Effects of the Diacylglycerol Kinase Inhibitor, R59022, on TSH-Stimulated Iodide Organification in Porcine Thyroid Cells

We and others have demonstrated that protein kinase C (PKC) activators such as the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibit differentiated thyroid function in vitro. However, phorbol-mediated PKC activation differs from that induced by analogues of the endogenous PKC activator, diacylglycerol (DAG). To explore the effects of endogenous PKC activation on differentiated thyroid function, we examined the effects of the DAG kinase inhibitor, R59022, on TSH-mediated iodide organification in porcine thyroid cells. Following incubation of the thyroid cells for 30 min, 20 and 50 mumol/l R59022 inhibited TSH-stimulated iodide organification by 20 and 41%, respectively. Prolonged exposure (36 h) to R59022 was also studied since similar treatment with TPA downregulates PKC activity. Inhibition of TSH-mediated iodide organification was observed with as little as 5 mumol/l R59022 (56% of control, p < 0.01) with maximal inhibition using 50 mumol/l R59022 to 13% of control values (p < 0.001). To demonstrate that these effects were mediated by PKC activation, PKC isoforms were measured by Western blotting following R59022 exposure (50 mumol/l, 30 min). Increased membrane-bound alpha- and zeta-PKC isozymes were observed. This is the first demonstration linking specific PKC isoforms to changes in differentiated thyroid function in vitro. The present data suggest that alpha- and/or zeta-PKC mediate the effects of R59022 on differentiated thyroid function in vitro. Further, a PKC inhibitor, chelerythrine (1 mumol/l) was able to partially reverse the effects of prolonged R59022 exposure on TSH-mediated iodide organification. These studies demonstrate that R59022 exposure inhibits TSH-mediated iodide organification in porcine thyroid cells and that these effects are mediated via endogenous PKC activation. These data are consistent with the concept that endogenous PKC acts as a negative modulator of differentiated thyroid function in the porcine thyroid cell.