World J. Surg. 22, 544 –551, 1998
WORLD Journal of
SURGERY © 1998 by the Socie´te´ Internationale de Chirurgie
Heterologous Desensitization in Neoplastic Thyroid Cells: Influence of the Phospholipase C Signal Transduction System on the Thyrotropin–adenylate Cyclase Signal Transduction System S. Tezelman, M.D.,1 T. Hoelting, M.D.,2 G.H. Jossart, M.D.,3 M.G. Wong, B.S.,3 A.E. Siperstein, M.D.,3 Q.-Y. Duh, M.D.,4 O.H. Clark, M.D.3 1
Department of Surgery, Istanbul Faculty of Medicine, Capa, Istanbul 34390, Turkey Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, D-69120, Heidelberg, Germany Department of Surgery, UCSF/Mount Zion Medical Center, 1600 Divisadero Street, San Francisco, California 94115, USA 4 Surgical Service of Veterans Affairs Medical Center and Department of Surgery, University of California San Francisco, San Francisco, California 94121, USA 2 3
Abstract. Desensitization is defined as a decreased functional response after continuous or repetitive stimulation of a receptor with its agonist. Thyrotropin (TSH) increases cAMP levels in normal and neoplastic thyroid tissue. The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) activates protein kinase C (PKC). The aim was to determine whether TPA induces heterologous desensitization of the TSH–adenylate cyclase (AC) signal transduction system. Three human thyroid neoplasms in culture for 6 months or longer (one papillary carcinoma, one Hurthle cell carcinoma, one follicular adenoma) were incubated with TSH (10 mU/ml) and TPA (1.6 3 1028 M) separately and together for various time periods (from 10 minutes to 24 hours). The mixture was subsequently incubated for 30 minutes with TSH. TPA alone had no effect on cAMP levels, but co-incubation of TPA and TSH caused a significant reduction in cAMP response when compared to the cAMP response that resulted after stimulation with only TSH (p < 0.001). cAMP levels in response to TSH decreased by 31%, 44%, and 57% after preincubation with TSH for 10 minutes, 4 hours, and 24 hours, respectively. cAMP levels in response to TSH decreased by 44%, 33%, and 29% after preincubation with TPA for 10 minutes, 4 hours, and 24 hours, respectively (p < 0.01; ANOVA). Co-incubation of cells with TPA and staurosporine (10 ng/ml), a PKC inhibitor, prevented the effect of TPA on desensitization at 10 minutes and blunted the effect at 4 hours. This is the first demonstration in human neoplastic thyroid cells that TPA induced heterologous desensitization of the cAMP response to TSH. This TPA-induced effect appears to involve PKC activation, as it can be blocked by staurosporine.
Hormones such as thyroid-stimulating hormone (TSH) and tumor promoters such as 12-0-tetra decanoyl-phorbol-13-acetate (TPA) influence thyroid cellular proliferation, differentiation, and protein phosphorylation. TSH is a major regulator of thyroid differentiated function and growth [1, 2]. TSH acts, through its receptor, on both the adenylate cyclase (AC)– cyclic adenosine monophosphate (cAMP) and phospholipase C (PLC)–phosphatiThis International Association of Endocrine Surgeons (IAES) article was presented at the 37th World Congress of Surgery International Surgical Week (ISW97), Acapulco, Mexico, August 24 –30, 1997. Correspondence to: O.H. Clark, M.D.
dylinositol–protein kinase C (PKC) systems [2– 4]. Numerous investigators have documented that continuous agonist stimulation of b-adrenergic receptors usually leads to a decrease in receptor-mediated adenylate cyclase activity. This reduced response to the same stimuli is called desensitization. Desensitization can be homologous (as a consequence of hormone binding to its specific receptor) or heterologous (stimulation of another receptor or of a postreceptor signal transduction site). Prior intermittent or continuous exposure of thyroid tissue to TSH in vivo or in vitro, results in a decreased adenylate cyclase response to subsequent TSH stimulation [5–10]. We have reported that both homologous and heterologous desensitization of TSH–AC– cAMP occur in Chinese hamster ovary cells transfected with human TSH receptor [11] and in well-differentiated thyroid cancer cells [12]. An increase in intracellular cAMP decreased the adenylate cyclase response to TSH [11, 12]. The tumor promoter TPA is a potent promoter of thyroid proliferation and increases PKC activity [13–15]. Roger et al. [16] documented that TPA induced DNA synthesis and proliferation of dog thyroid cells. In dog thyroid cells TPA enhanced the accumulation of c-myc mRNA after 3 and 6 hours [17]. TSH and TPA had no additive effect on the proliferation of dog thyroid cells [16, 18]. TPA, however, reduced the cAMP response to TSH when both agonists were incubated in dog and pig thyroid cells [19 –21]. TSH decreased cAMP-dependent PKA activity in dog thyroid cells [22], but the simultaneous presence of TPA and TSH, in contrast, inhibited TSH-induced down-regulation of PKA I [19]. The purpose of this investigation was to determine if there is “cross-talk” between the TSH–AC and PLC–PKC mitogenic signaling systems and if TPA induces heterologous desensitization of the TSH–AC signal transduction system in human thyroid neoplastic cells.
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Materials and Methods Thyroid Cell Culture
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production by stimulated cells 2 cAMP produced by desensitized cells)/cAMP production by stimulated cells] % Maximal adenylate cyclase 5 100 % 3 [cAMP production by stimulated cells)/cAMP production by unstimulated cells (basal condition)].
Three thyroid neoplasms (one papillary carcinoma, one Hurthle cell carcinoma, and one follicular adenoma) were maintained in tissue culture for more than 6 months. Thyroid tissues were obtained at surgery as approved by the UCSF Human Experimentation Committee. A sample of tumor tissue measuring 0.5 to 1.0 cm3 was mechanically dissociated and then washed three times with phosphate-buffered saline (PBS) pH 7.4. The cells were incubated in 0.5 ml of a 50:50 DME 21 and F-12 medium including 10% fetal calf serum, 0.1 % collagenase, TSH (10 mU/ml), insulin (10 mg/ml), Fungizone (2.5 mg/ml), streptomycin (100 mg/ml), and penicillin (100 U/ml). After 4 hours the single cell suspension was aspirated and placed in a 15-ml conical tube and centrifuged for 10 minutes at 2000 rpm. The supernatant was discarded, and the pellet was resuspended in tissue culture medium as described above. The cells were then grown in 75 cm2 Falcon tissue culture flasks at 37°C in a humidified atmosphere of 95% air/5% CO2. The medium was changed at intervals of 2 to 3 days until the cells reached 95% confluence. The cells were detached with 0.05% trypsin and counted using a hemocytometer and trypan blue dye exclusion (1:1 dilution). Cells were then transferred at a density of 3 3 104 cells per well onto 24-well plates (Costar, Cambridge, MA, USA). The experiments were performed in the 24-well plates when cells reached 95% confluence.
Cell proliferation was measured by dimethylthiazoldiphenyltetrazolium bromide (MTT) assay and by fluorometric DNA measurement. Cells were seeded at a density of 5 3 103/well in 96-well plates. After 24 hours the plating medium was replaced by medium including different concentrations of TSH (10 mU/ml) and TPA (1.6 3 1028 M), and TSH-free medium or medium including only 10% FCS. MTT in PBS was used in a concentration of 1 mg/ml. After removing the medium, 100 ml of MTT/PBS solution was added to the wells and incubated for 3 hours. A second incubation was then done with 150 ml of solubilizing solution for 30 minutes that included 0.04 N HCl/isopropanol and 3% lauryl sulfate. After incubation, the solution in each well was mixed 10 times and read in a Vmax kinetic microplate reader at 570/630 nm wavelength [25]. The pellet containing DNA was diluted with 500 ml of 0.1% SDS. The 10 ml stock Hoechst 33258 dye solution was diluted with 100 ml of 1 3 TNE buffer (100 mM Tris, 10 mM EDTA, 1.0 M NaCl, pH 7.4). The DNA-containing solution (2 ml) was added to 2 ml of dye solution and read with a TKO 100 dedicated minifluorometer [26].
Effect of TSH and TPA on Adenylate Cyclase
Statistical Analysis
Neoplastic thyroid cells were incubated with TSH (10 mU/ml) and TPA (1.6 3 1028 M) separately and together for various time periods (10 and 30 minutes and 1, 4, and 24 hours) with 1 mM 3-isobutyl-1-methylxanthine (IBMX). IBMX was used to inactivate phosphodiesterase. Staurosporine (10 ng/ml), a specific PKC inhibitor, was used to block the effect of TPA on PKC activity and to document the effect of the PKC signal transduction system on the TSH–AC signal transduction system.
Statistical calculations were performed by the Student’s unpaired t-test, Wilcoxon’s rank test, and ANOVA one-factor analysis; p , 0.05 was considered significant. The data are displayed as the mean 6 SD. Experiments were done at least three times in duplicate unless otherwise indicated in text.
Desensitization Neoplastic thyroid cells in 24-well Costar plates were rinsed 3 times with PBS pH 7.4 to remove TSH. They were then incubated for 24 hours with regular medium without TSH or fetal calf serum (FCS). On the following day, the first incubation (10 minutes, 4 hours, and 24 hours) was performed with and without TSH (10 mU/ml) or TPA (1.6 3 1028 M). The cells were again rinsed 3 times with PBS and were incubated (30 minutes) with or without TSH (10 mU/ml) in medium including IBMX (1 mM), maintained at 37°C and 95% O2/5% CO2. Staurosporine was added in some experiments to determine the effect of TPA on the cAMP response. Intracellular cAMP Measurement (Radioimmunoassay) Intracellular cAMP was measured as described previously [11, 12] using slight modifications of the method reported by Brooker et al. [23]. Desensitization was calculated as the percent decrease of the cAMP response [24]. % Desensitization 5 100% 3 [(cAMP
Cell Growth Assays
Materials Materials are obtained from the following sources: culture media and additives from University of California San Francisco Cell Culture Facility; bovine thyrotropin (TSH), MTT reagent, and TPA from Sigma Chemical Co. (St. Louis, MO, USA); cAMP Kit from Amersham (Arlington Heights, IL, USA); penicillin-streptomycin and trypan blue from GIBCO Laboratories (Grand Island, NY, USA); Hoechst dye 33258 from Polysciences, (Niles, IL, USA); and staurosporine from Boehringer Mannheim Biochemica (Mannheim, Germany). Results Effect of TPA and Staurosporine on Growth of Neoplastic Thyroid Cells Addition of TPA increased the proliferation of all three human thyroid neoplastic cells after 24 hours of incubation when compared to the growth observed in the same medium containing bovine insulin (10 mg/ml), human transferrin (5 mg/ml), and glycyl-L-histidyl-L-lysine acetate (2 ng/ml) without TSH (p , 0.02). Staurosporine (10 ng/ml) inhibited the growth of these cells. The
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Heterologous Desensitization and Prevention of Desensitization
Fig. 1. Time–response studies of the effect of plain medium (5H), TPA, and staurosporine (S) on the growth of three neoplastic thyroid cells in culture. Formazan production (MTT) was measured as optical density (OD). Results are expressed as percentage of OD.
TPA stimulation of cell growth and the inhibition of cell growth by staurosporine were greater when these cells were incubated with these agents for 10 days (p , 0.02) (Fig. 1).
Effect of TSH and TPA on Intracellular cAMP Levels The TSH increased cAMP levels threefold (290%) above basal levels in all three neoplastic thyroid cells studied (two carcinomas and one follicular adenoma) (Table 1). TPA had a biphasic temporal effect on the cAMP response to TSH. When neoplastic thyroid cells were incubated with both TSH and TPA for 10, 30, and 60 minutes, the effect of these agents were additive (Fig. 2). However, the cAMP response decreased significantly (p , 0.01) when cells were exposed to TSH and TPA together for longer periods (4 and 24 hours) (Fig. 2). The inhibitory effect of TPA was abolished when neoplastic thyroid cells were incubated with TSH, TPA, and staurosporine for 4 hours (Fig. 3). In fact, co-incubation of TSH, TPA, and staurosporine increased cAMP levels more than TSH and TPA alone. Because staurosporine did not affect the cAMP response of these cells at any time when they were incubated with staurosporine alone or with TSH (data not shown), it appears that activation of PKC by TSH is responsible for the decreased cAMP in neoplastic thyroid cells.
Preincubation of neoplastic thyroid cells for 10 minutes with either TSH or TPA (Fig. 4), 4 hours (Fig. 5), or 24 hours (Fig. 6) caused desensitization of the cAMP response to subsequent stimulation by TSH. The cAMP response to TSH (homologous desensitization) decreased by 31% at 10 minutes, by 44% at 4 hours, and by 57% at 24 hours (Table 1). TPA preincubation also induced a lower cAMP response to TSH (heterologous desensitization) with a decrease of 44% at 10 minutes, 33% at 4 hours, and 29% at 24 hours (p , 0.01) (Table 1). Thus the magnitude of desensitization increased with time in response to TSH but decreased with time in response to TPA (Table 1). This finding suggests that the activation of PKC is initially more effective than the activation of PKA for desensitization of adenylate cyclase at 10 minutes, whereas the activation of PKA is more effective at 24 hours. Thus heterologous desensitization appears to occur more rapidly than homologous desensitization in neoplastic thyroid cells. Homologous desensitization, however, is more effective than heterologous desensitization when neoplastic thyroid cells are exposed to TSH for periods of 4 hours or more. Overall maximal desensitization to homologous stimulation (57%) was slightly higher than heterologous desensitization (44%), but these results are not significantly different (p . 0.05). The TPA effect on cAMP levels (heterologous desensitization) was abolished by co-incubation with stauroporine at 10 minutes and was blunted at 4 hours. When these cells were incubated with TSH and staurosporine for 10 minutes, the inhibition of PKC activity appeared to prevent homologous desensitization and decreased cAMP levels (Fig. 4). Interestingly, staurosporine did not prevent homologous desensitization caused by TSH when neoplastic cells were co-incubated with TSH and staurosporine for 4 hours (Fig. 5). Staurosporine inhibited PKA activation through PKC activity. These investigations document that there is communication, or “cross-talk,” between TSH–AC–PKA and TSH–PLC–PKC systems during the induction of homologous or heterologous desensitization of the cAMP response to TSH. Discussion Our studies document that TSH causes homologous desensitization of the TSH–AC signal transduction system in both neoplastic human thyroid cells in long-term culture. The phorbol ester TPA had no direct effect on cAMP levels, but it did cause desensitization of AC in response to subsequent stimulation by TSH. Overall TPA had a biphasic effect on the cAMP response to TSH in neoplastic thyroid cells. When cells were incubated with both TPA and TSH for short periods (e.g., 10 minutes, 30 minutes, or 1 hour), TPA potentiated the TSH effect. When cells were incubated with both TPA and TSH for longer periods (e.g., 4 hours, or longer), TPA inhibited the subsequent cAMP response to TSH. Treatment of thyroid neoplastic cells with TPA alone induced heterologous desensitization of the TSH–AC– cAMP signal transduction system. TSH also causes homologous desensitization of the TSH–AC signal transduction system in neoplastic thyroid cells. The activation of PKC appears to be responsible for the heterologous desensitization because staurosporine, a potent PKC inhibitor, abolished (10 minutes) or inhibited (4 hours) the effect of TPA on desensitization. In addition, when neoplastic thyroid cells are co-incubated with TPA and TSH for 4 hours or
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Table 1. Maximal stimulation and desensitization of cAMP levels in response to TSH and TPA. 10 Minutes a
4 Hours
24 Hours
Parameter
TSH
TPA
TSH
TPA
TSH
TPA
Desensitization (%) cAMP (pmol) p
31.0 6 0.6 0.48 6 0.02 , 0.01
44.0 6 1.3 0.39 6 0.03 , 0.01
44.0 6 1.8 0.41 6 0.04 , 0.01
33.0 6 1.7 0.49 6 0.05 , 0.01
57.0 6 1.7 0.32 6 0.03 , 0.001
29.0 6 0.6 0.52 6 0.02 , 0.02
Results of pretreatment of neoplastic thyroid cells in culture by TSH or TPA. The agonists were used in the following concentrations: TSH 10 mU; TPA 1.6 3 1028. a Desensitization was calculated as the percent decrease in cAMP levels (described in the text). Basal cAMP was 0.25 6 0.04 (pmol/well). Maximal cAMP in response to TSH for 30 minutes was 0.74 6 0.05 (pmol/well) and 290%.
Fig. 2. Time curve for TPA effect on thyrotropin (TSH)-stimulated cAMP. When cells reached 95% confluence in the 24-well plates, the experiments were performed. Neoplastic cells were then washed with PBS (pH 7.4) and exposed to control medium (CON: without TSH, insulin, or fetal calf serum) and medium with TSH (10 mU/ml) or TPA (1.6 3 1028M) separately or TPA and TSH together for various time periods (10 minutes to 24 hours). TPA decreased the cAMP response to TSH when they were incubated together for 4 or 24 hours. (Each point is the mean 6 SD of six experiments.)
longer, TPA decreased the cAMP response compared to that when cells are stimulated with TSH alone. This effect of TPA was also abolished by co-incubation with staurosporine. Our results document that there is a biphasic effect apparently due to cross-talk between two signal transduction systems, the TSH–AC– PKA and TSH–PLC–PKC systems, in human neoplastic thyroid cells (Fig. 7). We have reported that TPA stimulated invasion and growth of follicular thyroid cells in vitro [27] and that TSH stimulated both the AC–PKA and PLC–PKC signal transduction systems. In our recent studies TSH appears to stimulate invasion and growth via activation of PKC rather than PKA [27]. A similar finding has
Fig. 3. Blocking of cross-talk between TPA- and TSH-stimulated cAMP. Thyroid neoplastic cells were incubated with control medium (CON: without TSH, FCS, or insulin); TSH (10 mU/ml) or TPA (1.6 3 1028 M) separately; or TSH and TPA together for 4 hours. The decreased cAMP response to TSH by TPA was abolished by co-incubation with staurosporine at 4 hours.
been described in animal thyroid cells [19 –21]. Omri et al. [20] documented that TPA had a biphasic effect on TSH-induced stimulation of cAMP production in pig thyrocytes. TPA potentiated the effect of TSH when pig thyroid cells were exposed simultaneously to TSH and TPA for 10 minutes, but after 20 minutes TPA inhibited the cAMP response to TSH [20]. TPA blocked the TSH effect and prevented cAMP-dependent PKA activity [20]. Our studies also document that TPA blocks the cAMP response to TSH at 4 hours in human neoplastic thyroid cells (Fig. 5). The precise mechanism of how TPA, an activator of PKC, decreases adenylate cyclase is unknown. This inhibition, however,
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Fig. 4. Homologous and heterologous desensitization of cAMP at 10 minutes. Neoplastic thyroid cells in 24-well Costar plates were rinsed 3 times with PBS (pH 7.4) to remove TSH. They were then incubated for 24 hours with regular medium without TSH or 10% FCS. On the following day, the first incubation (10 minutes) was performed with and without TSH (10 mU/ml) and TPA (1.6 3 1028 nM). After this first incubation the medium was removed, and the cells were rinsed 3 times with PBS. They were again incubated (second incubation) for 30 minutes with and without TSH (10 mU/ml) in media including IBMX (1 mM). TSH caused homologous desensitization, whereas TPA induced heterologous desensitization of the TSH receptor–AC– cAMP signal transduction system when cells were pretreated with TPA for 10 minutes. The desensitization was abolished by staurosporine. (Each point is the mean 6 SD of six experiments.)
seems to be specific, as staurosporine prevents the decreased cAMP response when cells are incubated with TPA, TSH, and staurosporine for 4 hours (Fig. 3). Staurosporine is a microbial alkaloid and a potent PKC inhibitor. It acts at the ATP binding site on protein kinase [28]. Staurosporine inhibits PKC and other protein kinases [29]. In porcine thyroid cells, the other PKC inhibitors including H7 [1-(5-isoquinolinesulfonyl-2-O-methylglycerol], and AMG-C16 (1-O-hexadecyl-2-O-methylglycerol), as well as staurosporine, enhanced TSH-stimulated iodide organification [30]. In the presence of staurosporine, TPA did not inhibit TSH-stimulated iodide organification [30]. A synergistic action on AC activity of TPA and forskolin (a diterpene that directly stimulates adenylate cyclase) on pancreatic islet cells has been also documented [31]. Our current investigation documents that staurosporine prevents heterologous desensitization induced by TPA in neoplastic thyroid cells at 10 minutes and blunts this effect at 4 hours. Omri
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Fig. 5. Homologous and heterologous desensitization of cAMP at 4 hours. The first incubation (4 hours) was performed with and without TSH (10 mU/ml) and TPA (1.6 3 1028 M). After this first incubation the medium was removed, and neoplastic cells were rinsed 3 times with PBS. They were subsequently stimulated for 30 minutes with and without TSH (10 mU/ml). TSH caused homologous desensitization, whereas TPA induced heterologous desensitization of the cAMP response. The desensitization was blunted by staurosporine. (Each point is the mean 6 SD of six experiments.)
et al. [20] suggested that TPA prevented TSH down-regulation of cAMP-dependent protein kinase activity. In vivo and in vitro studies suggest that receptor phosphorylation by at least two kinases is involved in desensitization and occurs rapidly in intact cells via phosphorylation of a cAMP-dependent protein kinase (PKA) and via phosphorylation of b-adrenergic receptor kinase (bAR kinase) [32–35]. Both PKA-dependent and PKA-independent pathways are important mechanisms for agonist-induced desensitization in intact cells [36]. PKA-mediated phosphorylation plays a major role in heterologous desensitization and is cAMP-dependent [36]. bAR kinase-mediated phosphorylation, on the other hand, causes homologous desensitization. This type of desensitization has been reported to be cAMP-independent [37]. Deery and Rani [38] documented that exposure of dog thyroid cells to TSH or TPA induced desensitization of adenylate cyclase. H-7, a general protein kinase inhibitor, prevented TPAinduced but not TSH-induced homologous desensitization. Although H-7 is a general kinase inhibitor, including protein kinases A and C, it failed to prevent homologous desensitization [38]. We found similar results in our current study; that is, despite the prevention of heterologous desensitization, staurosporine did not abolished homologous desensitization when neoplastic thyroid
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Fig. 7. Biphasic effect of TSH on PKA and PKC activity during early or long periods of exposure. TSH affects the PKC signal transduction system earlier than the PKA signal transduction system, whereas TSH activated PKA during the longer period of its stimulation.
TSH–AC– cAMP signal transduction system in human neoplastic thyroid cells, and that there is “cross-talk” or “back-talk” within these signal transduction systems. This investigation suggests that stimulation of a kinase distal to the TSH receptor–AC– cAMP signal transduction system results in desensitization of the cAMP response to TSH. This TPA-induced effect appears to involve PKC activation, as it can be blocked by staurosporine.
Re´sume´ Fig. 6. Homologous and heterologous desensitization of cAMP at 24 hours. The first incubation (24 hours) was performed with and without TSH (10 mU/ml) and TPA (1.6 3 1028 M). After this first incubation the medium was removed, and neoplastic cells were rinsed 3 times with PBS. They were subsequently stimulated for 30 minutes with and without TSH (10 mU/ml). TSH caused homologous desensitization, whereas TPA induced heterologous desensitization of the cAMP response. Homologous desensitization was more effective than heterologous desensitization. (Each point is the mean 6 SD of six experiments.)
cells were incubated with TSH and staurosporine for 4 hours. Staurosporine, in contrast, blunted homologous desensitization when these cells were incubated with TSH and staurosporine for 10 minutes. Pretreatment of cells with TPA for 10 minutes significantly decreased the cAMP response to TSH compared to the cAMP response to TSH stimulation after pretreatment with TSH for 10 minutes (Fig. 4). Heterologous desensitization seems to occur rapidly and perhaps more rapidly than does homologous desensitization of adenylate cyclase in both benign and malignant thyroid cells. Clark et al. [39] documented that heterologous desensitization in lymphoma cells requires an increase in intracellular cAMP level. However, after 4 hours the decrease in cAMP response to subsequent TSH stimulation was higher after pretreatment of cells with TSH than after pretreatment with TPA. Pretreatment of cells with TPA for 10 minutes decreased the subsequent response to TSH comparable or greater than the decrease that occurs after pretreatment with TSH. This study suggests that any agonist that increases cAMP levels results in TSH receptor desensitization. TPA did not increase cAMP but induced heterologous desensitization. This observation is consistent with the finding that not only the TSH receptor but also postreceptor sites are involved in desensitization of the
La de´sensibilisation se de ´finit comme une re ´ponse fonctionnelle diminue´e apre`s stimulation continue ou re ´pe´te´e d’un re´cepteur par son antagoniste. La thyrotropine (TSH) augmente les niveaux de CAMP dans le tissus normal et ne´oplasique de la thyroı¨de. Le carcinoge`ne 12-0-te ´trade´canoyl-phorbol-13-ace ´tate (TPA) est responsable de l’activation de la PKC. Le but de cette ´etude a ´ete´ de de´terminer si le TPA pouvait induire un de´sensibilisation he´t´erologue du syste`me signal de la transduction TSH-AC. On a mis en culture trois tumeurs thyroı¨diennes pendant 6 mois ou plus (un cancer papillaire, un cancer de cellules de Hurthle et un ade´nome folliculaire), qui ont ensuite ´ete´ incube´es avec de la TSH (10 mU/ml) et le TPA (1.6 3 1028M), se´pare´ment et ensemble pendant des pe´riodes d’observation allant de 10 min `a 24 h. On a ensuite proce´de´ `a une incubation de TSH pendant 30 min. Le TPA seul n’avait aucun effet sur les niveaux de CAMP, mais l’incubation de TPA et de la TSH ensemble ´etait responsable d’une re´duction significative dans la re ´ponse CAMP apre `s stimulation par la TSH seule (p , 0.001). Les niveaux de CAMP en re´ponse `a la TSH ont diminue´ de 31 %, de 44 % et de 57 % apre `s pre´incubation avec la TSH pendant, respectivement, 10 min, 4 h et 24 h. Les niveaux de CAMP en re´sponse `a la TSH ont diminue´ respectivement de 44 %, de 33 % et de 29 % apre`s pre´-incubation avec la TPA pendant, respectivement, 10 min, 4 h et 24 h (p , 0.01; ANOVA). L’incubation de cellules avec le TPA et la staurosporine (10 ng/ml), un inhibiteur de la PKC pre´vient les effets du TPA sur la de´sensibilisation `a 10 minutes et efface les effets `a 4 heures. En conclusion, ceci est la premie`re de´monstration de la de´sensibilisation he ´te´rologue de la re ´ponse CAMP `a la TSH, induite par le TPA, dans les cellules ne´oplasiques thyroı¨diennes humaines. Cet effet apparaıˆt ˆetre en rapport avec l’activation de la PKC puisque la re´action peut ˆetre bloque ´e par la staurosporine.
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Resumen Se define la desensibilizacio ´n como la disminucio ´n en la respuesta funcional luego de la estimulacio ´n repetitiva de un receptor por su agonista. La tirotropina (TSH) incrementa los niveles de CAMP en el tejido tiroideo normal y neopla´sico. El 12-0-tetradecanoilforbol-13-acetato (TPA), un promotor tumoral, activa la PKC. El propo ´sito del presente estudio fue determinar si el TPA induce desensibilizacio ´n homo ´loga del sistema TSH-Ac de transduccio ´n. Se incubaron tres neoplasmas tiroideos humanos en cultivo por 6 meses o ma´s (un carcinoma papilar, un carcinoma de ce´lulas de Hurthle y un adenoma folicular), con TSH (10 mU/ml) y TPA (1.6 3 1028 M) por separado y en forma conjunta durante periodos de tiempo variables (desde 10 minutos hasta 24 horas). Se hizo una incubacio ´n subsiguiente con TSH por 30 min. El TPA solo no exhibio ´ efecto sobre los niveles de CAMP, pero la coincubacio ´n de TPA y TSH causo ´ una disminucio ´n significativa en la respuesta de CAMP al compararla con la respuesta de CAMP resultante de la estimulacio ´n con so ´lo TSH (p , 0.001). Los niveles de CAMP en respuesta a TSH disminuyeron en 31%, 44% y 57% luego de preincubacio ´n con TSH por 10 min, 4 h y 24 h, respectivamente. Los niveles de CAMP en respuesta a TSH disminuyeron en 44%, 33% y 29% luego de preincubacio ´n con TPA por 10 min, 4h y 24 h, respectivamente, (p , 0.01; ANOVA). La coincubacio ´n de ce´lulas con TPA y estaurosporina (10 ng/ml), un agente inhibidor de PKC, previno el efecto del TPA sobre la desensibilizacio ´n a los 10 minutos y atenuo ´ el efecto a las 4 horas. En conclusio ´n, esta es la primera demostracio ´n de desensibilizacio ´n hetero ´loga de la respuesta de CAMP a TSH en ce´lulas neopla´sicas tiroideas humanas. Este efecto inducido por TPA parece involucrar la activacio ´n de PKC, puesto que puede ser bloqueado por la estaurosporina.
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Acknowledgments This study was supported in part by Mount Zion Health Systems, University of California San Francisco, the Ben Shane Fund, and the Medical Research Service of the Veterans Affairs Medical Center. References 1. Clark, O.H., Gerend, P.L., Nissenson, R.A.: Thyrotropin binding and adenylate cyclase activation of normal and neoplastic human thyroid tissue: lack of effect of thyroglobulin. J. Clin. Endocrinol. Metab. 54:1157, 1982 2. Laurent, E., Mockel, J., Van Sande, J., Graff, I., Dumont, J.E.: Dual activation by thyrotropin of the phospholipase C and cyclic AMP cascades in human thyroid. Mol. Cell. Endocrinol. 52:273, 1987 3. Van Sande, J., Perret, J., Lejeune, C., Maenheut, C., Vassardant, G., Dumont, J.E.: Thyrotropin activates both the cyclic AMP and the PIP2 cascades in CHO cells expressing the human DNA of TSH receptor. Mol. Cell. Endocrinol. 74:R1, 1990 4. Shaver, J.K., Tezelman, S., Siperstein, A.E., Duh, Q.Y., Clark, O.H.: TSH activates phospholipase C in normal and neoplastic thyroid. Surgery 114:1065, 1993 5. Shuman, S.J., Zor, U., Chayoth, R., Field, J.B.: Exposure of thyroid slices to thyroid stimulating hormone induces refractoriness of the cyclic AMP system to subsequent hormone stimulation. J. Clin. Invest. 53:1132, 1976 6. Rapoport, B., Adams, R.J.: Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. J. Biol. Chem. 251:6653, 1976 7. Avivi, A., Tramontano, D., Ambesi-Impiambato, F.S., Schlessinger, J.:
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