ORIGINAL PAPER

ERp29 deficiency affects sensitivity to apoptosis via impairmentof the ATF6–CHOP pathway of stress response

Irina Hirsch • Matthias Weiwad • Erik Prell •

David Michael Ferrari

Published online: 27 December 2013

� Springer Science+Business Media New York 2013

Abstract Endoplasmic reticulum protein 29 (ERp29)

belongs to the redox-inactive PDI-Db-subfamily of PDI-

proteins. ERp29 is expressed in all mammalian tissues

examined. Especially high levels of expression were

observed in secretory tissues and in some tumors. How-

ever, the biological role of ERp29 remains unclear. In the

present study we show, by using thyrocytes and primary

dermal fibroblasts from adult ERp29-/- mice, that ERp29

deficiency affects the activation of the ATF6–CHOP-

branch of unfolded protein response (UPR) without influ-

encing the function of other UPR branches, like the ATF4-

eIF2a-XBP1 signaling pathway. As a result of impaired

ATF6 activation, dermal fibroblasts and adult thyrocytes

from ERp29-/- mice display significantly lower apoptosis

sensitivities when treated with tunicamycin and hydrogen

peroxide. However, in contrast to previous reports, we

could demonstrate that ERp29 deficiency does not alter

thyroglobulin expression levels. Therefore, our study sug-

gests that ERp29 acts as an escort factor for ATF6 and

promotes its transport from ER to Golgi apparatus under

ER stress conditions.

Keywords Endoplasmic reticulum protein 29 (ERp29) �Unfolded protein response (UPR) � ATF6 � Thyroglobulin �Apoptosis

Abbreviations

ERp29 Endoplasmic reticulum protein 29

PDI Protein disulfide isomerase

UPR Unfolded protein response

ER Endoplasmic reticulum

ERAD ER associated degradation

ROS Reactive oxygen species

SD Standard deviation

Tg Thyroglobulin

Tu Tunicamycin

Introduction

The endoplasmic reticulum protein 29 (ERp29) is an ER

resident protein with a molecular weight of 24.5 kDa [1].

ERp29 consists of a thioredoxin-like D-domain and an a-

helical B-domain, which are connected by a flexible loop

that contains four conserved glycine residues, predicting

the high domain mobility [2]. Moreover, the protein con-

tains the C-terminal ER-retrieval signal KEEL suggesting

localization in the lumenal compartment. ERp29 belongs to

the family of PDI proteins, because both domains exhibit

up to 30 % sequence similarity to the members of this

family. However, ERp29 lacks a thioredoxin-box motive

(CGHC) and therefore does not exhibits redox-activity [1].

ERp29 was found to exist as homodimer in the ER [1, 2].

The protein is expressed in all tissues of Mammalia.

Enhanced levels of its expression were detected in secre-

tory tissues (pituitary, adrenal, mammary, thyroid, salivary

glands etc.) and in some tumors [3–6]. Therefore, some

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10495-013-0961-0) contains supplementarymaterial, which is available to authorized users.

I. Hirsch (&) � M. Weiwad � E. Prell � D. M. Ferrari

Max Planck Research Unit for Enzymology of Protein Folding,

Weinbergweg 22, 06120 Halle/Saale, Germany

e-mail: irina-hirsch@gmx.de

I. Hirsch

Institut 20, 06193 Wettin-Lobejun, Germany

123

Apoptosis (2014) 19:801–815

DOI 10.1007/s10495-013-0961-0

authors suggested the active involvement of ERp29 in the

malignant conversion of mammary epithelial cells [4, 5].

However the biological role of ERp29 remains unclear.

Some authors propose that ERp29 acts as secretion factor

and escort protein [7–9]. ERp29 probably stabilizes the

monomeric connexin 43, and promotes their transport from

ER to Golgi apparatus [9]. Thus, some authors suggest the

involvement of ERp29 in ER quality control of the secre-

tory protein thyroglobulin, which serves as precursor pro-

tein for T3 and T4 hormones [7, 8, 10, 11]. The ER quality

control machinery encompasses three groups of proteins:

folding helper enzymes, molecular chaperones, and lectins

(calnexin, calreticulin). The most significant members of

folding helper enzymes are peptidyl-prolyl cis–trans

isomerases and protein disulfide isomerases (PDI).

The molecular chaperones facilitate the protein folding

through shielding of unfolded regions, and preventing the

protein aggregation through interaction with hydrophobic

regions. ER molecular chaperones comprise HSP70 (BiP,

Lhs1p, GRp170) and HSP90 chaperones (GRp94/Endo-

plasmin) [12]. BiP participates in the translocation of

nascent polypeptides into the ER. GRp94 recognizes a

multitude of peptides, and facilitates the presentation of

immunogenic peptides. PDIs exhibit the characteristics of

folding helper enzymes as well as molecular chaperones.

PDIs oxidize Cys-residues in nascent proteins resulting

in the formation of intra- und intermolecular disulfide-

bonds [13]. The calnexin/calreticulin-cycle controls the

conformation of glycoproteins, and determines the pro-

tein transport to Golgi or ER associated degradation

(ERAD).

Many types of cellular stress, such as radioactivity [14],

toxicity, oxidative stress [15–17], provoke disturbance

between ER protein loading and ER folding capacity and

thus result in the accumulation of unfolded proteins. ER

stress triggers the activation of the evolutionary conserved

signal transduction pathway known as unfolded protein

response (UPR) [12, 18]. The activation of the UPR-cas-

cade involves three transmembrane proteins: IRE1, PERK,

ATF6. In their inactive state the lumenal domains of

transmembrane proteins type I (IRE1, PERK) are associ-

ated with BiP/GRP78. During ER stress BiP is released

from the lumenal domains of IRE1 and PERK and binds

preferentially unfolded proteins. Thereby, IRE1 and PERK

oligomerize, and activate their proximal signal transducers

[19]. Oligomerization of IRE1 results in a trans-phos-

phorylation leading to processing of unspliced XBP1

[XBP1(u)] [20]. Spliced XBP1 [XBP1(s)] encodes for the

transcription factor XBP1 that regulates transcription of

genes involved in the ER quality control, ER/Golgi-bio-

genesis, and redox homeostasis during oxidative stress [21,

22]. The activation of PERK results in an increased affinity

to eIF2a. PERK mediated phosphorylation of eIF2a

(Ser51) triggers the switch from normal translation to ER-

stress, and moreover, to cell cycle arrest in G1-phase [23].

Both isoforms of ATF6, ATF6a and ATF6b, were found

to exist as transmembrane proteins type II. In contrast to

PERK or IRE1, BiP binds to ATF6 and masks two Golgi-

localization sequences (GLS1 and GLS2) of the tran-

scription factor. In the absence of BiP during ER stress

GLS2 is the dominant sequence that leads to translocation

of the precursor protein p90ATF6 from ER to Golgi.

Translocated ATF6 is cleaved sequentially by site 1 pro-

tease (S1P) and site 2 protease (S2P) leading to the release

of the transcription factor domain from the Golgi mem-

brane. The N-terminal cytosolic ATF6-fragment

(p50ATF6) regulates the activity of transcriptional targets,

such as XBP1, chaperones, and moreover, increases the

ERAD [24–27]. Thus, ATF6 plays an important role during

the recovering from acute stress, and following cell adap-

tation to prolonged ER stress [12]. ER stress can activate

several death effectors such as BAK und BAX [28]. Their

upregulation leads to activation of caspase-12 that induces

the cleavage of procaspase-9 and procaspase-3 [29, 30].

The anti-apoptotic effect of Bcl-2 can be repressed trough

the transcriptional factor CHOP (GADD153). CHOP

expression during ER stress is upregulated by ATF6.

Moreover, increased eIF2a-phosphorylation by PERK also

promotes upregulation of CHOP [31]. CHOP upregulation

facilitates cell death, whereas its deficiency protects cells

against apoptosis [32].

Meanwhile, there are some studies about the role of

ERp29 during UPR. Some of these reports describe chan-

ges of ERp29 expression levels in cell lines as response to

ER stress [14–16]. It was shown that increased ERp29-

expression causes an increased splicing of XBP1-mRNA in

ERp29-transfected MDA-MB-231 cell line [33]. ERp29-

overexpression in these cells reduces the basal expression

of eIF2a but not its phosphorylation, and moreover,

downregulates cycline D1/D2 resulting in cell cycle arrest

in the G1-phase. Thus, ERp29-overexpression changes the

course of ER-stress, and leads to growth arrest [33]. Other

studies revealed that p38 and XBP1 decrease the ERp29-

expression in the MDA-MB-231 cell line [34]. Probably,

this regulation occurs through the p38-PERK-eIF2a-path-

way of UPR. The repression of this UPR branch in ERp29-

transfected cells blocks eIF2a-phosphorylation, and pro-

tects cells against caspase-3-mediated apoptosis [35].

The current state of scientific knowledge is gained by

studies on transfected cells, and therefore, leads to con-

troversial conclusions about the role of ERp29 in UPR

signaling. Therefore, it is necessary to study the ERp29

status during ER stress and its involvement in activation of

UPR cascade.

In this study we present first results concerning the

biological role of ERp29 by using two different types of

802 Apoptosis (2014) 19:801–815

123

primary adult cells prepared from ERp29-/- mice. We

found that ERp29 activates the ATF6–CHOP-brunch of

UPR during severe stress without influencing the function

of other UPR branches, like the ATF4-eIF2a-XBP1 sig-

naling pathway. As a result of impaired ATF6 activation,

dermal fibroblasts and adult thyrocytes from ERp29-/-

mice display significantly lower apoptosis sensitivities.

Materials and methods

Experimental animals

Experimental ERp29 knock out mice (strain C57BL/6N)

were generated by the Georg-August University (Gottin-

gen, Germany) using a conventional method of homolo-

gous recombination [36]. Mice were kept at a constant

temperature (22 �C) and light cycle (12 h light, 12 h dark),

and were provided with rat/mouse standard-diet (Altromin

#1324), and tap water ad libitum. Breeding of animals was

performed using 12–24 weeks old females and males. The

offspring of heterozygous matings were genotyped

3 weeks after birth by PCR from tail biopsy (see below).

Thereafter the littermates were separated according to their

sex.

Isolation of genomic DNA and genotype determination

by PCR

Genotype determination of ERp29 was performed with

genomic DNA isolated from either mouse tails or embryos.

Tails or embryos were resuspended in lysis buffer (50 mM

Tris–HCl, pH 8.0, 20 mM NaCl, 0.1 % SDS and 1 mg/ml

proteinase K), and sequentially incubated at 55 �C for 16 h

followed by 94 �C for 10 min for proteinase inactivation.

The lysates were then subjected to PCR analysis. Geno-

typing was carried out with two PCRs: first PCR for ERp29

knock out gene, second PCR for ERp29 wild type gene.

The primers and reaction conditions were used as described

by Guo [36]. The separation of PCR products using a 1 %

agarose gel electrophoresis gave fragments of 700 bp (wild

type) and 2,500 bp (mutant) (Sup. 1).

Cell culture

Primary cells isolated from 3 month old mice (strain

C57BL/6N) were used for cell culture.

Establishment of the primary dermal fibroblasts

from mouse skin

The primary skin fibroblasts were prepared from littermate

wild type (ERp29?/?) and ERp29 knock out (ERp29-/-)

males according to the modified protocol for human skin

fibroblasts described by Lichti et al. [37]. Skin fragments

from dorsum of 3 months old mice were used for prepa-

ration. Animals were sacrificed by CO2 asphyxiation, and

their dorsum was disinfected by 70 % ethanol. The coat in

this area was removed by a shaver. Skin fragments about

4 9 2 cm were cutted out and washed three times with

sterile PBS. The skin-pieces were washed with sterile PBS,

cutted in small squares by a scalpel and placed in 10 cm

culture plates.

The dermal fibroblasts out-migrate from dermis on the

bottom of culture plates. Thereby, the contact between

dermis and the culture plates plays an important role.

Therefore, the dermis fragments were allowed to stand in

the opened 10 cm culture plate to dry in the flow box for

about 15 min. Subsequently 10 ml fibroblasts medium

(DMEM high glucose (PAA) supplemented with 10 %

FCS (PAA), penicillin (PAA), streptomycin (PAA),

ampicillin (Sigma), buffered with HEPES pH 7.4 (Sigma)

was added drop by drop. Mouse dermal fibroblasts were

cultured in a 37 �C humidified incubator, under air/CO2

(95 %/5 %). Migration of skin fibroblasts starts after

3 days. After 7 days dermis-pieces can be removed from

culture plates. Every 3 days cells were harvested by

trypsinization, counted by trypan blue staining, and

transferred (1.0 9 105 cells) in the 6 cm culture plates in

5 ml medium. Cells were used for experiments at third

passage.

Isolation of follicles from mouse thyroid and culture

of primary thyrocytes

Primary thyrocytes were isolated from 3 months old mice

according to the protocol of Jeker et al. [38]. The littermate

ERp29?/? and ERp29-/- males were sacrificed by CO2

asphyxiation. Thyroid lobes were dissected aseptically

from the trachea, and cutted in small pieces. The fragments

were collected and transferred to a 1.5 ml tube containing

1 ml of digestion medium (MEM medium with 125 U/ml

of type I collagenase and 1.2 U/ml dispase). The enzymatic

digestion was carried out for 45 min in a 37 �C shaker at

400 rpm. Following the suspension was centrifuged for

3 min at 3009g. The supernatant containing single cells

was discarded. The pellet with thyroid follicles was

resuspended in 1 ml culture medium, and transferred in

6 cm culture plates containing 3 ml culture medium. The

thyroid culture medium is composed of DMEM nutrient

mixture F-12 Ham (Sigma), 2 mM L-glutamine (PAA),

100 U/ml penicillin (PAA), 100 lg/ml streptomycin

(PAA), 10 lg/ml insulin (Sigma), 10 nM hydrocortisone

(Sigma), 5 lg/ml transferrin (Sigma), 10 ng/ml gly-his-lys-

acetate (Sigma), 10 ng/ml somatostatin (Sigma), 1.0 mU/

ml TSH (Sigma), 10 % (v/v) FCS (PAA).

Apoptosis (2014) 19:801–815 803

123

In accordance to the protocol the medium was changed

1 day after the beginning of the culture, and then every

third day. Primary thyrocytes were cultured in a humidified

incubator at 37 �C, under 5 % CO2.

Treatment of primary thyrocytes with tunicamycin

In order to study the effects of UPR primary thyrocytes

were grown 7 days in the culture. Subsequently, the culture

medium was replaced against culture medium containing

two different concentrations of tunicamycin (Calbiochem);

1.5 lM (18 h treatment) and 10 lM (6 h treatment). Tu-

nicamycin is a nucleoside-antibiotics mix that inhibits the

GTPase, and therefore, inhibits the first step of glycopro-

tein-synthesis. Tunicamycin was dissolved as a 5 mg/ml

stock solution in DMSO. The treatment with the same

concentration of tunicamycin in culture medium serves as

control. Subsequently the thyrocytes were harvested by

trypsin-treatment, washed with PBS, and used for caspase-

3-activity assay or for Western blot analysis. However,

thyrocytes are only able to thyroglobulin-synthesis as

components of follicles [38]. In this case, methods for cell

suspensions are not applicable, which limits methods

available for analysis of cell viability.

Caspase-3-activity assay in thyrocytes

The caspase-3 activity assay was performed in lysates of

tunicamycin-treated thyrocytes according to manufac-

turer’s instructions (EnzChek�Caspase-3 assay kits II,

Molecular Probes). The principle of method is based on

enzymatic cleavage of the substrate rhodamine 110 bis-(N-

CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amid)

(Z-DEVD-R110) by caspase-3, and the measurement of the

released fluorescent dye rhodamine 110 (ext./em.

496/520 nm). Thereby, the fluorescence intensity correlates

with the quantity of cleaved substrate, and complies with

caspase-3-activity. The fluorescence intensity was mea-

sured using plate reader.

TCA precipitation of secreted thyroglobulin

from culture medium

Equal amounts of thyrocyte medium (5 ml) were collected

from dish after 18 h cultivation of cells, and concentrated

up to 1 ml using Vivaspin 15R (30,000 MWCO). 30 %

TCA was added to the concentrated medium to a final

concentration of 8 % and mixed quickly. The mixture was

incubated on ice for 30 min and centrifuged at 16,0009g,

4 �C for 15 min. The pellets were washed with 1 ml ice-

cold acetone, and centrifuged at 16,0009g, 4 �C for

10 min. Afterwards, the pellets were dried, and dissolved

into proper volume of lysis buffer. The secreted thyro-

globulin was analyzed by Western blot as described below.

Exposure of dermal fibroblasts to oxidative stress

and measurement of cell death by FACS

Dermal fibroblasts were inoculated in 6-well culture plates

at 1 9 105 cells per well and cultured for 24 h in complete

medium. Subsequently, the medium was replaced against

complete medium with 1 mM hydrogen peroxide (Sigma).

After 6 h exposure to the oxidizing agent, cells were col-

lected by trypsin treatment, washed with PBS and double

stained with a mix of 5 ll propidium iodide (PI) and 5 ll

fluorescein isothiocyanate (FITC)-labeled annexin V

(annexin V-FITC) for 15 min (Annexin V-FITC apoptosis

detection kit I, BD PharmingenTM). Subsequently, cell

death was analyzed by FACS measurement.

Western blot analysis

Cells were lysed in lysis buffer [7 M urea, 2 M thiourea,

4 % (w/v) CHAPS, 30 mM Tris–HCl pH 8.5]. Protein

amounts of whole cell extracts were quantified using the

Pierce� 660 nm protein assay kit (Thermo scientific).

Equivalent amounts of protein (20 lg) were mixed with

49 loading buffer (160 mM Tris–HCl pH 6.8, 12 % (w/v)

SDS, 20 % (v/v) glycerine, 2 % (w/v) bromophenol blue,

400 mM DTT), separated using 8 % for thyroglobulin

(24—205 kDa), 10 % (14—205 kDa), 12 % (14—66 kDa)

SDS-PAGE at 150 V, and transferred to nitrocellulose

membrane (Millipore Corporation) at 1 mA/cm2. Immu-

nodetection was performed using primary rabbit-anti-

mouse-antibodies against thyroglobulin (Abcam), BiP/

GRp78, which migrates as double-band (Sigma-Aldrich),

GRp94 (Sigma-Aldrich), ERp72 (MPG Gottingen), cal-

reticulin (MPG Gottingen), calnexin (MPG Gottingen),

ATF4 (Abcam), p90ATF6/p50ATF6 (Abcam), CHOP

(Santa Cruz Biotech.), XBP1(u)/XBP1(s) (Abcam), ERp29

(MPG Gottingen), PDI (MPG Gottingen), eIF2a and

eIF2a-P (phosphorylated at serine 51) (Cell Signaling

Technology). All primary antibodies were used at a 1:1,000

final dilution. Immunoreactive bands were visualized after

staining with secondary goat-anti-rabbit-IgG-cy5 or -cy3

conjugate (1:10,000) (Jackson laboratories Inc., USA)

using a Typhoon TRIO? scanner. The horseradish-perox-

idase-conjugated b-actin antibody (1:25,000) (Sigma-

Aldrich) serves as loading control. Signals were detected

using the CCD camera Fusion-SL 4.2 MP (chemilumi-

nescence system, Peqlab) after treatment with Supersignal

West Dura Extended Duration Substrate kit (Pierce). The

protein bands were related using PageRulerTM prestained

protein ladder (#SM0671, Fermentas).

804 Apoptosis (2014) 19:801–815

123

The signal intensities of protein bands were measured

using Adobe Photoshop CS3, and standardized according

to loading control (b-actin). The values were represented as

percentage of the control-experiment.

Statistical analysis

All experiments were repeated at least three times. The

data were expressed as mean ± SD (standard deviation).

Statistical analysis was performed by using Student0s t test.

The criteria for statistical significance were p \ 0.05.

Results

Initial characterization of ERp29 knock out mice

For the present experiments littermate wild type and

mutant mice from heterozygous matings were used in order

to reduce genetic variability. The analysis of genotype

distribution for embryos (embryonic day 15) and for off-

spring reveals a ratio as expected (1 ERp29?/? : 2

ERp29?/- : 1 ERp29-/-), indicating that the deletion of the

ERp29 gene did not result in embryonic lethality. The

ERp29-/- mice were apparently normal, and look similar

to wild type mice (Sup. 2). Male and female fertility in the

ERp29-/- mice appeared to be normal, as shown by the

analysis of matings of 3–6 month old mice, but litter size

was slightly affected. The mean litter sizes in ERp29?/?

and ERp29-/- pregnancies were 5.2 ± 3.1 and 4.6 ± 2.8,

respectively. A difference in body weight between 12 week

old wild type and mutant mice could not be observed:

26.3 ± 2.6 and 27.6 ± 2.3 g respectively.

Effect of ERp29-deficiency in primary thyrocytes

on responsiveness to ER stress

Adult thyrocytes grow in culture as a component of folli-

cles. The interior space of follicles contains a colloidal

substance that serves as storage for secreted proteins. The

ERp29-deficient follicles did not show any differences to

wild type cells in respect to their growth and viability in the

culture (Sup. 3). Moreover, thyroglobulin-synthesis in

thyrocytes decreases progressively 14 days after seeding

[38]. Therefore, cultured thyrocytes 7 days after seeding at

80 % confluence were used for experiments. Thyroglobulin

(Tg) forms a disulfide-connected complex with a molecular

weight over 2,000 kDa. The blocking of N-glycosylation

leads to accumulation of the protein in ER thus leading to

UPR. The UPR in thyrocytes was triggered by the use of

1.5 lg/ml tunicamycin for 18 h. The treatment conditions

were chosen according to previous studies that describe

triggering of UPR in primary thyrocytes isolated from

different species [39–41].

The stress response in cell lysate was studied using

Western blot. The analysis of Tg expression shows that

untreated ERp29-/- thyrocytes expressed about 20 % less

thyroglobulin than untreated ERp29?/? thyrocytes

(p = 0.03) (Fig. 1b–d). The inhibition of N-glycosylation

by tunicamycin-treatment leads to accumulation of Tg in

the cells. In the case of Tg expression no significant

genotype-related differences were observed. Expression

level of Tg after tunicamycin treatment increased about

50–70 % in both ERp29?/? as well as ERp29-/- cell lines

compared to control (DMSO-treated) cells. Moreover, the

ERp29-/- thyrocytes were able to secrete Tg into the

culture medium comparable to ERp29?/? thyrocytes

(Fig. 1b). In the case of Tu-treated cells, lower but

detectable amounts of Tg were observed in the culture

medium. There are two possible explanations for this

observation. First, the culture medium was collected over

the total treatment period of the experiment, whereas the

stress response occurs only after a time delay. Secondly, as

already mentioned, Tu-treatment leads to altered secretory

profiles of thyrocytes, but not to a complete inhibition of

Tg-secretion [42].

The folding and processing of Tg is assisted by several

chaperones of the ER quality control. In general, genotype-

independent upregulations of some chaperones in treated

cells were observed; GRp94 and calnexin were upregulated

about 50 %, and ERp72 twofold. In contrast, no changes in

expression level of calreticulin, PDI and ERp29 (in

ERp29?/? cells) were observed (Fig. 1a, c). Western blot

analysis shows a 70 % increase of BiP level in both cell

lines. An increase in BiP expression is a hallmark for ER

stress. BiP activates the UPR cascade in cooperation with

three transmembrane proteins IRE1, PERK and ATF6

(Fig. 2).

Tunicamycin-treatment of thyrocytes causes an upreg-

ulation of XBP1(u) expression and of the spliced form

XBP1(s) independently from genotype. XBP1 upregulation

points to an activation of the IRE1-branch, which mediates

cell survival. The activation of the PERK branch induces

the phosphorylation of the transcription factor eIF2a and

leads to ATF4 activation (Fig. 2a, b). However, ERp29

deficiency had no effect on activation of this UPR branch

(Fig. 2c). In the case of the ATF6 pathway genotype-

related differences could be observed. Tunicamycin treat-

ment of ERp29?/? thyrocytes leads to upregulation of

p90ATF6 (about 50 % of the respective control) as well as

to accumulation of its mature form p50ATF6. However,

expression analysis of ERp29-/- cells displayed no

increase in p90ATF6 after tunicamycin treatment. Never-

theless, Western blot analysis revealed an increase of its

mature form (Fig. 2a, b), which was however apparent

Apoptosis (2014) 19:801–815 805

123

lower compared to wild type cells (Fig. 2c). CHOP

(GADD153), which is involved in activation of apoptosis,

is a terminal signal transducer of UPR [43]. Probably, as a

consequence of the weak ATF6 activation, CHOP is

expressed to lower levels in treated ERp29-/- cells when

compared to ERp29?/? cells (Fig. 2).

Moreover, we were interested in the effect of ERp29

deficiency in thyrocytes during severe ER stress. As

referred, higher concentrations of tunicamycin (up to

10 lg/ml) cause massive cell death in the culture [44].

Therefore, cells were treated with 10 lg/ml tunicamycin

for 6 h. Western blot analysis showed a genotype-inde-

pendent accumulation of BiP about 40–50 % (Fig. 3).

However, independently of the genotype, there was no

activation of ATF6, ATF4, eIF2a-P and XBP1 under

these experimental conditions. Only CHOP was upregu-

lated in both cell lines (Fig. 3a, b). This increase was

much higher compared to gentle stress conditions

(1.5 lg/ml tunicamycin for 18 h) (Fig. 2). The CHOP

expression in ERp29-/- thyrocytes during severe stress

amounts 150 % of the control. Therefore, these results

represent a significant genotype-dependent difference

between ERp29-/- and ERp29?/? thyrocytes under

severe stress (Fig. 3b, c). CHOP activation leads to cas-

pase-3-mediated apoptosis. Caspase-3-activity was

determined in lysates from cells, which were subjected to

gentle and severe stress by treatment with different tu-

nicamycin concentrations. This analysis showed that

caspase-3-activation in thyrocytes occurs only during

severe stress (Fig. 4). Moreover, under these conditions,

caspase-3-activity in ERp29-/- thyrocytes was signifi-

cant lower compared to treated ERp29?/? cells also

complying with genotype-related differences in CHOP

expression (Fig. 3).

ERp29+/+ ERp29-/-

DMSO Tu DMSO Tu

ERp29+/+ ERp29-/-

DMSO Tu * DMSO Tu

calnexinGRp94ERp72

PDIcalreticulin

ERp29

β-actin

b

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Tg

calne

xin

GRp94

ERp72

Rat

io E

Rp2

9-/

- /ER

p29+

/+

controltreated

0

50

100

150

200

250

Tg

calne

xin

GRp94

ERp72

calre

ticuli

nPDI

ERp29

Rel

ativ

e ba

nd in

tens

ity (

%)

a

c

d

intracellular Tg

secreted Tg

Fig. 1 Expression of thyroglobulin and some ER chaperones in

thyrocytes after tunicamycin-induced ER stress. The thyrocytes were

treated with 1.5 lg/ml tunicamycin (Tu) for 18 h. Whole cell lysate

containing 20 lg protein per lane was resolved by 8–12 % SDS-

PAGE and transferred to a nitrocellulose membrane. Expression of

some ER chaperones (a), and of intracellular and secreted thyroglob-

ulin (Tg) (b) were analysed by Western blot. ERp29 was used as

genotype control. For the analysis of Tg-expression full length protein

about 300 kDa (marked with arrow) and its processing pattern were

used. Asterisk prestained protein ladder: 170, 130, 100, 70, 55, 40, 35,

25 kDa. c For densitometric quantification all signals were normal-

ized to the loading control b-actin. Relative expressions of proteins

are indicated as percentages (%) of control cells (DMSO-treated); in

black—control ERp29?/?, in dark grey—Tu-treated ERp29?/?, in

light grey—control ERp29-/-, in white—Tu-treated ERp29-/-. The

bars represent averages of signal intensity with their respective SD;

p [ 0.05. d Genotype-related differences of protein expression were

calculated as ratio ERp29-/- to ERp29?/?of integrated density of

normalized protein bands. The dashed line, that intersects the y axis at

the ratio = 1, indicates no changes of protein expression in ERp29-/-

compared to ERp29?/? cells. Bars under this value indicate

downregulation of protein expression. ERp29?/?: n = 5; ERp29-/-:

n = 5

806 Apoptosis (2014) 19:801–815

123

Effects of ERp29-deficiency on responsiveness

to oxidative stress in primary dermal fibroblasts

Adult diploid cells exhibit a limited proliferative potential

in vitro. For example, the murine dermal fibroblasts

undergo senescence and die after 5–7 passages in the cul-

ture [45, 46]. Therefore, experiments were performed with

primary dermal fibroblasts at third passage (Sup. 4). The

ERp29-/- dermal fibroblasts exhibit a typically shape with

branched cytoplasm surrounding a speckled nucleus. There

were no genotype-related differences between wild type

and ERp29-/- dermal fibroblasts (Sup. 5).

In order to study the effects of oxidative stress on adult

dermal fibroblasts, cells were treated with 1 mM hydrogen

peroxide for 6 h, followed by FACS analysis. Here, three

different cell populations were quantified; apoptotic, viable

and necrotic cells. During the early stage of apoptosis an

externalisation of phosphatidyl serine (PS) occurs, which

can be visualized by binding of FITC-coupled annexin-V

(Fig. 5). Late stages of apoptosis and necrosis are

characterized by a loss of membrane integrity and therefore

are positive for PI and annexin-V staining, whereas vital

cells are negative for both stains.

As shown in Fig. 5, Sup. 6, dermal fibroblasts subjected

to oxidative stress exhibit a constant necrosis rate of about

10–15 % independently from genotype, which is slightly

higher compared to the respective control experiments.

However, the apoptotic cell population rises after H2O2

treatment in dependence of the genotype. The quantitative

analysis of cell death shows that ERp29 deficiency leads to

decreased responsiveness to apoptosis caused by short-time

oxidative stress (Fig. 5a, b). The number of apoptotic

ERp29-/- cells was decreased about 20 % compared to

ERp29?/? cells (Sup. 6).

Western blot analysis of proteins involved in UPR cas-

cade was performed to elucidate apoptosis resistance in

ERp29-/- cells. The upregulation of BiP expression in

H2O2-treated cells indicates activation of UPR-cascade

(Fig. 6). The increase of about 20 % compared to respec-

tive control cells was independently from genotype

BiP

p90ATF6

p50ATF6

XBP1(u)XBP1(s)

ATF4

eIF2α

eIF2α-P

CHOP

ERp29

β-actin

ERp29+/+ ERp29-/- .DMSO Tu DMSO Tu

a

b

0

50

100

150

200

BiP

p90A

TF6

p50A

TF6

XBP1(u)

XBP1(s)

ATF4

CHOP

Rel

ativ

e ba

nd in

tens

ity (

%)

* ***

**

*

*

*

***

c

0

0.2

0.4

0.6

0.8

1

1.2

BiP

p90A

TF6

p50A

TF6

XBP1(u)

XBP1(s)

ATF4

CHOP

Rat

io E

Rp2

9-/

- /ER

p29

+/+

controltreated

elF2α

elF2α-P

elF2α

elF2α-P

Fig. 2 Expression of some UPR-proteins in thyrocytes after tunica-

mycin-induced ER stress. Thyrocytes were treated with 1.5 lg/ml

tunicamycin (Tu) for 18 h. Whole cell lysate containing 20 lg protein

per lane was resolved by 10–12 % SDS-PAGE and transferred to a

nitrocellulose membrane. a The expression of some UPR proteins

were analysed by Western blot. ERp29 was used as genotype control.

For densitometric quantification all signals were normalized to the

loading control b-actin. b Relative expression of proteins is indicated

as percentage (%) of control cells (DMSO-treated); in black—control

ERp29?/?, in dark grey—Tu-treated ERp29?/?, in light grey—

control ERp29-/-, in white—Tu-treated ERp29-/-. The bars repre-

sent averages of signal intensity with their respective SD; *p [ 0.05,

**p \ 0.05. c The genotype-related differences of protein expression

were calculated as ratio ERp29-/- to ERp29?/? of integrated density

of normalized protein bands. The dashed line, that intersects the y axis

at the ratio = 1, indicates no changes of protein expression in

ERp29-/- compared to ERp29?/? cells. Bars under this value

indicate downregulation of protein expression. ERp29?/?: n = 3;

ERp29-/-: n = 3

Apoptosis (2014) 19:801–815 807

123

(Fig. 6b, c). Western blot analysis of further UPR-proteins

revealed that basal level of p90ATF6 in unstressed

ERp29-/- cells were already about 30 % lower than those

of ERp29?/? cells (p = 0.03) (Fig. 6c). H2O2 treatment

caused an upregulation of about 80 % in ERp29?/? cells,

and of about 20 % in ERp29-/- cells, respectively. Thus,

the difference of p90ATF6 levels in stressed ERp29-/-

cells averages about 90 % of the expression of treated

ERp29?/? cells (Fig. 6b). The protein level of the mature

form of p50ATF6 in stressed wild type cells was twofold

upregulated compared to the stressed mutant cells indi-

cating different activation of ATF6 depending on the pre-

sence of ERp29 (Fig. 6c).

Moreover, Western blot analysis revealed a genotype-

related upregulation of CHOP expression in stressed cells.

Remarkably, the upregulation of CHOP in H2O2-treated

ERp29-/- cells was only about 20 % of the respective

control, whereas in ERp29?/? cells the difference in CHOP

levels amounts to 60 % (Fig. 6). An upregulation of other

UPR members, such as ATF4, XBP1, eIF2a-P (Fig. 6) as

well as upregulation of ER chaperones (Fig. 7) in stressed

dermal fibroblasts was not observed.

BiP

p90ATF6

p50ATF6

XBP1(u)XBP1(s)

ATF4

eIF2αeIF2α-P

CHOP

ERp29

β-actin

a

b

ERp29+/+ ERp29-/-

DMSO Tu DMSO Tu

0

50

100

150

200

BiP

p90A

TF6

XBP1(u)

ATF4

CHOP

Rel

ativ

e ba

nd in

tens

ity (

%)

*

**

c

0

0.2

0.4

0.6

0.8

1

1.2

p90ATF6R

atio

ER

p29

-/- /E

Rp2

9+

/+ control

treated

BiP CHOP

elF2α

elF2α-P

Fig. 3 Expression of some UPR proteins in thyrocytes after tunica-

mycin-induced severe ER stress. Thyrocytes were treated with 10 lg/

ml tunicamycin (Tu) for 6 h. Whole cell lysate containing 20 lg

protein per lane was resolved by 10–12 % SDS-PAGE and transferred

to a nitrocellulose membrane. a The expression levels of proteins

were analysed by Western blot. ERp29 was used as genotype control.

For densitometric quantification all signals were normalized to the

loading control b-actin. b Relative expression of proteins is indicated

as percentage (%) of control cells (DMSO-treated); in black—control

ERp29?/?, in dark grey—Tu-treated ERp29?/?, in light grey—

control ERp29-/-, in white—Tu-treated ERp29-/-. Bars represent

averages of signal intensity with their respective SD; *p [ 0.05,

**p \ 0.05. c The genotype-related differences of protein expression

were calculated as ratio ERp29-/- to ERp29?/? of integrated density

of normalized protein bands. The dashed line, that intersects the y axis

at the ratio = 1, indicates no changes of protein expression in

ERp29-/- compared to ERp29?/? cells. Bars under this value

indicate downregulation of protein expression. ERp29?/?: n = 3;

ERp29-/-: n = 3

Fluorescence intensity

1200

800

400

00 1.5 10

Tunicamycin, µg/ml

**

*

Fig. 4 Caspase-3-activity in thyrocytes after tunicamycin-induced

ER stress. The thyrocytes were treated with different concentrations

of tunicamycin: 0 lg/ml (18 h incubation in culture medium), 1.5 lg/

ml for 18 h, 10 lg/ml for 6 h. The fluorescence intensity was

measured in cell lysate, normalized against fluorescence intensity

from cells treated with equal amounts of DMSO for 18 and 6 h, and

subsequently plotted against corresponding tunicamycin concentra-

tions, in black—ERp29?/? cells, in red—ERp29-/- cells The

fluorescence intensity correlates with the quantity of cleavaged

bisamide-substrate, and complies with caspase-3-activity. Error bars

represent the standard deviation. *p [ 0.05, **p \ 0.05. Each sample

was obtained from a whole thyroid that was obtained from 3 month

old males. ERp29?/?: n = 3; ERp29-/-: n = 3 (Color figure online)

808 Apoptosis (2014) 19:801–815

123

Fig. 5 Responsiveness of

dermal fibroblasts to oxidative

stress triggered by treatment

with 1 mM H2O2. a FACS-

histogram of cells incubated

with FITC-coupled annexin-V

and PI. On the x axis

fluorescence intensity of FITC-

coupled annexin-V was plotted,

the y axis represents cell counts.

b Distribution of cell

populations according to the

FACS analysis. The bars

represent mean ± SD of three

independent experiments: in

black—control ERp29?/?, in

grey—treated ERp29?/?, in

red—control ERp29-/-, in

rose—treated ERp29-/- (Color

figure online)

BiP

p90ATF6

p50ATF6

XBP1(u)XBP1(s)

ATF4

eIF2αeIF2α-P

CHOP

β-actin

ERp29

ERp29+/+ ERp29-/-

0 1,0 0 1,0 H2O2, mM

0

0.2

0.4

0.6

0.8

1

1.2

BiP

p90A

TF6

p50A

TF6

CHOP

Rat

io E

Rp2

9-/

- /ER

p29

+/+ control

treated

a

c

b

0

50

100

150

200

250

300

BiP

p90A

TF6

p50A

TF6

XBP1(u)

ATF4

eIF2a

lfa

CHOPRel

ativ

e ba

nd in

tens

ity (

%)

*

**

****

Fig. 6 Comparison of the effect of oxidative stress in ERp29?/? and

ERp29-/- dermal fibroblasts on expression patterns of proteins

involved in UPR. a Dermal fibroblasts were treated with 1 mM H2O2

in DMEM/10 % FCS for 6 h. Cell lysate containing 20 lg protein per

lane was resolved by 10–12 % SDS-PAGE and transferred to a

nitrocellulose membrane. Western blot analysis was performed for

some proteins of unfolded protein response. b-actin serves as loading

control, and ERp29 as genotype control. Genotype-related differences

of protein expression are framed in black. b Relative expressions of

proteins are indicated as percentages (%) of untreated cells; in black—

untreated ERp29?/?, in dark grey—H2O2-treated ERp29?/?, in light

grey—untreated ERp29-/-, in white—H2O2-treated ERp29-/-. The

bars represent averages of signal intensity with their respective SD

*p [ 0.05, **p \ 0.05. c The genotype-related differences of protein

expression were calculated as ratio ERp29-/- to ERp29?/? of

integrated density of normalized protein bands. The dashed line, that

intersects the y axis at the ratio = 1, indicates no changes of protein

expression in ERp29-/- compared to ERp29?/? cells. Bars under this

value indicate downregulation of protein expression. ERp29?/?: n = 3;

ERp29-/-: n = 3

Apoptosis (2014) 19:801–815 809

123

Discussion

ERp29 knock out mouse

The aim of the present study was to reveal the physiolog-

ical role of ERp29. Therefore, gene knockout in mice was

performed to elucidate the function by analyzing the phe-

notype of the knock out mice. However, ERp29-deficient

mice did not exhibit any obvious phenotype. Hereupon, we

were interested in the effect of ERp29 deficiency on ER

stress in thyrocytes and dermal fibroblasts from knockout

mice to get further insights in the cellular function of the

protein.

Study of unfolded protein response in adult cells

UPR can be triggered trough many types of stress factors,

which lead to accumulation of unfolded proteins in ER.

The inhibition of different steps of the thyroglobulin syn-

thesis and processing in thyrocytes provides a popular

model for studying UPR [47–50]. Cultured primary thy-

rocytes more closely represent the in vivo physiological

state, maintaining highly differentiated functions, such as

the ability to secrete thyroglobulin [38]. Thyroglobulin

(Tg) is a glycoprotein that consists of 2,750-amino acid

residues, and is released in follicular lumen as a homodi-

mer [51]. It contains 60 intramolecular disulfide-bonds and

10–15 N-bonded oligosaccharides per Tg-molecule, which

causes a high potential for misfolding [52]. Therefore, the

Tg folding can be regarded as a challenging task for several

chaperones and folding enzymes, such as BiP, calnexin,

calreticulin, GRp94, and ERp72 [8, 10, 52, 53]. Previous

work on the thyroid cell line FRTL-5 proposed that ERp29

is connected with folding and/or secretion of Tg. Some

reports suggested that ERp29 displays a chaperon activity

[7, 54], because ERp29 and ER chaperones (BiP, GRp94,

PDI) were found to be upregulated by TSH-induced

physiological UPR. Based on co-immunoprecipitation of

ERp29 with denaturated Tg and ER chaperones, the

authors proposed the existence of ER-resident transient

multimolecular folding complexes. However, the ERp29

association was weak and of transient nature. If ERp29

plays an active role during the folding and processing of

Tg, its deficiency should affect Tg expression level, its

secretion, or should provoke some compensatory effects,

such as upregulation of aforementioned ER chaperones.

However, our studies revealed that ERp29 is not essential

for Tg expression, processing and secretion neither in

stressed nor in non-stressed thyrocytes. UPR, triggered by

tunicamycin treatment (1.5 lg/ml, 18 h), caused a geno-

type-independent intracellular Tg accumulation, and

moreover, an equal upregulation of some chaperones

(CNX, GRp94, ERp72) (Fig. 1). The expression levels of

ERp29 in non-stressed vs. stressed wild type cells remains

unaltered, and Tg expression levels were comparable to

that found in ERp29-/- thyrocytes. Furthermore, since

ERp29-/- mice did not show the occurrence of thyroid

dysfunctions, such as abnormalities of weight and repro-

ductivity, we exclude the hypothesis that ERp29 acts as an

essential chaperone for folding and processing of Tg, but a

possible escort function within ER-resident folding com-

plexes cannot be excluded.

The upregulation of chaperons occurs through activation

of UPR. Due to this evolutionary conserved cascade the

cell is able to regulate the synthesis-folding-capacity in ER

and to switch between two programs; i) ‘‘adaptation to

stress and survival’’ or ii) ‘‘elimination trough apoptosis’’.

Two arms of the UPR cascade were upregulated

GRp94

calnexin

ERp72

calreticulin

PDI

β-actin

ERp29

ERp29+/+ ERp29-/- .control H2O2 control H2O2

0

50

100

GRp94

calne

xin

ERp72

calre

ticuli

nPDI

Rel

ativ

e ba

nd in

tens

ity (

%)b a

Fig. 7 Comparison of the effect of oxidative stress in ERp29?/? and

ERp29-/- dermal fibroblasts on expression of some ER chaperones.

a Dermal fibroblasts were treated with 1 mM H2O2 in DMEM/10 %

FCS for 6 h. Cell lysate containing 20 lg protein per lane was

resolved by 10–12 % SDS-PAGE and transferred to a nitrocellulose

membrane. Western blot analysis was performed for some ER

chaperones. b-actin serves as loading control, and ERp29 as genotype

control. b Relative expressions of proteins are indicated as percent-

ages (%) of untreated cells; in black—untreated ERp29?/?, in dark

grey—H2O2-treated ERp29?/?, in light grey—untreated ERp29-/-,

in white—H2O2-treated ERp29-/-. Bars represent averages of signal

intensity with their respective SD p [ 0.05. ERp29?/?: n = 3;

ERp29-/-: n = 3

810 Apoptosis (2014) 19:801–815

123

genotype-independently in thyrocytes. In this connection

the accumulation of XBP1(s) transcription factor in both

cell types leads to aforementioned upregulation of chap-

erons. Our experiments using adult thyrocytes revealed

genotype-dependent differences in the activation of ATF6–

CHOP-pathway. The ATF6 (p90ATF6/p50ATF6) activa-

tion in stressed ERp29-/- thyrocytes were significant

lower than those in stressed ERp29?/? thyrocytes. Previous

studies suggested that activation of UPR results in accu-

mulation of p50ATF6 [24, 25], but changes in expression

of p90ATF6 were also described [55]. However, only

p50ATF6 is responsible for the activation of the tran-

scription factors XBP1(s) and CHOP [12]. In accordance

with these reports we found that expression levels of CHOP

in stressed ERp29-/- thyrocytes was lower than expression

levels of CHOP in stressed ERp29?/? cells. The proapo-

totic transcription factor CHOP induces the upregulation of

the BH3-only protein Bim, and therefore, activates via

caspase-12 the caspase-3-mediated apoptosis [29, 30].

Under these conditions we did not find an activation of

caspase-3 as a hallmark for cell death. These results are in

good agreement with previous reports [42, 44]. Thus, it can

be supposed that the treatment with 1.5 lg/ml Tu for 18 h

causes gentle ER stress resulting in cell survival. It is

proposed that cell survival during gentle ER stress is

facilitated through the instability of UPR-induced cell

death mediators, such as CHOP and GADD34. In accor-

dance with this hypothesis, the concentrations of these

proteins exceed a death threshold after prolonged or severe

ER stress only [43].

It is known that treatment with higher concentrations of

Tu (10 lg/ml for 6 h) causes severe ER stress [44]. Under

these conditions we did not detect an activation of XBP1 in

thyrocytes (Fig. 3), which regulate the folding capacity

of ER. However, we found an upregulation of CHOP

expression and caspase-3-activity in genotype-dependent

manner (Figs. 3, 4). The difference in genotype-related

CHOP upregulation during severe ER stress was higher

than those during gentle ER stress. In spite of upregulation

of the key activator of UPR signalling BiP, no upregulation

of ATF6, ATF4, eIF2a-P, XBP1(s) could be detected under

severe ER stress (Fig. 3). The reason for it can be found in

the different regulation through several mediators and

feedback mechanisms during varying degrees of stress [56,

57]. High expression levels of CHOP during severe ER

stress leads to the dephosphorylation of eIF2a by activation

of GADD34 [58]. The PERK activity in the later phase of

the ER stress response can be repressed by P58(IPK)

induction [59]. Terminated PERK signalling leads to

accumulation of transcripts of ATF6- and XBP1-targets,

and therefore affects likely the ATF6- and IRE1-XBP1-

signaling of UPR [60]. In contrast to previous studies that

described alteration of eIF2a-phosphorylation in ERp29-

transfected cells, and therefore, supposed the involvement

of ATF4-branch [35, 61], our experiments using ERp29-

deficient cells revealed no adverse effects on activation of

PERK-eIF2a-ATF4 as well as on activation of IRE1-

XBP1(s) pathways in adult thyrocytes.

The analysis of caspase-3 activity in stressed thyrocytes

showed that their activation occurs only during severe ER

stress (Fig. 4). Accordingly to CHOP levels, caspase-3

activity in ERp29-/- thyrocytes was lower compared to

ERp29?/? cells. These results indicate that ERp29 plays an

important role in UPR activation by affecting the apoptosis

sensitivity of adult thyrocytes during severe ER stress.

In order to verify cell and stress specificity of genotype-

related findings we studied severe ER stress in dermal

fibroblasts treated with 1 mM hydrogen peroxide. The pri-

mary culture of adult dermal fibroblasts is representative for

ploidy, contact inhibition and some other cell properties in

the body. Primary cells are responsive for different treat-

ments and are useful as model for studying cellular stress

response [62, 63]. Hydrogen peroxide is a membrane-per-

meable form of reactive oxygen species (ROS), which reg-

ulates the activity of calcium channels, and modify the redox

status in ER [64, 65]. Protein folding in ER, especially

disulfide-bond formation, is connected with generation of

ROS. Therefore, activation of UPR upon exposure to oxi-

dative stress serves as an adaptive mechanism for cell sur-

vival, whereas prolonged or severe oxidative stress leads to

alterations of redox status, protein misfolding, and the ini-

tiation of apoptotic cascades [65, 66].

In this work, we demonstrated that ERp29-defecient

dermal fibroblasts are less sensitive for oxidative stress

triggered by H2O2-treatment than wild type fibroblasts

(Fig. 5). As observed in the case of severe ER stress in

adult thyrocytes, ATF6 activation (p90ATF6/p50ATF6) in

stressed ERp29-/- fibroblasts was lower compared to

stressed ERp29?/? fibroblasts, whereas the upregulation of

other member of UPR-cascade and chaperones in both

stressed cell types remained unaltered versus their

expression in untreated cells (Figs. 6, 7). In accordance

with findings in thyrocytes, dermal fibroblasts also display

significant lower expression level of CHOP in stressed

ERp29-/- fibroblasts when compared to stressed ERp29?/

? cells, thus also explaining lower apoptosis sensitivity of

mutant cells [43].

In summary, the analysis of ER stress response in thy-

rocytes and dermal fibroblasts reveals genotype-related

differences during UPR. The ERp29 deficiency affected the

activation of ATF6–CHOP-pathway, and leads to lower

apoptosis sensitivity in adult cells (Fig. 8). In contrast to

previous reports, our results showed no influence of ERp29

on ATF4 activation, and eIF2a-phosphorylation [34, 35].

The PERK-pathway remained inactive after exposure to

severe ER stress independent from genotype.

Apoptosis (2014) 19:801–815 811

123

In this connection, ER stress-induced upregulation of

ERp29 [7, 15, 16, 54], which serves as argument for a

chaperone function of the protein, arouses special interest.

Our experiments show that the expression of ERp29 in

stressed ERp29?/? thyrocytes und dermal fibroblasts

remains unaltered, whereas all members of ER folding

machinery (BiP, calnexin/calreticulin, PDI, ERp72) were

up-regulated during gentle ER stress. Moreover, ERp29 has

no effect on upregulation levels of above mentioned

chaperones. An upregulation of ERp29 (but not of PDI and

BiP) was detected in cancer cell lines under ER stress

conditions [6, 67]. These data suggest that during ER

stress, ERp29 possesses another function or is differently

regulated than ER chaperones. In fact, a direct effect of ER

stress on ERp29-transcription could not be proven [1]. The

analysis of the erp29-promoter reveals several binding sites

for a number of transcription factors (GATA-1, Sp1, E2F,

CRE-BP1) that possibly together determine the ERp29

expression [68]. This interplay of these regulatory elements

could allow different expression pattern of ERp29 under

ER stress conditions dependent on tissue and cell type.

Putative function of ERp29 as escort factor in ER

ERp29 belongs to the redox-inactive PDI-Db-subfamily of

PDI proteins [1, 69]. The interaction of ERp29 with some

ER chaperones such as BiP was demonstrated by immu-

noprecipitation experiments, but the recognition mecha-

nism remained unclear. It was supposed that ERp29 is only

able to bind as homodimer [70]. Experiments with its

orthologous protein Wind from Drosophila melanogaster

gave indications for an ERp29-recognition motive. Wind is

required for the correct targeting of the proteoglycan

modifying enzyme Pipe from ER to Golgi apparatus. It was

shown that the D-domain from murine ERp29 can func-

tional replace the D-domain from Wind-protein for Pipe-

processing. Pipe targeting is determined through highly

conserved residues, whose mutagenesis in the Wind-pro-

tein prevents the Pipe transport to Golgi apparatus [71–74].

The study of Das et al. [9], which investigated the

trafficking of the gap junction protein connexin 43 (Cx43)

from ER to Golgi apparatus, also suggests an escort func-

tion of ERp29. Thus, it was supposed that the stabilization

of monomeric Cx43 is mediated through their interaction

with redox-inactive ERp29. ERp29 blocks intermolecular

cysteine residues, which connect Cx43-domains, and pre-

vents thereby their oligomerization in ER [9].

We demonstrated in two types of adult cells that ERp29

deficiency impairs ER stress-induced activation of ATF6.

In this regard we suppose an important role of ERp29 for

the relocation of p90ATF6 from ER to Golgi with fol-

lowing processing to the mature form p50ATF6. As yet the

detailed mechanism of ATF6 transport is not clear. It was

supposed that any anchor and escort proteins (such as BiP,

PDI and many of unidentified proteins) are involved in ER

stress-induced relocation of ATF6 [75]. This protein exists

in unstressed ER as monomer, dimer and oligomer, but

only the reduced monomeric form of ATF6 reaches the

Golgi apparatus [76–78]. Probably, ERp29 interacts with

monomeric form of ATF6, stabilizes it, and thus promotes

ATF6-relocation. Therefore, we suppose that ERp29 acts

as an escort factor for the p90ATF6 transport from ER to

Golgi apparatus under ER stress conditions. ERp29 defi-

ciency affects apoptosis sensitivity and probably adaptation

of cells to prolonged stress.

There are some interesting aspects about the role of

ERp29 that are important for further studies. ERp29 could

serve as escort factor within several protein transport

complexes from ER to Golgi. However, in this study we

did not detect an all-or-nothing response in ERp29-defi-

cient cells. Therefore, a further important aspect of this

field of research consists in the question, whether there are

rescue mechanisms for ERp29-deficiency during ER stress

either by increased expression levels of other ER members

[79] or by the emergence of proteins with similar function

[80].

Acknowledgments We are grateful to all of the members of Max

Planck Research Unit for Enzymology of Protein Folding (Halle/

Saale, Germany) for helpful discussion, and in part for technical

assistance. The present work was supported by grants from the

Exzellenznetzwerk Biowissenschaften (Sachsen-Anhalt).

References

1. Ferrari DM, Van Nguyen P, Kratzin HD, Soling HD (1998)

ERp28, a human endoplasmic reticulum luenal protein, is a

member of the protein disulfide isomerase family but lacks a

CXXC thioredoxin-box motif. Eur J Biochem 255(3):570–579

BiP

IRE1 ATF6 PERK

XBP1 eIF2α → eIF2α-PCHOP

ATF4

CHOP

ERAD caspase-3 caspase-3 chaperones apoptosis apoptosis

Fig. 8 Regulation of the UPR in differentiated adult cells during

oxidative stress. The UPR consists of three pathways, which can be

differently regulated during ER stress in dependence on stress-stimuli

and cell type. Our results suggest that the activity of the ATF6–

CHOP-axis is lowered during ER stress in ERp29-/- cells (in red)

compared to the wild type cells (Color figure online)

812 Apoptosis (2014) 19:801–815

123

2. Barak NN, Neumann P, Sevvana M, Schutkowski M, Naumann

K, Malesevic M, Reichardt H, Fischer G, Stubbs MT, Ferrari DM

(2009) Crystal structure and functional analysis of the protein

disulfide isomerase-related protein ERp29. J Mol Biol

385(5):1630–1642. doi:10.1016/j.jmb.2008.11.052

3. Mkrtchian S, Sandalova T (2006) ERp29, an unusual redox-

inactive member of the thioredoxin family. Antioxid Redox

Signal 8:325–337

4. Bambang IF, Xu S, Zhou J, Salto-Tellez M, Sethi SK, Zhang D

(2009) Overexpression of endoplasmic reticulum protein 29

regulates mesenchymal–epithelial transition and suppresses

xenograft tumor growth of invasive breast cancer cells. Lab

Invest 89:1229–1242

5. Mkrtchian S, Baryshev M, Sargsyan E, Chatzistamou I, Volakaki

AA, Chaviaras N et al (2008) ERp29, an endoplasmic reticulum

secretion factor is involved in the growth of breast tumor xeno-

grafts. Mol Carcinog 47(11):886–892. doi:10.1002/mc.20444

6. Mkrtchian S, Fang C, Hellman U, Ingelman-Sundberg M (1998)

A stress-inducible rat liver endoplasmic reticulum protein,

ERp29. Eur J Biochem 251:304–313

7. Sargsyan E, Baryshev M, Szekely L, Sharipo A, Mkrtchian S

(2002) Identification of ERp29, an endoplasmic reticulum

lumenal protein, as a new member of the thyroglobulin folding

complex. J Biol Chem 277:17009–17015. doi:10.1074/jbc.

M200539200

8. Baryshev M, Sargsyan E, Mkrtchian S (2006) ERp29 is an

essential endoplasmic reticulum factor regulating secretion of

thyroglobulin. Biochem Biophys Res Commun 340:617–624

9. Das S, Smith TD, Das Sarma J, Ritzenthaler JD, Maza J, Kaplan

BE, Cunningham LA, Suaud L, Hubbard MJ, Rubenstein RC,

Koval M (2009) ERp29 restricts Connexin43 oligomerization in

the endoplasmic reticulum. Mol Biol Cell 20:2593–2604. doi:10.

1091/mbc.E08-07-0790

10. Kuznetsov G, Chen LB, Nigam SK (1994) Several endoplasmic

reticulum stress proteins, including Erp72, interact with thyro-

globulin during its maturation. J Biol Chem 269:22990–22995

11. Kwon OY, Park S, Lee W, You K-H, Kim H, Shong M (2000)

TSH regulates a gene expression encoding ERp29, an endoplas-

mic reticulum stress protein, in the thyrocytes of FRTL-5 cells.

FEBS Lett 475:27–30

12. Schroder M, Kaufman RJ (2005) ER stress and the unfolded

protein response. Mutat Res 569:29–63

13. Sevier CS, Kaiser CA (2008) Ero1 and redox homeostasis in the

endoplasmic reticulum. Biochim Biophys Acta 1783:549–556

14. Zhang B, Wang M, Yang Y, Wang Y, Pang X, Su Y, Wang J, Ai

G, Zou Z (2008) ERp29 is a radiation responsive gene in IEC-6

cell. J Radiat Res 49:587–596

15. Dukes AA, Van Laar VS, Cascio M, Hastings TG (2008)

Changes in endoplasmic reticulum stress proteins and aldolase A

in cells exposed to dopamine. J Neurochem 106:333–346. doi:10.

1111/j.1471-4159.2008.05392.x

16. Hung YC, Wang PW, Pan TL, Bazylak G, Leu YL (2009) Proteo-

mic screening of antioxidant effects exhibited by radix Salvia mil-

tiorrhiza aqueous extract in cultured rat aortic smooth muscle cells

under homocysteine treatment. J Ethnopharmacol 124:463–474.

doi:10.1016/j.jep.2009.05.020

17. Buettner GR (2011) Superoxide dismutase in redox biology: the

roles of superoxide and hydrogen peroxide. Anticancer Agents

Med Chem 11:341–346

18. Verfaillie T, Salazar M, Velasco G, Agostinis P (2010) Linking

ER stress to Autophagy: potential implications for cancer ther-

apy. Int J Cell Biol. doi:10.1155/2010/930509

19. Liu CY, Wong HN, Schauerte JA, Kaufman RJ (2002) The

protein kinase/endoribonuclease IRE1 that signals the unfolded

protein response has a luminal N-terminal ligand independent

dimerization domain. J Biol Chem 277:18346–18356

20. Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP,

Clark SG, Ron D (2002) IRE1 couples endoplasmic reticulum

load to secretory capacity by processing the XBP-1 mRNA.

Nature 415:92–96

21. Lee AH, Chu GC, Iwakoshi NN, Glimcher LH (2005) XBP-1 is

required for biogenesis of cellular secretory machinery of exo-

crine glands. EMBO J 24:4368–4380

22. Acosta-Alvear D, Zhou Y, Blais A, Tsikitis M, Lents NH, Arias

C, Lennon CJ, Kluger Y, Dynlacht BD (2007) XBP1 controls

diverse cell type- and condition-specific transcriptional regulatory

networks. Mol Cell 6:53–66

23. DuRose JB, Scheuner D, Kaufman RJ, Rothblum LI, Niwa M

(2009) Phosphorylation of eucaryotic translation initiation factor

2a coordinates rRNA transcription and translation inhibition during

endoplasmic reticulum stress. Mol Cell Biol 15:4295–4307. doi:10.

1128/MCB.00260-09

24. Haze K, Yoshida H, Yanagi H, Yura T, Mori K (1999) Mam-

malian transcription factor ATF6 is synthesized as a transmem-

brane protein and activated by proteolysis in response to

endoplasmic reticulum stress. Mol Biol Cell 10:3787–3799

25. Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R,

Brown MS, Goldstein JL (2000) ER stress induces cleavage of

membranebound ATF6 by the same proteases that process

SREBPs. Mol Cell 6:1355–1364

26. Chen X, Shen J, Prywes R (2002) The luminal domain of ATF6

senses endoplasmic reticulum (ER) stress and causes transloca-

tion of ATF6 from the ER to the Golgi. J Biol Chem 277:

13045–13052

27. Okada T, Haze K, Nadanaka S, Yoshida H, Seidah NG, Hirano Y,

Sato R, Negishi M, Mori K (2003) A serine protease inhibitor

prevents endoplasmic reticulum stress-induced cleavage but not

transport of the membrane-bound transcription factor ATF6.

J Biol Chem 278:31024–31032

28. Wei MC, Zong WX, Cheng EH, Lindsten T, PanoutsakopoulouV, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Kors-

meyer SJ (2001) Proapoptotic BAX and BAK: a requisite gate-

way to mitochondrial dysfunction and death. Science 292:

727–730

29. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA,

Yuan J (2000) Caspase-12 mediates endoplasmic-reticulum-spe-

cific apoptosis and cytotoxicity by amyloid-b. Nature 403:98–103

30. Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko

Y (2002) An endoplasmic reticulum stress-specific caspase cas-

cade in apoptosis. Cytochrome c-independent activation of cas-

pase-9 by caspase-12. J Biol Chem 277:34287–34294

31. Harding H, Zhang Y, Bertolotti A, Zeng H, Ron D (2000) PERK

is essential for translational regulation and cell survival during

the unfolded protein response. Mol Cell 5:897–904

32. Zinszner H, Kuroda M, Wang X, Batchvarova N, Lightfoot RT,

Remotti H, Stevens JL, Ron D (1998) CHOP is implicated in

programmed cell death in response to impaired function of the

endoplasmic reticulum. Genes Dev 12:982–995

33. Zhang D, Putti TC (2010) Over-expression of ERp29 attenuates

doxorubicin-induced cell apoptosis through the upregulation of

Hsp27 in breast cancer cells. Exp Cell Res 316:3522–3531.

doi:10.1016/j.yexcr.2010.08.014

34. Bambang IF, Lu D, Li H, Chiu LL, Lau QC, Koay E, Zhang D

(2009) Cytokeratin 19 regulates endoplasmic reticulum stress and

inhibits ERp29 expression via p38 MAPK/XBP-1 signaling in

breast cancer cells. Exp Cell Res 315:1964–1974. doi:10.1016/j.

yexcr.2009.02.017

35. Gao D, Bambang IF, Putti TC, Lee YK, Richardson DR, Zhang D

(2011) ERp29 induces breast cancer cell growth arrest and sur-

vival through modulation of activation of p38 and upregulation of

ER stress protein p58(IPK). Lab Invest 92:200–213. doi:10.1038/

labinvest.2011.163

Apoptosis (2014) 19:801–815 813

123

36. Guo Ch (2003) Generation of endoplasmic reticulum protein 28

(ERp28) knock out mice, and structural and functional analysis of

its Drosophila Homologue, Wind. Dissertation, Georg-August

University, Gottingen

37. Lichti U, Anders J, Yuspa SH (2008) Isolation and short-term

culture of primary keratinocytes, hair follicle populations and

dermal cells from newborn mice and keratinocytes from adult

mice for in vitro analysis and for grafting to immunodeficient

mice. Nat Protoc 3:799–810. doi:10.1038/nprot.2008.50

38. Jeker LT, Hejazi M, Burek CL, Rose NR, Caturegli P (1999)

Mouse thyroid culture. Biochem Biophys Res Commun 257:

511–515

39. Bjorkman U, Ekholm R (1982) Effect of tunicamycin on thyro-

globulin secretion. Eur J Biochem 125:585–591

40. Eggo MC, Burrow GN (1983) Glycosylation of thyroglobulin—

its role in secretion, iodination, and stability. Endocrinology

113:1655–1663

41. Kim PS, Bole D, Arvan P (1992) Transient aggregation of nas-

cent thyroglobulin in the endoplasmic reticulum: relationship to

the molecular chaperone, BiP. J Cell Biol 118:541–549

42. Kuznetsov G, Chen LB, Nigam SK (1997) Multiple molecular

chaperones complex with misfolded large oligomeric glucopro-

teins in the endoplasmic reticulum. J Biol Chem 272:3057–3063.

doi:10.1074/jbc.272.5.3057

43. Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in

endoplasmic reticulum stress. Cell Death Differ 11:381–389

44. Zamarbide M, Martinez-Pinilla E, Ricobaraza A, Aragon T,

Franco R, Perez-Mediavilla A (2013) Phenyl acyl acids attenuate

the unfolded protein response in tunicamycin-treated neuroblas-

toma cells. PLoS One 8:1–7

45. Van Gansen P, Van Lerberghe N (1988) Potential and limitations

of cultivated fibroblasts in the study of senescence in animals. A

review on the murine skin fibroblasts system. Arch Gerantol

Geriatr 7:31–74

46. Jones GE, Wise C (1997) Establishment, maintenance, and

cloning of human dermal fibroblasts. Methods in molecular

biology. Methods Mol Biol 75:13–24

47. Kim PS, Arvan P (1991) Folding and assembly of newly syn-

thesized thyroglobulin occurs in a pre-Golgi compartment. J Biol

Chem 266:12412–12418

48. Wegrowski Y, Perreau C, Martiny L, Haye B, Maquart FX,

Bellon G (1999) Transforming growth factor beta-1 up-regulates

clusterin synthesis in thyroid epithelial cells. Exp Cell Res 247:

475–483

49. Park S, Hwang I, Shong M, Kwon OY (2003) Identification of

genes in thyrocytes regulated by unfolded protein response by

using disulfide bond reducing agent of dithiothreitol. J Endocrinol

Invest 26:132–137

50. Lorenz S, Eszlinger M, Paschke R, Aust G, Weick M, Fuhrer D,

Krohn K (2010) Calcium signaling of thyrocytes is modulated by

TSH through calcium binding protein expression. Biochim Bio-

phys Acta 1803:352–360. doi:10.1016/j.bbamcr.2010.01.007

51. Malthiery Y, Marriq C, Berge-Lefranc JL, Franc JL, Henry M,

Lejeune PJ, Ruf J, Lissitzky S (1989) Thyroglobulin structure and

function: recent advances. Biochemie 71:195–209

52. Kim PS, Arvan P (1995) Calnexin and BiP act as sequential

chaperones during thyroglobulin folding in the endoplasmic

reticulum. J Cell Biol 128:29–38

53. Christis C, Fullaondo A, Schildknegt D, Mkrtchian S, Heck A,

Braakman I (2010) Regulates increase in folding capacity pre-

vents unfolded protein stress in the ER. J Cell Sci 123:787–794.

doi:10.1242/jcs.041111

54. Sargsyan E, Baryshev M, Mkrtchian S (2004) The physiological

unfolded protein response in the thyroid epithelial cells. Biochem

Biophys Res Commun. 322:570–576

55. Garrido JL, Maruo S, Takada K, Rosendorff A (2009) EBNA3C

interacts with GADD34 and counteracts the unfolded protein

response. Virol J 6:231. doi:10.1186/1743-422X-6-231

56. Rutkowski DT, Arnold SM, Miller CN, Wu J, Li J, Gunnison

KM, Mori K, Sadighi Akha AA, Raden D, Kaufman RJ (2006)

Adaptation to ER stress is mediated by differential stabilities of

pro-survival and pro-apoptotic m RNAs and proteins. PLoS Biol.

doi:10.1371/journal.pbio.0040374

57. Kincaid MM, Cooper AA (2007) ERADicate ER stress or die

trying. Antioxid Redox Signal 9:2373–2387

58. Novoa I, Zeng H, Harding HP, Ron D (2001) Feedback inhibition

of the unfolded protein response by GADD34-mediated dephos-

phorylation of eIF2a. J Cell Biol 153:1011–1021

59. Yan W, Frank CL, Korth MJ, Sopher BL, Novoa I, Ron D, Katze

MG (2002) Control of PERK eIF2a kinase activity by the

endoplasmic reticulum stress-induced molecular chaperone

P58IPK. PNAS 99:15920–15925. doi:10.1073/pnas.252341799

60. Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001)

XBP1 mRNA is induced by ATF6 and spliced by IRE1 in

response to ER stress to produce a highly active transcription

factor. Cell 107:881–891

61. Farmaki E, Mkrtchian S, Papazian I, Papavassiliou AG, Kiaris H

(2011) ERp29 regulates response to doxorubicin by a PERK-medi-

ated mechanism. Biochim Biophys Acta 1813(6):1165–1171. doi:10.

1016/j.bbamcr.2011.03.003

62. Long H, Han H, Yang B, Wang Z (2003) Opposite cell density-

dependence between spontaneous and oxidative stress-induced

apoptosis in mouse fibroblast L-cells. Cell Physiol Biochem

13:401–414. doi:10.1159/000075128

63. Sen P, Mukherjee S, Bhaumika G, Dasb P, Ganguly S, Cho-

udhury N, Raha S (2003) Enhancement of catalase activity by

repetitive low-grade H2O2 exposures protects fibroblasts from

subsequent stress-induced apoptosis. Mutat Res 529:87–94

64. Akaishi T, Nakazawa K, Sato K, Saito H, Ohno Y, Ito Y (2004)

Hydrogen peroxide modulates whole cell Ca2? currents through

L-type channels in cultured rat dentate granule cells. Neurosci

Lett 356:25–28. doi:10.1016/j.bbrc.2013.08.087

65. Malhotra JD, Kaufman RJ (2007) Endoplasmic reticulum stress

and oxidative stress: a vicious cycle or a double-edged sword?

Antioxid Redox Signal 9:2277–2293

66. Tu BP, Weissman JS (2004) Oxidative protein folding in eukaryotes

mechanisms and consequences. J Cell Biol 164:341–346. doi:10.

1083/jcb.200311055

67. Shnyder SD, Mangum JE, Hubbard MJ (2008) Triplex profiling

of functionally distinct chaperones (ERp29/PDI/BiP) reveals

marked heterogeneity of the endoplasmic reticulum proteome in

cancer. J Proteome Res 7:3364–3372

68. Sargsyan E, Baryshev M, Backlund M, Sharipo A, Mkrtchian S

(2002) Genomic organization and promoter characterization of

the gene encoding a putative endoplasmic reticulum chaperone,

ERp29. Gene 285:127–139

69. Ferrari DM, Soling HD (1999) The protein disulphide-isomerase

family: unravelling a string of folds. Biochem J 339:1–10

70. Mkrtchian S, Baryshev M, Matvijenko O, Sharipo A, Sandalova

T, Schneider G, Ingelman-Sundberg M (1998) Oligomerization

properties of ERp29, an endoplasmic reticulum stress protein.

FEBS Lett 431:322–326

71. Kobayashi M, Habuchi K, Yoneda M, Habuchi O, Kimata K

(1997) Molecular cloning and expression of Chinese hamster

ovary cell heparan-sulfate 2-sulfotransferase. J Biol Chem 272:

13980–13985

72. Sergeev P, Streit A, Heller A, Steinman-Zwicky M (2001) The

Drosophila dorsoventral determinant PIPE contains ten copies of

a variable domain homologous to mammalian heparan sulfate

2-sulfotransferase. Dev Dyn 220:122–132

814 Apoptosis (2014) 19:801–815

123

73. Ma Q, Guo C, Barnewitz K, Sheldrick GM, Soling HD, Uson I,

Ferrari DM (2003) Crystal structure and functional analysis of

Drosophila Wind, a protein-disulfide isomerase-related protein.

J Biol Chem 278:44600–44607

74. Barnewitz K, Guo C, Sevvana M, Ma Q, Sheldrick GM, Soling H-D,

Ferrari DM (2004) Mapping of a substrate-binding site in the protein

disulfide isomerase- related chaperone Wind based on protein

function and crystal structure. J Biol Chem 279:39829–39837

75. Sato Y, Nadanaka S, Okada T, Okawa K, Mori K (2011) Lumenal

domain of ATF6 alone is sufficient for sensing endoplasmic

reticulum stress and subsequent transport to the Golgi apparatus.

Cell Struct Funct 36:35–47. doi:10.1247/csf.10010

76. Nadanaka S, Yoshida H, Kano F, Murata M, Mori K (2004)

Activation of mammalian unfolded protein response is compati-

ble with the quality control system operating in the endoplasmic

reticulum. Mol Biol Cell 15:2537–2548. doi:10.1091/mbc.E03-

09-0693

77. Nadanaka S, Yoshida H, Mori K (2006) Reduction of disulfide

bridges in the lumenal domain of ATF6 in response to glucose

starvation. Cell Struct Funct 31:127–134. doi:10.1247/csf.06024

78. Nadanaka S, Okada T, Yoshida H, Mori K (2007) Role of disulfide

bridges formed in the lumenal domain of ATF6 in sensing endo-

plasmic reticulum stress. Mol Cell Biol 27:1027–1043. doi:10.1128/

MCB.00408-06

79. Wright J, Birk J, Haataja L, Liu M, Ramming T, Weiss MA,

Appenzeller-Herzog C, Arvan P (2013) Endoplasmic Reticulum

Oxidoreductin-1a (Ero1a) Improves folding and secretion of

mutant proinsulin and limits mutant proinsulin-induced endo-

plasmic reticulum stress. J Biol Chem 288:31010–31018. doi:10.

1074/jbc.M113.510065

80. Rabeler R, Mittag J, Geffers L, Ruther U, Leitges M, Parlow AF, Visser

TJ, Bauer K (2004) Generation of thyrotropin-releasing hormone

receptor 1-deficient mice as an animal model of central hypothyroid-

ism. Mol Endocrinol 18:1450–1460. doi:10.1210/me.2004-0017

Apoptosis (2014) 19:801–815 815

123