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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cell fusion is a central phenomenon during the immune response that leads to formation of large elements called multinucleated giant cells (MGCs) of common occurrence at sites of granulomatous inflammation. We have previously reported on the involvement in this event of a novel receptor expressed to high level by mononuclear phagocytes, the purinergic P2X(7) receptor. Herein, we show that blockade of this receptor by a specific monoclonal antibody prevents fusion in vitro. In contrast, cell fusion is stimulated by addition of enzymes that destroy extracellular ATP (i.e., apyrase or hexokinase). Experiments performed with phagocytes selected for high (P2X(7) hyper) or low (P2X(7) hypo) P2X(7) expression show that fusion only occurs between P2X(7) hyper/P2X(7) hyper and not between P2X(7) hyper/P2X(7) hypo or P2X(7) hypo/P2X(7) hypo. During MGCs formation we detected activation of caspase 3, an enzyme that is powerfully stimulated by P2X(7). Finally, we observed that during MGCs formation, the P2X(7) receptor is preferentially localized at sites of cell-to-cell contact. These findings support the hypothesis originally put forward by our group that the P2X(7) receptor participates in multinucleated giant cell formation.
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PMID:P2X(7) receptor and polykarion formation. 1098 8

The cytotoxicities of lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium to a lymphocytic cell line, MOLT-4, and a monocytic cell line, HL-60, was upregulated by ATP added extracellularly in a dose-dependent manner. These lipoproteins induced ATP release and plasma membrane permeability increase in these cell lines. In addition, periodate-oxidized ATP, an antagonist for P2X purinergic receptors, suppressed the cytotoxicity of the lipoproteins, suggesting the possibility that P2X receptors for ATP play crucial roles in the cytotoxicity. Activation of caspase-3 induced by the lipoproteins, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase, was also upregulated and downregulated by extracellular ATP and periodate-oxidized ATP, respectively. On the basis of these results, this study suggests that mycoplasmal lipoproteins induce the permeability increase in lymphocytes and monocytes, by which ATP is released, and the ATP regulates the cytotoxicities of the lipoproteins to the cells, possibly by interaction with ATP receptors such as P2X purinergic receptors.
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PMID:Extracellular ATP regulates cell death of lymphocytes and monocytes induced by membrane-bound lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium. 1247 45

Extracellular ATP is a potent signaling factor that modulates a variety of cellular functions through the activation of P2 purinergic receptors. Extracellular ATP at higher concentrations exerts cytostatic as well as cytotoxic effects in a variety of cell systems, the mechanism of which is not fully understood. In this study, we used cultured human embryonic kidney (HEK) cells stably transfected with human P2X(7) receptors (HEK-P2X(7)) to investigate the mechanism of ATP-induced cell death. The cytotoxic effects of ATP in HEK-P2X(7) cells were dose- and time-dependent, whereas ADP, AMP, and UTP had no effect. ATP treatment induced a significant increase in apoptotic HEK-P2X(7) cells as ascertained by the terminal deoxynucleotidyl transferase dUTP nick-end labeling technique and flow cytometry. An ATP-induced decrease in the pro-apoptotic bax gene expression was detected by apoptosis-related cDNA microarray analysis, which correlated with a decrease of Bax protein expression. Western blot analysis revealed that ATP treatment resulted in the processing of pro-caspase 3 to its active form and cleavage of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP). Both ATP-induced molecular alterations in HEK-P2X(7) cells (i.e., decrease of Bax expression and increase of PARP cleavage) were blocked by the purinergic P2X(7) receptor antagonist oxidized ATP. In conclusion, we demonstrated the importance of the P2X(7) receptor in ATP induced cell death of HEK-P2X(7) cells, which seems to be independent of bax expression; however, the activation of caspases is required.
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PMID:Poly(ADP-ribose) polymerase activation and changes in Bax protein expression associated with extracellular ATP-mediated apoptosis in human embryonic kidney 293-P2X7 cells. 1260 81

Extensive labelling for the apoptotic markers calcium channel receptor P2X(7) and caspase-3 and telomerase activity was co-localized at a similar intensity in areas affected by superficial spreading melanoma obtained from 80 patients. Labelling for each of these markers also extended 2 microm from the melanoma into the keratinocyte layer of the adjacent normal epidermis. Conversely, the calcium-regulating receptors P2X(1-3) and P2Y(2) (found in normal but not neoplastic skin) were fully de-expressed within 2 microm of the melanoma but fully expressed beyond that distance. The cell adhesion protein E-cadherin (also only present in normal skin) was progressively de-expressed from a point 2 microm from the melanoma until full de-expression within the lesion. These results show that telomerase-induced proliferation and defensive apoptosis are co-localized and simultaneous processes in melanoma tissue. Melanoma cell proliferation appears to overwhelm the apoptotic defence, perhaps due to the anti-apoptotic effects of telomerase. In addition, keratinocyte regulation of the epidermis and dermis is severely compromised by the loss of E-cadherin and P2X(1-3) and P2Y(2) receptors, resulting in a lesion that is aggressive and malignant.
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PMID:Increased expression of apoptotic markers in melanoma. 1269 Feb 96

We have generated and characterised a clone of chicken DT40 lymphocytes stably transfected with the rat P2X(7) receptor (rP2X(7)). Successful transfection was confirmed by Western blotting. Under voltage clamp, P2X(7)-expressing cells responded to ATP and dibenzoyl-ATP (Bz-ATP) (a more potent P2X(7) receptor agonist) with a rapidly activating and sustained inward current. The EC(50) values for these agonists were 305 and 15 microM, respectively. Bz-ATP evoked Ca(2+) and Mn(2+) influx into transfected cells as determined by Fura-2 spectrofluorimetry. Responses to Bz-ATP were inhibited by pre-treatment of cells with oxidised ATP. Treatment of cells with Bz-ATP for up to 24hr produced time- and concentration-dependent cell death. This was associated with an increase in caspase-3-like activity, exposure of phosphatidylserine on the outside of cell membrane and DNA cleavage, indicating death by apoptosis. Pre-treatment with Z-VAD-fmk, a pan-caspase inhibitor, reduced the DNA fragmentation and phosphatidylserine externalisation, but did not affect overall rates of cell death at 24hr, implicating caspase-independent mechanisms. The properties of rP2X(7) receptors expressed in DT40 cells are similar to those described for other expression systems. Because DT40 cells lack functionally detectable endogenous P2 receptors and are highly amenable to genetic manipulation, stably transfected DT40 cells provide a novel and potentially useful model system in which to investigate the intracellular signal transduction pathways associated with P2X(7) receptor stimulation, in particular those involved in induction of cell death.
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PMID:Chicken DT40 cells stably transfected with the rat P2X7 receptor ion channel: a system suitable for the study of purine receptor-mediated cell death. 1290 40

Brief activation of the ATP-sensitive P2X(7) receptor (P2X(7)R) stimulates the maturation and release of interleukin 1beta (IL-1beta)in macrophages, whereas prolonged agonist activation induces the formation of cytolytic pores in cell membranes. The present study investigated potential downstream mechanisms associated with native human P2X(7)R activation in lipopolysaccharide and interferon-gamma differentiated THP-1 cells. 2,3-O-(4-Benzoylbenzoyl)-ATP (BzATP)-induced pore formation (EC(50) = 35 microM) was blocked by a selective P2X(7)R antagonist, 1[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) (IC(50) = 44 nM) and by pyridoxal phosphate-6-azophenyl-2-4-disulfonic acid (PPADS) (IC(50) = 344 nM). KN-62 and PPADS also blocked BzATP-induced IL-1beta release (EC(50) = 617 microM) with IC(50) values of 75 and 3500 nM, respectively. The selective p38 mitogen-activated protein kinase (MAPK) inhibitor, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB 202190), potently inhibited BzATP-induced pore formation (IC(50) = 75 nM) but did not alter P2X(7)-mediated calcium influx or IL-1beta release. SB 202190 and KN-62 also attenuated BzATP-mediated activation of phosphorylated p38 MAPK (pp38 MAPK). Two caspase inhibitors, YVAD (caspase 1) and DEVD (caspase 3), attenuated both BzATP-induced pore formation and IL-1beta release in a concentration-dependent fashion. Neither DEVD nor p38-MAPK inhibitors blocked cell membrane pore formation evoked by maitotoxin or by activation of human P2X(2a) receptors. These results indicate that P2X(7)R-mediated pore formation results from a coordinated cascade involving both the p38 MAPK and caspase pathways that is distinct from other cytolytic pore-forming mechanisms. In contrast, P2X(7)R-mediated IL-1beta release is dependent on caspase activity but not p38 MAPK. Taken together, these results support the hypothesis that downstream cellular signaling mechanisms, rather than channel dilation, mediate cytolytic pore formation after prolonged agonist activation, which underlies P2X(7) receptors.
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PMID:Mitogen-activated protein kinase and caspase signaling pathways are required for P2X7 receptor (P2X7R)-induced pore formation in human THP-1 cells. 1463 45

Guanosine has many trophic effects in the CNS, including the stimulation of neurotrophic factor synthesis and release by astrocytes, which protect neurons against excitotoxic death. Therefore, we questioned whether guanosine protected astrocytes against apoptosis induced by staurosporine. We evaluated apoptosis in cultured rat brain astrocytes, following exposure (3 h) to 100 nM staurosporine by acridine orange staining or by oligonucleosome, or caspase-3 ELISA assays. Staurosporine promoted apoptosis rapidly, reaching its maximal effect (approximately 10-fold over basal apoptotic values) in 18-24 h after its administration to astrocytes. Guanosine, added to the culture medium for 4 h, starting from 1 h prior to staurosporine, reduced the proportion of apoptotic cells in a concentration-dependent manner. The IC50 value for the inhibitory effect of guanosine is 7.5 x 10(-5) M. The protective effect of guanosine was not affected by inhibiting the nucleoside transporters by propentophylline, or by the selective antagonists of the adenosine A1 or A2 receptors (DPCPX or DMPX), or by an antagonist of the P2X and P2Y purine receptors (suramin). In contrast, pretreatment of astrocytes with pertussis toxin, which uncouples Gi-proteins from their receptors, abolished the antiapoptotic effect of guanosine. The protective effect of guanosine was also reduced by pretreatment of astrocytes with inhibitors of the phosphoinositide 3-kinase (PI3K; LY294002, 30 microM) or the MAPK pathway (PD98059, 10 microM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase-3beta (GSK-3beta) and induced an upregulation of Bcl-2 mRNA and protein expression. These data demonstrate that guanosine protects astrocytes against staurosporine-induced apoptosis by activating multiple pathways, and these are mediated by a Gi-protein-coupled putative guanosine receptor.
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PMID:The antiapoptotic effect of guanosine is mediated by the activation of the PI 3-kinase/AKT/PKB pathway in cultured rat astrocytes. 1509 66

Large amounts of adenosine 5'-triphosphate (ATP) released from cellular sources under pathological conditions such as ischemia may activate purinoceptors of the P2X and P2Y types. In the present study, the expression of the P2X7 receptor-subtype in the brain cortex of spontaneously hypertensive rats was investigated using a permanent focal cerebral ischemia model. Immunocytochemistry with antibodies raised against the intracellular C-terminus of the P2X7 receptor showed a time-dependent upregulation of labeled cells in the peri-infarct region after right middle cerebral artery occlusion (MCAO) in comparison to controls. Double immunofluorescence visualized with confooal laser scanning microscopy indicated the localization of the P2X7 receptor after ischemia on microglial cells (after 1 and 4 days), on tubulin betaIII-labeled neurons (after 4 and 7 days), and on glial fibrillary acidic protein (GFAP)-positive astrocytes (after 4 days). In the following experiments, changes occurring 4 days after MCAO were investigated in detail. Western blot analysis of the cortical tissue around the area of necrosis indicated an increase in the P2X7 receptor protein. Immunoelectron microscopy revealed the receptor localization on synapses (presynaptically), on dendrites, as well as on the nuclear membrane of neurons (postsynaptically) and glial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling in combination with P2X7 receptor immunocytochemistry indicated a co-expression on the apoptotic cells. Active caspase 3 was especially observed on GFAP-positive astrocytes. In conclusion, the present data demonstrate a postischemic, time-dependent upregulation of the P2X7 receptor-subtype on neurons and glial cells and suggest a role for this receptor in the pathophysiology of cerebral ischemia in vivo.
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PMID:P2X7 receptor expression after ischemia in the cerebral cortex of rats. 1529 Aug 94

P2X(7), a purinergic receptor, is expressed in renal collecting ducts as they undergo fulminant cystogenesis in the cpk/cpk mouse model of autosomal recessive polycystic kidney disease (ARPKD). Dissociated cpk/cpk kidneys generate cysts from cell aggregates within 24h of suspension culture and we demonstrate that BzATP, a P2X(7) agonist, reduces cystogenesis. This effect is P2X(7)-specific, because: (i) equimolar concentrations of other purinergic agonists, ATP and UTP, had lesser effects and (ii) the P2X(7) inhibitor, oxidized ATP, abrogated the BzATP-mediated reduction in cystogenesis. BzATP did not significantly affect total cell number, proliferation, LDH release or caspase 3 activity, and zVAD-fmk, a caspase blocker, failed to modulate BzATP effects. In addition, this P2X(7) agonist did not significantly alter cyst size, probably excluding altered vectorial transport. In vivo, ATP was detected in cyst fluid from cpk/cpk kidneys; moreover, P2X(7) protein was also upregulated in human fetal ARPKD epithelia versus normal fetal collecting ducts. Thus, ATP may inhibit pathological renal cyst growth through P2X(7) signaling.
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PMID:The P2X7 ATP receptor modulates renal cyst development in vitro. 1532 48

Exposure of endothelial cells to lipid A-containing molecules, such as lipopolysaccharide (LPS) or lipooligosaccharide (LOS), causes the release of purinergic compounds [e.g., adenosine 5'-triphosphate (ATP)] and can lead to apoptosis. The P2X family of purinergic receptors (e.g., P2X(7)) has been reported to modulate LPS signaling events and to participate in apoptosis. We investigated the role that P2X receptors play in the apoptosis that follows exposure of bovine endothelial cells to Haemophilus somnus LOS. Addition of P2X inhibitors, such as periodate-oxidized ATP (oATP) or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium, significantly reduced LOS-induced apoptosis. Incubation of endothelial cells with apyrase, which degrades ATP, diminished LOS-induced apoptosis of endothelial cells. Concomitant addition of P2X agonists [e.g., 2',3'-(4-benzoyl)-benzoyl ATP or ATP] to LOS-treated endothelial cells significantly enhanced caspase-3 activation. The P2X antagonist oATP significantly blocked caspase-8 but not caspase-9 activation in LOS-treated endothelial cells. Together, these data indicate that stimulation of P2X receptors enhances LOS-induced apoptosis of endothelial cells, possibly as a result of endogenous release of ATP, which results in caspase-8 activation.
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PMID:Stimulation of P2X receptors enhances lipooligosaccharide-mediated apoptosis of endothelial cells. 1572 16


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