Background/Purpose: RA fibroblast-like synoviocytes (FLS) display an aggressive phenotype, such as increased cytokine production and cell growth. Currently no therapeutics specifically target FLS. To this end, we have taken an unbiased integrative omics approach to identify therapeutic candidate gene by combining three different genome-wide assays: (i) GWAS in RA, (ii) differentially expressed genes in RA vs. osteoarthritis (OA) FLS and, (iii) differential DNA methylation in RA vs. OA FLS. We identified and characterized one gene present in all three databases, namely limb bud and heart development (LBH). LBH is a conserved putative protein with largely unknown functions. 

Methods: FLS cultures were established from RA and OA synovial tissues from joint replacement surgery. Gene expression was measured by qPCR. LBH gene expression was silenced using siRNA (mean 91% decrease) or over-expressed (mean 13 fold increase) using an LBH expression vector. Differentially expressed genes of 4 RA FLS lines with modified lbh expression were determined by microarray. The affected pathways were identifed using Ingenuity Pathway Analysis. Effects of modified lbhgene expression were assessed by cell migration (scratch-wound assay), cell growth (MTT), apoptosis (caspase 3/7 activity) and TNF-stimulated gene expression.

Results: LBH is constitutively expressed in RA FLS as determined by qPCR. TGFß stimulation (1 ng/ml, 18 hr) significantly increased LBH mRNA by 5.0±0.4 fold (P=0.02) and PDGF-BB (0.1 ng/ml,12 hr) significantly decreased LBH mRNA expression by 5.7±1.7 fold (P=0.04). Stimulating FLS with TNF, Wnt1, Wnt3a or Wnt 5a had no effect on LBH gene expression. LBH was then knocked down using siRNA, with no effects on cell migration or TNF-induced MMP3 or IL-6 expression. To determine potential functions, microarrays were performed using LBH-deficient FLS. Pathway analysis of gene expression profiles in LBH^low compared to control FLS identified “Cellular growth and proliferation” as the most significantly enriched pathway. Therefore, we performed cell growth assays.  LBH-deficiency increased FLS proliferation by 92±15% (p=0.025). Conversely, LBH-overexpressing FLS significantly inhibited cell growth by 62±10% (P=0.018). LBH did not alter apoptosis.

Conclusion: We identified LBH as a candidate gene for RA by integrating multiple omics datasets. Microarray experiments focused our attention on cell growth as a potential LBH-regulated function, and this was confirmed using functional assays. Furthermore, the gene is highly regulated by growth factors that modulate the cell cycle. The data suggest that LBH contributes to synovial intimal hyperplasia and joint damage in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-novel-rheumatoid-arthritis-ra-risk-gene-lbh-is-regulated-by-tgfs-and-pdgf-and-modulates-cell-growth-in-fibroblast-like-synoviocytes/