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

Shangling Zhu,Xiaoxue Feng,Junlong Dang,Yiming Shi,Jianlin Huang,Yuanmei Ye,Nancy Olsen,Song Guo Zheng,Fang Liu,Yingdi Chen

doi:10.3389/fimmu.2020.01300

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.

Highlights

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease characterized by inflammation and damage in the joints [1]
To measure the effects of agonist and antagonist of sonic hedeghog (SHH) signaling, SMO, Suppressor of Fused (SUFU), and GLI family zinc finger 1 (GLI1) expression was determined in the study since GLI1, which is regulated by SMO and SUFU, serves as the transcription activator of SHH signaling and controls the downstream target genes [13]
We observed that SHH agonist (SAG) significantly increased the expression of GLI1 protein in the nucleus and inhibition of SHH signaling with cyclopamine decreased the level of GLI1 expression (Figure 1B)

Summary

Introduction

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease characterized by inflammation and damage in the joints [1]. Downstream molecules of SMO facilitate the signaling transduction from cytoplasm to nucleus, leading to the activation of the transcriptional factors GLI family zinc finger 1–3 (GLI1-3). Among the three GLI factors, GLI1 functions as a transcriptional activator and a target gene, while GLI2 and GLI3 are the mediators of signaling transduction and can act as activators or repressors according to the cellular context [13]. Suppressor of Fused (SUFU) can directly bind to GLI transcription factors and act as a major negative regulator by promoting the generation of the repressor form of GLI or by preventing translocation to the nucleus [14, 15]

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