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.

15-Lipoxygenase-2 expression in benign and neoplastic sebaceous glands and other cutaneous adnexa.

15-Lipoxygenase-2 has a limited tissue distribution in epithelial tissues, with mRNA detected in skin, cornea, lung, and prostate. It was originally cloned from human hair rootlets. In this study the distribution of 15-lipoxygenase-2 was characterized in human skin using immunohistochemistry and in situ hybridization. Strong uniform 15-lipoxygenase-2 in situ hybridization (n = 6) and immunostaining (n = 16) were observed in benign cutaneous sebaceous glands, with expression in differentiated secretory cells. Strong 15-lipoxygenase-2 immunostaining was also observed in secretory cells of apocrine and eccrine glands. Variable reduced immunostaining was observed in skin-derived sebaceous neoplasms (n = 8). In the eyelid, Meibomian glands were uniformly negative for 15-lipoxygenase-2 in all cases examined (n = 9), and sebaceous carcinomas apparently derived from Meibomian glands were also negative (n = 12). The mechanisms responsible for differential expression in cutaneous sebaceous vs eyelid Meibomian glands remain to be established. In epidermis, positive immunostaining was observed in the basal cell layer in normal skin, whereas five examined basal cell carcinomas were negative. Thus, the strongest 15-lipoxygenase-2 expression is in the androgen regulated secretory cells of sebaceous, apocrine, and eccrine glands. This compares with the prostate, in which 15-lipoxygenase-2 is expressed in differentiated prostate secretory cells (and reduced in the majority of prostate adenocarcinomas). The product of 15-lipoxygenase-2, 15-hydroxyeicosatetraenoic acid, may be a ligand for the nuclear receptor peroxisome proliferator activated receptor-gamma, which is expressed in sebocytes, and contribute to secretory differentiation in androgen regulated tissues such as prostate and sebaceous glands.

Detection and cellular localization of 12R-lipoxygenase in human tonsils.

Formation of the 12R-lipoxygenase product, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE), has been detected previously only in human skin (Boeglin et al. (1998) Proc. Natl. Acad. Sci. USA 95, 6744). The unexpected appearance of an EST sequence (AA649213) for human 12R-lipoxygenase from germinal center B lymphocytes purified from human tonsils prompted our search for the existence of the enzyme in this novel source. Incubation of [1-14C]arachidonic acid with homogenates of human tonsillar tissue yielded mixtures of radiolabeled 12-HETE and 15-HETE. Stereochemical analysis showed varying ratios of 12S- and 12R-HETE, while 15-HETE was exclusively of the S-configuration. Using stereospecifically labeled [10S-3H]- and [10R-3H]arachidonic acid substrates we detected pro-R hydrogen abstraction at carbon 10 associated with formation of 12R-HETE. This mechanistic evidence implicates a 12R-lipoxygenase in the biosynthesis of 12R-HETE. The mRNA for the enzyme was identified in tonsils by RT-PCR and Northern analysis. The cellular distribution was established by in situ hybridization. Unexpectedly, hybridization was not observed in the lymphocytes of the germinal centers. Specific reaction was restricted to squamous epithelial cells, including the epithelium lining the tonsillar crypts. In this location the 12R-lipoxygenase might help regulate differentiation of the epithelium or participate in lymphocyte- epithelial cell interactions.

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 omega-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 omega-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Two novel sites of expression of NADPH cytochrome P450 reductase during murine embryogenesis: limb mesenchyme and developing olfactory neuroepithelia.

While all cells in eukaryotic organisms probably express the gene encoding NADPH cytochrome P450 reductase, we identified two novel sites which have the highest local concentrations of P450 reductase transcripts during murine embryogenesis. One site is in developing limbs, including lateral limb bud mesenchyme and condensing mesenchyme in the footplate which will form precartilage. A second site is in primitive neuroepithelia, including future olfactory epithelia and olfactory lobes of the brain. These high, local concentrations of P450 reductase transcripts revealed by in situ hybridization were transient and most prominent between embryonic (E) days 12.5-15.5. They cannot be explained by the known functions for P450 reductase. The precursor nature of the highest reductase-expressing cells suggests that differentiation-specific mechanisms regulate P450 reductase gene transcription during organogenesis. The data suggest this multifunctional protein might serve an important role in the formation of precartilage models from condensing limb mesenchyme and in the early development of joints that will form at apposed surfaces of these models.

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 phenotype in 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 enzyme in 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.

The regulation of 3 beta-hydroxysteroid dehydrogenase expression.

3 beta-Hydroxysteroid dehydrogenasel delta 5-->4-isomerase (3 beta-HSD) catalyzes the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids, an obligate step in the biosynthesis not only of androgens and estrogens but also of mineralocorticoids and glucocorticoids. The enzyme is expressed in the adrenal cortex and in steroidogenic cells of the gonads, consistent with this role. However, 3 beta-HSD is also expressed in many other tissues, such as the liver and kidney, where its function is not entirely clear. It is established that a family of closely related genes encode for 3 beta-HSD. The various 3 beta-HSD isoforms are expressed in a tissue-specific manner involving separate mechanisms of regulation. The human type I 3 beta-HSD is expressed at high levels in syncytial trophoblast and in sebaceous glands, and the type II isoform is almost exclusively expressed in the adrenal cortex and gonads. An important feature in liver and kidney (at least of hamster, mouse, rabbit, and rat) is the sexual dimorphic nature of 3 beta-HSD expression. We briefly review studies on the regulation of the human 3 beta-HSD I and II genes in human trophoblast and adrenal cortex and extend this to discuss the rat 3 beta-HSD I gene expressed in adrenals and gonads. The complexity of 3 beta-HSD expression through multiple signaling pathways acting on a multigene family of enzymes may contribute importantly to the diverse patterns and locations of steroid hormone biosynthesis.

Functional expression and cellular localization of a mouse epidermal lipoxygenase.

Three distinct murine lipoxygenase genes have been functionally characterized: 5-lipoxygenase (Chen, X.-S., Naumann, T. A., Kurre, U. , Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1995) J. Biol. Chem. 270, 17993-17999), platelet-type 12-lipoxygenase and leukocyte-type 12-lipoxygenase (Chen, X.-S., Kurre, U., Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1994) J. Biol. Chem. 269, 13979-13987). Here, we describe the cloning and functional characterization of a fourth lipoxygenase gene in mice. Using a polymerase chain reaction-based approach together with partial sequence information from a genomic clone, we isolated a novel lipoxygenase cDNA from the RNA of 3-6-day-old mouse epidermis. The open reading frame predicts a 662-amino acid lipoxygenase that displays 60% identity with both murine 12-lipoxygenase isozymes and 40% identity to 5-lipoxygenase; the sequence is identical to a genomic sequence reported recently (van Dijk, K. W., Steketee, K., Havekes, L., Frants, R., and Hofker, M. (1995) Biochim. Biophys. Acta 1259, 4-8). A full-length clone was expressed in human embryonic kidney 293 cells and homogenates from disrupted cells produced 12-hydroxyeicosatetraenoic acid (12-HETE) and minor amounts of 15-HETE from arachidonic acid. Chiral phase analysis indicated that the 12-HETE is exclusively the 12S enantiomer. In situ hybridization revealed highly specific expression of epidermal lipoxygenase in differentiated keratinocytes of the epidermis and in restricted regions of the root sheath and bulb of hair follicles. High expression was also detected in conjunctiva of the eyelid and in cells of Meibomian and preputial (sebaceous) glands. A 2. 4-kilobase mRNA was detected in mouse epidermis by Northern blot analysis and its abundance was not affected by phorbol ester treatment. The epidermal lipoxygenase gene (Aloxe) resides on mouse chromosome 11 closely linked with the two 12-lipoxygenase genes (Alox12p and Alox12l).

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), 17 alpha-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.

Developmentally regulated expression of adrenal 17 alpha-hydroxylase cytochrome P450 in the mouse embryo.

Corticosterone is the major circulating glucocorticoid in adult mice and rats, and this is explained, in part, by the absence of 17 alpha-hydroxylase cytochrome P450 (P450c17) in adrenal glands of these rodents. During embryonic development, however, we discovered transient expression of P450c17 in a subset of adrenocortical cells in the fetal mouse adrenal. This differs from cholesterol side-chain cleavage cytochrome P450 and adrenodoxin, which are expressed continuously in most fetal adrenocortical cells from onset of expression at embryonic days 11-12 (E11-12) until term. Adrenal P450c17 transcripts are detectable in situ at E12.5 and increase in abundance from E12.5 to E14.5. Transcripts are then lost between E16.5 and term (E18.5) and are undetectable in situ in adrenal glands of adult mice. These results are consistent with the presence of pregnenolone 17 alpha-hydroxylase activity in adrenal homogenates of fetal but not adult mice. By using polymerase chain reaction, we determined that murine fetal (E14.5-15.5) adrenal glands contain messenger RNAs (mRNAs) encoding all of the steroid hydroxylases required to produce cortisol and corticosterone but little aldosterone synthase mRNA. Adrenal glands from adult mice contain mRNAs encoding steroid hydroxylases required to produce corticosterone and aldosterone but not cortisol (little P450c17 mRNA). The spatial and temporal expression patterns of P450c17 and aldosterone synthase mRNA, which differ from those of cholesterol side-chain cleavage cytochrome P450 and adrenodoxin, suggest that multiple factors must be required to program cell type- and species-specific expression of these steroid hydroxylases during embryonic development.

Cholesterol side-chain cleavage cytochrome P450 gene expression in the primitive gut of the mouse embryo does not require steroidogenic factor 1.

In situ hybridization studies reveal novel sites of expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) during murine embryonic development. In addition to fetal adrenals and testes, P450scc transcripts localize in situ to the primitive gut and to a subset of unidentified cells in the dermal mesenchyme of embryonic skin. In the gut, transcripts are most abundant in luminal epithelia of the hindgut, which will form the colon. P450scc transcript abundance at these novel sites is a fraction of that in fetal adrenals or testes, suggesting a local rather than an endocrine function. Immunocytochemical analyses localize P450scc protein to the fetal hindgut, indicating that the transcripts are translated in vivo. RNA isolated from microdissected embryonic hindgut and skin was reverse transcribed and amplified by polymerase chain reaction. DNA sequence analyses of polymerase chain reaction products confirmed that specific hybridization in situ represents authentic P450scc gene (Cyp11A) transcripts and that 3 beta-hydroxysteroid dehydrogenase/delta 5-->delta 4-isomerase transcripts are also present, demonstrating the potential of these fetal tissues to produce pregnenolone and progesterone. P450scc transcripts are also detectable by in situ hybridization in primitive gut and skin of Fushi tarazu factor 1 null mice, which lack the nuclear receptor steroidogenic factor 1, proving that steroidogenic factor 1 is not required for steroid hydroxylase gene expression at these sites. The capacity for C21 steroid biosynthesis in primitive gut and skin during organogenesis raises the question whether local production of steroid hormones may be required for normal cellular growth and differentiation of these tissues during embryogenesis.

Perspectives in steroid hydroxylase gene expression: novel sites of expression during embryonic development.

CYP17 is not expressed in adult mouse adrenal glands but is expressed in a subset of fetal adrenocortical cells, indicating the potential to produce both corticosterone and cortisol during murine embryogenesis. CYP11A is expressed in the fetal adrenal but also in developing hindgut, which will form the colon, and in cells located beneath the skin of the embryo. Novel sites of expression of CYP17 and CYP11A in the mouse embryo suggest potential physiological roles for local production of steroids in diverse organs systems during development.

In vivo model for chronic direct intratesticular drug administration.

The efficacy and toxicity of a new method for chronic direct intratesticular drug infusion were assessed in a rat model. To this end, luteinizing hormone (LH) or buffer was infused via miniosmotic pumps for 14 days directly into the parenchyma of Copenhagen rat testes. The surgical manipulation and direct infusion of buffer did not have any apparent adverse effect upon either spermatogenesis or steroidogenesis as measured by testis weight, homogenization-resistant spermatid count, and in vitro response of the testes to a maximally stimulating concentration of LH. Histologic studies revealed only a localized inflammatory response in the testis around the Silastic tubing leading from the mini-osmotic pump to the testis. The biologic efficacy of direct infusion into the testis was assessed by determining the ability of mini-osmotic pump-infused LH to maintain steroidogenesis for 14 days in animals whose pituitary function was suppressed by simultaneous subcutaneous placement of testosterone/estradiol capsules. Steroidogenesis was found to be maintained quantitatively in testes infused with an appropriate dose of LH. At a given LH dose, the directly infused testes were found to produce fivefold more testosterone than contralateral testes and 10-fold more testosterone than testes from rats receiving systemic administration of the same dose of LH. We conclude that the miniosmotic pump system is a useful means to chronically administer a high concentration of LH, and presumably other agents, to the testis without any significant adverse effects.

Growth hormone transgenes regulate the expression of sex-specific isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse liver and gonads.

The sexually dimorphic pattern of GH secretion regulates the expression of several steroidogenic enzymes in rat liver, including a male-specific 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta HSD). Recently, we identified male-specific isoforms of immunoreactive 3 beta HSD in mouse liver [42 kilodaltons (kDa)] and gonads (47 kDa). To test whether GH can regulate the expression of these murine 3 beta HSDs, endogenous forms of 3 beta HSD were studied in transgenic mice expressing heterologous GH transgene products. Mice from five transgenic lines were used; two expressed GH transgenes encoding the phosphoenolpyruvate carboxykinase (PEPCK) promoter fused to either the human (h) GH (somatogenic and lactogenic) or bovine (b) GH (somatogenic) structural genes, and three expressed GH transgenes encoding the mouse metallothionein-1 (MT1) promoter fused to the hGH, hGH variant (hGHv), or bGH structural genes. Control mice were normal nontransgenic littermates. Expression of a male-specific (42 kDa) isoform of hepatic 3 beta HSD is dramatically suppressed in all transgenic mouse lines, as detected on Western immunoblots, without affecting a 47-kDa isoform expressed in livers of both male and female mice. This negative regulation was not observed in mouse kidney, which normally expresses two 3 beta HSD isoforms (in both sexes) with molecular masses similar to those in liver. Considering that PEPCK and MT1 promoters direct expression of GH fusion genes in both tissues, the inhibition of hepatic, but not renal, 3 beta HSD immunoreactivity suggests that GH affects sex-specific, rather than tissue-specific, expression of 3 beta HSD. As in the liver, sex-specific expression of 3 beta HSD in the testis is also suppressed by heterologous GH, but with one notable difference. Only human-derived GH (MT1-hGH and MT1-hGHv) effectively inhibits expression of the 47-kDa sex-specific isoform of testicular 3 beta HSD, without affecting the 44-kDa isoform expressed in gonads of both male and female mice. These results suggest that the negative effects of heterologous GH on sex-specific 3 beta HSDs may be mediated by PRL receptors in the testis and GH receptors in the liver. PEPCK-GH transgenes had little effect on testicular 3 beta HSD, possibly because this promoter (unlike MT1) is relatively inactive in this tissue. In the liver of male transgenics (PEPCK-hGH), loss of the sex-specific (42-kDa) 3 beta HSD has little effect on the Km for dehydroepiandrosterone (DHEA; 11 microM) compared with that in normal controls (16 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse tissues: male-specific isoforms are expressed in the gonads and liver.

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta HSD) are expressed in various mouse tissues in a tissue-specific, sex-specific, and developmental manner. Three distinct immunoreactive species [molecular masses, 47, 44, and 42 kilodaltons (kDa)] are detectable by Western immunoblot analysis using a 3 beta HSD antiserum. Different immunoreactive isoforms are expressed in steroidogenic (44 and 47 kDa in gonads) and nonsteroidogenic (42 and 47 kDa in liver and kidney) tissues. Two of these isoforms are sex-specific in the gonads (47 kDa) and liver (42 kDa), because they are detectable only in male mice. Sex-specific expression in the liver is developmentally regulated. Low levels of this male-specific hepatic isoform are first detectable at 23-25 days of age, but its level of expression increases progressively during sexual maturation to adult levels. NAD(+)-dependent 3 beta HSD activity is detectable in homogenates of all tissues examined, but the kinetic characteristics of this activity differ among tissues and are sexually dimorphic in the liver. Apparent Michaelis constants for dehydroepiandrosterone are much lower in steroidogenic (0.24 +/- 0.07 microM for testis) than in nonsteroidogenic (range, 10-100 microM for liver and kidney) tissues and are lower in male mouse liver (16 +/- 1 microM) than in female mouse liver (82 +/- 20 microM). Oligonucleotides with unique sequences but encoding homologous regions of the mouse type I, II, and III 3 beta HSD cDNAs were used for Northern blot analyses. A type I oligomer hybridizes with RNA from steroidogenic (adrenal, ovary, and testis) tissues, and a type III oligomer hybridizes with RNA from nonsteroidogenic (liver and kidney) tissues. A type II oligomer, however, hybridizes specifically with RNA from testis and liver of male mice, tissues that express a male-specific 3 beta HSD. These results suggest that type II-like transcripts may encode a 47-kDa sex-specific 3 beta HSD in testis and a 42-kDa sex-specific 3 beta HSD in liver of male mice. It is unclear how many members of subfamilies of the 3 beta HSD gene family will be discovered. The mouse may prove to be a valuable experimental model, as this is the first species in which multiple immunoreactive isoforms can be identified in a single tissue. This multiplicity makes it difficult to correlate the size and number of immunoreactive isoforms with the diverse kinetic characteristics of NAD(+)-dependent 3 beta HSD activities in tissue homogenates and to develop isoform-specific probes.(ABSTRACT TRUNCATED AT 400 WORDS)

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.