From the School of Pharmacy, the
§ Environmental Toxicology Center, the
Waisman
Center, and the ** Center for Neuroscience, University of Wisconsin,
Madison, Wisconsin 53706 and the ¶ Department of Pharmacology,
Toxicology, and Therapeutics, University of Kansas Medical Center,
Kansas City, Kansas 66160-7417
Received for publication, January 25, 2001, and in revised form, March 21, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The antioxidant-responsive element (ARE) plays an
important role in the induction of phase II detoxifying enzymes
including NADPH:quinone oxidoreductase (NQO1). We report herein that
activation of the human NQO1-ARE (hNQO1-ARE) by
tert-butylhydroquinone (tBHQ) is mediated by
phosphatidylinositol 3-kinase (PI3-kinase), not extracellular
signal-regulated kinase (Erk1/2), in IMR-32 human neuroblastoma cells.
Treatment with tBHQ significantly increased NQO1 protein without
activation of Erk1/2. In addition, PD 98059 (a selective
mitogen-activated kinase/Erk kinase inhibitor) did not inhibit
hNQO1-ARE-luciferase expression or NQO1 protein induction by tBHQ.
Pretreatment with LY 294002 (a selective PI3-kinase inhibitor), however, inhibited both hNQO1-ARE-luciferase expression and endogenous NQO1 protein induction. In support of a role for PI3-kinase in ARE
activation we show that: 1) transfection of IMR-32 cells with constitutively active PI3-kinase selectively activated the ARE in a
dose-dependent manner that was completely inhibited by
treatment with LY 294002; 2) pretreatment of cells with the PI3-kinase
inhibitors, LY 294002 and wortmannin, significantly decreased
NF-E2-related factor 2 (Nrf2) nuclear translocation induced by
tBHQ; and 3) ARE activation by constitutively active PI3-kinase was
blocked completely by dominant negative Nrf2. Taken together,
these data clearly show that ARE activation by tBHQ depends on
PI3-kinase, which lies upstream of Nrf2.
The antioxidant-responsive element
(ARE)1 plays an important
role in transcriptional activation of several phase II detoxifying enzymes such as NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) (1, 2). The consensus ARE core
sequence in the human NQO1 gene (5'-TGACTCAGC-3') is very similar to
the DNA binding sequence for NF-E2-related factor 2 (Nrf2,
5'-TGAGTCA-3'). Several lines of evidence suggest that Nrf2
binds to the ARE sequence (3-7). Nrf2 was originally cloned
using an AP1-NF-E2 tandem repeat as a recognition site probe and
belongs to the basic leucine zipper family of transcription factors
(8). Itho et al. (9) suggest that Nrf2 is sequestered
in the cytoplasm by Keap1 protein and that oxidative stress releases
Nrf2 from the Nrf2-Keap1 complex, resulting in nuclear
translocation of Nrf2. Recently our laboratory showed that
activation of the human NQO1-ARE depends on Nrf2 and that
tert-butylhydroquinone (tBHQ) dramatically induces
Nrf2 nuclear translocation in human neuroblastoma cells (10).
Although the role of Nrf2 in ARE activation seems evident, the
upstream regulatory mechanisms by which ARE-activating signals are
linked to Nrf2 and how this transcription factor is released
from the Nrf2-Keap1 complex remain to be elucidated.
Extracellular signal-regulated kinase (Erk1/2) is a member of the
mitogen-activated protein (MAP) kinases, a serine/threonine kinase
family (11, 12). Erk1/2 plays an important role in the regulation of
cell growth and differentiation (13-16). Activation of Erk1/2
culminates in the phosphorylation of downstream factors such as
p90RSK, c-Myc, and Elk-1, which control various
cellular processes (17-19). Although there are several reports
attempting to address the relationship between MAP kinases and ARE
activation, the role of MAP kinases in ARE activation remains
controversial, and the mechanism by which MAP kinases drive ARE
activation through Nrf2 is unresolved.
Phosphatidylinositol 3-kinase (PI3-kinase) phosphorylates
phosphatidylinositol at the D-3 position of the inositol ring
and has been shown to form a heterodimer consisting of a 85 kDa
(adapter protein) and 110 kDa (catalytic) subunit (20, 21). The role of
PI3-kinase in intracellular signaling has been underscored by its
implication in a plethora of biological responses such as cell growth,
differentiation, apoptosis, calcium signaling, and insulin signaling
(21-25). Among the downstream targets of PI3-kinase are phospholipase
C and the serine/threonine kinase Akt (22, 23, 26, 27). Akt (protein
kinase B), one of the most well known downstream targets of
PI3-kinases, protects cells from apoptosis by the phosphorylation and
inhibition of the Bad protein (28, 29). Based on these diverse effects
of PI3-kinase (especially protective effects) and because the induction
of phase II enzymes is thought to be a protective response in cells, we were interested in determining whether PI3-kinase is involved in ARE
regulation. The present investigation was designed, therefore, to
distinguish between the roles of Erk1/2 and PI3-kinase in ARE regulation using IMR-32 human neuroblastoma cells.
Materials--
tert-butylhydroquinone (tBHQ) was
obtained from Acros Organics (St. Louis, MO). PD 98059, LY 294002, wortmannin, and insulin were purchased from Calbiochem. Antibodies for
phospho-Erk1/2, Erk1/2, and phospho-GSK-3 Plasmids and Expression Vectors--
The reporter gene fusion
construct for human NQO1-ARE (hNQO1-ARE-luciferase;
5'-CTCAGCCTTCCAAATCGCAGTCACAGTGACTCAGCAGAATC-3') was made as described
previously (30). The mammalian expression vector for dominant negative
(DN) Nrf2 was described previously (10). Plasmids for
constitutively active PI3-kinase p110* (CA PI3-kinase) and
kinase-deficient PI3-kinase p110* Transient Transfection and Reporter Gene Activity
Assay--
IMR-32 human neuroblastoma cells (ATCC, CCL-127) were
plated at a density of 2.5 × 104 cells/well in
96-well plates and grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. Transient transfections were
performed using the calcium phosphate methods as described previously
(10). To investigate the role of PI3-kinase, IMR-32 cells were
cotransfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well), CMV- Preparation of Whole Cell and Nuclear Extracts--
IMR-32 human
neuroblastoma cells were plated at a density of 2.0 × 106 cells/10-cm dish and grown in 10 ml of Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine
serum. Cells were treated with various chemicals as described in
each figure legend. After washing two times with cold
phosphate-buffered saline, whole cell extracts and nuclear extracts
were prepared as described previously (10).
Immunocomplex Kinase Assay of Akt--
Akt enzymatic activity
was measured using a commercially available Akt-kinase assay kit (New
England Biolabs) using GSK-3 Western Immunoblot Analysis--
For Western immunoblot
analysis, a whole cell extract (for NQO1, phospho-Erk1/2, Erk1/2, and
phospho-GSK-3 Cytotoxicity Assay--
Cytotoxicity was measured using an MTS
cytotoxicity assay kit (Promega, Madison, WI) according to the protocol
provided by the manufacturer.
tBHQ Does Not Increase Erk1/2 Phosphorylation--
To investigate
the relationship between Erk1/2 activation and ARE activation, we
treated IMR-32 cells with a vehicle (0.01% Me2SO in
phosphate-buffered saline) or tBHQ (10 µM) and carried out time-course Western immunoblot analysis for the phosphorylation of
Erk1/2 as well as for the induction of NQO1. As shown in Fig. 1, A and B, tBHQ
did not increase phosphorylation of Erk1/2 compared with
vehicle-treated controls (Fig. 1A) or effect the level of Erk1/2 protein (Fig. 1B) up to 24 h. In contrast,
endogenous NQO1 protein induction was significant by 12 h in
tBHQ-treated groups (Fig. 1C). It should be noted that the
phosphorylation status of Erk1/2 varied with time in both vehicle- and
tBHQ-treated cells (Fig. 1A). We think the change of the
basal level of phospho-Erk1/2 might be caused by a cell cycle effect or
a challenge of temperature change during treatment. Irrespective of
this observation, tBHQ never consistently increased phospho-Erk1/2 when
compared with the appropriate vehicle-treated cells in repeated
experiments (data not shown). These data suggest that tBHQ treatment
increase endogenous NQO1 protein without changing Erk1/2 activity.
A Selective PI3-kinase Inhibitor, LY 294002, Inhibits ARE
Activation--
Initially, to evaluate the involvement of PI3-kinase
in ARE activation, we used a selective PI3-kinase inhibitor, LY 294002. As shown in Fig. 2, A and
B, pretreatment with LY 294002 inhibited both tBHQ-mediated
NQO1-ARE-luciferase expression and endogenous NQO1 increase in a
dose-dependent manner, suggesting a positive role for
PI3-kinase in ARE activation. These concentrations of LY 294002 had no
effect on cell viability as determined by the MTS cytotoxicity assay
(data not shown).
ARE Activation by tBHQ Is Not Inhibited by PD 98059--
To
evaluate the involvement of Erk1/2 in ARE regulation further, we
transiently transfected IMR-32 cells with hNQO1-ARE-luciferase and
treated them with PD 98059, a selective inhibitor of MAP/Erk kinase.
tBHQ (10 µM) treatment resulted in a 57-fold increase in
hNQO1-ARE-luciferase expression that was significantly inhibited by
pretreatment with LY 294002 (Fig.
3A). In contrast, pretreatment with PD 98059 (50 µM) did not inhibit ARE activation by
tBHQ (Fig. 3A). Similarly, endogenous NQO1 protein induction
by tBHQ (10 µM) was decreased by only LY 294002 pretreatment (Fig. 3B). tBHQ did not increase the level of
Erk1/2 phosphorylation, and PD 98059 completely blocked the basal level
of phospho-Erk1/2 (Fig. 3C), indicating that PD 98059 was
functioning as expected.
Erk1/2 Activation Does Not Correlate with Inhibition of ARE
Activation--
Interestingly, LY 294002 increased Erk1/2
phosphorylation (Fig. 4A),
raising the possibility that increased Erk1/2 activity may actually be
contributing to the inhibitory effect of LY 294002. To test this
hypothesis, IMR-32 cells were treated with nerve growth factor (NGF), a
potent activator of Erk1/2 (32, 33). NGF was very effective at
increasing Erk1/2 phosphorylation in IMR-32 cells (Fig. 4B),
but NGF treatment neither increased hNQO1-ARE reporter gene expression
nor inhibited ARE activation by tBHQ up to 500 ng/ml (Fig.
4C). Similarly, treatment with LY 294002 strongly activated
Erk1/2 (Fig. 4A), but LY 294002 significantly inhibited
hNQO1-ARE-luciferase expression as well as NQO1 protein increase by
tBHQ (Figs. 2 and 3).
tBHQ Does Not Increase Akt Activity or GSK-3 PI3-kinase Regulates ARE--
More direct evidence that PI3-kinase
is involved in ARE activation is presented in Figs.
7 and 8.
First, in IMR-32 cells transfected with
CA PI3-kinase or KD PI3-kinase, only the CA PI3-kinase increased hNQO1-ARE reporter gene expression (Fig. 7A), and that
induction was inhibited completely by treatment with LY 294002 (Fig.
7B). Second, we recently reported that tBHQ treatment
effectively induces nuclear translocation of Nrf2 in IMR-32
cells (10). Pretreatment of IMR-32 cells with the PI3-kinase inhibitors
LY 294002 or wortmannin significantly decreased Nrf2 nuclear
translocation induced by tBHQ (Fig. 8A). Finally, ARE
activation mediated by CA PI3-kinase was blocked completely by DN
Nrf2 (Fig. 8B), suggesting that Nrf2 is
downstream of PI3-kinase in IMR-32 cells. KD PI3-kinase did not show a
dominant negative effect on endogenous PI3-kinase, and ARE activation
by tBHQ was not inhibited by KD PI3-kinase (data not shown).
In this study, we clearly showed that activation of the hNQO1-ARE
by tBHQ is mediated by PI3-kinase, not Erk1/2, in IMR-32 human
neuroblastoma cells. tBHQ treatment increased hNQO1 protein without
changing phospho-Erk1/2, and inhibition of Erk1/2 phosphorylation did
not effect hNQO1-ARE-luciferase expression or hNQO1 protein induction.
Selective PI3-kinase inhibitors, however, significantly decreased both
ARE activation and nuclear translocation of Nrf2 by tBHQ. In
addition, ARE activation by constitutively active PI3-kinase was
blocked completely by dominant negative Nrf2, demonstrating the
critically important role for PI3-kinase in
Nrf2-dependent ARE activation.
Recent publications have looked at the relationship between MAP kinases
and the regulation of the ARE. Yu et al. (38) reported increased Erk2 activity by tBHQ and positive regulation of ARE by
Erk1/2 in HepG2 cells. In contrast, Alam et al. (39)
reported that cadmium increased phospo-Erk1/2, but inhibition of Erk1/2 did not effect heme oxygenase-1 induction in MCF-7 cells. Recently, Zipper and Mulcahy (4) published evidence that phospho-Erk1/2 was
increased by pyrrolidine dithiocarbamate and proposed a positive role
for Erk1/2 in the regulation of the Despite the controversy surrounding the MAP kinases, it is quite clear
that Nrf2 and its translocation to the nucleus are principal to
ARE activation in all cell types (3, 4, 10, 39, 42, 43). Nrf2
has been hypothesized to be sequestered by its cytoplasmic binding
partner Keap1 (9). We have shown that tBHQ treatment dramatically
increased Nrf2 nuclear translocation (10), suggesting that
Nrf2 is released from Keap1 by treatment with tBHQ in IMR-32
cells. However, the mechanism by which Nrf2 is released from the
Nrf2-Keap1 complex is not characterized fully. One hypothesis is
that protein modification (such as oxidation at cysteine residues) by
oxidative stress releases Nrf2 from the Nrf2-Keap1
complex (7, 9, 44). Previously, we demonstrated that pretreatment of
antioxidants or antioxidant enzymes did not inhibit hNQO1-ARE
activation by tBHQ in IMR-32 cells, implying that hNQO1-ARE activation
by tBHQ does not involve oxidative stress (10). A second hypothesis is
based on data from Huang et al. (42), who reported that tBHQ
or phorbol 12-myristate 13-acetate treatment induced the
phosphorylation of Nrf2 through a protein kinase
C-dependent mechanism leading the release of Nrf2.
Again, data from our laboratory suggest that protein kinase C is not involved in ARE activation in IMR-32 cells (30). A third hypothesis brings us back to the MAP kinases and the potential phosphorylation of
Nrf2 at several identified MAP kinase phosphorylation consensus sites throughout the Nrf2 protein (45). Although the relevance of these putative phosphorylation sites has not been demonstrated, Yu
et al. (46) have shown that dominant negative Nrf2
blocks MAP/Erk kinase kinase 1-mediated induction of heme oxygenase-1 and activation of the mouse GSTA1-ARE in HepG2 cells.
In this study, we implicate PI3-kinase, not MAP kinase, as a key
regulatory protein leading to Nrf2 nuclear translocation and
subsequent ARE activation in IMR-32 human neuroblastoma cells. Recently, Kang et al. (40) reported that selective
PI3-kinase inhibitors decreased rat GSTA2 mRNA induction by sulfur
amino acid deprivation in H4IIE rat hepatoma cells. Because the rat GSTA2 promoter contains an ARE, these data are consistent with our
finding that NQO1 induction by tBHQ is blocked by LY 294002. In
addition, we show that inhibitors of PI3-kinase block hNQO1-ARE reporter gene activation and nuclear translocation of Nrf2
induced by tBHQ. Furthermore, the data indicate that dominant negative Nrf2 completely blocked the increased hNQO1-ARE-luciferase
expression by constitutively active PI3-kinase. Insulin, a well known
activator of PI3-kinase (37), however, did not activate the ARE,
suggesting that not all activators of PI3-kinase result in ARE
activation. Therefore, we speculate that the PI3-kinase responsible for
ARE activation is an insulin-independent PI3-kinase or
phosphatidylinositol kinase-related kinase (21).
Our laboratory and others propose that increased expression of
ARE-driven genes contribute to the ability of cells to tolerate a
variety of chemical-induced stresses. Murphy and co-workers (47) have
demonstrated that pretreatment (24 h) of rodent neuroblastoma cells
with compounds that activate the ARE and induce NQO1 protects cells
from H2O2 and dopamine-induced cytotoxicity. In
addition, these investigators demonstrated that the stable
overexpression of NQO1 in this cell line did not confer resistance to
cytotoxicity, suggesting that the regulation of multiple genes is
required for protection. Preliminary data from our laboratory also
indicate that pretreatment (24 h) of IMR-32 human neuroblastoma cells
with tBHQ protects cells from H2O2
toxicity.3 Furthermore,
recently published studies using Nrf2 null mice show that these
mice are more sensitive to butylated hydroxytoluene-induced pulmonary
toxicity (48) and acetaminophen-induced hepatic toxicity (49). The
increased sensitivity to these chemicals was correlated with a lower
expression of ARE-driven genes in the respective tissues. These data
demonstrate the importance of understanding how different tissues or
cell types control the expression of ARE-driven genes and the potential
impact of modulating their expression on cell survival.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
were obtained from
New England Biolabs, Inc. (Beverly, MA). The Nrf2 antibody was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Tissue
culture supplies were purchased from Atlanta Biologics (Norcross, GA),
Life Technologies, Inc., and Midwest Scientific (St. Louis, MO). All
other reagents were purchased from Fisher.
kin (KD PI3-kinase) were kindly
provided by Dr. Anke Klippel (31).
-galactosidase (20 ng/well), and the CA PI3-kinase or
KD PI3-kinase plasmid. To investigate the effect of DN Nrf2 on
ARE activation by constitutively active PI3-kinase, IMR-32 cells were
cotransfected with the hNQO1-ARE-luciferase (80 ng/well),
CMV-
-galactosidase (20 ng/well), CA PI3-kinase (40 ng/well), and DN
Nrf2 (5 ng/well). After 24 h of transfection, the cells
were treated for another 24 h and harvested. Luciferase and
-galactosidase activity were determined as described
previously (10, 30). Data are expressed as the ratio of luciferase to
-galactosidase activity.
/
as a substrate.
/
) or nuclear fraction (for Nrf2) of IMR-32
cells was resolved by SDS-PAGE. The transferred membranes were probed
with an hNQO1 (10), phospho-Erk1/2, Erk1/2, phospho-GSK-3
/
, or
Nrf2 antibody. Chemiluminescence was produced using SuperSignal
West Pico chemiluminescent substrate (Pierce). Representative Western
blots are shown in the figures.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (58K):
[in a new window]
Fig. 1.
Effect of tBHQ on Erk1/2 phosphorylation and
NQO1 induction. A, to investigate
time-dependent activation of Erk1/2, IMR-32 human
neuroblastoma cells were treated with the vehicle ( ) or tBHQ (+, 10 µM) for the indicated times. After incubation, whole cell
extracts were prepared, and 50 µg of protein was analyzed for
phosphorylation of Erk1/2 using phospho-Erk1/2 antibody. B,
the same samples described in A were analyzed with control
the Erk1/2 antibody for loading control. C, the same samples
described in A were used for the analysis of
time-dependent NQO1 protein induction by tBHQ.
View larger version (29K):
[in a new window]
Fig. 2.
Effect of LY 294002 on ARE activation and
induction of NQO1. A, IMR-32 cells were transfected
with the hNQO1-ARE-luciferase reporter construct (80 ng/well) and
CMV- -galactosidase (20 ng/well). After 24 h, cells were
pretreated with increasing doses of LY 294002 (LY). After 30 min of pretreatment, cells were treated with the vehicle (
) or tBHQ
(
, 10 µM). After 24 h of treatment, the cells
were lysed, and luciferase and galactosidase activities were determined
as described under "Experimental Procedures." Data are expressed as
the ratio of luciferase to
-galactosidase activity. Each data point
represents the mean ± S.E. (n = 6). B,
IMR-32 cells were pretreated with increasing doses of LY 294002, followed by vehicle (
) or tBHQ (+, 10 µM) treatment.
After 24 h of treatment, whole cell extracts were prepared, and 50 µg of protein was resolved by SDS-PAGE. The transferred membrane was
probed with the NQO1 antibody.
View larger version (25K):
[in a new window]
Fig. 3.
Effects of PD 98059 and LY 294002 on
hNQO1-ARE activation in IMR-32 cells. A, IMR-32 cells
were transfected with the hNQO1-ARE-luciferase reporter construct (80 ng/well) and CMV- -galactosidase (20 ng/well). After 24 h, the
cells were pretreated for 30 min with the vehicle (V,
ethanol 0.1%), LY 294002 (LY, 20 µM), or PD
98059 (PD, 50 µM) and then treated with
vehicle or tBHQ (T, 10 µM). After 24 h of
treatment, the cells were lysed, and luciferase and galactosidase
activities were determined as described under "Experimental
Procedures." Data are expressed as the ratio of luciferase to
-galactosidase activity. Each bar represents the
mean ± S.E. (n = 6). B, IMR-32 cells
were pretreated with vehicle, LY 294002 (20 µM), or PD
98059 (50 µM) for 30 min followed by vehicle or tBHQ (10 µM) treatment. After 24 h, whole cell extracts were
prepared, and 50 µg of protein was used for Western immunoblot
analysis of NQO1 as described under "Experimental Procedures."
C, IMR-32 cells were pretreated with the vehicle or PD 98059 (50 µM) for 30 min followed by treatment with the vehicle
or tBHQ (10 µM) for another 30 min. Whole cell extracts
were prepared, and 50 µg of protein was used for Western immunoblot
analysis of phospho-Erk1/2 as described under "Experimental
Procedures."
View larger version (30K):
[in a new window]
Fig. 4.
Erk1/2 activation does not correlate with
inhibition of ARE activation. A, IMR-32 cells were
pretreated with the vehicle (V) or LY 294002 (LY,
20 µM) for 30 min and treated with vehicle or tBHQ (10 µM). Whole cell extracts were prepared, and 50 µg of
protein was resolved and probed with phospho-Erk1/2 antibody as
described under "Experimental Procedures." B, IMR-32
cells were treated with vehicle, NGF (100 ng/ml), or tBHQ (10 µM). After 30 min, whole cell extracts were prepared, and
50 µg of protein was resolved and probed with phospho-Erk1/2 antibody
as described under "Experimental Procedures." C, IMR-32
cells were transfected with the hNQO1-ARE-luciferase reporter construct
(80 ng/well) and CMV- -galactosidase (20 ng/well). After 24 h of
transfection, the cells were pretreated for 30 min with NGF (0-500
ng/ml) or LY 294002 (20 µM) and then treated with the
vehicle (open bar) or 10 µM tBHQ (filled
bar). After 24 h of treatment, the cells were lysed, and
luciferase and galactosidase activities were determined as described
under "Experimental Procedures." Data are expressed as the ratio of
luciferase to
-galactosidase activity. Each bar
represents the mean ± S.E. (n = 6).
Phosphorylation--
Recent findings linking PI3-kinase to Akt and
subsequent phosphorylation of GSK-3
(34-36) lead us to speculate
that tBHQ may activate this same pathway. Using insulin as a positive
control (37), the data show that tBHQ does not increase Akt activity (Fig. 5A) or lead to increased
phosphorylation of GSK-3
(Fig. 5B). As expected, the
effect of insulin on the phosphorylation of GSK-3
was blocked
completely by inhibitors of PI3-kinase activity (Fig. 5C).
Finally, insulin did not increase either hNQO1-ARE-luciferase expression (Fig. 6A) or hNQO1
protein (Fig. 6B), implying that not all activators of
PI3-kinase lead to ARE activation.
View larger version (46K):
[in a new window]
Fig. 5.
tBHQ does not activate Akt activity or
increase GSK-3 phosphorylation.
A, IMR-32 cells were treated with the vehicle, tBHQ (10 µM), or insulin (2.5 µg/ml) for 30 min. Akt activity
was measured using a commercially available Akt-kinase assay kit.
B, IMR-32 cells were treated with the vehicle, tBHQ (10 µM), or insulin (2.5 µg/ml) for 30 min. Whole cell
extracts were prepared, and 80 µg of protein was resolved by SDS-PAGE
as described under "Experimental Procedures." The transferred
membrane was probed with phospho-GSK-3
/
antibody. C,
IMR-32 cells were pretreated with the vehicle (V), LY 294002 (LY, 20 µM), or wortmannin (Wort, 1 µM). After 30 min, the cells were treated with the
vehicle or insulin (I, 2.5 µg/ml) for another 30 min.
Whole cell extracts were prepared, and 80 µg of protein was resolved
by SDS-PAGE as described under "Experimental Procedures." The
transferred membrane was probed with phospho-GSK-3
/
antibody.
View larger version (28K):
[in a new window]
Fig. 6.
Insulin does not activate ARE.
A, IMR-32 cells were transfected with the
hNQO1-ARE-luciferase reporter construct (80 ng/well) and
CMV- -galactosidase (20 ng/well). After 24 h of transfection,
the cells were treated with the vehicle, tBHQ (10 µM), or
insulin (2.5 µg/ml) for 30 min. After 24 h of treatment, the
cells were lysed, and luciferase and galactosidase activities were
determined as described under "Experimental Procedures." Data are
expressed as the ratio of luciferase to
-galactosidase activity.
Each bar represents the mean ± S.E. (n = 6). B, IMR-32 cells were treated with the vehicle, tBHQ
(10 µM), or insulin (2.5 µg/ml). After 24 h, whole
cell extracts were prepared, and 50 µg of protein was used for
Western immunoblot analysis of NQO1 as described under "Experimental
Procedures."
View larger version (17K):
[in a new window]
Fig. 7.
Constitutively active PI3-kinase activates
the ARE. A, IMR-32 cells were cotransfected with the
hNQO1-ARE-luciferase reporter construct (80 ng/well),
CMV- -galactosidase (20 ng/well), and the indicated amounts of the
PI3-kinase plasmid. After 24 h, the cells were harvested in lysis
buffer. Luciferase and galactosidase activities were measured as
described under "Experimental Procedures." Each data
point represents the mean ± S.E. (n = 6).
B, the cells were cotransfected with the
hNQO1-ARE-luciferase reporter construct (80 ng/well),
CMV-
-galactosidase (20 ng/well), and 40 ng/well of the PI3-kinase
plasmid. After 24 h, the cells were treated with the vehicle or LY
294002 (20 µM) for another 24 h. Luciferase and
-galactosidase activities were determined as described under
"Experimental Procedures." Each bar represents the
mean ± S.E. (n = 6).
View larger version (27K):
[in a new window]
Fig. 8.
PI3-kinase is linked to Nrf2.
A, IMR-32 cells were pretreated with the vehicle
(V), LY 294002 (LY, 20 µM) or
wortmannin (Wort, 1 µM) for 30 min and treated
with the vehicle or tBHQ (T, 10 µM). After
6 h, nuclear extracts were isolated and resolved by SDS-PAGE. The
transferred membrane was probed with the Nrf2 antibody.
B, the cells were cotransfected with the
hNQO1-ARE-luciferase reporter construct (80 ng/well),
CMV- -galactosidase (20 ng/well), CA PI3-kinase (40 ng/well), and DN
Nrf2 (5 ng/well). After 24 h, the cells were harvested, and
Luciferase and
-galactosidase activities were determined as
described under "Experimental Procedures." Each data bar
represents the mean ± S.E. (n = 6).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamylcysteine synthetase gene and its ARE in HepG2 cells. However, in the present study with
IMR-32 cells, modulation of Erk1/2 activity did not effect Nrf2-dependent ARE activation. Another MAP kinase,
p38 MAP kinase, also has been suggested to regulate the ARE positively
(4, 39, 40) or negatively (41). In our system, the inhibition of p38
MAP kinase by SB 203580 did not effect tBHQ-induced
hNQO1-ARE-luciferase expression.2 The data show
that strong Erk1/2 activators such as LY 294002 and NGF do not induce
ARE activation in IMR-32 cells. In fact, LY 294002 significantly
inhibited ARE activation in these neuroblastoma cells. Taken together,
these observations suggest that activation of Erk1/2 does not
necessarily lead to activation of the ARE in all cell types studied and
that the role of MAP kinases in regulating ARE-driven gene expression
is probably cell type-specific.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jawd Alam (Alton Ochsner Medical Foundation) for the DN Nrf2 expression vector and Dr. Anke Klippel (Chiron Corporation) for the PI3-kinase expression vector.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Environmental Health Sciences Grant ES08089 and the Burroughs Wellcome New Investigators in Toxicology Award.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: School of Pharmacy,
University of Wisconsin, 425 N. Charter St., Madison, WI 53706. Tel.:
608-262-2893; Fax: 608-262-3397; E-mail:
jajohnson@pharmacy.wisc.edu.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100734200
2 J. M. Lee and J. A. Johnson, unpublished observations.
3 J. Li and J. A. Johnson, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ARE, antioxidant
responsive element;
NQO1, NADPH:quinone oxidoreductase;
GST, glutathione S-transferase;
Nrf2, NF-E2-related factor
2;
Erk1/2, extracellular signal-regulated kinase;
MAP, mitogen-activated protein;
PI3-kinase, phosphatidylinositol 3-kinase;
tBHQ, tert-butylhydroquinone;
GSK, glycogen synthase
kinase;
h, human;
DN, dominant negative;
CA PI3-kinase, constitutively
active PI3-kinase p110*;
KD PI3-kinase, kinase-deficient PI3-kinase
p110*kin;
CMV, cytomegalovirus;
PAGE, polyacrylamide gel
electrophoresis;
NGF, nerve growth factor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Rushmore, T. H.,
and Pickett, C. B.
(1990)
J. Biol. Chem.
265,
14648-14653 |
2. | Prestera, T., and Talalay, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8965-8969[Abstract] |
3. |
Wild, A. C.,
Moinova, H. R.,
and Mulcahy, R. T.
(1999)
J. Biol. Chem.
274,
33627-33636 |
4. | Zipper, L. M., and Mulcahy, R. T. (2000) Biochem. Biophys. Res. Commun. 278, 484-492[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Venugopal, R.,
and Jaiswal, A. K.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14960-14965 |
6. | Venugopal, R., and Jaiswal, A. K. (1998) Oncogene 17, 3145-3156[CrossRef][Medline] [Order article via Infotrieve] |
7. | Itho, K., Chiba, T., Takahasi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., and Nabeshima, Y. (1997) Biochem. Biophys. Res. Comm. 236, 313-322[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Moi, P.,
Chan, K.,
Asunis, I.,
Cao, A.,
and Kan, Y. W.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9926-9930 |
9. |
Itho, K.,
Wakabayashi, N.,
Katoh, Y.,
Ishii, T.,
Igarashi, K.,
Engel, J. D.,
and Yamamoto, M.
(1999)
Genes Dev.
13,
76-86 |
10. | Lee, J. M., Moehlenkamp, J. D., Hanson, J. M., and Johnson, A. J. (2001) Biochem. Biophys. Res. Commun. 280, 286-292[CrossRef][Medline] [Order article via Infotrieve] |
11. | Crews, C. M., Alessandrini, A. A., and Erikson, R. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8845-8849[Abstract] |
12. |
Seger, R.,
and Krebs, E. G.
(1995)
FASEB J.
9,
726-735 |
13. | Hill, C. S., and Treisman, R. (1995) Cell 80, 199-211[Medline] [Order article via Infotrieve] |
14. | Hunter, T. (1995) Cell 80, 225-236[Medline] [Order article via Infotrieve] |
15. | Marshall, C. J. (1995) Cell 80, 179-185[Medline] [Order article via Infotrieve] |
16. |
Pang, L.,
Sawada, T.,
Decker, S. J.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
13585-13588 |
17. |
Davis, R. J.
(1993)
J. Biol. Chem.
268,
14553-14556 |
18. | Whitmarsh, A. J., and Davis, R. J. (1996) J. Mol. Med. 74, 589-607[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Gutkind, J. S.
(1998)
J. Biol. Chem.
273,
1839-1842 |
20. | Klippel, A., Escobedo, J. A., Hu, Q., and Williams, L. T. (1993) Mol. Cell. Biol. 13, 5560-5566[Abstract] |
21. | Shepherd, P. R., Withers, D. J., and Siddle, K. (1998) Biochem. J. 333, 471-490[Medline] [Order article via Infotrieve] |
22. | Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88, 435-437[Medline] [Order article via Infotrieve] |
23. |
Rameh, L. E.,
Rhee, S. G.,
Spokes, K.,
Kazlauskas, A.,
Cantley, L. C.,
and Cantley, L. G.
(1998)
J. Biol. Chem.
273,
23750-23757 |
24. |
Sabbatini, P.,
and McCormick, F.
(1999)
J. Biol. Chem.
274,
24263-24269 |
25. |
Jiang, B. H.,
Aoki, M.,
Zheng, J. Z.,
Li, J.,
and Vogt, P. K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2077-2081 |
26. |
Falasca, M.,
Logan, S. K.,
Lehto, V. P.,
Baccante, G.,
Lemmon, M. A.,
and Schlessinger, J.
(1998)
EMBO J.
17,
414-422 |
27. |
Le Good, J. A.,
Ziegler, W. H.,
Parekh, D. B.,
Alessi, D. R.,
Cohen, P.,
and Parker, P. J.
(1998)
Science
281,
2042-2045 |
28. | Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[Medline] [Order article via Infotrieve] |
29. |
Dudek, H.,
Datta, S. R.,
Franke, T. F.,
Birnbaum, M. J.,
Yao, R.,
Cooper, G. M.,
Segal, R. A.,
Kaplan, D. R.,
and Greenberg, M. E.
(1997)
Science
275,
661-665 |
30. | Moehlenkamp, J. D., and Johnson, J. A. (1999) Arch. Biochem. Biophys. 363, 98-106[CrossRef][Medline] [Order article via Infotrieve] |
31. | Hu, Q., Klippel, A., Muslin, A. J., Fantl, W. J., and Williams, L. T. (1995) Science 268, 100-102[Medline] [Order article via Infotrieve] |
32. |
Volente, C.,
Angelastro, J. M.,
and Greene, L. A.
(1993)
J. Biol. Chem.
268,
21410-21415 |
33. | York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and Stork, P. J. (1998) Nature 392, 622-626[CrossRef][Medline] [Order article via Infotrieve] |
34. | Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995) Nature 378, 785-789[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Moule, S. K.,
Welsh, G. I.,
Edgell, N. J.,
Foulstone, E. J.,
Proud, C. G.,
and Denton, R. M.
(1997)
J. Biol. Chem.
272,
7713-7719 |
36. |
Takata, M.,
Ogawa, W.,
Kitamura, T.,
Hino, Y.,
Kuroda, S.,
Kotani, K.,
Klip, A.,
Gingras, A. C.,
Sonenberg, N.,
and Kasuga, M.
(1999)
J. Biol. Chem.
274,
20611-20618 |
37. | Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1411-1415[Abstract] |
38. |
Yu, R.,
Lei, W.,
Mandlekar, S.,
Weber, M. J.,
Der, C. J.,
Wu, J.,
and Kong, A. T.
(1999)
J. Biol. Chem.
274,
27545-27552 |
39. |
Alam, J.,
Wicks, C.,
Stewart, D.,
Gong, P.,
Touchard, C.,
Otterbein, S.,
Choi, A. M. K.,
Burow, M. E.,
and Tou, J.
(2000)
J. Biol. Chem.
275,
27694-27702 |
40. |
Kang, K. W.,
Ryu, J. H.,
and Kim, S. G.
(2000)
Mol. Pharmacol.
58,
1017-1025 |
41. |
Yu, R.,
Mandlekar, S.,
Lei, W.,
Fahl, W. E.,
Tan, T. H.,
and Kong, A. T.
(2000)
J. Biol. Chem.
275,
2322-2327 |
42. |
Huang, H. C.,
Nguyen, T.,
and Pickett, C. B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
12475-12480 |
43. |
Alam, J.,
Stewart, D.,
Touchard, C.,
Boinapally, S.,
Choi, A. M.,
and Cook, J. L.
(1999)
J. Biol. Chem.
274,
26071-26078 |
44. |
Ishii, T.,
Itho, K.,
Takahasi, S.,
Sato, H.,
Yanagawa, T.,
Katoh, Y.,
Bannai, S.,
and Yamamoto, M.
(2000)
J. Biol. Chem.
275,
16023-16029 |
45. | Hayes, J. D., Ellis, E. M., Neal, G. E., Harrison, D. J., and Manson, M. M. (1999) in Cellular Responses to Stress (Downes, C. P. , Wolf, C. R. , and Lane, D. P., eds) , pp. 141-168, Portland Press, London, U. K. |
46. |
Yu, R.,
Chen, C.,
Mo, Y. Y.,
Hebbar, V.,
Owuor, E. D.,
Tan, T. H.,
and Kong, A. T.
(2000)
J. Biol. Chem.
275,
39907-39913 |
47. | Duffy, S., So, A., and Murphy, T. H. (1998) J. Neurochem. 71, 69-77[Medline] [Order article via Infotrieve] |
48. |
Chan, K.,
and Kan, Y. W.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12731-12736 |
49. |
Enomoto, A.,
Itoh, K.,
Nagayoshi, E.,
Haruta, J.,
Kimura, T.,
O'Connor, T.,
Harada, T.,
and Yamamoto, M.
(2001)
Toxicol. Sci.
59,
169-177 |