title: Identification of intestinal ion transport defects in microvillus inclusion disease.
abstract: Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl(-)) and sodium (Na(+)) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na(+)), CFTR (Cl(-)), and SLC26A3 (DRA) (Cl(-)/HCO3 (-)) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl(-) and Na(+) stool loss in MVID diarrhea.
authors.affiliation: Department of Pediatrics/Gastroenterology and Hepatology, Yale School of Medicine, New Haven, Connecticut;
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Kravtsov
authors.affiliation: Department of Pediatrics/Gastroenterology and Hepatology, Yale School of Medicine, New Haven, Connecticut;
authors.forename: Md Kaimul
authors.initials: MK
authors.lastname: Ahsan
authors.affiliation: Department of Pediatrics/Gastroenterology and Hepatology, Yale School of Medicine, New Haven, Connecticut;
authors.forename: Vandana
authors.initials: V
authors.lastname: Kumari
authors.affiliation: Department of Cell Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands;
authors.forename: Sven C D
authors.initials: SC
authors.lastname: van Ijzendoorn
authors.affiliation: Children's Hospital Pittsburgh of UPMC, Pittsburgh, Pennsylvania;
authors.forename: Miguel
authors.initials: M
authors.lastname: Reyes-Mugica
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; and.
authors.forename: Anoop
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; and.
authors.forename: Tarunmeet
authors.initials: T
authors.lastname: Gujral
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois; and.
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Dudeja
authors.affiliation: Department of Pediatrics/Gastroenterology and Hepatology, Yale School of Medicine, New Haven, Connecticut; Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut nadia.ameen@yale.edu.
authors.forename: Nadia A
authors.initials: NA
authors.lastname: Ameen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D018938:Caco-2 Cells; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D020895:Enterocytes / Q000378:metabolism* / Q000648:ultrastructure; D005786:Gene Expression Regulation; D006801:Humans; D017136:Ion Transport; D007583:Jejunum / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D008286:Malabsorption Syndromes / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008871:Microvilli / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D009081:Mucolipidoses / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D024701:Myosin Type V / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D000076988:Sulfate Transporters; D014157:Transcription Factors; D014162:Transfection; D000091102:YAP-Signaling Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C505032:CFTR protein, human; D027963:Chloride-Bicarbonate Antiporters; C505832:MYO5B protein, human; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; C081011:SLC26A3 protein, human; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000076988:Sulfate Transporters; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins; C088374:YAP1 protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D024701:Myosin Type V; D018995:Myosin Heavy Chains
keywords: CFTR; MVI; MVID; Myo5b; NHE3; brush border