Guanylate cyclase 2C agonism corrects CFTR mutants
Kavisha Arora, Yunjie Huang, Kyushik Mun, Sunitha Yarlagadda, Nambirajan Sundaram, Marco M. Kessler, Gerhard Hannig, Caroline B. Kurtz, Inmaculada Silos-Santiago, Michael Helmrath, Joseph J. Palermo, John P. Clancy, Kris A. Steinbrecher, Anjaparavanda P. Naren
Kavisha Arora, Yunjie Huang, Kyushik Mun, Sunitha Yarlagadda, Nambirajan Sundaram, Marco M. Kessler, Gerhard Hannig, Caroline B. Kurtz, Inmaculada Silos-Santiago, Michael Helmrath, Joseph J. Palermo, John P. Clancy, Kris A. Steinbrecher, Anjaparavanda P. Naren
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Research Article Gastroenterology

Guanylate cyclase 2C agonism corrects CFTR mutants

Abstract

Cystic fibrosis (CF) is a genetic disorder in which epithelium-generated fluid flow from the lung, intestine, and pancreas is impaired due to mutations disrupting CF transmembrane conductance regulator (CFTR) channel function. CF manifestations of the pancreas and lung are present in the vast majority of CF patients, and 15% of CF infants are born with obstructed gut or meconium ileus. However, constipation is a significantly underreported outcome of CF disease, affecting 47% of the CF patients, and management becomes critical in the wake of increasing life span of CF patients. In this study, we unraveled a potentially novel molecular role of a membrane-bound cyclic guanosine monophosphate–synthesizing (cGMP-synthesizing) intestinal enzyme, guanylate cyclase 2C (GCC) that could be targeted to ameliorate CF-associated intestinal fluid deficit. We demonstrated that GCC agonism results in functional rescue of murine F508del/F508del and R117H/R117H Cftr and CFTR mutants in CF patient–derived intestinal spheres. GCC coexpression and activation facilitated processing and ER exit of F508del CFTR and presented a potentially novel rescue modality in the intestine, similar to the CF corrector VX-809. Our findings identify GCC as a biological CFTR corrector and potentiator in the intestine.

Authors

Kavisha Arora, Yunjie Huang, Kyushik Mun, Sunitha Yarlagadda, Nambirajan Sundaram, Marco M. Kessler, Gerhard Hannig, Caroline B. Kurtz, Inmaculada Silos-Santiago, Michael Helmrath, Joseph J. Palermo, John P. Clancy, Kris A. Steinbrecher, Anjaparavanda P. Naren

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Figure 1

Validation of intestinal spheres as model to study GCC-stimulated fluid secretion.

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Validation of intestinal spheres as model to study GCC-stimulated fluid ...
RNA-seq analysis demonstrates relative abundance of transcripts of various guanylate cyclases in (A) ileum and (B) trachea isolated from WT/WT Cftr (n = 3) and F508del/F508del Cftr (n = 3) mice. (C) Bright-field images represent fluid accumulation in the lumen of intestinal spheres isolated from WT (Gucy2c+/+) and GCC- KO (Gucy2c–/–) mice in response to various CFTR activating agonists. (D) Bar graph shows quantitation of fluid secretion in intestinal spheres corresponding to basal or no stimulation and in response to STc (500 nM, 30 min), 8-Br-cGMP (250 μM, 30 min), and forskolin (FSK, 10 μM, 30 min), all added in the media and calculated from n = 5–10 enterospheres from 2 mice in each group. Data is ± SEM with P value by 1-way ANOVA with Bonferroni’s adjustment for multiple comparisons. (E) Confocal images of intestinal spheres showing GCC-specific immunostaining predominantly present on the apical side of the spheres. (F) Image of an intestinal sphere depicting microinjection of STc into the lumen of the structure. BL, basolateral; AP, apical. (G) Bar graph shows quantitation of fluid secretion in intestinal spheres corresponding to basal or no stimulation and in response to STc delivered in the media or microinjected into the lumen calculated from n = 5 enterospheres. Response to FSK was tested after adding the reagent into the media. Data is ± SEM with P value by 1-way ANOVA with Bonferroni’s adjustment for multiple comparisons. (H) Dot blot and line-graph depict fluid secretion calculated as the total area of intestinal spheres (n = 9–11 enterospheres) following activation of CFTR function by using STc (500 nM, 30 min) in the presence and absence of CFTRinh-172 (20 μM, 30 min) in an assay done in a high-content microscope. Data is ± SEM with P value by 2-tailed unpaired Student’s t test. (I) FSK- and STc-dependent swelling of intestinal spheres isolated from CFTR-mutant mice F508del/F508del Cftr was tested. A set of F508del/F508del Cftr intestinal spheres was subjected to low-temperature incubation (28°C, 24 h) and subsequently tested for FSK- (10 μM, 30 min) and STc-dependent (500 nM, 30 min) secretion. (J) Dot plot represents quantitation of the fluid secretion in n = 9–17 enterospheres per condition described in I. Temp. R, temperature rescue. Data is ± SEM with P value determined using 1-way ANOVA with Bonferroni’s test for multiple comparisons.