Identification of Nrf2-dependent airway epithelial adaptive response to proinflammatory
oxidant-hypochlorous acid (HOCl) challenge by transcription profiling
1Lingxiang Zhu, 1Jingbo Pi,3Shinichiro Wachi, 2Melvin E. Andersen, 3Reen Wu and 1Yin
1Division of Translational Biology, The Hamner Institutes for Health Sciences, NC
27709.2Division of Computational Biology, The Hamner Institutes for Health Sciences,
NC 27709.3Center for Comparative Respiratory Biology and Medicine and Division of
Pulmonary/Critical Care Medicine, University of California Davis, CA 95616
* Corresponding author: Yin Chen, Ph.D.
Division of Translational Biology
The Hamner Institutes for Health Sciences
6 Davis Dr., Research Triangle Park, NC 27709
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Articles in PresS. Am J Physiol Lung Cell Mol Physiol (December 21, 2007). doi:10.1152/ajplung.00310.2007
Copyright © 2007 by the American Physiological Society.
In the airway of inflammatory diseases, high level of HOCl (estimated to be as high as
8mM) can be generated through a reaction catalyzed by leukocyte granule enzyme-
Myeloperoxidase (MPO). HOCl, a potent oxidative agent, causes extensive tissue injury
through its reaction with various cellular substances including thiols, nucleotides and
amines. Besides its physiological source, HOCl can also be generated by chlorine gas
inhalation resulting from either an accident or potential terrorist attack. Despite the
important role of HOCl induced airway epithelial injury, underlying molecular
mechanism is largely unknown. In this study, we found that HOCl induced dose-
dependent toxicity in airway epithelial cells. By transcription profiling using Genechip, a
battery of HOCl inducible antioxidant genes was identified. And all of them have been
reported previously to be regulated by nuclear factor erythroid-2 related factor 2 (Nrf2), a
transcription factor that is critical to lung antioxidant response. In consistent to this
finding, Nrf2 was found to be activated both time- and dose- dependent of HOCl
treatment. Interestingly, although EGFR-MAPK pathway was also highly activated by
HOCl, it was not involved in Nrf2 activation and Nrf2-dpendent gene expression. Instead,
HOCl induced cellular oxidative stress appeared to directly lead to Nrf2 activation. To
further understand the functional significance of Nrf2 activation, small interference RNA
(si RNA ) was used to either knock down Nrf2 level by targeting Nrf2 or enhance the
nuclear accumulation of Nrf2 by targeting its endogenous inhibitor-Keap1. By both
methods, we conclude that Nrf2 directly protects airway epithelial cell from HCOL-
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Key words: HOCl, microarray, airway epithelium, Nrf2, anti-oxidant
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In the airway of inflammatory diseases such as bacterial infection, cystic fibrosis (CF)
and COPD, neutrophil is predominant inflammatory cell type in the airway. The activated
neutrophils generate reactive oxygen species such as superoxide anion radicals (O 2) and
hydrogen peroxide (H2O2),and secrete the enzyme myeloperoxidase (MPO)(24). In the
presence of physiological concentration of chloride (Cl-), MPO can utilize H2O2 to
generate the powerful oxidant hypochlorous acid (HOCl)(24). Because of its high
reactivity, accurate HOCl concentration in human airway is hard to measure. In CF
airway, based on published estimates of the number of neutrophils in the sputum and the
amount of HOCl produced by stimulated neutrophil, airway HOCl concentration was
estimated to be approximately 3 mM on average, with a maximum of ~8 mM(15). This
estimation was close to another report showing active neutrophils could produce up to
5mM HOCl (43). Because the pKa of HCOL is 7.53, under physiological pH, HOCl
exists as both neutral form (HOCl) and the ionized form (OCL-)(24). Throughout this
manuscript, we use HOCl to represent this mixture. HOCl is a very potent oxidant, and
can react with numerous molecules such as protein, lipids, nucleotides, thols and amines
of the proteins(24). Thus, besides its primary function as a pathogen-killer, HOCl also
causes tissue injuries that result in various human pathologies (24).
Other than the endogenous source, high level of HOCl can also be generated through
hydrolysis of inhaled chlorine gas(12). Chlorine gas is a widely used industrial chemical,
and it has been used as a chemical weapon dating back to World War I(12). Human
exposure to chlorine can be caused by occupational exposures(12), transportation
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accidents(12), misuse of cleaners(12), or perhaps a deliberated terrorist attack(see the list
Despite its importance, very few studies have been done on the molecular nature of HOCl
induced tissue injury. In human airway, there are enormous amount of neutrophils present
in the bronchoalveolar lavage (BAL) from the patients of acute or chronic diseases(24).
Likewise, MPO level are also highly elevated in airway lumen(24). Considering its
massive surface area, airway epithelia is likely to be one of the major targets of HOCl
generated by MPO catalyzed reaction. Under the scenario of chlorine inhalation, airway
epithelium is the first line of barrier that encounters inhaled chlorine; thus, it is also the
first target of the large amount of HOCl generated from chlorine hydrolytic reaction with
airway surface fluids (ASL). Because epithelial injury and its corresponding adaptive
response have significant impact on airway inflammation and remodeling, we seek to
understand the molecular basis of airway epithelia response to HOCl challenge.
To vividly mimic in vivo airway epithelium, we take advantage of state-of-the-art well-
differentiated epithelial cell culture system that grows and differentiates cells under
air/liquid interface(5). The resulting culture maintains the morphological and
physiological characteristics of the in vivo epithelia(45, 46). Combining with Genechip
profiling and genetic manipulation using epithelial cell line model, we have demonstrated
for the first time that a key Nrf2-dependent antioxidant pathway is activated by HOCl and
protects airway epithelial cells from HOCl induced toxicity.
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Materials and Methods:
1. Chemicals, inhibitors , antibodies
HOCl was purchased from Sigma-Ald rich ( St. Louis, MO). The concentration of HOCl
was determined by measuringits absorbance at 290 nm (13). Chemical inhibitors
(AG1478, U0126) werepurchased from Calbiochem (EMD Biosciences, Inc., San Diego,
CA). Nrf2 and Actin antibodies were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Antibodies against phospho-proteins (pEGFR, pERK, p38, pJNK) were
purchased from Cell signaling technology (Danvers, MA).
2. Cell culture and HOCl treatment
Differentiated primary cell culture: Human tracheobronchial tissues were obtained
from National DiseaseResearch Interchange, with an approved protocol. The Hamner
Institute Health and Safety Committee approvedall procedures involved in tissue
procurement. We have, in thepast, successfully established primary airway epithelial
culturesfrom these tissues(5, 46). Normally, primary cells were platedon a Transwell
(Corning Costar, Corning, NY) chamber (25 mm)at 1–2 x 104 cells/cm2, in a Ham's
F12:Dulbecco's modifiedEagle's medium (1:1) supplemented with eight factors,
including:insulin (5 µg/ml), transferrin (5 µg/ml), epidermalgrowth factor (10 ng/ml),
dexamethasone (0.1 µM), choleratoxin (10 ng/ml), bovine hypothalamus extract (15
µg/ml),BSA (0.5 mg/ml), and all-trans-retinoic acid (30 nM). Aftera week in immersed
culture condition, cultured cells were shiftedto an air–liquid interface culture condition.
Under thebiphasic culture condition, high transepithelial resistance(> 500 · cm2),
multiple cell layers, cilia beating, and the formation ofmucus-secreting granules were
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observed(5, 46). Normally, experimentswere performed at Day 21 or 2 wk after the
change of the culturecondition from immersed to air–liquid interface. Mediumwas
routinely changed once every other day.
Monolayer primary culture: primary epithelial cells were cultivated on regular tissue
culture dish in the same media as described above. Those cells were not differentiated
and morphologically similar to each other.
NCI-H292 cells: Cells were obtained from ATCC, and cultivated on regular tissue
culture dish in RPMI media plus 10% fetal bovine serum (FBS).
HOCl treatment : for the monolayer primary cells and NCI-H292 cells, HOCl was
diluted into culture media with desired concentration right before treatment. For
differentiated primary cell culture, 100 ul media containing desired concentration of
HOCl was only added on the top of cells, which mimicked apical side of airway epithelia.
3. Assay for cell viability
MTS assay (Promega, Inc., Madison, WI) wasused based on the manufacturer's
instructions to determine the HOCl induced toxicity. Monolayer culture was used for this
assay. Briefly, 2X104 primary epithelial cells or NCI-H292 cells were seeded into 96-well
plate and were grown until 90%-95% confluence. After washing three times with fresh
media, media containing different concentrations of HOCl were added, and incubated for
designated time periods (e.g. 6 hours or 24hours). Then, cells were washed three times
with media without HOCl, and incubated with the media containing MTS mixture made
freshly based on the manufacturer’s instruction. MTS was bioreduced by cells into a
formazan product that was soluble in tissue culture medium and the absorbance was
Page 7 of 37
measured at wavelength 490nm. The quantity of formazan product as measured by the
amount of 490nm absorbance is directly proportional to the number of living cells in
culture. To obtain cell viability, readings from non-treated cells were designated as
“100%”. The readings of treated cells were then compared with non-treated cells to get
their percentage. The final percentage reading represents the number of metabolically
viable (“living”) cells in the culture.
4. Transcription profiling by GenechipTM analysis
The HGU133A chip that contains 22,283 probe set was used, andall protocols used in
this study were based on the manufacturer'sinstruction (Affymetrix, Inc.). The double-
extracted total RNAwas submitted to the Gene Expression core facility of the Hamner
Institute. At this facility, RNA sampleswere prepared, hybridized to these array chips,
and the hybridizationsignals were scanned using the standard protocols suggestedby
Affymetrix. For quality control, the scanned images of eacharray were visually inspected
to be free of artifacts. Scatterplots of individual arrays were also used to assess the
overallquality of the array data.All array data sets (total 32) were deposited into NCBI
GEO database under ID: GSM245427-58.
Bioconductor(14), a biological data analysis packagebased on R statistical programming
language (Vienna Universityof Technology; http://www.r-project.org/), was used for
arraydata analysis and integration with other gene annotations. Signal intensity and noise
correction was performed using Robust Microarray Analysis (RMA)
algorithm(18). Differential expressions (DE) were determined using LIMMA package (R
package) by determining the genes with statistically significant difference using false
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detection rate (FDR) based adjusted p-value below 0.05, as calculated by method
described by Benjamini and Hochberg(3). The DE genes were then ordered using
hierarchical clustering method (using R package hclust), based on the Euclidean distance
measures of the average difference of normalized values between the experiment and the
5. Real-time PCR
Real-time PCR was performed as described previously(21). cDNAwas prepared from 3
µg of total RNA with Moloney murineleukemia virus (MoMLV)–reverse transcriptase
(Promega,Inc.) by oligo-dT primers for 90 min at 42°C in a 20-µlreaction solution, and
was then further diluted to 100 µlwith water for the following procedures. Two
microliters ofdiluted cDNA was analyzed using 2x SYBR Green PCR Master Mixby an
ABI 5700 or ABI Prism 7900HT Sequence Detection System(Applied Biosystems Inc.,
Foster City, CA), following the manufacturer’s protocol. Primers (Table.1) were used at
0.2µM. The PCR reaction was performed in 96-well opticalreaction plates, and each
well contained a 50-µl reactionmixture. The SYBR green dye was measured at 530 nm
during theextension phase. The relative mRNA amount in each sample wascalculated
based on the
Ct method using housekeeping gene GAPDH.The purity of amplified
product was determined from a singlepeak of a dissociation curve. Efficiency curves
were performedfor each gene of interest relative to the housekeeping gene,based on the
manufacturer's instructions. Results were calculatedas fold induction over control, as
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6. Measurement of intracellular ROS by live cell imaging
ROS production was determined using chloromethyl-2',7'-dichlorodihydrofluorescein
diacetate (CM-H2DCFDA; Molecular Probes, Eugene, OR) based on the manufacture’s
instruction. In brief, cells were rinsed two times with warm phenol red–free media, and
then loaded for 30 min with CM-H2DCFDA(3 µM) in media without phenol red. The
cells were rinsed three times to remove the extracellular dye, and replaced with media
without phenol red. Then the cells were loaded to a live cell imaging incubator (Carl
Zeiss, Thornwood, NY) with appropriate temperature (37°C) and CO2(5%). Live cell
images were continuously recoded before and after HOCl treatment by Confocal
Microscope (LSM 510 meta, Carl Zeiss, Thornwood, NY ).
7. Measurement of Cellular GSH and GSSG Levels
Reduced and oxidized thiols were measured by BioXYTECH® GSH/GSSG-412TM kit
from OxisResearch (Foster City, CA) based on manufacture’s instruction. This method
is essentially based on the enzymatic method for glutathione quantification developed by
Tietze F(39). The kit uses patented thiol-scavenging reagent (1-methyl-2-vinylpyridinium
trifluoromethanesulfonate (M2VP)), instead of N-ethylmaleimide (NEM) that also
inhibits glutathione reductase (GR), to rapidly scanvages GSH but does not interfere with
the GR reaction. Briefly,10 µl M2VP was added to 100 µl of cell extract and used for
GSSG sample preparation. For GSH sample preparation,50-µl cell extract without the
presence ofM2VP were used. The mixture of sample (or blank or standard), chromogen,
enzyme, and NADPH (200 µl of each) in a cuvette was examinedfor the change of
absorbance at 412 nm for 3 min with a spectrophotometer.The reaction rate and
Page 10 of 37
calibration curves were used to calculateconcentrations of GSH and GSSG, which will
be standardized with total protein concentration. The concentration of GSH or GSSG was
expressed as umol (GSH or GSSG)/g (protein). ∆GSSG or ∆GSH was calculated by
subtracting either GSSG or GSH value of non-treated cells from those of HOCl treated
8. Western blot
Total cellular protein was collected based on the methods describedpreviously(9).
Nuclear proteins were collected using nuclear protein extraction kit from Panomics
(Fremont, CA). The sources of antibodies have been described in the “Chemicals,
inhibitors, antibodies”. Equal protein load for both total and nuclear proteins was
confirmed using the staining of anti–actin antibody.
9. Small interference RNA (siRNA) and transient transfection
Control siRNA was purchased from Ambion (Austin, TX). siRNA against Nrf2
(GTAAGAAGCCAGATGTTAA) (35) or Keap1 (GGGCGTGGCTGTCCTCAAT)(35)
were synthesized by Ambion (Austin, TX). siRNA was transfected into cells using
lipofectamineTM 2000 (Invitrogen, Carlsbad, CA) based on manufacturer’s instruction.
Successful knockdown of the target was confirmed by realtime RT-PCR and western blot.
10. Statistical analysis
Experimental groups were compared using a two-sided Student'st test, with significance
level set as P < 0.05. When datawere not distributed normally, significance was assessed
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withthe Wilcoxon matched-pairs signed-ranks test, and P < 0.05was considered to be
significant. Matlab 6.0 with statisticstoolbox (MathWorks, Inc., Natick, MA) was used
for analysesof the data.
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1. HOCl induces dose-dependent epithelial cell toxicity
We first examined if HOCl could elicit any cyto-toxic effect on airway epithelial cells. In
this study, we used a MTS assay kit from promega, which is similar, but much
convenient than, traditional MTT assay. This assay, by measuring the number of
metabolic active (viable) cells in the culture, counts for both direct damage that kills the
cells and indirect toxicity that repress es metabolic activity of the cells and eventually lead
to cell growth inhibition and death. For the convenience, we are using the term-
“viability” to represent viable cells in the culture. As shown in Fig.1, HOCl induced a
dose-dependent toxicity on primary airway epithelial cells grown as monolayer. At 6h,
doses up to 2mM had no effect, while 4mM HOCl elicited 61% reduction of cell viability.
Interestingly, low dose HOCl, particularly at 0.8 mM, had a slight promoting effect on
cell viability. At 24h, doses higher than 0.8mM had significant toxic effect, which is also
consistent with the light microscopic observation of increasing number of floating cells.
At 4mM, most of cells were dead under light microscope (data not shown). Thus, HOCl
alone, which was estimated before to be as high as 8mM in severely inflamed
airways(15), should be sufficient to damage airway epithelial cells.
2. Nrf2-dependent antioxidant gene expression was identified by transcription gene
profiling in differentiated airway epithelial cell culture treated with HOCl.
To further understand the epithelial response to HOCl induced injury at molecular basis,
we took advantage of state-of-the-art Genechip technology to characterize transcription
profiling of HOCl treated cells comparing with non-treated controls. Our goal was to
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identify key genes or pathways that represented either cellular stress response that led to
epithelial injury and death, or the adaptive response that protected cells from damage. For
cell model, we utilized a differentiated cell culture model grown on air-liquid interface.
As we (5, 46) have demonstrated before that this model has major airway cell types and
is similar to in vivo epithelial layer both morphologically and physiologically.
Considering human to human variability, epithelial cells isolated from 4 different healthy
individuals were used as replicates. Accurately measuring viability (MTS assay) is
difficult in the differentiated cell culture with multi-layers, because the cell number in the
culture exceeds the maximum for linearity, and also because different layer experiences
different actual concentration of the treatment. Since the initial epithelial injury tends to
occur at the top layer, viability test in mono-layered epithelial cell as shown in Fig.1
should provide a best approximation. Thus, based on the cell viability measurements on
mono-layered epithelial cells (Fig.1), we chose three doses (0.4, 1 and 4 mM) for our
study. 0.4mM represented a dose having no obvious toxicity; 1mM caused some toxicity
at 24h but not at 6h, suggesting an underlying molecular event might have already been
triggered at earlier time point; 4 mM dose was the highest in our study which caused
significant toxicity even at early time point (6h). Any higher dose (e.g. 6, 8, 10mM) was
too toxic (Fig.1). Because we were particularly interested in the early response, we chose
the time 2h and 6h for this Genechip study. As shown in Fig.2A, total 83 differentially
regulated genes were identified. Hierarchical analysis revealed several very interesting
gene expression pattern changes depending on dose and time (Fig.2A). In early induced
genes (genes that were elevated in 2h but not in 6h), we found two transcription factors-
MAFF and MAFG were highly elevated by HOCl (Fig.2B). And in the late (genes that
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were elevated in 6h but not in 2h) or persistent (genes that were elevated both in 2h and
in 6h) induced genes, a battery of antioxidant genes, including TXNRD1, HMOX1,
ALDH1A3, NQO1 and GCLM, were also significantly elevated by HOCl (Fig.2C).
Interestingly, all those genes are related to Nrf2-dependent cellular antioxidant/adaptive
pathway. MAFF and MAFG are DNA binding partners of Nrf2 on ARE (antioxidant
response element) site(19), which can modulate Nrf2 binding preference and activity.
TXNRD1(37), HMOX1(31), ALDH1A3(11), NQO1(31) and GCLM(31) belong to phase
II detoxification enzyme and are regulated by Nrf2. Thus, activation of Nrf2 appeared to
be a major epithelial adaptive response to HOCl treatment. Because of the critical role of
Nrf2 in airway diseases(8), we focused our effort on the role of this pathway in HOCl-
induced epithelial injury.
3. HOCl induced Nrf2 activation is not dependent on EGFR-MAPK pathway.
We first decided to confirm if Nrf2 was indeed activated by HOCl. As shown in Fig.3A,
Nrf2 induced a time- and dose- dependent elevation of nuclear Nrf2, a hallmark of Nrf2
activation(23). In epithelial cells, without treatment, Nrf2 was very low both in the
nucleus (Fig.3A) and cytosol (data not shown). Upon treatment, nuclear Nrf2 level was
significantly elevated within 2h. At 6h, nuclear Nrf2 was decreased under low dose
treatment (1mM), but was further increased under high dose treatment (4mM), which
might reflect high oxidative burden under this dose.
Then, we seek to understand the upstream event that activated Nrf2. Besides the classic
Keap1 dissociation mechanism(40), which we will discuss later in Fig.5, phosphorylation
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events that caused by upstream kinases have been reported to activate Nrf2 in lung cells
challenged by other oxidant (29). In our model, we indeed found that HOCl dose-
dependently activated EGFR and MAP kinases (e.g. ERK1/2, p38 and JNK) (Fig3B). But,
only EGFR and ERK1/2 phosphorylations were correlated with Nrf2 activation, because
HOCl only activated p38 and JNK at 4mM but not at 1mM. To test if the activation of
either EGFR or ERK1/2 was responsible for Nrf2 activation, we used specific inhibitors-
AG1478 to inhibit EGFR activation, and U0126 to inhibit MEK1/2, the upstream kinase
of ERK1/2. Both inhibitors had no effects on either the level of nuclear Nrf2 (Fig.3C) or
Nrf2-dependent gene expression (HMOX1, NQO1 and GCLM were tested and had
similar responses. For simplicity, only HMOX1 was shown in Fig.3D). Thus, it appeared
that EGFR and MAPK pathways were not responsible for HOCl activated Nrf2 in airway
4. HOCl induced Nrf2 activation is dependent on cellular oxidative stress
Because HOCl is an oxidant, we took another direction to ask the question if HOCl could
induce the generation of reactive oxygen species (ROS), and if ROS could then activate
Nrf2. As shown in Fig 4A, HOCl treatment induced cellular oxidative stress indicated by
significant decrease of GSH (a reducing form of Glutathione) and increase of GSSG (an
oxidizing form of Glutathione). Substantial amount of intracellular ROS was also
detected by oxidizing-sensitive fluorescence dye in HOCl-treated cells (Fig.4B). When
cells were treated with N-acetylcysteine (NAC), an antioxidant, both nuclear Nrf2 level
(Fig.4C) and Nrf2 dependent HMOX1 expression (Fig.4D) were significantly decreased.
Thus, HOCl induced Nrf2 activation was dependent on ROS mediated signaling.
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5. Nrf2 protects epithelial cell from HOCl induced toxicity
Nrf2 has been shown to play a protective role in animal studies(1, 6, 32, 33, 38). Since
HOCl induced epithelial cell toxicity, we wanted to ask the question as of whether the
activation of Nrf2 provided any protection. Because primary cells are notoriously
resistant to regular transfection procedures, we decided to use an epithelial cell line-NCI-
H292 to test the protective role of Nrf2 in HOCl treated cells. Similar to primary cells,
HOCl induced Nrf2 activation (Fig.5A), Nrf2-dpendent gene expression (Fig.5B), and
dose-dependent cellular toxicity (Fig.5C). By using small interference RNA (siRNA)
approach (Fig.5A), we either specifically knocked down Nrf2 level by using siRNA
against Nrf2, or enhance nuclear Nrf2 level by using siRNA against Keap1, a cellular
inhibitor of Nrf2 whose function is believed to trap Nrf2 in cytosol and target Nrf2 to
proteasomal degradation(23). Western blot analysis confirmed that the change of Nrf2
was indeed consistent with our manipulation as shown in Fig.5A. As another
confirmation, modulating nuclear Nrf2 level also affected Nrf2-dependent HMOX1
expression which was correlated with the change of nuclear Nrf2 level (Fig.5B).
Interestingly, knocking down Nrf2 also reduced basal level (untreated) of HMOX1
expression, when Nrf2 level was very low. This was consistent with the Nrf2 knockout
mouse study, in which the expression of many Nrf2 dependent genes were significantly
lower than the wide-type even without any challenge(6).
We then tested if Nrf2 could protect HOCl induced epithelial toxicity. By using MTS
assay, we found that elevation of nuclear Nrf2 level by Keap1 knockdown could protect
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cells under 0.6 ~1.5 mM HOCl treatments. Consistently, loss of Nrf2 elicited
significantly exacerbated toxicity under similar treatment. At high does (e.g. 2 and 4mM),
there was no protection or exacerbation under both manipulations. Under such condition,
HOCl might already overwhelm all defense systems. Notably, NCI-H292 cells were more
susceptible to HOCl treatment than primary epithelial cells (Fig.1). Nevertheless, those
data have demonstrated the protective role of Nrf2 activation in HOCl-induced epithelial
Page 18 of 37
HOCl is a powerful oxidant, which can be generated from environmental exposure to
chlorine(12), or from catalytic reaction by major neutrophilic granular enzyme-MPO(24).
One of the major clinical manifestation of high-dose chlorine inhalation is acute lung
injury (ALI) and may lead to acute respiratory distress syndrome (ARDS)(25). Animal
study on Chlorine inhalation confirmed that the progress from ALI to ARDS was
correlated with inhaled chlorine dose(2). In addition, intra-tracheal infusion of glucose
peroxidase (as a H2O2source) and MPO into rats generated acute lung injury, in which
peroxidase or MPO alone didn’t have any effect(20). Under either condition, HOCl was
the major products of chemical/biochemical reactions. And the damage of epithelia by
HOCl might represent the first event that eventually led to inflammation and progressive
lung injury. Consistently, the levels of 3-chlorotyrosine(CL-Tyr), a specific marker for
HOCl reaction with protein, were high in infants who developed chronic lung diseases(4).
Although there are no reports on Cl-Tyr level in adult patients with lung injury, numerous
studies have demonstrated the positive correlation between HOCl-generating activated
neutrophils, as well as its catalyzing enzyme-MPO, and lung injury in various lung
diseases(24). Thus, HOCl is very likely to play an essential role in the epithelial injury in
those disease contexts.
The most important finding in this report is the involvement of a key antioxidant factor-
Nrf2 in the adaptive response to HOCl treatment. In addition to epithelial cells reported
in this study, we have also observed HOCl activated Nrf2 in macrophages(30). Thus,
HOCl appears to be Nrf2 activator in various cell types. Interestingly, Nrf2 was
Page 19 of 37
previously identified as an susceptible locus to hyperoxia induced lung injury (7). Later,
by using gene knockout approach, Nrf2 was found to be a master transcription factor that
regulates most of the phase II detoxification enzyme and appeared to be critical for
antioxidant response(8). Consistently, in HOCl treated cells, a battery of phase II genes
including TXNRD1, HMOX1, ALDH1A3, NQO1 and GCLM were highly regulated. All
those genes have been reported to be Nrf2 dependent (11, 31, 37) and have specific role
to detoxify different deleterious oxygen intermediates. TXND1, thioredoxin reductase 1,
is a selenoprotein that catalyzes the NADPH-dependent reduction of thioredoxin(TRX)
(27), an ubiquitous small peptidewith a redox active thiol group. TRX and TXND system
is a critical antioxidant system that is essential for intracellular signaling pathways by
catalyzing protein disulfide/dithiol exchange(28). Because HOCl is known to oxidize
protein thiol group(24), activation of TRX-TXND system may facilitate the reduction of
those oxidized thiols and restore the normal protein function. HMOX1, also called HO-
1, is the first enzyme in heme catabolism, producingCO, Fe2+, and biliverdin, which can
further be reduced to bilirubinby biliverdin reductase(47). Both biliverdin and bilirubin
are antioxidants that can prevent H2O2/O2.- -induced lipid peroxidation and cell death (22).
CO was identified asa signaling molecule that prevented NO toxicity inHeLa cells(34).
In HOCl treated cells, those HMOX1 related antioxidant products may help to eliminate
intracellular ROS. ALDH1A3, also known as ALDH6, play a major role in the
detoxification of aldehydes generated by lipid peroxidation, alcohol metabolism, etc.(16).
In our case, lipid peroxidation by HOCl is likely to be the source of aldehydes. NQO1 is
a member of the NAD(P)H dehydrogenase (quinone) family and encodes a cytoplasmic
2-electron reductase, which detoxifies toxic quinone by reduc ing it to hydroquinones(36).
Page 20 of 37
Because HOCl has no quinine-like structure, the substrate of NQO1 in our study is
currently unknown. But it is very likely that some toxic quinone or quinone like
molecules were generated from HOCl induced oxidative reaction on various cell
components. GCLM is the modulator unit of glutamate-cysteine ligase, also known as
gamma-glutamylcysteine synthetase, which is the first rate limiting enzyme of
glutathione synthesis (10). Glutathione system provides the most important antioxidant
capacity in both intracellular and extracellular environment. Because HOCl reduced GSH
level and increased GSSG level, upregulation of GCLM may contribute to re-balancing
the whole system to restore the cellular antioxidant capacity. Interestingly, GSH is a very
efficient scavenger of HOCl(44). Therefore, increased level of GSH may be directly used
to neutralize HOCl.
Despite the importance of Nrf2 signaling in HOCl effect, the upstream pathway that
triggered Nrf2 activation is not entirely clear. In lung cells, other studies demonstrated
that EGFR-MAPK mediated signaling was essential for hyperoxia-induced Nrf2
activation(29). Although HOCl appeared to both activate EGFR-MAPK pathway and
Nrf2, we failed to find any connection between these two events. Other studies(17, 29)
have also indicated the involvement of PI3K-AKT and PKC pathways in activating Nrf2.
However, specific PI3K inhibitor (LY203580, Calbiochem) and AKT inhibitors (AKT
inhibitor I and II, Calbiochem), as well as a pan-PKC (Calphostin C, Calbiochem) all
failed to inhibit Nrf2 activation (unpublished observation). Thus, HOCl induced Nrf2
activation appeared not to be associated with those pathways either. Our data only
indicated that the oxidant-dependent signaling upstream of Nrf2, whatever it is, could be
Page 21 of 37
inhibited by NAC. And this unknown signaling was likely to activate Nrf2 by disrupting
Keap1-Nrf2 interaction and facilitate the nuclear accumulation of Nrf2. This mechanism
was corroborated by our siRNA study on epithelial cell line. Because Keap1 itself can be
an oxidant sensor, it was very likely that HOCl activated Nrf2 by directly oxidizing key
cysteine residues in Keap1(41). However, at this stage, we can’t exclude the possibility
that Nrf2 activation by HOCl was mediated through some unidentified signaling pathway
other than EGFR-MAPK.
Consistent with the proposed protective role of Nrf2, increased sensitivity to lung
oxidative stress in Nrf2-deficient mice has been reported in several disease models(1, 6,
32, 33, 38). However, due to the nature of systematic protection, it is still unclear as of at
what level this protection occurs. To date, no study has been done to determine if Nrf2
can directly protect airway epithelia from oxidative injury. The present study has filled in
this knowledge gap by demonstrating that modulating Nrf2 activation by siRNA could
directly affect the sensitivity of epithelial cells to HOCl induced toxicity. To our
knowledge, this is the first study that conclusively demonstrates the causal link between
Nrf2-Keap1 signaling and the protection of epithelial cells from HOCl induced toxicity.
Most importantly, our model and approach can be readily applied to the study of
epithelial injury induced by other oxidants, which is very interesting topic to pursue in
the future. One weakness of our study is to use epithelial cell line-NCI-H292 cell to
substitute the primary cell, which is due to the difficulty of transfecting these cells.
Nonetheless, as we have shown in the result section, NCI-H292 cells could recapitulate
all HOCl responses of primary cells including dose-dependent toxicity, Nrf2 activation,
Page 22 of 37
and Nrf2 dependent gene activation. Thus, NCI-H292 provides the most convenient and
close surrogate of primary epithelial cell to study the detailed molecular mechanism of
HOCl induced epithelial response. In the next stage, we plan to apply this in vitro finding
to animal model to further investigate its physiological role in vivo.
Identifying epithelial Nrf2 activation in HOCl challenge has significant implications in
both the area of biomedicine and the area of risk assessment. In the area of biomedical
research, our study suggests that Nrf2 activation provides a critical protection against
HOCl induced airway epithelial injury. HOCl is abundantly present in the airways of
neutrophilic inflammation associated with many chronic diseases including COPD, cystic
fibrosis etc. Since it has been shown that Nrf2 polymorphism is directly associated with
the susceptibility to lung injury(26), it is tempting to develop therapeutic reagents, which
can modulate Nrf2 level, to prevent lung injury in those chronic diseases. In the area of
risk assessment, since the Nrf2 activation is dose-dependent and occurred much earlier
than the actual injury, it provides an early biomarker to detect lung injury induced by
chlorine inhalation. This is particularly important because the chlorine induced acute ling
injury (ALI) is delayed and has little symptom right after exposure (25). It is also
attractive to examine if other major toxicants, most of which generate ROS, induce Nrf2
activation, which will expand the scope of using Nrf2 as a valuable biomarker for risk
assessment of various oxidant-generating toxicants.
In summary, by using transcription profiling, we have identified the Nrf2-dependent
adaptive response that protects epithelial cells from HOCl induced injury. Further study
Page 23 of 37
of this pathway on animal model as well as extending it to other toxicant challenges will
have impacts on both biomedicine and risk assessment.
Page 24 of 37
The study was supported by Long Range Initiative (LRI) of American Chemistry Council
(Chen, Y) and NIH grant RO1AI061695 (Chen, Y). Shinichiro Wachi was supported by
NIH grant RO1HL077902 (Wu, R). Authors would like to express their thanks for the
technical assistance from the Hamner Institute Gene Expression Core on Genechip
processing and data retrieval; especially for Dr. Longlong Yang’s help on data deposit
into NCBI GEO database.
Page 25 of 37
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Fig.1: Dose-dependent epithelial (mono-layer primary cells) toxicity by HOCl treatment.
Method has been described in the Material and Methods. To obtain cell viability,
readings from non-treated cells were designated as “100%”. The readings of
treated cells were then divided by non-treated cells to get their percentage. The
final percentage represents the number of metabolically viable (“living”) cells in
6-hour HOCl treatment (6h); : 24-hour HOCl treatment
(24h). #: p< 0.05 when comparing with 6h control, n=4; *: p< 0.05 when
comparing with 24h control, n=4.
Fig.2: Transcriptional profiling of HOCl treated airway epithelial cells (differentiated
primary cells). Culture condition and HOCl treatment has been described in
Materials and Methods. A) Heatmap showing relative levels of expression of
genes differentially regulated treated with different doses (0.4, 1, 4 mM) of HOCl
for different length (2h, 6h) of time. Genes with an adjusted P value of less than
0.05 in the comparison between untreated cell and cells with any treatment were
clustered according to the Euclidean distance between their expression levels and
the average agglomeration method using the R package hclust. Expression values
are represented (in rows) for each gene in different treatment dose by color on a
scale from Green (underexpressed) through black (unchanged) to red
(overexpressed) by using the heatmap.2 function of the R package gplots (42) .
Genes that are further expanded in Fig2B, Fig.2C and also discussed in the text
are marked by B1-2, and C1-5. B) Dose dependent elevation of MAFF (B1) and
Page 29 of 37
MAFG (B2) at 2h time point. *: p< 0.05 when control, n=4. C) Dose dependent
expression of TXNRD1 (C1), HMOX1 (C2), ALDH1A3 (C3), NQO1 (C4),
GCLM (C5) at 6h time point. *: p< 0.05 when con trol , n=4. The box indicates a
commonly significant dose point.
Fig.3: HOCl activated Nrf2 in airway epithelial cells (differentiated primary cells) is not
dependent on EGFR and MAPK activation. A) HOCl induced Nrf2 nuclear
accumulation both time- and dose- dependently. B) HOCl activated EGFR and
MAP kinases. C) Nrf2 nuclear accumulation is not dependent on EGFR and
ERK1/2. C: Control; HOCl: HOCl treated; DMSO: Solvent of the inhibitors; AG:
AG1478 (2uM), EGFR inhibitor; U0126 (1uM): ERK1/2 inhibitor. D) Nrf2-
induced HMOX1 is not dependent on EGFR and ERK1/2.
Fig.4: HOCl activated Nrf2 in airway epithelial cells (differentiated primary cells) is
caused by cellular oxidant mediated signaling. A) HOCl induced imbalance of
GSH/GSSG. GSH or GSSG level was measured at 6h HOCl treatment, and the
protocol is described in Materials and Methods. GSH: reducing form of
glutathione. GSSG: oxidized form of glutathione. ∆GSSG or ∆GSH was
calculated by subtracting either GSSG or GSH value of non-treated cells from
HOCl treated cells. Values were expressed as umol per gram(g) cellular protein. *:
p<0.05 when comparing with non-treated controls. n=4. B) HOCl induced
intracellular oxidant production. Left image was recorded after 30min after dye
loading but without HOCl treatment. Right image was recorded 30min after HOCl
Page 30 of 37
treatment. Green Fluorescence indicates the generation of ROS that oxidized the
dye to emit fluorescence. C) HOCl induced Nrf2 nuclear accumulation was
blocked by antioxidant N-acetylcysteine (NAC). D) HOCl-induced HMOX1 was
also blocked by NAC treatment. *, #: p< 0.05, n= 4.
Fig.5: Nrf2 activation protects epithelial cell line (NCI-H292) from HOCl-induced
toxicity. A) siRNA specifically modulates Nrf2 nuclear accumulation. siRNA
transfection was described in Materials and Methods. Cells were treated with
0.8mM HOCl for 6h. siC: Cells transfected with control siRNA that targets no
specific mamalian sequences (Ambion); siNrf2: Cells transfected with siRNA
specifically targets Nrf2; siKeap1: Cells transfected with siRNA specifically
targets Keap1. upper panel is the representative western blot. Lower panel is the
summary from 3 western replicates. Band intensity was quantified and presented
as fold induction when comparing with siC transfection without treatment. *, #: p
<0.05, n= 3. B) HOCl-induced HMOX1 was modulated by Nrf2 in NCI-H292
cells. Cells, transfected with various siRNA, were treated with 0.8mM HOCl for
6h. Empty bar: siC transfected cells; streaked bar: siNrf2 transfected cells; filled
bar: siKeap1 transfected cells. *, #: p< 0.05, n= 4. C) Nrf2 protects epithelial
cells from HOCl induced toxicity. MTS assay similar to Fig.1. : siC
transfected cells; : siNrf2 transfected cells; : siKeap1 transfected cells. *,
#: p< 0.05, n= 4.
Page 31 of 37
Table.1. Realtime Primers
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0 0.40.6 0.812468 10
Cell Viability (%)
0.41 4 mM
0 1 4
0 1 4mM
0.4 1 4 mM
A) B) Download full-text
0 0.4 0.6 0.81 1.524
Cell Viability (%)