The process of apoptosis (programmed cell death) is regulated by signals generated when cytokines bind to their receptors. There are two types of cytokine-induced signals. The first is an inductive signal that initiates apoptosis. Cytokines producing an inductive signal include TNF-alpha, Fas/APO-1 ligand, and TRAIL/APO-2 ligand.1-4 The second is an inhibitory signal that suppresses apoptosis. Cytokines producing inhibitory signals include those required for cell survival.
Apoptosis proceeds through cleavage of vital intracellular proteins.5 The proteases that mediate this execution are called caspases, for Cytosolic Aspartate-Specific cysteine Proteases.1, 6 Caspases are inactive until a signal initiates activation of one, starting a cascade in which a series of other caspases are proteolytically activated. Although both signalling processes affect caspase activation, the mechanism differs.
Activation of caspases by the pro-apoptotic cytokines TNF-alpha and Fas ligand occurs in a protein complex formed on the cytoplasmic tail of the pro-apoptotic cytokine receptors. Oligomerization of the receptor recruits several proteins including inactive caspase 8 (FLICE/mach 1).7, 8 Caspase 8 is activated and activates other caspases.
|Figure 1. Removal of a cytokine required for cell viability leads to the following sequential events: i) loss of kinase (AKT) activity; ii) dephosphorylation of Bad (or some other regulator of Bcl-2/Bcl-x activity); iii) Bad binding to Bcl-x or Bcl-2; iv) loss of mitochondrial normal physiology; v) release of cytochrome c; vi) binding of cytochrome c by Apaf-1 with concomitant activation of caspase 9; vii) amplification by the caspase cascade; viii) cleavage of vital cellular proteins; ix) fission of cells into apoptotic bodies; and finally, x) disappearance of any traces of the cell when the apoptotic bodies are engulfed by either neighboring or phagocytic cells.
Loss of Inhibitory Signals
The suppressive signal appears to regulate the function of mitochondrial ion and/or water channels.9 Loss of the suppressive signal, when the anti-apoptotic cytokines are not available to the cell, leads to the failure of the channels to maintain the normal ion potential across the inner mitochondrial membrane and to the failure to maintain proper mitochondrial volume.9 Bcl-x and Bcl-2,10, 11 are channel forming proteins responsible for maintaining normal mitochondrial physiology.9 Regulation of their activities is central to apoptosis.
Inhibition of Bcl activities leads to altered mitochondrial membrane permeability resulting in the release of cytochrome c into the cytosol (reviewed in ref. 16).12-16 In the cytosol, cytochrome c is bound by the protein, Apaf-1, an acronym for apoptotic protease-activating factor,15, 17 which also binds caspase 918 and dATP.15, 17, 18 Binding of cytochrome c triggers activation of caspase 9, which then accelerates apoptosis by activating other caspases.
The mechanism for cytochrome c release from the mitochondria is becoming clearer. Inhibition of normal mitochondrial channel function causes mitochondria to swell, rupture and release cytochrome c.9 The inner mitochondrial membrane has more surface area than the outer membrane. Swelling of the matrix extends the inner membrane, which then appears to cause rupture of the outer membrane, releasing cytochrome c from the inter-membrane space. This is consistent with the findings by many groups that the majority of cytochrome c in apoptotic cells is in the cytosol. Inhibitors of caspase activity did not prevent outer mitochondrial membrane rupture, indicating that the membrane rupture did not depend on caspase activity.9
An involvement of permeability transition pores in apoptosis has also been suggested.19 These pores are multiprotein complexes that are present at sites where the inner mitochondrial membrane contacts the outer mitochondrial membrane. The relationship between the transition pores and members of the Bcl-2 family remain to be determined. Although the exact details require more investigation, it is clear that regulation of mitochondrial physiology by the Bcl-2 family and release of cytochrome c into the cytosol are critical events in the process of apoptosis that occurs in the absence of many viability-sustaining cytokines.
Could loss of mitochondria function, by itself cause cell death in vivo? If so, caspases may aid acceleration of the killing process and to conduct the program of cell suicide by an ordered program leading to the formation of apoptotic bodies. Since apoptotic bodies are phagocytosed by neighboring cells,20 an inflammatory response similar to that found when cells die through necrosis would be avoided.
What are the signalling molecules that inhibit normal Bcl-x and Bcl-2 function? There are a group of pro-apoptotic proteins that belong to the Bcl-2 family. These proteins lack important domains found in Bcl-2 and Bcl-x (reviewed in ref. 21 and 22). Cells in which these pro-apoptotic proteins interact with Bcl-2 undergo apoptosis. Thus, these proteins appear to act as regulatory molecules by inhibiting Bcl-2 activity. Two ways in which these proteins are prevented from interacting with Bcl-2 in living cells is low level expression and sequestration in the cytosol.
Bad induces apoptosis when it interacts with Bcl-2 or Bcl-x.23 It appears to be phosphorylated by protein kinase B (AKT).24 When phosphorylated, Bad interacts poorly with Bcl-x and is sequestered in the cytosol.25 Cytokines required for cell viability would appear, therefore, to ensure that Bad and possibly other pro-apoptotic Bcl-2 family members remain phosphorylated.
Expression of other pro-apoptotic Bcl-2 family members may increase under specific circumstances, possibly inhibiting Bcl-2 or Bcl-x activity and causing apoptosis. Bax-alpha is a member of the Bcl-2 family that induces apoptosis and dimerizes with Bcl-2 and Bcl-x in solution.26-29 Bax-alpha could form channels that alter normal mitochondrial physiology, since it has been demonstrated to form pores in synthetic membranes.30, 31 Or, it could act as a pore through which cytochrome c passes from the inter-mitochondrial membrane space to the cytosol. Bax-alpha appears to be a critical component linking the regulation of mitochondrial physiology and apoptosis to the surveilance of DNA integrity by p53 and/or other factors with activities similar to p53.32, 33
Understanding of the cell biology of apoptosis has ballooned in the last few years and it is now possible to explain how loss of a cytokine required for cell viability leads to apoptosis. The sequential events of apoptosis due to loss of a cytokine required for cell viability is summarized in figure 1. In vivo, cells might undergo a slow death if mitochondrial function were lost and there were no follow-up program of destruction.
Activation of caspases may ensure cell death, speed up the killing process, and initiate the program to remove all evidence of cell death without disrupting other cells in the surrounding area.
Now that a core of events is known, it will be of great interest to follow progress in determining the molecular mechanism of regulation of mitochondrial physiology by the inhibitory and inductive members of the Bcl-2 family and how phosphorylation and dephosphorylation events influence the process of apoptosis.
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