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. 2018 Mar 30;233(9):7415–7423. doi: 10.1002/jcp.26587

Alendronate induces osteoclast precursor apoptosis via peroxisomal dysfunction mediated ER stress

Ning Ding 1, Chuan Liu 2,3, Li Yao 3, Yun Bai 2, Peng Cheng 1, Zhilin Li 1, Keyu Luo 1, Tieniu Mei 4, Jianhua Li 5, Junchao Xing 1, Xiaoliang Gao 1, Qinyu Ma 1, Jianzhong Xu 1, Fei Luo 1,✉, Ce Dou 1,2,✉
PMCID: PMC13482122  PMID: 29600563

Abstract

Nitrogen‐containing bisphosphonates including alendronate (ALN) are the current first line antiresorptive drug in treating osteoporosis. In our study, we found that ALN administration impaired the secretion of platelet derived growth factor‐BB (PDGF‐BB), the most important angiogenic cytokines produced by preosteoclast (POC), in both sham and ovariectomized (OVX) mice. To further understand this phenomenon, we induced bone marrow macrophages (BMMs) to POCs in vitro and detected the effects of ALN particularly in POCs. The proapoptotic effect of ALN in POCs was confirmed by flow cytometry. On the molecular level, we found that farnesyl diphosphate synthase (FDPS) inhibition of ALN led to peroxisomal dysfunction and up regulation of cytoprotective protein glucose‐regulated protein (GRP) 78. Peroxisomal dysfunction further induced endoplasmic reticulum (ER) stress in POCs and finally resulted in cell apoptosis marked by reduced expression of B‐cell lymphoma 2 (Bcl‐2) and increased expressions of CCAAT/enhancer binding protein homologous protein (CHOP), Bcl2 associated X (Bax), and cleaved caspase‐3. We concluded that ALN has no selectivity in inhibiting POC and mature osteoclast. For POCs, ALN inhibition of FDPS leads to peroxisomal dysfunction, which further mediates ER stress and finally causes cell apoptosis. Considering that decreased angiogenesis is also an important issue in treating osteoporosis, how to preserve pro‐angiogenic POCs while depleting mature osteoclasts is a problem worthy to be solved.

Keywords: alendronate, apoptosis, ER stress, osteoclast, peroxisome

Alendronate inhibit both mature osteoclast and preosteoclast without selectivity. Avoiding POC depletion in nitrogen‐containing bisphosphonates based antiresorptive therapy is a potential strategy in improving the curative effects.

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1. INTRODUCTION

Bone homeostasis is maintained by delicate balance between osteoclast (OC)‐mediated bone resorption and osteoblast (OB)‐mediated bone formation. These two cell types collaborate together orchestrating bone remodeling throughout life. Derived from hematopoietic stem cells (HSCs), OCs are bone specific multinucleated cells. Two important regulating factors, receptor activator of nuclear factor κB ligand (RANKL) and macrophage‐colony stimulating factor (M‐CSF) are necessary for OC differentiation and survival (Lacey et al., 1998; Yasuda et al., 1998). Binding of RANKL to its ligand results in the initiation of the TNF receptor‐associated factor (TRAF) 6 (TRAF‐6) signaling, which eventually activates nuclear factor of activated T cells c1 (NFATc1), the master regulator in osteoclastogenesis (Hwang & Putney, 2011; Li et al., 2014), leading to OC differentiation and maturation (Roodman, 1996; Teitelbaum, 2000). Dysregulation of osteoclastogenesis can lead to various bone disorders and the most common one is osteoporosis (Le Goff, Berthelot, Maugars, & Heymann, 2013; Rachner, Khosla, & Hofbauer, 2011; Sobacchi, Schulz, Coxon, Villa, & Helfrich, 2013).

For treatment of osteoporosis, the most commonly used antiresorptive drug is bisphosphonate. Bisphosphonates have a very high affinity for bone mineral by binding to hydroxyapatite crystals thus inhibit hydroxyapatite breakdown, thereby effectively suppress bone resorption (Russell, Muhlbauer, Bisaz, Williams, & Fleisch, 1970). The presence of a nitrogen or amino group increases the bisphosphonate's antiresorptive potency by 10–10,000 relative to non–nitrogen‐containing bisphosphonates (Dunford et al., 2001). Nitrogen‐containing bisphosphonate such as alendronate (ALN) binds to and inhibit the activity of farnesyl diphosphate synthase (FDPS), a key regulatory enzyme in the mevalonic acid pathway critical to the production of cholesterol and isoprenoid lipids (Kavanagh et al., 2006). Accordingly, the prenylation of small guanosine triphosphate (GTP)‐binding proteins including Rab, Rac, and Rho is inhibited thus disrupting fiber assembly and membrane ruffling. The failure in forming actin ring and ruffle border eventually lead to cell dysfunction and apoptosis in mature osteoclast (Drake, Clarke, & Khosla, 2008; Kavanagh et al., 2006).

Activated from preosteoclast (POC), mature OC has a relatively short lifespan of about 2 weeks and then undergoes cell apoptosis (Manolagas, 2000). According to recent discoveries, POCs barely resorb bone matrix and are even beneficial for angiogenesis through secreting platelet derived growth factor‐BB (PDGF‐BB) (Kusumbe & Adams, 2014; Teti, 2013; Xie et al., 2014). However, nitrogen‐containing bisphosphonates including ALN seem to have no selectivity in depleting osteoclasts (Drake et al., 2008; Rachner et al., 2011). This potentially explained why current long‐term antiresorptive therapy is not satisfying and even increases risk of atypical fractures (Adler et al., 2016; Misof et al., 2015). We assumed that depletion of mature OCs while preserving POCs will be a more effective strategy in treating osteoporosis. The objective of this study is to investigate and understand the underlying mechanism of ALN effects in POCs for preservation of POCs and better treatment for osteoporosis.

In this study, for the first time, we found that the inhibition of FDPS by ALN causes peroxisome dysfunction and further induces endoplasmic reticulum (ER) stress in POCs. Excessive and prolonged ER stress eventually trigger cell apoptosis in POCs.

2. MATERIALS AND METHODS

2.1. Mice

C57BL/6 mice were provided by the animal center of Third Military Medical University. All experimental procedures were approved by Third Military Medical University and performed according to guidelines of laboratory animal care and use. All efforts were made to reduce the number of animals tested and their suffering. Bone marrow cells were separated and cultured with M‐CSF (50 ng/ml) for 24 hr to obtain bone marrow macrophages (BMMs). Mice were divided into four groups: Sham operated mice (sham, n = 8), ovariectomized (OVX) mice (control, n = 8), low dosage ALN treated OVX mice (10 mg/kg/week, n = 8) and high dosage ALN treated OVX mice (100 mg/kg/week, n = 8). Mice were weighed weekly and concentrations were calculated for the ALN administration for 4 weeks. Control groups received normal saline. Feed consumption and body weight were monitored weekly for 4 weeks. No adverse effects were detected. All treated mice were sacrificed by cervical dislocation after 1 week of last administration.

2.2. In vitro assays for osteoclast differentiation

Bone marrow cells were separated and cultured with M‐CSF (50 ng/ml) for 24 hr to obtain BMMs. Cells were cultured in α‐minimal essential medium (MEM) containing 10% FBS and 1% Penicillin‐streptomycin solution. For tartrate resistant acid phosphatase (TRAP) stain, cells were cultured in a 96‐well plate at a density of 5 × 103 cells/well with RANKL (100 ng/ml) and M‐CSF (50 ng/ml) for 24 hr. Cells were fixed in 4% paraformaldehyde for 20 min and then stained with TRAP staining solution (0.1 mg/ml of naphthol AS‐MX phosphate, 0.3 mg/ml of Fast Red Violet LB stain) according to the manufacturers’ instructions. TRAP stained cells counted with three or more nuclei were used to identify osteoclasts. Relative TRAP activity was measured by colorimetric analysis using software Image J (Ver 1.41). For actin cytoskeleton and focal adhesion stain, cells were cultured on glass sheet in a 12‐well plate at a density of 4 × 104 cells/well with RANKL (100 ng/ml) and M‐CSF (50 ng/ml) for 24 hr. Procedures were described in previous study (Dou, Zhang, et al., 2014). In brief, on day 4, cells were washed and fixed for permeabilization. After blocking, primary antibody was then diluted to a working concentration (1:300) in blocking solution, and cells were incubated for 1 hr at room temperature. Secondary antibody (Alexa Fluor 488/555 Goat Anti‐Mouse IgG (H + L) Antibody, Invitrogen, Carlsbad, CA) (1:500) and TRITC conjugated Phalloidin (1:500) was diluted in 1 × PBS and cells were incubated for 1 hr at room temperature. Nuclei counterstaining was performed by DAPI (1:1000) for 5 min followed by confocal microscopy observation.

2.3. Flow cytometry

Flow cytometry was used for analysis of cell cycle change and cell apoptosis. For cell cycle analysis, 1 × 106 BMMs were induced with RANKL (100 ng/ml) and M‐CSF (50 ng/ml) for 24 hr with ALN of different concentrations (0, 10, 30, and 50 µM). Cells were then washed in PBS for two times and fixed by 70% ethonal. After fixation, cells were washed with PBS for another time and stained with 10 µg propidiumiodide (PI, Sigma–Aldrich, St. Louis, MO) together with RNase treatment for 30 min (37 °C). A FACStar flow cytometer (BD, Triangle, NC) was adopted to measure the percentages of cells in the different cell cycle phases and cells having sub‐G1 DNA contents. The results were analyzed by Lysis II and Cellfit software (BD). Cell apoptosis was determined by Annexin V/PI staining as previously described. (Dou, Li, et al., 2014). In brief, BMMs were induced with RANKL (100 ng/ml) and M‐CSF (50 ng/ml) for 24 hr with ALN of different concentrations (0, 10, 30, and 50 µM). Cells were washed twice with cold PBS and then resuspended in 500 µl of binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2] at a concentration of 1 × 106 cells/ml. Cells were then stained with 5 µl of annexin V‐FITC (Life Technologies) and 10 µl of 20 µg/ml PI. Apoptosis was analyzed using a FACStar flow cytometer (BD). Viable cells were negative for both annexin V and PI; early apoptotic (still viable) were positive for annexin V and negative for PI; late apoptotic cells (non‐viable) cells were both positive for annexin V and PI; necrotic cells were positive for PI and negative for annexin V.

2.4. µCT analysis and immunofluorescence analysis

For µCT analysis (Bouxsein et al., 2010), Bruker MicroCT Skyscan 1272 system (Kontich city, Belgium) with an isotropic voxel size of 10.0 µm was used to image the whole femur. Scans were conducted in 4% paraformaldehyde and used an x‐ray tube potential of 60 kV, an x‐ray intensity of 166 µA, and an exposure time of 1700 ms. For trabecular bone analysis of the distal femur, an upper 3 mm region beginning 0.8 mm proximal to the most proximal central epiphysis of the femurwas contoured. For cortical bone analysis of femur (2D analysis), a 0.5 mm region beginning 4.5 mm proximal to the most proximal central epiphysis of the femur. Trabecular and cortical bones were threshholded at 86–255 (8bit gray scale bitmap). µCT scans of whole body of mice (except skull) were performed using isotropic voxel sizes of 148 µm. Reconstruction was accomplished by Nrecon (Ver. 1.6.10). 3D images were obtained from contoured 2D images by methods based on distance transformationof the gray scale original images (CTvox, Ver. 3.0.0). 3D and 2D analysis were performed using software CT Analyser (Ver. 1.15.4.0). All images presented are representative of the respective groups.

For the bone histological analysis (Dempster et al., 2013), femurs were dissected and fixed in 4% paraformaldehyde in PBS for 48 hr. Femurs were then decalcified by daily change of 15% tetrasodium EDTA for 2 weeks. We processed three longitudinally oriented sections of trabecular bone for immunofluorescence analysis in each experimental group. Briefly, bone sections were incubated with individual primary antibodies to mouse TRAP (Santa Cruz Biotechnology, Santa Cruz, CA 1:100), PDGF‐BB (Santa Cruz Biotechnology, 1:100) overnight at 4 °C. Subsequently, secondary antibodies conjugated with fluorescence were used at room temperature for 1 hr while avoiding light. Leica TCS SP8 confocal microscope was used for imaging samples.

2.5. Statistical analysis

All data are representative of at least three experiments of similar results performed in triplicate unless otherwise indicated. Data are expressed as mean ± SD. One‐way ANOVA followed by Student–Newman–Keuls post hoc tests was used to determine the significance of difference between results, with *p < 0.05, **p < 0.01 being regarded as significant.

The complete detailed methods are provided in the supplemental materials.

3. RESULTS

3.1. ALN impairs PDGF‐BB secretion in both sham and OVX mice

We performed ovariectomy in 6 week old female C57BL/6 mice to simulate the osteoporotic condition. OVX mice exhibited significant lower bone mineral density (BMD), trabecular bone volume fraction (BV/TV), and trabecular number (Tb.N) compared with sham mice (Figure 1a). ALN was then administered (10 and 100 mg/kg) for osteoporosis treatment. The results showed that both low and high dosages of ALN significantly increased the BMD, BV/TV, and Tb.N in OVX mice. For detailed trabecular bone analysis, an upper 3 mm region beginning 0.8 mm proximal to the most proximal central epiphysis of the femur was contoured and reconstructed (Figure 1b). Quantification analysis showed that ALN reduced trabecular separation (Tb.Sp) while trabecular thickness (Tb.Th) was not affected (Figure 1b). Further immunofluorescent staining of bone marrow sections characterizing TRAP and PDGF‐BB positive cells was performed (Figure 1c). The results revealed that TRAP+ cell number increased significantly in OVX and was reduced sharply by ALN administration. Although bone volume was rescued by ALN treatment, the decrease of PDGF‐BB+ cell number in OVX mice was not recovered by ALN. Furthermore, sham mice treated with ALN also exhibited a significant decrease in PDGF‐BB+ cell number (Supplemental Figure S1). These results suggested that ALN treatment in both OVX mice and sham mice impaired PDGF‐BB secretion. Considering that PDGF‐BB is mainly produced by POCs, we assumed that ALN inhibit both mature OC and POCs in treating osteoporosis.

Figure 1.

Figure 1

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ALN impairs PDGF‐BB secretion in treating osteoporosis in OVX mice. (a) Representative µCT images of longitudinal section femurs and quantification of bone mineral density (BMD), trabecular bone volume fraction (BV/TV), trabecular number (Tb. N), and cortical thickness (Ct. Th) (b) Reconstructed trabecular structure from distal femur, color scale bar represents bone mineral density level. Quantification of trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were shown. (c) Immunostaining of TRAP (red), PDGF‐BB (green) in distal femur bone marrow sections. Bar represents 100 µm with quantification of number of TRAP positive cells (N. TRAP +), and number of PDGF‐BB positive cells per respective trabecular bone (TB) (N. PDGF‐BB +). Images are representative of n = 5 independent experiments. The data in the figures represent the averages ± SD. Significant differences are indicated as ** (p < 0.01)

3.2. ALN reduces POC number and PDGF‐BB secretion in vitro

To study the effects of ALN in POCs particularly, bone marrow macrophages (BMMs) isolated from mouse femur were cultured in the presence of RANKL (100 ng/ml) and M‐CSF (50 ng/ml) for 24 hr to obtain POCs as we previously reported (Dou et al., 2016). Obtained POCs were then treated with ALN at different concentrations (0, 10, 30, and 50 µM) for another 24 hr. TRAP stain results and immunofluorescent staining of F‐actin of these cells were then performed (Figure 2a). The results showed that ALN dose dependently decreased TRAP positive POC number and relative TRAP activity. In addition, total cell count of POCs was also decreased by ALN treatment suggesting the increased cytotoxicity (Figure 2b). Western blot analysis showed an obvious reduction of PDGF‐BB treated with ALN (10 and 50 µM) in POCs (Figure 2c). Moreover, qPCR results suggested no significant change of PDGF‐BB expression while the ELISA results revealed a decrease secretion of PDGF‐BB in POCs treated with ALN (Figure 2d). The above results showed that ALN treatment reduced POC number and PDGF‐BB secretion in vitro.

Figure 2.

Figure 2

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ALN reduces POC number and PDGF‐BB secretion in vitro. (a) TRAP stain and immunostaining of F‐actin in BMMs or POCs treated with ALN (0, 10, 30, and 50 µM). Bar represents 200 µm. (b) Quantification of TRAP+ POC number per well, relative TRAP activity and total cell counts per well. (c) Western blot analysis of PDGF‐BB and β‐actin in POCs treated with ALN (0, 10, and 50 µM). (d) Relative mRNA expression of PDGFB and ELISA test for CM PDGF‐BB concentration of POCs treated with ALN (0, 10, and 50 μM). Images are representative of n = 3 independent experiments. The data in the figures represent the averages ± SD. Significant differences are indicated as ** (p < 0.01)

3.3. ALN induces cell apoptosis and cell cycle arrest in POCs

To explain why the cell number of POC decreased, we first investigated the effects of ALN on cell viability of POCs. POCs or BMMs were treated with ALN at different concentrations (0, 1, 5, 10, 20, 30, 40, and 50 µM) for 24 hr before evaluation of cell viability. The results showed that ALN became toxic when the concentration is greater than 20 µM in both BMMs and POCs (Figure 3a). For verification, FITC‐Annexin‐V/PI stain detected by flow cytometry (FCM) (Figure 3b) revealed that ALN of 50 µM significantly increased both early and late cell apoptosis rate in POCs (Figure 3c). In addition, we used FCM to detect the effects of ALN on cell cycle change of POCs (Figure 3d). The results showed that ALN of 50 µM significantly increased G0/G1 cell proportion and reduced G2/M cell proportion, suggesting the arrest of cell cycle (Figure 3e). Herein, we concluded that ALN induces cell apoptosis and cell cycle arrest in POCs.

Figure 3.

Figure 3

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ALN induces cell apoptosis and cell cycle arrest in POCs. (a) Cell viability of ALN (0, 1, 5, 10, 20, 30, 40, and 50 µM) in POCs using cell counting kit‐8. (b) Flow cytometry (FCM) analysis of annexin‐V/PI staining in POCs treated with ALN (0, 10, 30, and 50 µM). (c) Quantification or early and late cell apoptosis rate. (d) FCM analysis of cell cycle in POCs treated with ALN (0, 10, 30, and 50 µM). (e) Quantification of percentage of cells at G0/G1 and G2/M. Images are representative of n = 3 independent experiments. The data in the figures represent the averages ± SD. Significant differences are indicated as * (p < 0.05)

3.4. ALN causes peroxisomal dysfunction and upregulation of GRP78 in POCs

To further explore the reason of cell apoptosis and cell cycle arrest in POC induced by ALN, we then performed Western blot to detect 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A (HMG‐CoA) reductase and FDPS expression in POCs treated with ALN for 24 hr. As expected, ALN reduced its target protein FDPS expression in POCs while the expression of HMG‐CoA reductase was not affected (Figure 4a). Taking into consideration that FDPS is localized in peroxisomes (Biardi et al., 1994; Krisans et al., 1994), we asked whether peroxisome function might be affected by ALN treatment. For investigation, immunofluorescent stain of cytoplasmic catalase, normally a luminal peroxisomal marker was performed in POC treated with ALN for 24 hr. The results revealed that catalase (green) is present in peroxisomes in POCs of controls, but is localized in the cytoplasm in POCs treated with ALN suggesting a peroxisomal dysfunction (Figure 4b). Interestingly, further Western blot analysis showed that ALN treatment in POCs for 24 hr induced the cytoprotective protein GRP78 (BiP) expression (Figure 4c). Correspondingly, the increase of GRP78 mRNA expression was confirmed by qPCR detection at 12 hr after ALN was introduced (Figure 4d). As ER chaperone, the upregulation of GRP78 indicates the occurrence of ER stress.

Figure 4.

Figure 4

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ALN causes peroxisomal dysfunction and upregulation of GRP78 in POCs. (a) Western blot analysis of HMG‐CoA reductase, FDPS and GAPDH in POCs treated with ALN (0, 10, 30, and 50 µM). (b) Immunostaining of catalase in POCs treated with ALN (50 µM). Bar represents 30 µm. (c) Western blot analysis of GRP78 and β‐actin in POCs treated with ALN (0, 10, 30, and 50 µM). (d) Relative mRNA expression of GRP78 in POCs 12 hr after ALN treatment. Images are representative of n = 3 independent experiments. The data in the figures represent the averages ± SD. Significant differences are indicated as * (p < 0.05)

3.5. ALN mediated peroxisomal dysfunction further induces ER stress and cell apoptosis in POCs

To determine whether ER stress can be activated by ALN treatment in POCs, we detected the phosphorylation of ER stress response related proteins including protein kinase R (PKR)‐related ER kinase (PERK), eukaryotic initiation factor two alpha (eIF2α) and the downstream apoptosis mediator CCAAT/enhancer binding protein homologous protein (CHOP). Thapsigargin (Tg) and tunicamycin (Tm) were introduced as positive control groups for ER stress induction. The results showed that ALN increased the phosphorylation of PERK and eIF2α similarly with Tg and Tm suggesting the induction of ER stress. CHOP expression was also triggered by ALN, Tg, and Tm suggesting the induction of cell apoptosis (Figure 5a). It is worthy to notice that the increase of p‐PERK is not dose‐dependent in POCs treated with ALN (10, 30, and 50 µM). We assumed that the dose‐dependent increase of p‐PERK might be observed in POCs challenged by lower concentrations of ALN (lower than 10 µM). For verification, Western blot was performed to detect the expressions of anti‐apoptotic protein B‐cell lymphoma 2 (Bcl‐2), and pro‐apoptotic protein Bcl2 associated X (Bax) and cleaved caspase‐3. The results showed that both Bax and cleaved caspase‐3 were up regulated while Bcl‐2 was down regulated by ALN in POCs suggesting the induction of cell apoptosis (Figure 5b). On the mRNA level, the expression of more ER markers including Chop, Trib3, and Gadd34 were detected by qPCR revealing an increase by ALN treatment (Figure 5c). Further immunofluorescent stain in POCs confirmed the accumulation of CHOP induced by ALN (Figure 5d). Combining with previous FCM results, we concluded that ALN mediated peroxisomal dysfunction further induces ER stress and eventually cell apoptosis in POCs.

Figure 5.

Figure 5

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ALN mediated peroxisomal dysfunction further induces ER stress and cell apoptosis in POCs. (a) Western blot analysis of p‐PERK, PERK, p‐eIF2α, eIF2α, CHOP, and β‐actin in POCs treated with ALN (0, 10, 30, and 50 µM), Tg (1 µM), and Tm (10 µg/ml). (b) Western blot analysis of Bax, Bcl‐2, cleaved caspase‐3, and GAPDH in POCs treated with ALN (0, 10, 30, and 50 µM), Tg (1 µM), and Tm (10 µg/ml). (c) Relative mRNA expression of Chop, Trib3, and Gadd34 in POCs 12 h after treatment of ALN (0, 10, 30, and 50 µM), Tg (1 µM), and Tm (10 µg/ml). (d) Immunostaining of CHOP (green) and Phalloidin (red) in POCs treated with ALN (0, 10, 30, and 50 µM), Tg (1 µM), and Tm (10 µg/ml) and quantification of CHOP relative fluorescent intensity. Bar represents 30 µm. Images are representative of n = 5 independent experiments. The data in the figures represent the averages ± SD. Significant differences are indicated as ** (p < 0.01)

4. DISCUSSION

In this study, we found that ALN treatment in OVX mice significantly reversed bone mineral density and volume; however, ALN impaired PDGF‐BB positive cell number in both sham and OVX mice. As PDGF‐BB is mainly secreted by POC, we then studied whether and how ALN decreased cell number of POC. As expected, ALN induce cell apoptosis and cell cycle arrest in POCs. Furthermore, we found that ALN led to peroxisomal dysfunction by inhibiting FDPS expression in POCs. Poroxisomal dysfunction then induced ER stress and activated apoptotic mediator CHOP. For the first time, we discovered that ALN induces POC apoptosis via peroxisomal dysfunction mediated ER stress.

The importance of POC has been highlighted recently, studies revealed that POC‐releasing PDGF‐BB can induce osteoblastic differentiation of mesenchymal stem cells (MSCs) and promote angiogenesis from activating endothelial progenitor cells (EPCs) in vitro. Moreover, PDGF‐BB also induces CD31hiEmcnhi vessel subtype formation in coupling osteogenesis in vivo, which plays a crucial role in bone tissue healing and new bone formation (Xie et al., 2014). Recent studies suggest that increased osteoclastic bone resorption is not the only issue need to be solved in osteoporosis, decreased angiogenesis should also be taken into consideration (Roche et al., 2014; Spangenberg et al., 2016). So, the strategy to preserve POC while selectively depleting mature osteoclast is a more effective in treating osteoporosis. However, we found that ALN inhibit both POC and mature OC without selectivity. A recent study also reported that another widely used nitrogen‐containing bisphosphonate zoledronate suppresses angiogenesis and osteogenesis by inhibiting osteoclast formation and PDGF‐BB secretion (Gao et al., 2017). Although the authors did not investigate the effects of zoledronate on POC in particular, we hypothesized that the underlying mechanism might be similar with our study.

FDPS is a known target protein for nitrogen‐containing bisphosphonates including ALN (Fisher et al., 1999; Luckman et al., 1998). Upon FDPS inhibition, post‐translational prenylation of small GTP–binding proteins including Rab, Rac, and Rho was also dampened. These proteins play a key role in regulation of cytoskeletal organization, cell morphology, membrane ruffling, and endocytosis (Jedd, Richardson, Litt, & Segev, 1995; Machesky & Hall, 1997; Zhang et al., 1995). Most of the cell events are essential in mature OC, which explained that ALN inhibits mature OC function and finally causes cell apoptosis. As one of the most important enzyme located in peroxisome, we found that FDPS inhibition also led to perosisomal dysfunction. We further observed the upregulation of cytoprotective protein GRP78, which is also an ER stress marker. Although in a different system, a report showed that peroxisome deficiency can induce ER stress in liver of newborn mice (Kovacs et al., 2012). The authors also discussed and concluded that functional peroxisomes are necessary to prevent chronic ER stress and dysregulation of the endogenous sterol response pathway in their review (Faust & Kovacs, 2014).

ER stress is intimately related with cell apoptosis, excessive, and prolonged ER stress triggers cell suicide, usually in the form of apoptosis, representing a last resort of multicellular organisms to dispense of dysfunctional cells (Faust & Kovacs, 2014). We are not the first group reporting nitrogen‐containing bisphosphonates induce ER stress in cells. A recent study showed that zoledronic acid induce cell apoptosis in breast cancer cells though ER stress triggered REDD1‐mTOR pathway (Lan et al., 2013). In our study, we found that ER stress can also be induced by ALN treatment; we further explained that ER stress is mediated by peroxisomal dysfunction. It is worthy to notice that statins, which targeting FDPS upper stream enzyme HMG‐CoA reductase, can also inhibit protein lipidation and induce the unfolded protein response (UPR) in the non‐sterol producing nematode Caenorhabditis elegans (Morck et al., 2009). It is very interesting to notice that several ER stress and UPR related genes play a completely different role in the process of osteoclastogenesis. A recent study showed that the inositol‐requiring protein‐1α/X‐box–binding protein–mediated (IRE1α/XBP1‐mediated) branch of the UPR is transiently activated during osteoclastogenesis and that the abrogation of this pathway markedly suppresses osteoclast formation in vitro (Tohmonda et al., 2015). Another study showed that activating transcription factor 4 (ATF4), a member of the bZIP family of transcription factors regulating the promoters of several genes implicated in the UPR, is also critical in regulating OC differentiation (Cao et al., 2010). These studies suggested that early ER stress adaptation and UPR might play a different role in specifically in osteoclasts, although detailed investigation needs to be further made. Whereas prolonged ER stress mediated cell apoptosis is universal in all cell types including POC as we discovered. In our study, peroxisome dysfunction and ER stress initial response seemed to occur simultaneously. It is reasonable because that the ER stress itself is a time‐course event. Early ER stress adaptation can be detected very soon, through progression, the prolonged ER stress then cause cell apoptosis.

In summary, we found that ALN treatment induces cell apoptosis in POCs. On the molecular level, ALN inhibition of FDPS leads to peroxisomal dysfunction, which further mediates ER stress and finally causes cell apoptosis. Considering that decreased vascularization is also an important issue in dealing with osteoporosis, meanwhile, POC is beneficial in promoting angiogenesis. Avoiding POC depletion in nitrogen‐containing bisphosphonates based antiresorptive therapy is a potential strategy in improving the curative effects.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Supporting information

Additional Supporting Information may be found online in the supporting information tab for this article.

Figure S1. Immunostaining of TRAP (red), PDGF‐BB (green) in distal femur bone marrow sections. Bar represents 100 μm with quantification of number of TRAP positive cells (N. TRAP+), and number of PDGF‐BB positive cells per respective trabecular bone (TB) (N. PDGF‐BB+). Images are representative of n = 5 independent experiments. The data in the figures represent the averages ± SD. Significant differences are indicated as ** (p < 0.01).

Table S1. Primer sequences for qPCR.

JCP-233-7415-s001.docx (146.8KB, docx)

ACKNOWLEDGMENT

This work was funded by the first class General Financial Grant from the China Postdoctoral Science Foundation (2017M613315).

Contributor Information

Fei Luo, Email: luofly1009@21cn.com.

Ce Dou, Email: lance.douce@gmail.com.

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Supplementary Materials

Additional Supporting Information may be found online in the supporting information tab for this article.

Figure S1. Immunostaining of TRAP (red), PDGF‐BB (green) in distal femur bone marrow sections. Bar represents 100 μm with quantification of number of TRAP positive cells (N. TRAP+), and number of PDGF‐BB positive cells per respective trabecular bone (TB) (N. PDGF‐BB+). Images are representative of n = 5 independent experiments. The data in the figures represent the averages ± SD. Significant differences are indicated as ** (p < 0.01).

Table S1. Primer sequences for qPCR.

JCP-233-7415-s001.docx (146.8KB, docx)

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