RESUMEN
In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4alpha. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism.
Asunto(s)
Cumestrol/farmacología , Proteínas de Unión al ADN/agonistas , Glucosa/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Fitoestrógenos/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas , Transcripción Genética/efectos de los fármacos , Apolipoproteínas B/metabolismo , Línea Celular , Proteínas de Unión al ADN/metabolismo , Humanos , Ligandos , Metabolismo de los Lípidos/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Assessment of the risk of human exposure to man-made chemicals that bind to hormone receptors has emerged as a major public health issue. Among hormone receptors, nuclear receptors tend to be targets of xenobiotics because their endogenous ligands are small, fat-soluble molecules. Nuclear receptors are ligand-inducible transcriptional factors and regulate the transcriptional activity of various target genes. At the start of the initiation step of transcription, nuclear receptors interact with coactivators (TIF2, SRC1, ACTR, CBP/p300, etc.) in an agonist-dependent manner. Using the interaction of the nuclear receptor with a coactivator, we have developed a novel rapid ligand in vitro screening method that is easy to use and has high sensitivity. This method, called by us the CoA-BAP system, is applicable to most nuclear receptors and is suitable for high-throughput screening because the entire experimental operation can be carried out on a microplate. We used human TIF2 as a coactivator including LXXLL motifs expressed in Escherichia coli as a fusion protein with BAP and nuclear receptor LBD expressed in E. coli as a fusion protein with GST. On a GSH-coupled microplate these proteins were incubated with chemicals and the protein-protein interactions were detected as alkaline phosphatase activity. To date we have examined seven nuclear receptors (ERalpha/beta, TRalpha, RARalpha/gamma, RXRalpha,and VDR) and confirmed that the method works well.