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.

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.