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Molecular Endocrinology, doi:10.1210/me.2006-0328
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Molecular Endocrinology 21 (4): 921-932
Copyright © 2007 by The Endocrine Society

Reciprocal Negative Regulation between Thyrotropin/3',5'-Cyclic Adenosine Monophosphate-Mediated Proliferation and Caveolin-1 Expression in Human and Murine Thyrocytes

Maria José Costa, Maximin Senou, Fabienne Van Rode, Jean Ruf, Matteo Capello, Didier Dequanter, Philippe Lothaire, Chantal Dessy, Jacques Emile Dumont, Marie-Christine Many and Jacqueline Van Sande

Institut de Recherche Interdisciplinaire (M.J.C., F.V.R., J.E.D., J.V.S.), Faculté de Médecine, Université Libre de Bruxelles, 1070 Brussels, Belgium; Faculté de Médecine (M.S., C.D., M.-C.M.), Université Catholique de Louvain, 1200 Brussels, Belgium; Institut National de la Santé et de la Recherche Médicale Unité 555 (J.R.), Faculté de Médecine Timone, Université de la Méditerranée, 13284 Marseille, France; Hôpital Erasme (M.C.), 1070 Brussels, Belgium; and Institut Jules Bordet (D.D., P.L.), 1000 Brussels, Belgium

Address all correspondence and requests for reprints to: Jacqueline Van Sande, Institut de Recherche Interdisciplinaire, Campus Erasme, Université Libre de Bruxelles, 808 Route de Lennik, Building C, 1070 Brussels, Belgium. E-mail: jvsande{at}ulb.ac.be.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The expression of caveolins is down-regulated in tissue samples of human thyroid autonomous adenomas and in the animal model of this disease. Because several cell types present in thyroid express caveolins, it remained unclear if this down-regulation occurs in thyrocytes and which are the mechanism and role of this down-regulation in the tumor context. Here we show that prolonged stimulation of isolated human thyrocytes by TSH/cAMP/cAMP-dependent protein kinase inhibits caveolins’ expression. The expression of caveolins is not down-regulated by activators of other signaling pathways relevant to thyroid growth/function. Therefore, the down-regulation of caveolins’ expression in autonomous adenomas is a direct consequence of the chronic activation of the TSH/cAMP pathway in thyrocytes. The down-regulation of caveolin-1 occurs at the mRNA level, with a consequent protein decrease. TSH/cAMP induces a transcription-dependent, translation-independent destabilization of the caveolin-1 mRNA. This effect is correlated to the known proliferative role of that cascade in thyrocytes. In vivo, thyrocytes of caveolin-1 knockout mice display enhanced proliferation. This demonstrates, for the first time, the in vivo significance of the specific caveolin-1 down-regulation by one mitogenic cascade and its relation to a human disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE CAVEOLAR PROTEIN caveolin-1 (Cav-1) acts through its scaffolding domain as a negative modulator of several signal transduction proteins that can activate cell proliferation: G proteins (1), Src-like tyrosine kinases (2), tyrosine kinase receptors, cAMP-dependent protein kinase (PKA) (3, 4), and endothelial NO synthase (5). Conversely, activation of Neu/epidermal growth factor receptor-Ras-p44/42-MAPK cascade decreases Cav-1 expression (6, 7, 8).

Cav-1 and Cav-2 are down-regulated in sporadic follicular thyroid carcinoma (9) and in other tumors (10). In thyroid, this down-regulation is often associated with a loss of heterozygosity. Therefore, Aldred et al. (11) suggested that Cav-1 and Cav-2 may be tumor suppressor genes in follicular thyroid carcinoma.

Cav-1 is also down-regulated in human thyroid autonomous adenomas (12), as well as in the animal model of this pathology: the thyroid of adenosine A2-receptor mice (13). However, because both studies were conducted in whole thyroids, and because Cav-1 is expressed in thyrocytes (14), but also in fibroblasts and endothelial cells, it remained unclear whether the down-regulation in autonomous adenomas indeed takes place in the thyrocytes and which are its consequences for the tumor physiology. Autonomous adenomas are caused by a chronic stimulation of thyrocytes by cAMP, due most often to constitutively activating mutations of the TSH receptor (15) or of the {alpha}-subunit of the downstream Gs protein.

In the present work, we mimicked the prolonged cAMP stimulation in primary cultures of pure human thyrocytes, a good in vitro model of autonomous adenomas (16) and showed that the down-regulation of Cav-1 expression is a direct effect of the TSH-cAMP-PKA pathway, through a mechanism depending on RNA, but not protein, synthesis. Using Cav-1 knockout (Cav-1–/–) mice, we obtained evidence that shutdown of Cav-1 increases proliferation of thyrocytes, which demonstrates in vivo the physiological role of the TSH-induced down-regulation of Cav-1 expression in human thyrocytes. To our knowledge, this work constitutes the first in vitro study of regulation of caveolins’ expression in human cells. It also brings an explanation of the mechanism and role of the decreased caveolins’ expression in thyroid autonomous adenomas.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Prolonged TSH Stimulation Down-Regulates Cav-1 Protein Expression in Human Thyrocytes
The serum-free medium used in primary cultures of human thyrocytes does not support the adherence of fibroblasts and endothelial cells, which, in any case, are mostly eliminated in the preparation of thyroid follicles (17). This in vitro system, constituted of pure thyrocytes, is thus a good model with which to study specific Cav-1 expression. To mimic the chronic activation of TSH receptor in autonomous adenomas, thyrocytes were incubated with TSH for 4 d. This led to a decrease in Cav-1 protein expression levels, as assessed by immunoblot and in situ cell immunolabeling (Fig. 1Go, A and B). We checked the specificity of the bovine TSH effect on Cav-1 protein levels by comparing it to the action of the recombinant human TSH used clinically; it also induced a down-regulation of Cav-1 (Fig. 1Go, A and B). The well-characterized up-regulation of thyroperoxidase (TPO) in response to the TSH-cAMP cascade was used as a positive control in all these experiments (18). In contrast, the expression of Adaptin-ß, a protein involved in clathrin-dependent endocytosis, was not regulated by TSH (Fig. 1AGo). A down-regulation (50%) of Cav-1 expression was found 12–24 h after the beginning of stimulation (Fig. 1CGo).


Figure 1
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  		Fig. 1. TPO, Cav-1, and Cav-2 Protein Expression Levels in Human Thyrocytes in Response to Stimulation with TSH Was Measured by the Following Methods

A, Immunoblot with 3 µg total protein for Cav-1 and TPO, 10 µg protein for Cav-2 and Adaptin-ß. B, In situ cell immunolabeling; results (means ± SD, n = 4) are a ratio of RLU/sec of TPO, Cav-1, or Cav-2 to the RLU/sec of Adaptin-ß (the expression of which is not regulated by TSH, therefore providing a control for the cell amount). C, Kinetics of Cav-1 protein down-regulation mediated by bovine TSH and measured by immunoblot. All incubations started at the same moment (time zero) and lasted for different times. Graph, The OD of each immunoblot band was quantified and compared with time zero. Solid line on the graph indicates 50% of Cav-1 protein levels present at time zero. Adaptin-ß expression is shown as a protein loading control.

 
Cav-2 Protein Levels Are Also Decreased by TSH in Human Thyrocytes
Cav-2 is generally coexpressed and colocalized with Cav-1 (19), and it has been proposed that the Cav-2 protein expression needs the presence of Cav-1 protein to be stabilized through the formation of heterooligomers (20). Cav-2 protein is expressed in human thyrocytes, and its levels are also decreased upon prolonged stimulation by TSH (Fig. 1Go, A and B).

The Down-Regulation of Cav-1 and Cav-2 Protein Expression by TSH Is Mediated by cAMP
Autonomous adenomas are characterized by a chronic activation of the cAMP cascade. Therefore, we assessed whether forskolin (FSK), a direct activator of adenylate cyclase, could mimic the effects of TSH. FSK at concentrations of 3, 10, or 50 µM down-regulated Cav-1 and Cav-2 protein levels in a concentration-dependent way (Fig. 2Go, A and B). Within the same experiment, at the concentrations used, TSH drove a stronger down-regulation of Cav-1 expression than FSK (Fig. 2CGo, immunoblot and left graph), which was paralleled by higher intracellular cAMP levels induced by TSH (Fig. 2CGo, right graph). These results suggest that Cav-1 and Cav-2 expression levels are an inverse function of the cAMP concentration.


Figure 2
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  		Fig. 2. TPO, Cav-1, and Cav-2 Protein Expression Levels in Human Thyrocytes after 4 d Stimulation with Various Concentrations of FSK or TSH

A, Immunoblot of Cav-1, TPO (3 µg protein), Cav-2, and Adaptin-ß (10 µg protein). B, In situ cell immunolabeling; results (means ± SD, n = 4) are a ratio of RLU/sec of TPO, Cav-1, or Cav-2 to the RLU/sec of Adaptin-ß (the expression of which is not regulated by TSH-cAMP, therefore providing a control for the cell amount). C (Left panel), Immunoblot with 3 µg total protein, showing the expression levels of Cav-1 in human thyrocytes stimulated with various TSH and FSK concentrations. The OD of each band was quantified and plotted. C (Right panel), Quantification of the cAMP levels (means ± SD, n = 2) generated by those stimulations.

 
Down-Regulation of Cav-1 and Cav-2 by TSH-cAMP Is Dependent on Protein Kinase A
In thyroid, PKA does not account for all the cAMP-mediated effects (21). cAMP is also able to activate EPAC (exchange nucleotide protein activated by cAMP) (22). To identify the downstream cAMP target involved in the regulation of Cav-1 and Cav-2 expression, cells were incubated with cAMP analogs that specifically activate PKA [N6-monobutyryl-cAMP (MB-cAMP)] or EPAC [8-(4-chlorophenylthio)-2'-O-methyl-cAMP (CPT-cAMP)]. The specificity of the action of these cAMP analogs has been demonstrated before (23). Only MB-cAMP down-regulated Cav-1 and Cav-2 protein expression, suggesting that PKA is the target of cAMP mediating the down-regulation of caveolins’ expression (Fig. 3Go, A and B). As expected, MB-cAMP also increased TPO protein levels (24). A PKA inhibitor (H89) partially blocked the effect of TSH on Cav-1 and Cav-2 expression, further supporting a role for PKA as the mediator of cAMP on the down-regulation of caveolins (Fig. 3CGo).


Figure 3
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  		Fig. 3. TPO, Cav-1, and Cav-2 Protein Expression Levels in Human Thyrocytes after Incubation for 4 d in the Presence of cAMP Analogs that Stimulate PKA (500 µM MB-cAMP) or EPAC (20 µM CPT-cAMP) (A and B), and of an Inhibitor of PKA (H89, 10 µM) (C)

A and C, Immunoblots with 3 µg (Cav-1 and TPO) or 10 µg (Cav-2 and Adaptin-ß) total protein. TSH and FSK were used at concentrations of 1 mU/ml and 10 µM, respectively. Adaptin-ß immunoblot is shown as a protein loading control. B, In situ cell immunolabeling; results (means ± SD, n = 4) are a ratio of RLU/sec of TPO, Cav-1, or Cav-2 to the RLU/sec of Adaptin-ß (the expression of which is not regulated by TSH, cAMP, PKA, or EPAC, therefore providing a control for the cell amount).

 
Activation of p44/42-MAPK or Ca2+ Pathways Does Not Inhibit Cav-1 and Cav-2 Protein Expression in Human Thyrocytes
The down-regulation of Cav-1 protein levels by the activation of the MAPK cascade has been well documented in other cell types (25, 26). To determine whether the MAPK pathway could play such a role in the regulation of caveolins’ expression in human thyrocytes, cells were incubated with phorbol myristate acetate (PMA) (an activator of the MAPK pathway in thyrocytes) under the same conditions as with TSH (4 d). Although PMA induced phosphorylation of p44/42-MAPK (Fig. 4CGo) and mediated down-regulation of TPO expression (an index of its dedifferentiating action on thyrocytes, Fig. 4BGo) (18), it was unable to influence caveolins’ expression (Fig. 4Go, A and B). Therefore, our data do not support an involvement of the MAPK pathway in the regulation of caveolins’ expression in human thyrocytes. TSH itself does not activate MAPK pathway in human thyroid (27, 28). Challenging the thyrocytes with adenosine 5'-O-(3-thio)-triphosphate (ATP{gamma}S), a potent Ca2+-mobilizing agent in human thyrocytes (29, 30), did not reduce Cav-1 and Cav-2 protein levels (Fig. 4Go, A and B). In contrast, it did increase H2O2 generation by thyrocytes, an effect mediated by an increase of intracellular Ca2+ levels (Fig. 4DGo) (31).


Figure 4
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  		Fig. 4. TPO, Cav-1, and Cav-2 Protein Expression Levels in Human Thyrocytes after Incubation for 4 d in the Presence of Activators of the MAPK Pathway (PMA) or the Ca2+ Pathway (ATP{gamma}S)

A, Immunoblot of Cav-1 (3 µg total protein), Cav-2, and Adaptin-ß (10 µg) from cells stimulated with 1 mU/ml TSH, 100 ng/ml PMA, or 100 µM ATP{gamma}S. B, In situ cell immunolabeling; results (means ± SD, n = 4) are a ratio of RLU/sec of TPO, Cav-1, or Cav-2 to the RLU/sec of Adaptin-ß (the expression of which is not regulated by PMA or ATP{gamma}S, therefore providing a control for the cell amount). C, Immunoblot of phosphorylated-p44/p42-MAPK with 10 µg protein from cells stimulated, or not, with PMA for 15 min. Total p42-MAPK is shown as a protein-loading control. D, H2O2 production by cells stimulated, or not, with 100 µM ATP{gamma}S for 90 min.

 
TSH Stimulation Alone Is Sufficient to Drive the Inhibition of Caveolins’ Expression
Down-regulation of Cav-1 expression is considered to be a requisite for cell proliferation in several cell models, because Cav-1 seems to play a central role in suppressing mitogenic signaling and promoting cellular senescence (32, 33, 34, 35). In thyroid, TSH/cAMP is a costimulator of the proliferation of follicular cells together with the phosphatidyl inositol 3-phosphate kinase (PI3-K) pathway, which is activated by IGF-I (17, 36). The latter cascade leads to the up-regulation of cyclin D3 levels (37). Once cyclin D3 is expressed, cAMP stimulates the formation of a cyclin D3/cyclin-dependent kinase 4 complex and its nuclear translocation (38). At high concentrations (5 µg/ml), insulin activates the IGF-I receptor and the downstream PI3-K pathway. Because insulin is present in the regular medium at such concentrations, we wondered if the negative regulation of caveolins by TSH depends on a synergism with insulin activity. We observed that TSH is able to efficiently down-regulate Cav-1 and Cav-2 expression in the absence as in the presence of insulin, which did not modify TPO expression as well (Fig. 5Go, A and B). Thus, down-regulation of caveolins by TSH is not dependent on or amplified by the PI3-K pathway. As a positive control, stimulation of thyrocytes by insulin decreased the expression of its own receptor (39) (Fig. 5CGo).


Figure 5
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  		Fig. 5. Effect of 5 µg/ml Insulin and 1 mU/ml TSH on the Expression of TPO, Cav-1, and Cav-2 Proteins in Human Thyrocytes Stimulated for 4 d

A, Immunoblot of Cav-1, TPO (3 µg protein), Cav-2, Adaptin-ß (10 µg protein); Adaptin-ß immunoblot on the same samples is shown as a control of the protein load. B, In situ cell immunolabeling; results (means ± SD, n = 4) are a ratio of RLU/sec of TPO, Cav-1, or Cav-2 to the RLU/sec of Adaptin-ß (the expression of which is not regulated by TSH or insulin, and therefore it provides a control for the cell amount). C, Immunoblot of insulin receptor (ß-subunit) with 15 µg protein extracted from thyrocytes stimulated, or not, with 5 µg/ml insulin, for 4 d.

 
TSH Controls the Expression of Cav-1 at the mRNA Level
In autonomous adenomas, Cav-1 mRNA is decreased. Thus, we examined whether the down-regulation of Cav-1 protein in vitro was due to a decrease of its mRNA expression. Cells were incubated in control medium or stimulated with TSH, FSK, ATP{gamma}S, PMA, MB-cAMP, and CPT-cAMP. Cav-1 mRNA expression was analyzed by quantitative RT-PCR. Cav-1 mRNA expression was reduced (after 2 days) approximately 4-fold by TSH and about 2.5-fold by FSK and MB-cAMP (Fig. 6AGo). In contrast, CPT-cAMP, PMA, or ATP{gamma}S did not down-regulate Cav-1 mRNA level (Fig. 6AGo). These results paralleled the results at the protein level (Figs. 3Go, A and B, and 4, A and B). They were further confirmed by Northern blotting (Fig. 6BGo).


Figure 6
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  		Fig. 6. Regulation of Cav-1 mRNA Levels in Human Thyrocytes in Response to Stimulation

A and B, Thyrocytes were stimulated for 2 d with 1 mU/ml TSH, 10 µM FSK, 100 µM ATP{gamma}S, 100 ng/ml PMA, 500 µM MB-cAMP, 20 µM CPT-cAMP, or left in control medium. A, Relative expression of Cav-1 was evaluated by quantitative RT-PCR; results represent fold variation of Cav-1 expression in stimulated cells, relative to cells in control medium, and in comparison with NEDD-8 and TTC-1 expression (reference genes). Solid line on the plot indicates a significant 2-fold regulation, and dotted line indicates no change compared with the nonstimulated cells. B, Cav-1 Northern blotting with 8 µg total RNA extracted from thyrocytes stimulated in the same conditions as in panel A. rRNAs (28S and 18S) were stained with acridine orange, as a RNA loading control. C, The TSH receptor blocking serum (10% vol/vol) was added to the thyrocytes 1 h before TSH (1 mU/ml). Then, the relative expression of Cav-1 was evaluated after 24 h, by quantitative RT-PCR, as in panel A. FSK was 10 µM and human recombinant TSH was 1 mU/ml.

 
Thyrocytes were preincubated with human serum containing TSH receptor-inhibitory antibodies. Blocking of the TSH receptor prevented the inhibitory effect of TSH on Cav-1 mRNA expression (Fig. 6CGo). In addition, human recombinant TSH had the same effect as bovine TSH on Cav-1 mRNA expression (Fig. 6CGo). These results further support that the down-regulation of Cav-1 is specific to an activated TSH receptor and is not due to a contaminant present in the bovine TSH preparation.

TSH Down-Regulates Cav-1 mRNA Expression via a Mechanism that Involves Synthesis of RNA, But Not of Protein
To achieve further insight into the mechanism of Cav-1 mRNA shutdown, we compared the kinetics of Cav-1 mRNA down-regulation induced by TSH to the kinetics of the natural extinction of the Cav-1 mRNA in the presence of actinomycin D (ActD), a transcription inhibitor (40). As a positive control of the ActD effect, we checked the rate of degradation of c-myc mRNA (Fig. 7AGo, left panel). When TSH and ActD were used in parallel incubations, TSH was faster than ActD (transcription arrest) in producing a decrease of Cav-1 mRNA (Fig. 7AGo, graph). This kinetic difference suggests that TSH acts downstream of Cav-1 transcription. TSH may act through destabilization of the Cav-1 mRNA. In the same experiment, cells were incubated with both TSH and ActD. We found that the presence of ActD abolished the TSH effect on Cav-1 expression (Fig. 7AGo, graph). This result suggests that the effect of TSH on Cav-1 expression requires RNA synthesis which, in turn, leads to the destabilization of the Cav-1 mRNA. Then, we checked the effect of inhibition of protein synthesis on the Cav-1 mRNA destabilization induced by TSH. Two inhibitors, cycloheximide (CHX) and emetine, were used alone or combined with TSH for 6 h. In parallel, cells were treated with ActD alone or in combination with TSH. Despite a decrease of the protein synthesis of 92% with CHX and of 98% with emetine (but not with ActD; Fig. 7DGo), none of the protein synthesis inhibitors could reproduce the effect of ActD, i.e. a blunting of the TSH-induced destabilization of Cav-1 mRNA. CHX alone increased Cav-1 mRNA in our conditions, a classical superinduction effect (41) (Fig. 7Go, B and C). This difference was not due to differential toxic effects of protein synthesis inhibitors vs. ActD, because after both treatments, cells generated cAMP in response to TSH as control cells (Fig. 7EGo). These data suggest that TSH-induced destabilization of Cav-1 mRNA depends on RNA, but not on protein synthesis.


Figure 7
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  		Fig. 7. TSH Down-Regulates Cav-1 mRNA Expression via a Mechanism that Involves Synthesis of RNA, But Not of Protein

A (Left panel), Kinetics of degradation of c-myc mRNA after stop of transcription by ActD, evaluated by Northern blotting with 8 µg total RNA per lane; rRNAs (28S and 18S) were stained with acridine orange, as a RNA loading control. Graph, Kinetics of Cav-1 mRNA down-regulation in the presence of TSH, ActD, or both, measured by quantitative RT-PCR. Quantitative RT-PCR results represent fold variation of Cav-1 expression in treated cells, relative to the untreated control and in comparison with Tg expression (reference gene). Solid line on the plot indicates a significant 2-fold regulation relative to the untreated control cells (dotted line). Note: no remarkable differences were detected in the threshold PCR cycles between the control samples across the time (data not shown), and an average was considered in calculations. B, Effect of incubation with inhibitors of RNA (5 µg/ml ActD) or protein (10 µg/ml CHX and 20 µM emetine) synthesis, for 6 h on the down-regulation of Cav-1 mRNA induced by TSH and evaluated by quantitative RT-PCR; results are an average of five independent experiments. No normalization by reference genes was performed, because the expressions of Tg, on the one hand, and of TTC-1 and NEDD-8, on the other hand, are unstable in the presence of protein synthesis inhibitors and RNA synthesis inhibitors, respectively; however, the same total RNA amount was used in each condition. Solid line on the plot indicates a significant 2-fold regulation compared with the untreated control cells (dotted line). C, Effect of incubation with inhibitors of RNA (5 µg/ml ActD) or protein (10 µg/ml CHX and 20 µM emetine) synthesis, for 6 h on the down-regulation of Cav-1 mRNA induced by TSH and evaluated by Northern blotting. Graph, Quantification of OD of Northern blotting bands. D, Effect of incubation with inhibitors of RNA (5 µg/ml ActD) or protein (10 µg/ml CHX and 20 µM emetine) synthesis on the protein production, measured by incorporation of L-[35S]methionine in total protein; results are mean cpm (± SD; n = 2). E, Effect of incubation with inhibitors of RNA (5 µg/ml ActD) or protein (10 µg/ml CHX and 20 µM emetine) synthesis, for 6 h, on the cell condition: after incubation with ActD, CHX, emetine, or control medium, the ability of cells to generate intracellular cAMP in response to 1 mU/ml TSH was measured, as described in Materials and Methods.

 
Thyrocytes of Cav-1 Knockout Mice Display Increased Proliferation
Next, we asked what could be the role of Cav-1 down-regulation upon prolonged exposure to TSH/cAMP. We hypothesized that Cav-1 is a negative regulator of proliferation in follicular cells. The down-regulation of Cav-1 expression upon TSH stimulation would contribute to cell cycle induction. We compared the proliferation cell nuclear antigen (PCNA) immunostaining on thyroid follicular cells between wild-type and Cav-1–/– mice. We found that proliferation of Cav-1–/– thyrocytes is increased more than 4-fold, relatively to the Cav-1+/+ thyrocytes (Fig. 8AGo). This increase in proliferation was accompanied by an increase in apoptosis (Fig. 8BGo), the cause of which is currently under investigation. The result is an increased cell turnover: the increase in apoptosis compensates the high proliferation rate. This explains why Cav-1–/– mice do not spontaneously develop a goiter.


Figure 8
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  		Fig. 8. Thyrocytes of Cav-1–/– Mice Display Increased Cell Proliferation and Apoptosis

A (Left panel), PCNA immunostaining on thyroid sections of 2- to 3-month-old Cav-1+/+ and Cav-1–/– mice; original magnification, x200. Graph, Proliferation index of Cav-1+/+ and Cav-1–/– thyroid follicular cells upon quantification of PCNA-stained nuclei (n = 10 animals each group). B (Left panel), Activated caspase-6 immunostaining on the same thyroid samples as panel A; original magnification, x400. Graph, Apoptosis index of Cav-1+/+ and Cav-1–/– thyroid follicular cells upon quantification of activated caspase-6-stained cells (n = 10 animals each group).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The aim of this study was to delineate the mechanism and role of the in vivo down-regulation of Cav-1 and Cav-2 expression in human thyroid autonomous adenomas (12). The possibility of an involvement of the TSH-cAMP pathway in that phenotype was suggested by the evidence that mutations that constitutively increase TSH receptor or Gs protein activation of the cAMP pathway are a main cause of these adenomas (15). In addition, chronic stimulation of the thyroid by cAMP in the adenosine A2-receptor transgenic mice also leads to a decrease in the Cav-1 expression in this gland (13). In the human adenomas, as well as in the thyroids of these transgenic mice, constitutive activation of the cAMP cascade leads to increased thyroid hormone secretion, hyperthyroidism and, due to the negative feedback of these hormones on the pituitary, a shutdown of TSH secretion. However, as both studies were carried on whole-thyroid tissue samples, the decrease of caveolins’ expression could take place in thyrocytes, endothelial cells, or fibroblasts, three cell types that highly express Cav-1. Primary cultures of isolated follicles of human thyroid are a well-established in vitro model, in which these cells constitute 99% of the cell population (42). As such, they allow expression studies specifically in thyrocytes. Our study clearly demonstrated that prolonged stimulation of human thyrocytes with TSH, cAMP enhancers, or cAMP analogs that activate PKA, all induced a decrease in the expression of Cav-1 and Cav-2 proteins and of Cav-1 mRNA. The level of Cav-1 mRNA down-regulation found in our in vitro model is the same as that found in vivo (12). Therefore, with regard to caveolins’ expression, our in vitro system mimics the in vivo phenotype of autonomous adenomas. The demonstration that thyrocytes from Cav-1–/– mice have increased proliferation rate shows in vivo the physiological role of Cav-1 down-regulation in the induction of thyrocytes proliferation. A down-regulation of Cav-1 by the cAMP signaling cascade has also been found in some cell lines (25, 43) and in astrocytes in primary culture (44), but cell proliferation was not analyzed in these studies.

A number of reports have established a reciprocal negative regulation between Cav-1 expression and the epidermal growth factor receptor/Neu-Ras-p44/42-MAPK signaling cascade in vitro: in MTLn3 (45), NIH 3T3, and A431 (7) cell lines, in mouse embryonic fibroblasts derived from Cav-1–/– mice (46), and in human diploid fibroblasts (47). Similar results were found in vivo, in breast (6, 48, 49), skin (50), and parathyroid (8) tumors. In a striking contrast to other cell types, in human thyrocytes the activation of the MAPK pathway does not produce such down-regulation. Likewise, other signal transduction pathways relevant to thyrocytes growth and function, namely, the Ca2+ and IGF-I/PI3-K cascades, do not down-regulate Cav-1 and Cav-2 expression. Interestingly, the direct (EPAC) substrate, Rap1, is also activated by the MAPK and IGF-I pathways in thyrocytes in primary culture (22), and we show here that none of these three cascades leads to down-regulation of caveolins’ expression. Thus, among the major pathways controlling thyrocytes growth, only a prolonged activation of the TSH-cAMP-PKA cascade is able to down-regulate caveolins’ expression. The down-regulation of caveolins in autonomous adenomas is therefore likely a direct consequence of the chronic stimulation of follicular cells by cAMP, i.e. a pathological mechanism other than loss of heterozygosity.

To further dissect the mechanism of Cav-1 protein down-regulation by TSH, we investigated its regulation at the mRNA level. Down-regulation of Cav-1 mRNA in rat muscle-derived cell lines (43) and of Cav-1 protein in CHO cells (25) are mediated by cAMP and PKA, respectively. In our study, all the results obtained at the Cav-1 protein level were reproduced at the mRNA levels, as measured by quantitative RT-PCR and Northern blotting. The decrease in Cav-1 mRNA in response to TSH was detected earlier than the protein decrease, i.e. 6 h for mRNA and 12–24 h for protein. Furthermore, the reductions in the expression of Cav-1 mRNA and protein were roughly of the same magnitude. These results indicate a primary control of Cav-1 expression at the mRNA level.

In contrast with other known systems in which PKA (25) or hormone (26) induces down-regulation of Cav-1 promoter activity, TSH seems to act beyond Cav-1 transcription, because the disappearance of its mRNA is faster than after blocking of transcription by ActD. Moreover, TSH accelerates the destabilization of Cav-1 mRNA, via an ActD-sensitive, i.e. transcription-dependent, mechanism. Interestingly, this process is not prevented by protein synthesis inhibitors. Thus, our data raise the possibility of a TSH-induced microRNA that, in turn, would be responsible for the destabilization of Cav-1 mRNA and inhibition of protein synthesis.

The cellular levels and localization of Cav-2 protein seem to be dependent on the availability of Cav-1 protein, because they form heterooligomers that stabilize Cav-2 (20). Cav-2 protein is consistently down-regulated in human thyrocytes in all the same conditions that induce decrease of Cav-1 expression. Whether or not down-regulation of Cav-2 protein is, in thyrocytes, a consequence of Cav-1 down-regulation or a direct effect of TSH-cAMP-PKA on Cav-2 expression remains to be clarified.

The late kinetics of the down-regulation of Cav-1 protein expression by TSH provides some clues about the mechanism of this effect on proliferation. The earliest decrease at the protein level is after 12–24 h. At this stage, the whole mitogenic process is already well advanced, with the early immediate gene induction largely finished and the appearance of the first cyclin D3/cyclin-dependent kinase 4 complexes (51). The effect should therefore bear on the late steps of the G1 phase.

In conclusion, this work demonstrates that the down-regulation of caveolins’ expression in autonomous adenomas is a direct consequence of the chronic stimulation of thyrocytes by cAMP. Conversely, the increased cell proliferation of Cav-1–/– thyrocytes clearly shows an antimitogenic role of Cav-1 in thyroid. It also strongly suggests that Cav-1 is a tumor suppressor gene in the human thyroid, explaining the decrease of its expression in autonomous adenomas.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Bovine TSH, bovine insulin, rolipram, collagenase, ActD, CHX, PMA, and ATP{gamma}S were purchased from Sigma-Aldrich (St. Louis, MO). Forskolin is from Calbiochem (La Jolla, CA). Human recombinant TSH (Thyrogen) is from Genzyme Corp. (Cambridge, MA). MB-cAMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (CPT-cAMP) were purchased from BioLog Life Sciences Institute (Bremen, Germany). Sera inhibiting TSH receptor from patients with thyroid-autoimmune disease were a kind gift from Dr. Khoo (Singapore General Hospital). Mouse monoclonal antibodies against adaptin-ß (clone 74) and Cav-2 (clone 65) and rabbit antibodies against Cav-1 and insulin receptor were purchased from Becton Dickinson and Co. (Franklin Lakes, NJ) Mouse antiphosphorylated p44/p42-MAPK antibody (clone E10) was purchased from Cell Signaling Technology (Beverly, MA). The PCNA mouse antibody (clone PC10) was from DAKO Corp. (Carpinteria, CA). The rabbit antibody against p42-MAPK and the rabbit polyclonal antibody against activated caspase-6 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Noncommercial mouse antibody against human thyroperoxidase was previously described (52). Primers and probes for real-time PCR were designed using the PerkinElmer Primer Express software (PerkinElmer, Wellesley, MA). The sequences of used primers and probes are available upon request. All reagents for quantitative PCR were produced by Eurogentec (Seraing, Belgium).

Primary Cultures
Human thyroid tissue was obtained from patients undergoing surgery for partial or total thyroidectomy due to cold nodules, multinodular goiter, or Grave’s disease, following the rules of the University Hospital Ethical Committee. Isolation of thyroid follicles and primary culture of thyrocytes were as described elsewhere (17). Isolated follicles (2 µl follicles/ml) were incubated overnight. The next day, the medium was replaced and cells were stimulated or not for the times indicated in the figures. In kinetic experiments, all incubations started at the same time and lasted for the times indicated in the figures. MB-cAMP was used at 500 µM and CPT-cAMP was used at 20 µM, i.e. concentrations that potently and exclusively stimulate PKA, or the guanyl nucleotide EPAC, respectively (data not shown).

Total Protein Extraction and Immunoblotting
At the end of incubation, the medium was withdrawn, and total cell proteins were extracted, quantified (53), and subjected to semiquantitative immunoblotting, as previously described (14).

In Situ Cell Immunolabeling
Human thyrocytes (0.2 µl) were incubated in black 96-well plates. After incubations, all the procedures were performed at room temperature, unless otherwise stated. The media were removed, and cells were washed twice with PBS and fixed in PBS-4% paraformaldehyde (10 min). Cells were equilibrated in Tris-buffered saline (TBS) (50 mM Tris; 140 mM NaCl, pH 7.6) for 5 min, and permeabilized with TBS-0.1% Triton X-100 (10 min). The activity of endogenous peroxidases was blocked with methanol-0.6% H2O2 (7 min). Nonspecific binding was blocked in TBS-B (TBS-0.5% BSA) containing 20% (vol/vol) normal sheep serum (30 min). Primary antibodies diluted in TBS-B were incubated at 37 C for 1 h, followed by washing with TBS-0.05% Tween 20. Then, horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Buckinghamshire, UK) diluted in TBS-B were added (30 min). Cells were washed in TBS-0.05% Tween 20 and incubated with LumiGlo (Upstate Biotechnology, Inc., Lake Placid, NY). Relative light units (RLU) were measured in a luminometer (Berthold Technologies, Bad Wildbad, Germany) (10 sec /well), and the integrated results were obtained with the WinGlow software (Berthold). The expression of Cav-1, Cav-2, and TPO proteins in each condition was normalized by the expression of Adaptin-ß (ratios of the RLU/sec obtained for the protein of interest to the RLU/sec obtained for Adaptin-ß).

cAMP Measurements
Thyrocytes (2 µl pellet) were seeded in Petri dishes (3.5 cm diameter) and incubated in the basal or stimulation medium for 4 d, in the absence of phosphodiesterases inhibitors. To test the toxicity of RNA and protein synthesis inhibitors, after 6 h incubation with these drugs, cells were rinsed and equilibrated in Krebs-Ringer-HEPES medium (25 mM HEPES, pH 7.4; 1.25 mM KH2PO4, 124 mM NaCl, 1.25 mM MgSO4, 8 mM glucose, 1.45 mM CaCl2, and 5 mM KCl), at 37 C for 30 min and then incubated with fresh Krebs-Ringer-HEPES supplemented with 50 µM rolipram and with or without 1 mU/ml TSH (37 C, 2 h). At the end of incubations, cells were lysed in 0.1 M HCl, and the cellular acid extract was collected. Samples were dried in a speed vac concentrator (Jouan RC10.10; Jouan, Inc., Winchester, VA), and cAMP was measured by RIA (54).

Measurement of H2O2 Generation
The production of H2O2 by thyrocytes was measured by fluorimetry, as described elsewhere (55).

Quantitative RT-PCR and Northern Blotting
At the end of incubations, the cells media were removed and total RNA was extracted (TRIzol; Invitrogen, Carlsbad, CA), purified (Rneasy; QIAGEN, Chatsworth, CA), and treated with DNase (DNA Free; Ambion, Austin, TX). Total RNA (0.5 µg) was reverse transcribed with Superscript II reverse transcriptase H– (Invitrogen), using random primers (2.5 µg; Promega Corp., Madison, WI). Normalization genes are sample endogenous controls. Thyroglobulin (Tg), NEDD-8, and TTC-1 were validated as reference genes within the conditions used. TTC-1 is a gene involved in cell cycle and NEDD-8 is a gene implicated in ubiquitination. The amplification of TTC-1 and NEDD-8 used the SYBR green technology (Eurogentec). The amplifications of Cav-1 and Tg were performed using the Taqman method. The detection of fluorescence was done using an ABI Prism 7700 Applied Sequence Detector and the PE Sequence detector version 1.7 software (PerkinElmer). All PCRs were performed in duplicate, and the mean of the threshold cycles (Ct) was used in calculations. Controls without reverse transcriptase were performed. The deviation of Cav-1 expression (in comparison with a reference gene) between control and stimulated cells was calculated according to the model of Pfaffl (56). The Northern blotting experiments were performed on total RNA (57).

Inhibition of RNA and Protein Synthesis
Follicles (4 µl) were seeded in 3.5-cm diameter Petri dishes. The next day, the cells were incubated in fresh culture medium containing 20 µCi/ml L-[35S]methionine (Amersham Biosciences), with or without inhibitors of RNA (ActD) or protein synthesis (CHX and emetine) for 6 h. The media were removed, and the cells were rinsed twice with PBS (4 C) and lysed with 10% trichloroacetic acid (4 C). Precipitated proteins were scraped off the dish, washed twice with 10% trichloroacetic acid, dried, solubilized in 1 ml soluene (Packard Instruments, Meriden, CT), and transferred to scintillation vials, containing 9 ml of insta-fluor (Packard) and 50 µl 100% acetic acid. The radioactivity of the samples was evaluated in a scintillation counter (Packard).

Analysis of Cell Proliferation and Apoptosis in Thyrocytes of Cav-1 Knockout Mice
Cav-1–/– mice (C57/BL6) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) [strain 004585 (58)] and obtained from Dr. Kurzchalia (59). Results were similar with mice from both origins. Mice were kept in the animal facility under the 12-h dark, 12-h light cycle and were given water and AO3 mice breeding diet (Safe UAR, with normal iodine content, 0.4 mg/kg). All procedures respected regulations and guidelines of the Belgian state and European Union and were approved by the local ethical committee. Littermates Cav-1+/+ and Cav-1–/– mice (2–3 months old) from the two different origins were killed, and the thyroids were removed, fixed in buffered-formalin, and embedded in paraffin. Thyroid sections (4 µm) were mounted on poly-L-lysine glass slides, deparaffinated in xylene, and hydrated (ethanol, water). The sections were washed in PBS, containing 1% BSA (PBS-BSA). Blocking of nonspecific binding sites was performed by incubation in goat normal serum diluted 1:50 in PBS, for 20 min at room temperature. For the cell proliferation index, sections were incubated overnight (room temperature) with anti-PCNA antibody (10 µg/ml in PBS). For the apoptosis index, sections were microwave treated and washed, and nonspecific binding was blocked as above. Anti-activated-caspase-6 antibody (5 µg/ml in PBS) was added overnight at room temperature. Sections were washed in PBS-BSA and incubated with the appropriate immunoglobulin conjugated to peroxidase-labeled polymer (EnVision+; DAKO). For quantifications, 600–700 cells per thyroid were counted.


   ACKNOWLEDGMENTS
 
We thank Dr. Khoo (Singapore General Hospital, Singapore) and Dr. Costagliola (Institut de Recherche Interdisciplinaire, Brussels, Belgium), who provided the human sera inhibiting TSH receptor activity and the plasmid containing human caveolin-1, respectively. We also thank Dr. Mendive for helpful discussions and critical reading of the manuscript and C. Massart and C. Degraef for their excellent technical support.


   FOOTNOTES
 
This work was supported by Fundação para a Ciência e a Tecnologia (Portugal), Fonds National pour la Recherche Scientifique, Fonds de la Recherche Scientifique Médicale, Télévie, Ministère de la Politique Scientifique, Fondation David et Alice Van Buuren, and Fondation Rose et Jean Hoguet.

Present address for M.J.C.: Pacific Vascular Research Laboratory, University of California San Francisco, HSW1652, Box 0507, 513 Parnassus Avenue, San Francisco, California 94143-0507.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 3, 2007

Abbreviations: ActD, Actinomycin D; ATP{gamma}S, adenosine 5'-O-(3-thio)-triphosphate; Cav-1, caveolin-1; Cav-2, caveolin-2; CHX, cycloheximide; CPT-cAMP, 8-(4-chlorophenylthio)-2'-O-methyl-cAMP; EPAC, exchange nucleotide protein activated by cAMP; FSK, forskolin; MB-cAMP, N6-monobutyryl-cAMP; PCNA, proliferation cell nuclear antigen; PI-3K, phosphatidyl inositol 3-phosphate kinase; PKA, cAMP-dependent protein kinase; PMA, phorbol myristate acetate; RLU, relative light units; TBS, Tris-buffered saline; TBS-B, TBS-0.5% BSA; Tg, thyroglobulin; TPO, thyroperoxidase.

Received for publication August 9, 2006. Accepted for publication December 29, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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