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Nerve growth factor-induced differentiation of PC12 cells employs the PMA-insensitive protein kinase C-ζ isoform

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Abstract

To elucidate the role of protein kinase C (PKC) in nerve growth factor (NGF)-induced differentiation, PMA downregulation of pheochromocytoma (PC12) cells was undertaken. Prolonged treatment (2 d) of PC12 cells with PMA (1 µM) resulted in depleting the cells of α, β, δ, and ɛ-PKC isoforms, but had no effect on the expression of the atypical PKC isoform ζ. PC12 cells, which expressed only PKC ζ, were evaluated for their responses to NGF. Removal of the PMA-sensitive PKC isoforms enhanced the ability of NGF to promote neurite extension. Both the percentage cells with neurites and length of neurites were increased in the PMA-treated cells, whereas no effect was observed on the number of neurites per cell or branching of individual neurites. In addition, PMA downregulation resulted in an increase in the incorporation of3H-thymidine without any significant effect on the expression of c-fos. Addition of NGF to PC12 cells depleted of the PMA-sensitive PKC isoforms resulted in the activation of PKC ζ (Wooten et al., 1994). To test whether the transient activation of PKC ζ is a necessary component of the neuritogenetic pathway, antisense oligonucleotide strategy was utilized to remove this particular PKC isoform. The addition of a 20-bp antisense oligonucleotide directed against the 5′ coding sequence of PKC ζ attenuated NGF-induced neurite outgrowth in PC12 cells lacking PMA-sensitive PKC isoforms. Sense oligonucleotide directed at the same site was without effect on NGF responses. These data indicate that PKC ζ comprises a portion of the NGF pathway and underscores the importance of this isoform in neuronal differentiation. Moreover, these findings demonstrate that the PMA-insensitive pathway, which was previously characterized as PKC-independent, and the neurite induction pathway are synonymous and mediated by PKC ζ.

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References

  • Altin J. G., Wetts R., Riabowol K. T., and Bradshaw R. A. (1992) Testing the in vivo role of protein kinase C and c-fos in neurite outgrowth by microinjection of antibodies into PC12 cells.Mol. Cell. Biol. 3, 323–333.

    CAS  Google Scholar 

  • Baxter G. T., Miller D. L., Kuo R. C., Wada H. G., and Owicki J. C. (1992) PKCɛ is involved in granulocyte-macrophage colony-stimulating factor signal-transduction: evidence from microphysiometry and antisense oligonucleotide experiments.Biochemistry 31, 10,950–10,954.

    Article  CAS  Google Scholar 

  • Burstein D. E., Blumberg P. M., and Greene L. A. (1982) Nerve growth factor-induced neuronal differentiation of PC12 pheochromocytoma cells: lack of inhibition by a tumor promotor.Brain Res. 247, 115–119.

    Article  PubMed  CAS  Google Scholar 

  • Caporaso G. L., Gandy S. E., Buxbaum J. D., Ramabhadran T. V., and Greengard P. (1992) Protein phosporylation regulates secretion of Alzheimer β/A4 amyloid precursor protein.Proc. Natl. Acad. Sci. USA 89, 3055–3059.

    Article  PubMed  CAS  Google Scholar 

  • Contreras M. L. and Guroff G. (1987) Calcium-dependent nerve growth factor-stimulated hydrolysis of phosphoinositides in PC12 cells.J. Neurochem. 48, 1466–1472.

    Article  PubMed  CAS  Google Scholar 

  • Damon D. H., D’Amore P. A., and Wagner J. A. (1990) Nerve growth factor and fibroblast growth factor regulate neurite outgrowth and gene expression in PC12 cells via both protein kinase C- and cAMP-independent mechanisms.J. Cell Biol. 110, 1331–1339.

    Article  Google Scholar 

  • Dominguez I., Diaz-Meco M. T., Municio M. M., Berra E., de Herreros A. G., Cornet M. E., Sanz L., and Moscat J. (1992) Evidence for a role of protein kinase C ζ subspecies in maturation ofXenopus laevis oocytes.Mol. Cell Biol. 12, 3773–3783.

    Google Scholar 

  • Gamard C. J., Blobe G. C., Hannun Y. A., and Obeid L. M. (1994) Specific role for protein kinase C β in cell differentiation.Cell Growth Diff. 5, 405–409.

    PubMed  CAS  Google Scholar 

  • Glowaka D. and Wagner J. A. (1990) Role of the cAMP-dependent protein kinase and protein kinase C in regulating the morphological differentiation of PC12 cells.J. Neurosci. Res. 25, 453–462.

    Article  Google Scholar 

  • Greenberg M. E., Greene L. A., and Ziff E. B. (1985) Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells.J. Biol. Chem. 260, 14,101–14,110.

    CAS  Google Scholar 

  • Greene L. A., Sobeih M. M., and Teng K. (1991) Methodologies for the culture and experimental use of the PC12 rat pheochromocytoma cell line, inCulturing Nerve Cells (Banker G. and Goslin K., eds.), MIT, Boston, MA, pp. 207–226.

    Google Scholar 

  • Guroff G. (1993) Nerve growth factor as a neurotrophic agent.Ann. NY Acad. Sci. 692, 51–59.

    Article  PubMed  CAS  Google Scholar 

  • Hagiwara M., Uchida C., Usuda N., Nagata T., and Hidaka H. (1990) ζ-related protein kinase C in nuclei of nerve cells.Biochem. Biophys. Res. Comm. 168, 161–168.

    Article  PubMed  CAS  Google Scholar 

  • Halegoua S. and Patrick J. (1980) Nerve growth factor mediates phosphorylation of specific proteins.Cell 22, 571–581.

    Article  PubMed  CAS  Google Scholar 

  • Hall F. L., Fernyhough P., Ishii D. N., and Vulliet P. R. (1988) Suppression of nerve growth factor-directed neurite outgrowth in PC12 cells by sphingosine, an inhibitor of protein kinase C.J. Biol. Chem. 263, 4460–4466.

    PubMed  CAS  Google Scholar 

  • Heasley L. E. and Johnson G. L. (1989) Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochromocytoma cells.J. Biol. Chem. 264, 8648–8652.

    Google Scholar 

  • Hempstead B. L., Rabin S. J., Kaplan L., Reid S., Parada L. F., and Kaplan D. R. (1992) Overexpression of the trk tyrosine kinase rapidly accelerates nerve growth factor-induced differentiation.Neuron 9, 883–896.

    Article  PubMed  CAS  Google Scholar 

  • Hsu L., Natyzak D., and Laskin J. D. (1984) Effects of the tumor promotor 12-O-tetradecanoylphorbol-13-acetate on neurite outgrowth from chick embryo sensory ganglia.Cancer Res. 44, 4607–4614.

    PubMed  CAS  Google Scholar 

  • Hug H. and Sarre T. F. (1993) Protein kinase C isoenzymes: divergence in signal transduction?Biochem. J. 291, 329–343.

    PubMed  CAS  Google Scholar 

  • Ishii D. N. (1978) Effect of tumor promotors on the response of cultured embryonic chick ganglia to nerve growth factor.Cancer Res. 38, 3886–3893.

    PubMed  CAS  Google Scholar 

  • Ishii D. N., Fibach E., Yamasaki H., and Weinstein I. B. (1978) Tumor promotors inhibit morphological differentiation in cultured mouse neuroblastoma cells.Science 200, 556–559.

    Article  PubMed  CAS  Google Scholar 

  • Ito E., Sonnenberg J. L., and Narayanan R. (1989) Nerve growth factor-induced differentiation in PC12 cells is blocked byfos oncogene.Oncogene 4, 1193–1199.

    PubMed  CAS  Google Scholar 

  • Kochs G., Hummel R., Meyer D., Hug H., Marme D., and Sarre T. F. (1993) Activation and substrate specificity of the human protein kinase C α and ζ isoenzymes.Eur. J. Biochem. 216, 597–606.

    Article  PubMed  CAS  Google Scholar 

  • Lacal J. C., Cuadrado A., Jones J. E., Trotta R., Burstein D. E., Thomson T., and Pellicer A. (1990) Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC12 cells.Mol. Cell Biol. 10, 2983–2990.

    PubMed  CAS  Google Scholar 

  • Leli U., Parker P., and Shea T. B. (1992) Intracellular delivery of protein kinase C-α or -ɛ isofom-specific antibodies promotes acquisition of a morphologically differentiated phenotype in neurobastoma cells.FEBS Lett. 297, 91–94.

    Article  PubMed  CAS  Google Scholar 

  • Leli U., Shea T. B., Cataldo A., Hauser G., Grynspan F., Beermann M. L., Liepkalns V. A., Nixon R. A., and Parker P. (1993) Differential expression and subcellular localization of protein kinase α, β, γ, and ɛ isoforms in SH-SY5Y neuroblastoma cells: modifications during differentiation.J. Neurochem. 60, 289–298.

    Article  PubMed  CAS  Google Scholar 

  • Levi-Montalcini R. (1987) The nerve growth factor: thirty-five years later.EMBO J. 6, 1145–1154.

    PubMed  CAS  Google Scholar 

  • Mahadeo D., Kaplan L., Chao M. V., and Hempstead B. L. (1994) High-affinity nerve growth-factor binding displays a faster rate of association than p140trk binding: implications for multi-subunit polypeptide receptors.J. Biol. Chem. 269, 6884–6891.

    PubMed  CAS  Google Scholar 

  • Maher P. A. (1988) Nerve growth factor induces protein-tyrosine phosphorylation.Proc. Natl. Acad. Sci. USA 85, 6788–6791.

    Article  PubMed  CAS  Google Scholar 

  • Mehta S., Hsu L., Jeng A. Y., and Chen K. Y. (1993) Neurite outgrowth and protein phosphorylation in chick embryonic sensory ganglia induced by a brief exposure to 12-O-tetradecanoylphorbol 13-acetate.J. Neurochem. 60, 972–981.

    Article  PubMed  CAS  Google Scholar 

  • Milward E. A., Papadopoulos R., Fuller S. J., Moir R. D., Small D., Beyreuther K., and Masters C. L. (1992) The amyloid protein precursor of Alzheimer’s disease is a mediator of the effects of nerve growth factor on neurite outgrowth.Neuron 9, 129–137.

    Article  PubMed  CAS  Google Scholar 

  • Muir J. G. and Murray A. W. (1987) Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C.J. Cell. Physiol. 130, 382–391.

    Article  PubMed  CAS  Google Scholar 

  • Nakanishi H. and Exton J. H. (1992) Purification and characterization of the ζ isoform of protein kinase C from bovine kidney.J. Biol. Chem. 267, 16,347–16,354.

    CAS  Google Scholar 

  • Nakanishi N. and Guroff G. (1985) Nerve growth factor-induced increase in the cell-free phosphorylation of a nuclear protein in PC12 cells.J. Biol. Chem. 260, 7791–7799.

    PubMed  CAS  Google Scholar 

  • Nakanishi H., Brewer K. A., and Exton J. H. (1993) Activation of the ζ isoenzyme of protein kinase C by phosphatidyl inositol 3,4,5-trisphosphate.J. Biol. Chem. 268, 13–16.

    PubMed  CAS  Google Scholar 

  • Nikodijevic B. and Guroff G. (1991) Nerve growth factor-induced increase in calcium uptake by PC12 cells.J. Neurosci. Res. 28, 192–199.

    Article  PubMed  CAS  Google Scholar 

  • Nishizuka Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.Science 258, 607–614.

    Article  PubMed  CAS  Google Scholar 

  • Ohmichi M., Decker S. J., and Saltiel A. R. (1992) Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of thetrk proto-oncogene with src homology 2 domains.Neuron 9, 769–777.

    Article  PubMed  CAS  Google Scholar 

  • Ono Y., Fujii T., Ogita K., Kikkawa U., Igarishi K., and Nishizuka Y. (1989) Protein kinase C ζ subspecies from rat brain: its structure, expression, and properties.Proc. Natl. Acad. Sci. USA 86, 3099–3103.

    Article  PubMed  CAS  Google Scholar 

  • Pahlman S., Odelstad L., Larsson E., Grotte G., and Nilsson K. (1981) Phenotypic changes of human neuroblastoma cells in culture induced by 12-O-tetradecanoylphorbol-13 acetate.Int. J. Cancer 28, 583–589.

    Article  PubMed  CAS  Google Scholar 

  • Parrow V., Nanberg E., Heikkila J., Hammerling U., and Pahlman S. (1992) Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH-SY5Y human neuroblastoma cells.J. Cell. Physiol. 152, 536–544.

    Article  PubMed  CAS  Google Scholar 

  • Ponzoni M., Lucarelli E., Corrias M. V., and Cornaglia-Ferraris P. (1993) Protein kinase C isoenzymes in human neuroblasts: involvement of PKCɛ in cell differentiation.FEBS Lett. 322, 120–124.

    Article  PubMed  CAS  Google Scholar 

  • Reinhold D. S. and Neet K. E. (1989) The lack of a role for protein kinase C in neurite extension and in the induction of ornithine decarboxylase by nerve growth factor in PC12 cells.J. Biol. Chem. 264, 3538–3544.

    PubMed  CAS  Google Scholar 

  • Rudkin B. B., Lazarovici P., Levi B. Z., Abe Y., Fujita K., and Guroff G. (1989) Cell cycle specific action of nerve growth factor in PC12 cells: differentiation without proliferation.EMBO J. 8, 3319–3325.

    PubMed  CAS  Google Scholar 

  • Ryu S. H., Kim U., Wahl M. I., Brown A. B., Carpenter G., Huang K., and Rhee S. G. (1990) Feedback regulation of phospholipase C-β by protein kinase C.J. Biol. Chem. 265, 17,941–17,945.

    CAS  Google Scholar 

  • Selbie L. A., Schmitz-Peiffer C., Sheng Y., and Biden T. J. (1993) Molecular cloning and characterization of PKCl, an atypical isoform of protein kinase C derived from insulin-secreting cells.J. Biol. Chem. 268, 24,292–24,302.

    Google Scholar 

  • Skaper S. D., Selak I., and Varon S. (1983) Serum vulnerability and time-dependent stabilization of neurites induced by nerve growth factor in PC12 pheochromocytoma cells.J. Neurosci. Res. 10, 303–315.

    Article  PubMed  CAS  Google Scholar 

  • Soltoff S. P., Rabin S. L., Cantley L. C., and Kaplan D. R. (1992) Nerve growth factor promotes the activation of phosphatidyl-inositol 3-kinase and its association with thetrk tyrosine kinase.J. Biol. Chem. 267, 17,472–17,477.

    CAS  Google Scholar 

  • Spinelli W., Sonnenfeld K. H., and Ishii D. N. (1982) Effect of phorbol ester tumor promoters and nerve growth factor on neurite outgrowth in cultured human neuroblastoma cells.Cancer Res. 42, 5067–5073.

    PubMed  CAS  Google Scholar 

  • Wooten M. W., Seibenhener M. L., Soh Y., Ewald S. J., White K. R., Lloyd E. D., Olivier A., and Parker P. (1992) Characterization and differential expression of protein kinase C isoforms in PC12 cells: differentiation parallels an increase in PKC beta11.FEBS Lett. 298, 74–78.

    Article  PubMed  CAS  Google Scholar 

  • Wooten M. W., Zhou G., Seibenhener M. L., and Coleman E. S. (1994) A role for ζ protein kinase C in nerve growth factor-induced differentiation of PC12 cells.Cell Growth Diff. 5, 395–403.

    PubMed  CAS  Google Scholar 

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  1. Division of Zoology, Auburn University, 36849, Auburn, AL

    Elaine S. Coleman & Marie W. Wooten

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  1. Elaine S. Coleman
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Coleman, E.S., Wooten, M.W. Nerve growth factor-induced differentiation of PC12 cells employs the PMA-insensitive protein kinase C-ζ isoform. J Mol Neurosci 5, 39–57 (1994). https://doi.org/10.1007/BF02736693

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