Evidence for involvement of ROCK signaling in bradykinin-induced increase in murine blood–tumor barrier permeability
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We have previously shown that activation of RhoA by bradykinin (BK) is associated with cytoskeleton rearrangement, tight junction (TJ) protein disassembly, and an increase in blood–tumor barrier (BTB) permeability in rat brain microvascular endothelial cells (RBMECs). Subsequently, we investigated whether Rho-kinases (ROCKs), a family of downstream effectors of activated RhoA known to stimulate F-actin rearrangement, play a key role in the above-mentioned processes in RBMECs. Our study uses primary RBMECs as an in vitro BTB model and a specific ROCK inhibitor (Y-27632) and ROCK II small interfering RNA (siRNA) to establish whether ROCK plays a role in the process of TJ opening by BK. Y-27632 and ROCK II siRNA could partially inhibit endothelial leakage and restored normal transendothelial electric resistance (TEER) values in RBMECs. A shift in occludin and claudin-5 distribution from insoluble to soluble fractions was prevented by Y-27632. Additionally, Y-27632 inhibited BK-induced relocation of occludin and claudin-5 from cellular borders into the cytoplasm as well as stress fiber formation in RBMECs. A time-dependent increase in phosphorylated myosin light chain (p-MLC) and phosphorylated cofilin (p-cofilin) by BK was observed, which was also inhibited by Y-27632. An increase in ROCK activity by BK was inhibited by Y-27632. ROCK’s contribution to BK-induced stress fiber formation is associated with TJ disassembly and an increase in BTB permeability.
KeywordsROCK Bradykinin Blood–tumor barrier Tight junction Actin rearrangement Myosin light chain Cofilin
The authors are grateful to Lisa M. Abernathy, PhD pre-candidate (Department of Immunology and Microbiology, Wayne State University School of Medicine) and Mr. Arjun Dupati (College of Human Medicine, MSII Michigan State University) for critical reading of the manuscript. Contract grant sponsor: This work was supported by the Natural Science Foundation of China, under contract nos. 30800451, 30872656, 30973079, 30670723, 81001029, and 30570650, the special fund for Scientific Research of Doctor-degree Subjects in Colleges and Universities no. 20092104110015, and Scientific and Technological Planning Projects of Shenyang nos. F10-205-1-22, F10-205-1-37, and 1081266-9-00.
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