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Transcriptional activation of the macrophage-colony stimulating factor gene by minimally modified LDL. Involvement of
T B Rajavashisth1, H Yamada, N K Mishra
1Department of Medicine, Harbor-UCLA Medical Center, Torrance 90502-2064, USA.
Abstract:
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 micrograms/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 runon 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Minimally modified LDL (MM-LDL) increases macrophage-colony stimulating factor (M-CSF) mRNA in smooth muscle cells. A specific promoter region containing an NF-kappa B binding site is crucial for this MM-LDL-induced M-CSF gene transcription.
Area of Science:
- Cardiovascular Biology
- Molecular Biology
- Atherosclerosis Research
Background:
- Minimally modified LDL (MM-LDL) induces macrophage-colony stimulating factor (M-CSF) in endothelial cells.
- The response of other cell types to MM-LDL and the underlying mechanisms are not fully understood.
Purpose of the Study:
- To investigate the effect of MM-LDL on M-CSF mRNA expression in mouse L-cells and human aortic smooth muscle cells.
- To elucidate the molecular mechanisms by which MM-LDL upregulates M-CSF gene transcription.
Main Methods:
- Dose-dependent analysis of M-CSF mRNA levels in response to MM-LDL.
- Nuclear run-on assays to assess M-CSF gene transcription.
- Reporter gene assays using chimeric M-CSF promoter constructs in transfected L-cells.
Main Results:
- Both L-cells and human aortic smooth muscle cells exhibited dose-dependent increases in M-CSF mRNA upon MM-LDL treatment.
- MM-LDL activated M-CSF gene transcription.
- A promoter region from -406 to -344 of the human M-CSF gene, containing an NF-kappa B binding site, was essential for MM-LDL-mediated transcriptional activation.
Conclusions:
- MM-LDL upregulates M-CSF mRNA expression in various cell types, including smooth muscle cells.
- The human M-CSF gene promoter, specifically the region containing an NF-kappa B binding site, mediates the transcriptional response to MM-LDL.