Activation of phospholipase A2 and stimulation of prostaglandin E2 production by transforming growth factor-alpha in rat thymic epithelial cells requires influx of calcium.

The stimulation of both phospholipase A2 (PLA2) enzymic activity and the production of prostaglandin E2 (PGE2) by transforming growth factor-alpha (TGF-alpha) and Ca2+ ionophore A23187 in TEA3A1 rat thymic epithelial cells were studied. TGF-alpha by itself at various concentrations (5-200 ng/ml) had no effect on the stimulation of PGE2 production. A23187 (1 microgram/ml) by itself stimulated PGE2 production on average by 18-fold over the control. When TGF-alpha (50 ng/ml) was added to the cells in the presence of A23187, a synergistic stimulation (on average 45-fold) of PGE2 production was observed. Synergistic stimulation was also observed at the level of arachidonic acid released from phospholipid pools, suggesting the activation of PLA2 enzymic activity. We have found that this synergistic activation of PLA2 enzymic activity and subsequent stimulation of PGE2 production required the activation of epidermal growth factor (EGF) receptor tyrosine kinase and Ca2+ influx. This was shown by the fact that genistein, an inhibitor of tyrosine kinase, blocks the synergistic stimulation by TGF-alpha and A23187 and by the fact that the stimulation of PGE2 production by TGF-alpha and A23187 is dependent on the culture-medium Ca2+ concentrations. The requirement for Ca2+ influx instead of intracellular mobilization of Ca2+ was shown by the fact that PGE2 production was not stimulated when cells were treated with TGF-alpha and thapsigargin. Moreover, the synergistic stimulation of PGE2 production by TGF-alpha and A23187 was not affected in protein kinase C down-modulated cells. In addition, the synergistic stimulation was not observed in cells treated with either phorbol 12-myristate 13-acetate (PMA) and TGF-alpha or PMA and A23187, and in cells treated with TGF-alpha and thapsigargin. The requirement for the activation of receptor tyrosine kinase seems to be specific to the EGF receptor, since a synergistic stimulation of PGE2 production was not observed when cells are treated with either insulin-like growth factor-I or fibroblast growth factor-I in the presence of A23187.