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

Abstract

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.

Keywords: interleukin-6; osteoclast; reprogramming; rheumatoid arthritis; synovial fibroblasts.

Figure 1

Human RASFs in the presence of IL-6/IL-6R exhibit enhanced TRAP-positive staining and transform to an invasive phenotype. (A) RASFs grown with M-CSF (2 ng/ml)+ RANKL (10 ng/ml) (M+R) alone or with IL-6 and IL-6R (100 ng/ml each) for 12 days show an increased number of TRAP-positive RASFs compared with M-CSF/RANKL-differentiated and nonstimulated (NS) cell population. (B) IL-6+IL-6R treatment found to be almost 40% TRAP-positive cells which is more synergetic in combination with MCSF+RANKL where 70% of RASF cells are TRAP positive. (C) RASFs grown on calcium phosphate plates with M-CSF+ANKL alone or with IL-6+IL-6R for 12 days shows the increased pit formation on calcium phosphate-coated plates. (D) An average of three independent field quantifications of pits formed by RASFs from n = 3 patient donor samples reveal a threefold increase in the number of pits formed in calcium phosphate-coated plates when RASFs were stimulated with IL-6/IL-6R alone or in combination with M-CSF/RANKL as compared with nonstimulated RASFs, presented as mean ± SEM. ^* p < 0.05; ^** p < 0.01. Bar scale for images acquired is 150 µm.

Figure 2

Chronic exposure of IL-6/IL-6R induces cathepsin K, cathepsin B, and Ets2 expression in RASFs. (A) Western blot analysis of whole cell extracts prepared from human RASFs grown in the presence of IL-6+IL-6R for 12 days shows upregulation of endogenous CTSB, CTSK, RANKL, and Ets2. (B) Quantitative analysis of Ets2 expression reveals significant upregulation of Ets2 expression in response to IL-6+IL-6R treatment for 12 days. (C, D) CTSK, CTSB, and Ets2 were upregulated specifically in the presence of both IL-6 and IL-6R in human RASFs. (E) Human PBMC-derived osteoclast progenitor cells were stimulated with either M-CSF+RANKL or IL-6+IL-6R for 12 days. Western blot analysis of whole cell extracts prepared from these cells showed increased expression of CTSB and Ets2 in response to IL-6 trans-signaling. (F) CTSB, CTSK, and Ets2 expression was upregulated in mouse BMDMs in response to M-CSF+RANKL or IL-6+IL-6R. (G) Joint homogenates prepared from adjuvant-induced arthritis (AIA) rats show significant upregulation of CTSB, CTSK, RANKL, and Ets2 expression.**p < 0.01 while *p < 0.05.

Figure 3

MS-MS analysis of conditioned media identified uniquely expressed protein by IL-6/IL-6R treatment in human RASFs in vitro. (A) Conditioned media from four RASF donor cells treated with M-CSF/RANKL, IL-6/IL-6R, and M-CSF/RANKL plus IL-6/IL-6R (All) for 12 days were subjected to untargeted proteomics, which revealed 107 common proteins with differential expression. (B) Venn diagram shows the number of common and unique secretory proteins identified through untargeted proteomics in RASFs. (C) Increased secretion of CTSB, CTSK, and RANKL by transformed RASFs was confirmed through Western blotting of the conditioned media from the treated RASFs. (D) Cellular pathway changes brought about by chronic exposure of IL-6+IL-6 R in RASFs were analyzed using Metascape Gene Ontology studies, and (E) network analysis using ingenuity pathway analysis which predicted metabolic reprogramming in RASFs.

Figure 4

IL-6+IL-6R activates MAPK pathway in addition to canonical JAK-STAT signaling pathway. (A) Stimulation of RASFs with M+R (M-CSF+RANK), IL-6+IL-6R, and M+R+IL-6+IL-6R (All) activated the canonical JAK-STAT signaling pathway, evidenced by the increased phosphorylation of JAK1, JAK2, STAT3, and AKT, which was abrogated by tofacitinib and anti-IL-6 antibody treatment. (B) M-CSF+RANK, known to engage the MAPK signaling pathway, show cooperativity with IL-6+IL-6R and M+R+IL-6+IL-6R (All)-induced JAK-STAT signaling. Activation of key MAPK proteins by phosphorylation such as TAK1 seems unaffected by tofacitinib or anti-IL-6 antibody pretreatment. (C, D) Quantitative analysis of TAK1 phosphorylation at Thr184/187 and phospho-STAT3 Ser 705 shows M+R+IL-6+IL-6R (All) samples are unaffected for phosphorylation of TAK1 in presence of inhibitors. *p < 0.05.

Figure 5

IL-6/IL-6R-induced nuclear translocation of Ets2 and binds to the promoters of CTSK and CTSB genes to promote osteoclastogenesis in RASFs. (A) Ets2 translocates to the nucleus of RASFs within 4 h of IL-6+IL-6R stimulation alone or in combination with M-CSF+RANKL, which was inhibited by tofacitinib. (B) Bioinformatic and ChIP-seq analysis on tracks generated by GSE31621 identified the core-binding motif (GGAA/T) for Ets2 and the transcriptionally active regions in the promoters of CTSK and CTSB genes. (C) Chromatin immunoprecipitation using anti-Ets2 antibodies enriched CTSK and CTSB promoters from three independent human RASF donor cells upon stimulation with IL-6+IL-6R and M+R+IL-6+IL-6R (All). (D) Ets2 knockdown along with control (CTR), using siRNA, in RASFs treated with IL-6+IL-6R for 12 days resulted in the downregulation of endogenous CTSB and CTSK expression. Bar scale for images acquired is 150 µm. (E) Knockdown of Ets2 in RASFs obliterated the IL-6+IL-6R-induced TRAP-positive staining. *p < 0.05, **p < 0.05-p < 0.01, and ***p < 0.001.

Figure 6

IL-6+IL-6R trans-signaling activates both NLSFs and RASFs to an aggressive and invasive phenotype mediated by Ets2. (A) NLSFs and RASFs were differentiated in the presence of IL-6+IL-6R for 5 and 10 days followed by preparation of whole cell extracts that were then probed for osteoclast-specific markers. IL-6 trans-signaling was activated in both NLSFs and RASFs that led to the increased expression of Ets2 which in turn increased the expression of RANKL, CTSK, CTSB, and PDPN. (B) IL-6+IL-6R stimulation upregulated the expression of differentiation marker proteins Sox2, Oct4, KLF4, and Nanog higher in RASFs as compared with NLSFs. (C) IL-6+IL-6R-induced heterogeneity markers in RASFs, Thy1, and PDPN were downregulated in the absence of Ets2. (D) Quantitation of Thy1, PDPN, and Ets2 in 12-day differentiated RASFs confirms more then 80% knockdown of Ets2 as well as significant reduction in PDPN and Thy1 expression. *p < 0.05.