0013-7227/92/1304-2363$03.00/0

Endocrinology Copyright 0 1992 by The Endocrine Society

Basic Fibroblast of Rat Thyroid

Vol. 130, No. 4

Printed

Growth Follicular

ANN LOGAN, ELIZABETH G. BLACK, MARINO BUSCAGLIA, AND MICHAEL

Factor: An Autocrine Cells?*

in U.S.A.

Mitogen

ANA-MARIA GONZALEZ, C. SHEPPARD

Departments of Clinical Chemistry (A.L.) and Medicine (E.G.B., M.C.S.), University of Birmingham, Edgbaston, Birmingham, B15 2TT United Kingdom; and the Department of Molecular and Cellular Growth Biology, Whittier Institute of Diabetes and Endocrinology (A.-M.G., M.B.), Lu Jolla, California 92037

ABSTRACT. Basic fibroblast growth factor (FGF) is a mitogen for the rat thyroid cell line FRTL-5. A possible autocrine role for this growth factor has been investigated in rat thyroid follicular cells both in vitro and in uiuo. We report here the synthesis and localisation of basic FGF and one of its high affinity receptors (fig) in FRTL-5 cells, shown by Northern hvbridization anal&s. Western blotting, and immunohistochemistry. Two major species of basic FGF mRNA of approximately 212 and 7.0 kilobases and one major species off& mRNA of annroximatelv 4.2 kilobases were identified in FRTL-5 cells. The’ basic FGF” immunoreactivity observed histologically was attributed to a heparin-binding protein of approximately 20 kilodaltons mol wt. The physiological relevance of basic FGF to the thyroid is underlined by the demonstration of significant stores of immu-

F

noreactive protein, predominantly in the basement membrane of thyroid follicularcells, in paraffin sections of the normal rat thvroid. although basic FGF mRNA was not detected bv in situ Or-Northern hybridization analysis. The mitogenic response of FRTL-5 cells to human recombinant basic FGF has been further characterized, and the factor shown to stimulate with an ED,, of 4 rig/ml. The mitogenic effects of exogenously supplied and endogenously produced basic FGF were shown to be notentiated bv henarin. Examination of the mitogenic activity of both exogenous-and endogenous basic FGF and its immunoneutralization in vitro suggests that locally produced basic FGF may be an important autocrine regulator of thyroid follicular cell growth. (Endocrinology 130: 2363-2372, 1992)

nadal (6-g), and adrenal (10) tissues. Clearly, its angiogenic influences on endothelial cells (11,12) may denote its importance to these endocrine organs, helping to maintain the integrity of their complex capillary network. In some of these endocrine systems, basic FGF has trophic activity and may modulate differentiated endocrine function as well as cell proliferation (4). A role for basic FGF in the control of the complete brain-pituitary-thyroid axis has been suggested. Immunoreactive basic FGF has been demonstrated in the neurons of the paraventricular, supraoptic, and circular nuclei of the hypothalamus and in neurons of the hypothalamo-hypophyseal neuroendocrine pathway (13). Colocalization of a high affinity basic FGF receptor has also been demonstrated (14). Basic FGF has been localized in pituitary thyrotroph cells, and it potentiates TRHstimulated PRL and TSH secretion from cultured anterior pituitary cells (5). Little is known of the relevance of basic FGF to the thyroid, although the angiogenic activity of basic FGF suggests a connection with the pathogenesis of goiter and thyroid tumour growth via the stimulation of neovascularization of actively growing thyroid tissue. However, the influence of basic FGF on thyroid growth may be more direct than effects on vas-

OR MANY years, thyroid follicular cell growth and function were thought to be regulated principally by TSH and iodide availability. However, recently, the concept of paracrine or autocrine regulation of the thyroid has received much attention. It is now clear that locally produced peptide growth factors, such as the insulin-like growth factors (1) and epidermal growth factor (2), have potent effects on thyroid epithelial cell growth and function. Although basic fibroblast growth factor (FGF) was first isolated from brain and pituitary (3), it has now been demonstrated in most body tissues and is known to be multifunctional (for review, see Ref. 4). Basic FGF is implicated in the regulation of a number of highly vascular endocrine tissues, for example pituitary (5), goReceived August 27,1991. Address all correspondence and requests for reprints to: Dr. Ann Logan, Department of Clinical Chemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, B15 ZTH United Kingdom. * This work was supported by the Whittier/Erbamont Angiogenesis Research Proeram (to Andrew Baird). the NIH (Grant DK-18811 to Andrew Baird?, the Endowment Fund Medical Research Committee of the Birmingham Regional Hospitals (to MS.), and the University of Birmingham Scientific Projects Committee (to A.L.). 2363

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BASIC FGF IN THE THYROID

2364

cular endothelial cells. The presence of basic FGF within cultured adult porcine thyroid cells and in the rat thyroid follicular cell line FRTL-5

together

with

observations

of a mitogenic

response of FRTL-5 cells to human recombinant basic FGF were first reported in abstract form (15,16). Further investigations demonstrated lated iodide

in these two cell culture systems have that basic FGF can inhibit TSH-stimuuptake (17) and have characterized the mechanisms of the mitogenic action of basic FGF (18). A potential autocrine role for basic FGF in thyroid epithelial cells is, therefore, strongly suggested, whereby basic FGF regulates the growth and function of the thyroid follicular cells in which it has been produced. The studies described in this paper were designed to test the hypothesis that basic FGF is an autocrine regulator of the growth of rat thyroid cells. Although functional,

primary cultures of ovine and porcine thyroid cells do not grow well and, therefore, are not appropriate models for the study of thyroid cell growth. In contrast, FRTL5 cells make an ideal system in which to investigate thyroid cell growth regulation. We have examined the synthesis and localization of basic FGF and one of its

high affinity

receptors in FRTL-5

cells and normal

rat

thyroid in vivo by in situ and Northern blot hybridization analyses, immunohistochemistry, and Western blot

analysis. In addition, we have investigated the characteristics of the mitogenic activity of both endogenous and exogenous basic FGF in FRTL-5 cells using an antibasic FGF neutralizing antibody and heparin, which potentiates the influence of basic FGF.

Materials

and Methods

Materials

Tissueculture reagentswere obtained from Gibco Ltd. (Paisley, Scotland). FRTL-5 cells were a gift from Dr. L. Kohn (NIDDK, NIH, Bethesda,MD). All other reagentsnot specified were analytical grade from BDH Ltd. (Atherstone, United Kingdom) and Sigma Chemical Co. Ltd. (Poole, United Kingdom). Isotopes were suppliedby Amersham International plc (Aylesbury, United Kingdom). Northern

blot analysis

RNA was extracted from subconfluent cultures of FRTL-5 cells and from freshly excised normal adult rat thyroids and brains by the guanidinium isothiocyanate-cesium chloride method (19). Sampleswere homogenizedin 4 M guanidinium isothiocyanate solution. RNA was pelleted through 5.7 M cesium chloride, extracted with phenol-chloroform, precipitated with ethanol, and quantified by absorption at 260 nm. Samples of 20 pg total RNA were denatured for 5 min at 65 C and separatedon a 1% agarose-formaldehydegel. The RNA was blotted onto Hybond N hybridization membrane(Amersham) by the capillary transfer method and fixed to the membranes by a 5-min exposureto UV irradiation. Basic FGF mRNA was

Endo. Voll30.

1992 No 4

detected with a 32P-labeledrat basic FGF cDNA probe derived from the clone RobFGF103. For detection of fig mRNA, a LOkilobasepair fragment of DNA encoding the extracellular domain of fig (20) was used. Blots were hybridized overnight at 65 C with the 32P-labeledcDNA probes in 3.3 mM EDTA, 0.5 M Na phosphate, 6.7 M sodiumdodecyl sulfate (SDS), and 100 pg/ml denatured salmonspermDNA (pH 7.3). The filters were washedin 2 x standard salinecitrate @SC)-0.1%(wt/vol) SDS at room temperature twice for 10 min, then in 1 X SSC-0.1% (wt/vol) SDS at 65 C for 1 h, finishing with two lo-min washes in 0.1 X SSC-0.1%(wt/vol) SDS at room temperature. Hybridizing specieswere visualized by autoradiography. In situ hybridization

In situ hybridization of basicFGF usedthe NcoI-XhoI fragment of 0.477kilobasepairderived from the rat basicFGF clone RobFGF103, which wassubclonedinto pBluescript SK+ (Stratagene,San Diego, CA) and linearized with NcoI. The antisense strand of the coding sequencewastranscribed using T7 polymeraseand [35S]UTP (21). A [35S]UTP-labeledRNA probe encoding the sense strand of the 5’-noncoding sequencewas preparedwith T3 RNA polymeraseand usedfor control tissue sections. Mounted frozen sectionsof thyroid and brain, prepared as describedin the Histology section below, were digestedwith 10 pg/ml proteinase-K in 0.1 M Tris (pH 8.0) containing 50 mM EDTA at 37 C for 30 min. Sections were rinsed in deionized water, followed by incubation in 0.1 M triethanolamine, pH 8.0, for 3 min. Sectionswere then acetylated for 10 min with 0.25% (wt/vol) acetic anhydride in 0.1 M triethanolamine, rinsed in 2 x SSC, dehydrated through a gradedseriesof ethanol washes, and air dried under vacuum for 2 h before hybridization. Hybridizations with labeled basic FGF antisense or sense probes (1 X lo7 cpm/ml) were performed at 55 C overnight in 10 mM Tris (pH 8.0) containing 50% (wt/vol) formamide, 0.3 M NaCl, 1 mM EDTA, 10 mM dithiothreitol (DTT), 1 x Denhardt’s solution (19), and 10% (wt/vol) dextran sulfate. After hybridization, sectionswere rinsed for 1 h in 4~ SSC and treated with 25 rg/ml ribonucleaseA in 10 mM Tris (pH 8.0) containing 0.5 M NaCl and 1 mM EDTA at 37 C for 30 min. This wasfollowed by increasinghigh stringency washesof SSC containing 1 mM DTT, finishing with 0.1 X SSC at 65 C for 30 min. Slides were then dehydrated through a graded seriesof ethanol, dried under vacuum, and then exposedto @Max Hyperfilm (Amersham) for 5 days to examine grosschangesin mRNA. For microscopicanalysis,slideswere exposedto Kodak NTB-2 liquid autoradiographic emulsion (Eastman Kodak, Rochester,NY) for 3 weeksat 4 C, processedwith Kodak D19 developer,rinsed, and fixed with Kodak rapid fixer. The slides were rinsed for 30 min in tap water, counterstainedwith Harris’ hematoxylin, and examined by darkfield and brightfield microscopies. Histology

For the purposeof histology, maleSprague-Dawleyrats were deeply anesthetisedby ip injection of a mixture of acepromazine (1.875 mg/kg), ketamine (3.75 mg/kg), and xylazine (1.9 mg/ kg). They were perfused transcardially with 300 ml 0.9% (wt/ vol) salineand 250 ml 4% (wt/vol) paraformaldehyde(PFA) in

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BASIC

FGF IN THE THYROID

0.1 M acetate buffer, pH 6.5, followed by 500 ml 4% (wt/vol) PFA plus 0.05% (wt/vol) glutaraldehyde in 0.1 M borate buffer using the pH shift method (22). For in situ studies,the thyroids were postfixed overnight at 4 C in 4% (wt/vol) PFA in 0.1 M borate buffer containing 10% (wt/vol) sucrose.The thyroids were then rapidly frozen on powdereddry ice in Tissue Tek OCT compound (Miles Laboratories, Inc., Naperville, IL) and stored at -80 C. Rat brains were collected and treated identically for comparisonof basic FGF mRNA presence.Frozen sections (15 grn) were mounted on poly-L-lysine-coated slides, air dried, and stored at -80 C until use. Brains were processedas describedby Emoto et al. Cm. For immunohistochemistry studies, the fixed thyroids were rinsedin 70% (vol/vol) ethanol, dehydrated through ascending gradesof ethanol, and embeddedin paraffin. Sections (8 pm) were mounted on poly-L-lysine-coated slides. Basic FGF antibody

The primary polyclonal antibody against basic FGF was raisedagainstthe l-24 synthetic fragment of bovine basicFGF (antibody 773), generouslyprovided by Dr. Andrew Baird of. the Whittier Institute of Diabetes and Endocrinology (La Jolla, CA). Immunoglobulin G (IgG) antibody fractions were prepared by ammonium sulfate precipitation (30%, wt/vol), purified by protein-A-Sepharoseaffinity chromatography, and diluted to a concentration of 2.5 pg/ml in 2.5% (wt/vol) BSA. The antibody detects basicFGF and doesnot recognize acidic FGF, hst/ks, or FGF-5 (~1%). Its cross-reactivity with other FGFs is not known, but would not be predicted on the basisof sequencehomology (23). The antibody is known to block the mitogenic actions of exogenousbasic FGF on vascular endothelial cells in vitro (Baird, A., personalobservations). Immunofluorescent

staining

of FRTL-5

cells

Immunofluorescent staining for basic FGF was carried out with FRTL-5 cells grown to subconfluenceon Lab-Tek tissue culture chamber slides(Miles Laboratories) and kept for 48 h in 5H medium(for details, seecell culture section below). Cells were washedin situ twice with PBS (0.01 M phosphate buffer plus 0.15 M NaCl, pH 7.5) and fixed onto the slideswith 4% (wt/vol) paraformaldehyde in PBS for 10 min. After fixation, cells were washed twice in PBS, dehydrated through graded alcohols,and air dried before storageat -80 C. Before staining, cells were rehydrated through graded alcohols and washedin PBS, and nonspecific binding sites were blocked by a 30-min incubation with 1.5% (vol/vol) goat serum in PBS supplementedwith 0.3% (wt/vol) Triton X-100. After a l-h incubation at room temperature with protein-A-purified primary antibody 773 (6 rg/ml) diluted in PBS supplementedwith 0.3% (wt/vol) Triton X-100 and 5% (wt/vol) BSA, the cells were treated with 1:lOOgoat antirabbit IgG conjugated with fluorescein isothiocyanate (FITC; TAGO, Inc., Burlingham, CA) for 1 h. The cells were then washedin three changesof PBS and mounted in a nonquenching mountant. For controls, either the primary or secondaryantibody was omitted or cells were incubated with a primary antibody that had been passedthrough a basic FGFAffigel affinity column to deplete it of antibasic FGF IgG; all were negative.

Immunoperoxidase

2365 staining

of rat thyroid

Immunoperoxidasestaining for basic FGF in paraffin sections of adult rat thyroid glandsusedthe ABC Vectastain Elite kit (Vector Laboratories Ltd., Burlingame, CA) and has been describedin detail previously (23). Briefly, tissuesectionswere deparaffinized and hydrated. They were then washedin PBS, and the endogenousperoxidase was quenchedby incubation with 0.3% (vol/vol) hydrogen peroxide in PBS for 30 min. The sections were rinsed in PBS and incubated in 1.5% (vol/vol) goat serum, diluted in PBS containing 0.3% (vol/vol) Triton X-100, for 30 min to reduce nonspecific staining. After a 24-h incubation at 4 C with the protein-A-purified primary antibody 773 (2.5 pg/ml), diluted in PBS supplementedwith 0.3% (vol/ vol) Triton X-100 and 5% (vol/vol) BSA, the sections were treated with a 1:200dilution of biotinylated goat antirabbit IgG (Vector) for 1 h. This wasfollowed by a 30-min incubation with a biotin-avidin-peroxidase complex (Vector). Finally, the sections were treated for 5 min with 0.5 mg/ml diaminobenzidine in PBS containing 0.01% (vol/vol) hydrogen peroxide. All steps were separatedby buffer washesconsistingof PBS with 0.3% (vol/vol) Triton X-100. The sections were finally washed in PBS, counterstained with Harris’ hematoxylin, dehydrated, cleared, and mounted. The equivalent protein concentration of the flow-through from a basicFGF-Affigel affinity chromatography column (BioRad, Richmond, CA) was also diluted to 2.5 pg/ml in 5% (wt/ vol) BSA in PBS plus 0.3% (vol/vol) Triton and usedon control sections. Other controls used the primary antibody in the presenceof either 250 pg/ml of the peptide fragment bovine basicFGF-( l-24) or BSA. Sectionsprocessedwith all of these proceduresfailed to stain. Extraction

of basic FGF

Heparin-binding proteins wereextracted at 4 C from FRTL5 cellsby batch affinity chromatography. Sampleswerehomogenizedin an extraction buffer (five times; wt by vol) comprising 1% (vol/vol) Nonidet P-40,0.5% (wt/vol) deoxycholate, 20 mM Tris-HCl (pH 7.4), 1 mM phenylmethylsulfonylfluoride, 1 mM EGTA, 1 mM EDTA, 1 pg/ml aprotinin, 1 pg/ml leupeptin, 1 kg/ml pepstatin, and 2 M NaCl. After centrifugation at 20K rpm for 50 min to remove cellular debris,the supernatant was diluted to lessthan 1.0 M NaCl with 10 mM Tris-HCl, pH 7.4. A heparin-Sepharoseslurry wasaddedto the diluted material, which was incubated overnight on a rotator. The heparinSepharosewas spun down, and the pellet was washedthree times with 0.6 M NaCl-10 mM Tris-HCl, pH 7.4. The heparinbinding activity waseluted with 200~12.0 M NaCl. The protein concentration of eluant was assessed by the method of Lowry et al. (24). Western blot analysis

The molecular size of basic FGF immunoreactivity present in the heparin-Sepharoseextracts of FRTL-5 cellswasassessed by Western blot analysis. SDS-polyacrylamide gel electrophoresis of 10 pg protein of the heparin-Sepharoseextract on a 15% (wt/vol) polyacrylamide gelwasfollowed by electroblotting of the separatedproteins to nitrocellulose membrane(Bio-Rad Laboratories Ltd., Hemel Hempstead,United Kingdom). Re-

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1l-m

combinant human basic FGF (generous gift of Dr. P. Barr, Chiron Corp., Emeryville, CA) and low mol wt markers (BioRad) were also run and blotted simultaneously. After transfer, the membranes were washed in PBS for 15 min, followed by 50 ml 50 mM Tris-HCl containing 0.9% (wt/vol) NaCl and 5% (wt/vol) BSA for 2 h. Membranes were analyzed by incubation for 16 h at 4 C with 10 pg/ml protein-A affinity-purified rabbit polyclonal antibody to bovine basic FGF-( l-24) (antibody 773) or the same concentration of a similarly purified nonimmune rabbit serum as a control, which was dissolved in the same incubation buffer. After incubation, membranes were washed in 50 mM Tris-HCl containing 0.2% (vol/vol) Tween-20 and 250 mM NaCl. Detection of complexed 773 antibody on the membrane was achieved by incubation with 10” cpm/ml [“‘I] protein-A in incubation buffer for 2 h at 37 C, followed by further washing, as described above, and visualized by autoradiography. Cell culture and incorporation of fH]thymidine FRTL-5 cells were grown in g-cm tissue culture-treated petri dishes and maintained in 6H medium [a modified Ham’s F-12 medium supplemented with 5% (vol/vol) newborn calf serum and a mixture of 10 pg insulin/ml, 10 pmol cortisol/ml, 5 rg transferrin/ml, 10 ng glycyl-L-histidyl-L-lysine acetate/ml, 10 ng somatostatin/ml (all from Calbiochem, Novabiochem Ltd., Nottingham, United Kingdom), and 150 PU TSH (Thytropar)/ ml from Rorer Health Care Ltd. (Eastbourne, United Kingdom) at 37 C in an atmosphere of 5% CO,-95% air (25). For experiments, cells were removed from dishes with 2 ml Hanks’ Balanced Salt Solution containing 1.5 mg trypsin, 20 U collagenase, and 40 ~1 heat-inactivated chicken serum, washed with 10 ml 6H containing 7.6 mg EGTA, and resuspended in 6H medium to give 3-4 x lo” cells/ml. Aliquots of cell suspension (0.5 ml) were pipetted into each well of a 24-well tissue culture plate and allowed to grow for 4-7 days. Medium was then changed to 5H (6H medium from which TSH had been excluded) for 7 days. On the day of the experiment, medium was replaced with 1 ml fresh 5H medium/well, and test substances (basic FGF, heparin, or 773 antibody) were added in volumes of 10 ~1. To allow cells to complete at least two cell cycles [cell cycle time for FRTL-5 cells is 31 h (our observations)], cells were incubated with test substances and 1 &i [3H]thymidine/ml for 72 h. The experiment was terminated by removing medium, washing cells with 1 ml cold (4 C) PBS, and incubating for 10 min at 4 C with 1 ml 5% (wt/vol) trichloroacetic acid (TCA). TCA was discarded, and cells were scraped off wells with 1 ml PBS. Triton X-100 (5%, vol/vol; 10 ~1) and 4 M NaOH (40 ~1) were added to the cell suspension, which was then heated at 65 C for 30 min. A 500-~1 aliquot of this solution was added to 4.5 ml scintillant and counted in a Beckman P-counter (Palo Alto, CA) to give a measure of disintegrations per min/well. To confirm the ability of basic FGF to stimulate cell proliferation and validate the measurements of DNA synthesis ( [3H] thymidine incorporation), cells were exposed to 0.4-40 rig/ml basic FGF over 7 days, and cell number was estimated with a Coulter counter (model 2F, Coulter Electronics, Inc., Hialeah, FL).

!xldO * lYYZ Voll30. No 4

lHYl-wllJ

Statistical analysis In each cell culture experiment, cells from a single donor culture were plated onto 24-well plates, and the results were expressed as the mean disintegrations per min/well or as a percentage of the control value, + SEM. The results represent the pooled data from at least two experiments in each case. The number of separate cell populations tested in each experiment was between 3 and 6, and significance values were obtained using Student’s unpaired t test. Results Identification

of basic FGF and

fig mRNA

Northern hybridization analysis was used to detect the presence of basic FGF and fig mRNA in cultured FRTL5 cells. Figures 1 and 2 show two major hybridizing

species of basic FGF mRNA, 7.0 and 2.2 kilobases in length, and one major species of fig mRNA of 4.2 kilobases. These sizes are identical to those reported previously for rat basic FGF (21) and rat fZg(14). We could not detect any hybridizing mRNA of basic FGF in the normal adult rat thyroid by either Northern analysis of total and poly(A)+ RNA or in situ hybridization analysis. Tissue sections or RNA extracts of adult rat brain processed identically and simultaneously showed good levels of specific hybridization signal (data not shown).

bFGF

9-S D7-5 -

44

t

1*4 -

FIG. 1. Northern blot analysis of basic FGF mRNA derived from FRTL-5 cells. Total RNA (20 pg) was prepared from subconfluent cell cultures, electrophoretically separated, blotted onto hybridization membrane, and hybridized with the 32P-labeled rat basic FGF cDNA probe.

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BASIC FGF IN THE THYROID

FIG. 2. Northern blot analysis of f& receptor mRNA FRTL-5 cells. Total RNA (20 pg/ml) was prepared from cell cultures, electrophoretically separated, blotted onto membrane, and hybridized with the 32P-labeled fZg cDNA

2367

derived from subconfluent hybridization probe.

FIG. 3. Immunocytochemical localization of basic FGF in FRTL-5 cells. Immunocytochemical localization of basic FGF was examined in subconfluent monolayer cultures of FRTL-5 cells kept for 48 h in 5H medium. Cells were incubated with antibasic FGF antibody or the eluent of a basic FGF-Affigel column. Immunopositive cytoplasmic fluorescence corresponding to immunoreactive basic FGF was seen in all cells. Control cells incubated with the preparation of antibody depleted of antibasic FGF IgG were essentially immunonegative.

Immunocytochemical localization of basic FGF

Immunopositive staining for basic FGF was diffusely distributed in all cultured FRTL-5 cells, indicating that it is cytoplasmic or associated with the cell membrane (Fig. 3). Strong staining was also seen in tissue sections of the normal adult rat thyroid (Fig. 4A). Immunoreactive basic FGF was seen in the cytoplasm of thyroid follicular epithelial cells, but was most intense in the basement membrane, suggesting a site of storage. The staining was specific, since absorption of the antibody to Affigel-basic FGF beads abolished virtually all staining (Fig. 4B). Characterization of the FRTL-5 heparin-binding extract

Heparin-Sepharose affinity chromatography was used to prepare the FRTL-5 cell extract, which was subsequently submitted to SDS-polyacrylamide gel electrophoresis. Western blot analysis demonstrated that of the

FIG. 4. Immunocytochemical localization of basic FGF in the normal adult rat thyroid. Immunopositive peroxidase staining was seen in the follicular cell cytoplasm and also, most strongly, in the surrounding basement membrane, suggesting a site of storage, probably associated with glycosaminoglycans (A). Preincubation of antibasic FGF antibody with excess basic FGF completely abolished immunopositive staining US.

heparin-binding proteins extracted, only one species was detected by a specific antibasic FGF antibody, and this was shown to have a mol wt of approximately 20 kilodaltons (Fig. 5). Mitogenic activity of endogenous and exogenous basic FGF

Basic FGF stimulates [3H] thymidine incorporation into FRTL-5 cells in a dose-dependent manner between 0.04-400 rig/ml. The half-maximal response was achieved with basic FGF concentrations of the order of 4 rig/ml, and the stimulatory effect plateaued at basic FGF concentrations above 160 rig/ml in this experiment (Fig. 6). The increase in [3H]thymidine incorporation in response to basic FGF was accompanied by increased cell number. FRTL-5 cells cultured in the presence of 1 rig/ml exogenous basic FGF for 7 days showed an approximate doubling of cell number (Table 1).

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BASIC

2368

FGF

IN THE

THYROID

5H

Endo. Voll30.

0.04

0.4 Basic

4 40 FGF ha/ml)

160

1992 No 4

400

FIG. 6. Mitogenic activity of basic FGF on FRTL-5 cells. Mitogenic activity was assessed by measuring incorporation of [3H]thymidine into DNA of FRTL-5 cells incubated with increasing concentrations of recombinant human basic FGF. Values represent the mean f SEM for six replicate incubations. TABLE 1. Effects of neutralizing antibasic FGF antibody (773) on the mitogenic action of basic FGF in FRTL-5 cells

NRS

bFGF

FIG. 5. Western blot analysis of basic FGF present in heparin-sepharose extracts of FRTL-5 cells. Proteins were electrophoretically separated on a 15% polyacrylamide gel, transferred to nitrocellulose, incubated with antibasic FGF antibody (anti-bFGF) or nonimmune rabbit serum (NRS), and immunoreactivity was detected with [‘251]protein-A. A single species of immunoreactive protein was visualized with a mol wt of approximately 18 kilodaltons. Mol wt size markers are shown to the left.

The ability of 500 rig/ml of a protein-A-purified antibasic FGF antibody (no. 773) to block the mitogenic activity of basic FGF is demonstrated in Fig. 7. While the neutralizing activity of the antibody was apparent at 0.5 and 1 rig/ml basic FGF, higher concentrations (5 ng/ ml) overcame the blocking effects. This observation implies the specificity of the neutralizing antibody. When unstimulated FRTL-5 cells were incubated in 5H medium with 500 rig/ml of the same antibody (no. 773), a reproducible and statistically significant reduction in the basal rate of [3H] thymidine incorporation was seen compared to that in control cultures (P < 0.05; Table 1 and Fig. 7). In these experiments this concentration of antibody also significantly reduced [3H]thymidine incorporation into cultures exposed to exogenous basic

Cell no. Treatment 13H1Thvmidine (dnm/well) 4,649 + 195 1,427 + 92 Control 12,289 f 179” 3,363 + 405" Basic FGF 773 antibody 4,078& 179’ Basic FGF and 773 6,672 f495' 4.883 f 273 Nonimmune IeG Values represent the mean + SEM for 10 replicate incubations. Cells were incubated in 5H medium with 1 rig/ml recombinant human basic FGF and/or 500 rig/ml protein-A-purified antibasic FGF antibody (no. 773) or 500 rig/ml nonimmune rabbit IgG, and [3H]thymidine incorporation was measured for 72 h or cell proliferation for 7 days. a P < 0.001 vs. control incubations. b P < 0.05 vs. control incubations. ’ P < 0.005 vs. basic FGF incubations.

FGF (P < 0.005; Table 1 and Fig. 7). No change in incorporation was seen in the presence of equivalent protein concentrations of control nonimmune rabbit IgG (Table 1). In a separate experiment, cells were exposed to increasing doses of heparin between 0.01-200 rig/ml, with and without 2 rig/ml basic FGF (Fig. 8). When cells were simultaneously exposed to basic FGF and heparin (Fig. 8A), heparin potentiated the stimulatory effect of the growth factor in a dose-related manner; 200 rig/ml heparin increased the stimulation by 2 rig/ml basic FGF by some 2-fold over levels seen in its absence (P < 0.001). In this experiment heparin alone (Fig. 8B) had a biphasic effect on [3H]thymidine incorporation into FRTL-5 cells. At lower doses it significantly (P < 0.001) enhanced basal rates of incorporation, while at doses above 10 ng/ ml it significantly reduced it (P < 0.001). Discussion The results show that rat thyroid FRTL-5 cells contain basic FGF mRNA and localize a heparin-binding protein

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BASIC FGF IN THE THYROID

7. Effects of a neutralizing antibasic FGF antibody on the mitogenic action of increasing concentrations of basic FGF in FRTL-5 cells. The effect on DNA synthesis of increasing concentrations of basic FGF (between 0.5-5 ng/ ml) with and without 500 rig/ml proteinA-purified antibasic FGF antibody (no. 773) was assessed by measuring [SH]thymidine incorporation into the DNA of FRTL-5 cells over 72 h. Values represent the mean f SEM for nine replicate cdtures. FIG.

= 5 -7 200000 I

2369

El With antibody

& is 5 E 100000 2 zCL

0

with basic FGF immunoreactivity of approximately 20 kilodaltons mol wt. It is likely that this protein is basic FGF. Basic FGF may contribute to an autocrine control of thyroid epithelial cell mitogenesis, since these cells also express abundant mRNA encoding one of the high affinity FGF receptors, fZg (20). The receptors are presumably functional and help confer basic FGF responsiveness on FRTL-5 cells, since exogenously supplied basic FGF stimulates [3H]thymidine incorporation in a dose-dependent manner, with an EDs0 of 4 rig/ml. The increases in [3H]thymidine incorporation in response to basic FGF were accompanied by increased cell proliferation. The observation of FRTL-5 responsiveness to exogenous basic FGF illustrated here confirms and extends the previous reports of ourselves (18) and others (17). The observation that FRTL-5 cells can only be growth restricted and not growth arrested in 5H medium suggests the contribution of continued synthesis of endogenous growth factors. Strong evidence to support the hypothesis of an autocrine role for endogenously produced basic FGF in FRTL-5 cell growth comes from observations with a specific basic FGF neutralizing antibody. We have shown that this antibody (no. 773) specifically blocks recombinant basic FGF bioactivity in FRTL-5 cells in a dose-dependent manner. Since the basal proliferation rate of FRTL-5 cells in 5H medium is also reproducibly and significantly reduced by immunoneutralization of endogenous basic FGF, it is probable that this growth factor is synthesized and externalized by FRTL-5 cells and makes a significant autocrine contribution to the control of basal levels of thyroid cell growth. The effects of heparin on FRTL-5 cell growth provide further evidence for an autocrine role of basic FGF in FRTL-5 cells. Basic FGF binds with high affinity to heparin-related, highly sulfated glycosaminoglycans both in vivo and in vitro, and this acts to protect the growth

0.5

1 rig/ml basic FGF

factor from proteolysis, high temperature, or low pH and extend its half-life (26-28). Heparin is known to potentiate the effects of basic FGF in other cells in culture (29) and cause the release of sequestered basic FGF from heparin sulfate proteoglycans on cell surfaces (30). This action of heparin is confirmed in FRTL-5 cells, in which the ability of heparin to enhance the mitogenic activity of exogenous basic FGF is dose dependent. We predicted that if endogenously produced basic FGF is contributing to basal growth of FRTL-5 cells, then basal levels of DNA synthesis should also be enhanced in the presence of heparin. Interestingly, in these studies we observed a biphasic effect of heparin when added alone to nonstimulated cultures of FRTL-5 cells exhibiting a basal rate of DNA synthesis. As predicted, at concentrations between 0.01-10 rig/ml, heparin caused a significant enhancement of the basal rate of [3H]thymidine incorporation in 5H medium. Clearly, this may be due to potentiation of the autocrine influences of endogenously produced and bioactive basic FGF. Although the actions of heparin are not specific to basic FGF, but extend to other cationic heparin-binding growth factors, it is likely that, in this cell line at least, basic FGF is a major component of the endogenously produced growth factors that might be potentiated by heparin. In contrast, at higher doses of heparin (100 and 200 rig/ml), a significant reduction in the basal rate of incorporation was observed. This may be due to the inhibitory actions of higher concentrations of internalized free heparin on inositol trisphosphate receptors (31). These intracellular receptors are activated as a consequence of basic FGF binding to its high affinity, cell surface receptor(s) and the subsequent activation of phosphoinositide hydrolysis (32). As higher doses of free heparin are internalized they may be blocking the mitogenic signalling pathway of bioactive endogenous basic FGF or other autocrine factors that also use the phosphoinositide signal pathway. Thus, when the concentrations of basic

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BASIC

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B

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8. Effects of heparin in FRTL-5 cells. The effects of heparin were assessed by measuring the incorporation of [3H]thymidine into DNA of FRTL-6 cells incubated with increasing concentrations of heparin, with (A) and without (B) 2 rig/ml basic FGF. Values represent the mean + SEM for six replicate cultures. Note differences in scale between A and B. *, P < 0.05; tt, P < 0.001 (us. 4 rig/ml basic FGF alone in A and us. 5H control in B). FIG.

FGF are relatively low and the concentrations of heparin relatively high, inhibition of intracellular signal pathways by heparin overcomes potentiation of basic FGFstimulated mitogenesis. The biphasic response to heparin was not apparent in the presence of exogenously added basic FGF. Low doses of heparin seem insufficient to potentiate the relatively large quantities of basic FGF present in stimulated cultures. Neither was the inhibitory effect of high doses of heparin observed in the presence of exogenously supplied basic FGF; 100 and 200 rig/ml heparin significantly potentiated the effects of the exogenously supplied basic FGF. It is likely that most of the heparin in this case will have bound to the relatively large excess of extracellular basic FGF present, compromising the route of entry of heparin into the cell and its access to inositol trisphosphate receptors on intracellular membranes. Thus, in the presence of higher concentrations of basic FGF, potentiation by heparin of basic FGF actions outside the cell overcomes inhibition by heparin of intracellular signal pathways. Together, these results suggest that both endogenous and exogenous basic FGFs are capable of contributing to

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thyroid epithelial cell growth. The responses of thyroid follicular cells to basic FGF that have been observed seem to be distinct from the responses to other trophic factors that have also been implicated in regulation of thyroid growth and function. Basic FGF is known to stimulate growth and inhibit differentiated function of follicular cells (18, 17). Similarly, epidermal growth factor stimulates thyroid cell growth and inhibits iodide uptake (2). In contrast, while insulin-like growth factorI also stimulates growth, it has no apparent effect on differentiated function alone (33, 34), and transforming growth factor-p inhibits both parameters (35). TSH stimulates growth and thyroid cell function (36). These observations suggest that the physiological regulation of thyroid cell growth and function in uiuo is likely to be the result of a complex interactive network of trophic autocrine, paracrine, and endocrine factors. The physiological relevance of basic FGF to the autocrine control of adult rat thyroid follicular cells is underlined by our observation of immunoreactive basic FGF in tissue sections of the normal rat thyroid. The localization of strong immunopositive staining for basic FGF in the cytoplasm of follicular cells, particularly the intense staining seen in their underlying basement membrane, suggests the presence of a local storage depot of this growth factor. FGF-binding heparin sulfate proteoglycans are located in basement membranes and on the surface of many cell types (37). Thus, basic FGF bound in the extracellular matrix and to cell surface glycosaminoglycans could function as extracellular stored growth factor, which has an extremely prolonged halflife. Similar observations in other tissues have led Baird and Walicke (38) to postulate a mechanism for the chronic regulation of basic FGF bioactivity via its local sequestration and selective release and activation by proteases or heparinases (39). This may explain our inability, despite rigorous attempts, to demonstrate basic FGF mRNA in thyroid tissues in uiuo by hybridization analyses, since the presence of substantial long term local stores of the growth factor would negate the requirement for continual high levels of basic FGF gene expression. Low level expression and instability of basic FGF mRNA has been reported in many peripheral tissues that contain high concentrations of basic FGF protein (4). The report of basic FGF mRNA in cultured porcine thyroid cells (17) and our observation of its presence in FRTL-5 cells can be reconciled with our failure to detect mRNA signal in normal rat follicular cells in uiuo if we consider follicular cells to have been artificially and acutely activated by pseudoinjury or immortalization when placed into culture. We would predict that significant levels of basic FGF mRNA would only be detected in thyroid cells in uiuo when they are actively growing during development, after injury, or in disease.

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BASIC FGF IN THE THYROID In summary, we have demonstrated the synthesis, localization, and autocrine mitogenic activity of basic FGF in cultured rat thyroid FRTL-5 cells and further demonstrated the presence of significant stores of this growth factor in the follicular basement membrane of the normal adult rat thyroid in uiuo. These results suggest the relevance of basic FGF as an autocrine regulator of physiological and pathophysiological growth of the thyroid.

Acknowledgments The authors would like to thank Drs. Andrew Baird and David Hill for helpful discussionsduring the execution of these studiesand for critical review of the manuscript.

References 1. Maciel RMB, Moses AC, Villone G, Tramontano D, Ingbar SH 1988 Demonstration of the production and physiological role of insulin-like growth factor II in rat thyroid follicular cells in culture. J Clin Invest 82:X46-1553 2. Eggo MC, Bacharach LK, Fayet G, Errick J, Kudlow JE, Cohen MF, Burrow GN 1984 The effects of growth factors and serum on DNA synthesis and differentiation in thyroid cells in culture. Mol Cell Endocrinol38:141-150 3. Gospodarowicz D 1974 Localisation of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth. Nature 249123-127 4. Baird A. Bohlen P 1990 Fibroblast growth factors. In: Sporn MB, Roberts.AB (eds) Peptide Growth Factors and Their Receptors. I. Handbook of Experimental Pharmacology. Springer-Verlag, Berlin and Heidelberg, vol95:369-418 5. Baird A, Mormede P, Ying SY, Wehrenberg WB, Ueno N, Ling N, Guillemin R 1985 A non-mitoeenic nituitarv function of fibroblast growth factor: regulation of chyrotropin and prolactin secretion. Proc Nat1 Acad Sci USA 825545-5549 6. Adashi EY, Resnik CE, Croft CS, May JV, Gospodarowicz D 1988 Basic fibroblast growth factor as a regulator of ovarian granulosa cell differentiation: a novel non-mitogenic role. Mol Cell Endocrino1 55:7-14 7. Baird A, Hsueh AJW 1986 Fibroblast growth factor as an intraovarian hormone: differential regulation of steroidogenesis by an angiogenic factor. Regul Peptides l&243-250 8. Fauser B, Baird A, Hsueh AJW 1988 Fibroblast growth factor inhibits luteinizing hormone-stimulated androgen production by cultured rat testicular cells. Endocrinology 1232935-2941 9. Raeside JI, Berthelon M-C, Sanchez P, Saez JM 1988 Stimulation of aromatase activity in immature porcine Leydig cells by fibroblast growth factor (FGF). Biochem Biophys Res Commun 151:163-169 10. Gospodarowicz D, Ill CR, Homsby PJ, Gill G 1977 Control of bovine adrenal cortical cell proliferation by fibroblast growth factor: lack of effect of epidermal growth factor. Endocrinology 100:1080-1089 11. Esch F, Baird A, Ling N, Ueno N, Hill F, Denoroy L, Klepper R, Gospodarowicz D, Bohlen P, Guillemin R 1985 Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine acidic FGF. Proc Nat1 Acad Sci USA 82~6507-6511 12. Gospodarowicz D, Bialecki H, Thakral TK 1979 The angiogenic activity of the fibroblast and epidermal growth factor. Exp Eye Res 28501-514 13. Iwata H, Matsuyama A, Okumua N, Yoshida S, Lee Y, Imaizumi K, Shiosaka S 1991 Localization of basic FGF-like immunoreactivity in the hypothalamo-hypophyseal neuroendocrine axis. Brain Res 550:329-332 14. Wanaka A, Johnson EM, Milbrandt J 1990 Localization of FGF receptor mRNA in the adult rat central nervous system by in situ hybridization. Neurone 5:267-281

15. Black EG, Davis JRE, Logan A, Sheppard MC 1989 Basic fibroblast growth.factor (FGF) effects on cellular proliferation and gene expression in thyroid FRTL-5 and pituitary GH3 cell lines. J Endocrinol121:242 16. Emoto N, Arai M, Murakami H, Tsushima T, Identification of basic FGF (FGF) from adult porcine thyroid. 72nd Annual Meeting of The Endocrine Society, Atlanta GA, 1990, p 207 17. Emoto N, Isozaki 0, Arai M, Murakami H, Shizume K, Baird A, Tsushima T, Demura H 1991 Identification and characterization of basic fibroblast growth factor in porcine thvroids. Endocrinolozv-12858-64 18. Black EG, Logan A, Davis JRE, Sheppard MC 1990 Basic fibroblast growth factor affects DNA synthesis and cell function and activates multiple signalling pathways in rat thyroid FRTL-5 and pituitary GHI cells. J Endocrinol 127:39-46 19. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning-A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor 20. Lee PL, Johnson DE, Cousens LS, Fried VA, Williams LT 1989 Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor. Science 24557-60 21. Emoto N. Gonzalez AM. Walicke PA. Wada E. Simmons DM. Shimasaki S, Baird A 1989 Identification of specific loci of basic fibroblast growth factor synthesis in the rat brain. Growth Factors 2:21-29 22. Simmons DM, Arriza JL, Swanson LW 1989 A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radiolabeled single-stranded RNA probes. J Histotechno1 12:169-181 23. Gonzalez AM, Buscaglia M, Ong M, Baird A 1990 Immunohistochemical localisation of basic FGF in tissues of the 18&y foetal rat. J Cell Biol 110~347-358 24. Lowry OH, Rosebrough NJ, Farr AL, Randall RF 1951 Protein measurements with Folin phenol reagent. J Biol Chem 193:265275 25. Ambesi-Impiombato FS, Parks LAM, Coon HG 1980 Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc Nat1 Acad Sci USA 723255-3459 26. Baird A, Schubert D, Ling N, Guillemin R 1988 Receptor and heparin-binding domains of basic tibroblast growth factor. Proc Nat1 Acad Sci USA 85~2324-2328 27. Gospodarowicz D, Cheng J 1986 Heparin protects basic and acidic FGF from inactivation. J Cell Physiol 128475-484 28. Rosengart TK, Johnson WV, Friesel R, Clark R, Maciag T 1988 Heparin protects heparin-binding growth factor-l from proteolytic inactivation in vitro. Biochem Biophys Res Commun l&432-440 29. Hill DJ. Logan A. Interactions of nentide growth factors durine DNA s&h&is in. isolated ovine fetal gro$h plate chondrocytei Growth Regul, in press 30. Bashkin P, Doctrow S, Klagsbrun M, Suahn CM, Folkman J, Vlodavsky I 1989 Basic tibroblast growth factor binds to subendothelial extracellular matrix and is released by heperitinase and henarin-like molecules. Biochemistrv 281737-1743 31. Hii1 TD, Berrgren P-O, Boynton AL 1987 Heparin inhibits trisphosphate-induced calcium release from permeabilized rat liver cells. Biochem Bionhvs Res Commun 149:897-901 32. Logan A, McDermott EE, Logan SD 1990 Production of inositol phosphates in Balb/c 3T3 cells stimulated with basic fibroblast growth factor. Cell Signal 2~77-84 33. Toramontano D, Cushing GW, Moses AC, Ingbar SH 1986 Insulinlike growth factor-l stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Grave’s-IgG. Endocrinology 119940-942 34. Saji M, Tsushima T, Isozaki 0, Murakami H, Ohba Y, Sato K, Arai M. Shizume K 1987 Interaction of insulin-like mowth factor I with porcine thyroid cells cultured in monolayer. Endocrinology 121:749-756 35. Tsushima T, Arai M, Saji M, Ohba Y, Murakami H, Ohmura E, Sato K, Shizume K 1988 Effects of transforming growth factor-0 on deoxyribonucleic acid synthesis and iodine metabolism in porcine thyroid cells in culture. Endocrinology 123:1187-1194 36. Roger PP, Dumont JE 1984 Factors controlling proliferation and

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differentiation of canine thyroid cells cultured in reduced serum conditions: effects of thyrotropin, cyclic AMP and growth factors. Mol Cell Endocrinol36:79-83 37. Moscatelli D, Presta M, Joseph-Silver&in J, Rifkin DB 1987 Presence of basic tibroblast growth factor in a variety of cells and its binding to cells. In: Rifkin DB, Klagsbrun M (eds) Angiogenesis. Current Communications in Molecular Biology. Cold Spring Har-

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bor Laboratory, Cold Spring Harbor, pp 4’7-51 38. Baird A, Walicke PA 1989 Fibroblast growth factors. Br Med Bull 4243%452 39. Baird A, Ling N 1987 Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in uitro: implication for a role of heparinase-like enzymes in the neovascular response. Biochem Biophys Res Commun 142:428-435

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