In serum-starved NIH 3T3 clone 7 fibroblasts, choline phosphate (ChoP) (0.5-1 mM) and insulin synergistically stimulate DNA synthesis. Here we report that ATP also greatly enhanced the mitogenic effects of ChoP (0.1-1 mM) both in the absence and presence of insulin; maximal potentiating effects required 50 -100 M ATP. The co-mitogenic effects of ATP were mimicked by adenosine 5-O-(3-thiotriphosphate), adenosine 5-O-(2-thiodiphosphate), ADP, and UTP, but not by AMP or adenosine, indicating the mediatory role of a purinergic P 2 receptor. Externally added ChoP acted on DNA synthesis without its detectable uptake into fibroblasts, indicating that ChoP can be a mitogen only if it is released from cells. Extracellular ATP (10 -100 M) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the presence of ATP or insulin, on the activity state of p42/p44 mitogen-activated protein kinases, while in combination these agents stimulated the activity of phosphatidylinositol 3-kinase (PI 3-kinase). Expression of a dominant negative mutant of the p85 subunit of PI 3-kinase or treatments with the PI 3-kinase inhibitor wortmannin only partially (ϳ40 -50%) reduced the combined effects of ChoP, ATP, and insulin on DNA synthesis; in contrast, the pp70 S6 kinase inhibitor rapamycin almost completely inhibited these effects. ATP and insulin also potentiated, while rapamycin strongly inhibited, the mitogenic effects of sphingosine 1-phosphate (S1P). Furthermore, even maximally effective concentrations of ChoP and S1P synergistically stimulated DNA synthesis. The results indicate that in the presence of extracellular ATP and/or S1P, ChoP induces mitogenesis through an extracellular site by mechanisms involving the activation of pp70 S6 kinase and, to a lesser extent, PI 3-kinase.

Stimulation of phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho) by phorbol 12-myristate 13-acetate (PMA) has been shown to be mediated by the ␣-and ␤I-isoforms of protein kinase C (PKC). To determine the role of various PKC isozymes in the regulation of PLD-mediated phosphatidylethanolamine (PtdEtn) hydrolysis, MCF-7 human breast carcinoma cells overexpressing the ␣-and -isoforms, and R6 rat fibroblasts overexpressing the ␣-, ␤I-, and ⑀-isoforms were used. In the vector control MCF-7 cells, which contain low levels of PKC-␣, PMA (100 nM) had only small effects on the hydrolysis of PtdEtn (1.1-1.35-fold) and PtdCho (1.15-1.6-fold). Stable expression of PKC-␣ in MCF-7 cells, which was accompanied by increased levels of the ␤I-and -isoforms as well, greatly enhanced both PMA-induced PLD-mediated formation of phosphatidylethanol (ϳ5-fold) and the hydrolysis of PtdEtn (2.5-2.9-fold) and PtdCho (5.5-7.2-fold). The effects of PMA on the hydrolysis of PtdEtn (and PtdCho) in MCF-7/PKC-␣ cells were significantly inhibited by 0.5-3 M concentrations of Gö 6976, a selective inhibitor of the conventional PKC subfamily. Stable expression of PKC-␣ in R6 fibroblasts enhanced, at a shorter (10 min) incubation time, the effects of PMA on the hydrolysis of both PtdEtn and, to a lesser extent, PtdCho. In contrast, stable expression of PKC-␤I in R6 fibroblasts, which originally did not contain this enzyme, enhanced the effects of PMA only on PtdCho, but not PtdEtn, hydrolysis. Overexpression of either PKC-in MCF-7 cells or PKC-⑀ in R6 and NIH 3T3 fibroblasts had no detectable effects on PMA-induced hydrolysis of PtdEtn. Collectively, the results suggest that PKC-␣ has a major role in the mediation of phorbol ester action on PtdEtn hydrolysis, while PtdCho hydrolysis may be regulated by both the ␣ and ␤I isoforms.In many cell lines activators of phospholipase D (PLD), 1 including the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), appear to stimulate only the hydrolysis of phosphatidylcholine (PtdCho) (1, 2). In MadinDarby canine kidney cells (3) and in Swiss/3T3 cells (4) PKC-␣ was shown to be a major regulator of PtdCho hydrolysis. However, expression of PKC-␤I (5) or addition of PKC-␤I to isolated membranes (6, 7) also enhanced the effect of PMA on PtdCho hydrolysis. Interestingly, while PKC-␣ was a more effective mediator of PMA effect in lung fibroblast membranes than PKC-␤I (6), a reversed order of potency was observed in neutrophil membranes (7). Collectively, these data suggest that if expressed, both PKC-␣ and PKC-␤I may be able to regulate PtdCho hydrolysis.The role of increased PtdCho hydrolysis in the mediation of cellular actions of PKC activators is unknown. Recently, we presented evidence showing that in NIH 3T3 fibroblasts PtdCho hydrolysis is unlikely to play a major role in the mediation of mitogenic effects of PMA (8). In fact, choline phosphate, which in agonist-treated cells can be formed by the sequential actions of PLD and choline kinase, and PMA were found to sti...

In most cellular systems ethanol inhibits growth factor-induced cell growth. Here we examined the effects of ethanol on DNA synthesis and cell proliferation induced by insulin and phosphocholine (PCho) in NIH3T3 fibroblasts, Swiss 3T3 fibroblasts and mouse epidermal JB6 cells. In serum-starved low (12-18) passage NIH3T3 fibroblasts, 60 mM ethanol enhanced the mitogenic effect of insulin in the absence or presence of 25 microM zinc about 2- or 12-fold, respectively. In contrast, in serum-starved high (30-47) passage NIH3T3 cells 60 mM ethanol had large (20-40-fold) potentiating effects on insulin-induced DNA synthesis even in the absence of zinc. Furthermore, ethanol also enhanced the effects of PCho on DNA synthesis in both the absence and presence of insulin. The potentiating effects of ethanol on insulin- and PCho-induced DNA synthesis were associated with 1.2-1.3-fold stimulation of cell proliferation. Rapamycin, an inhibitor of p70 S6 kinase action, strongly inhibited the potentiating effects of ethanol on insulin- and PCho-induced mitogenesis. Unexpectedly, ethanol inhibited synergistic activation of p42/p44 mitogen-activated protein kinases by insulin and PCho. In both Swiss 3T3 and JB6 cells, ethanol potentiated insulin-induced DNA synthesis only in the presence of zinc. In these cells, ethanol also increased the effects of PCho on both DNA synthesis and cell proliferation in the co-presence of either insulin or ATP. The results indicate that in various cell lines physiologically relevant concentrations of ethanol can increase the ability of insulin and PCho to induce DNA synthesis and, to smaller extents, cell proliferation. In low passage NIH3T3 cells as well as in Swiss 3T3 and JB6 cells potentiation of insulin-induced DNA synthesis by ethanol requires the presence of zinc.