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E F Johnson

Publications and source records attributed to E F Johnson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Peroxisome proliferator activated receptor-alpha expression in human liver.

The peroxisome proliferator activated receptor alpha (PPAR) is a member of the steroid/hormone receptor superfamily that mediates the peroxisome proliferator-dependent transcriptional activation of genes encoding several peroxisomal and microsomal enzymes as well as peroxisome proliferation. Human liver is refractory to the pathological effects of peroxisome proliferators that are seen in mice. With the use of RNase protection assays, the ratio of hepatic PPAR alpha mRNA to beta-actin mRNA was found to be 1 order of magnitude lower in humans than that observed in mice. In addition, the isolation of human cDNA for PPAR alpha that does not encode a functional PPAR because it lacks exon 6 as a result of alternate RNA splicing suggested that this process might also diminish the expression of PPAR alpha. RNase protection analysis of total RNA revealed the presence of splice variants lacking exon 6 at significant levels in all 10 human liver samples examined. Supershift analysis using the CYP4A6-Z peroxisome proliferator response element and antisera specific for PPAR alpha revealed easily detectable amounts of PPAR alpha DNA binding activity in mouse liver lysates, whereas human liver lysates contained > 10-fold lower amounts of PPAR alpha DNA binding activity. In contrast to mouse lysates, the amount of PPAR alpha binding in human lysates was generally less than that of other unidentified proteins. These results suggest that although humans retain the coding potential for a functional receptor, the low levels of PPAR alpha expression in liver may be insufficient to compete effectively with other proteins that bind to peroxisome proliferator response elements.

Alternative Splicing↗
PubMed1998

Structure-function of cytochromes P450 and flavin-containing monooxygenases: implications for drug metabolism.

This article is a report on a symposium held at Experimental Biology '98 in San Francisco, California. Recent developments in site-directed mutagenesis, computer-modeling, and mechanistic analysis of cytochromes P450 and flavin-containing monooxygenases are described. A unifying theme is the elaboration of general approaches for understanding and predicting the function of individual forms of these enzymes. A related goal is the production of soluble forms of mammalian cytochromes P450 for X-ray crystallography.

Animals↗
PubMed1997

Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modification of its N-terminus.

A hydrophobic segment present in the N-terminus of microsomal P450s is thought to serve as a membrane anchor. A variant of P450 2C3 was constructed, P450 2C3d, that lacked the putative membrane-spanning segment of the N-terminus, residues 3-20. This construct also incorporated substitutions of an alanine for 2Asp to facilitate expression in Escherichia coli and of serines for 24His and 25Gly to introduce a restriction site. P450 2C3d is expressed at relatively high levels in E. coli, 800-1200 nmol/liter of culture medium. In contrast to P450 2C3mod, which retains a membrane-spanning N-terminal sequence modified for expression in E. coli, the subcellular distribution of P450 2C3d in E. coli is dependent on the ionic strength of the buffer used for cell disruption. In low ionic strength buffers, 2C3d was mainly localized in the membrane fraction, whereas in buffers containing 1 M NaCl or 0.5 M KPi, P450 2C3d was predominantly found in the soluble fraction, indicating that deletion of the hydrophobic segment converted the intrinsic membrane protein to an extrinsic one. P450 2C3d was further modified by the incorporation of four histidine residues at the C-terminus (P450 2C3dH), and this enzyme could be purified in the absence of detergent using immobilized metal affinity chromatography following extraction from isolated membranes in high salt buffers. The catalytic properties of the purified, modified enzymes are similar to those of the native enzyme. Size-exclusion chromatography indicated that 2C3dH and 2C3d are predominantly dimers, whereas 2C3 is a larger oligomer (> 8-mer). Moreover, the detergents sodium cholate and Chaps each dissociate the dimers of 2C3dH to monomers at concentrations that do not alter the aggregation state of 2C3. These modifications are likely to facilitate attempts to crystallize the catalytic domains of microsomal P450s.

Amino Acid Sequence↗
PubMed1997

Targeted antipeptide antibodies to cytochrome P450 2C18 based on epitope mapping of an inhibitory monoclonal antibody to P450 2C51.

The epitope recognized by the inhibitory monoclonal antibody designated 2F5, which was raised against P450 2C5, was mapped to amino acids 237-260 by immunoblotting using a combination of recombinant antigens and chimeric and partial fusion proteins constructed from rabbit P450s 2C2, 2C4, 2C5, and 2C16, which are recognized by 2F5, and from 2C1 and 2C3, which are not. When the sequence of the epitope for 2F5 (amino acids 237-260) was compared with those of other rabbit 2C P450s, a single lysine residue at position 253 appeared to be a likely determinant of 2F5 immunoreactivity. Substitution of lysine for glutamic acid 253 in P450 2C3 (2C3E253K) conferred immunoreactivity and the ability of 2F5 to inhibit progesterone metabolism catalyzed by P450 2C3E253K. Sequence alignment revealed that this epitope lies in close proximity to the epitope identified for LKM-1 autoantibodies to P450 2D6. Based on these results, an antipeptide antibody was raised to the corresponding region (amino acids 252-263) of human P450 2C18. The resulting antipeptide antiserum recognizes P450 2C18 but not P450 2C8, 2C9, or 2C19. However, the antipeptide 2C18 antiserum did not inhibit 2C18-catalyzed diazepam N-demethylation. Human 2C P450s were also quantitated by immunoblot analysis in a panel of six human liver microsomes using Escherichia coli expressed P450s as standards. Analysis of immunoblots indicated that, if present, P450 2C18 was expressed at very low levels (<2.5 pmol/mg), whereas P450s 2C8, 2C9, and 2C19 were easily detected.

Amino Acid Sequence↗
PubMed1997

Diazepam metabolism by cDNA-expressed human 2C P450s: identification of P4502C18 and P4502C19 as low K(M) diazepam N-demethylases.

The present study provides a detailed kinetic analysis of diazepam metabolism by all four known members of the human P4502C subfamily expressed from their cDNAs in Escherichia coli. Both P4502C18 and P4502C19 were found to be low K(M) diazepam N-demethylases with apparent K(M) values of 24 +/- 4 microM and 21 +/- 3 microM, respectively. These values closely resemble the low K(M) component of diazepam N-demethylase activity exhibited by human liver microsomes. In addition, P4502C19 also catalyzed diazepam 3-hydroxylation with a K(M) value of 21 +/- 9 microM. Although P4502C8 was essentially inactive in catalyzing diazepam metabolism, P4502C9 catalyzed the N-demethylation with a relatively high K(M) of 80 +/- 15 microM and an overall 3- to 6-fold lower catalytic efficiency, compared with P4502C18 and P4502C19, respectively. At a substrate concentration of 10 microM, diazepam N-demethylation in a panel of human liver microsomes was inhibited 42 +/- 12% (mean +/- SD, N = 6) by a polyclonal anti-CYP2C antibody. In the same experiment, 3-hydroxylation remained unaffected (<10% inhibition). 1 microM of the CYP3A inhibitor ketoconazole inhibited 37 +/- 19% of the N-demethylation and 86 +/- 5% of 3-hydroxylation. Estimates of relative contributions to diazepam N-demethylation of P4502C9 (8 +/- 4%), P4502C18 (<2%), and P4502C19 (33 +/- 14%) and to diazepam 3-hydroxylation of P4502C19 (9 +/- 3%) based on the kinetic parameters of the recombinant enzymes and on specific contents of the individual 2C P450s determined in immunoblots are consistent with the inhibition data. In conclusion, these data confirm that both P4502C19 and P4503A are major contributors to human liver microsomal diazepam N-demethylation at low substrate concentrations, whereas P4503A is the major enzyme responsible for 3-hydroxylation.

Aged↗
PubMed1997

Heterologous expression of human drug-metabolizing enzymes.

This article is a report on a symposium held at the March 1997 meeting of the American Society for Pharmacology and Experimental Therapeutics in San Diego. Current developments in the heterologous expression of cytochrome P450, NADPH-cytochrome P450 reductase, glutathione transferase, and UDP-glucuronosyltransferase enzymes are described. Systems include bacteria, insect cells, and transient and stable mammalian cells. Uses of the products are described for discernment of which enzymes are involved in metabolism of drugs, genotoxicity assays, mutagenesis (for structure-activity relationships), large scale production of enzyme products, antibody production, and production of proteins for biophysical studies.

Enzymes↗
PubMed1996

Relationship between representation of hindpaw and hindpaw barrel subfield (HBS) in layer IV of rat somatosensory cortex.

We describe the organization of the hindpaw barrel subfield (HBS) in layer IV of rat somatosensory cortex (SI) and relate this organization to the representation of the hindpaw. The ovoid-shaped, HBS is oriented anterior to posterior and comprises barrels and barrel-like structures, the most prominent of which consist of at least five anteriorly-located elongated barrel bands. Posterior to these elongated bands is a cluster of four barrels. Two additional barrels are found, one lateral, the other medial. The lateral border is formed by a nearly continuous band that overlaps portions of the anterior elongated bands and posterior barrels. The HBS shows considerable variability in size and shape; nevertheless, the overall pattern reflects a common plan of organization. Electrophysiological mapping confirmed that hindpaw representation is somatotopically organized. The glabrous toes are represented anteriorly, the pads posteriorly, and the dorsal hairy skin of the toes and hindpaw laterally. By aligning physiological and morphological (HBS) maps according to lesion sites, our data suggest that the elongated anteriorly-located barrel bands represent the hindpaw toes, the four toe pads are represented immediately posterior followed by barrels representing the plantar pads. The representations of dorsal hairy skin of toe and dorsal hindpaw form the lateral border; the heel and ankle are represented most posterior. We interpret our findings as support that individual barrels in the HBS are associated with discrete regions of the hindpaw; however, the precise relationship of structure and function reported between the vibrissae and posteromedial barrel subfield (PMBSF) and between the forepaw and the forepaw barrel subfield (FBS) were not observed.

Animals↗
PubMed1996

Electrical stimulation of a forepaw digit increases the physiological representation of that digit in layer IV of SI cortex in rat.

We studied the physiological representation of digit three (D3) in rat somatosensory cortex (SI) before and immediately after electrical stimulation (1.5x threshold for 2 h) of the glabrous tip of D3 in anesthetized animals (n = 6). Measurements of D3 representation were also made in anesthetized non-stimulated control animals (n = 2). The post-stimulation areal measurements of D3 representation in experimental animals were statistically significantly larger than both pre-stimulation measurements in experimental animals and post-stimulation measurements in control animals. Our results suggest that short-term electrical stimulation is sufficient to expand the D3 representation in each of the experimental animals, while the maps in non-stimulated controls showed little variation. The fact that these studies were carried out in anesthetized animals suggests that the results are independent of the state of the animal. The present findings emphasize the importance of afferent input in modulating cortical organization.

Afferent Pathways↗
PubMed1996

Regulation of the human bilirubin UDP-glucuronosyltransferase gene.

The human UGT1 gene is a single copy gene consisting of four common exons and more than 13 variable exons which span more than 200 kb of the human genome. A single variable exon is spliced to the four common exons to form the mRNA for synthesis of a single UDP-glucuronosyltransferase (UGT) isoenzyme. Treatment of humans or hepatoma cell lines with drugs such as phenobarbital causes the induction of hepatic bilirubin UGT by increased transcription from the UGT1 gene. The upstream region of UGT1*1 (bilirubin UGT) was sequenced and found to contain consensus sequences for several transcriptional regulatory elements including a 'BARBIE box'. An unusual 'TATA' promoter sequence A(TA)6TAA was also observed. The 5' region flanking the UGT1*1 exon when cloned into reporter constructs and transfected into four cells lines was capable of promoting reporter gene expression, but not when transfected into monkey kidney cell fibroblasts (COS-7 cells) indicating a cell specific expression. Sequential deletion of the 5' flanking region in the plasmid constructs did not cause any significant reduction in reporter expression. Treatment of cells transfected with these plasmid constructs with drugs did not cause a significant increase in reporter expression except with retinoic acid plus WY 14643. Introduction of an additional two base pairs (TA) into the 'TATA' box of the 5' gene sequence (as observed in Gilbert's patients) did not significantly change reporter expression levels. The regulation of the biliruibin UGT gene by drugs is not yet understood and it will be important to identify additional genetic elements possibly further than -2kb upstream of the UGT1*1 coding region, which regulate the expression of this gene.

Base Sequence↗
PubMed1996

Role of the peroxisome proliferator-activated receptor in cytochrome P450 4A gene regulation.

Cytochrome P450s of the 4A subfamily generally catalyze the omega-hydroxylation of fatty acids. The induction of P450 4A enzymes by peroxisome proliferators or fatty acids is mediated by peroxisome proliferator-activated receptors (PPARs), which are members of the nuclear receptor family that regulates the expression of genes that control fatty acid synthesis, storage, and catabolism. PPARs bind as heterodimers with another member of the nuclear receptor family, the retinoid X receptor (RXR), to peroxisome proliferator response elements (PPREs) in the P450 4A1 and 4A6 genes. PPREs comprise two overlapping motifs for nuclear receptor binding. One motif consists of an imperfect, direct repeat of two copies of the nuclear receptor core binding site, AGGTCA, separated by a single nucleotide (a DR1 motif) that is recognized by other dimeric nuclear receptor complexes such as HNF-4 or ARP-1. A consensus sequence flanking the DR1 motif together with the 5' core binding site of the DR1 motif constitutes a second, overlapping motif resembling recognition elements for monomeric nuclear receptors, such as Rev-ErbA and the melatonin receptors. PPARs bind to the latter motif. The tripartite nature of PPREs together with imperfections in the core sites of DR1 motif confers specificity for PPAR alpha/RXR alpha binding to PPREs relative to other nuclear receptors.

Animals↗
PubMed1995

A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli.

Human cytochrome P450s 2C8, 2C9, 2C18, and 2C19 and rabbit cytochrome P450s 2C1, 2C2, 2C4, 2C5, and 2C16 were expressed from their respective cDNAs in Escherichia coli as chimeric enzymes in which a portion of the N-terminal membrane anchor sequence was replaced with a modified sequence derived from P450 17A. For 2C1 and 2C2 removal of the extraneous 3'-untranslated sequence allowed the successful expression of constructs that were unproductive in its presence. The levels of expression varied from 180 to 1500 nmol/liter of culture and the addition of delta-aminolevulinic acid to the culture media increased the amount of spectrally detectable P450 for several of these enzymes 2- to 10-fold. The catalytic properties of the modified human 2C P450s expressed in E. coli were concordant with previously published data for several marker substrates including (S)-mephenytoin for P450 2C19, tolbutamide and tetrahydrocannabinol (THC) for P450 2C9, and taxol for P450 2C8. Interestingly, P450 2C19 catalyzed the 21-hydroxylation of progesterone and, to a lesser extent, catalyzed the formation of 16 alpha-hydroxyprogesterone. The rabbit enzyme P450 2C16 catalyzed the formation of 17 alpha- and 16 alpha-hydroxyprogesterone in addition to 21-hydroxylation. P450 2C19 also catalyzed the methylhydroxylation of tolbutamide and the 7-hydroxylation of THC at rates that were similar to or greater than that of P450 2C9. This work has identified important factors required for the high-level expression of 2C subfamily P450s in E. coli. The availability of these enzymes will facilitate detailed kinetic measurements for known and yet to be identified substrates.

Alleles↗
PubMed1995

Novel sequence determinants in peroxisome proliferator signaling.

The gene encoding cytochrome P-450 4A6 (CYP4A6) is transcriptionally activated by peroxisome proliferators. This response is dependent on a strong enhancer element (Z) and weaker elements (X and -27). The peroxisome proliferator response is mediated by the binding of heterodimers containing the peroxisome proliferator-activated receptor alpha (PPAR alpha) and the retinoid X receptor alpha (RXR alpha) to these elements. These peroxisome proliferator response elements (PPREs) contain imperfect direct repeats of the nuclear receptor consensus recognition sequence with a spacing of one nucleotide (DR1) (AGGTCA N AGGTCA). This DR1 motif is seen in the binding sites for other nuclear receptor complexes, such as ARP-1, HNF-4, and RXR alpha homodimers. Mutational analysis of the Z element reveals that the DR1 motif is required for the transcriptional activation of the CYP4A6 gene by peroxisome proliferators; however, deletion of sequences immediately upstream of this motif also abolishes this response. Oligonucleotides corresponding to truncated and mutated Z elements were assayed by gel retardation for binding to RXR alpha, PPAR alpha, and ARP-1. Deletions or mutations within six nucleotides 5' of the DR1 motif dramatically diminish PPAR alpha.RXR alpha binding without reducing the binding of either RXR alpha or ARP-1 homodimers, whereas mutation or deletion of the core DR1 sequences abolishes the binding of PPAR alpha.RXR alpha heterodimers and of RXR alpha or ARP-1 homodimers. Thus, the DR1 motif in the Z element is not sufficient to constitute a PPRE. Moreover, the binding of PPAR alpha.RXR alpha to the Z element requires sequences immediately 5' of the DR1. These sequences are conserved in natural PPREs and promote binding of PPAR alpha.RXR alpha heterodimers in preference to potential competitors such as ARP-1 and RXR alpha.

Animals↗
PubMed1995

Inactivation of Escherichia coli-expressed rabbit cytochrome P-450 2C enzymes by 17 beta-substituted steroids.

The specific inactivation of rabbit cytochromes P-450 2C by 17 beta-substituted steroids has been investigated by using purified, Escherichia coli-expressed enzymes. The expressed P-450s provided a means to characterize accurately the effects of 21,21-dichloroprogesterone, 21,21-dichloropregnenolone, 21-chloro-21-fluoropregnenolone, pregn-5,20-diene-3 beta-ol and pregn-4,20-diene-3-one on progesterone hydroxylation by P-450 2C5, 2C4, 2C3 and 2C3v. Previous studies using rabbit liver microsomes had suggested that 21-chloro-21-fluoropregnenolone is a selective inactivator of 2C5, a progesterone 21-hydroxylase. Studies of the expressed P-450 2C forms showed little selectivity of 21,21-dichloroprogesterone, pregn-5,20-diene-3 beta-ol or pregn-4,20-diene-3-one, whereas 21,21-dichloropregnenolone and 21-chloro-21-fluoropregnenolone preferentially inactivate 2C5. The data indicate the importance of progesterone 21-hydroxylase activity in facilitating selective mechanism-based inactivation of 2C subfamily P-450s by 21,21-dihalogenated steroids. Studies of the inactivation of P-450 2C16, a progesterone 16 alpha-hydroxylase, by the three dihalogenated steroids yielded results consistent with previous findings of 16 alpha-hydroxylase inactivation in rabbit liver microsomes from the inbred B/J strain. Additionally, two mutants, 2C3v:V113A and 2C3v:V113A, T364N were created which confer progesterone 21-hydroxylation on 2C3v. The single mutant, a 6 beta- and 21-hydroxylase, is inactivated rapidly by all three of the 21,21-dihalogenated steroids, whereas the double mutant, a 16 alpha- and 21-hydroxylase, is preferentially inactivated by 21,21-dichloroprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A single amino acid change in the mouse peroxisome proliferator-activated receptor alpha alters transcriptional responses to peroxisome proliferators.

The mouse peroxisome proliferator-activated receptor alpha (mP-PAR alpha) can activate transcription from the CYP4A6 promoter in transient cotransfection experiments in the absence (intrinsic transactivation) or presence of added peroxisome proliferator. However, mPPAR alpha-G, in which glycine is substituted for Glu282, exhibits very low intrinsic transactivation and responds fully to added peroxisome proliferators. The two receptors, when expressed in COS-1 cells, are nuclear in localization, are expressed at similar levels, have similar stability, and bind DNA in vitro with similar efficiency. The phenotypic difference in intrinsic transactivation is not altered by overexpression of the human retinoid X receptor alpha. The mPPAR alpha-G mutant receptor displays a higher EC50 for pirinixic acid and for 5,8,11,14-eicosatetraynoic acid than the wild-type PPAR alpha. This difference in the apparent EC50 value is independent of the cell lines used and indicates that the Glu282 to glycine substitution alters the response of mPPAR alpha to peroxisome proliferators. The EC50 values obtained for each receptor with the CYP4A6 reporter construct are lower than those for a reporter derived from the acyl-CoA oxidase gene. In general, an inverse relation is evident between the apparent EC50 values and the extent of intrinsic transactivation observed. The difference in intrinsic transactivation may reflect the presence of an endogenous activator at a concentration that is not sufficient to activate the mPPAR alpha-G but that is sufficient to effect the intrinsic transactivation seen for the wild-type mPPAR alpha.

Acyl-CoA Oxidase↗
PubMed1994

Alterations of the regiospecificity of progesterone metabolism by the mutagenesis of two key amino acid residues in rabbit cytochrome P450 2C3v.

A Ser/Thr difference at position 364 underlies a phenotypic difference between naturally occurring variants of microsomal cytochrome P450 2C3 in their capacity to catalyze the 6 beta-hydroxylation of progesterone, as well as in their sensitivity to the inhibitor 16 alpha-methyl-progesterone. Position 364 of P450 2C3 maps to a substrate contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450,101. In this study, Thr-364 of P450 2C3v, a progesterone 6 beta- and 16 alpha-hydroxylase, was replaced by Gly, Asp, Asn, Val, Leu, or Ile. The latter three amino acids did not alter the regiospecificity of P450 2C3v, whereas the Gly, Asp, and Asn substitutions each produced enzymes with properties that correspond closely to the Ser mutant that catalyzes 16 alpha-hydroxylation but not 6 beta-hydroxylation. The former are distinguished from the latter amino acids by their greater hydrophobicity and size. In contrast, the 16 alpha-hydroxylase activity could be greatly diminished by the introduction of an alanine replacement for Val-113. This mutation conferred progesterone 21- and 17 alpha-hydroxylase activity to P450 2C3v at the expense of 16 alpha-hydroxylase activity, leaving the 6 beta-hydroxylase activity largely unaffected. Compound mutants displayed the additive effects of the two mutations. These results are consistent with two distinct orientations for the binding of progesterone to P450 2C3v, resulting in 6 beta- and 16 alpha-hydroxylation, respectively. The binding of progesterone in these two orientations can be modulated relatively independently by modifications of the two key amino acid residues at 113 and 364.

Amino Acid Sequence↗
PubMed1994

Recognition of uridine diphosphate glucuronosyl transferases by LKM-3 antibodies in chronic hepatitis D.

Patients with chronic hepatitis D often have liver-kidney microsomal antibodies type 3 (LKM-3). These antibodies react with several microsomal antigens that have a molecular weight of 55 KDa and an isoelectric point of about 8. We studied the molecular nature of the antigen and, by immunoscreening a human liver cDNA expression library with KM-3 sera, found that uridine diphosphate glucuronosyl transferases (UGT) appeared as candidate antigens. We confirmed the identity of UGT as an antigen by reacting the sera with recombinant rabbit liver UGT proteins. Some sera reacted with rabbit UGT-2 proteins, but UGT-1 proteins were more sensitive and specific in detecting LKM-3 autoantibodies in patient sera. Anti-UGT-1 antibodies were detected in all LKM-3 positive sera from patients with hepatitis D and 1 out of 11 patients with autoimmune hepatitis type 2. Sera from patients who had hepatitis B only did not react with UGT proteins. The UGT proteins are part of the phase II enzymes of drug metabolism and are the first such enzymes to be identified as human autoantigens.

Antibody Specificity↗
PubMed1994

Interaction of the peroxisome proliferator-activated receptor alpha with the retinoid X receptor alpha unmasks a cryptic peroxisome proliferator response element that overlaps an ARP-1-binding site in the CYP4A6 promoter.

P450 4A6 is highly induced by peroxisome proliferators in vivo. Gene transfer experiments indicate that this induction can be mediated by the mouse peroxisome proliferator-activated receptor alpha (PPAR alpha) and that it is dependent on upstream enhancer elements in the CYP4A6 gene. However, as has been seen for other peroxisome proliferator response elements (PPREs), PPAR alpha does not bind directly to a previously characterized PPRE of the CYP4A6 gene in the absence of additional proteins such as the retinoid X receptor alpha (RXR alpha). When PPAR alpha and RXR alpha are coexpressed, the overall transcription of the CYP4A6 reporter is increased, and a synergistic response to both retinoids and peroxisome proliferators is evident that is dependent on the presence of both receptors. In addition, a cryptic response element is unmasked in constructs lacking the upstream enhancers. DNase I protection assays indicate that when present together, but not singly, PPAR alpha and RXR alpha bind to a site located within 29 base pairs upstream of the CYP4A6 transcription start site. This region contains a sequence similar to that found in the apolipoprotein CIII gene that has been shown to bind RXR alpha and the orphan nuclear receptor, ARP-1. The corresponding sequence in the CYP4A6 gene also binds ARP-1. A similar sequence found in the promoter region of the rat CYP4A1 gene does not, however, bind either PPAR alpha/RXR alpha or ARP-1. Transfection of increasing amounts of the ARP-1 expression vector blocks the PPAR alpha/RXR alpha-mediated induction of transcription from the CYP4A6 promoter. Mutations that prevent the binding of either PPAR alpha/RXR alpha or ARP-1 to a double-stranded oligonucleotide corresponding to the proximal enhancer eliminate the peroxisome proliferator-induced transcriptional response observed for the promoter construct in the presence of PPAR alpha/RXR alpha, but these mutations do not eliminate the response seen when the upstream enhancers are present. These results indicate that the PPREs of the CYP4A6 gene are recognized by multiple members of the nuclear receptor family that are likely to contribute to the regulation of CYP4A6 expression in both an agonistic (RXR alpha) and an antagonistic (ARP-1) manner.

Animals↗