Cathy Manning

ObjectiveTo investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor κB (NF-κB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines.To investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor κB (NF-κB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines.MethodsESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase–polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor κB (IκB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-κB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-κB family were conducted using the NF-κB site from the ESE-1 promoter as a probe.ESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase–polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor κB (IκB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-κB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-κB family were conducted using the NF-κB site from the ESE-1 promoter as a probe.ResultsImmunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1β and TNFα in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1β requires activation of NF-κB and binding of p50 and p65 family members to the NF-κB site in the ESE-1 promoter. Overexpression of IκB using an adenoviral vector blocked IL-1β–induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-κB binds to the ESE-1 promoter in vivo.Immunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1β and TNFα in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1β requires activation of NF-κB and binding of p50 and p65 family members to the NF-κB site in the ESE-1 promoter. Overexpression of IκB using an adenoviral vector blocked IL-1β–induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-κB binds to the ESE-1 promoter in vivo.ConclusionESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1β, TNFα, or LPS. This induction relies on the translocation of the NF-κB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-κB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.ESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1β, TNFα, or LPS. This induction relies on the translocation of the NF-κB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-κB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.

ObjectiveTo investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor κB (NF-κB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines.To investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor κB (NF-κB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines.MethodsESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase–polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor κB (IκB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-κB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-κB family were conducted using the NF-κB site from the ESE-1 promoter as a probe.ESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase–polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor κB (IκB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-κB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-κB family were conducted using the NF-κB site from the ESE-1 promoter as a probe.ResultsImmunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1β and TNFα in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1β requires activation of NF-κB and binding of p50 and p65 family members to the NF-κB site in the ESE-1 promoter. Overexpression of IκB using an adenoviral vector blocked IL-1β–induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-κB binds to the ESE-1 promoter in vivo.Immunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1β and TNFα in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1β requires activation of NF-κB and binding of p50 and p65 family members to the NF-κB site in the ESE-1 promoter. Overexpression of IκB using an adenoviral vector blocked IL-1β–induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-κB binds to the ESE-1 promoter in vivo.ConclusionESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1β, TNFα, or LPS. This induction relies on the translocation of the NF-κB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-κB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.ESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1β, TNFα, or LPS. This induction relies on the translocation of the NF-κB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-κB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.