66 research outputs found
GD3 ganglioside directly targets mitochondria and induces the release of apoptogenic factors
No full textLipid and glycolipid diffusible mediators
are involved in the intracellular progression and
amplification of apoptotic signals. GD3 ganglioside
is rapidly synthesized from accumulated ceramide
after the clustering of death-inducing receptors and
triggers apoptosis. Here we show that GD3 induces
dissipation of DCm and swelling of isolated mitochondria,
which results in the mitochondrial release
of cytochrome c, apoptosis inducing factor, and
caspase 9. Soluble factors released from GD3-
treated mitochondria are sufficient to trigger DNA
fragmentation in isolated nuclei. All these effects can
be blocked by cyclosporin A, suggesting that GD3 is
acting at the level of the permeability transition pore
complex. We found that endogenous GD3 accumulates
within mitochondria of cells undergoing apoptosis
after ceramide exposure. Accordingly, suppression
of GD3 synthase (ST8) expression in intact
cells substantially prevents ceramide-induced DCm
dissipation, indicating that endogenously synthesized
GD3 induces mitochondrial changes in vivo. Finally,
enforced expression of bcl-2 significantly prevents
GD3-induced mitochondrial changes, caspase 9 activation,
and apoptosis. These results show that mitochondria
are a key destination for apoptogenic GD3
ganglioside along the lipid pathway to programmed
cell death and indicate that relevant GD3 targets are
under bcl-2 control.—Rippo, M. R., Malisan, F.,
Ravagnan, L., Tomassini, B., Condo, I., Costantini,
P., Susin, S. A., Rufini, A., Todaro, M., Kroemer, G.,
Testi, R. GD3 ganglioside directly targets mitochondria
in a bcl-2-controlled fashion
UVB-induced apoptosis of human dendritic cells: contribution by caspase-dependent and caspase-independent pathways
No full textDendritic cells (DCs) play a central role in the initiation and regulation of the immune response. The modalities by which DCs are committed to undergo apoptosis are poorly defined. Here it is shown that, unlike death receptor ligands, UVB radiation triggers apoptosis of human DCs very efficiently. UVB exposure is followed by the activation of caspases 8, 9, and 3, by the loss of mitochondrial transmembrane potential (deltaPsim), and by cellular and nuclear fragmentation. Caspase inhibitors substantially prevented the occurrence of cellular and nuclear fragmentation but had no effect on UVB-induced deltaPsim dissipation. Importantly, mature DCs (MDCs) displayed relative resistance to UVB; UVB-induced caspase activation and apoptosis were substantially delayed compared to immature DCs (IDCs). Resistance correlated with the strong up-regulation of cellular FLIP and bcl2 observed in MDCs compared to IDCs
Triggering of human monocyte activation through CD69, a member of the natural killer cell gene complex family of signal transducing receptors.
No full textThe expression and function of CD69, a member of the natural killer cell gene complex family of signal transducing receptors, was investigated on human monocytes. CD69 was found expressed on all peripheral blood monocytes, as a 28- and 32-kD disulfide-linked dimer. Molecular crosslinking of CD69 receptors induced extracellular Ca2+ influx, as revealed by flow cytometry. CD69 cross-linking resulted also in phospholipase A2 activation, as detected by in vivo arachidonic acid release measurement from intact cells and by direct in vitro measurement of enzymatic activity using radiolabeled phosphatidylcholine vesicles. Prostaglandin E 2 alpha, 6-keto-prostaglandin F 1 alpha, and leukotriene B-4 were detected by radioimmunoassay in supernatants from CD69-stimulated monocytes, suggesting the activation of both cyclooxygenase and lipoxygenase pathways after CD69 stimulation. CD69 cross-linking, moreover, was able to induce strong nitric oxide (NO) production from monocytes, as detected by accumulation of NO oxydixed derivatives, and cyclic GMP. It is important to note that NO generation was responsible for CD69-mediated increase in spontaneous cytotoxicity against L929 murine transformed fibroblast cell line and induction of redirected cytotoxicity towards P815 FcRII(+) murine mastocytoma cell line. These data indicate that CD69 can act as a potent stimulatory molecule on the surface of human peripheral blood monocytes
BIOLOGICAL EFFECTS ON HUMAN CELLS OF CHARACTERIZED ASBESTOS FIBERS. AN IN VITRO STUDY.
No full textThe biological interaction of several mineral fibers with cells and tissue can generate fibrosis, lung cancer,
mesothelioma and represent an important interdisciplinary issue. These minerals are recognized as primarly factor in
the generation of reactive oxygen species (ROS) [1]. The thought that only fiber morphology and dimension
represent the carcinogenic factors sounds like naive at the today level of consciousness: it is evident that other
aspects such as the availability of metallic cations, structural defects and surface characteristics have to be
considered, as well. Aim of study was the mineralogical characterization of different asbestos fibres and the
evaluation of their effects on biological systems. Methos. UICC chrysotile, chrysotile from Val Malenco, erionite
from Nevada, UICC crocidolite fibers were characterized by Transmission Electron Microscopy with annexed
Energy Dispersive Spectroscopy. To explore the possibility of different behaviour of mineral fibers, two biological
environments (mesothelial and bronchoalveolar cells) representing their first target have been used. The fibres were
administrated for 6, 12, 24 and 48h in Beas2B and MeT5A cells at 50μg/ml to evaluate their cytotoxic effects.
Biofunctional parameters at time points were evaluated: % number of alive, death, apoptotic cells; % number of
cells with low, medium, high ROS content. Analysis were conducted by Guava Easycite flow cytometer, 488 nm
excitation wavelength (emissions recorded by the green channel for carboxy-DCF, the red and yellow channel for
Via count dye; fluorescence intensity recorded on an average of 5,000 cells from each sample). Experiments were
carried out at least in triplicates and results were analyzed by In-cyte software. DNA damage was evaluated by
Comet assay. Results. The morphological and dimensional study assessed typical fibrous morphology for all the
samples, their chemical composition match well with the expected for this minerals: both the chrysotile samples
show the presence of aluminum and iron as substitute of tetrahedral and octahedral typical cations; the crocidolite
bears an adding of calcium and the erionite has magnesium and iron cations normally unexpected in the general
formula. All the fibers showed a high degree of crystallinity in the diffraction patterns study, without evidence of
natural amorphization (e.g. weathering). Higher direct cytotoxic effects were exerted by UICC crocidolite and UICC
chrysotile since short times of contact. Increases in DNA damage at 24h was evident in bronchial cells with erionite
contact. Conclusions: the results of this work point out biological risks of erionite fibres and can be useful to the
pathologists and biochemists who use asbestos minerals and fibrous erionite in-vitro studies as positive cyto- and
geno-toxic standard references. [1] Schoonen M.A.A. et al. “Mineral-Induced Formation of Reactive Oxygen
Species”, Reviews in Mineralogy & Geochemistry, Vol. 64, pp. 179-221, 2006, M
APOPTOTIC SIGNALING THROUGH CD95 (FAS/APO-1) ACTIVATES AN ACIDIC SPHINGOMYELINASE
No full textIntracellular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monoclonal antibody was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase (SMase) in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell-permeant C-2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death
Requirement for GD3 ganglioside in CD95- and ceramide-induced apoptosis
No full textGangliosides participate in development and tissue differentiation, Cross-linking of the apoptosis-inducing CD95 protein (also called Fas or APO-1) in lymphoid and myeloid tumor cells triggered GD3 ganglioside synthesis and transient accumulation. CD95-induced GD3 accumulation depended on integral receptor ''death domains'' and on activation of a family of cysteine proteases called caspases. Cell-permeating ceramides, which are potent inducers of apoptosis, also triggered GD3 synthesis, GD3 disrupted mitochondrial transmembrane potential (Delta psi(m),), and induced apoptosis, in a caspase-independent fashion. Transient overexpression of the GD3 synthase gene directly triggered apoptosis, Pharmacological inhibition of GD3 synthesis and exposure to GD3 synthase antisense oligodeoxynucleotides prevented CD95-induced apoptosis. Thus, GD3 ganglioside mediates the propagation of CD95-generated apoptotic signals in hematopoietic cells
Lipopolysaccharide induces Jun N-terminal kinases activation in macrophages by a novel Cdc42/Rac-independent pathway involving sequential activation of protein kinase C z and phosphatidylcholine-dependent phospholipase C
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Where Metabolism Meets Senescence: Focus on Endothelial Cells
No full textDespite the decline in their proliferative potential, senescent cells display a high metabolic activity. Senescent cells have been shown to acquire a more glycolytic state even in presence of high oxygen levels, in a way similar to cancer cells. The diversion of pyruvate, the final product of glycolysis, away from oxidative phosphorylation results in an altered bioenergetic state and may occur as a response to the enhanced oxidative stress caused by the accumulation of dysfunctional mitochondria. This metabolic shift leads to increased AMP/ATP and ADP/ATP ratios, to the subsequent AMPK activation, and ultimately to p53-mediated growth arrest. Mounting evidences suggest that metabolic reprogramming is critical to direct considerable amounts of energy toward specific activities related to the senescent state, including the senescence-associated secretory phenotype (SASP) and the modulation of immune responses within senescent cell tissue microenvironment. Interestingly, despite the relative abundance of oxygen in the vascular compartment, healthy endothelial cells (ECs) produce most of their ATP content from the anaerobic conversion of glucose to lactate. Their high glycolytic rate further increases during senescence. Alterations in EC metabolism have been identified in age-related diseases (ARDs) associated with a dysfunctional vasculature, including atherosclerosis, type 2 diabetes and cardiovascular diseases. In particular, higher production of reactive oxygen species deriving from a variety of enzymatic sources, including uncoupled endothelial nitric oxide synthase and the electron transport chain, causes DNA damage and activates the NAD+-consuming enzymes polyADP-ribose polymerase 1 (PARP1). These non-physiological mechanisms drive the impairment of the glycolytic flux and the diversion of glycolytic intermediates into many pathological pathways. Of note, accumulation of senescent ECs has been reported in the context of ARDs. Through their pro-oxidant, pro-inflammatory, vasoconstrictor, and prothrombotic activities, they negatively impact on vascular physiology, promoting both the onset and development of ARDs. Here, we review the current knowledge on the cellular senescence-related metabolic changes and their contribution to the mechanisms underlying the pathogenesis of ARDs, with a particular focus on ECs. Moreover, current and potential interventions aimed at modulating EC metabolism, in order to prevent or delay ARD onset, will be discussed
MicroRNAs as Factors in Bidirectional Crosstalk Between Mitochondria and the Nucleus During Cellular Senescence
No full textMitochondria are essential organelles that generate most of the chemical energy to power the cell through ATP production, thus regulating cell homeostasis. Although mitochondria have their own independent genome, most of the mitochondrial proteins are encoded by nuclear genes. An extensive bidirectional communication network between mitochondria and the nucleus has been discovered, thus making them semi-autonomous organelles. The nucleus-to-mitochondria signaling pathway, called Anterograde Signaling Pathway can be deduced, since the majority of mitochondrial proteins are encoded in the nucleus, less is known about the opposite pathway, the so-called mitochondria-to-nucleus retrograde signaling pathway. Several studies have demonstrated that non-coding RNAs are essential “messengers” of this communication between the nucleus and the mitochondria and that they might have a central role in the coordination of important mitochondrial biological processes. In particular, the finding of numerous miRNAs in mitochondria, also known as mitomiRs, enabled insights into their role in mitochondrial gene transcription. MitomiRs could act as important mediators of this complex crosstalk between the nucleus and the mitochondria. Mitochondrial homeostasis is critical for the physiological processes of the cell. Disruption at any stage in their metabolism, dynamics and bioenergetics could lead to the production of considerable amounts of reactive oxygen species and increased mitochondrial permeability, which are among the hallmarks of cellular senescence. Extensive changes in mitomiR expression and distribution have been demonstrated in senescent cells, those could possibly lead to an alteration in mitochondrial homeostasis. Here, we discuss the emerging putative roles of mitomiRs in the bidirectional communication pathways between mitochondria and the nucleus, with a focus on the senescence-associated mitomiRs
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