GALNT1 drives aggressive phenotypes of rheumatoid synoviocytes via NEK9 O-glycosylation
Yaoyao Zou, Haobo Lin, Jianling Su, Jieying Wang, Qin Zeng, Tianxiao Feng, Yunxia Lei, Jianda Ma, Hudan Pan, Hanshi Xu, Lie Dai, Yang Li
Yaoyao Zou, Haobo Lin, Jianling Su, Jieying Wang, Qin Zeng, Tianxiao Feng, Yunxia Lei, Jianda Ma, Hudan Pan, Hanshi Xu, Lie Dai, Yang Li
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Research Article Bone biology

GALNT1 drives aggressive phenotypes of rheumatoid synoviocytes via NEK9 O-glycosylation

Abstract

Fibroblast-like synoviocytes (FLSs) are crucial in driving synovial inflammation and joint damage in rheumatoid arthritis (RA). This study explored the functions and underlying mechanisms of GALNT1-mediated O-glycosylation, which is markedly upregulated in RA FLSs, in synovial aggression and subsequent experimental joint damage. Targeted suppression of GALNT1 effectively curtailed migration and invasion in RA FLSs and mitigated arthritis severity in a collagen-induced arthritis model in rats. Mechanistically, NEK9 was identified as a pivotal substrate and downstream effector of GALNT1, affecting the aggressive phenotype of RA FLSs. In vitro experiments further demonstrated that O-glycosylation of NEK9, mediated by GALNT1, promotes the pathogenic phenotype of RA FLSs by promoting cytoskeleton reorganization and restraining excessive ER stress activation. Our study provides mechanistic insights into the activation of RA FLSs and identifies GALNT1 as a potential therapeutic target for RA.

Authors

Yaoyao Zou, Haobo Lin, Jianling Su, Jieying Wang, Qin Zeng, Tianxiao Feng, Yunxia Lei, Jianda Ma, Hudan Pan, Hanshi Xu, Lie Dai, Yang Li

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Figure 6

NEK9 regulates the migration and invasion of RA FLSs through the regulation of the ER stress pathway.

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NEK9 regulates the migration and invasion of RA FLSs through the regulat...
(A) GO clustering analysis of the differentially upregulated genes in the siNEK9 group. (B) Upregulated genes enriched in the GO term of response to ER stress after NEK9 knockdown. (C) HC FLSs and RA FLSs were transfected with siNC, siGALNT1, and siNEK9 and were observed under a transmission electron microscope. (D) RA FLSs were infected with vector or NEK9-OE or NEK9-mut and were observed with a transmission electron microscope. (E) Relative expression of HSPA5 mRNA in RA FLSs transfected with siNEK9 or siGALNT1. n = 4 independent experiments. (F) Relative expression of HSPA5 mRNA in RA FLSs transfected with NEK9-OE or NEK9-mut. n = 3 independent experiments. Data were normalized to the respective control (siNC for E, vector for F), and comparisons between each treatment and control were performed using paired t tests. (G) GRP78 expression in CIA rat synovium detected by IHC. (H) Semiquantitative analysis was performed for the evaluation of GRP78 expression in vector control group synovial tissues (n = 8) and shGalnt1 group synovial tissues (n = 10). Differences were analyzed using the Mann-Whitney U test. RA FLSs were transfected with siGALNT1 and siNEK9, and 4-PBA (2 mM, 24 hours) was used to inhibit ER stress in RA FLSs. The migration (I) and invasion (J) capabilities of RA FLSs were assessed using Transwell chamber migration assays and invasion assays, respectively. Data were analyzed by 1-way repeated-measures ANOVA with Greenhouse-Geisser correction, followed by Tukey’s post hoc test. n = 3 independent experiments. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 500 nm (C and D) and 25 μm (G).