Cell Death and Cell Renewal

Cell Death and Cell Renewal

Cell death and cell proliferation are balanced throughout the life of multi-cellular organisms. Animal development begins with the rapid prolifera­tion of embryonic cells, which then differentiate to produce the many spe­cialized types of cells that make up adult tissues and organs. Whereas the nematode C. elegans consists of only 959 somatic cells, humans possess a total of approximately 10¹⁴ cells, consisting of more than 200 differentiated cell types. Starting from only a single cell—the fertilized egg—all the diverse cell types of the body are produced and organized into tissues and organs. This complex process of development involves not only cell prolif­eration and differentiation but also cell death. Although cells can die as a result of unpredictable traumatic events, such as exposure to toxic chemi­cals, most cell deaths in multicellular organisms occur by a normal physi­ological process of programmed cell death, which plays a key role both in embryonic development and in adult tissues.

In adult organisms, cell death must be balanced by cell renewal, and most tissues contain stem cells that are able to replace cells that have been lost. Abnormalities of cell death are associated with a wide variety of ill­nesses, including cancer, autoimmune disease, and neurodegenerative disorders, such as Parkinson's and Alzheimer's disease. Conversely, the ability of stem cells to proliferate and differentiate into a wide variety of cell types has generated enormous interest in the possible use of these cells, particularly embryonic stem cells, to replace damaged tissues. The mechanisms and regulation of cell death and cell renewal have therefore become areas of research at the forefront of biology and medicine.

1.      Programmed Cell Death

Programmed cell death is carefully regulated so that the fate of individual cells meets the needs of the organism as a whole. In adults, programmed cell death is responsible for balancing cell proliferation and maintaining constant cell numbers in tissues undergoing cell turnover. For example, about 5 x 10¹¹ blood cells are eliminated daily in humans by programmed cell death, balancing their continual production in the bone marrow. In addition, programmed cell death provides a defense mechanism by which damaged and potentially dangerous cells can be eliminated for the good of the organism as a whole. Virus-infected cells frequently undergo programmed cell death, thereby preventing the production of new virus particles and limiting spread of the virus through the host organism. Other types of cellular insults, such as DNA damage, also induce programmed cell death. In the case of DNA damage, programmed cell death may eliminate cells carrying potentially harmful mutations, including cells with mutations that might lead to the development of cancer.

During development, programmed cell death plays a key role by elimi­nating unwanted cells from a variety of tissues. For example, programmed cell death is responsible for the elimination of larval tissues during amphib­ian and insect metamorphosis, as well as for the elimination of tissue between the digits during the formation of fingers and toes. Another well-characterized example of programmed cell death is provided by develop­ment of the mammalian nervous system. Neurons are produced in excess, and up to 50% of developing neurons are eliminated by programmed cell death. Those that survive are selected for having made the correct connec­tions with their target cells, which secrete growth factors that signal cell sur­vival by blocking the neuronal cell death program. The survival of many other types of cells in animals is similarly dependent on growth factors or contacts with neighboring cells or the extracellular matrix, so programmed cell death is thought to play an important role in regulating the associations between cells in tissues.

The Events of Apoptosis

In contrast to the accidental death of cells that results from an acute injury (necrosis), programmed cell death is an active process, which usually pro­ceeds by a distinct series of cellular changes known as apoptosis, first described in 1972 (Figure 17.1). During apoptosis, chromosomal DNA is usually fragmented as a result of cleavage between nucleosomes. The chro-matin condenses and the nucleus then breaks up into small pieces. Finally, the cell itself shrinks and breaks up into membrane-enclosed fragments called apoptotic bodies.

Apoptotic cells and cell fragments are efficiently recognized and phago-cytosed by both macrophages and neighboring cells, so cells that die by apoptosis are rapidly removed from tissues. In contrast, cells that die by necrosis swell and lyse, releasing their contents into the extracellular space and causing inflammation. The removal of apoptotic cells is mediated by the expression of so-called "eat me" signals on the cell surface. These sig­nals include phosphatidylserine, which is normally restricted to the inner leaflet of the plasma membrane (see Figure 13.2). During apoptosis, phos­phatidylserine becomes expressed on the cell surface where it is recognized by receptors expressed by phagocytic cells (Figure 17.2).

Pioneering studies of programmed cell death during the development of C. elegans provided the critical initial insights that led to understanding the molecular mechanism of apoptosis. These studies in the laboratory of Robert Horvitz initially identified three genes that play key roles in regulat­ing and executing apoptosis. During normal nematode development, 131 somatic cells out of a total of 1090 are eliminated by programmed cell death, yielding the 959 somatic cells in the adult worm. The death of these cells is highly specific, such that the same cells always die in developing embryos. Based on this developmental specificity, Robert Horvitz undertook a genetic analysis of cell death in C. elegans with the goal of identifying the genes responsible for these developmental cell deaths. In 1986 mutagenesis of C. elegans identified two genes, ced-3 and ced-4, that were required for developmental cell death. If either ced-3 or ced-4 was inactivated by muta tion, the normal programmed cell deaths did not take place. A third gene, ced-9, functioned as a negative regulator of apoptosis. If ced-9 was inacti­vated by mutation, the cells that would normally survive failed to do so. Instead, they also underwent apoptosis, leading to death of the developing animal. Conversely, if ced-9 was expressed at an abnormally high level, the normal programmed cell deaths failed to occur. Further studies indicated that the proteins encoded by these genes acted in a pathway with Ced-4 act­ing to stimulate Ced-3, and Ced-9 inhibiting Ced-4 (Figure 17.3). Genes related to ced-3, ced-4, and ced-9 have also been identified in Drosophila and mammals and found to encode proteins that represent conserved effectors and regulators of apoptosis induced by a variety of stimuli.

Caspases: The Executioners of Apoptosis

The molecular cloning and nucleotide sequencing of the ced-3 gene indi­cated that it encoded a protease, providing the first insight into the molecu­lar mechanism of apoptosis. We now know that Ced-3 is the prototype of a family of more than a dozen proteases, known as caspases because they have cysteine (C) residues at their active sites and cleave after aspartic acid (Asp) residues in their substrate proteins. The caspases are the ultimate effectors or executioners of programmed cell death, bringing about the events of apoptosis by cleaving more than 100 different cell target proteins (Figure 17.4). One key target of the caspases is an inhibitor of a DNase, which when activated is responsible for fragmentation of nuclear DNA. In addition, caspases cleave nuclear lamins, leading to fragmentation of the nucleus; cytoskeletal proteins, leading to disruption of the cytoskeleton, membrane blebbing, and cell fragmentation; and Golgi matrix proteins, leading to fragmentation of the Golgi apparatus. The translocation of phos-phatidylserine to the cell surface is also dependent on caspases, although the caspase target(s) responsible for this plasma membrane alteration have not yet been identified.

Ced-3 is the only caspase in C. elegans. However, Drosophila and mam­mals contain families of at least seven caspases, classified as either initiator or effector caspases, that function in a cascade to bring about the events of apoptosis. All caspases are synthesized as inactive precursors that can be converted to the active form by proteolytic cleavage, catalyzed by other cas­pases. Initiator caspases are activated directly in response to the various sig­nals that induce apoptosis, as discussed later in this chapter. The initiator caspases then cleave and activate the effector caspases, which are responsi­ble for digesting the cellular target proteins that mediate the events of apop­tosis (see Figure 17.4). The activation of an initiator caspase therefore starts off a chain reaction of caspase activation leading to death of the cell.

Genetic analysis in C. elegans initially suggested that Ced-4 functioned as an activator of the caspase Ced-3. Subsequent studies have shown that Ced-4 and its mammalian homolog (Apaf-1) bind to caspases and promote their activation. In mammalian cells, the key initiator caspase (caspase-9) is acti­vated by binding to Apaf-1 in a multisubunit complex called the apopto-some (Figure 17.5). Formation of this complex in mammals also requires cytochrome c, which is released from mitochondria by stimuli that trigger apoptosis (discussed in the following section). Once activated in the apop-tosome, caspase-9 cleaves and activates downstream effector caspases, such as caspase-3 and caspase-7, eventually resulting in cell death.

Central Regulators of Apoptosis: The Bcl-2 Family

The third gene identified as a key regulator of programmed cell death in C. elegans, ced-9, was found to be closely related to a mammalian gene called bcl-2, which was first identified in 1985 as an oncogene that contributed to .the development of human B cell lymphomas (cancers of B lymphocytes). In contrast to other oncogene proteins, such as Ras, that stimulate cell pro­liferation (see Molecular Medicine, Chapter 15), Bcl-2 was found to inhibit apoptosis. Ced-9 and Bcl-2 were thus similar in function, and the role of Bcl-2 as a regulator of apoptosis first focused attention on the importance of cell survival in cancer development. As discussed further in the next chapter, we now recognize that cancer cells are generally defective in the normal process of programmed cell death and that their inability to undergo apop­tosis is as important as their uncontrolled proliferation in the development of malignant tumors.

Mammals encode a family of approximately 20 proteins related to Bcl-2, which are divided into three functional groups (Figure 17.6). Some mem­bers of the Bcl-2 family (antiapoptotic family members)—like Bcl-2 itself— function as inhibitors of apoptosis and programmed cell death. Other mem­bers of the Bcl-2 family, however, are proapoptotic proteins that act to induce caspase activation and promote cell death. There are two groups of these proapoptotic proteins, which differ in function as well as in their extent of homology to Bcl-2. Bcl-2 and the other antiapoptotic family mem­bers share four conserved regions called Bcl-2 homology (BH) domains. One group of proapoptotic family members called the "multidomain" proapoptotic proteins have 3 BH domains (BH1, BH2, and BH3), whereas the second group, the "BH3-only" proteins, have only the BH3 domain

The fate of the cell—life or death—is determined by the balance of activ­ity of proapoptotic and antiapoptotic Bcl-2 family members, which act to regulate one another (Figure 17.7). The multidomain proapoptotic family members, such as Bax and Bak, are the downstream effectors that directly induce apoptosis. They are inhibited by interactions with the antiapoptotic family members, such as Bcl-2. The BH3-only family members are upstream members of the cascade, regulated by the signals that induce cell death (e.g., DNA damage) or cell survival (e.g., growth factors). When activated, the BH3-only family members antagonize the antiapoptotic Bcl-2 family members, activating the multidomain proapoptotic proteins and tipping the balance in favor of caspase activation and cell death.

In mammalian cells, members of the Bcl-2 family act at mitochondria, which play a central role in controlling programmed cell death (Figure 17.8). When activated, Bax and Bak form oligomers in the mitochondrial outer membrane. Formation of these Bax or Bak oligomers leads to the release of cytochrome c from the mitochondrial intermembrane space, either by forming pores or by interacting with other mitochondrial outer membrane proteins. The release of cytochrome c from mitochondria then triggers caspase activation. In particular, the key initiator caspase in mam­malian cells (caspase-9) is activated by forming a complex with Apaf-1 in the apoptosome. In mammals, formation of this complex also requires cytochrome c. Under normal conditions of cell survival, cytochrome c is localized to the mitochondrial intermembrane space (see Figure 11.10) while Apaf-1 and caspase-9 are found in the cytosol, so caspase-9 remains inactive. Activation of Bax or Bak results in the release of cytochrome c to the cytosol, where it binds to Apaf-1 and triggers apoptosome formation and caspase-9 activation.

Caspases are also regulated by a family of proteins called the IAP, for inhibitor of apoptosis, family. Members of the IAP family directly interact with caspases and suppress apoptosis by either inhibiting caspase activity or by targeting caspases for ubiquitination and degradation in the protea-some. IAPs are present in both Drosophila and mammals (but not C. elegans), and regulation of their activity or expression provides another mechanism for controlling apoptosis. Regulation of IAPs is particularly important in Drosophila, where initiator caspases are chronically activated but held in check by IAPs (Figure 17.9). Many signals that induce apoptosis in Drosophila function by activating proteins that inhibit the IAPs, thus leading to caspase activation. In mammalian cells, the permeabilization of mito chondria by Bax or Bak results not only in the release of cytochrome c but also of IAP inhibitors that may help to stimulate caspase activity.

Signaling Pathways that Regulate Apoptosis

Programmed cell death is regulated by the integrated activity of a variety of signaling pathways, some acting to induce cell death and others acting to promote cell survival. These signals control the fate of individual cells, so that cell survival or elimination is determined by the needs of the organism as a whole. The pathways that induce apoptosis in mammalian cells are grouped as intrinisic orextrinsic pathways, which differ in their involve­ment of Bcl-2 family proteins and in the identity of the caspase that initates cell death.

One important role of apoptosis is the elimination of damaged cells, so apoptosis is stimulated by many forms of cell stress, including DNA dam­age, viral infection, and growth factor deprivation. These stimuli activate the intrinsic pathway of apoptosis, which leads to release of cytochrome c from mitochondria and activation of caspase-9 (see Figure 17.8). As illustrated in the following examples, the multiple signals that activate this pathway con­verge on regulation of the BH3-only members of the Bcl-2 family.

DNA damage is a particularly dangerous form of cell stress, because cells with damaged genomes may have suffered mutations that can lead to the development of cancer. DNA damage is thus one of the principal triggers of programmed cell death, leading to the elimination of cells carrying poten­tially harmful mutations. As discussed in Chapter 16, several cell cycle checkpoints halt cell cycle progression in response to damaged DNA, allowing time for the damage to be repaired. In mammalian cells, a major pathway leading to cell cycle arrest in response to DNA damage is medi­ated by the transcription factor p53. The ATM and Chk2 protein kinases, which are activated by DNA damage, phosphorylate and stabilize p53. The resulting increase in p53 leads to transcriptional activation of p53 target genes. These include the Cdk inhibitor p21, which inhibits Cdk2/cyclin E complexes, halting cell cycle progression in G₁(see Figure 16.20). However, activation of p53 by DNA damage can also lead to apoptosis (Figure 17.10). The induction of apoptosis by p53 results, at least in part, from transcrip­tional activation of genes encoding the BH3-only proapoptotic Bcl-2 family members PUMA and Noxa. Increased expression of these BH3-only pro­teins leads to activation of Bax and Bak, release of cytochrome c from mito­chondria, and activation of caspase-9. Thus p53 mediates both cell cycle arrest and apoptosis in response to DNA damage. Whether DNA damage in a given cell leads to apoptosis or reversible cell cycle arrest may depend on the extent of damage and the resulting level of p53 induction, as well as the influence of other life /death signals being received by the cell.

Growth factor deprivation is another form of cell stress that activates the intrinsic pathway of apoptosis. In this case, apoptosis is controlled by sig­naling pathways that promote cell survival by inhibiting apopiosis in response to growth factor stimulation. These signaling pathways control the fate of a wide variety of cells whose survival is dependent on extracellu­lar growth factors or cell-cell interactions. As already noted, a well-charac­terized example of programmed cell death in development is provided by the vertebrate nervous system. About 50% of neurons die by apoptosis, with the survivors having received sufficient amounts of survival signals from their target cells. These survival signals are polypeptide growth fac­tors related to nerve growth factor (NGF), which induces both neuronal survival and differentiation by activating a receptor protein-tyrosine kinase. Other types of cells are similarly dependent upon growth factors or cell contacts that activate nonreceptor protein-tyrosine kinases associated with integrins. Indeed, most cells in higher animals are programmed to undergo apoptosis unless cell death is actively suppressed by survival signals from other cells.

One of the major intracellular signaling pathways responsible for pro­moting cell survival is initiated by the enzyme PI 3-kinase, which is acti­vated by either protein-tyrosine kinases or G protein-coupled receptors. PI 3-kinase phosphorylates the membrane phospholipid PIP₂ to form PIP₃, which activates the protein-serine/threonine kinase Akt (see Figures 15.30 and 15.31). Akt then phosphorylates a number of proteins that regulate apoptosis (Figure 17.11). One key substrate for Akt is the proapoptotic BH3-only Bcl-2 family member called Bad. Phosphorylation of Bad by Akt cre­ates a binding site for 14-3-3 chaperone proteins that sequester Bad in an inactive form, so phosphorylation of Bad by Akt inhibits apoptosis and pro­motes cell survival. Bad is similarly phosphorylated by protein kinases of other growth factor-induced signaling pathways, including the Ras/Raf/MEK/ERK pathway, so it serves as a convergent regulator of growth factor signaling in mediating cell survival.

Other targets of Akt, including the FOXO transcription factors, also play key roles in cell survival. Phosphorylation of FOXO by Akt creates a bind­ing site for 14-3-3 proteins, which sequester FOXO in an inactive form in the cytoplasm (see Figure 15.32). In the absence of growth factor signaling and Akt activity, FOXO is released from 14-3-3 and translocates to the nucleus, stimulating transcription of proapoptotic genes, including the gene encod­ing the BH3-only protein, Bim. Akt and its downstream target GSK-3 also regulate other transcription factors with roles in cell survival, including p53 and NF-fd3, which control the expression of additional Bcl-2 family mem­bers. In addition, the level of the antiapoptotic Bcl-2 family member Mcl-1 may be modulated via translational regulation by both GSK-3 and the mTOR pathway (see Figure 15.33). These multiple effects on members of the Bcl-2 family converge to regulate the intrinsic pathway of apoptosis, controlling the activation of caspase-9 and cell survival in response to growth factor stimulation.

In contrast to the cell stress and growth factor signaling pathways that regulate the intrinsic pathway of apoptosis, some secreted polypeptides activate receptors that induce cell death via the extrinsic pathway of apop­tosis. These receptors directly activate a distinct initiator caspase, caspase-8 (Figure 17.12). The polypeptides that signal cell death by this pathway belong to the tumor necrosis factor (TNF) family. They bind to members of the TNF receptor family, which can induce apoptosis in a variety of cell types. One of the best characterized members of this family is the cell sur­face receptor called Fas, which plays important roles in controlling cell death in the immune system. For example, apoptosis induced by activation of Fas is responsible for killing target cells of the immune system, such as cancer cells or virus-infected cells, as well as for eliminating excess lympho­cytes at the end of an immune response.

TNF and related family members consist of three identical polypeptide chains, and their binding induces receptor trimerization. The cytoplasmic portions of the receptor bind adaptor molecules that in turn bind caspase-8. This leads to activation of caspase-8, which can then cleave and activate downstrem effector caspases. In some cells, caspase-8 activation and subsequent activation of caspases-3 and -7 is sufficient to induce apoptpsis directl. In other cell, however, amplification of the signal is needed. This results from caspase-8 cleavage of the proapoptotic BH3-only protein Bid, leading to Bid activation, permeabilization of mitochondria, and activation of caspase-9, thus amplifying the caspase cascade initiated by direct activa­tion of caspase-8 at cell death receptors.

Alternative Pathways of Programmed Cell Death

Although apoptosis is the most common form of regulated or programmed cell death, recent research has shown that programmed cell death can also occur by alternative, non-apoptotic mechanisms. One of these alternative pathways of regulated cell death is autophagy. As discussed in Chapter 8, autophagy provides a mechanism for the gradual turnover of the cell's components by the uptake of proteins or organelles into vesicles (autophagosomes) that fuse with lysosomes (see Figure 8.45). In addition, autophagy promotes cell survival under conditions of nutrient deprivation. Under conditions of starvation, activation of autophagy serves to increase the degradation of cellular proteins and organelles, generating energy and allowing their components to be reutilized for essential functions.

In other circumstances, however, autophagy provides an alternative to apoptosis as a pathway of cell death. Autophagic cell death does not require caspases and, rather than possessing the distinct morphological features of apoptosis, the dying cells are characterized by an accumulation of lyso­somes. Autophagy has been shown to be an important pathway of pro­grammed cell death during salivary gland development in Drosophila and to be induced by infection with some viruses. In addition, autophagy appears to provide an alternative pathway to cell death when apoptosis is blocked. For example, cells of mutant mice lacking Bak and Bax fail to undergo apoptosis in response to stimuli such as DNA damage, as expected since Bak and Bax are required for permeabilization of mitochondria (see Figure 17.8). However, the Bak/Bax-deficient cells die by autophagy instead, suggesting that autophagy may also be activated by cellular stress and provide an alternative to apoptosis under these conditions.

It also appears that some forms of necrosis can be a programmed cellular response, rather than simply representing uncontrolled cell lysis as the result of an acute injury. In contrast to unregulated necrosis, these forms of regulated necrotic cell death are induced as a programmed response to stimuli such as infection or DNA damage, which also induce apoptosis. Under these conditions, regulated necrosis may provide an alternative pathway of cell death if apoptosis does not occur. For example, if apoptosis is inhibited, stimulation of the TNF receptor leads to cell death by necrosis. The importance of both autophagy and necrosis as alternatives to apoptosis remains to be fully explored, not only in normal cells but also in diseases such as cancer, heart disease, and neurodegeneration, which involve abnor­malities of cell survival.

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