IFN-GAMMA INDUCTION OF THE HUMAN MONOCYTE CHEMOATTRACTANT PROTEIN (HMCP)-1 GENE IN ASTROCYTOMA-CELLS - FUNCTIONAL INTERACTION BETWEEN AN IFN-GAMMA-ACTIVATED SITE AND A GC-RICH ELEMENT

We characterized regulation of the human monocyte chemoattractant prot ein-1 (hMCP-1) gene by IFN-gamma in astrocytoma cells, because astrogl ial cells express chemokines in several central nervous system inflamm atory states, It was found that IFN-gamma-induced hMCP-1 transcription was rapid, transient, and mediated by a 213-bp promoter-proximal regu latory region of the gene, Our studies on both in vitro and in vivo st ates of the hMCP-1 regulatory region established requirement of an IFN -gamma-activated site (GAS) and the presence of IFN-gamma-inducible GA S-binding activity involving at least STAT-la for IFN-gamma-induced hM CP-1 expression, Unexpectedly, in vivo genomic footprinting of the pro ximal regulatory region of the IFN-gamma-induced gene revealed protect ion of a GC-rich sequence (GC bos) with the same temporal pattern as t hat seen at the GAS; in vitro, this GC-rich element is associated with nuclear factor Sp1., These observations suggested a cooperative inter action between the GAS and the GC box element, Interestingly, site-spe cific mutations that abolished GC-box or GAS-element function produced clearly disparate results, Disruption of the GC box did not affect fo ld induction by IFN-gamma but reduced promoter-reporter expression by half. Conversely, GAS mutation abrogated induction hut did not affect the magnitude of expression. These results establish the importance of the GAS element for induction of hMCP-1 and further our understanding of IFN-gamma-mediated transcriptional induction by providing the firs t evidence in vivo for inducible signaling to the GC box by this cytok ine.