Bioactive iron oxide nanoparticles suppress osteoclastogenesis and ovariectomy-induced bone loss through regulating the TRAF6-p62-CYLD signaling complex

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Bioactive Iron Oxide Nanoparticles Suppress Osteoclastogenesis and Ovariectomy-induced Bone Loss through Regulating the TRAF6-p62-CYLD Signaling Complex Li Liu , Rongrong Jin , Jimei Duan , Li Yang , Zhongyuan Cai , Wencheng Zhu , Yu Nie , Jing He , Chunchao Xia , Qiyong Gong , Bin Song , James M. Anderson , Hua Ai PII: DOI: Reference:

S1742-7061(19)30851-7 https://doi.org/10.1016/j.actbio.2019.12.022 ACTBIO 6505

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Acta Biomaterialia

Received date: Revised date: Accepted date:

13 August 2019 21 November 2019 17 December 2019

Please cite this article as: Li Liu , Rongrong Jin , Jimei Duan , Li Yang , Zhongyuan Cai , Wencheng Zhu , Yu Nie , Jing He , Chunchao Xia , Qiyong Gong , Bin Song , James M. Anderson , Hua Ai , Bioactive Iron Oxide Nanoparticles Suppress Osteoclastogenesis and Ovariectomy-induced Bone Loss through Regulating the TRAF6-p62-CYLD Signaling Complex, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.12.022

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Bioactive

Iron

Oxide

Nanoparticles

Suppress

Osteoclastogenesis

and

Ovariectomy-induced Bone Loss through Regulating the TRAF6-p62-CYLD Signaling Complex Li Liu1, Rongrong Jin1*, Jimei Duan1, Li Yang1, Zhongyuan Cai1, Wencheng Zhu1, 5, Yu Nie1, Jing He1, Chunchao Xia2, Qiyong Gong2, Bin Song2, James M. Anderson3, 4, Hua Ai1, 2* 1

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China

2

Department of Radiology, West China Hospital, Sichuan University, Chengdu 610041, China

3

Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA

4

Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA

5

Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

* Corresponding author: Rongrong Jin, National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, P. R. China Phone: 86-28-85413991, Email: [email protected]; Hua Ai, National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, P. R. China Phone: 86-28-85413991, Email: [email protected] 1

Graphical Abstract

2

Abstract Iron oxide nanoparticles (IONPs) have been widely used as contrast agents for magnetic resonance imaging (MRI) and other biomedical applications in both clinical and preclinical cases. In the present study, we show that two clinically used IONPs, ferumoxytol and ferucarbotran, have an intrinsic inhibitory effect on receptor activator

NF-κB

ligand

(RANKL)-induced

osteoclastogenesis

of

bone

marrow-derived monocytes/macrophages (BMMs). IONPs significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts and functional actin ring structures. More importantly, the inhibitory effect was also verified in vivo by its capacity to rescue the bone loss of ovariectomized (OVX) mice after intravenous injection with IONPs. Mechanistically, we found that IONPs trigger the upregulation of p62 which result in recruitment of CYLD and enhanced deubiquitination of TRAF6, a master controller of RANKL signaling. The downstream activation of NF-κB and MAPK signals was accordingly attenuated, ultimately leading to reduced expression of osteoclatogenesis-related genes. Taken together, clinically used IONPs can inhibit osteoclastogenesis through regulating TRAF6-p62-CYLD signaling complex, and they may be considered as alternative options for treatment of osteoporosis. Key words: Iron oxide nanoparticles (IONPs), Osteoclastogenesis, Osteoporosis, p62, TRAF6

3

Statement of Significance Nanoparticles have been developed as drug delivery systems for treatment of osteoporosis, mostly an age-related health problem with risk of fractures. In this work, we show that two clinically used iron oxide nanoparticles (IONPs) ferumoxytol and ferucarbotran themselves can significantly reduce the osteoporosis of ovariectomized (OVX) mice through inhibiting Osteoclastogenesis. We found that IONPs trigger the upregulation of p62 which result in recruitment of CYLD and enhanced deubiquitination of TRAF6, a master controller of RANKL signaling. The downstream activation of NF-κB and MAPK signals was accordingly attenuated, leading to reduced expression of osteoclatogenesis-related genes. Taken together, clinically

used

IONPs

inhibit

osteoclastogenesis

through

regulating

TRAF6-p62-CYLD signaling complex, and they may be considered as alternative options for treatment of osteoporosis.

4

1. Introduction In recent years, nanoparticles have been developed as drug delivery systems for treatment of osteoporosis, mostly an age-related health problem with risk of fractures. Nanoparticles were chosen for improving the penetration and retention of small molecular therapeutic drugs in bone, including organic (liposome, polyester and polysaccharide, etc.), inorganic (silica, gold, iron oxide and hydroxyapatite, etc.) and hybrid nanocomposites (mPEG-PLGA/hydroxyapatite, chitosan/gold, etc.) [1–4]. Not only serving as delivery vehicles, some nanoparticles themselves exhibit therapeutic effects through participating in bone metabolism[5]. For example, silica and gold nanoparticles could increase bone mass of osteoporotic mouse by either slowing down excessive bone resorption or stimulating new bone formation [6–8].

Iron oxide nanoparticles (IONPs) have been studied extensively in both preclinical and clinical cases, either as imaging contrast agents for diagnosis, treating anemia, or as multifunctional carriers of both imaging and therapeutic agents [9–13]. In addition, the bone-targeting property of IONPs was discovered, especially the ones with smaller size and longer half-life in the bloodstream [14]. After systemic administration, the IONPs will be partially internalized by bone marrow monocytes/macrophages (BMMs) which belong to the reticular endothelial system (RES) [15]. However, the biological effects of IONPs on BMMs are barely studied. BMMs are critical to bone metabolism as they could differentiate and fuse into large multinucleated osteoclasts, which are responsible for bone resorption and remodeling. 5

Excessive or deficient function of osteoclasts will lead to bone diseases such as osteoporosis, cancer bone metastasis, arthritis, Paget’s disease, osteopetrosis, etc. [16, 17]. The process of BMMs differentiating into osteoclasts, called osteoclastogenesis, is precisely determined by multiple cytokines, including two crucial ones, macrophage colony-stimulating factor (M-CSF) and receptor activator NF-kB ligand (RANKL) [18]. RANKL activates many signaling pathways after binding to its receptor RANK on BMMs, including c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), p38 and nuclear factor-kB (NF-kB) by recruiting the signaling-adaptor molecule TNF receptor–associated factor 6 (TRAF6) [19, 20]. Among them, the ubiquitination of TRAF6 is a key process, involving important adaptor protein-p62 and de-ubiquitinase cylindromatosis (CYLD) [21]. Our previous study has shown that IONPs could up-regulate the expression of p62 protein through activation of toll-like receptor-4 (TLR4)-Nrf2 signal axis in BMMs, which may influence the transduction of RANKL-RANK signaling as well as the differentiation of osteoclast [22]. In this study, we investigated the responses of BMMs to clinically used IONPs ferumoxytol and ferucarbotran, including their proliferation, differentiation, bone resorption and related molecular mechanisms.

6

2. Materials and methods 2.1. Reagents and antibodies Clinically used IONPs Ferumoxytol (Feraheme, AMAG Pharmaceuticals Inc.) and Ferucarbotran (Resovist, Bayer Pharma) were used in this study. Ferumoxytol suspended in a solution containing 44 mg/ml mannitol, and Ferucarbotran suspended in a solution containing 40 mg/ml mannitol and 2 mg/ml of lactatic acid. The characterization of the nanoparticles can be found in the supplementary material (Fig. S1). Antibodies against JNK (#9252), phospho-JNK (#4668), p38 (#8690),

phospho-p38 (#4511), Erk1/2 (#4695), phospho-Erk1/2 (#4370), IκBα (#4814), p65 (#8242), phospho-p65 (#3033)，and CYLD (#8462) were bought from Cell Signaling Technology. Antibodies against β-actin (sc-47778) and TRAF6 (sc-8409) were purchased from Santa Cruz Biotechnology and antibodies against p62 (ab56416) and ubiquitin (linkage-specific K63) (ab179434) were obtained from Abcam. M-CSF and RANKL for cell culture were purchased from PeproTech. 2.2. Cell culture Raw264.7 macrophage cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and characterized by mycoplasma detection, DNA-fingerprinting, isozyme detection and cell vitality detection. Then it was cultured in RPMI 1640 medium contain 10% FBS, 2 mM glutamine, and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin). For primary osteoclast cell culture, bone marrow cells were firstly isolated from both femur and tibia of female BALB/c mice and cultured with the following 3-step procedure: 1) bone marrow cells from 7

two mice were seeded into a 10 cm dish overnight for elimination of adherent cells; 2) suspended cells were isolated and cultured in medium with 20 ng/mL M-CSF for another 48 hours to obtain bone marrow monocytes (BMMs); 3) BMMs were seeded into culture plate with 50 ng/mL M-CSF and 50 ng/mL RANKL for further osteoclastogenesis in appropriate cell density. 2.3. Prussian blue staining BMMs were seeded into a 48 well-culture plate (2 × 104 cells/well) and incubated for 24 h at the absence or presence of various concentrations of IONPs. After incubation, cells were washed with PBS twice and fixed with 4% paraformaldehyde. Then Prussian blue reagents were added to incubate for 30 min, followed by the counterstain of cellular nuclei with Nuclear Fast Red for 2 min as described previously [23]. After that, the stained cells were captured under a microscope. 2.4. Intracellular iron content assay To quantify the intracellular uptakes of IONPs, BMMs (2×104 cells/well) were seeded into a 48-well plate and incubated with different concentrations of SPIONs nanocomposites for 24 h. Then Cells were then washed with PBS for three times and then 100 μL/well of NaOH (50 mM) was added and incubate for 2 h to dissolve the cells, followed by addition of 100 μL HCl (10 mM) for neutralization. Iron-releasing reagents (mixture of equal volumes of 4.5% (w/v) KMnO4 and 1.4 M HCl) were added and incubated for another 2 h at 60 °C. Cooling to room temperature, then 30 μL iron detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, 2.5 mM ammonium acetate, 1 M ascorbic acid which dissolved in water) were added for 8

further reaction. Thirty mins later, the absorbance of reaction mixtures was detected by microplate reader at 550 nm. The intracellular iron contents were calculated through comparing the absorbance to that of a range of concentrations of equal volume. 2.5. Tartrate-resistant acid phosphatase assay BMMs (5 × 103/well) were seeded in 96-well plates and incubated with M-CSF (50 ng/mL) for 24 h, then stimulated with RANKL (50 ng/mL) in the presence or absence of different concentrations of IONPs. After 4 days, cells were fixed with 4% paraformaldehyde for 5 min and stained with tartrate-resistant acid phosphatase (TRAP) staining kit (387A-1KT, Sigma). Then the cells were observed under a light microscope, and TRAP-positive multinucleated cells containing more than 5 nuclei were counted as osteoclasts. 2.6. Actin ring formation assay BMMs (1 × 104/well) were seeded in 48-well plates incubated with M-CSF (50 ng/mL) for 24 h, then RANKL (50 ng/mL) was added to induce the formation of actin ring with or without the treatment of IONPs. After 4 days, cells were fixed with 4% paraformaldehyde for 15 min followed by three washings with PBS, then 0.1% Triton X-100

was

used

for

permeabilization.

The

cells

were

stained

with

rhodamine-conjugated phalloidin overnight (387A-1KT, Sigma), and the actin rings were observed and calculated under a fluorescence microscope.

9

2.7. Real-time PCR Total RNA was isolated from the treated cells with Trizol reagent (Thermo), then transcripted into cDNA with iScript™ cDNA synthesized kit (Bio-Rad). Real-Time PCR was applied using SosoFast EvaGreen Supermix (Bio-Rad). The expression of a specific gene was standardized by the expression of GAPDH and then shown as fold change from the control. And the primers of the examined genes were as the following: mNfatc1, 5'ATGGGGTCCCTATCAAGT -3' (sence) 5'AGAAGTGGGTGGAGTGGT-3' (antisense) mSrc, 5'TCTATCCCAGACACGACC-3' (sense) 5'-AAACCAGACAGTTGAGGC-3' (antisense) mCtsk, 5'-GACTTCCGCAATCCTTAC-3' (sense) 5'-CCACAAGATTCTGGGGACTC-3' (antisense) Acp5, 5'-ACGATGCCAGCGACAAGA-3' (sense) 5'-TGGACCAGATGGGGTAGTG-3' (antisense) mCalcr, 5'-GTAAGTGCCATTAGAGCG-3' (sense) 5'-GGTAGGAGCCTGAAGAAC-3' (antisense) mRANK, 5'-ATCATCTTCGGCGTTTAC-3' (sense) 5'-CTTCTTGCTGACTGGAGG-3' (antisense)

10

2.9. Western blot Cells were lysed by RIPA lysis buffer (Cell Signaling Technology) added with protease and phosphatase inhibitors (Roche), then the cell lysates were centrifuged at 12000 rpm for 15 min at 4C and the precipitates were discarded. Cellular protein concentration was determined by BCA protein assay kit (Thermo), then the supernatant of 20 μg protein was applied to SDS-PAGE electrophoresis and transferred to a PVDF membrane (Bio-Rad). 5% non-fat milk was used to block membranes for 1 h followed by the incubation of corresponding primary and appropriate secondary antibodies. The protein band was visualized by an ECL kit and ChemiDoc™ XRS+ System (Bio-Rad). 2.9. Immunoprecipitation Cells were lysed by RIPA lysis buffer added with protease inhibitors, then the cell lysates were centrifuged at 10000 rpm for 10 min at 4C. A TRAF6 primary antibody was added to the protein supernatant and incubated for 2 h at 4℃, then Protein G plus agarose beads (Santa Cruz Biotechnology) were added and incubated overnight at 4℃ with continuous rotation. Centrifugation was applied to collect the beads after two times washing with lysis buffer before performing the western blot assay. 2.10. Proliferation assay The proliferation of BMMs with or without treatment with IONPs were measured by Sulforhodamine B (SRB) assay. Briefly, BMMs (3 × 103/well) were seeded in 96-well plates incubated with M-CSF (50 ng/mL) for 24 h, then the cells were treated with various concentrations of IONPs for different incubation periods (1, 2, 3 and 4 days). 11

After incubation, cells were fixed with 10% (w/v) trichloroacetic acid (Sigma) at 4C for at least 4 h, following by thorough washing with deionized water. After complete drying, 0.4% SRB dye (Sigma) solution was added for staining for 10 min followed with thorough washing by 1% (w/v) acetic acid to remove superfluous dye. Tris-base lye (10mM, 100 μL/well) was added to dissolve dyes that bind to cellular proteins for 20 min after complete drying. Then the absorbance of samples was examined with a microplate reader at 540 nm. 2.11. In vivo experiments and Micro-CT All animal experiments were approved and conducted in accordance with accepted standard of the Biomedical Research Ethics Committee of West China Hospital of Sichuan University. Female 8-week-old C57 mice were purchased from Chengdu Dashuo Experimental Animals co. LTD, and divided into three groups of 5 mice each randomly: sham operated mice, ovariectomized (OVX) mice, and OVX mice treated with IONPs. One week after OVX or sham operation was performed, the mice were injected in caudal vein with 10 mg/kg of Ferumoxytol every two days in the treated group along with the saline injection in other two groups. Then, the femurs of all mice were isolated after 90 days and fixed with 4% paraformaldehyde for at least 1 week. The bone mass of distal femurs was analyzed by a Scanco vivaCT80 scanner (Scanco Medical) set to 55kVp and 145 μA, voxel size 8 μm. 250 micro-CT slices below the distal growth plate of femurs were evaluated for trabecular bone architecture by a software that comes with the micro-CT system.

12

2.12. MRI study of mouse femur MRI was performed under a clinical 3 T scanner (Achieva, Philips) with a mouse coil. MR images were acquired by T2-weighted spin echo sequence (TR = 2000 ms, TE = 95 ms; matrix = 192 × 192, field of vision = 50 × 50 mm, slice thickness = 1.0 mm). MR signal intensities at different TE values were measured to calculate the T2 value of each phantom by T -weighted (TR = 4000 ms, TE = 18-112 ms; matrix = 192 × 192, field of vision = 50 × 50 mm, slice thickness = 0.8 mm) 2.13. Histology Femurs of mice in each group were fixed in 4% paraformaldehyde for 7 days, decalcified in 10% EDTA for 7 days, and embedded in paraffin. Sections were cut and subjected to H&E and TRAP staining. 2.14. Statistical analysis All values are presented as means ± SD. A comparison between control and experimental groups was analyzed by unpaired Student’s t test under assumption of equal variance. The statistical analyses for bone indexes were determined using SPSS 25.0 with normally distributed data. A value of p < 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. IONPs inhibit RANKL-induced osteoclasts differentiation in BMMs BMMs are critical to bone metabolism as they can differentiate into osteoclasts. However, it is not clear if internalization of IONPs will have any impact on such a process. First, we observed the phagocytosis of macrophages to different 13

concentrations of IONPs. The cells were treated with various concentrations of IONPs for 24 hours, then Prussian blue staining (Fig. S2A) and intracellular iron content assay (Fig. S2B) were performed to evaluate the accumulation of IONPs in BMMs. The results indicated that BMMs could internalize IONPs and the uptake was a dose-dependent mode. Phagocytosis of BMMs to Ferucarbotran is significantly higher than Ferumoxytol when cells are exposed to the nanoparticles of the same iron concentration. As shown in Fig. S2, all cells could be efficiently labeled with Ferucarbotran at the concentration of 0.1 mg Fe/mL while Ferumoxytol needs a much higher concentration of 1 mg Fe/mL. Different physicochemical properties of the two nanoparticles such as the particle size and surface modification may contribute to this difference [24, 25]. Next, we investigated the differentiation of BMMs with the stimulation of M-CSF and RANKL in the presence or absence of IONPs. TRAP staining was employed for the detection as it is a commonly used marker of osteoclast formation, which can degrade bone minerals to achieve bone resorption [26]. As shown in Fig. 1A, the number of TRAP-positive multinucleated osteoclasts was significantly reduced in the presence of IONPs comparing to the non-treated group, and the reduction presented a dose-dependent manner. It was also found that the inhibitory effect of Ferucarbotran is obviously stronger than that of Ferumoxytol when cells were exposed to the same iron concentration of these two nanoparticles. This inhibitory effect may be positively related with the intracellular IONP uptake, indicating that the osteoclastogenesis may depend on the number of nanoparticles internalized by BMMs. 14

Studies have shown that osteoclast formation is a multi-stage process, including proliferation, differentiation, cell fusion, and maturation [27]. To determine the effect of IONPs on the proliferation of BMMs, we examined the cell proliferation treated with M-CSF by SRB assay in the presence or absence of IONPs. The result showed that IONPs have no effect on cell proliferation(Fig. S3). To further confirm at which stage IONPs block the differentiation of osteoclasts, we then treated BMMs with Ferumoxytol or Ferucarbotran at different stages of osteoclastogenesis as shown in Fig. 1B. It was found that both Ferumoxytol and Ferucarbotran displayed the highest inhibitory effects when added at day 0 but the inhibitory effects were drastically attenuated when added at day 3. The data suggest that IONPs inhibit the formation of TRAP-positive multinucleated cells mainly at the early stage. At the later stage, cells have committed into the osteoclast lineage, adding IONPs has very limited effect on reversing the process.

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Fig. 1. Inhibition of RANKL-induced osteoclatogenesis in BMMs by IONPS. (A) BMMs were incubated with the indicated concentrations of Ferumoxytol and Ferucarbotran in the presence of RANKL and M-CSF. After cultured for 4 days under the stimulation of RANKL, TRAP expression of cells was examined; (B) BMMs were incubated with RANKL and M-CSF, followed by treatment with 0.1 mg Fe/mL Ferumoxytol and 0.01 mg Fe/mL Ferucarbotran at the indicated time points. After stimulation with RANKL for 4 days, cells were stained for TRAP expression. Values are expressed as the mean, n = 4 (4 independent experiments); bars show SD. ***p＜ 0.001 (vs control group), Scale bars represent 200 μm. 3.2. IONPs suppress RANKL-induced actin ring formation

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Actin ring is a unique structure formed through the rearrangement of cytoskeletal proteins in mature osteoclasts, and it is also the prerequisite for bone resorption [28]. The formation of actin ring structures in BMMs were examined under the induction of RANKL with or without of IONPs. The formation of acting ring structures was largely suppressed both in size and number after treated with IONPs. The suppression showed a dose-dependent mode and the structures even could not form when the cells were treated with Ferucarbotran at concentrations above 0.01 mg/ml and Ferumoxytol at concentrations above 0.1 mg/mL (Fig. 2A). In addition, different from the results of TRAP staining, the formation of actin ring structures decreased dramatically when IONPs were added at day 3 during the process of osteoclastogenesis (Fig. 2B). It was shown that the IONPs could further inhibit maturation of osteoclasts by blocking the formation of actin ring. Together, these results suggest that IONPs could inhibit the cell fusion and the maturation of osteoclast precursors.

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Fig. 2. Suppression of RANKL-induced actin ring formation in BMMs by IONPs. (A) BMMs were incubated with indicated concentrations of Ferumoxytol and Ferucarbotran in the presence of RANKL and M-CSF. After 4 days, cells were stained with phalloidin to detect the formation of actin ring structure. (B) BMMs were incubated with RANKL and M-CSF, followed by treatment with 0.1 mg Fe/mL Ferumoxytol and 0.01 mg Fe/mL Ferucarbotran at the indicated time points. After 4 days, cells were stained for formation of actin ring structure. Values are expressed as the mean, n = 3 (3 independent experiments); bars show SD. ***p＜0.001. Scale bars represent 300 μm.

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3.3. IONPs prevent OVX-induced bone loss through inhibiting osteoclast differentiation Osteoclast have a crucial role in the bone remodeling, of which up-regulated activity can lead to osteoporosis. Since IONPs can significantly suppress the osteoclast differentiation in vitro, we therefore examined whether they can prevent bone loss in ovariectomized (OVX) mouse model in vivo. Ferumoxytol was chosen in this study as they have shown higher amount of accumulation in bone marrow than Ferucarbotran after systematic administration due to their longer half-life in bloodstream [30]. Magnetic resonance imaging (MRI) was used to visualize the accumulation of IONPs in bone marrow, which would disturbs the relaxation of nearby water protons, causing decreases of T2 relaxation times as well as the T2 signal intensity [31]. MR images of mice femur were acquired using T2-weighted spin echo sequence. In addition, T2-map imaging was also used to observe the T2 value changes of femoral bone marrow after IONPs injection. It was found that the MR signal intensity of femoral bone marrow was reduced by 69% after injection of IONPs (Fig. 3A). Consistently, the average T2 values of femoral bone marrow acquired by T2-map imaging was reduced from 65 ms to 33 ms (Fig. 3B and 3C). Micro-CT was employed to analyze the bone mass of distal femurs of all mice as it is the “gold standard’’ for evaluation of bone morphology and microarchitecture with high resolution at the micrometer scale. It was observed that the mice of OVX group exhibited severe bone loss compared to the sham group, and the bone loss was significantly prevented after treating with Ferumoxytol as shown in 3D micro-CT 19

reconstruction images (Fig. 3D). Accordingly, the trabecular bone morphometric parameters of different groups were then calculated through the 3D construction. The bone volume density (BV/TV), representing the change of bone mass, was reduced by almost half in OVX mice, while the OVX mice treated with Ferumoxytol exhibited a similar value to the sham group. In addition, the OVX mice also showed obvious abnormalities in other trabecular bone morphometric parameters, including decreased trabecular number (Tb.N) and connectivity density (Conn.D), but increased trabecular separation (Tb.Sp), together indicating a reduced and more separated trabecular bone network. After treatment with Ferumoxytol, the abnormalities of these parameters in OVX mice were significantly prevented. Especially Conn. D，which even show a similar level to sham group. (Fig. 3E). These data indicated that IONPs would accumulate in bone marrow by intravenous administration and subsequently prevent the bone loss of OVX mice. Moreover, the IONPs treatment have no effect on the body weight of mice compared with the sham group, suggesting little toxicity of this nanoparticle at the tested dosage (Fig. S4).

To further investigate the effect of IONPs in osteoclast formation in vivo, we performed histological analyses of femoral sections of mice. Consistent with the results of Micro-CT, H&E staining of femoral sections from OVX group showed a drastical reduction in trabecular bone compared with the sham group. The bone loss

was significantly prevented after treating with Ferumoxytol (Fig. 4A). In addition, TRAP staining revealed that the trabecular bone from OVX mice contained increased TRAP activity compared with the sham group. IONPs treatment significantly reduced 20

number of TRAP-positive osteoclast compared with the OVX group (Fig. 4B, 4C).

Fig. 3. Accumulation of IONPs in mouse femoral bone marrow and its prevention on OVX-induced bone loss. (A) T2-weighted images of mouse femur. (B) T2-map images of mouse femur. (C) T2 value distribution of mouse femur. T2-weight sequence: TR = 2000 ms, TE = 95 ms; matrix = 192 × 192, field of vision = 50 × 50 21

mm, slice thickness = 1.0 mm. T2 –map sequence: TR = 4000 ms, TE = 18-112; matrix = 192 × 192, field of vision = 50 × 50 mm, slice thickness = 0.8 mm. (D) 3D micro-CT reconstruction images of trabecular bone, representative images of Sham, OVX and OVX treated with Ferumoxytol. (E) Trabecular bone mass parameters, including bone volume density (BV/TV), Trabecular Number (Tb.N), trabecular spacing (Tb.Sp) and connectivity density (Conn. D). Values are expressed as the mean, n = 5 (5 mice in each group); bars show SD. *P < 0.05, **P<0.01, ***P < 0.001.

Fig. 4. (A) H&E staining of femoral sections in each group, showing reduced trabecular bone (yellow arrow). Scale bars represent 250 μm. (B) TRAP staining showing increased TRAP activity in OVX mice (black arrows). Scale bars represent 75 μm. (C) The number of TRAP-positive cells were counted. Values are expressed as the mean, n = 3 (3 mice in each group); bars show SD. *P < 0.05, **P<0.01.

3.4. IONPs suppress osteoclast-specific marker gene expression The formation and function of osteoclasts are regulated by several specific marker genes, including Nfatc1, a master regulator of osteoclastogenesis; Acp-5, Ctsk and Calcr, involved in the bone resorption; c-Src, responsible for the formation of actin ring and the maturation of osteoclast; and RANK, the receptor of RANKL, answering 22

for initiating the osteoclastogenesis. To investigate which marker gene’s expression was affected by IONPs, RT-qPCR was conducted to determine the transcription level of those genes after treated with IONPs at the different stages of osteoclastogenesis. The results suggested that the transcription of osteoclast-specific marker genes was significantly up-regulated under the stimulation of RANKL, however, they were markedly suppressed after treated with IONPs at day 0 (Fig. 5A, 5B). Consistent with the result of TRAP staining and actin ring formation assay, when IONPs were expose to BMMs at the latter stage of osteoclastogenesis, the inhibitory effect of IONPs on the expression of some marker genes were drastically attenuated (Fig 5C, 5D). IONPs have no effect on the expression of Nfatc1, Acp-5 and RANK when add at day 3. However, IONPs also display a significant inhibitory effect on the expression of Calcr, Ctsk, and c-Src, which are involved in the formation of actin ring and the bone resorbtion (Fig. 5D). These data indicated that IONPs could downregulated most of the downstream genes of Nfatc1 as well as itself, a crucial nuclei transcription factor during the osteoclastogenesis.

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Fig. 5. Suppression of osteoclastogenesis-related marker genes expression by IONPs. BMMs (1.2 × 105 cells) were seeded in 6 cm dishes added with M-CSF (50 ng/mL) for 24 h, then they were stimulated with RANKL (50 ng/mL) for osteoclatogenesis in presence or absence of IONPs. 1 mg Fe/mL Ferumoxytol and 0.1 mg Fe/mL Ferucarbotran were exposed to BMMs respectively at day 0 (A, B), day 1 (C) and day 3 (D). IONPs simultaneously renewed with culture medium every 2 days. Then cells were harvested at indicated time points. Total RNA of the cells was isolated and transcripted cDNA for further qPCR assays with indicated primers of osteoclastogenesis related genes, including Nfatc1, Acp-5, RANK, Calcr, Ctsk, and c-Src. Values are shown as fold change from control and expressed as the mean, n = 3 (a single sample analyzed in triplicate); bars show SD. *P < 0.05, **P<0.01, ***P < 0.001. 3.5. IONPs block RANKL-induced activation of NF-κB and MAPK signals As demonstrated previously, the transcription of Nfatc1 and its downstream genes were all decreased in osteoclasts after treatment with IONPs, which means that IONPs probably have effect on the upstream controller of Nfatc1. It has been well studied that the nuclei transportation of Nfatc1 were mostly motivated by NF-κB and MAPK (JNK, ERK, p38) signaling pathways when RANKL bound to RANK [32]. To confirm whether the inhibitory effects of IONPs on osteoclastogenesis were mediated by the blockage of NF-κB and MAPK signaling pathways, the activation of NF-κB and MAPK signals in BMMs and Raw264.7 cells were then examined after stimulating with RANKL for different duration in the absence or presence of IONPs. 25

As shown in Fig. 6, phosphorylation of JNK, ERK and p38 induced by RANKL were evidently suppressed in the presence of IONPs in both cells. In addition, RANKL induced degradation of IκB-α and followed phosphorylation of NF-κB were also inhibited by IONPs in BMMs and Raw264.7 cells. As a result, IONPs could inhibit RANKL induced osteoclastogenesis through blocking both NF-κB and MAPK signal pathways. The different degree of inhibition on the two cell types may be due to the fact these two cells are intrinsically different [33].

Fig. 6. Inhibition of RANKL-induced activation of NF-κB and MAPK signaling in BMMs and Raw264.7 cells by IONPs. (A) BMMs (2×105 cells/well) were seeded in 6-well plates and (B) Raw264.7 (8 × 104 cells/well) were seeded in 12-well plates. They were cultured in medium added with M-CSF (50 ng/mL) for 24 h. After adhesion, the cells were pretreated with 0.1 mg Fe/mL Ferucarbotran for 24 h and then stimulated with RANKL (100 ng/mL) at indicated times. Then cell lysates were subjected to western blot analysis with appropriate antibodies. 26

3.6. IONPs Suppress RANK signaling through regulating TRAF6-p62-CYLD Signaling Complex Both NF-κB and MAPK signals were suppressed after incubation with IONPs. It was possible that their upstream controller TRAF6 has been affected in some degree as it is an essential component in RANK signaling. The binding of TRAF6 to specific domains of RANK leads to subsequent conformational change of RANK receptor is a primary step for activation of NF-κB and MAPK signaling pathways. Studies have shown that the ubiquitination of TRAF6 is indispensable for RANK signaling transduction [34]. We therefore investigated whether IONPs would inhibit the ubiquitination of TRAF6. As shown in Fig. 7A, RANKL-induced ubiquitination of TRAF6 is significantly suppressed after treatment with IONPs. CYLD, a deubiquitinating enzyme, recently identified as a negative regulator of RANK signal through the interaction with TRAF6 may also play an important role in IONPs mediated de-ubiquitination of TRAF6 [21]. The association of CYLD/TRAF6 was studied by immunoprecipitation. The result showed that IONPs could greatly promote the recruitment of CYLD to TRAF6 (Fig. 7A). However, we found that the induction of CYLD mRNA by RANKL is markedly suppressed by IONPs, especially in the later stages of osteoclastogenesis (Fig. 7B). It indicated that the increase of CYLD recruited to TRAF6 was not resulting from the up-regulation of the total expression of CYLD. It is also reported that p62, a well-known adaptor protein, can interact with CYLD and facilitate the binding of CYLD to TRAF6 [35]. More importantly, we have demonstrated that IONPs could up-regulate the expression of 27

p62 through activating TLR4 signaling in BMMs [22]. We further examined whether p62 was involved in the IONPs-mediated blockade of RANK signaling. In the same way,

the

enhanced

binding

of

p62

to

TRAF6

was

identified

by

co-immunoprecipitation (Fig. 7A). In addition, qPCR assay was used to determine the p62 mRNA expression under the stimulation of RANKL in the presence or absent of IONPs. The results indicated that the mRNA level of p62 increased significantly (Fig. 7B). These findings suggest that IONPs can promote the recruitment of CYLD to TRAF6 through up-regulating the expression of p62, followed by inhibiting the RANKL-induced ubiquitination of TRAF6.

Fig. 7. IONPs regulate TRAF6-p62-CYLD signaling complex in BMMs. (A) Raw264.7 (2 × 105 cells) were seeded in 6 cm dishes and incubated for 24 h, then 0.1 mg Fe/mL Ferucarbotran was added to treat cells for 24 h in the presence or absence of RANKL (100 ng/mL). Then the cells were lysed and subjected to 28

immunoprecipitation analysis with TRAF6 primary antibody. The ubiquitination of TRAF6 and the association of CYLD/TRAF6, p62/TRAF6 were subsequently examined with western blot analysis. (B) mRNA transcription of CYLD and p62 in the process of BMMs differentiating into multinucleated osteoclasts were detected with or without the treatment of IONPs. Total RNA of the cells was isolated at indicated times and qPCR assays were then performed with indicated primers. Values are expressed as the mean, n = 3 (a single sample analyzed in triplicate); bars show SD. *P < 0.05, **P<0.01.

Fig. 8. Illustration of IONPs induced inhibitory effect on RANKL-induced osteoclastogenesis. (A) After systematic administration, IONPs are detained in bone marrow through phagocytosis of BMMs. IONPs could inhibit RANKL-induced osteoclastogenesis both at the early differentiation and late maturation stage. While 29

they have no effects on the cell’s proliferation and fusion stage. (B) Molecular mechanism

of

IONPs

induced

inhibitory

effect

on

RANKL-induced

osteoclastogenesis. IONPs upregulate p62 expression through activating TLR4-Nrf2 signal axis, followed by enhancing the binding of CYLD to the TRAF6-p62-CYLD complex, leading to deubiquitination of TRAF6 and blockage of RANKL induced sequential signal transduction such as NF-KB and MAPK signals. As a result, the osteoclastogenesis related genes’ transcription in downstream were obviously inhibited, leading to the deficiency of bone resorption.

Conclusions In summary, we evaluated an intrinsic effect of two clinically used IONPs ferumoxytol and ferucarbotran on RANKL-induced osteoclastogenesis. Meanwhile, the underline mechanism was also thoroughly investigated (Fig. 7). The IONPs could upregulate p62 expression to enhance the binding of CYLD to the TRAF6-p62 -CYLD complex, leading to deubiquitination of TRAF6 and blockage of RANKL induced sequential signal transduction such as NF-KB and MAPK signals. As a result, most of the predominant genes’ transcription during osteoclatogenesis were obviously inhibited. Administration of IONPs could partially reverse the bone loss in ovariectomized mouse with significant accumulation in bone marrow. It is possible that these clinically used imaging probes can be considered as alternative options for treatment of osteoporosis.

30

Acknowledgements This work was supported by Innovative Research Groups of the National Natural Science Foundation of China (81621003), National Key Basic Research Program of China (2013CB933903), Sichuan Province Science and Technology Program (No. 2019JDRC0103),

China

Postdoctoral

Science

Foundation

Funded

Project

(2015M572475), Sino-German cooperation group (GZ1512) and National Natural Science Foundation of China (81873921).

Declaration of Competing Interest The authors declare no conflict of interest.

31

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