Session Title: Rheumatoid Arthritis – Human Etiology and Pathogenesis I
Session Type: ACR Concurrent Abstract Session
Session Time: 2:30PM-4:00PM
Background/Purpose: LBH (Limb-bud and heart development) was recently identified as an RA risk gene that has abnormally methylated loci and a functional enhancer SNP in RA FLS. These genetic and epigenetic marks decrease LBH expression, which is associated with increased RA risk. Subsequent studies showed that LBH deficiency unexpectedly leads to S phase arrest. The present studies define the mechanism of S-phase arrest and assess the effect of LBH deficiency in an animal model of rheumatoid arthritis.
Methods: Cultured FLS were derived from RA synovium obtained at the time of arthroplasty. DNA damage was assayed using a comet assay, which is a single cell method that measures diffusion of DNA fragments from the nucleus. Male Rosa-26-Cre; Lbh+/+ (WT) and conditional knockout Rosa-26-Cre; Lbhloxp/loxp mice (LBH-/-) were injected i.p. with 80 μl of pooled adult K/BxN mice serum on day 0 and 2. The clinical scores were determined until day 12. Western blots qPCR assays were performed on synovial tissue and cultured FLS extracts to assay protein and mRNA levels. LBH deficiency in FLS (LBHlow) was induced using siRNA transfection.
Results: Because S-phase arrest is often due to DNA replication damage, we determined whether LBH deficiency in FLS increases DNA damage. Comet assays showed that LBHlow cells had comets with significantly longer tails than control cells, indicating increased in DNA strand breaks (p = 0.001). DNA damage in LBHlow cells led us to assess the activation status of the checkpoint kinases, CHK1 and CHK2, which are activated in response to DNA damage during S phase and induces growth arrest. Western blot analysis showed that CHK1 phosphorylation increased 4-fold greater in LBH deficient cells than control cells, while CHK2 was not activated (p=0.03). Three main polymerases are responsible for DNA synthesis in S phase and were assayed in control and LBHlow cells. LBH deficiency reduced POLA1 protein by 86% (p < 0.01) compared with control. However, POLD1 and POLE were not affected by LBH deficiency. To examine the contribution of LBH in inflammatory arthritis, passive K/BxN arthritis was studied in WT and LBH-/- mice. LBH-/- mice had a significantly increased peak clinical score of arthritis (mean = 8±3, p = 0.02) compared to WT (mean = 6±2), primarily in the later stages of the model. IL-1ß gene expression, which plays an essential role in this model, was significantly increased in the joints of the LBH-/- mice with arthritis (2.1±0.7) compared with arthritic WT mice on day 12 (0.7±0.1, p<0.05). To determine whether checkpoint arrest occurred in the joints of LBH-/- mice with arthritis, western blot analysis was performed for phospho-CHK1. LBH-deficient mice had 2±0.5-fold higher levels of phospho-CHK1 compared with WT in the arthritis model (p < 0.05).
Conclusion: These findings indicate that LBH regulates the cell cycle in vitro and induces cell cycle arrest due to decreased POL1A expression in response to DNA damage and decreases DNA repair. LBH deficiency allows DNA damage to accumulate in vivo leading to S phase arrest. Because accumulation of DNA fragments is known to exacerbate murine arthritis, the LBH RA risk allele likely contributes to disease severity in RA by suppressing LBH expression and increasing DNA damage.
To cite this abstract in AMA style:Matsuda S, Hammaker D, Topolewski K, Briegel K, Dowdy S, Boyle DL, Wang W, Firestein GS. Deficiency of the Novel Rheumatoid Arthritis (RA) Risk Gene, LBH, Induces Replication Stress in RA Fibroblast-like Synoviocytes (FLS) and Exacerbates Arthritis Severity [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/deficiency-of-the-novel-rheumatoid-arthritis-ra-risk-gene-lbh-induces-replication-stress-in-ra-fibroblast-like-synoviocytes-fls-and-exacerbates-arthritis-severity/. Accessed September 20, 2021.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/deficiency-of-the-novel-rheumatoid-arthritis-ra-risk-gene-lbh-induces-replication-stress-in-ra-fibroblast-like-synoviocytes-fls-and-exacerbates-arthritis-severity/