1 Division of Gastroenterology, Washington University, Saint Louis, Missouri; and 2 Division of Gastroenterology and Hepatology, University of Virginia, Charlottesville, Virginia
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ABSTRACT |
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Prostaglandins may play an important role in
regulating normal renewal of gastrointestinal epithelium, epithelial
injury repair, and initiation or progression of intestinal neoplasia. Synthesis of prostaglandins is catalyzed by either of two
cyclooxygenase isoforms, Cox-1 and Cox-2. Cox-1 is the predominant
cyclooxygenase isoform found in the normal intestine. In contrast,
Cox-2 is present at low levels in normal intestine but is elevated at
sites of inflammation and in adenomas and carcinomas. To determine
directly whether prostaglandins synthesized by Cox-1 or Cox-2 regulate crypt epithelial cell fate after genotoxic or cytotoxic injury, we
examined apoptosis, prostaglandin synthesis, and crypt stem cell
survival after -irradiation in Cox-1
/
and
Cox-2
/
mice. Cox-1
/
mice had increased
crypt epithelial cell apoptosis and decreased clonogenic stem cell
survival compared with wild-type littermates. PGE2
synthesis was also diminished in Cox-1
/
mice compared
with wild-type controls in unstressed intestine and after radiation
injury. In contrast, apoptosis, stem cell survival, and intestinal
PGE2 synthesis in Cox-2
/
mice after
irradiation were the same as in wild-type littermates. Crypt stem cell
survival after irradiation was inhibited by a highly specific
neutralizing antibody to PGE2, suggesting that this
prostaglandin mediates stem cell fate in vivo. These data suggest that
prostaglandins synthesized by Cox-1 regulate multiple steps that
determine the fate of crypt epithelial cell after genotoxic or
cytotoxic injury.
cyclooxygenase; ionizing radiation; prostaglandin E2; intestinal epithelium; gastrointestinal malignancy
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INTRODUCTION |
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ALTHOUGH PROSTAGLANDINS ARE thought to play an important role in normal turnover, injury repair, and neoplasia arising in the gastrointestinal epithelium, the molecular mechanisms through which they act are not yet clear. The synthesis of prostaglandins from arachidonic acid is catalyzed by either of two cyclooxygenase isoforms, Cox-1 and Cox-2 (6, 9). Although Cox-1 and Cox-2 both catalyze the synthesis of prostaglandins, these cyclooxygenase isoforms have different biological roles. In most circumstances Cox-1 is constitutively expressed. Prostaglandins produced through Cox-1 are thought to play a key role in protection of the gastrointestinal mucosa from injury and/or injury repair. In contrast, prostaglandins produced through Cox-2 are an important component of the inflammatory response. Cox-2 is expressed at low levels in normal gastric, small intestinal, and colonic epithelium but is induced in macrophages and other cell types at sites of inflammation and injury by proinflammatory cytokines (31-33). Cox-2 is also expressed at high levels in human colon cancers and adenomas and in spontaneously arising adenomas in mice that carry mutations in the APC gene (4, 5, 11, 31, 37). A biological role for Cox-2 in the formation or progression of intestinal adenomas was suggested by the observation that mice with the APC mutation that also lacked functional Cox-2 developed fewer adenomas than mice carrying the APC mutation alone (1, 19).
In the normal intestine and colon, differentiated epithelial cells are
continuously and rapidly replaced by replication of undifferentiated
cells within the crypt (26). These undifferentiated, replicating crypt epithelial cells are, in turn, derived from a small
number of multipotent stem cells located near the base of each crypt.
The restoration of normal intestinal epithelial architecture after
injury such as that seen with ingested toxins, chemical carcinogens,
chemotherapeutic drugs, or -irradiation, is a multistep process that
must ultimately involve changes in the dynamics of epithelial stem cell
replication within the crypt. The response of the intestinal epithelium
to
-irradiation injury has been the most extensively studied model
system for investigating the fate of crypt epithelial stem cells and
their descendants after injury (3, 21-25, 27, 28).
After epithelial injury, crypt epithelial cells undergo apoptosis or
cease replicating and are shed as they migrate up the intestinal villi
or onto the surface epithelium in the colon. 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
epithelium (21, 27). The fate of epithelial stem cells after injury and their capacity to regenerate the crypt epithelium primarily have been studied using the microcolony formation assay. The
number of regenerating crypts can be scored by microscopic appearance
3-4 days after injury and used as a surrogate measure of crypt
stem cell survival after radiation injury and injury induced by other
cytotoxic or genotoxic agents.
Regulation of crypt epithelial apoptosis and stem cell fate after potentially mutagenic or carcinogenic injury may also be an important factor in the initiation of gastrointestinal neoplasia. Gastrointestinal adenomas and carcinomas arise through the acquisition of multiple, independent genetic mutations and subsequent clonal expansion of mutated epithelial stem cells or other long-lived progenitor cells that reside within the crypt (7, 8). Programmed cell death, or apoptosis, is the predominant biological response of crypt epithelial cells to low levels of genotoxic and cytotoxic damage that occur chronically in the small intestine and colon (24, 28). This process results in the removal of individual genetically damaged cells from the crypt epithelium. Thus it has been suggested that apoptosis is an effective cellular strategy for decreasing the probability that any particular crypt epithelial cell will survive injury and acquire the set of multiple mutations necessary for malignancy to occur (25, 28).
Recently, we reported (3) that intestinal prostaglandin
synthesis is induced by -irradiation and that treatment of mice with
indomethacin, a nonselective cyclooxygenase inhibitor, reduced survival
of intestinal epithelial stem cells after radiation injury. In the
normal intestine, Cox-1 was expressed predominantly in crypt epithelial
cells. Radiation injury resulted in a marked increase of Cox-1 within
regenerating crypt epithelial cells and a concomitant increase in
intestinal PGE2 levels. Treatment with indomethacin in the
period from 24 to 48 h after irradiation decreased PGE2 levels and decreased the number of surviving crypts.
However, recent studies have demonstrated that nonsteroidal
anti-inflammatory drugs (NSAIDs), such as indomethacin, may affect cell
transformation, proliferation, and apoptosis through mechanisms
distinct from their effects on prostaglandin synthesis
(38). Thus it is difficult to make inferences about the
effects of endogenous prostaglandins on crypt epithelial cell fate from
experiments using these pharmacological inhibitors.
In this study, we have used mice genetically lacking functional Cox-1 or Cox-2 to define directly the role of prostaglandins synthesized by each of these cyclooxygenases in the response of the intestinal epithelium to radiation injury. We report here that 1) Cox-1 deficient mice had decreased intestinal PGE2 synthesis, increased crypt epithelial cell apoptosis, and decreased crypt stem cell survival after radiation injury; 2) the response of Cox-2-deficient mice to radiation injury was indistinguishable from the response of wild-type mice; and 3) crypt stem cell survival after irradiation was inhibited by a highly specific neutralizing antibody to PGE2.
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MATERIALS AND METHODS |
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Animals.
The animals used in these studies were cared for in accordance with
approved University of Virginia and American Association for
Accreditation of Laboratory Animal Care guidelines. Mice were fed
standard laboratory mouse chow ad libitum and maintained in filter-top
microisolator caging in a specific pathogen-free environment, on a 12-h
(6:00 AM-6:00 PM) light-dark cycle. Mouse lines with targeted
disruption of Cox-1 or Cox-2 were obtained from Dr. R. Langenbach
(National Institute of Environmental Health Sciences, Research Triangle
Park, NC; Refs. 13, 17). Mice homozygous for the
disrupted Cox-1 gene (Cox-1/
) or for the disrupted
Cox-2 gene (Cox-2
/
) were obtained by crossing males
that were either heterozygous or homozygous for the respective
disrupted cyclooxygenase gene with the corresponding heterozygous
females. Genotype analysis on the progeny was performed by PCR analysis
or Southern blot analysis of tail DNA as previously described
(13, 17). Littermates that were homozygous wild type at
both the Cox-1 and Cox-2 loci were used as controls. Mice were
irradiated at the age of 15-20 wk in a Gamacel 40 cesium
irradiator at 0.94 cGy/min. Some mice received
dimethyl-PGE2 (10 µg/mouse ip) at the times indicated. FVB/N mice (Taconic, Germantown, NY) were used to determine the effect
of neutralizing anti-PGE2 antibody on crypt stem cell
survival. There was no statistically significant difference in crypt
survival after irradiation in untreated FVB/N mice compared with
homozygous wild-type littermates of the Cox-1
/
or
Cox-2
/
mice (data not shown). For these experiments,
mice received a mouse monoclonal neutralizing anti-PGE2
antibody (2B5, 0.2 mg/mouse ip; gift of Dr. J. Portanova, Monsanto, St.
Louis, MO) or an isotype-matched control antibody, MOPC21 (0.2 mg/mouse
ip), 24 h before and 24 h after
-irradiation
(16).
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 (Sigma) to label the S-phase cells. Proximal
jejunum was obtained by rapid dissection from mice 84 h after
irradiation and fixed in Bouin's fixative. Paraffin sections (5 µm)
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 by goat anti-BrdU (2),
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 (3). The
number of surviving crypts per cross section was determined for each
mouse by scoring the number of surviving crypts in eight complete,
well-oriented cross sections and dividing the total by the number of
cross sections scored. Because differences in the size of regenerating
crypts can affect the probability that a regenerating crypt will appear
in a cross section (23, 27), we determined the width of 15 representative crypts for homozygous wild-type, Cox-1/
,
and Cox-2
/
mice in longitudinal sections of proximal
jejunum at the widest point in each crypt. No differences were found in
the size of regenerating crypts in Cox-1
/
or
Cox-2
/
mice compared with homozygous wild-type mice
after 14-Gy
-irradiation (data not shown). Thus differences observed
in crypt survival cannot be accounted for by variation in the size of
regenerating crypts in the Cox-1
/
or
Cox-2
/
mice after radiation injury.
Apoptosis. The proximal jejunum was obtained from mice 6 h after irradiation, fixed overnight in 10% neutral-buffered formalin, and embedded in paraffin. Sections were prepared from the proximal jejunum oriented so that the sections were cut 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 (24). Briefly, only well-oriented crypts in longitudinal sections containing Paneth cells, a crypt lumen, and an uninterrupted column of epithelial cells extending to the crypt-villus junction were scored. An apoptotic cell was defined as a cell containing a single large fragment of condensed chromatin or a group of smaller fragments clustered together within an area that was similar to the size of an adjacent epithelial cell (24). Twenty well-oriented crypts were analyzed in each of eight jejunal cross sections for each mouse. Apoptotic cells were also detected in situ using the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) assay (Boehringer Mannheim; Indianapolis, IN) following the manufacturer's instructions. Before the labeling reaction was initiated, the deparaffinized tissue sections were incubated for 10 min with proteinase K at 20 µg/ml. Incorporated fluorescein-dUTP was detected using horseradish peroxidase-conjugated anti-fluorescein antibody, and bound antibody was visualized with diaminobenzidine. The number of apoptotic cells per crypt as assessed with the TUNEL assay was ~40% higher than those assessed by morphological criteria, but the pattern comparing test groups was identical.
Measurement of PGE2 levels. Lipids were extracted by homogenizing flash-frozen tissue in cold ethanol-0.1 M sodium phosphate, pH 4.0 (70%/30%, vol/vol) followed by shaking incubation at room temperature. An aliquot of the extract was dried down under a stream of nitrogen, and the PGE2 concentration was determined by a PGE2-specific ELISA (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's directions.
Statistical methods. Pairwise t-tests using the pooled estimate of variance and Bonferroni's correction of the P values for multiple comparisons were used for analysis of the effects of cyclooxygenase genotypes on crypt survival and radiation-induced apoptosis. Pairwise Wilcoxon Mann-Whitney tests with Bonferroni's adjustment of P values for multiple comparisons were used for analysis of the effects of radiation and cyclooxygenase genotype on intestinal PGE2 levels.
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RESULTS |
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Radiation-induced apoptosis was increased in mice lacking
functional Cox-1.
In view of the apparent roles of both Cox-1 and Cox-2 in epithelial
injury repair and in the development of intestinal neoplasia as well as
the potential importance of apoptosis as a mechanism for removing
genetically damaged epithelial cells, we examined crypt epithelial cell
apoptosis in wild-type mice and in mice lacking functional Cox-1 or
Cox-2. Apoptosis induced by radiation injury was significantly
increased in intestinal crypt epithelial cells of
Cox-1/
mice compared with wild-type littermates (Fig.
1). Spontaneous apoptosis occurred
infrequently in the crypts of uninjured wild-type mice (0.21 apoptotic
cells/crypt), Cox-1
/
mice (0.28 apoptotic cells/crypt),
or Cox-2
/
mice (0.20 apoptotic cells/crypt). Radiation
injury resulted in a large induction of apoptosis within the crypt
epithelium of all groups. The number of apoptotic cells per crypt
6 h after 8-Gy
-irradiation was 1.9-fold higher in the
Cox-1
/
mice (5.0 apoptotic cells/crypt) than in
wild-type mice (2.6 apoptotic cells/crypt) (Fig. 1). The lack of
functional Cox-2 did not affect radiation-induced apoptosis; the number
of apoptotic cells in the crypts of Cox-2
/
mice after
irradiation (2.5 apoptotic cells/crypt) was not significantly different
from that observed in wild-type mice (2.6 apoptotic cells/crypt).
Although apoptotic cells were found throughout the crypts, the majority
of apoptotic cells were located in the lower half of each crypt both in
Cox-1
/
mice and wild-type littermates (Fig.
2). Apoptotic cells were scarce in the
villus epithelium of either Cox-1
/
mice or wild-type
littermates (data not shown). These data suggest that the increase in
apoptosis was not caused by a change in gradient of susceptibility to
apoptosis along the crypt-to-villus axis of the gut. Clearly, it was
not simply the presence or absence of Cox-1 that determined the ability
of epithelial cells to undergo radiation-induced apoptosis because
prior studies have shown that in wild-type mice, differentiation of the
epithelial cells as they emerge onto the villus is associated with both
loss of Cox-1 expression (3) and loss of the ability to
undergo apoptosis in response to radiation (21, 22, 24),
whereas we observed that disruption of the Cox-1 gene resulted in
increased radiation-induced apoptosis in crypt epithelial cells.
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Mice lacking functional Cox-1 had impaired crypt stem cell survival
after irradiation.
Our previous study (3) showed that indomethacin, a
nonspecific cyclooxygenase inhibitor, decreased crypt stem cell
survival when given from 24 to 48 h after irradiation. Crypt
survival was also markedly diminished in Cox-1/
mice
(4.9 surviving crypts/cross section) compared with wild-type littermates (10.1 surviving crypts/cross section) (Fig.
4). However, crypt survival in
Cox-2
/
mice was the same as in wild-type mice. The 52%
reduction in crypt survival seen in the Cox-1
/
mice is
similar to the ~60% reduction in crypt survival previously reported
in wild-type FVB/N mice receiving indomethacin in the period after
radiation compared with mice not receiving indomethacin (3). Thus the lack of functional Cox-1, but not Cox-2,
recapitulated the effects of indomethacin on intestinal crypt survival,
and this suggests that prostaglandins produced by Cox-1 mediate the survival of intestinal epithelial stem cells after irradiation.
|
Intestinal PGE2 synthesis was impaired in
Cox-1/
mice after irradiation.
We previously found (3) that
-irradiation induced a
progressive rise in intestinal PGE2 levels in wild-type
FVB/N mice. The induction of PGE2 synthesis by
-irradiation was associated with enhanced crypt stem cell survival.
In the present study, nonirradiated Cox-1
/
mice had
diminished intestinal PGE2 levels compared with their wild-type littermates (4.7 vs. 16.3 pg/mg; P = 0.006)
(Fig. 5). In contrast, intestinal
PGE2 levels were not affected by the absence of functional
Cox-2; PGE2 levels in nonirradiated Cox-2
/
mice were similar to those in wild-type mice (16.1 vs. 16.3 pg/mg; P = 0.94). In mice that were homozygous wild type for
both Cox-1 and Cox-2, intestinal PGE2 levels increased
3.8-fold by 3.5 days after 13.8-Gy
-irradiation (16.3 to 61.2 pg/mg;
P = 0.007), an induction of PGE2 synthesis
similar to that previously seen in FVB/N mice after
-irradiation
(4). A similar increase in PGE2 levels was
observed after irradiation of Cox-2
/
mice (Fig. 5).
However, intestinal PGE2 levels failed to rise after
irradiation in the Cox-1
/
mice. Rather,
PGE2 levels in the Cox-1
/
mice fell after
irradiation to levels that were significantly lower than present in
nonirradiated Cox-1
/
mice (2.7 vs. 4.7 pg/mg;
P = 0.041). These data suggest that the intestinal
mucosa could not fully compensate for the chronic absence of functional
Cox-1 by increased Cox-2-mediated prostaglandin synthesis.
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Crypt stem cell survival after irradiation was mediated by
PGE2.
To determine whether the diminished crypt survival in
Cox-1/
mice was specifically related to decreased
PGE2 levels as opposed to changes in the levels of other
eicosanoids, we treated wild-type mice with a neutralizing
anti-PGE2 antibody, 2B5, beginning 24 h before 14-Gy
-irradiation. This antibody is able to inhibit the biological
activity of PGE2 in several experimental models of
inflammation and has <1% cross-reactivity with other eicosanoids, with the exception of PGE1 (16, 20). Treatment
of mice with anti-PGE2 reduced crypt survival to 51% of
the level compared with mice treated with MOPC21, an isotype-matched
control antibody (Fig. 6). Thus treatment
with anti-PGE2 beginning before irradiation recapitulated
the effects of both Cox-1 gene disruption and indomethacin administration. This suggests that the decrease in crypt survival seen
in indomethacin-treated mice and in Cox-1
/
mice was
specifically caused by decreased levels of PGE2.
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DISCUSSION |
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We report here that Cox-1/
mice had increased
crypt epithelial apoptosis, decreased survival of clonogenic crypt stem
cells, and impaired induction of prostaglandin synthesis after
radiation injury. Whether the lack of functional Cox-1 would also
affect the epithelial response to other types of injury (e.g.,
bacterial infection, chemotherapeutic agents, cytokines, reactive
oxygen species) or the response to other agents that can induce
apoptosis (e.g., chemical carcinogens, tumor necrosis factor-
, fas
ligand) has not yet been determined. Cox-1 is present in far greater
abundance than Cox-2 in the intestine after radiation injury
(3). The enhanced radiation-induced apoptosis and
diminished crypt survival observed in Cox-1
/
mice
combined with the absence of effects on these processes in
Cox-2
/
mice suggest that the prostaglandins that
regulate these early events during the epithelial response to radiation
injury are produced through Cox-1. However, the relative importance of
prostaglandins synthesized by Cox-1 or Cox-2 in epithelial wound repair
may depend on the nature of the injury and whether it is accompanied by
an inflammatory response. In circumstances in which epithelial injury is accompanied by significant inflammation or in adenomatous polyps, levels of Cox-2 are much higher than are found in either the normal intestine or in the intestine after radiation injury (4, 5, 11,
29, 33, 37). Recent studies demonstrated that prostaglandins synthesized by Cox-2 can accelerate healing of gastric ulceration (15, 30). Prostaglandins derived from Cox-1 and/or Cox-2
appear to play a role in reducing epithelial injury in dextran sodium sulfate-induced colitis (18, 35). Thus it is possible that prostaglandins produced by Cox-2 can also prevent epithelial damage or
improve healing of epithelial injuries by suppressing crypt epithelial
apoptosis or enhancing stem cell survival. Nevertheless, an important
role for prostaglandins produced through Cox-1 in the regulation of
crypt epithelial apoptosis and stem cell survival during epithelial
injury repair would be consistent with the hypothesis that combined
Cox-1 and Cox-2 inhibitors such as indomethacin increase epithelial
cell death after injury or impair epithelial wound healing in addition
to the direct toxic effects of these NSAIDs on the gastric and
intestinal mucosa (29, 34).
After damage produced by -irradiation or other agents that produce
DNA damage, crypt epithelial cells can undergo apoptosis or can attempt
to repair the DNA damage (28). Mutations may result within
the surviving epithelial cells via errors in the DNA repair process.
Previous studies have suggested that neoplasia in the intestinal
epithelium requires acquisition of multiple, independent somatic
mutations within functionally anchored epithelial stem cells and other
long-lived progenitor cells in the crypt (7, 8). Apoptosis
induced by radiation injury, chemical carcinogens, or other
DNA-damaging agents occurs most prominently in the lower half of the
intestinal crypt epithelium, a region of the intestinal epithelium
where these epithelial progenitor cells reside (10, 14, 25,
28). Studies in mice that have a mutation in the mouse homolog
of the gene responsible for familial adenomatous polyposis
(APC) suggest that prostaglandins produced by both Cox-1 and
Cox-2 are important in regulating the development of adenomas arising
from the intestinal epithelium (1, 19). Cox-2 was not
present in the normal intestine or colon of mice bearing a mutant
APC but was induced at very early stages of adenoma formation (19). Mice that had a mutant APC and
were homozygous for a disrupted Cox-2 gene (Cox-2
/
)
developed fewer colonic adenomas than mice that have the mutant APC alone, suggesting that prostaglandins synthesized by
Cox-2 are important mediators of adenoma formation or growth
(19). However, Cox-1
/
/APC
mutant mice also developed fewer adenomas than mice with only the
APC mutation despite the presence of functional Cox-2 in
these mice (1). When that finding is viewed in the light of the data presented here, it raises the possibility that
Cox-1
/
/APC mutant mice had fewer adenomas in
part because the diminished level of PGE2 in these mice
resulted in increased loss of damaged crypt epithelial cells and/or
decreased numbers of surviving clonogenic stem cells with potential
mutations leading to adenoma formation. However, the relationship
between alterations in crypt epithelial cell fate after
radiation-induced or carcinogen-induced epithelial injury and changes
in the frequency of adenoma formation has not yet been determined.
Crypt survival is the result of the successful completion of a series
of steps after irradiation (21). First, intestinal stem
cells in the base of the crypt must survive radiation injury. These
surviving stem cells must then give rise to a more actively proliferating transit cell population and form a regenerative crypt.
Finally, the transit cells must proliferate to expand their numbers and
give rise to all of the differentiated cell types found in the small
intestinal or colonic epithelium. In the normal intestine, Cox-1 is
expressed in all crypt epithelial cells, including stem cells, transit
cells, and Paneth cells (3). After radiation injury, the
transit cells cease replicating and are rapidly lost from the crypt via
apoptosis or by migration onto the villus epithelium. Our previous
studies (3) with indomethacin demonstrated that inhibition
of prostaglandin synthesis had the greatest effect in decreasing crypt
survival during the period from 24 to 48 h after radiation, when
Paneth cells and replicating crypt transit cells have largely
disappeared from the injured crypt. Thus one possible explanation for
the decrease in crypt survival seen in the Cox-1/
mice
is that Cox-1-mediated prostaglandin synthesis in crypt stem cells
after irradiation plays an important role in maintaining maximal stem
cell survival.
We found that production of PGE2 by the intestinal
epithelium was suppressed in Cox-1/
mice both at
baseline and after radiation injury. Previous studies also found that
gastric PGE2 levels in Cox-1
/
mice are
<1% that of their wild-type littermates (13). Why, then,
does disruption of the Cox-1 gene in mice recapitulate the effects of
indomethacin in radiation injury although it is not associated with
gastric ulceration in the unstressed condition? The most likely
explanation is that prostaglandins produced through Cox-1 are not
required for the maintenance of epithelial integrity in the absence of
injury but are required for resistance of the epithelium to damage or
for effective injury repair. A recent study that showed that absence of
either functional Cox-1 or Cox-2 exacerbated dextran sodium
sulfate-induced colitis is consistent with this possibility
(18). An alternative explanation is that prostaglandins
produced by Cox-1 are important for both the maintenance of epithelial
integrity in the absence of injury and for the epithelial response to
injury but that the Cox-1
/
mouse has developed a
compensatory mechanism for the absence of prostaglandin synthesis by
Cox-1 only in the unstressed condition and not in response to injury.
Increases in Cox-2 activity or phospholipase A2 activity,
for example, could maintain prostaglandin levels, compensating for the
loss of functional Cox-1 in Cox-1
/
mice. However, our
data suggest that this latter possibility is unlikely because
prostaglandin levels in uninjured Cox-1
/
mice were
markedly reduced compared with levels present in wild-type mice and
prostaglandin levels failed to increase in the Cox-1
/
mice after radiation injury.
The mechanism by which decreased crypt epithelial cell PGE2
production reduces crypt stem cell survival after irradiation is not
yet clear. We found that Cox-1/
mice had both increased
crypt epithelial apoptosis and decreased crypt survival after radiation
injury. These events could be related if one mechanism for the decrease
in crypt survival in Cox-1
/
mice is enhanced loss of
crypt stem cells through apoptosis. However, it should also be noted
that Cox-1
/
mice had diminished intestinal
PGE2 levels both before and after irradiation. We found
that dimethyl-PGE2 suppressed apoptosis in both wild-type
and Cox-1
/
mice when given either 1 h before or
1 h after irradiation. Furthermore, we observed significantly
increased radiation-induced apoptosis in Cox-1
/
mice by
6 h after irradiation. Both of these observations demonstrate the
existence of a prostaglandin-sensitive step important for regulation of
apoptosis that occurs well before the critical time period identified
in our previous study (3) for inhibition of crypt stem
cell survival by indomethacin given after irradiation. Together with
our previous study, these data suggest that prostaglandins synthesized
by Cox-1 are important in regulating multiple, distinct events that
determine the fate of damaged epithelial cells within the crypt after
genotoxic and/or cytotoxic injury.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Frank Harrell for assistance with the statistical analysis and Dr. Jenny Buzan for critically reviewing this manuscript.
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FOOTNOTES |
---|
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-50924 (S. M. Cohn), R01-DK-33165 (W. F. Stenson), and R01-DK-55753 (W. F. Stenson) and a Robert Wood Johnson Minority Medical Faculty Development Award (C. W. Houchen).
Address for reprint requests and other correspondence: S. M. Cohn, Division of Gastroenterology and Hepatology, Univ. of Virginia Health System, PO Box 800708, Charlottesville, VA 22908-0708 (E-mail: sc6w{at}virginia.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.
Received 30 September 1999; accepted in final form 4 May 2000.
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