p38 MAPK regulates Bax activity and apoptosis in enterocytes at baseline and after intestinal resection

Abstract

Increased apoptosis in crypt enterocytes is a key feature of intestinal adaptation following massive small bowel resection (SBR). Expression of the proapoptotic factor Bax has been shown to be required for resection-induced apoptosis. It has also been demonstrated that p38-α MAPK (p38) is necessary for Bax activation and apoptosis in vitro. The present studies were designed to test the hypothesis that p38 is a key regulator of Bax activation during adaptation after SBR in vivo. Enterocyte expression of p38 was deleted by tamoxifen administration to activate villin-Cre in adult mice with a floxed Mapk14 (p38-α) gene. Proximal 50% SBR or sham operations were performed on wild-type (WT) and p38 intestinal knockout (p38-IKO) mice under isoflurane anesthesia. Mice were killed 3 or 7 days after operation, and adaptation was analyzed by measuring intestinal morphology, proliferation, and apoptosis. Bax activity was quantified by immunoprecipitation, followed by Western blotting. After SBR, p38-IKO mice had deeper crypts, longer villi, and accelerated proliferation compared with WT controls. Rates of crypt apoptosis were significantly lower in p38-IKO mice, both at baseline and after SBR. Levels of activated Bax were twofold higher in WT mice after SBR relative to sham. In contrast, activated Bax levels were reduced by 67% in mice after p38 MAPK deletion. Deleted p38 expression within the intestinal epithelium leads to enhanced adaptation and reduced levels of enterocyte apoptosis after massive intestinal resection. p38-regulated Bax activation appears to be an important mechanism underlying resection-induced apoptosis.

Fig. 1.

p38-α MAPK is successfully deleted in enterocytes. Intestinal crypts (C) and villi (V) were isolated from wild-type (WT) and inducible, enterocyte-specific p38-α MAPK-null [p38-IKO or knockout (KO)] mice 1 wk after tamoxifen injection (0.5 mg/day × 2 doses). The underlying intestinal smooth muscle was also collected. A: Western blot analysis of total p38 MAPK and the p38-α isoform. Actin was used as the loading control. B: quantitative real-time PCR analysis of Mapk14 (the gene encoding p38-α MAPK) gene expression in crypts, villi, and underlying smooth muscle. Values are means ± SE; n = 4 in WT and KO groups. #P < 0.001.

Fig. 2.

p38 deletion causes small bowel hyperplasia. WT and p38-IKO (KO) mice were killed 1 wk after tamoxifen injection (0.5 mg/day × 2 doses). A: small bowel length as measured with a standardized 5-g weight. The middle line represents the mean, the edges of the box represent the 75th percentile, and the error bars show the 95th percentile. B: a proliferative index was calculated from a ratio of 5-bromodeoxyuridine (BrdU+) cells to total crypt cells. An apoptotic index was calculated both from the number of apoptotic bodies per 100 crypts on routine hematoxylin and eosin sections (C), or the number of crypt cells positively staining for cleaved caspase 3 (CC3; D). E: crypt fission. A crypt fission index was calculated from the number of crypts undergoing fission per 100 crypts. Values are means ± SE. *P < 0.05, %P < 0.01, &P < 0.005, #P < 0.001.

Fig. 3.

p38 is absent in enterocytes for the duration of the postoperative period. Levels of p38-α were detected by Western immunoblotting in crypt and villus enterocyte protein isolated from WT and p38-IKO (KO) mouse intestine collected 3 (POD3) and 7 days after operation (POD7). Actin was used as the loading control.

Fig. 4.

p38 deficiency results in enhanced intestinal adaptation after small bowel resection (SBR). WT and p38-IKO mice (KO) underwent either 50% proximal SBR or sham operation (transection and reanastomosis of the bowel alone). Crypt depth and villus length (means ± SE) were measured from H&E-stained sections of ileum. Crypt depth (A) and villus length (B) from ileum harvested on POD3 are shown. Crypt depth (C) and villus length (D) from ileum taken on POD7 are shown. *P < 0.05, %P < 0.01, &P < 0.005, #P < 0.001.

Fig. 5.

p38 deficiency alters enterocyte kinetics after SBR. Enterocyte kinetics were measured in WT and p38-IKO (KO) mice on POD3 following SBR and sham operation. A: a proliferative index was calculated from a ratio of BrdU+ cells to total crypts cells. B: the crypt fission index was calculated from the number of crypt fission events per 100 well-oriented crypts. An apoptotic index was calculated from the number of apoptotic bodies per 100 crypts from H&E (C) or CC3 immunostained slides (D). Values are means ± SE. *P < 0.05, %P < 0.01, #P < 0.001.

Fig. 6.

Bax activity is augmented after SBR and is regulated by p38 MAPK. A: Bax activity was measured in WT and p38-IKO (KO) mice by immunoprecipitating the active form of Bax with an isoform-specific antibody (6A7), followed by Western blotting for total Bax. The amount of total Bax used as input is shown for control. B: the amount of active Bax relative to input was quantified and is shown in the graph adjacent; n = 3 in each group. C: cytochrome c was measured by Western blotting in cytoplasm fractions of isolated crypts from WT and KO mice. D: quantification of the cytochrome c level against actin; n = 8 for each group. E: C57/B6 mice underwent SBR or sham operation and were killed on POD3. F: the amount of active Bax was quantified against total Bax and is shown; n = 6 in each group. G: p38-IKO mice underwent either sham or SBR and were killed on POD3. H: quantification of active Bax against the input total Bax; n = 3 in each group. Values are means ± SE. *P < 0.05, &P < 0.005.