Involvement of Synthesis and Phosphorylation of Nuclear Protein Factors That Bind to the Positivecis-Acting Element in the Transcriptional Activation of the CYP2B1/B2 Gene by Phenobarbitonein Vivo

Nirodi, Chaitanya S.; Sultana, Shahana; Ram, N. V. Raghava; Prabhu, L.; Padmanaban, Govindarajan

doi:10.1006/abbi.1996.0285

Cited by 30 publications

(13 citation statements)

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“…Thus, the PB-induced dephosphorylation of CAR seems to be critical step in CYP2B induction . In support of this observation, previous reports have documented the importance of phosphorylation status in CYP2B induction (Sidhu and Omiecinski, 1995;Nirodi et al, 1996;. Importantly, nuclear translocation of mCAR in HepG2 cells is spontaneous and does not seem to be dependent on ligand binding and/or modification of phosphorylation status Sueyoshi et al, 1999).…”

supporting

confidence: 79%

Transcriptional Regulation of the Human CYP3A4Gene by the Constitutive Androstane Receptor

Goodwin

Hodgson

D'Costa

et al. 2002

Molecular Pharmacology

232

144

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Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in adult human liver, is both constitutively expressed and transcriptionally activated by a variety of structurally diverse xenochemicals. In this study, we examined the role of the constitutive androstane receptor (CAR), a member of the steroid/retinoid/ thyroid hormone receptor superfamily, in the transcriptional regulation of CYP3A4. Herein, we demonstrate that CAR is capable of trans-activating expression of the CYP3A4 gene, both in vitro and in vivo. Induction of CYP3A4 is dependent on cooperativity between elements within the promoter proximal region of the gene and the distal xenobiotic-responsive enhancer module. CAR responsiveness was shown to be primarily mediated by two high-affinity binding motifs located within the CYP3A4 gene 5Ј-flanking region, approximately 7720 and 150 bases upstream of the transcription initiation site. Importantly, the human CAR response elements also mediate trans-activation of CYP3A4 by the human pregnane X receptor, suggesting that interplay between these receptors is likely to be an important determinant of CYP3A4 expression.

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supporting

confidence: 79%

Transcriptional Regulation of the Human CYP3A4Gene by the Constitutive Androstane Receptor

Goodwin

Hodgson

D'Costa

et al. 2002

Molecular Pharmacology

232

144

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“…These findings explain why CYP2H1 mRNA is induced by OA to a certain level and that this OA induction is not changed when adding PB. Our results may explain why in the rat, OA inhibits CYP2B1/2 mRNA induction by PB but induces a CYP2B1/2 minigene containing the proximal sequence of the 5Ј-flanking region in reporter gene assays (39,49). The similarity of these data to the inhibition of the transfer of CAR to the nucleus in mouse liver suggests that a CAR-like receptor protein is involved in induction mediated by the 264-bp PBRU in the CYP2H1 5Ј-flanking region of the chicken.…”

Section: Discussionmentioning

confidence: 92%

A Conserved Nuclear Receptor Consensus Sequence (DR-4) Mediates Transcriptional Activation of the Chicken CYP2H1 Gene by Phenobarbital in a Hepatoma Cell Line

Handschin

Meyer

2000

Journal of Biological Chemistry

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Phenobarbital-responsive DNA elements were identified in the 5-flanking region of the chicken CYP2H1 gene by in reporter gene assays in a chicken hepatoma cell line (leghorn male hepatoma (LMH)). A 264-base pair (bp) enhancer sequence (phenobarbital-responsive unit (PBRU)) responded to phenobarbital and a variety of phenobarbital-type inducers. Analysis of putative transcription factor binding sites within the 264-bp element revealed a nuclear receptor half-site repeat (DR-4) neighboring a putative nuclear factor-1 site. This motif resembles phenobarbital response elements in the flanking regions of three phenobarbital-inducible genes, rat CYP2B2, mouse Cyp2b10, and human CYP2B6. Activation of the 264-bp element was eliminated after site-directed mutagenesis of the DR-4 hexamer halfsites. Evidence for evolutionary conservation of this recognition site was indicated by activation in LMH cells of a mouse Cyp2b10 phenobarbital-responsive enhancer by the same spectrum of inducers that activate the CYP2H1 264-bp PBRU. Inhibition of this activation by okadaic acid may explain the reported inhibitory effects on induction of CYP2B1/2 and Cyp2b10 by this phosphatase inhibitor. We show that this inhibition occurs directly on the 264-bp PBRU, whereas the proximal promoter of CYP2H1 is induced by okadaic acid in reporter gene assays. These experiments exploit the unique phenobarbital inducibility of the hepatoma-derived cell line LMH. The cytochrome P-450 (CYP)1 gene superfamily encodes proteins responsible for the metabolism of numerous xenobiotic and endobiotic substrates (1). In addition, the transcription of drug-metabolizing enzymes from CYP1, CYP2, and CYP3 families can be induced a variety of compounds, including substrates of these CYPs (2-7). Five different classes of prototypical inducer-drugs that activate distinct but overlapping classes of CYPs have been defined. For example, phenobarbital (PB) induces predominantly enzymes from the CYP2B, -2C, and -3A subfamilies (2, 5, 7). These CYPs, as well as more than 50 other genes, are affected by PB and a number of structurally unrelated compounds classified as PB-like inducers (8). PB induction is observed in a wide variety of species ranging from Bacillus megaterium (9) to human. In vertebrates, drug induction occurs predominantly in the liver and the intestine and to a lesser extent in other extrahepatic tissues, such as skin, kidney, lung, and brain (2, 5).Phosphorylation and dephosphorylation events, cytokines, and hormones affect enzyme induction by as yet unknown mechanisms (for a review, see Ref. 10). Until recently, little was known about the molecular mechanisms of PB-type induction and a receptor for these drugs remains elusive. A major breakthrough has been the recent observation in several laboratories that orphan nuclear receptors are involved in CYP regulation (for reviews, see Refs. 6, 11, and 12). These findings emerged from the discovery and analysis of well defined PBresponsive elements in the flanking region of rat CYP2B2 and CYP3A1 (13-16), mouse C...

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“…However, the latter as well as previous investigations of this issue all failed to provide 1) any measures of concentration effect on the induction process, 2) measure of the relative efficacy of their test inhibitors on de novo protein synthesis, or 3) use of any negative analogs. Typically, past conclusions regarding the requirement of de novo protein synthesis for the PB-induction process were made on the basis of a single in vivo injection (20,22), or single concentrations of an inhibitor in cultures of primary hepatocytes (7).…”

Section: Discussionmentioning

confidence: 99%

Protein Synthesis Inhibitors Exhibit a Nonspecific Effect on Phenobarbital-inducible Cytochome P450 Gene Expression in Primary Rat Hepatocytes

Sidhu

Omiecinski

1998

Journal of Biological Chemistry

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Previous investigations have indicated that de novo protein synthesis is a critical requirement for phenobarbital (PB) induction. We reexamined this issue in PBresponsive primary rat hepatocyte cultures using a broader array of protein synthesis inhibitors and experimental end points. Anisomycin, cycloheximide, emetine, puromycin, and puromycin aminonucleoside, a negative analog, were evaluated for their respective effects on protein synthesis and the PB-induction process. All of the inhibitors effectively repressed de novo protein synthesis in the cells in a concentration-dependent manner. However, anisomycin only minimally effected PB induction, ascertained though the measure of CYP2B1, CYP2B2, and CYP3A1 mRNA levels. The inactive agent, puromycin aminonucleoside, produced marked repression of the PB-induction response. Results from further experiments demonstrated that these protein synthesis inhibitors stimulated rapid and differential phosphorylation of the stress-activated protein kinase/c-Jun kinase (SAPK/JNK) pathway, indicating nonselective actions on cellular processes. Puromycin aminonucleoside was without effect on these pathways, despite its efficacy as an inhibitor of PB induction. These results demonstrate that de novo protein synthesis is not a requirement for PB induction, nor is activation of the SAPK/JNK kinase cascade responsible for down-regulating PB responsiveness in primary hepatocytes. Phenobarbital (PB)1 is well recognized for its pleiotropic effects on mammalian cells, including its ability to transcriptionally activate a variety of genes (1, 2). However, identification of the molecular mechanisms controlling these inductive responses has proven difficult. Certain cytochome P450s (CYP), including the rat CYP2B1 and CYP2B2 genes, are highly responsive to PB in liver hepatocytes, and provide excellent models for mechanistic studies of the induction process (1, 3). PB and other PB-like inducers do not share obvious structural homology or chemical enantioselectivity (4), as typically associated with classical receptor-mediated gene activation responses.Using a highly differentiated hepatocyte culture system, we previously provided evidence for involvement of distinct intracellular signaling pathways that act in concert to modulate PB induction. Elevation of intracellular cAMP/protein kinase A associated activity by physiological hormones, or other protein kinase A activators, completely repressed induction (5), implying a negative modulatory role for this pathway. Recently, we demonstrated that inhibition of a PP1/PP2A protein phosphatase pathway also effectively suppressed the PB-induction response (6). Thus, the latter pathway may serve as a positive signaling intermediate in the induction process. These results led us to hypothesize that a dephosphorylation event is a required trigger, upstream of PB-mediated transcriptional activation.In potential conflict with this line of reasoning, results of earlier studies have led some to conclude that another, distinct control mechanism is invol...

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Involvement of Synthesis and Phosphorylation of Nuclear Protein Factors That Bind to the Positivecis-Acting Element in the Transcriptional Activation of the CYP2B1/B2 Gene by Phenobarbitonein Vivo

Cited by 30 publications

References 0 publications

Transcriptional Regulation of the Human CYP3A4Gene by the Constitutive Androstane Receptor

Transcriptional Regulation of the Human CYP3A4Gene by the Constitutive Androstane Receptor

A Conserved Nuclear Receptor Consensus Sequence (DR-4) Mediates Transcriptional Activation of the Chicken CYP2H1 Gene by Phenobarbital in a Hepatoma Cell Line

Protein Synthesis Inhibitors Exhibit a Nonspecific Effect on Phenobarbital-inducible Cytochome P450 Gene Expression in Primary Rat Hepatocytes

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