Background/Purpose: Tight control of the two major catabolic pathways proteasomal degradation and autophagy is critical for the maintenance of cellular homeostasis and cell survival. Here we analyzed the individual functions of the two homolog histone acetyltransferases cAMP-response element binding protein binding protein (CBP) and p300 in regulating catabolic pathways in rheumatoid arthritis (RA) synovial fibroblasts (SF).

Methods: SF were obtained 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). 24 hours later, cells were stimulated with TNF-α (10 ng/ml, 24 hours). Transcriptomes were determined by RNA-seq (Illumina NovaSeq 6000, n=6). Pathway enrichment analysis   (fold change >1.5, FDR < 0.05) was performed using DAVID Bioinformatic Resources. Autophagy was assessed by Western blotting using LC3B conversion and p62 as autophagy markers (n=5) in presence and absence of the lysosomal inhibitor bafilomycin A1 (100 nM, 4h). Cell death (n=6) was analyzed using the CytoTox-Glo cytotoxicity assay. Proteasome activities were analyzed using Proteasome-Glo chymotrypsin-like, trypsin-like and caspase-like cell-based assays (n=5).

Results: The top pathway identified after silencing of p300 in SF in presence (p=3.57-14) of TNF-α was ‘proteasome’, which was also among the top three pathways in unstimulated SF (p=6.77×10-5). The expression of most genes encoding proteasome subunits was increased after silencing of p300 but unaffected by silencing of CBP. In contrast, chymotrypsin-like and trypsin-like proteasome activities were decreased by silencing of p300, indicating a compensatory upregulation of transcription of proteasome subunits under conditions of impaired proteasome function. Caspase-like proteasome activity was not affected by silencing of p300. Genes contributing to the biological process ‘autophagy’ (p=0.031) were enriched after silencing of CBP in presence of TNF-α, whereas genes contributing to ‘regulation of mitophagy’ were enriched after silencing of p300 in absence (p=0.02) and presence of TNF-α (p=0.06). In line with RNA-seq data, 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 (p< 0.05) increased cell death only in presence of TNF-α. Viability of SF was not affected by silencing of CBP.

Conclusion: Our data provide the first evidence that p300 but not CBP regulates proteasome activity in SF by post-translational modifications. CBP and p300 have diverging functions in regulating autophagy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/individual-functions-of-histone-acetyltransferases-cbp-and-p300-in-regulating-autophagy-and-proteasomal-degradation-in-synovial-fibroblasts/