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 protein-1 (hMCP-1) gene by IFN-gamma in astrocytoma cells, because astroglial cells express chemokines in several central nervous system inflammatory states. It was found that IFN-gamma-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-gamma-activated site (GAS) and the presence of IFN-gamma-inducible GAS-binding activity involving at least STAT-1alpha for IFN-gamma-induced hMCP-1 expression. Unexpectedly, in vivo genomic footprinting of the proximal regulatory region of the IFN-gamma-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-gamma 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-gamma-mediated transcriptional induction by providing the first evidence in vivo for inducible signaling to the GC box by this cytokine.
Zhou
Z H
ZH
Department of Neurosciences, Research Institute, Cleveland Clinic Foundation, OH 44195, USA.
Chaturvedi
P
P
Han
Y L
YL
Aras
S
S
Li
Y S
YS
Kolattukudy
P E
PE
Ping
D
D
Boss
J M
JM
Ransohoff
R M
RM
D001254:Astrocytoma / Q000235:genetics / Q000276:immunology; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018932:Chemokine CCL2 / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
D018932:Chemokine CCL2; D017931:DNA Primers; D004273:DNA, Neoplasm; D011994:Recombinant Proteins; D007371:Interferon-gamma