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Apoptosis in steroidogenic cells: Structure-function analysis
ELSEVIER
Apoptosis in steroidogenic cells: Structure-function analysis Abraham Amsterdam, Ada Dantes, Natarajagounder Selvaraj, and Dorit Aharoni Department o f Molecular Cell Biology, The Weizmann Institute o f Science, Rehovot, Israel
Granulosa cells are the main producers of the female sex steroid hormones, progesterone and estradiol, which are re~ponsible for the cyclicity in ovarian function. Programmed cell death in the ovary plays a crucial role in limiting the number of follicles that can ovulate and thus prevents the development of more embryos than can successfully complete pregnancy. Granulosa cell apoptosis is regulated by the concerted action of endocrine, paracrine, and autocrine factors. These factors lead to the developmental decision of whether the steroidogenic cell will luteinize and enter the pathway leading to programmed cell death, ot" whether the life span of the luteinized cell will be prolonged to continue secretion of progesterone, which is essential for the maintenance of pregnancy. At the level of the individual cell, we find that enhanced steroidogenesis can be maintained during the initial steps of apoptosis as long as the steroidogenic apparatus remains intact. This can be achieved by a unique mechanism of compartmentalization of steroidogenic organelles in the perinuclear region and migration of the multicatalytic proteinase, the proteasome, to the apoptotic blebs. Reorganization of the actin cytoskeleton during apoptosis may provide an efficient barrier between the proteolytic activity and the steroidogenic activity in the apoptotic cell. It is suggested that steroidogenesis can be maintained in the apoptotic cells as long as the steroidogenic organelles bearing the steroidogenic apparatus remain intact. © 1997 by Elsevier Science Inc. (Steroids 62:207-211, 1997)
Keywords:steroidogenesis: apoptosis; granulosacells; ovary
Introduction Granulosa cells, the main constituents of the ovarian follicle, nurse the eggs and serve as the main source for progesterone and estradiol biosynthesis, t-3 Out of about 400,000 follicle-enclosed oocytes present at puberty, very few will reach full maturation and ovulation, while the rest will degenerate in a process termed atresia. In each reproductive cycle the newly formed corpora lutea will develop following ovulation, and the corpora lutea of the previous cycle will r_e~gress and will be eliminated in a process termed luteolysis.l-- In contrast, the life span of the corpus luteum will be markedly prolonged during pregnancy. We and others found recently that both atresia and luteolysis have features characteristic of programmed cell death (PCD) or apoptosis, including condensation and fragmentation of DNA. 4-8 Therefore, it became evident that the control of granulosa PCD plays an important role in the maintenance of normal ovarian function during the estrus cycle as well as during pregnancy. Moreover, it becomes evident
Address reprint requests to Dr. AbrahamAmsterdam, Department of Molecular Cell Biology,The WeizmannInstitute of Science, Rehovot76100, Israel. Steroids 62:207-211, 1997 © 1997 by Elsevier Science Inc. 655 Avenueof the Americas. New York, NY 10010
that the initial steps of aooptosis are associated with enhanced steroidogenesis.6-'Since follicular and corpora luteal PCD is not a synchronized event among the total cell population, it is not clear whether the same cells that undergo apoptosis also maintain their steroidogenic activity. In primary cultures of granulosa cells, complete synchrony cannot be achieved because the ceils derived from different layers of granulosa cells demonstrate different degrees of differentiation. 2'9 Therefore, to obtain synchronization in the induction of apoptosis, we have established granulosa cell lines in which PCD can be induced in a synchronous manner. These cells were transfected with a temperature-sensitive mutant of the tumor suppressor gene p53 and retain their steroidogenic response to cAMP stimulation. However, when the temperature is shifted from 37°C to 32°C, which leads to the manifestation of the wild-type activity of p53, they undergo a massive and rapid apoptosis. 7
Apoptosis is controlled by multiple extraceilular and intracellular signals In primary cultures obtained from preantral rat follicles, follicle-stimulating hormone (FSH) as well as basic fibro0039-128X/97/$17.00 Pll S0039-128X(96)00182-8
Papers blastic growth factor (bFGF) serve as survival factors that protect the cells from undergoing apoptosis in serum-free medium. 6.m Both FSH and substances elevating intracellular cAMP, such as 8-Br-cAMP, forskolin, and cholera toxin, induce differentiation of granulosa cells derived from preantral follicles in serum-free medium in vitro. This differentiation involves de novo synthesis of luteinizing hormone receptors, ll'12 In contrast, primary cultures obtained from preovulatory follicles were more sensitive to serum deprivation, m Moreover, gonadotropic hormones could not prevent apoptosis induced by serum deprivation, and forskolin and 8-Br-cAMP can even enhance apoptosis in both human and rat preovulatory cells. 6A3'14 Interestingly, bFGF, which stimulates progesterone production in these cultures and synergizes with cAMP-generated signals in stimulation of progesterone, can reduce the incidence of apoptosis induced either by serum removal or by prolonged cAMP stimulation. 6,m Thus we can conclude the following: (A) Activation of tyrosine phosphorylation serves as a survival factor for granulosa cells. (B) Granulosa cells acquire different sensitivities to cAMP, depending on the degree of their maturation and both the amplitude and the duration of cAMP stimulation. (C) Cross-talk between these two major signaling pathways can control the developmental decision of whether the steroidogenic cell will undergo luteinization followed by programmed cell death, or will survive in its differentiated state during pregnancy.
Intracellular compartmentalization of steroidogenic organeiles permits progesterone production and apoptotic processes to coexist within the same cell We have demonstrated that induction of steroidogenesis in granulosa cells is associated with reorganization of the cytoskeleton. This involves centripetal movement of actin filaments and clustering of steroidogenic organelles such as lipid droplets and mitochondria. 19'2° This process was accompanied by down-regulation in the expression of actin and actin-binding proteins, such as vinculin, a-actinin, and the five isoforms of tropomyosin. 21'22 Ultrastructural analysis of primary granulosa cells (Figure 1), as well as immortalized cells (Figure 2), showed that the mitochondria, lipid droplets, lysosomes, and smooth endoplasmic reticulum (organelles that are associated with steroidogenesis) remain intact and highly clustered during the first 24 h of induction of apoptosis, concomitantly with secretion of high levels of progesterone. 6'7 The apoptotic blebs were almost completely devoid of steroidogenic organelles, and condensation of chromatin and initial breakdown of the nuclear membrane were evident in the vast majority of the cells] These data strongly suggest that compartmentalization of the steroidogenic organelles during initial steps of apoptosis plays an important role in the preservation of steroidogenesis and
Tumor suppressor and survival genes as modulators of apoptosis bcl-2 and dad-1 act as survival genes that prevent apoptosis in granulosa cells as well as in other cell types, ls'16 On the other hand, APO-t, p53, and its target genes, waf-1 and bax, induce apoptosis in numerous cell types.t6'17 Immunocytochemistry demonstrated that the level of p53 increases in apoptotic granulosa cells. ~8 Blocking the activity of the wild-type p53 by transfection of granulosa cells with SV40 DNA, which results in the expression of T antigen, blocks the cAMP induction of progesterone production as well as the induction of apoptosis by 8-Br-cAMP and forskolin. 6 To examine the direct effect of p53 on steroidogenesis and apoptosis, we transfected primary granulosa cells with a temperature-sensitive mutant of p53 (p53Val 135). At 37°C this mutant is not capable of binding DNA, and thus cannot function as a tumor suppressor. In contrast, at 32°C, it binds DNA, exhibiting tumor suppressor activity by the induction of waf-1/cip-1 gene expression. 7 These cells are not steroidogenic during their proliferative stage, but become highly steroidogenic, expressing the P450scc enzyme system upon cAMP stimulation. A temperature shift from 37°C to 32°C was sufficient to suppress cell growth, but not to induce apoptosis. However, at 32°C in the presence of cAMP, cells underwent massive and rapid apoptosis with temporary, but significant, elevation of progesterone production. 7 Thus, in the immortalized cell lines as well as in primary cells, induction of apoptosis involves temporary elevation of progesterone production. 6"7
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Figure 1 Various degrees of disorganization in apoptotic preovulatory granulosa cells treated with forskolin for 40 h at 37°C. (A) Large inclusions of chromatin are visible within the nuclear matrix (arrow). Electrondense mitochondria (m) and endoplasmic reticulum (er) are well preserved and a few autophagic vesicles (p) are visible. (B) Cell with numerous large vacuoles. Organelles involved in steroidogenesis (i.e., mitochondria and endoplasmic reticulum) are well preserved. (C) Cytoplasmic organization is heavily deteriorated including Golgi complexes (g) and clustered mitochondria. Condensed chromatin inclusion is visible (arrow). (D) Cell in progressive lysis with remnant of membrane structures, lipid droplets (d), and two large irregular aggregates of condensed chromatin (arrows). Bar, 2 pm. (Adapted from Ref. 6 with permission.)
Apoptosis in steroidogenic cells: Amsterdam et al. proteinase, the proteosome, ma~, be modulated during apoptosis in human lymphocytes.25 Therefore, we examined possible rearrangement of the actin cytoskeleton and spatial distribution of proteasomes during apoptosis. For this purpose we utilized immunocytochemical methods and a laser confocal microscope to reveal the spatial organization and 3D reconstruction of the steroidogenic organelles, the actin cytoskeleton, and the proteasomes, which are the most abundant nonlysomal proteases in mammalian cells. Using rhodamine phalloidin we found that the actin filaments in the apoptotic cells are reorganized in a sphere composed of actin networks, which serve as a barrier between the apoptotic blebs and the main bulk of the cell. 26 The proteasomes, located in both the cytoplasm and the nucleus of the nonapoptotic ceils, 27'2s migrate from the nucleus to the apoptotic blebs. By staining the mitochondria with antiadrenodoxin antibodies, we followed their distribution in the entire cell volume. We could verify (Figure 2) that the mitochondria were highly clustered in the perinuclear region, whereas the apoptotic blebs were essentially devoid of mitochondria. 26 Thus reorganization of the cytoskeleton provides an efficient barrier between the proteasomes in the apoptotic blebs and the mitochondria in the perinuclear region.
Expression of components controlling steroidogenesis in apoptotic blebs
Figure 2 Apoptosis in immortalized granulosa cells (GTS-5) expressing the temperature-sensitive mutant of p53. GTS-5 cells were stimulated for 24 h with 50 tiM forskolin at 320C (A). Note condensation of chromatin in the nucleus (arrow), concentration of mitochondria in the perinuclear region (m), and formation of numerous apoptotic blebs at the periphery of the cells containing mainly polyribosomes but essentially devoid of mitochondria. (B) Intensive clustering of steroidogenic organelles. Note initial condensation of chromatin in the nuclear periphery (arrow) and a massive aggregation of mitochondria (m), lipid droplets (d), Golgi complexes (g), and small vesicles of smooth membranes (s) characteristic of a highly steroidogenic cell. Apoptotic cytoplasmic blebs (asterisk) are devoid of steroidogenic organelles. Bar, 2 tJm. (With permission from Ref. 3.)
that progesterone production can be continued in the same cells that show progressive signs of apoptosis, such as chromatin condensation and bleb formation.
Actin cytoskeleton rearrangement during apoptosis Apoptosis involves activation of endonucleases as well as proteases. Recent investigations revealed that fodrin, one of the main actin-binding proteins localized at the cortex of the actin cytoskeleton, is associated with the cell membrane and is cleaved by calpain during apoptosis in lymphoid cells. 23 Moreover, actin can undergo limited proteolysis by interleukin-1 converting enzymes (ICE).24Ubiquitinated proteins, which can serve as a substrate to the multicatalytic
It was recently demonstrated that the steroidogenic transcription factor Ad4BP and the steroidogenic acute regulatory protein (STAR) are essential for steroidogenesis in steroidogenic cells. 29-32 We found that the immortalized granulosa cell lines established in our laboratory express both the Ad4BP and StAR protein only in steroidogenic cell lines transfected with SV40 DNA and the H a - r a s oncogene, whereas cells transfected with SV40 DNA alone lose their steroidogenic ability and do not express the Ad4BP 33 and StAR protein (our unpublished observations). This would suggest that ras p21 may play an important role in signal transduction pathways that may lead to the expression of these proteins during gonadal development. Our preliminary experiments suggest that Ad4BP, StAR protein, and adrenodoxin expression is not down-regulated during the initial phase of apoptosis, permitting steroidogenesis to continue during this process.
Prospective research It is still not clear whether the cellular and molecular events characteristic of granulosa cell apoptosis are common to other steroidogenic cells, such as those of the adrenal cortex and the testicular Leydig cells. If these mechanisms are common, what are the cell-specific signals that induce or prevent apoptosis? Another important question is, what is the physiological significance of continued and even enhanced steroidogenic activity during apoptosis? For the granulosa luteal cells, it would suggest that the corpus luteum during its peak of activity in progesterone production is already committed to the apoptotic process. This raises the question, what are the signals that extend the life span of
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Papers the corpus luteum during pregnancy? As for the cellular level, the mechanism by which the cytoskeleton acquires its new conformation during programmed cell death is a challenge for future research.
12.
13.
Conclusion Elucidation of the steroidogenic pathway at the molecular level has dramatically progressed in the last few years, mainly because of the cloning of two essential proteins regulating steroidogenesis, i.e., Ad4BP and StAR protein. 29-32 High-resolution microscopy, immunocytochemistry, and 3D reconstruction of the steroidogenic cell can permit detailed resolution of the 3D organization of the steroidogenic elements in the intact cell and the modulation of intracellular organization. Such an approach will provide us with an insight into the in situ regulation of steroidogenesis in the intact cell as well as in the whole organism.
14.
15.
16. 17.
Acknowledgments We thank Dr. M. Walker for helpful discussions and Ms. Vivienne Laufer for excellent secretarial assistance. This work was supported in part by grants from the Leo and Julia Forchheimer Center of Molecular Genetics and from the Dr. Joseph Cohn Minerva Center for Biomembrane Research at the Weizmann Institute of Science. A.A. is the incumbent of the Joyce and B. Eisenberg Professorial Chair of Molecular Endocrinology and Cancer Research at the Weizmann Institute of Science.
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20.
21.
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Hsueh AJW, Adashi EY, Jones PBC, Welsh JTH (1984). Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5:76-127. Amsterdam A, Rotmensch S (1987). Structure-function relationships during granulosa cell differentiation. Endocr Rev 8:309-337. Richards JS, Herin L (1988). Molecular aspects of hormone action in ovarian follicular development, ovulation and luteinization. Annu Rev Physiol 50:441-463. Tilly JL, Kowalski KI, Johnson AL, Hsueh AJW (1991). Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 129:2799-2801. Tilly JL (1993). Ovarian follicular atresia: a model to study the mechanisms of physiological cell death. Endocr J 1:67-72. Aharoni D, Dantes A, Oren M, Amsterdam A (1995). cAMPmediated signals as determinants for apoptosis in primary granulosa cells. Exp Cell Res 218:271-282. Keren-Tal I, Suh B-S, Dantes A, Lindner S, Oren M, Amsterdam A (1995). Involvement of p53 expression in cAMP-mediated apoptosis in immortalized granulosa cells. Exp Cell Res 218:283-295. Hsueh AJW, Billig H, Tsafriri A (1994). Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 15:707-724. Amsterdam A, Koch Y, Lieberman ME, Lindner HR (1975). Distribution of binding sites for human chorionic gonadotropin in the preovulatory follicle of the rat. J Cell Biol 67:894-900. Tilly JL, Billig H, Kowalski KI, Hsueh AJW (1992). Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol 6:1942-1950. Amsterdam A, Knecht M, Catt KJ (1981). Hormonal regulation of
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cytodifferentiation and intercellular communication in cultured granulosa cells. Proc Natl Acad Sci USA 78:3000-3004. Knecht M, Amsterdam A, Catt KJ (1981). The regulatory role of cyclic AMP in hormone induced granulosa cell differentiation. J Biol Chem 256:10628-10633. Breckwoldt M, Selvaraj N, Aharoni D, Barash A, Segal L, Insler V, Amsterdam A (1996). Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long term cultures of human granulosa cells. Mol Hum Reprod 2:391-400. Minami S, Chun SY, Eisenhauer K, Hsueh AJW (1996). Suppression of apoptosis in cultured preantral follicles following activation of the cGMP but not the cAMP pathway: gonadotropins are not survival factors during early follicle development, lnt Congr Endocrinol Abstr P2-702. Tilly JL, Tilly KI, Kenton ML, Johnson AL (1995). Expression of members of the Bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive Bcl-2 and Bcl-xlong messenger ribonucleic acid levels. Endocrinology 136: 232-241. Kroemer G, Petit P, Zamzami N, Vayssiere JL, Mignotte B (1995). The biochemistry of programmed cell death. FASEB J 9:1277-1287. Haffner R, Oren M (1995). Biochemical properties and biological effects of p53. Curr Opin Genet Dev 5:84-90. Tilly KI, Banerjee S, Banerjee PP, Tilly JL (1995). Expression of the p53 and Wilms' tumor suppressor genes in the rat ovary: gonadotropin repression in vivo and immunohistochemical localization of nuclear p53 protein to apoptotic granulosa cells of atretic follicles. Endocrinology 136:1394-1402. Amsterdam A, Rotmensch S, Ben-Zeev A (1989). Coordinated regulation of morphological and biochemical differentiation in a steroidogenic cell: the granulosa cell model. Trends Biochem Sci 14:377-382. Amsterdam A, Aharoni D (1994). Plasticity of cell organization during differentiation of normal and oncogene transformed granulosa cells. Microsc Res Tech 27:108-124. Ben-Ze'ev A, Amsterdam A (1987). In vitro regulation of granulosa cell differentiation. Involvement of cytoskeletal protein expression. .I Biol Chem 262:5366-5376. Ben-Ze'ev A, Baum G, Amsterdam A (1989). Regulation of tropomyosin expression in the maturing ovary and in primary granulosa cell cultures. Dev Biol 135:191-201. Martin SJ, O'Brien GA, Nishioka WK, McGahon AJ, Mahboubi A, Saido TC, Green DR (1995). Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J Biol Chem 270:6425~i428. Mashima T, Naito M, Fujita N, Noguchi K, Tsuruo T (1995). Identification of actin as a substrate of ICE and an ICE-like protease and involvement of an ICE-like protease but not ICE in VP-16 induced U937 apoptosis. Biochem Biophys Res Commun 217:1185-1192. Delic J, Morange M, Magdelenat H (1993). Ubiquitin pathway involvement in human lymphocyte gamma-irradiation-induced apoptosis. 114o1Cell Biol 13:4875-4883. Amsterdam A, Dames A, Fuchs T, Baumeister W, Pitzer F (1996). Maintenance of granulosa cell steroidogenesis during early stages of apoptosis. In: lOth International Congress of Endocrinology. Abstr. P2-774. Amsterdam A, Pitzer F, Baumeister W (1993). Changes in intracellular localization of proteasomes in immortalized ovarian granulosa cells during mitosis associated with a role in cell cycle control. Proc Natl Aead Sci USA 90:99-103. Amsterdam A, Pitzer F, Santarius U, Dantes A, Baumeister W (1994). Possible role of the multi catalytic proteinase (proteasome) in regulating of the cell cycle. In: The Cell Cycle '93. Regulators. Targets and Clinical Applications, XIIIth Washington International Spring Symposium, Hu, VW, (ed). Plenum Press, pp. 203-209. Lin D, Sugawara T, Strauss JFI, Clark BJ, Stocco DM, Saenger P, Rogol A, Miller WL (1995). Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267:18281831. Sugawara T, Holt JA, Driscoll D, Strauss JFI, Lin D. Miller, WL.
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Patterson D, Clancy KP, Hart IM, Clark BJ, Stocco DM (1995). Human steroidogenic acute regulatory protein: functional activity in COS 1 cells, tissue-specific expression and mapping of the structural gene to 8pl 1.2 and a pseudogene to chromosome 13. Proc Natl Acad Sci USA 92:4778-4782. Morohashi K, Honda S, Inomata Y, Handa H, Omura T (1992). A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s. J Biol Chem 267:17913-17919.
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Lynch JP, Lala DS, Peluso JJ, Luo W, Parker KL, White BA (1993). Steroidogenic factor 1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues. Mol Endocrinol 7:776-786. Keren-Tal I, Dantes A, Plehn-Dujowich D, Amsterdam A (1996). Expression of Ad4BP/SF-1 transcription factor determines steroidogenic activity in oncogene-transformed granulosa cells. In: lOth International Congress of Endocrinology. Abstr. P2-699.
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Apoptosis in steroidogenic cells: Structure-function analysis Abraham Amsterdam, Ada Dantes, Natarajagounder Selvaraj, and Dorit Aharoni Department o f Molecular Cell Biology, The Weizmann Institute o f Science, Rehovot, Israel
Granulosa cells are the main producers of the female sex steroid hormones, progesterone and estradiol, which are re~ponsible for the cyclicity in ovarian function. Programmed cell death in the ovary plays a crucial role in limiting the number of follicles that can ovulate and thus prevents the development of more embryos than can successfully complete pregnancy. Granulosa cell apoptosis is regulated by the concerted action of endocrine, paracrine, and autocrine factors. These factors lead to the developmental decision of whether the steroidogenic cell will luteinize and enter the pathway leading to programmed cell death, ot" whether the life span of the luteinized cell will be prolonged to continue secretion of progesterone, which is essential for the maintenance of pregnancy. At the level of the individual cell, we find that enhanced steroidogenesis can be maintained during the initial steps of apoptosis as long as the steroidogenic apparatus remains intact. This can be achieved by a unique mechanism of compartmentalization of steroidogenic organelles in the perinuclear region and migration of the multicatalytic proteinase, the proteasome, to the apoptotic blebs. Reorganization of the actin cytoskeleton during apoptosis may provide an efficient barrier between the proteolytic activity and the steroidogenic activity in the apoptotic cell. It is suggested that steroidogenesis can be maintained in the apoptotic cells as long as the steroidogenic organelles bearing the steroidogenic apparatus remain intact. © 1997 by Elsevier Science Inc. (Steroids 62:207-211, 1997)
Keywords:steroidogenesis: apoptosis; granulosacells; ovary
Introduction Granulosa cells, the main constituents of the ovarian follicle, nurse the eggs and serve as the main source for progesterone and estradiol biosynthesis, t-3 Out of about 400,000 follicle-enclosed oocytes present at puberty, very few will reach full maturation and ovulation, while the rest will degenerate in a process termed atresia. In each reproductive cycle the newly formed corpora lutea will develop following ovulation, and the corpora lutea of the previous cycle will r_e~gress and will be eliminated in a process termed luteolysis.l-- In contrast, the life span of the corpus luteum will be markedly prolonged during pregnancy. We and others found recently that both atresia and luteolysis have features characteristic of programmed cell death (PCD) or apoptosis, including condensation and fragmentation of DNA. 4-8 Therefore, it became evident that the control of granulosa PCD plays an important role in the maintenance of normal ovarian function during the estrus cycle as well as during pregnancy. Moreover, it becomes evident
Address reprint requests to Dr. AbrahamAmsterdam, Department of Molecular Cell Biology,The WeizmannInstitute of Science, Rehovot76100, Israel. Steroids 62:207-211, 1997 © 1997 by Elsevier Science Inc. 655 Avenueof the Americas. New York, NY 10010
that the initial steps of aooptosis are associated with enhanced steroidogenesis.6-'Since follicular and corpora luteal PCD is not a synchronized event among the total cell population, it is not clear whether the same cells that undergo apoptosis also maintain their steroidogenic activity. In primary cultures of granulosa cells, complete synchrony cannot be achieved because the ceils derived from different layers of granulosa cells demonstrate different degrees of differentiation. 2'9 Therefore, to obtain synchronization in the induction of apoptosis, we have established granulosa cell lines in which PCD can be induced in a synchronous manner. These cells were transfected with a temperature-sensitive mutant of the tumor suppressor gene p53 and retain their steroidogenic response to cAMP stimulation. However, when the temperature is shifted from 37°C to 32°C, which leads to the manifestation of the wild-type activity of p53, they undergo a massive and rapid apoptosis. 7
Apoptosis is controlled by multiple extraceilular and intracellular signals In primary cultures obtained from preantral rat follicles, follicle-stimulating hormone (FSH) as well as basic fibro0039-128X/97/$17.00 Pll S0039-128X(96)00182-8
Papers blastic growth factor (bFGF) serve as survival factors that protect the cells from undergoing apoptosis in serum-free medium. 6.m Both FSH and substances elevating intracellular cAMP, such as 8-Br-cAMP, forskolin, and cholera toxin, induce differentiation of granulosa cells derived from preantral follicles in serum-free medium in vitro. This differentiation involves de novo synthesis of luteinizing hormone receptors, ll'12 In contrast, primary cultures obtained from preovulatory follicles were more sensitive to serum deprivation, m Moreover, gonadotropic hormones could not prevent apoptosis induced by serum deprivation, and forskolin and 8-Br-cAMP can even enhance apoptosis in both human and rat preovulatory cells. 6A3'14 Interestingly, bFGF, which stimulates progesterone production in these cultures and synergizes with cAMP-generated signals in stimulation of progesterone, can reduce the incidence of apoptosis induced either by serum removal or by prolonged cAMP stimulation. 6,m Thus we can conclude the following: (A) Activation of tyrosine phosphorylation serves as a survival factor for granulosa cells. (B) Granulosa cells acquire different sensitivities to cAMP, depending on the degree of their maturation and both the amplitude and the duration of cAMP stimulation. (C) Cross-talk between these two major signaling pathways can control the developmental decision of whether the steroidogenic cell will undergo luteinization followed by programmed cell death, or will survive in its differentiated state during pregnancy.
Intracellular compartmentalization of steroidogenic organeiles permits progesterone production and apoptotic processes to coexist within the same cell We have demonstrated that induction of steroidogenesis in granulosa cells is associated with reorganization of the cytoskeleton. This involves centripetal movement of actin filaments and clustering of steroidogenic organelles such as lipid droplets and mitochondria. 19'2° This process was accompanied by down-regulation in the expression of actin and actin-binding proteins, such as vinculin, a-actinin, and the five isoforms of tropomyosin. 21'22 Ultrastructural analysis of primary granulosa cells (Figure 1), as well as immortalized cells (Figure 2), showed that the mitochondria, lipid droplets, lysosomes, and smooth endoplasmic reticulum (organelles that are associated with steroidogenesis) remain intact and highly clustered during the first 24 h of induction of apoptosis, concomitantly with secretion of high levels of progesterone. 6'7 The apoptotic blebs were almost completely devoid of steroidogenic organelles, and condensation of chromatin and initial breakdown of the nuclear membrane were evident in the vast majority of the cells] These data strongly suggest that compartmentalization of the steroidogenic organelles during initial steps of apoptosis plays an important role in the preservation of steroidogenesis and
Tumor suppressor and survival genes as modulators of apoptosis bcl-2 and dad-1 act as survival genes that prevent apoptosis in granulosa cells as well as in other cell types, ls'16 On the other hand, APO-t, p53, and its target genes, waf-1 and bax, induce apoptosis in numerous cell types.t6'17 Immunocytochemistry demonstrated that the level of p53 increases in apoptotic granulosa cells. ~8 Blocking the activity of the wild-type p53 by transfection of granulosa cells with SV40 DNA, which results in the expression of T antigen, blocks the cAMP induction of progesterone production as well as the induction of apoptosis by 8-Br-cAMP and forskolin. 6 To examine the direct effect of p53 on steroidogenesis and apoptosis, we transfected primary granulosa cells with a temperature-sensitive mutant of p53 (p53Val 135). At 37°C this mutant is not capable of binding DNA, and thus cannot function as a tumor suppressor. In contrast, at 32°C, it binds DNA, exhibiting tumor suppressor activity by the induction of waf-1/cip-1 gene expression. 7 These cells are not steroidogenic during their proliferative stage, but become highly steroidogenic, expressing the P450scc enzyme system upon cAMP stimulation. A temperature shift from 37°C to 32°C was sufficient to suppress cell growth, but not to induce apoptosis. However, at 32°C in the presence of cAMP, cells underwent massive and rapid apoptosis with temporary, but significant, elevation of progesterone production. 7 Thus, in the immortalized cell lines as well as in primary cells, induction of apoptosis involves temporary elevation of progesterone production. 6"7
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Figure 1 Various degrees of disorganization in apoptotic preovulatory granulosa cells treated with forskolin for 40 h at 37°C. (A) Large inclusions of chromatin are visible within the nuclear matrix (arrow). Electrondense mitochondria (m) and endoplasmic reticulum (er) are well preserved and a few autophagic vesicles (p) are visible. (B) Cell with numerous large vacuoles. Organelles involved in steroidogenesis (i.e., mitochondria and endoplasmic reticulum) are well preserved. (C) Cytoplasmic organization is heavily deteriorated including Golgi complexes (g) and clustered mitochondria. Condensed chromatin inclusion is visible (arrow). (D) Cell in progressive lysis with remnant of membrane structures, lipid droplets (d), and two large irregular aggregates of condensed chromatin (arrows). Bar, 2 pm. (Adapted from Ref. 6 with permission.)
Apoptosis in steroidogenic cells: Amsterdam et al. proteinase, the proteosome, ma~, be modulated during apoptosis in human lymphocytes.25 Therefore, we examined possible rearrangement of the actin cytoskeleton and spatial distribution of proteasomes during apoptosis. For this purpose we utilized immunocytochemical methods and a laser confocal microscope to reveal the spatial organization and 3D reconstruction of the steroidogenic organelles, the actin cytoskeleton, and the proteasomes, which are the most abundant nonlysomal proteases in mammalian cells. Using rhodamine phalloidin we found that the actin filaments in the apoptotic cells are reorganized in a sphere composed of actin networks, which serve as a barrier between the apoptotic blebs and the main bulk of the cell. 26 The proteasomes, located in both the cytoplasm and the nucleus of the nonapoptotic ceils, 27'2s migrate from the nucleus to the apoptotic blebs. By staining the mitochondria with antiadrenodoxin antibodies, we followed their distribution in the entire cell volume. We could verify (Figure 2) that the mitochondria were highly clustered in the perinuclear region, whereas the apoptotic blebs were essentially devoid of mitochondria. 26 Thus reorganization of the cytoskeleton provides an efficient barrier between the proteasomes in the apoptotic blebs and the mitochondria in the perinuclear region.
Expression of components controlling steroidogenesis in apoptotic blebs
Figure 2 Apoptosis in immortalized granulosa cells (GTS-5) expressing the temperature-sensitive mutant of p53. GTS-5 cells were stimulated for 24 h with 50 tiM forskolin at 320C (A). Note condensation of chromatin in the nucleus (arrow), concentration of mitochondria in the perinuclear region (m), and formation of numerous apoptotic blebs at the periphery of the cells containing mainly polyribosomes but essentially devoid of mitochondria. (B) Intensive clustering of steroidogenic organelles. Note initial condensation of chromatin in the nuclear periphery (arrow) and a massive aggregation of mitochondria (m), lipid droplets (d), Golgi complexes (g), and small vesicles of smooth membranes (s) characteristic of a highly steroidogenic cell. Apoptotic cytoplasmic blebs (asterisk) are devoid of steroidogenic organelles. Bar, 2 tJm. (With permission from Ref. 3.)
that progesterone production can be continued in the same cells that show progressive signs of apoptosis, such as chromatin condensation and bleb formation.
Actin cytoskeleton rearrangement during apoptosis Apoptosis involves activation of endonucleases as well as proteases. Recent investigations revealed that fodrin, one of the main actin-binding proteins localized at the cortex of the actin cytoskeleton, is associated with the cell membrane and is cleaved by calpain during apoptosis in lymphoid cells. 23 Moreover, actin can undergo limited proteolysis by interleukin-1 converting enzymes (ICE).24Ubiquitinated proteins, which can serve as a substrate to the multicatalytic
It was recently demonstrated that the steroidogenic transcription factor Ad4BP and the steroidogenic acute regulatory protein (STAR) are essential for steroidogenesis in steroidogenic cells. 29-32 We found that the immortalized granulosa cell lines established in our laboratory express both the Ad4BP and StAR protein only in steroidogenic cell lines transfected with SV40 DNA and the H a - r a s oncogene, whereas cells transfected with SV40 DNA alone lose their steroidogenic ability and do not express the Ad4BP 33 and StAR protein (our unpublished observations). This would suggest that ras p21 may play an important role in signal transduction pathways that may lead to the expression of these proteins during gonadal development. Our preliminary experiments suggest that Ad4BP, StAR protein, and adrenodoxin expression is not down-regulated during the initial phase of apoptosis, permitting steroidogenesis to continue during this process.
Prospective research It is still not clear whether the cellular and molecular events characteristic of granulosa cell apoptosis are common to other steroidogenic cells, such as those of the adrenal cortex and the testicular Leydig cells. If these mechanisms are common, what are the cell-specific signals that induce or prevent apoptosis? Another important question is, what is the physiological significance of continued and even enhanced steroidogenic activity during apoptosis? For the granulosa luteal cells, it would suggest that the corpus luteum during its peak of activity in progesterone production is already committed to the apoptotic process. This raises the question, what are the signals that extend the life span of
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Papers the corpus luteum during pregnancy? As for the cellular level, the mechanism by which the cytoskeleton acquires its new conformation during programmed cell death is a challenge for future research.
12.
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Conclusion Elucidation of the steroidogenic pathway at the molecular level has dramatically progressed in the last few years, mainly because of the cloning of two essential proteins regulating steroidogenesis, i.e., Ad4BP and StAR protein. 29-32 High-resolution microscopy, immunocytochemistry, and 3D reconstruction of the steroidogenic cell can permit detailed resolution of the 3D organization of the steroidogenic elements in the intact cell and the modulation of intracellular organization. Such an approach will provide us with an insight into the in situ regulation of steroidogenesis in the intact cell as well as in the whole organism.
14.
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16. 17.
Acknowledgments We thank Dr. M. Walker for helpful discussions and Ms. Vivienne Laufer for excellent secretarial assistance. This work was supported in part by grants from the Leo and Julia Forchheimer Center of Molecular Genetics and from the Dr. Joseph Cohn Minerva Center for Biomembrane Research at the Weizmann Institute of Science. A.A. is the incumbent of the Joyce and B. Eisenberg Professorial Chair of Molecular Endocrinology and Cancer Research at the Weizmann Institute of Science.
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