The intrinsic and extrinsic pathways of apoptosis are both naturally occurring processes by which a cell is directed to programmed cell death. Both pathways of apoptosis activate cell signaling cascades that are an indispensable part of the development and function of an organism.

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What is the difference between intrinsic and extrinsic pathway of apoptosis?

There are at least two broad signaling pathways that lead to apoptosis: the intrinsic pathway of apoptosis and the extrinsic pathway of apoptosis.  The extrinsic pathway of apoptosis begins outside a cell, when conditions in the extracellular environment determine that a cell must die. The intrinsic pathway of apoptosis pathway begins when an injury occurs within the cell and the resulting stress activates the apoptotic pathway. In both the intrinsic and extrinsic pathway of apoptosis, signaling results in the activation of a family of Cys (Cysteine) proteases, named caspases, that act in a proteolytic cascade to dismantle and remove the dying cell (1).

Intrinsic pathway of apoptosis

The intrinsic apoptosis pathway begins when an injury occurs within the cell. Intrinsic stresses such as oncogenes, direct DNA damage, hypoxia, and survival factor deprivation, can activate the intrinsic apoptotic pathway. p53 is a sensor of cellular stress and is a critical activator of the intrinsic pathway (See the p53 Pathway for Apoptosis Signaling). The DNA checkpoints proteins, ATM (Ataxia Telangiectasia Mutated protein), and Chk2 (Checkpoints Factor-2), directly phosphorylate and stabilize p53 and inhibit MDM2 (Mouse Double Minute-2 Homolog)–mediated ubiquitination of p53. MDM2 binds p53 and mediates the nuclear export. When bound to MDM2, p53 can no longer function as an activator of transcription. p53 initiates apoptosis by transcriptionally activating pro-apoptotic Bcl2 family members and repressing anti-apoptotic Bcl2 proteins and CIAPs. Other p53 targets include BAX, Noxa, PUMA (p53-Upregulated Modulator of Apoptosis), and the most recently identified, BID. p53 also transactivates other genes that may contribute to apoptosis, including PTEN (Phosphatase and Tensin Homolog Deleted On Chromosome-10), APAF1, Perp, p53AIP1 (p53-regulated Apoptosis-Inducing Protein-1), and genes that lead to increases in ROS (Reactive Oxygen Species). These ROS lead to generalized oxidative damage to all mitochondrial components. Damage to mitochondrial DNA disrupts mitochondrial oxidative phosphorylation, contributing to a number of human diseases (2-3).

Other proteins released from damaged mitochondria, such as SMAC (Second Mitochondria-Derived Activator of Caspase), Diablo, Arts, and Omi/HTRA2 (High Temperature Requirement Protein-A2), counteract the effect of IAPs (Inhibitor of Apoptosis Proteins), which normally bind and prevent activation of Caspase-3. The interaction between Bcl family members IAPs, SMAC, and Omi/HTRA2 is central to the intrinsic apoptosis pathway. Recent studies demonstrate that another nuclease, EndoG (Endonuclease-G), is specifically activated by apoptotic stimuli and is able to induce nucleosomal fragmentation of DNA independently of Caspase and DFF (DNA-Fragmentation Factor)/CAD (Caspase-Activated DNAse). EndoG is a mitochondrion-specific nuclease that translocates to the nucleus and cleaves chromatin DNA during apoptosis. Another protein, AIF (Apoptosis Inducing Factor), has also been attributed a role in apoptosis, becoming active upon translocation from mitochondria to nuclei, where it initiates chromatin condensation and large-scale DNA fragmentation (4).

Extrinsic pathway of apoptosis

The extrinsic pathway begins outside a cell, when conditions in the extracellular environment determine that a cell must die. Based on the triggering stimulus and nature of the components involved, at least two apoptotic pathways can be differentiated:

  • one involving receptor systems
  • one triggered by cytotoxic stress

Receptor-mediated apoptotic pathways

Receptor-mediated pathways include those activated by death ligands. Death Receptors (DRs) are cell surface receptors that transmit apoptotic signals initiated by specific ligands and play a central role in instructive apoptosis (see also the Death Receptor Pathway). These receptors activate Death Caspases (DCs) within seconds of ligand binding, causing an apoptotic demise of the cell within hours. DRs belong to the superfamily of TNFR (Tumor Necrosis Factor Receptor), which are characterized by a Cys-rich extracellular domain and a homologous intracellular domain known as the Death Domain (see also TNF Superfamily Pathway). Adapter-molecules like FADD (Fas-Associated via Death Domain), TRADD (Tumor Necrosis Factor Receptor-1-Associated Death Domain), or Daxx contain Death Domains so that they can interact with the DRs and transmit the apoptotic signal to the death-machinery. The best characterized Death Receptors are Fas and TNFR1 (Tumor Necrosis Factor Receptor-1). Other DRs include Apo2 and Apo3 (5).

FasL (Fas Ligand), a homotrimeric protein, acts as ligand for Fas and causes oligomerization of its receptor upon binding (see also the Fas Pathway). Associated with this is the clustering of the Death Domains and binding of co-factor FADD. The FADD protein binds via its DED (Death Effector Domain) motif to a homologous motif in Procaspase-8. The complex of Fas, FADD and ProCaspase-8 is called the DISC (Death Inducing Signaling Complex). The co-factor function of FADD, in turn, is blocked by interaction with the regulator FLIP (FLICE Inhibitory Protein). Upon recruitment by FADD, Procaspase-8 oligomerization drives its activation through self-cleavage. Active Caspase-8 then activates downstream caspases (Caspase-3 and -7), committing the cell to apoptosis.

Activated Caspase-8 activates Caspase-3 through two pathways. In the first pathway Caspase-8 cleaves BID (Bcl2 Interacting Protein), and its COOH-terminal part translocates to mitochondria where it triggers CytoC (Cytochrome-C) release. The released CytoC binds to APAF1 (Apoptotic Protease Activating Factor-1) together with dATP and Procaspase-9 and activates Caspase-9. The Caspase-9 cleaves Procaspase-3 and activates Caspase-3. Another pathway is that Caspase-8 cleaves Procaspase-3 directly and activates it. The Caspase-3 cleaves DNA fragmentation factor ICAD (Inhibitor of Caspase-Activated DNase) in a heterodimeric form consisting of CAD and cleaved ICAD. Cleaved ICAD dissociates from CAD, inducing oligomerization of CAD that has DNase activity. The active CAD oligomer causes the internucleosomal DNA fragmentation, which is an apoptotic hallmark indicative of chromatin condensation (6).

Recently, a nuclear pathway linked to apoptosis has also been suggested. (ZIPK) ZIP Kinase triggers apoptosis from nuclear PODs (PML (Promyelocytic Leukemia) Oncogenic Domains) and, in collaboration with Daxx and Par-4 (Prostate Apoptosis Response Protein-4), mediates a novel nuclear pathway for apoptosis. Another set of DRs, Apo2 and Apo3, has been characterized that share a different death ligand, known as Apo2L (Apo2 Ligand), Apo3L (Apo3 Ligand). DISC complex formation and BID cleavage downstream of Apo2/Apo3 is similar to the Fas pathway (7-8).

In the case of TNFR1, various Death Domain–containing proteins can form distinct complexes in a temporal manner once the receptor is activated. A TNFR1 complex possessing the DD-containing protein TRADD, TRAF2 (TNF Receptor Associated Factor-2), CIAP1 (Cellular Inhibitor of Apoptosis-1), and the kinase RIP1 (Receptor-Interacting Protein-1) assembles at the plasma membrane within minutes after activation in order to recruit IKK (I-KappaB-Kinase) leading to NF-KappaB (Nuclear Factor-KappaB) activation and survival. In a second step, Complex II is formed after the TRADD-based complex dissociates from the receptor and recruits FADD and the initiator Caspase-8. The balance of effects by complex I versus II rest with FLIP, an inhibitor of Caspase-8. When Complex I NF-KappaB activation is sufficient, adequate FLIP is expressed to inhibit Caspase-8 of complex II. Complex II can mediate apoptosis only when Complex I-mediated NF-KappaB activation is insufficient (9-10). Besides DRs, growth factors also influence apoptosis via PI3K (Phosphatidylinositde-3 Kinase) and the Akt Pathway (v-Akt Murine Thymoma Viral Oncogene Homolog). Growth factors binds to growth factor receptors and activate PI3K. Activation of PI3K pathways leads to Akt activation. Akt is very important in BAD (Bcl2-Antagonist of Cell Death) regulation, which is a pro-apoptotic member of Bcl2 (B-Cell Leukemia-2) family and is involved in mitochondrial apoptosis. PKC (Protein Kinase-C) may also play an important role in apoptosis by activating p90RSKs (Ribosomal-S6 Kinases), which inhibits BAD (11).

Cellular stress-mediated apoptotic pathways

In addition to receptor-mediated apoptosis, there is another pathway activated by various forms of cellular stress. Stress effects that can induce apoptosis are Gamma- and UV-radiation, treatment with cytotoxic drugs such as Actinomycin D, and removal of cytokines. Stress-induced apoptosis occurs by a mechanism that involves altering mitochondrial permeability and subsequent CytoC release and formation of the apoptosome, a catalytic multiprotein platform that activates Caspase-9. Activated Caspase-9 then cleaves Caspase-3, resulting in downstream events involved in cell death. Release of CytoC is regulated by Bcl2 family proteins. Bcl2L (Bcl2-Like), BclXL (Bcl2 Related Protein Long Isoform), and other anti-apoptotic Bcl2 family members reside in the outer mitochondrial membrane and prevent CytoC release. BAX (Bcl2 Associated X-protein), BID (BH3 Interacting Death Domain), and BIM (Bcl2-Interacting Protein) are initially inactive and must translocate to mitochondria to induce apoptosis, either by forming pores in mitochondria directly or by binding via BH3 domains to Bcl2, BclXL, and Bfl1, and antagonizing these anti-apoptotic proteins.

Mitochondrial Membrane Permeabilization (MMP) is clearly a pivotal event in the progression of apoptosis in many systems (See Assays for Mitochondria Function). At least two mechanisms of MMP have been described. First mechanism proposes that VDAC (Voltage-Dependent Anion Channel), ANT (Adenine Nucleotide Transporter), PBR (Peripheral-type Benzodiazepine Receptor), and CypD (Cyclophilin-D) come together to form the PTPC (Permeability Transition Pore Complex). This pore complex may also associate with BAX, BAK1 (Bcl2 Antagonist Killer-1), or BIM, which accelerate channel opening or BclXL, which causes closure. According to the second mechanism, BAX is released from its interaction with 14-3-3 and translocates from cytosol to mitochondria in response to diverse signals. Here it oligomerizes, forming protein pores. Pore formation also takes place in conjunction with the BH3-only Bcl-2 family member BID upon proteolysis to tBID (Truncated BID). BAK1 is located at mitochondria and has similar pore-forming properties to BAX, via its oligomerization and association with tBID. BID is activated by Caspase-8–induced cleavage during DR signaling, whereas BIM is released from its association with microtubules (12-13).

Apoptosis as a conserved pathway

Programmed cell death and its morphologic manifestation of apoptosis is a conserved pathway that in its basic tenets appears operative in all metazoans. Apoptosis also operates in adult organisms to maintain normal cellular homeostasis. This is especially critical in long-lived mammals that must integrate multiple physiological as well as pathological death signals, which, for example, includes regulating the response to infectious agents. Gain- and loss-of-function in the core apoptotic pathway indicate that the violation of cellular homeostasis can be a primary pathogenic event that results in disease. Evidence indicates that insufficient apoptosis can manifest as cancer or autoimmunity, while accelerated cell death is evident in acute and chronic degenerative diseases, immunodeficiency, and infertility (14).

Intrinsic and extrinsic pathways of apoptosis

Intrinsic and extrinsic pathways of apoptosis
Cartoon schematic showing what symbols used in the pathway schematic represent.
Intrinsic and extrinsic pathways of apoptosis
Cartoon schematic showing what symbols used in the pathway schematic represent.
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