ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17.
ABSTRACT The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H(2)O(2) participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y(2) receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y(2)R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y(2)R activation as a proximal step in EGFR transactivation and downstream signaling.
- [Show abstract] [Hide abstract]
ABSTRACT: Dual oxidase 1 (Duox1) is the NADPH oxidase responsible for the H2O2 gradient formed in tissues after injury to trigger the early recruitment of leukocytes. Little is known about the signals that modulate H2O2 release from DUOX1 and whether the H2O2 gradient can orchestrate the inflammatory response in vivo. In this study, we report on a dominant-negative form of zebrafish Duox1 that is able to inhibit endogenous Duox1 activity, H2O2 release and leukocyte recruitment after tissue injury, with none of the side effects associated with morpholino-mediated Duox1 knockdown. Using this specific tool, we found that ATP release following tissue injury activates purinergic P2Y receptors, and modulates Duox1 activity through phospholipase C (PLC) and intracellular calcium signaling in vivo. Furthermore, Duox1-derived H2O2 is able to trigger the NF-κB inflammatory signaling pathway. These data reveal that extracellular ATP acting as an early danger signal is responsible for the activation of Duox1 via a P2YR/PLC/Ca(2+) signaling pathway and the production of H2O2, which, in turn, is able to modulate in vivo not only the early recruitment of leukocytes to the wound but also the inflammatory response through activation of the NF-κB signaling pathway.The Journal of Immunology 05/2014; 192(12). · 5.36 Impact Factor
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ABSTRACT: We studied the involvement of calcium and calcium-activated NADPH oxidases in NLRP3 inflammasome activation and IL-1β release to better understand inflammasome signaling in macrophages.Inflammation Research 07/2014; 63(10). · 2.14 Impact Factor
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ABSTRACT: Airway mucus has a key role in protective innate immune responses, but excessive mucus production and secretion in proximal and in distal airways are associated with disabling symptoms (cough and sputum), lung function decline, exacerbations and mortality in patients with chronic obstructive pulmonary disease (COPD). Cellular and molecular mechanisms leading to mucin production and secretion have largely been identified using cultured epithelial cells and animal models. Cigarette smoke and microbial products are potent triggers of mucin production, which involves recognition of specific molecular patterns by cognate receptors and activation of metalloproteases at the epithelial cell surface, leading to epidermal growth factor receptor activation and mucin mRNA and protein synthesis. After mucin synthesis has occurred, mucins are tightly packed into intracytoplasmic granules. Many stimuli induce secretion of mucin granules from epithelial cells, but neutrophil serine proteases are the most potent inducers of mucin secretion. Neutrophils recruited to the airway epithelium also promote mucin production via neutrophil proteases and oxidative stress. Several drugs currently available for the treatment of COPD patients reduced mucus hypersecretion in preclinical models relevant to COPD, but their effects on mucus hypersecretion in humans have not been assessed. Testing the effects of these drugs and of novel molecules designed for reducing mucus production and/or secretion will require performing specifically designed clinical trials. These trials will be necessary to explore the hypothesis that reducing mucus hypersecretion is beneficial in COPD patients.Drugs 06/2014; · 4.13 Impact Factor
ATP-Mediated Transactivation of the Epidermal Growth
Factor Receptor in Airway Epithelial Cells Involves
DUOX1-Dependent Oxidation of Src and ADAM17
Derek Sham1, Umadevi V. Wesley2,3¤, Milena Hristova1, Albert van der Vliet1*
1Department of Pathology, College of Medicine, University of Vermont, Burlington, Vermont, United States of America, 2Department of Microbiology and Molecular
Genetics, College of Medicine, University of Vermont, Burlington, Vermont, United States of America, 3Vermont Lung Center, University of Vermont, Burlington, Vermont,
United States of America
The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely
mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on
previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial
wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H2O2
participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway
epithelial cells was found to involve purinergic P2Y2receptor stimulation, and both ligand-dependent mechanisms as well
as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-
dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands.
Activation of P2Y2R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable
shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and
downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR
transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In
aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or
injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y2R activation as a proximal
step in EGFR transactivation and downstream signaling.
Citation: Sham D, Wesley UV, Hristova M, van der Vliet A (2013) ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells
Involves DUOX1-Dependent Oxidation of Src and ADAM17. PLoS ONE 8(1): e54391. doi:10.1371/journal.pone.0054391
Editor: Masuko Ushio-Fukai, University of Illinois at Chicago, United States of America
Received August 17, 2012; Accepted December 11, 2012; Published January 18, 2013
Copyright: ? 2013 Sham et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a research grant from the National Institutes of Health to AvdV (R01 HL085646), and by a Fellowship from the Department
of Pathology to DS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Albert.van-der-Vliet@uvm.edu
¤ Current address: Department of Neurological Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, United States of
The respiratory epithelium forms a first line defense against
inhaled pathogens and pollutants, and has developed intricate
innate response mechanisms against diverse environmental
challenges to provide important initial host defense and to
protect airway structure and function. Many recent lines of
evidence indicate that airway epithelial surface signaling through
the epidermal growth factor (EGFR) represents a common
pathway in many such innate host responses, and plays a key
role in several protective epithelial responses to a range of
member of the ErbB family, which comprises four receptors
HER4/Erb4), of which EGFR, Erb2 and Erb3 are expressed
within human airway epithelia. Activation of ErbB receptors by
their cognate ligands results in receptor homo- or heterodimer-
ization leading to (auto)phosphorylation within the intrinsic
kinase domain and activation of downstream signaling. Howev-
er, EGFR activation in response various diverse environmental
EGFR isthe prototypical
or microbial stresses typically involves the initial stimulation of
various G-protein-coupled receptors (GPCR), which promotes
EGFR transactivationby as
mechanisms involving ligand-independent intracellular mechan-
isms as well as activation of EGFR ligands by ADAM (a
disintegrin and metalloproteinase) family sheddases [4,5,6,7].
One GPCR family of particular interest in the context of
epithelial injury and wound responses includes purinergic
receptors, which are activated by epithelial release of ATP in
response to both mechanical and molecular stresses [8,9], and
are critical in epithelial responses to injury or infection
promoting mucociliary clearance and stimulating cellular repair
mechanisms [8,10,11,12], and transactivation of EGFR has
been implicated in these ATP-mediated wound responses in
various cell systems [13,14,15].
The mechanisms by which GPCR stimulation results in
EGFR transactivation are diverse and incompletely understood,
but a number of reports implicate the contribution of regulated
production of H2O2[16,17,18]. Proposed mechanisms in H2O2-
dependent EGFR activation include oxidative inactivation of
PLOS ONE | www.plosone.org1 January 2013 | Volume 8 | Issue 1 | e54391
protein tyrosine phosphatase 1B to augment and prolong EGFR
[16,17], as well as oxidative modification of EGFR itself in
response to ligand stimulation . Moreover, H2O2or related
ROS are also thought to contribute to ADAM17 activation by
ATP or other stimuli, although the oxidative mechanisms of
ADAM17 activation are unclear and have been suggested to
involve oxidative cysteine switch activation of pro-ADAM17 at
the epithelial cell surface,
questioned [21,22,23,24]. Alternatively, ADAM17 activity may
be controlledby oxidative
extracellular domain of the mature enzyme [25,26], although
its relevance for ATP-mediated EGFR activation is unclear.
Another potential mechanism by which H2O2 may mediate
EGFR transactivation is by oxidative activation of non-receptor
tyrosine kinases of the Src family [27,28], which promote
EGFR phosphorylation at selected residues in a ligand-in-
dependent fashion [29,30]. The activity of Src is tightly
controlled by inhibitory tyrosine phosphorylation at Y527 and
by auto-phosphorylation at Y416 during activation, but recent
evidence indicate that Src family kinases are also regulated by
oxidation of conserved cysteine residues with the C-terminal
region [31,32,33], and such oxidative modification of Src
kinases have been implicated in cell adhesion and spreading
and in wound responses [31,34].
The oxidative mechanisms involved in EGFR activation also
critically depend on the origin of H2O2production. While some
studies have implicated mitochondria-derived H2O2 or related
activation , ATP-dependent production of H2O2by airway
epithelial cells originates primarily from the NADPH oxidase
homologs DUOX1 and DUOX2, which are prominently
expressed in the airway epithelium [24,35]. Indeed, DUOX1
has been implicated in epithelial EGFR activation in response
to variousstimuli including
mechanisms are incompletely understood. The present studies
were designed to detail the mechanisms by which extracellular
ATP promotes transactivation of EGFR within airway epithelial
cells, and to establish the involvement of DUOX1/2 as major
H2O2 sources within the respiratory epithelium. Our results
demonstrate that ATP-dependent EGFR transactivation within
airway epithelial cells involves DUOX1-dependent activation of
Src and ADAM17 by cysteine oxidation within these enzymes,
as initial events in EGFR phosphorylation subsequent epithelial
although thishas been
Extracellular ATP Promotes Epithelial Wound Responses
by P2Y2R-mediated Activation of EGFR and Src
We first confirmed previous findings indicating a role for EGFR
in epithelial wound responses (refs), using a blocking a-EGFR
antibody (mAb 225; Calbiochem) or the EGFR tyrosine kinase
inhibitor AG1478. Both approaches significantly inhibited epithe-
lial wound closure in H292 cells in a model of scratch injury
(Fig. 1A), and suppressed ATP-dependent induction of MMP-9 or
IL-8 (Fig. 1B), established mediators of epithelial wound repair
[1,37]. Analysis of tyrosine phosphorylation within EGFR using
phospho-specific antibodies showed that stimulation of H292 cells
with ATP resulted in dose-dependent phosphorylation at tyrosine
1068 (Y1068), a known autophosphorylation site after ligand-
dependent EGFR dimerization, and at Y845, a target for non-
receptor tyrosine kinases such as Src [28,30]. Both phosphoryla-
tion events were dose-dependently increased by ATP up to
100 mM (Fig. 1C), suggesting the involvement of purinergic
receptor stimulation. EGFR phosphorylation at Y845 implies the
involvement of Src, which was confirmed by assessing tyrosine
auto-phosphorylation at Y416, which indicated that ATP dose-
dependently enhanced Src phosphorylation at Y416 (Fig. 1D),
whereas phosphorylation at Y527, which is associated with
inhibition of Src, was not significantly affected by ATP (not
Among the major purinergic P2Y receptors expressed in the
airway epithelial cells  as well as H292 cells, the P2Y2R subtype
appeared to be the most important in ATP-dependent EGFR
activation, based on similar EGFR activation by the P2Y2R ligand
UTP, but not by ADP or UDP, which primarily activate P2Y1R or
P2Y6R (Fig. S1). Using siRNA silencing of P2Y2R, we confirmed
its critical importance in ATP-dependent activation of EGFR and
Src (Fig. 1E), in agreement with previous studies with other cell
ATP-mediated EGFR Phosphorylation Depends on EGFR
Ligand Binding As Well As Src Activation
A range of studies have demonstrated that EGFR transacti-
vation by GPCR is mediated by ADAM-dependent shedding of
EGFR ligands and ligand-dependent EGFR dimerization and
activation , and our previous studies demonstrated that
exogenous ATP is capable of promoting shedding of the EGFR
ligand TGF-a . The involvement of EGFR ligand binding
in EGFR transactivation by ATP was evaluated used a blocking
a-EGFR antibody, which was found to almost completely
prevent EGFR phosphorylation at both Y845 and Y1068, by
EGF as well as ATP (Fig. 2A), while similar incubation with
control IgG1 had no effect (not shown). ATP-mediated EGFR
phosphorylation at both Y845 and Y1068 was also abrogated
by the EGFR kinase inhibitor, AG1478 (10 mM), as expected
Since ATP-mediated EGFR phosphorylation at Y845 and
Y1068 was associated with activation of Src, we postulated that
EGFR transactivation by ATP also involves Src in a ligand-
independent mechanism. Indeed, ATP-mediated EGFR phos-
phorylation of Y845 as well as Y1068 was almost completely
prevented in the presence of the pharmacological Src inhibitor
PP2 (1 mM) (Fig. S2B) or after siRNA-dependent silencing of Src
(Fig. 2B). In contrast, blockade or silencing of Src only suppressed
EGF-induced EGFR phosphorylation at Y845 but did not affect
EGFR autophosphorylation at Y1068 by EGF, indicating that
ligand-dependent EGFR phosphorylation at Y845 depends on Src
whereas autophosphorylation at other residues such as Y1068 is
Src-independent. Collectively, these findings highlight the impor-
tance of Src activation as well as EGFR ligand shedding in EGFR
transactivation in airway epithelial cells in response to extracellular
Extracellular ATP Activates ADAM17 via Src As Well As
Ligand-dependent EGFR Stimulation
Based on reported involvement of ADAM17 in airway epithelial
EGFR activation in response to many stimuli [24,40], we
monitored ADAM17 activation using a fluorogenic ADAM17
substrate (Calbiochem). Indeed, stimulation of H292 cells with
ATP enhanced fluorescence, which was prevented after siRNA
silencing (Fig. S3), indicating that this fluorescence increase
reflects activation of ADAM17. Activation of ADAM17 was
observed in response to ATP as well as EGF, and was in both cases
completely prevented in the presence of the Src inhibitor PP2, or
after siRNA silencing of Src (Fig. 3A,B). Intriguingly, ADAM17
activation was also partially suppressed in the presence of a-EGFR
Mechanisms of DUOX1-Dependent EGFR Activation
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(Fig. 3C), suggesting that ligand-dependent EGFR activation
contributes to ADAM17 activation, possibly representing a positive
feed-forward loop resulting in enhanced shedding of EGFR-
activating ligands. The complete dependency of ADAM17
activation by either ATP or EGF on the presence of Src is in
agreement with a recent report indicating the importance of Src in
paracrine signaling of EGFR and ERK through ADAM17-
mediated ligand shedding . These collective findings indicate
that EGFR transactivation by ATP is critically dependent on
initial activation of Src, which in turn phosphorylates EGFR at
Y845 and activates ADAM17 to promote ligand-dependent
ATP-dependent EGFR Stimulation Occurs at the Apical
Surface in Polarized Epithelia
Our findings so far were based on studies in non-polarized
submerged cultures of H292 cells, which may not adequately
reflect EGFR-dependent signaling in normal polarized airway
epithelia, in which apically generated EGFR ligands are thought
to be segregated from their receptor(s) at the basolateral
membrane by epithelial junctions, allowing effective EGFR
activation only under conditions of epithelial injury [42,43]. Since
ATP release upon epithelial stimulation by diverse environmental
or microbial stresses occurs primarily at the apical surface, where
P2Y2receptors are largely localized [8,9], we questioned whether
such apically released ATP is capable of transactivating EGFR
even in intact polarized epithelia. To test this, we cultured
immortalized human bronchial epithelial (HBE1) cells on culture
inserts at air-liquid interface, to generate polarized epithelial
monolayers, and stimulated these with ATP or EGF either apically
or basolaterally. As shown in Fig. 4, apical stimulation of
polarized HBE1 cells with ATP or EGF resulted in enhanced
EGFR phosphorylation at Y845 and Y1068, while such responses
were significantly less pronounced upon basolateral stimulation.
These differences in responsiveness were largely abolished after
preincubation of polarized HBE1 cells with EGTA, which disrupts
epithelial tight junctions and facilitate diffusion of extracellular
mediators between apical and basolateral compartments .
These findings indicate that ATP is capable of promoting EGFR
transactivation in intact epithelia, and does not necessarily require
epithelial injury or disruption of tight junctions. Overall, these
findings indicate that extracellular ATP is capable of transactivat-
ing EGFR in intact epithelia, via P2Y2R activation and ligand
Figure 1. EGFR-dependent wound responses are mediated by ATP-dependent P2Y2R activation. Confluent H292 cells were serum-
starved overnight and subjected to scratch wounding or stimulated with exogenous ATP (100 mM) for 8 hrs, and wound closure (A) or fold increases
in mRNA expression of MMP-9 or IL-8 compared to non-stimulated cells (B) were determined. *: p,0.05 compared to corresponding control (n=4).
(C,D) H292 cells were stimulated with ATP for 10 min, and whole cell lysates were analyzed by Western blot using phospho-specific antibodies p-
EGFR (Y845 or 1068) and unphosphorylated (total) EGFR or with antibodies against p-Src (Y416) and total Src. (E) H292 cells were pre-incubated with
P2Y2R siRNA or control non-specific (NS) siRNA for 72 hrs, and stimulated with ATP (100 mM, 10 min) for analysis of p-EGFR, p-Src, or P2Y2R by
Western blot. Representative results of 2 independent experiments are shown.
Mechanisms of DUOX1-Dependent EGFR Activation
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shedding at the apical surface, representing an important response
mechanism of intact epithelia to cell activation by mechanical,
chemical or biological stimuli, resulting in induction of down-
stream stress or wound responses.
ATP-dependent Activation of Src/ADAM17/EGFR are
Mediated by DUOX1
Previous studies by us and others have suggested the importance
of DUOX1 in epithelial wound responses and EGFR activation by
various stimuli [10,24,36], although others have also suggested the
importance of DUOX2 in epithelial responses to certain
exogenous triggers . We confirmed the importance of
DUOX1 rather than DUOX2 in epithelial wound responses
and in ATP-dependent induction of MMP-9 and IL-8 in H292
cells using siRNA silencing (Fig. 5A,B), which indicated that these
EGFR-mediated outcomes are also dependent on DUOX1.
Similarly, ATP-dependent phosphorylation of EGFR in H292
cells was found to depend on activation of DUOX1, whereas it
was completely unaffected by silencing of DUOX2 (Fig. 5C,D).
Moreover, silencing of DUOX1, but not DUOX2, also signifi-
cantly attenuated ATP-dependent activation of Src, and also
largely prevented tyrosine phosphorylation of the transcription
factor STAT3, a downstream target of Src and/or EGFR involved
in epithelial wound responses  (Fig. 5C). Silencing of DUOX1
also suppressed ATP-dependent production of H2O2, which was
unaffected by DUOX2 siRNA, although the latter weakly
suppressed basal H2O2production (Table 1). Importantly, the
effects of DUOX1 siRNA silencing could not be attributed to
altered levels of Src or EGFR (Fig. 6A) or altered expression of
Figure 2. ATP-mediated EGFR phosphorylation is both ligand- and Src-dependent. (A) Confluent H292 cells were serum-starved overnight
and pre-treated with a-EGFR mAb (4 mg/ml) for 30 min prior to treatment with ATP (100 mM) or EGF (100 ng/ml) for 10 min, and lysates were
analyzed by Western blot using p-EGFR and total EGFR antibodies. (B) H292 cells were pre-incubated with c-Src siRNA or control non-specific (NS)
siRNA for 72 hrs, and subsequently serum-starved overnight and stimulated with ATP or EGF, and cell lysates were analyzed for phosphorylated EGFR
and total EGFR, c-Src and b-actin. Representative blots from 2–3 separate experiments are shown.
Mechanisms of DUOX1-Dependent EGFR Activation
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Figure 3. Exogenous ATP results in activation of ADAM17 via c-Src and EGFR ligand. H292 cells were preincubated with PP2 (A), Src-
specific or control siRNA (B), or a-EGFR mAb (C) as in Figs 2 and 3, and stimulated with ATP (100 mM) or EGF (100 ng/ml) in the presence of
a fluorogenic substrate specific for ADAM17, and changes in fluorescence were monitored after 2 hrs. *: p,0.05 compared to untreated control or NS
siRNA;#:p,0.05 compared with corresponding stimulation in the absence of PP2, Src siRNA, or a-EGFR mAb (n=4).
Figure 4. Apical ATP activates EGFR in intact polarized epithelium. Immortalized HBE1 cells were cultured in Transwell inserts until they
formed a functional polarized barrier (TER .150 V.cm2) and stimulated with ATP or EGF, which was administered either apically or basolaterally.
Similar stimulations were performed in cell monolayers that were pretreated with EGTA (1 mM) to disrupt tight junctions and TER. Cell lysates were
prepared after 10 min and analyzed for (phosphorylated) EGFR (A). Western blots from 2–3 separate experiments were quantified by densitometry
(B,C). *: p,0.05 compared to corresponding apical stimulation (n=4).
Mechanisms of DUOX1-Dependent EGFR Activation
PLOS ONE | www.plosone.org5 January 2013 | Volume 8 | Issue 1 | e54391
P2Y receptors or ADAM17 (Table SI). Collectively, these
findings indicate the critical importance of DUOX1-dependent
H2O2 production in activation of Src/EGFR and subsequent
Previous studies indicated that ATP-dependent activation of Src
is facilitated by direct association of Src with P2Y2R at its SH3
binding domain . Immunoprecipitation of Src from untreated
or ATP-stimulated H292 cells confirmed a similar interaction
between Src and P2Y2R in H292 cells in response to ATP
stimulation (Fig. 6A). Furthermore, ATP stimulation also pro-
moted a direct association between Src and/or P2Y2R with
DUOX1 (Fig. 6B), further illustrating the potential involvement
of DUOX1 in Src activation and downstream signaling in
response to P2Y2R stimulation.
To more definitively explore the importance of DUOX1 in
EGFR transactivation by ATP and establish the DUOX1-
dependent redox signaling mechanism(s), we generated H292 cell
lines in which DUOX1 is stably and almost completely eliminated,
by stable transfection with DUOX1-targeted shRNA (Fig. S4).
Thus generated H292-shDUOX1 cells were found to almost
completely lack DUOX1 mRNA and protein (Fig. 7A,B), and
although H292-shDUOX1 cells produced similar extracellular
H2O2levels compared to control transfected cells (H292-shCTL),
they failed to respond to exogenous ATP with respect to
extracellular H2O2 production (Fig. 7C), indicating that they
lack functional DUOX1. As expected, H292-shDUOX1 cells were
resistant to ATP-induced EGFR phosphorylation at Y845 and
Y1068 compared to corresponding control cells (H292-shCTL),
although EGF-dependent EGFR phosphorylation was largely
unaffected, and were also resistant to ATP- or EGF-dependent
activation of Src, STAT3 or ADAM17 (Fig. S5). Kinetic analysis
of ATP-induced EGFR activation indicated rapid phosphorylation
at both Y845 and Y1068 in H292-shCTL cells (peaking at 5–
10 min and returned to baseline after 30–60 min), whereas no
significant increase in EGFR phosphorylation was observed at any
time point in H292-shDUOX1 cells (Fig. 7D,E). These findings
Figure 5. ATP-mediated wound responses and EGFR activation depend on DUOX1. (A) H292 cells were pre-incubated with DUOX1- or
DUOX2-targeted siRNA or with control NS siRNA for 72 hrs, and subsequently serum-starved overnight and subjected to scratch wounding for
analysis of wound closure after 8 hrs (A), stimulated with exogenous ATP (100 mM) for 8 hrs for analysis of ATP-dependent increases in mRNA
expression of MMP-9 or IL-8 (B), or stimulated with ATP (100 mM) or EGF (100 ng/ml) for 10 min for analysis for phosphorylated and
unphosphorylated forms of EGFR, c-Src, or STAT3 (C). Representative blots from 2–3 separate experiments are shown. Efficiency of siRNA-dependent
silencing of DUOX1 or DUOX2 was evaluated by RT-PCR (D).
Table 1. Contribution of DUOX1/2 to basal or ATP-
dependent H2O2production by H292 cells.
H292 siCTL siDUOX1siDUOX2
ATP 2.0160.631.6860.28 0.01960.002b
H292 cells were transfected with indicated siRNA and cells were placed in HBSS
and stimulated for 15 min with ATP (100 mM) after which conditioned media
was mixed with tyrosine and LPO for HPLC analysis of dityrosine. Data are
expressed as nmol H2O2/106cells and mean values SEM are presented from 4
ap=0.07 compared to siCTL.
bp,0.05 compared to siCTL.
Mechanisms of DUOX1-Dependent EGFR Activation
PLOS ONE | www.plosone.org6 January 2013 | Volume 8 | Issue 1 | e54391
illustrate the importance of DUOX1 in initiation of EGFR
phosphorylation by extracellular ATP, which contrasts with the
recently reported regulation of EGFR by a different NADPH
oxidase, NOX4, which was primarily found to regulate the
duration of EGFR activation due to PTP1B inactivation without
significantly affecting initial EGFR activation by EGF .
Collectively, our findings illustrate a critical role for DUOX1 in
initiating EGFR transactivation by extracellular ATP, and possibly
by other environmental stimuli, related to activation of Src and/or
ATP-mediated DUOX1 Activation Results in Cysteine
Oxidation within c-Src and ADAM17
While our results so far illustrate the importance of DUOX1 in
activation of Src as a proximal mechanism in EGFR activation,
the precise molecular mechanisms by which DUOX1-derived
H2O2mediates such response is not yet known. In this regard,
various lines of evidence indicate that activation of Src is not only
controlled by phosphorylation/dephosphorylation, but is also
subject to redox control by reversible oxidation of critical cysteine
residues . Because of the reported role of P2Y2R in DUOX1
activation , and the direct association between P2Y2R, Src
and DUOX in response to cell stimulation (Fig. 6C), we
hypothesized that DUOX1-derived H2O2might directly oxidize
cysteine residues within Src and thereby promote its activity
[31,46]. To examine this, untreated or ATP-stimulated H292 cells
were lysed in oxygen-free lysis buffer (under N2atmosphere to
minimize cysteine auto-oxidation) containing the thiol-specific
biotin-labeling reagent, EZ-link iodoaceteyl-LC-Biotin (100 mM).
Analysis of purified biotinylated proteins with an a-Src antibody
demonstrated significant and time-dependent loss of biotinylated
Src in response to ATP stimulation (Fig. 8A,B), reflecting loss of
reduced cysteines within Src due to oxidation. Moreover, in
comparison to ATP-dependent Src oxidation in H292-shCTL
cells, no significant oxidation of Src was observed in ATP-
stimulated H292-shDUOX1 cells (Fig. 8C,D), indicating the
critical involvement of DUOX1 in Src oxidation. Based on
previous findings showing that oxidation of selected cysteine
residues within Src contributes to its full activation , our
findings indicate that ATP-mediated DUOX1 activation promote
Src activation by direct or indirect cysteine oxidation, and thereby
promote downstream activation of ADAM17 and EGFR.
Our studies indicate the critical importance of Src in ATP-
dependent activation of ADAM17 and EGFR ligand shedding.
Although the mechanisms involved in regulating ADAM17 are
complex and not fully understood [6,7], recent studies indicate the
presence of redox-sensitive cysteines within the extracellular region
of mature ADAM17, that appear to be subject to redox control
regulate ADAM17 activity by switching from a ‘‘closed’’ inactive
state to a more ‘‘open’’ active state at the cell surface [25,26]. We
considered a potential role of DUOX1 in similar redox regulation
of cysteine residues within mature ADAM17, and used labeling
with a cell-impermeable thiol-specific biotinylating agent, EZ-link
Maleimide-Biotin (MBP), to determine the cysteine status within
ADAM17 at the epithelial surface. As a loading control, cell-
surface ADAM17 was also labeled with amino-specific biotinyla-
tion with sulfo-NHS-biotin, which is insensitive to H2O2.
Biotinylated proteins were avidin-purified and probed by Western
blot analysis of ADAM17, which indicated loss of thiol-specific
biotinylation of ADAM-17 in response to ATP, as shown by
reduced band intensity at 120 kD and 100 kDa, which represent
proADAM17 and mature ADAM17, respectively (Fig. 9). Similar
analysis of amino-labeled ADAM17 with sulfo-NHS-biotin did not
show any change in response to ATP, illustrating selective
oxidation of cysteine residues within ADAM17 following ATP
stimulation. Moreover, whereas significant cysteine oxidation
within ADAM17 was observed in H292-shCTL cells in response
to ATP, no such alterations were seen in H292-shDUOX1 cells
(Fig. 9), indicating the critical involvement of DUOX1. Collec-
tively, these findings demonstrate that ATP-mediated activation of
DUOX1 results in specific cysteine oxidation of both Src and
ADAM17, thereby promoting their activity and subsequent
activation of EGFR signaling.
The present studies highlight the central involvement of the
chial epithelial cells in response to exogenous ATP, and thereby
further establish DUOX1 as a critical factor in innate defense
mediated by initial activation of P2Y2R purinergic receptors at the
epithelial surface, which have been implicated in regulation of
[8,12], wound responses that facilitate epithelial repair upon injury
[10,11,13], and inflammatory cytokine production [24,48]. Since
of EGFR signaling [1,2,49], the interactions between P2Y2R and
Our findings demonstrate the importance of the non-receptor
tyrosine kinase Src in ATP-dependent EGFR transactivation,
consistent with findings that Src can directly interact with P2Y2R
and EGFR to facilitate such signaling [30,38], as well as a critical
Figure 6. ATP stimulation promotes association between
P2Y2R, Src and DUOX1. (A) H292 cells were pre-incubated with
P2Y2R siRNA or control non-specific (NS) siRNA for 72 hrs, and
stimulated with ATP (100 mM, 10 min), and Src was immunoprecipi-
tated from cell lysates after which immunoprecipitated samples were
evaluated for the presence of P2Y2R or Src by Western blot. (B)
Untreated or ATP-treated H292 cells were immunoprecipitated with a-
Scr or control IgG and immunoprecipitates were analyzed by Western
blot for Src, P2Y2R, or DUOX1, in comparison with whole cell lysates
(input). Representative blots of 2 experiments are shown.
Mechanisms of DUOX1-Dependent EGFR Activation
PLOS ONE | www.plosone.org7 January 2013 | Volume 8 | Issue 1 | e54391
demonstrate the critical involvement of the NADPH oxidase
DUOX1 which promotes EGFR transactivation by oxidative
modification of critical cysteines within Src as well as ADAM17.
Our collective findings regarding the proposed role of DUOX1 in
ATP-mediated EGFR activation are schematically illustrated in
role of DUOX1 within the respiratory epithelium, which remains
a host defense enzyme that provides a source of extracellular H2O2
that contributes to oxidative microbial killing within the airway
lumen [53,54], findings from the present study further establish
DUOX1 as a mediator of redox signaling within the epithelium by
promoting discreet and reversible oxidative modifications within
downstream wound responses as indicated by activation of the
transcription factor STAT3 and induction of wound genes such as
MMP-9 or IL-8 [1,10,37,45]. Elegant studies in other organisms
have demonstrated similar roles for DUOX in host and wound
responses, highlighting the contribution of DUOX in specific
signaling pathways that orchestrate these responses [55,56,57],
suggesting that such functional properties of DUOX are highly
conserved. One complicating factor in mammalian systems,
however, is the presence of 2 separate DUOX isoforms rather than
a single DUOX gene, with distinct modes of regulation and cellular
localization [35,58,59,60]. Indeed, while recent studies implicate
DUOX2 in epithelial immune responses to TLR activating stimuli
[44,61], other reports suggest the importance of DUOX1 in
epithelial wound responses, mucus production, and cytokine
signaling [10,36,62], and our findings clearly establish that ATP-
dependent EGFR activation is exclusively mediated by DUOX1
and not by DUOX2. These various findings may suggest variable
interactions of specific DUOX isoforms with different GPCR or
TLR. Indeed, our findings in Fig. 6B indicate that DUOX1 is
directed associated with P2Y2R and Src in response to ATP
stimulation, although we did not address whether this interaction
was specific for DUOX1. The reported localization of Src  as
well as DUOX1  in lipid rafts in response to appropriate
activation further supports such a direct interaction in membrane
Although EGFR is primarily localized in the basolateral
membrane within the intact epithelium and its full activation by
ligand shedding, which occurs at the apical epithelial surface or
within the intracellular space, is thought to require mechanical
stress or disruption of epithelial junctions [42,65], our studies
indicate that luminal ATP was capable of activating EGFR in
intact epithelial monolayers without the need for disruption of
epithelial junctions, and disruption of tight junctions by EGTA-
mediated Ca2+chelation in fact diminished ATP responses. Such
findings are consistent with the reported location of P2Y2R and
DUOX at the apical epithelial surface, and suggest that Src,
Figure 7. Lack of ATP-dependent EGFR phosphorylation in H292-shDUOX1 cells. Stably transfected H292-shDUOX1 or H292-CTL cells were
evaluated for DUOX1 or DUOX2 RNA by qPCR (A), DUOX protein by western blot (B), or basal or ATP-stimulated H2O2production (15 min), measured
by HPLC analysis of peroxidase-catalyzed dityrosine (C). *: p,0.05 compared to untreated control; #:p,0.05 compared to H292-CTL (n=6).
Qualitatively similar findings were obtained using Amplex Red assay (not shown). Serum-starved H292-shDUOX1 or H292-CTL cells were stimulated
with ATP for 5–60 min and harvested for western blot analysis for phosho-EGFR (Y845), phospho-EGFR (Y1068) and total EGFR (D), and results from 2–
3 separate western blots were quantified (E).
Mechanisms of DUOX1-Dependent EGFR Activation
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ADAM17, and EGFR must be also be partially localized at the
apical epithelial surface to allow for such signaling. Alternatively,
ligand-independent EGFR activation may be transduced through
the cell by indirect EGFR-dependent ADAM-17 activation in
a feed-forward mechanism .
Our findings extend our understanding of the role of H2O2or
ROS in regulation of EGFR signaling. Various previous findings
have the demonstrated ability of ROS to activate EGFR and
indicated an important role for Src in such activation [27,28],
whereas others have demonstrated ROS-dependent EGFR ligand
shedding by activation of ADAM17 [24,36]. Oxidative regulation
of EGFR signaling may depend on whether EGFR activation
occurs in the context of global oxidative stress or as part of
a physiological signal, such as in GPRC-dependent EGFR
activation. Indeed, oxidative stress has been demonstrated to
result in aberrant EGFR activation and defective EGFR
trafficking in relation to Src activation [28,67]. In contrast,
endogenous H2O2production by NADPH oxidases appears to
promote EGFR activation by patho-physiological stimuli, as
a critical mechanism of physiological EGFR regulation [17,36].
Our studies indicate the involvement of DUOX1-derived H2O2in
EGFR activation by concerted oxidative activation of both Src
and ADAM17 in a pathophysiological setting of P2Y2R receptor
activation by extracellular ATP, which is readily released from
epithelial cells as a danger signal. The involvement of DUOX1 in
EGFR transactivation also differs from the previously reported
role of NOX4 in prolonging EGFR activation through inhibition
of PTP1B , and occurs at the level of initial activation of
EGFR signaling through Src and ADAM17.
Activation of Src appears to be central in ATP-mediated EGFR
transactivation, since it can directly phosphorylate EGFR at Y845
[30,68] and also contributes to activation of ADAM17 . While
the precise mechanism of Src activation by H2O2is currently
unknown, a number of reports indicate that Src and related
kinases, such as Lyn and Yes, are subject to redox regulation
which involves oxidation of one or more specific conserved
cysteine residues within a cysteine-cluster in the C-terminal region
[32,33]. It has been suggested that tyrosine auto-phosphorylation
at Y416 and cysteine oxidation within Src are successive events
during e.g. cell adhesion and spreading, and that cysteine
oxidation at C245 and C487 is necessary for full Src activation
. Our findings, however, indicate that Src phosphorylation at
Y416 in response to ATP depends on DUOX1, whereas EGF-
dependent phosphorylation of Src Y416 was more modest and
appeared to be independent of DUOX1 (Fig. S5). These results
suggest that DUOX1-dependent cysteine oxidation within Src also
contributes to its auto-phosphorylation of Y416, and is required
for its full activation, as was suggested previously . However, it
is important to point out that our studies did not elucidate the
critical cysteine residues involved, and various Cys residues have
been implicated in both positive and negative regulation of Src
activity [31,33,69]. Further studies with selected Src mutants will
be needed to more definitively address this. Also, our studies did
not address the potential activation of other Src family kinases
such as Fyn or Yes that may also be involved in innate epithelial
responses to pollutants or infectious stimuli [34,70].
ADAM17 plays a central role in the release of growth factors,
cytokines, and other membrane proteins, and is rapidly activated
by a variety of signaling pathways although the underlying
mechanisms are still poorly understood . Consistent with
a recent report , our results demonstrate the critical
importance of Src in ADAM17 activation by either ATP or
Figure 8. ATP stimulation induces DUOX1-dependent cysteine oxidation in Src. (A) Serum-starved H292 cells were stimulated with ATP
(100 mM) for indicated times and lysed in the presence of Iodoacetyl-LC-Biotin, followed by purification of biotin-labelled protein with NeutrAvidin
agarose, and western blot analysis of biotinylated proteins and whole lysates for Src, reflecting Src-SH and total Src, respectively. (B) Densitometry
analysis of Src-SH status from 3 separate experiments. *p,0.05 compared to untreated control. (C) Similar analysis of Src-SH status in H292-shDUOX1
or corresponding H292-CTL cells after 10-min stimulation with ATP (100 mM). (D) Densitometry analysis of Src-SH relative to total Src based on
Western blot analysis in panel C. Mean values of 4 replicates from 2 independent experiments are shown. *p,0.05 compared to untreated control.
Mechanisms of DUOX1-Dependent EGFR Activation
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Figure 9. ATP stimulation induced DUOX1-dependent oxidation of ADAM-17 at the cell surface. (A) Confluent H292-shDUOX1 or
corresponding H292-CTL cells were stimulated with ATP (10 min; 100 mM), and surface proteins were labeled with sulfo-NHS-biotin or MPB for
labeling of surface NH2groups or cysteine-SH residues, respectively. Avidin-purified biotinylated proteins were probed for ADAM-17 by western blot.
Representative blot of 2 separate experiments are shown. Densitometry analysis of SH labeling relative to NH2labeling of upper 120 kD bands (B) and
lower 100 kD bands (C) are shown from 4 replicates from 2 independent experiments. *p,0.05 compared to untreated control.
Figure 10. Schematic representation of proposed involvement of DUOX1 in ATP-mediated EGFR transactivation. ATP-mediated
activation of P2Y2R promotes Ca2+and protein kinase C (PKC) and thereby activates DUOX1-dependent H2O2production. DUOX1-derived H2O2
promotes activation of Src and ADAM-17 by cysteine oxidation (1), and Src activation also promotes ADAM-17 activity by phosphorylation and
directly activates EGFR by phosphorylation at Y845 (2). In addition, ADAM-17 activation promotes EGFR ligand shedding resulting in further EGFR
activation (3). Activated EGFR can in turn promote activation of Src and ADAM-17 in a feed-forward mechanism to enhance EGFR signaling and
downstream pathways such as ERK1/2 and STAT3 (4).
Mechanisms of DUOX1-Dependent EGFR Activation
PLOS ONE | www.plosone.org10 January 2013 | Volume 8 | Issue 1 | e54391
EGF, the latter finding indicating that EGFR-dependent activa-
tion of ADAM17 may serve to augment or prolong EGFR
activation in a feed-forward mechanism. In accordance with
several recent reports [25,26], we also provide evidence of cysteine
oxidation with ADAM17 in response to ATP stimulation in
a DUOX1-depedendent fashion. It was proposed that such
cysteine oxidation within the mature form of ADAM17 at the
cell surface alters its conformation to a more ‘‘open’’ state and
promotes its activity, and that such oxidation occurs via thiol-
disulfide exchange after initial oxidation of protein disulfide
isomerase (PDI) . We attempted to detect reduced or oxidized
PDI at the cell surface of H292 cells, but were unsuccessful.
In summary, our findings highlight a central role of the
NADPH oxidase DUOX1 in EGFR transactivation in airway
epithelial cells in response to P2Y2R stimulation by exogenous
ATP, and identify Src and ADAM17 as proximal targets for redox
regulation in response to DUOX1 activation. While our findings
provide further insight into the functional importance of DUOX1
in airway epithelial signaling and biology, our studies provoke
several questions. First, our studies did not address how such
DUOX1-dependent signaling is terminated, and future studies will
be necessary whether such signaling is controlled by common
reductases such as thioredoxins or glutaredoxins. Second, our
studies did not establish the oxidation products within Src or
ADAM17, or whether these enzymes are directly targeted by
DUOX1-derived H2O2. In this regard, kinetic arguments suggest
that H2O2-dependent protein cysteine oxidation is in most cases
too slow compared to reactions with H2O2-metabolizing enzymes,
and redox signaling by e.g. NADPH oxidase-derived H2O2 is
thought to be largely mediated by a select number of sensitive
H2O2sensor proteins that can then transfer cysteine oxidation to
less intrinsically susceptible proteins [71,72]. Indeed, the peroxir-
edoxin (Prx) thiol peroxidases present strong candidates for such
a H2O2 sensor role because of their cellular abundance and
exquisite reactivity towards H2O2, and several findings indeed
confirm the involvement of Prx in transducing oxidative signals
towards various signaling proteins via thiol-disulfide exchange
[71,73]. It remains to be established whether Prx plays a similar
role in P2Y2R-mediated oxidative activation of Src and ADAM17,
or whether Src could possess similar redox sensor properties.
Materials and Methods
Cell Culture and Treatments
carcinoma cell line, were originally obtained from the American
Type Culture Collection (ATCC) and grown in RPMI 1640
medium containing 10% fetal bovine serum and 1% penicillin/
streptomycin at 37uC and 5% CO2. For experimentation, cells
were seeded in 24-well plates, grown to confluence and serum-
starved for 24 hrs prior to treatments. Cells were placed in fresh
serum-free medium for 2 hrs and stimulated with either
exogenous ATP (Sigma, St. Louis, MO; 100 mM) or EGF
(Calbiochem; 100 ng/ml), and cell lysates were collected at
various time points by placing cells on ice in 100 ml lysis buffer
(Cell Signaling, Beverly, MA) per well for 20 min. Lysates were
collected by scraping, briefly sonicated, and cleared of insoluble
material by centrifugation (14,000 rpm, 5 min) for analysis. In
appropriate cases, inhibitors were added 30 min prior to cell
Additional experiments were performed with immortalized
human bronchial epithelial (HBE1) cells, originally provided by
Drs. Yankaskas and Wu, which were cultured in Ham’s F-12/
Dulbecco’s modified Eagle’s medium (50:50) supplemented with
a humanpulmonary mucoepidermoid
insulin (5 mg/ml), transferrin (5 mg/ml), EGF (10 ng/ml), dexa-
methasone (0.1 mM), cholera toxin (10 ng/ml), and bovine serum
albumin (0.5 mg/ml), and bovine hypothalamus extract (15 mg/
ml). HBE1 cells were seeded on 12-well tissue culture inserts
(Corning) coated with collagen (BD Biosciences; 50 mg/ml). After
culture for 5 days to confluence, cells were grown at air-liquid
interface conditions for an additional 14 days in the presence of
30 ng/ml retinoic acid to obtain polarized epithelia. One day
prior to experimentation, cells were kept overnight in full media
lacking EGF, and treatments with either ATP or EGF were added
either apically (in 0.5 mL media) or basolaterally, and cells were
collected as described above for analysis.
Analysis of Epithelial Wound Responses
Confluent serum-starved H292 cells in 24-well plates were
subjected to scratch injury using a sterile pipette tip, rinsed with
warm PBS to remove cellular debris, and placed in fresh full media
to monitor wound closure over 8 hrs. Initial and final wound areas
were imaged using Image J software (NIH) for calculation of %
wound closure. Alternatively, serum-starved H292 cells were
stimulated with ATP (100 mM) for 8 hrs and RNA was extracted
for analysis of mRNA levels of the common wound response genes
MMP-9 and IL-8.
Western Blot Analysis
Lysates containing equal amounts of protein (20–35 mg,
measured using BCA protein assay kit; Pierce) were loaded on
10% SDS-PAGE gels and transferred to nitrocellulose mem-
branes, and probed using antibodies against P2Y2R (Invitrogen;
1:250), or EGFR (C74B9; 1:1000), pEGFR Tyr845 (1:500),
pEGFR Tyr1068 (D7A5; 1:1000), p-Src Tyr 416 (1:1000), Src
(L4A1; 1:1000), p-STAT3 Tyr705 (D3A7; 1:1000), STAT3 (79D7;
1:1000), and b-actin (1:1000; Cell Signaling). Primarily antibodies
were probed with rabbit or mouse-specific secondary antibodies
conjugated with HRP (Cell Signaling) and detected by enhanced
SiRNA Silencing of P2Y2R, Src, ADAM17, or DUOX1/2
H292 cells were seeded at 70% confluence for siRNA trans-
fection in serum-free medium with siGENOME or ON-TAR-
GETplus SMARTpoolH siRNAs targeted against P2Y2R, Src, or
ADAM17 or Non-Targeting siRNA Pool#1 as control (Dharma-
con, Lafayette, CO) using DharmaFECT transfection reagent,
according to the manufacturer’s instructions. Silencing of DUOX1
or DUOX2 was performed by transfection with the following
siRNA reagents: DUOX1: (sense) GCUAUGCAGAUGGCGU-
GUAtt, (antisense) UACACGCCAUCUGCAUAGCtg; DUOX2:
GUAGACAUUGACUGCGtg, or with Silencer Negative Control
#1 siRNA (Invitrogen). Following overnight transfection, medium
was replaced with full RPMI 1640 medium (10% FBS & 1% pen/
strep) the following day and cells were grown for 48 hrs until
Analysis of H2O2Production
H292 cells were seeded at 150,000 cells/well in 24 plates and
media was replaced with 200 mL HBSS for cell stimulation with
ATP or EGF for 15 min, after which HBSS was removed and
mixed with 10 mg/ml lactoperoxidase (Sigma) and 1 mM tyrosine
for 15 min. Reactions were terminated by addition of 5% TCA for
analysis of dityrosine production by HPLC . Alternatively,
H2O2 was analyzed using the Amplex Red assay (Invitrogen)
according to the manufacturer’s instructions. H2O2production
Mechanisms of DUOX1-Dependent EGFR Activation
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was calculated using similar analysis of exogenous standards of
H2O2in HBSS, and expressed as nmol/106cells.
Analysis of ADAM17 Activity
Confluent H292 cells were serum-starved and treated with ATP
or EGF as described above, in the presence of a fluorogenic
ADAM17 substrate (Calbiochem; 10 mM). Media was collected
after 2 hrs, and ADAM17 activity was analyzed using a fluores-
cence plate reader. Inhibitors were added 30 min prior to
stimulation with ATP or EGF.
Generation of H292-shDUOX1 Cell Lines
H292 cells were transfected with pTET-On Advanced Vector
(Clontech, Mountainview, CA) using Lipofectamine 2000 and
transfected cells were selected using G418 (150 mg/ml). Selected
H292-TetOn cells were subsequently transfected with pRNATin-
H1.2/Hygro vector (GenScript, Piscataway, NJ) in which a short
hairpin RNA sequence targeting DUOX1 was inserted, according
to the manufacturer’s instructions (Fig. S4). Thus transfected
H292-shDUOX1 cells were selected by culturing in the presence
of hygromycin (1.5 mg/ml) for 2 weeks, and 6 colonies were picked
and expanded. Corresponding control cell lines were generated by
transfection of H292-TetOn cells with empty pRNATin-H1.2/
Hygro vector (H292-CTL) and selected similarly. Initial analysis of
selected H292-shDUOX1 cells showed a loss of DUOX protein
expression after culture in the presence of doxycycline. However,
upon further expansion of selected H292-shDUOX1 lines, levels
of DUOX1 mRNA were found to be dramatically reduced
compared to H292-CTL (Fig. S4), even in the absence of
doxycycline, most likely due to known some basal expression
associated with this 2ndgeneration TetOn expression system
(Clontech). Therefore, these cell lines were used in subsequent
studies without addition of doxycycline.
Immunoprecipitation of Src
Lysates of untreated or ATP-stimulated H292 cells containing
2 mg protein were pre-cleared with 50 ml protein A agarose beads
(Santa Cruz) in a total volume of 1 ml for 15 mins at 4uC.
Following brief centrifugation (3,000 rpm), supernatants were
incubated with 2.5 mg of Src monoclonal mouse antibody (Cell
Signaling) or equivalent amount of control IgG1, and incubated
overnight at 4uC under continuous rotation, and subsequently
mixed with protein A agarose beads (50 ml) for an additional 4 hrs
at 4uC. Beads were washed 3X with lysis buffer and resuspended
in 2X Laemmli’s sample buffer at boiled at 100uC for 10 min.
Samples were loaded on 10% SDS-PAGE gels for Western blot
analysis using rabbit antibodies against Src (L4A1, Cell Signaling),
P2Y2R (1:250; Invitrogen), or a polyclonal antibody against the
Arg618–His1044fragment of DUOX1 (kindly provided by Fran-
coise Miot, Brussels, Belgium) .
Total RNA was extracted from cell lysates using the RNAeasy
extraction kit according to the manufacturers protocol (Qiagen,
Inc, Valencia, CA) and cDNA was synthesized using M-MLV
reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen).
PCR amplifications were carried out for 30 cycles of denaturation
(94uC, 1 min) annealing (58uC, 30 s), and extension (72uC, 1 min),
using the following primer sets for DUOX1 (59-GCA GGA CAT
CAA CCC TGC ACT CTC-39 and 59-CTG CCA TCT ACC
ACA CGG ATC TGC-39) and DUOX2 (59-GAT GGT GAC
CGC TAC TGG TT-39 and 59-GCC ACC ACT CCA GAG
AGA AG-39), and the products were analyzed on 1% agarose gels.
Expression of genes of interest was also analyzed by qPCR relative
to GAPDH using SYBR Green PCR Supermix (BioRad) and the
following with appropriate primers (Table S2) and normalized to
GAPDH using the DDCTmethod.
Detection of Thiol Status of Src and ADAM-17
Following treatments, NCI-H292 cells were lysed in deoxygen-
ated lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% (vol/vol)
Triton X-100, 10 mg/ml of aprotinin and leupeptin), containing
100 mM EZ-link Iodoacetyl-LC-Biotin (Pierce), under N2atmo-
sphere to minimize artifactual cysteine oxidation during cell lysis
. Collected lysates were cleared by centrifugation (10 min,
14,000 rpm), and to remove excess iodoacetyl-LC-biotin, proteins
were precipitated with 10% TCA and protein pellets were washed
with cold acetone/HCl/H2O (at a ratio of 92:2:10) and
resuspended in lysis buffer containing 8 M urea. Samples contain-
ing equal protein concentrations (determined using TCA protein
assay; Thermo Scientific) were mixed overnight with NeutrAvidin
agarose beads (50 ml of 50/50 slurry; Pierce) at 4uC to collect
biotinylated proteins. Beads were washed 3X with PBS to remove
non-specific binding, and mixed with 2X reducing sample buffer
containing b-mercaptoethanol and heated at 95–100uC to elute
biotin-labeled protein from the resin. The amount of Src in these
Biotinylated samples and initial lysates (input controls) were
evaluated for Src content by SDS-PAGE and western blot analysis
using a rabbit antibody against Src (Cell Signaling).
Thiol status of cell-surface ADAM-17 in treated H292 cells was
analyzed essentially as described by Willems et al. . Briefly,
cells were incubated for 45 min with either EZ-Link sulfo-NHS (N-
hydroxysuccinimido)-biotin (100 mM; Pierce) in PBS, or with
100 mM MPB [3-(N-maleimido-propionyl) biocytin; Pierce] in
Hepes-buffered saline (21 mM Hepes, pH 7.05, 0.137 M NaCl,
5 mM KCl, 0.76 mM Na2HPO4?H2O and 5.56 mM dextrose), to
label amino groups or thiol groups, respectively. Excess EZ-Link
sulfo-NHS-biotin was quenched with 50 mM Tris/HCl (pH 7.5),
and excess MPB was quenched with 200 mM GSH, after which
cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl,
0.5% (vol/vol) Triton X-100, 10 mg/ml of aprotinin and
leupeptin) for collection of biotinylated proteins as described
above. Purified biotinylated proteins and original lysates were then
analyzed by SDS-PAGE and western blotting using a rabbit anti-
ADAM-17 antibody (Millipore; 1:1000).
Data Processing and Analysis
Western blots were semiquantified using densitometry analysis
by ImageJ, and quantified data are presented as mean 6 S.E.
Statistical differences were analyzed using Student’s t-test and
differences were considered significant at p,0.05.
dent EGFR activation. Confluent serum-starved H292 cells
were stimulated with either ADP, ATP, UDP, or UTP (100 mM
each) for 10 min, cell lysates were analyzed for phosphorylated
and unphosphorylated (total) EGFR by Western blot. Represen-
tative blots from 2 experiments are shown.
Pharmacological evaluation of P2Y-depen-
pends on Src. Confluent serum-starved H292 cells were pre-
treated with the EGFR kinase inhibitor AG1478 (10 mM; A) or
with the Src inhibitor PP2 (1 mM; B) for 15 min and subsequently
treated with ATP (100 mM) or EGF (100 ng/ml) for 10 min,
unless indicated otherwise. Cell lysates were analyzed for
ATP-dependent EGFR phosphorylation de-
Mechanisms of DUOX1-Dependent EGFR Activation
PLOS ONE | www.plosone.org12January 2013 | Volume 8 | Issue 1 | e54391
phosphorylated EGFR by Western blot, and representative blots
from 2–3 separate experiments are shown.
ADAM17 activity. Confluent H292 cells were pre-incubated
with ADAM17 siRNA or control non-specific (NS) siRNA
(Dharmacon) for 72 hrs, and ADAM17 mRNA expression was
evaluated by qPRC (left) and ATP-stimulated (100 mM) activation
of ADAM17 was determined using a fluorogenic substrate (right).
Evaluation of ATP-dependent increase in
Generation of stable H292-shDUOX1 cells using pTet-On
expression system (Clontech) and transfection with DUOX1-
targeted shRNA using pRNATin-H1.2/Hygro vector (Genscript).
(B) PCR analysis of DUOX1 or DUOX2 in H292-shDUOX1
cells and corresponding control cells (H292-CTL) grown in the
absence or presence of doxycycline.
Generation of stable H292-shDUOX1 cells. (A)
STAT3 is diminished in H292-shDUOX1 cells. Confluent
H292-shDUOX1 or corresponding H292-CTL cells were starved
ATP-dependent activation of EGFR, Src, or
and stimulated with either ATP (100 mM) or EGF (100 ng/ml) for
10 min, and cell lysates were analyzed for phosphorylated and
total EGFR, Src, or STAT3 (A). Western blots of phosphorylated
forms of EGFR from 3 separate experiments were quantified by
densitometry (B,C). ATP- or EGF-stimulated activation of
ADAM17 in H292-shDUOX1 or H292-CTL cells assessed using
fluorogenic ADAM17 substrate as in Fig. 4. (n=3). *: p,0.05
compared to untreated cells;
sponding stimulation in H292-CTL cells.
#:p,0.05 compared with corre-
on mRNA expression of P2YR and ADAM17.
Effects of DUOX siRNA silencing in H292 cells
qPCR primer sequences used in this study.
Conceived and designed the experiments: AvdV DS UVW. Performed the
experiments: DS MH UVW. Analyzed the data: AvdV DS MH.
Contributed reagents/materials/analysis tools: UVW. Wrote the paper:
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