A complex element regulates IFN-gamma-stimulated monocyte chemoattractant protein-1 gene transcription.
Monocyte chemoattractant protein-1 (MCP-1) is induced in chronic osseous inflammation, and is temporally and spatially correlated with monocyte recruitment. We investigated the mechanism of MCP-1 regulation in a human osteoblastic cell line in response to IFN-gamma, a potent mediator of the immune inflammatory response. Nuclear run-on and stability studies demonstrated that IFN-gamma stimulated MCP-1 transcription and did not enhance mRNA stabilization. Using MCP-1 promoter/reporter gene constructs, we determined that IFN-gamma-enhanced MCP-1 transcription is regulated by a 29-bp element located at -227 relative to the ATG start codon. This element contains a 13-bp CT-rich sequence (GCTTCCCTTTCCT) adjacent to a IFN-gamma activation site (GAS). Since deletion of the CT sequence enhanced both the magnitude and duration of IFN-gamma-stimulated, GAS-mediated transcription, we have termed it the IFN response-inhibitory sequence (IRIS). The combined IRIS/GAS sequence is highly conserved in mouse, rat, and bovine MCP-1 genes. In gel-shift assays, nuclear extracts from IFN-gamma-stimulated osteoblastic cells formed two specific inducible bands with labeled IRIS/GAS DNA. Both bands were supershifted by anti-STAT1 Abs, but not by Abs to STAT2, p48(ISGF-3y), IFN-regulatory factor-1, or IFN-regulatory factor-2. Formation of one of the bands required the presence of the IRIS moiety. IRIS/GAS DNA also formed a number of specific complexes with constitutively expressed factors, none of which were affected by the above Abs. These studies establish a mechanism for IFN-gamma-stimulated MCP-1 expression and identify a complex element that regulates MCP-1 gene transcription.
Valente
A J
AJ
Department of Medicine, University of Texas Health Science Center, San Antonio 78284-7870, USA. valente@uthscsa.edu
Xie
J F
JF
Abramova
M A
MA
Wenzel
U O
UO
Abboud
H E
HE
Graves
D T
DT
D001483:Base Sequence; D018932:Chemokine CCL2 / Q000235:genetics*; D017124:Conserved Sequence / Q000276:immunology; D004268:DNA-Binding Proteins / Q000378:metabolism; D019143:Evolution, Molecular; D015972:Gene Expression Regulation, Neoplastic / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000502:physiology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000276:immunology; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects / Q000276:immunology*; D014407:Tumor Cells, Cultured
D018932:Chemokine CCL2; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D007371:Interferon-gamma