Jody Ginsberg

Protein kinase C activators modulate differentiated thyroid function in vitro.

Exposure of porcine thyroid cells to the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) leads to inhibition of differentiated thyroid function. We investigated whether this effect is mediated via protein kinase C activation. TPA, phorbol 12,13‐didecanoate and phorbol 12,13‐dibutyrate inhibited TSH‐stimulated iodine organification in porcine thyroid cells by 98, 96 and 45%, respectively. Non‐tumour promoting phorbol esters had no effect. The diacylglycerol analogue, sn‐1,2‐dioctanoylglycerol had similar but quantitatively less activity than TPA. Dibutyryl cAMP could not reverse any inhibition noted. Under conditions that caused significant inhibition of differentiated function, TPA caused translocation of thyroidal protein kinase C from the cytosol to its membrane‐bound form. These data provide evidence that the mechanism of phorbol action on thyroid function in vitro includes activation of protein kinase C.

Protein Kinase C as a Mediator of TSH and Thyroid Autoantibody Action

Although it is well-established that TSH activates a cAMP-dependent pathway in the thyroid follicular cell leading to thyroid hormone synthesis and release, the present review provides new evidence that TSH also activates a non-cAMP-dependent signal transduction system. This cascade involves phosphoinositide (PI) turnover, diacylglycerol accumulation and protein kinase C (PKC) activation. Activation of this pathway leads to an inhibition of differentiated thyroid function in vitro. Recent evidence suggests that TSH can activate both pathways via a single transcription unit. Unlike TSH, TSH-receptor antibodies may selectively activate cAMP with no effects on PI turnover. In contrast, preliminary studies suggest TSH-blocking antibodies may activate PKC. PKC may be an important mediator of TSH and, possibly, thyroid autoantibody action.

Thyrotrophin blocking antibodies in the sera of mothers with congenitally hypothyroid infants.

We examined IgG from 15 mothers of infants with congenital hypothyroidism for their ability to block TSH‐stimulated iodine organification in vitro. Only one out of 15 of these IgGs exhibited TSH‐blocking activity. This IgG could inhibit TSH action by 30–50%. It could not block radiolabelled TSH from binding to its receptor but did inhibit dibutyryl cyclic AMP stimulation of iodine organification implying a unique effect on post‐receptor processes via a membrane binding site distant from the TSH binding site. Interestingly, there was no evidence of autoimmune thyroid disease in the mother. These results suggest that the transplacental passage of TSH‐blocking antibodies plays little, if any, role in the development of congenital hypothyroidism.

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.

Translocation of protein kinase C in porcine thyroid cells following exposure to thyrotropin

We have previously shown that protein kinase C activators modulate differentiated thyroid function in vitro; however, how protein kinase C may be activated physiologically is unknown. The present studies were undertaken in order to determine whether TSH could activate protein kinase C in vitro. Following exposure of porcine thyroid cells to TSH, translocation of protein kinase C from the cytosol to its membrane‐bound form was observed. Maximal translocation occurred at the lowest TSH concentration able to trigger this response (10 mU/ml) but persisted at higher concentrations (20–100 mU/ml). Time‐course studies revealed that translocation of protein kinase C was seen only after 40 min. TSH could also produce a similar translocation in human neutrophils (known to have TSH receptors). In thyroid cells pre‐treated with TSH, modulation of phorbol‐mediated protein kinase C translocation was noted. These results indicate that TSH causes the translocation of protein kinase C in porcine thyroid cells (and possibly other TSH receptor‐containing cells) and therefore may regulate the action of protein kinase C on differentiated thyroid function.

The characterization of phospholipase D in FRTL-5 thyroid cells.

We previously demonstrated that TSH activates phospholipase D (PLD) in Fischer rat thyroid line (FRTL)-5 cells. To date, two types of mammalian phosphatidylcholine-specific PLD cDNAs, designated as PLD-1 and PLD-2, have been cloned. The present study determined the PLD isoform composition in FRTL-5 thyroid cells and which isoform is regulated by TSH. PLD-1 is activated by small molecular weight G-proteins, such as ADP-ribosylation factor (ARF) and RhoA family members, while PLD-2 is relatively independent of such stimuli. We established the presence of PLD-1 and PLD-2 by Western blot analysis and compared PLD activity in cytosol, membranes and combined fractions in the presence and absence of GTPgammaS. The membrane fraction showed very little activity in the absence of GTPgammaS, but this activity increased approximately 5-fold (P<0.05, ANOVA) in the presence of GTPgammaS. Maximal PLD activity was seen with the combination of membrane plus cytosolic fractions (which contained ARF and RhoA) where the addition of GTPgammaS increased PLD activity approximately 8-fold (P<0.05, ANOVA). To determine the relative activities of PLD-1 and PLD-2 in FRTL-5 thyroid cells, cell-free PLD assays were performed in the presence of GTPgammaS or GDPbetaS with varying concentrations of phosphatidylinositol 4,5-bisphosphate (PIP(2)). PLD-2 contributed only approximately 19% of the total amount of PLD activity in the membranes and PLD-1 was the predominant PLD isoform. TSH stimulated PLD-1 activity by up to 2. 3-fold over control values (P<0.01, ANOVA). To establish the dependence of PLD-1 on small molecular weight G-proteins, the translocations of ARF and RhoA to the membrane fractions was determined after stimulation by TSH. Both ARF and RhoA were maximally translocated to the membrane fraction after 10 min incubation with 100 microU/ml TSH by approximately 1.7- and 2.3-fold over control values, respectively (P<0.02 and P<0.03, ANOVA). It is concluded that TSH stimulates PLD-1 activity in FRTL-5 thyroid cells and this is accompanied by the translocation of ARF and RhoA to the membrane fraction.

The effects of TSH receptor antibodies on protein kinase C in the thyroid

OBJECTIVE There is increasing evidence that TSH activates a non‐cyclic‐AMP‐dependent pathway in the thyroid resulting in protein kinase C activation. We have previously demonstrated that TSH activates protein kinase C by causing translocation of protein kinase C from an inactive cytosolic site to its active membrane‐bound form in porcine thyroid cells. In addition, TSH can modify the protein kinase C translocation induced by the phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate. We tested six TSH receptor antibodies for their ability to activate protein kinase C in vitro.

THE EFFECTS OF DITHIOTHREITOL ON THYROID STIMULATION IN VITRO

There is evidence that the porcine TSH receptor contains essential disulphide bridge(s) which can be disrupted by dithiothreitol (DTT). The aim of the present study was to determine whether exposure of intact thyroid cells to DTT leads to altered thyroid stimulation. TSH‐stimulated iodine organification in cultured porcine thyroid cells was studied following short‐term DTT exposure; a dose‐dependent inhibition was observed with DTT but not oxidized DTT. Cell viability, follicle formation, and total protein synthesis were preserved. A minimum of 30 min incubation with DTT was required for inhibition. However, under identical conditions, DTT had no effect on TSH or forskolin‐stimulated cyclic AMP (cAMP) production. These results suggest that DTT inhibition of organification is mediated by post‐receptor mechanisms likely involving thyroid peroxidase. The effects of DTT on thyroid stimulation in vitro does not appear to involve disruption of the disulphide bridge(s) in the TSH receptor.