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INTRODUCTION |
Vitamin A (retinol) and its biologically active derivatives
retinoids exert a wide range of biological effects on embryogenesis, neoplasia, and maintenance of normal tissues, especially epithelium (1). The crucial roles of retinoids in controlling cell function have
been elucidated especially using retinoic acid
(RA),1 the most notable
retinoid. Previous reports showed that RA has profound inhibitory
effects on tumorigenesis. It is via suppression of cell growth and
induction of cellular differentiation (1). It has also been reported
that RA induces apoptosis of various tumor cells in vitro
(2, 3), and it may contribute to the antitumor effect of RA. However,
the mode of action of RA on apoptosis is a little complicated. We
recently reported that all-trans-RA (t-RA) inhibited
H2O2-triggered apoptosis in mesangial cells and fibroblasts (4). The molecular mechanisms involved in the antiapoptotic effect of t-RA are not fully elucidated, but activator protein 1 (AP-1)
is a possible target. It is because 1) the AP-1 pathway plays a crucial
role in the H2O2-induced apoptosis (5, 6) and
2) t-RA is a known inhibitor of AP-1 in some cell types (7).
AP-1, mainly composed of heterodimers of c-Jun and c-Fos, binds to the
particular cis element,
12-O-tetradecanoylphorbol-13-acetate response element, and
initiates transcription of target genes (8). Several molecular events
are involved in the activation of AP-1; i.e. the
transacting potential of AP-1 depends on induction and phosphorylation
of AP-1 components by mitogen-activated protein (MAP) kinases (9). For
example, expression of c-fos is regulated by ternary complex
factors whose activity is regulated by extracellular signal-regulated
kinase, p38 MAP kinase, and c-Jun N-terminal kinase (JNK).
Expression of c-jun is regulated by c-Jun and activating transcription factor-2, which are phosphorylated by JNK and/or p38 MAP
kinase. Post-translational activation of AP-1 is also regulated by MAP
kinase-mediated phosphorylation. c-Jun is phosphorylated and activated
by JNK, and c-Fos is phosphorylated by a member of the MAP kinase
family, Fos-regulating kinase (9). To elucidate molecular events
involved in the antiapoptotic effect of t-RA, we previously
investigated the effect of t-RA on the expression of c-fos
and c-jun and activation of JNK triggered by
H2O2. Our data showed that t-RA inhibited AP-1
activation and that it was associated with suppression of both
c-fos/c-jun expression and JNK phosphorylation
(4).
The biological actions of RA, especially its transcriptional regulation
of target genes, are mediated by two families of nuclear receptors,
retinoic acid receptors (RAR-
, -
, and -
) and retinoid X
receptors (RXR-
, -
, and -
) (10). These receptors have
different ligand specificity; e.g. RARs are
activated by both t-RA and 9-cis RA, whereas RXRs are
activated only by 9-cis RA (10). After the ligand binding,
these receptors form homodimers or heterodimers and function as
transcriptional regulators. For example, t-RA binds to RARs and
activates RAR-RXR heterodimers, and the complex exerts its biological
effects via binding to particular cis elements, retinoic
acid response elements (11). However, currently, little is known about
the requirement of RAR and RXR for the regulation of apoptosis by RA.
In this report, we investigated whether RAR and RXR are essential for
the antiapoptotic effect of t-RA in H2O2-exposed mesangial cells. We further
examined whether these nuclear receptors are required for the
t-RA-mediated suppression of c-fos/c-jun
expression, JNK phosphorylation, and AP-1 activation.
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EXPERIMENTAL PROCEDURES |
Cells--
Mesangial cells (SM43) were established from
isolated glomeruli of a male Harlan Sprague-Dawley rat and identified
as being of the mesangial cell phenotype as described before (12).
Cells were maintained in Dulbecco's modified Eagle's medium/Ham's
F-12 (Life Technologies, Inc.) supplemented with 100 units/ml
penicillin G, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B,
and 10% fetal calf serum (FCS). Medium containing 1% FCS was
generally used for experiments.
Establishment of Stable Transfectants--
SM/
RAR8 and
SM/
RXR3 were created as follows. Using a modified calcium phosphate
coprecipitation method (13), SM43 cells were stably transfected with
expression plasmid pRSh/RAR
-403* (a gift of
Dr. R. M. Evans, The Salk Institute, San Diego, CA) or
pCDM/RXR
AF2 (a gift of Dr. J. W. Lee, Chonnam National
University, Kwangju, Korea), together with pcDNA3 (Invitrogen,
Groningen, The Netherlands). pRSh/RAR
-403* and pCDM/RXR
AF2
introduce a dominant-negative form of RAR-
(
RAR) and a
dominant-negative RXR-
(
RXR), respectively. These
dominant-negative mutants inhibit all subtypes of RARs or RXRs (14,
15). Stable transfectants were selected in the presence of G418 (750 µg/ml), and clones SM/
RAR8 and SM/
RXR3 were established.
Expression of transgenes was confirmed by Northern blot analysis. A
control clone SM/Neo was created by transfection of SM43 cells with
pcDNA3 alone.
Pharmacological Manipulation--
Confluent cells were
pretreated with or without RAR pan-antagonist AGN193109 (5 µM; a gift of Dr. R. A. S. Chandraratna, Allergan Pharmaceuticals, Irvine, CA) (16) or RXR pan-antagonist HX531 (2.5 µM; a gift of Dr. H. Kagechika, University of Tokyo,
Tokyo, Japan) (17) for 30 min, treated with or without t-RA (tretinoin, 2 µM; Sigma) for 0.5-2 h and then stimulated by
H2O2 (150 µM; Sigma) for
indicated time periods.
Assessment of Apoptosis--
After induction of apoptosis,
morphologic examination was performed by phase-contrast microscopy. For
fluorescence microscopic analysis, cells were fixed in 4% formaldehyde
for 10 min and stained by Hoechst 33258 (10 µg/ml; Sigma) for 1 h. Apoptosis was identified using morphological criteria including
shrinkage of the cytoplasm, membrane blebbing, and nuclear condensation
and/or fragmentation.
Transient Transfection with Dominant-Negative RAR and
RXR--
Transient transfection was performed using the calcium
phosphate coprecipitation method as described before (18). Cells cultured in 24-well plates were cotransfected with pRSh/RAR
-403*, pCDM/RXR
AF2, or a control vector (750 ng/well, respectively), together with pCI-
Gal (250 ng/well; a gift of Promega, Madison, WI).
pCI-
Gal introduces a
-galactosidase gene (lacZ) under
the control of the immediate early enhancer/promoter of human
cytomegalovirus. After incubation overnight, medium was replaced with
1% FCS. After 24 h, cells were pretreated with t-RA for 30 min,
stimulated by H2O2 (175 µM) for
4 h, and subjected to 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal) assay (13). The percentage
of shrunken/rounded blue cells against the total number of blue cells was calculated for each well, and the mean value of four wells was used
to compare data in different groups. Assays were performed in quadruplicate.
Reporter Assay--
The effect of t-RA on the activity of AP-1
was evaluated by a reporter assay as described before (5, 6). In brief,
cells cultured in 24-well plates were transiently transfected with AP-1 reporter plasmid pTRE-LacZ (350 ng/well) or control plasmid pCI-
Gal (350 ng/well). pTRE-LacZ introduces lacZ under the control
of tandemly repeated AP-1 binding sites (19). After the transfection, cells were incubated in 1% FCS for 36 h, pretreated with
AGN193109 or HX531 for 1 h, treated with or without t-RA for
1 h, and then stimulated by H2O2 (125 µM) for 30 h. After X-gal staining, the number of
X-gal-positive cells transfected with pTRE-LacZ was counted and
normalized by the number of X-gal-positive cells transfected with
pCI-
Gal. Assays were performed in quadruplicate.
Northern Blot Analysis--
Expression of c-fos,
c-jun, RAR-
, and RXR-
was examined by Northern blot
analysis (4). In brief, total RNA was extracted by the single-step
method (20) and subjected to analysis. To prepare probes, cDNAs for
c-Fos (21), c-Jun (22), RAR-
(23), and RXR-
(23) (gifts of
Dr. S. J. Collins, Fred Hutchison Cancer Research Center, Seattle,
WA) were labeled by the random priming method. Expression of
glyceraldehyde-3-phosphate dehydrogenase was used as a loading control.
JNK Assay--
Confluent cells cultured in 6-well plates in the
presence of 1% FCS for 24 h were pretreated with or without
AGN193109 or HX531 for 1 h, treated with t-RA for 2 h, and
exposed to H2O2 (200 µM) for 30 min. Activity of JNK was evaluated by the level of phosphorylated JNK,
using PhosphoPlus stress-activated protein kinase/JNK
(Thr182/Tyr185) antibody kit (Cell Signaling
Technology, Beverly, MA) following the protocol provided by the manufacturer.
Statistical Analysis--
Data were expressed as means ± S.D. Statistical analysis was performed using the nonparametric
Mann-Whitney U test to compare data in different groups. A
p value of < 0.05 was used to indicate a statistically
significant difference.
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RESULTS |
Roles of RAR and RXR in the Antiapoptotic Effect of t-RA--
To
examine roles of RAR and RXR in the antiapoptotic effect of t-RA, RAR
pan-antagonist AGN193109 and RXR pan-antagonist HX531 were used.
Mesangial cells were pretreated with AGN193109 or HX531, treated with
t-RA, and then stimulated by H2O2.
Phase-contrast microscopy showed that
H2O2-treated cells showed nuclear condensation and membrane blebbing typical of apoptosis (Fig.
1A). When pretreated with
t-RA, the apoptotic changes were suppressed. The antiapoptotic effect
of t-RA was abrogated by the pretreatment with either AGN193109 or
HX531 (Fig. 1A). The effects of receptor antagonists were
further confirmed quantitatively using Hoechst staining (Fig.
1B). Fluorescence microscopy showed that exposure of cells
to H2O2 induced nuclear condensation (% apoptosis, 81.0 ± 7.1%; mean ± S.D.), and t-RA protected
the cells from apoptosis (% apoptosis, 4.5 ± 2.9%;
p < 0.05). In contrast, the cells pretreated with
AGN193109 or HX531 exhibited significant apoptosis even in the presence
of t-RA (55.9 ± 10.4% in AGN193109-treated cells; 63.2 ± 12.0% in HX531-treated cells). Treatment with AGN193109 or HX531 alone
did not induce any apoptotic changes (data not shown).

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Fig. 1.
Roles of RAR and RXR in the
antiapoptotic effect of t-RA. A, phase-contrast
microscopy. Confluent rat mesangial cells were pretreated with RAR
pan-antagonist (AGN193109; 5 µM) or RXR pan-antagonist
(HX531; 2.5 µM) for 30 min, treated with (+) or without
( ) t-RA (2 µM) for 30 min and then stimulated by
H2O2 (150 µM) for 4 h.
B, Hoechst staining. After the induction of apoptosis, cells
were stained by Hoechst 33258 and examined by fluorescence microscopy
(top). The percentages of apoptotic cells were assessed
quantitatively and shown at the bottom. Data are expressed
as means ± S.D. Asterisks indicate statistically
significant differences (p < 0.05). C,
transient transfection. Cells were transfected with RAR, RXR, or
control vector (vector), together with a -galactosidase
gene. After the transfection, cell were pretreated with t-RA for 30 min, stimulated by H2O2 (175 µM)
for 4 h, and subjected to X-gal assay. The percentage of
shrunken/rounded blue cells against the total number of blue cells was
calculated for each well, and the mean value of four wells was used to
compare data in different groups. Assays were performed in
quadruplicate.
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The roles of RAR and RXR in the antiapoptotic effect of t-RA were
further examined using dominant-negative mutants. Mesangial cells were
transiently transfected with
RAR or
RXR, together with a
-galactosidase gene. When overexpressed, these mutant receptors
selectively and effectively inhibit the function of RARs or RXRs (14,
15). After the transfection, cells were pretreated with t-RA and
stimulated by H2O2. Consistent with the results
using pharmacological inhibitors, dominant-negative inhibition of RAR
or RXR significantly reduced the antiapoptotic effect of t-RA (Fig.
1C).
Roles of RAR and RXR in the Anti-AP-1 Effect of t-RA--
We
previously reported that the AP-1 pathway plays a crucial role in the
induction of apoptosis by H2O2 and that t-RA
inhibits H2O2-induced apoptosis by suppression
of AP-1 (4). To examine roles of RAR and RXR in the anti-AP-1 effect of
t-RA, reporter assay was performed. Cells were transfected with an AP-1
reporter plasmid or a control plasmid, pretreated with AGN193109 or
HX531, treated with t-RA, and then stimulated by
H2O2. As shown in Fig. 2, exposure of cells to
H2O2 significantly increased AP-1 activity (183.3 ± 24.0% versus untreated control, 100 ± 19.1%; p < 0.05). Pretreatment with t-RA suppressed
the AP-1 activation to 88.2 ± 15.6%. Both AGN193109 and HX531
significantly reversed the attenuated AP-1 activity (154.0 ± 27.4% and 149.7 ± 17.5%, respectively; p < 0.05).

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Fig. 2.
Roles of RAR and RXR in the anti-AP-1 effect
of t-RA. Cells were transfected with AP-1 reporter plasmid
pTRE-LacZ or control plasmid pCI- Gal, pretreated with AGN193109 or
HX531 for 1 h, treated with t-RA for 1 h, and then stimulated
by H2O2 (125 µM) for 30 h.
Relative activity of AP-1 was evaluated as described under
"Experimental Procedures." Data are expressed as means ± S.D.
Asterisks indicate statistically significant differences
(p < 0.05). Assays were performed in
quadruplicate.
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Roles of RAR and RXR in the Suppression of c-fos and c-jun
Expression by t-RA--
To further investigate roles of RAR and RXR in
the anti-AP-1 effect of t-RA, we examined effects of receptor
antagonists on the suppression of c-fos and c-jun
by t-RA (Fig. 3). Cells were pretreated
with AGN193109 or HX531, treated with t-RA, and then stimulated by
H2O2 for 2 h. Northern blot analysis
showed that exposure of cells to H2O2
substantially induced expression of c-fos and
c-jun and that treatment with t-RA inhibited this induction. Pretreatment with AGN193109 abrogated the suppressive effects of t-RA
on both c-fos and c-jun. In contrast, HX531
abolished the effect of t-RA on c-fos but not
c-jun.

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Fig. 3.
Roles of RAR and RXR in the suppression of
c-fos and c-jun expression by
t-RA. Cells were pretreated with AGN193109 or HX531 for 30 min,
treated with or without t-RA for 30 min, and then stimulated by
H2O2 (150 µM) for 2 h.
Expression of c-fos and c-jun was examined by
Northern blot analysis. Expression of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) is shown as a loading control.
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Roles of RAR and RXR in the Suppression of JNK Activation by
t-RA--
We further examined effects of receptor antagonists on the
suppression of JNK by t-RA (Fig. 4).
Cells were pretreated with AGN193109 or HX531, treated with t-RA, and
then stimulated by H2O2 for 30 min. Kinase
assay showed that stimulation by H2O2 induced
activation of JNKs (p54 and p46), and treatment with t-RA suppressed
JNK activation. Pretreatment with AGN193109 or HX531 failed to reverse
the suppressed JNK activity.

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Fig. 4.
Roles of RAR and RXR in the suppression of
JNK activation by t-RA: effects of receptor antagonists. Cells
cultured in 1% FCS for 24 h were pretreated with AGN193109 or
HX531 for 1 h, treated with t-RA for 2 h, and then stimulated
by H2O2 (200 µM) for 30 min.
After the treatment, cells were subjected to JNK assay. The amount of
JNK protein is shown at the bottom. The position of p54 and
p46 JNKs are shown on the right.
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The lack of involvement of RAR and RXR was further confirmed using
stable transfectants. Mesangial cells were stably transfected with the
dominant-negative constructs for
RAR and
RXR, which were used in
the transient transfection study (Fig. 1C). Abundant expression of transgenes in the established SM/
RAR8 and SM/
RXR3 cells was confirmed by Northern blot analysis (Fig.
5A). Using these clones,
activity of JNK was evaluated. Kinase assay revealed that, in response
to H2O2, JNK was activated similarly in these clones. Overexpression of
RAR or
RXR did not affect the
suppressive effect of t-RA on JNK (Fig. 5B).

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Fig. 5.
Roles of RAR and RXR in the suppression of
JNK activation by t-RA: effects of dominant-negative mutants.
A, expression of RAR and RXR in established
transfectants. Mesangial cells were stably transfected with RAR or
RXR, and clones SM/ RAR8 and SM/ RXR3 were established. The
expression of RAR and RXR was examined by Northern blot analysis.
SM/Neo is a mock-transfected, control clone. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase. B, effects
of RAR and RXR on H2O2-induced JNK
activation. Stable transfectants were treated with or without t-RA for
2 h and stimulated by H2O2 (200 µM) for 30 min. After the treatment, cells were subjected
to JNK assay.
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DISCUSSION |
In this report, we describe the roles of RAR and RXR in the
antiapoptotic effect of t-RA on oxidant-induced apoptosis. We found
that both nuclear receptors were required for the antiapoptotic effect
of t-RA, as well as its inhibitory effect on AP-1, the crucial molecule
involved in H2O2-induced apoptosis. However, the roles of RAR and RXR in the regulation of individual AP-1 components by t-RA were found to be different. RAR was necessary for
the suppressive effect of t-RA on both c-fos and
c-jun expression, whereas RXR was required only for its
inhibitory effect on c-fos. Furthermore, suppression of JNK
activation by t-RA was mediated by neither RAR nor RXR. To our
knowledge, this is the first to demonstrate selective roles of RAR and
RXR in the t-RA-induced, antiapoptotic pathway.
In general, RAR and RXR are required for various biological effects of
RA including transcriptional regulation, induction of apoptosis, growth
suppression, and cellular differentiation (1, 10). In some cases,
however, RA can also exert its effects without nuclear receptors. For
example, t-RA modulates the activity of protein kinase C by direct
binding to the retinoic acid binding site of protein kinase C-
(24).
Previous reports suggested that the proapoptotic effect of t-RA is
mediated by both receptor-dependent (25, 26) and
receptor-independent mechanisms (27). However, it has not been
determined whether RAR and RXR are involved in the antiapoptotic effect
of t-RA. In this report, we elucidated that the antiapoptotic effect of
t-RA also involved receptor-dependent and -independent
mechanisms. That is, the suppression of c-fos and
c-jun by t-RA was mediated by RAR and RXR, which is
consistent with a previous report showing that expression of
c-fos in response to epidermal growth factor was inhibited
by t-RA through an RAR/RXR-dependent pathway (28). In
contrast, we found that the inhibition of JNK by t-RA was nuclear
receptor-independent. It was confirmed by both pharmacological
inhibitors and dominant-negative mutants. This result is contrastive to
a previous report that showed receptor-dependent inactivation of JNK by t-RA in serum-stimulated epithelial cells (29).
The reason for this discrepancy is currently unclear, but it may be
because of differences in stimuli and downstream pathways to JNK
activation. Of note, Lee et al. (29) showed that t-RA
inhibited JNK activation in a bimodal pattern,
i.e. transient activation at early phase and
sustained activation at late phase and that nuclear receptors were
required for the late but not for the early suppression of JNK.
Currently, the mechanism involved in the RAR/RXR-independent
suppression of JNK is not determined. One possibility is that the
suppressive effect of t-RA observed here might be mediated by other
types of receptors. For example, it has been reported that t-RA can
bind to insulin-like growth factor-II receptor and enhances the primary
function of this receptor (30). Another previous report showed that the
insulin-like growth factor/insulin-like growth factor receptor axis can
inhibit the tumor necrosis factor-
-induced JNK activation via the
phosphatidylinositol 3-kinase pathway (31). The similar mechanism could
be involved in the RAR/RXR-independent suppression of JNK by t-RA.
It is known that t-RA binds to RAR but not to RXR. From this viewpoint,
our finding that RXR inhibitors attenuated the effects of t-RA is
interesting. Because t-RA exerts various effects through heterodimerization of RAR and RXR (10, 32), a simple explanation may be
that t-RA binds to RAR and facilitates their heterodimerization with
RXR, which is essential for its anti-AP-1 action. RXR is supposedly
indispensable for the antiapoptotic and anti-AP-1 effects of t-RA. The
facts that t-RA-mediated differentiation, growth suppression, and
inhibition of c-fos expression required not only RAR but
also RXR further support this hypothesis (26, 28).
Another interesting finding in this report is that the suppression of
c-jun expression by t-RA was reversed by RAR antagonist but
not by RXR antagonist. In contrast to its effect on c-fos, RXR was found to be unnecessary for the suppressive effect of t-RA on
c-jun. Expression of c-fos is regulated by
ternary complex factors whose activity is regulated mainly by
extracellular signal-regulated kinase and p38 MAP kinase. In contrast,
expression of c-jun is regulated by c-Jun and activating
transcription factor-2 that are activated mainly by JNK and/or p38 MAP
kinase (9). The different regulation of c-jun and
c-fos by RAR and RXR could be because of different roles of
these receptors in the regulation of individual MAP kinases.