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
View: Text | PDF
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

×

Figure 3

GALNT1 functions by interacting with NEK9.

Options: View larger image (or click on image) Download as PowerPoint
GALNT1 functions by interacting with NEK9. (A). Identification of co-IP ...
(A). Identification of co-IP products by GALNT1 antibody by mass spectrometry and GO analysis of the immunoprecipitated products. (B) GALNT1 antibody co-IP followed by Western blot was conducted to validate the binding of the indicated proteins to GALNT1. (C) The binding of GALNT1 to NEK9 was confirmed by NEK9 antibody co-IP. Visualization of the colocalizations between GALNT1 and NEK9 (D) and NEK9 and actin (E) are shown with representative images. Original magnification, ×1,000. RA FLSs were transfected with NEK9 siRNA and the rates of migration (F) and invasion (G) were evaluated by Transwell assays. n = 5 independent experiments. Secretion of IL-6 (H) and CCL-2 (I) by RA FLSs was measured by ELISA assays. n = 5 (IL-6) and n = 6 (CCL-2) independent experiments. Data were normalized to the siNC control and analyzed by a 2-tailed 1-sample t test (theoretical value = 1). GALNT1 overexpression RA FLSs were transfected with NEK9 siRNA and subjected to migration assays (J) and invasion assays (K). n = 5 independent experiments. Data were normalized to the siNC/vector control. One-way repeated-measures ANOVA with Greenhouse-Geisser correction; Tukey’s post hoc test. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.