1
SUPPLEMENTARY INFORMATION
Evidence for osteocyte regulation of bone homeostasis through
RANKL expression
Tomoki Nakashima1,2,3
, Mikihito Hayashi1,2,3
, Takanobu Fukunaga1,3
, Kosaku Kurata4,
Masatsugu Oh-hora1,3
, Jian Q. Feng5, Lynda F. Bonewald
6, Tatsuhiko Kodama
7, Anton
Wutz8, Erwin F. Wagner
9, Josef M. Penninger
10 &
Hiroshi Takayanagi
1,2,3,11
Correspondence and requests for materials should be addressed to H.T.
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Methods
Generation of mice carrying the Tnfsf11 conditional allele. All fragments for the
construction of the Tnfsf11 targeting vector were derived from a BAC clone
(RP23-68C13: BACPAC Resource Center) by restriction digestion and subcloned into a
pBluescript II vector. The homology arms were cloned into a targeting vector backbone
containing PGK-neo and DTA-positive and -negative selection cassettes (pLFNeo-DTA
vector) (Supplementary Fig. 5b). The long arm was excised as XhoI/AhdI fragment (6.8
kb) from the pBluescript II vector including all of the homology arms of Tnfsf11, and
cloned into the SacI sites of a pLFNeo-DTA vector upstream of the neo selection
cassette, which was flanked by frt sites. For construction of the middle arm, a fragment
encompassing exons 3 and 4 of Tnfsf11 was excised as the AhdI fragment (1.9 kb), and
cloned into the NheI site of the pLFNeo-DTA vector just upstream of the neo selection
cassette so as to flank exons 3 and 4 with loxP sites. The short arm was excised as the
AhdI/PmlI fragment (0.8 kb), then cloned into the EcoRV sites of the pLFNeo-DTA
vector upstream of the DTA cassette to give rise to the final targeting vector for the
Tnfsf11floxNeo
allele. The targeting vector was linearized with PciI and electroporated
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into A9 embryonic stem (ES) cells. A9 ES cells were derived from the 129 and
C57BL/6 hybrid strain (generated by A. Wutz, U. Möhle-Steinlein and E.F. Wagner).
After 8-9 days of G418 selection, resistant single colonies were picked and transferred
on primary embryonic fibroblast cells acting as feeder cells, and expanded to diagnose
homologous recombination. Potential recombinant clones were detected by genomic
PCR. The forward primer (PS1: the construct-specific primer,
5’-AGACTGCCTTGGGAAAAG-3’) binds within the neo selection cassette region, the
reverse primer (PS2: the locus-specific primer, 5’-AGGTGATTTCTCACCGTC-3’)
binds to a unique region outside the short arm of the targeting vector and within the
endogenous locus (indicated as arrowheads in Supplementary Fig. 5b). The loxP site
between the long and middle arms was confirmed using the primer pairs (P1 and P2:
described below). The 3’ flanking probe was used for Southern blot screening on
NcoI/AhdI-digested genomic DNA (probe; 863 bp fragment from outside the short arm
was amplified by PCR with the primers 5’-ATAGCATAGGCCATAGCG-3’ and
5’-ATGAGGCACGCTGAGGAC-3’). Approximately 1-2 out of 100 ES cell clones
were identified as correctly targeted by genomic PCR and Southern blot screening.
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Targeted ES cells were injected into C57BL/6 blastocysts to achieve initial germ-line
transmission. Chimeric male mice were crossed with C57BL/6 females to establish a
line for the Tnfsf11floxNeo
allele. Mice carrying the frt-flanked PGK-neo cassette were
crossed with C57BL/6 background FLPe transgenic mice to excise the PGK-neo
cassette and subsequently crossed to C57BL/6 mice to remove the FLPe recombinase
transgene to generate Tnfsf11flox
mice. In parallel, Tnfsf11flox
mice were also crossed to
C57BL/6 background β-actin-Cre ubiquitous deleter mice to generate mice carrying a
Tnfsf11Δ allele. Genotypes were confirmed by Southern blot using the probe indicated
above. Mice carrying the Tnfsf11flox
or Tnfsf11Δ
alleles were backcrossed 5 times to
C57BL/6 mice. These mice were then intercrossed to generate Tnfsf11flox/Δ
and
Tnfsf11+/Δ
mice before generating Tnfsf11flox/Δ
; Dmp1- or Lck-Cre mice15,21
. In all the
experiments, littermate control Tnfsf11flox/flox
mice were used that did not carry the Cre
recombinase; these controls behaved similar to wild-type mice. Tnfsf11flox/Δ
; Dmp1- or
Lck-Cre mice are viable, exhibit normal fertility, and are in general undistinguishable
from their control littermates. For mouse genotyping, genomic DNA from mouse tails
was isolated by phenol/chloroform extraction and ethanol precipitation, and amplified
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by PCR with three primers: P1, 5'- GATACCATTGGGAATCCC-3'; P2, 5'-
CTGAGGTCACATAAGGTC-3'; and P3, 5'-GTGATGACTACCTAGCAC-3'. The
PCR products were 198 bp (wild-type allele, primers P1/P2), 328bp (floxed allele,
primers P1/P2) and 303 bp (delta allele, primers P1/P3). The Cre transgene was
detected as a 455 bp PCR product by using the forward primer
5'-TCGCGATTATCTTCTATATCTTCAG-3' and reverse primer
5'-GCTCGACCAGTTTAGTTACCC-3'. The sex determination gene, Sry, was detected
as a 266 bp PCR product by using the forward primer
5'-GAGAGCATGGAGGGCCAT-3' and reverse primer
5'-CCACTCCTCTGTGACACT-3'. All of the animals were maintained in a specific
pathogen-free environment, and all animal experiments were performed with the
approval of the Institutional Animal Care and Use Committee of Tokyo Medical and
Dental University and conformed to relevant guidelines and laws.
Conventional isolation of osteoblast- and osteocyte-rich fractions. After the removal
of the sutures or bone marrow cells, minced neonatal calvarial or long bones of mice
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were subjected to 5 times sequential digestion in a mixture containing 0.1% collagenase
(Wako) and 0.2% dispase II (SANKOJUNYAKU) using a modified version of the
protocol11,12
. Cell fraction 2 (the osteoblast-rich fraction) and 5 (the osteocyte-rich
fraction) were collected and resuspended in PBS with 2 % FBS. Cells were cultured in
MEM containing 10% FBS.
Isolation of osteocyte population of high purity. Mice with osteocyte-specific
expression of EGFP were generated by crossing of CAG-CAT-EGFP14
and Dmp1-Cre
transgenic mice15
. Using fluorescence-activated cell sorting (FACS Aria III cell sorter),
EGFP-positive osteocytes were isolated from the cell fractions (fraction 2-5) obtained
by sequential enzymatic digestion of neonatal calvarial bones of the
CAG-CAT-EGFP/Dmp1-Cre double-transgenic mice. Long bones (femurs and tibiae)
were dissected from adult double-transgenic mice (over 12-weeks old). Periosteum and
bone marrow cells were completely removed and cell fractions were obtained by serial
enzymatic digestion. Isolated cells were collected and resuspended in PBS with 2 %
FBS, and plated in a cell culture dish (RepCell, Cell Seed Inc.) for flow cytometric
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analysis. EGFP-positive and -negative cells were visualized under fluorescence
microscopy.
Isolation of bone marrow stromal cells. Bone marrow stromal cells (BMSCs) derived
from long bones were isolated as described previously22,23
. Briefly, bone marrow cells
from each of the mice were isolated by flushing the femurs and tibiae with PBS, and
these cells were plated on plastic dishes with MEM containing 20% FBS.
Non-adherent cells were removed by replacing the media after 3 days. Adherent cells
were passaged and plated out in MEM containing 20% FBS. After the cells were
grown to 80% confluency, CD11b-positive and -negative cells were separated by
fluorescence-activated cell sorting (FACS Aria III cell sorter). CD11b-CD45
-Sca-1
+
cells were used as BMSCs.
Isolation and activation of T cells. Naïve CD4+ T cells were purified from the spleen
using the CD4+CD62L
+ T cell Isolation kit (Miltenyi Biotec) and stimulated with 20 ng
ml-1
phorbol 12-myristate 13-acetate (PMA, Calbiochem) and 0.2 μg ml-1
ionomycin
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(Sigma-Aldrich) for 1 day or 2 μg ml-1
anti-CD3 (145-2C11, BD Biosciences) and 0.2
μg ml-1
anti-CD28 (37.51, BD Biosciences) monoclonal antibodies for 3 days. T cells in
bone marrow were isolated by staining with a phycoerythrin (PE)-conjugated CD3e
monoclonal antibody (145-2C11, BD Biosciences) together with fluorescence-activated
cell sorting (FACS Aria III cell sorter).
Flow cytometric analysis. Cell surface expression of RANKL was confirmed by
staining with phycoerythrin (PE)-conjugated anti-RANKL monoclonal antibody
(IK22/5, eBioscience). For the characterization of BMSCs, the cells were stained with
anti-CD11b, CD45 and Sca1 antibodies. Stained cells were analyzed by FACSCanto II
(BD Biosciences) using FlowJo software (Tree Star).
Quantitative RT-PCR analyses. Total RNA and cDNA were prepared by ISOGEN
(Wako) and Superscript III reverse transcriptase (Invitrogen) according to the
manufacturers’ instructions. Quantitative PCR analysis was performed with a
LightCycler (Roche) using SYBR Green (Toyobo). Gene expression values were
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calculated based on the Ct method using Gapdh expression as an internal control.
The primer sequences were: Dmp1, 5’-CCCAGAGGGACAGGCAAATA-3’ and
5’-TCCTCCCCACTGTCCTTCTT-3’; Fmod, 5’-GGGCAACAGGATCAATGAGT-3’
and 5’-CTGCAGCTTGGAGAAGTTCAT-3’; Gapdh,
5’-GGATGCAGGGATGATGTTCT-3’ and 5’-AACTTTGCCATTGTGGAAGG-3’;
Kera, 5’-TCCCCCATCAACTTATTTTAGC-3’ and
5’-GGTTGCCATTACAGGACCTT-3’; Npy, 5’-CCGCTCTGCGACACTACAT-3’ and
5’-TGTCTCAGGGCTGGATCTCT-3’; Reln, 5’-CGTGCTGCTGGACTTCTCT-3’ and
5’- TCCATCTCGTGAAGCAAGGT-3’; Sost, 5’-TCCTGAGAAGAACCAGACCA-3’
and 5’-GCAGCTGTACTCGGACACATC-3’; Tnfrsf11b,
5’-GTTTCCCGAGGACCACAAT-3’ and 5’-CCATTCAATGATGTCCAGGAG-3’;
Tnfsf11, 5’-AGCCATTTGCACACCTCAC-3’ and
5’-CGTGGTACCAAGAGGACAGAGT-3’.
In vitro osteoclastogenesis. We followed the method for in vitro osteoclast
differentiation described previously with minor modification24,25
. Non-adhrerent bone
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marrow cells or spleen cells cultured with 10 ng ml-1
M-CSF (R&D system) for 2 days
were used as osteoclast precursor cells, which were further cocultured with osteoblasts
or osteocytes in the presence of 10 nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, Wako)
and 1 M prostaglandin E2 (PGE2, Cayman Chemical) for 5-6 days. To investigate the
role of cell-cell contact, osteoclast precursor cells were separated from osteoblasts or
osteocytes with a membrane filter (0.4 m, Transwell, Coster) 2. Osteoclast precursor
cells were cultured with the conditioned media (12.5-100%) obtained from osteoblasts
or osteocytes treated with 1, 25(OH)2D3 and PGE2 (ref. 2). Osteoclast induction by
soluble RANKL was performed by culturing osteoclast precursor cells with 25 ng ml-1
RANKL (Peprotech) in the presence of 10 ng ml-1
M-CSF for 3 days26,27
.
Osteoclastogenesis was evaluated by tartrate-resistant acid phosphatase (TRAP)
staining. TRAP-positive multinuclear cells (TRAP+ MNCs, more than three nuclei)
were counted.
Analysis of bone phenotype. Radiography was performed using a high resolution soft
X-ray system (SOFTEX). Microcomputed tomography (CT) scanning was performed
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using a ScanXmate-A100S Scanner (Comscantechno). Three-dimensional
microstructural image data were reconstructed and structural indices were calculated
using TRI/3D-BON software (RATOC). The bone mineral was calculated using
TRI/3D-BON-BMD-PNTM software (RATOC). Bone morphometric analyses were
performed as described25,28
. Skeletogenesis in newborns was evaluated by alizarin
red/alcian blue staining. Calcified tissues were stained red and cartilage was stained
blue as described28
.
Immunohistochemical staining. After fixation in 4% paraformaldehyde (PFA), bone
tissues were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) at 4°C for 2
weeks, and embedded in paraffin after dehydration. For immunohistochemical
staining, antigen retrieval was carried out with 10 mM citric acid (pH 6.0) at room
temperature for 2 hours. After quenching of endogenous peroxidase activity by
incubation with 3% H2O2 in methanol, the sections were incubated with anti-RANKL
(C-19, Santa Cruz Bioteclonogy) or anti-Sost antibody (AF1589, R&D system) in
immunoreaction enhancer solution (Can Get Signal immunostain solution B,
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TOYOBO) at 4°C for overnight. After washing with PBS, the sections were incubated
with peroxidase-conjugated secondary antibody according to the manufacturer's
instructions (Histofine Simple Stain Mouse MAX-PO, Nichirei Bioscience). The signals
were visualized with 3,3-diaminobenzidine tetrahydrochloride (DAB) and H2O2. Methyl
green or hematoxylin was used for nuclear counterstaining.
GeneChip analysis. Osteocyte-like cells (MLO-Y4) were cultured in a
three-dimensional gel-embedded system and were stimulated by mechanical force as
described previously29
. The total RNAs extracted from these cells were utilized for
cDNA synthesis by reverse transcription followed by synthesis of biotinylated cRNA
through in vitro transcription. After cRNA fragmentation, hybridization with mouse
genome 430 2.0 array (Affymetrix) was performed as described previously30
.
Statistical analysis. Statistical analysis was performed using the unpaired two-tailed
Student's t test (*P< 0.05; **P< 0.01; ***P< 0.005; NS, not significant; ND, not
detected, throughout the paper), unless otherwise described. All data are expressed as
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the mean ± s.e.m. (n = 3 or more). Results are representative of more than three
independent experiments.
References
21. Oh-hora, M., et al. Nat Immunol 9, 432-443 (2008).
22. Wieczorek, G., et al. Cell Tissue Res 311, 227-237 (2003).
23. Dawson, M.R., Chae, S.S., Jain, R.K. & Duda, D.G. Am J Cancer Res 1,
144-154 (2011).
24. Wada, T., et al. Nat Med 11, 394-399 (2005).
25. Shinohara, M., et al. Cell 132, 794-806 (2008).
26. Sato, K., et al. Nat Med 12, 1410-1416 (2006).
27. Nishikawa, K., et al. Proc Natl Acad Sci U S A 107, 3117-3122 (2010).
28. Nishikawa, K., et al. J Clin Invest 120, 3455-3465 (2010).
29. Kurata, K., Fukunaga, T., Matsuda, J. & Higaki, H. Int J Fatigue 29, 1010-1018
(2007).
30. Takayanagi, H., et al. Dev Cell 3, 889-901 (2002).
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Supplementary figure legends
Supplementary figure 1
Profiling of gene expression in osteoblast- and osteocyte-rich fractions.
Osteoblast- and osteocyte-rich fractions were obtained by conventional enzymatic
digestion of bone (quantitative RT-PCR analysis).
Supplementary figure 2
Gene expression in osteoblasts and osteocytes isolated using a high-purity method.
The expression of genes related to osteocyte/osteoblast distinction, including Reln
(encoding reelin), Npy (encoding neuropeptide Y) and Fmod (encoding fibromodulin)
examined by quantitative RT-PCR analysis. Error bars, mean ± s.e.m.; *P< 0.05; **P<
0.01.
Supplementary figure 3
RANKL expression in mouse embryonic fibroblasts and skin fibroblasts. Cell
surface expression of RANKL in mouse embryonic fibroblasts (MEFs) and skin
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fibroblasts was quantitated by FACS analyses. MFI, mean fluorescence intensity.
Supplementary figure 4
Analysis of osteocytes, osteoblasts and BMSCs derived from long bone. a, Isolation
and characterization of bone marrow stromal cells (BMSCs, CD11b-CD45
-Sca-1
+ cells)
derived from CAG-CAT-EGFP/Dmp1-Cre double-transgenic mice. b, Morphology and
EGFP expression of BMSCs, osteoblasts and osteocytes derived from the long bone of
CAG-CAT-EGFP/Dmp1-Cre double-transgenic mice. c, Expression of Dmp1, Kera,
Tnfsf11 and Tnfrsf11b in the BMSCs, osteoblasts and osteocytes (quantitative RT-PCR
analysis). d, Immunohistochemical analysis of RANKL expression in the central
marrow of the femur. Note that RANKL was under the detectable level in BMSCs
(arrows). B: bone, BM: bone marrow, V: vein. e, Osteoclastogenesis-supporting
ability of BMSCs, osteoblasts or osteocytes derived from long bone. Scale bars, b: 100
m, d: 80 m. Error bars, mean ± s.e.m.; *P< 0.05; **P< 0.01; ***P< 0.005 (c and e).
Supplementary figure 5
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Conditional gene-targeting of Tnfsf11. a, Representation of the functional
extracellular TNF-like domain of RANKL binding to its receptor, RANK. Cre-mediated
excision of exons 3 and 4 of the Tnfsf11 gene led to a loss of the TNF-like domain of
RANKL. Arrowhead: the cleavage site. TM: transmembrane region. E: exon. b,
Targeting strategy to generate Tnfsf11 conditional mutant mice. The genomic structure
of the wild-type Tnfsf11 gene, the targeting vector and the targeted alleles are indicated.
Exons 3 and 4 are flanked by loxP sequences; the Neo cassette is flanked by frt
sequences. The modified Tnfsf11 locus after homologous recombination (Tnfsf11floxNeo
allele), the Tnfsf11 gene after excision of the Neo cassette following expression of Flp
recombinase (Tnfsf11flox
allele), and the deleted Tnfsf11gene after Cre-mediated excision
of exons (Tnfsf11Δ allele) are shown. The 3’ flanking probe used for Southern blots is
indicated by the horizontal gray bar. The arrows below the diagram of the wild-type
allele indicate the positions of the primers (P1, P2 and P3) used for PCR genotyping.c,
Southern blot analysis of NcoI/AhdI-digested tail genomic DNA from mice. d,
Genotyping PCR of tail genomic DNA using the primer pairs shown in Supplementary
Fig. 5b.
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Supplementary figure 6
Analysis of global Tnfsf11-deficient mice generated by crossing Tnfsf11flox
mice and
β-actin-Cre ubiquitous deleter mice. a, Growth retardation in global Tnfsf11-deficient
mice (Tnfsf11Δ/Δ
) at 12 weeks of age. b, Body weight in the wild-type (Tnfsf11+/+
),
heterozygous mutant (Tnfsf11+/Δ
) and Tnfsf11Δ/Δ
during growth (male, n = 7-8). c,
Radiographic analysis of Tnfsf11+/+
and Tnfsf11Δ/Δ
littermates at 12 weeks of age. Note
the massive increase in overall bone density, enhanced curvature of spine (dotted lines),
shortening of the long bones and enlargement of the metaphysis (arrows) in Tnfsf11Δ/Δ
mice. d, Microcomputed tomography (CT) analysis of the femurs of wild-type
(Tnfsf11+/+
) and Tnfsf11Δ/Δ
mice at 12 weeks of age (male, n = 5-6). Upper: longitudinal
view, lower: axial view of the metaphyseal region. e, Histological analysis of the
proximal tibia of Tnfsf11+/+
and Tnfsf11Δ/Δ
littermates. Note the cartilage remnants
(arrowheads) and the complete lack of TRAP-positive osteoclasts in Tnfsf11Δ/Δ
mice. f,
Bone volume and the trabecular bone parameters in CT and bone morphometric
analysis. g, Splenomegaly in Tnfsf11Δ/Δ
mice. h, Osteoclastogenesis from spleen cells in
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response to RANKL and M-CSF. i, Lack of tooth eruption in Tnfsf11Δ/Δ
mice. j, CT
analysis of incisor and molar teeth (arrows). Scale bars, d: 1 mm, e: 100 m (top,
middle), 1 mm (bottom). Error bars, mean ± s.e.m.; *P< 0.05; **P< 0.01; ***P< 0.005;
NS, not significant (b, f, g and h).
Supplementary figure 7
Analysis of T cell-specific Tnfsf11-deficient mice. a, Successful Cre recombination in
genomic DNA of T cells in T cell-specific Tnfsf11-deficient mice (Tnfsf11flox/Δ
; Lck-Cre).
b, Expression of RANKL on the surface of T cells stimulated with PMA/inomycin or
anti-CD3/CD28 antibodies. c, CT analysis of the femurs of Tnfsf11flox/flox
, Tnfsf11flox/Δ
and Tnfsf11flox/Δ
; Lck-Cre littermates at 8 weeks of age (male, n = 3-6). Axial views of
the metaphyseal region. Scale bar, c: 1 mm. Error bars, mean ± s.e.m.; NS, not
significant (c).
Supplementary figure 8
Analysis of osteocyte-specific Tnfsf11-deficient mice. a, Genotyping PCR of genomic
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DNA of osteoblasts and osteocytes using primer pairs (shown in Supplementary Fig. 5b,
d) in Tnfsf11+/+
and Tnfsf11flox/Δ
mice with Dmp1-Cre and CAG-CAT-EGFP transgenes.
Using fluorescence-activated cell sorting, EGFP-positive osteocytes and EGFP-negative
osteoblasts were isolated from neonatal calvarial bones of Tnfsf11+/+
and Tnfsf11flox/Δ
;
Dmp1-Cre; CAG-CAT-EGFP mice. b, Gene expression in osteoblasts and osteocytes
derived from Tnfsf11flox/Δ
mice with Dmp1-Cre and CAG-CAT-EGFP transgenes
(quantitative RT-PCR analysis). c, Immunohistochemical analysis of RANKL
expression in Tnfsf11flox/Δ
; Dmp1-Cre mice. B: bone, BM: bone marrow. d, CT
analysis of incisor and molar teeth. e, Body weight in Tnfsf11flox/flox
, heterozygous
mutant (Tnfsf11flox/Δ
) and Tnfsf11flox/Δ
; Dmp1-Cre littermates during growth (male, n =
7-9). Scale bar, c: 40 m. Error bars, mean ± s.e.m.; *P< 0.05; **P< 0.01; NS, not
significant (b and e).
Supplementary figure 9
Decreased bone formation in osteocyte-specific Tnfsf11-deficient mice. a,
Osteoblastic parameters in adult osteocyte-specific Tnfsf11-deficient mice
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(12-week-old). b, Osteoblastic parameters and calcein double-labeling in young
osteocyte-specific Tnfsf11-deficient mice (4-week-old). c, Osteoclast surface per bone
surface in young osteoctyte-specific Tnfsf11 deficient mice (4-week-old). Error bars,
mean ± s.e.m.; *P< 0.05; **P< 0.01; ***P< 0.005 (a, b and c).
Supplementary figure 10
Normal expression of sclerostin in osteocyte-specific Tnfsf11-deficient mice. a,
Immunohistochemical analysis of sclerostin (Sost) expression in the femur. Note that
Sost is expressed exclusively in the bone-embedded osteocytes, and not in the
osteoblasts on the bone surface. B: bone, BM: bone marrow. b, Quantitative RT-PCR
analysis of Sost mRNA in isolated osteocytes. Scale bar, a: 40 m. Error bars, mean ±
s.e.m.; NS, not significant (b).
Supplementary figure 11
Growth and skeletogenesis in wild-type, global Tnfsf11-deficient and
osteocyte-specific Tnfsf11-deficient newborn mice. a, CT analysis of control
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(Tnfsf11+/+
and Tnfsf11flox/flox
), global Tnfsf11-deficient (Tnfsf11Δ/Δ
) and Tnfsf11flox/Δ
;
Dmp1-Cre newborn mice (postnatal day 1 [P1], male, n = 6-7). Upper: whole skeleton,
lower: axial view of the metaphyseal femur. b, c, Body weight (b) and alizarin
red/alcian blue staining (c) of control (Tnfsf11+/+
and Tnfsf11flox/flox
), global
Tnfsf11-deficient (Tnfsf11Δ/Δ
) and Tnfsf11flox/Δ
; Dmp1-Cre newborn mice (P1, male, n =
5-9). Scale bars, a: 5 mm (upper), 250 m (lower). Error bars, mean ± s.e.m.; NS, not
significant (b).
Supplementary figure 12
The mRNA expression of stress-response genes in an osteocyte-like cell line
MLO-Y4 cells. GeneChip analysis of stress-response genes in three-dimensional
gel-embedded MLO-Y4 cells after mechanical stimulation. Nos: nitric oxide synthase,
Ptgs2: prostaglandin-endoperoxide synthase 2, Vegfa: vascular endothelial growth factor
A. Note the induction of Tnfsf11 mRNA during in vitro mechanical stimulation.
Supplementary figure 13
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Crucial role of membrane-bound RANKL for osteoclastogenesis in the coculture
with osteocytes. a, Osteoclast formation in the culture of osteoclast precursor cells
separated from supporting cells using a membrane filter. b, Effect of osteoblast or
osteocyte conditioned media on osteoclast precursor cells without exogenous RANKL
and M-CSF. c, The expression level of soluble RANKL and OPG in osteocytes (ELISA
analysis). Error bars, mean ± s.e.m.; ***P< 0.005 (c).
Nature Medicine doi:10.1038/nm.2452
Osteocyte-rich fractionOsteoblast-rich fraction
0
2
4
6
8
10
0
2
4
6
8
10
0
0.2
0.4
0.6
0.8
1
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0
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4
6
8
10 R
ela
tive m
RN
A e
xpre
ssio
n
Sost Dmp1 Kera Tnfsf11 Tnfrsf11b Supplementary figure 1
Supplementary figure 2
Supplementary figure 3
**
*
**
0
10
20
30
40
0
2.5
5
7.5
10
0
5
10
15
20
Rela
tive m
RN
A e
xpre
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n Npy Reln Fmod
Osteocyte OsteoblastMEF
Skin fibroblast
0
2.5
5
7.5
10
MF
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tio
of R
AN
KL
MEF
MF
I ra
tio
of R
AN
KL
0
2.5
5
7.5
10
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Supplementary figure 4
Central marrow
Contr
ol Ig
GA
nti-R
AN
KL
B
BMV
BM
a
cb
ed
Num
ber
of T
RA
P+
MN
Cs (
cm
-2)
0
100
200
300
400
500
Osteoblast OsteocyteBMSC
***
***
Tnfrsf11b
0
100
200
300
400
500
0
20
40
60
80
100
0
50
100
150
200
250
0
10
20
30
40Dmp1 Kera Tnfsf11
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Rela
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RN
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xpre
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n
Osteocyte OsteoblastBMSC
***** *
**
Nature Medicine doi:10.1038/nm.2452
a
c
TNF-like domainN C1
TM316
E1 E2 E3 E4 E5
139
N CTM
E1 E2
Cre recombination
Wt :198bp
(P1/P2)
d
Δ : 303bp
(P1/P3)
Wt :198bp
(P1/P2)
flox : 328bp
(P1/P2)
Wt : 2571bp
Δ : 8384bp
flox : 10330bp
Supplementary figure 5
b
XhoI AhdI
AhdI/NheIAhdI/EcoRV
PmlI/EcoRV
AhdI/NheI
AhdI/SacII
frt
Tnfsf11 wild-type allele
Targeting vector
Long arm;
6854 bp
Middle arm;
1946 bp
Short arm;
816 bp
AhdI PmlI
XhoI/SacII
P1
loxP
NeoDT-A
NeoTnfsf11floxNeo allele
Tnfsf11Δ allele
Exon3-4
P2 P3
P1 P2
P3P1
P3
Tnfsf11flox allele
Cre recombination
Flp recombination
NcoI NcoI
PS2 PS1
Nature Medicine doi:10.1038/nm.2452
a b
Tnfsf11+/+
Tnfsf11Δ/Δ
c
0
5
10
15
20
25
30
35
Tnfsf11Δ/ΔTnfsf11+/+
Tota
l body
weig
ht
(g)
Tnfsf11+/+
Tnfsf11Δ/Δ
Tnfsf11+/Δ
2 4 8 12 (W)
***
NS
***
NS
***
NS
***NS
Supplementary figure 6
edTnfsf11+/+ Tnfsf11Δ/Δ
von K
ossa T
olu
idin
e b
lue T
RA
P
Tnfsf11Δ/ΔTnfsf11+/+
Nature Medicine doi:10.1038/nm.2452
i
Supplementary figure 6
j
Tnfsf11Δ/Δ
Tnfsf11+/+
Tnfsf11Δ/Δ
Tnfsf11+/+
Tnfsf11+/+ Tnfsf11Δ/Δ Tnfsf11Δ/+
f
Bone v
olu
me p
er
tissue
volu
me (
%)
Marr
ow
space s
tar
volu
me
(10
-2 m
m3)
0
20
40
60
80
100
120
140
160
Tra
becula
r separa
tion (mm
)
***
**
***
***
***
hg
Sple
en w
eig
ht
per
body
weig
ht (%
)
Num
ber
of T
RA
P+
MN
Cs
(cm
-2)
0
10
20
30
40
50
60
70
0
0.2
0.4
0.6
0.8
1
1.2
***
NS
NS
0
20
40
60
80
100
0
1
2
3
4
5
6
7
Oste
ocla
st n
um
ber
per
bone s
urf
ace (
mm
-1)
Oste
ocla
st surf
ace p
er
bone s
urf
ace (
%)
ND0
5
10
15
ND0
2
4
6
8
10
0
3
6
9
12
15
NDEro
ded s
urf
ace p
er
bone
surf
ace (
%)*
Nature Medicine doi:10.1038/nm.2452
b
a cflox Δ Cre
Tnfsf11flox/flox
Tnfsf11flox/Δ
Lck-Cre
Tnfsf11flox/flox Tnfsf11flox/Δ Lck-CreTnfsf11flox/Δ
0
20
40
60
80
100
120
140
160
0
20
40
60
80
100
0
20
40
60
80
100
Bone v
olu
me p
er
tissue
volu
me (
%)
Tra
becula
r separa
tion (mm
)
Tra
becula
r th
ickness (mm
)
NS
NS
NS
Tnfsf11flox/flox
Tnfsf11flox/Δ
Lck-CreTnfsf11flox/Δ
Tnfsf11flox/floxTnfsf11flox/Δ
Lck-Cre
RANKL
Control
PMA
ionomycin
aCD3
aCD28
Perc
enta
ge o
f m
axim
um
Supplementary figure 7
Nature Medicine doi:10.1038/nm.2452
a
To
tal b
od
y w
eig
ht
(g
)
2 4 8 12 (W)
0
5
10
15
20
25
30
35
e
NS NS
NS NS
NS NS
NS NS
Supplementary figure 8
c
Tnfsf11flox/flox
Tnfsf11flox/Δ
Dmp1-Cre
+ Δ flox Δ + Δ flox Δ
Tnfsf11+/+ Tnfsf11flox/Δ Tnfsf11+/+ Tnfsf11flox/Δ
Osteoblast Osteocyte
Dmp1-Cre CAG-CAT-EGFP
dTnfsf11flox/flox Tnfsf11flox/Δ Dmp1-Cre
Contr
ol Ig
GA
nti-R
AN
KL
Trabecular bone
B
BM
B
BM
b
Tnfsf11flox/Δ Dmp1-CreOsteocyte
Osteoblast
Tnfsf11flox/Δ Dmp1-Cre
Tnfsf11flox/Δ
Tnfsf11flox/flox
0
0.25
0.5
0.75
1
1.25
0
10
20
30
40
50
Rela
tive m
RN
A e
xpre
ssio
n Dmp1 Kera Tnfsf11
**
**
0
0.25
0.5
0.75
1
1.25
*
Nature Medicine doi:10.1038/nm.2452
Supplementary figure 9
Tnfsf11flox/flox Tnfsf11flox/Δ Dmp1-Cre
Oste
ocla
st surf
ace p
er
bone s
urf
ace (
%)
Oste
obla
st surf
ace p
er
bone s
urf
ace (
%)
Bone form
ation r
ate
per
bone
surf
ace (
mm
3m
m-2
year-
1)
0
100
200
300
400
500
600
700
800
0
10
20
30
40
50
0
5
10
15
20
Tnfsf11flox/flox
Tnfsf11flox/Δ Dmp1-Cre
b
a
Bone form
ation r
ate
per
bone
surf
ace (
mm
3m
m-2
year-
1)
c
0
100
200
300
400
500
600
700
800
***
Oste
obla
st surf
ace p
er
bone s
urf
ace (
%)
***
0
10
20
30
40
50
12-week-old
4-week-old
**
**
Nature Medicine doi:10.1038/nm.2452
Supplementary figure 10
Tnfsf11flox/flox Tnfsf11flox/Δ Dmp1-CreC
ontr
ol Ig
GA
nti-S
ost
Trabecular bone Cortical bone
Metaphysis
B
BM
B
BM
Trabecular bone Cortical bone
Metaphysis
B
BM
B
BM
b
a
0
5
10
15
20
Rela
tive e
xpre
ssio
n o
fS
ost
Tnfsf11+/+ Tnfsf11flox/Δ
Dmp1-Cre CAG-CAT-EGFP
NS
Osteocyte
Osteoblast
Nature Medicine doi:10.1038/nm.2452
b
c
Tota
l body
weig
th (
g)
Tnfsf11+/+ Tnfsf11Δ/Δ Tnfsf11flox/floxTnfsf11flox/Δ
Dmp1-Cre
0
0.5
1
1.5
2
2.5
NS NS NS
Tnfsf11+/+
Tnfsf11flox/Δ Dmp1-Cre
Tnfsf11Δ/Δ
Tnfsf11flox/flox
Supplementary figure 11
aTnfsf11+/+ Tnfsf11Δ/Δ Tnfsf11flox/flox
Tnfsf11flox/Δ
Dmp1-Cre
Nature Medicine doi:10.1038/nm.2452
mR
NA
exp
ressio
n
(ave
rag
e d
iffe
ren
ce
)
Supplementary figure 12
0
500
1000
1500
2000
0
10
20
30
40
50
0
5
10
15
20Nos1 Nos2 Ptgs2 Vegfa Tnfsf11
0 6 12 24 (h)
0
500
1000
1500
2000
0
500
1000
1500
2000
Nature Medicine doi:10.1038/nm.2452
Supplementary figure 13
Osteocyte
or Osteoblast
Osteoclast
precursor cell
a
c
Culture using conditioned medium
Separate coculture
Osteocyte
or Osteoblast
Osteoclast
precursor cell Num
ber
of T
RA
P+
MN
Cs
(cm
-2)
0
100
200
300
Num
ber
of T
RA
P+
MN
Cs
(cm
-2)
0
100
200
300
0
500
1000
1500
0
2000
4000
6000
8000OPG
Concentr
ation (
pg m
l-1)
Concentr
ation (
pg m
l-1)
sRANKL
Control 1, 25(OH)2D3/PGE2
***
***
b
Nature Medicine doi:10.1038/nm.2452
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