| 1. |
- Kågedal, Katarina, 1970-, et al.
(författare)
-
Sphingosine-induced apoptosis is dependent on lysosomal proteases
- 2001
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 359:2, s. 335-343
-
Tidskriftsartikel (refereegranskat)abstract
- We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes.
|
|
| 2. |
|
|
| 3. |
- Stoltenberg, M, et al.
(författare)
-
Bismuth-induced lysosomal rupture in J774 cells
- 2002
-
Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 110:5, s. 396-402
-
Tidskriftsartikel (refereegranskat)abstract
- Bismuth-containing drugs have several applications, one being their use against Helicobacter pylori-associated peptic ulcers, and bismuth has been discovered in macrophages at the base and margins of peptic ulcers. In the present study, the autometallographic technique for the histochemical demonstration of bismuth was applied, showing that bismuth citrate-exposed J774 cells accumulate the metal in their lysosomes. Such accumulations resulted in lysosomal rupture - assayed by the acridine orange uptake technique and flow cytofluorometry - and ensuing apoptotic cell death.
|
|
| 4. |
|
|
| 5. |
- Zhao, Ming, 1966-, et al.
(författare)
-
Bcl-2 phosphorylation is required for inhibition of oxidative stress-induced lysosomal leak and ensuing apoptosis
- 2001
-
Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 509:3, s. 405-412
-
Tidskriftsartikel (refereegranskat)abstract
- B-cell leukemia/lymphoma 2 (Bcl-2) blocks oxidant-induced apoptosis at least partly by stabilizing lysosomes. Here we report that phosphorylation of Bcl-2 may be required for these protective effects. J774 cells overexpressing wild-type Bcl-2 resist oxidant-induced lysosomal leak as well as apoptosis, and this protection is amplified by pretreatment with phorbol 12-myristate 13-acetate (which promotes protein kinase C (PKC)-dependent phosphorylation of Bcl-2). In contrast, cells overexpressing the Bcl-2 mutant S70A (which cannot be phosphorylated) are not protected in either circumstance. Transfection with Bcl-2(S70E), a constitutively active Bcl-2 mutant which does not require phosphorylation, is protective independent of PKC activation. In contrast, C2-ceramide, a putative protein phosphatase 2A activator, abolishes the protective effects of wild-type Bcl-2 overexpression but does not diminish protection afforded by Bcl-2(S70E). Additional results suggest that, perhaps as a consequence of lysosomal stabilization, Bcl-2 may prevent activation of phospholipase A2, an event potentially important in the ultimate initiation of apoptosis.
|
|
| 6. |
- Zhao, Ming, 1966-, et al.
(författare)
-
Delayed oxidant-induced cell death involves activation of phospholipase A2
- 2001
-
Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 509:3, s. 399-404
-
Tidskriftsartikel (refereegranskat)abstract
- Short-term (1 h) exposure of cells to a low steady-state concentration of H2O2 causes no immediate cell death but apoptosis occurs several hours later. This delayed cell death may arise from activation of phospholipases, in particular phospholipase A2 (PLA2), which may destabilize lysosomal and mitochondrial membranes. Indeed, the secretory PLA2 (sPLA2) inhibitor 4-bromophenacyl bromide diminishes both delayed lysosomal rupture and apoptosis. Furthermore, sPLA2 activation by mellitin, or direct micro-injection of sPLA2, causes lysosomal rupture and apoptosis. Finally, B-cell leukemia/lymphoma 2 (Bcl-2) over-expression prevents oxidant-induced activation of PLA2, delayed lysosomal destabilization and apoptosis. This supports a causal association between PLA2 activation and delayed oxidant-induced cell death and suggests that Bcl-2 may suppress apoptosis by preventing PLA2 activation.
|
|
| 7. |
- Zhao, Ming, 1966-, et al.
(författare)
-
Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis
- 2003
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:18, s. 3778-3786
-
Tidskriftsartikel (refereegranskat)abstract
- Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant proapoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 - but not cathepsins B or D - causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.
|
|
| 8. |
- Zhao, Ming, 1966-, et al.
(författare)
-
Protection against oxidant-mediated lysosomal rupture : a new anti-apoptotic activity of Bcl-2?
- 2000
-
Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 485:2-3, s. 104-108
-
Tidskriftsartikel (refereegranskat)abstract
- Bcl-2 antagonizes apoptosis through mechanisms which are not completely understood. We have proposed that apoptosis is initiated by minor lysosomal destabilization followed some time later by secondary massive lysosomal rupture. In J774 cells over-expressing Bcl-2, early oxidant-induced lysosomal destabilization is unaffected but secondary lysosomal rupture and apoptosis are suppressed, despite the fact that wild-type and Bcl-2 over-expressing cells degrade hydrogen peroxide at similar rates. It may be that Bcl-2 directly blocks the effects of released lysosomal enzymes and/or prevents downstream activation of unknown cytosolic pro-enzymes by released lysosomal hydrolases, suggesting a new and heretofore unknown activity of Bcl-2.
|
|
| 9. |
- Zhao, Ming, 1966-
(författare)
-
The Lysosomal-Mitochondrial Axis Theory of Apoptosis
- 2002
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In many cases, apoptosis may be initiated by a minor lysosomal destabilization, which some time later is followed by a secondary, more pronounced, lysosomal rupture. After exposure to low concentrations of sphingosine, a lysosomotropic detergent, Jurkat and J774 cells underwenr apoptotic cell death, while cells exposed to higher concentrations of this agent showed necrosis. Sphingosine-induced apoptosis was partly prevented by the inhibitors of lysosomal aspartic or cysteine proteases, pepstatin A or E64d. Under these conditions, caspase-3 like activity was reduced 40-55%, suggesting that lysosomal enzymes could be upstream activators of caspase-3.In J774 cells over-expressing Bcl-2, the early oxidant-induced lysosomal destabilization takes place, but the delayed secondary lysosomal rupture and ensuing apoptosis are both suppressed. Phosphorylation of Bcl-2 seems to be required for this anti-apoptotic effect because the protection is amplified by pre-treatment with phorbol 12-myristate 13-acetate, which promotes protein kinase C (PKC)-dependent phosphorylation of Bcl-2. In contrast, cells over-expressing the Bcl-2 mutant S70A (which cannot be phosphorylated and is inactive) are not protected. Transfection with Bcl-2(S70E), a constitutively active Bcl-2 mutant, which does not require phosphorylation, is protective independent of PKC activation. In contrast, C2- ceramide, a putative protein phosphatase 2A (PP2A)-activator, abolishes the protective effects of wild-type Bcl-2 over-expression but does not diminish protection afforded by Bcl-2(S70E).It may be that Bcl-2 directly blocks the effects of initially released lysosomal enzymes and/or prevents down-stream activation of cytosolic pro-apoptotic enzymes by released lysosomal hydrolases. Short-term (1 h) exposure of cells to a low steadystate concentration of H202 causes no immediate cell death, but apoptosis occurs several hours later when cells have been returned to standard culture conditions. This delayed cell death seems to arise from activation of phospholipases, in particular phospholipase A2 (PLA2), which may dcstabilize lysosomal as well as mitochondrial membranes. Indeed, the specific inhibition of PLA2 by 4-bromophenacyl bromide (BPB ), diminishes both delayed lysosomal rupture and apoptosis. Furthermore, PLA2 activation by mellitin, or direct micro-injection of PLA2, causes lysosomal rupture and apoptosis. Finally, Bcl-2 over-expression prevents oxidant-induced activation of PLA2, and delays lysosomal destabilization as well as apoptosis.Exogenous oxidative stress may induce apoptosis, but enhanced endogenous production of oxidants is also often found during apoptosis caused by other agonists, raising the question of whether this latter actually contributes to apoptosis or is simply a by-product. Our data show that leak to the cytosol of lysosomal enzymes results in mitochondria-mediated oxidative stress, release of cytochrome c, and further lysosomal rupture. In mixed lysosome-mitochondria preparations, the lysosomotropic detergent, 0-methyl-serine dodecylamide hydrochloride (MSDH), selectively lyses lysosomes, while PLA2 attacks lysosomes as well as mitochondria. Released lysosomal enzymes, and aclivated PLA2, cause mitochondria to produce enhanced amounts of hydrogen peroxide and to release cytochrome c. Purified lysosomal cathepsins B and D have the same effects on mitochondrial oxidant production but do not destabilize lysosomal membranes in these mixed preparations of mitochondria and lysosomes. In intact cells, MSDH induces lysosomal rupture, oxidative stress and apoptosis.These data allow us to propose the following lysosomal-mitochondrial axis theory of apoptosis:1. Limited lysosomal rupture induces activation of PLA2 (probably often mediated by the lysosomal enzyme, cathepsin B).2. Released lysosomal enzymes, and activated PLA2, cause enhanced mitochondrial production of reactive oxygen as wel1 as release of cytochrome c.3. This cascade of events is accompanied by further lysosomal rupture (by the combined effects of oxidative stress and PLA2), initiating full-blown apoptosis.4. Through presently unknown mechanisms, phosphorylated Bcl-2 preserves the integrity of both mitochondria and lysosomes, preventing further release of lysosomal enzymes and of mitochondrial pro-apoptotic proteins.
|
|
| 10. |
- Zhao, Ming, 1966-, et al.
(författare)
-
Vascular smooth muscle cell proliferation requires both p38 and BMK1 MAP kinases
- 2002
-
Ingår i: Archives of Biochemistry and Biophysics. - 0003-9861 .- 1096-0384. ; 400:2, s. 199-207
-
Tidskriftsartikel (refereegranskat)abstract
- Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of atherosclerosis. Induction of both c-fos (through the transcription factor Elk-1) and c-jun, both immediate early genes, is important for the stimulation of VSMC proliferation and migration. It was earlier found that p38 mitogen-activated protein (MAP) kinase upregulates c-jun gene transcription through phosphorylation of two myocyte enhancer factor 2 (MEF2) family transcription factors, MEF2A and MEF2C, while big MAP kinase 1 (BMK1) may upregulate c-jun gene transcription through MEF2A, MEF2C, and also MEF2D. Here, we report that inhibition of BMK1 by a dominant negative form of MEK5 or pharmacologic inhibition of p38 by SB 203580 additively suppress serum-induced VSMC proliferation. This additive effect of p38 and BMK1 inhibition implies that these two kinases coordinately regulate MEF2 transcription factors. The exclusive activation of MEF2D by BMK1 appears required for this cooperative upregulation of c-jun in VSMC, and coactivation of p38 and BMK1 also has additive effects on the activation of a reporter gene linked to the c-jun promoter in our experimental system. Thus, coordinate activity of both the p38 and BMK1 pathways appears necessary for optimal transcription of c-jun and, pari pasu, VSMC proliferation. These results may have implications for the future design of pharmacologic agents for inhibition of VSMC growth.
|
|
