Effect of cycloheximide on the expression of LPS-inducible iNOS, IFN-beta, and IRF-1 genes in J774 macrophages

The effect of cycloheximide (CHX) on the gene expression for inducible NO synthase (iNOS), interferon (IFN)-beta, and IFN regulatory factor (IRF)-1 was examined in LPS-stimulated J774 macrophages. LPS caused increased expression of mRNAs specific for iNOS, IFN-beta, and IRF-1 with different kinetics. Addition of CHX resulted in inhibition of the LPS-induced iNOS gene expression and parallel decrease in NO production. In contrast, expression of IFN-beta and IRF-1 genes in response to LPS was potentiated in the presence of CHX. These results indicate that de novo protein synthesis is not required for IFN-beta and IRF-1 gene expression and that ongoing protein synthesis including IFN-beta and IRF-1 may be involved in the induction process of iNOS in mouse macrophages.     

A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes

We have isolated a novel cDNA clone encoding interferon (IFN) consensus sequence-binding protein in adult T-cell leukemia cell line or activated T cells (ICSAT); this protein is the human homolog of the recently cloned Pip/LSIRF. ICSAT is structurally most closely related to the previously cloned ICSBP, a member of the IFN regulatory factor (IRF) family of proteins that binds to interferon consensus sequences (ICSs) found in many promoters of the IFN-regulated genes. Among T-cell lines investigated, ICSAT was abundantly expressed in human T-cell leukemia virus type 1 (HTLV-1)-infected T cells. When the HTLV-1 tax gene was expressed or phorbol myristake acetate-A23187 stimulation was used, ICSAT expression was induced in Jurkat cells which otherwise do not express ICSAT. When the binding of ICSAT to four different ICSs was tested, the relative differences in binding affinities for those ICSs were determined. To study the functional role of ICSAT, we performed cotransfection experiments with the human embryonal carcinoma cell line N-Tera2. ICSAT was demonstrated to possess repressive function over the gene activation induced by IFN stimulation or by IRF-1 cotransfection. Such repressive function is similar to that seen in IRF-2 or ICSBP. However, we have found that ICSAT has a different repressive effect from that of IRF-2 or ICSBP in some IFN-responsive reporter constructs. These results suggest that a novel mechanism of gene regulation by "differential repression" is used by multiple members of repressor proteins with different repressive effects on the IFN-responsive genes.