POTENTIATION BY CHOLESTEROL AND VITAMIN-D-3 OXYGENATED DERIVATIVES OF ARACHIDONIC-ACID RELEASE AND PROSTAGLANDIN E(2) SYNTHESIS INDUCED BY THE EPIDERMAL GROWTH-FACTOR IN NRK 49F CELLS - THE ROLE OF PROTEIN-KINASE-C

We have previously demonstrated that oxysterols and calcitriol potentiate arachidonic acid (AA) release and prostaglandin (PG) synthesis when NRK cells (fibroblastic clone 49F) are activated by foetal calf serum. As serum is essential for a full oxysterol effect, we hypothesized that these compounds could act on one or more of the events triggered by serum growth factor binding to their specific receptors and leading to PLA(2) activation; we showed that the oxysterol effect on AA release is synergistic with, but not fully dependent on, protein kinase C (PKC) activity and Ca2+ ion fluxes, suggesting that oxysterols could effect early events in the cell signalling pathway. In the present paper, we investigated the effect of some oxysterols and calcitriol on epidermal growth factor (EGF)-induced AA release and PGE(2) synthesis in NRK cells. The clear potentiation of EGF effect by most of the oxygenated sterols-chiefly when polyoxidized-cannot be explained by a modification of EGF high affinity binding site number which was only moderately increased after a 4 h incubation of cells with these compounds, and moreover was not related to the ability of a given oxysterol to increase PLA(2) activity; whatever the compound, the dissociation constant (K-d) of either a high or low affinity binding site was unchanged (respectively, 3.5 x 10(-11) M and 4.4 x 10(-10) M). Genistein, a known inhibitor of EGF receptor tyrosine kinase, changed neither the EGF effect on AA release nor its potentiation by oxysterol, whereas it inhibited PGE(2) synthesis in both situations. PKC activation by phorbol ester TPA increased the effect of EGF alone as well as the oxysterol potentiating effect, whereas PKC down-regulation strongly decreased both of these effects, showing that both are dependent on PKC activity. Nevertheless staurosporine, a PKC inhibitor, did not reproduce the effects of PKC down-regulation on EGF activation: stimulatory when AA release was induced by EGF alone, inhibitory when AA release is induced by TPA alone, this compound did not modify the oxysterol potentiating effect. In conclusion, the potentiating effect of oxysterols on AA release seems to be exerted downstream to the growth factor receptor (as demonstrated here with EGF) and probably at the PKC level, but not exclusively.