The bHLHPAS protein ARNT effects on ER signalling

1
The bHLH-PAS protein ARNT - effects on ER
signalling
2
Biological Effects of Dioxin

• Toxic Effects Wasting, lymph atrophy,
  endometriosis, etc
• Teratogenic Effects
• Tumor Promotion
• Enzyme Induction
• Interference with Hormonal signaling (EDC)

3
The bHLH-PAS proteins
Dimerization DNA- and hsp90 binding
Ligand- and hsp90 binding
Examples
Metabolism of xenobiotics
Partner for AhR and HIF-1a
CNS growth/differentiation
Regulation of circadian rhythm
4
AhR Nuclear Translocator ARNT

• Obligatory partner for DR and HIF-1a
• Ligand ? DR-ARNT dimers bind to xenobiotic
  response elements (XREs) of target genes (i.e.
  P450 1A1)
• ARNT is constitutively expressed
• Nuclear localization
• 3 isoforms identified

5
ARNT isoforms

• ARNT-1 ubiquitously expressed
• ARNT-2 in developing brain, kidney, heart
• bMAL brain, muscle

bHLH
PAS1
PAS2
mARNT-1 791 aa
57
95
78
mARNT-2 712 aa
39
34
63
bMAL/mARNT-3 632 aa
6
ARNT (AhR Nuclear Translocator)
ARNT , ARNT2 and ARNT3 (bMAL)
AhR - ARNT
HIF-1a - ARNT
Clock - bMAL
Mediate the biochemical mechanism of action of
TCDD
Mediate the transcriptional response of cells to
hypoxia
Circadian control
7
Activation process of the AhR
8
ARNT regulates multiple pathways
DR
A
B
ARNT
p23
hsp90
Other nuclear receptors? Other transcription
factors?
A
B
HIF-1a
9
ERa and ARNT interact
pSG5-Flag-
pSG5-Flag-ERa
pCMV-ARNT
-
-
E2
Tam
IPanti-Flag
ARNT
ARNT
WCEs 10 of input
Western anti-ARNT
10
ER? and ARNT interact
pSG5-
pSG5-ERb
pCMV-ARNT
-
-
E2
Tam
IPERb mAb
ARNT
ARNT
WCEs 10 input
Western anti-ARNT
11
ARNT stimulates ER? activity
ARNT
TIF-2
12
ARNT stimulates ER? activity
ARNT
TIF-2
13
ARNT isoforms
14
ARNT interacts with the LBD of ERa/b
15
The C-Terminal domain of ARNT is required for
co-activation
16
ARNT is recruited to the pS2 promoter
E2
PCR products
-54
-353
17
Hypoxia and TCDD inhibit ER transcriptional
activity in HC11 cells
18
Activation of the AhR inhibits recruitment of
ARNT to E2 regulated promoters
ARNT
19
Effects of ARNT Si RNA
20
3-MC not TCDD activates E2 trans-activation
21
SUMMARY

• ARNT interacts with ligand-activated ERa and ERb
• ARNT stimulates both ERa and ERb transcriptional
  activity
• ARNT is recruited to the E2 regulated promoters
  in an ligand-dependent manner

22
ARNT regulates multiple cellular pathways
DR
ERa/b
A
B
ARNT
p23
hsp90
Other nuclear receptors? Other transcription
factors?
A
B
HIF-1a
23
Ingemar Pongratz
Katarina Pettersson
Elin Swedenborg
Hanne Pettersen
Wen Cai
Petra Berg
Pauliina Pentinen