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

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.

Transcriptional Regulation

Phase I Enzymes

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 + +

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

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

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

Transcriptional Regulation

UDP-GLucuronosyltransferases

Ritter JK, 1992

UGT1A Gene Cluster and Putative Protein Structure in Humans

Radominska-Pandya A, 1999

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 + + + +

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)

Pregnane X Receptor (PXR) and Constitutive Androstane Receptor

(CAR)

Regulation of UGT1A1 and UGT1A6 Expression

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)

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

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.

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

Suppression of UGT2B7 mRNA expression by FXR and Lithocholic

Acid (LCA)

LCA Suppresses the UGT2B7 Gene Expression in Caco-2 Cells as Demonstrated By Real Time RT-

PCR

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

Localization of the FXR Response Region in the Human 1.3 kb UGT2B7 Promoter by Progressive

Deletion Analysis

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

Mutation of the NFRE Relieves UGT2B7 Inhibition

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.

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,

Practice Questions