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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 18, 2001 as doi:10.1096/fj.00-0708fje. |
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Division of Pathology II, Faculty of Health Sciences, Linköping University, Linköping, Sweden
2Correspondence: Division of Pathology II, University Hospital, S-581 85 Linköping, Sweden. E-mail: katka{at}inr.liu.se
SPECIFIC AIMS
The overall objective of the present study was to examine lysosomal stability during apoptosis and the relationship between caspase activation and the lysosomal proteases cathepsins D and B with regard to their effects on apoptosis.
PRINCIPAL FINDINGS
1. Naphthazarin-induced apoptosis is dependent on the lysosomal
protease cathepsin D
The caspase-3-like activity increased after 12 h of treatment
with the redox cycling quinone naphthazarine (NZ). By comparison, the
activity of cathepsin D was augmented after 4 h of NZ treatment
and peaked at 16 h (Fig. 1A
, B
). Moreover, increased levels of p53, a transcription
factor for cathepsin D, were detected after exposure to NZ for 4 h
(Fig. 1D
). The total protein level did not change in
response to p53 induction and was estimated to 8.2 ± 3.5,
8.0 ± 2.1, and 7.1 ± 1.4 µg protein/10 000 cells after 0,
12, and 20 h respectively. We also found that NZ caused a rapid
decrease in the activity of the lysosomal cysteine protease cathepsin B
(Fig. 1C
).
|
Pretreatment of fibroblasts with the cathepsin D inhibitor pepstatin A or the caspase-3-like inhibitor Ac-DEVD-CHO, but not with the cathepsin B inhibitor CA-074 Me, inhibited activation of caspase-3 (not shown). To investigate the link between cathepsin D and caspase-3, we measured cathepsin D activity while inhibiting caspase-3 with Ac-DEVD-CHO. The results show no significant change in cathepsin D activity (not shown). Furthermore, pepstatin A did not inhibit active recombinant caspase-3 (not shown).
2. The lysosomal proteases cathepsins D, B, and L are translocated
to the cytosol early during apoptosis
The role of cathepsin D in the apoptotic process has been
documented, although the subcellular localization of the protease
during such cell death was not established in these studies. Using
immunocytochemistry, we detected granular staining of cathepsin D in
control cells (Fig. 2A
). After 30 min of NZ treatment, fluorescence staining was
more diffuse, indicating that cathepsin D had started to translocate
from lysosomes to the cytosol; after 1 h, most of the cathepsin D
was detected in the cytosol (Fig. 2B
). These results are
consistent with a previous study in our laboratory, in which
immunotransmission electron microscopy revealed the presence of
cathepsin D in lysosomes in control cells and translocation of the
enzyme to the cytosol after NZ exposure. NZ-exposed cells tend to
shrink and the fluorescence appear to be nuclear. Since we have never
detected cathepsin D in nuclei by using immunotransmission electron
microscopy, we believe that the fluorescence is not truly nuclear but
arises from cytosolic cathepsin D. Figures 2C
–D
show that
treatment with pepstatin A or Ac-DEVD-CHO did not prevent cathepsin D
translocation to the cytosol. The cathepsins B and L were similarly
relocated from the lysosomes during the first hour of NZ exposure (Fig. 2E
–H
).
|
Early translocation of the lysosomal protease cathepsin D to the cytosol could be caused by the generation of free radicals and increased oxidative stress due to redoxcycling of NZ. The generation of ROS production in our experimental system, measured as luminol-enhanced chemiluminescence, was enhanced 1.5-fold after 15 min of NZ treatment; a simultaneous decrease in the intracellular concentration of GSH was detected. Thereafter, the chemiluminescence declined and the GSH level was restored. Cells pretreated with pepstatin A showed the same reaction pattern, which indicates that pepstatin A does not act as a free radical scavenger (not shown).
CONCLUSIONS AND SIGNIFICANCE
Cathepsin D is a major cellular aspartic protease that has numerous functions within the lysosomal compartment, the best known of which is proteolysis of endocytosed and autophagocytosed proteins at low pH. Earlier it was generally assumed that lysosomes were stable during apoptosis because they appear to be ultrastructurally intact in apoptotic cells. Lysosomal rupture has instead been considered to take place in necrosis. We found, however, that lysosomes participate in apoptosis through early translocation of the lysosomal protease cathepsin D to the cytosol. During NZ-induced apoptosis, we propose that lysosomal membranes are exposed to increased amounts of reactive oxygen species that might initiate lipid peroxidation reactions in intracellular membranes. One present hypothesis describes lysosomal membrane damage originating from intracellular production of hydrogen peroxide, which might diffuse into the lysosome. Inside the lysosomal apparatus, low molecular weight iron, the acidic milieu, and the occurrence of the reducing amino acid cysteine would promote iron reduction and Fenton-like chemistry, destabilizing the lysosomal membranes and thereby causing lysosomal leakage. It has also been found that atractyloside, which is commonly used to induce the mitochondrial permeability transition and release of cytochrome c from mitochondria, could induce release of cathepsin B from isolated lysosomes. This observation raises the possibility that similar mechanisms of pore opening might exist in mitochondria and lysosomes.
Free radicals were generated during treatment with NZ, but the overall
redox state of the cell decreased very slowly. We have previously
observed an initial and rapid fluctuation in both ATP and mitochondrial
membrane potential (
m) in fibroblasts
exposed to NZ. In that study, the cathepsin D inhibitor pepstatin A
blocked the initial depletion of ATP and release of cytochrome
c. The cytosolic targets for cathepsin D have not been
ascertained, although it is possible that this enzyme has an effect
exerted directly on mitochondrial function.
Cathepsin D showed augmented activity soon after it was released and was accompanied by an increased level of p53 protein, which is a cathepsin D transcription factor. The mechanism responsible for increase in CD activity might be an effect of increased synthesis regulated by p53. Both the release of cathepsin D and a significant increase in cathepsin D activity were detected before caspase-3 was activated.
Results reported in the literature indicate that an increase in the cytosolic concentration of cathepsin D may have a specific effect on apoptosis. First, unlike the cysteine lysosomal proteases, the aspartic proteases have no counteracting endogenous cytosolic inhibitors that limit extralysosomal proteolysis. Second, assays in vitro have shown that cathepsin D is stable in the pH range 1–9 and displays significant activity above pH 6.5. Consequently, cathepsin D may mediate apoptosis by cleaving cytosolic substrates, although our preliminary data indicate that caspase-3 is not activated directly by cathepsin D (K. Kågedal et al., unpublished data). In a recent study of cathepsins B, H, K, L, S, and X, no direct activation of caspase zymogenes was found. Instead, an indirect mode of caspase activation by lysosomal proteases was found through Bid cleavage. Bid was cleaved in the presence of lysosomal extracts, and incubation of mitochondria with Bid that had been cleaved by lysosomal extract resulted in cytochrome c release (Stoka et al., 2001)
In our study, examination of morphology and caspase-3 activity showed that apoptosis was prevented by the cathepsin D inhibitor pepstatin A and the caspase-3 inhibitor Ac-DEVD-CHO. If caspase-3 and cathepsin D are activated in parallel pathways, inhibition of cathepsin D will not affect caspase activation. However, such an effect did occur in our experiments, which strongly suggests a connection between cathepsin D and caspases in NZ-induced apoptosis and that cathepsin D apparently is involved upstream of the caspase cascade. Since apoptosis could be prevented for only 24 h using a caspase-3 inhibitor, we speculate that the loss of cytochrome c might cause cell death in this case. An earlier report from our group showed that inhibition of cathepsin D prevented cytochrome c release, which might explain the longer survival time using an cathepsin D inhibitor. These conclusions are further strengthened by our observation that Ac-DEVD-CHO did not inhibit the increase in cathepsin D activity. It is also possible that besides its role in apoptosis induction, cathepsin D further exacerbates the apoptosis process in the later stages due to an amplification loop.
Taken together our data strongly implicate the lysosomal protease cathepsin D in apoptosis induction and potentiation, and further support the emerging picture of cathepsin D as an important mediator of programmed cell death.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0708fje ; to cite this
article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0708fje 
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0.05, calculated by the
Mann-Whitney U test. C) Cathepsin B
activity analyzed using z-Arg-Arg-AMC as a substrate; 1 µ will
liberate 1 nmol/min at pH 6.0 at 40°C. D) Immunoblot
analysis of p53 during NZ treatment. Values are means ±
SD, n = 4. NZ-exposed cells (
);
unexposed cells (•).
