Increased SUMO-activating enzyme SAE1/UBA2 promotes glycolysis and pathogenic behavior of rheumatoid fibroblast-like synoviocytes
Cuicui Wang, Youjun Xiao, Minxi Lao, Jingnan Wang, Siqi Xu, Ruiru Li, Xuanxian Xu, Yu Kuang, Maohua Shi, Yaoyao Zou, Qingwen Wang, Liuqin Liang, Song Guo Zheng, Hanshi Xu
Cuicui Wang, Youjun Xiao, Minxi Lao, Jingnan Wang, Siqi Xu, Ruiru Li, Xuanxian Xu, Yu Kuang, Maohua Shi, Yaoyao Zou, Qingwen Wang, Liuqin Liang, Song Guo Zheng, Hanshi Xu
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Research Article Immunology

Increased SUMO-activating enzyme SAE1/UBA2 promotes glycolysis and pathogenic behavior of rheumatoid fibroblast-like synoviocytes

Abstract

Fibroblast-like synoviocytes (FLSs) are critical to joint inflammation and destruction in rheumatoid arthritis (RA). Increased glycolysis in RA FLSs contributes to persistent joint damage. SUMOylation, a posttranslational modification of proteins, plays an important role in initiation and development of many diseases. However, the role of small ubiquitin-like modifier–activating (SUMO-activating) enzyme 1 (SAE1)/ubiquitin like modifier activating enzyme 2 (UBA2) in regulating the pathogenic FLS behaviors is unknown. Here, we found an increased expression of SAE1 and UBA2 in FLSs and synovial tissues from patients with RA. SAE1 or UBA2 knockdown by siRNA and treatment with GA, an inhibitor of SAE1/UBA2-mediated SUMOylation, resulted in reduced glycolysis, aggressive phenotype, and inflammation. SAE1/UBA2-mediated SUMOylation of pyruvate kinase M2 (PKM2) promoted its phosphorylation and nuclear translocation and decreased PK activity. Moreover, inhibition of PKM2 phosphorylation increased PK activity and suppressed glycolysis, aggressive phenotype, and inflammation. We further demonstrated that STAT5A mediated SUMOylated PKM2-induced glycolysis and biological behaviors. Interestingly, GA treatment attenuated the severity of arthritis in mice with collagen-induced arthritis and human TNF-α transgenic mice. These findings suggest that an increase in synovial SAE1/UBA2 may contribute to synovial glycolysis and joint inflammation in RA and that targeting SAE1/UBA2 may have therapeutic potential in patients with RA.

Authors

Cuicui Wang, Youjun Xiao, Minxi Lao, Jingnan Wang, Siqi Xu, Ruiru Li, Xuanxian Xu, Yu Kuang, Maohua Shi, Yaoyao Zou, Qingwen Wang, Liuqin Liang, Song Guo Zheng, Hanshi Xu

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Figure 2

Effects of SAE1/UBA2-mediated SUMOylation inhibition on migration, invasion, and the expression of proinflammatory cytokines in RA FLSs.

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Effects of SAE1/UBA2-mediated SUMOylation inhibition on migration, invas...
RA FLSs were transfected with siRNA-1 and siRNA-3 for SAE1 (siSAE1-1, siSAE1-3) or UBA2 (siUBA2-1, siUBA2-3) or control siRNA (siC) or pretreated with ginkgolic acid (GA) (150 μM) for 24 hours. (A and B) Migration (A) and invasion (B) were measured with a Boyden chamber. An invasion assay was performed using inserts coated with a Matrigel basement membrane matrix, and 10% FBS was used as a chemoattractant. The migrated or invaded FLSs were stained violet using a Diff-Quik kit (left panel, original magnification, ×100). The migration or invasion index represents the number of migrated cells normalized to that in the siC or DMSO group. Data show the mean ± SD of samples from 3 patients with RA. (C) Effect of SAE1/UBA2 inhibition on lamellipodia formation. RA FLSs were plated overnight on coverslips and then fixed and stained with fluorescent phalloidin 6 hours after wounding to visualize polymerized actin in migrating cells. Arrows indicate lamellipodia formation. Graph indicates the number of RA FLSs with positive lamellipodia. (D) Effect of SAE1/UBA2 inhibition on proliferation of RA FLSs. An EdU incorporation assay was used to measure cell proliferation. Representative images show proliferation of RA FLSs labeled with EdU (red) and nuclei stained with Hoechst 33342 (blue) (original magnification, ×100). Graphs indicate the mean ± SD of 3 independent experiments involving 3 patients with RA. (E) Effect of SAE1/UBA2 inhibition on apoptosis of RA FLSs. The cellular apoptosis rate was evaluated by annexin V and propidium iodide (PI) staining and measured by flow cytometry. Representative flow plots are shown. Mean ± SD percentage of 3 independent experiments involving 3 patients with RA shown. (F) Effect of SAE1/UBA2 inhibition on the expression of IL-1β, IL-6, and IL-8. Cytokine expression was evaluated by RT-qPCR. Ct values were normalized to β-actin values. Data are presented as the mean ± SD of 4 independent experiments involving 4 patients with RA. *P < 0.05 versus siC or DMSO; #P < 0.05 versus TNF-α+siC or TNF-α+DMSO, by Student’s t test (for comparisons between DMSO and GA in panels A–E) or 1-way ANOVA (for panel F and other groups in panels A–E).