The Long Non-coding RNA HOTAIR Regulates BMP2 and Wnt Pathways in Synovial Fibroblasts

Muriel Elhai¹, Raphael Micheroli ¹, Mojca Frank-Bertoncelj ², Kerstin Klein ¹, Oliver Distler ³ and Caroline Ospelt ², ¹Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Switzerland., Zürich, Switzerland, ²University Hospital Zürich, Zürich, Switzerland, ³Dept. of Rheumatology, University Hospital Zürich, Zürich, Switzerland, Zürich, Switzerland

Meeting: 2019 ACR/ARP Annual Meeting

Keywords: Epigenetics, hotair, pathogenesis, rheumatoid arthritis, synovial cells, synovial fluid and WNT Signaling

Session Information

Date: Sunday, November 10, 2019

Title: 3S079: RA – Etiology & Pathogenesis I (815–820)

Session Type: ACR Abstract Session

Session Time: 2:30PM-4:00PM

Background/Purpose: Mechanisms involved in joint patterning of rheumatic diseases remain unknown. The long non-coding RNA HOTAIR is exclusively expressed in synovial fibroblasts (SF) from lower extremity joints. HOTAIR is known to be an important regulator of the epigenetic landscape by modulating H3K27me3 and H3K4me1 marks. Here, we analysed the function of HOTAIR in SF.

Methods: HOTAIR was silenced in knee SF from osteoarthritis (OA) patients by GapmeRs. ChIP-sequencing of H3K27me3 marks (Illumina HiSeq 2500) and RNA-sequencing (NovaSeq 6000) were performed in GapHOTAIR- and control-transfected SF at 48h after transfection (n=3). Enriched pathways and protein interactions were analyzed using DAVID and STRING networks, respectively. Regulated genes were confirmed by real-time PCR in SF from OA and rheumatoid arthritis (RA) patients (n=8) and changes after cycloheximide adjunction (10ug/ml 6h and 24h) were evaluated (n=3). Signaling pathways were studied by Western blotting (n=5). Effect of HOTAIR silencing on the canonical Wnt pathway was assessed using TOP/FOP reporter system (n=4). The expression of HOTAIR was studied after stimulation with TNF-α in SF (n=17) and correlated with TNF-α-expression in synovial tissues from RA patients (knee and ankle) (n=7).

Results: ChIP-sequencing showed 3225 loci, which lost H3K27me3 marks in HOTAIR silenced SF. Transcription of the genes at 492 of these loci was up-regulated in SF transfected with GapHOTAIR. Pathway enrichment showed that these transcripts were mainly involved in cancer, apoptosis and Wnt signaling. Among them, we confirmed the overexpression of BMP2 (1.7±0.5-fold; p< 0.01) and CTNNB1 (beta-catenin) (1.5±0.5-fold; p< 0.01) and showed a down-regulation of other genes belonging to the Wnt pathway (LGR5 (p=0.02), GSK3β (p=0.03) and a trend for LRP6 (p=0.06)). Blocking translation by adding cycloheximide did not change BMP2 and CTNNB1 expression, confirming a direct effect of HOTAIR on these genes, whereas the effect on other Wnt genes was indirect. The repressor of Wnt pathway SFRP1 was significantly enriched for H3K27me3 marks in controls as compared to GapHOTAIR SF, but its transcription was not changed in GapHOTAIR SF. The regulation of the canonical-Wnt pathway was confirmed by the TOP/FOP system by 1.8±0.2-fold decrease in Wnt activation after HOTAIR silencing (p=0.01). BMP2 regulation was mediated by non-canonical pathway through ERK1/2 phosphorylation (p=0.01). TNF-α stimulation led to a 2.1±0.3 decrease in HOTAIR expression in SF (p< 0.0001) and was inversely correlated with HOTAIR in the RA synovium (r=-0.79; p< 0.05).

Conclusion: HOTAIR regulates the non-canonical BMP2 and the canonical Wnt pathway by epigenetic and transcriptional mechanisms. Downregulation of HOTAIR during inflammation might influence the phenotype of chronic arthritis in joints of the lower extremities with implications for disease severity and therapy.

Disclosure: M. Elhai, None; R. Micheroli, None; M. Frank-Bertoncelj, Kurt und Senta Herrmann Foundation, 2, Promedica Foundation, 2; K. Klein, None; O. Distler, A. Menarini, 5, Abbvie, Acceleron, 5, Acceleron Pharma, 5, Actelion, 2, 5, 8, Actelion Pharmaceuticals, 2, 5, 8, 9, Amgen, 5, AnaMar, 2, 5, Bayer, 2, 5, 8, 9, Biogen Idec, 2, 5, Blade Therapeutics, 5, Boehringer Ingelheim, 2, 5, 8, 9, Catenion, 5, 9, ChemomAb, 2, 5, ChemomAB, 5, CSL Behring, 5, Ergonex, 5, espeRare Foundation, 2, 5, Genentech/Roche, 2, 5, GlaxoSmithKline, 5, GSK, 2, 5, Holds Patent mir-29 for the treatment of systemic sclerosis, 9, Inventiva, 2, 5, iQvia, 5, Italfarmaco, 2, 5, Italfarmco, 5, Lilly, 2, 5, med, 5, 8, medac, 5, Medac, 2, 5, MedImmune, 2, 5, Medscape, 5, 8, 9, Menarini, 8, Mepha, 8, Mitsubishi Tanabe, 2, 5, Mitsubishi Tanabe Pharma, 2, 5, MSD, 5, 8, Novartis, 2, 5, 8, 9, Patent, 9, Patent issued, 9, Pfizer, 2, 5, 8, Pharmacyclics, 2, 5, Roche, 5, 8, 9, Sanofi, 2, 5, Sinoxa, 2, 5, Target Bio Science, 5, Target BioScience, 5, UCB, 2, 5, 9, UCB in the area of potential treatments of scleroderma and its complications, 2, 5; C. Ospelt, Kurt und Senta Herrmann Foundation, 2, Promedica Foundation, 2.

To cite this abstract in AMA style:

Elhai M, Micheroli R, Frank-Bertoncelj M, Klein K, Distler O, Ospelt C. The Long Non-coding RNA HOTAIR Regulates BMP2 and Wnt Pathways in Synovial Fibroblasts [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/the-long-non-coding-rna-hotair-regulates-bmp2-and-wnt-pathways-in-synovial-fibroblasts/. Accessed .

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