Background/Purpose: The minor C allele variant of rs26232 SNP, located within the first intron of the Macrophage Immunometabolism regulator (MACIR) gene, is associated with both risk of developing rheumatoid arthritis (RA) and with more severe radiological joint damage. However, rs26232 genotype is not associated with either quantitative or qualitative differences in MACIR mRNA, suggesting that the genetic association is primarily related to another nearby gene. This work aims to determine the biological basis of the association of rs26232 with RA.

Methods: RA synovial fibroblasts (RASFs) were derived from biopsies of RA patients taken at arthroscopy. Gene expression was determined using qPCR. Gene knock-down was achieved using siRNA technology. Cellular invasiveness was determined using Matrigel-coated Boyden transwell chambers and migration was assayed using the scratch assay. Proliferation was quantified using BrdU ELISA and Caspase 3/7 levels were used to measure apoptosis. Transcriptome sequencing was carried out by Qiagen and differential expression evaluated using EdgeR. Synovial single cell transcriptome sequencing was performed on 4 different biopsy RA samples.

Results: rs26232 is an expression quantitative trait loci (eQTL) for peptidylglycine alpha-amidating monooxygenase (PAM) in fibroblasts as revealed by analysis of online eQTL databases (GTEx portal and Open Targets Genetics), as well as other tissue types. The Pathobiology of Early Arthritis database (https://peac.hpc.qmul.ac.uk/) revealed highest levels of PAM expression in the fibrous synovial pathotypes compared with lymphoid- or myeloid-rich synovial pathotypes, and lower PAM levels were associated with increased systemic markers (CRP levels) and synovial inflammation. Single cell RNAseq of RA synovial tissue revealed PAM expression to be restricted to RASFs, being highest in the recently identified tissue damaging F4 subtype. Inhibition of PAM resulted in increased RASF proliferation (+36.97%; p=0.001) and invasion (+18.02%; p=0.022), and decreased apoptosis (-23.73%; p=0.0003), compared to control siRNA treated RASFs. Transcriptomic analysis of RASFs treated with siPAM compared to non-targeting control siRNA treated cells revealed 24 genes significantly differentially expressed; notably PRG4 (Lubricin), which acts as a joint lubricant and regulates cell growth, was reduced in siPAM treated cells (Log fold-change -1.4; FDR p-value= 0.042). Western blotting confirmed PRG4 protein levels drop following siPAM treatment of RASFs.

Conclusion: Our data reveals that PAM modulates tissue destruction mediated by F4 RASFs and suggests that genetically determined lower levels increases damage to cartilage and bone via effects on PRG4. The primary role of PAM is peptide amidation, a post-translation modification that increases protein half-life and reactivity. Our ongoing work will concentrate on elucidating the molecular mechanisms by which influences RASF-mediated tissue damage.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-peptidylglycine-alpha-amidating-monooxygenase-as-a-regulator-of-tissue-damage-mediated-by-rheumatoid-arthritis-synovial-fibroblasts/