Nerve growth factor in combination with second messenger analogues causes neuronal differentiation of PC12 cells expressing a dominant inhibitory Rasprotein without inducing activation of extracellular signal-regulated kinases

In the present work, nerve growth factor (NGF) was used in combination with the calcium ionophore, ionomycin or dibutyryl cyclic AMP (dbcAMP), to stud y the connection between neuronal differentiation and extracellular signal- regulated kinase (ERK) activation of PC12 rat pheochromocytoma cells expres sing a dominant negative, Ha-Ras Asn17 protein. Due to the block of endogen ous Ras activity, neurite outgrowth in response to NGF is completely inhibi ted in these cells. However, this blockade can be bypassed by combined trea tment with NGF plus ionomycin or NGF plus dbcAMP. The mitogen-activated pro tein kinase (MAPK)/ERK kinase inhibitor, PD98069, proved to be insufficient in inhibiting the neurite outgrowth under these conditions. Moreover, alth ough both long-term ERK activation and nuclear translocation of ERKs are be lieved to be key events in neuronal differentiation, neither detectable ERK phosphorylation, nor nuclear translocation of these enzymes, occurred upon combination treatments in our experimental system. However, the neuritogen esis induced by either the combination of NGF/ionomycin or NGF/dbcAMP was i nhibited by the Trk inhibitor, K252a. Ras-independent pathways, originating from the NGF receptor, can thus synergize with second messenger analogues bypassing the ERK cascade but leading to the same biological result-neurite formation.