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 4

Effect of SAE1/UBA2-mediated SUMOylation inhibition on pyruvate kinase M2 activity in RA FLSs.

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Effect of SAE1/UBA2-mediated SUMOylation inhibition on pyruvate kinase M...
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) for 48 hours or pretreated with GA (150 μM) for 24 hours and then stimulated with or without TNF-α (10 ng/mL) for 24 hours. Protein expression was measured by Western blot. (A) Effect of SAE1/UBA2 inhibition on PK activity. Enzyme activity was detected by the indicated kits in the Methods section. Data are expressed as the mean ± SD from 4 independent experiments. (B) Effect of SAE1 or UBA2 inhibition on protein expression of HK2, PFKP, and PKM2. (C and D) Effect of SAE1 or UBA2 knockdown (C) or GA treatment (D) on TNF-α–induced PKM2 phosphorylation. (E) Effect of SAE1 or UBA2 inhibition on TNF-α–induced nuclear translocation of PKM2. For cellular IF staining, PKM2 (green) and nuclei (blue) were evaluated using fluorescence microscopy, and representative images from 3 independent experiments are shown. Original magnification, ×400. (F) Effect of SUMO-1 knockdown on TNF-α–induced PKM2 phosphorylation. (G and H) Effect of SAE1 (G) or UBA2 (H) knockdown on TNF-α–induced phosphorylation of PKM2 in SUMO-1–overexpressing RA FLSs. (I and J) Effect of SAE1 or UBA2 knockdown on PKM2 and SUMO-1 interaction. The immunoprecipitates were probed with anti-PKM2 (upper panels) or anti–SUMO-1 (lower panels) antibody. Immunoprecipitations were also performed with IgG as a negative control. Input controls are shown as indicated (left panel). (K) Effect of the K336R mutation on PKM2 SUMO-1 modification. MH7A, a human RA FLS cell line, was transfected with or without a PKM2 plasmid carrying a mutation of K336 to arginine (K336R). Input controls are shown as indicated (left panel). (L) Effect of the K336R mutation on TNF-α–induced PKM2 phosphorylation. Data (C, D, F, G, H, and L) are expressed as the mean ± SD of densitometry quantification of Western blot results from at least 3 independent experiments. *P < 0.05 versus siC or DMSO or VECTOR+siC or WT; #P < 0.05 versus TNF-α + siC or TNF-α + DMSO or TNF-α + OE SUMO-1 + siC or TNF-α + WT; &P < 0.05 versus OE SUMO-1+ siC, by Student’s t test (for comparisons between DMSO and GA in panel B) or 1-way ANOVA (for other groups in panel B and other panels).