An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct

摘要

Bone morphogenetic proteins (BMPs) control cell fate by regulating gene expression, especially inhibitor of differentiation (Id) genes. This property has been exploited to create a highly sensitive assay for quantification of active BMP. Embryonic mouse cells (C3H10T1/2) were stably transfected with an expression construct (BRE-Luc) containing a BMP-responsive element fused to the firefly luciferase reporter gene. BRE results from a multimerization of distinct sequences elements from a mouse Id1 promoter [15]. The addition of BMP2 (0.5-100 ng/ml) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. This new assay was 100-fold more sensitive than the classical alkaline phosphatase (ALP) activity assay (0.5-1 vs. 50-100ng/ml, respectively) as well as much more rapid (24 h vs. 3-6 days, respectively, of BM P treatment). This new assay is specific to BMPs (BMP-2, BMP-4, and BMP7) as evidenced by its relative insensitivity to TGF beta 1, bFGF, and VEGF. Because of its BMP specificity, this rapid, sensitive, nonradioactive, and easily performed assay could be used in monitoring the biological activity of BMP and, eventually, as a cell-based screening assay to identify and evaluate molecules that modulate BMP signaling in cells. (c) 2005 Elsevier Inc. All rights reserved.