Sonic Hedgehog Regulates Proliferation, Migration and Invasion of Synoviocytes in Rheumatoid Arthritis via JNK Signaling

Abstract

Activated fibroblast-like synoviocytes (FLSs) play a central role in the formation of synovial pannus and joint destruction in rheumatoid arthritis (RA). Targeting FLSs could be a potential therapeutic strategy. The objective of this study is to explore the role of c-Jun N-terminal kinase (JNK) in proliferation, migration and invasion of FLSs promoted by the sonic hedeghog (SHH) signaling pathway in patients with RA. Activation of SHH signaling was evaluated by real-time PCR and Western Blot. Levels of phosphorylation of JNK and c-Jun were detected by Western Blot. FLSs proliferation was quantified by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Cell migration and invasion were assessed by wound healing assay and Transwell chamber assay. Invasiveness of FLSs in vivo was evaluated using a humanized synovitis animal model. We observed that treatment of SHH agonist (SAG) significantly increased the levels of phosphorylation of JNK and c-Jun, while SHH antagonist (cyclopamine) significantly decreased the expression of phospho-JNK and phospho-c-Jun in FLSs. The elevated level of phospho-c-Jun stimulated by SAG was decreased in the presence of JNK inhibitor (SP600125) ( P < 0.001). FLSs proliferation, migration and invasion were promoted by SHH agonist ( P < 0.05). However, the enhanced aggressiveness of FLSs was abolished in the presence of JNK inhibitor ( P < 0.05). In vivo study showed that the invasion of FLSs into cartilage was increased by SHH overexpression and the excessive invasiveness was inhibited by blockade of JNK signaling ( P < 0.01). These results suggest that JNK is one of the downstream molecules mediating the effect of SHH signaling in FLSs. These findings indicate that SHH-JNK signaling could be a potential therapeutic target to suppress the aggressiveness of FLSs and prevent articular damage of RA.

ES-62 suppression of arthritis reflects epigenetic rewiring of synovial fibroblasts to a joint-protective phenotype

“…Perhaps the observed counter-regulation of Pde4c is particularly pertinent to current translational strategies given the recent strong interest in the therapeutic potential of PDE4 inhibitors in RA as well as a wide range of other chronic inflammatory conditions including psoriatic arthritis, for which Apremilast was recently approved as a treatment 90 : indeed, ES-62 further acts to promote cAMP signalling in SFs by inducing inverse methylation signatures in the 1 st Exon region of other important elements, including Adcy10 (adenylate cyclase 10), AKAPs (A-kinase anchoring proteins) and Prkacb (Protein kinase A catalytic subunit beta) to those of naïve- and CIA-SFs. Interestingly therefore, in the context of ES-62 targeting this anti- inflammatory pathway, cilia provide a spatiotemporal platform for compartmentalising elements of the cAMP signalsome to promote protein kinase A-mediated antagonism of Hedgehog signalling 91 , a key pathway regulating cell differentiation which has recently been implicated in the proliferation, migration and invasion of RA SFs 92 . Moreover, recent mathematical modelling of differential gene expression metadata from a number of studies in healthy and RA synovial tissue 93 identified a gene signature panel representing an interactive biological network of hub (STAT1, RAC2 and KYNU) proteins and effector molecules (PEPD receptor and NR4A1, MEOX2, KLF4, IRF1 and MYB transcription factors and miRs-146a, -299, -3659, -6882 and - 8078) that provides a platform for validation of pathogenic mechanisms and potentially, a predictive tool for diagnosis and development of effective treatment strategies for RA: apart from KYNU and miR-299 (miRs-3659, -6882 and -8078 were not identified in our methylome study), these “signature” genes were differentially methylated at one or more sites in naïve-, CIA- and ES-62-CIA-SFs (Supplementary Information Table 2), with CIA- and ES-62 SFs generally exhibiting inverse profiles of methylation.…”

Rosmanol and Carnosol Synergistically Alleviate Rheumatoid Arthritis through Inhibiting TLR4/NF-κB/MAPK Pathway

“…MAPKs are downstream of TLR4 and play important roles in the pathogenesis of RA, as the abnormal activation of JNK and p38 almost participate in all aspects of RA-related pathologies [ 21 , 22 , 23 , 24 ]. To study the effects of rosmanol, carnosol, and their combination on the MAPKs, the expression levels of JNK, p-JNK, p38, and p-p38 in synovial tissue were examined by Western blot on day 42 ( Figure 7 ).…”

“…MAPK family proteins, including the extracellular-signal-regulated kinases, c-Jun N-terminal kinase (JNK), and p38 in mammals, are tightly associated with RA pathogenesis [ 20 ]. JNK regulates the expression of matrix metalloproteinases (MMPs), as well as the proliferation, migration, and invasion of synoviocytes and the destruction of joints [ 21 , 22 ]. P38 is critical for RA pathogenesis, as its activation involves almost all aspects of RA-related pathologies, including the expression of pro-inflammatory cytokines, synovitis, cartilage degradation, bone destruction, and angiogenesis [ 23 , 24 ].…”

Sonic Hedgehog Induces Mesenchymal Stromal Cell Senescence-Associated Secretory Phenotype and Chondrocyte Apoptosis in Human Osteoarthritic Cartilage

“…SHH regulates progenitor cell proliferation and differentiation in a concentration-dependent manner ( Lin et al, 2014 ). In adult, SHH acts as an oncogene to transform adult stem cells to cancer stem cells and is involved in tumorigenesis of many types of cancer ( Ali et al, 2019 ; Zhu et al, 2020 ). However, aside from cancer, the role of SHH in adult human tissue degeneration and aging-associated degenerative diseases was not well documented.…”

“…The hedgehog protein family, including Sonic Hedgehog (SHH) and Indian Hedgehog (IHH), have been shown to play an important role during skeletal development and homeostasis ( Alman, 2015 ; Zhu et al, 2020 ). While SHH is expressed by mesenchymal progenitor cells during limb bud patterning ( Alman, 2015 ; Tan et al, 2018 ), IHH is expressed by pre-hypertrophic chondrocytes within the growth plate ( Zhou J. et al, 2014 ).…”