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Regulation of rat CYP2B1 gene expression: The role of nuclear receptors

Muangmoonchai, Roongsiri; (2000) Regulation of rat CYP2B1 gene expression: The role of nuclear receptors. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Cytochrome P450 (CYP) plays a central role in the metabolism of endogenous and exogenous compounds. This thesis aims to investigate the molecular mechanism which underlies CYP2B1 induction in response to xenobiotics, particularly phenobarbital (PB), by characterisation of the phenobarbital-responsive element (PBRE) of the CYP2B1 gene and identification of nuclear regulatory proteins, which bind to the PBRE. In vivo transfection studies by biolistic particle delivery, showed that the 5'-upstream region between -2301 to -2142 (159 bp) conferred PB responsiveness on a reporter gene. Within this region, there are two nuclear receptor binding sites; NR1 and NR2, flanking a nuclear factor-1 binding site. Gel mobility shift assays showed the heterodimerisation of constitutive androstane receptor-β (CAR-β) and retinoid X receptor α (RXRα) onto the NR1 site, but not the NR2 site. Cotransfection of the expression vector for CAR-β, with a CYP2B1 PBRE-Luc reporter construct confirmed both constitutive and xenobiotic-mediated transactivation of reporter gene expression in transfected liver and primary hepatocytes. However, HepG2, Hela, and CV-1 cell lines support only constitutive activation of gene expression by CAR-β. The co-activator SRC-1 enhances both constitutive and xenobiotic-mediated transactivation of a CYP2B1 PBRE-Luc reporter gene via CAR-β in primary hepatocytes. The SRC-1 stimulation of CAR-β transactivation is not observed when a reporter construct containing only the NR1 site was used. The regulation of the CYP2B1 gene in response to xenobiotics is therefore mediated by a nuclear receptor mechanism and involves the interaction between several transcription factors and co-activators. PB and pregnenolone 16α-carbonitrile (PCN) increase the expression of both CYP2B1 and CYP3A1 genes. PCN-induction of CYP3A1 is mediated by a pregnane X receptor (PXR). Both CAR-β and PXR were shown to bind to and transactivate via the same DNA element, either the CYP2B1 NR1 or the CYP3A1 PXRE, in primary hepatocytes and in vivo. Thus, the versatility of these promiscuous nuclear receptors in regulating of more than one CYP may enable an organism to efficiently respond to xenobiotics.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Regulation of rat CYP2B1 gene expression: The role of nuclear receptors
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; CYP2B1; Nuclear receptors
URI: https://discovery.ucl.ac.uk/id/eprint/10102738
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