Regulation of Dab2 expression in intestinal and renal epithelia by development

Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression.     

α-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine

The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and α-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether α-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with α-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to α-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with α-AP-2. siRNA silencing of α-AP-2 in cells significantly reduced CFTR endocytosis, further supporting α-AP-2 as the direct binding partner for CFTR. α-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snell''s Waltzer myosin VI^(sv/sv) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI^(sv/sv) enterocytes. The findings support α-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.     

Shigella targets epithelial tricellular junctions and uses a noncanonical clathrin-dependent endocytic pathway to spread between cells

Bacteria move between cells in the epithelium using a sequential pseudopodium-mediated process but the underlying mechanisms remain unclear. We show that during cell-to-cell movement, Shigella-containing pseudopodia target epithelial tricellular junctions, the contact point where three epithelial cells meet. The bacteria-containing pseudopodia were engulfed by neighboring cells only in the presence of tricellulin, a protein essential for tricellular junction integrity. Shigella cell-to-cell spread, but not pseudopodium protrusion, also depended on phosphoinositide 3-kinase, clathrin, Epsin-1, and Dynamin-2, which localized beneath the plasma membrane of the engulfing cell. Depleting tricellulin, Epsin-1, clathrin, or Dynamin-2 expression reduced Shigella cell-to-cell spread, whereas AP-2, Dab2, and Eps15 were not critical for this process. Our findings highlight a mechanism for Shigella dissemination into neighboring cells via targeting of tricellular junctions and a noncanonical clathrin-dependent endocytic pathway.     

Clint: a novel clathrin-binding ENTH-domain protein at the Golgi

We have characterized a novel clathrin-binding 68-kDa epsin N-terminal homology domain (ENTH-domain) protein that we name clathrin interacting protein localized in the trans-Golgi region (Clint). It localizes predominantly to the Golgi region of epithelial cells as well as to more peripheral vesicular structures. Clint colocalizes with AP-1 and clathrin only in the perinuclear area. Recombinantly expressed Clint interacts directly with the gamma-appendage domain of AP-1, with the clathrin N-terminal domain through the peptide motif (423)LFDLM, with the gamma-adaptin ear homology domain of Golgi-localizing, gamma-adaptin ear homology domain 2, with the appendage domain of beta2-adaptin and to a lesser extent with the appendage domain of alpha-adaptin. Moreover, the Clint ENTH-domain asssociates with phosphoinositide-containing liposomes. A significant amount of Clint copurifies with rat liver clathrin-coated vesicles. In rat kidney it is preferentially expressed in the apical region of epithelial cells that line the collecting duct. Clathrin and Clint also colocalize in the apical region of enterocytes along the villi of the small intestine. Apart from the ENTH-domain Clint has no similarities with the epsins AP180/CALM or Hip1/1R. A notable feature of Clint is a carboxyl-terminal methionine-rich domain (Met(427)-Met(605)), which contains >17% methionine. Our results suggest that Clint might participate in the formation of clathrin-coated vesicles at the level of the trans-Golgi network and remains associated with the vesicles longer than clathrin and adaptors.     

Albumin endocytosis via megalin in astrocytes is caveola- and Dab-1 dependent and is required for the synthesis of the neurotrophic factor oleic acid

The synthesis and release of the neurotrophic factor oleic acid requires internalization of albumin into the astrocyte, which is mediated by megalin. In this study, we show that the binding and internalization of albumin involve its interaction with megalin, caveolin-1, caveolin-2 and cavin, but not with clathrin in astrocytes from primary culture. Electron microscopy analyses revealed albumin-gold complexes localized in caveolae, but not in clathrin-coated vesicles. Neither chlorpromazine nor silencing clathrin expression modified albumin uptake. Silencing caveolin-1 strongly reduced the binding and internalization of albumin and the distribution of megalin in the plasma membrane. However, silencing caveolin-2 only decreased albumin internalization, suggesting that caveolin-1 is responsible for megalin recruitment to the caveolae and that caveolin-2 participates in caveolae internalization. In most tissues, the cytosolic adaptor protein disabled (Dab)-2 connects megalin to clathrin, astrocytes lack Dab-2; instead, they express Dab-1, which interacts with caveolin-1 and megalin and is required for albumin internalization. The transcytosis of albumin in astrocytes, including the passage through the endoplasmic reticulum, which is a compulsory step for oleic acid synthesis, was confirmed by electron microscopy analyses. Thus, whereas silencing clathrin did not modify the synthesis and release of oleic acid, the knock-down of caveolin-1, caveolin-2 and Dab-1 strongly reduced the synthesis and release of this neurotrophic factor. In conclusion, caveola-mediated endocytosis of albumin requires megalin and the adaptor protein Dab-1 in cultured astrocytes. Albumin endocytosis may be a key step in brain development because it stimulates the synthesis of oleic acid, which in turn promotes neuronal differentiation.     

The Clathrin Adaptor Proteins ARH,Dab2, and Numb Play Distinct Roles in Niemann-Pick C1-Like 1 Versus Low Density Lipoprotein Receptor-mediated Cholesterol Uptake

The uptake of circulating low density lipoproteins (LDL) is mediated by LDL receptor (LDLR) through clathrin-dependent endocytosis. At the early stage of this process, adaptor proteins ARH and Dab2 specifically bind the endocytic signal motif in LDLR and recruit clathrin/AP2 to initiate internalization. On the other hand, intestinal cholesterol is absorbed by Niemann-Pick C1-Like 1 (NPC1L1) through clathrin-dependent endocytosis. Another adaptor protein, Numb recognizes the endocytic motif in NPC1L1 C terminus and couples NPC1L1 to endocytic machinery. The ARH, Dab2, and Numb proteins contain a homogeneous phosphotyrosine binding (PTB) domain that directly binds endocytic motifs. Because ARH, Dab2, and Numb are all PTB domain family members, the emerging mystery is whether these adaptors act complementally in LDLR and NPC1L1 endocytosis. Here, we found that ARH and Dab2 did not bind NPC1L1 and were not required for NPC1L1 internalization. Similarly, Numb lacked the ability to interact with the LDLR C terminus and was dispensable for LDL uptake. Only the Numb isoforms with shorter PTB domain could facilitate NPC1L1 endocytosis. Besides the reported function in intestinal cholesterol absorption, Numb also mediated cholesterol reabsorption from bile in liver. We further identified a Numb variant with G595D substitution in humans of low blood LDL-cholesterol. The G595D substitution impaired NPC1L1 internalization and cholesterol reabsorption, due to attenuating affinity of Numb to clathrin/AP2. These results demonstrate that Numb specifically regulates NPC1L1-mediated cholesterol absorption both in human intestine and liver, distinct from ARH and Dab2, which selectively participate in LDLR-mediated LDL uptake.     

Apical location of ferroportin 1 in airway epithelia and its role in iron detoxification in the lung

Ferroportin 1 (FPN1; aka MTP1, IREG1, and SLC40A1), which was originally identified as a basolateral iron transporter crucial for nutritional iron absorption in the intestine, is expressed in airway epithelia and upregulated when these cells are exposed to iron. Using immunofluorescence labeling and confocal microscopic imaging techniques, we demonstrate that in human and rodent lungs, FPN1 localizes subcellularly to the apical but not basolateral membrane of the airway epithelial cells. The role of airway epithelial cells in iron mobilization in the lung was studied in an in vitro model of the polarized airway epithelium. Normal human bronchial epithelial cells, grown on membrane supports until differentiated, were exposed to iron, and the efficiency and direction of iron transportation were studied. We found that these cells can efficiently take up iron across the apical but not basolateral surface in a concentration-dependent manner. Most of the iron taken up by the cells is then released into the medium within 8 h in the form of less reactive protein-bound complexes including ferritin and transferrin. Interestingly, iron release also occurred across the apical but not basolateral membrane. Our findings indicate that FPN1, depending on its subcellular location, could have distinct functions in iron homeostasis in different cells and tissues. Although it is responsible for exporting nutrient iron from enterocytes to the circulation in the intestine, it could play a role in iron detoxification in airway epithelial cells in the lung.     

Na+-dependent glucose transporter SGLT1 is localized in the apical plasma membrane upon completion of tight junction formation in MDCK cells

SGLT1, an isoform of Na⁺-dependent glucose transporters, is localized at the apical plasma membrane in the epithelial cells of the small intestine and the kidney. In the present study we examined its location in SGLT1 cDNA-transfected MDCK cells, which form an epithelial sheet connected by tight junctions in culture. Formation of tight junctions was monitored by staining for occludin, an integral tight junction protein. In the cells demarcated by an uninterrupted occludin meshwork, SGLT1 was specifically localized at the apical plasma membrane, showing that SGLT1 has a signal to accomplish this restricted localization. In the cells with little or no occludin accumulation in the tight junction, however, SGLT1 was present along the entire aspect of the plasma membrane. Similar distribution of SGLT1 was observed in the cells as long as the occludin meshwork remained incomplete. These observations sugget that apical localization of SGLT1 occurs upon the completion of the uninterrupted meshwork of tight junctions.     

Proper E‐cadherin membrane location in colon requires Dab2 and it modifies by inflammation and cancer

We reported that Disabled‐2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E‐cadherin. Dab2 depletion in Caco‐2 cells led to E‐cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium‐induced colitis increased Dab2/E‐cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E‐cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E‐cadherin delocalization. Epithelial Dab2 decrement preceded that of E‐cadherin. We suggest that Dab2, by inhibiting E‐cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.     

STa and cGMP stimulate CFTR translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase G

The cystic fibrosis transmembrane conductance regulator (CFTR) is critical to cAMP- and cGMP-activated intestinal anion secretion and the pathogenesis of secretory diarrhea. Enterotoxins released by Vibrio cholerae (cholera toxin) and Escherichia coli (heat stable enterotoxin, or STa) activate intracellular cAMP and cGMP and signal CFTR on the apical plasma membrane of small intestinal enterocytes to elicit chloride and fluid secretion. cAMP activates PKA, whereas cGMP signals a cGMP-dependent protein kinase (cGKII) to phosphorylate CFTR in the intestine. In the jejunum, cAMP also regulates CFTR and fluid secretion by insertion of CFTR from subapical vesicles to the surface of enterocytes. It is unknown whether cGMP signaling or phosphorylation regulates the insertion of CFTR associated vesicles from the cytoplasm to the surface of enterocytes. We used STa, cell-permeant cGMP, and cAMP agonists in conjunction with PKG and PKA inhibitors, respectively, in rat jejunum to examine whether 1) cGMP and cGK II regulate the translocation of CFTR to the apical membrane and its relevance to fluid secretion, and 2) PKA regulates cAMP-dependent translocation of CFTR because this intestinal segment is a primary target for toxigenic diarrhea. STa and cGMP induced a greater than fourfold increase in surface CFTR in enterocytes in association with fluid secretion that was inhibited by PKG inhibitors. cAMP agonists induced a translocation of CFTR to the cell surface of enterocytes that was prevented by PKA inhibitors. We conclude that cAMP and cGMP-dependent phosphorylation regulates fluid secretion and CFTR trafficking to the surface of enterocytes in rat jejunum. small intestine; cystic fibrosis transmembrane conductance regulator; membrane traffic; phosphorylation     

Structural-functional analysis of diffusion in glucose absorption by rat small intestine enterocytes

To elucidate mechanisms providing transport of sugars across intestinal epithelium, on taking into account the current hypotheses (active transport, participation of paracellular transport and passive component of transcellular transport), it was important to reveal structural changes of tight junctions and distribution of the carriers of facilitated diffusion of GLUT2 and protein kinase C during absorption of glucose. On using confocal and electron microscopy, ultrastructural and immunocytochemical studies of enterocytes after perfusion of isolated rat small intestine fragment with 75 mM glucose (chronic experiment) have shown: 1) fluorescent labels of transporter GLUT2 and PKCbetaII are located in the apical area of enterocytes situated at the upper half of the villus. Antibodies against GLUT2, conjugated with gold, are revealed at the microvilli or apical membrane and in the area of terminal network; 2) no ultrastructural changes of the tight junction are detected on ultrathin sections and freeze--fracture replics. At the same time, fluorescent and gold labels against actin are concentrated in the vicinity of the lateral membrane in the tight junction area. The results obtained can serve a confirmation of a hypothesis that at high glucose concentrations GLUT2 participates in its transfer across the apical membrane.     

Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin-coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI–Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.     

Differential localization and regulation of two aquaporin-1 homologs in the intestinal epithelia of the marine teleost Sparus aurata

Aquaporin (AQP)-mediated intestinal water absorption may play a major osmoregulatory role in euryhaline teleosts, although the molecular identity and anatomical distribution of AQPs in the fish gastrointestinal tract is poorly known. Here, we have investigated the functional properties and cellular localization in the intestine of two gilthead seabream (Sparus aurata) homologs of mammalian aquaporin-1 (AQP1), named SaAqp1a and SaAqp1b. Heterologous expression in Xenopus laevis oocytes showed that SaAqp1a and SaAqp1b were water-selective channels. Real-time quantitative RT-PCR and Western blot using specific antisera indicated that abundance of SaAqp1a mRNA and protein was higher in duodenum and hindgut than in the rectum, whereas abundance of SaAqp1b was higher in rectum. In duodenum and hindgut, SaAqp1a localized at the apical brush border and lateral membrane of columnar enterocytes, whereas SaAqp1b was detected occasionally and at very low levels at the apical membrane. In the rectum, however, SaAqp1a was mainly accumulated in the cytoplasm of a subpopulation of enterocytes spread in groups over the surface of the epithelia, including the intervillus pockets, whereas SaAqp1b was detected exclusively at the apical brush border of all rectal enterocytes. Freshwater acclimation reduced the synthesis of SaAqp1a protein in all intestinal segments, but it only reduced SaAqp1b abundance in the rectum. These results show for the first time in teleosts a differential distribution and regulation of two functional AQP1 homologs in the intestinal epithelium, which suggest that they may play specialized functions during water movement across the intestine.     

Polarized localization of vitamin C transporters, SVCT1 and SVCT2, in epithelial cells

Messenger RNA of homologous sodium-vitamin C cotransporters, SVCT1 and SVCT2, were found in the intestine. Studies using cultured intestinal cells suggested an apical presence of SVCT1 but the function of SVCT2 was unknown. Here, we showed that enterocytes from heterozygous SVCT2-knockout mice had lower sodium-dependent vitamin C accumulation compared to those from the wildtype. Thus, SVCT2 appears to be functional in enterocytes. We then tested whether SVCT2 could have a redundant function as SVCT1 by constructing and expressing EGFP-tagged SVCTs in intestinal Caco-2 and kidney MDCK cells. In confluent epithelial cells, SVCT1 protein expressed predominantly on the apical membrane. SVCT2, in contrast, accumulated at the basolateral surface. Functionally, SVCT1 expression led to more transport activity from the apical membrane, while SVCT2 expression only increased the uptake under the condition when basolateral membrane was exposed. This differential epithelial membrane distribution and function suggests non-redundant functions of these two isoforms.     

Comparative analysis of SGLT1 and GLUT2 transporters distribution in rat small-intestine enterocytes and Caco-2 cells during hexose absorption

The distribution of SGLT1 and GLUT2 hexose transporters has been evaluated in enterocytes of an isolated loop of the small intestine and Caco-2 cell culture after absorption of hexoses at their high and low concentrations. The SGLT1 transporter was found to be located in enterocytes along the edge of the intestinal villus. The GLUT2 transporter after loading with high hexose concentrations is located in the apical part of enterocytes. In culture, Caco-2 cells form a characteristic of enterocytes microvilli and the cell junction complex. During the incubation of the culture in solutions of glucose and galactose, the absorption of these sugars from the incubation medium was observed. The SGLT1 transporter in the Caco-2 cells is located in the apical and perinuclear enterocyte parts and is organized in globules. After loading with hexoses at low concentrations, the GLUT2 transporter is in the basal cell area. The Caco-2 cell culture can serve a model for studying the transport of sugar in the intestinal epithelium.     

Keeping it tight: The relationship between bacterial dysbiosis,septate junctions,and the intestinal barrier in Drosophila

Maladaptive changes in the intestinal flora, typically referred to as bacterial dysbiosis, have been linked to intestinal aging phenotypes, including an increase in intestinal stem cell (ISC) proliferation, activation of inflammatory pathways, and increased intestinal permeability^1,2. However, the causal relationships between these phenotypes are only beginning to be unravelled. We recently characterized the age-related changes that occur to septate junctions (SJ) between adjacent, absorptive enterocytes (EC) in the fly intestine. Changes could be observed in the overall level of SJ proteins, as well as the localization of a subset of SJ proteins. Such age-related changes were particularly noticeable at tricellular junctions (TCJ)³. Acute loss of the Drosophila TCJ protein Gliotactin (Gli) in ECs led to rapid activation of stress signalling in stem cells and an increase in ISC proliferation, even under axenic conditions; a gradual disruption of the intestinal barrier was also observed. The uncoupling of changes in bacteria from alterations in ISC behaviour and loss of barrier integrity has allowed us to begin to explore the interrelationship of these intestinal aging phenotypes in more detail and has shed light on the importance of the proteins that contribute to maintenance of the intestinal barrier.     

Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.     

ApoER2 is endocytosed by a clathrin-mediated process involving the adaptor protein Dab2 independent of its Rafts' association

The apolipoprotein E receptor 2 (apoER2) is a member of the low-density lipoprotein receptor family which binds ligands such as reelin, apolipoprotein E and apolipoprotein J/clusterin and has been shown to play roles in neuronal migration during development and in male fertility. The function of apoER2 mainly depends on cellular signaling triggered by ligand binding. Although the receptor is internalized, the mechanism and functional significance of its endocytic trafficking remain unclear. Apolipoprotein E receptor 2 partitions into lipid rafts and interacts with caveolin-1, a feature that could modulate its endocytic behavior. Recent evidence also suggested that apoER2 might be endocytosed by a pathway independent of clathrin. Here, we show that despite a raft association, apoER2 internalization depends on its cytoplasmic FxNPXY motif that is similar to canonical motifs for clathrin-mediated endocytosis. This motif mediates receptor binding to the adaptor protein Dab2, which can interact directly with clathrin. Several inhibitory conditions of clathrin-mediated endocytosis, including expression of the dominant negative forms of eps15 and Dab2, decreased apoER2 internalization. In contrast, treatment with the drug nystatin, which blocks the caveolar/raft internalization pathway, has no effect on the receptor's endocytosis. Neither the transmembrane nor the proline-rich insert of the cytoplasmic domain, which has been previously reported to exclude the receptor from the clathrin-mediated pathway, altered apoER2 endocytic activity. These studies indicate that apoER2 internalizes through a clathrin-mediated pathway and that its association with caveolar and noncaveolar rafts does not determine its endocytosis.     

Numb modulates the paracellular permeability of intestinal epithelial cells through regulating apical junctional complex assembly and myosin light chain phosphorylation

Numb is highly expressed throughout the crypt-villus axis of intestinal mucosa and functions as cell fate determinant and integrator of cell-to-cell adhesion. Increased paracellular permeability of intestinal epithelial cells is associated with the epithelial barrier dysfunction of inflammatory bowel diseases (IBDs). The apical junctional complex (AJC) assembly and myosin light chain (MLC) phosphorylation regulate adherens junctions (AJ) and tight junctions (TJ). We determined whether and how Numb modulate the paracellular permeability of intestinal epithelial cells. Caco-2 intestinal epithelial cells and their Numb-interfered counterparts were used in the study for physiological, morphological and biological analyses. Numb, expressed in intestinal epithelial cells and located at the plasma membrane of Caco-2 cells in a basolateral to apical distribution, increased in the intestinal epithelial cells with the formation of the intestinal epithelial barrier. Numb expression decreased and accumulated in the cytoplasm of intestinal epithelial cells in a DSS-induced colitis mouse model. Numb co-localized with E-cadherin, ZO-1 and Par3 at the plasma membrane and interacted with E-cadherin and Par3. Knockdown of Numb in Caco-2 cells altered the F-actin structure during the Ca²⁺ switch assay, enhanced TNFα-/INF-γ-induced intestinal epithelial barrier dysfunction and TJ destruction, and increased the Claudin-2 protein level. Immunofluorescence experiments revealed that NMIIA and F-actin co-localized at the cell surface of Caco-2 cells. Numb knockdown in Caco-2 cells increased F-actin contraction and the abundance of phosphorylated MLC. Numb modulated the intestinal epithelial barrier in a Notch signaling-independent manner. These findings suggest that Numb modulates the paracellular permeability by affecting AJC assembly and MLC phosphorylation.