Diane S. Keeney

Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (−/−) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (−/−) mice and minimizes Cyp 4a12 expression and arachidonate ω-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (−/−) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 ω-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Organization, structure and evolution of the cyp2 gene cluster on human chromosome 19

The cytochrome P450 superfamily of mixed-function oxygenases has been extensively studied due to its many critical metabolic roles, and also because it is a fascinating example of gene family evolution. The cluster of genes on human chromosome 19 from the CYP2A, 2B, and 2F subfamilies has been previously described as having a complex organization and many pseudogenes. We describe the discovery of genes from three more CYP2 subfamilies inside the cluster, and assemble a complete map of the region. We comprehensively review the organization, structure, and expression of genes from all six subfamilies. A general hypothesis for the evolution of this complex gene cluster is also presented.

Differentiating Keratinocytes Express a Novel Cytochrome P450 Enzyme, CYP2B19, Having Arachidonate Monooxygenase Activity

The novel cytochrome P450, CYP2B19, is a specific cellular marker of late differentiation in skin keratinocytes. CYP2B19 was discovered in fetal mouse skin where its onset of expression coincides spatially (upper cell layer) and temporally (day 15.5) with the appearance of loricrin-expressing keratinocytes during the stratification stage of fetal epidermis. CYP2B19 is also present postnatally in the differentiated keratinocytes of the epidermis, sebaceous glands, and hair follicles. CYP2B19 mRNA is tightly coupled to the differentiated (granular cell) keratinocyte phenotypein vivo and in vitro. In primary mouse epidermal keratinocytes, it is specifically up-regulated and correlated temporally with calcium-induced differentiation and expression of the late differentiation genes loricrin and profilaggrin. Recombinant CYP2B19 metabolizes arachidonic acid and generates 14,15- and 11,12-epoxyeicosatrienoic (EET) acids, and 11-, 12-, and 15-hydroxyeicosatetraenoic (HETE) acids (20, 35, 18, 7, and 7% of total metabolites, respectively). Arachidonic acid metabolism was stereoselective for 11S,12R- and 14S,15R-EET, and 11S-, 12R-, and 15R-HETE. The CYP2B19 metabolites 11,12- and 14,15-EET are endogenous constituents of murine epidermis and are present in similar proportions to that generated by the enzymein vitro, suggesting that CYP2B19 might be the primary enzymatic source of these EETs in murine epidermis.

A Keratinocyte-specific Epoxygenase, CYP2B12, Metabolizes Arachidonic Acid with Unusual Selectivity, Producing a Single Major Epoxyeicosatrienoic Acid

The CYP monooxygenase, CYP2B12, is the first identified skin-specific cytochrome P450 enzyme. It is characterized by high, constitutive expression in an extrahepatic tissue, the sebaceous glands of cutaneous tissues. It is expressed exclusively in a subset of differentiated keratinocytes called sebocytes, as demonstrated by Northern blot analysis, in situ hybridization, and polymerase chain reaction. The onset of its expression coincides with the morphological appearance of sebaceous glands in the neonatal rat. Recombinant CYP2B12 produced in Escherichia coli epoxidizes arachidonic acid to 11,12- and 8,9-epoxyeicosatrienoic acids (80 and 20% of total metabolites, respectively). The identification of arachidonic acid as a substrate for this skin-specific CYP monooxygenase suggests an endogenous function in keratinocytes in the generation of bioactive lipids and intracellular signaling.

Preimplantation mouse blastocysts fail to express CYP genes required for estrogen biosynthesis

Implantation is initiated on day 4 in the mouse and on day 13 in the pig. The preimplantation pig blastocyst synthesizes steroid hormones, but whether preimplantation rodent embryos also have this ability has remained unresolved for the last two decades. In this study, the mRNAs encoding NADPH‐cytochrome P450 reductase (P450‐reductase), adrenodoxin, lanosterol 14‐demethylase P450 (CYP51), 17α‐hydroxylase P450 (CYP17), cholesterol side‐chain cleavage P450 (CYP11A1), sterol 27‐hydroxylase P450 (CYP27), and aromatase P450 (CYP19) were examined in day 4 mouse blastocysts (day 1 = vaginal plug) and in day 13 and 16 pig blastocysts using reverse transcription‐polymerase chain reaction (RT‐PCR). In mouse blastocysts, mRNAs of P450‐reductase, adrenodoxin, and CYP51, but not CYP17, CYP11A1, CYP27, and CYP19, were detected. In agreement with this finding, no aromatase protein could be detected by immunohistochemistry. By contrast, all these mRNAs were detected in the pig blastocyst. Furthermore, both the ovarian and placental types of aromatase (CYP19) mRNAs were detected in the pig blastocyst on days 13 and 16 of pregnancy, although the ovarian form was more abundant. Both forms of aromatase were much higher in day 13 than in day 16 pig blastocysts. The results provide definitive evidence that the preimplantation mouse blastocyst, as opposed to the pig blastocyst, has no ability to synthesize estrogen and no steroidogenic capacity. Maternal estrogen synthesis is essential for implantation of the mouse blastocyst.

The importance of the lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier

This review covers the background to discovery of the two key lipoxygenases (LOX) involved in epidermal barrier function, 12R-LOX and eLOX3, and our current views on their functioning. In the outer epidermis, their consecutive actions oxidize linoleic acid esterified in ω-hydroxy-ceramide to a hepoxilin-related derivative. The relevant background to hepoxilin and trioxilin biochemistry is briefly reviewed. We outline the evidence that linoleate in the ceramide is the natural substrate of the two LOX enzymes and our proposal for its importance in construction of the epidermal water barrier. Our hypothesis is that the oxidation promotes hydrolysis of the oxidized linoleate moiety from the ceramide. The resulting free ω-hydroxyl of the ω-hydroxyceramide is covalently bound to proteins on the surface of the corneocytes to form the corneocyte lipid envelope, a key barrier component. Understanding the role of the LOX enzymes and their hepoxilin products should provide rational approaches to ameliorative therapy for a number of the congenital ichthyoses involving compromised barrier function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Genes Involved in Androgen Biosynthesis and the Male Phenotype

A series of enzymatic steps in the testis lead to the conversion of cholesterol to the male sex steroid hormones, testosterone and 5 alpha-dihydrotestosterone. Mutations in any one of these steps are presumed to alter or block the development of the male phenotype. Most of the genes encoding the enzymes involved in this pathway have now been cloned, and mutations within the coding regions of these genes do, in fact, block development of the male phenotype.

Differentiation-Specific Factors Modulate Epidermal CYP1–4 Gene Expression in Human Skin in Response to Retinoic Acid and Classic Aryl Hydrocarbon Receptor Ligands

Human epidermal keratinocytes express subsets of cytochromes P450 (P450) (CYP gene products) that are strongly up-regulated, not regulated, or down-regulated by differentiation-specific factors. We investigated how drug exposure affects epidermal expression of CYP1–4 genes, which encode many drug-metabolizing P450s. Real-time polymerase chain reaction (PCR) assays measured CYP1–4 mRNA levels in epidermal keratinocytes differentiated in vitro in the presence of drug or vehicle for 6 days. We confirmed the spinous phenotype at day 6 by changes in cellular morphology and upregulation of cytokeratin 10 and transglutaminase (TGM)1 mRNA in the differentiating keratinocytes. Effects of drug exposure depended on the influence of differentiation-specific factors in controlling epidermal CYP1–4 expression. CYP2C18, 2C19, 2C9, 2W1, 3A4, and 4B1 are up-regulated by cellular differentiation; mRNA levels for these CYP genes were inhibited in differentiating keratinocytes exposed to retinoic acid and aryl hydrocarbon receptor (AhR) ligands. These same drugs effected ≤2-fold change or even augmented mRNA levels for CYP genes that are not regulated by differentiation (CYP2S1, 2J2, 1B1, 1A1, 1A2, 2E1, and 2D6) and for CYP2U1, which is expressed at highest levels in undifferentiated keratinocytes. The clinically relevant drugs miconazole, dexamethasone, rifampicin, and dapsone had little effect on CYP1–4 mRNA levels under assay conditions. The AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin also up-regulated keratinocyte TGM1 mRNA in a concentration- and time-dependent manner. This effect was blocked by the AhR antagonist resveratrol. These findings implicate AhR-dependent up-regulation of TGM1 mRNA in differentiating keratinocytes as one mechanism contributing toward chloracne in humans exposed to toxic levels of dioxin.

Regulation of steroid hydroxylase gene expression: Importance to physiology and disease

Steroid hydroxylase gene expression is multifactorial in nature, being regulated by tissue-specific, developmental, constitutive and signal transduction systems. The biochemistry of this complex pattern of regulation is not yet clearly elucidated, but studies in several laboratories have led to an understanding of specific aspects of regulation, particularly that involving signal transduction. The complexity of regulation appears to be necessary for normal human physiology because of the wide variety of steroid hormones produced by these enzymes. Genetic diseases associated with the steroid hydroxylases provide examples of how aberrant physiology can result from alterations in the multifactorial regulation of steroid hydroxylase gene expression.

Investigation of a Second 15S-Lipoxygenase in Humans and its Expression in Epithelial Tissues

The first reports of a lipoxygenase in animal cells, the 12S-lipoxygenase of platelets, were published in 1974–75 (Hamberg and Samuelsson, 1974; Nugteren, 1975). At nearly the same time, Rapoport and colleagues recognized the existence of the 15S-lipoxygenase in reticulocytes (Schewe et al., 1975), and discovery of the leukocyte 5S-lipoxygenase was reported in the following year (Borgeat et al., 1976). Subsequent metabolism studies and molecular analyses indicated the occurrence of the same enzymes in selected other tissues; for example, both the 12S-and 15S-lipoxygenases were expressed also in skin (Nugteren and Kivits, 1987; Hussain et al., 1994; Takahashi et al., 1993). For the past two decades it had appeared that the three enzymes could account for all known lipoxygenase activities in man.