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Lysosomal Membrane Permeabilization: A Cellular Suicide Stragegy
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved.

The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor.

Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention.

Abstract [en]

In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved.

The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor.

Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention.

Place, publisher, year, edition, pages
Linköping University Electronic Press, 2008. , 105 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1055
Keyword [en]
apoptosis, lysosomes, lysosomal membrane permeabilization, cathepsins, Bax, Bid, mitochondria, caspases
National Category
Dentistry
Identifiers
URN: urn:nbn:se:liu:diva-11614ISBN: 978-91-7393-940-9 (print)OAI: oai:DiVA.org:liu-11614DiVA: diva2:18029
Public defence
2008-05-15, Linden, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2008-04-17 Created: 2008-04-17 Last updated: 2017-08-30
List of papers
1. Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine
Open this publication in new window or tab >>Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine
2003 (English)In: Cell Death and Differentiation, ISSN 1350-9047, Vol. 10, no 11, 1253-1259 p.Article in journal (Refereed) Published
Abstract [en]

There is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 µM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation.

Keyword
apoptosis, caspases, cathepsin D, cytochrome c, fibroblasts, lysosomes
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13200 (URN)10.1038/sj.cdd.4401290 (DOI)
Available from: 2008-04-17 Created: 2008-04-17 Last updated: 2017-08-30
2. 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
3. Cathepsin D-mediated processing of Bid at Phe24, Trp48, and Phe183
Open this publication in new window or tab >>Cathepsin D-mediated processing of Bid at Phe24, Trp48, and Phe183
Show others...
2008 (English)In: International Journal of Experimental PathologyArticle in journal (Refereed) Submitted
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13202 (URN)
Available from: 2008-04-17 Created: 2008-04-17 Last updated: 2017-08-30
4. Role of lysosomal cathepsins in naphthazarin- and Fas-induced apoptosis in oral squamous cell carcinoma cells
Open this publication in new window or tab >>Role of lysosomal cathepsins in naphthazarin- and Fas-induced apoptosis in oral squamous cell carcinoma cells
2006 (English)In: Acta Oto-Laryngologica, ISSN 0001-6489, Vol. 126, no 1, 70-81 p.Article in journal (Refereed) Published
Abstract [en]

Conclusion. Intracellular cysteine cathepsins are pro-apoptotic factors involved in activation of caspases in two oral squamous cell carcinoma (SCC) cell lines.

Objective. To study the possible involvement of lysosomal cathepsins in oral SCC cell apoptosis.

Material and methods. Apoptosis was induced in the two human oral SCC cell lines UT-SCC-20A and UT-SCC-24A using naphthazarin or anti-Fas antibodies, and was studied by analysis of caspase activity and nuclear morphology. Involvement of lysosomal cathepsins was investigated using the cysteine cathepsin inhibitor z-FA-FMK and the cathepsin D inhibitor pepstatin A. The amounts of cellular and soluble Fas death receptor were determined by ELISA.

Results. Release of cathepsins from the lysosomes to the cytosol was observed early in apoptosis. Cysteine cathepsins were found to be involved in activation of caspases in response to treatment with naphthazarin or anti-Fas antibodies, but inhibition of cysteine cathepsin activity was not sufficient to prevent cell death. Moreover, inhibition of cysteine cathepsin activity resulted in increased expression of the Fas death receptor, suggesting involvement of extracellular cysteine cathepsins in death receptor shedding.

Keyword
Caspases; cathepsin D; cell death; cysteine cathepsins; Fas death receptor; lysosomes; shedding; soluble Fas
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13203 (URN)10.1080/00016480510043422 (DOI)
Available from: 2008-04-17 Created: 2008-04-17 Last updated: 2009-05-20

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