1. Proteasome Inhibition of Pathologic Shedding of Enterocytes to Defend Barrier Function Requires X-Linked Inhibitor of Apoptosis Protein and Nuclear Factor κB 
Derek M. Foster, Stephen H. Stauffer, Maria R. Stone, Jody L. Gookin 
Gastroenterology 
Volume 143, Issue 1, Pages e4 (July 2012)
DOI: /j.gastro
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2. Figure 1
Caspase-3 is cleaved by C parvum–infected epithelium without overt apoptosis. (A) Immunoblot of full-length (32 kilodaltons) and cleaved subunits (17 kilodaltons) of caspase-3 in control and infected piglet villous epithelial cells (n = 6 each). (B) Immunofluorescence shows villous cleavage of caspase-3 in infected but not control epithelium. (C) TUNEL and cleaved cytokeratin (M30 antigen) staining in only shedding cells from control and infected ileum. (D) Immunoblot of control and infected villous epithelial cells for XIAP, survivin, cIAP1, and cIAP2 (n = 4 each). cIAP1 and cIAP2 antibodies were confirmed to react with at least 1 of 4 positive control porcine tissues: liver, subcutaneous (SQ) fat, kidney, and peripheral blood mononuclear cells (PBMCs). Tissues were harvested (A, B, and D) immediately after the animals were killed at peak infection or (C) following incubation in Ussing chambers to enable direct visualization of shedding and shed epithelial cells. Bars, 25 μm.
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3. Figure 2
Shedding of enterocytes is more common in C parvum–infected vs control ileum. (A and B) Percent and location of villous enterocytes in the act of shedding in control (n = 5) and C parvum–infected (n = 9) ileum. (C) Enterocytes in the process of shedding categorized by the presence or absence of infection and apoptosis. A C parvum–infected, apoptotic enterocyte in the process of shedding from a villus tip (arrow) and numerous C parvum organisms (arrowheads) attached to the epithelium. *P ≤ .05, **P ≤ .01, ***P ≤ .001 vs tip, †P ≤ .05 vs control. Mean ± SEM. Bars, 5 μm.
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4. Figure 3
NF-κB is activated within enterocytes of C parvum–infected piglets. (A) Time course characterization of the light microscopic appearance, burden of epithelial parasitism, immunofluorescence localization of cleaved caspase-3, and NF-κB activity of villous epithelial cell lysates from control and C parvum–infected piglets on days 1, 3, 5, 8, and 11 after infection (n = 4 at each time point). (B) Immunostaining of control and infected ileum for intranuclear phospho-p65. Note the absence of phospho-p65 in enterocytes undergoing shedding (arrow). (C) Percent distribution of phospho-p65 among infected and uninfected resident and shedding villous enterocytes (n = 4). *P ≤ .05, **P ≤ .01, ***P ≤ Mean ± SEM. Bars, 5 μm.
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5. Figure 4
Proteasome activity represses enterocyte shedding in defense of barrier function in C parvum infection. (A) H&E and cytokeratin staining of control and infected ileal mucosa before chambering and after treatment with vehicle alone (dimethyl sulfoxide [DMSO]) or proteasome inhibitor (lactacystin 100 μmol/L). (B and C) Quantitative morphometry of DMSO- and lactacystin-treated control (n = 5) and C parvum–infected (n = 6–9) ileal mucosa. Barrier function as measured by (D) transepithelial electrical resistance and (E) mucosal to serosal flux of 3[H]labeled mannitol. *P ≤ .05, **P ≤ .01, ***P ≤ Mean ± SEM. Bar, 25 μm.
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6. Figure 5
Selective inhibition of NF-κB recapitulates effects of the proteasome on epithelial shedding and barrier function but not XIAP expression. (A and B) Quantitative morphometry of control and infected ileal mucosa treated with vehicle alone (DMSO) or selective NF-κB inhibitor (Bay ; 35 μmol/L). Barrier function as measured by (C) transepithelial electrical resistance and (D) mucosal to serosal flux of 3[H]labeled mannitol. (E) Immunoblot of intestinal mucosa after 5 hours of treatment with Bay shows no effects on IAP expression. *P ≤ .05, **P ≤ .01, ***P ≤ Mean ± SEM.
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7. Figure 6
XIAP expression is dependent on proteasome activity, and specific XIAP inhibition causes failure to contain enterocyte shedding and loss of barrier function. (A) Immunoblot of intestinal mucosa after 5 hours of treatment with lactacystin shows that only XIAP is acutely dependent on proteasome activity (representative of blots from 3–6 piglets). (B and C) Quantitative morphometry, (D) transepithelial electrical resistance, and (E) mucosal to serosal flux of 3[H]labeled mannitol of DMSO and XIAP inhibitor (LBW242)-treated control and C parvum–infected ileum (n = 6 each). *P ≤ .05, **P ≤ .01, ***P ≤ Mean ± SEM.
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8. Figure 7
XIAP coimmunoprecipitates with caspase-3, and caspase-3 inhibition mediates effects of the proteasome on control of enterocyte shedding and barrier function. (A) XIAP and (B) not survivin from infected villous epithelial cells coimmunoprecipitates with cleaved caspase-3. Negative controls for immunoprecipitation: immune mouse immunoglobulin G (lane 3), XIAP or survivin antibody with non-A/G agarose resin (lane 4), XIAP or survivin antibody with no protein lysate (lane 5). Failure to coimmunoprecipitate XIAP or survivin with caspase-3 in control pig lysates is shown in Supplementary Figure 2. Caspase-3 activity is attenuated by actions of the proteasome. (C) Caspase-3 activity in control (n = 7) and infected (n = 9) ileum mucosa treated with DMSO or lactacystin (100 μmol/L) for 5 hours. (D–F) Specific inhibition of caspase-3 (Z-DEVD-FMK; 50 μmol/L, n = 6) rescues control of cell shedding and barrier function after proteasome inhibition (data also shown in Figure 4B and C). *P ≤ .05, **P ≤ .01, ***P ≤ Mean ± SEM.
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9. Supplementary Figure 1
XIAP inhibition causes enterocyte shedding, failure to contain shedding to the villous tips, and loss of barrier function. (A) Number and (B) location of cell shedding and (C) effect on transepithelial electrical resistance after treatment of piglet ileal mucosa with normal Ringer (NR) vs the small molecule XIAP inhibitor JP1584 (1 μm; n = 6 each). †P = .01 vs control, *P ≤ .05, **P ≤ .01, ***P ≤ Mean ± SEM.
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10. Supplementary Figure 2
Neither XIAP nor survivin coimmunoprecipitates with cleaved caspase-3 in epithelial cell lysates from control piglet ileum. Negative controls for immunoprecipitation included nonimmune isotype IgG, XIAP, or survivin immunoprecipitation of cell lysate using a non-A/G agarose resin and XIAP or survivin immunoprecipitation in the absence of a cell lysate. Note that an excessive amount of control protein lysate was required to successfully IP XIAP from control piglets because of their low level of expression. The epithelial lysate lane was inadvertently overloaded relative to the infected lysate blot shown in Figure 7A.
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11. Supplementary Figure 3
Transepithelial electrical resistance (TER), transepithelial flux of 3H-mannitol, and light microscopic appearance of neonatal piglet ileum over time in Ussing chambers. TER was recorded at 30-minute intervals after placement of mucosa in Ussing chambers at time = 0 (n = 18 piglets), and 60-minute 3H mannitol fluxes were obtained at hours 2 and 4 of chambering (n = 4 piglets). Histologic specimens were obtained from an individual piglet before (time 0) and after 1, 2, 3, 4, or 5 hours in Ussing chambers. H&E stain. Mean ± SEM.
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