Fibroblast-like synoviocytes (FLSs) are the major effector cells that lead to rheumatoid arthritis (RA) synovitis and joint destruction. Our previous studies showed that Sonic Hedgehog (SHH) signaling pathway is involved in aberrant activation of RA-FLSs and inhibition of SHH pathway decreases proliferation and migration of RA-FLSs. The objective of this study was to investigate if the SHH pathway mediates proliferation and migration of RA-FLSs via the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway. SHH signaling was studied by using SHH agonist (Purmorphamine) and antagonist (Cyclopamine) targeting the Smoothened (SMO) in FLSs. U0126-EtOH was used to inhibit the MAPK/ERK signaling pathway. The phosphorylation of ERK 1/2 (p-ERKl/2) was examined by western blot. Cell viability was detected using cell proliferation and cytotoxicity kit-8 (CCK8), and cell cycle distribution and proliferating cells were evaluated by the flow cytometry. Cell migration was examined by Transwell assay. Results showed that, compared with the control group, Purmorphamine increased the levels of p-ERK1/2 in concentration-and time-dependent manners (P < 0.01). Co-treated with Purmorphamine and U0126-EtOH or Cyclopamine both decreased the levels of p-ERK1/2 (P < 0.05). RA-FLSs treated with Purmorphamine resulted in alteration of cell cycle distribution, increasing of proliferating cells, cell viability, and migration cells compared to controls (P < 0.01). However, the above phenomenon can be abolished by U0126-EtOH (P < 0.05). The findings suggest that SHH signaling pathway mediates proliferation and migration of RA-FLSs via MAPK/ERK pathway and may contribute to progression of RA. Targeting SHH signaling may have a therapeutic potential in patients with RA.

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.

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Background:Abnormal activation of sonic hedgehog (SHH) signaling has been found in synovium from patients with rheumatoid arthritis (RA). Inhibition of SHH signaling is reported to attenuate inflammation and cartilage damage in adjuvant-induced arthritis (AIA). Previously we have demonstrated that SHH signaling promoted the tumor-like behavior of fibroblast-like synoviocytes (FLSs) through p38 MAPKin vitro.Objectives:In the current study, we aim to further explore the role of SHH-p38 MAPK signaling in regulating synovial hyperplasia and bone erosion in experimental arthritis.Methods:Collagen-induced arthritis (CIA) mouse model was induced and the mice were injected with adenovirus associated virus (AAV) overexpressing SHH or treated with small molecule inhibitors GDC-0449. SB203580 was administrated for the inhibition of p38 MAPK. The severity of paw inflammation was graded and serum levels of TNFα, IL-6 were detected. The histological features of arthritis were evaluated by H&E staining. The bone erosion was identified by micro-CT assessment and the number and function of osteoclasts were determined.Results:Blockade of SHH by GDC-0449 significantly alleviated the symptoms and decreased the synovial hyperplasia, inflammatory infiltration, cartilage and bone damage in ankles of CIA. The bone erosions in the area of the metatarsophalangeal and ankle joints and production of TNFα, IL-6 were decreased by SHH inhibition. In addition, the administration of GDC-0449 significantly decreased the number of TRAP positive cells and the expression of NFATc1. On the contrary, SHH overexpression led to increased severity of arthritis and pathological changes. We also observed the accelerated bone injury accompanied with increased number and activity of osteoclasts and increased production of serum IL-6 in mice with upregulation of SHH expression. Of note, the administration of p38 MAPK inhibitor reversed the effects of SHH overexpression, with a reduction of joint swelling and histological scores. Inhibition of p38 MAPK prevented the bone erosion and decreased the number of TRAP positive cells and the expression of NFATc1, which were promoted by SHH overexpression.Conclusion:The study indicates that SHH promotes the synovial hyperplasia and bone erosion of CIA in a p38 MAPK-dependent manner. SHH-p38 MAPK signaling could be a potential target for the treatment of RA.Acknowledgments:This work was supported by grants from the National Natural Science Foundation of China (81571584, 81701609).Disclosure of Interests:None declared