Synergistic Potentiating Effects of Choline Phosphate and Ethanolamine on Insulin-Induced DNA Synthesis in NIH 3T3 Fibroblasts

Tomono, Masahiro; Crilly, Karan S.; Kiss, Zoltán

doi:10.1006/bbrc.1995.2225

Cited by 20 publications

(13 citation statements)

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“…We have previously reported [2] that in NIH 3T3 fibroblasts ethanolamine alone has no mitogenic effect, but it can significantly enhance the relatively modest stimulatory effect of insulin on DNA synthesis. As shown in Fig.…”

Section: Resultsmentioning

confidence: 97%

“…Recent reports from another [1] and our laboratory [2,3] demonstrated that in NIH 3T3 fibroblasts externally added choline phosphate can greatly enhance DNA synthesis particularly in the presence of insulin. Although the significance of this phenomenon still remains to be elucidated, it would be important to know whether other intermediates of phospholipid metabolism can also regulate cell growth, and if yes, how it is related to phospholipid synthesis.…”

Section: Introductionmentioning

confidence: 97%

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Ethanolamine analogues stimulate DNA synthesis by a mechanism not involving phosphatidylethanolamine synthesis

Kiss

Crilly

1996

FEBS Letters

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Dimethylethanolamine (0.5-1 mM), added to serumstarved N1H 3T3 fibroblasts, stimulated DNA synthesis 11-32-fold, and it also greatly enhanced the relatively modest (15-20-fold) mitogenic effect of insulin. Ethanolamine and momomethylethanolamine alone had no effects on DNA synthesis, but they also enhanced the stimulatory effect of insulin, although less effectively than dimethylethanolamine did. Lower concentrations (2.5-5 ~tglml) of compound D 609 (tricyclo-9-yl-xanthogenate), which had no effects on phospholipase activities, synergistically enhanced the combined effects of ethanolamine analogs and insulin on DNA synthesis without affecting the synthesis of ethanolamine phospholipids. These results suggest that ethanolamine and its analogues, formed by phospholipase D-mediated hydrolysis of ethanolamine phospholipids, may have growth regulatory functions independent of their role as phospholipid precursors.

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Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 97%

Ethanolamine analogues stimulate DNA synthesis by a mechanism not involving phosphatidylethanolamine synthesis

Kiss

Crilly

1996

FEBS Letters

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“…In growth factor-stimulated NIH 3T3 cells, the choline liberated is phosphorylated by choline kinase to produce phosphocholine (18). This step is thought to be essential for the activation of Raf-1 and MAP kinases, which are thought to be involved in the transmission of signals to the nucleus (18,19). Consequently, synergistic stimulation with Etn of the hepatocyte proliferation might be due to the enhanced production of these mediators in the hepatocytes that have been stimulated by a growth factor or factors.…”

Section: Discussionmentioning

confidence: 99%

Ethanolamine modulates the rate of rat hepatocyte proliferation in vitro and in vivo

Sasaki

Kume

Nemoto

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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A low molecular weight, heat-resistant hepatotrophic factor in an extract from the bovine intestinal mucosa was purified and identified as ethanolamine by structural analyses. The mode of action of ethanolamine in vitro and in vivo coincided with that of the crude extract of the tissue, indicating that ethanolamine is the active component. Ethanolamine synergistically elevated the stimulation of DNA synthesis in hepatocytes in primary culture when added together with a growth factor, such as epidermal growth factor, with the ED 50 being 20 M, although it showed little stimulatory effect by itself. Contrary to these in vitro results, the intraperitoneal administration of ethanolamine hydrochloride (24 mg of ethanolamine per kg of body weight) enhanced hepatocyte proliferation in regenerating rat livers after two-thirds hepatectomy without the administration of any growth factors. In the regenerating liver, hepatocyte proliferation may be initiated by an endogenous growth factor, but the supply of ethanolamine in circulation may not be sufficient for optimal hepatocyte proliferation; thus, the exogenous administration of ethanolamine may further enhance hepatocyte proliferation. Ethanolamine in circulation may be a humoral hepatotrophic factor.Previously we reported that the proliferation of hepatocytes in primary culture was synergistically stimulated by a low molecular weight (LMW, Ͻ1,000) and a high molecular weight (HMW, Ͼ10,000) factor that were derived from the bovine small intestine mucosa, separated by their solubility in methanol (1). The LMW factor synergistically enhanced the basal level of stimulation by the HMW factor, while it had little effect when added alone. Since the LMW factor could enhance the level of DNA synthesis in hepatocytes that were stimulated by epidermal growth factor (EGF) or hepatocyte growth factor (HGF), it acts by amplifying the effects of a growth factor. Contrary to the in vitro result, the LMW factor could stimulate hepatocyte proliferation in regenerating livers even when added alone. We speculate that an endogenous growth factor(s) is probably produced in the animals with the regenerating liver, but the supply of the LMW factor is below the optimal level, thus the exogenous supply of the LMW factor can further stimulate hepatocyte proliferation. The LMW factor may function as a humoral hepatotrophic factor for liver regeneration.We purified the LMW factor from the bovine small intestinal mucosa, and we identified it as ethanolamine (Etn). The commercially available Etn had effects identical to those of the LMW factor before purification; i.e., Etn synergistically stimulated the proliferation of hepatocytes in primary culture when added with the HMW factor, and it stimulated hepatocyte proliferation in the regenerating liver when administered alone. Etn can synergistically enhance DNA synthesis in hepatocytes when added with EGF, suggesting that it enhances the actions of hepatotrophic growth factors. Etn in circulation may function as a humoral hepatotrophic factor ...

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“…Ethanolamine (10), and particularly its methylated analogues (11,12), can greatly enhance insulin-induced DNA synthesis by a wortmannin-sensitive mechanism not involving PtdEtn synthesis. PMA also potentiates the mitogenic effect of insulin by a wortmannin-sensitive mechanism (9).…”

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confidence: 99%

Protein Kinase Cα Is a Major Mediator of the Stimulatory Effect of Phorbol Ester on Phospholipase D-mediated Hydrolysis of Phosphatidylethanolamine

Mukherjee

Chung

Ways

et al. 1996

Journal of Biological Chemistry

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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...

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Synergistic Potentiating Effects of Choline Phosphate and Ethanolamine on Insulin-Induced DNA Synthesis in NIH 3T3 Fibroblasts

Cited by 20 publications

References 0 publications

Ethanolamine analogues stimulate DNA synthesis by a mechanism not involving phosphatidylethanolamine synthesis

Ethanolamine analogues stimulate DNA synthesis by a mechanism not involving phosphatidylethanolamine synthesis

Ethanolamine modulates the rate of rat hepatocyte proliferation in vitro and in vivo

Protein Kinase Cα Is a Major Mediator of the Stimulatory Effect of Phorbol Ester on Phospholipase D-mediated Hydrolysis of Phosphatidylethanolamine

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