T H E J O U R N A L O F C E L L B I O L O G Y
© 2008 Chen et al.
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 181 No. 7 1129–1139
Correspondence to Kai Chen: email@example.com
Abbreviations used in this paper: EGFR, EGF receptor; ERK, extracellular signal-
regulated kinase; HAEC, human aortic endothelial cell; MEF, mouse embryonic
fi broblast; MSP, mitochondrial signal peptide; NO, nitric oxide; NOS, NO syn-
thase; Nox, NADPH oxidase; PTP, protein tyrosine phosphatase; ROS, reactive
oxygen species; VE, vascular endothelial.
There is now considerable evidence indicating that reactive
oxygen species (ROS) are involved in signal transduction. These
species include the three successive reduction products of molec-
ular oxygen such as superoxide ( • O 2 ? ), hydrogen peroxide
(H 2 O 2 ), and hydroxyl radical ( • OH). Each of these species pos-
sesses chemical properties that potentially impact their signal-
ing function. For example, OH • is the most unstable ROS with
a half-life of 10 ? 9 s ( Pryor, 1986 ), indicating that it will react
with any species within a radius of ? 30 Å , thereby limiting
its ability to transmit signals across any signifi cant distance.
Superoxide carries a negative charge that limits its membrane
permeability to anion channels. In contrast, H 2 O 2 is thought to
be freely permeable and, thus, could react with several intra-
and extracellular targets, limiting its specifi city; however, this
concept has recently been challenged ( Branco et al., 2004 ).
Thus, the chemical properties of ROS suggest signifi cant limi-
tations toward the production of specifi c cellular responses.
The prototypical NADPH oxidase (Nox) family member
is Nox2 (also known as gp91 phox ), which was initially found in
phagocytes and plays a role in host defense by giving an outward
burst of ROS ( Cheng et al., 2001 ). Over the last several years,
Nox2 along with its homologues, including Nox1, Nox3, Nox4,
Nox5, Duox1, and Duox2, have been identifi ed in nonphago-
cytes. It now appears that Noxs in nonphagocytes serve as a major
source of intracellular ROS that play important signaling roles.
However, the complexity of these isoforms in controlling ROS
production is increasingly apparent because each isoform has its
unique expression pattern, subcellular localization, and subunits
requirement. The specifi c mechanisms for specifi c cell signaling
responses are not known; therefore, the goal of this study is to
examine the mechanisms of ROS signaling specifi city.
Exogenous versus endogenous sources of
ROS initiate distinct signaling responses
ROS are known to mediate a variety of cellular signaling path-
ways, and experiments in vitro typically use an exogenous
source of ROS such as H 2 O 2 to directly initiate signaling re-
sponses. However, one must consider that exogenous applica-
tion of H 2 O 2 may not adequately refl ect authentic endogenous
ROS signaling. Indeed, exogenous H 2 O 2 produces broad signal-
ing responses, including the activation of extracellular signal-
regulated kinase (ERK), JNK, p38 MAPKs ( Fig. 1 A ), and Akt
( Thomas et al., 2002 ). In contrast, the EGF-stimulated signaling
response, which is known to be mediated by ROS in epithelial
cells ( Bae et al., 1997 ), was restricted to the mitogenic ERK path-
way ( Fig. 1 B ) in endothelial cells. We found that EGF-induced
diffusible ROS dictate specifi c cellular responses. In this
study, we demonstrate that nicotinamide adenine dinucle-
otide phosphate reduced oxidase 4 (Nox4), a major Nox
isoform expressed in nonphagocytic cells, including vascu-
lar endothelium, is localized to the endoplasmic reticulum
(ER). ER localization of Nox4 is critical for the regulation
of protein tyrosine phosphatase (PTP) 1B, also an ER resi-
eactive oxygen species (ROS) function as intracellu-
lar signaling molecules in a diverse range of bio-
logical processes. However, it is unclear how freely
dent, through redox-mediated signaling. Nox4-mediated
oxidation and inactivation of PTP1B in the ER serves as a
regulatory switch for epidermal growth factor (EGF) re-
ceptor traffi cking and specifi cally acts to terminate EGF
signaling. Consistent with this notion, PTP1B oxidation
could also be modulated by ER targeting of antioxidant
enzymes but not their untargeted counterparts. These data
indicate that the specifi city of intracellular ROS-mediated
signal transduction may be modulated by the localization
of Nox isoforms within specifi c subcellular compartments.
Regulation of ROS signal transduction by NADPH
oxidase 4 localization
Kai Chen , Michael T. Kirber , Hui Xiao , Yu Yang , and John F. Keaney Jr.
Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605
JCB • VOLUME 181 • NUMBER 7 • 2008 1130
lation induced by EGF, whereas Nox2 suppression had no mate-
rial impact ( Fig. 1 F ). Together, these data implicate endothelial
Nox4 as an endogenous source of ROS in mediating EGF-
induced signaling responses.
Nox4 is an ER-residing protein
To gain insight into the nature of Nox4, we examined its sub-
cellular localization. The topography of Nox4 was examined in
silico initially using PredictProtein (Columbia University), and
it possesses six putative membrane-spanning regions, predicting
ERK activation was attenuated by catalase overexpression ( Fig. 1,
C and D ), indicating a role for ROS and a distinction between
endogenous and exogenous ROS signaling.
To examine endogenous ROS responses in the endothe-
lium, we suppressed the expression of Nox4 and Nox2, the two
major Nox isoforms present in endothelial cells ( Sorescu et al.,
2002 ), using RNAi. As shown in Fig. 1 E , both Nox4 and Nox2
siRNA substantially reduced their respective mRNA and protein
levels without cross-reactivity by > 75% and 60%, respectively.
We found that suppression of Nox4 attenuated ERK phosphory-
Figure 1. A different source of ROS initiates distinct signaling responses in endothelial cells. (A) HAECs were treated with 100 μ M H 2 O 2 for the indicated
times, and total cell lysates were subjected to antibodies specifi c for pERK, ERK, pcJun, cJun, pp38, or p38. (B) HAECs were treated with 50 ng/ml EGF for the
indicated times followed by immunoblotting with antibodies as in A. (C and D) Cells were treated with control adenovirus (Ad-LacZ) or catalase-overexpressing
adenovirus (Ad-Cat) for 24 h before treatment with EGF followed by immunoblotting as in A. Error bars represent SD. *, P < 0.05. (E and F) HAECs were trans-
fected with siRNA against nontargeting control (NT), Nox4, or Nox2 for 48 h before extraction of total RNA for RT-PCR and Western blotting (E) or treatment with
EGF for 15 min followed by immunoblotting with pERK, ERK, Nox4, and Nox2 antibodies (F). Results are representative of four independent experiments.
1131NADPH OXIDASE 4 REGULATES REDOX SIGNALING • Chen et al.
when COS-7 cells were examined after transfection with either
V5- or myc-tagged Nox4, identical results were obtained regard-
less of whether the tag was C or N terminal (unpublished data).
Finally, using immunogold electron microscopy in HAECs trans-
fected with adenoviral V5-tagged Nox4 ( Fig. 2 C ), we observed
gold particles predominantly in the ER membrane. To confi rm
the aforementioned results, a biochemical analysis of sub-
cellular fractionation was performed in Nox4-V5 – overexpressing
cells and revealed that Nox4-V5 was mainly detected in the frac-
tion of membrane/organelle that coincides with the ER marker
GRP78 ( Fig. 2 D ) but not with other intracellular markers, in-
cluding SHP-2 as a cytoplasmic marker, histone H4 as a nuclear
marker, and vimentin as a cytoskeletal marker ( Higashi et al., 2002 ).
Similarly, analysis by nonlinear Nycodenz density gradient
an integral membrane protein similar to Nox2. Further in silico
analysis based on the Nox4 amino acid sequence using the
PSORT II program (Human Genome Center, Tokyo University)
and the k -nearest neighbor algorithm placed the probability of
Nox4 localizing to the ER at 67%, whereas the probability
of localization in plasma membrane, mitochondria, or the Golgi
was only 11% each. Consistent with this prediction, confocal
microscopy revealed a perinuclear distribution of endogenous
Nox4 with colocalization with the ER marker protein GRP78
( Fig. 2 A ). These data were confi rmed using transfection with
V5-tagged Nox4 by adenoviral vector in human aortic endo-
thelial cells (HAECs; Fig. 2 B ). There was no localization to
the plasma membrane, as evident by staining for the plasma
membrane marker vascular endothelial (VE) cadherin. Moreover,
Figure 2. Nox4 is localized to the ER.
(A) HAECs cultured in EBM2 medium were fi xed
and subjected to immunostaining with anti-
Nox4 (AlexaFluor488; green) and anti-GRP78
(AlexaFluor594; red) followed by two-photon
confocal microscopy. (B) HAECs were trans-
fected with adenoviral vector expressing Nox4-V5
(AdNox4-V5). After 24 h, cells were probed
with anti-V5 and VE-cadherin antibodies and
visualized with AlexaFluor594 (red) and Alexa-
Fluor488 (green), respectively. (C) Cells were
transfected with AdNox4-V5 as in B followed
by immunogold staining and were visualized by
electron microscopy. (D and E) Similarly, HAECs
with overexpression of Nox4-V5 were fraction-
ated by biochemical detergent method (D) or
Nycodenz gradient centrifugation (E). The ob-
tained fractions cytosolic (Cyto), membrane/
organelle (MO), nuclear (Nuc), and cytoskeletal/
matrix (CSK/MAT) in D or fractions from top to
bottom in E were subjected to immunoblotting
with antibodies. Bars: (A) 5 μ m; (B) 10 μ m.
JCB • VOLUME 181 • NUMBER 7 • 2008 1132
tyrosine phosphatases (PTPs). Because PTP1B is an ER-resident
protein and its C-terminal 35 amino acid residues are critical for
ER targeting ( Frangioni et al., 1992 ; Salmeen et al., 2003 ; van
Montfort et al., 2003 ), we fi rst overexpressed PTP1B wild type
(PTP1B) and a mutant lacking the C-terminal 35 amino acids
(PTP1B- ? 35) in COS-7 cells and observed their distinct cellular
localization. Although the wild-type PTP1B shows strong peri-
nuclear colocalization with Nox4, the mutant PTP1B exhibits
diffuse cytoplasmic distribution ( Fig. 3 A ) and predominant
centrifugation revealed that Nox4-V5 is present in fractions that
are enriched with the ER marker GRP78 but not the Golgi marker
GS-28 ( Fig. 2 E ). Collectively, these data establish that Nox4 is
localized to the ER of endothelial cells.
Nox4-dependent redox regulation of
PTP1B requires colocalization
Signaling via ROS is thought to be mediated by regulation of
the redox-sensitive cysteine of target proteins, including protein
Figure 3. PTP1B oxidation by Nox4 is spatially dependent. (A) COS7 cells were transfected with pcDNA3.1/PTP1B wild type or pcDNA3.1/PTP1B- Δ 35
in addition to pcDNA3.1/Nox4-V5. After 24 h, cells were fi xed and immunostained with anti-V5 (green) for Nox4-V5 and anti-PTP1B for PTP1B wild
type and C-terminal deleted PTP1B- Δ 35 (red). (B) Cells transfected as in A were also subjected to detergent fractionation followed by immunoblotting with
anti-PTP1B and anti-GRP78. Fractions include cytosolic (Cyto), membrane/organelle (MO), nuclear (Nuc), and cytoskeletal/matrix (CSK/MAT). (C) COS-7
cells treated as in A were lysed and labeled with biotin polyethylene oxide maleimide. Subsequent immunoprecipitation with avidin followed by immuno-
blotting with anti-PTP1B yields a band that represents the reduced, active form of PTP1B. (D) COS-7 cells were transfected with pcDNA3.1/Nox4-V5 or its
Nox4i-resistant version pcDNA3.1/Nox4-R in combination with/without pQ/Nox4i. PTP1B oxidation was assessed as in C, and the effi ciency of Nox4
knockdown was probed by V5. Total cellular PTP1B is shown in the bottom panel. (E and F) HAECs were transfected with Ad-control siRNA (Ad-Ctli) or
Ad-Nox4i for 48 h before lysis and labeling with biotin polyethylene oxide maleimide. Treatment of cells with H 2 O 2 at 100 μ M for 5 min served as a
positive control. Blots are representative of three independent experiments. Error bars represent SD. *, P < 0.05. Bar, 10 μ m.
1133 NADPH OXIDASE 4 REGULATES REDOX SIGNALING • Chen et al.
suggesting an involvement of Nox4 in EGFR signaling at the late
phase. To determine whether the regulation of EGFR phosphory-
lation by Nox4 is mediated by PTP1B, we transfected COS-7
cells with PTP1B in the presence or absence of Nox4. Over-
expression of PTP1B enhanced EGFR dephosphorylation at
30 min after EGF treatment. In contrast, the late component
of EGFR ( Fig. 4 B ) and ERK ( Fig. 4 C ) phosphorylation was
enhanced in cells with Nox4 overexpression compared with
PTP1B alone. Concerning the presence of endogenous PTP1B
in COS-7 cells, we further used immortalized PTP1B ? / ? mouse
embryonic fi broblasts (MEFs) along with PTP1B ? / ? (+WT) MEFs
in which PTP1B has been reconstituted in PTP1B ? / ? MEFs.
Consistent with the fi ndings in COS-7 cells, Nox4 overexpres-
sion prolonged EGFR phosphorylation in PTP1B ? / ? (+WT) MEFs
( Fig. 4 D ). These results suggest Nox4-mediated regulation of
EGFR dephosphorylation via PTP1B.
To address the spatial requirements of Nox4 in regulating
EGFR signaling, we overexpressed PTP1B- Δ 35 ( Frangioni et al.,
1993 ) and observed reduced EGFR phosphorylation ( Fig. 4 B ).
However, Nox4 overexpression did not modify the effect of
PTP1B- Δ 35 on EGFR dephosphorylation and subsequent ERK
activation status ( Fig. 4 C ). Further study with the cotransfec-
tion of Nox4 and PTP1B wild type/PTP1B- Δ 35 in PTP1B ? / ?
MEFs supported PTP1B wild type being the target of Nox4 in
regulation of EGFR dephosphorylation ( Fig. 4 E ). In contrast,
Nox4 was not effective in modulating the effect of PTP1B- Δ 35
( Fig. 4 B ). Thus, these data support the notion that colocalization
of PTP1B and Nox4 in the ER is essential for redox regulation
of EGFR signaling in various cell types, including endothelial
cells ( Fig. 4 F ).
Oxidation of PTP1B trapping mutant
by Nox4 attenuates its substrate-
To gain insight into the mechanism of Nox4-mediated modu-
lation of PTP1B activity, we used substrate-trapping PTP1B
mutant that retains substrate-binding activity but cannot com-
plete the catalytic cycle and release the substrate ( Tonks, 2003 ).
Indeed, COS-7 cell transfection with the PTPT1B trapping mu-
tant (D181A/Q262A) produces an increase in phosphorylation of
the EGFR that is enhanced after treatment with EGF ( Fig. 5 A ).
Because the mutant PTP1B(D181A/Q262A) features disrupted
trapping ability upon oxidation ( Salmeen et al., 2003 ), we then
sought to evaluate the involvement of Nox4-derived ROS in this
setting. Overexpression of Nox4 in this system attenuated EGFR
tyrosine phosphorylation at basal condition and after EGF
stimulation ( Fig. 5 A ). The localization of EGFR visualized by
using an enhanced GFP-EGFR expression vector was consistent
with EGFR mobilization to the ER induced by PTP1B(D181A/
Q262A) as a result of substrate trapping, which was reversed
by Nox4 overexpression ( Fig. 5 B ). In agreement with this
observation, the interaction between EGFR and PTP1B(D181A/
Q262A) was evident but greatly attenuated by the presence of
Nox4 as determined by pull-down assay ( Fig. 5 C ). Thus, these
data suggest that Nox4-derived ROS mediate the oxidation
status/trapping ability of PTP1B(D181A/Q262A) in relation
relocation to the cytosol by detergent fractionation ( Fig. 3 B ).
Other than the location, PTP1B- ? 35 has retained its enzymatic
activity and indeed has shown a slightly higher activity compared
with wild-type PTP1B ( Frangioni et al., 1992 ). This property
prompted us to determine whether PTP1B is subject to oxida-
tive modifi cation by Nox4 in a spatially dependent manner.
Using the preceding cotransfection system, we assessed the
PTP1B oxidation status with biotin maleimide as a function of co-
overexpressed Nox4 level. Biotin maleimide irreversibly alkylates
SH ? groups on the active cysteine of PTP1B, and, therefore,
its PTP1B incorporation refl ects the amount of reduced PTP1B.
As shown in Fig. 3 C , coexpression of Nox4 with both PTP1B
wild type and PTP1B- ? 35 led to less reduced PTP1B (PTP1B-S ? )
only in wild-type PTP1B, which is consistent with more oxi-
dation. However, there was no material change in PTP1B- ? 35
oxidation with or without Nox4 overexpression ( Fig. 3 C ),
suggesting that cytosolic PTP1B is not subject to oxidation
To further validate the Nox4-dependent PTP1B oxidation
by rescue experiment, we constructed an RNAi-producing vec-
tor targeting wild-type Nox4 (pQ/Nox4i) and an RNAi-resistant
Nox4 overexpression vector (pcDNA3.1/Nox4-R) containing
silent mutations in the RNAi target region but encoding wild-
type Nox4 protein. Immunoblot analysis verifi ed that Nox4 from
pcDNA3.1/Nox4-V5 could be effi ciently knocked down by
pQ/Nox4i, whereas pcDNA3.1/Nox4-R was indeed resistant to
Nox4i. Further examination of PTP1B wild-type redox status
revealed increased oxidation (less reduced form) in association
with Nox4 overexpression, which was attenuated by the pres-
ence of Nox4i. In contrast, the effect of Nox4-R, the Nox4i-
resistant version of wild-type Nox4, on PTP1B oxidation was
equivalent to that of the wild-type Nox4 but was unaltered by
Nox4i ( Fig. 3 D ), suggesting the specifi city of Nox4i and con-
sistency of Nox4-dependent PTP1B oxidation. To recapitulate
this interaction in HAECs, we used Ad-Nox4i originated from
pQ/Nox4i and found that suppression of Nox4 by RNAi in
HAECs was associated with more of the reduced form of PTP1B
(PTP1B-S ? ), whereas H 2 O 2 treatment attenuated the band inten-
sity ( Fig. 3, E and F ), indicating the Nox4-dependent oxidation
of PTP1B in endothelial cells. Together, these results further
support the notion that colocalization is essential for redox-
Nox4-dependent PTP1B oxidation is
involved in EGFR dephosphorylation
One action of PTP1B is the negative regulation of multiple
receptor tyrosine kinases, including the EGF receptor (EGFR;
Flint et al., 1997 ; Liu and Chernoff, 1997 ), in part through re-
ceptor endocytosis to a dephosphorylation compartment within
the ER ( Haj et al., 2002 ). To probe Nox4 involvement in regu-
lating EGFR phosphorylation, we initially transfected COS-7
cells with Nox4 and examined EGF-stimulated EGFR tyrosine
phosphorylation. As shown in Fig. 4 A , EGF treatment pro-
duced early robust EGFR phosphorylation at 5 min, which was
attenuated at 30 min. Overexpression of Nox4 enhanced the
phosphorylation of both EGFR and downstream ERK at 30 min
after EGF treatment with little impact on the early response,
JCB • VOLUME 181 • NUMBER 7 • 2008 1134
Figure 4. Nox4-dependent PTP1B oxidation regulates EGF signaling. (A) COS-7 cells were transfected with pcDNA3.1/Nox4-V5 for 24 h followed by
50 ng/ml EGF treatment for the indicated times. The lysates were probed for phosphorylated and total EGFR and ERK. *, P < 0.05 compared with the
respective control. (B) COS-7 cells were transfected with pcDNA3.1/PTP1B or pcDNA3.1/PTP1B- Δ 35 in addition to pcDNA3.1/Nox4-V5 and treated as
in A. Phosphorylation of EGFR was determined by immunoprecipitation with anti-EGFR followed by immunoblotting with antiphosphotyrosine. Total PTP1B
and Nox4 levels were examined by using anti-PTP1B and anti-V5, respectively. (C) ERK phosphorylation was measured at 30 min after EGF treatment by
immunoblotting with antiphospho-ERK. (D) PTP1B ? / ? and PTP1B ? / ? (+WT) MEFs were transfected with or without pcDNA3.1/Nox4 followed by treatment and
detection as in B. (B – D) *, P < 0.05. (E) PTP1B ? / ? MEFs were transfected with pcDNA3.1/PTP1B or pcDNA3.1/PTP1B- Δ 35 in addition to pcDNA3.1/Nox4.
Cells were collected for assay as in B. *, P < 0.05 compared with the respective control. (F) HAECs were transfected with Ad-control siRNA (Ad-Ctli) or
Ad-Nox4i for 48 h and were treated with EGF for the indicated times. Phospho-ERK, total ERK, and Nox4 were determined. *, P < 0.05 compared with
the respective Ad-Ctli. The blots are representative of three independent experiments. Error bars represent SD.
1135 NADPH OXIDASE 4 REGULATES REDOX SIGNALING • Chen et al.
ER-targeting antioxidants attenuate
Nox4-derived ROS signaling
If ER localization is critical for Nox4 to modulate EGFR signal-
ing, one might expect that ER targeting of antioxidants could
attenuate the effect of Nox4. To test this hypothesis, we used the
N-terminal 35 amino acid residues (N35) of Nox4 that confer
ER localization ( Fig. 6 A ) to target catalase (N35-Cat) to the
ER. N35-Cat exhibits perinuclear ER distribution distinct from
its original peroxisomal sites ( Fig. 6 B ). We chose to use intra-
cellular organelles targeting catalases for spatial-dependent
manipulation because overexpression of wild-type catalase might
leak into other intracellular compartments, including the ER,
that might not be clean systems. We found that COS-7 cells
transfected with catalase targeted to the ER (N35-Cat) or mito-
chondria (mitochondrial signal peptide [MSP] – Cat; Fig. 6 B ;
Bai et al., 1999 ) exhibited increased catalase activity ( Fig. 6 C ),
but only N35-Cat and not MSP-Cat attenuated Nox4 modula-
tion of EGFR signaling ( Fig. 6 D ). These results demonstrated
the effectiveness of ER-targeting antioxidants in regulating
ER-localized Nox4 ROS signaling.
Figure 5. Oxidation of PTP1B trapping mutant by Nox4 attenuates
its substrate-binding capacity. (A) COS-7 cells were transfected with ex-
pression vectors as indicated for 24 h followed by treatment with or with-
out 50 ng/ml EGF for 30 min. Cell lysates were immunoprecipitated
with EGFR antibody and immunoblotted with antiphosphotyrosine. Total
cell lysates were also immunoblotted with anti-EGFR as shown in the
bottom panel. (B) COS-7 cells were cultured on the glass coverslips
and transfected with expression vectors as indicated for 24 h. Imaging
was visualized with a two-photon confocal microscope. (C) COS-7 cells
were treated as in B, and cell lysates were immunoprecipitated with PTP1B
antibody followed by immunoblotting with anti-EGFR and anti-PTP1B.
Bar, 10 μ m.
Figure 6. ER-targeting antioxidant attenuates Nox4-dependent ROS
signaling. (A) COS-7 cells on coverslips were transfected with pcDNA3.1/
Nox4-V5 or pcDNA3.1/N35-V5. After 24 h, cells were fi xed and immuno-
stained with anti-V5 (green) for fusion proteins and anti-GRP78 (red). (B) COS-7
cells were transfected with overexpression vectors as indicated and were
immunostained with anticatalase antibodies. (C) COS-7 cells were transfected
with mitochondrial-targeting catalase (MSP-Cat) or ER-targeting catalase
(N35-Cat) as indicated for 24 h. Catalase activity was measured in cell
lysates using the Amplex red catalase kit. Error bars represent SD. *, P <
0.05 versus control. (D) The Nox4-overexpressing COS-7 cell line was
transfected with pcDNA3.1/PTP1B in combination with either MSP-catalase
or N35-catalase. After 24 h, cells were treated with 50 ng/ml EGF, and
phosphorylation of EGFR and ERK and PTP1B oxidation status were deter-
mined as described in Materials and methods. The blots are representative
of three independent experiments. Bars, 10 μ m.
JCB • VOLUME 181 • NUMBER 7 • 2008 1136
The notion that ROS are involved in cell signaling is not
new ( Finkel and Holbrook, 2000 ), and the diffusible nature of
H 2 O 2 is thought to be consistent with this role. In this regard,
previous experience with nitric oxide (NO) merits particular at-
tention. For example, NO was thought to readily diffuse to its
effector site, making its site of generation irrelevant. However,
it is now clear that cell responses vary dramatically depending
on the site of the NO synthase (NOS) enzymes. For example,
NOS1 in the sarcoplasmic reticulum of cardiomyocytes regu-
lates ryanodine receptor Ca 2+ release, whereas NOS3 in caveo-
lae regulates L-type Ca 2+ channels ( Barouch et al., 2002 ). The ER
localization of Nox4 may have some advantages for redox-
related signal transduction as a result of the relatively oxidized
state of the ER ( Tu and Weissman, 2004 ). The low antioxidative
capacity in the region may allow Nox4 to initiate signaling more
effi ciently or cause pathological events while dysregulated. Con-
sistent with this notion, Nox4 has recently been related to ER
stress and apoptosis in 7-ketocholesterol – treated human aortic
smooth muscle cells ( Pedruzzi et al., 2004 ). The data presented
here underlines the importance of subcellular localization as a
regulatory theme in redox signaling.
PTP1B is localized exclusively on the cytoplasmic face
of the ER and contains a small hydrophobic C-terminal anchor
sequence that is necessary and suffi cient to localize the enzyme
to the ER ( Frangioni et al., 1992 ). Haj et al. (2002 ) demonstrated
that the activated EGFR is internalized and transported to the
ER, where it is subject to dephosphorylation by PTP1B. The cata-
lytic activity of PTPs is dependent on a reduced active site Cys
residue, and the catalytic Cys is extremely susceptible to oxida-
tion by H 2 O 2 ( Meng et al., 2002 ). Here, we demonstrate that Nox4
is a source of H 2 O 2 that modulates the PTP1B redox state and,
as a consequence, the duration of EGFR signaling. Modulating
the duration of EGFR signaling had functional consequences, as
EGF-induced endothelial proliferation is
mediated by Nox4-dependent
To examine the functional implications of Nox4 in EGF-dependent
endothelial cell responses, we manipulated the endothelial Nox4
levels using adenoviral overexpression and RNAi. Both strategies
resulted in signifi cant changes in Nox4 expression at the mRNA
level ( Fig. 7 A ), and EGF-induced ROS production in these cells
was correlated with Nox4 expression ( Fig. 7 B ). Furthermore,
EGF-stimulated endothelial cell proliferation as determined by
either BrdU incorporation ( Fig. 7 C ) or cell counting ( Fig. 7 D )
was consistently enhanced by the overexpression of Nox4. Con-
versely, RNAi-mediated suppression of Nox4 attenuated cell pro-
liferation. Collectively, these data indicate that Nox4-derived ROS
play an important role in regulating PTP1B oxidation and EGF sig-
naling in a manner that modulates cell proliferation.
The principal fi nding of this study is that endothelial Nox4, an
endogenous source of ROS in HAECs, is localized to the ER and
appears involved in the regulation of another ER-residing pro-
tein, PTP1B, in a spatially dependent manner. Despite the diffus-
ible nature of H 2 O 2 , Nox4-dependent oxidative modifi cation
of PTP1B requires colocalization of both proteins in the ER.
We found that cytosolic PTP1B prevented its oxidation by Nox4.
Furthermore, we found that Nox4-mediated PTP1B oxidation
was relevant to EGF signaling and was associated with reduced
dephosphorylation of EGFR in proximity to the ER. We were
able to confi rm the importance of ER localization using ER tar-
geting of antioxidant enzymes such as catalase. Collectively, these
data provide a paradigm for Nox4-dependent redox signaling
that highlights spatial specifi city within the cell.
Figure 7. EGF-induced endothelial proliferation is
mediated by Nox4-dependent PTP1B oxidation. HAECs
were transfected with Ad-control siRNA (Ad-Ctli) or
Ad-Nox4i for 48 h or adenoviral vectors expressing
LacZ (AdLacZ)/Nox4 (AdNox4) for 24 h. (A and B)
Total RNA was extracted followed by RT-PCR for mea-
surement of Nox4 mRNA (A) and dichlorofl uorescein
assay for ROS production in response to 50 ng/ml
EGF for 15 min (B). For proliferation assay, HAECs
were transfected with adenoviral vectors as in A for
24 h and were seeded into 24-well plates at 2.5 × 10 4
cells/well. (C and D) Proliferation was measured by
BrdU incorporation (C), and cell number was counted
at 24 and 48 h after plating (D). Data are shown
as representative (gel picture) or statistics of three
independent experiments. Error bars represent SD.
*, P < 0.05.
1137NADPH OXIDASE 4 REGULATES REDOX SIGNALING • Chen et al.
The intracellular localization of Nox4 remains controversial.
Hilenski et al. (2004) reported Nox4 in the nucleus and focal
adhesion sites in rat vascular smooth muscle cells, whereas
other studies revealed perinuclear or nuclear distribution in COS-7,
HEK293, and endothelial cells ( Kuroda et al., 2005 ; Martyn
et al., 2006 ; Petry et al., 2006 ). It may not be surprising if different
cell types display distinct intracellular localization of the same
protein. However, a recent study that showed nuclear localiza-
tion of Nox4 in human VE cells ( Kuroda et al., 2005 ) confl icts
with the results here. Although the reason for the discrepancy
remains unclear, one explanation may be related to the anti-
bodies used in the study by Kuroda et al. (2005) , in which anti-
bodies were generated solely targeting the C terminus of Nox4.
It is plausible that these antibodies may recognize certain splice
variants of Nox4 because two out of four different splice vari-
ants have been found to be predom inantly C-terminal truncated
forms ( Goyal et al., 2005 ). Of note, we have constructed such a
truncated form of Nox4 (amino acids 418 – 578) and observed
a nuclear localization (unpublished data).
The current fi ndings demonstrate a new biological para-
digm for ROS signaling whereby the spatial confi nement of ROS
with redox-sensitive targets in proximity allows ROS signals to
keep targeting selectivity. As such, ROS oxidatively fi ne-tune the
PTP1B activity in this system that subsequently regulates recep-
tor traffi cking. In addition, we have shown the effectiveness of
ER-targeting catalase in the regulation of Nox4, a fi nding that
implicates the importance of manipulating ROS within specifi c
intracellular microdomains to ensure the effects. This may par-
tially explain the failure of antioxidants as therapeutic agents in a
series of clinical trials and points out the relevance of manipulation
at the subcellular level. ROS signaling appears to be operated in
a spatially restricted microenvironment with substrate specifi city.
It is worth noting that the Nox4-dependent PTP oxidative modi-
fi cation was evident across multiple cell types and held true for
the regulatory mechanism of other different receptors, including
the insulin receptor. Our data may provide a model for numerous
redox-sensitive signaling systems and have broad-ranging impli-
cations in cardiovascular diseases and cancer.
Materials and methods
A rabbit polyclonal antibody was raised against human Nox4 in our labo-
ratory ( Wendt et al., 2005 ). Anti-EGFR, GS-28, SHP-2, and VE-cadherin
were purchased from Santa Cruz Biotechnology, Inc. Anti-GRP78 was ob-
tained from BD Biosciences, anticatalase and anti – histone H4 were ob-
tained from Abcam, antivimentin was purchased from NeoMarkers, and
anti-V5, AlexaFluor488-, and AlexaFluor594-conjugated secondary anti-
bodies were obtained from Invitrogen. Phosphotyrosine antibody (4G10)
was obtained from Millipore, and all MAPK antibodies were purchased
from Cell Signaling Technology. Human recombinant EGF, anti-PTP1B, and
a BrdU cell proliferation assay kit were purchased from EMD. All other re-
agents were obtained from Sigma-Aldrich. Double-stranded siRNAs for
Nox4, Nox2, and nontargeting control were obtained from Dharmacon.
HAECs were obtained and cultured in EGM-2 (Cambrex) as described
previously ( Chen et al., 2001 ). COS-7 cells were purchased from the
American Type Culture Collection and maintained in DME supplemented
with 10% FBS. The COS-Nox4 cell line was established by transfection
of pcDNA3.1/Nox4-V5 in COS-7 cells followed by G418 selection. Im-
mortalized PTP1B ? / ? primary MEFs (PTP1B ? / ? MEFs) and PTP1B ? / ? MEFs
both downstream ERK signaling and endothelial cell prolifera-
tion were subject to Nox4-mediated control. Insofar as receptor
tyrosine kinase transport to sites of dephosphorylation is shared
among receptors, one might speculate that our observations
represent a general mechanism for regulating the duration of
receptor tyrosine kinase activity. Indeed, most studies of ROS
signaling have found only partial modulation of signaling rather
than a strict dependence of responses by manipulating ROS.
For example, a previous study demonstrated limited inhibition
of angiotensin-induced ERK activation by antioxidants tempol
or tiron in vascular smooth muscle cells exposed to angiotensin
for 5 min ( Touyz et al., 2004 ). Nevertheless, tempol has shown
signifi cant reduction in angiotensin-induced aortic vascular hyper-
trophy in mouse models, which tends to support the new para-
digm ( Dikalova et al., 2005 ).
The data presented here indicate that ROS-mediated sig-
naling responses are compartmentalized. Consistent with this
notion, EGF treatment has been shown to cause only selective
oxidation of the subcellularly localized thioredoxin pool but has
no material impact on the intracellular glutathione/glutathione
disulfi de redox pool ( Halvey et al., 2005 ). The precise mecha-
nisms of this signaling compartmentalization are not yet known
but may be maintained through either membrane barrier func-
tion and/or localization of antioxidant capacity close to the ROS
source. Indeed, our observations with ER-targeted antioxidants
tend to support the latter possibility. This contention is consistent
with the observation that compared with cytosol or mitochondria-
targeting peroxiredoxin 5, only nucleus-targeting peroxiredoxin 5
confers DNA protection from oxidative injury ( Banmeyer et al.,
2004 ). However, it remains unknown which intracellular anti-
oxidants might perform this function in the context of the
The functional implications of Nox4 have been diverse, in-
cluding antiproliferative effects in NIH3T3 cells ( Geiszt et al.,
2000 ; Shiose et al., 2001 ), proliferative effects in smooth muscle
cells ( Menshikov et al., 2006 ), maintenance of a differentiated
smooth muscle cell phenotype ( Clempus et al., 2007 ), and modu-
lation of insulin signaling in adipocytes ( Mahadev et al., 2004 ).
The proliferative effect of Nox4 in endothelial cells suggests a
common signaling role of intracellular ROS in nonphagocytic
cells. This fi nding is in agreement with previous studies indicat-
ing that ROS mediates peptide growth factor – induced signaling
and increased cell growth ( Sundaresan et al., 1995; Bae et al.,
1997 ). As an important intracellular ROS source, Nox1 was the
fi rst enzyme among the members of the Nox family found to par-
ticipate in physiological mitogenesis in response to growth factors
(such as platelet-derived growth factor) as well as pathological
hyperproliferation (such as cancer and atherosclerosis; Suh et al.,
1999 ). However, the assumption of Nox4 being proliferative
turned out to be untrue in early studies using overexpression
strategy in NIH3T3 fi broblasts ( Shiose et al., 2001 ). In agreement
with our results, the depletion of Nox4 resulted in a signifi cantly
decreased rate of proliferation in melanoma, pancreatic cancer,
and vascular smooth muscle cells ( Brar et al., 2002 ; Vaquero
et al., 2004; Djordjevic et al., 2005 ). Although the reasons for the
difference from these studies are not clear, an important clue to
the biological function of Nox4 links to its subcellular location.
JCB • VOLUME 181 • NUMBER 7 • 2008 1138
Fluor488 or -594 goat anti – rabbit or mouse IgG (Invitrogen) at 1:200
dilution. Fluorescence images were obtained using a 40 × 1.30 oil objective
(Nikon) on an inverted microscope (TE-2000; Nikon) with a camera (Cool-
SNAP HQ; Photometrics). Images were captured using NIS-Elements software
(Nikon) and processed with a 3D deconvolution plug-in (MediaCybernetics).
Immunogold electron microscopy
HAECs were transfected with adenoviral vector expressing Nox4-V5 for
24 h and were removed from the dish with 0.5 mM EDTA in PBS. The cell
suspension was layered on top of a cushion of 4% PFA and pelleted for 3 min
at 3,000 rpm. The pellet was further fi xed in fresh 4% PFA for 2 h followed
by wash with PBS containing 0.2 M glycine and infi ltration with 2.3 M
sucrose. Frozen samples were sectioned and subjected to anti-V5 (1:100)
followed by protein A gold labeling. The grids were examined in a transmission
electron microscope (Harvard Medical School EM Facility; 1200EX; JEOL).
Detection of PTP1B redox state
For detection of PTP1B at a reduced form (SH-PTP1B), cells were lysed on
ice for 10 min in lysis buffer containing 1 mM biotin polyethylene oxide
maleimide (Thermo Fisher Scientifi c) followed by precipitation with Ultra-
Link Immobilized NeutrAvidin (Thermo Fisher Scientifi c) and Western blot
analysis with PTP1B antibody ( Lee et al., 2002 ). The band detected here
represents the amount of PTP1B at a reduced form.
Extraction of total RNA and RT-PCR was performed as previously described
( Chen et al., 2003 ). The forward and reverse primers corresponding to Nox4,
Nox2, and glyceraldehyde-3-phosphate dehydrogenase were 5 ? -AAGCC-
GGAGAACCAGAAGAT-3 ? and 5 ? -GCTGCATTCAGTTCGACAAA-3 ? for
Nox4, 5 ? -GCTTGTGGCTGTGATAAGCA-3 ? and 5 ? -TCCCTGCTCCCACTAA-
CATC-3 ? for Nox2, and 5 ? -ACCCAGAAGACTGTGGATGG-3 ? and 5 ? -AGGC-
CATGCCAGTGAGCTT-3 ? for glyceraldehyde-3-phosphate dehydrogenase.
Measurement of ROS
Intracellular ROS production was measured by dichlorofl uorescein fl uo-
rescence as described previously ( Chen et al., 2000 ). Cells cultured in
6-well plates were changed to Hepes-buffered physiological saline solution
followed by the addition of 5 μ M H2DCFH-DA with/without EGF treatment
and further incubation for 20 min at 37 ° C. After treatment and wash, fl uo-
rescence intensity was quantifi ed by using a fl uorescence plate reader
(SpectraMax GeminMPS; MDS Analytical Technologies).
Catalase activity assay
Cells were transfected with control vector or catalase expression vectors for
24 h and lysed in 0.1 M Tris buffer, pH 7.4. Cell lysates were used for
measurement of catalase activity by using the Amplex Red Catalase Assay
kit (Invitrogen). The decomposition of hydrogen peroxide by catalase was
followed by reaction with 50 μ M Amplex red reagent in the presence of
0.2 U/ml horseradish peroxidase. Fluorescence was measured in a fl uo-
rescence microplate reader (MDS Analytical Technologies) using excitation
at 530 nm and emission at 590 nm.
All numerical data are presented as means ± SD. The Western blots
shown are representative of three or more independent experiments. For
parametric data, comparisons among treatment groups were performed
with one-way analysis of variance and an appropriate posthoc compari-
son. Instances involving only two comparisons were evaluated with a t test.
Instances involving more than two comparisons were evaluated using
analysis of variance. Statistical signifi cance was accepted if the null hypo-
thesis was rejected with P < 0.05.
We are grateful to Yongmei Pei, Xiaoyun Huang, and Adam Albano for their
K. Chen is the recipient of a Scientist Development grant from the Amer-
ican Heart Association, and this work was partially supported by National
Institutes of Health grant AG027081 to J.F. Keaney Jr.
Submitted: 10 September 2007
Accepted: 27 May 2008
Bae , Y.S. , S.W. Kang , M.S. Seo , I.C. Baines , E. Tekle , P.B. Chock , and S.G.
Rhee . 1997 . Epidermal growth factor (EGF)-induced generation of
reconstituted with wild-type PTP1B (PTP1B ? / ? (+WT) MEFs) were provided by
B. Neel (Harvard Medical School, Boston, MA; Haj et al., 2003 ).
Plasmid constructs and adenoviral vectors
CFP-EGFR and enhanced GFP-EGFR expression vectors were provided
by L. Samelson (National Institutes of Health, Bethesda, MD) and T. Jovin
(Max Planck Institute, G ö ttingen, Germany), respectively. pZeoSV2/MSP-
catalase vector was a gift from J. Andres Melendez (Albany Medical College,
Albany, NY). PTP1B vectors, including wild-type and mutant D181A/
Q262A, were provided by Z.-Y. Zhang (Indiana University, Bloomington,
IN). Expression vectors pcDNA3.1/Nox4-V5, pcDNA3.1/PTP1B wild type,
pcDNA3.1/PTP1B- Δ 35, pcDNA6.2/EmGFP-PTP1B (D181A/Q262A),
pcDNA3.1/SOD1, and pcDNA3.1/catalase were constructed by using
the PCR subcloning technique with pcDNA3.1/TOPO-V5 or pcDNA6.2/
EmGFP vectors (Invitrogen). Adenoviral Nox4 vector was a gift from
B. Goldstein (Thomas Jefferson University, Philadelphia, PA). For the gen-
eration of AdNox4-V5, full-length human Nox4 cDNA was subcloned
into pENTR/D-TOPO vector (Invitrogen) followed by recombination reac-
tion with pAd/CMV/V5-DEST (Invitrogen). The resulting clone was used for
the generation of adenoviral stock in 293A cells, and further purifi cation
was performed with cesium chloride ultracentrifugation. An MOI of 50 was
used throughout the experiments. For generation of Nox4-targeting RNAi
vector pQ/Nox4i, a sequence targeting Nox4 (nucleotides 418 – 436 of
human Nox4 from the start codon) was constructed within pQuiet-U6 vector
(Welgen). The construct was also ligated into a linearized adenoviral
genome for subsequent generation of adenoviral vector (Ad-Nox4i). To gen-
erate a Nox4-overexpressing vector (pcDNA3.1/Nox4-R) that is specifi -
cally resistant to the corresponding pQ/Nox4i for rescue experiments,
eight silent mutations were introduced into a parental pcDNA3.1/Nox4-V5
at the Nox4i target region without altering the encoding protein by using
the QuikChange Site-Directed Mutagenesis kit (Stratagene).
Plasmid and siRNA transfection
COS-7 cells were seeded at a density of 2 × 10 5 cells/ml in 6-well plates.
Transfection with overexpression plasmids was performed using Fugene 6
(Roche) in cells at 70% confl uency according to the manufacturer ’ s instruc-
tions. Typically, 2 μ g of plasmid per well was used for transfection, and
cells were ready for experiments 24 h later. Transfection in PTP1B ? / ? MEFs
was performed by using Lipofectamine 2000 (Invitrogen). Transfection of
HAECs with siRNA was performed using the TransMessenger Transfection
Reagent (QIAGEN) according to the manufacturer ’ s instructions. Cells were
incubated for 48 h after siRNA transfection before experiments.
Immunoprecipitation and Western blotting
Immunoprecipitation and Western blotting procedures were performed as
previously described ( Chen et al., 2001 ). Densitometric analysis of immuno-
blots was performed using ImageJ software (National Institutes of Health).
Detergent fractionation of subcellular organelles
Cell fractions were extracted by using the differential detergent fraction-
ation method as described previously ( Simpson, 2003 ). In brief, the deter-
gent fractionation involves the sequential extraction of cells with Pipes
buffers containing 0.015% digitonin, then 0.5% Triton X-100, and fi nally
1% Tween 40/0.5% deoxycholate, yielding three biochemically and electro-
phoretically distinct fractions composed of cytosolic, membrane/organelle,
and nuclear compartments, respectively. The fi nal cell residue from the
aforementioned procedures was the detergent-resistant cytoskeletal/
Nycodenz fractionation of subcellular organelles
Nycodenz gradient fractionation was performed essentially as described
previously ( Hammond and Helenius, 1994 ). Cells were harvested and
homogenized in 2 ml of ice-cold homogenizing buffer (10 mM Tris-HCl,
pH 7.4, 250 mM sucrose, 5 mM EDTA, and protease inhibitor mixture).
Postnuclear supernatant was obtained (3,000 rpm for 10 min at 4 ° C), and
a step gradient was created in centrifuge tubes (TLS-55; Beckman Coulter) by
loading top to bottom 0.5 ml of 10, 14.66, 19.33, and 24% Nycodenz
solution in saline buffer. The postnuclear supernatant was then layered
on top of the gradient and fractionated by centrifugation (169,000 g for
45 min at 15 ° C). After centrifugation, fractions were collected from the top
of the tube, and an aliquot of each fraction was resolved by SDS-PAGE and
Western blot analysis.
Cells were grown on glass cover slides and fi xed with 4% PFA. Primary anti-
bodies were diluted at 1:250, and secondary antibodies include Alexa-
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