REVIEW URRENT C OPINION
The multiple roles of guanylate cyclase C, a heat stable enterotoxin receptor Kris A. Steinbrecher
Purpose of review Guanylate cyclase C (GC-C) is a transmembrane receptor that is expressed primarily on intestinal epithelial cells. Activation of this receptor by its endogenous peptide ligands initiates cyclic guanosine monophosphate-dependent (cGMP) salt and water movement in the intestine. GC-C is targeted by the enterotoxigenic Escherichia coli heat-stable enterotoxin STa, which deregulates this pathway and causes secretory diarrhea. This review discusses current work on the physiological function of GC-C in the intestine. Recent findings Familial GC-C mutations demonstrate that epithelial cGMP signaling is critical to electrolyte and fluid balance in the neonatal intestine. Chronic deregulation of GC-C activity in early life increases susceptibility to a number of disorders, including obstruction and inflammatory bowel disease. Murine models indicate that GC-C regulates the composition of intestinal commensal microflora and that it suppresses bacterial infection and modulates colonic injury and inflammation. Therapeutic GC-C ligands are used to successfully treat constipation-predominant irritable bowel syndrome and recent studies show that extracellular cGMP is an important mechanism of reducing abdominal pain associated with this disorder. Summary Originally identified as a target of E. coli enterotoxin STa, GC-C is an important regulator of physiological salt and water homeostasis and may directly impact a wide range of intestinal disorders. Keywords cGMP, guanylate cyclase, infection, inflammatory bowel disease, irritable bowel syndrome
INTRODUCTION Diarrheal disease is a significant source of morbidity and mortality in the developing world and is often caused by enterotoxigenic Escherichia coli (ETEC). Recent estimates attribute ETEC as the cause of approximately 900 million episodes of diarrhea annually [1,2]. Enterotoxins secreted by ETEC include heat labile (LT) as well as heat stable (ST) variants. ETEC that produce ST enterotoxins elicit a robust secretory response in the intestine that is especially problematic in children. The importance of ST-producing ETEC to child health is evident in that the majority of the 300 000–500 000 deaths caused by this pathogen each year are of children less than 5 years old [2]. The isolation of ST enterotoxin led to an understanding of its mode of action within the intestine via deregulated epithelial ion flow [3,4]. ST-induced electrolyte movement is mediated by increases in the intracellular signaling molecule cyclic guanosine monophosphate (cGMP) that results in water secretion in the gut lumen.
Subsequent studies utilizing animals and human intestinal mucosa demonstrated binding of the STa enterotoxin variant at a significantly higher level during the neonatal period relative to the adult, and this may explain in part the debilitating disease caused by ETEC in infants [5–8]. The transmembrane receptor guanylate cyclase C (GC-C) was eventually identified as the target of ETEC STa and its expression is greatest during the first few years of life [9]. This review will highlight recent clinical and
Department of Pediatrics, University of Cincinnati College of Medicine, Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA Correspondence to Kris A. Steinbrecher, PhD, Cincinnati Children’s Hospital Medical Center, Division of Gastroenterology, Hepatology and Nutrition, MLC 2010, 3333 Burnet Ave, Cincinnati, Ohio 45220, USA. Tel: +1 513 636 4415; fax: +1 513 636 5581; e-mail: kris.stein [email protected] Curr Opin Gastroenterol 2014, 30:1–6 DOI:10.1097/MOG.0000000000000020
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KEY POINTS The critical importance of physiological GC-C signaling in maintaining intestinal fluid homeostasis has been confirmed by the identification of GC-C mutant kindreds who display familial diarrheal syndrome or non-CF meconium ileus. GC-C expression and activity in the intestine are greatest during the neonatal period and a crucial physiological role for GC-C at this age is strongly supported by human and animal model data. GC-C agonists are efficacious in the treatment of IBS and associated abdominal pain and recent work indicates that this is mediated through the release of cGMP across the basolateral membrane of epithelial cells. Disease caused by GC-C mutation in humans, as well as studies in laboratory animals, supports an emerging role for GC-C in enteric bacterial infection and IBD.
basic research reports indicating that GC-C signaling is important not only to enteric pathogen infection but to a number of intestinal disorders and that this receptor may have great potential as a therapeutic target to treat gastrointestinal disease.
LIGAND-INDUCED GC-C SIGNALING REGULATES ELECTROLYTE AND WATER FLOW INTO THE INTESTINAL LUMEN The presence of a specific lumen-oriented guanylate cyclase receptor that binds STa suggested the existence of an endogenous signaling system. Guanylin and uroguanylin are peptide ligands that are produced as prohormones in epithelia of the intestine. They are secreted into the lumen of the gut wherein they are activated by protease cleavage to signal through GC-C in a paracrine manner. Proper glycosylation of GC-C is essential for binding of these peptides to the extracellular portion of the receptor, which then triggers a well-controlled burst of cGMP within the cell [10]. A number of effector proteins are proposed to respond to cGMP production by GC-C, including phosphodiesterases, protein kinases and cyclic nucleotide-gated ion channels. The best-described downstream signaling component in enterocytes is the cGMP-dependent protein kinase II (PKG II). PKG II controls signaling cascades in many cell types in response to elevated cGMP levels and it is the critical intermediate that is essential to GC-Cregulated ion channel activation in the intestine [11–14]. PKG II regulates the cystic fibrosis transmembrane conductance regulator (CFTR) and Naþ Hþ exchanger 3 (NHE3) to increase chloride/ 2
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bicarbonate secretion and block sodium absorption. The resulting elevation in luminal salt increases water flow into the intestine and is important for hydration and bicarbonate-dependent unpackaging of secreted goblet cell mucins. This is the mechanistic basis of secretory diarrhea induced by ETEC STa. The primary sequence and overall structure of STa are very similar to guanylin and uroguanylin. Minor residue differences in STa result in superagonist activity that causes highly deregulated GC-C-mediated production of cGMP and massive, sustained salt and water secretion into the intestine. Although the vast majority of GC-C is present on the apical membrane of intestinal epithelial cells, there is some indication that GC-C may also be present on other cell types, including some regions of the brain [15,16]. Notably, GC-C ligands are present in the blood, and rodent studies indicate that the intestine is the primary source of these circulating prohormones [17]. The GC-C/ ligand-based gut–brain circuit is clearly physiologically relevant and this important new aspect of GC-C biology is reviewed elsewhere [18].
THE GC-C SIGNALING PATHWAY HAS A CRITICAL ROLE IN HYDRATING THE INTESTINAL LUMEN OF THE NEONATE Mutation in the human gene encoding GC-C, GUCY2C, causes familial intestinal disorders characterized by deregulated fluid secretion in the intestine. Following up on an initial report of familial meconium ileus not caused by cystic fibrosis (CF) [19], Romi et al. [20 ] describe two families with inactivating mutations in GUCY2C. Loss of GC-C function and diminished cGMP production in enterocytes of affected individuals initially caused intestinal obstruction at birth because of thickened meconium and this was often followed by diarrhea, enteric infection and sepsis during the first few months of life. An interesting aspect of the initial report is that most of these individuals were asymptomatic by 4–5 years of age [19]. Familial inheritance of activating GC-C mutations has also been recently described [21 ]. Fiskerstrand et al. [21 ] report a kindred in which GUCY2C mutation results in enhanced cGMP production upon activation, resulting in chronic diarrheal disease and dehydration that presents in infants and was often accompanied by metabolic acidosis and electrolyte imbalances. In addition, bowel obstruction due to volvulus and ileal inflammation was common in this group, as were inflammatory bowel disease (IBD) and infectious gastroenteritis. These reports provide a convincing answer to the long-held question of why the GC-C signaling system has been preserved in mammals even in the presence &&
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of negative selection pressure applied by ETEC infection. Although GC-C heterozygosity likely confers some resistance to diarrheal disease mediated by ETEC STa, GC-C and its ligands provide a key physiological function in humans by regulating intestinal secretion during the neonatal period and mediating a mechanism by which meconium is flushed from the gut [20 ]. While endpoint molecules in the GC-C signaling pathway such as NHE3 are proven modifiers of the meconium ileus CF phenotype, additional work will be necessary to determine whether the GC-C signaling pathway itself can affect CF intestinal disease severity as well [22]. It is notable that there is no mention of out of norm body mass, gastrointestinal cancer or behavioral issues in affected adults, although further long-term assessment may be necessary [20 ,21 ]. It is apparent that deregulated GC-C activity early in life is the defining event that elevates susceptibility to inflammation and infection in these individuals. This suggests that GC-C-regulated cGMP production is of the greatest physiological importance during the first few years of life. &&
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GC-C PROVIDES RESISTANCE TO ATTACHING/EFFACING LESION-FORMING BACTERIA The presence of GC-C on the apical surface of the enterocyte is highly detrimental when targeted by the ETEC STa enterotoxin. However, recent work indicates that GC-C signaling may provide resistance to bacteria that do not express STa [23 ]. Citrobacter rodentium are attaching/effacing lesionforming bacteria that cause a self-limiting infectious colitis and are used in rodents to model enteropathogenic and enterohemorrhagic E. coli [24]. Mice genetically deficient in GC-C (GC-C-/-) are more susceptible to infection with C. rodentium. Relative to wild-type mice, bacterial load at the height of the infection course is greater in GC-C-/- mice. Early after infection, prior to differences in mucosal apoptosis between wild-type and GC-C-/- mice, intestinal barrier defects are apparent in GC-C deficient mice, as is elevated cytokine expression in the liver. Mice lacking GC-C cannot contain the pathogen in the intestine as the infection progresses and have elevated bacteria in the liver and a corresponding increase in liver cytokines and histopathology. Relevant to the GC-C signaling pathway is the established role of CFTR and NHE3 in mucin dynamics and luminal pH [25–28]. These factors directly impact both the susceptibility to enteric pathogens as well as the numbers and composition of commensal microflora. Consistent with this, mice lacking GC-C have diminished levels of &
commensal bacteria colonization and a microflora community that is significantly altered in composition relative to wild-type mice [23 ]. Of interest, the family Enterobacteriaceae, of which E. coli and C. rodentium are members, is greatly expanded in GC-C-/- mice, and this suggests that diminished mucosal cGMP levels can lead to an environmental niche in which some pathogens can thrive [23 ,29]. This recent work indicates that, while GC-C provides an enterotoxin target that mediates infectious diarrheal disease, it may also be essential for wellregulated host–commensal interaction and suppression of bacterial pathogens that do not produce STa. &
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THERAPEUTIC AGONISTS OF GC-C ARE EFFICACIOUS IN THE TREATMENT OF BOTH CONSTIPATION AND VISCERAL PAIN ASSOCIATED WITH IRRITABLE BOWEL SYNDROME A number of studies very clearly demonstrate the therapeutic utility of GC-C agonists in treating constipation-predominant irritable bowel syndrome (IBS) and chronic idiopathic constipation [30–32]. An important aspect in the clinical use of GC-C agonists is that these peptides also diminish abdominal pain associated with IBS [33,34 ,35]. Developed independently, plecanatide and linaclotide are orally available GC-C agonists similar to guanylin and uroguanylin that activate lumenoriented GC-C and are not systemically absorbed. Linaclotide is FDA-approved and is effective in increasing fluid secretion in the small bowel and alleviating constipation and abdominal pain. Diminished pain perception occurs through a novel signaling mechanism in the intestine in which GC-C-dependent release of cGMP across the basolateral epithelial surface suppresses colonic nociception [36 ,37]. This work is particularly interesting not only because it provides an explanation for the analgesic effects of GC-C agonists but also because it ascribes a physiological role for extracellular cGMP in the intestine. Going forward, identifying the mechanism through which extracellular cGMP affects neuronal function will be critical [38]. GC-C agonist peptides are well tolerated and efficacious, and regulatory approval prompts consideration of their utility in treating additional gastrointestinal disorders, including intestinal inflammation. &&
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GC-C SIGNALING IS AN IMPORTANT MODIFIER OF GASTROINTESTINAL INFLAMMATION Mutation of the GUCY2C gene in some individuals is associated with the development of IBD
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[19,20 ,21 ]. Studies by multiple groups using murine models of intestinal inflammation suggest that GC-C and its ligands have a complex role in the development and mucosal response to inflammation and injury in the intestine. Mice lacking GC-C are resistant to dextran sodium sulfate (DSS) chemical-induced injury in the colon and this is due, in part, to diminished expression of resistinlike molecule beta (RELMb) [39]. RELMb is a proinflammatory goblet cell product that is necessary to drive cytokine expression and mucosal damage in ulcerating chemical colitis models [40,41]. However, Lin et al. [42] report contrasting data by suggesting that GC-C-/- mice have increased susceptibility to colonic injury by DSS. Although barrier function may influence the development of human IBD, this is not always the case in animal models of intestinal inflammation [40,41,43]. The GC-C-/- mice used in both of these studies have elevated intestinal permeability under some conditions but the
STa, Gn, Ugn Linaclotide, Plecanatide
mechanism through which GC-C signaling affects epithelial integrity is not defined [44–46]. Furthermore, barrier dysfunction may not always be relevant to the DSS colitis model because it relies on widespread ulceration of the colonic epithelia to initiate disease. It is notable that analysis of mice deficient in the GC-C/cGMP effector kinase PKG II demonstrated no difference in sensitivity to DSSinduced colonic injury [14]. Importantly, these apparently conflicting findings may be because of not only variation in experimental approach but also the differences in colony microflora, an aspect of murine disease modeling that is clearly relevant in interpreting colitis susceptibility phenotypes [47–49]. It should also be noted that, although use of chemical colitis models can be informative, they do not provide the most accurate means to model human IBD in laboratory animals. Spontaneous intestinal inflammation in mice lacking the immunosuppressive molecule interleukin 10
Intestinal Lumen CI+ HCO3–
H+
GC–C
Water
Extracellular Domain
CFTR
NHE3
Na+ Guanylate Cyclase Domain
PKG II Intestinal Epithelial cell
GTP cGMP
(CNG,PDE) Pro–Gn Pro–Ugn
cGMP
Reduced visceral pain
Brain (satiety, behavior) Kidneys (systemic salt regulation)
FIGURE 1. Role of GC-C in the epithelial cell of the intestine. Production of cGMP by GC-C regulates ion and water flow across the epithelial monolayer and may reduce visceral pain associated with irritable bowel syndrome. GC-C ligands are released from the intestine as prohormones (Pro-Gn, Pro-Ugn) and can regulate food intake, behavior, and systemic salt homeostasis. CFTR, cystic fibrosis transmembrane conductance regulator; CNG, cyclic nucleotide gated channel; Gn, guanylin; NHE3, sodium hydrogen exchanger 3; PDE, phosphodiesterase; PKG II, cGMP-dependent protein kinase II; Ugn, uroguanylin. 4
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(IL-10) more closely replicates the pathophysiology found in human colitis [50,51]. Consistently with elevated expression and activity of GC-C in the neonatal intestine, we have recently found that mice lacking both IL-10 and GC-C develop early onset, severe intestinal inflammation as compared with GC-C sufficient IL-10 null mice (manuscript in press). Furthermore, expression of guanylin is lost at very early stages of inflammation, prior to colitisinduced goblet cell depletion, and in-vitro studies indicate that tumor necrosis factor alpha is able to profoundly suppress both guanylin mRNA and protein production. Reduced expression of GC-C ligands has also been shown in human IBD and it is possible that loss of GC-C signaling during the early stages of intestinal inflammation accelerates disease progression [52]. This may be because of altered transmembrane sodium, chloride and/or bicarbonate movement, and the resulting defective water secretion and pH imbalance at the epithelial surface. A related finding can be seen in mice lacking CFTR, in which defective mucus hydration and Paneth cell granule release results in poorly controlled growth of commensal bacteria as well as loss of an effective physical barrier between the epithelial cell monolayer and the bacterial population [27,53,54]. Similarly, mice deficient in NHE3 have enhanced bacterial adhesion and translocation, are prone to spontaneous inflammation, and have more severe colitis in an IL-10 null setting [28,55,56]. In the context of loss of immunosuppression, as can be the case in IBD, the inability to properly regulate commensal microflora is a critically important aspect of disease initiation and severity [57,58]. It may be appropriate to view GC-C signaling to cell membrane transporters, as it relates to ion flow and water secretion, as a pathway critical to establishing a luminal microenvironment that facilitates a beneficial commensal microflora population. This is likely to be especially important during the neonatal/weaning period when the height of GC-C signaling activity coincides with the initial microflora colonization of the intestine and the subsequent rapid expansion in species composition and complexity.
CONCLUSION In addition to its long-standing role in secretory diarrhea, recent advances indicate that the GC-C signaling pathway is of surprisingly broad physiological importance (Fig. 1). The defining function for GC-C and its ligands, however, remains the regulation of salt and water homeostasis [20 ,21 ]. The identification of familial GC-C mutations and the clinical presentation of affected family members &&
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underscores the critical function for this epithelial receptor in regulation of intestinal hydration at birth and during the first few years of life. Future studies will be necessary to determine whether the success of therapeutic GC-C agonists in treating constipationpredominant intestinal disorders can be replicated in other disease conditions such as intestinal inflammation. Acknowledgements The author apologizes to those whose work could not be cited here due to space constraints. Work in this laboratory was supported by AI107274 and DK047318. Conflicts of interest There are no conflicts of interest.
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The multiple roles of guanylate cyclase C, a heat stable enterotoxin receptor Kris A. Steinbrecher
Purpose of review Guanylate cyclase C (GC-C) is a transmembrane receptor that is expressed primarily on intestinal epithelial cells. Activation of this receptor by its endogenous peptide ligands initiates cyclic guanosine monophosphate-dependent (cGMP) salt and water movement in the intestine. GC-C is targeted by the enterotoxigenic Escherichia coli heat-stable enterotoxin STa, which deregulates this pathway and causes secretory diarrhea. This review discusses current work on the physiological function of GC-C in the intestine. Recent findings Familial GC-C mutations demonstrate that epithelial cGMP signaling is critical to electrolyte and fluid balance in the neonatal intestine. Chronic deregulation of GC-C activity in early life increases susceptibility to a number of disorders, including obstruction and inflammatory bowel disease. Murine models indicate that GC-C regulates the composition of intestinal commensal microflora and that it suppresses bacterial infection and modulates colonic injury and inflammation. Therapeutic GC-C ligands are used to successfully treat constipation-predominant irritable bowel syndrome and recent studies show that extracellular cGMP is an important mechanism of reducing abdominal pain associated with this disorder. Summary Originally identified as a target of E. coli enterotoxin STa, GC-C is an important regulator of physiological salt and water homeostasis and may directly impact a wide range of intestinal disorders. Keywords cGMP, guanylate cyclase, infection, inflammatory bowel disease, irritable bowel syndrome
INTRODUCTION Diarrheal disease is a significant source of morbidity and mortality in the developing world and is often caused by enterotoxigenic Escherichia coli (ETEC). Recent estimates attribute ETEC as the cause of approximately 900 million episodes of diarrhea annually [1,2]. Enterotoxins secreted by ETEC include heat labile (LT) as well as heat stable (ST) variants. ETEC that produce ST enterotoxins elicit a robust secretory response in the intestine that is especially problematic in children. The importance of ST-producing ETEC to child health is evident in that the majority of the 300 000–500 000 deaths caused by this pathogen each year are of children less than 5 years old [2]. The isolation of ST enterotoxin led to an understanding of its mode of action within the intestine via deregulated epithelial ion flow [3,4]. ST-induced electrolyte movement is mediated by increases in the intracellular signaling molecule cyclic guanosine monophosphate (cGMP) that results in water secretion in the gut lumen.
Subsequent studies utilizing animals and human intestinal mucosa demonstrated binding of the STa enterotoxin variant at a significantly higher level during the neonatal period relative to the adult, and this may explain in part the debilitating disease caused by ETEC in infants [5–8]. The transmembrane receptor guanylate cyclase C (GC-C) was eventually identified as the target of ETEC STa and its expression is greatest during the first few years of life [9]. This review will highlight recent clinical and
Department of Pediatrics, University of Cincinnati College of Medicine, Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA Correspondence to Kris A. Steinbrecher, PhD, Cincinnati Children’s Hospital Medical Center, Division of Gastroenterology, Hepatology and Nutrition, MLC 2010, 3333 Burnet Ave, Cincinnati, Ohio 45220, USA. Tel: +1 513 636 4415; fax: +1 513 636 5581; e-mail: kris.stein [email protected] Curr Opin Gastroenterol 2014, 30:1–6 DOI:10.1097/MOG.0000000000000020
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Gastrointestinal infections
KEY POINTS The critical importance of physiological GC-C signaling in maintaining intestinal fluid homeostasis has been confirmed by the identification of GC-C mutant kindreds who display familial diarrheal syndrome or non-CF meconium ileus. GC-C expression and activity in the intestine are greatest during the neonatal period and a crucial physiological role for GC-C at this age is strongly supported by human and animal model data. GC-C agonists are efficacious in the treatment of IBS and associated abdominal pain and recent work indicates that this is mediated through the release of cGMP across the basolateral membrane of epithelial cells. Disease caused by GC-C mutation in humans, as well as studies in laboratory animals, supports an emerging role for GC-C in enteric bacterial infection and IBD.
basic research reports indicating that GC-C signaling is important not only to enteric pathogen infection but to a number of intestinal disorders and that this receptor may have great potential as a therapeutic target to treat gastrointestinal disease.
LIGAND-INDUCED GC-C SIGNALING REGULATES ELECTROLYTE AND WATER FLOW INTO THE INTESTINAL LUMEN The presence of a specific lumen-oriented guanylate cyclase receptor that binds STa suggested the existence of an endogenous signaling system. Guanylin and uroguanylin are peptide ligands that are produced as prohormones in epithelia of the intestine. They are secreted into the lumen of the gut wherein they are activated by protease cleavage to signal through GC-C in a paracrine manner. Proper glycosylation of GC-C is essential for binding of these peptides to the extracellular portion of the receptor, which then triggers a well-controlled burst of cGMP within the cell [10]. A number of effector proteins are proposed to respond to cGMP production by GC-C, including phosphodiesterases, protein kinases and cyclic nucleotide-gated ion channels. The best-described downstream signaling component in enterocytes is the cGMP-dependent protein kinase II (PKG II). PKG II controls signaling cascades in many cell types in response to elevated cGMP levels and it is the critical intermediate that is essential to GC-Cregulated ion channel activation in the intestine [11–14]. PKG II regulates the cystic fibrosis transmembrane conductance regulator (CFTR) and Naþ Hþ exchanger 3 (NHE3) to increase chloride/ 2
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bicarbonate secretion and block sodium absorption. The resulting elevation in luminal salt increases water flow into the intestine and is important for hydration and bicarbonate-dependent unpackaging of secreted goblet cell mucins. This is the mechanistic basis of secretory diarrhea induced by ETEC STa. The primary sequence and overall structure of STa are very similar to guanylin and uroguanylin. Minor residue differences in STa result in superagonist activity that causes highly deregulated GC-C-mediated production of cGMP and massive, sustained salt and water secretion into the intestine. Although the vast majority of GC-C is present on the apical membrane of intestinal epithelial cells, there is some indication that GC-C may also be present on other cell types, including some regions of the brain [15,16]. Notably, GC-C ligands are present in the blood, and rodent studies indicate that the intestine is the primary source of these circulating prohormones [17]. The GC-C/ ligand-based gut–brain circuit is clearly physiologically relevant and this important new aspect of GC-C biology is reviewed elsewhere [18].
THE GC-C SIGNALING PATHWAY HAS A CRITICAL ROLE IN HYDRATING THE INTESTINAL LUMEN OF THE NEONATE Mutation in the human gene encoding GC-C, GUCY2C, causes familial intestinal disorders characterized by deregulated fluid secretion in the intestine. Following up on an initial report of familial meconium ileus not caused by cystic fibrosis (CF) [19], Romi et al. [20 ] describe two families with inactivating mutations in GUCY2C. Loss of GC-C function and diminished cGMP production in enterocytes of affected individuals initially caused intestinal obstruction at birth because of thickened meconium and this was often followed by diarrhea, enteric infection and sepsis during the first few months of life. An interesting aspect of the initial report is that most of these individuals were asymptomatic by 4–5 years of age [19]. Familial inheritance of activating GC-C mutations has also been recently described [21 ]. Fiskerstrand et al. [21 ] report a kindred in which GUCY2C mutation results in enhanced cGMP production upon activation, resulting in chronic diarrheal disease and dehydration that presents in infants and was often accompanied by metabolic acidosis and electrolyte imbalances. In addition, bowel obstruction due to volvulus and ileal inflammation was common in this group, as were inflammatory bowel disease (IBD) and infectious gastroenteritis. These reports provide a convincing answer to the long-held question of why the GC-C signaling system has been preserved in mammals even in the presence &&
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Emerging roles for guanylate cyclase C in intestine Steinbrecher
of negative selection pressure applied by ETEC infection. Although GC-C heterozygosity likely confers some resistance to diarrheal disease mediated by ETEC STa, GC-C and its ligands provide a key physiological function in humans by regulating intestinal secretion during the neonatal period and mediating a mechanism by which meconium is flushed from the gut [20 ]. While endpoint molecules in the GC-C signaling pathway such as NHE3 are proven modifiers of the meconium ileus CF phenotype, additional work will be necessary to determine whether the GC-C signaling pathway itself can affect CF intestinal disease severity as well [22]. It is notable that there is no mention of out of norm body mass, gastrointestinal cancer or behavioral issues in affected adults, although further long-term assessment may be necessary [20 ,21 ]. It is apparent that deregulated GC-C activity early in life is the defining event that elevates susceptibility to inflammation and infection in these individuals. This suggests that GC-C-regulated cGMP production is of the greatest physiological importance during the first few years of life. &&
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GC-C PROVIDES RESISTANCE TO ATTACHING/EFFACING LESION-FORMING BACTERIA The presence of GC-C on the apical surface of the enterocyte is highly detrimental when targeted by the ETEC STa enterotoxin. However, recent work indicates that GC-C signaling may provide resistance to bacteria that do not express STa [23 ]. Citrobacter rodentium are attaching/effacing lesionforming bacteria that cause a self-limiting infectious colitis and are used in rodents to model enteropathogenic and enterohemorrhagic E. coli [24]. Mice genetically deficient in GC-C (GC-C-/-) are more susceptible to infection with C. rodentium. Relative to wild-type mice, bacterial load at the height of the infection course is greater in GC-C-/- mice. Early after infection, prior to differences in mucosal apoptosis between wild-type and GC-C-/- mice, intestinal barrier defects are apparent in GC-C deficient mice, as is elevated cytokine expression in the liver. Mice lacking GC-C cannot contain the pathogen in the intestine as the infection progresses and have elevated bacteria in the liver and a corresponding increase in liver cytokines and histopathology. Relevant to the GC-C signaling pathway is the established role of CFTR and NHE3 in mucin dynamics and luminal pH [25–28]. These factors directly impact both the susceptibility to enteric pathogens as well as the numbers and composition of commensal microflora. Consistent with this, mice lacking GC-C have diminished levels of &
commensal bacteria colonization and a microflora community that is significantly altered in composition relative to wild-type mice [23 ]. Of interest, the family Enterobacteriaceae, of which E. coli and C. rodentium are members, is greatly expanded in GC-C-/- mice, and this suggests that diminished mucosal cGMP levels can lead to an environmental niche in which some pathogens can thrive [23 ,29]. This recent work indicates that, while GC-C provides an enterotoxin target that mediates infectious diarrheal disease, it may also be essential for wellregulated host–commensal interaction and suppression of bacterial pathogens that do not produce STa. &
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THERAPEUTIC AGONISTS OF GC-C ARE EFFICACIOUS IN THE TREATMENT OF BOTH CONSTIPATION AND VISCERAL PAIN ASSOCIATED WITH IRRITABLE BOWEL SYNDROME A number of studies very clearly demonstrate the therapeutic utility of GC-C agonists in treating constipation-predominant irritable bowel syndrome (IBS) and chronic idiopathic constipation [30–32]. An important aspect in the clinical use of GC-C agonists is that these peptides also diminish abdominal pain associated with IBS [33,34 ,35]. Developed independently, plecanatide and linaclotide are orally available GC-C agonists similar to guanylin and uroguanylin that activate lumenoriented GC-C and are not systemically absorbed. Linaclotide is FDA-approved and is effective in increasing fluid secretion in the small bowel and alleviating constipation and abdominal pain. Diminished pain perception occurs through a novel signaling mechanism in the intestine in which GC-C-dependent release of cGMP across the basolateral epithelial surface suppresses colonic nociception [36 ,37]. This work is particularly interesting not only because it provides an explanation for the analgesic effects of GC-C agonists but also because it ascribes a physiological role for extracellular cGMP in the intestine. Going forward, identifying the mechanism through which extracellular cGMP affects neuronal function will be critical [38]. GC-C agonist peptides are well tolerated and efficacious, and regulatory approval prompts consideration of their utility in treating additional gastrointestinal disorders, including intestinal inflammation. &&
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GC-C SIGNALING IS AN IMPORTANT MODIFIER OF GASTROINTESTINAL INFLAMMATION Mutation of the GUCY2C gene in some individuals is associated with the development of IBD
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[19,20 ,21 ]. Studies by multiple groups using murine models of intestinal inflammation suggest that GC-C and its ligands have a complex role in the development and mucosal response to inflammation and injury in the intestine. Mice lacking GC-C are resistant to dextran sodium sulfate (DSS) chemical-induced injury in the colon and this is due, in part, to diminished expression of resistinlike molecule beta (RELMb) [39]. RELMb is a proinflammatory goblet cell product that is necessary to drive cytokine expression and mucosal damage in ulcerating chemical colitis models [40,41]. However, Lin et al. [42] report contrasting data by suggesting that GC-C-/- mice have increased susceptibility to colonic injury by DSS. Although barrier function may influence the development of human IBD, this is not always the case in animal models of intestinal inflammation [40,41,43]. The GC-C-/- mice used in both of these studies have elevated intestinal permeability under some conditions but the
STa, Gn, Ugn Linaclotide, Plecanatide
mechanism through which GC-C signaling affects epithelial integrity is not defined [44–46]. Furthermore, barrier dysfunction may not always be relevant to the DSS colitis model because it relies on widespread ulceration of the colonic epithelia to initiate disease. It is notable that analysis of mice deficient in the GC-C/cGMP effector kinase PKG II demonstrated no difference in sensitivity to DSSinduced colonic injury [14]. Importantly, these apparently conflicting findings may be because of not only variation in experimental approach but also the differences in colony microflora, an aspect of murine disease modeling that is clearly relevant in interpreting colitis susceptibility phenotypes [47–49]. It should also be noted that, although use of chemical colitis models can be informative, they do not provide the most accurate means to model human IBD in laboratory animals. Spontaneous intestinal inflammation in mice lacking the immunosuppressive molecule interleukin 10
Intestinal Lumen CI+ HCO3–
H+
GC–C
Water
Extracellular Domain
CFTR
NHE3
Na+ Guanylate Cyclase Domain
PKG II Intestinal Epithelial cell
GTP cGMP
(CNG,PDE) Pro–Gn Pro–Ugn
cGMP
Reduced visceral pain
Brain (satiety, behavior) Kidneys (systemic salt regulation)
FIGURE 1. Role of GC-C in the epithelial cell of the intestine. Production of cGMP by GC-C regulates ion and water flow across the epithelial monolayer and may reduce visceral pain associated with irritable bowel syndrome. GC-C ligands are released from the intestine as prohormones (Pro-Gn, Pro-Ugn) and can regulate food intake, behavior, and systemic salt homeostasis. CFTR, cystic fibrosis transmembrane conductance regulator; CNG, cyclic nucleotide gated channel; Gn, guanylin; NHE3, sodium hydrogen exchanger 3; PDE, phosphodiesterase; PKG II, cGMP-dependent protein kinase II; Ugn, uroguanylin. 4
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Emerging roles for guanylate cyclase C in intestine Steinbrecher
(IL-10) more closely replicates the pathophysiology found in human colitis [50,51]. Consistently with elevated expression and activity of GC-C in the neonatal intestine, we have recently found that mice lacking both IL-10 and GC-C develop early onset, severe intestinal inflammation as compared with GC-C sufficient IL-10 null mice (manuscript in press). Furthermore, expression of guanylin is lost at very early stages of inflammation, prior to colitisinduced goblet cell depletion, and in-vitro studies indicate that tumor necrosis factor alpha is able to profoundly suppress both guanylin mRNA and protein production. Reduced expression of GC-C ligands has also been shown in human IBD and it is possible that loss of GC-C signaling during the early stages of intestinal inflammation accelerates disease progression [52]. This may be because of altered transmembrane sodium, chloride and/or bicarbonate movement, and the resulting defective water secretion and pH imbalance at the epithelial surface. A related finding can be seen in mice lacking CFTR, in which defective mucus hydration and Paneth cell granule release results in poorly controlled growth of commensal bacteria as well as loss of an effective physical barrier between the epithelial cell monolayer and the bacterial population [27,53,54]. Similarly, mice deficient in NHE3 have enhanced bacterial adhesion and translocation, are prone to spontaneous inflammation, and have more severe colitis in an IL-10 null setting [28,55,56]. In the context of loss of immunosuppression, as can be the case in IBD, the inability to properly regulate commensal microflora is a critically important aspect of disease initiation and severity [57,58]. It may be appropriate to view GC-C signaling to cell membrane transporters, as it relates to ion flow and water secretion, as a pathway critical to establishing a luminal microenvironment that facilitates a beneficial commensal microflora population. This is likely to be especially important during the neonatal/weaning period when the height of GC-C signaling activity coincides with the initial microflora colonization of the intestine and the subsequent rapid expansion in species composition and complexity.
CONCLUSION In addition to its long-standing role in secretory diarrhea, recent advances indicate that the GC-C signaling pathway is of surprisingly broad physiological importance (Fig. 1). The defining function for GC-C and its ligands, however, remains the regulation of salt and water homeostasis [20 ,21 ]. The identification of familial GC-C mutations and the clinical presentation of affected family members &&
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underscores the critical function for this epithelial receptor in regulation of intestinal hydration at birth and during the first few years of life. Future studies will be necessary to determine whether the success of therapeutic GC-C agonists in treating constipationpredominant intestinal disorders can be replicated in other disease conditions such as intestinal inflammation. Acknowledgements The author apologizes to those whose work could not be cited here due to space constraints. Work in this laboratory was supported by AI107274 and DK047318. Conflicts of interest There are no conflicts of interest.
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