Complex Formation of the Interferon (IFN) Consensus Sequence-binding Protein with IRF-1 Is Essential for Murine Macrophage IFN-γ-induced iNOS Gene Expression

Huabao Xiong,Chen Zhu,Hongxing Li,Frank Chen,Lloyd Mayer,Keiko Ozato,Jay C Unkeless,Scott E Plevy

doi:10.1074/jbc.m209583200

Huabao Xiong, Chen Zhu + Show 6 more

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https://doi.org/10.1074/jbc.m209583200

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Abstract

This study describes the role of the interferon (IFN) consensus sequence-binding protein (ICSBP or IRF-8) in iNOS gene expression by murine macrophages. An ICSBP binding site in the iNOS promoter region (-923 to -913) was identified using an electrophoretic mobility shift assay and chromatin co-immunoprecipitation. Overexpression of ICSBP greatly enhanced IFN-gamma-induced iNOS promoter activation in RAW264.7 cells, and IFN-gamma-induced iNOS promoter activation was abolished in ICSBP-/- macrophages. Furthermore, transduction of retrovirus-ICSBP in ICSBP-/- macrophages rescued IFN-gamma-induced iNOS gene expression. However, transduction of retrovirus-ICSBP in the absence of IFN-gamma activation did not induce iNOS expression in either RAW264.7 cells or ICSBP-/- macrophages. Interestingly, ICSBP alone transduced into ICSBP-/- macrophages did not bind to IFN-stimulated response element site (-923 to -913) of the iNOS promoter region, although following activation with IFN-gamma, a DNA.protein complex was formed that contains ICSBP and IRF-1. Co-transduction of ICSBP with IRF-1 clearly induces nitric oxide production. In addition, interleukin-4 inhibits IFN-gamma-induced iNOS gene expression by attenuating the physical interaction of ICSBP with IRF-1. Complex formation of ICSBP with IRF-1 is essential for iNOS expression, and interleukin-4 attenuates the physical interaction of ICSBP with IRF-1 resulting in the inhibition of INOS gene expression.

Highlights

This study describes the role of the interferon (IFN) consensus sequence-binding protein (ICSBP or IFN regulatory factor (IRF)-8) in isoform of nitric oxide synthase (iNOS) gene expression by murine macrophages
The IFN-stimulated response elements (ISRE) site between Ϫ923 and Ϫ913 was selected for the analysis, because this site is important for full induction of iNOS gene expression by IFN-␥
RAW264.7 cells were activated with IFN-␥, LPS, or IFN-␥ plus LPS for 4 h, and nuclear proteins were extracted for Electrophoretic Mobility Shift Assays (EMSA), which was performed by using probe spanning Ϫ935 to Ϫ905 of the iNOS promoter region containing Ϫ923 to Ϫ913 of the ISRE site

Summary

Introduction

This study describes the role of the interferon (IFN) consensus sequence-binding protein (ICSBP or IRF-8) in iNOS gene expression by murine macrophages. ICSBP alone transduced into ICSBP؊/؊ macrophages did not bind to IFN-stimulated response element site (؊923 to ؊913) of the iNOS promoter region, following activation with IFN-␥, a DNA1⁄7protein complex was formed that contains ICSBP and IRF-1. The upstream portion of the iNOS promoter contains an enhancer region, with several transcription factor binding sites that mediate transcriptional responses to IFN-␥. These sites include a ␥-interferon-activated site element and two IFN-stimulated response elements (ISRE) [15, 16]. The molecular mechanism for the regulation of NO by ICSBP is not clear

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