Evidence for osteocyte regulation of bone homeostasis ... · reverse primer (PS2: the locus-specific primer, 5’-AGGTGATTTCTCACCGTC-3’) binds to a unique region outside the short

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.

([email protected]).

Nature Medicine doi:10.1038/nm.2452

mailto:[email protected]

2

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


3

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


11

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


13

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).


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.


RANKL expression in mouse embryonic fibroblasts and skin fibroblasts. Cell

surface expression of RANKL in mouse embryonic fibroblasts (MEFs) and skin


15

fibroblasts was quantitated by FACS analyses. MFI, mean fluorescence intensity.


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).



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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.


17


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).


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).


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).


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).


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).


Growth and skeletogenesis in wild-type, global Tnfsf11-deficient and

osteocyte-specific Tnfsf11-deficient newborn mice. a, CT analysis of control


21

(Tnfsf11+/+

and Tnfsf11flox/flox

), global Tnfsf11-deficient (Tnfsf11Δ/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


; 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).


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.



22

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).


Osteocyte-rich fractionOsteoblast-rich fraction

0

2

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8

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**

*

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0

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200

300

400

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***

***

Tnfrsf11b

0

100

200

300

400

500

0

20

40

60

80

100

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150

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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


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


a b

Tnfsf11+/+

Tnfsf11Δ/Δ

c

0

5

10

15

20

25

30

35

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(g)

Tnfsf11+/+

Tnfsf11Δ/Δ

Tnfsf11+/Δ

2 4 8 12 (W)

***

NS

***

NS

***

NS

***NS


edTnfsf11+/+ Tnfsf11Δ/Δ

von K

ossa T

olu

idin

e b

lue T

RA

P

Tnfsf11Δ/ΔTnfsf11+/+


i


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 (

%)*


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



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


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

*



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


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

**

**



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


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+/+


Tnfsf11Δ/Δ

Tnfsf11flox/flox


aTnfsf11+/+ Tnfsf11Δ/Δ Tnfsf11flox/flox

Tnfsf11flox/Δ

Dmp1-Cre


mR

NA

exp

ressio

n

(ave

rag

e d

iffe

ren

ce

)


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



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


Documents

Evidence for osteocyte regulation of bone homeostasis ... · reverse primer (PS2: the locus-specific primer, 5’-AGGTGATTTCTCACCGTC-3’) binds to a unique region outside the short