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Copyright © 2004, The American Society for Cell Biology Induction of Apoptosis in Starfish Eggs Requires Spontaneous Inactivation of MAPK (Extracellular Signal-regulated Kinase) Followed by Activation of p38MAPK Carl-Henrik Heldin, Monitoring Editor * Corresponding author. E-mail address: kchiba/at/cc.ocha.ac.jp. Received June 6, 2003; Revised November 17, 2003; Accepted November 17, 2003. ![]() | |||||||||||||||||
Abstract Mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase) prevents DNA replication and parthenogenesis in maturing oocytes. After the meiotic cell cycle in starfish eggs, MAPK activity is maintained until fertilization. When eggs are fertilized, inactivation of MAPK occurs, allowing development to proceed. Without fertilization, highly synchronous apoptosis of starfish eggs starts 10 h after germinal vesicle breakdown, which varies according to season and individual animals. For induction of the apoptosis, MAPK should be activated for a definite period, called the MAPK-dependent period, during which eggs develop competence to die, although the exact duration of the period was unclear. In this study, we show that the duration of the MAPK-dependent period was ~8 h. Membrane blebbing occurred ~2 h after the MAPK-dependent period. Surprisingly, when MAPK was inhibited by U0126 after the MAPK-dependent period, activation of caspase-3 occurred earlier than in the control eggs. Thus, inactivation of MAPK is a prerequisite for apoptosis. Also, even in the absence of the inhibitor, MAPK was inactivated spontaneously when eggs began to bleb, indicating that inactivation of MAPK after the MAPK-dependent period acts upstream of caspase-3. Inactivation of MAPK also resulted in the activation of p38MAPK, which may contribute to apoptotic body formation. | |||||||||||||||||
INTRODUCTION Apoptosis plays critical roles in development and in the maintenance of homeostasis. Once triggered, the apoptotic program induces activation of a series of biochemical events. The best characterized pathway of apoptosis involves the release of cytochrome c from mitochondria, leading to the activation of caspase-9. The caspase-9 cleaves and activates caspase-3, which is the key enzyme to execute apoptosis (reviewed by Chang and Yang, 2000 A mitogen-activated protein kinase kinase kinase phosphorylates and activates a MAP kinase kinase (MAPKK), which phosphorylates and activates mitogen-activated protein kinase (MAPK). In mammals, there are at least three genetically distinct groups of MAPK pathways, including extracellular signal-regulated kinase (ERK: MAPK), the c-Jun NH2-terminal kinase (JNK), and the p38MAPK (reviewed by Widmann et al., 1999 Fully grown starfish oocytes are arrested at prophase of meiosis I. Meiosis is reinitiated by 1-methyladenine (1-MA), which is released from follicle cells, causing germinal vesicle breakdown (GVBD) (Kanatani et al., 1969 Although starfish immature oocytes can live >1 wk in seawater, postmeiotic eggs synchronously and rapidly undergo apoptosis in <24 h after 1-MA treatment (Sasaki and Chiba, 2001 | |||||||||||||||||
MATERIALS AND METHODS Materials U0126 (Promega, Madison, WI) and U0124 and SB203580 (Calbiochem, La Jolla, CA) were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. 1-MA (1 mM), purchased from Kanto Kagaku Reagent Division (Tokyo, Japan), was dissolved in distilled water. These solutions were stored at -20°C.Animal Maintenance, Gamete Collection, and Culture Starfish A. pectinifera were collected on the Pacific coast of Honshu Island, Japan, and kept in laboratory aquaria supplied with circulating seawater at 10-17°C. To remove follicle cells, isolated ovaries were incubated in ice-cold Ca2+-free artificial seawater (480 mM NaCl, 10 mM KCl, 27 mM MgCl2, 29 mM MgSO4, 2 mM NaHCO3, or Ca2+-free Jamarin; Jamarin Laboratory, Osaka, Japan), and released oocytes were washed twice with Ca2+-free artificial seawater. Defolliculated oocytes were stored in artificial seawater (Ca2+-free Jamarin plus 9.2 mM CaCl2) at 20°C. Oocyte maturation was induced by the addition of 1 μM 1-MA. GVBD occurred around 20 min after 1-MA treatment. 1-MA was washed out of the culture 40-60 min after 1-MA treatment.Transmission Electron Microscopy Small portions of the egg suspension were sampled and fixed with OsO4 for 30 min on ice. The fixative contained 1% OsO4, 0.05% sodium cacodylate, and 1.5% potassium ferrocyanite in Ca2+-free artificial seawater. After dehydration in an ethanol series, the oocytes were embedded in Epon resin containing 14% Quetol653 (Okenshoji, Osaka, Japan), 23% ERL4206, 63% nonenyl succinic anhydride, and 0.5% S-1 (TAAB Laboratories Equipment, Berkshire, England). Ultrathin sections were mounted on a grid coated with Bioden mesh cement (Okenshoji) and then stained with uranyl acetate and lead citrate. Grids were examined using a JEOL-1230 electron microscope (JEOL, Tokyo, Japan).Measurement of DEVD-MCA Cleavage Activity Eggs were collected by centrifugation at 1800 × g for 2 min at 4°C. The eggs were resuspended in ice-cold buffer A (100 mM HEPES-NaOH, pH 7.5, 10 mM dithiothreitol; 0.05 μl/egg) and homogenized on ice. Egg homogenates were spun at 14,000 × g for 15 min at 4°C to obtain cytosolic extracts. Proteolytic reactions were carried out in 1 ml of buffer A, containing 50 μl of cytosolic extracts and 10 μM acetyl-Asp-Glu-Val-Asp- (4-methyl-coumaryl-7-amide) (Ac-DEVD-MCA; Peptide Institute, Osaka, Japan) at 20°C. The fluorogenic product substrate 7-amino-4-methylcoumarin was detected by excitation at 380 nm and emission at 460 nm with a fluorescence spectrophotometer (650-10 Sl; Hitachi, Tokyo, Japan), and the initial velocity of hydrolysis of the substrate by DEVDase was measured.SDS-PAGE and Western Blotting Eggs were pelleted by brief centrifugation to remove seawater. The egg pellet was resuspended in SDS-sample buffer at 0.33 μl/egg, heated to 95-100°C for 5 min, and subjected to gel electrophoresis. Typically, 10 μl of each sample (containing 30 eggs) was run on a 12.5% polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride transfer membrane (Immobilon-P; Millipore, Bedford, MA). The membrane was blocked with phosphate-buffered saline (PBS) containing 5% skim milk and incubated with an anti-rat ERK1 antibody (Seikagaku, Tokyo, Japan) for 45 min or an anti-active p38MAPK antibody (Promega) for 1 h at room temperature. After washing with PBS containing 0.05% Tween 20 (vol/vol), the membrane was incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody for 45 min at room temperature and then washed again. Bound antibody was detected using an ECL Western blotting analysis system (Amersham Biosciences, Piscataway, NJ) and an LAS-1000 lumino image analyzer (Fuji Photo Film, Tokyo, Japan). Western blots were sometimes stripped by using Western blot stripping buffer (Pierce Chemical, Rockford, IL) and reprobed with another antibody. When we use highly sensitive detection reagents (ECL+; Amersham Biosciences), the membrane was incubated overnight with an anti-active p38MAPK antibody at 4°C and then incubated for 1 h at room temperature. After washing with PBS containing 0.05% Tween 20 (vol/vol), the membrane was incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody for 1 h at room temperature.Preparation of Recombinant Protein To prepare glutathione S-transferase (GST), the pGEX-6P-3 vector was purchased from Amersham Biosciences. The GST-starfish Mos (GST-Mos) construct in the pGEX-4T-2 vector was kindly provided by Dr. Kazunori Tachibana (Tokyo Institute of Technology, Tokyo, Japan). The plasmids were transformed into the BL21 bacterial strain, followed by growth at 37°C for 1 h, chilled in ice water for 15 min, and then induced with 0.5 mM isopropyl β-d-thiogalactoside at 37°C for 3 h. Bacteria were then pelleted by centrifugation at 8000 × g for 5 min, resuspended in 50 ml of PBS containing 0.05% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) per liter culture, and disrupted by sonication. The GST-Mos and GST were purified with glutathione-Sepharose 4B (Amersham Biosciences) and concentrated to 1.6 mg/ml in buffer B (20 mM HEPES, pH 6.8, 88 mM NaCl, 7.5 mM MgCl2) with Centricon YM-50 and Microcon YM-50 (Millipore). Aliquoted proteins were frozen in liquid nitrogen and stored at -80°C.Microinjection Microinjection into an egg and quantitation of injection volumes were performed according to the methods of Hiramoto (1974 ![]() ![]() ![]() | |||||||||||||||||
RESULTS Morphological Changes during Starfish Egg Apoptosis Immature oocytes of the starfish A. pectinifera were treated with 1-MA to reinitiate meiotic maturation. Oocytes completed both meiotic divisions to yield haploid interphase-arrested eggs (Figure 1A, a). Although the timing of the occurrence of blebbing depends on the animals, synchronous blebbing always starts between 8 and 12 h after 1-MA treatment (Sasaki and Chiba, 2001 ![]() ![]() ![]()
Membrane blebbing and apoptotic body formation are hallmark morphological features of apoptosis. In addition, the key enzyme to execute apoptosis, caspase-3, is activated when starfish eggs initiate blebbing (Sasaki and Chiba, 2001 The MAPK-dependent Period for Induction of Apoptosis For induction of starfish egg apoptosis, MAPK should be activated for a definite period, called the MAPK-dependent period (Sasaki and Chiba, 2001 ![]() Thus, to measure the length of the MAPK-dependent period, we blocked MAPK by using mitogen-activated protein kinase kinase (MEK) inhibitor U0126 at various times after 1-MA treatment. Because 50% inhibitory concentration of MAPK was ~0.1 μM in starfish eggs (Figure 2A), which is comparable with that of in COS-7 cells (Favata et al., 1998
Using the results in Figure 2B, the MAPK-dependent period was estimated to continue until 7 h 18 min after 1-MA treatment (Figure 2C, a). Because GVBD occurred at 20 min after 1-MA treatment, the MAPK-dependent period should start from then. In this animal, the duration of MAPK-dependent period was ~7 h. The MAPK-dependent period depends on the animal and season. The duration of the period varied from 5.5 to 8.5 h. Frequently, the end of the MAPK-dependent period was 8 h after 1-MA treatment, and 50% blebbing occurred ~2 h after the end of MAPK-dependent period. When U0126 was added to the egg culture from 7 h after 1-MA treatment, 15% of eggs underwent apoptosis (Figure 2B). In these 15% of eggs, the MAPK-dependent period had probably finished. Interestingly, these eggs initiated membrane blebbing earlier than the control eggs treated with inactive analogue U0124. Similar acceleration of blebbing occurred when U0126 was administered immediately after MAPK-dependent period as shown in Figure 3A, a. U0126-treated and U0124-treated eggs revealed 50% blebbing at 8 h 05 min and 8 h 34 min after 1-MA treatment, respectively. Similar results were obtained using oocytes from different females (Figure 3, b and c). Also, U0126-treated eggs proceed from blebbing to apoptotic body formation within 1-1.5 h; essentially the same kinetics as eggs cultured in the absence of U0126 (Figure 3B). These results indicate that inactivation of MAPK after the MAPK-dependent period accelerates the initiation of blebbing but does not prolong or shorten the execution phase. These results also suggest that inactivation of MAPK after the MAPK-dependent period is prerequisite to blebbing initiation because spontaneous inactivation of MAPK occurs at about the same time as the eggs initiated membrane blebbing (Sasaki and Chiba, 2001
Inactivation of MAPK after the MAPK-dependent Period Is Required for Caspase-3 Activation In starfish eggs, execution of apoptosis (membrane blebbing and apoptotic body formation) is regulated by the activity of caspase-3 (Sasaki and Chiba, 2001 ![]()
Dynamics of MAPK and p38MAPK during Starfish Egg Apoptosis Next, we examined the timing of blebbing and dynamics of MAPK of starfish eggs without U0126 treatment. Exposure of oocytes to 1-MA led to the activation of MAPK at ~30 min. The active form of MAPK persists for almost 10 h after 1-MA treatment. Immediately before the onset of blebbing, a small proportion of the MAPK became inactive even in the absence of U0126 (Figure 5A). At 11 h, a large quantity of MAPK was spontaneously inactivated, followed by initiation of blebbing. Thus, it is likely that MAPK inactivation causes execution of apoptosis. Also, artificial inactivation of MAPK after the MAPK-dependent period resulted in caspase-3 activation (Figure 4). Thus, cell death probably occurs in the following order: persistent activation of MAPK during the MAPK-dependent period, MAPK inactivation, caspase-3 activation, and execution of apoptosis. Inactivation of MAPK does not act downstream of caspase-3 as shown in Figure 7B (see below).Another MAPK family protein, p38MAPK, plays critical roles in stress responses and apoptosis in many cell lines (reviewed by Widmann et al., 1999 SB203580, an inhibitor of p38MAPK but not of p38MAPK-kinase MKK3 or MKK6, prevents phosphorylation of p38MAPK in many experimental systems, presumably via inhibiting its autophosphorylation (Ge et al., 2002 Just around GVBD, MAPK was activated and p38MAPK was inactivated (Figure 5, A and B). Conversely, when eggs began to bleb, MAPK is inactivated and p38MAPK was activated, suggesting that there may be some connection between the two pathways. Also, because p38MAPK was highly activated when eggs initiated blebbing (Figure 5A), starfish p38MAPK is likely to participate to the execution of apoptosis. The role of weakly activated p38MAPK in GV oocytes is unknown. Microinjection of the GST-Starfish Mos Fusion Protein into Eggs Delayed Execution of Apoptosis If execution of apoptosis depends on the inactivation of MAPK, apoptosis should be inhibited by the microinjection of exogenous Mos, which activates the Mos/MEK/MAPK pathway. Indeed, when we injected recombinant GST-starfish Mos fusion protein (GST-Mos, 160 μg/ml final concentration) into the eggs 8-9 h after 1-MA treatment (before the initiation of blebbing), initiation of blebbing of injected eggs was delayed ~2 h (Figure 6, a-c). As a control, injection of GST alone had no effect on execution of apoptosis (Figure 7, g-l). These results indicate that continuous activation of the Mos/MEK/MAP kinase cascade after the MAPK-dependent period had inhibitory effects on execution of apoptosis. Because occurrence of blebbing was delayed but not completely blocked in GST-Mos-injected eggs, these eggs might have strong activity to inactivate the MAPK pathway, or there may be some another component to activate caspase-3 other than MAPK inactivation.
Inactivation of MAPK after the MAPK-dependent Period Is Followed by Activation of p38MAPK To determine whether activation of p38MAPK occurred after inactivation of MAPK, we treated the eggs with U0126 at 8 h 15 min after 1-MA treatment. As shown in Figure 7A, the timing of p38MAPK activation as well as blebbing initiation was accelerated by U0126 treatment. These results strongly suggested that inactivation of MAPK acts upstream of p38MAPK.Eggs injected with Ac-DEVD-CHO fail to undergo membrane blebbing and apoptotic body formation, because Ac-DEVD-CHO blocks caspase-3-dependent blebbing (Sasaki and Chiba, 2001 Involvement of p38MAPK in Apoptotic Body Formation To determine whether p38MAPK contributed some feature of apoptosis, we treated eggs with the specific p38MAPK inhibitor SB203580 just before the onset of blebbing (10.5 h after 1-MA treatment). As shown in Figure 8a, SB203580 did not block the blebbing. Apoptotic body formation, however, was severely inhibited in the SB203580-treated eggs, exhibiting an almost rounded morphology (Figure 8, b and c, arrows) with remaining small protrusions (Figure 8, b and c, arrowheads). The small protrusions were separated spontaneously from the rounded egg, and finally they degraded and released apoptotic body-like particles (Figure 8d, arrowhead). The egg still exhibited a rounded morphology even at 20 h after 1-MA treatment (Figure 8d, arrow). These results indicate that p38MAPK may contribute to apoptotic body formation.
Discussion In this study, we found that MAPK has both positive and negative functions in the induction of starfish egg apoptosis. During the MAPK-dependent period (~8 h after 1-MA treatment), inactivation of MAPK blocked apoptosis, indicating that it gives the death-activating signal. Conversely, after the MAPK-dependent period but before blebbing (~8-10 h after 1-MA treatment), inactivation of MAPK resulted in caspase-3 activation, causing apoptosis. Moreover, p38MAPK, which is generally considered as a death factor (Xia et al., 1995 ![]() ![]() During the MAPK-dependent period, starfish eggs are likely to develop competence to die, as reported in mammalian sympathetic neurons. After nerve growth factor deprivation, apoptosis of sympathetic neurons requires the activation of two events: a protein synthesis dependent, Bax-dependent release of mitochondrial cytochrome c and protein synthesis-independent, Bax-independent development of competence. Unlike most cells, cytosolic cytochrome c is not sufficient to induce cell death in nerve growth factor-maintained sympathetic neurons but can do so in the neurons that have developed competence (Deshmukh and Johnson, 1998 Usually in mammalian apoptosis, the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification to inhibit a component of the cell death machinery and the increased transcription of prosurvival genes. Because inactivation of MAPK after the MAPK-dependent period resulted in caspase-3 activation in starfish eggs (within 1 h after U0126 treatment; Figure 4B), MAPK may regulate the apoptotic machinery directly or indirectly, presumably via phosphorylation. It is reported that egg extracts prepared from the frog Xenopus laevis initiate and execute a full apoptotic program in vitro when egg extracts are “aged” on the bench (Newmeyer et al., 1994 In mammals, two proapoptotic Bcl-2-family proteins, Bad and Bim, are involved in apoptosis after withdrawal of survival factors. MAP kinase-activated kinase Rsk phosphorylates the proapoptotic protein BAD. Phosphorylated BAD is inactivated, and thus active Rsk prevents apoptosis by inhibiting BAD (Bonni et al., 1999 We also demonstrated in this study that U0126 treatment resulted in the activation of p38MAPK. This result strongly suggested that inactivation of MAPK acts upstream of p38MAPK activation. In addition, just after GVBD, MAPK is activated (Pelech et al., 1988 Because activation of p38MAPK occurred spontaneously even in the eggs injected with caspase-3 inhibitor Ac-DEVDCHO (Figure 7B), p38MAPK does not act downstream of caspase-3. Further studies are required to determine whether p38MAPK acts upstream of caspase-3. Regulation of actin dynamics is one of the functions of the p38MAPK pathway. After activation by p38MAPK, MAP kinase-activated protein kinase-2 phosphorylates HSP27, a protein that can modulate actin polymerization (Huot et al., 1998 We and others had demonstrated that postmeiotic starfish eggs undergo apoptosis, if they were not fertilized (Sasaki and Chiba, 2001 Moreover, apoptosis is a widespread event in oogenesis (reviewed by Matova and Cooley, 2001 The time after ovulation during which mammalian eggs can give rise to developmentally competent embryo is short. Under in vivo conditions, ovulated mouse eggs exhibit maximum ability to fertilize for only 4-6 h (Lewis and Wright, 1935 The target molecules of MAPK and p38MAPK for apoptotic cell death as well as cell survival have to be identified to further understand the molecular mechanism of determining the egg fate, i.e., whether the cells undergo development or apoptosis. | |||||||||||||||||
Acknowledgments We thank Dr. Kazunori Tachibana for providing GST-starfish Mos clones and for helpful advice. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a grant from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists, the Human Frontier Science Program, and by funds from the Cooperative Program provided by Ocean Research Institute, University of Tokyo. | |||||||||||||||||
Notes Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E03-06-0367. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03-06-0367. | |||||||||||||||||
References
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