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Relocation of lysosomal enzymes induces mitochondria-mediated oxidative stress, release of cytochrome c, and apoptosis
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Oxidative stress induces apoptosis, or necrosis, initiated by iron-catalyzed, intra-lysosomal oxidation leading to lysosomal rupture. Moderate lysosomal disruption induces apoptosis, while more extensive release of lysosomal contents results in necrosis. Enhanced cellular production of reactive oxygen (presumably of mitochondrial origin) also occurs during apoptosis caused by a variety of proapoptotic agonists, raising the question of whether increased oxidant generation is causal or consequential. In mixtures of rat liver lysosomes and mitochondria, selective rupture of the lysosomes by the lysosornotropic detergent 0-methyl-serine dodecylamide hydrochloride (MSDH) triggers augmented mitochondrial production of reactive oxygen species and release of cytochrome c. These mitochondrial effects are also caused by addition of purified cathepsins B and D, as well as phospholipase A2 (PLA2). We have earlier shown that PLA2 is activated by lysosomal rupture in cells undergoing apoptosis, and we now find that PLA2 - but not cathepsins B or D - causes destabilization of the membranes of semi-purified lysosomes, suggesting an amplification mechanism. In intact cultured fibroblasts, added MSDH induces lysosomal rupture, intracellular oxidant production, and apoptosis. These results suggest that initiation of the apoptotic cascade by agonists other than exogenous oxidants may involve early release of lysosomal constituents (such as cathepsins Band D) and activation of PLA2. These agents may act in concert to promote mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Thus, non-oxidant agonists of apoptosis may further amplify the process through oxidant mechanisms.

National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-81656OAI: oai:DiVA.org:liu-81656DiVA: diva2:555501
Available from: 2012-09-20 Created: 2012-09-20 Last updated: 2012-09-20Bibliographically approved
In thesis
1. The Lysosomal-Mitochondrial Axis Theory of Apoptosis
Open this publication in new window or tab >>The Lysosomal-Mitochondrial Axis Theory of Apoptosis
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2002. 54 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 747
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28091 (URN)12857 (Local ID)91-7373-187-0 (ISBN)12857 (Archive number)12857 (OAI)
Public defence
2002-12-18, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-20Bibliographically approved

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Zhao, MingEaton, John W.Brunk, Ulf T.

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