CELL SIGNALING

Cell signaling mechanisms

Five major types of cell-cell signaling are considered (Figure 3-1):

Figure 3-1 Mechanisms of hormone action

Mechanisms of action of cell signaling molecules

Cell signaling molecules exert their action after binding to receptors expressed by their target cells. Target cells, in turn, can determine either a negative or positive feedback action to regulate the release of the targeting hormone (Figure 3-2).

Figure 3-2 Positive and negative feedback

Cell receptors can be expressed on the cell surface of the target cells. Some receptors are intracellular proteins in the cytosol or the nucleus of target cells. Intracellular receptors require that the signaling molecules diffuse across the plasma membrane (Figure 3-3).

Figure 3-3 Mechanism of action of steroid hormones

Steroid hormones (Box 3-B) belong to this class of signaling molecules. Steroid hormones are synthesized from cholesterol and include testosterone, estrogen, progesterone, and corticosteroids.

Testosterone, estrogen, and progesterone are sex steroids and are produced by the gonads. Corticosteroids are produced by the cortex of the adrenal gland and include two major classes: glucocorticoids, which stimulate the production of glucose, and mineralocorticoids, which act on the kidneys to regulate water and salt balance.

There are three cell signaling molecules that are structurally and functionally distinct from steroids but act on target cells by binding to intracellular receptors after entering the cell by diffusion across the plasma membrane. They include thyroid hormone (produced in the thyroid gland to regulate development and metabolism), vitamin D₃ (regulates calcium metabolism and bone growth), and retinoids (synthesized from vitamin A to regulate development).

Steroid receptors are members of the steroid receptor superfamily. They act as transcription factors through their DNA binding domains, which have transcription activation or repression functions. Steroid hormones and related molecules can therefore regulate gene expression.

In the androgen insensitivity syndrome (also known as the testicular feminization syndrome [Tfm]), there is a mutation in the gene expressing the testosterone receptor such that the receptor cannot bind the hormone, and hence the cells do not respond to the hormone. Although genetically male, the individual develops the secondary sexual characteristics of a female. We discuss the androgen insensitivity syndrome in Chapter 21, Sperm Transport and Maturation.

Nitric oxide

Nitric oxide is a signaling molecule. It is a simple gas synthesized from the amino acid arginine by the enzyme nitric oxide synthase. It acts as a paracrine signaling molecule in the nervous, immune, and circulatory systems. Like steroid hormones, nitric oxide can diffuse across the plasma membrane of its target cells. Unlike steroids, nitric oxide does not bind to an intracellular receptor to regulate transcription. Instead, it regulates the activity of intracellular target enzymes. The following are relevant characteristics of nitric oxide:

Nitric oxide increases the activity of the second messenger cyclic guanosine monophosphate (cGMP; see later in this section) in smooth muscle cells, which then causes cell muscle relaxation and blood vessel dilation. Nitroglycerin, a pharmacologic agent used in the treatment of heart disease, is converted to nitric oxide, which increases heart blood flow by dilation of the coronary blood vessels.

Cell signaling molecules bind to cell surface receptors

A large variety of signaling molecules bind to cell surface receptors. Several groups are recognized

Pathways of intracellular signaling by cell surface receptors

When a cell-signaling molecule binds to a specific receptor, it activates a series of intracellular targets located downstream of the receptor. Several molecules associated with receptors have been identified:

1. G protein–coupled receptors (guanine nucleotide–binding proteins): Members of a large family of G proteins (more than 1000 proteins) are present at the inner leaflet of the plasma membrane (Figure 3-4).

Figure 3-4 C protein-coupled receptors

When a signaling molecule or receptor ligand binds to the extracellular portion of a cell surface receptor, its cytosolic domain undergoes a conformational change that enables binding of the receptor to a G protein. This contact activates the G protein, which then dissociates from the receptor and triggers an intracellular signal to an enzyme or ion channel. We return to the G protein when we discuss the cyclic adenosine monophosphate (cAMP) pathway.

2. Tyrosine kinases as receptor proteins (Figure 3-5): These surface receptors are themselves enzymes that phosphorylate substrate proteins on tyrosine residues. EGF, NGF, PDGF, insulin, and several growth factors are receptor protein tyrosine kinases. Most of the receptor protein tyrosine kinases consist of single polypeptides, although the insulin receptor and other growth factors consist of a pair of polypeptide chains.

Figure 3-5 Tyrosine kinases

Binding of a ligand (a growth factor) to the extracellular domain of these receptors induces receptor dimerization that results in receptor autophosphorylation (the two polypeptide chains phosphorylate one another). The autophosphorylation of the receptors determines the binding of the tyrosine kinase domain to downstream signaling molecules. Downstream signaling molecules bind to phosphotyrosine residues through domains called SH2 domains (for Src homology 2). Src (for sarcoma) is a gene present in the tumor-producing Rous sarcoma virus and encodes a protein that functions as a protein tyrosine kinase.

Clinical significance: Tyrosine kinases, targets for therapeutic agents

There are two main classes of tyrosine kinases: (1) receptor tyrosine kinases are transmembrane proteins with a ligand-binding extracellular domain and a catalyic intracellular kinase domain (see Figure 3-5), and (2) nonreceptor tyrosine kinases found in the cytosol, nucleus, and inner side of the plasma membrane.

The transmembrane receptor kinase subfamily belongs to the PDGF family, which includes c-kit. The subfamily of nonreceptor tyrosine kinases includes the Src family, the Fujinami poultry sarcoma/feline sarcoma (Fps/Fes), and Fes-related (Fer) subfamily.

In the absence of a ligand, receptor tyrosine kinases are unphosphorylated and monomeric. The nonreceptor tyrosine kinase is maintained in an inactive state by cellular inhibitor proteins. Activation occurs when the inhibitors are dissociated or by recruitment to transmembrane receptors that trigger autophosphorylation. Tyrosine kinase activity terminates when tyrosine phosphatases hydrolyze tyrosyl phosphates and by induction of inhibitory molecules.

The activity of tyrosine kinases in cancer cells can be disrupted by a protein that determines unregulated autophosphorylation in the absence of a ligand, by disrupting au to regulation of the tyrosine kinase, or by overexpression of receptor tyrosine kinase and/or its ligand. Abnormal activation of tyrosine kinases can stimulate the proliferation and anticancer drug resistance of malignant cells.

Tyrosine kinase activity can be inhibited by imatinib mesylate, a molecule that binds to the adenosine triphosphate (ATP)–binding domain of the tyrosine kinase catalytic domain. Imatinib can induce hematologic remission in patients with chronic myeloid leukemia and in tumors caused by activated receptor tyrosine kinase PDGF receptor (chronic myelomonocytic leukemia) and c-kit (systemic mastocytosis and mast cell leukemias). Imatinib has been successfully used in the treatment of gastrointestinal solid tumors.

Major pathways of intracellular cell signaling

Upon ligand binding, most cell surface receptors stimulate intracellular target enzymes to transmit and amplify a signal. An amplified signal can be propagated to the nucleus to regulate gene expression in response to an external cell stimulus.

The major intracellular signaling pathways include the cAMP and cGMP pathways, the phospholipase C–Ca²⁺ pathway, the NF-κB (for nuclear factor involved in the transcription of the κ light chain gene in B lymphocytes) transcription factor pathway, the Ca²⁺-calmodulin pathway, the MAP (for mitogen-activated protein) kinase pathway, and the JAK-STAT (for signal transducers and activators of transcription) pathway.

The cAMP pathway

The intracellular signaling pathway mediated by cAMP was discovered in 1958 by Earl Sutherland while studying the action of epinephrine, a hormone that breaks down glycogen into glucose before muscle contraction.

When epinephrine binds to its receptor, there is an increase in the intracellular concentration of cAMP. cAMP is formed from adenosine triphosphate (ATP) by the action of the enzyme adenylyl cyclase and degraded to adenosine monophosphate (AMP) by the enzyme cAMP phosphodiesterase. This mechanism led to the concept of a first messenger (epinephrine) mediating a cell-signaling effect by a second messenger, cAMP. The epinephrine receptor is linked to adenylyl cyclase by G protein, which stimulates cyclase activity upon epinephrine binding.

The intracellular signaling effects of cAMP (Figure 3-6) are mediated by the enzyme cAMP-dependent protein kinase (or protein kinase A). In its inactive form, protein kinase A is a tetramer composed of two regulatory subunits (to which cAMP binds) and two catalytic subunits. Binding of cAMP results in the dissociation of the catalytic subunits. Free catalytic subunits can phosphorylate serine residues on target proteins.

Figure 3-6 Cyclic adenosine monophosphate (cAMP) pathway

In the epinephrine-dependent regulation of glycogen metabolism, protein kinase A phosphorylates two enzymes:

Note that an elevation of cAMP results in two distinct events: the breakdown of glycogen and, at the same time, a blockage of further glycogen synthesis. Also note that the binding of epinephrine to a single receptor leads to a signal amplification mechanism during intracellular signaling mediated by many molecules of cAMP. cAMP signal amplification is further enhanced by the phosphorylation of many molecules of phosphorylase kinase and glycogen synthase by the catalytic subunits dissociated from protein kinase A. It is important to realize that protein phosphorylation can be rapidly reversed by protein phosphatases present in the cytosol and as transmembrane proteins. These protein phosphatases can terminate responses initiated by the activation of kinases by removing phosphorylated residues.

cAMP also has an effect on the transcription of specific target genes that contain a regulatory sequence called the cAMP response element (CRE). Catalytic sub-units of protein kinase A enter the nucleus after dissociation from the regulatory subunits. Within the nucleus, catalytic subunits phosphorylate a transcription factor called CRE-binding protein (CREB), which activates cAMP-inducible genes.

Finally, cAMP effects can be direct, independent of protein phosphorylation. An example is the direct regulation of ion channels in the olfactory epithelium. Odorant receptors in sensory neurons of the nose are linked to G protein, which stimulates adenylyl cyclase to increase intracellular cAMP.

cAMP does not stimulate protein kinase A in sensory neurons but acts directly to open Na⁺ channels in the plasma membrane to initiate membrane depolarization and nerve impulses.

The cGMP pathway

cGMP is also a second messenger. It is produced from guanosine triphosphate (GTP) by guanylate cyclase and degraded to GMP by a phosphodiesterase. Guanylate cyclases are activated by nitric oxide and peptide signaling molecules.

The best characterized role of cGMP is in photoreceptor rod cells of the retina, where it converts light signals to nerve impulses. Chapter 9, Sensory Organs: Vision and Hearing, in the eye section, provides a detailed description of this cell signaling process.

Phospholipase C–Ca²⁺ pathway

Another second messenger involved in intracellular signaling derives from the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂) present in the inner leaflet of the plasma membrane (Figure 3-7).

Figure 3-7 Phospholipase-protein kinase C-Ca²⁺ pathway

The hydrolysis of PIP₂ by the enzyme phospholipase C (PLC)—stimulated by a number of hormones and growth factors—produces two second messengers: diacylglycerol and inositol 1,4,5-trisphosphate (IP₃).

These two messengers stimulate two downstream signaling pathway cascades: protein kinase C and Ca²⁺ mobilization.

Two forms of PLC exist: PLC-β and PLC-γ. PLC-β is activated by G protein. PLC-γ contains SH2 domains that enable association with receptor protein tyrosine kinases. Tyrosine phosphorylation increases PLC-γ activity, which in turn stimulates the breakdown of PIP₂.

Diacylglycerol, derived from PIP₂ hydrolysis, activates members of the protein kinase C family (protein serine and threonine kinases).

Phorbol esters are tumor growth–promoting agents acting, like diacylglycerol, by stimulation of protein kinase C activities. Protein kinase C activates other intracellular targets such as protein kinases of the MAP kinase pathway to produce the phosphorylation of transcription factors leading to changes in gene expression and cell proliferation.

NF-κB transcription factor pathway

NF-κB is a transcription factor involved in immune responses in several cells and is stimulated by protein kinase C (Figure 3-8).

Figure 3-8 NF-κB transcription factor pathway

In its inactive state, the NF-κB protein heterodimer is bound to the inhibitory subunit I-κB and the complex is retained in the cytoplasm. The phosphorylation of I-κB—triggered by I-κB kinase—leads to the destruction of I-κB by the 26S proteasome and the release of NF-κB. The free NF-κB heterodimer translocates into the nucleus to activate gene transcription in response to immunologic and inflammatory signaling.

Ca²⁺-calmodulin pathway

Although the second messenger diacylglycerol remains associated with the plasma membrane, the other second messenger IP₃, derived from PIP₂, is released into the cytosol to activate ion pumps and free Ca²⁺ from intracellular storage sites. High cytosolic Ca²⁺ concentrations (from a basal level of 0.1 μM to an increased 1.0 μM concentration after cytosolic release) activate several Ca²⁺-dependent protein kinases and phosphatases.

Calmodulin is a Ca²⁺-dependent protein that is activated when the Ca²⁺ concentration increases to 0.5 μM. Ca²⁺-calmodulin complexes bind to a number of cytosolic target proteins to regulate cell responses. Note that Ca²⁺ is an important second messenger and that its intracellular concentration can be increased not only by its release from intracellular storage sites but also by increasing the entry of Ca²⁺ into the cell from the extracellular space.

MAP kinase pathway

This pathway involves evolutionarily conserved protein kinases (yeast to humans) with roles in cell growth and differentiation. MAP kinases are protein serine and threonine kinases activated by growth factors and other signaling molecules (Figure 3-9).

Figure 3-9 ERK-MAP kinase pathway

A well-characterized form of MAP kinase is the ERK family. Members of the ERK (for extracellular signal–regulated kinase) family act through either protein tyrosine kinase or G protein—associated receptors. Both cAMP and Ca²⁺-dependent pathways can stimulate or inhibit the ERK pathway in different cell types.

The activation of ERK is mediated by two protein kinases: Raf, a protein serine or threonine kinase, which, in turn, activates a second kinase called MEK (for MAP kinase or ERK kinase). Stimulation of a growth factor receptor leads to the activation of the GTP-binding protein Ras (for rat sarcoma virus), which interacts with Raf. Raf phosphorylates and activates MEK, which then activates ERK by phosphorylation of serine and threonine residues. ERK then phosphorylates nuclear and cytosolic target proteins.

In the nucleus, activated ERK phosphorylates the transcription factors Elk-1 (for E-26-like protein 1) and serum response factor (SRF), which recognize the regulatory sequence called serum response element (SRE).

In addition to ERK, mammalian cells contain two other MAP kinases called JNK and p38 MAP kinases. Cytokines and ultraviolet irradiation stimulate JNK and p38 MAP kinase activation mediated by small GTP-binding proteins different from Ras. These kinases are not activated by MEK but by a distinct dual kinase called MKK (for MAP kinase kinase).

A key element in the ERK pathway are the Ras proteins, a group of oncogenic proteins of tumor viruses that cause sarcomas in rats. Mutations in the Ras gene have been linked to human cancer. Ras proteins are guanine nucleotide–binding protein with functional properties similar to the G protein α subunits (activated by GTP and inactivated by guanosine diphosphate [GDP]).

A difference with G protein is that Ras proteins do not associate with βγ subunits. Ras is activated by guanine nucleotide exchange factors to facilitate the release of GDP in exchange for GTP. The activity of the Ras-GTP complex is terminated by GTP hydrolysis, which is stimulated by GTPase-activating proteins.

In human cancers, mutation of Ras genes results in a breakdown failure of GTP and, therefore, the mutated Ras protein remains continuously in the active GTP-bound form.

JAK-STAT pathway

The preceding MAP kinase pathway links the cell surface to the nucleus signaling mediated by a protein kinase cascade leading to the phosphorylation of transcription factors.

The JAK-STAT pathway provides a close connection between protein tyrosine kinases and transcription factors by directly affecting transcription factors (Figure 3-10).

Figure 3-10 JAK-phosphorylated STAT dimer pathway

STAT (for signal transducers and activators of transcription) proteins are transcription factors with an SH2 domain and are present in the cytoplasm in an inactive state. Stimulation of a receptor by ligand binding recruits STAT proteins, which bind to the cytoplasmic portion of receptor-associated JAK protein tyrosine kinase through their SH2 domain and become phosphorylated. Phosphorylated STAT proteins then dimerize and translocate into the nucleus, where they activate the transcription of target genes.

Transcription factor genes: SOX9

Genes encoding proteins that turn on (activate) or turn off (repress) other genes are called transcription factors. Many transcription factors have common DNA-binding domains and can also activate or repress a single target gene as well as other genes (a cascade effect). Therefore, mutations affecting genes encoding transcription factor have pleiotropic effects (Greek pleion, more; trope, a turning toward).

Examples of transcription factor genes include homeobox-containing genes, high mobility group (HMG)-box–containing genes, and the T-box family.

The HMG domain of Sox proteins can bend DNA, and facilitate the interaction of enhancers with a distantly located promoter region of a target gene. Several SOX genes act in different developmental pathways. For example, Sox9 protein is expressed in the gonadal ridges of both genders but is up-regulated in males and down-regulated in females before gonadal differentiation. Sox9 also regulates chondrogenesis and the expression of type II collagen (see Chapter 4, Connective Tissue). Mutations of the SOX9 gene cause skeletal defects (campomelic dysplasia), and sex reversal (XY females).

Stem cells: A multipotent cell population

Cells in the body show a remarkable range in ability to divide and grow. Some cells (for example, nerve cells and erythrocytes) reach a mature, differentiated state and usually do not divide. Such cells are referred to as postmitotic cells. Other cells, called stem cells, show continuous division throughout life (for example, epithelial cells lining the intestine and stem cells that give rise to the various blood cell types). Many other cells are intermediate between these two extremes and remain quiescent most of the time but can be triggered to divide by appropriate signals. Liver cells are an example. If the liver is damaged, cell division can be triggered to compensate for the lost cells.

Stem cells have three properties: self-renewal, proliferation, and differentiation. These properties depend on their specific microenvironment, called stem cell niche, that supplies the required factors for their development.

Stem cells have the potential to generate a large number of mature cells continuously throughout life. When stem cells divide by mitosis, some of the progeny differentiates into a specific cell type. Other progeny remains as stem cells (Figure 3-11). The intestinal epithelium, the epidermis of the skin, the hematopoietic system, and spermatogenic cells of the seminiferous epithelium share this property. We discuss in detail the significance of stem cells in each of these tissues in the appropriate chapters.

Figure 3-11 Properties of stem cells

Following stress and injury, other tissues, such as the liver, muscle, and the nervous system, can regenerate mature cells. For example, it has been shown that bone marrow stem cells can produce muscle tissue as well as hematopoietic tissue in an appropriate host system (see Chapter 7, Muscle Tissue). Cultured stem cells of the central nervous system are capable of hematopoiesis in transplanted irradiated mouse recipients.

Recall that embryonic stem cells, forming the inner cell mass (embryoblast) of the early embryo (the blastocyst), give rise to all the tissues and organs except the placenta. Embryonic stem cells provide an experimental source of medically useful differentiating tissues such as pancreatic islets for the treatment of diabetes, skin for the treatment of burns and wounds, regenerating cartilage for the treatment of arthritis, and endothelial cells for the repair of blood vessels affected by arteriosclerosis. A potential complication is that embryonic stem cells injected into mature mice develop an embryonic tumor called a teratoma.

In vitro cell proliferation, senescence, and telomerase

Cell culture techniques have been a powerful tool for examining the factors that regulate cell growth and for comparing the properties of normal and cancer cells.

Many cells grow in tissue culture, but some are much easier to grow than others. Culture medium contains salts, amino acids, vitamins, and a source of energy such as glucose. In addition, most cells require a number of hormones or growth factors for sustained culture and cell division. These factors are usually provided by addition of serum to the culture medium.

For some cell types the components supplied by serum have been identified, and these cells can be grown in serum-free, hormone and growth factor–supplemented medium. Some of these factors are hormones, such as insulin. A number of growth factors have been identified, for example, EGF, fibroblast growth factor (FGF), and PDGF.

When normal cells are placed in culture in the presence of adequate nutrients and growth factors, they will grow until they cover the bottom of the culture dish, forming a monolayer. Further cell division then ceases. This is called density-dependent inhibition of growth. The cells become quiescent but can be triggered to enter the cell cycle and divide again by an additional dose of growth factor or by replating at a lower cell density.

Cells cultured from a tissue can be kept growing and dividing by regularly replating the cells at lower density once they become confluent. After about 50 cell divisions, however, the cells begin to stop dividing and the cultures become senescent. The number of divisions at which this occurs depends on the age of the individual from which the initial cells were taken. Cells from an embryo will thus keep growing longer than cells taken from an adult.

In our discussion of mitosis (see Figure 1-53 in Chapter 1, Epithelium), we call attention to the role of telomerase, an enzyme that maintains the ends of chromosomes, or telomeres.

In normal cells, insufficient telomerase activity limits the number of mitotic divisions and forces the cell into senescence, defined as the finite capacity for cell division. Telomere shortening and the limited life span of a cell are regarded as potent tumor suppressor mechanisms. Most human tumors express human telomerase reverse transcriptase (hTERT). The ectopic expression of hTERT in primary human cells confers endless growth in culture. The use of telomerase inhibitors in cancer patients is currently being pursued.

Occasionally, cells that would normally stop growing become altered and appear to become immortal. Such cells are called a cell line. Cell lines are very useful experimentally and still show most of the phenotype and growth characteristics of the original cells.

An additional change known as transformation is associated with the potential for malignant growth. Transformed cells no longer show normal growth control and have many alterations, such as anchorage-independent growth. Normal cells can grow when anchored to a solid substrate.

Cells in culture can be transformed by chemical carcinogens or by infection with certain viruses (tumor viruses). Tumor viruses will also cause tumors in certain host animals, but in different species they may cause ordinary infections. Cancer cells cultured from tumors also show the characteristics of transformation. We will discuss at the end of this chapter the role of retroviruses in carcinogenesis.

Apoptosis, or programmed cell death

Cell death occurs by necrosis or apoptosis. Under normal physiologic conditions, cells deprived of survival factors, damaged, or senescent commit suicide through an orderly regulated cell death program called apoptosis (Greek apo, off; ptosis, fall).

Apoptosis (Figure 3-12) is different from necrosis. Necrosis is a nonphysiologic process that occurs after acute injury (for example, in an ischemic stroke). Necrotic cells lyse and release cytoplasmic and nuclear contents into the environment, thus triggering an inflammatory reaction.

Figure 3-12 Programmed cell death or apoptosis

Cells undergoing apoptosis lose intercellular adhesion, fragment the chromatin, and break down into small blebs called apoptotic bodies. Apoptotic bodies are phagocytosed by macrophages and inflammation does not occur.

Apoptotic cell death is observed during fetal development. For example, the formation of fingers and toes of the fetus requires the elimination by apoptosis of the tissue between them. During fetal development of the central nervous system, an excess of neurons, eliminated later by apoptosis, is required to establish appropriate connections or synapses between them (see Chapter 8, Nervous Tissue). Mature granulocytes in peripheral blood have a life span of 1 to 2 days before undergoing apoptosis. The clonal selection of T cells in the thymus (to eliminate self-reactive lymphocytes to prevent autoimmune diseases; see Chapter 10, Immune-Lymphatic System) and cellular immune responses involve apoptosis.

What a nematode worm told us about apoptosis

The genetic and molecular mechanisms of apoptosis emerged from studies of the nematode worm Caenorhabditis elegans, in which 131 cells are precisely killed and 959 remain. In this worm, four genes are required for the orderly cell death program: ced-3 (for cell death defective-3), ced-4, egl-1 (for egg laying-1), and ced-9. The products of the first three genes mediate cell death. The gene ced-9 is an inhibitor of apoptosis.

The proteins encoded by these four genes in the worm are found in vertebrates. Protein ced-3 is homologous to caspases, ced-4 corresponds to Apaf-1 (for apoptotic protease activating factor-1), ced-9 to Bcl-2 (for B-cell leukemia-2), and egl-1 is homologous to Bcl-2 homology region 3 (BH3)-only proteins.

External signals trigger apoptosis: Fas receptor/Fas ligand

External and internal signals determine cell apoptosis. External signals bind to cell surface receptors (for example, tumor necrosis factor-α and Fas ligand). Internal signals (for example, the release of cytochrome c from mitochondria) can trigger cell death.

Fas receptor (also known as APO-1 or CD95) is a cell membrane protein that belongs to the tumor necrosis factor (TNF) receptor family. Fas receptor has an intracellular cell death domain. Fas ligand binds to Fas receptor and causes its trimerization. Fas ligand initiates programmed cell death by binding to the Fas receptor and triggers a cell signaling cascade consisting of the sequential activation of procaspases into active caspases. The trimerized cell death domain recruits procaspase 8 through the FADD (for Fas-associated protein with death domain) adaptor and forms a DISC (for death-inducing signaling complex). DISC consists of Fas receptor, FADD, and procaspase 8.

Procaspase 8 autoactivated at DISC becomes active caspase 8. Active caspase 8 can do two things:

As we will discuss in Chapter 10, Immune-Lymphatic System, a cytotoxic T cell destroys a target cell (for example, a virus-infected cell) by first binding to the target cell and then releasing Fas ligand. Fas ligand binds to Fas receptor on the surface of the target cell and triggers the cell death cascade.

Caspases: Initiators and executioners of cell death

Caspases (for cysteine aspartic acid–specific proteases) exist as inactive precursors (procaspases), which are activated to produce directly or indirectly cellular morphologic changes during apoptosis.

Procaspases consist of two sub units (p10 and p20) and an N-terminal recruitment domain (see Figure 3-12). Activated caspases are heterotetramers consisting of two p10 sub units and two p20 sub units derived from two procaspases.

Caspases can be upstream initiators and downstream executioners. Upstream initiators are activated by the cell-death signal (for example, Fas ligand or TNF-α). Upstream initiator caspases activate downstream caspases, which directly mediate cell destruction.

Completion of the cell death process occurs when executioner caspases activate the DNA degradation machinery. Caspases cleave two DNA repair enzymes (poly-ADP-ribose polymerase [PARP], and DNA protein kinase), and unrestricted fragmentation of chromatin occurs.

As you realize, the key event in caspase-mediated cell death is the regulation of the activation of initiator caspases.

Upstream (initiator) procaspases include procaspases 8, 9, and 10 with along N-terminal prodomain called CARD (for caspase-recruiting domain). Downstream (executioner) procaspases comprise procaspases 3, 6, and 7 with a short N-terminal prodomain called DED (for death-effector domain).

Caspase activation takes place when a caspase-specific regulatory molecule (for example, FADD) binds to the CARD/DED domain. Caspase activation may become out of control and destroy the cell. To prevent this uncontrolled event, inhibitors of apoptosis are available to interact with modulators of cell death, thus preventing unregulated caspase activation.

Bcl-2 regulates the release of mitochondrial cytochrome c through Bax

Cytochrome c is a component of the mitochondria electron-transporting chain involved in the production of ATP, and also a trigger of the caspase cascade.

The cell death pathway can be activated when cytochrome c is released from the mitochondria into the cytoplasm. How does cytochrome c leave mitochondria? To answer this question, we need to consider aspects of members of the Bcl-2 family.

Bcl-2 family members can have proapoptotic or antiapoptotic activities. Bcl-2 and Bcl-xL have antiapoptotic activity. Bax, Bak, Bid, and Bad are proapoptotic proteins. Bcl-2 is associated with the outer mitochondrial membrane of viable cells and prevents Bax from punching holes in the outer mitochondrial membrane, causing cytochrome c to leak out. As you can see, a balance between proapoptotic Bax and antiapoptotic Bcl-2 proteins controls the release of cytochrome c.

In the cytoplasm, leaking cytochrome c, in the presence of ATP, soluble internal membrane proteins (SIMPs), and procaspase 9, binds to Apaf-1 to form a complex called an apoptosome.

The apoptosome determines the activation of caspase 9, an upstream initiator of apoptosis (Figure 3-13). Caspase 9 activates caspase 3 and caspase 7, leading to cell death.

Figure 3-13 Role of mitochondria in apoptosis

You can gather from this discussion that external activators such as Fas ligand and TNF-α, and the internal release of cytochrome c are two key triggers of apoptosis. However, AIF (for apoptosis-inducing factor) is a protein of the inter-mitochondrial membrane space that can be released into the cytoplasm, migrate to the nucleus, bind to DNA, and trigger cell destruction without participation of caspases.

Clinical significance of apoptosis: Apoptosis in the immune system

Mutations in the Fas receptor, Fas ligand, or caspase 10 genes can cause autoimmune lymphoproliferative syndrome (ALPS). ALPS is characterized by the accumulation of mature lymphocytes in lymph nodes and spleen causing lymphoadenopathy (enlargement of lymph nodes) and splenomegaly (enlargement of the spleen), and the existence of autoreactive lymphocyte clones producing autoimmune conditions such as hemolytic anemia (caused by destruction of red blood cells) and thrombocytopenia (reduced number of platelets).

Clinical significance of apoptosis: Neurodegenerative diseases

Neurologic diseases are examples of the mechanism of cell death. For example, an ischemic stroke can cause an acute neurologic disease in which necrosis and activation of caspase 1 are observed. Necrotic cell death occurs in the center of the infarction, where the damage is severe. Apoptosis may be observed at the periphery of the infarction, because the damage is not severe due to collateral blood circulation. Pharmacologic treatment with caspase inhibitors can reduce tissue damage leading to neurologic improvement.

Caspase activation is associated with the fatal progression of chronic neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) and Huntington’s disease are two examples.

ALS consists in the progressive loss of motor neurons in the brain, brainstem, and spinal cord. A mutation in the gene encoding superoxide dismutase 1 (SID1) has been identified in patients with familial ALS. Activated caspase 1 and caspase 3 have been found in spinal cord samples of patients with ALS. Motor neurons and axons die and reactive microglia and astrocytes are present. We come back to ALS in Chapter 9, Nervous Tissue.

Huntington’s disease is an autosomal dominant neurodegenerative disease characterized by a movement disorder (Huntington’s chorea). The disease is caused by a mutation in the protein huntingtin. Huntingtin protein fragments accumulate and aggregate in the neuronal nucleus and transcription of the caspase 1 gene is upregulated. Caspase 1 activates caspase 3 and both caspases cleave the allelic wild-type form of huntingtin, which becomes depleted. As the disease progresses, Bid is activated and releases mitochondrial cytochrome c. Apoptosomes are assembled and further caspase activation leads to neuronal death.

Three major cellular mechanisms are involved in proteolysis

In addition to the procaspase-caspase pathway activated by Fas ligand (see Figure 3-12), the intracellular degradation of residual or misfolded proteins (proteolysis) can occur by the classic endosomal-lysosomal pathway (see Figure 2-19), the apoptosis pathway (see Figure 3-12), and the ubiquitin-proteasome pathway (Figure 3-14). We have already seen that the endosomal-lysosomal mechanism operates within a membrane-bound acidic compartment. In contrast, the procaspase-caspase pathway and the ubiquitin-proteasome pathway carry out proteolysis in the cytosol.

Figure 3-14 Three proteolytic mechanisms

The ubiquitin–26S proteasome pathway involves four successive regulated steps:

The 26S proteasome is a giant (~2000 kd) multimeric protease present in the nucleus and cytoplasm. Structurally, the 26S proteasome consists of a barrel-shaped core capped by two structures that recognize ubiquitinated proteins. Protein degradation occurs within a chamber of the barrel-shaped core. Proteins degraded by the 26S proteasome include molecules involved in the regulation of the cell cycle (cyclins), transcription factors, and the processing of antigens I involved in the activation of inflammatory and immune responses.

Proto-oncogenes and oncogenes

Genes that cause cancer are called oncogenes (Greek onkos, bulk, mass; genos, birth). Most oncogenes originate from proto-oncogenes (Greek prõtos, first). Proto-oncogenes (Box 3-E) are involved in the four basic regulatory mechanisms of cell growth by expressing growth factors, growth factor receptors, signal transduction molecules, and nuclear transcription factors.

An oncogene results from the mutation of a proto-oncogene. Oncogenes express constantly active products leading to unregulated cell growth and differentiation. A cell becomes transformed when it changes from regulated to unregulated growth.

Although most animal viruses destroy the cells they infect, several types of viruses are able to establish a long-term infection, in which the cell is not killed. This stable virus–host cell interaction perpetuates the viral information in the cell, usually by direct insertion into cellular DNA.

The first oncogenes to be identified came from the study of retroviruses. All vertebrate animals, including humans, inherit genes related to retro viral genes and transmit them to their progeny. These are called endogenous proviruses, whereas those that infect a cell are called exogenous proviruses.

Cancer viruses isolated from every type of vertebrate animal induce a wide variety of tumors and belong to several virus types: RNA-containing tumor viruses, called retroviruses, and DNA-containing tumor viruses, including the polyomaviruses, the papillomaviruses, the adenoviruses, and the herpesviruses.

RNA-containing retroviruses have a distinct cell cycle. In the initial stages of infection, the viral RNA is copied into DNA by the viral enzyme reverse transcriptase. Once synthesized, the viral DNA molecule is transported into the nucleus and inserted randomly as a provirus at any one of the available sites of host chromosomal DNA. Proviruses contain signals for the regulation of their own viral genes, but such signals can be transmitted to the proto-oncogene, forcing it to produce larger than normal amounts of RNA and a protein.

Retroviruses and polyomaviruses have received the most attention because they carry one or two genes that have specific cancer-inducing properties: so-called viral oncogenes. Retroviruses and polyomaviruses like cellular genes, are subject to mutations. A group of such mutants of Rous sarcoma virus (RSV; species of origin: chicken) has proved useful for determining the role of the viral gene v-src. The src-like sequences in normal cells constitute a cellular gene called c-src, a proto-oncogene.

The viral src derives directly from the cellular src. A precursor of RSV seems to have acquired a copy of c-src during infection of a chicken cell. c-src is harmless but its close relative, v-src, causes tumors and transform cells after RSV infection. A chicken fibroblast produces about 50 times more src RNA and protein than an uninfected fibroblast containing only the c-src gene. The c-src gene assumed great significance when it was recognized that many other retroviruses carry oncogenes, often different from v-src. Each of these genes is also derived from a distinct, normal cellular precursor.

The classification of genes as proto-oncogenes is based on the understanding that mutant forms of these genes participate in the development of cancer (see Box 3-F). However, proto-oncogenes serve different biochemical functions in the control of normal growth and development. They can also undergo a variety of mutations that convert them to dominant genes capable of inducing cancers in the absence of viruses.

RSV-infected cells produce a 60-kd protein. This protein was identified as the product that the v-src gene uses to transform cells. It was designated p60^v-src. This protein can function as a protein kinase and, within a living cell, many proteins can be phosphorylated by Src kinase activity. The target for phosphorylation is tyrosine residues.

Cell transformation by the v-src oncogene causes a tenfold increase in total cellular phosphotyrosine in cellular target proteins restricted to the inner side of the cell membrane. Many other proteins encoded by proto-oncogenes or involved in control of cell growth function like the Src protein, such as protein kinases, are often specific for tyrosine.