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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 9 4472-4480
Copyright © 2003 by The Endocrine Society

Adenovirus-Mediated Expression of Dominant Negative Fibroblast Growth Factor (FGF) Receptor 1 in Thyroid Cells Blocks FGF Effects and Reduces Goitrogenesis in Mice

Emma L. Davies, James D. Ramsden, Helen Cocks, Peter F. Searle, John C. Watkinson, Hikaru Ueno, Ann Logan, Jayne A. Franklyn, Vivien Mautner and Margaret C. Eggo

Division of Medical Sciences (E.L.D., J.D.R., H.C., A.L., J.A.F., M.C.E.) and Cancer Research UK United Kingdom Institute for Cancer Research (P.F.S., V.M.), University of Birmingham, and Department of Otolaryngology, University Hospital Birmingham National Health Service Trust (J.C.W.), Birmingham, United Kingdom B15 2TT; and University of Occupational and Environmental Health School of Medicine (H.U.), Kitakyushu 807-8555, Japan

Address all correspondence and requests for reprints to: Dr. M. C. Eggo, The Medical School, University of Birmingham, Birmingham, United Kingdom B15 2TT. E-mail: m.c.eggo{at}bham.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Levels of fibroblast growth factor 2 (FGF-2) and its receptor, FGFR1, are elevated in goiter, but whether this is a direct effect of TSH is unknown. We have determined the regulation of FGF-2 and FGFR1 synthesis by TSH in a rat thyroid cell line (FRTL5) and have used a replication-defective adenovirus (RAd) expressing dominant negative FGFR1 (RAdDN-FGFR1) to examine the role of FGFR signaling in vitro and in goiter induced in mice. TSH induced FGF-2 and increased the expression of FGFR1 in FRTL5 cells. Infection of TSH-stimulated FRTL5 cells with RAdDN-FGFR1 inhibited growth and prevented FGF-2-mediated inhibition of 125I uptake. Similar effects were found in primary cultures of human thyroid follicular cells. For in vivo experiments, male BALB/c mice were injected systemically with RAdDN-FGFR1 or RAd encoding green fluorescent protein, and goiter was simultaneously induced. Mouse thyroid follicles were shown to be transduced with RAd encoding green fluorescent protein. Circulating TSH was elevated comparably in the two groups. In the RAdDN-FGFR1-injected animals, goiter induced over 14 d was significantly smaller, and the vascular volume increase seen in goiter was also diminished. We conclude that the FGF axis is important in thyroid growth and that RAdDN-FGFR1 effectively blocks FGF actions, offering a means to control goitrogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FIBROBLAST GROWTH FACTORS 1 and 2 (FGF-1 and FGF-2) are elevated in human thyroid hyperplasia (1, 2), and elevated levels of FGF-2 and its receptor (FGFR1) mRNAs and protein are found in thyroid follicular cells in goitrogen-treated rats (3, 4). We have recently shown that FGF-2 is a mitogen for normal human thyrocytes in vitro (5) as it is for the rat thyroid cell line, FRTL5 (6, 7). Consistent with this, in vivo studies in rats have shown that FGF-1 administration induces goiter (8). TSH is the prime mediator of thyroid growth and function and is elevated when circulating levels of thyroid hormones fall. Whether TSH directly mediates the increase in FGFR1 and FGF-2 production in thyroid hyperplasia or whether the effects are indirect is not known. Furthermore, the relevance of their elevated expression to goitrogenesis is as yet unproven.

FGFR1 is one of four high-affinity receptors for FGFs, having a common structure of extracellular immunoglobulin-like loops including a characteristic acid box region, a transmembrane portion, and an intracellular tyrosine kinase domain (9). Binding of the ligand (FGF-1 to -6 and FGF-10) to the receptor initiates receptor dimerization and stimulates a signal cascade mediated through the intracellular tyrosine kinase domain. Overexpression of FGFR1 lacking the tyrosine kinase domain results in receptors that dimerize, but can no longer signal. On ligand binding, the defective receptors form nonfunctional heterodimers with endogenous full-length receptors, thereby inhibiting receptor-mediated signal transduction. The dominant negative (DN) action of the protein encoded by a plasmid containing truncated FGFR1 was shown to be specific for FGFR when injected into Xenopus oocytes (10, 11). One aim of our study was to construct a replication-defective recombinant adenovirus encoding DN-FGFR1 for use in vivo and in vitro to determine the effects of blocking FGF signaling on thyroid follicular cell growth and function.

Adenovirus-mediated gene delivery offers several advantages over cell transfections. In vitro the efficiency of virus transduction can approach 100%, higher than that achieved with transfection; furthermore, in vivo the use of adenovirus constructs offers the possibility of gene therapy. Studies have shown that this mode of gene delivery is clinically safe, causing no adverse events when used at low/moderate doses (12). The replication-defective adenovirus DNA is not integrated into the host genome, but remains nuclear where its effects are relatively short-lived, especially in vivo and in a dividing population (13, 14). Differentiated thyroid tumors are the most common endocrine malignancy, and anaplastic thyroid carcinoma, although rare, is rapidly fatal. Nonmalignant goiter is common, affecting 1 in 20 in the population of the Western world, even in iodine-replete populations. To date, gene therapy approaches in vitro and in animal models have shown that thyroid cells are readily infected with viruses of different types (15, 16, 17), and that suicide genes, under the control of the rat thyroglobulin promoter, can be introduced into thyroid cells by adenovirus in a mouse model in vivo (18).

These studies demonstrate that goiter, either malignant or physiological, is an attractive potential target for gene therapy using agents that block follicular cell growth. FGF receptor signaling is important in angiogenesis, an essential component of tissue expansion in goiter, and we hypothesized that by blocking FGF signaling in the thyroid, both thyroid endothelial and thyroid follicular cell growth would be limited. For the in vitro studies to determine the role of FGF signaling in thyroid follicular cells, we used primary cultures of functional human thyroid cells and FRTL5 cells, a rat thyroid cell line. FRTL5 cells remain responsive to TSH, both as a mitogen and to stimulate differentiation, and are exquisitely sensitive to FGF-2 as a mitogen and as an inhibitor of 125I uptake (19). Human thyroid cells also show inhibition of thyroid function with FGF-2 and stimulation of thyroid growth (5). To determine the effects of blocking FGFR1 signaling on goitrogenesis in vivo, we used the DN-FGFR1 (RAdDN-FGFR1) virus in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

FRTL-5 cells were a gift of the Interthyr Foundation and were grown in 5% newborn bovine calf serum in the medium described by Ambesi-Impiombato et al. (20) supplemented with 1 mg/liter insulin and 0.3 nmol/liter TSH (equivalent to 300 mU/liter TSH using a conversion of 1 mg pure TSH = 30 IU). Human thyroid cells were isolated and cultured as described by Eggo et al. (21). In brief, thyroid tissue obtained at surgery in accordance with local ethical guidelines was used. Multinodular goiter removed to relieve tracheal compression or thyroid tissue removed as treatment of Graves’ disease was trimmed of extraneous connective tissue and chopped finely with scalpel blades. After 3-h digestion at 37 C in 0.1% collagenase to release follicles, the material was repeatedly washed by centrifugation. Follicles were plated initially in 1.0% calf serum in the medium described above for FRTL5 cells. At the first medium change, serum was omitted from the medium, and cells were cultured without serum thereafter. Medium was changed twice weekly. Cells were screened for their ability to take up and organify iodide and those cultures that showed TSH-dependent iodide uptake and organification (described later) were used in these studies. We used these parameters to confirm that the cells expressed full, differentiated function and were responsive to TSH.

Construction of recombinant Ad-CMV-FGFR1N virus (RAd-FGFR1N)

FGFR1 cDNA was provided by Dr. I. J. Mason (KCL, London, UK) in the expression vector pKC3 (22). PCR primers were designed to obtain a truncated (1.4-kb) fragment of the FGFR1 cDNA. The 5' primer introduced the sequence CCACC immediately upstream of the ATG initiation codon to favor optimal translation in eukaryotic cells (23). The 3' primer truncates FGFR1 at the junction of the transmembrane and intracellular tyrosine kinase domain, introducing the stop codon TGA following Asp468. This was immediately followed by the sequence GGATCC (BamHI site). Hot start PCR was carried out using Pfu polymerase (Promega, Southampton, UK) and an annealing temperature of 61 C with extension at 72 C for 2 min. The resulting 1.4-kb PCR product was gel purified, kinased, and inserted into the HpaI site downstream of the cytomegalovirus (CMV) immediate-early promoter in the retroviral vector plasmid pxLNCX (24) to yield plasmid pxLNC-DN-FGFR1 (DN truncation of FGFR1). The 2.2-kb BamHI fragment containing the CMV promoter and the DN-FGFR1 open reading frame were subcloned into the BamHI site of the adenovirus E1 region replacement vector pSW115a5 containing Ad5 sequences 1–359 and 3328–10594. This plasmid is similar to pPS971C5 (25), but with the EcoRI site at the left end of the viral genome converted to a SwaI site. The E1 region has been replaced by a unique BamHI cloning site and RNA splice and poly(A)/termination signals from human ß-globin and complement C2 genes, respectively. Recombinant virus was rescued in 911 cells by overlap cotransfection with a plasmid encoding the remainder of the Ad5 genome bearing an E3 deletion as previously described (26, 27). RAdDN-FGFR1 was plaque-purified, grown, and titrated as previously described (28) to give virus stocks for in vitro experiments. For in vivo experiments, virus stocks were purified by centrifugation on CsCl gradients and dialyzed into PBS containing 10% glycerol (10 mM Na2HPO4, 2.7 mM KCl, 137 mM NaCl, 10 mM CaCl2, 0.5 mM MgCl2, and 10% glycerol, pH 7.4) (29). The particle number was determined by assaying the DNA content of purified virus, denatured by heating to 56 C for 30 min, using a Picogreen DNA assay kit (Molecular Probes, Eugene, OR), and infectivity was determined by plaque assay. One microgram of DNA equals 2.7 x 1010 viral particles.

Recombinant adenovirus RAdFGF-2 codes for an FGF-2 that is modified by incorporating a signal sequence tag derived from FGF-4 so that its secretion is facilitated (30). The s-FGF-2 cDNA is under a CMV enhancer and chicken ß-actin promoter. We used two control recombinant adenovirus, viz. RAd35, which is an E1/E3-deleted adenovirus carrying the Escherichia coli lacZ gene encoding ß-galactosidase (31), a gift from Dr. Gavin Wilkinson (Cardiff University, Cardiff, UK), and an E1/E3-deleted adenovirus expressing green fluorescent protein (RAdGFP).

Assay for ß-galactosidase

Forty-eight hours after RAd35 treatment, cells on culture dishes were washed with Hanks’ Balanced Salt Solution (HBSS) and incubated for 10 min at 4 C in PBS containing 2% paraformaldehyde and 0.2% glutaraldehyde. Cells were washed twice with PBS and incubated in PBS containing 1 mg/ml 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside. The plate was left at room temperature for 4 h, at which time blue staining denoting ß-galactosidase activity was evident.

Infection of primary human thyrocytes

For studies of effects on cell growth, cells were plated at a density of 50,000 cells/cm2 and infected 48 h after isolation at a multiplicity of infection (MOI) of 20 plaque-forming units (pfu)/cell of RAd35 or RAdDN-FGFR1. Infection with RAd was performed in 0.2 ml serum-free medium in 2-cm2 culture wells for 90 min with occasional agitation. The virus-containing medium was removed and replaced with medium without serum but containing insulin and TSH. [Methyl-3H]thymidine (80 Ci/mmol) was added from 0–4 or 4–7 d after infection, when experiments were terminated by removing the cell medium, washing the cell layer with HBSS, and precipitating the cells with 6% trichloroacetic acid. Where used, epidermal growth factor (EGF; 10 nmol/liter) was included in the medium, added to the cells after the 90-min adsorption period.

For studies of effects on cell function, cells were cultured to 50–70% confluence for 3 d in 24-well plates. RAd-FGF-2 was used at a MOI of 0.1 pfu/cell; RAdDN-FGFR1 and RAd35 were used at MOI of 20 pfu/cell. The final MOI in all cultures was 20.1 pfu/cell. Infection with RAd was performed in 0.2 ml serum-free medium for 90 min with occasional agitation. The virus-containing medium was removed and replaced with complete medium, and cells were incubated for 3–4 d. Function was assayed by the addition of NaI (final concentration, 10-7 mol/liter) and 0.05 µCi/125I/well for 2 h. After incubation, medium was removed, and the cell layer was immediately and rapidly washed with HBSS. Cells were solubilized in 1% sodium dodecyl sulfate, and radioactivity was determined using a {gamma}-counter.

Western blotting

Cell layers were solubilized using 1% sodium dodecyl sulfate. Protein content was determined, and 50 µg were analyzed by SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride nylon membrane (Amersham Pharmacia Biotech, Little Chalfont, UK) and probed with a monoclonal antibody to the N terminus of the chicken FGFR1 (Upstate Biotechnology, Lake Placid, NY) that cross-reacts with rat FGFR1 or with polyclonal antibodies to the C terminus of FGFR1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Polyclonal antibodies specific for FGF-2 were obtained from Santa Cruz Biotechnology, Inc. Secondary antibody labeled with horseradish peroxidase (Santa Cruz Biotechnology, Inc.) was used to visualize bands by chemiluminescence using Lumiglow (Kirkegaarde & Perry Laboratories, Gaithersburg, MD). For controls, preincubation of antibody with blocking peptide (Santa Cruz Biotechnology, Inc.) prevented labeling of bands, confirming antibody specificity.

In vivo studies

All in vivo studies were undertaken in accordance with United Kingdom Home Office procedures and appropriate local controls. BALB/c male inbred mice were used for all experiments. They were 8 wk old at the start of the experiments and weighed between 20–30 g. Only full-grown, male mice were used to ensure that there was no active angiogenesis associated with estrus or growth occurring.

After acclimatization to their environment for 5 d, mice (minimum of four per group) were injected with 1010 viral particles (either RAdGFP or RAdDN-FGFR1) by tail vein injection. Diet was simultaneously changed to low iodine chow (<0.05 ppm iodide; Lillico Biotech, Surrey, UK) mixed with an equal weight of water containing 0.15% methimazole (MMI). The drinking water was supplemented with 1.0% sodium perchlorate, which we found was necessary to induce goiter. The weights and well-being of the mice were monitored daily. No animal lost more than 5.4% body weight during the course of these treatments. After 14 d, mice were anesthetized with flurane and 0.4 mg of the lectin, rhodamine giffonia (Vector Laboratories, Inc., Peterborough, UK), which binds to blood vessels, were injected via the tail vein as described by Debbage et al. (32). After 2 min, mice were killed by cervical dislocation, blood was collected, and tissues were removed for weighing. The thyroids were dissected out, washed, stored in HBSS at 4 C, blotted, and weighed within 1 h on a small piece of foil using a microbalance.

RIA of serum levels of total T3 and T4

Total T3, total T4, and TSH in serum of control and treated mice were measured after centrifugation of clotted blood samples. The supernatant was used in RIA kits supplied by ICN Pharmaceuticals (High Wycombe, UK). The rat TSH kit was used to measure mouse serum TSH values. This assay has a within-assay variation of 2.9% when measured in the midrange of the standard curve. The manufacturers report that there is no cross-reactivity with other pituitary glycoprotein hormones.

Determination of vascular volume density (Vv)

To analyze the alteration in vascularity in the developing mouse goiter, the mice were injected iv with 0.4 mg rhodamine-labeled lectin. Whole thyroid glands were optically sectioned within 2 h using a confocal microscope, and images were captured at 10-µm intervals to allow independence of separate sections. The vascular Vv was calculated using stereological techniques described by Howard and Reed (33). It is a dimensionless value of the proportion of the tissue that is composed of vasculature. To prevent overestimation of the vascular volume, the sections must be as thin as possible and be independent of each other so that the same blood vessel is not imaged in more than one section. The confocal microscope allows the sections to be less than 5 µm thick, and by sectioning every 10 µm, each section is independent from surrounding slices. For analysis, z-series of images were collected at x200 magnification and with 10-µm steps. The z-series were taken from at least three thyroid glands in each group, and for each gland three series of four sections were taken. The vascular volume in the first section was higher than sections within due to orientation effects of the vessels lying parallel to the surface. However, this effect is constant, so the first four sections were counted (corresponding to 0, 10, 20, and 30 µm deep into the thyroid) in all samples. In none of the thyroids analyzed was extrathyroidal tissue seen by confocal microscopy. The images of the thyroid vasculature were projected on a stereological grid. Grid intersections overlying the vasculature were point counted and divided by the total area of the sample to give the vascular Vv of the specimen.

Statistics

ANOVA with Student-Newman-Keuls post test was used to analyze the results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of TSH and cAMP on FGF-2 expression in FRTL5 cells

To determine whether FGF-2 expression is regulated by TSH and other agents that increase intracellular cAMP, cells were treated with TSH, forskolin (which directly activates adenylate cyclase), and bromo-cAMP (8BrcAMP), a cell-permeable analog of cAMP, and Western immunoblotting for FGF-2 of cell lysates was performed. A representative blot of three performed is shown in Fig. 1AGo. FGF-2 was detected as several bands between 32-18 kDa (lanes 1–6), which were also seen in the recombinant preparation of FGF-2 (lane 7). Forskolin (lanes 2 and 3), TSH (lane 4), and 8BrcAMP (lanes 5 and 6) all increased the expression of FGF-2 in the cell layer compared with that in control untreated cells (lane 1).



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  		FIG. 1. Effects of TSH and agents that increase intracellular cAMP on the expression of FGF-2 in FRTL5 cells. A, Cells were incubated in forskolin (10 and 100 µmol/liter; lanes 2 and 3), TSH (300 mU/liter; lane 4), and 8BrcAMP (1 and 5 mmol/liter; lanes 5 and 6) for 72 h before analysis of total cellular protein by Western blotting using antisera to FGF-2 (Santa Cruz Biotechnology, Inc.). Lane 7, Positive control of recombinant FGF-2; lane 1, control, unstimulated cells. B, Western blotting showing dose response to TSH after 72-h incubation in the indicated concentrations of TSH. The histogram shows data from ELISA of intracellular FGF-2 levels from cells incubated in the TSH concentrations shown for 3.5 d ({square}) or 7 d ({blacksquare}). The mean ± SEM are shown (n = 3). For all figures and tables, significance is denoted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
To examine the effects of TSH on FGF-2 expression in more detail, a dose response using varying concentrations of TSH was performed, and Western blotting and ELISAs were used. The results are shown in Fig. 1BGo. Western blotting showed a marked increase in FGF-2 expression when cells were treated for 72 h with 0.3 nM TSH. ELISAs of cell lysates shown in the histogram in Fig. 1BGo confirmed the Western blotting data. Two different experimental protocols were used. After 3.5-d culture in the TSH concentrations shown ({square}), FGF-2 expression was lowest in the control cells, and the TSH-treated cells showed a dose-dependent increase in FGF-2 expression. After 1 wk in culture without TSH ({blacksquare}), basal levels of FGF-2 were not measurable, and detectable amounts were found only in cells cultured in 100 mU/liter (0.1 nmol/liter) TSH and higher. At the highest TSH concentration (10 U/liter), the effects were reduced.

Effects of TSH and cAMP on FGFR1 expression in FRTL5 cells

Regulation of FGFR1 expression by TSH and other agents that increase intracellular cAMP was also examined by Western immunoblotting of cell lysates from cells treated with TSH, forskolin, and 8BrcAMP. FGFR1 was detected as the full-length polypeptide of 120 kDa and a fragment of 87 kDa in extracts of FRTL5 cells as shown in Fig. 2AGo. For the densitometry measurements, which are shown beneath the figure, the sum of the immunoreactive bands detected was used, and the histogram shows the results from three pooled experiments. After 72-h incubation, forskolin (lanes 2 and 3), TSH (lane 4), and 8BrcAMP (lane 5) had increased expression of FGFR1 compared with controls (lanes 1 and 6).



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  		FIG. 2. The effects of TSH and agents that elevate cAMP on FGFR1 expression in FRTL5 cells analyzed by Western blotting using polyclonal antisera to the C terminus of FGFR1 (Santa Cruz Biotechnology, Inc.). A, Cells were incubated in forskolin (10 and 100 µmol/liter; lanes 2 and 3), TSH (300 mU/liter; lane 4), and 8BrcAMP (5 mmol/liter) for 72 h before analysis of total cellular protein by Western blotting. Control unstimulated cells are shown in lanes 1 and 6. Densitometry is shown beneath the figure (n = 3; mean ± SEM). B, Western blotting showing a dose response to TSH after 72-h incubation in the indicated concentrations of TSH. Densitometry is shown beneath the figures (n = 3; mean ± SEM).

 
To examine the effects of TSH on FGFR1 expression in more detail, a dose response using varying concentrations of TSH was performed, and Western blotting was used with densitometry for quantitation. A representative blot is shown in Fig. 2BGo, and densitometry, which is shown in the histogram beneath the figure, shows the results from three pooled experiments that employed the sum of the immunoreactive bands detected. The predominant band is the 87-Da fragment with only faint labeling of the 120-kDa form. After 72-h incubation, TSH treatment dose-dependently increased FGFR1 expression.

Expression of RAdDN-FGFR1 in thyroid cells

To test the ability of FRTL5 cells and primary cultures of human thyroid cells to express a transgene from an adenovirus vector, cells were infected with RAd35 at various MOI, and the functional activity of ß-galactosidase was assayed in situ. Positive staining within cells is shown in Fig. 3AGo. Thyroid cell cultures were readily infected with adenovirus, and the follicular cells from both species showed similar multiplicity-dependent gene expression.



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  		FIG. 3. A, Expression of ß-galactosidase in FRTL5 and human thyroid cells infected with RAd35 at various MOIs. Enzyme activity was assayed, as described in Materials and Methods, on cells infected for 72 h with RAd35. B, Expression of truncated DN-FGRFR1 in RAdDN-FGFR1-infected human thyroid cells. Two preparations of human thyroid cells are shown. Lane 1, RAd35; lane 2, RAd35 plus RAdFGF-2; lane 3, RAdDN-FGFR1; lane 4, RAdDN-FGFR1 plus RAdFGF-2.

 
The expression of DN-FGFR1 in primary cultures of human thyroid cells 72 h after infection with RAdDN-FGFR1 was detected by Western blotting using a monoclonal antibody to the N terminus of chicken FGFR1 as shown in Fig. 3BGo. The antibody identified a single band of approximately 80 kDa, which was only detectable in cells infected with RAdDN-FGFR1 (lanes 3 and 4). Cells infected with RAd35 alone (lane 1) and with RAd35 and RAdFGF-2 (lane 2) did not express this protein. The expression of DN-FGFR1 was unaffected by coinfection with RAdFGF-2 (lane 4).

Effects of RAdFGF-2 and RAdDN-FGFR1 on FRTL-5 thyroid cell function

To examine the effects of RAdDN-FGFR1 expression on FRTL5 cell function, cells were infected with varying MOI of RAdDN-FGFR1 or RAd35 (Fig. 4Go). They were coinfected with RAdFGF-2 ({blacksquare}) or RAd35 ({square}) at an MOI of 0.1 and incubated for 3 d, at which time uptake of 125I was determined as described in Materials and Methods. Infection with RAdDN-FGFR1 or RAd35 did not inhibit 125I uptake even at an MOI of 60. Coinfection with RAdFGF-2 inhibited 125I significantly to 15% of the control values. RAdDN-FGFR1 at an MOI of 6 and higher reversed the inhibition of 125I uptake. whereas RAd35 had no effect on RAdFGF-2 inhibition. Addition of recombinant FGF-2 (PeproTech, London, UK; 30 ng/ml) in place of RAdFGF-2 had similar effects (data not shown).



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  		FIG. 4. Effect of RAdDN-FGFR1 on RAdFGF-2 effects on 125I uptake in FRTL5 cells. Cells were infected with RAd35 or RAdDN-FGFR1 at varying MOI, shown on the abscissa ({square}). Half of the cultures were also infected with RAdFGF-2 (MOI, 0.1 pfu/cell; {blacksquare}), and after 72-h incubation, 125I uptake was determined. Data are the mean ± SD (n = 4).

 
Effects of RAdDN-FGFR1 and RAdFGF-2 on human thyroid cell function

RAdDN-FGFR1-infected or RAd35-infected cultures of primary human thyroid cells were incubated for 3 d with or without RAdFGF-2, at which time uptake of 125I was determined as described in Materials and Methods (Fig. 5Go). When cells were transduced with both RAd35 and RAdFGF-2 ({blacksquare}), 125I uptake was inhibited significantly compared with that by cells transduced with RAD35 alone (Fig. 5AGo); however, when cells were transduced with RAdDN-FGFR1 and RAdFGF-2, the inhibition of 125I uptake seen with RAdFGF-2 was lost (Fig. 5AGo). RAdDN-FGFR1 did not inhibit 125I uptake. The same experiment was performed, substituting recombinant FGF-2 (30 ng/ml) for RAdFGF-2, and similar data were obtained.



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  		FIG. 5. A, Effect of RAdDN-FGFR1 on FGF-2 effects on human thyroid cell 125I uptake. Cells were infected with RAd35 or RAdDN-FGFR1 at the same MOI ({square}). Half of the cultures were also infected with RAdFGF-2 ({blacksquare}), and after 72-h incubation, 125I uptake was determined. Data are the mean ± SD (n = 4). B, Specificity of the effects of RAdDN-FGFR1 on human thyroid cells. Cells were infected with RAd35 or RAdDN-FGFR1 at the same MOI ({square}). After infection, fresh medium containing EGF (10 nmol/liter) was added to some cells ( ). After 72-h incubation, 125I uptake was determined. Data are the mean ± SD (n = 4).

 
Specificity of RAdDN-FGFR1 for FGF-2 signaling

To check that the effects of RAdDN-FGFR1 were specific for FGFR1, human thyroid cells were infected with RAdDN-FGFR1, and the effects of EGF, which, like FGF-2, inhibits thyroid function (21) were examined. EGF also acts via a dimerizing receptor coupled to tyrosine kinase. As shown in Fig. 5BGo, EGF (10 nmol/liter) () inhibited thyroid function measured by 125I uptake. RAdDN-FGFR1 did not prevent the inhibitory effects of EGF.

Effect of RAdDN-FGFR1 on human and FRTL5 cell growth

Human thyroid cells were infected with RAd35 or RAdDN-FGFR1 24–48 h after plating at 50,000 cells/cm2 when cells were growing rapidly. Growth was assessed by [methyl-3H]thymidine incorporation into cell DNA 4–7 d after infection. Significant decreases in [methyl-3H]thymidine incorporation into DNA were seen in RAdDN-FGFR1-transduced cells compared with RAd35-transduced cells, as shown in Fig. 6Go.



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  		FIG. 6. Effects of RAdDN-FGFR1 on incorporation of [methyl-3H]thymidine into primary cultures of human thyroid follicular cells and FRTL5 cells. Human cells were infected 48 h after isolation at an MOI of 20 pfu/cell of RAd35 or RAdDN-FGFR1. [Methyl-3H]thymidine (80 Ci/mmol) was included 4–7 d after infection. For FRTL5 cells, infection was the same as for human cells, but [methyl-3H]thymidine incorporation into DNA was measured 1–3 d after infection. Data are the mean ± SD (n = 4).

 
For experiments using FRTL-5 cells, cultures were infected in 6H medium (20), and growth was examined by [methyl-3H]thymidine incorporation, added 24 h postinfection and incubated for 72 h. There were significant decreases in [methyl-3H]thymidine incorporation into DNA, as shown in Fig. 6Go.

Effect of RAdDN-FGFR1 on morphology changes induced in FRTL5 cells

We observed that when cells were treated with either RAdFGF-2 or exogenous FGF-2, a change in FRTL5 cell morphology was apparent. Figure 7AGo shows control cells treated with RAd35 alone for 72 h, and Fig. 7BGo shows RAd35-infected cells treated with FGF-2 for 72 h. The cells at the periphery of the colonies of the FGF-2-treated were flattened. When cells were treated with RAdDN-FGFR1 colonies were smaller, consistent with the reduction in growth, although this is not evident on a micrograph of this magnification (Fig. 7CGo). When FGF-2 was added to RAdDN-FGFR1-infected cells, the morphology changes induced by FGF-2 were not seen (Fig. 7DGo).



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  		FIG. 7. Effects of FGF-2 on FRTL5 cell morphology and its reversal by RAdDN-FGFR1. FRTL5 cells were infected with RAd35 (A and B), RAdDN-FGFR1 (C and D), and recombinant FGF-2 (30 ng/ml; B and D) as described in Materials and Methods, and incubation was continued for 6 d. FGF-2 (and RAdFGF-2) both produced in FRTL5 cells a characteristic morphology, with larger colonies containing flattened, larger cells (B). Infection with RAdDN-FGFR1 prevented these changes (D). Magnification, x400.

 
Thyroid expression of adenovirus in BALB/c mice

For the in vivo studies, evidence that mouse thyroid can be infected with adenovirus administered via a tail vein injection was sought. We used RAdGFP for these studies to allow direct visualization of GFP expression with a fluorescence microscope. Mice received 1010 virus particles of RAdGFP and 60 h later were anesthetized and killed by cervical dislocation. Thyroids were removed, digested for 1 h with collagenase, and plated overnight on glass slides in the medium used for human thyroid cells. Slides were air-dried and examined under white light (Fig. 8Go, A and C) and fluorescence (Fig. 8Go, B and D) for the expression of GFP. GFP can be seen in the fluorescence micrographs of thyroid follicles from RAdGFP-infected mice (Fig. 8DGo), but not in the controls (Fig. 8BGo).



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  		FIG. 8. Light (A and C) and fluorescence (B and D) micrographs of mouse thyroid follicles after infection in vivo with RAdGFP for 60 h and culture for 1 d. A, Light micrograph of control mice thyroid follicular cells; B, fluorescence field of the cells shown in A; C, light micrograph of RAdGFP-infected mousethyroids; D, fluorescence micrograph of the same field as C. Magnification, x100.

 
Effect of RAdDN-FGFR1 infection on goiters induced in mice

To determine the role of the elevated FGF and FGFR1 in goitrogenesis, an in vivo model, described in Materials and Methods, was used. The effect of RAdDN-FGFR1 on goiter induction in mice, measured by the weight of thyroid lobes, is shown in Table 1Go. Mice treated with the goitrogenic regimen with or without RAdGFP showed a greater than 2-fold increase in thyroid weight. The difference between goiter weights in goitrogen-treated mice and goitrogen-treated mice with RAdGFP infection was small and not statistically significant. Mice infected with RAdDN-FGFR1 showed a statistically significant reduction of 59% in goiter weight compared with the RAdGFP, goitrogen-treated mice. Vascular Vv for the thyroids from control animals not treated with goitrogens and from goitrogen-treated animals treated simultaneously with RAdGFP and RAdDN-FGFR1 are shown in the Table 1Go. There was an almost 2-fold increase in vascular Vv in the goitrogen-treated animals, in addition to the 2-fold increase in thyroid weight in these animals. Animals injected with RAdDN-FGFR1 showed a reduction in vascular Vv, but this was not as great as the reduction in goiter weight, and it was significantly higher than the control value. Also shown in Table 1Go are the weights of the mice at the termination of the experiment, expressed as a percentage of the starting weight and the total T3, total T4, and serum TSH levels in the mice. All mice lost a little weight through the course of the experiment, but there was no significant difference between the mice infected with RAd and the goitrogen-treated controls. Parameters of thyroid function showed that TSH was elevated, and T3 and T4 were severely depressed in all goitrogen-treated mice, and there was no statistically significant difference in these parameters between any of the groups.


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  		TABLE 1. RAdGFP and RAdDN-FGFR1 effects in adult male mice with goiters induced with methimazole and low iodine diet

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies show that TSH, signaling through increases in intracellular cAMP, induces FGF-2 and increases FGFR1 expression in a rat thyroid cell line, consistent with the hypothesis that in rodents the FGF axis is important in goitrogenesis. The direct effects of TSH on thyroid growth may thus be complemented by modulating the activity of this mitogenic axis. As FGFR1 activation also has the effect of inhibiting thyroid function, shown previously (19) and here, the reductions in thyroid hormone synthesis in vivo could lead to sustained elevations of pituitary secretion of TSH. This would promulgate the effects, but there are several mechanisms by which the response could be limited. Although FGFR1 expression increased directly with TSH concentration, the effects of TSH on FGF-2 synthesis were not dose dependent; high doses became inhibitory. This would thus limit the mitogenic effect. This bell-shaped dose-response curve to TSH also occurs in assays of growth and function performed in vitro and has yet to be adequately explained mechanistically (34). In vivo the disparity in the regulation of the ligand and its receptor may in part explain the marked differences in goiter size observed in man. Another potential regulator of the FGF axis, and thus goiter size, is the thyroidal levels of T3 and T4, i.e. whether the patient is hypo-, eu-, or hyperthyroid. Alternatively, FGFR1 on follicular cells may desensitize to the effects of FGF.

The FGF-2 synthesized by the thyroid follicular cells in response to TSH will also have paracrine growth effects on the thyroid endothelium (35). As thyroid follicular cells also secrete vascular endothelial growth factor, whose synthesis is similarly regulated by TSH (36), and angiopoietins (37), the thyroid endothelium in TSH-stimulated animals is in a milieu well endowed with angiogenic factors, triggering growth and maturation. The efficacy of locally produced FGF-2 at activating FGFR1 may depend on its phosphorylation status and its bioavailability. cAMP-mediated phosphorylation of FGF-2 is reported to increase binding affinity to FGFR1 by 8-fold (38). Bioavailability will be controlled by ligand secretion and the presence of heparanases, proteases, and FGF-binding proteins able to release FGF-2 from the glycosaminoglycan-binding sites expressed on the cell surface (9, 39). Angiogenesis inhibitors (40, 41) may also determine the response of the endothelial cells.

We have constructed an Ad expressing truncated FGFR1 that is able to block FGF signaling in thyroid cells. Expression of this construct within the cells was shown and its specificity confirmed by demonstrating that it had no inhibitory effect on EGF signaling in human thyroid cells. The transgene product was able to block the inhibition of 125I uptake seen when the FGFR1 is activated by FGF-2 in both human follicular and FRTL5 cells and to block the morphological changes induced in FRTL5 cells with FGF-2. Using this construct alone, we found that 125I uptake was increased in several preparations of human thyroid cells compared with cells infected with RAd-35, indicating that autocrine FGF has a negative effect on thyroid function. As we found that both the human thyroid follicular cells (5) and FRTL5 cells express low levels of FGF-2, the possibility that other forms of FGF that interact with FGFR1 are synthesized by thyroid cells or that the FGF-2 that is expressed is sequestered near the receptor should be considered. Furthermore, Ueno et al. (10) showed that truncated FGFR1 expressed in Xenopus oocytes inhibited signal transduction by other types of FGFR, thus removing most, if not all, response to FGFs. We also found that in some human thyroid preparations, in particular those with low iodide uptake, RAd35 increased thyroid function (data not shown). Adenovirus infection has been reported to activate cAMP and thus protein kinase A in HeLa cells, which may explain this phenomenon (42). Adenoviral vectors were found to enhance the basal secretion of steroid hormones from primary cultures of bovine adrenocortical cultures, although, in contrast to our data with thyroid cells, ACTH-stimulated secretion was inhibited (43).

We also found that the DN-FGFR1 construct inhibited the growth of FRTL5 and human cells. This is consistent with the hypothesis that some of the effects of TSH on mitogenesis in thyroid cells are indirect and mediated through its ability to induce these proteins. The growth inhibitory effects of RAdDN-FGFR1 were not due to toxicity of the adenovirus per se on thyroid cells, as control incubations where RAd35 was used at the same MOI showed no inhibitory effects. Effects on growth in human follicular cells were only seen in the rapidly dividing cells shortly after isolation. After 1 wk of culture, when the cells were no longer sensitive to FGF-2 (5), these effects were not observed. In FRTL5 cells, the effect was seen within 4 d posttransduction with RAdDN-FGFR1 for cells incubated in 6H and 5% newborn calf serum, conditions under which growth is optimal.

Administration of RAdGFP to mice showed that the thyroid follicular cells expressed GFP and were thus infected with adenovirus. This is consistent with the findings of Zeiger et al. (15), who showed that more than 80% of thyrocytes from neonatal rats expressed ß-galactosidase when 2.5–5 x 109 pfu ß-galactosidase-expressing adenovirus were introduced by intracardiac injection. These studies show that the thyroid is a potential target for adenoviral-mediated gene therapy and that sufficient virus remains circulating before and even after hepatic clearance to infect the thyroid. Infection of mice with RAdDN-FGFR1 and control RAd expressing GFP produced no adverse effects on total body weight; the percent loss was no more that 5.4% through the duration of the study. The mice infected with control RAdGFP and simultaneously switched to a goitrogenic diet developed goiters equal in size to those in uninfected control mice fed the same diet. The mice infected with RAdDN-FGFR1 showed a significant reduction in goiter size. The parameters of thyroid function measured, i.e. total T3, total T4, and TSH, were not significantly different in the RAd-infected mice compared with the uninfected, but goitrogen-fed, controls. The vascular Vv, although reduced, was not reduced to the same extent as the goiter, implying that the greater effect of the RAdDN-FGFR1 was on the follicular cells.

We have shown that blocking FGF signaling exerts a significant effect on thyroid cell growth and function in vitro as well as a marked effect on goiter development in vivo. These findings suggest that a gene therapy approach, abrogating the role of FGF in the thyroid, may have therapeutic potential in man. Although benign goiter is likely to be managed by other means, recurrent and treatment-resistant differentiated thyroid cancer (or the less common, but rapidly fatal, anaplastic cancer) may be conditions in which such an approach is feasible and therapeutically effective.


   Acknowledgments
 
We thank Dr. Stuart Egginton (Department of Physiology) for suggesting the approach to measure thyroidal vascular volume, Dr. Nicola Green for help with viral work, Frances Turner for performing the ELISAs, Prof. Lawrence S. Young (Department of Cancer Studies) for initial helpful discussions, and Prof. Michael C. Sheppard for support and advice.


   Footnotes
 
This work was supported by a project grant from the Wellcome Trust, a fellowship award from the Medical Research Council (to J.D.R.), and a fellowship award from the GetAHead Campaign and the Royal College of Surgeons (to H.C.C.).

Abbreviations: 8BrcAMP, 8-Bromo-cAMP; CMV, cytomegalovirus; DN, dominant negative; EGF, epidermal growth factor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GFP, green fluorescent protein; HBSS, Hanks’ Balanced Salt Solution; MOI, multiplicity of infection; pfu, plaque-forming units; RAd, replication-defective adenovirus; Vv, volume density.

Received February 26, 2003.

Accepted June 4, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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