Prosurvival and antiapoptotic effects of PGE2 in radiation injury are mediated by EP2 receptor in intestine

Courtney W. Houchen1, Mark A. Sturmoski1, Shrikant Anant1, Richard M. Breyer2, and William F. Stenson1

1 Department of Medicine, Division of Gastroenterology, Washington University School of Medicine, St. Louis, Missouri 63110; and 2 Department of Medicine, Division of Nephrology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2372


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The biological activities of PGE2 are mediated through EP receptors (EP1-EP4), plasma membrane G protein-coupled receptors that differ in ligand binding and signal-transduction pathways. We investigated gastrointestinal EP2 receptor expression in adult mice before and after radiation injury and evaluated intestinal stem cell survival and crypt epithelial apoptosis after radiation injury in EP2 null mice. EP2 was expressed throughout the gut. Intestinal EP2 mRNA increased fivefold after gamma -irradiation. Crypt survival was diminished in EP2-/- mice (4.06 crypts/cross section) compared with wild-type littermates (8.15 crypts/cross section). Radiation-induced apoptosis was significantly increased in EP2-/- mice compared with wild-type littermates. Apoptosis was 1.6-fold higher in EP2 -/- mice (5.9 apoptotic cells/crypt) than in wild-type mice (3.5 apoptotic cells/crypt). The EP2 receptor is expressed in mouse gastrointestinal epithelial cells and is upregulated following radiation injury. The effects of PGE2 on both crypt epithelial apoptosis and intestinal crypt stem cell survival are mediated through the EP2 receptor.

prostaglandin; cyclooxygenase; prostaglandins; receptor signaling


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PG IS SYNTHESIZED from arachidonate by two isoforms of the cyclooxygenase enzyme (COX) (16, 19). COX-1 is constitutively expressed in the mouse gastrointestinal tract; it can, however, be induced by radiation injury (10). COX-2 is barely detectable in the gastrointestinal tract under normal conditions, but it is induced in response to a wide variety of proinflammatory cytokines and mitogenic stimuli, including IL-1beta and TNF-alpha (46, 48, 50), and is upregulated in some epithelial neoplasms (14, 15, 54). PGE2, the most abundant gastrointestinal PG, regulates many of the normal homeostatic functions of the gut, including motility and secretion (18, 31, 53). Furthermore, PGE2 influences mitogenesis (28), promotes growth of tumors (42), and stimulates gene transcription (47). PGE2 upregulates VEGF in activated macrophages through a receptor-mediated mechanism (3).

The biological activities of PGE2 are mediated through its interaction with plasma membrane G protein-coupled receptors termed EP receptors. The EP receptor subtypes are EP1, EP2, EP3, and EP4 (1, 12, 33, 36). These receptors differ in their ligand binding specificities and in their downstream signal-transduction pathways. EP1 stimulation results in increases in intracellular calcium. The EP2 and EP4 receptors are coupled to Gs and mediate increases in intracellular cAMP. Alternatively, EP3 negatively regulates adenylate cyclase and results in decreases in intracellular cAMP (33, 36). Alternatively spliced variants of EP3 can also result in increases in adenylate cyclase activity with subsequent increases in cAMP (5). PGE2 is metabolized quite rapidly in most tissues (32) and is therefore thought to act at or near its site of synthesis. The tissue distribution and subcellular expression of the EP receptors in the gastrointestinal tract is currently unclear. A survey of EP2 mRNA by Northern blot analysis suggested that EP2 was expressed at very low levels if at all in the rodent intestinal tract (13).

The murine response to radiation injury is the most extensively characterized model of intestinal injury. In this model, adult mice are exposed to lethal doses of total body gamma -irradiation. After epithelial injury, the rapidly proliferating transit cells in the intestinal crypts are eliminated via apoptosis or cease replicating and are shed as they migrate up the intestinal villi or onto the surface epithelium of the colon (40). If a crypt contains a surviving stem cell, it will proliferate to form a regenerative crypt, and cells from these regenerative crypts will ultimately repopulate the entire intestinal epithelium (44). The fate of epithelial stem cells after injury and their capacity to regenerate the crypt epithelium have been primarily studied using the microcolony formation assay. In this assay, the number of regenerating crypts can be scored by microscopic appearance 3-4 days after injury and can be used as a surrogate measure of crypt stem cell survival after radiation injury.

We have previously shown that COX-1 is markedly induced in the regenerative crypt epithelium 3.5 days after 13 Gy gamma -irradiation in adult mice and the PGE2 produced is synthesized primarily through COX-1 and regulates intestinal stem cell survival after radiation (10). In contrast, PGE2 synthesized in response to LPS stimulation is synthesized primarily through COX-2 (43) and mediates radioprotection. These two findings suggest an important role for PGE2 synthesis in response to radiation injury, albeit by different mechanisms.

Crypt epithelial cells respond to radiation injury by undergoing apoptosis. Recently, our laboratory (20) reported that mice, which lack the COX-1 gene, have diminished baseline intestinal PGE2 synthesis and have an impaired crypt epithelial response to radiation injury. These mice have decreased intestinal stem cell survival and increased crypt epithelial apoptosis in response to radiation injury. These studies, taken together, suggest that the epithelial response to radiation injury is mediated by PGs synthesized through COX-1.

In this study, we sought to define the EP receptors that mediate the effects of PGE2 in radiation injury. Here, we report that the EP2 receptor is expressed throughout the mouse gastrointestinal tract and is differentially expressed along the crypt-to-villus axis in both the small intestine and colon. Furthermore, EP2 expression is induced by radiation injury, and the effects of PGE2 on intestinal crypt epithelial apoptosis and crypt epithelial stem cell survival after radiation are mediated, at least in part, through the EP2 receptor.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Animals used in these studies were cared for and housed according to guidelines approved by Washington University and the American Association for Accreditation of Laboratory Animal Care. Mice were fed standard laboratory mouse chow ad libitum and maintained in a pathogen-free environment on a 12:12-h light-dark schedule. Mouse lines with targeted disruption of the EP2 were obtained from Dr. R Breyer (Vanderbilt University School of Medicine, Nashville, TN) (24, 26). Mice were irradiated at 12-14 wk of age in a Gamacel 40 cesium irradiator (Atomic Energy of Canada, Ottawa, Canada) at 0.96 cGy/min. Animals were killed at specified times after irradiation and were rapidly dissected as described previously (11). The proximal jejunum was fixed in Bouin's solution and divided into at least 8- to 5-mm segments before paraffin embedding and immunohistochemical analysis. The colon was divided into 2- to 5-mm sections before paraffin embedding. The remaining gastrointestinal tissues were snap frozen in liquid nitrogen for further analysis. Total cellular RNA was prepared for use in ribonuclease protection analysis from frozen tissues using TRIzol (GIBCO-BRL, Bethesda, MD) according to the manufacturer's directions.

Immunohistochemical studies. For immunohistochemical localization of mouse EP2, deparaffinized sections tissue fixed in Bouin's solution were incubated with a 1:500 dilution of a rabbit anti-human EP2 (a gift from Jeff Johnson Cayman Chemical). This antibody was generated against the peptide SLRTQDATQTSDASKQADL (30). After quenching of endogenous peroxidase activity, sections were washed. Bound anti-EP2 was detected by tyramide signal amplification (TSA direct; Dupont, NEN Life Science Products, Boston, MA) according to manufacturer's directions following incubation with biotin-labeled donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories) and subsequent incubation with streptavidin-horseradish peroxidase.

Measurement of EP2 and EP4 mRNA levels. RNA from stomach, proximal, and distal jejunum, ileum proximal colon, and distal colon was prepared as described previously. Samples (20 ug) of total RNA were hybridized with 32P antisense RNA probes corresponding to mouse EP2 (32), using the RPAII ribonuclease protection kit (Ambion, Austin, TX) according to manufacturer's directions. The EP2 probe hybridized to nucleotides 757-1158 of EP2 mRNA. The EP4 probe hybridized to nucleotides 941-1364 of mouse EP4 mRNA. The RNA hybrids were digested with T1 and RNase A at 37°C. Protected RNA fragments were separated by electrophoresis in 7.5 M urea/8% acrylamide sequencing gels. After the gel was dried, autoradiography was performed using Kodak BioMax MR film.

SDS PAGE and Western blot analysis of EP2. Intestines and colons from 10- to 12-wk-old mice were homogenized in cold lysis buffer containing 1 ml PBS + 10 mM EDTA, 1% Triton X-100, 0.5% deoxycholic acid, 1 mM diethyldithiocarbamic acid containing leupeptin (10 uM), pepstatin A (1.5 um), and aprotinin (0.2 U/ml). Samples were solubilized in SDS-PAGE sample buffer containing Tris (62.5 M), glycerol (10%), SDS (2%), bromophenol blue (1%), and beta -mercaptoethanol (5%), pH 6.8. Equal amounts of protein were loaded and separated by electrophoresis on 7.5% sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, the separated proteins were transferred to an Immobilon transfer membrane (Millipore, Bedford, MA). Rabbit antibodies against human EP2 (a gift from J. Johnson, Cayman Chemical, Ann Arbor, MI) were used to detect major bands corresponding to EP2. Bound antibody was visualized using a donkey anti-rabbit IgG linked to horseradish peroxidase and enhanced chemiluminescence (Amersham) with fluorographic detection on BioMax film.

Crypt survival. Two hours before death, each mouse received 120 mg/kg 5-bromo-2'-deoxyuridine (BrdU; Sigma, St. Louis, MO) and 12 mg/kg 5-fluoro-2'-deoxyuridine 9 (Sigma) to label S-phase cells. Proximal jejunum was obtained by rapid dissection 3.5 days after irradiation and fixed immediately in Bouin's fixative. Paraffin sections (5 um) were prepared from the proximal jejunum oriented so that the sections were cut perpendicular to the long axis of the intestine. Cells incorporating BrdU were detected using a goat anti-BrdU (9), and bound antibody was visualized using horseradish peroxidase-labeled donkey anti-goat IgG followed by staining with 3,3'- diaminobenzidine. A surviving crypt was defined as one containing five or more BrdU-positive cells as previously described (10). The number of surviving crypts per cross section was determined for each mouse by scoring the number of surviving crypts in at least eight complete, well-oriented cross sections and dividing the total by the number of cross sections scored. Because size differences in regenerative crypts may affect the probability that a regenerative crypt will appear in a cross section (38, 41) we determined the width of 20 representative crypts for EP2-/- mice and their wild-type littermates in longitudinal sections of proximal jejunum at their widest point in each crypt. We did not find any difference in the size of regenerative crypts in EP2-/- mice compared with wild type after 14 Gy gamma -irradiation (data not shown). Thus differences in crypt survival are not due to variation in the size of regenerating crypts after radiation injury.

Apoptosis. The proximal jejunum was obtained from mice that received a single dose of 12 Gy gamma - irradiation. To account for diurnal variation in the intestinal apoptotic response to radiation, animals were irradiated at 8:00 AM for all experiments. All animals were housed on a 12:12-h light-dark cycle in a pathogen-free animal facility. Animals were killed 6 h after irradiation for apoptosis studies. Tissues were fixed overnight in 10% neutral buffered formalin and embedded in paraffin. Sections were prepared from the proximal jejunum oriented so that the sections were perpendicular to the long axis of the intestine. Sections were stained with hematoxylin and eosin, and the number of apoptotic cells per crypt was assessed by morphological criteria as previously described by Potten and Grant (39). Twenty well-oriented crypts were analyzed in each of eight cross sections for each mouse.

Statistical methods. Pairwise t-tests using the pooled estimate of variance and Bonferroni's corrections for the P values for multiple comparisons were used for analysis of the effects of EP2 genotypes on crypt survival and radiation-induced apoptosis.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intestinal EP2 mRNA and protein are increased following radiation injury. Expression of EP2 mRNA along the duodenal-to-colonic axis in the adult FVB/n mouse gastrointestinal tract was investigated using RNase protection analysis (Fig. 1). Relatively uniform levels of EP2 mRNA expression were observed from the stomach to distal jejunum, with a small decline in expression in the ileum and cecum. EP2-receptor mRNA levels were higher in the proximal colon than in the distal small intestine.


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Fig. 1.   Regional distribution of EP2 mRNA in the mouse gastrointestinal tract. FVB/n mice were killed at 12 wk of age. Total cellular RNA was prepared from the tissues indicated. Small intestinal EP2 and actin mRNA levels were determined by RNase protection assays. Protected bands were separated by denaturing PAGE, and gels were analyzed by autoradiography. Autoradiographs for EP2 were exposed overnight and for actin for 4 h. A: RNase protection assays from gastrointestinal tissues: St, stomach; Du, duodenum; Pj, proximal jejunum; Dj, distal jejunum; Il, ileum; Ce, cecum; Pc, proximal colon; Dc, distal colon. B: quantitation of RNase protection assays for EP2 shown in A. The intensity of the major bands protected for EP2 mRNA was determined by scanning densitometry on autoradiographs of RNase protection assays. Data represent a pooled analysis of 3 mice for each region.

Radiation injury increases EP2 mRNA expression in the proximal jejunum (Fig. 2A). EP2 mRNA was detectable in the proximal jejunum of unirradiated mice; levels increased significantly by 24 h after 13 Gy gamma -irradiation and peaked at 96 h at levels approximately fivefold higher than those observed in unirradiated mice (Fig. 2A). There was no increase in EP2-receptor mRNA expression in the early time periods (1-12 h) after irradiation.


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Fig. 2.   Time course of EP2 mRNA expression after irradiation in the mouse small intestine and colon. FVB/n mice received 13 Gy gamma -irradiation in a cesium irradiator at 0.96 cGy/min. Total cellular RNA was prepared from the distal jejunum of mice killed at indicated times after irradiation. Intestinal EP2 and actin mRNA levels were determined by RNase protection assays. A: protected bands were separated by denaturing polyacrylamide gel electrophoresis, and gels were analyzed by autoradiography. Autoradiographs for EP2 were exposed overnight and actin for 4 h. Bottom: quantitation of RNase protection assays for EP2 shown at top. The intensity of the major bands protected for EP2 mRNA was determined by scanning densitometry on autoradiographs of RNase protection assays at each time point. Data were normalized for the abundance of actin mRNA present in each sample. Data are the average of EP2 mRNA levels determined from pooled analysis of 6 mice for each time point. B: time course of EP2 mRNA expression after irradiation in the mouse colon. FVB/n mice received 13 Gy gamma -irradiation in a cesium irradiator at 0.96 cGy/min. Total cellular RNA was prepared from the proximal colon of mice killed at indicated time points after irradiation. Colonic EP2 and actin mRNA levels were determined by RNase protection assays. Protected bands were separated by denaturing PAGE and gels were analyzed by autoradiography. Bottom: quantitation of RNase protection assays for EP2 shown at top. The intensity of the major bands protected for EP2 mRNA was determined by scanning densitometry on autoradiographs of RNase protection assays at each time. Data were normalized for the abundance of actin mRNA present in each sample. Data are average of EP2 mRNA levels determined from pooled analysis of 6 mice for each time point.

Western blot analysis of adult mouse jejunal tissues at similar time points following radiation injury revealed immunoreactive EP2 protein bands of ~56-kDa (Fig. 3). Relatively low levels of EP2 protein were present at baseline in unirradiated mice. Protein levels increased by 48 h and peaked at 72 h after irradiation. These levels were nearly fivefold higher than those observed in unirradiated mice (Fig. 3, A and B). Thus both EP2 mRNA and protein are upregulated in the mouse small intestine after radiation injury and peak at a time when early crypt morphogenesis and epithelial restitution are the major morphological features of the severely injured intestine.


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Fig. 3.   Western blot analysis of EP2 protein expression in small intestine after irradiation. Small intestinal lysates were prepared from nonirradiated FVB/n mice and from irradiated mice at times indicated after irradiation with a single dose of 13 Gy. Equal amounts (20 µg) of protein from nonirradiated and irradiated mouse intestinal lysates were separated by electrophoresis on 15% SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes, and bands were detected by enhanced chemiluminescence using rabbit polyclonal antibodies against EP2.

Localization of EP2 within the small intestine before and after radiation injury. The cell-specific EP2 expression in the uninjured mouse jejunum was investigated by immunohistochemistry. EP2 was detected in intestinal crypt epithelial cells (Fig. 4), with predominant staining in the nuclei (Fig. 4B). Immunoreactive EP2 was also expressed on villus epithelial cells, which exhibited distinct lateral membrane staining (Fig. 4, A and C). Epithelial cells at the crypt villus transition did not exhibit EP2 immunostaining (Fig. 4A, red arrow). A similar staining pattern was observed for EP2 at 84 h after irradiation (Fig. 5, A and B). At this time, regeneration of the crypt epithelium is the predominant morphological feature of the intestine, and regenerative crypts are easily identifiable by routine histological techniques (Fig. 5A). Immunoreactive EP2 was again detected in the nuclei of crypt epithelial cells (Fig. 5B). There was considerable nonepithelial cell EP2 staining in the lamina propria. The staining in regenerative crypt epithelial cells represents new synthesis of EP2 protein in crypt epithelial cells. These regenerative crypt epithelial cells will ultimately serve to repopulate the entire gastrointestinal epithelium and restore the normal functional characteristics of the gut as well as the normal morphological features of the crypt-to-villus axis.


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Fig. 4.   Immunohistochemical localization of EP2 in mouse small intestine. Cellular localization of EP2 was analyzed in uninjured FVB/N mice. Sections were incubated with rabbit anti-human EP2 IgG at a 1:500 dilution (A) or preimmune rabbit IgG (D), and bound antibody was visualized using biotin-conjugated tyramide amplification technique with 3,3'-diaminobenzidine as chromogenic substrate (brown pigment). A: EP2 immunoreactivity is seen in the smooth muscle layer and in scattered lamina propria cells (thin arrows). Abundant immunoreactive EP2 is also observed in the nuclei of crypt epithelial cells (arrowheads). Villus epithelial cells, however, exhibit lateral membrane and basilar cytoplasmic EP2 staining (large arrows). The transition between crypt nuclear epithelial staining and membranous villus epithelial staining is evident at the crypt villus junction (red arrow). No staining was observed when preimmune rabbit IgG was substituted for EP2 (D) or when primary antibody was omitted (data not shown). B: ×40 magnification of an intestinal crypt. C: ×40 magnification of intestinal villi.



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Fig. 5.   Immunohistochemical localization of EP2 in small intestinal regenerative crypts. Cellular localization of EP2 was analyzed in gamma -irradiated (13 Gy) FVB/n mice. Irradiated mice were killed 84 h after irradiation. Sections were incubated with 1:500 dilution of rabbit anti-human polyclonal antibody, and immunohistochemical detection for EP2 was performed as described. Regenerating crypts were readily detected by routine hematoxylin and eosin staining (data no shown). EP2 staining was observed in scattered nonepithelial cells in the lamina propria. A: abundant staining was observed in the nuclei of regenerative crypts. B: at higher magnification, nuclear staining in the regenerative crypt is evident (arrow). Immunoreactive EP2 is detected in occasional lamina propria cells (arrowhead). C: immunohistochemical localization of EP2 in mouse colon. Cellular localization of colonic EP2 was analyzed FVB/n mice. Sections were incubated with 1:500 dilution of rabbit anti-human EP2 polyclonal antibody. Bound antibody was visualized using biotin-conjugated tyramide amplification system as described earlier. EP2 immunoreactivity is present in the smooth muscle layer and in scattered lamina propria cells. EP2 is seen in the nuclei of colonic crypt epithelial cells (arrowhead); the surface epithelial cells exhibit cytoplasmic and lateral membrane staining for EP2 (arrow).

EP2 mRNA levels are increased in the mouse colon after radiation injury. The expression of EP2 mRNA in the colon after irradiation was examined by RNase protection analysis (Fig. 2B). In the colon, EP2 mRNA levels were low and constant for 48-72 h; however, at 96 h, there was nearly a 2.5-fold increase over levels observed in unirradiated mice. The radiation-induced increase in EP2 mRNA levels was not as pronounced as those observed in the small intestine and occurred later (96 h; Fig. 2B). These differences in EP2 expression may reflect differences in regeneration kinetics between the colon and small intestine following radiation injury (6).

Immunoreactive EP2 protein was detected in uninjured mouse colon. We observed a similar differential staining pattern to what was observed in the small intestine (Fig. 5C). In colonic crypt epithelial cells, EP2 is expressed in the nuclei (Fig. 5C, arrowhead). Staining was also observed in occasional lamina propria cells and in smooth muscle cells in the basement membrane. Surface epithelial cells exhibited basolateral membrane staining (Fig. 5C, arrow). Thus, in both the mouse small intestine and colon of FVB/N mice, the EP2 receptor is expressed differentially in crypt and villus/surface epithelial cells.

Intestinal EP4 mRNA levels are increased following radiation injury. In light of the increased epithelial expression of EP2 observed after radiation injury, we sought to determine whether the EP4 receptor was similarly upregulated following radiation injury. EP4 has been previously shown to be expressed in gastrointestinal epithelial cells (49), and activation of the EP4 receptor following ligand binding results in increases in intracellular cAMP similar to that observed following EP2 receptor stimulation (33). Radiation injury increases EP4 mRNA expression in the proximal jejunum (Fig. 6A) of adult mice. EP4 mRNA was detectable in the proximal jejunum of unirradiated mice; levels fell at 4 h but returned to baseline by 12 h after irradiation. EP4 mRNA levels peaked at 84 h after 13 Gy gamma -irradiation at levels nearly twofold higher than those in unirradiated mice (Fig. 6A). In the colon, however, there was a rapid upregulation of the EP4-receptor mRNA at 1 h after irradiation to levels approximately twofold higher than unirradiated controls. There was a gradual decrease, however, in EP4 mRNA between 4 and 96 h to levels ~50% below baseline (Fig. 6B).


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Fig. 6.   Time course of EP4 mRNA expression after irradiation in the mouse small intestine and colon. FVB/n mice received 13 Gy gamma -irradiation in a cesium irradiator at 0.96 cGy/min. Total cellular RNA was prepared from the distal jejunum (A) and colon (B) of mice killed at indicated times after irradiation. EP4 and actin mRNA levels were determined by RNase protection assays. Protected bands were separated by denaturing PAGE, and gels were analyzed by autoradiography. Autoradiographs for EP2 were exposed overnight and actin for 4 h. Bottom: quantitation of RNase protection assays for EP4 shown at top. The intensity of the major bands protected for EP4 mRNA was determined by scanning densitometry on autoradiographs of RNase protection assays at each time point. Data were normalized for abundance of actin mRNA present in each sample. Data are the average of EP4 mRNA levels determined from pooled analysis of 6 mice for each time point.

Radiation-induced apoptosis was increased in EP2-/- mice. In view of the apparent roles for both COX-1 and PGE2 in epithelial repair following radiation injury, as well as the potential importance of apoptosis in removing genetically damaged epithelial cells, we examined crypt epithelial cell apoptosis in mice lacking the EP2 receptor and their wild-type littermates. Spontaneous apoptosis occurred infrequently in both wild-type and in EP2-/- mice (Fig. 7B). Radiation-injury (12 Gy) resulted in a large increase in apoptosis in both groups of mice compared with unirradiated mice (Fig. 7, A and B). Radiation-induced apoptosis, however, was significantly increased in intestinal crypt epithelial cells of EP2-/- mice compared with their wild-type littermates' (5.47 vs. 3.02) apoptotic cells per crypt (±SE; n = 4 mice/group. P < 0.001 for wild-type vs. EP2-/-).


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Fig. 7.   The distribution of apoptotic cells in EP2-/- mice and wild-type littermates. Mice received 12 Gy of gamma -irradiation and were analyzed 6 h later. A: apoptotic cells within the intestinal epithelium of the jejunum are demonstrated by morphological criteria during routine hematoxylin and eosin staining (top). Note that apoptotic bodies are most abundant in the lower half of the crypt in both wild-type and EP2-/- mice. a, EP+/+; b, EP-/-. B: radiation-induced apoptosis in wild-type or EP2-/- mice. Mice received either 0 or 12 Gy of gamma -irradiation and were killed 6 h later. Apoptosis was quantified using previously described histologic criteria (37). Data are expressed as means ± SE; n = 4 mice/group. * P < 0.001 for wild-type vs. EP2-/-.

Mice lacking functional EP2 had impaired intestinal crypt stem cell survival after irradiation. We have previously shown that mice that lack functional COX-1 had significantly diminished crypt stem cell survival after 13 Gy gamma  -irradiation (4.9 surviving crypts/cross section) compared with wild-type littermates (10.1 surviving crypts/cross section) (20). Crypt survival in COX-2-/- mice, however, was the same as in wild-type mice. Crypt survival was diminished in EP2-/- mice (4.06 surviving crypts/cross section) compared with wild-type littermates (8.15 surviving crypts/cross section) (Fig. 8). The ~50% reduction in crypt stem cell survival (P < 0.003 for wild-type vs. EP2-/- mice) is similar to the 52% reduction observed in COX-1-/- mice and the ~60% reduction in crypt stem cell survival previously reported in FVB/n mice that received the nonspecific cyclooxygenase inhibitor indomethacin (10). Thus the lack of functional EP2 receptor recapitulated the effects of decreased intestinal PGE2 observed in COX-1-/- mice and in indomethacin-treated mice. Together, these data suggest that the effects of radiation injury on intestinal crypt survival and crypt epithelial apoptosis are mediated, at least in part, through the EP2 receptor.


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Fig. 8.   Crypt survival after gamma -irradiation in wild-type and EP2-/- mice. Mice received 13 Gy of total body gamma -irradiation and were killed 3.5 days later. All animals received 5-bromo-2'-deoxyuridine 2 h before death to label replicating epithelial cells. Crypt survival was measured using a modified microcolony assay (10). Data are expressed as means ± SE; n = 7 mice/group. *P < 0.003 for wild-type vs. EP2-/- mice.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have demonstrated that the EP2 receptor is expressed in intestinal epithelial cells and is induced by radiation. Moreover, EP2 mediates the prosurvival and antiapoptotic effects of PGE2 in the irradiated intestinal epithelium.

In intestinal crypt epithelial cells, EP2 is expressed primarily in the nuclei (Fig. 4, A and B); whereas in villus epithelial cells, EP2 exhibits a plasma membrane distribution. These differences in receptor expression parallel gut epithelial cell differentiation. In the less-differentiated crypt epithelial cells, the receptor is predominantly nuclear; as the epithelial cells migrate out of the crypt, EP2 expression is targeted to the plasma membrane. These differences in cellular distribution suggest potential functional differences for this receptor. Nuclear EP2 may be responsible for transducing PGE2 signals that turn on nuclear transcription pathways that protect crypt epithelial cells from genotoxic injury. The effects of dimethyl PGE2 both in enhancing crypt survival and diminishing apoptosis may be mediated through nuclear EP2 in crypt stem cells. In other cell types, including NIH3T3 cells, there is nuclear membrane localization of COX-1 and -2 and PGE2 (52). In addition, a transporter has been identified that mediates the influx of prostanoids across the plasma membrane (23, 29), and Ca-dependent cPLA2 can be transported to the nuclear envelope (45), Finally, growth factor-induced translocation of COX-2 to the nuclear envelope has been described (8). Furthermore, functional EP1, EP3, and EP4 are localized to the nucleus in porcine brain and rat liver (4).

The presence of EP2 on nonepithelial cells may also be significant in the regulation of intestinal crypt defense in vivo. For example, functional EP2 receptors have been described in corneal endothelial cells (21, 22), and endothelial cell signaling may play a role in the intestinal radiation syndrome (35). The appearance of EP2-expressing lamina propria cells after irradiation suggests a role for these cells in repair following severe injury. Further studies are planned to better define the specific cell populations that express EP2 that may play a significant role in epithelial defense against radiation injury. Although villus epithelial cell EP2 signaling is unlikely to be involved in promoting crypt survival or downregulating apoptosis, EP2 signaling in these cells may mediate other gastrointestinal effects of PGE2 and may be responsible for many of the gastrointestinal effects of PGE2 including nutrient absorption, secretion, and villus epithelial defense (2, 16, 18).

The time course and distribution of EP2 expression after radiation fits with the suggestion that EP2 is a component of an adaptive response to radiation injury. EP2 protein expression peaks at 48 h after radiation, a time when disruption of epithelial integrity and early crypt regeneration are the predominant morphological features of the intestine. This is also the time when COX-1 is expressed and PGE2 production in regenerative crypts increases. Forty-eight hours after radiation injury is also the time when exogenous dimethyl PGE2 increases crypt survival (10). The rapidly proliferating cells of the regenerative crypt express both COX-1 and EP2 at 48 h after radiation, raising the possibility that PGE2 produced in the cytoplasm of these cells binds to nuclear EP2 receptors resulting in enhanced proliferation and diminished apoptosis.

EP4-receptor mRNA increased following radiation injury; expression peaked at 84 h after irradiation. This is also the time when peak crypt regeneration is occurring in the small intestine. In the colon, a twofold increase in EP4-receptor mRNA was observed at 1 h, with declining expression seen throughout the postirradiation period. The significance of the different induction responses of EP4 in the intestine and colon is unclear. However, increased EP4 mRNA expression in the small intestine suggests a potential role for the EP4 receptor in mediating the intestinal response to radiation injury. The mechanism by which radiation injury induces EP2 and EP4 expression is not known. Ionizing radiation and other forms of DNA damage induce the expression of a number of transcription factors in mammalian cells such as (c-Fos, c-Jun, EGR-1, and NF-kappa b), which may be involved in inducing EP2 and/or EP4 expression (17, 25, 27, 55).

PGE2 plays a key role in regulating the epithelial response to radiation injury. Both endogenous and exogenous PGE2 are radioprotective in the intestine (10, 43). Endogenous PGE2 increases the number of surviving crypts after radiation injury and decreases the number of crypt epithelial cells undergoing radiation-induced apoptosis (10, 20). In EP2-/- mice, crypt survival after radiation is reduced and radiation-induced apoptosis is increased, demonstrating that the effects of PGE2 on crypt survival and apoptosis after radiation are mediated through EP2. PGE2 signaling through the EP2 receptor involves activation of adenyl cyclase and production of cAMP. It is likely that the effects of PGE2 on crypt survival and apoptosis after radiation are mediated through cAMP; however, the association of adenyl cyclase and cAMP was described with plasma membrane EP2 receptors. Whether the same signaling mechanisms with nuclear EP2 receptors use the same signaling mechanisms is unknown. We cannot completely rule out in this study the potential contribution of EP4 receptors in the regulation of the response to radiation.

PG production and tumorigenesis are strongly associated. Mice homozygous for a disrupted COX-2 gene and a mutant APC gene developed fewer intestinal tumors than mice that have an APC mutation alone (34). Similarly, mice homozygous for a disrupted COX-1 gene and a mutant APC have fewer intestinal tumors than mice with an APC mutation alone (7). A recent study suggests a role for EP2 in mediating the effects of PGE2 on intestinal tumorigenesis. Mice with a disrupted EP2 gene and a mutant APC gene developed smaller and fewer polyps than mice with a mutant APC gene alone (51). These data lend support to the hypothesis that PGE2 signaling through the EP2 receptor accelerates intestinal tumorigenesis. In the present study, we demonstrate that EP2-/- mice have larger numbers of apoptotic cells in response to radiation injury than wild-type mice. It may be that the decreased tumorigenesis seen in the EP2-compound mutant mice is due to increased apoptosis.

In this study, we have demonstrated that EP2 receptors are expressed in gastrointestinal epithelial cells in mice. EP2 mRNA and protein are upregulated following radiation injury in mice. EP2-/- mice have impaired intestinal crypt stem cell survival and increased epithelial apoptosis in response to radiation injury. Together, these studies suggest an important role for PGE2 acting through the EP2 receptor in mediating intestinal epithelial apoptosis in response to radiation injury.


    ACKNOWLEDGEMENTS

This work was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-02822 and a Robert Wood Johnson Foundation Minority Faculty Development Award (to C. W. Houchen), NIDDK Grants DK-52574 (to C. W. Houchen, S. Anant, and W. F. Stenson) and DK-33165 and DK-55753 (to W. F. Stenson), National Institutes of Health Grants GM-15431 and DK-46205 (to R. M. Breyer) and DK-62265 (to S. Anant). S. Anant is a Research Scholar of the American Gastroenterology Association.


    FOOTNOTES

Address for reprint requests and other correspondence: C. W. Houchen, Dept. of Medicine, Div. of Gastroenterology, Box 8124, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: chouchen{at}im.wustl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 13, 2002;10.1152/ajpgi.00240.2002

Received 20 June 2002; accepted in final form 6 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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