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Induction of the Human Monocyte Chemoattractant Protein (hMCP)-1 Gene in Astrocytoma Cells: Functional Interaction Between an IFN--Activated Site and a GC-Rich Element1
*
Department of Neurosciences, Research Institute, and
Department of Neurology and The Mellen Center for Multiple Sclerosis Treatment and Research, Cleveland Clinic Foundation, Cleveland, OH 44195;
Medical Biochemistry and Neurobiotechnology Center, The Ohio State University, Columbus, OH 43210; and
§
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
We characterized regulation of the human monocyte chemoattractant
protein-1 (hMCP-1) gene by IFN-
in astrocytoma cells, because
astroglial cells express chemokines in several central nervous system
inflammatory states. It was found that IFN--induced hMCP-1
transcription was rapid, transient, and mediated by a 213-bp
promoter-proximal regulatory region of the gene. Our studies on both in
vitro and in vivo states of the hMCP-1 regulatory region established
requirement of an IFN--activated site (GAS) and the presence of
IFN--inducible GAS-binding activity involving at least STAT-1
for
IFN--induced hMCP-1 expression. Unexpectedly, in vivo genomic
footprinting of the proximal regulatory region of the IFN--induced
gene revealed protection of a GC-rich sequence (GC box) with the same
temporal pattern as that seen at the GAS; in vitro, this GC-rich
element is associated with nuclear factor Sp1. These observations
suggested a cooperative interaction between the GAS and the GC box
element. Interestingly, site-specific mutations that abolished GC-box
or GAS-element function produced clearly disparate results. Disruption
of the GC box did not affect fold induction by IFN- but reduced
promoter-reporter expression by half. Conversely, GAS mutation
abrogated induction but 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--mediated
transcriptional induction by providing the first evidence in vivo for
inducible signaling to the GC box by this cytokine.
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