Nirogacestat suppresses RANKL-Induced osteoclast formation in vitro and attenuates LPS-Induced bone resorption in vivo

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Nirogacestat suppresses RANKL-Induced osteoclast formation in vitro and attenuates LPS-Induced bone resorption in vivo Xuzhuo Chena,1, Xinwei Chena,1, Zhihang Zhoua, Yi Maoa, Yexin Wanga, Zhigui Maa, Weifeng Xua,***, An Qinb,**, Shanyong Zhanga,* a Department of Oral Surgery, Ninth People's Hospital, College of Stomatology, Shanghai JiaoTong University School of Medicine, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, National Clinical Research Center of Stomatology, Shanghai, 200011, China b Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200011, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Nirogacestat Osteoclasts RANKL Bone resorption Notch2

Bone resorption, initiated by osteoclasts (OCs), plays an essential role in bone homeostasis. The abnormalities of bone resorption may induce a series of diseases, including osteoarthritis, osteoporosis and aseptic peri-implant loosening. Nirogacestat (PF-03084014, PF), a novel gamma-secretase inhibitor, has been used in phase II clinical trial for treatment of desmoid tumor. However, whether it has the therapeutic effect on abnormal bone resorption remains to be evaluated. In this study, we investigated the role of PF in the regulation of receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis in vitro, and the lipopolysaccharide (LPS)-induced bone resorption in vivo. It was found that PF could suppress the formation of osteoclasts from bone marrow macrophages (BMMs) without causing cytotoxicity, inhibit bone resorption and downregulate the mRNA level of osteoclast-specific markers, including calcitonin receptor (CTR), tartrate resistant acid phosphatase (TRAP), cathepsin K (CTSK), dendritic cell-specific transmembrane protein (Dc-stamp), Atp6v0d2 (VATPase d2) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Furthermore, Notch2 signaling, as well as RANKL-induced AKT signaling was significantly inhibited in BMMs. Consistent with in vitro observation, we found that PF greatly ameliorated LPS-induced bone resorption. Taken together, our study demonstrated that PF has a great potential to be used in management of osteolytic diseases.

1. Introduction Bone is a dynamic tissue that is being constantly reshaped by the formation of new bone and the elimination of old bone [1]. The concerted relationship between bone-forming osteoblasts (OBs) and boneresorbing osteoclasts (OCs) is one of the prerequisites for bone homeostasis [2]. Disequilibrium of these two types of cells may undermine the soundness of bone structure, triggering a series of osteolytic diseases including osteoarthritis, osteoporosis and the aseptic peri-implant loosening [3–5]. Thus, prophylactic agents, which can inhibit abnormal bone resorption caused by excessive formation of OCs, are in high demand for susceptible population such as the old, postmenopausal women, and patients undergoing alloplastic joint replacement with unsatisfactory bone condition [6,7].

The differentiation of OCs is the first step for bone resorptive process, induced by macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). M-CSF is essential for osteoclast precursor proliferation and the normal function of RANKL, while RANKL is indispensable for multinucleated OCs formation [8,9]. RANKL is one of the important members of tumor necrosis factor (TNF) family, which can activate the TNF receptor-associated factor 6 (TRAF6) by interacting with RANK, leading to continuous activation of the downstream signaling pathways like NF-κB pathway and mitogenactivated protein kinases (MAPKs) pathways [10–13]. Furthermore, nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is induced by the activation of c-Fos, contributing to the increasing expression of osteoclast-related genes like tartrate resistant acid phosphatase (TRAP) and cathepsin K (CTSK) [14,15].

*

Corresponding author. Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (W. Xu), [email protected] (A. Qin), [email protected] (S. Zhang). 1 These authors contributed equally to this work. **

https://doi.org/10.1016/j.yexcr.2019.06.015 Received 6 January 2019; Received in revised form 11 June 2019; Accepted 14 June 2019 0014-4827/ © 2019 Published by Elsevier Inc.

Please cite this article as: Xuzhuo Chen, et al., Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2019.06.015

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2.4. Osteoclast formation and TRAP staining assay

Nirogacestat (PF-03084014, PF), a novel gamma-secretase inhibitor which can be orally administrated, has been used in phase I and phase II clinical trials for treatment of desmoid tumors. However, whether this compound has the inhibitory effect on osteolysis remains unclear. Gamma-secretase is a multi-subunit protease complex, which has the function of activating notch signaling by producing the released notch intracellular domain (NICD) [16]. It is proved that Notch signaling, especially Notch2 pathway, plays a critical role in RANKL-induced osteoclastogenesis [17–19]. Previous study has shown that overexpression of NICD2 in osteoclasts could rescue the impaired bone resorption activity suppressed by Notch signaling inhibitor, indicating that the activation of Notch2 pathway may upregulate osteoclast formation [20,21]. Therefore, in this study, we attempted to evaluate whether PF could suppress RANKL-induced osteoclastogenesis of bone marrow macrophages (BMMs), and to further demonstrate the possible molecular mechanisms of this process. We found that PF inhibited osteoclast formation and function by suppressing Notch2 and AKT signaling, which was also supported by the in vivo results.

To investigate the effect of PF on osteogenesis, BMMs were seeded into 96-well plates at a density of 1 × 104 cells/well in triplicate. After 24 h, the cells were supplied with complete α-MEM, RANKL (50 ng/ mL), and M-CSF (30 ng/mL) to stimulate osteoclast differentiation in the presence of various concentrations of PF (0, 2.5, 5 and 10 μM). The culture medium was replaced every 2 days until the formation of osteoclasts was observed at day 5. The cells were then fixed with 4% paraformaldehyde for 20 min and stained with the TRAP staining solution at 37 °C for 1 h, according to the manufacturer's protocol. TRAPpositive cells with more than three nuclei were counted as osteoclasts, which were imaged using an optical microscope (Olympus, Tokyo, Japan) and counted using the Image J software (National Institutes of Health). 2.5. Podosome actin belt immunofluorescence BMMs were incubated in 48-well plates at a density of 8 × 104 cells/well for 5 days in complete α-MEM containing M-CSF (30 ng/mL) and RANKL (50 ng/mL), with different dose of PF (0, 2.5, 5 and 10 μM). On the day 5 when the osteoclast formation was observed, Cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and then permeabilized for 5 min with 0.2% Triton X–PBS. F-actin ring in the cells was incubated with FITC-labeled phalloidin for 30 min in darkness. After three times washing with PBS, the nuclei were stained at 37 °C for 10 min with 4’, 6-diamidino-2-phenylindole (DAPI) in darkness. And after further washing in PBS, the cells were observed by using a fluorescence microscope (Leica), and analyzed by using Image J software (National Institutes of Health).

2. Materials and methods 2.1. Reagents and antibodies The gamma-secretase inhibitor Nirogacestat (PF-03084014) was purchased from Selleck (Houston, TX, USA), and was dissolved in Dimethylsulfoxide (DMSO) at a concentration of 10 mM as a stock solution. Recombinant mouse M-CSF and RANKL were obtained from R& D Systems (Minneapolis, MN, USA). Minimal essential medium alpha (α-MEM) was purchased from Basalmedia (Shanghai, China), fetal bovine serum (FBS) was purchased from Gibco BRL (Sydney, Australia), penicillin was purchased from Gibco BRL (Gaithersburg, MD, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technology (Japan). TRAP staining kit was purchased from SigmaAldrich (St. Louis, MO, USA). The Prime Script RT reagent Kit and SYBR® Premix Ex Taq™ II were obtained from Takara Biotechnology (Otsu, Shiga, Japan). Primary antibodies against β-actin, phospho-AKT, AKT, phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, JNK and Hes1, as well as secondary antibody, were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Primary antibody against NFATc1, NICD2 were obtained from Absin Bioscience Inc (Shanghai, China).

2.6. Bone resorption assay For the bone resorption assay, BMMs were seeded into Corning Osteo Assay Surface plates (Corning, NY, USA) at a density of 2 × 104 cells/well, in triplicate, and cultured in complete α-MEM containing M-CSF (30 ng/mL). Twenty-four hours later, the cells were stimulated with M-CSF (30 ng/mL), RANKL (50 ng/mL), and different dose of PF (0, 2.5, 5 and 10 μM) for 9 days. The OCs were then removed by incubating with 5% sodium hypochlorite for 5 min. The total resorption pits were photographed using a BioTek Cytation 3 Cell Imaging Reader (BioTek, Winooski, VT) and analyzed using Image J software (National Institutes of Health). The bone resorption area was normalized by the number of osteoclasts in each group.

2.2. BMMs and culture system

2.7. Quantitative PCR analysis

As reported previously, BMMs were obtained and cultured [22–24]. Briefly, primary BMMs were extracted from the femurs and tibiae of 6week-old C57/BL6 male mice. The isolated cells were suspended in complete α-MEM (α-MEM supplemented with 10% FBS, 100 U/ml penicillin/streptomycin and 30 ng/ml M-CSF). The cell cultures were maintained at 37 °C in a humid environment with 5% CO2 for 5 days to obtain BMMs.

BMMs were seeded in 6-well plates at a density of 3 × 105 cells/well and cultured in complete α-MEM supplemented with M-CSF (30 ng/mL) and RANKL (50 ng/mL). Cells were treated with different doses of PF (0, 2.5, 5, and 10 μM) for 5 days. After the formation of OCs, total RNA was extracted using Axygen RNA Miniprep Kit (Axygen, Union City, CA, USA) according to the manufacturer's instructions. Reverse transcription was performed using the Prime Script RT reagent Kit to obtain cDNA form the RNA template. Subsequently, a real-time PCR assay was performed on an ABI 7500 Sequencing Detection System (Applied Biosystems, Foster City, CA) using the SYBR® Premix Ex Taq™ II. Briefly, 10 μl of SYBR® Premix Ex Taq™ II, 7.2 μl of ddH2O, 2 μl of cDNA, and 0.4 μl of each primer were mixed to make up a total volume of 20 μl for each PCR. Cycling conditions were: 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The specificity of amplification was verified by performing reverse transcription PCR (RT-PCR) and analyzing the melting curves. The comparative 2−ΔΔCT method was used to calculate the relative expression levels of each gene, as described previously [26]. GAPDH was included as housekeeping gene, and all reactions were run in triplicate. The Primers for osteoclastogenic genes used in this study were

2.3. Cell viability assay To evaluate whether PF exhibits cytotoxity to BMMs, BMMs were seeded into 96-well plates in triplicate at a density of 8 × 103 cells/ well, supplied with complete α-MEM and M-CSF (30 ng/mL), and increasing concentrations of PF (0, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM). Following treatment with PF, the cells were incubated for 48, 72, and 96 h, respectively. After treatment, 10 μl of CCK-8 solution was added to each well; the cells were then incubated for 4 h at a wavelength of 450 nm by using a microplate reader. The effect of PF on cell viability was expressed as percent cell viability, with the viability of the control cells set at 100% [25]. 2

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The calvarial osteolysis model was established to evaluate the inhibitory effect of PF on bone resorption in vivo, based upon previous reports [27,28]. Briefly, twenty-four 6-week-old C57/BL6 male mice (approximate weight 20 ± 2 g) were obtained and raised in the Department of Laboratory Animal Science, Shanghai Ninth People's hospital. The mice were divided into four groups with six animals per group: (1) Sham group (PBS); (2) LPS group (LPS treatment with 10 mg/kg and injection with 1 × PBS; (3) PF low-dose group (LPS treatment and injection with 500 μg/kg PF); (4) PF high-dose group (LPS treatment and injection with 2000 μg/kg PF). Gelatin Sponge (4 mm × 4 mm x 2 mm) soaked with PBS or LPS (200 μg) were implanted on the left side of calvaria under general anesthesia. According to the groups mentioned above, 1 × PBS and PF were intraperitoneally injected every other day over a 10-day period. All mice were euthanized at the end of the experiment. Then, whole calvaria bones were separated, then washed with PBS and fixed in 4% paraformaldehyde for 24 h for radiographic and histological analysis.

as follows: The Primers for osteoclastogenic genes used in this study were as follows: mouse NFATc1: forward, 5′-TGCTCCTCCTCCTGCTG CTC-3′ and reverse, 5′-GCAGAAGGTGGAGGTGCAGC-3’; mouse CTR: forward, 5′-TGCAGACAACTCTTGGTTGG-3′ and reverse, 5′-TCGGTTT CTTCTCCTCTGGA-3’; mouse CTSK: forward, 5′-CTTCCAATACGTGCA GCAGA-3′ and reverse, 5′-TCTTCAGGGCTTTCTCGTTC-3’; mouse VATPase d2: forward, 5′-AAGCCTTTGTTTGACGCTGT-3′ and reverse 5′-TTCGATGCCTCTGTGAGATG-3’; mouse TRAP: forward, 5′-CTTCCA ATACGTGCAGCAGA-3′ and reverse, 5′-CCCCAGAGACATGATGAAG TCA-3’; mouse DC-STAMP: Forward, 5′-AAAACCCTTGGGCTGTTCTT-3′ and Reverse, 5′-AATCATGGACGACTCCTTGG-3’; mouse GAPDH: forward, 5′-CACCATGGGAGAAGGCCGGGG-3′ and reverse, 3′-GACGGAC ACATTGGGGGTAG-5’. Notch2: forward, 5′- TCGCCTCATTCATCAGTT TGTG-3′ and reverse, 5′-CTGGCAGTGTTGTCTTCTTCATCT-3’; Hes1: forward, 5′-GCCAGTGTCAACACGACACCGG-3′ and reverse, 5′- TCAC CTCGTTCATGCACTCG-3’; 2.8. Western blotting analysis

2.11. Micro-computed tomography To analyze the protein expression of the long-term activated signaling, BMMs were seeded in 6-well plates at a density of 3 × 105 cells/ well and cultured in complete α-MEM supplemented with M-CSF (30 ng/mL) and RANKL (50 ng/mL). Cells were treated in the presence or absence of PF for 0,1,3 and 5 days, and the total protein of these specific time points was obtained respectively. For analysis of the expression of phosphorylated protein, BMMs were seeded in 6-well plates at a density of 5 × 105 cells/well with complete α-MEM containing MCSF (30 ng/mL). After 24 h, the BMMs were treated with a serum-free α-MEM with/without PF for 3 h, then stimulated with 50 ng/mL RANKL for 0, 5, 10, 20, and 30 and 60 min. After being washed twice in 1 × phosphate-buffered saline (PBS), total protein was extracted from the cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) with protease inhibitor cocktail (Sigma-Aldrich). The lysate was centrifuged at 12,000×g for 15 min and the protein in the supernatant was collected. Protein concentrations were determined using the bicinchoninic acid (BCA) assay. After dissolved in SDS-sample loading buffer, Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose filter membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membranes were blocked in 5% skim milk in 1 × TBST (Tris-buffered saline with Tween 20) at room temperature for 1 h and then incubated with the primary antibodies (â-actin, 1:1000; p-Akt, 1:1000; Akt, 1:1000; p-ERK, 1:1000; ERK, 1:1000; p-p38, 1:1000; p38, 1:1000; p-JNK, 1:1000; and JNK, 1:1000) overnight at 4 °C. Thereafter, the secondary antibodies were incubated for 1 h at room temperature and the antibody reactivity was visualized by using Odyssey V3.0 image scanning (Li-COR. Inc., Lincoln, NE, USA).

Micro-computed tomography (CT) scanning was performed using a high-resolution micro-CT (μCT-100, SCANCO Medical AG, Switzerland). The resolution of the scanning was 10 μm; the X-ray energy was set at 70 kv, 200 μA; and a fixed exposure time was 300 ms. The microstructure indicators of bone volume/tissue volume (BV/TV), were measured in a three-dimensional region of interest (ROI) using evaluation analysis software (Version: 6.5–3, SCANCO Medical AG, Switzerland). The number of pores and percentage of porosity for each sample were measured according to the previous reports [22–24]. 2.12. Histological staining and histomorphometric analysis After micro-CT imaging, the calvarial samples were decalcified in 10% EDTA (pH = 7.4) for 2 weeks and then embedded in paraffin. Histological sections were prepared for hematoxylin and eosin (H&E) and TRAP staining. Immunohistochemical (IHC) staining was accomplished with antibodies against RANKL, OPG, OCN and TNF-α (USA, Affinity; dilution 1:100). The TRAP-positive multinucleated cells were considered as OCs. The stained slices were examined and photographed under a high-quality microscope (Leica DM4000B). The number of TRAP stain-positive osteoclasts was quantified using Image J software. 2.13. Statistical analysis All values are presented as the mean ± standard deviation (SD). Differences between the experimental and control groups were evaluated by using Student's t-test. Results for multiple group comparisons were analyzed using Scheffe's test and one-way analysis of variance (ANOVA) with the SPSS 22.0 software (SPSS Inc., USA). Values were determined to be significant at *P < 0.05, **P < 0.01 and ***P < 0.001.

2.9. OBs culture and assays To explore the effect of PF on osteoblast differentiation, MC3T3-E1 cells were seeded into 24-well plates with 50 μg/mL ascorbic acid, 10 mM β-glycerophosphate and 10−7mM dexamethasone (DXM). The medium was added in 0, 2.5, 5 and 10 μM PF. Alkaline phosphatase (ALP) and Alizarin Red staining was performed using a staining kit (Beyotime, Shanghai, China) on day 7 and day 21 of culture, according to the manufacturer's instructions. The expression of OBs-related genes was evaluated by real-time PCR assay on days 14 of culture.

3. Results 3.1. PF inhibited RANKL-Induced osteoclastogenesis in vitro The effect of PF on RANKL-induced osteoclast differentiation in vitro was investigated first. As shown in Fig. 1A, a large number of trappositive multinucleated OCs formed after 5-day stimulation with M-CSF and RANKL in the control group. However, the treatment of PF with different dose (2.5, 5, and 10 μM) significantly reduced the number and area of OCs in dose dependent manner compared to the control group (Fig. 1B, C). Furthermore, cell viability test was performed to explore whether the inhibition was associated with cytotoxity of PF. The results showed that PF did not exhibit cellular toxicity even when the concentration reached 40 μM (Fig. 1D). Together, these results

2.10. LPS-induced calvarial osteolysis mice model The animal experiment was approved by the Animal Care and Experiment Committee of Shanghai Jiao Tong University School of Medicine. This study was carried out in terms of the guidelines for the Ethical Conduct in the Care and Use of Nonhuman Animals in Research by the American Psychological Association. 3

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Fig. 1. PF inhibited RANKL-induced osteoclastogenesis without cytotoxity in vitro. (A) BMMs were treated with 0, 2.5, 5, and 10 μM PF in the presence of 30 ng/mL M-CSF and 50 ng/mL RANKL for 5 days. Cells were fixed with 4% paraformaldehyde and stained for TRAP. (B) Number of TRAP-positive cells. (C) Area of TRAPpositive cells. (D) BMMs were treated with 0, 2.5, 5, and 10 μM PF in the presence of 30 ng/mL M-CSF for 48, 72, and 96 h to measure cell viability by the CCK-8 assay. The results showed that PF did not exhibit cellular toxicity even when the concentration reached 40 μM. (E) Chemical structure of PF, with a molecular formula of C27H41F2N5O and a molecular weight of 489.64 g/mol. The data are presented as the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

hydroxyapatite-coated Osteo Assay plates after 9-day culture of BMMs with M-CSF and RANKL (Fig. 2D). However, for the groups with different dose of PF (5 and 10 μM), the area of bone resorption pits was markedly reduced, in a dose-dependent manner (Fig. 2E). When treated with PF at 10 μM, it could be observed that the bone resorption area was less than 30% of that of the control group.

demonstrated that PF suppressed RANKL-induce osteoclast differentiation in a dose-dependent manner without cytotoxity even at 40 μM. 3.2. PF suppressed podosome actin belt formation and osteoclast precursor cell fusion As an actin structure, cytoskeletal podosome actin belt circumscribing the plasma of OCs, symbolizes the ability of osteoclast precursor cell fusion [24]. Therefore, we examined the effect of PF on cytoskeletal podosome actin belt formation. As expected, the immunofluorescence results showed that PF dose-dependently suppressed podosome actin belt formation of OCs (Fig. 2A–C). Moreover, BMMs treated with increasing concentrations of PF were primarily mononucleated compared with the control group. Together, these data suggested that PF substantially inhibited podosome actin belt formation as well as osteoclast precursor cell fusion in vitro.

3.4. PF depressed RANKL-Induced osteoclast genes expression To further explore the mechanism behind the inhibitory effect of PF on OCs, the expression of osteoclast genes in the mRNA level was analyzed by real-time quantitative PCR (qPCR). The genes markedly upregulated during osteoclastogenesis were measured, including NFATc1, CTR, CTSK, V-ATPase d2, TRAP and Dc-stamp. According to (Fig. 3A–F), it was found that PF dose-dependently suppressed the transcription of these genes, indicating that PF impaired the differentiation and function of OCs by suppressing RANKL-induced osteoclast genes expression in mRNA level. Meanwhile, we investigated the mRNA expression of Notch signaling related genes, including Notch2 and Hes1. We found that the mRNA level of Hes1 diminished markedly when treated with PF, while no significant changes were observed in the expression of Notch2 (Fig. 3G, H). This result indicated that PF inhibited the downstream

3.3. PF inhibited OCs-Mediated bone resorption activity Considering that PF significantly inhibited RANKL-induced osteoclastogenesis, we further investigated whether PF has the suppressive effect on bone resorption activity, regarded as an important osteoclast function. The results showed substantial resorption on the surface of 4

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Fig. 2. PF impaired podosome actin belt formation and osteoclast precursor cell fusion, and inhibited OCs-mediated bone resorption activity in vitro. (A) BMMs were seeded onto 48 well plates with 0, 2.5, 5, and 10 μM PF in the presence of 30 ng/mL M-CSF and 50 ng/mL RANKL for 5 days. Cells were fixed and stained for immunofluorescence. (B) Number of OCs with F-actin ring. (C) Area of OCs with F-actin ring. (D) BMMs were seeded onto hydroxyapatite-coated Osteo Assay plates after 9-day culture with M-CSF (30 ng/mL), RANKL (50 ng/mL), and with 0, 2.5, 5, and 10 μM PF. Representative scanning images of bone resorption pits are shown. (E) Area of the resorption pits. The data are presented as the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

expression was greatly attenuated especially on the day 3 and day 5. Likewise, Hes1, the downstream regulator of Notch2/NICD2, exhibited increasing expression over time with the treatment of RANKL, while its expression was almost hard to detect after the treatment of PF (Fig. 4C). Furthermore, we investigated whether PF has the inhibitory effect on the expression of NFATc1, one of the key transcription factors for osteoclast differentiation. The results of western blotting showed that the expression of NFATc1 increased gradually over time, staying in its peak after 3-day treatment of RANKL (Fig. 4D). However, for the PF-treated group, the expression level of NFATc1 reduced significantly and

regulators of Notch signaling in mRNA level. 3.5. PF inhibited osteoclastogenesis by downregulating Notch2 signaling pathways and the phosphorylation of AKT To investigate the detailed molecular mechanism on how PF influences RANKL-induced osteoclastogenesis, we first explored the effect of PF on Notch2 signaling pathway in BMMs. As shown in Fig. 4A, B, the expression of NICD2 was strongly upregulated over time, with the stimulation of RANKL. However, for the group treated with PF, the NICD2 5

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Fig. 3. PF depressed the expression of RANKL-induced osteoclast genes. BMMs were treated with M-CSF (30 ng/mL), RANKL (50 ng/mL) in the presence of 0, 2.5, 5, and 10 μM PF for 5 days. (A–H) Expression of the osteoclast-specific genes including NFATc1, CTR, CTSK, V-ATPase d2, TRAP and DC-STAMP, and the Notch signaling related genes including Notch2 and Hes1, were analyzed using quantitative real-time PCR. RNA expression levels were normalized to the expression of Gapdh. The data are presented as the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

group and the PF-treated groups. Meanwhile, cell viability test showed PF had no cytotoxity for the precursor of OBs (Fig. 5C). Furthermore, we investigated the effect of PF on osteoblast genes, including RANKL, OPG, ALP and OCN. It was observed that the expression of OBs-related genes was not affected by treatment of PF in 10 μM, nor the ratio of RANKL/OPG expression. Together, these results suggested that PF did not affect osteoclast differentiation, mineralization and gene expression.

reached its lowest level on the day 5, indicating the inhibitory effect of PF on osteoclastogenesis. Together, these results suggested that PF inhibited the osteoclast procedure by suppressing the activation of Notch2 signaling. Furthermore, we focused on several important signaling pathways that were confirmed to play essential roles in osteogenesis. The activations of AKT and MAPKs were examined by western blotting, with the presence or absence of PF. As shown in Fig. 4E, the RANKL-induced AKT phosphorylation was activated in the control group, reaching the peak at 10 min and 20 min. However, the activation was greatly depressed after the treatment with PF (Fig. 4F). Then, we checked whether PF influenced the activation of MAPKs signaling pathways. It was observed that the phosphorylation of p38, ERK and JNK was activated in the control groups, while no significant inhibitory effect was observed in the PF-treated groups. Combined with the previous results shown above, it was suggested that PF also inhibited osteoclastogenesis via downregulating the phosphorylation of AKT, apart from suppressing the activation of Notch2 signaling.

3.7. Administration of PF prevented LPS-Induced bone resorption without affecting osteoblast activity in vivo The in vitro study elucidated the inhibitory effect of PF on RANKLinduced osteoclast formation and function by studying the phenotype and mechanism. We then attempted to analyze whether PF exhibited protective effect in mice with LPS-induced bone resorption. As shown in Fig. 6A, the three-dimensional (3D) reconstruction of the micro-CT scanning showed that LPS induced severe bone resorption with numerous large and deep pits on the surface of calvaria. In contrast, the bone resorption activity was greatly ameliorated in the PF-treated groups, with fewer and smaller resorption pits. Interestingly, the quantitative analysis indicated that the inhibitory effect also exhibited in a dose-dependent manner in vivo (Fig. 6B–D). We detected the prominent reduction in BV/TV, and increased number of pores as well as the percentage of porosity in the LPS group compared with the sham group. However, the BV/TV almost returned to the normal level in the sham group after administration of PF. Moreover, the number of pores

3.6. PF did not affect osteoblast differentiation and OBs-related gene expression in vitro Bone is a dynamic tissue regulated by the equilibrium of OCs and OBs. The previous data identified the inhibitory effect of PF on OCs in vitro. Therefore, it should be further investigated whether PF influences the osteoblast differentiation. As shown in Fig. 5A, B, ALP and Alizarin Red staining showed no significant difference between the control 6

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Fig. 4. PF inhibited osteoclastogenesis by downregulating Notch2 signaling pathways and the phosphorylation of AKT. (A) BMMs were seeded at 3 × 105 cells/well in 6-well plates and stimulated with M-CSF (30 ng/mL) and RANKL (50 ng/mL), with or without PF (10 μM) for 0, 1, 3 and 5 days. Cells were lysed and subjected to western blotting using specific antibodies against NICD2, Hes1 and NFATc1. (B) PF treatment suppressed the expression of NICD2. (C) PF treatment suppressed the expression of Hes1. (D) PF treatment suppressed the expression of NFATc1. (E) BMMs were seeded at 5 × 105 cells/well in 6-well plates and stimulated with RANKL (50 ng/mL), with or without PF (10 μM) for 0, 5, 10, 20, 30, 60s. (F) PF treatment suppressed AKT phosphorylation expression. The data are presented as the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

TNF-α was not affected in the PF treatment groups compared with the LPS group (Fig. 7F, G). Together, these results indicated that PF attenuated LPS-induced bone resorption without affecting OBs activity in vivo.

and the percentage of porosity also reduced with the increasing concentration of PF. Histological analysis further confirmed that the protective effect of PF on LPS-induced osteolysis in vivo. Consistent with previous data, the results of HE staining revealed extensive osteolysis in the LPS group, whereas the reduced osteolytic level in PF-treated groups (Fig. 7A). Furthermore, the TRAP staining showed the increased number of OCs in the LPS group, while the trap-positive cells decrease dose-dependently after treatment with PF (Fig. 7B, C, H). Furthermore, the IHC staining showed the expression of RANKL and the RANKL/OPG ratio expression increased significantly in all the groups treated with LPS. However, no significant difference was detected between the LPS group and the PFtreated groups (Fig. 7D, E, I). Meanwhile, the expression of OCN and

4. Discussion Due to its dynamic nature, bone is constantly remodeled by the coordination between the bone-forming OBs and bone-resorbing OCs. In the remodeling process, overactivation of OCs may give rise to the imbalances of bone homeostasis, manifesting with a series of osteolytic diseases, including osteoarthritis, rheumatoid arthritis, osteoporosis and the aseptic peri-implant loosening [3–5,29]. Medications which can 7

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Fig. 5. Effect of PF on osteoblast differentiation in vitro. (A, B) MC3T3-E1 cells were treated with 0, 2.5, 5, and 10 μM PF in the presence of ascorbic acid (A.A), βglycerophosphate (β-gly) and dexamethasone (DXM) for 7, 21 days during osteoblastic differentiation. The ALP (Alkaline phosphatase) and Alizarin Red Staining demonstrated no significant difference between control and PF treated groups. (C) PF did not exhibit cytotoxic effect on osteoblast precursor cells. (D) PF did not affect the OBs-related gene expression. The data are presented as the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

resorption in vitro and in vivo. In the present study, we found that PF could inhibit RANKL-induced osteoclast formation and function in a dose-dependent manner without cytotoxity. It was shown that osteoclastogenesis was strongly inhibited, and the formation of podosome actin belt was dose-dependently suppressed after the treatment of PF. Meanwhile, almost no bone resorption pits were observed at the concentration of 10 μM, suggesting PF's inhibitory effect on osteoclast function [39]. For the transcriptional level, the reduced expression of osteoclast-specific genes further demonstrated the suppressive effect of PF on OCs, including NFATc1, CTR, CTSK, V-ATPase d2, TRAP and Dc-stamp. Interestingly, it was found that no significant difference was observed in mRNA level of Notch2, while the expression of Hes1, one of the important downstream regulators of Notch2, was reduced significantly. This data suggested that PF downregulated the activation of Notch2 signaling without affecting the expression of total Notch2. Based upon the results above, we investigated the effect of PF on Notch2 signaling pathway by western blotting. It was found that the production of NICD2, the activated form of Notch2, and Hes1 were markedly suppressed, indicating the inhibitory effect of PF on Notch2 signaling. Meanwhile, the attenuated expression of NFATc1 further indicated that PF had an anti-osteoclastogenic potential in RANKL-induced osteoclastogenesis. It is well established that NFATc1 is a master

help prevent bone-resorbing activity, are widely recommended for the management of these diseases, including bisphosphonates, calcitonin, selective estrogen receptor modulators (SERM) and the newly-advent cathepsin K inhibitors [30–32]. However, these therapies exhibit multiple side-effects, including increased risk of osteonecrosis, renal toxicity and high-allergic reaction [33,34]. Thus, it is highly imperative to develop new agents for the treatment and prevention of bone loss diseases. As one of the highly-conserved signaling in most multicellular organisms, Notch signaling plays an essential role in cell proliferation, differentiation and apoptosis [35]. Previous studies have confirmed the important role of Notch2 signaling in OCs-induced bone resorption [18–21]. Gamma-secretase, an integral membrane protein, cleaves multiple different transmembrane protein complexes to produce their activated forms. Therefore, the inhibitors towards this specific target have the great potential to be used in management of over-activated bone resorption. A series of gamma-secretase inhibitors were reported to exhibit the inhibitory effect on osteoclast formation and differentiation, including DAPT, Semagacestat (LY450139) and Dibenzazepine (YO-01027) [21,36,37]. Nirogacestat (PF), an oral, small molecule, selective gamma-secretase inhibitor, is currently in Phase II clinical trials for treatment of Desmoid tumors [38]. However, there were no reports discussing the role of PF in osteogenesis and bone 8

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Fig. 6. PF suppressed LPS-induced calvarial osteolysis in vivo. (A) Representative images of 3D reconstruction of micro-CT scanning. (B–D) Bone volume to tissue volume (BV/TV, %), number of porosity and the percentage of total porosity of each sample were measured and analyzed. The data are presented as the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

PF. Both the Micro-CT analysis and histological staining showed markedly reduced bone resorptive activity in PF-treated groups, reiterating the inhibitory effect of PF on bone resorption. In addition, The IHC staining demonstrated that administration of PF did not affect the expression of RANKL, OPG and OCN, suggesting the marginal effect of PF on OBs in vivo. Moreover, considering the induced effect of LPS on osteoclast-related cytokines, we further investigate whether PF affects the expression of these cytokines. It was found that the LPS-treated groups exhibited increased expression of RANKL and TNF-α compared with the sham group. However, no significant differences were observed in RANKL and TNF-α after the treatment of PF, indicating that PF may directly inhibit osteoclastogenesis without influencing these osteoclast-related cytokines. However, as a preliminary study investigating the effect of PF on OCs, this study exhibits several limitations. First, although this study clarified the effect of PF on the activation of Notch2 signaling and AKT, we have yet to explain the detailed relationship and interaction between these two signaling pathways, which are under further investigation now. Moreover, it is not suitable to perform repeated local injection for patients with osteolytic diseases. Whether oral administration of PF still exhibit the inhibitory effect on bone resorption remains to be explored. Furthermore, whether PF affects osteoblast activity and inflammatory process should be further explored in other animal models, such as the skull defect and osteoarthritis models. To put it in a nutshell, our data demonstrated PF could effectively suppress RANKL-induced osteoclastogenesis in vitro via downregulating

transcription regulator in formation and function of OCs, regulating the expression of osteoclast-specific genes, including TRAP, CTSK, VATPase-d2 [40]. The reduced expression of these genes in the mRNA level demonstrated the inhibitory effect of PF on the downstream effectors of NFATc1. Therefore, these data suggest that PF might suppress RANKL-induced osteoclast formation by inhibiting Notch2 signaling. The RANKL-induced AKT, ERK, p38 and JNK pathways are essential for the survival and differentiation of OCs [41,42]. Therefore, we attempted to explore the effect of PF on these important OCs-related pathways. Interestingly, PF markedly depressed the phosphorylation of AKT without influencing ERK, p38 and JNK, indicating that PF may inhibit RANKL-induced osteogenesis in a multi-target manner, including Notch2 signaling and AKT pathway. Moreover, because bone homeostasis is maintained by the equilibrium between OCs and OBs, we then examined whether PF may influence osteoblast differentiation and functions. It was found that PF did not affect osteoblast differentiation, mineralization and gene expression in the concentration of 10 μM, indicating that the treatment of PF inhibited osteoclastogenesis without affecting the normal function of OBs in vitro. Consistent with the anti-osteoclastogenic and anti-resorptive property in vitro, our in vivo results further clarified the protective effect of PF on LPS-induced osteolysis in vivo. The calvarial bone loss model induced by LPS is a widely-accepted experimental method for the investigation of osteolytic diseases [43,44]. In this study, we used the LPS-induced calvarial osteolytic model with intraperitoneal injection of 9

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Fig. 7. Histological analysis of the inhibitory effect of PF on LPS-induced bone resorption. (A–C) Representative images of HE and TRAP staining, showing the reduced osteolytic legion and TRAP-positive OCs in the PF-treated groups. (D–G) Representative images of IHC staining of RANKL, OPG, OCN and TNF-α. (H) TRAPpositive OCs number. (H) Quantitative analysis of the expression of RANKL, OPG and RANKL/OPG ratio. The data are presented as the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

Acknowledgments

Notch2 signaling and phosphorylation of AKT, and attenuates LPS-induced osteolysis in vivo. Consistent with in vitro observation, we found that PF greatly ameliorated LPS-induced bone resorption PF could suppress RANKL-induced osteoclastogenesis and has a great potential to be used in treatment of bone lytic diseases.

This work was supported by National Natural Science Foundation of China (Grant No. 81671010; 81572167; 81772373; 81800932); Shanghai Hospital Development Center (Grant No. 16CR3104B); Science and Technology Commission of Shanghai Municipality (Grant No. 16441908800); Research Fund of Medicine and Engineering of Shanghai Jiao Tong University (Grant No. YG2016QN04); and the Innovation Fund for Doctoral Program of Shanghai Jiao Tong University, School of Medicine (BXJ201931).

Conflicts of interest The authors declare no conflict of interest. 10

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References

[23] C. Wu, W. Wang, B. Tian, X. Liu, X. Qu, Z. Zhai, H. Li, F. Liu, Q. Fan, T. Tang, et al., Myricetin prevents titanium particle-induced osteolysis in vivo and inhibits RANKLinduced osteoclastogenesis in vitro, Biochem. Pharmacol. 93 (2015) 59–71. [24] H. Touaitahuata, A. Morel, S. Urbach, J. Mateos-Langerak, S. de Rossi, A. Blangy, Tensin 3 is a new partner of Dock5 that controls osteoclast podosome organization and activity, J. Cell Sci. 129 (2016) 3449–3461. [25] Z. Jiao, W. Xu, J. Zheng, P. Shen, A. Qin, S. Zhang, C. Yang, Kaempferide prevents titanium particle induced osteolysis by suppressing JNK activation during osteoclast formation, Sci. Rep. 7 (2017) 16665. [26] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25 (2001) 402–408. [27] Y.H. Cheon, J.Y. Kim, J.M. Baek, S.J. Ahn, H.S. So, J. Oh, Niclosamide suppresses RANKL-induced osteoclastogenesis and prevents LPS-induced bone loss, Biochem. Biophys. Res. Commun. 470 (2016) 343–349. [28] C.M. Wei, Q. Liu, F.M. Song, X.X. Lin, Y.J. Su, J. Xu, L. Huang, S.H. Zong, J.M. Zhao, Artesunate inhibits RANKL-induced osteoclastogenesis and bone resorption in vitro and prevents LPS-induced bone loss in vivo, J. Cell. Physiol. 233 (2018) 476–485. [29] E. Nevius, A.C. Gomes, J.P. Pereira, Inflammatory cell migration in rheumatoid arthritis: a comprehensive review, Clin. Rev. Allergy Immunol. 51 (2016) 59–78. [30] P. Hadji, N. Papaioannou, E. Gielen, M. Feudjo Tepie, E. Zhang, I. Frieling, P. Geusens, P. Makras, H. Resch, G. Möller, et al., Persistence, adherence, and medication-taking behavior in women with postmenopausal osteoporosis receiving denosumab in routine practice in Germany, Austria, Greece, and Belgium: 12month results from a European non-interventional study, Osteoporos. Int. 26 (2015) 2479–2489. [31] M. Hiligsmann, S.M. Evers, W. Ben Sedrine, J.A. Kanis, B. Ramaekers, J.Y. Reginster, S. Silverman, C.E. Wyers, A. Boonen, A systematic review of costeffectiveness analyses of drugs for postmenopausal osteoporosis, Pharmacoeconomics 33 (2015) 205–224. [32] K. Mukherjee, N. Chattopadhyay, Pharmacological inhibition of cathepsin K: a promising novel approach for postmenopausal osteoporosis therapy, Biochem. Pharmacol. 117 (2016) 10–19. [33] F.S. Hough, S.L. Brown, B. Cassim, M.R. Davey, W. de Lange, T.J. de Villiers, G.C. Ellis, S. Lipschitz, M. Lukhele, J.M. Pettifor, et al., The safety of osteoporosis medication, S. Afr. Med. J. 104 (2014) 279–282. [34] N.V. Hinchy, V. Jayaprakash, R.A. Rossitto, P.L. Anders, K.C. Korff, P. Canallatos, M.A. Sullivan, Osteonecrosis of the jaw-Prevention and treatment strategies for oral health professionals, Oral Oncol. 49 (2013) 878–886. [35] S. Artavanis-Tsakonas, M.D. Rand, R.J. Lake, Notch signaling: cell fate control and signal integration in development, Science 284 (1999) 770–776. [36] J.W. Ashley, J. Ahn, K.D. Hankenson, Notch signaling promotes osteoclast maturation and resorptive activity, J. Cell. Biochem. 116 (2015) 2598–2609. [37] E. Canalis, L. Schilling, S.P. Yee, S.K. Lee, S. Zanotti, Hajdu cheney mouse mutants exhibit osteopenia, increased osteoclastogenesis, and bone resorption, J. Biol. Chem. 291 (2016) 1538–1551. [38] B. Hoffner, A.D. Elias, V.M. Villalobos, Targeted therapies in the treatment of sarcomas, Targeted Oncol. 13 (2018) 557–565. [39] J.M. Baek, J.Y. Kim, C.H. Lee, K.H. Yoon, M.S. Lee, Methyl gallate inhibits osteoclast formation and function by suppressing Akt and btk-PLCγ2-Ca2+ signaling and prevents lipopolysaccharide-induced bone loss, Int. J. Mol. Sci. 18 (2017) 581. [40] F. Ikeda, R. Nishimura, T. Matsubara, S. Tanaka, J. Inoue, S.V. Reddy, K. Hata, K. Yamashita, T. Hiraga, T. Watanabe, et al., Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation, J. Clin. Investig. 114 (2004) 475–484. [41] M. Wu, W. Chen, Y. Lu, G. Zhu, L. Hao, Y.P. Li, Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signaling pathway, Nat. Commun. 8 (2017) 13700. [42] K. Lee, Y.H. Chung, H. Ahn, H. Kim, J. Rho, D. Jeong, Selective regulation of MAPK signaling mediates RANKL-dependent osteoclast differentiation, Int. J. Biol. Sci. 12 (2016) 235–245. [43] L. Li, M. Sapkota, S.W. Kim, Y. Soh, Herbacetin inhibits RANKL-mediated osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo, Eur. J. Pharmacol. 777 (2016) 17–25. [44] C.M. Wei, Y.J. Su, X. Qin, J.X. Ding, Q. Liu, F.M. Song, S.H. Zong, J. Xu, B. Zhou, J.M. Zhao, Monocrotaline suppresses RANKL-induced osteoclastogenesis in vitro and prevents LPS-induced bone loss in vivo, Cell. Physiol. Biochem. 48 (2018) 644–656.

[1] T.D. Rachner, S. Khosla, L.C. Hofbauer, Osteoporosis: now and the future, Lancet 377 (2011) 1276–1287. [2] B. Langdahl, S. Ferrari, D.W. Dempster, Bone modeling and remodeling: potential as therapeutic targets for the treatment of osteoporosis, Ther. Adv. Musculoskelet. Dis. 8 (2016) 225. [3] A. Bertuglia, M. Lacourt, C. Girard, G. Beauchamp, H. Richard, S. Laverty, Osteoclasts are recruited to the subchondral bone in naturally occurring posttraumatic equine carpal osteoarthritis and may contribute to cartilage degradation, Osteoarthritis Cartilage 2 (2016) 555–566. [4] K. Mukherjee, N. Chattopadhyay, Pharmacological inhibition of cathepsin K: a promising novel approach for postmenopausal osteoporosis therapy, Biochem. Pharmacol. 117 (2016) 10–19. [5] E.R. Kapasa, P.V. Giannoudis, X. Jia, P.V. Hatton, X.B. Yang, The effect of RANKL/ OPG balance on reducing implant complications, J. Funct. Biomater. 8 (2017) e42. [6] S.H. Tella, J.C. Gallagher, Prevention and treatment of postmenopausal osteoporosis, J. Steroid Biochem. Mol. Biol. 142 (2014) 155–170. [7] S. Alami, L. Hervouet, S. Poiraudeau, K. Briot, C. Roux, Barriers to effective postmenopausal osteoporosis treatment: a qualitative study of patients' and practitioners' views, PLoS One 11 (2016) e0158365. [8] D.M. Biskobing, X. Fan, J. Rubin, Characterization of MCSF-induced proliferation and subsequent osteoclast formation in murine marrow culture, J. Bone Miner. Res. 10 (1995) 1025–1032. [9] N. Udagawa, N. Takahashi, E. Jimi, K. Matsuzaki, T. Tsurukai, K. Itoh, N. Nakagawa, H. Yasuda, M. Goto, E. Tsuda, et al., Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/ RANKL but not macrophage colony-stimulating factor, Bone 25 (1999) 517–523. [10] K. Lee, Y.H. Chung, H. Ahn, H. Kim, J. Rho, D. Jeong, Selective regulation of MAPK signaling mediates RANKL-dependent osteoclast differentiation, Int. J. Biol. Sci. 12 (2016) 235–245. [11] S. Strickson, C.H. Emmerich, E.T.H. Goh, J. Zhang, I.R. Kelsall, T. Macartney, C.J. Hastie, A. Knebel, M. Peggie, F. Marchesi, et al., Roles of the TRAF6 and pellino E3 ligases in MyD88 and RANKL signaling, Proc. Natl. Acad. Sci. U.S.A. 114 (2017) E3481. [12] D.V. Novack, Role of NF-κB in the skeleton, Cell Res. 21 (2011) 169–182. [13] H. Huang, J. Ryu, J. Ha, E.J. Chang, H.J. Kim, H.M. Kim, T. Kitamura, Z.H. Lee, H.H. Kim, Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and Nf-kappaB transactivation by RANKL, Cell Death Differ. 13 (2006) 1879–1891. [14] Y. Zhang, S. Xu, K. Li, K. Tan, K. Liang, J. Wang, J. Shen, W. Zou, L. Hu, D. Cai, et al., mTORC1 inhibits NF-κB/NFATc1 signaling and prevents osteoclast precursor differentiation, in vitro and in mice, J. Bone Miner. Res. 32 (2017) 1829–1840. [15] K. Kim, T.H. Kim, H.J. Ihn, J.E. Kim, J.Y. Choi, H.I. Shin, E.K. Park, Inhibitory effect of purpurogallin on osteoclast differentiation in vitro through the downregulation of c-fos and NFATc1, Int. J. Mol. Sci. 19 (2018) 601. [16] T.E. Golde, E.H. Koo, K.M. Felsenstein, B.A. Osborne, L. Miele, γ-Secretase inhibitors and modulators, Biochim. Biophys. Acta 1828 (2013) 2898–2907. [17] B. Isidor, P. Lindenbaum, O. Pichon, S. Bézieau, C. Dina, S. Jacquemont, D. MartinCoignard, C. Thauvin-Robinet, M. Le Merrer, J.L. Mandel, et al., Truncating mutations in the last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis, Nat. Genet. 43 (2011) 306–308. [18] S. Zanotti, J. Yu, A. Sanjay, L. Schilling, C. Schoenherr, A.N. Economides, E. Canalis, Sustained Notch2 signaling in osteoblasts, but not in osteoclasts, is linked to osteopenia in a mouse model of Hajdu-Cheney syndrome, J. Biol. Chem. 292 (2017) 12232–12244. [19] H. Fukushima, K. Shimizu, A. Watahiki, S. Hoshikawa, T. Kosho, D. Oba, S. Sakano, M. Arakaki, A. Yamada, K. Nagashima, et al., NOTCH2 hajdu-cheney mutations escape SCFFBW7-dependent proteolysis to promote osteoporosis, Mol. Cell 68 (2017) 645–658. [20] H. Fukushima, A. Nakao, F. Okamoto, M. Shin, H. Kajiya, S. Sakano, A. Bigas, E. Jimi, K. Okabe, The association of Notch2 and NF-κB accelerates RANKL-induced osteoclastogenesis, Mol. Cell. Biol. 28 (2008) 6402–6412. [21] W.J. Jin, B. Kim, J.W. Kim, H.H. Kim, H. Ha, Z.H. Lee, Notch2 signaling promotes osteoclast resorption via activation of PYK2, Cell. Signal. 28 (2016) 357–365. [22] Z. Zhai, X. Qu, H. Li, K. Yang, P. Wan, L. Tan, Z. Ouyang, X. Liu, B. Tian, F. Xiao, et al., The effect of metallic magnesium degradation products on osteoclast-induced osteolysis and attenuation of NF-κB and NFATc1 signaling, Biomaterials 35 (2014) 6299–6310.

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