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Endocrinology Vol. 138, No. 9 3787-3796
Copyright © 1997 by The Endocrine Society

ARTICLES

Suppression of Thyrotropin Receptor-G Protein-Phospholipase C Coupling by Activation of Protein Kinase C in Thyroid Carcinoma Cells1

Martina Broecker, Georg W. Mayr and Michael Derwahl

Laboratories of Experimental Endocrinology, Department of Medicine, Ruhr University (M.B., M.D.), 44789 Bochum; and Institute for Physiological Chemistry, University Hospital Eppendorf (G.W.M.), 25421 Hamburg, Germany

Address all correspondence and requests for reprints to: Dr. Michael Derwahl, Laboratories of Experimental Endocrinology, University Clinic of Internal Medicine, Buerkle-de-la-Camp-Platz 1, 44789 Bochum, Germany.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In human thyroid follicular cells TSH exerts its action on growth and function at least via two distinct pathways, the adenylate cyclase cascade and the phospholipase Cß (PLCß)-mediated inositol phosphate generation. We investigated the effect of TSH on activation of phosphoinositide hydrolysis and inositol phosphate generation by PLCß in HTh74 thyroid carcinoma cells that express functional TSH receptors and in HTC-TSHr thyroid carcinoma cells that are devoid of endogenous TSH receptors but express recombinant human TSH receptors. In both cell lines, TSH up to concentrations of 300 mU/ml failed to stimulate myo-inositol 1,4,5-trisphosphate and myo-inositol-tetrakisphosphate generation, but led to a decrease in these compounds within 1 min of stimulation. However, ATP and bradykinin increased concentrations of inositol phosphates in both thyroid carcinoma cell lines. In contrast, in differentiated FRTL5 thyroid cell line and CHO-TSHr cell line expressing recombinant human TSH receptors, TSH elicited a significant increase in myo-inositol 1,4,5-trisphosphate and its metabolic derivatives. However, when HTC-TSHr cells were pretreated with calphostin C or staurosporine, inhibitors of protein kinase C, a TSH concentration of 20 mU/ml enhanced generation of inositol phosphates in these cells. From our data we conclude that in HTC-TSHr and HTh74 thyroid carcinoma cells, the coupling within the TSH receptor-Gq protein-PLCß signaling pathway is impaired compared to that in nontransformed cells. It is conceivable that this is at least in part dependent on the level of protein kinase C activation in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GROWTH of endocrine cells is controlled by various growth factors and hormones that are part of a complex endocrine, autocrine, and paracrine network. In normal thyroid cells TSH is generally accepted to be the main regulator of growth and function (reviewed in 1 . Whereas in human thyroid follicular cells, TSH exerts its action at least via two distinct pathways, the TSH receptor-Gs protein-adenylate cyclase cascade and the TSH receptor-Gq protein-phospholipase Cß (PLCß) cascade (2, 3, 4), activation by TSH of the latter pathway is still controversial in other species (5, 6, 7).

It is well established that most effects of TSH on the thyroid gland, including stimulation of proliferation, thyroid hormone synthesis, and expression of thyroid-specific genes, are transmitted by the adenylate cyclase pathway (reviewed in 8 . However, recent data on stimulation of the PLCß-mediated generation of inositol phosphates suggest that a simultaneous stimulation of this pathway may at least in part act as a negative regulator of thyroid hormone synthesis by inhibiting iodide organification and thyroid hormone secretion (9, 10).

To our knowledge, there are only two reports of an altered phosphatidylinositol/Ca2+ signaling pathway in neoplastic thyroid tissues. Kobayashi and co-workers (11) measured increased enzymatic activity of PLC in thyroid carcinomas. Shaver et al. (12) recently reported on the measurement of TSH-stimulated PLC activity in various thyroid tissues. They demonstrated that in about 50% of high risk thyroid cancer tissues, TSH failed to activate PLC. In addition, Hatada and co-workers (13) have recently demonstrated high levels of particulate protein kinase C (PKC) in papillary and follicular thyroid carcinomas. These data are in accordance with a recent report of Prévostel et al. (14), who demonstrated in human thyroid neoplasms a point mutation at amino acid position 294 of the PKC{alpha} isozyme that led to increased expression of other PKC isozymes.

The aim of this study was to investigate the influence of TSH, bradykinin, and ATP on the generation of inositol phosphates in two human thyroid carcinoma cell lines, HTh74 and HTC-TSHr cells. The HTh74 cell line (15) is derived from an anaplastic thyroid carcinoma, but expresses functional TSH receptors, whereas HTC-TSHr cells (16) are derived from a follicular thyroid carcinoma and express recombinant, but lack endogenous, TSH receptors. We demonstrate here that neither thyroid carcinoma cell line exhibits any increase in inositol phosphates in response to concentrations of TSH up to 300 mU/ml, but both display decreases in myo-inositol bisphosphate (InsP2), myo-inositol 1,3,4-trisphosphate [Ins(1, 3, 4)P3], Ins(1, 4, 5)P3, and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1, 3, 4, 5)P4], in marked contrast to other TSH receptor-expressing cell lines, such as FRTL5 rat thyroid cells and CHO-TSHr cells. On the other hand, bradykinin and ATP were found to activate the PLCß cascade in both carcinoma cell lines. However, when HTC-TSHr cells were pretreated with calphostin C or staurosporine, inhibitors of PKC, significant increases in InsP, InsP2, Ins(1, 3, 4)P3, Ins(1, 4, 5)P3, and InsP(1, 3, 4, 5)P4 in response to TSH were observed. From our data we conclude that in HTC-TSHr cells the TSH receptor-Gq-PLC signaling pathway is at least in part depressed by an activated PKC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Coon’s modified Ham’s F-12 medium, FCS, and trypan blue were purchased from Life Technologies (Grand Island, NY). Glycyl-histidyl-lysine, insulin, somatostatin, transferrin, hydrocortisone, ATP, bradykinin, N6-(2-phenylisopropyl)-adenosine (PIA), 8-bromo-cAMP, phorbol 12-myristate 13-acetate, and bovine calf serum were obtained from Sigma Chemical Co. (St. Louis, MO). Bovine TSH preparations from both Sigma and Calbiochem (La Jolla, CA) were used for stimulation experiments. Calphostin C and staurosporine were purchased from Calbiochem. Quickszint Flow 306 liquid scintilator was obtained from Zinsser Analytic (Frankfurt, Germany). Myo-[2-3H]inositol, the Biotrak PKC enzyme assay system, and [{gamma}-32P]ATP were purchased from Amersham-Buchler (Braunschweig, Germany). Mono-Q columns, diethylaminoethyl-Sepharose, and the PCR Template Prep for single strand DNA sequencing kit were obtained from Pharmacia (Uppsala, Sweden). The Sequenase 2.0 sequencing system was obtained from U.S. Biochemical Corp. (Cleveland, OH). HTh74 thyroid carcinoma cell line was provided by Dr. Heldin, Uppsala (15). The FRTL5 Fischer rat thyroid cell line (17) was previously obtained from American Type Culture Collection (Bethesda, MD).

Cell cultures
Monolayer cultures of the human thyroid carcinoma cell lines HTC-TSHr, clone h5–2 (16), and HTh74 (15) were grown in Coon’s modified Ham’s F-12 medium supplemented with 10% FCS and five hormones or growth factors (glycyl-histidyl-lysine, 10 ng/ml; insulin, 10 mg/ml; somatostatin, 10 ng/ml; transferrin, 5 mg/ml; hydrocortisone, 3.2 ng/ml). FRTL5 cells (17) were maintained in the same medium with or without 10 mU/ml TSH as indicated, but with 5% calf serum. CHO-TSHr cells, clone c7–6 (Derwahl. M., and M. Broecker, unpublished data), were grown in Coon’s modified Ham’s F-12 medium supplemented with 10% FCS.

Labeling of cells, stimulation, and extraction of inositol phosphates and phosphoinositides
Cells were labeled for 48 h in the appropriate medium without TSH containing 4 mCi/ml myo-[2-3H]inositol (SA, 100 Ci/mmol). The concentration of total inositol in the medium was about 140 mM. At the end of the labeling period, the cell monolayer was washed five times with prewarmed stimulation buffer (130 mM NaCl, 10 mM HEPES, 10 mM LiCl, 5 mM glucose, 1 mM MgCl2, 1 mM CaCl2, and 1 mM KPO4, pH 7.4). After adding 9 ml stimulation buffer, the petri dishes were preincubated at 37 C for 15 min with or without 10, 20, 50, 100, or 200 nM calphostin C or 0.15, 0.3, or 1.5 nM staurosporine. The indicated hormones or ligands were added in 1 ml stimulation buffer, and agonist-stimulated cells were kept for the indicated times. To stop stimulation, the petri dishes were placed on ice, and the buffer was quickly aspirated and replaced with 3 ml cold 10% trichloroacetic acid. After scraping with a rubber policeman and centrifugation, inositol phosphates in the supernatants were extracted four times with equal volumes of diethyl ether. Phospoinositides were made water soluble by mild deacylation (18, 19).

Separation of inositol phosphates and phospoinositides by HPLC
Inositol phosphates and deacylated phosphoinositides (glycerophosphoinositol phosphates) were separated by anion exchange HPLC using a guard column (0.5 x 5 cm) and a main column (0.5 x 20 cm) packed with Mono-Q. The elution protocol with the HCl gradients and the KCl gradients as well as the sample workup procedure were described previously (20). Retention times were established by analyzing nonradioactive mixtures of inositol phosphates and deacylated phospholipids by the metal dye detection-HPLC technique as previously described (20, 21). Fractions were collected at 30-sec intervals, and radioactivity of the whole fraction (0.5 ml) supplemented with 1.5 ml Quickszint Flow 306 was measured in a ß-counter by liquid scintillation counting. To calculate changes in phospholipids, the counts of the small fraction of inositol phosphates generated from phospholipids during the deacylation procedure (Fig. 2Go) were added to the counts of the corresponding glycerophosphoinositol phosphate peaks.



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  		Figure 2. HPLC elution profiles of deacylated phospholipid head groups marked with [3H]inositol. Phospholipids were extracted from unstimulated (left) and stimulated (right) CHO-TSHr and FRTL5 cells. CHO-TSHr and FRTL5 cells were stimulated with 300 mU/ml TSH for 1 min. a, GroPIns; b, InsP; c, GroPIns4P; d, InsP2; e, GroPIns(4,5)P2.

 
Measurement of PKC activities
Cells were washed three times with prewarmed stimulation buffer, preincubated at 37 C for 15 min, and then stimulated with the indicated concentrations of TSH and 8-bromo-cAMP at 37 C for 15 sec or 0.5, 1, 3, or 5 min. The cytosolic and solubilized particulate fractions were prepared on ice and then purified by diethylaminoethyl-Sepharose column chromatography as described by Fujimori et al. (22). PKC activity was determined by a commercial assay kit (Biotrak PKC enzyme assay, Amersham-Buchler). Protein concentrations were determined using the Bio-Rad protein assay kit (Richmond, CA) and BSA as the standard (23). All experiments were repeated at least three times in duplicate.

Isolation of DNA, PCR, and sequencing of PCR products
DNA was isolated from cells using standard methods (24). PCR reactions were performed according to the conditions described by Prévostel et al. (14). For nucleotide sequencing of PCR-generated fragments, we performed the method of Higuchi and Ochman (25) using the PCR Template Prep for single strand DNA sequencing (Pharmacia, Uppsala, Sweden) and the Sequenase 2.0 system (U.S. Biochemical Corp.).

Statistical analysis
Experiments were repeated at least three times. Statistical analysis was performed using Student’s t test; P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of TSH on inositol phosphate formation in HTC-TSHr, HTh74, FRTL5, and CHO-TSHr cells
PLCß-mediated inositol phosphate generation in response to TSH was measured in HTh74 and HTC-TSHr thyroid carcinoma cells and, as controls, in nonthyroid CHO-TSHr cells that are stably transfected with the human TSH receptor complementary DNA and in FRTL5 rat thyroid cells. Both cell lines showed TSH-dependent signaling, i.e. activation of the cAMP and PLCß cascades, similar to those observed in normal human thyroid cells (2, 3, 26, 27, 28). Although both HTC-TSHr and nonthyroid CHO-TSHr cell lines express recombinant human TSH receptors with numbers of receptors per cell within the range of normal thyroid cells (16), FRTL5 cells express about 2–3 times more TSH receptors (29) and (our unpublished data). Treatment of HTC-TSHr and HTh74 thyroid carcinoma cells with increasing concentrations of TSH up to 300 mU/ml did not result in stimulation of inositol phosphate generation (Fig. 1Go and Table 1Go), but significantly decreased InsP2 and Ins(1, 4, 5)P3 after 1 min of TSH stimulation in HTC-TSHr cells. In HTh74 cells Ins(1, 3, 4)P3, Ins(1, 4, 5)P3, and Ins(1, 3, 4, 5)P4 concentrations were diminished. After 5 min of stimulation of HTC-TSHr cells, concentrations of Ins(1, 3, 4)P3, Ins(1, 4, 5)P3, and Ins(1, 3, 4, 5)P4 were decreased, whereas in HTh74 cells, concentrationsof inositol monophosphate (InsP), InsP2, Ins(1, 3, 4)P3, Ins(1, 4, 5)P3, Ins(1, 3, 4, 6)P4, and Ins(1, 3, 4, 5)P4 were diminished (Table 1Go).



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  		Figure 1. HPLC elution profiles of [3H]inositol phosphates extracted from unstimulated (left) and stimu-lated (right) CHO-TSHr, FRTL5, and HTC-TSHr cells. Cells were stimulated with 300 mU/ml TSH for 5 min. a, InsP; b, InsP2; c, Ins(1,3,4)P3; d, Ins(1,4,5)P3; e, Ins(4,5,6)P3; f, Ins(1,3,4,6)P4; g, Ins(1,3,4,5)P4; h, Ins(1/3,4,5,6)P4; i, Ins(1,3,4,5,6)P5; j, InsP6.

 

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  		Table 1. Changes in inositol phosphates in CHO-TSHr, FRTL5, HTC-TSHr, and HTh74 cells after stimulation with 300 mU/ml TSH

 
The data given in Table 1Go represent changes in inositol phosphate concentrations after stimulation with a high dose of 300 mU/ml TSH, enabling an optimal evaluation of changes of InsP3 and InsP4 isomers. However, marked changes in inositol phosphates were achieved by as little as 10 mU/ml TSH (data not shown). The shortest time of TSH stimulation that led to a significant decrease in inositol phosphate levels was 30 sec (data not shown).

In contrast to both thyroid carcinoma cell lines, a remarkable increase in InsP2 and Ins(1, 4, 5)P3 generation within 1 min of TSH stimulation was observed in CHO-TSHr cells (Table 1Go), indicating a rapid activation of PLCß. After 3 min of stimulation, CHO-TSHr cells displayed significant increases in InsP, InsP2, Ins(1, 4, 5)P3, Ins(1, 3, 4, 6)P4, Ins(1, 3, 4, 5)P4, Ins(1/3,4,5,6)P4, and inositol pentakisphosphate [Ins(1, 3, 4, 5, 6)P5]. After 5 min of TSH stimulation, the concentrations of Ins(1, 3, 4)P3 and InsP6 were additionally elevated (Table 1Go).

FRTL5 thyroid cells showed a significant increase in inositol phosphates in response to TSH stimulation (Table 1Go). These results are in accordance with two previous reports (27, 28) of inositol phosphate accumulation after TSH stimulation in FRTL5 cells and in nontransformed human thyroid cells (2, 3). As FRTL5 cells exhibited PLC activation after TSH stimulation, we used these cells as a second control to specify in a thyroid cell line inositol phosphate isomer profiles elicited upon TSH stimulation. One minute of stimulation led to increased concentrations of Ins(1, 3, 4)P3, Ins(1, 3, 4, 6)P4, and Ins(1, 3, 4, 5)P4 in these cells. After 3 and 5 min of stimulation, concentrations of InsP, InsP2, Ins(1, 4, 5)P3, and Ins(1/3,4,5,6)P4 were also significantly increased. However, the elevation of InsP6 was slight, but not significant (Table 1Go).

Furthermore, in FRTL5 cells, concomitant with PLCß activation, TSH apparently led to a very rapid activation of Ins(1, 4, 5)P3 3-kinase, which generates Ins(1, 3, 4, 5)P4 from Ins(1, 4, 5)P3, and of Ins(1, 3, 4, 5)P4 5'-phosphatase, which converts Ins(1, 3, 4, 5)P4 into Ins(1, 3, 4)P3. Therefore, PLCß must have been stimulated within 1 min to generate Ins(1, 4, 5)P3, as otherwise a marked increase in InsP4 with an unchanged mass of Ins(1, 4, 5)P3 after 1 min of stimulation could not be explained. These data are in accordance with the very recent data of Singh et al. (30), who investigated inositol phosphate metabolism in FRTL5 cells after the addition of TSH. These researchers demonstrated an increase in an InsP3 isomer and two InsP4 isomers, but no increase in Ins(1, 4, 5)P3 during the first 2 min of stimulation (30).

Sho and co-workers (31) recently reported that PIA, a P1-purinergic agonist, modulates the sensitivity of PLCß activation and inositol phosphate generation in FRTL5 cells in response to TSH stimulation. Therefore, we conducted experiments to prove the effect of PIA in HTC-TSHr and HTh74 cells. However, treatment of carcinoma cells with 100–250 nM PIA did not affect inositol phosphate concentrations compared with those in untreated controls. Furthermore, stimulation of pretreated cells with up to 300 mU/ml TSH resulted in a decrease in inositol phosphate concentrations similar to that observed in TSH-stimulated cells without PIA preincubation (data not shown).

Inositol phosphate formation in response to ATP and bradykinin stimulation
To analyze the potential of other G protein-coupled receptors to activate PLCß in HTC-TSHr and HTh74 cells, we treated these thyroid carcinoma cells with 10 mM ATP and 1 mM bradykinin, which are known to stimulate phosphoinositide hydrolysis via a PLCß isozyme (reviewed in 32 . As indicated in Table 2Go, these agonists led to a rapid increase in inositol phosphates after 1 min of stimulation.


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  		Table 2. Changes in inositol phosphates in HTC-TSHr and HTh74 cells after stimulation with 10 mM ATP and 1 mM bradykinin for 1 min

 
Phosphoinositide changes in stimulated cells
In addition, we measured the alterations in phosphoinositide composition of the stimulated cells by measuring the radioactivities of water-soluble inositide head groups after deacylation of the latter. After TSH stimulation, CHO-TSHr and FRTL5 cells exhibited decreases in {alpha}-glycerophospho(1)-D-myo-inositol-4-phosphate (GroPIns4P) and {alpha}-glycerophospho(1)-D-myo-inositol-4,5-bisphosphate [GroPIns(4, 5)P2] in parallel with the elevation of inositol phosphates (Fig. 2Go).

In HTC-TSHr cells, a significant increase, but not a decrease, in phosphoinositides was seen after 1 min of stimulation with ATP and bradykinin (chromatograms not shown). In response to ATP and bradykinin, total phosphoinositide concentrations increased to 112% and 111%, respectively, and GroPIns(4, 5)P2 concentrations increased to 151% and 147% of control values, respectively. Similar results were obtained in HTh74 cells (data not shown). These data indicate that HTC-TSHr cells are able to rapidly resynthesize phosphoinositides to a higher extent than that hydrolyzed by ATP or bradykinin stimulation, probably by activation of PtdIns4P 5-kinase. This enzyme is reported to be activated by some G protein-coupled receptors, for example the N-formylmethionyl-leucylphenylalanine and platelet-activating factor receptor, and by nonhydrolysable GTP analogs (33).

Generation of inositol phosphates in response to TSH in HTC-TSHr cell after pretreatment with inhibitors of PKC
As a negative feedback between PKC activation and Gq-PLCß coupling has previously been reported (34, 35), we pretreated HTC-TSHr cells with either 100 nM calphostin C or 1.5 nM staurosporine, potent inhibitors of PKC, for 15 min at 37 C before stimulation with 10–300 mU/ml TSH. After preincubation with 100 nM calphostin C, concentrations of TSH as low as 20 mU/ml significantly enhanced the synthesis of Ins(1, 3, 4)P3, Ins(1, 4, 5)P3, and Ins(1, 3, 4, 5)P4 (Table 3Go). Lower concentrations of calphostin C did not affect inositol phosphate generation (data not shown). A more pronounced effect on Ins(1, 3, 4)P3 and Ins(1, 3, 4, 5)P4 generation was observed after a 1-min stimulation of HTC-TSHr cells with 300 mU/ml TSH. Stimulation of these cells for 3 or 5 min additionally enhanced the concentrations of InsP, InsP2, and Ins(1, 4, 5)P3 (Fig. 3Go). Similar results were obtained after preincubation with 1.5 nM staurosporine (data not shown).


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  		Table 3. Changes in inositol phosphates in HTC-TSHr cells pretreated with 100 nM calphostin C for 15 min and stimulated with various TSH concentrations for 5 min

 


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  		Figure 3. Inositol phosphates in HTC-TSHr cells pretreated with 100 nM calphostin C and stimulated with 300 mU/ml TSH for 1, 3, and 5 min. Changes in inositol phosphates are presented as a percentage of the control (mean ± SEM).

 
As described above, HTC-TSHr cells pretreated with PKC inhibitors showed an elevation of Ins(1, 3, 4)P3 and Ins(1, 3, 4, 5)P4 after a short stimulation. The data resemble those obtained in FRTL5 cells, indicating a rapid activation of Ins(1, 4, 5)P3 3-kinase and InsP4 5'-phosphatase and a concomitant activation of PLCß within the first minute of TSH stimulation in both thyroid cell lines.

Finally, phosphoinositide concentrations in calphostin C-pretreated and TSH-stimulated HTC-TSHr cells were slightly elevated compared with those in unstimulated cells, indicating a rapid resynthesis of these compounds (data not shown), as demonstrated in response to ATP and bradykinin stimulation.

PKC activities in TSH-stimulated cells
Additionally, we measured PKC activities in unstimulated and TSH-stimulated FRTL5, CHO-TSHr, HTh74, and HTC-TSHr cells by analyzing the phosphorylation of a PKC-specific peptide. As shown in Table 4Go, TSH stimulation led to PKC translocation to the particulate fraction in all cell lines. Translocation of PKC activity to the particulate fraction corresponded to an activation of the enzyme in thyroid cells as shown by others (36, 37) and in own experiments. Treatment of HTC-TSHr, HTh74, FRTL5, and CHO-TSHr cells with 500 nM phorbol 12-myristate 13-acetate for 5 min resulted in a redistribution of PKC activity, with over 95% of total PKC activity measured in the particulate fraction of all cell lines (data not shown).


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  		Table 4. PKC activity in CHO-TSHr, FRTL5, HTC-TSHr, and HTh74 cells after stimulation with 300 mU/ml TSH and 250 µM 8-bromo-cAMP for 5 min

 
However, whereas in FRTL5 and CHO-TSHr cells, about two thirds of the total PKC activity was found in the particulate fraction after stimulation with 300 mU/ml TSH for 5 min, in both carcinoma cell lines nearly 100% of PKC activity was measured in the particulate fraction, indicating a maximal activation of PKC in these tumor cell lines (Table 4Go). A significant translocation of PKC activity (HTC-TSHr cells, 586 ± 49 pmol/mg·min in the particulate fraction and 349 ± 24 pmol/mg·min in the cytosolic fraction; HTh74 cells, 570 ± 30 pmol/mg·min in the particulate fraction and 389 ± 37 pmol/mg·min in the cytosolic fraction) was already determined after 30 sec of TSH stimulation. Incubation of all cell lines with the stable cAMP analog 8-bromo-cAMP for 5 min did not result in a significant activation of PKC (Table 4Go).

To search for the mutation of the PKC{alpha} gene that has very recently been described by Prévostel et al. (14), we additionally amplified and sequenced a portion of the V3 region of the PKC{alpha} gene of HTC-TSHr and HTh74 cells. However, we could not detect a mutation of the PKC{alpha} gene in this region in either thyroid tumor cell line (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Coupling of a receptor to different effector systems is not only dependent on the specific properties of the receptor molecule, but also on the cell type in which it is expressed and on the presence of the appropriate coupling systems and effector enzymes in these cells. For instance, expression of dopaminergic D2 receptors in Ltk- fibroblasts resulted in ligand-induced phosphoinositide hydrolysis and activation of Ins(1, 4, 5)P3-induced intracellular Ca2+ release, whereas the same receptors expressed in pituitary GH4C1 cells failed to affect phosphoinositide hydrolysis and induced a decrease in cytosolic free Ca2+ concentrations (38).

Very recently, it was reported that the human TSH receptor can be coupled to at least 10 different G protein {alpha}-subunits (39). However, only two of the dependent effector systems, the adenylate cyclase system, activated by Gs{alpha}, and the phospholipase C cascade, activated by Gq/11{alpha}, have been characterized in detail (2, 3, 4). Activation of both effectors has been demonstrated for the endogenous TSH receptor in human thyroid slices (2) as well as for the recombinant human TSH receptor expressed in CHO cells (26).

Both thyroid carcinoma cell lines used in the present study were found to express functional TSH receptors that are coupled to the adenylate cyclase pathway, leading to a dose-dependent generation of intracellular cAMP in response to TSH (15, 16). However, in both cell lines TSH apparently failed to stimulate inositol phosphate generation, whereas ATP and bradykinin, via their G protein-coupled receptors, induced rapid inositol phosphate generation and phosphoinositide resynthesis.

It has been shown that the expression level of thyroid-specific genes, e.g. of TSH receptor messenger RNA, in thyroid tumors is related to the level of differentiation resulting in a complete loss of TSH receptor expression in poorly differentiated thyroid carcinomas (40, 41). HTh74 thyroid carcinoma cells are derived from an anaplastic thyroid carcinoma, but still express TSH receptors (15), albeit at a presumably low level. In this respect it might be argued that the number of TSH receptors in these cells is too low to induce phosphoinositide hydrolysis in response to TSH stimulation. However, in HTC-TSHr cells, TSH also failed to stimulate inositol phosphate generation. These cells are known to express 3000–5000 recombinant human TSH receptors/cell, which is in the range of normal thyroid cells (16).

In thyroid cells of various animal species, higher than physiological doses of TSH are required to stimulate PLCß-mediated phosphoinositide hydrolysis in vitro (2, 3), which makes a physiological role for this signaling pathway questionable. However, very recently it has been shown by Laglia and co-workers (42) that an enhanced concentration of cAMP attenuates TSH-induced inositol phosphate generation in FRTL5 thyroid cells. Furthermore, Sho et al. (31) demonstrated that in rat FRTL5 thyroid cells PIA, a P1-purinergic agonist, can increase the sensitivity of the phosphoinositide hydrolysis pathway to TSH stimulation. PIA inhibited TSH-induced cAMP accumulation and simultaneously enhanced TSH-induced activation of PLCß and Ins(1, 4, 5)P3-induced Ca2+ release in these cells. This switch is supposed to be due to cross-talk of a cAMP-dependent pathway and the ß-isozyme of PLC (43). However, using high doses of both PIA and TSH we were not able to detect any increase in inositol phosphate generation in either HTh74 or HTC-TSHr cells.

A recent report by Shaver et al. (12) supports our results concerning TSH-stimulated PLC activity in thyroid carcinoma cells. In about 50% of the high risk thyroid cancer tissues, these researchers could not detect generation of inositol phosphates in response to 300 mU/ml TSH.

In both HTh74 and HTC-TSHr cell lines, ATP and bradykinin were found to activate PLCß isozymes, resulting in a rapid increase in inositol phosphates. This demonstrates that in both cell lines, G protein-coupled receptors are able to affect the phosphatidylinositol signaling system. As there is a strong activation of PLCß signaling in response to ATP or bradykinin stimulation despite high basal PKC activity, it is likely that different combinations of members of the Gq protein family or G protein ß{gamma}-subunits and PLCß isozymes are used by TSH or ATP and bradykinin in thyroid cells. In fact, it has been demonstrated in various cell lines that G proteins of the Gq family as well as G protein ß{gamma}-subunits may couple to different PLCß isozymes with combinations that are more or less responsive (reviewed in 44 . Due to this variability in different G protein coupling systems, the ATP or bradykinin signaling pathway may be less sensitive to inhibition by high PKC activity than the TSH receptor-PLCß signaling pathway. Alternatively, the latter coupling pathway might be attenuated only by a combined action of PKC activation and a cAMP-dependent and, thus, TSH-dependent process. This presumption is in accordance with the recent data reported by Laglia et al. (42), who demonstrated an attenuation of inositol phosphate generation by cAMP in FRTL5 cells.

In addition, our data have to be reconciled with the recent finding of increased basal and GTP-stimulated PLC activity in neoplastic thyroid membranes (11). These researchers measured the total PLC activity, but not that of different PLC isozymes. As increased PLC activity may be due to a stimulation of both G protein-coupled receptors and growth factor receptors (reviewed in 32 , these results are not contradictory to our data.

On the ground of recent reports on increased PKC activity in thyroid carcinomas (13) and high levels of PKC in rapidly growing thyroid cells in vitro (36, 45), we conducted experiments to analyze the effect of PKC inhibition on inositol phosphate formation in response to TSH. By feedback regulation, activation of PKC is known to prevent activation of PLCß by G protein-coupled receptors (46, 47). We demonstrate here that short term inhibition of PKC activity markedly improved TSH receptor-G protein-PLCß signaling in response to TSH and that HTC-TSHr cells thereby regained TSH-dependent formation of inositol phosphates.

In addition, inhibition of PKC enabled us to determine the generation of different inositol phosphate isomers in HTC-TSHr cells and to compare the results with those measured in our controls, FRTL5 and CHO-TSHr cells. In all three cell lines TSH stimulated Ins(1, 4, 5)P3 3-kinase. Although in FRTL5 and pretreated HTC-TSHr cells, TSH rapidly activated Ins(1, 4, 5)P3 3-kinase and Ins(1, 3, 4, 5)P4 5'-phosphatase and thus stimulated Ins(1, 3, 4, 5)P4 and Ins(1, 3, 4)P3 generation, in nonthyroid CHO-TSHr cells we observed an elevation of Ins(1, 3, 4, 5)P4 not before 3 min and an increase in Ins(1, 3, 4)P3 not before 5 min of TSH stimulation. It is evident that in thyroid cells the rapid activation of Ins(1, 4, 5)P3 3-kinase and Ins(1, 3, 4, 5)P4 5'-phosphatase may depend on some cross-talk with other TSH-activated pathways, for example by an elevation of the intracellular Ca2+ concentration or the activation of protein kinase A or PKC, which are able to increase Ins(1, 4, 5)P3 3-kinase activity in some cell systems (reviewed in 48 .

Although the molecular mechanism that modulates the TSH receptor-Gq-PLCß signaling pathway has to be further elucidated in all details, it is obvious that an activated PKC in the HTC-TSHr tumor cell line plays a central role in suppressing this signaling pathway. As PLCß as well as PLC{gamma} activities are controlled by PKC via a negative feedback (49, 50), we measured PKC activity after TSH stimulation in both tumor cell lines and found an early increase in PKC activity that was independent of an elevation of the intracellular cAMP concentration. The more pronounced increase in membrane-bound PKC activity in tumor cells, alone or in combination with activation of other signaling pathways, must be responsible for the small, but significant, decrease in inositol phosphates subsequent to stimulation with TSH. Inhibition of basal PLC activity and/or a signal-responsive activation of InsP3 3-kinase and InsP3/Ins(1, 3, 4, 5)P4 5'-phosphatase may be sufficient to explain this unexpected behavior.

FRTL5 cells, which responded to high doses of TSH with an increase in Ins(1, 4, 5)P3 as well as an increase in products derived thereof by the known interconversions, showed the same unexpected changes. After 1 min of stimulation with 300 mU/ml TSH, but not thereafter, a slight decrease in Ins(1, 4, 5)P3 was observed in FRTL5 cells, although products apparently derived from Ins(1, 4, 5)P3 as Ins(1, 3, 4, 5)P4, Ins(1, 3, 4)P3, Ins(1, 3, 4, 6)P4, and even InsP5 and InsP6 are increased. The data reported by Singh et al. (30), who demonstrated the inositol phosphate metabolism up to 2 min are in agreement with our findings after 1 min of stimulation, whereas after 3 min we found an increase in Ins(1, 4, 5)P3.

As the early activation of PKC is, at least in thyroid carcinoma cell lines, independent of inositol phosphate generation and an elevated cAMP concentration, it is evident that it is mediated by an alternative, Gs- and Gq-independent signaling pathway. This conclusion fits with the recent finding that the activated TSH receptor can be coupled to 10 different G protein {alpha}-subunits in normal thyrocytes (39). Indeed, Fujimoto and Brenner-Gati (36) reported on a cAMP-dependent activation of PKC that is likely to be the result of PLD or PLA2 activation (51). In addition, Lejeune et al. (52) demonstrated an increase in phosphatidic acid accumulation in thyroid cells after TSH stimulation that was not mimicked by cAMP analogs and was not caused by PLD stimulation.

However, whereas Fujimoto and Brenner-Gati (36) measured the effects of TSH in TSH-deprived FRTL5 cells after a relatively long stimulation period of 24 h, our data indicate an immediate TSH-mediated PKC activation. Similar results of early PKC activation by G protein-coupled receptors have been reported by Fujimori et al. (22), who reported a rapid PKC translocation without Ins(1, 4, 5)P3 release in rat osteogenic sarcoma (UMR 100–01) cells after stimulation with PTH.

The results of our study demonstrated the crucial role of high basal PKC activities for the inhibition of TSH receptor-Gq-PLCß signaling. By treatment of HTC-TSHr cells with PKC inhibitors, this tight inhibition of TSH receptor-Gq-PLCß signaling was relieved and, in turn, the depressed signaling pathway was reconstituted. As high levels of PKC activities are not restricted to the thyroid carcinoma cell lines investigated in this study, but are also detectable in thyroid carcinoma tissues (13), the question of the molecular mechanisms that lead to high basal PKC activities is of particular interest. In a subset of thyroid neoplasms, high PKC activities may be due to a mutation of the PKC{alpha} gene that lead to an increased expression of PKC isozymes (14). However, in thyroid carcinoma cells that do not harbor such mutations, high PKC activities may at least in part be explained by autocrine stimulation of tyrosine kinase receptors such as epidermal growth factor and platelet-derived growth factor receptors that has been described in different thyroid carcinoma cell lines and is also detectable in HTC-TSHr cells (53, 54). Taken together, the present study supports the concept of the eminent importance of PKC in the pathogenesis of thyroid neoplasms.


   Acknowledgments
 
We acknowledge the expert technical assistance of Cornelia Tietz and Antje Zint. We are indebted to Dr. N.-E. Heldin, Institute of Pathology, University of Uppsala (Uppsala, Sweden), for generous gift of HTh74 thyroid carcinoma cells.


   Footnotes
 
1 This work is dedicated to Prof. H. Schatz on the occasion of his 60th birthday. This work was supported by Deutsche Forschungsgemeinschaft (Bonn, Germany), Grant De 407/7–1 (to M.D.), and a grant for project 16 (SFB 354; to G.W.M.). Back

Received February 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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