Abstract
Interferon (IFN)-gamma induces the expression of the gp91(phox) gene both during myeloid differentiation and also in mature phagocytes through several cis-elements and their binding proteins. To find new cis-elements for this induction, transient expression assays were performed using a reporter gene driven by serially truncated gp91(phox) promoters in U937 cells. The results suggest that a critical cis-element for induction exists in the region from bp -115 to -96 of the promoter. Site-directed mutagenesis showed that a gamma-activated sequence (GAS) element at bp -100 (-100GAS) of the gp91(phox) promoter plays a pivotal role for the IFN-gamma-dependent activity of the bp -115 to +12 region of the gp91(phox) promoter. Electrophoretic mobility shift assays using several GAS competitors and specific antibodies indicated that phosphorylated STAT-1alpha specifically binds to the -100GAS. Site-directed mutagenesis showed that an interferon-stimulated response element (ISRE) at bp -88 (-88ISRE) mediates the induction of the gene by IFN-gamma in cooperation with -100GAS. Electrophoretic mobility shift assay showed that IRF-1 dominantly binds to -88ISRE in an IFN-gamma-dependent fashion. These results demonstrate a new mechanism for IFN-gamma-induced transcription of the gp91(phox) gene by the cooperation of STAT-1alpha and IRF-1 binding to -100GAS and -88ISRE, respectively.
Highlights
Phagocytes, such as macrophages and granulocytes, generate superoxide anions by the phagocyte NADPH oxidase to kill ingested microorganisms [1]
Binding of STAT-1␣ to the Ϫ100GAS was detected within 15 min after IFN-␥ treatment and was not inhibited by cycloheximide, suggesting the rapid phosphorylation of pre-existing STAT-1␣ and its translocation from the cytosol into the nucleus by IFN-␥ treatment. Both Ϫ100 GAS and an interferon-stimulated response element (ISRE) Are Required for Maximal gp91phox Promoter Activity Inducted by IFN-␥—To determine whether Ϫ100GAS contributed to the IFN-␥-dependent gp91phox promoter activity through the bp Ϫ115 to Ϫ96 region, we examined the effect of a Ϫ100GAS mutation (TTCTGATAA to TGATGATAA), which abolishes the binding of STAT-1␣ to Ϫ100GAS (Fig. 5), on gp91phox promoter activity of pϪ115/Luc in IFN-␥-treated U937 cells
These results indicate that STAT-1␣ activated by IFN-␥ directly enhances the gp91phox promoter activity through binding to Ϫ100GAS
Summary
Phagocytes, such as macrophages and granulocytes, generate superoxide anions by the phagocyte NADPH oxidase to kill ingested microorganisms [1]. Enhancer elements for the expression of gp91phox gene in mature myeloid cells have been suggested to be in a 50-kbp region located upstream of a transcription start site of the gene [10], principal cis-regulatory elements are clustered in a 450-bp proximal promoter region of the gene. Coincident with induction of gp91phox gene expression, the binding activity of CDP to these binding sites decreases during differentiation of the cells [11,12,13,14]. Interferon regulatory factor (IRF)-2 activates the gp91phox promoter through two interferonstimulated response elements (ISRE) in the absence of CDP binding [15, 17]. We further show that IRF-1 participates in the STAT-1-mediated transcription through binding to the ISRE at bp Ϫ88 of the gene These results suggest a new mechanism for IFN-␥-induced transcription of the gp91phox gene in which STAT-1␣ and IRF-1 cooperate with each other
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