紧密连接蛋白claudins应用于肿瘤治疗的进展

Claudins蛋白家族是组成紧密连接(Tight junctions,TJs)必不可少的骨架蛋白,在维持上皮和内皮细胞中的细胞极性、细胞间的粘附固定、细胞旁路的离子运输等发挥重要作用。近年来大量的研究结果证明,claudins在许多人类恶性肿瘤中异常表达。因此,claudins也被作为癌症治疗的潜在靶标。文中就claudin蛋白家族在肿瘤中的表达情况及其相关药物的研究进展进行阐述。     

扣带素——调节血管内皮细胞屏障功能的新靶点

正血管内皮细胞间连接最顶端的紧密连接是构成旁细胞屏障的重要结构。紧密连接由跨膜蛋白和胞浆蛋白组成,其中胞浆蛋白作为桥梁连接跨膜蛋白和细胞骨架蛋白,因此在决定紧密连接结构的完整性中发挥了重要的作用。然而目前,关于血管内皮中特异性表达的重要胞浆蛋白的研究仍十分有限。扣带素(cingulin)是1988年发现的一种紧密连接胞浆蛋白,已有研究表明cingulin在上皮细胞中参与调控紧密连接跨膜蛋白的表达以及细胞的增殖能力。然而,cingulin在血管内皮中的作用     

PRR11在胃癌中的表达及其与预后的关系

检测PRR11蛋白在胃癌中的表达,分析其表达异常与胃癌临床指标及预后间的关系。用Western免疫印迹比较胃癌和正常组织中PRR11的表达。构建含167例胃癌的组织芯片,用免疫组化法检测PI汛11蛋白在胃癌组织中的表达,统计学分析其与肿瘤大小、肿瘤侵袭、组织分化、淋巴结转移、TNM分期及胃癌患者总生存期之间的关系。PRR11在胃癌组织中的表达高于癌旁组织,在胃癌中的表达率为50．9％（85／167）,而在癌旁黏膜中不表达或微弱表达。PRR11的表达与胃壁侵袭、淋巴结转移、疾病分期和组织分化呈正相关（P〈0．05）。单因素生存分析表明,PRRll蛋白阳性表达患者较阴性患者生存期短（45个月VS81个月,P〈0．001）。多因素生存分析也表明,PRR11蛋白阳性表达患者生存期短于阴性患者（95％CI：0．347～0．865,P=0．01）。高表达PRR11与胃癌的发生、进展及预后密切相关,是判断胃癌患者预后的重要指标之一。     

Wnt信号通路GS蛋白在胃癌中的表达和临床意义

目的：研究wnt信号通路的中GS蛋白（谷氨酰胺合成酶）在胃癌组织中的表达,探索其在胃癌发生、发展中的意义。方法：用免疫组织化学法测定胃癌组织（110例）、肠化生组织（30例）、不典型增生组织（20例）及慢性浅表性胃炎组织（60例）中GS蛋白表达。用快速尿素酶法与病理组织切片染色法检测上述各组织中HP感染的情况,并予统计学分析比较其差异。结果：胃癌组织GS高蛋白表达与组织分型、分化程度、淋巴结转移密切相关（P〈0．05）,与肿瘤大小、部位、TNM分期、Borrmann分型、性别、年龄等无明显相关（P〉0．05）。GS表达与HP感染密切相关。结论：GS蛋白高表达同胃癌生物学行为密切相关,在胃癌的发生、发展中起重要作用。     

PCNA、P27蛋白在老年胃癌中的表达及其临床意义

目的：探讨增殖细胞核抗原（PCNA）和P27蛋白（P27）在老年胃癌中表达及其临床意义。方法：采用免疫组织化学SP法检测58例老年胃癌组织中PCNA、P27蛋白表达情况,分析它们与老年胃癌临床生物学行为的关系。结果：PCNA在老年胃癌组中的阳性表达率明显高于对照组的阳性表达率,P27在老年胃癌组中的阳性表达率明显低于对照组的阳性表达率。PCNA蛋白阳性表达与老年胃癌是否有浆膜浸润、组织分化程度、淋巴结转移以及TNM分期密切相关（P〈0.01）,而P27蛋白阳性表达与老年胃癌类型、是否有浆膜浸润、组织分化程度、淋巴结转移以及TNM分期密切相关（P〈0.01）。老年胃癌组织中PCNA、P27的表达呈负相关（r=-0.536,P〈0.05）。结论：P27表达下调、PCNA表达增强在老年胃癌的发生、发展过程中具有重要的作用,且两者具有相互作用。     

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。     

SNCG在胃癌中的表达及意义

目的：探讨SNCG在胃癌及正常胃粘膜组织中的蛋白和mRNA表达及其与临床病理特征的关系。方法：采用免疫组织化学SP法检测90例胃癌及40例正常胃粘膜组织中SNCG蛋白表达情况,同时应用RT-PCR检测29例胃癌及15例正常胃粘膜组织中SNCG mRNA的表达情况。结果：SNCG蛋白在胃癌组中的表达高于正常胃粘膜组（Uc=7.149,P〈0.05）,胃癌组中SNCG蛋白阳性表达与癌组织的浸润深度以及有无淋巴结转移有关（Uc=2.742,Uc,3.970,P均〈0.05）,而与患者的性别、年龄、及癌组织的分化程度无关。SNCG mRNA在胃癌组织中的表达量明显高于正常胃粘膜组织（t=4.399,P〈0.01）。结论：SNCG在胃癌组织中的高表达与胃癌发生发展及浸润转移密切相关,可能会成为胃癌早期发现、早期诊断以及判断转移、预后的重要参考指标。     

Tenascin，β-catenin，TGF—β1在胃癌中的表达及意义

目的：探讨胃癌组织中Tenascin、β—catenin、TGF－β1的表达及意义。方法：采用免疫组织化学方法检测70例胃癌组织和20例癌旁正常组织中Tenascin、β-catenin、TGF—β1的表达。结果：①Tenascin主要表达于胃癌组织中癌相关纤维母细胞的胞质,且与胃癌的Lauren分型、分化程度、临床分期、淋巴结转移关系显著（P〈0．05）;②β－catenin在胃癌的异常表达与胃癌的Lauren分型及分化程度关系显著（P〈0．05）;③TGF—β1在胃癌组织中主要表达于肿瘤细胞的胞质,其表达强度与胃癌的临床分期、浸润深度及淋巴结转移显著相关（P〈0．05）;④Tenascin与β—catenin在胃癌中的表达呈负相关（r=－0．505,P〈0．05）。结论：胃癌组织中Tenascin、β—catenin、TGF—β1蛋白的表达与肿瘤的侵袭、转移关系密切,β-catenin对间质中Tenascin的聚集具有抑制效应。     

封闭蛋白的结构、功能及其与人类疾病

紧密连接（tight junction,TJ）是脊椎动物细胞间连接的一种主要形式,对介导上皮细胞间的黏合、维持上皮细胞的功能具有重要作用。TJ是由一系列跨膜蛋白和外周蛋白相互作用而形成的一个复杂的蛋白质体系,封闭蛋白（occludin）是构成TJ的主要成分之一。目前,已发现封闭蛋白与许多人类疾病有关。本文仅就封闭蛋白的结构、功能及其与人类疾病的关系做一综述。     

Snail基因及其蛋白、E-cadherin蛋白在胃癌中的表达及其临床意义

目的观察Snail mRNA及其蛋白、E-cadherin蛋白在胃癌组织中的表达及其与胃癌临床病理特征的关系,并探讨它们在胃癌发生、发展中的作用及其临床应用价值。方法收集96例手术切除胃癌标本,同时取80例癌旁组织作为对照。应用免疫组织化学S-P法检测胃癌组织、癌旁组织中snail蛋白、E-cadherin蛋白的表达;运用原位分子杂交技术检测胃癌组织、癌旁组织中Snail mRNA的表达。结果（1）Snail蛋白在胃癌组织阳性率（83.3%）显著高于癌旁组织（41.25%）（P〈0.05）;高、中分化组Snail蛋白阳性表达率显著低于低分化组（P〈0.05）;Snail蛋白的阳性表达率在乳头状腺癌、管状腺癌及低分化腺癌与黏液癌之间差异有显著性（P〈0.05）;Snail蛋白的表达与胃癌浸润深度、淋巴结转移及远处转移有关（P〈0.05）,与性别、年龄、肿瘤大小、肿瘤部位及临床分期无关（P〉0.05）;（2）胃癌组织中snail mR-NA的阳性率（76%）显著高于癌旁组织（30%）（P〈0.05）;高、中分化组Snail mRNA阳性表达率显著低于低分化组（P〈0.05）;Snail mRNA的阳性表达率在乳头状腺癌、管状腺癌及低分化腺癌与黏液癌之间差异有显著性（P〈0.05）;Snail mRNA的表达与浸润深度及淋巴结转移有关（P〈0.05）,与性别、年龄、肿瘤大小、肿瘤部位、临床分期及远处转移无关（P〉0.05）;（3）E-cadherin蛋白在胃癌组织阳性率（37.5%）显著低于癌旁组织（100%）（P〈0.05）;高、中分化组E-cadherin蛋白阳性率显著高于低分化组（P〈0.05）;E-cadherin蛋白阳性率在乳头状腺癌、管状腺癌及低分化腺癌与黏液癌之间差异有显著性（P〈0.05）;E-cadherin蛋白的表达与胃癌浸润深度、淋巴结转移、临床分期及远处转移有关（P〈0.05）,与性别、年龄、肿瘤大小、肿瘤部位均无关（P〉0.05）;（4）胃癌组织中snail mRNA和snail蛋白的表达呈正相关（r=0.594,P〈0.05）;Snail蛋白和E-cadherin蛋白的表达呈负相关（r=-0.234,P〈0.05）。结论（1）E-cadher-in蛋白低表达与Snail蛋白高表达可能是胃黏膜恶性转变以及胃癌发生浸润转移的重要生物学标志;联合检测E-cadherin蛋白与Snail蛋白对预测胃癌浸润转移有重要意义。（2）Snail蛋白可能在转录水平上调控E-cadherin蛋白的表达。     

The ubiquitin-proteasome pathway regulates claudin 5 degradation

The tight junctions (TJs) form continuous intracellular contacts, which help create selective barriers in epithelial and endothelial cell layers. The structures created by the TJs are very dynamic and can be rapidly remodeled in response to physiological and pathological signals. Claudin 5 is a membranal TJ protein which plays a critical role in determining the permeability of endothelial barriers. We describe the regulation of claudin 5 degradation by the ubiquitin-proteasome system (UPS). Our results indicate that claudin 5 has a relatively short half-life and can be polyubiquitinated on lysine 199. This ubiquitination appears to trigger the proteasome-dependent degradation of claudin 5. Other mechanisms also seem to be involved in the post-translational regulation of claudin 5, including a ubiquitin-independent and probably indirect lysosomal-dependent pathway. These findings provide evidence for the involvement of the UPS in the regulation of claudin 5 levels, and set the stage for further research to determine the involvement of this pathway in the modulation of the properties of TJs and cell-layer barriers.     

Tight junctions potentiate the insulative properties of small CNS myelinated axons

Claudin family proteins form the physical barriers of tight junctions (TJs) and regulate paracellular diffusion across polarized epithelia. In addition to these heterotypic TJs, claudin 11 forms autotypic TJs comprising the radial component of central nervous system myelin. The exact function of these TJs has been unclear, although their location at the membrane perimeter is well sited to regulate diffusion between the interstitium and intramyelinic space. In this study, we demonstrate that claudin 11 affords rapid nerve conduction principally for small diameter myelinated axons. Claudin 11–null mice have preserved myelin and axonal architecture, but as much as a 60% decrease in conduction. They also have increased action potential thresholds and activated internodal potassium channels. These data indicate that TJs modulate the biophysical properties of myelin. Computational modeling reveals that claudin 11 reduces current flow through myelin and moderates its capacitive charging. Together, our data shed new light on myelin structural components and our understanding of the biology and pathophysiology of this membrane.     

Double gene deletion reveals lack of cooperation between claudin 11 and claudin 14 tight junction proteins

Members of the claudin family of proteins are the main components of tight junctions (TJs), the major selective barrier of the paracellular pathway between epithelial cells. The selectivity and specificity of TJ strands are determined by the type of claudins present. An understanding of the cooperation between different claudins in various tissues is thus important. To study the possible cooperation between claudin 11 and claudin 14, we have generated claudin 11/claudin 14 double-deficient mice, which exhibit a combination of the phenotypes found in each of the singly deficient mutants, including deafness, neurological deficits, and male sterility. These two claudins have distinct and partially overlapping expression patterns in the kidney. Claudin 11 is located in both the proximal and distal convoluted tubules, whereas claudin 14 occurs in both the thin descending and thick ascending limbs of the loop of Henle and in the proximal convoluted tubules. Although daily urinary excretion of Mg(++), and to a lesser extent of Ca(++), tends to be higher in claudin 11/claudin 14 double mutants, these changes do not reach statistical significance compared with wild-type animals. Thus, under normal conditions, co-deletion of claudin 11 and claudin 14 does not affect kidney function or ion balance. Our data demonstrate that, despite the importance of each of these claudins, there is probably no functional cooperation between them. Generation of additional mouse models in which different claudins are abolished should provide further insight into the complex interactions between claudin proteins in various physiological systems.     

Junctions gone bad: claudins and loss of the barrier in cancer

The tight junctions (TJs) of epithelia are responsible for regulating the "fence and gate" function of polarized epithelial cells. It is now well-established that dysregulation of these functions contributes to initiation and progression of cancer. Recently, it has become clear that the Claudins, members of a large family of 27 closely related transmembrane proteins, play a crucial role in formation, integrity and function of TJs, the epithelial permeability barrier and epithelial polarization. A growing body of data indicates that Claudin expression is altered in numerous epithelial cancers in a stage- and tumor-specific manner. While a single universal mechanism is still lacking, accumulating evidence supports a role for epigenetic regulation of Claudin expression in tumorgenesis, with concomitant alterations in barrier function. We review here new insights and challenges in understanding Claudin function in normal physiology and cancer.     

Role of claudin interactions in airway tight junctional permeability

Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction.     

Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon

Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon (Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na(+)-K(+)-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium.     

Claudin 7 is reduced in uterine epithelial cells during early pregnancy in the rat

The non-receptive uterine luminal epithelium forms an intact polarised epithelial barrier that is refractory to blastocyst invasion. During implantation, organised dismantling of this barrier leads to a receptive state promoting blastocyst attachment. Claudins are tight junction proteins that increase in the uterine epithelium at the time of implantation. Claudin 7 is a member of this family but demonstrates a basolateral localisation pattern that is distinct from other claudins. The present study investigated the localisation, abundance and hormonal regulation of claudin 7 to elucidate a role for the protein during implantation. The results showed that claudin 7 demonstrates a distinct basal and lateral localisation in the uterine luminal and glandular epithelium throughout early pregnancy. On day 1, claudin 7 is abundantly present in response to ovarian estrogen. At the time of implantation, claudin 7 decreases in abundance. This decrease is not dependent on blastocyst presence, as shown by results in pseudopregnant animals. We propose that claudin 7 mediates intercellular adhesions in the uterine epithelium and also may be responsible for stabilising adhesion proteins at the basolateral cell surface. Thus, claudin 7 may function under the maintenance of the uterine luminal epithelial barrier, in the non-receptive state preventing implantation from occurring.     

Anterior and intermediate pituitary tissues express claudin 4 in follicle stellate cells and claudins 2 and 5 in endothelial cells

Follicle-stellate cells are pituitary non-granular cells that are arranged between secretory cells or organized in follicles with small lumens. Cells from the follicles exhibit the typical phenotype of a transporting epithelium, including apical microvilli with a cilium and tight junctions. Freeze-fracture electron microscopy images show that the tight junctions consist of 5–7 anastomosing strands and that cultured follicle-stellate cells develop a trans-epithelial electrical resistance characteristic of “tight” epithelia. Here, we investigate the molecular composition of the tight junction from follicle stellate cells. We found that the rat anterior pituitary lobe expresses mRNAs for claudins 2, 4 and 5; the proteins of all these claudins are observed in the anterior lobe, whereas the intermediate lobe expresses claudins 2 and 5 and the posterior lobe contains only claudin 5. Follicle-stellate cells, identified by their protein marker S100β, expresses claudin 4 in the apical membrane, in co-localization with dipeptidyl-peptidase and near acetylated β-tubulin. Claudin 4 partially co-localizes with E-cadherin, indicating that a fraction of the protein is located in the basolateral domain. Follicle-stellate-enriched cell cultures develop patches of polygonal cells expressing claudin 4 and E-cadherin, encircled by extensive monolayers of fusiform cells. Claudin 2 stains specifically blood vessels, identified by claudin 5 and VE-cadherin labels. Thus, follicles in the anterior pituitary consist of “tight” epithelia that can carry out intense vectorial transport, together with a high cation movement in blood vessels, possibly related to the ion requirements of excitable secretory cells for hormone secretion.     

Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.     

Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells

Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporated later into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculated M(r) of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs.