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
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ABSTRACT |
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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
-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
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INTRODUCTION |
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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-1 and TNF-
(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 -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
-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.
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MATERIALS AND METHODS |
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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
-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
-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 - 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.
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RESULTS |
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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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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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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 -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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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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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 -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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DISCUSSION |
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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-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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