| The monocyte chemoattractant protein-1 (MCP-1) is a member of the CC chemokine family that induces chemotaxis of monocytes and macrophages to areas of infection and inflammation. Expression of MCP-1 has been associated with a variety of human diseases, including atherosclerosis, rheumatoid arthritis, and prostate adenocarcinoma. Thus, proper regulation of MCP-1 is important for normal inflammatory responses. MCP-1 expression can be induced in numerous cell types by inflammatory mediators, of which tumor necrosis factor-α (TNF) is the most potent.; MCP-1 is regulated at the transcriptional level. To investigate how MCP-1 expression is regulated, both in vivo and in vitro experiments were employed. Deletion and mutational analysis were used to identify two regulatory regions, the proximal and the distal, which are separated by 2.1 kb of DNA sequence. The distal regulatory region which was necessary for TNF-induced MCP-1 expression, could be subdivided into four elements (Site A, &kgr;B-1, HS, and &kgr;B-2) of which the two NF-&kgr;B binding sites are the most important. With the exception of Site A, the distal regulatory region becomes occupied upon TNF treatment as shown by IVGF. Supershift assays, NF-&kgr;B knockout cell lines, and ChIP assays identified the &kgr;B binding factor to be a member of the NF-&kgr;B/Rel transcription factor family, of which the p65 subunit is required for TNF-induced MCP-1 expression. From the three sites within the proximal regulatory region (&kgr;B-3, Site B, and GC-box), the GC-box is essential for both constitutive and induced expression of MCP-1. The GC-box binding factor was shown to bind members of the Sp/KLF transcription factor family, particularly Sp1, by supershift assays, Sp1 knockout cell lines, and ChIP assays. The proximal and the distal regulatory region was shown by IVGF and ChIP to communicate over the 2.1 kb distance, where distal NF-&kgr;B-p65 binding was required for the binding of the proximal Sp1. Even though the proximal-binding factors are present in the nucleus of an unstimulated cell, they are only able to bind after TNF induction. To establish if chromatin structure plays a role in MCP-1 expression, treatment with the histone deacetylase inhibitor, thricostatin A (TSA), was shown to induce the expression of MCP-1. It was determined using ChIP and micrococcal nuclease sensitivity assays that TNF induces a novel mechanism by which chromatin modifications can regulate gene expression, in this case expression of the MCP-1 gene. Here, the binding of NF-&kgr;B to the distal regulatory region is necessary for the progressive acetylation of histones H3 and H4 throughout the MCP-1 promoter which subsequently results in the rearrangement of nucleosomes at the proximal regulatory region.; Together these results illustrate the complexity of MCP-1 regulation. This includes the binding of ligand to cell surface receptors, activation of signal transduction cascades, nuclear translocation of transcription factors, cooperative transcription factor binding, long distance protein-protein communication, histone modification, chromatin rearrangements, assembly of the transcription machinery, and ultimately transcription initiation. |
