Control of cytokine mRNA degradation by the histone deacetylase inhibitor ITF2357 in rheumatoid arthritis fibroblast-like synoviocytes: beyond transcriptional regulation

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease, characterized by the excessive activation of the immune system and the uncontrolled production of cytokines and other inflammatory mediators in synovial joints. Cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1β produced by macrophages and lymphocytes infiltrating the synovial tissue lead to the abnormal activation of fibroblast-like synoviocytes (FLS), which in turn causes bone and cartilage deterioration [1]. Regulation of inflammatory cytokines occurs at multiple levels and results from the intricate modulation of epigenetic regulatory mechanisms, activation of intracellular signaling pathways, control of messenger RNA (mRNA) stability and protein translation. The correct regulation of mRNA decay is critical for immune homeostasis, as it allows cells to quickly adjust the expression of inflammatory mediators, the overproduction of which could adversely affect the organism [2]. Conditions that interfere with stability of mRNA are associated with diverse diseases, including chronic inflammation and cancer [3].

Adenosine uridine (AU)-rich elements (AREs) represent one of the largest and most important groups of cis-acting mRNA stability determinants. AREs allow the recruitment of trans-acting ARE binding proteins (ARE-BP), which in turn mediate mRNA degradation [4]. Several human ARE-BP have been identified, such as tristetraprolin (TTP, or ZFP36), TTP family members BRF1 (ZFP36L1) and BRF2 (ZFP36L2), AU-rich binding factor-1 (AUF1, or HNRNPD), KH-type splicing regulatory protein (KHSRP), and Hu antigen R (HuR, or ELAVL1). The majority of ARE-BP promote the recruitment of ARE-containing mRNAs to the exosomes for eventual degradation, although some, such as HuR and Hu family members, act as mRNA stabilizing factors [5].

The expression of several ARE-BP was found to be dysregulated in RA, and their silencing shown to affect key regulatory mechanisms in arthritis pathogenesis, both in vitro and in vivo [6‐10]. To date, TTP is the ARE-BP that has been best characterized and associated with RA development and disease progression [11, 12]. TTP expression is altered in patients with synovium affected by RA [11] and TTP-deficient mice display a severe inflammatory phenotype that includes synovial pannus formation and erosive arthritis [10]. Remarkably, overexpression of endogenous TTP or mutations at TTP phosphorylation sites protect mice in experimental models of arthritis [11, 13].

Growing interest in the modulation of ARE-BP in RA pathology has thus promoted the search for novel inhibitory compounds that can reverse the aberrant expression and function of ARE-BP. Inhibitors of the mitogen-activated protein kinase (MAPK) p38, a critical regulator of the phosphorylation status and activity of multiple ARE-BP, have been extensively used to dampen uncontrolled production of pro-inflammatory cytokines resulting from dysregulated mRNA decay [14]. However, p38 inhibitors are currently not approved for RA treatment due to molecule-related adverse events, such as cutaneous toxicity, and limited clinical efficacy [15, 16]. HDACi represent a novel class of small molecule drugs that have shown promising results in vitro and in vivo in models of RA and immune-related diseases [17, 18] and have demonstrated initial clinical efficacy in the treatment of systemic-onset juvenile idiopathic arthritis [19]. Although the primary mechanism of action of HDACi is proposed to rely on the regulation of chromatin opening and transcription, studies have reported that HDACi can impair cytokine mRNA expression despite favoring their transcriptional activation [20, 21]. We previously reported that pan-specific HDACi ITF2357 (Givinostat) and trichostatin A (TSA) prevented IL-6 production in RA FLS and macrophages by promoting accelerated degradation of IL6 mRNA [22].

In this study, we aimed to dissect the transcriptional and post-transcriptional regulation of cytokine mRNA expression by ITF2357, and to identify whether, and through which mechanisms, HDACi can restore the balance in mRNA-stability mechanisms that are deregulated in RA.

In RA and in other immune-mediated inflammatory diseases (IMIDs), the excessive production and accumulation of cytokines and chemokines contributes to the perpetuation of chronic inflammation and immune responses [1]. Continuous exposure to pro-inflammatory stimuli drives RA FLS to develop an epigenetically imprinted, aggressive phenotype and inflammatory memory that promotes the degradation of synovial joints [39]. Development of immunomodulatory epigenetic inhibitors has emerged in recent years. Among these, HDACi comprise a class of small anti-inflammatory molecules that showed pre-clinical potential for the treatment of RA [18]. Despite extensive research in recent years, the mode of action of these compounds remains largely unknown.

Here we show that in RA FLS, pan-HDACi ITF2357 efficiently suppresses cytokine production independently of the kinetics of gene induction by IL-1β. As we previously found that IL-6, a key cytokine contributing to RA pathobiology, was suppressed by ITF2357 via acceleration of IL6 mRNA decay [22], we aimed to investigate whether post-transcriptional, rather than transcriptional, regulatory events could be the key factor explaining the broad anti-inflammatory effects of ITF2357. Of note, recent reports indicate that prolonged exposure to TNF leads to a gradual reshaping of the FLS transcriptome, which is largely dependent on mRNA stability processes [14], highlighting the importance of post-transcriptional regulatory mechanisms in maintaining the chronicity of inflammation in RA. We extended our analysis to additional IL-1β-induced cytokines (IL8, CXCL2) and mediators of inflammatory responses, matrix degradation, and cell survival (PTGS2, ADAMTS1, and BCL2L1). We confirmed that a subset of these genes, specifically IL8, CXCL2 and PTGS2, were subject to mRNA stability regulation by ITF2357. On the contrary, some targets displayed sustained stability (MMP1–3, CXCL10, CXCL5–6), while others rapidly decayed over time but were not further destabilized by ITF2357 (IRF1, FoxO1). More intriguingly, kinetics played an important role in the transcriptional or post-transcriptional regulation of genes affected by ITF2357. Indeed, while mostly affected at the transcriptional level after shorter exposure to ITF2357, the mRNA expression of IL6, IL8 and PTGS2 was post-transcriptionally regulated at later time points. These results indicate that the initial anti-inflammatory events mediated by ITF2357 occur by suppressing the nascent production of cytokine mRNA, while subsequent immune suppressive functions are related to the destabilization of their transcripts.

A key mechanism responsible for the post-transcriptional regulation of gene expression is ARE-BP-mediated mRNA decay [4]. We evaluated whether ARE-BP could mediate the effects of ITF2357 on cytokine mRNA stability in RA FLS and found that TTP mRNA expression rapidly increased after short exposure to IL-1β. After treatment with ITF2357, TTP mRNA was not stabilized despite being induced at all time-points, implying that a transcriptional component is responsible for the regulation of this ARE-BP by HDACi. To date, the mRNA-destabilizing TTP has been best described as a regulator of inflammatory processes [40]. TTP expression is significantly increased in the synovial joints in RA compared to non-inflamed joints, and it is abundant in macrophages and synovial fibroblasts [41], possibly indicating a relevant role for this ARE-BP in these cells. Studies of peripheral blood mononuclear cells in RA have reported a global reduction of TTP expression, compared to healthy controls and patients with osteoarthritis (OA) [12, 42]. In animal studies, knockout of TTP has been shown as sufficient to the development of a complex inflammatory phenotype, characterized by auto-immunity and polyarthritis [10] On the contrary, induction of TTP expression is protective in collagen-induced arthritis (CIA) [13].

In our study, we observed that besides affecting the transcriptional regulation of TTP, ITF2357 additionally reduced the abundance of a higher molecular-weight form of TTP. Treatment with phosphatase confirmed this as the phosphorylated form of the protein. The destabilizing effects of ITF2357 on TTP mRNA decay further support this finding, as dephosphorylated and active TTP would also cause its own mRNA to be degraded [13]. The equilibrium between the phosphorylated and the dephosphorylated pools of TTP has frequently been reported to be a critical feature in the determination of the inflammatory response [30]. Mice expressing a phosphorylation-deficient form of TTP, in which serines 52 and178 are converted to arginine residues, are protected from CIA, as a consequence of increased functionality of the protein [11, 30]. Also, activation or depletion of phosphatases that revert TTP to its dephosphorylated form, such as PP2A and Dusp1, reduce the production of cytokines such as IL6, IL8 and TNF, and increase broad pro-inflammatory gene expression, respectively [11, 43, 44].

Phosphorylation is the most common post-translational modification of TTP and other ARE-BP, but other modifications have been reported [45, 46]. Thus, it remains possible that ITF2357 may enhance the acetylation levels of TTP and subsequently reduce its phosphorylation. Indirect regulation of TTP phosphorylation by HDACi is yet another possibility. In fact, evidence from the literature suggests that HDAC1,2 and 3 can bind to and acetylate Dusp1 [47]. On the contrary, direct effects of HDACi on p38 activation are likely to be excluded, as we found pan-HDACi to leave p38 phosphorylation unaltered in IL-1β-stimulated FLS [22]. Similarly, ITF2357 may affect mRNA stability independently of the c-Jun N-terminal kinase (JNK) signaling pathway, as FoxO1 mRNA stability, previously shown to be mediated by JNK inhibition [48] was not affected by ITF2357.

We tested whether single silencing of multiple ARE-BP could result in the differential regulation of IL6, IL8, CXCL2 and PTGS2. Unlike other ARE-BP, TTP silencing caused increased expression of inflammatory mediators and proved to be a critical regulatory factor in RA FLS. Additionally, ITF2357 reduced IL6 and CXCL2 production in ZFP36^+/+ but not ZFP36^−/− murine fibroblasts, overall demonstrating crucial involvement of TTP in IL6 regulation by HDACi.

Recent studies suggested that the induced expression and the reduced phosphorylation of TTP could be beneficial in dampening inflammatory responses in arthritis [11]. Our results indicate that ITF2357 functions as an activator of TTP function and transcription in RA FLS, and provide novel understanding of how HDACi dictate not only the transcriptional, but also the post-transcriptional regulation of inflammatory genes. These findings provide rationale for further evaluation of HDACi as a therapeutic tool in RA and other chronic inflammatory diseases.