transcriptional regulation of udp-glucuronosyltransferase ... · transcriptional regulation of drug...
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Transcriptional Regulation of Drug Metabolizing Enzymes
Anna Radominska-Pandya
Department of Biochemistry and Molecular Biology
University of Arkansas for Medical Sciences
Little Rock, Arkansas, US
October 2010; Gdansk University of Technology
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Regulation of Drug Metabolizing Enzymes
• Transcriptional Regulation
– A transcription factor is a protein that binds to specific parts of DNA and is part of the system that controls the transcription of genetic information from DNA to RNA.
– Compounds can regulate these transcription factors by permitting (acting as an activator), or preventing (acting as a repressor) the presence of RNA polymerase, the enzyme which activates the transcription.
• Constitutive Regulation
– A constitutive gene or constitutive expression describes a gene that is transcribed continually and therefore, is present at a constant level in the cell.
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Transcriptional Regulation
Phase I Enzymes
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Regulation by Ligand Activated Transcriptions Factors: Phase-I Enzymes
Nrf2 AhR PPAR LXR FXR VDR PXR CAR GR
CYP1A1 + + Ind.
CYP1A2 + Ind.
CYP1B1 +
CYP2A6 + + Ind.
CYP2B1 +
CYP2B6 - + + + Ind.
CYP2C8 + +
CYP2C9 + + + +
CYP2C19 + + +
CYP3A4 - + + + + Ind.
CYP7A1 + - - -
ALDH1 + +
ALDH3 +
NQO1 + +
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CYP3A Regulation
• Diverse drugs activate through heterodimer complex
• Protect against xenobiotics
• Cause drug-drug interactions
T.M. Wilson, S. A. Kliewer 2002:1, 259-266
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CYP3A Inducers Activate PXR
rifampicin
PCN
dexamethasone
RU486
clotrimazole
Reporter activity (fold)
troglitazone
1 3 5 7 9 11 13 15 17 19
tamoxifen
Cell-basedreporter assay
S.A. Kliewer
Human
Rabbit
Rat
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Regulation of Target Genes by PPAR:RXR Heterodimers
• Fatty acids serve as transcriptional inducers and substrates of enzymes involved in the lipid homeostasis.
steroids
metabolism & excretion
acetyl-CoA
metabolism & excretion
bile acids
PXR/CAR
SF-1 LXRFXR
PXR/CAR
FXRPXR/CAR
LXR
CYP3AsCYP2CsCYP2Bs
CYP11ACYP11BCYP21
CYP7A1
CYP3AsCYP2CsCYP2Bs
Cholesterol
SREBP
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Transcriptional Regulation
UDP-GLucuronosyltransferases
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Ritter JK, 1992
UGT1A Gene Cluster and Putative Protein Structure in Humans
Radominska-Pandya A, 1999
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Regulation by Ligand Activated Transcriptions Factors: Phase-II Enzymes
Nrf2 AhR PPAR LXR FXR VDR PXR CAR GR
SULTs + +
SULT2A1 - + + + + +
UGT1A1 + + + +
UGT1A3 + +
UGT1A4 + +
UGT1A6 + + +
UGT1A9 + + +
UGT2B1 +
UGT2B4 + +
UGT2B7
GSTs + + + +
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Major Receptors Involved in UGT Regulation
Receptor/
PartnerLigands and Inducers
Target
UGT GeneReference
hPXR/RXRPregnanolone, Corticosterone (CCS), Bile Acids
(BA), Rifampicin (Rif), Dexamethasone (Dex)
1A1
1A6
1A3
1A4
Xie/Radominska/Tukey (2001)
Xie/Radominska/Tukey (2003)
Li/Radominska (Unpublished)
Li/Radominska (Unpublished)
CAR/RXR Phenobarbital, Androstane Metabolites () 1A1
1A6
Sugatani (2001)
Xie/Radominska/Tukey (2003)
LXR/RXR Oxysterols, Bile Acids 1A3 Barbier (2006)
AhR/ARNTPolycylic Aromatic Hydrocarbons (PAHs),
Halogenated Aromatic Hydrocarbons (HAHs), β-Naphthoflavone (BNP)
1A1
1A6
1A3
1A4
Yueh (2003)
Sugatani (2004)
Li/Radominska (Unpublished)
Li/Radominska (Unpublished)
PPAR/RXR
(α and γ)Fatty Acids, Oxidized Fatty Acids, Hypolipidemic
Fibrates, Glitazones1A9 Barbier (2003)
FXR/RXRBile Acids, Chenodeoxycholic acid (CDCA),
Lithocholic Acid (LCA)
2B4
2B7
Barbier (2003)
Radominska (2005)
ER/ER Estradiol (E2)
1A3
1A10
2B17Radominska (2007)
Abbreviations: PXR (Pregnane X Receptor); RXR (Retinoid X Receptor); CAR (Constitutive Androstane Receptor); FXR (FarnesoidX Receptor); AhR (Arylhydrocarbon Receptor); ARNT (AhR Nuclear Translocator); LXR (Liver X Receptor); PPAR (PeroxisomeProliferator-Activated Receptor); ER (Estrogen Receptor)
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Pregnane X Receptor (PXR) and Constitutive Androstane Receptor
(CAR)
Regulation of UGT1A1 and UGT1A6 Expression
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Xie, et al. Nature 2000. 406, 435-439
Xie, et al. PNAS 2001. 98, 3375-3380
Creation of Transgenic Mice Expressing hPXRand Constitutively Activated hPXR (VP-hPXR)
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Northern and Western Blot Analysis of UGT1A Expression
A. Activation of UGT1A6, UGT1As, and CYP3A11 mRNA in Alb-VP-hPXRtransgenic mice visualized by Northern Blot
B. Activation of UGT1A mRNA by Rif in Alb-hPXR transgenic mice visualized by Northern Blot
C. Liver microsomes were prepared from wild type and VP-hPXR mice and subjected to Western Blot analysis using anti-UGT1A and specific anti-UGT1A1 antibodies
Xie, et al. PNAS 2002.
C
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A. Substrates for UGT1A Family
B. Substrates for UGT1A9
Increased UGT1A Expression: Increased Glucuronidation
• Glucuronidation activities were measured for:
– Xenobiotics:• pNP, PhIP
– Corticosteroids:• Corticosterone (CCS),
Cortisone, Cortisol
– Thyroid hormones:• 3,3’,5’-triiodothyronine (rT3),
thyroxine (T4)
Xie, et al. PNAS 2002.
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Summary
• The mechanism of PXR/CAR mediated induction of UGT1A1 and 1A6 was delineated (Xie, et al. PNAS 2002.)
• VP-hPXR mice provide a “gain-of-function” in vivo model to help identify hPXR target genes
• UGT1A1 and 1A6 were identified as PXR/CAR target genes• Both phenobarbital and rifampicin induce UGT1A1 expression
via CAR and PXR– This explains the effectiveness of these drugs in treating Crigler-Nijjar
disease and Gilbert syndrome• Both diseases lead to hyperbilirubinemia (accumulation of bilirubin in serum)
• hPXR represents a key protective mechanism of UGT up-regulation to insure the elimination of:– Bilirubin– Thyroxine– Corticosteroids– Some carcinogens
• UGT1A3 and 1A4 may be gene targets for PXR
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Suppression of UGT2B7 mRNA expression by FXR and Lithocholic
Acid (LCA)
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LCA Suppresses the UGT2B7 Gene Expression in Caco-2 Cells as Demonstrated By Real Time RT-
PCR
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Suppression of UGT2B7 mRNA by atRA and 9-cis RA and the Effect of the RAR Antagonist, TTNPB
atRA - all trans Retinoic Acid
TTNPB - (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoic acid
RAR - Retinoic Acid Receptor
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Localization of the FXR Response Region in the Human 1.3 kb UGT2B7 Promoter by Progressive
Deletion Analysis
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NFRE in the UGT2B7 Promoter is Required for Suppression by FXR
• ApoA1 (Apoprotein A1)– Negative FXR Response element (NFRE) – Novel Element: GATCCTTTGAACTCT
• UGT2B7– Putative Negative FXR response element (NFRE)– Consensus sequence: GATCCTTGAACTCT - 148 bp upstream of the
transcription start site
GATCCTTGATATTA
LuciferaseP-1328
NFREBARE
P-1315 NFREΔLuciferaseBARE
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Mutation of the NFRE Relieves UGT2B7 Inhibition
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EMSA Binding Analysis Confirms the Role of NFRE in UGT2B7 Gene Suppression
A. Labeled NFRE ProbeB. Labeled IR-1 Probe C. Labeled NFRE Probe NFRE with Mutant
IR-1 and CREB oligonucleotides used as non-specific competitors
• Competition experiments were performed by adding a 0, 50, and 100-fold molar excess of the indicated unlabeled probes.
• Sites were 32P-labeled and used as probes and incubated with 5 ug of Caco-2 nuclear extract in gel mobility shift assay.
• The specific FXR/RXR complex is indicated by an arrow.
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Conclusions
• Human UGT2B7 is a target gene of LCA-activated FXR
– UGT2B7 transcription is suppressed by LCA
• LCA suppression occurs via FXR binding to a novel NFRE in the UGT2B7 promoter
• Suppression of UGT2B7 expression might occur under pathological conditions that are known to produce significant levels of LCA
– Ex) liver damage and/or cholestasis,
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Practice Questions