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. Low LBH expression has been observed in RA, which affects the cell cycle and contributes to aggressive behavior of RA FLS. We investigated the molecular mechanism of cell cycle regulation by LBH to understand why RA FLS exhibit this characteristic phenotype.

Methods: RA FLS were cultured from synovial tissues obtained at arthroplasty. LBH was knocked down using LBH siRNA, which decreased LBH expression by 88%. Gene expression was measured by qPCR and normalized to ß-actin. Cell cycle analysis was performed with propidium iodide staining and flow cytometry by synchronizing cells by serum starvation and then culturing FLS with 10% FBS. Western blot analysis was used to quantify protein expression and results were normalized to GAPDH.

Results: Cell cycle analysis of RA FLS showed that LBH siRNA silencing decreased S phase progression. The percentage of serum-starved cells in S phase was 2±1%, which increased to 18±4% in control FLS and from 2±1% to 15±4% in LBH-deficient FLS after adding complete medium for 24 hr. However, the percentage of control FLS in S phase decreased to 9±1% in control cells after 38 hr while LBH-silenced cells continued to increased to 25±4% (p<0.05 for control vs. LBH-deficient). These data suggest that LBH deficiency delays S phase progression. To understand the mechanism, we explored the influence of LBH on steps that activate the intra-S phase checkpoint. One key element is DNA replication stress, which can cause the DNA replication forks to stall. LBH-deficient FLS displayed 4±1-fold higher phospho-CHK1 at 24 hr compared to control FLS, which is induced by replication stress (p<0.05). DNA polymerase alpha catalytic subunit (POLA1) is required for initiation of DNA replication in S phase at the replication fork. LBH silencing reduced POLA1 mRNA by 38% (p<0.01) and POLA1 protein by 86% (p<0.01) compared with scrambled siRNA.

Conclusion:  These data show that LBH deficiency reduces POLA1 expression and induces replication stress, which leads to S phase arrest. Lack of progression through S phase arrest in RA FLS could alter the growth patterns of FLS in RA and contribute to their aberrant behavior. Targeting the cell cycle in RA could potentially overcome these pathogenic steps, allow an appropriate response to replication stress, and allow normal progression through the cell cycle.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/deficient-expression-of-the-novel-rheumatoid-arthritis-ra-risk-gene-lbh-induces-s-phase-arrest-in-ra-fibroblast-like-synoviocytes-fls/