Transactivation of a DR-1 PPRE by a human constitutive androstane receptor variant expressed from internal protein translation start sites

Downstream in-frame start codons produce amino-terminal-truncated human constitutive androstane receptor protein isoforms (ΔNCARs). The ΔNCARs are expressed in liver and in vitro cell systems following translation from in-frame methionine AUG start codons at positions 76, 80, 125, 128, 168 and 265 within the full-length CAR mRNA. The resulting CAR proteins lack the N-terminal DNA-binding domain (DBD) of the receptor, yielding ΔNCAR variants with unique biological function. Although the ΔNCARs maintain full retinoid X receptor alpha (RXRα) heterodimerization capacity, the ΔNCARs are inactive on classical CAR-inducible direct repeat (DR)-4 elements, yet efficiently transactivate a DR-1 element derived from the endogenous PPAR-inducible acyl-CoA oxidase gene promoter. RXRα heterodimerization with CAR1, CAR76 and CAR80 isoforms is necessary for the DR-1 PPRE activation, a function that exhibits absolute dependence on both the respective RXRα DBD and CAR activation (AF)-2 domains, but not the AF-1 or AF-2 domain of RXRα, nor CAR's DBD. A new model of CAR DBD-independent transactivation is proposed, such that in the context of a DR-1 peroxisome proliferator-activated response element, only the RXRα portion of the CAR-RXRα heterodimer binds directly to DNA, with the AF-2 domain of tethered CAR mediating transcriptional activation of the receptor complex

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

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

Transcription coactivator peroxisome proliferator-activated receptor-binding protein/mediator 1 deficiency abrogates acetaminophen hepatotoxicity

Peroxisome proliferator-activated receptor-binding protein (PBP), also known as thyroid hormone receptor-associated protein 220/vitamin D receptor-interacting protein 205/mediator 1, an anchor for multisubunit mediator transcription complex, functions as a transcription coactivator for nuclear receptors. Disruption of the PBP gene results in embryonic lethality around embryonic day 11.5 by affecting placental and multiorgan development. Here, we report that targeted deletion of PBP in liver parenchymal cells (PBP(Liv-/-)) results in the abrogation of hypertrophic and hyperplastic influences in liver mediated by constitutive androstane receptor (CAR) ligands phenobarbital (PB) and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, and of acetaminophen-induced hepatotoxicity. CAR interacts with the two nuclear receptor-interacting LXXLL (L, leucine; X, any amino acid) motifs in PBP in a ligand-dependent manner. We also show that PBP interacts with the C-terminal portion of CAR, suggesting that PBP is involved in the regulation of CAR function. Although the full-length PBP only minimally increased CAR transcriptional activity, a truncated form of PBP (amino acids 487-735) functioned as a dominant negative repressor, establishing that PBP functions as a coactivator for CAR. A reduction in CAR mRNA and protein level observed in PBP(Liv-/-) mouse liver suggests that PBP may regulate hepatic CAR expression. PBP-deficient hepatocytes in liver failed to reveal PB-dependent translocation of CAR to the nucleus. Adenoviral reconstitution of PBP in PBP(Liv-/-) mouse livers restored PB-mediated nuclear translocation of CAR as well as inducibility of CYP1A2, CYP2B10, CYP3A11, and CYP7A1 expression. We conclude that transcription coactivator PBP/TRAP220/MED1 is involved in the regulation of hepatic CAR function and that PBP deficiency in liver abrogates acetaminophen hepatotoxicity