INVOLVEMENT OF PHOSPHOLIPASE-D IN GANGLIOSIDE GQ(1B)-INDUCED BIPHASIC DIACYLGLYCEROL PRODUCTION IN HUMAN KERATINOCYTES

Ganglioside IV3 (NeuAc)(2), II3 (NeuAc)(2)-GgOse(4)Cer (GQ(1b)), which induces terminal differentiation in keratinocytes, was previously found to enhance the mass content of inositol 1,4,5-trisphosphate and intracellular calcium concentration ([Ca++](i)), peaking at 30 seconds. In the present study, the biphasic accumulation of 1,2 diacylglycerol, i.e., the first transient and the second sustained phase, was observed in cultured human keratinocytes stimulated by GQ(1b). On the other hand, II3 NeuAc-LacCer (GM(3)), which inhibits keratinocyte proliferation without inducing differentiation, did not cause diacylglycerol formation. Phosphatidylethanol, produced by transphosphatidylation and a potential marker for phospholipase D activity, was produced by the exposure to GQ(1b) in the presence of ethanol. The second sustained phase of diacylglycerol was repressed by ethanol, indicating that the diacylglycerol-formation pathway via phospholipase D followed by phosphatidic acid phosphohydrolase would in part account for the second diacylglycerol phase. Furthermore, this second phase of Gq(1b)-induced diacylglycerol generation was reduced by pretreatment with propranolol, an inhibitor of phosphatidic acid phosphohydrolase. In addition, the levels of [H-3]choline, a direct metabolite of the phospholipase D pathway, were elevated within 1 min after GQ(1b) addition and then sustained for at least 20 min. Taken together, the results suggest that the phospholipase D pathway may contribute to the second phase of diacylglycerol formation, which might be involved in differentiation.