Syntaxin 3 is necessary for cAMP- and cGMP-regulated exocytosis of CFTR: implications for enterotoxigenic diarrhea

Abstract

Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal Caco-2(BB)e cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized Caco-2(BB)e cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea.

Identification of intestinal ion transport defects in microvillus inclusion disease

“…Similar to the observations supporting polarity defects in humans (47), mouse (49), and cellular models of MVID morphological features of human villus MVID enterocytes, BB ion transporter redistribution, and functional defects in NHE3 and DRA-dependent uptake of Na ϩ and Cl Ϫ that can contribute to sodium and chloride loss in human MVID. The data here also support our characterization of ion transporters and apical trafficking machinery in C2BBe cells (11). Except for the presence of CHE cells (that lack NHE3) (27), IFL of BB transporters in Myo5b loss-of-function human MVID intestine corroborated our findings in C2BBe cells.…”

“…In addition, two intestinal epithelial cellular models, CaCo2 and the subclone C2BBe, have been invaluable in contributing to our understanding of enterocyte defects in MVID as they resemble villus enterocytes and are amenable to Myo5b silencing approaches. C2BBe cells are superior to CaCo2 as a cellular model for MVID because they possess a highly developed BB upon confluency and express apical proteins and transporters similar to native villus enterocytes of the small intestine (11,34), where the major morphological changes in MVID are observed. Similar to the observations supporting polarity defects in humans (47), mouse (49), and cellular models of MVID morphological features of human villus MVID enterocytes, BB ion transporter redistribution, and functional defects in NHE3 and DRA-dependent uptake of Na ϩ and Cl Ϫ that can contribute to sodium and chloride loss in human MVID.…”

“…Lysates were centrifuged at 12,000 rpm for 15 min at 4°C. Protein concentrations were determined from supernatants, samples were eluted with 2ϫ Laemmli sample buffer (BioRad, Hercules, CA), and equivalent protein loads were analyzed by PAGE as before (11,32).…”

“…In secretory diarrhea, second messengers stimulate exocytic insertion of CFTR from subapical vesicles to markedly increase its functional expression on the enterocyte BBM (4,6,18). A number of factors regulate CFTR function include apical recycling, cAMP, cGMP-regulated traffic, myosin motors including myosin VI (Myo6), myosin Ia (Myo1a), Rab GTPases, and STX3 (6,11,18,34,52,56).…”

“…C2BBe is a subclone of parental CaCo2 cells and an excellent model for studies of villus enterocyte BB development and assembly. This cell line expresses endogenous BB myosins, STX3, Rab11, absorptive apical hydrolases, and ion transporters such as sucrase isomaltase (SI) and CFTR that are found on the BBM of villus enterocytes in the native small intestine (11,34,43,58). Finally, the observations that MVID villus enterocytes possess disordered BB and resemble crypt cells led us to test the hypothesis that loss of Myo5b leads to enterocyte maturation defects and a prosecretory cryptlike phenotype due to alterations in the Hippo pathway, an evolutionary conserved signaling pathway that regulates tissue growth in organs (62).…”