supplementary materials for...in methyl methacrylate, and cut on a microtec rotation microtome (cvt...
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www.sciencetranslationalmedicine.org/cgi/content/full/10/466/eaau7137/DC1
Supplementary Materials for
Wnt1 is an Lrp5-independent bone-anabolic Wnt ligand
Julia Luther, Timur Alexander Yorgan, Tim Rolvien, Lorenz Ulsamer, Till Koehne, Nannan Liao, Daniela Keller,
Nele Vollersen, Stefan Teufel, Mona Neven, Stephanie Peters, Michaela Schweizer, Andreas Trumpp, Sebastian Rosigkeit, Ernesto Bockamp, Stefan Mundlos, Uwe Kornak, Ralf Oheim, Michael Amling*, Thorsten Schinke*, Jean-Pierre David*
*Corresponding author. Email: [email protected] (J.-P.D.); [email protected] (T.S.); [email protected] (M.A.)
Published 7 November 2018, Sci. Transl. Med. 10, eaau7137 (2018)
DOI: 10.1126/scitranslmed.aau7137
The PDF file includes:
Materials and Methods Fig. S1. Age-related decreased bone mass in the radius of patients with WNT1 mutation. Fig. S2. Inactivation of Wnt1 in osteoclasts does not affect bone remodeling. Fig. S3. Histology of the fractures in Runx2-cre;Wnt1fl/fl mice. Fig. S4. Wnt1 is a general bone-anabolic molecule. Fig. S5. Wnt1 induces bone formation in adult mice. Fig. S6. Wnt1 induction protects aging female from bone loss. Fig. S7. Wnt1 expression induces a rapidly increased bone mass in adult mice. Fig. S8. Wnt1 is not directly stimulating osteoblast differentiation. Fig. S9. Wnt1 is not up-regulated during osteoblast differentiation. References (50–58)
Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/10/466/eaau7137/DC1)
Table S1. Individual subject-level data (Excel file).
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Materials and Methods
Patients
In a subset of patients with early-onset osteoporosis from our osteologic outpatient clinic,
mutational analysis was carried out by enrichment using a custom designed SureSelect XT
gene panel (Agilent) containing all coding exons of 373 genes in which mutations have been
associated with changes in bone mass, skeletal dysplasias, dysostoses, or connective tissue
diseases [skeletal disorder associated genome (sDAG)]. The pathogenicity of the prioritized
variants was judged using MutationTaster (http://www.mutationtaster.org/).
To analyse the bone status, areal bone mineral density (aBMD) was evaluated for lumbar
spine (L1 to L4) and total hip using Dual Energy X-Ray absorptiometry (DXA, Lunar iDXA,
GE Healthcare), and converted to Z-scores and T-scores based on equipment-specific
reference values. Bone microstructure was determined using high-resolution peripheral
quantitative computed tomography (HR-pQCT; XtremeCT, Scanco Medical) at the
nondominant distal radius and tibia in a standardized procedure as described (50). Serum bone
turnover analyses including bone formation markers (osteocalcin and bone alkaline
phosphatase (BAP)) and bone resorption (desoxypyridinoline (DPD) cross-links per creatinine
in the urine) were assessed.
In patient no. 3, a diagnostic transiliac crest biopsy was obtained in the course of the surgery
of a femoral fracture. The specimen was fixed in 3.7% formaldehyde, dehydrated, embedded
in methyl methacrylate, and cut on a Microtec rotation microtome (CVT 4060E, Micro Tec).
5 µm sections were stained by Masson-Goldner. Histomorphometric analysis was performed
as described (51) according to the ASBMR standards (52).
Patient no. 1 had initially received an anti-resorptive denosumab treatment 60 mg
subcutaneous every 6 months (Prolia, Amgen), which is a human monoclonal RANKL
antibody. The patient was then treated with a daily subcutaneous administration of teriparatide
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20 g (Forsteo, Eli Lilly) for 24 months, and eventually again by denosumab according to the
DATA (53).
Informed consent was obtained from all patients for the presented data. The study was
approved by the Ethics Committee of the Hamburg Chamber of Physicians (agreement
number PV 5364).
Mouse lines
Wnt1 floxed mice (B6.B6--Wnt1tm2Hhtg
/Uke) were generated from ES-cell clones available at
EUCOMM (Wnt1tm1a(EUCOMM)Wtsi) by blastocyst injection. The modified Wnt1 allele
was transformed into a conditional floxed allele by mating with the B6;SJL-
Tg(ACTFLPe)9205Dym/J universal Flp-deleter line (Jackson Laboratory). In the resulting
Wnt1 allele the exons 2 up to 4 were flanked by loxP sites, allowing for a conditional, Cre-
mediated inactivation of the gene. Genotyping was performed using primers (5’-
CTGCCCAGCTGGGTTTCTACTACG-3’ and 5’-ACCAGCTGCAGACTCTTGGAATCCG-
3’) detecting a 696-bp wildtype or 800-bp floxed Wnt1 allele. Recombination events were
detected by using the primers 5’-AGTGAGCTAGTACGGGGTCC-3’ and 5’-
AGGACCATGAACTGATGGCG-3’ resulting in 368-bp product.
Lyz2-Cre (B6.129P2-Lyz2tm1(cre)Ifo
/J) and Runx2-Cre (Tg(Runx2-icre)1Jtuc) transgenic mice
and their genotyping have been described previously (54, 55). The osteoblast specific Wnt1Tg
mice were generated by crossing Col1a-rtTA mice with ptet-Wnt-1 mice (56). Unless
indicated, mice were kept under a doxycycline-containing diet (ssniff). Lrp5-deficient mice
were obtained from The Jackson Laboratory (B6.129P2-Lrp5tm1Dgen
/J).
All mice were kept in a specific pathogen-free environment with a 12-h light/dark cycle, 45-
65% relative humidity and 20-24 °C ambient temperature in open or individually ventilated
cages with wood shavings bedding and nesting material in groups not surpassing 6 animals.
The mice had access to tap water and standard rodent chow (1328P, Altromin Spezialfutter
https://www.jax.org/strain/005823
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GmbH & Co. KG) or doxycycline-containing diet ad libitum. After initiation of calcein
labeling, the welfare of the mice was assessed daily based on overall appearance and body
weight. All animal experiments were approved by the animal facility of the University
Medical Center Hamburg-Eppendorf and by the “Behörde für Soziales, Familie, Gesundheit
und Verbraucherschutz” (G14/035, Org529 and Org869) in accordance with the local
implementation of EU Directive 2010/63/EU for animal experiments.
Micro-computer tomography and X-ray analysis
All μCT scanning, 3D reconstruction and analysis was performed using a μCT 40 desktop
cone-beam microCT (Scanco Medical, CHE) with 10 μm voxel size (57). Structural
parameters of the trabecular bone were evaluated in the distal femoral metaphysis in a volume
situated 2000 μm to 500 μm proximal of the distal growth plate. Cortical parameters were
evaluated in a volume with a length of 1000 μm situated in the middle of the femoral
diaphysis. Contact X-ray was performed on fixed skeletons using a Faxitron X-ray cabinet at
35 kV for 2 seconds.
Cell culture
Primary osteoblasts were isolated by sequential digestion of calvariae of newborn mice with
0.5 mg/ml collagenase and 1 mg/ml dispase, differentiation was induced by addition of 50
µg/ml ascorbate and 10 mM ß-glycerophosphate to the medium (α-MEM supplemented with
10% FCS and 1% penicillin/streptomycin, ± 1 µg/ml doxycycline) (58).
Fixed cells (90% EtOH, 10 minutes) were stained with alizarin red (40 mM, pH 4.2, 10
minutes). For quantification, cells were incubated with 10% acetic acid for 30 minutes,
incubated at 85°C for 5 minutes and centrifuged at 13.000 rpm for 10 minutes. 10%
ammonium hydroxide solution was added to the supernatant (ratio 1:8), pH was adjusted with
acetic acid to 4.1-4.5 and OD was measured at 405 nm.
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Biomechanical Testing
Destructive three point bending tests were performed on explanted femora using a BZ2.5
materials testing machine and the testXpert software (both Zwick Roell). The femora were
placed centrally on two support bars placed 7mm apart with the posterior surface facing
down. A tip was lowered medially between the support bars at a constant speed of 2mm/min
onto the anterior surface of the femora and the force at the point of fracture was recorded.
Histology and hismorphometry
Skeletons were fixed in 3.5% Formalin in PBS for 2 days and vertebral bodies L2-5 and the
right tibia were dehydrated and embedded in methylmethacrylate. 4 µm sections, cut with a
Microtec rotation microtome (Techno-Med), were stained by von Kossa/van Gieson, toluidine
blue and Masson's Trichrome staining according to standard protocols. Static
histomorphometry was achieved by analyzing von Kossa stained sections using Bioquant
software (Bioquant Image Analysis Corporation), dynamic histomorphometry was performed
on toluidine blue stained sections as well as on unstained 12 µm sections of the vertebral
bodies of calcein labeled mice (labeling 9 and 2 days before scarifying) using the
OsteoMeasure system (Osteometrics Inc.). Analyses were performed according to American
Society for Bone and Mineral Research standards (52).
Gene expression analysis, Western and immunostaining
RNA was isolated using Trifast Reagent (Peqlab) and reverse transcribed using a Verso
cDNA kit (Thermo Scientific) according to the manufacturer's instructions. Quantitative real-
time PCR analyses were performed with a StepOnePlus system (Thermo Scientific) using
predesigned TaqMan gene expression assays (Thermo Scientific) or by SYBR Green.
Samples were normalized to GapDH expression level.
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Western blotting: protein was extracted by using RIPA buffer (150 mM NaCl, 2 mM EDTA,
10 mM sodium phosphate, 1% NP-40, 1% sodium desoxycholat, 0.1% SDS) at day 14 of
differentiation by incubation for 15 minutes and centrifugation at 16000rpm for 10 minutes at
4°C. Protein concentration was determined by Bradford assay (Bio-rad) and 90 µg of protein
were separated on a polyacrylamide gel and transferred to a nitrocellulose membrane.
Antibodies against α-Tubulin, non-P-ß-catenin, ß-catenin, P-S6, S6, Actin (all obtained from
Cell Signaling Technology) and Wnt1 (GeneTex), were used.
Tibiae from wt and Wnt1Tg mice were dissected and immersion fixed in 4% PFA in PBS, pH
7.2 overnight. Thereafter they were decalcified in 10% EDTA for two to three weeks. 60 µm
thick vibratom sections were prepared and washed in PBS several times. Fluorescence
immunohistochemistry was performed and non-specific binding sites were blocked for 30-60
minutes at RT (0.3% BSA, 10% horse serum, 0.3% Triton X-100 in PBS). The tissue was
subsequently incubated overnight at 4°C with the primary antibody (anti Wnt1 1:500) in
Carrier (0.2% BSA, 1% horse serum, 0.3% Triton X-100 in PBS). After several washes in
PBS sections were incubated for 1 to 2 hours with fluorochrome-coupled secondary
antibodies in Carrier, washed in PBS and mounted in ProLong Gold (Invitrogen). Images
were taken with an Olympus F1000 Confocal Microscope.
Serum protein concentrations were measured by ELISA for Wnt1 (Cusabio) according to
manufacturer’s instructions.
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Fig. S1. Age-related decreased bone mass in the radius of patients with WNT1 mutation.
(A) Representative scan of the distal radius by HR-pQCT. (B) Bone structural parameters and
percent change compared to age-matched controls at the distal radius measured by HR-pQCT.
(C) Age-related changes in BV/TV and (D) Ct.Th at the distal radius measured by HR-pQCT.
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Fig. S2. Inactivation of Wnt1 in osteoclasts does not affect bone remodeling.
(A) Genotyping for recombination in various tissues of the Lyz2-Cre;Wnt1fl/fl
and in in vitro
generated osteoclasts, the localization of the primers is shown in Fig. 2. (B) µCT imaging of
longitudinal section of femora in 24-week-old females. (C) Quantification of the bone
volume, trabecular thickness and cortical thickness. (D) Von Kossa staining of vertebrae and
(E) histomorphometric quantification of the bone volume, trabecular thickness and trabecular
numbers. N≥6 for all experiments. Data are the mean ± s.e.m. (unpaired t-test).
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Fig. S3. Histology of the fractures in Runx2-cre;Wnt1fl/fl
mice.
(A) Genotyping for recombination in various tissues of the Dmp1-Cre;Wnt1wt/fl
compared to
the Wnt1wt/fl
control. (B) Von Kossa staining of undecalcified section of the femora, and close
up on the indicated region, toluidine blue and Masson-Goldner staining of the same area, (C)
similar stainings were performed on fractured rib. (c: indicate the presence of a callus). (D)
ELISA quantification of the circulating levels of Wnt1 in 24-week-old Runx2-Cre negative or
positive Wnt1fl/fl
males (M) or females (F), n≥3. Data are the mean ± s.e.m. (unpaired t-test).
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Fig. S4. Wnt1 is a general bone-anabolic molecule.
(A) Histomorphometric analysis of the trabecular bone parameters in control and Wnt1Tg
female after releasing the doxycycline for 3 and 9 weeks from the age of 3 weeks, n≥3. (B)
CT scan of longitudinal (upper panels) and transversal sections at midshaft (lower panels) of
femora of 6-week-old control and Wnt1 transgenic mice 3 weeks after removing the
doxycycline. (C) CT quantification of the trabecular bone in the femora, n≥4. (D)CT
imaging of the skull (upper panels) and sagittal section of the calvariae (lower panels) of 12-
week-old control and induced Wnt1 transgenic mice 9 weeks after removing the doxycycline,
the arrows indicate the closure of cranial sutures in the transgenic. (E) Von Kossa staining of
the calvariae (upper panels) and close up pictures of a cranial suture (white arrows). Data are
the mean ± s.e.m. ****P
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Fig. S5. Wnt1 induces bone formation in adult mice.
(A) Von Kossa staining of sections of vertebra of 15-week-old mice of the indicated genotype
kept for 3 weeks under doxycycline-free food. (B) Histomorphometric analysis of the bone
parameters (BV/TV, Tb.Th and Tb.N). (C) CT imaging of the femora, longitudinal section,
of the trabecular bone and transversal section at midshaft are shown. (D) CT quantification
of the endochondral BV/TV, of the trabecular thickness and number. (E) Quantification of the
cortical thickness. (F) Force necessary to apply to fracture femora as determined by three
point bending test. (G) Histomorphometric quantification of osteoclast number and osteoclast
surface and (H) of osteoblast number and osteoblast surface. (I) Quantification of the bone
formation rate. N≥3 (B), n≥4 (D-I). Data are the mean ± s.e.m. ****P
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Fig. S6. Wnt1 induction protects aging female from bone loss.
(A) Von Kossa staining of sections of vertebrae and tibiae of 30-week-old Wnt1 transgenic
females after 4 weeks induction of the transgene compared to control littermates. (B)
Histomorphometric analysis of the bone parameters (BV/TV, Tb.Th and Tb.N) in the
vertebrae, n≥5. Data are the mean ± s.e.m. ****P
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Fig. S7. Wnt1 expression induces a rapidly increased bone mass in adult mice.
(A) CT imaging of tibiae of 30-week-old males 1 week after induction of the transgene
compared to control littermates. (B) Quantification of the bone parameters (BV/TV, Tb.N and
Tb.Th) in the femora, n≥4. (C) and (D) Histomorphometric quantification of the osteoclast
and osteoblast parameters, n=5. (E) Q-PCR analysis of the expression of component of the
Hippo pathway in the calvaria of of Wnt1Tg mice 2 and 7 days after removing the
Doxycycline, n≥3. Data are the mean ± s.e.m. ***P
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Fig. S8. Wnt1 is not directly stimulating osteoblast differentiation.
(A) Q-PCR analysis of the expression of Wnt1 in primary osteoblast culture isolated from
control or Wnt1Tg new born mice at day 14 of differentiation in the absence of doxycycline.
(B) ELISA quantification of Wnt1 in the supernatant of the culture, n≥3. (C) Western blot for
the expression of Wnt1 in primary osteoblast culture from control (C) and Wnt1Tg (Tg) mice
grown in the presence or absence of doxycycline, -Tubulin is used as a loading control. (D)
Representative pictures of alizarin-red stained wild type (wt) and Wnt1Tg primary osteoblast
culture grown for 14 days in the presence of mineralizing media with or without doxycycline.
(E) Quantification of the incorporated alizarin-red. (F) Q-PCR analysis of the expression of
markers of osteoblast differentiation and of typical Wnt/-Catenin target genes reported to the
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values of the wild type control (red line). (G) Western blot analysis of the expression of active
-Catenin (non-P--Cat), of the total -Catenin, the phosphorylated S6 peptide (P-S6) and the
total S6 peptide in the primary culture, Actin is used as a loading control. For cell culture
differentiations 3 independent experiments were performed. Data are the mean ± s.e.m.
*P
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Fig. S9. Wnt1 is not up-regulated during osteoblast differentiation.
Q-PCR analysis of the kinetic of expression of Wnt1 in primary osteoblast culture isolated
from wild-type new born compared with the kinetic of expression of early markers of
differentiation (Col1a1, Ibsp and Alpl), late marker of differentiation (Bglap) and osteocyte
markers (Dmp1 and Phex), n=3. Data are the mean ± s.e.m. ****P