| ObjectiveGlioma 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.Methods1. 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.Results1. 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.DiscussionIt 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). Conclusions1. 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.
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