TRANSCRIPTIONAL ACTIVATION OF THE MACROPHAGE-COLONY-STIMULATING FACTOR GENE BY MINIMALLY MODIFIED LDL - INVOLVEMENT OF NUCLEAR FACTOR-KAPPA-B

Minimally modified LDL (MM-LDL), obtained by mild iron oxidation or prolonged storage at 4 degrees C, has been shown to induce the expression of macrophage-colony stimulating factor (M-CSF) in cultured aortic endothelial cells. To examine whether other cell types also respond to MM-LDL, we investigated its effect on the expression of M-CSF mRNA in mouse L-cells and human aortic smooth muscle cells. Both L-cells and human aortic smooth muscle cells showed increased levels of M-CSF mRNA in response to 10 to 200 mu g/mL MM-LDL in a dose-dependent manner. This allowed us to use mouse L-cells as a model to study the mechanism involved in MM-LDL-mediated increase in M-CSF mRNA. Nuclear run-on assays showed that M-CSF gene transcription was activated by MM-LDL. In the present study, we identified specific elements that conferred MM-LDL-mediated transcriptional activation of the human M-CSF gene. Chimeric constructs containing sequential deletions in the 5'-promoter region of the M-CSF gene linked to a reporter chloramphenicol acetyltransferase (CAT) gene were transfected into mouse L-cells. The human M-CSF promoter region extending upstream from the transcription start site to nucleotide -406 showed maximum induction of CAT activity by MM-LDL. Induction of CAT activity was drastically reduced, with a deletion plasmid lacking the promoter region -406 to -344. A functional nuclear factor (NF)-kappa B binding site present in this critical region was required for MM-LDL-mediated induction of CAT activity since an internal deletion construct lacking this element showed significant loss of transcriptional activation. Similar results also were obtained with the use of bovine aortic endothelial cells, suggesting that part of the mechanism is shared in different cell types. Gel shift assays with bovine aortic endothelial cell nuclear extracts revealed that this element binds to MM-LDL-inducible nuclear protein(s) that exhibited DNA binding specificity of NF-kappa B and cross-reacted to NF-kappa B-specific antibodies. Taken together, these results are consistent with the involvement of NF-kappa B in the transcriptional activation of the human M-CSF gene by MM-LDL.