Sclerostin blockade inhibits bone resorption through PDGF receptor signaling in osteoblast lineage cells

While sclerostin-neutralizing antibodies (Scl-Abs) transiently stimulate bone formation by activating Wnt signaling in osteoblast lineage cells, they exert sustained inhibition of bone resorption, suggesting an alternate signaling pathway by which Scl-Abs control osteoclast activity. Since sclerostin can activate platelet-derived growth factor receptors (PDGFRs) in osteoblast lineage cells in vitro and PDGFR signaling in these cells induces bone resorption through M-CSF secretion, we hypothesized that the prolonged anticatabolic effect of Scl-Abs could result from PDGFR inhibition. We show here that inhibition of PDGFR signaling in osteoblast lineage cells is sufficient and necessary to mediate prolonged Scl-Ab effects on M-CSF secretion and osteoclast activity in mice. Indeed, sclerostin coactivates PDGFRs independently of Wnt/β-catenin signaling inhibition, by forming a ternary complex with LRP6 and PDGFRs in preosteoblasts. In turn, Scl-Ab prevents sclerostin-mediated coactivation of PDGFR signaling and consequent M-CSF upregulation in preosteoblast cultures, thereby inhibiting osteoclast activity in preosteoblast/osteoclast coculture assays. These results provide a potential mechanism explaining the dissociation between anabolic and antiresorptive effects of long-term Scl-Ab.


Introduction
Sclerostin, encoded by the SOST gene, is an osteocyte-secreted protein that antagonizes lowdensity lipoprotein receptor-related proteins (LRP) 5 and 6, thereby inhibiting canonical Wnt signaling in osteoblast lineage cells and bone formation (1)(2)(3)(4).Due to its restricted expression in the adult skeleton, sclerostin has emerged as an attractive therapeutic target to increase bone mass and strength in osteoporotic patients.Consequently, administration of antibodies targeting sclerostin (Scl-Ab) has been shown to augment bone mineral density and bone strength in humans, through transient elevation of bone formation and sustained reduction of bone resorption (5)(6)(7)(8)(9)(10).
Mechanistically, short-term Scl-Ab treatment induces a rapid and intense increases in serum procollagen type I N-terminal propeptide (PINP) and histomorphometric indices of bone formation, as well as a significant decrease in serum level of the bone resorption marker Cterminal telopeptides of type I collagen (CTX) in osteoporotic patients (7)(8)(9)(10).Preclinical investigations have shown that Scl-Ab simultaneously activate modeling-based bone formation by stimulating transition of bone lining cells into active osteoblasts and generate a positive bone balance at remodeling sites by enhancing the anabolic power (vigor) of each osteoblast (11-13).
In parallel, bone resorption surfaces are decreased and associated with lower receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) ratio, decreased expression of Csf1 (encoding macrophage colony-stimulating factor, M-CSF), essential for osteoclast differentiation and survival, and enhanced expression of Wisp1, a negative regulator of bone resorption (13,14).After 3 to 6 months of Scl-Ab treatment, serum PINP and bone formation indices return to initial values, whereas serum CTX and bone resorption parameters are maintained below baseline levels (7)(8)(9)(10).Although overall bone turnover eventually decreases, the positive bone mineral balance within bone remodeling units is maintained, but attenuated, allowing .CC-BY 4.0 International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 15, 2023.; https://doi.org/10.1101/2023.09.11.557168 doi: bioRxiv preprint significant bone mass gain to continue for the duration of therapy (1 year) (15).Counterregulation of bone formation with Scl-Ab administration can be explained by increased expressions of Wnt pathway inhibitors such as Sost and Dkk1 (encoding dickkopf-related protein 1, DKK1), and decreased number of osteoprogenitors (14,16).In this context, the reason why Scl-Ab exerts prolonged anti-catabolic effect despite attenuation of its bone anabolic activity remains unclear.A possible explanation is that sclerostin neutralization could reduce bone resorption independently of canonical Wnt signaling activation.
We previously showed that platelet-derived growth factor receptor (PDGFR)α and PDGFRβ in Osterix-positive cells redundantly control osteoclastogenesis and bone resorption by upregulating expression of Csf1 in mice (17), and that sclerostin can induce PDGFR signaling in osteoblast lineage cells in vitro (18).Therefore, we hypothesized that the prolonged anti-catabolic effect of Scl-Ab treatment could result from an inhibition of PDGFR signaling independently of its Wnt-activating properties in osteoblast lineage cells.

Scl-Ab transiently stimulates bone formation in both control and Pdgfr cKO mice
Osx-Cre;Pdgfra f/f ;Pdgfrb f/f (hereafter Pdgfr cKO) mice, in which Pdgfra and Pdgfrb genes were effectively deleted by the tetracycline-Off-controllable Osterix-dependent Cre expression system at 2 months of age, exhibit a progressive increase in trabecular bone mass.In these conditions, Csf1 expression by Osterix-positive cells is reduced, as well as osteoclast surfaces and bone resorption, whereas bone formation remains unchanged (17).To test the role of PDGFR signaling in osteoblast lineage cells on Scl-Ab effects, we treated control (Osx-Cre) and Pdgfr cKO mice (after deletions of Pdgfra and Pdgfrb genes induced one week prior to treatment, Fig. 1A, B) with Scl-Ab or its vehicle solution (Veh) for 2 and 6 weeks (a validated protocol in mice during which bone formation rate peaked after 2 weeks and returned to basal level after 6 weeks of Scl-Ab treatment) (19).In this case, due to the short duration of PDGFR deletion, Pdgfr cKO mice showed similar cortical and trabecular bone mass in comparison to Osx-Cre mice at baseline as well as after 2 and 6 weeks of Veh treatment (Fig. 1C, D).After 2 and 6 weeks of Scl-Ab, cortical bone volume at tibial midshaft, as well as trabecular number, thickness and bone volume at the proximal metaphysis were increased in both genotypes (Fig. 1C, D).However, Scl-Ab increased trabecular bone volume more in Pdgfr cKO mice than in Osx-Cre mice after 2 weeks, a trend which persisted after 6 weeks of treatment (Fig. 1D).
Dynamic histomorphometric analyses showed that, although 2-week Scl-Ab treatment stimulated the mineral apposition rate on trabecular bone surfaces equally in both genotypes, it increased trabecular mineralizing surfaces and bone formation rate more in Pdgfr cKO mice than in Osx-Cre mice (Fig. 1E).However, those parameters of bone formation returned to pretreatment levels after 6 weeks of Scl-Ab treatment in mice of both genotypes (Fig. 1E).These observations indicated that PDGFR signaling could attenuate the extent of bone-formation surfaces in response to Scl-Ab, but did not play a role in the counter-regulation of Scl-Ab effect on bone formation.

Self-regulation of Wnt signaling in response to Scl-Ab is independent of PDGFRs
To determine whether the decline of bone anabolism observed with prolonged Scl-Ab treatment is associated with a self-regulation of Wnt signaling, we measured expressions of selected Wnt target genes and Wnt pathway regulators in proximal tibial metaphysis isolated from Osx-Cre and Pdgfr cKO mice.2-week Scl-Ab treatment enhanced expressions of Wisp1 and Twist1, two Wnt target genes which are responsive to sclerostin neutralization (13,14), even more strongly in Pdgfr cKO mice than in Osx-Cre mice (Fig. 2A, B).Expressions of both Wnt target genes returned to basal levels following 6 weeks of Scl-Ab treatment in both genotypes, thereby confirming attenuation of Wnt signaling with prolonged exposure to Scl-Ab (Fig. 2A, B).This down-regulation of Wnt target gene expression was associated with a significant elevation of Sost (after 6 weeks) and Dkk1 (from 2 weeks) expressions in both genotypes (Fig. 2C, D).Moreover, Scl-Ab administration also quickly reduced expressions of Wnt1 and Wnt10b in bones of mice from both genotypes (Fig. 2E, F).Altogether, those results indicated that Wnt signaling attenuation following prolonged Scl-Ab treatment was mediated by a negative feedback mechanism involving increased expressions of Wnt signaling inhibitors and decreased expressions of Wnt1 class of ligands, independently of PDGFR signaling.

PDGFRs mediate Scl-Ab inhibitory effects on bone resorption and Csf1 expression
Quantitative histomorphometric analyses of femoral secondary spongiosa confirmed that suppression of PDGFRs in osteoblast lineage cells significantly decreased osteoclast number and surface (Fig. 3A-C), which was related to reduced Csf1 expression (Fig. 3D) (17).Scl-Ab also significantly reduced osteoclast number and surfaces in control mice after 2 and 6 weeks (Fig. 3A-C), while it tended to further diminish the already low osteoclast number and surfaces in Pdgfr cKO mice after 2 weeks, but not after 6 weeks of treatment (Fig. 3A-C).Ex vivo gene expression analyses indicated that sclerostin neutralization lowered Csf1 expression in control Osx-Cre mice but not in Pdgfr cKO mice after 2 and 6 weeks of treatment (Fig. 3D).In contrast, we found no significant differences in Rankl or Opg expression in response to Scl-Ab treatment and PDGFRs deletion (Fig. 3E, F).Thus, those data indicated that sclerostin and PDGFRs operated within the same signaling pathway to inhibit Csf1 expression and bone resorption.

PDGFRs expression
To confirm that Scl-Ab-mediated inhibition of bone resorption is associated with a concomitant inhibition of PDGFR signaling, we measured expressions of PDGFR target genes and PDGFR signaling components in proximal tibial metaphysis isolated from Osx-Cre and Pdgfr cKO mice treated with Veh and Scl-Ab for 2 and 6 weeks.Selective suppressions of Pdgfra and Pdgfrb in osteoblast lineage cells were associated with reduced expressions of c-Myc and Ccl2, two PDGFR target genes (Fig. 4A, B) (20,21), and confirmed by reduced expressions of both genes in Pdgfr cKO mice (Fig. 4C, D).In contrast to the transient effect of Scl-Ab on Wnt target gene expression (Fig. 2), Scl-Ab treatment decreased expressions of c-Myc and Ccl2 in Osx-Cre mice consistently after 2 and 6 weeks, but did not further reduce the already low expression levels in Pdgfr cKO mice (Fig. 4A, B).In contrast, Scl-Ab did not affect Pdgfra and Pdgfrb expression levels (Fig. 4C, D).Furthermore, both selective PDGFRs ablation in osteoblast lineage cells and Scl-Ab administration reduced expression of Pdgfb, encoding PDGF-B ligand (Fig. 4E), probably reflecting low number of Pdgfb-expressing osteoclasts under these conditions (17).

Sclerostin potentiates PDGF-BB-stimulated Csf1 expression and bone resorption in vitro by forming a sclerostin/LRP6/PDGFR ternary complex for ERK1/2 activation
To confirm and expand these in vivo observations, we analyzed the molecular mechanisms by which sclerostin could interfere with PDGFR signaling to regulate osteoclast development and function in vitro.Recombinant sclerostin did not have any effect on in vitro osteoclastogenesis in basal conditions, but enhanced calcitriol-induced osteoclastogenesis and resorption of a synthetic matrix in osteoblasts/osteoclasts co-cultures (Fig. 5A, B).These effects were blocked by Scl-Ab, M-CSF-targeting antibody or by PDGFR deletion in osteoblasts (Fig. 5A, B).In addition, sclerostin potentiated PDGF-BB-induced Csf1 expression and M-CSF secretion in osteoblast cultures (Fig. 5C, D).Again, those effects were blocked by either Scl-Ab treatment (Fig. 5C, D) or suppression of PGDFRs in osteoblasts (Fig. 5C).Consistent with those findings, sclerostin amplified PDGF-BB-induced phosphorylation of PDGFRs in osteoblast cultures after 15 minutes and elevation of M-CSF protein level after 24 hours, effects that were abrogated in the presence of Scl-Ab (Fig. 5E).Eventually, immunoprecipitation of proteins by anti-LRP6 antibody showed that sclerostin could intensify PDGF-BB-induced activation of PDGFRs by forming a ternary complex with LRP6 and PDGFRs in osteoblasts (Fig. 5F).Sclerostinmediated potentiating effects on PDGF-BB-dependent PDGFRs activation resulted in further stimulation of downstream PDGFR signaling pathways such as ERK1/2 and STAT3 in osteoblasts (Fig. 5G).

Sclerostin potentiates PDGF-BB-induced Csf1 expression independently of Wnt/β-catenin signaling in osteoblast cultures
To provide an explanation for the continuous inhibition of PDGFR signaling and Csf1 expression despite self-attenuation of Wnt signaling in response to prolonged Scl-Ab exposure (Fig. 4), we tested whether Wnt/β-catenin signaling activation or inhibition could influence sclerostin ability to co-activate PDGFRs and stimulate Csf1 expression in osteoblast cultures.

Discussion
Sclerostin blockade exerts dual effects on bone, resulting in a potent but transient stimulation of bone formation but a milder and sustained inhibition of bone resorption.Because Scl-Ab effects on bone formation are primarily mediated by stimulation of the Wnt/β-catenin signaling pathway in osteoblast lineage cells and this pathway is rapidly down-regulated by the overexpression of Wnt inhibitors including sclerostin itself and DKK1 (14,16), the persistent inhibition of bone resorption suggests that sclerostin and its pharmacological inhibitors control osteoclastogenesis by an alternative, Wnt-independent pathway, in osteoblast lineage cells.
Consistent with human data, we showed that, despite continuous cortical and trabecular bone mass gain, bone formation induced by Scl-Ab treatment in mice was transient.The decline of Scl-Ab osteoanabolic effects was associated with attenuation of Wnt signaling due to elevated expressions of Wnt signaling inhibitors, and decreased expressions of Wnt1 class of ligands.In contrast, bone PDGFR signaling, Csf1 expression and bone resorption were durably reduced by Scl-Ab in control mice.These parameters were also reduced in Pdgfr cKO mice and, although short-term Scl-Ab treatment tended to further decrease bone resorption in these mice, prolonged Scl-Ab exposure did not further reduce it.Scl-Ab abolished sclerostin-mediated co-activation of PDGFR signaling and consequent Csf1 up-regulation in osteoblast cultures, and calcitriolinduced bone resorption in osteoblasts/osteoclasts co-cultures.Eventually, we showed that sclerostin could potentiate PDGFRs activation, unlike DKK1 and independently of the presence of Wnt ligand, by forming a ternary complex with LRP6 and PDGFRs in osteoblast lineage cells.Genetic ablation of PDGFRs-encoding genes potentiated Scl-Ab-promoted trabecular bone mass gain.This effect was rather due to an amplified early bone anabolic response to sclerostin neutralization (Fig. 1), since bone resorption reached similar levels following Scl-Ab treatment either in presence or in absence of PDGFRs in osteoblast lineage cells (Fig. 3).
Consistent with it, osteoblast lineage-specific suppression of PDGFRs intensified the expressions of Wnt target genes, such as Wisp1 and Twist1, following short-term Scl-Ab treatment (Fig. 2).From a mechanistic point of view, deletion of PDGFRs-encoding genes in osteoblasts stimulated Wnt1-induced accumulation of active β-catenin and Wnt target gene expression in vitro (Fig. 6), showing that PDGFRs negatively regulate the Wnt/β-catenin signaling pathway (Fig. 7).Of note, the synergistic effect of osteoblast lineage-specific suppression of PDGFRs on short-term Scl-Ab-induced trabecular bone formation was solely caused by augmentations in mineralizing surfaces (Fig. 1).The fact that the extent of endosteal and periosteal mineralizing surfaces is a limiting factor for further stimulation of cortical bone formation in this context may explain the cancellous compartment-specific potentiation of bone formation.In addition, we cannot rule out that crosstalk between Wnt/β-catenin and PDGFR signaling may require specific Wnt proteins or PDGF ligands which are differentially expressed in the different bone compartments (17,22,23).
We confirmed that stimulation of trabecular bone formation by Scl-Ab therapy was only transient, as observed in clinical trials and preclinical studies (7-10, 14, 19), and that attenuation of Scl-Ab anabolic action in the cancellous compartment preceded that occurring in cortical bone (24).Counter-regulation of Scl-Ab-stimulated bone formation was associated with decreased expressions of Wnt target genes Wisp1 and Twist1, and increased expressions of Sost and Dkk1 (Fig. 2), which is consistent with previous findings (14,16,25).Unexpectedly, we also observed reduced expressions of Wnt1 and Wnt10b (Fig. 2), which could also contribute to taper LRP6 activation.These effects were similar in mice with osteoblast lineage-specific PDGFRs deletion, indicating that inhibitory action of PDGFRs on Wnt/β-catenin signaling in presence of Scl-Ab is overridden by DKK1 and low Wnt1 class availability (Fig. 7).
We found that Scl-Ab treatment exerted sustained inhibition of bone resorption in control mice, as observed previously in humans (7)(8)(9)(10).Scl-Ab administration transiently enhanced expression of Wisp1, a Wnt1-responsive negative regulator of osteoclastogenesis (26), and continuously reduced expression of Csf1, encoding an essential growth factor for osteoclastogenesis, which is consistent with previous observations in ovariectomized rats (14).
The differential regulation between Wisp1 and Csf1 expressions by Scl-Ab suggested that sclerostin could regulate Csf1 expression independently of Wnt/β-catenin signaling inhibition.
Interestingly, Scl-Ab treatment could not further reduce Csf1 expression in Pdgfr cKO mice (Fig. 3), thus indicating that PDGFR signaling is sufficient and necessary to explain sclerostin effects on Csf1 expression and bone resorption.At the cellular level, short-term Scl-Ab treatment induced a slight additional decrease in osteoclast surfaces while prolonged Scl-Ab exposure did not further reduce them in Pdgfr cKO mice (Fig. 3), thus reflecting the combination of a transient stimulatory effect on Wnt/β-catenin-mediated Wisp1 and possibly Opg expressions, and a continuous inhibitory effect on PDGFR-mediated Csf1 expression (14).
Altogether, those findings provide the cellular and molecular mechanisms involved in the biphasic inhibitory effects of Scl-Ab on bone resorption observed in clinical trials, i.e., a sharp decrease in serum CTX levels at 1 month, a return close to baseline at 3 months, followed by a progressive and continuous reduction at 6 and 12 months (7-9).
In line with the continuous inhibitory effect of Scl-Ab on PDGFR-mediated Csf1 expression, prolonged anti-catabolic effect of Scl-Ab was associated with diminished expressions of PDGFR target genes c-Myc and Ccl2 in bone (Fig. 4).The fact that Scl-Ab could prevent sclerostin-mediated potentiation of PDGF-BB-dependent PDGFRs activation and Csf1 expression in osteoblast cultures, and calcitriol-induced resorption of a synthetic matrix in osteoblasts/osteoclasts co-cultures also supports our in vivo observations (Fig. 5).Most importantly, we demonstrated that sclerostin, in contrast to DKK1, could function as a coactivator of PDGFRs and downstream ERK1/2 signaling pathways, independently of Wnt1 and Wnt/β-catenin signaling inhibitors (Fig. 6), thereby explaining why bone resorption remains inhibited by prolonged Scl-Ab treatment despite the down-regulation of bone formation (Fig. 7).At a molecular level, heterodimerization between PDGFRs and LRP6, as also observed in other mesenchymal stem cell-derived cell types (27,28), likely contributes to sclerostin effects on PDGFR signaling in osteoblasts (Fig. 7).
In conclusion, we identified a new pathway for sclerostin effects on bone resorption and provide an explanation for the dissociation of Scl-Ab long-term therapeutic efficacy on bone resorption versus formation.Indeed, Scl-Ab anti-catabolic effects on the skeleton occur by repressing PDGFR signaling and M-CSF expression in osteoblast lineage cells in a Wntindependent manner.In addition, although it remains to be formally shown that activating mutations of PDGFRβ in osteoprogenitors contributes to osteopenia and occurrence of fractures in patients with Penttinen syndrome or Kosaki overgrowth syndrome (29), Scl-Ab could be useful to treat those pathological conditions.Finally, our findings also suggest that combination of PDGFR inhibition and sclerostin neutralization could represent a powerful approach to rapidly increase bone mass and strength in patients with osteolytic lesions provoked by multiple myeloma or bone metastases involving excessive PDGFR activity (30,31).

Mice and Scl-Ab treatment
Pdgfr cKO mice in which Pdgfra and Pdgfrb genes can be selectively deleted under the control of an Osterix promoter following cessation of doxycycline treatment (Tet-Off system) were generated as previously described (17).Since both PDGFRs can compensate for the loss of the other and exert redundant function in osteoblast lineage cells (17), we used Pdgfr cKO mice in our experiments.Osx-Cre mice were used as control animals.All mice were on a C57BL/6J genetic background.16-week-old male Osx-Cre and Pdgfr cKO mice were randomly assigned to receive subcutaneous injections of 25 mg/kg Scl-Ab (r13c7, provided by UCB Pharma and Amgen Inc.) or an equivalent volume of saline solution twice a week for 2 weeks (bone formation rate peaked after 2 weeks of Scl-Ab treatment) or 6 weeks (bone formation rate returned to basal level after 6 weeks of Scl-Ab treatment) (Fig. 1A) (19).Cre expression and consequent Pdgfra and Pdgfrb inactivation were induced one week prior to the onset of Scl-Ab treatments by stopping doxycycline administration (Fig. 1A).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.Mice treatments (Veh or Scl-Ab) and endpoint measurements (by µCT and histomorphometry) were performed by different investigators.Investigators were blinded during endpoint measurements.

Bone phenotyping
Mice were sacrificed and their bones were excised for µCT analyses.Trabecular bone microarchitecture of proximal tibiae (100 slices from the beginning of secondary spongiosa), and cortical bone geometry of 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.

Osteoblast cultures
Primary osteoblast were isolated from long bones of Pdgfra f/f ;Pdgfrb f/f (Pdgfr f/f) mice as previously described (32).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.Pdgfr f/f osteoblasts were infected with 400 moi of empty or Cre-expressing adenoviruses (Vector Biolabs) to obtain Pdgfr f/f (control) and Pdgfr cKO osteoblasts.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) in the presence of vehicle (Veh), 100 ng/mL Wnt1-sFRP1 (R&D Systems) ± 500 ng/mL recombinant sclerostin (Peprotech).

Co-cultures
For co-culture experiments, primary Pdgfr f/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 Veh, 10 -8 M 1,25-dihydroxyvitamin D3 (calcitriol, Vit.D3), or/and 250 ng/mL recombinant murine sclerostin (Peprotech), with or without 1.25 µg/mL Scl-Ab or 500 ng/mL anti-M-CSF antibody (#AF416 from R&D Systems).After 8 days, co-cultures were fixed and stained, and multinucleated TRAP-positive cells were counted.To evaluate in vitro bone resorption, cocultures were performed under the same conditions in Corning® Osteo Assay Surface multiple well plates for 15 days.Multiple well plates were cleaned with a bleaching solution and observed under an inverted phase-contrast microscope (Nikon Eclipse TE2000).Synthetic matrix resorption was quantified using Image J software.

Immunoprecipitations
Confluent osteoblast cultures were pre-treated with Veh or 500 ng/mL recombinant sclerostin for 2 hours, and then treated with 25 ng/mL PDGF-BB for 15 minutes.Cell lysates were prepared by incubating osteoblast cultures in lysis buffer containing 1% NP-40 and phosphatase/protease inhibitors at 4 °C for 30 minutes.Cells were then scraped and sonicated on ice for 10 seconds.Lysates were then centrifuged at 6000g for 30 minutes at 4 °C.Cell lysates were incubated with 1:200 anti-LRP6 antibody (#3395 from Cell Signaling) or isotype control overnight at 4 °C.The day after, immunocomplexes were incubated with pre-washed Protein G Magnetic beads (#70024 from Cell Signaling) under agitation for 1 hour at room temperature.Then, beads were pelleted by using a magnetic separation rack and washed 3 times with cell lysis buffer.Immunocomplexes attached to beads were diluted with equal volumes of 2-fold concentrated loading buffer and heated at 70 °C for 30 minutes.Immunocomplexes were separated from magnetic beads by using a magnetic separation rack and collecting supernatants.
Finally, supernatants were subjected to western blot analyses as described below.

RNA isolation and real-time PCR
Total RNA was extracted from tibial metaphyses (whose bone marrow was removed by centrifugation at 16200g for 20 seconds) 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 primer sequences are described in supplementary table 1. Melting curve analyses performed at the completion of PCR amplifications revealed a single dissociation peak for each primer pair.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.

Statistics
A sample size of 5 mice/group was required in order to detect a difference of 30% in fractional trabecular osteoclast surface (SD=30%) between groups at the significance level of 0.01 and a power of 80%.In vitro experiments were performed in triplicate and independently repeated 3 or 4 times.Interactions between effects of treatments and those of genotypes, were analyzed by using 2-way ANOVA followed by Tukey post hoc tests.Interactions between effects of treatments, those of treatment durations and those of genotypes, were analyzed by using linear mixed-effects models followed by Tukey post hoc tests.

Study approval
All performed experiments were in compliance with the guiding principles of the Guide for the Care and Use of Laboratory Animals (8 th edition) and approved by the Ethical Committee of the University of Geneva School of Medicine and the State of Geneva Veterinarian Office.

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CC-BY 4.0 International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 15, 2023.; https://doi.org/10.1101/2023.09.11.557168 doi: bioRxiv preprint 34 or 25 ng/mL PDGF-BB for 15 minutes before evaluation of PDGFR signaling by western blot analyses.(D) Wildtype osteoblasts were pre-treated with DMSO or 5 µM WIKI4 (inhibitor of Wnt/β-catenin signaling), and Veh or 500 ng/mL SOST for 2 hours, and treated with Veh or 25 ng/mL PDGF-BB for 24 hours before measurements of Csf1 expression by quantitative RT-PCR.(E) Wildtype osteoblasts were pre-treated with Veh, 500 ng/mL SOST or 500 ng/mL DKK1 for 2 hours, and treated with Veh or 25 ng/mL PDGF-BB for 24 hours before measurements of Csf1 expression by quantitative RT-PCR.(F) Wildtype osteoblasts were pretreated with Veh or 500 ng/mL DKK1 for 2 hours, and treated with Veh or 25 ng/mL PDGF-BB for 15 minutes before evaluation of PDGFR signaling by western blot analyses.(G) Pdgfr f/f and Pdgfr cKO osteoblasts were pre-treated with Veh or 500 ng/mL SOST, and treated with Veh or 100 ng/mL Wnt1 for 2 hours before determination of Wnt/β-catenin signaling by western blot analyses.(H) Pdgfr f/f and Pdgfr cKO osteoblasts were pre-treated with Veh or 500 ng/mL SOST, and treated with Veh or 100 ng/mL Wnt1 for 24 hours before measurements of Wisp1 expression by quantitative RT-PCR.

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