摘要
目的
神经胶质瘤是一种最常见的中枢神经系统恶性肿瘤,恶性脑肿瘤患者的术后化疗是目前治疗肿瘤的主要手段。由于正常脑组织存在血脑屏障(blood-brain barrier, BBB),而肿瘤组织也存在着血肿瘤屏障(blood-tumor barrier, BTB),从而限制了抗肿瘤药物进入肿瘤组织。因此有效地增加肿瘤组织中抗肿瘤药物的浓度,是提高化疗药物疗效的关键。我们前期研究发现缓激肽(bradykinin, BK)能够选择性开放BTB,也对相关机制进行了初探,发现BK开放BTB过程中出现PKA下调,细胞骨架重排,神经型一氧化氮合酶升高等,但BK的作用是通过何种信号通路发挥作用以及深入的分子机制如何,尚未见报道。
RhoA/ROCK信号通路参与许多血管活性物质介导的内皮细胞通透性升高的过程。BK是通过与其2型受体(B2)作用开放BTB。B2受体属于G蛋白偶联性受体,BK、溶血磷脂酸和凝血酶是G蛋白偶联受体激动剂,可以使COS-7和成纤维细胞的G蛋白偶联性受体激活,进而通过RhoA信号通路介导丝状肌动蛋白(filamentous actin, F-actin)细胞骨架重排,引起紧密连接相关蛋白occludin、claudin-5和ZO-1重分布,紧密连接(tight junction, TJ)开放。另有研究证明RhoA/ROCK-MLC和RhoA/ROCK-LIMK-cofilin通路可以介导细胞骨架重排,开放BBB。上述信号转导通路是否参与BK开放BTB调节过程目前尚不清楚。
本研究的目的是明确BK与B2受体结合后是否能够通过RhoA/ROCK通路调节紧密连接相关蛋白重分布,进而开放BTB,以及RhoA/ROCK-MLC和RhoA/ROCK-cofilin信号通路是否参与了BK选择性开放BTB的调节过程。
方法
1、大鼠体外BTB模型的制备。
2、分别予以RhoA特异性抑制剂肉毒梭菌C3胞外酶(Clostridium botulinum C3 exoenzyme)和Rho激酶(Rho associated kinase, ROCK)特异性抑制剂Y-27632预处理体外BTB模型。
3、应用Millicell-ERS电阻测量系统测量体外BTB的跨内皮细胞阻抗值(transendothelial electric resistance, TEER)。
4、应用辣根过氧化物酶(horseradish peroxidase, HRP)实验检测体外BTB的通透性。
5、应用免疫荧光法检测BK作用后体外BTB模型上紧密连接相关蛋白occludin、claudin-5、ZO-1和细胞骨架蛋白F-actin的分布和表达水平的变化。
6、应用western blot法检测BK作用前后体外BTB模型上紧密连接相关蛋白occludn、claudin-5和ZO-1的蛋白质表达水平；检测磷酸化的肌球蛋白轻链(myosin light chain, MLC).肌动蛋白素(cofilin)和总MLC、cofilin的表达。
7、应用Pull-down方法检测RhoA活性。
结果
1、成功建立了大鼠体外BTB模型。
2、BK作用后,体外BTB模型的TEER值显著降低,HRP流量显著增加。C3 exoenzyme或Y-27632预处理使TEER值显著升高,HRP流量显著降低。
3、BK作用后,体外BTB模型上的内皮细胞紧密连接相关蛋白occludin、claudin-5和ZO-1由连续分布状态变为不连续分布状态,occludin和claudin-5由细胞膜向细胞质转移,ZO-1由内皮细胞的边缘向细胞质转移,occludin和claudin-5的可溶性片段(S)与不溶性片段(IS)IDV比值显著升高,ZO-1的蛋白表达显著降低；细胞骨架蛋白F-actin沿细胞边缘的分布减少,应力纤维增加,且分布在细胞中央区。C3 exoenzyme和Y-27632预处理后,occludin、claudin-5和ZO-1部分恢复连续分布状态,occludin和claudin-5由细胞膜向细胞质转移明显减少,ZO-1由内皮细胞的边缘向细胞质转移明显减少,occludin和claudin-5可溶性片段(S)与不溶性片段(IS)IDV比值显著降低；ZO-1的表达水平显著升高；F-actin沿细胞边缘的分布明显增多,细胞质中的分布减少,应力纤维明显减少。
4、BK作用后,体外BTB模型上的内皮细胞的RhoA活性显著升高。BK作用5 min时开始增加,10 min时达峰值,之后降低。C3 exoenzyme预处理使RhoA活性显著下降。
5、BK作用后,体外BTB模型上的内皮细胞的p-MLC和p-cofilin表达显著升高,BK作用5 min时开始增加,10 min时达峰值,之后降低。Y-27632预处理使p-MLC和p-cofilin表达显著下降。
讨论
我们的研究首次证明了在BK选择性增加BTB通透性的过程中,RhoA/ROCK信号通路对BK介导的细胞骨架重排,紧密连接相关蛋白occludin、claudin-5和ZO-1重分布和表达水平的变化起到了重要的调节作用。MLC和cofilin作为RhoA/ROCK通路的下游信号分子也参与了上述调节过程。
HRP是一种示踪剂,通过透过BTB的HRP流量来检测体外BTB通透性的变化；跨内皮细胞阻抗实验也是一种检测体外BTB通透性的实验。我们的HRP流量实验和跨内皮细胞阻抗实验的结果显示,BK作用后TEER值显著降低,HRP流量显著增加,与Easton和Abbott报道的研究结果相一致。本研究进一步发现应用RhoA特异性抑制剂C3 exoenzyme和ROCK特异性抑制剂Y-27632预处理后,BTB的通透性显著升高,证明RhoA/ROCK信号通路参与调节BK介导的BTB通透性增加的过程。
紧密连接是维持BBB完整性重要的结构基础。Occludin和claudin-5是构成紧密连接的主要跨膜蛋白,是维持紧密连接结构和功能重要组成部分。当occludin和claudin-5蛋白的可溶性片段(S)与不溶性片段(IS)IDV比值升高时,说明紧密连接结构变化,通透性增加。我们通过TritonX-100抽提技术和western blot技术同样证实了BK作用下occludin和claudin-5的可溶性片段(S)与不溶性片段(IS)IDV比值显著升高,说明BK作用下,紧密连接结构发生了变化,进而导致通透性增加。此结果与Nighot和Tenenbaum研究一致。本研究还发现C3 exoenzyme和Y-27632预处理后,显著抑制了occludin和claudin-5的可溶性片段(S)与不溶性片段(IS) IDV比值升高,提示RhoA/ROCK信号通路参与BK介导的TJ开放。
胞质附着蛋白Zona occludens家族中的ZO-1同样是构成TJ的一个主要蛋白,ZO-1作为连接跨膜蛋白occludin和claudin-5和细胞骨架蛋白F-actin的桥梁蛋白,ZO-1的分布和表达的变化同样影响TJ的结构和功能。我们的研究发现BK作用后,ZO-1表达显著下降,C3 exoenzyme和Y-27632预处理后,显著上调ZO-1表达,说明RhoA/ROCK信号通路参与BK介导的TJ开放。
为了进一步明确RhoA/ROCK信号通路与细胞骨架重排,紧密连接相关蛋白重分布,BTB通透性增高的关系,我们应用C3 exoenzyme和Y-27632预处理后,采用免疫荧光技术观察细胞骨架重排,紧密连接相关蛋白重分布的变化,明确RhoA/ROCK信号通路与细胞骨架重排,紧密连接相关蛋白重分布的关系。本研究发现BK作用后,occludin、claudin-5和ZO-1重分布,occludin、claudin-5由细胞膜向细胞质转移,ZO-1由细胞边缘向细胞质转移,同时F-actin由环状连续地分布于大鼠脑微血管内皮细胞(Rat brain microvascular endothelail cells, RBMECs)的边缘向细胞质中发生分布转移,同时伴有大量应力纤维形成。应力纤维收缩,牵拉ZO-1蛋白,使细胞膜上跨膜蛋白occludin和claudin-5重分布,紧密连接结构变化,使BTB通透性升高。C3 exoenzyme和Y-27632抑制了occludin、claudin-5和ZO-1的重分布、F-actin的重排和应力纤维的形成。说明RhoA／ROCK信号通路参与BK介导的细胞骨架重排,紧密连接相关蛋白重分布,BTB通透性增高的过程。
一些研究证明RhoA活性升高可以使紧密连接通透性升高。我们的结果证实了BK介导BTB的开放过程中RhoA活性升高,说明了RhoA/ROCK信号通路参与BK介导BTB的开放,但是RhoA活性升高的峰值先于紧密连接开放, BTB通透性增加的峰值,其可能的原因是信号分子从上游传导到下游需要一定的时间,或者还有其它信号分子介导BK开放BTB。
MLC和cofilin是ROCK重要的下游信号分子,ROCK通过直接磷酸化MLC或通过磷酸化肌球蛋白磷酸酶的肌球蛋白结合亚单位起作用,调节MLC的磷酸化,在调控细胞的动态改变中起着重要作用。ROCK还可以激活LIM激酶(LIM Kinase, LIMK), LIMK磷酸化cofilin, cofilin是肌动蛋白丝解聚所必需的,磷酸化后肌动蛋白脱聚合化活性受抑制,增加了应力纤维的稳定性。磷酸化的MLC和cofilin是介导细胞骨架重排的重要信号分子。我们的研究发现BK作用后,(?)-MLC与MLCIDV比值及p-cofilin与cofilin IDV比值呈时间依赖式显著升高,BK作用10 min时达峰值,以后逐渐下降。这个峰值先于细胞骨架重排,紧密连接开放,BTB通透性增加的峰值,而与RhoA活性的峰值一致,说明MLC和cofilin是F-actin、ZO-1、occludin和claduin-5上游信号分子。Y-27632预处理后,抑制p-MLC和p-cofilin表达,说明ROCK-MLC和ROCK-cofilin参与BK介导BTB开放。Chen SH证实新型隐球菌通过ROCK-LIMK-cofilin通路穿过BBB,导致脑膜脑炎；Li B同样证实小细胞肺癌细胞通过RhoA/ROCK-MLC和RhoA/ROCK-cofilin通路迁移穿过BBB,导致小细胞肺癌脑转移。
上述结果证明,RhoA/ROCK-MLC和RhoA/ROCK-cofilin信号通路对BK介导的细胞骨架重排,紧密连接相关蛋白occludin、claudin-5和ZO-1重分布和表达水平的变化起到了重要的调节作用。
结论
1、RhoA/ROCK信号途径参与BK选择性开放BTB的调节过程。
2、MLC参与BK选择性开放BTB的调节过程,且为RhoA/ROCK下游信号因子。
3、Cofilin参与BK选择性开放BTB的调节过程,且为RhoA/ROCK下游信号因子。
Objective
Glioma is one of the most common types of malignant tumors in the central nervous system, and post-operative chemotherapy is currently the main method to treat malignant brain tumors. Because of the existence of blood-brain barrier (BBB) in normal brain tissue and the blood-tumor barrier (BTB) in tumor brain tissue respectively, the delivering of anti-cancer drugs to the tumor tissues is limited. Thus the key point to improve therapeutic effect is to increase the concentration of anti-cancer drugs in tumor tissue efficiently. Our previous studies had found that bradykinin (BK) could open the BTB selectively, and the correlative mechanism was studied to find that the down-regulation of PKA, the rearrangement of F-actin and the increase of the nitricoxide synthase (NOS) were involved in the process of BK opening the BTB. But the concreted signaling pathway and the molecular mechanism about BK-induced the opening of BTB are not completely understood.
RhoA/ROCK pathway participates in the increase process of endothelial permeability induced by many vasoactive-related substances. BK opens the BTB by activating BK type-2 receptor (BK-2R). BK-2R belongs to the G-protein coupled receptor (GPCR). BK, lysophosphatidicacid and thrombin are the agonists of GPCR, which can activate GPCR to rearrange the filamentous actin (F-actin), and to redistribute the tight junction (TJ) associated proteins (occludin, claudin-5 and ZO-1), and to open TJ in fibroblasts and COS7 cells by RhoA pathway. In addition, the RhoA/ROCK-MLC and RhoA/ROCK-LIMK-cofilin pathways were identified to induce the rearrangement of cystoskeleton and open the BBB. It is unclear whether the above-mentioned pathways participate in the opening of BTB by BK.
This study is performed to determine whether the binding of BK and BK-2R can redistribute TJ associated proteins and open the BTB by RhoA/ROCK pathway, and whether RhoA/ROCK-MLC and RhoA/ROCK-cofilin pathways are involved in the process of BK opening the BTB selectively.
Methods
1. Establishment of the rat BTB model in vitro.
2. The BTB model was pretreated by RhoA specific inhibitor clostridium botulinum C3 exoenzyme and Rho associated kinase (ROCK) specific inhibitor Y-27632, respectively.
3. Millipore electrical resistance system (Millicell-ERS) was used to detect the transendothelial electric resistance (TEER) of BTB model in vitro.
4. Horseradish peroxidase (HRP) assay was used to measure the permeability of BTB model in vitro.
5. Before and after BK administering, immunofluorescence assay was used to determine the distribution of TJ associated proteins occludin, claudin-5, ZO-1 and cytoskeleton protein F-actin in BTB model in vitro.
6. Before and after BK administering, western blot assay was used to detect the proteins expression levels of TJ associated proteins (occludin, claudin-5 and ZO-1) in BTB model in vitro and the levels of p-MLC, p-cofilin and total MLC and cofilin in BTB model in vitro.
7. Pull-down assay was used to detect the activity of RhoA.
Results
1. The BTB model in vitro was established successfully.
2. TEER decreased and HRP flux increased significantly in BTB in vitro after BK infusion. After the pretreatment of C3 exoenzyme and Y-27632, the TEER increased and HRP flux decreased significantly.
3. In BTB model in vitro, after administrating BK, the TJ associated proteins (occludin, claudin-5 and ZO-1) changed from continuously distribution to discontinuously distribution. Occludin and claudin-5 relocated from cellular membrane into the cytoplasm. ZO-1 relocated from cellular borders into the cytoplasm. The IDV ratio of soluble fraction (S) and insoluble fraction (IS) of occludin and claudin-5 increased significantly and the protein expression level of ZO-1 decreased significantly. The F-actin on cellular boundaries decreased and the formation of stress fiber increased. After the pretreatment of C3 exoenzyme and Y-27632, occludin, claudin-5 and ZO-1 restored continuously distribution partly, and the relocation of occludin and claudin-5 from cellular membrane into the cytoplasm decreased. The IDV ratio of soluble fraction (S) and insoluble fraction (IS) of occludin and claudin-5 was decreased significantly. ZO-1 expression in RMBEC was increased significantly. The F-actin on cellular boundaries increased, and the F-actin in cytoplasm decreased, and the formation of stress fiber decreased.
4. In BTB model in vitro, after administrating BK, the activity of RhoA increased significantly. The activity increased after 5 min of BK administering, reached the peak at 10 min, and then decreased gradually. After the pretreatment of C3 exoenzyme, the activity of RhoA was decreased significantly.
5. In BTB model in vitro, after administrating BK, the protein expression levels of p-MLC and p-cofilin increased significantly after 5 min of BK administering, reached the peak at 10 min, then decreased gradually. After the pretreatment of Y-27632, the protein expression levels of p-MLC and p-cofilin were decreased significantly.
Discussion
It is the first time to demonstrate that RhoA/ROCK signal pathway was necessary and important for BK-induced rearrangement of actin cytoskeleton, and redistribution and expression of TJ-associated protein (occludin, claudin-5 and ZO-1) in the process of BK increasing the BTB permeability selectively. MLC and cofilin were involved in the above regulating process as the downstream signaling molecule of RhoA/ROCK pathway.
HRP is a kind of tracer, the change of the permeability of the BTB in vitro can be determined by detecting the flux of HRP permeating the BTB. TEER assay is also a common kind of experiments to measure the permeability of the BTB. Our results showed that BK decreased TEER and increased HRP flux significantly, which coincided well with Easton and Abbott's findings. Here we also found for the first time that inhibition of RhoA by C3 exoenzyme and inhibition of ROCK by Y-27632 prevented BK-induced increase in BTB permeability significantly; and RhoA/ROCK was required for BK-induced increase in BTB permeability.
TJ is an important structure to maintain the integrality of BBB. Occludin and claudin-5 are the major transmembrane protein to construct the TJ, are the important ingredients to maintain the structure and function of TJ. The increase of IDV ratio of soluble fraction (S) and insoluble fraction (IS) of occludin and claudin-5 suggested that TJ disassembled and the permeability increased. We confirmed the above viewpoint similarly through the TritonX-100 extraction technology and western blot assay. Our results coincided well with the study of Nighot and the Tenenbaum. In addition, we found that, after the pretreatment of C3 exoenzyme and Y-27632, an increase of IDV ratio of soluble fraction (S) and insoluble fraction (IS) of occludin and claudin-5 was inhibited significantly. This result suggested that RhoA/ROCK pathway was involved in BK-induced open of TJ.
ZO-1 belongs to Zona occludens family, is an important protein for TJ assemble. It acts as a scaffolding protein to organize the transmembrane proteins (such as occludin and claudin-5) and F-actin, which distribution and expression can impact the structure and function of TJ. Our study found that the protein expression of ZO-1 was decreased after BK administering, and the pretreatment of C3 exoenzyme and Y-27632 could up-regulated the expression of ZO-1 significantly. This result suggested that RhoA/ROCK pathway was involved in BK-induced open of TJ.
In order to clarify further the relation among the RhoA/ROCK pathway, cytoskeleton rearrangement, TJ associated proteins redistribution and the increase of the BTB permeability. After the pretreatment of C3 exoenzyme and Y-27632, immunofluorescence assay was used to detect the rearrangement of cytoskeleton and the redistribution of TJ associated proteins, and to identify the relationship between RhoA/ROCK pathway and them. Our studies found that occludin and claudin-5 relocated from cellular membrane into the cytoplasm, ZO-1 relocated from cellular borders into the cytoplasm, F-actin transferred from cell periphery into the cytoplasm, and formed a number of stress fibers at the same time. Stress fiber contraction produced a redistribution of trans-membrane protein from plasma lemma to cytoplasm through ZO-1, which resulted to the open of BTB. C3 exoenzyme and Y-27632 inhibited the redistribution of occludin, claudin-5 and ZO-1, and inhibited the rearrangement of F-actin and the formation of stress fiber. This result suggested that RhoA/ROCK was involved in BK-induced F-actin rearrangement, TJ associated proteins rearrangement and the increase of BTB permeability.
Recent researches implied the increase of RhoA/ROCK activity could increase the permeability of TJ. Our results suggested that RhoA/ROCK pathway was involved in the BK-induced the open of BTB. However, the peak of activity of Rho A preceded the peak of TJ open, it is possible that such a time differential represents signal transduction from upsteam to downsteam molecules. In addition, the authors believed that it was the possible role for other signal molecular mechanisms involved in these phenomena.
MLC and cofilin are two mainly downstream molecular for ROCK. ROCK can phosphorylate MLC directly, or phosphorylate MLC indirectly through phosphorylation of myosin light chain phosphorylated kinase (MLPK). MLC plays an important role to control the dynamic change of cells. ROCK also can activate LIM kinase (LIMK), which results to a phosphorylation of cofilin. Cofilin protein is necessary in the depolymerization of F-actin. The p-cofilin inhibits depolymerization of F-actin. The p-MLC and p-cofilin were two important signal molecules induced cytoskeleton rearrangement. Our study showed that the ratio of p-MLC and MLC IDV was increasing time-dependently significantly after BK administering, and the radio of p-cofilin and cofilin IDV was increasing at the same mode. The radios reached the peak after 10 min of BK administering, and then decreased gradually. This peak coincided with the activity of RhoA, and preceded the peak of cytoskeleton rearrangement, TJ open, and the increase of BTB permeability. The results demonstrated that MLC and cofilin were upsteam molecules of F-actin, ZO-1, occludin and claduin-5. Inhibition of ROCK prevented the expression of p-MLC and p-cofilin by BK. Our experimental results suggested that ROCK-MLC and ROCK-cofilin was involved in BK-induced the open of BTB. Chen SH found that Cryptococcus neoformans traversing the BBB resulted in meningocerebritis trough ROCK-LIMK-cofilin pathway. Li B also demonstrated that small cell lung cancer cells migration across BBB resulted in cancer brain metastases, which was mediated by RhoA/ROCK-MLC and RhoA/ROCK-cofilin pathway.
In summary, our present results suggested that RhoA/ROCK-MLC and RhoA/ROCK-cofilin signal pathways were important for BK-induced cytoskeleton rearrangement, and the redistribution and expression change of TJ associated proteins (occludin, claudin-5 and ZO-1).
Conclusions
1. Involvement of RhoA/ROCK signalling in BK opening BTB selectively.
2. MLC was a downstream molecule of RhoA/ROCK, and participated in BK opening BTB selectively.
3. Cofilin was a downstream molecule of RhoA/ROCK, and participated in BK opening BTB selectively.
引文
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