A rapid and sensitive bioassay to measure bone morphogenetic protein activity.
BACKGROUND
Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily and were originally identified as proteins that induce ectopic bone formation. BMPs were shown subsequently to be involved in several biological processes during development and in adult tissues through the regulation of the growth, differentiation and apoptosis of various cell types. An alkaline phosphatase (ALP)-based assay is the most widely used assay to evaluate BMP activity. However, the ALP assay is not rapid and not sensitive enough to measure BMP activity at physiological concentrations. In this paper, we describe a highly sensitive, rapid, and specific cell-based assay for the quantification of BMP activity.


RESULTS
Two cells lines, C2C12 and HepG2 were stably transfected with a reporter plasmid consisting of BMP-responsive elements from the Id1 promoter fused to a luciferase reporter gene. Exposure of cells containing this construct to BMPs induces the expression of luciferase, which can be quantified with a luminometer. The bioassay is specific for BMPs and can detect BMP-4 activity at a concentration as low as 3 pM. Related family members, such as TGF-beta1, TGF-beta2 and TGF-beta3, do not induce the reporter gene.


CONCLUSIONS
The assay is rapid (less than 24 hours) and can be used, as described in this paper, to measure BMP activity in complex solutions and in cell culture in a simple and efficient way.
Zilberberg
Lior
L
Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA. zilbel01@med.nyu.edu
ten Dijke
Peter
P
Sakai
Lynn Y
LY
Rifkin
Daniel B
DB
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D052004:Bone Morphogenetic Protein Receptors / Q000378:metabolism*; D019485:Bone Morphogenetic Proteins / Q000032:analysis* / Q000378:metabolism*; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000737:chemistry; D008163:Luminescent Measurements; D051379:Mice; D032446:Myoblasts / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000502:physiology; D051785:Smad Proteins / Q000737:chemistry; D015534:Trans-Activators
D019485:Bone Morphogenetic Proteins; D003470:Culture Media; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051785:Smad Proteins; D015534:Trans-Activators; D008156:Luciferases; D052004:Bone Morphogenetic Protein Receptors