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Cathepsin D released from lysosomes mediates apoptosis
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-28053Local ID: 12816ISBN: 91-7373-528-0 (print)OAI: oai:DiVA.org:liu-28053DiVA: diva2:248604
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
List of papers
1. The lysosomal protease cathepsin D mediates apoptosis induced by oxidative stress
Open this publication in new window or tab >>The lysosomal protease cathepsin D mediates apoptosis induced by oxidative stress
2001 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 15, no 7, 1592-1594 p.Article in journal (Refereed) Published
Abstract [en]

No abstract available.

Keyword
caspases, cell death, naphthazarin p53, pepstatin A
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-47905 (URN)10.1096/fj.00-0708fje (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-08-30Bibliographically approved
2. Microinjection of cathepsin D induces caspase-dependent apoptosis in fibroblasts
Open this publication in new window or tab >>Microinjection of cathepsin D induces caspase-dependent apoptosis in fibroblasts
2002 (English)In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 161, no 1, 89-96 p.Article in journal (Refereed) Published
Abstract [en]

Recent reports have indicated that enzymes such as cathepsins D and B are translocated from lysosomal compartments to the cytosol early during apoptosis. We have previously noted that a translocation of cathepsins D and B occur before cytochrome c release and caspase activation in cardiomyocytes and human fibroblasts during oxidative stress-induced apoptosis. In the present report, we use a microinjection technique to investigate if cytosolic location of the cathepsins D and B are important for induction of apoptosis. We found that microinjection of cathepsin D into the cytosol of human fibroblasts caused apoptosis, which was detected as changes in distribution of cytochrome c, cell shrinkage, activation of caspases, chromatin condensation, and formation of pycnotic nuclei. No apoptosis was, however, induced by microinjection of cathepsin B. Moreover, apoptosis was prevented in fibroblasts pretreated with a caspase-3-like inhibitor, and also when microinjected with cathepsin D mixed with the cathepsin D inhibitor, pepstatin A. These results show that cytosolic cathepsin D can act as a proapoptotic mediator upstream of cytochrome c release and caspase activation in human fibroblasts.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-48872 (URN)10.1016/S0002-9440(10)64160-0 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-08-30Bibliographically approved
3. Lysosomal membrane permeabilization during apoptosis: Involvement of Bax?
Open this publication in new window or tab >>Lysosomal membrane permeabilization during apoptosis: Involvement of Bax?
Show others...
2005 (English)In: International journal of experimental pathology (Print), ISSN 0959-9673, E-ISSN 1365-2613, Vol. 86, no 5, 309-321 p.Article in journal (Refereed) Published
Abstract [en]

Bcl-2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis-inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co-localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno-electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.

Place, publisher, year, edition, pages
John Wiley & Sons, 2005
Keyword
Bax, cathepsins, lysosomes, lysosomal membrane permeabilization, mitochondria
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13201 (URN)10.1111/j.0959-9673.2005.00442.x (DOI)
Note

The previous status of this article was Manuscript and the working title was Insertion of Bax into lysosomal membranes promotes release of lysosomal proteases during apoptosis.

Available from: 2008-04-17 Created: 2008-04-17 Last updated: 2017-08-30Bibliographically approved
4. Sphingosine-induced apoptosis is dependent on lysosomal proteases
Open this publication in new window or tab >>Sphingosine-induced apoptosis is dependent on lysosomal proteases
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27700 (URN)10.1042/0264-6021:3590335 (DOI)12438 (Local ID)12438 (Archive number)12438 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-09Bibliographically approved

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