From the Eicosanoids Biochemistry Section, Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
Received for publication, August 16, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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Troglitazone (TGZ) is a peroxisome
proliferator-activated receptor The peroxisome proliferator-activated receptors
(PPARs)1 are transcription
factors belonging to the nuclear hormone receptor gene superfamily (1).
Three isoforms ( There are several known ligands for PPAR TGZ has specific functions, in addition to being a PPAR The Egr-1 transcription factor (also known as NGFI-A, TIS8, Krox-24,
and Zif268) is a member of the immediate early gene family and
encodes a nuclear phosphoprotein involved in the regulation of cell
growth and differentiation in response to signals such as mitogens,
growth factors, and stress stimuli. However, many reports have recently
suggested Egr-1 as a tumor suppressor gene (20). Egr-1 activates PTEN
(phosphatase and tensin homolog) tumor
suppressor gene during UV irradiation (21), and re-expression of Egr-1
suppresses the growth of transformed cells both in soft agar and in
athymic nude mice (22). Egr-1 is induced very early in the apoptotic
process, where it mediates the activation of downstream regulators such
as p53 (23-25). However, Egr-1-induced apoptosis has been reported in
p53 In the present study, we examine the relationship between TGZ and Egr-1
expression and the effect of TGZ-induced apoptosis. We show that TGZ
induces Egr-1 expression by transcriptional and post-transcriptional
regulation. Egr-1 induction by TGZ results in the increase of binding
affinity and transactivation of the promoter containing Egr-1 consensus
sequences, thereby possibly inducing other anti-tumorigenic proteins.
Furthermore, Egr-1 induction by TGZ appears to be independent of
PPAR Cell Lines and Reagents--
Human colorectal carcinoma cells,
HCT-116, were purchased from ATCC (Manassas, VA) and maintained
in McCoy's 5A medium supplemented with 10% fetal bovine serum
and gentamicin (10 µg/ml). All of HCT-116 cell experiments were done
within passage 16. Rosiglitazone (BRL), azelaoyl PAF (PAF),
PGJ2, and ciglitazone were purchased from Cayman
Chemical (Ann Arbor, MI). TGZ was obtained from Parke-Davis Pharmaceutical Research. PD98059, SB203580, and herbimycin were purchased from Sigma. Egr-1 (sc-110), Egr-2 (sc-190), Egr-3
(sc-191), p53 (sc-263), PTEN (sc-7974), ERK1 (sc-94), ERK2 (sc-154),
PPAR Construction of Plasmids--
The full-length Egr cDNAs were
generated by PCR from human universal QUICK-Clone cDNA
(Clontech, Palo Alto, CA) using the following
primers: for the Egr-1, 5'-GACACCAGCTCTCCAGCCTGCTCGTCCAGG-3' (top strand) and 5'-TTCCCTTTAGCAAATTTCAATTGTCCTGGG-3' (bottom strand);
for the Egr-2, 5'-GTGCGAGGAGCAAATGATGACCGCCAAGGC-3' (top strand) and
5'-CAGCCTGAGTCTCATCTCAAGGTGTCCGGG-3' (bottom strand); for the Egr-3,
5'-CGGCGGCAGCTCGGGAGTGCTATGACCGGC-3' (top strand) and
5'-TCTGGGGGCCCGATCCTCAGGCGCAGGTGG-3' (bottom strand). The amplified products were cloned into pCR2.1 TOPO vector (Stratagene, CA)
and followed by cloning into pCDNA3.1/NEO expression vector (Invitrogen). The pEBS14luc construct was generously
provided by Dr. Gerald Thiel (University of Bari, Bari, Italy). The
Egr-1 promoter ( Apoptosis and Soft Agar Cloning Assay in the Presence of
TGZ--
The DNA content for sub-G1 population was
determined by flow cytometry. HCT-116 cells were plated at 3 × 105 cells/well in 6-well plates, incubated for 16 h,
and then treated with TGZ at different time points in the presence of
serum. The cells (attached and floating cells) were then harvested,
washed with phosphate-buffered saline, fixed by the slow addition of cold 70% ethanol to a total of 1 ml, and stored at 4 °C overnight. The fixed cells were pelleted, washed with ethanol (50%, then 30%), followed by phosphate-buffered saline, and stained in 1 ml of 20 µg/ml propidium iodide containing 1 mg/ml RNase in phosphate-buffered saline for 20 min. 7,500 cells were examined by flow cytometry using BD
Biosciences FACSort equipped with CellQuest software by gating
on an area versus width dot plot to exclude cell debris and
cell aggregates. Apoptosis was measured by the level of sub-diploid DNA
contained in cells following treatment with compounds using CellQuest software.
Soft agar assays were performed to compare the clonogenic potential of
HCT-116 cells in semisolid medium. HCT-116 cells were resuspended at
10,000 cells in 2 ml of 0.4% agarose-containing TGZ in McCoy's 5A
medium and plated on top of 1 ml of 0.8% agarose in 6-well plates.
Plates were incubated for 2-3 weeks at 37 °C. Cell colonies were
visualized by staining with 0.5 ml of p-iodonitrotetrazolium violet staining (Sigma).
Transfection and Luciferase Assay--
HCT-116 cells were plated
in 6-well plates at 2 × 105 cells/well in McCoy's 5A
medium supplemented with 10% fetal bovine serum. After growth for
16 h, plasmid mixtures containing 0.5 µg of promoter linked to
luciferase, 0.5 µg of expression vector, and 0.05 µg of pRL-null
(Promega) were transfected by LipofectAMINE (Invitrogen) according to the manufacturer's protocol. After 48 h of
transfection, the cells were harvested in 1× luciferase lysis buffer,
and luciferase activity was determined and normalized to the pRL-null
luciferase activity using a dual luciferase assay kit (Promega). For
the PPAR RNA Stability in the Presence of Actinomycin D--
When
reaching 60-80% confluence in 10-cm plates, the cells were grown in
the absence of serum for 24 h and then treated with either vehicle
or TGZ (5 µM) for 2 h. The transcription inhibitor, actinomycin D (5 µg/ml), was treated at the indicated time points. Total RNAs were isolated using TRIzol reagent (Invitrogen)
according to the manufacturer's protocol. For Northern blot analysis,
20 µg of total RNA was denatured at 55 °C for 15 min and separated in a 1.0% agarose gel containing 2.2 M formaldehyde and
transferred to Hybond-N membrane (Amersham Biosciences). After
fixing the membrane by UV, blots were prehybridized in hybridization
solution (Rapid-hyb buffer; Amersham Biosciences) for 1 h at
65 °C followed by hybridization with cDNA labeled with
[ Western Blot Analysis--
The level of protein expression was
evaluated using Western blot analysis with Egr-1, Egr-2, Egr-3,
PPAR Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assay (EMSA)--
Nuclear extracts were prepared as described
previously (30). For the gel shift assay, double stranded
oligonucleotides were end-labeled with [ Nuclear Run-on--
HCT-116 cells were grown in the absence of
serum for 24 h and treated with 5 µM TGZ or vehicle
for 3 h. Nuclei were isolated as described previously (30).
In vitro nuclear run-on transcription was carried out using
1 × 107 nuclei and 250 µCi of
[ Troglitazone Inhibits Clonogenic Growth and Induces Apoptosis
in HCT-116 Cells--
We examined the correlation between TGZ-induced
apoptosis and TGZ-induced anti-tumorigenesis in HCT-116 cells.
Anti-tumorigenic activity was measured by soft agar assay, whereas
apoptosis was measured by flow cytometry. As shown in Fig.
1A, TGZ treatment in HCT-116
cells dramatically inhibited the growth of HCT-116 cells on soft agar.
The growth inhibition by TGZ was concentration-dependent, and 5 µM TGZ completely inhibited the growth of HCT-116
cells in soft agar assays. To determine whether TGZ induces cell cycle arrest and/or apoptosis in HCT-116 cells, flow cytometry analysis was
performed (Fig. 1B). Apoptosis and G1 cell cycle
arrest were observed as early as 12 h after treatment with 5 µM TGZ. The stimulation of apoptosis and G1
cell cycle arrest by TGZ are consistent with previous publications (8,
31) reporting that TGZ induces apoptosis and cell cycle arrest in human
colon and breast cancer cells.
Expression of Anti-tumorigenic Proteins Mediated by TGZ--
One
logical mechanism by which TGZ exerts anti-tumorigenesis is the
up-regulation of anti-tumorigenic proteins. To address this question,
we measured the expression of known anti-tumorigenic proteins, Egr-1,
PTEN, and p53. HCT-116 cells were treated with 5 µM TGZ
at indicated time points, and Western analysis was performed. As shown
in Fig. 2A, Egr-1 is induced
dramatically within 3 h, and longer treatment, up to 24 h,
results in a decrease of Egr-1 expression. TGZ-induced Egr-1 expression
was also seen in Northern analysis using HCT-116 cells treated with 5 µM TGZ for 2 h (data not shown). We also measured
PTEN and p53 tumor suppressor gene expression. PTEN is only marginally
induced at an early time point, whereas p53 expression is not altered
by TGZ in HCT-116 cells. Taken together, these results suggest that
Egr-1 induction by TGZ may be pivotal to the anti-tumorigenic activity
of TGZ. Because TGZ is a PPAR TGZ Induces DNA Binding Affinity of Egr-1--
To address whether
Egr-1 protein, produced by TGZ-treated HCT-116 cells, has potential
binding activity to the Egr-1 consensus sequences, EMSA was performed.
Because Egr-1 protein increases in the presence of TGZ, the protein
should bind to the Egr-1 consensus binding site. As shown in Fig.
3A, oligonucleotides
containing two copies of Egr-1 binding sites were generated and used as
a probe. Nuclear extracts prepared from 5 µM TGZ
treatment at different time points (Fig. 3B, lanes
2-5) and the recombinant proteins, Egr-1, Egr-2, and Egr-3,
generated by in vitro translation (Fig. 3B,
lanes 6-8), were used for the EMSA. Using nuclear extracts from TGZ-treated HCT-116 cells and a probe corresponding to the Egr-1
binding site, results show multiple DNA·protein complexes with a
mobility shift (Fig. 3B, arrows TGZ Induces the Transactivation of Egr-1--
The transactivation
activity of Egr-1 in TGZ-treated HCT-116 cells was determined using an
Egr-1-responsive reporter. The plasmid pEBS14luc contained
four copies of Egr-1 response elements linked to the basal promoter
followed by a luciferase reporter gene (Fig. 4A) (35). To determine whether
the expression of Egr-1, Egr-2, and Egr-3 proteins could activate the
luciferase reporter, the plasmid pEBS14luc was transfected
into HCT-116 cells, in combination with empty, Egr-1, Egr-2, or Egr-3
expression vectors. As shown in Fig. 4B, the luciferase
activity was increased by overexpression of Egr-1, Egr-2, and Egr-3
compared with vector-transfected cells, suggesting that overexpression
of Egr family proteins is able to bind and transactivate the reporter
vector. Subsequently, to determine whether TGZ induced Egr-1 expression
would also transactivate the reporter vector containing Egr-1 binding
sites, we transfected the reporter vector and treated with varying
concentrations of TGZ. Indeed, TGZ induced luciferase activity in a
concentration-dependent manner, with 4- and 12-fold
induction observed with 10 and 20 µM TGZ treatment,
respectively. Because TGZ is a PPAR TGZ Induces Egr-1 at the Transcription Level but Not Other PPAR TGZ Induces Egr-1 at the Post-transcription Level--
Because TGZ
affects minimum induction at the Egr-1 promoter, and Egr-1 proteins
were dramatically induced in HCT-116 cells, it is possible that TGZ may
increase Egr-1 mRNA stability. HCT-116 cells were treated with
either TGZ or vehicle for 2 h and then 5 µg/ml of actinomycin D
was added at the indicated time points. Fig.
6, A and B
demonstrates that the half-life of Egr-1 mRNA in control HCT-116
cells was ~15 min compared with 48 min in TGZ-treated cells. These
results suggest that an increase in stability of Egr-1 mRNA is also
a major factor in the induction of Egr-1 proteins by TGZ. Therefore,
TGZ-induced Egr-1 promoter activity and increased Egr-1 mRNA
stability may explain the dramatic induction of Egr-1 at the protein
level.
Effect of TGZ on ERK1/2 Activation, PPAR Although the anti-tumorigenic activities of PPAR (PPAR
) ligand that has
pro-apoptotic activity in human colon cancer. Although TGZ binds to
PPAR
transcription factors as an agonist, emerging evidence suggests
that TGZ acts independently of PPAR
in many functions, including
apoptosis. Early growth response-1 (Egr-1) transcription factor has
been linked to apoptosis and shown to be activated by extracellular
signal-regulated kinase (ERK). We investigated whether TGZ-induced
apoptosis may be related to Egr-1 induction, because TGZ has been known
to induce ERK activity. Our results show that Egr-1 is induced
dramatically by TGZ but not by other PPAR
ligands. TGZ affects Egr-1
induction at least by two mechanisms; TGZ increases Egr-1 promoter
activity by 2-fold and prolongs Egr-1 mRNA stability by 3-fold.
Inhibition of ERK phosphorylation in HCT-116 cells abolishes the Egr-1
induction by TGZ, suggesting its ERK-dependent manner.
Further, the TGZ-induced Egr-1 expression results in increased promoter
activity using a reporter system containing four copies of Egr-1
binding sites, and TGZ induces Egr-1 binding activity to Egr-1
consensus sites as assessed by gel shift assay. In addition, TGZ
induces ERK-dependent phosphorylation of PPAR
, resulting
in the down-regulation of PPAR
activity. The fact that TGZ-induced
apoptosis is accompanied by the biosynthesis of Egr-1 suggests that
Egr-1 plays a pivotal role in TGZ-induced apoptosis in HCT-116 cells.
Our results suggest that Egr-1 induction is a unique property of TGZ
compared with other PPAR
ligands and is independent of PPAR
activation. Thus, the up-regulation of Egr-1 may provide an
explanation for the anti-tumorigenic properties of TGZ.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
/
, and
) have been identified and are
encoded by separate genes. Among them, PPAR
has been further
characterized into three subtypes, PPAR
1, PPAR
2, and PPAR
3 (2,
3). Each type plays an important role in cellular differentiation (4),
apoptosis (5), anti-inflammatory response (6), and lipid metabolism and
metabolic disease, such as glucose homeostasis (7).
, including the natural
prostaglandin 15-deoxy-
12,14-prostaglandin
J2 (PGJ2), the synthetic anti-diabetic
thiazolidinediones, and certain polyunsaturated fatty acids. PPAR
ligands are able to bind to the PPAR
transcription factor, which
then forms a heterodimeric complex with retinoid X receptor that
functions as a central regulator of differentiation, and modulator of
cell growth. Many reports present evidence for anti-tumorigenic
activity of PPAR
ligands (5, 6, 8-10). Among PPAR
ligands, the anti-tumorigenic activity of troglitazone (TGZ) has been well established. For example, TGZ significantly inhibits tumor growth of
human colorectal cancer cells (HCT-116), human breast cancer cells
(MCF-7), and human prostate cancer cells (PC-3) in immunodeficient mice
(11-13). However, the molecular mechanism of TGZ effects on anti-tumorigenesis, other than PPAR
activation, is not known.
agonist. For
example, TGZ up-regulates nitric oxide synthesis (14), induces the p53
pathway (15), inhibits cholesterol biosynthesis (16), induces p21
cyclin-dependent kinase inhibitor (17), has antioxidant
function (18), and activates extracellular signal-regulated protein
kinase (ERK) (19) in a PPAR
-independent manner. Thus, the molecular
mechanism of TGZ-induced anti-tumorigenesis may result from multiple mechanisms.
/
cells (26), indicating that Egr-1 induction occurs in both
p53-dependent and p53-independent manners. Moreover, Egr-1
is down-regulated in several types of neoplasia, as well as in an array
of tumor cell lines (27, 28). These results indicate that Egr-1 plays a
consistent role in growth suppression. Therefore, it is reasonable to
think that Egr-1 could be regulated, at least in part by TGZ, because
both TGZ and Egr-1 have anti-tumorigenic effects.
, because other PPAR
ligands do not induce Egr-1. These data
provide a novel mechanism for understanding how troglitazone exerts its
anti-tumorigenic activity.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (sc-7273), and actin (sc-1615) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-ERK antibody was obtained from Cell Signaling (Beverly, MA).
1260 to +35) linked to the luciferase gene was cloned
by PCR from human genomic DNA (Promega, Madison, WI) using the
following primers:
5'-CGGCTCGAGCGGGAGGAGGAGCGAGGAGGCGGCGG-3' (top
strand; XhoI site is underlined) and
5'-CCCAAGCTTGGGCGGCGGCGGCTCCCCAAGTTCTGCGGC-3' (bottom
strand; HindIII site is underlined). After PCR
amplification, the fragment was digested with XhoI and
HindIII and ligated into pGLBasic3 luciferase vector. All
plasmids were sequenced for verification.
ligand treatments, cells were treated with PPAR
ligand in the absence of serum for 24 h and assayed for luciferase activity.
-32P]dCTP using random primer extension (DECAprimeII
kit; Ambion). The probes used were full-length human Egr-1
fragment. After 4 h of incubation at 65 °C, the blots were
washed once with 2× SSC/0.1% SDS at room temperature and twice with
0.1× SSC/0.1% SDS at 65 °C. Messenger RNA abundance was estimated
by intensities of the hybridization bands of autoradiographs using
Scion Image (Scion Image Co.). Equivalent loading of RNA samples was
confirmed by hybridizing the same blot with a 32P-labeled
glyceraldehyde-3-phosphate dehydrogenase probe, which recognizes RNA of
~1.3 kb.
1, PTEN, and phospho-ERK antibodies. Cells were grown to
60-80% confluency in 10-cm plates followed by 24 h of additional
growth in the absence of serum. Cells were treated with indicated
compounds, and total cell lysates were isolated using 0.1 M
Tris/pH 8.0 containing proteinase inhibitors (Sigma). After sonication
of samples, proteins (30 µg) were separated by SDS-PAGE and
transferred for 1 h onto nitrocellulose membrane (Schleicher & Schuell). The blots were blocked overnight with 5% skim milk in TBS-T
(Tris-buffered saline/Tween 0.05%), and probed with each antibody for
2 h at room temperature. After washing with TBS-T, the blots were
treated with horseradish peroxidase-conjugated secondary antibody for
1 h and washed several times. Proteins were detected by the
enhanced chemiluminescence system (Amersham Biosciences). Western
analysis for p53 was described previously (29).
-32P]ATP by T4
polynucleotide kinase (New England Biolabs). Assays were
performed by incubating 10 µg of nuclear extracts in the binding
buffer (Geneka Biotechnology) containing 200,000 cpm of labeled
probe for 20 min at room temperature. To assure the specific binding of
transcription factors to the probe, the probe was chased by 50-fold
molar excess of cold wild type or mutant oligonucleotide. For the
supershift experiments, Egr-1 antibody (Geneka Biotechnology) was
incubated with nuclear extracts on ice for 30 min before adding to the
binding reaction. Samples were then electrophoresed on 5%
nondenaturing polyacrylamide gels with 0.5× TBE
(Tris/borate/EDTA), and gels were dried and subjected to autoradiography.
-32P]UTP in transcription-optimized buffer (P118A;
Promega). The reaction was performed at 30 °C for 30 min. Labeled
transcripts were purified using TRIzol reagent (Invitrogen) according
to manufacturer's protocol. A total of 1 × 107 cpm
elongated nascent RNA per assay was hybridized for 24 h at 65 °C to filter-immobilized 5 µg of plasmid DNA. The filters were washed with 2× SSC/0.1% SDS for 20 min, followed by 0.1× SSC/0.1% SDS for 40 min. The autoradiographs were subjected to densitometric analysis using Scion Image.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Treatment with troglitazone in HCT-116 cells
results in growth arrest and apoptosis induction. A,
TGZ effects on soft agar cloning assay. Soft agar cloning assay was
performed as described under "Materials and Methods" and
photographed. The figure shown here is representative of results from
three independent experiments. Original magnification, ×16.
B, apoptosis and cell cycle kinetics of TGZ-treated HCT-116
cells at different times. HCT-116 cells were plated in 6-well plates at
a density of 4 × 105 cells/well in 2 ml of medium,
incubated for 16 h, and treated with TGZ (5 µM) for
the indicated times. The cells were stained with propidium iodide and
analyzed by flow cytometry. 7,500 cells were examined by flow cytometry
by gating on an area versus width dot plot to exclude cell
debris and cell aggregates. Apoptosis is represented by the -fold
increase in sub-G1 population over 0 h of treatment.
All values represent mean ± S.D.
ligand, we next compared TGZ to other
PPAR
ligands with regard to Egr-1 induction. HCT-116 cells were
treated with several PPAR
ligands, BRL, PGJ2, PAF,
ciglitazone, and 13-hydroxyoctadecadienoic acid, for 3 h.
These PPAR
ligands are reported to bind and activate PPAR
transcription factor (32, 33). Consistent with previous data, Egr-1
induction was seen in TGZ-treated cells, but poor or no induction was
observed in other PPAR
ligand-treated cells (Fig. 2B),
suggesting that Egr-1 induction by TGZ may be independent of PPAR
.
In addition, the expression of other Egr family proteins, Egr-2 and
Egr-3, was not altered by TGZ and other PPAR
ligands, indicating
that the effect of TGZ is specific for Egr-1.
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Fig. 2.
TGZ induces anti-tumorigenic proteins, Egr-1
and PTEN, but not p53. A, HCT-116 cells were grown in
the absence of serum for 18 h and then treated with 5 µM TGZ for indicated times. The cell lysates were
isolated and subjected to Western analysis. Protein levels of Egr-1,
PTEN, and p53 were measured using specific antibodies described under
"Materials and Methods." Actin antibody was used for loading
control. B, HCT-116 cells were serum-starved for 18 h
and treated with indicated PPAR ligands for 3 h. Cell lysates
were isolated and subjected to Western analysis using the indicated
antibodies. Compounds used were as follows: Vehicle,
Me2SO 0.2%; TGZ, 5 µM;
BRL, 5 µM; PGJ2, 1 µM; PAF, 1 µM; ciglitazone
(CGZ) 1 µM; 13-HODE (HODE), 30 µM.
and
).
Compared with in vitro synthesized Egr proteins, the shifted
band
correspond to Egr-1 proteins, whereas
indicates Egr-3
proteins. Interestingly, a shifted band representing Egr-1 was seen
only at the 3-h time point, whereas Egr-3 was constitutively expressed
during the time course. A shifted band representing Egr-2 was not
detected in TGZ-treated HCT-116 cells. These results are consistent
with Egr-1 induction at the 3-h time point after TGZ treatment as
assessed by Western analysis. These bands represent a specific protein binding to the Egr-1 sequence elements, because complex formation was
diminished by the addition of 50 molar excess of non-radiolabeled identical competitor but not by addition of the identical
oligonucleotide in which the Egr-1 sites were point-mutated (Fig.
3C, lanes 2 and 3). To confirm that
Egr-1 binds to these sites, we performed a gel shift assay in the
presence of Egr-1 antibody to demonstrate supershifting. As shown in
Fig. 3C, the Egr-1 antibody supershifted the band
(lanes 4 and 6). This indicates that the shifted
bands (SS) contain Egr-1 protein. As a control, we examined
Egr-1 binding affinity using nuclear extracts from
12-O-tetradecanoylphorbol-13-acetate-treated K562 cells and
observed similar results as with the nuclear extracts from TGZ-treated
HCT-116 cells (Fig. 3C, lanes 5 and
6). It has been shown that Egr-1 is induced by
12-O-tetradecanoylphorbol-13-acetate-treated K562 cells
(34). Taken together, TGZ specifically induces Egr-1, and the induced
Egr-1 can bind to the Egr-1 consensus sequence as assessed by EMSA.
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Fig. 3.
Complex formation between labeled
oligonucleotide and nuclear extracts. A, comparison of
wild type (Wt) and mutant (Mu) oligonucleotide
sequences containing two copies of the Egr-1 binding site
(underlined). Mutated base pairs are indicated in each
mutated sequence. B, gel shift assay was performed using a
NUSHIFT Egr-1 kit (Geneka Biotechnology) with TGZ-treated HCT-116
nuclear extracts. Nuclear extracts were prepared from HCT-116 cells
after TGZ treatment. Ten µg of nuclear extracts from the indicated
time points were incubated with a 32P-labeled
double-stranded oligonucleotide corresponding to the two Egr-1 binding
sites (lanes 2-5). As a control, gel shift assay was
performed using in vitro translated (IVT) Egr-1,
Egr-2, and Egr-3 proteins (lanes 6-8). Arrow indicates Egr-1, whereas arrow
indicates Egr-3.
C, competitions were done in the presence of 50 molar excess
of non-radiolabeled oligonucleotide corresponding to the wild type
(Wt) or mutant Egr-1 oligonucleotide (Mu) shown
in A. The binding reactions were resolved by 5%
nondenaturing acrylamide electrophoresis. Supershift assays were
performed by a 30-min pre-incubation of the reaction mixture with 2 µg of Egr-1 antibody (Ab) (Geneka Biotechnology), prior to
the addition of radiolabeled probe. The arrows indicate
shifted bands, whereas SS indicates supershifted bands.
Nuclear extracts (NE) were used from two different cell
lines, H for 3 h of TGZ-treated HCT-116 cells, and
K for
12-O-tetradecanoylphorbol-13-acetate-treated K562
cells.
ligand, we tested whether other
PPAR
ligands would also induce luciferase activity. We examined BRL,
PGJ2, and PAF, which have been known to bind to PPAR
.
TGZ is the strongest Egr-1 inducer of luciferase activity (Fig.
4D), although the other compounds are better PPAR
ligands
in terms of PPAR
binding activity. However, in this system, 10 µM TGZ is required to see any significant luciferase
induction, compared with 5 µM TGZ being used for Western
and Northern analyses. This system is an artificial construct and may
require higher concentration of TGZ to see effects. Taken together with
previous results, these data demonstrate that TGZ not only induces
Egr-1 expression and binding activity but also transactivates Egr-1 responsive genes.
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Fig. 4.
Luciferase assay using
pEBS14luc. A, schematic diagram of reporter
plasmid, pEBS14luc, containing four copies of the Egr-1
binding site (EBS) (35). B, co-transfection of
pEBS14luc with Egr expression vectors. HCT-116 cells were
co-transfected with 0.5 µg of the pEBS14luc construct and
with 0.5 µg of Egr-1, Egr-2, Egr-3, or empty pCDNA3 vector.
Luciferase activity was assayed after 48 h as described under
"Materials and Methods." As an internal control, pRL-null vector
(0.05 µg) was used to adjust transfection efficiency. The results
shown here are the means ± S.D. of three independent
transfections. The y axis shows -fold induction of RLU
(firefly luciferase activity/Renilla luciferase
activity) compared with RLU of empty vector transfectants.
C, TGZ induces luciferase activity. pEBS14luc
construct (1 µg) was transfected into HCT-116 cells and then
transfected cells were treated with varying concentrations of TGZ for
24 h. As an internal control, pRL-null vector (0.05 µg) was used
to adjust for transfection efficiency. The results are the means ± S.D. of three independent transfections. The y axis shows
-fold induction of RLU compared with RLU of vehicle-treated cells.
D, HCT-116 cells were transfected with the
pEBS14luc construct (1 µg) and pRL-null vector (0.05 µg) and treated with 10 µM TGZ, 5 µM BRL,
1 µM PAF, or 1 µM PGJ2. After
24 h of incubation, luciferase activity was measured as described
above.
Ligands--
We performed nuclear run-on experiments to examine
whether the Egr-1 induction by TGZ is regulated at a transcriptional
level. HCT-116 cells were treated with serum-free medium for 24 h
followed by treatment with TGZ or vehicle for 3 h.
Radioactive-labeled nascent transcripts were analyzed by hybridization
to immobilized DNAs. Egr-1 gene transcription at 3 h after TGZ
treatment was increased 2-fold, as determined by triplicate
independent experiments. The gene for Sp1 transcription factor was used
as an internal control, because Sp1 expression is not altered by TGZ
treatment in HCT-116 cells (data not shown). Thus, Egr-1 transcripts
were induced by TGZ at the transcriptional level. Fig.
5A is a representative autoradiogram of three experiments. To confirm whether TGZ induces Egr-1 at the transcription level, the Egr-1 promoter was cloned into
the luciferase reporter gene. A plasmid, pEgr1260/LUC, was transfected
into HCT-116 cells, and several PPAR
ligands were treated. As shown
in Fig. 5B, TGZ enhances Egr-1 promoter activity 2-fold,
whereas the other PPAR
ligands do not enhance promoter activity
significantly. Taken together with the nuclear run-on experiments,
these results suggest that TGZ induces Egr-1 expression at the
transcription level by at least 2-fold. However, these data do not
fully explain the dramatic induction shown in Western analysis (Fig.
2), indicating that TGZ may be involved with other mechanisms of Egr-1
induction.
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Fig. 5.
Egr-1 is selectively and transcriptionally
induced by TGZ in HCT-116 cells. A, determination of
Egr-1 transcription levels by nuclear run-on experiment. HCT-116 cells
were grown in the absence of serum for 24 h and treated with
either 5 µM TGZ or vehicle (0.2% Me2SO) for
3 h. The nuclei were isolated, and nuclear run-on assay was
performed using 1 × 107 nuclei and 250 µCi of
[ -32P]UTP at 30 °C for 30 min. Labeled transcripts
were purified and hybridized to membrane containing 5 µg of plasmid
DNA. Sp1 transcripts were used as internal control. The data shown here
are representative of three independent experiments performed in
triplicate (p < 0.003, Student's t test).
B, Egr-1 promoter activity in the presence of several
PPAR
ligands. Human Egr-1 promoter was cloned as described under
"Materials and Methods." HCT-116 cells were transfected with
pEgr1260/LUC and then serum-starved for 24 h. Several PPAR
ligands were treated for 24 h, and luciferase activity was
measured. The internal control vector (pRL-null) was used to normalize
for transfection efficiency. The data represent means ± S.D. from
three different experiments. The concentration of PPAR
ligands used
were the same as for Fig. 2B.
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Fig. 6.
Effect of TGZ on the stability of Egr-1
mRNA. A, HCT-116 cells were treated with TGZ (5 µM) or vehicle (Me2SO) for 2 h and
subsequently with actinomycin D (5 µg/ml). At the indicated times,
total RNAs were isolated and examined by Northern blot analysis with
radiolabeled probe for human Egr-1 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Membrane from vehicle-treated samples
(left panel) was exposed to x-ray film for 20 h,
whereas membrane from TGZ-treated samples (right panel) was
exposed for 2 h. B, hybridization signals were
quantitated with the Scion Image program. The relative level of Egr-1
mRNA (relative to the level of glyceraldehyde-3-phosphate
dehydrogenase) was calculated, and the results were plotted as the
ratio of the mRNA level present at time 0 of actinomycin D
treatment.
Inactivation, and Egr-1
Induction--
TGZ activates ERK1/2 activity in smooth muscle cells
(19), and the activated ERK pathway induces Egr-1 activity (36, 37). In
addition, ERK phosphorylates PPAR
, which results in the inactivation of PPAR
activity (38-40). Therefore, we speculated that TGZ might increase ERK1/2 phosphorylation/activation, resulting in increased expression of Egr-1. In addition, the increase in ERK activity by TGZ
would down-regulate PPAR
activity. The dual phosphorylation of
threonine and tyrosine residues, which is necessary for ERK activation,
was evaluated using the anti-phosphorylated ERK antibody. To test
whether TGZ induces ERK1/2 activation in human colorectal HCT-116
cells, we treated with 5 µM TGZ at the indicated time points and measured the phosphorylation of ERK1/2 using a
phosphospecific antibody. Based on Western blot analysis with an
anti-phospho-ERK antibody, TGZ activates ERK1/2 as early as 30 min
after TGZ treatment in HCT-116 cells and then decreases at later time
points (Fig. 7A). However,
total levels of ERK proteins did not change. To examine whether PPAR
phosphorylation increases concomitant with ERK1/2 activation, PPAR
phosphorylation and expression were examined. PPAR
antibody
recognizes both phosphorylated and nonphosphorylated forms of PPAR
1
or PPAR
2. However, HCT-116 cells express only PPAR
1 (40). TGZ
induced maximum PPAR
1 phosphorylation at 30 min after TGZ treatment,
as measured by a shift in PPAR
1 mobility (Fig. 7B). This
result is consistent with a previous report that PPAR
is
phosphorylated after ERK activation in HCT-116 cells (40). The increase
in ERK activation could also be responsible, in part, for the Egr-1
induction. To investigate whether the activation of ERK1/2 is
associated with Egr-1 induction, HCT-116 cells were pre-treated with
protein kinase inhibitors, PD98059 (mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase inhibitor), SB203580 (p38 MAPK inhibitor), and herbimycin (tyrosine kinase inhibitor), for 1/2 h, followed by 3 h of TGZ treatment. A
significant induction of Egr-1 was detected as early as 3 h after
the initiation of treatment of HCT-116 cells with 5 µM
TGZ. However, the Egr-1 induction by TGZ was abolished by pre-treatment
with PD98059 (Fig. 7C, lane 6) but not by the
pre-treatment of SB203580 or herbimycin A (Fig. 7C,
lanes 7 and 8), indicating that Egr-1 induction
by TGZ is dependent on the ERK/MAPK signaling pathway. Furthermore, the
reporter construct containing four copies of Egr-1 responsive elements
was used to measure luciferase activity after protein kinase inhibitor
and TGZ treatment. As shown in Fig. 7D, PD98059 pre-treatment in HCT-116 cells inhibits TGZ-induced Egr-1 activation as
assessed by the luciferase assay. This result is consistent with
Western analysis for Egr-1 expression as shown in Fig. 7C and indicates that TGZ-induced Egr-1 transactivity is dependent on the
ERK pathway.
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Fig. 7.
ERK1/2 phosphorylation,
PPAR phosphorylation, and Egr-1 induction
after TGZ treatment. HCT-116 cells were serum-starved for 16 h and treated with 5 µM TGZ at indicated time points. The
cell lysates were harvested, and 30 µg of total proteins were
subjected to SDS-PAGE. After transfer, the blots were hybridized with
anti-phospho-ERK1/2 (P-ERK1/2) and anti-ERK1/2
(T-ERK1/2) antibodies (A), and anti-PPAR
and
anti-actin antibodies (B). STD represents 10 µg
of 3T3-L1 cells for PPAR
standard. The data shown here are
representative of three-five independent experiments. C,
HCT-116 cells were preincubated for 30 min with 10 µM
PD98059 (PD), 10 µM SB203580 (SB),
or 1 µM Herbimycin (Her) kinase inhibitors and
then treated with TGZ for an additional 3 h. Whole cell lysates
were isolated and immunoblotted with
-Egr-1. Blots were striped and
reprobed with actin antibody. D, luciferase activity of
promoter containing four Egr-1 binding sites in the presence of protein
kinase inhibitors. The pEBS14luc construct (1 µg) was
transfected into HCT-116 cells and treated with kinase inhibitors alone
or in combination with TGZ. As an internal control, pRL-null vector
(0.05 µg) was used to adjust for transfection efficiency. The results
shown here are the means ± S.D. of three independent
transfections. The concentrations of kinase inhibitors used were the
same as in C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ligands are
well established in human cancer, controversy exists in the literature
with regard to the relative contributions of nuclear receptor-dependent (13, 41, 42) and -independent mechanisms (43-45). In this report, we demonstrate that troglitazone, a PPAR
agonist, induces human colorectal cancer cells to undergo apoptosis and
inhibits their growth on soft agar. In an effort to better understand
the mechanisms responsible for these cellular responses, we measured
the expression of several tumor suppressor proteins and found that TGZ
but not other PPAR
ligands stimulates the expression of Egr-1, a
transcription factor involved in cell growth. TGZ uniquely stimulates
the ERK pathway that down-regulates the PPAR
receptor activity (Fig.
7B), indicating that the increased expression of Egr-1 is
not mediated by the activity of PPAR
receptor (Fig.
8). Furthermore,
TGZ-dependent expression of Egr-1 is attenuated by
inhibition of ERK activation indicating that ERK plays a pivotal role
to induce TGZ-induced Egr-1 expression.
View larger version (14K):
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Fig. 8.
Schematic diagram of TGZ effects on
anti-tumorigenic activity. TGZ affects several pathways shown in
this study. TGZ activates PPAR as a PPAR
agonist, thereby many
PPAR
responsive genes including anti-tumorigenic genes are induced.
TGZ also activates ERK phosphorylation, followed by the phosphorylation
of PPAR
. The phosphorylation of PPAR
leads to an inactivation of
PPAR
activity. In addition, TGZ induces Egr-1 expression by ERK
activation, which leads to induction of the Egr-1 promoter activity and
stabilization of the Egr-1 mRNA. Therefore, the balance of these
pathways may be an important factor to determine the TGZ effects on
cancer.
TGZ differs from other thiazolidinediones in that it has the ability to
induce Egr-1 (Fig. 2B). Of the PPAR agonists reported to
bind to PPAR
and activate target gene expression, TGZ is the only
ligand to induce Egr-1 significantly. One explanation for this
difference is that TGZ contains a vitamin E moiety, which is a unique
feature, compared with the other PPAR
ligands (18). One could think
that TGZ may exert its Egr-1 induction by virtue of its antioxidant
property. However, vitamin E treatment in HCT-116 cells resulted in no
significant induction of Egr-1 expression up to 100 µM
concentration (data not shown), suggesting that the Egr-1 induction by
TGZ is not dependent on antioxidant effect of TGZ. Thus, we propose the
model illustrated in Fig. 8. TGZ exerts it anti-tumorigenic activity
via the activation of the ERK signaling pathway that, in turn,
down-regulates the PPAR
receptor. However, the ERK pathway
up-regulates the expression of the Egr-1 transcription protein that
elicits the anti-tumorigenic action. In contrast, other PPAR
agonists that do not stimulate ERK pathway but instead activate the
PPAR
receptor and alter the expression of anti-tumorigenic proteins.
Thus, several mechanisms appear to be responsible for the
anti-tumorigenic activity of PPAR
agonists, both dependent and
independent of the PPAR
nuclear receptor. It also implies that a
different family of proteins may be responsible for the
anti-tumorigenic activities of the various PPAR
ligands.
Evidence is presented to support that up-regulation of Egr-1 by TGZ occurs by at least two mechanisms; one is at the transcriptional level, and the other is at the post-transcriptional level. The combination of these two mechanisms appears to be responsible for the dramatic increase in the level of Egr-1 protein observed after treatment with TGZ. The fact that Egr-1 induction by TGZ is regulated at the transcriptional level (Fig. 5) suggests that the Egr-1 promoter may contain a binding site downstream of ERK. Indeed, the human Egr-1 promoter contains five serum response elements that mediate signal-induced activation of Egr-1 gene transcription via the ternary response factor Elk1, which is an established substrate for ERK (35). Thus, ERK activation results in the activation of Elk1, which may induce Egr-1 expression. In addition, TGZ also increases mRNA stability of Egr-1 3-fold (Fig. 6). Stability of mRNA can be mediated by several mechanisms. The control of mRNA stability can involve A/U-rich elements (ARE) in the 3' untranslated region or specific RNA stem loop motifs (46). Increased stability might be because of a reduction in the level of proteins that destabilize Egr-1 mRNA or specific mRNA-binding proteins, including AUF1 (47). Interacting with ARE may protect Egr-1 mRNA from degradation by endo- and exonuclease, thereby increasing RNA stability. In the present study, TGZ-mediated induction of Egr-1 is dependent on ERK1/2 but not p38 MAPK or tyrosine kinase, because PD98059 inhibits the TGZ-induced Egr-1 expression. Although p38 MAPK has been reported to play a major role in the induction of mRNA stabilization through ARE in the 3' untranslated region (48), p38 MAPK pathway may not involve Egr-1 RNA stability in HCT-116 cells, because the inhibition of p38 MAPK pathway does not abolish the TGZ-induced Egr-1 expression (Fig. 7C). Recent studies demonstrate that ERK is also involved in the induction of mRNA stability (49, 50). Therefore, ERK or its downstream kinases are considered to affect the proteins that bind to the ARE and induce stabilization. Egr-1 has at least one ARE sequence in the 3' untranslated region. However, further studies are required to identify the exact molecular mechanism by which ERK activation induces the stabilization of Egr-1 mRNA and increases the transcriptional activity of the Egr-1 promoter.
Egr-1 is a member of the immediate early gene response family and encodes a nuclear phosphoprotein involved in the regulation of cell growth and differentiation in response to signals such as mitogens, growth factors, and stress stimuli. However, evidence was presented recently (20, 21, 35, 51) that Egr-1 is a pro-apoptotic protein, and the overexpression of Egr-1 results in increased susceptibility to apoptosis when induced by an apoptotic agent (25). The molecular mechanism by which Egr-1 induces apoptosis and/or anti-tumorigenesis has not been studied, but Egr-1 can be considered as a tumor suppressor gene (52). One logical mechanism by which Egr-1 exerts anti-tumorigenesis is the transcriptional up-regulation of anti-tumorigenic genes. Several known anti-tumorigenic proteins including PTEN are induced by Egr-1 expression (21). PTEN is induced modestly by TGZ along with robust Egr-1 expression (Fig. 2A). Egr-1 up-regulates transcription of the p53 tumor suppressor directly (25), but we do not observe the p53 induction after Egr-1 induction by TGZ in HCT-116 cells. Thus, the increased expression of this transcription factor, Egr-1, appears to play a role in the anti-tumorigenic activity of TGZ, but further evidence is required to fully support that conclusion.
The anti-tumorigenic effect of TGZ is not well understood, and there appears to be some dependence on species studied. Whereas TGZ significantly inhibits tumor growth of human tumors in, for example, HCT-116 colorectal cancer cells, MCF-7 breast cancer cells, and PC-3 prostate cancer cells in immunodeficient mice (11-13), TGZ stimulates colon polyp formation in Min mouse (53, 54). In our study, TGZ also induces Egr-1 in mouse colorectal cancer cell, CMT-93 (data not shown), suggesting that Egr-1 induction by TGZ may be, at least, a common effect in both human and mouse cells. There are several ongoing attempts to unravel this puzzle. One hypothesis is that human and mouse cells use different downstream signaling molecules, resulting in these two distinct outcomes. TGZ-induced apoptosis is mediated by ERK activation (55), whereas TGZ treatment in skeletal muscle cells does not alter ERK activity (56). Although the ERK pathway is linked to cell proliferation and tumorigenic activity, recent studies have shown that ERK activation can lead to arrest of cell growth by the activation of p21 cyclin-dependent kinase inhibitor. Because Egr-1 expression can be pro-tumorigenic or anti-tumorigenic, depending on target genes, TGZ may have opposite effects depending on the downstream targets of the Egr-1 pathway. Another hypothesis is that the cells are actually using the same signaling pathways, but then the cells selectively utilize different cofactors, which direct the basic signaling molecules to the appropriate final targets. In this regard, there are two Egr-1-binding proteins, NAB1 and NAB2 (57, 58), that function as transcriptional repressors of Egr-1. Therefore, either pro-apoptotic or anti-apoptotic effects of TGZ may be determined by cofactor expression. The competition and usage of these two proteins in the same region may be one of the determinations for the TGZ-induced anti-tumorigenic activity.
In conclusion, we have shown that the PPAR ligand TGZ,
independent of the nuclear receptor, stimulates the expression of the
transcription factor, Egr-1, a protein with established tumor suppressor activity. The expression is mediated by enhancement of ERK
signaling pathway and occurs via both transcriptional and post-transcriptional mechanisms. Further investigations are required to
elucidate an understanding of how changes in Egr-1 expression mediate
the anti-tumorigenic activity of TGZ.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Jeanelle M. Martinez and Jennifer B. Nixon of NIEHS, National Institutes of Health for comments and suggestions. We also thank Dr. Gerald Thiel (University of Bari, Bari, Italy) for providing the pEBS14luc construct and Scott M. Moore for providing technical assistance.
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FOOTNOTES |
---|
* 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: Laboratory of
Molecular Carcinogenesis, 111 TW Alexander Dr., Research Triangle Park,
NC 27709. Tel.: 919-541-3911; Fax: 919-541-0146; E-mail: Eling@niehs.nih.gov.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M208394200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PPAR, peroxisome proliferator-activated receptor;
Egr-1, early growth
response-1;
TGZ, troglitazone;
ERK, extracellular signal-regulated
protein kinase;
PGJ2, 15-deoxy-12,14-prostaglandin J2;
EMSA, electrophoretic mobility shift assay;
BRL, rosiglitazone;
PAF, azelaoyl
PAF;
MAPK, mitogen-activated protein kinase;
ARE, A/U-rich
elements.
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