Bax is required for resection-induced changes in apoptosis, proliferation, and members of the extrinsic cell death pathways

BACKGROUND AND 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 show increases 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 receptor-mediated 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.     

Humoral immune response to tissue transglutaminase is related to epithelial cell proliferation in celiac disease

BACKGROUND & AIMS: Tissue transglutaminase (tTG) autoantibodies are markers of celiac disease, and the enzyme is required for several crucial biological processes. The aim of this study was to determine whether these autoantibodies are involved in the pathogenesis of the mucosal lesion typical of celiac disease. METHODS: Using rhodamine-conjugated phalloidin staining, we evaluated whether tTG antibodies, both commercially available and cloned from patients with celiac disease, cause cytoskeletal changes in Caco-2, MCF7, and NIH 3T3 cells. We monitored cell levels of bromodeoxyuridine incorporation to determine whether tTG autoantibodies are able to induce NIH 3T3 fibroblasts and epithelial mucosal cells into S phase. RESULTS: Treatment with tTG antibodies caused a dose-dependent increase of membrane ruffling in Caco-2, MCF7, and NIH 3T3 cells. It also dose-dependently induced G(0)-synchronized NIH 3T3 fibroblasts into S phase but did not affect the rate of apoptosis. Similarly, tTG antibodies induced S-phase entry of epithelial cells in cultured intestinal biopsy specimens from patients with celiac disease. They did not affect biopsy specimens from patients without celiac disease. CONCLUSIONS: Our results suggest that tTG autoantibodies per se, by interacting with the extracellular membrane-bound transglutaminase, may play an important role in epithelial cell proliferation in celiac disease.     

Tumor necrosis factor alpha, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver.

BACKGROUND & AIMS: Apoptosis of hepatocytes is a central feature of ischemic injury in the liver. The aim of this study was to identify extracellular inducers of apoptosis in the murine ischemic liver. METHODS: Involvement of tumor necrosis factor (TNF)-alpha and Fas signaling was evaluated using various knockout mice (TNF-receptor 1 [TNF-R1]-/-, Fas[lpr]-/-, and Fas ligand[gld]-/-) and wild-type mice pretreated with pentoxifylline, an inhibitor of TNF-alpha synthesis. RESULTS: Expression of TNF-alpha was increased after ischemia and reperfusion in wild-type mice and TNF-R1-deficient mice when compared with sham-operated animals. Pentoxifylline prevented up-regulation of TNF-alpha expression. Inhibition of TNF-alpha resulted in significant decrease of serum aspartate aminotransferase levels and prolonged animal survival. Markers of apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, cytochrome C release, and caspase 3 activity) were consistently decreased, and animal survival was prolonged after blocking TNF-alpha. In contrast, inhibition of Fas signaling did not alter parameters of tissue injury or apoptosis, and animal survival remained unchanged. CONCLUSIONS: We identify TNF-alpha as a crucial inducer of apoptotic cell death in the ischemic liver. A role for Fas could not be identified. These findings may lead to novel strategies to prevent ischemic injury of the liver.     

The role of transforming growth factor beta-2, beta-3 in mediating apoptosis in the murine intestinal mucosa

BACKGROUND & AIMS: Apoptosis is especially relevant in the gastrointestinal tract because the mammalian intestinal mucosa undergoes continual epithelial regeneration. Most recently, we confirmed the proapoptotic role of endogenous transforming growth factor (TGF)-beta in the developing chick retina as well as in chick ciliary, dorsal root, and spinal motor neurons. In the present study, we determined to establish the role of TGF-beta2 and TGF-beta3 in mediating apoptosis in non-neuronal tissue by analyzing the intestinal mucosa of Tgfbeta2(+/-) and Tgfbeta3(+/-) heterozygous mice. METHODS: Intestinal localization of TGF-beta2 and TGF-beta3 isoforms and antiapoptotic molecules Bcl-xL and Bcl-2 was examined immunocytochemically and by Western blot analysis. Apoptosis was detected by enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and proliferation was detected by proliferating cell nuclear antigen stains. RESULTS: TGF-beta2 was detected in endocrine cells, whereas TGF-beta3 was predominantly found in goblet cells. Programmed cell death was significantly reduced in the intestinal mucosa of Tgfbeta2(+/-) and Tgfbeta3(+/-) heterozygous mice. This decrease in apoptosis was accompanied by an increase in villus length; proliferation, however, seemed to remain unchanged. The level of Bcl-xL and Bcl-2 was significantly up-regulated in Tgfbeta2(+/-) and Tgfbeta3(+/-) mice. CONCLUSIONS: Our data show that TGF-beta2 and TGF-beta3 play an important role in mediating apoptosis in the intestinal mucosa and regulating apoptosis-associated proteins Bcl-xL and Bcl-2 in vivo.     

Cysteine-rich domains of muc3 intestinal mucin promote cell migration, inhibit apoptosis, and accelerate wound healing

BACKGROUND & AIMS: Muc3 intestinal mucin contains an extracellular cysteine-rich domain with 2 epidermal growth factor (EGF)-like motifs. The aim of this study was to determine the functional properties of Muc3 proteins. METHODS: Glutathione S-transferase-fusion proteins containing both Muc3 EGF-like domains (m3EGF1,2) or truncated versions (m3EGF1 and m3EGF2) were purified from Escherichia coli. Mouse colon (young adult mouse colon) and human A431 and LoVo cells were examined for migration and tyrosine phosphorylation in response to recombinant proteins. LoVo cells were transfected with a human MUC3A transmembrane-EGF1,2 construct and a stable clone was isolated (LhM3c14). Endogenous MUC3A in LoVo was inhibited by specific small interfering RNA transfection. Apoptosis was quantitated by nuclear morphology or terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling assay. Colitis was induced in mice by oral 5% dextran sodium sulfate or rectal 5% acetic acid, followed by enema treatments. RESULTS: m3EGF1,2 stimulated cell migration in all cell lines, but did not induce proliferation. Migration was inhibited by a tyrosine phosphorylation inhibitor, genistein, but not by the EGF receptor inhibitor, tyrphostin (AG1478). Inhibition of endogenous MUC3A in LoVo reduced baseline migration. Tyrosine phosphorylation of ErbB receptors was not observed after treatment of cells with m3EGF1,2. LoVo cells pretreated with m3EGF1,2 and transfected LhM3c14 cells showed reduced apoptosis in response to tumor necrosis factor alpha or Fas-receptor stimulation. Administration of m3EGF1,2 per rectum significantly reduced mucosal ulceration and apoptosis in experimental acute colitis. Truncated proteins m3EGF1 and m3EGF2 had no effect. CONCLUSIONS: The Muc3 mucin cysteine-rich domain plays an active role in epithelial restitution, and represents a potential novel therapeutic agent for intestinal wound healing.     

Expression and regulation of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in human and mouse tissue

BACKGROUND & AIMS: Nonsteroidal anti-inflammatory drugs (NSAIDs) induce NSAID-activated gene 1 (NAG-1), which has proapoptotic and antitumorigenic activities. However, NAG-1 expression and its relationship with apoptosis in human and mouse intestinal tract have not been determined. METHODS: NAG-1 expression in human and mouse tissue was determined by immunohistochemistry, and apoptosis was estimated by in situ apoptosis detection. Apoptosis in NAG-1 overexpressing HCT-116 cells was examined with flow cytometry after cell sorting by green fluorescence protein. NAG-1 regulation in mouse cells was examined by Northern blot analysis, comparing sulindac-treated and nontreated mice. RESULTS: Apoptosis was higher in NAG-1 overexpressing cells compared with controls. Human NAG-1 protein was localized to the colonic surface epithelium where cells undergo apoptosis, and higher expression was observed in the normal surface epithelium than in most of the tumors. This localization and lower expression in tumors was similar to that in the Min mouse, in which NSAIDs were also shown to regulate the expression of NAG-1 in mouse cells. Sulindac treatment of mice increased the NAG-1 expression in the colon and liver. CONCLUSIONS: Based on these results, we propose that NAG-1 acts as a mediator of apoptosis in intestinal cells and may contribute to cancer chemoprevention by NSAIDs.     

Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners

BACKGROUND & AIMS: Viral hepatitis infection, which is a major cause of liver fibrosis, is associated with activation of innate immunity. However, the role of innate immunity in liver fibrosis remains obscure. METHODS: Liver fibrosis was induced either by feeding mice with the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet or by injecting them with carbon tetrachloride. The Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, was used to activate innate immunity cells and mediators, including natural killer cells and interferon gamma. RESULTS: In the mouse model of DDC-induced liver fibrosis, natural killer cell activation by polyinosinic-polycytidylic acid induced cell death to activated hepatic stellate cells and attenuated the severity of liver fibrosis. Polyinosinic-polycytidylic acid treatment also ameliorated liver fibrosis induced by carbon tetrachloride. The observed protective effect of polyinosinic-polycytidylic acid on liver fibrosis was diminished through either depletion of natural killer cells or by disruption of the interferon gamma gene. Expression of retinoic acid early inducible 1, the NKG2D ligand, was undetectable on quiescent hepatic stellate cells, whereas high levels were found on activated hepatic stellate cells, which correlated with the resistance and susceptibility of quiescent hepatic stellate cells and activated hepatic stellate cells to natural killer cell lysis, respectively. Moreover, treatment with polyinosinic-polycytidylic acid or interferon gamma enhanced the cytotoxicity of natural killer cells against activated hepatic stellate cells and increased the expression of NKG2D and tumor necrosis factor-related apoptosis-inducing ligand on liver natural killer cells. Blocking NKG2D or tumor necrosis factor-related apoptosis-inducing ligand with neutralizing antibodies markedly diminished the cytotoxicity of polyinosinic-polycytidylic acid-activated natural killer cells against activated hepatic stellate cells. CONCLUSIONS: Our findings suggest that natural killer cells kill activated hepatic stellate cells via retinoic acid early inducible 1/NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent mechanisms, thereby ameliorating liver fibrosis.     

Early pregnancy induced expression of prostaglandin E2 receptors EP2 and EP4 in the ovine endometrium and regulated by interferon tau through multiple cell signaling pathways

Prostaglandin E2 (PGE₂) plays pleiotropic roles at fetal-maternal interface during establishment of pregnancy. The objectives of the study were to: (i) determine regulation of PGE2 receptors EP1, EP2, EP3, and EP4 in the endometrium during the estrous cycle and early pregnancy; and (ii) understand endometrial epithelial and stromal cell-specific hormonal regulation of EP2 and EP4 in sheep. Results indicate that: (i) early pregnancy induces expression of EP2 and EP4 but not EP1 and EP3 proteins in the endometrium on days 12-16 compared to that of estrous cycle; (ii) intrauterine infusion of interferon tau (IFNT) increases expression of EP2 and EP4 proteins in endometrium; and (iii) IFNT activates distinct epithelial and stromal cell-specific JAK, EGFR, ERK1/2, AKT, or JNK signaling module to regulate expression of EP2 and EP4 proteins in the ovine endometrium. Our results indicate a role for EP2 and EP4-mediated PGE₂ signaling in endometrial functions and establishment of pregnancy in ruminants.