Functional cooperation between Stat-1 and ets-1 to optimizeicam-1gene transcription

Abstract

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the immune system, enabling the interactions between effector cells and target cells. It is also known to be involved in tumor growth and metastasis. Its expression is transcriptionally regulated by several proinflammatory cytokines including IFN-γ, which induces ICAM-1 transcription via the JAK–STAT signaling pathway in a Stat1-dependent fashion. The ICAM-1 promoter contains several cis-active regulatory elements including 2 Ets binding sites (EBSs) located at positions –158 and –138 relatively to the AUG, which were previously shown to play a role in the constitutive activity of the ICAM-1 promoter. In the present study, we have determined whether the EBSs are also involved in the regulation of ICAM-1 gene transcription by pro-inflammatory cytokines. Transient transfection assays were performed with reporter genes containing ICAM-1 promoter constructions cloned upstream from the firefly luciferase gene. Site-specific mutations of the EBS diminished the promoter activity stimulated by IFN-γ, although the IFN-γ responsive element (pIγRE), which binds Stat1, was intact. Stimulation of the transcriptional activity following IFN-γ treatment was significantly reduced when both EBSs were inactivated. Co-immunoprecipitation experiments provided evidence of a physical interaction involving Ets1 and Stat1. In COS-1 and HEK 293 cells cotransfected with CFP-Stat1 and YFP-Ets fusion protein, fluorescence resonance energy transfer experiments confirmed the close proximity of these 2 proteins in living cells following treatment with IFN-γ.