epidermal-growth-factor and phosphatidylethanol

The receptor-mediated activation of phospholipase D (PLD) is a major signaling pathway in several cell systems. This study determined the effects of epidermal growth factor (EGF) on PLD activity in normal rat osteoblastic cells. Primary cultures were obtained from fetal rat calvaria by sequential collagenase digestion and seeded in BGJb media supplemented with 10% fetal calf serum. PLD activity was assayed by the transphosphatidylation reaction in [H3]myristic acid (5 microCi/ml)-labeled cells treated with EGF in the presence of 5% ethanol and measuring the production of phosphatidylethanol (PEtOH). Lipids were extracted and separated by thin-layer chromatography, detected by iodine staining, and the areas of interest were scraped off and transferred to vials for scintillation counting. EGF significantly increased PEtOH production in a dose-dependent manner and at short (10-60 s) and long (up to 30 minutes) incubation periods (p < 0.05). Phosphatidic acid levels were also significantly increased (p < 0.05) compared with unstimulated controls, but the levels were approximately 60% less than those of PEtOH. 4b-phorbol 12-myristate, 13-acetate (PMA) also produced a significant increase in PEtOH levels when compared with unstimulated control cultures, but when PMA was added together with EGF, the production of PEtOH was reduced about 30%. Pretreatment of cells with the protein kinase C (PKC) inhibitor H-7 caused a significant increase in PEtOH levels, compared with cells stimulated with EGF alone. Preincubation of cells with pertussis toxin produced a partial decrease in PEtOH levels. This study demonstrates that EGF activates the PLD signaling cascade in normal rat osteoblastic cells and that the pathway appears to involve, at least in part, a PKC- and Gi protein-dependent mechanism.

Topics: Analysis of Variance; Animals; Cells, Cultured; Chromatography, Thin Layer; Enzyme Activation; Epidermal Growth Factor; Glycerophospholipids; Osteoblasts; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Rats; Signal Transduction

In response to epidermal growth factor (EGF), HeLa cells and A431 cells rapidly accumulate substantial amounts of phosphatidic acid (up to 0.16 and 0.2 micrograms/10(6) cells respectively), which represents approx. 0.17% of total phospholipid. Phosphatidic acid may be a potential product of diacylglycerol kinase and/or of phospholipase D. To evaluate the contribution of phospholipase D, the phosphatidyl-transfer reaction to a primary alcohol (mostly butan-1-ol; 0.2%) was measured; this reaction is known to be mediated exclusively by phospholipase D in intact cells. In HeLa and in A431 cells prelabelled with [1-14C]oleic acid, EGF (10 and 100 nM respectively) caused a 3-fold increase in radioactive phosphatidylbutanol within 5 min at the expense of labelled phosphatidic acid. Dose-response relationships showed 10 nM- and 100 nM-EGF to be maximally effective in HeLa cells and A431 cells respectively. Mass determinations showed that the phosphatidylbutanol formed within 5 min represented only part of the phosphatidic acid. Depletion of protein kinase C by pretreatment of A431 cells for 17 h with the phorbol ester phorbol 12-myristate 13-acetate (0.1 microM) did not impair EGF-induced formation of phosphatidylbutanol, thus indicating that the reaction was independent of this enzyme. Since phosphatidic acid is suggested to exert second-messenger functions as well as to induce biophysical changes in cellular membranes, its formation, including that via the phospholipase D pathway, may represent an important link between extracellular signals and intracellular targets.

Topics: Diglycerides; Enzyme Activation; Epidermal Growth Factor; Glycerophospholipids; HeLa Cells; Humans; Phosphatidic Acids; Phosphatidylinositols; Phospholipase D; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phos

Topics: Cells, Cultured; Diglycerides; Epidermal Growth Factor; Ethanol; Fibroblasts; Glycerophospholipids; Humans; Kinetics; Phosphatidic Acids; Phosphatidylcholines; Phospholipase D; Phospholipid Ethers; Skin; Type C Phospholipases