title: Apparent coactivation due to interference of expression constructs with nuclear receptor expression.
abstract: Transient cotransfection in COS-7 cells, a standard approach to demonstrate coactivation, was used to study the coactivation properties of NuRIP183, a new nuclear receptor interacting protein of 183 kDa, isolated by a yeast two-hybrid screening. Transfection with a NuRIP183 expression construct strongly increased the ligand-dependent response of reporter constructs for several nuclear receptors when compared to transfection with the empty expression vector. A more detailed study, however, revealed major changes in the expression level of the nuclear receptors in cotransfection experiments, indicating that the observed changes in receptor activity were not due to coactivation but to differences in receptor concentration caused by interference from the cotransfected expression constructs with the expression of the receptor. Such interference, which is inversely related to the length of the insert, was observed, not only in COS-7 cells but also in CV-1 and MCF-7 cells, using different transfection techniques (FuGENE-6 and calcium phosphate) and different expression vectors (pSG5, pcDNA1.1 and pIRESneo). These data cast some doubt on coactivation of nuclear receptors based on similar cotransfection experiments without measurement of receptor concentration. Moreover, it is recommended to limit the amounts of (co)transfected expression plasmid and to avoid the use of empty expression plasmid as a control. Finally, one should be aware of similar misleading results in other experimental set-ups based on cotransfection.
authors.affiliation: Laboratory for Experimental Medicine and Endocrinology, Faculty of Medicine, Catholic University of Leuven, B-3000, Leuven, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Hofman
authors.forename: J V
authors.initials: JV
authors.lastname: Swinnen
authors.forename: F
authors.initials: F
authors.lastname: Claessens
authors.forename: G
authors.initials: G
authors.lastname: Verhoeven
authors.forename: W
authors.initials: W
authors.lastname: Heyns
mesh_terms: D000818:Animals; D016477:Artifacts; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D015870:Gene Expression; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D020798:Two-Hybrid System Techniques
chemical_list: D009687:Nuclear Proteins; D011944:Receptors, Androgen; D018167:Receptors, Calcitriol; D011965:Receptors, Glucocorticoid