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Endocrinology Vol. 140, No. 4 1724-1730
Copyright © 1999 by The Endocrine Society

ARTICLES

Thyrotropin Regulates c-Jun N-Terminal Kinase (JNK) Activity through Two Distinct Signal Pathways in Human Thyroid Cells1

Takeshi Hara, Hiroyuki Namba, Noboru Takamura, Ting-Ting Yang, Yuji Nagayama, Shuji Fukata, Kanji Kuma, Naofumi Ishikawa, Kunihiko Ito and Shunichi Yamashita

Department of Nature Medicine (T.H., H.N., T.-T.Y., S.Y.), Department of International Health and Radiation Research (N.T., S.Y.), Atomic Bomb Disease Institute, Department of Pharmacology (Y.N.), Nagasaki University School of Medicine, Nagasaki, 852-8523, Japan; Kuma Hospital (S.F., K.K.), Kobe, 650, Japan; Ito Hospital (N.I., K.I.), Tokyo, 150, Japan

Address all correspondence and requests for reprints to: Hiroyuki Namba, MD, Associate Professor, Department of Nature Medicine, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, 1–12-4 Sakamoto, Nagasaki 852, Japan. E-mail: namba{at}net.nagasaki-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
c-Jun N-terminal kinases (JNK) participate in cellular responses to mitogenic stimuli and environmental stresses. We investigated whether and how TSH, which promotes the proliferation and differentiation of thyroid cells, regulates JNK activity in primary cultured human thyroid cells. TSH stimulated JNK activity in cytosolic fractions of thyroid cells measured by in vitro kinase assay. A low concentration of TSH (10-11 M) stimulated JNK activity but at a higher dose (10-8–10-7 M), TSH suppressed JNK activity without any change of JNK protein level. Activation of JNK by TSH was also observed in CHO cells stably transfected with TSH receptor complementary DNA (cDNA), suggesting a ligand-receptor specific interaction. TSH stimulated JNK activity through a pertussis toxin-sensitive pathway. We next elucidated the signal transduction pathways in TSH-induced JNK activation by examining the involvement of four distinct intracellular signal molecules; protein kinase C (PKC), cAMP, Ca2+, and PI3-kinase. The stimulation of JNK by TSH was blocked by two PKC inhibitors and suppressed by 8-bromo-cAMP or forskolin. These findings demonstrate that TSH regulates JNK activity biphasically in human thyroid cells through an interaction between Gi-PKC and cAMP-PKA pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TSH PLAYS AN important role in the regulation of thyroid cell proliferation and differentiation by binding to its receptor (TSHR) on the plasma membrane (1, 2). TSHR belongs to a family of seven transmembrane domain receptors and is coupled to heterotrimeric G proteins, including Gs, Gi/o, G12, and Gq/11 (3). Among of them, the roles of Gs and Gq/11 proteins in thyroid cells have been well studied (4). TSH activates both the adenyl cyclase-cAMP cascade and phospholipase C (PLC) pathway through Gs{alpha}-subunits (Gs{alpha}) and Gq/11 {alpha}-subunits (Gq{alpha}), respectively (5). Sequentially, the cAMP pathway activates cAMP-dependent protein kinase (PKA) and PLC pathway increases intracellular Ca2+ and activates protein kinase C (PKC). Although the involvement of Gs-cAMP cascade in the activity of TSH has been well described (6), the role of other pathways through Gi/o, G12 and Gq/11 proteins remains to be elucidated in thyroid cells. It is possible that various TSH actions are mediated through different pathways of intracellular signal transduction. In addition, the diverged signals may exhibit a cross-talk phenomenon in the cell. Recent studies by Laglia et al. (7) have demonstrated that high levels of cAMP inhibit the activity of PLC in thyroid cells transfected with cholera toxin A1 subunit gene, a process that results in persistent activation of Gs{alpha}.

c-Jun N-terminal kinase (JNK), a member of the mitogen activated protein kinase (MAPK) superfamily, binds to and phosphorylates c-Jun, resulting in the activation of the transcription factor AP-1 (8, 9). The JNK cascade is stimulated in various cells by hormones acting through G protein-coupled receptors (10, 11, 12). Furthermore, the intracellular signaling to JNK is mediated by diverse cytoplasmic molecules, such as heterotrimeric G protein ß{gamma}-subunits (11), small G proteins (13, 14), phosphatidylinositol 3-kinase (PI3-K) (15, 16), and intracellular Ca2+ (17), in various cell types. The intracellular molecules and cascades that activate JNK are cell-type and/or stimulus specific. For example, TGF-ß stimulates JNK activity through TGF-ß-activated protein kinase (TAK1) (18) and activation of JNK through Rho, a small G protein-coupled, is only observed in human kidney 293T cells (19). It has also been recently demonstrated in human thyroid cells that TSH increased mRNA expression in c-jun gene, which is regulated by AP-1 promoter elements (20).

In the present study, we investigated whether and how TSH regulates JNK activity in primary cultured human thyroid cells. Our results provide evidence for TSH activation of JNK mediated through a pertussis toxin (PTX)-sensitive and PKC-dependent pathway. A similar TSH-mediated stimulation of JNK was also reproduced in cells transfected with cDNA of TSH receptor. Furthermore, the activation of JNK was attenuated by increased intracellular cAMP, suggesting that activation of JNK in human thyroid cells is regulated by a cross-talk between cAMP and PKC pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Polyclonal antibodies to JNK were obtained from PharMingen (San Diego, CA). Phospho Plus c-Jun antibody kit was purchased from New England Biolabs, Inc. (Beverly, MA). Bovine TSH (bTSH), recombinant TSH, insulin-like growth factor-I (IGF-I), 1-(5-isoquinolinesulphonyl)-2,5-dimethylpiperazine 2HCl (H7), 8-bromoadenosine 3',5'-cyclicmonophosphate (8Br-cAMP), 12-O-tetradecanoylphorbol ß-acetate (TPA), forskolin, ethylene glycoltetraacetic acid (EGTA) and BSA were obtained from Sigma Chemical Co. Corp. (St. Louis, MO). Wortmannin, thapsigargin, 1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl-ester (BAPTA-AM) and G418 were from Wako Chemical Corp. (Osaka, Japan). Tissue culture media, PTX and lipofectin were from Life Technologies, Inc. (Rockville, MD). FBS was purchased from Intergen. Enhanced chemiluminescence system was from Amersham Corp. (Arlington Heights, IL). pFR-Luc and pFA-cJun plasmids (PathDetect c-Jun trans-reporting system) were from Stratagene (La Jolla, CA). Dual-luciferase reporter assay system was from Promega Corp. (Madison, WI).

Cells
For primary thyroid cell cultures, thyroid tissue samples were obtained by subtotal thyroidectomy from 22 patients with Graves’ disease who had received antithyroid drug therapy for several weeks and were euthyroid at the time of the operation. Thyroid cells were isolated and 99% of these cells were confirmed to be thyroid epithelial cells using the method described previously by our laboratory (21). The primary cells were cultured in 2:1 mixture of Hams’ F12 and DMEM supplemented with 20 µg/liter IGF-I, 100 mU/liter bTSH, 40 mg/liter vitamin C, and 3% FBS. CHO cells and 293 human embryonal kidney cells (ATCC, CRL 1573) were transfected with eukaryotic expression vector pCR-TSHRmyc, which contained epitope tagged TSHR cDNA, by Lipofectin method (22). Clonal cell lines were selected with 400–800 mg/liter G418 and isolated using cloning cylinders. The transfected CHO cells were incubated with Hams’ F12 containing 5% FBS and 293 cells with DMEM with 10% FBS. Before use in experiments, the cells were cultured overnight in a medium supplemented only with 0.3% BSA.

Jun kinase assay
After stimulation with various reagents in serum-free media, the culture medium was removed at the appropriate time. Phosphorylation of c-Jun was then measured using a Phospho Plus c-Jun antibody kit according to the protocol recommended by the manufacturer. In brief, the cells were washed three times with PBS and harvested with lysis buffer: 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4 and 1 mg/liter leupeptin. After extracting cell lysates, the kinase reaction was performed using GST-c-Jun (1–89) in the presence of cold 100 µM ATP. Samples were separated by electrophoresis in 15% SDS-PAGE, then blotted onto a nitrocellulose membrane. Immunodetection was performed by incubation of the membrane with a phospho-specific c-Jun antibody. Visualization was achieved by chemiluminescence reaction and autoradiography.

Immunoblotting
To detect the level of JNK protein, the cell lysates were separated by SDS-PAGE (15%), then blotted onto an immobilon membrane (Millipore Corp.). The latter was incubated for 30 min with an antibody against JNK (1:1000 dilution). The primary antibody was detected by horseradish peroxidase-conjugated second antibody (1:2000 dilution), which in turn was visualized using the enhanced chemiluminescence system.

Transient transfection and luciferase assay
A total of 1.5 x 106 CHO-TSHR cells were plated per well in 6-well plates the day before transfection. Cells were transfected with 1 mg pFR-Luc, 50 ng pFA-cJun and 10ng pPL-TK plasmids per well using lipofectin method. The pFR-Luc vector contains a synthetic promoter with five tandem repeats of the yeast GAL4 binding sites and luciferase gene. The pFA-cJun vector expressing the fusion trans-activator protein consists of the activation domain of the c-Jun fused with the DNA binding domain of the yeast GAL4. The pPL-TK plasmid contains the herpes simplex virus thymidine kinase promoter to provide moderate levels of Renilla luciferase expression. Cells were treated with the indicated dose of TSH at 24 h after transfection and incubated with serum-free medium. Then, cells were harvested 6 h after treatment and lysed in 250 µl passive lysis buffer (Promega Corp.). Ten microliters of cleared lysate was used for measuring the luciferase activity based on the manufacturer’s protocol (Dual-Luciferase Reporter Assay System; Promega Corp.). Firefly luciferase activity was normalized relative to Renilla luciferase activity.

Cellular cAMP measurements
For cAMP measurements, the cells, seeded in 6-cm diameter culture dish (5 x 105) and cultured overnight, were incubated for 30 min at 37 in serum-free medium with 0.5 mmol/liter 3-isobutyl-1-methylxanthine and the indicated concentrations of bTSH. The incubation was stopped by removal of medium, followed immediately by the addition of 0.1 N HCl to extract the cellular cAMP. Concentrations of cellular cAMP were determined with a commercial RIA kit (Yamasa, Tokyo, Japan). The experiment involved duplicate wells and was repeated twice.

Statistical analysis
Data in the luciferase assay were analyzed by one-way ANOVA, followed by Fisher’s least-significant-difference (LSD) tests. All analysis were performed by Statview 4.5, fitted for the Macintosh system. The level of significance were set at P < 0.05. Data are expressed as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To elucidate a possible effect of TSH on JNK activity in human thyroid cells, we first performed a dose-response study of TSH-induced JNK kinase activation using primary human thyroid cells (Fig. 1AGo). Phosphorylated Jun protein was detected (using a specific anti-phospho Jun protein antibody) after 30 min of TSH stimulation. Peak JNK activity (approximately 2.3-fold of control) occurred in the presence of 10-11 M TSH. However, a fall in JNK activity was observed at higher concentrations of TSH (10-8 to 10-7 M). Similar dose-dependent effect of JNK activation was observed in the experiment using recombinant human TSH. To determine the effect of JNK protein level after TSH stimulation, Western blot analysis was performed using the same samples with anti-JNK1 antibody. No significant change in JNK1 protein levels was observed. In terms of cellular cAMP response, TSH stimulated cAMP production in a dose-dependent manner (Fig. 1BGo). An inverse correlation was observed between JNK activation and cellular cAMP production induced by TSH. The highest activity of JNK in thyroid cells occurred 30 min after the addition of 10-11 U/liter TSH but the activity decreased thereafter and stabilized at 120 min to the basal level without any change in JNK1 protein levels (Fig. 2Go).



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  		Figure 1. Dose-response effect of TSH on JNK activity and cellular cAMP production in cultured human thyroid cells. Cells were stimulated with TSH for 30 min at the indicated concentrations. Cell lysates were used for the in vitro kinase assay with GST-c-Jun as the substrate. A, A representative immunoblot obtained with anti-phospho-c-Jun antibody is shown (upper panel). JNK1 protein levels were determined by immunoblotting with anti-JNK1 antibody (middle panel). Results of densitometric analysis using NIH Image 1.58 were shown (lower panel). Data were expressed as -fold increase relative to representing the unstimulated levels. Each column with bar shows the mean ± SE of three separate experiments. *, P < 0.05 vs. Basal. B, cAMP response to bTSH stimulation in cultured human thyroid cells. Data are representative of two separate experiments; each column represents the mean of duplicate values in duplicate dishes of cells.

 


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  		Figure 2. Kinetics of TSH-induced activation of JNK in cultured human thyroid cells. Cells were stimulated with TSH at 10-11 M for the indicated time intervals. Cell lysates were used for the in vitro kinase assay with GST-c-Jun as the substrate. A representative immunoblot obtained with anti-phospho-c-Jun antibody is shown (upper panel). JNK1 protein levels were determined by immunoblotting with anti-JNK1 antibody (middle panel). Results of densitometric analysis using NIH Image 1.58 were shown (lower panel). Data were expressed as -fold increase relative to representing the unstimulated levels. Each point with bar shows the mean ± SE of three separate experiments. *, P < 0.05 vs. Basal.

 
To establish that TSH-induced activation of JNK is mediated through ligand-receptor specific binding, we next used CHO and 293 cells to examine JNK activity after TSH stimulation. Both types of cells were stably transfected with human TSH receptor expression vector, designated CHO/TSHR cells and 293/TSHR cells, respectively. As shown in Fig. 3AGo, in CHO/TSHR cells, the highest activity of JNK was noted in the presence of 10-11 M TSH, but the activity became almost similar to basal levels at 10-9 M. Moreover, the presence of 10-8 M TSH resulted in a depression of JNK activity to levels as low as 50% of the control. To further assess JNK activity by TSH in CHO-TSHR cells, cells were transiently transfected both with pFA-cJun plasmid, which expresses the fusion trans-activator protein consisting of the activation domain of the c-Jun fused with the DNA binding domain of the yeast GAL4, and pFR-Luc plasmid, which contains a synthetic promoter with five tandem repeats of the yeast GAL4 binding sites and luciferase gene. After 6 h of treatment with various doses of TSH, the luciferase activity was significantly elevated at concentrations of 10-11 M TSH relative to control (P < 0.05) and attenuated with higher dose of TSH (Fig. 3BGo). TSH did not change JNK activity in nontransfected CHO cells (data not shown). On the other hand, in 293/TSHR cells, we did not observe any significant activation of JNK in the presence of TSH (data not shown).



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  		Figure 3. Dose-response effect of TSH on JNK activation in CHO cells transfected with TSH receptor cDNA. A, Cells were stimulated with TSH for 30 min at the indicated concentration while cell lysates were used for the in vitro kinase assay with GST-c-Jun as the substrate. A representative immunoblot obtained with anti-phospho-c-Jun antibody is shown (upper panel). JNK1 protein levels were determined by immunoblotting with anti-JNK1 antibody (middle panel). The c-Jun N-terminal phosphorylation activities observed in three separate experiments were quantified by densitometry (lower panel). Data are expressed as -fold increase relative to representing the unstimulated levels. Each column with bar shows the mean ± SE. B, Cells were transfected with pFR-Luc, pFA-cJun and pPL-TK plasmids and treated with indicated dose of TSH. Cell lysates harvested 24 h after treatment were used for measuring the luciferase activity. Data are expressed as -fold activation over control values (mean ± SD n = 6). *, P < 0.05 vs. Basal.

 
Several G protein-coupled receptors are known to stimulate JNK pathway through different G protein subunits. To investigate which type of G protein is involved JNK activation by TSH, thyroid cells were pretreated by 0.2 µg/ml PTX for 3.5 h, which completely ADP-ribosylates the cellular complement of PTX-sensitive Gi/o protein. As shown in Fig. 4Go, PTX inhibited the stimulation by TSH of JNK.



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  		Figure 4. Effects of PTX on TSH-induced JNK activation in cultured human thyroid cells. Cells were pretreated with PTX (0.2 µg/ml, 3.5 h) and further incubated with or without TSH (10-11 M) for 30 min. JNK activity was determined in the cell extracts. Representative immunoblots with anti-phospho-c-Jun antibody (upper panel) is shown. The c-Jun N-terminal phosphorylation activities of three separate experiments were quantified (lower panel). Data were expressed as -fold increase relative to representing the unstimulated levels. Each column with bar shows the mean ± SE *, P < 0.05 vs. Basal.

 
To further analyze the postG protein signal transduction pathway of TSH-regulated activation of JNK, we examined the involvement of cAMP pathway in this process. As shown in Fig. 5AGo, 0.1 mM 8-bromo-cAMP, a cAMP analog, attenuated TSH-induced JNK activation while a higher dose of 8-bromo-cAMP (1 mM) suppressed the basal and TSH-induced JNK activity. However, a lower dose of 8-bromo-cAMP (0.001 and 0.01 mM) failed to change JNK activity (data not shown). In addition, treatment with 50 µM forskolin, a diterpene that directly stimulates adenylate cyclase, suppressed the basal JNK activity and completely abolished TSH-induced JNK activation (Fig. 5BGo). In the next step, we investigated the role of PI3-kinase or Ca2+ pathway in TSH-induced JNK activation. As shown in Fig. 6BGo, wortmannin, a fungal metabolite that specifically inhibits PI3-K, did not attenuate TSH-induced JNK activation, suggesting that JNK activation is not likely to be mediated by PI3-K pathway. We also observed activation of JNK in cultured thyroid cells by 1 µM thapsigargin, a compound that raises intracellular Ca2+ (Fig. 6AGo). TSH additively enhanced thapsigargin-induced JNK activity. In addition, EGTA, an extracellular Ca2+ chelator and BAPTA-AM, an intracellular Ca2+ chelator, failed to attenuate TSH-induced JNK activity, suggesting that activation of JNK by TSH is mainly due to Ca2+-independent pathway (Fig. 6Go).



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  		Figure 5. Effects of 8-bromo-cAMP and forskolin on basal and TSH-induced JNK activities in cultured human thyroid cells. Cells were pretreated with 8-bromo-cAMP for 30 min at the indicated concentration (0.1 and 1.0 mM) (A) or with forskolin (50 µM) for 15 min (B) and further incubated with or without TSH (10-11 M) for 30 min. The activity of JNK in the cell extracts was determined by in vitro kinase assay. Representative immunoblots with anti-phospho-c-Jun antibody and anti-JNK1 antibody are shown in the upper and middle panels, respectively. The c-Jun N-terminal phosphorylation activities in three separate experiments were quantified by densitometry (lower panel). Data were expressed as -fold increase over control values. Each column with bar shows the mean ± SE *, P < 0.05 vs. Basal.

 


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  		Figure 6. Effects of thapsigargin, wortmannin and Ca2+ removal on TSH-induced JNK activation in cultured human thyroid cells. A, Cells were treated with TSH (10-11 M) and/or thapsigargin (1 µM) for 30 min. JNK activity was determined in each cell extract. Representative immunoblots with anti-phospho-c-Jun antibody (upper panel) or anti-JNK1 antibody (middle panel) are shown. The c-Jun N-terminal phosphorylation activities of three separate experiments were quantified (lower panel). Data are representative of two separate experiments; each column represents the mean of duplicate values in duplicate dishes of cells. B, Cells were pretreated with wortmannin (50 nM, 30 min), EGTA (1 mM, 30 min), or BAPTA-AM (50 mM, 30 min) and further stimulated with TSH (10-11 M) for 30 min. Data are expressed as -fold increase over control values. Each column with bar shows the mean ± SE *, P < 0.05 vs. Basal.

 
TSH stimulates the PLC pathway through a Gq/11 protein coupled to the TSH receptor (4), hence we explored in the next series of experiments whether the activation of JNK by TSH was mediated by PKC. As shown in Fig. 7Go, the effects of TPA mimicked those of TSH in activating JNK in primary cultured human thyroid cells. Combination with TSH and TPA does not change TSH or TPA-induced JNK activity statistically. Moreover, H7, a PKC inhibitor, and pretreatment with TPA, which leads to depletion of endogenous PKC activity, clearly attenuated TSH-induced JNK activation, suggesting that activation of JNK by TSH involves a PKC-dependent pathway in human thyroid cells.



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  		Figure 7. Effects of TPA and H7 on TSH-induced JNK activation in cultured human thyroid cells. Cells were pretreated with TPA (100 nM, 15 min), H7 (50 µM, 15 min) or TPA (5 µM, 18 h) and further incubated with or without TSH (10-11 M) for 30 min. JNK activity was determined in the cell extracts. Representative immunoblots with anti-phospho-c-Jun antibody (upper panel) or anti-JNK1 antibody (middle panel) are shown. Lower panel, The c-Jun N-terminal phosphorylation activities were quantified. Data are expressed as -fold increase over control values. Each column with bar shows the mean ± SE of three separate experiments. *, P < 0.05 vs. Basal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results demonstrating a stimulatory effect of low and physiological TSH concentrations on JNK activity imply a significant involvement of the JNK pathway in human thyroid cells. This activation of the JNK by TSH is also observed in CHO cells stably transfected with TSH receptor cDNA but not in control CHO cells, suggesting that it is a ligand-receptor specific interaction. Considered together, these results provide the first evidence for TSH regulation of JNK activity via TSH receptor in cells containing appropriate intracellular signaling components. Bi-directional regulation of JNK activity by TSH, low TSH concentration enhanced but higher dose of TSH suppressed JNK activity, was observed. Since TSH elicited the production of intracellular cAMP in a dose-dependent manner and treatments by forskolin or 8-bromo-cAMP suppressed JNK activity, the inhibitory effect of higher concentrations of TSH on JNK activity might be mediated through the cAMP pathway.

Concerning with the stimulating effect on JNK activity, several G protein-coupled receptors are known to activate the JNK pathway through different G protein subunits. It has been demonstrated that JNK is activated by stimulation of Gq/11-coupled m1 and Gi-coupled m2 muscarinic acetylcholine receptors (23, 24). An activated human TSH receptor in thyroid membranes can couple to at least 10 different G proteins-i.e. Gs short, Gs long, Gq, G11, Gi1, Gi2, Gi3, Go, G12, and G13 (3). PTX affected the stimulation of JNK by TSH, suggesting Gi/o proteins were implicated in this TSH effect. Coupling of the activated TSH receptor to subtypes of the PTX-sensitive G protein Gi/o may be functionally relevant by the release of ß{gamma}-subunits because JNK activates through heterotrimeric G proteins ß{gamma}-subunits (11, 25). In mammalian cells, Gß{gamma} has been shown to modulate the activities of adenylyl cyclases (26), phospholipase Cß (PLCß) isozymes (27, 28), PI3-K {gamma} (29), inward rectifier potassium channels (30, 31), and N-type and P/Q-type calcium channels (32, 33). To clarify the stimulatory pathway of JNK after coupling G protein with TSH receptor, the involvement of PI3-K, Ca2+ and PKC pathways were examined. Previous studies have demonstrated PI3-K-dependent activation of JNK pathway by PDGF in COS-7 cells transfected with PDGF receptor expression vector (17), Ca2+-dependent JNK activation by angiotensin II in rat liver epithelial cells (11) or PKC-independent activation of JNK by ligands, such as carbachol and angiotensin II, which bind to G coupled protein receptor (10, 23). In contrast with these studies, our findings indicate that the activation of JNK by TSH is not due to PI3-K or an intracellular Ca2+-independent pathway but due to a PKC-dependent pathway, suggesting the involvement of Ca2+-independnt PKC subgroup (novel PKC-i.e., PKC {delta}, {epsilon}, {eta}, {theta}, or µ) on JNK activation by TSH stimulation.

Taken together, two distinct signal pathways, Gs-cAMP and Gi/o-PKC, might be concomitantly stimulated by different doses of TSH via its receptor and conversely interacted with each other to an identical target molecule, JNK. Consistent with these results, Laglia et al. (7) demonstrated that a persistent activation of Gs{alpha} inhibits the activity of PLC in thyroid cells. On the other hand, Heinrich et al. (20) observed that TSH, forskolin and 8-bromo-cAMP inhibited c-jun and c-fos mRNA elicited by TPA or EGF, which act via PKC pathway.

Although the functional significance of TSH-induced activation of JNK in human thyroid cells remains to be elucidated, recent studies have implicated such activation in growth inhibition or apoptosis-triggering mechanisms in the presence of external stress or inflammatory cytokines, e.g. IL-1 and TNF-{alpha}. Induction of MEKK1, an upstream-signal molecule of JNK, results in growth arrest or apoptosis of fibroblasts (34, 35). In contrast to the inhibitory effect on cell growth, activation of JNK by agonists that bind to G protein-coupled receptor or tyrosine kinase receptor correlates with a proliferative response in the cells (10, 36). Microinjection of small G protein Rho, Rac, or Cdc42 into quiescent fibroblasts stimulated cell cycle progression that correlated well with JNK activation (37). It seems that activation of JNK pathway has, in general, at least two diverse functions; inhibition of cell growth (or apoptosis) and cell proliferation. TSH exerts proliferative and antiapoptotic activity in thyroid cells (38). Furthermore, Selzer et al. have demonstrated that the expression of Gi{alpha}-1 is under tight control of TSH in human thyroid cells in vivo and Gi{alpha}-1 is capable of transmitting mitogenic stimuli in human thyroid epithelial cells (39). Our recent observation also supports no relationship of JNK activation induced by ionizing radiation with thyroid cell apoptosis (40, 41). Although JNK phosphorylates c-Jun and sequentially activates AP-1, an increase in AP-1 activity has been shown to be associated with in vitro and in vivo thyroid cell transformation (42). Therefore, activation of JNK by TSH might serve to promote the mitogenic response rather than apoptosis.

In conclusion, we demonstrated in human thyroid cells that low concentrations of TSH stimulated JNK activity through PTX G protein Gi/o and PKC pathway but a higher concentration suppressed such activity through cAMP pathway. These results indicate the evidence of a cross-talk between Gi-PKC and cAMP-PKA pathways through two distinct G proteins at the postreceptor signal transduction system in human thyroid cells.


   Footnotes
 
1 This work was supported by a Grant-in-Aid for General Scientific Research from the Ministry of Education, Culture, and Science of Japan (to H.N., No. 09680529, and S.Y., No. 09558072) and a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, and Science of Japan (to S.Y., No. 09253242). Back

Received June 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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