Novel potential of tunicamycin as an activator of the aryl
hydrocarbon receptor – dioxin responsive element signaling pathway
Kyohei Horikawa, Naoki Oishi, Jin Nakagawa, Ayumi Kasai, Kunihiro Hayakawa,
Nobuhiko Hiramatsu, Yosuke Takano, Makiko Yokouchi, Jian Yao, Masanori Kitamura*
Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo,
Yamanashi 409-3898, Japan
Received 15 April 2006; revised 19 May 2006; accepted 19 May 2006
Available online 6 June 2006
Edited by Robert Barouki
cosylation and used as an inducer of endoplasmic reticulum (ER)
stress. We found that tunicamycin induced expression of cyto-
chrome P450 1A1 in a dose-dependent manner. Like dioxin,
the transcriptional induction was associated with dose-dependent
activation of the dioxin responsive element (DRE). This effect
was independent of inhibition of protein glycosylation or induc-
tion of ER stress. Pharmacological and genetic inhibition of
the aryl hydrocarbon receptor (AhR) significantly attenuated
activation of DRE by tunicamycin. These results elucidated the
novel potential of tunicamycin as an activator of the AhR –
DRE signaling pathway.
? ? 2006 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Tunicamycin is a well-known inhibitor of protein gly-
Keywords: Tunicamycin; Cytochrome P450; Dioxin responsive
element; Aryl hydrocarbon receptor
Tunicamycin has been extensively used as inhibitor of lipid
carrier-dependent protein glycosylation and, currently, is
widely used as an inducer of endoplasmic reticulum (ER) stress
. Tunicamycin was originally purified from Streptomyces
lysosuperificus as an antibiotic that inhibits cell wall polymer
synthesis . However, its therapeutic usefulness is limited be-
cause of toxicity in mammals. For example, tunicamycin is
hepatotoxic and causes a periportal pattern of damage result-
ing in denudation of hepatocytes into blood vessels, and for-
mation of pulmonary and cerebral emboli . ER stress may
be involved in the toxic effects of tunicamycin, but underlying
mechanisms have not been identified yet.
Cytochrome P450 1A1 (CYP1A1) is a member of the multi-
gene family of xenobiotic metabolizing enzymes . Beside its
CYP1A1 is also responsible for metabolic activation of polycy-
clic aromatic hydrocarbons and aromatic amines, leading to
generation of genotoxic substances . For example, benzo[a]-
pyrene induces CYP1A1 which in turn metabolizes benzo[a]-
pyrene per se to reactive, carcinogenic intermediates. Mice
having the higher CYP1 levels were, therefore, more suscepti-
ble to various aromatic hydrocarbons-induced pathologies
including liver injury .
regulated by the aryl hydrocarbon receptor (AhR), the ligand-
activating transcription factor. Binding of xenobiotic ligands
[e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD)] to
AhR triggers nuclear translocation of AhR and causes binding
of the AhR-ligand complex to the AhR nuclear translocator in
the nuclei. This molecular event results in binding of the com-
plexes to the dioxin responsive element (DRE, also termed as
xenobiotic responsive element; XRE) in the 50flanking region
of various target genes including cyp1a1 .
Recently, we unexpectedly found that tunicamycin induced
expression of cyp1a1 in a murine hepatoma cell line Hepa-
1c1c7. In the present report, we elucidate molecular mecha-
nisms involved in this novel potential of tunicamycin. Our
study especially focuses on involvement of distinct cellular
events that can be triggered by tunicamycin; i.e., protein hypo-
glycosylation, ER stress and activation of the AhR – DRE
rolein thedetoxificationof xenobiotics,
2. Materials and methods
2.1. Cells and reagents
Murine hepatoma cell line Hepa-1c1c7 was purchased from Amer-
ican Type Culture Collection (ATCC; Manassas, VA). The reporter
cell line HeXS34 was established by transfection of Hepa-1c1c7 cells
with pXRE-SEAP, as described previously . pXRE-SEAP encodes
secreted alkaline phosphatase (SEAP) under the control of four
copies of XRE/DRE . 2,3,7,8-TCDD and 2-deoxy-glucose were
obtained from Wako Pure Chemical Industries (Osaka, Japan), and
other reagents were purchased from Sigma–Aldrich, Japan (Tokyo,
Japan). All experiments were performed using a-minimum essential
medium (Invitrogen, Carlsbad, CA) supplemented with 1% fetal
2.2. Transient transfection
Using lipofectamine 2000 (Invitrogen), Hepa-1c1c7 cells were tran-
siently co-transfected with pGL3-XRE-Luc  together with pEFBOS
or pEFBOS-AhR(Arg39Ile) encoding a dominant-negative mutant of
AhR . pGL3-XRE-Luc encodes firefly luciferase under the control
Abbreviations: ER, endoplasmic reticulum; CYP1A1, cytochrome P4-
50 1A1; AhR, aryl hydrocarbon receptor; 2,3,7,8-TCDD, 2,3,7,8-tet-
rachlorodibenzo-p-dioxin; DRE, dioxin responsive element; SEAP,
secreted alkaline phosphatase; XRE, xenobiotic responsive element;
GRP78, 78-kD glucose-regulated protein; GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; AhRDN, dominant-negative mutant of
*Corresponding author. Fax: +81 55 273 8054.
E-mail address: email@example.com (M. Kitamura).
0014-5793/$32.00 ? 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 580 (2006) 3721–3725
of consensus sequences of XRE/DRE. Forty eight hours after the
transfection, the cells were treated with tunicamycin (10 lg/ml) or
2,3,7,8-TCDD (100 pM) for 6 h and subjected to luciferase assay, as
described later. Assays were performed in quadruplicate.
2.3. Northern blot analysis
Total RNA was extracted by the single-step method, and Northern
blot analysis was performed as described before . cDNAs for
CYP1A1 , SEAP (BD Biosciences, Palo Alto, CA) and 78-kDa
glucose-regulated protein (GRP78)  were used to prepare radio-la-
beled probes. Expression of glyceraldehyde-3-phosphate dehydroge-
nase (GAPDH) was used as a loading control. Densitometric
analysis was performed using Scion Image (Scion Corporation, Fred-
2.4. Luciferase assay
Activity of luciferase was evaluated by Luciferase Assay System
(Promega, Madison, WI). In brief, according to the manufacturer’s
protocol, cells were lysed by lysis buffer, and activity of luciferase
was evaluated in the presence of luciferin using a luminometer (Gene
Light 55; Microtech Nition, Chiba, Japan).
2.5. Statistical analysis
Data were expressed as means ± S.E. Statistical analysis was per-
formed 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.
3.1. Induction of cyp1a1 mRNA by tunicamycin
We first examined a dose-dependent effect of tunicamycin on
the expression of cyp1a1. Hepa-1c1c7 cells were exposed to 0–
5 lg/ml of tunicamycin for 6 h and subjected to Northern blot
analysis. As shown in Fig. 1A, expression of cyp1a1 mRNA
was induced by tunicamycin in a dose-dependent manner. Sub-
stantial induction was observed at concentrations higher than
0.5 lg/ml and peaked to maximum at 2.5–5 lg/ml. Time-lapse
experiments revealed that the induction of cyp1a1 was tran-
sient; i.e., it was peaked at 3 h and declined thereafter (Fig. 1B).
Fig. 2. Dose- and time-dependent activation of the dioxin responsive element (DRE) by tunicamycin. Hepa-1c1c7-derived HeXS34 cells that express
secreted alkaline phosphatase (SEAP) under the control of DRE consensus sequences were exposed to either indicated concentrations of tunicamycin
for 6 h (A) or 5.0 lg/ml tunicamycin for indicated time periods (B), and expression of SEAP was examined by Northern blot analysis.
Fig. 1. Dose- and time-dependent induction of cytochrome P450 1A1 (CYP1A1) mRNA by tunicamycin. Hepa-1c1c7 cells were exposed to either
indicated concentrations of tunicamycin for 6 h (A) or 5.0 lg/ml tunicamycin for indicated time periods (B), and expression of cyp1a1 was examined
by Northern blot analysis. The expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown at the bottom as a loading
Fig. 3. Lack of involvement of endoplasmic reticulum stress and protein hypoglycosylation in the effects of tunicamycin. (A) HeXS34 cells were
treated with 1–10 lg/ml tunicamycin for 6 h, and expression of SEAP and grp78 was examined by Northern blot analysis. (B) HeXS34 cells were
treated with 5 lg/ml tunicamycin, 2 mM castanospermine or 6 mg/ml 2-deoxy-glucose for 6 h, and expression of cyp1a1 and grp78 was examined.
K. Horikawa et al. / FEBS Letters 580 (2006) 3721–3725
Fig. 4. Involvement of aryl hydrocarbon receptor (AhR) in the activation of DRE and induction of cyp1a1 by tunicamycin. (A and B) Effects of
resveratrol on 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD)- and tunicamycin-induced activation of DRE and expression of cyp1a1. HeXS34
cells were pretreated with 0–30 lM resveratrol for 30 min, exposed to 25 pM 2,3,7,8-TCDD or 5 lg/ml tunicamycin for 3 h and subjected to Northern
blot analysis of SEAP and cyp1a1. Intensity of individual signals normalized by the levels of GAPDH is shown in (B). (C) Hepa-1c1c7 cells were
transiently co-transfected with pGL3-XRE-Luc together with pEFBOS (closed bars) or pEFBOS-AhR (Arg39Ile) encoding a dominant-negative
mutant of AhR (shaded bars). Forty-eight hours after the transfection, the cells were treated with tunicamycin (10 lg/ml) or 2,3,7,8-TCDD (100 pM)
for 6 h and subjected to luciferase assay. Assays were performed in quadruplicate. Data are expressed as means ± S.E. Asterisks indicate statistically
significant differences (P < 0.05). RLU, relative light unit.
K. Horikawa et al. / FEBS Letters 580 (2006) 3721–3725
3.2. Activation of DRE by tunicamycin
The 50-flanking region of the cyp1a1 gene contains DREs.
These DREs are essential for transcriptional induction of
cyp1a1 by aromatic hydrocarbons including dioxins [4,7]. We
next examined whether tunicamycin can trigger activation of
DRE. For this purpose, Hepa-1c1c7-derived HeXS34 cells
were used. This reporter line expresses SEAP under the control
of the DRE/XRE consensus sequences . As shown in
Fig. 2A, tunicamycin induced activation of DRE in a dose-
dependent manner, which was closely correlated with the
expression level of cyp1a1 (Fig. 1A). The activation was
peaked at 6 h and declined thereafter (Fig. 2B). The similar re-
sult was also obtained using a different Hepa-1c1c7-derived re-
porter cells, HeDS49 , that express SEAP under the control
of the truncated promoter of the mouse cyp1a1 gene that con-
tains 4 DREs (data not shown).
3.3. Lack of involvement of protein hypoglycosylation and ER
stress in the effects of tunicamycin
Tunicamycin inhibits transfer of N-acetylglucosamine-1-
phosphate from uridine 5-diphosphate-N-acetylglucosamine
to dolichol monophosphate in the first step of glycoprotein
synthesis and thereby blocks formation of protein-carbohy-
drate linkages of the N-glycosidic type . Accumulation of
hypoglycosylated proteins in the ER subsequently causes ER
stress and induces the so-called unfolded protein responses
including expression of GRP78 . We examined whether pro-
tein hypoglycosylation or ER stress is causative of induction of
cyp1a1 and activation of DRE by tunicamycin. First, HeXS34
cells were treated with different concentrations of tunicamycin,
and the levels of SEAPand grp78, an endogenous marker of
ER stress, were compared by Northern blot analysis. As
shown in Fig. 3A, the level of ER stress indicated by grp78
was not in parallel with the activation level of DRE indicated
by SEAP. For example, 1 lg/ml of tunicamycin fully induced
ER stress whereas activation of DRE was not obvious. This re-
sult indicated that activation of DRE by tunicamycin may be
independent of ER stress. To confirm this conclusion, HeXS34
cells were treated with different glycosylation inhibitors, cast-
anospermine and 2-deoxy-glucose, and expression of cyp1a1
and grp78 was examined. Like tunicamycin, both castanosper-
mine and 2-deoxy-glucose substantially induced ER stress indi-
cated by expression of grp78. However, both agents did not
cause induction of cyp1a1 (Fig. 3B). These results suggested
that neither protein hypoglycosylation nor ER stress was caus-
ative of activation of the DRE pathway by tunicamycin.
3.4. Involvement of AhR in the activation of DRE and expression
of cyp1a1 by tunicamycin
In general, DRE is activated via binding of AhR-xenobiotics
complexes [4,7]. We tested whether functional AhR is required
for the effects of tunicamycin on the activation of DRE and
expression of cyp1a1. For this purpose, we used resveratrol,
a pure antagonist of AhR . As shown in Fig. 4A and B,
2,3,7,8-TCDD induced activation of DRE and expression of
cyp1a1 in HeXS34 cells. This induction was inhibited by the
treatment with resveratrol modestly at 20 lM and markedly
at 30 lM. Like its suppressive effect on 2,3,7,8-TCDD, expres-
sion of cyp1a1 triggered by tunicamycin was substantially
inhibited by the treatment with resveratrol, and it was associ-
ated with suppression of DRE activation evidenced by attenu-
ated SEAP mRNA.
To further confirm the involvement of AhR in the effects of
tunicamycin, Hepa-1c1c7 cells were transiently transfected with
a dominant-negative mutant of AhR (AhRDN) together with a
luciferase-based DRE reporter plasmid. Northern blot analysis
confirmed high levels of expression of AhRDN in the transfec-
ted cells (data not shown). Using these cells, the effect of tunica-
affect the basal luciferase activity. As expected, functional sup-
pression of AhR by AhRDN significantly inhibited activation
of DRE triggered by 2,3,7,8-TCDD (35.0 ± 17.5% vs. 100% in
control, P < 0.05). AhRDN also substantially inhibited activa-
tionofDREtriggeredbytunicamycin(39.7 ± 19.8%vs.100%in
control, P < 0.05) (Fig. 4C). These results provided additional
evidence that tunicamycin has the potential for activation of
the AhR – DRE signaling pathway.
Tunicamycin has been used for many years as an inducer of
ER stress as well as an inhibitor of protein glycosylation in the
fields of cell biology and biochemistry. In the present report,
we elucidated the novel potential of tunicamycin as an activa-
tor of the AhR – DRE signaling pathway. A number of previ-
ous reports showed crucial roles of the AhR – DRE pathway
in various pathologies caused by halogenated and polycyclic
aromatic hydrocarbons [4,7]. These environmental substances
cause toxic effects via induction of DRE-regulated genes that
inhibit mitosis, induce apoptosis and generate toxic metabo-
lites. DREs are also located in the 50-regulatory regions of a
variety of inflammation-associated genes including: (1) inter-
leukins and chemokines; (2) cytokine receptors and adhesion
receptors; (3) enzymes involved in synthesis of nitric oxide,
prostaglandins and lipoxygenases; and (4) molecules involved
in the NF-jB pathway . Therefore, the current finding that
tunicamycin has the potential for activating the DRE – AhR
pathway raises a possibility that this agent may affect not only
protein glycosylation and ER function but also influence a di-
verse range of other cellular events. For example, in vivo toxic-
ity of tunicamycin in the liver and the lung  could be caused
by the activation of the AhR – DRE pathway. It should be
emphasized that the triggering effect of tunicamycin on the
AhR – DRE pathway must be considered carefully if this agent
is used as a ‘‘selective’’ inhibitor of protein glycosylation and/
or inducer of ER stress under in vitro and in vivo situations.
Acknowledgements: Kyohei Horikawa, Naoki Oishi and Jin Nakagawa
are medical students at University of Yamanashi and were equally con-
tributed to this work. We thank Dr. Shigeaki Kato (University of To-
kyo), Dr. Kazunori Imaizumi (Nara Institute of Science and
Technology) and Dr. Kazuhiro Sogawa (Tohoku University) for pro-
viding with reporter and expression plasmids. We also thank Dr.
Kaoru Nagai (University of Yamanashi) for helpful suggestions. This
work was supported in part by a grant from the Smoking Research
Foundation and by Grants-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and Technology,
Japan (Nos. 1639243 and 17651026 to M. Kitamura).
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