Ionizing radiation up-regulates cyclooxygenase-2 in I407 cells through p38 mitogen-activated protein kinase
Teresa G. Tessner1,4,
Filipe Muhale1,
Suzanne Schloemann1,
Steven M. Cohn3,
Aubrey R. Morrison2 and
William F. Stenson1
1 Division of Gastroenterology and 2 Department of Medicine and Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, MO 63110, USA and 3 Division of Gastroenterology and Hepatology, University of Virginia, Charlottesville, VA 22904, USA
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Abstract
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The epithelial cell line I407 up-regulates cyclooxygenase-2 (COX-2) mRNA and protein expression following ionizing radiation exposure. Prostaglandin E2 (PGE2) production is concomitantly up-regulated. Irradiation of I407 cells also results in phosphorylation of the p38 mitogen-activated protein kinase and the p38 inhibitor SB203580 abrogates radiation-induced PGE2 synthesis. Wild-type p38
(p38
WT) and dominant-negative p38
(p38
DN) stable-transfectant clones of I407 cells were used to examine the role of the p38 mitogen-activated protein kinase pathway in the events controlling PGE2 synthesis after ionizing radiation. Treatment of p38
WT clones with
-radiation resulted in increased COX-2 protein levels and PGE2 synthesis similar to treated control-transfected cells. In contrast, the p38
DN clones failed to up-regulate COX-2 protein or increase PGE2 synthesis when irradiated. Exogenous arachidonate did not restore PGE2 synthesis by p38
DN cells. Radiation increased COX-2 mRNA stability and the p38 inhibitor SB203580 attenuated COX-2 mRNA stability in irradiated I407 cells. In contrast, irradiation had no effect on transcription from a COX-2 promoter/luciferase reporter plasmid in the presence or absence of SB203580. The data demonstrate a crucial role for p38
in COX-2 expression and PGE2 synthesis in an irradiated transformed epithelial cell line. Furthermore, they indicate that p38 activity is required at a step distal to arachidonate release, most probably COX-2 up-regulation, since exogenous arachidonate did not restore PGE2 synthesis.
Abbreviations: COX, cyclooxygenase; DRB, dichlorobenzimidazole; IL-1ß, interleukin-1ß; MAPK, mitogen-activated protein kinase; p38
DN, dominant-negative p38
; p38
WT, wild-type p38
; PGE2, prostaglandin E2
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Introduction
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Radiation therapy is limited by the susceptibility of normal tissue within the exposed field to radiation damage. Both the bone marrow and gastrointestinal tract are sensitive to radiation injury and thus are often limiting in therapy. Modalities that reduce normal tissue radiosensitivity and/or increase tumor radioresponsiveness are of interest in clinical practice. Radiation injury in the mouse results in up-regulation of cyclooxygenase (COX) and a concomitant increase in prostaglandin E2 (PGE2) synthesis (1). We have shown that the ability of intestinal cells to synthesize PGE2 strongly impacts the survival of intestinal crypt stem cells after radiation: inhibition of prostanoid synthesis by indomethacin after radiation decreases the number of surviving crypts in a PGE2-reversible manner (1); COX-1 knockout mice exhibit decreased numbers of surviving crypts and increased apoptosis after radiation compared with their wild-type littermates (2); and lipopolysaccharide is radioprotective in the mouse intestine via a prostaglandin-mediated mechanism (3). Therefore, prostaglandin synthesis plays a significant role in modulating the response of normal gastrointestinal tissue to radiation.
COX expression is up-regulated in colon cancer and other tumor types (reviewed in 47). COX inhibitors have been shown to increase the radioresponsiveness of many cancer cells, both in vivo and in vitro (811), presumably by inhibiting prostanoid synthesis. A selective COX-2 inhibitor, NS-398, decreases the surviving fraction of irradiated rat intestinal epithelial cells expressing COX-2, RIE-S, but not rat intestinal epithelial cells transfected with an anti-sense COX-2 construct, and increases apoptosis following radiation of RIE-S cells (8). The radiosensitivity of the human glioma cell line U251, which expresses comparable COX-2 with the colon adenocarcinoma HT-29 is enhanced by the selective COX-2 inhibitor, sc-236 (10). Kishi et al. (9) and Milas et al. (11) have also demonstrated enhanced radioresponsiveness of sarcoma cell lines with elevated COX expression using sc-236. They found no adverse effect of sc-236 on jejunal crypt survival at doses of 9.513.5 Gy (9). However, these experiments did not directly determine if the inhibitor increased tumor sensitivity to within tolerable doses for the gut. In the prostate cancer cell line, PC-3, ionizing radiation increases COX-2 expression in a dose-dependent manner and is accompanied by increased PGE2 synthesis (12). However, in this cell line NS-398 had no effect on apoptosis or the cell cycle (12). Recently non-steroidal anti-inflammatory drugs have been shown to increase the expression of COX-2 in several carcinoma cell lines, most probably via activation of peroxisome proliferation-activated receptor
(1315). Furthermore, over-expression of COX-1 and COX-2, independent of their ability to produce prostanoids, has been associated with malignant transformation (16) and cell cycle alterations (17), respectively, in several cell lines. The data indicate that the use of COX inhibitors may be problematic on several fronts. First, COX expression, irrespective of activity, may significantly impact cell physiology. Secondly, COX inhibitors may have effects on gene expression, with previously unappreciated consequences. Thus, other means of modulating COX-2 expression and its products may be more effective in selectively enhancing the ability of normal tissue to survive radiation injury while targeting cancer cells.
-Radiation dose-dependently increases COX-2 protein expression by PC-3 cells that is accompanied by increased PGE2 synthesis when the irradiated cells are supplied with exogenous arachidonate (12). However, the signal transduction mechanisms responsible for up-regulating COX and PGE2 synthesis after ionizing radiation are unknown. The cellular response to radiation is a complex interplay between radiation sensors and existing signal transduction pathways in the cell. Reactive oxygen and reactive nitrogen species generated by ionizing radiation activate downstream signal transduction events (reviewed in 18). In mammalian cells three major mitogen-activated protein kinase (MAPK) pathways respond to stress and inflammation; these are the p38, JNK (SAPK) and ERK5/big MAPK-1 pathways (reviewed in 19). However, the growth responsive ERK MAPK module can also be activated by ionizing radiation (18). Indeed, activation of both the stress and growth responsive MAPK signal transduction pathways figures prominently in the mammalian cell response to ionizing radiation (18). The activation and interaction of the various MAPK modules elicits cell cycle arrest, DNA repair and apoptosis that ultimately determine the cell's fate after radiation (18,19).
Any of the MAPK modules may be involved in regulating COX-2 expression (20). The COX-2 promoter contains a number of potential regulatory elements; one of these elements, the ATF/CRE site, binds transcription factor complexes whose formation can be modulated by activation of p38 (20). Studies using small molecule inhibitors of p38 have demonstrated a requirement for p38 in up-regulated COX-2 expression (2130); the contribution of p38-dependent transcriptional activation to the observed increase in COX-2 expression has been explored less extensively. Post-transcriptional regulation plays a significant role in induced COX-2 expression (31,32). Run-on experiments indicate that increased transcription accounts for only a small component of the observed increase in COX-2 mRNA and that message stabilization is the primary mechanism in COX-2 induction in cells treated with inflammatory stimuli such as interleukin (IL)-1ß, lipopolysaccharide and tumor necrosis factor
(22,24,29,33,34). Not surprisingly, the stress-activated MAPK, p38, appears to be required for this enhanced stability (22,24,25,27,29). Stress stimuli such as UV radiation and ionizing radiation are known to activate p38 in some cells (reviewed in 35,36). A clear role for p38 activation has been demonstrated in COX-2 expression following UV irradiation in human keratinocytes (37) and in cells exposed to products probably produced following ionizing radiation such as reactive nitrogen generating systems (38), superoxide (39) and 4-hydroxy-nonenal (40). However, a requirement for p38 in modulating COX-2 expression and PGE2 production following ionizing radiation has not been explored. Given the association of COX activity and intestinal crypt stem cell survival after radiation injury and the association of up-regulated COX-2 with carcinomas, we have used an epithelial cell line, I407, to investigate the role of p38 MAPK in radiation-induced PGE2 synthesis.
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Materials and methods
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Cell culture and transfected cell lines
The human epithelial cell line I407 (American Type Culture Collection, Manassas, VA) was maintained in Basal Medium Eagle with Earle's salts (Gibco BRL, Grand Island, NY) supplemented with 6 mM glutamine, 10% heat-inactivated fetal calf serum and antibiotics (penicillin 50 U/ml and streptomycin 50 µg/ml). Control transfected, p38
wild-type (WT), and p38
dominant-negative (DN) stable-transfectant cell lines were maintained in the above media supplemented with G418 as the antibiotic, but were cultured 1 week in the absence of G418 prior to use in experiments. Briefly, 5080% confluent cultures of I407 cells seeded in 6-well tissue culture plates were transfected with FuGENE 6 reagent (Roche, Indianapolis, IN) according to the manufacturer's guidelines at a ratio of 3:2 FuGENE/DNA and 2 µg DNA/well. pCMV-p38
WT and pCMV-p38
DN (Thr180 and Tyr182 replaced with Ala and Phe, respectively) plasmids used for transfection were obtained from Dr Roger Davis, Howard Hughes Medical Institute. pcDNA 3.1 (Invitrogen, Carlsbad, CA) was used as the empty vector for control transfections. After overnight exposure to the DNA/FuGENE complex, the media was changed and 2 days later cultures were placed into media containing 1.2 mg/ml G418 for selection of stable-transfectant clones. Individual clones were assigned a letter/number code for identification.
COX-2 mRNA and protein levels
The effect of
-irradiation on COX-2 was determined by irradiating sub-confluent I407 cells with 812 Gy using a Gammacel 40 Cesium irradiator (Atomic Energy of Canada, Ottawa, Canada). At the indicated times, cells were lysed with Laemmli buffer for subsequent detection of COX-2 protein by western analysis or lysed with a guanidine thiocyanate-based solution (Ambion Direct Protect kit) for subsequent ribonuclease protection assays.
For western analysis, lysates were separated by SDSPAGE, blotted to Immobilon P membrane (Millipore, Bedford, MA), and COX-2 protein detected using an anti-COX-2 antibody from Santa Cruz Biotechnology (Santa Cruz, CA) according to the protocol suggested by the supplier. Immunoreactive protein was detected by chemiluminescence (ECL reagent, Amersham, Piscataway, NJ) and corrected to actin by re-probing the same blot (antibody from Santa Cruz). COX-1 protein expression was determined in the same manner, using a COX-1 antibody from Santa Cruz.
Steady-state COX-2 mRNA levels were evaluated using the Direct Protect ribonuclease protection kit from Ambion (Austin, TX). The 400 nt antisense human COX-2 32P-labeled probe was generated using SalI linearized plasmid (gift of Dr J.Masferrer, Pharmacia) and T7 polymerase (Maxiscript kit, Ambion). The 32P-labeled actin probe was generated using a template from Ambion. Following gel purification of the probes, they were combined with the cell lysate and the ribonuclease protection assay performed as directed in the kit instructions. Protected fragments were separated by electrophoresis and radioactivity was detected using autoradiography on Kodak Biomax MR film or using the PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA). The COX-2 signal was corrected to actin. COX-1 mRNA levels were determined in a similar manner. For COX-1 RPAs a 240 nt antisense COX-1 32P-labeled probe was generated from a EcoRV linearized plasmid from Dr J.Masferrer (Pharmacia) using T7 polymerase.
COX-2 mRNA stability
For experiments determining the stability of COX-2 mRNA, COX-2 mRNA levels were evaluated using real-time RTPCR. Cells were irradiated with 12 Gy and 3 h later treated with dichlorobenzimidazole (DRB; 100 µM) or DRB and the p38 inhibitor SB203580 (10 µM) for the indicated times. RNA was isolated using Trizol (Invitrogen) and reverse transcribed using Superscript II reverse transcriptase (Invitrogen) with random hexamers according to manufacturer's instructions. Real-time PCR was performed in an iCycler (Bio-Rad, Hercules, CA) using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). COX-2 primers (0.2 µM each) were, forward: 5'-ATC CTG AAT GGG GTG ATG AG-3'; reverse: 5'-GCC ACT CAA GTG TTG CAC AT-3'. GAPDH primers (0.3 µM each) were, forward: 5'-GAA GGT GAA GGT CGC AGT C-3'; reverse: 5'-GAA GGT GAT GGG ATT TC-3'. Reaction conditions were as follows: 10 min at 95°C, then 40 cycles of 95°C (15 s) and 60°C (60 s) with data acquisition during the 1 min 60°C step. Melt analysis was used to confirm PCR products.
Transcriptional activation of the COX-2 promoter by irradiation
I407 cells were transfected with a human COX-2 promoter/luciferase reporter plasmid containing the region -1432/+59 of the human COX-2 promoter in the pGL3 reporter plasmid (generously supplied by Dr Tadashi Tanabe; National Cardiovascular Research Institute, Japan). Control vectors for promoter activity were pGL3-basic (negative control; Promega, Madison, WI) and pGL3-control (positive control; Promega). pSV-ß-galactosidase vector (Promega) was co-transfected as a transfection control. Subconfluent cells in 12-well plates were transfected using FuGENE 6 as suggested by the manufacturer at a ratio of 3:1 FuGENE 6 to DNA. Each well of cells received a total of 0.3 µg DNA: 0.2 µg of the promoter reporter plasmid and 0.1 µg of the transfection control plasmid. Twenty-four hours later, the transfected cells were placed into fresh media and then received 12 Gy radiation or not. For experiments using the p38 MAPK inhibitor, SB203580 (10 µM) was added 1 h prior to the media change and was included during the subsequent period. At the indicated times after treatment, cells were lysed and luciferase and ß-galactosidase activity assayed using kits from Promega. As a positive control for the promoter activity of the COX-2 reporter plasmid, I407 cells were transfected as above with the pSV-ß-galactosidase and COX-2 promoter/luciferase plasmids. Approximately 30 h after starting the transfection, cells were serum-starved overnight (media containing 0.1% fetal calf serum), and then incubated with various stimuli for the indicated times. Luciferase and ß-galactosidase activities were then assayed as described above.
p38 activation
To determine the time course of p38 activation, equivalent amounts of protein from irradiated I407 cells were separated by SDSPAGE, blotted to Immobilon P, and probed with an antibody specific for the dually phosphorylated form of p38 (Cell Signaling Technology, Beverly, MA) using the protocol suggested by the supplier. Phosphorylated protein was corrected to total p38 protein using an anti-p38 antibody (Cell Signaling Technology).
PGE2 enzyme immunoassay
PGE2 levels were determined by analyzing the media from irradiated cells, using a kit from Cayman (Ann Arbor, MI). For time course experiments, the media was collected every 12 h and replaced with fresh media. Inhibitors were added at the time of stimulation and at the time of media replacement. In experiments testing the effect of exogenous arachidonate on PGE2 synthesis, cells were irradiated (8 Gy) and then 48 h later the media was replaced with fresh media containing vehicle or 10 µM arachidonate. Media was collected after 15 min and analyzed.
Statistics
Student's two-tailed t-test was used to determine if PGE2 production by irradiated I407 clones was significantly different than that of irradiated non-transfected I407 cells (Figure 5). The Student's two-tailed t-test was also used to analyze the hCOX-2 promoter data (Figures 8 and 9).

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Fig. 8. COX-2 promoter activity in irradiated I407 cells. I407 cells were transfected with a human COX-2 promoter/luciferase reporter plasmid containing the region -1432/+59 of the human COX-2 promoter in the pGL3 reporter plasmid along with the pSV-ß-galactosidase vector as a transfection control. Twenty-four hours later, the transfected cells were placed into fresh media and irradiated (12 Gy). At the indicated times after irradiation cells were lysed and luciferase and ß-galactosidase activity assayed. Data are the average of triplicate wells (±SE) and are representative of at least two separate experiments. *P < 0.01 compared with time zero (end of transfection).
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Fig. 9. COX-2 promoter activity by serum-starved I407 cells. I407 cells were transfected with a human COX-2 promoter/luciferase reporter plasmid containing the region -1432/+59 of the human COX-2 promoter in the pGL3 reporter plasmid along with the pSV-ß-galactosidase vector. Thirty hours after transfection cells were serum-starved overnight and then received no treatment (continued starve) or were stimulated with serum-starved media containing 10% serum (serum) or IL-1ß (20 ng/ml) in serum-starved media (IL-1ß). At the indicated times after stimulation cells were lysed and luciferase and ß-galactosidase activity assayed. Data are the average of quadruplicate wells (±SE) and are representative of at least two separate experiments. **P < 0.05 for either T0 (end of transfection/overnight starve) or to comparable time point of continued starved.
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Results
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Irradiating the human epithelial cell line I407 resulted in increased PGE2 synthesis (Figure 1). PGE2 production was biphasic with a small peak of synthesis in the 1224 h after irradiation and sustained synthesis from 36 through to 72 h. The increase in PGE2 synthesis coincided with increases in both COX-2 mRNA and protein (Figure 2). COX-2 mRNA increased 6-fold and peaked at 6 h following 12 Gy irradiation (Figure 2A). By 10 h post-irradiation COX-2 mRNA was half-maximal and was near baseline by 24 h following irradiation. COX-2 mRNA production was biphasic with levels once again elevated 48 h after radiation. COX-2 protein showed similar kinetics to COX-2 mRNA (Figure 2B). As observed for PGE2 synthesis and COX-2 mRNA, COX-2 protein was once again elevated at 48 h post-radiation after returning to near baseline levels at 24 h. Neither COX-1 mRNA nor protein was altered by irradiation (data not shown). Consistent with COX-2- catalyzed PGE2 synthesis, both indomethacin and NS-398 completely abrogated PGE2 production (Figure 3).

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Fig. 1. Time course of PGE2 production by I407 cells after irradiation. Cells were treated with media alone or received 12 Gy -irradiation. Media was collected every 12 h and replaced with fresh media. PGE2 in the media was determined as indicated in Materials and methods. Data are the average (±range) of replicate wells assayed in duplicate and are representative of at least two separate experiments.
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Fig. 3. Effect of COX or p38 inhibition on radiation-stimulated PGE2 production. I407 cells were irradiated with 8 or 12 Gy -irradiation in the presence of vehicle, indomethacin (10 µM), NS-398 (10 µM) or SB203580 (10 µM). At the end of 72 h, media was collected and analyzed for PGE2 as described under Materials and methods. Data are presented as the average (±range) of replicate wells assayed in duplicate and are representative of at least two separate experiments.
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Having demonstrated that radiation induces COX-2 expression, we next sought to determine if the effects of radiation on COX-2 expression are mediated through p38. Western analysis revealed that irradiation resulted in the rapid phosphorylation of p38 MAPK, with peak phosphorylation occurring at 3 h post-irradiation (Figure 4). Radiation-induced PGE2 synthesis was inhibited by the p38 MAPK inhibitor, SB203580 (Figure 3). To further address the role of p38 MAPK in radiation-induced COX-2 expression we produced stable transfectants of I407 cells containing either empty vector (control-transfected), p38
WT MAPK, or p38
DN MAPK. Several clones of both the wild-type and dominant-negative were selected and assigned letter/number codes for identification (Figure 5). Unirradiated clones produced little if any PGE2. Although p38
WT MAPK transfectants did not exhibit any further augmentation in PGE2 synthesis after radiation compared with non-transfected or control-transfected cells, the p38
DN MAPK clones showed attenuated PGE2 synthesis following radiation (Figure 5). However, expression of the p38
DN did not completely abrogate radiation-induced PGE2 synthesis. Two p38
WT MAPK (H11 and H6) clones and two p38
DN MAPK (A4 and E2) clones were used for further characterization. As observed in non-transfected and control-transfected I407 cells, 8 Gy irradiation resulted in increased COX-2 protein synthesis by both p38
WT MAPK clones (Figure 6). In contrast, the p38
DN MAPK clones failed to up-regulate COX-2 expression (Figure 6). Thus, radiation-induced PGE2 synthesis requires increased COX-2 expression, which is mediated through p38
MAPK activation.

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Fig. 4. Time-course of p38 phosphorylation following irradiation. p38 phosphorylation was monitored by western analysis using a phospho-specific antibody as described under Materials and methods. Cell lysates (equivalent amounts of protein) from duplicate wells were combined and analyzed and corrected to total p38. Data are presented as mean ± range for duplicate samples. The time course is representative of at least two separate experiments.
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The failure of p38
DN cells to produce PGE2 and to up-regulate COX-2 synthesis in response to ionizing radiation suggests that radiation-stimulated PGE2 synthesis requires COX-2 induction. However, even at baseline there is observable COX-2 protein and it is possible that this level of COX-2 is sufficient to generate the PGE2 observed after radiation. If this is the case, the failure of p38
DN clones to up-regulate PGE2 synthesis after radiation may be due to a defect in arachidonate release. Cytosolic phospholipase A2 (PLA2) can be phosphorylated by p38 and phosphorylation is a critical event in this phospholipase's activation (20,41). To investigate the possibility that the defect in radiation-induced PGE2 synthesis in p38
DN cells was due to diminished PLA2 activation, we examined the effect of exogenous arachidonate on PGE2 synthesis in irradiated cells. Cells received either no radiation or 8 Gy radiation and then 48 h later the media was removed and fresh media containing either vehicle or 10 µM arachidonate was added to the well. Fifteen minutes later the media was collected and analyzed. Exogenous arachidonate resulted in increased PGE2 synthesis by irradiated non-transfected, control-transfected and p38
WT-transfected cells (Figure 7). The data indicate that 48 h after radiation the induced level of COX-2 enzyme activity is sufficient to metabolize all the available endogenous substrate; thus arachidonate release is the limiting factor in PGE2 synthesis 48 h after radiation. Exogenous arachidonate was not able to overcome the defect in PGE2 synthesis in p38
DN cells. Thus, in I407 cells ionizing radiation stimulates PGE2 synthesis through a p38
-dependent mechanism that requires up-regulation of COX-2 protein.
COX-2 protein levels may be regulated by both transcriptional and post-transcriptional mechanisms. We investigated the effect of radiation on COX-2 transcription using a COX-2 promoter/luciferase reporter construct containing the region -1432/+59 of the human COX-2 promoter. I407 cells transfected with the luciferase vector without a promoter (pGL3-basic) averaged 3.8 x 104 relative light units (RLU) at the end of transfection and cells transfected with the luciferase vector under the control of the SV40 promoter (pGL3-control) averaged 4.6 x 107 RLU (data not shown). We found that the COX-2 promoter is active in unstimulated I407 cells (23 x 106 RLUs) and that promoter activity is not further increased by radiation (Figure 8). Treatment of transfected cells with SB203580 (1 h prior to and following irradiation) did not alter COX-2 promoter activity (data not shown). This experiment demonstrates that in I407 cells irradiation does not affect COX-2 expression by enhancing transcription. Furthermore, p38 activity is not required for COX-2 transcription under these conditions (data not shown). We confirmed our ability to detect changes in activity of the transfected COX-2 promoter/luciferase plasmid using serum and IL-1 as stimuli. As shown in Figure 9, activity of the COX-2 promoter was increased when serum-starved cells were incubated with media containing 10% fetal calf serum. Similarly, serum-starved cells stimulated with 20 ng/ml IL-1ß showed a detectable increase in promoter activity (Figure 9). C2-Ceramide (10 µM) did not stimulate promoter activity and phorbol myristate acetate (100 ng/ml) resulted in only a slight increase in promoter activity, which was not consistently statistically significant (data not shown).
We next determined if the observed p38-dependent net increase in COX-2 mRNA following radiation was due to message stabilization. For these experiments I407 cells received 0 or 12 Gy and then 3 h later were incubated with DRB with and without the p38 inhibitor SB203580. In untreated I407 cells COX-2 mRNA rapidly decayed, with a half-life of
15 min and 90% loss within the first 30 min after DRB addition (Figure 10). In irradiated cells COX-2 mRNA stability was increased to a t1/2 of 90 min (Figure 10). Inhibition of p38 with SB203580 reduced the half-life of COX-2 message in irradiated cells from 90 to
30 min (Figure 10). Thus, the major mechanism responsible for increased COX-2 mRNA levels in irradiated I407 cells is message stabilization, and p38 plays an important role in this stabilization.

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Fig. 10. COX-2 mRNA stability is increased in irradiated I407 cells and is attenuated by inhibition of p38. Three hours after 12 Gy irradiation, DRB or DRB plus 10 µM SB203580 was added to cells for the indicated times. The amount of COX-2 mRNA at each time point was determined by real-time PCR. Data are presented as the average of samples done in triplicate and are representative of at least two separate experiments done in duplicate.
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Discussion
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Intestinal sensitivity to radiation-induced damage is often limiting in radiation treatment of tumors. Recent data obtained in the mouse demonstrates a significant role for up-regulated COX expression and activity in modulating the ability of the normal intestine to respond to radiation injury. Many tumors exhibit high basal levels of COX-2 expression, which play an integral role in sustaining proliferation and reducing apoptosis. Therefore, an understanding of the mechanisms supporting the differential expression of COX may lead to cancer therapies with reduced toxicity to normal tissue. The signal transduction mechanisms that trigger PGE2 synthesis and COX up-regulation following ionizing radiation have not been described. Therefore, we have used a transformed epithelial cell line, I407, to determine if ionizing radiation induces COX expression and prostanoid synthesis and to elucidate the signal transduction mechanisms that are involved. Irradiation of I407 cells resulted in an increase within 12 h in PGE2 synthesis from endogenous substrate; either indomethacin or NS-398 inhibited this increase. Increased PGE2 synthesis was accompanied by elevated COX-2 protein expression. Steinauer et al. found that the prostate cancer cell line, PC-3, exhibited an enhanced ability to synthesize PGE2 when supplied with exogenous arachidonate 7 h after 15 Gy (12). Similar to our findings this synthesis was completely abrogated by NS-398 and accompanied by increased COX-2 protein expression (12). The magnitude of the increase in COX-2 protein reported for PC-3 cells was similar to our observation in I407 cells. Our findings in the I407 cell line indicate that this cell line may be used as a model for studying the signal transduction mechanisms that lead to increased COX-2 expression in tumors following radiation.
The p38 MAPK signaling module is variably activated by ionizing radiation. For example, ionizing radiation has been reported to induce p38 MAPK activity in U937 and HS Sultan multiple myeloma cells but not NIH 3T3 cells (42). Irradiation of the human glioma cell line U87 did not increase p38 MAPK phosphorylation (43). However, ionizing radiation does activate p38 in the human renal epithelial cell line 293T (44,45). Similarly, we found that treatment of I407 cells with 12 Gy radiation resulted in the transient phosphorylation of p38, with a maximum at 3 h post-radiation. This time course was similar to that observed in 293T cells (44).
In I407 cells the activation of p38 preceded an increase in PGE2 synthesis, COX-2 mRNA and COX-2 protein expression. The p38 inhibitor SB203580 also abrogated radiation-induced PGE2 synthesis by I407 cells. Thus, p38 activation appears to modulate radiation-induced PGE2 synthesis and COX-2 expression in I407 cells. A role for p38 MAPK in COX-2 induction by stimuli other than ionizing radiation has been established for several gastrointestinal epithelial cell lines and associated cell types (30,40,4650). However, the non-steroidal anti-inflammatory drugs flufenamic acid and sulindac sulfide induce COX-2 in the colonic cell line HT-29 in a p38 MAPK-independent manner (13).
To evaluate the role of p38 in radiation-induced COX-2 expression we developed I407 cell lines stably transfected with p38
WT and p38
DN. p38
WT transfected, non-transfected and control-transfected cells exhibited comparable induction of COX-2 following radiation. I407 cells transfected with p38
WT did not exhibit elevated basal levels of COX-2 or PGE2. This observation is similar to the findings of Guan et al. (21) with rat primary mesangial cells stably transfected with similar constructs. However, transient transfection of other cell lines with p38 WT did increase basal COX-2 promoter activity or protein expression (28,29,40,51). This difference in response may simply reflect experimental differences (i.e. different cell types, transient versus stable transfection) or may reflect differences in the basal activity of upstream modulators of p38 activity. Our data indicate that the p38
isoform plays a significant role in the ability of an epithelial cell line to up-regulate prostanoid synthesis following irradiation. This is the first demonstration that p38 MAPK is required for COX-2 up-regulation in response to ionizing radiation.
Increased COX-2 expression via a p38-dependent mechanism (37,39,40,48) has been reported for stress stimuli other than ionizing radiation, however, the contribution of transcriptional and post-transcriptional regulation to induced COX-2 expression has been explored less extensively for these stimuli. The contribution of transcriptional activation and mRNA stabilization to increased COX-2 expression following ionizing radiation have not been reported previously. Our data support a primary role for COX-2 message stabilization in the increased expression observed following irradiation. Stability studies indicated that in resting I407 cells COX-2 mRNA has a half-life of 15 min. COX-2 mRNA is similarly short-lived in rat intestinal epithelial cells (t1/2
13 min) (53). Following 12 Gy radiation the half-life of COX-2 mRNA increased to 90 min. In the presence of the p38 inhibitor SB203580, the COX-2 mRNA half-life following radiation was reduced to 45 min. In rat intestinal cells treated with transforming growth factor ß, COX-2 mRNA message was stabilized (t1/2
30 min) (53). In HeLa cells treated with IL-1, COX-2 mRNA half-life is
60 min and is reduced to
30 min in the presence of SB203580 (22). We were unable to detect transcriptional activation following irradiation of I407 cells. This may be due to robust transcription from the COX-2 promoter in unirradiated I407 cells. Kutchera et al. (54) have reported 5-fold higher transcription from a COX-2 promoter/luciferase construct in the colonic carcinoma cell line HCT-116 compared with transcription from the same construct placed in control epithelial cells (184A1 breast cells) or 3T3 cells. The colon carcinoma cell lines HCA-7 and LS-174 also have detectable basal transcription of the COX-2 gene as determined by nuclear run-on assays (55). COX-2 transcription in I407 cells appears to be independent of p38 activity as transcription from the reporter construct was unaffected in the presence of SB203580. Ridley et al. (22) reported that IL-1 induced transcription of COX-2 in HeLa cells is p38-independent. Our ability to detect changes in transcriptional activity from the COX-2 promoter/luciferase reporter construct was verified using serum-starved I407 cells stimulated with serum or IL-1ß. The modest increase in transcriptional activation we observed in response to serum and IL-1 is consistent with previous reports (20,22,29). Thus, the increase in COX-2 expression following radiation appears to be mediated through a p38
-dependent increase in message stability as in the absence of p38
activation irradiation fails to increase COX-2 expression, and mRNA stability in irradiated cells is attenuated by an inhibitor of p38.
p38 MAPK has been shown to regulate the activity of the cytosolic PLA2 responsible for releasing arachidonate from cellular stores (20,41). To show that the lack of prostanoid synthesis by p38
DN-transfected cells was not simply due to a lack of substrate, we determined the effect of exogenous arachidonate on PGE2 synthesis. Unstimulated I407 cells produce
8 pg/ml PGE2. When supplied with exogenous arachidonate, PGE2 synthesis increased
4-fold for unirradiated non-transfected, control-transfected, p38
WT and p38
DN cells, most probably reflecting metabolism by COX-1. PGE2 synthesis was elevated
10-fold in irradiated non-transfected, control-transfected and WT-transfected cell lines supplied with exogenous arachidonate compared with synthesis from endogenous arachidonate by irradiated cells indicating that the COX-2 activity induced following radiation exceeds the amount of endogenous substrate. In p38
DN I407 cells, irradiation does not stimulate PGE2 production, even in the presence of exogenous arachidonate. This demonstrates that the p38
-mediated increase in PGE2 synthesis induced by radiation requires COX-2 synthesis and is not mediated solely via arachidonate availability.
Our studies demonstrate that radiation increases COX-2 expression in a transformed human epithelial cell line that already expresses some COX-2 at baseline. Whether this would be the case for normal human intestinal epithelium, which does not appreciably express COX-2, remains unclear. We found that in the mouse, whole body irradiation induces COX-1 expression in the intestinal epithelium, but neither COX-2 mRNA nor protein was significantly elevated. The difference in COX-1 versus COX-2 induction following radiation may reflect a species difference (mouse versus human) or the difference between the epithelial cell response in situ versus isolated. However, it is more probable that the differential induction of COX-1 or COX-2 following radiation reflects the normal versus neoplastic phenotype.
Many cancers, especially colorectal cancers, have elevated COX-2 expression. Furthermore, intestinal cell lines over-expressing COX-2 appear to be especially sensitive to ambient prostaglandin levels as inhibition of COX decreases proliferation and increases apoptosis in these cells, while cell lines which do not over-express COX-2 are often insensitive to these inhibitors (5660). Inhibition of COX-2 activity in carcinoma cell lines with high levels of COX-2 expression enhances radioresponsiveness (811). Conversely, COX-2 inhibitors do not decrease prostaglandin production or crypt survival in the irradiated mouse (1) and COX-1 knockout mice exhibit increased apoptosis and decreased crypt survival following radiation injury (2). Thus, the response of normal intestinal epithelial cells to radiation injury is characterized by the induction of COX-1 rather than COX-2. This may explain why selective inhibition of COX-2 increases the radiosensitivity of a COX-2 expressing sarcoma cell line, FSA, implanted into mice while having no adverse effect on the intestine's sensitivity to radiation (9).
The ability of radiation to up-regulate COX-2 expression in a malignant epithelial cell line raises the possibility that radiation therapy may generate a population of surviving cancer cells with elevated COX-2 expression. The consequences of such up-regulation to the metastatic potential of surviving tumor cells, and the response of these cells to subsequent radiation therapy or chemotherapy are unknown and should be considered. The requirement for p38 activation for COX-2 up-regulation following radiation may represent an exploitable modality of using p38 inhibitors in combination with radiotherapy to prevent COX-2 up-regulation and thus enhance curability.
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Notes
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4 To whom correspondence should be addressed Email: stensnlb{at}im.wustl.edu 
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Acknowledgments
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grants R01-DK33165 and R01-DK55753 (W.F.S.); NIH RO1-DK50924 (S.M.C.); and DK-52574 (Washington University Digestive Disease Center).
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Received March 27, 2003;
revised August 12, 2003;
accepted September 17, 2003.