Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153

Patient recruitment

Synovial tissues were obtained from ten RA patients with a disease duration of 1.5–27 years and seropositive, erosive RA. All RA patients fulfilled the 2010 American College of Rheumatology (ACR) classification criteria for RA63. Tissue samples from matched healthy donors were included for the comparison.

Mice

Wildtype male C57BL/6JRj mice were supplied by Janvier Labs (Saint-Berthevin, France). Transgenic mice that overexpressed human TNFα (hTNFα tg)64,65, Ogt-floxed66, and LysM-Cre30,65 (The Jackson Laboratory, Bar Harbor, ME, USA) have been described previously. To knockout Ogt in myeloid lineage cells, we generated OgtΔLysM mice by cross-breeding Ogt-floxed and LysM-Cre. We used Ogt-floxed mice as littermate control for all the Ogt knockout experiments.

Bone marrow isolation and in vitro osteoclastogenesis

In vitro osteoclastogenesis was performed as previously described38,67 with minor modifications. In brief, bone marrow cells from 8 to 12-week old wildtype, Ogt-floxed, or OgtΔlysM mice were harvested by flushing the tibia and femur of mice with α-MEM (Gibco, New York, USA), followed by the lysis of red blood cells with RBC lysis buffer (Biolegend, San Diego, CA, USA). Afterwards, cells were cultured in in 10% FBS α-MEM overnight. The non-adherent cells were then seeded at the density of 2.5 × 105 cells per mL and expanded with 25 ng·mL−1 M-CSF (R&D Systems, Wiesbaden-Nordenstadt, Germany) for 3 days. Osteoclastogenesis was induced with 25 ng·mL−1 M-CSF, 50 ng·mL−1 RANKL (Peprotech, Hamburg, Germany) with or without 20 ng·mL−1 TNFα (R&D Systems) for 4 days. The culture medium was changed every 2 days.

Cellomics and TRAP activity for in vitro osteoclastogenesis

Cells were fixed in 4% formaldehyde and permeabilized in 0.2% Triton X-100 after 2 days (immature phase) or 4 days (mature phase) of in vitro osteoclastogenesis. After blocking with 5% horse serum in 2% BSA/PBS, cells were incubated with antibodies against F4/80 (Bio-Rad, Feldkirchen, Germany; clone Cl: A3-1) and O-GlcNAc (Invitrogen, Darmstadt, Germany, clone RL2). Corresponding Alexa Fluor-conjugated secondary antibodies and the dye HCS CellMask Deep Red Stain (both from Invitrogen) were used for detection of primary antibodies and for cytosol/nucleus staining, respectively. TRAP activity was visualized using a leukocyte acid phosphatase staining kit (Sigma-Aldrich, Steinheim, Germany) with ELF 97 Phosphatase Substrate (Invitrogen) as described68. Images and cellomics data were acquired by using a CellInsight CX5 High Content Screening Platform with SpotDetector.V4 BioApplication (Thermo Scientific, Darmstadt, Germany). For cells derived from bone marrow, TRAP+F4/80+ cells with a single nucleus were identified as immature osteoclasts69. TRAP+F4/80− cells with more than 3 nuclei were identified as mature osteoclasts. For cells derived from RAW264.7, immature and mature osteoclasts were identified by TRAP activity and nucleus count. Cellomics data were analyzed using the R software (version 3.6.1) with custom codes, and the images were processed by ImageJ software (NIH, version 1.52p).

Western blot analysis and ProteinSimple Wes immunoassay

Whole cell lysates from in vitro osteoclastogenesis were collected with Cell Lysis Buffer (Cell Signaling Technology, Frankfurt am Main, Germany). Western blots were performed as described previously70. Briefly, protein was separated by SDS-PAGE and transferred onto an Immobilon-P membrane (Millipore, Darmstadt, Germany). The membrane was incubated with antibodies against O-GlcNAc (Invitrogen, clone RL2), NFATc1 (Biolegend, clone 7A6), and β-actin (Sigma-Aldrich, clone AC-15) overnight. The blot was visualized with corresponding horseradish peroxidase-conjugated antibodies (Dako, Glostrup, Denmark) and Amersham ECL Prime Western Blotting Detection Reagent (Cytiva, Freiburg, Germany) and a ChemiDoc MP Imaging System (Bio-Rad Laboratories). The images of the bots were analyzed with Image Lab software (Bio-Rad Laboratories, version 6.0.1).

ProteinSimple Wes immunoassay was performed as described71 and analyzed by using a Wes system (ProteinSimple, Wiesbaden, Germany) with antibodies against OGT (OriGene Technologies, Herford, Germany), OGA (Sigma-Aldrich), and β-actin.

Histological and immunofluorescence (IF) analysis of synovial tissue

The histomorphometric analysis was performed as described previously72. Hind legs from mice were fixed in 4% formaldehyde and dehydrated in 50% ethanol, followed by decalcification in 500 mmol·L−1 acid-free EDTA and paraffin-embedding. Five µm sections of paws were stained with H&E for histology and TRAP for the quantification of the area of bone erosions and osteoclast counts using a leukocyte acid phosphatase staining kit (Sigma-Aldrich). Mature osteoclasts were identified as TRAP-positive multinucleated (nuclei ≥ 3) cells and the immature osteoclasts as TRAP-positive cells with a single nucleus. Images were captured using a Nikon Eclipse 80i microscope (Nikon Metrology, Alzenau, Germany) or a NanoZoomer S60 Digital Slide Scanner (Hamamatsu Photonics, Herrsching am Ammersee, Germany).

The IF analysis was performed as described73. Briefly, paw sections were deparaffinized and rehydrated, followed by heat-induced epitope retrieval using boiling 10 mmol·L−1 sodium citrate pH 6.0 buffer and Tris-EDTA buffer (10 mmol·L−1 Tris, 1 mmol·L−1 EDTA, 0.05% Tween-20, pH 9.0). After blocking with 5% horse serum in 2% BSA/PBS, sections were incubated with antibodies against TRAP (Abcam, Berlin, Germany; clone EPR15556 for human tissue; Abnova, Taipei City, Taiwan; polyclonal for mouse tissue), CD14 (Biorbyt, Eching, Germany; polyclonal for human tissue), F4/80 (Bio-Rad Laboratories, clone Cl: A3-1 for mouse tissue), and O-GlcNAc (Invitrogen, clone RL2), followed by alexa fluor-conjugated secondary antibodies (Invitrogen). Confocal images were acquired by using a Leica SP5 II confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany), and processed with the same contrast adjustment for comparison by using OMERO web platform (University of Dundee & Open Microscopy Environment, version 5.6.3). The quantification of O-GlcNAc in TRAP-positive cells on the articular surface of the tarsus was performed by measuring mean gray value of O-GlcNAc staining within semi-automatically generated region based on TRAP signal using ImageJ software (NIH, version 1.52p) for unbiased quantification.

Quantitative real-time PCR

Quantification of gene expression by quantitative real-time PCR was performed as described74. Briefly, RNA was isolated using a NucleoSpin RNA isolation kit (Macherey-Nagel, Düren, Germany). After reverse transcription, cDNA was mixed with SYBRGreen PCR Master Mix (Applied Biosystems, Darmstadt, Germany) and primer pairs described at Supplementary Table 1. Gene expression was quantified using the comparative CT method with Actb as a house keeping gene. Real-time PCR was performed using StepOnePlus Real-Time PCR System (Applied Biosystems). The data were analyzed with StepOne Software v2.3 (Applied Biosystems).

Quantification of the number of metabolically active cells

The number of metabolically active cells was determined using the Cell Counting Kit-8 (CCK-8, Sigma-Aldrich) following the instruction from the supplier. In brief, the CCK-8 reagent was added to the cells after 3 days of in vitro osteoclastogenesis for 2 h. Then the absorbance at 450 nm was measured by using a GloMax Discover Microplate Reader (Promega, Walldorf, Germany). The number of metabolically active cells was calculated using a standard curve generated by serial dilution of cell number when seeding.

In vitro bone resorption assay

The bone resorption assays were performed as previously described75. In brief, bone marrow cells from mice were seeded on bone slices (Immunodiagnostic Systems, Frankfurt am Main, Germany) with cytokines for in vitro osteoclastogenesis and with or without 20 µmol·L−1 OSMI-1 (Sigma-Aldrich) or 10 µmol·L−1 Thiamet-G (Tocris, Wiesbaden-Nordenstadt, Germany). The culture medium was changed every 2 days for 2 weeks. The resorption pits were visualized by staining with 1% toluidine blue (Sigma-Aldrich). Images were acquired using a Nikon Eclipse 80i microscope (Nikon Metrology) and then analyzed with ImageJ software (NIH, version 1.52p).

Arthritis mouse models and in vivo treatments

hTNFα tg mice spontaneously develop chronic inflammatory arthritis64. For OGA inhibition, hTNFα tg mice were injected with 6 mg·kg−1 Thiamet-G (Tocris) intraperitoneally (i.p.) every other day, starting at the age of four weeks. To knockout Ogt in the osteoclast precursors, bone marrow transplantation was performed as described11. In brief, four-week-old wildtype or hTNFα tg mice received total body irradiation at the dose of 10.5 Gy. After 24 h, the irradiated mice were transplanted with bone marrow cells from Ogt-floxed or OgtΔLysM mice by tail vein injection. The mice were euthanized at the age of nine weeks.

Serum-induced arthritis (SIA) was induced by injection of pooled serum from K/BxN mice as described72. Arthritis was initiated in mice at the age of eight weeks by i.p. injection of 200 µL K/BxN serum and boosted with the same amount of serum three days later. To inhibit OGT or OGA, mice were injected i.p. with 10 mg·kg−1 OSMI-1 (Sigma-Aldrich) or 6 mg·kg−1 Thiamet-G (Tocris), respectively, every other day. Mice were euthanized 14 days after the first serum injection.

Ovariectomy (OVX) -induced osteoporosis was performed by surgical removal of bilateral ovaries75. Female Ogt-floxed and OgtΔLysM mice were used to evaluate the effect of O-GlcNAc in osteoporosis. OVX and sham operation, in which ovaries were left intact, were performed when the mice reached 8 weeks of age. Mice were kept for five weeks after the operation before euthanization.

The development of arthritis was evaluated by measuring joint swelling at the paws and the grip strength of the mice as described75. Bone tissues from the hind legs of the mice were harvested for further analysis. All studies were approved by the government of Unterfranken, Germany.

Microcomputed tomography (µCT) and analysis

µCT analysis was performed as previously described76. Bones of tibiae and paws from mice were preserved in 50% ethanol. µCT images were acquired using a SCANCO Medical μCT 40 scanner (SCANCO Medical AG, Brüttisellen, Switzerland) with optimized settings for visualization of calcified tissue at 55 kVp at a current of 145 µA and 200 ms integration time for 500 projections/180°. The trabecular measurements were performed at a 1 680 µm region located around 400 µm below the middle of the tibia metaphysis. The volume segmentation for the microarchitectural quantification of the trabecular bone was performed using the SCANCO Evaluation Software (SCANCO Medical AG) with a voxel size of 8.4 µm.

RNA sequencing

RNA sequencing was performed by Novogene Co., Ltd (Cambridge, United Kingdom) on an Illumina NovaSeq platform using a paired-end 150 bp sequencing strategy. Quality assessment and gene mapping were also performed by Novogene Co., Ltd (Cambridge, United Kingdom). Briefly, reads pairs in FASTQ format were quality assessed by FastQC v0.11.5 and mapped to murine genome (GRCm38/mm10). On average, 41% of raw reads were uniquely mapped. Uniquely mapped reads were assigned to annotated genes with Tophat software (Johns Hopkins University). Read counts were normalized by trimmed mean of M values (TMM) method. Principal component analysis (PCA) plots, heatmaps, and volcano plots, and bubble plots were generated with ggplot2, pheatmap, EnhancedVolcano77 R packages. Differential gene expression analysis was performed by using R package edgeR with Quasi-likelihood F-tests for statistical significance and following thresholds for the identification of differential expression genes (DEGs): false discovery rate-corrected q value <0.05 and fold change > 1.5 (for Thiamet-G treated cells) or 2 (for OSMI-1 treated cells). DEGs were subsequently used for gene ontology (GO) gene functional analysis with clusterProfiler package78. Gene ratios of selected GO terms were plotted as bubble plots. Gene set enrichment analysis (GSEA) was performed using the GSEA software version 4.0.3 (Broad Institute) with a threshold of 0.25 for false discovery rate-corrected q value79. The curated gene sets for GSEA were downloaded from Bader Lab80 (release April 01 2020), which contains pathways from GO biological process81, Reactome82, Panther83, NetPath84, NCI85, MSigDB86 (C2, H collections). Gene sets from GSE1050087 and a published gene set for osteoclast differentiation36 were also included for GSEA analysis.

To identify distinct genes at different stages of osteoclast differentiation, we analyzed the microarray data from the previous study (accession number GSE138324)34 by the web tool GEO2R (NCBI). The unique DEGs at different time points of differentiation were identified with the threshold of P-value > 0.05 and fold change >1.5. The DEGs from microarray were further matched to our RNA sequencing data and visualized by heatmaps. To compare the transcriptome of osteoclasts with OSMI-1 treatment with those of MYC knockout osteoclasts, we employed the RNA-Seq data created by Bae et al. (accession number SRP096890) and analyzed as described above38.

The information of DEGs was submitted to Ingenuity Pathway Analysis (Qiagen) for upstream regulator analysis. To perform the motif enrichment analyses, the upstream sequences of DEGs were retrieved by RSAT tools88 then analyzed by matrix-scan89 and AME90 with motifs retrieved from JASPAR (2020) and ENCODE (2018-03) databases. Random sequences generated based on the upstream sequences were used as the background model for the motif enrichment analyses.

Identification of O-GlcNAcylated proteins by mass spectrometry

Mass spectrometry with O-GlcNAcylated protein was performed as described91. Briefly, whole cell lysates were collected from RAW264.7 cells treated with RANKL and TNFα. O-GlcNAcylated protein was further enriched by using Protein A/G agarose beads (Santa Cruz Biotechnology) coupled with antibodies against O-GlcNAc (Invitrogen, clone RL2). Following elution with 2× Lämmli buffer, the enriched protein was separated by SDS-PAGE and visualized by InstantBlue Coomassie staining (Abcam). The protein was in-gel digested with trypsin (Roche), extracted with 70% acetonitrile, 0.03% formic acid, lyophilized, and resuspended in 0.1% TFA (Trifluoroacetic acid). NanoLC-ESI-MS/MS experiments were performed on an Ultimate 3000 nano-RSLC (Thermo Fisher Scientific) coupled to a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific) using a Nanospray-Flex ion source (Thermo Fisher Scientific). Peptides were concentrated and desalted on a trap column (5 mm × 30 µm, Thermo Fisher Scientific) and separated on a 25 cm × 75 µm nanoEase MZ HSS T3 reversed-phase column (100 Å pore size, 1.8 µm particle size, Waters, USA) operating at constant temperature of 35 °C. Peptides were separated at a flow rate of 300 nL per min with the following gradient profile: 2%–15% solvent B in 37 min, 15%–30% solvent B in 30 min, 30%–45% solvent B in 23 min, 45%–95% solvent B in 20 min and isocratic at 95% solvent B for 15 min. Solvents used were 0.1% formic acid (solvent A) and 0.1% formic acid in acetonitrile/H2O (80/20, v/v, solvent B).

The Q Exactive HF-X was operated under the control of XCalibur 4.1.31.9 software. MS spectra (m/z = 300–1 800) were detected in the Orbitrap at a resolution of 60 000 (m/z = 200) using a maximum injection time (MIT) of 100 ms and an automatic gain control (AGC) value of 1 × 106. Internal calibration of the Orbitrap analyzer was performed using lock-mass ions from ambient air as described92. The 30 most abundant peptide precursor signals per MS scan were selected for MS/MS analysis. Peptides were fragmented using high energy collision dissociation (HCD) at a normalized collision energy of 27 and subsequently analyzed in the Orbitrap at a resolution of 15 000. Further settings for MS/MS spectra included an isolation width of 1.6 Da, a MIT of 100 ms and an AGC value of 5 × 105. The dynamic exclusion was set to 20 s.

The protein was identified using the Mascot 2.6.1 software (Matrix Science, London, UK). Spectra were searched against the mouse reference proteome sequence downloaded in FASTA-format from UniProt93. Search parameters specified trypsin as cleaving enzyme with three accepted missed cleavages, a 5 ppm mass tolerance for peptide precursors, and 0.02 Da for fragment ions. Carbamidomethylation of cysteine residues was defined as fixed modification. Methionine oxidation, S, T, Y phosphorylation, and O-HexNAc-glycosylation at S and T were considered as variable modifications. Quantitative analysis was done by Scaffold 4.10 software (Proteome Software, Protland, USA) with a threshold of peptide probability >65% and a spectrum counting method. Selected O-glycopeptides were further inspected manually. Neutral loss N-acetylglucosamine and the O-GlcNAc oxonium ion (m/z = 204.086) was used as O-GlcNAc diagnostic marker.

Determine O-GlcNAc levels on NUP153 by immunoprecipitation

Whole cell lysates from in vitro osteoclastogenesis were collected in Cell Lysis Buffer (Cell Signaling Technology) either two (early) or four (late) days after first RANKL stimulation. Following pre-clearing, 50 µg of the lysates were incubated with 0.5 µg antibodies against NUP153 (Santa Cruz Biotechnology, clone R3G1) or NFATc1 (BioLegend, clone 7A6) and pulled down by Protein A/G agarose beads (Santa Santa Cruz Biotechnology). After elution with 2× Lämmli buffer, O-GlcNAc levels were determined by Western blot. Images were analyzed by Image Lab software (Bio-Rad Laboratories, version 6.0.1) with levels normalized to MCSF-treated cells at individual time points.

Assessment of nuclear accumulation of MYC in osteoclast precursors

RAW264.7 cells were transfected with Nup153 siRNA and costimulated with RANKL and TNFα as described. To determine the level of chromatin-bound MYC, lysate was collected 2 days after the stimulation. The chromatin-bound faction was obtained using a Subcellular Protein Fractionation kit (Thermo Fisher Scientific) and subjected to Western blot analysis.

The nuclear levels of MYC were evaluated by immunofluorescence staining using anti-MYC (Cell Signaling Technology, clone D84C12), anti-NUP153 (Santa Cruz Biotechnology), and DAPI. Images were acquired using CellInsight CX5 High Content Screening Platform with Colocalization.V4 BioApplication (Thermo Fisher Scientific) for cellomics analysis and Leica SP5 II confocal laser scanning microscope (Leica Microsystems) for confocal microscopy. Nuclear volume was identified from the confocal images by an ImageJ plugin Colocalization Image Creator with Otsu thresholding on DAPI signal. The nuclear MYC and perinuclear NUP153 was quantified by ImageJ plugin MorphoLibJ94,95.

RNAi and osteoclastogenesis on RAW264.7

Nup153 knockdown was achieved by transfecting Nup153 siRNA (Thermo Fisher Scientific) with Lipofectamine RNAiMAX (Thermo Fisher Scientific) reagent following the manufacturer’s instruction. RAW264.7 cells were incubated overnight for post-transfection recovery. Cells were treated with 100 ng·mL−1 RANKL (Peprotech) and 20 ng·mL−1 TNFα (R&D Systems) to trigger osteoclastogenesis. The culture medium was changed every 2 days. Mature osteoclasts could be found after 4 days of RANKL stimulation.

Regulation of Ogt and Oga upon RANKL and TNFα costimulation

To determine the initial response triggered by osteoclastogenic stimulation, bone marrow-derived cells were pre-treated with 1 µmol·L−1 SB202190 (Tocris), 5 µmol·L−1 of FR180204 (Tocris), 1 µmol·L−1 SU6656 (Selleck Chemicals), 40 µmol·L−1 T-5224 (ApexBio Technology), or vehicle 2 hours before osteoclastogenic stimulation. Cells were lysed after 6 hours of RANKL + TNFα costimulation, and the Ogt and Oga mRNA levels were measured by quantitative real-time PCR.

Statistics

The results are presented as median ± interquartile range (IQR), if not stated elsewhere. The sample sizes (n) are shown in the figure legends. Quantification of cellomics data is shown as violin-histograms, in which the violin graph shows the data distribution within the group, and the histogram shows the proportion of the data from the whole dataset. Mann–Whitney U-tests were applied for the comparison between two groups. ANOVA was applied for multiple comparisons within more than two groups. The post hoc analysis was performed with the Bonferroni method. P-values lower than 0.05 were considered statistical significant. All graphs and statistics were generated using Prism9 (GraphPad Software) and the R software (version 3.6.1) with tidyverse and see packages.

Study approval

This study was approved by the ethical committee of the University of Erlangen-Nürnberg. All patients recruited in this study signed informed consent. The animal experiments were approved by the regional government (Regierung von Unterfranken, Würzburg, Germany).

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