Mice

Lysozyme M promoter-driven Cre transgene on the C57/BL6 background mice (known as LysMcre mice) and SREBP2flox/flox mice were purchased from The Jackson Laboratory. LysMcre mice were crossed with SREBP2flox/flox mice to generate Srebp2fl/flLysM-Cre (referred to as SREBP2ΔM) and Srebp2+/+LysM-Cre (referred to as SREBP2WT) mice. Cathepsin K promoter-driven Cre transgene on the C57/BL6 background mice (known as CtsKcre mice) were obtained from Dr. Takashi Nakamura.41SCAPflox/flox mice were also purchased from The Jackson Laboratory. CtsKcre mice were crossed with SCAPflox/flox mice to generate SCAPfl/flCtsK-Cre (referred to as SCAPΔOC mice). IRF7 deficient mice were obtained from Dr. Deng.26 Oligonucleotide sequences for genotype OCR are in Table S2. All animals were housed in a specific pathogen-free environment in the Weill Cornell Medicine vivarium and all the experiments conformed to the ethical principles and guidelines approved by the Institutional and Animal Care and Use Committee of Weill Cornell Medical College and Sloan Kettering Cancer Institute (IACUC #2015-0065).

Inflammatory bone destruction models

TNF-induced supracalvarial osteolysis model was performed as previously described. Briefly, recombinant human TNF-α was administrated daily injected at the dose of 40 µg/kg to calvarial periosteum of 12-week-old female control and SREBP2ΔM mice for five consecutive days. For K/BxN serum-induced arthritis model, K/BxN serum was collected as previously described.42 In brief, 80 µL of K/BxN sera were administered intraperitoneally to 14-week-old C57/BL6 female mice on days 0 and 2 and the mice were euthanized on day 14 after daily monitoring and measuring the thickness of wrist and ankle joints using calipers. The thickness of joints (total of 4 per mouse) was summed up and described in the graph.

Micro-CT and histomorphometry analysis

To analyze bone volume and architecture, μ-CT analysis was performed using micro-CT-35 instrument (Scanco Medical, Bruttisellen, Switzerland) as described previously,43,44 and all samples were included in the analysis conducted in a blinded manner. For μCT analysis, prior to decalcification, femurs were scanned by μCT, with an isotropic voxel resolution of 6 µm (μCT35, Scanco, Bruttisellen, Switzerland; 55kVp, 145μA, 600 ms integration time) to evaluate morphological changes in bone. Bone morphology in the femur was examined in two regions: the diaphysis and the metaphysis. For cortical bone, the volume of interest (VOI) encompassed cortical bone within a 100-slice section in the diaphysis. For trabecular bone, the VOI encompassed a 200-slice section in the metaphysis, proximal to the growth plate. To ensure exclusion of primary spongiosa in the growth plate, VOIs began 50 slices proximal to the median of the growth plate. Outcome parameters for cortical bone included thickness and tissue mineral density (TMD). Trabecular bone parameters included bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and trabecular number (Tb.N.). 3D reconstructions were generated by stacking thresholded 2D images from the contoured region. To measure a bone formation rate, the dynamic bone labeling analysis was performed as previously described. In brief, calcein (Sigma) was dissolved in 2% NaHCO3 and administered twice at the dose of 10 mg/kg with a 5-day interval. The right femurs were fixed in 4% PFA for 4 days and decalcified for 2 days, followed by 10% sucrose solution overnight incubation at 4 °C. After embedding in Tissue-Tek OCT compound (Sakura Finetek), frozen section at 9 µm thickness was performed. After mice were euthanized, femurs, calvarial bones, hind paws, and ankles were collected for histologic assessment. The bones were fixed in 4% paraformaldehyde (PFA) for 4 days followed by decalcification in 10% EDTA (Sigma Aldrich) solution for 4 weeks. Paraffin embedded samples were subjected for 8–9 µm sectioning followed by TRAP staining and methyl green counterstaining (Vector Laboratories) for clear visualization of osteoclast and bone. Trabecular analyses were confined to the secondary spongiosa of femurs (excluding cortical bone), covering approximately 1.5 mm2 tissue area. For the supracalvarial osteolysis model, the analyses were restricted to the right parietal (where TNF-α injected), excluding frontal, interparietal, and occipital regions. For the K/BxN model, left tarsal bones were subjected to analyses, including the surfaces of cuneiforms, navicular, and cuboid. Multinucleated TRAP+ cells that adhered to the bone surface were considered osteoclasts. All measurements were performed using Osteometric software (Osteomeasure) following the standard procedure.45

In vitro osteoclastogenesis

Mouse bone marrow macrophages (BMMs) were prepared as previously described.43 BMMs were committed to the pre-osteoclast lineage by treating them with 5% L929 cell supernatant, which served as a source of M-CSF conditioned medium (CM).46 In brief, flushed bone marrow cells from the femurs and tibiae were incubated for 1 min in ACK lysing buffer (Gibco) to remove red blood cells and cultured with CM for 3 days. Cultured cells were scraped, counted, and seeded at a density of 105/mL in an appropriate plate (triplicate wells on a 96-well plate for TRAP staining, single to double wells on a 6-well plate for RNA, single to double wells on a 24-well plate for protein). 5% CM and RANKL (50 ng/mL, Peprotech) were added in α-MEM (Thermo Fisher Scientific) supplemented with 10% FBS (R&D systems), 1% glutamine (200 mmol/L, Thermo Fisher Scientific), and 1% pen/strep antibiotics (Thermo Fisher Scientific) to induce osteoclasts with every 2 days replenishment.

For human osteoclast differentiation assay, the procedures were followed by previously described method.47 This protocol was approved by the Hospital for Special Surgery Institutional Review Board (IRB #2019-0681). Briefly, peripheral blood mononuclear cells (PBMCs) were collected from blood leukocyte preparations purchased from the New York Blood Center by density gradient centrifugation with Ficoll (Thermo Fisher Scientific). Anti-CD14 magnetic beads (0.6 µL/106 cells, Miltenyi Biotec) were used to select CD14+ monocytes. Cells were plated at a density of 106/mL in an appropriate plate (triplicate wells on a 96-well plate for TRAP staining, single to double wells on a 6-well plate for RNA, single to double wells on a 24-well plate for protein). M-CSF (20 ng/mL, Peprotech) and RANKL (40 ng/mL, Peprotech) were added in α-MEM (Thermo Fisher Scientific) supplemented with 10% FBS (Hyclone, GE Healthcare Life Sciences) and 1% glutamine (200 mmol/L, Thermo Fisher Scientific) to induce osteoclasts. Cells seeded on the 96 plates were fixed and stained for TRAP using the Acid Phosphatase Leukocyte Diagnositic Kit (Sigma Aldrich). Multinucleated TRAP+ cells with 3 or more nuclei were considered as osteoclasts. For bone resorption pit assay, mouse BMMs were seeded at a concentration of 2 ×105/mL in 96-well Corning Osteo Assay Surface Plates (Sigma). 5% CM and RANKL (50 ng/mL, Peprotech) were added in α-MEM supplemented with 10% FBS, 1% glutamine, and 1% pen/strep antibiotics to induce osteoclasts with every 2 days replenishment. Total culture days were 6 to 8 days for optimal resorption in the entire well. Cells were removed by 10% bleach solution followed by 1% toluidine blue staining for clear visualization of resorbed pits.

Quantitative real-time PCR

RNeasy Mini Kit (Qiagen) with DNase treatment was used to prepare DNA-free RNA. 100–800 ng total RNA was reverse transcribed using the First Strand cDNA synthesis kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Real-time PCR was performed in triplicate with Fast SYBR Green Master Mix (Applied Bisosystems) and ran on 7500 Fast Real-time PCR system (Applied Bisosystems). Human hypoxanthine guanine phosphoribosyl transferase (Hprt) and mouse TATA box-binding protein (Tbp) were used for housekeeping internal controls for human and mouse cells respectively. Oligonucleotide sequences are in Table S3.

RNA-sequencing

Total RNA was prepared by an RNeasy Mini Kit with DNase treatment. 200 ng RNAs were used to construct mRNA libraries using TruSeq Stranded mRNA kits (Illumina). Multiplexed barcode adapters were ligated to the libraries following the manufacturer’s protocols. Samples that were passed quality control analysis by a Bioanalyzer 2100 (Agilent) were submitted to the Weill Cornell Medical College Genomics Resources Core Facility. Paired-end reads (50 × 2 cycles, ~75 × 106 reads per sample) were obtained on an Illumina NovaSeq 6000.

Immunoblotting

Whole cell lysates were collected using 1x Laemmli Sample Buffer (Bio-Rad), and nuclear lysates were prepared by Buffer A solution (10 mmol/L Hepes, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 1x proteinase inhibitor cocktail). Then, boiled lysates were loaded onto 7.5% or 10% SDS-PAGE, followed by standard Western blotting protocols. Alpha-tubulin for whole cell lysates and Lamin B for nuclear lysates were used as loading control. Anti-SREBP2 (PA1-338) antibody was purchased from Thermo Fisher Scientific (Waltham, USA), anti-SREBP2 (ab30682) from Abcam (Cambridge, UK), and Lamin B (12987–1-AP) from Proteintech (Rosemont, USA).

Cholesterol quantitation

An Amplex Red Cholesterol Assay Kit (Invitrogen) was used to perform cholesterol quantitation. In brief, 5 × 105 cells were extracted with 100 µL of chloroform: isopropanol: NP-40/IGEPAL-CA-630 (7:11:0.1) in a Bioruptor (Diagenode) with 30 s on 30 s off on a high-power output for 10 cycles for homogenization. Then, samples were centrifuged at 13 000 × g for 10 min to remove insoluble materials. Next, organic phase was transferred to a new tube and air dried completely at 50 °C to remove chloroform and organic residues. Then, 1X reaction buffer (included in the kit) was added and further processed according to the manufacturer’s instructions. Fluorescence intensity was measured with Varioskan Flash Spectral Scanning (Thermo Scientific) and 5 × 105 cells were subjected for DNA quantitation using a DNeasy Blood & Tissue Kit (Qiagen) for normalization.

Fluorescence microscopy

BMMs from control and SREBP2ΔM mice were cultured with M-CSF and RANKL for three days, and then, cells were fixed with 4% formaldehyde for 10 min. Next, cells were stained with Phalloidin-iFluor 568 for 20 min at room temperature, followed by Filipin (Sigma F9765) staining for 2 h at room temperature. Stained cells were viewed using the red and blue filters equipped in the ZEISS LSM 880 with Airyscan or Nikon Ni-E Eclipse microscope. Randomly chosen 5 fields with regions of interest (mature osteoclasts) were subjected to analysis with the QuPath (version 0.2.3) program for blue color intensity in osteoclasts.

Adenoviral transduction

Recombinant adenoviral particles encoding human nuclear domain of SREBP2 with FLAG protein (Ad-CMV-2xFLAG-SREBP2) was custom built from Vector Biolabs and was also used in previous report.13 Recombinant adenoviral particles encoding human IRF7 with HA protein (Ad-GFP-h-IRF7.3xHA) was custom ordered from Vector Biolabs. Ad-CMV-GFP from Vector Biolabs was used as control adenoviral particles. For transduction experiment, mouse BMMs were seeded at a density of 2 ×105 cells/mL on a 6 well plate with daily addition of M-CSF (40 ng/mL) for 2 days. Then, the complete media was exchanged to low serum media (2% FBS with no added glutamine and antibiotics) containing M-CSF (20 ng/mL), followed by adenoviral particles (150 MOI) addition. Human monocytes were seeded at a density of 2 × 106 cells/mL on a 6 well plate with daily addition of M-CSF (40 ng/mL) for 4 days. Then, the complete media was exchanged to low serum media (2% FBS with no added glutamine) containing M-CSF (20 ng/mL), followed by adenoviral particles (30 MOI) addition. The plates were centrifuged at 1 600 r/min for 30 min at room temperature. After 12 h of transduction, fresh complete media was replaced and incubated for >8 h before further process. The green fluorescent was monitored for efficiency of transduction.

RNA interference

0.4 nmol of siRNA oligonucleotides targeting SREBP2 (Dharmacon Cat#L-009549-00-0005) or Ly6a (Dharmacon Cat#L-063198-00-0005) were nucelofected in human CD14+ monocytes using a Nucleofector kit (Lonza) as previously described.48 Human Monocyte Nucleofector buffer and the AMAXA Nucleofector System (Lonza) program Y001 were used following the manufacturer’s instructions.

Chromatin immunoprecipitation

ChIP experiments were performed as previously described. After cells were cultured with M-CSF and RANKL, cells were fixed with 1% formaldehyde (Thermo Scientific) for 5 mins. 0.125 mol/L glycine was added for quenching the formaldehyde residues. Next, cells were lysed/washed and sonicated sequentially using LB1, LB2, and LB3 (LB1, 50 mmol/L HEPES-KOH, pH 7.5, 140 mmol/L NaCl, 1 mmol/L EDTA, 10% glycerol, 0.5% NP-40 and 0.25% Triton X-100; LB2, 10 mmol/L Tris-HCl, pH 8.0, 200 mmol/L NaCl, 1 mmol/L EDTA and 0.5 mmol/L EGTA; LB3, 10 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5 mmol/L EGTA, 0.1% sodium deoxycholate and 0.5% N-lauroylsarcosine). Nuclear lysates were sonicated with Bioruptor using a cycle with 30 s on 30 s off on a high-power output for 8 cycles. The sonicated samples were centrifuged at 20 000 × g for 15 min at 4 °C. Supernatants were collected and 5% of supernatants were saved as input and DNA analysis and the rest was incubated overnight with 5 µg of anti-FLAG M2 antibody at 4 °C (Sigma Aldrich) on a rotator. After overnight incubation, the magnetic beads were washed in ice-cold condition as follow (once in low salt buffer, 150 mmol/L NaCl, 0.5% Na deoxycholate, 0.1% SDS, 1% Nonidet P-40, 1 mmol/L EDTA pH 8.0, 50 mmol/L Tris HCl pH 8.0; once in high salt buffer, 500 mmol/L NaCl, 0.5% Na deoxycholate, 0.1% SDS, 1% Nonidet P-40, 1 mmol/L EDTA pH 8.0, 50 mmol/L Tris HCl pH 8.0; once in LiCl wash buffer, 100 mmol/L LiCl, 0.5% Na deoxycholate, 0.1% SDS, 1% Nonidet P-40, 1 mmol/L EDTA pH 8.0, 50 mmol/L Tris HCl pH 8.0; once in TE wash buffer, 10 mmol/L Tris-HCl pH 8.0, 1 mmol/L EDTA, 50 mmol/L NaCl). Then, DNA was eluted with elution buffer (50 mmol/L Tris-HCl pH 8.0, 10 mmol/L EDTA, 1% SDS) with added 0.3 mol/L NaCl and incubated for overnight at 65°C. Next day, the samples were incubated with Rnase for an hour at 37 °C followed by Proteinase K incubation for 2 h at 55 °C. Then, the DNA was purified with PCR purification kit (Qiagen) and subjected for quantitative Real-Time PCR assays.

Pathway analysis using GSEA

Gene set enrichment analysis (GSEA) is used for analyzing the differentially expressed genes.49 Hallmark gene sets in MSigDB (molecular signature data base) were used for the analysis. Pathways were ranked based on P values.

Statistics

Data are presented as the mean ± SD of biological replicates. Animals for each experiment were randomly selected for further treatments. An unpaired two-tailed Student’s t test was used to assess differences between two groups. One-way or two-way analysis of variance with post hoc Bonferroni or Tukey’s test was used for analyses of multiple groups. All statistical tests were performed using GraphPad Prism 8 (GraphPad Software). A P value of less than 0.05 was considered statistically significant.

Study approval

Animal experiments were performed in compliance with ethical regulations and approved by the Hospital for Special Surgery and Weill Cornell Medical College Institutional Animal Care and Use Committee (IACUC #2015-0065). The protocol for human osteoclast differentiation assay was approved by the Hospital for Special Surgery Institutional Review Board (IRB #2019-0681).