Selective, potent blockade of the IRE1 and ATF6 pathways by 4-phenylbutyric acid analogues

Article abstract of DOI:10.1111/bph.12306

Background and Purpose 4-Phenylbutyric acid (4-PBA) is a chemical chaperone that eliminates the accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, its chaperoning ability is often weak and unable to attenuate the unfolded protein response (UPR) in vitro or in vivo. To develop more potent chemical chaperones, we synthesized six analogues of 4-PBA and evaluated their pharmacological actions on the UPR. Experimental Approach NRK-52E cells were treated with ER stress inducers (tunicamycin or thapsigargin) in the presence of each of the 4-PBA analogues; the suppressive effects of these analogues on the UPR were assessed using selective indicators for individual UPR pathways. Key Results 2-POAA-OMe, 2-POAA-NO₂ and 2-NOAA, but not others, suppressed the induction of ER stress markers GRP78 and CHOP. This suppressive effect was more potent than that of 4-PBA. Of the three major UPR branches, the IRE1 and ATF6 pathways were markedly blocked by these compounds, as indicated by suppression of XBP1 splicing, inhibition of UPRE and ERSE activation, and inhibition of JNK phosphorylation. Unexpectedly, however, these agents did not inhibit phosphorylation of PERK and eIF2α triggered by ER stress. These compounds dose-dependently inhibited the early activation of NF-κB in ER stress-exposed cells. 2-POAA-OMe and 2-POAA-NO₂ also inhibited ER stress-induced phosphorylation of Akt. Conclusion and Implications The 4-PBA analogues 2-POAA-OMe, 2-POAA-NO₂ and 2-NOAA strongly inhibited activation of the IRE1 and ATF6 pathways and downstream pathogenic targets, including NF-κB and Akt, in ER stress-exposed cells. These compounds may be useful for therapeutic intervention in ER stress-related pathological conditions.

Full text of DOI:10.1111/bph.12306

2013-BJP-0294-RP Zhang H et al.
Selective, Potent Blockade of the IRE1 and ATF6 Pathways by
4-Phenylbutyric Acid Analogues1
Hui Zhang,1 Shotaro Nakajima,1 Hironori Kato,1 Liubao Gu,1,4 Tatsuya Yoshitomi,1
Kaoru Nagai,3 Hideyuki Shinmori,2 Susumu Kokubo2 and Masanori Kitamura1
1Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine
and Engineering, University of Yamanashi, Chuo, Yamanashi, 409-3898, Japan;
2Division of Medical and Engineering Science, Interdisciplinary Graduate School of
Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi,
3
400-8511, Japan; Department of Epigenetic Medicine, Interdisciplinary Graduate
School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi,
4
409-3898, Japan; Diabetes Care and Research Center, Jiangsu Province Institute of
Geriatrics, Nanjing 210024, China
Running title: Selective blockade of UPR by 4PBA analogues
Address correspondence to: Masanori Kitamura, M.D., Ph.D., Department of
This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process, which may lead to differences between
this version and the Version of Record. Please cite this article as doi: 10.1111/bph.12306
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2013-BJP-0294-RP Zhang H et al.
Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering,
University of Yamanashi, Shimokato 1110, Chuo, Yamanashi 409-3898, Japan
Tel: +81-55-273-8054; Fax: +81-55-273-8054; E-mail: masanori@yamanashi.ac.jp
Summary
BACKGROUND AND PURPOSE
4-Phenylbutyric acid (4-PBA) is a chemical chaperone that eliminates accumulation of
unfolded proteins in the endoplasmic reticulum (ER). However, its chaperoning
potential is often weak and insufficient to attenuate the unfolded protein response (UPR)
in vitro and in vivo. Towards development of more potent chemical chaperones, we
synthesized six analogues of 4-PBA, and their pharmacological actions on the UPR
were evaluated.
EXPERIMENTALAPPROACH
NRK-52E cells were treated with ER stress inducers (tunicamycin or thapsigargin) in
the presence of individual 4-PBA analogues, and their suppressive effects on the UPR
were assessed using selective indicators for individual UPR pathways.
KEY RESULTS
2-POAA-OMe, 2-POAA-NO2 and 2-NOAA, but not others, suppressed induction of ER
stress markers GRP78 and CHOP. This suppressive effect was more potent than that of
4-PBA. Among three major UPR branches, the IRE1 and ATF6 pathways were
markedly blocked by these compounds, which was evidenced by suppression of XBP1
splicing, inhibition of UPRE and ERSE activation and inhibition of JNK
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2013-BJP-0294-RP Zhang H et al.
phosphorylation. Unexpectedly, however, these agents did not inhibit phosphorylation
of PERK and eIF2α triggered by ER stress. In ER stress-exposed cells, early activation
of NF-κB was inhibited by these compounds dose-dependently. 2-POAA-OMe and
2-POAA-NO2 also inhibited ER stress-induced phosphorylation of Akt.
CONCLUSIONS AND IMPLICATIONS
We developed 4-PBA analogues that strongly inhibit activation of the IRE1 and ATF6
pathways and downstream pathogenic targets including NF-κB and Akt in ER
stress-exposed cells. These compounds would be useful for efficient therapeutic
intervention in ER stress-related pathological processes.
Keywords: 4-phenylbutyric acid; chemical chaperone; endoplasmic reticulum (ER)
stress; unfolded protein response (UPR); IRE1; ATF6; PERK–eIF2α
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2013-BJP-0294-RP Zhang H et al.
Abbreviations: ER, endoplasmic reticulum; 4-PBA, 4-phenylbutyric acid; UPR,
unfolded protein response; ATF6, activating transcription factor 6; IRE1,
inositol-requiring enzyme 1; PERK, PKR-like ER kinase; XBP1, X-box-binding protein
1; ERSE, ER stress response element; UPRE, UPR element; GRP78, 78 kDa
glucose-regulated protein; eIF2α, eukaryotic translation initiation factor 2α; CHOP,
CCAAT/enhancer-binding protein-homologous protein; NF-κB, nuclear factor-κB; JNK,
c-Jun N-terminal kinase; 2-POAA, 2-phenoxyacetic acid; 2-POAA-OMe,
2-(4-methoxyphenoxy) acetic acid; 2-POAA-OMeNa, sodium 2-(4-methoxyphenoxy)
acetate; 2-POAA-NO2, 2-(4-nitrophenoxy) acetic acid; 2-PyA, 2-(pyridinium-1-yl)
acetate; 2-NOAA, 2-(2-naphathyloxy) acetic acid; TNF-α, tumor necrosis factor-α;
SubAB, AB5 subtilase cytotoxin; SAHA, suberoylanilide hydroxamic acid; VPA,
valproic acid; NaB, sodium butyrate; SV40, simian virus 40; ORP150, 150 kDa
oxygen-regulated protein; MCP-1, monocyte chemoattractant protein 1; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; MAP kinase, mitogen-activated protein
kinase; HDAC, histone deacetylase; TRAF2, TNF receptor-associated factor 2
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2013-BJP-0294-RP Zhang H et al.
Introduction
The endoplasmic reticulum (ER) is an intracellular compartment responsible for
post-translational modification and appropriate folding of newly synthesized proteins.
Pathophysiological insults such as hypoxia, redox imbalance, change of calcium
homeostasis and excessive protein synthesis disturb ER homeostasis, leading to
accumulation of unfolded and/or misfolded proteins in the ER, namely ER stress. ER
stress-mediated tissue dysfunction is implicated in a wide range of diseases including
cancers, infection, atherosclerosis, ischemic injury, neurodegenerative disorders and
metabolic diseases such as diabetes mellitus (Aridor and Balch, 1999). Seeking agents
that attenuate ER stress is a challenge towards effective treatment of ER stress-related
disorders.
Chemical chaperones are a group of compounds that have the potential to stabilize
proteins and/or to facilitate protein folding. Among these, 4-phenylbutyric acid (4-PBA)
is a well-known chemical chaperone. Several reports have described use of 4-PBA as a
chaperone to reverse the mislocalization and/or aggregation of proteins associated with
human diseases (Burrows et al., 2000; Rubenstein and Zeitlin, 2000; Perlmutter, 2002).
For example, 4-PBA reversed accumulation of misfolded proteins in the ER, including
ΔF508 cystic fibrosis transmembrane conductance regulator (Rubenstein and Zeitlin,
2000) and mutant α1-antitrypsin (Burrows et al., 2000). However, the chaperoning
potential of 4-PBA is often weak and insufficient to attenuate ER stress. More efficient
chemical chaperones would be required for effective attenuation of ER stress in vitro
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2013-BJP-0294-RP Zhang H et al.
and in vivo.
On the membrane of the ER, there are three major transducers for the unfolded
protein response (UPR); activating transcription factor 6 (ATF6), inositol-requiring
enzyme 1 (IRE1) and PKR-like ER kinase (PERK) (Rutkowski and Kaufman, 2004). In
response to ER stress, ATF6 transits to the Golgi where it is cleaved by S1P and S2P,
yielding a free cytoplasmic domain that functions as a transcription factor. Similarly,
activated IRE1α catalyzes removal of a small intron from X-box-binding protein 1
(XBP1) mRNA. This splicing creates a translational frameshift in XBP1 to produce an
active transcription factor. Activated ATF6 and XBP1 subsequently bind to the ER
stress response element (ERSE) and the UPR element (UPRE), leading to expression of
target genes including 78 kDa glucose-regulated protein (GRP78). Activation of PERK
leads to phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and
consequent
induction
of
ER
stress-responsive
molecules
including
CCAAT/enhancer-binding protein-homologous protein (CHOP) (Walter and Ron,
2011).
In the present report, we aimed at developing potent chemical chaperones for
effective attenuation of pathogenic UPR. We synthesized six analogues of 4-PBA,
evaluated their effects on individual UPR and identified three active compounds. Our
current results revealed that three 4-PBA analogues strongly inhibit induction of the
IRE1 and ATF6 pathways and downstream pathogenic events including activation of
nuclear factor-κB (NF-κB) and phosphorylation of Akt in ER stress-exposed cells.
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2013-BJP-0294-RP Zhang H et al.
Methods
Reagents
2-Phenoxyacetic acid (2-POAA), 2-(4-methoxyphenoxy) acetic acid (2-POAA-OMe),
sodium 2-(4-methoxyphenoxy) acetate (2-POAA-OMeNa), 2-(4-nitrophenoxy) acetic
acid (2-POAA-NO2), 2-(pyridinium-1-yl) acetate (2-PyA) and 2-(2-naphathyloxy)
acetic acid (2-NOAA) were synthesized by hydrolysis of corresponding acetic acid
ethyl esters in 24 - 75 % yields, which were obtained through a Williamson reaction or
quaternization between phenolic compounds or pyridine and ethyl bromoacetate
(Al-Amiery et al., 2011). The chemical structure and purity (95 - 99 %) of these
1
compounds were confirmed by H NMR analysis. Chemical structure of individual
reagents is shown in Figure 1. 4-PBA was purchased from Calbiochem (San Diego, CA),
tunicamycin and thapsigargin were from Sigma-Aldrich Japan (Tokyo, Japan), and
tumor necrosis factor-α (TNF-α) was from R&D Systems (Minneapolis, MN). AB5
subtilase cytotoxin (SubAB) (Paton et al., 2006) was kindly provided by Dr. James C.
Paton (University of Adelaide, Australia). Valproic acid (VPA) was purchased from MP
Biomedicals (Santa Ana, CA), sodium butyrate (NaB) was from Alfa Aesar (Ward Hill,
MA), and suberoylanilide hydroxamic acid (SAHA) was from Cayman Chemical (Ann
Arbor, MI).
Cell culture
The rat renal tubular epithelial cell line NRK-52E was purchased from American Type
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2013-BJP-0294-RP Zhang H et al.
Culture Collection (Manassas, VA). Cells were maintained in Dulbecco’s modified
Eagle’s medium//Ham’s F-12 (Gibco-BRL, Gaithersburg, MD) supplemented with 5 %
fetal bovine serum.
Establishment of stable transfectants
Using electroporation, NRK-52E cells were stably transfected with pcDNA3.1
(Invitrogen, Carlsbad, CA) together with pUPRE-Luc (provided by Dr. Laurie H.
Glimcher, Harvard Medical School), pERSE-Luc (provided by Dr. Laurie H Glimcher)
(Lee et al., 2003) or pNFκB-Luc (Panomics; Fremont, CA) that introduces a luciferase
gene under the control of UPRE, ERSE or the κB site, respectively. Stable transfectants
were selected by G418 (Sigma-Aldrich Japan), and reporter clones NRK/UPRE-Luc
cells, NRK/ERSE-Luc cells and NRK/NFκB-Luc cells were established.
Transient transfection
To evaluate cleavage of XBP1 mRNA, NRK-52E cells were transiently transfected with
pCAX-F-XBP1∆DBD-Luc (provided by Dr. Takao Iwawaki, Gunma University, Japan)
(Iwawaki and Akai, 2006) by electroporation. After 24 h, cells were seeded into 96-well
plates, incubated for 24 h and subjected to stimulation. As a control, NRK-52E cells
transfected with pSV40-Luc (pGL3-Control; Promega, Madison, WI) were used.
pSV40-Luc introduces a luciferase gene under the control of the simian virus 40 (SV40)
promoter/enhancer.
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2013-BJP-0294-RP Zhang H et al.
Luciferase assay
Activity of luciferase was evaluated by Luciferase Assay System (Promega), according
to the manufacturer’s protocol.
Formazan Assay
The number of viable cells was assessed by a formazan assay using Cell Counting Kit-8
(Dojindo Laboratory, Kumamoto, Japan).
Northern blot analysis
Total RNA was extracted by a single-step method, and Northern blot analysis was
performed as described before (Kitamura et al., 2011). cDNAs for GRP78 (provided by
Dr. Kazunori Imaizumi, Hiroshima University, Japan) (Katayama et al., 2001), CHOP
(provided by Dr. David Ron, University of Cambridge, UK) (Wang et al., 1998),
GRP94 (provided by Dr. Amy S Lee, University of South California) (Gazit et al.,
1999), 150 kDa oxygen-regulated protein (ORP150) (provided by Dr. Satoshi Ogawa,
Kanazawa University, Japan) (Ozawa et al., 1999), monocyte chemoattractant protein 1
(MCP-1) (Rollins et al., 1988) and firefly luciferase were used to prepare radio-labeled
probes. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and levels
of 28S ribosomal RNA were used as loading controls.
Western blot analysis
Western blot analysis was performed as described previously (Kato et al., 2012).
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2013-BJP-0294-RP Zhang H et al.
Primary antibodies used were; anti-phospho-eIF2α (Ser51) antibody from Cell
Signaling Technology (Beverly, MA), and anti-phospho-PERK (Thr980) antibody,
anti-PERK antibody, anti-eIF2α antibody, anti-ATF4 (CREB-2) and anti-XBP1
antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Levels of phosphorylated
Akt and c-Jun N-terminal kinase (JNK) were evaluated using PhosphoPlus Akt (Ser473)
Antibody Kit and PhosphoPlus SAPK/JNK (Thr183/Tyr185) Antibody Kit, according to
protocols provided by the manufacturer (Cell Signaling Biotechnology). As loading
controls, levels of β-actin and lamin B1 were evaluated using anti-β actin antibody
(Sigma-Aldrich Japan) and anti-lamin B1 antibody (Invitrogen, Carlsbad, CA). Nuclear
proteins were extracted using ProteoExtract Subcellular Proteome Extraction Kit
(Merck KGaA, Darmstadt, Germany).
RT-PCR
Reverse transcription was performed using Omniscript Reverse Transcriptase (Qiagen,
Valencia, CA). Splicing of XBP1 mRNA was examined using following primers;
5’-ACACGCTTGGGGATGAATGC-3’ and 5’-CCATGGGAAGATGTTCTGGG-3’
(Sigma-Aldrich Japan), as described before (Kato et al., 2012).
Analysis of histone deacetylase (HDAC) activity
HDAC activity was evaluated by Cyclex HDACs Deacetylase Fluorometric Assay Kit
(Cyclex, Nagano, Japan) under the manufacturer’s instruction. Briefly, each compound
was mixed with fluorescence-labeled substrate peptide, HDACs and lysylendopeptidase,
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2013-BJP-0294-RP Zhang H et al.
and incubated for 15 min. Fluorescence intensity was measured at excitation
wavelength 355 nm and emission wavelength 460 nm using a fluorescence microplate
reader (Molecular Devices Japan, Tokyo, Japan).
Statistical analysis
In reporter assays and microscopic analysis, experiments were performed in
quadruplicate, and data were expressed as means ± SE. Statistical analysis was
performed using the non-parametric Mann-Whitney U test to compare data in different
groups. P value < 0.05 was considered to indicate a statistically significant difference.
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2013-BJP-0294-RP Zhang H et al.
Results
Suppression of ER stress response by 4-PBA analogues
4-PBA has been used as a chemical chaperone to attenuate ER stress in vitro and in vivo.
However, based on our experience, the potential of 4-PBA is often weak and
insufficient to attenuate ER stress responses. Towards development of more efficient
chemical chaperones, we synthesized 6 analogues of 4-PBA, and their effects on the
UPR were examined. NRK-52E cells were stimulated by an ER stress inducer
thapsigargin (inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase) in the absence or
presence of 4-PBA analogues; 2-POAA, 2-POAA-OMe, 2-POAA-OMeNa,
2-POAA-NO2, 2-PyA and 2-NOAA. The cells were then subjected to Northern blot
analysis of endogenous ER stress markers, GRP78 and CHOP. As shown in Figures 2B,
2D and 2F, 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA suppressed the induction of
GRP78 and CHOP in a dose-dependent manner. Among these 3 compounds, 2-NOAA
was most effective. In contrast, the suppressive effect was not observed by other 4-PBA
analogues; 2-POAA, 2-POAA-OMeNa and 2-PyA (Figures 2A, 2C and 2E).
To further confirm this result, cells were exposed to another ER stress inducer
tunicamycin (inhibitor of protein glycosylation), and effects of the 4-PBA analogues
were re-tested. Consistent with the results in thapsigargin-treated cells, induction of
GRP78 and CHOP by tunicamycin was suppressed by 2-POAA-OMe, 2-POAA-NO2
and 2-NOAA (Figures 2G - 2I). Like the effects on thapsigargin-induced ER stress,
2-NOAA was most effective. The suppressive effect was not observed by other 4-PBA
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2013-BJP-0294-RP Zhang H et al.
analogues 2-POAA, 2-POAA-OMeNa and 2-PyA.
We compared the potential of 2-NOAA with that of 4-PBA to attenuate ER stress.
Cells were treated with thapsigargin in the presence of serial concentrations of 2-NOAA
or 4-PBA and subjected to Northern blot analysis. The induction of GRP78 and CHOP
by ER stress was suppressed by both 2-NOAA and 4-PBA in a dose-dependent manner.
However, the suppressive effect of 2-NOAA was more potent than that of 4-PBA
(Figure 2J). The similar, superior effects were also observed in 2-POAA-OMe and
2-POAA-NO2, when compared with 4-PBA (data not shown). The depression of ER
stress markers by 2-POAA-NO2 was associated with dramatic attenuation of
cytoplasmic vacuolation (a typical morphological feature of renal tubular cells under ER
stress in vitro and in vivo; Kitamura et al., 2011; Kato et al., 2012) (Figures 2K, 2L),
which was not obvious by the treatment with 4-PBA (our unpublished observation).
Influence of 4-PBA analogues on IRE1 pathway
ER stress causes induction of three major branches of the UPR; IRE1, ATF6 and
PERK–eIF2α pathways. In the IRE1 pathway, activation of IRE1α at its
endoribonuclease domain removes a small intron from XBP1 mRNA, leading to
production of functional XBP1 that triggers activation of UPRE. Activation of IRE1α at
its kinase domain also recruits TNF receptor-associated factor 2 (TRAF2) and thereby
causes phosphorylation of JNK (Walter and Ron, 2011). To examine effects of
2-POAA-OMe, 2-POAA-NO2 and 2-NOAA on the IRE1 pathway, cells were treated
with ER stress inducers in the presence of 4-PBA analogues and subjected to analysis of
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2013-BJP-0294-RP Zhang H et al.
XBP1 splicing, activity of UPRE and phosphorylation of JNK. As shown in Figure 3A,
splicing of XBP1 mRNA induced by tunicamycin was suppressed by the treatment with
2-POAA-OMe, 2-POAA-NO2 and 2-NOAA in a dose-dependent manner. Among these
compounds, 2-NOAA was most effective. Similarly, the three agents inhibited splicing
of XBP1 elicited by thapsigargin dose-dependently where the effect of 2-NOAA was
most evident (Figure 3B). RT-PCR analysis also showed that splicing of XBP1 mRNA
caused by ER stress was suppressed by the treatment with 2-NOAA (Figure 3C).
Nuclear translocation of XBP1 protein by ER stress was also blocked by this agent
(Figure 3D). Consistent with these results, treatment with three compounds inhibited
activation of UPRE by ER stress (Figure 3E). Quantitative analysis showed that
2-NOAA most effectively inhibited activation of UPRE by thapsigargin (IC50=1.59
mM) (Table 2). The inhibition was less by 2-POAA-OMe (IC50=1.92 mM) and by
2-POAA-NO2 (IC50=2.06 mM). The inhibitory potential of both agents was higher than
that of 4-PBA (IC50=2.31 mM). This effect was not ascribed to non-specific
suppression of transcription or translation, because activity of luciferase driven by the
SV40 promoter was not inhibited significantly by the treatment with these 4-PBA
analogues (Figure 3F).
The suppression of the IRE1 pathway by 4-PBA analogues was further confirmed
using JNK as an indicator. As shown in Figure 3G, thapsigargin-induced
phosphorylation of JNK was inhibited by all three compounds. The suppressive effect
was obvious and dose-dependent in 2-POAA-OMe and 2-POAA-NO2. Unexpectedly,
the suppressive effect of 2-NOAA, the most potent inhibitor of GRP78 and CHOP, was
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2013-BJP-0294-RP Zhang H et al.
weak. Paradoxically, the higher concentration of 2-NOAA was less effective.
Subsequent experiments revealed that treatment with 2-NOAA, but not others, induced
phosphorylation of JNK dose-dependently (Figure 3H). This effect was independent of
ER stress, because, 2-NOAA, as well as 2-POAA-OMe and 2-POAA-NO2, did not
induce expression of GRP78 and CHOP (Figure 3I) and activation of UPRE and ERSE
(Figures 3J, 3K).
Suppression of ATF6 pathway by 4-PBA analogues
Under ER stress conditions, ATF6 translocates to the Golgi apparatus where it is
cleaved into an active transcription factor. Activated ATF6 moves into the nucleus and
promotes ERSE activity, leading to expression of target genes including ER chaperones
GRP78, GRP94 and ORP150 (Yoshida et al., 1998). To examine effects of
2-POAA-OMe, 2-POAA-NO2 and 2-NOAA on the ATF6 pathway, cells were treated
with ER stress inducers in the presence of the 4-PBA analogues and subjected to
Northern blot analysis. As shown in Figures 4A and 4B, ER stress-triggered induction
of GRP78, GRP94 and ORP150 was suppressed by the treatment with these compounds.
Of note, 2-NOAA was most effective among the three agents.
To further confirm this result, activity of ERSE was evaluated using reporter cells.
Consistent with the results by Northern blot, treatment with all three agents completely
suppressed activation of ERSE by ER stress (Figure 4C). It indicated that the ATF6
pathway may be a preferential target for the suppressive effects of the 4-PBA analogues.
Indeed, a dose-response study showed that 2-NOAA significantly inhibited activation of
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2013-BJP-0294-RP Zhang H et al.
ERSE at 0.5 mM (Figure 4D). Consistent with the result in Northern blot analysis,
2-NOAA most effectively inhibited activation of ERSE by thapsigargin (IC50=0.48 mM)
(Table 2). The inhibition was less by 2-POAA-OMe (IC50=1.88 mM) and by
2-POAA-NO2 (IC50=1.93 mM). The inhibitory potential of both agents was higher than
that of 4-PBA (IC50=2.59 mM).
Lack of inhibition of PERK and eIF2α phosphorylation by 4-PBA analogues
The PERK–eIF2α pathway is another branch of the UPR triggered by ER stress. We
further examined effects of 4-PBA analogues on the PERK–eIF2α pathway. Cells were
treated with thapsigargin in the absence or presence of 2-POAA-OMe, 2-POAA-NO2 or
2-NOAA and subjected to Western blot analysis of phosphorylated PERK. The results
showed that, unexpectedly, none of the 4-PBA analogues inhibited phosphorylation of
PERK in thapsigargin-treated cells (Figures 5A - 5C). Consistent with this result,
treatment with these agents did not suppress phosphorylation of eIF2α by ER stress
(Figures 5D - 5F). To elucidate mechanisms underlying this unpredicted finding, we
examined effects of 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA on the activity of
PERK and eIF2α under non-stress conditions. We found that treatment with 4-PBA
analogues induced phosphorylation of PERK dose-dependently (Figure 5G). 2-NOAA
exhibited the most potent effect. Consistent with the result of PERK, phosphorylation of
eIF2α was also induced by these agents (Figure 5H). These results suggest that the three
4-PBA analogues differentially regulate the major branches of the UPR; i.e., these
compounds selectively block activation of the IRE1 and ATF6 pathways without
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2013-BJP-0294-RP Zhang H et al.
suppression of PERK and eIF2α.
Effects of 4-PBA analogues on ER stress-triggered NF-кB activation and Akt
phosphorylation
ER stress and the UPR contribute to pathological processes through several different
mechanisms. One of the most important downstream targets is NF-κB that plays a role
in the development of various inflammatory and malignant diseases (Zhang and
Kaufman, 2008; Kitamura, 2009). Previous reports demonstrated that NF-κB activation
is induced by ER stress, at least in part, through ATF6-mediated transient
phosphorylation of Akt (Yamazaki et al., 2009). We therefore examined whether and
how the 4-PBA analogues modulate NF-κB activation triggered by ER stress. Treatment
of reporter cells with thapsigargin induced activation of NF-κB, and it was suppressed
by 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA in a dose-dependent manner (Figure
6A). Among three agents, 2-NOAA was most effective. The suppressive effects of these
compounds on NF-κB were further confirmed by Northern blot analysis (Figure 6B).
Similar suppressive effects were also observed in the cells treated with a more selective
inducer of ER stress, SubAB. That is, treatment of reporter cells with SubAB induced
activation of NF-κB, and it was suppressed by 2-POAA-OMe, 2-POAA-NO2 and
2-NOAA dose dependently (Figure 6C). Like in thapsigargin-treated cells, 2-NOAA
was most effective. This result was further supported by Northern blot analysis of the
reporter gene (Figure 6D). Of note, treatment with 4-PBA analogues alone did not affect
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2013-BJP-0294-RP Zhang H et al.
basal NF-κB activity (Figure 6E). It is also worth noting that activation of NF-κB by
TNF-α was never suppressed by the 4-PBA analogues, except for modest inhibition by
the higher concentration of 2-NOAA (Figure 6F).
As described, activation of NF-κB by ER stress is, at least in part, through
modulation of Akt phosphorylation. Akt is a multifunctional serine/threonine protein
kinase that regulates cell metabolism, survival and proliferation and immune cell
functions including production of inflammatory cytokines. Because of this reason, Akt
plays important roles in the development of cancers and inflammation (Lee et al., 2011;
Cheung and Testa, 2013). We further tested whether activation of Akt by ER stress is
influenced by the 4-PBA analogues. Western blot analysis showed that treatment with
2-POAA-OMe and 2-POAA-NO2 suppressed Akt phosphorylation induced by ER stress.
However, 2-NOAA did not show any suppressive effects on Akt (Figure 6G). We found
that the lack of suppression of Akt by this compound was ascribed to its stimulatory
effect on Akt. As shown in Figure 6H, 2-NOAA, but not others, increased
phosphorylation of Akt under non-stress condition.
ER stress contributes to inflammatory processes and tumorigenesis via activation
of NF-κB and Akt. This current concept raises a possibility that 2-POAA-OMe,
2-POAA-NO2 and 2-NOAA may be useful for therapeutic intervention in ER
stress-related, NF-κB/Akt-mediated pathologies including inflammations and cancers.
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2013-BJP-0294-RP Zhang H et al.
Discussion
Various pathologic insults cause ER stress that is involved in the pathogenesis of a wide
range of diseases. Seeking substances that attenuate ER stress is an important challenge
for therapeutic intervention in ER stress-related disorders. Towards development of
potent inhibitors of the UPR, we synthesized analogues of 4-PBA, and their potential
was evaluated. Among the six compounds tested, 2-POAA-OMe, 2-POAA-NO2 and
2-NOAA were identified to be powerful, selective inhibitors of the IRE1 and ATF6
pathways and downstream pathogenic targets including NF-κB and Akt in ER
stress-exposed cells. Our current findings were summarized in Table 1.
In the tested compounds, the aromatic ring was bound to the carboxyl end group
by an ether linkage (pyridinium in the case of 2-PyA) having shorter alkyl chain than
that of 4-PBA. In particular, 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA contain the
polar group and condensed ring, respectively. Their suppressive effects on the UPR
might be correlated with the nonionic carboxyl group and the electronic state changed
by the polar group and condensed ring on the phenyl group.
ER stress transduces signals through the three major branches of the UPR; IRE1,
ATF6 and PERK pathways. In theory, chemical chaperones reduce the level of ER stress
and thereby attenuate activation of individual UPR branches under stress conditions.
Indeed, all three compounds efficiently suppressed the ATF6 pathway to the same extent.
However, their effects on the IRE1 pathway were somewhat different from substance to
substance. The kinase activity of IRE1α indicated by JNK phosphorylation was
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2013-BJP-0294-RP Zhang H et al.
inhibited by 2-POAA-NO2 most effectively, and 2-POAA-OMe and 2-NOAA were less
efficient. In contrast, the endoribonuclease activity of IRE1α, indicated by XBP1
splicing and UPRE activation, was inhibited by 2-NOAA most effectively, and the
suppressive effects of 2-POAA-NO2 and 2-POAA-OMe were modest. Interestingly,
2-NOAA, but not others, rather induced activation of IRE1α kinase without activation
of IRE1α endoribonuclease. 2-NOAA, but not others, also activated Akt. Currently, it is
unclear how 2-NOAA induces phosphorylation of JNK and Akt. However, like
2-NOAA, 4-PBA also have stimulatory effects on JNK and Akt. For example, Roque et
al. (2008) showed that 4-PBA induced expression of IL-8 via activation of JNK in lung
epithelial cells. Kim et al. (2012) reported that 4-PBA triggered phosphorylation of Akt
in MCF-7 cells. Like butyrate, 4-PBA is a well-known HDAC inhibitor that activates a
variety of signaling molecules (Davie, 2003). Previous reports indicated the potential of
HDAC inhibitors to activate JNK via generation of reactive oxygen species or
deacetylation-mediated inhibition of mitogen-activated protein kinase phosphatase 1
(Sharma et al., 2010; Yu et al., 2003; Chi and Flavell, 2008). Similar mechanisms might
be involved in the activation of JNK and Akt by 2-NOAA and 4-PBA. It is worthwhile
to note that, contrastive to 2-NOAA and 4-PBA, 2-POAA-NO2 and 2-POAA-OMe did
not activate JNK. Similarly, 2-NOAA activated Akt, whereas 2-POAA-NO2 and
2-POAA-OMe did not. In addition to their enhanced potential to inhibit the UPR
branches, the lack of JNK and Akt activation may be another important property of
these compounds, which certifies their superiority over the prototypic 4-PBA.
The current, most unexpected finding is that, although all three 4-PBA analogues
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2013-BJP-0294-RP Zhang H et al.
effectively inhibited ER stress-induced expression of GRP78 and CHOP, they did not
affect phosphorylation of PERK and eIF2α by ER stress. Subsequent experiments
revealed that these compounds per se up-regulated PERK and eIF2α phosphorylation.
We found that the similar phenomenon was also observed in the cells treated by the
prototypic 4-PBA (Supplemental Figures S1A, S1B). Currently, underlying
mechanisms are unknown. However, Kahali et al. recently proposed one possibility.
They identified that GRP78 is a possible molecular target of some HDAC inhibitor; i.e.,
treatment with SAHA led to GRP78 acetylation, dissociation, and subsequent activation
of its client protein PERK (Kahali et al., 2010). Downstream activation of eIF2α was
also observed. The similar mechanism could be ascribed to phosphorylation of the
PERK and eIF2α by the 4-PBA analogues and 4-PBA.
To examine whether or not the effects of 4-PBA analogues on PERK and eIF2α
are mediated by inhibition of HDAC, influences of these compounds on HDAC activity
were evaluated in vitro. 4-PBA and NaB were used as positive controls. Like 4-PBA and
NaB, 2-POAA-NO2 significantly inhibited HDAC activity. In contrast, 2-POAA-OMe
and 2-NOAA, both of which have the similar potential to activate PERK and eIF2α,
failed to inhibit HDAC (Supplemental Figure S2). This result suggests that activation of
PERK and eIF2α by the 4-PBA analogues is independent of HDAC activity. To further
confirm this result, cells were treated with known HDAC inhibitors VPA, NaB or
SAHA and subjected to analysis of phosphorylated PERK and eIF2α. Western blot
analysis showed that none of these HDAC inhibitors triggered activation of the
PERK–eIF2α pathway in NRK-52E cells (Supplemental Figure S3), confirming
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2013-BJP-0294-RP Zhang H et al.
HDAC-independent action of the 4-PBA analogues. To date, there is no information
regarding mechanisms underlying selective activation of PERK by 4-PBA and its
analogues, but they could facilitate dimerization of PERK directly or dissociation of
GRP78 from PERK, leading to its selective phosphorylation.
In general, activation of the PERK–eIF2α pathway contributes to the survival of
cells under ER stress conditions, mainly due to translational suppression (Harding et al.,
2000). However, the PERK–eIF2α pathway may also contribute to cellular damage by
induction and activation of the ATF4–CHOP pathway (Fawcett et al., 1999; Ma et al.,
2002). An interesting finding in the current report is that, although 4-PBA analogues
activated the PERK–eIF2α pathway, induction of CHOP by ER stress was markedly
suppressed by the treatment with these compounds. It was associated with suppression
of nuclear translocation of ATF4 (Supplemental Figure S4). In the regulation of the
PERK branch by the 4-PBA analogues, therefore, additional unknown mechanisms
should exist and underlie disruption of the link between phosphorylation of
PERK/eIF2α and activation of ATF4. 4-PBA analogues could suppress
transcription/translation of ATF4 or its nuclear transport. Further investigation is
required to elucidate this issue, which would be our next step of investigation.
ER stress is known to cause activation of NF-κB. We found that all 4-PBA
analogues suppressed ER stress-induced NF-κB activation. Previous reports showed
that some HDAC inhibitors may inhibit activation of NF-κB triggered by various
stimuli (Glauben et al., 2009). For example, SAHA can suppress NF-κB activation
triggered by TNF-α, IL-1β, lipopolysaccharide, phorbol myristate acetate and reactive
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2013-BJP-0294-RP Zhang H et al.
oxygen species (Takada et al., 2006). However, in our experimental setting, the
suppressive effect of the 4-PBA analogues on NF-κB was not via HDAC inhibition but
conceivably via attenuation of ER stress, because they did not suppress activation of
NF-κB triggered by TNF-α (Figure 6F).
ER stress is involved in the development of various diseases. Clinical utility of
4-PBA has been reported by experimental studies on the treatment of ER stress-related
hereditary and acquired disorders (Maestri et al., 1996; Rubenstein and Zeitlin, 1998;
Burrows et al., 2000). However, its clinical use is still limited because of its insufficient
potential for the regulation of the UPR. Our present data suggest that the 4-PBA
analogues, described here, may provide more efficient tools for the treatment of ER
stress-related, NF-κB/Akt-mediated pathologies including intractable inflammations
and malignant diseases.
23
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2013-BJP-0294-RP Zhang H et al.
Acknowledgments
We thank Dr. Laurie H. Glimcher, Dr. Kazunori Imaizumi, Dr. Takao Iwawaki, Dr. Amy
S Lee, Dr. Satoshi Ogawa and Dr. David Ron for providing us with plasmids. We also
appreciate a kind gift of SubAB by Dr. James Paton. This work was supported by
Strategic Research Project Grant from University of Yamanashi to M. Kitamura and H.
Shinmori, and in part, by Grant-in-Aids for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan (No. 20390235) to M.
Kitamura.
Conflicts of Interest
None
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2013-BJP-0294-RP Zhang H et al.
Table 1. Influences of 4-PBA analogues on ER stress responses
Non-stress condition
ER stress response
2-POAA-OMe
2-POAA-NO2
2-NOAA
Kinase
IRE1
→
→
→
↑
→
→
→
↑
↑
→
→
↑
Nuclease
ATF6
PERK-eIF2α
NF-κB
Akt
→
→
→
→
→
↑
ER stress condition
ER stress response
2-POAA-OMe
2-POAA-NO2
2-NOAA
Kinase
IRE1
↓
↓
↓
↓
↓
↓
Nuclease
ATF6
↓
↓
↓
PERK-eIF2α
NF-κB
Akt
→
↓
→
↓
→
↓
↓
↓
→
↑ up-regulation; ↓ down-regulation; → no change
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2013-BJP-0294-RP Zhang H et al.
Table 2. IC50 values of 4-PBA analogues and 4-PBA
2-POAA-OMe
(mM)
2-POAA-NO2
(mM)
2-NOAA
(mM)
4-PBA
(mM)
0.48
ERSE activity
UPRE activity
1.88
1.92
1.93
2.06
2.59
2.31
1.59
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2013-BJP-0294-RP Zhang H et al.
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