Redox modulation of the NMDA receptor

Redox modulation has been recognized to be an important mechanism of regulation for the N-methyl-D-aspartate (NMDA) receptor. Sulfhydryl reducing agents enhance, whereas oxidizing agents decrease, NMDA-evoked currents. Multiple cysteine residues located in different NMDA receptor subunits have been identified as molecular determinants underlying redox modulation. The NMDA receptor is also regulated by nitric oxide (NO)-related species directly, not involving cyclic GMP, but the molecular mechanism of this action has heretofore not been entirely clear. The confusion arose at least partly due to the fact that various redox forms of NO (NO+, NO*, NO-, each having an additional electron compared with the previous) have distinct mechanisms of action. Recently, a critical cysteine residue (Cys 399) on the NR2A subunit has been shown to react under physiological conditions with NO by S-nitrosylation (transfer of the NO+ to cysteine thiol) or by reaction with NO- (nitroxyl anion) to underlie this form of modulation.     

Proteases in apoptosis

The interleukin-1 β-converting enzyme (ICE)-like family proteases have recently been identified as key enzymes in apoptotic cell death. Among these proteases one can identify specific activities which may be involved in cytokine production or in resident protein cleavage. Several factors influence the constitutive apoptotic mechanism and may provide insight into the role of protease(s) in apoptosis. Although it appears that ICE family members play a most important role in promoting apoptotic cell death, evidence has been advanced that other proteases are also involved in sequential or parallel steps of apoptosis. Activation of a particular protease can lead to processing molecules either of the same or different proteases, leading to an activation of a protease cascade. Here we attempt to summarize the current thinking concerning these proteases and their involvement in apoptosis.     

Apoptotic and necrotic cell death induced by death domain receptors

Apoptosis and necrosis are two distinct forms of cell death. Caspases are indispensable as initiators and effectors of apoptotic cell death and are involved in many of the morphological and biochemical features of apoptosis. Major changes in mitochondrial membrane integrity and release of proapoptotic factors, such as cytochrome c from the mitochondrial intermembrane space, play an important sensor and amplifying role during apoptotic cell death. In vitro studies of cell death in cell lines have revealed that inhibition of the classical caspase-dependent apoptotic pathway leads in several cases to necrotic cell death. Thus, the same cell death stimulus can result either in apoptotic or necrotic cell death, depending on the availability of activated caspase. Therefore, death domain receptors may initiate an active caspase-independent necrotic signaling pathway. In this review, we describe what is known about the apoptotic and necrotic cell death pathways. Principal elements of necrosis include mitochondrial oxidative phosphorylation, reactive oxygen production, and non-caspase proteolytic cascades depending on serine proteases, calpains, or cathepsins.     

Hepatocyte growth factor-mediated attraction of mesenchymal stem cells for apoptotic neuronal and cardiomyocytic cells

Human bone marrow-derived mesenchymal stem cells (MSC) home to injured tissues and have regenerative capacity. In this study, we have investigated in vitro the influence of apoptotic and necrotic cell death, thus distinct types of tissue damage, on MSC migration. Concordant with an increased overall motility, MSC migrated towards apoptotic, but not vital or necrotic neuronal and cardiac cells. Hepatocyte growth factor (HGF) was expressed by the apoptotic cells only. MSC, in contrast, revealed expression of the HGF-receptor, c-Met. Blocking HGF bioactivity resulted in significant reduction of MSC migration. Moreover, recombinant HGF attracted MSC in a dose-dependent manner. Thus, apoptosis initiates chemoattraction of MSC via the HGF/c-Met axis, thereby linking tissue damage to the recruitment of cells with regenerative potential.     

Protein kinases mediate nitric oxide-induced apoptosis in the insect cell line IPLB-LdFB

The involvement of protein kinases (PKA, PKC and PKB) in nitric oxide (NO)-induced apoptosis with sodium nitroprusside plus N-acetyl-L-cysteine in the IPLB-LdFB cell line from the insect Lymantria dispar was investigated. The presence of protein kinase-like molecules was demonstrated by western blot analysis. The role of the kinases in programmed cell death was analysed in cytofluorimetric experiments by incubating the insect cells with H-89 (a specific inhibitor of PKA), calphostin C (an inhibitor of PKC) or wortmannin (an inhibitor of phosphatidylinositol 3-kinase). The results show that PKA is correlated with the induction and PKC and PKB with the prevention of NO-induced insect cell death. Moreover, NO-induced apoptosis involves the release of cytochrome c. Received 15 March 2002; accepted 25 March 2002     

Going up in flames: necrotic cell injury and inflammatory diseases

Recent evidence indicates that cell death can be induced through multiple mechanisms. Strikingly, the same death signal can often induce apoptotic as well as non-apoptotic cell death. For instance, inhibition of caspases often converts an apoptotic stimulus to one that causes necrosis. Because a dedicated molecular circuitry distinct from that controlling apoptosis is required for necrotic cell injury, terms such as “programmed necrosis” or “necroptosis” have been used to distinguish stimulus-dependent necrosis from those induced by non-specific traumas (e.g., heat shock) or secondary necrosis induced as a consequence of apoptosis. In several experimental models, programmed necrosis/necroptosis has been shown to be a crucial control point for pathogen- or injury-induced inflammation. In this review, we will discuss the molecular mechanisms that regulate programmed necrosis/necroptosis and its biological significance in pathogen infections, drug-induced cell injury, and trauma-induced tissue damage.     

Mitochondrial control of caspase-dependent and -independent cell death

Mitochondria control whether a cell lives or dies. The role mitochondria play in deciding the fate of a cell was first identified in the mid-1990s, because mitochondria-enriched fractions were found to be necessary for activation of death proteases, the caspases, in a cell-free model of apoptotic cell death. Mitochondrial involvement in apoptosis was subsequently shown to be regulated by Bcl-2, a protein that was known to contribute to cancer in specific circumstances. The important role of mitochondria in promoting caspase activation has therefore been a major focus of apoptosis research; however, it is also clear that mitochondria contribute to cell death by caspase-independent mechanisms. In this review, we will highlight recent findings and discuss the mechanism underlying the mitochondrial control of apoptosis and caspase-independent cell death.     

Reduced syncytin-1 expression in choriocarcinoma BeWo cells activates the calpain1–AIF-mediated apoptosis,implication for preeclampsia

Placentas associated with preeclampsia are characterized by extensive apoptosis in trophoblast lineages. Syncytin-1 (HERVWE1) mediates the fusion of cytotrophoblasts to form syncytiotrophoblasts, which assume the placental barrier, fetal–maternal exchange and endocrine functions. While decreased syncytin-1 expression has been observed in preeclamptic placentas, it is not clear if this alteration is involved in trophoblast apoptosis. In the current study, we found that siRNA-mediated knockdown of syncytin-1 led to apoptosis in choriocarcinoma BeWo, a cell line of trophoblastic origin. Characterization of the apoptotic pathways indicated that this effect does not rely on the activation of caspases. Rather, decreased syncytin-1 levels activated the apoptosis inducing factor (AIF) apoptotic pathway by inducing the expression, cleavage, and nuclear translocation of AIF. Moreover, calpain1, the cysteine protease capable of cleaving AIF, was upregulated by syncytin-1 knockdown. Furthermore, treatment with calpain1 inhibitor MDL28170 effectively reversed AIF cleavage, AIF nuclear translocation, and cell apoptosis triggered by syncytin-1 downregulation, verifying the specific action of calpain1–AIF pathway in trophoblast apoptosis. We confirmed that preeclamptic placentas express lower levels of syncytin-1 than normal placentas, and observed an inverse correlation between syncytin-1 and AIF/calpain1 mRNA levels, a result consistent with the in vitro findings. Immunohistochemistry analyses indicated decreased syncytin-1 and increased AIF and calpain1 protein levels in apoptotic cells of preeclamptic placentas. These findings have for the first time revealed that decreased levels of syncytin-1 can trigger the AIF-mediated apoptosis pathway in BeWo cells. This novel mechanism may contribute to the structural and functional deficiencies of syncytium frequently observed in preeclamptic placentas.     

Menadione-induced apoptosis and the degradation of lamin-like proteins in tobacco protoplasts

Detection of stereotypic hallmarks of apoptosis during cell death induced by menadione, including DNA laddering and the formation of apoptotic bodies, is reported. Comet assay and the TdT-mediated dUTP nick end labelling (TUNEL) procedure were also performed to detect DNA fragmentation. Inhibition of DNA fragmentation by Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and phenylmethylsulfosyl (PMSF) implicated the involvement of caspase-like proteases in menadione-induced apoptosis in plants. We further studied the cleavage of lamin-like proteins during apoptosis in menadione-treated tobacco protoplasts. In animals, it has been reported that the solubilization of nuclear lamina and lamin degradation occurs during apoptotic cell death. However, little is known about the fate of lamins in apoptotic plant cells. Our study provided evidence that lamin-like proteins degraded into 35-kDa fragments in tobacco protoplasts induced by menadione, and this preceded DNA fragmentation. The results thus indicated that proteolytic cleavage of nuclear lamins was also conserved in programmed cell death in plants. Received 16 November 1998; received after revision 21 December 1998; accepted 23 December 1998     

Chromosomal territory segmentation in apoptotic cells

The nuclear architecture of selected chromosomes in apoptotic nuclei of human leukemic cells K-562 and HL-60 was investigated. Etoposide and prolonged confluence were used for the induction of apoptosis. DAPI as well as TUNEL labeling of apoptotic nuclear bodies was combined with visualization of chromosomal territories by the FISH technique. Simultaneous vital staining by annexin V, propidium iodide, and Hoechst 33342 was applied to distinguish apoptotic, necrotic, and intact cell fraction of tested populations. Our FISH analyses revealed that the three-dimensional (3D) structure of apoptotic nuclei as well as the 3D structure of apoptotic bodies is preserved in formaldehyde-fixed cells. High-molecular-weight DNA fragmentation was determined in apoptotic K-562 cells in contrast to oligonucleosomal cleavage observed in apoptotic HL-60 cells. In K-562 populations, chromosomal territories were located separately either in one apoptotic body or underwent disassembly into chromosomal segments dispersed into single and/or several apoptotic bodies. The apoptotic disorganization of chromosomal territories was irregular, leading mainly to chromosomal segments of different sizes and, consequently, chromosomal disassembly was not observed at specific sites. In comparison with the control, an increased number of centromeric FISH signals were observed in prolonged confluence-treated K-562 cells induced to apoptosis. This finding can be explained either as a consequence of apoptosis or by polyploidization. Sequential staining of the same apoptotic nuclei by the FISH and TUNEL techniques revealed that chromosomal territory segmentation precedes the formation of nuclear apoptotic bodies.     

Melatonin and cell death: differential actions on apoptosis in normal and cancer cells

Melatonin is a natural compound synthesized by a variety of organs. It has been shown to function as a cell-protective agent. Since 1994, when the first paper was published documenting the role of melatonin in apoptosis, the number of reports in this area has increased rapidly. Much of the research conducted falls into three major categories: first, the role of melatonin in inhibiting apoptosis in immune cells; second, the role of melatonin in preventing neuronal apoptosis and finally, the role of melatonin in increasing apoptotic cell death in cancer cells. The mechanisms whereby melatonin influences apoptosis have not clarified, although a number of mechanistic options have been suggested. Apoptotic cell death is a physiological phenomenon related to homeostasis and proper functioning of tissues and organs; however, a failure in the apoptotic program is related to a number of diseases. The participation of melatonin in apoptosis in numerous cell types and its potential importance in a variety of diseases such as immunodeficiency, neurodegeneration and cancer is summarized in this review.Received 14 November 2002; received after revision 16 January 2003; accepted 10 February 2003     

BH3-only proteins in tumorigenesis and malignant melanoma

BH3-only proteins are a subset of the Bcl-2 family of apoptotic regulators. BH3-only proteins function as ‘damage sensors’ in the cell; they are activated in response to cellular stress or DNA damage, whereupon they initiate apoptosis. Apoptosis is the primary mechanism by which the body rids itself of genetically defective cells and is critical for preventing the accumulation of cells with tumorigenic potential. Therefore, dysregulation of BH3-only proteins may promote tumorigenesis. Furthermore, functional apoptosis pathways are required for the success of most cancer treatments, including chemotherapy. Resistance to chemotherapy, as seen with malignant melanoma, often reflects an inability of tumor cells to undergo apoptosis. By deciphering the roles of BH3-only proteins in tumorigenesis, we may learn how to manipulate cell death pathways to overcome apoptotic resistance. This review summarizes the current knowledge of BH3-only proteins and how they contribute to tumorigenesis, with particular attention given to studies involving melanoma. Received: 12 August 2006; received after revision: 2 October 2006; accepted 13 November 2006     

Tumor necrosis factor-mediated cell death: to break or to burst,that’s the question

In this review, we discuss the signal-transduction pathways of three major cellular responses induced by tumor necrosis factor (TNF): cell survival through NF-κB activation, apoptosis, and necrosis. Recruitment and activation of caspases plays a crucial role in the initiation and execution of TNF-induced apoptosis. However, experimental inhibition of caspases reveals an alternative cell death pathway, namely necrosis, also called necroptosis, suggesting that caspases actively suppress the latter outcome. TNF-induced necrotic cell death crucially depends on the kinase activity of receptor interacting protein serine-threonine kinase 1 (RIP1) and RIP3. It was recently demonstrated that ubiquitination of RIP1 determines whether it will function as a pro-survival or pro-cell death molecule. Deeper insight into the mechanisms that control the molecular switches between cell survival and cell death will help us to understand why TNF can exert so many different biological functions in the etiology and pathogenesis of human diseases.     

Unscheduled CDK1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells

Cyclin-dependent kinase 1 (CDK1) is a major component of the cell cycle progression engine. Recently, several investigations provided evidence demonstrating that unscheduled CDK1 activation may also be involved in apoptosis in cancerous cells. In this article, we demonstrate that X-ray irradiation induced G1 arrest in MOLT-4 lymphocytic leukemia cells, the arrest being accompanied by reduction in the activity of CDK2, but increased CDK1 activity and cell apoptosis in the G1 phase. Interestingly, this increase in CDK1 and apoptosis by ionizing radiation was prevented by pretreatment with the CDK1 inhibitor, roscovitine, suggesting that CDK1 kinase activity is required for radiation-induced apoptotic cell death in this model system. Furthermore, cyclin B1 and CDK1 were detected co-localizing and associating in G1 phase MOLT-4 cells, with the cellular lysates from these cells revealing a genotoxic stress-induced increase in CDK1 phosphorylation (Thr-161) and dephosphorylation (Tyr-15), as analyzed by postsorting immunoprecipitation and immunoblotting. Finally, X-irradiation was found to increase Bcl-2 phosphorylation in G1 phase cells. Taken together, these novel findings suggest that CDK1 is activated by unscheduled accumulation of cyclin B1 in G1 phase cells exposed to X-ray, and that CDK1 activation, at the wrong time and in the wrong phase, may directly or indirectly trigger a Bcl-2-dependent signaling pathway leading to apoptotic cell death in MOLT-4 cells. Received 30 March 2006; received after revision 23 June 2006; accepted 24 August 2006 J. Wu and Y. Feng contributed equally to this work.     

S-Nitrosylation of proteins

The transfer of a nitric oxide group to cysteine sulfhydryls on proteins, known as S-nitrosylation, is increasingly becoming recognized as a ubiquitous regulatory reaction comparable to phosphorylation. It represents a form of redox modulation in diverse tissues, including the brain. An increasing number of proteins have been found to undergo S-nitrosylation in vivo. These proteins are called S-nitrosothiols, and may play an important role in many processes ranging from signal transduction, DNA repair, host defense, and blood pressure control to ion channel regulation and neurotransmission. This review focuses on the importance of the S-nitrosylation reaction and describes some recently identified S-nitrosothiols in various fields of research.     

Redox-dependent thiol modifications: implications for the release of extracellular vesicles

Extracellular vesicles (EVs), including microvesicles and exosomes, are emerging as important regulators of homeostasis and pathophysiology. During pro-inflammatory and pro-oxidant conditions, EV release is induced. As EVs released under such conditions often exert pro-inflammatory and procoagulant effects, they may actively promote the pathogenesis of chronic diseases. There is evidence that thiol group-containing antioxidants can prevent EV induction by pro-inflammatory and oxidative stimuli, likely by protecting protein thiols of the EV-secreting cells from oxidation. As the redox state of protein thiols greatly impacts three-dimensional protein structure and, consequently, function, redox modifications of protein thiols may directly modulate EV release in response to changes in the cell’s redox environment. In this review article, we discuss targets of redox-dependent thiol modifications that are known or expected to be involved in the regulation of EV release, namely redox-sensitive calcium channels, N-ethylmaleimide sensitive factor, protein disulfide isomerase, phospholipid flippases, actin filaments, calpains and cell surface-exposed thiols. Thiol protection is proposed as a strategy for preventing detrimental changes in EV signaling in response to inflammation and oxidative stress. Identification of the thiol-containing proteins that modulate EV release in pro-oxidant environments could provide a rationale for broad application of thiol group-containing antioxidants in chronic inflammatory diseases.