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. 2019 Dec 15;30(26):3076-3089.
doi: 10.1091/mbc.E18-07-0415. Epub 2019 Oct 30.

Glucocorticoids and myosin5b loss of function induce heightened PKA signaling in addition to membrane traffic defects

Radia Forteza  1 , M Kaimul Ahsan  2 , Fernando Cartón-García  3 , Diego Arango  3 , Nadia A Ameen  2 , Pedro J Salas  1
Affiliations

Glucocorticoids and myosin5b loss of function induce heightened PKA signaling in addition to membrane traffic defects

Radia Forteza et al. Mol Biol Cell. .

Abstract

Loss-of-function mutations in the nonconventional myosin Vb (Myo5b) result in microvillus inclusion disease (MVID) and massive secretory diarrhea that often begins at birth. Myo5b mutations disrupt the apical recycling endosome (ARE) and membrane traffic, resulting in reduced surface expression of apical membrane proteins. ARE disruption also results in constitutive phosphoinositide-dependent kinase 1 gain of function. In MVID, decreased surface expression of apical anion channels involved in Cl- extrusion, such as cystic fibrosis transmembrane conductance regulator (CFTR), should reduce fluid secretion into the intestinal lumen. But the opposite phenotype is observed. To explain this contradiction and the onset of diarrhea, we hypothesized that signaling effects downstream from Myo5b loss of function synergize with higher levels of glucocorticoids to activate PKA and CFTR. Data from intestinal cell lines, human MVID, and Myo5b KO mouse intestine revealed changes in the subcellular redistribution of PKA activity to the apical pole, increased CFTR phosphorylation, and establishment of apical cAMP gradients in Myo5b-defective cells exposed to physiological levels of glucocorticoids. These cells also displayed net secretory fluid fluxes and transepithelial currents mainly from PKA-dependent Cl- secretion. We conclude that Myo5b defects result in PKA stimulation that activates residual channels on the surface when intestinal epithelia are exposed to glucocorticoids at birth.

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Figures

FIGURE 1:
FIGURE 1:
Myosin 5b knockdown and corticoids induce changes in the distribution of PKA activity in C2BBe intestinal epithelial cells. (A) C2BBe cells were transduced with lentivirus expressing a scrambled shRNA (scr, control) or anti-myosin 5b shRNA (Myo5b kd), extracted, and analyzed by immunoblot. (B) C2BBe cells (scr or Myo5b kd) were stained with fluorescent phalloidin (XZ plane from confocal stacks). Arrow, phalloidin-positive inclusion. Scale bar = 10 µm. (C) C2BBe cells were treated with 10 µM forskolin or vehicle (control) for 3 h. Immunoblots were processed with PKA p-substrate antibody (RRXpS/pT) or anti-GAPDH antibody. (D) Parallel cultures were processed for immunofluorescence with the same PKA p-substrate antibody (red channel, XZ plane). Scale bar = 10 µm.
FIGURE 2:
FIGURE 2:
Myosin 5b knockdown and dexamethasone increase apical PKA-phosphorylated targets in T84 intestinal cells. (A) Immunofluorescence localization of proteins phosphorylated by PKA in scr or Myo5b kd (only kd is labeled in the graph) T84 cells were incubated vehicle (control) or with 0.5 µM dexamethasone for 10 d. As a positive control, parallel cultures, expressing scr or kd shRNA were also incubated in 10 µM forskolin for 3 h. Confocal XY images typical of three experiments at the apical focal plane (see B) shown for PKA p-substrate antibody signal (red), and the corresponding images in the transnuclear plane below for DAPI signal. Yellow rectangle: Example of ROI used for quantification in D. Unbiased random distribution of ROIs was ensured by overlapping a grid and placing ROIs at every other intersection. Scale bar = 10 µm. (B) XZ sections from the same experiment. Yellow line shows the focal plane of ROIs shown in A and the approximate focal depth (vertical lines). Scale bar = 10 µm. (C) T84 cells without transduction or selection (wt) or transduced with lentiviral particles expressing scrambled (scr, control) or anti-Myo5b (kd) shRNAs and selected, were analyzed for Myo5b expression by immunoblot to determine knockdown efficacy. (D) Random ROIs were placed on the apical domain in confocal (XY) sections of monolayers as shown in A by overlapping grid intersections. Several ROIs per image were taken. The average pixel intensities were calculated and normalized to the average in scr control cells. n = 3; *, p < 0.05; **, p < 0.01.
FIGURE 3:
FIGURE 3:
Myo5b kd and dexamethasone induce a fluid secretory phenotype in live unstimulated intestinal epithelial cells. (A) Transepithelial net fluid secretion in Myo5b kd T84 cells treated with dexamethasone. Gravimetric determinations of net fluid transport in T84 cells (grown on filters) show secretion in Myo5b KD cells when incubated in 0.5 μM dexamethasone (green arrow). This effect was blocked by 10 µM CFTR172. Control vs. pharmacologic treatments estimated by ANOVA; *, p < 0.01; **, p < 0.02; ***, p < 0.05; n = 4. NS, not significant. (B) T84 cells expressing scrambled shRNA (scr) or anti-Myo5b shRNA were grown in Matrigel for 10 d. The volume of the spheroids was measured as maximum caliper diameter at 4 and 27 h after adding 0.5 µM dexamethasone (dexa) or vehicle to the medium. Each dot represents the change in one spheroid. Relative changes in diameter are expressed as the percentage of increase (or decrease, −). *, p < 0.001; **, p < 0.01 (Kruskal-Wallis). Box-and-whisker plots are shown for categories with n > 5.
FIGURE 4:
FIGURE 4:
Myo5b kd and dexamethasone induce a Cl− secretory phenotype in unstimulated intestinal cells. (A) Typical recordings of short circuit current (Isc) in confluent, differentiated T84 cells transduced with scrambled shRNA (blue and purple), or Myo5b antisense shRNA (green and black traces), grown on Snapwell filters and analyzed in an Ussing chamber in standard DMEM culture medium. Forskolin (0.5 µM) was added from the apical side only. Some cells had been incubated in the presence of 0.5 µM dexamethasone for 10 d (blue and black traces). (B) Typical recordings of Isc in confluent, differentiated T84 cells transduced with scrambled shRNA, or Myo5b antisense shRNA (colors as in A), grown on Snapwell filters and measured in an Ussing chamber in normal Cl− Ringer solution (129.1 mM Cl−), or low Cl− Ringer solution (128 mM sodium gluconate, 1.1 mM Cl−). For both Ringer formulations, the apical solution was supplemented with 10 mM mannitol while the basolateral solution contained 10 mM glucose. At the end of each experiment, the cells were incubated in normal Cl− solution in the presence of 40 µM Rp-cAMP. (C) Quantification of experiments like the one shown in B. Measurements were taken around 15 min after adding forskolin, in steady state. The bars represent the difference between Isc (in µA/cm2) in stimulated cells minus the baseline in the same experiment. Statistical significance was determined by t test; *, p < 0.05; n = 4. (D) Quantification of the experiments like the one in C. Bars represent the average difference between nonstimulated Isc (µA/cm2) in normal Cl− Ringer and low Cl− (gluconate) Ringer. *, p < 0.04; **, p < 0.02; n = 3.
FIGURE 5:
FIGURE 5:
PKA-phosphorylated substrates concentrate under the apical domain in intestinal epithelia defective in Myo5b in vivo. (A–C) Fetal mouse small intestine, stained with PKA p-substrate antibody (A, B) or isotype control, processed with a similar dilution of nonimmune rabbit serum (C). (B) Sample from Myo5b KO mouse. (A, C) Heterozygous control. (D, E) Human duodenum biopsies from unrelated disease (D, F) or MVID (E). Both patients (6 and 5 mo old, respectively) were subjected to endoscopy and duodenal biopsy as diagnostic procedures for chronic diarrhea. Diagnosis of MVID was reached by PAS and EM (unpublished data), confirming the presence of MI (arrowhead). Arrows point at apical PKA p-substrate signal. Scale bar = 20 µm.
FIGURE 6:
FIGURE 6:
CFTR and PKA p-substrate signal colocalize within molecular distances in the apical domain in cells lacking Myo5b grown in the presence of 0.5 µM dexamethasone for 10 d (dexa). T84 cells were fixed and stained with anti-PKA p-substrate (p-s) antibody and anti-CFTR monoclonal antibodies, followed by affinity-purified antibodies labeled with Cy3 or Cy5. (A) Confocal stacks including the apical surface of the cells were imaged in the XZ plane. Scale bar = 10 µm. (B) In similar experiments, the apicalmost confocal section (0.68 µm thick in the z-axis) showing microvilli was subjected to acceptor (Cy5) bleaching (200 cycles) in random ROI and imaged again after bleaching. Scale bar = 4 µm. (C) Average FRET efficiency from three experiments (30 ROIs) is shown for control (scr) and Myo5b kd cells, incubated or not in dexamethasone (dexa) for 10 d. *, p < 0.001; **, p < 0.04.
FIGURE 7:
FIGURE 7:
Myo5b loss of function induces redistribution of cAMP gradients in intestinal cells in the presence of GCs. Cyclic AMP was determined in vivo by transduction of a cAMP “upward” biosensor in T84 cells. Cells expressing a scrambled shRNA (scr) or Myo5b antisense shRNA (kd) were incubated in dexamethasone or vehicle as described before and transduced with a human-adapted baculovirus expressing cAMP biosensor. Some scr cell cultures (with no dexamethasone) were also preincubated with 10 µM forskolin for 30 min as a positive control for transduction and biosensor fluorescence. (A) 3D reconstruction of confocal stacks from live T84 cells show DNA counterstain (DAPI, blue) and biosensor fluorescence (red). Yellow rectangles are example focal planes of randomly localized apical (a) or basolateral (b) ROI in the XY sections used for quantification in B. Scale bar = 10 µm. (B) Biosensor fluorescence was quantified in positively transduced cells using random ROIs in the transnuclear/basolateral domain (b) and or in the apical domain (a) in XY confocal sections. Yellow rectangles show examples of the location of a and b confocal planes in XZ reconstructions. Arrows point at the only condition in which the apical/basolateral cAMP gradient is inverted in unstimulated (no forskolin) cells: Myo5b kd cells grown in dexamethasone. *, p < 0.001.
FIGURE 8:
FIGURE 8:
Biotinylated apical membrane protein epithelial cells deficient in Myo5b show increased phosphorylation. (A) C2BBe cells constitutively expressing scrambled shRNA (c and s) or Myo5b antisense shRNA (kd) were grown on filters, biotinylated from the apical side, extracted, pulled down with streptavidin-agarose, and visualized by blot and chemiluminescence with streptavidin. One set of cultures was not biotinylated (control, c). A fraction (10%) of the input was stained with Ponceau S red to show total protein (input). Stars indicate examples of biotinylated bands that remain exposed to the apical chamber in Myo5b-deficient cells (representative experiment out of three). (B) In similar experiments, T84 cells expressing scrambled RNA (s) or Myo5b shRNA (kd) were grown with or without 0.5 µM dexamethasone (dexa), and revealed with streptavidin-HRP. (C) A similar extract was pulled down with streptavidin beads and analyzed by immunoblot with PKA p-substrate antibody. A fraction (5%) of total cell extracts in Triton X-100 (input) was analyzed by immunoblot with anti-tubulin antibody as loading control. (D) Quantification of PKA p-substrate signal in two biotinylated bands. The PKA p-substrate signal relative to the average intensity of the bands in Myo5b-deficient cells with no dexamethasone treatment were plotted for the prominent 54 and 60 kDa bands shown in C. Box plots represent first, third quartile, and median for each group (each dot represents an independent observation). Kruskal-Wallis; *, p < 0.02. (E) T84 cells were treated as described in B. The cells were extracted in 2% Triton X-114 in the cold, and the extracts subjected to detergent condensation to enrich in membrane proteins and their binding partners. 10% of the input was analyzed by immunoblot (i.b.) with the antibodies listed on the right-hand side. The rest was immunoprecipitated with an anti–14-3-3 antibody recognizing multiple isoforms and analyzed for immunoblot with the same antibodies. For 14-3-3 blots, the signal was revealed with an anti-rabbit secondary antibody coupled to HRP that only recognizes the native conformation of IgG. Arrowheads in CFTR immunoblots point at the position of the immature (b) and mature (c) forms. Mr of standards are expressed ×10−3.
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