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Sphingosine-induced apoptosis is dependent on lysosomal proteases
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.
2001 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 359, no 2, 335-343 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2001. Vol. 359, no 2, 335-343 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-27700DOI: 10.1042/0264-6021:3590335Local ID: 12438OAI: oai:DiVA.org:liu-27700DiVA: diva2:248252
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically 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
2. Cathepsin D released from lysosomes mediates apoptosis
Open this publication in new window or tab >>Cathepsin D released from lysosomes mediates apoptosis
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Last year (2002), the Nobel Prize in Physiology or Medicine was awarded to three scientists who have conducted pioneer research on programmed cell death. In the human body, more than a thousand billion cells are created every day, and an equal number die, thus programmed cell death, or apoptosis, is an important mechanism for maintaining tissue homeostasis and protecting against disease. Malfunctioning apoptosis is associated with many pathological conditions, for example, excess apoptosis is characteristic of AIDS, stroke, neurodegenerative diseases, and myocardinal infarction, and insufficient apoptosis is seen in autoimmune conditions and cancer. Robert Horvitz, one of the mentioned Nobel Prize Laureates, was the first to identify death genes, namely ced-3, -4, and -9 in the nematode Caenorhabditis elegans, which were later discovered to have counterparts in humans.

The aim of this thesis is to clarify the participation of lysosomes and lysosomal proteases in the initiation of apoptosis. The lysosomal enzyme cathepsin D regulates the human homologue of ced-3, which encoded the caspase family of proteases. Moreover, the human homologue of ced-9 encodes the Bcl-2 family of proteins such as Bax, which was involved in regulating the release of cathepsin D from lysosomes during apoptosis. In the present studies, apoptosis was induced by various substances, all of which first caused damage to lysosomes with ensuing release of lysosomal proteases. Fibroblasts exposed either to free radicals generated by the redox cycling quinone naphthazarin or to the kinase inhibitor staurosporine exhibited rapid translocation of cathepsin D from lysosomes to the cytosol and subsequent apoptosis. Malignant macrophages (J774 cells) and T lymphocytes (Jurkat cells) exposed to the lysosomotropic detergent sphingosine displayed early lysosomal destabilization and later apoptosis. Sphingosine also destabilized isolated lysosomes. Moreover, mimicking the translocation of cathepsin D by microinjecting cathepsin D into the cytosol induced apoptosis in fibroblasts.

In the mentioned systems, lysosomes were destabilized before mitochondrial changes occurred and caspases were activated. Furthermore, apoptosis was prevented by inhibition of cathepsin D in the naphthazarin, staurosporine, and sphingosine systems and by inhibition of cysteine proteases such as cathepsins B and L in the sphingosine system. These results emphasize that cytosolic localization of lysosomal proteases is necessary for the ability of these enzymes to induce apoptosis.

The present results also demonstrate that, during apoptosis, lysosomal membranes are destabilized by the following: (i) free-radical-mediated lipid peroxidation; (ii) pore formation through the Bcl-2 family member Bax; (iii) the impact of the lysosomotropic detergent sphingosine. All three of these events have been implicated in numerous other apoptosis systems. Accordingly, the participation of lysosomal enzymes in apoptosis may be more widespread than previously assumed. This new perspective on lysosomes as regulators of apoptosis may lead to novel treatment strategies for diseases associated with malfunctioning apoptosis.

Abstract [sv]

År 2002 fick tre forskare Nobelpriset i fysiologi eller medicin för upptäckten att celldöd kan regleras genetiskt. I en människa bildas vruje dygn mer än tusen miljarder celler, och vmje dygn dör likamånga celler. Denna balans mellan celldöd, eller apoptos, och celldelning är viktig får vävnadsstabilitet och som skydd mot sjukdomar. AIDS, stroke, hjärtinfarkt och neurodegenerativa sjukdomar är tillstånd då apoptosfrekvensen är för hög, medan sjukdomar som autoimmuna tillstånd och cancer uppkommer vid för låg apoptosfrekvens. En av nobelpristagarna, Robert Horvitz, identifierade i nematoden Ceanorhabditis elegans de första så kallade dödsgenema, ced-3, -4 och -9, och motsvarande gener visade sig finnas hos människa.

Målsättningen med denna avhandling är att klargöra vilken roll lysosamer och lysosamala enzymer har vid initiering av apoptos. Avhandlingen visar att det lysosamala enzymet cathepsin D reglerade aktivering av caspaser, den humana motsvarigheten till ced-3. Vidare kodar den humana homologen till ced-9 för Bcl-2. I denna proteinfamilj ingår även Bax, som reglerade frisättningen av cathepsin D från lysasornerna vid apoptos. Fibroblaster som exponerats för fria radikaler, genererade genom redoxcykling av naphthazarin, eller för proteinkinashämmaren staurosporin, frisatte snabbt cathepsin D från lysosamer med påföljande apoptos som resultat. Dessutom destabiliserade den lysosomotropa detergenten sphingosin lysosamer i maligna makrofager och T-celler, vilket ledde till apoptos. För att efterlikna frisättningen av cathepsin D från lysosamer till cytosolen mikroinjicerades fibroblaster med cathepsin D, vilket också inducerade apoptos. En cytosolisk lokalisering av cathepsiner verkar vara avgörande för initiering av apoptos.

I dessa cellsystem skedde den lysosamala destabiliseringen innan mitokondriella förändringar och caspasaktivering kunde detekteras. I cellsystemet med naphthazarin, staurosporin och sphingosin förhindrades apoptos då cathepsin D hämmades. Hänming av cystein-cathepsiner som t. ex. cathepsin B och L förhindrade också apoptos i sphingosinsystemet.

Avhandlingen visar att permeabilisering av lysosamala membraner kan induceras på flera olika sätt: (i) genom lipidperoxidering medieract av fria radikaler, (ii) genom generering av porer via Bcl-2-medlemmen Bax, (iii) via effekten av den lysosomotropa detergenten sphingosin. Dessa tre fenomen finns alla representerade som signalvägar i många apoptossystem. Följaktligen kan deltagande av lysosamala enzym vid apoptos vara långt mer generellt än vad man tidigare förstått. Detta nya perspektiv, med lysosamer som regulatorer av apoptos, kan stimulera till att söka nya behandlingsstrategier för sjukdomar som beror på ett defekt apoptossystem.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2003. 80 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 771
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28053 (URN)12816 (Local ID)91-7373-528-0 (ISBN)12816 (Archive number)12816 (OAI)
Public defence
2003-01-30, Berzeliussalen, Campus US, Linköpings univeristet, Linköping, 13:00 (English)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-01-11Bibliographically approved

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Kågedal, KatarinaZhao, MingSvensson, IreneBrunk, Ulf

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