IL-6 plays a critical role in the development of RA [10, 15] and has been found to stimulate vascular endothelial growth factor production by vascular endothelial cells in affected joints, leading to joint swelling, synovial growth, and accumulation of synovial fluid . Several studies showing that Cyr61 increases invasion and angiogenesis in several types of tumours led us to hypothesize that Cyr61 might be functionally linked to IL-6 [16–18]. Furthermore, pro-inflammatory cytokines, such as IL-17, have been recently shown to affect the expression of CYR61 in FLSs . Our results reveal that CYR61 expression and protein synthesis in FLSs from RA patients were enhanced by IL-6, and that both Cyr61 and IL-6 enhanced migration and invasion of RA-FLSs.
Joint destruction with synovitis is a characteristic of affected joints in RA. Pannus formation results in direct contact of FLSs with bone and cartilage tissues, leading to cartilage and bone destruction. The pannus invades cartilage, the surface of which is covered by FLSs, after which the local invasion of the cartilage matrix by the pannus starts . We suspected that elevated levels of IL-6 in RA synovium might affect the FLS genotype and accelerate pannus formation. Transcriptome analysis was performed to identify genetic alterations of RA-FLSs after IL-6 treatment. In our study, we identified 315 DEGs between the RA samples and IL-6 treated RA samples, including 277 upregulated genes and 38 downregulated genes. GO enrichment analysis revealed that the upregulated genes were mainly involved in blood vessel development, regulation of cell adhesion, and chemotaxis. These GO terms included angiogenesis and regulation of epithelial cell migration that are associated with a cancer-like phenotype. In addition to TNF and the nuclear factor-kappa B (NF-κB) signalling pathway related to the inflammatory response, IL-6-induced FLS gene expression also regulates the MAPK signalling cascade. Our results suggest that increased IL-6 levels promote FLS phenotype transition to tumour-like patterns that are responsible for the vicious cycle of cytokine and chemokine production. These findings are consistent with the finding that IL-6 stimulates tumour-like proliferation of FLSs in RA  and indicate that tumour progression mechanisms could account for pannus formation and function.
The 38 downregulated DEGs found in our study are most closely associated with FOXO-mediated transcription. FOXO transcription factors regulate many cellular processes, including cell survival, apoptosis, and resistance to oxidative stress. In particular, FOXO proteins regulate bone cell survival, cell cycle, proliferation, and also participate in network control among different kinds of bone cells . Given that our results show that IL-6 downregulated FOXO-mediated transcription in FLSs, further research on RA pathogenesis is warranted.
IL-6 acts through the JAK/STAT, MAPK, and PI-3K/AKT pathways . It binds to plasma membrane receptor complexes or to soluble receptor complexes to trigger its association with the signal-transducing gp130 . Signal transduction involves activation of JAK kinases, leading to the activation of transcription factors of the STAT family, particularly STAT3 . To determine which pathway is responsible for promoting the expression of CYR61, we used known inhibitors of several pathways. Although the canonical pathway of IL-6 is reported to be the JAK/STAT pathway, our results suggest that IL-6 altered Cyr61 protein synthesis mainly through the ERK 1/2 pathway. Although IL-6 and IL-6R are known to be important targets for RA, our results suggest that targeting IL-6 specific downstream signalling proteins could also be beneficial for RA therapy.
Based on our DEG results, we selected candidates such as c-jun, NR4A1, ATF3, and EGR3 to determine whether they regulated IL-6-induced Cyr61 protein synthesis. Previous studies have suggested that the effect of IL-6 on target gene transcription may involve c-jun . Hence, we examined whether c-jun was involved in the IL-6-induced increase in Cyr61 protein synthesis and found that knocking down JUN expression did not affect IL-6-induced Cyr61 protein synthesis; NR4A1 and ATF3 also did not show any significant effects.
Interestingly, we found that EGR3 modulated IL-6-induced Cyr61 protein synthesis through the ERK 1/2 pathway. EGR3 is a member of the early growth response (EGR) gene family of transcription factors that regulates a wide range of biological processes in response to growth factors, cytokines, and mechanical forces. In human foreskin fibroblasts lacking EGR3, transforming growth factor-beta 2 (TGF-β2) induction of the fibrotic genes collagen alpha 1 (COL1A1), alpha-smooth muscle actin (ACTA2), TGFB1, connective tissue growth factor (CTGF), and plasminogen activator inhibitor-1 (SERPINE1) was significantly abrogated . Furthermore, in human T cells, EGR4 and EGR3 interact with NF-κB to control the transcription of genes encoding inflammatory cytokines such as IL-2 and TNF-α, as well as intercellular adhesion molecule 1 . Thus, EGR3 contributes to production of Cyr61 as a fibrotic or inflammatory mediator. Further studies are required to clarify the binding site for EGR3 on the CYR61 gene.
As a secreted ECM protein, Cyr61 is considered to mediate cell proliferation, adhesion, migration, and act on pro-inflammatory molecules that induce the production of several cytokines and chemokines . Moreover, activation of MMPs is essential for cells to migrate, through the rearrangement of ECM to facilitate cell migration . Given its autocrine and paracrine features, increased secretion of Cyr61 by FLSs after IL-6 stimulation were found to stimulate the expression of MMP2, suggesting a role of Cyr61 in the activation of FLS migration and invasion. These results support the hypothesis that expression of CYR61 leads to increased angiogenesis and invasion in joints in RA, leading to greater degrees of synovial inflammation and cartilage erosion.
To summarize, we report here that protein synthesis of Cyr61 was enhanced in the FLSs of RA patients compared to those from OA patients. Interestingly, IL-6 stimulated the expression of CYR61 through the ERK/EGR3 pathway in RA-FLSs and modulated the expression of genes associated with angiogenesis, cell migration, and chemotaxis. Cyr61 was produced and secreted into the ECM environment and acted on the FLSs in an autocrine/paracrine manner. MMP expression was consequently enhanced and contributed to FLS migration and invasion. As a result, RA FLS migration and invasion were stimulated due to an enhancement of CYR61 expression (Fig. 6).