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. 1990 Apr 1;110(4):1333–1339. doi: 10.1083/jcb.110.4.1333

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

PMCID: PMC2116059  PMID: 2157719

Abstract

Nerve growth factor (NGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) promote the survival and differentiation of a variety of peripheral and central neurons. The signal transduction mechanisms that mediate the actions of these factors in neuronal cells are not well understood. We examined the effect of a deficiency in protein kinase C (PKC) and/or cAMP second messenger systems on the actions of NGF, aFGF, and bFGF in the pheochromocytoma (PC12) cell line. Activation of PKC was not required for NGF, aFGF, and bFGF to maximally induce ornithine decarboxylase (ODC), transcription of the early response genes, d2 and d5, or neurite outgrowth. In a PC12 cell mutant that is deficient in cAMP responsiveness (A126-1B2), all three growth factors maximally induced the transcription of d5 and neurite outgrowth, but aFGF and bFGF did not induce significant increases in ODC. NGF and aFGF maximally induced the transcription of d2 in A126-1B2 cells, but bFGF-induced d2 transcription was attenuated. NGF, aFGF, and bFGF maximally induced neurite outgrowth and d5 transcription in A126 cells that were made deficient in PKC. The d2 transcriptional response was substantially reduced in cells deficient in both PKC and cAMP responsiveness. These observations lead us to conclude that (a) cAMP- and PKC-dependent events are, at least in part, causally linked to NGF, aFGF, and bFGF induction of both ODC and transcription of d2 and may control functionally redundant pathways; (b) NGF, aFGF, and bFGF can elicit neurite outgrowth and increase transcription of d2 and d5 in PC12 cells via mechanisms that are independent of both PKC and cAMP; (c) NGF, aFGF, and bFGF can induce ODC in the absence of PKC; and (d) aFGF and bFGF require cAMP responsiveness to induce ODC in PC12 cells.

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Selected References

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  1. Chan B. L., Chao M. V., Saltiel A. R. Nerve growth factor stimulates the hydrolysis of glycosylphosphatidylinositol in PC-12 cells: a mechanism of protein kinase C regulation. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1756–1760. doi: 10.1073/pnas.86.6.1756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Changelian P. S., Feng P., King T. C., Milbrandt J. Structure of the NGFI-A gene and detection of upstream sequences responsible for its transcriptional induction by nerve growth factor. Proc Natl Acad Sci U S A. 1989 Jan;86(1):377–381. doi: 10.1073/pnas.86.1.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cho K. O., Skarnes W. C., Minsk B., Palmieri S., Jackson-Grusby L., Wagner J. A. Nerve growth factor regulates gene expression by several distinct mechanisms. Mol Cell Biol. 1989 Jan;9(1):135–143. doi: 10.1128/mcb.9.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Christy B. A., Lau L. F., Nathans D. A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with "zinc finger" sequences. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7857–7861. doi: 10.1073/pnas.85.21.7857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Claude P., Parada I. M., Gordon K. A., D'Amore P. A., Wagner J. A. Acidic fibroblast growth factor stimulates adrenal chromaffin cells to proliferate and to extend neurites, but is not a long-term survival factor. Neuron. 1988 Nov;1(9):783–790. doi: 10.1016/0896-6273(88)90126-2. [DOI] [PubMed] [Google Scholar]
  6. Contreras M. L., Guroff G. Calcium-dependent nerve growth factor-stimulated hydrolysis of phosphoinositides in PC12 cells. J Neurochem. 1987 May;48(5):1466–1472. doi: 10.1111/j.1471-4159.1987.tb05687.x. [DOI] [PubMed] [Google Scholar]
  7. Coughlin S. R., Barr P. J., Cousens L. S., Fretto L. J., Williams L. T. Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo. J Biol Chem. 1988 Jan 15;263(2):988–993. [PubMed] [Google Scholar]
  8. Cremins J., Wagner J. A., Halegoua S. Nerve growth factor action is mediated by cyclic AMP- and Ca+2/phospholipid-dependent protein kinases. J Cell Biol. 1986 Sep;103(3):887–893. doi: 10.1083/jcb.103.3.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Djurhuus R. Ornithine decarboxylase (EC 4.1.1.17) assay based upon the retention of putrescine by a strong cation-exchange paper. Anal Biochem. 1981 May 15;113(2):352–355. doi: 10.1016/0003-2697(81)90088-9. [DOI] [PubMed] [Google Scholar]
  10. End D., Tolson N., Yu M. Y., Guroff G. Effects of 12-0-Tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: interactions with epidermal growth factor and nerve growth factor. J Cell Physiol. 1982 May;111(2):140–148. doi: 10.1002/jcp.1041110204. [DOI] [PubMed] [Google Scholar]
  11. Feinstein S. C., Dana S. L., McConlogue L., Shooter E. M., Coffino P. Nerve growth factor rapidly induces ornithine decarboxylase mRNA in PC12 rat pheochromocytoma cells. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5761–5765. doi: 10.1073/pnas.82.17.5761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friesel R., Burgess W. H., Maciag T. Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells. Mol Cell Biol. 1989 May;9(5):1857–1865. doi: 10.1128/mcb.9.5.1857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gospodarowicz D., Neufeld G., Schweigerer L. Fibroblast growth factor. Mol Cell Endocrinol. 1986 Aug;46(3):187–204. doi: 10.1016/0303-7207(86)90001-8. [DOI] [PubMed] [Google Scholar]
  14. Greenberg M. E., Greene L. A., Ziff E. B. Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells. J Biol Chem. 1985 Nov 15;260(26):14101–14110. [PubMed] [Google Scholar]
  15. Guroff G., Dickens G., End D. The induction of ornithine decarboxylase by nerve growth factor and epidermal growth factor in PC12 cells. J Neurochem. 1981 Aug;37(2):342–349. doi: 10.1111/j.1471-4159.1981.tb00461.x. [DOI] [PubMed] [Google Scholar]
  16. Hagag N., Halegoua S., Viola M. Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21. Nature. 1986 Feb 20;319(6055):680–682. doi: 10.1038/319680a0. [DOI] [PubMed] [Google Scholar]
  17. Hall F. L., Fernyhough P., Ishii D. N., Vulliet P. R. Suppression of nerve growth factor-directed neurite outgrowth in PC12 cells by sphingosine, an inhibitor of protein kinase C. J Biol Chem. 1988 Mar 25;263(9):4460–4466. [PubMed] [Google Scholar]
  18. Hama T., Huang K. P., Guroff G. Protein kinase C as a component of a nerve growth factor-sensitive phosphorylation system in PC12 cells. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2353–2357. doi: 10.1073/pnas.83.8.2353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hatten M. E., Lynch M., Rydel R. E., Sanchez J., Joseph-Silverstein J., Moscatelli D., Rifkin D. B. In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor. Dev Biol. 1988 Feb;125(2):280–289. doi: 10.1016/0012-1606(88)90211-4. [DOI] [PubMed] [Google Scholar]
  20. Heasley L. E., Johnson G. L. Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochromocytoma cells. J Biol Chem. 1989 May 25;264(15):8646–8652. [PubMed] [Google Scholar]
  21. Huang S. S., Huang J. S. Association of bovine brain-derived growth factor receptor with protein tyrosine kinase activity. J Biol Chem. 1986 Jul 25;261(21):9568–9571. [PubMed] [Google Scholar]
  22. Koizumi S., Contreras M. L., Matsuda Y., Hama T., Lazarovici P., Guroff G. K-252a: a specific inhibitor of the action of nerve growth factor on PC 12 cells. J Neurosci. 1988 Feb;8(2):715–721. doi: 10.1523/JNEUROSCI.08-02-00715.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee P. L., Johnson D. E., Cousens L. S., Fried V. A., Williams L. T. Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor. Science. 1989 Jul 7;245(4913):57–60. doi: 10.1126/science.2544996. [DOI] [PubMed] [Google Scholar]
  24. Levi-Montalcini R. The nerve growth factor: thirty-five years later. EMBO J. 1987 May;6(5):1145–1154. doi: 10.1002/j.1460-2075.1987.tb02347.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lim R. W., Varnum B. C., O'Brien T. G., Herschman H. R. Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways. Mol Cell Biol. 1989 Apr;9(4):1790–1793. doi: 10.1128/mcb.9.4.1790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lipton S. A., Wagner J. A., Madison R. D., D'Amore P. A. Acidic fibroblast growth factor enhances regeneration of processes by postnatal mammalian retinal ganglion cells in culture. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2388–2392. doi: 10.1073/pnas.85.7.2388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lobb R., Sasse J., Sullivan R., Shing Y., D'Amore P., Jacobs J., Klagsbrun M. Purification and characterization of heparin-binding endothelial cell growth factors. J Biol Chem. 1986 Feb 5;261(4):1924–1928. [PubMed] [Google Scholar]
  28. Logan A., Berry M., Thomas G. H., Gregson N. A., Logan S. D. Identification and partial purification of fibroblast growth factor from the brains of developing rats and leucodystrophic mutant mice. Neuroscience. 1985 Aug;15(4):1239–1246. doi: 10.1016/0306-4522(85)90266-0. [DOI] [PubMed] [Google Scholar]
  29. Maher P. A. Nerve growth factor induces protein-tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6788–6791. doi: 10.1073/pnas.85.18.6788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Matthies H. J., Palfrey H. C., Hirning L. D., Miller R. J. Down regulation of protein kinase C in neuronal cells: effects on neurotransmitter release. J Neurosci. 1987 Apr;7(4):1198–1206. doi: 10.1523/JNEUROSCI.07-04-01198.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Milbrandt J. A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor. Science. 1987 Nov 6;238(4828):797–799. doi: 10.1126/science.3672127. [DOI] [PubMed] [Google Scholar]
  32. Mobley W. C., Schenker A., Shooter E. M. Characterization and isolation of proteolytically modified nerve growth factor. Biochemistry. 1976 Dec 14;15(25):5543–5552. doi: 10.1021/bi00670a019. [DOI] [PubMed] [Google Scholar]
  33. Morrison R. S., Sharma A., de Vellis J., Bradshaw R. A. Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7537–7541. doi: 10.1073/pnas.83.19.7537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reinhold D. S., Neet K. E. 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. 1989 Feb 25;264(6):3538–3544. [PubMed] [Google Scholar]
  35. Richter-Landsberg C., Jastorff B. The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells. J Cell Biol. 1986 Mar;102(3):821–829. doi: 10.1083/jcb.102.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ruta M., Burgess W., Givol D., Epstein J., Neiger N., Kaplow J., Crumley G., Dionne C., Jaye M., Schlessinger J. Receptor for acidic fibroblast growth factor is related to the tyrosine kinase encoded by the fms-like gene (FLG). Proc Natl Acad Sci U S A. 1989 Nov;86(22):8722–8726. doi: 10.1073/pnas.86.22.8722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rydel R. E., Greene L. A. cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1257–1261. doi: 10.1073/pnas.85.4.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schubert D., Ling N., Baird A. Multiple influences of a heparin-binding growth factor on neuronal development. J Cell Biol. 1987 Mar;104(3):635–643. doi: 10.1083/jcb.104.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stemple D. L., Mahanthappa N. K., Anderson D. J. Basic FGF induces neuronal differentiation, cell division, and NGF dependence in chromaffin cells: a sequence of events in sympathetic development. Neuron. 1988 Aug;1(6):517–525. doi: 10.1016/0896-6273(88)90182-1. [DOI] [PubMed] [Google Scholar]
  40. Suissa M. Spectrophotometric quantitation of silver grains eluted from autoradiograms. Anal Biochem. 1983 Sep;133(2):511–514. doi: 10.1016/0003-2697(83)90117-3. [DOI] [PubMed] [Google Scholar]
  41. Thoenen H., Barde Y. A. Physiology of nerve growth factor. Physiol Rev. 1980 Oct;60(4):1284–1335. doi: 10.1152/physrev.1980.60.4.1284. [DOI] [PubMed] [Google Scholar]
  42. Togari A., Baker D., Dickens G., Guroff G. The neurite-promoting effect of fibroblast growth factor on PC12 cells. Biochem Biophys Res Commun. 1983 Aug 12;114(3):1189–1193. doi: 10.1016/0006-291x(83)90688-5. [DOI] [PubMed] [Google Scholar]
  43. Togari A., Dickens G., Kuzuya H., Guroff G. The effect of fibroblast growth factor on PC12 cells. J Neurosci. 1985 Feb;5(2):307–316. doi: 10.1523/JNEUROSCI.05-02-00307.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Unsicker K., Reichert-Preibsch H., Schmidt R., Pettmann B., Labourdette G., Sensenbrenner M. Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5459–5463. doi: 10.1073/pnas.84.15.5459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Van Buskirk R., Corcoran T., Wagner J. A. Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists. Mol Cell Biol. 1985 Aug;5(8):1984–1992. doi: 10.1128/mcb.5.8.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wagner J. A., D'Amore P. A. Neurite outgrowth induced by an endothelial cell mitogen isolated from retina. J Cell Biol. 1986 Oct;103(4):1363–1367. doi: 10.1083/jcb.103.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Walicke P. A. Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions. J Neurosci. 1988 Jul;8(7):2618–2627. doi: 10.1523/JNEUROSCI.08-07-02618.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Winicov I., Gershengorn M. C. Sphingosine inhibits thyrotropin-releasing hormone binding to pituitary cells by a mechanism independent of protein kinase C. J Biol Chem. 1988 Sep 5;263(25):12179–12182. [PubMed] [Google Scholar]

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