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 5

GCC agonism improves fluid secretion in CF patient–derived enterospheres.

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GCC agonism improves fluid secretion in CF patient–derived enterospheres...
(A) Representative images of CF patient 1–derived F508del/F508del CFTR enterospheres depict secretion in response to forskolin stimulation under various treatment conditions: STc (50 nM and 500 nM, 24 h), VX-809 (2 μM, 24 h), and temperature rescue (TR). (B) Dot plot depicts fluid secretion calculated from n = 15–32 enterospheres corresponding to the treatment conditions described in A. Data represents mean ± SEM with P value calculated using 1-way ANOVA with Bonferroni’s multiple comparisons test. (C) Representative traces of short-circuit current (Isc) (left) in primary human bronchial epithelial cells carrying F508del/F508del CFTR untreated or treated with STc (500 nM, 24 h) or VX-809 (2.5 μM, 24 h). CFTR-dependent chloride transport was stimulated using forskolin (10 μM) and IBMX (100 μM) and inhibited with CFTRinh-172 added at the end of the experiment. (D) Bar graph represents quantitation of Isc measured in above-described treatment conditions (n = 3 independent experiments) and normalized to the untreated control. Data represents mean ± SEM. P value calculated by 1-way ANOVA with Bonferroni’s adjustment. (E) Representative images of CF patient 2–derived F508del/R117H 7T/9T CFTR enterospheres depict secretion in response to forskolin stimulation under various treatment conditions: VX-809 (2 μM, 24 h), VX-770 (2 μM, 24 h), VX-770 + VX-809, STc (50 nM, 24 h), STc + VX-809, and STc + VX-770. (F) Bar graph depicts fluid secretion calculated from n = 5–14 enterospheres corresponding to the treatment conditions described in E. Data represents mean ± SEM with P value calculated using 1-way ANOVA with Bonferroni’s multiple comparisons test. (G) Representative images of CF patient 3–derived G542X/ R74W, V201M, D1270N CFTR enterospheres depict secretion in response to forskolin stimulation under various treatment conditions: VX-809 (2 μM, 24 h), VX-770 (2 μM, 24 h), VX-770 + VX-809, and STc (50 nM, 24 h). (H) Bar graph depicts fluid secretion calculated from n = 6–9 enterospheres corresponding to the conditions described in G. Yellow circles indicate luminal area. Data represents mean ± SEM with P value calculated using 1-way ANOVA with Bonferroni’s multiple comparisons.