The extrinsic pathway of apoptosis is a process whereby cells initiate programmed cell death in response to external signals, such as those from neighbouring cells or the immune system. This route is activated by the binding of particular ligands to cell surface death receptors, such as tumour necrosis factor (TNF) or Fas ligand.
The binding of the ligand to the receptor results in the recruitment of adaptor proteins and the creation of a signalling complex, which triggers an intracellular signalling cascade. This finally results in the activation of caspases, the primary effectors of apoptosis, a family of cysteine proteases.
Once activated, caspases break a number of intracellular substrates, such as cytoskeletal proteins and DNA repair enzymes, resulting in the distinctive morphological and biochemical alterations of apoptosis. These modifications include cell shrinkage, chromatin condensation, DNA fragmentation, and the creation of apoptotic bodies, which are then phagocytosed by surrounding cells.
The extrinsic pathway of apoptosis is essential for numerous physiological and pathological processes, including immune cell activity, tissue formation and maintenance, and the response to viral and bacterial infections. This pathway’s dysregulation has also been linked to a variety of illnesses, including autoimmune disorders, cancer, and neurodegenerative disorders.
What is Extrinsic Apoptosis Signaling Pathway?
- Apoptosis-initiating extrinsic apoptosis signalling pathways require transmembrane receptor-mediated interactions. These involve death receptors that are members of the gene superfamily for tumour necrosis factor (TNF) receptors.
- Members of the TNF receptor family have comparable cyteine-rich extracellular domains and a “death domain” of around 80 amino acids. This death domain is essential for the transmission of the death signal from the cell surface to intracellular signalling pathways. FasL/FasR, TNF-α/TNFR1, Apo3L/DR3, Apo2L/DR4, and Apo2L/DR5 are now the best-characterized ligands and death receptors, respectively.
- FasL/FasR and TNF-/TNFR1 are the models that best characterise the sequence of events that constitute the extrinsic phase of apoptosis. In these models, receptor clustering and binding with the homologous trimeric ligand are seen.
- Upon ligand interaction, cytplasmic adapter proteins with death domains that interact with the receptors are recruited.
- Fas ligand binding to Fas receptor results in the binding of the adapter protein FADD, whereas TNF ligand binding to TNF receptor results in the binding of the adapter protein TRADD and the recruitment of FADD and RIP. Upon dimerization of the death effector domain, FADD then interacts with procaspase-8. Upon formation of the death-inducing signalling complex (DISC), procaspase-8 is activated autocatalytically.
- Once caspase-8 is activated, the apoptosis execution phase is initiated. A protein called c-FLIP can block death receptor-mediated apoptosis by binding to FADD and caspase-8 and leaving them inactive.
- Toso, a protein that inhibits caspase-8 processing and has been found to prevent Fas-induced apoptosis in T cells, is an additional possible regulator of apoptosis.
Process and Regulation of Extrinsic Apoptosis Pathway
- A death ligand binding to a death receptor, such as TNF-α to TNFR1, triggers the extrinsic route that begins apoptosis. The TNFR family consists of 29 transmembrane receptor proteins arranged as homotrimers and activated by the interaction of their specific ligands (s). They have similar cysteine-rich extracellular domains and an 80-amino acid cytoplasmic domain known as the “death domain” (DD). This death domain is essential for the transmission of the death signal from the cell surface to intracellular signalling pathways. There are 19 members of the TNF ligand family, and depending on the adaptor proteins linked with the activated receptor, binding might result in proliferation, inflammation, or death.
- Through recruitment of RIP, TNFR may also promote pro-inflammatory pathways leading to NFκB activation. IB kinase (IKK) activation is dependent on the death domain kinase RIP. It has been identified that the binding of TNF-α and TNFR1 activates the NFkB pathway, which, depending on the cell type and biological setting, promotes both cell survival and death.
- Fas and DR4/DR5 participate in the process as death receptors and bind CD95 and TRAIL, respectively, in addition to TNFR1. With the assistance of the adapter proteins (FADD/ TRADD), all ligand binding to receptors will result in the recruitment, dimerization, and activation of a caspase cascade, and ultimately the cleavage of both cytoplasmic and nuclear substrates. CD95/Fas, TNF-α/TNFR1, Apo2L/DR4 and Apo2L/DR5 are currently the best-characterized ligands and death receptors.
- Receptor trimerization results in the recruitment and activation of caspase-8 and caspase-10, as well as the recruitment of several death domains. Active caspase-8 and caspase-10 either trigger apoptosis directly by cleaving and activating executioner caspase-3/6/7), or stimulates the intrinsic apoptotic pathway by cleaving the BID in order to induce effective cell death. Immunoblot research demonstrated that the caspase-6 inhibitor prevented the cleavage of lamin A/C, whereas the caspase-3/7 inhibitor prevented the cleavage of poly (ADP-ribose) polymerase (PARP). FLICE inhibitory protein can inhibit caspase-8 activation (FLIP).
- These findings imply that the extrinsic process of cell death involves the activation of caspases, the subsequent cleavage of lamin A/C and PARP, and the NFkB pathway.
Clinical Significance
Apoptosis is a critical process for the development and maintenance of the human body, which removes damaged and unwanted cells through programmed cell death. The extrinsic pathway of apoptosis is initiated by death receptors such as Fas, TNF receptor (TNFR), and TNF-related apoptosis-inducing ligand (TRAIL) receptor, which are expressed on the surface of cells. Binding of these receptors to their ligands activates caspases, leading to apoptotic cell death. The extrinsic apoptosis pathway has been shown to be a promising target for cancer therapy. However, the development of drug resistance and unwanted effects in other diseases are significant challenges. In this article, we will explore the potential of increasing the killing capabilities of the extrinsic apoptosis pathway and the opportunities and challenges it presents for cancer treatment and other diseases.
The Role of Extrinsic Apoptosis Pathway in Cancer Treatment
The extrinsic apoptosis pathway plays a crucial role in cancer therapy. Various anticancer drugs have been shown to increase Fas receptor expression in some tumor cell lines, leading to the activation of caspases and apoptotic cell death. TRAIL has also shown promise in selectively killing cancer cells by binding to multiple receptors, DR4 and DR5, leading to the activation of caspases. However, the resistance of cancer cells to TRAIL-induced apoptosis is a significant challenge. Many strategies are under investigation to overcome this challenge, including the use of combination therapies, increasing the expression of TRAIL receptors, and enhancing the intracellular signal transduction pathways.
The Challenges of Extrinsic Apoptosis Pathway in Other Diseases
While the extrinsic apoptosis pathway is beneficial in cancer treatment, it can be detrimental in other situations. For example, extrinsic apoptosis mediated by cytotoxic T lymphocytes (CTLs) has been postulated to cause the damaging effects of liver destruction in chronic viral hepatitis. In this situation, CTLs recognize and kill virus-infected hepatocytes through the extrinsic apoptosis pathway, leading to liver damage. CTLs also appear to be the cause of Type I diabetes, where the extrinsic apoptotic pathway initiated by CTLs eliminates the pancreatic beta cells. Moreover, both natural killer (NK) cells and CTLs can contribute to graft-versus-host disease, a significant barrier to the success of bone marrow transplantation.
Future Directions in Enhancing the Efficacy of Extrinsic Apoptosis Pathway
The challenges of the extrinsic apoptosis pathway in cancer treatment and other diseases highlight the need for developing more effective and targeted therapies. Future research should focus on identifying the mechanisms of drug resistance in cancer cells, developing strategies to overcome resistance, and enhancing the intracellular signal transduction pathways. For example, combination therapies with TRAIL and other drugs that enhance the apoptotic signal may increase the sensitivity of cancer cells to TRAIL-induced apoptosis. In addition, the development of new drug delivery systems, such as nanoparticles and liposomes, can improve the efficacy of apoptosis-inducing drugs by targeting cancer cells specifically. Moreover, the investigation of alternative ligands that selectively target cancer cells through death receptors can also provide new opportunities for cancer therapy.
In conclusion, the extrinsic apoptosis pathway presents a promising target for cancer therapy. However, the development of drug resistance and the unwanted effects in other diseases are significant challenges. Enhancing the efficacy of the extrinsic apoptosis pathway requires a deeper understanding of the mechanisms of drug resistance and the intracellular signal transduction pathways. Combining therapies with TRAIL and other drugs and developing new drug delivery systems can improve the efficacy of apoptosis-inducing drugs. The investigation of alternative ligands that selectively target cancer cells through death receptors can also provide new opportunities for cancer therapy. Moreover, further research is necessary to identify more specific and efficient targets in the extrinsic apoptosis pathway to overcome the drug resistance and improve the therapeutic efficacy. The extrinsic apoptosis pathway also plays a crucial role in the pathogenesis of other diseases, including chronic viral hepatitis, Type I diabetes, and graft-versus-host disease. Developing targeted therapies that avoid the detrimental effects of extrinsic apoptosis in these diseases is a significant challenge that requires further investigation.
Molecular Signaling Events
The TNF receptor family consists of a group of transmembrane proteins that mediate diverse cellular functions. These receptors can also trigger apoptosis, or programmed cell death, by activating caspases. Caspases are proteases that are involved in cell death and other cellular processes. In this article, we will explore the sequence of events that occur when TNFRs are activated, leading to the initiation of the caspase cascade and ultimately cell death.
Apoptosis, or programmed cell death, is a physiological process that plays a vital role in maintaining homeostasis and normal cellular functions. It is a tightly regulated process that eliminates cells that are no longer needed or have become damaged, without causing inflammation or harm to surrounding cells. Apoptosis can be initiated through two major pathways: the intrinsic pathway and the extrinsic pathway. In this article, we will focus on the extrinsic pathway of apoptosis, specifically exploring the role of soluble TNF family ligands TNF receptor apoptosis-inducing ligand (TRAIL), FasL, and TNF in this process.
The Role of TNF Family Ligands in Apoptosis
The TNF family of ligands consists of over 19 members, including TNF, FasL, and TRAIL. These ligands are involved in diverse biological processes, such as immune cell activation, inflammation, and apoptosis. TRAIL and FasL are known as death ligands because they have the ability to trigger apoptosis through the extrinsic pathway. TNF, on the other hand, can induce both apoptosis and survival, depending on the cellular context and signaling pathways involved.
The extrinsic pathway of apoptosis is initiated when death ligands bind to their cognate death receptors, which are members of the TNF receptor superfamily. The binding of death ligands to death receptors triggers a series of events that culminate in the activation of caspases, which are a family of proteases that cleave cellular proteins and initiate the process of apoptosis.
FasL and TRAIL
FasL and TRAIL are capable of inducing apoptosis by engaging their cognate death receptors, Fas and death receptor 4 (DR4) or death receptor 5 (DR5), respectively. When FasL or TRAIL binds to their receptors, the receptors undergo conformational changes that result in the assembly of a protein complex called the death-inducing signaling complex (DISC). The DISC consists of Fas-associated death domain (FADD) and procaspase 8, a precursor form of caspase 8.
Decoy receptor 1 (DcR1), DcR2, and DcR3 are also members of the TNF receptor superfamily and can bind to FasL and TRAIL with high affinity, but they do not induce apoptosis. The decoy receptors lack the cytoplasmic domain required for DISC assembly and caspase activation.
Once FADD is recruited to the DISC through its complementary death domains (DDs), it can then recruit procaspase 8 through their complementary death-effector domains (DEDs). The recruitment of procaspase 8 to the DISC leads to its autoproteolytic cleavage, releasing two subunits that form an active enzyme. In type I cells, such as lymphocytes and some tumor cells, the active caspase 8 is sufficient to cleave and activate effector caspases 3, 6, and 7, leading to apoptosis. In type II cells, such as hepatocytes and some other tumor cells, activated caspase 8 cleaves Bid, a member of the Bcl-2 family of proteins, which then stimulates the release of factors from the mitochondria, including cytochrome c. This activates the intrinsic pathway of apoptosis and augments active caspase 8.
TNF
TNF is a pleiotropic cytokine that can induce both apoptosis and survival depending on the cellular context and signaling pathways involved.
In summary, the extrinsic pathway of apoptosis is a tightly regulated mechanism that plays a crucial role in maintaining tissue homeostasis and eliminating damaged or infected cells. This pathway is initiated by the binding of TNF family ligands to their cognate death receptors, which results in the assembly of signaling complexes that activate the caspase cascade and ultimately lead to cell death.
FasL and TRAIL mainly activate the extrinsic pathway of apoptosis by binding to DR4, DR5, and Fas and forming the DISC. Meanwhile, TNF induces the extrinsic pathway of apoptosis by binding to TNF-R1 and recruiting TRADD and a complex of proteins containing RIP and TRAF2, leading to the formation of Complex I and II.
Overall, understanding the molecular mechanisms underlying the extrinsic pathway of apoptosis is crucial for the development of novel therapeutic approaches for diseases such as cancer, autoimmune disorders, and chronic inflammatory diseases. By targeting specific components of the extrinsic pathway of apoptosis, it may be possible to induce or inhibit cell death and modulate the immune response, providing new avenues for the treatment of these conditions.
The Death-Inducing Signaling Complex (DISC)
Upon binding ligand, TNFRs undergo conformational changes that result in the recruitment of a group of proteins known as the death-inducing signaling complex (DISC). The DISC was first described in FasL-Fas apoptotic signaling. The binding of TRAIL to its death-inducing receptors acts similarly to FasL. On the other hand, TNF-mediated signaling is more complex and will be discussed in further detail.
Fas-Associated DD (FADD)
The ligand-bound Fas or TRAIL death receptors recruit a DD-containing adapter protein, Fas-associated DD (FADD). FADD contains a second important death receptor-signaling motif, the death-effector domain (DED). It is the only protein in either the human or the mouse genome that contains both a DD and a DED. Bound FADD recruits initiator caspases 8 and 10 through complimentary DED domains.
Activation of Caspases
Recruitment of caspases 8 and 10 to the DISC leads to their autoproteolytic cleavage and release of two caspase subunits that form a mature active enzyme. If activated caspase 8/10 is present in sufficient abundance, it can cleave and activate effector caspases 3 and 7, thereby fully engaging the caspase cascade. In type I cells, activation of these effector caspases by activated caspase 8/10 alone is sufficient to induce apoptosis. In type II cells, activated caspase 8/10 stimulates the release of factors from the mitochondria, including cytochrome c, Smac/DIABLO, and Omi/Htr2A, thereby engaging the intrinsic pathway of apoptosis.
Type I vs. Type II Cells
Why cells behave in a type I or a type II manner is not well understood. Gene expression analysis comparing type I and type II cells using Fas activation has been performed. The expression analysis of type I cell lines showed a preponderance of mesenchymal-like genes, whereas the type II cell lines preferentially express epithelium-like markers. A chemical screen for growth inhibition of these cells revealed that actin-binding compounds selectively inhibited growth of type I cells and tubulin-interacting compounds inhibited growth of type II cells. The functional significance of this observation may become useful in chemotherapeutic treatment selection for cancers with these types of gene expression profiles.
Caspase 8/10 in Intrinsic and Extrinsic Pathways
Caspase 8/10 connects the intrinsic and extrinsic pathways by cleaving Bid, a BH3-only member of the Bcl-2 family, which can mediate destabilization of the outer mitochondrial membrane by interacting with other Bcl-2 family members. To date, Bid is the only known physiologic mediator that connects the extrinsic pathway with the release of apoptotic factors from mitochondria.
The Role of TNFRs in Inflammation
Apart from its role in cell death, TNFR signaling has a critical role in the regulation of inflammation. TNFα, the prototypical pro-inflammatory cytokine, is involved in a wide range of inflammatory processes, including acute and chronic inflammation, sepsis, autoimmune diseases, and cancer. The primary function of TNFα is to promote the recruitment of immune cells to the site of injury or infection, where they eliminate pathogens and promote tissue repair. However, excessive TNFα production can cause tissue damage and organ failure, as seen in septic shock. Therefore, the regulation of TNFα signaling is critical for maintaining a healthy balance between pro-inflammatory and anti-inflammatory responses.
TNFα signals through two receptors, TNFR1 and TNFR2, which are expressed on most cell types, including immune cells, endothelial cells, and epithelial cells. TNFR1 is the predominant mediator of TNFα-induced inflammation and cell death, whereas TNFR2 has been shown to have a more limited role in these processes. TNFα binding to TNFR1 leads to the recruitment of a cytoplasmic adaptor protein, TNFR1-associated death domain protein (TRADD), which mediates the assembly of a signaling complex known as the TNFR1 complex I. This complex activates multiple downstream signaling pathways, including the nuclear factor kappa B (NF-κB) pathway, the mitogen-activated protein kinase (MAPK) pathway, and the caspase cascade.
The NF-κB pathway is a critical mediator of TNFα-induced inflammation and cell survival. NF-κB is a transcription factor that regulates the expression of numerous pro-inflammatory genes, including cytokines, chemokines, and adhesion molecules (34). In resting cells, NF-κB is sequestered in the cytoplasm by a family of inhibitor proteins known as IκBs. Upon TNFα stimulation, the TNFR1 complex I activates a signaling cascade that leads to the phosphorylation and degradation of IκBs, allowing NF-κB to translocate to the nucleus and induce gene transcription.
The MAPK pathway is another critical mediator of TNFα-induced inflammation and cell survival. MAPKs are a family of serine/threonine kinases that regulate numerous cellular processes, including proliferation, differentiation, and apoptosis. TNFα stimulation leads to the activation of three major MAPK pathways: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. These pathways activate downstream transcription factors that induce the expression of pro-inflammatory genes.
The caspase cascade is the primary mediator of TNFα-induced cell death. TNFR1 activation leads to the recruitment of TRADD, FADD, and caspase 8, which form a signaling complex known as the TNFR1 complex II (36). Caspase 8 activation leads to the cleavage and activation of downstream effector caspases, such as caspase 3, which induce apoptotic cell death.
Function
Apoptosis, also known as programmed cell death, plays a critical role in maintaining immunological homeostasis. This process can be initiated via intrinsic or extrinsic pathways, and it is essential for the eradication of infected or potentially cancerous cells. In this article, we will delve deeper into the role of apoptosis in various aspects of the immune system.
The Importance of the Extrinsic Pathway in Eradicating Viral Infections
The extrinsic pathway of apoptosis is crucial for eradicating cells that are harboring an intracellular infection or are transformed and potentially malignant. This pathway is particularly important for the eradication of virally infected cells. Antibodies are effective at neutralizing extracellular viruses and preventing them from initiating an infection. However, they are not as effective at eliminating intracellular viruses that have already established an infection. By inducing apoptosis in virally infected cells, the dying cell does not lyse and release its contents into surrounding tissues, including intact infective virions that would be capable of infecting neighboring cells.
Cooperation Between the Extrinsic and Intrinsic Pathways in Maintaining T Cell Homeostasis
Both the extrinsic and intrinsic pathways of apoptosis play a crucial role in maintaining T cell homeostasis. After clonally expanding in response to the presence of a foreign antigen, the majority of clonally expanded lymphocytes die by apoptosis, leaving a small percentage of cells that function as memory or effector lymphocytes. Both death receptors of the extrinsic pathway and mitochondrial proteins of the intrinsic pathway contribute to the contraction phase of lymphocytes after successful clearance of a pathogen. This process prevents additional host damage induced by cytokines, and also permits more energy expenditure if a subsequent pathogen is encountered. Similarly, both pathways induce apoptosis in neutrophils after clearance of a pathogen, helping to subside the damaging inflammation that can occur in response to infection.
The Role of Apoptosis in Achieving Central and Peripheral Tolerance
Apoptosis is essential in achieving central tolerance and preventing autoimmune disease. Developing thymocytes that bind too strongly to MHC molecules presenting self-antigens in the thymus are eliminated in a process called negative selection. Negative selection requires the actions of caspases to initiate apoptosis in self-reactive thymocytes. However, negative selection is an imperfect process, and some self-reactive T lymphocytes will escape to the periphery. Peripheral tolerance occurs via multiple mechanisms, including the induction of apoptosis in self-reactive T cells in a process termed activation-induced cell death (AICD). Repeated stimulation of self-reactive T cells in the periphery results in the upregulation of the death receptor Fas, therefore making the auto-reactive T cell more susceptible to apoptosis by cells expressing FasL. Similar processes occur to eliminate developing self-reactive B cells in the bone marrow as well as mature B cells in the periphery.
The Role of Apoptosis in Immunological Privilege
Immunological privilege is a physiological mechanism of self-tolerance functioning to exclude certain peripheral tissues such as the eye, brain, and testes from the damaging effects of the immune system. The constitutive expression of FasL on privileged tissues represents a backup mechanism to protect privileged tissues from lymphocyte destruction, as the FasL initiates the extrinsic pathway of apoptosis in infiltrating lymphocytes expressing the Fas receptor.
In conclusion, apoptosis plays a critical role in maintaining immunological homeostasis. Understanding the various ways in which apoptosis is involved in the immune response can shed light on the development of new therapies for treating immune-related disorders.
FAQ
What is the extrinsic pathway of apoptosis?
The extrinsic pathway of apoptosis is a signaling pathway that is initiated by external signals such as the binding of death ligands to their receptors on the cell surface.
What are death ligands?
Death ligands are molecules that bind to specific receptors on the cell surface and trigger the extrinsic pathway of apoptosis. Examples of death ligands include tumor necrosis factor (TNF) and Fas ligand (FasL).
What is the role of death receptors in the extrinsic pathway?
Death receptors are specific cell surface receptors that bind to death ligands and initiate the signaling cascade that leads to apoptosis.
What are the key components of the extrinsic pathway?
The key components of the extrinsic pathway include death receptors, death ligands, adaptor proteins, initiator caspases (such as caspase-8 and caspase-10), and effector caspases (such as caspase-3 and caspase-7).
How does the extrinsic pathway differ from the intrinsic pathway of apoptosis?
The intrinsic pathway of apoptosis is initiated by internal signals such as DNA damage or cellular stress, while the extrinsic pathway is initiated by external signals such as the binding of death ligands to death receptors on the cell surface.
What is the function of initiator caspases in the extrinsic pathway?
Initiator caspases, such as caspase-8 and caspase-10, are responsible for cleaving and activating downstream effector caspases, which then cleave and activate other proteins in the cell to carry out the apoptotic program.
How does the extrinsic pathway contribute to immune system function?
The extrinsic pathway of apoptosis is involved in the regulation of immune system function by eliminating activated immune cells, such as T cells and natural killer cells, once their target cells have been eliminated.
What are some diseases that are associated with dysregulation of the extrinsic pathway?
Dysregulation of the extrinsic pathway has been implicated in a variety of diseases, including cancer, autoimmune disorders, and infectious diseases.
How do some cancer cells evade the extrinsic pathway of apoptosis?
Some cancer cells can evade the extrinsic pathway of apoptosis by downregulating death receptors or their ligands, or by overexpressing anti-apoptotic proteins that inhibit the activity of initiator and effector caspases.
Are there any therapies that target the extrinsic pathway of apoptosis?
Yes, there are several therapies that target the extrinsic pathway of apoptosis, including monoclonal antibodies that block death ligands or death receptors, and small molecule inhibitors that target initiator and effector caspases.
References
- Yanumula A, Cusick JK. Biochemistry, Extrinsic Pathway of Apoptosis. In: StatPearls. StatPearls Publishing, Treasure Island (FL); 2022. PMID: 32809646.
- Yanumula A, Cusick JK. Biochemistry, Extrinsic Pathway of Apoptosis. [Updated 2022 Aug 1]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK560811/
- Hongmei, Z. (2012). Extrinsic and Intrinsic Apoptosis Signal Pathway Review. Apoptosis and Medicine. doi: 10.5772/50129
- Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007 Jun;35(4):495-516. doi: 10.1080/01926230701320337. PMID: 17562483; PMCID: PMC2117903.
- https://biologydictionary.net/apoptosis/#extrinsic-pathway
- https://www.sciencedirect.com/science/article/abs/pii/S1876162321000134
- https://www.creative-diagnostics.com/extrinsic-apoptosis-pathway.htm
- https://www.thermofisher.com/in/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/cell-signaling-pathways/cellular-apoptosis-pathway.html#ExtrinsicPA
- https://www.sinobiological.com/research/signal-transduction/extrinsic-apoptosis
- https://www.slideshare.net/AananthaKumar/intrinsic-and-extrinsic-pathway-of-apoptosis
- https://www.easybiologyclass.com/extrinsic-pathway-of-apoptosis-apoptosis-molecular-mechanism-part-2/
- http://eknygos.lsmuni.lt/springer/520/31-54.pdf
