Cholesterol depletion alters detergent-specific solubility profiles of selected tight junction proteins and the phosphorylation of occludin

Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH)-depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent-resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [(3)H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH-dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH.     

The tight junctional protein occludin is found in the uterine epithelium of squamate reptiles

Occludin, an integral protein associated with the mammalian tight junction, has for the first time been identified in the uterus of squamate reptiles. The tight junction is made up of anastamosing strands and forms a selective barrier that regulates paracellular diffusion of solutes across uterine epithelium. Occludin exclusively labels tight junctional strands and is an excellent marker for tight junction permeability. Using western blotting and immunohistochemistry, occludin expression was examined in the uterine epithelium of five species of Australian skinks at different stages of gestation. More occludin was detected during late stage pregnancy/gravidity compared to the lower levels of occludin detected in vitellogenic and post-parturient females in three of the five species. We conclude that the paracellular permeability of the squamate uterine epithelium decreases as gestation progresses. As placental transport of ions and solutes to the embryo is highest during the last third of pregnancy in viviparous squamates, it is likely that a decrease in paracellular permeability is compensated by an upregulation of other transporting mechanisms such as histotrophy.     

Occludin 蛋白与细胞间紧密连接关系及其临床意义

细胞间紧密连接(tight junctions)广泛存在于上皮细胞及内皮细胞之间,其作用是保持细胞间结构的完整性,确保其功能的正常发挥,紧密连接上有很多种蛋白,occludin蛋白是其中主要蛋白之一,occludin蛋白的结构发生变化会导致紧密连接结构及功能的改变,而紧密连接结构与功能的紊乱是很多临床疾病共同的病理生理学特点,如肿瘤、中风及炎症性肺疾病。Occludin蛋白的结构及功能的改变受很多机制的调控,本文主要对occludin蛋白的结构、功能、调控机制及其与紧密连接之间的关系进行叙述。     

Casein kinase I epsilon associates with and phosphorylates the tight junction protein occludin

Occludin is an integral-membrane protein that contributes to tight junction function. We have identified casein kinase I epsilon (CKI epsilon) as a binding partner for the C-terminal cytoplasmic domain of occludin by yeast two-hybrid screening. CKI epsilon phosphorylated occludin and co-localised and co-immunoprecipitated with occludin from human endothelial cells. Amino acids 265-318 of occludin were sufficient for CKI epsilon binding and phosphorylation. Deletion of the C-terminal 48 amino acids of occludin increased CKI epsilon binding and phosphorylation, suggesting that this region inhibits CKI epsilon binding. These data identify CKI epsilon as a novel occludin kinase that may be important for the regulation of occludin.     

Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells

The blood-brain barrier (BBB) serves as a critical organ in the maintenance of central nervous system homeostasis and is disrupted in a number of neurological disorders, including stroke. We examined the effects of hypoxia/aglycemia on the expression and localization of tight junction proteins, and on the function of the BBB in an in vitro model system. A receptor-operated/store-operated calcium channel blocker, SKF 96365, was used to determine if calcium flux was important in mediating hypoxia/aglycemia effects on the BBB. Expression of the tight junction protein occludin increased after hypoxic/aglycemic stress when cells were exposed to SKF 96365; this was correlated with partial protection of membrane localization of occludin and inhibition of the hypoxia-induced increase in permeability. Actin expression was dramatically reduced by hypoxia/aglycemia. Treatment with SKF 96365 during hypoxic stress protected monolayer permeability of sucrose, but transendothelial electrical resistances decreased with exposure to hypoxic stress regardless of treatment. Therefore, the presence of occludin at the membrane is dependent in part on calcium-sensitive signaling cascades; this provides a target for therapeutic intervention to minimize BBB disruption after stroke.     

高温对原代支持细胞增殖能力和occludin紧密连接蛋白的影响

目的:研究高温对原代大鼠睾丸支持细胞增殖作用和对紧密连接蛋白的损伤机制.方法:体外分离培养雄性Wistar大鼠睾丸支持细胞(Sertoli cell,SC),免疫组化FasL鉴定.实验分为对照组(36℃)和实验组(37℃、38℃、39℃).在相应的温度下培养5d后,CCK-8法检测SC增殖作用,Western印迹法检测OCLN表达水平.结果:体外培养SC的纯度＞90％,CCK-8结果显示,细胞增殖活性在36℃时最强,37～39℃时逐渐降低;Western印迹显示,36℃时OCLN表达量最高;高于36℃时,OCLN表达量随着温度的增加而降低(P＜0.05).结论:高温影响SC增殖活性,破坏了支持细胞紧密连接蛋白的正常表达和血睾屏障的完整性,从而影响正常精子的形成.     

布拉酵母菌对胃黏膜上皮细胞损伤的保护作用及机制

目的 研究布拉酵母菌对胃黏膜上皮细胞损伤的保护作用。方法 建立人胃黏膜上皮细胞GES-1单层细胞模型,并将其分为对照组,布拉酵母菌上清培养组,阿司匹林处理组（18.32 mmol/L）,阿司匹林+布拉酵母菌上清液组。采用流式细胞术检测各组细胞凋亡率;采用免疫荧光、Western blot方法检测胃黏膜上皮细胞中Occludin和ZO-1蛋白、JNK蛋白、ERK蛋白、磷酸化JNK（p-JNK）蛋白、磷酸化ERK（p-ERK）蛋白表达水平。结果 流式细胞术显示阿司匹林+布拉酵母菌上清液组凋亡率低于阿司匹林处理组,差异有统计学意义（P<0.05）;免疫荧光结果显示,阿司匹林+布拉酵母菌上清液组Occludin蛋白、ZO-1蛋白表达较阿司匹林处理组增加,Western blot结果显示,阿司匹林+布拉酵母菌上清液组的该两种蛋白表达量较阿司匹林处理组增加,阿司匹林+布拉酵母菌上清液组p-ERK、p-JNK蛋白表达较阿司匹林处理组减少。结论 布拉酵母菌对阿司匹林造成的胃黏膜上皮细胞损伤有保护作用,其机制可能通过抑制MAPK信号通路中ERK、JNK的激活而上调Occludin和ZO-1蛋白的表达。     

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.     

High glucose stimulates macrophage SR-BI expression and induces a switch in its activity from cholesterol efflux to cholesterol influx

Aims

Diabetes is associated with atherogenesis and macrophage-foam cell formation, due in part to a decrease in HDL-mediated cholesterol efflux from macrophages. This study examined the expression of proteins involved in cholesterol transport, i.e. ABCA1 and SR-BI, under diabetic conditions.

Methods and results

ABCA1 expression was similar, whereas SR-BI expression (mRNA and protein) was significantly increased in mouse peritoneal macrophages (MPM) harvested from C57Bl/6 diabetic mice, compared to MPM from control non-diabetic mice. Similar results were obtained in vitro in J-774A.1 macrophage-like cell line incubated with high (30 mM) vs. low (5 mM) glucose concentrations. Accordingly, association and internalization of HDL to MPM from diabetic mice, or to J-774A.1 macrophages grown under diabetic conditions was significantly higher compared to control cells. Unexpectedly, however, increased macrophage SR-BI expression was associated with a substantial reduction in HDL-mediated cholesterol efflux from the macrophages. Moreover, total cellular cholesterol content was increased by 28% in macrophages incubated with HDL under high glucose concentrations, compared to low glucose concentrations. This effect was abolished by a rabbit polyclonal anti-SR-BI, which blocks binding to the receptor, or alternatively by using BLT1, a specific inhibitor of lipid transport via the SR-BI.

Conclusions

Diabetes stimulates the expression of SR-BI in macrophages and leads to a shift in its activity from HDL-mediated cholesterol efflux to HDL-mediated cholesterol influx. These effects may lead to increased foam cell formation and atherosclerosis development.     

Effect of estradiol and dihydrotestosterone on hypergravity-induced MAPK signaling and occludin expression in human umbilical vein endothelial cells

Female astronauts have been reported to have a higher incidence of post-flight orthostatic intolerance (POI) compared with that of their male counterparts. POI may result from increased permeability of the endothelial cell (EC) layer in the vasculature. The goal of this study has been to determine whether estradiol (E₂) and dihydrotesterone (DHT) alter human umbilical vein ECs (HUVECs) responses to short term (10 min) hypergravity (1–3 g) mimicking the g force experienced by astronauts during liftoff. E₂ and DHT rapidly (within 5 min) activated MAPK (mitogen-activated protein kinase) in HUVEC at 1 g in a receptor-dependent manner. Liftoff inhibited MAPK phosphorylation, and rapid E₂ and DHT activation of MAPK was blocked. Liftoff simulation or brief (5–90 min) treatment with E₂ or DHT at 1 g had no effect on the expression of the EC tight-junction protein occludin. However, 24-h pre-treatment of HUVECs with E₂ and DHT prior to liftoff simulation significantly increased occludin expression, and hypergravity exposure did not alter this increase. These data provide evidence for a possible protective effect of E₂ and DHT on EC function as indicated by increased occludin; this may help maintain the integrity of EC tight junction and could thus retard or reduce the incidence of POI.This work was supported by NASA grant NAG5-12874 to C.M.K. and R.S.K. and by American Heart Association Ohio Valley Affiliate grant 0555270B to C.M.K. The American Heart Association Ohio Valley supported W.K.S. with an affiliate post-doctoral fellowship (0425431B).     

Changes in the distribution of ZO-1, occludin,and claudins in the rat uterine epithelium during the estrous cycle

During the estrous cycle, the endometrium epithelium experiences marked cellular structural changes. For fertilization to proceed, maintenance of an adequate uterine environment by ovarian hormones is essential. Epithelial cells lining the uterine lumen are associated with each other by tight junctions (TJs), which regulate the passage of ions and molecules through the paracellular pathway. The aim of the present study was to assess by confocal immunofluorescence the distribution pattern of the TJ proteins ZO-1, occludin, and claudins 1–7 in the rat uterus during the estrous cycle. Our results reveal that on proestrus, the day when mating takes place, ZO-1, occludin, and claudins 1 and 5 are located in the TJs, while claudins 3 and 7 display a basolateral distribution. In contrast, on metestrus day, when no sexual mating occurs and the uterine lumen is devoid of secretions, none of these proteins were detected in the TJ region, and only a diffuse cytosolic staining was observed for some of the proteins. On estrus and diestrus days, an intermediate situation was encountered, since ZO-1 localized in the TJs, whereas occludin was no longer detectable in the TJs. The distribution of claudins during these stages varied from the lowermost portion of the basolateral membrane to its apex. In conclusion, the results show that the protein composition of TJs present in the luminal epithelial cells of the uterus changes during the different days of the estrous cycle, and suggest that the expression of TJ proteins participates in providing an adequate environment for a successful fertilization.This work was supported by grants PAPIIT (IN210902, IX228504) and PAIP (6190-08) from the National Autonomous University of Mexico (UNAM), and by grants G34511-M and 37846-N from the Mexican National Council on Science and Technology (CONACYT).     

Tight junctions and the modulation of barrier function in disease

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease.     

The C-terminal domain of apolipoprotein A-I is involved in ABCA1-driven phospholipid and cholesterol efflux

ABCA1, a member of the ATP-binding cassette family, mediates the efflux of cellular lipids to free apolipoproteins, mainly apoA-I. The role of the C-terminal domain of apoA-I in this process has been evaluated by measuring the efflux capacity of a truncated form (apoA-I-(1-192)) versus intact apoA-I in different cellular models. In stimulated J774 macrophages, cholesterol efflux to apoA-I-(1-192) was remarkably lower than that to the intact apoA-I. The truncated apoA-I, lacking an important lipid-binding domain, was also significantly less efficient in removing phospholipids from stimulated macrophages. No difference was detected with stimulated Tangier fibroblasts that do not express functional ABCA1. The C-terminal domain of apoA-I is clearly involved in ABCA1-driven lipid efflux. Independent of the interaction with the cell surface, it may be the decreased ability of the truncated apoA-I to recruit membrane phospholipids that impairs its capacity to promote cell cholesterol efflux.     

The intriguing links between prominin-1 (CD133), cholesterol-based membrane microdomains, remodeling of apical plasma membrane protrusions, extracellular membrane particles, and (neuro)epithelial cell differentiation

Prominin-1 (CD133) is a cholesterol-interacting pentaspan membrane protein concentrated in plasma membrane protrusions. In epithelial cells, notably neuroepithelial stem cells, prominin-1 is found in microvilli, the primary cilium and the midbody. These three types of apical membrane protrusions are subject to remodeling during (neuro)epithelial cell differentiation. The protrusion-specific localization of prominin involves its association with a distinct cholesterol-based membrane microdomain. Moreover, the three prominin-1-containing plasma membrane protrusions are the origin of at least two major subpopulations of prominin-1-containing extracellular membrane particles. Intriguingly, the release of these particles has been implicated in (neuro)epithelial cell differentiation.     

Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells

F9 murine embryonal carcinoma cells provide an attractive system for facilitating molecular mechanisms for epithelial morphogenesis, since they have the capability of differentiating into polarized epithelial cells bearing an apical junctional complexes. We previously showed that a specific retinoid X receptor-retinoic acid receptor heterodimer transduced retinoid signals for biogenesis of functional tight junctions in F9 cells (Exp. Cell Res. 263, (2001) 163). In the present study we generated F9 cells expressing doxycycline-inducible hepatocyte nuclear factor (HNF)-4alpha, a nuclear receptor. We herein show that induction of HNF-4alpha initiates differentiation of F9 cells to polarized epithelial cells, in which tight-junction proteins occludin, claudin-6, claudin-7, and ZO-1 are concentrated at the apical-most regions of lateral membranes. Expression of occludin, claudin-6, and claudin-7 was induced in the cells by doxycycline treatment in a dose- and time-dependent manner, in terms of the amount of HNF-4alpha. In contrast, expression levels of ZO-1, ZO-2, E-cadherin, and beta-catenin were not altered by HNF-4alpha. We also demonstrate, by analysis of diffusion of labeled sphingomyelin, that the fence function of tight junctions is achieved by induction of HNF-4alpha. These findings indicate that HNF-4alpha triggers de novo formation of functional tight junctions and establishment of epithelial cell polarity.     

Distinct roles of Rab3B and Rab13 in the polarized transport of apical,basolateral, and tight junctional membrane proteins to the plasma membrane

Regulated transport of proteins to distinct plasma membrane domains is essential for the establishment and maintenance of cell polarity in all eukaryotic cells. The Rab family small G proteins play a crucial role in determining the specificity of vesicular transport pathways. Rab3B and Rab13 localize to tight junction in polarized epithelial cells and cytoplasmic vesicular structures in non-polarized fibroblasts, but their functions are poorly understood. Here we examined their roles in regulating the cell-surface transport of apical p75 neurotrophin receptor (p75NTR), basolateral low-density lipoprotein receptor (LDLR), and tight junctional Claudin-1 using transport assay in non-polarized fibroblasts. Overexpression of Rab3B mutants inhibited the cell-surface transport of LDLR, but not p75NTR and Claudin-1. In contrast, overexpression of Rab13 mutants impaired the transport of Claudin-1, but not LDLR and p75NTR. These results suggest that Rab3B and Rab13 direct the cell-surface transport of LDLR and Claudin-1, respectively, and may contribute to epithelial polarization.     

Redistribution and Phosphorylation of Occludin During Opening and Resealing of Tight Junctions in Cultured Epithelial Cells

We studied the expression, distribution, and phosphorylation of the tight junction (TJ) protein occludin in confluent MDCK cell monolayers following three procedures for opening and resealing of TJs. When Ca²⁺ is transiently removed from the culture medium, the TJs open and the cells separate from each other, but the occludin band around each cell is retained. When Ca²⁺ is reintroduced, the TJs reseal. When the monolayers are exposed to prolonged Ca²⁺ starvation the cells maintain contact, but occludin disappears from the cell borders and can be detected only in a cytoplasmic compartment. When Ca²⁺ is reintroduced, new TJs are assembled and the transepithelial electrical resistance (TER) is reestablished in about 20 hr. Monolayers treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) show a different pattern of TJ opening: the cell-cell contact is maintained but the TJ strand network, as seen in freeze-fracture replicas, becomes discontinuous. Occludin is still localized at the cell periphery, but in a pattern of distribution that matches the discontinuous TJ. These TJs do not reseal even 24 hr after removal of the TPA. Western blot analysis showed that the 62–65 kD double band of occludin did not change with these treatments. However, in vivo phosphorylation analysis showed that the TPA treatment reduced the phosphorylation levels of occludin, while the prolonged Ca²⁺ starvation completely dephosphorylated the two occludin bands. In addition, a highly phosphorylated 71 kD band that immunoprecipitates with occludin is not present when TJ is opened by the Ca²⁺ removal. Phosphoaminoacid analysis showed that the 62–65 kD occludin bands are phosphorylated on serine and threonine, while the 71 kD band was phosphorylated exclusively on serine. Our results provide further evidence that phosphorylation of occludin is an important step in regulating TJ formation and permeability. Received: 28 December 1998/Revised: 8 April 1999     

The blood-testis barrier: the junctional permeability, the proteins and the lipids

The elucidation of how individual components of the Sertoli cell junctional complexes form and are dismantled to allow not only individual cells but whole syncytia of germinal cells to migrate from the basal to the lumenal compartment of the seminiferous epithelium without causing a permeability leak in the blood-testis barrier is amongst the most enigmatic yet, challenging and timely questions in testicular physiology. The intriguing key event in this process is how the barrier modulates its permeability during the periods of formation and dismantling of individual Sertoli cell junctions. The purpose of this review is therefore to first provide a reliable account on the normal formation, maintenance and dismantling process of the Sertoli cells junctions, then to assess the influence of the expression of their individual proteins, of the cytoskeleton associated with the junctions, and of the lipid content in the seminiferous tubules on the regulation of the their permeability barrier function. To help focus on the formation and dismantling of the Sertoli cell junctions, several considerations are based on data gleaned not only from rodents but from seasonal breeders as well because these animal models are characterized by exhaustive periods of junction assembly during development and the onset of the seasonal re-initiation of spermatogenesis as well as by an extensive junction dismantling period at the beginning of testicular regression, something unavailable in normal physiological conditions in continual breeders. Thus, the modulation of the permeability barrier function of the Sertoli cell junctions is analyzed in the physiological context of the blood-epidydimis barrier and in particular of the blood-testis barrier rather than in the context of a detailed account of the molecular composition and signalisation pathways of cell junctions. Moreover, the considerations discussed in this review are based on measurements performed on seminiferous tubule-enriched fractions gleaned at regular time intervals during development and the annual reproductive cycle.     

Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1

Expansion of the neocortex requires symmetric divisions of neuroepithelial cells, the primary progenitor cells of the developing mammalian central nervous system. Symmetrically dividing neuroepithelial cells are known to form a midbody at their apical (rather than lateral) surface. We show that apical midbodies of neuroepithelial cells concentrate prominin-1 (CD133), a somatic stem cell marker and defining constituent of a specific plasma membrane microdomain. Moreover, these apical midbodies are released, as a whole or in part, into the extracellular space, yielding the prominin-1-enriched membrane particles found in the neural tube fluid. The primary cilium of neuroepithelial cells also concentrates prominin-1 and appears to be a second source of the prominin-1-bearing extracellular membrane particles. Our data reveal novel origins of extracellular membrane traffic that enable neural stem and progenitor cells to avoid the asymmetric inheritance of the midbody observed for other cells and, by releasing a stem cell membrane microdomain, to potentially influence the balance of their proliferation versus differentiation.