pdgf receptor signaling in osteoblast lineage cells

University of Southern Denmark

PDGF Receptor Signaling in Osteoblast Lineage Cells Controls Bone Resorption ThroughUpregulation of Csf1 Expression

Brun, Julia; Andreasen, Christina Møller; Ejersted, Charlotte; Andersen, Thomas Levin;Caverzasio, Joseph; Thouverey, Cyril

Published in:Journal of Bone and Mineral Research

DOI:10.1002/jbmr.4150

Publication date:2020

Document version:Accepted manuscript

Citation for pulished version (APA):Brun, J., Andreasen, C. M., Ejersted, C., Andersen, T. L., Caverzasio, J., & Thouverey, C. (2020). PDGFReceptor Signaling in Osteoblast Lineage Cells Controls Bone Resorption Through Upregulation of Csf1Expression. Journal of Bone and Mineral Research, 35(12), 2458-2469. https://doi.org/10.1002/jbmr.4150

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https://doi.org/10.1002/jbmr.4150

https://doi.org/10.1002/jbmr.4150

https://portal.findresearcher.sdu.dk/en/publications/b4c53db4-f48c-4008-af9d-9844b0e32da1

Andreasen Christina (Orcid ID: 0000-0002-2624-5677) Andersen Thomas (Orcid ID: 0000-0002-6981-7276) Thouverey Cyril (Orcid ID: 0000-0002-2741-1825) PDGF receptor signaling in osteoblast lineage cells controls bone resorption through up-

regulation of Csf1 expression

Julia Brun1, Christina Møller Andreasen2, Charlotte Ejersted3, Thomas Levin Andersen2,

Joseph Caverzasio1, Cyril Thouverey1

1 Service of Bone Diseases, Department of Medicine, University Hospital of Geneva, 1205

Geneva, Switzerland

2 Clinical Cell Biology, Pathology Research Unit, Odense University Hospital, DK-5000

Odense C, Denmark; Dept. of Molecular Medicine, University of Southern Denmark, DK-5000

Odense C, Denmark; Dept. of Clinical Research, University of Southern Denmark, DK-5000

Odense C, Denmark.

3 Dept. of Endocrinology, Odense University Hospital, DK-5000 Odense C, Denmark

Running title: PDGFRs in OB lineage controls bone resorption

Corresponding author: Cyril Thouverey - Service of Bone Diseases, Department of Medicine,

University Hospital of Geneva, 64 Avenue de la Roseraie, 1205 Geneva, Switzerland - E-mail

address: [email protected] - Tel: +41 22 379 46 76

Disclosures: The authors state that they have no conflicts of interest. This work was supported

by the Swiss National Science Foundation (310030-166341), by the Fondation pour la

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2

Recherche sur l’Ostéoporose et les Maladies Osseuses, by the Novartis Foundation for medical-

biological research (Project number 16C212 - Basel, Switzerland), by the VELUX

FOUNDATION (VELUX25723), and by the Region of Southern Denmark Research Fund

(18/17871).

Abstract

The physiological functions of platelet-derived growth factor receptors (PDGFRs) α and β in

osteoblast biology and bone metabolism remain to be established. Here, we show that PDGFRA

and PDGFRB genes are expressed by osteoblast-lineage canopy and reversal cells in close

proximity to PDGFB-expressing osteoclasts within human trabecular bone remodeling units.

We also report that, while removal of only one of the two PDGFRs in Osterix-positive cells

does not affect bone phenotype, suppression of both PDGFRs in those osteoblast lineage cells

increases trabecular bone volume in male mice as well as in female gonad-intact and

ovariectomized mice. Furthermore, osteoblast lineage-specific suppression of PDGFRs reduces

Csf1 expression, bone marrow level of M-CSF, number of osteoclasts and therefore, bone

resorption, but does not change bone formation. Finally, abrogation of PDGFR signaling in

osteoblasts blocks PDGF-induced ERK1/2-mediated Csf1 expression and M-CSF secretion in

osteoblast cultures, and calcitriol-mediated osteoclastogenesis in co-cultures. In conclusion, our

results indicate that PDGFR signaling in osteoblast lineage cells controls bone resorption

through ERK1/2-mediated Csf1 expression.

Key words: osteoblast lineage cells, PDGF receptors, ERK1/2, bone resorption, M-CSF


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Introduction

Bone development, repair and maintenance are regulated by a myriad of locally secreted

factors. Among them, platelet-derived growth factors (PDGFs) have emerged as important

regulators of multiple aspects of bone physiology (1). PDGF ligands consist in four distinct

polypeptide chains (PDGF-A, -B, -C and –D) that assemble in homodimers (PDGF-AA, -BB,

-CC and -DD) or heterodimers (PDGF-AB) (2,3). PDGFs act via two receptor tyrosine kinases,

PDGFRα and PDGFRβ. Ligand binding promotes receptor dimerization, autophosphorylation

and initiation of signaling (2,3). Depending on distinct ligand affinities and patterns of receptor

expression, different receptor dimers can form. Indeed, PDGFRαα is activated by PDGF-AA, -

AB, -BB and -CC, PDGFRαβ is activated by PDGF-AB, -BB and -CC, and PDGFRββ is

activated by PDGF-BB and -DD (2,3). PDGFRs play essential roles in embryonic development

as well as in different types of fibrosis and malignancies since it regulates proliferation,

chemotaxis and survival of cells of mesenchymal origin (3). In general, both PDGFRs exert

overlapping and distinct functions (3).

Numerous cells such as mesenchymal stem cells, osteoblasts, pre-osteoclasts, osteoclasts and

endothelial cells can produce PDGFs in the bone microenvironment (1). PDGF-AA, -AB and -

BB are most abundant PDGF members in bone and are mainly produced by osteoclast-lineage

cells in physiological condition (4-6). PDGFRs are expressed by perivascular mesenchymal

stem cells (MSCs) in metaphysis, endosteum and periosteum, and by

osteoprogenitors/osteoblasts located on trabecular, endosteal and periosteal surfaces (7-10).

Mechanistically, osteoclast-lineage cells couple angiogenesis to bone formation by releasing

PDGF-BB (6,9). In turn, elevated peri-osseous PDGF-BB concentration releases perivascular


4

MSCs from vessel wall and enhances their proliferation and recruitment to bone surfaces, where

they are going to provide bone-forming osteoblasts (6,9,10).

PDGF-BB/PDGFRβ is a potent mitogenic and chemoattractant signal for skeletal MSCs and

committed osteoprogenitors in vitro (5,10,11). Accordingly, conditional suppression of

PDGFRβ in nestin-positive or leptin receptor-positive MSCs impairs periosteal and trabecular

bone formation due to defective self-renewal and migratory capacities (9). Conditional ablation

of PDGFRβ in osterix-positive cells does not affect bone formation in normal condition, but

decreases osteoprogenitor proliferation, trafficking and angiotropism during fracture healing

(10). On the other hand, PDGF-BB/PDGFRβ strongly inhibits osteoblast differentiation in vitro

(4,10-12). Although PDGFRα probably exerts redundant functions with PDGFRβ in bone

formation, it may play distinct roles since Pdgfra-deficient mice exhibit defective skeletal

patterning and maturation (13).

A growing body of evidence suggests that PDGF-BB/PDGFRβ also regulates

osteoclastogenesis and bone resorption. For instance, targeted Pdgfb overexpression in

endosteum and metaphyseal bone marrow stimulates bone resorption by elevating expressions

of Rankl (encoding receptor activator of nuclear factor κB ligand, RANKL) and Csf1 (encoding

macrophage colony-stimulating factor, M-CSF) (14). Moreover, PDGFRβ inhibition increases

expression of Opg (encoding osteoprotegerin, OPG, the RANKL decoy receptor) and reduces

that of Rankl by MSC and osteoblast cell lines in vitro (15). Finally, aberrant PDGF-

BB/PDGFRβ signaling due to absence of low-density lipoprotein receptor-related protein

(LRP)-1 in osteoblasts stimulates RANKL production and bone resorption (16).


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Osteoprogenitors and osteoblasts express PDGFRα and β. Although PDGF-BB/PDGFRβ

signaling stimulates their proliferation and migration, and inhibits their maturation in vitro, its

predominant in vivo function remains to be clarified. Suppression of PDGFRβ in Osterix-

positive cells does not affect osteoprogenitors or osteoblasts in physiological condition (10),

suggesting that PDGFRα plays overlapping functions with PDGFRβ. To investigate

physiological functions of PDGFRs in osteoblast biology and bone remodeling, we localized

expressions of PDGFRA and PDGFRB in human bone tissue by in situ hybridization, and we

analyzed mice in which PDGFRα, PDGFRβ or both receptors were deleted from osteoblast

lineage cells under the control of an inducible Osterix promoter.

Materials and Methods

Human bone specimens

3-mm needle biopsy specimens were obtained from the posterior iliac crest of four volunteers

without known musculoskeletal disease (two men, aged 27 and 41 years and two women, aged

33 and 49 years) using an 8G needle under local anesthesia. The biopsies were formalin-fixed,

decalcified and paraffin-embedded before serial sectioning. All volunteers gave their written

informed consent. The study was conducted according to the World Medical Association

Declaration of Helsinki and approved by the Danish National Committee on Biomedical

Research Ethics, journal no. S-20110112.

In situ hybridization


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3.5-mm-thick serial paraffin sections were in situ hybridized using an enhanced version of

RNAScope 2.5 high definition procedure (310035, ACD Bioscience) followed by tartrate-

resistant acid phosphatase (TRAP) immunostaining, as previously described (17). Sections

were rehydrated, deparaffinized, pretreated with pepsin digestion and heat treatment, and

hybridized overnight at 40°C with 20-ZZ-pair probes for human PDGFB mRNA (406701, ACD

Bioscience), PDGFRA mRNA (604481, ACD Bioscience) or PDGFRB mRNA (548991, ACD

Bioscience) diluted 1:1 in probe diluent (449819, ACD Bioscience) or only with probe diluent

(negative controls). The branch amplification was conducted according to the instructions

provided by the manufacturer, but enhanced with digoxigenin (DIG)-conjugated tyramide

(NEL748001KT, PerkinElmer) followed by alkaline phosphatase-conjugated sheep anti-DIG

FAB fragments (11093274910, Roche) and visualization with Liquid Permanent Red (Dako,

Denmark). The stained sections were then immunostained with mouse anti-TRAP antibodies

(clone 9C5, MABF96, Merck Millipore), which were labelled with horseradish peroxidase-

conjugated anti-mouse IgG polymers (BrightVision, Immunologic) and visualized with Deep

Space Black (Biocare Medical) and counterstained with Mayer’s hematoxylin.

Mice

Mice in which the expression of a tetracycline-Off-controllable GFP::Cre fusion protein is

transcriptionally regulated by an Osterix promoter (Osx-GFP::Cre, hereafter Osx-Cre) were on

a C57BL/6J genetic background and obtained from the Jackson Laboratory (#006361) (18).

Mice harboring floxed Pdgfra (#006492) (19) and floxed Pdgfrb (#010977) (20) were also

purchased from the Jackson Laboratory and bred to C57BL/6J mice for 3 and 6 generations,


7

respectively. Then, those mouse strains were used to generate Osx-Cre;Pdgfraf/f, Osx-

Cre;Pdgfrbf/f and Osx-Cre;Pdgfraf/f;Pdgfrbf/f (hereafter Pdgfra cKO, Pdgfrb cKO and Pdgfra

cKO;Pdgfrb cKO) mice. Osx-Cre mice were used as control animals. In a first experiment,

since Osx-Cre mice were shown to exhibit a slight growth delay by 1 month of age (21), Cre

expression and consequent Pdgfra or/and Pdgfrb inactivation was induced at 6 weeks of age by

stopping doxycycline treatment, and male mice were sacrificed at 18 weeks of age. In a second

experiment, 12-week-old Osx-Cre and Pdgfra cKO;Pdgfrb cKO female mice were randomly

subjected to sham-operation (Sham) or Ovx, and sacrificed 8 weeks after surgeries. Pdgfra and

Pdgfrb inactivations were induced one week prior to surgeries. Mice (3 to 6 animals per cage)

were maintained under standard non-barrier conditions, exposed to a 12-hour light/12-hour dark

cycle and had access to mouse diet RM3 containing 1.24 % calcium and 0.56 % available

phosphorus (SDS, Betchworth, UK) and water ad libitum. Experimental units were single

animals. Investigators were blinded during endpoint measurements. All performed experiments

were in compliance with the guiding principles of the Guide for the Care and Use of Laboratory

Animals (8th edition) and approved by the Ethical Committee of the University of Geneva

School of Medicine and the State of Geneva Veterinarian Office.

Bone phenotyping

Mice were sacrificed and their bones were excised for micro-computed tomography (µCT)

analyses. Trabecular bone microarchitecture of proximal tibiae and fifth lumbar vertebral

bodies (100 slices from the beginning of secondary spongiosa), and cortical bone geometry of


8

tibial midshafts (50 slices) were assessed using µCT (Viva-CT40, Scanco Medical,

Switzerland) employing a 12-μm isotropic voxel size.

To measure dynamic indices of bone formation, mice received subcutaneous injections of

calcein (10 mg/kg body weight; Sigma) at 9 and 2 days before euthanasia. Formalin-fixed

undecalcified femurs were embedded in methylmethacrylate (Merck). 8-µm transversal

sections of midshafts and 8-µm sagittal sections of distal femurs were cut and mounted

unstained for fluorescence visualization. Additional sagittal sections were stained with Goldner

trichrome for osteoblast counting or with tartrate-resistant acid phosphatase (TRAP) substrate

for osteoclast counting. Histomorphometric measurements were carried out using a Nikon

Eclipse microscope and the BioQuant software. The nomenclature for µCT and

histomorphometric analyses followed recommendations of published ASBMR guidelines

(22,23).

Immunohistochemistry

Femurs were fixed in 4% paraformaldehyde overnight, demineralized in 17% EDTA for 2

weeks, washed in demineralized water, and then incubated in 30% Sucrose for 48 hours at 4 °C

under agitation. Femurs were then frozen and embedded in NEG50 medium (Thermo Fischer

Scientific) and 10-µm frozen sections were cut using a Leica cryostat CM3050S (Leica

Biosystems). Cryosections were blocked with 3% bovine serum albumin at room temperature

for 30 minutes and then incubated at 4 °C overnight with primary antibodies: anti-PDGFRα

(#3174) and anti-PDGFRβ (#3169) (from Cell Signaling Technology), and anti-Endomucin

(Ab106100 from Abcam). Cryosections were washed, incubated with appropriate secondary


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antibodies: anti-rabbit (Ab150075) or anti-rat (Ab150155) conjugated with Alexa Fluor 647.

Samples were visualized using a Zeiss Axio Imager M2 microscope.

Osteoblast cultures

Primary osteoblast were isolated from long bones of Pdgfraf/f;Pdgfrbf/f mice as previously

described (24). Briefly, bone chips were prepared from cleaned long bones and digested in 1

mg/mL collagenase II (Sigma) for 90 minutes at 37 °C. Bone pieces were washed several times

and incubated in α-MEM (Amimed, Bioconcept) containing 10% FBS (Gibco) for 9 days to

allow cell migration from bone fragments. At that point, cells and bone chips were trypsinized

(with trypsin/EDTA from Sigma) and passaged at a split ratio of 1:3. At the second passage,

bone chips were removed. Medium was changed every 2-3 days. Osteoblasts at passages 3-4

were used for in vitro experiments. Pdgfraf/f;Pdgfrbf/f osteoblasts were infected with 400 moi

of empty or Cre-expressing adenoviruses (Vector Biolabs) to obtain Pdgfraf/f;Pdgfrbf/f (control)

and ΔPdgfra;ΔPdgfrb osteoblasts. Cell proliferation was assessed using a cell counter. Cell

migration was evaluated by wound scratch assays. Osteoblast differentiation was determined

by incubating confluent osteoblast cultures in osteogenic medium containing α-MEM, 10%

FBS, 0.05 mM L-ascorbate-2-phosphate (Sigma) and 10 mM β-glycerphosphate (AppliChem

GmbH) for 6 days for the measurement of alkaline phosphatase (ALP) activity and for 14 days

for the measurement of matrix mineralization by Alizarin Red-S staining (Sigma).

Co-cultures


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For co-culture experiments, primary Pdgfraf/f;Pdgfrbf/f osteoblasts infected with 400 moi of

empty or Cre-expressing adenoviruses were seeded at 30000 cells per well in 24-well plates.

The day after, non-adherent bone marrow cells isolated from wildtype mice were seeded over

osteoblasts at 300000 cells per well in α-MEM supplemented with 10% FBS and treated with

vehicle (Veh), 10 ng/mL PDGF-BB (Peprotech) or/and 10-8 M 1,25-dihydroxyvitamin D3

(calcitriol, Vit.D3). After 8 days, co-cultures were fixed and stained, and multinucleated TRAP-

positive cells were counted.

Western Blot

Pdgfraf/f;Pdgfrbf/f and ΔPdgfra;ΔPdgfrb osteoblasts were treated with Veh or 25 ng/mL PDGF-

BB at indicated times, rapidly frozen in liquid nitrogen and stored at -80 °C until their use for

analysis. Cell lysates were prepared by incubating cell cultures in RIPA buffer containing

phosphatase and protease inhibitors at 4 °C for 30 minutes. Lysates were then centrifuged at

6000g for 30 minutes. Lysate supernatants were diluted with equal volumes of 2-fold

concentrated reducing sample buffer. Those mixtures were then heated at 70 °C for 30 minutes

and subjected to gel electrophoresis on 6% to 15% gels. Proteins were electro-transferred to

Immobilon P membranes and immunoblotted with specific antibodies: anti-PDGFRα (#3174),

anti-PDGFRβ (#3169), anti-p-PDGFRs (#3170), anti-p-AKT (#4060), anti-p-SRC (Tyr416)

(#6943), anti-p-ERK1/2 (#9106), anti-ERK1/2 (#4695), anti-p-PLCγ2 (#3874) and anti- PLCγ2

(#3872) (all from Cell Signaling Technology). Detection was performed using peroxidase-

coupled secondary antibody, enhanced chemiluminescence reaction, and visualization by using


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G:Box gel analysis system (Syngene). Reprobed membranes were stripped according to the

manufacturer’s protocol.

Biochemistry

Serum levels of C-terminal telopeptide fragments of type I collagen (CTX), type I procollagen

N-terminal propeptide (PINP) and osteocalcin (OCN) were determined by using RatLaps

immunoassay kits (Immunodiagnostic Systems Ltd). RANKL, OPG and M-CSF protein levels

were determined in bone marrow supernatants (obtained by centrifugation at 1000 g for 10

minutes) or in cell culture medium using Quantikine ELISA kits (R&D Systems).

RNA isolation and real-time PCR

Total RNA was extracted from proximal tibial metaphyses (without bone marrow) or primary

osteoblast cultures using Tri Reagent® (Molecular Research Center) and purified using a

RNeasy Mini Kit (Qiagen). Single-stranded cDNA was synthesized from 2 µg of total RNA

using a High-Capacity cDNA Archive Kit (Applied Biosystems) according to the

manufacturer’s instructions. Real-time PCR was performed to measure the relative mRNA

levels using the QuantStudio™ 5 Real-Time PCR System with SYBR Green Master Mix

(Applied Biosystems). The mean mRNA levels were calculated from triplicate analyses of each

sample. Obtained mRNA level for a gene of interest was normalized to β2-microglobulin

mRNA level in the same sample.

Statistical analysis


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A sample size of 6 mice/group was required in order to detect a difference of 45% in trabecular

bone volume (SD=20%) between groups at the significance level of 0.01 (generally necessary

for Holm-Sidak tests) and a power of 80%. In vitro experiments were performed in triplicate

and independently repeated three times. Statistical analyses were performed using unpaired t-

test for the comparison of 2 groups, and one-way ANOVA followed by post hoc tests (Holm-

Sidak method) for the comparison of multiple genotypes. Interaction between genotype and

Ovx, or between genotype and in vitro treatments were analyzed by using two-way ANOVA.

Results

PDGFRA and PDGFRB are expressed by osteoprogenitors in close proximity to PDGFB-

expressing osteoclasts within human trabecular bone remodeling units

First, we localized expressions of genes encoding the components of the PDGF/PDGFR system

in human bone tissues. As previously shown, PDGFB was expressed by megakaryocytes and

endothelial cells in the bone marrow (Fig. 1A,B) (1). In addition, PDGFB was also expressed

by almost all osteoclasts at the surface of cancellous bone (Fig. 1D,E). More interestingly,

PDGFRA and PDGFRB were expressed by most of osteoprogenitor canopy cells that had been

shown to separate bone remodeling compartments from the bone marrow cavity (Fig. 1C,F,H)

(25). PDGFRA and PDGFRB were also expressed by most osteoblast lineage reversal cells on

the eroded surfaces in the vicinity of PDGFB-expressing TRAP-positive osteoclasts within

trabecular bone remodeling units (Fig. 1C,G,I) (26,27). PDGFRA and PDGFRB mRNA

expression was more abundant in reversal cells present on eroded surfaces than in bone lining

cells on quiescent surfaces (Fig. 1C,G,I).


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Postnatal osteoblast lineage-specific suppression of both PDGFRs increases trabecular

bone mass in male mice

Pdgfra and Pdgfrb mRNAs were highly expressed in primary osteoblasts that expressed Col1a1

(encoding type I collagen; early marker) and Alpl (encoding tissue non-specific alkaline

phosphatase; intermediate marker), but progressively declined as those cells differentiated into

mature osteoblasts expressing Bglap (encoding osteocalcin; late marker) (Fig. S1A).

Accordingly, PDGFRα and PDGFRβ proteins were abundant in early osteoblasts, but no longer

expressed in mature osteoblasts (Fig. S1B). To investigate physiological functions of PDGFRs

in osteoblast biology and bone metabolism, we generated murine models in which tetracycline-

Off-controllable Cre expression and consequent inactivation of floxed Pdgfra or/and Pdgfrb

genes in Osterix-positive cells were induced from 6 weeks to 18 weeks of age by stopping

doxycycline treatment. Conditional deletion of Pdgfra or Pdgfrb was confirmed by real-time

PCR in the osteoblast-rich fraction of tibiae and in primary osteoblasts isolated from Pdgfra

cKO and Pdgfrb cKO mice (Fig. S1C). Consistent with this, intense immunostainings of

PDGFRα and PDGFRβ on trabecular bone surfaces were absent in Pdgfra cKO;Pdgfrb cKO

mice (Fig. S1D). µCT analyses revealed that Pdgfra cKO and Pdgfrb cKO male mice did not

show any changes in trabecular or cortical bone mass and microarchitecture (Fig. 2A-D).

Although Pdgfra cKO;Pdgfrb cKO male mice did not display any change in cortical bone at

tibial midshaft (Fig. 2C,D), they exhibited significant increase in trabecular bone volume at the

proximal metaphysis of tibiae (+53%; Fig. 2A,B) and at the fifth lumbar vertebrae (+47%; Fig.


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S2). Increased trabecular bone mass in Pdgfra cKO;Pdgfrb cKO male mice was associated with

elevated number of trabeculae (Fig. 2B and Fig. S2).

Postnatal osteoblast lineage-specific suppression of both PDGFRs decreases bone

resorption in male mice

Quantitative histomorphometric analyses of femoral secondary spongiosa showed that,

although Pdgfra cKO and Pdgfrb cKO mice did not present changes in parameters of bone

resorption, Pdgfra cKO;Pdgfrb cKO mice demonstrated significant decreases in osteoclast

number and surfaces (Fig. 3A,B). Significant reductions in serum CTX levels and expression

of cathepsin K-encoding gene (CtsK) in bones of Pdgfra cKO;Pdgfrb cKO mice confirmed that

suppression of both PDGFRs in osteoblast lineage cells decreased bone resorption (Fig. S3).

Further ex vivo gene expression analyses indicated that expressions of pro-osteoclastogenic

genes Rankl and Csf1 were diminished in bones of Pdgfra cKO;Pdgfrb cKO mice whereas that

of Opg remained unchanged (Fig. 3C). However, only the protein level of M-CSF in the bone

marrow was significantly reduced in Pdgfra cKO;Pdgfrb cKO mice in comparison to that of

control mice (Fig. 3D).

Postnatal osteoblast lineage-specific suppression of both PDGFRs does not affect bone

formation in male mice

Dynamic histomorphometric analyses showed that neither Pdgfra cKO, Pdgfrb cKO nor Pdgfra

cKO;Pdgfrb cKO mice displayed modification in mineral apposition rate, mineralizing surfaces

or bone formation rate at trabecular, endosteal and periosteal bone envelopes in comparison to

control mice (Fig. 4A,B and Fig. S4). Furthermore, Pdgfra cKO, Pdgfrb cKO and Pdgfra


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cKO;Pdgfrb cKO mice did not demonstrate any changes in osteoblast number on trabecular

bone surfaces (Fig. 4C) or in serum levels of bone formation marker PINP (Fig. 4D). Moreover,

Pdgfra cKO;Pdgfrb cKO mice showed similar distribution of endomucin-positive blood vessels

in the metaphysis in comparison to control mice (Fig. 4E). Finally, Pdgfra cKO;Pdgfrb cKO

mice had comparable number of osteocytes per trabecular bone surfaces compared with that of

control mice (Fig. 4F), which is coherent with the absence of PDGFRs in osteocytes (Fig.

S1A,B).

Osteoblast lineage-specific suppression of both PDGFRs increases trabecular bone mass

in both gonad-intact and ovariectomized female mice

To test the effect of osteoblast lineage-specific abrogation of PDGFR signaling on ovariectomy-

induced bone loss, we took advantage of the inducible Cre expression in Osx-Cre mice to

suppress PDGFRα and PDGFRβ in osteoblast lineage cells at the time of sham-surgeries and

ovariectomies. Both Osx-Cre and Pdgfra cKO;Pdgfrb cKO female mice experienced similar

trabecular bone loss at proximal metaphysis of tibiae following Ovx (Fig. 5A), as a result of

increased osteoclast number (Fig. 5B). Interestingly, suppression of PDGFR signaling in

osteoblast lineage cells increased trabecular bone volume and trabecular number in both gonad-

intact and Ovx female mice (Fig. 5A). This increase in trabecular bone volume in Sham and

Ovx Pdgfra cKO;Pdgfrb cKO female mice was associated with reduced osteoclast number (Fig.

5B) and unchanged osteoblast number (Fig. 5C). Moreover, decreased bone resorption in Sham

and Ovx Pdgfra cKO;Pdgfrb cKO female mice could be explained by significant reduction in

bone marrow level of M-CSF (Fig. 5D).


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Suppression of PDGFR signaling in osteoblasts inhibits M-CSF production and M-CSF-

mediated osteoclastogenesis in vitro

To confirm and expand in vivo observations, we analyzed the molecular mechanisms by which

PDGFR signaling in osteoblasts could control osteoclastogenesis in vitro. PDGF-AA treatment

did not alter expressions of Csf1, Rankl or Opg in osteoblast cultures (Fig. 6A and Fig. S4). On

the other hand, although PDGF-AB and PDGF-BB did not affect expressions of Rankl and Opg

(Fig. S4), they significantly elevated that of Csf1 (Fig. 6A). In agreement with it, PDGF-BB

stimulated secretion of M-CSF in osteoblast cultures (Fig. 6B). Removal of both PDGFRs in

osteoblasts prevented PDGF-AB- and PDGF-BB-induced Csf1 expression and M-CSF

secretion (Fig. 6A,B). PDGF-BB did not have any effect in basal condition but slightly

enhanced osteoclastogenesis in osteoblasts/osteoclasts co-cultures (Fig. 6C). Conversely,

abrogation of PDGFR signaling in osteoblasts blocked osteoclastogenesis in basal condition, in

presence of calcitriol, or in presence of calcitriol and PDGF-BB (Fig. 6C,D). Interestingly,

defective osteoclastogenesis in absence of PDGFR signaling in osteoblasts could be rescued by

supplementation with recombinant M-CSF (Fig. 6C). Suppression of both PDGFRs in

osteoblasts stopped PDGF-BB-stimulated activation of PI3K-AKT, PLCγ-PKC, SRC and

ERK1/2 signaling pathways (Fig. 6E). While selective inhibitions of PI3K, PKC and SRC did

not affect PDGF-BB-induced expressions of Csf1, selective inhibition of MEK1/2 (mitogen-

activated kinase kinases upstream of ERK1/2) blocked it (Fig. 6F). Together, those results

indicate that PDGF-BB promoted Csf1 expression through PDGFR-ERK1/2 signaling pathway.


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Suppression of PDGFR signaling inhibits osteoblast proliferation and migration, but

stimulates osteoblast differentiation in vitro

Finally, we analyzed effects of PDGFR signaling on osteoblast proliferation, migration and

differentiation in vitro. PDGF-AA treatment did not have any effect on those parameters (Fig.

7A-C). PDGF-AB and PDGF-BB treatments enhanced osteoblast proliferation and migration

(Fig. 7A,B), and inhibited osteoblast differentiation, as shown by decreased ALP activity (Fig.

7C). In contrast, removal of PDGFRs in osteoblasts inhibited osteoblast proliferation and

migration (Fig. 7A,B), but stimulated osteoblast differentiation and BMP2-induced

mineralization (Fig. 7C,D).

Discussion

Osteoprogenitors and osteoblasts express PDGFRα and β, but overlapping and distinct

functions of those two receptor tyrosine kinases in osteoblast biology and bone remodeling

remained to be elucidated. Here, we report that PDGFRA and PDGFRB were expressed by

osteoprogenitors in close proximity to PDGFB-expressing osteoclasts within human trabecular

bone remodeling units. Moreover, we show that postnatal genetic ablation of single PDGFRα

or β under the control of the Osx promoter did not affect bone metabolism, but that postnatal

removal of both PDGFRs in osteoblast lineage cells increased trabecular bone mass by lowering

Csf1 expression, M-CSF secretion, osteoclast number and therefore bone resorption, without

having any effect on bone formation. Abrogation of PDGFR signaling in osteoblasts prevented

calcitriol-mediated osteoclastogenesis in co-cultures, while its activation promoted ERK1/2-

mediated Csf1 expression.


18

The identification of osteoclasts as a cellular source of PDGF-BB, and osteoblast lineage

canopy and reversal cells as PDGF-responsive cells at the surface of human cancellous bone

indicates that the PDGF/PDGFR signaling system is operative within trabecular bone

remodeling compartment. To investigate functions of PDGFRs in osteoblast lineage cells, we

generated mice in which PDGFRs-encoding genes were selectively deleted in Osx-positive

cells. Surprisingly, while Pdgfra cKO or Pdgfrb cKO mice did not exhibit any bone phenotype,

mice lacking both receptors in Osx-positive cells displayed increased trabecular bone mass due

to reduced bone resorption and unchanged bone formation. The absence of bone phenotype in

Pdgfrb cKO mice is consistent with previous findings (10,11). Given that PDGF-AA only binds

to PDGFRαα, the absence of bone phenotype in Pdgfra cKO mice indicates that PDGF-AA

plays minor role in osteoblast development and function, which is coherent with the absence of

PDGF-AA effect on osteoblast cultures. Altogether, our results also show that either PDGFRα

or PDGFRβ can compensate for the loss of the other receptor in osteoblast lineage cells and

that both receptors redundantly regulate osteoclastogenesis and bone resorption. The fact that

absence of both PDGFRs in osteoblast lineage cells only affected cancellous bone in axial and

appendicular skeletal sites may be explained by its predominant effect on bone resorption and

by cellular sources of PDGFs, i.e. osteoclasts, which are more abundant in the trabecular

compartment than in cortical one (4-6).

Postnatal removal of PDGFRs in Osx-positive cells decreased osteoclast number and bone

resorption, an effect that was associated with reduced M-CSF production, indicating that

PDGFRα and PDGFRβ in osteoblast lineage cells positively regulate osteoclastogenesis and

bone resorption by elevating Csf1 expression. This notion is further supported by our in vitro


19

investigations. First, suppression of PDGFR signaling in osteoblasts prevented calcitriol-

mediated osteoclastogenesis in co-cultures. Second, supplementation with M-CSF in this

situation was sufficient to restore osteoclast formation. Third, PDGF-AB and PDGF-BB could

stimulate Csf1 expression in osteoblasts. Our findings are in agreement with increased Csf1

expression and osteoclast number in mice transplanted with Pdgfb-overexpressing

hematopoietic stem cells that preferentially colonize bone marrow niches (14). Expression of

Rankl was also reduced in bones of mice lacking both PDGFRs in Osx-positive cells. This

observation could be consistent with stimulated Rankl expression induced by PDGF-BB in

osteoblast-like cell lines, or by Pdgfb overexpression or aberrant osteoblastic PDGFR signaling

in mice (14-16), but this has not been confirmed in vitro or at the protein level in vivo. Moreover,

ERK1/2 signaling pathway was required for PDGF-BB-induced Csf1 expression in osteoblast

cultures. That information provides a molecular mechanism by which ERK1/2 signaling in

osteoprogenitors regulates bone resorption (28,29). Our data are also in accordance with the

requirement of osteoblast lineage-derived M-CSF for osteoclast development in the bone

microenvironment (30). In addition, our results suggest that osteoclasts can control their own

optimal development and maintenance as well as those of neighboring osteoclasts within the

same bone remodeling unit by releasing PDGF-BB. In this context, however, a role of

osteoblast lineage-secreted M-CSF in the regulation of osteoclast migration, activity and

survival cannot be ruled out (31).

Unexpectedly, abrogation of PDGFR signaling in osteoblast lineage cells did not affect bone

formation. First, distribution of endomucin-positive blood vessels remained unchanged in bone

metaphyses of mice lacking PDGFRs in Osx-positive cells, indicating that PDGFR signaling in


20

osteoblast lineage cells is not required for the formation of local blood vessels (6,9). Second,

osteoblast number was not affected in mice lacking both PDGFRs in Osx-positive cells,

showing that PDGFR signaling in osteoblast lineage cells is not involved in osteoprogenitor

proliferation and recruitment to bone surfaces during physiological bone modeling and

remodeling. Those data confirm that PDGFR signaling governs osteoprogenitor expansion and

recruitment by acting in earlier progenitors than Osx-positive cells in physiological condition

(9,32). Third, bone formation remained unchanged on trabecular surfaces despite diminished

bone resorption in mice lacking PDGFRs in osteoblast lineage cells, suggesting an uncoupling

between bone resorption and formation. That may be explained by a cell-autonomous

stimulatory effect of PDGFR suppression on osteoblast differentiation, as observed in our in

vitro analyses and in previous investigations (4,12). This explanation is supported by a recent

report showing that deletion of PDGFRβ in Osx-positive cells leads to accelerated osteoblast

differentiation and callus mineralization during fracture healing (10).

Conditional suppression of PDGFRα and β in Osx-positive cells decreased bone marrow level

of M-CSF, osteoclast number and bone resorption, and increased trabecular bone mass in both

gonad-intact and Ovx female mice, thus confirming results obtained with male mice. However,

it did not prevent Ovx-induced bone loss, which is somehow coherent with the literature.

Indeed, estrogen deficiency reduces bone marrow level of PDGF-BB, and supplementation with

PDGF-BB in this situation averts cancellous bone loss by restoring pericyte-mediated pro-

osteogenic vascularization (6,33). Perhaps more conceivable, local administration of PDGF-

BB could be used to enhance fracture repair in diabetic and osteoporotic patients (10,34,35). In

that context, our results imply that such pharmacological intervention must respect adequate


21

timing to stimulate callus vascularization, expansion and recruitment of osteoprogenitors,

without affecting later callus mineralization and remodeling (10). Finally, excessive PDGFR

activity is involved in several cancers, including those that give rise to osteolytic lesions such

as multiple myeloma and bone metastases from breast and prostate cancers (2,36-38). Our

findings reveal that PDGFR inhibition could have additional beneficial effect by lowering the

occurrence of osteolytic lesions associated with those types of cancers.


22

Acknowledgments

We would like to thank Pierre Apostolides for his expert technical assistance. This work was

supported by the Swiss National Science Foundation (310030-166341), by the Fondation pour

la Recherche sur l’Ostéoporose et les Maladies Osseuses, and by the Novartis Foundation for

medical-biological research (Project number 16C212 - Basel, Switzerland), by the VELUX

FOUNDATION (VELUX25723), and by the Region of Southern Denmark Research Fund

(18/17871).

Authors’ roles: Study design: CT. Study conduct: JB, CT, CE and CMA. Data collection: JB,

CT, CMA and TLA. Data analysis: JB, CT, CMA and TLA. Data interpretation: JB, CT, CMA

and TLA. Drafting manuscript: JB, CT, CMA and TLA. Revising manuscript content: JC, CT,

CE, CMA and TLA. Approving final version of manuscript: JB, JC, CT, CE, CMA and TLA.

CT takes responsibility for the integrity of the data analysis.


23

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29

Figure legends

Fig. 1. PDGFB mRNA expression in TRAP-positive osteoclasts and PDGFRA and PDGFRB

mRNA expression in proximate canopy and reversal cells in iliac crest bone biopsy specimens

from four human volunteers without known musculoskeletal disease. The mRNA expression

was visualized by in situ hybridization (red dots) combined with TRAP immunostaining (black

stain). Images are representative of those obtained from the four patients. Scale bars are 20 µm.

(A, B) PDGFB mRNA expression in megakaryocytes (MKC) and vascular structures (VS)

within bone marrow. (C) Overview image showing the location of the osteoclastic zone

illustrated in adjacent sections in D, F and H. (D, E) PDGFB mRNA expression in TRAP-

positive osteoclasts (OC). Note the red dots within the black osteoclasts. (F, G) PDGFRA

mRNA expression in elongated canopy cells (red arrows) above osteoclasts (OC) and reversal

cells (black arrows) on eroded surfaces close to osteoclasts. (H, I) PDGFRB mRNA expression

in elongated canopy cells (red arrows) above osteoclasts and reversal cells (black arrows) on

eroded surfaces close to osteoclasts. (J, K) Images from negative controls.

Fig. 2. Postnatal osteoblast lineage-specific suppression of PDGFR signaling increases

trabecular bone mass in male mice. (A) Representative reconstructed µCT images of proximal

tibiae from 18-week-old male Osx-Cre, Pdgfra cKO, Pdgfrb cKO and Pdgfra cKO;Pdgfrb cKO

mice, in which Cre expression and consequent conditional gene deletion were induced (by

stopping doxycycline treatment) from 6 weeks to 18 weeks of age. (B) Trabecular bone

microarchitecture measured at proximal tibiae. µCT parameters include BV/TV: bone

volume/total volume, Tb.Th: trabecular thickness, Tb.N: trabecular number. (n=6 per group).


30

(C) Representative reconstructed µCT images of tibial midshafts from 18-week-old male Osx-

Cre, Pdgfra cKO, Pdgfrb cKO and Pdgfra cKO;Pdgfrb cKO mice, in which Cre expression and

consequent conditional gene deletion was induced from 6 weeks to 18 weeks of age. (D)

Cortical bone microarchitecture measured at tibial midshaft. µCT parameters include Ct.TV:

cortical total volume, Ct.BV: cortical bone volume and Ct.Th: cortical thickness (n=6 per

group). Comparisons between the multiple genotypes were analyzed by one-way ANOVA

followed by Holm-Sidak post hoc tests.

Fig. 3. Postnatal osteoblast lineage-specific suppression of PDGFR signaling decreases bone

resorption. (A) Histomorphometric parameters of trabecular bone resorption measured at the

secondary spongiosa of distal femurs from 18-week-old male Osx-Cre, Pdgfra cKO, Pdgfrb

cKO and Pdgfra cKO;Pdgfrb cKO mice (n=6 per group). Cre expression and consequent

conditional gene deletion were induced (by stopping doxycycline treatment) from 6 weeks to

18 weeks of age. N.Oc/Pm: osteoclast number/bone perimeter, Oc.S/BS: osteoclast

surface/bone surface. (D) Representative images of TRAP-stained histological sections of distal

femurs (n=6 per group). (E) Quantitative RT-PCR analyses of Rankl, Opg and Csf1 expressions

in proximal tibial metaphyses (n=6 per group). (F) Levels of RANKL, OPG and M-CSF

detected by ELISA in bone marrow supernatants (n=6 per group). Comparisons between the

multiple genotypes were analyzed by one-way ANOVA followed by Holm-Sidak post hoc tests.

Fig. 4. Postnatal osteoblast lineage-specific suppression of PDGFR signaling does not affect

bone formation. (A-C) Histomorphometric parameters of trabecular bone formation measured


31

at the secondary spongiosa of distal femurs from 18-week-old male Osx-Cre, Pdgfra cKO,

Pdgfrb cKO and Pdgfra cKO;Pdgfrb cKO mice (n=6 per group). Cre expression and consequent

conditional gene deletion were induced (by stopping doxycycline treatment) from 6 weeks to

18 weeks of age. (A) Tb.MAR: trabecular mineral apposition rate, Tb.MS/BS: trabecular

mineralizing surfaces/bone surfaces, Tb.BFR: trabecular bone formation rate. (B)

Representative images of calcein-labeled histological sections of distal femurs. (C) Ob.N/Pm:

osteoblast number/bone perimeter. (D) Serum levels of PINP detected by ELISA (n=6 per

group). (E) Immunohistochemical staining of endomucin (red) and nuclei (blue) on histological

sections of distal femurs and quantification of endomucin-positive area using ImageJ software

(n=3 per group). (F) N.Ocy/BS: number of osteocytes/bone surface (n=5 per group).

Comparisons between the multiple genotypes were analyzed by one-way ANOVA followed by

Holm-Sidak post hoc tests.

Fig. 5. Postnatal osteoblast lineage-specific suppression of PDGFR signaling increases

trabecular bone mass in both gonad-intact and ovariectomized female mice. Twelve-week-old

female Osx-Cre and Pdgfra cKO;Pdgfrb cKO mice were Sham-operated or Ovx and sacrificed

8 weeks later. Cre expression and consequent conditional gene deletion were induced (by

stopping doxycycline treatment) one week prior Sham and Ovx operations. (A) Trabecular bone

microarchitecture measured at proximal tibiae (n=6 per group). µCT parameters include

BV/TV: bone volume/total volume, Tb.Th: trabecular thickness, Tb.N: trabecular number,

Tb.Sp: trabecular separation. (B, C) Histomorphometric parameters of trabecular bone

resorption (B) and formation (C) measured at the secondary spongiosa of distal femurs (n=6


32

per group). (F) Levels of RANKL, OPG and M-CSF detected by ELISA in bone marrow

supernatants (n=6 per group). The two-way ANOVA did not detect significant interaction

between effects of genotype and those of Ovx. The comparison between the two genotypes (p

values in bold) or between Sham and Ovx operations (p values in italic) was performed using

unpaired t-test.

Fig. 6. Suppression of PDGFR signaling in osteoblasts inhibits M-CSF production and M-CSF-

mediated osteoclastogenesis in vitro. Primary osteoblasts were isolated from long bones of

Pdgfraf/f;Pdgfrbf/f mice and infected with empty or Cre-expressing adenoviruses to obtain

Pdgfraf/f;Pdgfrbf/f (control) and ΔPdgfra;ΔPdgfrb (without PDGFRs) osteoblasts respectively.

(A) Quantitative RT-PCR analysis of Csf1 expression in confluent cultures of Pdgfraf/f;Pdgfrbf/f

and ΔPdgfra;ΔPdgfrb osteoblasts treated with 25 ng/mL PDGF-AA, PDGF-AB or PDGF-BB

or their vehicle (Veh) solution for 8 hours. (B) Levels of M-CSF detected by ELISA in the

culture medium of Pdgfraf/f;Pdgfrbf/f and ΔPdgfra;ΔPdgfrb osteoblasts treated with Veh or 25

ng/mL PDGF-BB for 48 hours. (C) Number of TRAP-positive multinucleated cells in

osteoblasts/osteoclasts co-cultures treated with Veh, 10 ng/mL PDGF-BB or/and 10-8 M 1,25-

dihydroxyvitamin D3 (Vit.D3), and supplemented or not with 20 ng/mL M-CSF for 8 days. (D)

Representative images of TRAP-positive multinucleated cells in co-cultures with Veh or 10-8

M Vit.D3 for 8 days. (E) Western blot analyses of PDGFR signaling in Pdgfraf/f;Pdgfrbf/f and

ΔPdgfra;ΔPdgfrb osteoblasts treated with Veh or 25 ng/mL PDGF-BB at indicated times. (F)

Quantitative RT-PCR analysis of Csf1 expression in confluent cultures of Pdgfraf/f;Pdgfrbf/f

osteoblasts pretreated with 5 µM LY294002 (PI3K inhibitor), 5 µM U0126 (MEK1/2 inhibitor),


33

5 µM GO6983 (PKC inhibitor) and 5 µM SU6656 (Src family kinase inhibitor) for 30 minutes

and then, treated with Veh or 25 ng/mL PDGF-BB for 8 hours. Data are represented as mean ±

SD. # p≤0.002 versus Veh, * p≤0.002 versus Pdgfraf/f;Pdgfrbf/f.

Fig. 7. Suppression of PDGFR signaling in osteoblasts stimulates osteoblast differentiation in

vitro. Primary osteoblasts were isolated from long bones of Pdgfraf/f;Pdgfrbf/f mice and infected

with empty or Cre-expressing adenoviruses to obtain Pdgfraf/f;Pdgfrbf/f (control) and

ΔPdgfra;ΔPdgfrb (without PDGFRs) osteoblasts respectively. (A) Number of primary

Pdgfraf/f;Pdgfrbf/f and ΔPdgfra;ΔPdgfrb osteoblasts treated with Veh, 25 ng/mL PDGF-AA,

PDGF-AB or PDGF-BB for 3 days. (B) Quantification of cell migration rates in scratched sub-

confluent cultures of Pdgfraf/f;Pdgfrbf/f and ΔPdgfra;ΔPdgfrb osteoblasts treated with Veh, 25

ng/mL PDGF-AA, PDGF-AB or PDGF-BB for 3 days. (C) Alkaline phosphatase (ALP)

activity of Pdgfraf/f;Pdgfrbf/f and ΔPdgfra;ΔPdgfrb osteoblasts incubated in osteogenic

medium and treated with Veh, 25 ng/mL PDGF-AA, PDGF-AB or PDGF-BB for 6 days. (D)

Representative images of matrix mineralization by Pdgfraf/f;Pdgfrbf/f and ΔPdgfra;ΔPdgfrb

osteoblasts treated with Veh or 200 ng/mL BMP2 for 14 days. Data are represented as mean ±

SD. # p≤0.001 versus Veh, * p≤0.001 versus Pdgfraf/f;Pdgfrbf/f.


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pdgf receptor signaling in osteoblast lineage cells

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