FRI0513 HISTONE-ACETYLTRANSFERASES CBP AND P300 REGULATE AUTOPHAGY AND PROTEASOMAL DEGRADATION IN SYNOVIAL FIBROBLASTS
Authors: Monika Krosel, Marcel Gabathuler, Christoph Kolling, Matija Tomsic, Oliver Distler, Caroline Ospelt, Kerstin Klein
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Proteasomal degradation and autophagy are the major catabolic pathways that maintain the homeostasis of cells and are associated with cell survival. The histone acetyltransferases cAMP-response element binding protein binding protein (CBP) and p300 are close homologues and widely accepted as redundant proteins. <h3>Objectives</h3> To analyse individual functions of CBP and p300 in catabolic pathways in rheumatoid arthritis (RA) synovial fibroblasts (SF). <h3>Methods</h3> SF were isolated from knee, shoulder and hand joints of RA patients undergoing joint replacement surgery. The expression of CBP and p300 was silenced by transfection of antisense LNA gapmeRs (12,5 nM). 24h after transfection cells were stimulated with TNF-α (10 ng/ml, 24h). Transcriptomes were determined by RNA-seq (Illumina NovaSeq 6000, n=6). Pathway enrichment analysis of RNA-seq data (fold change &gt;1.5, FDR &lt;0.05) was performed using DAVID bioinformatic resources. Autophagy was assessed by Western blotting using LC3B conversion and p62 as autophagy markers (n=4) in presence and absence of bafilomycin A1 (100 nM, 4h), a lysosomal inhibitor. Cell death was analysed using the CytoTox-Glo cytotoxicity assay. <h3>Results</h3> The top pathway identified after silencing of p300 in SF in presence (p=1.33x10<sup>-10</sup>) and absence of TNF-α (p=1.76x10<sup>-10</sup>) was 'proteasome', with an enrichment of genes contributing to 'proteasome assembly' and 'proteasome regulation'. The expression of several genes encoding proteasome subunits was increased after p300 silencing but unaffected by silencing of CBP. Genes contributing to the biological processes 'autophagy' (p&lt;0.05) and 'regulation of autophagy' (p&lt;0.05) were enriched after silencing of CBP and p300, whereas 'autophagy assembly' was only affected by CBP silencing. In RNA-seq data, the expression of MAP1LC3B, encoding the autophagy marker LC3B, and the autophagy substrate p62, was increased by p300 silencing but slightly decreased by CBP silencing. In line with the RNA expression, silencing of CBP reduced the conversion of LC3B and the protein expression of p62 in presence and absence of TNF-α. Results were similar in presence of bafilomycin A1, indicating a decrease in autophagosome synthesis. In contrast, the conversion of LC3B and p62 expression were increased after silencing of p300 in unstimulated SF, indicating increased autophagy. This effect was lost for LC3B after treatment with TNF-α, and LC3B conversion was even decreased in presence of bafilomycin A1. This indicates a late stage block of autophagy after silencing of p300 in TNF-α-stimulated SF. In line with this, silencing of p300 in SF (n=6, p&lt;0.05) increased cell death only in presence of TNF-α. Viability of SF was not affected by silencing of CBP. <h3>Conclusion</h3> Here we identified p300 as a major regulator of the proteasome in SF and provide first evidence for individual functions of CBP and p300 in regulating autophagy in SF. <h3>Disclosure of Interests</h3> Monika Krosel: None declared, Marcel Gabathuler: None declared, Christoph Kolling: None declared, Matija Tomsic: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Caroline Ospelt: None declared, Kerstin Klein: None declared