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Carcinogenesis Advance Access originally published online on April 22, 2006
Carcinogenesis 2006 27(11):2170-2179; doi:10.1093/carcin/bgl053
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Reciprocal regulation of cyclooxygenase-2 and 15-hydroxyprostaglandin dehydrogenase expression in A549 human lung adenocarcinoma cells

Min Tong{dagger}, Yunfei Ding{dagger} 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: htail{at}uky.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
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) 1ß, tumor necrosis factor-{alpha} (TNF-{alpha}) 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-1ß or TNF-{alpha} induced synergistically the expression of COX-2. Interestingly, cytokines and cytokine plus PMA-induced 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 adenovirus-mediated approach led to a decrease in 15-PGDH expression, indicating the specificity of COX-2. Furthermore, downregulation of the IL-1ß-induced expression of COX-2 by silencing RNA (siRNA) approach resulted in an increase in the expression of 15-PGDH by COX-2-siRNA but not by COX-1-siRNA, indicating that it was indeed the expression of COX-2 attenuating the expression of 15-PGDH. The IL-1ß-induced reduction of the expression of 15-PGDH was shown not to be mediated by COX-2-derived 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-1ß-induced expression of COX-2. On the contrary, downregulation of 15-PGDH expression by 15-PGDH-siRNA or 15-PGDH-antisense approach resulted in an increase in IL-1ß-induced expression of COX-2 but not that of COX-1. In fact, it was further observed that A549 clones expressing different degrees of 15-PGDH showed also different levels of COX-2 expression after IL-1ß induction. The levels of IL-1ß-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.

Abbreviations: 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; COX, cyclooxygenase; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate 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


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
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) 1ß and tumor necrosis factor-{alpha} (TNF-{alpha}), 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 3'-untranslated region (3'-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 anti-inflammatory steroids in HEL cells (12) and in human lung adenocarcinoma A549 cells (14), by androgens and other steroid hormones in prostate cancer cells (15), by 1,25-dihydroxyvitamin 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 anti-inflammatory 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-1ß and TNF-{alpha}, in villous and chorionic trophoblasts (18). This latter observation is particularly interesting since IL-1ß and TNF-{alpha} 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
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
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-1ß and TNF-{alpha} 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 x 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 (5'-AACTGCTCAACACCGGAATTTTT-3'). The target sequence for the COX-1-siRNA was bases 1533–1555 (5'-AAGTGCCATCCAAACTCTATCTT-3'). 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 517–537 (5'-AACAGTGGTGTGAGACTGAAT-3'). 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 µg/ml leupeptin, 1 µg/ml soybean trypsin inhibitor and 0.5 mM PMSF) for half an hour on ice. Approximately 50–150 µg 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 1x 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-1ß 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 (5'-CCGGAATTCATGCTCGCCCGCGCCCTGCTGC-3' and 5'-CCGCTCGAGCTACAGTTCAGTCGAACGTT-3'). 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
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Stimulation of A549 cells with IL-1ß 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-1ß, 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-1ßstimulation (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-1ß 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 time- and 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-1ß 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 dose-dependent studies (Figure 2B). Similarly, other cytokines such as TNF-{alpha}, 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-1ß 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-1ß were combined as shown in Figure 3A. A similar observation was made when PMA and increasing concentrations of TNF-{alpha} were combined as shown in Figure 3B. Interestingly, this finding was also found to be true when IL-1ß and TNF-{alpha} 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-1ß and TNF-{alpha} 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 the decreased expression of 15-PGDH, the expression of COX-2 induced by IL-1ß was specifically suppressed by COX-2-siRNA. Figure 4 shows that inhibition of IL-1ß-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 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-1ß, 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-1ß-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-1ß. Figure 7A shows that IL-1ß 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-1ß-induced COX-2 expression was observed. It seems that the endogenous level of 15-PGDH activity and expression inhibited the IL-1ß-induced expression of COX-2. Again, the expression of COX-2 was increased by IL-1ß, 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 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-1ß was significantly increased, as shown in Figure 7B. Similarly, when stable A549 cells expressed more 15-PGDH, the level of COX-2 expression induced by IL-1ß 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 IL-1ß as expected. However, the cells infected with adenovirus alone also exhibited decreasing expression of COX-2 induced by IL-1ß albeit to a lesser degree, indicating that infection with adenovirus interfered with the expression of COX-2 to some extent. Inhibition of IL-1ß-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-1ß-induced COX-2 expression, as shown by western blot (Figure 8B) and by RT–PCR (Figure 8C).


Figure 1
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  		Fig. 1 Expression and activity of COX-2 in IL-1ß-stimulated A549 cells. After starvation with 0.1% FBS for 24 h, A549 cells were treated with 0.5 ng/ml IL-1ß (filled circle) or vehicle (filled square) for the indicated time (A), and the indicated concentrations of IL-1ß 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 µg 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.

 

Figure 2
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  		Fig. 2 Expression and activity of 15-PGDH in IL-1ß-stimulated A549 cells. After starvation with 0.1% FBS for 24 h, A549 cells were treated with 0.5 ng/ml IL-1ß for the indicated time (A) and the indicated concentrations of IL-1ß 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 µg 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.

 

Figure 3
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  		Fig. 3 Expression of COX-2 and 15-PGDH induced by IL-1ß, TNF-{alpha} 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-1ß either alone or in combination with PMA at 20 nM for 24 h. A total of 100 µg 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-{alpha} either alone or in combination with PMA at 20 nM for 24 h. A total of 100 µg 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-1ß (0.5 ng/ml) or TNF-{alpha} (10 ng/ml) either alone or in various combinations for 24 h. A total of 100 µg 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.

 

Figure 4
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  		Fig. 4 Inhibition of IL-1ß-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 µg/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-1ß (0.5 ng/ml) for 24 h. Cells were lysed for 15-PGDH activity assay, and a total of 100 µg 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.

 

Figure 5
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  		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 µg 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 µg 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.

 

Figure 6
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  		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-1ß for 4 h. Cells were lysed for 15-PGDH activity assay, and a total of 100 µg 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.

 

Figure 7
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  		Fig. 7 Correlation of the IL-1ß-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-1ß (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 µg 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-1ß (0.5 ng/ml) for 6 h. A total of 100 µg 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.

 

Figure 8
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  		Fig. 8 Overexpression of 15-PGDH in A549 cells resulted in a decreasing expression of COX-2 induced by IL-1ß. 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-1ß for 8 h. (A) A total of 50 µg 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 µg 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. ß-actin mRNA was used as a loading control.

 
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-1ß. 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 IL-1ß 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-1ß was due to the COX-2 protein expressed. The molecular mechanism of attenuation of 15-PGDH expression by COX-2 awaits further investigation.


Figure 9
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  		Fig. 9 Effect of COX-2 inhibitors and COX-2-derived products on IL-1ß-induced attenuation of 15-PGDH expression in A549 cells. A549 cells were treated with IL-1ß (0.5 ng/ml) or without IL-1ß as shown in Figure 1 for 24 h in the absence and presence of COX-2 inhibitors, SC-236 and DuP697, at 5 µM each. Cells were also treated with PGE2 (3 µM) or thromboxane receptor agonist, I-BOP (0.1 µM). 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
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 15-PGDH was clearly observed under resting conditions. Increased expression of COX-2 induced by IL-1ß and TNF-{alpha} 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-1ß or TNF-{alpha}, 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 the expression of COX-2 induced by IL-1ß 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-1ß, the level also continued to increase and began to reach plateau around 1.0 ng/ml of IL-1ß, 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-1ß 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-1ß. 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-1ß. 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-1ß. In fact, when the level of 15-PGDH expression was reduced to a minimum by transfection with pcDNA3 encoding anti-sense 15-PGDH, the level of COX-2 expression was enhanced to a maximum following the stimulation of IL-1ß. It is interesting to note that increased expression of an inactive 15-PGDH mutant also impeded the expression of COX-2 induced by IL-1ß 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. 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 3'-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 3'-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 ß{gamma}ß 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 3'-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-1ß 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-1ß. 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-1ß-induced COX-2 expression by COX-2-siRNA. At the same time, we employed COX-1-siRNA and control siRNA to examine the specificity and to serve as a control. The reversal of attenuation of 15-PGDH expression induced by IL-1ß was only seen with COX-2-siRNA 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-1ß 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-1ß-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.


   Footnotes
 
{dagger}The first two authors contributed equally to this work. Back


   Acknowledgments
 
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.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 15, 2005; revised March 21, 2006; accepted April 13, 2006.


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