Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice

Craig M. Coopersmith1,2, David O'Donnell1, and Jeffrey I. Gordon1

Departments of 1 Molecular Biology and Pharmacology and 2 Surgery, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Little is known about the effects of ischemia-reperfusion on the inductive, commitment, or execution phases of apoptosis. We have created a genetically defined model to study the response of small intestinal epithelial cells to ischemia-reperfusion injury as a function of their proliferative status and differentiation. Occlusion of the superior mesenteric artery for 20 min in adult FVB/N or C57BL/6 mice results in the appearance of TUNEL-positive apoptotic cells in the jejunal epithelium within 4 h, with a maximum response occurring at 24 h. Stimulation of apoptosis is greater in postmitotic, differentiated epithelial cells located in the upper portions of villi compared with undifferentiated, proliferating cells in the crypts of Lieberkühn (7-fold vs. 2-fold relative to sham-operated controls). Comparisons of p53+/+ and p53-/- mice established that the apoptosis is p53 independent. To further characterize this response, we generated FVB/N transgenic mice that express human Bcl-2 in epithelial cells distributed from the base of crypts to the tips of their associated villi. The fivefold elevation in steady-state Bcl-2 concentration is not accompanied by detectable changes in the levels or cellular distributions of the related anti-apoptotic regulator Bcl-xL or of the proapoptotic regulators Bax and Bak and does not produce detectable effects on basal proliferation, differentiation, or death programs. The apoptotic response to ischemia-reperfusion is reduced twofold in the crypts and villi of transgenic mice compared with their normal littermates. These results suggest that both undifferentiated and differentiated cells undergo a commitment phase that is sensitive to Bcl-2. Forced expression of Bcl-2 also suppressed the p53-dependent death that occurs in proliferating crypt epithelial cells following gamma -irradiation. Thus suppressibility by Bcl-2 operationally defines a common feature of the apoptosis induced in the crypt epithelium by these two stimuli.

programmed cell death; intestinal epithelial differentiation; gamma -irradiation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EXPERIMENTAL MODELS have shown that ischemia-reperfusion causes postmitotic cell lineages in the brain, myocardium, kidney, and adrenal cortex to undergo apoptosis (e.g., Refs. 18, 50, 55, and 70). These tissues do not allow evaluation of the effects of proliferative status or state of differentiation on the apoptotic response to this inductive stimulus. The intestinal epithelium provides an excellent system for such an analysis.

Renewal of the adult mouse small intestinal epithelium takes place continuously in anatomically distinct crypt-villus units. The epithelium is replaced on average every 60 h (8, 9). Proliferation is restricted to mucosal invaginations known as crypts of Lieberkühn. All epithelial cells in each crypt are derived from an uncertain number of multipotent stem cells located at or near the base of the crypt (2, 3, 6, 7, 10-12, 42, 67). Descendants of the stem cells are amplified through approximately four to six rounds of cell division, forming a rapidly cycling transit cell population in the midportion of each crypt (53). Three of the four principal epithelial cell lineages descended from the stem cell differentiate as they move from a crypt up an adjacent villus: absorptive enterocytes (representing >80% of all epithelial cells), mucus-producing goblet cells, and enteroendocrine cells. It takes 2-5 days for members of these lineages to travel from the crypt to the apex of the villus (6, 10-12) where they are removed by apoptosis and/or exfoliation (27). Members of the Paneth lineage differentiate during a downward migration from the stem-cell zone to the base of the crypt where they reside for ~20 days (2, 7).

Apoptosis has been divided into three phases (25): 1) an initial induction phase that is dependent on the signal transduction repertoire possessed by a given cell lineage and on the nature of the inductive stimulus, 2) a commitment phase that is dependent at least in part on the translocation of cytochrome c from mitochondria to the cytoplasm (19, 34, 36, 64), and 3) a final execution phase that leads to death and is characterized by stereotyped morphological changes regardless of the inductive signal. The execution phase appears to have been highly conserved during evolution, is completed in ~1 h, and is associated with degradation of cellular proteins by members of the caspase family of cysteine proteases (21, 22, 31, 44, 47, 56, 58, 68). Attention has been paid to some aspects of the execution phase of ischemia-reperfusion-stimulated programmed cell death in other tissues. Given the anatomically well-defined stratification of proliferation and differentiation programs along its crypt-villus axis, the self-renewing intestinal epithelium provides an opportunity to consider whether these programs affect the induction or commitment phases. We have addressed this issue by first characterizing the temporal and spatial features of the apoptotic response that follows transient occlusion of the principal artery supplying the small intestine. We then examined the effects of forced expression of Bcl-2 on the response.

We chose Bcl-2 because of the "position" it occupies in programmed cell death. Augmented expression of Bcl-2 or the related protein Bcl-xL blocks translocation of cytochrome c following application of a death-inducing stimulus (20, 34, 36, 64, 69), whereas augmented expression of Bax promotes translocation (32, 57). Cytochrome c binds Apaf-1 and procaspace-9. In the presence of dATP, active caspace-9 is generated, allowing activation of other capases and proteolytic processing of their substrates (37, 40, 41, 58). These findings suggest that Bcl-2 and related family members function as anti-apoptotic regulators by preventing or delaying release of cytochrome c, perhaps through their ability to influence the creation, maintenance, or function of mitochondrial membrane channels (e.g., Ref. 39). This conceptualization places Bcl-2 in a position to regulate the commitment phase of programmed cell death. Forced expression of Bcl-2 could be viewed as delaying commitment to programmed cell death or cellular competency to progress to the execution phase (17). Such a delay, in turn, may allow cells to marshall additional responses that oppose death. Our studies now establish that ischemia-reperfusion induces a p53-independent apoptosis in the intestinal epithelium that is greater on the villus than in the crypt and is partially suppressible at both sites by Bcl-2.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of Fabpl-Bcl-2 transgenic mice. A plasmid containing a full-length human Bcl-2 cDNA (p3194 from Stanley Korsmeyer, Washington University) was digested with EcoR I, releasing an 820-bp fragment containing the Bcl-2 open reading frame. pLf-SV40 Bluescript contains nucleotides -596 to +21 of a rat fatty acid binding protein gene (Fabpl) linked to a 0.9-kb fragment composed of simian virus (SV)-40 early splice and late polyadenylation signals (35). The 0.8-kb EcoR I Bcl-2 fragment was ligated to EcoR I-digested pLf-SV40 Bluescript yielding pLf-Bcl-2-SV40 Bluescript. Its Fabpl-596 to +21-Bcl-2-SV40 insert was excised as a 2.3-kb fragment using Sal I and Xba I. The fragment was purified and injected into FVB/N oocytes using standard techniques (29). Liveborn animals (n = 93) were screened for the presence of the Bcl-2 transgene using PCR with tail DNA as the template and primer pairs that amplify a 356-bp portion of the SV40 splice-polyadenylation sequence (5'-ATGAATGGGAGCAGTGGTG-3' and 5'-GCAGACACTCTATGCCTGTGTGG-3'). Nine FVB/N Fabpl-Bcl-2 founders were identified. Pedigrees were established from two of these founders and maintained by crosses to their normal FVB/N littermates.

Housing of mice. C57BL/6 mice homozygous for wild-type or null alleles of the p53 gene were purchased from GenPharm International. These animals, as well as Fabpl-Bcl-2 mice and their normal littermates, were kept in microisolator cages under a strict light cycle (lights on at 0600 and off at 1800). Animals were given a standard irradiated chow diet (Pico rodent chow 20, Purina Mills) ad libitum. Routine screens for hepatitis, minute, lymphocytic choriomeningitis, ectromelia, polyoma, sendai, pneumonia, and mouse adenoviruses, enteric bacterial pathogens, and parasites were negative.

Confirmation of transgene expression by Western analysis. Proteins were extracted from a 2-cm segment taken from the junction of the proximal and distal halves of the small intestine. The segments were lyophilized, pulverized, and resuspended in extraction buffer [40 mM Tris (pH 6.8), 2% 2-mercaptoethanol, 1% SDS, 5% glycerol, 10 mM EDTA, 50 µg/ml aprotinin, 50 µg/ml leupeptin, 500 µg/ml 4-(2-aminoethyl)-benzene sulfonyl fluoride, and 10 µg/ml pepstatin A; 50 µg lyophilized tissue/ml extraction buffer]. Samples were then boiled for 5 min, and insoluble debris was removed by centrifugation for 3 min at 12,000 g. The protein concentrations of the supernatants were determined using the DC protein assay kit (Bio-Rad). Cellular proteins (75 µg/sample) were fractionated by SDS-PAGE (38) and transferred electrophoretically to polyvinylidene difluoride membranes (Amersham). Membranes were stained with Ponceau red to verify equivalent transfer of samples from each lane. Blots were pretreated in blocking buffer (1% gelatin, 0.2% Tween 20 in PBS) for 1 h at 23°C and then incubated in the same buffer for 2 h at 23°C with one of the following antibodies (each diluted 1:500 in blocking buffer): 1) affinity-purified rabbit anti-human Bcl-2 [raised against a peptide encompassing residues 4-21 of Bcl-2 (absolutely conserved in the orthologous mouse and human proteins); Santa Cruz Biotechnology], 2) affinity-purified rabbit antibodies raised against a peptide spanning residues 43-61 of mouse and/or human Bax (obtained from Stanley Korsmeyer), and 3) rabbit anti-Bcl-x [residues 2-19 (also absolutely conserved in the human and mouse proteins); Santa Cruz Biotechnology]. Antigen-antibody complexes were visualized with alkaline phosphatase-conjugated secondary antibodies and a chemiluminescent substrate (Western Light kit; Tropix). Blocking controls were performed by preincubating antibodies with a 10-fold weight excess of their peptide antigens overnight at 4°C.

After being probed with antibodies directed against these regulators of apoptosis, all blots were stripped (15), washed in PBS, incubated overnight in blocking buffer, and then reprobed with rabbit anti-actin sera (1:5,000; Sigma). Signals produced by the various apoptotic regulators were quantitated and compared with the signal produced by the internal actin standard (15, 16).

Intestinal ischemia. Postnatal day 42 (P42) to P60 transgenic mice and their normal littermates were anesthetized and placed under a 37°C heating lamp, and a midline abdominal incision was made. The superior mesenteric artery (SMA) was identified, and a microaneurysm clip (Roboz Surgical) was placed across the vessel at its origin to occlude blood flow. Ischemia was maintained for 5, 10, 15, 20, or 25 min (n = 5-12 mice/time point). During the course of the ischemia, the abdomen was kept moist by repeated irrigation with 0.15 M NaCl (prewarmed to 37°C). After release of the clip and return of normal bowel color (~10-15 s later), the abdominal incision was closed. Animals were kept under a cage warmer for 4 h to preserve body temperature and given 1-ml injections of 0.15 M NaCl (37°C) via tail vein every 20 min during the first postoperative hour to ensure that their intravascular volume was maintained. Mice were allowed access to food and water as soon as they were fully active. They were killed 24 h after wound closure, and apoptosis in their jejunal epithelium was quantitated.

Two control groups of mice were analyzed: untreated and sham-operated (n = 10/group). In the sham-operated group the abdomen was opened, the SMA was exposed but not occluded, and the wound was closed after 20 min. The postoperative treatment protocol was the same for this group as it was for the experimental group.

Vascular occlusions and sham operations were always performed at the same time of day (between 0900 and 1100). Animals in control and experimental groups were killed at the same time of day to minimize possible circadian variations in the response to ischemia-reperfusion injury.

gamma -Irradiation. P42 transgenic mice and their normal littermates received a total dose of 6 Gy at a rate of 0.94 Gy/min using a Gammacell 40 irradiator equipped with a 137Cs source (Atomic Energy of Canada). Animals were killed 4 h later, and apoptotic cells were quantitated in their jejunal epithelium.

Quantitation of apoptosis. The small intestine was removed immediately after death, and a 2-cm segment was taken at the junction of the proximal and distal halves. This segment was frozen in liquid nitrogen for subsequent Western blot analysis of steady-state levels of regulators of apoptosis. The proximal half of the intestine was then opened along the length of its cephalocaudal axis, washed in 10% buffered Formalin to remove luminal contents, and then fixed in the same solution for 4 h at 23°C. After fixation, the intestine was rolled up from its proximal to distal end with the luminal side facing outward. The resulting "Swiss roll" was cut in half, parallel to its cephalocaudal axis, and placed in a tissue cassette with the cut edge of one half facing down and the cut edge of the other half facing up. After they were embedded in paraffin, serial 5-µm thick sections were prepared (n = 15-20/mouse).

Apoptotic cells were identified using the terminal deoxynucleotidyltransferase-mediated, dUTP nick end-labeling (TUNEL) assay and by their morphological appearance after staining with hematoxylin and eosin (assays done in parallel; see Refs. 15 and 27). For TUNEL assays, incorporation of digoxigenin-labeled dUTP was detected using peroxidase-conjugated sheep anti-digoxigenin Fab fragments (Boehringer Mannheim, diluted 1:500 in PBS-blocking buffer) and the Vector VIP kit (Vector Laboratories). Sections were counterstained with methyl green (Zymed).

Serial sections were scored for apoptotic cells in a single-blinded fashion. A minimum of 200 well-oriented crypt-villus units were scored per section. Well-oriented was defined as a crypt sectioned parallel to the crypt-villus axis with Paneth cells at the crypt base and an unbroken epithelial column extending from the crypt base to the villus tip. Only those jejunal crypt-villus units located in the second quarter of the small intestine were surveyed. This corresponds to the region of the cephalocaudal axis of the gut where transgene expression is highest. Statistical analysis was performed using Student's t-test (SigmaPlot).

Immunohistochemical studies. Normal and transgenic mice that were either untreated, irradiated, sham-operated, or subjected to intestinal ischemia-reperfusion were killed, and their small intestines were fixed at 23°C in Formalin for 4 h or in Bouin's solution for 6-12 h. Swiss rolls of the proximal half of the small intestine were embedded in paraffin. Sections were prepared (5 µm thick), deparaffinized in xylene and isopropanol, and subjected to antigen unmasking (see Ref. 15). Sections were subsequently washed in PBS, placed in histo-blocking buffer (1% wt/vol BSA, 0.3% Triton X-100, 0.2% powdered skim milk in PBS) for 15 min at 23°C and then incubated overnight at 4°C with one of the following antibodies: 1) affinity-purified rabbit anti-human Bcl-2 (final dilution 1:500), 2) affinity-purified rabbit anti-mouse Bcl-x (1:500; note that the adult FVB/N small intestine only contains Bcl-xL, cf. Ref. 15), 3) mouse monoclonal antibodies to Bak that react with the orthologous mouse and human proteins (1:100, Calbiochem), or 4) goat anti-5-bromo-2'-deoxyuridine [BrdU; 1:1,000, Ref. 14; used for a subset of mice that had received an intraperitoneal injection of an aqueous solution of BrdU (120 mg/kg) and 5'-fluoro-2'-deoxyuridine (12 mg/kg) 1.5 h before death].

Antigen-antibody complexes were detected using indocarbocyanine (Cy3)-labeled donkey anti-rabbit Ig, Cy3-sheep anti-mouse Ig, or Cy3-labeled donkey anti-goat Ig (all at 1:500; Jackson ImmunoResearch). Blocking controls were performed by preincubating the various primary antibodies with a 10-fold weight excess of their peptide antigens overnight at 4°C.

Cellular differentiation was assessed by staining sections with members of a panel of lectins that have been used to characterize glycoconjugate production in the gastrointestinal epithelium of normal developing and adult FVB/N mice (23, 28).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transient Occlusion of SMA Induces an Increase in Apoptosis That Is Greater in Villus Compared With Crypt Epithelium

A surgical protocol was developed that produced transient ischemia followed by reperfusion of the adult mouse small intestine without high immediate mortality. Six-week-old FVB/N mice were anesthetized, and the SMA was clamped near its origin from the aorta. The result was dramatic, with the entire length of the small intestine assuming a dark purple, cyanotic color within 30 s (Fig. 1, A and B). The effect of duration of ischemia on intestinal epithelial apoptosis was initially defined by clamping the SMA for 5, 10, 15, 20, or 25 min, releasing the clamp to allow reperfusion of the gut, immediately closing the wound, and then allowing animals free access to food and water for 24 h. The survival rate was 80-90% when the SMA was occluded for as long as 20 min, whether survival was assessed 24 or 72 h after wound closure (n = 12). Survival for sham-operated controls was 100% (n = 10). A 25-min occlusion reduced survival to 25% at 24 h (n = 12).


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Fig. 1.   A and B: transient occlusion of superior mesenteric artery (SMA) induces apoptosis in small intestinal epithelial cells. Intestine of normal postnatal day 42 (P42) FVB/N mouse before (A) and 1 min after (B) clamping SMA is shown. C: apoptosis in jejunal crypts and villi of FVB/N mice killed 24 h after 20-min SMA occlusion or in sham-operated controls. Apoptotic cells were quantitated using TUNEL assay. Values are means ± SE. There were no statistically significant differences in jejunal crypt and villus apoptosis between unoperated and sham-operated controls (data not shown). D: hematoxylin and eosin-stained section of jejunal villus from mouse killed 24 h after 20-min SMA occlusion. Note presence of apoptotic cells in upper portion of this villus (arrows). Inset: another villus section that has been subjected to TUNEL assay and counterstained with methyl green. TUNEL-positive cells appear dark purple. Bars, 25 µm.

Apoptosis was measured by TUNEL assay and by morphological appearance (after hematoxylin and eosin staining) in serial sections prepared from the middle quarter of the small intestine (jejunum). TUNEL assay and morphological analysis gave similar results.

SMA occlusion for 5 or 10 min produced no statistically significant increases in the number of apoptotic cells compared with sham-operated or untreated aged-matched controls when assayed 24 h after wound closure (data not shown). Statistically significant increases in apoptosis were observed after a 20-min occlusion. The augmentation was evident along the length of jejunal crypt-villus units but was greater on the villus; i.e., the average increase was sevenfold for villi [154 ± 24 TUNEL-positive cells/200 villus sections (experimental group) vs. 24 ± 4 cells (controls P < 0.05)] and twofold for crypts [57 ± 9 cells/200 crypt sections (experimental) vs. 34 ± 9 cells (controls, P < 0.05; Fig. 1C)]. Highest levels of apoptosis were noted in the upper quarter of the villus (Fig. 1D).

The time animals were killed after a 20-min SMA occlusion was varied to further define the apoptotic response. Surveys of mice killed 1 and 20 min and 1, 4, 12, 24, and 36 h after the abdomen was closed (n = 4/time point) revealed that statistically significant increases in TUNEL-positive cells were not detectable until 4 h after the occlusion and were maximal at 24 h (data not shown). These differences could not be ascribed to any appreciable differences in postoperative food intake among the various groups.

Apoptosis Produced by Ischemia-Reperfusion Does Not Require p53

p53 is induced in the crypt epithelium following ischemia-reperfusion (19). To determine whether this protein plays a role in the apoptotic response, we compared the effects of a 20-min occlusion of the SMA in C57BL/6 mice homozygous for p53 wild-type or null alleles. Control experiments had indicated that the time course, magnitude, and crypt-villus distribution of the apoptotic response to ischemia-reperfusion were similar in P42-P60 C57BL/6 and FVB/N mice homozygous for wild-type p53 (data not shown). C57BL/6 p53+/+ and p53-/- mice have no statistically significant differences in their apoptotic response to a 20-min SMA occlusion when assayed 24 h after wound closure (n = 3-6 mice/genotype) (Fig. 2).


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Fig. 2.   Apoptosis following ischemia-reperfusion is p53 independent. P42 C57BL/6 mice homozygous for wild- type (+) and null (-) alleles of p53 gene were subjected to 20-min ligation of their SMA. Animals were killed 24 h later, and apoptotic cells were quantitated in the epithelium of jejunal crypt-villus units.

Forced Expression of Bcl-2 in Jejunal Epithelium Suppresses Apoptosis Induced by Ischemia-Reperfusion

In creating a transgenic mouse model to test the effects of Bcl-2 on the apoptotic response of the intestinal epithelium to ischemia-reperfusion, we took advantage of the fact that transcriptional regulatory elements from a fatty acid binding protein gene (Fabpl) have been identified that can be used to force expression of gene products in all four of its epithelial cell lineages (63). Expression of Fabpl-reporter transgenes is sustained throughout the course of the differentiation-maturation of these lineages (e.g., Ref. 65) from the time that the epithelium first forms in late fetal life through at least the first 2 yr of adulthood. Highest levels of Fabpl-reporter expression occur in the jejunum.

Forced expression of Bcl-2 does not perturb intestinal epithelial homeostasis. Two pedigrees of FVB/N transgenic mice were generated that express human Bcl-2 under the control of these Fabpl transcriptional regulatory elements. Western blots of total jejunal proteins established that the steady-state level of immunoreactive Bcl-2 was four- to fivefold higher in P42 Fabpl-Bcl-2 mice belonging to both pedigrees compared with their age-matched normal littermates (n = 2-3 mice/genotype/pedigree) (Fig. 3A).


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Fig. 3.   Apoptosis produced by transient SMA occlusion is suppressed in Fabpl-Bcl-2 mice. A: Western blot of total jejunal proteins (75 µg/lane) showing that steady-state levels of Bcl-2 but not Bax are increased in a P42 Fabpl-Bcl-2 mouse compared with its aged-matched normal littermate. Blot was sequentially probed with affinity-purified antibodies to Bcl-2 and Bax. B: comparison of cellular levels of Bcl-2 in jejunal crypt-villus units of a Fabpl-Bcl-2 transgenic mouse and its age-matched normal littermate (and cage mate). The section was incubated with affinity-purified rabbit anti-Bcl-2 (visualized with Cy3 donkey anti-rabbit Ig) and fluorescein isothiocyanate-conjugated Ulex europaeus agglutinin type 1 (UEA-1). Immunoreactive Bcl-2 appears red-orange. UEA-1 lectin recognizes Fuc-alpha 1,2Galbeta -containing glycoconjugates produced in a subset of goblet cells located in crypts and villi (e.g., open arrows) and in Paneth cells positioned at crypt base (e.g., solid arrows). Junctions between crypts and villi are indicated by dashed lines. Augmentation of steady-state Bcl-2 levels in the transgenic gut epithelium does not affect goblet or Paneth cell differentiation, as judged by their ability to make fucosylated glycoconjugates recognized by the lectin. C: quantitation of crypt and villus apoptosis 24 h after a 20-min SMA ligation in P42 FVB/N normal and Fabpl-Bcl-2 transgenic mice. Mean values ± SE for TUNEL-positive cells per 200 villus sections: 154 ± 24 (operated nontransgenic mice) vs. 91 ± 11 cells (operated transgenic littermates). Mean values ± SE per 200 crypt sections: 56 ± 7 (operated nontransgenic mice) vs. 35 ± 9 (operated transgenic littermates). Bars in B, 25 µm.

Immunoreactive Bcl-2 is undetectable or barely detectable in the crypt epithelium of normal adult FVB/N mice. Levels increase markedly at the crypt-villus junction and are sustained during the subsequent migration of epithelial cells to the villus tip (Fig. 3B). Immunohistochemical analysis confirmed that an increase in steady-state Bcl-2 levels occurred in epithelial cells distributed throughout the length of jejunal crypt-villus units (n = 4 mice/genotype/pedigree; e.g., Fig. 3B). Moreover, Western blot and immunohistochemical studies of jejunal samples prepared from normal and transgenic mice indicated that forced expression of Bcl-2 was not accompanied by detectable changes in the steady-state level or cellular distribution of the pro-apoptotic regulators Bak (confined to the crypt) and Bax (Fig. 3A) or the anti-apoptotic regulator Bcl-xL (undetectable in the crypt, induced at the crypt-villus junction with no subsequent alteration in expression as cells complete their journey to the villus tip; data not shown).

Comparisons of P42 transgenic and normal littermates failed to disclose any statistically significant differences in their body weight, small intestinal weight, or small intestinal length. There were no significant quantitative differences in the basal levels of jejunal crypt or villus epithelial apoptosis, whether judged by TUNEL assay or by morphological criteria (n = 15 mice/genotype). There were no significant differences in the number of S-phase cells per jejunal crypt section (identified by pulse-labeling animals with BrdU 90 min before they were killed). There were no significant differences in crypt-villus morphology. The differentiation programs of each of the four epithelial lineages was also unperturbed: glycoconjugate production is a sensitive marker of these programs (23) and was monitored by staining sections with a previously characterized panel of lectins (n = 8-10 mice/genotype; e.g., Fig. 3B).

Although Kamada et al. (33) reported that Bcl-2-/- mice have a statistically significant reduction in the number of cells in M phase per crypt section and an increase in basal crypt apoptosis, several other groups failed to identify any intestinal abnormalities in these animals (48, 49, 66). Our findings indicate that an overabundance of Bcl-2 has no detectable effects on intestinal epithelial homeostasis, providing additional support for the notion that the normal functioning of this cell population is not dependent on or sensitive to this protein.

Ischemia-reperfusion-induced apoptosis is suppressible by Bcl-2. Forced expression of human Bcl-2 reduces the apoptotic response observed 24 h after a 20-min occlusion of the SMA. There is a statistically significant (P < 0.05) approximately twofold reduction in both crypt and villus apoptosis in transgenic mice compared with their nontransgenic littermates (Fig. 3C). There were no appreciable differences in food consumption between SMA-occluded transgenic mice and their normal littermates.

Immunohistochemical and Western blot analysis of jejunums harvested from normal FVB/N mice revealed that the crypt-villus and intracellular distributions, as well as the steady-state levels of Bcl-2, were not appreciably different before or 4-24 h after SMA occlusion (data not shown). This was also true in their transgenic littermates, although at both times the concentration of immunoreactive Bcl-2 was greater in transgenic compared with nontransgenic mice. In addition, there were no detectable differences in the levels or cellular patterns of accumulation of Bcl-xL or Bak before or 24 h after SMA occlusion, either within or between the two groups of mice (data not shown). These data support the notion that the suppression of apoptosis observed in transgenic mice is directly related to sustained, augmented expression of Bcl-2 in their small intestinal epithelium.

Forced Expression of Bcl-2 Also Suppresses p53-Dependent Apoptotic Response to gamma -Irradiation

We examined whether forced expression of Bcl-2 affected other types of induced apoptotic responses in the intestinal epithelium. The best characterized of these responses is the one that occurs in the crypt following gamma -irradiation. Comparisons of C57BL/6 mice homozygous for wild-type and null alleles of the p53 gene revealed that this response is p53 dependent (13, 45). Previous studies of P42 nontransgenic FVB/N mice established that 4 h following 6 Gy of whole body gamma -irradiation, there is a 40-fold increase in apoptosis in the crypt epithelium but no increase in villus apoptosis (16). Comparisons of P42 FVB/N Fabpl-Bcl-2 mice and their normal littermates revealed that 4 h after a 6-Gy dose, forced expression of Bcl-2 results in a statistically significant (P < 0.05) twofold reduction in crypt apoptosis (from 603 ± 75 to 304 ± 54 cells/200 crypt sections; see Fig. 4, A-C).


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Fig. 4.   Forced expression of Bcl-2 suppresses crypt apoptosis induced by gamma -irradiation. A: comparison of crypt apoptosis in normal and Fabpl-Bcl-2 transgenic mice 4 h after 6 Gy of gamma -irradiation. B and C: hematoxylin- and eosin-stained sections of jejunal crypts prepared from a normal mouse (B) and its transgenic littermate (C) killed 4 h after gamma -irradiation. Apoptotic cells are readily apparent in crypts of normal FVB/N mouse (e.g., arrows). Bars, 25 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The lack of genetically defined and manipulated in vivo models has made it difficult to study the regulation of apoptosis within the intestinal epithelium. Such models are necessary because it has not been possible to recreate ex vivo the continuous renewal of its component cell lineages; the signaling pathways that mediate the apoptotic responses of cultured intestinal epithelial cells to various inductive stimuli may not operate in the intact gut (e.g., Refs. 26 and 59).

Noda and co-workers (51) reported recently that ischemia-reperfusion induces an apoptotic response in the adult rat intestinal epithelium, although they did not quantitate this response along the crypt-villus axis. Our studies indicate that transient occlusion of the SMA induces apoptosis in undifferentiated epithelial cells located in the proliferative compartment of the adult mouse small intestine, as well as in its differentiated villus-associated cells. The greatest response occurs in the upper portion of the villus, the region farthest from submucosal vessels that supply blood to the mesenchymal core of the villus. The asymmetric distribution of apoptosis may reflect the fact that hypoxia or other inductive signals generated by SMA occlusion is (are) greater in epithelial cells located in the upper villus compared with the villus base or crypts. However, it is also possible that the signaling pathways that mediate the induction differ qualitatively or quantitatively between undifferentiated proliferating cells in the crypt and postmitotic differentiated cells in the upper villus. To address this issue, the inducing signal must first be identified and the corresponding signaling pathways characterized.

Comparisons of tumors produced from oncogenically transformed p53+/+ and p53-/- mouse embryonic fibroblasts indicated that hypoxia-induced apoptosis involves p53 (24). However, comparisons of p53+/+ and p53-/- mice showed that the apoptosis induced in postmitotic cardiomyocytes during acute myocardial infarction does not require p53 (1). The p53 requirement for the apoptotic response induced by ischemia-reperfusion has not been reported in other systems. Our results establish that the apoptosis which follows application of this death stimulus is p53-independent both in proliferating undifferentiated crypt epithelial lineages as well as in their postmitotic, differentiated villus descendants.

Studies of transformed fibroblasts and other cell lines have shown that hypoxia induces a programmed cell death that occurs through a pathway which does not involve reactive oxygen species and that the apoptotic response is suppressible by forced expression of Bcl-2 (24, 30, 61, 62). Levels of anti-apoptotic regulators such as Bcl-2 and Bcl-x are increased in cortical neurons that survive ischemia-reperfusion injury (5). Although the effects of forced expression of Bcl-2 on ischemia-reperfusion-induced neuronal apoptosis had not been described previously, Martinou et al. (43) found that when Bcl-2 was placed under the control of neuron-specific transcriptional regulatory elements the area of brain infarction caused by permanent vascular occlusion in transgenic animals was reduced approximately twofold. Our studies indicate that forced expression of Bcl-2 produces a twofold reduction in the number of TUNEL positive cells that appear following transient SMA occlusion in both crypt and villus epithelial cells. These results are compatible with the notion that for this type of death stimulus, both undifferentiated and differentiated cells undergo a commitment phase that is sensitive to Bcl-2, and the observed reduction in execution reflects a delay in commitment.

As noted previously, the best studied induced apoptotic response in the gut is that produced by gamma -irradiation (52, 54). This response does not depend on Bcl-2; the number and distribution of apoptotic cells are indistinguishable in the small intestinal crypts of mice homozygous for wild type or null alleles of Bcl-2 (46). Nonetheless, we found that the crypt apoptosis provoked by gamma -irradiation is also suppressed when Bcl-2 levels are increased. Moreover, the extent of reduction is similar to that observed after ischemia-reperfusion. Thus suppressibility by overexpression of Bcl-2 operationally defines a common feature of the apoptotic responses of the crypt to ischemia-reperfusion and gamma -irradiation. We do not know the extent to which the inductive phases of these death programs are similar or even if the cellular targets are the same in the crypt. However, our results suggest that their commitment phases share common components.

Therapeutic implications and tests. Ischemia of the adult human small intestine, whether due to physical occlusion of components of its mesenteric arterial supply, arterial vasospasm, or impaired venous drainage, results in high mortality (4). Because the intestinal epithelium is self-renewing, it is capable of repairing itself after injury. Our findings raise the possibility that interventions that elevate Bcl-2 levels or enhance its function may be useful for reducing the damage produced by ischemia-reperfusion.

Fabpl-Bcl-2 mice and their normal littermates could also be used to test the concept that augmenting Bcl-2 levels or function may ameliorate the hypoperfusion and/or reperfusion injury that accompanies intestinal transplantation (60) or the epithelial injury that accompanies hypoperfusion brought about by sepsis. One limitation of this transgenic mouse model is that Bcl-2 levels are "constitutively" elevated, so it is not possible to determine whether enhancement of Bcl-2 levels or function is needed before, during, or immediately after these injuries.

Finally, the radiosensitivity of proliferating crypt epithelial cells is the major limiting factor in the use of radiotherapy for abdominal cancers (54). The fact that increased expression of Bcl-2 has no effect on basal intestinal proliferation, differentiation, or death programs in Fabpl-Bcl-2 transgenic mice raises the possibility that agents that elevate Bcl-2 levels or augment its function may be useful for prophylactic treatment before irradiation.


    ACKNOWLEDGEMENTS

We thank Maria Karlsson, Sabrina Wagoner, and Kathy Fredrick for technical assistance, and Eugene Johnson for helpful discussions.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. I. Gordon, Dept. of Molecular Biology and Pharmacology, Box 8103, Washington Univ. School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110 (E-mail: jgordon{at}pharmsun.wustl.edu).

Received 6 October 1998; accepted in final form 25 November 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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