Recognition of Apoptotic Cells by Epithelial Cells: CONSERVED VERSUS TISSUE-SPECIFIC SIGNALING RESPONSES*

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (mϕ). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence mϕ survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like mϕ, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and mϕ. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and mϕ. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in mϕ, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from mϕ. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.     

Phagocytosis of apoptotic cells by macrophages induces novel signaling events leading to cytokine-independent survival and inhibition of proliferation: activation of Akt and inhibition of extracellular signal-regulated kinases 1 and 2

Recent evidence indicates that phagocytic clearance of apoptotic cells, initially thought to be a silent event, can modulate macrophage (M phi) function. We show in this work that phagocytic uptake of apoptotic cells or bodies, in the absence of serum or soluble survival factors, inhibits apoptosis and maintains viability of primary cultures of murine peritoneal and bone marrow M phi with a potency approaching that of serum-supplemented medium. Apoptotic uptake also profoundly inhibits the proliferation of bone marrow M phi stimulated to proliferate by M-CSF. While inhibition of proliferation is an unusual property for survival factors, the combination of increased survival and decreased proliferation may aid the M phi in its role as a scavenger during resolution of inflammation. The ability of apoptotic cells to promote survival and inhibit proliferation appears to be the result of simultaneous activation of Akt and inhibition of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and ERK2 (ERK1/2). While several activators of the innate immune system, or danger signals, also inhibit apoptosis and proliferation, danger signals and necrotic cells differ from apoptotic cells in that they activate, rather than inhibit, ERK1/2. These signaling differences may underlie the opposing tendencies of apoptotic cells and danger signals in promoting tolerance vs immunity.     

Inhibition of death receptor signaling by bacterial gut pathogens

Gastrointestinal bacterial pathogens such as enteropathogenic Escherichia coli, Salmonella and Shigella control inflammatory and apoptotic signaling in human intestinal cells to establish infection, replicate and disseminate to other hosts. These pathogens manipulate host cell signaling through the translocation of virulence effector proteins directly into the host cell cytoplasm, which then target various signaling pathways. Death receptors such as TNFR1, FAS and TRAIL-R induce signaling cascades that are crucial to the clearance of pathogens, and as such are major targets for inhibition by pathogens. This review focuses on what is known about how bacterial gut pathogens inhibit death receptor signaling to suppress inflammation and prevent apoptosis.     

Dysregulated interleukin 11 in primary Sjögren's syndrome contributes to apoptosis of glandular epithelial cells

The purpose of this study was to explore the potential function of interleukin‐11 (IL‐11) in the pathogenesis of primary Sjögren's syndrome (pSS) patients. Real‐time polymerase chain reaction was performed to examine IL‐11 expression in the labial glands of 30 pSS patients and 30 healthy controls. Immunohistochemistry was conducted to assess the distribution of IL‐ll‐positive cells in labial glands. The human salivary gland (HSG) cell line was used to study the effects of IL‐11 on gland epithelial cells in vitro. Cell viability and cell proliferation were examined by CCK‐8 kit and EdU assay, respectively. The population of apoptotic cells was detected in flow cytometry followed by Annexin V/PI and Hoechst staining. We found that the expression levels of IL‐11 were remarkably decreased in pSS labial glands and were positively correlated with C‐reactive protein levels and negatively correlated with rheumatoid factor levels. Fewer numbers of glandular epithelial cells were observed to be positively stained with IL‐11 antibody in labial glands from pSS patients than those in healthy control patients. After IL‐11 treatment, the viability and proliferation of HSG cells were significantly higher than those in the control group. The total apoptotic and necrotic rates of HSG cells in the group after IL‐11 treatment were significantly lower. In conclusion, the results indicated that IL‐11 promoted viability and proliferation and inhibited apoptotic and necrotic rates of glandular epithelial cells. In pSS, downregulated IL‐11 might contribute to the apoptosis of salivary gland epithelial cells. However, it might be a potential target to alleviate the pathological atrophy of glandular epithelial cells in pSS patients.     

Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation

Apoptotic cell death has been observed in many in vivo and in vitro models of ischemia. However, the molecular pathways involved in ischemia-induced apoptosis remain unclear. We have examined the role of Bcl-2 family of proteins in mediating apoptosis of PC12 cells exposed to the conditions of oxygen and glucose deprivation (OGD) or OGD followed by restoration of oxygen and glucose (OGD-restoration, OGD-R). OGD decreased mitochondrial membrane potential and induced necrosis of PC12 cells, which were both prevented by the overexpression of Bcl-2 proteins. OGD-R caused apoptotic cell death, induced cytochrome C release from mitochondria and caspase-3 activation, decreased mitochondrial membrane potential, and increased levels of pro-apoptotic Bax translocated to the mitochondrial membrane, all of which were reversed by overexpression of Bcl-2. These results demonstrate that the cell death induced by OGD and OGD-R in PC12 cells is potentially mediated through the regulation of mitochondrial membrane potential by the Bcl-2 family of proteins. It also reveals the importance of developing therapeutic strategies for maintaining the mitochondrial membrane potential as a possible way of reducing necrotic and apoptotic cell death that occurs following an ischemic insult.     

Recent advances in apoptosis, mitochondria and drug resistance in cancer cells

Defective or inefficient apoptosis is an acquired hallmark of cancer cells. Thus, a thorough understanding of apoptotic signaling pathways and insights into apoptosis resistance mechanisms are imperative to unravel novel drug targets for the design of more effective and target selective therapeutic strategies. This review aims at providing an overview of the recent understanding of apoptotic signaling pathways, the main mechanisms by which cancer cells resist apoptotic insults, and discusses some recent attempts to target the mitochondrion for restoring efficient cell death signaling in cancer cells.     

Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy

Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis. In necrotic caspase-independent modes of death, the role and extent of phagocytosis is not well documented. To address this issue, we studied at the ultrastructural level the phagocytic response to dying cells in an in vitro phagocytosis assay with a mouse macrophage cell line (Mf4/4). As target cells, murine L929sAhFas cells were induced to die by TNFR1-mediated necrosis or by Fas-mediated apoptosis. Apoptotic L929sAhFas cells are taken up by complete engulfment of apoptotic bodies as single entities forming a tight-fitting phagosome, thus resembling the "zipper"-like mechanism of internalization. In contrast, primary and secondary necrotic cells were internalized by a macropinocytotic mechanism with formation of multiple ruffles by the ingesting macrophage. Ingestion of necrotic cellular material was invariably taking place after the integrity of the cell membrane was lost and did not occur as discrete particles, in contrast to apoptotic material that is surrounded by an intact membrane. Although nuclei of necrotic cells have been observed in the vicinity of macrophages, no uptake of necrotic nuclei was observed. The present report provides a basis for future studies aimed at discovering molecular pathways that precede these diverse mechanisms of uptake.     

Apoptotic cell removal

Ingestion by professional or amateur phagocytes is the fate of most cells that undergo apoptosis. Studies in both Caenorhabditis elegans and mammals are now converging to reveal some of the key mechanisms and consequences of this removal process. At least seven corpse removal genes in nematodes have mammalian equivalents, and represent elements of signaling pathways involved in uptake. In mammals, a wide variety of apoptotic cell recognition receptors has been implicated and appears to be divided into two categories, involved in tethering the apoptotic cell or triggering an uptake mechanism related to macropinocytosis. Apoptotic cell removal is normally efficient and non-inflammatory. By contrast, the process may become subverted by parasites to yield a more favorable growth environment, or in other cases lead to fibrosis. Removal may also clinch the apoptotic process itself in cells not yet completely committed to death.     

Thioredoxin-1 redox signaling regulates cell survival in response to hyperoxia

The most common form of newborn chronic lung disease, bronchopulmonary dysplasia (BPD), is thought to be caused by oxidative disruption of lung morphogenesis, which results in decreased pulmonary vasculature and alveolar simplification. Although cellular redox status is known to regulate cellular proliferation and differentiation, redox-sensitive pathways associated with these processes in developing pulmonary epithelium are unknown. Redox-sensitive pathways are commonly regulated by cysteine thiol modifications. Therefore two thiol oxidoreductase systems, thioredoxin and glutathione, were chosen to elucidate the roles of these pathways on cell death. Studies herein indicate that thiol oxidation contributes to cell death through impaired activity of glutathione-dependent and thioredoxin (Trx) systems and altered signaling through redox-sensitive pathways. Free thiol content decreased by 71% with hyperoxic (95% oxygen) exposure. Increased cell death was observed during oxygen exposure when either the Trx or the glutathione-dependent system was pharmacologically inhibited with aurothioglucose (ATG) or buthionine sulfoximine, respectively. However, inhibition of the Trx system yielded the smallest decrease in free thiol content (1.44% with ATG treatment vs 21.33% with BSO treatment). Although Trx1 protein levels were unchanged, Trx1 function was impaired during hyperoxic treatment as indicated by progressive cysteine oxidation. Overexpression of Trx1 in H1299 cells utilizing an inducible construct increased cell survival during hyperoxia, whereas siRNA knockdown of Trx1 during oxygen treatment reduced cell viability. Overall, this indicated that a comparatively small pool of proteins relies on Trx redox functions to mediate cell survival in hyperoxia, and the protective functions of Trx1 are progressively lost by its oxidative inhibition. To further elucidate the role of Trx1, potential Trx1 redox protein–protein interactions mediating cytoprotection and cell survival pathways were determined by utilizing a substrate trap (mass action trapping) proteomics approach. With this method, known Trx1 targets were detected, including peroxiredoxin-1 as well as novel targets, including two HSP90 isoforms (HSP90AA1 and HSP90AB1). Reactive cysteines within the structure of HSP90 are known to modulate its ATPase-dependent chaperone activity through disulfide formation and S-nitrosylation. Whereas HSP90 expression is unchanged at the protein level during hyperoxic exposure, siRNA knockdown significantly increased hyperoxic cell death by 2.5-fold, indicating cellular dependence on HSP90 chaperone functions in response to hyperoxic exposure. These data support the hypothesis that hyperoxic impairment of Trx1 has a negative impact on HSP90-oxidative responses critical to cell survival, with potential implications for pathways implicated in lung development and the pathogenesis of BPD.     

Engulfment mechanism of apoptotic cells

Apoptotic cells are engulfed and removed by phagocytes. This ensures proper development of the organism and can modulate immune responses. Recent studies have examined molecules on apoptotic cells, such as phosphatidylserine, which may signal for engulfment through multiple receptors. Apoptotic recognition mechanisms may vary with the apoptotic and engulfing cell type, and even with the age of the corpse.     

Transformation by oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by up-regulating death receptor 4 and death receptor 5 through a MEK-dependent pathway

RAS oncogenes play a major role in cancer development by activating an array of signaling pathways, most notably mitogen-activated protein kinases, resulting in aberrant proliferation and inhibition of apoptotic signaling cascades, rendering transformed cells resistant to extrinsic death stimuli. However, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill specific tumor cells through the engagement of its receptors, death receptor 4 (DR4) and death receptor 5 (DR5), and the activation of apoptotic pathways, providing promising targets for anticancer therapies. In this study, we show that TRAIL induces cell death in human colon adenocarcinoma cells in a MEK-dependent manner. We also report a prolonged MEK-dependent activation of ERK1/2 and increased c-FOS expression induced by TRAIL in this system. Our study reveals that transformation of the colon cell line Caco-2 by Ki- and mainly by Ha-ras oncogenes sensitizes these cells to TRAIL-induced apoptosis by causing specific MEK-dependent up-regulation of DR4 and DR5. These observations taken together reveal that RAS-MEK-ERK1/2 signaling pathway can sensitize cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 and overall imply that TRAIL-based therapeutic strategies using TRAIL agonists could be used in cases of human colon cancers bearing RAS mutations.     

Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency

Apoptotic and necrotic blebs elicited by H(2)O(2) were compared in terms of dynamics, structure and underlying biochemistry in HeLa cells and Clone 9 cells. Apoptotic blebs appeared in a few minutes and required micromolar peroxide concentrations. Necrotic blebs appeared much later, prior to cell permeabilization, and required millimolar peroxide concentrations. Strikingly, necrotic blebs grew at a constant rate, which was unaffected throughout successive cycles of budding and detachment. At 1 microm diameter, the necks of necrotic and apoptotic blebs were almost identical. ATP depletion was discarded as a major factor for both types of bleb. Inhibition of ROCK-I, MLCK and p38MAPK strongly decreased apoptotic blebbing but had no effect on necrotic blebbing. Taken together, these data suggest the existence of a novel structure of fixed dimensions at the neck of both types of plasma membrane blebs in epithelial cells. However, necrotic blebs can be distinguished from apoptotic blebs in their susceptibility to actomyosin kinase inhibition.     

High cell surface death receptor expression determines type I versus type II signaling

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.     

Clearance of apoptotic and necrotic cells and its immunological consequences

The ultimate and most favorable fate of almost all dying cells is engulfment by neighboring or specialized cells. Efficient clearance of cells undergoing apoptotic death is crucial for normal tissue homeostasis and for the modulation of immune responses. Engulfment of apoptotic cells is finely regulated by a highly redundant system of receptors and bridging molecules on phagocytic cells that detect molecules specific for dying cells. Recognition of necrotic cells by phagocytes is less well understood than recognition of apoptotic cells, but an increasing number of recent studies, which are discussed here, are highlighting its importance. New observations indicate that the interaction of macrophages with dying cells initiates internalization of the apoptotic or necrotic targets, and that internalization can be preceded by “zipper”-like and macropinocytotic mechanisms, respectively. We emphasize that clearance of dying cells is an important fundamental process serving multiple functions in the regulation of normal tissue turnover and homeostasis, and is not just simple anti- or pro-inflammatory responses. Here we review recent findings on genetic pathways participating in apoptotic cell clearance, mechanisms of internalization, and molecules involved in engulfment of apoptotic versus necrotic cells, as well as their immunological consequences and relationships to disease pathogenesis. Katharina D’Herde and Peter Vandenabeele share senior authorship. This study was supported by Ghent University GOA grant No. 12050502, IUAP-V/12-12.0C14.02, FWO-Vlaanderen 3G.0218.06, and Flanders Interuniversity Institute for Biotechnology (VIB).     

Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells

One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cells through cell cycle inhibition or induction of apoptosis without affecting normal cells. In this study, employing normal human prostate epithelial cells (PrEC), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate cancer cells (LNCaP, DU145, and PC-3), we evaluated the growth-inhibitory and apoptotic effects of tocotrienol-rich fraction (TRF) extracted from palm oil. TRF treatment to PrEC and PZ-HPV-7 resulted in almost identical growth-inhibitory responses of low magnitude. In sharp contrast, TRF treatment resulted in significant decreases in cell viability and colony formation in all three prostate cancer cell lines. The IC(50) values after 24h TRF treatment in LNCaP, PC-3, and DU145 cells were in the order 16.5, 17.5, and 22.0 microg/ml. TRF treatment resulted in significant apoptosis in all the cell lines as evident from (i) DNA fragmentation, (ii) fluorescence microscopy, and (iii) cell death detection ELISA, whereas the PrEC and PZ-HPV-7 cells did not undergo apoptosis, but showed modestly decreased cell viability only at a high dose of 80 microg/ml. In cell cycle analysis, TRF (10-40 microg/ml) resulted in a dose-dependent G0/G1 phase arrest and sub G1 accumulation in all three cancer cell lines but not in PZ-HPV-7 cells. These results suggest that the palm oil derivative TRF is capable of selectively inhibiting cellular proliferation and accelerating apoptotic events in prostate cancer cells. TRF offers significant promise as a chemopreventive and/or therapeutic agent against prostate cancer.     

Serglycin proteoglycan promotes apoptotic versus necrotic cell death in mast cells

The mechanisms that govern whether a cell dies by apoptosis or necrosis are not fully understood. Here we show that serglycin, a secretory granule proteoglycan of hematopoietic cells, can have a major impact on this decision. Wild type and serglycin(-/-) mast cells were equally sensitive to a range of cell death-inducing regimens. However, whereas wild type mast cells underwent apoptotic cell death, serglycin(-/-) cells died predominantly by necrosis. Investigations of the underlying mechanism revealed that cell death was accompanied by leakage of secretory granule compounds into the cytosol and that the necrotic phenotype of serglycin(-/-) mast cells was linked to defective degradation of poly(ADP-ribose) polymerase-1. Cells lacking mouse mast cell protease 6, a major serglycin-associated protease, exhibited similar defects in apoptosis as observed in serglycin(-/-) cells, indicating that the pro-apoptotic function of serglycin is due to downstream effects of proteases that are complex-bound to serglycin. Together, these findings implicate serglycin in promoting apoptotic versus necrotic cell death.