Ets-2 Propagates IL-6 Trans-Signaling Mediated Osteoclast-Like Changes in Human Rheumatoid Arthritis Synovial Fibroblast

Rheumatoid arthritis synovial fibroblasts (RASFs) contribute to synovial inflammation and bone destruction by producing a pleiotropic cytokine interleukin-6 (IL-6). However, the molecular mechanisms through which IL-6 propels RASFs to contribute to bone loss are not fully understood. In the present study, we investigated the effect of IL-6 and IL-6 receptor (IL-6/IL-6R)-induced trans-signaling in human RASFs. IL-6 trans-signaling caused a significant increase in tartrate-resistant acid phosphatase (TRAP)-positive staining in RASFs and enhanced pit formation by ~3-fold in the osteogenic surface in vitro. IL-6/IL-6R caused dose-dependent increase in expression and nuclear translocation of transcription factor Ets2, which correlated with the expression of osteoclast-specific signature proteins RANKL, cathepsin B (CTSB), and cathepsin K (CTSK) in RASFs. Chromatin immunoprecipitation (ChIP) analysis of CTSB and CTSK promoters showed direct Ets2 binding and transcriptional activation upon IL-6/IL-6R stimulation. Knockdown of Ets2 significantly inhibited IL-6/IL-6R-induced RANKL, CTSB, and CTSK expression and TRAP staining in RASFs and suppressed markers of RASF invasive phenotype such as Thy1 and podoplanin (PDPN). Mass spectrometry analysis of the secretome identified 113 proteins produced by RASFs uniquely in response to IL-6/IL-6R that bioinformatically predicted its impact on metabolic reprogramming towards an osteoclast-like phenotype. These findings identified the role of Ets2 in IL-6 trans-signaling induced molecular reprogramming of RASFs to osteoclast-like cells and may contribute to RASF heterogeneity.

Cells were grown in complete RPMI medium with or without 100ng/ml IL6 +100ng/ml IL6R for 12 days.
To maintain continuous knockdown while differentiation of RASFs every transfection step was repeated on consecutive 3 times on day 3, 6 and 9. On day 11 of transfection, RASFs were serum starved overnight along with 100ng/ml IL6 +100ng/ml IL6R simulation for overnight.

Pit formation assay analysis:
For analysis of the pit in the calcium phosphate coated plate used image-J Fiji software which is freely available from the NIH. briefly we have a pit (which is stained with toluidine blue for the visibility) we draw a random boundary based on the pit area for bigger or smaller or any size which we can see blue color we drew hands free lines. We added all the area (size of area in field) from all the hand drawn pit outlines and divided it from overall size of the field (that gives us total blue area in overall field which is also proportional to cumulative of pit area and multiplied by 100 to get percentage value. We repeated this at least in an N-9 independent field to get % of pit relative to each other. Western Immunoblotting. To study the effect on RASF differentiation and phenotypic switch, the whole-cell extracts were prepared using RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1% Triton X100, 1mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS) containing Complete miniprotease inhibitor and Phospho-Stop tablets (Roche, Indianapolis, IN). The amount of protein was measured using the Bio-Rad DC method (Bio-Rad, CA). Equal amounts of protein (25 μg) were loaded and separated by SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (EMD Millipore). Blots were probed using respective primary blots followed by strip and re-probed for other primary antibodies or β-actin as an endogenous control. Densitometric analysis of the relative expression of each protein was done as described earlier (22). For determining soluble proteins, the Western blotting was performed on the conditioned media collected from the treated samples at different time points and concentrated 500 µL using a 3-kDa cutoff Amicon filters (Millipore).
Chromatin immunoprecipitation assay (ChIP). Human RASFs crosslinking was performed by adding formaldehyde to a final concentration of 1% at room temperature for 10 minutes, and the reaction stopped by the addition of 125 mM glycine. Cells were washed with ice-cold phosphatebuffered saline containing 0.1 mM PMSF. Cell pellet was resuspended in 1 mL of ChIP sonication buffer (50 mM Tris-HCl, pH 8, 1% Triton X-100, 0.5% Deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.3% SDS, Complete mini and Phos STOP protease inhibitors) and placed on ice for 30 minutes. DNA was sheared by sonication, and the cell debris was pelleted by centrifugation at 15,000 g for 15 minutes. Forty µl of clear lysate was taken out to check quality controls of sonication. Sonication quality was checked on an agarose gel, and leftovers were stored as input for further use. The supernatant was collected and pre-cleared with 50 µl of Protein G Sepharose (Roche, IN) equilibrated with Baker's Yeast tRNA (Sigma) for 60 minutes at 4 o C. The pre-cleared whole-cell extract was equally divided into three parts and three times diluted in Chip dilution buffer (Chip Sonication buffer without SDS). The diluted samples were incubated with 5 µg of Ets2 or IgG antibody at 4 o C overnight. 10% of the volume was frozen and kept aside as input. The immune complex was precipitated by incubation of the samples with 50µl Protein G Sepharose equilibrated with tRNA at 4 o C for 4 hours. The samples were centrifuged at 500 g at 4 o C for 1 minute, and the supernatant was discarded. The Protein G Sepharose beads were washed with cold ChIP dilution buffer containing protease inhibitors followed by a wash with low salt wash buffer (1% TritonX-100, 0.1% sodium deoxycholate, 50 mM Tris pH 8.0, 150 mM NaCl and 5 mM EDTA), high salt wash buffer (1% Triton X-100, 0.1% sodium deoxycholate, 50 mM Tris pH 8.1, 500 mM NaCl and 5 mM EDTA), LiCl wash buffer (0.25 M LiCl, 0.5% Triton X100, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris pH 8.1) and finally with 1X TE pH 8.
The immune complex was eluted with 1% SDS, 0.1 M NaHCO3. Crosslinking was reversed by incubating at 65 o C overnight with 2µg RNAse A followed by 2 hours of 5µl proteinase K (20 mg/mL) treatment at 65 o C. After Proteinase K digestion, immunoprecipitated DNA was subjected to ethanol precipitation overnight at -20 o C. Extracted DNA was further purified using a PCR purification column (Qiagen). PCR was performed using corresponding distal, mid, and proximal sequences to Cathepsin K and Cathepsin B gene promoters. IDT and the sequences of the synthesized primers are listed in Table S1. Additionally preliminary bioinformatics analysis was performed on CTSK and CTSB promoter by generating track hubs for the chip datasets which are available at http://www.ag-rehli.de. GSE31621 from NCBI GEO data were used for temple to generate track.
Untargeted proteomics of RASFs. Two μl of the sample (~100 ng/μl protein) was injected into a 100 μm ID Integrafrit trap (New Objective) packed with Reprosil-Pur C18-AQ 120 Å 5 µm material to a bed length of 2.5 cm at a flow rate of 2 µL/min. After loading and desalting for 10 min with 0.1% formic acid plus 2% acetonitrile, the trap was brought in-line with a pulled fused- For untargeted proteomics profiling, tandem mass spectra were analyzed using a variety of bioinformatics tools. All the raw data were converted to mzXML files for database searching and further analyzed by Trans-Proteomic Pipeline using PeptideProphet (Comet), iProphet, Protein Prophet, and StPeter. A target protein database was created by using the UniProt human database.
All searches were configured to use a variable modification of methionine oxidation and carbamidomethylation of cysteine as a fixed modification. The results were filtered to a less than 1% protein false discovery rate (FDR) with the requirement for a minimum of one signature peptide per reported protein. The parameters used include: i) minimum peptide length of 6 amino acid, ii) maximum FDR of 1% and protein identification probability more than 0.85, iii) minimum unique peptides per protein of 1, and modifications to cysteine or methionine were not considered distinct from the unmodified peptides and, iv) tryptic digestion with maximum of two miscleavages allowed. STRING was used for pathway analysis.
Animal experiments. Five wild type control C57BL/6 female mice (8-week-old) were provided by Washington State University animal care facility at Spokane, WA. Briefly, animals were euthanized by CO2 asphyxiation followed by which hind limbs were harvested and stored in PBS solution. Before pursuing cell isolation from tibia and femur bone were soaked with 70% ethanol for 2-3 minutes followed by cutting of ends using a surgical grade scissors. Bone marrow was flushed out using a 25-gauge needle using PBS in a sterile tube containing penicilliumstreptomycin containing RPMI without serum. Collected cells were dispersed using 5 ml pipettes followed by ficoll density centrifugation at 400 g rpm in a swinging bucket rotor. Further buffy phases containing all mononuclear suspension cells were collected using 10 ml pipette. Cells were further washed 2 to 3 times in plain RPMI-1640 and counted before proceeding to various treatments as mentioned in the method section for Cell culture and stimulation.
To determine protein expression in rat inflammatory arthritis, joint homogenates were considered from our previously published study [DOI: 10.1002/art.39447] for only naïve and adjuvant-induced arthritis (AIA) joint homogenates to be used for determining the expression levels of CTSB, CTSK, RANKL, Ets2 and β-actin.    Figure S1: (A) Culture of bone marrow derived macrophages from C56BL/6 mice with IL-6+IL-6R for 12 day (B) IL-6+IL-6R along with M+R cotreatment shows an increased number of TRAPpositive osteoclast progenitor cells (n=3; p<0.05). Representative image acquired using 40X and the size of scale is 300 µm. Figure S2: CD14 positive cells were shorted using anti-CD14 antibody (mouse monoclonal) on human THP1 monocytic cell line as well as on 3 independent human patients derived RASFs using Alexa Fluor anti mouse 488 antibody. All 3 human RASF were more than passage 5. Human THP1 cell line were found to be enriched with CD14+ cells for more than 17% cell populations. While human RASFs subjects had less than 1% to no stating for CD14 antibodies. Experimental RASF which is grown more than passage 5 are devoid of any visible contamination of CD14+ cells.  Figure S3: Gene Ontology study of affected biological function from RASFs were analyzed using metascape.org gene ontology platform. S1A) Human RASFs stimulated with M-CSF/RANKL with 261 secreted proteins; S1B) Human RASFs stimulated with IL-6/IL-6R with 288 proteins and S1C) Human RASFs stimulated M-CSF/RANKL/IL-6/IL-6R (All) with 199 secretory proteins.