Analysis of signal transduction pathways in macrophages using expression vectors with CMV promoters: a cautionary tale.
The cytomegalovirus (CMV) major immediate-early promoter is a strong promoter used for both in vitro and in vivo expression of proteins in signal transduction and gene therapy studies. CMV activity is induced by external stimuli such as endotoxin from Gram-negative bacteria (LPS), TNF-alpha and phorbol esters. This inducibility poses problems when this promoter is used to drive the expression of either wild type or dominant negative mutated proteins as tools in signal transduction studies. This report draws attention to the problem associated with this widely used approach. The role of NF-kappaB and Hypoxia Inducible Factor-1alpha (HIF-1alpha) in the transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) in macrophages was investigated using CMV-promoter-driven expression of either wild type or dominant negative proteins involved in these pathways. Difficulties encountered while interpreting the data due to the inducibility of the CMV promoter by LPS are highlighted in this report and provide a cautionary note for the evaluation of data acquired using this approach.
Ramanathan
Madhuri
M
Department of Cell Biology and Molecular Medicine & The Cardiovascular Research Institute, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103, USA.
Haskó
György
G
Leibovich
Samuel Joseph
SJ
D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008070:Lipopolysaccharides; D016328:NF-kappa B; D007371:Interferon-gamma