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

University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Nucleic Acids Research (Impact Factor: 9.11). 02/2007; 35(7):2177-90. DOI: 10.1093/nar/gkm090
Source: PubMed


Downstream in-frame start codons produce amino-terminal-truncated human constitutive androstane receptor protein isoforms (DeltaNCARs). The DeltaNCARs 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 DeltaNCAR variants with unique biological function. Although the DeltaNCARs maintain full retinoid X receptor alpha (RXRalpha) heterodimerization capacity, the DeltaNCARs 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. RXRalpha 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 RXRalpha DBD and CAR activation (AF)-2 domains, but not the AF-1 or AF-2 domain of RXRalpha, 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 RXRalpha portion of the CAR-RXRalpha heterodimer binds directly to DNA, with the AF-2 domain of tethered CAR mediating transcriptional activation of the receptor complex.

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    • "Furthermore, CAR-RXR heterodimers from mice and humans can bind and trans-activate at a PPAR-RXR DNA–binding site, and in humans, this transactivation does not require the CAR DNA–binding domain (Guo et al., 2007; Stoner et al., 2007). Further work is required to clarify the functional significance of PPARa and CAR antagonistic effects. "
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    ABSTRACT: Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor (PPAR) alpha. Recent studies indicate that the plasticizer di-(2-ethylhexyl) phthalate (DEHP) increased the incidence of liver tumors in PPARalpha-null mice. We hypothesized that some PPC, including DEHP, induce transcriptional changes independent of PPARalpha but dependent on other nuclear receptors, including the constitutive-activated receptor (CAR) that mediates phenobarbital (PB) effects on hepatocyte growth and liver tumor induction. To determine the potential role of CAR in mediating effects of PPC, a meta-analysis was performed on transcript profiles from published studies in which rats and mice were exposed to PPC and compared the profiles to those produced by exposure to PB. Valproic acid, clofibrate, and DEHP in rat liver and DEHP in mouse liver induced genes, including Cyp2b family members that are known to be regulated by CAR. Examination of transcript changes by Affymetrix ST 1.0 arrays and reverse transcription-PCR in the livers of DEHP-treated wild-type, PPARalpha-null, and CAR-null mice demonstrated that (1) most (approximately 94%) of the transcriptional changes induced by DEHP were PPARalpha-dependent, (2) many PPARalpha-independent genes overlapped with those regulated by PB, (3) induction of genes Cyp2b10, Cyp3a11, and metallothionine-1 by DEHP was CAR dependent but PPARalpha-independent, and (4) induction of a number of genes (Cyp8b1, Gstm4, and Gstm7) was independent of both CAR and PPARalpha. Our results indicate that exposure to PPARalpha activators including DEHP leads to activation of multiple nuclear receptors in the rodent liver.
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    ABSTRACT: Activating transcription factor (ATF) 5 is a member of the ATF/cAMP response element-binding protein family, which has been associated with differentiation, proliferation, and survival in several tissues and cell types. However, its role in the liver has not yet been investigated. We show herein that ATF5 is a highly abundant liver-enriched transcription factor (LETF) whose expression declines in correlation with the level of dedifferentiation in cultured human hepatocytes and cell lines. Re-expression of ATF5 in human HepG2 cells by adenoviral transduction resulted in a marked selective up-regulation of CYP2B6. Moreover, adenoviral cotransfection of ATF5 and constitutive androstane receptor (CAR) caused an additive increase in CYP2B6 mRNA. These results were confirmed in cultured human hepatocytes, where the cooperation of ATF5 and CAR not only increased CYP2B6 basal expression but also enhanced the induced levels after phenobarbital or 6-(4-chloropheny-l)-imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Comparative sequence analysis of ATF5 and ATF4, its closest homolog, showed a large conservation of the mRNA 5'-untranslated region organization, suggesting that ATF5 might be up-regulated by stress responses through a very similar translational mechanism. To investigate this possibility, we induced endoplasmic reticulum stress by means of amino acid limitation or selective chemicals, and assessed the time course response of ATF5 and CYP2B6. We found a post-transcriptional up-regulation of ATF5 and a parallel induction of CYP2B6 mRNA. Our findings uncover a new LETF coupled to the differentiated hepatic phenotype that cooperates with CAR in the regulation of drug-metabolizing CYP2B6 in the liver. Moreover, ATF5 and its target gene CYP2B6 are induced under different stress conditions, suggesting a new potential mechanism to adapt hepatic cytochrome P450 expression to diverse endobiotic/xenobiotic harmful stress.
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