GeneChip analysis

Total RNA was extracted from BMMs cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL and used for cDNA synthesis by reverse-transcription. Biotinylated cDNA was synthesized with Ambion Illumina RNA amplification kit (Ambion). Microarray was performed according to the Illumina GeneChip manual (outsourced to Macrogen).

Cell culture

BMMs were obtained from tibial and femoral bone of male mice. Bone marrow cells were cultured in α-MEM (Hyclone, USA) containing 10% FBS (Hyclone). After 1 day, non-adherent cells were cultured with 30 ng/mL M-CSF (R&D Systems, USA) for 3 days, as described previously.68 Osteoclasts were generated by culturing BMMs with 30 ng/mL M-CSF and 100 ng/mL RANKL (PeproTech, UK).

Bone marrow derived dendritic cells (BMDCs) were obtained from 6–8-week-old mice. Bone marrow cells were isolated and seeded in DMEM (Hyclone) containing 10% FBS with 20 ng/mL GM-CSF. On days 3 and 5, cells were treated with 20 ng/mL rmIL-4. Then on day 7, cells were harvested after incubation with or without 100 ng/mL LPS for 24 h.

Bone marrow stromal cells (BMSCs) were obtained from 4-week-old mice as described previously.69 The tibial and femoral bone were cut into small pieces and incubated for 90 min at 37 °C with shaking in a solution containing collagenase type II (C6885, Sigma-Aldrich, USA). The cells were cultured in a 100 mm culture dish.

Primary osteoblasts were harvested from calvaria as described previously.70 The calvariae were digested for 15 min in a solution containing collagenase type II (Sigma-Aldrich) and dispase II (Roche) at 37 °C with shaking. The cells were cultured in a 100 mm culture dish. HEK293T cells and HeLa cells were cultured in DMEM (Hyclone).

Mice

Whole-body Rufy4 knockout mice were generated using CRISPR/Cas9 technology (outsourced to Macrogen) and backcrossed to a C57BL/6 J background for more than five generations. C57BL/6 J mice were purchased from Jackson Laboratory. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Laboratory Animal Center.

Micro‑computed tomography (micro-CT) analysis

The left hind limb was harvested, fixed, and scanned using a micro-CT (Skyscan1176, Bruker microCT, Kontich, Belgium). The scanning parameters were set to a voltage of 75 kV, a scanning current of 333 μA, a resolution of 18 μm, an exposure time of 260 ms, and a rotation step of 0.7 deg. The raw data was converted into 2D cross-sectional imageS using NRecon (Brucker micro-CT, Kontich, ver.1.6.9.3, Belgium). Reconstructed images were geometrically aligned with DataViewer (Brucker micro-CT, Kontich, ver.1.5.1.2, Belgium), after which the structural parameters of the tibial and femoral bone were evaluated by CT Analyzer (CT-AN ver.1.10.9.0, Brucker, Belgium). The structural trabecular bone variables are as follows: bone mineral density (BMD, g/cm3), which reflects the extent of bone mineral in the bone tissue; bone volume fraction (BV/TV, %); trabecular number (Tb.N, mm−1); trabecular bone pattern factor (Tb.Pf, mm−1), which is related to connectivity of trabecular bone; trabecular thickness (Tb.Th, mm). The structural cortical bone variables are as follows: BMD (g/cm3); bone volume fraction (BV/TV, %); cross-sectional thickness (Ct.Th, mm); cortical bone area (Ct.Ar, mm2); total cross-sectional area inside the periosteal envelope (Tt.Ar, mm2).

Histology and immunohistochemistry

The right hind limb was harvested, fixed in 10% formalin for 3 days and decalcified in 0.5 mol/L EDTA for 10 days. 5 μm-thick paraffin sections were stained with ALP or TRAP (Wako, Japan). For immunohistochemistry, sections were incubated with anti-CTSK antibody (sc-48353, Santa Cruz, USA) overnight. Images were obtained under an Olympus BX51 microscope (Olympus) with Olympus DP72 camera (Olympus). Osteoclastic and osteoblastic variables (N.OC/BS, OC-bone contact length, resorption pit depth, N.OB/BS) were analyzed using ImageJ (NIH, USA).

Bone resorption pit assay

Pre-osteoclasts were seeded on bone slices and cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL for 3 days. The cells adhering to bone slices were removed by mechanical agitation before staining the bone slices with hematoxylin (Sigma-Aldrich) or 20 μg/mL WGA-Alexa 488 conjugate (W11261, Invitrogen, USA) for 30 min. The resorption pit area was visualized using Olympus CKX53 inverted microscope (Olympus, Japan) with microscope digital camera. The resorbed pit area was quantified with Image-Pro Plus (Media Cybernetics, USA). The resorption pit depth was detected with confocal microscope (LSM 880 with Airyscan, Carl Zeiss, Germany) and measured using IMARIS (Bitplane, Switzerland).

Enzyme-linked immunosorbent assay (ELISA)

Supernatants or serum were collected and the concentrations of CTX-1 or PINP were detected with CrossLaps for Culture CTX-1 ELISA kit (AC-07F1, IDS, UK), RatLaps CTX-1 EIA (AC-06F1, IDS), or Rat/Mouse PINP EIA (AC-33F1, IDS) according to the manufacturer’s instructions.

Calcium colorimetric assay

Amounts of calcium released after bone resorption were measured with calcium colorimetric assay kit (ab102505, Abcam, USA) according to the manufacturer’s instructions.

Immunoblot analysis

Cells were lysed in a RIPA buffer containing 0.5% Na-deoxycholate, 50 mmol/L Tris-Cl (pH 8.0), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP40, supplemented with protease inhibitors (1 mmol/L PMSF and 1 μg/mL leupeptin and aprotinin) and phosphatase inhibitors (1 mmol/L NaVO4 and 1 mmol/L NaF) after vortexing 5 times for 30 min on ice. After centrifugation at 14 000 r/min for 20 min at 4 °C, the supernatants were boiled in 6X SDS buffer containing 0.6 mol/L DTT. The cell lysates or immunoprecipitated proteins from co-IP experiments were separated by 10% SDS-polyacrylamide gels and transferred electrophoretically onto a polyvinylidene difluoride membrane (Millipore, USA). The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 and then immunoblotted with the indicated primary antibodies (Table S1) and secondary antibodies conjugated to HRP. Proteins were detected using ECL detection kit (Biorad, USA).

Immunofluorescence staining

Cells on glass coverslips or bone slices were fixed with 4% paraformaldehyde in PBS for 15 min. For F-actin staining, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min and incubated with fluorescent dye-conjugated phalloidin (Invitrogen) for 30 min. For LAMP2 staining, cells were permeabilized with 0.2% saponin in PBS for 1 h and blocked with 0.05% saponin/1% BSA/PBS for 30 min followed by an incubation with primary antibodies overnight. For other immunofluorescence staining, cells were permeabilized and blocked with 5% BSA/0.3% Triton X-100/PBS for 1 h prior to adding primary antibodies (Table S2) in 1% BSA/0.3% Triton X-100/PBS overnight. Following primary antibody incubation, cells were incubated with the respective fluorescent dye-conjugated secondary antibodies (Invitrogen) for 1 h. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole. Fluorescence images were obtained under confocal microscope and the mean intensity per cell or actin ring was measured using IMARIS.

Measurement of endo-lysosomal pH

Endo-lysosomal pH in osteoclasts was measured as described previously.43 Briefly, osteoclasts were incubated with 20 μg/mL pH-sensitive pHrodo green dextran (Thermo Fisher Scientific, USA) or 20 μg/mL pH-insensitive dextran with Alexa Fluor 546 (Thermo Fisher Scientific) for 3 h at 37 °C. The cells were washed 3 times with cold PBS, detached and collected for FACS analysis. Intracellular dextran fluorescence was measured using BD FACS Calibur (BD Biosciences, USA) and analyzed by FlowJo (TreeStar, USA).

Lysosomal intracellular activity assay

Lysosomal activity was measured with Lysosomal Intracellular Activity kit (Abcam) according to the manufacturer’s instructions. Briefly, osteoclasts were incubated in fresh medium supplemented with 0.5% FBS and 15 μg/mL self-quenched substrate for 1 h at 37 °C. After incubation, the cells were collected, washed twice with Assay buffer, and resuspended in PBS. The intracellular immunofluorescence was measured with BD FACS Calibur (BD Biosciences) and analyzed by FlowJo (TreeStar).

Transmission electron microscopy

Osteoclasts cultured on bone slices were fixed with 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (pH 7.4) for 1 h at 4 °C. The bone slices were decalcified in 5% EDTA/0.1% glutaraldehyde for 3 days and post-fixed with osmium tetroxide for 40 min at 4 °C. The samples were dehydrated in a graded series of ethanol, treated with graded propylene oxide series, and embedded into Epon. The sections were then cut into ultra-thin 80 nm-thick sections and placed on a copper grid. The samples were stained with uranyl acetate and lead citrate and then observed using transmission electron microscope (JEOL-2100F, USA).

Flow cytometry analysis

Single-cell suspensions in FACS buffer (PBS containing 2% FBS) were prepared and incubated with anti-mouse CD16/CD32 for blocking non-specific Fc receptors binding. The cells were then stained with primary antibodies (Table S3). After 30 min of incubation on ice in the dark, the cells were washed and resuspended in FACS buffer. Flow cytometry was performed on a BD LSRFortessa.

Constructs

Full-length Rufy4 cDNA (NM_001170641.1) was amplified from osteoclasts. After PCR amplification, Rufy4 and its deletion mutants were subcloned into p3xFlag-CMV-13 (Addgene). 3xFlag-tagged RUFY4 and its mutants were subcloned into pMX-Puro plasmid (provided by T. Kitamura, University of Tokyo). pEGFP-N3 and pEBG vectors were obtained from Addgene. pGL3 luciferase reporter vector was obtained from Promega. CMV-CFP-Rab7 vector was provided by Dongmin Kang (Ewha Womans University). LAMP2 expression vector in the pCMV6 vector was obtained from Origene. Amplified LAMP2 from pCMV6-LAMP2 was subcloned into p3xFlag-CMV-13. The primers used are listed in Table S4.

Retroviral infection

Platinum-E (Plat-E) packaging cells were transfected with various pMX-puro construct DNAs using PEI transfection reagent (Sigma-Aldrich). BMMs were infected with the retroviruses as previously described.71 The pMX-puro vector and Plat-E cells were provided by T. Kitamura (University of Tokyo). After viral infection, BMMs were cultured with 30 ng/mL M-CSF and 2 μg/mL puromycine for 3 days. Puromycine-resistant BMMs were used for further analysis.

Plasmid transfection

HEK293T cells were transfected with the indicated expression vectors using PEI transfection reagent (Sigma-Aldrich) and then subjected to GST-pull down assays or co-IP.

GST-pull down and co-IP assays

Cells were lysed on ice in RIPA buffer supplemented with protease inhibitors (1 mmol/L PMSF and 1 μg/mL leupeptin and aprotinin). For GST-pull down assay, cell lysates were pulled down by glutathione-Sepharose 4B (GE Healthcare Life Science, USA) for 4 h. After washing three times with RIPA lysis buffer, the beads were boiled with 2X SDS loading buffer. For co-IP assay, cell lysates were incubated with the indicated primary antibodies overnight and were further incubated with protein A-agarose (Millipore) for 1 h at 4 °C with rotation. After washing three times with lysis buffer, the beads were boiled with 2X SDS loading buffer. Immunoblot analysis was then conducted.

RNA isolation and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted using TRIzol (Invitrogen) and reverse transcribed with RTase kit (Biofact). Polymerase chain reaction (PCR) amplification was performed with SYBR Green Master kit (Bioline) using ABI PRISM 7300 system (Applied Biosystems). Actin primers were used for normalization. The gene-specific primers are listed in Table S5.

Transcription factor binding site prediction and luciferase reporter assay

NFATc1 and NF-κB p65 binding site sequences were obtained from JASPAR database (https://jaspar.genereg.net/). The prediction of the NF-κB p65 binding site in Rufy4 promoter was obtained from ALGGEN-PROMO (http://alggen.lsi.upc.es). Each of the predicted Rufy4 promoter regions (total 4.2 kb: −3 735 ~ −2 338, −2 337 ~ −1 776, −1 775 ~ −90, −455 ~ +462) was cloned into pGL3-basic luciferase vector. HEK293T cells were seeded on 12-well culture plate and then transfected with 900 ng of the above reporter constructs and 0.2 ng of the Renilla luciferase construct with 100 ng of pcDNA-Nfatc1, pcDNA-p65, or a mock plasmid using PEI transfection reagent (Thermo Fisher Scientific) for 24 h. Transfected cells were harvested for dual-luciferase reporter gene assay kit (Promega) according to the manufacturer’s instructions. Firefly and Renilla luciferase were measured with VICTOR X Light Luminescence Plate Reader (PerkinElmer). The reporter activity of all samples was normalized with Renilla luciferase.

TRAP staining

BMMs were cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL on 48-well culture plate for 4–5 days. The cells were fixed with 4% paraformaldehyde for 10 min and stained with TRAP (Wako).

Colony-forming unit fibroblast (CFU-F) assay

300 BMSCs were seeded on 6-well culture plate and cultured for 14 days. The cells were fixed with 4% paraformaldehyde for 10 min and stained with crystal violet solution (V5265, Sigma-Aldrich). Colonies that contained more than 50 cells were counted.

ALP and ARS staining

BMSCs or calvarial osteoprogenitors were cultured with 50 μg/mL L-ascorbic acid, 10 mmol/L β-glycerophosphate, 10 nmol/L dexamethasone, and 50 ng/mL rhBMP2 on 48-well culture plate for 7 or 14 days. The cells were then fixed with 4% paraformaldehyde for 10 min and stained with ALP (Wako). The mineralized bone matrix was stained with 2% ARS after fixation with ethanol.

Calcein double-labeling

8–9-week-old male mice were intraperitoneally injected with calcein (25 mg/kg, C0875, Sigma-Aldrich) twice at intervals of 3 days. Femurs and tibias of the mice were harvested 4 days after the last injection, fixed with 10% formalin, and dehydrated in a graded series of ethanol. The tissues were then embedded in plastic (Osteo-Bed Bone Embedding kit, Sigma-Aldrich) and sectioned into 7 μm-thick slices. The mineral apposition rate (MAR), mineralizing surface (MS/BS), and bone formation rate (BFR/BS) of femur were analyzed under a confocal microscope.

LPS-induced calvarial bone destruction

An animal model of LPS-induced bone loss was generated as previously described.72 Briefly, PBS or LPS (12.5 mg/kg) was injected into the subcutaneous tissue overlying calvaria of 5–6-week-old male mice. The calvaria were harvested 5 days after the initial injection, fixed, and decalcified, and stained with TRAP.

OVX-induced postmenopausal osteoporosis

OVX or sham surgery was conducted in 10–11-week-old female mice. Femurs and tibias of the mice were harvested 7 weeks after the surgery and subjected to micro-CT scanning and TRAP staining.

Statistical analysis

Student’s two-tailed t-test or one-way or two-way ANOVA was performed using Prism 8.0.2 (GraphPad) as indicated in the figure legends. P < 0.05 was considered statistically significant.