Carcinogenesis vol.27 no.11 pp.2170–2179, 2006
doi:10.1093/carcin/bgl053
Advance Access publication April 22, 2006
Reciprocal regulation of cyclooxygenase-2 and 15-hydroxyprostaglandin
dehydrogenase expression in A549 human lung adenocarcinoma cells
Min Tong , Yunfei Ding and Hsin-Hsiung Tai
†
†
Department of Pharmaceutical Sciences, College of Pharmacy,
University of Kentucky, Lexington, KY 40536-0082, USA
To whom correspondence should be addressed at: Department of
Pharmaceutical Sciences, College of Pharmacy, University of Kentucky,
Lexington, KY 40536-0082, USA. Tel: +1 859 257 1837;
Fax: +1 859 257 7585;
Email: [email protected]
Human lung adenocarcinoma cells, A549, possess the capacity of expressing both cyclooxygenase-2 (COX-2) and
NAD+-linked 15-hydroxyprostaglandin dehydrogenase
(15-PGDH). Resting cells express little COX-2 but significant levels of 15-PGDH. Interleukin (IL) 1b, tumor necrosis
factor-a (TNF-a) or phorbol ester [phorbol 12-myristate
13-acetate (PMA)] induced the expression of COX-2, as
revealed by western blot analysis. Combination of PMA
and IL-1b or TNF-a induced synergistically the expression
of COX-2. Interestingly, cytokines and cytokine plus PMAinduced expression of COX-2 were accompanied by a
downregulation of 15-PGDH. This was evident from both
the western blot analysis and activity assay of 15-PGDH.
It appears that the higher the expression of COX-2 was
induced, the lower the expression of 15-PGDH was found.
This was further supported by the observation that overexpression of COX-2 but not COX-1 by adenovirusmediated approach led to a decrease in 15-PGDH expression, indicating the specificity of COX-2. Furthermore,
downregulation of the IL-1b-induced expression of
COX-2 by silencing RNA (siRNA) approach resulted in
an increase in the expression of 15-PGDH by COX-2siRNA but not by COX-1-siRNA, indicating that it was
indeed the expression of COX-2 attenuating the expression
of 15-PGDH. The IL-1b-induced reduction of the expression of 15-PGDH was shown not to be mediated by COX-2derived products since the presence of COX-2 inhibitors
did not block the attenuation of the expression of
15-PGDH. Exogenous PGE2 also did not induce the reduction of the expression of 15-PGDH. However, overexpression of 15-PGDH by transfection with recombinant
plasmid encoding 15-PGDH or adenovirus-mediated
approach attenuated IL-1b-induced expression of COX2. On the contrary, downregulation of 15-PGDH expression by 15-PGDH-siRNA or 15-PGDH-antisense approach
resulted in an increase in IL-1b-induced expression of
COX-2 but not that of COX-1. In fact, it was further
observed that A549 clones expressing different degrees
Abbreviations: 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; COX,
cyclooxygenase; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3phosphate dehydrogenase; HRP, horseradish peroxidase; IL, interleukin;
MOI, multiplicity of infection; mPGE, microsomal PGE; PG, prostaglandin;
PMA, phorbol 12-myristate 13-acetate; RT–PCR, reverse transcription–
polymerase chain reaction; siRNA, silencing RNA; TNF, tumor necrosis
factor.
†
The first two authors contributed equally to this work.
#
of 15-PGDH showed also different levels of COX-2 expression after IL-1b induction. The levels of IL-1b-induced
COX-2 expression appeared to correlate inversely with
those of 15-PGDH expression in the cells. These results
support the contention that COX-2 and 15-PGDH are
regulated reciprocally in A549 cells.
Introduction
Prostaglandins (PGs) are biosynthesized from arachidonic acid
through the cyclooxygenase (COX) pathway. Two isoforms of
COX have been recognized. COX-1 is generally constitutively
expressed as a housekeeping enzyme in most tissues and cells
and mediates homeostatic functions such as cytoprotection of
the stomach and regulation of platelet aggregation (1). COX-2,
which is encoded by an immediate early gene, can be induced
by pro-inflammatory agents such as lipopolysaccharides,
cytokines such as interleukin (IL) 1b and tumor necrosis
factor-a (TNF-a), and mitogenic factors such as phorbol
ester and growth factors (2,3). COX-2 has been shown to
be the isoform primarily responsible for the synthesis of
PGs involved in pathological processes such as cancer and
inflammatory states (4). Regulation of COX-2 expression is
achieved not only at the transcriptional level but also at the
post-transcriptional level. The latter aspect of regulation is
related to the presence of several AU-enriched elements in
the 30 -untranslated region (30 -UTR) of COX-2 mRNA,
which confers message instability (5). Several RNA-binding
proteins have been discovered to bind to these elements and to
stabilize the message either facilitating or inhibiting the translation, resulting in an increased or a decreased expression of
COX-2 (6,7).
PGs and lipoxins are rapidly metabolized by the initial
oxidation of 15(S)-hydroxyl group catalyzed by NAD+-linked
15-hydroxyprostaglandin dehydrogenase (15-PGDH) followed by the reduction of 13,14-double bond generating
13,14-dihydro-15-keto-prostaglandins and lipoxins (8). The
initial products, 15-keto-prostaglandins and lipoxins, exhibit
greatly reduced biological activities rendering 15-PGDH, the
key enzyme responsible for biological inactivation of PGs and
lipoxins (8). The enzyme is expressed in most of the mammalian tissues, and lung is one of the most active organs (9).
The enzyme utilizes PGs as well as lipoxins as substrates (10).
It is intriguing that the enzyme metabolizes and inactivates
pro-inflammatory PGs as well as anti-inflammatory lipoxins.
This may be related to the presence of this enzyme in specific
cell types for unique function. Expression of 15-PGDH was
induced by phorbol ester in human promyelocytic leukemia
HL-60 cells, human erythroleukemia (HEL) cells and
promonocytic U-937 cells (11–13). Expression of this enzyme
could be also induced by dexamethasone and other antiinflammatory steroids in HEL cells (12) and in human lung
adenocarcinoma A549 cells (14), by androgens and other
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Reciprocal regulation of COX-2 and 15-PGDH
steroid hormones in prostate cancer cells (15), by 1,25dihydroxyvitamin D3 in human neonatal monocytes (16)
and by high concentrations of indomethacin in human tumoral
C cells (17). In addition to anti-inflammatory steroids, an antiinflammatory cytokine, IL-10, may also regulate the expression of 15-PGDH by antagonizing the decrease in 15-PGDH
expression induced by pro-inflammatory cytokines, IL-1b and
TNF-a, in villous and chorionic trophoblasts (18). This latter
observation is particularly interesting since IL-1b and TNF-a
are known to induce the expression of COX-2 in many cell
lines (19,20). It appears that there is an interplay between the
expression of COX-2 and that of 15-PGDH.
Increased expression of COX-2 was commonly found in
lung tumors as well as in many other tumors (21,22). Similarly,
enhanced expression of an inducible microsomal PGE (mPGE)
synthase, an enzyme preferentially coupled to COX-2 for
PGE2 synthesis, was also reported in lung tumor (23). On
the contrary, decreased expression of 15-PGDH was recently
described in lung (24,25), bladder (26) and colorectal (27,28)
tumors. It has been well documented that proliferative and
immunosuppressive PGE2 is greatly elevated in tumors
(29). Obviously, increased expression of synthetic enzymes,
COX-2 and mPGE synthase, in lung tumors can contribute to
increased tissue levels of PGE2. Underexpression of the catabolic enzyme 15-PGDH or expression of the defective enzyme
in lung tumors may synergize with the overexpression of synthetic enzymes to amplify the tissue levels of PGE2. Very
recently, we demonstrated that 15-PGDH might behave as a
tumor suppressor in lung cancer (25). This is consistent with
the finding that 15-PGDH is underexpressed in lung tumors.
Whether the expression of COX-2 and 15-PGDH occurs in the
same cells or in different type of cells resulting in elevated
tumor levels of PGE2 remains to be determined. Utilizing
human lung adenocarcinoma A549 cells as a model system,
we describe in this report that both enzymes can be expressed
in the same cells and are reciprocally regulated. Increased
expression of COX-2 is inversely related to the expression
of 15-PGDH and vice versa. Furthermore, we show that
enhanced expression of 15-PGDH may lead to decreased accumulation of PGE2, suggesting that the gene delivery of
15-PGDH into tumors may be valuable for gene therapy.
Materials and methods
Materials
Human non-small cell lung carcinoma (NSCLC) cell line A549 (adenocarcinoma) and AD293 cell line were obtained from the American Type Culture
Collection. The plasmids encoding 15-PGDH cDNA and its mutant cDNA
(Y151F) were obtained as reported previously (30,31). The pcDNA3 expression vector was from Invitrogen. AdEasyTM XL Adenoviral Vector System
was purchased from Stratagene Co. MessageMuterTM shRNAi Production
Kit and Fast-LinkTM DNA Ligation Kit were obtained from Epicentre. Taq
DNA polymerase, all restriction endonucleases, geneticin selective antibiotic
(G418) and heat-inactivated fetal bovine serum (FBS) were from Gibco BRL.
QIAprep Spin Plasmid Miniprep Kit, QIAquick PCR Purification Kit and QIA
Quick Gel Extraction Kit were from QIAGEN. Gentamicin, lipofectamine
2000 transfection reagent and superscript one-step RT–PCR system were
supplied by Life Technologies. Sodium dodecyl sulfate (SDS), dithiothreitol
(DTT), leupeptin, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride
(PMSF), phorbol 12-myristate 13-acetate (PMA) and RPMI-1640 were
obtained from Sigma Chemical Co. Polyvinylidene fluoride (PVDF) membrane was obtained from the Millipore Corp. Electro-chemiluminescence
(ECL+) plus Western Blotting Detection System RPN 2132 was obtained
from Amersham Pharmacia Biotech. Rabbit antiserum against human placental 15-PGDH was generated as described previously (32). Rabbit antisera
against human COX-1 N-terminal (LLPPLPVLLADPGAPTPV) and COX-2
C-terminal (NASSSRSGLDDINPTVLLK) specific sequences were generated
using glutathione-S-transferase fusion protein as an antigen (15). Rabbit antiserum against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was generated as reported previously from our laboratory (33). Horseradish peroxidase
(HRP) labeled goat anti mouse IgG was supplied by Transduction Laboratories
(Lexington, KY). HRP-labeled goat anti rabbit IgG was from Zymed. PGE2
and SC-236 were supplied by Cayman Chemical Co. DuP697 was a gift of
du Pont Co. PGE2 antisera and PGE2–HRP conjugate used for enzyme immunoassay were prepared in-house as described previously (34). 15(S)-[15-3H]
PGE2 was prepared according to a previously published procedure (35).
IL-1b and TNF-a were supplied by PeproTech. Other reagents were obtained
from the best commercial sources.
Cell culture
Cells were maintained in RPMI-1640 medium (A549 cells) or DMEM medium
(AD293 cells) supplemented with 10% fetal calf serum (FBS) and 1 mg/100 ml
gentamicin in a humidified atmosphere containing 5% CO2 at 37 C. Cells were
plated in 12-well plate (2 ml per well) at about 105 cells per well in duplicate
and grown for 24 h before the cells were starved for 24 h in a medium
containing 0.1% FBS. The cells were treated with the stimulant or transfected
with the plasmid or infected with the adenovirus, as indicated in the figure
legends.
Recombinant adenovirus
The wild-type COX-2 cDNA, 15-PGDH cDNA or mutant 15-PGDH (Y151F)
cDNA was cloned into an adenoviral shuttle vector (pshuttle-IRES-hrGFP1) in
the AdEasyTM XL Adenoviral Vector System. The preparation of recombinant
adenovirus wild-type 15-PGDH (Ad-w) and mutant 15-PGDH (Ad-m) or the
adenovirus-shuttle (Ad-s) was performed according to the manufacturer’s
instructions. Recombinant adenovirus-COX-1 was kindly supplied by
Dr Kenneth Wu of the University of Texas Health Science Center at Houston
(36). Adenoviruses were subsequently expanded by sequential rounds of infection on AD293 cells and purified by the CsCl gradient method. Viral titers were
estimated by using two different methods, that is, optical density measurement
and tissue culture infectious dose 50 (TCID50) method. Titration was always
run in duplicate, to assure that equal amounts of recombinant and control
adenovirus were used in all experiments. The expression of reporter gene,
green fluorescent protein in the adenoviral shuttle vector, was also used as a
reference to control the amount of different viruses used in all experiments.
Viral infection
The cells were infected with various adenovirus vectors encoding COX-1,
COX-2, 15-PGDH and mutant 15-PGDH or no insert at a multiplicity of
infection (MOI) of 1000–2000 viral particles per cell. After infection for
2–3 days, cells were harvested to carry out experiments.
Stable expression of the wild-type 15-PGDH
Human 15-PGDH cDNA was cloned into the mammalian expression vector
pcDNA3 at BamHI and XhoI sites. The insertion was confirmed by DNA
sequencing. To create cell lines stably expressing the wild-type 15-PGDH,
pcDNA3 expression vector containing the cDNA of the wild-type 15-PGDH
was transfected into A549 cells using lipofectamine 2000 transfection reagent
according to the manufacturer’s directions. To isolate permanent transfectants,
G418-resistant cells were selected in complete culture medium containing
1 mg/ml G418 as described previously (37). Expression of the wild-type
15-PGDH was monitored by the western blotting analysis and the activity
assay of the enzyme.
15-PGDH assay
Cells were sonicated in 50 mM Tris–HCl, pH 8.0, containing 0.1 mM DTT for
2 · 10 s The crude extract was used for 15-PGDH assay. 15-PGDH activity
was routinely determined by measuring the transfer of tritium from 15(S)[15-3H]-PGE2 to glutamate by coupling 15-PGDH with glutamate dehydrogenase as described previously (35).
PGE2 enzyme immunoassay
PGE2 was analyzed by enzyme immunoassay using PGE2–HRP conjugate as
an enzyme label as described previously (34).
Silencing RNAs (siRNAs)
The target sequences for the COX-1 and COX-2-siRNA have been described
earlier by Denkert et al. (38). The target sequence for the COX-2-siRNA was
bases 291–313 (50 -AACTGCTCAACACCGGAATTTTT-30 ). The target
sequence for the COX-1-siRNA was bases 1533–1555 (50 AAGTGCCATCCAAACTCTATCTT-30 ). siRNA against COX-1 and COX-2
were constructed as described by Elbashir et al. (39) and synthesized using
MessageMuterTM shRNAi Production Kit according to the manufacturer’s
instruction. The target sequence for the 15-PGDH-siRNA was bases
2171
M.Tong, Y.Ding and H.-H.Tai
517–537 (50 -AACAGTGGTGTGAGACTGAAT-30 ). 15-PGDH-siRNA and
control siRNA were kindly supplied by Dr Sanford Markowitz of the Case
Western Reserve University. The oligo for control siRNA against luciferase
was provided in the kit. A549 cells were transfected with siRNA using lipofectamine 2000 reagent.
Immunoblotting
To determine the expression of various proteins in the lung cancer cells following 15-PGDH overexpression, western blot analysis was performed as
described previously (15). Briefly, cells were harvested by trypsinization,
washed and lysed in lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM
NaCl, 2 mM EDTA, 1 mM DTT, 0.1% SDS, 1% Nonidet P-40, 1 mg/ml
leupeptin, 1 mg/ml soybean trypsin inhibitor and 0.5 mM PMSF) for half
an hour on ice. Approximately 50–150 mg of protein extracts were then loaded
on a 12% polyacrylamide gel. Next, the separated proteins were electrophoretically blotted from gel onto PVDF membrane and then blocked with a
blocking buffer (5% non-fat dry milk in 1· TBST, that is, 20 mM Tris–
HCl, pH 7.6, containing 0.8% NaCl and 0.1% Tween-20) at room temperature
for 1 h. The membranes were incubated with the primary antibodies in blocking buffer, followed by incubation with HRP-labeled secondary antibodies.
Bands were visualized using ECL western blotting detection system.
RT–PCR
Relative quantitative RT–PCR was performed on RNA isolated from
A549 cells infected without or with adenoviral vector carrying wild-type or
mutant 15-PGDH cDNA at MOI of 1000 viral particles per cell. A549 cells
were infected for 24 h, changed to the medium with 0.1% FBS for 24 h and
then stimulated with 0.5 ng/ml IL-1b for 8 h. Total RNA was isolated from
infected A549 cells by using Tri-Reagent. Reverse transcription was accomplished with reverse transcriptase and a random hexamer according to the
manufacturer’s protocol. COX-2 mRNA levels were determined by
PCR using Taq DNA polymerase and COX-2 sequence-specific oligonucleotides (50 -CCGGAATTCATGCTCGCCCGCGCCCTGCTGC-30 and
50 -CCGCTCGAGCTACAGTTCAGTCGAACGTT-30 ). The PCR products
were separated by gel electrophoresis on 1% agarose and visualized with
ethidium bromide staining.
Statistical analysis
Each enzyme sample was performed in duplicate. The data were expressed as
the mean ± SE. Statistical significance was assessed by Student’s t-test using a
P-value of <0.05. Each figure is a representative of two to four replications.
Results
Stimulation of A549 cells with IL-1b indicated a time- and
dose-dependent increase in COX-2 activity and expression, as
shown in Figure 1. COX-2 activity was assayed by the release
of PGE2 into the medium, while COX-2 expression was
determined by the immunoreactivity of COX-2 in the cells.
The release of PGE2 is an indication of COX-2 activity since
arachidonic acid, which is induced to release from membrane
phospholipids by IL-1b, is oxygenated by COX-2 to PGH2,
which is in turn isomerized to PGE2 by mPGE synthase. The
level of PGE2 began to increase at 4 h following IL-1b
stimulation (Figure 1A). The increase continued to persist
even after 24 h of stimulation. This could be due to COX-2
expression appearing to reach plateau after 6 h of stimulation
and remaining elevated at 24 h, and 15-PGDH expression was
still downregulated (Figure 2A). Similarly, the level of COX-2
expression was increased as the concentration of IL-1b was
elevated (Figure 1B). Near-maximal stimulation was achieved
at 1 ng/ml as shown by the western blot and by the release of
PGE2 in the medium. However, when these cells were assayed
for 15-PGDH activity and protein expression, there were timeand dose-dependent decrease in 15-PGDH activity and
immunoreactivity, as shown in Figure 2. The 15-PGDH
activity appeared to level off after 6 h of IL-1b stimulation
(Figure 2A). As the expression of COX-2 began to appear,
the expression of 15-PGDH started to decrease. This observation appeared to be true for both time course and
2172
Fig. 1. Expression and activity of COX-2 in IL-1b-stimulated A549 cells.
After starvation with 0.1% FBS for 24 h, A549 cells were treated with
0.5 ng/ml IL-1b (filled circle) or vehicle (filled square) for the indicated
time (A), and the indicated concentrations of IL-1b for 24 h (B) to induce
the expression of COX-2 and endogenous synthesis of PGE2. The PGE2 in
the medium was assayed by enzyme immunoassay as described in the
‘Materials and methods’ section. A total of 100 mg protein from cell
extracts was resolved by electrophoresis on a 12% polyacrylamide gel. The
protein level of COX-2 was assessed by western blot analysis. GAPDH
was used as a loading control.
dose-dependent studies (Figure 2B). Similarly, other cytokines
such as TNF-a, which induced COX-2 expression in A549
cells, also decreased 15-PGDH expression. PMA, which also
induced COX-2 expression, however, did not appear to downregulate 15-PGDH expression. This is due to the fact that PMA
alone also induced 15-PGDH expression to some extent, as
shown in Figure 3. When A549 cells were stimulated with
increasing concentrations of IL-1b in the absence and presence
of PMA, the level of 15-PGDH expression was decreased as
the level of COX-2 expression was increased as shown by the
western blot. This is particularly evident when PMA and
increasing concentrations of IL-1b were combined as shown
Reciprocal regulation of COX-2 and 15-PGDH
Fig. 2. Expression and activity of 15-PGDH in IL-1b-stimulated A549
cells. After starvation with 0.1% FBS for 24 h, A549 cells were treated
with 0.5 ng/ml IL-1b for the indicated time (A) and the indicated
concentrations of IL-1b for 24 h (B) to suppress the expression and activity
of 15-PGDH. The assay for 15-PGDH activity was described in the
‘Materials and methods’ section. A total of 100 mg protein from cell
extracts was resolved by electrophoresis on a 12% polyacrylamide gel. The
protein level of 15-PGDH was assessed by western blot analysis. GAPDH
was used as a loading control.
in Figure 3A. A similar observation was made when PMA and
increasing concentrations of TNF-a were combined as shown
in Figure 3B. Interestingly, this finding was also found to be
true when IL-1b and TNF-a were combined as shown in
Figure 3C. Enzyme activity assay was also made to confirm
that these combinations led to a fall in 15-PGDH activity. It
appears that any combinations of PMA, IL-1b and TNF-a
stimulated COX-2 expression significantly higher than any
agent alone. These combinations also decreased 15-PGDH
expression much more than by a single agent alone. The
expression of COX-2 was found to correlate with the expression of 15-PGDH in a reciprocal manner. In order to verify that
it is the increased expression of COX-2 that is responsible for
Fig. 3. Expression of COX-2 and 15-PGDH induced by IL-1b, TNF-a or
PMA in A549 cells either alone or in combinations. A549 cells were
prepared and treated in the same manner as described in Figure 1.
(A) A549 cells were stimulated with the indicated concentrations of IL-1b
either alone or in combination with PMA at 20 nM for 24 h. A total of
100 mg protein from cell extracts was resolved by electrophoresis on a 12%
polyacrylamide gel. The expression of COX-2 and 15-PGDH was determined by western blot analysis. (B) A549 cells were stimulated with the
indicated concentrations of TNF-a either alone or in combination with
PMA at 20 nM for 24 h. A total of 100 mg protein from cell extracts was
resolved by electrophoresis on a 12% polyacrylamide gel. The expression
of COX-2 and 15-PGDH was determined by western blot analysis.
(C) A549 cells were stimulated with PMA (20 nM), IL-1b (0.5 ng/ml) or
TNF-a (10 ng/ml) either alone or in various combinations for 24 h. A total
of 100 mg protein from cell extracts was resolved by electrophoresis on a
12% polyacrylamide gel. The expression of COX-2 and 15-PGDH and also
the activity of 15-PGDH were determined as described in the ‘Materials
and methods’ section. Each enzyme sample was performed in duplicate.
The data were expressed as the mean ± SD. GAPDH was used as a loading
control.
the decreased expression of 15-PGDH, the expression of
COX-2 induced by IL-1b was specifically suppressed by
COX-2-siRNA. Figure 4 shows that inhibition of
IL-1b-induced expression of COX-2 by COX-2-siRNA resulted in the enhancement of the expression and the activity of
15-PGDH. This enhancement was not observed by
2173
M.Tong, Y.Ding and H.-H.Tai
15-P GD H A ctivity
R elative to C ontrol (fold)
A
1
0.5
0
Ad-s
Ad-COX2
COX-2
PGDH
GAPDH
Fig. 4. Inhibition of IL-1b-induced COX-1 and COX-2 expression by
COX-1-siRNA and COX-2-siRNA, respectively, on the expression of
15-PGDH in A549 cells. A549 cells were transfected with 0.5 mg/ml
COX-1-siRNA, COX-2-siRNA or control siRNA for 6 h before the
medium was changed to 0.1% FBS and the incubation continued for
another 24 h before the cells were stimulated with IL-1b (0.5 ng/ml) for
24 h. Cells were lysed for 15-PGDH activity assay, and a total of 100 mg
protein from cell extracts was prepared for western blot analysis of
15-PGDH, COX-1 and COX-2 as described in the ‘Materials and methods’
section. Each enzyme sample was performed in duplicate. The data were
expressed as the mean ± SD. GAPDH was used as a loading control.
15-PGDH Activity
Relative to Control (fold)
B
1
0.5
0
Ad-s
COX-1-siRNA and control siRNA. On the contrary, overexpression of COX-2 by adenovirus-mediated approach resulted
in a significant decrease in the expression of 15-PGDH as
predicted (Figure 5A). However, overexpression of
COX-1 appeared to show limited effect on the expression
of 15-PGDH (Figure 5B). In order to provide evidence for
the endogenous expression of 15-PGDH that may affect the
expression of COX-2 induced by IL-1b, the expression of
15-PGDH was specifically suppressed by 15-PGDH-siRNA.
Figure 6 shows that suppression of the expression of 15-PGDH
led to a further increase in the IL-1b-induced expression of
COX-2 but not that of COX-1. The reciprocal regulation of
COX-2 and 15-PGDH was further illustrated by a separate
study in which five stable clones of A549 cells expressing
different levels of 15-PGDH were stimulated with IL-1b.
Figure 7A shows that IL-1b stimulated COX-2 expression
in a manner that was dependent on the endogenous levels
of 15-PGDH. The higher the endogenous level of 15-PGDH
was found, the lower the IL-1b-induced COX-2 expression
was observed. It seems that the endogenous level of
15-PGDH activity and expression inhibited the IL-1b-induced
expression of COX-2. Again, the expression of COX-2 was
increased by IL-1b, and the expression of endogenous
15-PGDH was decreased as shown by the western blot and
the activity assay. When stable A549 cells devoid of 15-PGDH
2174
Ad-COX1
COX-1
PGDH
GAPDH
Fig. 5. Effect of the overexpression of COX-2 and COX-1 on the
expression of 15-PGDH in A549 cells. (A) A549 cells infected without
(control) or with adenovirus alone (Ad-s) or with the COX-2 cDNA insert
(Ad-COX-2) at MOI of 2000 viral particles per cell for 16 h were
changed to the medium without FBS for 72 h, and then harvested for
15-PGDH activity assay, and a total of 70 mg protein from cell extracts
was prepared for western blot analysis of COX-2 and 15-PGDH as
described in the ‘Materials and methods’ section. (B) A549 cells infected
with adenoviral vector alone (Ad-s) or with the COX-1 cDNA insert
(Ad-COX-1) at MOI of 2000 viral particles per cell for 16 h were
changed to the medium without FBS for 72 h, and then harvested for
15-PGDH activity assay, and a total of 50 mg protein from cell extracts
was prepared for western blot analysis of COX-1 and 15-PGDH, and as
described in the ‘Materials and methods’ section. Each enzyme sample
was performed in duplicate. The data were expressed as the mean ± SD.
GAPDH was used as a loading control.
were obtained by transfection with plasmid expression vector
encoding anti-sense 15-PGDH cDNA and further selection by
G418, the level of COX-2 expression induced by IL-1b was
significantly increased, as shown in Figure 7B. Similarly,
when stable A549 cells expressed more 15-PGDH, the
Reciprocal regulation of COX-2 and 15-PGDH
A
PGDH
1
GAPDH
L-1β
0.5
5# 12# 14# 17# 21# 5# 12# 14# 17# 21#
- + +
+ + +
25
0
Control
siRNA
PGDH
siRNA
PGDH
15-PGDH Activity
Relative to Control (fold)
15-PGDH Activity
Relative to Control (fold)
COX-2
20
15
10
5
0
COX-2
IL-1β
COX-1
5# 12# 14# 17# 21# 5# 12# 14# 17# 21#
- +
+ + + +
B
A549
GAPDH
Vector
Anti-sense
PGDH
Sense
PGDH
COX-2
Fig. 6. Inhibition of 15-PGDH expression by 15-PGDH-siRNA on the
expression of COX-1 and COX-2 in A549 cells. A549 cells were
transfected without or with 20–60 pmol/l 15-PGDH-siRNA or control
siRNA for 6 h before the medium was changed to 0.1% FBS and the
incubation continued for another 24 h before the cells were stimulated with
0.5 ng/ml IL-1b for 4 h. Cells were lysed for 15-PGDH activity assay, and
a total of 100 mg protein from cell extracts was prepared for western blot
analysis of 15-PGDH, COX-1 and COX-2 as described in the ‘Materials
and methods’ section. Each enzyme sample was performed in duplicate.
The data were expressed as the mean ± SD. GAPDH was used as a loading
control.
level of COX-2 expression induced by IL-1b was significantly curtailed. This finding was further supported by
overexpression studies in which 15-PGDH was induced to
express in an increasing manner by infection with an increasing dose of adenovirus carrying 15-PGDH cDNA. Figure 8A
shows that an increasing expression of 15-PGDH in A549 cells
resulted in a decreasing expression of COX-2 induced by IL1b as expected. However, the cells infected with adenovirus
alone also exhibited decreasing expression of COX-2 induced
by IL-1b albeit to a lesser degree, indicating that infection with
adenovirus interfered with the expression of COX-2 to some
extent. Inhibition of IL-1b-induced COX-2 expression by
15-PGDH was apparently not due to its catalytic activity
since infection with adenovirus carrying mutant 15-PGDH
also inhibited IL-1b-induced COX-2 expression, as shown
by western blot (Figure 8B) and by RT–PCR (Figure 8C).
The mechanism of reciprocal regulation of COX-2 and
15-PGDH expression was further examined. It was suspected
that COX-2-derived products might mediate the inhibition of
15-PGDH expression induced by IL-1b. Figure 9 indicates that
15-PGDH expression was neither affected by the addition of
COX-2-derived products nor their mimetics such as PGE2 and
TXA2 analog, I-BOP, nor by inhibitors of COX-2 (SC-236 and
DuP 697). Attenuation of 15-PGDH expression induced by
PGDH
GAPDH
Fig. 7. Correlation of the IL-1b-stimulated COX-2 expression and the
endogenous levels of 15-PGDH in stable subclones of A549 cells. (A) Each
of the five stable subclones of A549 cells expressing different levels of
15-PGDH was starved with 0.1% FBS for 24 h before the cells were
stimulated with or without IL-1b (0.5 ng/ml) for 6 h. Enzyme activity
assay of 15-PGDH was determined in five stable subclones of A549 cells,
and a total of 100 mg protein from each cell extracts was prepared for
western blot analysis of COX-2 and 15-PGDH as described in the
‘Materials and methods’ section. (B) Stable subclones of A549 cells
derived from cells transfected with either an expression vector alone or
with an expression vector encoding anti-sense 15-PGDH cDNA or with an
expression vector encoding wild-type 15-PGDH cDNA were starved with
0.1% FBS for 24 h before the subclones were each stimulated with IL-1b
(0.5 ng/ml) for 6 h. A total of 100 mg protein from cell extracts was
prepared for western blot analysis of COX-2 and 15-PGDH expression as
described in the ‘Materials and methods’ section. GAPDH was used as a
loading control.
IL-1b was not reversed by any of the COX-2 inhibitors, indicating that the suppression was not due to the COX-2 activity
and its derived products. It appears that the attenuation of
15-PGDH expression induced by IL-1b was due to the
COX-2 protein expressed. The molecular mechanism of
attenuation of 15-PGDH expression by COX-2 awaits further
investigation.
Discussion
A549 cells were shown to have the capacity of expressing both
COX-2 and 15-PGDH. Expression of COX-2 was rarely seen
in non-stimulated state. However, constitutive expression of
2175
M.Tong, Y.Ding and H.-H.Tai
A
IL-1β
Ad-s
-
+
-
+
500
+
1000
+
1500
+
2000
MOI
PGDH
COX-2
GAPDH
IL-1β
Ad-w
-
+
-
+
500
+
1000
+
1500
+
2000
MOI
PGDH
COX-2
GAPDH
B
IL-1 β
Adenovirus
-
+
-
+
Ad-s
+
+
Ad-m Ad-w
PGDH
COX-2
GAPDH
C
IL-1β
Adenovirus
-
+
-
+
+
+
Ad-s Ad-m Ad-w
COX-2
β-ACTIN
Fig. 8. Overexpression of 15-PGDH in A549 cells resulted in a decreasing
expression of COX-2 induced by IL-1b. A549 cells infected without or
with adenoviral vector (Ad-s), adenoviral vectors carrying the wild-type
15-PGDH cDNA (Ad-w) or mutant 15-PGDH (Y151F) cDNA (Ad-m) at
the indicated MOI for 24 h were changed to the medium with 0.1% FBS
for 24 h and then treated with 0.5 ng/ml IL-1b for 8 h. (A) A total of
50 mg protein from cell extracts was resolved by electrophoresis on a 12%
polyacrylamide gel. Then western blot analysis of COX-2 and 15-PGDH in
A549 cells infected with an increasing dose of adenovirus either alone or
with wild-type 15-PGDH cDNA insert was carried out as described in the
‘Materials and methods’ section. (B) A total of 80 mg protein from cell
extracts was resolved by electrophoresis on a 12% polyacrylamide gel.
Then western blot analysis of COX-2 and 15-PGDH in A549 cells infected
with different types of recombinant adenoviruses at the MOI of 1000 was
carried out as described in the ‘Materials and methods’ section. GAPDH
was used as a loading control. (C) The levels of COX-2 mRNA in (B)
were determined by RT–PCR assay as described in the ‘Materials and
methods’ section. b-actin mRNA was used as a loading control.
15-PGDH was clearly observed under resting conditions.
Increased expression of COX-2 induced by IL-1b and TNFa was found to result in a diminished expression of 15-PGDH.
PMA, which also induced COX-2 expression, did not appear to
downregulate 15-PGDH. This is primarily due to the fact that
PMA is also a stimulator of 15-PGDH expression in A549
cells, although it is a relatively weak one as shown in this
study. Previously, PMA was shown to induce 15-PGDH
expression significantly in HL-60, HEL and U-937 cells
(11–13). However, when PMA was combined with IL-1b or
TNF-a, synergistic induction of COX-2 expression and diminished 15-PGDH expression were observed. The ability of PMA
to induce 15-PGDH appeared to be overpowered by the greatly
enhanced expression of COX-2. It is interesting to note that as
2176
Fig. 9. Effect of COX-2 inhibitors and COX-2-derived products on IL-1binduced attenuation of 15-PGDH expression in A549 cells. A549 cells were
treated with IL-1b (0.5 ng/ml) or without IL-1b as shown in Figure 1 for
24 h in the absence and presence of COX-2 inhibitors, SC-236 and
DuP697, at 5 mM each. Cells were also treated with PGE2 (3 mM) or
thromboxane receptor agonist, I-BOP (0.1 mM). Cells were then lysed and
prepared for 15-PGDH assay as described in the ‘Materials and methods’
section. Each enzyme sample was performed in duplicate. The data were
expressed as the mean ± SD.
the expression of COX-2 induced by IL-1b reached a plateau,
the attenuation of the expression of 15-PGDH was also slowly
leveled off. However, when the release of PGE2 was analyzed
as a function of time, the level continued to increase even at
24 h following the stimulation. This indicates that the metabolism of PGE2 by 15-PGDH was overtaken by the synthesis
of PGE2 by COX-2 and mPGES, leading to a continued accumulation of PGE2. When the release of PGE2 was analyzed in
response to an increasing dose of IL-1b, the level also continued to increase and began to reach plateau around 1.0 ng/ml
of IL-1b, where 15-PGDH activity also became leveled off. It
appears that the amount of COX-2 expression determines the
level of attenuation of 15-PGDH expression. On the contrary,
when the level of 15-PGDH expression was increased
by transfection with pcDNA3 or adenovirus encoding
15-PGDH, the level of COX-2 expression induced by
IL-1b was decreased in a manner dependent on the level of
15-PGDH expression. It seems that increased expression of
15-PGDH may impede the expression of COX-2 induced by
IL-1b. This is further illustrated by using stable clones of A549
cells expressing different levels of 15-PGDH to induce the
expression of COX-2 by IL-1b. The lower the endogenous
level of 15-PGDH expression was present in A549 cells,
the higher the level of induced COX-2 expression was
found. This was further supported by the observation that
suppression of 15-PGDH expression by 15-PGDH-siRNA
led to an increase in COX-2 expression induced by IL-1b.
In fact, when the level of 15-PGDH expression was reduced
to a minimum by transfection with pcDNA3 encoding antisense 15-PGDH, the level of COX-2 expression was enhanced
to a maximum following the stimulation of IL-1b. It is interesting to note that increased expression of an inactive
15-PGDH mutant also impeded the expression of COX-2
induced by IL-1b just as effective as that of wild-type
15-PGDH, indicating that inhibition of COX-2 expression
was not dependent on the catalytic activity of the enzyme.
Other mechanisms appear to be operative.
Molecular mechanism of the regulation of the COX-2
expression by the 15-PGDH protein remains to be determined.
Reciprocal regulation of COX-2 and 15-PGDH
Regulation of COX-2 expression at the transcriptional and
post-transcriptional levels was intensively studied in the
past decade. Transcriptional mechanism involving various
transcriptional factors interacting with different response elements in the promoter region to induce COX-2 expression has
been presented (40). An equally significant mechanism that
regulates the stability of COX-2 mRNA and hence the level of
COX-2 expression has been recognized (5). This mechanism
is related to the existence of several AU-rich sequences in the
30 -UTR of COX-2 mRNA. These sequences have been shown
to be important for enhancing message translation as well as
for translational silencing (5). A number of RNA-binding proteins have been identified to bind to these sequences. They are
either to stabilize COX-2 mRNA and enhance COX-2 expression such as HuR (41), or to destabilize mRNA and curtail
COX-2 expression such as TIA-1 (7) and tristertraprolin (42).
Additionally, there are also proteins that bind to these
sequences and stabilize COX-2 mRNA but, paradoxically,
inhibit its translation such as CUG-binding protein 2 (6).
Although the potential binding of 15-PGDH to COX-2
30 -UTR remains to be determined, binding of a few NAD+dependent dehydrogenases to RNA has been demonstrated
(43). Nagy et al. (44) showed that NAD+-linked GAPDH
bound AU-rich sequences primarily through its N-terminal
first mononucleotide-binding bgb fold. 15-PGDH is also
known to contain this mononucleotide-binding domain in its
N-terminal region (10). It is very likely that increased expression of 15-PGDH may bind to the COX-2 30 -UTR through its
AU-rich sequences and destabilize mRNA or block translation.
Further verification of this hypothesis is under way in our
laboratory.
Although cytokines or cytokine plus PMA are able to
increase the expression of COX-2 and decrease the expression
of 15-PGDH, it is not clear if the increase of COX-2 expression
leads to the suppression of 15-PGDH expression since COX-2
is an immediate early gene while 15-PGDH is not (40). We
examined this issue from two perspectives. It is possible that
the COX-2-derived products, such as PGE2 and TXA2, may
mediate the suppression of 15-PGDH expression. However,
either PGE2 or stable TXA2 analog, I-BOP, did not induce the
attenuation of the 15-PGDH expression. Addition of COX-2
inhibitors, SC-236 and DuP 697, during the stimulation by
IL-1b did not cause the reversal of 15-PGDH expression.
Therefore, it is not the COX-2 activity or the COX-2-derived
products that are responsible for the attenuation of 15-PGDH
expression induced by IL-1b. The other alternative is that the
expression of COX-2 itself is responsible for the attenuation of
15-PGDH expression. To provide evidence for this possibility,
we tried to suppress IL-1b-induced COX-2 expression by
COX-2-siRNA. At the same time, we employed COX-1siRNA and control siRNA to examine the specificity and to
serve as a control. The reversal of attenuation of 15-PGDH
expression induced by IL-1b was only seen with COX-2siRNA but not with COX-1-siRNA, indicating that it
was the expression of COX-2 that caused the attenuation of
15-PGDH expression. This was further supported by the finding that adenovirus-mediated overexpression of COX-2 but
not that of COX-1 attenuated the expression of 15-PGDH.
The finding that the expression of COX-2 protein and not
the activity is responsible for attenuation of 15-PGDH expression is reminiscent of the situation in which it is the expression
of 15-PGDH protein and not the activity that inhibits COX-2
expression, as illustrated in Figure 8.
Molecular mechanism of the regulation of the 15-PGDH
expression by the COX-2 protein remains to be elucidated.
Regulation of 15-PGDH expression at the transcriptional and
post-transcriptional levels was much less understood. There
has been little report on the regulation of 15-PGDH expression
at the post-transcriptional level. Regulation at the transcriptional level is also limited in publications. Using 15-PGDH
promoter-luciferase construct, we first demonstrated that the
promoter activity was stimulated by PMA (45). Greenland
et al. (46) further suggested that the PMA acted through
AP-1 in Jurkat leukemia T cells. Other transcriptional factors
such as Ets-1, Ets-2, PEA-3 and CREB were implicated in
regulating promoter activity in myometrial smooth muscle
cells. Lennon et al. (47) showed that 8-bromo-cAMP, which
stimulates COX-2 expression and presumably acts through
CREB, diminished the expression and activity of 15-PGDH
in human trophoblast cells. Backlund et al. (28) demonstrated
that epidermal growth factor (EGF), which stimulated COX-2
expression, also decreased 15-PGDH expression and activity
significantly in human colon cancer cells, HCT-15 and HCA-7.
How increased expression of COX-2 induced by IL-1b or EGF
inhibited the expression of 15-PGDH at the promoter level is
not clear. Apparently, it is not due to COX-2-derived product
such as PGE2 and TXA2 since PGE2 and TXA2 mimetic did
not attenuate 15-PGDH expression and COX-2 inhibitors did
not abolish IL-1b-induced attenuation of 15-PGDH expression
as shown in this study. However, it is still possible that the
expressed COX-2 protein and not the activity may attenuate
15-PGDH expression, although the mechanism of attenuation
remains elusive.
In summary, we have found that expressions of COX-2 and
15-PGDH are regulated reciprocally. Increased expression of
COX-2 attenuates the expression of 15-PGDH and enhanced
expression of 15-PGDH also decreases the expression of
COX-2. Since the expression of COX-2 is generally dormant
and the expression of 15-PGDH is constitutive, induced
expression of COX-2 by growth-promoting signals will downregulate the expression of 15-PGDH to ensure that the level of
PGE2 is elevated and the cells are stimulated to proliferate.
When cells are stimulated to grow to a desired state, the
expression of 15-PGDH is resumed and the expression of
COX-2 is downregulated. The reciprocal regulation of
COX-2 and 15-PGDH may provide a plausible biochemical
mechanism for the control of cell growth and metastasis. Furthermore, our finding that COX-2 and 15-PGDH are regulated
reciprocally also provides a logical explanation for lung tumor
and possibly other tumors overexpressing COX-2 and underexpressing 15-PGDH.
Acknowledgements
We are indebted to Dr Kenneth Wu of the University of Texas Health Science
Center at Houston for the gift of adenoviral vector encoding COX-1 and to
Dr Sanford Markowitz of the Case Western Reserve University for the gift of
15-PGDH-siRNA. This work was supported in part by grants from the NIH
(HL-46296) and the Kentucky Lung Cancer Research Program.
Conflict of Interest Statement: None declared.
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Received November 15, 2005; revised March 21, 2006;
accepted April 13, 2006
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