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Regulation of heme oxygenase-1 expression through the
phosphatidylinositol 3 kinase/Akt pathway and the Nrf2 transcription factor
in response to the antioxidant phytochemical, carnosol.
Daniel Martin1, Ana I. Rojo1, Marta Salinas1, Raquel Diaz1,3, German Gallardo3,
Jawed Alam2, Carlos M. Ruiz de Galarreta3, and Antonio Cuadrado1*.
1 Instituto de Investigaciones Biomédicas and Departamento de Bioquímica
Facultad de Medicina, Universidad Autónoma de Madrid. 28029 Madrid, Spain,
2Department of Biochemistry and Molecular Biology, Louisiana State University
Health Science Center, New Orleans, Louisiana 70112. USA, 3 Departamento de
Bioquímica, Facultad de Medicina, Universidad de las Palmas de Gran Canaria,
35016 Gran Canaria, Spain.
Running title: Carnosol induction of HO-1 through PI3K and Nrf2.
Keywords: heme oxygenase, carnosol, Nrf2, PI3K, Akt, AREs.
* To whom correspondence should addressed to:
Dr. Antonio Cuadrado Departamento de Bioquímica, Universidad Autónoma de Madrid Arzobispo Morcillo 4 28029 Madrid Spain. Tel: 3491 497 5327 FAX 3491 585 4401 E-mail: antonio.cuadrado@uam.es
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on December 19, 2003 as Manuscript M309660200 by guest on February 26, 2020
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ABSTRACT
The phosphatidylinositol 3 kinase (PI3K)/Akt pathway elicits a survival
signal against multiple apoptotic insults. In addition, phase II enzymes, such as
heme oxygenase-1 (HO-1), protect cells against diverse toxins and oxidative
stress. In this report, we describe a link between these defense systems at the
level of transcriptional regulation of the antioxidant enzyme HO-1. The herb-
derived phenol, carnosol, induced HO-1 expression at both messenger RNA and
protein levels. Luciferase reporter assays indicated that carnosol targets the
mouse ho1 promoter at two enhancer regions comprising the antioxidant
response elements (AREs). Moreover, carnosol increased the nuclear levels of
Nrf2, a transcription factor governing AREs. Electrophoretic mobility shift assays
and luciferase reporter assays with a dominant negative Nrf2 mutant, indicated
that carnosol increases the binding of Nrf2 to ARE and induces Nrf2-dependent
activation of the ho1 promoter. While investigating the signaling pathways
responsible for HO-1 induction, we observed that carnosol activates the ERK,
p38 and JNK pathways as well as the survival pathway driven by PI3K. Inhibition
of PI3K reduced the increase in Nrf2 protein levels and activation of the ho1
promoter. Expression of active PI3K-CAAX was sufficient to activate AREs. The
use of dominant negative mutants of PKCζ and Akt1, two downstream kinases
from PI3K, demonstrated a requirement of active Akt1 but not PKCζ. Moreover,
the long-term antioxidant effect of carnosol was partially blocked by PI3K or HO-
1 inhibitors, further demonstrating that carnosol attenuates oxidative stress
through a pathway that involves PI3K and HO-1.
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INTRODUCTION
High levels of reactive oxygen species (ROS) cause damage to cells and
are involved in several human pathologies including neurodegenerative disorders
and cancer (1, 2). Therefore, the use of compounds with anti-oxidant properties
may help prevent or alleviate diseases in which oxidative stress is a primary
cause (3). Carnosol, a diterpene derived from the herb rosemary, is a
representative member of a family of plant-derived phenols, which also include
curcumin, carnosic acid, phenylethyl isothiocyanate, epigallocatechin gallate and
other green tea polyphenols. These bioactive phytochemicals exhibit Michael
acceptor function and therefore behave as antioxidants (4). In addition, being
themselves xenobiotic compounds, they activate a xenobiotic response in the
target cells affecting the expression of phase II enzymes such as
NAD(P)H:quinone oxidoreductase, aldo-keto reductase, glutathione S-
transferase, gamma-glutamylcysteine synthetase, glutathione synthetase or
heme oxygenase-1 (HO-1) (5, 6, 7).
Heme oxygenase isozymes, HO-1 and HO-2, catalyze the stepwise
degradation of heme to release free iron and equimolar concentrations of carbon
monoxide (CO) and the linear tetrapyrrol, biliverdin, which is converted to
bilirubin by the enzyme biliverdin reductase (8). The HO-1 isozyme is a phase II
enzyme that is transcriptionally regulated by a large variety of stimuli. These
include its substrate, heme (9, 10), oxidative stress (11, 12), the signaling
proteins NGF, TNF-α, interleukin1β and interferon -γ (13, 14, 15) and phenolic
compounds such as curcumin and caffeic acid (16). Many reports have
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demonstrated the potent antioxidant activity of the heme-derived metabolites
generated by HO-1 catalysis, biliverdin and bilirubin, and the cytoprotective
actions of CO on vascular endothelium and nerve cells (17, 18, 19, 20).
Moreover, both HO-1-defficient mice (21) and a human case of genetic HO-1
deficiency (22) exhibit a serious impairment of iron metabolism leading to liver
and kidney oxidative damage and inflammation. Therefore, it is now widely
accepted that induction of HO-1 expression represents an adaptive response that
increases cell resistance to oxidative injury.
The signaling mechanisms used to activate transcription of phase II genes
such as HO-1 are poorly defined. Most studies have focused on the activation of
the mitogen-activated protein kinases (MAPKs) related to cell growth and stress
response. In vertebrates, the three major MAPK pathways are represented by
kinase cascades leading to activation of extracellular signal-regulated kinase
(ERK), c-jun N-terminal kinase (JNK) and p38 (for a review see 23). All three
pathways appear to be involved to some extent in the up-regulation of HO-1
expression in response to diverse stimuli. For instance, in rat hepatocytes,
sodium arsenite appears to regulate HO-1 expression via activation of Ras and
the JNK pathway but not the ERK pathway (24). On the other hand, arsenite
induces HO-1 expression via ERK and p38 pathways in LMH chicken hepatoma
cells (25). Ischemia-reperfusion in the lung activates HO-1 through JNK and p38
(26). Finally, regarding phytophenols, curcumin up-regulates HO-1 through the
p38 pathway in porcine renal epithelial cells (27).
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However, the role of the survival signaling pathway represented by
PI3K/Akt on the regulation of HO-1 expression is still not defined. An initial study
by Lee et al (28) suggested a role of PI3K in regulation of the phase II enzyme,
NADPH:quinone oxidoreductase. In addition, we previously showed that NGF
induces the expression of HO-1 by a still unknown PI3K-dependent mechanism
(13).
It is accepted that induction of HO-1 expression is mediated through cis-
regulatory DNA sequences located at their promoter region. Among the large
variety of putative regulatory sites found in the promoter 5’-flanking region of
human, rat, mouse and chicken HO-1 genes (29), most investigations have
focused on those which exhibit inducible response to oxidative signals (30).
Several AP-1 binding sites have been shown to be involved in the transcriptional
induction of HO-1 by MAPKs, cyclic nucleotides, protein phosphatase inhibitors
and the polyphenol curcumin (31, 32, 33).
More recently, a new class of AP-1 related site has been shown to
mediate oxidative stress responsiveness of phase II genes. These regions are
termed stress-response elements (StREs) or antioxidant response elements
(AREs) and are tightly regulated by the redox-sensitive transcription factor Nrf2
(NF-E2-related factor 2) (34). Nrf2 is a member of the Cap’n’Collar family of
transcription factors (35). Under non-stimulated conditions Nrf2 is sequestered in
the cytoplasm by binding to Keap1, an actin binding protein (36). Several stimuli,
which include oxidative stress, lead to the disruption of this complex freeing Nrf2
for translocation to the nucleus and dimerization with basic leucine zipper
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transcription factors such as Maf and Jun family members (37). The mechanism
by which Nrf2 is liberated from the Nrf2-Keap1 complex remains controversial but
probably involves phosphorylation and stabilization of the Nrf2 protein (38, 39)
which otherwise has a half life of just 30 min and is degraded via the ubiquitin-
proteasome pathway (39, 40, 41).
In this study we have analyzed the effect of carnosol on expression of the
antioxidant enzyme HO-1 and we have dissected the signaling pathways leading
to HO-1 regulation. Our results indicate that carnosol activates HO-1 expression
probably by increasing Nrf2 protein levels in a PI3K/Akt-dependent manner.
Therefore, in addition to its intrinsic antioxidant nature, carnosol activates the
PI3K/Akt survival pathway and up-regulates expression of the antioxidant
enzyme HO-1.
EXPERIMENTAL PROCEDURES
Cell culture, transfections and reagents. Rat phaeochromocytoma PC12
cells were seeded on poly D-lysine coated plates and grown in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 7.5 % fetal bovine serum
(FBS), 7.5 % heat-inactivated horse serum (HS) and 80 µg/ml gentamicin. Sub-
confluent cell cultures were maintained overnight (16 h) in DMEM medium
supplemented with 1% FBS and 1% HS, an experimental condition that does not
compromise viability in our PC12 cells (13, 42). For luciferase assays, transient
transfections of PC12 cells were performed with the expression vectors pGL3bv,
pHO1-15Luc, pHO1-15-Luc?E1, pHO1-15Luc?E2, pHO1-15Luc(?E1+?E2),
pEF-Nrf2(DN), pEF18-MafG(DN) (43), pCEFL(X-)Akt1(K179M) (42), pcDNA3-
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HA-PKCζ-DN (kindly provided by Dr. J. Moscat, Centro de Biología Molecular,
Madrid), pcDNA3 and pcDNA3-ß-gal. Lipofectamine Reagent (Invitrogen,
Carlsbad, CA, USA), was used according to manufacturer instructions. NGF and
carnosol were purchased from Calbiochem (San Diego, CA, USA), and hemin,
LY294002, PD98059, SB203580, SP600125 and MG132 from Sigma-Aldrich (St
Louis, Mo, USA) These drugs were dissolved in DMSO and used to a final
concentration of vehicle below 0.2%.
Analysis of mRNA levels by semi-quantitative reverse transcriptase-PCR.
Cells were plated on 60 mm dishes and total cellular RNA was extracted using
TRIzol reagent (Invitrogen). Equal amounts (1 µg) of RNA from the different
treatments were reverse-transcribed (75 min, 42 ºC) using 5 units of AMV-RT
(Promega, Madison, WI, USA) in the presence of 20 units of RNAsin (Promega).
Amplification of cDNA was performed in 25 µl of PCR buffer (10 mM Tris-HCl, 50
mM KCl, 5 mM MgCl2, 0.1 % Triton X-100, pH 9.0) containing 0.6 units of Taq
DNA polymerase (Promega), and 30 pmol of synthetic gene-specific primers for
HO-1 (forward, 5’-AAGGCTTTAAGCTGGTGATGG-3’; and reverse, 5’-
AGCGGTGTCTGGGATGAACTA-3’). To ensure that equal amounts of cDNA
were added to the PCR, the ß-actin gene was amplified using synthetic
oligonucleotides (forward 5’-TGTTTGAGACCTTCAACACC-3’; and reverse 5’-
CGCTCATTGCCGATAGTGAT-3’). After an initial denaturation step (4 min, 94
ºC), amplification of each cDNA was performed at the minimal number of cycles
that allowed detection of basal HO-1 mRNA levels in control cells (data not
shown); 19 cycles for ß-actin and 24 cycles for HO-1, using a thermal profile of 1
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min at 94 ºC (denaturalization), 1 min at 58 ºC (annealing), and 1 min at 72 ºC
(elongation). The amplified PCR products were resolved by 1.8% agarose gel
electrophoresis and stained with ethidium bromide.
Real-time quantitative PCR. Quantitative PCR was performed with 20 ng
cDNA obtained as indicated above, in 25 µl containing 0.5 µM primers (HO-1
forward: 5’-GCCTGCTAGCCTGGTTCAAG-3’, HO-1 reverse: 5’-
AGCGGTGTCTGGGATGAACTA-3’, β-actin forward: 5’-
GCCTCACTGTCCACCTTCCA-3’, β-actin reverse: 5’-
CCCGGCCTGAGTAGCATGA-3’) and the nucleotides, buffer and Taq
polymerase included in SYBR Green I Mastermix (PE Applied Biosystems).
Amplification was conducted in an ABI Prism 7700 sequence detection system.
PCR conditions were: 50 ºC for 2 min, 95 ºC for 10 min, 40 cycles at 94 ºC for 15
s, and 60 ºC for 1 min. HO-1 expression was estimated in duplicate samples and
was normalized with β-actin expression levels.
Preparation of nuclear extracts. 5x106 PC12 cells were washed three
times with cold phosphate-buffered saline and harvested by centrifugation at
1100 rpm during 10 min. The cell pellet was carefully resuspended in three pellet
volumes of cold buffer A (20 mM HEPES pH 7.0, 0.15 mM EDTA, 0.015 mM
EGTA, 10 mM KCl, 1% Nonidet-40, 1 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM
Na3VO4). The homogenate was then centrifuged at 500 g for 20 min and the
nuclear pellet was resuspended in five pellet volumes of cold buffer B (10 mM
HEPES pH 8.0, 25 % glycerol, 0.1 M NaCl, 0.1 mM EDTA, 1 µg/ml leupeptin, 1
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mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1
mM Na3VO4). After centrifugation at 500 g for 20 min, nuclei were resuspended
in two pellet volumes of hypertonic cold buffer C (10 mM HEPES pH 8.0, 25%
glycerol, 0.4 M NaCl, 0.1 mM EDTA, 1 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM
Na3VO4), and incubated during 30 min at 4 ºC on a rotating wheel. Nuclear
debris was removed by centrifugation at 900 g for 20 min at 4ºC; one part of the
supernatant was resolved in SDS-PAGE, and submitted to immunoblot analysis
using anti-Nrf2 and anti-Sp1 antibodies. The rest of the supernatant was used for
EMSA. Protein concentration was determined with the Dc Protein Assay kit
(BioRad, Hercules, CA, USA).
Electrophoretic mobility shift assays (EMSAs). The double stranded wild-
type oligonucleotide used as Nrf2 probe was 5’-TTTTATGCTGTGTCATGGTT-3’.
Complementary strand was annealed by incubation at a concentration of 250
ng/µl in STE buffer (100 mM NaCl, 10 mM Tris pH8.0, 1 mM EDTA), at 80 ºC for
2 min. The mixture was then slowly cooled down to 4 ºC with a thermal profile of
1 ºC/min in a thermal incubator. Annealed oligonucleotides were diluted to 25
ng/µl in STE buffer. The 5’ end labeling was performed with T4 polynucleotide
kinase (Promega), using 25 ng of double-stranded oligonucleotide and 25 µCi of
[?32P]-ATP (3000 Ci/mmol, Amersham Biosciences, Little Chalfont,
Buckinghamshire, England). The labeled probes were purified using a G-25 spin
column (Amersham Biosciences). The binding reaction mixture contained 5 µg of
nuclear extract in buffer containing 40 mM HEPES pH 8.0, 50 mM KCl, 0.05 %
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Nonidet-40, 1% dithiothreitol and 10 µg/ml poly(dI)-poly(dC), in a total volume of
20 µl. Anti-Nrf2 antibody (2 µg) or unlabeled competitor probe (25 ng) was added
as indicated, and the reaction was incubated for 1 h at 25 ºC. Labeled DNA (0.25
ng) was added to the mixture and submitted to an additional 20 min incubation at
25 ºC. Samples were resolved at 4 ºC, in a 5 % non-denaturing poly-acrylamide
gel in 0.5x Tris-Borate/EDTA. After electrophoresis, the gel was dried and
autoradiographed.
Immunoblotting. 30 µg cell lysate were resolved in SDS-PAGE and
transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). Blots
were analyzed with the appropriate antibodies: anti-Akt1C20 (1:500), anti-ERK2
(1:1000), anti-p38 (1:1000), anti-Nrf2 (1:16000), anti-Sp1 (1:1000) (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), anti-phosphoAkt1S473 (1:1000), anti-
phospho-ERK2 (1:1000), anti-phospho-p38 (1:1000) (Cell Signaling Technology,
Inc, Beverly, Ma, USA), anti-phospho-JNK (1:1000) (Upstate-Biotech, Lake
Placid, NY, USA), anti-PDI (Protein disulfide isomerase, kind gift of Dr. González
Castaño, Universidad Autónoma de Madrid, Spain), anti-HO-1 (1:1000) or anti-
HO-2 (1:1000) (Stressgene, Victoria, British Columbia, Canada). Appropriate
peroxidase-conjugated secondary antibodies (1:10000) were used to detect the
proteins of interest by enhanced chemiluminiscence.
Flow cytometry. A FACScan flow cytometer (Becton-Dickinson, San Jose,
CA, USA) was used to analyze intracellular ROS with the fluorescence probe
2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes, Leiden,
Netherland) that passively diffuses into the cell and is cleaved and oxidized to
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2',7'-dichloro fluorescein (DCF) (BP 530/25 nm). Cells were submitted to
carnosol, Zn-protoporphyrin IX (ZnPPIX), LY294002 (as indicated for each
experiment) and H2O2 (5 min) and then detached mechanically from the plates,
washed once with cold PBS and analyzed immediately. Three independent
samples of 10.000 cells were analyzed for each experimental condition.
Statistics. Student’s t test was used to assess differences between groups.
A p value <0.05 was considered significant. All luciferase and flow cytometry
experiments were performed at least three times using triplicate or quadruplicate
samples per group. The values in graphs correspond to the average of at least
three samples + SEM. Densitometric analyses were performed with the NIH
Image V2.51 software on representative immunoblots from experiments that
were repeated 2-5 times.
RESULTS
Carnosol induces expression of HO-1.
We investigated the possibility that carnosol, a natural diterpene derived
from the herb rosemary, might alter the expression of the antioxidant enzyme
HO-1. PC12 cells were maintained for 16 h in Dubeccos modified Eagle’s
medium supplemented with 7.5% fetal bovine serum plus 7.5% horse serum
(high serum medium) or with 1% fetal bovine serum plus 1% horse serum
(thereafter low serum). Under both growing conditions cells retained a similar
viability as determined by cytometric analysis of propidium iodide incorporation
and annexin-V PE staining, at least within the time-frame of these experiments
(data not shown and 42). Then, cells were stimulated with 10 µM carnosol for 6 h
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or an equivalent volume of DMSO vehicle (final concentration less than 0.2%)
and analyzed for the amount of HO-1 protein. In agreement with a recent study
(44), cells grown in low serum exhibited a slight decrease in HO-1 protein levels
as compared to complete serum-growing cells (Fig 1A). Interestingly, carnosol
induced a significant increase of HO-1 protein that was more evident in cells
submitted to low serum conditions. To further study the effect of carnosol on
induction of HO-1 expression, PC12 cells were maintained in low serum for 16 h
and then treated for 6 h with carnosol concentrations ranging from 1 to 100 µM.
Real-time PCR (Fig. 1B) indicated that carnosol induces a maximal increase
(over 200 fold) of HO-1 mRNA at 10 µM. Moreover, as shown in Fig. 1C, semi-
quantitative PCR also evidenced a dose-dependent increase in HO-1 mRNA
levels. This increase was not as high as that found with real-time PCR because
amplicon size and primers were different (see Experimental Procedures) and
because, in order to detect basal mRNA levels in the carnosol-untreated cells, it
was necessary to increase PCR cycles over the linear range in the carnosol-
induced cells. In addition, carnosol also produced a parallel increase in HO-1
protein levels, while HO-2 levels corresponding to the other isozyme
constitutively expressed in these cells, remained without significant alteration (Fig.
1D). Consistent with these data, 10 µM carnosol also induced a time-dependent
increase in HO-1 mRNA levels which was apparent as early as 2 h after
treatment and was followed by a robust increase in HO-1 protein levels at 4 h
(Fig. 1E and 1F). Similar results were obtained in other cell lines of human and
mouse origin (data not shown). Therefore, these results suggest that carnosol
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specifically up-regulates expression of the inducible heme oxygenase isozyme,
HO-1.
Carnosol activates HO-1 through the antioxidant response elements
(AREs)
The mouse ho1 promoter contains two AREs that might represent
potential targets of regulation by carnosol. To investigate this possibility, PC12
cells were transfected with luciferase expression vectors carrying the wild type 15
kb mouse promoter or deletion mutant versions lacking either the proximal ARE
(∆E1) or distal ARE (∆E2) or both (∆E1+∆E2) regulatory sequences. Cells were
maintained in low serum for 16 h and then stimulated with 10 µM carnosol for 16
h. As shown in Fig. 2, deletion of ARE sequences E1 or E2 resulted in 30% and
36% decrease in carnosol induction, respectively. Interestingly, deletion of both
AREs abolished the response to carnosol. Therefore, these results suggest that
carnosol activates HO-1 expression through both AREs.
Carnosol increases the levels of the Nrf2 transcription factor.
Several transcription factors have been reported to interact with AREs.
However, considering that induction of HO-1 expression by several inducers
requires de novo protein synthesis (45, 46) we analyzed the possibility that this
molecule would operate on Nrf2, a transcription factor of very short half life (39,
40, 41) that regulates AREs. PC12 cells were maintained in medium with low
serum for 16 h and then stimulated for 3 h with the carnosol concentrations
indicated in Fig. 3A. As a control, cells were also treated with the proteasome
inhibitor MG132. In agreement with previous studies, blockage of proteolysis with
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MG132 resulted in a large accumulation of Nrf2 protein within 3 h, even in the
absence of carnosol. Interestingly, carnosol increased Nrf2 levels in a dose-
dependent manner with maximal effect at 10 µM in our PC12 cells. Additionally,
we analyzed the time-course of Nrf2 accumulation in the presence of 10 µM
carnosol. As shown in Fig. 3B, the accumulation of Nrf2 protein levels was
already detectable after 30 min in the presence of 10 µM carnosol and increased
for at least 3 h during treatment. Of note, the combined treatment with the
proteasome inhibitor MG132 and carnosol did not significantly increase Nrf2
levels over those obtained with each drug separately, suggesting that carnosol
prevents degradation of Nrf2 via the ubiquitin-proteasome pathway.
Next, we analyzed the nuclear levels of carnosol-induced Nrf2 protein. As
shown in Fig. 4A, both MG132 and carnosol induced a strong accumulation of
Nrf2 in the nucleus, while the levels of the house keeping transcription factor Sp1
were stable. Moreover, we investigated whether the carnosol-induced increase of
Nrf2 at the nucleus was sufficient to promote binding of protein complexes
containing this transcription factor to the ARE (E1 sequence) corresponding to
the mouse ho1 promoter. EMSA reactions were performed with nuclear extracts
from cells exposed to carnosol or MG132. As shown in Fig. 4B, among the
several retarded complexes formed, one of them was significantly increased in
intensity upon treatment with carnosol or MG132. Moreover, when the EMSA
reaction was conducted in the presence of an anti-Nrf2 antibody, the intensity of
this complex dropped dramatically, indicating the presence of Nrf2 in the complex.
Taken together these results indicate that carnosol increases both total and
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nuclear protein levels of Nrf2 resulting in increased binding to the ARE promoter
sequence of HO-1.
To verify the functional relevance of Nrf2 binding to the ho1 ARE, PC12
cells were co-transfected with pHO1-15-Luc and expression vectors for dominant
negative mutants of either Nrf2 or one of its dimerization partners, MafG. As
shown in Fig. 4C, the carnosol-induced activation of the ho1 promoter was
significantly decreased in the presence of dominant negative Nrf2 or MafG or
both mutants. These results further suggest that Nrf2 mediates the carnosol-
induced activation of the ho1 promoter.
Carnosol activates the canonical MAPK cascades and the PI3K/Akt
pathway.
In order to identify the signaling pathways used by carnosol to induce HO-
1 expression, we first analyzed the effect of this phytophenol on the three MAPK
kinase cascades, leading to activation of ERK, p38 and JNK, and on the survival
pathway represented by the PI3K and Akt kinases. The activation of these
pathways was analyzed with activation-specific antibodies that selectively
recognize the active and phosphorylated form of ERK, p38, JNK and Akt (see
Experimental Procedures). Cells under low serum conditions were treated with
10 µM carnosol for the indicated times and then cell lysates were immunoblotted
with the anti-phospho-specific antibodies. As shown in Fig. 5, carnosol activated
the three MAP kinase cascades as well as the PI3K/Akt pathway with different
kinetics. Maximal activation ERK was observed after 30 min stimulation while
maximal activation of Akt, JNK and p38 required at least 1 h. Moreover, ERK
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activation was more transient. Therefore, we analyzed which of these pathways
might be involved in channeling the stimulus for HO-1 up-regulation. Low serum
growing cells were pre-incubated for 15 min with inhibitors of each pathway
LY294002 (PI3K), PD98059 (MEK), SB203580 (p38) and SP600125 (JNK), and
then stimulated with 10 µM carnosol for 6 h. The effectiveness of these inhibitors
was confirmed in carnosol-treated cells with antibodies specific for phospho-ERK,
phospho-Jun, phospho-ATF2 and phospho-Akt1 (data not shown). As shown in
Fig. 6A, HO-1 protein levels increased following incubation with 10 µM carnosol,
as expected. Inhibition of the ERK and JNK pathways had little or no effect on
HO-1 protein levels, suggesting that these MAP kinases are not required for HO-
1 up-regulation by carnosol. Curiously, inhibition of both PI3K and, to a lower
extent, p38 pathways dramatically reduced the capacity of carnosol to increase
HO-1 protein levels.
To further confirm these observations, we analyzed the effect of carnosol
on the upstream regulatory sequences of the mouse ho1 gene. PC12 cells were
co-transfected with expression vectors for β-galactosidase, used for
normalization, and pHO1-15-Luc. After serum-starvation, cells were pre-treated
with the MAP kinase and the PI3K inhibitors for 15 min and then incubated with
10 µM carnosol for 16 h. As shown in Fig. 6B, carnosol increased luciferase
activity, consistent with the notion that it activates HO-1 expression. Moreover,
the stimulation of this promoter fragment was significantly reduced in the
presence of the p38 and the PI3K inhibitors while remained unaltered in the
presence of the ERK and JNK inhibitors. Therefore, these observations on the
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mouse promoter correlate with the induction of the endogenous rat HO-1 protein
and strongly suggest that carnosol requires PI3K and, to a lower extent, p38
kinases to induce HO-1 expression in PC12 cells.
Since other groups have described the involvement of the p38 in
activation of HO-1 in the presence of polyphenols (27), we focused our study on
the role of PI3K. Cells maintained in low-serum medium were pretreated with
several concentrations of the PI3K inhibitor, LY294002, and then submitted to 10
µM carnosol for 6 h. As a control for the effectiveness and specificity of the
inhibitor, we monitored the activation of the PI3K downstream effector Akt1, after
30 min of carnosol addition, with activation specific anti-phospho-Akt antibodies.
As shown Fig. 7A, LY294002 concentrations in the low micromolar range
successfully inhibited carnosol-induced activation of Akt and also blocked, at
least partially, the increase in HO-1 and Nrf2 protein levels. Therefore, these
results indicate that PI3K is required to induce HO-1 expression and suggest that
the Nrf2 transcription factor may mediate at least in part this response.
Then, we analyzed whether PI3K alone was sufficient to activate the ho1
promoter, and, in such case, which was the role of AREs. We co-transfected
PC12 cells with pcDNA3-β-gal, an expression vector for constitutively active PI3K
(PI3K-CAAX), and the luciferase expression vectors pHO1-15-Luc or deletion
mutants ∆E1, ∆E2 or (∆E1+∆E2). As shown in Fig. 7B, active PI3K was sufficient
to induce the activity of the ho1 wild type promoter. However, this activation was
significantly decreased on the deletion mutants lacking the E1 or E2 sites and
was drastically reduced in the double mutant (E1+E2). In addition, we analyzed
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the requirement of Nrf2 for PI3K-induced activation of the ho1 promoter. PC12
cells were co-transfected with pHO1-15-Luc, pcDNA3-β-gal, and PI3K-CAAX
together with the expression vectors for dominant negative Nrf2 and MafG
mutants. In several independent experiments, the stimulatory effect of PI3K-
CAAX on the ho1 promoter was smaller in this experimental setting, implying co-
transfection of four plasmids, as compared to the previous experiments
performed by co-transfection of two or three plasmids. Nevertheless, as shown in
Fig. 7C, dominant negative Nrf2 and MafG alone or in combination significantly
blocked PI3K-induced activation of the ho1 promoter. These results indicate that
PI3K is sufficient to activate the ho1 promoter through the AREs in a Nrf2-
dependent manner.
We also analyzed the contribution of kinases regulated by PI3K to the
carnosol-induced up-regulation of HO-1. PC12 cells were transfected with the
expression vector pHO1-15-Luc and HA-tagged, dominant negative versions of
the protein kinases Akt1 and PKCζ, two well known effectors of PI3K. Over-
expression of these proteins was determined by immunoblot analysis with anti-
HA antibodies (Fig. 8C). As shown in Fig. 8A, transfection with dominant
negative PKCζ (K281W) did not have a significant effect on the response to
carnosol. By contrast, as shown in Fig. 8B, dominant negative Akt1(K179M),
significantly blocked the induction of HO-1. Moreover, co-transfection of both
expression vectors inhibited carnosol induction to the same extent as dominant
negative Akt1 alone (data not shown). These results suggest that Akt1, rather
than PKCζ, is responsible for at least part of the carnosol induced up-regulation
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of HO-1. Moreover, as shown in Fig. 8D, PC12 cells, stably transfected with
active PI3K and Akt1 versions, displayed increased HO-1 protein levels, further
indicating that the PI3K/Akt axis is sufficient to up-regulate HO-1 expression.
Finally, we studied whether carnosol attenuates oxidative stress through
the induction of HO-1 expression an the relevance of the PI3K pathway in such
induction. PC12 cells, maintained in low serum for 16 h, were preloaded (1h at
37ºC) with 8 µM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), a probe
that passively diffuses into the cells and is cleaved and oxidized in the
intracellular environment to the green fluorescence emitting compound, 2',7'-
dichlorofluorescein (DCF) (47). Then, cells were treated with DMSO vehicle
(control) or carnosol for 30 min, and finally exposed to 250 µM H2O2 for 5 min.
Then, cells were immediately analyzed by flow cytometry. As shown in Fig. 9A
and 9B, micromolar concentrations of carnosol dose-dependently prevented the
intracellular oxidation of the fluorescent probe despite the presence of H2O2.
Moreover, as shown in Fig. 9C, 10 µM carnosol fully prevented the oxidation of
H2DCFDA by H2O2 concentrations ranging from 50 µM to 500 µM. Since the
length of carnosol incubation (just 30 min) was probably insufficient to elicit a
significant effect on gene expression, these results support the intrinsic
antioxidant nature of this phytochemical, in agreement with other studies (48).
Then, we analyzed the long-term effect of carnosol, after 6 h-incubation, a
time consistent with the induction of HO-1 expression through the PI3K/Akt
pathway described in this study. The antioxidant effect of carnosol was compared
in the presence of the HO-1 inhibitor, ZnPPIX, and the PI3K inhibitor, LY294002.
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PC12 cells, maintained in low-serum medium for 16 h, were pre-treated with
vehicle, 10 µM LY294002 or 100 µM ZnPPIX, as indicated in Fig. 10A and 10B,
and then incubated with 10 µM carnosol for 30 min or 6 h. Finally, cells were
challenged with 250 µM H2O2 for 5 min and immediately analyzed by flow
cytometry. As shown in Fig. 10A, the HO-1 inhibitor (ZnPPIX) did not significantly
alter the antioxidant effect of carnosol after 30 min incubation. By contrast,
ZnPPIX partially, but not completely, blocked the long-term antioxidant effect of
carnosol after 6 h incubation. Similarly, as shown in Fig. 10B, inhibition of PI3K
(10 µM LY294002) did not significantly alter the antioxidant effect of carnosol
after 30 min but partially reduced its antioxidant effect at 6 h incubation. These
results indicate that induction of HO-1 by carnosol through the PI3K pathway is
essential to elicit at least part of its long-term antioxidant effects (but see
Discussion).
DISCUSSION
In this study, we show the involvement of the PI3K/Akt survival pathway in
the up-regulation of HO-1, a prototypical phase II enzyme, in response to the
herb-derived diterpene, carnosol. We have found that carnosol up-regulates HO-
1 expression by targeting the ARE sequences found in the mouse ho1 gene
promoter. The regulation of AREs by carnosol was mediated, at least in part,
through an increase in Nrf2 protein levels in a PI3K- and Akt- dependent manner.
To our knowledge this is the first study reporting the activation and the relevance
of the PI3K/Akt1 pathway to the induction of phase II enzymes by food
phytophenols with therapeutic potential.
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Most studies on the regulation of phase II gene expression have focused
on the role of the MAPK pathways. Our results indicate that, although the ERK,
p38 and JNK pathways are activated by carnosol, they do not participate equally
in the induction HO-1 in PC12 cells. Thus, ERK and JNK were dispensable for
HO-1 up-regulation while inhibition of p38 significantly reduced the response to
carnosol. The role of MAPKs in ARE activation remains controversial. For
instance, in agreement with our data, dominant negative mutants of JNK did not
prevent cadmium-induction of the mouse ho1 promoter (43). However, in rat
hepatocytes, the glutathione depletor pherone and arsenite promoted a JNK-
dependent induction of ho1 gene expression (24, 49). Balogun et al (27)
described the effect of curcumin on induction of HO-1 expression by promoting
inactivation of the Nrf2-Keap1 complex in a p38-dependent manner and
subsequent binding of Nrf2 to the AREs. Accordingly, arsenate induced a p38-
dependent activation of the ho1 promoter in the LMH chicken hepatoma cell line
(50) but inhibition of p38 had no effect on cadmium, arsenate and hemin-
dependent HO-1 mRNA induction in HeLa cells (51). Our results are consistent
with the requirement of p38 for full activation of the ho1 promoter by carnosol
since a generic inhibitor of p38 significantly reduced Nrf2 protein levels (data not
shown) and HO-1 protein levels and promoter activity in response to carnosol.
Finally, the ERK pathway was dispensable of HO-1 induction in our system and
in rat hepatoma cells challenged with arsenite (24) but, it was necessary in the
arsenite-induced response in LMH cells (50). One possible interpretation for
these diverging observations may stem from the diverse assortment and intensity
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of the signaling pathways activated by different inducers in different cell types. In
this regard, it is interesting to note that carnosol failed to induce HO-1 at the
highest concentration tested (100 µM) despite the fact that this concentration
yielded the strongest activation of the MAPK pathways (data not shown),
therefore suggesting that specificity may rely not only on the nature of the
inducer and cell type but also on the relative potencies of these pathways.
We found that carnosol increases the levels of Nrf2 transcription factor in
the nucleus and its binding to the AREs. The mechanisms leading to nuclear
translocation of Nrf2 are poorly defined but certainly include the release from
Keap1 at the cytosol. However, since the half life of this transcription factor is
very short (39, 40, 41), these mechanisms must necessarily rely on the
stabilization of Nrf2 protein. Our results demonstrate that, indeed, carnosol
increases the levels of Nrf2 as does the proteasome inhibitor MG132, suggesting
that this phytochemical blocks Nrf2 degradation. The increase in Nrf2 protein
levels induced by carnosol was partially dependent of the activation of PI3K since
LY294002 concentrations that inhibited PI3K and the subsequent
phosphorylation and activation of Akt1 decreased the carnosol-induced
accumulation Nrf2 protein. However, inhibition of PI3K did not fully prevent the
carnosol-induced increase in Nrf2 and HO-1 protein levels, suggesting that other
elements, probably p38 and other PI3K-independent pathways, are also involved
in carnosol signaling to Nrf2 and HO-1. Interestingly, active PI3K was sufficient to
activate the ho1 promoter through AREs and constitutive expression of active
PI3K or Akt yielded cells with increased HO-1 levels.
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In search for the downstream kinases that might be responsible for the
increase Nrf2 protein levels we analyzed the effect of PKCζ. A dominant negative
version of this kinase only produced a slight decrease in the carnosol-induced
activation of the ho1 promoter, suggesting a very minor role of this atypical PKC
on HO-1 transcriptional regulation. However, a recent study by Numazawa et al
(52) has indicated that PKCι, another member of the atypical PKCs,
phosphorylates Nrf2 at Ser 40, leading to its release from Keap1 and nuclear
translocation. Whether the dispensability of PKCζ is due to a lack of involvement
in the regulation of Nrf2 or to redundancy with other atypical PKCs, such as PKCι,
remains to be determined.
On the other hand, a dominant negative mutant of Akt1 significantly
reversed carnosol-induced activation of the ho-1 gene promoter. Contrary to
PKCι, direct phosphorylation of Nrf2 or Keap1 by Akt seems unlikely because
these proteins do not have a consensus phosphorylation sequence for Akt. Since
Nrf2 is regulated primarily at the level of stability we propose that Akt might be
acting on the ubiquitin ligases involved in its tagging for degradation. In fact, at
least two ubiquitin ligases, Mdm2 (53) and BRCA1/BARD1 (54) have been
reported to be substrates of Akt.
Interestingly, the activation of Akt by carnosol was a transient event that
subsided by 2 h. However, Nrf2 protein levels increased beyond 3 h. Therefore,
transient activation of Akt must result in persistent activation/inhibition of effector
substrates that regulate Nrf2, resulting in increased protein levels. Thus,
phosphorylation of the Nrf2-specific ubiquitin ligase might result in its persistent
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inhibition leading to sustained increase in Nrf2 protein levels. Alternatively, the
transient activation of Akt might activate transcription of stable proteins that
contribute to increase Nrf2 protein levels.
Plant-derived phenols exhibit strong antioxidant properties due to Michael
reaction acceptor function. Indeed, we have observed the intrinsic antioxidant
nature of carnosol since 30 min pre-treatment with this drug completely abolished
the oxidant effect of as much as 500 µM H2O2, as determined by the blockage in
the conversion of H2DCFDA to its oxidized, green fluorescent form, DCF. More
importantly for this study, inhibition of the PI3K pathway, leading to activation of
Akt and up-regulation of HO-1 expression, impaired the long-term, but not the
short-term, antioxidant function of carnosol, therefore evidencing the relevance of
this pathway for carnosol to confer persistent antioxidant protection. Interestingly,
a fraction of about half of the antioxidant effect of carnosol was insensitive to
inhibition of PI3K and HO-1 at 6 h. This observation is consistent with the
possibility that at this time there is still enough non-metabolized carnosol to
maintain its intrinsic antioxidant effect. In fact, the PI3K and HO-1 inhibitors
dropped H2DCFDA oxidation to similar levels as those observed at 30 min.
However, we favor the non-exclusive hypothesis that carnosol also up-regulates
other intracellular antioxidant systems that do not belong to the PI3K/HO-1
module and that also contribute to the long-term antioxidant properties of this
compound. In fact, preliminary microarray data from mouse neuroblastoma cells
indicates that carnosol also up-regulates the expression of mitochondrial, Mn-
dependent superoxide dismutase and glutathione peroxidase (data not shown).
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A corollary of this study is that pharmacological activation of the PI3K/Akt
pathway by this food-related compound, leading to increase of Nrf2 and HO-1,
efficiently protects cells from oxidative stress and should be evaluated as a new
therapeutic approach in degenerative processes, such as Alzheimer’s and
Parkinson diseases, that correlate with oxidative damage.
ACKNOWLEDGEMENTS
We thank Ms Beatriz Palacios for her excellent technical assistance. This
work was supported by grants SAF2001-0545 from Ministerio de Ciencia y
Tecnología (AC), 08.5/0048/2001 from Comunidad Autónoma de Madrid (AC),
FEDER 97/0602 (CMRG) and PI-2002/168 from Comunidad Autónoma de
Canarias (CMRG).
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LEGEND TO FIGURES
Figure 1.- Carnosol induces HO-1 expression. A. Effect of serum and
carnosol on the protein levels of HO-1 and HO-2 isoenzymes. PC12 cells were
maintained for 16 h in DMEM supplemented with 1% fetal calf serum and 1%
horse serum (low serum) or in 7.5% fetal calf serum and 7.5% horse serum (high
serum) and then stimulated with vehicle (DMSO) or 10 µM carnosol for 6 h.
Upper panel, immunoblot with anti-HO-1 antibodies; middle panel, immunoblot
with anti-HO-2 antibodies; lower panel, densitometric quantification of relative
HO-1 protein levels. B. Real-time PCR determination of HO-1 mRNA levels in the
presence of carnosol. PC12 cells, maintained for 16 h in low serum conditions,
were incubated with the indicated concentrations of carnosol and analyzed as
indicated in Experimental Procedures. C. Semi-quantitative reverse-transcriptase
PCR showing a dose-dependent induction of HO-1 mRNA by carnosol. Upper
panel, HO-1 mRNA; middle panel, β-actin mRNA used for normalization; lower
panel, densitometric quantification of relative HO-1 mRNA levels. D. Dose-
dependent induction of HO-1 protein by carnosol. Upper panel, immunoblot with
anti-HO-1 antibodies; middle panel, immunoblot with anti-HO-2 antibodies; lower
panel, densitometric quantification of relative HO-1 protein levels. For C and D,
PC12 cells, maintained in low serum of 16 h, were treated with the indicated
carnosol concentrations for 6 h, or with 50 µM hemin, a well-established induced
of HO-1, and then analyzed for HO-1 mRNA and protein levels. E. Time-course
of HO-1 mRNA expression by carnosol. Upper panel, HO-1 mRNA; middle panel,
β-actin mRNA used for normalization; lower panel, densitometric quantification of
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relative HO-1 mRNA levels. F. Time-course of HO-1 protein expression by
carnosol. Upper panel, immunoblot with anti-HO-1 antibodies; middle panel,
immunoblot with anti-HO-2 antibodies; lower panel: densitometric quantification
of relative HO-1 protein levels. For E and F, PC12 cells were maintained in low
serum for 16 h, and then submitted to 10 µM carnosol for the indicated times or
with 50 µM hemin for 6 h.
Figure 2.- Carnosol targets the mouse ho1 gene promoter at the two
AREs. PC12 cells were transfected with expression vectors for luciferase under
the control the 15 kb 5’ promoter region of mouse ho1 (WT) or the same
promoter with deletions of either E1 (∆E1) or E2 (∆E2) or both regions (∆E1+∆E2)
comprising the AREs. In addition, cells were co-transfected with pcDNA3-β-gal
for normalization. 16 h after transfection, cells were maintained in low serum for
16 h and then stimulated with 10 µM carnosol for additional 16 h. Then cells were
lysed and analyzed for luciferase and β-galactosidase activity.
Figure 3.- Carnosol increases the levels of Nrf2 transcription factor. Dose-
effect (A) and time-course (B) of carnosol-induced increase of Nrf2 protein levels.
Upper panels: blots with anti-Nrf2 antibodies; middle panels: blots with anti-
protein disulfide isomerase (PDI) used for normalization; lower panels:
densitometric quantification of relative Nrf2 levels. In A, Low serum growing
PC12 cells were treated for 3 h with the indicated carnosol concentrations, or
with MG132 (20 µM) or both MG132 (20 µM) and carnosol (10 µM) as indicated.
In B PC12 cells, maintained in low serum for 16 h, were treated with 10 µM
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carnosol for the indicated times or with MG132 or with MG132 plus carnosol for 3
h.
Figure 4.- Carnosol stimulates activation of ho1 promoter AREs by Nrf2. A.
Increase of Nrf2 protein levels in nuclear extracts of cells treated with 10 µM
carnosol or 20 µM MG132 for 3h. B. EMSA reactions using a double strand
oligonucleotide corresponding to the mouse ho1 ARE and nuclear extracts from
cells treated with 10 µM carnosol or 20 µM MG132. The last two lanes
correspond to cell extracts from carnosol-treated cells pre-incubated without (-)
or with (+) anti-Nrf2 antibody. The arrow points the position of the complex that is
not formed in the presence of the anti-Nrf2 antibody. C. Requirement of Nrf2 and
MafG for activation of the mouse ho1 promoter by carnosol. PC12 cells were co-
transfected with pHO1-15-Luc, pcDNA-β-gal, and expression vectors for
dominant negative versions of Nrf2 or MafG, as indicated. Following 16 h after
transfection cells were kept in low serum for 16 h and then stimulated with 10 µM
carnosol. After 6 h cells were lysed and analyzed for luciferase and β-
galactosidase activity.
Figure 5.- Carnosol activates the MAPK and PI3K pathways. PC12 cells in
low serum were stimulated with 10 µM carnosol for the indicated times and then
immunoblotted with activation-specific antibodies that recognize the
phosphorylated kinases (P-Akt1, P-ERK2, P-p38 and P-JNK). Parallel
immunoblots were analyzed for total kinase levels with the indicated antibodies
(Akt1, ERK, p38, and JNK). Lower graphics correspond to a densitometric
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quantification of P-Akt, P-ERK2, P-JNK and P-p38 levels relative to total kinase
levels.
Figure 6.- Effect of MAPK and PI3K inhibitors on induction of HO-1 by
carnosol. A. Upper panel: immunoblot with anti-HO-1 antibodies. Lower panel:
immunoblot with anti-PDI antibodies used for normalization. PC12 cells were
maintained in low serum for 16 h and then were pre-incubated with 40 µM
LY294002 (LY), 50 µM PD98059 (PD), 5 µM SB203580 (SB), and 10 µM
SP600125 (SP) for 15 min. Then, cells were submitted to 10 µM carnosol for 6 h.
B. Carnosol induced luciferase activity in the mouse ho1 promoter in the
presence of the MAPK and PI3K inhibitors. PC12 cells were co-transfected with
pHO1-15-Luc and pcDNA3-β-gal vectors. After 16 h cells were transferred to low
serum in the presence of the inhibitors and then stimulated with 10 µM carnosol
for 16 h. Then cells were lysed and analyzed for luciferase and β-galactosidase
activity.
Figure 7.- The PI3K/Akt axis is involved in the carnosol-induced increase
in Nrf2 protein levels and on ho1 promoter activation. A. Inhibition of PI3K
prevents the increase of HO-1 and Nrf2 protein levels in response to carnosol.
PC12 cells, maintained in low serum for 16 h, were pre-incubated with the
indicated LY294002 concentrations for 15 min and then with 10 µM carnosol for
either 30 min (immunoblot with P-Akt) or 6 h (immunoblots with HO-1, Nrf2 and
PDI). B. PI3K induces HO-1 expression through regulation of AREs. PC12 cells
were transfected with pHO1-15-Luc, pHO-∆E1-Luc, pHO1-∆E2-Luc and pHO1-
(∆E1+∆E2) together with either empty vector or expression vector for active PI3K
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Martin et al, 2003
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(PI3K-CAAX). In addition, cells were co-transfected with pcDNA3-β-gal for
normalization. Following 16h from transfection, cells were transferred to low
serum medium for 16 h and analyzed for luciferase and β-galactosidase activity.
C. Induction of HO-1 expression by PI3K is attenuated by interfering with Nrf2
function. PC12 cells were co-transfected with pHO1-15-Luc, empty vector
(pcDNA3), PI3K-CAAX, pcDNA-β-gal, and expression vectors for dominant
negative versions of Nrf2 or MafG, as indicated. Following 16 h after transfection
cells were kept in low serum for 16 h and then stimulated with 10 µM carnosol.
After 6 h cells were lysed and analyzed for luciferase and β-galactosidase activity.
Figure 8.- Carnosol requires Akt1 but not PKCζ to activate the ho1
promoter. Cells were transfected with pHO1-15-Luc and the indicated vectors for
expression of dominant negative Nrf2 mutant or the indicated amounts in A of
dominant negative PKCζ (PKCζ (DN)) or in B of dominant negative Akt1
(Akt1(DN)). Cells were also transfected with pcDNA3-β-gal for normalization.
Following 16 h from transfection, cells were maintained in low serum medium for
16 h and analyzed for luciferase and β-galactosidase activity. C. Detection of HA-
tagged dominant negative versions of PKCζ and Akt1 by immunoblot with anti-
HA antibodies. Cells were treated in parallel with the luciferase experiments
indicated in A and B, were lysed and immunoprecipitated with anti-HA antibodies.
The immune-complexes were resolved in PAGE and immunoblotted with anti-HA
antibodies. The lower band (IgG H) corresponds to the immunoglobulin heavy
chain of the anti-HA antibody used during immunoprecipitation. D. Immunoblots
showing HO-1 protein levels in cells stably transfected with empty vector
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(pcDNA3), membrane-anchored active PI3K (PI3K-CAAX), and membrane-
anchored active myristylated Akt1 (myr-Akt1). Upper panel immunoblot with anti-
HO-1 antibodies. Lower panel, immunoblot with anti PDI antibodies.
Figure 9. - Carnosol prevents oxidative stress. PC12 cells were
maintained in low serum for 16 h, and then loaded for 1 h with 8 µM H2DCFDA
and simultaneously treated with vehicle (controls) or 10 µM carnosol for 30 min.
Finally cells were exposed to 250 µM H2O2 for 5 min and immediately analyzed
by flow cytometry. A. A representative sample of 10.000 cells is shown for
vehicle-treated cells (control), or cells treated with H2O2 alone or in combination
with carnosol. B. Dose-dependent, antioxidant effect of carnosol. PC12 cells
were loaded with H2DCFDA and treated with DMSO vehicle (control) or the
indicated carnosol concentrations for 30 min. During the last 5 min cells were
exposed to 250 µM H2O2, as indicated, and rapidly used for flow cytometry
analysis. C. Carnosol-mediated antioxidant protection against micromolar
concentrations of H2O2. PC12 cells were loaded with H2DCFDA as indicated
above, incubated for 30 min with vehicle or 10 µM carnosol and treated for the
last 5 min with increasing concentrations of H2O2, as shown.
Figure 10.- HO-1 and PI3K inhibitors attenuate long- but not short-term
antioxidant protection of carnosol. A. Effect of the HO-1 inhibitor, ZnPPIX, on the
antioxidant activity of carnosol. B. Effect of the PI3K inhibitor LY294002 on the
antioxidant activity of carnosol. PC12 cells were maintained in low serum for 16 h
and then loaded for 1 h with H2DCFDA. Then, cells were pre-pretreated with
either 100 µM ZnPPIX (A) or 10 µM LY294002 (B) for 15 min and submitted to
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carnosol for 30 min and 6 h as indicated. During the last 5 min of carnosol
incubation, cells were challenged with 250 µM H2O2 and then analyzed by flow
cytometry.
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DC
carnosol ( M)µ
0
2
12
4
6
hemin100 10030µM: 50
carnosol
HO-1
HO-2
3
0 10020 40 60 80
carnosol ( M)µ
0
heminµM: 50
carnosol
HO-1
β-actin
12345
100 100303
0 10020 40 60 80
Figure 1
FE
time (h):
10 M carnosolµ
HO-1
β-actin
10 420.5 6
time (h)
HO-1
HO-2
0
15
36
129
time (h):
10 M carnosolµ
10 420.5 6
time (h)
250
200
150
100
2
00 3 10 30 100
carnosol Mµ
B
HO-1
HO-2
high serumlow serum
vehicle:carnosol:
--
+-
-+
--
+-
-+
A
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18
16
14
12
10
8
6
4
0
untreated
p
∆E1 ∆E2 ∆(E1+E2)WT pGL3bv
HO1-15-Luc
carnosol
Figure 2
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carnosol ( M)µ
0
Nrf2
PDI
12345
0 10 20 30
1010 3 30µM:
carnosol
6
Nrf2
PDI
10.50 2hours:
carnosol
3
Time (h)
012345
0 1 2 3
6
A B
Figure 3
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- +
α-Nrf2
B
Nrf2
Sp1
A
0
4
2
6
8
10
12
14
16
carnosoluntreated
Nrf2(DN):
MafG(DN):
pHO1-15-Luc:
- -
- -
+ +
+
- -
+ +
+
- -
+ +
+
+ +
+
C
Figure 4
+
+ +
+
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ERK2Akt
P-Akt1 P-ERK2
P-p38
p38
P-JNK
P-JNK
60100 120min: 30 60100 120min: 30
60100 120min: 30 60100 120min: 30
Time (min)
0123456
30 60 1200 30 60 1200 30 60 1200 30 60 1200
P-Akt P-ERK2
JNK
P-p38
Figure 5
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HO-1
PDI
Carnosol: - - -+ + + - -+ +
LY PD SB SP
A untreatedcarnosol
18
16
14
12
10
8
6
4
0LY PD SB SP-
B
Figure 6
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A
vectorPI3K-CAAX
p
∆E1 ∆E2 ∆(E1+E2)WT pGL3bv
HO1-15-Luc
25
20
15
10
5
2.5
0
B
Figure 7
HO1
Nrf2
P-Akt1
PDI
- + + + + -+Carnosol:LY ( M):µ - - 103 30 100 100
3
2
1
0
C
pcDNA3
vectorPI3K-CAAX
Nrf2(DN) MafG(DN)Nrf2 (DN)
+MafG(DN)
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12
10
8
6
4
0
untreatedcarnosol
12
10
8
6
4
0
untreatedcarnosol
A B
Akt1(DN)
PKC (DN)ζ
IgG H
PKC (DN)ζ Akt1(DN)
C
Figure 8
PDI
HO-1
D
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100
010 10 10 10
0 1 2 3
H DCFDA fluorescence2
controlH OH O + carnosol
2 2
2 2
10
5
1 3 10 30
Carnosol ( M)µ
0
H O + carnosol2 2
50
H O ( M)µ2 2
10
5
0125 250 500
H O + carnosol2 2H O2 2
A
B
C
Figure 9
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-+- -
- -
- -
+-
-
-
+
+-
-
+
+-
+
+-
-
-
+-
+
-
+-
+
+
0
5
10
15
carnosol 30 min:
carnosol 6 h:
ZnPPIX:
H O :22
+-
-
-
-
-
-
-
+-
-
-
+
+-
-
+
+-
+
+-
-
-
+-
+
-
+-
+
+
0
5
10
15
carnosol 30 min:
carnosol 6 h:
LY294002:
H O :22
A
B
Figure 10
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Carlos M. Ruiz de Galarreta and Antonio CuadradoDaniel Martin, Ana I. Rojo, Marta Salinas, Raquel Diaz, German Gallardo, Jawed Alam,
antioxidant phytochemical, carnosolkinase/Akt pathway and the Nrf2 transcription factor in response to the
Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3
published online December 19, 2003J. Biol. Chem.
10.1074/jbc.M309660200Access the most updated version of this article at doi:
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