Apoptosis is a highly preserved and controlled mechanism to achieve tissue homeostasis through targeted elimination of single cells without disrupting the biological functionality of the tissue. The morphological changes associated with apoptosis include nuclear condensation, cell shrinkage, and plasma membrane blebbing resulting in apoptotic bodies (see Fig. 1) (1). Rapid engulfment of these through neighboring cells or macrophages prevents an inflammatory response as observed with necrotic cell death in most cases. However, recent studies of cholestatic liver injury have indicated a role of resident macrophages in the liver, Kupffer cells, in the pathogenesis of a sustained inflammatory response following apoptotic cell death (2). Apoptosis can be initiated through an extrinsic or an intrinsic pathway depending on the initial site of activation of the cell death process. The extrinsic pathway is initiated after binding of trimeric ligand molecules to corresponding preassembled receptor multimers (see Fig. 2) (3, 4). Most prominent among the cytokines to induce hepatocellular apoptosis are the members of the tumor necrosis factor (TNF) ligand superfamily CD95L, TNF and TNF-related apoptosis-inducing ligand (TRAIL) (3). These cytokines exert a physiological function through their cognate receptors, namely the CD95 receptor (Apo1/Fas receptor), TNF receptor type 1 (TNF-R1, p55/65, CD120a) and type 2 (TNF-R2, p75/80, CD120b), TRAIL receptor type 1 and type 2 (5). Ligand–receptor interactions occur at the plasma membrane and result in conformational changes of the receptor, initiating the assembly of an early intracellular signaling complex, to which downstream signaling molecules are subsequently recruited (6). The intracellular domains of death receptors are devoid of intrinsic kinase activity and therefore depend on homophilic protein–protein interactions for the initiation of cell signaling (7). The apoptotic cell death pathway is activated following recruitment of the Fas-associated death domain (FADD/MORT1), which results in activation of the caspase (cysteine aspartate protease) family of enzymes (8). Caspases are capable of cleaving substrates after a loosely specific motif of four amino acids that contain aspartate in the first position. Crucial to their catalytic activity is the presence of a cysteine residue in the active center of the molecule (9). The family can be divided into upstream initiator caspases, such as caspase-8, -9, -10 and downstream effector caspases, caspase-3 and -6. The effector caspases are responsible for the cleavage of proteins whose functional loss induces apoptosis (10). Caspases are constitutively expressed as inactive zymogens or procaspases that require cleavage into smaller active subunits. A total of 13 caspases, 11 of which are expressed in humans, have been cloned, but not all of them participate in apoptosis (11). Upon recruitment and colocalization with FADD, high local concentrations of procaspases-8 undergo autoproteolytic cleavage, releasing activated caspase-8. This complex has been termed the death-inducing signaling complex (DISC), and the mode of activation is referred to as the induced proximity model of activation (12). The central role of the adapter molecules could be shown in mice expressing dominant-negative (dn) FADD protein. These mice are protected from liver injury following activation of CD95 and TNF signaling (13). Expression of the FLICE inhibitory proteins (FLIP-long and -short) can block apoptosis at this point and prevent caspase-8 activation by inhibition of the recruitment and processing of procaspases-8 at the level of the DISC (14). Recent studies have implied a dichotomous role for the long isoform of FLIP in the context of cell death. While the antiapoptotic function prevents execution of the cell death process, mice with defective FADD exhibited, in addition, an impaired proliferative response upon 2/3 hepatectomy, implying a potential role of the apoptotic pathways in liver regeneration (15). The intrinsic pathway of apoptosis involves mitochondria and caspase-9 activation. Cleavage of the Bcl-2 family member Bid by caspase-8 results in truncated Bid (tBid), which triggers oligomerization of the proapoptotic Bcl-2 family members Bax and Bak (16). These molecules then insert into the mitochondrial membrane, resulting in mitochondrial permeability transition (MPT) and release of mitochondrial proteins including cytochrome c (17). This function of Bid has been demonstrated to mediate hepatocyte death from TNF. Hepatocytes deficient for Bid show resistance to TNF and CD95-induced cell death associated with the prevention of mitochondrial depolarization and cytochrome c release (18). In vivo, Bid-deficient mice were partially protected from toxic liver injury (19). In accordance with these observations, inhibition of cytochrome c release from mitochondria by the MPT inhibitor cyclosporin A prevents hepatocyte apoptosis at a point downstream of the adapter molecule FADD, but upstream of caspase-3 activation (20). Following release into the cytosol, cytochrome c triggers formation of the apoptosome, a complex with apoptosis protease-activating factor-1 and procaspases-9. Caspase-9 becomes activated and in turn activates caspase-3, leading to degradation of structural proteins and resulting in apoptosis (21). Hepatocytes are dependent on the mitochondrial death pathway and have been termed type II cells. In contrast, type I cells generate high levels of caspase-8 activity that directly cleave caspase-3 (22). Accordingly, expression of the antiapoptotic factors Bcl-2 and Bcl-XL that inhibit Bid and Bax activation prevents apoptosis in type II, but not type I cells (22). Supporting the concept of the hepatocyte as a type II cell is that in vivo Bcl-2 or Bcl-XL overexpression is partially effective in preventing liver injury from TNF (23, 24). Another mechanisms involving the intrinsic pathway in hepatocytes is the activation of the lysosomal cysteine protease cathepsin B. In vitro and in vivo TNF-induced apoptosis is dependent on the release of cathepsin B from vesicles (25). The proapoptotic effect of cathepsin B is above the level of mitochondrial cytochrome c release as this process is blocked in TNF-treated cathepsin B null hepatocytes. Additional evidence of an effect of this protease on mitochondria is that in a cell-free system, cathepsin B induced mitochondrial cytochrome c release (26).
Figure 2. Apoptosis receptor signaling pathways. Binding of corresponding ligand and receptor induces recruitment of proteins through homophilic interactions of conserved domains. In the death pathway (right-hand side of the figure), autoproteolytic activation of procaspases-8 results in the DISC. Activated caspase-8 cleaves Bid producing a truncated and active tBid. tBid in turn activates the proapoptotic Bcl-2 family members Bax and Bak. Their oligomerization and integration into the mitochondrial membrane result in cytochrome c release. In a complex consisting of cytochrome c and APAF-1, procaspases-3 is recruited and activated, resulting in apoptosis. Additionally, caspase-8 activation causes release of the lysosomal enzyme cathepsin B that acts in an undefined fashion to activate the mitochondrial death pathway. NF-κB-regulated gene products act to block the TNF apoptotic death pathway and TNF also mediates cellular proliferation involving the TNF-R1 (left-hand side of the figure). Activation of IKKs that phosphorylate IκB leads to proteasome-dependent degradation of phosphorylated IκB, releasing NF-κB heterodimers that translocate to the nucleus and activate genes that may be necessary for TNF-induced hepatocyte proliferation. Activation of the JNK/c-Jun/AP-1 pathway can promote hepatocyte proliferation through AP-1-dependent gene expression, but sustained AP-1 activation may alternatively serve to trigger the apoptotic death pathway. DISC, death-inducing signaling complex; tBid, truncated Bid; TNF, tumor necrosis factor; NF-κB, nuclear factor-κB; IKK, IκB kinase; APAF-1, apoptosis protease activating factor-1; AP-1, activator protein-1; JNK, c-Jun N-terminal kinase.
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Recent findings related to the regulation of apoptosis signaling pathways have implied a central role of mitogen-activated protein kinases (MAPK) in apoptosis signaling through TNF and to a lesser extent through CD95 and TRAIL (27–29). A prominent member of the MAPK family is the c-Jun N-terminal kinase (JNK). Activated JNK phosphorylates substrates that include the activator protein-1 (AP-1) transcription factor subunit c-Jun, leading to increased AP-1 transcriptional activity (30). Transient JNK/AP-1 activation occurs after TNF stimulation and may in part mediate the proliferative effects of TNF (31); however, with nuclear factor-κB (NF-κB) inhibition, TNF induces a prolonged activation of JNK and AP-1 in hepatocytes and non-hepatic cells in association with cell death (32, 28). Death from TNF following NF-κB inactivation can be blocked in hepatocytes by inhibition of c-Jun, suggesting that an AP-1 gene product activates the TNF mitochondrial death pathway (28).
TNF, as opposed to other apoptosis-inducing ligands, mediates pleiotropic effects on tissue homeostasis, apoptosis and proliferation. The transcription factor NF-κB has been implicated in the modulation of apoptosis induced by TNF, and inhibition of NF-κB induces apoptosis in hepatocytes following TNF exposure (33). NF-κB activation is achieved through degradation of its inhibitor IκB following its phosphorylation through IκB kinases (IKKs) (34). TNF production in the liver can be observed after partial hepatectomy within 1 h and neutralizing antibodies to TNF inhibit the regenerative stimulus of this cytokine (35, 36). TNF-mediated regeneration involves activation of IL-6 and STAT3 (37, 38). The role of NF-κB in regeneration is less clear, and adenoviral inhibition NF-κB did not affect liver regeneration in vivo (39).