Dual specificity phosphatases 8 directly interacts with mitogen-activated protein kinase 1 to regulate the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synovial cells / 中华风湿病学杂志

ABSTRACT

Objective:To explore the interaction between dual specificity phosphatases 8 (DUSP8) and mitogen-activated protein kinase 1 (MAPK1) in rheumatoid arthritis fibroblast-like synovial (RA-FLS), and its effect on the proliferation and apoptosis of RA-FLSs.Methods:RA-FLS and normal fibroblast-like synovial cells (FLS) were cultured. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the expression levels of DUSP8 mRNA and protein in the two groups of cells. DUSP8 overexpression cell lines and DUSP8 silencing cell lines were constructed using cell transfection technology and RNA interference technology, respectively. Cell counting kit 8 (CCK-8) method was used to detect cell proliferation in each group, and flow cytometry was used to detect cell apoptosis in each group. Western blotting was used to detect the expression levels of DUSP8, MAPK1, p-MAPK1, ki-67 and Bax protein in each group. The indirect immunofluorescence experiment was used to analyze the spatial co-localization of DUSP8 and MAPK1, and the co-immunoprecipitation experiment was used to analyze whether there was interaction between DUSP8 and MAPK1 protein. The t-test was used to compare the means of the two groups. One-way analysis of variance was used to compare the mean values of the three groups of samples, and then the LSD- t tests were used to compare the two groups. Results:In RA-FLS, both mRNA [(2.4±0.6) vs (11.2±0.8), t=21.63, P<0.001] and protein levels [(0.24±0.04) vs (0.74±0.08), t=9.45, P<0.001] of DUSP8 were significantly lower than FLS. Compared with the blank control group and the overexpression control group, RA-FLS cells transfected with pcDNA3.1-Myc-DUSP8 could inhibit the proliferation of RA-FLS cells [(90.5±5.6) vs (92.5±1.8) vs (56.4±4.4), F=138.60, P<0.001], increase the rate of apoptosis significantly [(12.7±1.4)% vs (12.6±1.3)% vs (27.5±3.0)%, F=16.98, P<0.001], increase the expression levels of DUSP8 [(0.49±0.05) vs (0.45±0.04) vs (0.73±0.07), Bax (0.39±0.06) vs (0.36±0.05) vs (0.89±0.10)] and down-regulate the expression levels of ki-67 [(1.07±0.12) vs (1.11±0.16) vs (0.70±0.08), and p-MAPK1/MAPK1 [(0.59±0.06) vs (0.65±0.07) vs (0.39±0.03) (all P<0.001). Compared with the blank control group and the silent control group, RA-FLS cells transfected with siRNA-DUSP8 could promote the proliferation of RA-FLS cells [(90.5±5.6) vs (91.1±2.9) vs (128.3±4.6), F=137.50, P<0.001) and decrease apoptosis rate [(12.7±1.4) vs (13.2±1.2) vs (5.4±0.7), F=16.98, P<0.001], down-regulate the expression levels of DUSP8 [(0.492±0.048) vs (0.432±0.051) vs (0.102±0.024)], Bax [(0.391±0.062) vs (0.411±0.058) vs (0.090±0.011)], and up-regulate the expression levels of ki-67 [(1.07±0.12) vs (1.11±0.15) vs (1.93±0.22)], p-MAPK1/MAPK1 [(0.59±0.06) vs (0.68±0.06) vs (0.93±0.11)] (all P<0.001). The results of indirect immunofluorescence tests showed that both DUSP8 and MAPK1 were ex-pressed in the cytoplasm and nucleus of RA-FLS. The co-immunoprecipitation study verified that DUSP8 and MAPK1 protein could interact with each other. Conclusion:DUSP8 can bind to MAPK1 and regulate the abundance of active phospho-MAPK1 through its phosphatase activity and by inhibiting the proliferation of RA-FLS and promoting apoptosis.