HOMEOBOX D13 TRANSCRIPTION FACTOR MODULATES THE FORMATION OF THE PRIMARY CILIUM IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS

BackgroundDifferences in gene expression and functions of synovial fibroblasts between distinct joints might lead to site-specific activation of arthritis-relevant pathways. We previously showed that the transcription factors HOX-D10, -D11 and -D13 are higher expressed in synovial fibroblasts from hands and feet compared to shoulder and knee, and that this expression pattern is epigenetically imprinted1.ObjectivesTo investigate the functional role of HOXD13 in synovial fibroblasts.MethodsSynovial fibroblasts from patients with rheumatoid arthritis (n=2) were cultured and transfected with HOXD13 antisense LNA GapmeRs (12.5 nM). Transcriptomes were determined by RNA-seq (Illumina NovaSeq 6000). Pathway enrichment analysis of RNA-seq data was performed using web-based tools (ToppGene, EnrichR and Cytoscape) (± fold change > 1.5, FDR < 0.05). The expression of histone deacetylases (HDACs) and sirtuins was measured by quantitative real-time PCR and immunofluorescence staining. Ciliogenesis of synovial fibroblasts was assessed by measuring the expression of PKD2, ARL13B, KIF3A, and IFT88 using qPCR. Acetylation of alpha-tubulin was assessed by immunofluorescence staining. Imaging was performed with the CellInsight™ CX7 platform. Primary cilia were manually counted. Approximately 500 cells for each replicate were recorded for the presence of a primary cilium. The length of primary cilia was measured using Image J (Version 1.53o). Synovial fibroblasts were treated with Trichostatin A (2µM), an inhibitor of HDACs (HDACi), for 24h.ResultsPathway enrichment analysis of genes changed after HOXD13 silencing of synovial fibroblasts showed that HOXD13 regulated genes are involved in primary cilia related pathways. We confirmed lower mRNA expression of the ciliary proteins, PKD2, KIF3A and IFT88 after HOXD13 silencing (n=4; PKD2: 0.65 ± 0.4, p<0.05; KIF3a: 0.6 fold ± 0.3, p<0.05; IFT88: 0.7 fold ± 0.3, p<0.05), and higher expression of ARL13B (ARL13B: 1.8 fold ± 0.4, p<0.05). An increase in the number and length of primary cilia, as well as acetylation of alpha- tubulin, a characteristic feature of primary cilia, was seen after HOXD13 silencing (n=4). Accordingly, expression of HDAC4, -5 and -7 was decreased after HOXD13 silencing (n=4; HDAC4: 0.25 fold ± 0.1, p<0.05; HDAC5: 0.4 fold ± 0.1, p<0.05; HDAC7: 0.3 fold ± 0.2, p<0.05). SIRT1 expression was higher (n=4; 1.7 fold ± 0.4, p<0.05), but SIRT2 expression was lower (n=4; 0.1 fold ± 0.03, p<0.05) in HOXD13 silenced synovial fibroblasts. Trichostatin A treatment increased the acetylation level of alpha tubulin (n=4) as well as the expression of ARL13B, KIF3A, and IFT88 in synovial fibroblasts (n=4; ARL13B: 2.3 fold ± 0.9, p<0.05; IFT88: 1.5 fold ± 0.3, p<0.05; KIF3A: 3.7 fold± 1.8, p<0.05). The length and number of primary cilia was not changed by Trichostatin A treatment.ConclusionOur data show that HOXD13 plays a role in regulating the assembly of primary cilia in synovial fibroblasts. This effect seems partly mediated by the expression of HDACs and partly by the expression of ciliary proteins. The primary cilium is a sensory organelle mediating reactions to mechanical and chemical signals from the environment. Thus, our results suggest joint specific regulation of synovial fibroblasts in response to stimulation via the primary cilium.References[1]Klein K, et al. Ann Rheum Dis 2018;77: P126Disclosure of InterestsMasoumehalsadat Mirrahimi: None declared, Eva Camarillo: None declared, Kerstin Klein: None declared, Miranda Houtman: None declared, Oliver Distler Consultant of: Abbvie, Caroline Ospelt: None declared