An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.

BACKGROUND: Simple epithelia encase developing embryos and organs. Although these epithelia consist of only one or two layers of cells, they must provide tight barriers for the tissues that they envelop. Apoptosis occurring within these simple epithelia could compromise this barrier. How, then, does an epithelium remove apoptotic cells without disrupting its function as a barrier? RESULTS: We show that apoptotic cells are extruded from a simple epithelium by the concerted contraction of their neighbors. A ring of actin and myosin forms both within the apoptotic cell and in the cells surrounding it, and contraction of the ring formed in the live neighbors is required for apoptotic cell extrusion, as injection of a Rho GTPase inhibitor into these cells completely blocks extrusion. Addition of apoptotic MDCK cells to an intact monolayer induces the formation of actin cables in the cells contacted, suggesting that the signal to form the cable comes from the dying cell. The signal is produced very early in the apoptotic process, before procaspase activation, cell shrinkage, or phosphatidylserine exposure. Remarkably, electrical resistance studies show that epithelial barrier function is maintained, even when large numbers of dying cells are being extruded. CONCLUSIONS: We propose that apoptotic cell extrusion is important for the preservation of epithelial barrier function during cell death. Our results suggest that an early signal from the dying cell activates Rho in live neighbors to extrude the apoptotic cell out of the epithelium.     

P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium

To preserve epithelial barrier function, dying cells are squeezed out of an epithelium by “apoptotic cell extrusion.” Specifically, a cell destined for apoptosis signals its live neighboring epithelial cells to form and contract a ring of actin and myosin II that squeezes the dying cell out of the epithelial sheet. Although most apoptotic cells extrude apically, we find that some exit basally. Localization of actin and myosin IIA contraction dictates the extrusion direction: basal extrusion requires circumferential contraction of neighboring cells at their apices, whereas apical extrusion also requires downward contraction along the basolateral surfaces. To activate actin/myosin basolaterally, microtubules in neighboring cells reorient and target p115 RhoGEF to this site. Preventing microtubule reorientation restricts contraction to the apex, driving extrusion basally. Extrusion polarity has important implications for tumors where apoptosis is blocked but extrusion is not, as basal extrusion could enable these cells to initiate metastasis.     

Colocalization of BAX and BCL-2 in small intestine and kidney biopsies with different degrees of DNA fragmentation

Morphological changes associated with apoptosis are closely correlated with the expression of specific proteins. However, the cause-effect relationships between the expression of these proteins and DNA degradation are barely known. For studying expression of apoptosis-related proteins in relation to different degrees of DNA fragmentation, the small intestine with its spatially organized continuum of proliferation, differentiation and death is a very useful preparation. Enterocytes towards the apex of the villi become increasingly susceptible to apoptosis. Here, this "apoptotic gradient" is used to demonstrate the presence of BAX and BCL-2 proteins in the cytoplasm of cells at the onset of apoptosis. In semithin serial sections of the small intestine, BAX, BCL-2 and DNA fragmentation were demonstrated. BAX and BCL-2 are always colocalized and only in cells with fragmented DNA. The gradient of BAX or BCL-2 staining is similar to the gradient of DNA fragmentation. Immunoreactivity for BCL-2 or BAX is most intense in cells that are prone to become apoptotic next in the course of cellular turnover but not in cells in an advanced apoptotic state, showing strongly condensed chromatin. When using the same technique on semithin sections of kidney biopsies, containing epithelia with low cellular turnover, we found DNA fragmentation mainly in the epithelial cells of the distal tubules. Similar to the situation in the enterocytes, BAX staining was confined to the cytoplasm of epithelial cells with a moderate degree of DNA fragmentation and reduced in epithelial cells with a high degree of DNA fragmentation. In contrast to the situation in the small intestine, very low levels of BCL-2 were found. The results suggest that expression of BCL-2 and BAX is related to cell damage as indicated by DNA fragmentation but not to advanced stages of cellular death, as indicated by chromatin condensation and cellular shrinkage.     

Epithelial apoptosis

Apoptosis is an essential part of the normal cellular phenotype repertoire. In the absence of appropriate survival factors, apoptosis is activated through specific signalling cassettes. Epithelia form distinctive three-dimensional cohesive structures that depend on adhesive interactions in order for these tissues to carry out their specialised roles, such as secretion and reproduction. The cellular programme that triggers apoptosis in epithelial cells has not yet been shown to differ from that in other cell types, yet the unique characteristics of epithelia endow them with specific determinants for survival. In particular, cell-matrix and cell-cell interactions are required to prevent entry of epithelial cells into apoptosis, and soluble factors that have profound effects on epithelia, such as steroid hormones or hepatocyte growth factor, also influence their survival. The regenerative capacity of certain epithelia is controlled by intrinsic expression of survival genes within stem cell populations, and may regulate the susceptibility of different epithelial tissues to undergo carcinogenesis.     

Blockade of TGF-beta accelerates mucosal destruction through epithelial cell apoptosis

To clarify the protective role of transforming growth factor (TGF)-beta for the intestinal epithelial injury in vivo, the effect of antibodies against TGF-beta on epithelial destruction and apoptosis was assessed in dextran sulfate sodium (DSS)-induced colitis by histological analysis of colonic sections, account of apoptotic epithelial cells. To evaluate the pathways of epithelial apoptosis, we analyzed the activities of caspases, the level of Fas and cellular FLICE-inhibitory protein (cFLIP) expression in epithelial cells. Apoptotic epithelial cells were increased prior to the onset of ulceration in DSS-induced colitis, and the neutralization of TGF-beta exacerbated epithelial apoptosis and histological damage score. The up-regulation of caspase-8 activity and Fas expression and reduced cFLIP expression were observed in intestinal epithelial cells from anti-TGF-beta antibody-treated mice. The present study revealed that suppression of TGF-beta deteriorated epithelial apoptosis, and the increase of apoptotic epithelial cells may amplify the inflammation in gut mucosa.     

Mating modifies apoptosis pattern in epithelial cells of the rat uterus

Estrous cycle in mammals includes marked epithelial changes in reproductive tract, regulated by sex steroid hormones. In the present work we studied the activation of caspases and apoptotic pattern in uterine epithelial cells during proestrus and estrus, and the effect of mating in this process. In addition, we investigated the role of seminal vesicle secretions on apoptosis of uterine epithelia. Apoptotic index was evaluated by TUNEL assay, caspases‐8, ‐9, and ‐3 activation was detected by Western blot and active caspase‐3 expression was detected by immunohistochemistry. Our results show that mating during proestrus and estrus transition induced changes in the apoptotic pattern of uterine luminal epithelium during estrus, characterized by a delay in the onset of apoptosis as compared with that observed in nonmated rats. No differences in the apoptotic pattern in the glandular epithelium between mated and nonmated rats were observed. Seminal vesicle secretions inhibited luminal epithelium apoptosis, while no changes in glandular epithelium apoptosis were observed. We also demonstrate that activation of caspases‐8, ‐9, and ‐3 occurred in both mated and nonmated rats. Active caspase‐3 was detected in the luminal and glandular epithelium in both nonmated and mated rats. The overall results indicate that mating delays but does not prevent the cellular death of the rat uterine luminal epithelium and seminal vesicle secretions are involved in this delay. Finally, the activation of both the mitochondrial and the membrane receptor pathways of cell death are implicated in the molecular mechanism of uterine apoptosis. Mol. Reprod. Dev. 76: 564–572, 2009. © 2008 Wiley‐Liss, Inc.     

Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals

Cataract is a major ocular disease that causes blindness in many developing countries of the world. It is well established that various factors such as oxidative stress, UV, and other toxic agents can induce both in vivo and in vitro cataract formation. However, a common cellular basis for this induction has not been previously recognized. The present study of lens epithelial cell viability suggests such a general mechanism. When lens epithelial cells from a group of 20 cataract patients 12 to 94 years old were analyzed by terminal deoxynucleotidyl transferase (TdT) labeling and DNA fragmentation assays, it was found that all of these patients had apoptotic epithelial cells ranging from 4.4 to 41.8%. By contrast, in eight normal human lenses of comparable age, very few apoptotic epithelial cells were observed. We suggest that cataract patients may have deficient defense systems against factors such as oxidative stress and UV at the onset of the disease. Such stress can trigger lens epithelial cell apoptosis that then may initiate cataract development. To test this hypothesis, it is also demonstrated here that hydrogen peroxide at concentrations previously found in some cataract patients induces both lens epithelial cell apoptosis and cortical opacity. Moreover, the temporal and spatial distribution of induced apoptotic lens epithelial cells precedes development of lens opacification. These results suggest that lens epithelial cell apoptosis may be a common cellular basis for initiation of noncongenital cataract formation.     

Apoptosis in endometrium of mouse during estrous cycle

The present study was carried out to evaluate apoptosis in endometrium and to correlate these changes with the circulating levels of estradiol and progesterone in the mouse. Apoptosis was observed in various compartments of mouse uterus i.e. stroma, glandular epithelium and luminal epithelium depending on the stage of cycle. Stromal cell apoptosis was observed during various stages of cyclicity except on estrus day. Luminal epithelial cells showed apoptotic changes during all stages of cyclicity except on diestrus day. During metestrus, apoptosis was observed in glandular and luminal epithelia as well as stromal cells. Steroid antagonists such as tamoxifen and onapristone altered the apoptotic changes in the uterus. The results suggest that epithelial cell apoptosis is regulated by estrogen while stromal cell apoptosis is under the control of progesterone.     

Apoptosis in human embryo development: 2. Cerebellum

We analysed the spatial and temporal distribution of apoptosis in human cerebellum development, during embryonic and fetal periods. Cerebella excised from two human embryos (8 weeks old) and eight fetuses (12-22 weeks old), were paraffin embedded and serially sectioned. Apoptotic cells were identified by propidium iodide staining, and TUNEL. In addition, immunohistochemistry for suicide receptor Fas(APO-1/CD95) was performed. We determined the distribution and percentage of apoptotic cells as well as Fas(APO-1/CD95)-positive cells in different regions and stages of development. Apoptotic cells were seen in both proliferative zones and postmitotic regions along the migratory pathways as well as in the developing cerebellar cortex in all examined stages. The Fas(APO-1/CD95) immunoreactivity was present in all examined stages in a small population of apoptotic cells: either neuroblasts or differentiated cells in postmitotic zones. These findings suggest that apoptosis drives the selection of the cells which are committed to differentiate during the early stages of cerebellar development. The differences between apoptotic cells distribution and Fas receptor expression suggest that cell selection is driven by different apoptotic pathways.     

Forced involution of the functionally differentiated mammary gland by overexpression of the pro-apoptotic protein bax

The mammary gland is a developmentally dynamic, hormone-responsive organ that undergoes proliferation and differentiation within the secretory epithelial compartment during pregnancy. The epithelia are maintained by pro-survival signals (e.g., Stat5, Akt1) during lactation, but undergo apoptosis during involution through inactivation of cell survival pathways and upregulation of pro-apoptotic proteins. To assess if the survival signals in the functionally differentiated mammary epithelial cells can override a pro-apoptotic signal, we generated transgenic mice that express Bax under the whey acidic protein (WAP) promoter. WAP-Bax females exhibited a lactation defect and were unable to nourish their offspring. Mammary glands demonstrated: (1) a reduction in epithelial content, (2) hallmark signs of mitochondria-mediated cell death, (3) an increase in apoptotic cells by TUNEL assay, and (4) precocious Stat3 activation. This suggests that upregulation of a single pro-apoptotic factor of the Bcl-2 family is sufficient to initiate apoptosis of functionally differentiated mammary epithelial cells in vivo.     

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion¹. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.Download video file.^{(59M, mov)}     

Compartmentalization of Fas and Fas ligand may prevent auto- or paracrine apoptosis in epithelial cells

Oligomerization of Fas receptor by its ligand, FasL, activates a signaling cascade that leads to apoptosis of Fas bearing cells. Interestingly, many epithelia coexpress Fas and FasL, yet FasL does not trigger Fas present on the same or neighboring cells to induce spontaneous apoptosis. Here, we show that Fas and FasL are segregated from each other to different cellular compartments in kidney epithelial MDCK cells. While Fas is restricted to the basolateral surface, FasL is sequestered to an intracellular compartment and, a lesser extent, the apical surface. This spatial segregation of Fas and FasL may explain how epithelial cells can constitutively express a functional Fas pathway but avoid auto- or paracrine cell death. Compromising this spatial segregation in physiological or pathological situations may play a so far underestimated role in initiating apoptosis of epithelial cells.     

Leukocyte elastase induces epithelial apoptosis: role of mitochondial permeability changes and Akt

During acute inflammation, neutrophil-mediated injury to epithelium may lead to disruption of epithelial function, including the induction of epithelial apoptosis. Herein, we report the effects of neutrophil transmigration and of purified leukocyte elastase on epithelial cell survival. Neutrophil transmigration induced apoptosis of epithelial cells [control monolayers: 5 +/- 1 cells/25 high-power fields (HPF) vs. neutrophil-treated monolayers: 29 +/- 10 cells/HPF, P < 0.05, n = 3 as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay] as did low concentrations (0.1 U/ml) of purified leukocyte elastase (control monolayers: 6.4 +/- 2.5% apoptotic vs. elastase: 26.2 +/- 2.9% apoptotic, P < 0.05, as determined by cytokeratin 18 cleavage). Treatment with elastase resulted in decreased mitochondrial membrane potential, release of cytochrome c to the cytosol, and cleavage of caspases-9 and -3 as determined by Western blot analysis, implicating altered mitochondrial membrane permeability as a primary mechanism for elastase-induced apoptosis. Additionally, incubation of epithelial cells with leukocyte elastase resulted in an early increase followed by a decrease in the phosphorylation of epithelial Akt, a serine/threonine kinase important in cell survival. Inhibition of epithelial Akt before elastase treatment potentiated epithelial cell apoptosis, suggesting that the initial activation of Akt represents a protective response by the epithelial cells to the proapoptotic effects of leukocyte elastase. Taken together, these observations suggest that epithelial cells exhibit a dual response to cellular stress imposed by leukocyte elastase with a proapoptotic response mediated via early alterations in mitochondrial membrane permeability countered by activation of the survival pathway involving Akt.     

Tissue inhibitor of metalloproteinase-1 protects human breast epithelial cells against intrinsic apoptotic cell death via the focal adhesion kinase/phosphatidylinositol 3-kinase and MAPK signaling pathway

Tissue inhibitor of metalloproteinase (TIMP-1) is a natural protease inhibitor of matrix metalloproteinases (MMPs). Recent studies revealed a novel function of TIMP-1 as a potent inhibitor of apoptosis in mammalian cells. However, the mechanisms by which TIMP-1 exerts its anti-apoptotic effect are not understood. Here we show that TIMP-1 activates cell survival signaling pathways involving focal adhesion kinase, phosphatidylinositol 3-kinase, and ERKs in human breast epithelial cells to TIMP-1. TIMP-1-activated cell survival signaling down-regulates caspase-mediated classical apoptotic pathways induced by a variety of stimuli including anoikis, staurosporine exposure, and growth factor withdrawal. Consistently, down-regulation of TIMP-1 expression greatly enhances apoptotic cell death. In a previous study, substitution of the second amino acid residue threonine for glycine in TIMP-1, which confers selective MMP inhibition, was shown to obliterate its anti-apoptotic activity in activated hepatic stellate cells suggesting that the anti-apoptotic activity of TIMP-1 is dependent on MMP inhibition. Here we show that the same mutant inhibits apoptosis of human breast epithelial cells, suggesting different mechanisms of TIMP-1 regulation of apoptosis depending on cell types. Neither TIMP-2 nor a synthetic MMP inhibitor protects breast epithelial cells from intrinsic apoptotic cell death. Furthermore, TIMP-1 enhances cell survival in the presence of the synthetic MMP inhibitor. Taken together, the present study unveils some of the mechanisms mediating the anti-apoptotic effects of TIMP-1 in human breast epithelial cells through TIMP-1-specific signal transduction pathways.     

Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans

Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.     

PI3K Acts in synergy with loss of PKC to elicit apoptosis via the UPR

It is known that Ras mutations, together with loss of PKC, are apoptotic in various types of mammalian cells. The mechanism of how aberrant Ras transmits this apoptotic signaling remains unclear. Using three V12‐Ha‐ras loop mutants that preferentially bind to and activate one of Ras effectors, we tested the role of Ras downstream pathways in the induction of apoptosis in rat lung epithelia, human lung or prostate cancer cells. After PKC inhibition, the activation of PI3K/Akt renders the susceptibility of cells to apoptosis. We also demonstrate that the amount of ROS is moderately increased in the cells ectopically expressing V12C40 and dramatically elevated by suppression of PKC, which leads to apoptosis through the activation of UPR. Thus, our study suggests that after PKC abrogation, PI3K functions downstream of Ras to perturb the state of cellular redox and signals to ER stress‐regulated apoptotic machinery. J. Cell. Biochem. 107: 76–85, 2009. © 2009 Wiley‐Liss, Inc.     

胞外ATP在呼吸道中的作用

ATP不但是各种细胞的能量来源,而且更是一种自分泌或旁分泌的胞外信使,参与细胞一系列的生物学效应。ATP从呼吸道上皮细胞中释放,在调节呼吸道表面液体量的平衡、黏膜纤毛清除能力和呼吸道防御功能方面起重要作用,并参与呼吸道疾病及炎症的发生。本文对ATP从呼吸道上皮释放的途径,ATP调节呼吸道上皮离子转运的机制,ATP对呼吸道平滑肌的双重调节作用,以及ATP参与呼吸道疾病和炎症的发生机制等方面予以综述。     

Overexpression of beta-catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators

beta-Catenin promotes epithelial architecture by forming cell surface complexes with E-cadherin and also interacts with TCF/LEF-1 in the nucleus to control gene expression. By DNA transfection, we overexpressed beta-catenin and/or LEF-1 in NIH 3T3 fibroblasts, corneal fibroblasts, corneal epithelia, uveal melanoma cells, and several carcinoma cell lines. In all cases (with or without LEF-1), the abundant exogenous beta-catenin localizes to the nucleus and forms distinct nuclear aggregates that are not associated with DNA. Surprisingly, we found that with time (5-8 d after transfection) cells overexpressing beta-catenin all undergo apoptosis. LEF-1 does not need to be present. Moreover, LEF-1 overexpression in the absence of exogenous beta-catenin does not induce apoptosis, even though some endogenous beta-catenin moves with the exogenous LEF-1 into the nucleus. TOPFLASH/FOPFLASH reporter assays showed that full-length beta-catenin is able to induce LEF-1-dependent transactivation, whereas Arm beta-catenin totally abolishes the transactivating function. However, Arm beta-catenin, containing deletions of known LEF-1-transactivating domains, has the same apoptotic effects as full-length beta-catenin. Overexpressed beta-catenin also induces apoptosis in cells transfected with nuclear localization signal-deleted LEF-1 that localizes only in the cytoplasm. Thus, the apoptotic effects of overexpressed exogenous beta-catenin do not rely on its transactivating function with nuclear LEF-1. Overexpressed delta-catenin, containing 10 Arm repeats, induces only minor apoptosis, suggesting that the major apoptotic effect may be due to domains specific to beta-catenin as well as to Arm repeats. The absence of p53, Rb, cyclin D1, or E2F1 does not affect the apoptotic effect of overexpressed beta-catenin, but Bcl-x(L) reduces it. We hypothesize that in vivo apoptosis of cells overexpressing beta-catenin might be a physiological mechanism to eliminate them from the population.     

Recognition-dependent signaling events in response to apoptotic targets inhibit epithelial cell viability by multiple mechanisms: implications for non-immune tissue homeostasis

Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival. Generally, apoptotic and necrotic targets have opposite effects with apoptotic targets inhibiting and necrotic targets stimulating the activity of these pathways. Here we explore the consequences of these signaling differences. We show that recognition of apoptotic targets induces a profound decrease in PTEC viability through increased responder cell death and decreased proliferation. In contrast, necrotic targets promote viability through decreased death and increased proliferation. Both target types mediate their effects through a network of Akt-dependent and -independent events. Apoptotic targets modulate Akt-dependent viability in part through a reduction in cellular β-catenin and decreased inactivation of Bad. In contrast, Akt-independent modulation of viability occurs through activation of caspase-8, suggesting that death receptor-dependent pathways are involved. Apoptotic targets also activate p38, which partially protects responders from target-induced death. The response of epithelial cells varies depending on their tissue origin. Some cell lines, like PTEC, demonstrate decreased viability, whereas others (e.g. breast-derived) show increased viability. By acting as sentinels of environmental change, apoptotic targets allow neighboring cells, especially non-migratory epithelial cells, to monitor and potentially adapt to local stresses.