Download O - Belgian Thyroid Club

Thyroid and hydrogen peroxide (H2O2): Duox1 and
Duox2 as novel actors in the thyroid pathophysiology
Xavier De Deken
IRIBHM
Université Libre de Bruxelles (U.L.B)
Brussels, Belgium
• Part 1 : Duox H2O2 generators: Tonic control of the
thyroid hormone synthesis
• Part 2 : Duox2 deficiency: New cause of congenital
hypothyroidism
De Deken X.
29/05/2010
• Part 1 : Duox H2O2 generators: Tonic control of the
thyroid hormone synthesis
• Part 2 : Duox2 deficiency: New cause of congenital
hypothyroidism
De Deken X.
29/05/2010
Thyroid hormone synthesis
Tg
TgI
Apical Pole
O2
H2O2
I-
TPO
e-
I-
NADP+ + H+
I-
NADPH
Pentose Cycle
Na+
Basal Pole
NIS
K+
I-
Na+/K+
ATPase
Na+
K+
T4 + T3
De Deken X.
29/05/2010
The thyroid H2O2 generating system
• 1971 : Measurement of thyroid H2O2 (Bénard, Brault) / NADPH-dependent (Dumont)
• 1981 : Cyto-localization of H2O2 at the apex of thyrocytes (Björkman, Van sande)
• 1984 : Calcium-dependent H2O2 system (Virion)
• 1985 : NADPH oxidase complex (Nakamura)
• 1989 : Primary product is H2O2 not superoxide (Dupuy)
• 1994 : FAD containing complex (diphenyleneiodonium-sensitive) (Dupuy)
• 1999 : Purification and cloning from pig thyroid particles of p138Tox (partial Duox2
sequence) (Dupuy)
• 2000 : Screening of human thyroid cDNA libraries with the Nox2 cDNA: Thyroid
Oxidases ThOX1 and ThOX2 (De Deken)
• 2001 : Human Genome Organization International Committee : Dual Oxidase - Duox
De Deken X.
29/05/2010
The NADPH oxidase family (1999-2001)
Nox1-1999
Function
Tissue
Colon, Prostate Cell proliferation ?
Uterus
Proton channel ?
Nox2-1986
Phagocytes
Immune defense
Nox3-2001
Inner ear
Fetal kidney
Otoconia formation
Embryogenesis ?
Nox4-2000
Kidney, skin
Oxygen sensor ?
Osteoclast
Bone resorption ?
Endothelial cells Vascular tone ?
Nox5-2001
Spleen
Testis
Immune system ?
Fertilization ?
Duox1-2000
Thyroid
Lung
Iodide organification
Host defense
Duox2-2000
Thyroid
Digestive tract
Iodide organification
Host defense
Duox are not thyroid specific marker !!
De Deken X.
29/05/2010
Regulation of thyroid hormone synthesis
•
TPO activity is dependent on substrates availability:
 Iodide concentrated by NIS
 Tg secreted in the follicular lumen
 H2O2 generated by Duox
•
Under sufficient iodide supply, H2O2 availability constitute the limiting step
for thyroid hormone synthesis (Corvilain, 1991)
•
TSH increase expression of NIS, TPO and TG but not DUOX
•
TPO activity not directly regulated by TSH through post-translational
modifications
 Duox enzymatic activity must be acutely regulated
De Deken X.
29/05/2010
2006 - Identification of the Duox maturation factors: DuoxA1 / DuoxA2
From 2000: No functional reconstitution of
Duox activity in non-thyroid cells
 Additional factor absolutely required
Outside
Inside
Chromosome: 15q15.3
DUOX2
34
DUOXA2
1
21.500 bp
6 8
4.000 bp
DUOX1
DUOXA1
1
12.000 bp
34
35.000 bp
De Deken X.
29/05/2010
DuoxA-based functional assay (Rigutto, 2007)
Specific activity of Duox enzyme: H2O2 production normalized to Cos-7 cell surface
expression of the Duox proteins
Cellular transfection :
Duox + DuoxA cDNA
Stimulation by different agents
Protein expression by the cells
H2O2 quantification in the incubation medium :
FACS analysis of cells :
Fluorimetric
measurement
De Deken X.
29/05/2010
Thyroid hormone synthesis
Tg
TgI
Apical Pole
O2
H2O2
I-
TPO
Ionomycin
+
e-
I-
NADP+
+
H+
Ca2+
NADPH
DAG
I-
Pentose Cycle
IP3
Na+
Basal Pole
NIS
K+
I-
+
cAMP
PMA
Na+/K+
ATPase
Na+
Forskolin
K+
T4 + T3
De Deken X.
29/05/2010
Calcium regulation
• Absence of Ca2+ in the reaction buffer  No H2O2 production
• Mutation of Glu to Gln at position 12 of EF-Hand abolish the H2O2 generation:
I=1µM Ionomycin (Ca2+ ionophore)
 Duox1 and Duox2 activities are Ca2+-dependent
De Deken X.
29/05/2010
PMA – PKC regulation : Duox2
• Dose/response with increasing PMA concentrations:
• Phosphorylation status on Duox2 with 32P Labeling:
C
F
PMA 5µM 100 10 1
DA2
10 20 30 nM nM nM
32P
250
150
AntiDuoxs
 Duox2 is activated by PMA – EC50=0.8nM
 PMA increase phosphorylation of Duox2
De Deken X.
29/05/2010
cAMP-dependent activation of Duox1
H2O2 production of Duox/DuoxA cells stimulated with 1µM ionomycin, 1µM forskolin, 50µM 6MBcAMP or transfected with PKA catalytic subunit expressing plasmid :
 Duox1 activity is stimulated by PKA activation with an EC50=0.1µM Fsk
 Duox2 activity is not modulated by cAMP
De Deken X.
29/05/2010
PKA-mediated Duox1 phosphorylation
Analysis of Duox1 phosphorylation using anti-phospho-PKA substrate antibody (RXXS/T) :
 Constitutive phosphorylation of the mature form of Duox1
 Phosphorylation increased by cAMP-dependent protein kinase
De Deken X.
29/05/2010
PKA-mediated Duox1 Phosphorylations
Motif scanning [RK](2)-x-[ST] identify 3 potential PKA phospho-residues in Duox1 :
S955 – T1007 – S1217
1007
1217
K-K-V-T
R-R-R-S
955
R-R-A-S
 S955 residue phosphorylated by PKA
 S955 necessary for Duox1 response to cAMP
activation
De Deken X.
29/05/2010
Thyroid H2O2: Duox1 or Duox2 ?
•
•
Thyroid gland: Organ expressing the highest amount of Duox1 and Duox2
TSH 4d
mRNA (De Deken, 2000)
-
+
Are both proteins expressed ? YES
Duox1
Duox specific antibodies (U. Knaus, 2009)
TPO
150kDa
100kDa
Duox2
•
Are both enzymes functional ? yes
 cAMP (Duox1) and DAG (Duox2)
stimulate H2O2 generation associated
with Duox phosphorylation (Rigutto, 2009)
But: Ca2+ necessary
 Rat thyroid cell line, Pccl3, Duox1 is the
main H2O2 generator
De Deken X.
29/05/2010
Thyroid H2O2: Duox1 or Duox2 ?
•
Duox2 mRNA 2-5x > Duox1 (Pachucki, 2004)
•
PMA responsiveness of Duox2 in nanomolar range: Constitutive tonic H2O2
generation (Rigutto, 2009)
 Duox2 is the principal H2O2 generator
•
If TSH blood levels increased: Duox activity stimulated mainly via Ca2+ +
phosphorylations cAMP/DAG-dependent
 Tight regulation to avoid oxidative stress
•
Duox1: Emergency program revealed in case of Duox2 deficiency
De Deken X.
29/05/2010
• Part 1 : Thyroid hormone synthesis: Implication of the
Duox H2O2 generators
• Part 2 : Duox2 deficiency: New cause of congenital
hypothyroidism
De Deken X.
29/05/2010
Alteration of the thyroid metabolism
Hypothyroidism
 Iodide deficiency
 High iodide intake (Wolff-Chaikoff
effect)
 Auto-immune diseases (Hashimoto's
thyroiditis)
 Anti-thyroid drugs
 Pituitary (TSH) or hypothalamus (TRH)
defect
 …
Hyperthyroidism





Auto-immune diseases (Grave’s)
Toxic thyroid adenoma (Hot nodule)
Toxic multinodular goiter
Pregnancy (hCG TSH-like effect)
…
De Deken X.
29/05/2010
Congenital hypothyroidism
1/4.000 newborns
80% dysembryogenesis – 20% dyshormonogenesis
IOD (iodide organification defect): TPO / NIS / Tg
Perchlorate discharge test: >10% and <90% PIOD // >90% TIOD
25
NaClO4
20
normal
123 I uptake (%)
•
•
•
•
15
Patient 1
Patient 2
Patient 3
10
Patient 4
TIOD
5
0
0
30
60
90
120
time (min)
150
180
(Moreno, 2001)
De Deken X.
29/05/2010
DUOX2 mutations associated with congenital hypothyroidism
•
•
•
•
From 2002: 27 mutations found in ± 30 patients
Only one homozygous mutation (R434X) causing TIOD (Moreno, 2002)
11 mutations in ECD – 11 mutations in first intracellular loop – 1 in catalytic domain
Before 2008: Bi-allelic defect : Permanent CH - Mono-allelic defect : Transient CH
De Deken X.
29/05/2010
DUOX2 defect in Italy (Vigone et al. 2005)
Permanent congenital hypothyroidism with
PIOD
Proband
Neonatal Diagnosis
Age (Days)
7
380
390
400
410
420
430
440
(368) 368
TSH (0.5-8.7 mU/L)
173.2
ThOX2_h (365) QALRVCNNYWIRE---------------------NPNLNSTQEVNELLLGMASQISELEDNIVVEDLRDYWPGPGKFSRTDY
FT4 (1.5-2.4 ng/dL)
0.5
DUOX2_p (365) PALRVCNSYWIRE---------------------NPNLNSAEAVNQLLLGMASQISELEDWIVVEDLRDYWPGPGKFSRTDY
Thyroid Ab (anti-TPO, TRAb)
Neg
DUOX2_r (365) PALRVCNNYWIRE---------------------NPSLKTAQDVDQLLLGMASQISELEDRIVIEDLRDYWPGPDRYSRTDY
Ultrasonography
Enlarged
DUOX2_m (365) PALRVCNSYWIRE---------------------NPNLKTAQDVDQLLLGMASQISELEDRIVIEDLRDYWPGPERFSRTDY
Urinary
iodine
(100-400
µg/L)
-----DUOX2_ch (353) PAMRLCNSYWSRE----------------------SIEMQQEDVDDLLLGMSSQIAEREDSIVVEDLQDYWYGPLKYSRADY
Treatment (L-T4)
+
ThOX2_d (131) PALRVCNSYWLRE---------------------NANLNSAQAVDQLLLGMASQISELEDRIVVEDLRDYWPGSGKFSRTDY
ThOX1_h (359) RALRVCNSYWSRE---------------------HPSLQSAEDVDALLLGMASQIAEREDHVLVEDVRDFWPGPLKFSRTDH
Age (Days)
------DUOX1_p (359) RALRVCNSYWSRE---------------------HPNLQRAEDVDALLLGMASQIAEREDHMVVEDVQDFWPGPLKFSRTDH
TSH (mU/L)
------DUOX1_r (359) GALRVCNSYWSRE---------------------NPKLQRAEDVDALLLGMASQIAEREDHLVVEDVQDFWPGPLKFSRTDY
Urinary iodine (µg/L)
------DUOX1_m (359) GALRVCNSYWSREREPGAQTLVLLAMSVFSPLLKHPKLQRAEDVDALLLGMASQIAEREDHVVVEDMQDFWPGPLKFSRTDY
Treatment (L-T4)
------ThOX1_d (359) RALRVCNSYWSRK---------------------HPNLRRAEDVDALLLGMASQIAEREDHVVVEDVLDFWPGSLKFSRTDH
Reevaluation (1 month without treatment)
DUOX-dr (334) -AVRLCSTWWDSS-----------------------GFFADTSVEEVLMGLASQISEREDPVLCSDVRDKLFGPMEFTRRDL
Age (Years)
4
DUOX_ce (356) PALRLCQNWWNAQ-----------------------DIVKEYSVDEIILGMASQIAERDDNIVVEDLRDYIFGPMHFSRLDV
TSH (0.25-5.0 PmU/L)
6.3
Consensus (368) ALRVCNSYW RE
LQ AEDVD LLLGMASQIAERED IVVEDLRDYWPGPLKFSRTDY
FT4 (0.7-1.7 ng/dL)
1.2
Urinary iodine (µg/L)
139
Mutant protein inactive
ClO4 discharge test (<10%)
28
in functional assay
Genotype
Allele 1
Allele 2
p.R376W
p.R842X
De Deken X.
29/05/2010
DUOX2 defect in Italy (Vigone et al. 2005)
Permanent congenital hypothyroidism with
PIOD
Proband Brother
Neonatal Diagnosis
Age (Days)
7
11
380
390
400
410
420
430
440
(368) 368
TSH (0.5-8.7 mU/L)
173.2
9.6
ThOX2_h (365) QALRVCNNYWIRE---------------------NPNLNSTQEVNELLLGMASQISELEDNIVVEDLRDYWPGPGKFSRTDY
FT4 (1.5-2.4 ng/dL)
0.5
1.8
DUOX2_p (365) PALRVCNSYWIRE---------------------NPNLNSAEAVNQLLLGMASQISELEDWIVVEDLRDYWPGPGKFSRTDY
Thyroid Ab (anti-TPO, TRAb)
Neg
Neg
DUOX2_r (365) PALRVCNNYWIRE---------------------NPSLKTAQDVDQLLLGMASQISELEDRIVIEDLRDYWPGPDRYSRTDY
Ultrasonography
Enlarged
Normal
DUOX2_m (365) PALRVCNSYWIRE---------------------NPNLKTAQDVDQLLLGMASQISELEDRIVIEDLRDYWPGPERFSRTDY
Urinary
iodine
(100-400
µg/L)
-----550
DUOX2_ch (353) PAMRLCNSYWSRE----------------------SIEMQQEDVDDLLLGMSSQIAEREDSIVVEDLQDYWYGPLKYSRADY
Treatment (L-T4)
+
------ThOX2_d (131) PALRVCNSYWLRE---------------------NANLNSAQAVDQLLLGMASQISELEDRIVVEDLRDYWPGSGKFSRTDY
ThOX1_h (359) RALRVCNSYWSRE---------------------HPSLQSAEDVDALLLGMASQIAEREDHVLVEDVRDFWPGPLKFSRTDH
Age (Days)
------45
DUOX1_p (359) RALRVCNSYWSRE---------------------HPNLQRAEDVDALLLGMASQIAEREDHMVVEDVQDFWPGPLKFSRTDH
TSH (mU/L)
------18.4
DUOX1_r (359) GALRVCNSYWSRE---------------------NPKLQRAEDVDALLLGMASQIAEREDHLVVEDVQDFWPGPLKFSRTDY
Urinary iodine (µg/L)
------350
DUOX1_m (359) GALRVCNSYWSREREPGAQTLVLLAMSVFSPLLKHPKLQRAEDVDALLLGMASQIAEREDHVVVEDMQDFWPGPLKFSRTDY
Treatment (L-T4)
------+
ThOX1_d (359) RALRVCNSYWSRK---------------------HPNLRRAEDVDALLLGMASQIAEREDHVVVEDVLDFWPGSLKFSRTDH
Reevaluation (1 month without treatment)
DUOX-dr (334) -AVRLCSTWWDSS-----------------------GFFADTSVEEVLMGLASQISEREDPVLCSDVRDKLFGPMEFTRRDL
Age (Years)
4
4
DUOX_ce (356) PALRLCQNWWNAQ-----------------------DIVKEYSVDEIILGMASQIAERDDNIVVEDLRDYIFGPMHFSRLDV
TSH (0.25-5.0 PmU/L)
6.3
5.6
Consensus (368) ALRVCNSYW RE
LQ AEDVD LLLGMASQIAERED IVVEDLRDYWPGPLKFSRTDY
FT4 (0.7-1.7 ng/dL)
1.2
1.4
Urinary iodine (µg/L)
139
181
Mutant protein inactive
ClO4 discharge test (<10%)
28
12
in functional assay
Genotype
Allele 1
Allele 2
p.R376W
p.R842X
p.R376W
p.R842X
Phenotypic variability between the two sibling : Use of iodine-containing mouthwash during
pregnancy of the brother
De Deken X.
29/05/2010
DUOX2 defect in Argentina (Varela et al. 2006)
Permanent congenital hypothyroidism with PIOD
ThOX2_h
DUOX2_p
DUOX2_r
DUOX2_m
DUOX2_ch
ThOX2_d
ThOX1_h
DUOX1_p
DUOX1_r
DUOX1_m
ThOX1_d
DUOX-dr
DUOX_ce
Consensus
(30)
(30)
(30)
(30)
(30)
(19)
(1)
(24)
(24)
(24)
(24)
(24)
(2)
(23)
(30)
30
40
50
60
70
80
Case
1
90
Case 2
100
110
SLPWEVQRYDGWFNNLRHHERGAVGCRLQRRVPANYADGVYQALEEPQLPNPRRLSNAATRGIAGLPSLHNRTVLGVFFGYHV
Reevaluation (4 weeks without treatment)
SLTWEVQRYDGWFNNLRQHEHGAAGSPLRRLVPANYADGVYQALGEPLLPNPRQLSHTTMRGPAGLRSIRNRTVLGVFFGYHV
Age (Years)
5
4.5
SLPREVQRYDGWFNNLKYHQRGAAGSQLRRLVPANYADGVYQALQEPLLPNARLLSDAVSKGKAGLPSAHNRTVLGLFFGYHV
TSH (0.25-5.0 mU/L)
156
>100
SLPWEVQRYDGWFNNLKYHQRGAAGSRLRRLIPANYADGVYQALEEPLLPNPRRLSDAVAKGKAGLPSVHNRTVLGVFFGYHV
Serum T4 (nmol/L)
37.3
<12.87
NITWEVQRYDGWYNNLQHRSRGSVGSRLLRLLPANYADGVYQALQEPHVPNARQLSNAVARGPSGLPSKRNTTVLAVFFGFHV
Serum TG (µg/L)
431
131
----------------------------------------------------------------------------------ClO4- discharge test (<10%)
46
60
PISWEVQRFDGWYNNLMEHRWGSKGSRLQRLVPASYADGVYQPLGEPHLPNPRDLSNTISRGPAGLASLRNRTVLGVFFGYHV
Treatment (L-T4)
+
+
SISWEVQRFDGWYNNLMEHKWGSKGSRLQRLVPASYADGVYQPLGEPHLPNPRDLSNTAMRGPAGQASLRNRTVLGVFFGYHV
Genotype
PVSWEVQRFDGWYNNLMEHRWGSKGSRLQRLVPASYADGVYQPLREPYLPNPRHLSNRVMRGPAGQPSLRNRTVLGVFFGYHV
Allele 1
p.Q36H
p.G418fsX65
SISWEVQRFDGWYNNLMEHRWGSKGSRLQRLVPASYADGVYQPLKEPYLPNPRHLSNRVMRGSAGQPSLRNRTVLGVFFGYHV
Allele 2
p.S965fsX30
p.R885fsX3
PISWEVQRFDGWYNNLMEHKWGSKGSRLQRLVPASYADGVYQPLGEPHLPNPRDLSNAAMRGPAGQASLRNRTVLGVFFGYHV
YSQTEKQRYDGWYNNLAHPDWGSVDSHLVRKAPPSYSDGVYAMAGAN-RPSTRRLSRLFMRGKDGLGSKFNRTALLAFFGQLV
QQNEEFQRYDGWYNNLANSEWGSAGSRLHRDARSYYSDGVYSVNNSL--PSARELSDILFKGESGIPNTRGCTTLLAFFSQVV
ISWEVQRYDGWYNNL H WGS GSRL RLVPA YADGVYQ L EP LPNPR LSN V RG AGLPSLRNRTVLGVFFGYHV
Mutant protein inactive
in functional assay
De Deken X.
29/05/2010
DUOX2 defect in Japan (Maruo et al. 2008)
Transient
mild
congenital
hypothyroidism
associated with complete DUOX2 inactivation
Case 1
Neonatal Diagnosis
Age (Days)
TSH (0.1-4.3 mU/L)
FT4 (0.97-1.7 ng/dL)
Ultrasonography
Reevaluation (stop treatment)
Age (Years)
TSH (0.25-5.0 mU/L)
FT4 (0.7-1.7 ng/dL)
Genotype
Allele 1
Allele 2
15
95.4
0.43
Mild Enlargement
11
2.16
1.35
p.L479fsX3
p.K628fsX11
De Deken X.
29/05/2010
DUOX2 defect in Canada (Hoste et al. 2010)
•
•
•
•
First missense mutation associated with partial DUOX2 deletion
First published inactivating mutation in the catalytic domain of DUOX2: G1518S
Complete inactivation of DUOX2
Transient congenital hypothyroidism
Patient
Neonatal Diagnosis
Age (Days)
TSH (<5 mU/L)
FT4 (11-24 pmol/L)
Ultrasonography
Reevaluation (stop treatment)
Age (Years)
TSH (0.1-6.2 mU/L)
FT4 (7.6-15.6 pmol/L)
Genotype
Allele 1
Allele 2
31
23
3.9
Mild Enlargement
18.5
2.65
8.5
p.G1518S
p.G1172_F1548del
NADPH
De Deken X.
29/05/2010
DUOX2 defect in Canada (Hoste et al. 2010)
WT
25%
G1518S
• G1518S mutant functionally inactive in reconstituted
•
system
Correctly processed at the cell surface
12%
De Deken X.
29/05/2010
Transient or permanent congenital hypothyroidism ?
•
Genetic inter-population variability ?
•
Partial compensation by Duox1 oxidase :
•


Inactivation of DUOX2 cause mainly a PIOD (29/30 cases)

But: Duox1 mRNA 2-5x < than Duox2
Duox1 is functionally active in rat and human thyroid models (Rigutto, 2007 and
2009)
Environmental factors such as different iodine supply :

WHO: ± 2 billion in 2007 peoples concerned by iodide intake insufficient

? Deficiency in Italy and Argentina: Permanent CH

? High intake in Japan and Canada: Transient CH
 Iodide: alternative treatment to L-T4 ?
De Deken X.
29/05/2010
Conclusions
 Under sufficient iodide supply, H2O2 production is the limiting step for
thyroid hormone synthesis
 T3/T4 synthesis controls :
 Acute: Duox activity
 Chronic: TPO, NIS, Tg gene expression
 DUOX2 inactivating mutations quite frequent in Pt with CH and PIOD
(7Pt/20 - Persani, 2008)  Treatment follow-up
 Transient or persistent CH phenotype: not directly correlated to the number
of DUOX2 inactivated alleles
 Homozygous DUOXA2 deficiency in Pt with mild CH (Zamproni, 2007)
 Physiological role of Duox1 ?
De Deken X.
29/05/2010
ACKNOWLEDGMENTS
IRIBHM – ULB :
 J.E. Dumont and G. Vassart
 F. Miot
 B. Corvilain









University of Chicago :
 S. Refetoff
 H. Grasberger
U.C.L :
 M.C. Many
S. Rigutto
C. Hoste
D. Wang
Y. Song
M. Milenkovic
J. Pachucki
C. Degraef
B. Bournonville
J. Van Sande
Montreal University :
 G. van Vliet
ARC
De Deken X.
29/05/2010