An Assay for Transforming Growth Factor-Beta Using Cells Transfected With a Plasminogen Activator inhibitor-1 Promoter-Luciferase Construct

Anal Biochem. 1994 Feb 1;216(2):276-84. doi: 10.1006/abio.1994.1042.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • CHO Cells / physiology
  • Cricetinae
  • Epithelial Cells
  • Epithelium / enzymology
  • Epithelium / physiology
  • Growth Substances / pharmacology
  • Guinea Pigs
  • HeLa Cells
  • Humans
  • Luciferases / genetics*
  • Luciferases / metabolism
  • Lung / cytology
  • Lung / enzymology
  • Lung / physiology
  • Mink
  • Plasminogen Activator Inhibitor 1 / genetics*
  • Promoter Regions, Genetic / genetics*
  • Radioligand Assay
  • Recombinant Fusion Proteins / genetics
  • Sensitivity and Specificity
  • Transfection
  • Transforming Growth Factor beta / analysis*
  • Transforming Growth Factor beta / pharmacology

Substances

  • Growth Substances
  • Plasminogen Activator Inhibitor 1
  • Recombinant Fusion Proteins
  • Transforming Growth Factor beta
  • Luciferases