scholarly journals A novel adhesive complex at the base of intestinal microvilli

2021 ◽  
Author(s):  
Christian Hartmann ◽  
Eva-Maria Thüring ◽  
Birgitta E. Michels ◽  
Denise Pajonczyk ◽  
Sophia Leußink ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Pdz Domain ◽  
Basal Region ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Pdz Domains ◽  
Ternary Complex Formation ◽  
Adhesion Complex

AbstractIntestinal epithelial cells form dense arrays of microvilli at the apical membrane to enhance their functional capacity. Microvilli contain a protocadherin-based intermicrovillar adhesion complex localized at their tips which regulates microvillar length and packaging. Here, we identify a second adhesive complex in microvilli of intestinal epithelial cells. This complex is localized at the basal region of microvilli and consists of the adhesion molecule TMIGD1, the phosphoprotein EBP50 and the F-actin – plasma membrane cross-linking protein ezrin. Ternary complex formation requires unmasking of the EBP50 PDZ domains by ezrin binding and is strongly enhanced upon mutating Ser162 located in PDZ domain 2 of EBP50. Dephosphorylation of EBP50 at S162 is mediated by PP1α, a serine/threonine phosphatase localized at the microvillar base and involved in ezrin phosphocycling. Importantly, the binding of EBP50 to TMIGD1 enhances the dynamic turnover of EBP50 at microvilli in a Ser162 phosphorylation-dependent manner. We identify an adhesive complex at the microvillar base and propose a potential mechanism that regulates microvillar dynamics in enterocytes.

scholarly journals Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3

2015 ◽  
Vol 26 (11) ◽  
pp. 2030-2043 ◽  
Author(s):  
Varsha Singh ◽  
Jianbo Yang ◽  
Boyoung Cha ◽  
Tiane-e Chen ◽  
Rafiquel Sarker ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Pdz Domain ◽  
Surface Expression ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Pdz Domains ◽  
Sorting Nexin ◽  
C Terminus ◽  
Cargo Proteins

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain–dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.

scholarly journals Positive cross talk between protein kinase D and β-catenin in intestinal epithelial cells: impact on β-catenin nuclear localization and phosphorylation at Ser552

2016 ◽  
Vol 310 (7) ◽  
pp. C542-C557 ◽  
Author(s):  
Jia Wang ◽  
Liang Han ◽  
James Sinnett-Smith ◽  
Li-Li Han ◽  
Jan V. Stevens ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Transgenic Mice ◽  
Nuclear Localization ◽  
Cross Talk ◽  
Intestinal Epithelial Cells ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Ang Ii ◽  
Potent Inducer

Given the fundamental role of β-catenin signaling in intestinal epithelial cell proliferation and the growth-promoting function of protein kinase D1 (PKD1) in these cells, we hypothesized that PKDs mediate cross talk with β-catenin signaling. The results presented here provide several lines of evidence supporting this hypothesis. We found that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II (ANG II), a potent inducer of PKD activation, promoted endogenous β-catenin nuclear localization in a time-dependent manner. A significant increase was evident within 1 h of ANG II stimulation ( P < 0.01), peaked at 4 h ( P < 0.001), and declined afterwards. GPCR stimulation also induced a marked increase in β-catenin-regulated genes and phosphorylation at Ser552 in intestinal epithelial cells. Exposure to preferential inhibitors of the PKD family (CRT006610 or kb NB 142-70) or knockdown of the isoforms of the PKD family prevented the increase in β-catenin nuclear localization and phosphorylation at Ser552 in response to ANG II. GPCR stimulation also induced the formation of a complex between PKD1 and β-catenin, as shown by coimmunoprecipitation that depended on PKD1 catalytic activation, as it was abrogated by cell treatment with PKD family inhibitors. Using transgenic mice that express elevated PKD1 protein in the intestinal epithelium, we detected a marked increase in the localization of β-catenin in the nucleus of crypt epithelial cells in the ileum of PKD1 transgenic mice, compared with nontransgenic littermates. Collectively, our results identify a novel cross talk between PKD and β-catenin in intestinal epithelial cells, both in vitro and in vivo.

scholarly journals Lipid rafts mediate internalization of β1-integrin in migrating intestinal epithelial cells

2008 ◽  
Vol 295 (5) ◽  
pp. G965-G976 ◽  
Author(s):  
Elena V. Vassilieva ◽  
Kirsten Gerner-Smidt ◽  
Andrei I. Ivanov ◽  
Asma Nusrat
Keyword(s):  
Epithelial Cells ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Intestinal Epithelial Cells ◽  
Focal Adhesions ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Caveolin 1 ◽  
Cell Matrix

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of β1-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, β1-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent β1-integrin internalization. However, β1-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized β1-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased β1-integrin endocytosis. Our data suggest that, in migrating IEC, β1-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

scholarly journals Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b-dependent mechanism

2020 ◽  
Vol 318 (4) ◽  
pp. C732-C739
Author(s):  
Fangyi Liu ◽  
Xiao Wang ◽  
Hua Geng ◽  
Heng-Fu Bu ◽  
Peng Wang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Intestinal Epithelial Cells ◽  
Specific Inhibitor ◽  
In Silico Analysis ◽  
Interferon Γ ◽  
Luciferase Assay ◽  
Epithelial Cell Line ◽  
Dependent Manner ◽  
Intestinal Epithelial

Sirtuin 6 (Sirt6) is predominantly expressed in epithelial cells in intestinal crypts. It plays an important role in protecting intestinal epithelial cells against inflammatory injury. Previously, we found that colitis is associated with the downregulation of Sirt6 protein in the intestines. Here, we report that murine interferon-γ (Ifnγ) inhibits Sirt6 protein but not mRNA expression in young adult mouse colonocytes (YAMC, a mouse colonic epithelial cell line) in a dose- and time-dependent manner. Using microRNA array analysis, we showed that Ifnγ induces expression of miR-92b in YAMC cells. With in silico analysis, we found that the Sirt6 3′-untranslated region (UTR) contains a putative binding site for miR-92b. Luciferase assay showed that Ifnγ inhibited Sirt6 3′-UTR activity and this effect was mimicked by miR-92b via directly targeting the miR-92b seed site in the 3′-UTR of Sirt6 mRNA. Furthermore, Western blot demonstrated that miR-92b downregulated Sirt6 protein expression in YAMC cells. Blocking miR-92b with a specific inhibitor attenuated the inhibitory effect of Ifnγ on Sirt6 protein expression in the cells. Collectively, our data suggest that Ifnγ inhibits Sirt6 protein expression in intestinal epithelial cells via a miR-92b-mediated mechanism. miR-92b may be a novel therapeutic target for rescuing Sirt6 protein levels in intestinal epithelial cells, thereby protecting against intestinal mucosal injury caused by inflammation.

scholarly journals Clostridium perfringens Delta-Toxin Damages the Mouse Small Intestine

Toxins ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 232 ◽  
Author(s):  
Soshi Seike ◽  
Masaya Takehara ◽  
Keiko Kobayashi ◽  
Masahiro Nagahama
Keyword(s):  
Epithelial Cells ◽  
Clostridium Perfringens ◽  
Intestinal Epithelial Cells ◽  
Cell Damage ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Fluid Accumulation ◽  
Ileal Loop ◽  
Junction Protein ◽  
E Cadherin

Clostridium perfringens strains B and C cause fatal intestinal diseases in animals. The secreted pore-forming toxin delta-toxin is one of the virulence factors of the strains, but the mechanism of intestinal pathogenesis is unclear. Here, we investigated the effects of delta-toxin on the mouse ileal loop. Delta-toxin caused fluid accumulation and intestinal permeability to fluorescein isothiocyanate (FITC)-dextran in the mouse ileal loop in a dose- and time-dependent manner. Treatment with delta-toxin induced significant histological damage and shortening of villi. Delta-toxin activates a disintegrin and metalloprotease (ADAM) 10, leading to the cleavage of E-cadherin, the epithelial adherens junction protein, in human intestinal epithelial Caco-2 cells. In this study, E-cadherin immunostaining in mouse intestinal epithelial cells was almost undetectable 1 h after toxin treatment. ADAM10 inhibitor (GI254023X) blocked the toxin-induced fluid accumulation and E-cadherin loss in the mouse ileal loop. Delta-toxin stimulated the shedding of intestinal epithelial cells. The shedding cells showed the accumulation of E-cadherin in intracellular vesicles and the increased expression of active caspase-3. Our findings demonstrate that delta-toxin causes intestinal epithelial cell damage through the loss of E-cadherin cleaved by ADAM10.

Fructooligosaccharide Inhibits the Absorption of β-conglycinin (A Major Soybean Allergen) in IPEC-J2

2018 ◽  
Vol 15 (1-2) ◽  
Author(s):  
Yuan Zhao ◽  
Shiyao Zhang ◽  
Xiaodong Zhang ◽  
Li Pan ◽  
Nan Bao ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Enzymatic Hydrolysis ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Time Dependent ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Intracellular Accumulation ◽  
Novel Therapeutic ◽  
Soybean Allergen

AbstractDissecting the inhibited variation of allergen absorption could contribute to the development of novel therapeutic or preventive treatments for food/feed allergies. This study investigated the effects of fructooligosaccharide (FOS) on the absorption, intracellular accumulation of intact or hydrolysed β-conglycinin in porcine intestinal epithelial cells (IPEC-J2). As demonstrated by ELISA and immunoblotting, β-conglycinin was absorbed in a dose- and time-dependent manner (p < 0.05). Actually, β-conglycinin was easily transported and absorbed after enzymatic hydrolysis. Three peptides (52 kDa, 30 kDa and 25 kDa) were produced during transcellular absorption of intact or hydrolysed β-conglycinin. FOS inhibited the absorption of β-conglycinin, especially the 52 and 30 kDa peptides. The immunoreactive peptides derived from the 52, 35 or 22 kDa peptides were the substrings of the known epitopes determined by mass spectrometry and bioinformatic analyses. These results indicate that FOS can efficiently inhibit the absorption of 52 and 30 kDa peptides derived from β-conglycinin.

Effects of Stearic Acid on Proliferation, Differentiation, Apoptosis, and Autophagy in Porcine Intestinal Epithelial Cells

2020 ◽  
Vol 20 (2) ◽  
pp. 157-166
Author(s):  
Yuan Yang ◽  
Jin Huang ◽  
Jianzhong Li ◽  
Huansheng Yang ◽  
Yulong Yin
Keyword(s):  
Cell Differentiation ◽  
Epithelial Cells ◽  
Stearic Acid ◽  
Er Stress ◽  
Intestinal Epithelial Cells ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
High Concentrations ◽  
Induced Apoptosis ◽  
Dose Dependent

Background: Stearic acid (SA), a saturated long-chain fatty acid consisting of 18 carbon atoms, is widely found in feed ingredients, such as corn, soybeans, and wheat. However, the roles of SA in the renewal of intestinal epithelial cells remain unclear. Methods and Results: In the present study, we found that 0.01-0.1 mM SA promoted IPEC-J2 cell differentiation and did not affect IPEC-J2 cell viability. In addition, the results showed that the viability of IPEC-J2 cells was inhibited by SA in a time- and dose-dependent manner at high concentrations. Flow cytometry and western blot analysis suggested that SA induced apoptosis, autophagy and ER stress in cells. In addition, the amounts of triglyceride were significantly increased upon challenge with SA. Moreover, the decrease in the viability of cells induced by SA could be attenuated by 4-PBA, an inhibitor of ER stress. Conclusion: In summary, SA accelerated IPEC-J2 cell differentiation at 0.01-0.1 mM. Furthermore, SA induced IPEC-J2 cell apoptosis and autophagy by causing ER stress.

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