ADAM8 silencing suppresses the migration and invasion of fibroblast-like synoviocytes via FSCN1/MAPK cascade in osteoarthritis

Abstract

Objective: Osteoarthritis (OA) is a degenerative joint disease that affects elderly populations worldwide, causing pain and disability. Alteration of the fibroblast-like synoviocytes (FLSs) phenotype leads to an imbalance in the synovial inflammatory microenvironment, which accelerates the progression of OA. Despite this knowledge, the specific molecular mechanisms of the synovium that affect OA are still unclear.

Methods: Both in vitro and in vivo experiments were undertaken to explore the role of ADAM8 playing in the synovial inflammatory of OA. A small interfering RNA (siRNA) was targeting ADAM8 to intervene. High-throughput sequencing was also used.

Results: Our sequencing analysis revealed significant upregulation of the MAPK signaling cascade and ADAM8 gene expression in IL-1β-induced FLSs. The in vitro results demonstrated that ADAM8 blockade inhibited the invasion and migration of IL-1β-induced FLSs, while also suppressing the expression of related matrix metallomatrix proteinases (MMPs). Furthermore, our study revealed that inhibiting ADAM8 weakened the inflammatory protein secretion and MAPK signaling networks in FLSs. Mechanically, it revealed that inhibiting ADAM8 had a significant effect on the expression of migration-related signaling proteins, specifically FSCN1. When siADAM8 was combined with BDP-13176, a FSCN1 inhibitor, the migration and invasion of FLSs was further inhibited. These results suggest that FSCN1 is a crucial downstream factor of ADAM8 in regulating the biological phenotypes of FLSs. The in vivo experiments demonstrated that ADAM8 inhibition effectively reduced synoviocytes inflammation and alleviated the progression of OA in rats.

Conclusions: ADAM8 could be a promising therapeutic target for treating OA by targeting synovial inflammation.

Keywords: ADAM8; Fibroblast-like synoviocytes; Inflammation; MAPK; Osteoarthritis.

Fig. 1

The MAPK signaling cascade is involved in the processes in FLSs treated with IL-1β. A Differentially expressed mRNAs in FLSs in response to IL-1β are illustrated as a heat map. B Volcano map showing differentially expressed mRNAs. C-D Gene ontology classification. E KEGG pathway classification. F Bubble map of KEGG enrichment analysis (Total). G Bubble map of KEGG enrichment analysis (Up) and correlational heatmap of different samples and MAP kinase activity and bone resorption-related markers in different samples

Fig. 2

ADAM8 is up-regulated in FLSs after treatment with IL-1β. A, D Wound healing assays and quantitative analysis of the migration abilities of FLSs with different concentrations of IL-1β. B, E Transwell assays and quantitative analysis of the migration ability of FLSs with different concentrations of IL-1β. C, F Transwell assays and quantitative analysis of the invasion abilities of FLSs with different concentrations of IL-1β. G Western blot and quantitative analysis of IL-6, TNF-α, and COX-2 in FLSs after treatment with IL-1β. H-J qRT-PCR analysis of the mRNA expression levels of IL-6, TNF-α, and COX2. K qRT-PCR analysis of the mRNA expression levels of ADAM8. L Immunofluorescence staining of ADAM8 in FLSs. Scale bar = 200 µm. *P < 0.05; **P < 0.01

Fig. 3

The expression of ADAM8 was found to be increased in the synovium of patients with OA. A H&E staining of synovial tissues in ACL injury and OA patients. B-D TNF-α, IL-1β, and ADAM8 IHC staining of synovial tissues in ACL injury and OA patients. E–G Quantification of IHC staining for TNF-α, IL-1β, and ADAM8. Scale bars, 100 µm. **P < 0.01

Fig. 4

ADAM8 blockade suppresses FLS migration and invasion by inhibiting MMPs. A qRT-PCR analysis of the mRNA expression levels of ADAM8. B ADAM8 transfection efficiency was detected by fluorescence assay. C Immunofluorescence staining of ADAM8. D, G Wound healing assays and quantitative analysis of the migration ability of FLSs. E, H Transwell assays and quantitative analysis of the migration ability of FLSs. F, I Transwell assays and quantitative analysis of the invasion ability of FLSs with different concentrations of IL-1β. J Western blot and quantitative analysis of MMP-1 and MMP-13. K qRT-PCR analysis of the mRNA expression levels of MMP-1 and MMP-13. Scale bar = 200 µm. *P < 0.05; **P < 0.01

Fig. 5

ADAM8 blockade suppresses FLS inflammation by inhibiting MAPK signaling cascades. A Western blot and quantitative analysis of TNF-α, IL-6 and COX2 after treatment with siADAM8. H qRT-PCR analysis of the mRNA expression levels of TNF-α, IL-6, COX2, and PGE2. D-F Immunofluorescence staining and quantitative analysis of TNF-α and IL-6 after treatment with siADAM8. G Western blot of P-JNK, JNK, P-ERK, ERK, P-P38, and P38 after treatment with siADAM8. H Quantitative analysis of P-JNK/JNK, P-ERK/ERK, and P-P38/P38. *P < 0.05; **P < 0.01

Fig. 6

ADAM8 blockade prevent IL-1β-stimulated biological phenotypes specific of FLSs by targeting FSCN1. A The differentially expressed mRNAs in FLSs between IL-1β group and IL-1β + siADAM8 are illustrated as a heat map. B Gene ontology classification. C The heat map shows the detected differential genes associated with cell migration. D, G Wound healing and quantitative analysis of the migration ability of FLSs. E, H Transwell assays and quantitative analysis of the migration ability of FLSs. F, I Transwell assays and quantitative analysis of the invasion ability of FLSs with different concentrations of IL-1β. J qRT-PCR analysis of the mRNA expression levels of FSCN1, MMP-1, and MMP-13. Scale bar = 200 µm. *P < 0.05; **P < 0.01

Fig. 7

Inhibition of ADAM8 alleviates OA progression in vivo. A Schematic diagram of the in vivo experiments. B-C H&E staining and OARSI score of rat knee joint. D-E Safranin O staining of rat knee joint. F-G Immunohistochemical staining and quantitative analysis for ADAM8 of decalcified bone sections. Scale bars, 200 µm. *P < 0.05; **P < 0.01

Fig. 8

Inhibition of ADAM8 alleviates synovitis and MMPs in vivo. A, B IL-1β IHC staining and quantitative analysis of synovial tissues in OA rats. C, D IL-6 IHC staining and quantitative analysis of synovial tissues in OA rats. E, F TNF-α IHC staining and quantitative analysis of synovial tissues in OA rats. G, H MMP-1 IHC staining and quantitative analysis of synovial tissues in OA rats. I, J MMP-13 IHC staining and quantitative analysis of synovial tissues in OA rats. Scale bars, 200 µm. *P < 0.05; **P < 0.01