Cell-cycle arrest in TrkA-expressing NIH3T3 cells involves nitric oxide synthase.
We have examined nerve growth factor (NGF)-triggered signaling in two NIH3T3 cell lines exogenously expressing the NGF receptor, TrkA. TRK1 cells cease to proliferate and extend long processes in response to NGF, while E25 cells continue to proliferate in the presence of NGF. These two cell lines express similar levels of TrkA and respond to NGF with rapid elevation of mitogen-activated protein kinase (MAPK) activity. MAPK activation is slightly more sustained for E25 cells than for TRK1 cells, although sustained activation of MAPK has been suggested to cause cell-cycle arrest. As judged by NADPH-diaphorase staining, nitric oxide synthase (NOS) activity is increased in TRK1 cells upon exposure to NGF. In contrast, diaphorase staining in E25 cells is unaffected by NGF treatment. Immunocytochemistry shows that levels of the brain NOS (bNOS) isoform are increased in TRK1, but not E25, cells exposed to NGF. Furthermore, Western blots show that NGF elevated cyclin-dependent kinase inhibitor, p21(WAF1), in TRK1 cells only. NGF-induced p21(WAF1) expression, cell-cycle arrest and process extension are abolished by N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS. The inactive enantiomer, D-NAME, did not inhibit these responses. Furthermore, even though E25 cells do not respond to NGF or nitric oxide donors, they do undergo a morphological change in response to NGF plus a nitric oxide donor. Therefore, NOS and p21(WAF1) are induced only in the TrkA-expressing NIH3T3 cell line that undergoes cell-cycle arrest and morphological changes in response to NGF. These results demonstrate that sustained activation of MAPK is not the sole determining factor for NGF-induced cell-cycle arrest and implicate NO in the cascade of events leading to NGF-induced morphological changes and cell-cycle arrest.
Bulseco
D A
DA
Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Poluha
W
W
Schonhoff
C M
CM
Daou
M C
MC
Condon
P J
PJ
Ross
A H
AH
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D005434:Flow Cytometry; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D020917:Receptor, trkA / Q000378:metabolism*
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D020030:Nitric Oxide Donors; D019001:Nitric Oxide Synthase; D020917:Receptor, trkA; D020928:Mitogen-Activated Protein Kinases