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Endocrinology Vol. 141, No. 2 722-730
Copyright © 2000 by The Endocrine Society

ARTICLES

The 3',5'-Cyclic Adenosine Monophosphate Response Element Binding Protein (CREB) Is Functionally Reduced in Human Toxic Thyroid Adenomas1

Antonio Brunetti, Eusebio Chiefari, Sebastiano Filetti and Diego Russo

Cattedra di Endocrinologia (A.B., E.C., S.F.), Dipartimento di Medicina Sperimentale e Clinica, Facolta di Medicina e Chirurgia; and Cattedra di Farmacologia (D.R.), Facolta di Farmacia, Universita degli Studi di Catanzaro, 88100 Catanzaro, Italy

Address all correspondence and requests for reprints to: Sebastiano Filetti, M.D., Cattedra di Endocrinologie, Dipartimento di Medicina Sperimentale e Clinica, via T. Campanella, 88100 Catanzaro, Italy. E-mail: filetti{at}tin.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In human normal thyrocytes, the cAMP-responsive signaling pathway plays a central role in gene regulation, cell proliferation, and differentiation. Constitutive activation of the cAMP signal transduction system has been documented in thyroid autonomously hyperfunctioning adenomas in which activating mutations in either the TSH receptor gene or the Gs{alpha} protein gene (gsp oncogene) have been described. The molecular mechanism whereby cAMP induces thyrocyte proliferation is unknown, but recent evidence suggests that the transcription factor cAMP response element binding protein (CREB) may serve as an important biochemical intermediate in this proliferative response. Herein we have investigated the expression of CREB in normal and tumoral thyroid tissues from a series of ten unrelated patients with autonomously hyperfunctioning adenomas, previously screened for mutations in the TSH receptor and Gs{alpha} genes. In all tumors examined, the expression of the activated, phosphorylated form of CREB was markedly reduced compared with that of the corresponding paired normal thyroid tissue, and this reduction was independent of the presence of mutations in the TSH receptor gene and Gs{alpha} gene. Moreover, no correlation was observed in these tissues between CREB phosphorylation and either protein kinase A activity or protein phosphatase expression. Thus, these data suggest that in human hyperfunctioning thyroid adenomas, the PKA/CREB system does not play a role in cell proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AUTONOMOUSLY hyperfunctioning thyroid adenomas are responsible for 5–30% of cases of thyrotoxicosis and have a higher prevalence in iodine deficient areas. Histologically, they are well encapsulated benign tumors characterized by TSH-independent growth, iodine uptake, and function and by a large spectrum of clinical manifestations, such as weight loss, sweating, nervousness, irritability, fine tremor, fatigue, and tachycardia (1).

Studies indicate that the cAMP signal transduction pathway is essential in mediating signaling in thyroid cells (2). In thyrocytes, intracellular levels of cAMP are regulated primarily by adenylate cyclase, which is in turn modulated by extracellular stimuli mediated by TSH receptor (TSHR) and G proteins at the cell surface (2). While in most cell types cAMP inhibits cell proliferation by interfering with signaling through the mitogen-activated protein kinase pathway (3), in normal thyrocytes TSH activation of its receptor through an increase of cAMP levels stimulates both cell proliferation and differentiation, as demonstrated by in vitro studies, mostly using nonhuman thyroid cell cultures (4). In human thyroid cells, a growth promoting effect of cAMP has not unequivocally demonstrated. Moreover, in thyroid tumors the role of TSHR-adenylate cyclase system is still unclear. Although TSH is considered a growth promoter factor in thyroid tumors, studies indicate that cAMP may act as a growth inhibitor in some human thyroid tumoral cell lines (5, 6, 7, 8, 9). Recently, activating mutations in either the TSHR gene or the Gs{alpha} protein gene (the oncogene gsp), responsible for the constitutive activation of the cAMP signal transduction system, have been documented in hyperfunctioning thyroid adenomas with different frequency (10) and have been proposed to play a role in the etiology of the disease. Constitutively activated cAMP pathway has also been implicated in the formation of human pituitary adenomas, in which both mutant gsp and overexpression of Gs{alpha} protein have been described (11).

The molecular mechanism whereby cAMP stimulates both proliferation and differentiation in human normal thyrocytes, as well as in other endocrine systems, is still unknown. cAMP is known to stimulate the cAMP-dependent protein kinase A (PKA), which in turn phosphorylates cytoplasmic and nuclear target proteins. One of the best characterized PKA substrates is the nuclear transcription factor cAMP response element binding protein (CREB), which stimulates the transcription of cAMP-responsive genes after its phosphorylation by PKA (12, 13). Recent evidence in vitro suggests that CREB is essential for a normal rate of growth of the FRTL-5 thyroid follicular cells (14). Therefore, examination of CREB in thyroid adenomas may represent an important point in the characterization of the molecular mechanisms that are involved in the generation of thyroid tumors. We have investigated CREB phosphorylation in a series of human toxic thyroid adenomas previously examined for the presence of either TSHR or gsp mutations (15, 16). We report herein that CREB phosphorylation is markedly reduced in all thyroid adenomas relative to that in normal thyroid tissues, and this reduction appears to be independent by cAMP/PKA pathway and phosphatase mediated dephosphorylation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
BSA, HEPES, phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, Nonidet P-40, and T4 polynucleotide kinase were obtained from Sigma-Aldrich S.r.l. (Milan, Italy); radioisotopes, nitrocellulose membranes, autoradiography film and enhanced chemiluminescence (ECL) Western blotting detection reagents were from Amersham Italia S.r.l. (Milan, Italy); protein assay reagent and dithiothreitol (DTT) from Bio-Rad Laboratories, Inc. (Segrate, Milan, Italy); colorimetric protein kinase A (PKA) assay kit from Pierce Chemical Co., Prodotti Gianni spa (Milan, Italy); poly(dI-dC) from Pharmacia Biotech Italia (Cologno Monzese, Milan, Italy); antibodies against CREB and CREB-PSer133, and 27-mer double stranded consensus oligonucleotides containing DNA binding sites for CREB transcription factor were purchased from Santa Cruz Biotechnology, Inc. (Segrate, Milan, Italy); antihuman protein phosphatase 1 (PP1) polyclonal antibody and antihuman protein phosphatase 2A (PP2A) monoclonal antibody were from Upstate Biotechnology, Inc. (Segrate, Milan, Italy); consensus oligonucleotide for OCT-1 transcription factor from Promega Corp. (Madison, WI). Basic laboratory procedures were performed according to standard protocols (17) unless otherwise stated.

Patients
Ten unrelated patients with hyperfunctioning thyroid adenomas were studied (Table 1Go). Tissue specimens, obtained at the time of surgery and frozen in liquid nitrogen, were taken from the tumor and from the healthy surrounding tissue. The thyroid nodules were classified as autonomous toxic/hyperfunctioning adenomas on the basis of clinical thyrotoxicosis, elevated serum levels of free thyroid hormones, and undetectable TSH levels, associated with a predominant 131I uptake in the nodule as shown by 131I-scanning. All tumors were classified as adenomas according to conventional pathological criteria (18). This study was approved by the local ethical committee.


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  		Table 1. Clinical and molecular characteristics of patients1

 
Nuclear protein extraction
Nuclei from 100 mg human thyroid were prepared by homogenizing the tissue with a Tekmar homogenizer SD-45K homogenizer (19) in two volumes of lysis buffer (10 mM HEPES pH 7.9, 25 mM KCl, 0.6 M sucrose, 1 mM EDTA, 10% glycerol with 20 µg/ml aprotinin, 1 mM PMSF, 10 mM Na pyrophosphate, 1 mM Na3VO4, and 1 mM NaF). The homogenate was further treated with a glass Dounce (type B), filtered through polypropylene mesh and centrifuged at 14,000 x g at 4 C for 20 min. The crude nuclear pellet was washed twice with two volumes of lysis buffer, resuspended in two volumes of the same buffer (19) and nuclear extracts were prepared following the procedure of Dignam et al., as previously described (20). Equal number of nuclei were homogenized and final protein concentrations in the extracts were determined using the colorimetric assay of Bradford (19). The extraction of nuclear protein (1–2 mg/ml) was the same for all of the samples analyzed. Nuclear extracts were normalized by use of the octamer (OCT-1) nuclear protein, a member of the OCT family that is apparently ubiquitous in mammalian cells (21). Consensus oligonucleotide for OCT-1 transcription factor was 5' end labeled with ({gamma}-32P)ATP and T4 polynucleotide kinase and used for gel retardation assays under conditions suggested by the supplier (Promega Corp.).

Western blot analysis
Ten micrograms of nuclear protein derived from thyroid tissues were separated by SDS-PAGE (10% resolving gel) using a Minigel apparatus (Bio-Rad Laboratories, Inc.), and transferred to nitrocellulose membranes using transfer buffer containing 20% methanol, 25 mM Tris base, and 192 mM glycine and a Mini-transelectrophoretic transfer cell (Bio-Rad Laboratories, Inc.) (120 V, 1 h). After blocking the membranes for 1.5 h at room temperature in PBS solution containing 5% BSA, the membranes were incubated at 4 C overnight in TBS buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 1:5000 dilution of polyclonal anti-CREB antibody specific for the total CREB-protein, or 1:2000 dilution of polyclonal anti-CREB-PSer133 antibody specific for the 43-kDa phosphorylated CREB-protein and washed three times (10 min each time) in TBS with 0.2% Tween-20 (TBS-T). The membranes were then incubated for 1 h at room temperature in TBS containing 1:5000 dilution of goat antirabbit IgG antibody coupled to horseradish peroxidase, followed by two 30 min washings with TBS-T. Immunoreactive bands were visualized by incubation with luminol and exposed to autoradiography film. Quantification was achieved by densitometric scanning.

The same procedure was used for the immunodetection of PP1 and PP2A protein phosphatases in the nuclear extracts from normal and tumoral thyroid tissues.

Measurement of adenylate cyclase and PKA activities
Adenylate cyclase activity in normal and tumoral thyroid tissues was assessed by measuring the amount of (32P)cAMP generated from (32P)ATP, following previously published procedures (22). For the PKA assay, thyroid tissues were washed with cold PBS, and homogenized in a buffer containing 20 mM HEPES pH 7.5, 10 mM EGTA, 40 mM ß-glycerophosphate, 1% Nonidet P-40, 2.5 mM MgCl2, 1 mM dithiothreitol, 2 mM sodium vanadate, 1 mM PMSF, 20 µg/ml aprotinin, and 20 µg/ml leupeptin. The activity of PKA was determined in each sample using a commercial nonradioactive PKA assay Kit (SpinZyme, Pierce Chemical Co.) following the manufacturer’s instructions.

Gel retardation assay
Binding reactions were performed as previously described (20). Briefly, 10 µg of nuclear extracts from both normal and tumoral thyroid tissues were incubated with 2 ng of radiolabeled probe, in the presence of 0.5 µg poly(dI-dC) which was used as competitor DNA for nonspecific DNA binding proteins in the nuclear extracts. After 30 min of incubation at 20 C, reaction products were separated by electrophoresis through a nondenaturing 6% polyacrylamide gel, and free and bound DNA were detected by autoradiography (20).

27-mer double stranded consensus oligonucleotides containing wild-type (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') and mutated (5'-AGAGATTGCC TGTGGTCAGAGAGCTAG-3') cAMP response element (CRE) motif were 5' end labeled with ({gamma}-32P)ATP and T4 polynucleotide kinase and used for gel retardation assays under conditions suggested by the supplier (Santa Cruz Biotechnology, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of OCT-1 nuclear protein in thyroid tissues
In preliminary experiments, nuclear extracts from normal and tumoral thyroid tissues were tested in a gel retardation analysis, using a probe that interacted with the ubiquitous transcription factor OCT-1 nuclear protein (23). This experiment was necessary to verify the quantity of nuclear protein in each nuclear extract preparation. By measuring the concentration of this nuclear protein, nuclear extracts were normalized. Figure 1Go and Table 2Go indicate that OCT-1 nuclear protein is expressed and present in a similar concentration in all of the nuclear extracts used for this study.



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  		Figure 1. Protein-binding activity of consensus oligonucleotides containing DNA binding sites for the OCT-1 nuclear protein. Nuclear extracts from thyroid tissues of either normal (n = 10) or tumoral (n = 10) specimens were incubated with radiolabeled probe and analyzed by gel retardation assay. Arrows show the position of free (DNA) and bound (DNA-P) probe. A representative of three separated assays is shown.

 

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  		Table 2. Protein-binding activity of consensus oligonucleotide containing DNA binding site for the OCT-1 nuclear protein

 
Detection of phosphorylated and unphosphorylated CREB-protein
In initial experiments, Western blot analyses were carried out with nuclear extracts from the pheochromocytoma cell line PC12. In these cells, CREB becomes newly phosphorylated on Ser133 when the cells are stimulated with forskolin to activate adenylate cyclase (24). Anti-PCREB did not recognize CREB in extracts of unstimulated cells but did specifically recognize CREB present in PC12 cells minutes after treatment with forskolin. In particular, a major band of PCREB that migrated with the 43K molecular weight marker was revealed (data not shown).

We next performed Western blot analyses of nuclear extracts of thyroid tissues from patients with thyroid adenomas, in either the absence or presence of THSR and/or Gsp mutations. CREB-protein was detected by either an anti-CREB antibody, specific for the total CREB-protein, or an anti-PCREB antibody specific for the 43-kDa phosphorylated CREB-protein. Anti-CREB, which recognizes CREB regardless of the phosphorylation state of Ser133, revealed comparable amounts of the 43-kDa CREB protein in extracts of both normal and tumoral thyroid tissues (Fig. 2AGo). Western blot analysis of phosphorylated form of CREB revealed that PCREB content was significantly lower in tumoral thyroid tissue compared with that of the surrounding normal tissue, indicating that CREB phosphorylation was specifically reduced in thyroid adenomas (Fig. 2BGo). Levels of PCREB protein in thyroid tumors were approximately 70% less than that of normal thyroid (Table 3Go). In addition to recognizing CREB, anti-PCREB detected one other (lower) band in thyroid extracts that may represent a phosphorylated form of CREB-related protein which shares the same antigenic motif in the region that includes Ser133. Like the 43-kDa PCREB, it was markedly reduced in nuclear extracts from thyroid adenomas. No correlation was observed between CREB protein levels and the presence of either TSHR or gsp mutations.



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  		Figure 2. Levels of PCREB are reduced in human thyroid adenomas compared with normal thyroid. A, Western blot analysis of nuclear extracts from normal and tumoral tissues, using a polyclonal anti-CREB antibody specific for total CREB protein. B, Western blots of nuclear extracts using a polyclonal anti-PCREB antibody specific for phospho-CREB protein. The 43-kDa product represents full-length PCREB protein. A representative of three separated assays is shown. N, Normal thyroid tissue (n = 10); T, tumoral thyroid tissue (n = 10).

 

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  		Table 3. CREB protein levels and DNA-binding activity to CREB oligonucleotide in normal and tumoral thyroid tissues

 
Detection of adenylate cyclase and PKA activities in normal and tumoral thyroid tissues
The importance of the adenylate cyclase in the regulation of intracellular levels of cAMP as well as the crucial role played by PKA in the activation of CREB prompted us to determine whether the reduced phosphorylation of CREB observed in thyroid adenomas reflected a reduced adenylate cyclase and/or PKA activities in these tissues. In accordance with the reports of others (25), adenylate cyclase activity was only slightly higher in all toxic adenomas when compared with that of paired normal thyroid tissues (Fig. 3AGo), whereas there were no measurable differences in PKA activity in normal and tumoral tissues (Fig. 3BGo).



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  		Figure 3. Adenylate cyclase and PKA activities in normal and tumoral thyroid tissues. A, Adenylate cyclase activity. Activity of adenylate cyclase was performed by measuring the amount of (32P)cAMP generated from (32P)ATP, following previously published procedures (22 ). Each value represents the mean of at least two determinations in triplicate. B, PKA activity. Activity of PKA was measured by spectrophotometric analysis of the samples (see methods). Results are the mean ± SEM for three separated assays. , Normal thyroid tissue (n = 10); , Tumoral thyroid tissue (n = 10).

 
Detection of PP2A protein phosphatase expression in normal and tumoral thyroid tissues
CREB is known to be dephosphorylated by PP1 and/or PP2A protein phosphatases (26, 27). We measured PP2A protein expression in nuclear extracts. As detected by Western blot analysis, comparable amounts of the 36-kDa PP2A protein were observed in both normal and tumoral thyroid tissues (Fig. 4Go, Table 4Go). Similar results were obtained with the 34-kDa PP1 protein (data not shown).



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  		Figure 4. Levels of PP2A protein phosphatase in normal and tumoral thyroid tissues. Western blot analyses were carried out with nuclear extracts from thyroid tissues using a mouse monoclonal antibody (1:2000 dilution) specific for the human protein phosphatase 2A (PP2A). The 36-kDa PP2A protein is shown. A representative of three separated assays is shown. N, Normal thyroid tissue (n = 10); T, tumoral thyroid tissue (n = 10).

 

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  		Table 4. PP2A protein phosphatase levels in normal and tumoral thyroid tissues

 
Interaction of nuclear proteins from thyroid tissue with CREB consensus oligonucleotide
Nuclear extracts from thyroid tissue were incubated with 32P-labeled CREB consensus oligonucleotide and tested for the presence of specific DNA-binding factors using a gel retardation assay (20). In initial experiments using normal thyroid tissue, 10 µg of nuclear extracts were incubated with labeled consensus oligonucleotide, in the presence of increasing amounts of the synthetic polynucleotide poly(dI-dC) (which was used as competitor DNA for non specific DNA binding proteins in the nuclear extracts). In vitro DNA-binding activity of nuclear extracts was supported by the detection of a retarded DNA-protein complex (Fig. 5Go).



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  		Figure 5. Effect of the polynucleotide poly(dI-dC) on gel retardation assays with human thyroid nuclear extracts, using 27-bp CREB consensus oligonucleotide as probe. End-labeled CREB probe was incubated with 10 µg of nuclear extracts from normal thyroid tissues in the presence of increasing amounts of poly(dI-dC) (0.1–20 µg) and DNA protein complexes were resolved on a nondenaturing 6% polyacrylamide gel. Arrows show the position of the DNA protein complexes.

 
DNA binding activity to CREB consensus oligonucleotide in normal and tumoral thyroid tissues
We next determined binding of 32P-labeled CREB consensus oligonucleotide to nuclear proteins from normal and tumoral thyroid tissues. In tumoral tissue, DNA binding activity of nuclear extracts was significantly reduced (Figs. 6Go and 7Go). Laser densitometric scanning of the autoradiograms revealed that nuclear extracts from thyroid adenomas had approximately 30% of DNA binding activity when compared with that of the surrounding normal thyroid tissue (Table 3Go). The decrease in DNA binding activity in nuclear extracts from thyroid adenomas paralleled the decrease in PCREB protein abundance as detected by Western blot analysis. This reduction did not reflect the levels of adenylate cyclase activity observed in all tumoral thyroid tissues examined, suggesting that inhibition of DNA binding of CREB was unlikely due to increased expression of ICERs (inducible cAMP early repressors) that can be up-regulated, as part of feedback loop, by cAMP (28).



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  		Figure 6. Protein binding activity of CREB DNA to nuclear extracts from thyroid tissues. CREB DNA was labeled, incubated in the presence of 0.5 µg poly(dI-dC) with nuclear extracts from normal and tumoral thyroid tissues, and DNA protein complexes were resolved as in Fig. 5Go. Lanes: C, probe alone; N, probe plus nuclear extract from normal thyroid tissue; T, probe plus nuclear extract from tumoral thyroid tissue. Arrows show the position of free (DNA) and bound (DNA-P) probe. A representative of three separated assays from each patient is shown.

 


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  		Figure 7. Competition for binding between thyroid nuclear proteins and CREB consensus oligonucleotide. CREB consensus oligonucleotide was 5' end-labeled and used as probe in gel retardation assays with 10 µg of extracts from thyroid tissues under the same conditions as in Fig. 6Go. Specificity of DNA-protein binding was determined by using either a 50-fold molar excess of unlabeled CREB consensus oligonucleotide, or a labeled CREB mutant oligonucleotide. Lanes: 1, probe alone; 2, probe plus nuclear extract from tumoral thyroid tissue; 3, probe plus nuclear extract from normal thyroid tissue; 4, probe plus nuclear extract from normal thyroid tissue in the presence of a 50-fold molar excess of unlabeled CREB consensus oligonucleotide. In lane 5, nuclear extract from normal thyroid tissue were incubated with a CREB mutant oligonucleotide as probe. In lane 6, probe plus nuclear extract from normal thyroid tissue were incubated in the presence of a specific anti-CREB antibody, able to induce a supershift of the complex. Arrows show the position of free (DNA) and bound (DNA-P) probes.

 
Specificity of DNA-protein binding
To determine specificity of DNA-protein binding, competition assays were performed. The binding of thyrocyte nuclear proteins to 32P-labeled CREB consensus oligonucleotide was almost abolished when a 50-fold excess of unlabeled CREB consensus oligonucleotide was included in the binding reaction (Fig. 7Go). Moreover, DNA binding activity of nuclear extracts was prevented by using a 27-mer double stranded synthetic oligonucleotide probe containing a mutated CRE motif (Fig. 7Go).

To confirm that PCREB protein was specifically reduced in thyroid adenomas, we next determined binding of a 27-mer double stranded synthetic oligonucleotide containing DNA binding site for TTF-1 transcription factor (29) to nuclear proteins from normal and tumoral thyroid tissues. As detected by gel retardation assays, nuclear extracts from tumor tissue specimens demonstrated 5- to 10-fold enhanced DNA-binding activity when compared with that of the surrounding normal tissue (Fig. 8Go).



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  		Figure 8. Protein binding activity of TTF-1 DNA to nuclear extracts from thyroid tissues. TTF-1 DNA was labeled, incubated with nuclear extracts from normal (N) and tumoral (T) thyroid tissues, and DNA protein complexes were resolved as in Fig. 6Go. Arrows show the position of free (DNA) and bound (DNA-P) probe. The ubiquitous protein UFA (30 ) migrating below the TTF-1/DNA complex is visible. A representative of three separated assays from each patient is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Based on a variety of in vitro data, mostly obtained with nonhuman thyroid cells, cAMP is considered to play a key role as a second messenger of TSH in stimulating both thyrocyte proliferation and differentiation (2). Therefore, it has been postulated that in thyroid tumors, at least in the more differentiated ones, an alteration in cAMP signal transduction could participate in the development of the tumorigenic process. This alteration may be the consequence of mutations or other defects present in some of the genes participating in cAMP pathway. Somatic activating mutations of TSHR and Gs{alpha} genes have been recently described in autonomously hyperfunctioning thyroid adenomas, in which a constitutive activation of the cAMP cascade has been demonstrated (10, 31). However, although the constitutive activation of the cAMP cascade has been experimentally shown to cause increased proliferation of thyroid cells in vitro (32) and in vivo (33, 34), studies indicate that this mechanism may be alone not sufficient to generate toxic thyroid adenomas (35), and far more complex mechanisms may be effective in the pathogenesis of these tumors.

The importance of the nuclear regulatory protein CREB in mediating cAMP-dependent proliferative signals in thyroid cells has been recently documented, and recent evidence suggests that CREB may serve as an important biochemical intermediate in this proliferative response (14). In this study, we have investigated the expression of CREB in a series of ten thyroid hyperfunctioning adenomas previously screened for the presence of TSHR and Gs{alpha} protein gene alterations. By using Western blot analysis, we measured the content of CREB protein in thyroid tumors. We found that in all toxic adenomas examined the amounts of PCREB protein were 3- to 4-fold lower than that observed in nuclear extracts of the surrounding normal tissue.

As a step toward understanding the molecular basis of the impaired CREB phosphorylation observed in thyroid adenomas, we investigated whether this defect was accompanied by an altered adenylate cyclase and/or PKA activity and/or protein phosphatase expression in these tissues. While the adenylate cyclase activity was significantly higher in all toxic adenomas compared with normal tissues, no significant differences in PKA activity were found. The observed discrepancy between adenylate cyclase activity and PKA activity may be related to the activation of cyclic nucleotide phosphodiesterases in tumoral tissues with persistent activation of adenylate cyclase. In this regard, increase in phosphodiesterase activity following the activation of the adenylate cyclase has been described either in vitro, in the rat thyroid cell line FRTL-5 expressing a constitutively active Gs{alpha} protein, or in vivo in human thyroid tissue (Clark, personal communication) (32). It has been proposed that the occurring phosphodiesterase feedback loop may counteract the effects in the abnormal growth induced by the constitutive activation of adenylate cyclase (37, 38). Moreover, we have data showing that in toxic thyroid adenomas phosphodiesterase activity is higher in tumoral tissue compared with that of the adjacent normal tissue. Discrepancy between adenylate cyclase and PKA activities has also been described in human brain tissue, where impaired G protein-stimulated adenylate cyclase activity is not accompanied by reduced cAMP-dependent PKA activity (39). On the other hand, our data concerning protein phosphatase expression indicate that the decreased phosphorylation of CREB in thyroid adenomas is not affected by dephosphorylation process. These findings indicate, therefore, that the reduced phosphorylation of CREB in thyroid adenomas does not reflect a reduced level of adenylate cyclase and/or PKA activities and suggest that CREB phosphorylation can be modulated by molecular mechanisms apparently independent by cAMP/PKA pathway (40). For example, phosphorylation of CREB through mechanisms independent of cAMP and PKA has been reported in primary cell cultures from ovine pars tuberalis (41), and in the murine B lymphoma cell line BAL-17, in which it has been shown that CREB Ser133 phosphorylation occurs apparently without the intervention of PKA, and CREB responds poorly to increased levels of cAMP (42). In addition, the existence of cAMP-dependent/PKA-independent transcriptional activation pathways, (with effects on differentiation and/or proliferation markers), has been postulated in two recent studies, in which the exchange protein directly activated by cAMP (Epac) is able to activate Rap 1 protein in a cAMP-dependent but PKA-independent manner (43, 44), and the NIS upstream enhancer (NUE) in the 5' flanking region of the rat sodium iodide symporter (NIS) gene, mediates the cAMP-dependent transcription through a novel PKA-independent mechanism involving the thyroid specific factor Pax-8 (45).

There is some controversy in the field concerning whether phosphorylation at Ser133 is necessary for transcriptional activation of CREB and its DNA binding activity (26, 46). In our study, we tested nuclear extracts from normal and tumoral thyroid tissues for their binding to consensus oligonucleotides containing DNA binding sites for the regulatory protein CREB. Gel retardation analysis revealed that DNA binding of PCREB was significantly reduced in nuclear extracts obtained from tumoral thyroid tissues, and this reduction paralleled the decrease in PCREB protein levels as detected by Western blot analysis. Therefore, these data indicate that in human hyperfunctioning thyroid adenomas cell proliferation does not correlate with the activation of the cAMP/CREB pathway, suggesting the existence of other transforming events perhaps not dependent on the CREB family, as reported in BALB/c3T3 fibroblast cells, in which CREB had no effect on cell growth, either in the presence or absence of elevated cAMP (14). The possibility of cross-talk among intracellular signaling pathways at a level before CREB phosphorylation has been postulated (42). Reduced amounts of CREB has been recently described also in proliferating, activated hepatic stellate cells (47).

Our data are in contrast with the results previously described in human GH-secreting tumors (11). In this study, CREB phosphorylation was elevated in pituitary adenomas expressing the mutant gsp oncogene, and in tumors with overexpression of Gs{alpha} protein relative to nonfunctioning adenomas. Instead, in our work, levels of CREB have been studied in thyroid samples obtained from the tumor and from the healthy surrounding tissue, and no correlations were observed between CREB levels and the mutation of either TSHR or gsp.

We believe that this is the first report describing a quantitative abnormality in CREB phosphorylation in thyroid tissues of human origin. The mechanism for this effect is still unexplained, and it is possible that reduced levels of PCREB may occur for differences in the cell cycle kinetics between the adenoma and the normal tissue as observed in dog thyroid cells (28). Studies aimed at better defining the mechanism(s) by which functionally reduced CREB is related to the process of thyroid tumorigenesis are in progress.


   Acknowledgments
 
We thank Dr. F. Arturi for his collaboration in overviewing the clinical data of the patients and Prof. G. Damante for providing the consensus oligonucleotide containing DNA binding site for TTF-1 nuclear protein.


   Footnotes
 
1 This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro and MURST (to S.F.). Back

Received July 15, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hay ID, Morris JC 1991 Autonomously functioning thyroid nodules. In: Braverman LE, Utiger RD (eds) The Thyroid, A Fundamental and Clinical Text. JB Lippincott Company, Philadelphia, pp 698–704
  2. Dumont JE, Lamy F, Roger P, Maenhaut C 1992 Physiological and pathological regulation of thyroid cell proliferation and differentiation by thyrotropin and other factors. Physiol Rev 72:667–697[Free Full Text]
  3. Wu J, Dent P, Jelinek T, Wolfman A, Weber M, Sturgill T 1993 Inhibition of the EGF-activated MAP kinase signalling pathway by adenosine 3',5' monophosphate. Science 262:1065–1069[Abstract/Free Full Text]
  4. Dremier S, Coppée F, Delange F, Vassart G, Dumont JE, Van Sande J 1996 Thyroid autonomy: mechanism and clinical effects. J Clin Endocrinol Metab 81:4187–4192[CrossRef][Medline]
  5. Helding NE, Cvejic D, Smeds S, Westermark B 1991 Expression of functionally active receptors for thyrotropin and platelet-derived growth factor in human thyroid carcinoma cells. Endocrinology 129:2187–2193[Abstract/Free Full Text]
  6. Kimura H, Yamashita S, Namba H, Tominaga T, Tsuruta M, Yokoyama N, Izumi M, Nagataki S 1992 Interleukin-1 inhibits human thyroid carcinoma cell growth. J Clin Endocrinol Metab 75:596–602[Abstract]
  7. Derwahl M, Kuemmel M, Goretzki P, Schatz H, Broecker M 1993 Expression of the human thyroid carcinoma cell line that lacks an endogenous TSH receptor: growth inhibition by cAMP. Biochem Biophys Res Commun 191:1131–1138[CrossRef][Medline]
  8. Hishinuma A, Yamanaka T, Kasai T, So S, Bamba N, Shimoda S 1994 Growth regulation of the human thyroid cancer cell line by protein tyrosine kinase and cAMP-dependent protein kinase. Endocr J 41:399–407[Medline]
  9. Ohta K, Pang XP, Berg L, Hershman JM 1997 Growth inhibition of new human thyroid carcinoma cell lines by activation of adenylate cyclase through the ß-adrenergic receptor. J Clin Endocrinol Metab 82:2633–2638[Abstract/Free Full Text]
  10. Russo D, Arturi F, Chiefari E, Filetti S 1997 Molecular insights into TSH receptor abnormality and thyroid disease. J Endocrinol Invest 20:36–47[Medline]
  11. Bertherat J, Chanson P, Montminy M 1995 The cyclic adenosine 3',5'-monophosphate-responsive factor CREB is constitutively activated in human somatotroph adenomas. Mol Endocrinol 7:777–783
  12. Della Fazia MA, Servillo G, Sassone-Corsi P 1997 Cyclic AMP signalling and cellular proliferation: regulation of CREB and CREM. FEBS Lett 410:22–24[CrossRef][Medline]
  13. Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675–680[CrossRef][Medline]
  14. Woloshin PI, Walton KM, Rehfuss RP, Goodman RH, Cone RD 1992 3',5'-cyclic adenosine monophosphate-regulated enhancer binding (CREB) activity is required for normal growth and differentiated phenotype in the FRTL5 thyroid follicular cell line. Mol Endocrinol 6:1725–1733[Abstract/Free Full Text]
  15. Russo D, Arturi F, Wicker R, Chazenbalk GD, Schlumberger M, DuVillard JAD, Caillou B, Monier M, Rapoport B, Filetti S, Suarez HG 1995 Genetic alterations in thyroid hyperfunctioning adenomas. J Clin Endocrinol Metab 80:1347–1351[Abstract]
  16. Russo D, Arturi F, Suarez HG, Schlumberger M, DuVillard JAD, Crocetti U, Filetti S 1996 Thyrotropin receptor gene alterations in thyroid hyperfunctioning adenomas. J Clin Endocrinol Metab 81:1548–1551[Abstract]
  17. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  18. Hedinger C, Williams ED, Sabin LH 1989 The WHO histological classification of thyroid tumors: a commentary on the second edition. Cancer 63:908–911[CrossRef][Medline]
  19. Yamamoto KK, Gonzalez GA, Biggs III WH, Montminy M 1988 Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature 334:494–498[CrossRef][Medline]
  20. Brunetti A, Foti D, Goldfine ID 1993 Identification of unique nuclear regulatory proteins for the insulin receptor gene, which appear during myocyte and adipocyte differentiation. J Clin Invest 92:1288–1295
  21. Brunetti A, Brunetti L, Foti D, Accili D, Goldfine ID 1996 Human diabetes associated with defects in nuclear regulatory proteins for the insulin receptor gene. J Clin Invest 97:258–262[Medline]
  22. Derwahl M, Hamacher C, Russo D, Broecker M, Manole D, Schatz H, Kopp P, Filetti S 1996 Constitutive activation of the Gs-{alpha} protein-adenylate cyclase pathway may not be sufficient to generate toxic thyroid adenomas. J Clin Endocrinol Metab 81:1898–1904[Abstract]
  23. O’Neill EA, Fletcher C, Burrow CR, Hentz N, Roeder RG, Kelly TJ 1988 Transcription factor OTF-1 is functionally identical to the DNA replication factor NF-III. Science 241:1210–1213[Abstract/Free Full Text]
  24. Ginty DD, Kornhauser JM, Thompson MA, Bading H, Mayo KE, Takahashi JS, Greenberg ME 1993 Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260:238–241[Abstract/Free Full Text]
  25. O’Sullivan C, Barton CM, Staddon SL, Brown CL, Lemoine NR 1991 Activating point mutations of the gsp oncogene in human thyroid adenomas. Mol Carcinog 4:345–349[Medline]
  26. Montminy M 1997 Transcriptional regulation by cyclic AMP. Annu Rev Biochem 66:807–822[CrossRef][Medline]
  27. Wadzinski BE, Wheat WH, Jaspers S, Peruski Jr LF, Lickteig LR, Johnson GL, Klemm DJ 1993 Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 13:2822–2834[Abstract/Free Full Text]
  28. Uyttersprot N, Costagliola S, Dumont JE, Miot F 1999 Requirement for cAMP-response element (CRE) binding protein/CRE modulator transcription factors in thyrotropin-induced proliferation of dog thyroid cells in primary culture. Eur J Biochem 259:370–378[Medline]
  29. Damante G, Di Lauro R 1994 Thyroid gene expression. Biochim Biophys Acta 1218:255–266[Medline]
  30. Acebron A, Aza-Blanc P, Rossi DL, Lamas L, Santisteban P 1995 Congenital human thyroglobulin defect due to low expression of the thyroid-specific transcription factor TTF-1. J Clin Invest 96:781–785
  31. Poertl S, Kirner J, Mann K, Hoermann R 1996 Enhanced functional activity in thyroid adenomas in vitro. Exp Clin Endocrinol Diabetes [Suppl 4] 104:39–40
  32. Porcellini A, Ruggiano G, Pannain S, Ciullo I, Amabile G, Fenzi G, Avvedimento EV 1997 Mutations of thyrotropin receptor isolated from thyroid autonomous functioning adenomas confer TSH-independent growth to thyroid cells. Oncogene 15:781–789[CrossRef][Medline]
  33. Ledent C, Dumont JE, Vassart G, Parmentier M 1992 Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism. EMBO J 11:537–542[Medline]
  34. Michiels F, Caillou B, Talbot M, Dessarps-Freichey F, Maunaury M, Schlumberger M, Merken L, Monier R 1994 Oncogenic potential of guanine nucleotide stimulatory factor {alpha} subunit in thyroid gland of transgenic mice. Proc Natl Acad Sci USA 91:10488–10492[Abstract/Free Full Text]
  35. Derwahl M 1996 TSH receptor and Gs-{alpha} gene mutations in the pathogenesis of toxic thyroid adenomas - a note of caution. J Clin Endocrinol Metab 81:2783–2785[Abstract/Free Full Text]
  36. Nemoz G, Sette C, Hess M, Muca C, Vallar L, Conti M 1995 Activation of cyclic nucleotide phosphodiesterases in FRTL-5 thyroid cells expressing a constitutively active Gs{alpha}. Mol Endocrinol 9:1279–1287[Abstract/Free Full Text]
  37. Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM 1991 Activating mutations of the stimulatory G protein in the McCune-Albright syndrome. N Engl J Med 325:1688–1695[Abstract]
  38. Schwindinger WF, Francomano CA, Levine MA 1992 Identification of a mutation in the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase in McCune-Albright syndrome. Proc Natl Acad Sci USA 89:5152–5156[Abstract/Free Full Text]
  39. Bonkale WL, Fastbom J, Wiehager B, Ravid R, Winblad B, Cowburn RF 1996 Impaired G-protein-stimulated adenylyl cyclase activity in Alzheimer’s disease brain is not accompanied by reduced cyclic-AMP-dependent protein kinase A activity. Brain Res 737:155–161[CrossRef][Medline]
  40. Armstrong R, Wen W, Meinkoth J, Taylor S, Montminy M 1995 A refractory phase in cyclic AMP-responsive transcription requires down regulation of protein kinase A. Mol Cell Biol 15:1826–1832[Abstract]
  41. McNulty S, Ross AW, Shiu KY, Morgan PJ, Hastings MH 1996 Phosphorylation of CREB in ovine pars tuberalis is regulated both by cyclic AMP-dependent and cyclic AMP-independent mechanisms. J Neuroendocrinol 8:635–645[CrossRef][Medline]
  42. Xie H, Wang Z, Rothstein TL 1996 Signaling pathways for antigen receptor-mediated induction of transcription factor CREB in B lymphocytes. Cell Immunol 169:264–270[CrossRef][Medline]
  43. Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM 1998 A family of cAMP-binding proteins that directly activate Rap1. Science 282:2275–2279[Abstract/Free Full Text]
  44. deRooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL 1998 Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396:474–477[CrossRef][Medline]
  45. Ohno M, Zannini M, Levy O, Carrasco N, Di Lauro R 1999 The paired-domain transcription factor Pax8 binds to the upstream enhancer of the rat sodium/iodide symporter gene and participates in both thyroid-specific and cyclic-AMP-dependent transcription. Mol Cell Biol 19:2051–2060[Abstract/Free Full Text]
  46. Schmid W, Nitsch D, Boshart M, Schutz G 1993 Role of cyclic AMP in the control of cell-specific gene expression. Trends Endocrinol Metab 4:204–209[CrossRef][Medline]
  47. Houglum K, Kwan SL, Chojkier M 1997 Proliferation of hepatic stellate cells is inhibited by phosphorylation of CREB on serine 133. J Clin Invest 99:1322–1328[Medline]



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