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Lysosomal Membrande Stability and Cathepsins in Cell Death
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Lysosomes are acidic organelles that are critically involved in a number of physiological processes, including macromolecule degradation, endocytosis, autophagy, exocytosis and cholesterol homeostasis. Several pathological conditions, such as cancer, neurodegenerative disorders and lysosomal storage diseases, involve lysosomal disturbances, indicating the importance of the organelle for correct cellular function. The aim of this thesis was to investigate the role of lysosomes in cell death signaling.

Previous studies have shown that permeabilization of the lysosomal membrane and release of hydrolytic enzymes such as cathepsin D to the cytosol occurs during apoptosis. We identified Bid and 14-3-3 proteins as cytosolic targets of cathepsin D in human fibroblasts. Truncated Bid, generated by cathepsin D proteolytic cleavage, stimulates Bax-mediated release of pro-apoptotic factors from the mitochondria, thereby engaging the intrinsic pathway to apoptosis.

Since the presence of cathepsins in the cytosol is sufficient to induce apoptosis, the permeability of the lysosomal membrane influences the fate of the cell. In this thesis, we demonstrated that the stability of the lysosomal membrane can be manipulated by altering the lysosomal cholesterol content. Cells with high lysosomal cholesterol content were less prone to undergo apoptosis when challenged with stimuli known to induce lysosome-mediated cell death. In addition, cholesterol accumulation was associated with increased expression of lysosome-associated membrane proteins and storage of other lipids; however, these factors did not contribute to lysosomal stabilization.

Lysosomal membrane permeabilization and cathepsins contribute to ultraviolet (UV) irradiation-induced apoptosis. We demonstrate plasma membrane damage induced by UVA irradiation to be rapidly repaired by lysosomal exocytosis in human keratinocytes. Despite efficient plasma membrane resealing, the cells underwent apoptosis, which was dependent on early activation of caspase-8. The activation of caspase-8 was lysosome-dependent and occurred in vesicles positive for lysosomal markers.

This thesis demonstrates the importance of lysosomal stability for apoptosis regulation and that this stability can be influenced by drug intervention. Modulation of the lysosomal membrane permeability may have potential for use as a therapeutic strategy in conditions associated with accelerated or repressed apoptosis.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. , 160 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1325
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-85008ISBN: 978-91-7519-803-3 (print)OAI: oai:DiVA.org:liu-85008DiVA: diva2:563534
Public defence
2012-11-28, Eken, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2012-10-30 Created: 2012-10-30 Last updated: 2017-08-30Bibliographically approved
List of papers
1. Lysosome-Mediated Apoptosis is Associated with Cathepsin D-Specific Processing of Bid at Phe24,Trp48, and Phe183
Open this publication in new window or tab >>Lysosome-Mediated Apoptosis is Associated with Cathepsin D-Specific Processing of Bid at Phe24,Trp48, and Phe183
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2012 (English)In: Annals of Clinical and Laboratory Science, ISSN 0091-7370, E-ISSN 1550-8080, Vol. 42, no 3, 231-242 p.Article in journal (Refereed) Published
Abstract [en]

Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts. Lysosome-mediated cell death is associated with degradation of Bax sequestering 14-3-3 proteins, cleavage of the Box activator Bid, and translocation of Box to mitochondria, all of which were cathepsin D-dependent. Processing of Bid could be reproduced by enforced lysosomal membrane permeabilization, using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride (MSDH). We identified three cathepsin D-specific cleavage sites in Bid, Phe24, Trp48, and Phe183. Cathepsin D-cleaved Bid induced Bax-mediated release of cytochrome c from purified mitochondria, indicating that the fragments generated are functionally active. Moreover, apoptosis was associated with cytosolic acidification, thereby providing a more favorable environment for the cathepsin D-mediated cleavage of Bid. Our study suggests that cytosolic cathepsin D triggers Bax-mediated cytochrome c release by proteolytic activation of Bid.

Place, publisher, year, edition, pages
Institute for Clinical Science, 2012
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80794 (URN)000307091500001 ()
Note

Funding Agencies|Swedish Cancer Society||Swedish Research Council||Swedish Society for Medical Research||County Council of Ostergotland||foundation of Lars Hierta||foundation of Tore Nilson||foundation of Magn||foundation of Bergvall||foundation of Stohne||foundation of Hedberg||

The original title of this article in Manuscript was: Cathepsin D-specific processing of Bid at Phe24, Trp48, and Phe183

Available from: 2012-08-30 Created: 2012-08-30 Last updated: 2017-12-07Bibliographically approved
2. Attenuation of the Lysosomal Death Pathway by Lysosomal Cholesterol Accumulation
Open this publication in new window or tab >>Attenuation of the Lysosomal Death Pathway by Lysosomal Cholesterol Accumulation
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2011 (English)In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 178, no 2, 629-639 p.Article in journal (Refereed) Published
Abstract [en]

In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent 0-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.

Place, publisher, year, edition, pages
American Society for Investigative Pathology (ASIP), 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-66151 (URN)10.1016/j.ajpath.2010.10.030 (DOI)000287264400018 ()
Available from: 2011-03-04 Created: 2011-03-04 Last updated: 2017-12-11Bibliographically approved
3. Sensitivity to Lysosome-Dependent Cell Death is Directly Regulated by Lysosomal Cholesterol Content
Open this publication in new window or tab >>Sensitivity to Lysosome-Dependent Cell Death is Directly Regulated by Lysosomal Cholesterol Content
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11Article in journal (Refereed) Published
Abstract [en]

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determined the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

Place, publisher, year, edition, pages
Public Library of Science, 2012
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-85004 (URN)10.1371/journal.pone.0050262 (DOI)000311885300096 ()23166840 (PubMedID)
Note

Funding Agencies|Swedish Research Council|2010-3463|Deutsche Forschungsgemeinschaft||foundation of Olle Engqvist||foundation of Ake Wiberg||

Available from: 2012-10-30 Created: 2012-10-30 Last updated: 2017-12-07Bibliographically approved
4. Lysosomal exocytosis and caspase-8-mediated apoptosis in UVA-irradiated keratinocytes
Open this publication in new window or tab >>Lysosomal exocytosis and caspase-8-mediated apoptosis in UVA-irradiated keratinocytes
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2013 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 126, no 24, 5578-5584 p.Article in journal (Refereed) Published
Abstract [en]

Ultraviolet (UV) irradiation is a major environmental carcinogen involved in the development of skin cancer. To elucidate the initial signaling during UV-induced damage in human keratinocytes, we investigated lysosomal exocytosis and apoptosis induction. UVA, but not UVB, induced plasma membrane damage, which was repaired by Ca2+-dependent lysosomal exocytosis. The lysosomal exocytosis resulted in extracellular release of cathepsin D and acid sphingomyelinase (aSMase). Two hours after UVA irradiation, we detected activation of caspase-8, which was reduced by addition of anti-aSMAse. Furthermore, caspase-8 activation and apoptosis was reduced by prevention of endocytosis and by the use of cathepsin inhibitors. We conclude that lysosomal exocytosis is part of the keratinocyte response to UVA and is followed by cathepsin-dependent activation of caspase-8. The findings have implications for the understanding of UV-induced skin damage and emphasize that UVA and UVB initiate apoptosis through different signaling pathways in keratinocytes.

Place, publisher, year, edition, pages
Company of Biologists, 2013
Keyword
Keratinocyte; UV irradiation; Lysosome; Cathepsin; Endocytosis; Apoptosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-103290 (URN)10.1242/jcs.130633 (DOI)000328686600005 ()
Note

The previous status of this article was manuscript with the title Lysosomal exocytosis repairs the plasma membrane after UVA and is followed by caspase-8 induced apoptosis.

Available from: 2014-01-17 Created: 2014-01-16 Last updated: 2017-08-30

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