Myosin 5b loss of function leads to defects in polarized signaling: implication for microvillus inclusion disease pathogenesis and treatment

Abstract

Microvillus inclusion disease (MVID) is an autosomal recessive condition resulting in intractable secretory diarrhea in newborns due to loss-of-function mutations in myosin Vb (Myo5b). Previous work suggested that the apical recycling endosomal (ARE) compartment is the primary location for phosphoinositide-dependent protein kinase 1 (PDK1) signaling. Because the ARE is disrupted in MVID, we tested the hypothesis that polarized signaling is affected by Myo5b dysfunction. Subcellular distribution of PDK1 was analyzed in human enterocytes from MVID/control patients by immunocytochemistry. Using Myo5b knockdown (kd) in Caco-2BBe cells, we studied phosphorylated kinases downstream of PDK1, electrophysiological parameters, and net water flux. PDK1 was aberrantly localized in human MVID enterocytes and Myo5b-deficient Caco-2BBe cells. Two PDK1 target kinases were differentially affected: phosphorylated atypical protein kinase C (aPKC) increased fivefold and phosohoprotein kinase B slightly decreased compared with control. PDK1 redistributed to a soluble (cytosolic) fraction and copurified with basolateral endosomes in Myo5b kd. Myo5b kd cells showed a decrease in net water absorption that could be reverted with PDK1 inhibitors. We conclude that, in addition to altered apical expression of ion transporters, depolarization of PDK1 in MVID enterocytes may lead to aberrant activation of downstream kinases such as aPKC. The findings in this work suggest that PDK1-dependent signaling may provide a therapeutic target for treating MVID.

Mechanisms of Cell Polarity–Controlled Epithelial Homeostasis and Immunity in the Intestine

“…Atypical PKC and Par6B show aberrant localization in IEC of individuals with microvillus inclusion disease carrying MYO5B mutations (Dhekne et al 2014;Kravtsov et al 2014;Michaux et al 2015), which is in agreement with a reported role for apical recycling endosomes in the regulation of the subcellular distribution of these polarity proteins (Golachowska et al 2010;Dhekne et al 2014). The mislocalization of aPKC-iota from the subapical domain of IEC was associated with impaired activation of ezrin at the apical surface and consequent impaired brush-border development (Dhekne et al 2014).…”

“…In addition, the spatial distribution of endosomal system provides polarized signaling platforms (Bryant et al 2010;Dhekne et al 2014;Kravtsov et al 2014) that control the activation of ezrin at the apical surface of intestinal epithelial cells and, thereby, the development of the apical brush-border membrane (Dhekne et al 2014). Within the complex endosomal system (Perret et al 2005;Bay et al 2015), two endosomal subcompartments have been identified that appear particularly important for the correct sorting and trafficking of apical and basal proteins: the apical recycling endosome (ARE) and the common recycling endosome (CRE) (Fig.…”

Identification of intestinal ion transport defects in microvillus inclusion disease

“…MYO5B KD cells were generated with MYO5B mRNA targeted shRNA (NM_ 001080467) delivered by a pLKO.1-Puro-based lentiviral system and maintained with a selection marker puromycin (12 g/ml; Cayman Chemical, Ann Arbor, MI). Cells were transduced to stably express either scrambled or MYO5B-targeting shRNA (32). C2BBe cells at passage number 15 or less were grown on 100-mm dishes (Corning, Corning, NY) to ϳ70 -90% confluency and then subjected to reverse transduction (cells were seeded in the titrated viral medium supplemented with 8 g/ml Polybrene at 1:2 ratio; Millipore, Waltham, MA).…”

“…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).…”

An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking

“…MYO5B functions as a homodimer and has three functional domains: an N-terminal motor domain, a calmodulin-binding domain, and a C-terminal tail, which binds cargo through association with the small GTPases RAB8A and/or RAB11A (12, 13). Altered expression of myosin Vb affects the apical membrane trafficking mechanism in epithelial cells, causing mislocalization of apical brush border proteins, such as villin (vil), CD10, or alkaline phosphatase (ALP) in the cytoplasm of duodenal enterocytes (2, 3, 5), and an increased apical localization of transferrin receptor (5,14).Although mouse models mimicking certain features of MVID have previously been described, such as Rab8 (15), Cdc42 (16, 17), and Rab11a knockout (KO) mice (18,19), no mutations in the coding regions of those genes have been reported in human MVID patients. Current in vitro models to study apical trafficking and polarization-associated diseases such as MVID are the parental Caco2 cell line, Caco-BBE, and LS174 W4 cells, in which polarization can be induced in vitro (4,8,12, 20).…”