ABSTRACT
The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.
Subject(s)
Blood Platelets , Megakaryocytes , Animals , Humans , Mice , ATP-Binding Cassette Transporters/metabolism , Blood Platelets/metabolism , Cell Differentiation , Megakaryocytes/metabolism , Mercaptopurine/pharmacology , Mercaptopurine/metabolism , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolismABSTRACT
Identifying the molecular mechanisms by which genome-wide association study (GWAS) loci influence traits remains challenging. Chromatin accessibility quantitative trait loci (caQTLs) help identify GWAS loci that may alter GWAS traits by modulating chromatin structure, but caQTLs have been identified in a limited set of human tissues. Here we mapped caQTLs in human liver tissue in 20 liver samples and identified 3,123 caQTLs. The caQTL variants are enriched in liver tissue promoter and enhancer states and frequently disrupt binding motifs of transcription factors expressed in liver. We predicted target genes for 861 caQTL peaks using proximity, chromatin interactions, correlation with promoter accessibility or gene expression, and colocalization with expression QTLs. Using GWAS signals for 19 liver function and/or cardiometabolic traits, we identified 110 colocalized caQTLs and GWAS signals, 56 of which contained a predicted caPeak target gene. At the LITAF LDL-cholesterol GWAS locus, we validated that a caQTL variant showed allelic differences in protein binding and transcriptional activity. These caQTLs contribute to the epigenomic characterization of human liver and help identify molecular mechanisms and genes at GWAS loci.
Subject(s)
Chromatin/metabolism , Liver/metabolism , Quantitative Trait Loci , Amino Acid Motifs , Binding Sites , Chromatin Assembly and Disassembly , Enhancer Elements, Genetic , Genetic Variation , Genome-Wide Association Study , Humans , Promoter Regions, Genetic , Protein Binding , Transcription Factors/chemistry , Transcription Factors/metabolism , TranscriptomeABSTRACT
Bile salt export pump (BSEP) adenosine triphosphate-binding cassette B11 (ABCB11) is a liver-specific ABC transporter that mediates canalicular bile salt excretion from hepatocytes. Human mutations in ABCB11 cause progressive familial intrahepatic cholestasis type 2. Although over 150 ABCB11 variants have been reported, our understanding of their biological consequences is limited by the lack of an experimental model that recapitulates the patient phenotypes. We applied CRISPR/Cas9-based genome editing technology to knock out abcb11b, the ortholog of human ABCB11, in zebrafish and found that these mutants died prematurely. Histological and ultrastructural analyses showed that abcb11b mutant zebrafish exhibited hepatocyte injury similar to that seen in patients with progressive familial intrahepatic cholestasis type 2. Hepatocytes of mutant zebrafish failed to excrete the fluorescently tagged bile acid that is a substrate of human BSEP. Multidrug resistance protein 1, which is thought to play a compensatory role in Abcb11 knockout mice, was mislocalized to the hepatocyte cytoplasm in abcb11b mutant zebrafish and in a patient lacking BSEP protein due to nonsense mutations in ABCB11. We discovered that BSEP deficiency induced autophagy in both human and zebrafish hepatocytes. Treatment with rapamycin restored bile acid excretion, attenuated hepatocyte damage, and extended the life span of abcb11b mutant zebrafish, correlating with the recovery of canalicular multidrug resistance protein 1 localization. CONCLUSIONS: Collectively, these data suggest a model that rapamycin rescues BSEP-deficient phenotypes by prompting alternative transporters to excrete bile salts; multidrug resistance protein 1 is a candidate for such an alternative transporter. (Hepatology 2018;67:1531-1545).
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 11/metabolism , Bile/metabolism , Cholestasis, Intrahepatic/genetics , Hepatocytes/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics , Animals , Autophagy/genetics , Cholestasis, Intrahepatic/pathology , Female , Humans , Immunosuppressive Agents/pharmacology , Infant , Liver/pathology , Male , Mutation , Sirolimus/pharmacology , Zebrafish/metabolismABSTRACT
OBJECTIVES: Smoking patterns and cessation rates vary widely across smokers and can be influenced by variation in rates of nicotine metabolism [i.e. cytochrome P450 2A6 (CYP2A6), enzyme activity]. There is high heritability of CYP2A6-mediated nicotine metabolism (60-80%) owing to known and unidentified genetic variation in the CYP2A6 gene. We aimed to identify and characterize additional genetic variants at the CYP2A6 gene locus. METHODS: A new CYP2A6-specific sequencing method was used to investigate genetic variation in CYP2A6. Novel variants were characterized in a White human liver bank that has been extensively phenotyped for CYP2A6. Linkage and haplotype structure for the novel single nucleotide polymorphisms (SNPs) were assessed. The association between novel five-SNP diplotypes and nicotine metabolism rate was investigated. RESULTS: Seven high-frequency (minor allele frequencies ≥6%) noncoding SNPs were identified as important contributors to CYP2A6 phenotypes in a White human liver bank (rs57837628, rs7260629, rs7259706, rs150298687 (also denoted rs4803381), rs56113850, rs28399453, and rs8192733), accounting for two times more variation in in-vitro CYP2A6 activity relative to the four established functional CYP2A6 variants that are frequently tested in Whites (CYP2A6*2, *4, *9, and *12). Two pairs of novel SNPs were in high linkage disequilibrium, allowing us to establish five-SNP diplotypes that were associated with CYP2A6 enzyme activity (rate of nicotine metabolism) in-vitro in the liver bank and in-vivo among smokers. CONCLUSION: The novel five-SNP diplotype may be useful to incorporate into CYP2A6 genotype models for personalized prediction of nicotine metabolism rate, cessation success, and response to pharmacotherapies.
Subject(s)
Cytochrome P-450 CYP2A6/genetics , High-Throughput Nucleotide Sequencing/methods , Nicotine/metabolism , Polymorphism, Single Nucleotide , Sequence Analysis, DNA/methods , Gene Frequency , Genome-Wide Association Study , Humans , In Vitro Techniques , Linkage Disequilibrium , Liver/chemistry , Tissue Banks , White People/geneticsABSTRACT
We investigated whether pheophorbide A (PhA) could serve as a selective breast cancer resistance protein (BCRP) substrate (victim) to screen in vivo using fluorescent live animal imaging for transporter-mediated interactions with orally administered inhibitors (perpetrators), and whether that could be coupled with serum sampling to measure the systemic concentration of PhA with a fast-throughput in vitro fluorescent assay. PhA is a breakdown product of chlorophyll and is highly fluorescent in the near-infrared (NIR) spectrum. Whole-body NIR fluorescence was greater in the Bcrp KO compared with wild-type (WT) mice fed a regular diet containing chlorophyll and PhA, with fluorescence in WT mice confined to the intestine. PhA intestinal enterocyte fluorescence, after removing lumen contents, was greater in Bcrp knockout (KO) mice versus WT mice due to PhA enterocyte absorption and lack of PhA efflux by Bcrp. This difference was eliminated by maintaining the mice on an alfalfa (chlorophyll/PhA)-free diet. The area under the fluorescence ratio-time curve up to 6 hours (AUCFL 0-6 h) of orally administrated PhA was 3.5 times greater in the Bcrp KO mice compared with WT mice, and the PhA serum concentration was 50-fold higher in KO mice. Pretreatment with known BCRP inhibitors lapatinib, curcumin, elacridar, pantoprazole, and sorafenib, at clinically relevant doses, significantly increased PhA AUCFL 0-6 h by 2.4-, 2.3-, 2.2-, 1.5-, and 1.4-fold, respectively, whereas the area under PhA serum concentration-time curve calculated up to 6 hours (AUCSerum 0-6 h) increased by 13.8-, 7.8-, 5.2-, 2.02-, and 1.45-fold, respectively, and corresponded to their hierarchy as in vitro BCRP inhibitors. Our results demonstrate that live animal imaging using PhA can be used to identify BCRP inhibitors and to assess the potential for BCRP-mediated clinical drug-drug interactions.
Subject(s)
Chlorophyll/analogs & derivatives , Drug Interactions/physiology , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Animals , Biological Transport/physiology , Cell Line , Chlorophyll/metabolism , Dogs , Fluorescence , Madin Darby Canine Kidney Cells , Male , Mice , Mice, KnockoutABSTRACT
To understand the systemic impact of breast cancer resistance protein (Bcrp) and P-glycoprotein (Pgp) deletion, untargeted metabolomics was performed on cerebral spinal fluid (CSF) and plasma of wild-type (WT) and Pgp and Bcrp double-knockout (dKO) rats anesthetized with ketamine-xylazine. We unexpectedly found elevated ketamine levels in both CSF and plasma of dKO versus WT rats. Therefore, the effect of these transporters was investigated on the 1) oral and intraperitoneal serum pharmacokinetics (PK) of ketamine, using a liquid chromatography method (high-performance liquid chromatography with ultraviolet detection), and 2) the anesthetic effect of ketamine using a duration of loss-of-righting reflex (dLORR) test in WT, Bcrp knockout (KO), Pgp KO, and Pgp/Bcrp dKO mice. The PK data demonstrated a significantly increased oral bioavailability and serum exposure of ketamine in dKO > Pgp KO > Bcrp KO mice compared with WT mice. Intraperitoneal ketamine-induced dLORR was significantly longer in dKO > Pgp KO > Bcrp KO > WT mice compared with WT mice. Inhibition of Bcrp and Pgp in WT mice using the dual Pgp/Bcrp inhibitor elacridar increased the ketamine-induced dLORR compared with vehicle-treated mice. The ketamine intracellular concentration was significantly decreased in Madin-Darby canine kidney II BCRP/PGP cells compared with the parental cells. In total, these results demonstrate that ketamine appears to be a dual Pgp/Bcrp substrate whose PK and pharmacodynamics are affected by Pgp and Bcrp-mediated efflux.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Ketamine/pharmacology , Ketamine/pharmacokinetics , Membrane Transport Proteins/metabolism , Animals , Biological Availability , Biological Transport/physiology , Cell Line , Dogs , Madin Darby Canine Kidney Cells , Male , Mice , Mice, Knockout , Rats , Rats, Sprague-DawleyABSTRACT
ATP-binding cassette (ABC) transporters are transmembrane efflux transporters mediating the extrusion of an array of substrates ranging from amino acids and lipids to xenobiotics, and many therapeutic compounds, including anticancer drugs. The ABC transporters are also recognized as important contributors to pharmacokinetics, especially in drug-drug interactions and adverse drug effects. Drugs and xenobiotics, as well as pathologic conditions, can influence the transcription of ABC transporters, or modify their activity or intracellular localization. Kinases can affect the aforementioned processes for ABC transporters as do protein interactions. In this review, we focus on the ABC transporters ABCB1, ABCB11, ABCC1, ABCC4, and ABCG2 and illustrate how kinases and protein-protein interactions affect these transporters. The clinical relevance of these factors is currently unknown; however, these examples suggest that our understanding of drug-drug interactions will benefit from further knowledge of how kinases and protein-protein interactions affect ABC transporters.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents/metabolism , Drug Interactions/physiology , Phosphotransferases/metabolism , Animals , Biological Transport/physiology , HumansABSTRACT
Metabolism of 25-hydroxyvitamin D3 (25OHD3) plays a central role in regulating the biologic effects of vitamin D in the body. Although cytochrome P450-dependent hydroxylation of 25OHD3 has been extensively investigated, limited information is available on the conjugation of 25OHD3 In this study, we report that 25OHD3 is selectively conjugated to 25OHD3-3-O-sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD3, 25OHD3-3-O-sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD3 sulfonation was not inducible by the potent human pregnane X receptor agonist, rifampicin. The 25OHD3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common single nucleotide variant within intron 1 of SULT2A1 (rs296361; minor allele frequency = 15% in whites) and liver cytosolic SULT2A1 content as well as 25OHD3-3-O-sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to interindividual differences in vitamin D homeostasis. Finally, 25OHD3-3-O-sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile but not in urine samples. Thus, circulating concentrations of 25OHD3-3-O-sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD3 in vivo, and contribute indirectly to the biologic effects of vitamin D.
Subject(s)
Calcifediol/blood , Calcifediol/metabolism , Sulfates/metabolism , Sulfotransferases/metabolism , Vitamin D/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Cytochrome P-450 Enzyme System/metabolism , Female , Humans , Hydroxylation/physiology , Infant , Kinetics , Liver/metabolism , Male , Middle Aged , Pregnane X Receptor , Receptors, Steroid/metabolism , Young AdultABSTRACT
The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes (n = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (â¼10-fold) with â¼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures.
Subject(s)
Androgens/metabolism , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Liver/metabolism , Minor Histocompatibility Antigens/genetics , Minor Histocompatibility Antigens/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , DNA Copy Number Variations/genetics , Female , Genotype , Humans , Inactivation, Metabolic/genetics , Infant , Infant, Newborn , Male , Microsomes, Liver/metabolism , Middle Aged , Polymorphism, Single Nucleotide/genetics , Testosterone/metabolism , Young AdultABSTRACT
Cytochrome P450 2A6 CYP2A6: metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47-0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine.
Subject(s)
Cytochrome P-450 CYP2A6/genetics , Cytochrome P-450 CYP2A6/metabolism , Genetic Variation , Liver/enzymology , Tissue Banks , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Gene Expression Regulation, Enzymologic/drug effects , Genotype , Humans , Infant , Infant, Newborn , Male , Middle Aged , NADPH-Ferrihemoprotein Reductase/metabolism , Nicotine/pharmacology , Oxidoreductases/genetics , RNA, Messenger/genetics , Umbelliferones/pharmacology , Young AdultABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 engineering of the CYP3A5 *3 locus (rs776746) in human liver cell line HuH-7 (CYP3A5 *3/*3) has led to three CYP3A5 *1 cell lines by deletion of the exon 3B splice junction or point mutation. Cell lines CYP3A5 *1/*3 sd (single deletion), CYP3A5 *1/*1 dd (double deletion), or CYP3A5 *1/*3 pm (point mutation) expressed the CYP3A5 *1 mRNA and had elevated CYP3A5 mRNA (P < 0.0005 for all engineered cell lines) and protein expression compared with HuH-7. In metabolism assays, HuH-7 had less tacrolimus (all P < 0.05) or midazolam (MDZ) (all P < 0.005) disappearance than all engineered cell lines. HuH-7 had less 1-OH MDZ (all P < 0.0005) or 4-OH (all P < 0.005) production in metabolism assays than all bioengineered cell lines. We confirmed CYP3A5 metabolic activity with the CYP3A4 selective inhibitor CYP3CIDE. This is the first report of genomic CYP3A5 bioengineering in human cell lines with drug metabolism analysis.
Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Cytochrome P-450 CYP3A/genetics , Hepatocytes/metabolism , Midazolam/metabolism , Tacrolimus/metabolism , Cell Line , Humans , Liver/metabolism , Male , Middle Aged , Point Mutation/genetics , RNA, Messenger/genetics , Sequence Deletion/geneticsABSTRACT
Determining appropriate pharmacotherapy in young children can be challenging due to uncertainties in the development of drug disposition pathways. With knowledge of the ontogeny of drug-metabolizing enzymes and an emerging focus on drug transporters, the developmental pattern of the uptake transporters organic anion transporting polypeptide (OATP) 1B1 and 1B3 was assessed by relative protein quantification using Western blotting in 80 human pediatric liver specimens covering an age range from 9 days to 12 years. OATP1B3 exhibited high expression at birth, which declined over the first months of life, and then increased again in the preadolescent period. In comparison with children 6-12 years of age, the relative protein expression of highly glycosylated (total) OATP1B3 was 235% (357%) in children <3 months of age, 33% (64%) in the age group from 3 months to 2 years, and 50% (59%) in children 2-6 years of age. The fraction of highly glycosylated to total OATP1B3 increased with age, indicating ontogenic processes not only at the transcriptional level but also at the post-translational level. Similar to OATP1B3, OATP1B1 showed high interindividual variability in relative protein expression but no statistically significant difference among the studied age groups.
Subject(s)
Aging/metabolism , Liver-Specific Organic Anion Transporter 1/metabolism , Solute Carrier Organic Anion Transporter Family Member 1B3/metabolism , Age Factors , Aging/genetics , Child , Child, Preschool , Female , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Glycosylation , Humans , Infant , Infant, Newborn , Liver-Specific Organic Anion Transporter 1/genetics , Male , Protein Processing, Post-Translational , Solute Carrier Organic Anion Transporter Family Member 1B3/geneticsABSTRACT
The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Genetic Variation/genetics , Inactivation, Metabolic/genetics , Xenobiotics/metabolism , Animals , Drug Interactions/genetics , Humans , PhenotypeABSTRACT
P-glycoprotein (Pgp) [the product of the MDR1 (ABCB1) gene] at the blood-brain barrier (BBB) limits central nervous system (CNS) entry of many prescribed drugs, contributing to the poor success rate of CNS drug candidates. Modulating Pgp expression could improve drug delivery into the brain; however, assays to predict regulation of human BBB Pgp are lacking. We developed a transgenic mouse model to monitor human MDR1 transcription in the brain and spinal cord in vivo. A reporter construct consisting of â¼10 kb of the human MDR1 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line (MDR1-luc). Fluorescence in situ hybridization localized the MDR1-luciferase transgene on chromosome 3. Reporter gene expression was monitored with an in vivo imaging system following D-luciferin injection. Basal expression was detectable in the brain, and treatment with activators of the constitutive androstane, pregnane X, and glucocorticoid receptors induced brain and spinal MDR1-luc transcription. Since D-luciferin is a substrate of ABCG2, the feasibility of improving D-luciferin brain accumulation (and luciferase signal) was tested by coadministering the dual ABCB1/ABCG2 inhibitor elacridar. The brain and spine MDR1-luc signal intensity was increased by elacridar treatment, suggesting enhanced D-luciferin brain bioavailability. There was regional heterogeneity in MDR1 transcription (cortex > cerebellum) that coincided with higher mouse Pgp protein expression. We confirmed luciferase expression in brain vessel endothelial cells by ex vivo analysis of tissue luciferase protein expression. We conclude that the MDR1-luc mouse provides a unique in vivo system to visualize MDR1 CNS expression and regulation.
Subject(s)
Brain/metabolism , Genes, Reporter/physiology , Luciferases, Firefly/biosynthesis , Spine/metabolism , Transcription, Genetic/physiology , ATP Binding Cassette Transporter, Subfamily B/biosynthesis , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Diagnostic Imaging , Female , Humans , Luciferases, Firefly/genetics , Mice , Mice, TransgenicABSTRACT
CYP2C19 rs12769205 alters an intron 2 branch point adenine leading to an alternative mRNA in human liver with complete inclusion of intron 2 (exon 2B). rs12769205 changes the mRNA reading frame, introduces 87 amino acids, and leads to a premature stop codon. The 1000 Genomes project (http://browser.1000genomes.org/index.html) indicated rs12769205 is in linkage disequilibrium with rs4244285 on CYP2C19*2, but found alone on CYP2C19*35 in Blacks. Minigenes containing rs12769205 transfected into HepG2 cells demonstrated this single nucleotide polymorphism (SNP) alone leads to exon 2B and decreases CYP2C19 canonical mRNA. A residual amount of CYP2C19 protein was detectable by quantitative proteomics with tandem mass spectrometry in CYP2C19*2/*2 and *1/*35 liver microsomes with an exon 2 probe. However, an exon 4 probe, downstream from rs12769205, but upstream of rs4244285, failed to detect CYP2C19 protein in livers homozygous for rs12769205, demonstrating rs12769205 alone can lead to complete loss of CYP2C19 protein. CYP2C19 genotypes and mephenytoin phenotype were compared in 104 Ethiopians. Poor metabolism of mephenytoin was seen in persons homozygous for both rs12769205 and rs4244285 (CYP2C19*2/*2), but with little effect on mephenytoin disposition of CYP2C19*1/*2, CYP2C19*1/*3, or CYP2C19*1/*35 heterozygous alleles. Extended haplotype homozygosity tests of the HapMap Yorubans (YRI) showed both haplotypes carrying rs12769205 (CYP2C19*35 and CYP2C19*2) are under significant natural selection, with CYP2C19*35 having a higher relative extended haplotype homozygosity score. The phylogenetic tree of the YRI CYP2C19 haplotypes revealed rs12769205 arose first on CYP2C19*35 and that rs4244285 was added later, creating CYP2C19*2. In conclusion, rs12769205 is the ancestral polymorphism leading to aberrant splicing of CYP2C19*35 and CYP2C19*2 alleles in liver.
Subject(s)
Cytochrome P-450 CYP2C19/genetics , Liver/enzymology , Acetylation , Adult , Alleles , Cytochrome P-450 CYP2C19/metabolism , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Ethiopia/epidemiology , Exons , Female , Gene Frequency , Haplotypes , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Mephenytoin/pharmacokinetics , Phenotype , Phylogeny , Polymorphism, Single Nucleotide , RNA Splicing , Tandem Mass SpectrometryABSTRACT
When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.
Subject(s)
Allopurinol/pharmacology , Cytosol/enzymology , Enzyme Inhibitors/pharmacology , Liver/enzymology , Oxypurinol/pharmacology , Xanthine Oxidase/metabolism , Aldehyde Oxidase/metabolism , Allopurinol/analysis , Allopurinol/metabolism , Chromatography, High Pressure Liquid , Cytosol/drug effects , Enzyme Inhibitors/analysis , Enzyme Inhibitors/metabolism , Female , Humans , Limit of Detection , Liver/drug effects , Male , Oxypurinol/analysis , Oxypurinol/metabolism , Perfusion , Tandem Mass Spectrometry , Tissue Culture Techniques/methods , Xanthine Oxidase/antagonists & inhibitorsABSTRACT
CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may serve to engage each E2-E3 complex. Collectively, our findings underscore the interplay between protein phosphorylation and ubiquitination in ERAD and, to our knowledge, provide the very first example of gp78 substrate recognition via protein phosphorylation.
Subject(s)
Cytochrome P-450 CYP3A/metabolism , Liver/enzymology , Receptors, Autocrine Motility Factor/metabolism , Serine/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Amino Acid Sequence , Animals , Chromatography, Liquid , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A Inhibitors , Endoplasmic Reticulum-Associated Degradation , Hep G2 Cells , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Molecular Sequence Data , Mutation/genetics , Phosphorylation , Protein Kinase C/metabolism , Proteomics , RNA Interference , RNA, Small Interfering/genetics , Rats , Serine/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , UbiquitinationABSTRACT
Human liver gene regulatory (Bayesian) network analysis was previously used to identify a cytochrome P450 (P450) gene subnetwork with Aldo-keto reductase 1D1 (AKR1D1) as a key regulatory driver of this subnetwork. This study assessed the biologic importance of AKR1D1 [a key enzyme in the synthesis of bile acids, ligand activators of farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), known transcriptional regulators of P450s] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. We resequenced AKR1D1 from 98 donor livers and identified a 3'-untranslated region (UTR) (rs1872930) single nucleotide polymorphism (SNP) significantly associated with higher AKR1D1 mRNA expression. AKR1D1 3'-UTR-luciferase reporter studies showed that the variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. Consistent with AKR1D1's putative role as a driver of the P450 subnetwork, the AKR1D1 3'-UTR SNP was significantly associated with increased hepatic mRNA expression of multiple P450s (CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6) and CYP3A4, CYP2C8, CYP2C19, and CYP2B6 activities. After adjusting for multiple testing, the association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA expression and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy and adverse drug events. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of the P450 network.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Gene Expression Regulation, Enzymologic , Oxidoreductases/genetics , Alternative Splicing , Genetic Variation , Hep G2 Cells , Hepatocytes/enzymology , Humans , Liver/enzymology , Oxidoreductases/physiology , Polymorphism, Single Nucleotide , RNA, Messenger/analysis , RNA, Messenger/chemistryABSTRACT
The subarachnoid space, where cerebrospinal fluid (CSF) flows over the brain and spinal cord, is lined on one side by arachnoid barrier (AB) cells that form part of the blood-CSF barrier. However, despite the fact that drugs are administered into the CSF and CSF drug concentrations are used as a surrogate for brain drug concentration following systemic drug administration, the tight-junctioned AB cells have never been examined for whether they express drug transporters that would influence CSF and central nervous system drug disposition. Hence, we characterized drug transporter expression and function in AB cells. Immunohistochemical analysis showed P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in mouse AB cells but not other meningeal tissue. The Gene Expression Nervous System Atlas (GENSAT) database and the Allen Mouse Brain Atlas confirmed these observations. Microarray analysis of mouse and human arachnoidal tissue revealed expression of many drug transporters and some drug-metabolizing enzymes. Immortalized mouse AB cells express functional P-gp on the apical (dura-facing) membrane and BCRP on both apical and basal (CSF-facing) membranes. Thus, like blood-brain barrier cells and choroid plexus cells, AB cells highly express drug transport proteins and likely contribute to the blood-CSF drug permeation barrier.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Arachnoid/cytology , Blood-Brain Barrier/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/biosynthesis , ATP-Binding Cassette Transporters/genetics , Animals , Biological Transport/genetics , Brain/metabolism , Cell Line , Gene Expression , Haplorhini , Humans , Mice , Mice, Knockout , Neoplasm Proteins/metabolism , Spinal Cord/metabolismABSTRACT
The effects of sex, ethnicity, and genetic polymorphism on hepatic CYP2B6 (cytochrome P450 2B6) expression and activity were previously demonstrated in vitro. Race/ethnic differences in CYP2B6 genotype and phenotype were observed only in women. To identify important covariates associated with interindividual variation in CYP2B6 activity in vivo, we evaluated these effects in healthy volunteers using bupropion (Wellbutrin SR GlaxoSmithKline, Research Triangle Park, NC) as a CYP2B6 probe substrate. A fixed 150-mg oral sustained-release dose of bupropion was administered to 100 healthy volunteers comprising four sex/ethnicity cohorts (n = 25 each): Caucasian men and Caucasian, African American, and Hispanic women. Blood samples were obtained at 0 and 6 hours postdose for the measurement of serum bupropion (BU) and hydroxybupropion (HB) concentrations. Whole blood was obtained at baseline for CYP2B6 genotyping. To characterize the relationship between CYP2B6 activity and ethnicity, sex, and genotype when accounting for serum BU concentrations (dose-adjusted log(10)-transformed), analysis of covariance model was fitted in which the dependent variable was CYP2B6 activity represented as the log(10)-transformed, metabolic ratio of HB to BU concentrations. Several CYP2B6 polymorphisms were associated with CYP2B6 activity. Evidence of dependence of CYP2B6 activity on ethnicity or genotype-by-ethnicity interactions was not detected in women. These results suggest that CYP2B6 genotype is the most important patient variable for predicting the level of CYP2B6 activity in women, when measured by the metabolism of bupropion. The bupropion metabolic ratio appears to detect known differences in CYP2B6 activity associated with genetic polymorphism, across different ethnic groups. Prospective studies will be needed to validate the use of bupropion as a probe substrate for clinical use.