An Adenoviral Vector System for Functional Identification of Nuclear Receptor Ligands

Abstract

A recombinant adenovirus system has been designed that confers glucocorticoid responsiveness upon infected cells in culture. Two mutually dependent viruses are required: a trans-activator virus containing the human glucocorticoid receptor transcription unit and a second receptor virus harboring a glucocorticoid response element linked to the firefly luciferase gene. Another reciprocal pair of viruses has been generated; one member expresses the rat thyroid hormone receptor alpha, while the other contains the luciferase gene regulated by a thyroid hormone-responsive DNA element. Corticosteroid- or thyroid hormone-induced transcription can be efficiently and accurately quantitated from cells coinfected with the appropriate complementary virus pair 20 h after infection in 96-well microtiter plates. This coinfection assay offers a convenient way to measure transcriptional activation by nuclear receptors and has certain key advantages over the commonly used cotransfection method. Its sensitivity and precision make it a practical approach to rapidly identify substances extracted from complex biological samples activating candidate "orphan" nuclear receptor molecules.

Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.

“…That is, the intracellular concentrations of ligands must be within the ranges of receptor binding affinities and activation potencies. For example, T3 and T4 circulate in human plasma at 2 and 100 nM, respectively (Scully et al, 1980), but T4 activates the thyroid hormone receptor with a 50-fold reduced potency, as compared with T3 (Shih et al, 1991). Consequently, a low-affinity receptor ligand such as T4 should not be dismissed as nonphysiological on the basis of potency alone, provided that its total effector activity is similar to that of a less abundant but more active one like T3.…”

“…Circulating levels of steroid and thyroid hormones, retinoids, and vitamin D in animal sera are within the ranges of their receptor-activation potencies. Therefore, orphan receptor activators have been sought from biological tissues such as plasma (Shih et al, 1991;Banner et al, 1993) as well as by a survey of chemical compounds. Chromatographic separation methods can be used to compare the candidate chemically synthesized receptor activators with those extracted from biological tissues to assess their physiological significance.…”

Expression of the human androgen receptor in eukaryotic cells using a recombinant adenovirus vector yields high levels of the soluble, functional receptor protein

“…Function of the AR expressed in CV1 cells following infection with the Ad His hAR adenovirus. Cells were infected with varying MOIs of the recombinant adenovirus encoding the hAR and a constant quantity (MOI 250) of a second adenovirus carrying the MMTV luciferase reporter cassette (Shih et al 1991). Twenty-four hours following infection, the medium was removed and replaced with medium containing 5% charcoal-stripped serum containing either no hormone or mibolerone.…”

“…The incubations were continued for an additional 48 h. At the time, the cells were lysed and assayed to determine the levels of luciferase activity. The means the standard deviations of three independent measurements are presented (Shih et al 1991). Twenty-four hours following infection, the medium was removed and replaced with medium containing 5% charcoal-stripped serum containing either no hormone or 2 nM Mb.…”

“…CV1 cells were co-infected with the Ad His hAR and MMTV luciferase (Shih et al 1991) adenoviruses in various proportions. Twenty-four hours following infection, the medium was removed and replaced with fresh medium containing 5% charcoal-stripped serum containing either no hormone or 2 nM Mb.…”