The long non-coding RNA HOTAIR contributes to joint-specific gene expression in rheumatoid arthritis.

Although patients with rheumatoid arthritis (RA) typically exhibit symmetrical joint involvement, some patients develop alternative disease patterns in response to treatment, suggesting that different molecular mechanism may underlie disease progression depending on joint location. Here, we identify joint-specific changes in RA synovium and synovial fibroblasts (SF) between knee and hand joints. We show that the long non-coding RNA HOTAIR, which is only expressed in knee SF, regulates more than 50% of this site-specific gene expression in SF. HOTAIR is downregulated after stimulation with pro-inflammatory cytokines and is expressed at lower levels in knee samples from patients with RA, compared with osteoarthritis. Knockdown of HOTAIR in knee SF increases PI-Akt signalling and IL-6 production, but reduces Wnt signalling. Silencing HOTAIR inhibits the migratory function of SF, decreases SF-mediated osteoclastogenesis, and increases the recruitment of B cells by SF. We propose that HOTAIR is an important epigenetic factor in joint-specific gene expression in RA.

BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts.

BACKGROUND: Individual functions of members of the bromodomain (BRD) and extra-terminal (BET) protein family underlying the anti-inflammatory effects of BET inhibitors in rheumatoid arthritis (RA) are incompletely understood. Here, we aimed to analyze the regulatory functions of BRD3, an understudied member of the BET protein family, in RA synovial fibroblasts (FLS). METHODS: BRD3 was silenced in FLS prior to stimulation with TNF. Alternatively, FLS were treated with I-BET. Transcriptomes were analyzed by RNA sequencing (RNAseq), followed by pathway enrichment analysis. We confirmed results for selective target genes by real-time PCR, ELISA, and Western blotting. RESULTS: BRD3 regulates the expression of several cytokines and chemokines in FLS, and positively correlates with inflammatory scores in the RA synovium. In addition, RNAseq pointed to a profound role of BRD3 in regulating FLS proliferation, metabolic adaption, and response to stress, including oxidative stress, and autophagy. CONCLUSIONS: BRD3 acts as an upstream regulatory factor that integrates the response to inflammatory stimuli and stress conditions in FLS and executes many functions of BET proteins that have previously been identified using pan-BET inhibitors.

The histone acetyl transferases CBP and p300 regulate stress response pathways in synovial fibroblasts at transcriptional and functional levels.

The activation of stress response pathways in synovial fibroblasts (SF) is a hallmark of rheumatoid arthritis (RA). CBP and p300 are two highly homologous histone acetyl transferases and writers of activating histone 3 lysine 27 acetylation (H3K27ac) marks. Furthermore, they serve as co-factors for transcription factors and acetylate many non-histone proteins. Here we showed that p300 but not CBP protein expression was down regulated by TNF and 4-hydroxynonenal, two factors that mimic inflammation and oxidative stress in the synovial microenvironment. We used existing RNA-sequencing data sets as a basis for a further in-depth investigation of individual functions of CBP and p300 in regulating different stress response pathways in SF. Pathway enrichment analysis pointed to a profound role of CBP and/ or p300 in regulating stress response-related gene expression, with an enrichment of pathways associated with oxidative stress, hypoxia, autophagy and proteasome function. We silenced CBP or p300, and performed confirmatory experiments on transcriptome, protein and functional levels. We have identified some overlap of CBP and p300 target genes in the oxidative stress response pathway, however, with several genes being regulated in opposite directions. The majority of stress response genes was regulated by p300, with a specific function of p300 in regulating hypoxia response genes and genes encoding proteasome subunits. Silencing of p300 suppressed proteasome enzymatic activities. CBP and p300 regulated autophagy on transcriptome and functional levels. Whereas CBP was indispensable for autophagy synthesis, silencing of p300 affected late-stage autophagy. In line with impaired autophagy and proteasome function, poly-ubiquitinated proteins accumulated after silencing of p300.

Bromodomain Protein Inhibitors Reorganize the Chromatin of Synovial Fibroblasts.

Bromodomain- and extra-terminal domain (BET) proteins are epigenetic reader proteins that regulate transcription of their target genes by binding to acetylated histone side chains. Small molecule inhibitors, such as I-BET151, have anti-inflammatory properties in fibroblast-like synoviocytes (FLS) and in animal models of arthritis. Here, we investigated whether BET inhibition can also affect the levels of histone modifications, a novel mechanism underlying BET protein inhibition. On the one hand, FLSs were treated with I-BET151 (1 µM) for 24 h in absence and presence of TNF. On the other hand, FLSs were washed with PBS after 48 h of I-BET151 treatment, and the effects were measured 5 days after I-BET151 treatment or after an additional 24 h stimulation with TNF (5 d + 24 h). Mass spectrometry analysis indicated that I-BET151 induced profound changes in histone modifications, with a global reduction in acetylation on different histone side chains 5 days after treatment. We confirmed changes on acetylated histone side chains in independent samples by Western blotting. I-BET151 treatment reduced mean TNF-induced levels of total acetylated histone 3 (acH3), H3K18ac, and H3K27ac. In line with these changes, the TNF-induced expression of BET protein target genes was suppressed 5 d after I-BET151 treatment. Our data indicate that BET inhibitors not only prevent the reading of acetylated histones but directly influence overall chromatin organization, in particular after stimulation with TNF.

Individual functions of the histone acetyl transferases CBP and p300 in regulating the inflammatory response of synovial fibroblasts.

Chromatin remodeling, and a persistent histone 3 lysine 27 acetylation (H3K27ac) in particular, are associated with a sustained inflammatory response of synovial fibroblasts (SF) in rheumatoid arthritis (RA). Here we investigated individual functions of the writers of H3K27ac marks, the homologues histone acetyl transferases (HAT) CBP and p300, in controlling the constitutive and inflammatory gene expression in RA SF. We applied a silencing strategy, followed by RNA-sequencing and pathway analysis, complemented with the treatment of SF with inhibitors targeting the HAT (C646) or bromo domains (I-CBP) of CBP and p300. We showed that CBP and p300 undertook overlapping and, in particular at gene levels, distinct regulatory functions in SF. p300 is the major HAT for H3K27ac in SF and regulated more diverse pathways than CBP. Whereas both factors regulated genes associated with extracellular matrix remodeling, adhesion and proliferation, p300 specifically controlled developmental genes associated with limb development. Silencing of CBP specifically down regulated the TNF-induced expression of interferon-signature genes. In contrast, silencing of p300 resulted in anti- and pro-inflammatory effects. Integration of data sets derived from RNA-sequencing and chromatin immunoprecipitation sequencing for H3K27ac revealed that changes in gene expression after CBP or p300 silencing could be only partially explained by changes in levels of H3K27ac. Inhibition of CBP/p300 using HAT and bromo domain inhibitors strongly mirrored effects obtained by silencing of p300, including anti- and pro-inflammatory effects, indicating that such inhibitors are not sufficient to be used as anti-inflammatory drugs.