Background/Purpose: The SNP rs26232 is associated with both risk and severity of rheumatoid arthritis (RA), with the C allele associated with the susceptibility to RA, and with more severe radiological joint damage. rs26232 is located within the first intron of MACIR, but is not associated with either quantitative or qualitative differences in MACIR gene expression. The link between rs26232 and RA pathology is therefore likely due to regulation of another nearby gene. This work aims to demonstrate the mechanisms by which rs26232 is associated with RA severity.

Methods: Gene knock-down was achieved using siRNA technology. Invasiveness was determined using Matrigel-coated Boyden transwell chambers and migration was assayed using scratch-assay. Proliferation was quantified using BrdU ELISA and apoptosis was measured by caspase 3/7 assay. Synovial single cell transcriptome sequencing (scRNAseq) was performed on 4 different biopsy RA samples. Transcriptome sequencing was carried out by Eurofins. Hypoxia impact on PAM expression was determined using western blotting. Confocal Microscopy was carried out using ZEISS LSM 800.

Results: rs26232 is an eQTL for peptidylglycine alpha-amidating monooxygenase (PAM) in primary RASF cell lines, with lower PAM levels associated with the RA risk C allele, this data was replicated in multiple tissue in the GTEx database.  The Pathobiology of Early Arthritis database (https://peac.hpc.qmul.ac.uk/) revealed highest expression in the fibroblast-rich pathotype and an inverse correlation with CRP levels and ultrasound assessed inflammation. scRNAseq of RA synovium revealed PAM expression restricted to fibroblasts, with highest expression in the tissue damaging F4 subtype. siRNA-mediated inhibition of PAM resulted in increased RASF proliferation (+36.97%; p=0.001), invasion (+18.02%; p=0.022), and decreased apoptosis (-23.73%; p=0.0003). PAM carries out the post-translational amidation of secretory pathway proteins. RASF incubation with the amidation inhibiting drug 4-phenyl-3-butenoic acid (PBA) resulted in effects similar to those of PAM siRNAs, indicating an essential role for enzymatic activity in this phenotype.  siRNA mediated knockdown of PAM in a collagen induced arthritis (CIA) mouse model demonstrated a significant increase in clinical score at weeks 4 and 5 compared to non-targeting control treated mice (p = 0.004). Western blot analysis revealed PAM to be induced by hypoxia (3% O₂) and confocal microscopy shows PAM localised to the golgi apparatus consistent with a role in proteins in the secretory pathway.

Conclusion: A genetically mediated decrease in PAM expression in RASFs increases their tissue damaging activities. This effect is likely mediated by the secretion of an amidated peptide or protein.  The identification of the amidated secretome is the focus of our ongoing work.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/genetically-determined-peptidylglycine-alpha-amidating-monooxygenase-pam-mediated-amidation-regulates-tissue-damage-by-rheumatoid-arthritis-synovial-fibroblasts/