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G protein-coupled receptor kinase 5 deletion suppresses synovial inflammation in a murine model of collagen antibody-induced arthritis.

ABSTRACT: G protein-coupled receptor kinase 5 (GRK5) regulates inflammatory responses via the nuclear factor-kappa B (NF-κB) pathway. This study investigated the functional involvement of GRK5 in the pathogenesis of inflammatory arthritis. Immunohistochemically, rheumatoid arthritis (RA) synovium had a significantly higher proportion of GRK5-positive cells in the synovial lining layer than healthy control synovium. Gene expression and NF-κB activation in lipopolysaccharide-stimulated human SW982 synovial cells were significantly suppressed by silencing of the GRK5 gene. Similarly, GRK5 kinase activity inhibition in human primary RA synovial cells attenuated gene expressions of inflammatory factors. In a murine model of collagen antibody-induced arthritis, arthritis scores and serum IL6 production of GRK5 knockout (GRK5^-/-) mice were significantly lower than those of wild-type mice. Histologically, the degree of synovitis and cartilage degeneration in GRK5^-/- mice was significantly lower than in wild-type mice. In in vitro analyses using activated murine macrophages and fibroblast-like synoviocytes, gene expression of inflammatory factors and p65 nuclear translocation were significantly lower in GRK5^-/- mice compared to wild-type mice. In conclusion, our results suggested that GRK5 is deeply involved in the pathogenesis of inflammatory arthritis, therefore, GRK5 inhibition could be a potential therapeutic target for types of inflammatory arthritis such as RA.

SUBMITTER: Toya M

PROVIDER: S-EPMC8131379 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCircular RNAs (circRNAs) are important regulators on the onset and progression of rheumatoid arthritis (RA). Our purpose is to explore the role and underpin mechanism of circ_0000396 in RA progression.MethodsRA patients (n = 39) and healthy volunteers (n = 33) were recruited from the Affiliated Hospital of Shaanxi University of Chinese Medicine for the present work. Circ_0000396, microRNA-574-5p (miR-574-5p) and R-spondin 1 (RSPO1) RNA levels were analyzed by reverse transcription-quantitative polymerase chain reaction. Cell proliferation was analyzed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine (EDU) assay. Cell apoptosis was assessed by flow cytometry. Protein expression levels of proliferating cell nuclear antigen (PCNA), Cyclin D1, Cyclin E1, BCL2-associated × protein (Bax), B-cell lymphoma-2 (Bcl2), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and RSPO1 were detected by western blot assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the secretion of pro-inflammatory cytokines including IL-1β and TNF-α. The interaction between miR-574-5p and circ_0000396 or RSPO1 was confirmed by dual-luciferase reporter assay and RNA-pull down assay.ResultsCirc_0000396 expression was notably down-regulated in RA patients compared with healthy controls. Circ_0000396 overexpression suppressed the proliferation and inflammatory response and triggered the apoptosis of RA synovial fibroblasts (RASFs), accompanied by decreases in PCNA, Cyclin D1, Cyclin E1, Bcl2, IL-1β and TNF-α protein expression and an increase in Bax protein expression. Circ_0000396 acted as a molecular sponge for miR-574-5p, and circ_0000396 overexpression-mediated protective effects on RASFs dysfunction were largely reversed by the introduction of miR-574-5p mimics. miR-574-5p interacted with the 3' untranslated region (3'UTR) of RSPO1, and miR-574-5p negatively regulated RSPO1 expression in RASFs. Circ_0000396 could up-regulate the expression of RSPO1 by sponging miR-574-5p in RASFs. RSPO1 interference largely overturned circ_0000396 overexpression-mediated effects in RASFs.ConclusionCirc_0000396 restrained the proliferation and inflammation and induced the apoptosis of RASFs by mediating miR-574-5p/RSPO1 axis, which provided novel potential targets for RA treatment.

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G protein-coupled receptor kinase 5 deletion suppresses synovial inflammation in a murine model of collagen antibody-induced arthritis.

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