p66Shc Expression in Proliferating Thyroid Cells Is Regulated by Thyrotropin Receptor Signaling
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内分泌学杂志 2005年第5期
Department of Internal Medicine (Y.J.P., T.Y.K., S.H.L, S.W.K., B.Y.C., D.J.P.) and General Surgery (Y.K.Y), Seoul National University College of Medicine, Seoul 110-799; Department of Internal Medicine (Y.J.P), Seoul National University Bundang Hospital, Seongnam 463-707; Clinical Research Institute (B.Y.C., D.J.P.), Seoul National University Hospital, Seoul 110-744; Department of Internal Medicine (H.K., M.S.), School of Medicine, Chungnam National University, Taejon 301-747; and Thyroid Cancer Clinic (S.W.K), National Cancer Center, Goyang 411-769 Korea
Address all correspondence and requests for reprints to: Do Joon Park, M.D., Ph.D., Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-dong Chongno-gu, Seoul 110-744, Korea. E-mail: djpark@snu.ac.kr.
Abstract
It is almost unanimously accepted that thyrocyte proliferation is synergistically activated by TSH and insulin/IGF-I. Moreover, it was recently suggested that p66Shc, which is an adaptor molecule of the IGF-I receptor, might play a critical role in this synergistic effect. In this study, we undertook to confirm the role and the mechanism underlying the regulation of p66Shc expression via TSH receptor in thyrocytes. We have found that p66Shc expression is elevated in proliferating human thyroid tissues, including adenomatous goiter, adenoma, Graves’ disease, and thyroid cancer, but not in normal thyroid. Among growth factors, TSH increased p66Shc expression both in vivo and in vitro; however, IGF-I, epidermal growth factor, or insulin did not. TSH and Graves’ Ig increased the p66Shc expression via the TSH receptor-Gs-cAMP pathway. However, interestingly, IGF-I or epidermal growth factor increased the tyrosine phosphorylations of p66Shc, and this was enhanced by TSH pretreatment. A similar synergism was observed during the DNA synthesis. When we measured the p66Shc levels induced by individual Igs from 130 patients with Graves’ disease, TSH receptor stimulating activity and goiter size showed a weak correlation. We conclude that the expression of p66Shc is regulated by signaling through the TSH receptor in proliferating thyroid cells and that p66Shc appears to be an important mediator of the synergistic effect between TSH and IGF-I with respect to thyrocyte proliferation. Moreover, we suggest that TSH potentiates the regulatory effect of IGF-I on thyrocyte growth, at least in part, by increasing the expression of p66Shc.
Introduction
THYROID GLAND ENLARGEMENT, goiter, is frequently found in various thyroid diseases. Although goiter itself seldom produces serious clinical problems, it frequently causes cosmetic problem in many patients. However, the precise mechanism of goitrogenesis in terms of thyrocyte growth regulation is yet to be understood. Many growth factors are thought to be involved in the goitrogenesis in patients with autoimmune thyroid diseases, and the elevated levels of TSH are considered to play an important role in the goitrogenesis.
It is known that TSH stimulates cell cycle progression and proliferation in cooperation with insulin or IGF-I in various thyrocyte culture systems, including rat thyroid cell lines (FRTL-5, WRT, and PC Cl3) and in primary cultures of rat, dog, sheep, and human thyroid cells. In the case of FRTL-5 cells, a 12- to 24-h preincubation with TSH or a cAMP enhancer shortens the G1 phase and strongly amplifies the DNA synthesis response induced by adding insulin or IGF-I (1, 2, 3, 4, 5).
The continuous presence of TSH is dispensable during the cell cycle progression triggered and supported by insulin/IGF-I (2, 3, 4). TSH is thus referred to as a competence factor that exerts a priming effect that facilitates the action of the progression factor insulin/IGF-I; however, the mechanism is not fully understood. As reported by Takahashi and colleagues (2, 6, 7), TSH pretreatment does not increase the number or activity of IGF-I receptors, but rather it potentiates the IGF-I-dependent tyrosine phosphorylation of the insulin receptor substrate (IRS)-2 and the activation of phosphatidylinositol 3-kinase. In addition, it potentiates the phosphorylation and up-regulation of Src homology collagen (Shc), which increases growth-factor-receptor-bound protein 2 to Shc binding and activates p42/p44 MAPKs. This finding suggests that IGF-I receptor substrates, including IRS-2 and p66Shc, mediate the synergistic effect of TSH and IGF-I. In the present study, we found that the expression of p66Shc is increased in proliferating thyroid tissues and cells and that this increased expression of p66Shc may mediate TSH action on thyrocyte proliferation. Thus we investigated the mechanism by which TSH regulates p66Shc expression.
Materials and Methods
Materials
Rabbit polyclonal anti-Shc antibodies, IRS-2 antibodies, and phosphotyrosine antibodies, 4G10, were purchased from Santa Cruz Biotechnology, Inc. (Delaware, CA) and antirabbit antibodies from New England Biolabs, Inc. (Beverly, MA). Bovine TSH, cholera toxin, pertussis toxin, IGF-I, epidermal growth factor (EGF), insulin, methimazole, and phorbol myristate acetate were obtained from Sigma Chemical Co. (St. Louis, MO). Forskolin, 8-bromoadenosine 3',5'-cyclic-monophosphate (8-bromo cAMP), GF109203X, and H89 were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). [3H]Thymidine was obtained from NEN Life Science Products (Boston, MA). All other reagents were obtained from Sigma unless otherwise stated.
Study subjects and samples
Thyroid tissue samples were obtained at the time of surgery from 27 patients with various forms of goiter (six with adenomatous goiter, one with adenoma, three with Graves’ disease, three with follicular thyroid cancer, and 14 with papillary thyroid cancer; 12 males and 15 females with age range of 30–68 yr and with no history of thyroid autoimmunity). Tissue samples were snap-frozen in liquid nitrogen in the operating room. Frozen sections were prepared from each sample and reviewed by a single pathologist for a confirmatory histological diagnosis. Sera were obtained from 130 Graves’ patients for IgG preparations.
Informed consent was obtained from all subjects, and all experiments were conducted in accordance with the guidelines proposed in The Declaration of Helsinki (http://www.wma.net) involving humans and the use of laboratory animals, and in addition, all experiments were approved by the Institutional Review Board of the Clinical Research Institute at Seoul National University Hospital.
Cell culture
A fresh subclone of FRTL-5 rat thyroid cells was obtained from the Interthyr Research Foundation (Dr. Kohn, Ohio University, Athens, OH). The doubling time of these cells was 36 ± 6 h when cultured in the presence of TSH, but they did not proliferate in the absence of TSH. Cells were grown in 6H medium, which consisted of Coon’s modified F-12 medium supplemented with 5% calf serum, 1 mM nonessential amino acids, and a mixture of six hormones: bovine TSH (10 U/liter), insulin (10 mg/liter), hydrocortisone (0.4 mg/liter), human transferrin (5 mg/liter), glycyl-L-histidyl-L-lysine acetate (10 μg/liter), and somatostatin (10 μg/liter). Before the experiments and after the cells were about 80% confluent, the FRTL-5 rat thyroid cells were grown for 7 d in 5H medium depleted of TSH. Human thyroid carcinoma cell lines (FRO and NPA) were cultured in RPMI 1640 (Life Technologies, Inc., Cergy Pontoise, France) supplemented with 7% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD) at 37 C in 5% CO2.
Animals
Male Sprague Dawley rats (120–130 g) were fed with normal chow and supplied water containing 0.025% methimazole for 2 wk. After 2 wk of methimazole treatment, the rats were supplied with distilled water for 4 wk and then killed for analysis. Tissue samples of rat thyroid glands were fixed in 10% buffered formalin or frozen in liquid nitrogen. For histological examination, tissues were fixed in formalin and embedded in paraffin. Three-micrometer-thick sections were cut from the paraffin blocks and stained with hematoxylin and eosin.
Preparation of Ig and measurements of TSH receptor autoantibody activity
Sera were obtained from 130 Graves’ patients. A diagnosis of Graves’ disease was made based on conventional clinical and laboratory criteria, including elevated serum thyroid hormone levels, undetectable TSH by a RIA, and a diffuse goiter with increased 99mTcO4 uptake at scintiscan. One single physician measured goiter sizes by palpation. TSH receptor-stimulating antibody (TSAb) and TSH receptor-blocking antibody (TRBAb) activities were measured as previously described (8). TSH-binding inhibitory Ig (TBII) activity was expressed as the percentage inhibition of [125I]TSH binding; a TBII activity exceeding 15%, which was 2 SD above the mean value of 64 normal samples, was considered positive. The intra- and interassay variances of TBII activity were 1.7–8.0% and 3.7–10.5%, respectively.
IgG was extracted on an individual basis from the sera of Graves’ patients by affinity chromatography using protein A-Sepharose CL-4B columns; IgG was lyophilized and stored at –20 C until assay. IgGs were also extracted from normal pooled sera (n = 15), Graves’ pooled sera (GP) (n = 15), Graves’ pooled sera with high TBII activity (HGP) (n = 15; TBII activity more than 70%), and Graves’ pooled sera with low TBII activity (LGP) (n = 15; TBII activity less than 15%).
Protein extraction and immunoblot analysis
FRTL-5 cells were incubated at 37 C under the conditions indicated, and then they were scraped and lysed [20 mM Tris (pH 7.4), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM glycerophosphate, 2 mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 4 μg/ml aprotinin], and SDS sample buffer was added [62.5 mM Tris-HCl (pH 6.8), 6% SDS, 30% glycerol, 125 mM dithiothreitol, 0.03% bromophenol blue] for Western blot analysis.
Thyroid tissue (90 mg) was homogenized with 1 ml of the above lysis buffer, and lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes by electrotransfer for 2 h. Membranes were soaked with blocking buffer (Tris-buffered saline, 0.1% Tween 20 with 5% milk as blocking reagent) for 2 h and incubated with primary antibodies Shc, IRS-3, and 4G10 overnight at 4 C. Blots were visualized using horseradish peroxidase-linked antirabbit secondary antibody and a chemiluminescent detection system (Phototope-HRP Western Blot Detection Kit; New England Biolabs).
Immunoprecipitation
The following immunoprecipitation procedures were carried out at 4 C. FRTL-5 cells grown on 100-mm dishes were washed with PBS twice before lysis with radioimmunoprecipitation analysis buffer containing the protease inhibitors (20 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml chymostatin, 2 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Cell lysates were collected, triturated, and centrifuged at 1000 x g for 10 min. To preclear the cell lysates, supernatants were mixed with 20 μl protein A/G beads (Santa Cruz), incubated for 30 min with rocking, and centrifuged for 15 min at 1000 x g. Precleared samples were incubated with the primary antibodies Shc and IRS-2 for 2 h with rocking, protein A/G beads were then added and incubated overnight, and samples were centrifuged at 1000 x g. Immunoprecipitates were collected and washed three times with radioimmunoprecipitation analysis buffer and subjected to PAGE.
RNA isolation and Northern blot analysis
Total RNA was extracted from frozen thyroid tissues and FRTL-5 thyroid cells using TriZol RNA isolation kits (Life Technologies, Inc., Carlsbad, CA) and was diluted to 1 μg/μl in ribonuclease-free water, after RNA concentration determinations at 260 nm. cDNA was synthesized from 1 μg RNA by RT-PCR (Superscript first-strand synthesis kit; Life Technologies, Inc., Rockville, MD). The primers used to amplify p66Shc cDNA were 5'-TACAACCCACTTCGGAATGGTCT-3' and 5'-ATGTACCGAACCAAGTAGG-3' (GenBank accession no. U46956).
Total RNA (15 μg) was separated on a 1% agarose gel containing formaldehyde and transferred to a nylon filter. A p66Shc RNA probe was prepared by labeling cDNAs with Rediprime II (Amersham Pharmacia Biotech, San Francisco, CA). The filters were hybridized with the probes and washed, and mRNA signals were analyzed using x-ray film.
[3H]Thymidine incorporation assay
[3H]Thymidine incorporation assays were performed as follows. Confluent FRTL-5 thyroid cells in 100-mm dishes were detached by trypsinization, resuspended in 6H growth medium, seeded at a density of 3 x 104 cells per well in 24-well plates, and incubated for 2–3 d until 80% confluent. The medium was changed to 5H medium and incubated for an additional 7 d. TSH, forskolin, cAMP, and/or IGF-I and H89 were added to the quiescent cells. The cells were then incubated for 24 h, and 2 μCi/ml [3H]thymidine was added to pulse the cells for an additional 12 h. The cells were then washed four times with ice-cold PBS, precipitated twice with ice-cold 10% trichloroacetic acid for 30 min on ice, briefly washed once with ice-cold ethanol, lysed with 0.2 N NaOH in 0.5% SDS, and incubated at 37 C for at least 30 min. Levels of radioactivity were determined by liquid scintillation spectrometry (Beckman Instruments, Fullerton, CA), and results were expressed as the number of counts per minute per well. Each experimental data point represents triplicate wells from at least four different experiments.
Results
Expression of p66Shc is increased in thyroid goiter
We performed immunoblotting analysis using Shc antibody (Fig. 1A) and found the expression of p66Shc to be increased in all kinds of proliferating thyroid tissues, Hurthle cell adenoma (1 of 1), adenomatous goiter (6 of 6), Graves’ disease (3 of 3), follicular cancer (3 of 3), and papillary cancer (14 of 14), but not in the normal thyroid tissue of the same patients. In contrast, p46 and 52Shc proteins were expressed in all thyroid tissues, including normal thyroid tissues. To confirm these results, we analyzed p66Shc protein expression in some thyroid cell lines, human papillary thyroid carcinoma cell lines (NPA), and follicular carcinoma cell lines (FRO). p66Shc was found to be expressed in NPA and FRO cells but not in normal thyroid FRTL-5 cells (Fig. 1B), which suggests that p66Shc protein may play an important role in thyrocyte proliferation.
FIG. 1. A, The expression of p66Shc protein in the tissues of proliferating thyroid disease. Tissue lysates were subjected to immunoblotting with anti-Shc antibody. The expressions of p66Shc were found increased in the tissues of Huerthle cell adenoma (HA) (1 of 1), adenomatous goiter (AG) (6 of 6), Graves’ disease (GD) (3 of 3), and papillary cancer (PC) (14 of 14) but not in the normal thyroid tissues (N) of the same patients. B, Human papillary thyroid carcinoma cell lines (NPA) and follicular carcinoma cell lines (FRO) were cultured in RPM1. p66Shc was expressed in NPA and FRO cells without TSH treatment but not in normal thyroid FRTL-5 cells.
TSH increased the expression of p66Shc in thyroid cells
We then attempted to identify the growth factors that increase the expression of p66Shc in thyroid cells. Thus, we tested the effects of the well-known thyroid cell growth factors TSH, IGF-I, insulin, and EGF. After treating FRTL-5 cells for 24 h with these factors, only TSH was found to increase the expression of p66Shc (Fig. 2A). We also confirmed this effect of TSH on p66Shc expression in vivo. It is well known that serum TSH levels are elevated in Sprague Dawley rats after 8–10 d of methimazole treatment (9); therefore, we treated rats with methimazole at 0.025% in drinking water for 2 wk to induce serum TSH. Consequently, thyroid weights were increased 2-fold vs. the controls (26.0 ± 7.00 g vs. 12.3 ± 1.53 g), and the number of thyroid follicles also increased. However, when methimazole-containing water was substituted for distilled water after the 4-wk treatment period, the weight of thyroid tissue was reduced to basal levels (14.3 ± 2.89 g) (Fig. 2B). Interestingly, the expression of p66Shc mRNA was also increased in methimazole-treated rats, and this expression also decreased after removing methimazole from drinking water (Fig. 2C). Thus, the above experiments confirmed TSH as primarily responsible for increasing the expression of p66Shc in thyroid cells.
FIG. 2. A, The expression of p66Shc after stimulation with growth factors. FRTL-5 cells were grown to 80% confluency in complete 6H medium (see Materials and Methods) with 5% serum, and cells were maintained for 5–7 d with TSH-free 5H medium. The medium was replaced with 3H medium (see Materials and Methods) containing 0.5% serum 12 h before each experiment. In these experiments, medium was replaced with fresh 3H medium containing TSH (1 mU/ml), IGF-I (100 ng/ml), EGF (100 ng/ml), and insulin (10 mg/ml). Total cell lysates were prepared at 24 h after treatment and subjected to Western blotting with an anti-Shc antibody. TSH stimulated the expression of p66Shc, whereas IGF-I, EGF, or insulin failed to increase its expression (four experiments were performed with similar results). B, Sprague Dawley rats were treated with 0.025% methimazole for 2 wk to induce elevated serum TSH levels. After treating animals with methimazole in drinking water (MMI) for 2 wk, the number of thyroid follicles also increased but reduced to basal levels after the methimazole-containing water was replaced with distilled water (DW) (hematoxylin and eosin; x200). C, Total RNA was isolated and analyzed by Northern blotting (20 μg/lane) using probes for p66Shc and rat ?-actin. p66Shc mRNA levels were also increased in methimazole-treated rats, and these also decreased after removing methimazole from drinking water (Fig. 2C). 4w, 4 wk; 2w, 2 wk.
TSH-dependent expression of p66Shc in FRTL-5 cells
To investigate p66Shc regulation by TSH, we performed a time-response study on TSH-induced p66Shc expression in FRTL-5 thyroid cells. The increased expression of p66Shc could be observed after 8 h of TSH treatment. This increase peaked at 24 h and was sustained until 72 h. The expression of p66Shc mRNA was also elevated from 2–12 h after TSH treatment (Fig. 3A). In a dose-response study, p66Shc expression was increased by 0.01 mU/ml TSH, and the largest response was obtained by 1 mU/ml TSH (Fig. 3B). As a next step, we investigated changes in the expression of p66Shc after removing TSH from media. It was found that the TSH-induced expression of p66Shc was reduced to the basal level 72 h after removing TSH (Fig. 3C). These findings suggest that TSH is the major regulator of the expression of p66Shc in FRTL-5 cells.
FIG. 3. TSH-dependent expression of p66Shc. A, The expression of p66Shc according to TSH incubation time. FRTL-5 cells were cultured as mentioned in Fig. 2. In this experiment, cells were treated with TSH (1 mU/ml) for the indicated times. Similar results were obtained for three independent experiments. p66Shc expression increased obviously after 8 h of TSH treatment, and this peaked at 24 h. The expression of p66Shc mRNA also increased after 2–12 h of TSH treatment. B, p66Shc expression according to the concentration of TSH. In this experiment, cells were treated with the indicated concentrations of TSH for 24 h. p66Shc protein levels were increased even at a TSH concentration of 0.01 mU/ml and peaked at 1 mU/ml. p66Shc mRNA expression also increased after treatment with 0.1 mU/ml TSH. C, Changes in p66Shc expression after TSH removal. TSH-induced p66Shc expression was reduced to the basal level 72 h after removing TSH.
TSH receptor autoantibodies also can regulate the expression of p66Shc
Because TSH acts via the TSH receptor in thyroid cells, we considered that an agonist of the TSH receptor other than TSH could also increase p66Shc expression. To test this hypothesis, we examined the effects of TSAbs on the expression of p66Shc (TSAbs are agonists of the TSH receptor and are believed to be related to goitrogenesis in Graves’ disease). When we measured the influence of IgGs obtained from Graves’ patients, IgGs from the pooled sera of Graves’ patients were found to increase p66Shc expression in a time- and dose-dependent manner (Fig. 4A). Moreover, the extent of p66Shc expression seemed to be increased with TBII activity. Thus, we compared p66Shc expression using the IgGs obtained from HGP or LGP, which had different TBII activities (see Materials and Methods). The amount of p66Shc protein was higher in HGP-treated cells than in LGP-treated cells, although the amount of HGP-induced p66Shc was not as large as that induced by TSH (Fig. 4B). To verify the relation between TSAb activity and the expression of p66Shc, we measured the intensities of p66Shc bands induced by the IgGs of individual Graves’ patients by immunoblotting and then compared these with their TBII activities. The extent of p66Shc expression after stimulation by individual IgGs was highly variable between individuals (Fig. 4C). Relative amounts of p66Shc protein were determined by dividing the intensity of the p66 band stimulated by an individual IgG by the intensity of the p66 band stimulated by TSH. To observe the correlation between p66Shc expression and TSAb activity, we isolated IgGs from the patients’ sera and measured TSAb activity. A weak but clear linear correlation was observed between the TSAb level and the expression of p66Shc protein (P = 0.04; r = 0.26; Fig. 4D), which supported the notion that TSH receptor activation is important for the elevation of p66Shc protein in thyroid cells. Next, we examined whether the amount of p66Shc stimulated by TSAbs correlated with goiter size. Accordingly, we graded the relative intensities of p66Shc bands into quartiles (i.e. <25, 25–50, 50–75, and >75%) and compared these with goiter size. Interestingly, of the six patients who showed high p66Shc expression (over 75%), five (83.3%) had a large goiter of over 40 g (mean size, 56 ± 29 g), although this was not significant vs. patients with lower expression levels (percentage over 40 g was 61.5, 56.2, and 48.1% for the <25, 25–50, and 50–75 groups, respectively; mean size, 45 ± 21, 45 ± 22, and 39 ± 17 g, respectively). We concluded that there might be a relation between p66Shc expression and thyroid cell proliferation.
FIG. 4. FRTL-5 cells and IgGs were prepared as described in Materials and Methods. A, IgGs from pooled Graves’ patients sera (GP) up-regulated p66Shc expression in a time- and dose-dependent manner. B, This up-regulation seemed to be enhanced by TBII. NP, the pooled IgGs of normal controls. C, The diverse expression of p66Shc observed after stimulating with the individual IgGs. The relative amounts of p66Shc protein were determined by dividing the intensity of the p66 band stimulated by individual IgGs by the intensity of the corresponding p66 band stimulated by TSH. D, A weak correlation was observed between the amount of p66Shc and TBII activity (P = 0.04; r = 0.26).
TSH increased the expression of p66Shc via Gs protein and cAMP- and protein kinase A (PKA)-dependent pathways in FRTL-5 thyroid cells
To verify whether the binding of TSH to its receptor is required for p66Shc expression, we examined the effects of TRBAbs, which are known to inhibit TSH receptor binding and block TSH-induced p66Shc expression. We found that TRBAbs at 10 and 20 mg/ml reduced TSH-induced p66Shc expression (Fig. 5A). As a next step, we investigated the G protein subtypes involved in the expression of p66Shc by TSH. Thyroid cells were pretreated for 24 h before TSH stimulation with either 50 ng/ml pertussis toxin, a Gi/o inhibitor, or with 100 ng/ml cholera toxin, a permanent Gs stimulant. As shown in Fig. 5B, TSH-induced p66Shc expression was not inhibited by pertussis toxin. Pretreatment with cholera toxin, however, induced p66Shc expression. These data indicate that Gs but not Gi/o protein is involved in the induction of p66Shc by TSH.
FIG. 5. TSH receptor/Gs protein/cAMP-mediated expression of p66Shc. A, IgGs (TRBAb) from primary myxedema patients were pooled as described in Materials and Methods. Cells were preincubated with TRBAb for 4 h and further incubated with TSH (1 mU/ml) for 24 h. TRBAb blocked TSH-induced increments in the expression of p66Shc. B, Cells were treated with cholera toxin (200 ng/ml) or pertussis toxin (50 ng/ml) with or without TSH (1 mU/ml) for 24 h. p66Shc expression was up-regulated by cholera toxin, but no further increment was observed by adding TSH. Pertussis toxin had no effect on p66Shc expression. C, Cells were treated with TSH (1 mU/ml), forskolin (10 μM), or 8-bromo cAMP (8-Br-cAMP) (10 mM) for 24 h. Stimulating FRTL-5 cells with forskolin and 8-Br-cAMP increased the expression of p66Shc and TSH. D, Cells were pretreated with H89 (20 μM) for 2 h and then treated with TSH (1 mU/ml) for 24 h. H89 inhibited the effect of TSH on p66Shc expression. E, Cells were pretreated with GF109203X (GF) (200 nM) or PMA (10 μM) for 2 h and then treated with TSH (1 mU/ml) for 24 h. GF and PMA did not affect the expression of p66Shc induced by TSH.
The cAMP-PKA pathway is the main signaling pathway by which thyrocytes respond to TSH. Therefore, we investigated whether the activation of adenylate cyclase/cAMP mediates p66Shc up-regulation in response to TSH. Cells were treated with 1 mM 8-bromo cAMP, a cAMP analog, or 10 μM forskolin, a stimulant of adenylate cyclase, and both treatments increased p66Shc expression like TSH (Fig. 5C). We next investigated the involvement of PKA, a downstream effector of Gs and cAMP, in TSH-induced p66Shc expression. FRTL-5 cells were treated with H89 (20 μM) (a PKA inhibitor), which inhibited the TSH-induced expression of p66Shc (Fig. 5D).
TSH also activates the protein kinase C (PKC) pathway through a Gq/11 protein coupled to TSH receptor (10). Therefore, we investigated whether the PKC pathway mediates the expression of p66Shc induced by TSH. As shown in Fig. 5E, 24 h of 200 nM phorbol-12 myristate 13-acetate (PMA) (an activator of PKC) treatment did not induce p66Shc expression. Moreover, treatments with 200 nM PMA or 5 μM GF109203X (both general PKC inhibitors) had no influence on the TSH-induced expression of p66Shc. These data suggest that the TSH-induced expression of p66Shc is mediated by a PKA-dependent pathway, and not by a PKC-dependent pathway.
Effects of TSH pretreatment on the activation of p66Shc stimulated by IGF-I
The question arises as to what role TSH-induced p66Shc fulfills in thyroid cells. Because Shc is an adaptor protein of growth factor receptors, we tested whether TSH-induced p66Shc has some role in the signal transduction process after growth factor receptor activation in thyroid cells.
To confirm that p66Shc up-regulation by TSH pretreatment affects the growth factor receptor signal transduction process after IGF-I stimulation, we first examined the physical association between p66Shc and IRS-2 in response to IGF-I by immunoprecipitating with an antibody against IRS-2 followed by immunoblotting with an antibody against Shc. Physical association of IRS-2 and p66Shc was observed, and this association increased as FRTL-5 cells were treated with IGF-I (Fig. 6A).
FIG. 6. FRTL-5 cells were cultured as described in Materials and Methods. In this experiment, cells were pretreated with or without TSH (1 mU/ml) for 24 h and then stimulated with IGF-I (100 ng/ml) for 5 min. Total cell lysates were prepared as described in the text and immunoprecipitated with IRS-2 or anti-Shc antibody. Immunoprecipitated complexes were separated by SDS-PAGE and immunoblotted. Similar results were obtained for three experiments. A, p66Shc bound to IRS-2, and the up-regulation of p66Shc by TSH increased the IGF-I-induced binding to IRS-2. B, TSH pretreatment potentiated the tyrosine phosphorylation of p66Shc induced by IGF-I by up-regulating p66Shc protein expression. IP, Immunoprecipitation.
Because tyrosine phosphorylation is important for the signaling in IRS-1- and Shc-mediated signal transduction, we then studied the tyrosine phosphorylation of Shc in response to IGF-I. FRTL-5 cells were preincubated with or without TSH, and the tyrosine phosphorylation of p66Shc was analyzed by immunoprecipitation using an antibody against Shc and immunoblotting with an antiphosphotyrosine antibody, and we observed IGF-I-induced phosphorylation of p66Shc. TSH pretreatment significantly potentiated the tyrosine phosphorylation of p66Shc induced by IGF-I (Fig. 6B), thus suggesting increased activity of the IGF-I signaling pathway. However, the amount of p66Shc protein in the immunoprecipitates was also increased by TSH pretreatment, indicating that an increase in the phosphorylation of p66Shc was mainly a result of the up-regulation of p66Shc expression.
Furthermore, as shown in Fig. 7, pretreatment with TSH or cAMP/PKA potentiated the effects of IGF-I on [3H]thymidine uptake, which suggested that p66Shc might be responsible for the synergistic effect between TSH and IGF-I.
FIG. 7. FRTL-5 cells were cultured as described in Materials and Methods. In this experiment, cells were pretreated with or without TSH (1 mU/ml), forskolin (10 μM), or 8-bromo cAMP (8-Br-cAMP) (10 mM) for 24 h, and then IGF-I (100 ng/ml) was added at the indicated conditions for an additional 24 h. [3H]Thymidine uptake was measured as a measure of DNA synthesis, as previously described in Materials and Methods. Pretreatment with either TSH, forskolin, or cAMP potentiated the effects of IGF-I on [3H]thymidine uptake.
Discussion
The present study demonstrates that p66Shc protein levels are up-regulated in proliferating thyroid tissues and in FRTL-5 cells and also shows that the expression of p66Shc is regulated by signaling via the TSH receptor in vitro.
The pretreatment of FRTL-5 rat thyroid cells with TSH was found to markedly potentiate the mitogenic response to IGF-I (1, 2), which is consistent with our results. Therefore, it was suggested that cAMP and IGF-I act synergistically with respect to thyroid cell growth and that this synergism may be related to changes in IGF-I-dependent phosphorylation. Takahashi et al. (6) reported that the pretreatment of FRTL-5 thyroid cells with cAMP potentiated the IGF-I-dependent tyrosine phosphorylation of 175-, 120- to125-, and 90- to 100-kDa proteins. In addition, they recently reported changes in the tyrosine phosphorylation of IGF-I receptor substrates, which included IRS-2 and Shc (7).
p66Shc is an isoform of mammalian ShcA protein and is an adaptor molecule of the IGF-I receptor. In the present study, it was consistently observed that p66Shc expression is regulated in a TSH-dependent manner both in vivo and in vitro and that p66Shc is up-regulated in all proliferating thyroid tissues, which suggests that p66Shc has a role in mediating the synergistic effect between TSH and IGF-I with respect to thyrocyte proliferation. p66Shc is transiently phosphorylated at tyrosine residues in response to growth factor stimulation (11, 12), and this activation of p66Shc may be physiologically relevant to thyroid cell proliferation. However, the functional significance of p66Shc activation in thyroid cells remains to be clarified.
TRBAb can block TSH receptor activity and the binding of TSH to its receptor and was found to inhibit TSH-induced p66Shc expression in a dose-dependent manner, suggesting that the specific binding of TSH and TSHR may be essential for p66Shc expression in response to TSH.
After TSH receptor activation, TSH induced p66Shc expression in a Gs protein and cAMP pathway-dependent manner, but independently of PKC, in FRTL-5 thyroid calls. Moreover, pertussis toxin (a Gi/o inhibitor) did not block p66Shc expression, but cholera toxin (a Gs stimulator) increased its expression, which suggests that Gs protein is involved in the induction of p66Shc by TSH. In addition, intracellular cAMP up-regulation by forskolin or by the addition of 8-bromo cAMP induced p66Shc expression, and this was similar to the effect of TSH on p66Shc expression. Moreover, when H89 was used to block PKA, p66Shc expression was down-regulated, but the PKC inhibitors, PMA or GF109203X, did not affect p66Shc up-regulation by TSH.
In addition to TSH, TSAb also up-regulated p66Shc, and the amount of this up-regulation was correlated with TSAb activity, although this correlation was not strong. These results are explicable because TSAb activity is determined by the ability of the in vivo system to generate cAMP and because they are similar to the TSH dose-dependent up-regulation of p66Shc. In Graves’ disease, TSAb is believed to be the main cause of the development of goiter, because it can facilitate the growth of thyroid follicles by continuously stimulating the TSH receptor. In fact, it was observed that Graves’ Ig, which contains TSAb, can stimulate thyrocyte proliferation (13, 14). Therefore, we examined the correlation between TSAb-induced p66Shc expression and goiter size in Graves’ patients. However, because of the limited number of patients involved, no significant correlation was found. Another explanation of no significant correlation is that the role of other growth factors that can stimulate goiter formation, especially IGF-I or IGF-I receptor-stimulating substances. Recently, Prichard et al. (15) reported that patients with Graves’ disease have evidence for another potentially pathogenic Graves’ disease IgG that can recognize the IGF-I receptor on fibroblasts and activate that receptor and thus stimulates hyaluronan synthesis in orbital fibroblasts (16). Because we did not measure the IGF-I level or IGF-I receptor-stimulating activity of Igs from the patient’s sera, we could not exclude the possibility that the different status of other growth factors and antibodies may contribute to the poor correlation. Furthermore, we measured the goiter size by palpation but not by ultrasonography, which may contribute to the poor correlation, too.
Nevertheless, it was notable that 83% of patients whose IgGs induced strong p66Shc up-regulation had a large goiter. This finding supports a possible relation between p66Shc up-regulation and thyroid cell proliferation.
Unlike the other isoforms of ShcA (p46 or p52), p66Shc does not transform mouse fibroblasts, nor does it induce MAPK activation, although p66Shc is transiently phosphorylated at tyrosine residues in response to growth factor stimulation (9, 10). However, p66Shc has been shown to play an important role in the signaling events that lead to cell death in response to oxidative damage (17). In the present study, we could not resolve the influence of p66Shc up-regulation on the growth or apoptosis of thyrocytes. However, the IGF-I-induced binding of p66Shc to IRS-2, IGF-I-induced tyrosine phosphorylation, and the insulin-induced serine phosphorylation of p66Shc were increased by TSH pretreatment; thus, we speculate that TSH-induced p66Shc mediates and potentiates the effects of growth factor signals via various pathways and that during these processes, TSH-induced p66Shc regulates thyrocytes growth. It will be interesting to determine the downstream effects of TSH-induced p66Shc in thyroid cells.
One thing to note in our data is that although p66Shc expression is regulated by TSH or TSAb, it is also up-regulated in thyroid tumor tissues. In proliferating thyroid tumor, p66Shc expression was increased regardless of TSH level, which means that not only TSH but also unknown factors can clearly increase the expression of p66Shc in thyrocytes. If p66Shc protein mediates signaling through TSH receptor and thus activates thyrocyte growth, we may speculate that this increased p66Shc protein in thyroid tumor might accentuate cell growth with relatively normal TSH stimulation.
In summary, we have demonstrated that p66Shc, an adaptor molecule of IGF-I receptor, is up-regulated in proliferating thyroid cells both in vivo and in vitro. TSH or TSAb signals through the TSH receptor-Gs protein-cAMP cascade were found to be responsible for the incrementation or regulation of p66Shc expression, and TSH-induced p66Shc seemed to potentiate the effects of growth factors in thyroid cells. Our results suggest that p66Shc might be an important mediator of the synergism between TSH and IGF-I and that TSH might potentiate the regulatory effect of IGF-I on thyrocyte growth, and that it accomplishes this at least in part, by increasing the expression of p66Shc.
References
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Address all correspondence and requests for reprints to: Do Joon Park, M.D., Ph.D., Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-dong Chongno-gu, Seoul 110-744, Korea. E-mail: djpark@snu.ac.kr.
Abstract
It is almost unanimously accepted that thyrocyte proliferation is synergistically activated by TSH and insulin/IGF-I. Moreover, it was recently suggested that p66Shc, which is an adaptor molecule of the IGF-I receptor, might play a critical role in this synergistic effect. In this study, we undertook to confirm the role and the mechanism underlying the regulation of p66Shc expression via TSH receptor in thyrocytes. We have found that p66Shc expression is elevated in proliferating human thyroid tissues, including adenomatous goiter, adenoma, Graves’ disease, and thyroid cancer, but not in normal thyroid. Among growth factors, TSH increased p66Shc expression both in vivo and in vitro; however, IGF-I, epidermal growth factor, or insulin did not. TSH and Graves’ Ig increased the p66Shc expression via the TSH receptor-Gs-cAMP pathway. However, interestingly, IGF-I or epidermal growth factor increased the tyrosine phosphorylations of p66Shc, and this was enhanced by TSH pretreatment. A similar synergism was observed during the DNA synthesis. When we measured the p66Shc levels induced by individual Igs from 130 patients with Graves’ disease, TSH receptor stimulating activity and goiter size showed a weak correlation. We conclude that the expression of p66Shc is regulated by signaling through the TSH receptor in proliferating thyroid cells and that p66Shc appears to be an important mediator of the synergistic effect between TSH and IGF-I with respect to thyrocyte proliferation. Moreover, we suggest that TSH potentiates the regulatory effect of IGF-I on thyrocyte growth, at least in part, by increasing the expression of p66Shc.
Introduction
THYROID GLAND ENLARGEMENT, goiter, is frequently found in various thyroid diseases. Although goiter itself seldom produces serious clinical problems, it frequently causes cosmetic problem in many patients. However, the precise mechanism of goitrogenesis in terms of thyrocyte growth regulation is yet to be understood. Many growth factors are thought to be involved in the goitrogenesis in patients with autoimmune thyroid diseases, and the elevated levels of TSH are considered to play an important role in the goitrogenesis.
It is known that TSH stimulates cell cycle progression and proliferation in cooperation with insulin or IGF-I in various thyrocyte culture systems, including rat thyroid cell lines (FRTL-5, WRT, and PC Cl3) and in primary cultures of rat, dog, sheep, and human thyroid cells. In the case of FRTL-5 cells, a 12- to 24-h preincubation with TSH or a cAMP enhancer shortens the G1 phase and strongly amplifies the DNA synthesis response induced by adding insulin or IGF-I (1, 2, 3, 4, 5).
The continuous presence of TSH is dispensable during the cell cycle progression triggered and supported by insulin/IGF-I (2, 3, 4). TSH is thus referred to as a competence factor that exerts a priming effect that facilitates the action of the progression factor insulin/IGF-I; however, the mechanism is not fully understood. As reported by Takahashi and colleagues (2, 6, 7), TSH pretreatment does not increase the number or activity of IGF-I receptors, but rather it potentiates the IGF-I-dependent tyrosine phosphorylation of the insulin receptor substrate (IRS)-2 and the activation of phosphatidylinositol 3-kinase. In addition, it potentiates the phosphorylation and up-regulation of Src homology collagen (Shc), which increases growth-factor-receptor-bound protein 2 to Shc binding and activates p42/p44 MAPKs. This finding suggests that IGF-I receptor substrates, including IRS-2 and p66Shc, mediate the synergistic effect of TSH and IGF-I. In the present study, we found that the expression of p66Shc is increased in proliferating thyroid tissues and cells and that this increased expression of p66Shc may mediate TSH action on thyrocyte proliferation. Thus we investigated the mechanism by which TSH regulates p66Shc expression.
Materials and Methods
Materials
Rabbit polyclonal anti-Shc antibodies, IRS-2 antibodies, and phosphotyrosine antibodies, 4G10, were purchased from Santa Cruz Biotechnology, Inc. (Delaware, CA) and antirabbit antibodies from New England Biolabs, Inc. (Beverly, MA). Bovine TSH, cholera toxin, pertussis toxin, IGF-I, epidermal growth factor (EGF), insulin, methimazole, and phorbol myristate acetate were obtained from Sigma Chemical Co. (St. Louis, MO). Forskolin, 8-bromoadenosine 3',5'-cyclic-monophosphate (8-bromo cAMP), GF109203X, and H89 were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). [3H]Thymidine was obtained from NEN Life Science Products (Boston, MA). All other reagents were obtained from Sigma unless otherwise stated.
Study subjects and samples
Thyroid tissue samples were obtained at the time of surgery from 27 patients with various forms of goiter (six with adenomatous goiter, one with adenoma, three with Graves’ disease, three with follicular thyroid cancer, and 14 with papillary thyroid cancer; 12 males and 15 females with age range of 30–68 yr and with no history of thyroid autoimmunity). Tissue samples were snap-frozen in liquid nitrogen in the operating room. Frozen sections were prepared from each sample and reviewed by a single pathologist for a confirmatory histological diagnosis. Sera were obtained from 130 Graves’ patients for IgG preparations.
Informed consent was obtained from all subjects, and all experiments were conducted in accordance with the guidelines proposed in The Declaration of Helsinki (http://www.wma.net) involving humans and the use of laboratory animals, and in addition, all experiments were approved by the Institutional Review Board of the Clinical Research Institute at Seoul National University Hospital.
Cell culture
A fresh subclone of FRTL-5 rat thyroid cells was obtained from the Interthyr Research Foundation (Dr. Kohn, Ohio University, Athens, OH). The doubling time of these cells was 36 ± 6 h when cultured in the presence of TSH, but they did not proliferate in the absence of TSH. Cells were grown in 6H medium, which consisted of Coon’s modified F-12 medium supplemented with 5% calf serum, 1 mM nonessential amino acids, and a mixture of six hormones: bovine TSH (10 U/liter), insulin (10 mg/liter), hydrocortisone (0.4 mg/liter), human transferrin (5 mg/liter), glycyl-L-histidyl-L-lysine acetate (10 μg/liter), and somatostatin (10 μg/liter). Before the experiments and after the cells were about 80% confluent, the FRTL-5 rat thyroid cells were grown for 7 d in 5H medium depleted of TSH. Human thyroid carcinoma cell lines (FRO and NPA) were cultured in RPMI 1640 (Life Technologies, Inc., Cergy Pontoise, France) supplemented with 7% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD) at 37 C in 5% CO2.
Animals
Male Sprague Dawley rats (120–130 g) were fed with normal chow and supplied water containing 0.025% methimazole for 2 wk. After 2 wk of methimazole treatment, the rats were supplied with distilled water for 4 wk and then killed for analysis. Tissue samples of rat thyroid glands were fixed in 10% buffered formalin or frozen in liquid nitrogen. For histological examination, tissues were fixed in formalin and embedded in paraffin. Three-micrometer-thick sections were cut from the paraffin blocks and stained with hematoxylin and eosin.
Preparation of Ig and measurements of TSH receptor autoantibody activity
Sera were obtained from 130 Graves’ patients. A diagnosis of Graves’ disease was made based on conventional clinical and laboratory criteria, including elevated serum thyroid hormone levels, undetectable TSH by a RIA, and a diffuse goiter with increased 99mTcO4 uptake at scintiscan. One single physician measured goiter sizes by palpation. TSH receptor-stimulating antibody (TSAb) and TSH receptor-blocking antibody (TRBAb) activities were measured as previously described (8). TSH-binding inhibitory Ig (TBII) activity was expressed as the percentage inhibition of [125I]TSH binding; a TBII activity exceeding 15%, which was 2 SD above the mean value of 64 normal samples, was considered positive. The intra- and interassay variances of TBII activity were 1.7–8.0% and 3.7–10.5%, respectively.
IgG was extracted on an individual basis from the sera of Graves’ patients by affinity chromatography using protein A-Sepharose CL-4B columns; IgG was lyophilized and stored at –20 C until assay. IgGs were also extracted from normal pooled sera (n = 15), Graves’ pooled sera (GP) (n = 15), Graves’ pooled sera with high TBII activity (HGP) (n = 15; TBII activity more than 70%), and Graves’ pooled sera with low TBII activity (LGP) (n = 15; TBII activity less than 15%).
Protein extraction and immunoblot analysis
FRTL-5 cells were incubated at 37 C under the conditions indicated, and then they were scraped and lysed [20 mM Tris (pH 7.4), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM glycerophosphate, 2 mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 4 μg/ml aprotinin], and SDS sample buffer was added [62.5 mM Tris-HCl (pH 6.8), 6% SDS, 30% glycerol, 125 mM dithiothreitol, 0.03% bromophenol blue] for Western blot analysis.
Thyroid tissue (90 mg) was homogenized with 1 ml of the above lysis buffer, and lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes by electrotransfer for 2 h. Membranes were soaked with blocking buffer (Tris-buffered saline, 0.1% Tween 20 with 5% milk as blocking reagent) for 2 h and incubated with primary antibodies Shc, IRS-3, and 4G10 overnight at 4 C. Blots were visualized using horseradish peroxidase-linked antirabbit secondary antibody and a chemiluminescent detection system (Phototope-HRP Western Blot Detection Kit; New England Biolabs).
Immunoprecipitation
The following immunoprecipitation procedures were carried out at 4 C. FRTL-5 cells grown on 100-mm dishes were washed with PBS twice before lysis with radioimmunoprecipitation analysis buffer containing the protease inhibitors (20 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml chymostatin, 2 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Cell lysates were collected, triturated, and centrifuged at 1000 x g for 10 min. To preclear the cell lysates, supernatants were mixed with 20 μl protein A/G beads (Santa Cruz), incubated for 30 min with rocking, and centrifuged for 15 min at 1000 x g. Precleared samples were incubated with the primary antibodies Shc and IRS-2 for 2 h with rocking, protein A/G beads were then added and incubated overnight, and samples were centrifuged at 1000 x g. Immunoprecipitates were collected and washed three times with radioimmunoprecipitation analysis buffer and subjected to PAGE.
RNA isolation and Northern blot analysis
Total RNA was extracted from frozen thyroid tissues and FRTL-5 thyroid cells using TriZol RNA isolation kits (Life Technologies, Inc., Carlsbad, CA) and was diluted to 1 μg/μl in ribonuclease-free water, after RNA concentration determinations at 260 nm. cDNA was synthesized from 1 μg RNA by RT-PCR (Superscript first-strand synthesis kit; Life Technologies, Inc., Rockville, MD). The primers used to amplify p66Shc cDNA were 5'-TACAACCCACTTCGGAATGGTCT-3' and 5'-ATGTACCGAACCAAGTAGG-3' (GenBank accession no. U46956).
Total RNA (15 μg) was separated on a 1% agarose gel containing formaldehyde and transferred to a nylon filter. A p66Shc RNA probe was prepared by labeling cDNAs with Rediprime II (Amersham Pharmacia Biotech, San Francisco, CA). The filters were hybridized with the probes and washed, and mRNA signals were analyzed using x-ray film.
[3H]Thymidine incorporation assay
[3H]Thymidine incorporation assays were performed as follows. Confluent FRTL-5 thyroid cells in 100-mm dishes were detached by trypsinization, resuspended in 6H growth medium, seeded at a density of 3 x 104 cells per well in 24-well plates, and incubated for 2–3 d until 80% confluent. The medium was changed to 5H medium and incubated for an additional 7 d. TSH, forskolin, cAMP, and/or IGF-I and H89 were added to the quiescent cells. The cells were then incubated for 24 h, and 2 μCi/ml [3H]thymidine was added to pulse the cells for an additional 12 h. The cells were then washed four times with ice-cold PBS, precipitated twice with ice-cold 10% trichloroacetic acid for 30 min on ice, briefly washed once with ice-cold ethanol, lysed with 0.2 N NaOH in 0.5% SDS, and incubated at 37 C for at least 30 min. Levels of radioactivity were determined by liquid scintillation spectrometry (Beckman Instruments, Fullerton, CA), and results were expressed as the number of counts per minute per well. Each experimental data point represents triplicate wells from at least four different experiments.
Results
Expression of p66Shc is increased in thyroid goiter
We performed immunoblotting analysis using Shc antibody (Fig. 1A) and found the expression of p66Shc to be increased in all kinds of proliferating thyroid tissues, Hurthle cell adenoma (1 of 1), adenomatous goiter (6 of 6), Graves’ disease (3 of 3), follicular cancer (3 of 3), and papillary cancer (14 of 14), but not in the normal thyroid tissue of the same patients. In contrast, p46 and 52Shc proteins were expressed in all thyroid tissues, including normal thyroid tissues. To confirm these results, we analyzed p66Shc protein expression in some thyroid cell lines, human papillary thyroid carcinoma cell lines (NPA), and follicular carcinoma cell lines (FRO). p66Shc was found to be expressed in NPA and FRO cells but not in normal thyroid FRTL-5 cells (Fig. 1B), which suggests that p66Shc protein may play an important role in thyrocyte proliferation.
FIG. 1. A, The expression of p66Shc protein in the tissues of proliferating thyroid disease. Tissue lysates were subjected to immunoblotting with anti-Shc antibody. The expressions of p66Shc were found increased in the tissues of Huerthle cell adenoma (HA) (1 of 1), adenomatous goiter (AG) (6 of 6), Graves’ disease (GD) (3 of 3), and papillary cancer (PC) (14 of 14) but not in the normal thyroid tissues (N) of the same patients. B, Human papillary thyroid carcinoma cell lines (NPA) and follicular carcinoma cell lines (FRO) were cultured in RPM1. p66Shc was expressed in NPA and FRO cells without TSH treatment but not in normal thyroid FRTL-5 cells.
TSH increased the expression of p66Shc in thyroid cells
We then attempted to identify the growth factors that increase the expression of p66Shc in thyroid cells. Thus, we tested the effects of the well-known thyroid cell growth factors TSH, IGF-I, insulin, and EGF. After treating FRTL-5 cells for 24 h with these factors, only TSH was found to increase the expression of p66Shc (Fig. 2A). We also confirmed this effect of TSH on p66Shc expression in vivo. It is well known that serum TSH levels are elevated in Sprague Dawley rats after 8–10 d of methimazole treatment (9); therefore, we treated rats with methimazole at 0.025% in drinking water for 2 wk to induce serum TSH. Consequently, thyroid weights were increased 2-fold vs. the controls (26.0 ± 7.00 g vs. 12.3 ± 1.53 g), and the number of thyroid follicles also increased. However, when methimazole-containing water was substituted for distilled water after the 4-wk treatment period, the weight of thyroid tissue was reduced to basal levels (14.3 ± 2.89 g) (Fig. 2B). Interestingly, the expression of p66Shc mRNA was also increased in methimazole-treated rats, and this expression also decreased after removing methimazole from drinking water (Fig. 2C). Thus, the above experiments confirmed TSH as primarily responsible for increasing the expression of p66Shc in thyroid cells.
FIG. 2. A, The expression of p66Shc after stimulation with growth factors. FRTL-5 cells were grown to 80% confluency in complete 6H medium (see Materials and Methods) with 5% serum, and cells were maintained for 5–7 d with TSH-free 5H medium. The medium was replaced with 3H medium (see Materials and Methods) containing 0.5% serum 12 h before each experiment. In these experiments, medium was replaced with fresh 3H medium containing TSH (1 mU/ml), IGF-I (100 ng/ml), EGF (100 ng/ml), and insulin (10 mg/ml). Total cell lysates were prepared at 24 h after treatment and subjected to Western blotting with an anti-Shc antibody. TSH stimulated the expression of p66Shc, whereas IGF-I, EGF, or insulin failed to increase its expression (four experiments were performed with similar results). B, Sprague Dawley rats were treated with 0.025% methimazole for 2 wk to induce elevated serum TSH levels. After treating animals with methimazole in drinking water (MMI) for 2 wk, the number of thyroid follicles also increased but reduced to basal levels after the methimazole-containing water was replaced with distilled water (DW) (hematoxylin and eosin; x200). C, Total RNA was isolated and analyzed by Northern blotting (20 μg/lane) using probes for p66Shc and rat ?-actin. p66Shc mRNA levels were also increased in methimazole-treated rats, and these also decreased after removing methimazole from drinking water (Fig. 2C). 4w, 4 wk; 2w, 2 wk.
TSH-dependent expression of p66Shc in FRTL-5 cells
To investigate p66Shc regulation by TSH, we performed a time-response study on TSH-induced p66Shc expression in FRTL-5 thyroid cells. The increased expression of p66Shc could be observed after 8 h of TSH treatment. This increase peaked at 24 h and was sustained until 72 h. The expression of p66Shc mRNA was also elevated from 2–12 h after TSH treatment (Fig. 3A). In a dose-response study, p66Shc expression was increased by 0.01 mU/ml TSH, and the largest response was obtained by 1 mU/ml TSH (Fig. 3B). As a next step, we investigated changes in the expression of p66Shc after removing TSH from media. It was found that the TSH-induced expression of p66Shc was reduced to the basal level 72 h after removing TSH (Fig. 3C). These findings suggest that TSH is the major regulator of the expression of p66Shc in FRTL-5 cells.
FIG. 3. TSH-dependent expression of p66Shc. A, The expression of p66Shc according to TSH incubation time. FRTL-5 cells were cultured as mentioned in Fig. 2. In this experiment, cells were treated with TSH (1 mU/ml) for the indicated times. Similar results were obtained for three independent experiments. p66Shc expression increased obviously after 8 h of TSH treatment, and this peaked at 24 h. The expression of p66Shc mRNA also increased after 2–12 h of TSH treatment. B, p66Shc expression according to the concentration of TSH. In this experiment, cells were treated with the indicated concentrations of TSH for 24 h. p66Shc protein levels were increased even at a TSH concentration of 0.01 mU/ml and peaked at 1 mU/ml. p66Shc mRNA expression also increased after treatment with 0.1 mU/ml TSH. C, Changes in p66Shc expression after TSH removal. TSH-induced p66Shc expression was reduced to the basal level 72 h after removing TSH.
TSH receptor autoantibodies also can regulate the expression of p66Shc
Because TSH acts via the TSH receptor in thyroid cells, we considered that an agonist of the TSH receptor other than TSH could also increase p66Shc expression. To test this hypothesis, we examined the effects of TSAbs on the expression of p66Shc (TSAbs are agonists of the TSH receptor and are believed to be related to goitrogenesis in Graves’ disease). When we measured the influence of IgGs obtained from Graves’ patients, IgGs from the pooled sera of Graves’ patients were found to increase p66Shc expression in a time- and dose-dependent manner (Fig. 4A). Moreover, the extent of p66Shc expression seemed to be increased with TBII activity. Thus, we compared p66Shc expression using the IgGs obtained from HGP or LGP, which had different TBII activities (see Materials and Methods). The amount of p66Shc protein was higher in HGP-treated cells than in LGP-treated cells, although the amount of HGP-induced p66Shc was not as large as that induced by TSH (Fig. 4B). To verify the relation between TSAb activity and the expression of p66Shc, we measured the intensities of p66Shc bands induced by the IgGs of individual Graves’ patients by immunoblotting and then compared these with their TBII activities. The extent of p66Shc expression after stimulation by individual IgGs was highly variable between individuals (Fig. 4C). Relative amounts of p66Shc protein were determined by dividing the intensity of the p66 band stimulated by an individual IgG by the intensity of the p66 band stimulated by TSH. To observe the correlation between p66Shc expression and TSAb activity, we isolated IgGs from the patients’ sera and measured TSAb activity. A weak but clear linear correlation was observed between the TSAb level and the expression of p66Shc protein (P = 0.04; r = 0.26; Fig. 4D), which supported the notion that TSH receptor activation is important for the elevation of p66Shc protein in thyroid cells. Next, we examined whether the amount of p66Shc stimulated by TSAbs correlated with goiter size. Accordingly, we graded the relative intensities of p66Shc bands into quartiles (i.e. <25, 25–50, 50–75, and >75%) and compared these with goiter size. Interestingly, of the six patients who showed high p66Shc expression (over 75%), five (83.3%) had a large goiter of over 40 g (mean size, 56 ± 29 g), although this was not significant vs. patients with lower expression levels (percentage over 40 g was 61.5, 56.2, and 48.1% for the <25, 25–50, and 50–75 groups, respectively; mean size, 45 ± 21, 45 ± 22, and 39 ± 17 g, respectively). We concluded that there might be a relation between p66Shc expression and thyroid cell proliferation.
FIG. 4. FRTL-5 cells and IgGs were prepared as described in Materials and Methods. A, IgGs from pooled Graves’ patients sera (GP) up-regulated p66Shc expression in a time- and dose-dependent manner. B, This up-regulation seemed to be enhanced by TBII. NP, the pooled IgGs of normal controls. C, The diverse expression of p66Shc observed after stimulating with the individual IgGs. The relative amounts of p66Shc protein were determined by dividing the intensity of the p66 band stimulated by individual IgGs by the intensity of the corresponding p66 band stimulated by TSH. D, A weak correlation was observed between the amount of p66Shc and TBII activity (P = 0.04; r = 0.26).
TSH increased the expression of p66Shc via Gs protein and cAMP- and protein kinase A (PKA)-dependent pathways in FRTL-5 thyroid cells
To verify whether the binding of TSH to its receptor is required for p66Shc expression, we examined the effects of TRBAbs, which are known to inhibit TSH receptor binding and block TSH-induced p66Shc expression. We found that TRBAbs at 10 and 20 mg/ml reduced TSH-induced p66Shc expression (Fig. 5A). As a next step, we investigated the G protein subtypes involved in the expression of p66Shc by TSH. Thyroid cells were pretreated for 24 h before TSH stimulation with either 50 ng/ml pertussis toxin, a Gi/o inhibitor, or with 100 ng/ml cholera toxin, a permanent Gs stimulant. As shown in Fig. 5B, TSH-induced p66Shc expression was not inhibited by pertussis toxin. Pretreatment with cholera toxin, however, induced p66Shc expression. These data indicate that Gs but not Gi/o protein is involved in the induction of p66Shc by TSH.
FIG. 5. TSH receptor/Gs protein/cAMP-mediated expression of p66Shc. A, IgGs (TRBAb) from primary myxedema patients were pooled as described in Materials and Methods. Cells were preincubated with TRBAb for 4 h and further incubated with TSH (1 mU/ml) for 24 h. TRBAb blocked TSH-induced increments in the expression of p66Shc. B, Cells were treated with cholera toxin (200 ng/ml) or pertussis toxin (50 ng/ml) with or without TSH (1 mU/ml) for 24 h. p66Shc expression was up-regulated by cholera toxin, but no further increment was observed by adding TSH. Pertussis toxin had no effect on p66Shc expression. C, Cells were treated with TSH (1 mU/ml), forskolin (10 μM), or 8-bromo cAMP (8-Br-cAMP) (10 mM) for 24 h. Stimulating FRTL-5 cells with forskolin and 8-Br-cAMP increased the expression of p66Shc and TSH. D, Cells were pretreated with H89 (20 μM) for 2 h and then treated with TSH (1 mU/ml) for 24 h. H89 inhibited the effect of TSH on p66Shc expression. E, Cells were pretreated with GF109203X (GF) (200 nM) or PMA (10 μM) for 2 h and then treated with TSH (1 mU/ml) for 24 h. GF and PMA did not affect the expression of p66Shc induced by TSH.
The cAMP-PKA pathway is the main signaling pathway by which thyrocytes respond to TSH. Therefore, we investigated whether the activation of adenylate cyclase/cAMP mediates p66Shc up-regulation in response to TSH. Cells were treated with 1 mM 8-bromo cAMP, a cAMP analog, or 10 μM forskolin, a stimulant of adenylate cyclase, and both treatments increased p66Shc expression like TSH (Fig. 5C). We next investigated the involvement of PKA, a downstream effector of Gs and cAMP, in TSH-induced p66Shc expression. FRTL-5 cells were treated with H89 (20 μM) (a PKA inhibitor), which inhibited the TSH-induced expression of p66Shc (Fig. 5D).
TSH also activates the protein kinase C (PKC) pathway through a Gq/11 protein coupled to TSH receptor (10). Therefore, we investigated whether the PKC pathway mediates the expression of p66Shc induced by TSH. As shown in Fig. 5E, 24 h of 200 nM phorbol-12 myristate 13-acetate (PMA) (an activator of PKC) treatment did not induce p66Shc expression. Moreover, treatments with 200 nM PMA or 5 μM GF109203X (both general PKC inhibitors) had no influence on the TSH-induced expression of p66Shc. These data suggest that the TSH-induced expression of p66Shc is mediated by a PKA-dependent pathway, and not by a PKC-dependent pathway.
Effects of TSH pretreatment on the activation of p66Shc stimulated by IGF-I
The question arises as to what role TSH-induced p66Shc fulfills in thyroid cells. Because Shc is an adaptor protein of growth factor receptors, we tested whether TSH-induced p66Shc has some role in the signal transduction process after growth factor receptor activation in thyroid cells.
To confirm that p66Shc up-regulation by TSH pretreatment affects the growth factor receptor signal transduction process after IGF-I stimulation, we first examined the physical association between p66Shc and IRS-2 in response to IGF-I by immunoprecipitating with an antibody against IRS-2 followed by immunoblotting with an antibody against Shc. Physical association of IRS-2 and p66Shc was observed, and this association increased as FRTL-5 cells were treated with IGF-I (Fig. 6A).
FIG. 6. FRTL-5 cells were cultured as described in Materials and Methods. In this experiment, cells were pretreated with or without TSH (1 mU/ml) for 24 h and then stimulated with IGF-I (100 ng/ml) for 5 min. Total cell lysates were prepared as described in the text and immunoprecipitated with IRS-2 or anti-Shc antibody. Immunoprecipitated complexes were separated by SDS-PAGE and immunoblotted. Similar results were obtained for three experiments. A, p66Shc bound to IRS-2, and the up-regulation of p66Shc by TSH increased the IGF-I-induced binding to IRS-2. B, TSH pretreatment potentiated the tyrosine phosphorylation of p66Shc induced by IGF-I by up-regulating p66Shc protein expression. IP, Immunoprecipitation.
Because tyrosine phosphorylation is important for the signaling in IRS-1- and Shc-mediated signal transduction, we then studied the tyrosine phosphorylation of Shc in response to IGF-I. FRTL-5 cells were preincubated with or without TSH, and the tyrosine phosphorylation of p66Shc was analyzed by immunoprecipitation using an antibody against Shc and immunoblotting with an antiphosphotyrosine antibody, and we observed IGF-I-induced phosphorylation of p66Shc. TSH pretreatment significantly potentiated the tyrosine phosphorylation of p66Shc induced by IGF-I (Fig. 6B), thus suggesting increased activity of the IGF-I signaling pathway. However, the amount of p66Shc protein in the immunoprecipitates was also increased by TSH pretreatment, indicating that an increase in the phosphorylation of p66Shc was mainly a result of the up-regulation of p66Shc expression.
Furthermore, as shown in Fig. 7, pretreatment with TSH or cAMP/PKA potentiated the effects of IGF-I on [3H]thymidine uptake, which suggested that p66Shc might be responsible for the synergistic effect between TSH and IGF-I.
FIG. 7. FRTL-5 cells were cultured as described in Materials and Methods. In this experiment, cells were pretreated with or without TSH (1 mU/ml), forskolin (10 μM), or 8-bromo cAMP (8-Br-cAMP) (10 mM) for 24 h, and then IGF-I (100 ng/ml) was added at the indicated conditions for an additional 24 h. [3H]Thymidine uptake was measured as a measure of DNA synthesis, as previously described in Materials and Methods. Pretreatment with either TSH, forskolin, or cAMP potentiated the effects of IGF-I on [3H]thymidine uptake.
Discussion
The present study demonstrates that p66Shc protein levels are up-regulated in proliferating thyroid tissues and in FRTL-5 cells and also shows that the expression of p66Shc is regulated by signaling via the TSH receptor in vitro.
The pretreatment of FRTL-5 rat thyroid cells with TSH was found to markedly potentiate the mitogenic response to IGF-I (1, 2), which is consistent with our results. Therefore, it was suggested that cAMP and IGF-I act synergistically with respect to thyroid cell growth and that this synergism may be related to changes in IGF-I-dependent phosphorylation. Takahashi et al. (6) reported that the pretreatment of FRTL-5 thyroid cells with cAMP potentiated the IGF-I-dependent tyrosine phosphorylation of 175-, 120- to125-, and 90- to 100-kDa proteins. In addition, they recently reported changes in the tyrosine phosphorylation of IGF-I receptor substrates, which included IRS-2 and Shc (7).
p66Shc is an isoform of mammalian ShcA protein and is an adaptor molecule of the IGF-I receptor. In the present study, it was consistently observed that p66Shc expression is regulated in a TSH-dependent manner both in vivo and in vitro and that p66Shc is up-regulated in all proliferating thyroid tissues, which suggests that p66Shc has a role in mediating the synergistic effect between TSH and IGF-I with respect to thyrocyte proliferation. p66Shc is transiently phosphorylated at tyrosine residues in response to growth factor stimulation (11, 12), and this activation of p66Shc may be physiologically relevant to thyroid cell proliferation. However, the functional significance of p66Shc activation in thyroid cells remains to be clarified.
TRBAb can block TSH receptor activity and the binding of TSH to its receptor and was found to inhibit TSH-induced p66Shc expression in a dose-dependent manner, suggesting that the specific binding of TSH and TSHR may be essential for p66Shc expression in response to TSH.
After TSH receptor activation, TSH induced p66Shc expression in a Gs protein and cAMP pathway-dependent manner, but independently of PKC, in FRTL-5 thyroid calls. Moreover, pertussis toxin (a Gi/o inhibitor) did not block p66Shc expression, but cholera toxin (a Gs stimulator) increased its expression, which suggests that Gs protein is involved in the induction of p66Shc by TSH. In addition, intracellular cAMP up-regulation by forskolin or by the addition of 8-bromo cAMP induced p66Shc expression, and this was similar to the effect of TSH on p66Shc expression. Moreover, when H89 was used to block PKA, p66Shc expression was down-regulated, but the PKC inhibitors, PMA or GF109203X, did not affect p66Shc up-regulation by TSH.
In addition to TSH, TSAb also up-regulated p66Shc, and the amount of this up-regulation was correlated with TSAb activity, although this correlation was not strong. These results are explicable because TSAb activity is determined by the ability of the in vivo system to generate cAMP and because they are similar to the TSH dose-dependent up-regulation of p66Shc. In Graves’ disease, TSAb is believed to be the main cause of the development of goiter, because it can facilitate the growth of thyroid follicles by continuously stimulating the TSH receptor. In fact, it was observed that Graves’ Ig, which contains TSAb, can stimulate thyrocyte proliferation (13, 14). Therefore, we examined the correlation between TSAb-induced p66Shc expression and goiter size in Graves’ patients. However, because of the limited number of patients involved, no significant correlation was found. Another explanation of no significant correlation is that the role of other growth factors that can stimulate goiter formation, especially IGF-I or IGF-I receptor-stimulating substances. Recently, Prichard et al. (15) reported that patients with Graves’ disease have evidence for another potentially pathogenic Graves’ disease IgG that can recognize the IGF-I receptor on fibroblasts and activate that receptor and thus stimulates hyaluronan synthesis in orbital fibroblasts (16). Because we did not measure the IGF-I level or IGF-I receptor-stimulating activity of Igs from the patient’s sera, we could not exclude the possibility that the different status of other growth factors and antibodies may contribute to the poor correlation. Furthermore, we measured the goiter size by palpation but not by ultrasonography, which may contribute to the poor correlation, too.
Nevertheless, it was notable that 83% of patients whose IgGs induced strong p66Shc up-regulation had a large goiter. This finding supports a possible relation between p66Shc up-regulation and thyroid cell proliferation.
Unlike the other isoforms of ShcA (p46 or p52), p66Shc does not transform mouse fibroblasts, nor does it induce MAPK activation, although p66Shc is transiently phosphorylated at tyrosine residues in response to growth factor stimulation (9, 10). However, p66Shc has been shown to play an important role in the signaling events that lead to cell death in response to oxidative damage (17). In the present study, we could not resolve the influence of p66Shc up-regulation on the growth or apoptosis of thyrocytes. However, the IGF-I-induced binding of p66Shc to IRS-2, IGF-I-induced tyrosine phosphorylation, and the insulin-induced serine phosphorylation of p66Shc were increased by TSH pretreatment; thus, we speculate that TSH-induced p66Shc mediates and potentiates the effects of growth factor signals via various pathways and that during these processes, TSH-induced p66Shc regulates thyrocytes growth. It will be interesting to determine the downstream effects of TSH-induced p66Shc in thyroid cells.
One thing to note in our data is that although p66Shc expression is regulated by TSH or TSAb, it is also up-regulated in thyroid tumor tissues. In proliferating thyroid tumor, p66Shc expression was increased regardless of TSH level, which means that not only TSH but also unknown factors can clearly increase the expression of p66Shc in thyrocytes. If p66Shc protein mediates signaling through TSH receptor and thus activates thyrocyte growth, we may speculate that this increased p66Shc protein in thyroid tumor might accentuate cell growth with relatively normal TSH stimulation.
In summary, we have demonstrated that p66Shc, an adaptor molecule of IGF-I receptor, is up-regulated in proliferating thyroid cells both in vivo and in vitro. TSH or TSAb signals through the TSH receptor-Gs protein-cAMP cascade were found to be responsible for the incrementation or regulation of p66Shc expression, and TSH-induced p66Shc seemed to potentiate the effects of growth factors in thyroid cells. Our results suggest that p66Shc might be an important mediator of the synergism between TSH and IGF-I and that TSH might potentiate the regulatory effect of IGF-I on thyrocyte growth, and that it accomplishes this at least in part, by increasing the expression of p66Shc.
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