From the Departments of Pharmacology,
§ Medicine, ¶ Surgery, and
Cancer Biology,
Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-6602
Received for publication, January 2, 2003, and in revised form, January 30, 2003
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ABSTRACT |
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In human colorectal adenocarcinoma cell lines, we
found two major transcripts of cyclooxygenase-2, the full-length
mRNA and a short polyadenylation variant (2577 kb) lacking the
distal segment of the 3'-untranslated region. Tristetraprolin, an
mRNA-binding protein that promotes message instability, was shown
to bind the cyclooxygenase-2 mRNA in the region of the
3'-untranslated region between nucleotides 3125 and 3432 and to reduce
levels of the full-length mRNA. During cell growth and confluence,
the expression of tristetraprolin mRNA was inversely correlated
with that of the full-length cyclooxygenase-2 transcript, and
transfection of tristetraprolin into HCA-7 cells reduced the level of
full-length cyclooxygenase-2 mRNA. However, the truncated
transcript escaped tristetraprolin binding and downregulation.
Cyclooxygenases (prostaglandin H2 synthases, EC
1.14.99.1) are enzymes that catalyze conversion of arachidonic acid to
prostaglandin H2. Two isozymes have been described:
COX-11 is constitutively
expressed, whereas COX-2 is mitogen-inducible (1, 2). COX-2 is the
product of an immediate-early gene (3) that is induced by growth
factors and cytokines (4, 5) and is related to cell proliferation (6,
7). Levels of COX-2 expression are increased in 85-90% of human
colorectal adenocarcinomas (8). COX-2 is also expressed in gastric
cancer (9) as well as in esophageal carcinoma (10), pancreatic
carcinoma (11), prostate carcinoma (12), lung cancer (13), and mammary cancer (14). The use of nonsteroidal anti-inflammatory drugs is
associated with reduced mortality from colorectal cancer (15, 16).
COX-2-derived prostaglandin can promote the multistep sequence of
events that lead to colon cancer (17, 18). Constitutive expression of
COX-2 leads to increased metastatic potential of colon cancer cells
(19) and regulates angiogenesis in colon tumors (20). Moreover,
COX-2-selective inhibitors have been shown to reduce tumor growth
in vivo by inhibiting angiogenesis (21, 22) and by inducing
apoptosis (23). As a result of altered regulation of its expression,
COX-2 is overexpressed in a number of cancer cell lines, and its
expression in the human colorectal carcinoma cell line HCA-7 is among
the highest.
In HCA-7 cells, we found and characterized two major COX-2 transcripts.
One is the full-length mRNA (4465 nt), and the second is a 2577-nt
polyadenylation variant in which the terminal 1888 nt of the
3'-untranslated region (UTR) are not present. During cell growth and
confluence, the levels of the full-length mRNA declined, whereas
those of the truncated mRNA increased. Truncated COX-2 mRNAs
characterized as polyadenylation variants have been described
previously in other cell types (2, 24-26). Ristimaki et al.
(24) provided evidence that deletion of the distal portion of the
3'-UTR in a polyadenylation variant of COX-2 mRNA confers increased
stability to the mRNA. Because we found disparate regulation of the
levels of the two COX-2 transcripts with different 3'-UTRs in HCA-7
cells and in the context of increasing evidence that the 3'-UTR of a
number of mRNAs can regulate their stability, we considered the
hypothesis that the distal 1888 nt of the 3'-UTR of COX-2 mRNA
could participate in post-transcriptional regulation of message abundance.
Post-transcriptional mechanisms have been shown to play an important
role in the regulation of COX-2 expression during carcinogenesis (27).
p38 mitogen-activated protein kinase increases COX-2 mRNA stability
(28-30), and inhibition of the kinase by anti-inflammatory glucocorticoids such as dexamethasone leads to a decrease in COX-2 mRNA stability (31).
The 3'-UTRs of some cytokines (e.g. TNF- TTP (also known as TIS11 and Nup475) (41, 42) is a product of an
"immediate-early gene." TTP-deficient mice have a severe inflammatory syndrome (43) and show increased stability of the mRNAs for TNF- We report that TTP is expressed in HCA-7 cells, that its transcription
is up-regulated with cell confluence, and that levels of its RNA vary
inversely with those of the full-length COX-2 mRNA. TTP binds to
the 3'-UTR of COX-2 mRNA in a region present in the full-length
mRNA, but deleted from the truncated polyadenylation variant.
Transfection of the TTP cDNA into HCA-7 cells leads to a decrease
in the full-length COX-2 mRNA, but does not affect the 2577-nt mRNA.
Cell Lines
The human colon cancer cell line HCA-7, which was established
from a patient with well differentiated mucoid adenocarcinoma of the
colon (49, 50), was kindly provided by Dr. Susan Kirkland (University
of London). The moderately differentiated human colon cancer cell line
Moser (50, 51) was provided by Dr. Harold Moses (Vanderbilt
University). Human umbilical vein endothelial cells (HUVECs) were a
generous gift from Dr. Douglas Vaughan (Vanderbilt University). HCA-7
cells were maintained in Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 10% heat-inactivated fetal calf serum
(BioWhittaker, Inc., Walkersville, MD) and 100 units/ml penicillin, 100 units/ml streptomycin, and 250 ng/ml amphotericin B (Invitrogen). Moser
cells were cultured in McCoy's 5A medium (Invitrogen) supplemented
with 10% fetal calf serum, 100 units/ml penicillin, 100 units/ml
streptomycin, and 250 ng/ml amphotericin B. HUVECs were grown in Medium
199 (Invitrogen) supplemented with 15% fetal calf serum, 25 µg/ml
endothelial growth mitogen, 90 µg/ml heparin, 100 units/ml
penicillin, 100 units/ml streptomycin, and 250 ng/ml amphotericin B. These cell lines were free of mycoplasma.
HCA-7 cells were grown at 37 °C to 50, 70, 90, and 100% confluence
or overconfluence for 3 days. When indicated, HCA-7 cells at 50%
confluence were stimulated with 10 ng/ml LPS in serum-free Dulbecco's
modified Eagle's medium for 0, 1, 2, 4, or 8 h. When indicated,
HUVECs were activated with IL-1 Northern Blotting
Total RNA was extracted from cells using TRI Reagent
(Molecular Research Center, Inc., Cincinnati, OH). mRNA was
purified from total RNA using a MicroPoly(A) Pure kit (Ambion
Inc., Austin, TX). Total RNA (15 µg each) or mRNA (450 ng each)
was denatured in 50% formamide and 6% formaldehyde and resolved by
electrophoresis on a 1.0% formaldehyde-agarose gel in MOPS buffer. The
RNA was subsequently transferred to a nylon membrane
(Hybond-N+, Amersham Biosciences) and hybridized with
32P-labeled cDNAs from human COX-1, COX-2, TTP, and
5'- and 3'-RACE
RACE was performed using a SMART RACE cDNA amplification kit
(Clontech, Palo Alto, CA). Total RNA was extracted
from HCA-7 cells using TRI Reagent, and mRNA was purified from
total RNA using the MicroPoly(A) Pure kit.
Primer Design--
The gene-specific primers were designed in
exon 10 of COX-2 with the following criteria: 23-28 nt, 50-70% GC,
and Tm > 65 °C. We chose exon 10 because it has
the least homology to the COX-1 sequence.
First-strand cDNA Synthesis--
Poly(A)+ RNA
(mRNA) from HCA-7 cells under confluent conditions (1 µg) was
incubated with 5'- or 3'-cDNA synthesis primer and Moloney murine
leukemia virus reverse transcriptase (Superscript II, Invitrogen) at
42 °C for 1.5 h.
RACE--
The 5'-RACE PCRs were performed using the
gene-specific primer at position 1899 in exon 10 (GSP1,
5'-CTAGTCCGGAGCGGGAAGAACTTGCA) and the template-primer (SMART RACE
cDNA amplification kit) and Advantage-GC 2 polymerase
(Clontech) to amplify the GC-rich region. The
3'-RACE PCRs were performed using the gene-specific primer at position
1591 in exon 10 (GSP2, CTGTGGAGCTGTATCCTGCCCTTCTGGT) and the
template-primer (SMART RACE cDNA amplification kit) and Advantage
2 polymerase (Clontech). The RACE products
were resolved by electrophoresis on a 1.3% agarose gel. The bands of
5'- and 3'-RACE products were cut out from the gel and purified using a
QIAquick gel extraction kit (QIAGEN, Hilden, Germany). The isolated fragments were cloned directly into a T/A cloning vector (pGEM-T-Easy vector system, Promega, Madison, WI). The identified clones were fully
sequenced at the Vanderbilt-Ingram Cancer Center Sequencing Core
Facility by the dideoxy chain termination method using an ABI 3700 automated DNA sequencer (Applied Biosystems, Foster City, CA).
Ribonuclease Protection Assay
The expression of each of the variants of COX-2 mRNA was
evaluated by ribonuclease protection assay (RPA III kit, Ambion Inc.) with human cyclophilin mRNA as an internal standard following the
manufacturer's protocol. To prepare the template for human COX-2
riboprobes, target fragments were amplified by PCR. Each template was
designed to overlap the end of each of the variants determined by
3'-RACE (A, nt 1950-2341; B, nt 2322-2747; C, nt 2748-3169; D, nt
3168-3579; E, nt 3561-3988; F, nt 3816-4223; and G, nt 3989-4457).
The sense primers were designed to contain a BamHI site at
their 5'-ends, and the antisense primers were designed to contain the
T3 promoter sequence at their 3'-ends to generate antisense RNA probes.
The PCR fragments were resolved by electrophoresis, isolated using the
QIAquick gel extraction kit, and ligated with the pGEM-T-Easy vector.
To synthesize radiolabeled antisense riboprobes, the plasmids were
linearized with BamHI (New England Biolabs Inc., Beverly,
MA) and transcribed with T3 RNA polymerase (MAXIscript, Ambion Inc.) in
the presence of 80 µCi of [ Western Blotting
Following RNA extraction, the proteins were extracted
using TRI Reagent. The protein extract was denatured at 70 °C in the presence of NuPAGETM lithium dodecyl sulfate loading
buffer and loaded on a 10% acrylamide NuPAGETM BisTris gel
from Novex (San Diego, CA). The proteins were separated by
electrophoresis using the NuPAGETM MES running buffer from
Novex. At the end of the electrophoresis, the proteins were transferred
onto polyvinylidene difluoride membranes at 30 V for 1 h. The
nonspecific sites were blocked with 5% dry skim milk in Tris-buffered
saline with Tween 20 (Sigma), and the blot was incubated with
anti-human COX-2 antibody or anti-human COX-2 Activity Assay
COX-2 activity in HCA-7 cells was evaluated by measurement of
prostaglandin E2.
[2H8]Arachidonic acid (Sigma) was added to
fresh serum-free Dulbecco's modified Eagle's medium at concentration
of 20.0 µM. After 15 min at 37 °C, the medium was
removed. [2H4]Prostaglandin E2 (2 ng) was added to the samples as an internal standard. Prostaglandins
were isolated and derivatized for analysis by gas
chromatography/negative ion chemical ionization/mass spectrometry, monitoring selected ions, as described previously (53). The signal for
the internal standard ([2H4]prostaglandin
E2) is m/z 528. To account for the
deuterium-protium exchange at C-12 of
[2H7]prostaglandin E2, summation
of the signals obtained at m/z 530, 531, and
m/z 532 was performed (54).
RNA-Protein Cross-linking and Immunoprecipitation
The interactions between TTP protein and COX-2 RNA were
evaluated by RNA-protein cross-linking and immunoprecipitation. To prepare the template for human COX-2 sense riboprobes, target fragments
were amplified by PCR. Each template was designed to correspond to the
end of each variant determined by 3'-RACE: nt 1941-2211, 2189-2599,
2581-2858, 2842-3151, 3125-3432, 3409-3822, 3801-4225, and
4207-4446. The sense primers were designed to contain the T3 promoter
at their 5'-ends, and the antisense primers were designed to contain
BamHI sequence at their 3'-ends to generate sense RNA
probes. The PCR fragments were resolved by electrophoresis, isolated
using the QIAquick gel extraction kit, and ligated with the pGEM-T-Easy
vector. To synthesize radiolabeled sense riboprobes, the plasmids were
linearized with BamHI and transcribed with T3 RNA polymerase
(MAXIscript) in the presence of 80 µCi of [ UV cross-linking experiments were performed as described previously
(55). In brief, the cell lysates (10 µg) were incubated with 1 × 105 cpm of sense RNA probe in RNA binding buffer (20 mM HEPES (pH 7.5), 3 mM MgCl2, 40 mM KCl, 1 mM dithiothreitol, and 5% glycerol) in a total volume of 40 µl. The reaction mixtures were UV-irradiated in 96-well plates using a Stratalinker 9600 (Stratagene) for 5 min and
then incubated with 10 µg of RNase A and 5 units of RNase T1 for 30 min at 37 °C. The samples were analyzed by SDS-PAGE, and the
radioactivity associated with proteins was visualized by autoradiography.
Immunoprecipitation was performed using 200 µl of UV-cross-linked
samples. Three affinity-purified goat polyclonal antibodies specific
for TTP (Santa Cruz Biotechnology) were used: G-20, raised against the
amino terminus of the mouse protein; P-20, raised against the carboxyl
terminus of the human protein; and N-18, raised against the amino
terminus of the human protein. The antibodies (2 µg) were added to
the samples in immunoprecipitation buffer (50 mM HEPES (pH
7.5), 150 mM NaCl, 5 mM MgCl2, 1%
Nonidet P-40, 1 mM dithiothreitol, and 10% glycerol) and
incubated for 5 h at 4 °C with agitation. Then, 20 µl of
protein G PLUS-agarose (Santa Cruz Biotechnology) was added to
the mixplus the samples were incubated overnight at 4 °C with
agitation. The pellets were collected by centrifugation at 2500 × g for 2 min and washed four times with phosphate-buffered
saline. The immunoprecipitates were analyzed by SDS-PAGE, and the
radioactivity associated with the proteins was visualized by autoradiography.
TTP Transfection
The pcDNA3.1-FLAG-TTP expression construct (under the
control of the cytomegalovirus promoter) containing the human TTP
coding region (nucleotides 10-990) was a generous gift from Dr.
William Rigby (Dartmouth University) (56). Transient transfection of HCA-7 cells was performed using Cellfectin (Invitrogen) following the
manufacturer's instructions. Briefly, 50% confluent HCA-7 cells in
T-75 flasks were washed with Opti-MEM I reduced serum medium
(Invitrogen). The TTP DNA (7.5 µg) in 500 µl of Opti-MEM I and
Cellfectin (15 µl) in 500 µl of Opti-MEM were combined and added to
the cells. After 18 h, the DNA-containing medium was replaced with
Dulbecco's modified Eagle's medium with 10% fetal calf serum and
incubated for 6 h at 37 °C. RNA and proteins were extracted
from the cells using TRI Reagent.
Statistical Analysis
The data are presented as means ± S.E. and were compared
by unpaired t test or by one-way ANOVA followed by Fisher's
PLSD procedure. The criterion for statistical significance was
p < 0.05.
COX-2 Transcripts in HCA-7 and Moser Colon Cancer Cells and
HUVECs--
Northern blot analysis of mRNA from HCA-7 cells using
a COX-2-specific probe showed two bands migrating at ~4.5 and 2.6 kb (Fig. 1A). Interestingly, the
smaller species was the more abundant in confluent HCA-7 cells. By
comparison, the longest species at 4.5 kb was the main transcript in
Moser cells (Fig. 1B) (50, 51). HUVECs grown under standard
conditions did not express COX-2. However, after activation by IL-1 Cloning of the Variants of COX-2 mRNA in HCA-7 Cells by 5'- and
3'-RACE--
We performed 5'- and 3'-RACE using the gene-specific
primer located in the coding region of exon 10 because it has the least homology to COX-1. Analysis of HCA-7 mRNA by 5'-RACE generated a
lone product at 1.9 kb, indicating no splice variant in this portion of
the coding region. On the other hand, analysis of HCA-7 mRNA by
3'-RACE generated nine products at 2.9, 2.6, 2.3, 1.8, 1.6, 1.3, 1.0, 0.9, and 0.6 kb. Each product was subcloned in a T/A cloning vector and
sequenced. The results of sequencing were compared with the published
sequence of the human COX-2 gene (57). The 5'-RACE product started at
Characterization of the Small RNA Transcript--
To characterize
the two transcripts observed by Northern blot analysis, we designed
probes specific for each COX-2 3'-RACE product (A, nt 1846-2116; B, nt
2269-2525; C, nt 2578-2852; D, nt 2896-3097; E, nt 3135-3366; F, nt
3544-3834; G, nt 3927-4134; and H, nt 4204-4446). Total RNA and
mRNA from HCA-7 cells were transferred to nylon membranes and
examined by Northern blot analysis using these probes. Probes A and B
recognized the short mRNA species, indicating two possible
polyadenylation variants at 2443 and 2577 bp (Fig.
2A). Probe C-H recognized
only the long mRNA transcript, identifying it as the full-length
transcript at 4465 bp (Fig. 2A). To differentiate between
the two polyadenylation sites at 2443 and 2577 bp, we analyzed the
total RNA by ribonuclease protection assay. Antisense RNA probes ~400
nucleotides long were synthesized to span either side of the different
polyadenylation sites (A, nt 1950-2341; B, nt 2322-2747; C, nt
2748-3169; D, nt 3168-3579; E, nt 3561-3988; F, nt 3816-4223; and
G, nt 3989-4457). Hybridization with probe B generated two fragments
corresponding to the fully protected probe (426 nt) and to the
partially protected probe (256 nt) (Fig. 2B). This indicates
that the antisense probe B hybridizes with an mRNA that corresponds
to the COX-2 sequence through nt 2577. The 3'-terminal sequence of the
polyadenylated 2577-nt mRNA determined from sequencing the 3'-RACE
product is depicted in Fig. 2C, indicating the
polyadenylation signal located 19 nt proximal to nt 2577. The five
adenines at nt 2573-2577 are present in the COX-2 3'-UTR sequence. The
generation of the fully protected fragments when probes C-G were used
confirmed the identity of the long transcript as the full-length
mRNA at 4465 nt (Fig. 2B).
COX-2 and TTP mRNAs in HCA-7 Cells Related to Cell
Confluence--
HCA-7 cells were harvested at 50, 70, 90, and 100 confluence or at 3 days post-confluence. Total RNA extracted from cells was analyzed by Northern blotting with probe B (nt 2269-2525), and the
presence of COX-2 protein was analyzed by Western blotting. Northern
blot analysis revealed that, before confluence, the 4465-nt COX-2
mRNA was the main transcript in HCA-7 cells; the level of this
variant was then reduced as the cells became overconfluent. The 2577-nt
COX-2 mRNA represented a less abundant species before confluence,
but its level was increased after confluence (Fig. 3A). Densitometric analysis of
the Northern blots revealed that the differences between the values
obtained before and after confluence for both transcripts were
statistically significant as ascertained by ANOVA followed by Fisher's
PLSD procedure (Fig. 3B).
The expression of TTP was also examined by Northern blotting using
total RNA from HCA-7 cells. The analysis revealed that TTP was
expressed in HCA-7 cells and that its transcription was increased with
cell confluence. The densitometric analysis of Northern blots revealed
that the differences between the values obtained before and after
confluence were statistically significant as ascertained by ANOVA
followed by Fisher's PLSD procedure (Fig. 3C).
Interestingly, the increase in TTP transcription correlated with the
decrease in the full-length COX-2 transcript, consistent with a
possible role for TTP in the post-transcriptional regulation of COX-2.
These results also indicate that these two COX-2 transcripts are
differentially regulated.
Analysis of COX-2 protein expression by Western blotting revealed that,
at the stage of confluence when the full-length RNA was down-regulated,
the amount of protein produced was increased (Fig.
4, A and B).
Analysis of the cyclooxygenase activity showed that production of
prostaglandin E2 increased as the cells grew from 50% to
overconfluence and while levels of the full-length transcript decreased
(Fig. 4C). These results suggest that the short COX-2
polyadenylation variant is translationally competent.
Binding of TTP Protein to COX-2 mRNAs--
To characterize the
interactions between TTP protein and COX-2 mRNAs, we assessed
whether TTP expressed in HCA-7 cells at 50% confluence and
overconfluent for 3 days would bind to the COX-2 3'-UTR. We performed
UV cross-linking of radiolabeled sense RNA probes spanning the COX-2
3'-UTR to soluble proteins from HCA-7 cells. Each probe was designed to
hybridize with COX-2 RNA at the end of each variant determined by
3'-RACE: nt 1941-2211, 2189-2599, 2581-2858, 2842-3151, 3125-3432,
3409-3822, 3801-4225, and 4207-4446. As shown in Fig.
5A, numerous proteins that
were bound to the different RNA probes were detected. Because only the
full-length COX-2 transcript was down-regulated with confluence, we
focused our attention on proteins that bound to the probes located
distal to nucleotide 2577 and that were up-regulated between 50%
confluence and 3 days post-confluence. Several proteins fulfilled these
criteria, but one prominent protein binding between nucleotides 3125 and 3432 also had an apparent molecular mass of 43 kDa, which corresponds to human TTP. To determine whether TTP can bind this region
of the full-length COX-2 mRNA, we performed another cross-linking experiment in which TTP was immunoprecipitated using three different antibodies specific for TTP after the UV cross-linking step. The immunoprecipitates were then analyzed by SDS-PAGE to detect bound TTP.
As shown in Fig. 5B, only one band was detected with a
molecular mass of 43 kDa and was strongly up-regulated with confluence. These results demonstrate that TTP protein expressed in HCA-7 cells can
functionally bind to COX-2 RNA between positions 3125 and 3432.
Effect of TTP Transfection on COX-2 mRNA Stability--
To
determine the effect of TTP expression on COX-2 post-transcriptional
regulation, we transfected 50% confluent HCA-7 cells with a plasmid
containing the human FLAG-tagged TTP coding region (nucleotides
10-990) regulated by the cytomegalovirus promoter. We examined COX-2
mRNA in the cells after transient transfection with this plasmid
(Fig. 6). Northern blot analysis
confirmed TTP transcription in cells transfected with the plasmid
coding for the human protein. In the same time, we observed a
statistically significant decrease in the amount of 4465-nt COX-2
mRNA in cells transfected with TTP compared with mock transfection.
By comparison, the 2577-nt COX-2 mRNA was not decreased under the
same conditions. As TTP transfection had no effect on the truncated
polyadenylation variant, these data suggest that the reduction in the
levels of the full-length COX-2 mRNA results from a destabilizing
effect of TTP that is targeted to the long transcript containing the identified TTP-binding site.
Inhibition of gene transcription with actinomycin D has been used to
assess the rates of decay of some mRNAs. However, this approach is
not possible for assessing the rate of mRNA decay if its regulation
is influenced by an inducible transactivating protein because
actinomycin D will arrest the transcription of the transactivator (24).
As expected, we found that the levels of TTP mRNA fell rapidly
after actinomycin D treatment (data not shown).
Correlation of TTP and COX-2 Transcription in HCA-7
Cells--
HCA-7 cells at 50% confluence were stimulated with 10 ng/ml LPS for 0, 1, 2, 4, or 8 h. Total RNA extracted from the
cells was analyzed by Northern blotting using probe B (nt 2269-2525) (Fig. 7). TTP RNA was examined by
Northern blotting following stimulation with LPS. The results indicate
that LPS induced TTP transcription, with the maximum effect reached at
1 h (Fig. 7). The level of RNA was already decreased at 2 h
and was back to the basal level at 4 h. This transient induction
of TTP transcription is consistent with previous findings (39, 41). The
expression of both COX-2 transcripts was seen with LPS stimulation,
with the maximum induction reached after 1 h. The 2577-nt mRNA
of COX-2 was still elevated at 4 h. On the other hand, the 4465-nt
mRNA of COX-2 was only sustained until 2 h and then rapidly
decreased; it was back to the basal level at 4 h. The greater
reduction of the 4465-nt mRNA at 4 h is significant at the
p < 0.05 level.
As an immediate-early gene, the induction of COX-2 transcription by LPS
is rapid and brief. Because both the 4465- and 2577-nt COX-2 mRNAs
derive from transcription of the COX-2 gene, termination of
transcription after the brief burst elicited by LPS leads to concurrent
arrest of formation of both the full-length and variant transcripts.
Thus, a difference in the rate of decline in the concentration of the
two COX-2 mRNAs reflects a difference in the rate of their degradation.
This report provides evidence that TTP binds to the 3'-UTR of
COX-2 mRNA. The investigations leading to this finding were informed by the finding that a polyadenylation variant of COX-2 mRNA is regulated differently from the full-length COX-2 transcript.
We found two major transcripts of COX-2 in the human colorectal
adenocarcinoma cell lines HCA-7 and Moser. This is of particular interest because of the importance of COX-2 in the development of
colorectal adenocarcinoma and because of the role COX-2 overexpression has in growth and tumorigenicity in the HCA-7 and Moser cell lines (50,
51). Accordingly, we fully characterized these two COX-2 transcripts.
The larger was found to be the full-length mRNA, 4465 nt in length.
The smaller is 2577 nt in size and represents a polyadenylation variant
that lacks the distal segment 2578-4465 of the 3'-UTR (24, 25). It is
polyadenylated downstream from the non-canonical polyadenylation
signal, AUUAAA (58).
The consequences of deletion of the distal 1888 nt of the 3'-UTR may be
considered in the context of evidence that the 3'-UTR participates in
the regulation of the stability of a number of mRNA species,
including that of COX-2 itself. The 3'-UTR of COX-2 mRNA contains
22 copies of a conserved ARE, the AUUUA pentamer. This pentamer,
frequently located in or near a U-rich region, has been associated with
the regulation of mRNA stability (57) in a number of mRNAs,
including those of proto-oncogenes (59, 60) and cytokines (37).
Recently, Dixon et al. (55) reported the importance of AREs
in the post-transcriptional regulation of COX-2 mRNA. A number of
ARE-binding proteins have been identified, including HuR (33),
AUF1/heterogeneous nuclear ribonucleoprotein D (34, 35), TIA-1 (36),
and tristetraprolin (37). Binding of these proteins to AREs in the
3'-UTR can exert either positive or negative effects on stability,
translation, and subcellular localization of the mRNA. For example,
HuR binds to the ARE present in the proximal segment of the
COX-2 3'-UTR to enhance stability of the mRNA (38); both
transcripts of COX-2 contain the HuR-binding site. By contrast, AREs
present at the distal end of the 3'-UTR proto-oncogenes have been shown
to participate in destabilization of the mRNA. For example,
truncation of 61 bases containing AREs that are 24 nucleotides before
the poly(A)+ addition signal in the myc
proto-oncogene mRNA increases the half-life of the
mRNA by at least 5-fold (59).
The evidence that AREs in the 3'-UTR region can influence the stability
and translation of mRNA and the presence of 15 AREs in the deleted
sequence between bases 2578 and 4465 provided the basis for the
hypothesis that the regulation of the stability of the 2577-nt COX-2
mRNA could be different from that of the full-length transcript. As
one approach toward assessing whether lack of sequence 2578-4465
altered the content of COX-2 mRNA or COX-2 expression and activity,
we determined the effect of confluence of the cells on the amount of
each of the mRNA species as well as COX-2 protein and prostaglandin
production. One of the characteristics of cancer cells is the loss of
contact inhibition. The HCA-7 cell line shares this characteristic, and
cells continue to grow for several days after reaching confluence. We
observed that, as the cell culture progressed from 50% confluence to a
post-confluent state, the ratio of 4465-nt to 2577-nt mRNA
decreased. This change in the ratio resulted from both a decrease in
the abundance of 4465-nt mRNA and an increase in the abundance of
2577-nt mRNA. One explanation for these results is that confluence
is associated with the synthesis of a cellular factor that signals the
rapid degradation of the mRNA by binding to region 2578-4465 of
the 3'-UTR. Despite the reduction in the level of the full-length transcript, COX-2 protein and activity were increased, indicating that
the 2577-nt mRNA is translationally competent.
The TTP gene is a member of the immediate-early gene family (41, 61).
The TTP protein binds to AREs and promotes destabilization of mRNAs
of oncogenes and cytokines such as TNF- To further characterize the differential regulation of the two COX-2
messages, we analyzed the proteins binding to the COX-2 3'-UTR. We
observed that several proteins bound to the 3'-UTR distal to nucleotide
2577. Among these proteins, four were up-regulated with confluence, and
one of these migrated on SDS-polyacrylamide gel at ~45 kDa. We
addressed the question of whether that protein was TTP by
immunoprecipitating the RNA-protein complexes cross-linked by UV,
employing three different antibodies specific for TTP. The results
demonstrate that TTP bound to the COX-2 3'-UTR between nucleotides 3125 and 3432. Analysis of the nucleotide sequence of COX-2 message in this
region showed an ARE starting at nucleotide 3369 with the consensus
sequence UAUUUA. Recently, Worthington et al. (47) showed
that addition of Us on each side of the consensus sequence AUUUA
increased the affinity of the ARE for TTP. Although the highest
affinity was reached with the palindromic sequence UUAUUUAUU, they
showed that TTP had a higher affinity for UAUUUAU than for AUUUA.
Because the UAUUUA at nt 3369 is the only ARE in that region of the
COX-2 3'-UTR, it is likely that TTP binds to the COX-2 3'-UTR by
association with the ARE present between nucleotides 3369 and 3374.
Binding of TTP to ARE-rich 3'-UTRs has resulted in accelerated
degradation of the mRNA. To determine whether TTP increases the
removal of the full-length COX-2 mRNA, we transfected HCA-7 cells
at 50% confluence with a plasmid coding for the human TTP. The levels
of the 4.5-kb COX-2 mRNA were significantly reduced after the
transfection. The absence of any change in the levels of the 2.6-kb
message lacking the TTP-binding site provides evidence that TTP did not
alter transcription, as the polyadenylation variant and full-length
mRNAs both derive from a common transcription process (24). This
finding is consistent with the hypothesis that the transactivating
protein TTP binds to the distal portion of the 3'-UTR of COX-2 mRNA
and facilitates its degradation.
We then examined the post-transcriptional regulation of the 4.5-kb
mRNA in a pathophysiological model in which TTP levels are
increased (44, 62). LPS induced a short burst of transcription of both
TTP and COX-2; and in this TTP-induced environment, the removal of the
COX-2 transcript containing the TTP-binding site was accelerated
relative to that of the concurrently transcribed mRNA with a
truncated 3'-UTR lacking the TTP-binding domain.
In conclusion, the transactivating protein tristetraprolin binds to the
3'-UTR of COX-2 mRNA in the region between nucleotides 3125 and
3432. This segment of the 3'-UTR contains the ARE UAUUUA, beginning at
nucleotide 3369. Transfection of the TTP gene demonstrated that TTP can
decrease the level of the full-length COX-2 mRNA, but it does not
affect the truncated 2577-nt polyadenylation variant that lacks the
TTP-binding site. This polyadenylation variant, which escapes
down-regulation by TTP, is prominent in a colon cancer cell line. We
also hypothesize that TTP up-regulation at confluence provides evidence
for a role of the protein in the mechanism by which cells down-regulate
specific mRNAs in the process of contact inhibition.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
granulocyte/macrophage colony-stimulating factor) and proto-oncogenes
(e.g. c-fos and c-myc) contribute
importantly to the post-transcriptional regulation of message
abundance. These 3'-UTRs contain AU-rich elements (AREs) that are the
binding sites for proteins that interact with the 3'-UTR to regulate
mRNA de-adenylation and decay (32), and the 3'-UTR of COX-2
contains many AREs. A number of ARE-binding proteins have been
identified, including HuR (33), AUF1/heterogeneous nuclear
ribonucleoprotein D (34, 35), TIA-1 (36), and tristetraprolin (TTP) (37). Binding of some of these proteins to AREs in the 3'-UTR can
enhance stability of the message, and HuR has been shown to stabilize
COX-2 mRNA (38). Alternatively, proteins that bind to a 3'-UTR can
accelerate mRNA degradation, as is the case with TTP (37, 39, 40).
Because our findings suggested destabilization of COX-2 mRNA by a
protein binding to the distal 1888 nt of the 3'-UTR, we explored the
possibility that this protein might be TTP.
and granulocyte/macrophage colony-stimulating factor (44, 45). TTP binds to AREs from RNAs coding for immediate-early genes such as c-fos, interleukin-3, and TNF-
(39, 44, 46, 47) and confers instability to these messages. TTP also has a
physiological role in the induction of apoptosis (48).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1 ng/ml; Sigma) for 16 h as
described previously (52).
-actin. COX-2 cDNAs were obtained by PCR amplification of
regions located on the end of each variant determined by 3'-rapid
amplification of cDNA ends (RACE): A, nt 1846-2116; B, nt
2269-2525; C, nt 2578-2852; D, nt 2896-3097; E, nt 3135-3366; F, nt
3544-3834; G, nt 3927-4134; and H, nt 4204-4446. TTP
cDNAs were obtained from American Type Culture Collection
(Manassas, VA). cDNAs from human COX-1 (Oxford Biomedical
Research, Inc., Oxford, MI) and COX-2, TTP, and
-actin (Ambion
Inc.) were labeled with 32P using a random prime labeling
system (Rediprime, Amersham Biosciences). The blots were washed five
times with 1× SSC with 1% SDS at room temperature and then exposed to
x-ray film at
70 °C. RNA bands were quantified by densitometry
using Scion Image (Scion Corp., Frederick, MD). Total RNA was extracted
from three independent series of cells, and Northern blotting was
performed twice with each RNA.
-32P]UTP at 37 °C for
1 h. The full-length radiolabeled riboprobes were purified by
preparative electrophoresis on an 8 M urea and 5%
acrylamide denaturing gel and eluted into 0.5 M ammonium
acetate, 1 mM EDTA, and 0.2% SDS at 37 °C for 16 h. For ribonuclease protection assay, 10 µg of total RNA was
hybridized with [
-32P]UTP-labeled antisense RNA probes
complementary to human COX-2 mRNA or human cyclophilin mRNA
(Ambion Inc.). After hybridization overnight at 45 °C and digestion
with 7.5 units of RNase T1 (Ambion Inc.), the protected mRNA
fragments were denatured and then resolved on an 8 M urea
and 5% acrylamide gel. The gels were exposed to x-ray film at
70 °C. COX-2 mRNA abundance in each sample was normalized to
the abundance of constitutively expressed cyclophilin mRNA to
control for RNA loading.
-actin antibody (both from
Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room
temperature. After incubation with the secondary antibody coupled to
horseradish peroxidase (1:2000 dilution; Santa Cruz Biotechnology) for
1 h at room temperature, the proteins were detected with luminous
ECL reagent (Amersham Biosciences).
-32P]UTP
at 37 °C for 1 h. Cytoplasmic cell lysates were prepared from
HCA-7 cells grown in T-75 flasks at 37 °C to 50, 70, 90, and 100%
confluence or overconfluence for 3 days. They were washed twice with
phosphate-buffered saline, and 2 ml of lysis buffer (25 mM
Tris-HCl (pH 7.5) and 0.5% Nonidet P-40) was added. The cells were
frozen at
70 °C. The thawed cells were scraped from the plate,
vortexed shortly, and centrifuged at 14,000 × g for 10 min. The supernatant was assayed for protein concentration and keep
frozen at
70 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
HUVECs also expressed two transcripts of COX-2, the main one being 4.5 kb long. Because full-length COX-1 mRNA is 2.5 kb long, we
investigated whether the small transcripts in HCA-7 and Moser cells
were due to cross-reactivity of our probe with COX-1 RNA by analyzing
the same total RNA by Northern blotting using a probe specific for
COX-1. As shown in Fig. 1C, a band at ~2.5 kb was observed
in HUVECs grown under normal conditions or after activation with
IL-1
. On the contrary, both HCA-7 and Moser cells did not
express COX-1. This indicates that the small transcript of 2.5 kb
present in HCA-7 is specific to COX-2.
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Fig. 1.
Expression of COX-1 and COX-2 RNAs in HUVECs,
HUVECs activated with IL-1 , and HCA-7 and
Moser cells upon Northern blot analysis. HCA-7 and Moser cells and
HUVECs were grown as described under "Experimental Procedures."
HUVECs were activated with or without 1 ng/ml IL-1
for 16 h.
Cells were harvested at confluence, total RNA was extracted, and
mRNA was purified on a poly(A) affinity column. 450 ng of mRNA
(A) or 15 µg of total RNA (B and C)
was used for Northern blot analysis. Membranes were hybridized with
COX-2-specific (A and B) or COX-1-specific
(C) probes. The lower panels show Northern blots
for
-actin as a control of equal loading of RNA.
2 and ended at the gene-specific primer for 5'-RACE located in exon
10 (position 1899). The 3'-RACE products started at the gene-specific
primer for 3'-RACE located in exon 10 (position 1591) and ended with a
poly(A) tail starting at positions 4465, 4208, 3902, 3408, 3109, 2871, 2577, 2443, and 2187.
View larger version (50K):
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Fig. 2.
Characterization of the two COX-2
transcripts. A, Northern blots of COX-2 RNA. mRNA
(450 ng each) was hybridized with each COX-2 probe as described under
"Experimental Procedures." Probes A-H used for each panel are
indicated (A, nt 1846-2116; B, nt 2269-2525;
C, nt 2578-2852; D, nt 2896-3097; E,
nt 3135-3366; F, nt 3544-3834; G, nt
3927-4134; and H, nt 4204-4446). The signals corresponding
to the long and short COX-2 mRNAs are indicated by the
arrowheads. B, RNase protection assay of COX-2
RNA. Antisense RNA probes ~400 nucleotides long were synthesized.
Total RNA (10 µg) was hybridized with
[ -32P]UTP-labeled antisense RNA probes designed to
span either side of the putative polyadenylation site of interest
(A, nt 1950-2341; B, nt 2322-2747;
C, nt 2748-3169; D, nt 3168-3579; E,
nt 3561-3988; F, nt 3816-4223; and G, nt
3989-4457). After digestion with RNase T1, the protected mRNA
fragments were denatured and separated on polyacrylamide gel. Results
obtained with probes B and G are represented. Lanes 1,
protected fragments; lanes 2, negative control using yeast
RNA; lanes 3, full-length probe without RNase digestion. The
lengths of the protected fragments are indicated next to the
arrowheads in nucleotides. The lengths of the corresponding
transcripts are indicated in parentheses. C,
sequence of the 52 final nucleotides of 2577-nt mRNA. The
non-canonical polyadenylation signal is underlined. The
polyadenylation tail is indicated in boldface. The end of
the sequence matching the COX-2 cDNA at position 2577 is
indicated.
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Fig. 3.
Relative abundance of the two major COX-2
mRNAs and TTP mRNA under different conditions of
confluence. HCA-7 cells were harvested at 50, 70, 90, and
100% confluence or at 3 days post-confluence. Total RNA (15 µg each)
was analyzed by Northern blotting. A, Northern blots for
COX-2, TTP, and -actin are represented. Lanes 1-4, 50, 70, 90, and 100% confluence, respectively; lane 5, 3 days
post-confluence. B, RNA bands for 4.5-kb (
) and 2.6-kb
(
) COX-2 were quantified by densitometry and corrected by the amount
of
-actin in the same sample by one-way ANOVA followed by Fisher's
PLSD procedure. The asterisks indicate statistical
significance compared with 50% confluence (*, p < 0.05; **, p < 0.01; n = 3).C, RNA bands for TTP were integrated, corrected by the
amount of
-actin in the same sample, and compared by one-way ANOVA
followed by Fisher's PLSD procedure. The asterisks indicate
statistical significance (p < 0.01; n = 3) between every condition and each other.
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Fig. 4.
Catalytic activity and expression of COX-2 in
HCA-7 cells as a function of confluence. HCA-7 cells were
harvested at 50% confluence or at 3 days post-confluence.
A, proteins (50 µg) were analyzed by Western blotting
after electrophoresis on a 10% polyacrylamide gel under denaturing
conditions. B, the amount of protein was integrated and
corrected by the amount of -actin in the same sample. The
asterisk indicates statistical significance compared with
50% confluence (p < 0.01; n = 4).
C, [2H8]arachidonic acid (20 µM) was added to the cells in fresh serum-free medium.
After 15 min of incubation at 37 °C, the medium was harvested, and
prostaglandin E2 (PGE2) was analyzed by
gas chromatography/mass spectrometry. The asterisk indicates
statistical significance compared with 50% confluence
(p < 0.01; n = 4).
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Fig. 5.
Analysis of TTP protein and COX-2 RNA
interactions by RNA-protein cross-linking and immunoprecipitation.
Human COX-2 sense riboprobes labeled with [ -32P]UTP
were designed to bracket the end of each variant determined by 3'-RACE:
nt 1941-2211, 2189-2599, 2581-2858, 2842-3151, 3125-3432,
3409-3822, 3801-4225, and 4207-4446. Cytoplasmic cell lysates were
prepared from HCA-7 cells harvested at 50% confluence or at 3 days
post-confluence. A, shown are the results from RNA-protein
cross-linking experiments. The cell lysates (10 µg) were incubated
with 1 × 105 cpm of sense RNA probe, UV-irradiated
for 5 min, and then incubated with RNase. The samples were analyzed by
SDS-PAGE, and the radioactive proteins were visualized by
autoradiography. Lanes 1, 50% confluence; lanes
2, 3 days post-confluence. B, for immunoprecipitation,
the three different TTP antibodies (Ab) were added to the
UV-cross-linked samples, and the mixture was incubated for 5 h at
4 °C. Then, protein G PLUS-agarose was added to the mixture,
and the samples were incubated overnight at 4 °C. The pellets were
collected by centrifugation and washed. The immunoprecipitates were
analyzed by SDS-PAGE followed by autoradiography. The results using
probe 3125-3432 are represented. Lanes 1, 50% confluence;
lanes 2, 3 days post-confluence.
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Fig. 6.
Transcription of COX-2 and TTP in HCA-7 cells
after transfection with human TTP. HCA-7 cells at 50%
confluence were transiently transfected with the
pcDNA3.1-FLAG-TTP expression construct using Cellfectin.
RNA was extracted from the cells using TRI reagent. A,
Northern blot analysis for COX-2, TTP, and -actin. Lane
1, mock transfection; lane 2, TTP transfection.
B, RNA bands for 4.5- and 2.6-kb COX-2 and TTP were
quantified by densitometry and corrected by the amount of
-actin in
the same samples. The asterisks indicate statistical
significance (p < 0.05; n = 3) with
mock transfection. The black and white bars
indicate mock transfection and TTP transfection,
respectively.
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Fig. 7.
Relative abundance of the two major COX-2
mRNAs and TTP mRNA upon LPS stimulation. HCA-7 cells at
50% confluence were incubated with LPS (10 ng/ml) for 0, 1, 2, 4, and
8 h. Total RNA (15 µg each) was analyzed by Northern blotting
for COX-2 and TTP transcription. A, the results of Northern
blotting for COX-2, TTP, and -actin after induction with LPS are
represented. Lane 1, 0 h of incubation; lane
2, 1 h; lane 3, 2 h; lane 4,
4 h; lane 5, 8 h. B, RNA bands for
4.5-kb (
) and 2.6-kb (
) COX-2 and TTP (
) were quantified by
densitometry, corrected by the amount of
-actin in the same sample,
and compared by one-way ANOVA followed by Fisher's PLSD procedure. The
asterisks indicate statistical significance compared with
0 h (p < 0.05; n = 3).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(37, 39, 40). Because of the
similarities between the 3'-UTRs of TNF-
and COX-2, we
hypothesized that TTP could be responsible for full-length COX-2
mRNA down-regulation in HCA-7 cells. We demonstrated that the TTP
gene was transcribed in HCA-7 cells and that its transcription was
up-regulated with confluence. Its up-regulation correlated with the
down-regulation of 4.5-kb COX-2 message.
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ACKNOWLEDGEMENTS |
---|
We thank Elizabeth Shipp for excellent technical assistance and Joseph Covington for providing the HUVECs. We thank Dr. Ronald Emeson for valuable advice.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants CA68485, CA77839, and GM15431 and by a grant from Merck-Frosst Canada & Co.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.
** Thomas F. Frist, Sr. Professor of Medicine.
To whom correspondence should be addressed: Dept. of
Pharmacology, Vanderbilt University, 514 RRB, 23rd Ave. S. at Pierce, Nashville, TN 37232-6602. Tel.: 615-343-7398; Fax: 615-322-4707; E-mail: olivier.boutaud@vanderbilt.edu.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M300016200
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ABBREVIATIONS |
---|
The abbreviations used are:
COX, cyclooxygenase;
nt, nucleotide(s);
UTR, untranslated region;
TNF-, tumor necrosis
factor-
;
ARE, AU-rich element;
TTP, tristetraprolin;
HUVECs, human
umbilical vein endothelial cells;
LPS, lipopolysaccharide;
IL-1
, interleukin-1
;
MOPS, 3-(N-morpholino)propanesulfonic
acid;
RACE, rapid amplification of cDNA ends;
BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
MES, 2-(N-morpholino)ethanesulfonic acid;
ANOVA, analysis of
variance.
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REFERENCES |
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