A COMPLEX ELEMENT REGULATES IFN-GAMMA-STIMULATED MONOCYTE CHEMOATTRACTANT PROTEIN-1 GENE-TRANSCRIPTION

Monocyte chemoattractant protein-1 (MCP-1) is induced in chronic osseo us inflammation, and is temporally and spatially correlated with monoc yte recruitment, We investigated the mechanism of MCP-1 regulation in a human osteoblastic cell line in response to IFN-gamma, a potent medi ator of the immune inflammatory response. Nuclear run-on and stability studies demonstrated that IFN-gamma stimulated MCP-1 transcription an d did not enhance mRNA stabilization. Using MCP-1 promoter/reporter ge ne constructs, we determined that IFN-gamma-enhanced MCP-1 transcripti on 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 (GCTTCCCT TTCCT) adjacent to a IFN-gamma activation site (GAS), Since deletion o f the CT sequence enhanced both the magnitude and duration of IFN-gamm a-stimulated, GAS-mediated transcription,,ve have termed it the IPN re sponse-inhibitory sequence (IRIS). The combined IRIS/GAS sequence is h ighly conserved in mouse, rat, and bovine MCP-1 genes. In gel-shift as says, nuclear extracts from IFN-gamma-stimulated osteoblastic cells fo rmed two specific inducible bands with labeled IRIS/GAS DNA. Both band s were supershifted by anti-STAT1 Abs, but not by Abs to STAT2, p48(IS GF-3 gamma), IFN-regulatory factor-1, or IFN-regulatory factor-2. Form ation of one of the bands required the presence of the IRIS moiety, IR IS/GAS DNA also formed a number of specific complexes with constitutiv ely expressed factors, none of which were affected by the above Abs, T hese studies establish a mechanism for IFN-gamma-stimulated MCP-1 expr ession and identify a complex element that regulates MCP-1 gene transc ription.