation of macrophages into osteoclasts. It was also

reported that RANKL-induced expression of nuclear factor of

activated T cells c1 (NFATc1), a master regulator of osteoclastic

differentiation, is tightly regulated by Fos (Ishida et al., 2002;

Matsuo et al., 2004; Takayanagi et al., 2002). Therefore, Fos

in osteoclast precursors is believed to play a role in

osteoclastogenesis as part of the RANKL-induced pathway.

We have examined the characteristics and behavior of

osteoclast precursors in vivo. Immunohistochemical analysis

revealed that the precursors exist along the bone surface in

RANKL2/2 mice, as RANK-positive cells (Mizoguchi et al.,

2009). We named these osteoclast precursors ‘cell-cycle-arrested

quiescent osteoclast precursors’ (QOPs), because QOPs rapidly

differentiate into osteoclasts in response to RANKL without cell-

cycle progression. In contrast to RANKL2/2 mice, Fos2/2 mice

had no RANK-positive cells in bone tissues (Mizoguchi et al.,

2009). These results suggest that Fos is also necessary for the

occurrence of RANK-positive cells along the bone surface.

In the present study, we examined the role of Fos in RANK

expression in osteoclast precursors. We show that the bone

environment is involved in the upregulation of RANK expression

in osteoclast precursors, and the expression of Fos in the

precursors is necessary for the upregulation. Our results suggest

that osteoblast-derived factors such as CSF-1 are involved in the

Fos-dependent upregulation of RANK expression in osteoclast

precursors, which makes it possible that osteoclast precursors use

only RANKL as an osteoclast differentiation factor under

physiological conditions. The physiological importance of the

upregulation of RANK expression in osteoclast precursors is

discussed in detail.

ResultsMany tartrate-resistant acid phosphatase (TRAP)-positive

osteoclasts were observed along the surfaces of proximal tibiae

in wild-type mice but not in RANKL2/2 mice or Fos2/2 mice

(Fig. 1A). RANK-positive osteoclasts or osteoclast precursors

were also detected with similar distributions to TRAP-positive

cells in wild-type mice (Fig. 1B). As we reported previously,

RANK-positive cells existed along the bone surface as osteoclast

precursors in RANKL2/2 mice, but not in Fos2/2 mice (Fig. 1B)

(Mizoguchi et al., 2009). By contrast, CSF-1R-positive cells were

similarly distributed in all the genotypes, and the difference in

expression of CSF-1R mRNA in bone tissues of RANKL2/2 and

Fos2/2 mice was not significant (Fig. 1B,C). The number of

RANK-positive cells and CSF-1R-positive cells in wild-type

Fig. 1. Distribution of RANK-positive cells and CSF-1R-positive cells in bone in wild-type, RANKL2/2 and Fos2/2 (c-Fos2/2) mice. (A,B) Sections of

tibiae were prepared from 6-week-old wild-type, RANKL2/2 and Fos2/2 mice. (A) Sections were stained for TRAP. TRAP-positive cells appear pink.

(B) Sections were stained for RANK (green, upper panels) and CSF-1R (green, lower panels). Nuclei were detected by DAPI staining (blue). Numbers of RANK-

positive cells and CSF-1R-positive cells in 0.135 mm2 of the central area just under the growth plate (rectangles) were counted in three images prepared from three

RANKL2/2 mice and Fos2/2 mice (right panel). Results are expressed as the means 6 s.d. for three images; aP,0.01. Representative images are shown in the left

panel. (C) Total RNA was extracted from tibiae of RANKL2/2 mice and Fos2/2 mice. Expression levels of RANK and CSF-1R mRNAs were estimated by

quantitative real-time RT-PCR. Results are expressed relative to the levels in RANKL2/2 mice. Results are expressed as the means 6 s.d. for three mice;aP,0.01. (D) Bone lysates were prepared from tibiae of RANKL2/2 mice and Fos2/2 mice, and subjected to western blot analysis using anti-RANK antibody.

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mice are not shown in Fig. 1B, right panel, because both

multinucleated osteoclasts and osteoclast precursors express

RANK and CSF-1R. Most RANK-positive cells express CSF-

1R in both wild-type and RANKL2/2 mice (Mizoguchi et al.,

2009). Real-time PCR and western blot analyses confirmed that

the mRNA and protein levels of RANK were much lower in bone

tissues obtained from Fos2/2 mice than those from RANKL2/2

mice (Fig. 1C,D).

Osteoclast precursors have been shown to exist in spleen in

wild-type mice (Takahashi et al., 1988). We therefore examined

the distribution of RANK-positive cells in comparison with that of

CSF-1R-positive cells in spleen of wild-type, RANKL2/2 and

Fos2/2 mice (Fig. 2A). CSF-1R-positive cells were observed in

spleen in all the genotypes of mice. By contrast, RANK-positive

cells were detected in spleen in wild-type and RANKL2/2 mice

but not in Fos2/2 mice (Fig. 2A). However, the level of RANK

expression in spleen was much lower than that in bone in wild-type

and RANKL2/2 mice, because in order to obtain the similar

fluorescence intensity of the positive cells, the camera exposure

period for spleen sections needed to be much longer (more than ten

times) than that for bone sections. Nevertheless, RANK-positive

cells were not detected in spleen, as well as bone, in Fos2/2 mice.

Medullary thymic epithelial cells and intestinal antigen-

sampling M cells have been reported to express RANK in

order to accomplish RANKL-ordered functions (Akiyama et al.,

2008; Hikosaka et al., 2008; Knoop et al., 2009). A similar

distribution of RANK-positive cells was observed in medullary

thymic epithelium and in intestinal epithelium situated over

Peyer’s patches in wild-type, RANKL2/2 and Fos2/2 mice

(Fig. 2B). These results suggest that Fos-induced upregulation of

RANK expression is cell-type specific.

To confirm that cell autonomous expression of Fos in

osteoclast precursors is necessary for the upregulation of

RANK expression on bone surfaces, we used a bone marrow

transplantation model. When wild-type bone marrow cells were

inoculated into Fos2/2 mice that had been myelosuppressed with

busulfan, RANK-positive cells appeared along the bone surface

(Fig. 3A). Immunostainable RANK-positive cells were not

observed in soft tissues around the bone (data not shown).

These results suggest that Fos is required for the upregulation of

RANK expression, and bone-derived factors are involved in the

process.

We then examined whether RANK expression in precursors is

upregulated in cocultures with osteoblasts. When wild-type and

Fos2/2 spleen cells were cocultured for 1 or 5 days with wild-type

osteoblasts, the expression of RANK was increased in wild-type

spleen cells but not in Fos2/2 spleen cells (Fig. 3B,C). The

expression of CSF-1R in Fos2/2 spleen cells was increased on day

5. RANK expression in wild-type spleen cells was also increased,

even by coculturing with RANKL2/2 osteoblasts (Fig. 3B,C). Most

RANK-positive cells were positive for CSF-1R (Fig. 3B). CSF-1

is reported to enhance RANK expression in osteoclast precursors

(Arai et al., 1999). AFS98, an anti-CSF-1R monoclonal antibody,

has been shown to inhibit the interaction between CSF-1 and CSF-

1R (Sudo et al., 1995). Therefore, we added AFS98 to cocultures

of wild-type osteoblasts and wild-type spleen cells. AFS98

inhibited the appearance of both CSF-1R-positive cells and

RANK-positive cells (Fig. 3B,C). These results suggest that

RANK is expressed by CSF-1-expressing cells. We also

observed that RANK-positive cells were scarce in bone in CSF-

1-deficient op/op mice (Y.N., unpublished data). Furthermore,

many CSF-1R-positive cells in bone tissues were observed to be in

direct contact with CSF-1-expressing osteoblastic cells in Fos2/2

mice (Fig. 3D). These results suggest that CSF-1 but not RANKL

expressed by osteoblasts plays a role in the upregulation of RANK

expression in osteoclast precursors.

We next examined the effect of CSF-1 stimulation on RANK

expression in wild-type and Fos2/2 macrophages. It was reported

that the binding of CSF-1 to CSF-1R induced the downregulation

of CSF-1R mRNA expression within 6 hours in human

monocytes (Sariban et al., 1989). In accordance with the

previous finding, CSF-1 treatment suppressed CSF-1R

Fig. 2. Distribution of RANK-positive cells in spleen, thymus and intestinal Peyer’s patches in wild-type, RANKL2/2 and Fos2/2 mice. (A) Sections of

spleen were prepared from 6-week-old wild-type, RANKL2/2 and Fos2/2 mice, and stained for RANK (green, upper panels) and CSF-1R (green, lower panels).

Nuclei were detected by DAPI staining (blue). Numbers of RANK-positive cells and CSF-1R-positive cells in 0.135 mm2 of the red pulp region (rectangles) were

counted in three images of each tissue type (right panel). Results are expressed as the means 6 s.d. for three images; aP,0.01. Representative images are shown in

the left panel. (B) Sections of thymus and intestinal Peyer’s patches were prepared from 6-week-old wild-type, RANKL2/2 and Fos2/2 mice. Sections were

stained for RANK (green). Nuclei were detected by DAPI staining (blue). Peyer’s patches are indicated by dashed circles in lower panels.

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expression in both wild-type and Fos2/2 macrophages (Fig. 4A,

upper panel), suggesting that CSF-1R-mediated signaling is

normally transduced in Fos2/2 macrophages. CSF-1 also

stimulated Fos mRNA expression in wild-type macrophages

(Fig. 4A, middle panel). In culture, the expression of RANK

mRNA in Fos2/2 macrophages is comparable with that of wild-

type macrophages. The expression of RANK mRNA was

upregulated by CSF-1 in wild-type macrophages but not in

Fos2/2 macrophages (Fig. 4A, lower panel). Western blot

analysis confirmed that CSF-1 stimulated Fos protein

expression in wild-type macrophages (Fig. 4B). Wild-type and

Fos2/2 macrophages similarly expressed RANK protein at basal

levels, and the CSF-1-induced upregulation of RANK protein

expression was observed only in wild-type macrophages. Spleen,

as well as bone, was shown to strongly express CSF-1 (Wiktor-

Jedrzejczak et al., 1990). These results suggest that

immunohistochemically stainable levels of RANK protein are

expressed in osteoclast precursors in bone and spleen in wild-type

mice but not Fos2/2 mice.

Then we examined the effect of transfection of Fos on RANK

expression in Fos2/2 macrophages. Overexpression of Fos in

Fos2/2 macrophages increased the level of RANK (Fig. 4C).

These results suggest that upregulation of Fos expression in

macrophages is crucial for the expression of RANK.

Furthermore, these results raised the possibility that if RANK

is highly expressed in Fos2/2 osteoclast precursors, these

precursors may differentiate into osteoclasts even in the

absence of Fos.

Next, spleen-derived wild-type and Fos2/2 macrophages were

prepared and infected with a retrovirus carrying cDNA for

RANK (pMX-RANK; Fig. 5). Phosphorylation of ERK in

Fos2/2 macrophages was increased by the transfection with

RANK cDNA even in the absence of RANKL, and was further

enhanced in response to RANKL (Fig. 5A). Wild-type

macrophages transfected with RANK cDNA spontaneously

differentiated into TRAP-positive cells even in the absence of

RANKL (Fig. 5B). The treatment with RANKL enhanced

TRAP-positive cell formation in both RANK-transfected and

empty vector-transfected macrophages, although the number of

TRAP-positive multinucleated cells induced by RANKL was

substantially higher in RANK-transfected cultures (Fig. 5B). By

contrast, overexpression of RANK in Fos2/2 macrophages failed

Fig. 3. A bone environment is required for the upregulation of RANK expression in osteoclast precursors. (A) Wild-type mouse bone marrow cells were

injected into the left cardiac ventricle of Fos2/2 mice myelosuppressed with busulfan. After 18 days, sections of tibiae were prepared and stained for RANK

(green). Nuclei were detected by DAPI staining (blue). A high power view of the boxed region is shown in the right panel. (B) Primary osteoblasts were prepared

from calvariae of wild-type and RANKL2/2 mice. Osteoblasts were cocultured for 1 day (upper panels) or 5 days (lower panels) with wild-type or Fos2/2 spleen

cells. Anti-CSF-1R antibody (AFS98) was also added to some cocultures of wild-type osteoblasts and spleen cells. Cells were fixed and double-stained for

RANK (green) and CSF-1R (red). Nuclei were detected by DAPI staining (blue). Arrows indicate cells double positive for CSF-1R and RANK (yellow).

Representative images of three independent experiments are shown. (C) Numbers of RANK-positive cells (green, yellow) and CSF-1R-positive cells (red, yellow),

shown in B, were counted. Results are expressed as the means 6 s.d. of three cultures; aP,0.01. (D) Tibiae were recovered from 6-week-old Fos2/2 mice.

Sections of tibiae were prepared and subjected to double staining of CSF-1 (green) and CSF-1R (red). Nuclei were detected by DAPI staining (blue). Arrows

indicate CSF-1R-positive cells that are in contact with CSF-1-expressing osteoblastic cells.

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to induce their differentiation into TRAP-positive cells in the

presence or absence of RANKL. Expression of NFATc1 was

induced in response to RANKL in wild-type macrophages but not

Fos2/2 macrophages transfected with and without RANK cDNA

(Fig. 5C). These results suggest that Fos plays essential roles, not

only in the upregulation of RANK expression in osteoclast

precursors, but also in the differentiation of RANK-positive

precursors into osteoclasts (Fig. 5D).

DiscussionFos has been thought to act on osteoclastic differentiation only

down-stream of the RANKL–RANK signaling pathway

(Grigoriadis et al., 1994; Matsuo et al., 2004). In the present

study, we showed that the upregulation of RANK expression

during osteoclastogenesis is cell-type specific and requires Fos.

Although RANK-positive cells were observed in both spleen and

bone, the expression level was much lower in spleen than in

bone. The upregulation of RANK expression in osteoclast

precursors occurred in the bone tissues, suggesting the boneenvironment to be essential: that is, the Fos-dependent

upregulation of RANK expression made it possible forosteoclast precursors to use RANKL for their osteoclasticdifferentiation. RANK overexpression in Fos2/2 macrophagesfailed to induce osteoclastic differentiation. Importantly, this

finding provides suggests that Fos is involved in two steps duringosteoclastogenesis: induction of RANK expression in osteoclastprecursors and transduction of osteoclast-inducing signals from

RANK and CSF-1R.

Immunohistochemical studies have shown that RANK isnormally expressed by medullary thymic epithelial cells and

intestinal antigen-sampling cells in Fos2/2 mice. The basalexpression of RANK in osteoclast precursors was not influencedby the presence or absence of Fos. One abnormality observedin Fos2/2 mice is the absence of any increase in RANK in

osteoclast precursors. CSF-1R-positive cells were observed inspleen of Fos2/2 mice but they failed to express RANK. Theseresults suggest that RANK expression in osteoclast precursors

is upregulated in a Fos-dependent manner. The upregulationappears to be related to physiological osteoclastogenesis, andrequires an increase in Fos expression. Nevertheless,

overexpression of RANK in Fos2/2 macrophages did not leadto the development of osteoclasts. These results suggest that inosteoclastogenesis, Fos is required first for bone- and spleen-

induced RANK production in osteoclast precursors, and secondfor the RANKL-induced upregulation of NFATc1 expression inosteoclast precursors (Matsuo et al., 2004).

Cells that strongly express RANK were detected in bone

tissues in RANKL2/2 mice and wild-type mice but not in Fos2/2

mice. These results suggest that bone-derived factors areinvolved in the upregulation of RANK expression in osteoclast

precursors. Arai et al. previously showed, for the first time, thatCSF-1 acted on osteoclast precursors and induced RANKexpression (Arai et al., 1999). We also confirmed their finding

and further showed that the CSF-1-induced upregulation ofRANK expression is dependent on Fos. Cells positive for CSF-1R were always detected near or in contact with CSF-1-expressing osteoblasts or bone marrow stromal cells in bone.

These results suggest that CSF-1 is one of the bone-derivedfactors that induce the upregulation of RANK expression inosteoclast precursors.

IL-34 is a newly discovered cytokine that binds to CSF-1R andexerts its action in the same way as CSF-1 (Lin et al., 2008). Onexploring the mechanism of osteoclastogenesis in op/op mice, we

found that IL-34 is involved in the development of osteoclastprecursors in spleen (Y.N., unpublished data). IL-34 is highlyexpressed in spleen but not in bone. Cells double positive forCSF-1R and RANK were detected in spleen in op/op mice as

well as wild-type mice. However, the expression level of RANKin osteoclast precursors in spleen was much lower than that inbone in op/op mice and wild-type mice. These results suggest that

additional factors other than CSF-1 expressed by osteoblasts arenecessary for the increase in RANK in osteoclast precursors inbone tissues. Recently we have observed that noncanonical Wnt

signaling in osteoclast precursors induces RANK expression(Y.K., unpublished data). Wnt5a produced by osteoblastsenhanced RANK expression in osteoclast precursors through

the receptor Ror2. These results suggest that the upregulation ofRANK expression in osteoclast precursors is induced by severalfactors expressed by osteoblasts.

Fig. 4. CSF-1 upregulates RANK expression in osteoclast precursors.

(A) Spleen cells obtained from wild-type and Fos2/2 mice were cultured for 2

days in the presence of CSF-1 (104 units/ml) to prepare macrophages. Splenic

macrophages were further cultured in the absence of CSF-1 for 16 hours.

Then, cells were treated for 0 and 8 hours with CSF-1 (104 units/ml), and total

cellular RNA was prepared. Levels of CSF-1R, Fos and RANK mRNAs were

estimated by quantitative real-time RT-PCR. Results are expressed relative to

levels in the wild-type macrophages at 0 hours (control). Results are

expressed as the means 6 s.d. for three cultures; aP,0.01; n.d., not

detectable. (B) Wild-type and Fos2/2 spleen macrophages were cultured for

0, 8 and 24 hours in the presence of CSF-1 (104 units/ml). Cell lysates were

then prepared and subjected to western blot analysis using anti-Fos antibody

and anti-RANK antibody. (C) Spleen macrophages were prepared from

Fos2/2 mice and infected with empty pMX retrovirus (pMX-empty) or pMX

retrovirus expressing Fos (pMX-Fos). Infected macrophages were cultured

with CSF-1 (104 units/ml) for 48 hours, and cell lysates were prepared and

subjected to Western blot analysis using anti-Fos and anti-RANK antibodies.

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Kobayashi et al. were the first to report that TNF-a stimulatedosteoclastic differentiation independent of RANKL–RANK

signaling (Kobayashi et al., 2000). By contrast, Li et al.

showed that although the injection of TNF-a to the calvariae ofRANKL2/2 mice could generate osteoclasts, the number of

osteoclasts produced was very low (Li et al., 2000). These results

raise the question of why RANKL cannot be replaced with TNF-

a during osteoclastogenesis in vivo. Treatment of macrophageswith CSF-1 upregulated the expression of RANK (Fig. 4A) but

not TNF receptor type 1 and TNF receptor type 2 (data not

shown). The overexpression of RANK in macrophages induced

osteoclastic differentiation even in the absence of the addition of

RANKL. The upregulation of RANK expression in osteoclast

precursors must lower the threshold for the RANKL-induced

osteoclastogenesis. These results suggest that, under

physiological conditions in vivo, the upregulation of RANK

expression in osteoclast precursors is an important requirement

for RANKL-induced but not TNF-a-induced osteoclastogenesis.We previously reported that osteoclasts were generated in bone

in response to an injection of RANKL in RANKL2/2 mice, as

they were in wild-type mice (Yamamoto et al., 2006). Osteoclasts

were not observed in the soft tissues around the bone in RANKL-

injected RANKL2/2 mice, indicating that the expression of

RANKL is not involved in determining the correct site for

osteoclastogenesis. The present study showed that osteoblasts

play a role in the upregulation of RANK expression in osteoclast

precursors. These results suggest that the distribution of

osteoclast precursors expressing high levels of RANK

determines the site for osteoclastogenesis. Thus, we have

uncovered the physiological importance of the increase in

RANK expression in osteoclast precursors and the role of Fos

in this process. Further experiments should reveal the molecularmechanism by which Fos regulates RANK expression inosteoclast precursors.

Materials and MethodsAnimals

C57BL/6 mice were obtained from Japan SLC (Tokyo, Japan). RANKL2/2 mice(C57BL/6) were generated in the laboratory of J.P. (Kong et al., 1999). Fos2/2

mice (C57BL/6) were obtained from Jackson Laboratory (Bar Harbor, ME). Allexperiments were conducted in accordance with the guidelines for studies withlaboratory animals of the Matsumoto Dental University Experimental AnimalCommittee.

Antibodies

The antibodies used for the immunohistochemical analysis were biotin-conjugatedanti-RANK and anti-CSF-1R and anti-CSF-1R from R&D Systems (Minneapolis,MN) and anti-CSF-1 (EP1179Y) from Abcam (Cambridge, UK). CSF-1Rneutralizing antibody (AFS98) was obtained from eBioscience (San Diego, CA).The antibodies used for the western blot analysis were anti-RANK, anti-Fos, anti-extracellular signal-regulated kinase (ERK), anti-phosphorylated ERK and anti-NFTAc1 antibodies from Cell Signaling Technology (Danvers, MA), and anti-b-actin antibody (AC-74) from Sigma-Aldrich (St. Louis, MO).

Real-time PCR

Cultured cells and whole bone tissue homogenized with Tissue Lyser II (Qiagen,Hilden, Germany) were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA), andtotal RNA was extracted from the lysate using a PureLink RNA Mini Kit (Ambion,Austin, TX). cDNA was synthesized from the total RNA using reversetranscriptase (ReverTra Ace; Toyobo, Osaka, Japan), and subjected to a two-step real-time PCR in Applied Biosystems StepOnePlusTM (Applied Biosystems,Foster City, CA). Results were normalized with respect to the amount ofglyceraldehyde-3-phosphate dehydrogenase (G3PDH) in the same sample. Thefold-change ratios between test and control samples were calculated. All primerswere purchased from TAKARA BIO (Shiga, Japan). The primer IDs were asfollows: RANK, MA075536; CSF-1R, MA072804; Fos, MA05419; G3PDH,MA05371.

Fig. 5. Overexpression of RANK in Fos2/2 macrophages failed to induce their osteoclastic differentiation. (A) Fos2/2 spleen macrophages were infected

with empty pMX retrovirus (pMX-empty) or pMX retrovirus expressing RANK (pMX-RANK), and incubated with RANKL for 0 and 15 minutes. Then, cell

lysates were prepared and subjected to western blot analysis using anti-RANK, anti-ERK and anti-phosphorylated ERK antibodies. (B) Wild-type and

Fos2/2 spleen macrophages were infected with pMX-empty or pMX-RANK. Infected macrophages were cultured for 3 days with CSF-1 (104 units/ml) in the

presence or absence of RANKL (5 nM). Cells were then fixed and stained for TRAP. (C) Wild-type and Fos2/2 spleen macrophages were infected with pMX-

empty and pMX-RANK, respectively. Infected macrophages were cultured for 3 days with CSF-1 (104 units/ml) in the presence or absence of RANKL (5 nM).

Then cell lysates were prepared and subjected to western blot analysis using anti-NFATc1 and b-actin antibodies. (D) A schematic model of osteoclastogenesis

along the bone surface. The expression of RANK in osteoclast precursors is upregulated by factors in the bone environment such as CSF-1 produced by osteoblasts

in a Fos-dependent manner. Osteoclast precursors that express high levels of RANK differentiate into osteoclasts in response to RANKL and CSF-1. This

differentiation process also requires Fos as an essential transcription factor.

Regulation of RANK expression by Fos 2915

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Histochemical analysis

Paraffin-embedded 4-mm thick sections were stained for TRAP, and counterstainedwith Hematoxylin. For immunofluorescent staining, tissues were frozen in hexaneusing a cooling apparatus (PSL-1800; Tokyo Rikakikai, Tokyo, Japan) andembedded in a 5% carboxymethyl cellulose (CMC) gel. Sections, 5-mm thick, wereprepared using Kawamoto’s film method (Cryofilm transfer kit; Finetec, Tokyo,Japan) (Kawamoto and Shimizu, 2000). The sections and cultured cells were fixedin ice-cold 5% acetic acid in ethanol, and subjected to staining for RANK, CSF-1Rand CSF-1 using specific antibodies. Biotin-conjugated antibodies were used forRANK and CSF-1R staining (R&D Systems). Anti-CSF-1R antibody was labeledwith FITC using a commercially available kit (Dojindo, Kumamoto, Japan)for double staining for RANK and CSF-1R. Horseradish peroxidase (HRP)-conjugated streptavidin (PerkinElmer, Boston, MA), HRP-conjugated anti-FITC(PerkinElmer), NorthernLights (NL) 557-conjugated anti-sheep IgG (R&DSystems) and Histofine Simple Stain Mouse MAX-PO (Rabbit; NichireiBiosciences, Tokyo, Japan) were used as the secondary antibodies. Nuclei weredetected by 4,49-diamidino-2-phenylindole (DAPI) staining (Vector Laboratories,Burlingame, CA). RANK- or CSF-1R-positive cells in bone were counted in threeimages of 0.135 mm2 (3186425 mm) of the central area just under the growth platewith AxioVision 4.8.1 Mosaix software (Carl Zeiss, Jena, Germany). RANK- orCSF-1R-positive cells in spleen were also counted in randomly selected areas(0.135 mm2) (3186425 mm) of the red pulp region. Three images of bone andspleen were prepared from three different mice of each genotype using amicroscope (Axiovert 200; Carl Zeis) with a digital camera (AxioCamHRc; CarlZeiss). Images were captured with AxioVision 3.1 (Carl Zeiss). The constructionof figures using the images was performed with Photoshop software (Adobe, SanJose, CA).

Western blot analysis

Whole bone tissue homogenized with Tissue Lyser II (Qiagen) was lysed withTRIzol reagent (Invitrogen), and protein lysates were prepared according to theQiagen protocol. In brief, the phenol supernatant containing the protein fraction wasincubated with isopropanol and subsequently incubated with 0.3 M guanidine inethanol. Protein was dissolved using 10 M urea and 50 mM dithiothreitol (DTT).Cultured cells were lysed in 0.1% NP-40 lysis buffer [20 mM Tris (pH 7.5), 50 mMb-glycerophosphate, 150 mM NaCl, 1 mM EDTA, 25 mM NaF, 1 mM Na3VO4, 16protease inhibitor cocktail (Sigma-Aldrich), 16 phosphatase inhibitor cocktail I(Sigma-Aldrich) and phosphatase inhibitor cocktail II (Sigma-Aldrich)]. Lysateswere electrophoresed on a SDS-PAGE gel, transferred onto a PVDF membrane(Clear blot P membrane, Atto, Tokyo, Japan), blotted with antibodies to specificproteins, and visualized using ECL (Amersham, Piscataway, NJ).

Cultures of spleen macrophages

RANKL2/2 spleen cells were used as control (wild-type) cells against Fos2/2

spleen cells for the following reasons. (1) The total number of hematopoieticprogenitor cells in spleen is lower in wild-type mice than Fos2/2 and RANKL2/2

mice, because osteopetrotic mice exhibit splenic extramedullary hematopoiesisbecause of the lack of a bone marrow environment (Kong et al., 1999; Okada et al.,1994). (2) RANKL is not expressed by macrophages (Kong et al., 1999; Suda et al.,1999; Yasuda et al., 1998). Spleen cells were prepared from 6-week-old maleFos2/2 and RANKL2/2 mice, and layered onto a lympholyte-M (CedarlaneLaboratories, Burlington, ON, Canada) gradient. After centrifugation,mononuclear cells were collected and cultured in a-MEM (Sigma-Aldrich)containing 10% FBS (JRH Bio-sciences, Lenexa, KS) in the presence of 104 units/ml CSF-1 (Kyowa Hakko Kirin, Tokyo, Japan). After 16 hours, nonadherentspleen cells were harvested. Spleen cells (16106) were incubated with 104 units/mlCSF-1 for 2 days in 6-well plates, and used as spleen macrophages. Old mediumwas replaced with fresh medium without FBS and CSF-1. After culturing for16 hours, spleen macrophages were further cultured with 104 units/ml CSF-1 forgiven periods.

Coculture of primary osteoblasts with spleen cells

To isolate primary osteoblasts from either wild-type or RANKL2/2 mice, calvariaefrom 2-day-old mice (male and female) were separately subjected to samplepreparation. Briefly, each calvaria was cut into small pieces and cultured for 5 daysin type I collagen gel (cell matrix type-IA; Nitta Gelatin, Osaka, Japan) prepared ina-MEM containing 10% FBS. Osteoblasts grown from the calvariae were collectedby treating the collagen gel cultures with collagenase (Wako, Osaka, Japan) and16105 were cocultured with 56104 nonadherent spleen cells in a-MEM containing10% FBS in 96-well plates for specific periods. Cells were cultured for 1 and 5days without osteoclastogenesis-stimulating factors. In some experiments, an anti-CSF-1R monoclonal antibody (AFS98; 10 mg/ml) was added to the culturemedium.

Overexpression experiments in spleen macrophages

Spleen macrophages (26105 cells) were infected with empty pMX retrovirus, Fos-expressing retrovirus or RANK-expressing retrovirus and cultured with 104 units/ml

CSF-1. After 1 day, infected cells were further cultured in the presence of 104 units/ml CSF-1 with or without 5 nM RANKL (GST-RANKL) for given periods. GST-RANKL was generated in the laboratory of H.Y. (Oriental Yeast, Shiga, Japan).

Bone marrow cell transfer

Bone marrow cells were prepared from 6-week-old male wild-type mice,and layered onto a lympholyte-M (Cedarlane Laboratories) gradient. Aftercentrifugation, mononuclear cells (56106) were collected, and injected into theleft cardiac ventricle of 8-week-old male Fos2/2 mice. The recipient mice weretreated with busulfan (25 mg/kg/day) for 2 days before the transplantation of wild-type bone marrow mononuclear cells (Ashizuka et al., 2006). After 18 days, micewere killed, and tibiae were removed and subjected to RANK staining.

Statistical analysis

Stat View 5.0 software (SAS Institute, Inc., Cary, NC) was used for all statisticalanalyses. Data were evaluated by a one-way analysis of variance (ANOVA)followed by Fisher’s PLSD test. Experiments were performed three times andsimilar results were obtained. The results were expressed as means 6 s.d. for threecultures or three photographs. P,0.01 was considered statistically significant. Onerepresentative result of each experiment was shown in the manuscript unlessotherwise noted.

FundingThis work was supported by Grants-in-Aid for Scientific Researchfrom the Ministry of Education, Culture, Sports, Science andTechnology of Japan [grant numbers 22791804 to T.M., 20200019to T.M., 23792455 to A.A. and 22390351 to N.T.]; and by a grantfrom the Naito Foundation for Natural Science [grant number 2009-972 to T.M.].

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