Cell Death

9.1.1

Morphology of Apoptosis

In apoptosis, the cell is dismantled from within in a manner that minimizes damage and disruption to neighboring cells. The full course of apoptosis takes several hours. During apoptosis the cell undergoes a series of morphologic changes. Early changes include condensation of nuclear chromatin along the perimeter of the nucleus and separation of the cell from surrounding cells in the tissue and from the extracellular matrix ( Fig. 9.1 ). As apoptosis progresses, the cell becomes rounded and shrinks. The nucleus first compacts and then fragments. Finally, the cell disintegrates into apoptotic bodies which may contain any part of the cellular material surrounded by a piece of plasma membrane. In the final step, apoptotic bodies are phagocytosed by macrophages and other cells and digested through the lysosomal pathway. The defining morphologic feature of apoptosis is that the cell contents remain membrane bound and it is this feature that explains the absence of an active immune response.

9.1.2

Biochemistry of Apoptosis

Apoptosis involves a complex series of biochemical events mediated by caspases, a family of cysteine proteases present in the cytoplasm as proenzymes (procaspases). When apoptosis is initiated, procaspases are cleaved and the active caspases break down cellular proteins leading to the morphologic changes seen in apoptosis. Two biochemical pathways lead to apoptosis: the extrinsic pathway and the intrinsic pathway.

9.1.2.1

Extrinsic Pathway of Apoptosis

The extrinsic pathway of apoptosis is typically induced by extracellular stress ligands of the TNF family that are sensed by specific transmembrane “death receptors.” Two ligand-receptor interactions initiating the extrinsic pathway are TNFα binding to TNFR1 and FAS ligand (FASL) binding to FAS. Members of the TNF family acting through the extrinsic pathway play a dominant role in IEC apoptosis in the context of inflammation as is seen in enteric infections, inflammatory bowel disease (IBD), and graft versus host disease. All of these conditions are associated with the production of TNFα. Antibodies to TNF are an important component of IBD therapy.

On FASL binding to FAS, the cytoplasmic tails of FAS trimerize and recruit FAS-associated protein with a death domain (FADD). TNFα binding to TNFR1 causes TNFR1 to trimerize and induces the assembly Complex I which includes trimerized TNFR1, TNFR-associated death domain (TRADD), TNF receptor-associated factors (TRAFs), cellular inhibitors of apoptosis proteins (cIAPs), and kinase receptor-interacting protein 1 (RIP1) ( Fig. 9.2 ). If RIP1 is ubiquitinated then the inhibitor of nuclear factor (NF) κ-B kinase (IKK) is recruited to Complex I inducing a classical NF-κB activated response leading to cell survival. If RIP1 is not ubiquitinated then Complex I is destabilized and internalized into the cytosol to form Complex IIa or the cytosolic death-inducing signaling complex (DISC). Procaspase 8, FADD, and the long isoform of FLICE-like inhibitory protein (FLIP) are recruited to the DISC resulting in the cleavage of procaspase 8 to yield caspase 8 which triggers caspase 3 and caspase 7 leading to apoptosis. The ubiquitination of RIP1 is a key regulatory event in deciding between cell survival and apoptosis. RIP1 is ubiquitinated by E3 ubiquitin ligases including the linear ubiquitination assembly complex (LUBAC) and cIAP1 and cIAP2. RIP1 deubiquitination is promoted by inhibition of E3 ubiquitin ligases and by the induction of deubiquitinases such as A20 and cylindromatosis deubiquitinating enzyme (CYLD).

TNFα, a pleiotropic cytokine, activates a number of biochemical pathways some of which are proapoptotic but others are prosurvival. The likelihood of TNFα binding to TNFR1 inducing cell survival or apoptosis depends on the cell type and the context. In most cell types, the dominant effects of TNFα are prosurvival and TNFα-induced apoptosis is uncommon. In IECs, the proapoptotic effects of TNFα are dominant and, as a consequence, IECs are more sensitive to TNFα-induced apoptosis than most other cell types. NF-κB activation induced by TNFα binding to TNFR1 protects most cells from apoptosis; translocation of NF-κB to the nucleus induces the transcription of genes for the antiapoptotic proteins Bcl-2, Bcl-X _L , and various IAPs ( Fig. 9.2 ) which directly or indirectly inhibit the activation of caspases and insure that cells are not killed by spontaneous activation of these enzymes. These molecules that control caspase activation play critical roles in IEC apoptosis.

Members of the IAP family have received increasing interest due to their position as regulators of the balance between life and death signals in the intestinal epithelium. One of the IAPs, cIAP1, is a key regulator of TNFα-induced IEC death and survival. TNFα induces increased levels of cell death in IEC lines and human intestinal organoids in which cIAP1 levels are depleted. Similarly, TNFα induces high levels of IEC death in cIAP-1 null mice compared with wild-type mice. The X-linked inhibitor of apoptosis (XIAP), exerts its apoptotic inhibitory function by directly binding to the catalytic domain of caspases. Activation of NF-kB with the subsequent production of IAPs explains why TNFα binding to TNFR1 usually does not result in apoptosis.

Further evidence that NF-κB activation is essential to prevent TNFα-induced apoptosis in IECs comes from studies in mice lacking NF-κB essential modulator (NEMO) in IECs (NEMO ^IEC-KO ). NEMO is essential for NF-κB activation; mice lacking NEMO in IECs develop spontaneous colitis. The requirement for TNFR1 signaling for the development of colitis NEMO ^IEC-KO mice is demonstrated by the absence of colitis in NEMO ^IEC-KO mice that lack TNFR1.

9.1.2.2

Intrinsic Pathway of Apoptosis

The second major biochemical pathway of apoptosis is the intrinsic or mitochondrial pathway. The intrinsic pathway is activated by DNA damage as is seen with radiation or chemotherapy ( Fig. 9.3 ). Under homeostatic conditions p53 is bound to Mdm2, a ubiquitin ligase, resulting in the ubiquitination and inactivation of p53. DNA damage releases p53 from Mdm2. Free p53 in turn promotes the expression of a series of proapoptotic proteins including members of the Bcl-2 family. The central event in the intrinsic pathway is the formation of pores that permeabilize the mitochondrial outer membrane resulting in the release of cytochrome C and other proteins from the intermembrane space to the cytosol. In the cytosol, cytochrome C binds apoptotic protease-activating factor (APAF-1) to form the apoptosome which recruits and activates procaspase 9.

The Bcl-2 family of proteins, which includes both pro- and antiapoptotic members, regulates mitochondrial permeabilization. Proapoptotic members of the Bcl-2 family including BAX, Puma, Noxa, and Siva promote pore formation whereas antiapoptotic members such as Bcl-2 and Bcl-X _L block pore formation. The balance between antiapoptotic and proapoptotic members determines either apoptosis or cell recovery.

Withdrawal of growth factors initiates apoptosis in some cells. In the presence of growth factors, BAD is sequestered in the cytoplasm by binding to 14-3-3, a cytosolic protein. That sequestration is dependent on serine phosphorylation of BAD which is dependent on Akt activation by growth factors. Withdrawal of growth factors frees BAD from 14-3-3. Free BAD moves to the mitochondria and inactivates Bcl-2 and Bcl-X _L resulting in apoptosis.

The intrinsic and extrinsic pathways are not completely separate. Bid, a proapoptotic member of the Bcl-2 family, mediates cross talk between the intrinsic and extrinsic pathways. Activation of the extrinsic pathway leads to the activation of caspase 8 which cleaves Bid to its active form t-Bid. The binding of Bcl-2 by t-Bid inactivates Bcl-2 leading to apoptosis through the intrinsic pathway.

The final common pathway of the extrinsic and intrinsic pathways is the activation of caspase 3, caspase 6, and caspase 7 which cleave structural proteins leading to apoptosis. Biochemical events correlate with morphologic events in apoptosis. Caspase 3 and caspase 6 cleave lamin and various nuclear proteins responsible for the structural integrity of the nucleus resulting in chromatin condensation and nuclear fragmentation. DNA fragmentation is the result of the activation of caspase substrates including caspase-activated DNase and the DNA fragmentation factor (DFF). Both of these enzymes are constitutively present bound to inhibitory proteins and are released by the action of caspases. Caspases mediate the proteolysis of cell-cell adhesion proteins resulting in cell retraction and detachment. Proteolysis of ROCK1 leads to contraction of the active cytoskeleton resulting in plasma membrane blebbing. There is also a defined biochemical event leading to the phagocytosis of apoptotic bodies. Early during the apoptotic process, phosphatidylserine moves from the internal leaflet of the plasma membrane to the external leaflet providing a phagocytotic (Eat Me) signal to other cells. Annexin V binds to phosphatidylserine on the external leaflet of the plasma membrane. Fluorescent assays for Annexin V on the cell surface are standard methods for quantifying apoptosis.

9.1.3

Spontaneous Apoptosis

Apoptosis occurs at a low rate in intestinal crypts unrelated to any known stimulus. The highest frequency of spontaneous apoptosis is seen at positions 4 and 5 at the base of the crypt, the presumed location of the stem cells. Neither p53 nor Bcl-2 is involved in the spontaneous apoptosis of small intestinal crypt IECs. Spontaneous apoptosis in the small intestinal crypts occurs at the same rate in p53-deficient mice as in wild-type animals; thus, p53 does not play a role in regulating the number of stem cells in the small intestine. Bcl-2 is not expressed in small IECs ; Bcl-2-deficient mice have the same rate of spontaneous apoptosis in small IECs as do wild-type mice. In contrast, the proapoptotic protein Bax is strongly expressed in the stem cell region of the small intestine and in enterocytes at the villus tip. Thus, Bax may stimulate the removal of extra stem cells and play a role in the shedding of villus epithelial cells. Little or no Bax is found in the large intestine. Apoptosis in the colonic epithelium occurs at a lower rate than in the small intestine and it is not confined to the crypts. Instead, colonic epithelial cells are spontaneously eliminated at low rates irrespective of their position.

Knockout studies have shown that spontaneous apoptosis in the colonic epithelium occurs independently of both p53 and Bax. On the other hand, Bcl-2 is expressed in the colonic epithelium and knocking out Bcl-2 results in an increased rate of spontaneous apoptosis which occurs primarily in crypt positions 1 and 2, the location of colonic stem cells. Bcl-2 is believed to be playing an important role in the regulation of cell numbers in the colonic epithelium and its presence may explain why the colonic mucosa is more susceptible to cancer than the mucosa of the small intestine.

9.1.4

Anoikis

Anoikis is a form of programmed cell death in which the central event is detachment from the extracellular matrix and from adjacent cells. Anoikis is not just a component of cell senescence but rather is an active highly regulated process. Cells undergoing anoikis detach from the extracellular matrix prior to detaching from adjacent cells. An early event in cell shedding is redistribution of the tight junction protein zonula occludens 1 (ZO1). A few minutes prior to cell shedding ZO1 distributes to the apical and then to the basolateral region of the cell. This redistribution appears to be required for maintaining barrier function while the cells are being shed.

There is morphologic evidence that IECs undergo apoptosis prior to their being shed into the lumen; however, it is not clear if the early stages of detachment occur before or after the initiation of apoptosis. The biochemical mechanism by which detachment initiates anoikis at the villus tip under physiologic conditions is not clear. However, activation of either the intrinsic pathway or extrinsic pathway can initiate widespread anoikis in response to injury. Detachment of adherent epithelial cell lines leads to a Bcl-2 family-member-dependent form of cell death which is consistent with detachment activating the intrinsic pathway. One proapoptotic member of the Bcl-2 family, Bim, is upregulated by detachment in IEC-18 cells, an IEC line. TNFα can trigger the detachment and apoptosis of mature IECs from an epithelial layer via activation of TNFR1. It appears that TNFα-induced apoptosis occurs prior to detachment as evidenced by the finding of caspase-3 positive IECs in their normal position in the monolayer.

The importance of IEC apoptosis in homeostasis is brought into question by studies of mice deficient for proteins important in apoptosis. Mice deficient in caspase-3, caspase-8, or FADD all have morphologically normal intestines. These studies are consistent with the suggestion that cell shedding at the villus tip is driven by cell crowding and that cell death is the product of cell shedding rather than the cause.