Objective. To investigate the role of microRNA (miRNA) as posttranscriptional regulators of profibrotic genes in systemic sclerosis (SSc).Methods. MicroRNA, which target collagens, were identified by in silico analysis. Expression of miRNA-29 (miR-29) was determined by TaqMan real-time polymerase chain reaction analysis of skin biopsy and fibroblast samples from SSc patients and healthy controls as well as in the mouse model of bleomycininduced skin fibrosis. Cells were transfected with precursor miRNA (pre-miRNA)/anti-miRNA of miR-29 using Lipofectamine. Collagen gene expression was also studied in luciferase reporter gene assays. For stimulation, recombinant transforming growth factor ␤ (TGF␤), platelet-derived growth factor B (PDGF-B), or interleukin-4 (IL-4) was used. The effects of inhibiting PDGF-B and TGF␤ signaling on the levels of miR-29 were studied in vitro and in the bleomycin model.Results. We found that miR-29a was strongly down-regulated in SSc fibroblasts and skin sections as compared with the healthy controls. Overexpression in SSc fibroblasts significantly decreased, and accordingly, knockdown in normal fibroblasts increased, the levels of messenger RNA and protein for type I and type III collagen. In the reporter gene assay, cotransfection with pre-miR-29a significantly decreased the relative luciferase activity, which suggests a direct regulation of collagen by miR-29a. TGF␤, PDGF-B, or IL-4 reduced the levels of miR-29a in normal fibroblasts to those seen in SSc fibroblasts. Similar to human SSc, the expression of miR-29a was reduced in the bleomycin model of skin fibrosis. Inhibition of PDGF-B and TGF␤ pathways by treatment with imatinib restored the levels of miR-29a in vitro and in the bleomycin model in vivo.Conclusion. These data add the posttranscriptional regulation of collagens by miR-29a as a novel aspect to the fibrogenesis of SSc and suggest miR-29a as a potential therapeutic target.

Abstract-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. Key Words: pulmonary hypertension Ⅲ BMPR2 Ⅲ miR-17/92 Ⅲ interleukin-6 Ⅲ STAT3 P ulmonary hypertension is a devastating condition defined by the sustained elevation of pulmonary vascular resistance that leads rapidly to right heart failure and death when left untreated. 1 The pathogenesis of pulmonary hypertension is characterized by vascular remodeling and vasoconstriction. 2 Many chemotactic and inflammatory factors have been associated with these vascular changes including interleukin (IL)-6 and transforming growth factor (TGF)␤. [3][4][5] In familial pulmonary arterial hypertension, germline mutations in the gene encoding the type II receptor of the bone morphogenetic protein (BMPR2) comprise a genetic hallmark of the disease. 6 BMPR2 is a surface protein receptor that belongs to the transforming growth factor (TGF)␤ family. Its expression on endothelial and vascular smooth muscle cells mediates binding of bone morphogenetic proteins (BMPs) that have been identified as inhibitors of vascular smooth muscle cell proliferation while inducing cell death. 7 Thus, it was suggested that the downregulation of BMPR2 might lead to significant alterations in these signaling cascades and, ultimately, to remodeling of the pulmonary vascular bed. 8 Of interest, alterations in the surface expression of BMPR2 ha...

Objective. Rheumatoid arthritis (RA) is a chronic inflammatory disorder of unknown origin. Histone deacetylase (HDA) activity is considered to play a major role in the transcriptional regulation of proinflammatory genes. We undertook this study to investigate the balance of histone acetylase and HDA activity in synovial tissue from RA patients compared with that from patients with osteoarthritis (OA) and normal controls.Methods. Activity of histone acetylases and HDAs was measured in nuclear extracts of total synovial tissue samples, which were obtained from RA and OA patients undergoing surgical joint replacement, and compared with the activity in synovial tissues from patients without arthritis. Tissue expression of HDAs 1 and 2 was quantified by Western blotting. In addition, immunohistochemistry was performed for HDA-2.Results. Mean ؎ SEM HDA activity in synovial tissue samples derived from patients with RA was measured as 1.5 ؎ 0.3 moles/g, whereas the activity levels in OA (3.2 ؎ 0.7 moles/g) and normal (7.1 ؎ 4.2 moles/g) synovial tissue samples were significantly higher. Histone acetylase activity reached similar levels in RA and OA tissues and in normal tissues. The ratio of HDA activity to histone acetylase activity in RA synovial tissue was significantly reduced (12 ؎ 2%) compared with that in OA synovial tissue (26 ؎ 3%). The activity ratio in normal control samples was arbitrarily set at 100 ؎ 40%. In addition, the tissue expression of HDA-1 and HDA-2 proteins was clearly lower in RA samples than in OA samples.Conclusion. The balance of histone acetylase/ HDA activities is strongly shifted toward histone hyperacetylation in patients with RA. These results offer novel molecular insights into the pathogenesis of the disease that might be relevant to the development of future therapeutic approaches.

Pulmonary hypertension is an “umbrella term” used for a spectrum of entities resulting in an elevation of the pulmonary arterial pressure. Clinical symptoms include dyspnea and fatigue which in the absence of adequate therapeutic intervention may lead to progressive right heart failure and death. The pathogenesis of pulmonary hypertension is characterized by three major processes including vasoconstriction, vascular remodeling and microthrombotic events. In addition accumulating evidence point to a cytokine driven inflammatory process as a major contributor to the development of pulmonary hypertension.This review summarizes the latest clinical and experimental developments in inflammation associated with pulmonary hypertension with special focus on Interleukin-6, and its role in vascular remodeling in pulmonary hypertension.

This is the first report showing that miR-20a can be specifically targeted in an in vivo model for pulmonary hypertension. Our data emphasize that treatment with antagomiR-20a restores functional levels of BMPR2 in pulmonary arteries and prevents the development of vascular remodelling.

Objective. To elucidate whether the microRNA (miRNA) cluster miR-17-92 contributes to the activated phenotype of rheumatoid arthritis synovial fibroblasts (RASFs).Methods. RASFs were stimulated with tumor necrosis factor ␣ (TNF␣), and the expression and regulation of the miR-17-92 cluster were studied using realtime quantitative PCR (PCR) and promoter activity assays. RASFs were transfected with single precursor molecules of miRNAs from miR-17-92 and the expression of matrix-degrading enzymes and cytokines was measured by quantitative PCR and enzyme-linked immunosorbent assay. Potential miRNA targets were identified by computational prediction and were validated using reporter gene assays and Western blotting. The activity of NF-B signaling was determined by reporter gene assays.Results. We found that TNF␣ induces the expression of miR-17-92 in RASFs in an NF-B-dependent manner. Transfection of RASFs with precursor molecules of single members of miR-17-92 revealed significantly increased expression levels of matrix-degrading enzymes, proinflammatory cytokines, and chemokines in precursor miR-18a (pre-miR-18a)-transfected RASFs. Using reporter gene assays, we identified the NF-B pathway inhibitor TNF␣-induced protein 3 as a new target of miR-18a. In addition, pre-miR-18a-transfected RASFs showed stronger activation of NF-B signaling, both constitutively and in response to TNF␣ stimulation.Conclusion. Our data suggest that the miR-17-92-derived miR-18a contributes to cartilage destruction and chronic inflammation in the joint through a positive feedback loop in NF-B signaling, with concomitant up-regulation of matrix-degrading enzymes and mediators of inflammation in RASFs.

OBJECTIVES: /st> 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: /st> 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: /st> 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: /st> 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. Expression and function of EZH2 in synovial fibroblasts: epigenetic repression of the Wnt inhibitor SFRP1 in rheumatoid arthritis ResultsBy studying expression and function of EZH2 in SF we found that EZH2 is overexpressed in RASF and further induced by tumour necrosis factor alpha through the NF-B and Jun kinase pathways. As a target gene of EZH2 we identified secreted frizzled related protein (SFRP)1, 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. ConclusionsOur data strongly suggest that the chronic inflammatory environment of the RA joint induces EZH2and, thus, might cause changes in the epigenetic programmes of SF.

The acute-phase response is an inflammatory process triggered mainly by the cytokine IL-6. Signaling of IL-6 is transduced by activation of STAT3 (signal transducer and activator of transcription 3), which rapidly induces the production of acute-phase proteins such as haptoglobin and fibrinogen. Another target of the IL-6/STAT3 signal transduction pathway is the microRNA cluster miR-17/92. Here, we investigated the interplay of miR-17/92 and STAT3 signaling and its impact on the acute-phase response in primary human hepatocytes and hepatoma (HepG2) cells. Employing a reporter gene system consisting of STAT3-sensitive promoter sequences, we show that the miR-17/92 cluster member miR-18a enhanced the transcriptional activity of STAT3. IL-6 stimulation experiments in miR-18a-overexpressing hepatocytes and HepG2 cells revealed an augmented acute-phase response indicated by increased expression and secretion of haptoglobin and fibrinogen. This effect was due, at least in part, to repression of PIAS3 (protein inhibitor of activated STAT, 3), a repressor of STAT3 activity, which we identified as a novel direct target of miR-18a. Finally, we demonstrate that the expression of miR-17/92 in primary hepatocytes and HepG2 cells is modulated by IL-6. Our data reveal, for the first time, a microRNA-mediated positive feedback loop of IL-6 signal transduction leading to an enhanced acute-phase response in human hepatocytes.Severe inflammation is observed in several conditions, including overwhelming infection, systemic inflammatory response syndrome, sepsis, and septic shock, and results in the activation of a signaling cascade that is commonly referred to as the acute-phase response. Acute-phase proteins are involved in a great variety of physiological and biochemical processes, including growth inhibition of microbes (e.g. complement factors), blood coagulation (e.g. fibrinogen), and binding of proteins (e.g. haptoglobin-mediated binding of hemoglobin) (1).During inflammation, the production of plasma proteins by hepatocytes is altered either by increasing the levels of plasma proteins (positive acute-phase reaction) or by decreasing their levels (negative acute-phase reaction).IL-6 is one of the most important cytokines that orchestrates the hepatic production of acute-phase proteins (2). For signal transduction, IL-6 binds to a membrane-associated receptor complex, which subsequently leads to the phosphorylation of Tyr-705 of STAT3 (signal transducer and activator of transcription 3) (3). Phosphorylated STAT3 is actively transported to the nucleus, where it activates the transcription of IL-6 target genes such as the acute-phase genes haptoglobin (Hp) 2 (4) and fibrinogen ␥-chain (FGG) (5). Several negative feedback loops antagonize the activation of STAT3 to avoid persistent downstream signaling and subsequent dysregulation in the production of acute-phase proteins. A well characterized feedback pathway includes the blocking of the phosphorylation of STAT proteins by the action of the cytokine-inducible family of SOCS (sup...