Objective
To investigate the role of autophagy in the regulation of cell death in rheumatoid arthritis synovial fibroblasts (RASFs).
Methods
RASFs and osteoarthritis synovial fibroblasts (OASFs) were ...treated with thapsigargin (TG), an inducer of endoplasmic reticulum (ER) stress, and MG132, a proteasome inhibitor. Then, 3‐methyladenine was used as an autophagy inhibitor and bafilomycin A1 as a lysosome inhibitor. Polyubiquitinated proteins, p62, and autophagy induction were evaluated by immunoblotting, immunofluorescence microscopy, and immunohistochemistry, respectively. OASFs were transfected with small interfering RNA targeting autophagy‐linked FYVE protein (ALFY). Cell death was evaluated by flow cytometry and a caspase 3 activity assay.
Results
In RASFs, the induction of autophagy by TG and MG132 was increased compared to that in OASFs. Whereas autophagy promoted a caspase 3–independent induction of cell death under ER stress, autophagy had a protective role in apoptosis induced by proteasome inhibition. Treatment of RASFs with 3‐methyladenine blocked TG‐induced cell death. ER stress induced a strong accumulation of p62‐positive polyubiquitinated protein aggregates, accompanied by the formation of large vacuoles in RASFs but not OASFs. Furthermore, TG‐induced p62 protein expression was increased, whereas TG‐induced ALFY expression was reduced, in RASFs compared to OASFs. ALFY knockdown promoted the accumulation of p62, the formation of polyubiquitinated protein aggregates, and cell death.
Conclusion
Our data provide the first evidence of a dual role of autophagy in the regulation of death pathways in RASFs. A reduced expression of ALFY and the formation of p62‐positive polyubiquitinated protein aggregates promote cell death in RASFs under severe ER stress.
A number of human diseases, such as arthritis and atherosclerosis, include characteristic pathology in specific anatomical locations. Here we show transcriptomic differences in synovial fibroblasts ...from different joint locations and that HOX gene signatures reflect the joint-specific origins of mouse and human synovial fibroblasts and synovial tissues. Alongside DNA methylation and histone modifications, bromodomain and extra-terminal reader proteins regulate joint-specific HOX gene expression. Anatomical transcriptional diversity translates into joint-specific synovial fibroblast phenotypes with distinct adhesive, proliferative, chemotactic and matrix-degrading characteristics and differential responsiveness to TNF, creating a unique microenvironment in each joint. These findings indicate that local stroma might control positional disease patterns not only in arthritis but in any disease with a prominent stromal component.
ObjectiveTo investigate the effects of BET bromodomain protein inhibition on inflammatory activation and functional properties of rheumatoid arthritis synovial fibroblasts (RASF).MethodsThe ...expression of the BET bromodomain proteins BRD2, BRD3 and BRD4 was analysed in synovial tissue by immunohistochemistry. RASF were stimulated with tumour necrosis factor (TNF)-α, interleukin (IL)-1β and toll-like receptor (TLR) ligands (Pam3, pIC and lipopolysaccharide (LPS)) in the presence or absence of the BET inhibitor I-BET151, or siRNA targeting BRD2, BRD3 and BRD4. RASF expression of inflammatory mediators, including MMP1, MMP3, IL-6 and IL-8, was measured by q-PCR, q-PCR array and ELISA. Cellular viability, apoptosis, proliferation and chemoattractive properties of RASF were investigated using MTT, cell apoptosis ELISA, BrdU-based proliferation and transwell migration assays.ResultsBRD2, BRD3 and BRD4 proteins were detected in rheumatoid arthritis (RA) synovial tissue, expressed in both RASF and macrophages. I-BET151 suppressed cytokine and TLR ligand-induced secretion of MMP1, MMP3, IL-6 and IL-8, and mRNA expression of more than 70% of genes induced by TNF-α and IL-1β. Combined silencing of BRD2, BRD3 and BRD4 significantly reduced cytokine and TLR ligand-induced expression of a subset of gene products targeted by I-BET151, including MMP1, CXCL10 and CXCL11. I-BET151 treatment of RASF reduced RASF proliferation, and the chemotactic potential for peripheral blood leucocytes of RASF conditioned medium.ConclusionsInhibition of BET family proteins suppresses the inflammatory, matrix-degrading, proliferative and chemoattractive properties of RASF and suggests a therapeutic potential in the targeting of epigenetic reader proteins in RA.
Dysregulated expression of bone morphogenetic protein receptor type II (BMPR2) is a pathogenetic hallmark of pulmonary hypertension. Downregulation of BMPR2 protein but not mRNA has been observed in ...multiple animal models mimicking the disease, indicating a posttranscriptional mechanism of regulation. Because microRNAs (miRNAs) regulate gene expression mainly through inhibition of target gene translation, we hypothesized that miRNAs may play a role in the modulation of BMPR2. Performing a computational algorithm on the BMPR2 gene, several miRNAs encoded by the miRNA cluster 17/92 (miR-17/92) were retrieved as potential regulators. Ectopic overexpression of miR-17/92 resulted in a strong reduction of the BMPR2 protein, and a reporter gene system showed that BMPR2 is directly targeted by miR-17-5p and miR-20a. By stimulation experiments, we found that the miR-17/92 cluster is modulated by interleukin (IL)-6, a cytokine involved in the pathogenesis of pulmonary hypertension. Because IL-6 signaling is mainly mediated by STAT3 (signal transducer and activator of transcription 3), the expression of STAT3 was knocked down by small interfering RNA, which abolished the IL-6–mediated expression of miR-17/92. Consistent with these data, we found a highly conserved STAT3-binding site in the promoter region of the miR-17/92 gene (C13orf25). Promoter studies confirmed that IL-6 enhances transcription of C13orf25 through this distinct region. Finally, we showed that persistent activation of STAT3 leads to repressed protein expression of BMPR2. Taken together, we describe here a novel STAT3–miR-17/92—BMPR2 pathway, thus providing a mechanistic explanation for the loss of BMPR2 in the development of pulmonary hypertension.
Objectives To study the expression, regulation and function of the histone methyltransferase enhancer of zeste homologue 2 (EZH2) in synovial fibroblasts (SF) from patients with rheumatoid arthritis ...(RA) and osteoarthritis (OA). Methods SF were obtained from RA and OA patients undergoing joint surgery. Expression levels were assessed by quantitative real-time PCR and western blot. Kinase inhibitors and reporter gene assays were employed to study signalling pathways. Functional analyses included EZH2 overexpression by plasmid transfection and gene silencing by small interfering RNA. Chromatin immunoprecipitation assay was used to analyse histone methylation within distinct promoter regions. Results By studying the expression and function of EZH2 in SF the authors found that EZH2 is overexpressed in rheumatoid arthritis synovial fibroblasts (RASF) and further induced by tumour necrosis factor alpha through the nuclear factor kappa B and Jun kinase pathways. As a target gene of EZH2 the authors identified secreted frizzled-related protein 1 (SFRP1), an inhibitor of Wnt signalling, which is associated with the activation of RASF, and show that SFRP1 expression correlates with the occupation of its promoter with activating and silencing histone marks. Conclusions These data strongly suggest that the chronic inflammatory environment of the RA joint induces EZH2 and thus might cause changes in the epigenetic programmes of SF.
Background
The benefit of thoracic lymphadenectomy in the treatment of resectable non-small cell lung cancer (NSCLC) continues to be debated. We hypothesized that the number of lymph nodes (LNs) ...removed for patients with pathologic node-negative NSCLC would correlate with survival.
Methods
The National Cancer Data Base (NCDB) was queried for resected, node-negative, NSCLC patients treated between 2004 and 2014. Patients were grouped according to the number of LNs removed (1–4, 5–8, 9–12, 13–16, and ≥17). Patients with <10 LNs removed were also compared with those with ≥10 LNs removed. A Cox regression analysis was performed and hazard ratios (HRs) calculated, with 95 % confidence intervals (CIs).
Results
Of 1,089,880 patients with NSCLC reported to the NCDB during the study period, 98,970 (9.0 %) underwent resection without evidence of pathologic nodal involvement. Lobectomy was performed in 83.9 %, sublobar resection was performed in 12.7 % and pneumonectomy was performed in 2.8 % of patients. The number of LNs removed correlated with increasing tumor size and extent of resection. On multivariate analysis, increasing age, male sex, white ethnicity, high tumor grade, larger tumor size, pneumonectomy, and positive surgical margins were all negatively correlated with overall survival. The number of LNs removed and lobectomy/bi-lobectomy correlated with improved survival. The removal of <10 LNs was associated with a 12 % increased risk of death (HR: 1.12, 95 % CI 1.09–1.14;
p
< 0.001).
Conclusion
Survival of early-stage NSCLC patients is associated with the number of LNs removed. The surgical management of early-stage NSCLC should include thoracic lymphadenectomy of at least 10 nodes.
Rheumatoid arthritis (RA) is a chronic autoimmune-disease of unknown origin that primarily affects the joints and ultimately leads to their destruction. The involvement of immune cells is a general ...hallmark of autoimmune-related disorders. In this regard, macrophages, T cells and their respective cytokines play a pivotal role in RA. However, the notion that RA is a primarily T-cell-dependent disease has been strongly challenged during recent years. Rather, it has been understood that resident, fibroblast-like cells contribute significantly to the perpetuation of disease, and that they may even play a role in its initiation. These rheumatoid arthritis synovial fibroblasts (RASFs) constitute a quite unique cell type that distinguishes RA from other inflammatory conditions of the joints. A number of studies have demonstrated that RASFs show alterations in morphology and behaviour, including molecular changes in signalling cascades, apoptosis responses and in the expression of adhesion molecules as well as matrix-degrading enzymes. These changes appear to reflect a stable activation of RASFs, which occurs independently of continuous exogenous stimulation. As a consequence, RASFs are no longer considered passive bystanders but active players in the complex intercellular network of RA.
Objective
To investigate the expression and effect of the microRNA‐34 (miR‐34) family on apoptosis in rheumatoid arthritis synovial fibroblasts (RASFs).
Methods
Expression of the miR‐34 family in ...synovial fibroblasts with or without stimulation with Toll‐like receptor (TLR) ligands, tumor necrosis factor α (TNFα), interleukin‐1β (IL‐1β), hypoxia, or 5‐azacytidine was analyzed by real‐time polymerase chain reaction (PCR). Promoter methylation was studied by combined bisulfite restriction analysis. The effects of overexpression and silencing of miR‐34a and miR‐34a* on apoptosis were analyzed by annexin V/propidium iodide staining. Production of X‐linked inhibitor of apoptosis protein (XIAP) was assessed by real‐time PCR and immunohistochemistry analysis. Reporter gene assay was used to study the signaling pathways of miR‐34a*.
Results
Basal expression levels of miR‐34a* were found to be reduced in synovial fibroblasts from RA patients compared to osteoarthritis patients, whereas levels of miR‐34a, miR‐34b/b*, and miR‐34c/c* did not differ. Neither TNFα, IL‐1β, TLR ligands, nor hypoxia altered miR‐34a* expression. However, we demonstrated that the promoter of miR‐34a/34a* was methylated and showed that transcription of the miR‐34a duplex was induced upon treatment with demethylating agents. Enforced expression of miR‐34a* led to an increased rate of FasL‐ and TRAIL‐mediated apoptosis in RASFs. Moreover, levels of miR‐34a* were highly correlated with expression of XIAP, which was found to be up‐regulated in RA synovial cells. Finally, we identified XIAP as a direct target of miR‐34a*.
Conclusion
Our data provide evidence of a methylation‐specific down‐regulation of proapoptotic miR‐34a* in RASFs. Decreased expression of miR‐ 34a* results in up‐regulation of its direct target XIAP, thereby contributing to resistance of RASFs to apoptosis.
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