ABSTRACT
The monocyte chemoattractant protein-1 gene (MCP-1) is induced by the inflammatory cytokine tumor necrosis factor through the coordinate assembly of an NF-kappaB-dependent distal regulatory region and a proximal region that has been suggested to bind Sp1 as well as other factors. To provide a genetic correlation for Sp1 activity in this system, a cell line homozygous for a targeted truncation of the Sp1 gene was derived and examined. We found that the lack of Sp1 binding activity resulted in the inability of both the distal and proximal regions to assemble in vivo even though the binding of NF-kappaB to distal region DNA was unaffected in vitro. We also found that Sp1 and NF-kappaB were the minimal mammalian transcription factors required for efficient activity when transfected into Drosophila Schneider cells. Additionally, Sp3 was able to compensate for Sp1 in the Drosophila tissue cell system but not in the Sp1(-/-) cell line suggesting that Sp1 usage is site-specific and is likely to depend on the context of the binding site. Together, these data provide genetic and biochemical proof for Sp1 in regulating the MCP-1 gene.
Subject(s)
Chemokine CCL2/genetics , DNA-Binding Proteins/physiology , Promoter Regions, Genetic , Protein Kinases/physiology , Tumor Necrosis Factor-alpha/metabolism , 3T3 Cells , Animals , Base Sequence , Blotting, Northern , Cell Line , Chemokine CXCL10 , Chemokines, CXC/metabolism , DNA Footprinting , Drosophila/genetics , Gene Expression Regulation , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutagenesis , NF-kappa B/metabolism , Protein Binding , Recombinant Proteins/metabolism , Time Factors , TransfectionABSTRACT
Proper regulation of the CC chemokine MCP-1 (monocyte chemoattractant protein-1) is important for normal inflammatory responses. MCP-1 is regulated by a wide variety of agents, including platelet-derived growth factor-BB (PDGF-BB) and tumor necrosis factor-alpha (TNF). Using both in vivo and in vitro assays, the elements required for expression between these two cytokines were compared. In vivo genomic footprinting showed that PDGF-BB induction occurred through the occupancy of the proximal regulatory region, and unlike TNF induction, no changes in the NF-kappaB binding, distal regulatory region occurred. Treatment of cells with trans-retinoic acid, an inhibitor of PDGF-BB activity, resulted in a 50% reduction in PDGF-BB-mediated induction and a concomitant block in the assembly of the proximal regulatory region. trans-Retinoic acid had minimal effect on TNF induction or promoter occupancy. An inhibitor of histone deacetylation was found to stimulate expression of MCP-1 in a manner that correlated with increased accessibility to the proximal regulatory region. These results show that the mechanisms of PDGF-BB and TNF activation of MCP-1 are distinct, although they both require the proximal regulatory region Sp1 binding site. The results also suggest that part of the mechanism used by both of these cytokines involves a process that regulates transcription factor access to the regulatory regions.
Subject(s)
Chemokine CCL2/genetics , Platelet-Derived Growth Factor/pharmacology , Promoter Regions, Genetic , Sp1 Transcription Factor/metabolism , Tretinoin/pharmacology , 3T3 Cells , Animals , Becaplermin , Binding Sites , DNA-Binding Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Fibroblasts/drug effects , Fibroblasts/metabolism , Mice , Proto-Oncogene Proteins c-sis , Sp3 Transcription Factor , Transcription Factors/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Zinc FingersABSTRACT
TNF-alpha transcriptionally regulates murine monocyte chemoattractant protein-1 (MCP-1) expression. Three approaches were used to determine the mechanism by which TNF regulates MCP-1. Mutation analysis showed that two distal kappa B sites, a novel dimethylsulfate-hypersensitive sequence, and a promoter proximal SP-1 site were required for TNF induction. Although the kappa B sites and the hypersensitive sequence function as a NF-kappa B-mediated enhancer, regulating induction by TNF, stereospecific alignment of the kappa B sites was not critical. Trans-activation studies conducted by cotransfection of p50 and/or p65 expression vectors with MCP-1 constructions showed that TNF regulates MCP-1 through NF-kappa B. Examination of MCP-1 induction in NF-kappa B-disrupted embryonic fibroblasts showed that p65 was necessary for both the induction and the TNF-induced protein occupancy of the enhancer in vivo. The action of the antioxidant inhibitor of NF-kappa B activation, pyrrolidine dithiocarbamate, in wild-type and NF-kappa B mutant cells was examined. The results suggested that TNF activates NF-kappa B through both pyrrolidine dithiocarbamate-sensitive and -insensitive mechanisms. This study illustrates the crucial role for NF-kappa B p65 in the induction of the MCP-1 gene by TNF and in the assembly of a NF-kappa B dependent enhancer in vivo.
