Turnover of messenger RNA, apoprotein and haem of cytochrome <i>P</i>-450b + e induced by phenobarbitone in rat liver

Ravishankar, H. M.; Padmanaban, Govindarajan

doi:10.1042/bj2290073

Cited by 13 publications

(7 citation statements)

References 34 publications

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“…added P-450b/e gene fragment, and the result correlates well with changes in P-450b/e mRNA levels in vivo and run-on transcription rates in vitro reported earlier (8,10). Because transcription of the added gene fragment should involve fresh initiation, the upstream sequence (from position -179) probably has sites for interaction with protein factors modulating transcription of P450b/e genes.…”

Section: Resultssupporting

confidence: 64%

“…(ii) Effects of these inhibitors can be counteracted by exogenous hemin. (iii) Changes in the mRNA levels in vivo correlate with their run-on transcription rates with nuclei isolated from treated rat livers (7)(8)(9)(10). In addition, we have also shown that cycloheximide, a translation inhibitor, specifically blocks the transcriptional activation of P-450b/e and -c/d genes by phenobarbitone and 3-MC, respectively (11).…”

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confidence: 99%

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Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor.

Rangarajan

Padmanaban

1989

Proc. Natl. Acad. Sci. U.S.A.

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The cloned DNA fragment of the cytochrome P-450b/e gene containing the upstream region from position -179 through part of the first exon is faithfully transcribed in freeze-thawed rat liver nuclei. Phenobarbitone treatment of the animal strikingly increases this transcription, and the increase is blocked by cycloheximide (protein synthesis inhibitor) or CoCl2 (heme biosynthetic inhibitor) treatment of animals. This picture correlates very well with the reported cytochrome P-450b/e mRNA levels in vivo and run-on transcription rates in vitro under these conditions. The upstream region (from position -179) was assessed for protein binding with nuclear extracts by nitrocellulose filter binding, gel retardation, DNase I treatment ("footprinting"), and Western blot analysis. Phenobarbitone treatment dramatically increases protein binding to the upstream region, an increase once again blocked by cycloheximide or CoCl2 treatments. Addition of heme in vitro to heme-deficient nuclei and nuclear extracts restores the induced levels of transcription and protein binding to the upstream fragment, respectively. Thus, drugmediated synthesis and heme-modulated binding of a transcription factor(s) appear involved in the transcriptional activation of the cytochrome P-450b/e genes, and an 85-kDa protein may be a major factor in this regard.

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Section: Resultssupporting

confidence: 64%

mentioning

confidence: 99%

Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor.

Rangarajan

Padmanaban

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…Earlier studies from this laboratory have shown that CoCl 2 treatment leads to a striking decrease in the heme pool (13)(14)(15)(16). The present study has also made use of this reagent to precipitate heme deficiency.…”

Section: Resultsmentioning

confidence: 97%

“…Earlier studies from this laboratory have also implicated that heme, the prosthetic group of cytochrome P-450, is a positive modulator of CYP2B1/B2 gene transcription (13)(14)(15)(16)). This conclusion is based on the fact that inhibitors of heme biosynthesis block induction of CYP2B1/B2 mRNA by PB and its run-on transcription in isolated nuclei.…”

mentioning

confidence: 99%

A 65-kDa Protein Mediates the Positive Role of Heme in Regulating the Transcription of CYP2B1/B2 Gene in Rat Liver

Sultana

Nirodi

Ram

et al. 1997

Journal of Biological Chemistry

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Heme deficiency precipitated by CoCl 2 administration to rats leads to a striking decrease in the inducibility of CYP2B1/B2 mRNA levels and its transcription by phenobarbitone (PB), besides decreasing the basal levels. Exogenous hemin administration counteracts the effects of CoCl 2 administration. The binding of nuclear proteins to labeled positive cis-acting element (؊69 to ؊98 nucleotides) in the near 5-upstream region of the gene is inhibited by CoCl 2 administration to saline or PB-treated rats, as assessed in gel shift assays. Administration of exogenous hemin to the animal or addition in vitro to the extracts is able to overcome the effects of CoCl 2 treatment. The protein mediating this effect has been purified from CoCl 2 administered nuclear extracts by heparin-agarose, positive element oligonucleotide affinity, and heme affinity column chromatography. This 65-kDa protein manifests very little binding to the positive element, but in the presence of certain other nuclear proteins, shows a strong heme-responsive binding. The purified protein binds heme. It is also able to stimulate transcription of a minigene construct of the CYP2B1/B2 gene containing ؊179 nucleotides of the 5-upstream region and the I exon in a cell-free system, manifesting heme response. It is concluded that the 65-kDa protein mediates the constitutive requirement of heme for the transcription of CYP2B1/B2 gene.The transcriptional regulation of the cytochrome P-450 (CYP) supergene family is of considerable interest. The CYP1A1 gene of rat liver, induced by the prototype drug 3-methylcholanthrene, has been studied in detail. It involves interaction of the ligand, 3-methylcholanthrene, with the Ah receptor, translocation of the receptor to the nucleus, and interaction with specific upstream elements (1, 2). The details regarding the mechanism of transcriptional activation of the CYP2B1/B2 gene (2B1 and B2 are 97% homologous and hence treated as a unit) by the prototype drug, phenobarbitone (PB), 1 are beginning to emerge. Studies in this laboratory have led to the identification of a positive cis-acting element (Ϫ69 to Ϫ98 nt) and a negative cis-acting element (Ϫ126 to Ϫ160 nt) in the near 5Ј-upstream region of the CYP2B1/B2 gene (3-5). The positive element includes the 17-base pair PB-responsive consensus element, referred to as Barbie Box, identified first by Fulco and co-workers (6 -8) in Bacillus megaterium, rat, mice, and other organisms of barbiturate inducible cytochrome P-450 and other proteins. There have also been other PB-responsive sequences identified in the CYP2B1/B2 gene of the rat. Shephard et al. (9) have identified two sequences, located between Ϫ183 to Ϫ199 nt and Ϫ31 to Ϫ72 nt, to be PB-responsive, although these do not include the "Barbie" elements. In addition, Trottier et al. (10) have located a sequence between Ϫ2155 and Ϫ2318 nt to be PB-responsive and Ramsden et al. (11) have identified an upstream enhancer as far as Ϫ20 kilobases. Studies in this laboratory have led to the development of a model which propose...

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“…5 A shows that only PB induced these mRNA. Maximal induction with PB was attained by [16][17][18][19][20][21][22][23][24] h, as was previously…”

Section: Quantitative Assessmentmentioning

confidence: 99%

Demonstration by in situ hybridization of the zonal modulation of rat liver cytochrome P-450b and P-450e gene expression after phenobarbital.

Wojcik¹,

Dvorak²,

Chianale³

et al. 1988

J. Clin. Invest.

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The various physiological processes that constitute liver function are compartmentalized within the hepatic acinus. The molecular mechanisms modulating the development and maintenance of this hepatocyte heterogeneity have not been defined. The objective of this study was to determine whether transcriptional or posttranscriptional zonal modulation of cytochromes P450b,e gene expression was responsible for the heterogeneous induction of the P450 proteins, which is observed after phenobarbital (PB) administration. The exact localization in liver tissue of hepatocytes responding to PB with induction of either P450b,e mRNA or proteins was established by in situ hybridization and by immunofluorescence, respectively. As demonstrated by quantitative assessment of autoradiographs of -20 hepatocytes located between a terminal portal venule and a hepatic venule, PB induced the P450b,e mRNA up to sixfold in the 12-15 hepatocytes located closer to the hepatic venules (zones 2 and 3). In contrast, there was only a twofold induction in the 4-6 hepatocytes surrounding the terminal portal venules (zone 1). Quantitative immunofluorescence using an MAb showed that the acinar distribution of PB-induced P450b,e proteins was similar to that of the mRNA. This combined approach indicated that, most likely, an increased rate of transcription of cytochromes P450b,e genes in hepatocytes of zones 2 and 3 concomitantly, with a relative lack of activation, or repression, of these genes in hepatocytes of zone 1, were responsible for the heterogeneous phenotype observed after PB administration. Therefore, modulation of gene expression among hepatocytes of the liver acinus is one mechanism by which the functional heterogeneity of hepatocytes is attained. Experiments in which the induction of cytochromes P450b,e genes was studied after administration of either PB or para-hydroxyphenobarbital, a main hepatic metabolite of PB, suggested that the species involved in the inductive process is the parent PB molecule rather than para-hydroxyphenobarbital.

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Turnover of messenger RNA, apoprotein and haem of cytochrome P-450b + e induced by phenobarbitone in rat liver

Cited by 13 publications

References 34 publications

Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor.

Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor.

A 65-kDa Protein Mediates the Positive Role of Heme in Regulating the Transcription of CYP2B1/B2 Gene in Rat Liver

Demonstration by in situ hybridization of the zonal modulation of rat liver cytochrome P-450b and P-450e gene expression after phenobarbital.

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