DBD

bHLH transcription factors:

HIF-1, AHR

HIF-1Hypoxia inducible factor

oxidative stresshypoxia

O2 homeostasis

O2

consumption (oxidative phosphorylation)

O2

delivery(tissue capillaries)

O2

concentration

„global” regulator:hypoxia inducible factor 1 HIF-1

cardiac diseasesmalignant tumourscerebrovascular diseaseschronic pulmonar diseases

changes in O2 homeostasis

basic helix - loop - helix (bHLH) proteineukaryote transcription factor super gene family

HIF - 1

II class subunit

I class subunit

ARNT 2ARNT 3 (MOP 3)MOP 9

ARNT (aryl hydrocarbon receptor nuclear translocator) HIF - 1

CLOCKAHR (aryl hydrocarbon receptor)

HIF - 1

HIF - 2 (EPAS 1, HLF, HRF, MOP 2)HIF - 3 NPAS 1 (MOP 5)NPAS 2 (MOP 4)SIM 1SIM 2

Heterodimer bHLH proteins

II class subunit

I class subunit

ARNT 2ARNT 3 (MOP 3)MOP 9

ARNT (aryl hydrocarbon receptor nuclear translocator) HIF - 1

CLOCKAHR (aryl hydrocarbon receptor)

HIF - 1

HIF - 2 (EPAS 1, HLF, HRF, MOP 2)HIF - 3 NPAS 1 (MOP 5)NPAS 2 (MOP 4)SIM 1SIM 2

Heterodimer bHLH proteins

HIF - 1: HIF -1 / HIF - 1 heterodimer in every human tissues

basic helix - loop - helix (bHLH) proteineukaryote transcription factor super gene family

HIF - 1

bHLH domain: DNA binding dimerisation

PAS (PER, ARNT, SIM) domain:substrate bindingdimerisation transactivation

HRE: hypoxia response element

HIF - 1

5’ - RCGTG - 3’ other TF binding site

HNF 4 (hepatocyte nuclear factor 4)CBP (CREB binding protein)p53p300SRC - 1TIF 2

+

Binding of HIF- 1 to DNA

oxygen concentrationredox status

+ ?

hypoxia

normoxia

expression of O2 sensitive genes

O2

sensorHIF - 1

increase

HIF - 1

HIF - 1

HIF - 1

dimer formation

hypoxia signaling

hypoxia

normoxiadegradation

HIF - 1 HIF - 1

HIF - 1

HIF - 1

expression of O2 sensitive genes

Formation of HIF-1 heterodimer is dependent on O2 dependent degradation of HIF-1

hypoxia

normoxia

HIF - 1

HIF - 1

HIF - 1

HIF - 1

VHL

VHLvon Hippel-Lindau tumor

szupresszor fehérje

O2 Pro hydroxylase Asn hydroxylase

HIF - 1 HO

HO

HIF-1signaling

proteolysisHIF - 1 VHLHO

HO

ubi

expression of O2 sensitive genes

~ 70 genes directly 1% of the human genome indirectlyvascular tone and O2 delivery:

erythropoetinVEGF (vascular endothelial growth factor)VEGF receptor FLT - 1NO synthase 2

glucose metabolism and transport:hexokinaselactate dehydrogenasepyruvate kinase

GLUT 1GLUT 3

hypoxia signaling, expression of O2 sensitive genes

Module 4: Figure HIF functions Module 4: Figure HIF functions

Cell Signalling Biology www.cellsignallingbiology.org 2007 Cell Signalling Biology www.cellsignallingbiology.org 2007

AHRAryl hydrocarbon receptor

II class subunit

I class subunit

ARNT 2ARNT 3 (MOP 3)MOP 9

ARNT (aryl hydrocarbon receptor nuclear translocator) HIF - 1

CLOCKAHR (aryl hydrocarbon receptor)

HIF - 1

HIF - 2 (EPAS 1, HLF, HRF, MOP 2)HIF - 3 NPAS 1 (MOP 5)NPAS 2 (MOP 4)SIM 1SIM 2

Heterodimer bHLH proteins

W2005 HLTH 340 Lecture B6

Nuclear receptor mediated toxicity -P450 enzyme induction by dioxins and PCBs

• polyaromatic hydrocarbons (PAH)– hydrocarbon molecules made of multiple

benzene rings– products of incomplete combustion– co-planar, lipophilic, unreactive– readily metabolized by P450 enzymes– many PAH compounds are genotoxic

• halogenated PAH (HPAH)– PAH with substituent halogen atoms

(chlorine, bromine, etc.)– rings connected by intervening dioxin ring,

furan ring, C-C bond (PCBs)– co-planar, lipophilic, unreactive– often resist Phase-1 metabolism by P450

enzymes– many HPAH are endocrine disruptor

chemicals (EDCs)

W2005 HLTH 340 Lecture B6

Dioxin contamination of 2,4,5-T herbicide (Agent Orange) in Vietnam war (1964-73)

• dioxin (TCDD) first discovered as trace contaminant in defoliant herbicide

– 2,4,5-T used to defoliate trees in VN– TCDD toxic by-product created in production

• TCDD is potent teratogenic agent– birth-defects and spontaneous abortions in

exposed VN civilians (2.1 million directly)– anencephaly, cleft palate, spinal bifida– interferes with regulatory control of embryo

CNS and head development– disrupts retinoid hormone systems (vit.A)

• also abnormal reproductive function– acts as EDC by antiestrogen effects– excessive rate of estrogen degradation

• non-genotoxic (epigenetic) carcinogen– does not react with DNA (not mutagen)– disrupts normal cell regulation

W2005 HLTH 340 Lecture B6

Genetic induction of CYP 1A1 via transcriptional activation by the ligand/AhR/ARNT complex

XRE= xenobiotic response element DRE = dioxin response element

• AhR– aryl hydrocarbon

receptor protein– binds PAH or HPAH

xenobiotics as ligands

• HSP90– heat-shock protein– chaperone protein acts

as stabilizer of AhR

• ARNT– AhR nuclear

transporter protein– forms heterodimer

complex with AhR– complex binds to

DREs

Nqo1: NAD(P)H:quinone oxidoreductaseAldh3a1: cytoplasmic aldehyde-dehydrogenase 3Ugt1a6: UDP-glucuronosyltransferase 1A6Gsta1: glutathione-transferase

„Ah gene battery”