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Activation of dual oxidases Duox1 and Duox2: Differential regulation mediated by cAMP-dependent protein kinase and protein kinase C-dependent phosphorylation


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Dual oxidases were initially identified as NADPH oxidases producing H(2)O(2) necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC(50) = 0.1 microm) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H(2)O(2) generation (phorbol 12-myristate 13-acetate EC(50) = 0.8 nm). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1-2 proteins and reveal additional phosphorylation-mediated regulation.
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Activation of Dual Oxidases Duox1 and Duox2
Received for publication, September 5, 2008, and in revised form, January 12, 2009 Published, JBC Papers in Press, January 14, 2009, DOI 10.1074/jbc.M806893200
Sabrina Rigutto
, Candice Hoste
, Helmut Grasberger
, Milutin Milenkovic
, David Communi
Jacques E. Dumont
, Bernard Corvilain
, Franc¸oise Miot
, and Xavier De Deken
From the
Institut de Recherche Interdisciplinaire en Biologie Humaine et Mole´culaire and the
Department of Endocrinology,
Hoˆpital Erasme, Universite´ Libre de Bruxelles, Campus Erasme, 1070 Brussels, Belgium and the
Department of Medicine, University
of Chicago, Chicago, Illinois 60637
Dual oxidases were initially identified as NADPH oxidases
producing H
necessary for thyroid hormone biosynthesis.
The crucial role of Duox2 has been demonstrated in patients
suffering from partial iodide organification defect caused by bi-
allelic mutations in the DUOX2 gene. However, the Duox1 func-
tion in thyroid remains elusive. We optimized a functional assay
by co-expressing Duox1 or Duox2 with their respective matura-
tion factors, DuoxA1 and DuoxA2, to compare their intrinsic
enzymatic activities under stimulation of the major signaling
pathways active in the thyroid in relation to their membrane
expression. We showed that basal activity of both Duox isoen-
zymes depends on calcium and functional EF-hand motifs.
However, the two oxidases are differentially regulated by activa-
tion of intracellular signaling cascades. Duox1 but not Duox2
activity is stimulated by forskolin (EC
M) via protein
kinase A-mediated Duox1 phosphorylation on serine 955. In
contrast, phorbol esters induce Duox2 phosphorylation via pro-
tein kinase C activation associated with high H
(phorbol 12-myristate 13-acetate EC
0.8 nM). These results
were confirmed in human thyroid cells, suggesting that Duox1 is
also involved in thyroid hormonogenesis. Our data provide, for
the first time, detailed insights into the mechanisms controlling
the activation of Duox1–2 proteins and reveal additional phos-
phorylation-mediated regulation.
Dual oxidases (Duox1 and Duox2) belong to the family of
NADPH oxidases (Nox), which is composed of five additional
enzymes: Nox1–5 (1–3). These transmembrane proteins are
characterized by a COOH-terminal NADPH oxidase catalytic
core responsible for reactive oxygen species synthesis. The
best characterized NADPH oxidase Nox2 is involved in the
leukocyte respiratory burst and activated by invading patho-
gens (4). The mechanisms controlling the Nox-mediated reac-
tive oxygen species production are multiple and complex. The
activation of Nox1 (5, 6), Nox2 (3, 7), and Nox3 (8, 9) requires
the coordinated assembly of several subunits: the association
with the transmembrane protein p22
and the recruitment
of three cytosolic proteins, the small G protein Rac, p47
NOXO1), and p67
(or NOXA1).
Duox1 and Duox2 isoenzymes are large members of the Nox/
Duox family. In addition to the catalytic core, Duox1 and
Duox2 proteins are NH
-terminally extended by an extracellu-
lar peroxidase-like domain followed by a membrane-spanning
segment and an intracellular domain comprising two canonical
EF-hand motifs (2). Duox1 and Duox2 are the sole proteins
directly generating H
(10) outside the cells, whereas small
Nox homologues are mostly superoxide generators (3). Duox
isoforms do not need to be associated with cytosolic factors to
be active but undergo a critical maturation process necessary to
acquire their active conformation at the apical cell surface of
the thyrocytes (11). The immature nonfunctional form (180
kDa) is not properly glycosylated and maintained in the endo-
plasmic reticulum compartment. Only the co-expression of the
Duox maturation factors (DuoxA1 and DuoxA2) allows func-
tional reconstitution. They are N-glycosylated proteins permit-
ting the endoplasmic reticulum exit of properly folded Duox
enzymes (12, 13). The DUOXA genes are localized near their
respective DUOX gene in a head to head orientation on chro-
mosome 15 and co-expressed with their Duox counterpart in
the same tissues (12).
Dual oxidases expressed at the apical side of surface epithelia
exposed to microorganisms, like the airways or the digestive
tract, are supposed to function as components of the innate
host defense system (14–16). However, Duox1 and Duox2
isoenzymes were initially identified as H
-generating thyroid
oxidases (1, 2). The main function of the thyroid is the uptake
and concentration of iodide from the bloodstream to synthesize
thyroid hormones (T3 and T4) in the follicular lumen (17).
produced at the apical pole of the thyrocyte is utilized by
thyroperoxidase as an electron acceptor to oxidize iodide,
covalently link oxidized iodide to tyrosines of thyroglobulin,
*This work was supported by the “Fonds National pour la Recherche Me´ di-
cale,” “Actions de Recherches Concerte´ es de la Communaute´ Franc¸aise de
Belgique,” the “Fonds National de la Recherche Scientifique,” and the Fon-
dation Van Buren. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
The on-line version of this article (available at contains
supplemental Table S1 and Figs. S1–S6.
To whom correspondence should be addressed: IRIBHM, Universite´ Libre
de Bruxelles, Campus Erasme, Bat. C., 808 route de Lennik, B-1070
Bruxelles, Belgium. Tel.: 32-2-5554151; Fax: 32-2-5554655; E-mail:
Recipients of a fellowship from the “Fonds pour la Formation a` la Recherche
dans l’Industrie et l’Agriculture.”
Recipient of research grants from the Cancer Research Foundation and the
American Thyroid Association.
Senior Research associate at the Fonds National de la Recherche
Postdoctoral researcher at the Fonds National de la Recherche Scientifique.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 11, pp. 6725–6734, March 13, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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and couple iodinated tyrosyl residues to form protein-bound
iodothyronines (T3 and T4) (18). Under physiological iodide
supply, hormonogenesis is rate-limited by the availability of
hydrogen peroxide (19). Patients presenting iodide organifica-
tion defect caused by mutations in their DUOX2 gene suffer
from transient or permanent hypothyroidism depending on the
mono- or bi-allelic character of the mutation, demonstrating
the crucial function of Duox2 in thyroid hormone synthesis
(20–25). However, the physiological meaning of the co-exist-
ence of the two dual oxidases and their respective maturation
factors in the thyroid tissue remains an open question.
In this study, we analyzed the mechanisms of activation of
Duox1 and Duox2 using Duox/DuoxA co-transfected cells
stimulated by agonists known to regulate the thyroid metabo-
lism (26, 27). Our results indicate that Duox1 and Duox2 activ-
ities are mainly calcium-dependent NADPH oxidases. More-
over, additional mechanisms governing their intrinsic activity
are different; Duox1 is positively regulated by the cAMP-de-
pendent protein kinase A (PKA)
cascade, whereas Duox2 is
highly induced by activation of protein kinase C (PKC) with
very low concentrations of PMA.
Plasmids and Mutagenesis—The wild type untagged versions
of human Duox1 (accession number AF230495) and Duox2
(accession number AF230496) cDNA were cloned from ATG to
stop codon into the vector pcDNA3 (Invitrogen). The NH
terminal hemagglutinin epitope-tagged human Duox2 (HA-
Duox2-pcDNA3.1) and the COOH-terminal c-Myc epitope-
tagged human DuoxA1 and DuoxA2 (DuoxA1-Myc-pcDNA3.1
and DuoxA2-Myc-pcDNA3.1) were described elsewhere (12). We
added an additional Rho tag to Duox1 to be able to distinguish it
from HA-Duox2 in future experiments. The Duox1 native signal
peptide was replaced by the TSH receptor signal peptide to facili-
tate the cloning step. The Rho-HA-Duox1-pcDNA3 construct
was generated as follows. The 23 first amino acids (residue 1 cor-
responding to the initiation methionine) of the human Duox1 pro-
tein (accession number AAF73921) were replaced by the signal
peptide of the human TSH receptor (MRPADLLQLVLLLDL-
PRDLGG) (accession number CAA02195) and the first 19 resi-
dues of the bovine rhodopsin (Rho) (accession number P02699)
(MNGTEGPNFYVPFSNKTGVV; two putative glycosylation sites
are underlined) (28). The cDNA corresponding to the NH
nal HA-tagged Duox1 protein (24–1551 amino acids) was cloned
just downstream of the Rho tag by insertion of an EcoRI site. The
presence of the TSH receptor signal peptide, Rho, and HA tags at
the NH
terminus of Duox1 did not affect its expression nor its
peroxide generating activity (supplemental Fig. S1). Furthermore,
the extra N-linked sugars present on the Rho tag have been very
useful to separate the two glycosylated forms of Duox1 for mass
spectrometry analysis, because it is mainly the mature highly gly-
cosylated form of Duox1 that is phosphorylated upon stimulation.
Mutations in Duox1–2 were introduced by directed mutagenesis
with the QuikChange system (Stratagene, La Jolla, CA) (primers
are described in supplemental Table S1). All of the con-
structs were verified by Big Dye Terminator cycle sequenc-
ing on an automated ABI Prism 3100 sequencer (Applied
Biosystems, Foster City, CA).
Cell Culture and Transfection—Cos-7 cells were cultured in
Dulbecco’s modified Eagle’s medium (Invitrogen) with 10% fetal
bovine serum (Invitrogen), 2% streptomycin-penicillin, 1% fungi-
zone, and 1% sodium pyruvate. For H
assay, adherent cells at
50 60% confluence were transfected in 6-well plates using
FuGENE 6 reagent (Roche Applied Science) according to the
manufacture’s protocol (ratio: 1
g of DNA for 3
l of FuGENE
6) with 500 ng of Rho-HA-Duox1-pcDNA3 and 500 ng of
DuoxA1-Myc-pcDNA3.1 or with 500 ng of HA-Duox2-
pcDNA3.1 and 500 ng of DuoxA2-Myc-pcDNA3.1. Under opti-
mal conditions, the transfection efficiency reached 20 –30% of cells
expressing Duox proteins at the cell surface detected by FACS. For
immunoprecipitation experiments, the cells seeded in 10-cm-di-
ameter dishes were transfected with 8
g of DNA and 24
Human Thyroid Primary Culture—Human thyroid tissue
was obtained from patients undergoing partial or total thyroid-
ectomy for resection of solitary cold nodules or multinodular
goiters. Only healthy, normal-looking, non-nodular tissue was
used within 30 min after surgical removal. Thyrocytes in pri-
mary culture obtained from follicles isolated by collagenase
digestion and differential centrifugation were cultured in
Dulbecco’s modified Eagle’s medium/Ham’s F-12/MCDB104
(2:1:1) medium (Invitrogen) with 1% sodium pyruvate, 40
g/ml ascorbic acid, 5
g/ml insulin, 2% streptomycin-penicil-
lin, and 1% fungizone (29, 30). Thyrocytes were seeded 5 days
before the assay. The protocol has been approved by the hospi-
tal ethics committee.
Measurement and Flow Immunocytometry Analysis
Production of H
was determined by the sensitive fluorimet-
ric method of Be´nard and Brault (31) slightly modified as pre-
viously described (11). The peroxide released from transfected
cells (Duox1/DuoxA1 or Duox2/DuoxA2) into Krebs-Ringer-
Hepes medium was accumulated in the presence of stimulating
agents for 2.5 h at 37 °C. After removing the medium, cell sur-
face expression of Duox1–2 proteins was measured by flow
cytometry (FACS). Briefly, the cells detached with phosphate-
buffered saline EDTA/EGTA (5 mM) were incubated sequen-
tially with anti-HA antibody (clone 3F10; Roche Applied Sci-
ence) and fluorescein-conjugated anti-rat IgG, both diluted
1/100 in phosphate-buffered saline, 0.1% bovine serum albu-
min. Propidium iodide (5
g/ml) staining in the second incu-
bation step was used to exclude damaged cells from subsequent
analysis. Fluorescence was analyzed using cell sorting (FACS-
can; Becton Dickinson, Erembodegem, Belgium) counting
20,000 events/sample. Relative protein expression was deter-
mined by calculating the differences in total fluorescence inten-
sity (Arbitrary Unit) between the samples and an equal-sized
population of control cells expressing only Duox1 or Duox2
constructs without their respective maturation factors. With-
out the latter, no cell surface expression of Duox could be
detected (11). H
production was normalized to cell surface
The abbreviations used are: PKA, cAMP-dependent protein kinase; PKC, pro-
tein kinase C; PMA, phorbol 12-myristate 13-acetate; WT, wild type; HA,
hemagglutinin; TSH, thyroid-stimulating hormone; FACS, fluorescence-ac-
tivated cell sorter; Fsk, forskolin; 6-MB-cAMP, N
3,5-cyclic monophosphate.
Activation Mechanisms of Duox1–2 Isoenzymes
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expression of each construct and reported as pg of H
For cultures of human thyrocytes, H
released over 90 min in
the presence of various agents was normalized to total proteins
extracted in Laemmli buffer and quantified by paper dye bind-
ing assay (ng of H
g of protein) (32).
Immunoprecipitation and Western Blot Analysis—Proteins
were extracted in lysis buffer (10 mMTris-HCl, pH 7.5, 150 mM
KCl, 0.5% Nonidet P-40, 120 mM
-mercaptoethanol, 100 mM
NaF, 2 mMEDTA, pH 8.0, 50 nMokadaic acid, 1 mMvanadate)
supplemented with a mixture of protease inhibitors (Complete;
Roche Applied Science) for1hat4°C.Thelysate was centri-
fuged 15 min at 10,000 rpm, and the supernatant was pre-
cleared with Sepharose beads (GE Healthcare). Duox1 com-
plexes were immunoprecipitated with anti-Duox antibody
(1/100) conjugated to Sepharose beads (2) and Duox2 proteins
with monoclonal anti-HA antibody precoated on agarose beads
(Clone HA-7; Sigma-Aldrich). Proteins of the immunoprecipi-
tate were separated by SDS/PAGE and transferred to nitrocel-
lulose as previously described (2). Phosphorylated Duox pro-
teins were detected using a rabbit polyclonal antibody raised
against phospho-(Ser/Thr) PKA substrate (1/1,000; Cell Signal-
ing, Danvers, MA), which is directed to the phospho motif
RXX(S/T). Fluorescent secondary antibodies (1/10,000; IRDye
800 anti-rabbit from LI-COR, Lincoln, NE) were used for image
acquisition and quantification with the Odyssey infrared imag-
ing system (LI-COR). The membrane was stripped and immu-
noblotted with the anti-Duox antibody (1/16,000) and fluores-
cent secondary antibodies (1/10,000; IRDye 680 anti-rabbit) to
quantify total Duox proteins.
Radioactive Phosphorus Incorporation—Cells maintained
24 h in serum-free phosphate-depleted medium were incu-
bated 2 h with 500
Ci (Cos-7) or 1mCi (thyrocytes) of
P]orthophosphate. The proteins were prepared as described
above, and phosphorylated proteins were detected and quanti-
fied with a Storage Phosphor Screen (GE Healthcare) scanned
with Typhoon Trio(GE Healthcare). Total Duox proteins for
each condition were measured by Odyssey infrared imaging
system coupled with the polyclonal anti-Duox antibody.
In Vitro PKA Phosphorylation Assay—Cos-7 Duox1/DuoxA1
transfected cells were cultured 24 h without serum. The pro-
teins were prepared in lysis buffer, and Duox1 complexes were
immunoprecipitated overnight with monoclonal anti-HA anti-
body precoated on agarose beads as described above. Immuno-
precipitated proteins were incubated for 10 min at 37 °C in a
l final volume that contained 20 mMHepes, pH 7.4, 0.1 mM
dithiothreitol, 10 mMMgCl
, 0.1 mM[
P]ATP (5
and 100 ng of purified catalytic subunit of protein kinase A
(Calbiochem, Gibbstown, NJ). After SDS/PAGE and Western
blotting, radioactive signals were quantified with Typhoon
Trio; Duox proteins were immunodetected with the anti-
Duox antibody and visualized with Odyssey imaging system.
Mass Spectrometry Analysis—Cos-7 cells transfected with
wild type Duox1 and DuoxA1 constructs were cultured 24 h
without serum and stimulated 30 min with 10
Duox1 complexes were immunoprecipitated with anti-Duox
antibody as described above. Duox1 proteins were separated by
7%-polyacrylamide gel electrophoresis and stained with colloi-
dal Coomassie Blue. After excision of the Duox gel bands, the
proteins were in-gel digested with trypsin or chymotrypsin, and
the resulting peptides were extracted from the gel (33). The
digested peptides were separated onto a C18 reverse phase 1
50 mm column (Vydac; Alltech Associates, Lokeren, Belgium)
and deposited onto a stainless steel target. Mass spectrometry
analysis was performed on a Quadrupole-time of flight Ultima
Global mass spectrometer equipped with a matrix-assisted
laser desorption ionization source (Micromass, Waters, Zellik,
Belgium) calibrated using the monoisotopic masses of tryptic
and chymotryptic peptides from bovine serum albumin.
Statistical Analysis—The data are presented as the means
S.D. The results were analyzed using the unpaired Student’s t
test, and p0.05 was considered statistically significant (**, p
0.01; ***, p0.001).
DuoxA-based Functional Assay to Study Duox-mediated
Generation—Heterologous systems combining DuoxA
expression have already been successfully used to characterize
Duox activity of human Duox2 natural mutants (13) and to
reconstitute a functional H
-generating system in a lung
cancer cell line (34). The originality of our study is to analyze
the specific activity of Duox1 and Duox2 isoenzymes by nor-
malizing the hydrogen peroxide production to the cell surface
expression of the respective proteins. Insertion of an HA tag in
the Duox1–2 ectodomain provides an effective means to reli-
ably estimate Duox membrane expression and compare the
activities of the two enzymes. We first validated our heterolo-
gous system: 1) Measurement of H
produced after 1
ionomycin stimulation of cells transfected with Duox/DuoxA
(constant DuoxA quantity, 50 ng) was proportional to the
quantity of transfected Duox plasmids (25–500 ng) with a pla-
teau reached at 250 –500 ng of Duox DNA probably caused by a
limited amount of DuoxA (Fig. 1A). 2) We observed a linear
relationship between Duox-mediated H
generation and the
membrane expression of Duox as measured by FACS (Fig. 1B).
The specific activity of both Duox enzymes was comparable,
and no H
was detected in cells expressing Duox or DuoxA
alone (Fig. 1A). 3) We verified that the addition of the tags did
not modify the expression and activity of Duox1–2 proteins
(supplemental Fig. S1). In all experiments, the specific activity
of the Duox enzymes is represented as the amount of H
produced normalized to their surface expression (pg H
In humans, the thyroid metabolism is under the control of
the phosphatidylinositol 4,5-bisphosphate cascade and the
cAMP cascade (26, 27). In our functional assay, 1
increased the activity of both Duox1 and Duox2 enzymes to the
same level, but raising concentrations of ionomycin from 2 to 4
Mresulted in higher activity of Duox1 than Duox2 (Fig. 2A).
The adenylate cyclase agonist, forskolin (Fsk), raised the
amount of H
generated by Duox1 but not by Duox2 with a
50% effective concentration (EC
M(Fig. 2B). More-
over PMA, a PKC activator, differentially regulated activities of
Duox1 and Duox2 (Fig. 2, Cand D). Although micromolar con-
centrations of PMA were needed to increase Duox1 activity
with an EC
of 1.8
M, a maximal activation of Duox2 enzyme
was already observed with nanomolar PMA concentrations
Activation Mechanisms of Duox1–2 Isoenzymes
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0.8 nM). The addition of Fsk or PMA to ionomycin in
the incubation medium provoked a release of H
sponding to the sum of the amounts generated by the cells
treated with the agents separately (Iono versus IonoFsk for
Duox1: p0.01; Iono versus IonoPMA for Duox1: p0.001;
Iono versus IonoPMA for Duox2: p0.01) (Fig. 2E). In con-
trast to Duox1, raising calcium concentration was not sufficient
to activate Duox2 by Fsk. In all of the conditions, 1
nyleneiodonium, a flavoprotein inhibitor, reduced the H
level produced by the two enzymes, indicating that it derives
from an NADPH oxidase (data not shown).
Regulation of Duox Activity by Calcium—Duox-mediated
generation is presumed to be regulated by binding of
to the two EF-hand motifs, located in the cytosolic por-
tion spanning transmembrane domain 1 and 2 (13, 34, 35).
Indeed, the absence of calcium ions in the H
assay abolished the Duox1–2 activity (data not shown). The
other calcium-dependent NADPH oxidase, NOX5, possesses
four calcium-binding sites arranged in two functional pairs
(36). To study the role of the two Duox EF-hands, the crucial
glutamate residue was replaced by a glutamine in the twelfth
position of the EF-hand sequence. Duox2 mutants E843Q and
E879Q completely lost the ability to produce H
in basal or
stimulated conditions but maintained a cell surface expression
similar to the wild type (WT) protein (Fig. 3). Similarly, inacti-
vation of one of the two EF-hands in Duox1 (E839Q and E875Q
Duox1 mutants) was sufficient to inactivate Duox1, although a
correct processing to the membrane occurred as for Duox2
(supplemental Fig. S2). These
results strongly suggest that the two
EF-hand motifs operate as one
functional pair necessary for Duox
activation in response to an
increase of intracellular calcium
Duox1 Activity Is Positively Modu-
lated through the cAMP Pathway
As shown in Fig. 2, Duox1-depend-
ent H
generation was positively
controlled by Fsk, contrary to
Duox2. To establish the role of the
protein kinase A, N
ladenosine-3,5-cyclic monophos-
phate (6-MB-cAMP), a site-selec-
tive PKA agonist, was used. H
produced by Duox1 was signifi-
cantly increased with either 50
6-MB-cAMP or 1
MFsk (Fig. 4A).
We also co-transfected a vector
encoding the
isoform of the PKA
catalytic subunit with WT Duox/
DuoxA constructs (37, 38). Overex-
pression of PKA for 48 h, verified by
Western blotting (data not shown),
also induced an increase of Duox1
activity. Co-expression of the PKA
subunit had only a minor effect on
Duox2 activity, and treatment with
the 6-MB-cAMP did not increase H
production by Duox2.
Motif scanning for PKA substrates based on the consensus
sequence (R/K)2X(S/T) identifies three potential PKA phos-
phorylation sites in Duox1 (Ser
, Thr
, and Ser
) (sup-
plemental Fig. S3). We analyzed the Duox1 phosphorylation
state with an anti-RXX(pS/pT) antibody that could potentially
recognize phosphorylation on Ser
and Ser
but not on
. Under basal conditions, the mature form of Duox1
(200 kDa) was already phosphorylated in Cos-7 cells (supple-
mental Fig. S4). It is noteworthy that the slower migrating form
of Duox1 (mature form) and the immature form were shifted to
200 and 190 kDa, respectively, instead of the wild type 190- and
180-kDa proteins. This phenomenon can be explained by the
addition of extra N-linked sugars on the two putative N-glyco-
sylation sites present in the Rho tag sequence as previously
described for similar TSH receptor constructs (28). Duox1
phosphorylation was stimulated by Fsk in a time-dependent
way with a maximum reached after 30 min of stimulation (sup-
plemental Fig. S4). Increased Duox1 phosphorylation was also
observed in cells treated 30 min with 6-MB-cAMP or cells over-
expressing the PKA catalytic subunit (Fig. 4B). The stimulatory
effect of Fsk was also observed on H
accumulation for 30
min (data not shown). Constitutive and PKA-induced phos-
phorylation of Duox1 was evident, in
P incorporation exper-
iments, with the detection of a major radioactive band corre-
sponding to the high molecular mass form (200 kDa) of Duox1
(supplemental Fig. S5). No increase of Duox2 phosphorylation
FIGURE 1. Intrinsic activity of Duox isoenzymes in reconstituted systems. A, measurement of H
mulation (ng) produced by Duox1/DuoxA1 or Duox2/DuoxA2 co-transfected Cos-7 cells for 2.5 h in the pres-
ence of 1
Mionomycin. Constant amount of DuoxA-Myc-pcDNA3.1 DNA (50 ng) was transfected with increas-
ing DNA amounts of Rho-HA-Duox1-pcDNA3 (white) or HA-Duox2-pcDNA3.1 (black) (0 –500 ng). Each
measurement corresponds to a transfection experiment performed in duplicate (means S.D.). B, plot of H
production against the relative Duox expression level at the cell surface. Membrane expression of Rho-HA-
Duox1 () and HA-Duox2 (f) proteins were quantified by FACS using the anti-HA antibody (A.U, arbitrary unit
of fluorescein isothiocyanate fluorescence intensity). Inset, representative histograms of a FACS experiment;
the gray areas represent cells transfected with the Duox construct alone. The percentage of cells expressing
Duox at the cell surface is indicated for each construct.
Activation Mechanisms of Duox1–2 Isoenzymes
by guest on January 13, 2016 from
could be detected under Fsk stimulation by
P incorporation
experiments (data not shown).
Identification of PKA Target Sites in Duox1—To identify the
phosphorylated sites by PKA, Duox1 protein was isolated from
Fsk-stimulated Cos-7 cells co-transfected with Duox1/DuoxA1
and subjected to mass spectrometry analysis. After trypsin or
chymotrypsin digestions, two phosphorylated peptides were
isolated and microsequenced: the peptide
, which encloses the serine 955 (underlined), and the
including the serine 1217. Three
peptides containing the threonine 1007 (
, and
) were also isolated but were not
To address the role of these potential phosphorylated resi-
dues in Duox1 activity, we replaced them with the nonphos-
phorylatable amino acid, alanine. Three single mutants (S955A,
T1007A, and S1217A) and one double mutant (S955A/S1217A)
were constructed. Cos-7 cell surface expression of T1007A and
S1217A mutants was similar to membrane expression of WT
protein, whereas S955A and S955A/S1217A were less ex-
pressed than the WT Duox1 (Fig. 5A,inset). The basal activity
of S955A Duox1 was severely impaired and was no longer stim-
ulated after Fsk treatment, whereas it still responded to iono-
mycin and PMA (Fig. 5A). Mutant T1007A presented a slightly
lower activity than the WT protein but was still positively reg-
ulated by all agonists. Interestingly, mutation S1217A gener-
ated an enzyme with increased basal activity, and the double
mutant S955A/S1217A produced a similar H
amount as
WT Duox1 in basal and stimulated conditions, except for a loss
of response to Fsk.
Phosphorylation state of Duox1 mutants in basal condition
or after Fsk stimulation was analyzed by radioactive phospho-
rus incorporation (Fig. 5B). Basal phosphorylation of the 200-
kDa mature form of S955A and S1217A Duox1 was highly
decreased compared with the WT Duox1 but was still stimu-
lated by Fsk. The T1007A mutant presented the same Fsk-de-
pendent phosphorylation pattern as WT Duox1. On the other
hand, the S955A/S1217A double mutant showed a very low
basal phosphorylation no longer stimulated by Fsk. The same
experiment was performed using the anti-phospho PKA sub-
strate antibody. Unfortunately, this antibody was unable to
recognize the Ser
and Thr
as demonstrated by the com-
plete absence of phosphorylation of the S955A mutant (supp-
lemental Fig. S6). Nevertheless, the results confirmed the
Fsk-mediated phosphorylation of serine 955. Direct phospho-
rylation of Duox1 was measured in vitro. In the presence of
purified PKA, WT and T1007A Duox1 showed robust phos-
generation in response to ionomycin, Fsk, or PMA. Meas-
urement of H
produced by Cos-7 cells co-expressing Rho-HA-Duox1/
DuoxA1 () or HA-Duox2/DuoxA2 (f) for 2.5 h normalized to Duox mem-
brane expression. The cells were stimulated during the 2.5-h period with
increasing concentrations of ionomycin (A), Fsk (B), or PMA (Cand D). Each
measurement corresponds to a transfection experiment performed in dupli-
cate (means S.D.). E,H
production of Cos-7 cells co-expressing Rho-HA-
Duox1/DuoxA1 (white) or HA-Duox2/DuoxA2 (black) stimulated during the
2.5-h period with 1
Mionomycin in combination with 1
MFsk or PMA (5
for Duox1 and 0.5 nMfor Duox2). H
produced in basal condition was con-
sidered as 100% (means S.D., n4). Statistical significances compared with
basal are indicated. **, p0.01; ***, p0.001.
FIGURE 3. Loss of Duox2 activity by mutations in EF-hand motifs. H
accumulation was performed for 2.5 h at 37 °C in the presence of 1
mycin (Iono), 1
MFsk, or 1 nMPMA from cells expressing DuoxA2 with wild
type (black), E843Q (gray), or E879Q (white) HA-Duox2. The graphs show the
means S.D. (n4). Expression of mutated Duox2 constructs at the cell
surface relative to WT is shown in the inset.
Activation Mechanisms of Duox1–2 Isoenzymes
by guest on January 13, 2016 from
phorylation with the appearance of an additional highly
phosphorylated form corresponding to the immature 190-
kDa protein (Fig. 5C). As expected, the PKA-dependent
phosphorylation of S955A, S1217A, and S955/S1217A
mutants was clearly decreased. Taken together, these results
demonstrate that Ser
and Ser
are two key residues
phosphorylated by PKA controlling Duox1 activity.
Duox2 Activity Is Modulated by the PKC—We have shown
that Duox2 activity was stimulated with nanomolar concentra-
tions of PMA (Fig. 2). To determine whether PMA-dependent
activation of Duox2 involves PKC-dependent phosphorylation,
Cos-7 cells expressing Duox2/DuoxA2 were incubated with
PKC inhibitors. One
MRo318220 or Go¨6976 specifically
inhibited PMA-stimulated H
production by 50% with no
effect in basal and ionomycin stimulated conditions (Fig. 6A).
Analysis of radioactive phosphorus incorporation showed a
time-dependent phosphorylation of Duox2, already visible
after 10 min of exposure to PMA that was prevented by
Ro318220 (Fig. 6, Band C). A 30-min treatment with 1 nMPMA
significantly increased H
generation from Duox2/DuoxA2
co-transfected cells (data not shown). No increase of Duox1
phosphorylation could be observed after PMA treatment, and
the inhibitor Ro318220 did not inhibit PMA-stimulated Duox1
activity (data not shown). These results suggest that physiolog-
ically PKC-
or -
1 isoforms participate in the phosphorylation
and activation of Duox2 and not Duox1.
Regulation of H
Generation in Human Thyroid Primary
Cultures—In the functional assay, we found that Duox1 and
Duox2 activities are both calcium-dependent but differentially
regulated by PKA and PKC. To determine the physiological
relevance of these results, we analyzed the modulation of H
production in human thyrocytes under similar stimulation
conditions. H
measurements from primary cultures
showed that basal H
accumulation (0.31 ng of H
protein) was increased by ionomycin (4.08 ng of H
protein) and not by Fsk alone (Fig. 7A). Combining Fsk and
ionomycin treatments significantly increased the H
duction to 7.16 ng of H
g of protein (Iono versus
IonoFsk, p0.001), demonstrating a permissive effect of
calcium rather an additive effect observed in the heterologous
system (Fig. 2E). Ionomycin also increased the H
amount in
response to PMA to a similar extent (Iono versus IonoPMA,
p0.001). The 190-kDa mature form of Duox proteins was
phosphorylated in basal condition and showed increased phos-
phorylation level after stimulation with Fsk or PMA as esti-
mated by
P incorporation (Fig. 7B). This Duox phosphoryla-
tion was reproduced by 1 or 10 milliunits/ml of the thyrotropin
hormone (TSH), concentrations described to stimulate either
the adenylate cyclase-cAMP pathway or the phospholipase
C-diacylglycerol-calcium cascade, respectively (27).
The biochemical function of hydrogen peroxide in thyroid
hormone synthesis has been known for decades (18). A fla-
voprotein complex was predicted to produce directly H
outside the cell with an activity mediated by NADPH and stim-
ulated by calcium (10, 35, 39, 40). In 2000, the cloning of two
cDNAs encoding novel calcium-dependent NADPH oxidases,
Duox1 and Duox2, revealed the molecular nature of the pre-
sumed thyroid H
-generating system (1, 2, 11). However,
functional studies on dual oxidases have been conducted only
recently thanks to the discovery of their maturation factors,
DuoxA1 and DuoxA2, allowing correct processing of Duox1–2
isoenzymes in heterologous systems (12, 13). In this work, we
improved a DuoxA-based functional assay to compare the specific
activities of Duox1 and Duox2, and we uncovered mechanisms of
activation that discriminate between the two oxidase activities.
Each Duox protein possesses two canonical EF-hand motifs
that are involved in the main regulation step exerted by calcium
(2, 13, 34, 41). Co-expression of Duox/DuoxA proteins in Cos-7
cells reconstitutes an H
generator with an activity acutely
stimulated by an elevation of the intracellular calcium concen-
tration. We have demonstrated that intact EF-hand sequences
are required to maintain functional Duox proteins. Moreover,
FIGURE 4. cAMP-dependent activation of Duox1. A, Cos-7 cells expressing
Rho-HA-Duox1/DuoxA1 (white) or HA-Duox2/DuoxA2 (black) were stimu-
lated for 2.5 h with 1
Mionomycin (Iono), 1
MFsk, or 50
corresponding to the time of H
accumulation. In the PKA condition, cells
transfected with a third vector coding for the PKA catalytic subunit. H
production was normalized to Duox cell surface expression, and the resulting
specific activities are expressed relative to the basal condition (set to 100). The
data are shown as the values S.D. for n indicated measurements (***, p
0.001). B, immunodetection of Duox1 phosphorylation mediated by agents
activating the cAMP cascade. The cells were treated for 30 min with either 1
MFsk or 50
M6-MB-cAMP. After anti-Duox immunoprecipitation, PKA
phosphorylation was immunodetected with the anti-RXX(pS/pT) antibody
(P-PKA) and total Duox1 with anti-Duox polyclonal antibody. In the top panel,
the columns represent PKA-mediated Duox1 phosphorylation corrected to
the total amount of immunoprecipitated Duox1. The level of phosphoryla-
tion is expressed relative to the basal phosphorylation set to 100. The PKA
condition was performed in a different experiment from Fsk and 6-MB-cAMP
Activation Mechanisms of Duox1–2 Isoenzymes
by guest on January 13, 2016 from
whereas the two dual oxidases exhibit similar activity at low
ionomycin concentration (1
M), Duox1 seems to be more effi-
cient than Duox2 in cells treated with higher ionomycin con-
centrations. This effect might be
explained by the differences in the
amino acid sequence of the Duox1
second EF-hand motif.
Recent reports have shown that,
in addition to calcium, the Nox5
enzymatic activity is also regulated
through PKC-mediated phosphory-
lations on serine and threonine res-
idues enhancing its sensitivity to
calcium (42). Using radioactive
phosphorus incorporation and anti-
phospho-PKA substrate antibody,
we demonstrated that Duox1 is also
a phosphoprotein. The enzyme is
constitutively phosphorylated, and
activation of the PKA pathway
increases its phosphorylation state
as well as its activity. Immature
Duox1–2 proteins generate essen-
tially superoxide inside the cell and
become H
generators only at the
cell surface (41). In transfected cells,
in vivo
P incorporation shows that
mainly the mature form of Duox1 is
phosphorylated in basal and stimu-
lated conditions, even if the imma-
ture form of Duox1 is more abun-
dant (Fig. 5B). These data suggest
that, in intact cells, Duox isoen-
zymes might undergo conforma-
tional changes during their process-
ing from the endoplasmic reticulum
to the plasma membrane modifying
the accessibility of key residues to
the kinase. In in vitro PKA phospho-
rylation experiments, these amino
acids could be artificially demasked
by detergents showing elevated
phosphorylation intensity for the
immature form of Duox1 (Fig. 5C).
Mass spectrometry analyses
revealed Duox1 phosphopeptides
containing the residues Ser
in co-transfected Cos-7 cells
in response to Fsk. Systematic
mutations of potential PKA-medi-
ated phosphorylation sites have
identified Ser
and Ser
major residues responsible for the
basal phosphorylation state of
Duox1. These two serines are con-
served among human, dog, pig,
mouse, and rat species. However,
rendering these amino acids non-
phosphorylatable reveals distinct effects on Duox1 specific
activity. The S1217A variant presents higher constitutive activ-
ity than the WT enzyme and still responds to cAMP cascade
FIGURE 5. Identification of PKA-mediated phosphorylation sites in Duox1. A, Duox1 activity. Cos-7 cells
expressing DuoxA1 in combination with WT (black bars), S955A (vertically hatched bars), T1007A (gray bars),
S1217A (horizontally hatched bars), or S955A/S1217A (open bars) Rho-HA-Duox1 were stimulated for 2.5 h with
Mionomycin (Iono), 1
MFsk, or 5
MPMA and H
accumulation normalized to Duox membrane expres-
sion. The inset shows the cell surface expression for each constructs relative to WT Duox1. The graph corre-
sponds to one representative experiment from four independent experiments (means S.D., n2). B, Duox1
phosphorylation. Cells expressing DuoxA1 with WT or mutant Rho-HA-Duox1 were
P-labeled and treated for
30 min with either 1
MFsk () or solvent (). In the top panel, relative densitometry of phosphorylated Duox1
corrected to the total Duox1 immunoprecipitated (non stimulated WT Duox1 considered as 100%). C,in vitro
phosphorylation of Duox1 by PKA. Each construct was tested in duplicate. The relative Duox1 phosphorylation
corrected to the total amount of Duox1 is showed on the top of the figure.
Activation Mechanisms of Duox1–2 Isoenzymes
by guest on January 13, 2016 from
activation, whereas the S955A substitution decreases basal and
Fsk-stimulated Duox1 activity. These results demonstrate that
serine 955 is the crucial residue positively regulating Duox1
activity through PKA-mediated phosphorylation. It could
increase the sensitivity of Duox1 to lower level of intracellular
calcium as has been demonstrated for Nox5 enzyme (42). The
hyperactivity of the mutant S1217A was more surprising and
suggests an inhibitory effect on the overall Duox1 activity
driven by its phosphorylation state. Our hypothesis is that in
resting cells, constitutive phosphorylation on Ser
the enzyme to limit peroxide generation, whereas PKA-medi-
ated phosphorylation on Ser
stimulates Duox1-dependent
generation. Absence of phosphorylation on this residue
in the S955A variant would decrease its sensitivity to calcium,
explaining its lower basal activity. Combination of S955A and
S1217A leads to a Duox1 phospho-null mutant with WT-like
constitutive activity but without response to PKA stimulation.
However, we cannot exclude the possibility that these muta-
tions could also be associated with conformational changes,
inactivating the enzyme as suggested by low cell surface expres-
sion of the S955A and S955A/S1217A proteins.
Duox2/DuoxA2 co-transfected cells treated with nanomolar
concentrations of PMA show increased H
generation asso-
ciated with PMA-mediated Duox2 phosphorylations. Duox2
enzyme is 1,000 times more sensitive to PMA than Duox1, sug-
gesting that in physiological conditions, the resting levels of
diacylglycerol combined with basal calcium concentration
would be sufficient to stimulate Duox2 but not Duox1. There-
fore, Duox2 would be the predominant enzyme in the thyroid
conferring constitutive activity to the system. A survey of the
Duox2 primary sequence reveals 11 (S/T)X(R/K) motifs as
potential target sites for PKC. Among them, six are not con-
served in Duox1 and constitute good candidates for PKC-me-
diated phosphorylations. Their implication on Duox2 activity
will be investigated.
The crucial role of Duox2 in the thyroid hormone biosynthe-
sis has been demonstrated in permanent congenital hypothy-
roidism caused by bi-allelic DUOX2 gene mutations (20–25).
Mono-allelic inactivation of DUOX2 gene has also been linked
FIGURE 6. PMA stimulates the activity and phosphorylation of Duox2.
A, cells co-transfected with HA-Duox2/DuoxA2 were preincubated 30 min in
Krebs-Ringer-Hepes medium containing vehicle (black bars) or PKC inhibitors:
MRo318220 (gray bars)or1
MGo¨ 6976 (open bars) before 2.5 h of stimu-
lation with 1
Mionomycin (Iono)or1nMPMA. H
accumulation was nor-
malized to Duox2 expression at the plasma membrane. The level of H
represented as a percentage of the value obtained in basal condition without
PKC inhibitor (means S.D., n6). Statistically significant inhibition is indi-
cated. ***, p0.001. B, phosphorylation by
P incorporation measured after
10, 20, or 30 min of treatment with 5
MPMA. On the top of the Western blot,
the relative amount of phosphorylated Duox2 corrected to total Duox2 pro-
tein (basal phosphorylation was considered as 100%). C, inhibition of PMA-
mediated Duox2 phosphorylation by Ro318220. The cells were preincubated
or not with 1
MRo318220 before stimulated with 100 nMPMA. Total Duox2
proteins were detected with anti-Duox antibody, and the relative Duox2
phosphorylation corrected to total Duox2 protein is represented at the top of
the figure (basal phosphorylation without PKC inhibitor was considered as
measurement and Duox phosphorylation in human thy-
rocytes. A, cells were incubated 90 min at 37 °C with 1
Mionomycin (Iono),
MFsk, or 5
MPMA. The peroxide production was normalized to the total
amount of proteins. The graphs show means S.D. of three independent
experiments performed in duplicate (***, p0.001). B, Duox1–2 phosphoryl-
ation after stimulation by TSH, Fsk, and PMA. Thyrocytes were stimulated 30
min with 1 milliunits/ml TSH or 10
MFsk or 5 min with 10 milliunits/ml TSH or
MPMA. Quantification of Duox phosphorylation corrected to total immu-
noprecipitated protein represented at the top. Basal condition performed in
Activation Mechanisms of Duox1–2 Isoenzymes
by guest on January 13, 2016 from
to milder and transient cases of neonatal hypothyroidism (43).
Recently, DUOXA2 bi-allelic mutations in a patient suffering
from permanent dyshormonogenesis have been described,
reinforcing the role of Duox2/DuoxA2 in thyroid metabolism
(44). However, the role of existing Duox1/DuoxA1 beside
Duox2 proteins in the same tissue remains obscure. Complete
inactivation of DUOX2 generally leads to partial iodide organi-
fication defect meaning that Duox1/DuoxA1 could compen-
sate the impairment of Duox2 (13, 21, 22, 24, 25). Recently, we
have demonstrated that Duox1 is the main source of hydrogen
peroxide in the rat thyroid cell line: PCCl3 (45). Our experi-
ments in human thyroid cells showing that elevation of intra-
cellular cAMP concentration activates hydrogen peroxide pro-
duction reinforce the concept of a thyroid function for Duox1.
However, this effect is observed only in combination with iono-
mycin treatment. During the completion of this manuscript,
Pacquelet et al. (46) have demonstrated an inhibitory effect on
Duox activity by the Nox1 co-activator, NOXA1, which is
relieved by calcium binding on Duox. They showed that cell
lines devoid of NOXA1 protein exhibit high basal activity of
Duox compared with the airway cells. The absence of Noxa1 in
Cos-7 cells could explain why Fsk and PMA are able to stimu-
late Duox activity independently of ionomycin treatment. The
NOXA1 transcript has been detected in thyrocytes (47), where
it might also act as a down-regulator of the thyroid H
erating system.
In humans, thyroid metabolism is under the control of the
thyrotropin hormone (TSH) through its G protein-coupled
receptor linked to adenylate cyclase-cAMP and phospholipase
C-diacylglycerol-calcium cascades (26, 27, 48–50). Because
Duox2 transcript is 2 to 5 times more abundant than Duox1
(51), we propose the following model in which the Duox2 sys-
tem would ensure the constitutive tonic generation of H
make use of any available iodide. When thyroid hormone levels
in the blood decrease, TSH concentration augments and trig-
gers the release of both G
and G
proteins followed by intra-
cellular increase of cAMP, diacylglycerol, and Ca
tions. The latter constitute the primary activator of the dual
oxidase function, which is sustained by additional phosphoryl-
ations mediated by PKA for Duox1 and PKC for Duox2. Duox1
would represent the emergency program revealed in the case of
hypothyroidism with partial iodide organification defect linked
to inactive mutated Duox2. Nevertheless, Duox1 cannot fully
take over the impairment of Duox2, probably because of too
low protein expression.
In conclusion, we have used molecular approaches to char-
acterize the mechanisms regulating the function of Duox1 and
Duox2 proteins. We have demonstrated that the activity of the
two dual oxidases is controlled via two different phosphoryla-
tion pathways, and we provide the first experimental argument
in favor of a thyroid function for the Duox1 enzyme.
Acknowledgments—We thank Chantal Degraef, Bernadette Bourn-
onville, and Virginie Imbault for excellent technical assistance and
Dr. M. Cappelo for providing the thyroid tissue.
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Activation Mechanisms of Duox1–2 Isoenzymes
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Corvilain, Françoise Miot and Xavier De
Communi, Jacques E. Dumont, Bernard
Grasberger, Milutin Milenkovic, David
Sabrina Rigutto, Candice Hoste, Helmut
Activation of Dual Oxidases Duox1 and
Enzyme Catalysis and Regulation:
doi: 10.1074/jbc.M806893200 originally published online January 14, 2009
2009, 284:6725-6734.J. Biol. Chem.
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... This ROS is generated mainly by the intracellular family of NADPH oxidases (NOX). Hydrogen peroxide, which is an essential factor for thyroid hormone biosynthesis, is produced in the thyroid gland by two isoform enzymes, i.e., dual oxidase 1 Endocrine (DUOX1) and 2 (DUOX2), belonging to NOX family, with the most convincing experimental evidence found for DUOX2 [137][138][139]. Both of them are expressed in the apical plasma membrane of thyroid follicular cells (thyrocytes) [137,138]. ...
... Hydrogen peroxide, which is an essential factor for thyroid hormone biosynthesis, is produced in the thyroid gland by two isoform enzymes, i.e., dual oxidase 1 Endocrine (DUOX1) and 2 (DUOX2), belonging to NOX family, with the most convincing experimental evidence found for DUOX2 [137][138][139]. Both of them are expressed in the apical plasma membrane of thyroid follicular cells (thyrocytes) [137,138]. The next enzyme which also plays an important role in H 2 O 2 synthesis in the thyroid is NOX4, acting intracellulary [139]. ...
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Purpose Iodine is an essential micronutrient required for thyroid hormone biosynthesis. However, overtreatment with iodine can unfavorably affect thyroid physiology. The aim of this review is to present the evidence that iodine—when in excess—can interfere with thyroid hormone synthesis and, therefore, can act as a potential endocrine-disrupting chemical (EDC), and that this action, as well as other abnormalities in the thyroid, occurs—at least partially—via oxidative stress. Methods We reviewed published studies on iodine as a potential EDC, with particular emphasis on the phenomenon of oxidative stress. Results This paper summarizes current knowledge on iodine excess in the context of its properties as an EDC and its effects on oxidative processes. Conclusion Iodine does fulfill the criteria of an EDC because it is an exogenous chemical that interferes—when in excess—with thyroid hormone synthesis. However, this statement cannot change general rules regarding iodine supply, which means that iodine deficiency should be still eliminated worldwide and, at the same time, iodine excess should be avoided. Universal awareness that iodine is a potential EDC would make consumers more careful regarding their diet and what they supplement in tablets, and—what is of great importance—it would make caregivers choose iodine-containing medications (or other chemicals) more prudently. It should be stressed that compared to iodine deficiency, iodine in excess (acting either as a potential EDC or via other mechanisms) is much less harmful in such a sense that it affects only a small percentage of sensitive individuals, whereas the former affects whole populations; therefore, it causes endemic consequences.
... Working with the NMJ in the Drosophila larva as an experimental in vivo model system, we demonstrated that both NADPH oxidases, Nox and Duox, are required for activity-induced growth (Figure 1). Both enzymes are endowed with N-terminal calcium binding EF-hand motifs, linking their activity to intracellular calcium levels, as shown for Drosophila Duox (Ha et al., 2009;Rigutto et al., 2009;Razzell et al., 2013) and the vertebrate homolog, Nox5 (Bánfi et al., 2004;Millana Fañanás et al., 2020). Conversely, overexpression of either enzyme is sufficient to phenocopy such presynaptic terminal growth ( Figure 2). ...
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Neurons respond to changes in the levels of activity they experience in a variety of ways, including structural changes at pre- and postsynaptic terminals. An essential plasticity signal required for such activity-regulated structural adjustments are reactive oxygen species (ROS). To identify sources of activity-regulated ROS required for structural plasticity in vivo we used the Drosophila larval neuromuscular junction as a highly tractable experimental model system. For adjustments of presynaptic motor terminals, we found a requirement for both NADPH oxidases, Nox and dual oxidase (Duox), that are encoded in the Drosophila genome. This contrasts with the postsynaptic dendrites from which Nox is excluded. NADPH oxidases generate ROS to the extracellular space. Here, we show that two aquaporins, Bib and Drip, are necessary ROS conduits in the presynaptic motoneuron for activity regulated, NADPH oxidase dependent changes in presynaptic motoneuron terminal growth. Our data further suggest that different aspects of neuronal activity-regulated structural changes might be regulated by different ROS sources: changes in bouton number require both NADPH oxidases, while activity-regulated changes in the number of active zones might be modulated by other sources of ROS. Overall, our results show NADPH oxidases as important enzymes for mediating activity-regulated plasticity adjustments in neurons.
... Increased ROS levels, due to imbalances between ROS production and cellular responses to counteract their actions, is one of the hallmarks of cancer. ROS accumulation and the resulting oxidative stress contribute to carcinogenesis, tumor aggressiveness, and antitumor treatment resistance by causing DNA damage and changing intracellular signaling pathways via post-translational modifications [27][28][29]. Thus, it can be hypothesized that DUOX2-dependent oxidative stress affects treatment outcomes in rectal cancer patients treated with CCRT. ...
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High-throughput mass-spectrometry-based quantitative proteomic analysis was performed using formalin-fixed, paraffin-embedded (FFPE) biopsy samples obtained before treatment from 13 patients with locally advanced rectal cancer (LARC), who were treated with concurrent chemoradiation therapy (CCRT) followed by surgery. Patients were divided into complete responder (CR) and non-complete responder (nCR) groups. Immunohistochemical (IHC) staining of 79 independent FFPE tissue samples was performed to validate the predictive ability of proteomic biomarker candidates. A total of 3637 proteins were identified, and the expression of 498 proteins was confirmed at significantly different levels (differentially expressed proteins—DEPs) between two groups. In Gene Ontology enrichment analyses, DEPs enriched in biological processes in the CR group included proteins linked to cytoskeletal organization, immune response processes, and vesicle-associated protein transport processes, whereas DEPs in the nCR group were associated with biosynthesis, transcription, and translation processes. Dual oxidase 2 (DUOX2) was selected as the most predictive biomarker in machine learning algorithm analysis. Further IHC validation ultimately confirmed DUOX2 as a potential biomarker for predicting the response of nCR to CCRT. In conclusion, this study suggests that the treatment response to RT may be affected by the pre-treatment tumor microenvironment. DUOX2 is a potential biomarker for the early prediction of nCR after CCRT.
... DUOX2 a été identifié comme étant l'enzyme impliquée dans la synthèse des hormones thyroïdiennes, du fait que des mutations du gène DUOX2 étaient associées à des cas d'hypothyroïdie congénitale due à des anomalies de l'organification de l'iode (Grasberger 2010 Wang et al. 2000) . Les protéines kinases PKA et PKC, qui activent respectivement DUOX1 et DUOX2 via la phosphorylation, sont toutes les deux régulées par la TSH via des voies de signalisation couplées à la protéine Gs et à la protéine Gq (Rigutto, Hoste et al. 2009). Bien que ces voies modulent l'activité intrinsèque des DUOX, le Ca 2+ est l'activateur principal des deux enzymes. ...
A l’issue d’un accident nucléaire, les produits de fission de l’uranium tel les iodes radioactifs sont dispersés dans l’environnement. L’homme est susceptible d’être exposé à ces éléments majoritairement via l’inhalation d’air et/ ou l’ingestion d’aliments contaminés. L’iode 131 est connu pour être responsable de l’augmentation de l’incidence du cancer de la thyroïde. Une des contremesures pour prévenir cette pathologie est l’ingestion de dose unique de comprimés d’iodure de potassium (KI) à fin de saturer la glande thyroïde par de l’iode stable et d’éviter ainsi l’accumulation de l’iode radioactif. Les scénarios de rejets réitérés d’iodes radioactifs lors des deux accidents majeurs Tchernobyl et Fukushima ont mis en évidence les limites de cette mesure, des prises répétées de KI pour protéger dans le temps les populations s’avèrent nécessaires. Dans la littérature on dispose de peu de données clinique et préclinique sur la prise répétée d’iode stable, quant à son usage ça n’a pas été décrit. La doctrine iode ainsi que l’autorisation de mise sur le marché (AMM) du KI envisage seulement la prise unique à renouveler exceptionnellement chez la population adulte. L’iode est connu pour être un élément clé de la fonction thyroïdienne, on jouant un double rôle à la fois de substrat de régulateur de la thyroïde. Si sa présence est indispensable à la formation des hormones thyroïdienne, son excès exerce un effet inhibiteur transitoire de cette synthèse connu sous le nom de l’effet Wolff-Chaikoff. Les hormones thyroïdiennes jouent un rôle majeur dans le développement et la fonction de presque tous les organes du corps (cerveau, cœur, os...), la moindre variation de leurs niveau peut impacter l’homéostasie du corps. Ainsi, il est difficile d’appliquer la prise répétée du KI en absence connaissances biologiques et toxicologiques. Pour combler ses lacunes et proposer une solution de prophylaxie répétée en cas d’exposition réitérée, le programme de recherche français PRIODAC : PRophylaxie répétée par l’IODe stable en situation ACcidentelle (ANR/RSNR), dont fait partie cette thèse vise à exploiter les modalités d’administration répétées du KI chez toutes les tranches d’âge (in utero, adulte et âgé), et d’évaluer la toxicologie de la prise répétée de KI sur les grandes fonctions physiologiques de l’organisme. Trois modèles de rats Wistar ont fait l’objet de ce travail de thèse : le modèle de référence rat adulte (âgé de 3 mois), le modèle à risque, organisme en développement (exposé durant la gestation) et un autre modèle à risque le rat âgé (âgé de 12 mois), ces trois modèles ont reçus 8 prise consécutive de KI 1mg/kg/24h. Les effets biologiques de ce traitement pendant 8 jours ont été évalués à long-terme (30 jours post-prophylaxie). Concernant, le modèle de référence ont n’a pas observé d’impact néfaste à long-terme de la prise répétée du KI (Lebsir, Cohen et al. 2018; Lebsir, Manens et al. 2018) par contre sur les modèles à risque plusieurs effets à long-terme ont été mis en évidence. Chez la progéniture exposée in utero, la coordination motrice ainsi que l’expression de quelques gènes clés du cerveau ont été négativement modifiées par le traitement. Chez le rat âgé la biochimie clinique, l’expression de quelques gènes clés de la fonction cardiovasculaire ainsi que le système rénine-angiotensine-aldostérone ont été significativement impacté par le traitement. En conclusion, les résultats obtenus montrent l’innocuité sur le plan toxicologique du KI administré à 1mg/kg toutes les 24h pendant 8 jours chez le modèle adulte et la nocuité de ce schéma prophylactique chez les modèles à risque in utero et âgé. Ces résultats ont été communiquées à la pharmacie centrale des armées (producteur et détentrice de l’AMM) afin de servir de données d’entrée pour des études de bonne pratique de laboratoire notamment pour le modèle adulte et également, afin de contribuées à l’évolution de la doctrine de l’iode en terme de radioprotection.
... In congenital hypothyroidism, TH production by the thyroid gland is insufficient [1]. Causes of deficient TH synthesis include thyroid gland maldevelopment (dysgenesis), followed by genetic defects in TH synthesis [2][3][4]. Both thyroid gland development and the function of gene products involved in TH synthesis are critically dependent upon the development and maintenance of thyroid epithelial polarity. ...
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Congenital hypothyroidism is a genetic condition in which the thyroid gland fails to produce sufficient thyroid hormone (TH), resulting in metabolic dysfunction and growth retardation. Xb130−/− mice exhibit perturbations of thyrocyte cytoskeleton and polarity, and develop postnatal transient growth retardation due to congenital hypothyroidism, leading ultimately to multinodular goiter. To determine the underlying mechanisms, we performed transcriptomic analyses on thyroid glands of mice at three age points: week 2 (W2, before visible growth retardation), W4 (at the nadir of growth); and W12 (immediately before full growth recovery). Using gene set enrichment analysis, we compared a defined set of thyroidal genes between Xb130+/+ and Xb130−/− mice to identify differentially enriched gene clusters. At the earliest postnatal stage (W2), the thyroid glands of Xb130−/− mice exhibited significantly downregulated gene clusters related to cellular metabolism, which continued to W4. Additionally, mutant thyroids at W4 and W12 showed upregulated gene clusters related to extracellular matrix, angiogenesis, and cell proliferation. At W12, despite nearly normal levels of serum TH and TSH and body size, a significantly large number of gene clusters related to inflammatory response were upregulated. Early postnatal TH deficiency may suppress cellular metabolism within the thyroid gland itself. Upregulation of genes related to extracellular matrix and angiogenesis may promote subsequent thyroid growth. Chronic inflammatory responses may contribute to the pathogenesis of multinodular goiter in later life. Some of the pathoadaptive responses of Xb130−/− mice may overlap with those from other mutations causing congenital hypothyroidism.
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Neurons respond to changes in the levels of activity they experience in a variety of ways, including structural changes at pre- and postsynaptic terminals. An essential plasticity signal required for such activity-regulated structural adjustments are reactive oxygen species (ROS). To identify sources of activity-regulated ROS required for structural plasticity in vivo we used the Drosophila larval neuromuscular junction as a highly tractable experimental model system. For adjustments of presynaptic motor terminals, we found a requirement for both NADPH oxidases, Nox and Dual Oxidase (Duox), that are encoded in the Drosophila genome. This contrasts with the postsynaptic dendrites from which Nox is excluded. NADPH oxidases generate ROS to the extracellular space. Here, we show that two aquaporins, Bib and Drip, are necessary ROS conduits in the presynaptic motoneuron for activity regulated, NADPH oxidase dependent changes in presynaptic motoneuron terminal growth. Our data further suggest that different aspects of neuronal activity-regulated structural changes might be regulated by different ROS sources: changes in bouton number require both NADPH oxidases, while activity-regulated changes in the number of active zones might be modulated by other sources of ROS. Overall, our results show NADPH oxidases as important enzymes for mediating activity-regulated plasticity adjustments in neurons.
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Background: Thyroid hormones (TH) thyroxine (T4) and triiodothyronine (T3) are known to be essential for maintaining optimal cognitive ability in adults. TH deprivation (hypothyroidism) or excess (hyperthyroidism) may alter signaling pathways involved in adult hippocampal neurogenesis and synaptic plasticity, thus increasing the risk of neurodegenerative disorders and leading to changes in cognitive performance. Aim: This study aimed to explore the effect of experimental hypo- and hyperthyroidism on the lipid peroxidation and glutathione levels, and the activities of antioxidant system enzymes SOD, catalase (CAT) and glutathione peroxidase (GPx) and glutathione reductase (GR) in the rat hippocampus. Material and Methods: The study included 72 adult male Wistar albino rats which were grouped as follows: (1) control; (2) hypothyroidism [hypothyroidism was induced by intraperitoneal injection of 10 mg/kg/day propylthiouracil (PTU) for 4 weeks]; (3) hyperthyroidism [hyperthyroidism was induced by 4-weeks' thyroxine injection (0.3 mg/kg/day)]. The levels of lipid peroxidation, glutathione, antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in the hippocampus of rats with hypo- and hyperthyroidism were examined using ELISA techniques. Results: The ELISA analysis results of our study show that in the hyperthyroid and hypothyroid rats, the lipid peroksidasyon levels in the hippocampus did not differ significantly from the control groups (P
Atherosclerosis is a metabolic disorder characterized by chronic inflammation associated with progressive thickening and hardening of the large to medium-sized arteries due to plaque formation. The study aims to evaluate the antioxidative, anti-inflammatory, and hypolipidemic efficacy of Opuntia ficus-indica (OFI) fruit extract against the high-fat-diet associated atherosclerotic rat model. In-vitro qualitative and quantitative phytochemical screening of OFI fruit extract revealed the significant presence of total phenolic content and total flavonoid contents. In-vitro antioxidant activity of fruit extract was determined through 2,2-diphenyl-1-picrylhydrazyl and FRAP assays that have shown their protective efficacy against the overproduction of reactive oxygen species. Results revealed that the level of total oxidant stress was significantly (P < 0.05) reduced and down expression levels of dual oxidases (Duox, Duoxa-1, and Duox-2) in all the treatment groups (I, II, III) as compared with positive control were observed. The total antioxidant capacity was significantly (P < 0.05) increased in all treatment groups in comparison with the positive control group and higher expression level of the Nrf-2 signaling pathway (Nfe-212, NFR-1, and Keap-1) was observed in all the treatment groups compared with the positive control group. Histopathological examination of the aorta showed that high-fat diet markedly increased endothelial lining and thickness of tunica media and adventitia, with irregular media segments having wavy laminae, and a significant increase in entropy of fibers disposition was observed. Conclusively, OFI fruit extract has shown promising protective, anti-oxidative, and anti-inflammatory efficacy through the restoration of normal parenchyma in high-fat dieting-associated oxidative stress and endothelial inflammation.
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Thyroid hormones (THs), including T4 and T3, are produced and released by the thyroid gland under the stimulation of thyroid-stimulating hormone (TSH). The homeostasis of THs is regulated via the coordination of the hypothalamic-pituitary-thyroid axis, plasma binding proteins, and local metabolism in tissues. TH synthesis and secretion in the thyrocytes-containing thyroid follicles are exquisitely regulated by an elaborate molecular network comprising enzymes, transporters, signal transduction machineries, and transcription factors. In this article, we synthesized the relevant literature, organized and dissected the complex intrathyroidal regulatory network into structures amenable to functional interpretation and systems-level modeling. Multiple intertwined feedforward and feedback motifs were identified and described, centering around the transcriptional and posttranslational regulations involved in TH synthesis and secretion, including those underpinning the Wolff-Chaikoff and Plummer effects and thyroglobulin-mediated feedback regulation. A more thorough characterization of the intrathyroidal network from a systems biology perspective, including its topology, constituent network motifs, and nonlinear quantitative properties, can help us to better understand and predict the thyroidal dynamics in response to physiological signals, therapeutic interventions, and environmental disruptions.
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The bioinformatic pipeline previously developed in our research laboratory is used to identify potential general and specific deregulated tumor genes and transcription factors related to the establishment and progression of tumoral diseases, now comparing lung cancer with other two types of cancer. Twenty microarray datasets were selected and analyzed separately to identify hub differentiated expressed genes and compared to identify all the deregulated genes and transcription factors in common between the three types of cancer and those unique to lung cancer. The winning DEGs analysis allowed to identify an important number of TFs deregulated in the majority of microarray datasets, which can become key biomarkers of general tumors and specific to lung cancer. A coexpression network was constructed for every dataset with all deregulated genes associated with lung cancer, according to DAVID’s tool enrichment analysis, and transcription factors capable of regulating them, according to oPOSSUM´s tool. Several genes and transcription factors are coexpressed in the networks, suggesting that they could be related to the establishment or progression of the tumoral pathology in any tissue and specifically in the lung. The comparison of the coexpression networks of lung cancer and other types of cancer allowed the identification of common connectivity patterns with deregulated genes and transcription factors correlated to important tumoral processes and signaling pathways that have not been studied yet to experimentally validate their role in lung cancer. The Kaplan–Meier estimator determined the association of thirteen deregulated top winning transcription factors with the survival of lung cancer patients. The coregulatory analysis identified two top winning transcription factors networks related to the regulatory control of gene expression in lung and breast cancer. Our transcriptomic analysis suggests that cancer has an important coregulatory network of transcription factors related to the acquisition of the hallmarks of cancer. Moreover, lung cancer has a group of genes and transcription factors unique to pulmonary tissue that are coexpressed during tumorigenesis and must be studied experimentally to fully understand their role in the pathogenesis within its very complex transcriptomic scenario. Therefore, the downstream bioinformatic analysis developed was able to identify a coregulatory metafirm of cancer in general and specific to lung cancer taking into account the great heterogeneity of the tumoral process at cellular and population levels.
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Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. Pig and human thyroid plasma membrane contain a Ca(2+)-dependent NAD(P)H oxidase that generates H(2)O(2) by transferring electrons from NAD(P)H to molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138(Tox)). Human and porcine p138(Tox) have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a Caenorhabditis cDNA and with rbohA, a protein involved in the Arabidopsis NADPH oxidase. p138(Tox) shows also similarity to the p65(Mox) and to the gp91(Phox) in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91(Phox), p138(Tox) shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91(Phox) with the p47(Phox) cytosolic factor are absent in p138(Tox). The expression of porcine p138(Tox) mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138(Tox) is a differentiation marker of thyrocytes. The gene of human p138(Tox) has been localized on chromosome 15q15.
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In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
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Mutations in dual oxidase (DUOX2) have been proposed as a cause of congenital hypothyroidism. Previous reports suggest that biallelic mutations of DUOX2 cause permanent congenital hypothyroidism and that monoallelic mutations cause transient congenital hypothyroidism. To clarify the inheritance of hypothyroidism, we looked at the DUOX2 gene in patients with transient congenital hypothyroidism. DUOX2, thyroid peroxidase, Na+/I- symporter and dual oxidase maturation factor 2 genes were analyzed in eight patients with transient congenital hypothyroidism, using the PCR-amplified direct sequencing method. The eight patients were found by a neonatal screening program. Six of these patients belonged to two independent families; the other two were unrelated. Their serum TSH values varied from 24.8-233.0 mU/liter. Six of the eight patients had a low serum freeT4 level (0.19-0.84 ng/dl). Seven of the eight patients were treated with thyroid hormone replacement therapy, which ceased to be necessary by 9 yr of age. Eight novel mutations were detected in the DUOX2 gene. Four patients in one family were compound heterozygous for p.L479SfsX2 and p.K628RfsX10. Two patients in a second family were compound heterozygous for p.K530X and p.[E876K;L1067S]. The two remaining unrelated patients were also compound heterozygous, for p.H678R/p.L1067S and p.A649E/p.R885Q, respectively. All eight patients had biallelic mutations in the DUOX2 gene. We find that loss of DUOX2 activity results in transient congenital hypothyroidism and that transient congenital hypothyroidism caused by DUOX2 mutations is inherited as an autosomal recessive trait.
Hydrogen peroxide (H2O2) is an essential compound for the synthesis of thyroid hormone. Its presence in the follicular lumen is required by thyroperoxidase for the iodination of tyrosil residues of thyroglobulin, the initial step in the synthesis of T3 and T4. The biochemical requirement of H2O2 for thyroid hormone production has been known for decades and an H2O2-generating system was predicted to exist in the thyroid gland. In recent years, different research groups have unraveled the molecular nature of the system. Two homologous proteins, the dual oxidases 1 and 2, DUOX1 and DUOX2 (formerly THOX1 and 2, for thyroid oxidases), were identified and shown to contain functional domains typical of NADPH oxidoreductases. However, in vitro reconstitution of H2O2 production could not be obtained in nonthyroidal cell lines expressing these proteins. Evidence of DUOX involvement in thyroidal H2O2 production came from the identification of a DUOX2 nonsense homozygote mutation, which resulted in the absence of all functional domains of the protein, in a patient with permanent and severe congenital hypothyroidism (CH). Recently, an experimental demonstration of H2O2 production by DUOX2 was achieved by coexpression with a novel ’maturation factor’ for DUOX2 (DUOXA2). Transient CH has also been linked to heterozygote nonsense DUOX2 mutations, showing for the first time that transitory CH can have a genetic origin. These findings also establish that partial dyshormonogenetic defects can behave biochemically as transient forms of CH. Novel missense and splice-site DUOX2 mutations in compound heterozygosity have been recently reported in association with a wide spectrum of hypothyroid phenotypes, ranging from very mild to severe. Functional analysis of these point mutations using available assays opens now the possibility to ascertain whether transiency or permanency of DUOX2 phenotypes relate exclusively to monoallelic or biallelic inactivation of the gene, or if the degree of pathogenic severity of mutations may also influence the timely outcome of this type of hypothyroidism.
Primary cultures of dog thyroid cells have been established. The cells originated from follicles and displayed differentiation characteristics of such cells: iodide trapping and organification, responsiveness of iodide organification and cyclic AMP accumulation to thyrotropin (TSH), induction of a two-dimensional follicular structure by TSH. TSH also stimulated the multiplication of these cells. The effect of TSH was detected with concentrations as low as 100 μU/ml and was reproduced with purified TSH. It was reproduced by cholera toxin (10 ng/ ml) and dibutyryl cyclic AMP (10−5 M). The data show that TSH, which stimulates the function of thyroid tissue, in vivo and in vitro, activates the multiplication of differentiated dog-thyroid follicular cells in primary culture, which suggests that this trophic effect is, partly at least, mediated by cyclic AMP.
We have recently shown that adenosine triphosphate (ATP), bradykinin and thyrotropin-releasing hormone (THR) increase the ([Ca2+]i) of human thyrocytes in primary culture. We show here that these agents also stimulate the generation of [3H]-inositol monophosphate (IP1), inositol bisphosphate (IP2) and inositol trisphosphate (IP3). The stimulation of IP3 generation followed two distinct kinetics: it was sustained when the cells were triggered with ATP and transient when the response was elicited by TRH or bradykinin. In addition, we have shown that under the appropriate experimental conditions, high thyroid-stimulating hormone (TSH) concentrations were also able to stimulate human thyrocyte IP1, IP2 and IP3 accumulation and to increase their [Ca2+]i. These data suggest that ATP, bradykinin, TRH and high TSH concentrations activate the Ca(2+)-phosphatidylinositol cascade of human thyrocytes. Since this cascade plays a crucial role in the control of protein iodination, ATP, TRH and bradykinin could be important regulators of thyroid hormone synthesis in human thyrocytes.
Iodide oxidation and binding to proteins require a thyroperoxidase and an ill defined H2O2-generating system. The NADP⁺ supply and, thus, NADPH oxidation are the limiting steps of the pentose phosphate pathway. The purpose of this work was to test the hypothesis that H2O2 generation is a limiting step of iodination and NADPH oxidation and, therefore, of the pentose phosphate pathway. H2O2 produced by dog thyroid slices was measured with the homovanillic fluorescence assay. Our data show that H2O2 generation is stimulated by both the cAMP cascade [as activated by TSH, forskolin and (Bu)2cAMP] and the Ca²⁺-phosphatidylinositol cascade (as activated by carbamylcholine, ionomycin, and 12-O-tetradecanoylphorbol-13- acetate). We used several physiological and pharmacological agents that modulate iodide organification. In all cases there was a strict parallelism between effects on H2O2 generation, iodide binding to proteins, and pentose phosphate pathway activity. Moreover, in TSH- or carbamylcholine-stimulated slices, glucose or Ca²⁺ depletion, which greatly depressed H2O2 generation, also greatly decreased iodide organification and the activity of the pentose phosphate pathway. The glutathione peroxidase-catalyzed H2O2 reduction in the cytosol, which involves NADPH oxidation and, therefore, increases the NADP supply, also enhances the activity of the pentose phosphate pathway. All of these data strongly support the hypothesis that H2O2 generation in dog thyroid controls iodination of proteins; through the NADPH oxidation resulting from H2O2 production and reduction, hydrogen peroxide also regulates the activity of the pentose phosphate pathway. (Endocrinology 128: 779–785, 1991)