Monocyte-macrophages play a key role in the pathogenesis of atherosclerosis, notably as precursors of the cholesteryl-ester rich foams cells, but also as mediators of tissue lysis and remodeling. Accumulation of monocytes in the intima is one of the earliest events in atherogenesis and recruitment may continue throughout progression of the disease. A mechanism believed to be important in this process is the generation of monocyte chemoattractants in the artery wall, in particular, monocyte chemotactic protein-1 (MCP-1). MCP-1 mediates its function on monocytes via specific high affinity cell surface receptors. Soon after isolation, MCP- 1 binding activity begins to diminish and monocytes begin to express the MCP-1 gene. This transformation from an MCP-1-responsive to an MCP-1- unresponsive state and the spontaneous expression of MCP-1 may be an important component of the maturation/differentiation process experienced by the cells after recruitment from the circulation. The identification of the expression of MCP-1 by monocyte-macrophages in atheromatous lesions and other chronic inflammatory foci suggests an active role for these cells in regulating the further recruitment of monocytes from the blood. The overall aim of this project is to elucidate the mechanisms of MCP-1 gene regulation in monocytic cells and to define the regulatory elements which are specific to this cell type.
Three specific aims are proposed.
In Specific Aim 1 the regulatory factors mediating the MCP-1 response to alpha interferon and to gamma interferon, a potent activator of monocyte- macrophage function will be investigated. We have already cloned and sequenced 1874 nucleotides of the 5' flanking region of the MCP-1 gene and identified several potential DNA response elements. The responsive region of the gene will be determined by transient transfection and functional assays in the monocytic cell line THP-1. DNase 1 footprinting and electrophoretic mobility shift assays (EMSA) will be used to identify the DNA elements and transcription factors mediating the response to the two types of interferon:
In Specific Aim 2, the role of monocyte-B-cell specific, ets oncogene-related, transcription factors PU.1 and Spi-B on the control of MCP- l transcription in THP-l cells will be determined. Several PU.l recognition sites (GAGGAA) have been identified in the 5' flanking region and preliminary footprint and EMSA studies indicate that some actively bind nuclear proteins from THP- l but not HeLa cells. These studies will carefully define which of these sites are functional in controlling MCP- l expression in monocyte-macrophages and identify the accessory factors involved.
In Specific Aim 3, the information gained in Sp Aims l and 2, together with footprinting and EMSA will be used to identify the cis and trans acting factors responsible for the spontaneous induction of MCP- l expression in isolated human monocytes. The proposed experiments will identify some of the tissue-specific and possibly novel mechanisms regulating MCP-l gene expression in monocyte-macrophages and will provide valuable insights into one of the basic mechanisms regulating chronic inflammation, and the inflammatory component of atherosclerosis.
| Valente, A J; Xie, J F; Abramova, M A et al. (1998) A complex element regulates IFN-gamma-stimulated monocyte chemoattractant protein-1 gene transcription. J Immunol 161:3719-28 |
| Li, S L; Valente, A J; Zhao, S J et al. (1997) PU.1 is essential for p47(phox) promoter activity in myeloid cells. J Biol Chem 272:17802-9 |
