The mammalian interleukin-1 beta-converting enzyme (ICE) has sequence similarity to the C. elegans cell death gene ced-3. We show here that overexpression of the murine ICE (mICE) gene or of the C. elegans ced-3 gene causes Rat-1 cells to undergo programmed cell death. Point mutations in a region homologous between mICE and CED-3 eliminate the ability of mICE and ced-3 to cause cell death. The cell death caused by mICE can be suppressed by overexpression of the crmA gene, a specific inhibitor of ICE, as well as by bcl-2, a mammalian oncogene that can act to prevent programmed cell death. Our results suggest that ICE may function during mammalian development to cause programmed cell death.

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We report here the inactivation of a member of the Ice/Ced-3 (caspase) family of cell death genes, casp-11, by gene targeting. Like Ice-deficient mice, casp-11 mutant mice are resistant to endotoxic shock induced by lipopolysaccharide. Production of both IL-1alpha and IL-1beta after lipopolysaccharide stimulation, a crucial event during septic shock and an indication of ICE activation, is blocked in casp-11 mutant mice. casp-11 mutant embryonic fibroblast cells are resistant to apoptosis induced by overexpression of ICE. Furthermore, we found that pro-caspase-11 physically interacts with pro-ICE in cells, and the expression of casp-11 is essential for activation of ICE. Our data suggest that caspase-11 is a component of ICE complex and is required for the activation of ICE.

Mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are in a high-flux metabolic state, with a high dependence on threonine catabolism. However, little is known regarding amino acid metabolism in human ESCs/iPSCs. We show that human ESCs/iPSCs require high amounts of methionine (Met) and express high levels of enzymes involved in Met metabolism. Met deprivation results in a rapid decrease in intracellular S-adenosylmethionine (SAM), triggering the activation of p53-p38 signaling, reducing NANOG expression, and poising human iPSC/ESCs for differentiation, follow by potentiated differentiation into all three germ layers. However, when exposed to prolonged Met deprivation, the cells undergo apoptosis. We also show that human ESCs/iPSCs have regulatory systems to maintain constant intracellular Met and SAM levels. Our findings show that SAM is a key regulator for maintaining undifferentiated pluripotent stem cells and regulating their differentiation.

Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER lumen, and is associated with vascular and neurodegenerative diseases. Although the connection between ER stress and some disease-related proteins has been studied using animal models of these diseases, no in vivo data concerning ER stress are available. Here we report a new method for monitoring ER stress in vivo, based on XBP-1 mRNA splicing by inositol requiring-1 (IRE-1) during ER stress. The stress indicator was constructed by fusing XBP-1 and venus, a variant of green fluorescent protein. During stress, the spliced indicator mRNA is translated into an XBP-1-venus fusion protein, which can be detected by its fluorescence. We used transgenic animals expressing the ER stress indicator to show that it can be used to monitor physiological and pathological ER stress in vivo.

Summary Oncogenic alterations that confer proliferative advantages in epithelial tissues also often trigger apoptosis, suggesting an evolutionary mechanism by which organisms eliminate aberrant cells from epithelia. However, the underlying mechanism of how these tissues eliminate oncogenic cells remains to be elucidated. In Drosophila imaginal epithelia, clones of cells mutant for evolutionarily conserved tumor suppressors, such as scrib or dlg, lose their epithelial integrity and are eliminated by JNK-dependent cell death. Here, we show that Eiger, a Drosophila member of the tumor necrosis factor (TNF) superfamily, behaves like a tumor suppressor that eliminates oncogenic mutant cells from epithelia. In the absence of Eiger, these mutant clones are no longer eliminated; instead, they grow aggressively and develop into tumors. Our analysis shows that Eiger is translocated to endocytic vesicles in scrib mutant clones, which leads to activation of apoptotic Eiger-JNK signaling in endosomes. Furthermore, we show that Eiger’s tumor suppressor-like function is dependent on its endocytosis, as blocking endocytosis prevents both JNK activation and elimination of these clones. Our data indicate that TNF signaling and the endocytic machinary could be components of an evolutionarily conserved fail-safe mechanism by which animals maintain epithelial integrity to protect against neoplastic development.

The death receptor Fas transduces apoptotic death signaling mediated by caspases. In the present study, human hepatoma HepG2 cells showed the Fas-mediated apoptosis mediated by caspase, especially caspase 3, only in the presence of actinomycin D. Interestingly, cytosolic proteins extracted from intact HepG2 cells induced caspase 3 inactivation. Our results reveal that this inactivation was triggered by the direct inhibition of activated caspase 3 by IAP gene family ILP. In addition, a 53 kDa protein was co-immunoprecipitated with antihuman caspase 3 antibody from intact HepG2 cells. This protein was a complex-protein of procaspase 3 and the cell cycle regulator p21 WAF1 (p21). P21 bound to only procaspase 3, but not to activated caspase 3. We also demonstrate that p21 protein-loaded HepG2 cells resist to Fas-mediated apoptosis even in the presence of actinomycin D. Here we report that caspase 3 inactivation for the resistance to Fas-mediated apoptosis is induced by a procaspase 3/p21 complex formation and direct inhibition of activated caspase 3 by ILP.

Caspase-1 activated in inflammasomes triggers a programmed necrosis called pyroptosis, which is mediated by gasdermin D (GSDMD). However, GSDMD-deficient cells are still susceptible to caspase-1-mediated cell death. Therefore, here, we investigate the mechanism of caspase-1-initiated cell death in GSDMD-deficient cells. Inflammasome stimuli induce apoptosis accompanied by caspase-3 activation in GSDMD-deficient macrophages, which largely relies on caspase-1. Chemical dimerization of caspase-1 induces pyroptosis in GSDMD-sufficient cells, but apoptosis in GSDMD-deficient cells. Caspase-1-induced apoptosis involves the Bid-caspase-9-caspase-3 axis, which can be followed by GSDME-dependent secondary necrosis/pyroptosis. However, Bid ablation does not completely abolish the cell death, suggesting the existence of an additional mechanism. Furthermore, cortical neurons and mast cells exhibit little or low GSDMD expression and undergo apoptosis after oxygen glucose deprivation and nigericin stimulation, respectively, in a caspase-1- and Bid-dependent manner. This study clarifies the molecular mechanism and biological roles of caspase-1-induced apoptosis in GSDMD-low/null cell types.