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Rao JN, Wang JY. Regulation of Gastrointestinal Mucosal Growth. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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Regulation of Gastrointestinal Mucosal Growth.

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Peptide Growth Factors in GI Mucosal Growth

The intestinal tissues express a variety of peptide growth factors that modulate several functional properties of different intestinal cell populations, including the intestinal epithelium and lamina propria cell populations. These peptide growth factors are characterized by relatively low molecular weight of less than 25 kDa, and they generally exert their effects through binding to specific high-affinity cell-surface receptors present on their respective target cells [95,97,174,175]. In contrast to classical peptide hormones that are released to circulatory system for delivery to distant target organs or cells, peptide growth factors tend to act locally on adjacent cells (paracrine or juxtacrine action) or on the same cells that have expressed the peptide factors (autocrine action) (Figure 9). Although the full variety of peptide growth factors that are implicated in the control of the intestinal epithelium and nonepithelial compartment of the intestine remains to be demonstrated, an increasing body of evidence shows the diversity of these peptides and their importance in the regulation of GI mucosal growth. These peptides include members of epidermal growth factor (EGF) family, the transforming growth factor-β (TGF-β) family, fibroblast growth factor (FGF) family, the insulin-like growth factor (IGF) family, the trefoil factor family (TFF), and few other peptides described in Table 1. These growth factors are generally produced by intestinal mesenchymal tissues and regulate epithelium and nonepithelial tissue functions, such as cellular proliferation, differentiation, migration, and cytoprotection.

FIGURE 9. Ectodomain shedding of EGFR ligands and its consequences for signaling.

FIGURE 9

Ectodomain shedding of EGFR ligands and its consequences for signaling. Membrane-bound molecules can activate the EGFR of neighbor cells (juxtacrine mechanism). Following proteolytic release, the soluble EGF module activates the EGFR of neighbor cells (more...)

TABLE 1. Peptide growth factors and their target cells in the gastrointestinal system.

TABLE 1

Peptide growth factors and their target cells in the gastrointestinal system.

As shown in Table 1, the constituents of this network possess multiple functional properties and exhibit pleiotropism in their cellular sources and targets; they are highly redundant in several dimensions. For example, each cell type appears to produce more than one peptide growth factors, whereas each peptide may be produced by multiple different cell populations within the intestinal tract. In addition, most cell populations express receptors specific for more than one peptide growth factors, while receptors for a single growth factor are present on multiple cell types. Furthermore, functional effects of a certain growth factor are modulated by the co-presence of other factors, and structurally related multiple members of a peptide growth factor may interact with a single receptor. In this chapter, we briefly describe the roles and mechanisms of peptide growth factors of EGF family, TGF-β family, IGF family, and FGF family in the regulation of GI mucosal growth.

EGF FAMILY

The EGF family consists of different peptides including EGF, TGF-α, amphiregulin, heparin-binding EGF (HB-EGF), betacellulin, epiregulin, and neuregulin. All of them exhibit mitogenic activity upon binding to four different high-affinity receptors: EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4. Members of the EGF family are characterized by three properties: (i) the ability to bind to the EGF receptors, (ii) the capacity to mimic the biological activities of EGF, and (iii) amino acid sequence similarity to EGF. In fact, the overall identity of sequences within all members of the EGF family is approximately 20% [176]. EGF and TGF-α are the prototype members of the EGF family. In the GI tract, EGF is produced in submaxillary glands and Brunner's glands in the duodenum. Small amounts of EGF are also produced within exocrine pancreas; it is also present in gastric juice and within the intestinal lumen. In the early postnatal life, the breast milk is also a major source of EGF [177]. EGF produces a variety of biological responses, most of which are involved in the regulation of cell proliferation and/or differentiation, cell movement, and survival in epidermal as well as epithelial tissues [177,178]. The EGFR/ErbB1 is well characterized and possibly the most biologically important receptor for EGF family members in the GI epithelium, although additional EGFRs are also identified in normal and fetal GI tissues. In the GI epithelium, EGF promotes development of the intestinal mucosa, promotes cell proliferation and differentiation, and also enhances mucosal healing after injury [27,179].

EGF is a potent stimulator of cell division in epithelial and nonepithelial cell types in the GI tract, and specific EGF receptors are widely distributed in many cell types. The widespread distribution of EGFRs, including a variety of cells committed to terminal differentiation, suggests that EGF and/or another member of the EGF family have a range of biological functions beyond their mitogenic activity. EGF is shown to modulate the expression of enzymes involved in the production of cellular polyamines, to up-regulate intestinal electrolyte and nutrient transport in the enterocyte, to stimulate expression of brush border enzymes, to attenuate intestinal damage, and to enhance GI mucosal healing after injury [180,181]. For example, Berlanga-Acosta et al. [182] reported that the continuously infusing EGF for long periods (14 days) by implanting osmatic minipump subcutaneously in rats increases intestinal epithelial cell proliferation and induces the crypt and villus areas in the small intestine. This stimulatory effect of EGF occurs as early as 24 h after EGF infusion, but its maximal stimulation is observed 6 days thereafter. Another study conducted by Cellini et al. [183] revealed that infusing EGF directly into intra-amniotic fluid in pregnant rats during the last 8 days of gestational period results in a significant increase in fetal weight, intestinal villus height, and DNA synthesis within the crypts.

Recently, Kang et al. [184] demonstrated that oral administration of recombinant EGF together with probiotic bacteria for 14 days stimulates intestinal development and reduces the incidence of pathogen infection and diarrhea in pigs. Intestinal length, jejunal and duodenal villus heights are greater in animals treated with EGF and probiotic bacteria compared to controls and animals treated with EGF or probiotic bacteria alone. Immunohistochemistry with antibodies against proliferating cell nuclear antigen (PCNA) revealed that the proliferation of intestinal cells was significantly greater in the EGF+ probiotic bacteria administered group. Studies conducted by these authors in their earlier observations [186] also showed that the administration of recombinant EGF increased mean villus height, crypt depth, and enterocyte proliferation compared to control mice fed with phosphate-buffered saline. EGF also has a beneficial effect on the intestinal development and growth of newly weaned mice. Based on these observations, it has been suggested that the combination of EGF with probiotic approach could provide the possibility for formulating dietary supplements for children during their weaning transition stages [184,185].

TGF-α has been characterized as a product of many cell types, including most epithelial cells in the GI tract. The mRNA and protein levels of TGF-α have been identified in human and rodent stomach, small intestinal and colonic epithelium. The biological activities of TGF-α are mediated through the same receptor as EGF. In 1999, Montaner et al. [188] showed the immunolocalization of TGF-α in the rat gastroduodenal region. In the stomach, the surface and gastric pit cells showed increased immunostaining of TGF-α in the cytoplasm and basolateral and apical membranes. In the duodenum, the enterocytes co-express both TGF-α and EGFR in the supranuclear area. These immunolocalization studies demonstrate that the co-expression of TGF-α and EGFR in the rat GI tract suggests a functional role in the establishment and maintenance of the epithelial renewal. Simultaneously, in vivo studies conducted in rats showed that TGF-α administration resulted in a significant increase in mucosal weight, DNA and protein content, and villus height in jejunum and ileum and also induced crypt depth in jejunum and ileum. Important functions of TGF-α in the GI tract include trophic effects on mucosa, stimulator of epithelial and nonepithelial cell proliferation, alterations of expression involved in the mucosal development, promotion of growth of intestinal neoplasia, enhancement of epithelial restitution, and stimulation of angiogenesis.

The biological actions of EGF and its family of peptides are mediated via interaction with EGFR, which is detected throughout the fetal and neonatal GI tract. EGFR is predominantly expressed in the villus tip cells in young pigs still fed on maternal milk, but after weaning, it was more concentrated in Brunner gland and in goblet cells [189]. Kuwada et al. [190] reported that total cellular EGFR protein and mRNA transcript levels are relatively unchanged during cell differentiation in vitro, but the expression of surface EGFR and patterns of expressed EGF-ligand changed significantly. It is likely that EGFR system is regulated during intestinal epithelial cell differentiation primarily at the level of ligand expression. In addition, it has been shown that integrin α5/β1 mediates fibronectin-induced epithelial cell proliferation through the activation of the EGFR.

In another study conducted by Duh et al. [191], they have demonstrated the specific roles of EGFR during embryonic gut development by using EGFR knock-out mouse model. EGFR activation appears to accelerate the maturation rate of goblet cells and to induce differential crypt/villus proliferation pattern in early embryonic mouse gut. Moreover, the human milk induces fetal small intestinal cell proliferation through the mechanism involving different tyrosine kinase signaling pathways via the EGFR [192] (Figure 9). Taylor et al. [193] showed that the activation of EGFR enhances intestinal adaptation after massive small-bowel resection as indicated by taller villi, deeper crypt areas, and augmented enterocyte proliferation. Defective EGFR signaling in mutant mice exhibits increased apoptosis and reduction in bcl-2 family gene expression [194,195].

TGF-β FAMILY

TGF-β is a family of structurally homologous dimeric proteins consisting of at least three isoforms, TGF-β1, TGF-β2, and TGF-β3 [196]. The prototypic member of the TGF-β family in the GI tract is TGF-β1, although other two isoforms of TGF-β may be also detected in all GI tract tissues and accessory organs. TGF-β is synthesized as a large precursor propeptide [197]. Despite intracellular cleavage, the TGF-β1 dimer remains in a biological inactive complex with the two propeptide segments through noncovalent association, the so-called latent form. The biological processes regulating the bioactivation of TGF-β from its latent state have not been completely defined. TGF-β1 has been found to bind to several specific cell surface TGF-β receptors (TβRs) localized in responsive cells. There are five different types of receptors (TβRI through TβRV), and among them, TβRI and II isoforms are Ser/Thr-specific protein kinases that are believed to be primarily responsible for TGF-β induced cellular responses in the GI tract [198]. It has been shown that TβRI and TβRII work in a cooperative fashion: ligand binding to the TβRI facilitates activation of the associated TβRII, which then activates the intracellular signaling machine via Smad proteins [199]. Within the small intestine, TGF-β expression has been found in lamina propria and almost all the epithelial cells [200]. The major activity of TGF-β is to inhibit the growth of most cell types, including epithelial and endothelial cells, but in some instances, TGF-β also stimulates the growth of certain mesenchyme cells, such as in skin fibroblasts [196]. The GI mucosal growth inhibitory responses to TGF-β have been intensively investigated in vitro as well as in vivo, although the mechanism underlying the inhibitory effects of TGF-β remains to be fully understood.

Targeted disruption of the TGF-β gene by gene knock-out technology results in multiple focal inflammatory cell infiltration and/or necrosis, indicating the role of TGF-β in both inflammation and tissue repair. Mice homozygous for the mutated TGF-β allele exhibit no gross developmental abnormalities at birth, but they develop severe and multifocal inflammatory diseases that affect several organs, including diffuse inflammation in the stomach and intestine. In fact, TGF-β deficiency leads to severe pathology, causing death at about 20 days of age associated with dysfunction of the immune and inflammatory system, showing its essential role as a potent regulator of the immune system. Increased expression of TGF-β is also found in the GI mucosa after acute epithelial injury and in patients with active inflammatory bowel diseases.

Results from our laboratory and others show that increasing the levels of TGF-β inhibits intestinal epithelial cell proliferation through activating TβRI/Smad signaling cascade following polyamine depletion (Figure 10) [201203]. The addition of TGF-β to the culture medium significantly decreased the rate of DNA synthesis and final cell number. Increased activation of endogenous TGF-β/TβRI signaling in polyamine-deficient cells is also associated with an inhibition of intestinal epithelial cell growth, which is partially prevented by the addition of immunoneutralizing anti-TGF-β antibody or inactivation of TβRI activity [202,203]. We have further demonstrated that Smad proteins are the immediate downstream effectors of activated TGF-β/TβRI signaling since Smad silencing prevents inhibitory effects of exogenous TGF-β treatment or activated endogenous TGF-β/TβRI pathway via polyamine depletion on intestinal epithelial cell proliferation [201].

FIGURE 10. Schematic diagram depicting the role of TGF-β/Smad signaling pathway in the inhibition of normal intestinal cell proliferation following polyamine depletion.

FIGURE 10

Schematic diagram depicting the role of TGF-β/Smad signaling pathway in the inhibition of normal intestinal cell proliferation following polyamine depletion. In this model, polyamines are the negative regulators for expression of the TGF-β (more...)

Gebhardt et al. reported that TGF-β acts as a novel potent inhibitor of human intestinal mast cells [204]. In this study, mast cells were isolated from the human intestinal mucosa, purified, and cultured in the presence of stem cell factor (SCF) with or without TGF-β1. TGF-β1 was found to dose-dependently inhibit SCF-dependent growth of human intestinal mast cells by decreasing proliferation and enhancing apoptosis. In another study, the prenatal porcine intestine is shown to have low levels of endogenous TGF-β ligand and receptor density, which is associated with an induction in trophic response to enteral diets [205]. There is also a reporter showing that in fetal pigs, the TGF-β ligands are predominantly localized to the crypt epithelium, but staining intensity increased markedly just before term and shifted to the villus epithelium in newborn pigs [206].

In addition, TGF-β and gastrin-releasing peptides (GRP) jointly regulate intestinal epithelial cell division and differentiation [207210]. The treatment with TGF-β together with GRP is found to inhibit intestinal epithelial cell growth and to induce apoptosis much higher than those observed in cells exposed to TGF-β or GRP alone. This combined treatment also induces an induction in cycloxygenase-2 expression and prostaglandin E2 production through activating p38MAPK pathway in cells stably transfected with GRP receptor. In another study, TGF-β transcriptional activity was found to be upregulated in the small intestine after infection of mice with a parasite Trichinella spiralis, which leads to small intestinal inflammation [211]. The TGF-β signaling pathway also plays an essential role in intestinal stem cell development and organogenesis [207,212,213].

IGF FAMILY

IGF family is constituted by two ligands, IGF-I and IGF-II, which are single chain polypeptides consisting of 70 and 67 amino acids, respectively [214]. IGFs are secreted as small peptides (7.5 kDa) that are structurally related to insulin and display multiform effects on cell growth and metabolism in the GI tract [215,216]. Both IGF-I and IGF-II exert their mitogenic activities through interaction with specific IGF-receptors (mainly IGFR-I and IGFR-II), and they are capable of modulating epithelial cell kinetics by stimulating proliferation and inhibiting apoptosis [214]. In rodents, IGFI and IGFII are expressed in diverse sites with intrinsic biological activities. IGF-I is produced by intestinal mesenchymal cells in rats, and it is shown to increase proliferation of intestinal epithelial cells [217]. IGF-II is expressed at high levels in the fetus and lower levels at the adult stages, and its synthesis has been observed in adult liver and extrahepatic tissues [218].

The stimulatory effects of IGFs on the intestine had been identified over almost two decades ago. Recently, therapeutic indications are also defined for a range of candidate bowel disorders and diseases in which accelerated intestinal repair is desirable. IGFs are the potent stimulator of cell proliferation in the intestinal crypts, spurring progression through G1- to the S-phase of the cell cycle. Exogenous administrations of IGFs pharmacologically and systemically to neonatal or adult rats increase intestinal mucosal growth [219]. Continuous administration of IGF-I to adult rats for 14 days causes preferential growth of the GI organs, increase gut weight as a fraction of body weight by up to ~32%, which is accompanied by increases in crypt cell population and villus cell density. Consistently, overexpression of the IGF transgenically increases growth at the intestinal muscle layers [220]. IGFs have been also demonstrated to promote wound healing in the GI mucosa to exert trophic effects within the intestine and to enhance tumor growth through autocrine mechanism. There is also evidence that IGFs are important in the pathogenesis of fibrosis in Crohn's disease.

Experiments of IGF-I administration in vivo have shown both linear and cross-sectional growth of the GI organs affecting the mucosal and muscularis layers proportionally [221]. These findings suggest clinical application in bowel conditions characterized by impaired growth and repair processes [214,222224]. It is likely that bowel resection, chemotherapy-induced intestinal mucositis, radiation enteritis, and the inflammatory bowel diseases are candidate target conditions that may be beneficial from IGF administration in the first instance [225].

FGF FAMILY

Members of huge FGF family are 16–18 kDa proteins that control normal growth and differentiation of mesenchymal, epithelial, and neuroectodermal cell types [226]. FGFs also play key roles in growth and survival of stem cells during embryogenesis, tissue regeneration, and carcinogenesis. Both acidic and basic FGFs are the best-characterized members of the FGF family. Although there are limited results available on the expression and physiological functions of FGFs and their receptors in the GI tract, several studies have described the presence of FGF family peptides and their specific receptors in the intestine [174,227]. It has been shown that FGFs appear to act as autocrine growth factors and stimulate intestinal mucosal growth and epithelial cell division. In cultured IEC-6 cells, administration of FGFs increases cell proliferation; and this effect is further enhanced by the addition of heparin. This treatment mimics the in vivo situation of growth factors binding to the extracellular matrix.

Furthermore, FGFs are also shown to promote intestinal epithelial restitution after wounding through TGF-β-dependent pathway in vitro [228]. Helicobacter pylori are the major pathogen for peptic ulcers and chronic atrophic gastritis, and they are also implicated in the pathogenesis of gastric cancer [229]. FGF2 is one of the pro-angiogenic factors and shown to enhance healing of gastric mucosal damage associated with Helicobacter pylori infection. CagA protein and peptide glycan of Helicobacter pylori are phosphorylated by SRC family protein kinases to activate SHP2 phosphatase when they are injected to human gastric epithelial cells [229]. Since SHP2 is a component of docking protein complex for FGF signaling, SHP2 activation results in FGF signaling activation.

FGF signaling pathway networks with Wnt signaling pathway during a variety of cellular processes including early embryogenesis and gastrointestinal morphogenesis [174,230]. Expression analyses on different FGFs revealed that FGF-18 and FGF-20 isoforms are predominantly expressed in epithelial cells derived from the GI tract, and they seem to be the direct targets of the canonical Wnt signaling pathway. Wnt signals are transduced through Frizzled seven-transmembrane-type receptors and activate the β-catenin pathway. Wnt-induced transcriptional complex activates transcription of target genes. It has been reported that the promoter of FGF-18, FGF-20, or FGF-7 contains several TCF/β-catenin binding sites, but the exact role of Wnt signal in the regulation of FGF gene transcription remains to be fully defined.

OTHER FACTORS

Several other peptide growth factors and cytokines have also been found to play an important role in maintaining mucosal cell growth under biological and various pathological conditions. These factors include Trefoil factor (TFF) family [231,232], hepatocyte growth factor (HGF) [233], and colony-stimulating factors (CSF) [234]. All TFF, HGF, and CSF are shown to stimulate GI mucosal growth, promote wound healing, modulate epithelial cell apoptosis, and protect the epithelial integrity from damage in response to stressful environments.

Copyright © 2011 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK54096
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