Induction of airway allergic inflammation by ...

JBC Papers in Press. Published on November 18, 2016 as Manuscript M116.746909 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.746909

Airway allergic inflammation by hypothiocyanite

Induction of Airway Allergic Inflammation by Hypothiocyanite via Epithelial Cells Shoichi Suzuki1, Masahiro Ogawa1, Shoichiro Ohta1, Satoshi Nunomura1, Yasuhiro Nanri1, Hiroshi Shiraishi1, Yasutaka Mitamura1, Tomohito Yoshihara1, James J. Lee2, & Kenji Izuhara1‡ From 1Division of Medical Biochemistry, Department of Biomolecular Sciences, Saga Medical School, Saga 849-8501, Japan, and 2Division of Pulmonary Medicine, Mayo Clinic Arizona, Scottsdale, Arizona 85259, USA. ‡

To whom correspondence should be addressed: Prof. Kenji Izuhara

5-1-1, Nabeshima, Saga, 849-8501, Japan. Tel.: +81-952-34-2261; Fax: +81-952-34-2058; Email: [email protected]. Running title: Airway allergic inflammation by hypothiocyanite Keywords: anion transport, asthma, epithelial cell, hydrogen peroxide, hypothiocyanite, inflammation, NF-kappa B, pendrin, peroxidase, protein kinase A (PKA)

molecules would lead to airway

ABSTRACT Hypothiocyanite (OSCN-) serves as

inflammation. In this study, we examined

a potent innate defense system against

whether OSCN- produced via the

microbes in the lungs. OSCN- is generated by

pendrin/peroxidase/Duox pathway causes

the catalysis of peroxidases using thiocyanate

inflammation via airway epithelial cells. In

transported via several anion transporters,

an in vitro OSCN- production system,

including pendrin/SLC26A4 and hydrogen

OSCN-, but not H2O2, activated NF-B, a

peroxide (H2O2) generated by Duox1 and

transcription factor critical for inflammatory

Duox2. We previously demonstrated that

responses, in the airway epithelial cells.

expression of pendrin, peroxidases, and

OSCN- was sensed by protein kinase A

Duox1/Duox2 is upregulated in bronchial

(PKA) followed by formation of the

asthma patients and/or asthma model mice

dimerization of PKA. The dimerized PKA,

and that these molecules are important in

the active form, was critical in activating NF-

accelerating airway inflammation. However,

B. Detoxifying H2O2, mainly by catalase,

it remained unclear how activating these

enabled the dominant abilities of OSCN- to 1

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Division of Medical Biochemistry, Department of Biomolecular Sciences, Saga Medical School,


direct effects of IL-13 on airway epithelial

dimerize PKA and activate NF-B, compared -

to untreated H2O2. Furthermore, OSCN in

cells among various IL-13–targeted cells;

high doses caused necrosis of the cells,

ectopic expression of IL-13 in airway

inducing release of IL-33, a trigger to initiate

epithelial cells led to asthma-like phenotypes

type 2 inflammation. These results

(8). Moreover, selective expression of

-

demonstrate that OSCN in low doses

STAT6, a transcriptional factor critical for the

activates NF-B via PKA in airway epithelial

IL-13 signals, to airway epithelial cells in

-

STAT6-deficient mice recovered IL-13–

necrosis, suggesting an important role in

inducing asthma-like phenotypes (9).

airway allergic inflammation for the

However, it is not fully understood how IL-

production of OSCN- via the

13’s actions on epithelial cells would lead to

pendrin/peroxidase/Duox pathway.

airway allergic inflammation. Hypothiocyanite (OSCN-) serves as

Introduction

a potent innate defense system against

Asthma is a common disease affecting up to

microbes in lungs as do lysozyme, defensin,

10% of adults and 30% of children in the

cathelicidin peptides, lactoferrin, and

Western world (1). Type 2 immunity is

leukocyte protease inhibitors (10,11). In the

dominant in the pathogenesis of asthma, and

OSCN- production system, thiocyanate

it is estimated that 50-70% of adult asthma

(SCN-), a pseudohalide, is first incorporated

patients have type 2 immunity (2,3). Among

from the basal side via the Na+I- symporter

type 2 cytokines, IL-13 plays a central role in

(NIS)/the solute carrier family (SLC)5A5,

the pathogenesis of asthma; inhalation of IL-

into airway epithelial cells. SCN- is then

13 alone causes asthma-like phenotypes in

actively transported into pulmonary lumens

mice, whereas blocking the IL-13 signals

at the apical side via several anion

diminishes asthma-like phenotypes in

transporters—cystic fibrosis transmembrane

ovalbumin-induced asthma model mice (4,5).

conductance regulator (CFTR) and

Based on such an immunological

pendrin/SLC26A4. SCN- together with

background, several anti-asthma drugs

hydrogen peroxide (H2O2) generated by

targeting IL-13 are now under development

Duox1 and Duox2, members of the

(6).

Nox/Duox family, is catalyzed by peroxidases into OSCN-. Three

IL-13 is a multi-functional cytokine; it acts on both immune cells (e.g.,

peroxidases—myeloperoxidase (MPO),

eosinophils and mast cells) and resident cell

eosinophil peroxidase (EPX), and

(e.g., epithelial cells, fibroblasts, and smooth

lactoperoxidase (LPO)—are involved in this

muscle cells) (7). Analyses of the gene-

reaction in lung tissues. OSCN- is thought to

manipulated mice showed the importance of

be the major product of these peroxidases 2


cells, whereas OSCN in high doses causes


because SCN- is abundant in the airway

that the blockage of peroxidases by its

surface liquid and is the most preferred

inhibitors, widely used as thyroid

substrate (10-13). OSCN- has potent

pharmaceuticals, improves airway

antimicrobial properties against bacteria,

inflammation ((17), Suzuki unpublished

viruses, and fungi. The importance of the

observations). Given that much of the

-

OSCN production system in pulmonary

machinery of the OSCN- production system

defense is shown in cystic fibrosis patients in

is induced by the asthmatic condition and

whom susceptibility to chronic respiratory

show important roles in airway inflammation,

infections increases in proportion to impaired

we hypothesized that the OSCN- produced

CFTR function (14). In contrast to the

via the pendrin/peroxidase/Duox pathway

powerful effects of OSCN- on microbes, it is

would be involved in airway inflammation.

obscure how the OSCN production system

In this study, we examined the

disorders homeostasis of hosts leading to

molecular mechanism of how OSCN-

pathological conditions.

activates airway epithelial cells using an in vitro OSCN- production system. We found

We previously searched for IL-13– inducible genes in airway epithelial cells by a

that OSCN- in low doses is sensed by protein

comprehensive method using DNA

kinase A (PKA) followed by activation of

microarray, finding that pendrin is involved

NF-B, a transcription factor critical for

in those cells (15). We and others then

inflammatory responses, in the cells, whereas

showed the significance of pendrin in airway

OSCN- in high doses causes necrosis. These

allergic inflammation using model mice:

results suggest that OSCN- has deleterious

overexpression of pendrin in bronchial

effects on airway epithelial effects leading to

tissues causes mucus hyper-production,

airway inflammation.

enhanced airway reactivity, infiltration of inflammatory cells, and up-regulated

Results

expression of inflammatory mediators (15).

Activation of NF-B by OSCN-, but not by

Reciprocally, pendrin deficiency decreases

H2O2, in airway epithelial cells - We first

airway reactivity and infiltration of

established an in vitro OSCN- production

inflammatory cells in bronchoalveolar lavage

system to investigate the underlying

fluid (16). These findings suggest to us that

molecular mechanism of how OSCN- causes

anions transported by pendrin, or their

inflammation by acting on airway epithelial

derivatives, play an important role in airway

cells (Figure 1A). In this system, H2O2 was

allergic inflammation. Furthermore, we

first generated by glucose oxidase (GOX).

recently found that expression of peroxidases

Then SCN- and H2O2 were catalyzed in the

and Duox1/Duox2 is up-regulated in both

presence of LPO into OSCN-. H2O2 was

asthma model mice and asthma patients and

generated from -D-glucose in a dose3


-


dependent manner with up to 16 mU/mL of

examined the effects of inhibitors against

GOX in the absence of LPO (Figure 1B).

PKA (H-89), p38 (SB203580), MEK1/2

H2O2 production continuously increased up

(PD98059), and JNK (SP600125), signal-

to 9 h. When we added 10 g/mL LPO in this

transducing molecules potentially located in

system, OSCN- production also behaved in a

the upstream pathway of NF-B, on NF-B

dose-dependent manner with GOX, reaching

activation by OSCN-. H-89 diminished NF-

a peak at 3 h (Figure 1C). Therefore, we

B activation in a dose-dependent manner,

performed the following experiments with 16

whereas SB203580 and PD98059 did not

mU/mL GOX in the presence or absence of

change NF-B activation (Figure 3A).

10 g/mL LPO.

SP600125 inhibited NF-B activation

We next examined whether H2O2 or

(Figure 3A); however, we could detect

OSCN activates NF-B, a transcription

phosphorylation of p38, but not JNK by

factor critical for inflammatory responses, in

OSCN- (Figure 3B, C). These results

the human airway epithelial cell line H292

demonstrate that PKA plays an important

using the in vitro OSCN- production system.

role in NF-B activation by OSCN-. It has been reported that PKA is a

When we incubated the cells with GOX or LPO alone for 5 h in the system, no

sensor of oxidants in the cells and that upon

activation of NF-B was observed (Figure

stimulation, an oxidant generates a dimer, the

2A, 2B). In the former condition, ~1 mM

active form, by forming an intermolecular

H2O2 was generated (Figure 1B). In contrast,

disulfide bond independently of cAMP

when the cells were incubated in the presence

(18,19). We then examined whether OSCN-

of both GOX and LPO, the condition that

is sensed by PKA followed by activation of

~350 M OSCN- was generated (Figure 1C),

PKA in airway epithelial cells. When we

activation of NF-B was clearly observed.

exposed H292 cells with OSCN- in the

The supershift assay showed that NF-B

presence of LPO, PKA was dimerized in a

-

activated by OSCN in H292 cells contained

dose-dependent manner (Figure 3D). In

p50, but not p52, p65, Rel B, or c-Rel (Figure

contrast, when H292 cells were exposed with

-

2B). These results suggest that OSCN , but

H2O2 in the absence of LPO, no dimerization

not H2O2, activates the formation of the p50

of PKA was observed. To confirm whether

homodimer in H292 cells.

dimerized PKA by OSCN- is the active form, we then examined phosphorylation of PKA

PKA senses OSCN leading to NF-B

substrates by OSCN-. Phosphorylation of

activation in airway epithelial cells - We

PKA substrates was up-regulated in the

next investigated the molecular mechanism

presence of OSCN- as well as isoprenaline, a

of NF-B activation by OSCN- in airway

-agonist, and phosphorylation of PKA

epithelial cells. To address this question, we

substrates by both OSCN- and isoprenaline

-

4


-


We next examined the effects of the

was inhibited by H-89 (Figure 3E). Although isoprenaline caused 100-fold elevation of

detoxification system for H2O2 on

cAMP, OSCN- did not cause any changes of

dimerization of PKA in H292 cells. Three

it throughout the time course of the culture,

detoxification systems—catalase, the

excluding the possibility that OSCN-

glutathione peroxidase/reductase system, and

activates PKA by up-regulating the cAMP

the peroxiredoxin/thioredoxin/thioredoxin

concentrations (data not shown). These

reductase system—were expected to function

-

results demonstrate that OSCN is sensed by

in the cells (Figure 4B). We first examined

PKA in the airway epithelial cells, causing

which detoxification system was dominant

PKA dimerization followed by activation of

for H2O2 in H292 cells using inhibitors

NF-B independently of cAMP.

against catalase (aminotriazole), glutathione

H2O2 is detoxified mainly by catalase in

chloroethyl)-1-nitrosourea (BCNU)), and

airway epithelial cells - H2O2 has a potent

thioredoxin reductase (auranofin). Adding

oxidation activity so that it has a potential to

aminotriazole recovered the H2O2

-

dimerize PKA as well as OSCN (19).

concentrations in the medium, whereas

However, our present finding showed that

BCNU or auranofin did not (Figure 4C).

H2O2 has much weaker oxidation activity

Accordingly, adding aminotriazole to the in

than OSCN- in airway epithelial cells. We

vitro H2O2 production system significantly

hypothesized that the different abilities of

enhanced the formation of PKA dimer to the

OSCN- and H2O2 for dimer formation of

same level of OSCN- (Figure 4D). Moreover,

PKA in airway epithelial cells would be due

when we added more than 10 mM H2O2 in

to the difference in their clearance from these

the system, PKA was dimerized as the same

cells. To address this question, we first

level of OSCN- (Figure 4E), suggesting that

analyzed the kinetics of the OSCN- and H2O2

excess amounts of H2O2 overcome the

concentrations in the medium in the presence

detoxification system in H292 cells. These

of H292 cells. H2O2 was promptly removed

results demonstrate that catalase is

from the medium in the presence of H292

dominantly involved in the detoxification

cells, and mostly disappeared by 1 h (Figure

system for H2O2 in H292 cells and that in

-

4A). In contrast, clearance of OSCN was

airway epithelial cells, the detoxification of

slow even in the presence of H292 cells,

H2O2 contributes to the dominant abilities of

almost the half of the initial activity

OSCN- for dimerization of PKA compared to

remaining after 1 h. These results suggest

H2O2.

-

that H2O2, but not OSCN , would be rapidly incorporated into H292 cells or detoxified in

Induction of necrosis in airway epithelial

H292 cells.

cells by high concentrations of OSCN- 5


reductase (carmustine; 1,3-Bis(2-


Epithelial desquamation is a typical

cells.

histological feature of asthma reflecting Discussion

(20,21). Furthermore, necrosis of epithelial

In this study, we demonstrated that OSCN-

cells causes release of IL-33, which triggers

produced via the pendrin/peroxidase/Duox

type 2 immune responses targeting mainly

pathway activates NF-B via PKA in

the innate lymphoid cell type 2 (ILC2), from

epithelial cells, which would lead to the onset

their nuclei (22,23). On the other hand, it has

of airway inflammation (Figure 6). In this

been reported that OSCN- induces both

pathway, SCN- is first actively transported

apoptosis and necrosis in macrophage cell

into pulmonary lumens via NIS/SLC5A5 at

lines, but apoptosis in endothelial cells

the basal side and via anion transporters

(24,25). Lastly, we evaluated the effects of

including CFTR and pendrin/SLC26A4 at the

high-dose OSCN- on airway epithelial cells

apical side in airway epithelial cells (10,11).

to determine if the observed effects were

OSCN- is then generated by the catalysis of

necrotic and apoptotic in character. This

peroxidases composed of MPO, EPX, and

evaluation was carried out in two ways—

LPO using SCN- and H2O2 generated by

fractions estimated by annexin V/propidium

Duox1/Duox2. It has been known that many

iodide (PI) and quantitation of DNA content.

extrinsic or intrinsic factors activate NF-B

H292 cells treated with cycloheximide and

via pattern recognition receptors (PRRs) on

TNF- caused apoptosis in a time-dependent

epithelial cells (26); however, to our

manner, estimated by the increases of the

knowledge, OSCN- is the first anion to

annexin V+PI- fraction and the low-DNA–

activate NF-B in epithelial cells.

content fraction (Figure 5A, B, lower panels).

We have recently demonstrated that

In contrast, high-dose OSCN (≥ 32 mU/mL

expression of pendrin, peroxidases, and

GOX) caused necrosis (increase of the

Duox1/Duox2, is enhanced, resulting in that

-

+

+

annexin V PI fraction and no change in the

the OSCN- production would be up-regulated

low-DNA–content fraction) in H292 cells

in the airways of allergic inflammation in

(Figure 5A, B, upper panels). A trace amount

asthma model mice and asthma patients

of IL-33 (0.063 ± 0.022 pg/mL) was detected

((17), Suzuki unpublished observations). The

in the medium of H292 cells treated with

present data demonstrate that NF-B

high-dose OSCN-. In low-dose OSCN- (≤ 16

activation by OSCN- in airway epithelial

mU/mL GOX) in which PKA dimerization

cells would be one underlying mechanism of

occurred, the cells were still intact. These

airway allergic inflammation. Given that

-

results demonstrate that high-dose OSCN

OSCN- has harmful effects on various

causes necrosis, whereas low-dose OSCN-

microbes (10,11) and that type 2 immune

activates NF-B via PKA in airway epithelial

responses are equipped to be a defense 6


damage and injury in airway epithelial cells


Our data showed that OSCN- is

mechanism against parasites (27,28), we hypothesize that whereas the OSCN-

sensed by PKA followed by activation of NF-

production system may be an innate host

B in airway epithelial cells. It has also been

defense mechanism in the lung, this

shown that oxidative stress affects

misplaced production of OSCN- is a likely

mobilization of several signaling

contributor to pulmonary inflammation,

molecules—PKA, nucleotide diphosphate

causing deleterious effects in response to

kinase B, serine/threonine protein

airway allergen provocation.

phosphatase, and PKC /—in myocytes

Various extrinsic or intrinsic danger

(18). Among these molecules, upon stimulation of oxidants, the regulatory

via PRRs or cause necrosis followed by

subunit of type I PKA is dimerized through a

release of IL-33 in epithelial cells (29,30).

disulfide bond followed by increased affinity

Activation of NF-B in airway epithelial

for the substrates, demonstrating that

cells is important for production of

oxidative stress is changed to intracellular

chemokines and inflammatory cytokines and

signaling through PKA, independently of

expression of adhesion molecules

cAMP (19). Our present study shows PKA

accelerating type 2 immunity (29). IL-33 acts

acting as a dominant sensor for OSCN-,

on several immune cells—ILC2, mast cells,

leading to activation of NF-B in airway

basophils, eosinophils, Th2 cells, NKT and

epithelial cells (Figure 3). A previous report

NK cells (30). IL-33 acts on ILC2 inducing

that inhibition of PKA by H-89 abolished

production of IL-5 and IL-13. Thus,

airway allergy inflammation in model mice

activation of NF-B in epithelial cells and

supports our suggested importance of the

release of IL-33 from epithelial cells play

PKA mediated pathway (31).

critical roles in the transition from innate

The present finding showed that the

immunity to acquired immunity in the

detoxification system of H2O2 mainly by

pathogenesis of asthma. However, our

catalase causes the more potent abilities of

present study has shown that OSCN-, a

OSCN- for dimerization of PKA and

downstream molecule of the IL-13 signals,

activation of NF-B, compared with H2O2

activates NF-B in epithelial cells or induce

(Figure 4). H2O2, abundant in the

release of IL-33 from epithelial cells,

environmental milieu, is easily incorporated

suggesting that IL-13, OSCN-, and IL-33

into the cells and is toxic to living organisms,

may make a vicious circle. These results

causing DNA damage (32). Therefore, the

suggest the potential that type 2 inflammation

detoxification system of H2O2 including

is exaggerated or prolonged by this vicious

catalase is found widely among microbes,

circle involving the OSCN- production

plants, and animals. Although it has been

pathway in airway epithelial cells.

reported that OSCN- is detoxified by 7


signals including allergens activate NF-B


thioredoxin reductase (33), it is unclear that

can be a novel therapeutic target for

-

the detoxification system of OSCN is as

bronchial asthma using antagonists against

ubiquitous as that of H2O2. Actually, it is

peroxidases (17). Elucidation of the

likely that H292 cells are susceptible to

molecular mechanisms underlying OSCN-

OSCN-, which is at least partially explained

production and the activation of epithelial

by the lack of a detoxification system for

cells by OSCN- has promising clinical

OSCN- (Figure 4A). In microbes, this lack

significance for developing novel treatments

becomes advantageous in that animals can

for bronchial asthma.

use OSCN- as an effector of their innate Experimental Procedures

becomes disadvantageous for hosts because

In vitro production of H2O2 and OSCN- - A

of the high susceptibility to insults by

human lung carcinoma cell line, H292, was

OSCN-.

purchased from ATCC. H292 cells (1.8×106 In addition to its application to the

cells/well) were stimulated for 5 h with

pathogenesis of type 2 immunity, the present

phenol red-free RPMI1640 medium (1.5

finding can be also applied to airway

mL/well) containing 11 mM -D-glucose

inflammation in smokers. Plasma SCN-

(Life Technologies), 3 mM NaSCN (Wako,

levels in smokers are almost three times

Japan), and the indicated concentrations of

higher than in non-smokers (130-140 M vs.

GOX (Wako) in the presence or absence of

40-50 M) (34,35). It is well known that in

10 g/mL LPO (Calzyme Laboratories). For

asthma, smoking is associated with poor

the experiments to inhibit signal transducing

control, decrease of lung function, and

molecules, the indicated concentrations of

enhanced corticosteroid resistance (36,37).

inhibitors against PKA (H89, Merck

However, no report has yet shown any

Millipore), p38 (SB203580, Wako), MEK1/2

deleterious effects of SCN- or OSCN- derived

(PD98059, Cell Signaling Technology), and

from tobacco on lungs. The present results

JNK (SP600126, Wako) were added. H2O2

suggest the possible involvement of the

was measured by BIOXYTECH H2O2-

OSCN production system in how smoking

560™ (Percipio Biosciences), and OSCN-

affects asthma or other smoking-related

was measured by reacting with 5-thio-2-

pulmonary diseases.

nitrobenzoic acid and by estimating the

-

The present findings indicate that

decrease of the absorbance at 412 nm as

-

all of the machineries of the OSCN

previously described (38). For the

production system can be listed as novel

experiments to inhibit H2O2 detoxification

therapeutic targets for bronchial asthma

systems, inhibitors against catalase (100 mM

downstream of type 2 immunity. We have

aminotriazol, Sigma-Aldrich), glutathione

recently shown that among them, peroxidase

reductase (1 mM BCNU, Sigma-Aldrich), 8


defense system against microbes. In turn, it


and thioredoxin reductase (1 M auranofin,

Technology). The proteins were visualized by

Sigma-Aldrich) were added.

SuperSignal West Pico Substrate (Life Technologies).

Electrophoretic Mobility Shift Assay Cell death assay - H292 cells treated with

were prepared as described previously

phenol red-free RPMI1640 medium (1.5

(39,40). The sequence of the used probe was

mL/well) containing 11 mM -D-glucose, 3

5’-AGTTGAGGGGACTTTCCCAGGC-3’

mM NaSCN, 10 g/mL LPO, and the

and biotin was conjugated with the 5’ end of

indicated concentrations of GOX or with 50

the sense probe. To detect supershifted bands,

g/mL cycloheximide (Wako) and 50 ng/mL

antibodies against p50, p52, p65, Rel B, and

TNF-(PeproTech) for the indicated times

c-Rel (Santa Cruz Biotechnology) were

were used. Cell death was quantified in two

added to the assay mixtures.

ways. One was double-staining of antiannexin V antibodies (Apoptosis Detection

Western blotting - The procedure was

Kit I, BD Biosciences) and propidium iodide

performed as described previously (41) with

(PI). The other was quantitation of nuclear

modification. For blotting of PKA RI subunit

DNA (43), performed using FACSCalibur

dimer, cells were lysed by non-reducing

and Cell Quest (BD Biosciences). IL-33 was

RIPA buffer containing 100 mM maleimide

measured with a commercial ELISA kit (Bio-

(Sigma-Aldrich). The proteins were blotted

Techne).

by antibodies against PKA RI (Cell Signaling Technology), phospho-p38 (Cell Signaling

cAMP assay

Technology), and phospho-JNK (Cell

Intracellular cAMP levels were measured

Signaling Technology). In some experiments,

using the DetectX direct cyclic AMP enzyme

the cells were incubated with H2O2 (Wako).

immunoassay kit (Arbor Assay) according to

For blotting of phosphorylation of PKA

the manufacturer’s protocol.

substrates, cells were treated with 10%(w/v) trichloroacetic acid (Wako) in

Acknowledgements - We thank Dr. Dovie

0.15M NaCl (Wako) for 15min on ice and

R. Wylie for the critical review of this

then lysed as described previously (42).

manuscript. This work was supported in part

In some experiments, 100 nM isoprenaline

by Grants-in-Aid for Scientific Research

(Sigma-Aldrich) and/or 10 M H-89

from the Japan Society for the Promotion of

(Calbiochem) were added. The proteins were

Science.

blotted by antibodies against phospho(Ser/Thr) PKA substrate (Cell Signaling

Conflict of Interest - The authors declare no

Technology) or GAPDH (Cell Signaling

conflict of interest. 9


(EMSA) - Nuclear extraction and the EMSA


L., and Donaldson, D. D. (1998) Author Contributions - SS, MO, SO, YN,

Interleukin-13: central mediator of

HS, YM, and TY performed the experiments.

allergic asthma. Science 282, 2258-

SS and KI contributed to the experimental

2261

design. JJL and KI wrote the manuscript.

6.

Izuhara, K., Matsumoto, H., Ohta, S., Ono, J., Arima, K., and Ogawa, M.

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Nat Protoc 1, 1458-1461

band. All experiments were performed at least more than twice.

Figure Legends Figure 1 - Activation of NF-B by OSCN-,

Figure 3 - PKA causes NF-B activation by

but not by H2O2, in airway epithelial cells -

OSCN- in airway epithelial cells - (A) H292

(A) Chemical reactions of in vitro H2O2 and

cells were stimulated for 5 h with medium containing 11 mM -D-glucose, 3 mM

containing 11 mM -D-glucose, 3 mM

HSCN, 16 mU/mL GOX, and the indicated

HSCN, and GOX (closed square: 1 mU/mL,

concentration of H-89, SB203580, PD98059,

open triangle: 2 mU/mL, closed triangle: 4

or SP600126 in the presence or the absence

mU/mL, open circle: 8 mU/mL, and closed

of 10 g/mL LPO. The nuclear extracts were

circle: 16 mU/mL) was incubated without (B)

applied to the EMSA. (B, C) H292 cells were

or with (C) 10 g/mL LPO for the indicated

stimulated for 5 h with medium containing 11

times. Produced H2O2 (B) or OSCN- (C) is

mM -D-glucose, and 3 mM HSCN with the

depicted. All experiments were performed at

indicated concentrations of GOX, in the

least more than twice.

presence or the absence of 10 g/mL LPO. The cell lysates were applied to Western

Figure 2 - Activation of NF-B by OSCN-,

blotting using anti–phospho-JNK antibodies

but not by H2O2, in airway epithelial cells -

(B) or anti–phospho-p38 antibodies (C). (D,

(A) H292 cells were stimulated for 5 h with

E) The cell lysates prepared in B or C were

medium containing 11 mM -D-glucose, 3

applied to Western blotting under non-

mM HSCN, and in the presence or absence of

reducing conditions using anti-PKA

16 mU/mL GOX or 10 g/mL LPO. The

antibodies (D) or antibodies against

nuclear extracts were applied to the EMSA.

phosphorylated PKA substrates (pPKAS) or

Excess amounts of cold wild competitors

GAPDH (E). In E, 10 H-89 was added

(WT) or mutated competitors (MT) were

10 min before stimulation. As positive

added. (B) H292 cells were incubated as (A)

control, cells were stimulated with 100 nM

in the presence or absence of 16 mU/mL

isoprenaline for 5min. Asterisks indicate the

GOX or 10 g/mL LPO. The nuclear extracts

phosphorylated PKA substrates. All

were applied to the EMSA. Excess amounts

experiments were performed at least more

of cold WT, cold MT, or the antibodies, were

than twice.

added in the nuclear extracts from the cells stimulated in the presence of GOX or LPO.

Figure 4 - H2O2 is detoxified mainly by 14


OSCN production. (B, C) The medium

-


catalase in airway epithelial cells - (A) The

conditions using anti-PKA antibodies. All

medium was prepared with 11 mM -D-


glucose, 3 mM HSCN, 16 mU/mL GOX in

than twice.

the presence (left) or absence (right) of 10 g/mL LPO. The medium was incubated

Figure 5 - High concentrations of OSCN-

with H292 cells (open circle) or without

cause necrosis in human pulmonary

H292 cells (closed circle) for 5 h. Produced

epithelial cells - H292 cells were treated for 5 h with 11 mM -D-glucose, 3 mM HSCN,

The consumed amounts (closed triangle)

the indicated concentrations of GOX and 10

were calculated by subtracting the

g/mL LPO, or with 50 g/mL

concentrations in the presence of H292 cells

cycloheximide and 50 ng/mL TNF- for the

from the concentrations in the absence of

indicated times. The populations of the

H292 cells. (B) Chemical reactions of three

annexin V+PI+ fraction (necrosis) and the

H2O2 detoxification systems. (C) H292 cells

annexin V+PI- fraction (apoptosis) are shown

were incubated for 5 h with 100 mM

in (A) and the populations of the low-DNA–

aminotriazol (closed triangle) or 1 mM

content fraction (apoptosis) in (B). All

BCNU (open triangle) or 1 M auranofin


(closed square) or no inhibitor (open circle)

than twice.

using the medium prepared in A. The medium alone, without the cells, is depicted

Figure 6 - Schematic model of the OSCN-

as a closed circle. The residual H2O2 ratios

production and its activation of NF-B via

compared to the initial amounts at the

PKA in airway epithelial cells - SCN- is

indicated times are depicted. (D) H292 cells

actively transported into pulmonary lumens

were stimulated for 5 h with medium

via NIS/SLC5A5 at the basal side and via

containing 11 mM -D-glucose, and 3 mM

several anion transporters including CFTR

HSCN with the indicated concentrations of

and pendrin/SLC26A4 at the apical side in

GOX in the presence or the absence of 10

airway epithelial cells. SCN- together with

g/mL LPO and/or 100 mM aminotriazol.

H2O2 generated by Duox1 and Duox2 is

(E) H292 cells were stimulated with medium

catalyzed by peroxidases into OSCN-. Three

containing the indicated concentrations of

peroxidases including MPO, EPX, and LPO

H2O2 alone or 11 mM -D-glucose, 3 mM

are involved in this reaction. The produced

HSCN, and 10 g/mL LPO with the

OSCN- activates NF-B via PKA or causes

indicated concentrations of GOX for 5 h. In

necrosis in airway epithelial cells. All

D and E, The cell lysates were applied to


Western blotting under non-reducing

than twice.

15


OSCN (left) and H2O2 (right) is depicted.

-

A β-D-glucose + O2

GOX

SCN + H2O2

H2O2 + D-glucono-1,5-lactone

LPO

OSCN- + H2O

-

B 1,500

H2O2 (µM)

1,200 900 600 300 0 0 0 1

3

6

9

Time (h)

C

OSCN- (µM)

500 400 300 200 100 0

0 1

3

6

9

Time (h) Suzuki et al. Figure 1

A GOX

-

-

+

+

LPO

-

+

+

-

Competitor

- WT MT -

WT MT - WT MT

-

WT MT

NF-κB

B GOX

-

+

+

+

+

+

+

+

+

LPO

-

+

+

+

+

+

+

+

+

Competitor

-

-

-

-

-

-

-

Antibody

-

-

WT MT -

-

p50 p52 p65 RelB c-Rel

Supershift NF-κB

Suzuki et al. Figure 2

LPO

PD98059 (µ M)

SB203580 (µ M)

H-89 (µ M)

GOX (mU/mL)

SP600125 (µ M)

-

-

5

10

20

-

-

10

20

40

-

5

10

20

40

-

5

10

20

40

-

+

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

+

+

0

LPO

4

8

16

0

4

8

+

16

32

64

-

IL-1/3T3

B

A

75 kDa

NF-κB

50 kDa

p-JNK

37 kDa

D

C GOX (mU/mL)

0

4

LPO

8

16

0

4

8

+

16

32

GOX (mU/mL) 0

64

-

LPO

100 kDa

4

8 +

16

0

4

8

16

32

-

PKA RI dimer

75 kDa 50 kDa p-p38

37 kDa

E

GOX

-

-

+

+

+

-

-

-

LPO

-

+

-

+

+

-

-

-

H-89 Isoprenaline

-

-

-

-

+

-

+

+

-

-

-

-

-

+

+

-

PKA RI monomer


25 kDa

50 kDa

150 kDa 100 kDa 75 kDa

* *

pPKAS 50 kDa

25 kDa

GAPDH Suzuki et al. Figure 3

B

A 600

600

500

500

(i)

Catalase 2H2O2

Glutathione peroxidase

400

400 H2O2 (µM)

OSCN- (µM)

(ii)

300 200

0

0

50

100

125

2GSSG + NADPH + H+ (iii)

0

50

100

Peroxyredoxin (reduced) + Thioredoxin (oxidized) Thioredoxin (reduced) + NADP+

Time (min)

GOX (mU/mL) LPO Aminotriazol

120 100

0

8

16 32 -

0

8

16 32 +

4

8 + -

16

80 Downloaded from http://www.jbc.org/ by guest on November 28, 2016

Residual H2O2 (%)

Peroxyredoxin (oxidized) + 2H2O

Thioredoxin (oxidized) + NADPH + H+

125

D

60 40 20 0

0

30

90

60

120

Time (min)

E 10

1

LPO

-

GOX (mU/mL)

-

100 kDa

Peroxyredoxin (reduced) + H2O2

Thioredoxin reductase

C

0

2GSH + NADP+

Peroxyredoxin (oxidized) + Thioredoxin (reduced)

Time (min)

H2O2 (mM)

GSSG + 2H2O

Glutathione reductase

200 100

0

2GSH + H2O2

300

100

2H2O + O2

100

100 kDa 75 kDa 50 kDa

PKA RI dimer PKA RI monomer

+ 0

8

16

PKA RI dimer

75 kDa 50 kDa

PKA RI monomer


Annexin V

A

Propidium Iodide 0

GOX (mU/mL) + LPO

3.7

16 16.8

8.2

0

Time (h) + Cycloheximide /TNF-α

7.1

32 21.1

0.1

2 11.2

15.8

64 93.6

4 13.5

33.1

17.9

Count

16

32

+ LPO

2.3%

1.3%

0

Time (h)

1.3%

2

40.3


DNA content 0

95.3

6

B

GOX (mU/mL)

0.6

14.3

64

1.1%

4

6

+ Cycloheximide /TNF-α 7.0%

19.4%

47.5%

57.9%


EPX LPO SCN-

+

H 2 O2

OSCN-

H 2 O2

SCN-

Pendrin

DUOX1

CFTR Cl-

+

H 2O

DUOX2 PKA

Airway epithelial cell


MPO

Necrosis

NF-κB NIS SCN-

Na+

Izuhara et al. Figure 6

Induction of airway allergic inflammation by hypothiocyanite via epithelial cells Shoichi Suzuki, Masahiro Ogawa, Shoichiro Ohta, Satoshi Nunomura, Yasuhiro Nanri, Hiroshi Shiraishi, Yasutaka Mitamura, Tomohito Yoshihara, James J. Lee and Kenji Izuhara J. Biol. Chem. published online November 18, 2016

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