Background/Purpose: Fibroblast-like synoviocytes (FLS) contribute to the pathogenesis of rheumatoid arthritis (RA), especially cartilage damage and cytokine production. These cells display an aggressive phenotype in RA. Activation of the transcription factor NF-κB, which is mediated by proteasome degradation of its inhibitor IκB, is a hallmark of RA. We evaluated gene expression and the epigenetic marks of genes involved with ubiquitination in RA FLS. This process is, in part, regulated by neddylation, which conjugates NEDD8 to the cullin (CUL)-ring E3 ubiquitin ligases (CRLs). We hypothesized that dysregulated neddylation in RA FLS contributes to the RA FLS behavior.

Methods: RA or osteoarthritis (OA) FLS lines were obtained from synovial tissues at arthroplasty and used at passage 4-8. Analysis of ubiquitination pathway epigenetic marks in RA and OA FLS was performed using public ChIPseq data. RT-qPCR and Western blot analysis were used to assess gene and protein expression, respectively. NUB1 was overexpressed using an expression vector encoding NUB1 isoform 2 with a CMV promoter. NF-κB activation was assessed in vitro by stimulating FLS with IL-1β (IκBα degradation, NF-κB (p65) translocation, NF-κBreporter assay and IL6 gene expression). MLN4924 (a neddylation inhibitor) and bortezomib (a proteasome inhibitor) were used to evaluate the neddylation, NF-κB activation in RA FLS and its migration (wound healing assay). In vivo effect of MLN4924 was evaluated in the K/BxN serum transfer arthritis model.

Results: Epigenetic analysis identified an enhanced H3K27ac and H3K27me3 peaks in the promoter region of NUB1, a protein that inhibits neddylation, in OA FLS vs RA FLS, which indicates a poised region in the former. NUB1 was constitutively expressed by FLS and induction by IL-1β was greater in OA than RA FLS (6.6±0.5 fold and 3.4±0.8-fold increase for OA and RA, respectively; p< 0.0001). We then explored the neddylation pathway in FLS. The ratio of neddylated CUL1 (N8-CUL1) to non-neddylated CUL1 was lower in OA FLS than RA FLS in non-stimulated condition (0.30±0.04 and 0.40±0.09, respectively, p=0.02). NUB1 overexpression (NUB1 OE) decreased the neddylation ratio in non-stimulated RA FLS (56% decrease, p=0.02), decreased NF-κB nuclear translocation (30% inhibition, p=0.04), and IL-6 mRNA (58% inhibition, p=0.0007) in IL-1β stimulated RA FLS. MLN4924 decreased the neddylation ratio of CUL1 (55% inhibition, p=0.03), NF-κB nuclear translocation (65% inhibition, p=0.04) and IL-6 mRNA (99 % inhibition, p=0.002) in IL-1β stimulated RA FLS. Administration of MLN4924 (20 mg/kg) decreased the arthritis scores in K/BxN serum-transfer arthritis by 34% compared to vehicle alone (p≤0.0002).

Conclusion: We identified differential regulation of a novel gene, NUB1, in RA FLS after cytokine stimulation. Resultant abnormalities in ubiquitination, NF-κB activation, and cytokine expression in RA FLS suggest that neddylation system contributes to the pathogenesis of RA and regulation of neddylation could be a novel therapeutic approach.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dysregulated-nub1-and-neddylation-enhances-rheumatoid-arthritis-fibroblast-like-synoviocyte-inflammatory-responses/