Cell Death and Cell Renewal 18 Cell Death and Cell Renewal

18 Cell Death and Cell Renewal Programmed Cell Death Stem Cells and the Maintenance of Adult Tissues Pluripotent Stem Cells, Cellular Reprogramming and Regenerative Medicine

Most cell death occurs by a normal process of programmed cell death. Introduction Cell death and proliferation are balanced throughout the life of multicellular organisms. Animal development involves cell proliferation and differentiation and also cell death. Most cell death occurs by a normal process of programmed cell death.

Introduction Most tissues have stem cells that can replace cells that have been lost. Abnormalities of cell death are involved in cancers, autoimmune diseases, and neurodegenerative disorders. The ability of stem cells to proliferate and differentiate makes them a promising mechanism for replacing damaged tissues.

Programmed cell death is carefully regulated. In adults, it balances cell proliferation and maintains constant cell numbers. It also eliminates damaged and potentially dangerous cells (e.g., virus- infected cells).

Programmed Cell Death During development, programmed cell death plays a key role by eliminating unwanted cells from many tissues. Examples: Elimination of larval tissues during amphibian and insect metamorphosis. Elimination of tissue between the digits in the formation of fingers and toes.

Programmed Cell Death In development of the mammalian nervous system, up to 50% of developing neurons are eliminated by programmed cell death. Those that survive have made the correct connections with their target cells, which secrete growth factors that signal cell survival by blocking the neuronal cell death program. 7

Necrosis: Accidental cell death from acute injury. Programmed Cell Death Necrosis: Accidental cell death from acute injury. Apoptosis: Programmed cell death; an active process. Characterized by: DNA fragmentation Chromatin condensation Fragmentation of the nucleus and cell

Figure 18.1 Apoptosis (Part 1)

Figure 18.1 Apoptosis (Part 2)

Figure 18.1 Apoptosis (Part 3)

Programmed Cell Death Apoptotic cells and cell fragments are recognized and phagocytosed by macrophages and neighboring cells, and rapidly removed from tissues. Necrotic cells swell and lyse; the contents are released into the extracellular space and cause inflammation.

Apoptotic cells express “eat me” signals, such as phosphatidylserine. Programmed Cell Death Apoptotic cells express “eat me” signals, such as phosphatidylserine. In normal cells, phosphatidylserine is restricted to the inner leaflet of the plasma membrane.

Figure 18.2 Phagocytosis of apoptotic cells

C. elegans development includes the death of 131 specific cells. Programmed Cell Death Studies of C. elegans by the Robert Horvitz lab identified three genes with key roles in apoptosis. C. elegans development includes the death of 131 specific cells. Their experiments used mutant strains in which the cell death did not occur.

Key Experiment, Ch. 18, p. 694 (2)

The genes ced-3 and ced-4 are required for developmental cell death. Programmed Cell Death The genes ced-3 and ced-4 are required for developmental cell death. A third gene, ced-9, is a negative regulator. These genes are the central regulators and effectors of apoptosis that are highly conserved in evolution.

Figure 18.3 Programmed cell death in C. elegans

Ced-3 is the prototype of the caspases family of proteases. Programmed Cell Death Ced-3 is the prototype of the caspases family of proteases. Caspases have cysteine (C) residues at their active sites and cleave after aspartic acid (Asp) residues in their substrate proteins.

Programmed Cell Death Caspases are the ultimate executioners of programmed cell death by cleaving over 100 different target proteins. Activation of an initiator caspase starts a chain reaction of caspase activation leading to death of the cell.

Figure 18.4 Caspase targets

Programmed Cell Death Caspases are synthesized as inactive precursors (procaspases) that convert to active forms by proteolytic cleavage, catalyzed by other caspases. Initiator caspases are activated in response to various signals. They then cleave and activate effector caspases, which digest the cellular target proteins.

Programmed Cell Death ced-9 in C. elegans is closely related to the mammalian gene bcl-2, which was first identified as an oncogene. Bcl-2 inhibits apoptosis. Cancer cells are unable to undergo apoptosis.

Some inhibit apoptosis; others induce caspase activation. Programmed Cell Death Mammals encode about 20 proteins related to Bcl-2, in three functional groups. Some inhibit apoptosis; others induce caspase activation. The fate of the cell is determined by the balance of activity of proapoptotic and antiapoptotic Bcl-2 family members.

Figure 18.5 The Bcl-2 family

They are inhibited by antiapoptotic Bcl-2. Programmed Cell Death Bax and Bak are the downstream effectors that directly induce apoptosis. They are inhibited by antiapoptotic Bcl-2. BH3-only members are upstream; when activated by cell death signals, they antagonize the Bcl-2 family and activate Bax and Bak. 26

Figure 18.6 Regulatory interactions between Bcl-2 family members

Programmed Cell Death In mammal cells, Bcl-2 proteins act at mitochondria, which play a central role in controlling programmed cell death. Bax and Bak induce release of cytochrome c which triggers caspase activation.

Programmed Cell Death Caspase-9 is activated by forming a complex with Apaf-1 and cytochrome c in a complex called the apoptosome. Under normal conditions, cytochrome c is in the mitochondrial intermembrane space; Apaf-1 and caspase-9 are in the cytosol, so caspase-9 remains inactive.

Figure 18.7 The mitochondrial pathway of apoptosis (Part 1)

Figure 18.7 The mitochondrial pathway of apoptosis (Part 2)

Programmed Cell Death Caspases are also regulated by the IAP (inhibitor of apoptosis) family. They either inhibit caspase activity or target caspases for ubiquitylation and degradation in a proteasome. In Drosophila, initiator caspases are chronically activated but held in check by IAPs.

Figure 18.8 Regulation of caspases by IAPs in Drosophila

Intrinsic pathways are activated by DNA damage and other cell stress. Programmed Cell Death Regulation of programmed cell death is mediated by integrated signaling pathways; some induce cell death and others promote cell survival. Intrinsic pathways are activated by DNA damage and other cell stress. Extrinsic pathways are activated by signals from other cells.

Programmed Cell Death DNA damage can lead to cancer development; it is a principal trigger of programmed cell death. A major pathway leading to cell cycle arrest in response to DNA damage is mediated by the transcription factor p53.

Programmed Cell Death ATM and Chk2 protein kinases phosphorylate and stabilize p53, resulting in rapid increases in p53 levels. p53 then activates transcription of genes encoding the proapoptotic BH3-only proteins PUMA and Noxa, leading to cell death. 36

Figure 18.9 Role of p53 in DNA damage-induced apoptosis

Akt phosphorylates a number of proteins that regulate apoptosis. Programmed Cell Death A major signaling pathway that promotes cell survival is initiated by PI 3-kinase, which phosphorylates PIP2 to form PIP3, which activates the serine/ threonine kinase Akt. Akt phosphorylates a number of proteins that regulate apoptosis. 38

Programmed Cell Death Phosphorylation of the BH3-only protein Bad and FOXO transcription factors maintain them in an inactive state. In the absence of Akt signaling, Bad promotes apoptosis and FOXO stimulates transcription of another proapoptotic BH3-only protein, Bim. 39

Figure 18.10 The PI 3-kinase pathway and cell survival

Receptors activate an initiator caspase, caspase-8. Programmed Cell Death Extrinsic pathway: Polypeptides in the tumor necrosis factor (TNF) family are the signals. Receptors activate an initiator caspase, caspase-8. Caspase-8 can cleave and activate effector caspases and Bid, which leads to activation of caspase-9.

Figure 18.11 Cell death receptors

Programmed Cell Death Programmed cell death can also occur by non-apoptotic mechanisms such as autophagy. In normal cells, autophagy is a mechanism for gradual turnover of cell components. In starvation conditions, degradation of components provides energy and recycles materials.

Programmed Cell Death Autophagy and apoptosis both eliminate larval tissue in metamorphosis of Drosophila. Autophagic cell death does not require caspases; dying cells are characterized by an accumulation of lysosomes.

Programmed Cell Death Some forms of necrosis (necroptosis) can be a programmed cell response to stimuli such as infection or DNA damage. Regulated necrosis may provide an alternative pathway of cell death if apoptosis does not occur.

Programmed Cell Death Stimulation of the TNF receptor leads to cell death by necroptosis as well as apoptosis. Receptor interacting protein kinase-3 (RIPK3) is stimulated and MLKL is phosphorylated. Phosphorylated MLKL forms oligomers that disrupt the plasma membrane, causing cell death by necroptosis. 46

Figure 18.12 Necroptosis

Stem Cells and the Maintenance of Adult Tissues In early development, cells proliferate rapidly, then differentiate to form the specialized cells of tissues and organs. To maintain a constant number of cells in adult tissues, cell death must be balanced by cell proliferation.

Stem Cells and the Maintenance of Adult Tissues Most differentiated cells in adult animals are no longer capable of proliferation. If these cells are lost they are replaced by proliferation of cells derived from self-renewing stem cells.

Stem Cells and the Maintenance of Adult Tissues Some differentiated cells retain the ability to proliferate as needed, to repair damaged tissue throughout the life of the organism. Fibroblasts in connective tissue can proliferate quickly in response to platelet-derived growth factor (PDGF) released at the site of a wound.

Figure 18.13 A skin fibroblast

Stem Cells and the Maintenance of Adult Tissues Endothelial cells that line blood vessels can proliferate to form new blood vessels for repair and regrowth of damaged tissue. Proliferation is triggered by vascular endothelial growth factor (VEGF), which is produced by cells that lack oxygen.

Figure 18.14 Endothelial cells

Figure 18.15 Proliferation of endothelial cells

Stem Cells and the Maintenance of Adult Tissues The epithelial cells of some internal organs are also able to proliferate to replace damaged tissue. Liver cells, normally arrested in the G0 phase of the cell cycle, are stimulated to proliferate if large numbers of liver cells are lost (e.g., by surgical removal).

Figure 18.16 Liver regeneration

Stem Cells and the Maintenance of Adult Tissues Stem cells are less differentiated, self- renewing cells present in most adult tissues. They retain the capacity to proliferate and replace differentiated cells throughout the lifetime of an animal.

Stem Cells and the Maintenance of Adult Tissues The key property of stem cells: They divide to produce one daughter cell that remains a stem cell and one daughter cell that divides and differentiates. Stem cells are self-renewing and serve as a source of differentiated cells throughout life.

Figure 18.17 Stem cell proliferation

Stem Cells and the Maintenance of Adult Tissues Many types of cells have short life spans and must be continually replaced by proliferation of stem cells: Blood cells, sperm, epithelial cells of the skin and in the lining of the digestive tract.

Stem Cells and the Maintenance of Adult Tissues Hematopoietic (blood-forming) stem cells were the first to be identified. There are several types of blood cells with specialized functions; all are derived from the same population of stem cells. 100 billion human blood cells are lost every day and are continually produced from stem cells in the bone marrow.

Figure 18.18 Formation of blood cells

Stem Cells and the Maintenance of Adult Tissues Epithelial cells that line the intestines live only a few days before they die by apoptosis. New cells are derived from the continuous but slow division of stem cells at the bottom of intestinal crypts. New cells proliferate for three to four cell divisions and then differentiate.

Figure 18.19 Renewal of the intestinal epithelium (Part 1)

Figure 18.19 Renewal of the intestinal epithelium (Part 2)

Figure 18.19 Renewal of the intestinal epithelium (Part 3)

Stem Cells and the Maintenance of Adult Tissues Skin and hair are also renewed by stem cells. The epidermis, hair follicles, and sebaceous glands are all maintained by their own stem cells.

Figure 18.20 Stem cells of the skin (Part 1)

Figure 18.20 Stem cells of the skin (Part 2)

Stem Cells and the Maintenance of Adult Tissues Stem cells also play a role in repair of damaged tissue. Skeletal muscle normally has little cell turnover but can regenerate rapidly in response to injury or exercise. Satellite cells (stem cells of adult muscle) are normally arrested in G0, but proliferate in response to injury.

Figure 18.21 Muscle satellite cells (Part 1)

Figure 18.21 Muscle satellite cells (Part 2)

Stem Cells and the Maintenance of Adult Tissues Most adult tissues have stem cells, which reside in distinct microenvironments or niches (e.g., intestinal crypts). Niches provide the signals that maintain stem cells throughout life and control the balance between self-renewal and differentiation.

Stem Cells and the Maintenance of Adult Tissues Identification of stem cells and their niches is a major challenge in stem cell biology. Stem cells in intestinal crypts were first identified by Clevers et al. in 2007. Wnt polypeptides secreted by adjacent epithelial cells and fibroblasts control proliferation of these stem cells. 74

Figure 18.22 The intestinal stem cell niche

Stem Cells and the Maintenance of Adult Tissues Adult stem cells have potential utility in clinical medicine. In principal, stem cells could be used to replace damaged tissue and treat a variety of disorders, such as diabetes, muscular dystrophy, Parkinson’s or Alzheimer’s.

Stem Cells and the Maintenance of Adult Tissues Hematopoietic stem cell transplantation (bone marrow transplantation) is already important in the treatment of many cancers to replace cells damaged by toxic chemotherapy drugs. 77

Figure 18.23 Hematopoietic stem cell transplantation

Stem Cells and the Maintenance of Adult Tissues Epithelial stem cells are used in the form of skin grafts to treat burns, wounds, and ulcers. Epidermal skin cells can be cultured and then transferred to the patient. Because the patient’s own skin is used, it eliminates the problem of rejection by the immune system.

Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine Embryonic stem cells can be grown indefinitely as pure stem cell populations that have pluripotency— the capacity to develop into all of the different types of cells in adult tissues. Thus, there is enormous interest in embryonic stem cells for both basic research and clinical applications.

Embryonic stem cells were first cultured from mouse embryos in 1981. Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine Embryonic stem cells were first cultured from mouse embryos in 1981. They can be propagated indefinitely and are an important experimental tool: They can introduce altered genes into mice. They provide a model system to study cell differentiation.

Figure 18.24 Culture of mammalian embryonic stem cells

Key Experiment, Ch. 18, p. 713 (2)

Human embryonic stem cell lines were first established in 1998. Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine Human embryonic stem cell lines were first established in 1998. This raised the possibility of using embryonic stem cells in clinical transplantation therapies.

If LIF is removed, the cells aggregate and differentiate. Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine Mouse embryonic stem cells are grown with growth factor LIF to maintain the cells in the undifferentiated state. If LIF is removed, the cells aggregate and differentiate. Stem cells will differentiate along specific pathways if appropriate growth factors are added.

Figure 18.25 Differentiation of embryonic stem cells

Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine Mouse and human stem cells have been used to develop cardiomyocytes, neurons, and insulin-producing pancreatic β cells. These cells have been used for treatment of mouse models of various diseases.

In 1997, Ian Wilmut and colleagues cloned Dolly the sheep. Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine In 1997, Ian Wilmut and colleagues cloned Dolly the sheep. Dolly arose by a process called somatic cell nuclear transfer. Other mammals have since been cloned, but it is a difficult and inefficient process.

Figure 18.26 Cloning by somatic cell nuclear transfer

This would bypass the problem of tissue rejection. Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine In therapeutic cloning, a nucleus from an adult human cell would be transferred to an enucleated egg. The resulting embryo could produce differentiated cells for transplantation therapy. This would bypass the problem of tissue rejection.

Figure 18.27 Therapeutic cloning (Part 1)

Figure 18.27 Therapeutic cloning (Part 2)

Problems to be overcome: Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine Problems to be overcome: Low efficiency of generating embryos by somatic cell nuclear transfer (1 or 2% of embryos survive). Ethical concerns with respect to the possibility of cloning human beings (reproductive cloning) and with respect to the destruction of embryos.

Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine These difficulties may be overcome by using induced pluripotent stem cells: reprogramming somatic cells to resemble embryonic stem cells. Only four transcription factors introduced by retrovirus vectors are needed to reprogram adult mouse somatic cells.

Figure 18.28 Induced pluripotent stem cells

Adult human fibroblasts can also be reprogrammed to pluripotency. Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine Adult human fibroblasts can also be reprogrammed to pluripotency. This provides a new route to the derivation of pluripotent stem cells for use in transplantation therapy. 96

Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine Several combinations of transcription factors have recently been shown to induce pluripotency. They activate a transcriptional program that is also expressed in embryonic cells. Transcription factors Oct4, Sox2, and Nanog play central roles in this program.

Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine These factors form an autoregulatory loop, positively regulating each other’s expression. They also activate other genes that maintain the pluripotent state, while repressing genes that enable differentiation.

Figure 18.29 Pluripotency transcriptional program

Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine The positive autoregulation maintains pluripotency while allowing the cells to undergo differentiation. This helps overcome major obstacles: transcription factors originally used to reprogram fibroblasts can act as oncogenes, and the retroviral genes can also cause mutations.

This would bypass the need for pluripotent stem cells. Pluripotent Stem Cells, Cellular Reprogramming, and Regenerative Medicine Transdifferentiation: reprogramming somatic cells into other types of differentiated cells (e.g., fibroblasts to muscle cells or neurons). This would bypass the need for pluripotent stem cells. Mouse fibroblasts have been turned into heart muscle cells and nerve cells using only three transcription factors.

Figure 18.30 Transdifferentiation