ORL 2019;81:101–110

DOI: 10.1159/000496227 © 2019 S. Karger AG, Basel

Received: October 1, 2018 www.karger.com/orl
Accepted: December 11, 2018
Published online: April 29, 2019

Research Article

Expression of Endoplasmic
Reticulum Stress-Related mRNAs
in Chronic Otitis Media
Sung Hwa Dong a Young Il Kim b Myung Gu Kim c Youn-Jung Kim d
       

Jae Yong Byun a Moon Suh Park a Sang Hoon Kim a Seung Geun Yeo a, b

       

a
  Department of Otorhinolaryngology – Head and Neck Surgery, School of Medicine,
Kyung Hee University, Seoul, South Korea; b Medical Science Research Institute, Kyung
 

Hee University Medical Center, Seoul, South Korea; c Department of Otorhinolaryngology,

 

Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwon,

South Korea; d Department of Basic Nursing Science, College of Nursing Science, Kyung Hee
 

University, Seoul, South Korea

Keywords
Endoplasmic reticulum stress · Unfolded protein response · Chronic otitis media ·
Cholesteatoma

Abstract
Objectives: Unfolded proteins in the endoplasmic reticulum (ER) cause an ER stress response
and can result in various pathologic conditions, including inflammation. Otitis media is the
most common disease in otolaryngology and is associated with inflammation. The pathophys-
iology of chronic otitis media is not well understood; we therefore investigated the expression
pattern of ER stress-related mRNAs in chronic otitis media. Methods: Specimens were ob-
tained from 47 patients with chronic otitis media over a period of 2 years. Expression levels of
6 ER stress transcription factors were quantitatively assessed using real-time RT-PCR. Results:
The mRNA expression of sXBP1 was significantly higher in the otorrhea present group than in
the otorrhea absent group (p < 0.05). ATF6 expression was significantly higher in the ossicle
destruction group than in the ossicle intact group (p < 0.05). mRNA expression of the 6 ER
stress-related genes did not differ significantly between those patients with positive micro-
bial cultures versus those with negative cultures (p > 0.05) or those with facial nerve dehis-
cence versus those without facial nerve dehiscence (p > 0.05). Conclusions: sXBP1 appears to
be involved in chronic otitis media-associated inflammation, including otorrhea. ATF6 is asso-
ciated with the destruction of ossicles. Our results suggest that certain ER stress-related genes
are expressed in chronic otitis media-associated inflammation. © 2019 S. Karger AG, Basel

Seung Geun Yeo, MD, PhD

Department of Otorhinolaryngology – Head and Neck Surgery
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School of Medicine, Kyung Hee University, 23, Kyungheedae-ro

Auckland University of Technology

Dongdaemun-gu, Seoul (South Korea)

E-Mail yeo2park @ gmail.com
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Dong et al.: ER Stress in Chronic Otitis Media

Introduction

Otitis media is a common term for inflammatory changes that occur in the middle ear.
It is one of the most common diseases in otorhinolaryngology and can be classified according
to how rapid the onset of disease is, clinical features, cause of the disease, and the under-
lying pathology. Based on how rapid the onset of symptoms is, otitis media can be classified
as acute otitis media (onset within 3 weeks), subacute otitis media (onset between 3 weeks
and 3 months), and chronic otitis media (COM) (onset after 3 months). Otitis media is
caused by a combination of factors, but the most important factors are dysfunction of the
eustachian tube and infection by microorganisms [1]. After inflammatory lesions have
developed in the middle ear cavity, immune responses occur and various inflammatory
mediators are released [2]. These actions are believed to play a role in the recurrence and
chronicity of otitis media. Recurrent otitis media or serous otitis media with persistent
inflammation of the middle ear becomes COM. Some research has been performed on the
pathophysiology of COM. However, the exact pathophysiology of COM is still under inves-
tigation.
Inflammation associated with otitis media is caused mainly by bacteria. Bacterial entry
into eukaryotic cells generally results in bacteria residing within phagosomes, which leads to
antigen presentation and development of adaptive immunity [3]. Bacteria can also modulate
phagosomes to interact with the endoplasmic reticulum (ER). ER stress is likely to be asso-
ciated with inflammation. Unfolded proteins expose hydrophobic amino acid residues usually
located inside the protein, resulting in the formation of protein aggregates. To alleviate this
stress, eukaryotic cells activate self-defense mechanisms such as the ER stress or unfolded
protein response [4–11]. Protein aggregates are highly cytotoxic and induce apoptotic cell
death, and ER stress is associated with diseases such as Crohn’s disease and type 2 diabetes
[12–14].
In this study, we investigated the expression patterns of ER stress-related genes in
patients with COM.

Subjects and Methods

Subjects
From August 2015 to September 2017, 47 patients who visited the Otorhinolaryngology Department of
a single tertiary medical center and underwent surgery for COM were enrolled. Physical examination, pure
tone audiometry, impedance audiometry, and preoperative temporal bone computed tomography were
performed on admission to the outpatient clinic. Specimens were used only after patients provided informed
consent to participate in this study. Granulation tissue from 47 patients with COM was sampled. The collected
tissues were immediately frozen in liquid nitrogen, placed in sterilized tubes, and stored at –70 ° C until
    

experiments. This study was approved by the Kyung Hee University Clinical Research Ethics Committee
(KMC IRB 2017-12-030).

Clinical Data
Clinical records were retrospectively reviewed for age, sex, date of onset, direction, microbial culture
results, and revision surgery data. Data on the first operation of cases that underwent revision surgery were
excluded from the analysis because these medical records were not retrieved. Surgical records were reviewed
to determine the extent of ossicular destruction and the presence of facial nerve dehiscence.

Pure Tone Audiometry

Pure tone audiometry was performed by measuring air conduction and bone conduction hearing at the
time of diagnosis. Hearing threshold was calculated using a quadratic method with a weighting of two times
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at 1,000 Hz using average frequencies of 500, 1,000, and 2,000 Hz. Sensorineural hearing loss was defined as
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Table 1. Primer sequences used

in real-time PCR Name Sequence Size of
target,
bp

β-Actin 5′-GCGAGAAGATGACCCAGATC-3′ 77
5′-GGATAGCACAGCCTGGATAG-3′
IRE1α 5’-GCGAACAGAATACACCATCAC-3’ 147
5’-ACCAGCCCATCACCATTG-3’
sXBP1 5’-TGGATTCTGGCGGTATTGAC-3’ 146
5’-TCCTTCTGGGTAGACCTCTG-3’
PERK 5’-GAACCAGACGATGAGACAGAG-3’ 150
5’-GGATGACACCAAGGAACCG-3’
CHOP 5’-GTACCTATGTTTCACCTCCTGG-3’ 150
5’-TGGAATCTGGAGAGTGAGGG-3’
ATF6 5’-CCTGTCCTACAAAGTACCATGAG-3’ 148
5’-CCTTTAATCTCGCCTCTAACCC-3’
BiP 5′-CCTGGGTGGCGGAACCTTCGATGTG-3′ 358
5′-CTGGACGGGCTTCATAGTAGACCGG-3′

IRE1α, inositol-requiring enzyme 1α; sXBP1, X-box-binding protein

1; PERK, endoplasmic reticulum kinase; CHOP, C/EBP-homologous
protein; ATF6, activating transcription factor 6; BiP, immunoglobulin
heavy chain-binding protein.

a bone conduction threshold greater than 30 dB, while conductive hearing loss was defined as an air-bone
gap greater than 10 dB and a bone conduction threshold less than 25 dB. Patients with mixed hearing loss
(bone conduction ≥25 dB with an air-bone gap ≥10 dB) were included in the sensorineural hearing loss
group [15].

Microbial Culture
Bacterial cultures were identified by Gram stain, catalase test, and oxidase test. Colonies were cultured
at 37 ° C in a 5% CO2 atmosphere for 3 days. Strains were separated by a microorganism automation analyzer
    

Vitek 1 system (BioMerieux, France) GPI card, and GNI cards were used for final identification. Character-
ization of colonies, Gram staining, and catalase and oxidase tests were performed. When fungi were grown
on blood agar medium, final identification was performed with Sabouraud medium.

RNA Extraction and Real-Time RT-PCR

Total RNA was purified from homogenized tissue samples using TRIzol reagent according to the manu-
facturer’s protocol (Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis was performed with 1 μg of
total RNA, which was transcribed to cDNA using a reverse transcription system with random hexamers
(Promega, Madison, WI, USA) according to the manufacturer’s protocol. Primer sequences are shown in
Table 1. Real-time RT-PCR was performed on a StepOnePlus real-time PCR system with Power SYBR Green
PCR Master Mix (Applied Biosystems, Foster City, CA, USA). PCR was performed with 1 µL of cDNA in a 20-µL
reaction mixture containing 10 µL of Power SYBR Green PCR Master Mix, 2 µL of primers, and 7 µL of PCR-
grade water. Reaction conditions were denaturation at 95 ° C for 10 min followed by 40 cycles of 95 ° C for 15
         

s and 60 ° C for 1 min. The crossing points of the target genes with β-actin were calculated using the formula
    

2–(target gene – β-actin), and relative amounts were quantified.

Statistics
To determine whether the differences between groups were statistically significant, Student’s t test
was used. The Mann-Whitney U test was used when normality was not observed. IBM SPSS version 20 (IBM
Corp., Armonk, NY, USA) was used for statistical analyses, and p values less than 0.05 were considered
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significant.
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Table 2. Demographic and

clinical characteristics of patients
with chronic otitis media
Clinical characteristics
Age (mean ± SD), years 52.85±14.71
Sex (male:female), n (%) 20:27 (42.6:57.4)
Disease onset (mean ± SD), months 9.31±10.10
Affected side (Rt:Lt:Both), n (%) 24:23:0 (51.4:48.9:0)
Culture-positive, n (%) 22 (46.8)
Otorrhea-positive, n (%) 35 (74.5)
Revision surgery, n (%) 7 (14.9)
Ossicle destruction, n (%) 25 (53.1)
Facial canal dehiscence, n (%) 26 (55.3)
Audiologic configuration
PTA(AC) (mean ± SD), dB 52.63±20.91
PTA(BC) (mean ± SD), dB 26.05±16.40
Hearing loss type
Conductive hearing loss, n (%) 19 (40.1)
Sensorineural hearing loss, n (%) 13 (27.6)
Normal, n (%) 15 (31.9)
Serviceable hearing, n (%) 32 (68.0)

PTA, pure tone audiometry; AC, air conduction; BC, bone conduction.
* p < 0.05.

Table 3. Microbiology results

No growth 25 (53.2%)
Growth 22 (46.8%)
Pseudomonas aeruginosa 7
MRSA 6
Staphylococcus aureus 3
Coagulase-negative staphylococci 3
Candida 2
Achromobacter xylosoxidans 1

MRSA, methicillin-resistant Staphylococcus aureus. * p < 0.05.

Results

Clinical Data
Among patients with COM, 20 were male and 27 were female, and the mean age was
52.9 ± 14.7 years (Table 2). There were a similar number of cases located on the right and
left sides.
There were 22 positive bacterial culture results (46.8%), and otorrhea occurred in 35
(74.5%) patients. Seven (14.9%) patients were revision cases who had undergone a previous
COM operation. Ossicular destruction and facial canal dehiscence were observed in 25
(53.1%) and 26 (55.3%) patients, respectively.
Air conduction threshold was 52.6 ± 20.9 dB HL and bone conduction threshold was
26.1 ± 16.4 dB HL. Hearing loss type was divided into conductive hearing loss and senso-
rineural hearing loss. A total of 19 patients (40.1%) had conductive hearing loss, 13
patients (27.6%) had sensorineural hearing loss, and 15 (31.9%) patients had normal
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hearing.
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Table 4. Expression of mRNA

Gene Culture-negative Culture-positive p value
encoding ER stress-related genes
(n = 25) (n = 22)
according to a positive culture
result
IRE1α 0.006±0.007 0.044±0.009 0.628
sXBP1 0.204±0.194 0.431±0.443 0.221
PERK 0.015±0.021 0.008±0.014 0.505
CHOP 0.059±0.085 0.076±0.116 0.863
ATF6 0.066±0.069 0.026±0.031 0.300
BiP 0.074±0.090 0.021±0.030 0.076

ER, endoplasmic reticulum; IRE1α, inositol-requiring enzyme 1α;

sXBP1, X-box-binding protein 1; PERK, endoplasmic reticulum kinase;
CHOP, C/EBP-homologous protein; ATF6, activating transcription
factor 6; BiP, immunoglobulin heavy chain-binding protein.

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0
Fig. 1. Expression of mRNAs en- IRE1 sXBP1 PERK CHOP ATF6 Bip
coding ER stress-related genes in
all COM patients.

Microorganism Culture
A total of 22 (46.8%) patients had positive microorganism cultures (Table 3). The most
frequently detected species was Pseudomonas aeruginosa. Other detected microbes included
methicillin-resistant Staphylococcus aureus, coagulase-negative staphylococci, Candida, and
Achromobacter xylosoxidans.

Expression of mRNAs Encoding ER Stress-Related Genes

Although expression of mRNAs involved in ER stress was observed in all samples, the
expression level of sXBP1 was higher than that of other mRNAs, albeit not at a significant level
(Fig. 1, p > 0.05).

Expression of mRNAs Encoding ER Stress-Related Genes in Patients with Positive

Microbial Culture Results
Transcript expression did not differ significantly between culture-positive and culture-
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negative groups (Table 4, p > 0.05).

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Table 5. Expression of mRNA

Gene Otorrhea absent Otorrhea present p value
encoding ER stress-related genes
(n = 12) (n = 35)
in the presence or absence of
otorrhea
IRE1α 0.006±0.006 0.005±0.008 0.447
sXBP1 0.118±0.107 0.395±0.391 0.037*
PERK 0.022±0.025 0.008±0.014 0.304
CHOP 0.038±0.040 0.078±0.113 0.600
ATF6 0.055±0.073 0.041±0.048 0.849
BiP 0.059±0.067 0.041±.070 0.461

ER, endoplasmic reticulum; IRE1α, inositol-requiring enzyme 1α;

sXBP1, X-box-binding protein 1; PERK, endoplasmic reticulum kinase;
CHOP, C/EBP-homologous protein; ATF6, activating transcription
factor 6; BiP, immunoglobulin heavy chain-binding protein. * p < 0.05.

Table 6. Expression of mRNA

encoding ER stress-related genes Gene Ossicles intact Ossicles destructed p value
in patients with ossicle (n = 22) (n = 25)
destruction and those without
IRE1α 0.008±0.009 0.0009±0.0009 0.127
sXBP1 0.312±0.332 0.327±0.389 0.537
PERK 0.017±0.021 0.003±0.004 0.097
CHOP 0.061±0.084 0.074±0.117 0.686
ATF6 0.060±0.057 0.028±0.050 0.049*
BiP 0.054±0.082 0.032±0.040 0.698

ER, endoplasmic reticulum; IRE1α, inositol-requiring enzyme 1α;

sXBP1, X-box-binding protein 1; PERK, endoplasmic reticulum kinase;
CHOP, C/EBP-homologous protein; ATF6, activating transcription
factor 6; BiP, immunoglobulin heavy chain-binding protein. * p < 0.05.

Expression of mRNAs Encoding ER Stress-Related Genes in the Presence of Otorrhea

Transcript expression of sXBP1 was significantly higher in the otorrhea present group than
the otorrhea absent group (Table 5, p < 0.05). Expression of the other 5 mRNAs was not signif-
icantly different between the otorrhea present group and otorrhea absent group (p > 0.05).

Expression of mRNAs Encoding ER Stress-Related Genes in Patients with Ossicle

Destruction
Expression of ATF6 was significantly higher in the ossicle destruction group than the
ossicle intact group (Table 6, p < 0.05). Expression of other mRNAs was not significantly
different between the ossicle destruction group and ossicle intact group (p > 0.05).

Expression of mRNAs Encoding ER Stress-Related Genes in Patients with Facial Nerve

Dehiscence
mRNA expression did not differ significantly with regard to the presence or absence of
facial nerve dehiscence (Table 7, p > 0.05).
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Table 7. Expression of mRNA

Gene Facial canal intact Facial canal dehiscent p value
encoding ER stress-related genes
(n = 21) (n = 26)
in patients with facial nerve
dehiscence versus those without
IRE1α 0.007±0.009 0.003±0.004 0.821
sXBP1 0.308±0.364 0.332±0.364 0.964
PERK 0.016±0.021 0.006±0.010 0.812
CHOP 0.075±0.128 0.061±0.066 0.297
ATF6 0.057±0.059 0.033±0.050 0.389
BiP 0.061±0.089 0.030±0.036 0.448

ER, endoplasmic reticulum; IRE1α, inositol-requiring enzyme 1α;

sXBP1, X-box-binding protein 1; PERK, endoplasmic reticulum kinase;
CHOP, C/EBP-homologous protein; ATF6, activating transcription
factor 6; BiP, immunoglobulin heavy chain-binding protein.

Table 8. Expression of mRNA

encoding ER stress-related genes Gene CHL SNHL Normal p value
in patients with conductive (n = 19) (n = 13) (n = 15)
hearing loss, sensorineural
IRE1α 0.006±0.009 0.001±0.001 0.005±0.005 0.442
hearing loss, or normal hearing
sXBP1 0.306±0.367 0.351±0.369 0.323±0.397 0.993
PERK 0.015±0.021 0.002±0.002 0.014±0.013 0.336
CHOP 0.054±0.081 0.084±0.134 0.110±0.124 0.830
ATF6 0.047±0.061 0.038±0.050 0.052±0.048 0.757
BiP 0.035±0.051 0.062±0.110 0.062±0.032 0.344

ER, endoplasmic reticulum; CHL, conductive hearing loss; SNHL,

sensorineural hearing loss; IRE1α, inositol-requiring enzyme 1α; sXBP1,
X-box-binding protein 1; PERK, endoplasmic reticulum kinase; CHOP,
C/EBP-homologous protein; ATF6, activating transcription factor 6;
BiP, immunoglobulin heavy chain-binding protein.

Expression of mRNAs Encoding ER Stress-Related Genes in Patients with Conductive

Hearing Loss, Sensorineural Hearing Loss, or Normal Hearing
mRNA expression did not differ significantly between patients with conductive hearing
loss, sensorineural hearing loss, or normal hearing (Table 8, p > 0.05).

Expression of mRNAs Encoding ER Stress-Related Genes in Patients with or without

Serviceable Hearing Loss
mRNA expression did not differ significantly between those patients that had serviceable
hearing and those that did not (Table 9, p > 0.05).

Discussion

Various factors affect ER stress, but three different pathways have been identified as ER
stress sensors [9]. ER stress sensors such as activating transcription factor 6 (ATF6), inositol-
requiring protein 1α (IRE1α), and protein kinase RNA-like ER kinase (PERK) are involved in
signal transduction [8, 16]. Spliced XBP1 (XBP1s) is a signaling substance of IRE1, C/EBP-
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homologous protein (CHOP) is the final signaling molecule in the PERK signaling cascade, and
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Table 9. Expression of mRNA

Gene CHL SNHL Normal p value
encoding ER stress-related genes
(n = 19) (n = 13) (n = 15)
in patients with conductive
hearing loss, sensorineural
IRE1α 0.006±0.009 0.001±0.001 0.005±0.005 0.442
hearing loss, or normal hearing sXBP1 0.306±0.367 0.351±0.369 0.323±0.397 0.993
PERK 0.015±0.021 0.002±0.002 0.014±0.013 0.336
CHOP 0.054±0.081 0.084±0.134 0.110±0.124 0.830
ATF6 0.047±0.061 0.038±0.050 0.052±0.048 0.757
BiP 0.035±0.051 0.062±0.110 0.062±0.032 0.344

ER, endoplasmic reticulum; CHL, conductive hearing loss; SNHL,

sensorineural hearing loss; IRE1α, inositol-requiring enzyme 1α;
sXBP1, X-box-binding protein 1; PERK, endoplasmic reticulum kinase;
CHOP, C/EBP-homologous protein; ATF6, activating transcription
factor 6; BiP, immunoglobulin heavy chain-binding protein.

BiP is a chaperone involved in overall ER stress. For these reasons, we chose to examine the
expression of genes encoding 6 proteins in this study (ATF6, IRE1, XBP1s, CHOP, PERK, and
Bip). In cells with ER stress, ATF6 interacts with the coat protein II (COPII) complex and is
transferred to the Golgi. Activated IRE1α processes mRNA encoding the transcription factor
X box-binding protein 1 (XBP1) and cleaves a 26-neuron intron to move the coding reading
frame of this mRNA. XBP1 encodes an active and stable transcription factor called spliced
XBP1 (XBP1s) that translocates to the nucleus to induce upregulation of the target gene. Acti-
vation of PERK inhibits general protein translation through phosphorylation of eukaryotic
translation initiator factor 2α (eIF2α). The ER chaperone BiP (also known as GRP78 and
HSPA5) inhibits spontaneous biogenesis and activation by binding to IRE1α and PERK in
mammalian cells [10, 11, 17–20].
Bacterial culture is necessary to select appropriate antibiotics in cases of COM. The most
common aerobic bacteria in COM are P. aeruginosa, S. aureus, and Klebsiella pneumomiae. The
most common anaerobic bacteria are Bacteroides and Peptococcus. Combined infection with
aerobic bacteria and anaerobic bacteria is more common than single infection by aerobic
bacteria or anaerobic bacteria. In this study, P. aeruginosa and MRSA were the most frequently
identified bacteria in cultures of patients with COM. These two organisms are the most
frequently isolated bacteria from otorrhea of patients with COM [21]. In addition, P. aeru-
ginosa and MRSA are well known to form biofilms [22, 23]. The pathophysiology of COM is
thought to involve various factors, including the formation of biofilms. Biofilm formation
induces inflammation because polymicrobial colonies secrete substances such as exopolysac-
charide matrix to protect against host attack [22].
In this study, the expression of ER stress-related mRNAs in all samples indicated the
involvement of the ER stress response in the pathogenesis of COM. Among the ER stress-
related factors that we examined, sXBP1 expression was the highest and IRE1 expression was
the lowest, suggesting that ER-associated degradation is more active than translational atten-
uation and transcriptional induction in signaling pathways of the unfolded protein response
in COM. In addition, expression of sXBP1, CHOP, and BiP mRNA, which are induced by tran-
scription, was higher than that of PERK, IRE1α, and ATF6 mRNA during the early stage of ER
stress, suggesting that ER stress is already active in COM.
Expression of CHOP and sXBP1 mRNA was higher in the culture-positive group and
otorrhea-positive group, and sXBP1 levels were significantly higher in the otorrhea-
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positive group than the otorrhea-negative group. XBP1s binds to the ESRE (ER stress
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responsive element) present in the vicinity of the promoter region of genes and mainly
regulates protein folding and ERAD-related gene transcription. sXBP1 has directly been
implicated in chronic diseases such as intestinal inflammation and osteoarthritis [13, 22].
The results of this study suggest that sXBP1 mRNA expression is closely related to active
inflammation in COM.
ATF6 mRNA level was significantly higher in the ossicle destruction group than the
ossicle intact group. ATF6, like XBP1s, increases transcription of genes encoding chaperone
molecules and ERAD, and is also known to be associated with apoptosis of cartilage in osteo-
arthritis [22]. Osteoclasts play an important role in bone destruction, and the relationship
between osteoclasts and ER stress has been studied. When osteoclasts are activated on the
bone surface, bone resorption results and is characteristic of chronic inflammatory bone
diseases such as periodontitis and rheumatoid arthritis [23, 24]. ATF6 also mediates proin-
flammatory synergy between ER stress and TLR activation in the pathogenesis of liver
ischemia-reperfusion injury [25]. ATF6 is associated with apoptosis or ischemia in other
diseases and we therefore hypothesized that it would play a role in ossicle destruction.
Consistent with this hypothesis, recent studies have shown that ER stress plays an important
role in the differentiation of monocytes into osteoclast precursors [23].
There was no statistically significant difference in ER stress gene expression in those
patients with facial canal dehiscence and those without. Unlike ossicle destruction, facial
canal dehiscence can be congenital or related to inflammation. The fact that we did not distin-
guish between congenital or acquired facial canal dehiscence in this study may have
contributed to the lack of difference in gene expression between these two groups.
In patients with COM, hearing loss is an important determinant of quality of life. For this
reason, we compared mRNA expression levels of various ER stress-related genes among
groups with different causes of hearing changes. Although ATF6 expression was associated
with ossicle destruction, there was no significant difference in the expression of mRNA related
to the ER stress response according to type of hearing loss or hearing threshold.
This study had some limitations. First, levels of only 6 ER stress-related mRNAs were
assessed. Second, we did not confirm that elevated mRNA levels result in elevated protein
levels and vice versa, and it is therefore possible that protein expression levels may not have
reflected mRNA expression levels. Therefore, additional immunohistochemistry or Western
blotting results and comparative analysis are needed. Third, because of the small number of
samples examined and limited clinical information, a more diverse clinical analysis should be
performed to obtain robust results. Fourth, because of ethical issues, we did not sample
normal tissue from healthy individuals and so this study lacked a normal control group
without COM.

Conclusions

The ER stress-related gene XBP1 is likely to be involved in COM-associated inflammation.

ATF6 is associated with destruction of ossicles. Different ER stress mRNAs were involved in
the pathophysiology of COM and specific clinical features of this disease, such as otorrhea and
ossicle destruction.

Acknowledgements

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean
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government (MSIT, No. 2017R1C1B5076098; NRF-2018R1A6A1A03025124).

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Statement of Ethics

This study protocol was approved by the Institutional Review Board of Kyung Hee Medical Center
(2017-12-030). Written informed consent was obtained before study commencement after providing
patients with an explanation of the study purposes and how their samples would be used.

Disclosure Statement

The authors have no conflicts of interest or funding information to declare.

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