Effects of phorbol esters and various growth factors on prostaglandin E2 synthesis by cultured porcine thyroid cells

Abstract

The effects of phorbol esters and various growth factors on prostaglandin E2 (PGE2) synthesis by cultured porcine thyroid cells were examined. Phorbol 12-myristate, 13-acetate (PMA), phorbol 12, 13-dibutylate, epidermal growth factor (EGF) and fetal bovine serum (FBS) stimulated PGE2 production in a dose-related fashion. PMA stimulated PGE2 production over fifty-fold with a dose of 10(-7) M compared with controls during 4h incubation. EGF (10(-7) M) also stimulated it about ten-fold. The ED50 values of PMA and EGF were around 1 X 10(-9) M and 5 X 10(-10) M respectively. FBS also clearly stimulated it about ten-fold with a concentration of 20%. Thyroid stimulating hormone (TSH) and inactive phorbol, however, did not stimulate PGE2 production. The release of radioactivity from [3H]-arachidonic acid (A.A.) prelabeled cells was also stimulated by PMA, EGF and FBS. These results indicate that PMA, EGF and FBS are potent stimulators of PGE2 production, associated with the activity to stimulate A.A. release in thyroid cells. Secondly, in order to elucidate some mechanism(s) of the stimulation by these factors of PGE2 production, effects of various agents such as EGTA, Ca2+-ionophore (A-23187), cycloheximide, hydrocortisone and para-bromophenacylbromide (p-BPB) were examined on PGE2 production and/or [3H]-A.A. release by thyroid cells. Chelation of Ca2+ by EGTA, treatment of cells with hydrocortisone, an inducer of lipocortin which may inhibit phospholipase A2 (PLA2) activity, or inhibition of PLA2 activity by p-BPB clearly inhibited PGE2 production and/or [3H]-A.A. release induced by PMA, EGF and FBS. Cycloheximide also blocked the PMA-or EGF-stimulated [3H]-A.A. release and PGE2 synthesis. On the other hand, Ca2+-ionophore (A-23187) potentiated PMA- or EGF-stimulation. Furthermore, the Ca2+-ionophore alone-induced stimulation was clearly inhibited by the treatment with hydrocortisone or p-BPB. These data indicate that an increase of Ca2+ in cytosol is important to PLA2 activation, and also that PLA2 may be activated by PMA, EGF and FBS in thyroid cells. The inhibition of PMA- or EGF-induced [3H]-A.A. release and PGE2 synthesis by cycloheximide suggests that both factors-induced stimulation may be sensitive to regulation by short-lived protein(s). Finally, other growth factors such as insulin (0.1-10 micrograms/ml), insulin like growth factor I (IGF-1) (10(-9)-10(-7) M) and interleukin 1 alpha (1-100 ng/ml) also stimulated PGE2 production by thyroid cells in the presence or absence of EGF (10(-10)-10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)