Linköping University Medical Dissertations No. 1055
LYSOSOMAL MEMBRANE PERMEABILIZATION – A CELLULAR SUICIDE STRATEGY ANN­CHARLOTTE JOHANSSON Department of Clinical and Experimental Medicine,
Faculty of Health Sciences, Linköping University,
SE-581 85 Linköping, Sweden
Linköping 2008
© Ann-Charlotte Johansson, 2008
ISBN 978-91-7393-940-9
ISSN 0345-0082
Published articles have been reprinted with permission from the publishers:
Paper I © 2003 Nature Publishing group
Paper II © 2005 Blackwell Publishing Ltd
Paper IV © 2006 Taylor & Francis Group
Printed by LiU-Tryck, Linköping 2008
Till Mira och Isa ABSTRACT 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
isiolated 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 extra-cellular 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 therapeutic intervention.
TABLE OF CONTENTS LIST OF PAPERS ............................................................................... 9 ABBREVIATIONS .......................................................................... 11 INTRODUCTION ............................................................................ 13 LYSOSOMES ............................................................................................................... 13 CATHEPSINS ................................................................................................................................. 14 APOPTOSIS ................................................................................................................. 15 APOPTOSIS, NECROSIS, AND AUTOPHAGIC CELL DEATH ‐ WHAT IS THE DIFFERENCE? ............................................................................................................................... 16 CLEARANCE OF APOPTOTIC CELLS ................................................................................... 16 GENETIC CONTROL ................................................................................................................... 17 INITIATION OF APOPTOSIS ................................................................................................... 18 THE BCL‐2 FAMILY .................................................................................................................... 20 PROTEOLYTIC REGULATION OF APOPTOSIS ................................................................ 28 CANCER ....................................................................................................................... 34 APOPTOSIS IN CANCER DEVELOPMENT ......................................................................... 34 CATHEPSINS IN CANCER ........................................................................................................ 35 AIMS ................................................................................................. 37 MATERIALS AND METHODS ..................................................... 39 CELLS ............................................................................................................................ 39 INDUCERS OF APOPTOSIS................................................................................... 40 PROTEASE INHIBITORS ....................................................................................... 41 DETECTION OF APOPTOSIS ............................................................................... 42 ASSESSMENT OF CATHEPSIN RELEASE ....................................................... 43 MICROINJECTION ................................................................................................... 44 SUBCELLULAR LOCALIZATION OF BAX ........................................................ 44 ISOLATION OF RAT LIVER LYSOSOMES ....................................................... 45 STATISTICAL ANALYSIS ....................................................................................... 45 ETHICAL CONSIDERATIONS .............................................................................. 45 RESULTS .......................................................................................... 47 PAPERS I, II, AND III ............................................................................................... 47 PAPER IV ..................................................................................................................... 51 DISCUSSION .................................................................................... 55 LYSOSOMAL MEMBRANE PERMEABILIZATION ....................................... 55 PROMOTERS OF LYSOSOMAL MEMBRANE PERMEABILIZATION ....................... 56 SAFEGUARDS OF LYSOSOMAL MEMBRANE INTEGRITY ......................................... 62 MECHANISMS BY WHICH CATHEPSINS PROMOTE APOPTOSIS ....... 65 DIRECT ACTIVATION OF CASPASES .................................................................................. 65 CLEAVAGE OF BID ..................................................................................................................... 66 OTHER MECHANISMS .............................................................................................................. 68 CATHEPSIN D‐MEDIATED DEATH SIGNALING – DEPENDENT ON CATALYTIC ACTIVITY OR NOT? .................................................................................................................... 70 THE DUAL FUNCTION OF CATHEPSINS IN CANCER ............................... 71 INTRA‐CELLULAR MEDIATORS OF APOPTOSIS ........................................................... 71 EXTRA‐CELLULAR PROMOTERS OF TUMOR GROWTH AND INVASION ........... 72 CONCLUSIONS ................................................................................ 75 TACK ................................................................................................. 77 REFERENCES .................................................................................. 79 LIST OF PAPERS This thesis is based on the following papers, which will be referred to
in the text by their Roman numerals:
I.
Ann-Charlotte Johansson, Håkan Steen, Karin Öllinger,
and Karin Roberg (2003) Cathepsin D mediates
cytochrome c release and caspase activation in human
fibroblast apoptosis induced by staurosporine. Cell Death
and Differentiation 10; 1253-59
II.
Katarina Kågedal, Ann-Charlotte Johansson, Uno
Johansson, Gerd Heimlich, Karin Roberg, Nancy S. Wang,
Juliane M. Jürgensmeier, and Karin Öllinger (2005)
Lysosomal membrane permeabilization during apoptosis –
involvement of Bax? International Journal of Experimental
Pathology 86; 309-21
III.
Ann-Charlotte Johansson, Hanna Mild, Uno Johansson,
Cathrine Nilsson, Bruno Antonsson, Katarina Kågedal, and
Karin Öllinger (2008) Cathepsin D-mediated processing of
Bid at Phe24, Trp48, and Phe183. International Journal of
Experimental Pathology, submitted
IV.
Ann-Charlotte Johansson, Lena Norberg-Spaak, and Karin
Roberg (2006) Role of lysosomal cathepsins in
naphthazarin- and Fas-induced apoptosis in oral squamous
cell carcinoma cells. Acta Oto-Laryngologica 126; 70-81
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ABBREVIATIONS Ahr
aryl hydrocarbon receptor
AIF
apoptosis-inducing factor
ANT
adenine nucleotide translocator
Apaf-1
apoptosis protein activating factor-1
BCECF
2’,7’-bis(carboxyethyl)-5,6-carboxyfluorescein
BH
Bcl-2 homologue
CAD
caspase-activated DNAse
CARD
caspase activation and recruitment domain
cyp D
cyclophilin D
DD
death domain
DED
death effector domain
DFF45
DNA fragmentation factor 45 kDa subunit
DIABLO
Direct IAP-binding protein with a low pI
DISC
death-inducing signaling complex
DPPD
N,N-diphenyl-1,4-phenylene-diamine
ER
endoplasmic reticulum
FADD
Fas-associated death domain
FasL
Fas death receptor ligand
Hsp
heat shock protein
IAP
inhibitor of apoptosis proteins
ICAD
inhibitor of CAD
JNK
c-Jun N-terminal kinase
LAMP
lysosome-associated membrane protein
LAPF
lysosome-associated apoptosis-inducing protein
containing the pleckstrin homology and FYVE
domains
LDH
lactate dehydrogenase
LEDGF
lens epithelium-derived growth factor
11
LIMP
lysosomal integral membrane protein
LMP
lysosomal membrane permeabilization
MEFs
mouse embryonic fibroblasts
MMP
mitochondrial membrane permeabilization
MSDH
O-methyl serine dodecylamide hydrochloride
NAG
N-acetyl-β-glucosaminidase
NF-κB
nuclear factor-κB
PARP
poly(ADP-ribose) polymerase
PBS
phosphate-buffered saline
pep A
pepstatin A
PLA2
phospholipase A2
PT
permeability transition
ROS
reactive oxygen species
SCC
squamous cell carcinoma
siRNA
short interfering RNA
Smac
second mitochondrial activator of caspases
STS
staurosporine
SV40
simian virus 40
tBid
truncated Bid
TNF
tumor necrosis factor
TRADD
TNF-receptor-associated death domain
TRAIL
TNF-related apoptosis-inducing ligand
UV
ultraviolet
VDAC
voltage-dependent anion channel
wt
wild type
XIAP
X-linked inhibitor of apoptosis proteins
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INTRODUCTION
INTRODUCTION LYSOSOMES Lysosomes are acidic degradative organelles present in all eukaryotic
cells (de Duve, 1983). The endolysosomal system is composed of
early endosomes, with an intra-lumenal pH of 6.0-6.6, late endosomes,
with a more acidic pH (~5), and lysosomes with the most acidic
compartment (pH ~4.5). Lysosomes are extraordinarily diverse in
shape and size, and occupy up to 15% of the total cell volume.
Lysosomes were first described by de Duve and his collaborators and
for their discovery they were in 1974 awarded the Nobel Prize in
Physiology or Medicine. The term ‘lysosome’ is derived from the
Greek word for ‘digestive body’ and first appeared in the literature in
1955 (de Duve et al., 1955).
Lysosomes are surrounded by a single membrane unique in
composition. The membrane contains highly glycosylated lysosomalassociated membrane proteins (LAMPs) and lysosomal integral
membrane proteins (LIMPs), that constitute about 50% of all
lysosomal membrane proteins (Winchester, 2001). LAMPs and
LIMPs form a coat on the inner surface of the membrane, and are
thought to protect the membrane from attack by the numerous
hydrolytic enzymes retained within lysosomes. The lysosomal
membrane also harbors a proton pump that utilizes the energy from
ATP hydrolysis to pump H+ into the lumen, thereby creating the acidic
pH characteristic of lysosomes (Ohkuma and Poole, 1978; Schneider,
1981; Ohkuma et al., 1982).
The primary function of lysosomes is degradation of extra- and intracellular material. For this purpose, lysosomes are filled with
hydrolases (phosphatases, nucleases, glycosidases, proteases,
peptidases, sulphatases, and lipases) whose function is dependent on
the acidic pH of the lysosomal interior (de Duve, 1983). The material
to be digested is delivered to the endolysosomal compartment via
autophagocytosis, endocytosis, and in specialized cells, through
phagocytosis (Bagshaw et al., 2005; Ciechanover, 2005; Luzio et al.,
2007). The final degradation products are translocated via membrane
transporter proteins from the intra-lysosomal compartment across the
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INTRODUCTION
lysosomal membrane, and are released into the cytosol for metabolic
re-use.
CATHEPSINS The term “cathepsin” was introduced in 1920 and stands for
“lysosomal proteolytic enzyme” (Willstätter and Bamann, 1929;
Chwieralski et al., 2006). Since proteases are generally classified
based on their catalytic mechanisms, cathepsins are sub-divided
according to their active site amino acids, which confers the catalytic
activity, into cysteine (cathepsins B,C, F, H, K, L, N, O, S, T, U, W
and X), serine (cathepsins A and G), and aspartic cathepsins
(cathepsins D and E). Cathepsins were long believed to be involved
primarily in intra-lysosomal protein degradation. However, several
cathepsins have now been assigned specific and individual functions
(Turk et al., 2002). For example, cathepsin K is involved in bone
remodeling, cathepsins S, L and F in processing of the major
histocompatibility complex (MHC) class II-associated invariant chain,
and cathepsin C in the activation of a number of granular serine
proteases, such as granzymes A and B (Turk et al., 2002).
Cathepsins are synthesized in the endoplasmic reticulum (ER) as
inactive pre-pro-enzymes (Erickson, 1989). After folding,
glycosylation and removal of the signaling peptide, cathepsins leave
the ER and enter the Golgi network. In the cis-Golgi, one or several
carbohydrates are modified to mannose-6-phosphate moieties,
allowing cathepsins to be sorted from other proteins and directed
toward lysosomes by interaction with mannose-6-phosphate receptors
in the trans-Golgi network (Ghosh et al., 2003). Cathepsins are
released from their receptors in endosomes as a consequence of the
acidification associated with organelle maturation. The receptors are
recycled back to the trans-Golgi network, or in some cases, delivered
to the cell surface where they collect proteins for transport to the
endolysosomal compartment.
Cathepsins are activated in the acidic endolysosomal compartment via
proteolytic removal of the N-terminal pro-peptide, which in the proform blocks the active site cleft (Turk et al., 2001). Cathepsin D is
further cleaved into a two-chain form, however, this step is cell type
specific and does not seem to affect the enzyme activity (Erickson,
1989). It should be noted that the pro-forms of cysteine cathepsins are
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INTRODUCTION
catalytically active, since their pro-peptide can be partially removed
from the active site (Turk et al., 2001).
Cathepsins B, D and L, which are involved in apoptosis signaling and
therefore of special interest in this thesis, are all endo-peptidases, i.e.
enzymes that cleave their substrates internally (Bond and Butler,
1987; Turk et al., 2001). Cathepsin B is also an exo-peptidase and
cleaves its substrates in the C-terminal end. The activity of cysteine
cathepsins, including cathepsins B and L, is controlled by potent
endogenous inhibitors of the cystatin family (Turk et al., 2002). The
stefins, which are the major intra-cellular cystatins, are believed to
protect cells from accidental release of cathepsins to the cytosol. The
pro-peptide split off of pro-cathepsin D during activation, α2macroglobulin and DNA fragments are the only known endogenous
inhibitors of cathepsin D (Gacko et al., 2007). However, their
inhibitory effect is slight and observed only under special conditions.
APOPTOSIS Cellular suicide, apoptosis, has emerged as an important physiological
process. Many vital functions, such as termination of inflammatory
reactions, wound healing, physiologic tissue renewal, and elimination
of cells with irreparable damage depend on intact apoptosis (Böhm
and Schild, 2003). The deregulation of this process has disastrous
consequences, and is involved in many diseases such as cancer,
autoimmunity, and neurodegeneration (Nicholson, 2000) (Figure 1).
Figure 1: Homeostasis is maintained by a balance between cell proliferation and
cell death. Many severe diseases are associated with excessive or insufficient cell
death. Modified from: Thompson, C. B. (1995) Science 267: 1456-1462.
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INTRODUCTION
APOPTOSIS, NECROSIS, AND AUTOPHAGIC CELL DEATH ­ WHAT IS THE DIFFERENCE? There are three major types of cell death, apoptosis, autophagic cell
death, and necrosis, that can be distinguished from each other by
morphological and biochemical criteria (Lockshin and Zakeri, 2004).
Already in 1858 it was recognized that there exists more than one
form of cell death (Virchow and Chandler, 1859), and a century later
the word “apoptosis” was introduced by Kerr, Wyllie and Currie (Kerr
et al., 1972). The term is derived from an ancient Greek word meaning
“falling off of leaves from a tree in autumn” and refers to the
formation of apoptotic bodies, which is a typical morphological
feature of apoptotic cells. Apoptosis most often involve activation of
proteases from the caspase family, and is characterized not only by
formation of apoptotic bodies, but a series of distinct morphological
features (Kroemer et al., 2005). These were first described by Kerr
and colleagues and include loss of adhesion, cell shrinkage, chromatin
condensation, nuclear fragmentation, and membrane blebbing, leading
to formation of apoptotic bodies (Kerr et al., 1972; Ziegler and
Groscurth, 2004). Autophagic cell death refers to all cell deaths
associated with autophagy (Kroemer et al., 2005). This definition is
less clear-cut as the formation of autophagic vacuoles need not
necessarily promote cell death (Debnath et al., 2005). In fact,
autophagy may instead, under certain circumstances, prevent cells
from dying. Finally, necrosis is defined as cell death occurring without
signs of apoptosis or autophagy (Kroemer et al., 2005). This is, in
comparison, a more acute form of cell death, which is characterized
by cell swelling, ultimately leading to loss of plasma membrane
integrity and leakage of cellular contents into the surrounding tissue.
Apart from the three types of cell death defined above, there also exist
intermediate forms with mixed phenotypes, for instance with signs of
both apoptosis and necrosis (Jäättelä, 2002; Zhivotovsky, 2004).
CLEARANCE OF APOPTOTIC CELLS An important difference between apoptosis and necrosis in a
physiological context is that necrotic cell death is associated with local
inflammation (Ziegler and Groscurth, 2004). Cells dying from
apoptotic cell death, on the other hand, do not elicit an inflammatory
response due to containment of the cellular constituents by an intact
membrane and clearance of cell remnants by phagocytosis.
Phagocytosis is triggered by “eat me”-signals, such as the
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INTRODUCTION
phospholipid phosphatidylserine, given by the apoptotic cell.
Phosphatidylserine is normally present on the cytoplasmic side of the
cell membrane, but is exposed on the cell surface when cells undergo
apoptosis (Conradt, 2002). If the dying cell is not engulfed it will
eventually undergo degradation that resembles necrosis; a process
referred to as secondary necrosis (Ziegler and Groscurth, 2004). This
phenomenon is common in cell culture, but is not likely to occur in the
body which is patrolled by phagocytic cells.
GENETIC CONTROL Robert Horvitz, Sydney Brenner, and John Sulston were in 2002
awarded the Nobel Prize in Physiology or Medicine for their
pioneering work in defining the genetic pathway for cell death. Their
research is based on studies in the nematode Caenorhabditis elegans,
in which deletion of exactly 131 of 1 090 cells occurs during its
development (Hengartner and Horvitz, 1994). Thus, it provides a
simple model for studying the genetic basis of cell death. Several C.
elegans genes implicated in developmental cell death has been
identified [reviewed in (Metzstein et al., 1998; Lettre and Hengartner,
2006)]. The protein products of three of these genes; EGL-1 (egg
laying defective), CED-3 (cell death abnormal), and CED-4 are
required for cell death to occur, while CED-9 protects from cell death
(Figure 2).
Figure 2: C. elegans cell death proteins and their mammalian counterparts.
Modified from: Jin, Z. and El-Deiry, W. S. (2005) Cancer Biol. Ther. 4: 139-163.
17
INTRODUCTION
In 1993, researchers discovered that the ced-3 gene had remarkable
sequence similarity to interleukin-1β-converting enzyme (now called
caspase-1), a mammalian protease responsible for the proteolytic
maturation of pro-interleukin-1β. This finding lead to the
identification of several proteases of the caspase family, and suggested
that they might function during apoptosis. CED-4 was later on found
to be related to mammalian apoptotic protease activating factor-1
(Apaf-1), while CED-9 corresponds to Bcl-2. Finally, EGL-1 has
functional and molecular similarity to the BH3-only proteins of the
Bcl-2 family in mammalian cells. Thus, the core genetic program of
cell death shows high evolutionary conservation, emphasizing the
importance of a functional cell death program for survival of the
species.
INITIATION OF APOPTOSIS Signaling for apoptosis is initiated either from outside the cell
(extrinsic or death receptor pathway) or from inside the cell (intrinsic
or mitochondrial pathway) (Figure 3). In both pathways, signaling
results in activation of proteases of the caspase family, responsible for
dismantling and removal of the dying cell.
The intrinsic pathway Apoptosis is initiated via the intrinsic pathway in response to a variety
of different cellular stresses, such as starvation, ionizing radiation,
hypoxia, and exposure to various chemicals.
The intrinsic pathway is characterized by mitochondrial dysfunction
and mitochondrial membrane permeabilization (MMP) with release of
pro-apoptotic proteins to the cytosol (Garrido et al., 2006). One of
these proteins, cytochrome c, is essential for cell survival as it serves
as an electron transporter in the respiratory chain. However, it is also
generally considered as one of the most important molecules in
apoptosis signaling. After release to the cytosol, cytochrome c triggers
activation of caspases via formation of a signaling complex called the
apoptosome (see below for more detailed information). Other
mitochondrial factors promote cell death by disruption of caspase
inhibition. The cytosolic protein X-linked inhibitor of apoptosis
proteins (XIAP) is known to sequester caspases-3, -7, and -9 and keep
them inactive (Deveraux et al., 1997; Eckelman et al., 2006). This
interaction is disrupted, and caspases released from inhibition, by
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INTRODUCTION
binding of XIAP to one of two proteins released from mitochondria;
second mitochondrial activator of caspases (Smac), also known as
direct IAP binding protein with low pI (DIABLO), or HtrA2/Omi (Du
et al., 2000; Verhagen et al., 2000; Suzuki et al., 2001; Hegde et al.,
2002; Martins et al., 2002; van Loo et al., 2002; Verhagen et al.,
2002). Apoptosis-inducing factor (AIF) and endonuclease G, which
are also harbored within the inter-membrane space of mitochondria,
promote apoptosis independent of caspases by inducing fragmentation
of DNA after translocation to the nucleus (Susin et al., 1999; Li et al.,
2001).
The extrinsic pathway The extrinsic pathway is initiated at the cell surface through ligation
of death receptors [reviewed in (Ashkenazi and Dixit, 1998; Peter and
Krammer, 2003; Khosravi-Far and Esposti, 2004; Falschlehner et al.,
2007)]. To date, there are three death receptor ligands identified; Fas
ligand (FasL), tumor necrosis factor-α (TNF-α), and TNF-related
apoptosis-inducing ligand (TRAIL). FasL and TRAIL are expressed
mainly in cells of the immune system, and are involved in the
termination of immune responses by deletion of activated T-cells.
They also mediate killing of virus-infected cells and cancer cells.
TNF-α is produced by T-cells and macrophages in response to
infection. By binding to its receptors, TNFR-1 and TNFR-2, TNF-α
elicit a number of cellular responses, including apoptosis.
The main death receptors are TNFR-1, Fas/CD95, TRAIL-R1/DR4
and TRAIL-R2/DR5, which all belong to the TNF receptor superfamily. These trans-membrane death receptors are characterized by an
intra-cellular death domain (DD) essential for recruitment of
downstream apoptotic proteins. The extra-cellular cysteine-rich
domains that mediate ligand binding are another common feature.
Death receptor activation leads to assembly of a signaling complex
called the DISC (death-inducing signaling complex), to which
caspases are recruited for activation.
The intrinsic and extrinsic pathways are inter-connected by Bid, a proapoptotic member of the Bcl-2 family. Caspases activated in response
to death receptor signaling cleave Bid, generating a pro-apoptotic
truncated form that engages the intrinsic pathway by promoting MMP.
Apoptosis initiated via the extrinsic pathway may, depending on the
cell type, proceed with or without engagement of mitochondria. A few
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INTRODUCTION
cell types (type I cells) can efficiently activate caspases via the DISC,
while in the majority of cells (type II cells), amplification of the death
signal via the intrinsic pathway is required.
Figure 3: Apoptosis is initiated either via the extrinsic or the intrinsic pathway,
characterized by death receptor activation and mitochondrial membrane
permeabilization, respectively.
THE BCL­2 FAMILY The mechanisms of MMP have not been fully elucidated, but it is
clear that members of the Bcl-2 family of proteins are the main
regulators of the intrinsic pathway (Garrido et al., 2006; Adams and
Cory, 2007; Danial, 2007; Youle and Strasser, 2008).
Structure and function The Bcl-2 family includes both pro- and anti-apoptotic proteins that
share sequence homology within one or several highly conserved
regions, known as Bcl-2 homology (BH) domains [reviewed in
(Garrido et al., 2006; Adams and Cory, 2007; Danial, 2007; Youle
and Strasser, 2008)]. Bcl-2 proteins are divided into three main groups
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INTRODUCTION
on the basis of their BH domains and their function (Figure 4). The
first group consists of Bcl-2 itself, Bcl-XL, Bcl-w, Mcl-1, and A1,
which share homology in four domains (BH1-BH4) and act as
inhibitors of apoptosis. The members of the remaining two groups are
promoters of apoptosis; Bax, Bak and Bok carry three BH domains
(BH1-BH3), whereas Bim, Bad, Bid, Bik, Bmf, Puma, Noxa, and Hrk
are so-called BH3-only proteins, sharing homology within only one
domain. In addition, members of the first two groups and some of the
BH3-only proteins possess a C-terminal transmembrane domain that
enables association with cellular membranes, including those of
mitochondria, ER, nucleus, and, as will be discussed later, possibly
lysosomes.
Figure 4: Classification of Bcl-2 proteins. Anti-apoptotic members of the Bcl-2
family generally share homology within four domains (Mcl-1 is an exception),
while their pro-apoptotic relatives are either multi-domain proteins, containing
BH1-BH3, or BH3-only proteins, possessing only one of the four BH domains.
Many of the Bcl-2 proteins also contain a transmembrane (TM) domain.
Mechanisms of Bax/Bak­mediated MMP Cells that are doubly deficient for Bax and Bak are resistant to death
stimuli that activate the intrinsic pathway (Wei et al., 2001). Hence,
Bax and Bak seem to be crucial for MMP and the subsequent release
of apoptogenic factors from the inter-membrane space of
mitochondria. It is commonly thought that Bax and Bak themselves
mediate MMP by formation of pores in the outer mitochondrial
membrane (Jürgensmeier et al., 1998; Basanez et al., 1999; Kuwana et
al., 2002). While Bak is constitutively located in the mitochondrial
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INTRODUCTION
membrane, Bax is mainly a cytosolic protein in its inactive form and
migrates to mitochondria when the cell receives a death signal (Wolter
et al., 1997; Goping et al., 1998). The activity of Bax and Bak is
regulated by the pro-survival multidomain Bcl-2 proteins and the prodeath BH3-only proteins (Adams and Cory, 2007; Danial, 2007; Leber
et al., 2007; Youle and Strasser, 2008). The BH3-only proteins are
cellular sensors that are activated in response to various stress signals
and initiate MMP by heterotypic interactions with pro- and/or antiapoptotic multidomain Bcl-2 proteins [reviewed in (Puthalakath and
Strasser, 2002; Strasser, 2005; Willis and Adams, 2005)]. Some of the
activated BH3-only proteins, such as Bad and Noxa, bind only a
subset of the anti-apoptotic Bcl-2 proteins (see Figure 5). By contrast,
Bid, Bim and Puma bind all their pro-survival relatives, and possibly
also the pro-apoptotic multidomain proteins Bax and Bak, making
them the most potent triggers of MMP. The precise mechanism of Bax
and Bak activation is still largely unknown and the role of BH3-only
proteins in this process is highly controversial.
Figure 5: BH3-only protein binding specificity.
The direct activation model According to the “direct activation” theory (Figure 6) there are two
categories of BH3-only proteins; (1) activators, which bind both proand anti-apoptotic multidomain partners, and (2) sensitizers that
interact only with the anti-apoptotic Bcl-2 members [reviewed in
(Adams and Cory, 2007; Danial, 2007; Leber et al., 2007)]. Bid, Bim,
and possibly also Puma, are activators that via transient interaction
with Bax and Bak initiate their intra-membraneous oligomerization,
resulting in pore formation. Sensitizers, on the other hand, release
activators from their inhibitory interaction with anti-apoptotic Bcl-2
proteins, whose main function, according to this model, is
sequestration of BH3-only proteins.
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INTRODUCTION
Figure 6: The “direct activation” model. See text for details.
The indirect activation model In the “indirect activation” model (Figure 7), the primary function of
anti-apoptotic Bcl-2 proteins is inhibition of Bax and Bak [reviewed in
(Adams and Cory, 2007; Danial, 2007; Leber et al., 2007)]. To initiate
apoptosis, BH3-only proteins displace Bax and Bak from these prosurvival proteins. This is mediated by insertion of their BH3 domain
into a hydrophobic cleft on their anti-apoptotic relatives. After release,
Bax and Bak oligomerize and permeabilize the membrane. The
prominent feature of this model is that Bax and Bak are constitutively
active and must be continuously bound and inhibited by the antiapoptotic Bcl-2 family members for cells to survive.
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INTRODUCTION
Figure 7: The “indirect activation” model. See text for details.
The embedded together model There is increasing evidence that not merely protein-protein
interactions, but also protein-lipid interactions influence the ability of
Bax and Bak to oligomerize and form pores in the outer mitochondrial
membrane. Notably, it was reported that Bax-mediated
permeabilization of synthetic liposomes requires presence of
cardiolipin, a mitochondrial membrane phospholipid (Kuwana et al.,
2002). Furthermore, proteins other than those of the Bcl-2 family have
been shown to regulate the function of Bax and Bak at the surface of
mitochondria (Cuddeback et al., 2001; Tan et al., 2001; Zhang et al.,
2005; Ott et al., 2007).
A more recently proposed model, termed “embedded together”
(Figure 8), incorporates some aspects of both the direct and indirect
activation models, and proposes that Bax and Bak go through
multiple, regulated conformational changes before assuming a final
conformation necessary for membrane permeabilization (Leber et al.,
2007). The transition between conformations depends on interaction
not only with select BH3-only molecules, but also lipids and other
proteins. This model proposes that the inactive cytoplasmic form of
Bax is in equilibrium with a form that binds with low affinity to the
surface of membranes. Upon association with the membrane, Bax
undergoes a conformational change resulting in exposure of an
otherwise hidden N-terminal epitope. This is suggested to facilitate
interaction of Bax with activated BH3-only proteins. In contrast to the
“direct activation” model, the “embedded together” model postulates
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INTRODUCTION
that BH3-only proteins activate both pro- and anti-apoptotic
multidomain Bcl-2 family members. Activation leads to
conformational changes and insertion of helices 5 and 6 into the
membrane. The activated anti-apoptotic Bcl-2 proteins, which are
themselves unable to oligomerize, prevent Bax oligomerization by
binding to active Bax monomers. Upon sustained activation of
BH3-only proteins the number of inserted Bax molecules may be
sufficient to overcome this inhibition. In analogy with the “direct
activation” theory, this model also includes the possibility that antiapoptotic Bcl-2 proteins, before insertion of helices 5 and 6, prevent
apoptosis by sequestration of BH3-only molecules.
Figure 8: The “embedded together” model. For simplicity, the sequential
conformational change of Bax is not illustrated. See text for details.
Modulation of the permeability transition pore Bcl-2 family members may also govern MMP by regulating the
function of resident mitochondrial proteins (Figure 9). Apoptogenic
factors can be released from the inter-membrane space of
mitochondria as a result of opening of the so-called permeability
transition (PT) pore [reviewed in (Grimm and Brdiczka, 2007;
Schwarz et al., 2007)]. It is currently believed that the major
components of the PT pore are the adenine nucleotide translocator
25
INTRODUCTION
(ANT), located in the inner mitochondrial membrane, the voltagedependent anion channel (VDAC), resident in the outer mitochondrial
membrane, and cyclophilin D (cyp D), located in the matrix. Opening
of the PT pore leads to influx of solutes and water into the matrix.
This results in expansion of the extensively folded inner membrane
and, eventually, rupture of the outer mitochondrial membrane.
Opening of the PT pore can be stimulated by the ANT agonist
atractyloside, while bongkreic acid and cyclosporine A act as
inhibitors of this process and, thus, prevent cell death. Bax and Bcl-2
have been shown to interact with ANT and stimulate versus suppress
opening of the PT pore in response to atractyloside (Marzo et al.,
1998a; Marzo et al., 1998b). Association of Bax and Bak with VDAC
has also been reported (Narita et al., 1998). In that study, recombinant
Bax and Bak were found to trigger MMP in isolated mitochondria that
could be prevented by bongkreic acid and cyclosporine A, suggesting
involvement of the PT pore.
Figure 9: Bax may promote cytochrome c release via opening of the PT pore.
Activation of BH3­only proteins In response to a variety of cellular stresses, BH3-only proteins are
activated either by transcriptional upregulation or by various
posttranslational modifications [reviewed in (Puthalakath and Strasser,
2002; Willis and Adams, 2005)].
For example, Bad is activated by loss of phosphorylation following
starvation (Zha et al., 1996). In cells stimulated by growth factors,
Bad is phosphorylated and sequestered by 14-3-3 proteins. Upon
26
INTRODUCTION
growth factor withdrawal, there is accumulation of dephosphorylated
Bad which is free to interact with anti-apoptotic Bcl-2 family
members. Bim and Bmf, on the other hand, are regulated by
sequestration to cytoskeletal structures (Puthalakath et al., 1999;
Puthalakath et al., 2001). In resting cells, Bim is associated with the
dynein motor complex, while Bmf is sequestered to actin-myosin
motor complexes. Upon certain stress stimuli Bim and Bmf are
released, enabling their interaction with multidomain Bcl-2 proteins.
Bim may also be activated by transcriptional upregulation or
dephosphorylation (Dijkers et al., 2000; Puthalakath et al., 2007).
Dephosphorylation prevents ubiquitination and proteasomal
degradation, and thus, results in increased levels of Bim. Puma and
Noxa are both transcriptionally induced by the tumor suppressor p53
in response to DNA damage (Oda et al., 2000; Nakano and Vousden,
2001; Yu et al., 2001).
Bid is the only BH3-only protein known to be activated by proteolytic
cleavage [reviewed in (Yin, 2006)]. The resulting active C-terminal
fragment is often referred to as truncated Bid (tBid). Initially, Bid was
found to be cleaved by caspase-8 following death receptor activation,
and was, thus, thought to be specific for the death receptor pathway
(Li et al., 1998; Luo et al., 1998). However, more recent studies have
demonstrated that Bid can be cleaved by caspase-3 (Bossy-Wetzel and
Green, 1999), granzyme B (Barry et al., 2000; Sutton et al., 2000),
calpains (Chen et al., 2001; Mandic et al., 2002) , and lysosomal
cathepsins (Stoka et al., 2001; Cirman et al., 2004; Heinrich et al.,
2004) as well, suggesting that Bid is a sensor of protease-mediated
death signals. Proteolysis most often occur in the unstructured flexible
loop connecting helices α2 and α3, which seems to function as a “baitloop” for active proteases (see Figure 10). The BH3 domain, which is
essential for interaction with the core Bcl-2 proteins, is located in the
α3 helix. Cleavage in the flexible loop is believed to result in a
conformational change that relieve the BH3 domain from inhibitory
interaction with another BH3-like domain localized within the α2
helix (McDonnell et al., 1999; Tan et al., 1999).
Interestingly, Bid is subject to a second regulatory modification,
N-myristoylation, at a site exposed following activation by caspase-8
(Zha et al., 2000). This event appears to facilitate the targeting of
active Bid to mitochondria, and potentiates Bid-induced cytochrome c
release and cell death.
27
INTRODUCTION
Figure 10: The BH3-only protein Bid is activated via proteolytic processing by a
number of cellular proteases.
Cytosolic Bax inhibitors It has been suggested that the monomeric form of Bax is bound to
cytosolic factors that prevent its activation. One such example is
Ku70, a protein otherwise involved in DNA repair (Sawada et al.,
2003). Overexpression of Ku70 prevents Bax-mediated cytochrome c
release, while its downregulation has the opposite effect. Binding of
Ku70 to Bax seems to be associated with reduced translocation to
mitochondria. In analogy, it was demonstrated that the small peptide
Humanin suppresses Bax-induced MMP by sequestration of Bax in
the cytosol (Guo et al., 2003). Finally, 14-3-3 proteins, which are
known to keep the BH3-only protein Bad inactive in the cytosol,
might regulate the activity of Bax as well. Several of the 14-3-3
isoforms were found to bind Bax and prevent its activation (Samuel et
al., 2001; Nomura et al., 2003). Interestingly, upon induction of
apoptosis, 14-3-3θ was cleaved by caspases, releasing Bax from the
inhibitory interaction (Nomura et al., 2003).
PROTEOLYTIC REGULATION OF APOPTOSIS Signaling pathways use proteolysis, phosphorylation, acetylation,
ubiquitylation or glycosylation to alter protein properties. Those that
require proteolysis are irreversible, as cells are unable to re-ligate cut
peptide bonds. Apoptosis is such a pathway, and a number of
proteases are involved in the signaling cascade. Caspases are generally
considered the core components of the apoptosis machinery; however,
28
INTRODUCTION
there is increasing evidence that other proteases, such as calpains,
granzyme B, and lysosomal cathepsins, play significant roles as well.
Caspases Caspases are expressed in virtually all cells as inactive pro-caspases
[reviewed in (Kumar, 2007; Rupinder et al., 2007)]. When the cell
receives an apoptotic signal, caspases are activated in a cascade-like
fashion and cleave a number of cellular proteins. In this way, caspases
drive forward the biochemical events that culminate in death and
dismantling of the cell.
Caspases are cysteinyl aspartate proteinases, i.e. cysteine proteases
that cleave their substrates following an Asp residue. The caspase
family consists of 14 members divided into two functional groups.
Caspases-2, -3, -6, -7, -8, -9, and -10 are apoptosis-related enzymes,
while caspases-1, -4, -5, -11, -12, -13, and -14 are involved in
cytokine processing. All pro-caspases contain a highly homologous
protease domain as well as a pro-domain of variable length. Caspases
involved in apoptosis can be sub-divided into initiator and effector
caspases based on the length of their pro-domain (Figure 11). Initiator
caspases (caspases-2, -8, -9, and -10) bear long pro-domains essential
for their activation and “initiate” apoptosis by activating the
downstream effector caspases (caspases-3, -6, and -7). Effector
caspases, on the other hand, have short pro-domains and ultimately
execute apoptotic cell death.
Figure 11: Structure of initiator and effector caspases.
29
INTRODUCTION
Caspase activation Initiator and effector caspases are activated by distinct mechanisms
[reviewed in (Bao and Shi, 2007; Kumar, 2007; Riedl and Salvesen,
2007; Rupinder et al., 2007)]. Effector caspases are normally cleaved
and activated by active initiator caspases. They can, however, under
certain conditions be activated by other proteases. Activation of
effector caspases is a two-step process in which proteolysis of the
inter-domain linker, separating the two catalytic subunits, is followed
by proteolytic removal of the pro-domain (Figure 12). As this process
is mediated by cleavage at Asp residues, mature caspases may cleave
and activate their precursors as well as other caspases. The mature
caspase is a hetero-tetramer, composed of two hetero-dimers derived
from two precursor molecules.
Figure 12: Effector caspase activation. Proteolytic separation of the large (p20)
and small (p10) catalytic subunits is followed by removal of the pro-peptide. The
mature enzyme is a hetero-tetramer composed of two large and two small
catalytic units.
Activation of initiator caspases occurs via dimerization rather than
proteolytic cleavage. Cleavage is, however, often observed and is
thought to stabilize the active dimer. Initiator caspases are activated in
death signaling complexes, to which they are recruited by interaction
with adapter molecules. This interaction is dependent on conserved
motifs within their long pro-domain, more specifically the caspase
activation and recruitment domain (CARD) of caspases-9 and -2 and
the death effector domain (DED) of caspases-8 and -10. The same
domains are present in the adaptor molecules and binding is mediated
by their homotypic interaction. Activation of caspase-9 is mediated by
the apoptosome complex involving Apaf-1, cytochrome c and the
cofactor dATP/ATP. Formation of this complex is initiated by release
of cytochrome c from mitochondria. Binding of Apaf-1 to
30
INTRODUCTION
cytochrome c results in oligomerization and structural rearrangements
leading to exposure of the CARD domain. Finally, procaspase-9 is
recruited to the complex via interaction with the Apaf-1 CARD
domain. Caspases-8 and -10 are both activated in response to death
receptor activation, after formation of the DISC. Death receptor
activation is associated with trimerization of the receptor. This leads
to clustering of the intra-cellular death domains which, in turn, enables
the recruitment of DD-containing adaptor molecules via homotypic
interactions. Fas associated death domain (FADD) is such an adaptor
molecule. FADD also contains a DED domain which mediates
interaction with procaspases-8 and -10. Another adaptor protein, TNF
receptor associated death domain (TRADD), is involved in TNF-α
mediated death signaling. TRADD, which contains two death
domains, binds to the receptor with one death domain and recruits
FADD to the DISC with the other. Activation of caspase-2 is less well
characterized, but is also thought to occur via dimerization, mediated
by a protein complex known as the PIDDosome (Festjens et al., 2006;
Bao and Shi, 2007).
Cellular caspase substrates Several hundred cellular caspase substrates have been identified thus
far, including mediators and regulators of apoptosis, structural
proteins, and proteins involved in DNA repair [reviewed in (Fischer et
al., 2003; Jin and El-Deiry, 2005; Kumar, 2007; Rupinder et al., 2007;
Timmer and Salvesen, 2007)]. The most obvious example of the first
category is effector caspases themselves, which are activated through
cleavage by initiator caspases. The Bcl-2 family protein Bid (Li et al.,
1998; Luo et al., 1998), and inhibitor of caspase-activated DNAse
(ICAD), also called DNA fragmentation factor 45 kDa subunit
(DFF45) (Liu et al., 1997; Enari et al., 1998) are other examples of
substrates involved in the initiation and execution of apoptosis. In
resting cells, ICAD is bound to caspase-activated DNAse (CAD) and
thereby keeps it inactive. This inhibitory interaction is disrupted by
cleavage of ICAD, leading to CAD-mediated inter-nucleosomal DNA
degradation, a characteristic feature of apoptosis.
Caspases are responsible for many of the morphological changes seen
during apoptosis. Cleavage of the structural proteins fodrin and
gelsolin results in disruption of the actin filament network with loss of
cell shape and detachment from the matrix as a consequence.
31
INTRODUCTION
Cleavage of nuclear lamins leading to nuclear shrinkage and budding
is another example (Jin and El-Deiry, 2005).
DNA-repair, which is an energy-demanding process, is shut down
during apoptosis in order to avoid depletion of cellular energy stores
(Fischer et al., 2003). This is essential, as apoptotic cell death requires
energy in order to be executed. In the absence of a sufficient amount
of ATP, cell death shifts toward necrosis. Caspase substrates involved
in DNA repair include Poly(ADP-ribose) polymerase (PARP), which
was one of the first cellular caspase substrates to be identified
(Kaufmann et al., 1993; Lazebnik et al., 1994; Nicholson et al., 1995;
Tewari et al., 1995). PARP is activated in response to DNA breaks
and facilitates repair by catalyzing the attachment of ADP-ribose
polymers to multiple nuclear factors. Caspase-mediated cleavage
results in inactivation of PARP with suppression of DNA repair as a
consequence.
Cathepsins Lysosomal destabilization Lysosomes have long been referred to as ‘suicide bags’ as they
contain a wide range of potentially harmful hydrolytic enzymes that
were originally thought to inevitably trigger necrotic cell death when
released to the cytosol. This assumption was proven wrong by studies
showing that a moderate lysosomal destabilization leads to apoptosis,
while more pronounced lysosomal rupture results in necrosis (Brunk
and Svensson, 1999; Li et al., 2000; Antunes et al., 2001; Kågedal et
al., 2001b). In recent years it has become evident that
permeabilization of the lysosomal membrane occurs in cell death
induced by many different stimuli, and is important for execution of
cell death [reviewed in (Leist and Jäättelä, 2001; Jäättelä, 2002;
Guicciardi et al., 2004; Chwieralski et al., 2006)].
So far, the cause of lysosomal membrane permeabilization (LMP) is
not completely understood. Several mechanisms have, however, been
proposed that could contribute, probably in a stimulus- and cell-typedependent fashion, to permeabilization of the lysosomal membrane
(Guicciardi et al., 2004; Chwieralski et al., 2006). Since the question
to how the lysosomal membrane is permeabilized during apoptosis is
addressed in paper II, these mechanisms will be described in the
“discussion” section.
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INTRODUCTION
Cathepsins ­ regulators of apoptotic cell death As a consequence of LMP, cathepsins are released to the cytosol, and
there is compelling evidence that at least cathepsins B, D and L are
components of the cellular suicide machinery (Chwieralski et al.,
2006). It has been shown that cathepsins B, D and L, under certain
circumstances, may have an anti-apoptotic function (Castino et al.,
2002; Zheng et al., 2004; Gondi et al., 2006; Zajc et al., 2006). These
reports are, however, outnumbered by those demonstrating their role
as positive mediators of apoptosis.
The cysteine protease cathepsin B is essential in several models of
apoptosis, and some of the earliest findings emphasized its importance
in toxic bile salt-induced hepatocyte apoptosis (Roberts et al., 1997;
Jones et al., 1998; Faubion et al., 1999), PC12 cell apoptosis after
serum deprivation (Shibata et al., 1998), and TNF-α-induced
apoptosis of primary hepatocytes and tumor cells (Guicciardi et al.,
2000; Foghsgaard et al., 2001; Guicciardi et al., 2001). There are
fewer reports implicating cathepsin L as a death factor. It has,
however, been suggested that cathepsin L is involved in, for instance,
ultraviolet- (UV)-induced keratinocyte cell death (Tobin et al., 2002;
Welss et al., 2003) and P39 cell death following etoposide treatment
(Hishita et al., 2001).
The first evidence pointing to a role for cathepsin D in apoptotic cell
death was presented by Adi Kimchi and co-workers in 1996 (Deiss et
al., 1996). By random inactivation of genes via transfections with
antisense cDNA expression libraries, cathepsin D was identified as a
pro-apoptotic factor. Downregulation of cathepsin D, using antisense
RNA, as well as chemical inhibition employing pepstatin A, was
found to protect from cell death induced by interferon-γ, Fas, and
TNF-α. Conversely, overexpression of cathepsin D induced cell death
without any external stimuli. Two years later, cathepsin D was
identified as a protein up-regulated during p53-dependent apoptosis,
and two p53 DNA-binding sites were found located in the cathepsin
D-promoter (Wu et al., 1998). The involvement of cathepsin D in p53dependent apoptosis was confirmed by the observation that cathepsin
D-/- cells were more resistant to killing by adriamycin and etoposide as
compared to their wild type (wt) equivalents. The subcellular location
of cathepsin D during apoptosis was not considered in these
publications. This question was first addressed by my two supervisors,
Karin Öllinger and Karin Roberg, who developed a technique for
33
INTRODUCTION
immuno cytochemical detection of cathepsin D by electron
microscopy (Roberg and Öllinger, 1998b). Using this method,
cathepsin D was found to be released from lysosomes to the cytosol
during oxidative stress-induced apoptosis triggered by the redoxcycling drug naphthazarin (Roberg and Öllinger, 1998a). Release of
cathepsin D was independent of caspases and inhibition of cathepsin
D activity abrogated caspase activation and reduced cell death
(Roberg et al., 1999; Öllinger, 2000; Kågedal et al., 2001a; Roberg,
2001). Based on these findings, it was hypothesized that the mere
presence of cathepsin D in the cytosol is sufficient to induce
apoptosis, and by microinjection of cathepsin D into the cytosol of
human fibroblasts, this hypothesis was proven correct (Roberg et al.,
2002). A series of observations from our laboratory clarified the
temporal relationship between lysosomal release of cathepsin D and
mitochondrial events: (i) oxidative stress-induced release of
cytochrome c and decrease of the mitochondrial transmembrane
potential were both prevented by cathepsin D inhibition (Roberg et al.,
1999), (ii) release of cathepsin D from lysosomes was observed at a
slightly earlier time point than mitochondrial release of cytochrome c
(Roberg, 2001), and (iii) cytochrome c was released upon
microinjection of cathepsin D (Roberg et al., 2002). Collectively,
these data place cathepsin D upstream mitochondria in the signaling
cascade leading to apoptotic cell death.
A number of potential cytosolic cathepsin substrates, whose cleavage
in one way or another promote apoptosis, has emerged during the last
decade. The mechanisms by which cathepsins transmit the apoptotic
signal will be discussed later in this thesis.
CANCER Tumorigenesis is a multi-step process in which a series of genetic
alterations result in an imbalance between cell division and cell death
(Hanahan and Weinberg, 2000). Self-sufficiency in growth signals,
insensitivity to anti-growth signals, limitless replicative potential,
sustained angiogenesis, tissue invasion, and metastasis are all
hallmarks of cancer.
APOPTOSIS IN CANCER DEVELOPMENT Acquired defects in signaling pathways leading to programmed cell
death is another characteristic of cancer cells (Hanahan and Weinberg,
34
INTRODUCTION
2000). One of the most common strategies for evasion of apoptosis is
inactivation of the pro-apoptotic protein p53. Mutations in the p53
tumor suppressor gene, leading to loss of pro-apoptotic function of the
p53 protein and resistance towards DNA damage-induced cell death,
are found in up to 50% of all human cancers (Beroud and Soussi,
1998). Apoptosis can be avoided also by overexpression of antiapoptotic factors. Upregulation of the bcl-2 oncogene, a common
feature in follicular lymphoma, is one example (Vaux et al., 1988).
CATHEPSINS IN CANCER In addition to their role in apoptosis signaling, cathepsins B, D, and L
have been implicated in cancer progression and metastasis [reviewed
in (Liaudet-Coopman et al., 2006; Mohamed and Sloane, 2006;
Gocheva and Joyce, 2007; Vasiljeva and Turk, 2008)]. Increased
expression of cathepsins has been detected in many human tumors,
including brain, breast, lung, gastrointestinal, prostate, and melanoma.
Upregulation of certain cathepsins has been shown to have prognostic
value for patients with several types of cancer. For example,
overexpression of cathepsin D correlates with poor prognosis in breast
cancer (Rochefort, 1992; Westley and May, 1999). Similarly,
increased expression of cathepsin B was associated with shorter
survival rates in lung, breast, ovarian, brain, melanoma and head and
neck cancer [reviewed in (Berdowska, 2004; Jedeszko and Sloane,
2004)]. Cathepsins are also frequently secreted from cancer cells, and
have been assigned specific extra-cellular functions that promote
cancer growth and invasion.
Cysteine cathepsins facilitate metastasis by degradation of
components of the extra-cellular matrix, including laminin,
fibronectin, and type IV collagen (Mohamed and Sloane, 2006;
Gocheva and Joyce, 2007; Vasiljeva and Turk, 2008). They can also
activate other proteases, such as metalloproteases and urokinase-type
plasminogen activator, which are involved in remodeling of the extracellular matrix. Cathepsin-mediated cleavage of E-cadherin, which
mediates cell-cell adhesion, is another mechanism by which invasion
could be achieved.
Cathepsin D stimulates cancer cell proliferation and tumor
angiogenesis, and may do so either by acting as a protease or as a
binding protein (Liaudet-Coopman et al., 2006). It has been suggested
that catalytically active cathepsin D is involved in activation of growth
35
INTRODUCTION
factors as well as inactivation of growth inhibitors (Briozzo et al.,
1991; Liaudet et al., 1995). Interestingly, it was demonstrated that
cathepsin D may stimulate cancer cell growth independent of its
catalytic activity, by binding of the pro-peptide to an as yet
unidentified cell surface receptor (Fusek and Vetvicka, 1994). The
latter is supported by a study in which a mutated form of cathepsin D,
devoid of catalytic activity, was found to be mitogenic (Glondu et al.,
2001).
36
AIMS
AIMS The general objective of the studies in this thesis was to investigate
the role of lysosomal membrane permeabilization and lysosomal
proteases, cathepsins, in apoptosis signaling.
SPECIFIC AIMS OF PAPERS I, II, AND III • To evaluate the participation of cathepsins in the signaling cascade
in untransformed cells (human fibroblasts).
• To study the temporal relationship between cathepsin release and
central apoptotic events, such as release of cytochrome c from
mitochondria and caspase activation, during staurosporine-induced
cell death.
• To explore the mechanism(s) by which cathepsins escape the
lysosomal compartment.
• To identify cytosolic cathepsin substrates, whose processing could
promote propagation of the apoptotic signal.
• To examine the cytosolic pH of dying cells, and its effect on
cathepsin activity.
SPECIFIC AIMS OF PAPER IV • To study the involvement of lysosomes and cathepsins in cancer
cell death (oral squamous cell carcinoma cells).
• To evaluate if cathepsins secreted from cancer cells can modulate
death receptor signaling.
37
38
MATERIALS AND METHODS
MATERIALS AND METHODS CELLS HUMAN FIBROBLASTS, AG­1518 Papers I, II, and III are based mainly on studies using the
untransformed, apparently healthy, human fibroblast cell line AG1518, which is derived from a foreskin explant. These cells were
chosen due to their untransformed phenotype. As compared to many
cancer cell lines, these cells are slow-growing, and can be cultivated
only for a limited number of passages. For experiments presented in
this thesis, cells in passages 12-24 were used.
BID ­/­ MOUSE EMBRYONIC FIBROBLASTS In order to confirm results obtained in the human fibroblast model,
mouse embryonic fibroblasts (MEFs), kindly provided by Stanley
Korsmeyer (Dana-Farber Cancer Institute, Harvard Medical School,
Boston, MA, USA), were used in paper III. These cells were derived
from wt and Bid-/- mice, generated as described by Yin and coworkers
(Yin et al., 1999) and transformed by introduction of the simian virus
40 (SV40) T antigen. While primary cells can be passaged only a
limited number of times, MEFs expressing large T antigen are
immortal and can be propagated in culture indefinitely (Ahuja et al.,
2005). This is due to inhibitory interactions of large T antigen with the
p53 and Rb proteins.
ORAL SQUAMOUS CELL CARCINOMA CELLS Involvement of cathepsins in cancer cell apoptosis was studied in
paper IV. For this purpose, two squamous cell carcinoma (SCC) cell
lines (UT-SCC-20A and UT-SCC-24A), derived from primary oral
tumors, were used (passages 15-25). These cells were found suitable
for this study as cathepsins B, D, and L are frequently overexpressed
in SCC of the head and neck, and as their role in SCC apoptosis is
largely unknown.
39
MATERIALS AND METHODS
INDUCERS OF APOPTOSIS STAUROSPORINE Staurosporine (STS) was used in papers I, II (0.1 µM), and III (0.2
µM) to induce apoptosis in human fibroblasts. The difference in
concentrations used reflects a lower potency of the batch of STS used
in paper III. The concentration was increased to give an apoptotic
frequency comparable to that seen in previous studies.
STS is a broad spectrum protein kinase inhibitor, which has proven to
be a very potent death-inducer. It blocks the ATP-binding domain of
kinases with loss of catalytic activity as a consequence (Prade et al.,
1997). STS has been evaluated as an anti-cancer drug, but proven
useless as such, due to its low specificity, and the involvement of
protein kinases in multiple cellular functions.
MSDH MSDH (O-methyl serine dodecylamide hydrochloride) was used in
papers III and IV in order to study apoptosis triggered by lysosomal
destabilization. MSDH is a lysosomotropic detergent, i.e. an amine
that bears a long hydrophobic chain and becomes concentrated inside
lysosomes (Dubowchik et al., 1995). Upon protonation within the
lysosome it acquires detergent properties, causing disruption of the
lysosomal membrane with release of lysosomal proteases and ensuing
apoptosis (Li et al., 2000; Terman et al., 2002; Zhao et al., 2003).
NAPHTHAZARIN Apoptosis evoked by the redox-cycling drug naphthazarin (5 µM), a
structural analogue to the anti-cancer drug doxorubicin, was studied in
paper IV. The cytotoxic effect of naphthazarin is mainly due to
generation of reactive oxygen species (Öllinger and Brunmark, 1991).
The toxicity may, however, also be mediated by formation of adducts
with various cellular compounds.
FAS ANTIBODY In paper IV, cell death was induced also by engagement of the death
receptor pathway using agonistic anti-Fas antibodies. This pathway is
normally activated by trimerization of the receptor following binding
of FasL; however, some antibodies have been found to have the same
effect. Fas death receptor signaling is associated not only with intra40
MATERIALS AND METHODS
cellular pro-death, but also pro-survival signals. The latter depend on
protein synthesis, thus, the death-inducing effect of the Fas antibody
was enhanced by co-treatment with the protein synthesis inhibitor
cyclohexamide (1 µg/ml).
PROTEASE INHIBITORS CASPASE INHIBITORS Several synthetic peptide caspase inhibitors have been developed
based on the peptide sequence preceding the cleavage site of known
cellular caspase substrates (Callus and Vaux, 2007). One example is
the potent inhibitor of caspases-3 and -7, Ac-DEVD-CHO. The
tetrapeptide sequence DEVD, which perfectly matches the specificity
of caspases-3 and -7, is derived from PARP, one of the first identified
caspase substrates. The tetrapeptides are conjugated to different
chemical groups that improve cell permeability, stability and efficacy.
Peptides linked to aldehydes, nitriles or ketones are reversible
inhibitors that compete with the substrate for enzyme binding.
Peptides linked to a leaving group, such as fluoromethyl ketone
(FMK), chemically alter the enzyme and are, thus, irreversible
inhibitors. The synthetic caspase-3 and -7 inhibitor Ac-DEVD-CHO
(50µM) and the caspase-8 inhibitor Ac-IETD-CHO (50µM) were used
in paper I. These are both reversible inhibitors due to the aldehyde
group, while the N-terminal acetyl group serves to facilitate their entry
into the cell.
CATHEPSIN INHIBITORS The activity of cysteine cathepsins B and L was in paper I and IV
suppressed using the chemical inhibitor z-FA-FMK (100 µM). In
analogy with the caspase inhibitors described above, this compound
acts as an irreversible inhibitor due to its FMK group.
Pepstatin A, which in papers I, III and IV was used to block the
activity of cathepsin D, is a pentapeptide that is produced and secreted
by Streptomyces species. Pepstatin A contains the rare amino acid
Statin, which reacts with the catalytic-site residues of aspartic
proteases, such as renin, pepsin, cathepsin E and cathepsin D.
However, as renin and pepsin are extra-cellular proteases and
cathepsin E is expressed mainly in cells of the immune system,
pepstatin A is assumed to be a specific inhibitor of cathepsin D in our
41
MATERIALS AND METHODS
experimental setup. Pepstatin A, which is not cell permeable, is
believed to enter cells via endocytosis. Due to the inefficient transport
across the cell membrane, the relatively high concentration of 100 µM
was administered to cell cultures.
DETECTION OF APOPTOSIS MORPHOLOGICAL EVALUATION Cells were examined by light microscopy following staining with
Giemsa. Cells with reduced cell volume and pycnotic nuclei were
considered apoptotic. In paper I, cell morphology was also assessed by
electron microscopy. Using this method, it was confirmed that cell
death was accompanied by chromatin condensation, nuclear
fragmentation and formation of apoptotic bodies, and thus, could be
classified as apoptosis. Nuclear morphology was, in papers II and IV,
examined by fluorescence microscopy in DAPI-stained cells. Necrotic
cell death was assessed by the trypan blue exclusion test, which is
based on the inability of this dye to pass an intact plasma membrane.
Hence, only necrotic cells, which have lost their membrane integrity,
are stained.
ASSESSMENT OF CASPASE ACTIVATION In analogy with the caspase inhibitors described above, synthetic
caspase substrates have been engineered based on the structure of
known cellular substrates (Callus and Vaux, 2007). The tetrapeptide
sequence has, in this case, been linked to a chemical group that, after
being cleaved off by the caspase of interest, can be detected and
quantified. The caspase substrates used in the papers of this thesis are
conjugated to the fluorescent groups 7-amino-4- methyl coumarin
(AMC) or 7-amino-4 trifluoromethyl coumarin (AFC). However,
peptides can also be linked to groups that allow colorimetric detection.
Substrates are designed to match the substrate specificity of certain
caspases, but some cross-reactivity can be expected. Activation of
caspase-3 (and -7) was assessed using the substrate Ac-DEVD-AMC,
while Ac-IETD-AFC and Ac-LEHD-AFC were employed for
detection of caspase-8 and -9 activation, respectively. Caspase activity
was analyzed in cell lysates and gave a relative measure of caspase
activation when correlated to total protein and compared to the
activity of control cells.
42
MATERIALS AND METHODS
As caspase activation is most often associated with cleavage of the
protein, activation can also be assessed by gel electrophoresis
followed by immuno blot detection of active caspases. This method
was used in paper I as a complement to substrate-based activity
measurements of caspases-9 and -3. Procaspase-3 (32 kDa) is
activated by proteolytic cleavage, resulting in two fragments of 11 and
17 kDa. Similarly, activation of procaspase-9 (46 kDa) is associated
with generation of 10-, 17-, and 37-kDa fragments. Activation was
determined after visualization of the active 37- and 17-kDa fragments
of caspase-9 and -3, respectively. The advantage of this method is that
cross-reactivity can be ruled out. On the other hand, false negative
results could be obtained since proteolysis is not always required for
activation to occur.
ASSESSMENT OF CATHEPSIN RELEASE Release of cathepsins from lysosomes to the cytosol was studied by
immuno fluorescence microscopy in cells immuno labeled for
cathepsins B or D. The staining is punctate in control cells where
cathepsins are harbored within the lysosomes. Release is detected as a
diffuse cytosolic staining.
Cathepsin release was detected by western blot analysis of isolated
cytosol as well, and gave similar results. The advantage of this method
is that it gives an objective measure of the release, and that the relative
amount of cathepsins released can be determined. The extractionprocedure is based on the cholesterol-solubilizing agent digitonin,
which at the optimum concentration permeabilizes the cholesterol-rich
plasma membrane, but leaves the cholesterol-poor lysosomal
membrane intact. Cathepsins are, thus, detected only in cytosol
extracted from cells which display loss of lysosomal membrane
integrity. For optimization, release of cytosol was quantified by
measuring the activity of the cytosolic enzyme lactate dehydrogenase
(LDH), while the activity of N-acetyl-β-glucosaminidase (NAG) was
determined in order to reflect release of lysosomal constituents.
Control and treated cells were equally sensitive to digitonin when
analyzing release of LDH, suggesting that cathepsins are detected in
the cytosol of treated cells not merely due to an increased sensitivity
to digitonin.
Release of cathepsins from isolated lysosomes was assessed by
western blot analysis of cathepsins B and D, and by measurement of
43
MATERIALS AND METHODS
cathepsin B and L activity using the fluorescent substrate z-FA-AMC.
In addition, release of NAG was studied by activity analysis
employing the substrate 4-methyl-umbelliferyl-2-acetoamido-2deoxy-β-D-glucopyranoside, which upon cleavage releases the
fluorescent product 4-methylumbelliferone.
MICROINJECTION It is possible to inject substances into the cytosol of single cells if
using an extremely thin needle. The microinjection technique was
established in our laboratory in 2001 by one of my supervisors, Karin
Roberg. It was first used to show that presence of cathepsin D in the
cytosol is sufficient to induce apoptosis (Roberg et al., 2002). In paper
III, microinjection was employed in order to verify that cytosolic
cathepsin D triggers translocation of Bax to mitochondria. The method
is described only briefly in this paper, thus, more detailed information
about the procedure is given here.
Microinjection was performed on the stage of a Zeiss Axiovert (Zeiss,
Gena, Germany) inverted microscope, using a pressure injector from
Eppendorf (model 5246; Eppendorf, Hamburg, Germany) and an
Injectman micromanipulator (Eppendorf). Microinjection needles
(Femtotips II, Eppendorf), which had an inner diameter of less than
0.5 µm, were filled with the injectate using microloaders (Eppendorf).
Cells were injected (100 hPa, 1.5 s) with Dulbecco’s phosphatebuffered saline (PBS; pH 5.5) with or without cathepsin D (0.25
mg/ml; C8696, Sigma, St Louis, MO, USA). To enable identification
of injected cells the injectate contained also 0.25 mg/ml Alexa Fluor
546-conjugated dextran (Mw = 10 kDa; Molecular Probes, Eugene,
OR, USA). In each cell culture dish, approximately 100 cells were
injected, and the specimens were subjected to immuno cytochemistry
4 h later.
SUBCELLULAR LOCALIZATION OF BAX The intra-cellular localization of Bax was assessed by confocal
microscopy of immuno labeled cells (papers II and III). Colocalization
of Bax with lysosomes and mitochondria was evaluated by
concomitant immuno staining of the lysosomal membrane protein
LAMP-2 or labeling of mitochondria using Mito Tracker® Red,
respectively. No colocalization of lysosomes and mitochondria was
detected; however, a certain overlap between lysosomes and
44
MATERIALS AND METHODS
mitochondria cannot be completely excluded with this method.
Therefore, immuno electron microscopy was employed to verify the
lysosomal location of Bax.
ISOLATION OF RAT LIVER LYSOSOMES As the method for isolation of lysosomes is only briefly described in
paper IV, more detailed information is given here. Livers of SpragueDawley rats were washed, minced, and homogenized in ice cold
buffer (250 mM sucrose and 20 mM Pipes, pH 7.2). Nuclei and
cellular debris were removed by centrifugation of the homogenate for
10 min at 540 x g. The supernatant was cleared from mitochondria by
a 5-min incubation with CaCl2 (1 mM) at 37˚C, resulting in disruption
of the mitochondrial membrane. After centrifugation at 18 000 x g for
10 min (4˚C) the heavy membrane pellet was collected. The integrity
of lysosomes was evaluated by analysis of activity of the lysosomal
enzymes NAG and cathepsins B and L in the supernatant and the
pellet (with or without addition of 0.2% v/v Triton X-100). Only if
more than 80% of lysosomes remained intact after this procedure
purification was continued. After washing in sucrose-Pipes buffer, the
heavy membrane pellet was resuspended in a 40% w/v mixture of
Percoll and sucrose-Pipes buffer and centrifuged at 44 000 x g for 30
min. A 1-ml fraction, enriched in lysosomes, was collected from the
bottom of the tube. Mitochondrial contamination was evaluated by
measurement of succinic p-iodonitroteterazolium reductase activity,
western blot analysis of cytochrome c oxidase, and electron
microscopy. The lysosomal fraction was washed in sucrose-Pipes
buffer and pelleted by centrifugation at 17 000 x g for 10 min.
STATISTICAL ANALYSIS All experiments were repeated at least three times. Data were
statistically evaluated using the Mann-Whitney U-test (paper IV) and
the Kruskal-Wallis multiple comparison test (papers I-IV).
Differences were considered significant when p ≤ 0.05.
ETHICAL CONSIDERATIONS Lysosomes (paper II) and mitochondria (paper III) were isolated from
rat liver. These experiments were approved by the local research
ethics committee at Linköping University.
45
46
RESULTS
RESULTS Results presented in papers I, II, and III are based mainly on studies in
human fibroblast in which apoptosis was induced by STS. The role of
cathepsins in apoptosis of non-transformed cells was evaluated in this
experimental setup, while, in paper IV, their involvement in cancer
cell apoptosis was studied. For this purpose, two oral squamous cell
carcinoma cell lines were used, and apoptosis was initiated via the
intrinsic or the extrinsic pathway using naphthazarin and anti-Fas
antibodies, respectively.
PAPERS I, II, AND III Studies on apoptosis are often performed in cancer cell lines.
However, as evasion of apoptosis is a hallmark of cancer, there are
often alterations in the cell death program in cancer cells. For this
reason, the principal mechanisms of cell death is best studied in nontransformed cells, such as human fibroblasts, which were used in
papers I, II, and III. STS invoked human fibroblast cell death with
morphological features typical for apoptosis. Cell death was
associated with mitochondrial release of cytochrome c and activation
of caspases. The involvement of lysosomal cathepsins in the signaling
cascade was evaluated using chemical inhibitors, namely pepstatin A
and z-FA-FMK, for inhibition of cathepsin D and cysteine cathepsins,
respectively. While z-FA-FMK had no effect, pepstatin A protected
cells from STS-induced death (see Figure 13). In cells pretreated with
pepstatin A, cytochrome c remained in mitochondria and the caspase
activity was reduced by more than half. Cathepsin D, thus, seems to
be working upstream of mitochondria to promote cell death. It should,
however, be noted that cathepsin D inhibition did not suffice to rescue
cells after prolonged exposure to STS, suggesting that alternative
pathways may compensate for loss of cathepsin D activity.
Importantly, cathepsin D was found to be present in the cytosol
already 1 h after STS exposure, indicating that lysosomal release of
cathepsins is one of the initial events during apoptotic cell death.
Release to the cytosol is likely a critical event, allowing cathepsin D
to encounter its downstream targets. We, therefore, set out to explore
the mechanisms that enable cathepsins to escape the lysosomal
compartment. The results from this part of the study are presented
47
RESULTS
mainly in paper II. We first evaluated if cathepsin D could mediate its
own release, as has been shown for cathepsin B (Werneburg et al.,
2002; Feldstein et al., 2004). Since pepstatin A did not prevent
lysosomal destabilization this possibility was excluded. LMP due to
oxidative events was also ruled out, as the lipophilic antioxidant
DPPD (N, N-diphenyl-1, 4-phenylene-diamine) had no protective
effect. It has been suggested that caspase-8 is required for release of
cathepsins under certain circumstances (Guicciardi et al., 2000), and
that this process may involve cleavage of Bid (Werneburg et al.,
2004). Caspase-8 activation was, however, detected first after 8 h of
STS treatment, and a specific caspase-8 inhibitor, Ac-IETD-CHO
offered no protection, excluding caspase-8 as an important
executioner of LMP in this type of cell death. The ability of active
caspase-8 to permeabilize lysosomes was further investigated in a cell
free system using lysosomes isolated from rat liver. Caspase-8 was
unable to cause any significant release of intra-lysosomal proteins, and
no additive effect was seen when combined with recombinant Bid.
Accordingly, caspase-8 or truncated Bid alone is not sufficient to
trigger LMP. Atractyloside, an activator of the ANT protein present in
mitochondrial membranes was earlier found to release cathepsin B
from isolated lysosomes (Vancompernolle et al., 1998). For that
reason, the effect of atractyloside was evaluated in the cell free
system. Contrary to earlier results, only a relatively high concentration
of this compound induced partial release of lysosomal proteins.
Next, we hypothesized that Bax, known to be involved in release of
cytochrome c from mitochondria, could promote not only MMP but
LMP as well. This would explain how caspase-8, via activation of
Bid, could engage the lysosomal death pathway. A prerequisite for a
Bax-dependent release mechanism would be presence of Bax in the
lysosomal membrane. We, thus, investigated whether Bax could be
found in lysosomes following STS exposure. Indeed, upon apoptosis
induction, Bax was relocated from the cytosol to mitochondria, and
partially to lysosomes. The possible involvement of Bax in LMP was
further investigated in the cell free system using recombinant
oligomeric Bax. A truncated form of Bax [Bax(-TM)], lacking the Cterminal membrane-anchoring domain, was often used in early studies
on mitochondria, and was therefore used in parallel with wild type
Bax in these experiments. Bax(-TM) and wtBax were both found to
associate with lysosomal membranes and could not be removed by
alkali extraction, suggesting that they were inserted rather than loosely
48
RESULTS
attached. More importantly, co-incubation of lysosomes and Bax
resulted in release of lysosomal constituents, including cathepsins B
and D, and NAG. In this regard, wtBax was much more efficient than
Bax(-TM), indicating that the C-terminal domain of Bax may be of
critical importance. Notably, recombinant Bax was unable to release
hemoglobin from erythrocytes, suggesting that Bax-mediated
permeabilization is restricted to certain membranes, such as those of
mitochondria and lysosomes. Collectively, these data suggest that Bax
could be a mediator of LMP, regulating the release of cathepsins to
the cytosol.
Next, we sought to unravel the mechanism by which cathepsin D
triggers downstream apoptotic events. In a previous study it was
demonstrated that lysosomal extracts could cleave Bid (Stoka et al.,
2001). For that reason, we hypothesized that cathepsin D could have
this effect. In paper I, however, this possibility was ruled out as no Bid
cleavage was detected until 8 hours after STS exposure, long after
release of cytochrome c from mitochondria, which was detected at
1 h. In our search for downstream targets for cathepsin D we found
that pepstatin A prevented STS-induced translocation of Bax to
mitochondria. Moreover, microinjection of cathepsin D triggered
relocation of Bax without any external stimuli. Surprisingly, it was
also observed that Bid, in a similar fashion, relocated from the cytosol
to mitochondria upon microinjection of cathepsin D. Thus, it became
clear that we may have jumped into conclusions when ruling out Bid
as a cathepsin D substrate. This was largely due to a methodological
problem that prevented us from detecting the truncated form of Bid at
that time. When having solved this problem, it was found that there
was, in fact, an early increase in the truncated form of Bid, without
any detectable reduction in the full-length form. Furthermore,
cleavage of Bid was, indeed, found to be dependent on cathepsin D. In
a cell free system it was confirmed that cathepsin D cleaves Bid,
resulting in a 15-kDa and a 19-kDa fragment, both of which are
observed also in the human fibroblast model. By N-terminal sequence
analysis of cleaved fragments, three cathepsin D-specific cleavage
sites, at Phe24, Trp48 and Phe183, were identified. Importantly,
cathepsin D-cleaved Bid released cytochrome c from isolated
mitochondria in the presence of Bax, suggesting that cleavage was
associated with activation of Bid. The importance of Bid in lysosomemediated apoptosis was further established using Bid-/- MEFs. It was
demonstrated that cells deficient in Bid were less susceptible to
49
RESULTS
apoptosis induced by the lysosomotropic detergent MSDH, as
compared to their wt equivalents.
Figure 13: Summary of results presented in papers I-III. Cathepsin D released
from lysosomes promotes cell death in human fibroblasts by proteolytic
activation of Bid.
Finally, we investigated the pH-dependence of the cathepsin Dmediated Bid cleavage, and estimated the cytosolic pH of dying cells.
Bid cleavage was observed at pH 7.3, but was more efficient at pH 6.5
and 5.7, suggesting that this process would be favored by a
50
RESULTS
concomitant decrease in the cytosolic pH. By using the pH-sensitive
probe 2’,7’-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), it
was shown that the cytosol was, indeed, acidified in cells exposed to
STS (6.6±0.9) and MSDH (6.0±0.6) as compared to control cells
(7.4±0.4).
In summary, cathepsin D was shown to be an important death factor in
human fibroblast cell death in response to STS. Apoptosis was
associated with release of cathepsin D to the cytosol, possibly
mediated by the Bcl-2 family protein Bax. Once in the cytosol,
cathepsin D promoted downstream apoptotic events through cleavage
of Bid.
PAPER IV The expression and sorting of lysosomal cathepsins are often altered
in cancer cells (Rochefort and Liaudet-Coopman, 1999; Roshy et al.,
2003). As cathepsins B, D and L are frequently overexpressed in
SCCs of the head and neck (Zeillinger et al., 1992; Kos et al., 1995),
we aimed to investigate their involvement in oral SCC cell death. In
the two oral SCC cell lines used (UT-SCC-20A and UT-SCC-24A),
cathepsins B and D were released to the cytosol already 1 h after
exposure to the redox cycling drug naphthazarin, which is a structural
analogue to the anti-cancer drug doxorubicin. Naphthazarin induced
oxidative stress with approximately 30% cell death, as assessed by
apoptotic nuclear morphology, and a 4-fold increase in caspase
activity at 24 h of exposure. Neither caspase-3 activation nor cell
death was significantly affected in pepstatin A-pretreated cells,
suggesting that cathepsin D was not required for propagation of the
apoptotic signal (see Figure 14). The cysteine cathepsin inhibitor
z-FA-FMK, on the other hand, reduced caspase-3 activity by about
half in UT-SCC-20A cells, and almost completely blocked activation
in UT-SCC-24A cultures. Somewhat unexpected, z-FA-FMK
nonetheless failed to protect from cell death. Importantly,
destabilization of lysosomes using MSDH was sufficient to trigger
caspase activation and cell death, suggesting that the lysosomal death
pathway remain intact in these cancer cells.
51
RESULTS
Figure 14: Summary of results presented in paper IV. After release to the cytosol,
cathepsin B contributes to activation of the caspase cascade, but is not required
for oral SCC cell death.
Cathepsins are frequently secreted from cancer cells, and it has been
reported that extra-cellular cathepsins may favor tumor growth and
metastatic spread (Rochefort and Liaudet-Coopman, 1999; Berchem et
al., 2002; Roshy et al., 2003). We speculated that extra-cellular
cathepsins, as opposed to intra-lysosomal cathepsins, may have a
negative influence on tumor cell apoptosis, and thereby favor tumor
growth. We confirmed, by enzyme-linked immunosorbent assay
(ELISA) detection, that cathepsin B was secreted from the two oral
52
RESULTS
SCC cell lines to the culture medium, and then evaluated the possible
influence of cathepsin inhibitors on the cell surface expression of the
Fas death receptor. A slight, but statistically insignificant, increase in
soluble Fas was detected in pepstatin A treated cultures. The
expression of membrane-bound Fas remained unchanged, suggesting
that extra-cellular cathepsin D does not participate in shedding of the
death receptor, but may degrade soluble Fas after removal from the
cell surface. The opposite results were obtained with z-FA-FMK,
which tended to increase the amount of membrane-bound Fas in
UT-SCC-20A cultures, without affecting the level of soluble Fas.
Incubation at pH 7.2 of recombinant cathepsin B with UT-SCC-20A
cells in suspension reduced the amount of full length Fas as detected
by western blot analysis. We, thus, propose that extra-cellular
cathepsin B may engage in proteolytic removal of Fas from the
plasma membrane (Figure 15). However, under these conditions, this
was not sufficient to suppress cell death induced by activating anti-Fas
antibodies.
Figure 15: Cathepsin B secreted from cancer cells may engage in shedding of the
Fas death receptor from the cell surface, and possibly protect cancer cells from
FasL-induced cell death.
53
RESULTS
In summary, it was demonstrated that although cathepsins are released
to the cytosol, they are not required for oxidative stress- or Fasinduced apoptosis in the two oral SCC cell lines studied. It should,
however, be noted that MSDH-induced LMP was sufficient to elicit
an apoptotic response. Moreover, it was proposed that extra-cellular
cathepsin B may suppress cancer cell death by shedding of the Fas
death receptor.
54
DISCUSSION
DISCUSSION Cathepsins are key regulators of apoptosis induced by a variety of
different stimuli, including TNF-α (Guicciardi et al., 2000;
Foghsgaard et al., 2001), TRAIL (Vigneswaran et al., 2005; Nagaraj
et al., 2006), Fas (Wille et al., 2004), STS (paper I) (Bidère et al.,
2003), UV irradiation (Bivik et al., 2006), ischemia (Yamashima et
al., 1998), and oxidative stress (Roberg and Öllinger, 1998a; Öllinger,
2000; Kågedal et al., 2001a). The relative importance of different
cathepsins seems to vary with the death stimulus and cell type. This is
intriguing, as they are, most often, all released to the cytosol upon
induction of LMP. It can be argued that the relative expression of
different cathepsins is a major determinant, but this does not account
for all differences observed, as illustrated by the following example.
Inhibition of cathepsin D, but not cysteine cathepsins, protects from
STS- (paper I) and oxidative stress-induced human fibroblast cell
death (Kågedal et al., 2001a). More importantly, microinjection of
cathepsin D triggers cell death in human fibroblasts, while
microinjection of cathepsin B does not (Roberg et al., 2002). Clearly,
cathepsin B is of minor importance in these cells, and a low
expression of cathepsin B is not a satisfying explanation. In this case,
it is possible that the relative contribution of these death factors is
determined by the cytosolic concentration of naturally occurring
cathepsin inhibitors. Human fibroblasts may express high levels of
cytosolic inhibitors, such as stefins, that prevent cysteine cathepsins
from interacting with their downstream targets. However, no such
potent endogenous cathepsin D inhibitors, that could explain the
opposite situation, are known to date. Finally, it could be speculated
that the expression and/or compartmentalization of downstream
targets could be of importance, provided that these are specific for
individual cathepsins. Despite numerous reports on the involvement of
cathepsins in cell death, the mechanism(s) are still not clear, except
that they need to be released into the cytosol in order to exert their
pro-apoptotic activity.
LYSOSOMAL MEMBRANE PERMEABILIZATION As already mentioned, apoptosis is associated with a limited release of
lysosomal constituents (Brunk and Svensson, 1999; Li et al., 2000;
Antunes et al., 2001; Kågedal et al., 2001b) It is, however, unclear
55
DISCUSSION
whether only a subset of lysosomes are affected, or if all lysosomes
are targeted but releases only part of their contents. Upon induction of
apoptosis, cathepsins escape into the cytosol, and so do several of the
dyes that accumulate in lysosomes with intact membranes, e.g.
Acridine Orange (Brunk and Svensson, 1999), Lucifer Yellow
(Kågedal et al., 2001b), and LysoTracker (Werneburg et al., 2002).
This points to an unspecific release mechanism, such as pore
formation, rather than a transport process across an intact membrane,
that would be expected to be specific for a single group of molecules.
Lysosomal release may, however, be size-selective, as suggested by a
study in which it was demonstrated that 10-kDa and 40-kDa FITCdextran molecules were released from lysosomes during STS-induced
T-cell apoptosis, while 70-kDa and 250-kDa FITC-dextran was
retained within lysosomes (Bidère et al., 2003). Although true in this
experimental setting, it may not apply for all death models. Indeed, as
presented in paper II, recombinant Bax caused release of the 150 kDa
protein NAG from isolated lysosomes. In line with these data,
lysosomal release of NAG was observed during microbe-induced
neutrophil apoptosis (Blomgran et al., 2007). Conceivably, LMP may
be governed by several mechanisms, each with its own characteristics
(Figure 16).
PROMOTERS OF LYSOSOMAL MEMBRANE PERMEABILIZATION Non­mammalian proteins There are a few examples of non-mammalian proteins that, when
entering mammalian cells, permeabilize the lysosomal membrane. The
human immunodeficiency virus type 1 (HIV-1) protein Nef is one
interesting example. Upon overexpression, Nef elicits an apoptotic
response in CD4+ T lymphocytes via disruption of lysosomal integrity
(Laforge et al., 2007). The progressive and massive destruction of
CD4+ T lymphocytes characteristic of HIV-1-related disease may,
thus, in part be due to Nef-induced LMP.
Another example is the diphtheria toxin, which is known to create
pores in the lysosomal membrane through which the ADP-ribosylating
A-subunit of the toxin is transported. The pore-forming domain of this
toxin was found to show high structural similarity to some of the
Bcl-2 family members (Muchmore et al., 1996; Suzuki et al., 2000),
leading to the hypothesis that Bcl-2 proteins could release
56
DISCUSSION
cytochrome c from mitochondria through pore formation. In analogy,
based on these results, we hypothesized that Bax could permeabilize
the lysosomal membrane, and thereby promote cell death by release of
lysosomal cathepsins to the cytosol.
Bax In paper II we propose a novel mechanism for LMP with the Bcl-2
family protein Bax in the leading role. Upon STS treatment of human
fibroblasts, Bax was translocated from the cytosol to the lysosomal
membrane. Furthermore, recombinant Bax inserted into the membrane
of isolated rat liver lysosomes with release of lysosomal constituents
as a result. This paper provided the first evidence that Bax may be
mechanistically involved in LMP. These data are now supported by a
growing number of publications demonstrating involvement of
multiple pro- and anti-apoptotic Bcl-2 proteins in lysosomal
destabilization. Bax has been reported to be activated and relocated to
lysosomes in hepatocytes upon treatment with free fatty acids
(Feldstein et al., 2004; Feldstein et al., 2006), as well as in
cholangiocarcinoma cells in response to TRAIL (Werneburg et al.,
2007). In both cases, small interference RNA- (siRNA)-mediated
downregulation of Bax prevented LMP, confirming the mechanistical
importance of the lysosomal location (Feldstein et al., 2006;
Werneburg et al., 2007). Furthermore, siRNA-mediated
downregulation of Bim was found to prevent TRAIL-induced LMP in
cholangiocarcinoma cells, while suppression of Bid or Bak expression
had no such effect (Werneburg et al., 2007). Bim, which was activated
upon TRAIL treatment and colocalized with Bax in mitochondria,
was, thus, suggested to trigger Bax-mediated LMP. Interestingly, Bid,
which is another BH3-only protein known to promote Bax-mediated
MMP, was found to be involved in TNF-α-induced lysosomal
destabilization in hepatocytes (Werneburg et al., 2004). Upon TNF-α
treatment, Bid was detected in lysosomes, and cathepsin B was
released to the cytosol. However, no such release was seen in Bid-/cells. Even though Bax was not considered in this study, it could be
speculated that LMP was caused by Bid-mediated activation of Bax.
In a recent report, BH3 domain peptides were found to permeabilize
isolated lysosomes in the presence of Bax (Werneburg et al., 2007).
Conceivably, triggering of LMP could be a general function of proapoptotic Bcl-2 proteins. Interestingly, Bak has also been reported to
57
DISCUSSION
be present in lysosomes (Feldstein et al., 2004), but there are as yet no
reports of Bak-induced LMP.
In our model of apoptosis, Bax may not be responsible for the initial
release of cathepsins, but rather constitute a feed-back mechanism for
amplification of the apoptotic signal. This speculation is based on the
fact that cleavage of Bid and subsequent activation of Bax seems to be
dependent on cathepsin D in this system. Cathepsin D may, thus,
initially translocate to the cytosol by an alternative mechanism, and
promote release of more cathepsins from lysosomes, as well as release
of cytochrome c from mitochondria, through cleavage of Bid.
As presented in paper II, Bax did not release hemoglobin from
isolated erythrocytes, suggesting that Bax is selectively targeted to
certain cellular membranes, such as those of lysosomes and
mitochondria. This selectivity may depend on interaction with specific
membrane proteins, or possibly the membrane lipid composition.
Indeed, Bax-mediated MMP has been suggested to require interaction
with the negatively charged phospholipid cardiolipin, present in the
inner mitochondrial membrane (Kuwana et al., 2002). In analogy, the
structurally related phospholipid bis-(monoacylglylceryl)phosphate
(also called lysobisphosphatidic acid) present in lysosomes could be
essential for Bax-mediated LMP (Brotherus and Renkonen, 1977).
Finally, Bax has been observed not only in mitochondria and
lysosomes, but also in the ER (Gajkowska et al., 2001; Scorrano et al.,
2003), and it is certainly tempting to speculate that Bax may govern
release of pro-apoptotic factors from all of these compartments.
Caspases Caspase-8 seems to play a significant role in the engagement of the
lysosomal death pathway in response to death receptor activation
(Guicciardi et al., 2000; Werneburg et al., 2004; Gyrd-Hansen et al.,
2006; van Nierop et al., 2006; Werneburg et al., 2007). Caspase-8induced destabilization of the lysosomal membrane is likely mediated
by activation of a cytosolic substrate(s). The Bcl-2 family member
Bid, which presumably would trigger Bax-mediated LMP, has been
presented as a possible candidate (Werneburg et al., 2004). In our
experiments, neither caspase-8 nor activated Bid alone could
permeabilize the membrane of isolated lysosomes, suggesting that
additional factors, such as Bax or Bak, are indeed required. In another
intriguing study, caspase-8 was found to cleave and activate caspase-9
58
DISCUSSION
in an apoptosome-independent fashion (Gyrd-Hansen et al., 2006). By
using genetically modified MEFs caspase-9 was proven indispensable
for triggering of LMP, but the exact mode of action remained elusive.
Interestingly, in the same study, depletion of Bid did not prevent loss
of lysosomal membrane integrity, suggesting the existence of at least
two different mechanisms by which caspase-8 induces LMP.
Aryl hydrocarbon receptor The lysosomal pathway of apoptosis may be engaged upon death
receptor activation even in the absence of active initiator caspases
(Caruso et al., 2006). In a murine hepatoma cell line where TNF-αinduced cell death was found to be independent of caspase-8, LMP
was dependent on the aryl hydrocarbon receptor (Ahr). This receptor
is a ligand-activated transcription factor whose target genes are
involved in many cellular functions, including apoptosis. It has also
been suggested that the Ahr has ligand-independent activities. Ahr
deficiency prevents disruption of lysosomes induced not only by TNFα treatment, but also following photodynamic therapy employing the
photosensitizer NPe6 that accumulate in lysosomes. This suggests that
the Ahr somehow influences the permeability of the lysosomal
membrane, rather than interferes with TNF signaling.
p53 Permeabilization of the lysosomal membrane has been shown to occur
during p53-induced apoptosis (Yuan et al., 2002), and in a recent
study it was suggested that p53 itself localizes to lysosomes and
trigger LMP in a transcription-independent manner (Li et al., 2007). It
was proposed that p53 phosphorylated at Ser15 may be recruited to
the lysosomal membrane by a recently discovered protein called
LAPF (Lysosome-associated apoptosis-inducing protein containing
the pleckstrin homology and FYVE domains). This protein, which
upon induction of apoptosis associates with lysosomes, was found to
be essential for cell death induced by TNF-α, ionizing irradiation, and
the DNA-damaging drugs 5-fluorouracil and oxaliplatin (Chen et al.,
2005; Li et al., 2007). Downregulation of either p53 or LAPF
prevented TNF-α-induced LMP (Li et al., 2007). Interestingly,
silencing of LAPF expression abrogated lysosomal translocation of
phospho-p53Ser15, whereas silencing of p53 expression had no effect
on lysosomal translocation of LAPF, indicating that LAPF may trigger
LMP by acting as an adaptor protein for phospho-p53Ser15. In line
59
DISCUSSION
with these data, phospho-p53Ser15 was reported to localize to
lysosomes in cultured cortical neurons after exposure to the
psychoactive ingredient of marijuana, ∆9-tetrahydrocannabiol
(Gowran and Campbell, 2008). Also in this model of apoptosis, LMP
was abrogated by siRNA-mediated downregulation or chemical
inhibition of p53. p53 has been observed also at the mitochondrial
membrane during apoptosis, and has been shown to trigger MMP by
interacting with Bcl-2 family members (Mihara et al., 2003; Chipuk et
al., 2004). It is tempting to speculate that a similar mechanism could
govern p53-induced LMP. It is possible that p53, via transcriptional
upregulation of the BH3-only proteins Puma and Noxa, can engage
the lysosomal death pathway also without translocating to lysosomes.
Puma and Noxa may, in turn, elicit Bax-mediated LMP, as has been
described for Bid and Bim. In a very recent study from our laboratory,
UVB irradiation was found to trigger p53-dependent LMP in
melanocytes (Wäster and Öllinger, 2008). This was likely mediated by
a transcription-dependent mechanism as p53 could not be detected in
lysosomes, and as cell death was associated with an increase in Puma
and Noxa expression.
ANT Already in 1998 it was demonstrated that the ANT agonist
atractyloside, which triggers opening of the mitochondrial PT pore
and release of mitochondrial cytochrome c, also induces release of
cathepsin B from isolated lysosomes (Vancompernolle et al., 1998).
This was discovered by coincidence by researchers aiming at
identifying factors released from a mitochondrial fraction, apparently
contaminated with lysosomes. This finding suggests the possibility
that ANT-like proteins may be present in the lysosomal membrane
and regulate its permeability. We aimed to confirm these results, but
in our hands, only a very high concentration of atractyloside
permeabilized the membrane of isolated rat liver lysosomes to a
certain extent.
Cathepsin B It has also been suggested that lysosomal proteases, in particular
cathepsin B, may contribute to their own release (Werneburg et al.,
2002; Feldstein et al., 2004; Guicciardi et al., 2007). However, it is
not clear whether lysosomal or cytosolic cathepsin B catalyzes this
event. As cathepsin B is constitutively active inside lysosomes, an
60
DISCUSSION
extra-lysosomal trigger must exist for lysosomal cathepsin B to be
involved. Alternatively, this could be a feed-back loop, where a small
amount of released cathepsin B triggers more extensive LMP from
outside the lysosome. The latter is supported by the finding that
nuclear factor-κB- (NF-κB)- mediated upregulation of serine protease
inhibitor 2A, a potent inhibitor of cathepsin B located in the cytosol
and nucleus, reduces lysosomal breakdown (Liu et al., 2003). As
presented in paper I, pepstatin A did not prevent LMP, suggesting that
cathepsin D, under these circumstances, does not contribute to its own
release. The involvement of cathepsin B in STS-induced lysosomal
leak was not evaluated. However, as z-FA-FMK did not prevent cell
death, it is unlikely that cathepsin B is required for LMP to occur in
human fibroblasts.
Sphingosine LMP can also be mediated by the sphingolipid sphingosine (Kågedal
et al., 2001b; Werneburg et al., 2002). Ceramide is produced upon
activation of sphingomyelinase, for example in response to TNF-α
treatment, and is further processed into sphingosine by ceramidase
(Schutze et al., 1999). Sphingosine has lysosomotropic properties and,
thus, accumulates inside lysosomes where it permeabilizes the
membrane in a detergent-like fashion, resulting in cell death (Kågedal
et al., 2001b). Under certain circumstances, this process seems to be
dependent on cathepsin B, as sphingosine failed to permeabilize
lysosomes from cathepsin B-/- hepatocytes (Werneburg et al., 2002).
There is, thus, a possibility that sphingosine may serve as a trigger of
cathepsin B-mediated intra-lysosomal events leading to LMP.
Calpains Calpains have been suggested to catalyze lysosomal breakdown
during neuronal cell death (Yamashima et al., 1996; Yamashima et
al., 2003; Yap et al., 2006). Cell death induced by ischaemia or HOCl
exposure was associated with an increase in cytosolic Ca2+, activation
of calpains, and LMP. Immuno cytochemical studies showed that
activated µ-calpain located to the lysosomal membrane prior to release
of cathepsin B to the cytosol, indicating its possible involvement in
LMP (Yamashima et al., 1996; Yamashima et al., 1998; Yamashima
et al., 2003). Furthermore, HOCl-induced permeabilization of the
lysosomal membrane was abrogated by pharmacological inhibition of
calpains (Yap et al., 2006).
61
DISCUSSION
Reactive oxygen species The integrity of the lysosomal membrane could be compromised by
production of reactive oxygen species (ROS). It has been proposed
that intra-lysosomal iron originating from degraded proteins can react
with H2O2 via Fenton-type chemistry to generate oxygen radicals that
induce permeabilization of the lysosomal membrane (Antunes et al.,
2001; Persson et al., 2003; Yu et al., 2003). This is supported by the
fact that anti-oxidants and iron chelators efficiently protect from
lysosomal destabilization under certain circumstances (Öllinger and
Brunk, 1995; Persson et al., 2001a; Persson et al., 2001b; Persson et
al., 2003; Yu et al., 2003; Persson et al., 2005).
Phospholipase A2 According to one report, phospholipase A2 (PLA2) may be involved
in apoptosis-associated lysosomal destabilization. In response to shortterm exposure to low concentrations of H2O2, lysosomes were
permeabilized in a PLA2-dependent fashion (Zhao et al., 2001a).
Intriguingly, activation of PLA2 was dependent on an early minor
release of lysosomal contents, suggesting an alternative mechanism
for the initial release, and PLA2 to engage in an amplifying feed-back
loop. Notably, microinjection of PLA2 was sufficient to trigger LMP
in human fibroblasts, and incubation of PLA2 with rat liver lysosomes
resulted in leakage of lysosomal constituents.
Kinases LMP may be preceded by a cascade of signaling events, which, not
surprisingly, have been found to involve kinases. C-Jun N-terminal
kinase (JNK) promotes lysosomal breakdown in response to TNF-α,
TRAIL, and UVB irradiation, possibly via activation of the BH3-only
protein Bim (Dietrich et al., 2004; Werneburg et al., 2007; Bivik and
Öllinger, 2008). By contrast, p42/44 mitogen-activated protein kinase
(MAPK) preserves lysosomal membrane integrity, and thereby
contributes to inhibition of TRAIL-induced apoptosis (Guicciardi et
al., 2007). How this is accomplished is still unclear.
SAFEGUARDS OF LYSOSOMAL MEMBRANE INTEGRITY It has been suggested that the stability of the lysosomal membrane
could be modulated in different ways, and that this could contribute to
determination of cell fate. Apoptosis of germinal center B62
DISCUSSION
lymphocytes constitute an interesting example of physiological
importance. In germinal centers, B-cells with high affinity B-cell
receptors are selected, and B-cells with unwanted specificities are
removed through apoptosis. Anti-apoptotic signals provided by
follicular dendritic cells are essential in this selection process. It was
recently demonstrated that upon interaction with follicular dendritic
cells, B-cells become resistant to LMP (van Nierop et al., 2006). The
underlying mechanism was not unraveled in this study, but molecules
involved in stabilization of the lysosomal membrane have been
suggested by others.
Heat shock proteins Heat shock proteins (Hsps) are molecular chaperones essential for the
ability of cells to cope with environmental stress (Jäättelä, 1999).
Interestingly, Hsp70-1 is found in the lysosomal membrane of many
tumor cells and stressed cells and has been shown to prevent LMP
induced by TNF-α, etoposide, H2O2, and UVB irradiation (Nylandsted
et al., 2004; Bivik et al., 2007). Hsp70-1 is frequently overexpressed
in tumors and its tumorigenic potential could, in part, be due to
stabilization of lysosomes (Gyrd-Hansen et al., 2004). Also another
Hsp70 family member, Hsp70-2, has been shown to prevent
permeabilization of the lysosomal membrane (Daugaard et al., 2007).
In this case, the protective effect appears to depend on Hsp70-2mediated upregulation of lens epithelium-derived growth factor
(LEDGF). Knockdown of LEDGF in cancer cells induced
destabilization of lysosomal membranes and cathepsin-mediated cell
death, while ectopic expression stabilized lysosomes and prevented
cancer cell death. However, as LEDGF could not be detected in
lysosomes, the protective mechanism remains obscure.
Bcl­2 proteins Anti-apoptotic members of the Bcl-2 family are well-known
regulators of mitochondrial membrane permeabilization that, during
recent years, have gained attention also as possible modulators of
lysosomal membrane stability. Our finding that LMP may be induced
by activation of Bax is supported by the fact that overexpression of
Bcl-2 has been found to prevent lysosomal destabilization (Zhao et al.,
2000; Zhao et al., 2001b; Paris et al., 2007). In two of these studies, it
was suggested that Bcl-2 stabilizes lysosomes by preventing activation
of PLA2 (Zhao et al., 2000; Zhao et al., 2001b). However, with more
63
DISCUSSION
recent data from our group and others in hand, it is tempting to
speculate that the protective effect of Bcl-2 was, at least in part, due to
neutralization of pro-apoptotic members of the Bcl-2 family. It is,
unfortunately, unknown whether Bax localized to the lysosomal
membrane under these experimental conditions. Data in support of the
theory that anti-apoptotic Bcl-2 proteins are safeguards of lysosomal
membrane integrity has been provided by Gregory Gores and coworkers (Feldstein et al., 2006; Werneburg et al., 2007). In these
studies, Mcl-1 and Bcl-XL were found to prevent LMP induced by
TRAIL and free fatty acids, respectively. In both cases, Bax was
activated and translocated from the cytosol to lysosomes upon
apoptosis induction. Furthermore, overexpression of Mcl-1 and BclXL, as well as siRNA-mediated downregulation of Bax, prevented
destabilization of the lysosomal membrane.
Figure 16: Upon apoptosis induction, there is permeabilization of the lysosomal
membrane and release of cathepsins to the cytosol. Several factors that regulate
lysosomal membrane integrity have been identified. See text for details.
64
DISCUSSION
MECHANISMS BY WHICH CATHEPSINS PROMOTE APOPTOSIS Cathepsin-mediated cell death most often proceed via the
mitochondrial pathway with subsequent engagement of the caspase
cascade, as is the case in STS-induced human fibroblast apoptosis.
There is also evidence for a role of cathepsins in the execution of cell
death independent of caspase activation, resulting in an apoptosis-like
morphology (Foghsgaard et al., 2001; Li and Pober, 2005). In line
with this, cathepsin B is able to induce apoptotic morphology in
isolated nuclei (Vancompernolle et al., 1998). This suggests the
existence of multiple extra-lysosomal cathepsin targets, of which
some, already identified, will be presented below (Figure 18).
DIRECT ACTIVATION OF CASPASES It was initially hypothesized that cathepsins may promote apoptosis by
direct activation of effector caspases. Cathepsin B was found to
efficiently process and activate the inflammatory caspases-1 and -11,
and various apoptotic caspases to a lesser extent (Vancompernolle et
al., 1998). It was, however, later demonstrated by Stoka and coworkers that caspases-3 and -7 are poor substrates for lysosomal
extracts and cysteine cathepsins (Stoka et al., 2001). Cysteine
cathepsin-dependent cleavage of caspase-3 by lysosomal extracts was
observed by Ishisaka and co-workers, but was found to require yet
another lysosomal factor (Ishisaka et al., 1998; Ishisaka et al., 1999).
It was suggested that cathepsin L activates a lysosomal enzyme, called
lysoapoptase by the authors, which is capable of activating caspase-3
after release to the cytosol (Katunuma et al., 2001).
More recent reports suggest the possible involvement of cathepsins in
the activation of initiator caspases. Interestingly, a study from the
beginning of this year presents evidence that cathepsin D can activate
caspase-8 by cleavage at Leu237 (Conus et al., 2008). During
neutrophil apoptosis, cathepsin D was released from azurophilic
granules, and triggered downstream events via caspase-8 activation in
the absence of death receptor signaling. Cathepsin-mediated activation
of caspase-2 has also been reported (Guicciardi et al., 2005). In
hepatocytes, caspase-2 was activated in a cathepsin B-dependent
manner in response to TNF-α treatment.
65
DISCUSSION
Although direct activation of caspases may occur under certain
circumstances, it appears that cathepsin-mediated caspase activation
occurs predominantly through engagement of mitochondria.
CLEAVAGE OF BID There is compelling evidence that cathepsins can transmit the
apoptotic signal to mitochondria by proteolytic activation of Bid. This
was first suggested in a study by Stoka and co-workers where
lysosomal extracts were found to activate Bid, which in turn caused
release of cytochrome c from isolated mitochondria (Stoka et al.,
2001). In a later paper from the same group it was shown that several
cysteine cathepsins could process Bid at pH 7.2 in vitro. Cathepsins B,
L, S, and K cleaved Bid preferably at Arg65, while cathepsin H
cleaved at Arg71 (Cirman et al., 2004). Both of these sites are located
within the flexible loop connecting helices 2 and 3 along with the
caspase-8 cleavage site (Asp59) (see Figure 10). In addition, a number
of minor cleavage sites, including Tyr47 and Gln57, were found.
Interestingly, transfection of HEK 293 cells with plasmids encoding
variants of tBid generated by cleavage at Asp65, Arg71, Tyr47, or
Gln57 all induced apoptosis. Cysteine cathepsin-mediated activation
of Bid has been observed in several models of apoptosis, including
HeLa cell apoptosis induced by the lysosomotropic agent LeuLeuOMe
(Cirman et al., 2004), microbe-induced apoptosis in neutrophils
(Blomgran et al., 2007), and U937 cell death following treatment with
the anti-cancer agent miltefosine (Paris et al., 2007).
While Bid cleavage by cysteine cathepsins seems to be generally
accepted, the ability of cathepsin D to activate Bid is still a matter of
debate. Cathepsin D-mediated activation of Bid was first proposed by
Heinrich and colleagues, who observed that inhibition of cathepsin D
prevented TNF-α-induced Bid cleavage in HeLa cells (Heinrich et al.,
2004). Moreover, activation of Bid was absent in cathepsin D-/- MEFs,
but could be restored by reintroduction of cathepsin D. In paper III,
we present data indicating that cathepsin D cleaves Bid upon STS
treatment of human fibroblasts. Pepstatin A-inhibitable Bid activation
has been observed also during oxidative stress-induced apoptosis in
retinal photoreceptor cells (Sanvicens and Cotter, 2006), as well as
after TNF-α treatment in a murine hepatoma cell line unable to
activate caspase-8 due to lack of FADD (Caruso et al., 2006).
66
DISCUSSION
In cell free experiments, performed by Heinrich and co-workers,
cathepsin D was found to cleave Bid in a pH-dependent fashion
(Heinrich et al., 2004). Processing of Bid was efficient at a pH
ranging from 4.2 to 6.2, but almost no cleavage was detected at pH
7.0. Contrary to these results, Cirman and colleagues argued that
cathepsin D is unable to process Bid in vitro (Cirman et al., 2004).
These experiments were, however, performed at pH 7.2 that in the
above mentioned study was found to be sub-optimal (Heinrich et al.,
2004). Results presented in paper III provide additional evidence that
Bid is indeed a cathepsin D substrate. In contrast to data from both of
the above mentioned studies, significant cathepsin D-mediated
cleavage of recombinant Bid was observed at near neutral pH (7.3),
but was, nevertheless, more efficient at a moderately acidic pH (6.5
and 5.7). Importantly, the same two fragments (15 and 19 kDa) that
were generated by cathepsin D in test tube experiments were detected
in STS-treated human fibroblasts. We present, for the first time,
cathepsin D-specific cleavage sites in the Bid sequence, located at
Phe24, Trp48 and Phe183. As judged by the molecular mass, the 15kDa fragment could be generated by sequential cleavage at Trp48 and
Phe183, while the 19-kDa form is produced by processing at Phe24
(Figure 17).
Figure 17: Cathepsin D cleaves Bid at Phe24, Trp48, and Phe182, resulting in
15- and 19-kDa fragments.
Notably, in the presence of recombinant Bax, cathepsin D-cleaved Bid
induced release of cytochrome c from isolated mitochondria,
confirming activation of Bid. It is, however, unclear whether both
67
DISCUSSION
variants of tBid are active, or if the apoptogenic effect can be ascribed
only one of them. One of the cleavage sites, Trp48, is located in the
‘bait-loop’, only one residue downstream of a minor cysteine
cathepsin cleavage site that was previously found to produce active
Bid (Cirman et al., 2004). This clearly argues that the 15-kDa
fragment could be of physiological importance. Interestingly, cleavage
of Bid in the N-terminal region close to Phe24 (the cleavage site was
not identified) was earlier found to produce a Bid variant capable of
inducing release of Smac/DIABLO, but not cytochrome c, from
mitochondria (Deng et al., 2003). Accordingly, the 19-kDa fragment
generated by cathepsin D may also have apoptogenic effects.
OTHER MECHANISMS Bid-independent cathepsin D-mediated activation of Bax has also
been reported, and the authors speculated that cathepsin D may be
involved in the inactivation of cytosolic Bax inhibitors (Bidère et al.,
2003). Indeed, recent data from our laboratory suggests that cathepsin
D may cleave 14-3-3 proteins, which are known to sequester Bax and
Bad in the cytosol (unpublished results).
Degradation of anti-apoptotic proteins from the Bcl-2 family may be
an alternative mechanism for cathepsin-mediated MMP. Interestingly,
according to unpublished data briefly mentioned in a recent review,
Bcl-2, Bcl-XL and Mcl-1 are all cysteine cathepsin substrates (Turk
and Stoka, 2007).
Activation of caspase-2 seems to be yet another way for cathepsin B
to induce MMP (Guicciardi et al., 2005). In hepatocytes, TNF-αinduced activation of caspase-2 was attenuated upon pharmacological
inhibition of cathepsin B. Moreover, hepatocytes from cathepsin B
knockout mice failed to generate active caspase-2 in response to TNFα. Interestingly, caspase-2 was found to be required for downstream
mitochondrial events, which appeared to be independent of Bax.
Cathepsin B may promote apoptosis also by proteolytic inactivation of
the anti-apoptotic enzyme sphingosine kinase-1 (Taha et al., 2005).
Sphingosine kinase is involved in clearance of sphingolipids by
catalyzing the conversion of sphingosine to sphingosine-1-phosphate
(Taha et al., 2006a). While ceramide and sphingosine cause growth
arrest and apoptosis, sphingosine-1-phosphate mediates proliferation.
Notably, siRNA-mediated downregulation of sphingosine kinase-1
induces apoptosis in MCF-7 cells via activation of the intrinsic
68
DISCUSSION
pathway (Taha et al., 2006b). In response to TNF-α treatment,
sphingosine kinase-1is degraded in a cathepsin B-dependent manner,
and cathepsin B was found to cleave and inactivate this enzyme in
vitro (Taha et al., 2005).
It has been suggested that cathepsins, after release to the cytosol, is
involved in activation of PLA2 (Zhao et al., 2001a; Foghsgaard et al.,
2002). Chemical inhibition of cathepsin B prevented TNF-α-induced
PLA2 activation in several cancer cell lines (Foghsgaard et al., 2002).
Moreover, activation of PLA2 was significantly reduced in cathepsin
B-/- MEFs. Interestingly, oxidative stress- and TNF-α-induced
apoptosis was prevented upon inhibition of PLA2, while
microinjection of PLA2 into the cytosol of human fibroblasts
triggered apoptosis without any external stimuli (Zhao et al., 2001a;
Foghsgaard et al., 2002). Thus, this may be a major pathway to cell
death following LMP.
Figure 18: After release from lysosomes, cathepsins promote apoptosis by
cleaving a number of cytosolic substrates. See text for details.
69
DISCUSSION
It is generally believed that cathepsins need to be released into the
cytosol in order to exert their pro-apoptotic effect. It has, however,
been suggested that cathepsin D can promote apoptosis also from
within the lysosomal compartment (Haendeler et al., 2005). During
H2O2-induced endothelial cell apoptosis the anti-oxidant thioredoxin-1
was translocated to lysosomes for degradation. Inactivation of
thioredoxin-1 was found to be dependent on cathepsin D, and essential
for execution of cell death. In this model of apoptosis, there were no
signs of lysosomal destabilization. Thus, cathepsin D may, in some
cases, promote oxidative stress-induced apoptosis through intralysosomal inactivation of anti-oxidants.
CATHEPSIN D­MEDIATED DEATH SIGNALING – DEPENDENT ON CATALYTIC ACTIVITY OR NOT? Results from papers I and III, as well as from previous studies from
our group and others (Deiss et al., 1996; Öllinger, 2000; Roberg et al.,
2002; Heinrich et al., 2004; Caruso et al., 2006; Sanvicens and Cotter,
2006), suggest that the pro-apoptotic effect of cathepsin D is
dependent on its catalytic activity. There has, however, been a great
deal of controversy whether cathepsin D (and other lysosomal
cathepsins) can function outside the lysosome. Apart from their low
pH optimum, cathepsins have a short half-time at cytosolic pH due to
unfolding-induced inactivation (Turk et al., 1995). This clearly speaks
against their involvement in proteolytic signaling outside lysosomes,
but apparently, these obstacles are somehow overcome. The cytosolic
acidification often associated with apoptosis may serve to stabilize
cathepsins released to the cytosol, and prolong their life-time to a
certain extent. As presented in paper III, there is a significant
reduction in the cytosolic pH (7.4±0.4 in control fibroblasts) upon
treatment of fibroblasts with either STS or MSDH. The mean value
for STS-treated cells is 6.6±0.9 and for cells exposed to MSDH
6.0±0.6, however, as some cells are unaffected by the treatment, there
is an even lower cytosolic pH in certain individual cells. Certainly,
this could have a stimulatory effect on the cathepsin D-mediated Bid
cleavage observed in this system.
An alternative explanation is provided by a study in which the crystal
structure of cathepsin D was obtained at both neutral and acidic pH
(Lee et al., 1998). At neutral pH, cathepsin D was inactivated due to
structural rearrangements, leading to insertion of the N-terminal
section of the protein into the active site cleft. Interestingly, under
70
DISCUSSION
these conditions, the enzyme adopted its active conformation upon
interaction with pepstatin A. Therefore, it was suggested that the
conformational switch could serve as a regulatory mechanism,
allowing only substrates with the highest affinity to be cleaved by
cathepsin D outside lysosomes. This remains to be proven, but poses
an explanation as to how cathepsin D could contribute to cell death via
limited and specific proteolysis of cytosolic death factors, even in the
absence of pronounced cytosolic acidification. Indeed, in our cell free
experiments cathepsin D-mediated cleavage of Bid was observed at
near neutral conditions (pH 7.3), even though it was more efficient at
lower pH. Furthermore, cathepsin D-mediated cleavage at neutral pH
has previously been observed for Tau, a protein associated with
Alzheimer’s disease (Kenessey et al., 1997).
Contrary to our results, it has been proposed that the stimulatory effect
of cathepsin D on cell death, in some cases, is independent of its
catalytic activity. The most compelling evidence is provided by two
recent studies, the first showing that a mutant catalytically inactive
form of cathepsin D is as potent in enhancing apoptosis as wt
cathepsin D (Beaujouin et al., 2006), and the other demonstrating that
microinjection of the inactive pro-form of cathepsin D into the cytosol
of human fibroblasts and HeLa cells triggers an apoptotic response
(Schestkowa et al., 2007). Cathepsin D may, thus, under certain
circumstances function as an activating adaptor protein, or
alternatively, as an inhibitor of anti-apoptotic factors.
Collectively, these data suggest that cathepsin D is a multi-functional
apoptosis-promoting protein, probably with multiple substrates and
binding-partners still to be discovered.
THE DUAL FUNCTION OF CATHEPSINS IN CANCER INTRA­CELLULAR MEDIATORS OF APOPTOSIS Many anti-cancer therapies trigger cell death by induction of DNA
damage. Consequently, their effect is greatly impaired in cells
defective in p53 (Gasco and Crook, 2003). This is far from optimal as
the p53 gene is mutated in up to 50% of all human tumors (Beroud
and Soussi, 1998). There is, however, redundancy in cell death
pathways, and cancer cells may, thus, be killed by alternative
therapies targeting pathways that remain intact. Consequently,
71
DISCUSSION
elucidation of the new cell death pathways could reveal novel
strategies for combating cancer.
In paper IV, the involvement of lysosomes and cathepsins in oral SCC
cell death was investigated. Collectively, data from this study suggest
that even though cathepsins are released from lysosomes and
contribute to caspase activation, they are not required for Fas- or
oxidative stress-induced cell death to occur. Importantly, oral SCC
cell death could be triggered by MSDH-induced LMP. Thus, despite
being of minor importance in death invoked by Fas and oxidative
stress, the lysosomal death pathway remains intact in both cancer cell
lines. The involvement of lysosomal cathepsins is largely unexplored,
however, cathepsin B was recently found to be involved in TRAILinduced oral SCC cell death (Vigneswaran et al., 2005; Nagaraj et al.,
2006). Interestingly, it was found that primary oral cancer cells were
more susceptible to TRAIL-induced cell death than their metastatic
counterparts, probably due to an increased expression of cystatins in
metastatic cells (Vigneswaran et al., 2005).
There is considerable evidence that cathepsin D is involved in cell
death triggered by multiple conventional anti-cancer agents, such as
adriamycin (Wu et al., 1998), VP-16, cisplatin and 5-fluorouracil
(Emert-Sedlak et al., 2005). Moreover, when screening for new
potential anti-cancer drugs, several compounds that induced cancer
cell death independent of p53 were found to trigger LMP (Erdal et al.,
2005). Since cancer cells seem to be more sensitive to lysosomemediated killing the lysosomal permeabilization pathway could offer a
therapeutic window between cancer cells and normal tissue
(Fehrenbacher et al., 2004).
EXTRA­CELLULAR PROMOTERS OF TUMOR GROWTH AND INVASION Cathepsins secreted from tumor cells are known to participate in
cancer progression by stimulating cancer cell growth and angiogenesis
and facilitating metastatic spread (Liaudet-Coopman et al., 2006;
Mohamed and Sloane, 2006; Gocheva and Joyce, 2007; Vasiljeva and
Turk, 2008) . In paper IV we propose a novel potential tumorpromoting function of cathepsin B. Inhibition of cysteine cathepsin
activity gave a slight reduction in membrane bound Fas in one of the
oral SCC cell lines studied. The ability of cathepsin B to remove Fas
from the cell surface was confirmed by western blot analysis after
72
DISCUSSION
incubation of cells with exogenous cathepsin B. As the human
immune system eliminates cancer cells via ligation of death receptors,
including Fas, on the target cell, shedding of these receptors may
protect from death signals delivered by the immune system. Extracellular metallo- and serine proteases have been implicated in
shedding of Fas and TNF death receptors (Björnberg et al., 1995;
Hino et al., 1999; Xia et al., 2002; Paland et al., 2008), but cathepsinmediated death receptor shedding has, to my knowledge, not earlier
been reported on. Interestingly, Fas death receptor shedding has been
associated with suppression of apoptosis in rat hepatocytes following
partial hepatectomy, suggesting that this could be a process of
physiological importance also in normal tissue (Xia et al., 2002).
Even though cathepsins have two opposing roles in cancer, these
processes occur in different cellular compartments, enabling the
possibility of their selective targeting (Vasiljeva and Turk, 2008).
While cathepsins promote cancer progression when present in the
extra-cellular space, their pro-apoptotic effect is clearly intra-cellular
and relies on their release to the cytosol. Consequently, cancer cell
death could be induced by agents that trigger LMP, while non cellpermeable cathepsin inhibitors could suppress cancer growth and
metastasis in vivo.
73
74
CONCLUSIONS
CONCLUSIONS The following conclusions can be drawn from results presented in this
thesis:
• Lysosomes are destabilized upon STS treatment of human
fibroblasts, and cathepsins are released to the cytosol.
• Cathepsin D, but not cysteine cathepsins, is involved in
propagation of the apoptotic signal in the fibroblast model.
• Bax is translocated to lysosomes upon apoptosis induction, and
may participate in permeabilization of the lysosomal membrane.
• Cytosolic cathepsin D cleaves Bid at Phe24, Trp48, and Phe183,
and thereby promotes Bax-mediated release of cytochrome c,
which in turn leads to caspase activation and cell death.
• Release of cathepsins to the cytosol is accompanied by cytosolic
acidification, which may have a regulatory effect on the cytosolic
cathepsin activity.
• Cathepsins are released to the cytosol of oral SCC cells in response
to oxidative stress, but neither cathepsin D, nor cysteine cathepsins,
are essential for the cell death that follows.
• MSDH-induced release of lysosomal constituents to the cytosol is
sufficient to trigger oral SCC cell death.
• Extra-cellular cathepsin B may engage in shedding of the Fas death
receptor from the cell surface, but inhibition of cysteine cathepsin
activity does not suppress Fas-induced oral SCC cell death.
75
76
TACK
TACK Ett stort tack till alla er som på ett eller annat sätt bidragit till att den
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avhandlingsarbetet! Efter lite kollektivt úk/c#g0r1v$m så
lyckades vi ju till och med att få ordning på arbete 3!
Katarina och Jenny, som guidade mig in i doktorandvärlden. Tack för
er vänskap; utan den hade det inte varit värt att genomlida de ”svarta”
åren. Jag är otroligt glad för att ni ville ställa upp som toastmastrar på
min disputationsfest trots att ni båda har fullt upp. Efter denna
disputation är vår toastmasteri-cirkel sluten☺. Tack också, Katarina,
för att du försökt att ”sälja” mig till diverse forskare på HU nu under
våren när jag själv inte haft ork att engagera mig i mitt liv efter
disputationen (finns det ett sådant?).
Cathrine, som varit ett ovärderligt ”bollplank” och som delat (stökigt)
rum med mig vid diverse konferenser. Rimforsa, Åbo, New York; alla
lika trevliga med dig som ressällskap. Och snart bär det iväg på nya
äventyr…. Får jag en pedant till rumskamrat i Shanghai så flyttar jag
in hos Pigge och dig☺.
Hanna, som redan under sin tid som student imponerade på mig med
sin kunskap, noggrannhet och skarpsinnighet. Tack för att du i min
frånvaro har slitit med att färdigställa arbete III, och för att du med
beundransvärd noggrannhet korrekturläst kappan! Du är med all
säkerhet den enda som någonsin kommer att läsa min bok från pärm
77
TACK
till pärm inte bara en, utan två gånger. Jag lovar att betala tillbaka min
skuld när det om några år blir din tur.
Britt-Marie, som inte bara håller ordning på labben, utan också sprider
sin omtanke till alla som befinner sig i dem. Vad ska det bli av
patologen när du försvinner?!
Alla medarbetare på patologen, plan 9, för intressanta diskussioner,
avkopplande fikastunder och trevliga pub-, pärlpyssel- och
matlagningsträffar.
Camilla, som varit min följeslagare genom biologi- och
forskarskolestudierna. Tack för alla ”terapisamtal” både av
yrkesmässig och privat karaktär. Det ska bli kul att snart ha er tillbaka
på den här sidan jordklotet!
Alla mina vänner, som hjälpt mig att ”vila hjärnan” under
avhandlingsarbetet. Ni har blivit försummade på senare tid, men jag
lovar bättring!
Mamma och pappa, som alltid intresserat lyssnar på mina utläggningar
om med- och motgångar på labbet. Ni har alltid stöttat och uppmuntrat
mig. Bättre föräldrar än ni kan inte finnas! Jag ser verkligen fram
emot att återigen kunna tillbringa mer tid med tjejerna hos ”mormor
och morfar” i Kättilstad.
Familjen Wiström, som från första dagen behandlade mig som en i
familjen. Ryda är verkligen ett toppenställe där kan jag koppla av och
släppa tankarna på jobbet.
Och sist, men inte minst, min familj; Nicke, Mira och Isa, som alltid
finns vid min sida. Jag älskar er! ♥
Denna studie har genomförts med ekonomiskt stöd från
Cancerfonden, Vetenskapsrådet, Östergötlands läns landsting, Lions
forskningsfond mot folksjukdomar, Svenska Sällskapet för Medicinsk
Forskning (Curth Nilssons stiftelse) och Knut och Alice Wallenbergs
jubileumsfond.
78
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