0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
Autoimmunity, 1992, Vol. 13, pp. 51-59 Reprints available directly from the publisher Photocopying permitted by license only
PROTEIN KINASE C AS A MEDIATOR OF TSH AND THYROID AUTOANTIBODY ACTION JODY GINSBERG Division of Endocritio[ogy, 362 Heritage M e d i c ~ Research l Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
Autoimmunity Downloaded from informahealthcare.com by Chulalongkorn University on 01/05/15 For personal use only.
(Received March 24,1992) 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 activale 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.
KEY WORDS: Thyroid, thyrotropin, protein kinase c, phosphoinositide, TSH-receptor antibodies
phorbol 13-acetate (TPA), which contain a side-chain linkage structurally similar to the naturally-occurring activator, diacylglycerol (DG)'. Although the effects of TPA on differentiated thyroid function in vitro vary according to the concentration, incubation time, species studied and tissue preparation, the overall effect is an inhibition of TSH-stimulated differentiated function. For example, Bachrach et demonstrated that TPA caused a potent inhibition of iodine uptake and organification in TSH-stimulated cultured ovine thyroid cells. Dibutyryl cAMP ((BU)~CAMP) could not overcome this inhibition of iodine metabolism, implying that the site of TPA action involved a post-receptor locus. We observed similar findings in porcine thyroid cells (Figure 2)'. TPA also inhibited TSH and ( B U ) ~ C A Mmediated P iodine metabolism in the FRTL-5 rat thyroid cell line'. A similar inhibition of differentiated function was noted in canine thyroid where TPA inhibited TSH- and (Bu)?cAMP-mediated thyroglobulin mRNA expression9. In addition, TPA inhibits the stimulation by TSH, forskolin and ( B u ) ~ c A M Pof butanol extractable radioiodine release from prelabelled canine thyroid slices which depends on colloid endocytosis and thyroglobulin hydrolysis and is reflective of thyroid hormone secretion". The anomolous findings of increased free T3 secretion induced by low concentrations of TPA in mouse thyroid lobes" and the potentiation of forskolin-induced iodine organification in mouse open follicles" may represent a species variation. Alternatively, since cytoskeletal proteins are recognized substrates for PKC in other tissues", these differences in response to TPA may depend on whether intact tissue or dispersed cells are studied. Although TPA has effects similar to epidermal
INTRODUCTION It is well-established that thyroid hormone synthesis is stimulated by pituitary thyrotropin (thyroid-stimulating hormone, TSH) via the receptor-mediated accumulation of the second messenger, 3',5'-cyclic adenosine monophosphate (CAMP) which activates its respective CAMP-dependent protein kinase(s), protein kinase A'. In 1953, Morton and Schwartz had demonstrated that TSH stimulated phospholipid turnover in bovine thyroid slices2. While subsequent studies indicated that this effect was independent of cAMP3, the significance of this observation was not immediately apparent. Since this initial observation investigations in other model systems have demonstrated that a noncAMP dependent signal transduction system involving receptor-mediated phosphoinositide turnover, diacylglycerol (DG) accumulation and activation of a Ca++,phospholipid-sensitive enzyme, protein kinase C (PKC), is involved in the regulation of many eukaryotic cells including endocrine cells (Figure 1) (review')). The present manuscript will review the evidence that PKC plays a role in the regulation of differentiated thyroid function in vitro and will explore its activation by TSH. In addition, the ability of TSH receptor and TSH blocking antibodies to interact with this pathway will be discussed. PHORBOL ESTERS AND THYROID FUNCTION PKC can be activated in vitro and in vivo by tumorpromoting phorbol esters, e.g. 12-0-tetradecanoylAddress all correspondence to Dr Ginsberg at the above address.
51
Autoimmunity Downloaded from informahealthcare.com by Chulalongkorn University on 01/05/15 For personal use only.
52
J. GINSBERG
A3fx.a.
r
Figure 1 Schematic representation of the bifurcatingpathway of signal transduction. Various extracellular informational signals appear to be passed from the cell surface into the cell interior by two routes, protein kinase C activation and Ca2' mobilization. Both routes usually become available as the result of an interaction of a single agonist and a receptor, and subsequent hydrolysis of phosphatidylinositol4,5-bisphosphate. The resulting products, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, act as second messengers for protein kinase C activation and Cazi mobilization, respectively. These two routes can also be opened experimentally by exogenous application of a permeable diacylglycerol or tumor-promoting phorbol ester and Caz'-ionophore, respectively. PIP2, phosphatidylinositol 4,5-bisphosphate; DG, 1,2-diacylglycerol; IP,, inositol 1,4,5-trisphosphate; and PKC, protein kinase C (From Nishizuka Y. Studies and perspectives of the protein kinase C family for cellular regulation. Cancer 1989; 63: 1892-1903, with permission).
growth factor (EGF) on differentiated thyroid function incubation with TPA had no effect on cAMP levels; after a 120-min incubation, a significant inhiin v i m , these agents would appear to act d i ~ t i n c t l y ~ . ~while . Although TPA is known to affect EGF binding to its bition of TSH-stimulated cAMP was observed. A receptor14, its effects on the thyroid do not appear to similar time-course effect was noted in porcine thybe mediated in this fashion6. Similarly, TPA mimics roid cells". The noted lack of an effect of TPA on some, and inhibits other, effects of the muscarinic TSH-stimulated cAMP generation in some studies agonist carbachol on differentiated thyroid function". may therefore be due to insufficient TPA exposure. In The effects of TPA on more proximal intracellular several studies involving many species including the events are more varied. Basal iodine organification is FRTL5 rat cell line, TPA had no effect on basal cAMP stimulated by TPA in porcine thyroid cells", canine generation6..8.IO,I2,15.16 and in mouse open follicIesl2. In contrast, Phorbol esters exert their initial effects at the other investigators reported that basal iodine metab- plasma membrane and can alter some types of recepolism is unaffected" or decreased',' following TPA tor binding. EGF receptor autophosphorylation is exposure. The reason for these differences is induced by phorbol esters resulting in a reduction in unknown. ligand binding'4319.A decrease in binding to transAlthough TPA inhibits TSH and forskolin-stimu- ferrin receptors in HL60 cells is also observed followlated cAMP levels in ovine6 and FRTL-5 thyroid ing TPA2". In preliminary studies, we observed no cellsx, its inhibition of iodine organification is not effect of TPA on binding of radiolabelled TSH to its fully explained by this effect. No effects of TPA on receptor". TSH or forskolin-stimulated cAMP levels are seen in Thus, although isolated studies may differ, the vast mouse open follicles12, dog thyroid slices" or porcine majority of observations regarding the effects of TPA thyroid cells"; in the latter, significant inhibition of on TSH-stimulated differentiated thyroid function in TSH-stimulated iodine organification is observed. In vitro have revealed inhibition at multiple levels another study utilizing dog thyroid slicesI6, a 20-min including cAMP release, iodine organification and
PROTEIN KINASE C AND THE THYROID
T
T
Autoimmunity Downloaded from informahealthcare.com by Chulalongkorn University on 01/05/15 For personal use only.
T
-
** **
m
Figure 2 The effects of TPA and 4P-phorbol on TSH-stimulated iodine organification (expressed as organified '''1 cpm) in cultured porcine thyroid cells. The phorbol esters were dissolved in absolute ethanol at 2 mM and the final concentrations used are shown, **P< 0.001 (From Ginsberg J, Murray PG. Protein kinase C activators modulate differentiated thyroid function in vitro. FEBS Lett 1986; 206: 309-3 12, with permission).
thyroid hormone secretion. Although species and methodological variations may explain some of the different results observed, the reasons for the heterogeneous response to phorbol esters in the thyroid likely awaits identification of the endogenous substrates of PKC and an understanding of their interactions with intracellular events. EVIDENCE THAT PKC MEDIATES THE EFFECTS OF PHORBOL ESTERS ON DIFFERENTIATED THYROID FUNCTION It is well recognized that tumor-promoting phorbol esters such as TPA may have effects which are not mediated by PKC". Suppression of globin gene expression in Friend erythroleukemia cells by TPA occurs in spite of PKC down-regulation22. In HL60 leukemic cells, the DG analogue, 1-0leoy1-2-acetylglycerol, cannot mimic the cell differentiating action of TPA even though both agents similarly activate PKC2'. In porcine and ovine thyroid cells, the DG analogue, sn- 1-2-PKC2'. In porcine and ovine thyroid cells, the DG analogue, sn- 1,2-dioctanoylglycero1
53
cannot fully mimic the effects of TPA on iodide organification in vitro617.Several pieces of evidence have, however, recently emerged consistent with the idea that the effects of phorbol esters on the thyroid are mediated via PKC. Non-tumor-promoting phorbol esters have not been able to reproduce the effects of tumor-promoting phorbol esters on differentiated thyroid function in vitro6-I2.In addition, the ability of various tumor-promoting phorbol esters to activate PKC in vitro have paralleled their action on differentiated thyroid function6-I0. We observed that the non-phorbol diterpene, 12-hydroxy-daphnetoxin (mezerein) which is capable of activating PKC in vitro and in vivo2', is also capable of inhibiting TSH-stimulated iodide organification in porcine thyroid cells in a similar fashion to TPAIX.Similar findings were noted in FTRL-5 thyroid cells exposed to mezerein'. W e have also demonstrated that exogenous phospholipase C, which generates DG24, can also mimic the effects of TPA on differentiated thyroid function2'. PKC is activated when it is translocated from an inactive site in the cytosol to an active membranebound form4. We demonstrated that TPA, in concentrations which affect differentiated thyroid function, caused PKC translocation in porcine thyroid cell extracts (Table 1)'. Similar PKC translocation is induced in porcine thyroid cell extracts by phospholipase C2' and mezerein". It is interesting to note that mezerein affects differentiated thyroid function in v i m only at concentrations which cause PKC translocation in vitro". Several studies have utilized PKC inhibitors to determine the mechanism of phorbol ester action on differentiated thyroid function in vitro. The PKC inhibitor, 1-(.5-isoquinolinesulfonyl)-2-methylpiperazine (H7) has been shown to partially reverse the effects of TPA on differentiated thyroid function in vitro2'. Similar results have been shown with another PKC inhibitor, staurosporine26.In addition, staurosporine and the PKC inhibitor, l-O-hexadecyl-2-Omethyglycerol (AMG-C16), are capable of enhancing TSH-stimulated iodide organification in vitro2' implying that endogenous PKC acts as an inhibitor of differentiated thyroid function. Table 1 Protein kinase C activity in thyroid cell extracts (pmol/min per mg protein). Treatment
Cytosol Membrane
P*
4b-Phorhol
TPA
170(f5)** 1 1 l(f28) N.S.
53(+15) 466(&55) P
Autoimmunity, 1992, Vol. 13, pp. 51-59 Reprints available directly from the publisher Photocopying permitted by license only
PROTEIN KINASE C AS A MEDIATOR OF TSH AND THYROID AUTOANTIBODY ACTION JODY GINSBERG Division of Endocritio[ogy, 362 Heritage M e d i c ~ Research l Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
Autoimmunity Downloaded from informahealthcare.com by Chulalongkorn University on 01/05/15 For personal use only.
(Received March 24,1992) 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 activale 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.
KEY WORDS: Thyroid, thyrotropin, protein kinase c, phosphoinositide, TSH-receptor antibodies
phorbol 13-acetate (TPA), which contain a side-chain linkage structurally similar to the naturally-occurring activator, diacylglycerol (DG)'. Although the effects of TPA on differentiated thyroid function in vitro vary according to the concentration, incubation time, species studied and tissue preparation, the overall effect is an inhibition of TSH-stimulated differentiated function. For example, Bachrach et demonstrated that TPA caused a potent inhibition of iodine uptake and organification in TSH-stimulated cultured ovine thyroid cells. Dibutyryl cAMP ((BU)~CAMP) could not overcome this inhibition of iodine metabolism, implying that the site of TPA action involved a post-receptor locus. We observed similar findings in porcine thyroid cells (Figure 2)'. TPA also inhibited TSH and ( B U ) ~ C A Mmediated P iodine metabolism in the FRTL-5 rat thyroid cell line'. A similar inhibition of differentiated function was noted in canine thyroid where TPA inhibited TSH- and (Bu)?cAMP-mediated thyroglobulin mRNA expression9. In addition, TPA inhibits the stimulation by TSH, forskolin and ( B u ) ~ c A M Pof butanol extractable radioiodine release from prelabelled canine thyroid slices which depends on colloid endocytosis and thyroglobulin hydrolysis and is reflective of thyroid hormone secretion". The anomolous findings of increased free T3 secretion induced by low concentrations of TPA in mouse thyroid lobes" and the potentiation of forskolin-induced iodine organification in mouse open follicles" may represent a species variation. Alternatively, since cytoskeletal proteins are recognized substrates for PKC in other tissues", these differences in response to TPA may depend on whether intact tissue or dispersed cells are studied. Although TPA has effects similar to epidermal
INTRODUCTION It is well-established that thyroid hormone synthesis is stimulated by pituitary thyrotropin (thyroid-stimulating hormone, TSH) via the receptor-mediated accumulation of the second messenger, 3',5'-cyclic adenosine monophosphate (CAMP) which activates its respective CAMP-dependent protein kinase(s), protein kinase A'. In 1953, Morton and Schwartz had demonstrated that TSH stimulated phospholipid turnover in bovine thyroid slices2. While subsequent studies indicated that this effect was independent of cAMP3, the significance of this observation was not immediately apparent. Since this initial observation investigations in other model systems have demonstrated that a noncAMP dependent signal transduction system involving receptor-mediated phosphoinositide turnover, diacylglycerol (DG) accumulation and activation of a Ca++,phospholipid-sensitive enzyme, protein kinase C (PKC), is involved in the regulation of many eukaryotic cells including endocrine cells (Figure 1) (review')). The present manuscript will review the evidence that PKC plays a role in the regulation of differentiated thyroid function in vitro and will explore its activation by TSH. In addition, the ability of TSH receptor and TSH blocking antibodies to interact with this pathway will be discussed. PHORBOL ESTERS AND THYROID FUNCTION PKC can be activated in vitro and in vivo by tumorpromoting phorbol esters, e.g. 12-0-tetradecanoylAddress all correspondence to Dr Ginsberg at the above address.
51
Autoimmunity Downloaded from informahealthcare.com by Chulalongkorn University on 01/05/15 For personal use only.
52
J. GINSBERG
A3fx.a.
r
Figure 1 Schematic representation of the bifurcatingpathway of signal transduction. Various extracellular informational signals appear to be passed from the cell surface into the cell interior by two routes, protein kinase C activation and Ca2' mobilization. Both routes usually become available as the result of an interaction of a single agonist and a receptor, and subsequent hydrolysis of phosphatidylinositol4,5-bisphosphate. The resulting products, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, act as second messengers for protein kinase C activation and Cazi mobilization, respectively. These two routes can also be opened experimentally by exogenous application of a permeable diacylglycerol or tumor-promoting phorbol ester and Caz'-ionophore, respectively. PIP2, phosphatidylinositol 4,5-bisphosphate; DG, 1,2-diacylglycerol; IP,, inositol 1,4,5-trisphosphate; and PKC, protein kinase C (From Nishizuka Y. Studies and perspectives of the protein kinase C family for cellular regulation. Cancer 1989; 63: 1892-1903, with permission).
growth factor (EGF) on differentiated thyroid function incubation with TPA had no effect on cAMP levels; after a 120-min incubation, a significant inhiin v i m , these agents would appear to act d i ~ t i n c t l y ~ . ~while . Although TPA is known to affect EGF binding to its bition of TSH-stimulated cAMP was observed. A receptor14, its effects on the thyroid do not appear to similar time-course effect was noted in porcine thybe mediated in this fashion6. Similarly, TPA mimics roid cells". The noted lack of an effect of TPA on some, and inhibits other, effects of the muscarinic TSH-stimulated cAMP generation in some studies agonist carbachol on differentiated thyroid function". may therefore be due to insufficient TPA exposure. In The effects of TPA on more proximal intracellular several studies involving many species including the events are more varied. Basal iodine organification is FRTL5 rat cell line, TPA had no effect on basal cAMP stimulated by TPA in porcine thyroid cells", canine generation6..8.IO,I2,15.16 and in mouse open follicIesl2. In contrast, Phorbol esters exert their initial effects at the other investigators reported that basal iodine metab- plasma membrane and can alter some types of recepolism is unaffected" or decreased',' following TPA tor binding. EGF receptor autophosphorylation is exposure. The reason for these differences is induced by phorbol esters resulting in a reduction in unknown. ligand binding'4319.A decrease in binding to transAlthough TPA inhibits TSH and forskolin-stimu- ferrin receptors in HL60 cells is also observed followlated cAMP levels in ovine6 and FRTL-5 thyroid ing TPA2". In preliminary studies, we observed no cellsx, its inhibition of iodine organification is not effect of TPA on binding of radiolabelled TSH to its fully explained by this effect. No effects of TPA on receptor". TSH or forskolin-stimulated cAMP levels are seen in Thus, although isolated studies may differ, the vast mouse open follicles12, dog thyroid slices" or porcine majority of observations regarding the effects of TPA thyroid cells"; in the latter, significant inhibition of on TSH-stimulated differentiated thyroid function in TSH-stimulated iodine organification is observed. In vitro have revealed inhibition at multiple levels another study utilizing dog thyroid slicesI6, a 20-min including cAMP release, iodine organification and
PROTEIN KINASE C AND THE THYROID
T
T
Autoimmunity Downloaded from informahealthcare.com by Chulalongkorn University on 01/05/15 For personal use only.
T
-
** **
m
Figure 2 The effects of TPA and 4P-phorbol on TSH-stimulated iodine organification (expressed as organified '''1 cpm) in cultured porcine thyroid cells. The phorbol esters were dissolved in absolute ethanol at 2 mM and the final concentrations used are shown, **P< 0.001 (From Ginsberg J, Murray PG. Protein kinase C activators modulate differentiated thyroid function in vitro. FEBS Lett 1986; 206: 309-3 12, with permission).
thyroid hormone secretion. Although species and methodological variations may explain some of the different results observed, the reasons for the heterogeneous response to phorbol esters in the thyroid likely awaits identification of the endogenous substrates of PKC and an understanding of their interactions with intracellular events. EVIDENCE THAT PKC MEDIATES THE EFFECTS OF PHORBOL ESTERS ON DIFFERENTIATED THYROID FUNCTION It is well recognized that tumor-promoting phorbol esters such as TPA may have effects which are not mediated by PKC". Suppression of globin gene expression in Friend erythroleukemia cells by TPA occurs in spite of PKC down-regulation22. In HL60 leukemic cells, the DG analogue, 1-0leoy1-2-acetylglycerol, cannot mimic the cell differentiating action of TPA even though both agents similarly activate PKC2'. In porcine and ovine thyroid cells, the DG analogue, sn- 1-2-PKC2'. In porcine and ovine thyroid cells, the DG analogue, sn- 1,2-dioctanoylglycero1
53
cannot fully mimic the effects of TPA on iodide organification in vitro617.Several pieces of evidence have, however, recently emerged consistent with the idea that the effects of phorbol esters on the thyroid are mediated via PKC. Non-tumor-promoting phorbol esters have not been able to reproduce the effects of tumor-promoting phorbol esters on differentiated thyroid function in vitro6-I2.In addition, the ability of various tumor-promoting phorbol esters to activate PKC in vitro have paralleled their action on differentiated thyroid function6-I0. We observed that the non-phorbol diterpene, 12-hydroxy-daphnetoxin (mezerein) which is capable of activating PKC in vitro and in vivo2', is also capable of inhibiting TSH-stimulated iodide organification in porcine thyroid cells in a similar fashion to TPAIX.Similar findings were noted in FTRL-5 thyroid cells exposed to mezerein'. W e have also demonstrated that exogenous phospholipase C, which generates DG24, can also mimic the effects of TPA on differentiated thyroid function2'. PKC is activated when it is translocated from an inactive site in the cytosol to an active membranebound form4. We demonstrated that TPA, in concentrations which affect differentiated thyroid function, caused PKC translocation in porcine thyroid cell extracts (Table 1)'. Similar PKC translocation is induced in porcine thyroid cell extracts by phospholipase C2' and mezerein". It is interesting to note that mezerein affects differentiated thyroid function in v i m only at concentrations which cause PKC translocation in vitro". Several studies have utilized PKC inhibitors to determine the mechanism of phorbol ester action on differentiated thyroid function in vitro. The PKC inhibitor, 1-(.5-isoquinolinesulfonyl)-2-methylpiperazine (H7) has been shown to partially reverse the effects of TPA on differentiated thyroid function in vitro2'. Similar results have been shown with another PKC inhibitor, staurosporine26.In addition, staurosporine and the PKC inhibitor, l-O-hexadecyl-2-Omethyglycerol (AMG-C16), are capable of enhancing TSH-stimulated iodide organification in vitro2' implying that endogenous PKC acts as an inhibitor of differentiated thyroid function. Table 1 Protein kinase C activity in thyroid cell extracts (pmol/min per mg protein). Treatment
Cytosol Membrane
P*
4b-Phorhol
TPA
170(f5)** 1 1 l(f28) N.S.
53(+15) 466(&55) P
