Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes

Abstract

Rheumatoid arthritis (RA) is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS) invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdown of cGAS inhibited the proliferation of RA FLS. cGAS overexpression enhanced the production of proinflammatory cytokines and matrix metalloproteinases (MMPs) as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. In contrast, cGAS silencing inhibited production of proinflammatory cytokines and matrix metalloproteinases (MMPs) as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. These results suggest that cGAS activates the AKT and ERK pathways to promote the inflammatory response of RA FLS, and the development of strategies targeting cGAS may have therapeutic potential for human RA.

Figure 1

Expression of cGAS is enhanced in RA-FLS compared with OA FLS. (a) qRT-PCR analysis of cGAS expression in 8 RA-FLS and 8 OA FLS. Quantitative analysis of cGAS expression was normalized to β-actin expression. (b) Western blotting identified the expression of cGAS in RA-FLS and OA FLS.

Figure 2

The expression of cGAS gene in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) was inhibited by tumor necrosis factor α (TNFα) inflammatory stimulation. (a) cGAS mRNA was upregulated by TNFα stimulation in a time-dependent manner. RA FLS (2 × 10⁵) were cultured in 6-well plates and then treated with TNFα (100 ng/mL) for different times (0, 15, 30, 60, and 120 min). (b) The level of cGAS protein was determined by Western blot. Values are the mean and SEM (standard error of the mean). Results are representative of 3 independent experiments. ^∗ P < 0.01 versus 0 min.

Figure 3

Effects of cGAS on RA FLS proliferation. (a) The transfection of cGAS expression vector enhanced cGAS protein levels, while the transfection of cGAS shRNA markedly reduced the protein levels of cGAS in the presence of TNFα stimulation for 30 min. (b) and (c) MTS assay showed that cGAS overexpression dramatically increased the proliferation of RA FLS, whereas cGAS knockdown reduced the proliferation of FLS. ^∗ P < 0.01.

Figure 4

Effect of cGAS overexpression on TNFα-induced inflammatory responses in RA FLS. (a) FLS infected with cGAS-vector produced significantly higher amounts of IL-1β, IL-6, MMP-1, and MMP-3 than did FLS infected with control (^∗ P < 0.01) when exposed to TNFα stimulation for 30 min. (b) Overexpression of cGAS significantly increased TNFα-mediated ERK1/2 and AKT phosphorylation by Western blotting.

Figure 5

Effect of cGAS silencing on TNFα-induced inflammatory responses in RA FLS. (a) FLS infected with cGAS shRNA produced significantly lower amounts of IL-1β, IL-6, MMP-1, and MMP-3 than did FLS infected with control (^∗ P < 0.01) when exposed to TNFα stimulation for 30 min. (b) Knockdown of cGAS significantly reduced TNFα-mediated ERK1/2 and AKT phosphorylation by Western blotting in the presence of TNFα stimulation for 30 min.