Control of cytokine mRNA degradation by the histone deacetylase inhibitor ITF2357 in rheumatoid arthritis fibroblast-like synoviocytes: beyond transcriptional regulation
Histone deacetylase inhibitors (HDACi) suppress cytokine production in immune and stromal cells of patients with rheumatoid arthritis (RA). Here, we investigated the effects of the HDACi givinostat (ITF2357) on the transcriptional and post-transcriptional regulation of inflammatory markers in RA fibroblast-like synoviocytes (FLS).
The effects of ITF2357 on the expression and messenger RNA (mRNA) stability of IL-1β-inducible genes in FLS were analyzed using array-based qPCR and Luminex. The expression of primary and mature cytokine transcripts, the mRNA levels of tristetraprolin (TTP, or ZFP36) and other AU-rich element binding proteins (ARE-BP) and the cytokine profile of fibroblasts derived from ZFP36+/+ and ZFP36−/− mice was measured by qPCR. ARE-BP silencing was performed by small interfering RNA (siRNA)-mediated knockdown, and TTP post-translational modifications were analyzed by immunoblotting.
ITF2357 reduced the expression of 85% of the analyzed IL-1β-inducible transcripts, including cytokines (IL6, IL8), chemokines (CXCL2, CXCL5, CXCL6, CXCL10), matrix-degrading enzymes (MMP1, ADAMTS1) and other inflammatory mediators. Analyses of mRNA stability demonstrated that ITF2357 accelerates IL6, IL8, PTGS2 and CXCL2 mRNA degradation, a phenomenon associated with the enhanced transcription of TTP, but not other ARE-BP, and the altered post-translational status of TTP protein. TTP knockdown potentiated cytokine production in RA FLS and murine fibroblasts, which in the latter case was insensitive to inhibition by ITF2357 treatment.
Our study identifies that regulation of cytokine mRNA stability is a predominant mechanism underlying ITF2357 anti-inflammatory properties, occurring via regulation of TTP. These results highlight the therapeutic potential of ITF2357 in the treatment of RA.
KeywordsRheumatoid arthritis Fibroblast-like synoviocytes Inflammation mRNA stability Tristetraprolin Histone deacetylase inhibitor ITF2357
Adenosine uridine-rich elements
AU-rich binding factor-1
Dulbecco’s Modified Eagle Medium
Fetal bovine serum
Histone deacetylase inhibitor
Hu antigen R
c-Jun N-terminal kinase
KH-type splicing regulatory protein
Mitogen-activated protein kinase
Tumor necrosis factor
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 . 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 . Conditions that interfere with stability of mRNA are associated with diverse diseases, including chronic inflammation and cancer .
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 . 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 .
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, 7, 8, 9, 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  and TTP-deficient mice display a severe inflammatory phenotype that includes synovial pannus formation and erosive arthritis . 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 . 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 . 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 .
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.
Patient material and FLS isolation
FLS were derived from synovial tissue specimens obtained from patients with RA by needle arthroscopy, as previously described , cultured in medium supplemented with 10% fetal bovine serum (FBS, Invitrogen), and used between passages 4 and 10. All patients fulfilled the criteria for the classification of RA and had active disease including clinical arthritis of the joint from which the synovial biopsies were obtained .
FLS treatment and stimulation
FLS were cultured overnight in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) containing 1% FBS prior to incubation with cytokines. Cells were pre-incubated for 30 min with either 250 nM pan-HDAC inhibitor ITF2357 (Italfarmaco) or 5 μM p38 inhibitor (SB202190, Sigma) and stimulated with 1 ng/ml IL-1β (R&D Systems). Information about the specificity of the HDACi is published .
RNA extraction and gene expression profiling
RNeasy Micro Kit (Qiagen) was used for RNA extraction. Quantity and purity of RNA was assessed using a Nanodrop spectrophotometer (Nanodrop Technologies). RNA was reverse-transcribed using a First-Strand complementary DNA (cDNA) synthesis kit (Thermo Scientific) and quantitative (q)PCR was performed using Sybr Select PCR Master Mix (Applied Biosystems). For qPCR array analysis, RNA was reverse-transcribed using an RT2 HT First Strand Kit (Qiagen), cDNA was mixed with Sybr Green qPCR Master Mix (Qiagen) and expression of 83 genes involved in FLS activation was analyzed using RT2 Profiler customized qPCRarrays. qPCR reactions were performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) and relative mRNA expression was calculated using StepOne Software V.2.1 (Applied Biosystems). Sequences of the primers used are listed in Additional file 4. The ratio between the gene of interest and the expression of human GAPDH or murine ACTB housekeeping genes, or the expression of five housekeeping genes (B2M, HPRT1, RPL13A, GAPDH and ACTB) was calculated for qPCR and qPCR arrays, respectively.
Protein extraction and immunoblotting
FLS were lysed in Laemmli’s buffer and protein content was quantified with a BCA Protein Assay Kit (Pierce). Equivalent amounts of total protein lysate were then mixed with loading buffer and boiled at 95 °C for 5 min. Proteins were resolved by electrophoresis on either 4–12% Bis-Tris SDS NuPAGE gels (Invitrogen) for 1 h at constant 200 V, or on 10% SDS-PAGE gels for 5 h at constant 70 V for better separation of immunoreactive bands ranging between 26 and 55 kDa. Gels were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories), membranes were blocked in Tris-buffered saline (pH 8.0) containing 0.05% Tween-20 (Bio-Rad) and 4% milk (Bio-Rad), washed and probed overnight at 4 °C with antibodies recognizing TTP (Cell Signaling), histone 3 (H3) (Cell Signaling) or tubulin (Sigma-Aldrich). After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated swine anti-rabbit or goat anti-mouse immunoglobulin secondary antibody (Dako), and protein visualization was performed using a ChemiDoc MP system (Bio-Rad).
RA FLS were left unstimulated or were treated with 250 nM ITF2357 for 30 min prior to stimulation with 1 ng/ml IL-1β for 24 h. Supernatants were harvested and IL-8, matrix metalloproteinase (MMP)-3, CXCL-10, CXCL-5 and CXCL-6 protein secretion determined by Luminex (BioRad) according to the manufacturer’s instructions at the core facility of the Academic Medical Center (AMC).
Analysis of mRNA stability
FLS were left unstimulated or were treated with 250 nM ITF2357 for 30 min prior to stimulation with 1 ng/ml IL-1β. After 2 h of stimulation culture medium was discarded, cells were washed and fresh medium containing 10 μg/ml actinomycin D (ActD) (Sigma-Aldrich) was added. Cells were then harvested at 0, 2 and 5 h following the addition of ActD, RNA was isolated, and the rates of mRNA degradation in the presence or absence of HDACi assessed using a customized RT2 Profiler™ PCR Array set (SABiosciences) as described above. Transcripts displaying at least 1.5-fold change in the rate of degradation, compared to IL-1β-stimulated controls were analyzed.
Lambda phosphatase treatment
FLS were lysed in 1 × NEBuffer (New England Biolabs) supplemented with 1 mM MnCl2. Cell lysate was incubated on ice for 20 min, spun down, and supernatant was collected and incubated with 10,000 U/ml lambda (λ) phosphatase (New England Biolabs) at 30 °C for 30 min. Protein lysate was added to loading buffer and boiled at 95 °C for 5 min, and further processed for immunoblotting as described above.
RA FLS were transfected using DharmaFECT1 (Thermo Scientific). The day before transfection, cells were incubated with DMEM containing 10% FBS which was then replaced with OPTI-MEM serum-reduced medium. AUF1, BRF1, BRF2, KHSRP, HuR and TTP specific small interfering RNA (siRNA) (20 nM) and control non-targeting siRNA (20 nM), (Thermo Scientific) were mixed with DharmaFECT1 and incubated for 20 min at room temperature prior to transfection; 24 h after transfection, medium was replaced with DMEM containing 10% FBS and this was left for another 24 h.
TTP wild-type and knockout MEF
Mouse embryonic fibroblasts (MEF) were derived from littermate E14.5 Zfp36 (TTP) +/+ and Zfp36 (TTP)− /− embryos, as previously described . Zfp36−/− mice were generated by inserting a targeting vector containing a neomycin resistance gene (neo) in the TTP protein-coding region, which generated multiple stop codons and precluded synthesis of the functional protein. MEFs were maintained in medium containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine. Zfp36−/− cells were regularly maintained for one passage in feeding medium containing 0.3 mg/ml of the selection antibiotic Geneticin (G418, Thermo Fisher Scientific).
Data are presented as mean ± SEM unless otherwise indicated. One-way analysis of variance (ANOVA) was used for analyzing sets of data requiring multiple comparisons. Wilcoxon matched pairs test and the ratio t test was used for all other paired comparisons. Data were analyzed using GraphPad software 7 with p values < 0.05 considered statistically significant.
ITF2357 rapidly suppresses the expression of IL-1β-induced inflammatory genes
Reduced expression of a subset of ITF2357-regulated genes is associated with mRNA decay
In order to further dissect the transcriptional and post-transcriptional effects of ITF2357 on mRNA expression, we quantified unspliced primary transcripts of the genes that were either affected (IL6, IL8 and PTGS2) or unaffected (MMP1) by ITF2357 at the level OF mRNA decay, and compared them with their respective mature transcripts (Fig. 2b). The difference between primary and mature transcript expression of IL6, IL8 and PTGS2 after 4 h treatment with ITF2357 was modest; however, it became more prominent at 8 h and marked a pronounced reduction in the expression of the mature transcript. Conversely, MMP1 primary and mature transcript rates were similar. These results confirmed that genes found unaffected by ITF2357 in terms of mRNA decay (e.g. MMP1) are predominantly regulated at the transcriptional level, while those destabilized by ITF2357 treatment are post-transcriptionally regulated.
ITF2357 leads to TTP transcriptional and post-translational changes
Following inflammatory or growth factor-driven p38 MAPK activation, TTP protein is phosphorylated on different amino acid residues, becoming inactivated . We therefore examined whether ITF2357 could also affect TTP activity by altering its post-translational status. After 2 h IL-1β stimulation, we observed increased intensity of an immunoreactive band (45 kDa) at the expected mobility for TTP (Fig. 3e). A band with higher molecular weight of approximately 47 kDa was detected after 4 h, and confirmed by higher immunoblot resolution (Additional file 3A), suggesting phosphorylation of the protein . ITF2357 significantly reduced the intensity of the higher band (Fig. 3e and Additional file 3A), similarly to p38 MAPK inhibition (Additional file 3B). Additionally, protein lysate treatment with λ-phosphatase reduced the intensity of TTP band upon IL-1β stimulation but did not further reduce TTP signal in samples treated with either ITF2357 or p38 inhibitor (Additional file 3C). Together, these results suggest that ITF2357 has a dual role in the regulation of TTP, by inducing mRNA expression at the transcriptional level, and by preventing TTP phosphorylation and subsequent inactivation.
ITF2357 suppresses cytokine production independently of AUF1, BRF1, BRF2, KHSRP and HuR
TTP silencing causes pro-inflammatory responses in RA FLS and is required to prevent ITF2357-dependent IL6 suppression in murine fibroblasts
Because TTP protein domains are remarkably conserved within vertebrates, and share a common regulatory mechanism [33, 34], we made use of fibroblasts derived from wild-type (ZFP36+/+) and TTP knockout (ZFP36−/−) mice , and stimulated them with IL-1β to mimic experimental conditions used in RA FLS. Higher expression of IL6, CXCL2 and PTGS2 mRNA in ZFP36−/− fibroblasts indicated that these cytokines are TTP targets, as previously described [36, 37, 38]. In addition, while in wild-type fibroblasts IL6 mRNA expression was significantly reduced by ITF2357, no significant suppression was observed in ZFP36−/− fibroblasts (Fig. 5d). A similar trend was also observed for CXCL2, while PTGS2 was not reduced by ITF2357 in wild-type fibroblasts. Our findings indicate that TTP is an important regulator of cytokine mRNA expression in RA FLS, and suggest that this ARE-BP is responsible for mediating part of the anti-inflammatory properties of ITF2357.
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 . 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 . 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 . 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 , 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 , 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 . 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 . 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 , 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  On the contrary, induction of TTP expression is protective in collagen-induced arthritis (CIA) .
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 . 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 . 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 . 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 . 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  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 . 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.
The authors would like to thank Dr R Lutter and the Luminex core facility (Academic Medical Center, University of Amsterdam) for performing the measurement of soluble proteins in RA FLS supernatants.
AM Grabiec is currently supported by the National Science Center, Poland (POLONEZ fellowship UMO-2015/19/P/NZ7/03659); this project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie (grant agreement number 665778KA). WS Lai and PJ Blackshear are supported by the Intramural Research Program of the NIEHS, NIH. Reedquist is supported by the Dutch Arthritis Association (NR 11–1–403); TR Radstake is supported by a grant from the European Research Council (ERC); DL Baeten is supported by a VICI grant from the Netherlands Scientific Organization (NWO) and a Consolidator grant from the ERC.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
CA, PAK and AMG contributed to research design, performed experiments, analyzed data and contributed to writing the paper; WSL, GF, PM, CS and PJB interpreted data and contributed to writing the manuscript. MR, KAR, DLB and TRDJR designed the research, interpreted data and contributed to writing the manuscript. All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
FLS isolation was approved by the medical ethics committee (METC) of the Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands (METC 2013_069). Informed written consent was obtained from patients prior to inclusion in the study.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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