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Abstract Number: 0018

Caspase-8 Variant G Regulates Rheumatoid Arthritis Fibroblast-Like Synoviocyte Aggressive Behavior

Cecilia Ansalone1, Gyrid Nygaard1, Richard Ainsworth1, Rizi Ai1, Deepa Hammaker1, Narayanan Perumal2, Ken Weichert2, Frances Tung2, Lalitha Kodandapani2, Michael Sauder2, Elisabeth Mertsching2, Robert Benschop3, Wei Wang1, David Boyle1 and Gary Firestein1, 1University of California San Diego, San Diego, CA, 2Eli Lilly and Company, San Diego, CA, 3Eli Lilly and Company, Indianapolis, IN

Meeting: ACR Convergence 2021

Keywords: Fibroblasts, Synovial, rheumatoid arthritis

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Session Information

Date: Saturday, November 6, 2021

Session Title: RA – Etiology & Pathogenesis Poster (0011–0045)

Session Type: Poster Session A

Session Time: 8:30AM-10:30AM

Background/Purpose: Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) display an aggressive phenotype, including abnormal migration and invasion. Using data from our previous studies defining the epigenetic landscape of RA FLS, CASP8 (encoding caspase-8) was identified as a gene of interest in RA that might be implicated in this behavior due to abnormal epigenetic marks. Caspase-8 has proteolytic functions that regulate apoptosis and non-proteolytic functions that affect cell movement. However, the specific isoforms responsible for the latter are unknown. In this study, we identified the mechanisms of caspase-8 function in RA FLS and identified the specific isoform responsible.

Methods: RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5-8 (n=20). CASP8 deficiency was induced with siRNA to deplete either all isoforms or individual isoforms; non-targeting siRNA was used as control. RT-qPCR and western blot were used to assess gene and protein expression, respectively. PDGF-induced migration (scratch assay), invasion (through Matrigel membrane), and adhesion to fibronectin and collagen type II in CASP8-deficient and control cells were assessed. Talin cleavage was measured by western blot and calpain cleavage activity assessed via fluorescent assay. Analysis of CASP8 epigenetic marks and transcript isoforms in RA and osteoarthritis (OA) FLS was evaluated using public databases (ChIPseq and RNAseq). Crystal structures of variant B and G were determined.

Results: Silencing total CASP8 reduced RA FLS migration (36.7%±7.2, n=6, P=0.008) and invasion (41.6±6.3%, n=4, P=0.01) after PDGF stimulation, and lower adhesion to fibronectin and collagen type II (n=4; P=0.002 and P=0.005 respectively). Decreased migration after PDGF stimulation was associated with decreased talin cleavage and calpain activity. The caspase enzymatic inhibitor Z-IETD-FM did not affect FLS migration nor invasion. Of the 20 known alternatively spliced variants of caspase-8, only variant C and G are expressed in FLS, with G > >C. Epigenetic analysis found enhanced H3K4me1 and H3K27ac peaks in the promoter region of variant G and C in RA FLS compared with OA. Expression of caspase-8 variant G, but not other variants, was induced by PDGF (1.5±0.1 fold increase at 6h, n=6, P=0.003). Selective silencing of variant G showed that this isoform is solely responsible for the effect of caspase-8 on FLS invasion and calpain activity (n=6, P≤0.01). The crystal structures of caspase-8 isoform G and B were identical aside for a unique unstructured 59aa N-terminal domain in variant G.

Conclusion: Non-enzymatic functions of caspase-8 regulate key activities associated with aggressive RA FLS behavior. RA FLS have a restricted pattern of CAPS8 alternative splicing, and variant G is solely responsible for this function. Because caspase-8 variant G has a unique amino acid tail, selective targeting could potentially inhibit FLS invasion without the deleterious effects of blocking caspase-8-mediated apoptosis.


Disclosures: C. Ansalone, None; G. Nygaard, None; R. Ainsworth, None; R. Ai, None; D. Hammaker, None; N. Perumal, Eli Lilly and Company, 3; K. Weichert, Eli Lilly & Company, 3; F. Tung, Eli Lilly & Company, 3; L. Kodandapani, None; M. Sauder, Eli Lilly & Company, 3; E. Mertsching, Eli Lilly, 3; R. Benschop, Eli Lilly & company, 3, 10, 11; W. Wang, None; D. Boyle, None; G. Firestein, Eli Lilly, 5.

To cite this abstract in AMA style:

Ansalone C, Nygaard G, Ainsworth R, Ai R, Hammaker D, Perumal N, Weichert K, Tung F, Kodandapani L, Sauder M, Mertsching E, Benschop R, Wang W, Boyle D, Firestein G. Caspase-8 Variant G Regulates Rheumatoid Arthritis Fibroblast-Like Synoviocyte Aggressive Behavior [abstract]. Arthritis Rheumatol. 2021; 73 (suppl 9). https://acrabstracts.org/abstract/caspase-8-variant-g-regulates-rheumatoid-arthritis-fibroblast-like-synoviocyte-aggressive-behavior/. Accessed January 30, 2023.
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