The evolutionary divergence of receptor guanylyl cyclase C has implications for preclinical models for receptor-directed therapeutics.

Mutations in receptor guanylyl cyclase C (GC-C) cause severe gastrointestinal disease, including meconium ileus, early onset acute diarrhea, and pediatric inflammatory bowel disease that continues into adulthood. Agonists of GC-C are US Food and Drug Administration-approved drugs for the treatment of constipation and irritable bowel syndrome. Therapeutic strategies targeting GC-C are tested in preclinical mouse models, assuming that murine GC-C mimics human GC-C in its biochemical properties and downstream signaling events. Here, we reveal important differences in ligand-binding affinity and GC activity between mouse GC-C and human GC-C. We generated a series of chimeric constructs of various domains of human and mouse GC-C to show that the extracellular domain of mouse GC-C contributed to log-orders lower affinity of mouse GC-C for ligands than human GC-C. Further, the Vmax of the murine GC domain was lower than that of human GC-C, and allosteric regulation of the receptor by ATP binding to the intracellular kinase-homology domain also differed. These altered properties are reflected in the high concentrations of ligands required to elicit signaling responses in the mouse gut in preclinical models and the specificity of a GC inhibitor towards human GC-C. Therefore, our studies identify considerations in using the murine model to test molecules for therapeutic purposes that work as either agonists or antagonists of GC-C, and vaccines for the bacterial heat-stable enterotoxin that causes watery diarrhea in humans.

Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

cGMP controls many cellular functions ranging from growth, viability, and differentiation to contractility, secretion, and ion transport. The mammalian genome encodes seven transmembrane guanylyl cyclases (GCs), GC-A to GC-G, which mainly modulate submembrane cGMP microdomains. These GCs share a unique topology comprising an extracellular domain, a short transmembrane region, and an intracellular COOH-terminal catalytic (cGMP synthesizing) region. GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure/volume and energy balance. GC-B is activated by C-type natriuretic peptide, stimulating endochondral ossification in autocrine way. GC-C mediates the paracrine effects of guanylins on intestinal ion transport and epithelial turnover. GC-E and GC-F are expressed in photoreceptor cells of the retina, and their activation by intracellular Ca(2+)-regulated proteins is essential for vision. Finally, in the rodent system two olfactorial GCs, GC-D and GC-G, are activated by low concentrations of CO2and by peptidergic (guanylins) and nonpeptidergic odorants as well as by coolness, which has implications for social behaviors. In the past years advances in human and mouse genetics as well as the development of sensitive biosensors monitoring the spatiotemporal dynamics of cGMP in living cells have provided novel relevant information about this receptor family. This increased our understanding of the mechanisms of signal transduction, regulation, and (dys)function of the membrane GCs, clarified their relevance for genetic and acquired diseases and, importantly, has revealed novel targets for therapies. The present review aims to illustrate these different features of membrane GCs and the main open questions in this field.

Palmitic acid induces guanylin gene expression through the Toll-like receptor 4/nuclear factor-κB pathway in rat macrophages.

Recently, we showed that double-transgenic rats overexpressing guanylin (Gn), a bioactive peptide, and its receptor, guanylyl cyclase-C (GC-C), specifically in macrophages demonstrate an antiobesity phenotype and low-expression levels of proinflammatory cytokines in the mesenteric fat even when fed a high-fat diet. Here, we examined the levels and mechanism of Gn and GC-C transcription following saturated fatty acid and lipopolysaccharide (LPS), an activator of Toll-like receptor 4 (TLR4), exposure by using the NR8383 macrophage cell line. In addition, the levels of guanylin and cGMP were increased by addition of either palmitic acid or LPS. Next, we investigated the interaction of the gene transcription and nuclear factor-κB (NF-κB) by using an NF-κB inhibitor and chromatin immunoprecipitation assay. We showed that palmitic acid induced Gn gene expression via TLR4 and NF-κB. Moreover, we demonstrated that NF-κB binding to the Gn promoter was responsible for the induction of gene transcription by palmitic acid or LPS. Our results indicate that saturated fatty acids such as palmitic acid activate Gn gene expression via the NF-κB pathway, raising the possibility that the activated Gn-GC-C system may contribute to the inhibition of high-fat diet-induced proinflammatory cytokines in macrophages.

Prime-Boost Immunization Eliminates Metastatic Colorectal Cancer by Producing High-Avidity Effector CD8+ T Cells.

Heterologous prime-boost immunization with plasmid DNA and viral vector vaccines is an emerging approach to elicit CD8+ T cell-mediated immunity targeting pathogens and tumor Ags that is superior to either monotherapy. Yet, the mechanisms underlying the synergy of prime-boost strategies remain incompletely defined. In this study, we examine a DNA and adenovirus (Ad5) combination regimen targeting guanylyl cyclase C (GUCY2C), a receptor expressed by intestinal mucosa and universally expressed by metastatic colorectal cancer. DNA immunization efficacy was optimized by i.m. delivery via electroporation, yet it remained modest compared with Ad5. Sequential immunization with DNA and Ad5 produced superior antitumor efficacy associated with increased TCR avidity, whereas targeted disruption of TCR avidity enhancement eliminated GUCY2C-specific antitumor efficacy, without affecting responding T cell number or cytokine profile. Indeed, functional TCR avidity of responding GUCY2C-specific CD8+ T cells induced by various prime or prime-boost regimens correlated with antitumor efficacy, whereas T cell number and cytokine profile were not. Importantly, although sequential immunization with DNA and Ad5 maximized antitumor efficacy through TCR avidity enhancement, it produced no autoimmunity, reflecting sequestration of GUCY2C to intestinal apical membranes and segregation of mucosal and systemic immunity. Together, TCR avidity enhancement may be leveraged by prime-boost immunization to improve GUCY2C-targeted colorectal cancer immunotherapeutic efficacy and patient outcomes without concomitant autoimmune toxicity.

Guanylate cyclase-C as a therapeutic target in gastrointestinal disorders.

Functional gastrointestinal disorders (FGIDs) and IBDs are two of the most prevalent disorders of the GI tract and consume a significant proportion of healthcare resources. Recent studies have shown that membrane-bound guanylate cyclase-C (GC-C) receptors lining the GI tract may serve as novel therapeutic targets in the treatment of FGIDs and IBDs. GC-C receptor activation by its endogenous paracrine hormones uroguanylin and guanylin, and the resulting intracellular production of its downstream effector cyclic GMP, occurs in a pH-dependent manner and modulates key physiological functions. These include fluid and electrolyte homeostasis, maintenance of the intestinal barrier, anti-inflammatory activity and regulation of epithelial regeneration. Studies of the GC-C paracrine signalling axis have revealed the therapeutic potential of these receptors in treating GI disorders, including chronic idiopathic constipation and irritable bowel syndrome-constipation. This review focuses on the evolving understanding of GC-C function in health and disease, and strategies for translating these principles into new treatments for FGIDs and IBDs.

Absence of Receptor Guanylyl Cyclase C Enhances Ileal Damage and Reduces Cytokine and Antimicrobial Peptide Production during Oral Salmonella enterica Serovar Typhimurium Infection.

Nontyphoidal Salmonella disease contributes toward significant morbidity and mortality across the world. Host factors, including gamma interferon, tumor necrosis factor alpha, and gut microbiota, significantly influence the outcome of Salmonella pathogenesis. However, the entire repertoire of host protective mechanisms contributing to Salmonella pathogenicity is not completely appreciated. Here, we investigated the roles of receptor guanylyl cyclase C (GC-C), which is predominantly expressed in the intestine and regulates intestinal cell proliferation and fluid-ion homeostasis. Mice deficient in GC-C (Gucy2c-/-) displayed accelerated mortality compared with that for wild-type mice following infection via the oral route, even though both groups possessed comparable systemic Salmonella infection burdens. Survival following intraperitoneal infection remained similar in both groups, indicating that GC-C offered protection via a gut-mediated response. The serum cortisol level was higher in Gucy2c-/- mice than wild-type (Gucy2c+/+) mice, and an increase in infection-induced thymic atrophy with a loss of immature CD4+ CD8+ double-positive thymocytes was observed. Accelerated and enhanced damage in the ileum, including submucosal edema, epithelial cell damage, focal tufting, and distortion of the villus architecture, was seen in Gucy2c-/- mice concomitantly with a larger number of ileal tissue-associated bacteria. Transcription of key mediators of Salmonella-induced inflammation (interleukin-22/Reg3ß) was altered in Gucy2c-/- mice in comparison to that in Gucy2c+/+ mice. A reduction in fecal lactobacilli, which are protective against Salmonella infection, was observed in Gucy2c-/- mice. Gucy2c-/- mice cohoused with wild-type mice continued to show reduced amounts of lactobacilli and increased susceptibility to infection. Our study, therefore, suggests that the receptor GC-C confers a survival advantage during gut-mediated Salmonella enterica serovar Typhimurium pathogenesis, presumably by regulating Salmonella effector mechanisms and maintaining a beneficial microbiome.

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes.

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues.

Atrial Natriuretic Peptide　- Old But New Therapeutic in Cardiovascular Diseases.

With the discovery of atrial natriuretic peptide (ANP), the heart as an endocrine organ was established. Basic science revealed that ANP, through the particulate guanylyl cyclase A receptor and cGMP, plays a fundamental role in cardiorenal biology. This work has led to the development of ANP as a therapeutic, especially in heart failure (HF). Human genomics has strengthened our understanding of ANP, revealing specific ANP gene variants that may be associated with biological dysfunction, but also may mediate protective properties, including in metabolic syndrome. Advances in understanding the processing and degradation of ANP molecular forms have resulted in therapeutic breakthroughs, especially inhibition of ANP degradation by neprilysin inhibitors. Although ANP is administered intravenously for acute HF, a novel therapeutic strategy is its chronic delivery by subcutaneous injection. An innovative therapeutic development is engineering to develop ANP-based peptides for chronic use. These interconnected topics of ANP biology and therapeutics will be reviewed in detail.

Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C.

OBJECTIVE: Congenital sodium diarrhoea (CSD) refers to a form of secretory diarrhoea with intrauterine onset and high faecal losses of sodium without congenital malformations. The molecular basis for CSD remains unknown. We clinically characterised a cohort of infants with CSD and set out to identify disease-causing mutations by genome-wide genetic testing. DESIGN: We performed whole-exome sequencing and chromosomal microarray analyses in 4 unrelated patients, followed by confirmatory Sanger sequencing of the likely disease-causing mutations in patients and in their family members, followed by functional studies. RESULTS: We identified novel de novo missense mutations in GUCY2C, the gene encoding receptor guanylate cyclase C (GC-C) in 4 patients with CSD. One patient developed severe, early-onset IBD and chronic arthritis at 4 years of age. GC-C is an intestinal brush border membrane-bound guanylate cyclase, which functions as receptor for guanylin, uroguanylin and Escherichia coli heat-stable enterotoxin. Mutations in GUCY2C were present in different intracellular domains of GC-C, and were activating mutations that enhanced intracellular cyclic guanosine monophosphate accumulation in a ligand-independent and ligand-stimulated manner, following heterologous expression in HEK293T cells. CONCLUSIONS: Dominant gain-of-function GUCY2C mutations lead to elevated intracellular cyclic guanosine monophosphate levels and could explain the chronic diarrhoea as a result of decreased intestinal sodium and water absorption and increased chloride secretion. Thus, mutations in GUCY2C indicate a role for this receptor in the pathogenesis of sporadic CSD.

Intestinal Enteroids Model Guanylate Cyclase C-Dependent Secretion Induced by Heat-Stable Enterotoxins.

Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease.

Molecular Mechanisms of Reception and Perireception in Crustacean Chemoreception: A Comparative Review.

This review summarizes our present knowledge of chemoreceptor proteins in crustaceans, using a comparative perspective to review these molecules in crustaceans relative to other metazoan models of chemoreception including mammals, insects, nematodes, and molluscs. Evolution has resulted in unique expansions of specific gene families and repurposing of them for chemosensation in various clades, including crustaceans. A major class of chemoreceptor proteins across crustaceans is the Ionotropic Receptors, which diversified from ionotropic glutamate receptors in ancient protostomes but which are not present in deuterostomes. Representatives of another major class of chemoreceptor proteins-the Grl/GR/OR family of ionotropic 7-transmembrane receptors-are diversified in insects but to date have been reported in only one crustacean species, Daphnia pulex So far, canonic 7-transmembrane G-protein coupled receptors, the principal chemoreceptors in vertebrates and reported in a few protostome clades, have not been identified in crustaceans. More types of chemoreceptors are known throughout the metazoans and might well be expected to be discovered in crustaceans. Our review also provides a comparative coverage of perireceptor events in crustacean chemoreception, including molecules involved in stimulus acquisition, stimulus delivery, and stimulus removal, though much less is known about these events in crustaceans, particularly at the molecular level.

An activating gucy2c mutation causes impaired contractility and fluid stagnation in the small bowel.

OBJECTIVE: Familial GUCY2C diarrhoea syndrome (FGDS) is caused by an activating mutation in the GUCY2C gene encoding the receptor guanylate cyclase C in enterocytes. Activation leads to increased secretion of fluid into the intestinal lumen. Twenty percent of the patients have increased risk of Crohn's disease and intestinal obstruction (CD, 20%) and the condition resembles irritable bowel syndrome with diarrhoea. We aimed to describe fluid content, contractility, peristaltic activity and bowel wall thickness in the intestine in fasting FGDS patients, using ultrasound, with healthy volunteers serving as controls. METHODS: Twenty-three patients with FGDS and 22 healthy controls (HC) were examined with a Logiq E9 scanner in a fasting state. Bowel wall thickness was measured and fluid-filled small bowel loops were counted using three-dimensional (3D) magnetic positioning navigation. The HC ingested 500 ml PEG solution, an electrolyte balanced, non-absorbable solution, in order to investigate the contractions of the small bowel. RESULTS: The fasting 23 FGDS patients had significantly higher number of fluid-filled small bowel segments compared to 22 fasting HC, p < 0.001. A high number of non-occlusive contractions in the ileum was observed, which was significant when compared to HC after ingesting PEG solution, p < 0.016. An increase in intestinal wall thickness or other signs of CD were not observed. CONCLUSIONS: FGDS is characterised by multiple, fluid-filled small bowel loops with incomplete contractions and fluid stagnation in fasting state. These findings may play a role in the increased risk of bowel obstruction as well as IBS-like symptoms observed in these patients.

Inhibition of Heat-Stable Toxin-Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors.

BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. METHODS: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. RESULTS: Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. CONCLUSIONS: We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.

Intestinal cell proliferation and senescence are regulated by receptor guanylyl cyclase C and p21.

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c(-/-), mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence.

Meconium ileus caused by mutations in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C.

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.

Familial diarrhea syndrome caused by an activating GUCY2C mutation.

BACKGROUND: Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS: We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS: We identified a heterozygous missense mutation (c.2519G→T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS: Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest [Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.).

Selective antigen-specific CD4(+) T-cell, but not CD8(+) T- or B-cell, tolerance corrupts cancer immunotherapy.

Self-tolerance, presumably through lineage-unbiased elimination of self-antigen-specific lymphocytes (CD4(+) T, CD8(+) T, and B cells), creates a formidable barrier to cancer immunotherapy. In contrast to this prevailing paradigm, we demonstrate that for some antigens, self-tolerance reflects selective elimination of antigen-specific CD4(+) T cells, but preservation of CD8(+) T- and B-cell populations. In mice, antigen-specific CD4(+) T-cell tolerance restricted CD8(+) T- and B-cell responses targeting the endogenous self-antigen guanylyl cyclase c (GUCY2C) in colorectal cancer. Although selective CD4(+) T-cell tolerance blocked GUCY2C-specific antitumor immunity and memory responses, it offered a unique solution to the inefficacy of GUCY2C vaccines through recruitment of self-antigen-independent CD4(+) T-cell help. Incorporating CD4(+) T-cell epitopes from foreign antigens into vaccines against GUCY2C reconstituted CD4(+) T-cell help, revealing the latent functional capacity of GUCY2C-specific CD8(+) T- and B-cell pools, producing durable antitumor immunity without autoimmunity. Incorporating CD4(+) T-cell epitopes from foreign antigens into vaccines targeting self-antigens in melanoma (Trp2) and breast cancer (Her2) produced similar results, suggesting selective CD4(+) T-cell tolerance underlies ineffective vaccination against many cancer antigens. Thus, identification of self-antigens characterized by selective CD4(+) T-cell tolerance and abrogation of such tolerance through self-antigen-independent T-cell help is essential for future immunotherapeutics.

MRP4 Modulation of the Guanylate Cyclase-C/cGMP Pathway: Effects on Linaclotide-Induced Electrolyte Secretion and cGMP Efflux.

MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.

The differential role of renoguanylin in osmoregulation and apical Cl-/HCO3- exchange activity in the posterior intestine of the Gulf toadfish (Opsanus beta).

The guanylin family of peptides are effective regulators of intestinal physiology in marine teleosts. In the distal intestinal segments, they inhibit or reverse fluid absorption by inhibiting the absorptive short-circuit current (Isc). The present findings demonstrate that mRNA from guanylin and uroguanylin, as well as at least one isoform of the guanylin peptide receptor, apical guanylyl cyclase-C (GC-C), was highly expressed in the intestine and rectum of the Gulf toadfish (Opsanus beta). In the posterior intestine, GC-C, as well as the cystic fibrosis transmembrane conductance regulator and basolateral Na(+)/K(+)/2Cl(-) cotransporter, which comprise a Cl(-)-secretory pathway, were transcriptionally upregulated in 60 parts per thousand (ppt). The present study also shows that, in intestinal tissues from Gulf toadfish held in 35 ppt, renoguanylin (RGN) expectedly causes net Cl(-) secretion, inhibits both the absorptive Isc and fluid absorption, and decreases HCO3(-) secretion. Likewise, in intestinal tissues from Gulf toadfish acclimated to 60 ppt, RGN also inhibits the absorptive Isc and fluid absorption but to an even greater extent, corresponding with the mRNA expression data. In contrast, RGN does not alter Cl(-) flux and, instead, elevates HCO3(-) secretion in the 60-ppt group, suggesting increased apical Cl(-)/HCO3(-) exchange activity by SLC26a6. Overall, these findings reinforce the hypotheses that the guanylin peptide system is important for salinity acclimatization and that the secretory response could facilitate the removal of solids, such as CaCO3 precipitates, from the intestine.

Guanylin-Guanylyl cyclase-C signaling in macrophages regulates mesenteric fat inflammation induced by high-fat diet.

Guanylin (Gn), a bioactive peptide, and its receptor, guanylyl cyclase-C (GC-C), are primarily present in the intestine and maintain homeostasis in body fluids. Recently, rats whose macrophages overexpress Gn and GC-C were found to be resistant to diet-induced obesity. Considering that obesity is strongly related to a chronic inflammatory state in white adipose tissues, it is possible that Gn-GC-C macrophages contribute to the regulation of inflammation. In the present study, we investigated the inflammatory state of mesenteric fat in rats transgenic for both Gn and GC-C (double-transgenic [dTg] rats) by evaluating the levels of cyclic guanosine monophosphate (cGMP), a second messenger of Gn-GC-C, cGMP-dependent protein kinase (PKG), and phosphorylated vasodilator-stimulated phosphoprotein (VASP), a target protein of PKG. The levels of cGMP in dTg rats was higher than in WT rats fed the same diet. Although there were no significant differences in levels of PKG and phosphorylated VASP between WT and dTg rats fed a standard diet (STD), these levels in dTg rats fed a high fat diet (HFD) were markedly increased compared with levels in HFD WT rats. Furthermore, mRNA levels of proinflammatory factors in mesenteric fat were lower in HFD dTg rats than in HFD WT rats and were similar to levels in STD WT and dTg rats. These results indicate that the Gn-GC-C system in macrophages regulates the cGMP-PKG-VASP pathway and controls obesity through the downregulation of proinflammatory factors.