Molecular cloning and characterization of a gene selectively induced by interferon-gamma in human macrophages

Abstract

Interferon-{dollar}\gamma{dollar} (IFN-{dollar}\gamma{dollar}), the principal lymphokine known to activate macrophages, regulates a variety of biological functions. In studies of the molecular mechanisms by which these cells are regulated by IFN-{dollar}\gamma{dollar}, a cDNA clone encoding an IFN-{dollar}\gamma{dollar} inducible mRNA in human cells of the macrophage lineage was isolated and characterized. The corresponding gene, {dollar}\gamma{dollar}.1, is selectively induced by IFN-{dollar}\gamma{dollar}, responding a hundred-fold greater to IFN-{dollar}\gamma{dollar} than to IFN-{dollar}\alpha{dollar}. The induction is rapid and transient with maximal mRNA accumulation about 3h and decline to the basal level after 48h. Transcriptional activation can be detected as early as five minutes after IFN-{dollar}\gamma{dollar} stimulation and accounts entirely for the mRNA accumulation. In addition, {dollar}\gamma{dollar}.1 is efficiently induced by phorbol 12-myristate 13-acetate (PMA), which is known to activate protein kinase C (PKC). Both stimulators induce the translocation of PKC from the cytosol to a membrane fraction. Two selective inhibitors of PKC, H7 and sphingosine, suppressed induction of {dollar}\gamma{dollar}.1 by IFN-{dollar}\gamma{dollar} as well as by PMA. Cyclic AMP dependent protein kinase (PKA) exerts negative regulation, since {dollar}\gamma{dollar}.1 is inhibited by cholera toxin and 8-bromo-cAMP, which are known to activate PKA. The induction of {dollar}\gamma{dollar}.1 by IFN-{dollar}\gamma{dollar} is cell-type restricted, being seen thus far only in macrophages and endothelial cells. Phorbol ester-induced differentiation of promyelocytic HL-60 and promonocytic THP-1 cells renders the {dollar}\gamma{dollar}.1 gene inducible by IFN-{dollar}\gamma{dollar}. The 1.0 kb {dollar}\gamma{dollar}.1 cDNA sequence encodes a small predicted polypeptide of 38 amino acids and has sequences associated with rapidly turning over mRNAs in the 3{dollar}\sp\prime{dollar} untranslated region. In vitro translation of the {dollar}\gamma{dollar}.1 transcript yielded a 4,000 Da polypeptide. The 2.2 kb EcoR1 fragment containing {dollar}\gamma{dollar}.1 genomic DNA was sequenced and the initiation site of transcription was mapped. The 600 bp DNA fragment 5{dollar}\sp\prime{dollar} of {dollar}\gamma{dollar}.1 confers IFN-{dollar}\gamma{dollar} inducible expression of a 3{dollar}\sp\prime{dollar} reporter gene in transfected U937 cells. Two features of the 5{dollar}\sp\prime{dollar} regulatory region are notable: there are four AP-1 binding sites at {dollar}-370{dollar}, {dollar}-280{dollar}, {dollar}-160{dollar} and {dollar}-57{dollar} which could account for PMA inducibility, and a 13 nucleotide sequence at {dollar}-159{dollar} to {dollar}-171{dollar} with high homology to the interferon response sequences found in the 5{dollar}\sp\prime{dollar} regulatory regions of other IFN-inducible genes.