ETHANOLAMINE, BUT NOT PHOSPHOETHANOLAMINE, POTENTIATES THE EFFECTS OFINSULIN, 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 pho sphalidylethanolamine (PtdEtn) which produces ethanolamine (Etn) in re sponse to a variety of growth regulating agents. The main objective of this work was to evaluate the effects of Etn on mitogenesis and to de termine whether these effects require its metabolism to phosphoethanol amine (PEtn) or PtdEtn. To increase conversion of Etn to PEtn, an Etn- specific kinase derived from Drosophila was highly expressed in NM 3T3 cells. Overexpression of this Etn kinase resulted in large (10-12.5-f old) increases in PEtn formation, but only in modest (1.2-1.7-fold) in creases in PtdEtn synthesis. In both vector control and Etn kinase ove rexpressor cells, Etn had biphasic effects on insulin-induced DNA synt hesis with maximal (approximate to 2-fold) potentiating effects being observed at 0.5-1 mM concentrations, followed by an inhibitory phase a t higher Etn concentrations. In the Etn kinase overexpressor Lines, th e 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, phosphocho line (PCho) and insulin synergistically stimulated DNA synthesis; thei r effects were further enhanced by physiologically relevant (5-60 mu M ) concentrations of Etn by a mechanism independent of mitogen-activate d protein (MAP) kinase. Concentrations of Etn >50 mu M also enhanced t he effects of both PCho and the synergistic effects of PCho plus ATP; however, in the latter case 20 mu M 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 vecto r control and Etn kinase overexpressor cells; they were associated wit h stimulation and inhibition, respectively, of p42 MAP kinase activity . The results indicate that in NM 3T3 cells Etn exerts significant eff ects on DNA synthesis which, except inhibition of insulin-induced DNA synthesis by higher concentrations of Etn, do not correlate with the m etabolism of Etn to PEtn or PtdEtn.