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Fibroblast Growth Factors 1 and 2 and Fibroblast Growth Factor Receptor 1 Are Elevated in Thyroid Hyperplasia

Departments of ENT Surgery (S.D.T., J.C.W.) and Medicine (J.A.F., J.M.V., M.C.S., M.C.E.), University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, United Kingdom

Abstract

We have previously reported increased expression of fibroblast growth factor (FGF-1 and FGF-2) in benign and malignant human thyroid neoplasia. To determine the role of these factors in thyroid hyperplasia we have examined their expression in multinodular goiter and compared findings with those in normal thyroid tissue. Because the effects of FGF-1 and FGF-2 are predominantly mediated through the FGF receptor-1 (FGFR-1), its expression has also been examined. Immunocytochemistry was performed on sections from multinodular goiters (n = 18) and normal thyroid (n = 7). Cytoplasmic staining for FGF-1, FGF-2, and FGFR-1 was scored on a scale of 0 (no staining) to 3 (heavy staining) and expressed as a percentage of total cells stained. Confocal microscopy of immunofluorescent staining for FGF-1, FGF-2, and FGFR-1 in sections of multinodular goiter (n = 3) and normal thyroid (n = 3) provided quantitation of immunostaining. FGF-1 expression was significantly increased in multinodular goiter when compared with normal. A mean of 74% of follicular cells in multinodular goiter compared with 9% of follicular cells in normal thyroid expressed FGF-1 (P < 0.0001). When expression of FGF-2 was examined, 77% of the follicular cells in multinodular goiter compared with 5% in normal thyroids were immunopositive (P < 0.0001). Confocal microscopy revealed that the intensity was 160 times greater in follicular cells in sections of multinodular goiters when compared with normal. When expression of FGFR-1 was analyzed, 89% of the follicular cells in multinodular goiter stained positively, compared with 15% of follicular cells in sections of normal thyroid. Confocal microscopy revealed a 6-fold increase in intensity of FGFR-1 expression in follicular cells of multinodular goiter (P < 0.05). In addition, there was significant nuclear expression of FGFR-1 in multinodular goiter contrasting with negligible expression in normal thyroid. These data show that enhanced expression of FGF-1, FGF-2, and FGFR-1 accompany thyroid hyperplasia and are not exclusively associated with the neoplastic state. These factors may be involved in the pathogenesis of uncontrolled thyroid growth observed in these conditions.

ACIDIC and basic fibroblast growth factors (FGF-1 and -2) are prototype members of a family of ten growth factors, some of which are oncoproteins. FGFs were initially isolated from bovine pituitary and were found to stimulate division of fibroblasts in vitro (1). Their growth effects are not limited to cells of the mesenchyme, however, and FGFs are known to be mitogens for epithelial cells. They have been shown to be present in many tissues in the body, and they have important roles in embryogenesis (2), angiogenesis (3), and wound healing (4). In addition, expression of members of the FGF family is frequently elevated in solid tumors (5), including those of the thyroid (6). FGF-1 displays considerable sequence homology to FGF-2 (7). A novel feature of their structure is the lack of a classical signal sequence directing secretion. The mechanism of FGF secretion is poorly understood, but it may be released as a result of proteolysis following cell wounding or damage analagous to FGF-1 (8). Certain tumors are known to undergo a degree of necrosis during their natural history, and this process may in turn lead to the release of FGFs. Low affinity FGF membrane proteins have also been described that may be associated with extracellular trafficking of FGF (9).

Four high affinity tyrosine kinase receptors for FGF have been characterized. They have three components viz. an extracellular section that comprises three domains (immunoglobulin domains I, II, and III), a transmembrane section, and an intracellular domain that contains the tyrosine kinase. The ligand specificity of the receptor is dependent on differences in the extracellular domains in FGFR-1–FGFR-4. Multiple isoforms of the receptor also arise as a result of variant messenger RNA splicing within the extracellular immunoglobulin domain III. This feature is not uncommon in growth factor receptors generally, but the number of isoforms in the FGFR is unusually high (10). FGF binding proteins have also been described and are postulated to be truncated forms of the intact FGF receptor (11), analagous to the situation with growth hormone binding proteins. The principal ligands for FGFR-1 are FGF-1 and FGF-2 (10), and therefore this variant of receptor was examined in this study.

In sections of human thyroid tissue we have previously shown elevated expression of FGF-1 and FGF-2 in human papillary and follicular tumors (5). FGF-1 in a rat model has been shown to stimulate the formation of colloid goiter (12) and, along with FGF-2, to stimulate 5'-deiodinase activity (13). FGF-2 is known to be a potent mitogen for a rat thyroid cell line (14) and to be elevated during methimazole-induced goiter (15), implying a role for FGF-2 in thyroid follicular cell hyperplasia. In this study we wished to determine if FGF-1 and FGF-2 were implicated in the genesis of multinodular goiter in humans. The etiology of idiopathic multinodular goiter is essentially unknown, but is presumed to arise from chronic low level stimulation by TSH because of inadequate thyroid hormone levels, caused either by iodide deficiency and/or by defects in thyroid hormonogenesis (16). Histologically, multinodular goiter is characterized by large lakes of colloid interspersed by areas of follicular cell hyperplasia in addition to normal follicular cells (17) and is therefore an important and relevant pathological state in which to assess FGF-1 and FGF-2 expression in thyroid hyperplasia. Because the responses of cells to FGF-1 and FGF-2 are determined by the presence of their high affinity receptors, we also evaluated FGFR-1 expression in normal tissue and in thyroid disease. The expression of FGF-1, FGF-2 and FGFR-1 in human multinodular goiters has been compared with levels in normal tissue.

Materials and Methods

ABC (avidin-biotin complex) immunostaining

Archival formaldehyde-fixed, paraffin-embedded blocks from histologically normal thyroid sections were obtained from laryngectomy resections (n = 7) and from histologically-confirmed multinodular goiters removed for compressive or cosmetic reasons (n = 18). Five µm sections were cut and were progressively dewaxed and rehydrated by immersion in xylene, absolute alcohol, and water. The sections were prepared for immunostaining as previously described (5). Each primary antibody was diluted in phosphate-buffer saline (PBS). FGF-1 and FGF-2 were used at titres of 1:500. These antibodies were kindly supplied by Professor A. Baird of Prizm Pharmaceuticals, La Jolla, CA. Polyclonal antibody to FGFR-1 (Santa Cruz Chemicals, CA) was used at a dilution of 1:100. Antisera were added to each section and, after washing in PBS, secondary biotinylated goat antimouse/rabbit serum was added for 30 mins. Detection was performed with the avidin-biotin complex (ABC kit supplied by DAKO Ltd., High Wickham, UK). Staining was visualized using diaminobenzidine (DAB) substrate. Sections were counterstained with Mayers Haematoxylin blue and were dehydrated by passing through absolute alcohol and xylene and mounted using DPX. Assessment of cytoplasmic staining was carried out by identifying 100 cells within a randomly selected light microscopic field (magnification x 400). The cytoplasm of those positive cells was then semiquantitatively scored on a scale of zero (no staining), 1 (mild staining), 2 (moderate staining), and 3 (heavy staining) by two independent observers (S.D.T. and J.V.), and the results were expressed as a percentage of total cells examined.

Fluorescent immunostaining and quantification by confocal microscopy

The confocal microscope employs laser technology in the illumination and detection of fluorescence at a single point in the specimen. This eliminates out-of-focus interference, thereby allowing structures to be sectioned optically (18). The technique allows accurate subcellular localization of fluorescence and also provides a means for objective quantitation by calculation of image pixel intensity. Typical sections of multinodular goiter (n = 3) and normal thyroid (n = 3) were dewaxed and rehydrated as described for the immunoperoxidase-diaminobenzidine method above. Sections were blocked by incubation with 100 µL of 10% goat serum for 30 min and washed in PBS for 30 min. After dilution of the primary antibody in PBS (FGF-1 and -2 1:300, FGFR-1 1:100) 100 µL was added to each section and incubated at room temperature for 1 h. Sections were washed in PBS for 15 min and 100 µL of fluorescein-labeled goat antirabbit secondary antibody applied (Calbiochem Novobiochem UK, Nottingham, UK) at a dilution 1:50 for 30 min. After a further wash with PBS for 30 minutes, nuclei were labeled by incubating the sections in propidium iodide (1 µg/mL) for 1 min. Sections were subsequently washed and mounted with cover slips using 2.5% solution of 1–4 diazabicyclooctane (DABCO) in glycerol (19) as a mountant before confocal microscopic analysis. In our analysis, a random microscopic field was selected and intensity of immunopositivity assessed by measurement of pixel intensity per unit field, as previously used by ourselves (5). Sections incubated in secondary antibody alone were used as background controls.

Results

Immunocytochemistry

Comparisons of mean cytoplasmic expression of FGF-1, FGF-2, and FGFR-1 in normal thyroid and multinodular goiter using the immunoperoxidase-diaminobenzidine technique are shown in Fig. 1. Nine percent of the follicular cells in normal thyroid sections stained positively but weakly for FGF-1 (grade 1 staining). In multinodular goiter, 75% of the cells were immunopositive for FGF-1, of which approximately two thirds was moderate (grade 2) staining and the remainder weak (P < 0.0001 using Students’ alternative t test). Five percent of the follicular cells in normal thyroid tissue were positive for FGF-2 expression, all of this staining being weak (grade 1). In contrast, in multinodular goiter, 77% of the follicular cells were positive for FGF-2 (P < 0.0001 using Students’ alternative t test), of which approximately half (38%) was moderate (grade 2) staining. There was no grade 3 staining for either FGF-1 or FGF-2 in normal thyroid or goiter tissue.

View larger version (22K): [in this window] [in a new window]   Figure 1. Percentage immunopositivity (immunoperoxidase technique) of follicular cells in normal thyroid (n = 7) and multinodular goiter (n = 18). Results are shown as mean ± SE with intensity of staining indicated by shading of histogram bars. = grade 1, = grade 2, = grade 3. *P < 0.0001 by Students’ alternative t test, compared with normal thyroid.

Representative photomicrographs of sections of normal thyroid and goiter immunostained for FGF-1, FGF-2, and FGFR-1 using the immunoperoxidase technique are shown in Fig. 2, A–C. They show increased cytoplasmic immunopositivity for FGF-1, FGF-2, and FGFR-1 in goiters compared with normal thyroid sections. In normal sections positive staining is predominantly found in the stromal tissue, and examination of the follicular cells in these sections shows little positive staining for FGF-1, FGF-2, and FGFR-1. There was no nuclear staining for either FGF-1 or FGF-2. Marked nuclear expression of FGFR-1 can be seen. Varying intensities of staining are visible for FGF-1, FGF-2, and FGFR-1 in each of the fields shown, which reflects the heterogeneity of thyrocytes.

View larger version (142K): [in this window] [in a new window]   Figure 2. Expression of FGF-1 (a), FGF-2 (b), and FGFR-1 (c) in representative sections of goiter (upper panels) and normal thyroid (lower panels) using the immunoperoxidase stain. Immunopositivity is shown as a brown stain. Nuclear staining is colored blue.

Randomly selected sections of normal thyroid and goiter were analyzed further using the confocal microscope to allow subcellular localization and quantitation of immunofluorescence after subtraction of background staining. As found with the immunoperoxidase staining, analysis of FGF-2 in normal thyroid tissue demonstrated negligible FGF-2 expression, but in the multinodular goiter there was a 160-fold increase in intensity per unit field (mean pixel intensity per unit field ± SE: normal thyroid 1.0 x 10⁵ ± 0.5 x 10⁵ versus multinodular goiter 16.7 x 10⁶ ± 2.5 x 10⁶ P < 0.02 using Students’ alternative unpaired t test). When expression of FGFR-1 intensity was analyzed, there was a 6-fold increase in follicular cell expression in multinodular goiter when compared with normal (mean pixel intensity per unit field ± SE: normal thyroid 0.05 x 10⁶ ± 0.03 x 10⁶ = 3, versus multinodular goiter 0.3 x 10⁶ ± 0.1 x 10⁶, n = 3; P < 0.05 using Students’ unpaired t test).

Representative confocal micrographs of FGF-2 and FGFR-1 expression in normal thyroid and goiter are shown in Fig. 3. There was increased intensity of FGF-2 and FGFR-1 expression in goiter when compared with normal, located mainly in the cytoplasm of the cells.

View larger version (185K): [in this window] [in a new window]   Figure 3. Representative confocal micrographs depicting intensity of expression of FGF-2 (a) and FGFR-1 (b) in sections of goiter (upper panels) and normal thyroid (lower panels). Differences in intensity are shown as blue—weakest, to red—most intense.

In this study we have shown increased expression of FGF-1, FGF-2, and FGFR-1 in multinodular goiter when compared with normal thyroid tissue. Both the number of cells staining positively and the total amount of FGFs in the tissue were increased. These findings are compatible with a role for FGF-1 and -2 in the genesis of multinodular goiter. In suggesting a potential role for FGF-1 and FGF-2 in goitrogenesis two mechanisms are possible. First, it may act as a mitogen to thyrocytes producing hyperplasia, and/or second, in an adjunctive capacity by virtue of its role as an angiogenic agent. A study of human thyroid cells in culture showed no growth in response to treatment with exogenous FGF-2 in primary cell culture (20), however thyrocytes in cell culture may be sensitive to contact inhibition and growth effects may be compromised in vitro. FGF-1 in in vivo rat studies has been shown to induce the formation of colloid lakes in the thyroid (12) gland and also to induce multinodular goiter (21). The increased expression in multinodular goiter in human thyroid tissue shown in our study suggests a similar role in human goitrogenesis.

Neovascularization is a feature known to accompany goitrogenesis (22). The production of FGFs by cells in the thyroid may also promote an angiogenic response (23) inducing endothelial cell migration and capillary formation, providing the neovascularization necessary to sustain an increase in cell mass. Similarly, FGF effects on stromal cells may be expected.

Other studies on expression of FGF-1 and FGF-2 in different human thyroid pathologies have produced conflicting data that may reflect differences in the methods employed to expose the antigen, as well as differences with the antisera (24). One immunohistochemical study, comparing expression of various growth factors in normal and abnormal thyroid tissue found almost no follicular cell expression of FGF-2 in goiter. The expression of FGFR-1 was not examined (25). Another immunohistochemical study (6) reported cytoplasmic expression of both FGF-1 and FGF-2 in 16% of follicular cells in sections of multinodular goiter, but again expression of the receptor was not examined.

In the present study expression of the FGFR-1 receptor was evident in the normal thyroid, confirming previous reports (26) and suggesting a role for FGF-1 and FGF-2 in normal thyrocyte homeostasis. Significantly increased expression of FGFR-1 was observed in multinodular goiter when compared with normal. This indicates that FGF-1 and FGF-2 may be acting in an autocrine manner in vivo, as their activity may be regulated by the expression of FGFR-1, as well as FGF release from the cell matrix (27). In addition to the cytoplasmic staining observed, approximately one third of the nuclei in multinodular goiter stained positively for FGFR-1. The significance of this is unknown but truncated forms of the receptor have been identified that are postulated to act as FGF binding proteins (11). The negligible nuclear expression of FGF-1 and FGF-2 in either the normal thyroid or goiter would argue against FGFR acting as a binding protein. However, following internalization, processing may occur which could result in loss of immunoreactivity to the antiserum we used.

Current understanding of goitrogenesis in iodide replete areas suggests that chronic low level stimulation by TSH leads to nodule formation. Individual thyrocytes are postulated to differ markedly in their responses to TSH both in terms of growth and function, ranging from near autonomy, to those displaying exquisite sensitivity to stimulation by TSH (28). FGF production may be regulated by TSH or may act in a cooperative fashion with TSH and aid in the production of goiter. FGF has been described as a "competence factor," stimulating a shift of cells from the resting G₀ phase of the cell cycle into G₁ and the subsequent commitment to cell replication (29). This would serve to increase the available cell population upon which other factors leading to thyroid neoplasia such as genetic mutations may act.

We have previously shown increased expression of FGF-1 and FGF-2 in both benign and malignant thyroid neoplasia (5). Our current data showing increased expression of FGF-1 and FGF-2 in multinodular goiter provide evidence that these growth factors are implicated in hyperplastic as well as neoplastic growth of thyroid cells. The question of whether the incidence of hyperplasia and neoplasia in the thyroid is linked remains contentious (30). If such a link were to be established then the action of FGFs mediated by FGFR, may provide the explanation.

Acknowledgments

We gratefully acknowledge the help provided by the following: The Wellcome Trust, Get-Ahead Charity appeal, and The Royal College of Surgeons of Edinburgh for providing the financial support enabling this work to be completed, and Ms. Debbie Hardy for her assistance with the confocal microscopic analysis.

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