Browsing School, Graduate by Subject "5' UTR"
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Identification and characterization of tissue-specific alternative promoter usage for Bcrp1/Abcg2 in mice for establishing a murine in vivo model of dietary transcriptional regulation of human small intestinal BCRP/ABCG2 expressionOral bioavailability of certain drugs may be limited by the expression of the efflux transporter BCRP/ABCG2 (Breast Cancer Resistance Protein/ ATP–Binding Casette Transporter G2) on the apical surface of the human small intestine. So far, the factors regulating BCRP expression in the human intestine have not been identified. Based on the presence of an estrogen response element (ERE) in the BCRP promoter coupled with previous reports for ERα expression in the human intestine I screened dietary estrogens for their potential regulation of BCRP expression. I identified that genistein, a dietary estrogen, upregulates BCRP expression in the exogenously ERα expressing ERα (−) cell line MDA–MB–231. To support in vitro studies with a suitable in vivo model, I simultaneously characterized Bcrp1 expression as well as regulation along the mouse intestine. Initially I in silico characterized alternative promoter usage for Bcrp1 in mice, identifying four first exons (72kb, 58kb, 15kb and 10kb) upstream of Bcrp1 translational start site which I designated as E1u, E1a, E1b and E1c respectively. Using real time quantitative PCR, I confirmed the predominant expression of the E1b Bcrp1 mRNA in all regions of the mouse small intestine. Next, using luciferase reporter assays, I established that the promoter upstream of Bcrp1 E1b first exon is a functional promoter. The core promoter upstream of E1b spans −231/−42 bps in relation to its transcriptional start site. However, the absence of an ERE in the mouse E1b promoter suggests that mouse models might not be suitable for studying dietary estrogenic regulation of human intestinal BCRP expression. Since BCRP regulation of drug disposition is not limited to the intestine, I simultaneously characterized tissue-specific Bcrp1 expression as well as regulation. I identified E1c as the major Bcrp1 mRNA isoform expressed in mouse kidney and liver, the tissues with high Bcrp1 protein expression. Additionally I for the first time identified the predominant expression of the E1u Bcrp1 mRNA isoform along with the expression of a novel truncated Bcrp1 protein in the mouse testis. In conclusion, by identifying predominant expression of E1b and E1u Bcrp1 mRNA isoforms in the mouse small intestine and mouse testis respectively, I have confirmed tissue–specific transcriptional regulation of Bcrp1 expression.