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Department of Biological Sciences, The University of Notre Dame, Notre Dame, Indiana 46556
Address all correspondence and reprint requests to: A. L. Johnson, Department of Biological Sciences, P.O. Box 369, The University of Notre Dame, Notre Dame, Indiana 46556. E-mail: johnson.128{at}nd.edu
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Introduction |
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As many as five additional antiapoptotic proteins (Bcl-xLong, Bcl-w, A1/BFL-1, Boo/Diva, and Mcl-1) have now been characterized in mammals (4, 5, and references therein), each of which may be expressed in cell/tissue-specific as well as differentiation-stage-specific patterns. A prime example of selective tissue expression is the recently described Boo/Diva, which appears to be localized exclusively within the female ovary and male epididymus, implying a select role for this protein in germ cell maturation and/or survival (6).
Expression of Bcl-2-related antiapoptotic proteins is widespread among mammalian species thus far investigated, and their functional significance is implied by the evolutionary conservation observed within nonmammalian vertebrates. For instance, Bcl-2 (7, 8), Bcl-xLong (9, 10), A1 (11), and Mcl-1 (11) homologs have each been described in various tissues from avian species. Curiously, the novel avian nr-13 gene found in both the quail (12) and chicken (13) currently has no mammalian counterpart. Two genes from Xenopus closely resembling bcl-2 and bcl-x (14) and the zebrafish (Danio rerio) homolog to mcl-1 reported in the present issue of Endocrinology by Leo and colleagues (15) further illustrate conserved expression of antiapoptotic Bcl-2 family members. Although there are, to date, comparatively few reports of bcl-2-related genes in additional nonmammalian vertebrates, the screening of expressed sequence tag (EST) data bases, as conducted for mcl-1 by Leo and colleagues, is predicted to remedy this apparent deficiency in the near future.
By comparison, mammalian proapoptotic genes include bax, bcl-xShort, bak, and bok/mtd (3, 4, and references therein). Of these, only bcl-xShort (9, 10) and bak (GenBank Accession No. AI981098) have thus far been identified in a nonmammalian species (chicken); however, as is the case with antiapoptotic proteins, this likely reflects insufficient research efforts in this area rather than the actual absence of nonmammalian homologs.
A common feature among both antiapoptotic and proapoptotic Bcl-2 family
proteins is the conservation of one or more Bcl-2 homology (BH) domains
(BH1 through BH4) that are arranged as 
-helices. Based upon NMR
analysis, the -helix conformation is predicted to be critical for
regulating activity and protein-protein interactions among family
members. Deletion of either the BH1 or BH2 domain abolishes the ability
of Bcl-2 to heterodimerize with Bax and block Bax-induced cell death
(16). By comparison, deletion of the BH4 domain of Bcl-2 nullifies its
antiapoptotic function and homodimerization, but does not impair
binding to the proapoptotic protein Bax.
Bcl-2 family members expressing BH1 and BH2 domains also have the capacity to become integral membrane proteins by virtue of a carboxy-terminal hydrophobic domain. The functional significance of these domains is related to either the ability to form active pores or to serve as regulators of existing ion channels (e.g. 17). It is also of significance that, in the absence of a death-inducing signal, many antiapoptotic proteins are localized to mitochondria, endoplasmic reticulum or nuclear membranes, whereas proapoptotic proteins remain as monomers within the cytosol (5). Following a death-inducing signal, proapoptotic proteins containing BH1, BH2, and BH3 domains acquire the capacity to dimerize and translocate to intracellular compartments, such as mitochondria, for the purpose of membrane integration. Changes in mitochondrial function as a result of such integration can include alterations in membrane potential, production of reactive oxygen species, and the release of cytochrome c (5, 18). In turn, cytochrome c, combined with the cofactor dATP, interact with Apaf-1 (the mammalian homolog to C. elegans Ced-4) (19) to facilitate activation of procaspase-9 and initiate the proteolytic caspase cascade (reviewed in Ref. 18).
There is also data to suggest that another antiapoptotic action of Bcl-2-related proteins may occur via interaction with Apaf-1, thereby directly preventing caspase-9 activation. Accordingly, activated proapoptotic proteins are hypothesized to prevent or displace Apaf-1/antiapoptotic protein interactions, thereby facilitating activation of procaspase-9 (6). More recent data, however, have failed to confirm that antiapoptotic or proapoptotic proteins routinely interact with Apaf-1 (20).
Several independent lines of evidence suggest that the BH3 domain represents the primary death-inducing domain of proapoptotic family members. For instance, mutations within the BH3 domain may prevent dimerization with antiapoptotic proteins and abolish proapoptotic activity. The importance of the BH3 domain is further illustrated by the fact there exists a separate group of proapoptotic Bcl-2-related proteins classified on the basis that they contain only the BH3 domain. Such proteins include Bid, Bad, Bik, Blk, Bim, Hrk, Nip3, and Nix/Bnip3 (4,5 and references therein). Although in response to death-inducing signals these proteins are unable to integrate within cell membranes, they are apparently capable of translocation to mitochondria. Taken together, these data suggest that BH3-only-molecules may dimerize with and/or influence pore formation by other Bcl-2-related proteins.
Activity of several Bcl-2 family proteins is also altered by posttranslational modifications other than dimerization and membrane integration, often in the form of phosphorylation. Following phosphorylation by cell survival factors, proapoptotic Bad becomes sequestered by the cytosolic protein 14–3-3. Dephosphorylation of Bad in response to death-promoting signals results in exposure of the BH3 domain, thus permitting dimerization with, and neutralization of, antiapoptotic Bcl-xLong. Bcl-2 phosphorylation has been reported to enhance (21, 22) or diminish (23) its antiapoptotic actions. Similarly, phosphorylation of Bcl-xLong is observed following exposure to cell survival factors (e.g. gonadotropins, growth factors) (10), yet has also been associated with proapoptotic cell death (24). Reasons for apparent functional differences related to phosphorylation state have yet to be resolved.
Of central importance is the question why multiple antiapoptotic and proapoptotic Bcl-2 family members are expressed within the same cell type, either simultaneously or in stage-specific patterns. Insights emerging from studies of antiapoptotic Mcl-1 have provided clues to resolve this query.
The mcl (myeloid cell leukemia)-1 member of the bcl-2 gene family was originally identified as an immediate-early gene rapidly induced by a phorbol ester in ML-1 human myeloblastic leukemia cells undergoing differentiation (25). The predicted coding region encodes a protein of 37 kDa (350 AA) containing all four conserved BH domains plus a transmembrane domain. However, unlike other Bcl-2 family members, the N-terminal region contains two PEST sequences (enriched in proline, glutamic acid, serine and threonine) together with four pairs of arginines, which are indicative of a protein that is transiently expressed and rapidly turned over. Moreover, the intracellular distribution of Mcl-1 appears to be more widespread than Bcl-2 because, in addition to heavy localization to mitochondrial membranes, Mcl-1 is also distributed to a variety of nonmitochondrial compartments (26). In light of the apparent central importance of the mitochondria in mediating many forms of apoptotic cell death, the significance of Mcl-1 localization to additional organelle membranes is as yet unclear. There are as yet no reports of whether posttranslational phosphorylation alters the activity of the Mcl-1 protein.
In cells of hematopoietic lineage, the group in which Mcl-1 has best been characterized, Mcl-1 is up-regulated by cell survival cytokines such as macrophage colony-stimulating factor, interleukin (IL)-1ß, and IL-3 (27, 28). By contrast, some death-inducing signals (e.g. growth factor withdrawal, sodium salicylate treatment) rapidly down-regulate Mcl-1 expression (29). Significantly, a notable difference in the pattern of expression between Bcl-2 and Mcl-1 is observed following DNA damage induced by ionizing or UV radiation, or alkylating agents. While bcl-2 messenger RNA (mRNA) levels decrease within the first hours of exposure, mcl-1 mRNA levels are rapidly, but transiently, increased (30). Gene transfer experiments have demonstrated that Mcl-1 overexpression can prolong the survival of cells following exposure to apoptosis-inducing stimuli; however, the duration of survival is short-term, and the efficacy substantially less than when the same cells are transfected with Bcl-2 (31).
Leo and colleagues (15) now extend our understanding of Mcl-1’s antiapoptotic function to cells of nonhematopoietic origin by characterizing patterns of expression and its potential for dimerization with additional Bcl-2 family members within the rat ovary. Ovarian follicles, like the more commonly studied immune system, represent an excellent and clinically relevant model system for studying endocrine and molecular pathways regulating apoptosis. Greater than 99% of all follicles undergo atresia via apoptosis during the lifespan of the female, and this process of elimination begins during gestation (32, 33). In the human female, maximal numbers of germ cells (approximately 7 x 106) occur by about 20 weeks of pregnancy, and by parturition two-thirds of these are lost. The exact signal for such death early during ontogeny, whether preprogrammed, or originating from somatic (pregranulosa) cells or the oocyte itself, is not well established. Moreover, the loss of ovarian follicles following birth of the human female continues over the next 50 or so years such that as reproductive senescence (menopause) approaches there are fewer than 1 x 103 oocytes remaining (34). Thus, from a clinical perspective, an abnormally high rate of follicle atresia can contribute to sporadic or chronic subfertility/infertility as well as to premature menopause.
Apoptosis in postnatal oocytes has been observed within the resting pool of primordial follicles as well as during the earliest (e.g. the primary and secondary) stages of follicle development (35). Unfortunately, this model system has not been extensively studied due to the difficulty in obtaining large numbers of follicles during the earliest stages of follicle growth and development. However, a recent study of Bax-deficient mice has provided evidence for a dramatically reduced incidence of apoptosis within oocytes from primordial follicles at the time of puberty (35). Such results provide evidence not only for a definitive role of apoptosis in determining the number of available germ cells at puberty, but also the first evidence for an intrinsic pattern of cell death mediated via the proapoptotic protein, Bax. According to proposed paradigms to describe the delicate balance between cell survival and death (e.g. 36–38), it is also predicted that an excess of antiapoptotic factor(s) would tip the balance in favor of cell survival. Indeed, transgenic mice overexpressing Bcl-2 targeted to the granulosa layer exhibit reduced follicle atresia and increased ovulation rates following puberty (39).
To date, the most frequently studied system for follicle atresia, with associated germ cell loss, is the antral and preovulatory stages of development. Again, postnatal oocyte loss can be attributed directly to apoptosis within the oocyte or indirectly to the loss of the supporting granulosa cell layer via apoptosis. In studies of early antral and preovulatory follicles, gonadotropins, estrogen, and several growth factors and cytokines have been found to suppress apoptosis and prevent follicle atresia (40, 41). In situ analysis of early atretic follicles (by end-labeling for oligonucleosome formation) consistently demonstrates that widespread apoptosis occurs within the granulosa, but not theca, layer. Accordingly, much of the effect of these physiological factors is attributed to their actions on the granulosa cell layer.
The cellular pathway(s) by which gonadotropins protect granulosa cells from apoptosis has(have) yet to be elucidated. In rat ovarian follicles, gonadotropins have thus far proven to be ineffective in regulating expression of antiapoptotic proteins such as Bcl-2 and Bcl-xLong (38). Therefore, the finding by Leo and colleagues that human CG (hCG) and FSH each increase levels of mcl-1 mRNA perhaps begins to shed light on molecular mechanisms by which gonadotropins may act as fertility-enhancing drugs.
The rapid but short-lived increase in mcl-1 mRNA observed in ovarian cells (15), and in mcl-1 mRNA and Mcl-1 protein within hematopoietic cells (26, 27), suggests a function that may complement the actions of additional Bcl-2 family members. Specifically, it is proposed that the potential for rapid induction of this antiapoptotic protein within the follicle granulosa layer could serve to protect cells during stages of differentiation when they are particularly susceptible to apoptosis, or to acute perturbations (i.e. DNA damage, cytotoxic agents) that would otherwise result in apoptotic cell death. By comparison, levels of mcl-1 mRNA appear to be constitutively expressed at higher levels within the surrounding thecal layer, where apoptosis occurs with a much lesser incidence.
Of additional relevance is a recent publication, which reports that
activation of the phosphatidylinositol 3-kinase/Akt cell survival
pathway also results in rapid up-regulation of mcl-1 mRNA in
TF-1 myeloid cells (42). As several growth factors (e.g.
insulin-like growth factor I, epidermal growth factor, transforming
growth factor-) are known to activate Akt and to serve as cell
survival factors in granulosa cells (Johnson, A. L., and J. T.
Bridgham, unpublished data; 40, 43), it is reasonable to propose
that such growth factors will also be found to up-regulate Mcl-1
expression.
Finally, Leo and colleagues establish the preferential binding of Mcl-1 to a wide range of proapoptotic (including Bax, Bad, Bak, Bok, Bik, and Bod), but not antiapoptotic, Bcl-2 family members. Given that each of these proapoptotic proteins is expressed within the mammalian ovary, these data further reinforce the potential magnitude of complexity for protein-protein interactions which ultimately determine granulosa cell (and thus follicle) viability or death.
In summary, by virtue of its rapid, inducible expression, Mcl-1 may serve to influence granulosa cell viability on a short-term basis. By comparison, additional members of this family, such as Bcl-2 and particularly Bcl-x (9), likely exert more long-term actions to affect cell viability. One implication that has yet to be fully explored within this or any other tissue is that subtle alterations in the expression of wild-type Bcl-2 or Bcl-x may have more serious pathologic consequences, either accelerated death or prolonged survivability, compared with the more labile Mcl-1 protein. Future studies will undoubtedly unravel the intricacies that characterize the process of programmed cell death, and to do so will surely open many new avenues to the treatment of infertility as well as ovarian cancers.
Received October 5, 1999.
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