Camilla Machado1, Hyeonjeong Lee2, Sho Sendo3, Narayanan Perumal4, Wei Wang5, David Boyle5 and Gary S. Firestein6, 1UCSD, San Diego, CA, 2University of California San Diego, San Diego, CA, 3University California, San Diego (UCSD), La Jolla, CA, 4Eli Lilly Company, San Diego, 5University of California San Diego, San Diego, 6University of California, San Diego, San Diego, CA
Background/Purpose: Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) display an aggressive behavior. Previous studies have implicated cadherins in FLS function in this phenotype, which are type I transmembrane proteins involved in cell-cell interactions. Cadherin 11, in particular, is a key adhesion molecule that regulates FLS homotypic aggregation in the synovial intimal lining. We evaluated the expression and epigenetic marks of other cadherins in FLS and identified cadherin 6 (CDH6) as a gene of interest in RA. CDH6 is a type II cadherin and is associated with high expression in renal carcinomas. It also plays a role in cancer progression by regulating migration, invasion, and cell growth. To understand a potential role for CDH6 in RA, we investigated the expression, regulation, and function of CDH6 in RA FLS.
Methods: FLS were cultured from RA and osteoarthritis (OA) synovium collected at arthroplasty and were used in passages 5-8 (n=17 and n=6, respectively). Analysis of CDH6 epigenetic marks in RA and OA FLS was performed using our previous reported FLS public databases, including ChIPseq and RNAseq. CDH6 protein and gene expression were determined by Western blot analysis and RT-qPCR (reported as Relative Expression Units (REU)). For functional studies, CDH6 was silenced using siRNA (inhibition of 95% transcript and 73% protein) and non-targeted siRNA was used as a control (CT). RA FLS function was determined after knockdown in the following assays: migration (wound healing assay), cell growth (MTT assay), and invasion (Matrigel membrane). MMP-1, MMP-3, and IL-6 expression were determined in TNF-stimulate FLS (50ng/ml at 6h) by qPCR.
Results: The epigenetic analysis of RA and OA FLS showed a differential histone mark (H3K4me3) peak the CDH6 promoter in RA FLS compared with OA FLS. This mark is associated with an active promoter and is located near the transcription start site. CDH6 gene was expressed in cultured FLS and was significantly higher in RA FLS (2.9±1.7 REU) compared with OA FLS (0.08±0.02 REU) (P=0.002). We then confirmed that CDH6 protein and mRNA are expressed in RA synovial tissue extracts. TGFβ (20ng/ml) increased CDH6 expression after 6h in cultured RA FLS (3.2±0.6 fold increase, P< 0.05). In medium alone, silencing CDH6 inhibited RA FLS migration (26±5% inhibition, P< 0.01), cell growth (30±8.3% inhibition, P< 0.01), and invasion (18±6% inhibition, p=0.03). After PDGF (10ng/ml) stimulation, CDH6 deficiency also significantly decreased FLS migration (34±7% inhibition, P< 0.01), and cell growth (23±7% inhibition, p< 0.01). However, CDH6 knockdown did not change TNF-stimulated MMP-1, MMP-3, or IL-6 gene expression.
Conclusion: Using unbiased epigenetic and transcriptomic data, we discovered that the novel adhesion molecule CDH6 is highly expressed by RA FLS. It also is poised for further induction due to a histone mark near the gene promoter. This novel cadherin also promotes RA FLS migration, cell growth, and invasion in vitro. These data suggest that CDH6 contributes to RA pathogenesis by regulating FLS aggressive behavior.
Disclosures: C. Machado, None; H. Lee, None; S. Sendo, None; N. Perumal, Eli Lilly; W. Wang, None; D. Boyle, None; G. Firestein, Eli Lilly.
