Apoptosis Signaling Pathway

The stages of apoptosis: initiation and execution

Apoptosis can be divided into two general stages: initiation and execution. In the initiation stage of programmed cell death, the cell receives a signal that activates the apoptosis pathway, either from internal stresses or extracellular triggers. In the execution stage, a series of molecular and cellular changes unfold, ultimately resulting in death of the cell.

The execution stage of apoptosis can be broken down further into three distinct phases: early, mid and late.

• In the early phase initiator caspases (caspase-8, caspase-9) activate the executioner caspases (caspase-3, caspase-6 and caspase-7). These caspases cleave hundreds of protein substrates to disassemble the cell. One critical event is cleavage of ICAD, releasing CAD (caspase-activated DNase) to fragment the cell’s DNA.
• During the mid phase, DNA condenses and begins to fragment, the cytoskeleton breaks down and the cell shrinks. The plasma membrane forms bubble-like protrusions referred to as blebs or blebbing.
• In the late phase, the cell is dismantled into membrane-enclosed apoptotic bodies, which are then engulfed and cleared by phagocytes without triggering inflammation.

This stage is also regulated by Inhibitors of Apoptosis Proteins (IAPs), such as XIAP, which bind and inhibit active caspases. In intrinsic apoptosis, mitochondrial proteins like SMAC/DIABLO and HTRA2 are released to neutralize IAPs, ensuring full caspase activation.

Apoptosis utilizes two main pathways with different mechanisms: intrinsic and extrinsic.  

The intrinsic pathway of apoptosis: The role of Bcl-2, mitochondria and cytochrome c

The intrinsic apoptosis pathway, also known as mitochondrial-mediated apoptosis, is a central mechanism of programmed cell death. It is activated by internal stress signals such as DNA damage, radiation, oxidative stress or cytokine withdrawal. This pathway relies on precise cell death regulation via interactions between Bcl-2 family proteins, mitochondrial integrity and the controlled release of apoptogenic factors.

Normally, anti-apoptotic Bcl-2 family proteins (including  Bcl-2, Bcl-xL, Mcl-1) maintain mitochondrial outer membrane stability and suppress pro-apoptotic proteins like BAX and BAK.

When stress signals activate the intrinsic of apoptosis pathway, BAX and BAK oligomerize within the mitochondrial outer membrane, causing mitochondrial outer membrane permeabilization (MOMP). This process allows cytochrome c to escape from the mitochondrial intermembrane space into the cytosol.

Once in the cytosol, cytochrome c binds to apoptotic protease-activating factor 1 (Apaf-1) in the presence of dATP, forming the apoptosome. The apoptosome functions as a signaling platform that activates initiator caspase-9, which subsequently triggers executioner caspases such as caspase-3. This caspase activation cascade marks the execution phase of apoptosis and results in orderly cell dismantling.

Regulation of the intrinsic apoptosis pathway involves multiple molecular checkpoints. The tumor suppressor p53 promotes apoptosis by upregulating pro-apoptotic genes (BAX, PUMA, NOXA) and repressing anti-apoptotic Bcl-2 family members in response to DNA damage. In contrast, cell survival pathways like PI3K/Akt and MAPK/ERK can inhibit apoptosis by phosphorylating and inactivating pro-apoptotic proteins.

Additionally, endoplasmic reticulum (ER) stress can feed into the intrinsic pathway. Persistent activation of ER stress sensors (PERK, IRE1, ATF6) leads to induction of the transcription factor CHOP, which increases pro-apoptotic Bcl-2 family expression, further promoting MOMP and cytochrome c release.

In conclusion, the intrinsic apoptosis pathway integrates stress signals through a network of Bcl-2 family protein regulation, mitochondrial outer membrane permeabilization, apoptosome assembly and caspase activation to determine cell fate. Its precise control is essential for tissue homeostasis and the prevention of uncontrolled cell death or tumorigenesis.

The extrinsic pathway of apoptosis 

The extrinsic apoptosis pathway is triggered by extracellular signals from other cells, often mediated by members of the tumor necrosis factor (TNF) receptor family, such as FAS (CD95), TNFR1 (TNFRSF1A) and TRAIL-R (TNFRSF10A).

When these receptors are engaged by their ligands (e.g., FasL, TNF-α, TRAIL), they recruit adaptor proteins such as FADD and TRADD through their death domains. These adaptors scaffold the assembly of the Death-Inducing Signaling Complex (DISC), which brings procaspase-8 and/or procaspase-10 into close proximity, enabling their activation through death effector domain (DED) interactions.

Once activated, caspase-8 initiates the executioner caspase cascade (caspase-3, -6 and -7). Caspase-8 can also cleave the pro-apoptotic BH3-only protein BID into truncated BID (tBID), which translocates to the mitochondria to activate BAX and BAK. This serves as a key point of crosstalk between the extrinsic and intrinsic apoptosis pathways, amplifying the death signal through mitochondrial permeabilization.

It is important to note that TNFR1 signaling can also activate NF-κB-mediated survival pathways through TRADD and other intermediates. Only when FADD is recruited does the pathway switch fully toward apoptosis.

Apoptosis in the context of immunity and cellular stress

Apoptosis is closely tied to immune regulation and stress responses. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells induce apoptosis in infected or abnormal cells via two additional extrinsic mechanisms:

Perforin/granzyme delivery: Perforin forms pores in the target cell membrane, allowing granzyme B to enter and directly activate caspases or cleave BID, linking to the intrinsic pathway of apoptosis. Granzyme A can trigger a caspase-independent form of apoptosis through mitochondrial and ER stress pathways.

FasL–Fas interactions: CTLs express Fas ligand (FasL) to activate death receptors on target cells, initiating the extrinsic pathway of apoptosis through caspase-8.

Chronic ER stress can also induce apoptosis independently by activating CHOP through PERK, IRE1 and ATF6.

Finally, apoptosis does not occur in isolation. It shares regulatory networks with other forms of cell death such as autophagy and ferroptosis, which can converge or diverge depending on the cellular context. Mitochondria act as key signaling hubs, integrating inputs from reactive oxygen species, metabolic stress and death receptor pathways.

References

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2. Green DR, Llambi F. Cell death signaling. Cold Spring Harb Perspect Biol. 2015;7(12):a006080.
3. Tian X, Srinivasan PR, Tajiknia V, Sanchez AF, Uruchurtu S, et al. Targeting apoptotic pathways for cancer therapy. J Clin Invest. 2024;134(14):e179570.