Rabbit Prostaglandin ω-Hydroxylase (CYP4A4): Gene Structure and Expression

doi:10.1006/abbi.1993.1093

Archives of Biochemistry and Biophysics

Volume 300, Issue 2, 1 February 1993, Pages 670-676

https://doi.org/10.1006/abbi.1993.1093 Get rights and content

Abstract

Genomic DNA containing the entire gene encoding P450 4A4, a cytochrome P450 that is elevated during pregnancy, was isolated on two overlapping recombinant phage. The isolated gene, CYP4A4, encodes a protein that differs at one amino acid position from the predicted sequence of a previously isolated cDNA. The intron/exon structure is highly conserved in relation to the clofibrate inducible genes CYP4A1, CYP4A2, and CYP4A6. However, the 5′ flanking sequences of these genes exhibit little identity. Two transcriptional start sites of the CYP4A4 gene have been determined by RNase protection analysis and are located 37 and 40 nucleotides upstream of the initiation codon. Like other CYP4A genes the CYP4A4 promoter does not contain a TATA consensus sequence. CYP4A4 mRNAs are not detected in the lungs, liver, and kidneys of untreated rabbits by RNase protection assays. However, CYP4A4 mRNA is present to varying degrees in all three of these tissues during pregnancy with the greatest abundance observed in the lung and the lowest in the kidney. Treatment of male rabbits with dexamethasone also increases the levels of CYP4A4 mRNA in the lung and liver, but the levels are eightfold less than those seen for pregnant rabbits. Consensus recognition sequences for either the glucocorticoid or progesterone receptors were not found in 1086 bp of sequence upstream of the start of transcription.

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Cited by (20)

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Cardiovascular disease (CVD) is a human health crisis that remains the leading cause of death worldwide. The cytochrome P450 (CYP) class of enzymes are key metabolizers of both xenobiotics and endobiotics. Many CYP enzyme families have been identified in the heart, endothelium and smooth muscle of blood vessels. Furthermore, mounting evidence points to the role of endogenous CYP metabolites, such as epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraenoic acids (HETEs), prostacyclin (PGI₂), aldosterone, and sex hormones, in the maintenance of cardiovascular health. Emerging science and the development of genetic screening have provided us with information on the differences in CYP expression among populations and groups of individuals. With this information, a link between CYP expression and activity and CVD, such as hypertension, coronary artery disease (CAD), myocardial infarction, heart failure, stroke, and cardiomyopathy and arrhythmias, has been established. In fact many currently used therapeutic modalities in CVD owe their therapeutic efficacy to their effect on CYP metabolites. Thus, the evidence for the involvement of CYP in CVD is numerous. Concentrating on treatment modalities that target the CYP pathway makes ethical sense for the affected individuals and decreases the socioeconomic burden of this disease. However, more research is needed to allow the integration of this information into a clinical setting.
Evaluation of cytochrome P450 4 family as mediator of phospholipase D activation in aortic vascular smooth muscle cells
2005, Life Sciences
Norepinephrine (NE) stimulates phospholipase D (PLD) activity via phospholipase A₂-dependent arachidonic acid release in rabbit aortic vascular smooth muscle cells (VSMC). We have previously shown that exogenous 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid generated through the cytochrome P450 (CYP) 4A pathway in vivo, stimulates PLD activity. Whether endogenous CYP4-derived arachidonic acid metabolites act as intracellular mediators of NE-induced PLD activation in VSMC is not known. In rabbit aortic VSMC, prototypical hepatic/renal CYP4A inducers such as fenofibrate and Wy 14643 inhibited both basal and NE-induced PLD activity after 48 h of exposure. The level of CYP4F, and to a lesser extent CYP4A, was also decreased by these agents. The expression levels of rabbit aortic VSMC CYP4A and CYP4F isoforms were reduced by antisense oligonucleotides treatment for 48 hours as measured by RTQ-PCR or Western blotting. This reduction in CYP4A or CYP4F levels did not change NE-induced PLD activation. The corresponding CYP4A scrambled and CYP4F sense oligonucleotides did not alter CYP levels. PLD activity was increased by ~ 70% after 15 min of stimulation with NE, whereas lauric acid ω-hydroxylase activity, a measure of fatty acid ω-hydroxylation, was unchanged. Inhibition of ω-hydroxylation with DDMS and HET0016, selective ω-hydroxylase inhibitors, and 20-HEDE, an antagonist of 20-HETE, increased PLD activity in a concentration-dependent manner and did not alter NE-induced PLD activation. These data suggest that PLD activation by NE is independent of the CYP4A/4F enzymes in rabbit aortic VSMC.
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2005, Archives of Biochemistry and Biophysics
Citation Excerpt :
Real-time PCR identified the progesterone receptor positive T47-D cells as potential cell systems for the examination of CYP4Z1 regulation (Fig. 1). Previous work by our laboratory and others demonstrated increased renal and pulmonary P450 4A activities in rabbits during pregnancy or in response to either glucocorticoid or progesterone treatments [1,23,24]. CYP4Z1 mRNA is expressed in mammary tissue and elevated levels were found in two out of five tested samples, suggesting conditional regulation of the CYP4Z1 gene.
Cytochrome P450 genes (CYPs) encoding two new subfamilies designated CYP4X1 and CYP4Z1 were identified in the human genome and the Expressed Sequence Tags database. Partial cDNAs encoding both P450s were isolated from human kidney and used to determine tissue distribution. CYP4X1 was predominantly expressed in trachea and aorta, whereas CYP4Z1 mRNA was preferentially expressed in mammary tissue. In T47-D cells, CYP4Z1 mRNA levels were induced by dexamethasone (14-fold) or by progesterone (10-fold). The induction by these compounds was suppressed by co-treatment with the progesterone and glucocorticoid receptor antagonist mifepristone (RU486). In the progesterone receptor negative MCF-7 cells, CYP4Z1 mRNA was induced by dexamethasone but not by progesterone treatment. CYP4Z1 mRNA levels were unaffected by 17β-estradiol. In confluent cultures of human hepatoma HepG2 cells that stably express a mouse peroxisome proliferator activated receptor-α (PPARα) mutant, CYP4X1 mRNA was undetectable in vehicle-treated cells but was readily detectable following addition of the PPARα agonist Wy14643. This suggests that PPARα activation can affect human CYP4X1 gene transcription. These results demonstrate selective tissue expression and implicate PPARα in CYP4X1 regulation, and the glucocorticoid and progesterone receptors in CYP4Z1 gene activation.
Rabbit CYP4B1 engineered for high-level expression in Escherichia coli: Ligand stabilization and processing of the N-terminus and heme prosthetic group
2003, Archives of Biochemistry and Biophysics
Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless α-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hemoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme.
Differential regulation of human CYP4A genes by peroxisome proliferators and dexamethasone
2003, Archives of Biochemistry and Biophysics
HepG2 cells that stably overexpress PPARα were used to examine the regulation of the two known human CYP4A genes by Wy14643. Specific PCR amplification across intron 5 and restriction endonuclease analysis indicated that HepG2 cells possess genes corresponding to both the CYP4A11 cDNA and a more recently characterized gene, CYP4A22, that exhibits 95% identity to CYP4A11 in the coding region. These are unlikely to represent alleles because both genes were present in DNA samples from 100 of 100 individuals. Quantitative real-time PCR determined that CYP4A22 mRNA is expressed at significantly lower levels than CYP4A11 mRNA in human liver samples. The PPARα agonist Wy14643 induced CYP4A11 mRNA in confluent cultures of HepG2 cells stably expressing the murine PPARα-E282G mutant. This mutant exhibits a significantly decreased ligand-independent trans-activation and can be activated by Wy14643 to a level similar to that of wild-type PPARα. Dexamethasone induced CYP4A11 mRNA in both control and PPARα- E282G-expressing HepG2 cells, indicating that the induction of CYP4A11 by dexamethasone is independent of elevated PPARα expression. Wy14643 or dexamethasone induction of CYP4A22 mRNA was not evident in either control or PPARα -E282G-expressing HepG2 cells. The results indicate that CYP4A11 expression can be induced by glucocorticoids and peroxisome proliferators.
Induction of rabbit lung CPY4A4 prostaglandin ω-hydroxylase by various steroid hormones
2001, Archives of Biochemistry and Biophysics
CytochromeP4504A4 (CYP4A4) is expressed at low basal levels in adult rabbit lungs, but is significantly induced during pregnancy by an unknown mechanism. As the gradual rise in CYP4A4 levels appears to coincide with the progressive increase in several steroid hormones throughout pregnancy, we examined the induction of CYP4A4 after treatment with various steroid hormones by monitoring both the CYP4A4 mRNA level and the CYP4A4-specific prostaglandin E₁ (PGE₁) ω-hydroxylation reaction in rabbit lung microsomes. Treatment with progesterone and/or a synthetic glucocorticoid (dexamethasone) resulted in a significant increase in PGE₁ ω-hydroxylase activity, whereas estradiol, aldosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate did not. These studies indicated that dexamethasone was a more potent inducer of CYP4A4 than progesterone. Simultaneous injection of dexamethasone and glucocorticoid/progesterone antagonists (RU38486, RU40555, or RU43044) inhibited the increase in PGE₁ ω-hydroxylase activity as well as mRNA levels by approximately 50%. In addition, simultaneous treatment with both dexamethasone and progesterone did not result in an additive or synergistic effect on PGE₁ ω-hydroxylase activity. These data indicate that, while distinctive receptors for glucocorticoid and/or progesterone are involved, induction may also require common or interacting regulatory elements (yet to be determined) in the CYP4A4 gene. These findings implicate both of these steroid receptors (PR/GR) in the induction of CYP4A4 in rabbit lung.

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Regular ArticleRabbit Prostaglandin ω-Hydroxylase (CYP4A4): Gene Structure and Expression

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Rabbit Prostaglandin ω-Hydroxylase (CYP4A4): Gene Structure and Expression