IFN-γ Induction of the Human Monocyte Chemoattractant Protein (hMCP)-1 Gene in Astrocytoma Cells: Functional Interaction Between an IFN-γ-Activated Site and a GC-Rich Element

Abstract

We characterized regulation of the human monocyte chemoattractant protein-1 (hMCP-1) gene by IFN-γ in astrocytoma cells, because astroglial cells express chemokines in several central nervous system inflammatory states. It was found that IFN-γ-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-γ-activated site (GAS) and the presence of IFN-γ-inducible GAS-binding activity involving at least STAT-1α for IFN-γ-induced hMCP-1 expression. Unexpectedly, in vivo genomic footprinting of the proximal regulatory region of the IFN-γ-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-γ 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-γ-mediated transcriptional induction by providing the first evidence in vivo for inducible signaling to the GC box by this cytokine.

Regulation of monocyte chemoattractant protein (MCP)‐1 transcription by interferon‐gamma (IFN‐γ) in human astrocytoma cells: postinduction refractory state of the gene, governed by its upstream elements

“…CAT and luciferase assays were performed using standardized protocols (22). ␤-Galactosidase activity was measured in cell lysates with the ␤-galactosidase enzyme assay system kit (Promega, Madison, Wis.).…”

“…Further functional analyses of the hMCP-1 promoter by transient transfection of a series of site-directed mutants indicated a cooperative interaction between the GAS element and the GC box. Electrophoresis mobility shift assays (EMSA) showed that Sp1 was constitutively abundant in nuclear extracts of CRT cells and that levels of Sp1 were unaltered at any time point after IFN-␥ treatment (22). These observations led us to propose the hypothesis that hMCP-1 induction by varied stimuli would produce changes in access to the regulatory regions of the promoter, allowing binding of inducible transcription factors (STATs, NFB family members) to their cognate sites and recruitment of ubiquitous and constitutive Sp1 to the GC-rich element (22).…”

“…We previously described the mechanism whereby IFN-␥ treatment induced transcription of the hMCP-1 gene in astrocytic cells (22). For these experiments, we used CRT astrocytoma cells, which closely mimic the cytokine response characteristics of human astrocytes in primary culture (23,24).…”

“…For these experiments, we used CRT astrocytoma cells, which closely mimic the cytokine response characteristics of human astrocytes in primary culture (23,24). Initial studies established requirement of a cis-acting gamma-activated site (GAS) and the trans-acting factor signal transducer and activator of transcription (STAT)-1␣ for IFN-␥-induced MCP-1 transcription (22). Analysis of the proximal promoter region revealed a GC-rich consensus binding site for the ubiquitous transcription factor, Sp1, centered ϳ90 bp downstream of the GAS site.…”

“…Analysis of the proximal promoter region revealed a GC-rich consensus binding site for the ubiquitous transcription factor, Sp1, centered ϳ90 bp downstream of the GAS site. In vivo genomic footprinting (IVGF) revealed that the hMCP-1 GC box was not protected until 15-30 min after IFN-␥ treatment, temporally coincident both with inducible methylation-resistance of the GAS site and transcriptional activation of hMCP-1 (22). Further functional analyses of the hMCP-1 promoter by transient transfection of a series of site-directed mutants indicated a cooperative interaction between the GAS element and the GC box.…”