Sex-associated expression of mouse hepatic and renal CYP2B enzymes by glucocorticoid hormones1
Introduction
The P450 genes encode a superfamily of heme-thiolate proteins responsible for the oxidative metabolism of chemically diverse compounds of both endogenous and exogenous origin [1], [2]. In some cases, the toxicity of certain xenobiotics, such as mutagens and carcinogens, is enhanced by P450-dependent metabolism. Expression of P450 is influenced by endocrine factors [3], [4], such as growth hormone, sex hormones, and glucocorticoid hormones. CYP2B1 and CYP2B2 in rats, as well as CYP2B9 and CYP2B10 in mice, are major CYP2B isoenzymes expressed constitutively and inducibly [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. Their hepatic constitutive expression is sexually dimorphic, namely male > female in rats and female > male in mice [17], [18], [19], [20]. This sexual dimorphism in rats for CYP2B1 and CYP2B2 can be explained by a sex-dependent secretion of growth hormone, i.e. expression is suppressed more in female than in male rats by growth hormone [16], [21], [22], [23], [24]. However, a previous study did not support the notion that the secretion profile of growth hormone universally explains the sex-dependent difference in mRNA expression levels [17], although hypophysectomy has been shown to increase the expression of CYP2B mRNA in male mouse liver [16]. Therefore, although the secretion profile of growth hormone in mice is the same as that in rats, this explanation is not applicable to the dimorphism observed in the case of Cyp2b9 and Cyp2b10 genes in mice.
PB, PCN, and DDT, as well as Dex, a synthetic glucocorticoid, have been shown to modulate the expression of the CYP2B subfamily in murine liver [16], [17], [18], [19], [20], [24], [25], [26]. In rats, PB induced hepatic expression of some CYP2B subfamily isoform mRNAs [8], [10], [11], [16], [19], whereas it did not evoke renal expression of these isoforms [27], [28], [29], [30]. Additionally, no comparative information about the different actions of PB on these two organs in mice was obtained. However, by using a mouse-model system, the regulatory mechanism involving PB has been identified to occur through a key regulatory factor, the liver-enriched orphan nuclear receptor CAR. CAR interacts with the PBREM, a PB-responsive element that is localized in the 5′-flanking region of the Cyp2b10 gene, and confers PB-inducible Cyp2b10 gene transcription [31], [32], [33], [34]. However, although PBREM seems to be a general PB-responsive enhancer because of its responsiveness to numerous PB-type inducers, a presumable PB receptor or a cellular molecule where PB directly interacts has yet to be identified [34]. Dex also affects CYP2B gene expression [20], and the presence of a functional glucocorticoid response element in the CYP2B2 gene that might play a role in the well-established Dex dependency of PB induction has been reported [35], [36]. However, the regulatory mechanism used by Dex appears to be complicated [19], [31], [35], [37]. One possibility is that Dex induces these isoforms by a mechanism different from that involving the classical GR pathway [19], [35], [37]. In this regard, several GREs were predicted recently in the 5′-flanking region of the rat CYP2B2 gene [35] and the mouse Cyp2b10 gene [32]. However, these findings do not explain the regulatory mechanism of Dex completely. Receptor molecules involved in the Dex regulation of liver CYP2B isoforms have not been clearly established, although other Dex-inducible P450 genes, such as CYP3A1,CYP3A2, and CYP3A23, were suggested to be mediated via the orphan nuclear receptor PXR [25], [38]. Since Dex is widely used as a clinical drug and administered concurrently with medications, information related to the Dex-inducible CYP2B subfamily is valuable. For example, it is important to delineate how sex-specific expression of Cyp2b9 and Cyp2b10 genes is regulated or modified by endocrine factors.
This study deals with the constitutive and inducible expression of Cyp2b9 and Cyp2b10 genes in mouse kidney and liver, as well as in cultured hepatocytes. Dex induced only CYP2B10 in the kidneys of both sexes, although the level of induction was higher in males than females. In contrast to Dex, PB and compounds know to mimic PB (e.g. DDT and PCN) did not induce the expression of the CYP2B gene in kidneys, but did induce both CYP2B9 and CYP2B10 genes in liver independent of gender. These observations suggest that the mechanism by which mouse CYP2B expression is regulated by Dex differs somewhat from that of the other inducers.
Section snippets
Chemicals
Materials for culturing hepatocytes were purchased from ICN Biomedicals Inc., Collaborative Research Inc., and Kyokuto Seiyaku. Percoll and collagenase (Type I) were products of Pharmacia Biotech AB and the Sigma Chemical Co., respectively. A partial cDNA clone of mouse CYP2B10 was a gift from Dr. M. Negishi, NIEHS. Restriction endonucleases, an RNA PCR kit version 2.1, and T4 polynucleotide kinase were obtained from TaKaRa Biomedicals. [α-32P]dCTP (3000 Ci/mol) was a product of ICN
Induction of CYP2B expression by glucocorticoid and PB in mouse kidneys and liver
The constitutive expression level of CYP2B mRNAs in kidney was higher in female (4.4-fold) than in male mice as also observed in the mouse liver on northern blots (Fig. 1A). PB and Dex induced the expression of hepatic CYP2B mRNAs in both sexes to the same extent after normalization with GAPDH as a standard. In contrast, Dex, but not PB, induced the expression of CYP2B mRNAs in kidney to a far greater extent in male (8.0-fold induction) than in female mice. Western blots of microsomal proteins
Discussion
The present investigations revealed that Dex dose-dependently induced both hepatic and renal CYP2B mRNA expression in both male and female mice; the level of induction was similar in the liver of both sexes, but was far higher in the male than in the female kidneys. CYP2B10 was predominantly induced by Dex in the kidney. In contrast, PB induced the expression of these two isoforms in the liver but not in the kidney. These observations suggest that the two inducers mediate activation of the
Acknowledgements
This work was supported by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sport, and Science, the Nagase Science and Technology Foundation, the Smoking Research Foundation, as well as a Sasagawa Scientific Research Grant. The authors express special thanks to Dr. M. Negishi, NIEHS, NIH, for supplying the plasmid containing cDNA for the mouse Cyp2b10 gene.
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Abbreviations: Dex, dexamethasone; PB, phenobarbital; PCN, pregnenolone-16α-carbonitrile; DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane; P450, cytochrome P450; CAR, constitutive androstane receptor; PBREM, phenobarbital-responsive enhancer module; GR, glucocorticoid receptor; GRE, glucocorticoid-responsive element; PXR, pregnane X receptor; RT–PCR, reverse transcriptase–polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NR1, nuclear receptor binding site 1; SSC, standard saline citrate; and RXR, retinoid X receptor.