Tight Junctions and the Experimental Modifications of Lipid Content

Tight junctions (TJs) are cell-to-cell contacts made of strands, which appear as ridges on P faces and complementary furrows on E faces on freeze fracture replicas. Evidences and opinions on whether these strands are composed of either membrane-bound proteins or lipid micelles are somewhat varied. In the present work we alter the lipid composition of Madin-Darby canine kidney monolayers using a novel approach, while studying (i) their transepithelial electrical resistance, a parameter that depends on the degree of sealing of the TJs; (ii) the apical-to-basolateral flux of 4 kD fluorescent dextran (J_DEX), that reflects the permeability of the intercellular spaces; (iii) the ability of TJs to restrict apical-to-basolateral diffusion of membrane lipids; and (iv) the pattern of distribution of endogenous and transfected occludin, the sole membrane protein presently known to form part of the TJs. We show that changing the total composition of phospholipids, sphingolipids, cholesterol and the content of fatty acids, does not alter TER nor the structure of the strands. Interestingly, enrichment with linoleic acid increases the J_DEX by 631%. The fact that this increase is not reflected in a decrease of TER, suggests that junctional strands do not act as simple resistive elements but may contain mobile translocating mechanisms. Received: 7 November 1997/Revised: 20 March 1998     

Maintenance of the epithelial barrier in a bronchial epithelial cell line is dependent on functional E-cadherin local to the tight junctions

Tight junctions (TJ) are essential components of polarized epithelia, and E-cadherin is important for their formation and maintenance. The bronchial epithelial cell line, 16HBE14o- expresses E- and P-cadherin, but not N-cadherin. E- and P-cadherin levels changed during culture, the former increasing after confluence, and the latter were markedly reduced. All detectable E-cadherin was bound to β- and γ-catenins. We investigated involvement of E-cadherin with epithelial integrity using an E-cadherin specific, function-blocking antibody, SHE78-7. Surprisingly, apical SHE78-7 exposure caused a prompt fall in transepithelial resistance (TER), while TER remained unchanged for 8 hrs after basal exposure then dropped. SHE78-7 exposure increased epithelial permeability to mannitol, inulin, and 9.5 kDa and 77 kDa dextrans and caused fragmentation and loss of the tight junction protein, ZO-1, from the cell borders in some areas. Ultrastructural studies showed that all junctional intercellular contact was lost in the center of SHE78-7 induced lesions. Near the lesion periphery, epithelial structure was maintained, but TJs were dysfunctional as shown by ruthenium red penetration. Analysis of epithelial penetration by SHE78-7 revealed discrete, local defects in the apical barrier at the top of some cell hills that permitted rapid access of the antibody to E-cadherin near the apical surface. In contrast, after basal exposure, antibody initially engaged with E-cadherin nearer the basal surface and only accessed apical E-cadherin later. Taken together with the TER measurements, these data suggest compartmentalization of E-cadherin function within 16HBE14o- cells, with only the apical E-cadherin adjacent to the tight junctions contributing to the function of the latter.     

Annexin 2 regulates endothelial morphogenesis by controlling AKT activation and junctional integrity

Sprouting angiogenesis is a multistep process that involves endothelial cell activation, basement membrane degradation, proliferation, lumen formation, and stabilization. In this study, we identified annexin 2 as a regulator of endothelial morphogenesis using a three-dimensional in vitro model where sprouting angiogenesis was driven by sphingosine 1-phosphate and angiogenic growth factors. We observed that sphingosine 1-phosphate triggered annexin 2 translocation from the cytosol to the plasma membrane and its association with vascular endothelial (VE)-cadherin. In addition, annexin 2 depletion attenuated Akt activation, which was associated with increased phosphorylation of VE-cadherin and endothelial barrier leakage. Disrupting homotypic VE-cadherin interactions with EGTA, antibodies to the extracellular domain of VE-cadherin, or gene silencing all resulted in decreased Akt (but not Erk1/2) activation. Furthermore, expression of constitutively active Akt restored reduced endothelial sprouting responses observed with annexin 2 and VE-cadherin knockdown. Collectively, we report that annexin 2 regulates endothelial morphogenesis through an adherens junction-mediated pathway upstream of Akt.     

Protein Kinase D Promotes Airway Epithelial Barrier Dysfunction and Permeability through Down-regulation of Claudin-1

At the interface between host and external environment, the airway epithelium serves as a major protective barrier. In the present study we show that protein kinase D (PKD) plays an important role in the formation and integrity of the airway epithelial barrier. Either inhibition of PKD activity or silencing of PKD increased transepithelial electrical resistance (TEER), resulting in a tighter epithelial barrier. Among the three PKD isoforms, PKD3 knockdown was the most efficient one to increase TEER in polarized airway epithelial monolayers. In contrast, overexpression of PKD3 wild type, but not PKD3 kinase-inactive mutant, disrupted the formation of apical intercellular junctions and their reassembly, impaired the development of TEER, and increased paracellular permeability to sodium fluorescein in airway epithelial monolayers. We further found that overexpression of PKD, in particular PKD3, markedly suppressed the mRNA and protein levels of claudin-1 but had only minor effects on the expression of other tight junctional proteins (claudin-3, claudin-4, claudin-5, occludin, and ZO-1) and adherent junctional proteins (E-cadherin and β-catenin). Immunofluorescence study revealed that claudin-1 level was markedly reduced and almost disappeared from intercellular contacts in PKD3-overexpressed epithelial monolayers and that claudin-4 was also restricted from intercellular contacts and tended to accumulate in the cell cytosolic compartments. Last, we found that claudin-1 knockdown prevented TEER elevation by PKD inhibition or silencing in airway epithelial monolayers. These novel findings indicate that PKD negatively regulates human airway epithelial barrier formation and integrity through down-regulation of claudin-1, which is a key component of tight junctions.     

Interactions of tight junctions with membrane channels and transporters

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.     

Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail

In vertebrates, the positioning of the internal organs relative to the midline is asymmetric and evolutionarily conserved. A number of molecules have been shown to play critical roles in left-right patterning. Using representational difference analysis to identify genes that are differentially expressed on the left and right sides of the chick embryo, we cloned chick Claudin-1, an integral component of epithelial tight junctions. Here, we demonstrate that retroviral overexpression of Claudin-1, but not Claudin-3, on the right side of the chick embryo between HH stages 4 and 7 randomizes the direction of heart looping. This effect was not observed when Claudin-1 was overexpressed on the left side of the embryo. A small, but reproducible, induction of Nodal expression in the perinodal region on the right side of the embryo was noted in embryos that were injected with Claudin-1 retroviral particles on their right sides. However, no changes in Lefty,Pitx2 or cSnR expression were observed. In addition, Flectin expression remained higher in the left dorsal mesocardial folds of embryos with leftwardly looped hearts resulting from Claudin-1 overexpression on the right side of the embryo. We demonstrated that Claudin-1's C-terminal cytoplasmic tail is essential for this effect: mutation of a PKC phosphorylation site in the Claudin-1 C-terminal cytoplasmic domain at threonine-206 eliminates Claudin-1's ability to randomize the direction of heart looping. Taken together, our data provide evidence that appropriate expression of the tight junction protein Claudin-1 is required for normal heart looping and suggest that phosphorylation of its cytoplasmic tail is responsible for mediating this function.     

肠道微生物与1型糖尿病的相关性研究

1型糖尿病(type 1 diabetes, T1D)是一种自身免疫性疾病,越来越多的证据支持肠道菌群是T1D发病的重要因素。在T1D发生前,观察到肠道通透性发生改变,使抗原透过肠黏膜进而攻击胰岛β细胞。患有T1D宿主体内的肠道微生物也与健康宿主的微生物有别,其调节性T细胞、Toll样受体(toll-like receptor, TLR)、丁酸、粘蛋白、胰高血糖素样肽-1(glucagon-likepeptide1,GLP-1)等可能与T1D有关。尽管通过细菌定植促进自身免疫的机制在糖尿病动物模型中虽已发现,但有些问题目前尚不清楚。现就肠道黏膜通透性与T1D的关系、宿主体内微生物组成的变化、肠道微生物与T1D的相关性作一阐述。