Nox4 mediates RANKL-induced ER-phagy and Osteoclastogenesis via activating ROS/PERK/eIF-2α/ATF4 Pathway

Background: Receptor activator of nuclear factor-κB ligand (RANKL) has been found to induce osteoclastogenesis and bone resorption. However, the underlying molecular mechanisms remain unclear. Results: Inhibitor chloroquine (CQ) was used to veried the role of autophagy in RANKL-induced osteoclastogenesis; Via downregulating Nox4 with inhibitor (DPI) and retrovirus-conveyed shRNA, we further explored the importance of Nox4 in RANKL-induced autophagy and osteoclastogenesis, as well as the regulatory effects of Nox4 on nonmitochondrial reactive oxygen species (ROS) and PERK/eIF-2α/ATF4 pathway.Intracellular ROS scavenger (NAC), mitochondrial-targeted antioxidant (Mito-TEMPO) and inhibitor of PERK (GSK2606414) were also employed to investigate the role of ROS and PERK/eIF-2α/ATF4 pathway in RANKL-induced autophagy and osteoclastogenesis. RANKL markedly increased autophagy, while CQ treatment caused reduction of RANKL-induced autophagy and osteoclastogenesis. Consistent with the increased autophagy, the protein levels of Nox4 were signicantly increased, and Nox4 was selectively localized within the endoplasmic reticulum (ER) after RANKL stimulation. DPI and shRNA eciently decreased the protein level and (or) activity of Nox4 in the ER and inhibited RANKL-induced autophagy and osteoclastogenesis. Mechanistically, we found that Nox4 regulates RANKL-induced autophagy activation by stimulating the production of nonmitochondrial ROS. Additionally, Nox4-derived nonmitochondrial ROS dramatically activate PERK/eIF-2α/ATF4, which is a critical unfolded protein response (UPR)-related signaling pathway. Blocking the activation of the PERK/eIF-2α/ATF4 signaling pathway either by Nox4 shRNA, ROS antioxidant or PERK inhibitor (GSK2606414) treatment signicantly signaling pathway and then suppressed autophagy and ER-phagy during RANKL-induced osteoclastogenesis. Our results provide new insight into the molecular mechanisms of RANKL-induced osteoclastogenesis and will help the development of new therapeutic strategies for osteoclastogenesis-related diseases. suppressed RANKL-induced autophagy osteoclastogenesis. that Nox4 may the of autophagy ER-derived ROS during RANKL-induced by the


Background
Throughout life, bone homeostasis is maintained through elaborate remodeling via coordinated bone formation and bone resorption [1]. Osteoclasts (OCs) are the principal cells responsible for bone resorption [2]. Osteoclasts, characterized as tartrate-resistant acid phosphatase (TRAP)-positive, are derived from the hematopoietic monocyte/macrophage lineage, and fuse to form multinucleated cells by an orchestrated process [3,4]. The excessive differentiation of osteoblasts is the pathological basis of a variety of osteolytic diseases, such as postmenopausal osteoporosis, Paget disease of bone and in ammatory arthritis [5,6]. Therefore, there is no doubt that identifying pharmacological inhibitors targeting osteoclasts differentiation will help the development of new prophylactic and therapeutic strategies for osteolytic bone lesions [7]. The differentiation and maturation of osteoclasts is a complicated process that is regulated by various cytokines [8]. RANKL, a member of the tumor necrosis factor superfamily [9], has been demonstrated to interact with the RANK receptor expressed on osteoclast precursors to activate multiple osteoclastogenesis-related signaling pathways (NF-κB, Src, MAPK, etc.), releasing nuclear transcription factors (NFATc1, AP-1, etc.) and regulating the expression of osteoclastogenesis-related genes, which induce the differentiation and maturation of osteoclasts in the bone microenvironment [10,11]. Moreover, denosumab, a monoclonal antibody with activity against RANKL, has been demonstrated to be effective in the prevention and treatment of osteolytic disease [12].
However, the detailed mechanisms of osteoclastogenesis induced by RANKL remain unclear.
Previous studies have revealed that the upregulation of autophagy contributes to osteoclastogenesis in response to hypoxic conditions, glucocorticoid treatment and microgravity in vitro [20][21][22]. Moreover, several key autophagy-regulated proteins such as LC3 and Atg5 have been shown to participate in osteoclast bone resorption by directing lysosomal content secretion into the extracellular space [23].
However, it is still unclear whether autophagy is involved in RANKL-induced osteoclastogenesis.
Here, we found for the rst time that the increase in Nox4 protein levels in the ER contributes to the activation of autophagy during RANKL-induced osteoclastogenesis. Nonmitochondrial ROS, but not mitochondrial ROS, are closely associated with the critical roles of Nox4 in regulating RANKL-induced autophagy activation and osteoclastogenesis. Inhibiting Nox4 expression via shRNA or blocking ROS inhibited the activation of the PERK/eIF-2α/ATF4 signaling pathway and then inhibited autophagy and ER-phagy during RANKL-induced osteoclastogenesis. Our ndings provide new insight into the processes of RANKL-induced osteoclastogenesis and will facilitate the development of new therapeutic strategies for osteoclastogenesis-related diseases.

RANKL induces osteoclastogenesis and bone resorption via autophagy
Consistent with that reported in previous studies, we found that RANKL enhanced the proportion of fused multinuclear cells (see Additional le 1: part A) and the expression levels of osteoclastogenesis-related genes (TRAP, Cath K and MMP-9; see Additional le 1: part B), inducing the formation of TRAP-positive multinuclear (≥ 3) osteoclasts and bone resorption pits (see Additional le 1: part C) in RAW264.7 cells. These results indicate that RANKL induced the differentiation and subsequent bone resorption activity of osteoclasts in vitro, which is consistent with previous ndings [24].
Autophagy has been demonstrated to play critical roles in enhanced osteoclastogenesis under many conditions, such as hypoxia, oxidative stress and microgravity [22,25,26]. First, we assayed the level of autophagy after RANKL treatment. Western blot analysis showed that the LC3-II/LC3-I ratio was signi cantly upregulated from day 3 in a time-dependent manner during RANKL-induced osteoclastogenesis (Fig. 1A). The TEM showed that the number of autophagic vacuoles was dramatically increased after 3 days of treatment with RANKL (Fig. 1C). Then, the autophagic ux activity was further determined by using the adenovirus-mRFP-GFP-tagged LC3 system. The data showed that the number of yellow and red puncta in merged images was signi cantly increased after 3 days of RANKL-induced differentiation, indicating the activation of both autophagosome formation and lysosomal degradation in the RANKL-treated group (Fig. 1D). Collectively, these observations indicate that autophagy is activated during RANKL-induced osteoclastogenesis. Second, using a pharmacological inhibitor of autophagy (CQ), we explored whether autophagy is essential in RANKL-induced osteoclastogenesis. The results showed that CQ treatment markedly increased the LC3-II/LC3-I ratio (Fig. 1B) and inhibited autolysosomal degradation (Fig. 1C, D). More importantly, we found that CQ treatment suppressed the RANKL-induced upregulation of osteoclastogenesis-related genes (TRAP, Cath K and MMP-9; Fig. 1E), reduced the number of TRAP-positive multinuclear (≥ 3) osteoclasts and reduced the area of the bone resorption pits (Fig. 1F). Taken together, the above data reveal that RANKL induces osteoclastogenesis and bone resorption through autophagy.

Pharmacological inhibition of Nox4 suppresses RANKL-induced autophagy and osteoclastogenesis
The above experiments showed that RANKL induced osteoclastogenesis and bone resorption through autophagy; we next uncovered the molecular mechanism of RANKL-induced autophagy. It is well known that Nox family proteins promote the activation of autophagy in many cell types by generating ROS [27]. Recent evidence indicated that RANKL increases the generation of intracellular ROS by promoting the expression and activity of intracellular Nox family proteins during osteoclastogenesis [28][29][30]. Therefore, we explored whether Nox family proteins are involved in RANKL-induced autophagy.
Western blot analysis showed that RANKL time-dependently upregulated the levels of Nox1 and Nox4 proteins and decreased the levels of Nox2 protein but had no signi cant in uence on the levels of Nox3 protein ( Fig. 2A). To further investigate whether Nox family proteins are involved in RANKL-induced autophagy and osteoclastogenesis, the Nox pharmacological inhibitor DPI was utilized in the presence of RANKL. The data showed that DPI treatment obviously downregulated the RANKL-induced increase in the LC3-II/LC3-I ratio (Fig. 2B). To further investigate which Nox isoforms are involved in RANKL-induced autophagy and osteoclastogenesis, pharmacological inhibitors targeting speci c Nox isoforms were used. The data showed that the inhibition of Nox1 or Nox4 (not Nox2) separately by ML171 and 5-Omethyl quercetin signi cantly inhibited the RANKL-induced increase in the LC3-II/LC3-I ratio and osteoclastogenesis-related gene (TRAP, Cath K and MMP-9) expression and reduced the number of TRAPpositive multinuclear (≥ 3) osteoclasts and the area of the bone resorption pits (Fig. 2C-F). Importantly, the inhibitory effect of 5-O-methyl quercetin on RANKL-induced autophagy, osteoclastogenesis and bone resorption was signi cantly greater than that of ML171. Therefore, compared with Nox1, Nox4 may play a leading role in autophagy activation induced by RANKL. We selected Nox4 for the subsequent experiments.

Knockdown of Nox4 suppresses RANKL-induced autophagy and osteoclastogenesis
To further determine the functional signi cance of Nox4 in RANKL-induced autophagy and osteoclastogenesis, retroviruses encoding three different Nox4 shRNAs or scrambled shRNA were utilized. The results showed that the expression levels of Nox4 were signi cantly decreased after the transfection of sh-Nox4-1, sh-Nox4-2 and sh-Nox4-3 (Fig. 3A). Importantly, sh-Nox4-2 had the greatest Nox4 silencing effect. Therefore, sh-Nox4-2 was selected for the subsequent experiments. The knockdown of Nox4 markedly inhibited the RANKL-induced increase in the LC3-II/LC3-I ratio, autophagic ux activity, and expression of osteoclastogenesis-related genes (TRAP, Cath K and MMP-9) and reduced the number of TRAP-positive multinuclear (≥ 3) osteoclasts and bone resorption pit area ( Fig. 3B-E). Collectively, the above results indicate that knockdown of Nox4 suppresses RANKL-induced autophagy and osteoclastogenesis.

RANKL speci cally upregulates the level of Nox4 protein in the ER
Recent studies have indicated that Nox4 is localized on intracellular membranes, mainly in mitochondria and the ER [31]. To determine in which subcellular compartment Nox4 protein is located during RANKLinduced osteoclastogenesis, we investigated alterations in Nox4 protein expression levels in the ER and mitochondria in RAW264.7 cells cultured under RANKL induction conditions. Western blot analysis showed that RANKL treatment markedly increased the protein level of Nox4 in the ER (not in the mitochondria) of RAW264.7 cells (Fig. 4A-B). As shown in Fig. 4C, Nox4 shRNA treatment signi cantly downregulated the RANKL-induced increase in the Nox4 protein level in the ER of RAW264.7 cells (Fig. 4C). To further ascertain the ER localization of Nox4 protein induced by RANKL, an immuno uorescence staining assay was utilized. The results showed that RANKL treatment signi cantly enhanced the localization of Nox4 in the ER, which was markedly suppressed by Nox4 silencing (Fig. 4D). Collectively, the above data reveal that RANKL speci cally upregulates the level of Nox4 protein in the ER. This nding is consistent with the fact that membrane proteins are mainly synthesized in the ER and with the evidence that the N-terminal portion of Nox4 contains multiple ER-speci c signal sequences [32,33].

Nox4 promotes RANKL-induced autophagy activation and osteoclastogenesis by generating nonmitochondrial ROS
It has been reported that Nox family proteins can promote the production of intracellular ROS [34]. Therefore, the level of ROS was assayed by a uorescence staining assay. As shown in Fig. 5A, RANKL treatment markedly enhanced the level of intracellular ROS and ER ROS in RAW264.7 cells, which was reduced by Nox4 silencing. As shown in Fig. 5C, the level of mitochondrial ROS was increased in RAW264.7 cells during RANKL-induced osteoclastogenesis. As ROS have been reported to play an important role in autophagy regulation [35], we explored whether ROS are involved in RANKL-induced autophagy and osteoclastogenesis. The data showed that intracellular ROS scavenger (NAC) treatment signi cantly inhibited the RANKL-induced accumulation of intracellular ROS and ER ROS (Fig. 5B). Mitochondrial-targeted antioxidant (Mito-TEMPO) treatment signi cantly inhibited RANKL-induced mitochondrial ROS accumulation (Fig. 5C). Importantly, Mito-TEMPO treatment did not affect the RANKLinduced increase in the LC3-II/LC3-I ratio (Fig. 5D), whereas NAC treatment obviously reduced the RANKLinduced increase in the LC3-II/LC3-I ratio (Fig. 5D) and the number of yellow and red puncta in merged images, which implies an impairment in autophagic ux activity (Fig. 5E). Additionally, NAC treatment also signi cantly downregulated the RANKL-induced upregulation of the expression of osteoclastogenesis-related genes (TRAP, Cath K and MMP-9; Fig. 5F) and reduced the number of TRAPpositive multinuclear (≥ 3) osteoclasts and bone resorption pit area (Fig. 5G). In summary, these data indicate that Nox4 promotes RANKL-induced autophagy activation and osteoclastogenesis by generating nonmitochondrial ROS. Furthermore, we also found that the majority of Nox4-derived ROS colocalize with ER-Tracker (Fig. 5A). These results suggest that Nox4 may promote the activation of autophagy via the generation of ER-derived ROS during RANKL-induced osteoclastogenesis.

RANKL promotes ER-phagy via activating
Nox4/ROS/PERK/eIF-2α/ATF4 pathway Recent evidence has indicated that the activation of the UPR can selectively induce ER-phagy [38]. Therefore, we further validated whether UPR-related signaling pathways (PERK/eIF-2α/ATF4) induce ERphagy during RANKL-induced osteoclastogenesis. The results showed that RANKL treatment markedly increased the ratio of LC3-II/LC3-I in the ER and the number of GFP-LC3 puncta colocalized with the ER, which was reduced by Nox4 silencing (Fig. 7A-C). These observations indicate that RANKL promotes ERphagy by upregulating the protein level of Nox4 in RAW264.7 cells. Moreover, we found that NAC and PERK inhibitor (GSK2606414) treatment can separately reduce the RANKL-induced increase in the LC3-II/LC3-I ratio in the ER and number of GFP-LC3 puncta colocalized with the ER (Fig. 7D-G). Collectively, these results indicate that RANKL promotes ER-phagy by activating the Nox4/ROS/PERK/eIF-2α/ATF4 pathway.

Discussion
Autophagy, an evolutionarily conserved and dynamic catabolic process, plays a critical role in maintaining bone homeostasis [39]. However, it is still unclear whether autophagy is involved in RANKLinduced osteoclastogenesis. In the present study, we identi ed a novel mechanism of autophagy regulation during RANKL-induced osteoclastogenesis. Speci cally, ER-resident Nox4 promotes RANKLinduced autophagy activation and osteoclastogenesis by stimulating an increase in nonmitochondrial ROS. Nox4 shRNA or ROS antioxidant treatment inhibited the activation of the PERK/eIF-2α/ATF4 signaling pathway and then suppressed autophagy and ER-phagy during RANKL-induced osteoclastogenesis. Our results provide new insight into the molecular mechanisms of RANKL-induced osteoclastogenesis and will help the development of new therapeutic strategies for osteoclastogenesisrelated diseases.
Nox, which is widely distributed in various tissues and organs, is the key enzyme of redox signaling [40].
The Nox family is composed of ve different isoforms (Nox1, Nox2, Nox3, Nox4, Nox5) of a kind of transmembrane protein [41]. The protein levels of Nox1 and Nox4 are increased, the protein level of Nox2 is decreased, and the protein level of Nox3 remains unchanged during RANKL-induced osteoclastogenesis [42]. Compared with the other isoforms, Nox1 plays more prominent roles in stimulating RANKL-induced osteoclastogenesis [28]. In this study, we found that RANKL treatment caused similar Nox protein expression patterns. However, we found that Nox4 plays a more critical role in regulating RANKL-induced autophagy activation than other Nox isoforms, including Nox1. This speci c role of Nox4 in regulating autophagy may be dependent upon its intracellular localization. In contrast to Nox1, which is mainly located on the plasma membrane, Nox4 is localized to intracellular membranes, particularly in the ER and mitochondria [31].
Nox4 is composed of conserved transmembrane domains, FAD-and NADPH-binding domains in the Cterminal region, and two heme groups [43,44]. ER-localized Nox4 has been found to promote the proliferation, migration, differentiation and survival of cells [45,46]. The activity of ER-localized Nox4 in the regulation of cellular processes may be dependent upon its ability to produce H 2 O 2 , which can be a stable and diffusible signaling molecule, through its E-loop portion [47]. Here, we observed for the rst time that the level of ER-localized (not mitochondria-localized) Nox4 was dramatically increased during RANKL-induced osteoclastogenesis. This result may be explained by the fact that Nox4 is a membranebound protein and, therefore, is mainly translated in the ER through a cotranslational translocation mechanism [32]. However, it is very likely that the preferential cellular localization of Nox4 is stimulus dependent. Recent evidence suggests that alternative splicing of Nox4 mRNA may drive Nox4 synthesis in different subcellular compartments [48]. It is reasonable to speculate that posttranslational modi cations of nascent Nox4 protein or other unknown mechanisms may be involved in this selective activation of Nox4 in the ER of RAW264.7 cells cultured under RANKL induction conditions [49].
Nox proteins are considered the most important source of ROS from different parts of the cell, including mitochondria [50], the ER [27] and the cytomembrane [26]. Increased intracellular ROS accumulation induces autophagy in various cell types [51,52]. Mitochondria, as the sites of oxidative respiration in cells, are the main production sites of intracellular ROS [53]. Previously, we and others have found that RANKL treatment increases mitochondrial ROS in osteoclast precursors [54]; however, mitochondrialtargeted antioxidants do not block RANKL-induced autophagy activation. These results indicate that Nox4 promotes the activation of autophagy by generating nonmitochondrial ROS during RANKL-induced osteoclastogenesis. Furthermore, we also found that the majority of Nox4-derived ROS were colocalized with ER-Tracker. Inhibiting the increase in Nox4 protein levels e ciently reduced ROS, reversed the activation of the PERK/eIF-2α/ATF4 pathway and suppressed RANKL-induced autophagy and osteoclastogenesis. These results suggest that Nox4 may promote the activation of autophagy by generating ER-derived ROS during RANKL-induced osteoclastogenesis. This nding is supported by the ndings of a recent study in which an increase in Nox4-dependent ROS accumulation in the ER of cardiomyocytes was found to promote the activation of autophagy and survival during energy deprivation [27].
ER-phagy, a selective form of autophagy in which portions of the ER are sequestered within autophagosomes and transported to the lysosomes for degradation [55], is considered to play an important role in the ER quality control system by removing excess or damaged ER components [56].
Previous studies have shown that under ER stress conditions, ER-phagy is required for ER turnover and cell survival [57,58]. Activation of the UPR can selectively induce ER-phagy to promote recovery after ER stress and maintain ER homeostasis [59]. However, there are a variety of ER-phagy regulatory mechanisms under different stimulation conditions, suggesting the complexity of ER-phagy. In the present study, we found that the Nox4-dependent accumulation of ROS promotes ER-phagy by activating the UPR-related signaling pathway (PERK/eIF-2α/ATF4) during RANKL-induced osteoclastogenesis. To the best of our knowledge, this is the rst study to reveal the novel role of the PERK/eIF-2α/ATF4 pathway in regulating ER-phagy and enables us to explore novel molecular mechanisms of ER-phagy.

Conclusions
We identi ed a novel role and mechanism of Nox4 in regulating autophagy and ER-phagy during RANKLinduced osteoclastogenesis. Speci cally, ER-localized Nox4 promotes RANKL-induced autophagy and ERphagy by increasing ER-derived ROS and activating the UPR-related signaling pathway (PERK/eIF-2α/ATF4). These ndings may provide new insight into the processes of RANKL-induced osteoclastogenesis and help the development of new potential therapeutic strategies for osteoclastogenesis-related diseases. Future studies are needed to ascertain the functional role of Nox4- Signaling Technology. Primary antibodies against Nox1 (ab131088) and Nox2/gp91phox (ab129068) were obtained from Abcam.

Cell culture
The RAW264.7 mouse monocyte/macrophage cell line was purchased from the Cell Culture Center of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modi ed Eagle's medium (Gibco, 11995065) supplemented with 10% fetal bovine serum (Gibco, 10091148). Containing 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, 15070063). The cells were incubated in a humidi ed atmosphere with 95% air and 5% CO 2 at 37 °C. To induce osteoclast differentiation, RAW264.7 cells were stimulated with 100 ng/mL RANKL and further cultured for the indicated times.

Retrovirus-mediated stable knockdown of Nox4
RAW264.7 cells were plated and cultured in 35-mm dishes. When the con uence reached 50%, the cells were transfected with retrovirus encoding Nox4 shRNAs or scrambled shRNA at a multiplicity of infection (MOI) of 100 for 24 h according to the manufacturer's instructions. The nucleotide sequences were as follows: sh-Nox4-1, 5′-GCAGGAGAACCAGGAGATTGT-3′; sh-Nox4-2, 5′-GCATGGTGGTGGTGCTATT CC-3′; sh-Nox4-3, 5′-GGTATACTCATAACCTCTTCT-3′; and sh-NC, 5′-TTCTCCGAA CGTGTCACGT-3′. RAW264.7 cells with stable knockdown of Nox4 expression were screened by the addition of 2 µg/mL puromycin to the culture medium for 48 h. Then, the stable cells were digested with 0.25% trypsin and seeded on 35mm dishes at a density of 8 × 10 4 cells/dish and incubated overnight for attachment. The next day, adherent cells were treated with or without RANKL (100 ng/mL) for 3 days. The knockdown e ciency of the three Nox4 shRNAs was measured using western blotting, and the most effective was selected for use in subsequent experiments.

Osteoclast differentiation assay
Osteoclast formation was measured by quantifying cells positively stained with TRAP. Brie y, RAW264.7 cells were incubated at a density of 1 × 10 4 cells/well in 24-well plates overnight. After stimulation with RANKL (100 ng/mL) and various concentrations of different pharmacological reagents for 6 days, the cells were xed with 4% paraformaldehyde for 30 min at room temperature and then stained by using a Tartrate Resistant Acid Phosphatase Assay Kit (Beyotime Biotechnology, P0332) according to the manufacturer's instructions. TRAP-positive and multinucleated cells containing three or more nuclei were considered osteoclasts. For each well, the osteoclasts were observed under a light microscope (Leica, Wetzlar, Germany).

Osteoclast bone resorption pit formation assay
To con rm the bone resorption ability of differentiated osteoclasts, RAW264.7 cells were seeded at a density of 2 × 10 4 cells/well overnight in 24-well Osteo Assay Surface plates (Corning, New York, NY, USA) coated with hydroxyapatite matrix. Then, the cells were incubated with RANKL (100 ng/mL) or in the presence of various concentrations of different pharmacological reagents. The medium was replaced every 3 days. After 7 days of culture, the cells were removed using a 10% sodium hypochlorite solution, and the wells were stained with 1% toluidine blue. The plate was washed twice with distilled water and air dried at room temperature. The areas of bone resorption pits in each well were determined using a light microscope (Leica).

Quantitative real-time PCR (qRT-PCR)
Total cellular RNA was extracted using RNAiso plus reagent (Takara, 9108) according to the manufacturer's instructions. Subsequently, the total RNA concentration was determined with a NanoDrop 2.0 spectrophotometer (Thermo Fisher Scienti c, Pittsburgh, PA, USA), and the RNA was reverse transcribed to cDNA using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara, RR047A) according to the manufacturer's instructions. Subsequently, qRT-PCR assays were performed by using a SYBR Premix Ex Taq™ II (2×) kit (Takara, RR820A) according to the manufacturer's instructions and run on an ABI 7500 Real-Time PCR Detection System (Foster City, CA, USA). The reactions were performed using the following parameters: 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primer nucleotide sequences used for PCR are listed in Table 1. All primer sets for mRNA ampli cation were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The relative expression levels of the target gene were normalized with respect to the levels of β-actin expression and calculated using the 2 −△△CT method. RAW264.7 cells were cultured with the indicated treatments for 3 days. Then, the cells were digested with 0.25% trypsin, centrifuged (2000 rpm) for 10 min and xed with 2.5% glutaraldehyde overnight at 4 °C. Subsequently, the cells were post xed with 1% osmium tetroxide for 1.5 h, washed and stained in 3% aqueous uranyl acetate for 1 h. Thereafter, the samples were washed again, dehydrated with a graded series of increasing ethanol concentrations to 100% and embedded in Epon-Araldite resin. Subsequently, the ultrathin sections were cut using a Reichert ultramicrotome (Reichert, New York, NY, USA) and counterstained with 0.3% lead citrate. Then, the ultrastructure of autophagic vacuoles (autophagosomes and autolysosomes) was observed under a Philips EM420 transmission electron microscope (Philips, UK), and images were captured.

Autophagic ux assessment
After growth to 50% con uence in 35-mm dishes, the cells were transfected with adenovirus expressing mRFP-GFP-LC3 (HanBio) for 24 h using a multiplicity of infection of 1000, according to the manufacturer's instructions. Then, the cell growth medium was replaced with fresh complete medium for another 24 h. Afterward, the transfected cells were digested with 0.25% trypsin and seeded on confocal Petri dishes (NEST, Wuxi, Jiangsu, China) at a density of 5 × 10 4 cells/dish and incubated overnight for attachment. Thereafter, adherent cells were treated with the various indicated treatments for 3 days. The treated cells were washed with phosphate buffer saline (PBS) and viewed with a laser scanning confocal microscope (Leica). GFP loses its uorescence in acidic lysosomal conditions, whereas mRFP does not.
Therefore, yellow (merged GFP signal and RFP signal) puncta represent early autophagosomes, whereas puncta detectable only as red (RFP signal alone) indicate late autolysosomes that are formed by autophagosome fusion with lysosomes. Autophagic ux was ultimately assessed by quantifying the mRFP and GFP puncta per cell. The number of GFP and mRFP puncta was determined by manually counting 30 cells randomly in 5 elds per dish, and the average number of puncta per cell was calculated.
2.9. ER-Tracker staining in living cells RAW264.7 cells (5 × 10 4 ) were plated on confocal Petri dishes (NEST) and allowed to attach overnight. Then, the cells were cultured with the indicated treatments for the indicated times. Next, ER-Tracker (Thermo Fisher Scienti c, E34250) was added directly to the culture medium at 500 nM and incubated with cells for 30 min in a 37 °C humidi ed incubator containing 5% CO 2 . Then, the cells were washed with PBS and immediately observed under a laser scanning confocal microscope (Leica).

Immuno uorescence staining for Nox4 localization
The colocalization of Nox4 with the ER was detected by double-labeling immuno uorescence. Brie y, RAW264.7 cells were seeded on confocal Petri dishes (NEST) at a density of 5 × 10 4 cells/dish overnight.
Then, the cells were incubated with or without RANKL (100 ng/mL) for 3 days. Thereafter, the cells were stained with ER-Tracker (the detailed experimental procedure is described in step 2.9) and xed in 4% 2.12. Western blot analysis RAW264.7 cells were lysed in RIPA buffer that contained a protease and phosphatase inhibitor cocktail. After centrifugation at 14000 × g for 5 min at 4 °C, the concentrations of protein were measured using a BCA protein assay kit (Beyotime Biotechnology, P0010). Subsequently, equal amounts of protein (40 µg) were separated by 6%, 8% or 12%/5% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Determination of intracellular and ER ROS
The intracellular production of ROS was detected by staining cells with a Reactive Oxygen Species Assay kit (Beyotime Biotechnology, S0033S). Brie y, RAW264.7 cells (5 × 10 4 ) were seeded in confocal Petri dishes (NEST) overnight. Then, the adherent cells were cultured under conditions with various treatments for the indicated times. Subsequently, the cells were stained with ER-Tracker (the detailed experimental procedure is described in step 2.9) and washed with PBS. Then, 2′,7′-dichloro uorescein diacetate, which was added directly to serum-free medium, was diluted to a nal concentration of 10 µM and incubated with cells for 30 min at 37 °C in a humidi ed incubator containing 5% CO 2 . 2′,7′-dichloro uorescein diacetate diffuses into cells and is deacetylated by cellular esterases to non uorescent (DCFH), which can be oxidized by ROS to produce highly uorescent 2′,7′-dichloro uorescein. The green uorescence intensity is proportional to the levels of ROS within a cell. The cells were then washed three times with PBS, and the uorescence intensity was observed using a laser scanning confocal microscope (Leica). The data are expressed as the mean ± SD of three independent experiments. The difference in means between 2 groups was compared using Student's t-test. The data for multiple groups were analyzed by Availability of data and materials

Measurement of mitochondrial ROS
Most data generated or analyzed during this study are included in this published article and its supplementary information les, and the rest are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. to the conception and design of this work. All authors read and approved the nal manuscript and approved its submission. All authors have agreed both to be personally accountable for the their own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.