Apoptosis: function and pathways

Introduction
Apoptosis is a process of programmed cell death that occurs in multicellular organisms. It is a highly regulated and controlled process that occurs normally during development and aging as a homeostatic mechanism to maintain cell populations in tissues [1]. For example, the separation of fingers and toes in a developing human embryo occurs because cells between the digits undergo apoptosis. Apoptosis also occurs as a defence mechanism such as in immune reactions or when cells get damaged by disease or by noxious agents [2]. During the early process of apoptosis, cell shrinkage and pyknosis are visible by light microscopy [3]. With cell shrinkage, the cytoplasm becomes dense and the organelles are more tightly packed. Pyknosis is the result of chromatin condensation and this is the most characteristic feature of apoptosis. At a later stage, apoptosis produces cell fragments called apoptotic bodies that phagocytic cells engulf and quickly remove before the contents can spill out onto surrounding cells to cause inflammation [1].

There is a wide variety of both physiological and pathological stimuli and conditions that can trigger apoptosis. However, not all cells will necessarily die in response to the same stimulus. For example, irradiation or drugs used for cancer chemotherapy results in DNA damage, which can lead to apoptotic death through a p53-dependent pathway, but not in all cells. Some hormones, such as corticosteroids, may lead to apoptotic death in some cells (e.g. thymocytes) although other cells are unaffected. Some cells express Fas or TNF receptors that can facilitate apoptosis induction via ligand binding and protein cross-linking. Other cells have a default death pathway that must be blocked by a survival factor such as a hormone or growth factor [1]. There is also the issue of distinguishing apoptosis from necrosis, two processes that can occur independently, sequentially, as well as simultaneously [4, 5]. In some cases, it’s the type of stimuli and/or the degree of stimulation that determines if cells die by apoptosis or necrosis. At low doses, a variety of injurious stimuli such as heat, radiation, hypoxia and cytotoxic anti-cancer drugs can induce apoptosis but these same stimuli can result in necrosis at higher doses. Finally, apoptosis is a coordinated and energy-dependent process that involves the activation of a group of cysteine proteases called caspases and a complex cascade of events that link the initiating stimuli to the final structured demise of the cell.

Pathways
Apoptosis can be initiated through one of three pathways. In the intrinsic or mitochondrial pathway, the cell kills itself because it senses cell stress, while in the extrinsic pathway the cell is instructed to kill itself through signal transduction stimulators from other cells. The Perforin/Granzyme pathway is mediated by cytotoxic T cells. In this third pathway, apoptosis is induced via either Granzyme B or Granzyme A. All three initiation pathways (apart from Granzyme A) induce cell death through the Execution pathway that involves the activation of caspase-3 [1].

Intrinsic Pathway
The stimuli that initiate the intrinsic pathway produce intracellular signals that may act in either a positive or negative fashion. Negative signals involve the absence of certain growth factors, hormones and cytokines that can interfere with the suppression of cell death programs, thereby triggering apoptosis. In other words, there is the withdrawal of factors, loss of apoptotic suppression, and subsequent activation of apoptosis. Positive signals include, but are not limited to, radiation, toxins, hypoxia, hyperthermia, viral infections, and free radicals. These stimuli cause changes in the inner mitochondrial membrane that results in an opening of the mitochondrial permeability transition (MPT) pore, loss of the mitochondrial transmembrane potential and release of two main groups of normally sequestered pro-apoptotic proteins from the intermembrane space into the cytosol [6].

The first group consists of cytochrome c, Smac/DIABLO, and the serine protease HtrA2/Omi [7-9]. Cytochrome c binds and activates Apaf-1 as well as procaspase-9, forming an “apoptosome” [10, 11]. The clustering of procaspase-9 in this manner leads to caspase-9 activation. Smac/DIABLO and HtrA2/Omi are reported to promote apoptosis by inhibiting IAP (Inhibitors of Apoptosis Proteins) activity [9, 12]. IAP supresses caspases [13].

The second group consists of AIF (Apoptosis-Inducing Factor), endonuclease G and CAD (Caspase-Activated DNAse). AIF transfers to the nucleus and causes DNA fragmentation into ∼50–300 kb pieces and condensation of peripheral nuclear chromatin [14]. This early form of nuclear condensation is referred to as Stage I condensation [15]. Endonuclease G also transfers to the nucleus where it cleaves nuclear chromatin to produce oligo-nucleosomal DNA fragments [16]. AIF and endonuclease G both function in a caspase-independent manner. CAD is subsequently released from the mitochondria and transfers to the nucleus where, after cleavage by caspase-3, it leads to oligo-nucleosomal DNA fragmentation and a more pronounced and advanced chromatin condensation [17]. This later and more pronounced chromatin condensation is referred to as Stage II condensation [15].

Extrinsic pathway
The Extrinsic initiation pathway involves receptors of the TNFR (Tumour Necrosis Factor Receptor) family. In the extrinsic, death receptor pathway of apoptosis, ligation of death receptors on the cell surface leads to caspase activation. This pathway relies on the formation of a Death-Inducing Signalling Complex (DISC), which always includes FADD and caspase-8. The death receptors known so far are TNFR1 (ligand TNF-alpha), CD95 (ligand FasL), and TRAILR1 plus TRAILR2 (ligand TRAIL). In the case of ligated TNFR1, adapter TRADD (Tnf Receptor type 1-Associated Death Domain) is first engaged, which in turn recruits FADD. TRADD is only required for apoptosis when induced by TNF-alpha. The other ligated death receptors engage FADD directly [18]. Fas associated via death domain (FADD) protein is responsible for the recruitment of caspase-8 to form the DISC. The presence of the FLICE-like inhibitory protein (cFLIP) in the FADD-caspase-8-cFLIP complex determines if and how cells die. As such, cFLIP is a switch that determines cell fate [19]. cFLIP comes in two major isoforms, the long isoform cFLIPL and the short isoform cFLIPS. The cFLIPS protein is an inhibitor of caspase-8 and blocks DISC-dependent procaspase-8 activation. The cFLIPL protein regulates the extent of activation and possibly substrate specificity of procaspase-8. Low levels of cFLIPL can enhance apoptotic signaling, whereas apoptosis is inhibited when cFLIPL levels are high [19].

CD95 and TRAIL-R ligation induces apoptosis by direct recruitment of FADD-caspase-8 in a complex called the DISC [19]. As described above, isoform levels of cFLIP then determine whether apoptosis is blocked or engaged. Ligation of TNFR1 can both induce caspase-8-mediated apoptosis as well as block apoptosis via the NF-κB-induced expression of cFLIP in a feedback loop. Receptor-interacting serine/threonine kinases 1 (RIPK1) is key in regulating TNFR1-induced FADD-caspase-8-mediated apoptosis. TNFR1 ligation leads to the recruitment of TRADD, TRAF2, cIAP1/2, and RIPK1 (complex I). RIPK1 ubiquitination by cIAP1/2 mediates activation of NF-κB and the production of pro-inflammatory and pro-survival gene expression. RIPK1 is deubiquitinated by CYLD and leaves complex I to recruit FADD to form the ripoptosome (complex IIa) involving caspase-8 and cFLIPL. Homodimerization and activation of caspase-8 on FADD induces apoptosis. One of the expressed pro-survival genes, cFLIPL, heterodimerizes with caspase-8, resulting in inhibition of caspase-8 activation and apoptosis. Alternatively, RIPK1 interacts with RIPK3 to either stimulate RIPK3 oligomerization in the necrosome (complex IIb), or to suppress it [20]. Oligomerized RIPK3 is a prerequisite to trigger phosphorylation of the downstream mediator Mixed-Lineage Kinase-Like (MLKL) that triggers necrosis. Thus, death receptor TNFR1 provides two separate pathways downstream the TRADD recruitment: deubiquitinated RIPK1 either allows the activation of DISC in the ripoptosome (complex IIa) igniting apoptosis, or it interacts with RIPK3 to activate MLKL in the necrosome (complex IIb). MLKL is the effector protein that, once activated, transfers to the plasma membrane where it induces rupture and subsequent cell death. This regulated form of necrosis is also known as necroptosis. The release of cellular components this way results in an inflammatory response.

Perforin/Granzyme pathway
One aspect of the adaptive immune system is recognizing and eliminating target cells through the induction of apoptosis, involving CD8+ cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. CTLs recognize target cells by the presentation of major histocompatibility complex class I (MHC I) molecules on surface of the target cell, while NK cells recognize target cells by the loss of class I molecules, of killer cell immunoglobulin receptors (KIRs), and of killer cell lectin like receptors (KLRs). Although there is a difference in the process of target cell recognition, once identified, both CTLs and NK cells execute similar effector function to remove the target cell. This is achieved through the release of cytotoxic granules containing perforin, granzymes, and granulysin, which work together to induce apoptosis in the target cells. This effector-killing of target cells enables eradication of intracellular pathogens and malignancies [21].

A key protein in the cytotoxic granule is perforin. Classically, perforin is known to form a pore in cell membranes, allowing passage of granzymes into the cell to inducing apoptosis. Granzyme B can either directly, or through caspase-10 activation, convert pro-form caspase-3 into active caspase-3 to initiate the Apoptosis Execution pathway. The Granzyme A pathway evades the Execution pathway, leading to DNA cleavage through the SET complex. Once gaining entry to the cell, granzyme A activates a DNA nicking process via DNAse NM23-H1, a tumour suppressor gene product [22]. This DNAse has an important role in immune surveillance by prevention of cancer cell metastasis through the induction of tumour cell apoptosis. The nucleosome assembly protein SET normally inhibits the NM23-H1 gene. Granzyme A protease cleaves the SET complex thus releasing inhibition of NM23-H1, resulting in apoptosis inducing DNA degradation [23].

Execution pathways
Both extrinsic and intrinsic apoptosis induction pathways lead to an end-point execution phase, considered the final pathway of apoptosis.  Execution caspases activate cytoplasmic endonuclease, which degrades nuclear material, and proteases that degrade the nuclear and cytoskeletal proteins. Caspase-3, caspase-6, and caspase-7 function as effector or “executioner” caspases, cleaving various substrates including cytokeratins, PARP, the plasma membrane cytoskeletal protein Fodrin alpha, the nuclear protein NuMA and others, that ultimately cause the morphological and biochemical changes seen in apoptotic cells [23]. Caspase-3 is the most important of the executioner caspases and is activated by any of the initiator caspases (caspase-8, caspase-9, or caspase-10). Caspase-3 specifically activates the endonuclease CAD. In proliferating cells CAD is complexed with its inhibitor, ICAD. In apoptotic cells, activated caspase-3 cleaves ICAD to release CAD [24]. CAD then degrades chromosomal DNA within the nuclei causing chromatin condensation. Caspase-3 also induces cytoskeletal reorganization and disintegration of the cell into apoptotic bodies through the cleavage of Gelsolin. The cleaved fragments of Gelsolin, in turn, cleave actin filaments in a calcium independent manner. This results in disruption of the cytoskeleton, intracellular transport, cell division, and signal transduction [25].

Phagocytic uptake of apoptotic cells is the last component of apoptosis. Phospholipid asymmetry and externalization of phosphatidylserine on the surface of apoptotic cells and their fragments is the hallmark of this phase. The appearance of phosphatidylserine on the outer leaflet of apoptotic cells facilitates noninflammatory phagocytic recognition, followed up quickly by their early uptake and disposal [26]. This process of early and efficient uptake, with no release of intracellular constituents, essentially eliminates the probability of potentially catastrophic inflammatory events.

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