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. 2016 Jul 1;311(1):G142-55.
doi: 10.1152/ajpgi.00041.2016. Epub 2016 May 26.

Identification of intestinal ion transport defects in microvillus inclusion disease

Dmitri V Kravtsov  1 , Md Kaimul Ahsan  1 , Vandana Kumari  1 , Sven C D van Ijzendoorn  2 , Miguel Reyes-Mugica  3 , Anoop Kumar  4 , Tarunmeet Gujral  4 , Pradeep K Dudeja  4 , Nadia A Ameen  5
Affiliations

Identification of intestinal ion transport defects in microvillus inclusion disease

Dmitri V Kravtsov et al. Am J Physiol Gastrointest Liver Physiol. .

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.

Keywords: CFTR; MVI; MVID; Myo5b; NHE3; brush border.

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Figures

Fig. 1.
Fig. 1.
Myo5bKD C2BBe cells model MVID villus enterocytes. A: immunoblots of Myo5b, Myo6, Myo1e, Myo1a, Sec 8, NHE3, EBP50, NHERF2, and GAPDH (for protein load) from equivalent protein loads of lysates prepared from fully polarized mature control (scrambled) and Myo5bKD C2BBe cells. B: transmission electron micrographs (TEM) of mature Myo5bKD C2BBe cells show apical microvillus inclusion (MVI) with brush border (black asterisk) at low-power (left, scale bar 2 μm) and high-power (middle, scale bar 800 nm) magnification and basolateral MVI with BB (black asterisk) at low-power (right, scale bar 2 μm) magnification. BB, brush border; BLM, basolateral membrane. C: confocal (XZ and XY, scale bar 20 μm) and gSTED images of a bracketed XY confocal area shows actin labeling in green. Asterisk marks MVIs. Scale bar, 2.5 μm. D: quantification in fully polarized Transwell-grown C2BBe cells reveals increased number of inclusions in Myo5bKD C2BBe cells (expressed as a total number per ×40 power field of view).
Fig. 2.
Fig. 2.
Polarized 3D cysts of Myo5bKD C2BBe cells in culture display MVIs and F-actin redistribution. 3D cysts were cultured, immunolabeled for F-actin, and imaged by confocal microscopy as described in materials and methods. Control C2BBe cells (A), scrambled C2BBe cells (B), and Myo5bKD C2BBe cells (C) display varying degrees of BB disorganization. Top: images of F-actin (red) and nuclei (blue). Middle: images of F-actin-only-labeled cysts. Brackets circumscribe the areas inserted under higher magnification; asterisks and arrows indicate inclusions and brush borders, respectively. Inset, scale bar 5 μm; main panel, scale bar 20 μm. Bottom: representative phase contrast micrographs of polarized cysts. Images were taken on a phase-contrast inverted microscope at ×400 magnification.
Fig. 3.
Fig. 3.
Brush border disorganization in Myo5bKD C2BBe cells. A–D: TEM demonstrates significant BB disorganization following Myo5bKD. Control C2BBe cells (A) and Myo5bKD C2BBe cells (B–D) with various degrees of BB disorganization. Scale bar, 400 nm. E–I: actin IFL in polarized C2BBe cells. gSTED Z-stack images demonstrate the dependence of fluorescence intensity on the degree of BB disorganization. XY planes are at the mid-BB level. E: control cells. F: Myo5bKD cells. G–I: representative confocal microscopy images of large areas of apical actin-stained scans used for quantification. Scale bar 20 μm. G: WT cells. H: scrambled control cells. I: Myo5bKD cells. J: quantification of apical actin immunofluorescence intensity.
Fig. 4.
Fig. 4.
NHE3 localization, expression, and function in the human duodenum and C2BBe cells. A: confocal microscopy images of NHE3 (green) IFL in the human duodenum are shown in relation to F-actin (red); nuclei are stained in blue. Brackets circumscribe the areas inserted under higher magnification. Inset, scale bar 2.5 μm; main panel, scale bar 5 μm. B: bar graph shows NHE3 mRNA levels expressed relative to GAPDH in the mature polarized C2BBe cells. C: quantification of NHE3 protein relative to GAPDH in lysates of mature polarized C2BBe cells. D: NHE3 exchanger function assayed by 22Na uptake by mature polarized C2BBe cells represents EIPA-sensitive but HOE 694-insensitive 22Na uptake.
Fig. 5.
Fig. 5.
DRA localization, expression, and function in the human duodenum, control, and Myo5bKD C2BBe cells. A: confocal microscopy images of DRA (green) IFL in cryosections from normal control and MVID human duodenum are shown in relation to F-actin (red); nuclei are stained in blue. Brackets circumscribe the areas inserted under higher magnification, arrowheads indicate location of DRA label in merge images. Inset, scale bar 2.5 μm; main panel, scale bar 5 μm. B: confocal Z-stack XY and XZ projections of Transwell-grown C2BBe monolayer cells. DRA (green) staining is shown in relation to F-actin (red); nuclei are stained in blue. Scale bar, 2.4 μm. C: DRA mRNA levels relative to GAPDH in mature polarized C2BBe cells. D: quantification of DRA protein relative to GAPDH in lysates of mature polarized C2BBe cells. E: Cl−/HCO3− exchange using DIDS-sensitive 125I uptake in mature polarized C2BBe cells.
Fig. 6.
Fig. 6.
CFTR localization, expression, and ion transport in the human duodenum, C2BBe, and T84 cells. A: confocal microscopy images of CFTR (green) IFL in cryosections of normal and MVID human duodenum are shown in relation to F-actin (red); nuclei are stained in blue. Arrowheads indicate BB IFL; arrows indicate CFTR High Expressor (CHE) cells. Scale bar, 5 μm. B: immunoblot analysis of Myo5b and CFTR in lysates of mature polarized C2BBe cells relative to GAPDH. C: comparison of GAPDH-corrected CFTR expression in mature polarized C2BBe cells. D: immunoblot analysis of Myo5b and CFTR in lysates of mature polarized T84 cells relative to GAPDH. Cells were analyzed following Isc measurements. E: CFTR short-circuit delta current (ΔIsc) measurements in the polarized T84 cells. Cl− secretion was stimulated with forskolin and inhibited with CFTR(inh)-172 inhibitor. F–Q: immunohistochemical localization of CFTR in normal and MVID human intestine. Horseradish peroxidase staining detects CFTR in small intestine sections from paraffin-embedded blocks of normal control (G–I) and two independent nongenetically characterized but confirmed MVID patients, MVID 1 (J–M), and MVID 2 (N–Q). F, J, and N are controls for negative staining. Nuclei are stained blue. Scale bar 25 μm. G: staining of CFTR in the BB (arrowheads) and supranuclear cytoplasm of villus enterocytes. H: intense BB and subapical CFTR staining in villus CHE cells (arrows). I: apical CFTR staining in crypt (black arrowheads). K: BB CFTR stain (arrowheads). L: intense BB and subapical CFTR staining in villus CHE cells (arrows). M: apical CFTR stain in crypt. O–Q: IHC stain for CFTR in MVID 2. O–P: BB (arrowheads) and subapical stain for CFTR. Q: apical CFTR stain in crypt (arrowhead).
Fig. 7.
Fig. 7.
Myo1a localization in the human duodenum and C2BBe cells. Confocal microscopy images of Myo1a (green) IFL is shown in relation to F-actin (red); nuclei are stained in blue. A: control human and MVID duodenum cryosections. Insets, scale bar 2.5 μm; main panel, scale bar 5 μm. Brackets circumscribe the areas inserted under higher magnification. Arrowheads indicate Myo1a labeling in the microvilli of the normal brush border (control villus) and MVIs (MVID villus). No difference is visible in the Myo1a labeling pattern in crypts of control and MVID duodenum. B: confocal XY and XZ projections of Transwell-grown C2BBe monolayers. Arrowheads indicate Myo1a labeling in the microvilli of the normal brush border (scrambled C2BBe) and MVIs (Myo5bKD C2BBe). Scale bar, 2.4 μm. C: immunoblot analysis of Myo1a protein expression in immature (D0) mature (D21) control and Myo5bKD C2BBe cells. Total protein load (TPL) is shown.
Fig. 8.
Fig. 8.
YAP1 localization, expression, and phosphorylation in preconfluent and mature polarized day 21 C2BBe cells. IFL of pYAP1 distribution in normal and MVID human intestine and BB enterocyte compensatory responses in MVID. A: confocal microscopy images of YAP1 (green) and nuclear DAPI (red, arrow) IFL in control and Myo5bKD preconfluent (D0) and mature polarized (D21) C2BBe cells. Black arrowhead indicates cytosolic label. Scale bar, 25 μm. B: immunoblots of YAP1 and phospho-YAP1 (pYAP1) in lysates of prepolarized day 0 (D0) and polarized day 21 (D21) C2BBe cells is shown relative to the total protein load (TPL). C: IFL of pYAP1 (green) in crypt (arrowhead) and villus (arrow) of normal (left) and MVID (right) human intestine. Scale bar, 100 μm. D–F: confocal images of IFL of BB proteins in villus sections of human MVID duodenum. F-actin is shown in red and nuclei in blue in all panels. D: Myosin1a (green) labels the BB in two MVID enterocytes (arrows) and decorates an MVI marked with an asterisk. E: DRA (green) IFL in the BB of cells marked (arrow) is higher than in its neighbors. F: NHE3 (green) IFL in the BB of some enterocytes is significantly higher (arrow) than neighboring cells. Scale bar, 5 μm.
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