Skip to main content

Advertisement

SpringerLink
Log in

Role of RhoA/ROCK Signaling in Endothelial-Monocyte-Activating Polypeptide II Opening of the Blood–Tumor Barrier

Role of RhoA/ROCK Signaling in EMAP II Opening of the BTB

  • Published:
Journal of Molecular Neuroscience Aims and scope Submit manuscript

Abstract

The purpose of the present study was to determine the potential for RhoA/ROCK signaling to play a role in endothelial-monocyte-activating polypeptide (EMAP) II-induced increase in blood–tumor barrier (BTB) permeability in rat brain microvascular endothelial cells (RBMECs). In the present study, we used an in vitro BTB model, a RhoA inhibitor (C3 exoenzyme) and a ROCK inhibitor (Y27632) to determine whether RhoA/ROCK pathway play a role in the process of TJ disassembly, stress fiber formation, MLC and cofilin phosphorylation, as well as increase of BTB permeability induced by EMAP II. The results revealed that BTB permeability was increased by EMAP II induction, and C3 exoenzyme or Y27632 could partially inhibit the EMAP II-induced increase of BTB permeability. The significant down-regulations in tight junction (TJ)-associated proteins occludin, claudin-5 and ZO-1 and stress fiber formation by EMAP II administration were observed, which were partly prevented by C3 exoenzyme or Y27632 pretreatment. Moreover, the significant increases in RhoA activity, myosin light chain (MLC) and cofilin phosphorylation by EMAP II administration were observed, MLC and cofilin phosphorylation were partly inhibited by C3 exoenzyme or Y27632 pretreatment. The present study demonstrates that the activation of RhoA/ROCK signaling in RBMECs was required for the increase of BTB permeability and these effects are related with the ability for RhoA/ROCK to mediate TJ disassembly and stress fiber formation by phosphorylating cofilin and MLC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Adamson RH, Curry FE, Adamson G, Liu B, Jiang Y, Aktories K, Barth H, Daigeler A, Golenhofen N, Ness W, Drenckhahn D (2002) Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice. J Physiol 539(Pt 1):295–308

    Article  PubMed  CAS  Google Scholar 

  • 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(34):20246–20249

    Article  PubMed  CAS  Google Scholar 

  • Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y, Kaibuchi K (1997) Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 275(5304):1308–1311

    Article  PubMed  CAS  Google Scholar 

  • Berger AC, Tang G, Alexander HR, Libutti SK (2000) Endothelial monocyte-activating polypeptide II, a tumor-derived cytokine that plays an important role in inflammation, apoptosis, and angiogenesis. J Immunother 23(5):519–527

    Article  PubMed  CAS  Google Scholar 

  • Biegel D, Pachter JS (1994) Growth of brain microvessel endothelial cells on collagen gels: applications to the study of blood-brain barrier physiology and CNS inflammation. Vitro Cell Dev Biol Anim 30A(9):581–588

    Article  CAS  Google Scholar 

  • Biegel D, Spencer DD, Pachter JS (1995) Isolation and culture of human brain microvessel endothelial cells for the study of blood-brain barrier properties in vitro. Brain Res 692(1–2):183–189

    Article  PubMed  CAS  Google Scholar 

  • Black KL, Ningaraj NS (2004) Modulation of brain tumor capillaries for enhanced drug delivery selectively to brain tumor. Cancer Control 11(3):165–173

    PubMed  Google Scholar 

  • Carbajal JM, Schaeffer RC Jr (1999) RhoA inactivation enhances endothelial barrier function. Am J Physiol 277(5 Pt 1):C955–C964

    PubMed  CAS  Google Scholar 

  • Carbajal JM, Gratrix ML, Yu CH, Schaeffer RC Jr (2000) ROCK mediates thrombin’s endothelial barrier dysfunction. Am J Physiol Cell Physiol 279(1):C195–C204

    PubMed  CAS  Google Scholar 

  • Clements RT, Minnear FL, Singer HA, Keller RS, Vincent PA (2005) RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability. Am J Physiol Lung Cell Mol Physiol 288(2):L294–L306

    Article  PubMed  CAS  Google Scholar 

  • 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(1–2):157–169

    Article  PubMed  CAS  Google Scholar 

  • 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(1):37–47

    Article  PubMed  CAS  Google Scholar 

  • Gloor SM, Wachtel M, Bolliger MF, Ishihara H, Landmann R, Frei K (2001) Molecular and cellular permeability control at the blood-brain barrier. Brain Res Rev 36(2–3):258–264

    Article  PubMed  CAS  Google Scholar 

  • Hall A (1998) Rho GTPases and the actin cytoskeleton. Science 279(5350):509–514

    Article  PubMed  CAS  Google Scholar 

  • Harhaj NS, Antonetti DA (2004) Regulation of tight junctions and loss of barrier function in pathophysiology. Int J Biochem Cell Biol 36(7):1206–1237

    Article  PubMed  CAS  Google Scholar 

  • 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(13):10423–10431

    Article  PubMed  CAS  Google Scholar 

  • Hou Y, Plett PA, Ingram DA, Rajashekhar G, Orschell CM, Yoder MC, March KL, Clauss M (2006) Endothelial-monocyte-activating polypeptide II induces migration of endothelial progenitor cells via the chemokine receptor CXCR3. Exp Hematol 34(8):1125–1132

    Article  PubMed  CAS  Google Scholar 

  • Hurst RD, Fritz IB (1996) Properties of an immortalised vascular endothelial/glioma cell co-culutre model of the blood-brain barrier. J Cell Physiol 167(1):81–88

    Article  PubMed  CAS  Google Scholar 

  • Ivanov AI, McCall IC, Parkos CA, Nusrat A (2004) Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex. Mol Biol Cell 15(6):2639–2651

    Article  PubMed  CAS  Google Scholar 

  • Keezer SM, Ivie SE, Krutzsch HC, Tandle A, Libutti SK, Roberts DD (2003) Angiogenesis inhibitors target the endothelial cell cytoskeleton through altered regulation of heat shock protein 27 and cofilin. Cancer Res 63(19):6405–6412

    PubMed  CAS  Google Scholar 

  • Kemper EM, Boogerd W, Thuis I, Beijnen JH, Van Tellingen O (2004) Modulation of the blood–brain barrier in oncology: therapeutic opportunities for the treatment of brain tumours? Cancer Treat Rev 30(5):415–423

    Article  PubMed  Google Scholar 

  • Kniesel U, Wolburg H (2000) Tight junctions of the blood–brain barrier. Cell Mol Neurobiol 20(1):57–76

    Article  PubMed  CAS  Google Scholar 

  • Lai CH, Kuo KH, Leo JM (2005) Critical role of actin in modulating BBB permeability. Brain Res Brain Res Rev 50(1):7–13

    Article  PubMed  CAS  Google Scholar 

  • Lee NP, Cheng CY (2003) Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3′,5′-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study. Endocrinology 144(7):3114–3129

    Article  PubMed  CAS  Google Scholar 

  • Lee HS, Namkoong K, Kim DH, Kim KJ, Cheong YH, Kim SS, Lee WB, Kim KY (2004) Hydrogen peroxide-induced alterations of tight junction proteins in bovine brain microvascular endothelial cells. Microvasc Res 68(3):231–238

    Article  PubMed  CAS  Google Scholar 

  • Leung T, Chen XQ, Manser E, Lim L (1996) The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol 16(10):5313–5327

    PubMed  CAS  Google Scholar 

  • 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(17):4252–4260

    Article  PubMed  CAS  Google Scholar 

  • Liu Y, Chen BP, Lu M, Zhu Y, Stemerman MB, Chien S, Shyy JY (2002) Shear stress activation of SREBP1 in endothelial cells is mediated by integrins. Arterioscler Thromb Vasc Biol 22(1):76–81

    Article  PubMed  Google Scholar 

  • Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu A, Obinata T, Ohashi K, Mizuno K, Narumiya S (1999) Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285(5429):895–898

    Article  PubMed  CAS  Google Scholar 

  • Mark KS, Davis TP (2002) Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol 282(4):H1485–H1494

    PubMed  CAS  Google Scholar 

  • McKenzie AJ, Ridley AJ (2007) Roles of Rho/ROCK and MLCK in TNF-alpha-induced changes in endothelial morphology and permeability. J Cell Physiol 213(1):221–228

    Article  PubMed  CAS  Google Scholar 

  • Rubin LL, Staddon JM (1999) The cell biology of the blood–brain barrier. Annu Rev Neurosci 22:11–28

    Article  PubMed  CAS  Google Scholar 

  • Salama NN, Eddington ND, Fasano A (2006) Tight junction modulation and its relationship to drug delivery. Adv Drug Deliv Rev 58(1):15–28

    Article  PubMed  CAS  Google Scholar 

  • Scott PA, Bicknell R (1993) The isolation and culture of microvascular endothelium. J Cell Sci 105(Pt 2):269–273

    PubMed  Google Scholar 

  • Shalak V, Guigou L, Kaminska M, Wautier MP, Wautier JL, Mirande M (2007) Characterization of p43(ARF), a derivative of the p43 component of multiaminoacyl-tRNA synthetase complex released during apoptosis. J Biol Chem 282(15):10935–10943

    Article  PubMed  CAS  Google Scholar 

  • Stamatovic SM, Keep RF, Kunkel SL, Andjelkovic AV (2003) Potential role of MCP-1 in endothelial cell tight junction ‘opening’: signaling via Rho and Rho kinase. J Cell Sci 116(Pt22):4615–4628

    Article  PubMed  CAS  Google Scholar 

  • Sun H, Breslin JW, Zhu J, Yuan SY, Wu MH (2006) Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability. Microcirculation 13(3):237–247

    Article  PubMed  CAS  Google Scholar 

  • Takai Y, Sasaki T, Tanaka K, Nakanishi H (1995) Rho as a regulator of the cytoskeleton. Trends Biochem Sci 20(6):227–231

    Article  PubMed  CAS  Google Scholar 

  • Tandle AT, Mazzanti C, Alexander HR, Roberts DD, Libutti SK (2005) Endothelial monocyte activating polypeptide-II induced gene expression changes in endothelial cells. Cytokine 30(6):347–358

    Article  PubMed  CAS  Google Scholar 

  • Tiruppathi C, Minshall RD, Malik AB (2003) RhoA interaction with inositol 1,4,5-trisphosphate receptor and transient receptor potential channel-1 regulates Ca2+ entry. Role in signaling increased endothelial permeability. J Biol Chem 278(35):33492–33500

    Article  PubMed  Google Scholar 

  • Tsai BM, Wang M, Clauss M, Sun P, Meldrum DR (2004) Endothelial monocyte-activating polypeptide II causes NOS-dependent pulmonary artery vasodilation: a novel effect for a proinflammatory cytokine. Am J Physiol Regul Integr Comp Physiol 287(4):R767–R771

    Article  PubMed  CAS  Google Scholar 

  • Van Aelst L, D'Souza-Schorey C (1997) Rho GTPases and signaling networks. Genes Dev 11(18):2295–2322

    Article  PubMed  Google Scholar 

  • Wickstrom SA, Alitalo K, Keski-Oja J (2003) Endostatin associates with lipid rafts and induces reorganization of the actin cytoskeleton via down-regulation of RhoA activity. J Biol Chem 278(39):37895–37901

    Article  PubMed  Google Scholar 

  • Wojciak-Stothard B, Potempa S, Eichholtz T, Ridley AJ (2001) Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci 114(Pt 7):1343–1355

    PubMed  CAS  Google Scholar 

  • Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 38(6):323–337

    Article  PubMed  CAS  Google Scholar 

  • 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(2):446–455

    Article  PubMed  CAS  Google Scholar 

  • Xie H, Xue YX, Liu LB, Liu YH (2010) Endothelial-monocyte-activating polypeptide II increases blood-tumor barrier permeability by down-regulating the expression levels of tight junction associated proteins. Brain Res 1319:13–20

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work is supported by grants from the Natural Science Foundation of China (nos. 30670723, 30800451, 30872656, 30973079, 81001029, 81072056), the special fund for Scientific Research of Doctor-degree Subjects in Colleges and Universities, No. 20092104110015, and Shenyang Science and Technology Plan Projects (nos. F-10-205-1-22, F-10-205-1-37).

Author information

Authors and Affiliations

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

    Hui Xie, Yi-xue Xue, Li-bo Liu & Ping Wang

  2. Institute of Pathology and Pathophysiology, China Medical University, Shenyang, People’s Republic of China, 110001

    Hui Xie, Yi-xue Xue, Li-bo Liu & Ping Wang

  3. Department of Neurosurgery, Shengjing Hospital, China Medical University, Shenyang, People’s Republic of China, 110001

    Yun-hui Liu

Authors
  1. Hui Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yi-xue Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Li-bo Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yun-hui Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ping Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yi-xue Xue.

Additional information

Hui Xie and Yi-xue Xue contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xie, H., Xue, Yx., Liu, Lb. et al. Role of RhoA/ROCK Signaling in Endothelial-Monocyte-Activating Polypeptide II Opening of the Blood–Tumor Barrier. J Mol Neurosci 46, 666–676 (2012). https://doi.org/10.1007/s12031-011-9564-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12031-011-9564-9

Keywords

Search

Navigation