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NF-AT5 is a critical regulator of inflammatory arthritis.

ABSTRACT: To investigate the role of NF-AT5, an osmoprotective transcription factor, in synovial hyperplasia and angiogenesis in patients with rheumatoid arthritis (RA).The expression of NF-AT5 in synovial tissue and synoviocytes from RA patients was examined by immunohistochemistry and Western blot analysis, respectively. Messenger RNA (mRNA) in RA synoviocytes and human umbilical vein endothelial cells (HUVECs) transfected with dummy small interfering RNA (siRNA) or NF-AT5 siRNA were profiled using microarray technology. Assays to determine synoviocyte apoptosis and proliferation were performed in the presence of NF-AT5 siRNA. VEGF???-induced angiogenesis was assessed by measuring the proliferation, tube formation, and wound migration of HUVECs. Experimental arthritis was induced in mice by injection of anti-type II collagen antibody.NF-AT5 was highly expressed in rheumatoid synovium, and its activity was increased by proinflammatory cytokines, such as interleukin-1? and tumor necrosis factor ?. The mRNA profiling of synoviocytes and HUVECs transfected with NF-AT5-targeted siRNA revealed 3 major changes in cellular processes associated with the pathogenesis of RA: cell cycle and survival, angiogenesis, and cell migration. Consistent with these results, NF-AT5 knockdown in RA synoviocytes and HUVECs inhibited their proliferation/survival and impeded angiogenic processes in HUVECs. Mice with NF-AT5 haploinsufficiency (NF-AT5(+/-)) developed a very limited degree of synovial proliferation, as seen on histologic analysis, and decreased angiogenesis, and they exhibited a nearly complete suppression of experimentally induced arthritis.NF-AT5 regulates synovial proliferation and angiogenesis in chronic arthritis.

SUBMITTER: Yoon HJ

PROVIDER: S-EPMC3084342 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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NF-AT5 is a critical regulator of inflammatory arthritis.

Yoon Hyung-Ju HJ You Sungyong S Yoo Seung-Ah SA Kim Nam-Hoon NH Kwon H Moo HM Yoon Chong-Hyeon CH Cho Chul-Soo CS Hwang Daehee D Kim Wan-Uk WU

Arthritis and rheumatism 20110701 7

<h4>Objective</h4>To investigate the role of NF-AT5, an osmoprotective transcription factor, in synovial hyperplasia and angiogenesis in patients with rheumatoid arthritis (RA).<h4>Methods</h4>The expression of NF-AT5 in synovial tissue and synoviocytes from RA patients was examined by immunohistochemistry and Western blot analysis, respectively. Messenger RNA (mRNA) in RA synoviocytes and human umbilical vein endothelial cells (HUVECs) transfected with dummy small interfering RNA (siRNA) or NF- ...[more]

PMID: 21717420

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Project description:BACKGROUND:Overexpression of FAT1 gene and its oncogenic effects have been reported in several cancers. Previously, we have documented upregulation of FAT1 gene in glioblastoma (GBM) tumors which was found to increase the expression of proinflammatory markers, HIF-1?, stemness genes and EMT markers in glioma cells. Here, we reveal NF?B (RelA)/RelA/p65 as the transcriptional regulator of FAT1 gene in GBM cells. METHODS:In-silico analysis of FAT1 gene promoter was performed using online bioinformatics tool Promo alggen (Transfac 8.3) to identify putative transcription factor(s) binding motifs. A 4.0?kb FAT1 promoter (-?3220?bp to +?848?bp w.r.t. TSS?+?1) was cloned into promoter less pGL3Basic reporter vector. Characterization of FAT1 promoter for transcriptional regulation was performed by in-vitro functional assays using promoter deletion constructs, site directed mutagenesis and ChIP in GBM cells. RESULTS:Expression levels of NF?B (RelA) and FAT1 were found to be increased and positively correlated in GBM tumors (n?=?16), REMBRANDT GBM-database (n?=?214) and TCGA GBM-database (n?=?153). In addition to glioma, positive correlation between NF?B (RelA) and FAT1 expression was also observed in other tumors like pancreatic, hepatocellular, lung and stomach cancers (data extracted from TCGA tumor data). A 4.0?kb FAT1-promoter-construct [-?3220?bp/+?848?bp, transcription start site (TSS)?+?1, having 17 NF?B (RelA) motifs] showed high FAT1 promoter luciferase-activity in GBM cells (U87MG/A172/U373MG). FAT1 promoter deletion-construct pGL3F1 [-?200?bp/+?848?bp, with 3-NF?B (RelA)-motifs] showed the highest promoter activity. Exposure of GBM cells to known NF?B (RelA)-activators [severe-hypoxia/TNF-?/ectopic-NF?B (RelA)?+?IKBK vectors] led to increased pGL3F1-promoter activity and increased endogenous-FAT1 expression. Conversely, siRNA-mediated NF?B (RelA) knockdown led to decreased pGL3F1-promoter activity and decreased endogenous-FAT1 expression. Deletion of NF?B (RelA)-motif at -?90?bp/-?80?bp [pGL3F1?1-construct] showed significant decrease in promoter activity. Site directed mutagenesis at -90?bp/-?80?bp and ChIP assay for endogenous-NF?B (RelA) confirmed the importance of this motif in FAT1 expression regulation. Significant reduction in the migration, invasion as well as colony forming capacity of the U87MG glioma cells was observed on siRNA-mediated knockdown of NF?B (RelA). CONCLUSION:Since FAT1 and NF?B (RelA) are independently known to promote pro-tumorigenic inflammation and upregulate the expression of HIF-1?/EMT/stemness in tumors, targeting the NF?B (RelA)-FAT1 axis may attenuate an important tumor-promoting pathway in GBM. This may also be applicable to other tumors.

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NF-AT5 is a critical regulator of inflammatory arthritis.

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NF-AT5 is a critical regulator of inflammatory arthritis.

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