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Evidence for involvement of ROCK signaling in bradykinin-induced increase in murine blood–tumor barrier permeability

  • Laboratory Investigation - Human/Animal Tissue
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Abstract

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.

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References

  1. Black KL, Ningaraj NS (2004) Modulation of brain tumor capillaries for enhanced drug delivery selectively to brain tumor. Cancer Control 11:165–173

    PubMed  Google Scholar 

  2. Samoto K, Perng GC, Ehtesham M, Liu Y, Wechsler SL, Nesburn AB, Black KL, Yu JS (2001) A herpes simplex virus type 1 mutant deleted for gamma 34.5 and LAT kills glioma cells in vitro and is inhibited for in vivo reactivation. Cancer Gene Therapy 8:269–277

    Article  PubMed  CAS  Google Scholar 

  3. Fenstermacher JK, Cowles AS (1977) Theoretic limitations of intracarotid infusions in brain tumor chemotherapy. Cancer Treat Rep 61:519–526

    PubMed  CAS  Google Scholar 

  4. Muldoon LL, Soussain C, Jahnke K, Johanson C, Siegal T, Smith QR, Hall WA, Hynynen K, Senter PD, Peereboom DM, Neuwelt EA (2007) Chemotherapy delivery issues in central nervous system malignancy: a reality check. J Clin Oncol 25:2295–2305

    Article  PubMed  CAS  Google Scholar 

  5. Lockman PR, Mittapalli RK, Taskar KS, Rudraraju V, Gril B, Bohn KA, Adkins CE, Roberts A, Thorsheim HR, Gaasch JA, Huang S, Palmieri D, Steeg PS, Smith QR (2010) Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer. Clin Cancer Res 16:5664–5678

    Article  PubMed  CAS  Google Scholar 

  6. Liu LB, Xue YX, Liu YH, Wang YB (2008) Bradykinin increases blood–tumor barrier permeability by down-regulating the expression levels of ZO-1, occludin, and claudin-5 and rearranging actin cytoskeleton. J Neurosci Res 86:1153–1168

    Article  PubMed  CAS  Google Scholar 

  7. Ma T, Xue Y (2010) RhoA-mediated potential regulation of blood-tumor barrier permeability by bradykinin. J Mol Neurosci 42:67–73

    Article  PubMed  Google Scholar 

  8. Yin J, Yu FS (2008) Rho kinases regulate corneal epithelial wound healing. Am J Physiol Cell Physiol 295:C378–C387

    Article  PubMed  CAS  Google Scholar 

  9. Walsh SV, Hopkins AM, Chen J, Narumiya S, Parkos CA, Nusrat A (2001) Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia. Gastroenterology 121:566–579

    Article  PubMed  CAS  Google Scholar 

  10. Li B, Zhao WD, Tan ZM, Fang WG, Zhu L, Chen YH (2006) Involvement of Rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells. FEBS Lett 580:4252–4260

    Article  PubMed  CAS  Google Scholar 

  11. Calaminus SD, Auger JM, McCarty OJ, Wakelam MJ, Machesky LM, Watson SP (2007) MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability. J Thromb Haemost 5:2136–2145

    Article  PubMed  CAS  Google Scholar 

  12. Wojciak-Stothard B, Ridley AJ (2002) Rho GTPases and the regulation of endothelial permeability. Vascul Pharmacol 39:187–199

    Article  PubMed  CAS  Google Scholar 

  13. Essler M, Staddon JM, Weber PC, Aepfelbacher M (2000) Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling. J Immunol 164:6543–6549

    PubMed  CAS  Google Scholar 

  14. van Nieuw Amerongen GP, van Delft S, Vermeer MA, Collard JG, van Hinsbergh VW (2000) Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases. Circ Res 87:335–340

    PubMed  Google Scholar 

  15. Aepfelbacher M, Essler M (2001) Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators: a role for Rho/Rho kinase. Cell Microbiol 3:649–658

    Article  PubMed  CAS  Google Scholar 

  16. Hirase T, Kawashima S, Wong EY, Ueyama T, Rikitake Y, Tsukita S, Yokoyama M, Staddon JM (2001) Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms. J Biol Chem 276:10423–10431

    Article  PubMed  CAS  Google Scholar 

  17. Wojciak-Stothard B, Potempa S, Eichholtz T, Ridley AJ (2001) Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci 114:1343–1355

    PubMed  CAS  Google Scholar 

  18. Hurst RD, Fritz IB (1996) Properties of an immortalised vascular endothelial/glioma cell co-culture model of the blood-brain barrier. J Cell Physiol 167:81–88

    Article  PubMed  CAS  Google Scholar 

  19. Easton AS, Abbott NJ (2002) Brandykinin increases permeability by calcium and 5-lipoxygenase in the ECV304/C6 cell culture model of the blood-brain barrier. Brain Res 953:157–169

    Article  PubMed  CAS  Google Scholar 

  20. Wong D, Dorovini-Zis K, Vincent SR (2004) Cytokines, nitric oxide, and cGMP modulate the permeability of an in vitro model of the human blood-brain barrier. Exp Neurol 190:446–455

    Article  PubMed  CAS  Google Scholar 

  21. Fuller E, Duckham C, Wood E (2007) Disruption of epithelial tight junctions by yeast enhances the paracellular delivery of a model protein. Pharm Res 24:37–47

    Article  PubMed  CAS  Google Scholar 

  22. Nunes KP, Rigsby CS, Webb RC (2010) RhoA/Rho-kinase and vascular diseases: what is the link? Cell Mol Life Sci 67:3823–3836

    Article  PubMed  CAS  Google Scholar 

  23. Liao JK, Seto M, Noma K (2007) Rho kinase (ROCK) inhibitors. J Cardiovasc Pharmacol 50:17–24

    Article  PubMed  CAS  Google Scholar 

  24. Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105

    Article  PubMed  CAS  Google Scholar 

  25. Narumiya ST, Ishizaki M, Uehata (2000) Use and properties of ROCKspecific inhibitor Y-27632. Methods Enzymol 325:273–284

    Article  PubMed  CAS  Google Scholar 

  26. Ridley AJ (2001) Rho family proteins: coordinating cell responses. Trends Cell Biol 11:471–477

    Article  PubMed  CAS  Google Scholar 

  27. Nakagawa O, Fujisawa K, Ishizaki T, Saito Y, Nakao K, Narumiya S (1996) ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice. FEBS Lett 392:189–193

    Article  PubMed  CAS  Google Scholar 

  28. Di Cunto F, Imarisio S, Hirsch E, Broccoli V, Bulfone A, Migheli A, Atzori C, Turco E, Triolo R, Dotto GP, Silengo L, Altruda F (2000) Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis. Neuron 28:115–127

    Article  PubMed  CAS  Google Scholar 

  29. Wei L, Roberts W, Wang L, Yamada M, Zhang S, Zhao Z, Rivkees SA, Schwartz RJ, Imanaka-Yoshida K (2001) Rho kinases play an obligatory role in vertebrate embryonic organogenesis. Development 128:2953–2962

    PubMed  CAS  Google Scholar 

  30. Liu PY, Liao JK (2008) A method for measuring Rho kinase activity in tissues and cells. Methods Enzymol 39:181–189

    Article  Google Scholar 

  31. Rubenstein NM, Callahan JA, Lo DH, Firestone GL (2007) Selective glucocorticoid control of Rho kinase isoforms regulate cell–cell interactions. Biochem Biophys Res Commun 354:603–607

    Article  PubMed  CAS  Google Scholar 

  32. Mong PY, Wang Q (2009) Activation of Rho kinase isoforms in lung endothelial cells during inflammation. J Immunol 182:2385–2394

    Article  PubMed  CAS  Google Scholar 

  33. Harhaj NS, Antonetti DA (2004) Regulation of tight junctions and loss of barrier function in pathophysiology. Int J Biochem Cell Biol 36:1206–1237

    Article  PubMed  CAS  Google Scholar 

  34. Collares-Buzato CB, Jepson MA, Simmons NL, Hirst BH (1998) Increased tyrosine phosphorylation causes redistribution of adherens junction and tight junction proteins and perturbs paracellular barrier function in MDCK epithelia. Eur J Cell Biol 76:85–92

    PubMed  CAS  Google Scholar 

  35. Keita AV, Söderholm JD (2010) The intestinal barrier and its regulation by neuroimmune factors. Neurogastroenterol Motil 22:718–733

    Article  PubMed  CAS  Google Scholar 

  36. Kubota K, Furuse M, Sasaki H, Sonoda N, Fujita K, Nagafuchi A, Tsukita S (1999) Ca(2 +)-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions. Curr Biol 9:1035–1038

    Article  PubMed  CAS  Google Scholar 

  37. Mitic LL, Van Itallie CM, Anderson JM (2000) Molecular physiology and pathophysiology of tight junctions I. Tight junction structure and function: lessons from mutant animals and proteins. Am J Physiol Gastrointest Liver Physiol 279:G250–G254

    PubMed  CAS  Google Scholar 

  38. Olivera D, Knall C, Boggs S, Seagrave J (2010) Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins are associated with mainstream cigarette smoke-induced permeability of airway epithelium. Exp Toxicol Pathol 62:133–143

    Article  PubMed  CAS  Google Scholar 

  39. Olivera DS, Boggs SE, Beenhouwer C, Aden J, Knall C (2007) Cellular mechanisms of mainstream cigarette smoke-induced lung epithelial tight junction permeability changes in vitro. Inhal Toxicol 19:13–22

    Article  PubMed  CAS  Google Scholar 

  40. Chen SH, Stins MF, Huang SH, Chen YH, Kwon-Chung KJ, Chang Y, Kim KS, Suzuki K, Jong AY (2003) Cryptococcus deformans induces alterations in the cytoskeleton of human brain microvascular endothelial cells. J Med Microbiol 52:961–970

    Article  PubMed  CAS  Google Scholar 

  41. Tenenbaum T, Matalon D, Adam R, Seibt A, Wewer C, Schwerk C, Galla HJ, Schroten H (2008) Dexamethasone prevents alteration of tight junction-associated proteins and barrier function in porcine choroid plexus epithelial cells after infection with Streptococcus suis in vitro. Brain Res 1229:1–17

    Article  PubMed  CAS  Google Scholar 

  42. Li Q, Zhang Q, Wang C, Liu X, Qu L, Gu L, Li N, Li J (2009) Altered distribution of tight junction proteins after intestinal ischaemia/reperfusion injury in rats. J Cell Mol Med 13:4061–4076

    Article  PubMed  Google Scholar 

  43. Nighot PK, Moeser AJ, Ryan KA, Ghashghaei T, Blikslager AT (2009) ClC-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum. Exp Cell Res 315:110–118

    Article  PubMed  CAS  Google Scholar 

  44. Dudek SM, Garcia JG (2001) Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 91:1487–1500

    PubMed  CAS  Google Scholar 

  45. Birukova AA, Smurova K, Birukovm KG, Kaibuchi K, Garcia JG, Verin AD (2004) Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction. Microvasc Res 67:64–77

    Article  PubMed  CAS  Google Scholar 

  46. Breslin JW, Yuan SY (2004) Involvement of RhoA and Rho kinase in neutrophil-stimulated endothelial hyperpermeability. Am J Physiol Heart Circ Physiol 286:H1057–H1062

    Article  PubMed  CAS  Google Scholar 

  47. Müller SL, Portwich M, Schmidt A, Utepbergenov DI, Huber O, Blasig IE, Krause G (2005) The tight junction protein occludin and the adherens junction protein alpha-catenin share a common interaction mechanism with ZO-1. J Biol Chem 280:3747–3756

    Article  PubMed  Google Scholar 

  48. Hopkins AM, Walsh SV, Verkade P, Boquet P, Nusrat A (2003) Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function. J Cell Sci 116:725–742

    Article  PubMed  CAS  Google Scholar 

  49. Niwa R, Nagata-Ohashi K, Takeichi M, Mizuno K, Uemura T (2002) Control of actin reorganization by slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108:233–246

    Article  PubMed  CAS  Google Scholar 

  50. Brown M, Adyshev D, Bindokas V, Moitra J, Garcia JG, Dudek SM (2010) Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium. Microvasc Res 80:75–88

    Article  PubMed  CAS  Google Scholar 

  51. Ikebe M, Hartshorne DJ (1985) Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. J Biol Chem 260:10027–10031

    PubMed  CAS  Google Scholar 

  52. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K (1996) Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273:245–248

    Article  PubMed  CAS  Google Scholar 

  53. Moyer RA, Wendt MK, Johanesen PA, Turner JR, Dwinell MB (2007) Rho activation regulates CXCL12 chemokine stimulated actin rearrangement and restitution in model intestinal epithelia. Lab Invest 87:807–817

    Article  PubMed  CAS  Google Scholar 

  54. Lui WY, Lee WM, Cheng CY (2003) Sertoli-germ cell adherens junction dynamics in the testis are regulated by RhoB GTPase via the ROCK/LIMK signaling pathway. Biol Reprod 68:2189–2206

    Article  PubMed  CAS  Google Scholar 

  55. Bamburg JR (1999) Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu Rev Cell Dev Biol 15:185–230

    Article  PubMed  CAS  Google Scholar 

  56. Nagumo Y, Han J, Bellila A, Isoda H, Tanaka T (2008) Cofilin mediates tight-junction opening by redistributing actin and tight-junction proteins. Biochem Biophys Res Commun 377:921–925

    Article  PubMed  CAS  Google Scholar 

  57. Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K (1996) Phosphorylation and activation of myosin by Rho-associated kinase(Rho-kinase). J Biol Chem 271:20246–20249

    Article  PubMed  CAS  Google Scholar 

  58. Easton AS, Abbott NJ (1997) The effects of bradykinin on a cell culture model of the blood-brain barrier. J Physiol 505:49–50

    Google Scholar 

  59. Manning TJ Jr, Parker JC, Sontheimer H (2000) Role of lysophosphatidic acid and rho in glioma cell motility. Cell Motil Cytoskeleton 5:185–199

    Article  Google Scholar 

  60. Liu C, Zuo J, Janssen LJ (2006) Regulation of airway smooth muscle RhoA/ROCK activities by cholinergic and bronchodilator stimuli. Eur Respir J 28:703–711

    Article  PubMed  Google Scholar 

  61. Arita R, Hata Y, Nakao S, Kita T, Miura M, Kawahara S, Zandi S, Almulki L, Tayyari F, Shimokawa H, Hafezi-Moghadam A, Ishibashi T (2009) Rho kinase inhibition by fasudil ameliorates diabetes-induced microvascular damage. Diabetes 58:215–226

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

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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  1. Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang, 110001, Liaoning, People’s Republic of China

    Teng Ma, Libo Liu, Ping Wang & Yixue Xue

  2. Institute of Pathology and Pathophysiology, China Medical University, Shenyang, People’s Republic of China

    Libo Liu

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  1. Teng Ma
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Correspondence to Yixue Xue.

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Ma, T., Liu, L., Wang, P. et al. Evidence for involvement of ROCK signaling in bradykinin-induced increase in murine blood–tumor barrier permeability. J Neurooncol 106, 291–301 (2012). https://doi.org/10.1007/s11060-011-0685-3

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