NGF-dependent formation of ruffles in PC12D cells required a different pathway from that for neurite outgrowth.
Two signaling pathways, phosphoinositide 3-kinase (PI-3k)/Akt and Ras/MAPK, are major effectors triggered by nerve growth factor (NGF). Rac1, Cdc42 and GSK-3beta are reported to be targets of PI-3k in the signal transduction for neurite outgrowth. Immediately after NGF was added, broad ruffles were observed temporarily around the periphery of PC12 cells prior to neurite growth. As PC12D cells are characterized by a very rapid extension of neurites in response to various agents, the signaling pathways described above were studied in relation to the NGF-induced formation of ruffles and outgrowth of neurites. Wortmannin, an Akt inhibitor (V), and GSK-3beta inhibitor (SB425286) suppressed the neurite growth in NGF-treated cells, but not in dbcAMP-treated cells. The outgrowth of neurites induced by NGF but not by dbcAMP was inhibited with the expression of mutant Ras. But upon the expression of dominant-negative Rac1, cells often extended protrusions, incomplete neurites, lacking F-actin. Intact neurites were observed in cells with dominant-negative Cdc42. These results suggest that NGF-dependent neurite outgrowth occurs via a mechanism involving activation of the Ras/PI-3K/Akt/GSK-3beta pathway, while dbcAMP-dependent neurite growth might be induced in a distinct manner. However, inhibitors for GSK-3beta and PI-3k (wortmannin) did not suppress the NGF-dependent formation of ruffles. In addition, the formation of ruffles was not inhibited by the expression of mutant Ras. On the other hand, it was suppressed by the expression of dominant-negative Rac1 or Cdc42. These results suggest that the NGF-induced ruffling requires activation of Rac1 and Cdc42, but does not require Ras, PI-3k, Akt and GSK-3beta. Taken together, the NGF-dependent formation of ruffles might not require Ras/PI-3k/Akt/GSK-3beta, but these pathways might contribute to the formation of intact neurites due to combined actions including Rac1.
Jin
Erika
E
Department of Biology, Kyoto Prefectural University of Medicine, Taisyogun Nishitakatsukasa-cho 13, Kita-ku, Kyoto 603-8334, Japan. erika-j@koto.kpu-m.ac.jp
Nosaka
Kazuto
K
Sano
Mamoru
M
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D022081:Cell Surface Extensions / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000187:drug effects / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D009154:Mutation / Q000235:genetics; D020932:Nerve Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D020764:cdc42 GTP-Binding Protein / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000187:drug effects / Q000378:metabolism
D004791:Enzyme Inhibitors; D020932:Nerve Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D018631:ras Proteins