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

Ganglioside IV3 (NeuAc)(2), II3 (NeuAc)(2)-GgOse(4)Cer (GQ(1b)), which induces terminal differentiation in keratinocytes, was previously fou nd to enhance the mass content of inositol 1,4,5-trisphosphate and int racellular calcium concentration ([Ca++](i)), peaking at 30 seconds. I n 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 ha nd, II3 NeuAc-LacCer (GM(3)), which inhibits keratinocyte proliferatio n without inducing differentiation, did not cause diacylglycerol forma tion. Phosphatidylethanol, produced by transphosphatidylation and a po tential marker for phospholipase D activity, was produced by the expos ure to GQ(1b) in the presence of ethanol. The second sustained phase o f diacylglycerol was repressed by ethanol, indicating that the diacylg lycerol-formation pathway via phospholipase D followed by phosphatidic acid phosphohydrolase would in part account for the second diacylglyc erol phase. Furthermore, this second phase of Gq(1b)-induced diacylgly cerol generation was reduced by pretreatment with propranolol, an inhi bitor of phosphatidic acid phosphohydrolase. In addition, the levels o f [H-3]choline, a direct metabolite of the phospholipase D pathway, we re elevated within 1 min after GQ(1b) addition and then sustained for at least 20 min. Taken together, the results suggest that the phosphol ipase D pathway may contribute to the second phase of diacylglycerol f ormation, which might be involved in differentiation.