Spleen serves as a reservoir of osteoclast precursors
through vitamin D-induced IL-34 expression in
osteopetrotic op/op mice
Yuko Nakamichia, Toshihide Mizoguchia, Atsushi Araia, Yasuhiro Kobayashia, Masahiro Satob, Josef M. Penningerc,
Hisataka Yasudad, Shigeaki Katoe, Hector F. DeLucaf,1, Tatsuo Sudag, Nobuyuki Udagawah, and Naoyuki Takahashia,1
aDivision of Hard Tissue Research, Institute for Oral Science, and Departments ofbConservative Dentistry andhBiochemistry, School of Dentistry, Matsumoto Dental
University, Nagano 399-0781, Japan;cInstitute of Molecular Biotechnology, Austrian Academy of Sciences, A-1030, Vienna, Austria;dOriental Yeast Co., Tokyo
174-8505, Japan;eLaboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan;fDepartment of
Biochemistry, University of Wisconsin, Madison, WI 53706; andgResearch Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241, Japan
Contributed by Hector F. DeLuca, May 7, 2012 (sent for review December 9, 2011)
Osteoclasts are generated from monocyte/macrophage-lineage
precursors in response to colony-stimulating factor 1 (CSF-1) and
receptor activator of nuclear factor-κB ligand (RANKL). CSF-1–mu-
tated CSF-1op/opmice as well as RANKL−/−mice exhibit osteopet-
rosis (OP) caused by osteoclast deficiency. We previously identified
RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive
cells as osteoclast precursors (OCPs), which existed in bone in
RANKL−/−mice. Here we show that OCPs do not exist in bone
but in spleen in CSF-1op/opmice, and spleen acts as their reservoir.
IL-34, a newly discovered CSF-1R ligand, was highly expressed in
vascular endothelial cells in spleen in CSF-1op/opmice. Vascular en-
dothelial cells in bone also expressed IL-34, but its expression level
was much lower than in spleen, suggesting a role of IL-34 in the
splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1–in-
duced osteoclastogenesis in CSF-1op/opmice. Osteoclasts appeared
in aged CSF-1op/opmice with up-regulation of IL-34 expression in
spleen and bone. Splenectomy blocked the age-associated appear-
ance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-
1α,25(OH)2D3(2MD), a potent analog of 1α,25-dihidroxyvitamin
D3, into CSF-1op/opmice induced both hypercalcemia and osteoclas-
togenesis. Administration of 2MD enhanced IL-34 expression not
only in spleen but also in bone through a vitamin D receptor-medi-
ated mechanism. Either splenectomy or siRNA-mediated knock-
down of IL-34 suppressed 2MD-induced osteoclastogenesis. These
results suggest that IL-34 plays a pivotal role in maintaining the
to diverse stimuli, in CSF-1op/opmice. The present study also sug-
gests that the IL-34 gene in vascular endothelial cells is a unique
target of vitamin D.
platelet endothelial cell adhesion molecule 1-positive cells|osteoblasts|
cyte/macrophage-lineage precursors. The differentiation of
osteoclast precursors (OCPs) into osteoclasts is regulated by
bone-forming osteoblasts. Osteoblastic cells express two cyto-
kines responsible for osteoclastogenesis: one is colony-stimulat-
ing factor 1 [CSF-1, also called macrophage colony-stimulating
factor (M-CSF)] and the other is receptor activator of nuclear
factor-κB ligand (RANKL). OCPs express CSF-1 receptor (CSF-
1R, also called c-Fms) and RANK (receptor for RANKL) and
differentiate into osteoclasts in response to CSF-1 and RANKL.
The expression of RANKL is up-regulated by osteoclast-in-
ducing factors such as parathyroid hormone (PTH) and 1α,25-
dihydroxyvitamin D3[1α,25(OH)2D3] (1, 2).
CSF-1 is the most potent growth factor for monocytes/macro-
phages (3), but its synthesis by osteoblasts occurs independently
of PTH and 1α,25(OH)2D3(2). CSF-1op/opmice cannot produce
a functionally active CSF-1 (4), and therefore, exhibit mono-
cytopenia and osteopetrosis (OP) (5, 6). However, several curious
phenomena have been observed in CSF-1op/opmice. First,
steoclasts are bone-resorbing cells generated from mono-
osteoclasts are totally absent in young CSF-1op/opmice, but appear
in aged CSF-1op/opmice (7). Second, osteopetrotic characteristics
of CSF-1R−/−mice are more severe than those of CSF-1op/opmice
(8). Third, F4/80+[F4/80(+)] macrophages exist in the splenic
red pulp in CSF-1op/opmice as well as in WT mice, and their
number is regulated by a mechanism independently of CSF-1 (9,
10). Fourth, the administration of vascular endothelial growth
factor (VEGF) rescues osteopetrosis in CSF-1op/opmice (11, 12),
but VEGF cannot substitute for CSF-1 to induce osteoclast for-
mation in vitro (13).
Recently, Lin et al. (14) discovered IL-34, as a new ligand for
CSF-1R. The amino acid sequence of IL-34 was quite different
from that of CSF-1, but IL-34 promoted macrophage colony
formation like CSF-1 did. IL-34 was specifically expressed in
splenic tissues, predominantly in the red pulp region. When IL-
34 was expressed under the control of the CSF-1 promotor in
CSF-1op/opmice, the osteopetrotic phenotype was rescued (15).
IL-34 in combination with RANKL induced osteoclastic differ-
entiation of progenitor cells in mouse (16, 17) and human (17)
cell culture systems. However, it remains unclear why IL-34
cannot substitute for CSF-1 in CSF-1op/opmice in vivo.
Using RANKL−/−mice and CSF-1op/opmice, we identified cell-
cycle–arrested RANK/CSF-1R double-positive [RANK(+)/CSF-
1R(+)] cells as the direct OCPs in vivo (18). When RANKL was
administered to RANKL−/−mice and CSF-1 to CSF-1op/opmice,
OCPs similarly differentiated into osteoclasts in bone tissue
without cell cycle progression. OCPs were detected in the vicinity
of osteoblastic cells in RANKL−/−mice, suggesting the existence
of OCPs in bone in WT mice. However, our preliminary
experiments showed that OCPs were not present in bone in CSF-
The active form of vitamin D3[1α,25(OH)2D3] regulates cal-
cium homeostasis by acting on various types of cells such as in-
testinal endothelial cells, renal tubular cells, and osteoblastic
cells (19). Shevde et al. (20) reported that 2-methylene-19-nor-
(20S)-1α,25(OH)2D3(2MD), a highly potent analog of 1α,25
(OH)2D3, strongly enhanced osteoblastic cell-mediated osteo-
clast formation and also induced bone formation in vitro and
in vivo. We have synthesized a derivative of 2MD at carbon 2
(2α-methyl-19-nor-(20S)-1α,2β,25(OH)3D3, 2-methyl-2MD), and
showed that osteoclastic bone resorption is indispensable for the
hypercalcemic action of the 2MD analog in vivo (21). Recently,
increasing evidence has been accumulating in showing that 1α,25
(OH)2D3directly regulates activities of vascular endothelial cells
Author contributions: Y.N. and N.T. designed research; Y.N., T.M., A.A., and M.S. per-
formed research; J.M.P., H.Y., and S.K. contributed new reagents/analytic tools; Y.N.,
Y.K., and N.U. analyzed data; and Y.N., H.F.D., T.S., and N.T. wrote the paper.
Conflict of interest statement: H.Y. is an employee of the Oriental Yeast Co.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| June 19, 2012
| vol. 109
| no. 25www.pnas.org/cgi/doi/10.1073/pnas.1207361109
through vitamin D receptors (22–24). These results suggest that
1α,25(OH)2D3plays a unique role in the vascular system beyond
the classical role in calcium homeostasis.
In the present study, we examined how osteoclasts were
formed in CSF-1op/opmice in response to various stimuli. We
found that OCPs existed in spleen but not in bone in CSF-1op/op
mice. OCPs in CSF-1op/opmice were transferred from spleen to
bone and differentiated into osteoclasts in response to CSF-1,
VEGF, and 2MD administrations, and also to aging. IL-34
appeared to play a pivotal role in the generation and storage of
OCPs in spleen and osteoclastogenesis in CSF-1op/opmice. In
addition, we have shown that the IL-34 gene in the vascular
endothelial cells is a unique target of vitamin D.
Immunohistochemical analysis showed that RANK(+) cells and
CSF-1R(+) cells were present in the proximal region of tibiae
obtained from RANKL−/−mice as well as from WT mice (Fig.
1A). Most of the RANK(+) cells expressed CSF-1R in WT mice.
Cells double positive for RANK and CSF-1R [RANK(+)/CSF-
1R(+) cells] were also detected in RANKL−/−mice and identi-
fied as the direct OCPs in vivo (18). Conversely, neither RANK
(+) cells nor CSF-1R(+) cells were detected in bone tissue in
CSF-1op/opmice (Fig. 1A, Right). In contrast, RANK(+) cells,
CSF-1R(+) cells, and RANK(+)/CSF-1R(+) cells (arrows)
were detected in spleen in CSF-1op/opmice as well as in WT mice
and RANKL−/−mice (Fig. 1B, Right). RANK(+)/CSF-1R(+)
cells were mainly observed in the red pulp region and marginal
zones. These results suggest that OCPs do not exist in bone but
do exist in spleen in CSF-1op/opmice.
We then examined the tissue distribution of IL-34 mRNA
(Fig. 2A). Consistent with the previous report (14), IL-34 was
expressed predominantly in spleen but slightly in bone (Fig. 2A).
The osteoblast level of IL-34 mRNA was much lower compared
with that of CSF-1 mRNA (Fig. 2B). Little IL-34 mRNA or CSF-
1 mRNA was expressed in bone marrow (BM) macrophages and
osteoclastic cells (Fig. 2B). IL-34(+) cells were mainly distrib-
uted in the splenic red pulp region and marginal zones (Fig. 2C),
where RANK(+)/CSF-1R(+) cells were detected (Fig. 1B). IL-
34(+) cells were similarly distributed in the spleen in WT mice
(Fig. S1 A and B). Most IL-34(+) cells expressed platelet en-
dothelial cell adhesion molecule 1 (PECAM-1), a marker of
vascular endothelial cells (Fig. 2 C–E and Fig. S1 A and B). Cells
double positive for IL-34 and PECAM-1 [IL-34(+)/PECAM-1
(+) cells] were detected as endothelial cells in blood vessels.
Most of the endothelial cells found in white pulp also expressed
IL-34 (Fig. 2 C–E). Notably, endothelial cells in central arterioles
robustly expressed IL-34 (Fig. 2D). Next, we examined the dis-
tribution of IL-34 expression in bone. PECAM-1(+) cells
expressed IL-34 but alkaline phosphatase (ALP)(+) osteoblastic
cells did not (Fig. 2F and Fig. S1C). The endothelial cell
1op/opmice. (A) Localization of RANK (red) and CSF-1R (green) in proximal
tibiae in WT, RANKL−/−, and CSF-1op/opmice. Nuclei were stained with DAPI
(blue). Lower panels show magnified views of the boxed areas in the Upper
panels. Dashed lines represent bone surface. Arrows indicate RANK(+)/CSF-
1R(+) cells. [Scale bar, 400 μm (Upper), 5 μm (Lower).] (B) Localization of
RANK (red) and CSF-1R (green) in spleen in WT, RANKL−/−, and CSF-1op/op
mice. Dashed circles represent the white pulp. Lower panels show magnified
views of the boxed areas in the Upper panels. WP; white pulp, RP; red pulp.
[Scale bar, 400 μm (Upper), 10 μm (Lower).]
Distribution of OCPs in bone and spleen in WT, RANKL−/−, and CSF-
Bone Liver Spleen
Bone Liver Spleen
Osteoblasts Macrophages Osteoclasts
PECAM-1 IL-34/PECAM-1 IL-34
: PECAM-1(+) cells
: IL-34(+) cells
time RT-PCR measurements of IL-34 mRNA expression in bone, liver, and
spleen in 3-wk-old WT and CSF-1op/opmice. Data obtained from triplicate
PCRs using RNA from different mice are expressed as the mean ± SD (n = 3).
(B) Real-time RT-PCR measurements of IL-34 and CSF-1 mRNA expression in
osteoblastic cells, BM macrophages, and osteoclasts. Data obtained from
triplicate PCRs are expressed as the mean ± SD *P < 0.01. (C) Localization of
IL-34 (red) and PECAM-1 (green) in spleen in CSF-1op/opmice. Dashed circles
represent the white pulp. The outsides of the circles show the marginal
zones and red pulp. (Scale bar, 100 μm.) (D) Magnified views of the boxed
areas [(a) red pulp and (b) white pulp] in C. Nuclei were stained with DAPI
(blue). Arrows indicate IL-34(+)/PECAM-1(+) cells. (Scale bar, 20 μm.) (E)
Number of IL-34(+) cells and PECAM-1(+) cells in the red and white pulp.
Values in red bars represent percentages of IL-34(+) cells among PECAM-1(+)
cells. Data are expressed as the mean ± SD for four optical fields. (F) Lo-
calization of IL-34 (red) and PECAM-1 (green) (Left) and that of IL-34 (red)
and ALP activity (green) (Right) in the proximal tibiae in CSF-1op/opmice.
Dashed lines represent bone surface. Arrows indicate IL-34(+)/PECAM-1(+)
cells. Arrowheads indicate ALP(+) osteoblastic cells. (Scale bar, 20 μm.)
Distribution of IL-34 expression in WT and CSF-1op/opmice. (A) Real-
Nakamichi et al.PNAS
| June 19, 2012
| vol. 109
| no. 25
population was much lower in bone than in spleen, consistent
with the result of real-time RT-PCR (Fig. 2A). These results
suggest that IL-34 is involved in the generation of OCPs in
spleen in CSF-1op/opmice.
Then, we examined the biological activities of IL-34 in several
assays and found them to be similar to those of CSF-1 (Fig. S2).
Consistent with the previous reports (14–17), IL-34 promoted
not only the proliferation of BM macrophages (Fig. S2A), but
also the formation of osteoclasts (Fig. S2B), both of which were
similarly inhibited by adding αCSF-1R Ab. IL-34 as well as CSF-1
supported the survival of osteoclasts, which was similarly inhibi-
ted by adding αCSF-1R Ab (Fig. S2C). Thus, IL-34 is concluded
to stimulate osteoclastogenesis through CSF-1R. These results
suggest that splenic OCPs are transferred from spleen to bone in
response to CSF-1/IL-34 administration in CSF-1op/opmice.
We then examined the effect of splenectomy (SPX) on oste-
oclast formation in CSF-1op/opmice (Fig. 3A). Three-week-old
CSF-1op/opmice were subjected to SPX or a sham operation
(Sham) and injected with CSF-1 4 d after the surgery. The CSF-1
injection produced tartrate-resistant acid phosphatase (TRAP, an
osteoclast marker)(+) osteoclasts in bone in Sham CSF-1op/op
mice, but not in SPX CSF-1op/opmice. RANKL−/−mice were also
subjected to SPX or Sham and examined for osteoclastogenesis in
response to RANKL (Fig. 3B). The RANKL injection produced
osteoclasts in bone both in Sham and SPX RANKL−/−mice.
RANKL appeared to induce osteoclasts to form from RANK
(+)/CSF-1R(+) cells preexisting in bone in RANKL−/−mice.
The administration of VEGF improved the phenotype of
osteopetrosis in CSF-1op/opmice (11, 12), and a deficiency of
VEGFR1 worsened it (12). VEGF may stimulate the growth and
IL-34 synthesis of vascular endothelial cells. Consistent with the
previous reports(11, 12),the
(VEGF120) into CSF-1op/opmice increased the appearance of
TRAP(+) osteoclasts (Fig. S3A). SPX prevented VEGF-A120–
induced osteoclastogenesis in CSF-1op/opmice. However, the
VEGF120 injection did not increase the expression of IL-34
mRNA in bone, liver, or spleen in CSF-1op/opmice (Fig. S3B).
These results suggest that VEGF induces loosening of the en-
dothelial cell contacts (25), and the subsequent entry of OCPs
into the blood stream.
We next examined the possibilities of whether the spleen acts
as a reservoir of OCPs in aged CSF-1op/opmice and whether IL-34
is involved in this process as well (Fig. 4). TRAP(+) osteoclasts
were detected in bone in 10-wk-old and 10-mo-old CSF-1op/op
mice (Fig. 4A). The expression of IL-34 mRNA in bone and
spleen but not in the liver increased with aging in CSF-1op/opmice
(Fig. 4B). WT mice also exhibited an age-associated increase in
IL-34 expression in bone. CSF-1 mRNA expression showed no
correlation with aging in WT mice (Fig. 4B). Then, 5-wk-old
CSF-1op/opmice were subjected to SPX or Sham. Five weeks
later, tibiae were recovered and examined for osteoclastogenesis
(Fig. 4C). Histomorphometric analysis of tibiae showed that SPX
suppressed the age-associated appearance of osteoclasts, erosion
surface/bone surface (ES/BS), and increased bone volume/tissue
volume (BV/TV) in aged CSF-1op/opmice (Fig. 4C). These results
suggest that spleen acts as a reservoir of OCPs in the age-asso-
ciated appearance of osteoclasts in CSF-1op/opmice.
We previously reported that a large amount of a 2MD analog,
2-methyl-2MD, induced hypercalcemia in WT mice, but not in c-
Fos−/−mice (21). The potency of 2-methyl-2MD as well as 2MD
in inducing osteoclastogenesis was 100 times higher than that of
1α,25(OH)2D3(21). In the course of investigating further, we
found that administration of 2-methyl-2MD to CSF-1op/opmice
induced hypercalcemia (Fig. S4). We, then, examined whether
administration of the original 2MD induces osteoclastogenesis in
CSF-1op/opmice. CSF-1op/opmice subjected to SPX or Sham were
injected with a large amount of 2MD (Fig. 5A). The 2MD in-
jection induced the appearance of TRAP(+) osteoclasts in bone
in Sham but not in SPX CSF-1op/opmice. 2MD increased erosion
surface in parallel with the increase of osteoclast number in
Sham but not in SPX CSF-1op/opmice (Fig. 5A). 2MD is known
to induce RANKL expression in osteoblastic cells (20, 21). Then,
RANKL was injected into CSF-1op/opmice, but neither the os-
teoclast formation nor the increase in erosion surface (ES/BS)
was observed (Fig. 5A). The administration of 2MD stimulated
the expression of IL-34 mRNA in spleen and bone in CSF-1op/op
mice (Fig. 5B). The number of IL-34(+)/PECAM-1(+) cells and
that of RANK(+)/CSF-1R(+) cells were also increased in re-
sponse to 2MD administration (Fig. 5 C and D). We then per-
fluorescence-labeled control siRNA, we confirmed that the
siRNA was successfully delivered to spleen and bone (Fig. S5A).
Then IL-34 siRNA or control siRNA was injected into CSF-1op/op
mice 24 h before the administration of 2MD. The expression
of IL-34 mRNA in spleen was reduced by up to 80% by adding
IL-34 siRNA (Fig. S5B). IL-34 siRNA but not control siRNA
suppressed the 2MD-induced osteoclastogenesis in CSF-1op/op
mice (Fig. 5E). These results indicate that IL-34 is involved in
the 2MD-induced mobilization of OCPs from spleen to bone in
CSF-1op/opmice. We finally examined whether 2MD-induced up-
regulation of IL-34 expression is mediated by the vitamin D re-
ceptor (VDR) using VDR−/−mice. Although comparable levels
of IL-34 mRNA expression were detected in bone and spleen in
VDR−/−mice, the IL-34 expression was not enhanced by 2MD
administration (Fig. 5F).
We have shown that spleen in CSF-1op/opmice acts as a reservoir
of OCPs, which are transferred to bone and differentiate into
osteoclasts in response to diverse stimuli (Fig. 6). The existence
of OCPs in spleen seems to be supported by IL-34 expressed in
vascular endothelial cells. The mysterious phenomena observed
in CSF-1op/opmice (7–12) may be explained by the transfer of
OCPs from spleen to bone.
The exclusive localization of OCPs in spleen was observed in
CSF-1op/opmice but not in RANKL−/−mice. SPX prevented
osteoclastogenesis in 2MD-induced osteoclastogenesis in CSF-
1op/opmice. However, 2MD-induced osteoclastogenesis in nor-
mal mice was not impaired by SPX (Fig. S6). Therefore, it is
unlikely that spleen acts as a reservoir of OCPs in osteoclasto-
genesis under the physiological condition in normal animals.
However, 2MD administration increased the number of OCPs as
Vehicle RANKL RANKL
mice. Three-week-old CSF-1op/opmice (A) and 8-wk-old RANKL−/−mice (B)
were subjected to SPX or Sham. Four days later, CSF-1op/opmice and RANKL−/−
mice were administered CSF-1 (107units/kg) and RANKL (2.5 mg/kg), re-
spectively, daily for 4 d and killed 24 h after the last injection. Sections of
tibiae were double stained for TRAP and methyl green (Left), and the
number of TRAP(+) osteoclasts was counted (Right). Arrows indicate TRAP(+)
osteoclasts. Data are expressed as the mean ± SD for four optical fields from
four mice. *P < 0.01. (Scale bar, 50 μm.)
Effects of SPX on osteoclastogenesis in CSF-1op/opmice and RANKL−/−
| www.pnas.org/cgi/doi/10.1073/pnas.1207361109Nakamichi et al.
well as the expression of IL-34 in spleen in CSF-1op/opmice. SPX
suppressed the age-associated appearance of osteoclasts in CSF-
1op/opmice. These results suggest that OCPs are generated and
maintained in spleen through IL-34 during the lifetime of CSF-
OCPs were released from spleen into the blood stream in
CSF-1op/opmice. Using an in vivo system of bone morphogenetic
protein 2 (BMP-2)–induced ectopic bone formation in RANKL−/−
mice, we demonstrated that OCPs existed in the peripheral
blood as well as in bone marrow (26). Circulating OCPs were
cell-cycle–arrested cells committed to the osteoclast lineage.
When CSF-1 was injected into CSF-1op/opmice, osteoclasts
detected in bone were generated from cell-cycle–arrested OCPs
(18). Our findings also support the notion that the lineage-
committed OCPs circulate in the blood stream and fix to the
correct site for osteoclastogenesis. Ishii et al. (27) reported that
an agonist of sphingosine 1 phosphate (S1P) increased the mi-
gration of OCPs between blood and bone. CSF-1 administration
increased the mobilization of OCPs from spleen to bone, sug-
gesting that CSF-1 as well as IL-34 plays important roles not only
in the osteoclastic differentiation of OCPs in bone but also in the
mobilization of OCPs from spleen to bone in CSF-1op/opmice.
Future studies will further clarify the mechanism by which OCPs
are transferred from spleen into blood and home to bone.
The expression level of IL-34 was much lower in bone than in
spleen. This may explain why OCPs and osteoclasts are absent in
bone in young CSF-1op/opmice. Osteoclasts appeared in aged
CSF-1op/opmice with concomitant up-regulation of IL-34 ex-
pression in bone. 2MD administration also enhanced IL-34
expression in bone. These results suggest that IL-34 generated by
vascular endothelial cells contributes to osteoclastogenesis in-
duced by aging and 2MD administration in CSF-1op/opmice.
VEGF also induced osteoclastogenesis in CSF-1op/opmice.
However, the ability of VEGF to induce osteoclastogenesis was
much weaker than that of CSF-1 and 2MD, and VEGF failed to
up-regulate IL-34 expression. These results suggest that IL-34
expressed in vascular endothelial cells in bone is essentially in-
volved in osteoclastogenesis in CSF-1op/opmice.
Administration of 2MD induced osteoclastogenesis with up-
regulation of IL-34 expression in CSF-1op/opmice. The RNA
interference experiment further supported the notion that IL-34
is involved in the 2MD-induced osteoclastogenesis in CSF-1op/op
mice. When 2MD was administered to WT mice, the expression
of IL-34 mRNA was significantly increased in WT mice (Fig. S7).
The stimulatory effect of 2MD on IL-34 expression was not
observed in VDR−/−mice. Administration of a large amount of
1α,25(OH)2D3into CSF-1op/opmice also induced osteoclasto-
genesis (Fig. S8). These findings suggest that the IL-34 gene is
a unique target of 1α,25(OH)2D3. Using the program Pattern
Search for Transcription Factor Binding Sites (PATCH 1.0), we
found five putative binding sites of VDR within the 2 kb up-
stream of the transcription start site of the mouse IL-34 gene.
The expression level of IL-34 mRNA in bone and spleen in
VDR−/−mice was comparable to that in wild-type mice. These
results suggest that VDR-mediated signals are not essential for
IL-34 expression, but are involved in the up-regulation of IL-34
expression in endothelial cells.
: 3-week-old: 10-week-old : 10-month-old
clastogenesis in CSF-1op/opmice. (A) Appearance
of osteoclasts in CSF-1op/opmice in an age-de-
pendent manner. Sections of tibiae obtained
from CSF-1op/opmice at different ages were dou-
ble stained for TRAP and methyl green (Left), and
the number of osteoclasts was counted (Right).
Arrows indicate TRAP(+) osteoclasts. Data are
expressed as the mean ± SD for four optical fields
from four mice. (B) Expression of IL-34 and CSF-1
mRNAs in bone, liver, and spleen in CSF-1op/op
mice and WT mice at various ages. Data were
obtained from triplicate PCRs using RNA from
three mice and expressed as the mean ± SD. (C)
Effect of splenectomy on age-associated osteo-
clastogenesis. Five-week-old CSF-1op/opmice were
subjected to SPX or Sham. Five weeks later, they
were killed. Sections of tibiae were double
stained for TRAP and methyl green (Left). Osteo-
clast number (number/mm), erosion surface/bone
surface (ES/BS, %), and bone volume/tissue vol-
ume, (BV/TV, %) were measured (Right). Data are
expressed as the mean ± SD for four optical fields
from four mice. (Scale bar, 50 μm.) *P < 0.01. NS,
Role of spleen for age-associated osteo-
Nakamichi et al.PNAS
| June 19, 2012
| vol. 109
| no. 25
We have not succeeded in showing the stimulation of IL-34
expression by 2MD in mouse splenic endothelial cell cultures.
Vascular microenvironment or blood vascular networks may be
required for 2MD-induced up-regulation of IL-34 expression.
Vascular endothelial cells are shown to regulate the recruitment
of monocyte/macrophage lineage cells, which play a role in an-
giogenesis (28–31). At the amino acid sequence level, IL-34 gene
is more conserved than the CSF-1 gene during evolution (32).
These results suggest that the vitamin D system and IL-34 may
fundamentally work in normal angiogenesis.
Recently, it was reported that IL-34 was expressed in synovial
tissues obtained from rheumatoid arthritis patients, and tumor
necrosis factor α (TNFα) stimulated IL-34 expression in those
synovial fibroblasts in culture (33, 34). Giant cell tumors of bone
have been shown to express IL-34 (17). Therefore, we examined
the effects of osteotropic factors such as 1α,25(OH)2D3, 2MD,
TNFα, interleukin 1β (IL-1β) and prostaglandin E2(PGE2) on
IL-34 expression in mouse osteoblastic cells in culture (Fig. S9).
Real-time RT-PCR analysis showed that TNFα, IL-1β, and
PGE2, respectively, failed to increase IL-34 mRNA expression in
osteoblastic cells in our cell culture conditions. 2MD and 1α,25
(OH)2D3significantly increased IL-34 mRNA expression in os-
teoblastic cells. However, it is unlikely that IL-34 expressed by
osteoblastic cells in response to 1α,25(OH)2D3can substitute for
CSF-1 for osteoclastogenesis, because 1α,25(OH)2D3failed to
support osteoclast formation in cocultures of hematopoietic os-
teoclast precursors and CSF-1op/opmouse-derived osteoblastic
cells (35). At present, the cause of the difference between our
result and the results reported previously is not known. Further
studies will elucidate the regulatory mechanism of IL-34 ex-
pression in fibroblastic cells as well as endothelial cells.
Both CSF-1op/opand RANKL−/−osteopetrotic mice develop
splenic extramedullary hematopoiesis due to the impaired bone
microenvironment. This suggests that spleen acts as the reservoir
of hematopoietic precursors under pathological conditions such
as osteopetrosis. Recently, Miyamoto et al. (36) reported that
the mobilization of hematopoietic stem and progenitor cells
(HSPCs) occurring after granulocyte colony-stimulating factor
(G-CSF) injection was comparable or even increased in osteo-
petrotic CSF-1op/op, RANKL−/−and c-Fos−/−mice, compared
with that in WT mice. Contrary to OCPs, the mobilization of
HSPCs was not suppressed by SPX in CSF-1op/opmice in the G-
CSF treatment (36). These results suggest that spleen cannot act
as the reservoir of HSPCs in CSF-1op/opmice, although HSPCs
exist in the enlarged spleen. Swirski et al. (37) identified unique
monocytes in spleen, which exited the spleen en masse, accu-
mulated in the injured tissue, and participated in wound healing,
in response to ischemic myocardial injury. CSF-1 has been shown
to be involved in the coordinated dynamics of the tissue distri-
bution of macrophages and dendritic cells (38). Thus, spleen
plays important roles in the maintenance and tissue distribution
of monocyte–macrophage lineage cells through IL-34 expression.
In conclusion, IL-34 plays pivotal roles in the maintenance and
mobilization of splenic OCPs in CSF-1op/opmice. IL-34 and CSF-
1 play dominant roles in determining the distribution of OCPs.
The IL-34 gene in vascular endothelial cells is a unique target of
Vehicle 2MD Vehicle 2MD
Vehicle 2MD Vehicle 2MD
: PECAM-1(+) cells
: IL-34(+) cells
Vehicle 2MD Vehicle
osteoclastogenesis in CSF-1op/opmice. (A) Effects of 2MD
and RANKL on osteoclastogenesis. Three-week-old CSF-
1op/opmice were subjected to SPX or Sham. Four days
later, mice were injected with 2MD (2 nmol/kg) twice
2 d apart, and killed 48 h after the second injection.
Sham mice were administered daily RANKL (2.5 mg/kg)
daily for 4 d, and killed 24 h after the last injection.
Sections of tibiae were double stained for TRAP and
methyl green (Left), and osteoclast number (Center) and
erosion surface (ES/BS) (Right) were measured. Arrows
indicate TRAP(+) osteoclasts. Data are expressed as the
mean ± SD for four optical fields from four mice. (Scale
bar, 50 μm.) (B–D) Effects of 2MD on IL-34 expression (B
and C) and RANK(+)/CSF-1R(+) cells (D). Three-week-old
CSF-1op/opmice were injected with vehicle or 2MD (2
nmol/kg) and killed 24 h after the injection. (B) Effects of
2MD on IL-34 mRNA expression. Expression of IL-34
mRNA in bone, liver, and spleen was examined by real-
time RT-PCR. Data were obtained from triplicate PCRs
using RNA from two mice and expressed as the mean ±
SD. (C) Effects of 2MD on IL-34 protein expression. Spleen
sections were stained for IL-34 (red) and PECAM-1
(green) (Left). Number of IL-34(+)/PECAM-1(+) cells in the
red pulp was counted (Right). Values in red bars repre-
sent percentages of IL-34(+) cells among PECAM-1(+)
cells. Data are expressed as the mean ± SD for four
optical fields. (D) Effects of 2MD on RANK(+)/CSF-1R(+)
cells in spleen. Spleen sections were stained for RANK
(red) and CSF-1R (green) (Left). Number of RANK
(+)/CSF-1R(+) cells in the red pulp was counted (Right).
Data are expressed as the mean ± SD for four optical
fields. (Scale bar, 10 μm.) (E) Effects of IL-34 siRNA in-
jection on osteoclastogenesis. Three-week-old CSF-1op/op
mice were i.v. injected with control siRNA or IL-34 siRNA
(10 mg/kg) or its control. One day later, they were
injected with 2MD (2 nmol/kg) twice 2 d apart and kil-
led 48 h after the second injection. Sections of tibiae
were double stained for TRAP and methyl green (Left),
and the number of osteoclasts was counted (Right).
Arrows indicate TRAP(+) osteoclasts. Data are expressed
as the mean ± SD for four optical fields from four mice.
*P < 0.01. (Scale bars, 50 μm.) (F) Effects of 2MD on IL-34 mRNA expression in VDR−/−mice. Eight-week-old VDR−/−mice were injected with vehicle or 2MD (2
nmol/kg) and killed 24 h after the injection. Data obtained from triplicate PCRs using RNA from three mice are expressed as the mean ± SD (n = 3).
Effects of SPX and IL-34 siRNA on 2MD-induced
| www.pnas.org/cgi/doi/10.1073/pnas.1207361109 Nakamichi et al.
vitamin D. Clarifying how splenic OCPs enter the blood stream Download full-text
and reach bone may provide a unique strategy to control bone
Materials and Methods
Detailed protocols are given in SI Materials and Methods.
Animals. Breeding pairs of CSF-1op /+mice (B6C3Fe genetic background) were
purchased from The Jackson Laboratory and F2mice were raised in our
laboratory. Homozygous CSF-1op/opmice, identified by a lack of incisors at
postnatal day 10, and RANKL−/−mice (39) (C57BL/6 background) were fed
a softened rodent chow (Oriental Yeast) with water after weaning. VDR−/−
mice (C57BL/6 background) were generated by cross-breeding of VDR-floxed
mice with CMV-Cre mice (40). VDR−/−mice were fed a high calcium diet (CE-2
supplemented with 2% (wt/wt) calcium, 1.25% (wt/wt) phosphate, and 20%
(wt/wt) lactose; CLEA Japan) to normalize serum calcium level. Eight-week-
old male ddY mice and newborn ddY mice (Japan SLC) were used as WT mice
for preparation of BM cells and osteoblastic cells, respectively. All experi-
ments were conducted in accordance with the guidelines for studies with
laboratory animals of the Matsumoto Dental University Experimental
Splenectomy. Mice were anesthetized with isoflurane (Isoflu; Dainippon
Sumitomo Pharma) usingavaporizer(DSPharma Biomedical). The spleen was
identified after a transverse laparotomy incision just to the left of the spinal
cord and removed after appropriate blood vessel ligation. Sham-operated
animals underwent the laparotomy without a splenectomy.
Statistics. Statistical analyses were performed using the one-tailed Student
t test and Fisher’s exact probability test, as appropriate. P < 0.05 was con-
sidered statistically significant.
ACKNOWLEDGMENTS. This work was supported in part by Ministry of
Education, Culture, Sports, Science, and Technology of Japan Grants-in-Aid
21791817 (to Y.N.), 22791804 (to T.M.), 21390551 (to Y.K.), and 22390351 (to
N.T.) and by a grant from the Naito Foundation for Natural Science (to T.M.).
1. Boyle WJ, Simonet WS, Lacey DL (2003) Osteoclast differentiation and activation.
2. Suda T, et al. (1999) Modulation of osteoclast differentiation and function by the new
3. Stanley ER, Berg KL, Einstein DB, Lee PS, Yeung YG (1994) The biology and action of
colony stimulating factor-1. Stem Cells 12(Suppl 1):15–24, discussion 25.
4. Yoshida H, et al. (1990) The murine mutation osteopetrosis is in the coding region of
the macrophage colony stimulating factor gene. Nature 345:442–444.
5. Wiktor-Jedrzejczak WW, Ahmed A, Szczylik C, Skelly RR (1982) Hematological char-
acterization of congenital osteopetrosis in op/op mouse. Possible mechanism for
abnormal macrophage differentiation. J Exp Med 156:1516–1527.
6. Kodama H, et al. (1991) Congenital osteoclast deficiency in osteopetrotic (op/op) mice
is cured by injections of macrophage colony-stimulating factor. J Exp Med 173:269–272.
7. Begg SK, et al. (1993) Delayed hematopoietic development in osteopetrotic (op/op)
mice. J Exp Med 177:237–242.
8. Dai XM, et al. (2002) Targeted disruption of the mouse colony-stimulating factor 1
receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased
primitive progenitor cell frequencies, and reproductive defects. Blood 99:111–120.
9. Cecchini MG, et al. (1994) Role of colony stimulating factor-1 in the establishment and
regulation of tissue macrophages during postnatal development of the mouse. De-
10. Yamamoto T, et al. (2008) Macrophage colony-stimulating factor is indispensable for re-
population and differentiation of Kupffer cells but not for splenic red pulp macrophages
in osteopetrotic (op/op) mice after macrophage depletion. Cell Tissue Res 332:245–256.
11. Niida S, et al. (1999) Vascular endothelial growth factor can substitute for macro-
phage colony-stimulating factor in the support of osteoclastic bone resorption. J Exp
12. Niida S, et al. (2005) VEGF receptor 1 signaling is essential for osteoclast development
and bone marrow formation in colony-stimulating factor 1-deficient mice. Proc Natl
Acad Sci USA 102:14016–14021.
13. Lean JM, Fuller K, Chambers TJ (2001) FLT3 ligand can substitute for macrophage
colony-stimulating factor in support of osteoclast differentiation and function. Blood
14. Lin H, et al. (2008) Discovery of a cytokine and its receptor by functional screening of
the extracellular proteome. Science 320:807–811.
15. Wei S, et al. (2010) Functional overlap but differential expression of CSF-1 and IL-34 in
their CSF-1 receptor-mediated regulation of myeloid cells. J Leukoc Biol 88:495–505.
16. Chen Z, Buki K, Vääräniemi J, Gu G, Väänänen HK (2011) The critical role of IL-34 in
osteoclastogenesis. PLoS ONE 6:e18689.
17. Baud’huin M, et al. (2010) Interleukin-34 is expressed by giant cell tumours of bone
and plays a key role in RANKL-induced osteoclastogenesis. J Pathol 221:77–86.
18. Mizoguchi T, et al. (2009) Identification of cell cycle-arrested quiescent osteoclast
precursors in vivo. J Cell Biol 184:541–554.
19. DeLuca HF (2004) Overview of general physiologic features and functions of vitamin
D. Am J Clin Nutr 80(6 Suppl):1689S–1696S.
20. Shevde NK, et al. (2002) A potent analog of 1alpha,25-dihydroxyvitamin D3selectively
induces bone formation. Proc Natl Acad Sci USA 99:13487–13491.
21. Sato M, et al. (2007) New 19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 analogs
strongly stimulate osteoclast formation both in vivo and in vitro. Bone 40:293–304.
22. Wong MS, Delansorne R, Man RY, Vanhoutte PM (2008) Vitamin D derivatives acutely
reduce endothelium-dependent contractions in the aorta of the spontaneously hy-
pertensive rat. Am J Physiol Heart Circ Physiol 295:H289–H296.
23. Mantell DJ, Owens PE, Bundred NJ, Mawer EB, Canfield AE (2000) 1 α,25-dihydrox-
yvitamin D(3) inhibits angiogenesis in vitro and in vivo. Circ Res 87:214–220.
24. Hisa T, et al. (1996) Vitamin D inhibits endothelial cell migration. Arch Dermatol Res
25. Broermann A, et al. (2011) Dissociation of VE-PTP from VE-cadherin is required for
leukocyte extravasation and for VEGF-induced vascular permeability in vivo. J Exp
26. Muto A, et al. (2011) Lineage-committed osteoclast precursors circulate in blood and
settle down into bone. J Bone Miner Res 26:2978–2990.
27. Ishii M, et al. (2009) Sphingosine-1-phosphate mobilizes osteoclast precursors and
regulates bone homeostasis. Nature 458:524–528.
28. Grunewald M, et al. (2006) VEGF-induced adult neovascularization: Recruitment, re-
tention, and role of accessory cells. Cell 124:175–189.
29. Bergmann CE, et al. (2006) Arteriogenesis depends on circulating monocytes and mac-
rophage accumulation and is severely depressed in op/op mice. J Leukoc Biol 80:59–65.
30. Lobov IB, et al. (2005) WNT7b mediates macrophage-induced programmed cell death
in patterning of the vasculature. Nature 437:417–421.
31. Kubota Y, et al. (2009) M-CSF inhibition selectively targets pathological angiogenesis
and lymphangiogenesis. J Exp Med 206:1089–1102.
32. Garceau V, et al. (2010) Pivotal Advance: Avian colony-stimulating factor 1 (CSF-1), in-
terleukin-34 (IL-34), and CSF-1receptor genes and geneproducts. J Leukoc Biol 87:753–764.
33. Chemel M, et al. (2012) Interleukin 34 expression is associated with synovitis severity
in rheumatoid arthritis patients. Ann Rheum Dis 71:150–154.
34. Hwang SJ, et al. (2012) Interleukin-34 produced by human fibroblast-like synovial cells
in rheumatoid arthritis supports osteoclastogenesis. Arthritis Res Ther 14:R14.
35. Tanaka S, et al. (1993) Macrophage colony-stimulating factor is indispensable for both
proliferation and differentiation of osteoclast progenitors. J Clin Invest 91:257–263.
36. Miyamoto K, et al. (2011) Osteoclasts are dispensable for hematopoietic stem cell
maintenance and mobilization. J Exp Med 208:2175–2181.
37. Swirski FK, et al. (2009) Identification of splenic reservoir monocytes and their de-
ployment to inflammatory sites. Science 325:612–616.
38. Tagliani E, et al. (2011) Coordinate regulation of tissue macrophage and dendritic cell
population dynamics by CSF-1. J Exp Med 208:1901–1916.
39. Kong YY, et al. (1999) OPGL is a key regulator of osteoclastogenesis, lymphocyte
development and lymph-node organogenesis. Nature 397:315–323.
40. Li M, et al. (2006) Topical vitamin D3and low-calcemic analogs induce thymic stromal
lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis. Proc Natl
Acad Sci USA 103:11736–11741.
CSF-1, VEGF, Aging
CSF-1, VEGF, Aging
exist as RANK(+)/CSF-1R(+) cells in spleen but not in bone in CSF-1op/opmice.
IL-34 expressed by endothelial cells appears to be involved in the differen-
tiation of OCPs from hematopoietic progenitor cells. Administration of CSF-1,
2MD, or VEGF into CSF-1op/opmice produces osteoclasts in bone. Osteoclasts
also develop in bone in aged CSF-1op/opmice. OCPs in spleen are mobilized
into the circulation in response to diverse stimuli. Circulating OCPs settle
down in bone and differentiate into osteoclasts in response to RANKL and
CSF-1 or IL-34. IL-34 expression is increased in spleen and bone with aging or
2MD administration. IL-34 appears to play pivotal roles in the development
in spleen and transfer of OCPs from spleen to bone in CSF-1op/opmice.
A hypothetical model for osteoclastogenesis in CSF-1op/opmice. OCPs
Nakamichi et al.PNAS
| June 19, 2012
| vol. 109
| no. 25