Bax Is Required for Resection-Induced Changes in Apoptosis, Proliferation, and Members of the Extrinsic Cell Death Pathways

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1 GASTROENTEROLOGY 2004;126: Bax Is Required for Resection-Induced Changes in Apoptosis, Proliferation, and Members of the Extrinsic Cell Death Pathways YUZHU TANG,* DEBORAH A. SWARTZ BASILE,* ELZBIETA A. SWIETLICKI,* LU YI,* DEBORAH C. RUBIN,* and MARC S. LEVIN*, *Department of Medicine, Washington University School of Medicine; and Department of Specialty Care, St. Louis VA Medical Center, St. Louis, Missouri Background & Aims: To define better the homeostatic mechanisms contributing to small intestinal adaptation following partial resection, the relative contributions of apoptosis, cell proliferation, and enterocyte migration and the comparative roles of the intrinsic (mitochondrial) and extrinsic (death receptor-mediated) apoptotic pathways were assessed. Methods: After 50% jejunoileal resections or transections, adaptation was analyzed in duodenal-jejunal and ileal segments from C57BL/6 Bax / (16, 48, and 168 hours postoperative) and Bax / mice (168 hours). Results: Basal apoptotic rates were equivalent in all mice. By 1-week postresection, villus heights and crypt depths were increased in the duodenal-jejunal and ileal remnants of both genotypes. In Bax / mice, adaptation occurred in concert with increased crypt proliferative and apoptotic indices. Bax / mice did not showincreases in proliferation or apoptosis, yet adaptive increases in villus height were enhanced relative to Bax / mice. Enterocyte migration increased in both genotypes. Postresection, the expression of caspases and genes involved in death receptormediated apoptosis was decreased in Bax / compared with Bax / mice. Conclusions: Postresection adaptation involves parallel changes in crypt proliferation and apoptosis, but, as observed in Bax / mice, it can occur without increased proliferation. These studies demonstrate that spontaneous gut apoptosis is Bax independent, whereas adaptation-related apoptosis is Bax-dependent. Differences between resected Bax / and Bax / mice suggest that apoptosis in the adapting gut utilizes the extrinsic pathway, but this requires linkage to the mitochondrial pathway via Bax. The increased adaptive response in Bax / mice indicates that modulation of apoptosis may be useful for enhancing adaptation. The small intestinal epithelium is continually renewed by a dynamic process of rapid cell proliferation, migration along the crypt-villus axis, differentiation, and cell death. Following the loss of small bowel absorptive surface area, the residual intestine undergoes compensatory structural and functional changes, characterized by increased villus height, crypt depth, and cellular hyperplasia. Previous studies of the adaptive response have mainly focused on identifying the factors that mediate an increase in crypt cell proliferation, the hallmark of this response. However, programmed cell death is now recognized to be an important mechanism underlying cell loss in the gastrointestinal tract (reviewed in Hall et al. 1 ). Thus, the regulation of the balance between cell proliferation and programmed cell death appears to be crucial for maintaining normal intestinal growth and structure (reviewed in Potten et al. 2, Ramachandran et al. 3, and Watson and Pritchard 4 ). Crypt apoptosis functions to eliminate damaged cells and to maintain stem cell numbers, thereby regulating the number of cells migrating up the crypt-villus axis. Apoptosis may also be a major mechanism responsible for the removal of epithelial cells along the length of the villus and from the extrusion zone. 1 Apoptotic indices are increased in the early stages following small bowel resection in the mouse, 5 7 rat, 8 and rabbit. 9 This increase occurs in conjunction with a shift toward greater expression of proapoptotic (relative to prosurvival) members of the Bcl-2 family 5,7 and is independent of p Although studies using genetically engineered mice have begun to address the role of p53 and Bcl-2 family members in this process, the molecular mechanisms controlling enterocytic apoptosis as well as the intestinal adaptive response are not fully defined. For example, others have shown that the increase in crypt apoptosis following small bowel resection in mice is abolished in Bax / mice when studied at 72 Abbreviations used in this paper: 5-BrdU, 5-bromodeoxyuridine; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling by the American Gastroenterological Association /04/$30.00 doi: /j.gastro

2 January 2004 MECHANISMS OF SMALL INTESTINAL ADAPTATION 221 hours after resection. 11 However, the full phenotype of the adaptive response is not expressed until at least 1 week after resection, so the effects of eliminating Bax expression on other features of the morphologic adaptive response have not yet been studied. In addition, analyses of the contribution of other apoptotic regulatory pathways at early and late time points after resection have been limited. Thus, the present study was initiated to characterize further the temporal regulation of the adaptive response by examining crypt cell proliferation and apoptotic indices as well as epithelial cell migration at various times after resection and to address the roles of the intrinsic (or mitochondrial) and the extrinsic (or death receptor) apoptotic pathways in intestinal adaptation. Materials and Methods Animals, Diet, and Experimental Design For the time course experiments, male C57BL/6 mice aged 6 7 weeks were obtained from the Jackson Laboratory (Bar Harbor, ME) and placed on chow diets until 5 days before surgery. They were then switched to an AIN93G liquid diet (Dyets, Inc., Bethlehem, PA) and were maintained on the liquid diet for the duration of the study. Mice were subjected to either 50% intestine resection or transection and reanastomosis (n 8 per group). Anesthesia was induced with ketamine HCl (87 mg/kg)/xylazine HCl (13 mg/kg), atropine (0.04 mg/kg), and inhalational methoxyflurane. Bax / mice were killed 16, 48, or 168 hours postoperatively. To investigate the role of Bax, C57BL/6 Bax / mice were bred using founders provided by S. Korsmeyer. 12 Genotypes were determined by PCR using tail DNA as previously described. 13 Pentobarbital and inhalational methoxyflurane were used for anesthesia. Bax / mice were more sensitive to pentobarbital, and, thus, it was necessary to utilize a lower dose (28.5 mg/kg for Bax / and 14.3 mg/kg for Bax / mice). To improve postoperative survival, all mice were maintained on the liquid diet. 14 Bax / and Bax / were killed postoperatively at 168 hours for comparative studies. All animal experimentation was conducted in conformity with the Guiding Principles for Research Involving Animals and was approved by the Washington University Animal Studies Committee. Intestinal Resection and Tissue Procurement Bax / and Bax / mice were weighed and assigned to resected and sham-resected groups (n each group). Control mice (sham resected) underwent jejunal transection 2 3 cm distal to the ligament of Treitz, followed by reanastomosis without resection. Experimental mice underwent 50% small bowel resection, extending from 2 3 cm distal to the ligament of Treitz to 8 cm proximal to the ileocecal valve. An end-to-end anastomosis with the remaining jejunum and the distal ileum was then constructed using interrupted 9-0 silk sutures. Gentamicin (0.2 mg in 0.5 ml saline) was administered intraperitoneally. Postoperative analgesia was obtained by buprenorphine 0.3 mg/kg subcutaneously. Mice were killed by cervical dislocation while under methoxyflurane anesthesia. For resected mice, the remnant intestine proximal to the anastomosis was divided into duodenal and jejunal segments, and the remnant distal to the anastomosis was subdivided into equal proximal and distal ileal segments. The equivalent segments were harvested from the sham-resected mice. Each individual segment was subdivided into 3 pieces. The middle one was fixed by immersion in Bouin s solution for hematoxylin and eosin and immunohistochemical analysis. The remainders were immediately frozen in liquid nitrogen for RNA isolation. The remnant ileum was subjected to more in-depth analyses because functional and morphologic adaptation is greatest in this segment after middle small bowel resection when the transection control cut is in the jejunum Morphologic Assessment To assess the morphometric adaptive response at 168 hours (1 week) postresection, villus height, villus cell number (i.e., the number of cells in a vertical column from the villus base to tip), crypt depth, and crypt cell number, which reflect changes in cell number and absorptive area, were measured in the duodenum, jejunum, and proximal and distal ileum in 10 well-oriented, full-length crypt-villus units in hematoxylinstained sections with the aid of a slide micrometer or with AxioVision analysis of digitally acquired images (Carl Zeiss, version 2.0). Average villus and crypt enterocyte heights were estimated by measuring the heights of 3 midvillus or midcrypt cells. Cell widths were estimated by dividing the villus or crypt lengths by the villus or crypt cell number. Cell volumes were then calculated from cell heights and widths using the formula for a cylinder. 19 Crypt Cell Proliferation and Migration Crypt cell proliferation was determined using 5-bromodeoxyuridine (5-BrdU) incorporation into DNA. Mice were injected subcutaneously with 120 mg/kg 5-BrdU (8 g/l 5-bromodeoxyuridine and 0.8 g/l 5-fluorodeoxyuridine) 1.5 hours before being killed. 5-BrdU was detected with a monoclonal anti-brdu antibody (Zymed Laboratories, South San Francisco, CA) and streptavidin-biotin amplification (Accurate Chemical & Scientific Corporation, Westbury, NY). Proliferation rates are reported as the percentage of labeled cells in full-length crypts. For measurement of enterocyte migration rates, a doublelabeling technique using 5-BrdU was developed. Each mouse was injected with 5-BrdU at 49.5 hours (4.8 mg/kg) and 1.5 hours (120 mg/kg) before being killed. In preliminary studies using single injections, it was determined that, at these doses and time intervals, there were no significant overlaps in the position of BrdU-positive cells (i.e., after 1.5 hours, all of the labeled cells were in the crypt, and, after 49.5 hours, labeled

3 222 TANG ET AL. GASTROENTEROLOGY Vol. 126, No. 1 cells were rarely found in the crypt or lower part of the villus). Furthermore, cells at the villus tip were rarely labeled, indicating that extrusion of BrdU-positive cells did not result in spuriously low migration rates. Thus, with this technique, the enterocyte migration rate could be determined in individual mice. Each cell position from the base of the crypt to the tip of the villus was scored for BrdU staining. When plotted against cell position, there were 2 distinct labeling peaks (crypt and villus). The differences between the leading edge median peak labeling positions on the crypt and villus distribution curves were used to calculate the migration rates. 20 Documentation of Apoptosis by Morphologic Assessment and TUNEL Assay The identification and quantification of apoptotic cells were performed using morphologic criteria to evaluate H&Estained tissue sections and using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays (In situ Cell Death Detection Kit; Roche Diagnostics, Brussels, Belgium) on Bouin s fixed, paraffin-embedded proximal ileal sections. Only full-length crypts were used. Apoptotic indices are reported as the apoptotic cells per 100 crypt cells rather than per crypt to normalize for postoperative changes in crypt depth. The presence of TUNEL positivity and morphologic changes including nuclear condensation, perinuclear clearing, and cell shrinkage were the criteria for identification of apoptotic cells. Ribonuclease Protection Assays Total cellular RNA was extracted from full-thickness intestinal segments with Trizol Reagent (Life Technologies, Grand Island, NY). Ribonuclease protection assays were carried out using murine multiprobe template kits (Riboquant mapo-1, 2, and 3; BD Pharmingen, Franklin Lakes, NJ) to detect members of the Bcl-2 (i.e., Bcl-W, Bfl-1, Bcl-X L, Bcl-X S, Bak, Bax, Bcl-2, and Bad), caspase (i.e., caspases 1, 2, 3, 6, 8, 11, and 12) and tumor necrosis factor receptor (TNFR) signaling cascades (i.e., caspase 8, Fas ligand (FasL), Fas, Fasassociated death domain protein (FADD), Fas-associated factor (FAF), Fas-associated phosphatase (FAP), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TNFRp55 (i.e., TNFR-R1), TNFR-associated death domain protein (TRADD), and receptor interacting protein (RIP) families. Protected fragments were quantified using a Molecular Dynamics 425S phosphorimager (Molecular Dynamics, Sunnyvale, CA). The expression of each gene was normalized by dividing probe-specific signal to that obtained for ribosomal L32 RNA. Detection of Fas and Caspase-8 With Immunoblots Intestinal extracts were prepared from tissues that had been embedded in OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and snap frozen in liquid nitrogen. Protein aliquots (100 g) were electrophoresed on 10% SDS-polyacrylamide gels and transferred to PVDF-plus membranes (Osmonics Inc., Westborough, MA). The membranes were blocked overnight at 4 C and then incubated for 2 hours at room temperature with primary antibody (1:10,000 rabbit anti-hsp 40, StressGen Biotechnologies Corp, Victoria, BC, Canada; or 1:200 goat anti-caspase 8 or rabbit anti-fas, Santa Cruz Biotechnology, Inc. Santa Cruz, CA). Blots were washed then incubated for 1 hour at room temperature with horseradish peroxidase-conjugated anti-igg antibodies (1:10,000 antirabbit [Amersham Biosciences Corp., Piscataway, NJ ] or 1:5000 anti-goat [Jackson ImmunoResearch, West Grove, PA]) and washed 3 times before addition of chemiluminescent peroxidase substrate (ECL Western Blotting Kit; Amersham Biosciences Corp.). Blots were directly exposed to Kodak BioMax Film (Eastman Kodak Co., Rochester, NY). Equal loading and transfer were confirmed by detection of Hsp 40. Statistical Analysis One-way ANOVA and 2-way ANOVA (GraphPad Prism, Version 3.0 for Windows; Graphpad Software, San Diego, CA) with Tukey s HSD posttest ( obg.cuhk.edu.hk/researchsupport/least_sig_diff_tukey.asp) were used for data analysis, including identification of significant interactions between the Bax genotype and intestinal surgery. Paired t tests were used for comparing preoperative to postoperative changes in the same mouse. P values less than 0.05 were considered statistically significant. Results Characterization of the Adaptive Response Following Partial Small Bowel Resection in Bax / Mice Morphometric response. The adaptive response in Bax / mice after small bowel resection was characterized from 16 hours until 1 week postoperative. Compared with sham-resected controls, villus heights and crypt depths in the proximal ileum were significantly increased by 1 week after resection (Figure 1). Ileal crypt cell proliferation rates were also significantly increased by 16 hours after resection (1.5-fold) and remained elevated at 48 hours (1.5-fold) and 168 hours (1.2-fold, P 0.05 for all time points, see ( / ) bars in Figure 1 and data not shown). Crypt apoptotic indices were also significantly higher in the remnant ileum at 48 hours (2-fold) and 168 hours postoperative (2.6-fold, P 0.05 for both time points, Figure 1 and data not shown). Expression of Bcl-2 family members following resection. Because of the change in apoptotic indices following resection, the expression of Bcl-2 family members was determined. By ribonuclease protection assay, the relative expression of proapoptotic to prosurvival members of the Bcl-2 family was increased at 48 hours.

4 January 2004 MECHANISMS OF SMALL INTESTINAL ADAPTATION 223 Figure 1. Adaptive changes in the proximal ileum in Bax / and Bax / mice 1 week following 50% partial small intestinal resection (RE) or sham-resection (SHR). Representative sections from the proximal ileum of Bax / (A and B) and Bax / (C and D) mice demonstrating changes in villus height and crypt depth at 1 week following 50% small intestinal resection (RE) as compared with shamresection (SHR). The intestine was fixed in Bouin s and stained with H&E. (E) Villus heights, crypt depths, crypt apoptosis, crypt cell proliferation, and enterocyte migration rates were quantified as in the Materials and Methods section. Data are means SEM *P 0.05 or **P 0.01 RE vs. SHR. #P 0.05 Bax / RE vs. Bax / RE. For example, proapoptotic Bak, Bax, and Bad tended to increase ( fold, P 0.05 for Bak, P 0.08 for Bax and Bad), whereas the levels of prosurvival genes such as Bfl-1, Bcl-2, Bcl-W, and Bcl-X L were unchanged (data not shown). At 1 week, only the prosurvival Bfl-1 gene was increased (1.3-fold, P 0.05). Comparison of the Adaptive Response at 1 Week Postoperative in Bax / and Bax / Mice Morphometric response. Both Bax / and Bax / mice tolerated intestinal surgery equally well, with an overall survival rate of 64% for resected mice and

5 224 TANG ET AL. GASTROENTEROLOGY Vol. 126, No. 1 88% for sham-resected mice. One-week postoperative Bax / mice displayed the characteristic morphologic changes of adaptation with the maximal response in the proximal ileum (1.4-fold increase in villus length compared with sham controls; Figure 1). Furthermore, in the absence of Bax, adaptation following resection was significantly enhanced in the proximal ileum as indicated by an increase in villus length compared with Bax / mice (Figure 1). Apoptosis, Crypt Cell Proliferation, and Cell Migration Crypt cell proliferation and apoptosis were evaluated in Bax / compared with Bax / mice to assess their relative contributions to the observed morphologic changes (Figure 1). In the proximal ileum at 1 week postoperative, sham-resected Bax / and Bax / mice had similar apoptotic indices (0.5 and 0.8 apoptotic bodies per 100 crypt cells, respectively, P NS). This suggests that the low level of basal apoptotic activity in the ileum is not Bax dependent. In Bax / mice after 50% small bowel resection, apoptosis in the crypts increased 2.6-fold compared with sham-resected controls, whereas no significant changes occurred in Bax / mice. The apoptotic indices of resected Bax / mice were less than one third of resected Bax / mice. These data indicate that the increase in crypt cell apoptosis seen at 1 week postresection is Bax dependent. Villus apoptotic cells were rarely seen, and the frequency appeared to be equivalent across experimental groups (data not shown). In contrast to Bax / animals, crypt cell proliferation rates did not increase in Bax / mice by 1 week postresection when compared with sham-resected mice and were only 80% of Bax / rates (Figure 1). Thus, in Bax / mice, inhibition of apoptosis was accompanied by an inhibition of the normal increase in crypt cell proliferation that occurs postresection. Enterocyte migration rates were also assessed as an additional potential mechanism contributing to the adaptive phenotype and to differences between Bax / and Bax / mice (Figures 1 and 2). Following resection, epithelial cells migrated significantly faster (1.3-fold) in the jejunal ( fold) and proximal ileal ( fold) remnants of both genotypes. Migration rates were similar for resected Bax / and Bax / mice in the proximal ileum, whereas, in the jejunum, migration was slightly faster (1.1-fold) in the Bax / mice (P 0.05). Analyses of cell number and estimates of cell volumes (Figure 3) suggest that, (1) for both genotypes, the elongated villi resulted from increases in both cell size and number and (2) the deeper crypts resulted from hyperplasia in Bax / mice and from cell hypertrophy in Bax / mice. Figure 4 demonstrates the morphologic response to resection in the duodenum, jejunum, and proximal and distal ileum at 7 days postoperative. In Bax / mice, villus heights were significantly increased in the duodenum and proximal and distal ileum. In addition, jejunal villi were significantly longer than the adjacent resected preoperative segment (data not shown). Villus heights were significantly increased in all segments in Bax / mice. Crypt depths were significantly increased in the duodenal, jejunal, and proximal ileal remnants of both genotypes (Figure 4). These data demonstrate that homeostatic shifts in rates of cell proliferation and programmed cell death that facilitate the adaptive response are not limited to the ileum but also occur in the duodenum and jejunum. Expression of Apoptosis-Related Genes After Small Bowel Resection The apoptotic pathways responsible for the increased apoptotic index in the adapting remnant intestine at 1 week postresection were first assessed by comparing the expression of proapoptotic (Bad, Bak, Bax, and Bcl-X S ) and prosurvival (Bcl-2, Bcl-W, Bcl-X L, and Bfl-1) Bcl-2 family members in control and resected Bax / and Bax / mice. In Bax / resected mice, despite an increased apoptotic index, the only significant change in Bcl-2-related genes was increased expression of the prosurvival Bfl-1 gene (1.3-fold, P 0.05). In Bax / mice, consistent with the inhibition of adaptation-induced apoptosis, compensatory increases in the expression of other proapoptotic genes did not occur. Furthermore, expression of the prosurvival Bfl-1 and Bcl-w genes was repressed in Bax / compared with Bax / resected mice ( 23% P 0.05; 29% P 0.01, respectively). In Bax / mice, the discordance at 1 week postoperative between the persistently elevated apoptotic indices and the expression of Bcl-2 gene family members implied that other gene families and pathways are also important mediators of apoptosis in the adapting remnant intestine. Thus, components of the TNFR-FAS extrinsic signaling pathways were analyzed (Figure 5 and data not shown). There were no differences between sham-resected Bax / and Bax / mice for any of these genes. However, after resection, many of these genes were increased in Bax / but not in Bax / mice compared with respective sham-resected controls. Thus, when compared with resected Bax / mice, the resected Bax / mice had significantly reduced levels of Fas, caspase 8, FADD, FAF, TRAIL, and TRADD mrna

6 January 2004 MECHANISMS OF SMALL INTESTINAL ADAPTATION 225 Figure 2. Regional changes in small intestinal epithelial cell migration rates 1 week following 50% resection (RE) or sham-resection (SHR) in Bax / and Bax / mice. Panels A to D: Representative hematoxylin and BrdU-stained sections used to measure migration rates in the proximal ileum from Bax / SHR (A) and RE (B) and from Bax / SHR (C) and RE (D) mice injected with BrdU at 49.5 hours and 1.5 hours before death. Lower panels are percentage BrdU labeling at each cell position in jejunum (Jej) and proximal ileum (Pil) of RE (open circles) and SHR (solid circles) mice. The connected lines are moving averages over 3 cell positions. (Figure 5). The observed changes in Fas and caspase 8 mrna levels were further investigated by using immunoblots to detect immunoreactive Fas and caspase 8 in the proximal ileum. In agreement with the RNA and apoptosis data, the highest levels of Fas and caspase 8 were present after resection in Bax / mice (Figure 6). These protein and mrna data imply that, after resection, proapoptotic signaling is mediated by death domain containing receptors belonging to the TNFR super family, and this pathway is down-regulated in Bax / mice. To define further the apoptotic pathways utilized in the adapting remnant intestine, the expression of initiator, executioner, and cytokine processor caspases were also examined (summarized in Table 1). Consistent with the differences in apoptotic indices postresection, most of

7 226 TANG ET AL. GASTROENTEROLOGY Vol. 126, No. 1 Figure 3. Postoperative changes in cell number and volume in proximal ileal crypt and villus columns from Bax / and / mice. All of the cells on intact crypt and villus columns were counted (left panel). Cell width and height were measured and cell volumes estimated as described in the Materials and Methods section (right panel). Data are means SEM, n 3 4. *P 0.05, **P 0.01 for resected (RE) vs. sham-resected controls (SHR). Spontaneous apoptosis in the small intestine occurs primarily in the stem cell region of the crypt (cell positions 4 6 from the base) at a rate corresponding to approximately 1 apoptotic cell per every fifth crypt. 21,22 At 7 days postoperative, apoptotic rates were comparable in the proximal ileum of sham-resected Bax / and Bax / mice (Figure 1). Thus, as was observed in the small intestine or midcolon of unoperated mice, 23 Bax expression was not required to maintain basal apoptotic rates. However, in agreement with Stern et al. 11 finding in early adaptation (i.e., 72 hours postoperative), at 7 days following partial small bowel resection, apoptosis was markedly increased in the remnant gut of Bax / but unchanged in Bax / mice. These data demonstrate that Bax expression is essential for the increased apoptotic rates occurring in intestinal adaptation. Although Bax is required for the increase in apoptosis occurring in the resection model of intestinal adaptation, the examined caspases were increased in Bax / compared with Bax / mice (range, fold higher, P 0.05 for caspase 1, 3, 7, 8, and 11). Discussion Intestinal adaptation following small bowel resection has been viewed predominantly as a hyperplastic response, with increased crypt cell proliferation producing compensatory crypt deepening and villus elongation. However, in the small intestine, normal crypt and villus cell numbers are maintained by balancing rates of cell proliferation, migration, apoptosis, and anoikis (i.e., apoptosis preceded by loss of attachment to the extracellular matrix). 1 Therefore, the present study addressed the contributions of changes in the set point balancing cell proliferation and programmed cell death, as well as changes in enterocyte migration, to the full expression of the adaptive phenotype during the first 7 days following 50% resections of the small bowel of Bax / and Bax / mice. In Bax / mice, enterocyte proliferation increased as early as 16 hours postresection and peaked at 48 hours. This was followed by an increase in crypt cell apoptosis that began at 48 hours and peaked by 168 hours postresection. These data suggest that apoptosis is increased in parallel with enterocyte proliferation, perhaps as a mechanism to maintain the homeostatic balance between cell proliferation and cell death. Conversely, apoptosis could be induced to prevent survival and propagation of aberrant cells that may be more prevalent because of the hyperplastic response. Figure 4. Morphologic changes in Bax / and Bax / mice 1 week after 50% partial small intestinal resection or sham resection. Data shown are means SEM. (*P 0.05, **P 0.01, resected [RE] vs. sham resected [SHR]; #P 0.05 Bax / RE vs. Bax / RE; n per group) Villus heights and crypt depths were significantly increased in most segments of the resected mice compared with the sham-resected mice. In addition, the villi of resected Bax / mice were significantly longer in the JEJ and PIL.

8 January 2004 MECHANISMS OF SMALL INTESTINAL ADAPTATION 227 Table 1. Changes in Caspase Expression in Resected and Sham-Resected Bax / and Bax / Mice Fold change a,b Figure 5. Steady-state levels of Fas and TNF family member mrna in proximal ileum. One-week postresection (RE) or sham-resection (SHR), Bax / and Bax / mice were killed, and total RNA was isolated from full-thickness intestinal segments. RNA was analyzed using ribonuclease protection assays (see Materials and Methods section) to detect caspase 8 (Casp 8), Fas, Fas-associated death domain protein (FADD), Fas-associated factor (FAF), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), tumor necrosis factor receptor p55 (TNFR-R1), TNFR-associated death domain protein (TRADD), and receptor interacting protein (RIP). Signals from radiolabeled protected fragments were quantitated using a phosphorimager (Molecular Dynamics, Sunnyvale, CA) and normalized to the expression of the ribosomal L32 mrna. Results presented are means SEM, **P 0.01 RE vs. SHR, #P 0.05 Bax / RE vs. Bax / RE. In addition, the values for the Bax / data are presented as a percentage of / data when the differences are significant or as NS when not significant. Figure 6. Immunoblot analysis of caspase 8 and Fas in proximal ileum of Bax / and Bax / mice after partial small bowel resection. Protein extracts from pools of 2 or 3 mice per experimental group were electrophoresed, transferred, and used for immunodetection of caspase 8, Fas, and Hsp 40 (to control for protein loading) as described in the Materials and Methods section. Bax / resected/sham Bax / resected/sham Resected Bax / /Bax / Initiator Caspase 2 NS NS NS Caspase NS 1.4 Executioner Caspase Caspase 6 NS 1.5 NS Caspase 7 NS Cytokine processor Caspase 1 NS Caspase NS, No significant difference. a Fold change represents the increased or decreased (shown as minus) caspase expression level for the indicated comparisons. b Only significant changes (P 0.05) are shown. as discussed above, it is clearly not essential for spontaneous apoptosis in the small intestine. Thus, of the 2 major apoptosis pathways, the extrinsic or cell surface death receptor pathway and the intrinsic or mitochondria-mediated pathway (reviewed in Kaufmann and Hengartner 24 ), it appears that the former is the predominant mediator of spontaneous apoptosis in the small intestine. In the extrinsic pathway, ligand-induced receptor clustering and recruitment of FADD and procaspase-8 lead to formation of the Fas-death-inducing signaling complex (Fas-DISC) and caspase-8 activation. Activation of downstream executioner caspases causes apoptosis via a mitochondrial-independent (i.e., Bax-independent) pathway (Figure 7). The observed requirement for Bax in conjunction with the changes in the expression of Fas-DISC components, suggest that both intrinsic and extrinsic pathways of apoptosis are activated in this adaptation model. Under some circumstances, death signals transmitted via the extrinsic pathways can cause truncation and activation of Bid, which can result in activation of mitochondrialdependent (i.e., Bax-dependent) apoptosis (reviewed in Hengartner 25 and Ranger et al. 26 ). In our study using Bax / mice, expression of each of the DISC components including caspase 8 was up-regulated in proximal ileum 1 week postresection. Impairing the mitochondrial pathway by knocking out the Bax gene blocked adaptationinduced apoptosis without producing compensatory increases in caspase 8 or other DISC components, all of which were, instead, down-regulated. These data suggest that adaptation-induced apoptosis utilizes the Fas-DISC pathway, but this requires linkage to the mitochondrial pathway via Bax. Bax-mediated apoptosis results in the release of mitochondrial cytochrome C and SmaC that leads to activation of caspase 9 and then executioner caspases including caspase 3. Concordant with changes in apoptosis, many of these downstream effectors were increased following

9 228 TANG ET AL. GASTROENTEROLOGY Vol. 126, No. 1 Figure 7. Proposed apoptosis pathways after resection. The panels illustrate the changes in expression of several components responsible for programmed cell death occurring after partial small bowel resection of Bax / (left panel) and Bax / (right panel) mice. In the Bax / resection induced apoptosis correlated with the increased expression of Fas-DISC components (Fas, FADD, and caspase-8). Activated caspase-8 can proteolyze downstream caspases and Bid. Truncated Bid translocates to the mitochondria and can interact with proapoptotic Bax and Bad leading to the release of cytochrome c (cytoc) and SmaC and the ultimate cleavage of caspase-9, which amplifies the apoptotic effect. In Bax / mice, the elimination of the proapoptotic Bax gene switches the balance of pro- and antiapoptotic Bcl-2 family genes to prosurvival. In addition, decreased sensitivity to death receptor-induced apoptosis is suggested by the failure of Fas-DISC expression to increase and by decreased expression of caspases-3, 6, and 7. Key: genes increased, decreased, or unchanged by resection are colored green, red, or black, respectively. Genes colored gray were not studied. resection of Bax / mice and were decreased in Bax / mice. Consistent with the observation that Bcl-2 can block caspase-3 activation in mammalian cells, 27,28 increasing the Bcl-2 to Bax ratio by eliminating Bax expression resulted in down-regulated caspase-3, 6, and 7 expression in resected intestine. In the Bax / remnant gut, enhanced apoptosis was confined to the crypts and occurred in conjunction with increased cell proliferation. In Bax / mice, correlating with the blunted apoptotic response, crypt proliferation was not increased at 7 days postoperative despite resection of 50% of the small intestine. Thus, in both Bax / and Bax / mice, indices of proliferation and programmed cell death corresponded in the remnant gut following partial small bowel resection. This correlation in regulation of apoptosis and proliferation has the teleological benefit of maintaining normal cellularity of crypt villus units. Thus, although adaptation still occurred in Bax / mice, the response was only modestly increased compared with Bax / mice. Furthermore, the augmented adaptive response in Bax / mice occurring without increased crypt cell proliferation suggests that adaptation-induced Bax-dependent apoptosis may be the primary factor limiting the magnitude of the adaptive response in Bax / mice. In addition, the data from Bax / mice suggest that there are redundancies in cell survival and cell death regulatory pathways that prevent elimination of a single proapoptotic factor, such as Bax, from completely disrupting the balance between cell proliferation and apoptosis. For example, the expression of Bfl-1, which promotes cell survival and may stimulate cell proliferation, 29 was increased in the remnant intestine of Bax / mice but was relatively decreased in Bax / mice. Therefore, in Bax / mice, increased expression of Bfl-1 may be proadaptive by enhancing mucosal cellularity. In contrast, in Bax / mice, a relative decrease in expression may help to compensate for the impaired intrinsic cell

10 January 2004 MECHANISMS OF SMALL INTESTINAL ADAPTATION 229 death pathway, by reducing growth resulting from the prosurvival and proliferative effects of this gene. Although we observed that apoptosis and proliferation were inhibited in Bax / mice at 7 days postoperative, crypt cell proliferation was reported to be increased at 3 days postresection when assessed by detection of proliferating cell nuclear antigen (PCNA). 11 Using PCNA as a surrogate for crypt proliferation can overestimate proliferation rates compared with 5-BrdU because PCNA can be detected in cells in late G1 in addition to S-phase cells. 30,31 In addition, at 3 days postresection, an increase in crypt depths but not villus heights occurred in resected Bax / mice, compared with resected Bax / mice. These differences further demonstrate that the adaptive response is temporally regulated 17,32 and confirm the importance of studying both early and late time points to determine whether modulating apoptosis may provide a pharmacologic approach to enhancing the adaptive process. Enterocyte migration rates were significantly increased postresection in the adapting intestinal remnant of both Bax / and Bax / mice. Thus, although there were marked differences in rates of cell proliferation and apoptosis between the 2 Bax genotypes, enhanced enterocyte migration contributed to an adaptive phenotype in both. These observations indicate that cell migration rates are not solely determined by crypt cell proliferation and apoptosis rates. These data also suggest that analysis of migration rates could be an additional useful parameter for gauging the potency of putative modulators of adaptation. Our previous results with resected vitamin A deficient rats are consistent with these conclusions. 8,33 In conclusion, the studies reported here show that (1) adaptation following loss of small intestinal absorptive surface area is modestly enhanced in Bax / mouse intestine compared with Bax / control mouse gut and that this occurs without increased crypt cell proliferation and that (2), in addition to the inhibition of apoptosis mediated by the intrinsic (i.e., mitochondrial) pathway, extinction of Bax expression down-regulated the death receptor pathway postresection, suggesting coordinate regulation of the 2 pathways during the adaptive process. These studies indicate that successful therapeutic strategies to augment adaptation may derive from targeting multiple processes rather than a single one such as cell proliferation. References 1. Hall PA, Coates PJ, Ansari B, Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. 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11 230 TANG ET AL. GASTROENTEROLOGY Vol. 126, No. 1 lation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia. J Cell Sci 1995;108: Potten CS. The significance of spontaneous and induced apoptosis in the gastrointestinal tract of mice. Cancer Metastasis Rev 1992;11: Pritchard DM, Potten CS, Korsmeyer SJ, Roberts S, Hickman JA. Damage-induced apoptosis in intestinal epithelia from bcl-2-null and bax-null mice: investigations of the mechanistic determinants of epithelial apoptosis in vivo. Oncogene 1999;18: Kaufmann SH, Hengartner MO. Programmed cell death: alive and well in the new millennium. Trends Cell Biol 2001;11: Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407: Ranger AM, Malynn BA, Korsmeyer SJ. Mouse models of cell death. Nat Genet 2001;28: Armstrong RC, Aja T, Xiang J, Gaur S, Krebs JF, Hoang K, Bai X, Korsmeyer SJ, Karanewsky DS, Fritz LC, Tomaselli KJ. Fas-induced activation of the cell death-related protease CPP32 Is inhibited by Bcl-2 and by ICE family protease inhibitors. J Biol Chem 1996;271: Shimizu S, Eguchi Y, Kamiike W, Matsuda H, Tsujimoto Y. Bcl-2 expression prevents activation of the ICE protease cascade. Oncogene 1996;12: Bae J, Hsu SY, Leo CP, Zell K, Hsueh AJ. Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis. Apoptosis 2001;6: Coltrera MD, Gown AM. PCNA/cyclin expression and BrdU uptake define different subpopulations in different cell lines. J Histochem Cytochem 1991;39: Cameron HA. UNIT 3.9 quantitative analysis of in vivo cell proliferation. In: Crawley JN, Gerfen CR, Rogawski MA, Sibley DR, Skolnick P, Wray S, eds. Current protocols in neuroscience. New York: John Wiley & Sons, Dodson BD, Wang JL, Swietlicki EA, Rubin DC, Levin MS. Analysis of cloned cdnas differentially expressed in adapting remnant small intestine after partial resection. Am J Physiol Gastrointest Liver Physiol 1996;271:G347 G Swartz-Basile DA, Rubin DC, Levin MS. Vitamin A status modulates intestinal adaptation after partial small bowel resection. JPEN J Parenter Enteral Nutr 2000;24: Received September 6, Accepted October 9, Address requests for reprints to: Marc S. Levin, M.D., Washington University School of Medicine, Department of Medicine, Campus Box 8124, 660 South Euclid Avenue, St. Louis, Missouri mlevin@wustl.edu; fax: (314) Supported by National Institutes of Health grants RO1 DK50466, R01 DK46122, and P30 DK52574 and Institutional National Research Services Award T32 DK07130 and the ASPEN Rhoads Research Foundation Maurice Shils Grant (to D.A.S.-B.). The authors thank Karen Hutton and Randal May of the Morphology Core of the Washington University Digestive Diseases Research Core Center for providing outstanding technical support and Alan Davis, Hristo Iordanov, and Lihua Wang for their excellent technical contributions. D.A.S.-B. s current address is: Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin