Skip to main content
Download PDF

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

Cellular & Molecular Biology Letters volume 11, pages 12–29 (2006)Cite this article

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.

    PubMed  CAS  Article  Google Scholar 

  2. 2.

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

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    CAS  Article  Google Scholar 

  14. 14.

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

    PubMed  CAS  Article  Google Scholar 

  15. 15.

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

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  18. 18.

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

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  CAS  Article  Google Scholar 

  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.

    PubMed  Article  Google Scholar 

  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.

    PubMed  Article  Google Scholar 

Download references

Author information

Affiliations

  1. Centers for Neuroproteomics and Biomarkers Research, University of Florida, P.O. Box 100256, 100 S. Newell Drive, Gainesville, Florida, 32610, USA

    Zhiqun Zhang, Andrew K. Ottens, Firas H. Kobeissy & Kevin K. W. Wang

  2. Traumatic Brain Injury Studies, University of Florida, P.O. Box 100256, 100 S. Newell Drive, Gainesville, Florida, 32610, USA

    Zhiqun Zhang, Andrew K. Ottens, Stephen F. Larner, Firas H. Kobeissy, Ronald L. Hayes & Kevin K. W. Wang

  3. Departments of Neuroscience, University of Florida, P.O. Box 100256, 100 S. Newell Drive, Gainesville, Florida, 32610, USA

    Zhiqun Zhang, Andrew K. Ottens, Stephen F. Larner, Firas H. Kobeissy, Melissa L. Williams, Ronald L. Hayes & Kevin K. W. Wang

  4. Psychiatry, McKnight Brain Institute, University of Florida, P.O. Box 100256, 100 S. Newell Drive, Gainesville, Florida, 32610, USA

    Ronald L. Hayes & Kevin K. W. Wang

Authors
  1. Zhiqun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew K. Ottens
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stephen F. Larner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Firas H. Kobeissy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Melissa L. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ronald L. Hayes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kevin K. W. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kevin K. W. Wang.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zhang, Z., Ottens, A.K., Larner, S.F. et al. Direct Rho-associated kinase inhibiton induces cofilin dephosphorylation and neurite outgrowth in PC-12 cells. Cell. Mol. Biol. Lett. 11, 12–29 (2006). https://doi.org/10.2478/s11658-006-0002-x

Download citation

Key words

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

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us