Smoothened Regulates Migration of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via Activation of Rho GTPase Signaling.

Wei-Xiang Peng,Yi-Ming Shi,Jian-Lin Huang,Shang-Ling Zhu,Xiao-Xue Feng,Bai-Yu Zhang,Fang Liu,Song Guo Zheng

doi:10.3389/fimmu.2017.00159

Abstract

Fibroblast-like synoviocytes (FLSs) acquire aggressive phenotypes characterized with enhanced migration abilities and inherent invasive qualities in rheumatoid arthritis (RA). Smoothened (Smo) is a key component of sonic hedgehog (Shh) signaling and contributes to tumor cell invasion and metastasis. The objective of this study is to investigate the role of Smo in the modulation of cell migration and explore the underlying molecular mechanism(s). FLSs were isolated from RA synovium. Shh levels were regulated by a Smo agonist (purmorphamine), Smo antagonist (KAAD-cyclopamine), or small interfering RNA targeting the Smo gene (Smo-siRNA) in RA-FLSs. Expression of Smo was detected by real-time PCR and western blot analysis. Cell migration was examined by Transwell assay and activation of Rho GTPases was measured by pull-down assays. Incubation with purmorphamine resulted in a significant increase of cell migration and activation of Rho GTPase signaling compared to controls (P < 0.05). However, treatment with KAAD-cyclopamine or transfection with Smo-siRNA suppressed migration of RA-FLSs and showed an inhibitory effect of Rho GTPase signaling. Together, these results suggest that Smo plays an important role in RA-FLSs migration through activation of Rho GTPase signaling and may contribute to progression of RA, thus, targeting Shh signal may have a therapeutic potential in patients with RA.

Highlights

Activated fibroblast-like synoviocytes (FLSs) comprising the major cell population of the hyperplastic synovial linings have a central role in the pathogenesis of rheumatoid arthritis (RA)
In order to examine the property of migration of Fibroblast-like synoviocytes (FLSs) from patients of RA, OA, and knee trauma, we conducted Transwell assay and observed that the numbers of migrated cells were significantly higher in RA-FLSs (133.00 × 104 ± 4.73 × 104) than that in OA-FLSs (81.33 × 104 ± 1.45 × 104) and FLSs from patients with traumatic injury (45.67 × 104 ± 4.48 × 104) (P < 0.05) (Figure 2C)
To further investigate whether the higher levels of migration rates of RA-FLSs were associated with activation of Rho GTPases, we determined the activation of RhoA and Rac1 using pull-down assays

Summary

Introduction

Activated fibroblast-like synoviocytes (FLSs) comprising the major cell population of the hyperplastic synovial linings have a central role in the pathogenesis of rheumatoid arthritis (RA). Smoothened Modulates Inflamed FLS in RA behavior of RA synoviocytes contributes to synovial hypertrophy and formation of invasive pannus tissue and leading to joint destruction [3]. Shh signaling is launched by binding of Shh ligand to the transmembrane receptor, patched, and the suppression of Smoothened (Smo) is reversed, resulting in the activation of Gli family of zinc-finger transcription factors and expression of downstream target genes [4]. In the non-canonical Shh signaling pathway, Smo signals through heterotrimeric G proteins and stimulates the activation of Rho GTPases, and the signaling is independent of Gli activation [5]. Smo is a key component in both the canonical and non-canonical Shh signaling pathway

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