An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.
Transforming growth factor-beta (TGF-beta) is a potent regulator of cellular differentiation, proliferation, migration, and protein expression. These properties have been exploited to create a variety of bioassays for detecting the mature growth factor. In this paper, we describe a highly sensitive and specific, nonradioactive quantitative bioassay for TGF-beta based on its ability to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mink lung epithelial cells (MLEC) were stably transfected with an expression construct containing a truncated PAI-1 promoter fused to the firefly luciferase reporter gene. Addition of TGF-beta (0.2 to > 30 pM) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. Although responsive to TGF-beta, this promoter fragment was only minimally influenced by other known inducers of PAI-1 expression. When compared to the widely used MLEC assay, this assay demonstrated greater sensitivity and specificity, allowing quantification of TGF-beta in complex biological solutions.
Abe
M
M
Department of Cell Biology, New York University Medical Center, New York.
Harpel
J G
JG
Metz
C N
CN
Nunes
I
I
Loskutoff
D J
DJ
Rifkin
D B
DB
D000818:Animals; D016466:CHO Cells / Q000502:physiology; D006224:Cricetinae; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology / Q000502:physiology; D006133:Growth Substances / Q000494:pharmacology; D006168:Guinea Pigs; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000201:enzymology / Q000502:physiology; D008907:Mink; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000032:analysis* / Q000494:pharmacology
D006133:Growth Substances; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D008156:Luciferases