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 4

GALNT1-mediated O-glycosylation of NEK9 in RA FLSs promotes migration and invasion.

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GALNT1-mediated O-glycosylation of NEK9 in RA FLSs promotes migration an...
(A) Expression of NEK9 in RA synovial tissues (n = 8) and HC synovial tissues (n = 8) by IHC. Original magnification, ×400. (B) Semiquantitative NEK9 analysis; Mann-Whitney U test. (C) Expression of NEK9 in RA FLSs (n = 6) and HC FLSs (n = 3) by Western blot; 2-tailed 1-sample t test. (D) GALNT1 siRNA-transfected RA FLSs analyzed by Western blot; 2-tailed 1-sample t test (n = 5 independent experiments, normalized to siNC). (E) Lysates of RA FLSs were treated with neuraminidase or PNGase F, pulled down by VVL agarose beads, and then analyzed by Western blot with an anti-NEK9 antibody. Total lysate was used as a loading control. (F) Lysates of HC FLSs and RA FLSs were pulled down by VVL agarose beads and then analyzed by Western blot. (G) Lysates of GALNT1 knockdown or GALNT1 overexpression RA FLSs were pulled down by VVL agarose beads and then analyzed by Western blot. (H) Lysates of GALNT1 knockdown or GALNT1 overexpression RA FLSs were immunoprecipitated with an anti-NEK9 antibody, and the products were analyzed with VVL lectin blot. NEK9 overexpression RA FLSs were treated with O-glycosylation inhibitor benzyl-α-GalNAc (2 mM, 10 days) and then evaluated by migration assay (I) and invasion assay (J). Data were normalized to the vector control. Data were analyzed by 1-way repeated-measures ANOVA with Greenhouse-Geisser correction, followed by Tukey’s post hoc test. n = 5 independent experiments. RA FLSs were infected with vector or NEK9-OE or NEK9-mut and then evaluated by migration assay (K) and invasion assay (L). Data were normalized to the vector control. Comparisons of NEK9-OE and NEK9-mut against vector were performed using 2-tailed paired t tests. n = 5 independent experiments. Data are mean ± SD. *P < 0.05; **P < 0.01.