Chondroptosis: A variant of apoptotic cell death in chondrocytes?

Evidence has accumulated in recent years that programmed cell death (PCD) is not necessarily synonymous with the classical apoptosis, as defined by Kerr and Wyllie, but that cells use a variety of pathways to undergo cell death, which are reflected by different morphologies. Although chondrocytes with the hallmark features of classical apoptosis have been demonstrated in culture, such cells are extremely rare in vivo. The present review focuses on the morphological differences between dying chondrocytes and classical apoptotic cells. We propose the term 'chondroptosis' to reflect the fact that such cells are undergoing apoptosis in a non-classical manner that appears to be typical of programmed chondrocyte death in vivo. Unlike classical apoptosis, chondroptosis involves an initial increase in the endoplasmic reticulum and Golgi apparatus, reflecting an increase in protein synthesis. The increased ER membranes also segment the cytoplasm and provide compartments within which cytoplasm and organelles are digested. In addition, destruction occurs within autophagic vacuoles and cell remnants are blebbed into the lacunae. Together these processes lead to complete self-destruction of the chondrocyte as evidenced by the presence of empty lacunae. It is speculated that the endoplasmic reticulum pathway of apoptosis plays a greater role in chondroptosis than receptor-mediated or mitochondrial pathways and that lysosomal proteases are at least as important as caspases. Because chondroptosis does not depend on phagocytosis, it may be more advantageous in vivo, where chondrocytes are isolated within their lacunae. At present the initiation factors or the molecular pathways involved in chondroptosis remain unclear.     

Different modes of programmed cell death during oogenesis of the silkmoth Bombyx mori

It is increasingly recognized that programmed cell death includes not only apoptosis and autophagy, but also other types of nonapoptotic cell death, such as paraptosis, which are all characterized by distinct morphological features. Our findings indicate that all three types of programmed cell death occur in the ovarian nurse cell cluster during late vitellogenesis (formation of the egg yolk) of Bombyx mori (Lepidoptera), whereas middle vitellogenesis is exclusively characterized by the presence of a nonapoptotic type of cell death, known as paraptosis. During middle vitellogenesis, nurse cells exhibit clearly cytoplasmic vacuolization, as revealed by ultrastructural examination performed through conventional light and transmission electron microscopy, while no signs of apoptotic or autophagic features are detectable. Moreover, nurse cells of developmental stages 7, 8 and 9 contain autophagic compartments, as well as apoptotic characteristics, such as condensed chromatin, fragmented DNA and activated caspases, as revealed by in vitro assays. We propose that paraptosis precedes both apoptosis and autophagy during vitellogenesis, since its initial activation is detectable during middle vitellogenesis, whereas no apoptotic nor autophagic features are observed. In contrast, at the late stages of Bombyx mori oogenesis, paraptosis, autophagy and apoptosis operate synergistically, resulting in a more efficient elimination of the degenerated nurse cells.     

Proteolytic specificity of caspases is required to signal the appearance of apoptotic morphology

The caspase family of cysteine proteases is essential for implementation of physiological cell death. Since a wide variety of cellular proteins is cleaved by caspases during apoptosis, it has been predicted that digestion of proteins crucial to maintaining the life of a cell is central to apoptosis. To assess the role of the proteolytic destruction during apoptosis, we introduced the non-specific protease proteinase K into intact cells. This introduction led to extensive digestion of cellular proteins, including physiological caspase-substrates. Caspase-3-like activity was induced rapidly, followed by morphological signs of apoptosis such as membrane blebbing and nuclear condensation. The caspase inhibitor Z-VAD-fmk inhibited the appearance of these morphological changes without reducing the extent of intracellular proteolysis by proteinase K. Loss of integrity of the cell membrane, however, was not blocked by Z-VAD-fmk. This system thus generated conditions of extensive destruction of caspase substrates by proteinase K in the absence of apoptotic morphology. Taken together, these experiments suggest that caspases implement cell death not by protein destruction but by proteolytic activation of specific downstream effector molecules.     

Ars moriendi: Proteases as sculptors of cellular suicide

The Ars moriendi, which translates to “The Art of Dying,” encompasses two Latin texts that gave advice on how to die well and without fear according to the Christian precepts of the late Middle Ages. Given that ten to hundred billion cells die in our bodies every day, it is obvious that the concept of a well and orderly (“regulated”) death is also paramount at the cellular level. In apoptosis, as the most well-studied form of regulated cell death, proteases of the caspase family are the central mediators. However, caspases are not the only proteases that act as sculptors of cellular suicide, and therefore, we here provide an overview of the impact of proteases in apoptosis and other forms of regulated cell death.     

Apoptosis and autophagy function cooperatively for the efficacious execution of programmed nurse cell death during Drosophila virilis oogenesis

Programmed cell death consists of two major types, apoptotic and autophagic, both of which are mainly defined by morphological criteria. Our findings indicate that both types of programmed cell death occur in the ovarian nurse cells during middle- and late-oogenesis of Drosophila virilis. During mid-oogenesis, the spontaneously degenerated egg chambers exhibit typical characteristics of apoptotic cell death. Their nurse cells contain condensed chromatin and fragmented DNA, whereas active caspase assays and immunostaining procedures demonstrate the presence of highly activated caspases. Distinct features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining and ultrastructural examination performed by transmission electron microscopy. Additionally, atretic egg chambers exhibit an accumulation of lysosomal proteases. At the late stages of D. virilis oogenesis, apoptosis and autophagy coexist, manifesting cell death features that are similar to the ones described above, being also escorted by the involvement of an altered cytochrome c conformational display. We propose that apoptosis and autophagy operate synergistically during D. virilis oogenesis for a more efficient elimination of the degenerated nurse cells.     

Different modes of programmed cell death during oogenesis of the silkmoth Bombyx mori

It is increasingly recognized that programmed cell death includes not only apoptosis and autophagy, but also other types of nonapoptotic cell death, such as paraptosis, which are all characterized by distinct morphological features. Our findings indicate that all three types of programmed cell death occur in the ovarian nurse cell cluster during late vitellogenesis (formation of the egg yolk) of Bombyx mori (Lepidoptera), whereas middle vitellogenesis is exclusively characterized by the presence of a nonapoptotic type of cell death, known as paraptosis. During middle vitellogenesis, nurse cells exhibit clearly cytoplasmic vacuolization, as revealed by ultrastructural examination performed through conventional light and transmission electron microscopy, while no signs of apoptotic or autophagic features are detectable. Moreover, nurse cells of developmental stages 7, 8 and 9 contain autophagic compartments, as well as apoptotic characteristics, such as condensed chromatin, fragmented DNA and activated caspases, as revealed by in vitro assays. We propose that paraptosis precedes both apoptosis and autophagy during vitellogenesis, since its initial activation is detectable during middle vitellogenesis, whereas no apoptotic nor autophagic features are observed. In contrast, at the late stages of Bombyx mori oogenesis, paraptosis, autophagy and apoptosis operate synergistically, resulting in a more efficient elimination of the degenerated nurse cells.

Addendum to: Mpakou VE, Nezis IP, Stravopodis DJ, Margaritis LH, Papassideri IS. Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori. Dev Growth Differ 2006; 48:419–28.     

Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases

IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.     

alpha-fetoprotein causes apoptosis in tumor cells via a pathway independent of CD95, TNFR1 and TNFR2 through activation of caspase-3-like proteases.

alpha-Fetoprotein (AFP) is an oncoembryonal protein with multiple cell growth regulating, differentiating and immunosuppressive activities. Previous studies have shown that treatment of tumor cells in vitro with 1-10 microM AFP produces significant suppression of tumor cell growth by inducing dose-dependent cytotoxicity, but the molecular mechanisms underlying these AFP functions are obscure. Here, we show that AFP cytotoxicity is closely related to apoptosis, as shown by cell morphology, nuclear DNA fragmentation and caspase-3-like activity resulting in cleavage of poly(ADP-ribose) polymerase. Apoptosis was significantly inhibited by a CPP32 family protease inhibitor whereas a general caspase inhibitor had no inhibitory effect, showing some enhancement of AFP-mediated cell death. Using fluorogenic caspase substrates, we found that caspase-3-like proteases were activated as early as 4 h after treatment of Raji cells with 15 microM AFP, whereas caspase-1, caspase-8, and caspase-9-like activity was not detected during the time interval 0.5-17 h. AFP treatment of Raji cells increased Bcl-2 protein, showing that AFP-induced apoptosis is not explained by downregulation of the Bcl-2 gene. This also suggests that AFP operates downstream of the Bcl-2-sensitive step. AFP notably decreased basal levels of soluble and membrane-bound Fas ligand. Incubation of AFP-sensitive tumor cells (HepG2, Raji) with neutralizing anti-Fas, anti-tumor necrosis factor receptor (TNFR)1 or anti-TNFR2 mAb did not prevent AFP-induced apoptosis, demonstrating its independence of Fas-dependent and TNFR-dependent signaling. In addition, it was found that cells resistant to TNF-induced (Raji) or Fas-induced (MCF-7) apoptosis are, nevertheless, sensitive to AFP-mediated cell death. In contrast, cells sensitive to Fas-mediated cell death (Jurkat) are completely resistant to AFP. Taken as a whole, our data demonstrate that: (a) AFP induces apoptosis in tumor cells independently of Fas/Fas ligand or TNFR/TNF signaling pathways, and (b) AFP-mediated cell death involves activation of the effector caspase-3-like proteases, but is independent of upstream activation of the initiator caspase-1, caspase-8, and caspase-9-like proteases.     

Mitochondria,the killer organelles and their weapons

Apoptosis is a cell-autonomous mode of death that is activated to eradicate superfluous, damaged, mutated, or aged cells. In addition to their role as the cell's powerhouse, mitochondria play a central role in the control of apoptosis. Thus, numerous pro-apoptotic molecules act on mitochondria and provoke the permeabilization of mitochondrial membranes. Soluble proteins contained in the mitochondrial intermembrane space are released through the outer membrane and participate in the organized destruction of the cell. Several among these lethal proteins can activate caspases, a class of cysteine proteases specifically activated in apoptosis, whereas others act in a caspase-independent fashion, by acting as nucleases (e.g., endonuclease G), nuclease activators (e.g., apoptosis-inducing factor), or serine proteases (e.g., Omi/HtrA2). In addition, mitochondria can generate reactive oxygen species, following uncoupling and/or inhibition of the respiratory chain. The diversity of mitochondrial factors participating in apoptosis emphasizes the central role of these organelles in apoptosis control and unravels novel mechanisms of cell death execution.     

Expression of cyclin-dependent kinase 5 and its activator p35 in models of induced apoptotic death in neurons of the substantia nigra in vivo

Cyclin-dependent kinase 5 is predominantly expressed in postmitotic neurons and plays a role in neurite elongation during development. It has also been postulated to play a role in apoptosis in a variety of cells, including neurons, but little is known about the generality and functional significance of cdk5 expression in neuronal apoptosis in living brain. We have therefore examined its expression and that of its known activators, p35, p39 and p67, in models of induced apoptosis in neurons of the substantia nigra. We find that cdk5 is expressed in apoptotic profiles following intrastriatal injection of 6-hydroxydopamine and axotomy. It is expressed exclusively in profiles which are in late morphologic stages of apoptosis. In these late stages, derivation of the profiles from neurons, and localization of expression to the nucleus, can be demonstrated by co-labeling with a neuron-specific nuclear marker, NeuN. In another model of induced apoptotic death in nigra, produced by developmental striatal lesion, kinase activity increases in parallel with cell death. While mRNAs for all three cdk5 activators are expressed in nigra during development, only p35 protein is expressed in apoptotic profiles. We conclude that cdk5/p35 expression is a general feature of apoptotic neuron death in substantia nigra neurons in vivo.     

毛细血管扩张性共济失调突变蛋白在细胞凋亡过程中被Caspase┐3降解

新近的研究揭示：ｃａｓｐａｓｅ蛋白酶在细胞凋亡中起着死亡执行者的重要功能．一些蛋白相继被证明在细胞凋亡中可被ｃａｓｐａｓｅ特异切割,其中参与ＤＮＡ损伤修复过程的聚ＡＤＰ核糖聚合酶（ＰＡＲＰ）以及ＤＮＡ依赖的蛋白激酶（ＤＮＡ－ＰＫ）,在细胞凋亡过程中被ｃａｓｐａｓｅ选择性切割具有特殊的功能意义．为探索与ＤＮＡ－ＰＫ催化亚基有较高同源性,含有ｃａｓｐａｓｅ切割位点,且功能上目前也被认为是感受ＤＮＡ损伤和参与信号传导途径的ＡＴＭ（Ａｔａｘｉａｔｅｌａｎｇ－ｉｅｃｔａｓｉａｍｕｔａｔｅｄ）蛋白,是否在凋亡过程中也可被切割而降解？应用体外转录与翻译系统获得ＡＴＭ蛋白的ＰＩ３Ｋ结构域,同时通过建立无细胞反应体系获得含ｃａｓｐａｓｅ活性的细胞抽提液,将两者在体外共同保温．结果发现：ＡＴＭ蛋白与ｃａｓｐａｓｅ－３能免疫共沉淀,ＡＴＭ蛋白的ＰＩ３Ｋ结构域可被ｃａｓｐａｓｅ－３特异切割,并观察到辐射诱发细胞调亡中ＡＴＭ蛋白的降解．从而进一步证实了ＤＮＡ损伤修复的抑制,促进细胞凋亡的发生．     

Proteolytic mechanisms in necrotic cell death and neurodegeneration

Programmed neuronal cell death is required during development to achieve the accurate wiring of the nervous system. However, genetic or accidental factors can lead to the premature, non-programmed death of neurons during adult life. Inappropriate death of cells in the nervous system is the cause of multiple neurodegenerative disorders. Pathological neuronal death can occur by apoptosis, by necrosis or by a combination of both. Necrotic cell death underlies the pathology of devastating neurological diseases such as neurodegenerative disorders, stroke or trauma. However, little is known about the molecular mechanisms that bring about necrotic cell death. Proteases play crucial roles in neuron degeneration by exerting both regulatory and catabolic functions. Elevated intracellular calcium is the most ubiquitous feature of neuronal death with the concomitant activation of cysteine calcium-dependent proteases, calpains. Calpains and lysosomal, catabolic aspartyl proteases, play key roles in the necrotic death of neurons. In this review, we survey the recent literature on the role of cysteine and aspartyl proteases in necrosis and neurodegeneration, aiming to delineate common proteolytic mechanisms mediating cellular destruction.     

Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60(v-src) tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60(v-src), we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60(v-src) inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60(v-src) inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.     

The role of programmed cell death in Plasmodium-mosquito interactions

Many host-parasite interactions are regulated in part by the programmed cell death of host cells or the parasite. Here we review evidence suggesting that programmed cell death occurs during the early stages of the development of the malaria parasite in its vector. Zygotes and ookinetes of Plasmodium berghei have been shown to die by programmed cell death (apoptosis) in the midgut lumen of the vector Anopheles stephensi, or whilst developing in vitro. Several morphological markers, indicative of apoptosis, are described and evidence for the involvement of a biochemical pathway involving cysteine proteases discussed in relationship to other protozoan parasites. Malaria infection induces apoptosis in the cells of two mosquito tissues, the midgut and the follicular epithelium. Observations on cell death in both these tissues are reviewed including the role of caspases as effector molecules and the rescue of resorbing follicles resulting from inhibition of caspases. Putative signal molecules that might induce parasite and vector apoptosis are suggested including nitric oxide, reactive nitrogen intermediates, oxygen radicals and endocrine balances. Finally, we suggest that programmed cell death may play a critical role in regulation of infection by the parasite and the host, and contribute to the success or not of parasite establishment and host survival.     

Mechanisms of B cell receptor induced apoptosis

B cell receptor (BCR)-mediated apoptosis plays a key role in the negative selection (deletion) of autoreactive B cells. Mechanisms of BCR-mediated apoptosis have been widely studied in cell lines representing both immature (bone marrow) and mature (germinal center) B cells. However, there is much inconsistency and controversy concerning the possible mechanisms of BCR-mediated apoptosis, which may reflect differences in the origin or the maturational stage of the cell line used. Based on recent studies, collapse of mitochondrial membrane potential (Delta Psi m) seems to be an essential event for BCR-mediated apoptosis in both mature and immature cells. The collapse of Delta Psi m is dependent on the synthesis of new proteins, which are involved in the permeability change of mitochondrial membranes. Mitochondrial dysfunction induces activation of caspases, cysteine proteases, which play a central role in apoptosis. However, instead of caspases, other effector proteases, such as cathepsins or calpains, may also be responsible for the organized destruction of cell components seen during BCR-mediated apoptosis.     

Apoptosis and Autophagy: Decoding Calcium Signals that Mediate Life or Death

Calcium is a versatile and dynamic 2nd messenger that is essential for the survival of all higher organisms. In cells that undergo activation or excitation, calcium is released from the endoplasmic/sarcoplasmic reticulum to activate calcium-dependent kinases and phosphatases, thereby regulating numerous cellular processes; for example, apoptosis and autophagy. In the case of apoptosis, endogenous ligands or pharmacological agents induce prolonged cytosolic calcium elevation, which in turn leads to cell death. In contrast, there is now evidence that calcium regulates autophagy by several mechanisms, and these may be important for maintaining cell survival. Here we summarize what is known about how calcium regulates these life and death decisions. We pay particular attention to pathways that have been described in lymphocytes and cardiomyocytes, as these systems provide optimal models for understanding calcium signaling in the context of normal cell physiology.Apoptosis is a process of programmed cell death or suicide that occurs when cells have undergone irreversible stress or damage. It is required to maintain normal cell homeostasis or to eliminate a population of cells that may be harmful to the organism or unnecessary during organ development (Green 2003). For example, it is the primary mechanism by which potentially autoreactive T cells are eliminated from the immune system. There are two conventional apoptosis pathways: the extrinsic pathway, which is typically initiated by death receptors (e.g., Fas) on the plasma membrane and the intrinsic (mitochondrial) pathway, which involves permeabilization of the outer mitochondrial membrane followed by the release of cytochrome c. In this review, we primarily focus our attention on the intrinsic pathway due to the importance of intracellular calcium in the regulation of this process.In brief, cytochrome c release stimulates apoptosis via its interaction with the protein Apaf-1, which in turn activates the initiator caspase-9 and the executioner caspase-3 (Green 2005). Caspases comprise a family of cysteine proteases that are essential for the classically observed cellular and biochemical characteristics of apoptosis, which include (but are not limited to) membrane blebbing, chromatin condensation, and DNA fragmentation. Another class of cysteine proteases, calpains, require calcium for their activation and are important mediators of apoptosis following ER stress. As discussed later in this review, calpains are reported to directly activate caspases, thus promoting apoptotic cell death independent of mitochondrial cytochrome c release. The following sections provide a more detailed explanation of the varied ways in which calcium signals induce cell death and are themselves regulated.