Ethanolamine, but not phosphoethanolamine, potentiates the effects of insulin, phosphocholine, and ATP on DNA synthesis in NIH 3T3 cells--role of mitogen-activated protein-kinase-dependent and protein-kinase-independent mechanisms.

NIH 3T3 fibroblasts express a phospholipase D activity hydrolyzing phosphatidylethanolamine (PtdEtn) which produces ethanolamine (Etn) in response to a variety of growth regulating agents. The main objective of this work was to evaluate the effects of Etn on mitogenesis and to determine whether these effects require its metabolism to phosphoethanolamine (PEtn) or PtdEtn. To increase conversion of Etn to PEtn, an Etn-specific kinase derived from Drosophila was highly expressed in NIH 3T3 cells. Overexpression of this Etn kinase resulted in large (10-12.5-fold) increases in PEtn formation, but only in modest (1.2-1.7-fold) increases in PtdEtn synthesis. In both vector control and Etn kinase overexpressor cells, Etn had biphasic effects on insulin-induced DNA synthesis with maximal (approximately 2-fold) potentiating effects being observed at 0.5-1 mM concentrations, followed by an inhibitory phase at higher Etn concentrations. In the Etn kinase overexpressor lines, the inhibitory phase was elicited by lower Etn concentrations and it was partially blocked by 5 mM choline due to decreased formation of PEtn. In both vector control and Etn kinase overexpressor cells, phosphocholine (PCho) and insulin synergistically stimulated DNA synthesis; their effects were further enhanced by physiologically relevant (5-60 microM) concentrations of Etn by a mechanism independent of mitogen-activated protein (MAP) kinase. Concentrations of Etn >50 microM also enhanced the effects of both PCho and the synergistic effects of PCho plus ATP; however, in the latter case 20 microM Etn was inhibitory. The magnitude of both the potentiating and inhibitory effects of Etn on PCho-induced as well as PCho + ATP-induced DNA synthesis were similar in the vector control and Etn kinase overexpressor cells; they were associated with stimulation and inhibition, respectively, of p42 MAP kinase activity. The results indicate that in NIH 3T3 cells Etn exerts significant effects on DNA synthesis which, except inhibition of insulin-induced DNA synthesis by higher concentrations of Etn, do not correlate with the metabolism of Etn to PEtn or PtdEtn.