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 2

GALNT1 promotes aggressiveness of RA FLSs and severity of CIA in rats.

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GALNT1 promotes aggressiveness of RA FLSs and severity of CIA in rats. (...
(A) The cell scratch assay was performed for the measurement of in vitro migration of RA FLSs. Original magnification, ×50. n = 5 independent experiments. Cell migration assays (B) and invasion assays (C) by Transwell chambers were carried out in RA FLSs. Scale bars: 100 μm. Original magnification, ×200. n = 5 independent experiments. Data were normalized to the siNC control and analyzed by a 2-tailed 1-sample t test (theoretical value = 1). (D) The actin cytoskeleton in RA FLSs was stained with phalloidin and visualized under a fluorescence microscope. The white arrow indicates the formation of lamellipodia; yellow arrow indicates the formation of filopodia. Original magnification, ×1,000. Secretion of IL-6 (E) and CCL-2 (F) by RA FLSs was measured by assays. Data were normalized to the siNC control and analyzed by a 2-tailed 1-sample t test (theoretical value = 1). n = 4 independent experiments. For in vivo CIA experiments, rats were divided into normal control group (n = 3), CIA+vector group (n = 10), and CIA+shGalnt1 group (n = 10). (G) Effect of adenovirus-mediated knockdown of Galnt1 on joint swelling (first column), GALNT1 protein expression (second column, IHC staining, original magnification, ×200), synovial inflammation (third column, HE staining, original magnification, ×100), and cartilage erosion (fourth column, safranin O–fast green staining, original magnification, ×100) in rat paws. Arthritis index scores (H) and ankle circumferences (I) of each group after treatment are shown. Two-way repeated-measures ANOVA with Greenhouse-Geisser correction for sphericity violation, followed by Tukey’s post hoc test. (J) Histological scores of synovial inflammation and cartilage erosions were accessed under a microscope. Mann-Whitney U test. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus siNC or vector.