21 Regulation of Cell Death in the Gastrointestinal Tract

1. INTRODUCTION

The gastrointestinal (GI) tract begins with the mouth, leads to the esophagus, and extends through the stomach, small intestine (including duodenum, jejunum, and ileum), and large intestine (divided into cecum and colon), to end at the anus. In addition, the GI tract includes three accessory organs: liver, gallbladder, and pancreas. The liver produces bile, a fluid containing molecules (bile acids) that help the digestion of lipids, and, via numerous canaliculi forming the biliary system, secretes it into the gallbladder, where it is stored and concentrated. Upon eating, bile is discharged into the small intestine. The pancreas is a dual-function gland, working as both an endocrine and an exocrine gland. The exocrine pancreas secretes pancreatic juice containing bicarbonate and several enzymes, including trypsin, chymotrypsin, lipase, and pancreatic amylase, into the small intestine. Both the liver and the pancreas aid in the digestive process.

The gastrointestinal epithelium is characterized by rapid proliferation of stem cells that differentiate to become mature cells. At the same rate, older and/or damaged cells are eliminated by apoptosis, and the resulting apoptotic bodies are shed into the lumen and/or engulfed by adjacent epithelial cells and subepithelial macrophages. This highly regulated balance between cell proliferation and apoptosis ensures the maintenance of tissue function and architecture. Conversely, alterations in the rate of cell proliferation or cell death result in the development of pathologic states. Indeed, apoptosis has been shown to play an important role in the pathophysiology of several gastrointestinal diseases. Many infectious and immune-mediated diseases, such as gastritis, viral hepatitis, and inflammatory bowel diseases, may be triggered by excessive cell

death, whereas prolonged cell survival due to apoptosis inhibition, together with unregulated proliferation, can promote cancer development. This chapter reviews the current knowledge of the role and mechanisms of apoptosis in the organs of the GI tract under physiologic and pathological conditions.

2. ESOPHAGUS

The esophageal epithelium is a nonkeratinized, stratified squamous epithelium, with scattered submucosal glands that produce mucus and provide lubrication. The esophageal epithelial cells normally undergo a rapid turnover to eliminate and replace cells mechanically and chemically damaged during the transit of food. New cells are generated by the division of stem cells located in the basal compartment of the squamous epithelium; at each cell division, these cells give rise to one stem cell (to maintain the stem cell pool) and one daughter cell, which differentiates into mature epithelial cell and eventually undergoes apoptosis after a number of divisions, ensuring a functional tissue homeostasis. However, when the balance between cell proliferation and cell death is lost, the epithelial integrity and architecture are altered, with serious pathological consequences. A common example is represented by a condition known as Barrett’s esophagus (BE), during which the squamous epithelium is transformed into a metaplastic simple columnar epithelium, which resembles that of gastric mucosa or of intestinal mucosa (vide infra). BE is considered a premalignant condition and is associated with an increased risk for the development of esophageal adenocarcinoma (ADCA). This pathology is often the consequence of chronic inflammation caused by gastroesophageal reflux disease (GERD), a condition characterized by backflow of the gastric contents into the

esophagus. The two major components of the reflux responsible for the development of BE are gastric acid and bile acids. Bile acids cause apoptosis and, in an acidic environment, they are able to rapidly induce oxidative stress and oxidative DNA damage. Therefore, esophageal cells exposed to the refluxate are initially subjected to a faster turnover, mainly controlled by the tumor suppressor genes p16 (also known as CDKN2A or p16[INK4]) and p53, which regulate cell cycle arrest and DNA damage-induced apoptosis, respectively. A persistent exposure ultimately increases the risk of genetic instability, resulting in clonal selection of a cell population bearing alterations in gene expression that promote increased cell division, apoptosis resistance, invasion, and metastasis. Indeed, normal squamous epithelium is sensitive to bile acid-induced apoptosis, whereas BE metaplastic cells become resistant. Consistently, inactivating mutations of p16 and p53 genes through promoter methylation, gene mutation, or loss of heterozygosity are common early events in the progression from BE to ADCA. Other factors contributing to increased apoptosis-resistance of metaplastic cells include overexpression of the antiapoptotic proteins Bcl-XL and Mcl-1, interleukin (IL)-6, and cyclo-oxygenase-2 (COX2); decreased cell-surface expression of Fas (CD95) and increased Fas ligand (CD95L) expression. In particular, acid exposure has been shown to increase COX2 expression through activation of both extracellular signalregulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways, which cause a significant increase in COX2 promoter activity. COX2 expression is also induced by the activation of nuclear factor kappa B (NF-κB), a transcription factor constitutively activated in chronic inflammatory conditions. Moreover, specific bile acids may also directly activate the PI3 kinase/Akt signaling pathway, which stimulates cell growth and inhibits apoptosis in Barrett’s adenocarcinoma cells, therefore promoting neoplastic progression of BE. Thus dysregulation of apoptosis plays a central role in the progression to a malignant phenotype.

3. STOMACH

The gastric epithelium is made up of a single layer of cells indented into numerous short gastric pits. The epithelium consists of only one cell type, the surface mucous cells, which secrete mucus to protect the stomach surface from digestive acid and enzymes. Beneath the gastric pits, the mucosa is filled with long contiguous tubular glands divisible into isthmus, neck, and base regions. The gastric glands consist primarily of

two cell types: (1) the acid-secreting parietal (oxyntic) cells, found mainly in the neck region; and (2) the pepsinogen-secreting chief cells, usually located in the base region. The glands also contain mucous neck cells (in the neck area) and stem cells, located at the top of the glands (isthmus), where they open into the pits. The stem cells (or progenitor cells) undergo frequent mitosis to propagate themselves and to generate new gland cells and surface mucous cells. The newly generated cells migrate either outward into the pit, mature into surface mucous cells, and proceed toward the surface where they are eventually eliminated, or inward to the neck region where they differentiate into mucous neck cells, parietal cells, and chief cells. The turnover of surface epithelial cells is fairly rapid, with the entire epithelium replaced within 3 to 5 days, whereas parietal and chief cells die at a lower frequency. Under normal conditions, surface mucous cells constantly undergo apoptosis, and this rapid self-renewal of the epithelium serves as a host defense mechanism to limit bacterial colonization. However, some bacteria have developed the capacity to evade the defense mechanisms by interacting with the host epithelium. The most remarkable example is Helicobacter pylori, a Gram-negative spiral bacterium that chronically infects up to 50% of the human population, the infection of which has been associated with severe gastric pathologies, including gastritis and peptic ulcer. Chronic H. pylori infection is also the strongest known risk factor for the development of gastric cancer. This microorganism is able to invade and colonize human stomach by directly interacting with gastric epithelial cells, resulting in alterations of cell cycle and apoptosis in the host cell. H. pylori inhibits apoptosis in the directly infected gastric epithelial cells to facilitate its persistence within the human stomach, contributing to pit hyperplasia and persistent infection of the stomach. At the same time, the chronic infection and subsequent inflammatory response lead to loss of uninfected parietal cells and chief cells, resulting in oxyntic atrophy and gastric metaplasia, both pathological conditions predisposing to gastric cancer. H. pylori infection results in activation of ERKand NF-κB–mediated prosurvival signaling pathways, leading to growth factor upregulation (in particular gastrin and COX2), gastric epithelial proliferation, cell–cell dissociation and increased cell motility, and over-expression of the antiapoptotic protein Mcl-1 in the gastric pits. These effects are mediated by the cytotoxin cytotoxin-associated antigen A (CagA), which is injected by the bacterium into the gastric epithelial cell. Consistently, infections with CagA-positive strains of H. pylori are associated with

the highest risk of developing gastric cancer. Chronic H. pylori infection also triggers a persistent immune response, resulting in chronic inflammation with production of inflammatory cytokines (especially IL-6 family cytokines) and oxygen-free radicals, the latter produced by polymorphonuclear cells and macrophages infiltrating the gastric mucosa. The inflammatory milieu favors the onset of genetic mutations via direct DNA damage and impaired DNA repair and predisposes to neoplastic transformation through inhibition of apoptosis, resistance to immune response, and stimulation of angiogenesis.

4. SMALL AND LARGE INTESTINE

The simple columnar epithelium of the intestine is made up of highly specialized cells (enterocytes or intestinal epithelial cells [IECs]) whose primary function is to absorb and transport nutrients across the epithelial lining while maintaining a physical barrier to the external environment. Scattered among the enterocytes are other cell types, including goblet cells (specialized in secretion of mucus to facilitate passage of material through the bowel), Paneth cells (similar to neutrophils and providing host defense against microbes), enteroendocrine cells, and occasional infiltrating lymphocytes and eosinophils. To help maintain a barrier, the epithelial cells are joined by tight junctions on their lateral borders, thus limiting the passage of luminal contents across the cell. Within the small intestine, efficient absorption is facilitated through finger-like projections called villi, which greatly enhance the surface area. Unlike the small intestine, the large intestine does not contain villi. Throughout the intestine, flask-like structures called crypts contain rapidly proliferative cells responsible for maintaining epithelial integrity through constant production of new cells. In the small intestine, the crypts are located around the base of the villi, and new cells generated from the stem cells located at the bottom of the crypt move up the crypt–villus axis while undergoing the differentiation process. In the colon, the new cells migrate from the crypts toward the table region. Under normal conditions, spontaneous apoptosis is observed at two locations: (1) at the base of the small intestinal crypts, where it is believed to occur to control the stem cell population by removing excess and/or damaged cells; and (2) at the top of the villi (in the small intestine) or toward the top of the colonic crypt (in the large intestine), where aging and/or damaged epithelial cells are eliminated mainly by an apoptotic process triggered by the loss of cell–matrix interactions

during the progressive detachment of the cell, a form of cell death referred to as anoikis.

The expression of several members of the Bcl-2 family has been studied throughout the normal intestinal tissue and has been found to correlate with the levels of spontaneous apoptosis. For example, the antiapoptotic protein Bcl-2 is strongly expressed at the base of the colonic crypts, where virtually no spontaneous apoptosis of stem cells occurs, whereas it is absent in the crypts of the small intestine, where levels of spontaneous apoptosis are significantly higher. Conversely, the proapoptotic proteins Bax and Bak are highly expressed in the crypts of the small intestine, but weakly expressed within the colonic crypts. The distribution of these proand antiapoptotic Bcl-2 proteins may explain, at least in part, the increased risk of developing cancer in the large intestine as compared with the small intestine. Spontaneous apoptosis is, indeed, more frequent in the small intestine and provides a tight regulation of stem-cell homeostasis, preventing the generation of hyperplastic crypts with higher disposition to neoplastic transformation.

To avoid compromising the epithelial integrity, the enterocytes have developed mechanisms to sustain the epithelial barrier function during spontaneous apoptosis. However, excessive apoptosis can lead to depletion of crypt stem cells, shortening of the crypt-villus axis due to inability to compensate the cell loss at the villus tip, and ultimately, epithelium destruction and intestinal atrophy, which is associated with several gastrointestinal diseases. This represents a significant therapeutic problem for the use of abdominal and pelvic radiotherapy and chemotherapy-based treatments of cancer patients, as these treatments are known to induce severe intestinal damage as a result of intestinal stemcell apoptosis. Although the precise mechanisms that regulate repair and survival of the intestinal crypt are still elusive, a recent study established a critical role for the BH3-only p53-upregulated modulator of apoptosis (PUMA) protein in radiation-induced apoptosis of intestinal progenitor and stem cells and subsequent intestinal damage (Figure 21-1). Moreover, high concentrations of cytokines such as tumor necrosis factoralpha (TNF-α) and interferon-γ, as found in an inflammatory milieu, can directly induce epithelial apoptosis and disrupt tight junction formation, thereby weakening barrier function. Indeed, dysregulated apoptosis and changes in enterocytic junctions have been involved in the pathogenesis of inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis. In patients with IBD, increased apoptosis is found in the acute inflammatory sites throughout the entire

Villus

Crypt

Crypt base stem cells

Migration

Paneth cells

crypt–villus axis. This increased epithelial apoptosis is caused by chronically activated lamina propria T lymphocytes of the intestinal mucosa, which can directly kill the intestinal epithelial cells mainly via the Fas/FasL pathway and also produce high levels of proinflammatory cytokines such as TNF-α, IL-6, and interferon-γ, resulting in chronic mucosal inflammation and colonic tissue damage. Moreover, recent studies demonstrated that constant interfacing with microbes in the gut lumen results in endoplasmic reticulum (ER) stress and triggers a consequent unfolded protein response in the intestinal epithelial cells to restore ER homeostasis. Mutations in one key mediator of this ER stress response, X-box- binding protein 1 (XBP1), have been associated with increased apoptosis and development of IBD, suggesting that ER stress-mediated apoptosis plays a crucial role in the pathogenesis of IBD and that intestinal epithelial cells may perform homeostatic functions in the gut in addition to the immune cells (Figure 21-2). Persistent intestinal epithelial cell apoptosis eventually leads to disruption of the epithelial barrier function, facilitating the invasion of pathogenic microorganisms.

Conversely, an imbalance between cell proliferation and apoptosis in favor of proliferation predispose to the development of colorectal carcinoma. This cancer progresses through a multistep transformation of normal colonic epithelium to an adenomatous polyp and, ultimately, to invasive carcinoma, characterized by an accumulation of genetic alterations leading to an increasingly malignant phenotype. These mutations generally affect genes regulating cell proliferation and apoptosis in cells that ultimately acquire resistance to cell death and accumulate at the top of the crypt and surface epithelium, contributing to neoplastic transformation. Indeed, spontaneous apoptosis is progressively decreased as the colonic cell progresses from normal epithelium to sporadic adenoma to carcinoma. One of the most commonly mutated genes involved in regulation of cell cycle and apoptosis is p53, which is absent or mutated in 75% to 85% of all human colon cancers. Mutations in the p53 gene occur late in the adenoma-to-carcinoma sequence of colon cancer progression and may allow the growing tumor with multiple genetic alterations to evade cell cycle arrest and apoptosis. Induction of wild-type

Crypt

Surface epithelium

Shedding cells

Stem cells

Efficient unfolded

Protein response ER homeostasis

Defective unfolded Unresolved stress Protein response

Inflammation (No XBP1) Enterocyte apoptosis

IBD

p53 results in both cell cycle arrest by transcriptional upregulation of the cyclin kinase-dependent cell cycle inhibitor p21Waf1/Cip1 and apoptosis by upregulation of proapoptotic genes and direct induction of mitochondrial permeabilization via activation of Bax. Another genetic change often observed in colorectal carcinoma is over-expression of Bcl-2, which is no longer restricted to the crypt base, but it becomes detectable throughout the entire malignant epithelium. Bcl-2 expression increases only in the early stages of the progression from adenoma to carcinoma, contributing to the inhibition of apoptosis during the initial stages of tumorigenesis, and decreases thereafter. However, apoptosis remains impaired even in the presence of reduced Bcl-2 due to the onset of other antiapoptotic changes, including p53 mutations and over-expression of Bcl-XL. Overexpression of cellular FLIP (cFLIP), a potent inhibitor of death receptor-mediated apoptosis, is also a frequent event in the development of colon carcinoma, contributing to the progression from adenoma to carcinoma and increasing resistance to chemotherapy-induced apoptosis. In addition to inhibiting apoptosis, cFLIP has also

been shown to directly activate ERK-mediated survival pathways and to promote tumorigenesis by activating the Wnt/β-catenin pathway. Other genes involved in the regulation of cell proliferation and/or apoptosis are also commonly mutated in colorectal carcinomas. Among those, inactivating mutations of both alleles of the adenomatous polyposis coli (APC) gene, a tumor suppressor involved in regulation of the adherens junction protein β-catenin, are a frequent early event in the development of sporadic colorectal cancers. Mutations in the K-ras proto-oncogene also occur early in the adenoma stage and can increase proliferation and inhibit apoptosis. Finally, death receptor–mediated apoptosis, in particular Fasand TNF-related apoptosis-inducing ligand (TRAIL)–mediated apoptosis, is crucial to eliminate cells bearing genetic alterations by the immune cells. However, colon cancer cells have been found to express Fas ligand early in the adenoma-to-carcinoma sequence, which allows them to eliminate Fas-expressing lymphocytes and create sites of immune privilege. Moreover, despite the expression of Fas, most colon cancer cells are resistant to Fas-mediated apoptosis due