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DEVELOPMENTAL DYNAMICS 246:344–352, 2017 DOI: 10.1002/DVDY.24491

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Fibroblast Growth Factors in the Gastrointestinal Tract: Twists and Turns a

Soula Danopoulos,1,2 Christopher R. Schlieve,1,2 Tracy C. Grikscheit,1,2 and Denise Al Alam1,2*

DEVELOPMENTAL DYNAMICS

1 Developmental Biology and Regenerative Medicine Program, Department of Pediatric Surgery, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, CA 2 Keck School of Medicine, University of Southern California, Los Angeles, CA

Fibroblast growth factors (FGFs) are a family of conserved peptides that play an important role in the development, homeostasis, and repair processes of many organ systems, including the gastrointestinal tract. All four FGF receptors and several FGF ligands are present in the intestine. They play important roles in controlling cell proliferation, differentiation, epithelial cell restitution, and stem cell maintenance. Several FGFs have also been proven to be protective against gastrointestinal diseases such as inﬂammatory bowel diseases or to aid in regeneration after intestinal loss associated with short bowel syndrome. Herein, we review the multifaceted actions of canonical FGFs in intestinal development, homeostasis, and repair in rodents C 2017 Wiley Periodicals, Inc. and humans. Developmental Dynamics 246:344–352, 2017. V Key words: FGF; intestine; development; homeostasis; repair Submitted 11 October 2016; First Decision 2 February 2017; Accepted 6 February 2017; Published online 15 February 2017

The gastrointestinal tract derives from interactions between the embryonic endoderm and mesoderm germ layers. The gut epithelium derives from the endoderm layer, whereas the mesenchyme and mesenteric system derive from the mesoderm (Wells and Melton, 2000). The gut tube starts to form in late gastrulation; then anterior-posterior domain patterning occurs in response to

nodal and FGF4, respectively. Several signaling pathways are involved in the patterning of the gut thereafter. Opposing Wnt signals allow the patterning of the foregut, midgut, and hindgut, which will form the gastrointestinal tract. Intestinal development continues after birth, when the stem cells arrange into crypt units and the differentiation of the intestinal cells continues. The gastrointestinal tract has a high cellular turnover that includes signaling pathways regulating an array of stem and progenitor cells within the intestinal epithelium. In brief, there is a balance between the proliferation of stem/progenitor cells at the base of the crypts and differentiation in the transit amplifying (TA) zone, with eventual cell loss at the top of the villus, which is supplied by multiple crypts (Bach et al., 2000; Barker et al., 2010; Leblond and Messier, 1958; Leblond and Stevens, 1948; Quastler and Sherman, 1959; Walker and Leblond, 1958) An exception to this upward movement from the TA zone is the Paneth cell, which moves down to its location in the base of the crypt between the columnar base columnar (CBC) stem cells (Clevers, 2013). CBC cells were originally identified by electron microscopy (Barker et al., 2012; Cheng and Leblond, 1974) and then by transgenic mice expressing Wnt target leucine-rich G protein-coupled receptor 5 (LGR5) (Barker et al., 2007). LGR5-positive cells are highly proliferative and damage-sensitive, and also express olfactomedin-4 (OLFM4) and Achaete scute-like 2 (ASCL2) (Barker, 2014). The LGR5-positive cells are the rapidly cycling stem cells of the intestine and are complemented by another population of quiescent stem cells located in the TA zone, termed “þ4 cells”

*Correspondence to: Denise Al Alam, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, CA 90027. Email: dalalam@chla. usc.edu

Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24491/abstract C 2017 Wiley Periodicals, Inc. V

Introduction Fibroblast growth factors (FGFs) belong to a large family of 22 structurally related peptides. FGFs are classified into seven subfamilies based on gene locus, mode of action, and phylogenetics. These subfamilies are divided into three groups based on their mechanism of action: canonical FGFs, intracellular FGFs, and hormone-like FGFs. FGF subfamilies, groups, and receptor-/ ligand-binding specificity have been recently reviewed in detail by Ornitz and Itoh (Ornitz and Itoh, 2015). FGFs play an important role in the development, homeostasis, and repair of many organ systems such as the lung, liver, kidney, and gastrointestinal tract. Herein we will review the role of FGF signaling in the development of the small intestine and in intestinal injury and repair. Additionally, this review will cover the regulation of intestinal stem cells by FGF signaling in Zebrafish, mouse and humans.

Formation and Maintenance of the Gastrointestinal Tract

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because of the usual location 4 cells above the base of the crypt (Barker et al., 2007; Potten et al., 2003). þ 4 cells appear to be damage-resistant and relatively quiescent, and they express polycomb complex protein BMI1 (Yan et al., 2012), pan-ErbB inhibitor leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1), homeodomain-only protein (HOPX), and telomerase reverse transcriptase (TERT) (Barker, 2014). These cell populations have been recently reviewed by Barker (Barker, 2014). Among the signaling webs that promote differentiation of the intestinal stem and progenitor cells of the intestinal epithelium, the FGF family exerts important roles that will be reviewed herein.


FGF Ligands and Receptors The human and mouse FGF families each contain 22 members. FGFs are highly conserved, with most mouse and human FGFs being orthologs, except mouse Fgf15, which is instead orthologous to human FGF19 (as Fgf15 does not exist in humans) (Itoh 2007) (Table 1). However, the Zebrafish FGF family is composed of 27 members, 21 of which are orthologs to human FGFs, and six of which are paralogs (Itoh 2007) (Table). FGFs bind one of four receptors: FGFR1, FGFR2, FGFR3, or FGFR4. FGFR1, FGFR2, and FGFR3 have two splice variants each, IIIb and IIIc, that determine ligand binding specificity. Therefore, seven receptor proteins are present in human and mouse: FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c, and FGFR4. However, FGFR4 has a soluble splice variant found in human intestinal epithelial cells (Takaishi et al., 2000). In Zebrafish, only five receptors are present: fgfr1a, fgfr1b, fgfr2, fgfr3, and fgfr4. Several FGF ligands and receptors are expressed in the gastrointestinal tract during development and in adult tissue. All four FGF receptors are expressed in the intestine. We recently showed that FGFR1 and FGFR2 are expressed in the human ileum and throughout adult mouse intestine (Al Alam et al., 2015). FGFR3 and FGFR4 are also expressed in the mouse intestine, with FGFR3 expressed in the lower half of the intestinal crypts. FGFR4 expression is restricted to the epithelium of the embryonic gut (Stark et al., 1991). Several FGF ligands and receptors are also expressed in the intestine and mediate FGF signaling in the intestine. FGF1, which binds all 4 receptors, is expressed in the developing mouse intestine in utero and after birth, as well as in adult intestine (Al Alam et al., 2015; Madiai and Hackshaw, 2002; Vidrich et al., 2002). FGF1 is expressed in enterochromaffin cells and smooth muscle cells, as well as in the enteric nervous system (La Rosa et al., 1997; Capetandes et al., 2000). FGF2 is expressed near the basement membrane surrounding the crypts and the lower part of the villus epithelium (Houchen et al., 2000). FGF2 binds FGFR2IIIb, FGFR2IIIc, FGFR3IIIb, FGFR3IIIc, and FGFR4. In addition, Fgf1, Fgf7, and Fgf10 are expressed in the adult mouse intestine, but not Fgf3, Fgf20 and Fgf22 (Al Alam et al., 2015). FGF7 and FGF10 are also expressed in the human ileum (Al Alam et al., 2015). During human fetal development, FGF10 is expressed mainly in the apical epithelium of the hindgut until 7 weeks of gestation, after which FGF10 is no longer expressed in the epithelium (Yin et al., 2013). Fgf9 is expressed in the epithelium of the embryonic small intestine, cecum, and colon in mouse (Zhang et al., 2006; Colvin et al., 1999; Al Alam et al., 2012). Fgf9 reaches its peak expression in the mouse jejunum between day 7 and day 14 postnatally then decreases in adulthood, similar to FGF1 and FGF2 (Vidrich et al., 2004). FGF8

TABLE 1. FGF Ligands in Human and Their Orthologs in Mouse and Zebrafish Zebrafish/Human Mouse/ Human Human Ortholog FGF1 FGF2 FGF3 FGF4 FGF5 FGF6 FGF7 FGF8 FGF9 FGF10 FGF11 FGF12 FGF13 FGF14 FGF16 FGF17 FGF18 FGF19 FGF20 FGF21 FGF22 FGF23

Fgf1 Fgf2 Fgf3 Fgf4 Fgf5 Fgf6 Fgf7 Fgf8 Fgf9 Fgf10 Fgf11 Fgf12 Fgf13 Fgf14 Fgf16 Fgf17 Fgf18 Fgf15 Fgf20 Fgf21 Fgf22 Fgf23

fgf1 fgf2 fgf3 fgf4 fgf5 fgf6b fgf7 fgf8 fgf10a fgf11 fgf12 fgf13 fgf14 fgf16 fgf17b fgf18a fgf19 fgf20b fgf21 fgf22 fgf23

Paralog

Group

Canonical Canonical Canonical Canonical Canonical fgf6a Canonical Canonical fgf17a Canonical Canonical fgf10b Canonical Intracellular Intracellular Intracellular Intracellular Canonical Canonical fgf18b, fgf24 Canonical Endocrine fgf20a Canonical Endocrine Canonical Endocrine

is expressed in the epithelial cells of human colon and human duodenum and could signal to the muscularis by binding to FGFR1 and FGFR2 (Zammit et al., 2002). Furthermore, FGF18 is expressed in the smooth muscle cells of the mouse small intestine starting embryonic day (E) 15.5 (Hu et al., 1998). FGF5, FGF6, FGF16, and FGF17 do not seem to be expressed in the small intestine or colon. In summary, the FGFs ligands expressed in the intestine are Fgf1, Fgf2, Fgf7, Fgf8, Fgf9, Fgf10, and Fgf18.

FGF Signaling in Intestinal Development Establishing a fully functional gut requires a great deal of cross talk between the epithelium and mesenchyme. The gut starts off as a simple tube divided into three distinct regions known as the foregut (to become pharynx, esophagus, and stomach), midgut (to become small intestine), and hindgut (to become large intestine/colon) (de Santa Barbara et al., 2003) at around E8 (Zhang et al., 2006). Within late gestation (E15.5–E18.5), the epithelium of the primitive gut begins to differentiate from a pseudostratified cuboidal epithelium into a simple columnar epithelium (Zhang et al., 2006; Vidrich et al., 2004; de Santa Barbara et al., 2003; Schmidt et al., 1988), allowing the “tube” to undergo the process of vilification, during which the connecting epithelial and smooth muscle layers of the tube undergo a series of folding and zigzag patterning that results in projections of the columnar epithelium called villi (Shyer et al., 2013). However, the organization of progenitor cells into crypt units starts postnatally. All of these morphogenic processes depend on an array of fate-determining transcription and growth factors interacting between the gut’s epithelium and mesenchyme, including FGFs.

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Fig. 1. Fgf ligand expression in the developing mouse intestine. Several Fgf ligands are expressed in the developing mouse intestine and mediate Fgf signaling in the intestine. Fgf1 is expressed in the mouse intestine in utero through adulthood. Fgf2, Fgf4, Fgf7, Fgf15, and Fgf18 are expressed in the adult mouse intestine, but not Fgf3, Fgf20 and Fgf22. Fgf9 is expressed in the embryonic mouse small intestine, cecum and colon and reaches its peak expression in the mouse jejunum between postnatal days 7 to 14, then decreases in adulthood, similar to Fgf1 and Fgf2. Fgf10 is mostly expressed in the mesenchyme of the duodenum and distal colon, with minimal expression in the jejunum and ileum.

FGF10 has been demonstrated to play a critical role in gut organogenesis where it controls cell proliferation, survival, and differentiation. In mouse small intestine, Fgf10 is mostly expressed in the mesenchyme of the duodenum, with minimal expression in the jejunum and ileum (Al Alam et al., 2015; Nyeng et al., 2011; Kanard et al., 2005) (Figure 1). In human small intestine, we recently showed that FGF10 is present only in the ileum (Al Alam et al., 2015). Fgf10-/- mice demonstrated colon and duodenal atresia, alongside a failure of cecal formation. Furthermore, these animals demonstrated premature cellular differentiation that led to epithelial hypoplasia (Nyeng et al., 2011). Fgf10-/- embryos also display anorectal malformations and cecal atresia; there is mesenchymal expansion but no epithelial proliferation in the area where the cecal bud normally forms, between the ileum and large intestine (Al Alam, et al. 2012; Burns et al., 2004). Similar results are seen following Fgfr2b deletion in mice (Burns et al., 2004). Although binding of Fgf10 to Fgfr2b was not necessary for the induction of the cecum, it was necessary for the epithelial cell proliferation responsible for its elongation and development (Burns et al., 2004). In addition, FGF9 is also required for the induction of the cecal bud. In the intestine, FGF9 is expressed in the epithelium and acts upon the mesenchyme (Figure 1). In mesenchymal-specific and global deletion of Fgf9, the cecum is severely hypoplastic, with a more severe phenotype in the case of global deletion (Al Alam et al., 2015). Thus, epithelial and mesenchymal Fgf9 are important for the initial formation of the cecum, which in turn is thought to induce Pitx2 expression. Pitx2 then activates Fgf10 and promotes elongation of the intestine (Al Alam et al., 2015; Zhang et al., 2006). In addition to cecal atresia, mice lacking Fgf9 demonstrate shortening of the small intestine starting at E14.5 (Al Alam et al., 2015; Zhang et al., 2006). This shortening is accompanied by decreased mesenchymal proliferation, as well as premature differentiation of fibroblasts into myofibroblasts (Geske et al., 2008). Therefore, Fgf9 regulates the proliferative and differentiation rates of fibroblasts in the mesenchyme, allowing the gut to lengthen. Furthermore, when the mesenchymal receptors (Fgfr1 and Fgfr2) of Fgf9 were deleted using Dermo1-Cre, similar results were obtained, with the most comparable being when both receptors were deleted simultaneously (Geske et al., 2008). Another canonical FGF important for intestinal development is FGF2, which is expressed

mainly in the mesenchyme, where it binds to FGFR1 (Gonzalez et al., 1996) (Figure 1). Inhibition of FGF2 using anti-FGF2 antibody in mouse intestinal transplants impairs intestinal growth and slows the differentiation rate of the epithelial cells. However, supplementing the explants with exogenous FGF2 does not affect the growth of the explants (Liu et al., 1990). Studies using loss and gain of function of FGF4 (through tyrosine kinase inhibitors or FGF4-soaked beads, respectively) have shown that FGF4 regulates the anterior-posterior axis of the intestine by both a concentration- and temporal-dependent manner, determining the boundaries for the gut tube domains as fore-, mid-, and hindgut (Dessimoz et al., 2006). In summary, FGF signaling is required for normal gut development. In early development, FGF4 allows the specification of the gut tube, whereas Fgf10 and Fgf9 play an important role in later domain specification, intestinal proliferation, elongation, and differentiation.

FGF Signaling in Intestinal Homeostasis Once the adult gut has been organized and the intestine has developed villi and crypts (where rapidly cycling stem cells are located), the structure must then be maintained. A common enteric progenitor produces the four different cell types found in the intestine: Paneth cells, goblet cells, enteroendocrine cells, and absorptive enterocytes (Nyeng et al., 2011). Since the intestine is a very dynamic organ and has a high cellular turnover, a robust means of regulation must be present to monitor the types of cells produced. FGF10 is important not only in the developmental processes of the intestine, but also during homeostasis. Fgf10 overexpression in adult intestines increases crypt depths and villus heights throughout the small intestine. It also induces goblet cell differentiation at the expense of Paneth cells, which decrease in number (Al Alam et al., 2015). On the other hand, reducing Fgfr2b ligands by overexpressing a soluble form of Fgfr2b in the adult mouse intestine does not affect cell proliferation or differentiation (Al Alam et al., 2015). Also, FGFR3 plays an antagonistic role to FGF10 in Paneth cells. FGFR3 has been shown to be located on the basolateral surface of the epithelium in the lower half of the crypt, where the stem cells are located (Vidrich et al., 2004). Fgfr3-/- mice have half the number of intestinal crypts



overexpression, suggesting that Fgfr3 plays a role in regulating the number of Paneth cells (Vidrich et al., 2009). When injected into normal adult mice, FGF18, a ligand of FGFR3, promotes cell proliferation in the intestinal crypt and the mucosa (Hu et al., 1998). Systemic administration of FGF7 to adult rats results in increased cell proliferation and increased goblet cell differentiation (Zeeh et al., 1996; Iwakiri and Podolsky, 2001). An in vitro assay of human colonic epithelial cells showed that this increase in goblet cell differentiation was a result of the induction of the goblet cell–specific transcription factor GCSI binding protein, which regulates intestinal trefoil factor (ITF) (Iwakiri and Podolsky, 2001). FGF7 also increases villus height and crypt depth in the intestine (Cai et al., 2013). FGF7, secreted by colonic fibroblasts and/ or intraepithelial lymphocytes, enhances the proliferation and differentiation of intestinal epithelial cells (Visco et al., 2009). Several FGF ligands and receptors are therefore required for gut homeostasis and stem cell maintenance (Figure 2).

FGF and Intestinal Stem Cells

Fig. 2. The role of FGF in intestinal homeostasis and inflammation. Several FGF ligands and receptors are expressed in the gastrointestinal tract during development and in adult tissue. All four FGF receptors are expressed in the intestine, with Fgfr4 expression restricted to the epithelium of the embryonic gut and Fgfr3 expressed in the lower half of the intestinal crypts in mice. Fgf1 is expressed in enterochromaffin cells, myocytes, and the enteric nervous system. Fgf2 is expressed near the basement membrane surrounding the crypts. FGF8 is expressed in the epithelial cells of human duodenum and colon. Fgf9 is expressed in the epithelium of the embryonic small intestine, cecum and colon in mouse. Fgf18 is expressed in the smooth muscle cells of the mouse small intestine starting embryonic day 15.5.

compared to controls, resulting in a decreased number of stem cells (Vidrich et al., 2009). These mice also have reduced Paneth cell specification and differentiation, also seen following Fgf10

While FGFs are mostly known to induce cell proliferation and migration, they also play an important role in cell differentiation and maintenance of stem cells. FGF2 maintains the stem cells in a pluripotent undifferentiated status, and thus is used to maintain the pluripotency of human and mouse stem cells in vitro (Lotz et al., 2013; Yang et al., 2015). FGF4, along with WNT3a, is required to drive the differentiation of mouse pluripotent stem cells into hindgut, ultimately giving rise to all the cells of the intestinal epithelium and mesenchyme except for components of the enteric nervous system (Spence et al., 2011; Aurora and Spence, 2016). However, the role of FGF signaling in driving the differentiation of human embryonic stem cells (hESC) into intestinal epithelial cells remains controversial. Some studies report that synergy between FGF4 and WNT3a is required for the specification of hindgut (Spence et al., 2011; Ameri et al., 2010), while others reported that FGF signaling inhibits the differentiation of hESC into intestinal epithelial cells and as such is dispensable for the generation of intestinal cells (Ogaki et al., 2013; Tamminen et al., 2015). Intestinal stem cells are located at the base of the crypts. The intestinal epithelium has a rapid turnover and hence is severely affected by therapies targeting highly proliferative cells, such as radiation. FGF4 prevents radiation-induced apoptosis in mouse intestine and increases the survival of crypt intestinal cells (Sasaki et al., 2004). FGF2, expressed in the mesenchymal cells surrounding the intestinal crypts, enhances the survival of the stem cells contained in the crypts following radiation injury (Houchen et al., 1999). However, administration of FGF1 in vivo to irradiated mice did not show any significant effect on cell proliferation in the intestinal crypts (Potten et al., 1995). Although exogenous FGF1 does not modulate crypt cell proliferation, FGF2 and FGF4 both enhance cell survival following radiation. On the other hand, we showed that overexpression of Fgf10 in mouse-derived organoid units enhances the growth of tissueengineered intestine in vivo (Torashima et al., 2016). In an in vitro model of mouse intestinal enteroids, we also showed that FGF10 increases goblet cell differentiation at the expense of Paneth cells. Exogenous FGF10 resulted in the decrease of several stem cell markers, including Lgr5, Lrig1, Hopx, Ascl2, and Sox9, and increased the transient amplifying cells double positive for


Mmp7 and Muc2 (Al Alam et al., 2015). In Zebrafish intestine, inhibition of fgfr2c results in decreased goblet and enteroendocrine cell differentiation (Liu et al., 2013). In a mouse model of doxorubicin-induced damage, increased levels of Fgf9 and Fgf18 were found to correlate with expansion of Paneth cells (King et al., 2013), suggesting a role for Fgf9 and Fgf18 in Paneth cell differentiation. Fgf9 and Fgf18 bind to Fgfr3, which is expressed only in undifferentiated cells of the mouse intestinal crypts (Vidrich et al., 2004). Lack of Fgfr3 in mice resulted in increased crypt depths, while administration of FGF18 decreased cell proliferation in the crypts (Arnaud-Dabernat et al., 2008). In Caco-2 cells, the expression of FGFR3IIIb decreases as the cells become more differentiated (Kanai et al., 1997). Therefore, signaling through Fgfr3 and Fgfr2 is essential for the maintenance and differentiation of the intestinal stem cells.


FGF Signaling in Intestinal Metabolism The FGF19 subfamily, which consists of FGF15/19, FGF21, and FGF23, is considered endocrine FGFs due to its endocrine function. These FGFs act like circulating hormones, being synthesized by one organ and acting upon another (Degirolamo et al., 2016). Unlike the canonical FGFs, endocrine FGFs do not bind to FGFRs with high affinity. Therefore, they require the assistance of a single transmembrane glycoprotein co-receptor known as klotho (a or b) to dimerize to the FGFR, which stabilizes the interaction to the FGF of interest (Degirolamo et al., 2016). The limited location of the different klotho receptors determines where the different endocrine FGFs end up (Degirolamo et al., 2016). Within the FGF19 subfamily, the only one that is of importance to the intestine is FGF15/19. Mouse Fgf15, which is the ortholog to human FGF19, plays an important role in intestinal metabolism. It is mainly produced in the small intestine enterocytes and is secreted once food is introduced into the system following bile acid absorption in the ileum (Kuro-o, 2008). From there, FGF15/19 is released into the hepatic portal circulation so that it may reach the liver, where it binds FGFR4/bklotho. Furthermore, it has been shown that FGF15, FGFR4, and bklotho knockout mice display an increase in bile acid synthesis (Kuro-o, 2008; Potthoff et al., 2012). FGF15/19 has also been shown to act directly upon the gallbladder, causing it to fill with bile, thus limiting the amount of bile that is released into the intestine, eventually causing a decrease in the amount of FGF15/19 that is released from the intestinal enterocytes (Kuro-o, 2008). In summary, upon the introduction of food, bile is secreted into the lumen of the intestine via the enterohepatic circulation, where it binds to the nuclear farnesoid X receptor (FXR) of the small intestine (Kuro-o, 2008). The FXR receptor then heterodimerizes with retinoid X receptor (RXR), allowing an increase in FGF15/19 expression, which then travels to the liver and commences its aforementioned activities (Kuro-o, 2008).

FGF Signaling in Intestinal Injury/Repair FGFs in Inﬂammatory Bowel Disease In early reports of the role of FGFs in intestinal injury and repair, Dignass et al. showed that FGF1 and FGF2, also known as acidic FGF and basic FGF, respectively, enhanced epithelial cell proliferation of IEC-6, Caco-2 and ,-29 cell lines. Moreover, they

promoted epithelial cell restitution through the regulation of TGF-beta (Dignass et al., 1994). Interestingly, a mutant form of FGF1, which lost mitogenic activity, proved to be more efficient at reducing cell apoptosis in the gut epithelium of rats following ischemia-reperfusion injury (Fu et al., 2004). In an experimental model using dextran sulphate sodium (DSS) to induce colitis, Fgf2-/- animals displayed a more severe phenotype than wildtype animals treated with DSS (Song et al., 2015). DSS is widely used to induce acute or chronic inflammation and epithelial cell death in the colon, hallmarks of inflammatory bowel disease (IBD). It was determined that regulatory T (Treg) cells produce FGF2 upon the regulation of TGFbeta1. FGF2 ultimately allows for a cascade of genes to be activated to help repair the damaged epithelium (Song et al., 2015), thus maintaining the homeostatic environment of the intestine. In the presence of the cytokine IL-2, Caco-2 cells produced higher amounts of FGFR3IIIb, indicating an important role of FGFR3IIIb in the response to inflammation (Kanai et al., 1997). Inflammatory bowel diseases are characterized by chronic inflammation and defective intestinal barrier. Several FGFs were studied in experimental models of IBD. In a model of experimental colitis in rats, Zeeh et al. showed that FGF7 (also known as KGF) administered after but not before colitis induction decreased epithelial cell death and ulceration in rat intestines (Zeeh et al., 1996). Another study showed similar effects for FGF7 in a model of DSS-colitis in mice (Egger et al., 1999). In contrast, whether FGF7 is able to protect the intestine from chemotherapy-induced mucositis remains controversial (Gibson et al., 2002; Farrell et al., 2002). Fgf7-null mice are more susceptible to DSS injury and demonstrated a reduction in mucosal barrier repair compared to wild-type animals (Chen et al., 2002). Moreover, FGF10 (KGF-2) and its equivalent, repifermin (a truncated peptide), reduced the severity, death, and weight loss in mice with DSS-induced colitis in both a prophylactic and a therapeutic manner (Miceli et al., 1999; Greenwood-Van Meerveld et al., 2003). Hence, FGF10 decreases the severity of acute DSS-induced colitis, ameliorates indomethacin-induced ulceration in rats, and reduces the secretion of inflammatory cytokines (IL-6, TNF, and IL-8) in these injury models (Miceli et al., 1999; Sandborn et al., 2003; Hamady et al., 2010). In a model of duodenal ulcers, administration of FGF2 significantly improved the ulcer bed and significantly accelerated the healing process in the duodenum (Folkman et al., 1991). Other FGFs, such as FGF20, are also protective against experimental IBD. FGF20 has been shown to reduce the severity of DSS-induced colitis in mice by reducing cell death and inflammation (Jeffers et al., 2001). Several FGFs (FGF2, FGF7, FGF10, and FGF20) promote healing and decrease inflammation in colitis models. Therapeutic effects of FGF ligands or FGF signaling targets need to be explored in further detail.

Short Bowel Syndrome Following a massive injury to the intestine, or in the case of congenital anomalies where more than 75% of the small intestine has been resected, short bowel syndrome (SBS) may result. Intestines of SBS patients have limited absorption capacity because of the reduction in length and undergo a process termed adaptation, characterized by increased villus height and crypt depth, lengthening of the intestine, and increased proliferation, thought to lead to improved absorption (Goulet, 1998). Since many FGFs are known as mitogens, they are thought to play a role in intestinal



adaptation. FGF7 and FGFR1 expression is significantly increased in mouse intestines following massive small bowel resection (Haxhija et al., 2007). In a rat model of SBS, FGF7 enhanced growth in the duodenum, jejunum, and ileum of SBS animals. Increases in crypt depth, villus height, mucosal thickness, and goblet cell differentiation were observed 4 days following surgical resection and FGF7 administration (Johnson et al., 2000; Washizawa et al., 2004). In a mouse model of acute small bowel resection, Fgf10, which is normally absent in the small intestine, starts to be expressed in the epithelial crypts during adaptation, suggesting an important role for Fgf10 in intestinal adaptation (Tai et al., 2009). Because of the low survival rate in rodent models of small bowel resection, we developed a new model of SBS in adult Zebrafish, in which adaptation takes place for up to 4 weeks following resection (Schall et al., 2015). We later showed that inhibiting FGF signaling in Zebrafish severely impairs adaptation (Schall et al., 2015). Hence, Fgf7 and Fgf10, likely through their binding to FGFR1, promote repair following small bowel resection.

Ischemia/Reperfusion Injury Reperfusion injury is the re-oxygenation of the intestine after a period of ischemia or hypoxia. Absence of oxygen and nutrients to an area of the intestine results in inflammation and oxidative stress. The pathology of intestinal ischemia reperfusion (I/R) is characterized by loss of intestinal barrier function, causing local or, in some cases, systemic inflammation. Exogenous FGF1 and FGF2 have been shown to be protective in rat models of intestinal I/R (Fu et al., 2003; Chen et al., 2005) via the regulation of P53 and ERK/p38 MAPK, respectively. FGF2 also improves healing in rat colonic anastomoses through fibroblast activation, collagen deposition, and angiogenesis (Adas et al., 2011). Similarly, FGF7 attenuates I/R injury in mouse by reducing cell apoptosis and disruption of tight junctions (Cai et al., 2012a). FGF7 up-regulates IL-7 expression in the I/R intestines. IL-7 controls the intraepithelial lymphocytes necessary for epithelial cell growth (Cai et al., 2012b). Blocking FGFR2 abolishes the up-regulation of IL-7, suggesting that FGF7 regulates IL-7 via FGFR2 signaling pathway (Cai et al., 2012b). FGF7 also promotes healing of colonic anastomoses by increasing cell proliferation and mucus production and reducing inflammation (Egger et al., 1998). Similar to colitis models, FGF1, FGF2, and FGF7 have protective roles in I/R, where they also reduce inflammation.

FGF Signaling in Human Intestinal Diseases Mutations in FGFR1 and FGFR2 have been associated with human diseases such as Crouzon and Pfeiffer syndromes, both of which feature craniosynostosis, or premature fusion of the bones of the skull due to prolonged and inappropriate signaling. In Pfeiffer syndrome, there are three types; type 1 is associated with a normal intelligence and life span and a mutation in either FGFR1 or FGFR2. Types 2 and 3 are more severe and are only associated with FGFR2 mutations (Gonzales et al., 2005; Rutland et al., 1995). A Ser351Cys mutation in FGFR2 was found in a patient with Pfeiffer syndrome type 3 who also was found to have intestinal nonrotation, a fault in the normal process of intestinal development (Gripp et al., 1998). Heterozygous somatic mutations in FGFR2 and FGFR3 were identified in gastric cancers (Jang et al., 2001). Mutations in FGFR3 and FGFR4 are also

associated with colon cancer (Jang et al., 2001; Bange et al., 2002). Analyses of genetic mutations in 20 patients with anorectal malformations did not show any mutations in FGF10, suggesting it may not be genetically causative of these malformations in humans (Kruger et al., 2008). No other studies have associated mutations or single nucleotide polymorphisms (SNPs) in FGF ligands or receptors to intestinal abnormalities. Mutations in FGF ligands or receptors in human diseases such as duodenal and colonic atresia still require further investigation. To date, several reports have established links between levels of FGFs and intestinal diseases. In intestinal biopsies of IBD patients, FGF7 RNA and protein levels are elevated and positively correlate with the degree of inflammation (Finch et al., 1996; Brauchle et al., 1996). FGF7 levels are higher in ulcerative colitis compared to those in Crohn’s disease (Bajaj-Elliott et al., 1997). FGF7 is also increased in celiac disease (Salvati et al., 2001). FGF2 levels are elevated in blood samples from Crohn’s disease and ulcerative colitis patients, and the number of neutrophils expressing FGF2 in the intestine correlates with stenosis in ulcerative colitis patients, a usual outcome of excessive inflammation (Yamagata et al., 2011; Kanazawa et al., 2001). However, FGF2 is reduced at the RNA and protein levels in mucosa from tumor tissue obtained from patients with colorectal cancer compared to normal mucosa (Sundlisaeter et al., 2009), whereas FGF20 is upregulated in human colorectal cancers (Jeffers et al., 2001). Amplification of FGFR2 has been reported in colorectal cancer (Mathur et al., 2014). Our current knowledge of the implication of FGFs in human diseases is very limited and will require careful investigation, as this family of growth factors plays an important role in many cellular, molecular, and physiological events.

Therapeutic Use of FGF and FGF Inhibitors in Intestinal Diseases Palifermin, a truncated from of recombinant FGF7, is clinically used to treat oral mucositis. Despite the protective role of FGF7 shown in mouse and rat models of IBD, its use in humans to treat IBD is still under debate, as patients with IBD present a high risk of developing colorectal cancers and the administration of a mitogen could increase that risk. Repifermin, a truncated form of FGF10, was tested in a randomized clinical trial that included 88 patients with active ulcerative colitis. Repifermin did not prove efficacious in reducing the disease onset at the chosen times and doses (Sandborn et al., 2003). Targeting of FGF signaling using small molecule inhibitors to treat cancer, including colorectal cancers, has been reviewed in a comprehensive review by Turner and Grose (Turner and Grose, 2010). To date, there are no FGF targeting approaches in clinical practice for colorectal cancer treatment. Further investigations and trials are required in order to develop FGF pathway targeting approaches to treat human diseases.

Conclusion and Future Perspectives In summary, FGFs are key players in intestinal development, homeostasis, repair, and regeneration. Many studies have focused on the effect of FGF ligands and receptor signaling on the maintenance of the intestinal epithelial crypt cells. Epithelialmesenchymal interactions are essential for intestinal



development, homeostasis, and repair. Further studies are required to better understand the role of specific ligands/receptors in determining epithelial cell fate. Several other questions remain unanswered. How does FGF signaling control the maintenance and regeneration of intestinal mesenchymal stem cells? Furthermore, FGFs play an important role against inflammation-induced injury not only in the intestine but in other organ systems as well. Does FGF signaling directly regulate the immune cell response in the intestine? FGF signaling has been shown to be important in neural development; does it play a role in the formation of the enteric nervous system? Importantly, the role of canonical FGFs in the development of the human gastrointestinal tract and human intestinal regeneration is poorly understood. Bearing in mind the limitation of human studies, it would be important to address this gap of knowledge in order to develop successful targeted therapies.

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