ESE-1 is a novel transcriptional mediator of angiopoietin-1 expression in the setting of inflammation.

Angiogenesis is a critical component of the inflammatory response associated with a number of conditions. Angiopoietin-1 (Ang-1) is an angiogenic growth factor that promotes the chemotaxis of endothelial cells and facilitates the maturation of new blood vessels. Ang-1 expression is up-regulated in response to tumor necrosis factor-alpha (TNF-alpha). To begin to elucidate the underlying molecular mechanisms by which Ang-1 gene expression is regulated during inflammation, we isolated 3.2 kb of the Ang-1 promoter that contain regulatory elements sufficient to mediate induction of the promoter in response to TNF-alpha, interleukin-1beta, and endotoxin. Surprisingly, sequence analysis of this promoter failed to reveal binding sites for transcription factors that are frequently associated with mediating inflammatory responses, such as NF-kappaB, STAT, NFAT, or C/EBP. However, putative binding sites for ETS and AP-1 transcription factor family members were identified. Interestingly, among a panel of ETS factors tested in a transient transfection assay, only the ETS factor ESE-1 was capable of transactivating the Ang-1 promoter. ESE-1 binds to specific ETS sites within the Ang-1 promoter that are functionally important for transactivation by ESE-1. ESE-1 and Ang-1 are induced in synovial fibroblasts in response to inflammatory cytokines, with ESE-1 induction slightly preceding that of Ang-1. Mutation of a high-affinity ESE-1 binding site leads to a marked reduction in Ang-1 transactivation by ESE-1, inducibility by inflammatory cytokines, and DNA binding to the ESE-1 protein. Transcriptional profiling of cells transiently transfected with an ESE-1 expression vector demonstrates that the endogenous Ang-1 gene is directly inducible by ESE-1. Finally, Ang-1 and ESE-1 exhibit a similar and strong expression pattern in the synovium of patients with rheumatoid arthritis. Our results support a novel paradigm for the ETS factor ESE-1 as a transcriptional mediator of angiogenesis in the setting of inflammation.

Responses to the proinflammatory cytokines interleukin-1 and tumor necrosis factor alpha in cells derived from rheumatoid synovium and other joint tissues involve nuclear factor kappaB-mediated induction of the Ets transcription factor ESE-1.

OBJECTIVE: 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 kappaB (NF-kappaB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines. METHODS: 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-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor kappaB (IkappaB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-kappaB 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-kappaB family were conducted using the NF-kappaB site from the ESE-1 promoter as a probe. RESULTS: 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-1beta and TNFalpha 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-1beta requires activation of NF-kappaB and binding of p50 and p65 family members to the NF-kappaB site in the ESE-1 promoter. Overexpression of IkappaB using an adenoviral vector blocked IL-1beta-induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-kappaB binds to the ESE-1 promoter in vivo. CONCLUSION: 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-1beta, TNFalpha, or LPS. This induction relies on the translocation of the NF-kappaB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-kappaB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.