Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily

CYP2C enzymes epoxidize arachidonic acid (AA) to metabolites involved in the regulation of vascular and renal function. We tested the hypothesis that eicosapentaenoic acid (EPA), a n-3 polyunsaturated fatty acid, may serve as an alternative substrate. Human CYP2C8 and CYP2C9, as well as rat CYP2C11 and CYP2C23, were co-expressed with NADPH-CYP reductase in a baculovirus/insect cell system. The recombinant enzymes showed high EPA and AA epoxygenase activities and the catalytic efficiencies were almost equal comparing the two substrates. The 17,18-double bond was the preferred site of EPA epoxidation by CYPs 2C8, 2C11, and 2C23. 17(R),18(S)-Epoxyeicosatetraenoic acid was produced with an optical purity of about 70% by CYPs 2C9, 2C11, and 2C23 whereas CYP2C8 showed the opposite enantioselectivity. These results demonstrate that EPA is an efficient substrate of CYP2C enzymes and suggest that n-3 PUFA-rich diets may shift the CYP2C-dependent generation of physiologically active eicosanoids from AA- to EPA-derived metabolites.     

Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 microM, 400 microl for 4 days) by 1.59+/-0.2-fold (p<0.05). ATRA treatment (10 microM) resulted in a 59.9+/-9.8% increase (p<0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.     

Physiological insights into all-trans-retinoic acid biosynthesis

All-trans-retinoic acid (atRA) provides essential support to diverse biological systems and physiological processes. Epithelial differentiation and its relationship to cancer, and embryogenesis have typified intense areas of interest into atRA function. Recently, however, interest in atRA action in the nervous system, the immune system, energy balance and obesity has increased considerably, especially concerning postnatal function. atRA action depends on atRA biosynthesis: defects in retinoid-dependent processes increasingly relate to defects in atRA biogenesis. Considerable evidence indicates that physiological atRA biosynthesis occurs via a regulated process, consisting of a complex interaction of retinoid binding-proteins and retinoid recognizing enzymes. An accrual of biochemical, physiological and genetic data have identified specific functional outcomes for the retinol dehydrogenases, RDH1, RDH10, and DHRS9, as physiological catalysts of the first step in atRA biosynthesis, and for the retinal dehydrogenases RALDH1, RALDH2, and RALDH3, as catalysts of the second and irreversible step. Each of these enzymes associates with explicit biological processes mediated by atRA. Redundancy occurs, but seems limited. Cumulative data support a model of interactions among these enzymes with retinoid binding-proteins, with feedback regulation and/or control by atRA via modulating gene expression of multiple participants. The ratio apo-CRBP1/holo-CRBP1 participates by influencing retinol flux into and out of storage as retinyl esters, thereby modulating substrate to support atRA biosynthesis. atRA biosynthesis requires the presence of both an RDH and an RALDH: conversely, absence of one isozyme of either step does not indicate lack of atRA biosynthesis at the site. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

The Sulfolobus solfataricus electron donor partners of thermophilic CYP119: an unusual non-NAD(P)H-dependent cytochrome P450 system

CYP119 from Sulfolobus solfataricus is the first well-characterized thermophilic cytochrome P450 enzyme. The endogenous substrate for this enzyme is not known but it hydroxylates lauric acid in a reaction supported by surrogate mesophilic electron donors. However, reconstitution of a high-temperature catalytic system requires identification of the normal thermophilic electron donor partners of CYP119. Here, we describe cloning, expression in Escherichia coli, and characterization of the requisite electron donor partners from S. solfataricus. One is a thermostable ferredoxin and the second a 2-oxoacid-ferredoxin oxidoreductase that utilizes pyruvic acid rather than NAD(P)H as the source of reducing equivalents. CYP119 is the only cytochrome P450 to date known to obtain electrons from a non-NAD(P)H-dependent protein. The two thermophilic partners have been used to reconstitute a catalytic system that hydroxylates lauric acid at 70 degrees C, and the optimal conditions for this system have been defined. This first high-temperature in vitro catalytic system represents an important step in the development of industrially relevant catalysts.     

Lipid metabolism in mammalian tissues and its control by retinoic acid

Evidence has accumulated that specific retinoids impact on developmental and biochemical processes influencing mammalian adiposity including adipogenesis, lipogenesis, adaptive thermogenesis, lipolysis and fatty acid oxidation in tissues. Treatment with retinoic acid, in particular, has been shown to reduce body fat and improve insulin sensitivity in lean and obese rodents by enhancing fat mobilization and energy utilization systemically, in tissues including brown and white adipose tissues, skeletal muscle and the liver. Nevertheless, controversial data have been reported, particularly regarding retinoids' effects on hepatic lipid and lipoprotein metabolism and blood lipid profile. Moreover, the molecular mechanisms underlying retinoid effects on lipid metabolism are complex and remain incompletely understood. Here, we present a brief overview of mammalian lipid metabolism and its control, introduce mechanisms through which retinoids can impact on lipid metabolism, and review reported activities of retinoids on different aspects of lipid metabolism in key tissues, focusing on retinoic acid. Possible implications of this knowledge in the context of the management of obesity and the metabolic syndrome are also addressed. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Anionic phospholipid-induced regulation of reactive oxygen species production by human cytochrome P450 2E1

We suggest that the cytochrome P450 2E1 (CYP2E1)-induced formation of reactive oxygen species (ROS) can be regulated by anionic phospholipids and the presence of the N-terminal region of the enzyme. When the content of cardiolipin (CL) in membranes at the expense of phosphatidylcholine matrix was increased, the ROS produced by recombinant human CYP2E1 was decreased as a function of CL concentration. On the contrary, the N-terminally truncated CYP2E1 had a decreased effect on the lipid-induced reduction of ROS formation. These results suggest that specific phospholipids can regulate the function of CYP2E1 by interaction with the enzyme including the N-terminal region(s).     

Cloning of rat cytochrome P450RAI (CYP26) cDNA and regulation of its gene expression by all-trans-retinoic acid in vivo

A novel retinoic acid (RA)-inducible cytochrome P450 (P450 RAI or CYP26), previously cloned from human, zebra fish, and mouse, functions in the metabolism of all-trans-RA to polar metabolites including 4-hydroxy-RA and 4-oxo-RA. To further study CYP26 in the rat model, we first cloned rat CYP26 cDNA. The nucleotide sequence predicts a 497-amino-acid protein whose sequence is 95% identical to mouse and 91% homologous to human CYP26. Animal studies showed that CYP26 mRNA expression is very low (0.01+/-0.008;P<0.05) in vitamin-A-deficient rats compared to pair-fed vitamin-A-sufficient rats (defined as 1.0). In a kinetic study, vitamin-A-deficient rats were treated with approximately 100 microg of all-trans-RA and liver was collected after 3-72 h for analysis of CYP26 mRNA by quantitative real-time PCR. Liver CYP26 mRNA increased to nearly 10-fold above control after 3 h (P<0.01), reaching a peak of about 2000-fold greater around 10 h (P<0.001) and then decreased rapidly. The CYP26 dose response to RA was nearly linear (R(2)=0.9638). Additionally, significant regulation of CYP26 gene expression was observed in the vitamin-A-deficient, control, and RA-treated condition in lung, testis, and small intestine. We conclude that CYP26 mRNA expression is dynamically regulated in vivo by diet and RA in hepatic and extrahepatic tissues. The long-term down-regulation of CYP26 in retinoid deficiency may be critical for conserving RA, while the acute up-regulation of CYP26 may be important for preventing a deleterious overshoot of RA derived from either dietary or exogenous sources.     

Induction of the mRNA for CYP6B2, a pyrethroid inducible cytochrome p450, in Helicoverpa armigera (Hubner) by dietary monoterpenes

A cDNA clone encoding a cytochrome P450 from H. armigera was used to examine the expression of two homologous cytochrome P450 mRNAs, one of which, CYP6B2, is probably involved in pyrethroid metabolism. The mRNA for CYP6B2 in particular can be induced up to tenfold by peppermint oil and the monoterpene α-pinene as well as to a smaller extent by menthol, butylated hydroxyanisole, and piperonyl butoxide. Both mRNAs are present in the major larval tissues, midgut, fat body, and cuticle, although only the mRNAs in the midgut and fat body are inducible by peppermint oil. The induction is a rapid process occurring within a period of 4 h with a similarly rapid rate of decrease in the absence of the inducing compounds. During normal development both mRNAs are absent from eggs and increase during the larval stage, reaching a maximum during mid fifth instar. Both mRNAs then decrease to undetectable levels during pupation and remain undetectable in the adult stage. © 1997 Wiley-Liss, Inc.     

Active site mutations of cytochrome p450cam alter the binding, coupling, and oxidation of the foreign substrates (R)- and (s)-2-ethylhexanol

Three factors are of primary importance with respect to designing efficient P450 biocatalysts. (1) The substrate must be oxidized at a significant rate. (2) The regioselectivity must heavily favor the desired product. (3) The enzyme must use the majority of the reducing equivalents from NADH or NADPH to produce product. The reaction we chose to study was oxidation of 2-ethylhexanol to 2-ethylhexanoic acid by P450cam. We examined four active site mutations: F87W, Y96W, T185F, and L244A. The mutations were chosen to improve 2-ethyhexanoic acid production by decreasing active site volume, increasing active site hydrophobicity, and improving stereoselectivity. The F87W and Y96W mutations improved regioselectivity, giving almost exclusively the desired product. The T185F mutation improved coupling of NADH to product formation. The L244A mutation altered the stereoselectivity of 2-ethylhexanoic acid production. These results indicate that active site mutations of P450cam can alter catalysis of 2-ethylhexanol.     

Marked change in the balance between CYP27A1 and CYP46A1 mediated elimination of cholesterol during differentiation of human neuronal cells

Cholesterol metabolism in the brain is distinct from that in other tissues due to the fact that cholesterol itself is unable to pass across the blood-brain barrier. Elimination of brain cholesterol is mainly dependent on a neuronal-specific cytochrome P450, CYP46A1, catalyzing the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), which is able to pass the blood-brain barrier. A suitable model for studying this elimination from human neuronal cells has not been described previously. It is shown here that differentiated Ntera2/clone D1 (NT2) cells express the key genes involved in brain cholesterol homeostasis including CYP46A1, and that the expression profiles of the genes observed during neuronal differentiation are those expected to occur in vivo. Thus there was a decrease in the mRNA levels corresponding to cholesterol synthesis enzymes and a marked increase in the mRNA level of CYP46A1. The latter increase was associated with increased levels of CYP46A1 protein and increased production of 24OHC. The magnitude of the secretion of 24OHC from the differentiated NT2 cells into the medium was similar to that expected to occur under in vivo conditions. An alternative to elimination of cholesterol by the CYP46A1 mechanism is elimination by CYP27A1, and the product of this enzyme, 27-hydroxycholesterol (27OHC), is also known to pass the blood-brain barrier. The CYP27A1 protein level decreased during the differentiation of the NT2 cells in parallel with decreased production of 27OHC. The ratio between 24OHC and 27OHC in the medium from the cultured cells increased, by a factor of 13, during the differentiation process. The results suggest that progenitor cells eliminate cholesterol in the form of 27OHC while neurogenesis induces a change to the CYP46A1 dependent pathway. Furthermore this study demonstrates that differentiated NT2 cells are suitable for studies of cholesterol homeostasis in human neurons.     

Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites

Recently, several mutants of cytochrome P450 BM3 (CYP102A1) with high activity toward drugs have been obtained by a combination of site-directed and random mutagenesis. In the present study, the applicability of these mutants as biocatalysts in the production of reactive metabolites from the drugs clozapine, diclofenac and acetaminophen was investigated. We showed that the four CYP102A1 mutants used in this study formed the same metabolites as human and rat liver microsomes, with an activity up to 70-fold higher compared to human enzymes. Using these CYP102A1 mutants, three novels GSH adducts of diclofenac were discovered which were also formed in incubations with human liver microsomes. This work shows that CYP102A1 mutants are very useful tools for the generation of high levels of reference metabolites and reactive intermediates of drugs. Producing high levels of those reactive metabolites, that might play a role in adverse drug reactions (ADRs) in humans, will facilitate their isolation, structural elucidation, and could be very useful for the toxicological characterization of novel drugs and/or drug candidates.     

Cytochrome P450cin (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s

P450_cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450_cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450_cin D241N mutant has been produced and found to be analogous to the P450_cam D251N mutant. P450_cin catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450_cin D241N mutant also hydroxylated cineole to produce only (1R)-6β-hydroxycineole, was moderately well coupled (31 ± 3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450_cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450_cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase.     

A role for p21 (WAF1) in the cAMP-dependent differentiation of F9 teratocarcinoma cells into parietal endoderm

Combined treatment of teratocarcinoma F9 cells with retinoic acid and dibutyryl-cAMP induces the differentiation into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation, concomitantly with the appearance of p21 in the immunoprecipitated CDK2-cyclin E complex. The induction of differentiation markers could not be achieved by expression of ectopic p21 alone and still required treatment with differentiation agents. Clones of F9 cells transfected with sense or antisense p21 cDNA constructs revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker, or increased fraction of cells in sub-G1 phase of the cell cycle, respectively. Consistent with this observation, whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1-1303) fusion protein on the GAL sites-containing TM promoter. This implies that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. As differentiation of F9 cells into parietal endoderm-like cells requires the cAMP signaling, the results together suggest that the cyclin-dependent kinase inhibitor p21 may promote specifically this pathway in F9 cells.     

Transcriptional suppression of nephrin in podocytes by macrophages: roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1beta and TNF-alpha markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.