Interleukin-6 and tumour necrosis factor-α cooperatively promote cell cycle regulators and proliferate rheumatoid arthritis fibroblast-like synovial cells

• Published in: Scandinavian journal of rheumatology 2019-09, Vol.48 (5), p.353-361
• Main Authors: Kaneshiro, K, Sakai, Y, Suzuki, K, Uchida, K, Tateishi, K, Terashima, Y, Kawasaki, Y, Shibanuma, N, Yoshida, K, Hashiramoto, A
• Format: Article
• Language: English
• Subjects: Arthritis, Rheumatoid - genetics; Arthritis, Rheumatoid - metabolism; Arthritis, Rheumatoid - pathology; Blotting, Western; Cell Cycle; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Fibroblasts - metabolism; Fibroblasts - pathology; Gene Expression Regulation; Humans; Interleukin-6 - biosynthesis; Interleukin-6 - genetics; RNA - genetics; Synoviocytes - metabolism; Synoviocytes - pathology; Tumor Necrosis Factor-alpha - biosynthesis; Tumor Necrosis Factor-alpha - genetics
• ISSN: 0300-9742; 1502-7732
• DOI: 10.1080/03009742.2019.1602164

Objective: To elucidate the roles of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in cell cycle regulation and proliferation of rheumatoid arthritis fibroblast-like synovial cells (RA-FLSs). Methods: Under stimulation with IL-6/soluble interleukin-6 receptor (sIL-6R) and TNF-α, we examined the expression of cell cycle regulators [p16 INK4a , p21 Cip1 , p27 Kip1 , cyclin-dependent kinase-4 (CDK4), CDK6, Cyclin D, Cyclin E, and retinoblastoma protein (pRB)] by quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining. The expression of pRB, with or without 10% foetal bovine serum, was examined by Western blotting. DNA synthesis and cell viability were examined by the BrdU assay and WST-8 assay, respectively. After transfection with siRNA/p16 INK4a , siRNA/p21 Cip1 , siRNA/p27 Kip1 , siRNA/CDK4, or siRNA/CDK6, RA-FLSs were successively stimulated with or without IL-6/sIL-6R or TNF-α to determine cell viability. Results: IL-6/sIL-6R significantly decreased the expression of p16 INK4a , and increased p21 Cip1 , Cyclin E1, CYCLIN D, and pRB. TNF-α decreased the expression of CDK4, and significantly increased p27 Kip1 , CDK6, Cyclin E1/E2, CYCLIN D, CYCLIN E, pRB, and phosphorylated pRB (phospho-pRB). By immunofluorescence staining, CYCLIN D and phospho-pRB were simultaneously stained in the single cell. In serum-free culture, the expression of pRB was apparently decreased. DNA synthesis and cell viability were significantly increased by IL-6/sIL-6R and TNF-α. Silencing of CDK6 attenuated the cell viability induced by IL-6 and TNF-α. Conclusion: The results indicate that IL-6 and TNF-α interact with each other in regulating the cell cycle and accelerate the proliferation of RA-FLSs.