Assembly Of Epithelial Tight Junctions Research Articles

A prebiotic fructo-oligosaccharide promotes tight junction assembly in intestinal epithelial cells via an AMPK-dependent pathway

Tight junctions play an important role in maintaining barrier integrity of intestinal epithelia. Activation of AMP-activated protein kinase (AMPK) promotes tight junction assembly in intestinal epithelial cells (IEC). Fructo-oligosaccharides (FOS), well-known prebiotics, have previously been shown to alleviate inflammation-associated intestinal epithelial disruption although the mechanisms were unclear. This study aimed to investigate any effect of FOS on AMPK activity and tight junction assembly under non-inflammatory and inflammatory conditions using T84 cells as an IEC model. As analyzed by western blot, FOS induced AMPK activation through a calcium sensing receptor (CaSR)-phospholipase C (PLC)- Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) pathway. Calcium switch assays and immunofluorescence staining of zonula occludens-1 (ZO-1) revealed that FOS induced tight junction assembly via an CaMKKβ-AMPK-dependent mechanism in IEC. Interestingly, FOS reversed the suppressive effect of lipopolysaccharide (LPS) on AMPK activity and tight junction assembly via a CaMKKβ pathway. Taken together, these findings uncover a prebiotic-independent effect of FOS in promoting intestinal epithelial tight junction assembly through AMPK activation, which may have implications for the treatment of diseases whose pathogenesis involves impaired intestinal barrier function.

GPR40 receptor activation promotes tight junction assembly in airway epithelial cells via AMPK-dependent mechanisms

ABSTRACTTight junctions play key roles in the regulation of airway epithelial barrier function and promotion of tight junction integrity is beneficial to lung health. G-protein coupled receptor (GPR) 40 has been identified as a receptor of polyunsaturated fatty acids. This study aimed to investigate the function of GPR40 in regulating tight junction assembly in human airway epithelial cells (Calu-3 cells) using GW9508, a GPR40 agonist. Immunoblotting and immunofluorescence analyses showed that Calu-3 cells expressed both types of polyunsaturated fatty acid receptors including GPR40 and GPR120. Intracellular Ca2+ measurements confirmed that GW9508 stimulated GPR40, but not GPR120. In Ca2+ switch assays, GW9508 promoted the recovery of transepithelial electrical resistance and re-localization of zonula occludens (ZO)-1 to intercellular areas. These effects were suppressed by inhibitors of GPR40 and phospholipase C (PLC). Interestingly, GW9508 enhanced tight junction assembly in an AMP-activated protein kinase (AMPK)-dependent manner. The effect of GW9508 on inducing tight junction assembly was also confirmed in 16HBE14o- cells. Our results indicate that GPR40 stimulation by GW9508 leads to AMPK activation via calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Collectively, this study reveals an unprecedented role of GPR40 in facilitating airway epithelial tight junction assembly via PLC-CaMKKβ-AMPK pathways. GPR40 represents a novel regulator of airway epithelial integrity and its stimulation may be beneficial in the treatment of airway diseases.

Mesenchymal Stromal Cells Accelerate Epithelial Tight Junction Assembly via the AMP-Activated Protein Kinase Pathway, Independently of Liver Kinase B1.

Background Mesenchymal stromal cells (MSC) are fibroblast-like multipotent cells capable of tissue-repair properties. Given the essentiality of tight junctions (TJ) in epithelial integrity, we hypothesized that MSC modulate TJ formation, via the AMP-activated kinase (AMPK) pathway. Liver kinase-β1 (LKB1) and Ca2+-calmodulin-dependent protein kinase kinase (CaMKK) represent the main kinases that activate AMPK. Methods The in vitro Ca2+ switch from 5 μM to 1.8 mM was performed using epithelial Madin-Darby canine kidney (MDCK) cells cultured alone or cocultured with rat bone marrow-derived MSC or preexposed to MSC-conditioned medium. TJ assembly was measured by assessing ZO-1 relocation to cell-cell contacts. Experiments were conducted using MDCK stably expressing short-hairpin-RNA (shRNA) against LKB1 or luciferase (LUC, as controls). Compound STO-609 (50 μM) was used as CaMKK inhibitor. Results Following Ca2+ switch, ZO-1 relocation and phosphorylation/activation of AMPK were significantly higher in MDCK/MSC compared to MDCK. No difference in AMPK phosphorylation was observed between LKB1-shRNA and Luc-shRNA MDCK following Ca2+ switch. Conversely, incubation with STO-609 prior to Ca2+ switch prevented AMPK phosphorylation and ZO-1 relocation. MSC-conditioned medium slightly but significantly increased AMPK activation and accelerated TJ-associated distribution of ZO-1 post Ca2+ switch in comparison to regular medium. Conclusions MSC modulate the assembly of epithelial TJ, via the CaMKK/AMPK pathway independently of LKB1.

MICAL-like1 mediates epidermal growth factor receptor endocytosis.

Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein-Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.

Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin-Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.

Lymphocytes Accelerate Epithelial Tight Junction Assembly: Role of AMP-Activated Protein Kinase (AMPK)

The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-α. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK.

Signal Transducer and Activator of Transcription 5 in Enterocytes Promotes Intestinal Mucosal Wound Healing by Regulation of Epithelial Tight Junction Assembly

MethodsWe generated intestinal epithelial‐specific STAT5 knock out mice (STAT5 IEC KO) using cre‐mediated recombination to delete stat5 locus in IEC. STAT5 IEC KO mice were then treated with 3 % dextran sodium sulfate (DSS) either for seven days or for five days followed by 5 days of water. STAT5 IEC KO mice also exposed to NSAIDs chow for two weeks. We significantly knocked down GM‐CSF signaling and STAT5 expression in Caco‐2 and HT‐29 monolayer.ResultsSTAT5 IEC KO mice revealed an increased para‐cellular permeability and bacterial translocation in mesenteric lymph nude. STAT5 IEC KO mice were more susceptible to NSAIDs‐induced ileitis and DSS‐induced colitis, precluding mucosal healing after stopping DSS administration compared to littermate control mice (STAT5 IEC WT). Intestinal epithelial cells (IEC) monolayer exhibited a hyperpermeability under either GM‐CSF signaling or STAT5 knocking down; phosphoserine‐MLC was elevated and tight junction (TJ) assembly was disrupted in either IEC monolayer with STAT5 knock down or STAT5‐depleted IEC.ConclusionSTAT5 signaling in enterocytes is required for maintaining intestinal mucosal barrier function and promotes intestinal mucosal wound healing. Targeting on the activity of STAT5 signaling in enterocytes may be a novel therapeutic approach for treating intestinal barrier dysfunction in the sepsis and inflammatory bowel disease. This work was supported by Crohns and Colitis Foundation of America (XH), CCHMC Trustee Award (XH) and NIH clinical and translational research award KL2 (RR026315, XH).

Butyrate Enhances the Intestinal Barrier by Facilitating Tight Junction Assembly via Activation of AMP-Activated Protein Kinase in Caco-2 Cell Monolayers

Butyrate, one of the SCFA, promotes the development of the intestinal barrier. However, the molecular mechanisms underlying the butyrate regulation of the intestinal barrier are unknown. To test the hypothesis that the effect of butyrate on the intestinal barrier is mediated by the regulation of the assembly of tight junctions involving the activation of the AMP-activated protein kinase (AMPK), we determined the effect of butyrate on the intestinal barrier by measuring the transepithelial electrical resistance (TER) and inulin permeability in a Caco-2 cell monolayer model. We further used a calcium switch assay to study the assembly of epithelial tight junctions and determined the effect of butyrate on the assembly of epithelial tight junctions and AMPK activity. We demonstrated that the butyrate treatment increased AMPK activity and accelerated the assembly of tight junctions as shown by the reorganization of tight junction proteins, as well as the development of TER. AMPK activity was also upregulated by butyrate during calcium switch-induced tight junction assembly. Compound C, a specific AMPK inhibitor, inhibited the butyrate-induced activation of AMPK. The facilitating effect of butyrate on the increases in TER in standard culture media, as well as after calcium switch, was abolished by compound C. We conclude that butyrate enhances the intestinal barrier by regulating the assembly of tight junctions. This dynamic process is mediated by the activation of AMPK. These results suggest an intriguing link between SCFA and the intracellular energy sensor for the development of the intestinal barrier.

478 ZO-1 C-Terminal Domains Are Essential for Tight Junction Assembly and Maintenance

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.

479 Myosin Light Chain Kinase Inhibition Decreases the Incidence of Severe Bowel Injury in a Neonatal Mouse Model of Necrotizing Enterocolitis

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.

482 The Human Milk Factor Erythropoietin Protects Against Necrotizing Enterocolitis (NEC) in Preterm Rats By Maintaining Barrier Function Through Upregulation of Tight Junction Protein Expression

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.

481 The RNA-Binding Protein HuR Regulates Occludin Translation Through Chk2-Dependent HuR Phosphorylation Modulating Intestinal Epithelial Barrier Function

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.

C‐terminal domains are required for ZO‐1 stabilization and tight junction assembly

Zonula occludens (ZO) proteins direct assembly of epithelial tight junctions (TJs). While critical functions have been defined for N‐terminal PDZ domains, a central SH3‐GUK module, and the C‐terminal actin‐binding region (ABR), the remaining ZO‐1 C‐terminus has not been characterized. The aim of this study was to determine if the ZO‐1 C‐terminus participates in TJ assembly. Epithelial cells were stably transfected with full‐length ZO‐1 tagged with GFP at the N‐terminus (GFP‐ZO‐1) as well as a GFP‐tagged deletion mutant lacking the ABR (ZO‐1δABR) and a GFP‐tagged truncation mutant lacking the ABR and remaining C‐terminus (ZO‐11‐1152). ZO‐11‐1152 markedly delayed epithelial barrier development as well as peak transepithelial resistance (TER) achieved. In contrast, ZO‐1δABR did not delay assembly and only slightly diminished peak TER. Morphologic analysis showed that GFP‐ZO‐11‐1152, but not GFP‐ZO‐1 or GFP‐ZO‐1δABR, localized to the TJ inefficiently and interfered with junctional recruitment of endogenous ZO‐1 and β‐catenin. We have previously used fluorescence recovery after photobleaching to show that GFP‐ZO‐1 stability at the TJ increases as monolayers mature. GFP‐ZO‐11‐1152 stabilization did not occur with maturation, but GFP‐ZO‐1δABR stabilized normally. These data suggest that undefined domains within the ZO‐1 C‐terminus are essential for TJ assembly and maturation.

Unique Role of Junctional Adhesion Molecule-A in Maintaining Mucosal Homeostasis in Inflammatory Bowel Disease

Junctional adhesion molecule-A (JAM-A) is localized at the tight junctions and controls leukocyte migration into the tissues. However, its functional role in inflammatory bowel disease (IBD) is unexplored. Control, Crohn's disease (CD), and ulcerative colitis (UC) tissue specimens were studied for JAM-A expression, as well as the colon of mice given dextran sodium sulfate (DSS). Wild-type and JAM-A(-/-), Tie-2-Cre-JAM-A(-/-) (endothelial/hematopoietic-specific JAM inactivation) mice were studied for susceptibility to DSS. Disease activity and colonic inflammation were assessed using a disease activity index histology and endoscopy, and mucosal cytokines were measured by enzyme-linked immunosorbent assay. JAM-A function was investigated by RNA silencing in epithelial cells, and apoptosis was measured. In both CD and UC, as well as in experimental colitis, there is a loss of epithelial but not endothelial JAM-A expression. Deletion of JAM-A results in a dramatic increase in susceptibility to DSS colitis, as assessed by weight loss, disease activity index, histologic and endoscopic severity, and strikingly high mortality rates. This is not caused by the absence of JAM-A in the endothelial or hematopoietic compartments because Tie-2-Cre-JAM-A(-/-) mice are no more susceptible to DSS colitis than wild-type animals. JAM-A(-/-) mice displayed increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis. Silencing of JAM-A in intestinal epithelial cells resulted in increased permeability in vitro. Our results show a nonredundant and novel role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function.

Mammalian Scribble regulates the assembly of epithelial tight junctions

Scribble, Disc‐large (DLG), and Lethal giant larvae (LGL) tumor suppressor proteins have been implicated in the regulation of epithelial cell polarity. However, the mechanisms by which these proteins contribute to epithelial polarity are not well understood. Since tight junctions (TJs) play an important role in regulating intestinal epithelial cell (IEC) polarity and barrier function, we examined if these proteins participate in TJ dynamics and in the establishment of the IEC barrier function. Model IECs, SK‐CO15 and T84 cells were used for these studies, and TJ assembly/disassembly was analyzed using the calcium switch assay. Fluorescence labeling/confocal microscopy demonstrated that Scribble and LGL1 but not DLG1 co‐localized with TJ proteins, occludin and zonula occludens‐1. Additionally, Scribble and LGL1 co‐internalized with the TJ proteins during TJ disassembly induced by calcium depletion. Furthermore, siRNA‐mediated downregulation of Scribble, but not LGL1 or DLG1 significantly attenuated the functional and morphologic assembly of TJs during calcium repletion. Downregulation of Scribble did not influence expression of DLG1 or LGL1 and vise versa, and these proteins did not co‐associate as determined by immunoprecipitation. In conclusion, we demonstrate a unique role of mammalian Scribble in regulation of IEC TJ assembly and in the development of epithelial barrier function.Supported by CCFA Career Development Award (AII), NIH DK‐59888 (AN), and NIH DK‐55567 (AN)

The PAR-6 Polarity Protein Regulates Dendritic Spine Morphogenesis through p190 RhoGAP and the Rho GTPase

The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. Here we show that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, we further show that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity.

Regulation of epithelial tight junction assembly and disassembly by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that plays an important role in maintaining cellular energy balance. The activity of AMPK is modulated both by the cellular AMP-to-ATP ratio and by upstream kinases. Recently, AMPK was shown to be phosphorylated and activated by LKB1, a protein kinase that plays a conserved role in epithelial polarity regulation in mammals and Drosophila. Here, we investigate the involvement of AMPK in the regulation of epithelial tight junction assembly and cell polarization in MDCK cells. We show that the level of AMPK phosphorylation increases during calcium-induced tight junction assembly and cell polarization and that this increase depends on the kinase activity of LKB1. Expression of a kinase-dead mutant of AMPK inhibits tight junction assembly as indicated by measurement of transepithelial resistance and analysis of ZO-1 localization to the tight junction after calcium switch. Conversely, 5-aminoimidizole-4-carboxamide riboside, an activator of AMPK, promotes transepithelial resistance development and tight junction assembly upon calcium switch. Furthermore, 5-aminoimidizole-4-carboxamide riboside partially protects the tight junctions from disassembly induced by calcium depletion. These results support an important role of AMPK in the regulation of epithelial tight junction assembly and disassembly and suggest an intriguing link between cellular energy status and tight junction function.

AMP-activated protein kinase regulates the assembly of epithelial tight junctions

AMP activated protein kinase (AMPK), a sensor of cellular energy status in all eukaryotic cells, is activated by LKB1-dependent phosphorylation. Recent studies indicate that activated LKB1 induces polarity in epithelial cells and that this polarization is accompanied by the formation of tight junction structures. We wished to determine whether AMPK also contributes to the assembly of tight junctions in the epithelial cell polarization process. We found that AMPK is activated during calcium-induced tight junction assembly. Activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside facilitates tight junction assembly under conditions of normal extracellular Ca2+ concentrations and initiates tight junction assembly in the absence of Ca2+ as revealed by the relocation of zonula occludens 1, the establishment of transepithelial electrical resistance, and the paracellular flux assay. Expression of a dominant negative AMPK construct inhibits tight junction assembly in MDCK cells, and this defect in tight junction assembly can be partially ameliorated by rapamycin. These results suggest that AMPK plays a role in the regulation of tight junction assembly.

Tyrosine phosphorylated Par3 regulates epithelial tight junction assembly promoted by EGFR signaling

The conserved polarity complex, comprising the partitioning-defective (Par) proteins Par3 and Par6, and the atypical protein kinase C, functions in various cell-polarization events and asymmetric cell divisions. However, little is known about whether and how external stimuli-induced signals may regulate Par3 function in epithelial cell polarity. Here, we found that Par3 was tyrosine phosphorylated through phosphoproteomic profiling of pervanadate-induced phosphotyrosine proteins. We also demonstrated that the tyrosine phosphorylation event induced by multiple growth factors including epidermal growth factor (EGF) was dependent on activation of Src family kinase (SFK) members c-Src and c-Yes. The tyrosine residue 1127 (Y1127) of Par3 was identified as the major EGF-induced phosphorylation site. Moreover, we found that Y1127 phosphorylation reduced the association of Par3 with LIM kinase 2 (LIMK2), thus enabling LIMK2 to regulate cofilin phosphorylation dynamics. Substitution of Y1127 for phenylalanine impaired the EGF-induced Par3 and LIMK2 dissociation and delayed epithelial tight junction (TJ) assembly considerably. Collectively, these data suggest a novel, phosphotyrosine-dependent fine-tuning mechanism of Par3 in epithelial TJ assembly controlled by the EGF receptor-SFK signaling pathway.

Direct interaction of two polarity complexes implicated in epithelial tight junction assembly.

Tight junctions help establish polarity in mammalian epithelia by forming a physical barrier that separates apical and basolateral membranes. Two evolutionarily conserved multi-protein complexes, Crumbs (Crb)-PALS1 (Stardust)-PATJ (DiscsLost) and Cdc42-Par6-Par3-atypical protein kinase C (aPKC), have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. Here we identify a biochemical and functional link between these two complexes that is mediated by Par6 and PALS1 (proteins associated with Lin7). The interaction between Par6 and PALS1 is direct, requires the amino terminus of PALS1 and the PDZ domain of Par6, and is regulated by Cdc42-GTP. The transmembrane protein Crb can recruit wild-type Par6, but not Par6 with a mutated PDZ domain, to the cell surface. Expression of dominant-negative PALS1-associated tight junction protein (PATJ) in MDCK cells results in mis-localization of PALS1, members of the Par3-Par6-aPKC complex and the tight junction marker, ZO-1. Similarly, overexpression of Par6 in MDCK cells inhibits localization of PALS1 to the tight junction. Our data highlight a previously unrecognized link between protein complexes that are essential for epithelial polarity and formation of tight junctions.