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Endocrinology, doi:10.1210/en.2003-0438
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Endocrinology Vol. 144, No. 9 4018-4030
Copyright © 2003 by The Endocrine Society

Biological Activity of Activating Thyroid-Stimulating Hormone Receptor Mutants Depends on the Cellular Context

D. Fuhrer, M. D. Lewis, F. Alkhafaji, K. Starkey, R. Paschke, D. Wynford-Thomas, M. Eggo and M. Ludgate

Department of Medicine (Endocrinology, Metabolism and Diabetes Section) (D.F., M.D.L., F.A., K.S., M.L.) and Department of Pathology (D.W.-T.), University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom; Medizinische Klinik III (R.P.), Universität Leipzig, Philipp-Rosenthal Strasse 27, 04103 Leipzig, Germany; and Department of Medicine (M.E.), University of Birmingham, Queen Elizabeth Hospital, Birmingham B15 2TH, United Kingdom

Address all correspondence and requests for reprints to: Marian Ludgate, Ph.D., Department of Medicine (Endocrine Section), University of Wales College of Medicine, Cardiff, Heath Park, Cardiff CF14 4XN, United Kingdom. E-mail: ludgate{at}cf.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activating TSH receptor (TSHR) mutations are a major cause of toxic thyroid adenoma and familial hyperthyroidism, and more than 37 such mutations have been described. Previously their functional activity had been assessed in terms of cAMP and inositol phosphate production and predominantly in transiently transfected COS-7 (monkey embryonic kidney cells), a model that does not reflect effects on thyrocyte proliferation and function. Here we have performed a systematic comparison of wild-type and seven gain-of-function TSHR mutants, introduced into rat FRTL-5 and human thyrocytes, using retroviral vectors. Our results show that 1) biological potency of TSHR mutants in thyroid cells does not correlate with their cAMP levels in transfected COS cells, highlighting the importance of cellular context and level of expression when assessing biological effects of oncogenic mutations; 2) dissociation between stimulation of function and growth occurs with thyrocyte differentiated functions more readily stimulated than growth; 3) TSHR mutants show a similar order of potency in FRTL-5 cells and human thyrocytes; 4) mutants inducing the highest stimulation of adenylyl cyclase may paradoxically fail to induce proliferation; and 5) biological effects of cAMP activating TSHR mutants are attenuated by complex counterregulatory mechanisms at least at the level of phosphodiesterases and cAMP regulatory element modulator isoforms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GROWTH AND function of the thyroid gland are controlled by TSH (1), a pituitary-derived glycoprotein that exerts its effect via the G protein-coupled TSH receptor (TSHR). Unlike the other glycoprotein hormone receptors, the TSHR is noisy so that in vitro models involving expression of even the wild-type (WT) receptor result in an increase in cAMP levels (2). The TSHR is also unique in being the target of autoantibodies, which mimic the action of its physiological ligand and is home to an impressively broad spectrum of gain-of-function mutations (3, 4). Both of these are pathogenic mechanisms resulting in hyperthyroidism, which manifests as Graves’ disease or toxic thyroid nodule, respectively, and act predominantly via the cAMP pathway. In the thyroid, cAMP controls both thyroid growth and function, which is further exemplified by a phenotype of toxic thyroid hyperplasia in transgenic mice with chronic cAMP stimulation in the thyroid (e.g. due to adenosine A2 receptor overexpression) and hyperthyroidism and goiter in individuals with germline gain-of-function TSHR mutations (1, 4, 5, 6). Conversely, hypothyroidism and thyroid hypoplasia occur with inactivation of the cAMP pathway due to nonfunctional TSHR mutations in humans and mice (hyt/hyt mouse) (4, 5, 6).

Gain-of-function TSHR mutations are predominantly located in the membrane spanning domain, which is responsible for signal transduction and shares a common architecture with all G protein-coupled receptors, consisting of three extra- and intracellular loops and seven membrane spanning segments (Fig. 1Go). The existence of a wide range of naturally occurring activating mutations has provided a valuable tool for structure/function analysis of the TSHR. These have highlighted the importance of an alanine in the third intracellular loop (A623) for coupling to Gs (7), a tyrosine residue (Y601) at the cytosolic end of the fifth transmembrane (TM) region for coupling to Gq (8), and an asparagine residue (N674) in the seventh TM region necessary for TSH-induced receptor activation (9, 10). However, regarding the biological effects of TSHR mutations, in terms of stimulation of proliferation and function, the picture is less clear. The system most often employed for functional TSHR studies is to transiently overexpress the receptors, in a nonthyroid, nonhuman cell line (COS-7; monkey embryonic kidney cells) and to assess biochemical parameters, i.e. cAMP and inositol phosphate production, for the mutants in comparison with the WT TSHR (3, 4, 6, 11). This system, however, cannot be relied upon to generate data representative of effects in normal human thyroid cells because 1) it involves gross artefactual overexpression of the receptors, whereas expression of native TSHR in thyroid follicular cells, and particularly mutant forms, is remarkably low (12, 13); 2) signaling in COS-7 cells may not accurately reflect the situation in thyroid follicular cells due to the specificity of cell signaling (14); and 3) investigation of direct biological effects on growth, differentiation, and function is not possible in a cell system, in which gene expression is only transient.



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  		FIG. 1. Localization of the studied TSHR mutants in the exon 10 encoded TSHR transmembrane domain. Some mutants, e.g. L629F and M453T, occur as somatic mutations in toxic thyroid nodules and as germline mutations in autosomal dominant nonautoimmune hyperthyroidism. In addition, the M453T mutation has also been identified in a toxic papillary cancer.

 
Only a very limited number of activating TSHR mutations, largely restricted to the sixth TM, have been assessed in a more specific context using the rat thyroid FRTL-5 cell line (15, 16). Recently, we have successfully exploited for the first time a human thyrocyte model using retroviral vectors to compare the biological activity of a single TSHR mutant (A623I) with a G{alpha}s mutant (Q227L) (17). We now report the functional assessment of seven gain-of-function TSHR mutations, spread throughout the TM region, comprising somatic and germline events and having variable activation of Gs/adenylyl cyclase and/or Gq/phospholipase C when measured in COS-7 cells. We have used retroviral infection to introduce the TSHR mutants into thyrocytes, having first characterized the retroviral vectors in nonthyroidal cells to ensure they are able to induce expression of functional TSHR at the cell surface. Subsequently, we have used the vectors to introduce the TSHR mutants into FRTL-5 cells and human primary thyrocytes to compare their stimulation of proliferation and function and whether this correlates with the levels of cAMP they induce in COS cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells used and culture conditions
Primary human thyrocytes were obtained by protease/dispase digestion of histologically normal thyroid tissue (n = 2), multinodular goiter (n = 2), and Graves’ disease thyroid tissue (n = 2) as previously described (18). All primary thyroid cultures were freshly disaggregated. Cells were plated at a density of 2 x 106/35-mm dish. Monolayer cultures were either propagated in 10% fetal calf serum (FCS)/RPMI 1640 (GIBCO Life Technologies, Paisley, Scotland, UK) or 1% FCS/RPMI supplemented with 10 µg/ml insulin (Sigma, Poole, UK) for 48 h before retroviral infection.

FRTL-5 cells (SB5 sub-clone, selected for TSH-dependent growth and function) (19) were maintained in a 2:1:1 mixture of DMEM:Ham’s F12:MCDB104 (all from GIBCO Life Technologies) supplemented with 5% calf serum (GIBCO Life Technologies), 10 µg/ml insulin, 0.4 µg/ml hydrocortisone (Calbiochem, UK), 45 µg/ml ascorbic acid (Sigma), 5 µg/ml transferrin (Calbiochem), and 5 mU/ml bovine TSH (Sigma), referred to as "5H medium."

COS-7 cells were propagated in 10% FCS/DMEM; A431 cells and hORI3 were propagated in 10% FCS/RPMI 1640 as described (17, 20).

Production of mutant TSHR retroviral vectors
To construct TSHR retroviral vectors, the cDNAs of the six following TSHR mutants were subcloned from the pSVL vector into the retroviral pLNSX vector as previously described (17): M453T (21); V509A and C672Y (22); L629F (23); Del613-621 and V656F (24). The pLNSX plasmid contains a G418 resistance gene. The simian virus 40 early promoter drives expression of the gene insert in the pLNSX vector. Correct insertion of the TSHR mutant cDNAs was checked by test restriction with BstEII (New England Biolabs, Beverly, MA) and by sequencing the entire insert and 100 bp of the adjacent vector regions using dRhodamine Dye Terminator Sequencing chemistry (Applied Biosystems, Warrington, UK).

Retroviral supernatants were generated using the ‘Phoenix’ transient retroviral producer packaging cell line (25). Retroviral supernatants were harvested at 48 h after transfection and assessed for 1) retroviral titers in human epithelial cell line A431 (17, 26); 2) TSHR expression by specific [125-I]TSH binding of G418 selected A431 colonies (17); and 3) cell surface expression in the human thyroid cell line hORI3 by flow cytometry using the polyclonal TSHR Ab p60 (27).

Retroviral gene transfer into human primary thyrocytes, FRTL-5 cells, and COS-7 cells
Human thyrocytes.
Forty-eight hours after plating, triplicate dishes of human thyrocytes (2 x 106 cells/35-mm dish) were pretreated with 8 µg/ml polybrene for 1 h and then infected twice with pooled batches (n = 2) of the retroviral supernatants in the presence of 8 µg/ml polybrene for 3 h each (17, 26). G418 selection was started 24 h after infection. For experiments in 1% FCS, triplicate dishes were infected with the M453T, the WT TSHR, or the neo supernatants. The experiments were performed on four and two independently disaggregated thyroids for normal (10% FCS) and low (1% FCS) serum conditions, respectively.

FRTL-5 cells and COS-7 cells.
Infections of the rat thyroid FRTL-5 cell line and COS-7 cells, with all mutant and WT TSHR and neo, were performed in duplicates at 25–40% confluence/35-mm dish. COS-7 cell clones were selected by G418 resistance only, the resulting clones in each experiment (n = 2) were pooled for cAMP measurement.

Twenty-four hours after retroviral infection, FRTL-5 cells were passaged and selected under two conditions: 1) G418 resistance and 2) G418 resistance plus TSH withdrawal (4H medium). In each of four separate infection experiments, all FRTL-5 clones/retroviral construct (>100/35-mm dish with G418 selection, 25–100 with TSH withdrawal plus G418 selection for most mutants) were pooled and used for functional characterization at early passage numbers (n < 5).

Investigation of proliferation, signal transduction, and function in FRTL-5 cells
Four independently generated mixed pools of FRTL-5 cells were studied for each mutant/WT TSHR and the neo control, selected with G418 and TSH withdrawal. The mixed pools were cultured in the presence of TSH to increase cell numbers but then maintained in 4H medium, i.e. without TSH for 72 h before all measurements, which were all performed in triplicate (unless otherwise stated). Depending on the parameter being analyzed, the mixed pools were then cultured in 4H or 5H medium and with or without 250 µM isobutylmethylxanthine (IBMX) for varying periods of time. Growth curves and DNA synthesis provided a measure of proliferation and function was assessed by measuring iodide uptake. The signal transduction properties, TSHR transcript levels, and cell cycle profiles of the mixed pools were also compared.

Growth curves.
FRTL-5 cell pools were plated at a density of 5 x 104 cells/well in 24-well plates and cell numbers determined in a Coulter counter on d 3, 6, 8, 10, and 12.

DNA synthesis.
FRTL-5 cell pools were plated at a density of 5 x 104 cells/coverslip. Cells were labeled with 10 µM BrdU (5-bromo-2'deoxyuridine-5-monophosphate) for 24 h when immunostaining was performed using a monoclonal anti-BrdU antibody (1:200) and a peroxidase-labeled second antibody (1.100; all from Dako, Ely, UK) as described (17).

125-I uptake.
FRTL-5 cell pools were plated at a density of 1 x 105 cells/well in 24-well plates. Iodide uptake was measured after 72 h as described by Weiss et al. (28). Briefly, cells were washed twice with 1 ml Hanks’ balanced salt solution and were incubated in the presence of 0.1µCi Na125I + 20 µM NaI (n = 3/construct) or 20 µM NaI + 80 µM NaClO4 (n = 3/construct) in 500 µl Hanks’ balanced salt solution for 45 min in an incubator at 37 C. Iodine uptake was determined in a {gamma}-counter. Differences in growth rates were corrected by counting cell numbers on the day of analysis.

cAMP pathway.
FRTL-5 cell pools were plated at a density of 5 x 104 cells/well in 24-well plates. cAMP accumulating over 4 h was measured in the cell extracts using an in-house RIA (29). Differences in growth rate were corrected by protein content (30).

RT-PCR and real-time.
FRTL-5 cell pools were maintained in 4H medium for 72 h before standard RNA extraction (31). RT-PCR and real-time PCR (Lightcycler, Roche, Indianapolis, IN) were performed using exon spanning primers for the extracellular TSHR domain as previously described (32, 33). Transcript copy numbers were calculated from a standard curve obtained from cloned TSHR cDNA excised from pSVL as described (33).

Western blotting.
Pooled FRTL-5 cell clones for M453T, L629F, Del613-621 were propagated in duplicates in six-well plates until they were 80–90% confluent (7–14 d). Protein extraction was performed in Laemmli buffer containing 1 mM sodium orthovanadate and 1 mM phenylmethylsulfonyl fluoride. Samples were subjected to standard Western blotting. Blots were probed with anti phospho-cAMP response element binding protein (CREB), which detects only the phosphorylated form of CREB (Ser133, 1:2000 overnight; Cell Signaling Technology, Beverly, MA) and anti-total-CREB, which also detects activating transcription factor 1 (ATF-1), cAMP regulatory element modulator (CREM) isoforms, and inducible cAMP early repressor (ICER) proteins (1:1000, room temperature for 1 h; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Binding was demonstrated using an antirabbit IgG-horseradish peroxidase conjugate (1:5000, room temperature for 1 h, Dako) and the ECL Plus chemiluminescent chemistry system (Amersham Pharmacia Biosciences, Amersham, Buckinghamshire, UK). Quantification of staining was determined by densitometry analysis with the Alpha Imager 1200 system (Flowgen, Lichfield, UK).

Cell cycle analysis.
FRTL-5 cell pools were plated in duplicates at a density of 1 x 105 cells/well in 24-well plates. After 72 h, trypsinized cells were stained with 25 µM Draq 5 (Vinci Biochem, Rome, Italy) for 10 min and a minimum of 10,000 events was collected on a FACScan (Becton Dickinson, Franklin Lakes, NJ). Results were analyzed using WinMDI and Cylchred software (University of Wales College of Medicine, Cardiff, UK).

Investigation of cAMP generation in transiently transfected and retroviral infected COS-7
COS-7 cells (3 x 105 COS-7 cells/six-well plate) were transiently transfected in triplicates with 100 ng of the various mutant and WT pSVL TSHR constructs or the empty pSVL vector, as previously described (34), and assayed for basal and TSH- (100 mU/ml; 1 h) stimulated cAMP generation in the presence of 250 µM IMBX at 4 h. Pools of COS-7 cells infected with retroviral supernatants (see above) were grown to confluence in triplicates and were assayed for basal and TSH-dependent (5 mU/ml) cAMP generation in the presence of 250 µM IMBX using an in-house RIA (29).

Investigation of proliferation and thyroglobulin (Tg) expression in human thyrocytes
Four different primary thyroid cultures were infected with each mutant/WT TSHR and neo and selected for 28 d with G418. The thyrocyte cultures were then fixed in ice-cold methanol:acetone (1:1; -20 C; 20-min), air-dried (26), and the number and morphology of G418-resistant colonies determined using light microscopy. In addition cell numbers in colonies with epithelial morphology were counted to assess population doubling as an estimate for growth stimulation (17). Data are expressed as mean ± SEM of triplicates.

Tg immunochemistry.
After inhibition of endogenous peroxidase (0.025% periodic acid for 1 min), G418-resistant colonies were incubated with a monoclonal anti-Tg antibody (1:100; Dako) for 1 h at room temperature. Detection of antibody binding was carried out with the VECTASTAIN ABC Kit (Vector Laboratories, Burlingame, CA) (17, 26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Validation of WT and mutant TSHR retroviral supernatants in different cell lines
Before applying the retroviral supernatants, obtained from the Phoenix cell line, to rat or human thyrocytes, they were characterized for retroviral titers and transduction of TSH receptor expression: 1) Similar retroviral titers (3–5 x 105 colony-forming units/ml) assessed by induction of G418 resistance in nonthyroid epithelial A431 cells, were obtained for all retroviral supernatants. 2) All seven mutant and WT TSHR supernatants transduced receptor cell surface expression, as demonstrated by specific 125-I TSH binding on A431 cells and by flow cytometry on the human ORI3 thyroid cell line, which lacks endogeneous TSHR expression (data not shown). 3) Expression of functional TSH receptors was evidenced by a 1.5- to 3.5-fold increase in TSH-induced cAMP production in COS-7 cells stably transduced with mutant or WT TSHR retroviral vectors compared with controls (neo only) (Fig. 2BGo).



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  		FIG. 2. Differences in constitutive activity of 7 activating TSHR mutants in COS-7 cells after transient transfection or retroviral infection. A, A total of 3 x 105 COS-7 cells/six-well plate were transfected with 100 ng of the seven mutant and WT pSVL TSHR constructs or the empty pSVL vector and assayed for basal and TSH- (100 mU/ml; 1 h) stimulated cAMP generation in the presence of 250 µM IMBX. B, A total of 1.0 x 105 COS-7 cells were infected with retroviral supernatants, and pooled G418-resistant clones were assayed for basal and TSH-stimulated (5 mU/ml, 4 h) cAMP generation in the presence of 250 µM IMBX. A similar order of constitutive activity was observed for the investigated mutants in COS-7 cells irrespective of the mode of gene transfer. Considerably lower cAMP levels in the stably transfected populations are consistent with a low receptor expression in contrast to gross artefactual overexpression by transient transfection. Results are expressed in picomoles cAMP/well (±SEM) of duplicates in one representative of three independent experiments.

 
Comparison of TSHR mutants in FRTL-5 cells
Morphology of transduced FRTL-5 cells.
A subclone (SB5) of the FRTL-5 cell line selected for TSH-dependent growth and function was used (19) and, after infection, was subjected to two different selection procedures: 1) G418 resistance or 2) G418 resistance plus TSH independence (expected to be conferred by at least some activating TSHR mutants). Using only the first selection criterion, similar numbers of FRTL-5 clones (>100/35 mm dish) exhibiting the characteristic cobblestone morphology were obtained in all mutant, WT and the neo infected FRTL-5 cells consistent with the comparable titers observed when using epithelial A431 cells as recipients. In contrast, when TSH independence was used as an additional selection criterion, the resulting FRTL-5 cell clones differed considerably in their number (e.g. 80–100/35-mm dish for M453T; 20–35/35-mm dish for C672Y), morphology, and hormone dependence.

In TSH-free medium, the M453T and A623I FRTL-5 clones fully mimicked the epithelial phenotype, whereas the V656F and L629F FRTL-5 clones comprised a mixture of cells with an epithelial phenotype and others with a flattened appearance similar to FRTL-5 cells in TSH-free medium. The Del613-621, V509A, and C672Y FRTL-5 clones required the addition of the phosphodiesterase inhibitor IBMX to achieve epithelial morphology, but this treatment did not modify the morphology of WT or neo infected FRTL-5 cells (data not shown).

These variations in morphology were reproduced in three of three further infected FRTL-5 cell batches and were thus unlikely to result from random differences in expression levels.

To minimize potential methodological bias, we performed all functional studies on cell pools selected by both G418 resistance and TSH independence (n = 4 pools for the 7 mutants, WT TSHR and neo in four infection experiments), rather than just a few individual clones per mutant. Furthermore, all experiments were conducted on low passage numbers (n < 5) and each population was further characterized for the level of human (h) TSHR expression.

TSHR expression in transduced FRTL-5 cells
hTSHR expression was investigated at the mRNA and protein level in the seven mutant, WT TSHR and neo FRTL-5 populations. 1) mRNA expression was demonstrated by standard RT-PCR and was further quantified by real-time PCR using exon-spanning primers specific for amplification of the human TSHR but not the endogenous rat TSHR. Determination of the copy numbers showed that the majority of mutant as well as WT TSHR expressing FRTL-5 pools have 24–66 receptor transcripts per 50 ng input RNA (Table 1Go). Exceptions were the two populations with complete TSH independence in cell culture, i.e. the M453T FRTL-5 population having 290 receptor transcripts per 50 ng input RNA and the A623I population, where hTSHR transcripts were at the limit of detection (Table 1Go). As expected, no human TSHR transcripts were detected in the control (neo only) FRTL-5 cell pools.


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  		TABLE 1. hTSHR copy numbers and induction of DNA synthesis in infected FRTL-5 cells

 
Using a range of different TSHR antibodies, which are able to detect both exogenous human and endogenous rat TSHR, we did not observe differences in the TSHR protein cell surface expression levels in the transduced compared with untransduced FRTL5 cells by flow cytometry (data not shown). This suggests that mutant TSHR proteins are expressed at a low level at the cell surface and argues against artefactual overexpression, e.g. as a result of the retroviral promoter.

Effects on signal transduction
Constitutive activation of the cAMP pathway in transduced FRTL-5 cells was determined by measurement of basal and TSH-stimulated cAMP generation in the seven mutant, WT TSHR and neo populations propagated in different cell culture conditions:

1) In the absence of TSH a 1.5-, 1.7-, or 4-fold increase in basal cAMP production relative to the WT TSHR pool, was observed for the A623I, V656F, and M453T FRTL-5 pools, respectively (Fig. 3AGo).



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  		FIG. 3. Different effects on signal transduction in FRTL-5 cells infected with seven activating TSHR mutants, the WT hTSHR or the neo retroviral vectors. Basal and TSH- (5 mU/ml) stimulated cAMP levels (±250 µM IBMX) were measured in the different FRTL-5 populations plated at 5 x 104 cells/well. A, In the absence of TSH a 1.5-, 1.7-, or 4-fold increase in basal cAMP production relative to the WT TSHR pool, was observed for the A623I, V656F, and M453T FRTL-5 pools, respectively. In the presence of IBMX (routinely added to transfected COS-7 cells for cAMP measurement) higher intracellular cAMP levels were obtained in all mutants compared with the WT TSHR transduced FRTL-5 cells. B, In the presence of TSH, differences in cAMP generation were less pronounced between the studied mutant, WT TSHR and neo pools. However, addition of IMBX caused a dramatic increase in cAMP levels, predominantly in the mutant populations, underlining their constitutively activating nature. Results are expressed as pmol cAMP/well (±SEM) of duplicates in one representative of four independent infection experiments, corrected for cell numbers on the day of analysis.

 
2) Measurement of cAMP production in transiently transfected COS-7 cells is always performed in the presence of IBMX and all mutant FRTL-5 populations exhibited higher intracellular cAMP levels compared with the WT TSHR in this condition. The largest increase in cAMP levels was observed in M453T and the lowest in V509A (3.5- and 1.3-fold increase relative to WT, respectively) (Fig. 3AGo).

3) In the presence of TSH differences in cAMP generation were less pronounced between mutant, WT TSHR and neo FRTL-5 cells clones, reaching at most a 2.5-fold increase relative to WT TSHR levels in M453T and A623I FRTL-5 populations (Fig. 3BGo).

Effects on growth
The growth stimulatory potency of the seven mutant, WT TSHR, and neo FRTL-5 cell pools, was first investigated by determining changes in cell numbers over a period of 12 d in different culture conditions: 1) In the absence of TSH highest cell numbers were obtained in the M453T FRTL-5 population followed by A623I, and V656F. The L629F and C672Y FRTL-5 pools showed only a small increase, whereas no change was observed for the Del613-621 and V509A FRTL-5 cells (Fig. 4AGo). 2) Addition of IBMX increased cell numbers in all but one mutant population (Del613-621), the most dramatic effects being observed for the M453T and A623I mutants, which approached those seen in the presence of TSH (Fig. 4Go, B and C). 3) When cells were grown in the presence of TSH, similar increases in cell numbers were observed for all mutant, WT TSHR, and the neo FRTL-5 pools (Fig. 4CGo).



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  		FIG. 4. Difference in growth stimulation of FRTL-5 cells infected with seven activating TSHR mutants, the WT hTSHR or the neo retroviral vectors. Cell numbers were counted at d 3, 6, 8, 10, and 12 after plating 5 x 104 cells/well in three different culture conditions: A, 4H medium (without TSH), B, 4H medium + 250 µM IBMX; and C, 4H medium + 5 mU/ml TSH (5H medium). TSH independence of growth was most pronounced in the M453T, the A623I, and the V656F mutant, whereas constitutive stimulation of proliferation in the L629F and C672Y mutants is only obvious in the presence of IBMX. Similar growth dynamics were observed when cells were maintained in the presence of TSH. Results are expressed as mean numbers (103)/well of triplicates (which agree to within 5% errors too small to be detected at this scale) in one representative of four independent infection experiments.

 
To assess whether these differences in cell numbers were paralleled by induction of DNA synthesis, indicative of TSH-independent growth stimulation, BrdU labeling indices were determined in the FRTL-5 cell pools. 1) In the absence of TSH the highest percentages of BrdU-positive nuclei were obtained for M453T and A623I FRTL-5 pools followed by V656F. Low BrdU staining was observed for other mutants, the WT TSHR and neo populations (Table 1Go). 2) In the presence of IBMX induction of DNA synthesis was markedly increased, particularly in FRTL-5 pools with low basal BrdU labeling, e.g. L629F, Del613-621 (Table 1Go). This contrasted with the low or absent increase in cell numbers in the respective pools (Fig. 4BGo), suggesting either apoptosis or failure to complete the cell cycle.

Effects on function
In the absence of TSH, increased basal 125-I uptake was observed in all FRTL-5 pools stably expressing mutant TSHR (Fig. 5Go), consistent with a thyroid hot nodule detected by radioiodine scan. The highest 125-I uptake was obtained for the M453T mutant followed by the A623I, V509A, and Del613-621 FRTL-5 populations. 2) In the presence of IBMX, 125-I uptake was further enhanced with the highest uptake observed in the M453T mutant followed by C672Y, V656F, and Del613-621 (Fig. 5Go). In the presence of TSH similar 125-I uptakes were observed in all FRTL-5 cell populations, except for the L629F mutant, which exhibited marginally lower uptake than the WT and neo FRTL-5 pools (Fig. 5Go).



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  		FIG. 5. Difference in 125-I uptake in FRTL-5 cells infected with seven activating TSHR mutants, the WT hTSHR or the neo retroviral vectors—in vitro model of a hot thyroid nodule. 125-I uptake was investigated in the different FRTL-5 cells populations in 4H medium (basal), 4H medium + 250 µM IBMX, and 4H medium + 5 mU/ml TSH (5H medium). Increased basal 125-I uptake was observed in all mutant FRTL-5 pools compared with the WT TSHR and neo populations, consistent with the phenotypic scintiscan appearance of a hot thyroid nodule harboring a TSHR mutation. 125-I uptake was further enhanced in the presence of the phosphodiesterase inhibitor IBMX. Results are expressed as 103 cpm/well of triplicates in one representative of four independent infection experiments, corrected for cell numbers on the day of analysis (control, 125-I uptake in presence of NaClO4).

 
Effects on down stream signal transduction
Because an obvious dissociation was observed between constitutive cAMP generation and biological activity, notably stimulation of function but not growth, in some mutants, we started to investigate whether this could be related to altered down-stream signaling in cAMP-protein kinase A (PKA) targets. We studied protein expression of activating and repressing transcription factors of the CRE (cAMP-responsive element) binding protein CREB/CREM/ATF family in three of the mutant FRTL-5 populations, M453T, L629F, and Del613-621, representing the spectrum of TSH independence/dependence. 1) In the absence of TSH, the highest levels of p-CREB occurred in the Del613-621 mutant and were further increased 1.4-fold in the presence of IBMX, but in the presence of TSH were down-regulated from 2.6 to 1.6 relative to control neo clones (Fig. 6Go). In contrast, in M453T, p-CREB levels were fairly constant in all culture conditions, whereas L629F, in common with the neo population, required the addition of IBMX or TSH to increase p-CREB levels.



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  		FIG. 6. Differences in downstream cAMP signaling in transduced FRTL-5 cells with total (Del613-621) or partial TSH dependence (L629F) and complete TSH independence (M453T) of growth. Mutant and neo FRTL-5 populations were propagated in 4H medium (-/-), 4H medium + 250 µM IBMX (-/+), 4H medium + 5 mU/ml TSH (+/-), and 4H medium + 250 µM IBMX + 5 mU/ml TSH (+/+) until they were 80–90% confluent (7–14 d). Blots (10 µg protein/lane) were probed with an anti-Ser133-phospho-CREB antibody (top panel) or with an anti-total CREB antibody, which also detects CREM splicing isoforms, e.g. the transcriptional repressor CREMß and the inducible cAMP early repressor ICER (bottom panel). The highest basal levels of p-CREB were observed in the Del613-621 population; however, up-regulation of the CRE binding transcriptional repressors was also more pronounced and persisted, whereas p-CREB levels decreased with addition of IBMX and/or TSH.

 
To determine up-regulation of possible counterregulatory elements, blots were probed with an antibody to total CREB (43 kDa), which also reacts with a number of CREM splicing isoforms between 18 and 40 kDa. We detected a protein at approximately 25 kDa, corresponding to the transcription repressor CREMß. In the absence of TSH, this protein was most abundant in Del613-621 and L629F and least abundant in M453T (2.6-, 2.9-, and 1.2-fold compared with neo control clones, respectively; Fig. 6Go).

Although preliminary, these data suggest that, whereas high basal levels of transcriptional activator p-CREB were found in the Del613-621, indicative of its constitutive activity, inhibitory CRE binding transcriptional repressors are also up-regulated (compared with other mutants studied).

Effects on cell cycle progression
To further assess whether the apparent lack of growth stimulation in some mutants, e.g. Del613-621 or V509A, could be attributed to increased apoptosis or endoreduplication, cell cycle progression was analyzed by flow cytometry in four mutant (Del613-621, V509A, A623I, M453T), WT TSHR and neo FRTL-5 populations. In the absence of TSH the neo control consisted of 75% cells in G1 phase, similar results were obtained for the WT TSHR transduced cells. In the Del613-621 and V509A infected FRTL-5 cells, cell cycling was shifted toward G1 with approximately 86% in this phase. In contrast, in the A623I FRTL-5 pools 67% of the population were in G1 and similar results were obtained for the M453T FRTL-5 pools, indicating a shift toward proliferation. FRTL-5 pools for the other mutant TSHR were between these extremes. There was no evidence of polyploid cells beyond the G2 peak and pre-G1 apoptotic cells were 1–2%, irrespective of the presence of mutant or WT TSHR. In the presence of IBMX, the percentage of cells in G1 phase decreased to 63% in the neo and WT TSHR FRTL-5 pools. A similar effect was observed for Del613-621 and V509A FRTL-5 populations. However, in contrast to the neo, the percentage of pre-G1 cells increased to 6%/8% in V509A and Del613-621, i.e. suggesting increase of apoptosis. Of note, the cell cycle profiles of A623I and M453T FRTL-5 pools were minimally changed in the presence of IBMX and there was no evidence of polyploidy (data not shown).

These preliminary findings suggest that the lack of growth stimulation in Del613-621 and V509A, may be due to an increase in apoptosis and is compatible with results obtained from BrdU labeling.

Investigation of TSHR mutants in human primary thyrocytes
Induction of G418 resistant colonies and colony size in transduced human thyrocytes.
In 10% FCS/RPMI, three different types of colonies were observed after retroviral infection (n = 3 per retroviral construct) of four different preparations of human primary thyroctes and G418 selection, 1) epithelial; 2) variant; and 3) striated, as previously described (35).

A higher number of epithelial colonies (mean: 5.9 ± 0.8/dish) was obtained in thyrocytes transduced with mutant TSHR compared with WT TSHR [mean: 3.8 ± 0.3/dish) or neo supernatants (<1/dish)] (Table 2AGo). In contrast, similar numbers of variant colonies (mean: 2.4 ± 0.2/dish) were obtained with all retroviral supernatants. To assess the mutants’ effect on thyroid growth stimulation, the size of the epithelial colonies was determined by cell counting after 28 d of G418 selection and was found to vary considerably. The smallest epithelial colonies were obtained in the neo and WT TSHR transduced thyrocytes with mean sizes of 98 ± 8 and 127 ± 12 cells/epithelial colony, respectively, corresponding to six to seven population doublings (PD). Significantly larger epithelial colonies were observed in all mutant TSHR transduced thyrocytes: seven to eight PD (266 ± 29 cells/epithelial colony) in Del613-621, seven to nine PD (320 ± 26 cells/epithelial colony) for V509A, C672Y, and L629F and 8–11 PD (1580 ± 195 cells/epithelial colony) for M453T, A623I, and V656F infected thyrocytes.


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  		TABLE 2. Differences in colony induction and Tg staining in human primary thyrocytes infected with seven activating TSHR mutants, the WT hTSHR, or the neo retroviral supernatants

 
In 1% FCS/RPMI, no G418-resistant colonies were obtained in two independent preparations of primary human thyrocytes infected in triplicates with either the M453T, the WT TSHR, or the neo retroviral supernatants.

Effects on differentiation in transduced human thyrocytes
The thyroid origin of the G418-resistant human thyrocytes colonies was demonstrated by Tg immunocytochemistry. 1) Positive Tg staining was observed exclusively in colonies with epithelial morphology. 2) The highest number of Tg stained colonies/total number of epithelial colonies was obtained for the M453T followed by A623I, V656F, and C672Y compared with the WT TSHR transduced thyrocytes (Table 2AGo). 3) A marked difference in the degree of Tg staining (percentage of stained cells/Tg-positive colony) was observed among the neo, WT TSHR, and mutant TSHR colonies. Whereas the neo and WT epithelial colonies showed only very weak staining of less than 20% cells/colony, strong Tg staining in more than 80% thyrocytes was observed in the M453T and A623I infected thyrocytes (Fig. 7AGo, Table 2BGo). Moderate staining (30–65% of thyrocytes/colony) was obtained for the Del613-621 and the C672Y mutants followed by the L629F mutant (Fig. 7BGo, Table 2BGo). Only weak staining (20–30% cells/colony) was present in the V509A infected cells (Fig. 7CGo, Table 2BGo). The highest variablity of Tg staining was observed in V656F infected thyrocytes.



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  		FIG. 7. Difference in functional stimulation (Tg expression) in primary human thyrocytes infected with seven activating mutant, WT TSHR or neo retroviral vectors. Tg expression was investigated by immunocytochemistry and the number of Tg-stained cells/colony was determined by cell counting. Tg staining was only observed in colonies with epithelial morphology. A, High Tg expression in more than 80% of cells/colony was found in the M453T. B, Moderate staining (30–65% cells/colony) was observed in the Del613-621. C, Weak staining (20–30% cells/colony) was present in the V509A infected cells.

 
Unfortunately, the heterogeneity of clones obtained following retroviral infection of human primary thyrocytes precluded biochemical measurements, e.g. of cAMP. However, by assessment of growth stimulation (reported as epithelial colony size) and stimulation of differentiated function (reported as Tg staining) we found a similar order of biological activity for the different TSHR mutants in both the human and rat FRTL-5 thyroid context (Table 3Go).


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  		TABLE 3. Summary of results comparing biological properties of activating TSHR mutations in nonthyroid COS-7 cells (transient transfection and retroviral infection) and in rodent and human thyroid follicular cells (retroviral infection)

 
Investigation of TSHR mutants in transfected and transduced COS-7 cells
The seven TSHR mutants and the WT TSHR were transiently expressed in COS-7 cells. Compared with the WT TSHR all seven TSHR mutants induced increased basal cAMP generation in agreement with their previously reported characteristics: the Del613-621 and V656F mutants exhibited the strongest constitutive activity, whereas the C672Y and V509A mutants showed the weakest (Fig. 2AGo). A similar order was observed, when the seven TSHR mutants were stably expressed in COS-7 cells after retroviral infection, with the Del613-621 and V656F causing the strongest and C672Y the weakest constitutive cAMP stimulation (Fig. 2BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vitro, TSHR mutants differ in their degree of constitutive activation of the cAMP and inositol phosphate cascades (4, 5, 6). However, the in vitro potency does not correlate with the severity of the clinical phenotype either in toxic nodules or familial nonautoimmune hyperthyroidism. Furthermore, evaluating how closely the TSHR activation mechanisms observed in a nonthyroid cell system mimic the in vivo situation is relevant not only for TSHR modeling but also for the development of pharmaceutical TSHR superantagonist or inverse agonists, which can quench TSHR activation (36, 37).

Thus, the aim of this study was to investigate the biological activity of a panel of gain-of-function TSHR mutations in the correct thyroid cellular context and to compare these data with the mutants‘ constitutive activity in the standard nonthyroid COS-7 cell system. Introduction of the mutant TSHR into COS cells using transfection or retroviral infection produced a similar order of constitutive activity with Del613-621 and V656F displaying the strongest stimulation and C672Y the weakest. In contrast, in human and rat thyroid cells mutants A623I and M453T emerged as the most potent stimulators of proliferation and function with V509A as the least. The in vitro behavior of the TSHR mutants in human thyrocytes mirrored the biological characteristics of the same mutants in FRTL-5 (with the exception of Del613-621; Table 3Go). Furthermore, in thyrocytes, stimulation of function by the mutant TSHR was achieved more readily than increased proliferation and the functional activity correlated with the level of constitutive cAMP activation in FRTL-5 cells. This concurs with experimental data on dog thyrocytes, suggesting that lower levels of cAMP may be sufficient to maintain thyroid function than proliferation (14). In addition, the discrepancy might explain the clinical observation that thyrotoxic patients harboring an activating TSHR mutation do not always have a goiter (4, 5). In a similar context, Deleu et al. (38) recently described ex vivo characteristics of metabolism and gene expression in a series of 51 TTNs. In this study, a consistent finding was that of considerable proliferative heterogeneity as opposed to a more uniform stimulation of thyroid function assessed as thyroperoxidase immunoreactivity in the lesions.

The biological behavior of the TSHR mutants did not correlate, in human thyrocytes or FRTL-5 cells, with their constitutive activity in COS-7 cells. The observation that this discrepancy was not a result of gross mutant overexpression by transient transfection but was also sustained in retrovirally transduced COS-7 cells indicates a cell-specific effect, e.g. phosphodiesterase (PDE). Direct measurements of adenylate cyclase and PKA activity in toxic adenomas have revealed that increases in the former are not matched in the latter (39). This has been confirmed and found to be predominantly the result of increased IBMX insensitive PDE4 (40). Furthermore, PDE isoforms may even be thyroid specific, e.g. PDE8B (41). These findings emphasize the necessity for mutational studies in the correct cellular context, if the biological impact is of interest.

Four other studies have been dedicated to the elucidation of biological consequences of activating TSHR or Gs {alpha} protein mutations (gsp) in thyroid follicular cells: Muca et al. (42) have demonstrated that retroviral infection of FRTL-5 cells with a mutant gsp (Q227L) results in autonomous proliferation of FRTL-5 cells and autonomous function measured as iodide uptake, yet the extent of activation did not reach the level of TSH-dependent stimulation of untransduced FRTL-5 cells. This observation was confirmed in a study by Fournes et al. (15), comparing the in vitro behavior of a TSHR mutant (M453T) with another gsp (R201S) mutant in transfected FRTL-5 cells. In a previous study we have further investigated this important discrepancy in the context of primary human thyrocytes, clearly demonstrating that only constitutive TSHR activation (A623I) but not Gs {alpha} protein activation (Q227L) is sufficient to induce sustained proliferation in human thyrocytes (17). Several possible explanations were considered: firstly, activation of signal transduction in gsp is limited to the cAMP cascade, whereas TSHR action in thyrocytes can also involve stimulation of the phospholipase C-protein kinase C pathway (1, 14). In the present study, the TSHR mutants known to stimulate the inositol phosphate pathway in COS-7 cells, e.g. A623I and Del613-621 (3, 24), showed marked and moderate stimulation, respectively, of growth and differentiated function in human thyrocytes. This contrasted with the phenotype of the Del613-621 mutant in FRTL-5 cells. However, the finding does not entirely clarify the discrepancy of gsp and mutant TSHR effects in FRTL-5 because several studies have suggested absence of the classic phospholipase C-protein kinase C pathway in the rat cell line (43, 44, 45). Secondly, the discrepancy between gsp and mutant TSHR phenotype in human thyrocytes may be attributed to the putative ability of the TSHR to activate other oncogenic pathways (besides PKA and phospholipase C), e.g. PKA-independent MAPK (44). However, whereas the presence of this pathway is now established for the FRTL-5 cells and other rodent thyroid cell lines, the situation is less clear in human thyrocytes (45). Thus, one likely explanation for the discrepant effect of an activating Gs {alpha} and TSHR mutant on human thyroid follicular cells may be the relative lack of Gs ß- and {gamma}-subunits in the gsp-infected thyroid cells. Although the functional significance of these Gs subunits has not been extensively studied in the thyroid, experiments in other cell lines have suggested a role in growth regulation, linking Gs ß- and {gamma}-subunits to ras and putatively PI3K signal transduction (47).

In another study Porcellini et al. (16) reported that functional properties of four activating TSHR mutants (F631C, T623I, D633Y, D633E) were similar in transfected FRTL-5 cells and COS-7 cells. However, their selection of FRTL-5 cell clones was based on G418 resistance alone, which in our experiments yielded large numbers of clones even in the control populations. Furthermore, we have used a subclone of the FRTL-5 cell line reselected for TSH-dependent growth and function because this characteristic can be lost by FRTL-5 over time (48). The experiments of Porcellini et al. (16) and ourselves were performed on pooled FRTL-5 cell clones to correct for the random process of gene insertion. This was important because 1) disruption of a critical signaling/cell regulatory molecule might occur, which per se could induce changes in cellular function and 2) variable TSHR expression might be induced. To rule out further artefacts by stable gene transfer, an attempt was made to quantify mutant TSHR expression in the FRTL-5 cell clones. No differences were observed at the protein level by flow cytometry compatible with a low level of both endogenous and exogenous TSHR expression. Quantitative real-time PCR analysis confirmed that biological effects of the mutant TSHR constructs were not correlated with the level of the respective mutant TSHR transcripts.

In terms of in vitro behavior, the deletion mutation of nine amino acids (Del613-621) in the third intracellular TSHR loop shows the most apparent dissociation between the thyroid and nonthyroid cell context. It exhibited the strongest constitutive activity (level of cAMP generation plus stimulation of the IP pathway) in COS-7 cells, albeit at very low cell surface expression (49). However, only moderate functional activity and no stimulation of growth was observed in the Del613-621 FRTL-5 populations and only moderate induction of epithelial colony formation occurred in human primary thyrocytes. Because mRNA levels were comparable with other TSHR mutants in infected FRTL-5 cells reasoning from these findings suggested, that stimulation of counterregulatory signaling pathways by the Del613-621 could account for this discrepancy. Because addition of IBMX did not increase the proliferation in the Del613-621 FRTL5, the counteracting effects most likely arise from interference with signaling further downstream in the cAMP pathway. This hypothesis was tested in three TSHR mutants displaying a spectrum of TSH dependence. Our preliminary results indicate that the lack of proliferation in Del613-621 occurs despite having higher levels of pCREB than M453T. However, this is accompanied by increased expression of inhibitory CREM isoforms in Del613-621 compared with M453T. The results support the hypothesis of either cell cycle blockade or apoptosis as an explanation for the low proliferation of Del613-621.

Flow cytometry analysis of different FRTL-5 cell populations indicated that the lack of growth stimulation in some mutants, e.g. Del613-621 or V509A, could be attributed to an increase in pre-G1 cells in the presence of IBMX. Because pre-G1 cells can correspond to an apoptotic cell populations the observed discrepancy may be explained by up-regulation of apoptosis. It is also compatible with the results of increased DNA synthesis observed with BrdU labeling, which again were not reflected in growth stimulation. This does not, however, argue against a role for the Del613-621 and V509A mutants, which exhibited an effect on iodine uptake and so will produce a toxic adenoma albeit slowly.

In summary, this is the first study describing functional and morphological aspects of a panel of different activating TSHR mutations in vitro in rat and human primary thyrocytes. Data obtained in these models suggest that different biological properties of the TSHR mutants may result in different in vivo phenotypes. Thus, whereas the final outcome presenting to the clinicians will undoubtedly be modified by additional not yet fully understood genetic and epigenetic events, it is highly likely that TSHR mutants may, at the minimum, trigger thyroid autonomy at different levels. Our study highlights that biological effects of oncogenic mutations must be investigated in the correct cellular context and that net effect cannot be deduced purely on second messenger analysis. It will be challenging to apply these findings to TSHR modeling, since biochemical characteristics of gain-of-function TSHR mutants in COS-7 cells are clearly not representative of the physiological or pathophysiological scenario in the thyroid.


   Acknowledgments
 
We are indebted to BRAHMS Diagnostica GmbH (Berlin, Germany) for generously providing us with radiolabeled TSH tracer and to Professors Gilbert Vassart (Brussels, Belgium) and Edwin Milgrom (Paris, France) for the gift of some of the mutant TSH receptors used in this study.


   Footnotes
 
D.F. is supported by a research grant from BASF/Studienstiftung des Deutschen Volkes.

Abbreviations: BrdU, 5-Bromo-2'deoxyuridine-5-monophosphate; CRE, cAMP-responsive element; CREB, cAMP response element binding protein; CREM, cAMP regulatory element modulator; FCS, fetal calf serum; hTSHR, human TSHR; IBMX, isobutylmethylxanthine; ICER, inducible cAMP early repressor; PD, population doubling; PDE, phosphodiesterase; PKA, protein kinase A; Tg, thyroglobulin; TM, transmembrane; TSHR, TSH receptor; WT, wild-type.

Received April 8, 2003.

Accepted for publication May 7, 2003.


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 Results
 Discussion
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R. Moreno-Reyes, B.-N.-T. Tang, A. Seret, S. Goldman, C. Daumerie, and B. Corvilain
Impaired Iodide Organification in Autonomous Thyroid Nodules
J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4719 - 4724.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Endocrinol. Metab.Home page
I. Cozma, L. Zhang, J. Uddin, C. Lane, A. Rees, and M. Ludgate
Modulation of expression of somatostatin receptor subtypes in Graves' ophthalmopathy orbits: relevance to novel analogs
Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1630 - E1635.
[Abstract] [Full Text] [PDF]

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L. Zhang, G. Baker, D. Janus, C. A. Paddon, D. Fuhrer, and M. Ludgate
Biological Effects of Thyrotropin Receptor Activation on Human Orbital Preadipocytes
Invest. Ophthalmol. Vis. Sci., December 1, 2006; 47(12): 5197 - 5203.
[Abstract] [Full Text] [PDF]