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

Abstract

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...

Cytosolic persistence of mouse brain CYP1A1 in chronic heme deficiency

“…The mechanism by which impaired heme synthesis decreases CYP expression is not completely understood and is still the subject of some controversy. Several reports demonstrate that heme stabilizes the mRNA of target genes and is therefore required for effective gene transcription, e.g., for CYP1A1, CYP2B1/2 and HO-1 (Kloepper-Sams and Stegeman, 1994;Sultana et al, 1997;Alam et al, 2003). Consistent with these studies, we speculate that heme deficiency may lead to impaired CYP1A1 mRNA stability, which results in reduced gene transcription.…”

Expression and Function of Cytochrome P450 in Brain Drug Metabolism

“…We believe that the heme effect is isoform-specific. Besides CYP1A1, several other P450 isoforms are affected by the availability of heme, like CYP2A5, CYP2B1/2 and HO-1 [54,[56][57][58]. However, CYP2B10, and other hemoproteins, namely neuronal nitric oxide synthase and soluble guanylate cyclase, are reported to function normally with limited heme [54].…”

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats

“…It was reported that the heme moiety stimulated the transcription of the CYP2B1/2B2 gene, and that an unknown 65-kDa protein mediated the role of heme in this stimulation of CYP2B1/2B2 gene transcription. 22) At present, we cannot ignore the possibility that ascorbic acid deficiency might suppress the transcription of the CYP2B1/2B2 gene in liver by reducing cellular heme content. As still another possibility, the mechanism that ascorbic acid deficiency induce HO-1 gene expression might also cause the repression of CYP2B1/2B2 expression.…”