Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Aug;17(8):4633–4643. doi: 10.1128/mcb.17.8.4633

Differentiation of central nervous system neuronal cells by fibroblast-derived growth factor requires at least two signaling pathways: roles for Ras and Src.

W L Kuo 1, K C Chung 1, M R Rosner 1
PMCID: PMC232316  PMID: 9234720

Abstract

To evaluate the role of mitogen-activated protein (MAP) kinase and other signaling pathways in neuronal cell differentiation by basic fibroblast-derived growth factor (bFGF), we used a conditionally immortalized cell line from rat hippocampal neurons (H19-7). Previous studies have shown that activation of MAP kinase kinase (MEK) is insufficient to induce neuronal differentiation of H19-7 cells. To test the requirement for MEK and MAP kinase (ERK1 and ERK2), H19-7 cells were treated with the MEK inhibitor PD098059. Although the MEK inhibitor blocked the induction of differentiation by constitutively activated Raf, the H19-7 cells still underwent differentiation by bFGF. These results suggest that an alternative pathway is utilized by bFGF for differentiation of the hippocampal neuronal cells. Expression in the H19-7 cells of a dominant-negative Ras (N17-Ras) or Raf (C4-Raf) blocked differentiation by bFGF, suggesting that Ras and probably Raf are required. Expression of dominant-negative Src (pcSrc295Arg) or microinjection of an anti-Src antibody blocked differentiation by bFGF in H19-7 cells, indicating that bFGF also signals through a Src kinase-mediated pathway. Although neither constitutively activated MEK (MEK-2E) nor v-Src was sufficient individually to differentiate the H19-7 cells, coexpression of constitutively activated MEK and v-Src induced neurite outgrowth. These results suggest that (i) activation of MAP kinase (ERK1 and ERK2) is neither necessary nor sufficient for differentiation by bFGF; (ii) activation of Src kinases is necessary but not sufficient for differentiation by bFGF; and (iii) differentiation of H19-7 neuronal cells by bFGF requires at least two signaling pathways activated by Ras and Src.

Full Text

The Full Text of this article is available as a PDF (738.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alessi D. R., Cuenda A., Cohen P., Dudley D. T., Saltiel A. R. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem. 1995 Nov 17;270(46):27489–27494. doi: 10.1074/jbc.270.46.27489. [DOI] [PubMed] [Google Scholar]
  2. Anderson N. G., Li P., Marsden L. A., Williams N., Roberts T. M., Sturgill T. W. Raf-1 is a potential substrate for mitogen-activated protein kinase in vivo. Biochem J. 1991 Jul 15;277(Pt 2):573–576. doi: 10.1042/bj2770573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bare D. J., Lauder J. M., Wilkie M. B., Maness P. F. p59fyn in rat brain is localized in developing axonal tracts and subpopulations of adult neurons and glia. Oncogene. 1993 Jun;8(6):1429–1436. [PubMed] [Google Scholar]
  4. Barone M. V., Courtneidge S. A. Myc but not Fos rescue of PDGF signalling block caused by kinase-inactive Src. Nature. 1995 Nov 30;378(6556):509–512. doi: 10.1038/378509a0. [DOI] [PubMed] [Google Scholar]
  5. Bixby J. L., Jhabvala P. Tyrosine phosphorylation in early embryonic growth cones. J Neurosci. 1993 Aug;13(8):3421–3432. doi: 10.1523/JNEUROSCI.13-08-03421.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burgess W. H., Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem. 1989;58:575–606. doi: 10.1146/annurev.bi.58.070189.003043. [DOI] [PubMed] [Google Scholar]
  7. Campbell J. S., Wenderoth M. P., Hauschka S. D., Krebs E. G. Differential activation of mitogen-activated protein kinase in response to basic fibroblast growth factor in skeletal muscle cells. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):870–874. doi: 10.1073/pnas.92.3.870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cowley S., Paterson H., Kemp P., Marshall C. J. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell. 1994 Jun 17;77(6):841–852. doi: 10.1016/0092-8674(94)90133-3. [DOI] [PubMed] [Google Scholar]
  9. D'Arcangelo G., Halegoua S. A branched signaling pathway for nerve growth factor is revealed by Src-, Ras-, and Raf-mediated gene inductions. Mol Cell Biol. 1993 Jun;13(6):3146–3155. doi: 10.1128/mcb.13.6.3146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Debus E., Weber K., Osborn M. Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides. Differentiation. 1983;25(2):193–203. doi: 10.1111/j.1432-0436.1984.tb01355.x. [DOI] [PubMed] [Google Scholar]
  11. Doherty P., Williams E., Walsh F. S. A soluble chimeric form of the L1 glycoprotein stimulates neurite outgrowth. Neuron. 1995 Jan;14(1):57–66. doi: 10.1016/0896-6273(95)90240-6. [DOI] [PubMed] [Google Scholar]
  12. Eves E. M., Kwon J., Downen M., Tucker M. S., Wainer B. H., Rosner M. R. Conditional immortalization of neuronal cells from postmitotic cultures and adult CNS. Brain Res. 1994 Sep 12;656(2):396–404. doi: 10.1016/0006-8993(94)91484-2. [DOI] [PubMed] [Google Scholar]
  13. Eves E. M., Tucker M. S., Roback J. D., Downen M., Rosner M. R., Wainer B. H. Immortal rat hippocampal cell lines exhibit neuronal and glial lineages and neurotrophin gene expression. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4373–4377. doi: 10.1073/pnas.89.10.4373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grant S. G., O'Dell T. J., Karl K. A., Stein P. L., Soriano P., Kandel E. R. Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science. 1992 Dec 18;258(5090):1903–1910. doi: 10.1126/science.1361685. [DOI] [PubMed] [Google Scholar]
  15. Heasley L. E., Storey B., Fanger G. R., Butterfield L., Zamarripa J., Blumberg D., Maue R. A. GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases. Mol Cell Biol. 1996 Feb;16(2):648–656. doi: 10.1128/mcb.16.2.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ignelzi M. A., Jr, Miller D. R., Soriano P., Maness P. F. Impaired neurite outgrowth of src-minus cerebellar neurons on the cell adhesion molecule L1. Neuron. 1994 Apr;12(4):873–884. doi: 10.1016/0896-6273(94)90339-5. [DOI] [PubMed] [Google Scholar]
  17. Iwasaki S., Hattori A., Sato M., Tsujimoto M., Kohno M. Characterization of the bone morphogenetic protein-2 as a neurotrophic factor. Induction of neuronal differentiation of PC12 cells in the absence of mitogen-activated protein kinase activation. J Biol Chem. 1996 Jul 19;271(29):17360–17365. doi: 10.1074/jbc.271.29.17360. [DOI] [PubMed] [Google Scholar]
  18. Kmiecik T. E., Shalloway D. Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell. 1987 Apr 10;49(1):65–73. doi: 10.1016/0092-8674(87)90756-2. [DOI] [PubMed] [Google Scholar]
  19. Kuo W. L., Abe M., Rhee J., Eves E. M., McCarthy S. A., Yan M., Templeton D. J., McMahon M., Rosner M. R. Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells. Mol Cell Biol. 1996 Apr;16(4):1458–1470. doi: 10.1128/mcb.16.4.1458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kuo W. L., Gehm B. D., Rosner M. R. Regulation of insulin degradation: expression of an evolutionarily conserved insulin-degrading enzyme increases degradation via an intracellular pathway. Mol Endocrinol. 1991 Oct;5(10):1467–1476. doi: 10.1210/mend-5-10-1467. [DOI] [PubMed] [Google Scholar]
  21. Landgren E., Blume-Jensen P., Courtneidge S. A., Claesson-Welsh L. Fibroblast growth factor receptor-1 regulation of Src family kinases. Oncogene. 1995 May 18;10(10):2027–2035. [PubMed] [Google Scholar]
  22. Lim K., Chae C. B. A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase. Biotechniques. 1989 Jun;7(6):576–579. [PubMed] [Google Scholar]
  23. Liu J. J., Chao J. R., Jiang M. C., Ng S. Y., Yen J. J., Yang-Yen H. F. Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells. Mol Cell Biol. 1995 Jul;15(7):3654–3663. doi: 10.1128/mcb.15.7.3654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lowell C. A., Soriano P. Knockouts of Src-family kinases: stiff bones, wimpy T cells, and bad memories. Genes Dev. 1996 Aug 1;10(15):1845–1857. doi: 10.1101/gad.10.15.1845. [DOI] [PubMed] [Google Scholar]
  25. Maness P. F., Aubry M., Shores C. G., Frame L., Pfenninger K. H. c-src gene product in developing rat brain is enriched in nerve growth cone membranes. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5001–5005. doi: 10.1073/pnas.85.14.5001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Marshall C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995 Jan 27;80(2):179–185. doi: 10.1016/0092-8674(95)90401-8. [DOI] [PubMed] [Google Scholar]
  27. Mukhopadhyay D., Tsiokas L., Zhou X. M., Foster D., Brugge J. S., Sukhatme V. P. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature. 1995 Jun 15;375(6532):577–581. doi: 10.1038/375577a0. [DOI] [PubMed] [Google Scholar]
  28. Pang L., Sawada T., Decker S. J., Saltiel A. R. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem. 1995 Jun 9;270(23):13585–13588. doi: 10.1074/jbc.270.23.13585. [DOI] [PubMed] [Google Scholar]
  29. Peng X., Greene L. A., Kaplan D. R., Stephens R. M. Deletion of a conserved juxtamembrane sequence in Trk abolishes NGF-promoted neuritogenesis. Neuron. 1995 Aug;15(2):395–406. doi: 10.1016/0896-6273(95)90043-8. [DOI] [PubMed] [Google Scholar]
  30. Pumiglia K., Chow Y. H., Fabian J., Morrison D., Decker S., Jove R. Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction. Mol Cell Biol. 1995 Jan;15(1):398–406. doi: 10.1128/mcb.15.1.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Qui M. S., Green S. H. PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity. Neuron. 1992 Oct;9(4):705–717. doi: 10.1016/0896-6273(92)90033-a. [DOI] [PubMed] [Google Scholar]
  32. Qureshi S. A., Cao X. M., Sukhatme V. P., Foster D. A. v-Src activates mitogen-responsive transcription factor Egr-1 via serum response elements. J Biol Chem. 1991 Jun 15;266(17):10802–10806. [PubMed] [Google Scholar]
  33. Ray J., Peterson D. A., Schinstine M., Gage F. H. Proliferation, differentiation, and long-term culture of primary hippocampal neurons. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3602–3606. doi: 10.1073/pnas.90.8.3602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Renfranz P. J., Cunningham M. G., McKay R. D. Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain. Cell. 1991 Aug 23;66(4):713–729. doi: 10.1016/0092-8674(91)90116-g. [DOI] [PubMed] [Google Scholar]
  35. Rusanescu G., Qi H., Thomas S. M., Brugge J. S., Halegoua S. Calcium influx induces neurite growth through a Src-Ras signaling cassette. Neuron. 1995 Dec;15(6):1415–1425. doi: 10.1016/0896-6273(95)90019-5. [DOI] [PubMed] [Google Scholar]
  36. Sakai R., Iwamatsu A., Hirano N., Ogawa S., Tanaka T., Mano H., Yazaki Y., Hirai H. A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J. 1994 Aug 15;13(16):3748–3756. doi: 10.1002/j.1460-2075.1994.tb06684.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Samuels M. L., McMahon M. Inhibition of platelet-derived growth factor- and epidermal growth factor-mediated mitogenesis and signaling in 3T3 cells expressing delta Raf-1:ER, an estradiol-regulated form of Raf-1. Mol Cell Biol. 1994 Dec;14(12):7855–7866. doi: 10.1128/mcb.14.12.7855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Samuels M. L., Weber M. J., Bishop J. M., McMahon M. Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase. Mol Cell Biol. 1993 Oct;13(10):6241–6252. doi: 10.1128/mcb.13.10.6241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shihabuddin L. S., Hertz J. A., Holets V. R., Whittemore S. R. The adult CNS retains the potential to direct region-specific differentiation of a transplanted neuronal precursor cell line. J Neurosci. 1995 Oct;15(10):6666–6678. doi: 10.1523/JNEUROSCI.15-10-06666.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Soriano P., Montgomery C., Geske R., Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. doi: 10.1016/0092-8674(91)90499-o. [DOI] [PubMed] [Google Scholar]
  41. Sugrue M. M., Brugge J. S., Marshak D. R., Greengard P., Gustafson E. L. Immunocytochemical localization of the neuron-specific form of the c-src gene product, pp60c-src(+), in rat brain. J Neurosci. 1990 Aug;10(8):2513–2527. doi: 10.1523/JNEUROSCI.10-08-02513.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vaillancourt R. R., Heasley L. E., Zamarripa J., Storey B., Valius M., Kazlauskas A., Johnson G. L. Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation. Mol Cell Biol. 1995 Jul;15(7):3644–3653. doi: 10.1128/mcb.15.7.3644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vicario-Abejón C., Johe K. K., Hazel T. G., Collazo D., McKay R. D. Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons. Neuron. 1995 Jul;15(1):105–114. doi: 10.1016/0896-6273(95)90068-3. [DOI] [PubMed] [Google Scholar]
  44. Williams N. G., Roberts T. M., Li P. Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2922–2926. doi: 10.1073/pnas.89.7.2922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yan M., Templeton D. J. Identification of 2 serine residues of MEK-1 that are differentially phosphorylated during activation by raf and MEK kinase. J Biol Chem. 1994 Jul 22;269(29):19067–19073. [PubMed] [Google Scholar]
  46. Zhan X., Plourde C., Hu X., Friesel R., Maciag T. Association of fibroblast growth factor receptor-1 with c-Src correlates with association between c-Src and cortactin. J Biol Chem. 1994 Aug 12;269(32):20221–20224. [PubMed] [Google Scholar]
  47. van der Geer P., Hunter T., Lindberg R. A. Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol. 1994;10:251–337. doi: 10.1146/annurev.cb.10.110194.001343. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES