Circadian expression of steroidogenic cytochromes P450 in the mouse adrenal gland--involvement of cAMP-responsive element modulator in epigenetic regulation of Cyp17a1

The cytochrome P450 (CYP) genes Cyp51, Cyp11a1, Cyp17a1, Cyb11b1, Cyp11b2 and Cyp21a1 are involved in the adrenal production of corticosteroids, whose circulating levels are circadian. cAMP signaling plays an important role in adrenal steroidogenesis. By using cAMP responsive element modulator (Crem) knockout mice, we show that CREM isoforms contribute to circadian expression of steroidogenic CYPs in the mouse adrenal gland. Most striking was the CREM-dependent hypomethylation of the Cyp17a1 promoter at zeitgeber time 12, which resulted in higher Cyp17a1 mRNA and protein expression in the knockout adrenal glands. The data indicate that products of the Crem gene control the epigenetic repression of Cyp17 in mouse adrenal glands.     

Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction

The effect of xenobiotics (phenobarbital and atrazine) on the expression of Drosophila melanogaster CYP genes encoding cytochromes P450, a gene family generally associated with detoxification, was analyzed by DNA microarray hybridization and verified by real-time RT-PCR in adults of both sexes. Only a small subset of the 86 CYP genes was significantly induced by the xenobiotics. Eleven CYP genes and three glutathione S-transferases (GST) genes were significantly induced by phenobarbital, seven CYP and one GST gene were induced by atrazine. Cyp6d5, Cyp6w1, Cyp12d1 and the ecdysone-inducible Cyp6a2 were induced by both chemicals. The constitutive expression of several of the inducible genes (Cyp6a2, Cyp6a8, Cyp6d5, Cyp12d1) was higher in males than in females, and the induced level similar in both sexes. Thus, the level of induction was consistently higher in females than in males. The female-specific and hormonally regulated yolk protein genes were significantly induced by phenobarbital in males and repressed by atrazine in females. Our results suggest that the numerous CYP genes of Drosophila respond selectively to xenobiotics, providing the fly with an adaptive response to chemically adverse environments. The xenobiotic inducibility of some CYP genes previously associated with insecticide resistance in laboratory-selected strains (Cyp6a2, Cyp6a8, Cyp12d1) suggests that deregulation of P450 gene expression may be a facile way to achieve resistance. Our study also suggests that xenobiotic-induced changes in P450 levels can affect insect fitness by interfering with hormonally regulated networks.     

Insulin is a dominant suppressor of sterol 12 alpha-hydroxylase P450 (CYP8B) expression in rat liver: possible role of insulin in circadian rhythm of CYP8B

Sterol 12 alpha-hydroxylase (CYP8B) is a key enzyme for regulating the cholic acid/chenodeoxycholic acid ratio in bile acid biosynthesis. The hepatic CYP8B level was elevated in streptozotocin-induced diabetic rats, and the elevated CYP8B was suppressed by insulin administration [Ishida, H. et al. (1999) J. Biochem. 126, 19-25]. The streptozotocin-induced elevation of hepatic CYP8B mRNA concomitantly responded to the decrement of the serum insulin level. The CYP8B mRNA level in the cultivated rat hepatoma H4TG cells was strongly suppressed by insulin, although it was affected by dibutyryl cAMP or thyroxine to lesser extents. These observations demonstrate that CYP8B expression is dominantly regulated by the direct action of insulin on hepatocytes. A marked circadian rhythm (maximum at 13:00-16:00 and minimum at 1:00) was observed both on the mRNA level and the activity of CYP8B. This rhythm was shifted from that of cholesterol 7 alpha-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis, showing a maximum at 22:00 and a minimum at 10:00, and this shift might oscillate the cholic acid/chenodeoxycholic acid ratio, which is increased in the late afternoon and decreased at midnight. The rhythm of CYP8B was the inverse of the circadian variation of serum insulin level and was similar to the circadian rhythm of glucose 6-phosphatase. These facts and the potent suppressive effect of insulin on CYP8B indicate that the oscillation of the serum insulin may be a factor in producing the circadian rhythm of CYP8B.     

Induction of drug metabolism enzymes and transporters by oltipraz in rats

Coordinate regulation of Phase-I and -II enzymes with xenobiotic transporters has been shown after treatment with microsomal enzyme inducers. The chemopreventive agent oltipraz (OPZ) induces Phase-I and -II drug-metabolizing enzymes such as CYP2B and NQO1. The purpose of this study was to examine the regulation of drug-metabolizing enzymes and transporters in response to OPZ treatment and to investigate a potential role for constitutive androstane receptor (CAR) in OPZ-mediated induction. Sprague-Dawley rats treated with OPZ exhibited increased mRNA and protein levels of both Nqo1 and Cyp2b1/2 by 24 h. To examine whether OPZ activates transporter gene expression via CAR, sexually dimorphic male and female Wistar-Kyoto (WKY) rats were treated with OPZ and mRNA levels quantified by bDNA signal amplification. OPZ induced Ugt1a6 and Ugt2b1 in males significantly higher than in females, indicating a CAR-dependent mechanism of induction. However, OPZ induced microsomal epoxide hydrolase, NAD(P)H quinone oxidoreductase, and Cyp3a1/23 equally in both genders, indicating a CAR-independent mechanism of induction of these genes. Similarly, the transporters Mdr1a, Mdr1b, Mrp3, and Mrp4 were induced by OPZ without any apparent difference between genders. In summary, OPZ coordinately increases multiple hepatic xenobiotic transporter mRNA levels, along with Phase-I and -II enzymes some of which may occur through CAR-dependent mechanisms.     

The murine Cyp1a1 gene is expressed in a restricted spatial and temporal pattern during embryonic development

In adult mice the cytochrome P450 Cyp1a1 gene is not constitutively expressed but is highly inducible by foreign compounds acting through the aryl hydrocarbon (Ah) receptor. However, the expression profile of the Cyp1a1 gene in the developing embryo is not well under-stood. Using established transgenic mouse lines where 8.5 kb of the rat CYP1A1 promoter is cloned upstream of the lacZ reporter gene (1), we describe the expression of the CYP1A1-driven reporter gene in all tissues through-out stages E7-E14 of embryonic development. In contrast to the absence of constitutive Cyp1a1 and lacZ transgene expression in tissues of the adult mouse, a constitutive cell-specific and time-dependent pattern of CYP1A1 promoter activity was observed in the embryo. This expression pattern was confirmed as reflecting the endogenous gene by measuring Cyp1a1 mRNA levels and protein expression by immunohistochemistry. The number of cells displaying endogenous CYP1A1 activity could be increased in the embryo upon xenobiotic challenge, but only within areas where the CYP1A1 promotor was already active. When reporter mice were bred onto a genetic background expressing a lower affinity form of the Ah receptor (DBA allele), transgene and murine Cyp1a1 protein expression were both attenuated in the adult mouse liver upon xenobiotic challenge. By comparison, constitutive CYP1A1 promoter activity in the embryo was identical in the presence of either the high or low affinity Ah receptor. These novel data suggest that the Cyp1a1 protein may play a role in murine development and that regulation of the Cyp1a1 gene during this period is either through the action of a high affinity Ah receptor ligand or by an alternative regulatory pathway.     

Clock mutation facilitates accumulation of cholesterol in the liver of mice fed a cholesterol and/or cholic acid diet

Cholesterol (CH) homeostasis in the liver is regulated by enzymes of CH synthesis such as 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and catabolic enzymes such as cytochrome P-450, family 7, subfamily A, and polypeptide 1 (CYP7A1). Since a circadian clock controls the gene expression of these enzymes, these genes exhibit circadian rhythm in the liver. In this study, we examined the relationship between a diet containing CH and/or cholic acid (CA) and the circadian regulation of Hmgcr, low-density lipoprotein receptor (Ldlr), and Cyp7a1 gene expression in the mouse liver. A 4-wk CA diet lowered and eventually abolished the circadian expression of these genes. Not only clock genes such as period homolog 2 (Drosophila) (Per2) and brain and muscle arnt-like protein-1 (Bmal1) but also clock-controlled genes such as Hmgcr, Ldlr, and Cyp7a1 showed a reduced and arrhythmic expression pattern in the liver of Clock mutant mice. The reduced gene expression of Cyp7a1 in mice fed a diet containing CA or CH + CA was remarkable in the liver of Clock mutants compared with wild-type mice, and high liver CH accumulation was apparent in Clock mutant mice. In contrast, a CH diet without CA only elevated Cyp7a1 expression in both wild-type and Clock mutant mice. The present findings indicate that normal circadian clock function is important for the regulation of CH homeostasis in the mouse liver, especially in conjunction with a diet containing high CH and CA.     

Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14alpha-demethylase (Cyp51) resembles Antley-Bixler syndrome

Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.     

Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis

The liver and intestine play crucial roles in maintaining bile acid homeostasis. Here, we demonstrate that fibroblast growth factor 15 (FGF15) signals from intestine to liver to repress the gene encoding cholesterol 7alpha-hydroxylase (CYP7A1), which catalyzes the first and rate-limiting step in the classical bile acid synthetic pathway. FGF15 expression is stimulated in the small intestine by the nuclear bile acid receptor FXR and represses Cyp7a1 in liver through a mechanism that involves FGF receptor 4 (FGFR4) and the orphan nuclear receptor SHP. Mice lacking FGF15 have increased hepatic CYP7A1 mRNA and protein levels and corresponding increases in CYP7A1 enzyme activity and fecal bile acid excretion. These studies define FGF15 and FGFR4 as components of a gut-liver signaling pathway that synergizes with SHP to regulate bile acid synthesis.     

Alternate pathways of bile acid synthesis in the cholesterol 7alpha-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding

Bile acids are synthesized via the classic pathway initiated by cholesterol 7alpha-hydroxylase (CYP7A1), and via alternate pathways, one of which is initiated by sterol 27-hydroxylase (CYP27). These studies used mice lacking cholesterol 7alpha-hydroxylase (Cyp7a1(-/-)) to establish whether the loss of the classic pathway affected cholesterol homeostasis differently in males and females, and to determine if the rate of bile acid synthesis via alternate pathways was responsive to changes in the enterohepatic flux of cholesterol and bile acids. In both the Cyp7a1(-/-) males and females, the basal rate of bile acid synthesis was only half of that in matching Cyp7a1(+/+) animals. Although bile acid pool size contracted markedly in all the Cyp7a1(-/-) mice, the female Cyp7a1(-/-) mice maintained a larger, more cholic acid-rich pool than their male counterparts. Intestinal cholesterol absorption in the Cyp7a1(-/-) males fell from 46% to 3%, and in the matching females from 58% to 17%. Bile acid synthesis in Cyp7a1(+/+) males and females was increased 2-fold by cholesterol feeding, and 4-fold by cholestyramine treatment, but was not changed in matching Cyp7a1(-/-) mice by either of these manipulations. In the Cyp7a1(-/-) mice fed cholesterol, hepatic cholesterol concentrations increased only marginally in the males, but rose almost 3-fold in the females. CYP7A1 activity and mRNA levels were greater in females than in males, and were increased by cholesterol feeding in both sexes. CYP27 activity and mRNA levels did not vary as a function of CYP7A1 genotype, gender, or dietary cholesterol intake. We conclude that in the mouse the rate of bile acid synthesis via alternative pathways is unresponsive to changes in the enterohepatic flux of cholesterol and bile acid, and that factors governing gender-related differences in bile acid synthesis, pool size, and pool composition play an important role in determining the impact of CYP7A1 deficiency on cholesterol homeostasis in this species.