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Direct Rho-associated kinase inhibiton induces cofilin dephosphorylation and neurite outgrowth in PC-12 cells

Abstract

Axons fail to regenerate in the adult central nervous system (CNS) following injury. Developing strategies to promote axonal regeneration is therapeutically attractive for various CNS pathologies such as traumatic brain injury, stroke and Alzheimer’s disease. Because the RhoA pathway is involved in neurite outgrowth, Rho-associated kinases (ROCKs), downstream effectors of GTP-bound Rho, are potentially important targets for axonal repair strategies in CNS injuries. We investigated the effects and downstream mechanisms of ROCK inhibition in promoting neurite outgrowth in a PC-12 cell model. Robust neurite outgrowth (NOG) was induced by ROCK inhibitors Y-27632 and H-1152 in a time-and dose-dependent manner. Dramatic cytoskeletal reorganization was noticed upon ROCK inhibition. NOG initiated within 5 to 30 minutes followed by neurite extension between 6 and 10 hours. Neurite processes were then sustained for over 24 hours. Rapid cofilin dephosphorylation was observed within 5 minutes of Y-27632 and H-1152 treatment. Re-phosphorylation was observed by 6 hours after Y-27632 treatment, while H-1152 treatment produced sustained cofilin dephosphorylation for over 24 hours. The results suggest that ROCK-mediated dephosphorylation of cofilin plays a role in the initiation of NOG in PC-12 cells.

Abbreviations

Y-27632:

(R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide

ROCK:

Rho-associated kinase

NGF:

nerve growth factor

NOG:

neurite outgrowth

DAG:

dorsal root ganglion

MAG:

myelin associated glycoprotein

CSPG:

chondroitin sulfate proteoglycan

OMgp:

oligodendrocyte myelin

FGF:

fibroblast growth factor

BDNF:

brain-derived neurotrophic factor

NT-3:

neurotrophin-3

PRK:

protein kinase C-related kinase

LIMK:

LIM-kinase

FITC:

Fluorescein isothiocyanate

CNS:

central nervous system

References

  1. 1.

    McKerracher, L., David, S., Jackson, D.L., Kottis, V., Dunn, R.J. and Braun, P.E. Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth. Neuron 13 (1994) 805–811.

  2. 2.

    McKerracher, L. and David, S. Easing the brakes on spinal cord repair. Nat. Med. 10 (2004) 1052–1053.

  3. 3.

    Wang, K.C., Koprivica, V., Kim, J.A., Sivasankaran, R., Guo, Y., Neve, R.L. and He, Z. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 417 (2002) 941–944.

  4. 4.

    Fournier, A.E., Takizawa, B.T. and Strittmatter, S.M. Rho kinase inhibition enhances axonal regeneration in the injured CNS. J. Neurosci. 23 (2003) 1416–1423.

  5. 5.

    Lehmann, M., Fournier, A., Selles-Navarro, I., Dergham, P., Sebok, A., Leclerc, N., Tigyi, G. and McKerracher, L. Inactivation of Rho signaling pathway promotes CNS axon regeneration. J. Neurosci. 19 (1999) 7537–7547.

  6. 6.

    Yamashita, T., Higuchi, H. and Tohyama, M. The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho. J. Cell Biol. 157 (2002) 565–570.

  7. 7.

    Yamashita, T., Fujitani, M., Yamagishi, S., Hata, K. and Mimura, F. Multiple signals regulate axon regeneration through the nogo receptor complex. Mol. Neurobiol. 32 (2005) 105–112.

  8. 8.

    Bonini, S., Rasi, G., Bracci-Laudiero, M.L., Procoli, A. and Aloe, L. Nerve growth factor: neurotrophin or cytokine? Int. Arch. Allergy Immunol. 131 (2003) 80–84.

  9. 9.

    Ebadi, M., Bashir, R.M., Heidrick, M.L., Hamada, F.M., Refaey, H.E., Hamed, A., Helal, G., Baxi, M.D., Cerutis, D.R. and Lassi, N.K. Neurotrophins and their receptors in nerve injury and repair. Neurochem. Int. 30 (1997) 347–374.

  10. 10.

    Petruska, J.C. and Mendell, L.M. The many functions of nerve growth factor: multiple actions on nociceptors. Neurosci. Lett. 361 (2004) 168–171.

  11. 11.

    Ozdinler, P.H. and Erzurumlu, R.S. Regulation of neurotrophin-induced axonal responses via Rho GTPases. J. Comp. Neurol. 438 (2001) 377–387.

  12. 12.

    Yamaguchi, Y., Katoh, H., Yasui, H., Mori, K. and Negishi, M. RhoA inhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway. J. Biol. Chem. 276 (2001) 18977–18983.

  13. 13.

    Kwon, B.K., Borisoff, J.F. and Tetzlaff, W. Molecular targets for therapeutic intervention after spinal cord injury. Mol. Intervent. 2 (2002) 244–258.

  14. 14.

    Amano, M., Fukata, Y. and Kaibuchi, K. Regulation and functions of Rho-associated kinase. Exp. Cell Res. 261 (2000) 44–51.

  15. 15.

    Hall, A. Rho GTPases and the control of cell behaviour. Biochem. Soc. Trans. 33 (2005) 891–895.

  16. 16.

    Somlyo, A.P. and Somlyo, A.V. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J. Physiol. 522 Pt 2 (2000) 177–185.

  17. 17.

    Kawano, Y., Fukata, Y., Oshiro, N., Amano, M., Nakamura, T., Ito, M., Matsumura, F., Inagaki, M. and Kaibuchi, K. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J. Cell Biol. 147 (1999) 1023–1038.

  18. 18.

    Riento, K. and Ridley, A.J. Rocks: multifunctional kinases in cell behaviour. Nat. Rev. Mol. Cell Biol. 4 (2003) 446–456.

  19. 19.

    Greene, L.A. and Tischler, A.S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. 73 (1976) 2424–2428.

  20. 20.

    Park, Y.H., Kantor, L., Guptaroy, B., Zhang, M., Wang, K.K. and Gnegy, M.E. Repeated amphetamine treatment induces neurite outgrowth and enhanced amphetamine-stimulated dopamine release in rat pheochromocytoma cells (PC12 cells) via a protein kinase C-and mitogen activated protein kinase-dependent mechanism. J. Neurochem. 87 (2003) 1546–1557.

  21. 21.

    Sebok, A., Nusser, N., Debreceni, B., Guo, Z., Santos, M.F., Szeberenyi, J. and Tigyi, G. Different roles for RhoA during neurite initiation, elongation, and regeneration in PC12 cells. J. Neurochem. 73 (1999) 949–960.

  22. 22.

    Tojima, T. and Ito, E. Signal transduction cascades underlying de novo protein synthesis required for neuronal morphogenesis in differentiating neurons. Prog. Neurobiol. 72 (2004) 183–193.

  23. 23.

    Dent, E.W. and Gertler, F.B. Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40 (2003) 209–227.

  24. 24.

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

  25. 25.

    Ohashi, K., Nagata, K., Maekawa, M., Ishizaki, T., Narumiya, S. and Mizuno, K. Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. J. Biol. Chem. 275 (2000) 3577–3582.

  26. 26.

    Hashimoto, R., Nakamura, Y., Goto, H., Wada, Y., Sakoda, S., Kaibuchi, K., Inagaki, M. and Takeda, M. Domain-and site-specific phosphorylation of bovine NF-L by Rho-associated kinase. Biochem. Biophys. Res. Commun. 245 (1998) 407–411.

  27. 27.

    Amano, M., Kaneko, T., Maeda, A., Nakayama, M., Ito, M., Yamauchi, T., Goto, H., Fukata, Y., Oshiro, N., Shinohara, A., Iwamatsu, A. and Kaibuchi, K. Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase. J. Neurochem. 87 (2003) 780–790.

  28. 28.

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

  29. 29.

    Ikenoya, M., Hidaka, H., Hosoya, T., Suzuki, M., Yamamoto, N. and Sasaki, Y. Inhibition of rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor. J. Neurochem. 81 (2002) 9–16.

  30. 30.

    Nakajima, M., Hayashi, K., Egi, Y., Katayama, K., Amano, Y., Uehata, M., Ohtsuki, M., Fujii, A., Oshita, K., Kataoka, H., Chiba, K., Goto, N. and Kondo, T. Effect of Wf-536, a novel ROCK inhibitor, against metastasis of B16 melanoma. Cancer Chemother. Pharmacol. 52 (2003) 319–324.

  31. 31.

    Ishizaki, T., Uehata, M., Tamechika, I., Keel, J., Nonomura, K., Maekawa, M. and Narumiya, S. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol. Pharmacol. 57 (2000) 976–983.

  32. 32.

    Christensen, A.E., Selheim, F., de Rooij, J., Dremier, S., Schwede, F., Dao, K.K., Martinez, A., Maenhaut, C., Bos, J.L., Genieser, H.G. and Doskeland, S.O. cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension. J. Biol. Chem. 278 (2003) 35394–35402.

  33. 33.

    Hundle, B., McMahon, T., Dadgar, J. and Messing, R.O. Overexpression of epsilon-protein kinase C enhances nerve growth factor-induced phosphorylation of mitogen-activated protein kinases and neurite outgrowth. J. Biol. Chem. 270 (1995) 30134–30140.

  34. 34.

    Obara, Y., Aoki, T., Kusano, M. and Ohizumi, Y. Beta-eudesmol induces neurite outgrowth in rat pheochromocytoma cells accompanied by an activation of mitogen-activated protein kinase. J. Pharmacol. Exp. Ther. 301 (2002) 803–811.

  35. 35.

    Birkenfeld, J., Betz, H. and Roth, D. Inhibition of neurite extension by overexpression of individual domains of LIM kinase 1. J. Neurochem. 78 (2001) 924–927.

  36. 36.

    Fujita, A., Hattori, Y., Takeuchi, T., Kamata, Y. and Hata, F. NGF induces neurite outgrowth via a decrease in phosphorylation of myosin light chain in PC12 cells. Neuroreport 12 (2001) 3599–3602.

  37. 37.

    Kishida, S., Yamamoto, H. and Kikuchi, A. Wnt-3a and Dvl induce neurite retraction by activating Rho-associated kinase. Mol. Cell Biol. 24 (2004) 4487–4501.

  38. 38.

    Sasaki, Y., Suzuki, M. and Hidaka, H. The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathway. Pharmacol. Ther. 93 (2002) 225–232.

  39. 39.

    Braun, H., Schafer, K. and Hollt, V. BetaIII tubulin-expressing neurons reveal enhanced neurogenesis in hippocampal and cortical structures after a contusion trauma in rats. J. Neurotrauma 19 (2002) 975–983.

  40. 40.

    Aizawa, H., Wakatsuki, S., Ishii, A., Moriyama, K., Sasaki, Y., Ohashi, K., Sekine-Aizawa, Y., Sehara-Fujisawa, A., Mizuno, K., Goshima, Y. and Yahara, I. Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse. Nat. Neurosci. 4 (2001) 367–373.

  41. 41.

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

  42. 42.

    Ambach, A., Saunus, J., Konstandin, M., Wesselborg, S., Meuer, S.C. and Samstag, Y. The serine phosphatases PP1 and PP2A associate with and activate the actin-binding protein cofilin in human T lymphocytes. Eur. J. Immunol. 30 (2000) 3422–3431.

  43. 43.

    Revenu, C., Athman, R., Robine, S. and Louvard, D. The co-workers of actin filaments: from cell structures to signals. Nat. Rev. Mol. Cell Biol. 5 (2004) 635–646.

  44. 44.

    Zhou, Y., Su, Y., Li, B., Liu, F., Ryder, J.W., Wu, X., Gonzalez-DeWhitt, P.A., Gelfanova, V., Hale, J.E., May, P.C., Paul, S.M. and Ni, B. Nonsteroidal anti-inflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho. Science 302 (2003) 1215–1217.

  45. 45.

    Ellezam, B., Dubreuil, C., Winton, M., Loy, L., Dergham, P., Selles-Navarro, I. and McKerracher, L. Inactivation of intracellular Rho to stimulate axon growth and regeneration. Prog. Brain. Res. 137 (2002) 371–380.

  46. 46.

    Brabeck, C., Beschorner, R., Conrad, S., Mittelbronn, M., Bekure, K., Meyermann, R., Schluesener, H.J. and Schwab, J.M. Lesional expression of RhoA and RhoB following traumatic brain injury in humans. J. Neurotrauma 21 (2004) 697–706.

  47. 47.

    Brabeck, C., Mittelbronn, M., Bekure, K., Meyermann, R., Schluesener, H.J. and Schwab, J.M. Effect of focal cerebral infarctions on lesional RhoA and RhoB expression. Arch. Neurol. 60 (2003) 1245–1249.

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Correspondence to Kevin K. W. Wang.

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Key words

  • Neurite outgrowth
  • ROCK
  • Y-27632
  • PC-12
  • Cofilin
  • Actin dynamics