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. 1995 Aug 1;130(3):701–710. doi: 10.1083/jcb.130.3.701

Stimulation of neurite outgrowth in PC12 cells by EGF and KCl depolarization: a Ca(2+)-independent phenomenon

PMCID: PMC2120542  PMID: 7622569

Abstract

MAP kinase activity is necessary for growth factor induction of neurite outgrowth in PC12 cells. Although NGF and EGF both stimulate MAP kinase activity, EGF does not stimulate neurite extension. We report that EGF, in combination with KCl, stimulates neurite outgrowth in PC12 cells. This phenomenon was independent of intracellular Ca2+ increases and not due to enhancement of MAP kinase activity over that seen with EGF alone. However, EGF plus KCl increased intracellular cAMP, and other cAMP elevating agents acted synergistically with EGF to promote neurite outgrowth. Stimulation of neurite outgrowth by cAMP and EGF was blocked by inhibitors of transcription suggesting that synergistic regulation of transcription by the cAMP and MAP kinase pathways may stimulate neurite growth.

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Selected References

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  1. Ahn N. G., Seger R., Krebs E. G. The mitogen-activated protein kinase activator. Curr Opin Cell Biol. 1992 Dec;4(6):992–999. doi: 10.1016/0955-0674(92)90131-u. [DOI] [PubMed] [Google Scholar]
  2. Ball R. L., Tanner K. D., Carpenter G. Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells. J Biol Chem. 1990 Aug 5;265(22):12836–12845. [PubMed] [Google Scholar]
  3. Bokoch G. M., Katada T., Northup J. K., Hewlett E. L., Gilman A. G. Identification of the predominant substrate for ADP-ribosylation by islet activating protein. J Biol Chem. 1983 Feb 25;258(4):2072–2075. [PubMed] [Google Scholar]
  4. Boonstra J., Moolenaar W. H., Harrison P. H., Moed P., van der Saag P. T., de Laat S. W. Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells. J Cell Biol. 1983 Jul;97(1):92–98. doi: 10.1083/jcb.97.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bothwell M. A., Schechter A. L., Vaughn K. M. Clonal variants of PC12 pheochromocytoma cells with altered response to nerve growth factor. Cell. 1980 Oct;21(3):857–866. doi: 10.1016/0092-8674(80)90449-3. [DOI] [PubMed] [Google Scholar]
  6. Bravo R., Neuberg M., Burckhardt J., Almendral J., Wallich R., Müller R. Involvement of common and cell type-specific pathways in c-fos gene control: stable induction of cAMP in macrophages. Cell. 1987 Jan 30;48(2):251–260. doi: 10.1016/0092-8674(87)90428-4. [DOI] [PubMed] [Google Scholar]
  7. Budnik L. T., Mukhopadhyay A. K. Epidermal growth factor, a modulator of luteal adenylate cyclase. Characterization of epidermal growth factor receptors and its interaction with adenylate cyclase system in bovine luteal cell membrane. J Biol Chem. 1991 Jul 25;266(21):13908–13913. [PubMed] [Google Scholar]
  8. Budnik L. T., Mukhopadhyay A. K. Pertussis toxin can distinguish the augmentary effect elicited by epidermal growth factor from that of phorbol ester on luteal adenylate cyclase activity. Endocrinology. 1993 Jul;133(1):265–270. doi: 10.1210/endo.133.1.8319575. [DOI] [PubMed] [Google Scholar]
  9. Burstein D. E., Greene L. A. Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6059–6063. doi: 10.1073/pnas.75.12.6059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chao M. V. Growth factor signaling: where is the specificity? Cell. 1992 Mar 20;68(6):995–997. doi: 10.1016/0092-8674(92)90068-n. [DOI] [PubMed] [Google Scholar]
  11. Cohen S. The stimulation of epidermal proliferation by a specific protein (EGF). Dev Biol. 1965 Dec;12(3):394–407. doi: 10.1016/0012-1606(65)90005-9. [DOI] [PubMed] [Google Scholar]
  12. Connolly J. L., Green S. A., Greene L. A. Comparison of rapid changes in surface morphology and coated pit formation of PC12 cells in response to nerve growth factor, epidermal growth factor, and dibutyryl cyclic AMP. J Cell Biol. 1984 Feb;98(2):457–465. doi: 10.1083/jcb.98.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Frödin M., Peraldi P., Van Obberghen E. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells. J Biol Chem. 1994 Feb 25;269(8):6207–6214. [PubMed] [Google Scholar]
  15. Fu X. Y., Zhang J. J. Transcription factor p91 interacts with the epidermal growth factor receptor and mediates activation of the c-fos gene promoter. Cell. 1993 Sep 24;74(6):1135–1145. doi: 10.1016/0092-8674(93)90734-8. [DOI] [PubMed] [Google Scholar]
  16. Ginty D. D., Bonni A., Greenberg M. E. Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell. 1994 Jun 3;77(5):713–725. doi: 10.1016/0092-8674(94)90055-8. [DOI] [PubMed] [Google Scholar]
  17. Ginty D. D., Glowacka D., DeFranco C., Wagner J. A. Nerve growth factor-induced neuronal differentiation after dominant repression of both type I and type II cAMP-dependent protein kinase activities. J Biol Chem. 1991 Aug 15;266(23):15325–15333. [PubMed] [Google Scholar]
  18. Ginty D. D., Kornhauser J. M., Thompson M. A., Bading H., Mayo K. E., Takahashi J. S., Greenberg M. E. Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science. 1993 Apr 9;260(5105):238–241. doi: 10.1126/science.8097062. [DOI] [PubMed] [Google Scholar]
  19. Gotoh Y., Nishida E., Yamashita T., Hoshi M., Kawakami M., Sakai H. Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells. Eur J Biochem. 1990 Nov 13;193(3):661–669. doi: 10.1111/j.1432-1033.1990.tb19384.x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Greenberg M. E., Siegfried Z., Ziff E. B. Mutation of the c-fos gene dyad symmetry element inhibits serum inducibility of transcription in vivo and the nuclear regulatory factor binding in vitro. Mol Cell Biol. 1987 Mar;7(3):1217–1225. doi: 10.1128/mcb.7.3.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  24. Gunning P. W., Landreth G. E., Bothwell M. A., Shooter E. M. Differential and synergistic actions of nerve growth factor and cyclic AMP in PC12 cells. J Cell Biol. 1981 May;89(2):240–245. doi: 10.1083/jcb.89.2.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hashimoto S. K-252a, a potent protein kinase inhibitor, blocks nerve growth factor-induced neurite outgrowth and changes in the phosphorylation of proteins in PC12h cells. J Cell Biol. 1988 Oct;107(4):1531–1539. doi: 10.1083/jcb.107.4.1531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Heidemann S. R., Joshi H. C., Schechter A., Fletcher J. R., Bothwell M. Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells. J Cell Biol. 1985 Mar;100(3):916–927. doi: 10.1083/jcb.100.3.916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Huff K., End D., Guroff G. Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor. J Cell Biol. 1981 Jan;88(1):189–198. doi: 10.1083/jcb.88.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Huffaker T., Corcoran T., Wagner J. A. Adenosine inhibits cell division and promotes neurite extension in PC12 cells. J Cell Physiol. 1984 Aug;120(2):188–196. doi: 10.1002/jcp.1041200212. [DOI] [PubMed] [Google Scholar]
  29. Hughes A. R., Martin M. W., Harden T. K. Pertussis toxin differentiates between two mechanisms of attenuation of cyclic AMP accumulation by muscarinic cholinergic receptors. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5680–5684. doi: 10.1073/pnas.81.18.5680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Impey S., Wayman G., Wu Z., Storm D. R. Type I adenylyl cyclase functions as a coincidence detector for control of cyclic AMP response element-mediated transcription: synergistic regulation of transcription by Ca2+ and isoproterenol. Mol Cell Biol. 1994 Dec;14(12):8272–8281. doi: 10.1128/mcb.14.12.8272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Katada T., Ui M. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci U S A. 1982 May;79(10):3129–3133. doi: 10.1073/pnas.79.10.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Knecht M., Catt K. J. Modulation of cAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem. 1983 Mar 10;258(5):2789–2794. [PubMed] [Google Scholar]
  33. Kuo C. J., Chung J., Fiorentino D. F., Flanagan W. M., Blenis J., Crabtree G. R. Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature. 1992 Jul 2;358(6381):70–73. doi: 10.1038/358070a0. [DOI] [PubMed] [Google Scholar]
  34. Kurokawa M., Doctrow S. R., Klagsbrun M. Neutralizing antibodies inhibit the binding of basic fibroblast growth factor to its receptor but not to heparin. J Biol Chem. 1989 May 5;264(13):7686–7691. [PubMed] [Google Scholar]
  35. Machida C. M., Scott J. D., Ciment G. NGF-induction of the metalloproteinase-transin/stromelysin in PC12 cells: involvement of multiple protein kinases. J Cell Biol. 1991 Sep;114(5):1037–1048. doi: 10.1083/jcb.114.5.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miyasaka T., Chao M. V., Sherline P., Saltiel A. R. Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2. J Biol Chem. 1990 Mar 15;265(8):4730–4735. [PubMed] [Google Scholar]
  37. Nakafuku M., Kaziro Y. Epidermal growth factor and transforming growth factor-alpha can induce neuronal differentiation of rat pheochromocytoma PC12 cells under particular culture conditions. FEBS Lett. 1993 Jan 11;315(3):227–232. doi: 10.1016/0014-5793(93)81169-z. [DOI] [PubMed] [Google Scholar]
  38. Noda M., Ko M., Ogura A., Liu D. G., Amano T., Takano T., Ikawa Y. Sarcoma viruses carrying ras oncogenes induce differentiation-associated properties in a neuronal cell line. Nature. 1985 Nov 7;318(6041):73–75. doi: 10.1038/318073a0. [DOI] [PubMed] [Google Scholar]
  39. Pettmann B., Louis J. C., Sensenbrenner M. Morphological and biochemical maturation of neurones cultured in the absence of glial cells. Nature. 1979 Oct 4;281(5730):378–380. doi: 10.1038/281378a0. [DOI] [PubMed] [Google Scholar]
  40. Puck T. T. Cyclic AMP, the microtubule-microfilament system, and cancer. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4491–4495. doi: 10.1073/pnas.74.10.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ran W., Dean M., Levine R. A., Henkle C., Campisi J. Induction of c-fos and c-myc mRNA by epidermal growth factor or calcium ionophore is cAMP dependent. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8216–8220. doi: 10.1073/pnas.83.21.8216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ritchie A. K. Catecholamine secretion in a rat pheochromocytoma cell line: two pathways for calcium entry. J Physiol. 1979 Jan;286:541–561. doi: 10.1113/jphysiol.1979.sp012636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rosen L. B., Ginty D. D., Weber M. J., Greenberg M. E. Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras. Neuron. 1994 Jun;12(6):1207–1221. doi: 10.1016/0896-6273(94)90438-3. [DOI] [PubMed] [Google Scholar]
  44. Rosenberg A., Noble E. P. EGF-induced neuritogenesis and correlated synthesis of plasma membrane gangliosides in cultured embryonic chick CNS neurons. J Neurosci Res. 1989 Dec;24(4):531–536. doi: 10.1002/jnr.490240411. [DOI] [PubMed] [Google Scholar]
  45. Rudkin B. B., Lazarovici P., Levi B. Z., Abe Y., Fujita K., Guroff G. Cell cycle-specific action of nerve growth factor in PC12 cells: differentiation without proliferation. EMBO J. 1989 Nov;8(11):3319–3325. doi: 10.1002/j.1460-2075.1989.tb08493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Saffell J. L., Walsh F. S., Doherty P. Direct activation of second messenger pathways mimics cell adhesion molecule-dependent neurite outgrowth. J Cell Biol. 1992 Aug;118(3):663–670. doi: 10.1083/jcb.118.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Salomon Y., Londos C., Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974 Apr;58(2):541–548. doi: 10.1016/0003-2697(74)90222-x. [DOI] [PubMed] [Google Scholar]
  48. Seger R., Seger D., Reszka A. A., Munar E. S., Eldar-Finkelman H., Dobrowolska G., Jensen A. M., Campbell J. S., Fischer E. H., Krebs E. G. Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells. Evidence that MAPKK involvement in cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and VIII. J Biol Chem. 1994 Oct 14;269(41):25699–25709. [PubMed] [Google Scholar]
  49. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stallcup W. B. Sodium and calcium fluxes in a clonal nerve cell line. J Physiol. 1979 Jan;286:525–540. doi: 10.1113/jphysiol.1979.sp012635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sun H., Seyer J. M., Patel T. B. A region in the cytosolic domain of the epidermal growth factor receptor antithetically regulates the stimulatory and inhibitory guanine nucleotide-binding regulatory proteins of adenylyl cyclase. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2229–2233. doi: 10.1073/pnas.92.6.2229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Thomas S. M., DeMarco M., D'Arcangelo G., Halegoua S., Brugge J. S. Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases. Cell. 1992 Mar 20;68(6):1031–1040. doi: 10.1016/0092-8674(92)90075-n. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Traverse S., Gomez N., Paterson H., Marshall C., Cohen P. Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem J. 1992 Dec 1;288(Pt 2):351–355. doi: 10.1042/bj2880351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Traverse S., Seedorf K., Paterson H., Marshall C. J., Cohen P., Ullrich A. EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curr Biol. 1994 Aug 1;4(8):694–701. doi: 10.1016/s0960-9822(00)00154-8. [DOI] [PubMed] [Google Scholar]
  56. Treisman R. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors. Cell. 1986 Aug 15;46(4):567–574. doi: 10.1016/0092-8674(86)90882-2. [DOI] [PubMed] [Google Scholar]
  57. Verma I. M., Sassone-Corsi P. Proto-oncogene fos: complex but versatile regulation. Cell. 1987 Nov 20;51(4):513–514. doi: 10.1016/0092-8674(87)90115-2. [DOI] [PubMed] [Google Scholar]
  58. Wayman G. A., Impey S., Wu Z., Kindsvogel W., Prichard L., Storm D. R. Synergistic activation of the type I adenylyl cyclase by Ca2+ and Gs-coupled receptors in vivo. J Biol Chem. 1994 Oct 14;269(41):25400–25405. [PubMed] [Google Scholar]
  59. Wong Y. H., Federman A., Pace A. M., Zachary I., Evans T., Pouysségur J., Bourne H. R. Mutant alpha subunits of Gi2 inhibit cyclic AMP accumulation. Nature. 1991 May 2;351(6321):63–65. doi: 10.1038/351063a0. [DOI] [PubMed] [Google Scholar]
  60. Wood K. W., Qi H., D'Arcangelo G., Armstrong R. C., Roberts T. M., Halegoua S. The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for cellular raf kinases in neuronal growth factor signal transduction. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5016–5020. doi: 10.1073/pnas.90.11.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wood K. W., Sarnecki C., Roberts T. M., Blenis J. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 1992 Mar 20;68(6):1041–1050. doi: 10.1016/0092-8674(92)90076-o. [DOI] [PubMed] [Google Scholar]

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