From the Sealy Center for Cancer Cell Biology and the Departments of Pharmacology and Toxicology, Human Biological Chemistry and Genetics and Surgery, The University of Texas Medical Branch, Galveston, Texas 77555-1048
Received for publication, November 8, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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Elevated expression of protein kinase C Cancer has been described as a disease of aberrant signal
transduction (1). Carcinogenesis is a multistep process characterized by progressive changes in the amounts or activity of proteins that
regulate cellular proliferation, differentiation, and survival (1, 2).
These changes can be mediated through both genetic and epigenetic
mechanisms. Protein kinase C
(PKC)1 is a family of
ubiquitously expressed serine/threonine protein kinases whose members
play central roles in cell proliferation, differentiation, and
apoptosis (reviewed in Ref. 3). The discovery that PKC is a major
cellular target for the tumor-promoting phorbol esters suggested a role
for aberrant PKC signaling in tumor initiation and progression (4).
However, the relative contribution of individual PKC isozymes to
carcinogenesis is not well understood. Ultimately, the role of
individual PKC isozymes in carcinogenesis will be understood through
identification of downstream targets that participate in specific
aspects of the transformed phenotype.
We have focused our recent efforts on deciphering the role of specific
PKC isozymes in the development of colon cancer (5-7). We have shown
that colon carcinogenesis is accompanied by changes in PKC isozyme
expression, including a dramatic increase in the level of PKC Cell Culture and Cell Treatments--
RIE-1 cells and
derivatives were grown in 5% fetal bovine serum in Dulbecco's
modified Eagle's medium as previously described (14). HEK293 cells
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum. Mid-log-phase cultures were used for all
experiments unless otherwise specified. Construction of RIE/PKC Immunoblot Analysis--
For immunoblot analysis, cells were
washed twice with ice-cold phosphate-buffered saline and lysed in
protein lysis buffer consisting of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 0.1% SDS, and protease
inhibitor mixture (Sigma) for 30 min on ice. After removing particulate
matter by centrifugation at 10,000 × g for 20 min,
aliqouts of total cellular protein (50 µg) were electrophoresed in
10% acrylamide Tris-glycine gels (Invitrogen) and electrophoretically
transferred to polyvinylidene difluoride membrane (Bio-Rad). The
membranes were incubated with 5% nonfat dried milk in 10 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.05%
Tween 20 (TBST) overnight at 4 °C to block excess protein sites.
Membranes were incubated with rabbit polyclonal antibodies against
PKC Treatment of Transgenic Mice with Celecoxib and Isolation of
Colonic Epithelium--
Transgenic PKC Northern Blot Analysis--
Total RNA from RIE-1, RIE/PKC Determination of PGE2 Production--
PGE2 analysis was
conducted on equal numbers of RIE-1, RIE/PKC Analysis of Cox-2 Promoter and TGF- Gene Microarray Analysis--
Gene-profiling analysis was
performed on RIE-1 and RIE/PKC Real-time Reverse Transcriptase-Polymerase Chain Reaction
Analysis of Gene Expression--
Real-time reverse
transcriptase-polymerase chain reaction (real time RT-PCR) assays were
used to determine gene expression using TaqMan technology on an Applied
Biosystems 7000 sequence detection system. Applied Biosystems
Assays-By-Design containing a 20× assay mix of primers and TaqMan MGB
probes (FAMTM dye-labeled) were used for all target genes and the
endogenous control, rat
The efficiency of target amplification was validated using a reference
amplification reaction. Absolute values of the slope of log input RNA
amount versus Our recent studies demonstrated that elevated expression of
PKCII
(PKC
II) is an early promotive event in colon carcinogenesis
(Gokmen-Polar, Y., Murray, N. R., Velasco, M. A., Gatalica,
Z., and Fields, A. P. (2001) Cancer Res. 61, 1375-1381). Expression of PKC
II in the colon of transgenic mice
leads to hyperproliferation and increased susceptibility to colon
carcinogenesis due, at least in part, to repression of transforming
growth factor beta type II receptor (TGF-
RII) expression (Murray,
N. R., Davidson, L. A., Chapkin, R. S., Gustafson,
W. C., Schattenberg, D. G., and Fields, A. P. (1999)
J. Cell Biol., 145, 699-711). Here we report that
PKC
II induces the expression of cyclooxygenase type 2 (Cox-2) in rat intestinal epithelial (RIE) cells in vitro and in
transgenic PKC
II mice in vivo. Cox-2 mRNA increases
more than 10-fold with corresponding increases in Cox-2 protein and
PGE2 production in RIE/PKC
II cells. PKC
II activates the Cox-2
promoter by 2- to 3-fold and stabilizes Cox-2 mRNA by at least
4-fold. The selective Cox-2 inhibitor Celecoxib restores expression of
TGF-
RII both in vitro and in vivo and restores TGF
-mediated transcription in RIE/PKC
II cells. Likewise, the
-3 fatty acid eicosapentaenoic acid (EPA), which inhibits PKC
II activity and colon carcinogenesis, causes inhibition of Cox-2
protein expression, re-expression of TGF-
RII, and restoration of
TGF-
1-mediated transcription in RIE/PKC
II cells. Our data demonstrate that PKC
II promotes colon cancer, at least in
part, through induction of Cox-2, suppression of TGF-
signaling, and establishment of a TGF-
-resistant, hyperproliferative state in the
colonic epithelium. Our data define a procarcinogenic PKC
II
Cox-2
TGF-
signaling axis within the colonic epithelium, and
provide a molecular mechanism by which dietary
-3 fatty acids and
nonsteroidal antiinflammatory agents such as Celecoxib suppress colon carcinogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II
expression (5). PKC
II protein levels are elevated in preneoplastic
lesions in the colon, aberrant crypt foci, and are further elevated in
colon tumors (5). To determine whether elevated PKC
II levels
contribute to colon carcinogenesis, we developed transgenic PKC
II
mice that express elevated PKC
II in the colonic epithelium to levels
comparable with those observed in carcinogen-induced colon tumors (6,
7). These animals exhibit hyperproliferation of the colonic epithelium
and enhanced susceptibility to carcinogen-induced carcinogenesis (6).
We recently characterized the transforming growth factor
receptor type II (TGF-
RII) as a target for PKC
II-mediated transcriptional repression in intestinal epithelial cells and in the colonic epithelium of transgenic PKC
II mice (7). PKC
II-induced inhibition of TGF-
RII renders intestinal epithelial cells insensitive to growth inhibition by TGF-
and accounts, at least in part, for the colonic hyperproliferation and increased sensitivity to colon carcinogenesis characteristic of transgenic PKC
II mice (6, 7). Our studies to date
indicate that PKC
II plays a critical role in the early stages of
colon carcinogenesis by inducing the loss of TGF-
responsiveness, thereby imposing a hyperproliferative phenotype, two prominent characteristics of colon cancer. We reasoned that the cellular phenotype induced by PKC
II is mediated through changes in gene expression. Thus, we have initiated a genomic analysis to identify PKC
II target genes in rat intestinal epithelial (RIE-1) cells. Among
the gene targets induced by PKC
II is the inducible form of
cyclooxygenase, Cox-2. Cox-2 was originally cloned as a phorbol ester-inducible gene (8, 9), and it has been implicated in the etiology
of colon cancer in rodents and humans (10-13). Our present data
demonstrate that Cox-2 is a specific genomic target of PKC
II and
that PKC
II-mediated repression of TGF-
RII depends on Cox-2.
Finally, we show that the chemopreventive
-3 polyunsaturated fatty
acid eicosapentaenoic acid (EPA), a known PKC
II inhibitor in
vivo and in vitro (7), inhibits Cox-2 expression, induces TGF-
RII expression, and restores TGF-
responsiveness in
RIE/PKC
II cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II
cells has been described elsewhere (7). RIE/PKC
cells were produced
by infection of RIE-1 cells with a retrovirus containing the
full-length human PKC
cDNA. RIE/H-Ras and RIE/Cox-2 cells were
generous gifts of Drs. Hongmiao Sheng and Ray DuBois, Vanderbilt
University (11). In some experiments, cells were incubated with the
-3 fatty acid EPA (Cayman Biochemicals) at the concentrations
and for the times indicated in the figure legends. In some cases, cells
were incubated with 25 µM Celecoxib (UTMB Pharmacy)
and/or 120 pM TGF-
1 (BD Biosciences) in the
culture medium for the times indicated in the figure legends. EPA and Celecoxib were solubilized in dimethyl sulfoxide
(Me2SO). A final Me2SO concentration of
0.1% was used for all treatments, and 0.1% Me2SO was used
as a diluent control. The stability of the Cox-2 mRNA was
determined in RIE-1 and RIE/PKC
II cells by treatment of cells with
25 µM 5,6-dichlorobenzimidazole riboside to inhibit RNA
polymerase II. Total cellular RNA was isolated as described previously
(14) at various times after dichlorobenzimidazole riboside exposure and
subjected to real-time RT-PCR analysis for Cox-2 mRNA expression as
described below.
I (Santa Cruz; 1:2,000 dilution), PKC
II (Santa Cruz; 1:6,000
dilution), Cox-2 (Cayman; 1:1,000 dilution), TGF-
RII (Santa Cruz;
1:1,000 dilution), or actin (Santa Cruz; 1:10,000 dilution) in TBST at room temperature for 3 h, after which the membranes were washed in
TBST three times for 15 min each. The membranes were incubated with
horseradish peroxidase-conjugated goat anti-rabbit antibody (Kirkegaard
and Perry Laboratories,1:125,000 dilution) in TBST for 1 h at room
temperature. The membranes were washed three times in TBST for 15 min
each, and antigen-antibody complexes were detected using
chemiluminescence (Amersham Biosciences) according to the manufacturer's instructions.
II mice expressing PKC
II
in the colonic epithelium were characterized previously (6, 7).
Transgenic PKC
II mice and nontransgenic littermates were
administered 6 mg/kg Celecoxib by oral gavage twice daily for 3 days.
As controls, some mice were administered an equivalent volume of
diluent (0.5% carboxylmethylcellulose). Mice were terminated on the
morning of the fourth day. The colonic epithelium was isolated by
scraping and subjected to immunoblot analysis for TGF-
RII expression
as described previously (7).
II,
and RIE/Cox-2 cells was isolated by the
guanidinium-thiocyanate-phenol-chloroform method (15). Total RNA (10 µg) from each cell line was electrophoresed in 1% agarose gels
containing 0.66 M formaldehyde and electrophoretically transferred to nitrocellulose membranes (Intermountain Scientific Corp). After incubation at 80 °C for 2 h, the membranes were
prehybridized for 2h in a solution containing 50% formamide in 5×
Denhardt's solution, 0.1% SDS, 5× SSPE (750 mM NaCl, 50 mM NaH2PO4 pH 7.4, 5 mM
EDTA), and 100 µg/ml single-stranded sperm DNA and then hybridized overnight at 42 °C with a radiolabeled cDNA probe to the rat
Cox-2 gene consisting of 0.81 kb of the rat Cox-2 cDNA excised from PCB7-cox2. The probe was labeled with [
-32P]dCTP (800 Ci/mM, PerkinElmer) by the random priming method (Amersham Biosciences). After hybridization, membranes were washed three times in
0.1× SSPE, 0.1% SDS for 20 min at 55 °C and exposed to Eastman
Kodak X-Omat AR film at 80 °C. The intensity of the autoradiographic signal was quantified by a Lynx densitometer (Applied Imaging). To
verify equivalency of RNA loading of individual samples, the blot was
stripped of radioactivity and rehybridized with an 18S rRNA probe as
described previously (14).
II, and RIE/Cox-2 cells
cultured in 100-mm tissue culture plates in Dulbecco's modified
Eagle's medium containing 5% fetal bovine serum for 2 days. Aliquots
of culture medium (50 µl) were collected and subjected to a specific
enzyme-linked immunosorbent assay for PGE2 (Amersham Biosciences)
following the manufacturer's instructions. Culture medium was diluted
or concentrated appropriately to achieve values within the linear range
of the assay (50-6400 pg/ml). The range of the standard PGE2 curve was
from 2.5 pg to 320 pg/well. Triplicate samples were analyzed in each
experiment, and the results were expressed as pg of PGE2/ml of culture medium.
-dependent
Transcriptional Activity--
Cox-2 promoter activity and TGF-
transcriptional responses were assessed by transient transfection of
RIE-1, RIE/PKC
II, or RIE/H-Ras cells with either a Cox-2 promoter or
TGF-
-responsive promoter construct linked to a luciferase reporter
gene, respectively. Cox-2 promoter activity was determined using 4 kb
of the mouse Cox-2 promoter cloned into the PGL3 reporter plasmid (16).
TGF-
transcriptional responses were assessed using the 3TP-Luc
reporter as described previously (7). The appropriate expression vector was cotransfected with TK-pRL (Renilla luciferase
transcribed from the HSV TK promoter) into 70-80% confluent RIE-1,
RIE/PKC
II, or RIE/H-ras cell lines in 6-well plates using Tfx-50
(Promega) at a DNA:liposome ratio of 1:3 as described previously (7). Fresh medium was added 3 h after transfection, and cells were harvested after 24 h. Total cell extracts were prepared for
dual-luciferase assay according to the manufacturer's instructions
(Promega) using a Monolight 2010 Luminometer (Analytical Luminescence
Laboratory). The activity of Renilla luciferase was used as
an internal control. Results are expressed as the mean of triplicate
determinations ± standard deviation.
II cells using RG-U34A Gene Chips®
microarrays (Affymetrix). Total RNA (25 µg) was used for first-strand
cDNA synthesis using a T7-(dT)24 oligomer
(5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-dT24-3') and
SuperScript II reverse transcriptase (Invitrogen). The cDNA was
converted to double-stranded DNA by transcription in vitro. cRNAs were synthesized using bacteriophage T7 RNA polymerase in the
presence of biotinylated nucleotides. Biotin-labeled target RNAs were
fragmented to a mean size of 200 bases according to the manufacturer's
protocol. Hybridization of the rat RG-U34A microarrays was performed at
45 °C for 16 h in 0.1 M 2-morpholinoethanesulfonic acid (MES) pH 6.6, 1 M NaCl, 0.02 M EDTA, and
0.01% Tween 20. Microarrays were washed using both nonstringent (1 M NaCl, 25 °C) and stringent (1 M NaCl,
50 °C) conditions prior to staining with phycoerythrin-labeled
streptavidin (10 µg/ml final concentration). Data were collected
using a Gene Array Scanner (Hewlett Packard) and analyzed using the
Affymetrix Gene Chip Analysis Suite 5.0 software.
-actin. These assays were designed using
primers that span exon-exon junctions so as not to detect genomic DNA.
All primer and probe sequences were searched against the Celera data
base to confirm specificity. The primer and probe sequences used were
as follows: rat PAI1-probe spanning exon8 CCAACAGAGACAATCC,
forward primer ACCGATCCTTTCTCTTTGTGGTT, reverse primer
CATCAGCTGGCCCATGAAG; rat Cox-2-probe spanning exon8
CCCAGCAACCCGG, forward primer GAGTCATTCACCA-GACAGATTGCT, reverse primer
GTACAGCGATTGGAACATTCCTT; human PKC
II-probe spanning the
PKC
I/PKC
II alternative splice junction TCGCCCACAAGCT, forward primer AAACTTGAACGCAAAGAGATCCA, reverse primer ATCGGTCGAAGTTTTCAGCATT.
CT were <0.1 in all experiments. One-step
RT-PCR reactions were performed on 20 ng of input RNA for both target
genes and endogenous controls using the TaqMan one-step RT-PCR master
mix reagent kit (Applied Biosystems). The cycling parameters were as
follows: reverse transcription, 48 °C for 30 min; AmpliTaq
activation, 95 °C for 10 min; denaturation, 95 °C for 15 s;
and annealing/extension, 60 °C for 1 min for 40 cycles. Duplicate CT
values were analyzed in Microsoft Excel using the comparative
CT(
CT) method as described by the manufacturer (Applied Biosystems).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II in the colonic epithelium is an early, promotive event in
colon carcinogenesis (5-7). To elucidate the molecular mechanisms by
which PKC
II mediates increased colon carcinogenesis, we established a cell model system in which to explore PKC
II-mediated signaling. RIE-1 cells are immortalized but not transformed and consequently express abundant PKC
I but little or no detectable PKCBII protein (Fig. 1A). This pattern of
expression of PKC
I and PKC
II is consistent with that observed in
the colonic epithelium in vivo (5). To assess the cellular
and genomic effects of PKC
II expression, we created the RIE/PKC
II
cell line that expresses abundant human PKC
II (Fig. 1A).
A real-time RT-PCR assay for human PKC
II mRNA demonstrated that
RIE/PKC
II cells expressed human PKC
II mRNA at levels somewhat
lower than those observed in human HEK293 cells, which express abundant
endogenous PKC
II protein (Fig. 1B). We previously
demonstrated that the growth rate of RIE/PKC
II cells is
indistinguishable from that of RIE-1 cells (7), indicating that
overexpression of PKC
II has no significant effect on proliferation or apoptosis of RIE-1 cells in culture. Similarly, no changes in gross
cellular morphology were noted in RIE/PKC
II cells, nor were these
cells able to form colonies in soft agar, indicating that expression of
PKC
II is not sufficient to cause cellular transformation (data not
shown).
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Fig. 1.
Establishment of RIE cells expressing human
PKC II. RIE-1 cells were transfected with a retrovirus
containing the full-length human PKC
II cDNA as described under
"Experimental Procedures." A, immunoblot analysis of
RIE-1, RIE/PKC
II, and HEK293 cells with antibodies to PKC
I,
PKC
II, and actin. B, quantitative real time RT-PCR assay
for human PKC
II mRNA in HEK293, RIE-1, and RIE/PKC
II cells.
Results are expressed as relative PKC
II mRNA abundance and
represent the mean of triplicate determinations ± S.E.
Affymetrix Gene Chips® were used to identify genes that are either
induced or inhibited in RIE/PKCII cells when compared with RIE-1
cells. Significance analysis of microarrays (17) was used to compare
expression profiles of RNA extracted from control RIE-1 cells
(n = 7) and RIE/PKC
II cells (n = 3).
Among the probe sets whose expression changed by >2.0-fold in
RIE/PKC
II cells was that encoding the inducible form of
cyclooxygenase, Cox-2. Because Cox-2 expression can be induced by
phorbol esters (8, 9), and because of the strong association between
Cox-2 expression and colon cancer (10-13), we focused our analysis on this gene. We confirmed our microarray analysis using a quantitative real-time RT-PCR assay specific for Cox-2 mRNA (Fig.
2A). The mean signal
intensities obtained from Cox-2 probe sets in microarrays from RIE-1
(n = 7) and RIE/PKC
II (n = 3) cells
(gray bars) correlated well with the level of Cox-2 mRNA
detected by real time RT-PCR (black bars), providing
independent confirmation of elevated expression of Cox-2 RNA in
RIE/PKC
II cells. Northern blot analysis indicated that Cox-2
mRNA expression was increased >10-fold in RIE/PKC
II cells when
compared with RIE-1 cells (Fig. 2B), providing an
independent confirmation of the microarray and real-time RT-PCR data.
As a positive control, RIE/Cox2 cells, which express a Cox-2 transgene that is deleted of the 3'-untranslated region (18), was used to compare
the abundance of Cox-2 mRNA in RIE/PKC
II cells expressing the
endogenous, full-length Cox-2 transcript. The data in Fig. 2B indicate that Cox-2 mRNA is even more abundant in
RIE/PKC
II cells than in RIE/Cox2 cells. Real-time RT-PCR analysis
confirmed that RIE/PKC
II cells express 1.4-fold more PKC
II
mRNA than RIE/Cox-2 cells. Taken together, these data provide
conclusive evidence that Cox-2 mRNA is induced by PKC
II in RIE-1
cells.
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Immunoblot analysis was used to assess the level of Cox-2 protein
expression in RIE-1, RIE/PKCII, and RIE/Cox2 cells (Fig. 3A). Cox-2 protein was
expressed at nearly undetectable levels in RIE-1 cells. In contrast,
RIE/PKC
II cells expressed abundant Cox-2 protein comparable with the
amount of Cox-2 protein expressed in RIE/Cox2 cells. To assess whether
induction of Cox-2 expression is specific for PKC
II expression, we
assayed Cox-2 protein levels in RIE/PKC
cells, which were engineered
to overexpress transgenic human PKC
(Fig. 3B). RIE/PKC
cells expressed very low levels of Cox-2 (which could be observed only
after very long exposures of the immunoblots) comparable with those
observed in RIE-1 cells. These results are consistent with our
microarray analysis of RIE/PKC
cells, which did not identify Cox-2
as a potential transcriptional target of PKC
(data not shown). These
results indicate that induction of Cox-2 is not a general response to
the expression of any PKC isozyme, but rather is a specific response
to PKC
II expression.
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To assess whether the Cox-2 protein expressed in RIE/PKCII cells was
functional, RIE-1, RIE/PKC
II, and RIE/Cox-2 cells were assayed for
production of PGE2, a product of Cox-2 enzyme activity (Fig.
3C). Enzyme-linked immunosorbent assay analysis of culture supernatants demonstrated that RIE/PKC
II cells, like RIE/Cox2 cells,
secreted 3- to 4-fold higher levels of PGE2 than RIE-1 cells. These
results demonstrate that PKC
II induced the expression of active
Cox-2 enzyme in RIE/PKC
II cells.
We next wished to determine whether Cox-2 is also a target for PKCII
regulation in the colonic epithelium in vivo. We have developed transgenic PCK
II mice overexpressing PKC
II in the colonic epithelium that exhibit an increased sensitivity to
azoxymethane-mediated colon carcinogenesis (6, 7). Immunoblot analysis
of colonic epithelium from nontransgenic and transgenic PKC
II mice
demonstrate that transgenic PKC
II mice expressed significantly more
PKC
II and Cox-2 protein than their nontransgenic littermates (Fig.
3D). These data demonstrate that Cox-2 is a significant
genomic target of PKC
II both in RIE-1 cells in culture and in the
colonic epithelium in vivo.
We next assessed the mechanism by which PKCII leads to elevated
Cox-2 mRNA levels in RIE-1 cells. For this purpose, a Cox-2 promoter/luciferase reporter gene was transiently cotransfected into
RIE-1 cells along with a PKC
II expression vector to assess the
effect of PKC
II on Cox-2 promoter activity (Fig.
4A). The activity of the
Cox-2/luciferase reporter was increased by 2- to 3-fold in RIE-1 cells
in which a PKC
II expression vector was simultaneously introduced.
These experiments utilized a human Cox-2 promoter construct consisting
of 4 kb of 5'-flanking sequence, but consistent results were also
obtained using promoters containing 7 kb or 1.4 kb of 5'-flanking
sequence (data not shown). To independently assess the effect of
PKC
II expression on Cox-2 promoter activity, the Cox-2
promoter/luciferase reporter was transfected into RIE-1, RIE/PKC
II,
and RIE/Ras cells, and the activity of the reporter was assessed by
luciferase assay (Fig. 4B). RIE/Ras cells express an
activated H-Ras allele previously shown to activate Cox-2 transcription (11, 19) and served as a positive control for activation of Cox-2
promoter activity. Consistent with the transient cotransfection data
shown in Fig. 4A, the Cox-2 promoter was 2-3 times more
active in RIE/PKC
II cells than in RIE-1 cells. As expected, the
Cox-2 promoter was also more active, by about 5-fold, in RIE/Ras cells, which are known to express high levels of Cox-2 (11). These data
demonstrate that PKC
II causes a 2- to 3-fold increase in Cox-2
promoter activity in RIE cells. Although this represents a significant
and reproducible increase in Cox-2 promoter activity, the magnitude of
the effect was clearly not sufficient to account for the dramatic
increase in Cox-2 mRNA expression observed in RIE/PKC
II cells.
Taken together, these data indicate that an additional mechanism(s) may
be responsible for the effects of PKC
II on Cox-2 mRNA levels in
RIE-1 cells.
|
The Cox-2 gene is known to be regulated not only at the transcriptional
level but also at the level of mRNA stability (20-22). Therefore,
Cox-2 mRNA stability was compared in RIE-1 and RIE/PKCII cells
by measuring mRNA abundance by quantitative real-time RT-PCR as a
function of time after addition of the nonspecific RNA polymerase II
inhibitor dichlorobenzimidazole riboside (Fig. 4C). Cox-2
mRNA in RIE-1 cells was relatively unstable with an estimated
t1/2 of degradation of
~12-16 min. In contrast, the apparent
t1/2 of degradation of Cox-2
mRNA in RIE/PKC
II cells was greater than 50 min, indicating that
PKC
II expression results in significant stabilization of Cox-2
mRNA. Thus, PKC
II-mediated elevation of Cox-2 mRNA levels
result from a combined effect of PKC
II on Cox-2 gene transcription
and Cox-2 mRNA stability.
We recently showed that PKCII inhibits expression of TGF-
RII both
in the colonic epithelium of transgenic PKC
II mice and in
RIE/PKC
II cells (7). Therefore, we assessed whether the effect of
PKC
II on TGF
RII expression is mediated through Cox-2 activation
(Fig. 5). RIE-1 cells expressed abundant
TGF
RII protein as assessed by immunoblot analysis (Fig.
5A, lane 1), whereas RIE/PKC
II cells expressed
significantly lower levels of TGF
RII protein (Fig. 5A,
lane 2). Treatment of RIE/PKC
II cells with 25 µM Celecoxib for 0, 24, or 48 h led to a
time-dependent increase in TGF-
RII protein expression
(Fig. 5A, lanes 3-5). We have also observed that
treatment of RIE-1 cells with Celecoxib leads to increased TGF-
RII
protein expression, indicating that Cox-2 exerts a tonic suppressive
effect on TGF-
RII expression in RIE-1 cells that can be reversed by
inhibition of the enzyme(data not shown).
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RIE/PKCII cells exhibit a profound loss of TGF-
-mediated
transcriptional activity as a consequence of PKC
II-mediated
repression of TGF-
RII expression (7). We therefore assessed the
ability of Celecoxib to restore TGF-
-mediated transcriptional
activity in RIE/PKC
II cells (Fig. 5, B and C).
RIE/PKC
II cells were transiently transfected with a
TGF-
-reponsive luciferase reporter plasmid and then treated with
either TGF-
1, Celecoxib, or both (Fig. 5B). RIE/PKC
II
cells exhibited little or no transcriptional response to TGF-
1,
consistent with our previous results (7). However, when these cells
were treated with Celecoxib prior to exposure to TGF-
1, they
exhibited a robust transcriptional response. Celecoxib treatment had no
effect in the absence of TGF-
1, demonstrating that the observed
transcriptional effects of Celecoxib are TGF-
-dependent. Consistent with these results, the level of the mRNA for the
endogenous TGF-
1-responsive gene, plasminogen activator inihibitor-1
(PAI-1) (23), was dramatically induced when RIE/PKC
II cells were
treated with Celecoxib for 24 or 48 h prior to exposure to
TGF-
1 (Fig. 5C).
To determine whether the PKCII-mediated repression of TGF-
RII
expression is Cox-2-dependent in vivo, we
assessed the effect of treating transgenic PKC
II mice with Celecoxib
on TGF-
RII expression in the colonic epithelium (Fig.
5D). Treatment of transgenic PKC
II mice with Celecoxib
led to reexpression of TGF-
RII as assessed by immunoblot analysis.
Therefore, PKC
II-mediated repression of TGF-
RII expression and
TGF-
-responsiveness are dependent on Cox-2 activity both in
intestinal epithelial cells in vitro and in the colonic
epithelium in vivo
Chemopreventive dietary -3 fatty acids such as EPA block Cox-2
induction in azoxymethane-treated mice (24). We and others have shown
that azoxymethane induces colonic PKC
II expression (5, 25). We have
found that a diet high in
-3 fatty acids inhibits colonic PKC
II
activity, induces TGF-
RII expression, and blocks PKC
II-mediated
colon carcinogenesis in transgenic PKC
II mice (7). These
observations lead to the hypothesis that the chemopreventive effects of
-3 fatty acids are mediated through inhibition of a PKC
II
Cox-2
TGF-
signaling pathway. To test this hypothesis, we
treated RIE-1 and RIE/PKC
II cells with EPA, an
-3 fatty acid
found in fish oil, and measured TGF-
RII and Cox-2 expression by
immunoblot analysis (Fig. 6A).
Treatment of RIE/PKC
II cells with EPA led to a
dose-dependent increase in TGF-
RII expression and a
concomitant decrease in Cox-2 expression (Fig. 6A,
left panel). This effect was dependent on PKC
II
expression because no significant changes in either TGF-
RII or Cox-2
expression were observed in RIE-1 cells treated with EPA (Fig.
6A, right panel). Quantitative analysis of these
data revealed that Cox-2 expression was inhibited by >50% in
RIE/PKC
II cells but was unaffected in RIE-1 cells (Fig.
6B). On the other hand, TGF-
RII was induced in
RIE/PKC
II cells but not in RIE-1 cells treated with EPA (Fig. 6C). These data are consistent with our recent observation
that a diet high in
-3 fatty acids induces TGF-
RII expression in the colonic epithelium of transgenic PKC
II mice and blocks
PKC
II-mediated colon carcinogenesis (7). They also establish a
direct link between cancer-preventive dietary
-3 fatty acids,
PKC
II activity, Cox-2 expression, and TGF-
signaling in
vitro and in vivo.
|
![]() |
DISCUSSION |
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---|
We have focused our recent attention on the role of individual PKC
isozymes in the development of colon cancer (5-7). We have found that
PKCII, which is expressed at very low levels in the proliferative
zone of normal colonic epithelium, is rapidly induced in the colonic
epithelium of azoxymethane-treated rodents and is abundantly expressed
in both preneoplastic aberrant crypt foci and colon tumors that form
following azoxymethane treatment (5). Transgenic PKC
II mice
overexpressing PKC
II in the colonic epithelium exhibit
hyperproliferation and increased susceptibility to azoxymethane-induced
colon carcinogenesis (6, 7), demonstrating that PKC
II plays a
critical promotive role in colon carcinogenesis.
We also recently demonstrated that PKCII is an important cellular
target for the cancer-preventive activity of dietary
-3 fatty acids
(7). Diets high in
-3 fatty acids have been shown to block colon
carcinogenesis in rodent models (7, 26-29), and epidemiologic studies
indicate that
-3 fatty acids have chemopreventive effects against
colon cancer in humans (30-33). We found that
-3 fatty acids, which
are abundant in dietary fish oils, inhibit colonic PKC
II activity
and suppress the hyperproliferative and cancer-prone phenotype of
transgenic PKC
II mice (7). In those studies, we also established
that the TGF-
RII gene is a target for repression by PKC
II
(7).
The present studies were initiated in an effort to further elucidate
the molecular mechanism(s) by which PKCII promotes colon carcinogenesis. The data presented herein identify the Cox-2 gene as a
prominent target for PKC
II-mediated regulation in intestinal epithelial cells in vitro and the colonic epithelium
in vivo. PKC
II causes induction of Cox-2 expression by at
least two distinct mechanisms. First, PKC
II leads to a modest, 2- to
3-fold induction of Cox-2 gene transcription. However, the more
dramatic effect of PKC
II on Cox-2 gene expression appears to be
through stabilization of Cox-2 mRNA. The half-life of Cox-2
mRNA in RIE/PKC
II cells is more than 50 min. This represents a
significant stabilization of the Cox-2 mRNA, which exhibits a
half-life of 12-16 min in RIE-1 cells. Taken together, these two
actions of PKC
II provide a plausible mechanism by which PKC
II
leads to the dramatic increase in Cox-2 mRNA, protein, and activity
levels observed in RIE/PKC
II cells.
There is abundant evidence that both Cox-2 and PKCII are involved in
colon carcinogenesis, and several interesting parallels exist between
these two genes. First, the Cox-2 gene was originally described as an
immediate early gene that is induced by either serum or phorbol ester
stimulation of quiescent cells (8, 9), suggesting a connection between
Cox-2 gene regulation and PKC signaling. Second, both Cox-2 and
PKC
II are elevated in the colonic epithelium of rodents exposed to
the colon carcinogen azoxymethane and in aberrant cryptic foci and
colon tumors that develop in azoxymethane-treated animals (5, 34, 35),
indicating that these genes are involved in early events in the
carcinogenic process.
The data presented here provide the first direct mechanistic link
between elevated PKCII and Cox-2 expression during colon carcinogenesis. Based on our current and previously published data, we
propose a model for how PKC
II and Cox-2 promote colon cancer (Fig.
7). In this model, the tissue-selective
carcinogen azoxymethane induces PKC
II expression in the colonic
epithelium, an event that has been well documented by our group and
others (5, 25). Our current data show that PKC
II induces Cox-2 expression in intestinal epithelial cells in vitro and in
the colonic epithelium in vivo. We have also demonstrated
that PKC
II leads to repression of TGF-
RII expression and TGF-
1
signaling in RIE cells and to a loss of TGF-
RII expression in the
colonic epithelium of transgenic PKC
II mice (7). In the present
study, we demonstrate that PKC
II-mediated repression of TGF-
RII
expression and signaling can be reversed by the Cox-2 inhibitor
Celecoxib, indicating that PKC
II mediates its effects on TGF-
signaling through Cox-2. These data are consistent with the recent
association between elevated Cox-2 expression and loss of TGF-
1
responsiveness and TGF-
RII expression in RIE cell variants selected
for loss of TGF-
1 responses (36).
|
The effects of Cox-2 in intestinal epithelial cells and in colon tumors
are thought to be mediated through production of prostaglandins, particularly PGE2 (37). PGE2 in turn signals through binding to
specific PGE2 receptors, of which there are four well characterized members, termed EP 1-4 (reviewed in Ref. 38). The EPs are members of
the G-protein-coupled receptor family whose downstream effectors include adenylate cyclase and phosphatidylinositol-phospholipase C
(38). Accumulating evidence demonstrates that EP1 plays a pivotal role
in colon carcinogenesis (39-41). Specifically, two different
pharmacologic inhibitors selective for EP1 have been shown to inhibit
formation of preneoplastic aberrant cryptic foci in
azoxymethane-treated mice and of intestinal polyps in
APCmin mice (39-41). Furthermore, mice that are
nullizygous for EP1 exhibit suppressed colon carcinogenesis (39).
It is well documented that EP1 signals through activation of PI-PLC and
generation of the second messengers diacylglycerol and inositol
trisphosphate, which in turn lead to intracellular calcium mobilization
and PKC activation (38). Based on these observations, and our present
data, it is attractive to suggest that PGE2-mediated activation of EP1
leads to activation of PKCII, a classical PKC isozyme, generating an
autocrine positive-feedback loop. In this model, activated PKC
II in
turn causes repression of TGF-
RII function by an as-yet-unidentified
mechanism. It should be noted, however, that the role of EPs in colon
cancer development appears to be complex. In this regard, both EP2 (42)
and EP4 (43) have also been implicated in colon carcinogenesis,
indicating that PGE2 can activate multiple signaling pathways in the
colonic epithelium. The complex role of EPs, as well as other possible Cox-2 products, in PKC
II-mediated effects in intestinal epithelial cells will require further experimentation.
In recent studies, we demonstrated that the chemopreventive effects of
dietary -3 fatty acids are mediated, at least in part, through
inhibition of PKC
II activity in the colonic epithelium (7).
Furthermore, we demonstrated that
-3 fatty acids can block the
hyperproliferation and enhanced colon carcinogenesis exhibited by
transgenic PKC
II mice through inhibition of colonic PKC
II
activity (7). In this study we show that
-3 fatty acids inhibit
Cox-2 expression and induce TGF-
RII by a mechanism that depends on
PKC
II expression. These data are consistent with the model proposed
in Fig. 7, because there is abundant evidence that dietary
-3 fatty
acids prevent azoxymethane-induced elevation of Cox-2 expression in the
colonic epithelium (24).
-3 fatty acids such as EPA have been shown
in some cell systems to inhibit Cox-2 activity (44, 45). However, our
data indicate that EPA does not induce TGF-
RII expression in
RIE/PKC
II cells through inhibiton of Cox-2 for the following
reasons. First, both RIE-1 and RIE/PKC
II cells express active Cox-2
enzyme (Fig. 3C). Despite that fact, EPA induces TGF-
RII
expression in RIE/PKC
II cells, but not in RIE-1 cells (Fig. 6).
Treatment of RIE-1 cells with Celecoxib however, leads to elevated
expression of TGF-
RII in both RIE-1 and RIE/PKC
II cells. If EPA
were acting through inhibition of Cox-2 activity, it should induce
TGF-
RII expresion in both cell lines. Therefore, it is unlikely that
the effects of EPA on TGF-
RII expression in RIE/PKC
II cells are
caused by Cox-2 inhibition.
The accumulating evidence that PKCII and Cox-2 expression and
activity can be regulated in a similar fashion by dietary components that modulate colon cancer risk further suggests a mechanistic link
between these two cancer-promoting genes. Our model is attractive in
that it reconciles many seemingly disparate observations in the
literature regarding the role of PKC
II, Cox-2, and TGF-
signaling
in colon carcinogenesis. Furthermore, it provides a paradigm within
which to understand the mechanism(s) by which dietary compounds can
modulate colon cancer risk. We are currently using our transgenic cell
and animal models to explore the mechanism(s) by which azoxymethane and
dietary factors such as EPA regulate PKC
II expression and activity
and to assess whether PKC
II expression is required for Cox-2 gene
induction and colon carcinogenesis in vivo.
In summary, we have shown that PKCII induces Cox-2 expression both
in vitro and in vivo. Cox-2 is intimately linked
to the development of colon cancer, and our studies provide a molecular mechanism by which induction of PKC
II expression during
azoxymethane-induced carcinogenesis, or overexpression of PKC
II in
transgenic mice, predisposes mice to colon cancer. The elucidation of a
PKC
II
Cox-2
TGF-
signaling axis, which is operative in
both intestinal epithelial cells in culture, and the colonic epithelium
in vivo, provides an important mechanistic link that can
explain how changes in PKC
II expression promotes colon
carcinogenesis and how dietary lipids and nonsteroidal antiinflammatory
drugs can modulate colon cancer risk.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the members of the Fields laboratory for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the National Cancer Institute (to A. P. F.) (CA81436 and CA56869).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: The Sealy Center for
Cancer Cell Biology, The University of Texas Medical Branch, 301 University Blvd., MRB 9.104, Galveston, TX 77555-1048; Tel.: 409-747-1935; Fax: 409-747-1938; E-mail: afields@utmb.edu.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M211424200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PKC, protein kinase
C;
TGF-RII, transforming growth factor beta type II receptor;
Cox-2, cyclooxygenase type 2;
RIE, rat intestinal epithelial;
PG, prostaglandin;
EPA, eicosapentaenoic acid.
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