PLA2 activity is required for nuclear shrinkage in caspase-independent cell death

Apoptosis is defined on the basis of morphological changes like nuclear fragmentation and chromatin condensation, which are dependent on caspases. Many forms of caspase-independent cell death have been reported, but the mechanisms are still poorly understood. We found that hypoxic cell death was independent of caspases and was associated with significant nuclear shrinkage. Neither Bcl-2 nor Apaf-1 deficiency prevented hypoxic nuclear shrinkage. To understand the molecular mechanism of the nuclear shrinkage, we developed an in vitro system using permeabilized cells, which allowed us to purify a novel member of the phospholipase A2 (PLA2) family that induced nuclear shrinkage. Purified PLA2 induced nuclear shrinkage in our permeabilized cell system. PLA2 inhibitors prevented hypoxic nuclear shrinkage in cells and cell death. Hypoxia caused elevation of PLA2 activity and translocation of intracellular PLA2s to the nucleus. Knockdown of the Ca2+-independent PLA2 delayed nuclear shrinkage and cell death. These results indicate that Ca2+-independent PLA2 is crucial for a caspase-independent cell death signaling pathway leading to nuclear shrinkage

Vital staining for cell death identifies Atg9a-dependent necrosis in developmental bone formation in mouse

Programmed cell death has a crucial role in various biological events, including developmental morphogenesis. Recent evidence indicates that necrosis contributes to programmed cell death in addition to apoptosis, but it is unclear whether necrosis acts as a compensatory mechanism for failure of apoptosis or has an intrinsic role during development. In contrast to apoptosis, there have been no techniques for imaging physiological necrosis in vivo. Here we employ vital staining using propidium iodide to identify cells with plasma membrane disruption (necrotic cells) in mouse embryos. We discover a form of necrosis at the bone surface, which does not occur in embryos with deficiency of the autophagy-related gene Atg9a, although it is unaffected by Atg5 knockout. We also find abnormalities of the bone surface in Atg9a knockout mice, suggesting an important role of Atg9a-dependent necrosis in bone surface formation. These findings suggest that necrosis has an active role in developmental morphogenesis

Bcl2 Deficiency Activates FoxO through Akt Inactivation and Accelerates Osteoblast Differentiation

Osteoblast apoptosis plays an important role in bone development and maintenance, and is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging. Although Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis, the physiological significance of Bcl2 in osteoblast differentiation has not been fully elucidated. To investigate this, we examined Bcl2-deficient (Bcl2(-/-)) mice. In Bcl2(-/-) mice, bromodeoxyuridine (BrdU)-positive osteoblasts were reduced in number, while terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive osteoblasts were increased. Unexpectedly, osteoblast differentiation was accelerated in Bcl2(-/-) mice as shown by the early appearance of osteocalcin-positive osteoblasts. Osteoblast differentiation was also accelerated in vitro when primary osteoblasts were seeded at a high concentration to minimize the reduction of the cell density by apoptosis during culture. FoxO transcription factors, whose activities are negatively regulated through the phosphorylation by Akt, play important roles in multiple cell events, including proliferation, death, differentiation, longevity, and stress response. Expressions of FasL, Gadd45a, and Bim, which are regulated by FoxOs, were upregulated; the expression and activity of FoxOs were enhanced; and the phosphorylation of Akt and that of FoxO1 and FoxO3a by Akt were reduced in Bcl2(-/-) calvariae. Further, the levels of p53 mRNA and protein were increased, and the expression of p53-target genes, Pten and Igfbp3 whose proteins inhibit Akt activation, was upregulated in Bcl2(-/-) calvariae. However, Pten but not Igfbp3 was upregulated in Bcl2(-/-) primary osteoblasts, and p53 induced Pten but not Igfbp3 in vitro. Silencing of either FoxO1 or FoxO3a inhibited and constitutively-active FoxO3a enhanced osteoblast differentiation. These findings suggest that Bcl2 deficiency induces and activates FoxOs through Akt inactivation, at least in part, by upregulating Pten expression through p53 in osteoblasts, and that the enhanced expression and activities of FoxOs may be one of the causes of accelerated osteoblast differentiation in Bcl2(-/-) mice

Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Aβ-induced cell death

Recent studies have suggested that neuronal death in Alzheimer's disease or ischemia could arise from dysfunction of the endoplasmic reticulum (ER). Although caspase-12 has been implicated in ER stress-induced apoptosis and amyloid-β (Aβ)–induced apoptosis in rodents, it is controversial whether similar mechanisms operate in humans. We found that human caspase-4, a member of caspase-1 subfamily that includes caspase-12, is localized to the ER membrane, and is cleaved when cells are treated with ER stress-inducing reagents, but not with other apoptotic reagents. Cleavage of caspase-4 is not affected by overexpression of Bcl-2, which prevents signal transduction on the mitochondria, suggesting that caspase-4 is primarily activated in ER stress-induced apoptosis. Furthermore, a reduction of caspase-4 expression by small interfering RNA decreases ER stress-induced apoptosis in some cell lines, but not other ER stress-independent apoptosis. Caspase-4 is also cleaved by administration of Aβ, and Aβ-induced apoptosis is reduced by small interfering RNAs to caspase-4. Thus, caspase-4 can function as an ER stress-specific caspase in humans, and may be involved in pathogenesis of Alzheimer's disease

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Housing conditions affect enterocyte death mode and turnover rate in mouse small intestine

Abstract Small intestinal enterocytes are continuously renewed. Shedding/death of enterocytes involves receptor-interacting protein kinase 1 (RIPK1)-dependent (but RIPK3-independent) necrotic death, but the regulatory mechanism of the processes is not fully understood. Here, we show that mouse housing conditions, such as the type of bedding material and the presence or absence of a Shepherd Shack, affect enterocyte turnover rate and determine whether enterocyte shedding/death is RIPK1-independent or -dependent. Mice housed with ALPHA-dri (αDri, hard paper chip) bedding material without a Shepherd Shack had a higher, largely RIPK1-dependent enterocyte turnover rate and higher blood corticosterone levels, suggesting the involvement of minor stress, whereas mice housed with αDri plus a Shepherd Shack or with Soft Chip had a lower, RIPK1-independent turnover rate and lower blood corticosterone levels. Corticosterone administration to a small intestine culture derived from mice housed with αDri plus a Shepherd Shack or with Soft Chip increased enterocyte shedding/death and turnover. By using kinase inhibitors and knockout mice, we showed that the switch from RIPK1-independent to RIPK1-dependent enterocyte shedding/death and turnover involves suppression of TANK-binding kinase 1. Our results demonstrate that housing conditions may cause minor stress, which alters the mode of enterocyte shedding/death and enterocyte turnover rate in mice