P48 STAT-1-ALPHA-CONTAINING COMPLEXES PLAY A PREDOMINANT ROLE IN INDUCTION OF IFN-GAMMA-INDUCIBLE PROTEIN, 10 KDA (IP-10) BY IFN-GAMMA ALONE OR IN SYNERGY WITH TNF-ALPHA/

Human IFN-gamma-inducible protein, 10 kDa (hIP-10) and murine IP-10 (m IP-10) genes are induced by IFN-gamma alone, and synergistically induc ed by TNF-alpha and IFN-gamma, Upstream regions of the human and murin e genes contain conserved regulatory motifs, including an IFN-stimulat ed response element (ISRE), which governs response of the mIP-10 gene to IFN-gamma, Trans-acting factors mediating the IFN-gamma response vi a ISRE remain incompletely defined, We examined ISRE-binding factors i n the regulation of the hIP-10 gene. The requirement of p48 for hIP-10 induction by IFN-gamma, with or without TNF-alpha, was demonstrated u sing p48-deficient U2A cells, An hIP-10 promoter-reporter mutant (mISR E3) that was relatively deficient for binding a related factor, IFN re gulatory factor-1 (IRF-1) but competent for binding p48, was induced a s well as the wild-type hip-ill promoter, supporting the interpretatio n that p48 played a necessary and sufficient role in hIP-10 transcript ion. Genomic in vivo footprinting revealed IFN-gamma/TNF-alpha-inducib le binding at the ISRE consistent with the presence of p48 and associa ted factors, but not with IRF-1, induction of hIP-10 by TNF-alpha/IFN- gamma also required NF kappa B binding sites, which were protected in vivo and bound p65 homodimeric NF kappa B in vitro. These results docu mented the essential role of p48 (complexed with STAT-1 alpha) for ind uction and sustained transcription of the IP-10 gene, strongly suggest ing that IRF-1 is not required for IP-10 induction by these inflammato ry cytokines.