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* Metabolism and Cancer Susceptibility Section, Basic Research Laboratory, Division of Basic Sciences, National Cancer Institute and
§ Intramural Research Support Program, SAIC-Frederick, NCI-FCRDC, Frederick, Maryland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
lipopolysaccharide (LPS) were greatly reduced by two selective NOS II
inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2
overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and
found that they caused PGHS-2 expression and PGE2 production. This
effect was abolished by hemoglobin, a NO scavenger. Using
electrophoretic mobility shift assays, we found that both NOR-1 and
SNAP caused ß-catenin/LEF-1 DNA complex formation. Super-shift by
anti-ß-catenin antibody confirmed the presence of ß-catenin in the
complex. Cell fractionation studies indicated that NO donors caused an
increase in free soluble cytoplasmic ß-catenin. This is further
corroborated by the immunocytochemistry data showing the redistribution
of ß-catenin from the predominantly membrane localization into the
cytoplasm and nucleus after treatment with NO donors. To further
explore the possible connection between PGHS-2 expression and
ß-catenin/LEF-1 DNA complex formation, we studied IMCE
(ApcMin/+) cells, a sister cell line of YAMC
with similar genetic background but differing in Apc
genotype and, consequently, their ß-catenin levels. We found that
IMCE cells, in comparison with YAMC cells, had markedly higher
ß-catenin/LEF-1 DNA complex formation under both resting conditions
as well as after induction with NO. In parallel fashion, IMCE cells
expressed significantly higher levels of PGHS-2 mRNA and protein, and
generated more PGE2. Overall, this study suggests that NO may be
involved in PGHS-2 overexpression in conditionally immortalized mouse
colonic epithelial cells. Although the molecular mechanism of the link
is still under investigation, this effect of NO appears directly or
indirectly to be a result of the increase in free soluble ß-catenin
and the formation of nuclear ß-catenin/LEF-1 DNA complex.—Mei,
J. M., Hord, N. G., Winterstein, D. F., Donald, S. P., and Phang, J. M. Expression of prostaglandin endoperoxide H
synthase-2 induced by nitric oxide in conditionally immortalized murine
colonic epithelial cells.
Key Words: PGHS-2 • NOS II • nitric oxide • colonic epithelial cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
. Moreover, high PGHS-2 expression in
human colon cancer cells is closely associated with increased
metastatic potential (6)
. In contrast, PGHS-1, a
constitutively expressed PGHS family member with high-sequence homology
to PGHS-2, is not induced during colorectal neoplastic transformation,
supporting the hypothesis that the inducible expression of the PGHS-2
isozyme may be important in the development of colon adenoma and
cancer. Nonsteroid anti-inflammatory drugs that inhibit PGHS activity,
such as aspirin and the newly developed selective PGHS-2 inhibitors,
have well-documented preventive effects against precancerous colon
polyp formation and subsequent malignant transformation
(7
8
9)
. Furthermore, it has been reported that intestinal
polyp formation was dramatically reduced by ablation of the PGHS-2 gene
(10)
. Although PGHS-2 protein catalyzes the first step in
the production of a host of bioactive arachidonate metabolites, the
production of prostaglandin E2 (PGE2) is probably the most important in
carcinogenesis. This prostanoid has been shown to have antiapoptotic
and mitogenic activities that may enhance neoplastic progression
(11
, 12)
.
The role of the adenomatous polyposis coli gene (Apc) as a
‘gate-keeper’ in colonic carcinogenesis and its link with the
downstream transcription regulator ß-catenin/Tcf-LEF complexes has
been recently established (13
14
15
16
17
18)
. It has been
hypothesized, although not proven, that PGHS-2 may be one of the genes
responsive to ß-catenin accumulation and possibly the
ß-catenin/Tcf-LEF transcription pathway (19
, 20)
. PGHS-2
mRNA and protein are highly overexpressed in human and mouse colonic
adenomas compared with normal tissue (3
4
5)
. However,
evidence is mounting to support the contributory role for inducible
nitric oxide synthase (NOS II) expression in colorectal tumorigenesis.
Overexpression of NOS II frequently occurs in inflammatory bowel
diseases that predispose the subjects to colon cancer (21
, 22)
. Like PGHS-2, NOS II mRNA and protein are overexpressed in
colonic adenomas compared with normal tissue (23)
. These
studies, taken together with in vitro studies of PGHS-2
catalysis by NO, link the production of NO to the regulation of PGHS-2
catalytic activity as well as transcriptional activation of the PGHS-2
gene. Indeed, NO-releasing drugs have been shown to either activate the
catalytic function of PGHS-2 or transcription of the PGHS-2 gene,
resulting in accumulation of PGHS-2 protein (24
25
26
27)
. But,
it has not yet been established whether NOS II and PGHS-2 expression
are coincident, temporally distinct, or causally related biological
events, and the possible mechanism(s) involved remain unknown.
To gain insight into the interconnection between PGHS-2 and NOS II and
the potential mechanisms involved, we examined the expression of PGHS-2
and NOS II as well as the production of PGE2 and NO in the
nontransformed and nontumorigenic murine colonic epithelial cells.
These conditionally immortal cells are designated YAMC
(Apc+/+) derived from a SV40LT antigen
parental mouse; IMCE (ApcMin/+) are derived
from the F1 hybrids resulting from the mating of
ApcMin/+ and SV40LT antigen transgenic
mice. This pair of nontransformed murine colon epithelial cell lines of
similar genetic background includes one that carries the
ApcMin/+ mutation and consequently expresses
higher ß-catenin levels. Because both YAMC and IMCE express the
heat-labile SV40LT antigen that allows them to proliferate at 33°C,
they revert to a nontransformed phenotype at the restrictive
temperature of 39°C, at which the proliferation of these cells ceases
(28
29
30)
. The epithelial nature of these cells was
demonstrated by staining with anti-keratin antisera. The genotype and
expression of adematous polyposis coli (APC) protein of
these cell types have been confirmed by allele-specific polymerase
chain reaction and by western immunoblotting, respectively (28
, 29)
. Furthermore, these cells have been used to demonstrate that
the ApcMin/+ mutation in IMCE cells can
cooperate with stably transduced oncogenic ras to produce
the transformed, tumorigenic phenotype (e.g., growth in soft agar;
tumor formation in athymic mice) (31)
. In contrast to most
transformed cells or cell lines derived from colon tumors, these cells
have undetectable (YAMC) or little (IMCE) constitutive PGHS-2
expression by northern and Western blot analysis. On stimulation with
IFN-/LPS or NO donors at 39°C, they express high levels of PGHS-2
and generate PGE2. This stimulation by LPS and IFN- or NO mimics the
exposure of colonic epithelial cells to products of bacterial
metabolism and immune cells in vivo. Data from the present
study suggest that the presence of NO caused by IFN-/LPS stimulation
or NO-releasing drugs may play an important contributory role in PGHS-2
overexpression in these conditionally immortalized, nontumor-derived,
colonic epithelial cells under nontransforming conditions. Furthermore,
this overexpression of PGHS-2 is associated, either directly or
indirectly, with the accumulation of ß-catenin in the cytoplasm and
nucleus, and possibly the formation of the ß-catenin/LEF-1 DNA
complex.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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from Life Technologies-BRL (Grand Island,
N.Y.); neonatal calf serum from Gemini (Calabasas, Calif.), ITS+ from
Collaborative (Bedford, Mass.). An ELISA (enzyme-linked immunosorbent
assay) test kit for the quantitative analysis of PGE2 was purchased
from Cayman (Ann Arbor, Mich.). NOS II inhibitors S-methylisothiourea
sulfate (SMT), and L-N6-(1-iminoethyl) lysine (L-NIL) and NO donors
(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide
(NOR-1) and S-nitroso-N-acetylpenicillamine (SNAP) were purchased from
Calbiochem (San Diego, Calif.). Bovine hemoglobin was purchased
from Sigma and then prepared by dissolving in H2O
and reacting with 1 mol excess of sodium dithionite, followed by
purification with gel-chromatography (Sephadex G 25, Pharmacia,
Piscataway, N.J.). All other chemicals and reagents were purchased from
Sigma unless indicated otherwise.
Cell culture
Experiments were carried out using the conditionally
immortalized murine colonic epithelial cell YAMC (young adult mouse
colon, gift of Dr. Robert Whitehead at Ludwig Institute for Cancer
Research, Melbourne, Australia). Both YAMC and IMCE cells express the
heat-labile SV40 large T antigen with an IFN--inducible promoter
(28
, 29
, 31)
. The temperature-sensitive SV40 large T
antigen with IFN--inducible promoter is active only at 33°C. It
becomes inactivated and nonfunctional once cells are transferred to
39°C. All cells were grown on 75 cm2 culture
flask coated with type I collagen (5 µg/cm2) in
RPMI 1640 media supplemented with 5% neonatal calf serum, ITS+
(insulin 6.25 µg/ml, transferrin 6.25 µg/ml, selenious acid 6.25
ng/ml, linoleic acid 5.35 mg/ml, and bovine serum albumin 1.25 mg/ml),
5 IU/ml of murine IFN-, 100,000 IU/l penicillin and 100 mg/l
streptomycin. They were cultured under transforming (permissive)
conditions in a 33°C incubator with 5% CO2
plus all the aforementioned supplements in the media. All cells were
then transferred on attaining confluency into a 39°C incubator under
nontransforming (nonpermissive) conditions in serum-free and
IFN--free media for 72 h before each experiment. For NOS II and
PGHS-2 induction by inflammatory stimuli, cells in the treatment groups
were stimulated by incubation in media containing LPS (1 µg/ml) and
IFN- (100 IU/ml) for the desired period of time as indicated in each
experiment.
Nitrite assay
Culture media from treated cells were incubated for 60 min at
25°C in the presence of 3.5 mU/ml of nitrate reductase and 0.18 nM
NADPH to reduce nitrate to nitrite. Nitrite levels were then measured
in culture media by adding equal volumes of Griess reagent (1%
sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride,
2.5% phosphoric acid) and absorbance was read at 550 nm with a
microplate reader (Molecular, Sunnyvale, Calif.). Sodium nitrite was
used as the standard.
Analysis of PGE2
An ELISA test kit (Cayman) for the quantitative analysis of PGE2
was used that operates on the basis of competition between the enzyme
conjugate and PGE2 in the sample for a limited number of binding sites
on the monoclonal antibody-coated plate. The sample (culture media from
treated cells) or standard solution was first added to the microplate.
Next, the diluted enzyme conjugate was added, and the mixture was
shaken and incubated at room temperature for 1 h. The plate was
then washed, removing all unbound material. The bound enzyme conjugate
was detected by the addition of K-Blue Substrate, which generated color
intensity after 30 min. Quantitative test results were obtained by
measuring and comparing the absorbance reading of the sample wells
against the standards with a microplate reader at 650 nm. The extent of
color development was inversely proportional to the amount of PGE2 in
the sample or standard.
Northern blotting
As described previously (30)
, total RNA was
prepared from YAMC cells by the Trizol method (Life Technologies-BRL).
We electrophoresed 10 µg RNA on a 1% agarose/6.66% formaldehyde gel
at 25V for 18 h with 1x MOPS buffer containing 0.2 M MOPS (pH
7.0), 0.08 M sodium acetate, and 0.01 M ethylenediaminetetraacetate
(EDTA) (pH 8.0). The RNA was transferred onto Nytran (Schleicher &
Schuell, Keene, N.H.) membrane in 20x SSC overnight. The murine cDNA
of PGHS-2 (1.9 kb) probe was radiolabeled by random priming using
Pharmacia Labeling Beads (dCTP) (Pharmacia). The probe was then
hybridized to the RNA blot using 2 x 106
cpm/ml in ExpressHyb (Clontech, Palo Alto, Calif.) hybridization
solution at 65°C for 1 h. Blots were washed at a stringency of
50°C in 0.2x SSC/0.1% sodium dodecyl sulfate (SDS).
Western blotting
Cells were washed twice with cold phosphate-buffered saline
(PBS) and harvested under either denaturing conditions by scraping in
boiling 2x Laemmli sample buffer (Bio-Rad, Hercules, Calif.) or
nondenaturing conditions by using a RIPA Buffer Set (Boehringer
Mannheim). For total cell lysates under denaturing conditions, samples
were heated at boiling temperature for an additional 5 min. Homogenates
were then prepared in the Laemmli sample buffer by sonication (1 min
each). After centrifugation at 2000 g for 5 min, the
supernatants were used as the protein source. To make protein
preparations that contain only soluble cytosolic fractions, cells were
lysed in RIPA buffer by a micro-homogenizer (Sonifier, Branson,
Danbury, Conn.) under nondenaturing conditions at 4°C. The
homogenized soluble supernatant was then prepared by centrifugation at
100,000 g for 30 min at 4°C. The protein concentration was
determined by the BCA method (Pierce, Rockford, Ill.). Electrophoresis
samples were prepared by mixing the respective protein preparations
with 2x Laemmli sample buffer. To each well of SDS-polyacrylamide gel,
15–30 µl of cell lysate (~15–30 µg total protein) was applied
and electrophoresed in 0.75 mm thick 7.5% Tris-glycine gel (Bio-Rad).
Transfer to nitrocellulose membrane was done using a semi-dry blotter
(Bio-Rad) at 15V for 30 min. Blots were then probed with the respective
primary antibodies at the manufacturers’ suggested dilution, followed
by a secondary anti-mouse IgG antibody conjugated to horseradish
peroxidase (1:2000). Detection was done using an ECL kit (Amersham,
Arlington Heights, Ill.). Blots were routinely stripped by Encore Blot
Stripping Kit (Novus, San Diego, Calif.) and reprobed with anti-actin
monoclonal antibody to serve as controls.
Preparation of nuclear extracts and electrophoretic mobility shift
assays
Nuclear extracts were prepared from YAMC cells according to the
method by Dignam et al. with modifications (30
, 32)
. Cells
were rinsed once with cold PBS, followed by trypsinization. After
centrifugation at 1000 g for 5 min, they were resuspended in
5 pellet volumes of hypotonic buffer containing 0.2 mM PMSF and 0.5 mM
DTT. They were then chilled on ice for 10 min, followed by lysis with a
PT 3000 Polytron (Brinkmann, Littau, Switzerland) for 30 s and
centrifuged at 4000 g for 15 min. The pellet was resuspended
in 0.5 pellet volumes of low-salt buffer. An equal volume of high-salt
buffer was added dropwise to the gently stirred suspension. The nuclear
extracts were subjected to centrifugation at 16,000 g for 30
min, followed by dialysis overnight. We added 5 µg nuclear protein to
a 20 µl reaction mix containing 300 ng poly dI-dC; binding buffer (10
mM Hepes, pH 7.6; 60 µM KCl; 1 mM EDTA; 1 mM DTT; 12% glycerol);
with or without double-stranded mouse LEF-1 oligonucleotide (Life
Technologies-BRL), CACCCTTTGAAGCTC with 5' overhang, as a specific
competitor. Samples were incubated on ice for 10 min. Then, LEF-1
oligonucleotide, radio-labeled using T4 kinase (Life Technologies-BRL)
and 32P ATP (NEN, Boston, Mass.), was added at
1.5–2 x 104 cpm per reaction and incubated
at RT for 30 min. DNA loading dye (Quality Biological, Gaithersburg,
Md.) was added to stop the reaction. Samples were run on a 4%
polyacrylamide (37.5:1) (Protogel, National Diagnostics, Atlanta, Ga.)
gel at 189V for 2.5 h in 0.5x TBE running buffer. Gels were dried
and exposed to XAR-5 film (Kodak). For super-shift studies, 3–5 µg
nuclear lysate was mixed in a 20 µl reaction mixture as described for
electrophoretic mobility shift assays (EMSA) and incubated on ice for
10 min. Antibodies, 12 µg polyclonal anti-ß-catenin and 500 ng
monoclonal anti-E-cadherin, or preimmune IgG, were then added to the
respective reaction tubes. Reactions were incubated on ice for 15 min.
32P-labeled murine LEF-1 oligonucleotide probe
was added at 1.5–2 x 104 cpm per reaction
and incubated at RT for 30 min.
Immunocytochemical staining
Cells grown on Lab-Tec chambered coverslips (Nunc, Naperville,
Ill.) were fixed in 95% Ethanol at room temperature for 10 min and
air-dried. Immunocytochemical staining was performed following the
protocol from a Vectastain ABC kit (Vector, Burlingame, Calif.).
Coverslips were rinsed in TBS for 10 min before being blocked in 2%
nonimmune mouse serum for 20 min. Negative control coverslips were kept
in PBS without exposure to the primary antibody, monoclonal mouse
anti-ß-catenin antibody (Transduction), whereas the remaining
coverslips were incubated for 30 min with the primary antibody at 1:50
dilution. The coverslips were then incubated with 0.5% mouse
biotinylated secondary antibody for 30 min and stained with
biotin-avidin reagents (Vectorstain Elite ABC kit, Vector) for 30 min,
followed by 3,3'-diaminobenzidine tetrahydrochloride staining for 5
min. The coverslips were then counterstained with Gill’s hematoxylin
for 6 s.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and LPS (100 IU/ml) and LPS (1 µg/ml),
the classic inflammatory stimuli for PGHS-2 and NOS II induction
(Fig. 1A
). Actin served as a control. We found that not only PGHS-2
was induced, as previously reported (30)
, but also NOS II
was induced by the same stimulation. To ascertain the functionality of
these proteins, their enzymatic products PGE2 and nitrite (the stable
derivative of NO) were measured (Fig. 1B
). Furthermore, the
generation of PGE2 and NO was inhibited by the respective inhibitors of
PGHS-2 (sulindac sulfide) and NOS II (L-NIL and SMT), demonstrating the
specific functional activity of the induced PGHS-2 and NOS II.
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Attenuation of PGHS-2 expression and PGE2 generation by selective
NOS II inhibitors
The close interconnection between NO and the enzymatic activity of
PGHS-2 has been reported in the literature (24
25
26
27)
. NO
has been found to stimulate the generation of PGE2 through the
activation of PGHS-2 activity. To examine this effect of endogenously
produced NO in YAMC, we tested two selective NOS II inhibitors, L-NIL
and SMT, on the expression of PGHS-2 and the generation of PGE2 induced
by IFN- and LPS. We found that the inducible expression of PGHS-2
was greatly reduced by both L-NIL and SMT (Fig. 2A, B
). As expected, L-NIL and SMT also
significantly decreased PGE2 generation by blocking endogenous NO
production (Fig. 2C
). This experiment not only confirmed
that NO increases the enzymatic activity of PGHS-2 but also implied
that NO may play a role in regulating the expression of PGHS-2.
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Induction of PGHS-2 by NO-releasing compounds NOR-1 and SNAP
The above findings suggest that NO may exert an induction effect
on PGHS-2 overexpression in response to the stimulation of IFN- and
LPS. In an attempt to determine whether exogenous NO could generate the
same effect, NO donors, NOR-1 and SNAP, were used at several different
concentrations to treat these cells. The expression of PGHS-2 was then
examined by northern and western blotting (Figs. 3
, 4
). In addition, the enzymatic activity of PGHS-2 was studied by
measuring PGE2 production (Fig. 5
). As demonstrated in these experiments, both NOR-1 and SNAP induced
PGHS-2 mRNA expression in YAMC cells in a time-dependent manner (Fig. 3)
. NOR-1 and SNAP also caused a dose-dependent induction of PGHS-2
protein (Fig. 4)
. These effects were specifically caused by
NO released spontaneously from these two chemically distinct compounds
as they were abolished in the presence of hemoglobin, a potent NO
scavenger (Figs. 4
, 5)
. These experiments demonstrated that NO, either
endogenously produced or exogenously supplied, can greatly enhance the
expression of PGHS-2 in colonic epithelial cells.
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Formation of DNA/ß-catenin/LEF-1 complex induced by NO-releasing
compounds NOR-1 and SNAP
It has been suggested that the promoter region of the PGHS-2
gene contains Tcf-LEF binding sites. Therefore, PGHS-2 has been
proposed to be under the control of the ß-catenin/Tcf-LEF complex,
although definitive proof is still lacking (19)
. We found
that the mouse PGHS-2 gene promoter does indeed contain potential
binding sites of the consensus sequence for Tcf-LEF (see Discussion).
We set out to explore the possibility that the expression of PGHS-2 by
NO donors in these colonic epithelial cells may be associated with,
directly or indirectly, the ß-catenin/Tcf-LEF DNA complex formation.
Unlike cell lines derived from colon tumors that often express
constitutively high levels of ß-catenin/Tcf-LEF activity, the
nontransformed and nontumorigenic YAMC cells express very little
ß-catenin/LEF-1 complex when cultured under nontransforming
conditions (see Materials and Methods). As shown in Fig. 6
, the formation of the ß-catenin/LEF-1 complex was greatly enhanced by
two NO donors, NOR-1 and SNAP. Furthermore, the amount of
ß-catenin/LEF-1 complex induced by both NOR-1 (1, 5, and 10 µM) and
SNAP (5, 10, and 20 µM) appeared to be concentration-dependent.
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Time-dependence of DNA/ß-catenin/LEF-1 complex formation induced
by NO-releasing compounds NOR-1 and SNAP
To further confirm that NO released by NOR-1 and SNAP specifically
caused DNA/ß-catenin/LEF-1 complex formation in YAMC cells, a time
course study was carried out. Cells were treated with either NOR-1 (5
µM) or SNAP (10 µM) for 10, 30, 60, and 120 min (Figs. 7A, B
). The formation of ß-catenin/LEF-1 DNA complex induced
by NOR-1 and SNAP occurred in a time-dependent fashion.
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Super-shifting by anti-ß-catenin antibody of the
DNA/ß-catenin/LEF-1 complexes induced by NOR-1 or SNAP
The suggested participation of ß-catenin in the DNA binding
complex was confirmed by a series of super-shift assays using an
anti-ß-catenin antibody (Figs. 8A, B
). Cells were first treated with either NOR-1 (5 µM) or
SNAP (10 µM) for 30 min before nuclear extracts were harvested.
Nonspecific antibodies, namely, rabbit preimmune IgG and
anti-E-cadherin antibody, were used as controls in the super-shift
assays and did not cause any super-shifting of the bands. This study
directly demonstrates the presence of ß-catenin in the DNA binding
complex in conjunction with LEF-1.
|
Increase of free ß-catenin in the soluble cytosolic fraction in
response to NOR-1 or SNAP treatment
The localization of ß-catenin is normally associated with the
transmembrane glycoprotein E-cadherin. The resting ß-catenin level
remains very low because of the constant degradation by APC and
GSK-3ß in conjunction with Axin (34
, 35)
. We attempted
to explore the source of the increased nuclear ß-catenin present in
the DNA binding complex. The amount of total ß-catenin in whole cell
lysate was examined and did not change after various treatments with NO
donors, NOR-1 (5 µM) and SNAP (10 µM) (Fig. 9A
). In contrast, the amount of free ß-catenin in the
soluble cytosolic fraction showed a dramatic increase from
nondetectable in control cells to easily appreciated levels in cells
treated with NO donors for 2 h (Fig. 9B
). Although this
finding does not reveal the mechanism responsible for the increase in
the free cytoplasmic pool of ß-catenin, it implies that NO donors may
facilitate the nuclear ß-catenin/LEF-1 complex formation by
increasing the amount of free ß-catenin available for its nuclear
translocation.
|
Subcellular localization and redistribution of ß-catenin
ß-Catenin was primarily localized in the membrane in YAMC cells
after culturing under nontransforming condition for 48 h,
presumably bound to the transmembrane protein E-cadherin (Fig. 10A
). There was very little, if any, staining of ß-catenin in
the nucleus and cytoplasm. However, pronounced nuclear staining of
ß-catenin was detected when cells were exposed to NOR-1 (5 µM)
overnight (16 h) (Fig. 10B
), indicating a redistribution and
nuclear translocation of subcellular ß-catenin in response to NO
treatment. Corroborating the findings demonstrated in Fig. 9A, B
, immunocytochemical studies showed the increase of ß-catenin
in cytoplasm (Fig. 10B
). Similar findings were observed in
the redistribution of ß-catenin with SNAP treatment (data not shown).
|
Differential formation of ß-catenin/LEF-1 DNA binding complex and
induction of PGHS-2 mRNA and protein as well as production of PGE2 in
YAMC and IMCE cells contrasting in Apc genotype
Although the findings with NO treatment suggested a link between
PGHS-2 expression and the formation of ß-catenin/LEF-1 DNA binding
complex, the effects of NO on PGHS-2 may be mediated by distinct
NO-activated pathways. To implicate the participation of ß-catenin in
the NO effect on PGHS-2, we compared YAMC and IMCE cells differing in
ß-catenin degradation because of their genetic difference in
Apc. We found that IMCE cells expressed markedly higher
ß-catenin/LEF-1 DNA binding complex than YAMC cells (Fig. 11A
). The difference in ß-catenin/LEF-1 DNA complex between
these two cell lines and the corresponding differential expression of
PGHS-2 mRNA and protein were both amplified after treatment with SNAP
(Fig. 11B, C
). Furthermore, PGE2 production in IMCE was
significantly higher than that in YAMC cells (Fig. 11D
).
Although this is not definitive proof that ß-catenin/LEF DNA complex
is directly responsible for the expression of PGHS-2, it strongly
indicates that they are associated either directly or indirectly.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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29
30)
. Using
the classical stimuli for NOS II and PGHS-2, IFN- and LPS, we used
these cells to investigate the association between NO generation and
PGHS-2 expression as well as the possible roles of soluble free
ß-catenin accumulation and ß-catenin/Tcf-LEF complex formation. It
is important to note that the effect of IFN- and LPS during the
treatment is unrelated to the IFN- inducible promoter activity
because the temperature-sensitive mutant SV40 large T antigen is
inactivated and remains nonfunctional at 39°C during the experiment
(data not shown) (28
29
30)
. The link between PGHS-2 and NO
was suggested by the finding that selective NOS II inhibitors, L-NIL
and SMT, greatly reduced the overproduction of PGE2 induced by IFN-
and LPS under the above-described conditions (Fig. 2C
). It
is unlikely that these two chemically distinct selective NOS inhibitors
nonspecifically inhibited the activity of PGHS-2. Instead, it was
likely that perturbations of NO generation modulated PGHS-2 catalytic
activity as previously reported (24
25
26
27)
. However, to our
surprise, we found that both L-NIL and SMT decreased the expression of
PGHS-2 induced by IFN- and LPS (Fig. 2A
, 2B
).
To confirm this apparent connection between endogenous NO and PGHS-2
expression, we used two NO-releasing compounds, NOR-1 and SNAP, which
do not share any structural similarity; both released NO spontaneously
when dissolved in aqueous solution. We found that not only did NOR-1
and SNAP increase PGE2 production (Fig. 5)
, they also induced the
expression of PGHS-2 at both mRNA and protein levels in YAMC cells
(Figs. 3
, 4)
. When these cells were co-treated with hemoglobin, an avid
scavenger of NO, the induction of PGHS-2 and generation of PGE2 by NO
was abolished, indicating the specificity of NO-induced PGHS-2
expression (Figs. 4
, 5)
. Furthermore, the induction of PGHS-2 by either
NOR-1 or SNAP appeared both time and concentration dependent (Figs. 3
, 4)
. To explore the possible role of the ß-catenin/Tcf-LEF pathway in
PGHS-2 induction, electrophoretic mobility shift assays were carried
out. We found that NOR-1 and SNAP increased DNA binding
ß-catenin/LEF-1 complex formation in these cells (Figs. 6
, 7
, 11
A) and unlabeled probe competed away this complex (Fig. 7A, B
; Fig. 11A
). The amount of the
ß-catenin/LEF-1 complex formed in response to NO donors, either NOR-1
or SNAP, increased with either dose of the drugs applied or duration of
exposure (Figs. 6
, 7
, 11A
). The participation of ß-catenin
in the DNA binding complex was confirmed by super-shifting with an
anti-ß-catenin antibody, but not by either preimmune IgG or the
anti-E-cadherin antibody (Fig. 8)
. Further studies indicate that NO may
promote ß-catenin/LEF-1 DNA binding complex formation by increasing
the level of free soluble ß-catenin but not the total amount of whole
cell ß-catenin (Fig. 9)
. This finding is corroborated by
immunocytochemistry studies showing the redistribution of ß-catenin
into the nucleus and cytoplasm (Fig. 10)
.
Our demonstration that NO is involved in the accumulation of
ß-catenin and activation of the ß-catenin/LEF-1 DNA binding in
nontransformed mouse colonic epithelial cells may help elucidate the
early molecular events during colorectal carcinogenesis. Several
investigators have emphasized the importance of Apc
mutations and their effects on ß-catenin in the early phase of
colorectal tumor development (13
14
15
16
17
18
, 35
36
37)
. From
transfection studies in cultured cells and analysis of normal and
neoplastic human colorectal tissues, a model has emerged (19
, 20
, 34
, 35)
. APC, along with interacting proteins GSK-3ß and Axin,
actively degrades ß-catenin and maintains cytoplasmic ß-catenin at
very low levels. Direct or indirect disruption of the degradation
process resulting in accumulation of cytoplasmic ß-catenin leads to
the formation of a transcriptionally active ß-catenin/Tcf-LEF complex
(38)
. These activities have been associated with the
transformation of normal colonic epithelium to adenomas and
adenocarcinomas (40
41
42
43)
. Although the genes that are
controlled by the ß-catenin/Tcf-LEF pathway have not been fully
elucidated, the role of ß-catenin in the early stage of colon cancer
development is now well demonstrated (35
36
37
38
, 43
44
45)
.
Thus, our demonstration that NO may activate the ß-catenin/LEF-1
complex may be of considerable importance.
Although the free ß-catenin is being kept at a very low level in the
cytoplasm and nucleus as a result of APC-mediated degradation, an
abundant amount of ß-catenin is normally associated with the
transmembrane protein E-cadherin. The E-cadherin–bound ß-catenin is
also called the ‘insoluble’ ß-catenin because it is present in the
cytoplasm and nucleus at very low or undetectable levels (Figs. 9
, 10)
.
It has been reported, however, that the dissociation of ß-catenin
from E-cadherin can increase free soluble ß-catenin in the cytoplasm.
As a matter of fact, the association of ß-catenin and E-cadherin can
be affected by a variety of signals directly or indirectly
(46
47
48)
. Our data, as demonstrated in Figs. 9
and 10
,
suggest that NO may be able to facilitate such dissociation and
increase free ß-catenin in the cytoplasm and nucleus. The
morphological changes of NOR-1-treated cells (Fig. 10)
, namely, the
enlargement as well as swelling and blebbing of the cells, could be
because of the dissociation of the ß-catenin and E-cadherin complex
at the membrane and the subsequent disruption of the adherens junctions
at the cell–cell border (49
50)
. But, it remains unknown
how NO works to dissociate ß-catenin from the membrane-bound
cadherins.
It is clearly demonstrated that NO is involved in the release and/or
accumulation of ß-catenin into the cytosol and the formation of
ß-catenin/LEF-1 DNA binding complex in the nucleus. But, the
association of ß-catenin either directly or indirectly to the
expression of PGHS-2 required additional evidence. The use of an
oncogenic ß-catenin construct in transfection studies was attractive,
but we were unsuccessful in transfecting these nontransformed,
nonmalignant colonic epithelial cells with the constructs we tried.
Therefore, we took advantage of cells differing in Apc
genotype. IMCE cells, with their truncated, nonfunctional APC, are
defective in their degradation of ß-catenin. Thus, one would expect
higher levels of ß-catenin under resting and stimulated conditions.
Our finding that IMCE expressed markedly higher ß-catenin/LEF-1 DNA
binding complex formation than YAMC is a manifestation of the genotypic
difference between these two cell lines (Fig. 11A
). The
amplification of the difference after treatment with NO is consistent
with the defective degradation machinery for ß-catenin in IMCE cells.
The differential formation of ß-catenin/LEF-1 DNA binding complex
corresponds to the differential expression of PGHS-2 mRNA and protein
as well as the generation of PGE2 (Fig. 11B
,C
,D
). Although NO
may also cause PGHS-2 expression and ß-catenin/LEF-1 DNA complex
formation by other independent pathways, our data suggests that these
two events are functionally connected.
Both PGHS-2 and NOS II genes share a common regulatory pathway in their
transcriptional expression; both are under the regulatory control of
NF
B mediated response, either through inflammatory stimulation or
hypoxia (51
52
53)
. Recently, it has been suggested that the
up-regulation of PGHS-2 in colon polyp and adenoma as well as
adenocarcinoma may be a result of activation of the ß-catenin/Tcf-LEF
pathway (19)
. Although the evidence is still inconclusive
regarding the precise role of ß-catenin/Tcf-LEF, it clearly indicates
that Apc through the Wnt-signaling pathway may be involved
in the expression of PGHS-2 (20)
. We found that the
promoter region of murine PGHS-2 gene (Accession Number s82456,
GenBank) does contain several potential binding sites for LEF-1 with
close homology (seven out of nine bases as compared with the murine
LEF-1 consensus sequence CCTTTGAAG). Two of these tentative LEF-1
binding sites are identical to the consensus sequence within the seven
core bases at the center (positions -1963 to -1955: ACTTTGAAT; -2974
to -2966: TCTTTGAAT). Although further studies are needed to confirm
that the ß-catenin/Tcf-LEF heterodimeric complex directly modulates
transcriptional activation of the PGHS-2 gene, data presented in this
paper support that ß-catenin/LEF-1 is at least indirectly involved in
this process. Furthermore, this study indicates that PGHS-2 expression
may be regulated by NO, presumably through the
Apc/ß-catenin pathway, a feature that may represent a
level of integration in epithelial cells. Although, in circulating
immune cells, NFB is known as the primary transcriptional regulator
for inflammatory response, the overexpression of PGHS-2 that is often
seen in precancerous adenomas as well as malignant carcinomas, may be
the result of both pathways. This may help explain the findings
reported in animal studies that NOS II inhibitors are potent
chemopreventive agents against cancer (54
55
56
57)
.
The expression of both NOS II and PGHS-2 has been associated with
vascular injury, inflammation, and cellular proliferation in
pathological colonic conditions (58
59
60
61
62)
. More
specifically, in addition to its widespread detection in various
malignancies, NOS II is known to be highly expressed in invasive
inflammatory bowel diseases and precancerous colonic adenomas as well
as carcinomas (21
22
23)
. NO can cause DNA damage directly
or indirectly through the formation of carcinogenic compounds in the
body (59
, 60)
. Selective NOS II inhibitors have been
demonstrated to exert anti-tumor and chemopreventive effects in various
animal models (54
55
56
57)
. NOS II expression is relatively
decreased in colon adenocarcinomas compared with adenomas, indicating
that NO may contribute to colon cancer progression at the transition of
colon adenoma to carcinoma in situ (23)
. This
evidence for a temporally distinct expression pattern in humans shows
that the role of NOS II in colon tumor progression may be
stage-specific.
The role of PGHS-2 in the cellular inflammatory response and
carcinogenesis also has been established. Increased PGHS-2 expression
is closely correlated with the malignancies of the gastrointestinal
tract, colonic adenoma and carcinoma in particular (3
4
5)
.
Aspirin, a nonselective inhibitor of both PGHS-1 and PGHS-2, has shown
a preventive effect against polyp formation in FAP patients. Recently,
the newly developed selective PGHS-2 inhibitors have shown potent
chemopreventive effects against colon tumor growth (7
8
9)
.
The chemopreventive activity of these drugs can be explained, at least
partially, by the finding that PGHS-2 and its eicosanoid products have
potent anti-apoptotic, mitogenic, and proliferative effects (11
, 12
, 62)
.
Our data indicate that one of the consequences associated with elevated NOS II expression in murine colon epithelial cells is the NO-associated induction of PGHS-2 and accumulation of PGE2. Evidence presented in this study supports the notion that these events may be causally linked. Furthermore, they are probably associated with, albeit indirectly, the accumulation of free soluble ß-catenin in the cytoplasm and the formation of ß-catenin/Tcf-LEF DNA binding complex in the nucleus. This is the first direct demonstration in nontransformed and nontumorigenic colonic epithelial cells that the induction of NOS II activity and subsequent NO generation contributes to PGHS-2 overexpression and PGE2 accumulation in response to inflammatory stimuli. Our study suggests that the expression of NOS II and the presence of NO under these pathological conditions may contribute to the overexpression of PGHS-2 that has been frequently observed in colonic epithelium of precancerous adenomas as well as adenocarcinomas. Taken together, these findings lend biological plausibility to the hypothesis that NOS II-derived NO may play an important role in the early stages of colorectal carcinogenesis.
|
ACKNOWLEDGMENTS |
|---|
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FOOTNOTES |
|---|
2 These authors contributed equally to this work.
3 Present address: Department of Food Science and Human Nutrition, Michigan State University, 2100 Anthony Hall, East Lansing, MI 48824-1225.
Received for publication August 3, 1999. Revised for publication November 18, 1999.
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REFERENCES |
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