- Email: [email protected]
A role for the lysosomal protease cathepsin B in zebrafish follicular apoptosis
Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
A role for the lysosomal protease cathepsin B in zebrafish follicular apoptosis Angela J. Eykelbosh, Glen Van Der Kraak ⁎ Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1
a r t i c l e
i n f o
Article history: Received 6 January 2010 Received in revised form 4 February 2010 Accepted 8 February 2010 Available online 16 February 2010 Keywords: Zebrafish reproduction Ovary Caspase-3 Cathepsin B Atresia Apoptosis
a b s t r a c t This study presents evidence that cathepsin B, a lysosomal protease, may be involved in the regulation of apoptosis during serum-starvation in teleost follicles. Zebrafish vitellogenic follicles were isolated, incubated under serum-free conditions and homogenized. The follicle extracts demonstrated caspase-3-like activity using the fluorogenic substrate DEVD-AMC, indicating the onset of apoptosis. Cathepsin B activity as measured using the fluorogenic cathepsin B substrate, Z-Arg-Arg-AMC was elevated within the first 6 h of incubation in serum-free media and coincided with the onset of apoptosis. This increase in cathepsin B activity was sensitive to the cathepsin B inhibitor, CA-074-ME. Furthermore, adding CA-074-ME to the follicle incubation blocked caspase-3-like activation, suggesting that cathepsin B activity is a positive regulator of the apoptotic cascade during serum-starvation. Interestingly, the increase in cathepsin-B-like activity was not preceded by an increase in cathepsin B mRNA transcription, suggesting that regulation of this enzyme is at a level other than of the gene. These results suggest a regulatory role for cathepsin B during follicular apoptosis in zebrafish ovarian follicles. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Vitellogenesis is a key process in fish oogenesis that involves the synthesis of vitellogenin in the liver and its transport to and internalization within developing oocytes. After internalization, vitellogenin is processed by lysosomal proteases (e.g., cathepsins B, D, and/or L) into several functionally important yolk proteins (lipovitellins, phosvitins, the β'-component, and the C-terminal peptide; Hiramatsu et al., 2002; Matsubara et al. 2003). These are in turn important for the hydration of oocytes during maturation, the subsequent buoyancy of spawned eggs, and as an energy source for nascent embryos (Carnevali et al., 2006; Hiramatsu et al. 2006; Raldua et al. 2006). Thus, lysosomal proteases play dual roles in oogenesis and contribute to overall reproductive success. It is important to note, however, that not every oocyte is destined to become an embryo. In fish, as in all other vertebrates, the vast majority of ovarian follicles are prematurely culled in a process known as follicular atresia; this culling is thought to promote the energetic investiture and ovulation of the most viable follicles. Atresia is a controlled process that proceeds via an apoptotic mechanism, resulting in the elimination of “doomed” follicles without inflammatory damage to the surrounding tissues. However, because teleost oocytes contain large quantities of yolk and are produced in large numbers, the degradation and recovery of this valuable energy store is of key interest. Indeed, there is a visible liquefaction of the yolk proteins during the first stages of follicular atresia (Saidapur, 1978). This observation led investigators to postulate
⁎ Corresponding author. Tel.: + 1 519 824 4120x53424; fax: + 1 519 837 2075. E-mail address: [email protected] (G. Van Der Kraak). 1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2010.02.005
that the initial stages of teleost atresia may require proteolytic enzymes, such as cathepsins B, D, and L, to cleave yolk proteins before atresia can proceed (Wood and Van Der Kraak, 2003). In sea bream eggs, apoptotic activation was associated with increased cathepsin D activity (Carnevali et al., 2003) and serum-starved, apoptotic rainbow trout follicles showed a rise in cathepsin L-like activity and increased free amino acid concentrations (Wood and Van Der Kraak, 2003). Furthermore, it was shown that the mRNA expression of a novel salmonid oocyte cysteine protease inhibitor (OCPI) decreased during apoptosis (Wood et al., 2004) and this protein was previously shown to inhibit calpain and cathepsins B and L in chum salmon eggs (Yamashita and Konagaya, 1991). This connection between apoptosis and lysosomal protease activation is interesting given the accumulating evidence that cathepsin B plays a direct role in the regulation of the apoptotic cascade. Research in mammals has shown that cathepsin B contributes to cell death by either activating apoptotic factors upstream of executioner caspases (Stoka et al., 2001) or by binding to and activating caspases directly (Schotte et al., 1998; Vancompernolle et al., 1998; Canu et al., 2005). In addition, cathepsin B appears to be involved in caspase-independent cell death. In non-small cell lung cancer cells, it was found that cathepsin B activation was necessary for the induction of cell death, whereas caspase activation was not (Broker et al., 2004). Finally, cathepsin B may assume the function of caspases in the cleavage and deactivation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP-1) (Gobeil et al., 2001). Taken together, the evidence from both caspase-dependent and caspase-independent cell death research indicates that cathepsin B plays a major regulatory role in multiple aspects of mammalian cell death.
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223
219
Adult male and female zebrafish (Danio rerio) were obtained from DAP International (Etobicoke, ON, Canada). Fish were kept at 25–28 °C under a photoperiod of 14L:18D. Fish were fed daily with a commercial pellet food for salmon fry (Martin Mills, Elmira, ON, Canada), with a weekly supplementation of blood-worms (Oregon Desert Brine Shrimp Co., Lakeview, OR, USA). Females were segregated at sexual maturity.
generated in the caspase activity assay was deemed “caspase-3-like” because the effects of other caspases cannot be excluded by this assay. For the caspase-3 assay, samples of follicles were incubated 2 to 3 min on ice with 175 µL of lysis buffer (50 mM HEPES, 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA, pH 7.4). They were then homogenized with a Kontes hand-held homogenizing gun and centrifuged for 10 min at 10 000 g. The supernatant was drawn off and put into a fresh tube. A 50-µL aliquot of each sample was plated on ice into a 96-well opaque plate (Fisher Scientific, Toronto ON, Canada). A 50-µL aliquot of assay buffer was added (50 mM HEPES, 0.1% CHAPS, 10 mM DTT, 1.0 mM EDTA, 100 mM NaCl, 0.1% glycerol, pH 7.4) followed by 100 µL of the substrate solution (0.06 mM DEVD-AMC, made up in assay buffer). Cathepsin B activity was assayed under slightly different conditions. The assay was based on an earlier cathepsin B assay (Barrett, 1980), which was then adapted and optimized for micro-scale work using the substrate benzyloxycarbonyl–Arg–Arg-7-amino-4-methylcoumarin (Z-RR-AMC; Sigma). Samples of follicles were incubated 2 to 3 min on ice with 325 µL of lysis buffer (352 mM KH2PO4, 0.1% Brij-35, 48 mM Na2HPO4, 4 mM EDTA, pH 6.0). These were then homogenized with a Kontes hand-held homogenizer and centrifuged for 10 min at 10,000 g. The supernatant was drawn off and put into a fresh tube. A 100-µL aliquot of each sample was plated on ice into a 96-well opaque plate. A 50-µL aliquot of assay buffer was added (352 mM KH2PO4, 48 mM Na2HPO4, 4 mM EDTA, 24 mM Cys-base, pH 6.0) followed by 50 µL of substrate solution (0.04 mM Z–RR–AMC, made up in lysis buffer). The plates were read at 28 °C using a FLx Microplate Fluorescence Reader (Bio-Tek Instruments, Winooski, VT, USA). The caspase-3 assay was measured for 100 min and the cathepsin B assay for 60 min. Measurements were taken at 5-min intervals and the activity of each well was expressed as a maximum slope (using values for the linear range only). Fluorescence of the samples was measured in triplicate and normalized against protein content using the Bio-Rad protein assay (Bio-Rad Laboratories, Mississauga, ON, Canada).
2.3. Follicle isolation and incubation
2.5. Assessment of selected mRNA expression by real time RT-PCR
Female fish were sacrificed and their ovaries removed and placed in L-15 medium (Gibco, Burlington, ON, Canada) warmed to 28 °C and supplemented with a final concentration of 100 µg/mL streptomycin and 100 U/mL penicillin to discourage bacterial growth. Follicles were separated and sorted according to size range and physical characteristics. Vitellogenic follicles (opaque, unbroken follicles, 0.4–0.7 mM in diameter) were pooled together and then groups of 25 were allotted to treatment groups. Information on staging zebrafish follicles was taken from Selman et al. (1993). Follicles were incubated in 24-well sterile polystyrene tissue culture plates with 1 mL of L-15 medium. The medium was “serum-free”, meaning it contained no growth factors, hormones or other survival factors. Serum-free incubation is a widely accepted model for inducing atresia and has been shown to cause apoptosis in vitellogenic follicles from rainbow trout (Wood and Van Der Kraak, 2001) and zebrafish (Eykelbosh and Van Der Kraak, submitted for publication). Samples of pooled follicles were allotted to treatment groups, incubated in the dark for the specific period at 28 °C, and then snapfrozen until analysis. Follicles were incubated for 6 h for all experiments where cathepsin B-like activity is measured, and for 8 h where caspase-3-like activity was measured.
RNA was extracted using the guanidium thiocyanate-phenolchloroform method as previously described (Chomczynski and Sacchi, 1987) and processed as described by Ings and Van Der Kraak (2006). Yield was quantified by absorbance at 260 nm and the purity assessed as a ratio between the absorbances at 260 and 280 nm. For the reverse transcriptase reaction, one microgram (1 µg) of RNA in diethyl pyrocarbonate (DEPC) water (10 µL) was incubated at 70 °C for five min with 0.1 ng (2 µL) of random hexamer primer. The samples were then placed on ice while 13 µL of reaction cocktail was added to them. The constituents of the cocktail and their final concentrations in 25 µL are as follows: dNTPs (0.5 mM), 5 × RT buffer (50 mM Tris–HCl, 75 mM KCl, 3 mM MgCl2), dithiothreitol (10 mM), RNase Inhibitor (40 U), Moloney murine leukemia virus reverse transcriptase (M-MLV 200 U), and DEPC water. The samples were incubated at 37 °C for 1 h and then at 95 °C for 5 min. The RT product was stored at −20 °C until real time PCR was performed. All real time PCR analyses were carried out on an ABI Prism Sequence Detection System 7000 using the accompanying SYBR Green PCR master mix. Very briefly, 5 µL of cDNA, 10 µL of the SYBR Green master mix, and 5 µL of primers (final concentration of 50 nM each) were plated out in specialized 96-well Thermowell Gold PCR plates. The plates were sealed using optical adhesive covers and run in the machine on the following program: 2 min at 50 °C, 10 min at 95 °C for polymerase activation, and then 40 cycles of 15 s at 95 °C for denaturation and 1 min at 60 °C for annealing and extension. Cathepsin B, D, and L mRNA expression was measured and normalized against zebrafish β-actin expression. All products mentioned above, with the exception of the cDNA and primers, were designed to be compatible with the ABI Prism 7000 system and are available from
The research described above suggests that cathepsin B may be doubly important for teleost follicular apoptosis, both as a regulator of the apoptotic cascade and as a facilitator of energy resorption. This study examined the activity and expression of cathepsin B to determine whether this enzyme is involved in the regulation of apoptosis in zebrafish vitellogenic follicles. Isolated follicles were incubated under serum-free conditions which are known to induce caspase-3 activation and apoptosis (Wood and Van Der Kraak, 2001; Eykelbosh and Van Der Kraak, submitted for publication) and the resulting cathepsin B enzyme activity was measured. To functionally link cathepsin B activity to caspase activation, follicles were incubated with the cell-permeable specific cathepsin B inhibitor Ca-074-Me and then caspase-3-like activity was measured. Finally, we used real time RT-PCR to determine whether apoptosis was associated with increased mRNA expression of cathepsins B, D, and L in follicles following serum-free incubation. 2. Materials and methods 2.1. Chemicals All substrates, hormones, inhibitors and human cathepsin B were obtained from Sigma (Oakville, ON, Canada), unless otherwise noted. Ca-074-Me, a specific cell-permeable inhibitor of cathepsin B, was obtained from the Peptide Institute (Osaka, Japan). 2.2. Animal maintenance
2.4. Enzyme activity assays Activity of the executioner enzyme caspase-3 was used as an endpoint to measure the induction of apoptosis. This involved use of the fluorogenic substrate acetyl–Asp–Glu–Val–Asp-7-amino-4methylcoumarin (DEVD-AMC; Sigma), which is known to be cleaved by recombinant zebrafish caspase-3 (Yabu et al., 2001). The activity
220
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223
Applied Biosystems (Foster City, CA, USA). Primers were generated based on known zebrafish sequences using Primer Express (Applied Biosystems). The sequences and origins of these primers are listed in Table 1. Efforts were also made to amplify OCPI using real time RT-PCR. 2.6. Statistical analyses In an experiment with multiple treatment groups, the differences between control and treatment values were analyzed using a one-way ANOVA followed by a Tukey or Dunnett's t-test. The values presented are mean ± standard error. Where a Tukey t-test is used to detect differences between treatments, letter labels are used to group treatments that are not significantly different from each other. Where a Dunnett's t-test is used, an asterisk indicates that the treatment value is significantly different compared to the control. On the sole occasion where data were not normally distributed, a non-parametric Kruskal– Wallis test, followed by a Mann–Whitney test, was used to detect differences between the control and treatment groups. For all tests, the differences are considered significant at p b 0.05. Where only two groups were compared, the difference between control and treatment values was tested using a two-tailed t-test. The values presented are the mean ± standard error, and an asterisk in the graphs is used to indicate that a treatment value was significantly different from its corresponding control value. Two-tailed t-tests were considered significant when p b 0.05. Furthermore, statistical analysis was also applied to repetitions of the same experiment. In this case, a paired t-test compares each treatment value against its own control, and then determines if the mean difference in control vs. treatment is greater than zero over a series of experiments. Therefore, a pair-wise-test measures how consistent responses are throughout the series of experiments. Pair-wise t-tests were considered significant when p b 0.05.
Fig. 1. Effect of serum-starvation on cathepsin B-like activation in zebrafish follicles incubated for 6 h under serum-free conditions. Cathepsin-B-like activity was measured and compared to an un-incubated control (T0) by means of an independent samples t-test. An asterisk indicates that control and 6 h values are significantly different within one experiment. Experiments were repeated four times, and the difference across the series of experiments was compared using a paired t-test. ppaired = 0.003. Data are presented as the mean caspase-3-like activity± standard error, n = 3–6.
3.2. Prevention of cathepsin B-like activity with survival factors To investigate the effect of survival factors on cathepsin-B-like activation, follicles were incubated with or without 15 IU/mL human chorionic gonadotropin (hCG) and 1 ng/mL 17β-estradiol (E2). These doses were chosen based on previous studies (Eykelbosh and Van Der Kraak, submitted) showing that these hormones prevent caspase 3-activation. Neither hCG nor E2 caused a significant change in cathepsin B-like activity (Fig. 3). 3.3. Caspase-3-like activity after incubation with Ca-074-Me
3. Results 3.1. Cathepsin B activity in serum-starved follicles Fig. 1 shows that there is a significant increase in cathepsin B activity over six h of serum-starvation, compared to a time zero (T0) control. The rise in activity was consistent over four separate experiments (ppaired = 0.003). Based on these results, all further experiments looking at cathepsin B activation used an incubation period of 6 h. In order to confirm that this was indeed cathepsin B activation, samples were incubated as usual and then cathepsin B-like activity was assayed with or without 25 µM Ca-074-Me, a specific cathepsin B inhibitor in mammals (Buttle et al., 1992). Fig. 2 demonstrates that treatment with inhibitor abolished only the changes seen over time. This suggests that while there is some non-specific cleavage of the substrate, the changes observed are likely due to cathepsin B activity.
To investigate the possibility of a functional link between cathepsin B activation and caspase activity, follicles were incubated with or without 10 µM Ca-074-Me for 8 h at 28 °C and then the samples were assayed for caspase-3 like activity. Addition of Ca-074-Me caused a significant decrease in caspase-3-like activity compared to 8 h untreated follicles (Fig. 4). This suggests that cathepsin B may play a role in the activation of the executioner caspase-3.
Table 1 Primer sequences for the genes of interest and the Accession numbers of the sequences from which they were derived. Gene of interest
Accession #
Primer sequence (F = forward, R = reverse)
Cathepsin B
AY398331
Cathepsin D
AS278268
Cathepsin L
NM_131198
OCPI
AY094359
β-Actin
AF025305
F: TGAT GCAA GAGA GCAG TGGC R: AGAT AGCT TCGG CAGC TCCA F: TTGG CACT CCTG TCCA GACC R: CAAG CAGGCGAT GTCA GTCA F: ATGT TTGC TTTG CTCG TCAC R: TGCT GGCT CTTC CAGG AGTT F: GGAA GAGT GCAG AAGG AGAC R: AGCA GTGC CTGG TGGA GTCT F: ACAG GGAA AAGA TGAC ACAG ATCA R: CAGC CTGG ATGG CAAC GTA
Fig. 2. Representative graph depicting the effect of the cathepsin B inhibitor Ca-074-Me (25 µM) on cathepsin B-like activity in zebrafish follicles. Follicles were incubated for 6 h under serum-free conditions and then the cathepsin B assay was performed with or without 25 µM Ca-074-Me. Data were analyzed using a one-way ANOVA followed by a Tukey t-test. Similar letter labels indicate that values are not significantly different from each other. Data are presented as the mean caspase-3-like activity ± standard error, n = 3.
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223
221
3.4. Expression of lysosomal proteases in the follicle Follicles were incubated for 2 h in serum-free medium and then snap-frozen. This incubation period was chosen to accommodate the translation and protein processing that would presumably occur before the observed activation at 6 h. RNA isolation and real time PCR were performed. Cathepsin B, D, and L expressions were apparent at time zero; however, there was no significant change in expression for any of the cathepsins over the 2-h incubation period (Fig. 5). In order to investigate this further, follicles were incubated for extended time periods (0, 4, 8, 16, and 24 h). Once again, cathepsin B mRNA expression decreased over time, but this difference did not become significant until 24 h of serum-starvation (Fig. 6). These results demonstrate that the increase in cathepsin B enzyme activity is not due to an increase in gene expression.
3.5. Cloning of OCPI Given that we found an increase in cathepsin B activity without a concomitant increase in cathepsin B expression, we considered the possibility that increased cathepsin B activity may be due to decreased expression of an inhibitor such as OCPI. However, primers designed from the recently cloned rainbow trout gene (Wood et al., 2004; Table 1) failed to identify any homologous sequences in zebrafish.
Fig. 3. Effect of human chorionic gonadotropin (hCG; A) and 17β-estradiol (E2; B) on cathepsin B-like activation in zebrafish follicles incubated for 6 h under serum-free conditions. Data were analyzed using a one-way ANOVA followed by a Tukey t-test. Similar letter labels indicate that values are not significantly different from each other. Experiments were repeated 1–2 times. Data are presented as the mean caspase-3-like activity ± standard error. For hCG experiments, n = 6–8. For E2 experiments, n = 3. Fig. 5. Effect of serum-starvation on cathepsin mRNA expression in zebrafish follicles incubated for 2 h under serum-free conditions. Data were analyzed by means of an independent samples t-test and no significant differences were found between control (0 h) and 2 h of serum-free incubation. Data points show mean normalized gene expression ± standard error, n = 6.
Fig. 4. Effect of the cathepsin B inhibitor Ca-074-Me on caspase-3-like activity in zebrafish follicles incubated for 8 h under serum-free conditions, with or without addition of 10 µM Ca-074-Me. Caspase-3-like activity in treated samples was measured and compared to the 0 h control and the untreated 8 h control. Data were analyzed using a one-way ANOVA followed by a Tukey t-test. Similar letter labels indicate that values are not significantly different from each other. Data are presented as the mean caspase-3-like activity ± standard error, n = 4–6.
Fig. 6. Effect of serum-starvation on cathepsin B mRNA expression in zebrafish follicles incubated for various times under serum-free conditions. Because data were not normally distributed, a non-parametric Kruskal–Wallis test followed by a Mann– Whitney test was used to detect differences between the control (0 h) and the other time points. An asterisk indicates a significant difference. Data points show mean normalized gene expression ± standard error, n = 4.
222
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223
4. Discussion 4.1. Cathepsin B activity increases with serum-starvation A common hurdle encountered in comparative physiology is the validity of methods used to compare processes between different species. In this case, the consideration was whether using a specific mammalian cathepsin B substrate would give reliable results in zebrafish. The choice of substrate is reliable for two reasons. First, the protein sequence shared between zebrafish and rat cathepsin B is highly conserved (84% similarity). Secondly, assaying the samples with a specific cathepsin B inhibitor abolished the change in activity observed over 6 h. The 25 µM dose of Ca-074-Me was not sufficient to abolish all activity, meaning that some non-specific cleavage remained; however, cathepsin B inhibitor treatment consistently reduced starvation-induced activity to baseline. This observation and the similarity between protein sequences suggest that the activity shown here is due to cathepsin B. Further study is required to more fully characterize zebrafish cathepsin B and to develop an assay that would exclude non-specific activity. 4.2. Role of cathepsin B in apoptotic signalling in serum-starved zebrafish follicles Mammalian studies have clearly shown that cathepsin B is an active regulator of apoptosis. In a human cell line, incubation with the purified cathepsin B led to the cleavage of the pro-apoptotic protein Bid, increased cytochrome-c release, and caspase activation (Stoka et al. 2001). However, very little information is available regarding the apoptotic mechanism at work in serum-starved follicles in fish. In the present study, cathepsin B activity showed an increase at 6 h, prior to the rise in caspase activity at 8 h observed in previous studies (Eykelbosh and Van Der Kraak, submitted). Furthermore, the application of a specific inhibitor completely abolished caspase-3-like activation, indicating cathepsin B activation is involved in the onset of apoptosis. These results agree with a previous study showing that cathepsin B release from the lysosome leads to cytochrome-c release and caspase activation (Guicciardi et al., 2000). Moreover, Canu et al. (2005) found that activation of autophagolysosomes results cathepsin B activation and colocalization with procaspase-3 in the cytosol, demonstrating that caspase-3 activation was cathepsin B-dependent. In previous studies, however, caspase-3-like activation in serumstarved zebrafish follicles could be avoided through the application of specific survival factors, such as 17β-estradiol, and this anti-apoptotic effect appeared to be mediated through the α isoform of the estrogen receptor in a manner involving inhibition of the mitogen-activated protein kinase, ERK (Eykelbosh and Van Der Kraak, submitted). In the present study, cathepsin B activation was apparently insensitive to survival factors (hCG and 17β-estradiol), suggesting that the mentioned survival factors exert their effects downstream of cathepsin involvement. Thus, although results provide further evidence of a functional link between cathepsin B and caspase activation, the signalling pathway connecting these two events remains to be elucidated. Additional studies are required to determine at which point survival factors disrupt this cell death pathway. 4.3. Cathepsin B expression is unaffected by serum-starvation Cathepsin B mRNA did not show an increase in expression with serum-starvation; rather, mRNA expression showed a strong declining trend that became significant after 24 h of serum-starvation. The observation that, despite the lack of change in expression, cathepsin B enzyme activity still increased after 6 h of serum-starvation indicates that regulation is not at the level of gene transcription. Other studies have suggested that ovarian cathepsins may be regulated post translationally and, accordingly, transcript abundance may not
correlate with cathepsin enzymatic activity. For example, Kwon et al. (2001) reported that cathepsin B and L were highly expressed during oogenesis, and yet previous studies in the same model (rainbow trout) did not detect cathepsin B or L activity during the same period (Sire et al. 1994). These results may reflect the early accumulation of cathepsin B mRNAs or precursor proteins that is later followed by regulation at a higher level or release from the lysosome. There is evidence that the lysosome becomes permeable during the early stages of apoptosis (Roberg et al. 1999; Kagedal et al., 2001) and other studies have demonstrated that cathepsin B is translocated to the cytosol during apoptosis (Boya et al., 2003; Canu et al. 2005; Foghsgaard et al. 2005). Many levels of regulation are possible and relatively few details are known; however, this study argues against increased cathepsin B mRNA transcription (leading to cathepsin B activation) during serum-starvation induced apoptosis. Another explanation for an increase in enzymatic activity without gene transcription may be that serum-starvation causes a decrease in the transcription or translation of endogenous inhibitors. We were unable to clone OCPI out of zebrafish vitellogenic follicles, perhaps indicating that OCPI is a uniquely salmonid protein (Bobe and Goetz, 2001; Yabu et al., 2001; Wood et al., 2004). However, there are a number of other potential endogenous inhibitors for cathepsin B activity in the follicle, including members of the cystatin and the thyrotropin families; these are reviewed in Turk and Bode (1991) and Lenarčič and Bevec (1998), respectively. Cystatins that are homologous to the mammalian proteins have been cloned out of rainbow trout (Li et al., 2000) and carp (Tsai et al., 1996), and have shown inhibitory activity against papain, a cysteine protease closely related to cathepsin B. A better insight into cathepsin B regulation and apoptosis may be gained by studying the expression of these inhibitors. 4.4. Conclusions This study has demonstrated that zebrafish follicles subjected to serum-starvation show an increase in cathepsin B-like activity at a point in the time scale that would permit potential modulation of caspase-3-like activity. In confirmation of this, incubation with a specific cathepsin B inhibitor decreased caspase-3-like activity. This cathepsin B activity was not sensitive to survival factors, suggesting that cathepsin B activation is upstream of the checkpoint regulated by these survival factors. Finally, this study suggests that regulation of cathepsin B activity during apoptosis is not at the level of gene transcription; instead, it may be that cathepsin B is regulated either at the level of protein processing, release of the enzyme, or by a change in expression of an endogenous inhibitor. This work provides the first example of cathepsin B activation during atresia and regulation of caspase-3-like activity in teleosts. Acknowledgements This work was supported by an NSERC Discovery Grant to GVDK. AJE was supported by an NSERC Postgraduate Scholarship. Thanks are also due to Dr. R. Mosser and the lab for the use of their equipment. References Barrett, A., 1980. Fluorimetric assays for cathepsin B and cathepsin H with methycoumarylamide substrates. Biochem. J. 187, 909–912. Bobe, J., Goetz, F.W., 2001. Cysteine protease inhibitor is specifically expressed in preand early-vitellogenic oocytes from the brook trout peri-ovulatory ovary. Mol. Reprod. Dev. 60, 312–318. Boya, P., Gonzalez-Polo, R., Poncet, D., Andreau, K., La Vieira, H., Roumier, T., Perfettini, J., Kroemer, G., 2003. Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine. Oncogene 22, 3927–3936. Broker, L.E., Huisman, C., Span, S.W., Rodriguez, J.A., Kruyt, F.A.E., Giaccone, G., 2004. Cathepsin B mediates caspase-independent cell death induced by microtubule stabilizing agents in non-small cell lung cancer cells. Cancer Res. 64, 27–30. Buttle, D.J., Murata, M., Knight, C.G., Barrett, A.J., 1992. CA074 methyl ester: a proinhibitor for intracellular cathepsin B. Arch. Biochem. Biophys. 299, 377–380.
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223 Canu, N., Tufi, R., Serafino, A.L., Amadora, G., Ciotti, M.T., Calissano, P., 2005. Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells. J. Neurochem. 92, 1228–1242. Carnevali, O., Polzonetti, V., Cardinali, M., Pugnaloni, A., Natalini, P., Zmora, N., Mosonci, G., Polzonetti-Magni, A.M., 2003. Apoptosis in Sea Bream Sparus aurata eggs. Mol. Reprod. Dev. 66, 291–296. Carnevali, O., Cionna, C., Tosti, L., Lubzens, E., Maradonna, F., 2006. Role of cathepsins in ovarian follicle growth and maturation. Gen. Comp. Endocrinol. 146, 195–203. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. Eykelbosh, A, J., Van Der Kraak, G. Apoptosis in serum-starved zebrafish ovarian follicles. Mol. Reprod. Dev. (submitted for publication). Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bastholm, L., Boes, M., Elling, F., Leist, M., Jäättelä, M., 2005. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J. Cell Biol. 153, 999–1009. Gobeil, S., Boucher, C.C., Nadeau, D., Poirier, G.G., 2001. Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases. Cell Death Differ. 8, 588–594. Guicciardi, M.E., Deussing, J., Miyoshi, H., Bronk, S.F., Svingen, P.A., Peters, C., Kaufmann, S.H., Gores, G.J., 2000. Cathepsin B contributes to TNF-α-mediated hepatocytes apoptosis by promoting mitochondrial release of cytochrome-c. J. Clin. Invest. 106, 1127–1137. Hiramatsu, N., Ichikawa, N., Fukada, H., Fujita, T., Sullivan, C.V., Hara, A., 2002. Identification and characterization of proteases involved in specific proteolysis of vitellogenin and yolk proteins in salmonids. J. Exp. Zool. 292, 11–25. Hiramatsu, N., Matsubara, T., Fujita, T., Sullivan, C.V., Hara, A., 2006. Multiple piscine vitellogenins: biomarkers of fish exposure to estrogenic endocrine disruptors in aquatic environments. Mar. Biol. 149, 35–47. Ings, J.S., Van Der Kraak, G., 2006. Characterization of the expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles. Mol. Reprod. Dev. 73, 943–954. Kagedal, L., Zhao, M., Svensson, I., Brunk, U.T., 2001. Sphingosine induced apoptosis is dependent on lysosomal proteases. Biochem. J. 359, 335–343. Kwon, J.Y., Prat, F., Randall, C., Tyler, C.R., 2001. Molecular characterization of putative yolk processing enzymes and their expression during oogenesis and embryogenesis in rainbow trout (Oncorhynchus mykiss). Biol. Reprod. 65, 1701–1709. Lenarčič, B., Bevec, T., 1998. Thyrotropins — new structurally related proteinase inhibitors. J. Biol. Chem. 379, 105–111. Li, F., An, H., Seymour, T.A., Barnes, D.W., 2000. Rainbow trout (Oncorhynchus mykiss) cystatin C: expression in Escherichia coli and properties of the recombinant protease inhibitor. Comp. Biochem. Physiol. B 125, 493–502. Matsubara, T., Nagae, M., Ohkubo, N., Andoh, T., Sawaguchi, S., Hiramatsu, N., Sullivan, C.V., Hara, A., 2003. Multiple vitellogenins and their unique roles in marine teleosts. Fish Physiol. Biochem. 28, 295–299.
223
Raldua, D., Fabra, M., Bozzo, M.G., Weber, E., Cerda, J., 2006. Cathepsin B-mediated yolk protein degradation during killifish oocyte maturation is blocked by an H+-ATPase inhibitor, effects on the hydration mechanism. Am. J. Physiol. Regul. Comp. Integr. Comp. Physiol. 290, R456–R466. Roberg, K., Johansson, U., Ollinger, K., 1999. Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial trans-membrane potential during apoptosis induced by oxidative stress. Free Radic. Biol. Med. 27, 1228–1237. Saidapur, S.K., 1978. Follicular atresia in the ovaries of non-mammalian vertebrates. Int. Rev. Cytol. 54, 225–244. Schotte, P., Van Criekinge, W., Van de Craen, M., Van Loo, G., Desmedt, M., Grooten, J., Cornelissen, M., De Ridder, L., Vandekerckhove, J., Fiers, W., Vandenabeele, P., Beyaert, R., 1998. Cathepsin B mediated activation of the pro-inflammatory caspase-11. Biochem. Biophys. Res. Commun. 251, 379–387. Selman, K., Wallace, R.A., Sarka, A., Qi, X., 1993. Stages of oocyte development in the zebrafish, Brachydanio rerio. J. Morphol. 218, 203–224. Sire, M.F., Babin, P.J., Vernier, J.M., 1994. Involvement of the lysosomal system in yolk protein deposit and degradation during vitellogenesis and embryonic development in trout. J. Exp. Biol. 269, 69–83. Stoka, V., Turk, B., Schendel, S.L., Kim, T., Cirman, T., Snipas, S.J., Ellerby, L.M., Bedesen, D., Freeze, H., Abrahamsom, M., Bromme, D., Krajewski, S., Reed, J.C., Yin, X., Turk, V., Salvesen, G.S., 2001. Lysosomal protease pathways to apoptosis. J. Biol. Chem. 276, 3149–3157. Tsai, Y., Chang, G., Huang, C., Chang, Y., Huang, F., 1996. Purification and molecular cloning of carp ovarian cystatin. Comp. Biochem. Physiol. B 113, 573–580. Turk, V., Bode, W., 1991. The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett. 285, 213–219. Vancompernolle, K., Van Herreweghe, F., Pynaert, G., Van de Craen, M., De Vos, K., Totty, N., Sterling, A., Fiers, W., Vandenabeele, P., Grooten, J., 1998. Atracylosideinduced release of cathepsin B, a protease with caspase-processing ability. FEBS Lett. 438, 150–158. Wood, A.W., Van Der Kraak, G., 2001. Apoptosis and ovarian function: novel perspectives from the teleosts. Biol. Reprod. 64, 264–271. Wood, A.W., Van Der Kraak, G., 2003. Yolk proteolysis in rainbow trout oocytes after serum-free culture: evidence for a novel biochemical mechanism of atresia in oviparous vertebrates. Mol. Reprod. Dev. 65, 219–227. Wood, A.W., Matsumoto, J.Van, Der Kraak, G., 2004. Thyroglobulin type-1 domain protease inhibitors exhibit specific expression in the cortical ooplasm of vitellogenic rainbow trout oocytes. Mol. Reprod. Dev. 69, 205–214. Yabu, T., Kishi, S., Okazaki, T., Yamashita, M.Y., 2001. Characterization of zebrafish caspase-3 and induction of apoptosis through ceramide generation in fish fathead minnow tail-bud cell sand zebrafish embryo. Biochem. J. 360, 39–47. Yamashita, M., Konagaya, S., 1991. Cysteine protease inhibitor in egg of chum salmon. J. Biochem. 110, 762–766.
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
A role for the lysosomal protease cathepsin B in zebrafish follicular apoptosis Angela J. Eykelbosh, Glen Van Der Kraak ⁎ Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1
a r t i c l e
i n f o
Article history: Received 6 January 2010 Received in revised form 4 February 2010 Accepted 8 February 2010 Available online 16 February 2010 Keywords: Zebrafish reproduction Ovary Caspase-3 Cathepsin B Atresia Apoptosis
a b s t r a c t This study presents evidence that cathepsin B, a lysosomal protease, may be involved in the regulation of apoptosis during serum-starvation in teleost follicles. Zebrafish vitellogenic follicles were isolated, incubated under serum-free conditions and homogenized. The follicle extracts demonstrated caspase-3-like activity using the fluorogenic substrate DEVD-AMC, indicating the onset of apoptosis. Cathepsin B activity as measured using the fluorogenic cathepsin B substrate, Z-Arg-Arg-AMC was elevated within the first 6 h of incubation in serum-free media and coincided with the onset of apoptosis. This increase in cathepsin B activity was sensitive to the cathepsin B inhibitor, CA-074-ME. Furthermore, adding CA-074-ME to the follicle incubation blocked caspase-3-like activation, suggesting that cathepsin B activity is a positive regulator of the apoptotic cascade during serum-starvation. Interestingly, the increase in cathepsin-B-like activity was not preceded by an increase in cathepsin B mRNA transcription, suggesting that regulation of this enzyme is at a level other than of the gene. These results suggest a regulatory role for cathepsin B during follicular apoptosis in zebrafish ovarian follicles. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Vitellogenesis is a key process in fish oogenesis that involves the synthesis of vitellogenin in the liver and its transport to and internalization within developing oocytes. After internalization, vitellogenin is processed by lysosomal proteases (e.g., cathepsins B, D, and/or L) into several functionally important yolk proteins (lipovitellins, phosvitins, the β'-component, and the C-terminal peptide; Hiramatsu et al., 2002; Matsubara et al. 2003). These are in turn important for the hydration of oocytes during maturation, the subsequent buoyancy of spawned eggs, and as an energy source for nascent embryos (Carnevali et al., 2006; Hiramatsu et al. 2006; Raldua et al. 2006). Thus, lysosomal proteases play dual roles in oogenesis and contribute to overall reproductive success. It is important to note, however, that not every oocyte is destined to become an embryo. In fish, as in all other vertebrates, the vast majority of ovarian follicles are prematurely culled in a process known as follicular atresia; this culling is thought to promote the energetic investiture and ovulation of the most viable follicles. Atresia is a controlled process that proceeds via an apoptotic mechanism, resulting in the elimination of “doomed” follicles without inflammatory damage to the surrounding tissues. However, because teleost oocytes contain large quantities of yolk and are produced in large numbers, the degradation and recovery of this valuable energy store is of key interest. Indeed, there is a visible liquefaction of the yolk proteins during the first stages of follicular atresia (Saidapur, 1978). This observation led investigators to postulate
⁎ Corresponding author. Tel.: + 1 519 824 4120x53424; fax: + 1 519 837 2075. E-mail address: [email protected] (G. Van Der Kraak). 1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2010.02.005
that the initial stages of teleost atresia may require proteolytic enzymes, such as cathepsins B, D, and L, to cleave yolk proteins before atresia can proceed (Wood and Van Der Kraak, 2003). In sea bream eggs, apoptotic activation was associated with increased cathepsin D activity (Carnevali et al., 2003) and serum-starved, apoptotic rainbow trout follicles showed a rise in cathepsin L-like activity and increased free amino acid concentrations (Wood and Van Der Kraak, 2003). Furthermore, it was shown that the mRNA expression of a novel salmonid oocyte cysteine protease inhibitor (OCPI) decreased during apoptosis (Wood et al., 2004) and this protein was previously shown to inhibit calpain and cathepsins B and L in chum salmon eggs (Yamashita and Konagaya, 1991). This connection between apoptosis and lysosomal protease activation is interesting given the accumulating evidence that cathepsin B plays a direct role in the regulation of the apoptotic cascade. Research in mammals has shown that cathepsin B contributes to cell death by either activating apoptotic factors upstream of executioner caspases (Stoka et al., 2001) or by binding to and activating caspases directly (Schotte et al., 1998; Vancompernolle et al., 1998; Canu et al., 2005). In addition, cathepsin B appears to be involved in caspase-independent cell death. In non-small cell lung cancer cells, it was found that cathepsin B activation was necessary for the induction of cell death, whereas caspase activation was not (Broker et al., 2004). Finally, cathepsin B may assume the function of caspases in the cleavage and deactivation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP-1) (Gobeil et al., 2001). Taken together, the evidence from both caspase-dependent and caspase-independent cell death research indicates that cathepsin B plays a major regulatory role in multiple aspects of mammalian cell death.
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223
219
Adult male and female zebrafish (Danio rerio) were obtained from DAP International (Etobicoke, ON, Canada). Fish were kept at 25–28 °C under a photoperiod of 14L:18D. Fish were fed daily with a commercial pellet food for salmon fry (Martin Mills, Elmira, ON, Canada), with a weekly supplementation of blood-worms (Oregon Desert Brine Shrimp Co., Lakeview, OR, USA). Females were segregated at sexual maturity.
generated in the caspase activity assay was deemed “caspase-3-like” because the effects of other caspases cannot be excluded by this assay. For the caspase-3 assay, samples of follicles were incubated 2 to 3 min on ice with 175 µL of lysis buffer (50 mM HEPES, 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA, pH 7.4). They were then homogenized with a Kontes hand-held homogenizing gun and centrifuged for 10 min at 10 000 g. The supernatant was drawn off and put into a fresh tube. A 50-µL aliquot of each sample was plated on ice into a 96-well opaque plate (Fisher Scientific, Toronto ON, Canada). A 50-µL aliquot of assay buffer was added (50 mM HEPES, 0.1% CHAPS, 10 mM DTT, 1.0 mM EDTA, 100 mM NaCl, 0.1% glycerol, pH 7.4) followed by 100 µL of the substrate solution (0.06 mM DEVD-AMC, made up in assay buffer). Cathepsin B activity was assayed under slightly different conditions. The assay was based on an earlier cathepsin B assay (Barrett, 1980), which was then adapted and optimized for micro-scale work using the substrate benzyloxycarbonyl–Arg–Arg-7-amino-4-methylcoumarin (Z-RR-AMC; Sigma). Samples of follicles were incubated 2 to 3 min on ice with 325 µL of lysis buffer (352 mM KH2PO4, 0.1% Brij-35, 48 mM Na2HPO4, 4 mM EDTA, pH 6.0). These were then homogenized with a Kontes hand-held homogenizer and centrifuged for 10 min at 10,000 g. The supernatant was drawn off and put into a fresh tube. A 100-µL aliquot of each sample was plated on ice into a 96-well opaque plate. A 50-µL aliquot of assay buffer was added (352 mM KH2PO4, 48 mM Na2HPO4, 4 mM EDTA, 24 mM Cys-base, pH 6.0) followed by 50 µL of substrate solution (0.04 mM Z–RR–AMC, made up in lysis buffer). The plates were read at 28 °C using a FLx Microplate Fluorescence Reader (Bio-Tek Instruments, Winooski, VT, USA). The caspase-3 assay was measured for 100 min and the cathepsin B assay for 60 min. Measurements were taken at 5-min intervals and the activity of each well was expressed as a maximum slope (using values for the linear range only). Fluorescence of the samples was measured in triplicate and normalized against protein content using the Bio-Rad protein assay (Bio-Rad Laboratories, Mississauga, ON, Canada).
2.3. Follicle isolation and incubation
2.5. Assessment of selected mRNA expression by real time RT-PCR
Female fish were sacrificed and their ovaries removed and placed in L-15 medium (Gibco, Burlington, ON, Canada) warmed to 28 °C and supplemented with a final concentration of 100 µg/mL streptomycin and 100 U/mL penicillin to discourage bacterial growth. Follicles were separated and sorted according to size range and physical characteristics. Vitellogenic follicles (opaque, unbroken follicles, 0.4–0.7 mM in diameter) were pooled together and then groups of 25 were allotted to treatment groups. Information on staging zebrafish follicles was taken from Selman et al. (1993). Follicles were incubated in 24-well sterile polystyrene tissue culture plates with 1 mL of L-15 medium. The medium was “serum-free”, meaning it contained no growth factors, hormones or other survival factors. Serum-free incubation is a widely accepted model for inducing atresia and has been shown to cause apoptosis in vitellogenic follicles from rainbow trout (Wood and Van Der Kraak, 2001) and zebrafish (Eykelbosh and Van Der Kraak, submitted for publication). Samples of pooled follicles were allotted to treatment groups, incubated in the dark for the specific period at 28 °C, and then snapfrozen until analysis. Follicles were incubated for 6 h for all experiments where cathepsin B-like activity is measured, and for 8 h where caspase-3-like activity was measured.
RNA was extracted using the guanidium thiocyanate-phenolchloroform method as previously described (Chomczynski and Sacchi, 1987) and processed as described by Ings and Van Der Kraak (2006). Yield was quantified by absorbance at 260 nm and the purity assessed as a ratio between the absorbances at 260 and 280 nm. For the reverse transcriptase reaction, one microgram (1 µg) of RNA in diethyl pyrocarbonate (DEPC) water (10 µL) was incubated at 70 °C for five min with 0.1 ng (2 µL) of random hexamer primer. The samples were then placed on ice while 13 µL of reaction cocktail was added to them. The constituents of the cocktail and their final concentrations in 25 µL are as follows: dNTPs (0.5 mM), 5 × RT buffer (50 mM Tris–HCl, 75 mM KCl, 3 mM MgCl2), dithiothreitol (10 mM), RNase Inhibitor (40 U), Moloney murine leukemia virus reverse transcriptase (M-MLV 200 U), and DEPC water. The samples were incubated at 37 °C for 1 h and then at 95 °C for 5 min. The RT product was stored at −20 °C until real time PCR was performed. All real time PCR analyses were carried out on an ABI Prism Sequence Detection System 7000 using the accompanying SYBR Green PCR master mix. Very briefly, 5 µL of cDNA, 10 µL of the SYBR Green master mix, and 5 µL of primers (final concentration of 50 nM each) were plated out in specialized 96-well Thermowell Gold PCR plates. The plates were sealed using optical adhesive covers and run in the machine on the following program: 2 min at 50 °C, 10 min at 95 °C for polymerase activation, and then 40 cycles of 15 s at 95 °C for denaturation and 1 min at 60 °C for annealing and extension. Cathepsin B, D, and L mRNA expression was measured and normalized against zebrafish β-actin expression. All products mentioned above, with the exception of the cDNA and primers, were designed to be compatible with the ABI Prism 7000 system and are available from
The research described above suggests that cathepsin B may be doubly important for teleost follicular apoptosis, both as a regulator of the apoptotic cascade and as a facilitator of energy resorption. This study examined the activity and expression of cathepsin B to determine whether this enzyme is involved in the regulation of apoptosis in zebrafish vitellogenic follicles. Isolated follicles were incubated under serum-free conditions which are known to induce caspase-3 activation and apoptosis (Wood and Van Der Kraak, 2001; Eykelbosh and Van Der Kraak, submitted for publication) and the resulting cathepsin B enzyme activity was measured. To functionally link cathepsin B activity to caspase activation, follicles were incubated with the cell-permeable specific cathepsin B inhibitor Ca-074-Me and then caspase-3-like activity was measured. Finally, we used real time RT-PCR to determine whether apoptosis was associated with increased mRNA expression of cathepsins B, D, and L in follicles following serum-free incubation. 2. Materials and methods 2.1. Chemicals All substrates, hormones, inhibitors and human cathepsin B were obtained from Sigma (Oakville, ON, Canada), unless otherwise noted. Ca-074-Me, a specific cell-permeable inhibitor of cathepsin B, was obtained from the Peptide Institute (Osaka, Japan). 2.2. Animal maintenance
2.4. Enzyme activity assays Activity of the executioner enzyme caspase-3 was used as an endpoint to measure the induction of apoptosis. This involved use of the fluorogenic substrate acetyl–Asp–Glu–Val–Asp-7-amino-4methylcoumarin (DEVD-AMC; Sigma), which is known to be cleaved by recombinant zebrafish caspase-3 (Yabu et al., 2001). The activity
220
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223
Applied Biosystems (Foster City, CA, USA). Primers were generated based on known zebrafish sequences using Primer Express (Applied Biosystems). The sequences and origins of these primers are listed in Table 1. Efforts were also made to amplify OCPI using real time RT-PCR. 2.6. Statistical analyses In an experiment with multiple treatment groups, the differences between control and treatment values were analyzed using a one-way ANOVA followed by a Tukey or Dunnett's t-test. The values presented are mean ± standard error. Where a Tukey t-test is used to detect differences between treatments, letter labels are used to group treatments that are not significantly different from each other. Where a Dunnett's t-test is used, an asterisk indicates that the treatment value is significantly different compared to the control. On the sole occasion where data were not normally distributed, a non-parametric Kruskal– Wallis test, followed by a Mann–Whitney test, was used to detect differences between the control and treatment groups. For all tests, the differences are considered significant at p b 0.05. Where only two groups were compared, the difference between control and treatment values was tested using a two-tailed t-test. The values presented are the mean ± standard error, and an asterisk in the graphs is used to indicate that a treatment value was significantly different from its corresponding control value. Two-tailed t-tests were considered significant when p b 0.05. Furthermore, statistical analysis was also applied to repetitions of the same experiment. In this case, a paired t-test compares each treatment value against its own control, and then determines if the mean difference in control vs. treatment is greater than zero over a series of experiments. Therefore, a pair-wise-test measures how consistent responses are throughout the series of experiments. Pair-wise t-tests were considered significant when p b 0.05.
Fig. 1. Effect of serum-starvation on cathepsin B-like activation in zebrafish follicles incubated for 6 h under serum-free conditions. Cathepsin-B-like activity was measured and compared to an un-incubated control (T0) by means of an independent samples t-test. An asterisk indicates that control and 6 h values are significantly different within one experiment. Experiments were repeated four times, and the difference across the series of experiments was compared using a paired t-test. ppaired = 0.003. Data are presented as the mean caspase-3-like activity± standard error, n = 3–6.
3.2. Prevention of cathepsin B-like activity with survival factors To investigate the effect of survival factors on cathepsin-B-like activation, follicles were incubated with or without 15 IU/mL human chorionic gonadotropin (hCG) and 1 ng/mL 17β-estradiol (E2). These doses were chosen based on previous studies (Eykelbosh and Van Der Kraak, submitted) showing that these hormones prevent caspase 3-activation. Neither hCG nor E2 caused a significant change in cathepsin B-like activity (Fig. 3). 3.3. Caspase-3-like activity after incubation with Ca-074-Me
3. Results 3.1. Cathepsin B activity in serum-starved follicles Fig. 1 shows that there is a significant increase in cathepsin B activity over six h of serum-starvation, compared to a time zero (T0) control. The rise in activity was consistent over four separate experiments (ppaired = 0.003). Based on these results, all further experiments looking at cathepsin B activation used an incubation period of 6 h. In order to confirm that this was indeed cathepsin B activation, samples were incubated as usual and then cathepsin B-like activity was assayed with or without 25 µM Ca-074-Me, a specific cathepsin B inhibitor in mammals (Buttle et al., 1992). Fig. 2 demonstrates that treatment with inhibitor abolished only the changes seen over time. This suggests that while there is some non-specific cleavage of the substrate, the changes observed are likely due to cathepsin B activity.
To investigate the possibility of a functional link between cathepsin B activation and caspase activity, follicles were incubated with or without 10 µM Ca-074-Me for 8 h at 28 °C and then the samples were assayed for caspase-3 like activity. Addition of Ca-074-Me caused a significant decrease in caspase-3-like activity compared to 8 h untreated follicles (Fig. 4). This suggests that cathepsin B may play a role in the activation of the executioner caspase-3.
Table 1 Primer sequences for the genes of interest and the Accession numbers of the sequences from which they were derived. Gene of interest
Accession #
Primer sequence (F = forward, R = reverse)
Cathepsin B
AY398331
Cathepsin D
AS278268
Cathepsin L
NM_131198
OCPI
AY094359
β-Actin
AF025305
F: TGAT GCAA GAGA GCAG TGGC R: AGAT AGCT TCGG CAGC TCCA F: TTGG CACT CCTG TCCA GACC R: CAAG CAGGCGAT GTCA GTCA F: ATGT TTGC TTTG CTCG TCAC R: TGCT GGCT CTTC CAGG AGTT F: GGAA GAGT GCAG AAGG AGAC R: AGCA GTGC CTGG TGGA GTCT F: ACAG GGAA AAGA TGAC ACAG ATCA R: CAGC CTGG ATGG CAAC GTA
Fig. 2. Representative graph depicting the effect of the cathepsin B inhibitor Ca-074-Me (25 µM) on cathepsin B-like activity in zebrafish follicles. Follicles were incubated for 6 h under serum-free conditions and then the cathepsin B assay was performed with or without 25 µM Ca-074-Me. Data were analyzed using a one-way ANOVA followed by a Tukey t-test. Similar letter labels indicate that values are not significantly different from each other. Data are presented as the mean caspase-3-like activity ± standard error, n = 3.
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223
221
3.4. Expression of lysosomal proteases in the follicle Follicles were incubated for 2 h in serum-free medium and then snap-frozen. This incubation period was chosen to accommodate the translation and protein processing that would presumably occur before the observed activation at 6 h. RNA isolation and real time PCR were performed. Cathepsin B, D, and L expressions were apparent at time zero; however, there was no significant change in expression for any of the cathepsins over the 2-h incubation period (Fig. 5). In order to investigate this further, follicles were incubated for extended time periods (0, 4, 8, 16, and 24 h). Once again, cathepsin B mRNA expression decreased over time, but this difference did not become significant until 24 h of serum-starvation (Fig. 6). These results demonstrate that the increase in cathepsin B enzyme activity is not due to an increase in gene expression.
3.5. Cloning of OCPI Given that we found an increase in cathepsin B activity without a concomitant increase in cathepsin B expression, we considered the possibility that increased cathepsin B activity may be due to decreased expression of an inhibitor such as OCPI. However, primers designed from the recently cloned rainbow trout gene (Wood et al., 2004; Table 1) failed to identify any homologous sequences in zebrafish.
Fig. 3. Effect of human chorionic gonadotropin (hCG; A) and 17β-estradiol (E2; B) on cathepsin B-like activation in zebrafish follicles incubated for 6 h under serum-free conditions. Data were analyzed using a one-way ANOVA followed by a Tukey t-test. Similar letter labels indicate that values are not significantly different from each other. Experiments were repeated 1–2 times. Data are presented as the mean caspase-3-like activity ± standard error. For hCG experiments, n = 6–8. For E2 experiments, n = 3. Fig. 5. Effect of serum-starvation on cathepsin mRNA expression in zebrafish follicles incubated for 2 h under serum-free conditions. Data were analyzed by means of an independent samples t-test and no significant differences were found between control (0 h) and 2 h of serum-free incubation. Data points show mean normalized gene expression ± standard error, n = 6.
Fig. 4. Effect of the cathepsin B inhibitor Ca-074-Me on caspase-3-like activity in zebrafish follicles incubated for 8 h under serum-free conditions, with or without addition of 10 µM Ca-074-Me. Caspase-3-like activity in treated samples was measured and compared to the 0 h control and the untreated 8 h control. Data were analyzed using a one-way ANOVA followed by a Tukey t-test. Similar letter labels indicate that values are not significantly different from each other. Data are presented as the mean caspase-3-like activity ± standard error, n = 4–6.
Fig. 6. Effect of serum-starvation on cathepsin B mRNA expression in zebrafish follicles incubated for various times under serum-free conditions. Because data were not normally distributed, a non-parametric Kruskal–Wallis test followed by a Mann– Whitney test was used to detect differences between the control (0 h) and the other time points. An asterisk indicates a significant difference. Data points show mean normalized gene expression ± standard error, n = 4.
222
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223
4. Discussion 4.1. Cathepsin B activity increases with serum-starvation A common hurdle encountered in comparative physiology is the validity of methods used to compare processes between different species. In this case, the consideration was whether using a specific mammalian cathepsin B substrate would give reliable results in zebrafish. The choice of substrate is reliable for two reasons. First, the protein sequence shared between zebrafish and rat cathepsin B is highly conserved (84% similarity). Secondly, assaying the samples with a specific cathepsin B inhibitor abolished the change in activity observed over 6 h. The 25 µM dose of Ca-074-Me was not sufficient to abolish all activity, meaning that some non-specific cleavage remained; however, cathepsin B inhibitor treatment consistently reduced starvation-induced activity to baseline. This observation and the similarity between protein sequences suggest that the activity shown here is due to cathepsin B. Further study is required to more fully characterize zebrafish cathepsin B and to develop an assay that would exclude non-specific activity. 4.2. Role of cathepsin B in apoptotic signalling in serum-starved zebrafish follicles Mammalian studies have clearly shown that cathepsin B is an active regulator of apoptosis. In a human cell line, incubation with the purified cathepsin B led to the cleavage of the pro-apoptotic protein Bid, increased cytochrome-c release, and caspase activation (Stoka et al. 2001). However, very little information is available regarding the apoptotic mechanism at work in serum-starved follicles in fish. In the present study, cathepsin B activity showed an increase at 6 h, prior to the rise in caspase activity at 8 h observed in previous studies (Eykelbosh and Van Der Kraak, submitted). Furthermore, the application of a specific inhibitor completely abolished caspase-3-like activation, indicating cathepsin B activation is involved in the onset of apoptosis. These results agree with a previous study showing that cathepsin B release from the lysosome leads to cytochrome-c release and caspase activation (Guicciardi et al., 2000). Moreover, Canu et al. (2005) found that activation of autophagolysosomes results cathepsin B activation and colocalization with procaspase-3 in the cytosol, demonstrating that caspase-3 activation was cathepsin B-dependent. In previous studies, however, caspase-3-like activation in serumstarved zebrafish follicles could be avoided through the application of specific survival factors, such as 17β-estradiol, and this anti-apoptotic effect appeared to be mediated through the α isoform of the estrogen receptor in a manner involving inhibition of the mitogen-activated protein kinase, ERK (Eykelbosh and Van Der Kraak, submitted). In the present study, cathepsin B activation was apparently insensitive to survival factors (hCG and 17β-estradiol), suggesting that the mentioned survival factors exert their effects downstream of cathepsin involvement. Thus, although results provide further evidence of a functional link between cathepsin B and caspase activation, the signalling pathway connecting these two events remains to be elucidated. Additional studies are required to determine at which point survival factors disrupt this cell death pathway. 4.3. Cathepsin B expression is unaffected by serum-starvation Cathepsin B mRNA did not show an increase in expression with serum-starvation; rather, mRNA expression showed a strong declining trend that became significant after 24 h of serum-starvation. The observation that, despite the lack of change in expression, cathepsin B enzyme activity still increased after 6 h of serum-starvation indicates that regulation is not at the level of gene transcription. Other studies have suggested that ovarian cathepsins may be regulated post translationally and, accordingly, transcript abundance may not
correlate with cathepsin enzymatic activity. For example, Kwon et al. (2001) reported that cathepsin B and L were highly expressed during oogenesis, and yet previous studies in the same model (rainbow trout) did not detect cathepsin B or L activity during the same period (Sire et al. 1994). These results may reflect the early accumulation of cathepsin B mRNAs or precursor proteins that is later followed by regulation at a higher level or release from the lysosome. There is evidence that the lysosome becomes permeable during the early stages of apoptosis (Roberg et al. 1999; Kagedal et al., 2001) and other studies have demonstrated that cathepsin B is translocated to the cytosol during apoptosis (Boya et al., 2003; Canu et al. 2005; Foghsgaard et al. 2005). Many levels of regulation are possible and relatively few details are known; however, this study argues against increased cathepsin B mRNA transcription (leading to cathepsin B activation) during serum-starvation induced apoptosis. Another explanation for an increase in enzymatic activity without gene transcription may be that serum-starvation causes a decrease in the transcription or translation of endogenous inhibitors. We were unable to clone OCPI out of zebrafish vitellogenic follicles, perhaps indicating that OCPI is a uniquely salmonid protein (Bobe and Goetz, 2001; Yabu et al., 2001; Wood et al., 2004). However, there are a number of other potential endogenous inhibitors for cathepsin B activity in the follicle, including members of the cystatin and the thyrotropin families; these are reviewed in Turk and Bode (1991) and Lenarčič and Bevec (1998), respectively. Cystatins that are homologous to the mammalian proteins have been cloned out of rainbow trout (Li et al., 2000) and carp (Tsai et al., 1996), and have shown inhibitory activity against papain, a cysteine protease closely related to cathepsin B. A better insight into cathepsin B regulation and apoptosis may be gained by studying the expression of these inhibitors. 4.4. Conclusions This study has demonstrated that zebrafish follicles subjected to serum-starvation show an increase in cathepsin B-like activity at a point in the time scale that would permit potential modulation of caspase-3-like activity. In confirmation of this, incubation with a specific cathepsin B inhibitor decreased caspase-3-like activity. This cathepsin B activity was not sensitive to survival factors, suggesting that cathepsin B activation is upstream of the checkpoint regulated by these survival factors. Finally, this study suggests that regulation of cathepsin B activity during apoptosis is not at the level of gene transcription; instead, it may be that cathepsin B is regulated either at the level of protein processing, release of the enzyme, or by a change in expression of an endogenous inhibitor. This work provides the first example of cathepsin B activation during atresia and regulation of caspase-3-like activity in teleosts. Acknowledgements This work was supported by an NSERC Discovery Grant to GVDK. AJE was supported by an NSERC Postgraduate Scholarship. Thanks are also due to Dr. R. Mosser and the lab for the use of their equipment. References Barrett, A., 1980. Fluorimetric assays for cathepsin B and cathepsin H with methycoumarylamide substrates. Biochem. J. 187, 909–912. Bobe, J., Goetz, F.W., 2001. Cysteine protease inhibitor is specifically expressed in preand early-vitellogenic oocytes from the brook trout peri-ovulatory ovary. Mol. Reprod. Dev. 60, 312–318. Boya, P., Gonzalez-Polo, R., Poncet, D., Andreau, K., La Vieira, H., Roumier, T., Perfettini, J., Kroemer, G., 2003. Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine. Oncogene 22, 3927–3936. Broker, L.E., Huisman, C., Span, S.W., Rodriguez, J.A., Kruyt, F.A.E., Giaccone, G., 2004. Cathepsin B mediates caspase-independent cell death induced by microtubule stabilizing agents in non-small cell lung cancer cells. Cancer Res. 64, 27–30. Buttle, D.J., Murata, M., Knight, C.G., Barrett, A.J., 1992. CA074 methyl ester: a proinhibitor for intracellular cathepsin B. Arch. Biochem. Biophys. 299, 377–380.
A.J. Eykelbosh, G. Van Der Kraak / Comparative Biochemistry and Physiology, Part A 156 (2010) 218–223 Canu, N., Tufi, R., Serafino, A.L., Amadora, G., Ciotti, M.T., Calissano, P., 2005. Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells. J. Neurochem. 92, 1228–1242. Carnevali, O., Polzonetti, V., Cardinali, M., Pugnaloni, A., Natalini, P., Zmora, N., Mosonci, G., Polzonetti-Magni, A.M., 2003. Apoptosis in Sea Bream Sparus aurata eggs. Mol. Reprod. Dev. 66, 291–296. Carnevali, O., Cionna, C., Tosti, L., Lubzens, E., Maradonna, F., 2006. Role of cathepsins in ovarian follicle growth and maturation. Gen. Comp. Endocrinol. 146, 195–203. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. Eykelbosh, A, J., Van Der Kraak, G. Apoptosis in serum-starved zebrafish ovarian follicles. Mol. Reprod. Dev. (submitted for publication). Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bastholm, L., Boes, M., Elling, F., Leist, M., Jäättelä, M., 2005. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J. Cell Biol. 153, 999–1009. Gobeil, S., Boucher, C.C., Nadeau, D., Poirier, G.G., 2001. Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases. Cell Death Differ. 8, 588–594. Guicciardi, M.E., Deussing, J., Miyoshi, H., Bronk, S.F., Svingen, P.A., Peters, C., Kaufmann, S.H., Gores, G.J., 2000. Cathepsin B contributes to TNF-α-mediated hepatocytes apoptosis by promoting mitochondrial release of cytochrome-c. J. Clin. Invest. 106, 1127–1137. Hiramatsu, N., Ichikawa, N., Fukada, H., Fujita, T., Sullivan, C.V., Hara, A., 2002. Identification and characterization of proteases involved in specific proteolysis of vitellogenin and yolk proteins in salmonids. J. Exp. Zool. 292, 11–25. Hiramatsu, N., Matsubara, T., Fujita, T., Sullivan, C.V., Hara, A., 2006. Multiple piscine vitellogenins: biomarkers of fish exposure to estrogenic endocrine disruptors in aquatic environments. Mar. Biol. 149, 35–47. Ings, J.S., Van Der Kraak, G., 2006. Characterization of the expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles. Mol. Reprod. Dev. 73, 943–954. Kagedal, L., Zhao, M., Svensson, I., Brunk, U.T., 2001. Sphingosine induced apoptosis is dependent on lysosomal proteases. Biochem. J. 359, 335–343. Kwon, J.Y., Prat, F., Randall, C., Tyler, C.R., 2001. Molecular characterization of putative yolk processing enzymes and their expression during oogenesis and embryogenesis in rainbow trout (Oncorhynchus mykiss). Biol. Reprod. 65, 1701–1709. Lenarčič, B., Bevec, T., 1998. Thyrotropins — new structurally related proteinase inhibitors. J. Biol. Chem. 379, 105–111. Li, F., An, H., Seymour, T.A., Barnes, D.W., 2000. Rainbow trout (Oncorhynchus mykiss) cystatin C: expression in Escherichia coli and properties of the recombinant protease inhibitor. Comp. Biochem. Physiol. B 125, 493–502. Matsubara, T., Nagae, M., Ohkubo, N., Andoh, T., Sawaguchi, S., Hiramatsu, N., Sullivan, C.V., Hara, A., 2003. Multiple vitellogenins and their unique roles in marine teleosts. Fish Physiol. Biochem. 28, 295–299.
223
Raldua, D., Fabra, M., Bozzo, M.G., Weber, E., Cerda, J., 2006. Cathepsin B-mediated yolk protein degradation during killifish oocyte maturation is blocked by an H+-ATPase inhibitor, effects on the hydration mechanism. Am. J. Physiol. Regul. Comp. Integr. Comp. Physiol. 290, R456–R466. Roberg, K., Johansson, U., Ollinger, K., 1999. Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial trans-membrane potential during apoptosis induced by oxidative stress. Free Radic. Biol. Med. 27, 1228–1237. Saidapur, S.K., 1978. Follicular atresia in the ovaries of non-mammalian vertebrates. Int. Rev. Cytol. 54, 225–244. Schotte, P., Van Criekinge, W., Van de Craen, M., Van Loo, G., Desmedt, M., Grooten, J., Cornelissen, M., De Ridder, L., Vandekerckhove, J., Fiers, W., Vandenabeele, P., Beyaert, R., 1998. Cathepsin B mediated activation of the pro-inflammatory caspase-11. Biochem. Biophys. Res. Commun. 251, 379–387. Selman, K., Wallace, R.A., Sarka, A., Qi, X., 1993. Stages of oocyte development in the zebrafish, Brachydanio rerio. J. Morphol. 218, 203–224. Sire, M.F., Babin, P.J., Vernier, J.M., 1994. Involvement of the lysosomal system in yolk protein deposit and degradation during vitellogenesis and embryonic development in trout. J. Exp. Biol. 269, 69–83. Stoka, V., Turk, B., Schendel, S.L., Kim, T., Cirman, T., Snipas, S.J., Ellerby, L.M., Bedesen, D., Freeze, H., Abrahamsom, M., Bromme, D., Krajewski, S., Reed, J.C., Yin, X., Turk, V., Salvesen, G.S., 2001. Lysosomal protease pathways to apoptosis. J. Biol. Chem. 276, 3149–3157. Tsai, Y., Chang, G., Huang, C., Chang, Y., Huang, F., 1996. Purification and molecular cloning of carp ovarian cystatin. Comp. Biochem. Physiol. B 113, 573–580. Turk, V., Bode, W., 1991. The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett. 285, 213–219. Vancompernolle, K., Van Herreweghe, F., Pynaert, G., Van de Craen, M., De Vos, K., Totty, N., Sterling, A., Fiers, W., Vandenabeele, P., Grooten, J., 1998. Atracylosideinduced release of cathepsin B, a protease with caspase-processing ability. FEBS Lett. 438, 150–158. Wood, A.W., Van Der Kraak, G., 2001. Apoptosis and ovarian function: novel perspectives from the teleosts. Biol. Reprod. 64, 264–271. Wood, A.W., Van Der Kraak, G., 2003. Yolk proteolysis in rainbow trout oocytes after serum-free culture: evidence for a novel biochemical mechanism of atresia in oviparous vertebrates. Mol. Reprod. Dev. 65, 219–227. Wood, A.W., Matsumoto, J.Van, Der Kraak, G., 2004. Thyroglobulin type-1 domain protease inhibitors exhibit specific expression in the cortical ooplasm of vitellogenic rainbow trout oocytes. Mol. Reprod. Dev. 69, 205–214. Yabu, T., Kishi, S., Okazaki, T., Yamashita, M.Y., 2001. Characterization of zebrafish caspase-3 and induction of apoptosis through ceramide generation in fish fathead minnow tail-bud cell sand zebrafish embryo. Biochem. J. 360, 39–47. Yamashita, M., Konagaya, S., 1991. Cysteine protease inhibitor in egg of chum salmon. J. Biochem. 110, 762–766.