EGFR-Activated Signaling and Actin Remodeling
Regulate Cyclic Stretch–Induced NRF2-ARE Activation
Srinivas Papaiahgari, Adinarayana Yerrapureddy, Paul M. Hassoun, Joe G. N. Garcia, Konstantin G. Birukov,
and Sekhar P. Reddy
Division of Physiology, Department of Environmental Health Sciences, Division of Pulmonary and Critical Medicine, and Department of
Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland; and University of Chicago, Chicago, Illinois
Cyclic stretch (CS) associated with mechanical ventilation (MV) can
cause excessive alveolar and endothelial distention, resulting in
lung injury and inflammation. Antioxidant enzymes (AOEs) play a
major role in suppressing these effects. The transcription factor
Nrf2, via the antioxidant response element (ARE), alleviates pulmo-
nary toxicant- and oxidant-induced oxidative stress by up-regulating
the expression of several AOEs. Although gene expression profiling
has revealed the induction of AOEs in the lungs of rodents exposed
to MV, the mechanisms by which mechanical forces, such as CS,
regulate the activation of Nrf2-dependent ARE-transcriptional re-
sponses are poorly understood. To mimic mechanical stress associ-
ated with MV, we have cultured pulmonary alveolar epithelial and
endothelial cells on collagen I–coated BioFlex plates and subjected
them to CS. CS exposure stimulated ARE-driven transcriptional re-
sponses and subsequent AOE expression. Ectopic expression of a
dominant-negative Nrf2 suppressed the CS-stimulated ARE-driven
responses. Our findings suggest that actin remodeling is necessary
but not sufficient for high-level CS-induced ARE activation in both
epithelial and endothelial cells. We also found that inhibition of
EGFR activity by a pharmacologic agent ablated the CS-induced
ARE transcriptional response in both cell types. Additional studies
revealed that amphiregulin, an EGFR ligand, regulates this process.
stream effector of EGFR and regulates CS-induced ARE-activation
in an oxidative stress–dependent manner. Collectively, these novel
findings suggest that EGFR-activated signaling and actin remodel-
ing act in concert to regulate the CS-induced Nrf2-ARE transcrip-
tional response and subsequent AOE expression.
Keywords: oxidativestress; MAPkinases; mechanical stress; antioxidant
response element; lung
Mechanical ventilation (MV) and supplemental oxygen therapy
patients with acute lung injury. MV can improve intrapulmonary
shunting in patients with acute respiratory distress syndrome
(ARDS). However, it can also cause excessive alveolar disten-
tion, resulting in lung injury and increased pulmonary vascular
permeability as well as an increase in the production of pro-
inflammatory mediators (1–3) known to be associated with pro-
duction of reactive oxygen species (ROS), a condition generally
referred to as oxidative stress. These events may then serve to
initiate and/or potentiate an inflammatory response, leading to
a vicious cycle of inflammation either locally or systemically (4, 5).
(Received in original form March 31, 2006 and in final form September 25, 2006)
This work was supported by an NHLBI award SCCOR-1P50HL073994 (S.P.R.,
Leader of Project 6).
Correspondenceandrequests forreprints shouldbeaddressed toSekharP. Reddy,
The Johns Hopkins University, Department of Environmental Health Sciences,
Division ofPhysiology, Rm. E7610, 615 North Wolfe Street, Baltimore, MD 21205.
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2006-0131OC on September 28, 2006
Internet address: www.atsjournals.org
Vol 36. pp 304–312, 2007
Our findings may have particular physiologic importance
in clinical syndromes such as ventilator-induced lung injury
where inflammation and oxidative stress (an imbalance be-
tween antioxidant and oxidant systems) are thought to play
a preponderant role.
ical stress in general can cause cell deformation, leading to alter-
ation in the structureand function of a number of tissues, includ-
ing the lung (6). Furthermore, in vitro and in vivo studies have
shown that both the degree and pattern of CS are important in
determining cell responses (4). CS has been shown to differen-
tially regulate gene expression, in part through the activation of
MAP kinase signaling in lung epithelial cells (4, 7). Preliminary
results have demonstrated that administration of antioxidant
decreased lung neutrophil influx in rats exposed to MV, indicat-
ing a role for oxidative stress in the development of ventilator-
induced lung injury (8). Although these studies have suggested
the involvement of both molecular and cellular alterations, the
exact mechanisms involved in the pathogenesis of MV-induced
of antioxidant enzymes (AOEs) and their mechanisms of activa-
tion in response to CS.
acts as one of the “biosensors” that participate in cellular switch-
ing of the genetic program in response to various oxidative and
toxic stimuli. Nrf2 binds to the DNA sequence 5?-TGACN
NNGC-3?, known as the antioxidant response element (ARE),
and regulates the expression of a network of integrated AOEs
involved in cellular detoxification process, thereby protecting
cells from the deleterious effects of ROS (see recent reviews in
Refs. 9, 10). We recently demonstrated that Nrf2-deficient mice
are more susceptible than wild-type mice to inflammatory and
hyperpermeability responses in response to hyperoxic exposure
(11). Both basal and hyperoxia-inducible expression of mRNAs
encoding several AOEs, such as glutathione peroxidase 2
(Gpx2), glutamate-cysteine ligase catalytic subunit (Gclc), and
glutamate cysteine ligase modifier subunit (Gclm), are signifi-
cantly lower in Nrf2-knockout mice than in wild-type mice (11,
12). Consistent with these findings, studies from other labora-
tories have shown an important role for Nrf2 in the regulation of
AOE expression in response to various oxidative and cytotoxic
insults in many cells and tissues (9, 10).
Gene expression profiling has demonstrated that MV modu-
lates the expression of prototypical Nrf2 target genes, such as
Gclc and Gclm, in the lungs of animals in various experimental
models (13), further suggesting a role for Nrf2-dependent ARE-
ciated with conventional MV exacerbates lung injury and in-
Papaiahgari, Yerrapureddy, Hassoun, et al.: Mechanisms of Nrf2 Activation by Cyclic Stretch305
flammation, deciphering the mechanisms of CS-induced cellular
responses, especially the induction of AOEs, is critical to devel-
oping strategies aimed at minimizing MV-related stress. The
upstream signaling pathways that control the activation of Nrf2
by CS remain unclear. We have therefore used in vitro studies
to examine the mechanism of activation of the Nrf2-dependent
ARE-mediated transcriptional response in pulmonary epithelial
and endothelial cells subjected to CS. Here we report for the
first time that actin remodeling and EGFR-activated PI3K-Akt
signalingarenecessary for theregulation of Nrf2-dependent ARE-
mediated transcriptional responses elicited by CS. Moreover,
we demonstrate that oxidative stress regulates this process, sug-
gesting the existence of a regulatory feedback mechanism for
MATERIALS AND METHODS
Horseradish peroxidase–conjugated secondary antibodies were ob-
tained from Amersham GE (Piscataway,NJ). Native antibodies specific
for amphiregulin (R&D Systems, Minneapolis, MN) and anti-ERK1/2
(Santa Cruz Biotech, Santa Cruz, CA) and phosphospecific anti-ERK1/2
and anti-Akt antibodies (Cell Signaling, Danvers, MA) were obtained
from various commercial sources as indicated. The pharmacologic in-
hibitors AG1478 and LY 294,002 were obtained from Calbiochem (La
tems (Foster City, CA).
Cell Culture and CS Exposures
A murine nonmalignant alveolar type II–like epithelial cell line, C10
(14), was cultured in CMRL medium supplemented with 10% fetal
bovine serum (FBS) and antibiotics. Rat pulmonary microvascular en-
dothelial cells (RPMECs) were cultured in RPMI medium supple-
mented with 10% FBS and antibiotics. Cells were seeded onto collagen
I–coated BioFlex plates, and once confluence was reached, the medium
was replaced with fresh complete medium 2 h before CS exposure.
Plates weremounted onto theFX-4000TFlexercell TensionPlus system
(Flexercell International, McKeesport, PA) equipped with a 25-mm
BioFlex loading station. This system provides uniform radial and cir-
cumferential strain across a membrane surface along all radii (more
details at http://www.flexcellint.com).Cells were subjectedto 18% elon-
gation at 24 cycles per minute for various time points. Cells grown on
BioFlex plates and simultaneously placed in a cell culture incubator
were considered as static controls.
Measurement of ROS
diacetate (DCFH-DA; Molecular Probes, Eugene, OR) as detailed
elsewhere. In brief, cells were cultured and subjected to either static
or cyclic stretch conditions for 15 min or 6 h. After the exposure, cells
were rinsed two times with warm serum and phenol red–free MEM
and loaded for 30 min with DCFH-DA (3 ?M) in MEM without phenol
red. After treatment, the cells were rinsed and then incubated with
phenol red–free MEM. Images were acquired on an inverted Nikon
digital CCD camera. Five individual images were captured per well
using Spot advanced software 4.1 (Diagnostic Instruments, Inc, Sterling
Heights, MI). The total number of cells showing the oxidized dichloro-
fluorescein (DCF) staining were counted under a green fluorescence
field and compared with the total number of cells observed under the
phase-contrast field for five images per sample. Representative fields
with % DCF staining are shown.
Measurement of Cellular GSH and GSSG Levels
Reduced and oxidized thiols were measured essentially as described
elsewhere (15). For GSH measurement, 10 ?l of cell lysate was mixed
with 80 ?l potassium phosphate-EDTA (1 mM, pH 8.0) and 10 ?l of
o-phthalaldehyde (OPT, 1 mg/ml). After a 15-min incubation at room
temperature, the fluorescence of the sample was read at 360 nm excita-
tion and 465 nm emission using a fluoroscence plate reader (HT7000;
Perkin-Elmer, Wellesley, MA). For determination of oxidized glutathi-
EDTA (1mM, pH 8.0) and incubated with 4 ?l of 40 mM N-ethylmalei-
mide (which reduces the oxidized GSH) for 30 min. The reaction was
stopped with 6 ?l of 0.1 N NaOH and incubated with 10 ?l of OPT (1
mg/ml) for 15 min, and the amount of fluorescence was measured as
detailed above. The values are expressed as % oxidized glutathione
over reduced glutathione for the respective samples, with the value for
the static control group set at 100%.
Immunofluorescent detection of F-actin was determined as described
previously (16). C10 cells grown on coverslips were treated either with
dimethyl sulfoxide (DMSO) or LA (1 ?M) for 30 min, fixed with cold
methanol for 10 min, and permeabilized with PBS buffer containing
0.1% Triton X. Permeabilized cells were washed and blocked in 3%
bovine serum albumin for 30 min. Cells were washed three times,
and F-actin was stained using Texas Red X-phalloidin (1:200) and
Western Blot Analysis
ted using native and phosphospecific antibodies as previously described
(17). The blots were then visualized with the ECL Western blot detec-
Transfections and Reporter Gene Analyses
Transient transfections were performed using the ARE Luciferase re-
porter construct (hereafter referred as ARE-Luc) as described pre-
viously (18). To normalize transfection efficiency between wells, the
cells were cotransfected with 1 ng of the Renilla luciferase plasmid
tions or CS for 5 h, cell extracts were assayed for firefly and Renilla
luciferase activities using a dual luciferase kit (Promega Corp.). Firefly
luciferase activity was normalized to that of Renilla luciferase.
Electrophoretic Mobility Shift Assays
Nuclear extracts were isolated according to the manufacturer’s instruc-
tions, using an NE-PER nuclear and cytoplasmic extraction reagent kit
(#78833) from Pierce Biotechnology (Rockford, IL), and electropho-
retic mobility shift assays (EMSAs) were performed as described pre-
viously (19) using nuclear extracts (2.5 ?g) and a32P-labeled double-
stranded ARE oligonucleotide probe. In super-shift assays, nuclear
extracts were incubated on ice with 2 ?g of anti-Nrf2 antibody (sc-
722X) and IgG (sc-2025) (Santa Cruz Biotechnology) for 2 h before
addition of the probe.
TaqMan gene expression assays detecting mRNAs encoding mouse
Gapdh (Mm99999915_g1), and Actb (Mm00607939_s1) were purchased
from Applied Biosystems, and mRNA levels were quantified in tripli-
cate according to the supplier’s recommendations. The absolute values
for each gene were normalized to that of Gapdh and/or Actb, and
the relative value for the static or vehicle-treated control group was
considered as equal to one arbitrary unit (AU).
Data are expressed as the mean ? SE. Statistical significance was
determined using t test and accepted at P ? 0.05. All transfections were
performed in triplicate, and each experiment was repeated at least
twice. Data are presented as the mean luciferase activity ? SD (n ?
3–5) for a representative experiment. All Western blots and gel shift
analyses were performed twice in duplicate. Real-time PCR was per-
formed in triplicate (n ? 3) and was repeated to obtain reproducible
results. The statistical significance of the differences between groups
was determined using Student’s t test, and P ? 0.05 was considered
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