Effects of prolonged EGF treatment on phospholipid turnover and DAG formation in murine keratinocytes

In murine keratinocytes, the cellular diacylglycerol (DAG) content was considerably elevated following a 48-h exposure to epidermal growth factor (EGF), while formation of inositol phosphates (InsP) was not stimulated. A similar loss of InsP production upon stimulation of keratinocytes with 1.4 mM Ca2+ was seen after pretreatment with R59022, a DAG kinase inhibitor. These data suggest that accumulated endogenous DAG has an inhibitory feedback effect on PLC activity. To elucidate the possible phospholipid source of elevated DAG in keratinocytes, cells were first labeled with [3H]-choline and then exposed to EGF for 24 h or TPA, a protein kinase C activator, for 8 h. As expected, TPA increased [3H]-choline release into the culture medium, whereas EGF decreased the release, suggesting that EGF treatment does not result in sustained stimulation of phosphatidylcholine turnover. The release of [14C]-dihomo-gamma-linolenic acid (DHGLA), predominately bound to the 2-positions of phospholipids, was also stimulated by 8 h of TPA treatment but not by 24 h of EGF treatment. The distribution of DHGLA in various phospholipid subclasses was not influenced by EGF. These results indicate that prolonged EGF treatment does not markedly activate phospholipid A2 (PLA2) or lysophospholipase, and that the DAG accumulation after prolonged EGF exposure is apparently not associated with stimulated breakdown of any specific lipid pool. It is concluded that changes in keratinocyte lipid turnover induced by prolonged EGF treatment differ from those associated with short-term EGF exposure.