Cell-cycle arrest in TrkA-expressing NIH3T3 cells involves nitric oxide synthase

We have examined nerve growth factor (NGF)-triggered signaling in two NIH3T 3 cell lines exogenously expressing the NGF receptor, TrkA. TRK1 cells ceas e to proliferate and extend long processes in response to NGF, while E25 ce lls continue to proliferate in the presence of NGF. These two cell lines ex press similar levels of TrkA and respond to NGF with rapid elevation of mit ogen-activated protein kinase (MAPK) activity. MAPK activation is slightly more sustained for E25 cells than for TRK1 cells, although sustained activa tion of MAPK has been suggested to cause cell-cycle arrest. As judged by NA DPH-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 level s 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-d ependent kinase inhibitor, p21(WAF1), TRK1 cells only. NGF-induced p21(WAF1 ) expression, cell-cycle arrest and process extension are abolished by N-ni tro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS. The i nactive enantiomer, D-NAME, did not inhibit these responses. Furthermore, e ven 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 NIH3 T3 cell line that undergoes cell-cycle arrest and morphological changes in response to NGF. These results demonstrate that sustained activation of MAP K 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. (C) 2001 Wiley-Liss, Inc.