肿瘤坏死因子alpha增加肠上皮细胞屏障通透性的机制研究
|
摘要
肿瘤坏死因alpha增加肠上皮细胞屏障通透性的机制研究
目的
肠黏膜屏障是机体最重要的免疫防御屏障，将机体与肠道内的外源性物质隔离开来，避免病原微生物的侵袭和抗原分子的损伤。肠黏膜屏障包括肠上皮细胞屏障、免疫屏障和微生物屏障，肠上皮细胞屏障是最重要的一道屏障，是肠黏膜屏障具有选择性通透的基础。目前研究发现肠上皮细胞屏障通透性增高参与多种疾病的发生(如炎症性肠病、败血症、烧伤、终末期肝病、重症胰腺炎等)，并且在这些情况下，血清肿瘤坏死因子alpha(TNFα)明显升高，与肠上皮细胞屏障的损害程度呈正相关，抗TNFα抗体可以恢复受损的肠上皮细胞屏障。因此认为TNFα是增加肠上皮细胞屏障通透性的重要因素。
虽然目前已经形成共识：TNFα可以增加肠上皮细胞屏障的通透性，但TNFα的具体作用机制和方式还存在争议。早期认为与TNFα引起的细胞凋亡密切相关，近年来则倾向于TNFα引起肠上皮细胞屏障损伤是凋亡非依赖性的。另外TNFα引起肠上皮细胞屏障通透性增高的具体作用环节也不十分清楚，可能与蛋白激酶C(PKC)、核因子kB(NF-kappaB)、肌球蛋白轻链激酶(MLCK)、丝裂原活化的蛋白激酶(MAPK)等信号途径有关。因此我们应用Caco-2细胞株建立体外肠上皮细胞屏障模型，观察TNFα对肠上皮细胞屏障通透性和肠上皮细胞间紧密连接的影响，并在此基础上探讨了TNFα对调控紧密连接装配的蛋白酶活化受体-3／非典型蛋白激酶C／蛋白酶活化受体-6(Par-3／aPKC／Par-6)复合体的影响和可能信号通路，以明确病理条件下，TNFα增加肠上皮细胞屏障通透性的作用机制。
材料和方法
1、体外肠上皮细胞屏障模型的建立
应用Caco-2细胞株建立体外肠上皮细胞屏障模型，并应用跨上皮细胞电阻(TEER)和荧光黄透过率两种指标检测肠上皮细胞屏障的形成情况。
2、TNFα对肠上皮细胞屏障通透性的影响
加入不同浓度的TNFα(10μ／L或100μg／L)作用24h或加入TNFα100pg／L作用不同时间(2、4、8、24h)，观察加入TNFα前后Caco-2细胞屏障跨上皮细胞电阻的改变及TNFα对Caco-2细胞荧光黄透过率的影响。
3、TNFα对肠上皮细胞间紧密连接的影响
透射电镜下观察加入TNFα100μtg／L作用24h后，Caco-2细胞间紧密连接形态学的改变。
4、TNFα对肠上皮细胞紧密连接蛋白occludin定位和表达的影响
制备Caco-2细胞NP-40可溶性和NP-40不溶性蛋白框架，应用蛋白印迹杂交(Western blot)技术，检测加入不同浓度的TNFα(10μg／L或100μg／L)作用24h后，不同蛋白框架内肠上皮细胞紧密连接蛋白occludin表达的变化情况。并应用免疫荧光和实时定量PCR技术检测occludin蛋白的定位和mRNA表达的变化情况。
5、TNFα对肠上皮细胞Par-3表达和定位的影响
加入TNFα100μg／L作用不同时间(0、4、8、24h)后，应用免疫荧光、Western blot和逆转录-聚合酶链反应(RT-PCR)技术检测TNFα对肠上皮细胞Par-3表达和定位的影响。
6、TNFα对肠上皮细胞非典型蛋白激酶C亚型PKλ丸和PKCζ活性的影响
应用抗PKCζ／λ(Thr410／403)磷酸化抗体检测加入TNFα100μg／L作用不同时间(0、4、8、24h)后，PKCλ／ζ(活性的改变。
7、应用免疫共沉淀的方法检测Caco-2细胞内与Pat-3结合的信号分子
应用抗Par-3抗体沉淀Caco-2细胞裂解产物，然后应用抗T淋巴瘤侵袭转移因子(Tiaml)抗体对沉淀产物进行蛋白印迹杂交，明确Par-3是否与Tiaml结合。
8、TNFα对肠上皮细胞T淋巴瘤侵袭转移因子表达和定位的影响
加入TNFα100μg／L作用不同时间(0、4、8、24h)后，应用免疫荧光、免疫电镜、Western blot和RT-PCR技术检测TNFα对肠上皮细胞Tiaml表达和定位的影响。
9、TNFα对肠上皮细胞Racl活性的影响
应用配体免疫沉淀法检测加入TNFα100μg／L作用不同时间(0、4、8、24h)后，Racl活性的改变。
10、Tiaml在TNFα降低紧密连接蛋白occludin表达中的作用
应用小核糖核酸干扰(siRNA)技术沉默Caco-2细胞内Tiaml基因，观察在Tiaml低表达的Caco-2细胞内，TNFα对紧密连接蛋白occludin表达的影响。
结果
1、TNFα增加肠上皮细胞屏障的通透性
与对照组相比，10μg／L TNFα即可降低肠上皮细胞屏障的TEER(P＜0.0005)，增加荧光黄透过率(P＜0.0005)，100μg／L TNFα这种作用更加明显(P＜0.0005)。在作用时间上，TNFα100μg／L作用4h后，TEER值即开始下降(P＜0.0005)，荧光黄透过率开始升高(P=0.001)，24h这种作用达到最强(P＜0.0005)。
2、TNFα破坏肠上皮细胞间的紧密连接
正常CaCo-2细胞，紧密连接结构完整，呈致密的带状结构。加入TNFα100μg／L作用24h后，紧密连接断裂，细胞间隙增宽。
3、TNFα引起肠上皮细胞紧密连接蛋白occludin的定位异常
正常Caco-2细胞，occludin蛋白沿细胞膜分布；加入TNFα10μg／L作用24h后，occludin蛋白呈锯齿状分布，并且荧光信号减弱；加入TNFαt100μg／L作用24h后，锯齿状分布更加明显，荧光信号进一步减弱，并且在胞浆内可见阳性染色。
4、TNFα引起occludin蛋白磷酸化减少
与对照组比较，加入TNFα100μg／L作用24h后，NP-40不溶性蛋白框架内occludin蛋白(即磷酸化occludin蛋白)的相对含量明显减少(P=0.016)，而NP-40可溶性蛋白框架内occludin蛋白(即非磷酸化occludin蛋白)的相对含量没有变化(P=0.99)。
5、TNFα不影响occludin mRNA的表达
与对照组比较，不同浓度的TNFα或TNFα作用不同时间，均不能引起occludin mRNA的明显改变(P=0.85，P=0.99)。
6、TNFα不影响Par-3的表达和定位
正常Caco-2细胞，Par-3定位在细胞膜上，与紧密连接蛋白的定位一致，加入TNFα不能改变Par-3蛋白的定位。Western blot和RT-PCR结果提示加入TNFα后，Par-3蛋白和mRNA的相对含量与正常对照组比较没有差别(P=0.99，P=0.86)。
7、TNFα下调PKCλ/ζ活性
与正常对照组相比，TNFα100μg/L作用8h后，PKCλ/ζ活性开始下降(P=0.014)，24h达到最低(P=0.001)。
8、Par-3与Tiam1免疫共沉淀
应用抗Tiam1抗体对Par-3预沉淀产物进行蛋白印迹杂交后，发现在170kDa处出现特异性蛋白条带，证明在Caco-2细胞内，Par-3与Tiam1结合。
9、TNFα增加Tiam1表达，促进Tiam1活化
在正常Caco-2细胞，Tiam1分布在胞浆内，加入TNFα100μg/L作用24h后，Tiam1向细胞膜转移。Western blot和RT-PCR结果显示，与正常对照组比较，TNFα100μg/L作用24h后，Tiam1蛋白和mRNA的相对含量明显升高(P=0.029，P=0.011)。
10、TNFα促进Rac1的活化
与正常对照组比较，加入TNFα后，总Rac1的含量没有明显变化(P=0.99)。但活性Rac1(GTP-Rac1)的含量在TNFα100μg/L作用24h后明显升高(P=0.002)。
11、Tiam1介导TNFα引起的紧密连接蛋白occludin磷酸化减少
在Tiam1正常表达的Caco-2细胞，TNFα可以引起occludin蛋白磷酸化的减少，而在Tiam1低表达的Caco-2细胞，TNFα的这种作用消失，两者相比有明显的统计学差异(P=0.001)。
1、TNFα增加肠上皮细胞屏障的通透性。
2、TNFα破坏肠上皮细胞间的紧密连接，这是其增加肠上皮细胞屏障细胞旁通透性的作用位点。
3、TNFα引起紧密连接蛋白occludin的异常分布，使紧密连接蛋白occludin磷酸化减少，但不影响未磷酸化的occludin蛋白表达。
4、TNFα不影响occludin mRNA的表达，提示TNFα对occludin的调节发生在蛋白水平，而不是转录水平。
5、TNFα不影响Par-3的表达和定位，但能使PKCλ／ζ的活性降低，提示TNFα对于调控紧密连接装配的Par-3／aPKC／Par-6复合体的定位没有影响，但能抑制Par-3／aPKC／Par-6复合体的功能。
6、Tiam1-Rac1是Par-3／aPKC／Par-6复合体的上游信号因子。
7、TNFα能够增加Tiam1蛋白和mRNA水平的表达，促进Tiam1和Rac1活化。
8、TNFα引起Caco-2细胞紧密连接蛋白occludin磷酸化减少需要Tiam1的参与。
The mechanism of increased intestinal epithelial barrier permeability induced by tumor necrosis factor alpha
Objective
In addition to being the organ responsible for digestion and absorption of nutrients, the intestine serves a barrier function that is a critical component of the innate immune system. Only a single layer of epithelial cells separates the luminal contents from effector immune cells in the lamina propria and the internal milieu of the body. Breakdown of the barrier is implicated in bacterial translocation, leading to sepsis, and in the pathogenesis of several illnesses such as inflammatory bowel disease, liver failure, acute severe pancreases and multiple organ system failure. Studies in rodents show that tumor necrosis factor alpha(TNFα) can lead to increased ileal permeability, and anti-TNFαantibody can prevent the intestinal permeability disorders.
The mechanisms of TNFα-related epithelial breakdown are unclear, but regulation of paracellular pathways, especially via the interepithelial tight junction (TJ) proteins, and subsequent stimulation of highly immunoreactive submucosal cells are likely to play a significant role. Therefore, we detect the effect of TNFαon intestinal permeability and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells, and study the mechanisms involved in tight junction discharged assembly induced by TNFα.
Materials and methods
1、Cell cultures
Caco-2 cells were grown in a culture medium composed of DMEM with 4.5 mg/ml glucose, 50 U/ml penicillin, 50 U/ml streptomycin, 4 mmol/1 glutamine, and 10% FBS. In order to establish in vitro intestinal epithelial barrier model, Caco-2 cells were plated on Transwell filters and monitored regularly by visualization with an inverted microscope and by epithelial resistance measurements.
2、Determination of epithelial monolayer resistance and paracellular permeability
The electrical resistance of the filter-grown Caco-2 intestinal monolayers was measured by using an epithelial voltohrn-meter (EVOM) as previously reported. For determination of the effect of TNFαon Caco-2 monolayer paracellular permeability, the filter-grown Caco-2 cells by 3 wk postplating were incubated with TNFα10μg/L or 100μg/L for 24 hours or with TNFα100μg/L for indicated times(2、4、8、24h), and the mucosal-to-serosal flux rates of the established paracellular marker luminal yellow was determined.
3、Transmission electron microscopy
Cells were treated with TNFα100μg/L for 24 hours and ultrathin sections were made and stained with saturated uranyl acetate and Reynold's lead citrate. Ultrastructure of junctional complexes were observated by transmission electron microscopy.
4、Assessment of occludin protein localization and expression
Cells were treated with TNFα(10μg/L or 100μg/L) for 24 hours and the localization and expression of occludin protein were detected by immunofluorescence and Western blot analysis. SYBR-Green-based real-time PCR was used to measure the expression of occluding mRNA.
5、Assessment of Par-3 protein localization and expression
Cells were treated with TNFα100μg/L for indicated hours(4、8、24h) and the localization and expression of Par-3 protein were detected by immunofluorescence and Western blot analysis. Reverse-transcription PCR was used to measure the expression of Par-3 mRNA.
6、PKCλand PKCξkinase analysis
Cells were treated with TNFα100μg/L for indicated hours(4、8、24h) and the endogenous PKCξ/λactivity was detected by immunoblotting with anti-phos-PKCξ/λ(Thr410/403) antibody.
7、Immunoprecipitation
In order to judge the endogenous complexes of Par-3 and Tiaml, Caco-2 cells lysates were immunoprecipitated with anti-Par-3 antibody and immunoprecipitates were immunoblotted with anti-Tiaml antibody.
8、Assessment of Tiaml protein localization and expression
Cells were treated with TNFα100μg/L for indicated hours (4、8、24h) and the localization and expression of Tiaml protein were detected by immunofluorescence, immunogold electron microscopy and Western blot analysis. Reverse-transcription PCR was used to measure the expression of Tiaml mRNA.
9、Racl activity assays
After treatment with TNFα100μg/L for indicated hours (4、8、24h), Caco-2 cells lysates were prepared and Racl activity was determined as described previously using a biotinylated Racl interactive binding metifpeptide of PAK1.
10、RNA interference for Tiara1 gene
Silence the Tiarnl gene in Caco-2 cells use small interfering RNA specific for Tiaml. Detect the effect of TNFαon action occludin protein expression in Tiaml knock down cells.
Results
1、TNFαincrease intestinal epithelial paracellularpermeability
TNFαtreatment of filter-grown Caco-2 monolayers (0-100μg/L)produced a concentration and time-dependent drop in Caco-2 transepithelial electrical resistance (TEER) during the 24-h experimental period (P＜0.0005). The TNFαeffect on Caco-2 paracellular permeability examined by using the paracellular markers luminal yellow indicated that TNFαproduced a progressive concentration and time-dependent increase in transepithelial permeability to luminalyellow (P＜0.0005).
2、TNFαdisrupts the interepithelial tight junction
The presence of electron-dense material in the space between cells near the brush border reflects the TJ. In cells without TNFα, the TJ displayed an intact structure. When the cells were incubated with TNFα100μg/L for 24 hours, the TJ complex appeared reduced and contained less electron-dense material.
3、TNFαcauses tight junction protein occludin distributed
In the control Caco-2 monolayers, occludin proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders. TNFα(100μg/L) caused a progressive disturbance in the continuity of occludin localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contact.
4、TNFαdecreases the expression of action occludin protein
TNFαproduced a progressive decrease in higher molecular form, a phosphorylated form (85 kDa) of occludin protein expression (P=0.016), whereas the low molecular form, a nonphosphorylated form (65 kDa), was no significant change (P=0.99).
5、TNFαdoes not affect the expression of occludin mRNA
TNFαtreatment does not cause a significant decrease in occludin mRNA production compared with controls considering the difference of the concentration and time of TNFαtreatment (P=0.85, P=0.99).
6、TNFαdoes not affect the expression and localization of Par-3 protein
In the control Caco-2 monolayers, Par-3 proteins were localized at the apical cellular borders, consistent with the location of occludin protein. TNFα(100μg/L) didn't cause a significant change in the Par-3 protein localization. Western blot and RT-PCR analysis demonstrated that TNFαtreatment had on effect on the expression of Par-3 protein and mRNA (P=0.99, P=0.86).
7、TNFαinhibits PKCλ/ζactivity
TNFα100μg/L caused a progressive decrease in PKCλ/ζactivity over 8h period (P=0.014). The maxiam drop in PKCλ/ζactivity occurred at 24h (P=0.001).
8、Par-3 immunoprecipitated with Tiaml
Immunoprecipitates with anti-Par-3 antibody showed specific protein belt by immunoblotted with anti-Tiaml antibody.
9、TNFαincreases the expression of Tiaml protein
Tiaml proteins were localized at the cytoplasm in the control Caco-2 cells. TNFα can cause a migration of Tiaml protein from cytoplasm to cellular borders. Furthrmore, Western blot and RT-PCR assays demonstrated that TNFαincresed the expression of Tiaml protein and mRNA at 24-h time point (P=0.029, P=0.011).
10、TNFαstimulates Rac1 activity
TNFαhad no significant effect on the expression of total Rac1 (P=0.99), whereas the active Rac1 (GTP-Rac1) decreased at 24-h time point after TNFαtreatment (P=0.002).
11、TNFαdecreases action occludin protein expression through a Tiaml-dependent manner
TNFαdown regulated the phosphorylated form of occludin protein expression in Caco-2 cells. Moreover, absence of Tiaml rescued the impaired action occludin protein expression induced by TNFα(P=0.001).
Conclusion
1、TNFαdisrupts the interepithelial tight junction and increases Caco-2 intestinal epithelial permeability.
2、TNFαcauses a disturbance in the continuity of occludin localization at the cellular borders and produces a decrease in action occludin protein expression.
3、TNFαtreatment does not cause a significant decrease in occludin mRNA production.
4、TNFαdoes not affect the expression and localization of Par-3 protein, but inhibits the activity of PKCλ/ξ.
5、Tiaml-Pac1 is the upstream factor of Par3-aPKC-Par6 complex.
6、TNFαcauses an increase in Tiaml protein and mRNA expression, and stimulates Rac1 constitutively activated.
7、TNFαdownregulates action occludin protein expression via a Tiaml-dependent process.
目的
肠黏膜屏障是机体最重要的免疫防御屏障，将机体与肠道内的外源性物质隔离开来，避免病原微生物的侵袭和抗原分子的损伤。肠黏膜屏障包括肠上皮细胞屏障、免疫屏障和微生物屏障，肠上皮细胞屏障是最重要的一道屏障，是肠黏膜屏障具有选择性通透的基础。目前研究发现肠上皮细胞屏障通透性增高参与多种疾病的发生(如炎症性肠病、败血症、烧伤、终末期肝病、重症胰腺炎等)，并且在这些情况下，血清肿瘤坏死因子alpha(TNFα)明显升高，与肠上皮细胞屏障的损害程度呈正相关，抗TNFα抗体可以恢复受损的肠上皮细胞屏障。因此认为TNFα是增加肠上皮细胞屏障通透性的重要因素。
虽然目前已经形成共识：TNFα可以增加肠上皮细胞屏障的通透性，但TNFα的具体作用机制和方式还存在争议。早期认为与TNFα引起的细胞凋亡密切相关，近年来则倾向于TNFα引起肠上皮细胞屏障损伤是凋亡非依赖性的。另外TNFα引起肠上皮细胞屏障通透性增高的具体作用环节也不十分清楚，可能与蛋白激酶C(PKC)、核因子kB(NF-kappaB)、肌球蛋白轻链激酶(MLCK)、丝裂原活化的蛋白激酶(MAPK)等信号途径有关。因此我们应用Caco-2细胞株建立体外肠上皮细胞屏障模型，观察TNFα对肠上皮细胞屏障通透性和肠上皮细胞间紧密连接的影响，并在此基础上探讨了TNFα对调控紧密连接装配的蛋白酶活化受体-3／非典型蛋白激酶C／蛋白酶活化受体-6(Par-3／aPKC／Par-6)复合体的影响和可能信号通路，以明确病理条件下，TNFα增加肠上皮细胞屏障通透性的作用机制。
材料和方法
1、体外肠上皮细胞屏障模型的建立
应用Caco-2细胞株建立体外肠上皮细胞屏障模型，并应用跨上皮细胞电阻(TEER)和荧光黄透过率两种指标检测肠上皮细胞屏障的形成情况。
2、TNFα对肠上皮细胞屏障通透性的影响
加入不同浓度的TNFα(10μ／L或100μg／L)作用24h或加入TNFα100pg／L作用不同时间(2、4、8、24h)，观察加入TNFα前后Caco-2细胞屏障跨上皮细胞电阻的改变及TNFα对Caco-2细胞荧光黄透过率的影响。
3、TNFα对肠上皮细胞间紧密连接的影响
透射电镜下观察加入TNFα100μtg／L作用24h后，Caco-2细胞间紧密连接形态学的改变。
4、TNFα对肠上皮细胞紧密连接蛋白occludin定位和表达的影响
制备Caco-2细胞NP-40可溶性和NP-40不溶性蛋白框架，应用蛋白印迹杂交(Western blot)技术，检测加入不同浓度的TNFα(10μg／L或100μg／L)作用24h后，不同蛋白框架内肠上皮细胞紧密连接蛋白occludin表达的变化情况。并应用免疫荧光和实时定量PCR技术检测occludin蛋白的定位和mRNA表达的变化情况。
5、TNFα对肠上皮细胞Par-3表达和定位的影响
加入TNFα100μg／L作用不同时间(0、4、8、24h)后，应用免疫荧光、Western blot和逆转录-聚合酶链反应(RT-PCR)技术检测TNFα对肠上皮细胞Par-3表达和定位的影响。
6、TNFα对肠上皮细胞非典型蛋白激酶C亚型PKλ丸和PKCζ活性的影响
应用抗PKCζ／λ(Thr410／403)磷酸化抗体检测加入TNFα100μg／L作用不同时间(0、4、8、24h)后，PKCλ／ζ(活性的改变。
7、应用免疫共沉淀的方法检测Caco-2细胞内与Pat-3结合的信号分子
应用抗Par-3抗体沉淀Caco-2细胞裂解产物，然后应用抗T淋巴瘤侵袭转移因子(Tiaml)抗体对沉淀产物进行蛋白印迹杂交，明确Par-3是否与Tiaml结合。
8、TNFα对肠上皮细胞T淋巴瘤侵袭转移因子表达和定位的影响
加入TNFα100μg／L作用不同时间(0、4、8、24h)后，应用免疫荧光、免疫电镜、Western blot和RT-PCR技术检测TNFα对肠上皮细胞Tiaml表达和定位的影响。
9、TNFα对肠上皮细胞Racl活性的影响
应用配体免疫沉淀法检测加入TNFα100μg／L作用不同时间(0、4、8、24h)后，Racl活性的改变。
10、Tiaml在TNFα降低紧密连接蛋白occludin表达中的作用
应用小核糖核酸干扰(siRNA)技术沉默Caco-2细胞内Tiaml基因，观察在Tiaml低表达的Caco-2细胞内，TNFα对紧密连接蛋白occludin表达的影响。
结果
1、TNFα增加肠上皮细胞屏障的通透性
与对照组相比，10μg／L TNFα即可降低肠上皮细胞屏障的TEER(P＜0.0005)，增加荧光黄透过率(P＜0.0005)，100μg／L TNFα这种作用更加明显(P＜0.0005)。在作用时间上，TNFα100μg／L作用4h后，TEER值即开始下降(P＜0.0005)，荧光黄透过率开始升高(P=0.001)，24h这种作用达到最强(P＜0.0005)。
2、TNFα破坏肠上皮细胞间的紧密连接
正常CaCo-2细胞，紧密连接结构完整，呈致密的带状结构。加入TNFα100μg／L作用24h后，紧密连接断裂，细胞间隙增宽。
3、TNFα引起肠上皮细胞紧密连接蛋白occludin的定位异常
正常Caco-2细胞，occludin蛋白沿细胞膜分布；加入TNFα10μg／L作用24h后，occludin蛋白呈锯齿状分布，并且荧光信号减弱；加入TNFαt100μg／L作用24h后，锯齿状分布更加明显，荧光信号进一步减弱，并且在胞浆内可见阳性染色。
4、TNFα引起occludin蛋白磷酸化减少
与对照组比较，加入TNFα100μg／L作用24h后，NP-40不溶性蛋白框架内occludin蛋白(即磷酸化occludin蛋白)的相对含量明显减少(P=0.016)，而NP-40可溶性蛋白框架内occludin蛋白(即非磷酸化occludin蛋白)的相对含量没有变化(P=0.99)。
5、TNFα不影响occludin mRNA的表达
与对照组比较，不同浓度的TNFα或TNFα作用不同时间，均不能引起occludin mRNA的明显改变(P=0.85，P=0.99)。
6、TNFα不影响Par-3的表达和定位
正常Caco-2细胞，Par-3定位在细胞膜上，与紧密连接蛋白的定位一致，加入TNFα不能改变Par-3蛋白的定位。Western blot和RT-PCR结果提示加入TNFα后，Par-3蛋白和mRNA的相对含量与正常对照组比较没有差别(P=0.99，P=0.86)。
7、TNFα下调PKCλ/ζ活性
与正常对照组相比，TNFα100μg/L作用8h后，PKCλ/ζ活性开始下降(P=0.014)，24h达到最低(P=0.001)。
8、Par-3与Tiam1免疫共沉淀
应用抗Tiam1抗体对Par-3预沉淀产物进行蛋白印迹杂交后，发现在170kDa处出现特异性蛋白条带，证明在Caco-2细胞内，Par-3与Tiam1结合。
9、TNFα增加Tiam1表达，促进Tiam1活化
在正常Caco-2细胞，Tiam1分布在胞浆内，加入TNFα100μg/L作用24h后，Tiam1向细胞膜转移。Western blot和RT-PCR结果显示，与正常对照组比较，TNFα100μg/L作用24h后，Tiam1蛋白和mRNA的相对含量明显升高(P=0.029，P=0.011)。
10、TNFα促进Rac1的活化
与正常对照组比较，加入TNFα后，总Rac1的含量没有明显变化(P=0.99)。但活性Rac1(GTP-Rac1)的含量在TNFα100μg/L作用24h后明显升高(P=0.002)。
11、Tiam1介导TNFα引起的紧密连接蛋白occludin磷酸化减少
在Tiam1正常表达的Caco-2细胞，TNFα可以引起occludin蛋白磷酸化的减少，而在Tiam1低表达的Caco-2细胞，TNFα的这种作用消失，两者相比有明显的统计学差异(P=0.001)。
1、TNFα增加肠上皮细胞屏障的通透性。
2、TNFα破坏肠上皮细胞间的紧密连接，这是其增加肠上皮细胞屏障细胞旁通透性的作用位点。
3、TNFα引起紧密连接蛋白occludin的异常分布，使紧密连接蛋白occludin磷酸化减少，但不影响未磷酸化的occludin蛋白表达。
4、TNFα不影响occludin mRNA的表达，提示TNFα对occludin的调节发生在蛋白水平，而不是转录水平。
5、TNFα不影响Par-3的表达和定位，但能使PKCλ／ζ的活性降低，提示TNFα对于调控紧密连接装配的Par-3／aPKC／Par-6复合体的定位没有影响，但能抑制Par-3／aPKC／Par-6复合体的功能。
6、Tiam1-Rac1是Par-3／aPKC／Par-6复合体的上游信号因子。
7、TNFα能够增加Tiam1蛋白和mRNA水平的表达，促进Tiam1和Rac1活化。
8、TNFα引起Caco-2细胞紧密连接蛋白occludin磷酸化减少需要Tiam1的参与。
The mechanism of increased intestinal epithelial barrier permeability induced by tumor necrosis factor alpha
Objective
In addition to being the organ responsible for digestion and absorption of nutrients, the intestine serves a barrier function that is a critical component of the innate immune system. Only a single layer of epithelial cells separates the luminal contents from effector immune cells in the lamina propria and the internal milieu of the body. Breakdown of the barrier is implicated in bacterial translocation, leading to sepsis, and in the pathogenesis of several illnesses such as inflammatory bowel disease, liver failure, acute severe pancreases and multiple organ system failure. Studies in rodents show that tumor necrosis factor alpha(TNFα) can lead to increased ileal permeability, and anti-TNFαantibody can prevent the intestinal permeability disorders.
The mechanisms of TNFα-related epithelial breakdown are unclear, but regulation of paracellular pathways, especially via the interepithelial tight junction (TJ) proteins, and subsequent stimulation of highly immunoreactive submucosal cells are likely to play a significant role. Therefore, we detect the effect of TNFαon intestinal permeability and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells, and study the mechanisms involved in tight junction discharged assembly induced by TNFα.
Materials and methods
1、Cell cultures
Caco-2 cells were grown in a culture medium composed of DMEM with 4.5 mg/ml glucose, 50 U/ml penicillin, 50 U/ml streptomycin, 4 mmol/1 glutamine, and 10% FBS. In order to establish in vitro intestinal epithelial barrier model, Caco-2 cells were plated on Transwell filters and monitored regularly by visualization with an inverted microscope and by epithelial resistance measurements.
2、Determination of epithelial monolayer resistance and paracellular permeability
The electrical resistance of the filter-grown Caco-2 intestinal monolayers was measured by using an epithelial voltohrn-meter (EVOM) as previously reported. For determination of the effect of TNFαon Caco-2 monolayer paracellular permeability, the filter-grown Caco-2 cells by 3 wk postplating were incubated with TNFα10μg/L or 100μg/L for 24 hours or with TNFα100μg/L for indicated times(2、4、8、24h), and the mucosal-to-serosal flux rates of the established paracellular marker luminal yellow was determined.
3、Transmission electron microscopy
Cells were treated with TNFα100μg/L for 24 hours and ultrathin sections were made and stained with saturated uranyl acetate and Reynold's lead citrate. Ultrastructure of junctional complexes were observated by transmission electron microscopy.
4、Assessment of occludin protein localization and expression
Cells were treated with TNFα(10μg/L or 100μg/L) for 24 hours and the localization and expression of occludin protein were detected by immunofluorescence and Western blot analysis. SYBR-Green-based real-time PCR was used to measure the expression of occluding mRNA.
5、Assessment of Par-3 protein localization and expression
Cells were treated with TNFα100μg/L for indicated hours(4、8、24h) and the localization and expression of Par-3 protein were detected by immunofluorescence and Western blot analysis. Reverse-transcription PCR was used to measure the expression of Par-3 mRNA.
6、PKCλand PKCξkinase analysis
Cells were treated with TNFα100μg/L for indicated hours(4、8、24h) and the endogenous PKCξ/λactivity was detected by immunoblotting with anti-phos-PKCξ/λ(Thr410/403) antibody.
7、Immunoprecipitation
In order to judge the endogenous complexes of Par-3 and Tiaml, Caco-2 cells lysates were immunoprecipitated with anti-Par-3 antibody and immunoprecipitates were immunoblotted with anti-Tiaml antibody.
8、Assessment of Tiaml protein localization and expression
Cells were treated with TNFα100μg/L for indicated hours (4、8、24h) and the localization and expression of Tiaml protein were detected by immunofluorescence, immunogold electron microscopy and Western blot analysis. Reverse-transcription PCR was used to measure the expression of Tiaml mRNA.
9、Racl activity assays
After treatment with TNFα100μg/L for indicated hours (4、8、24h), Caco-2 cells lysates were prepared and Racl activity was determined as described previously using a biotinylated Racl interactive binding metifpeptide of PAK1.
10、RNA interference for Tiara1 gene
Silence the Tiarnl gene in Caco-2 cells use small interfering RNA specific for Tiaml. Detect the effect of TNFαon action occludin protein expression in Tiaml knock down cells.
Results
1、TNFαincrease intestinal epithelial paracellularpermeability
TNFαtreatment of filter-grown Caco-2 monolayers (0-100μg/L)produced a concentration and time-dependent drop in Caco-2 transepithelial electrical resistance (TEER) during the 24-h experimental period (P＜0.0005). The TNFαeffect on Caco-2 paracellular permeability examined by using the paracellular markers luminal yellow indicated that TNFαproduced a progressive concentration and time-dependent increase in transepithelial permeability to luminalyellow (P＜0.0005).
2、TNFαdisrupts the interepithelial tight junction
The presence of electron-dense material in the space between cells near the brush border reflects the TJ. In cells without TNFα, the TJ displayed an intact structure. When the cells were incubated with TNFα100μg/L for 24 hours, the TJ complex appeared reduced and contained less electron-dense material.
3、TNFαcauses tight junction protein occludin distributed
In the control Caco-2 monolayers, occludin proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders. TNFα(100μg/L) caused a progressive disturbance in the continuity of occludin localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contact.
4、TNFαdecreases the expression of action occludin protein
TNFαproduced a progressive decrease in higher molecular form, a phosphorylated form (85 kDa) of occludin protein expression (P=0.016), whereas the low molecular form, a nonphosphorylated form (65 kDa), was no significant change (P=0.99).
5、TNFαdoes not affect the expression of occludin mRNA
TNFαtreatment does not cause a significant decrease in occludin mRNA production compared with controls considering the difference of the concentration and time of TNFαtreatment (P=0.85, P=0.99).
6、TNFαdoes not affect the expression and localization of Par-3 protein
In the control Caco-2 monolayers, Par-3 proteins were localized at the apical cellular borders, consistent with the location of occludin protein. TNFα(100μg/L) didn't cause a significant change in the Par-3 protein localization. Western blot and RT-PCR analysis demonstrated that TNFαtreatment had on effect on the expression of Par-3 protein and mRNA (P=0.99, P=0.86).
7、TNFαinhibits PKCλ/ζactivity
TNFα100μg/L caused a progressive decrease in PKCλ/ζactivity over 8h period (P=0.014). The maxiam drop in PKCλ/ζactivity occurred at 24h (P=0.001).
8、Par-3 immunoprecipitated with Tiaml
Immunoprecipitates with anti-Par-3 antibody showed specific protein belt by immunoblotted with anti-Tiaml antibody.
9、TNFαincreases the expression of Tiaml protein
Tiaml proteins were localized at the cytoplasm in the control Caco-2 cells. TNFα can cause a migration of Tiaml protein from cytoplasm to cellular borders. Furthrmore, Western blot and RT-PCR assays demonstrated that TNFαincresed the expression of Tiaml protein and mRNA at 24-h time point (P=0.029, P=0.011).
10、TNFαstimulates Rac1 activity
TNFαhad no significant effect on the expression of total Rac1 (P=0.99), whereas the active Rac1 (GTP-Rac1) decreased at 24-h time point after TNFαtreatment (P=0.002).
11、TNFαdecreases action occludin protein expression through a Tiaml-dependent manner
TNFαdown regulated the phosphorylated form of occludin protein expression in Caco-2 cells. Moreover, absence of Tiaml rescued the impaired action occludin protein expression induced by TNFα(P=0.001).
Conclusion
1、TNFαdisrupts the interepithelial tight junction and increases Caco-2 intestinal epithelial permeability.
2、TNFαcauses a disturbance in the continuity of occludin localization at the cellular borders and produces a decrease in action occludin protein expression.
3、TNFαtreatment does not cause a significant decrease in occludin mRNA production.
4、TNFαdoes not affect the expression and localization of Par-3 protein, but inhibits the activity of PKCλ/ξ.
5、Tiaml-Pac1 is the upstream factor of Par3-aPKC-Par6 complex.
6、TNFαcauses an increase in Tiaml protein and mRNA expression, and stimulates Rac1 constitutively activated.
7、TNFαdownregulates action occludin protein expression via a Tiaml-dependent process.
引文
1 Bjarnason I, MacPherson A, Hollander D. Intestinal permeability. An overview.Gastroenterology. 1995; 108:1566-1581.
2 Pinsky MR, Vincent JL, Deviere J, et al. Serum cytokine levels in human septic shock. Relation to multiple-system organ failure and mortality. Chest.l993;103:565-575.
3 Grotz MR, Deitch EA, Ding J, et al. Intestinal cytokine response after gut ischemia: role of gut barrier failure. Ann Surg. 1999;229:478-486.
4 Schmitz H, Fromm M, Bentzel CJ, et al. Tumor necrosis factor-alpha (TNFα) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci.l999;112: 137-146.
5 Gitter AH, Bendfeldt K, Schmitz H, et al. Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha. Ann NY Acad Sci. 2000;915: 193-203.
6 Ma TY, Boivin M, Ye D, et al. Mechanism of TNF-(?) modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol. 2005;288: G422-G430.
7 Ma TY, Iwamoto G, Hoa NT, et al. TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-κB activation. Am J Physiol Gastrointest Liver Physiol.2004;286: G367-G376.
8 Suenaert P, Bulteel V, Lemmen L, et al. Anti- tumor necrosis factor treatment restores the gut barrier in Crohn's disease. Am J Gastroenterol.2002;97(8): 2000-2004.
9 Rutgeerts P, Feagan BG, Lichtenstein GR, et al. Comparison of scheduled and episodic treatment strategies of infliximab in Crohn's disease. Gastroenterology. 2004;126(2):402-13
10 Bruewer M, Luegering A, Kucharzik T, et al. Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms. The Journal of Immunology. 2003;171(11): 6164-6172.
11 Ye D, Ma I, Ma TY. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol. 2006 ;290(3):G496-504.
12 Wang F, Graham WV, Wang Y, et al. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol. 2005; 166:409-419.
13 Patrick DM, Leone AK, Shellenberger JJ, et al. Proinfiammatory cytokines tumor necrosis factor-a and interferon-γ modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling. BMC Physiol. 2006; 6: 2.
14 Clark EC, Patel SD, Chadwick PR, et al. Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate. Gut.2003;52:224-230.
15 Taylor CT, Dzus AL, Colgan SP. Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha. Gastroenterology. 1998; 114:657-668.
16 Fish SM, Proujansky R, Reenstra WW. Synergistic effects of interferon (?) and tumour necrosis factor α on T84 cell function. Gut. 1999;45:191.
17 Wang Q, Guo XL, Noel G, et al. Heat shock stress ameliorates cytokine mixture-induced permeability by downregulating the nitric oxide and signal transducer and activator of transcription pathways in Caco-2 cells. Shock. 2007;27(2):179-185.
18 Madara JL. Regulation of the movement of solutes across tight junctions. Annu Rev Physiol.1998;(60pp): 143-159.
19 Van Itallie C, Rahner C, Anderson JM. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest. 2001; 107:1319-1327.
20 Claude P. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J Membr Biol. 1978;39: 219-232.
21 Furuse M, Fujita K, Hiiragi T, et al. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol.1998;141(7):1539-1550.
22 Furuse M, Hirase T, Itoh M, et al. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol. 1993;123(6 Pt 2):1777-1788.
23 Morita K, Itoh M, Saitou M, et al. Subcellular distribution of tight junction-associated proteins (occludin, ZO-1, ZO-2) in rodent skin. J Invest Dermatol. 1998; 110(6):862-866.
24 Haskins J, Gu L, Wittchen ES, et al. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol. 1998;141(l):199-208.
25 Huber JD, Witt KA, Horn S, et al. Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol. 2001;280(3):H1241-248.
26 Luabeya MK, Dallasta LM, Achim CL, et al. Blood-brain barrier disruption in simian immunodeficiency virus encephalitis. Neuropathol Appl Neurobiol. 2000;26(5):454-462.
27 Papadopoulos MC, Saadoun S, Davies DC, et al. Emerging molecular mechanisms of brain tumour oedema. Br J Neurosurg. 2001;15 (2): 101-108.
28 Vorbrodt AW. Molecular anatomy of intercellular junctions in brain endothelial and epithelial barrier. Brain Research Rev. 2003;42: 221-242.
29 Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol. 1999;147: 891-903.
30 Ilja Vietor, Thomas Bader, Karin Paiha, et al. Perturbation of the tight junction permeability barrier by occludin loop peptides activates β-catenin/TCF/LEF-mediated transcription. Huber EMBO Reports. 2001; 4: 306-312.
31 Hirase T, Staddon JM, Saitou M, et al. Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci. 1997; 110: 1603-1613.
32 Saitou M, Ando-Akatsuka Y, Itoh M, et al. Mammalian occludin in epithelial cells: its expression and subcellular distribution. Eur J Cell Biol. 1997;73: 222-231.
33 Fujimoto K. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes. J Cell Sci. 1995; 108: 3443-3449.
34 Furuse M, Itoh M, Hirase T, et al. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol. 1994;127: 1617-1626.
35 McCarthy KM, Skare IB, Stankewich MC, et al. Occludin is a functional component of the tight junction. J Cell Sci. 1996;109: 2287-2298.
36 Balda MS, Whitney JA, Flores C, et al. Functional dissociation of paracellular permeability from electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant membrane protein of tight junctions. J Cell Biol. 1996;134: 1031-1049.
37 Chen YH, Merzdorf C, Paul DL, et al. COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos. J Cell Biol. 1997;138.
38 Saitou M, Fujimoto K, Doi Y, et al. Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol. 1998;141: 397-408.
39 Han X, Fink MP, Delude RL. Proinflammatory cytokines cause NO*-dependent and-independent changes in expression and localization of tight junction proteins in intestinal epithelial cells. Shock. 2003;19(3):229-237.
40 Sappington PL, Han X, Yang R, et al. Ethyl pyruvate ameliorates intestinal epithelial barrier dysfunction in endotoxemic mice and immunostimulated caco-2 enterocytic monolayers. J Pharmacol Exp Then 2003;304(l):464-476.
41 Tsukamoto T, Nigam SK. Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins. Am J Physiol Renal Physiol. 1999;276: F737-F750.
42 Chen YH, Lu Q, Goodenough DA, et al. Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell. 2002;13:1227-1237.
43 Sakakibara A, Furuse M, Saitou M, et al. Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol. 1997;137: 1393-1401.
44 Poritz LS, Garver KI, Tilberg AF, et al. Tumor necrosis factor alpha disrupts tight junction assembly. J Surg Res. 2004;116(1):14-18.
45 Quesnell RR, Erickson J, Schultz BD. Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium. Am J Physiol Cell Physiol. 2007 ;292(l):C305-318.
46 Joachim Mankertz, Jorg Stefan Waller, Bernd Hillenbrand, et al. Gene expression of the tight junction protein occludin includes differential splicing and alternative promoter usage. Biochemical and Biophysical Research Communications. 2002; 298(5): 657-666.
47 Mankertz J, Tavalali S, Schmitz H, et al. Expression from the human occludin promoter is affected by tumor necrosis factor alpha and interferon gamma. J Cell Sci. 2000;113 ( Pt 11):2085-2090.
48 Yamanaka T, Horikoshi Y, Suzuki A, et al. PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. Genes Cells.2001; 6: 721-731.
49 Matter K, Balda MS. Signalling to and from tight junctions. Nat Mol Cell Biol. 2003;4:225-236.
50 Balda MS, Matter K. Epithelial cell adhesion and the regulation of gene expression. Trends Cell Biol. 2003; 13: 310-318.
51 Roh MH, Margolis B. Composition and function of PDZ protein complexes during cell polarization. Am J Physiol Renal Physiol. 2003;285: F377-F387.
52 Medina E, Lemmers C, Lane-Guermonprez L, et al. Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells. Biol Cell.2002;94:305-313.
53 Tepass U, Tanentzapf G, Ward R, et al. Epithelial cell polarity and cell junctions in Drosophila. Annu Rev Genet. 2001;35: 747-784.
54 Knust E ,Bossinger O. Composition and formation of intercellular junctions in epithelial cells. Science. 2002; 298: 1955-1959.
55 Hirose T, Izumi Y, Nagashima Y, et al. Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation . J Cell Sci. 2002; 115: 2485-2495.
56 Suzuki A, Ishiyama C, Hashiba K, et al. aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization. J Cell Sci.2002;115: 3565-3573.
57 Ren XD, Schwartz MA. Determination of GTP loading on Rho. Methods Enzymol.2000;325:264-272.
58 Schneeberger EE, Lynch RD. The tight junction: a multifunctional complex. Am J Physiol Cell Physiol, 2004;286: C1213-C1228.
59 Bilder D. Cell polarity: squaring the circle. Curr Biol. 2001; 11 :R132 -R135.
60 Ohno S. Intercellular junctions and cellular polarity: the PAR-aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Cur Opin Cell Biol. 2001; 13:641 -648.
61 Macara IG. Par proteins: partners in polarization. Curr Biol. 2004; 14:R160-R162.
62 Macara IG. Parsing the polarity code. Nat Rev Mol Cell Biol. 2004; 5:220-231.
63 Itoh M, Sasaki H, Furuse M, et al. Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J Cell Biol. 2001;154:491-197.
64 Ebnet K, Suzuki A, Meyer zu Brickwedde MK, et al. The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). EMBO J. 2001;14: 3738-3748.
65 Chen X, IG Macara. Par-3 controls tight junction assembly through the Rac exchange factor Tiaml. Nat Cell Biol. 2005; 7:262-269.
66 Ritchie E, Saka M, Mackenzie C, et al. Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells. Br J Pharmacol. 2007; 150(8):1044-1054.
67 Abe K, Aslam A, Walls AF, et al. Up-regulation of protease-activated receptor-2 by bFGF in cultured human synovial fibroblasts. Life Sci. 2006;79(9):898-904.
68 Suzuki A, Yamanaka T, Hirose T, et al. Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J Cell Biol. 2001; 152:1183 -1196.
69 Joberty G, Petersen C, Gao L, et al. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat Cell Biol. 2000; 2: 531-539.
70 Nagai-Tamai Y, Mizuno K, Hirose T, et al. Regulated protein-protein interaction between aPKC and PAR-3 plays an essential role in the polarization of epithelial cells. Genes Cells. 2002; 7:1161-1171.
71 Rojas R, Ruiz WG, Leung SM, et al. Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells. Mol Biol Cell. 2001;12:2257-2274.
72 Lin D, Edwards AS, Fawcett JP, et al. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Racl and aPKC signalling and cell polarity. Nat Cell Biol. 2000; 2: 540-547.
73 Nunbhakdi-Craig V, Machleidt T, Ogris E, et al. Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol. 2002;158: 967-978.
74 Lozano E, Betson M, Braga VM. Tumor progression: small GTPases and loss of cell-cell adhesion. Bioessays. 2003;25:452-463.
75 Nishimura T, Yamaguchi T, Kato K, et al. PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiaml. Nat Cell Biol. 2005; 7:270-277.
76 Mertens AE, Rygiel TP, Olivo C, et al. The Rac activator Tiaml controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex. J Cell Biol. 2005; 170(7):1029-1037.
77 Hurd TW, Margolis B. Pars and polarity: taking control of Rac. Nat Cell Biol, 2005; 7:205-207.
78 Wiggin GR, Fawcett JP, Pawson T. Polarity proteins in axon specification and synaptogenesis. Dev Cell. 2005;8: 803-816.
79 Sander EE, van Delft S, ten Klooster JP, et al. Matrix-dependent Tiaml/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J Cell Biol. 1998; 143:1385-1398.
1 Furuse M, Fujita K, Hiiragi T.Claudin-1 and claudin-2:novel integral membrane protein localizing a tight junction with no sequence similarity to occluding. J Cell Biol. 1998; 141(7):1539-1550.
2 Furuse M, Sasaki H, Tsukito S. Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol. 1999; 147(4):891-903.
3 Morita K, et al. Molecular architecture of tight junctions of periderm differs from that of the maculae occludentes of epidermis. J Invest Dermatol. 2002; 118(6):1073-9.
4 ZHANG B-y, YAO G-y. Research progress of tight junction prote in s-claudins. Journal of International Pathology and ClinicalMedicine. 2006; 26(1): 3.
5 Martin-Padura I, et al. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol. 1998; 142(1): 117-27.
6 Nusrat A, et al. Tight junctions are membrane microdomains. J Cell Sci. 2000; 113 ( Pt 10):1771-81.
7 Mark KS, Davis TP. Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol. 2002; 282(4): H1485-94.
8 Cruzalegui, FH, Bading H. Calcium-regulated protein kinase cascades and their transcription factor targets. Cell Mol Life Sci. 2000; 57(3): 402-10.
9 Duffy HS, et al. Reciprocal regulation of the junctional proteins claudin-1 and connexin43 by interleukin-1 beta in primary human fetal astrocytes. J Neurosci. 2000; 20(23): RC114.
10 Gitter AH, et al. Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha. Ann N Y Acad Sci. 2000; 915: 193-203.
11 McKay DM, Singh PK. Superantigen activation of immune cells evokes epithelial (T84) transport and barrier abnormalities via IFN-gamma and TNF alpha: inhibition of increased permeability, but not diminished secretory responses by TGF-beta2. J Immunol. 1997; 159(5):2382-90.
12 Mullin JM, Snock KV. Effect of tumor necrosis factor on epithelial tight junctions and transepithelial permeability. Cancer Res. 1990; 50(7): 2172-6.
13 Rodriguez P, et al. Tumour necrosis factor-alpha induces morphological and functional alterations of intestinal HT29 cl.19A cell monolayers. Cytokine. 1995; 7(5): 441-8.
14 Ma TY, et al. TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation. Am J Physiol Gastrointest Liver Physiol. 2004; 286(3):G367-76.
15 Schmitz H, et al. Tumor necrosis factor-alpha (TNFalpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci. 1999; 112 (Pt 1): 137-46.
16 Ma TY, et al. Mechanism of TNF-{alpha} modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol. 2005; 288(3): G422-30.
17 Wang F, et al. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol. 2005; 166(2): 409-19.
18 Hanada S, et al. Tumor necrosis factor-alpha and interferon-gamma directly impair epithelial barrier function in cultured mouse cholangiocytes. Liver Int. 2003; 23(1): 3-11.
19 Matsushita K, et al. Intramolecular interaction of SUR2 subtypes for intracellular ADP-Induced differential control of K(ATP) channels. Circ Res. 2002; 90(5): 554-61.
20 Izumi Y, et al. An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol. 1998; 143(1): 95-106.
21 Ohno S. Intercellular junctions and cellular polarity: the PAR-aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Curr Opin Cell Biol. 2001; 13(5): 641-8.
22 Nelson WJ. Adaptation of core mechanisms to generate cell polarity. Nature. 2003; 422(6933):766-74.
23 Macara IG. Parsing the polarity code. Nat Rev Mol Cell Biol. 2004; 5(3): 220-31.
24 Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol. 2001; 2(4): 285-93.
25 Matter K, Balda MS. Signalling to and from tight junctions. Nat Rev Mol Cell Biol. 2003; 4(3):225-36.
26 Knust E, Bossinge O. Composition and formation of intercellular junctions in epithelial cells. Science. 2002; 298(5600): 1955-9.
27 Medina E, et al. Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells. Biol Cell. 2002; 94(6): 305-13.
28 Tepass U, et al. Epithelial cell polarity and cell junctions in Drosophila. Annu Rev Genet. 2001;35: 747-84.
29 Macara IG. Par proteins: partners in polarization. Curr Biol. 2004; 14(4): R160-2.
30 Rose LS, Kemphues KJ. Early patterning of the C. elegans embryo. Annu Rev Genet. 1998; 32:521-45.
31 Suzuki A, et al. Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J Cell Biol. 2001; 152(6): 1183-96.
32 Wodarz A, et al. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts. J Cell Biol. 2000; 150(6): 1361-74.
33 Mizuno K, et al. Self-association of PAR-3-mediated by the conserved N-terminal domain contributes to the development of epithelial tight junctions. J Biol Chem. 2003; 278(33):31240-50.
34 Joberty G, et al. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat Cell Biol. 2000; 2(8): 531-9.
35 Lin D, et al. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Racl and aPKC signalling and cell polarity. Nat Cell Biol. 2000; 2(8): 540-7.
36 Ebnet K, et al. The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). Embo J. 2001; 20(14): 3738-48.
37 Itoh M, et al. Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J Cell Biol. 2001; 154(3): 491-7.
38 Chen X, Macara IG. Par-3 controls tight junction assembly through the Rac exchange factor Tiam1. Nat Cell Biol. 2005; 7(3): 262-9.
39 Suzuki A, et al. aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization. J Cell Sci. 2002; 115(Pt 18):3565-73.
40 Yamanaka T, et al. PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. Genes Cells. 2001; 6(8): 721-31.
41 Eckert JJ, et al. PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo. Reproduction. 2004; 127(6): 653-67.
42 Gao L, Joberty G, Macara IG. Assembly of epithelial tight junctions is negatively regulated by Par6. Curr Biol. 2002; 12(3): 221-5.
43 Nagai-Tamai Y, et al. Regulated protein-protein interaction between aPKC and PAR-3 plays an essential role in the polarization of epithelial cells. Genes Cells. 2002; 7(11): 1161-71.
44 Rojas R, et al. Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells. Mol Biol Cell. 2001; 12(8): 2257-74.
45 Hirose T, et al. Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation. J Cell Sci. 2002;l 15(Pt 12): 2485-95.
46 Qiu RG, Abo A, Steven Martin G. A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation. Curr Biol. 2000; 10(12):697-707.
47 Johansson A, Driessens M, Aspenstrom P. The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Racl. J Cell Sci. 2000; 113 (Pt 18): 3267-75.
48 Noda Y, et al. Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C. Genes Cells. 2001; 6(2): 107-19.
49 Roh MH, Margolis B. Composition and function of PDZ protein complexes during cell polarization. Am J Physiol Renal Physiol. 2003; 285(3): F377-87.
50 Bachmann A, et al. Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature. 2001; 414(6864): 638-43.
51 Hong Y, et al. Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. Nature. 2001; 414(6864): 634-8.
52 Roh MH, et al. The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J Cell Biol. 2002; 157(1): 161-72.
53 Tepass U, Theres C, Knust E. crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell. 1990;61(5): 787-99.
54 Izaddoost S, et al. Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. Nature. 2002; 416(6877): 178-83.
55 Pellikka M, et al. Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis. Nature. 2002; 416(6877): 143-9.
56 Nam SC, Choi KW. Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila. Development. 2003; 130(18): 4363-72.
57 Makarova O, et al. Mammalian Crumbs3 is a small transmembrane protein linked to protein associated with Lin-7 (Palsl). Gene. 2003; 302(1-2): 21-9.
58 Roh MH, et al. The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. J Cell Sci. 2003; 116(Pt 14): 2895-906.
59 Anderson JM. Cell signalling: MAGUK magic. Curr Biol. 1996; 6(4): 382-4.
60 Kamberov E, et al. Molecular cloning and characterization of Pals, proteins associated with mLin-7. J Biol Chem. 2000; 275(15): 11425-31.
61 Straight SW, et al. Loss of PALS1 expression leads to tight junction and polarity defects. Mol Biol Cell. 2004; 15(4): 1981-90.
62 Lemmers C, et al. hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells. J Biol Chem. 2002; 277(28): 25408-15.
63 Roh MH, et al. The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions. J Biol Chem. 2002; 277(30): 27501-9.
64 Shin K, Straight S, Margolis B. PATJ regulates tight junction formation and polarity in mammalian epithelial cells. J Cell Biol. 2005; 168(5): 705-11.
65 Michel D, et al. PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells. J Cell Sci. 2005; 118(Pt 17): 4049-57.
66 Hurd TW, et al. Direct interaction of two polarity complexes implicated in epithelial tight junction assembly. Nat Cell Biol. 2003; 5(2): 137-42.
2 Pinsky MR, Vincent JL, Deviere J, et al. Serum cytokine levels in human septic shock. Relation to multiple-system organ failure and mortality. Chest.l993;103:565-575.
3 Grotz MR, Deitch EA, Ding J, et al. Intestinal cytokine response after gut ischemia: role of gut barrier failure. Ann Surg. 1999;229:478-486.
4 Schmitz H, Fromm M, Bentzel CJ, et al. Tumor necrosis factor-alpha (TNFα) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci.l999;112: 137-146.
5 Gitter AH, Bendfeldt K, Schmitz H, et al. Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha. Ann NY Acad Sci. 2000;915: 193-203.
6 Ma TY, Boivin M, Ye D, et al. Mechanism of TNF-(?) modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol. 2005;288: G422-G430.
7 Ma TY, Iwamoto G, Hoa NT, et al. TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-κB activation. Am J Physiol Gastrointest Liver Physiol.2004;286: G367-G376.
8 Suenaert P, Bulteel V, Lemmen L, et al. Anti- tumor necrosis factor treatment restores the gut barrier in Crohn's disease. Am J Gastroenterol.2002;97(8): 2000-2004.
9 Rutgeerts P, Feagan BG, Lichtenstein GR, et al. Comparison of scheduled and episodic treatment strategies of infliximab in Crohn's disease. Gastroenterology. 2004;126(2):402-13
10 Bruewer M, Luegering A, Kucharzik T, et al. Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms. The Journal of Immunology. 2003;171(11): 6164-6172.
11 Ye D, Ma I, Ma TY. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol. 2006 ;290(3):G496-504.
12 Wang F, Graham WV, Wang Y, et al. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol. 2005; 166:409-419.
13 Patrick DM, Leone AK, Shellenberger JJ, et al. Proinfiammatory cytokines tumor necrosis factor-a and interferon-γ modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling. BMC Physiol. 2006; 6: 2.
14 Clark EC, Patel SD, Chadwick PR, et al. Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate. Gut.2003;52:224-230.
15 Taylor CT, Dzus AL, Colgan SP. Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha. Gastroenterology. 1998; 114:657-668.
16 Fish SM, Proujansky R, Reenstra WW. Synergistic effects of interferon (?) and tumour necrosis factor α on T84 cell function. Gut. 1999;45:191.
17 Wang Q, Guo XL, Noel G, et al. Heat shock stress ameliorates cytokine mixture-induced permeability by downregulating the nitric oxide and signal transducer and activator of transcription pathways in Caco-2 cells. Shock. 2007;27(2):179-185.
18 Madara JL. Regulation of the movement of solutes across tight junctions. Annu Rev Physiol.1998;(60pp): 143-159.
19 Van Itallie C, Rahner C, Anderson JM. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest. 2001; 107:1319-1327.
20 Claude P. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J Membr Biol. 1978;39: 219-232.
21 Furuse M, Fujita K, Hiiragi T, et al. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol.1998;141(7):1539-1550.
22 Furuse M, Hirase T, Itoh M, et al. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol. 1993;123(6 Pt 2):1777-1788.
23 Morita K, Itoh M, Saitou M, et al. Subcellular distribution of tight junction-associated proteins (occludin, ZO-1, ZO-2) in rodent skin. J Invest Dermatol. 1998; 110(6):862-866.
24 Haskins J, Gu L, Wittchen ES, et al. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol. 1998;141(l):199-208.
25 Huber JD, Witt KA, Horn S, et al. Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol. 2001;280(3):H1241-248.
26 Luabeya MK, Dallasta LM, Achim CL, et al. Blood-brain barrier disruption in simian immunodeficiency virus encephalitis. Neuropathol Appl Neurobiol. 2000;26(5):454-462.
27 Papadopoulos MC, Saadoun S, Davies DC, et al. Emerging molecular mechanisms of brain tumour oedema. Br J Neurosurg. 2001;15 (2): 101-108.
28 Vorbrodt AW. Molecular anatomy of intercellular junctions in brain endothelial and epithelial barrier. Brain Research Rev. 2003;42: 221-242.
29 Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol. 1999;147: 891-903.
30 Ilja Vietor, Thomas Bader, Karin Paiha, et al. Perturbation of the tight junction permeability barrier by occludin loop peptides activates β-catenin/TCF/LEF-mediated transcription. Huber EMBO Reports. 2001; 4: 306-312.
31 Hirase T, Staddon JM, Saitou M, et al. Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci. 1997; 110: 1603-1613.
32 Saitou M, Ando-Akatsuka Y, Itoh M, et al. Mammalian occludin in epithelial cells: its expression and subcellular distribution. Eur J Cell Biol. 1997;73: 222-231.
33 Fujimoto K. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes. J Cell Sci. 1995; 108: 3443-3449.
34 Furuse M, Itoh M, Hirase T, et al. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol. 1994;127: 1617-1626.
35 McCarthy KM, Skare IB, Stankewich MC, et al. Occludin is a functional component of the tight junction. J Cell Sci. 1996;109: 2287-2298.
36 Balda MS, Whitney JA, Flores C, et al. Functional dissociation of paracellular permeability from electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant membrane protein of tight junctions. J Cell Biol. 1996;134: 1031-1049.
37 Chen YH, Merzdorf C, Paul DL, et al. COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos. J Cell Biol. 1997;138.
38 Saitou M, Fujimoto K, Doi Y, et al. Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol. 1998;141: 397-408.
39 Han X, Fink MP, Delude RL. Proinflammatory cytokines cause NO*-dependent and-independent changes in expression and localization of tight junction proteins in intestinal epithelial cells. Shock. 2003;19(3):229-237.
40 Sappington PL, Han X, Yang R, et al. Ethyl pyruvate ameliorates intestinal epithelial barrier dysfunction in endotoxemic mice and immunostimulated caco-2 enterocytic monolayers. J Pharmacol Exp Then 2003;304(l):464-476.
41 Tsukamoto T, Nigam SK. Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins. Am J Physiol Renal Physiol. 1999;276: F737-F750.
42 Chen YH, Lu Q, Goodenough DA, et al. Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell. 2002;13:1227-1237.
43 Sakakibara A, Furuse M, Saitou M, et al. Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol. 1997;137: 1393-1401.
44 Poritz LS, Garver KI, Tilberg AF, et al. Tumor necrosis factor alpha disrupts tight junction assembly. J Surg Res. 2004;116(1):14-18.
45 Quesnell RR, Erickson J, Schultz BD. Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium. Am J Physiol Cell Physiol. 2007 ;292(l):C305-318.
46 Joachim Mankertz, Jorg Stefan Waller, Bernd Hillenbrand, et al. Gene expression of the tight junction protein occludin includes differential splicing and alternative promoter usage. Biochemical and Biophysical Research Communications. 2002; 298(5): 657-666.
47 Mankertz J, Tavalali S, Schmitz H, et al. Expression from the human occludin promoter is affected by tumor necrosis factor alpha and interferon gamma. J Cell Sci. 2000;113 ( Pt 11):2085-2090.
48 Yamanaka T, Horikoshi Y, Suzuki A, et al. PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. Genes Cells.2001; 6: 721-731.
49 Matter K, Balda MS. Signalling to and from tight junctions. Nat Mol Cell Biol. 2003;4:225-236.
50 Balda MS, Matter K. Epithelial cell adhesion and the regulation of gene expression. Trends Cell Biol. 2003; 13: 310-318.
51 Roh MH, Margolis B. Composition and function of PDZ protein complexes during cell polarization. Am J Physiol Renal Physiol. 2003;285: F377-F387.
52 Medina E, Lemmers C, Lane-Guermonprez L, et al. Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells. Biol Cell.2002;94:305-313.
53 Tepass U, Tanentzapf G, Ward R, et al. Epithelial cell polarity and cell junctions in Drosophila. Annu Rev Genet. 2001;35: 747-784.
54 Knust E ,Bossinger O. Composition and formation of intercellular junctions in epithelial cells. Science. 2002; 298: 1955-1959.
55 Hirose T, Izumi Y, Nagashima Y, et al. Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation . J Cell Sci. 2002; 115: 2485-2495.
56 Suzuki A, Ishiyama C, Hashiba K, et al. aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization. J Cell Sci.2002;115: 3565-3573.
57 Ren XD, Schwartz MA. Determination of GTP loading on Rho. Methods Enzymol.2000;325:264-272.
58 Schneeberger EE, Lynch RD. The tight junction: a multifunctional complex. Am J Physiol Cell Physiol, 2004;286: C1213-C1228.
59 Bilder D. Cell polarity: squaring the circle. Curr Biol. 2001; 11 :R132 -R135.
60 Ohno S. Intercellular junctions and cellular polarity: the PAR-aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Cur Opin Cell Biol. 2001; 13:641 -648.
61 Macara IG. Par proteins: partners in polarization. Curr Biol. 2004; 14:R160-R162.
62 Macara IG. Parsing the polarity code. Nat Rev Mol Cell Biol. 2004; 5:220-231.
63 Itoh M, Sasaki H, Furuse M, et al. Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J Cell Biol. 2001;154:491-197.
64 Ebnet K, Suzuki A, Meyer zu Brickwedde MK, et al. The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). EMBO J. 2001;14: 3738-3748.
65 Chen X, IG Macara. Par-3 controls tight junction assembly through the Rac exchange factor Tiaml. Nat Cell Biol. 2005; 7:262-269.
66 Ritchie E, Saka M, Mackenzie C, et al. Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells. Br J Pharmacol. 2007; 150(8):1044-1054.
67 Abe K, Aslam A, Walls AF, et al. Up-regulation of protease-activated receptor-2 by bFGF in cultured human synovial fibroblasts. Life Sci. 2006;79(9):898-904.
68 Suzuki A, Yamanaka T, Hirose T, et al. Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J Cell Biol. 2001; 152:1183 -1196.
69 Joberty G, Petersen C, Gao L, et al. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat Cell Biol. 2000; 2: 531-539.
70 Nagai-Tamai Y, Mizuno K, Hirose T, et al. Regulated protein-protein interaction between aPKC and PAR-3 plays an essential role in the polarization of epithelial cells. Genes Cells. 2002; 7:1161-1171.
71 Rojas R, Ruiz WG, Leung SM, et al. Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells. Mol Biol Cell. 2001;12:2257-2274.
72 Lin D, Edwards AS, Fawcett JP, et al. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Racl and aPKC signalling and cell polarity. Nat Cell Biol. 2000; 2: 540-547.
73 Nunbhakdi-Craig V, Machleidt T, Ogris E, et al. Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol. 2002;158: 967-978.
74 Lozano E, Betson M, Braga VM. Tumor progression: small GTPases and loss of cell-cell adhesion. Bioessays. 2003;25:452-463.
75 Nishimura T, Yamaguchi T, Kato K, et al. PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiaml. Nat Cell Biol. 2005; 7:270-277.
76 Mertens AE, Rygiel TP, Olivo C, et al. The Rac activator Tiaml controls tight junction biogenesis in keratinocytes through binding to and activation of the Par polarity complex. J Cell Biol. 2005; 170(7):1029-1037.
77 Hurd TW, Margolis B. Pars and polarity: taking control of Rac. Nat Cell Biol, 2005; 7:205-207.
78 Wiggin GR, Fawcett JP, Pawson T. Polarity proteins in axon specification and synaptogenesis. Dev Cell. 2005;8: 803-816.
79 Sander EE, van Delft S, ten Klooster JP, et al. Matrix-dependent Tiaml/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J Cell Biol. 1998; 143:1385-1398.
1 Furuse M, Fujita K, Hiiragi T.Claudin-1 and claudin-2:novel integral membrane protein localizing a tight junction with no sequence similarity to occluding. J Cell Biol. 1998; 141(7):1539-1550.
2 Furuse M, Sasaki H, Tsukito S. Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol. 1999; 147(4):891-903.
3 Morita K, et al. Molecular architecture of tight junctions of periderm differs from that of the maculae occludentes of epidermis. J Invest Dermatol. 2002; 118(6):1073-9.
4 ZHANG B-y, YAO G-y. Research progress of tight junction prote in s-claudins. Journal of International Pathology and ClinicalMedicine. 2006; 26(1): 3.
5 Martin-Padura I, et al. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol. 1998; 142(1): 117-27.
6 Nusrat A, et al. Tight junctions are membrane microdomains. J Cell Sci. 2000; 113 ( Pt 10):1771-81.
7 Mark KS, Davis TP. Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol. 2002; 282(4): H1485-94.
8 Cruzalegui, FH, Bading H. Calcium-regulated protein kinase cascades and their transcription factor targets. Cell Mol Life Sci. 2000; 57(3): 402-10.
9 Duffy HS, et al. Reciprocal regulation of the junctional proteins claudin-1 and connexin43 by interleukin-1 beta in primary human fetal astrocytes. J Neurosci. 2000; 20(23): RC114.
10 Gitter AH, et al. Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha. Ann N Y Acad Sci. 2000; 915: 193-203.
11 McKay DM, Singh PK. Superantigen activation of immune cells evokes epithelial (T84) transport and barrier abnormalities via IFN-gamma and TNF alpha: inhibition of increased permeability, but not diminished secretory responses by TGF-beta2. J Immunol. 1997; 159(5):2382-90.
12 Mullin JM, Snock KV. Effect of tumor necrosis factor on epithelial tight junctions and transepithelial permeability. Cancer Res. 1990; 50(7): 2172-6.
13 Rodriguez P, et al. Tumour necrosis factor-alpha induces morphological and functional alterations of intestinal HT29 cl.19A cell monolayers. Cytokine. 1995; 7(5): 441-8.
14 Ma TY, et al. TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation. Am J Physiol Gastrointest Liver Physiol. 2004; 286(3):G367-76.
15 Schmitz H, et al. Tumor necrosis factor-alpha (TNFalpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci. 1999; 112 (Pt 1): 137-46.
16 Ma TY, et al. Mechanism of TNF-{alpha} modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol. 2005; 288(3): G422-30.
17 Wang F, et al. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol. 2005; 166(2): 409-19.
18 Hanada S, et al. Tumor necrosis factor-alpha and interferon-gamma directly impair epithelial barrier function in cultured mouse cholangiocytes. Liver Int. 2003; 23(1): 3-11.
19 Matsushita K, et al. Intramolecular interaction of SUR2 subtypes for intracellular ADP-Induced differential control of K(ATP) channels. Circ Res. 2002; 90(5): 554-61.
20 Izumi Y, et al. An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol. 1998; 143(1): 95-106.
21 Ohno S. Intercellular junctions and cellular polarity: the PAR-aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Curr Opin Cell Biol. 2001; 13(5): 641-8.
22 Nelson WJ. Adaptation of core mechanisms to generate cell polarity. Nature. 2003; 422(6933):766-74.
23 Macara IG. Parsing the polarity code. Nat Rev Mol Cell Biol. 2004; 5(3): 220-31.
24 Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol. 2001; 2(4): 285-93.
25 Matter K, Balda MS. Signalling to and from tight junctions. Nat Rev Mol Cell Biol. 2003; 4(3):225-36.
26 Knust E, Bossinge O. Composition and formation of intercellular junctions in epithelial cells. Science. 2002; 298(5600): 1955-9.
27 Medina E, et al. Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells. Biol Cell. 2002; 94(6): 305-13.
28 Tepass U, et al. Epithelial cell polarity and cell junctions in Drosophila. Annu Rev Genet. 2001;35: 747-84.
29 Macara IG. Par proteins: partners in polarization. Curr Biol. 2004; 14(4): R160-2.
30 Rose LS, Kemphues KJ. Early patterning of the C. elegans embryo. Annu Rev Genet. 1998; 32:521-45.
31 Suzuki A, et al. Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J Cell Biol. 2001; 152(6): 1183-96.
32 Wodarz A, et al. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts. J Cell Biol. 2000; 150(6): 1361-74.
33 Mizuno K, et al. Self-association of PAR-3-mediated by the conserved N-terminal domain contributes to the development of epithelial tight junctions. J Biol Chem. 2003; 278(33):31240-50.
34 Joberty G, et al. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat Cell Biol. 2000; 2(8): 531-9.
35 Lin D, et al. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Racl and aPKC signalling and cell polarity. Nat Cell Biol. 2000; 2(8): 540-7.
36 Ebnet K, et al. The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). Embo J. 2001; 20(14): 3738-48.
37 Itoh M, et al. Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J Cell Biol. 2001; 154(3): 491-7.
38 Chen X, Macara IG. Par-3 controls tight junction assembly through the Rac exchange factor Tiam1. Nat Cell Biol. 2005; 7(3): 262-9.
39 Suzuki A, et al. aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization. J Cell Sci. 2002; 115(Pt 18):3565-73.
40 Yamanaka T, et al. PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. Genes Cells. 2001; 6(8): 721-31.
41 Eckert JJ, et al. PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo. Reproduction. 2004; 127(6): 653-67.
42 Gao L, Joberty G, Macara IG. Assembly of epithelial tight junctions is negatively regulated by Par6. Curr Biol. 2002; 12(3): 221-5.
43 Nagai-Tamai Y, et al. Regulated protein-protein interaction between aPKC and PAR-3 plays an essential role in the polarization of epithelial cells. Genes Cells. 2002; 7(11): 1161-71.
44 Rojas R, et al. Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells. Mol Biol Cell. 2001; 12(8): 2257-74.
45 Hirose T, et al. Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation. J Cell Sci. 2002;l 15(Pt 12): 2485-95.
46 Qiu RG, Abo A, Steven Martin G. A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation. Curr Biol. 2000; 10(12):697-707.
47 Johansson A, Driessens M, Aspenstrom P. The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Racl. J Cell Sci. 2000; 113 (Pt 18): 3267-75.
48 Noda Y, et al. Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C. Genes Cells. 2001; 6(2): 107-19.
49 Roh MH, Margolis B. Composition and function of PDZ protein complexes during cell polarization. Am J Physiol Renal Physiol. 2003; 285(3): F377-87.
50 Bachmann A, et al. Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature. 2001; 414(6864): 638-43.
51 Hong Y, et al. Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. Nature. 2001; 414(6864): 634-8.
52 Roh MH, et al. The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J Cell Biol. 2002; 157(1): 161-72.
53 Tepass U, Theres C, Knust E. crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell. 1990;61(5): 787-99.
54 Izaddoost S, et al. Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. Nature. 2002; 416(6877): 178-83.
55 Pellikka M, et al. Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis. Nature. 2002; 416(6877): 143-9.
56 Nam SC, Choi KW. Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila. Development. 2003; 130(18): 4363-72.
57 Makarova O, et al. Mammalian Crumbs3 is a small transmembrane protein linked to protein associated with Lin-7 (Palsl). Gene. 2003; 302(1-2): 21-9.
58 Roh MH, et al. The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. J Cell Sci. 2003; 116(Pt 14): 2895-906.
59 Anderson JM. Cell signalling: MAGUK magic. Curr Biol. 1996; 6(4): 382-4.
60 Kamberov E, et al. Molecular cloning and characterization of Pals, proteins associated with mLin-7. J Biol Chem. 2000; 275(15): 11425-31.
61 Straight SW, et al. Loss of PALS1 expression leads to tight junction and polarity defects. Mol Biol Cell. 2004; 15(4): 1981-90.
62 Lemmers C, et al. hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells. J Biol Chem. 2002; 277(28): 25408-15.
63 Roh MH, et al. The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions. J Biol Chem. 2002; 277(30): 27501-9.
64 Shin K, Straight S, Margolis B. PATJ regulates tight junction formation and polarity in mammalian epithelial cells. J Cell Biol. 2005; 168(5): 705-11.
65 Michel D, et al. PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells. J Cell Sci. 2005; 118(Pt 17): 4049-57.
66 Hurd TW, et al. Direct interaction of two polarity complexes implicated in epithelial tight junction assembly. Nat Cell Biol. 2003; 5(2): 137-42.