Receptor Guanylyl Cyclase Research Articles

The Impact of Natriuretic Peptides on Heart Development, Homeostasis, and Disease.

During mammalian heart development, the clustered genes encoding peptide hormones, Natriuretic Peptide A (NPPA; ANP) and B (NPPB; BNP), are transcriptionally co-regulated and co-expressed predominately in the atrial and ventricular trabecular cardiomyocytes. After birth, expression of NPPA and a natural antisense transcript NPPA-AS1 becomes restricted to the atrial cardiomyocytes. Both NPPA and NPPB are induced by cardiac stress and serve as markers for cardiovascular dysfunction or injury. NPPB gene products are extensively used as diagnostic and prognostic biomarkers for various cardiovascular disorders. Membrane-localized guanylyl cyclase receptors on many cell types throughout the body mediate the signaling of the natriuretic peptide ligands through the generation of intracellular cGMP, which interacts with and modulates the activity of cGMP-activated kinase and other enzymes and ion channels. The natriuretic peptide system plays a fundamental role in cardio-renal homeostasis, and its potent diuretic and vasodilatory effects provide compensatory mechanisms in cardiac pathophysiological conditions and heart failure. In addition, both peptides, but also CNP, have important intracardiac actions during heart development and homeostasis independent of the systemic functions. Exploration of the intracardiac functions may provide new leads for the therapeutic utility of natriuretic peptide-mediated signaling in heart diseases and rhythm disorders. Here, we review recent insights into the regulation of expression and intracardiac functions of NPPA and NPPB during heart development, homeostasis, and disease.

Phosphorylation-Dependent Regulation of Guanylyl Cyclase (GC)-A and Other Membrane GC Receptors.

Receptor guanylyl cyclases (GCs) are single membrane spanning, multidomain enzymes, that synthesize cGMP in response to natriuretic peptides or other ligands. They are evolutionarily conserved from sea urchins to humans and regulate diverse physiologies. Most family members are phosphorylated on 4 to 7 conserved serines or threonines at the beginning of their kinase homology domains. This review describes studies that demonstrate that phosphorylation and dephosphorylation are required for activation and inactivation of these enzymes, respectively. Phosphorylation sites in GC-A, GC-B, GC-E, and sea urchin receptors are discussed, as are mutant receptors that mimic the dephosphorylated inactive or phosphorylated active forms of GC-A and GC-B, respectively. A salt bridge model is described that explains why phosphorylation is required for enzyme activation. Potential kinases, phosphatases, and ATP regulation of GC receptors are also discussed. Critically, knock-in mice with glutamate substitutions for receptor phosphorylation sites are described. The inability of opposing signaling pathways to inhibit cGMP synthesis in mice where GC-A or GC-B cannot be dephosphorylated demonstrates the necessity of receptor dephosphorylation in vivo. Cardiac hypertrophy, oocyte meiosis, long-bone growth/achondroplasia, and bone density are regulated by GC phosphorylation, but additional processes are likely to be identified in the future.

Association of natriuretic peptides and receptor activity with cardio-metabolic health at middle age

Natriuretic peptides (NP) have multiple actions benefitting cardiovascular and metabolic health. Although many of these are mediated by Guanylyl Cyclase (GC) receptors NPR1 and NPR2, their role and relative importance in vivo is unclear. The intracellular mediator of NPR1 and NPR2, cGMP, circulates in plasma and can be used to examine relationships between receptor activity and tissue responses targeted by NPs. Plasma cGMP was measured in 348 participants previously recruited in a multidisciplinary community study (CHALICE) at age 50 years at a single centre. Associations between bio-active NPs and bio-inactive aminoterminal products with cGMP, and of cGMP with tissue response, were analysed using linear regression. Mediation of associations by NPs was assessed by Causal Mediation Analysis (CMA). ANP’s contribution to cGMP far exceed those of other NPs. Modelling across three components (demographics, NPs and cardiovascular function) shows that ANP and CNP are independent and positive predictors of cGMP. Counter intuitively, findings from CMA imply that in specific tissues, NPR1 responds more to BNP stimulation than ANP. Collectively these findings align with longer tissue half-life of BNP, and direct further therapeutic interventions towards extending tissue activity of ANP and CNP.

Feedforward and feedback mechanisms cooperatively regulate rapid experience-dependent response adaptation in a single thermosensory neuron type

Sensory adaptation allows neurons to adjust their sensitivity and responses based on recent experience. The mechanisms that mediate continuous adaptation to stimulus history over seconds- to hours-long timescales, and whether these mechanisms can operate within a single sensory neuron type, are unclear. The single pair of AFD thermosensory neurons in Caenorhabditis elegans exhibits experience-dependent plasticity in their temperature response thresholds on both minutes- and hours-long timescales upon a temperature upshift. While long-term response adaptation requires changes in gene expression in AFD, the mechanisms driving rapid response plasticity are unknown. Here, we show that rapid thermosensory response adaptation in AFD is mediated via cGMP and calcium-dependent feedforward and feedback mechanisms operating at the level of primary thermotransduction. We find that either of two thermosensor receptor guanylyl cyclases (rGCs) alone is sufficient to drive rapid adaptation, but that each rGC drives adaptation at different rates. rGC-driven adaptation is mediated in part via phosphorylation of their intracellular domains, and calcium-dependent feedback regulation of basal cGMP levels via a neuronal calcium sensor protein. In turn, cGMP levels feedforward via cGMP-dependent protein kinases to phosphorylate a specific subunit of the cGMP-gated thermotransduction channel to further regulate rapid adaptation. Our results identify multiple molecular pathways that act in AFD to ensure rapid adaptation to a temperature change and indicate that the deployment of both transcriptional and nontranscriptional mechanisms within a single sensory neuron type can contribute to continuous sensory adaptation.

Fundamental Neurochemistry Review: The role of enteroendocrine cells in visceral pain.

While visceral pain is commonly associated with disorders of the gut-brain axis, underlying mechanisms are not fully understood. Dorsal root ganglion (DRG) neurons innervate visceral structures and undergo hypersensitization in inflammatory models. The characterization of peripheral DRG neuron terminals is an active area of research, but recent work suggests that they communicate with enteroendocrine cells (EECs) in the gut. EECs sense stimuli in the intestinal lumen and communicate information to the brain through hormonal and electrical signaling. In that context, EECs are a target for developing therapeutics to treat visceral pain. Linaclotide is an FDA-approved treatment for chronic constipation that activates the intestinal membrane receptor guanylyl cyclase C (GUCY2C). Clinical trials revealed that linaclotide relieves both constipation and visceral pain. We recently demonstrated that the analgesic effect of linaclotide reflects the overexpression of GUCY2C on neuropod cells, a specialized subtype of EECs. While this brings some clarity to the relationship between linaclotide and visceral analgesia, questions remain about the intracellular signaling mechanisms and neurotransmitters mediating this communication. In this Fundamental Neurochemistry Review, we discuss what is currently known about visceral nociceptors, enteroendocrine cells, and the gut-brain axis, and ongoing areas of research regarding that axis and visceral pain.

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 FDA-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 guanylyl cyclase 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 when compared to human GC-C. Further, the Vmax of the murine guanylyl cyclase 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 guanylyl cyclase 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.

Sex-based differences in metabolic protection by the ANP genetic variant rs5068 in the general population.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are produced in the heart and secreted into the circulation. As hormones, both peptides activate the guanylyl cyclase receptor A (GC-A), playing a role in blood pressure (BP) regulation. A significant role for ANP and BNP includes favorable actions in metabolic homeostasis. Sex-based high prevalence of risk factors for cardiovascular disease in males compared with females is well established, but sex-based differences on cardiometabolic protection have not been investigated in relation to ANP (NPPA) and BNP (NPPB) gene variants. We included 1,146 subjects in the general population from Olmsted County, Minnesota. Subjects were genotyped for the ANP gene variant rs5068 and BNP gene variant rs198389. Cardiometabolic parameters and medical records were reviewed. In the presence of the minor allele of rs5068, diastolic BP, creatinine, body mass index (BMI), waist measurement, insulin, and prevalence of obesity and metabolic syndrome were lower, whereas HDL was higher in males with only trends observed in females. We observed no associations of the minor allele with echocardiographic parameters in either males or females. Regarding rs198389 genotype, the minor allele was not associated with any BP, metabolic, renal, or echocardiographic parameters in either sex. In the general community, the minor allele of the ANP gene variant rs5068 is associated with a favorable metabolic phenotype in males. No associations were observed with the BNP gene variant rs198389. These studies support a protective role of the ANP pathway on metabolic function and underscore the importance of sex in relationship to natriuretic peptide responses.NEW & NOTEWORTHY Males are characterized by lower ANP and BNP with greater prevalence of cardiometabolic disease. The ANP genetic variant rs5068 was associated with less metabolic dysfunction in males, whereas no metabolic profile was related to the BNP genetic variant rs198389 in the general population. ANP may play a more biological role in metabolic homeostasis compared with BNP in the general population with greater physiological metabolic actions in males compared with females.

Molecular insights into recognition of GUCY2C by T-cell engaging bispecific antibody anti-GUCY2CxCD3

The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody’s activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb.

Structural insight into guanylyl cyclase receptor hijacking of the kinase–Hsp90 regulatory mechanism

Membrane receptor guanylyl cyclases play a role in many important facets of human physiology, from regulating blood pressure to intestinal fluid secretion. The structural mechanisms which influence these important physiological processes have yet to be explored. We present the 3.9 Å resolution cryo-EM structure of the human membrane receptor guanylyl cyclase GC-C in complex with Hsp90 and its co-chaperone Cdc37, providing insight into the mechanism of Cdc37 mediated binding of GC-C to the Hsp90 regulatory complex. As a membrane protein and non-kinase client of Hsp90–Cdc37, this work shows the remarkable plasticity of Cdc37 to interact with a broad array of clients with significant sequence variation. Furthermore, this work shows how membrane receptor guanylyl cyclases hijack the regulatory mechanisms used for active kinases to facilitate their regulation. Given the known druggability of Hsp90, these insights can guide the further development of membrane receptor guanylyl cyclase-targeted therapeutics and lead to new avenues to treat hypertension, inflammatory bowel disease, and other membrane receptor guanylyl cyclase-related conditions.

Structural insight into guanylyl cyclase receptor hijacking of the kinase-Hsp90 regulatory mechanism.

Membrane receptor guanylyl cyclases play a role in many important facets of human physiology, from regulating blood pressure to intestinal fluid secretion. The structural mechanisms which influence these important physiological processes have yet to be explored. We present the 3.9 Å resolution cryo-EM structure of the human membrane receptor guanylyl cyclase GC-C in complex with Hsp90 and its co-chaperone Cdc37, providing insight into the mechanism of Cdc37 mediated binding of GC-C to the Hsp90 regulatory complex. As a membrane protein and non-kinase client of Hsp90-Cdc37, this work shows the remarkable plasticity of Cdc37 to interact with a broad array of clients with significant sequence variation. Furthermore, this work shows how membrane receptor guanylyl cyclases hijack the regulatory mechanisms used for active kinases to facilitate their regulation. Given the known druggability of Hsp90, these insights can guide the further development of membrane receptor guanylyl cyclase-targeted therapeutics and lead to new avenues to treat hypertension, inflammatory bowel disease, and other membrane receptor guanylyl cyclase-related conditions.

Structural studies of the human particulate guanylyl cyclase receptor A (Pgc-A) to support therapeutics for cardiovascular diseases

Structural studies of the human particulate guanylyl cyclase receptor A (Pgc-A) to support therapeutics for cardiovascular diseases

Evolution of visual guanylyl cyclases and their activating proteins with respect to clade and species-specific visual system adaptation.

Membrane guanylyl cyclase receptors are important regulators of local cGMP production, critically influencing cell growth and differentiation as well as ion transport, blood pressure and calcium feedback of vertebrate phototransduction. Currently, seven different subtypes of membrane guanylyl cyclase receptors have been characterized. These receptors have tissue specific expression and are activated either by small extracellular ligands, changing CO2 concentrations or, in the case of visual guanylyl cyclases, intracellularly interacting Ca2+-dependent activating proteins. In this report, we focus on the visual guanylyl cyclase receptors (GCs) GC-E (gucy2d/e) and GC-F (gucy2f) and their activating proteins (GCAP1/2/3; guca1a/b/c). While gucy2d/e has been detected in all analyzed vertebrates, GC-F receptors are missing in several clades (reptiles, birds, and marsupials) and/or individual species. Interestingly, the absence of GC-F in highly visual sauropsida species with up to 4 different cone-opsins is compensated by an increased number of guanylyl cyclase activating proteins, whereas in nocturnal or visually impaired species with reduced spectral sensitivity it is consolidated by the parallel inactivation of these activators. In mammals, the presence of GC-E and GC-F is accompanied by the expression of one to three GCAPs, whereas in lizards and birds, up to five different GCAPs are regulating the activity of the single GC-E visual membrane receptor. In several nearly blind species, a single GC-E enzyme is often accompanied by a single variant of GCAP, suggesting that one cyclase and one activating protein are both sufficient and required for conferring the basic detection of light.

Epitope-tagged and phosphomimetic mouse models for investigating natriuretic peptide-stimulated receptor guanylyl cyclases.

The natriuretic peptide receptors NPR1 and NPR2, also known as guanylyl cyclase A and guanylyl cyclase B, have critical functions in many signaling pathways, but much remains unknown about their localization and function in vivo. To facilitate studies of these proteins, we developed genetically modified mouse lines in which endogenous NPR1 and NPR2 were tagged with the HA epitope. To investigate the role of phosphorylation in regulating NPR1 and NPR2 guanylyl cyclase activity, we developed mouse lines in which regulatory serines and threonines were substituted with glutamates, to mimic the negative charge of the phosphorylated forms (NPR1-8E and NPR2-7E). Here we describe the generation and applications of these mice. We show that the HA-NPR1 and HA-NPR2 mice can be used to characterize the relative expression levels of these proteins in different tissues. We describe studies using the NPR2-7E mice that indicate that dephosphorylation of NPR2 transduces signaling pathways in ovary and bone, and studies using the NPR1-8E mice that indicate that the phosphorylation state of NPR1 is a regulator of heart, testis, and adrenal function.

Guanylyl cyclase C as a diagnostic and therapeutic target in colorectal cancer.

Colorectal cancer remains a major cause of mortality in theUSA, despite advances in prevention and screening. Existing therapies focus primarily on generic treatment such as surgical intervention and chemotherapy, depending on disease severity. As personalized medicine and targeted molecular oncology continue to develop as promising treatment avenues, there has emerged a need for effective targets and biomarkers of colorectal cancer. The transmembrane receptor guanylyl cyclase C (GUCY2C) regulates intestinal homeostasis and has emerged as a tumor suppressor. Further, it is universally expressed in advanced metastatic colorectal tumors, as well as other cancer types that arise through intestinal metaplasia. In this context, GUCY2C satisfies many characteristics of a compelling target and biomarker for gastrointestinal malignancies.

Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

Particulate Guanylyl Cyclase Receptor A (pGC-A) is a natriuretic peptide membrane receptor, playing a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop methods to regulate pGC-A, structural information on the full-length form is needed. However, structural data on the transmembrane and intracellular domains are lacking. This work presents expression and optimization using baculovirus, along with the first purification of functional full-length human pGC-A. In vitro assays revealed the pGC-A tetramer was functional in detergent micelle solution. Based on our purification results and previous findings that dimer formation is required for functionality, we propose a tetramer complex model with two functional subunits. Previous research suggested pGC-A signal transduction is an ATP-dependent, two-step mechanism. Our results show the binding ligand also moderately activates pGC-A, and ATP is not crucial for activation of guanylyl cyclase. Furthermore, crystallization of full-length pGC-A was achieved, toward determination of its structure. Needle-shaped crystals with 3 Å diffraction were observed by serial crystallography. This work paves the road for determination of the full-length pGC-A structure and provides new information on the signal transduction mechanism.

Receptor Guanylyl Cyclase C and Cyclic GMP in Health and Disease: Perspectives and Therapeutic Opportunities.

Receptor Guanylyl Cyclase C (GC-C) was initially characterized as an important regulator of intestinal fluid and ion homeostasis. Recent findings demonstrate that GC-C is also causally linked to intestinal inflammation, dysbiosis, and tumorigenesis. These advances have been fueled in part by identifying mutations or changes in gene expression in GC-C or its ligands, that disrupt the delicate balance of intracellular cGMP levels and are associated with a wide range of clinical phenotypes. In this review, we highlight aspects of the current knowledge of the GC-C signaling pathway in homeostasis and disease, emphasizing recent advances in the field. The review summarizes extra gastrointestinal functions for GC-C signaling, such as appetite control, energy expenditure, visceral nociception, and behavioral processes. Recent research has expanded the homeostatic role of GC-C and implicated it in regulating the ion-microbiome-immune axis, which acts as a mechanistic driver in inflammatory bowel disease. The development of transgenic and knockout mouse models allowed for in-depth studies of GC-C and its relationship to whole-animal physiology. A deeper understanding of the various aspects of GC-C biology and their relationships with pathologies such as inflammatory bowel disease, colorectal cancer, and obesity can be leveraged to devise novel therapeutics.

A β‐catenin‐TCF‐sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer

BackgroundColorectal cancer most commonly arises from mutations in the tumor suppressor APC, or its downstream degradation target β‐catenin, initiating an oncogenic program of β‐catenin/TCF‐dependent nuclear transcription. Mechanisms coupling these mutations to tumorigenesis continue to be refined. The tumor suppressor axis regulated by the intestinal receptor guanylyl cyclase C (GUCY2C) and its paracrine ligands, guanylin and uroguanylin, contributes to intestinal homeostasis by regulating the proliferation, migration, and differentiation programs that maintain intestinal epithelial architecture. Furthermore, GUCY2C agonists oppose transformation in multiple mouse models of intestinal tumorigenesis. This signaling axis is among the earliest pathways silenced in tumorigenesis, reflecting an evolutionarily conserved pattern of GUCY2C retention, but ligand loss, in tumors. These observations suggest a pathophysiological model in which transformation orphans the receptor, silencing its signaling and lifting a block on tumorigenesis. Here, we examined the hypothesis that GUCY2C ligand loss arises from transcriptional silencing by β‐catenin/TCF.MethodsWe mapped the β‐catenin/TCF‐transcriptional program by RNA‐seq in four human colon cancer cell models. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, ChIP‐seq, CRISPR Cas9 knockout, and CRISPR epigenome editing to identify gene enhancers mediating GUCY2C ligand loss.ResultsRNA‐seq revealed a core β‐catenin/TCF‐transcriptional program of 1,289 genes, of which guanylin and uroguanylin were the seventh and twelfth most sensitive transcripts, reflecting transcriptional silencing. ChIP‐seq analyses of colon tissue revealed an evolutionarily conserved genomic locus encompassing the GUCY2C ligand genes, containing multiple regions of DNase hypersensitivity and H3K27ac enrichment, hallmarks of genetic enhancer loci. These markers were observed in normal colon, but not cancer tissue, consistent with enhancer inactivation and transcriptional silencing during transformation. ChIP‐seq in a colon cancer cell line revealed RNA Polymerase II and H3K27ac recruitment to the regions identified in human tissue, reflecting poised transcriptional machinery. Incorporation of the putative enhancer DNA into luciferase reporter constructs revealed a 2,683 bp locus control region (LCR) responsible for coordinated β‐catenin/TCF‐sensitive control of both GUCY2C ligand promoters. Both CRISPR‐Cas9 deletion of the LCR and epigenetic inactivation with a dCas9.KRAB repressor construct abolished β‐catenin/TCF‐sensitivity of GUCY2C ligand expression. Finally, LCR activation with a dCas9.VP64 transcriptional activator construct reconstituted GUCY2C ligand expression, overcoming gene silencing by β‐catenin/TCF.ConclusionsThis study reveals DNA elements regulating co‐repression of GUCY2C ligand transcription by β‐catenin/TCF, reflecting a novel step in tumorigenesis, and offers unique strategies to re‐establish hormone expression to oppose transformation.

The pseudokinase domain in receptor guanylyl cyclases.

Cyclic GMP is produced by enzymes called guanylyl cyclases, of which the membrane-associated forms contain an intracellular pseudokinase domain that allosterically regulates the C-terminal guanylyl cyclase domain. Ligand binding to the extracellular domain of these single transmembrane-spanning domain receptors elicits an increase in cGMP levels in the cell. The pseudokinase domain (or kinase-homology domain) in these receptors appears to be critical for ligand-mediated activation. While the pseudokinase domain does not possess kinase activity, biochemical evidence indicates that the domain can bind ATP and thereby allosterically regulate the catalytic activity of these receptors. The pseudokinase domain also appears to be the site of interaction of regulatory proteins, as seen in the retinal guanylyl cyclases that are involved in visual signal transduction. In the absence of structural information on the pseudokinase-guanylyl cyclase domain organization of any member of this family of receptors, biochemical evidence has provided clues to the physical interaction of the pseudokinase and guanylyl cyclase domain. An α-helical linker region between the pseudokinase domain and the guanylyl cyclase domain regulates the basal activity of these receptors in the absence of a stimulatory ligand and is important for stabilizing the structure of the pseudokinase domain that can bind ATP. Here, we present an overview of salient features of ATP-mediated regulation of receptor guanylyl cyclases and describe biochemical approaches that allow a clearer understanding of the intricate interplay between the pseudokinase domain and catalytic domain in these proteins.

Gut-associated cGMP mediates colitis and dysbiosis in a mouse model of an activating mutation in GUCY2C.

Activating mutations in receptor guanylyl cyclase C (GC-C), the target of gastrointestinal peptide hormones guanylin and uroguanylin, and bacterial heat-stable enterotoxins cause early-onset diarrhea and chronic inflammatory bowel disease (IBD). GC-C regulates ion and fluid secretion in the gut via cGMP production and activation of cGMP-dependent protein kinase II. We characterize a novel mouse model harboring an activating mutation in Gucy2c equivalent to that seen in an affected Norwegian family. Mutant mice demonstrated elevated intestinal cGMP levels and enhanced fecal water and sodium content. Basal and linaclotide-mediated small intestinal transit was higher in mutant mice, and they were more susceptible to DSS-induced colitis. Fecal microbiome and gene expression analyses of colonic tissue revealed dysbiosis, up-regulation of IFN-stimulated genes, and misregulation of genes associated with human IBD and animal models of colitis. This novel mouse model thus provides molecular insights into the multiple roles of intestinal epithelial cell cGMP, which culminate in dysbiosis and the induction of inflammation in the gut.

Sphingomyelin nanosystems loaded with uroguanylin and etoposide for treating metastatic colorectal cancer

Colorectal cancer is the third most frequently diagnosed cancer malignancy and the second leading cause of cancer-related deaths worldwide. Therefore, it is of utmost importance to provide new therapeutic options that can improve survival. Sphingomyelin nanosystems (SNs) are a promising type of nanocarriers with potential for association of different types of drugs and, thus, for the development of combination treatments. In this work we propose the chemical modification of uroguanylin, a natural ligand for the Guanylyl Cyclase (GCC) receptor, expressed in metastatic colorectal cancer tumors, to favour its anchoring to SNs (UroGm-SNs). The anti-cancer drug etoposide (Etp) was additionally encapsulated for the development of a combination strategy (UroGm-Etp-SNs). Results from in vitro studies showed that UroGm-Etp-SNs can interact with colorectal cancer cells that express the GCC receptor and mediate an antiproliferative response, which is more remarkable for the drugs in combination. The potential of UroGm-Etp-SNs to treat metastatic colorectal cancer cells was complemented with an in vivo experiment in a xenograft mice model.