0013-7227/91/1281-0058$02.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 1
Printed in U.S.A.
Identification and Characterization of Basic Fibroblast Growth Factor in Porcine Thyroids* NAOYA EMOTO, OSAMU ISOZAKI, MARIKO ARAI, HITOMI MURAKAMI, KAZUO SHIZUME, ANDREW BAIRD, TOSHIO TSUSHIMA, AND HIROSHI DEMURA Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College (N.E., O.I., T.T., H.D.), Tokyo 162, Institute of Growth Science (MA., H.M., K.S.), Tokyo 162 Japan; and Department of Molecular and Cellular Growth Biology, The Whittier Institute for Diabetes and Endocrinology (A.B), La Jolla, California 92037
of this conclusion, basic FGF mRNA was detected in poly A+ RNA isolated from cultured porcine thyroid cells. On the other hand, recombinant human basic FGF at the concentration of 0.1-10 ng/ml increased the incorporation of 3 H-thymidine into FRTL-5 cells and porcine thyroid follicular cells in culture. The stimulatory effects were also observed when the biologically active fractions of heparin-sepharose column of the thyroid extract were added to the culture. In addition, both recombinant human basic FGF at 10 ng/ml and heparin binding fractions from porcine thyroids inhibited TSH-induced iodide uptake by porcine thyroid cells in culture. These results suggest that basic FGF may be one of the important local modulators of thyroid function, cell growth, and angiogenesis. (Endocrinology 128: 58-64,1991)
ABSTRACT. Fibroblast growth factor (FGF) is one of the most potent endothelial cell growth factors and has been found in almost all tissues in the body. However, there is no definitive report showing that FGF exists in the thyroid. In this report, we describe heparin binding endothelial cell growth factor activity in porcine thyroids. The extract from normal adult porcine thyroids was loaded onto a heparin-sepharose affinity column. The mitogenic activity on endothelial cells was found to elute from the column with 0.9 M-2.0 M NaCl. Polyacrylamide gel electrophoresis and an immunoblotting of bioactive fractions showed the basic FGF immunoreactivity in a double band with a molecular weight of 18K and 20K. These results strongly imply that the adult porcine thyroid-derived heparin binding endothelial cell growth factor may be authentic basic FGF and one of its high molecular weight forms. In support
I
T HAS been described that thyroid enlargement is accompanied by angiogenesis (1). In general, peptides called angiogenic factors are considered to be key factors in the process of angiogenesis (2). Among many reported angiogenic factors, fibroblast growth factor (FGF) has been considered as one of the most potent angiogenic factors. Although FGFs were originally found in the mesoderm and neuroectodermal tissues, recent biochemical and immunohistochemical data revealed the presence of basic FGF in essentially almost all tissues in the body except serum and thyroid (3, 4). Goodman et al. (5) and Greil et al. (6) reported angiogenic factors from rat thyroid and porcine thyroid, respectively. These reports suggested that these factors may be distinct from FGFs; however, they could not exclude a possibility that FGFs may exist in thyroids with other angiogenic factors.
On the other hand, recent reports have suggested that peptide growth factors, including FGFs, are multi-functional (7). These factors are originally found as mitogenic factors for certain cells, and those for endothelial cells are considered to be angiogenic factors. However, the functions of these factors are not limited to cell growth. These factors seem to modulate the function of the organ in which they are found.
Materials
Received August 6,1990. Address all correspondence and requests for reprints to: Dr. Naoya Emoto, Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162 Japan. * This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, a grant from the Ministry of Health and Welfare, and a grant from the Foundation for Growth Science.
Fetal calf serum (FCS) was obtained from Filtron PTY Ltd. (Victoria, Australia); calf serum (CS) was obtained from Nakarai Company (Kyoto, Japan). F-12 medium was purchased from Flow Laboratories (McLean, VA), and the multiwell tissue culture plates were from Coster (Cambridge, MA). Recombinant basic FGF was obtained from Chiron Corporation (Emery Bille, CA). Other hormones and chemicals were
In the present study, we demonstrate the existence of basic FGF and its high molecular weight form in normal adult porcine thyroid tissues and suggest that basic FGF in porcine thyroids may be an important local modulator of thryoid function, cell growth, and angiogenesis.
Materials and Methods
58
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
FGF IN THE THYROID purchased from Sigma Chemical Company (St. Louis, MO), unless otherwise indicated. Extraction of porcine thyroid FGF Normal adult porcine thyroids (100 g) were obtained from a local slaughterhouse. Tissues were homogenized by polytron homogenizer in 2.0 M NaCl, 10 mM Tris-Cl, 1 mM EDTA, 1 mM EGTA, pH 2.5 with 1 mM phenylmethylsulfonylfluoride. The pH was readjusted to 2.5 with 10 N HCl. The suspension was centrifuged, and the pH of the supernatant was adjusted to 7.4 with 1 N NaOH. This crude extract was diluted with 10 mM Tris, 1 mM EDTA, 1 mM EGTA, pH 7.4 to less than 0.6 M as the salt concentration, and was applied to a heparin-sepharose column (1.6 X 1.0 cm, bed vol 2 ml). After the column was washed with 10 mM Tris (pH 7.4), 0.6 M NaCl, the protein was eluted sequentially by the stepwise addition of 0.9 M, 1.25 M, and 2.0 M NaCl in 10 mM Tris (pH 7.4). Endothelial cell proliferation assays The mitogenic activity of the column fractions and purified samples was determined using cloned vascular endothelial cells derived from the bovine pulmonary artery. Cells were seeded at an initial density of 1.0 X 104 cells per well in 12-well culture plates containing 0.5 ml F12 medium supplemented with 10% FCS. Six h later, the medium was changed to F12 medium supplemented with 15% CS. Ten-microliter aliquots of the appropriate diluation of each fraction (with F12 medium-0.1% BSA) were added to the wells every other day. After 4 days, plates were trypsinized, and final cell densities were determined by counting cells in a Coulter counter (Coulter Electronics, Inc., Hialeah, FL). Porcine thyroid cells and iodide uptake Isolated porcine thyroid cells were prepared using Dispase (Godo Susei Co., Tokyo, Japan) as described by Isozaki et al. (8). The cells were suspended in F-12 medium supplemented with 5% FCS at a concentration of 2 X 106 cells/ml, and 0.5 ml of the suspension was dispensed into the wells of 12-well tissue culture plates (3.8 cm2/well). The cells were cultivated at 37 C in a humidified atmosphere of 5% CO2 in air. Within 24 h, the cells attached to the plastic surface. The contamination of fibroblast was less than 1% during experiments, as described previously (9). On the second day of culture, medium was switched to F-12 medium containing 0.5% FCS. The thyroid cells were incubated with the indicated concentrations of TSH and test materials for 3 days, unless otherwise indicated. After that, the medium was replaced by fresh F-12 medium with 0.1% BSA, and the cells were exposed to 0.1 fid Na126I (New England Nuclear Corp., Boston, MA) with 10"7 M Nal for 1 h. The medium was rapidly removed, and the cultures were washed with ice-cold Hank's Balanced Salt Solution (HBSS). The cells were then solubilized with 0.1 N NaOH, and an aliquot of the solution was counted for radioactivity. Iodide uptake was defined as total intracellular radioactivity as described previously (10).
59
3
H-Thymidine incorporation into the cultured porcine thyroid cells The thyroid cells were cultured in F-12 medium containing 5% FCS for 1 day, and in F-12 medium containing 0.5% FCS and 0.1% BSA for 3 days, then the agents and (methyl3 H)thymidine (0.1 Ci/well; New England Nuclear Corp.) were added to the cultures. After 3 days the medium was aspirated, and the cells were washed with PBS twice. Then, the cells were precipitated with 10% trichloroacetic acid (TCA) and solubilized by 0.5 ml 1 N NaOH and neutralized with 0.5 ml 1 N HCl. The radioactivity of the solutions was counted by a /3-counter using liquid scintillation counting solution ACSII (Amersham, Arlington Heights, IL). FRTL-5 cells culture and 3H-thymidine incorporation assay The functional rat thyroid follicular cell line FRTL-5 cells were cultured in modified Ham's F-12 medium supplemented with 5% CS and a six-hormone preparation (6H medium) consisting of insulin (10 /ug/ml), hydrocortisone (10 nM), transferrin (5 mg/ml), glycyl-L-lysine acetate (10 ng/ml), somatostatin (10 ng/ml), and bovine TSH (1.0 mU/ml) as described previously (11). The cells (0.5 X 105 cells/well) were plated in 12-well plates and cultured for 3 days in 6H medium at 37 C in a humidified atmosphere (95% air, 5% CO2). The medium was then replaced by 5H medium (6H medium minus TSH) supplemented with 5% CS, and the cells were cultured for 2 days. On the fifth day, the 5H medium (5% CS) was replaced with the fresh 5H medium, and the test materials and 3H-thymidine (0.1 jiCi/ well) were added to the cultures. After 3 days, the cells were washed twice with PBS and precipitated with 10% trichloroacetic acid. The radioactivity of 3H-thymidine incorporated into the cells was determined by the same method described in the porcine thyroid cell experiments. SDS/PAGE and immunoblotting Fractions of heparin-sepharose column were dialyzed, concentrated by lyophilization, and applied to a 15% polyacrylamide resolving gel with a 3% stacking gel and electrophoresed. Samples were blotted from gels onto nitrocellulose sheets by electrotransfer. Blots were then incubated overnight in the buffer containing the antiserum. Immunoreactive proteins were revealed by successive incubations with goat antirabbit immunoglobulin conjugated with horse radish peroxidase (Bio-Rad Laboratories, Richmond, CA) and 4-chrol-l-naphthol/H2O2. Northern blot analyses Northern blot analyses were performed as previously described (12, 13). Briefly, total RNA was prepared by the conventional guanidine isothiocyanate method, and poly A+ RNA was selected by oligo(dT) cellulose column. RNA samples were separated on a 1% agarose gel containing 0.66 M formaldehyde and transfered to Hybond blotting membrane (Amersham). Membranes were hybridized with a 32P-labeled 0.5 kilobase rat bovine FGF complementary DNA (cDNA) (14). Hybridization and washing were performed as described by Church and Gil-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
FGF IN THE THYROID
60
bert (15); final washing was carried out at 65 C in 1 x SSPE (0.15 M NaCl, 0.01 M NaH 2 PO 4 ,1 mM EDTA, pH 7.4). Statistical analysis To analyze the data, analysis of variance was first performed, and statistical analysis of differences between groups was carried out using Student's t test if the variation in the data was uniform. When the variation in the data was not uniform, Cochran-Cox's test was employed.
Results Identification and characterization of the porcine thyroidderived heparin-binding endothelial cell growth factor The porcine thyroid-derived endothelial cell growth factor was isolated by using heparin-sepharose affinity chromatography. The crude extract of the porcine thyroid homogenate was diluted and applied onto a heparinsepharose column. In the preliminary study, the mitogenic activity was eluted with 2.0 M NaCl in 10 mM Tris, pH 7.4 (data not shown). Therefore, we examined the stepwise addition of 0.9 M, 1.25 M, and 2.0 M NaCl in 10 mM Tris-Cl. The volume of each elution was 30 ml. The elution profile of the extract after heparin-sepharose column is shown in Fig. 1. The mitogenic activity for endothelial cells was detected in all the fractions eluted with 0.9 M and 1.25 M NaCl, but in only the first several fractions eluted with 2.0 M NaCl. After completion of the chromatography, the protein profile was separated into five distinct pools (Pl-5, shown in Fig. 1, the vol of each pool was 6 ml), dialyzed, lyophilized, and resuspended in 200 (A 10 mM Tris-Cl (pH 7.4).
Endo• 1991 Voll28»Nol
Five pools were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot was performed with polyclonal antibasic FGF antibody raised against the synthetic bovine basic FGF fragment 1-24 (773) (Fig. 2). The basic FGF immunoreactivity was detected in two different forms with molecular mass of 18K and 20K. Both 18K and 20K bands were seen in the pool 3 and 4 (1.25 M NaCl elution), but the 18K band was predominant in pool 5 (2.0 M NaCl elution). In pool 2, two bands were detectable in a lower intensity, and there was no immunoreactivity in pool 1. We tried immunoblots using antibodies raised against synthetic bovine acidic FGF fragments 1-8, 1-15, and 132-140, but we could not detect any acidic FGF immunoreactivity in any of five pools. Effect of basic FGF on the growth of FRTL-5 cells It has been reported that FGFs stimulate the proliferation of a variety of cells. We examined the effect of basic FGF on the growth of rat thyroid follicle-derived cell line FRTL-5 cells. Figure 3 (top panel, solid line) shows the effect of recombinant human basic FGF on the 3H-thymidine incorporation into FRTL-5 cells. Basic FGF stimulated the DNA synthesis in FRTL-5 cells in a dose-dependent manner. 10 ng/ml of basic FGF stimulated the thymidine incorporation 100-fold over controls. We also examined the effect of the porcine thyroidderived heparin-binding growth factor on the growth of
1810
15
Fraction
20
25
30
35
Number
FlG. 1. Purification of porcine thyroid-derived endothelial cell growth factors by heparin-sepharose affinity chromatography. The porcine thyroid extract was chromatographed on a heparin-sepharose column, as described in Materials and Methods. The column was eluted stepwise with 0.6, 0.9, 1.25, and 2.0 M NaCl in 10 mM Tris-HCl, pH 7.4, as described in the text. Five-microliter aliquots of each fraction were added to bovine pulmonary artery endothelial cell cultures. Five zones of mitogenic activity were labeled as pools 1-5 (PI, P2, P3, P4, and P5).
3
A
FIG. 2. Western blots of five pools of fractions eluted from heparinsepharose column. Five pools (Pl-5 shown in Fig. 1; lane 1-5) were subjected to SDS-PAGE, and immunoblot was performed with antibasic FGF antibody as described in the text. Lane 6 is recombinant human basic FGF.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
61
FGF IN THE THYROID
0.001
Q01
0.1
1.0
1Q0
tion into porcine thyroid follicular cells in culture. Basic FGF stimulated the cell growth of porcine follicular cells estimated by 3H-thymidine incorporation in a dose-dependent manner. Compared to the effect on FRTL-5 cells, the magnitude of the response was much smaller in the porcine thyroid primary culture. Ten nanograms per ml basic FGF stimulated DNA synthesis in porcine follicular cells 4-fold over controls. The minimum effective dose was 100 pg/ml. We also examined the effect of heparin binding fractions of porcine thyroid extracts. The fractions eluted at 0.9 M, 1.25 M, and 2.0 M stimulated 3H-thymidine incorporation into cultured porcine thyroid cells (Fig. 4)
100.0
FGF (ng/ml)
S x „
a
7
125M
Q9M
2.0M
Effect of basic FGF on iodide uptake into the cultured porcine thyroid cells
6
3
10
20
30
FlG. 3. Mitogenic effects of basic FGF and porcine thyroid extracts on thyroid cells. Top panel, Concentration-dependent effects of recombinant human basic FGF on DNA synthesis in FRTL-5 cells (•—•) and cultured porcine thyroid cells ( • • ) . FRTL-5 cells were cultured for 3 days in 5H supplemented with 5% CS with various concentrations of recombinant human basic FGF. Porcine thyroid cells were cultured for 3 days in F-12 medium containing 1% FCS and 0.1% BSA with various concentrations of recombinant human basic FGF. Bottom panel, Effect of heparin-binding fractions of the thyroid extract on DNA synthesis in FRTL-5 cells. The column was eluted stepwise with 0.6, 0.9, 1.25, and 2.0 M NaCl in 10 mM Tris-HCl, pH 7.4. Fivemicroliter aliquots of each fraction were added to FRTL-5 cells cultured in 5H supplemented with 5% CS. The cultures were labeled with 3Hthymidine (3H-TdR) as described in the text. The data are expressed as cpm of 3H-TdR incorporated into TCA-insoluble cellular fractions. The values are the mean ± SE of triplicate wells from one representative experiment out of three.
Recent studies have suggested that FGFs are multifunctional factors (7). In order to clarify the physiological role of FGF in the thyroid, we examined the effect of basic FGF on thyroid function other than cell growth. Figure 5 (top panel) shows the effect of recombinant basic FGF on iodide uptake into the cultured porcine thyroid follicles. Basic FGF alone had no effect on the iodide uptake, but it inhibited the TSH-stimulated iodide uptake at the concentration of 10 ng/ml. The same inhibitory effect was seen when the heparin-sepharose column chromatography fractions of porcine thyroid extracts were used (Fig. 5, bottom panel). Northern blot analyses We could not detect any hybridizing mRNA of basic FGF in Northern blot analysis of poly A+ RNA isolated from adult porcine thyroid tissues (data not shown). We 3.0
2.0
FRTL-5 cells. Figure 3 (bottom panel) indicates the mitogenic effect of the porcine thyroid extract after a heparin-sepharose column chromatography. The mitogenic activity on FRTL-5 cells was seen in the fractions starting from 0.9 M-2.0 M NaCl elution. This profile was almost identical with the mitogenic effect for endothelial cells (Fig. 1). Effect of basic FGF on the growth of primary thyroid follicular cells in culture
porcine
We next examined the effect of basic FGF on the growth of normal thyroid follicular cells in culture. Figure 3 (top panel, broken line) shows the effect of human recombinant basic FGF on the 3H-thymidine incorpora-
1.0
Control
bFGF
0.6M
0.9M
1.25M
2.0M
(lOng/ml)
FlG. 4. Effect of heparin-binding fractions of the thyroid extract on DNA synthesis in porcine thyroid cells in culture. Five-microliter aliquots of fractions eluted at 0.6, 0.9,1.25, and 2.0 M NaCl were added to porcine thyroid cells and cultured for 3 days. The cultures were labeled with 3H-TdR as described in the text. The data are expressed as cpm of 3H-TdR incorporated into TCA-insoluble cellular fractions. The values are the mean ± SE of triplicate wells from one representative experiment out of three.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
FGF IN THE THYROID
62
Q01
0.1
1.0
100
1000
basic FGF (ng/ml) 0.6M
-
Q9M
Endo • 1991 Voll28«Nol
2 8S
2.0M
125M
5 "
18S
1 -
15
20
Fraction Number
FIG. 5. Top panel, Inhibitory effect of recombinant human basic FGF on TSH-induced iodide uptake in porcine thyroid cells. Thyroid cells were cultured with 50 /iU/ml TSH for 3 days in the presence or absence of various concentrations of basic FGF and tested for iodide uptake as described in the text. Bottom panel, Inhibitory effect of heparin-sepharose column fractions of the porcine thyroid extract on TSH-induced iodide uptake in porcine thyroid cells in culture. The column was eluted stepwise with 0.6, 0.9, 1.25, and 2.0 M NaCl in 10 mM Tris-HCl, pH 7.4. Five-microliter aliquots of each fraction were added to porcine thyroid cells cultured with 50 /iU/ml TSH. The cells were cultured for 3 days and tested for iodide uptake as described in the text. The results shown are the mean ± SE of triplicate wells from one representative experiment out of three.
next examined RNA isolated from cultured porcine thyroid cells. The cells were cultured in F-12 medium containing 0.1% BSA for two days, and then RNA was isolated. Two hybridizing basic FGF mRNA, 7.0 and 3.7 kilobases in length, were detected in poly A+ RNA (3 ng) (Fig. 6). These sizes are identical with those of reported human and bovine basic FGF mRNA (16).
Discussion In this report, we demonstrated the existence of heparin-binding endothelial cell growth factor in normal adult porcine thyroid. Mitogenic activity for endothelial cells was present in fractions that eluted from heparinsepharose column in broad peaks from 0.9-2.0 M NaCl. These fractions have basic FGF immunoreactivities. The immuno-blotting study of SDS-PAGE indicated a double band with a molecular weight of 18K and 20K. The 18K band was exactly identical in its size with recombinant
FIG. 6. Northern blot analysis of the expression of basic FGF in porcine thyroid cells in culture. Poly A+ RNA (3 fig) was prepared, as described in Materials and Methods, from porcine thyroid cells in culture. The membrane was hybridzed to rat basic FGF cDNA probe.
human basic FGF. There is high sequence homology between basic FGFs of different species. Among human, bovine, and rat, the sequence of basic FGFs are almost identical, except for several substitutes out of 155 or 154 amino acids (14). Thus, it seems reasonable to expect that the molecular weight of porcine basic FGF is the same as human. On the other hand, the 20K band may be a high molecular weight form of basic FGF. Recent studies show that there are several AT-terminal extended high molecular weight forms (22-25K) of basic FGF isolated from hepatoma cells (17), guinea pig (18), rat brain (19), and H-ras-transformed Rat-1 cells (20). The existence of the high molecular weight form of basic FGF had been questioned, because no AUG codon in cDNA frame has been found upstream of the putative start codon (AUG) from which synthesis of a 155-amino acid (18K) is thought to start. However, Florkiewicz et al. (21) and Prats et al. (22) showed that the high molecular weight forms of basic FGF are translationally initiated at unusual nonAUG codons. It may well be that the same mechanism could yield the high molecular weight form of basic FGF in porcine thyroid. In addition, cultured porcine thyroid cells expressed mRNA hybridized with bovine basic FGF cDNA. Thus, we conclude that porcine thyroid-derived heparin binding endothelial cell growth factor is at least partly, if not all, basic FGF, although there is a possibility that acidic FGF or other heparin binding growth factors also may
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
FGF IN THE THYROID exist in porcine thyroids. The immuno-blotting study of SDS-PAGE indicated another intriguing result. Both 18K and 20K form of immunoreactive porcine basic FGF started to elute at 0.9 M NaCl, but amount of eluted peptides was very low. At 1.25 M elution, two forms of basic FGF were clearly seen; however, the 18K band was dominant in the fractions eluted at 2.0 M. Furthermore, 0.9-1.25 M NaCl elution of heparin-sepharose column yielded broad peaks in the mitogenic activity for endothelial cells, but all the mitogenic activities came out by the wash with 2.0 M NaCl. It is generally accepted that basic FGF elute from heparin-sepharose column with 1.4-1.6 M NaCl and acidic FGF elute with 0.9-1.1 M NaCl (3), and that N-terminalextended high mol wt form of basic FGF have the same biological activities and the same affinity to heparin (21). However, Quinkler et al. (23) reported that some of canine and porcine heart FGF eluted at salt concentrations between 1.2 M-1.3 M. These results suggest that there may be a microheterogeneity in the affinity of porcine basic FGF to heparin. Actually, Sommer et al. (24) reported the existence of the placental basic FGF which consists of 157 amino acids, three amino acids longer at N-terminus than the one reported before. If this small diversity exists in porcine thyroid basic FGF, it may be difficult to differentiate these peptides in our SDS-PAGE. In addition, the high mol wt form (20K) of basic FGF seems to have a relatively low affinity to heparin, because not much 20K basic FGF retained to the column after the 1.25 M NaCl wash. Thus, there is a possibility that the N-terminal extended sequence may affect the chemical character of the molecule. Although further studies are necessary, the establishment of the complete primary structure of high mol wt form of porcine thyroid-derived basic FGF can be expected to clarify the significance of heparin-binding domains in the sequence of FGF family. Basic FGF was initially isolated from many tissues as a fibroblast and endothelial cell growth factor. However, within a short period of time, it was revealed that its function is not limited to the growth of these cells. In endocrine tissues, there are evidences that it is playing a role in modulating endocrine function through regulating the hormone secretion and/or the growth of endocrine cells. It potentiates TRH-stimulated PRL and TSH secretion from cultured anterior pituitary cells (25), affects the hormone synthesis in reproductive tissues (26-28), and stimulates the proliferation of adrenocortical cells in vitro (29). In this report, we demonstrated the multifunctional effect of basic FGF on the thyroid. It stimulates the growth of thyroid follicular cells and inhibits iodide uptake by these cells. These results suggest that basic FGF is one of the local function modulators in thyroid tissues. It has been reported that growth factors
63
have diverse effects on the function and growth of thyroid tissues. EGF stimulates the thyroid follicular cell growth but inhibits the iodide uptake by these cells (30-33). On the other hand, TGF-0 inhibits both (34, 35), and IGF-I stimulates the growth and has no effect on the iodide uptake (9, 36). Although the function of basic FGF seems similar to that of EGF, basic FGF is unique because of its angiogenic effect. Thus, it may well be that in the process of thyroid enlargement, basic FGF may promote the vascular formation, stimulate the thyroid cell growth, and inhibit the thyroid function. It may be reasonable to hypothesize that basic FGF may play important roles in the pathogenesis of goiter, but this relationship remains to be clarified. On the other hand, it should be noted that basic FGF exists in normal thyroid tissues in which neither active angiogenesis nor thyroid cell growth is taking place. Thus, in normal circumstances, basic FGF should exist as an inactive form. Folkman et al. (37) indicated that FGFs were detected in the basement membranes and speculated that FGFs bind to heparan sulfate proteoglycans, structurally related to heparin, present in extracellular matrix, making these factors inaccessible to their receptors. Baird and Ling (38) suggested the enzymatic degradation of extracellular matrix by proteases or heparanases, secreted by inflammatory and immune cells, may be able to release basic FGF. Although the exact mechanism of the activation of basic FGF in many tissues remains unclear, these data strongly imply that the inflammatory response in the thyroid, which is usually seen in autoimmune thyroid diseases, could activate stored basic FGF. In summary, we demonstrated the existence of basic FGF in normal adult porcine thyroid. It promotes the angiogenesis, stimulates the thyroid cell growth, and inhibits the iodide uptake in vitro. These data suggest the importance of basic FGF in thyroids as one of the local growth and function modulators. The possible involvement of basic FGF in the pathogenesis of goiter remains to be clarified. References 1. Wollman SH, Herverg JP, Zeligs JD, Ericson LE 1978 Blood capillary enlargement during the development of thyroid hyperplasia in the rat. Endocrinology 103:2306-2314 2. Folkman J, Klagsbrun M1987 Angiogenic factors. Science 235:442447 3. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G 1987 Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 8:95-114 4. Baird A, Walicke PA 1989 Fibroblast growth factors. Br Med Bull 45:438-452 5. Goodman AL, Rone JD 1987 Thyroid angiogenesis: endotheliotropic chemoattractant activity from rat thyroid cells in culture. Endocrinology 121:2131-2140 6. Geil W, Rafferzeder M, Bechtner G, Gartner R 1989 Release of an endothelial cell growth factor from cultured porcine thyroid folli-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
64
FGF IN THE THYROID
cles. Mol Endocrinol 3:858-867 7. Sporn MB, Roberts AB 1988 Peptide growth factors are multifunctional. Nature 332:217-219 8. Isozaki 0 , Tsushima T, Shizume K, Saji M, Ohba Y, Emoto N, Sato K, Sato Y, Kusakabe K 1985 Thyroid-stimulating antibody bioassay using porcine thyroid cells cultured in follicles. J Clin Endocrinol Metab 61:1105-1111 9. Saji M, Tsushima T, Isozaki 0 , Murakami H, Ohba Y, Sato K, Arai M, Shizume K 1987 Interraction of insulin-like growth factor I with porcine thyroid cells cultured in monolayer. Endocrinology 121:749-756 10. Tsuchiya Y, Saji M, Isozaki O, Arai M, Tsushima T, Shizume K 1990 Effect of lithium on deoxyribonucleic acid synthesis and iodide uptake in porcine thyroid cells in culture. Endocrinology 126:460-465 11. Saji M, Isozaki O, Tsushima T, Arai M, Miyakawa M, Ohba Y, Tsuchiya Y, Sano T, Shizume K 1988 The inhibitory effect of iodide on growth of rat thyroid (FRTL-5) cells. Acta Endocrinol (Copenh) 119:145-151 12. Emoto N, Gonzalez A-M, Walicke PA, Wada E, Simmons DM, Shimasaki S, Baird A 1989 Basic fibroblast growth factor (FGF) in the central nervous system: identification of specific loci of basic FGF expression in the rat brain. Growth Factors 2:21-29 13. Isozaki O, Kohn LD, Kozak CA, Kimura S 1989 Thyroid peroxidase: rat cDNA sequence, chromosomal localization in mouse, and regulation of gene expression by comparison to thyroglobulin in rat FRTL-5 cells. Mol Endocrinol 3:1681-1692 14. Shimasaki S, Emoto N, Koba A, Mercado M, Shibat F, Cooksey K, Baird A, Ling N 1988 Complementary DNA cloning and sequencing of rat ovarian basic fibroblast growth factor and tissue distribution study of its mRNA. Biochem Biophys Res Commun 157:256-263 15. Church GM, Gilbert W 1984 Genomic sequencing. Proc Natl Acad Sci USA 81:1991-1995 16. Schweigerer L, Neufeld G, Friedman J, Abraham JA, Fiddes JC, Gospodarowicz D 1987 Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature 325:257-259 17. Presta M, Moscatelli D, Joseph-Silverstein D, Rifkin DB 1986 Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration. Mol Cell Biol 6:4060-4066 18. Moscatelli D, Joseph-Silverstein J, Manejias R, Rifkin DB 1987 Mr 25,000 heparin binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc Natl Acad Sci USA 84:5778-5782 19. Presta M, Rusnati M, Maier JAM, Ragnotti G 1988 Purification of basic fibroblast growth factor from rat brain: identification of an Mr 22,000 immunoreactive form. Biochem Biophys Res Commun 155:1161-1172 20. Iberg N, Rogelj S, Fanning P, Klagsbrun M 1989 Purification of 18 kDa and 22 kDa forms of basic fibroblast growth factor from rat cells transformed by the ras oncogene. J Biol Chem 264:1995119955 21. Florkiewicz R, Sommer A 1989 Human basic fibroblast growth factor encodes four polypeptides: three initiate translation from non-AUG codons. Proc Natl Acad Sci USA 86:3978-3981 22. Prats H, Kaghad M, Prats AC, Klagsbrun M, Lelias JM, Leauzun P, Chalon P, Tauber JP, Amalric F, Smith JA, Caput D 1989 High molecular weight forms of basic fibroblast growth factor are initi-
23.
24.
25.
26.
27. 28.
29.
30.
31.
32.
33.
34.
35.
36.
Endo«1991 Voll28«Nol
ated by alternative CUG codons. Proc Natl Acad Sci USA 86:18361840 Quinkler W, Maasberg M, Bernotat-Danielowski S, Luthe N, Sharma HS, Schaper W 1989 Isolation of heparin-binding growth factors from bovine, porcine and canine hearts. Eur J Biochem 181:67-73 Sommer A, Brewer MT, Thompson RC, Moscatelli D, Presta M, Rifkin DB 1987 A form of human basic fibroblast growth factor with an extended amino terminus. Biochem Biophys Res Commun 144:543-550 Baird A, Mormede P, Ying SY, Wehrenberg WB, Ueno N, Ling N, Guillemin R 1985 A non-mitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proc Natl Acad Sci USA 82:5545-5549 Adashi EY, Resnick 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 Endocrinol 55:7-14 Baird A, Hsueh AJW 1986 Fibroblast growth factor as an intraovarian hormone: differential regulation of steroidogenesis by an angiogenic factor. Regul Pept 10:309-317 Raeside JI, Berthelon M-C, Sanchez P, Saiz JM 1988 Stimulation of aromatase activity in immature porcine Leydig cells by fibroblast growth factor (FGF). Biochem Biophys Res Commun 151:163-169 Gospodarowicz D, 111 CR, Hornsby 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 Roger PP, Dumont JE 1982 Epidermal growth factor controls the proliferation and the expression of differentiation of canine thyroid cells in primary culture. FEBS Lett 144:209-212 Westermark K, Karlsson FA, Westermark B 1983 Epidemal growth factor modulates thyroid growth and function in culture. Endocrinology 112:1680-1686 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 Endocrinol 38:141-150 Bacharach LK, Eggo MC, Mak WW, Burrow GN 1985 Phorbol esters stimulate growth and inhibit differentiation in cultured thyroid cells. Endocrinology 116:1603-1609 Tsushima T, Arai M, Saji M, Ohba Y, Murakami H, Ohmura E, Sato K, Shizume K 1988 Effects of transforming growth factor-/? on deoxyribonucleic acid synthesis and iodine metabolism in porcine thyroid cells in culture. Endocrinology 123:1187-1194 Grubeck-Loebenstein B, Buchan G, Sadeghi R, Kissonerghis M, Londei M, Turner M, Pirich K, Roka R, Niederle B, Kassal H, Werner W, Feldmann M 1989 Transforming growth factor-/? regulates thyroid growth. J Clin Invest 83:764-770 Toramontano D, Cushing GW, Moses AC, Ingbar SH 1986 Insulin-
like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Grave's-IgG. Endocrinology 119:940-942 37. Folkman J, Klagsbrun M, Sasse J, Wadsinski M, Ingber D, Vlodavsky I 1988 A heparin-binding angiogenic protein—basic fibroblast growth factor—is stored within basement membrane. Am J Pathol 130:393-400 38. Baird A, Ling N 1987 Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implication for a role of heparinase-like enzymes in the neovascular response. Biochem Biophys Res Commun 142:428-435
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
Vol. 128, No. 1
Printed in U.S.A.
Identification and Characterization of Basic Fibroblast Growth Factor in Porcine Thyroids* NAOYA EMOTO, OSAMU ISOZAKI, MARIKO ARAI, HITOMI MURAKAMI, KAZUO SHIZUME, ANDREW BAIRD, TOSHIO TSUSHIMA, AND HIROSHI DEMURA Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College (N.E., O.I., T.T., H.D.), Tokyo 162, Institute of Growth Science (MA., H.M., K.S.), Tokyo 162 Japan; and Department of Molecular and Cellular Growth Biology, The Whittier Institute for Diabetes and Endocrinology (A.B), La Jolla, California 92037
of this conclusion, basic FGF mRNA was detected in poly A+ RNA isolated from cultured porcine thyroid cells. On the other hand, recombinant human basic FGF at the concentration of 0.1-10 ng/ml increased the incorporation of 3 H-thymidine into FRTL-5 cells and porcine thyroid follicular cells in culture. The stimulatory effects were also observed when the biologically active fractions of heparin-sepharose column of the thyroid extract were added to the culture. In addition, both recombinant human basic FGF at 10 ng/ml and heparin binding fractions from porcine thyroids inhibited TSH-induced iodide uptake by porcine thyroid cells in culture. These results suggest that basic FGF may be one of the important local modulators of thyroid function, cell growth, and angiogenesis. (Endocrinology 128: 58-64,1991)
ABSTRACT. Fibroblast growth factor (FGF) is one of the most potent endothelial cell growth factors and has been found in almost all tissues in the body. However, there is no definitive report showing that FGF exists in the thyroid. In this report, we describe heparin binding endothelial cell growth factor activity in porcine thyroids. The extract from normal adult porcine thyroids was loaded onto a heparin-sepharose affinity column. The mitogenic activity on endothelial cells was found to elute from the column with 0.9 M-2.0 M NaCl. Polyacrylamide gel electrophoresis and an immunoblotting of bioactive fractions showed the basic FGF immunoreactivity in a double band with a molecular weight of 18K and 20K. These results strongly imply that the adult porcine thyroid-derived heparin binding endothelial cell growth factor may be authentic basic FGF and one of its high molecular weight forms. In support
I
T HAS been described that thyroid enlargement is accompanied by angiogenesis (1). In general, peptides called angiogenic factors are considered to be key factors in the process of angiogenesis (2). Among many reported angiogenic factors, fibroblast growth factor (FGF) has been considered as one of the most potent angiogenic factors. Although FGFs were originally found in the mesoderm and neuroectodermal tissues, recent biochemical and immunohistochemical data revealed the presence of basic FGF in essentially almost all tissues in the body except serum and thyroid (3, 4). Goodman et al. (5) and Greil et al. (6) reported angiogenic factors from rat thyroid and porcine thyroid, respectively. These reports suggested that these factors may be distinct from FGFs; however, they could not exclude a possibility that FGFs may exist in thyroids with other angiogenic factors.
On the other hand, recent reports have suggested that peptide growth factors, including FGFs, are multi-functional (7). These factors are originally found as mitogenic factors for certain cells, and those for endothelial cells are considered to be angiogenic factors. However, the functions of these factors are not limited to cell growth. These factors seem to modulate the function of the organ in which they are found.
Materials
Received August 6,1990. Address all correspondence and requests for reprints to: Dr. Naoya Emoto, Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162 Japan. * This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, a grant from the Ministry of Health and Welfare, and a grant from the Foundation for Growth Science.
Fetal calf serum (FCS) was obtained from Filtron PTY Ltd. (Victoria, Australia); calf serum (CS) was obtained from Nakarai Company (Kyoto, Japan). F-12 medium was purchased from Flow Laboratories (McLean, VA), and the multiwell tissue culture plates were from Coster (Cambridge, MA). Recombinant basic FGF was obtained from Chiron Corporation (Emery Bille, CA). Other hormones and chemicals were
In the present study, we demonstrate the existence of basic FGF and its high molecular weight form in normal adult porcine thyroid tissues and suggest that basic FGF in porcine thyroids may be an important local modulator of thryoid function, cell growth, and angiogenesis.
Materials and Methods
58
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
FGF IN THE THYROID purchased from Sigma Chemical Company (St. Louis, MO), unless otherwise indicated. Extraction of porcine thyroid FGF Normal adult porcine thyroids (100 g) were obtained from a local slaughterhouse. Tissues were homogenized by polytron homogenizer in 2.0 M NaCl, 10 mM Tris-Cl, 1 mM EDTA, 1 mM EGTA, pH 2.5 with 1 mM phenylmethylsulfonylfluoride. The pH was readjusted to 2.5 with 10 N HCl. The suspension was centrifuged, and the pH of the supernatant was adjusted to 7.4 with 1 N NaOH. This crude extract was diluted with 10 mM Tris, 1 mM EDTA, 1 mM EGTA, pH 7.4 to less than 0.6 M as the salt concentration, and was applied to a heparin-sepharose column (1.6 X 1.0 cm, bed vol 2 ml). After the column was washed with 10 mM Tris (pH 7.4), 0.6 M NaCl, the protein was eluted sequentially by the stepwise addition of 0.9 M, 1.25 M, and 2.0 M NaCl in 10 mM Tris (pH 7.4). Endothelial cell proliferation assays The mitogenic activity of the column fractions and purified samples was determined using cloned vascular endothelial cells derived from the bovine pulmonary artery. Cells were seeded at an initial density of 1.0 X 104 cells per well in 12-well culture plates containing 0.5 ml F12 medium supplemented with 10% FCS. Six h later, the medium was changed to F12 medium supplemented with 15% CS. Ten-microliter aliquots of the appropriate diluation of each fraction (with F12 medium-0.1% BSA) were added to the wells every other day. After 4 days, plates were trypsinized, and final cell densities were determined by counting cells in a Coulter counter (Coulter Electronics, Inc., Hialeah, FL). Porcine thyroid cells and iodide uptake Isolated porcine thyroid cells were prepared using Dispase (Godo Susei Co., Tokyo, Japan) as described by Isozaki et al. (8). The cells were suspended in F-12 medium supplemented with 5% FCS at a concentration of 2 X 106 cells/ml, and 0.5 ml of the suspension was dispensed into the wells of 12-well tissue culture plates (3.8 cm2/well). The cells were cultivated at 37 C in a humidified atmosphere of 5% CO2 in air. Within 24 h, the cells attached to the plastic surface. The contamination of fibroblast was less than 1% during experiments, as described previously (9). On the second day of culture, medium was switched to F-12 medium containing 0.5% FCS. The thyroid cells were incubated with the indicated concentrations of TSH and test materials for 3 days, unless otherwise indicated. After that, the medium was replaced by fresh F-12 medium with 0.1% BSA, and the cells were exposed to 0.1 fid Na126I (New England Nuclear Corp., Boston, MA) with 10"7 M Nal for 1 h. The medium was rapidly removed, and the cultures were washed with ice-cold Hank's Balanced Salt Solution (HBSS). The cells were then solubilized with 0.1 N NaOH, and an aliquot of the solution was counted for radioactivity. Iodide uptake was defined as total intracellular radioactivity as described previously (10).
59
3
H-Thymidine incorporation into the cultured porcine thyroid cells The thyroid cells were cultured in F-12 medium containing 5% FCS for 1 day, and in F-12 medium containing 0.5% FCS and 0.1% BSA for 3 days, then the agents and (methyl3 H)thymidine (0.1 Ci/well; New England Nuclear Corp.) were added to the cultures. After 3 days the medium was aspirated, and the cells were washed with PBS twice. Then, the cells were precipitated with 10% trichloroacetic acid (TCA) and solubilized by 0.5 ml 1 N NaOH and neutralized with 0.5 ml 1 N HCl. The radioactivity of the solutions was counted by a /3-counter using liquid scintillation counting solution ACSII (Amersham, Arlington Heights, IL). FRTL-5 cells culture and 3H-thymidine incorporation assay The functional rat thyroid follicular cell line FRTL-5 cells were cultured in modified Ham's F-12 medium supplemented with 5% CS and a six-hormone preparation (6H medium) consisting of insulin (10 /ug/ml), hydrocortisone (10 nM), transferrin (5 mg/ml), glycyl-L-lysine acetate (10 ng/ml), somatostatin (10 ng/ml), and bovine TSH (1.0 mU/ml) as described previously (11). The cells (0.5 X 105 cells/well) were plated in 12-well plates and cultured for 3 days in 6H medium at 37 C in a humidified atmosphere (95% air, 5% CO2). The medium was then replaced by 5H medium (6H medium minus TSH) supplemented with 5% CS, and the cells were cultured for 2 days. On the fifth day, the 5H medium (5% CS) was replaced with the fresh 5H medium, and the test materials and 3H-thymidine (0.1 jiCi/ well) were added to the cultures. After 3 days, the cells were washed twice with PBS and precipitated with 10% trichloroacetic acid. The radioactivity of 3H-thymidine incorporated into the cells was determined by the same method described in the porcine thyroid cell experiments. SDS/PAGE and immunoblotting Fractions of heparin-sepharose column were dialyzed, concentrated by lyophilization, and applied to a 15% polyacrylamide resolving gel with a 3% stacking gel and electrophoresed. Samples were blotted from gels onto nitrocellulose sheets by electrotransfer. Blots were then incubated overnight in the buffer containing the antiserum. Immunoreactive proteins were revealed by successive incubations with goat antirabbit immunoglobulin conjugated with horse radish peroxidase (Bio-Rad Laboratories, Richmond, CA) and 4-chrol-l-naphthol/H2O2. Northern blot analyses Northern blot analyses were performed as previously described (12, 13). Briefly, total RNA was prepared by the conventional guanidine isothiocyanate method, and poly A+ RNA was selected by oligo(dT) cellulose column. RNA samples were separated on a 1% agarose gel containing 0.66 M formaldehyde and transfered to Hybond blotting membrane (Amersham). Membranes were hybridized with a 32P-labeled 0.5 kilobase rat bovine FGF complementary DNA (cDNA) (14). Hybridization and washing were performed as described by Church and Gil-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
FGF IN THE THYROID
60
bert (15); final washing was carried out at 65 C in 1 x SSPE (0.15 M NaCl, 0.01 M NaH 2 PO 4 ,1 mM EDTA, pH 7.4). Statistical analysis To analyze the data, analysis of variance was first performed, and statistical analysis of differences between groups was carried out using Student's t test if the variation in the data was uniform. When the variation in the data was not uniform, Cochran-Cox's test was employed.
Results Identification and characterization of the porcine thyroidderived heparin-binding endothelial cell growth factor The porcine thyroid-derived endothelial cell growth factor was isolated by using heparin-sepharose affinity chromatography. The crude extract of the porcine thyroid homogenate was diluted and applied onto a heparinsepharose column. In the preliminary study, the mitogenic activity was eluted with 2.0 M NaCl in 10 mM Tris, pH 7.4 (data not shown). Therefore, we examined the stepwise addition of 0.9 M, 1.25 M, and 2.0 M NaCl in 10 mM Tris-Cl. The volume of each elution was 30 ml. The elution profile of the extract after heparin-sepharose column is shown in Fig. 1. The mitogenic activity for endothelial cells was detected in all the fractions eluted with 0.9 M and 1.25 M NaCl, but in only the first several fractions eluted with 2.0 M NaCl. After completion of the chromatography, the protein profile was separated into five distinct pools (Pl-5, shown in Fig. 1, the vol of each pool was 6 ml), dialyzed, lyophilized, and resuspended in 200 (A 10 mM Tris-Cl (pH 7.4).
Endo• 1991 Voll28»Nol
Five pools were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot was performed with polyclonal antibasic FGF antibody raised against the synthetic bovine basic FGF fragment 1-24 (773) (Fig. 2). The basic FGF immunoreactivity was detected in two different forms with molecular mass of 18K and 20K. Both 18K and 20K bands were seen in the pool 3 and 4 (1.25 M NaCl elution), but the 18K band was predominant in pool 5 (2.0 M NaCl elution). In pool 2, two bands were detectable in a lower intensity, and there was no immunoreactivity in pool 1. We tried immunoblots using antibodies raised against synthetic bovine acidic FGF fragments 1-8, 1-15, and 132-140, but we could not detect any acidic FGF immunoreactivity in any of five pools. Effect of basic FGF on the growth of FRTL-5 cells It has been reported that FGFs stimulate the proliferation of a variety of cells. We examined the effect of basic FGF on the growth of rat thyroid follicle-derived cell line FRTL-5 cells. Figure 3 (top panel, solid line) shows the effect of recombinant human basic FGF on the 3H-thymidine incorporation into FRTL-5 cells. Basic FGF stimulated the DNA synthesis in FRTL-5 cells in a dose-dependent manner. 10 ng/ml of basic FGF stimulated the thymidine incorporation 100-fold over controls. We also examined the effect of the porcine thyroidderived heparin-binding growth factor on the growth of
1810
15
Fraction
20
25
30
35
Number
FlG. 1. Purification of porcine thyroid-derived endothelial cell growth factors by heparin-sepharose affinity chromatography. The porcine thyroid extract was chromatographed on a heparin-sepharose column, as described in Materials and Methods. The column was eluted stepwise with 0.6, 0.9, 1.25, and 2.0 M NaCl in 10 mM Tris-HCl, pH 7.4, as described in the text. Five-microliter aliquots of each fraction were added to bovine pulmonary artery endothelial cell cultures. Five zones of mitogenic activity were labeled as pools 1-5 (PI, P2, P3, P4, and P5).
3
A
FIG. 2. Western blots of five pools of fractions eluted from heparinsepharose column. Five pools (Pl-5 shown in Fig. 1; lane 1-5) were subjected to SDS-PAGE, and immunoblot was performed with antibasic FGF antibody as described in the text. Lane 6 is recombinant human basic FGF.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
61
FGF IN THE THYROID
0.001
Q01
0.1
1.0
1Q0
tion into porcine thyroid follicular cells in culture. Basic FGF stimulated the cell growth of porcine follicular cells estimated by 3H-thymidine incorporation in a dose-dependent manner. Compared to the effect on FRTL-5 cells, the magnitude of the response was much smaller in the porcine thyroid primary culture. Ten nanograms per ml basic FGF stimulated DNA synthesis in porcine follicular cells 4-fold over controls. The minimum effective dose was 100 pg/ml. We also examined the effect of heparin binding fractions of porcine thyroid extracts. The fractions eluted at 0.9 M, 1.25 M, and 2.0 M stimulated 3H-thymidine incorporation into cultured porcine thyroid cells (Fig. 4)
100.0
FGF (ng/ml)
S x „
a
7
125M
Q9M
2.0M
Effect of basic FGF on iodide uptake into the cultured porcine thyroid cells
6
3
10
20
30
FlG. 3. Mitogenic effects of basic FGF and porcine thyroid extracts on thyroid cells. Top panel, Concentration-dependent effects of recombinant human basic FGF on DNA synthesis in FRTL-5 cells (•—•) and cultured porcine thyroid cells ( • • ) . FRTL-5 cells were cultured for 3 days in 5H supplemented with 5% CS with various concentrations of recombinant human basic FGF. Porcine thyroid cells were cultured for 3 days in F-12 medium containing 1% FCS and 0.1% BSA with various concentrations of recombinant human basic FGF. Bottom panel, Effect of heparin-binding fractions of the thyroid extract on DNA synthesis in FRTL-5 cells. The column was eluted stepwise with 0.6, 0.9, 1.25, and 2.0 M NaCl in 10 mM Tris-HCl, pH 7.4. Fivemicroliter aliquots of each fraction were added to FRTL-5 cells cultured in 5H supplemented with 5% CS. The cultures were labeled with 3Hthymidine (3H-TdR) as described in the text. The data are expressed as cpm of 3H-TdR incorporated into TCA-insoluble cellular fractions. The values are the mean ± SE of triplicate wells from one representative experiment out of three.
Recent studies have suggested that FGFs are multifunctional factors (7). In order to clarify the physiological role of FGF in the thyroid, we examined the effect of basic FGF on thyroid function other than cell growth. Figure 5 (top panel) shows the effect of recombinant basic FGF on iodide uptake into the cultured porcine thyroid follicles. Basic FGF alone had no effect on the iodide uptake, but it inhibited the TSH-stimulated iodide uptake at the concentration of 10 ng/ml. The same inhibitory effect was seen when the heparin-sepharose column chromatography fractions of porcine thyroid extracts were used (Fig. 5, bottom panel). Northern blot analyses We could not detect any hybridizing mRNA of basic FGF in Northern blot analysis of poly A+ RNA isolated from adult porcine thyroid tissues (data not shown). We 3.0
2.0
FRTL-5 cells. Figure 3 (bottom panel) indicates the mitogenic effect of the porcine thyroid extract after a heparin-sepharose column chromatography. The mitogenic activity on FRTL-5 cells was seen in the fractions starting from 0.9 M-2.0 M NaCl elution. This profile was almost identical with the mitogenic effect for endothelial cells (Fig. 1). Effect of basic FGF on the growth of primary thyroid follicular cells in culture
porcine
We next examined the effect of basic FGF on the growth of normal thyroid follicular cells in culture. Figure 3 (top panel, broken line) shows the effect of human recombinant basic FGF on the 3H-thymidine incorpora-
1.0
Control
bFGF
0.6M
0.9M
1.25M
2.0M
(lOng/ml)
FlG. 4. Effect of heparin-binding fractions of the thyroid extract on DNA synthesis in porcine thyroid cells in culture. Five-microliter aliquots of fractions eluted at 0.6, 0.9,1.25, and 2.0 M NaCl were added to porcine thyroid cells and cultured for 3 days. The cultures were labeled with 3H-TdR as described in the text. The data are expressed as cpm of 3H-TdR incorporated into TCA-insoluble cellular fractions. The values are the mean ± SE of triplicate wells from one representative experiment out of three.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
FGF IN THE THYROID
62
Q01
0.1
1.0
100
1000
basic FGF (ng/ml) 0.6M
-
Q9M
Endo • 1991 Voll28«Nol
2 8S
2.0M
125M
5 "
18S
1 -
15
20
Fraction Number
FIG. 5. Top panel, Inhibitory effect of recombinant human basic FGF on TSH-induced iodide uptake in porcine thyroid cells. Thyroid cells were cultured with 50 /iU/ml TSH for 3 days in the presence or absence of various concentrations of basic FGF and tested for iodide uptake as described in the text. Bottom panel, Inhibitory effect of heparin-sepharose column fractions of the porcine thyroid extract on TSH-induced iodide uptake in porcine thyroid cells in culture. The column was eluted stepwise with 0.6, 0.9, 1.25, and 2.0 M NaCl in 10 mM Tris-HCl, pH 7.4. Five-microliter aliquots of each fraction were added to porcine thyroid cells cultured with 50 /iU/ml TSH. The cells were cultured for 3 days and tested for iodide uptake as described in the text. The results shown are the mean ± SE of triplicate wells from one representative experiment out of three.
next examined RNA isolated from cultured porcine thyroid cells. The cells were cultured in F-12 medium containing 0.1% BSA for two days, and then RNA was isolated. Two hybridizing basic FGF mRNA, 7.0 and 3.7 kilobases in length, were detected in poly A+ RNA (3 ng) (Fig. 6). These sizes are identical with those of reported human and bovine basic FGF mRNA (16).
Discussion In this report, we demonstrated the existence of heparin-binding endothelial cell growth factor in normal adult porcine thyroid. Mitogenic activity for endothelial cells was present in fractions that eluted from heparinsepharose column in broad peaks from 0.9-2.0 M NaCl. These fractions have basic FGF immunoreactivities. The immuno-blotting study of SDS-PAGE indicated a double band with a molecular weight of 18K and 20K. The 18K band was exactly identical in its size with recombinant
FIG. 6. Northern blot analysis of the expression of basic FGF in porcine thyroid cells in culture. Poly A+ RNA (3 fig) was prepared, as described in Materials and Methods, from porcine thyroid cells in culture. The membrane was hybridzed to rat basic FGF cDNA probe.
human basic FGF. There is high sequence homology between basic FGFs of different species. Among human, bovine, and rat, the sequence of basic FGFs are almost identical, except for several substitutes out of 155 or 154 amino acids (14). Thus, it seems reasonable to expect that the molecular weight of porcine basic FGF is the same as human. On the other hand, the 20K band may be a high molecular weight form of basic FGF. Recent studies show that there are several AT-terminal extended high molecular weight forms (22-25K) of basic FGF isolated from hepatoma cells (17), guinea pig (18), rat brain (19), and H-ras-transformed Rat-1 cells (20). The existence of the high molecular weight form of basic FGF had been questioned, because no AUG codon in cDNA frame has been found upstream of the putative start codon (AUG) from which synthesis of a 155-amino acid (18K) is thought to start. However, Florkiewicz et al. (21) and Prats et al. (22) showed that the high molecular weight forms of basic FGF are translationally initiated at unusual nonAUG codons. It may well be that the same mechanism could yield the high molecular weight form of basic FGF in porcine thyroid. In addition, cultured porcine thyroid cells expressed mRNA hybridized with bovine basic FGF cDNA. Thus, we conclude that porcine thyroid-derived heparin binding endothelial cell growth factor is at least partly, if not all, basic FGF, although there is a possibility that acidic FGF or other heparin binding growth factors also may
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
FGF IN THE THYROID exist in porcine thyroids. The immuno-blotting study of SDS-PAGE indicated another intriguing result. Both 18K and 20K form of immunoreactive porcine basic FGF started to elute at 0.9 M NaCl, but amount of eluted peptides was very low. At 1.25 M elution, two forms of basic FGF were clearly seen; however, the 18K band was dominant in the fractions eluted at 2.0 M. Furthermore, 0.9-1.25 M NaCl elution of heparin-sepharose column yielded broad peaks in the mitogenic activity for endothelial cells, but all the mitogenic activities came out by the wash with 2.0 M NaCl. It is generally accepted that basic FGF elute from heparin-sepharose column with 1.4-1.6 M NaCl and acidic FGF elute with 0.9-1.1 M NaCl (3), and that N-terminalextended high mol wt form of basic FGF have the same biological activities and the same affinity to heparin (21). However, Quinkler et al. (23) reported that some of canine and porcine heart FGF eluted at salt concentrations between 1.2 M-1.3 M. These results suggest that there may be a microheterogeneity in the affinity of porcine basic FGF to heparin. Actually, Sommer et al. (24) reported the existence of the placental basic FGF which consists of 157 amino acids, three amino acids longer at N-terminus than the one reported before. If this small diversity exists in porcine thyroid basic FGF, it may be difficult to differentiate these peptides in our SDS-PAGE. In addition, the high mol wt form (20K) of basic FGF seems to have a relatively low affinity to heparin, because not much 20K basic FGF retained to the column after the 1.25 M NaCl wash. Thus, there is a possibility that the N-terminal extended sequence may affect the chemical character of the molecule. Although further studies are necessary, the establishment of the complete primary structure of high mol wt form of porcine thyroid-derived basic FGF can be expected to clarify the significance of heparin-binding domains in the sequence of FGF family. Basic FGF was initially isolated from many tissues as a fibroblast and endothelial cell growth factor. However, within a short period of time, it was revealed that its function is not limited to the growth of these cells. In endocrine tissues, there are evidences that it is playing a role in modulating endocrine function through regulating the hormone secretion and/or the growth of endocrine cells. It potentiates TRH-stimulated PRL and TSH secretion from cultured anterior pituitary cells (25), affects the hormone synthesis in reproductive tissues (26-28), and stimulates the proliferation of adrenocortical cells in vitro (29). In this report, we demonstrated the multifunctional effect of basic FGF on the thyroid. It stimulates the growth of thyroid follicular cells and inhibits iodide uptake by these cells. These results suggest that basic FGF is one of the local function modulators in thyroid tissues. It has been reported that growth factors
63
have diverse effects on the function and growth of thyroid tissues. EGF stimulates the thyroid follicular cell growth but inhibits the iodide uptake by these cells (30-33). On the other hand, TGF-0 inhibits both (34, 35), and IGF-I stimulates the growth and has no effect on the iodide uptake (9, 36). Although the function of basic FGF seems similar to that of EGF, basic FGF is unique because of its angiogenic effect. Thus, it may well be that in the process of thyroid enlargement, basic FGF may promote the vascular formation, stimulate the thyroid cell growth, and inhibit the thyroid function. It may be reasonable to hypothesize that basic FGF may play important roles in the pathogenesis of goiter, but this relationship remains to be clarified. On the other hand, it should be noted that basic FGF exists in normal thyroid tissues in which neither active angiogenesis nor thyroid cell growth is taking place. Thus, in normal circumstances, basic FGF should exist as an inactive form. Folkman et al. (37) indicated that FGFs were detected in the basement membranes and speculated that FGFs bind to heparan sulfate proteoglycans, structurally related to heparin, present in extracellular matrix, making these factors inaccessible to their receptors. Baird and Ling (38) suggested the enzymatic degradation of extracellular matrix by proteases or heparanases, secreted by inflammatory and immune cells, may be able to release basic FGF. Although the exact mechanism of the activation of basic FGF in many tissues remains unclear, these data strongly imply that the inflammatory response in the thyroid, which is usually seen in autoimmune thyroid diseases, could activate stored basic FGF. In summary, we demonstrated the existence of basic FGF in normal adult porcine thyroid. It promotes the angiogenesis, stimulates the thyroid cell growth, and inhibits the iodide uptake in vitro. These data suggest the importance of basic FGF in thyroids as one of the local growth and function modulators. The possible involvement of basic FGF in the pathogenesis of goiter remains to be clarified. References 1. Wollman SH, Herverg JP, Zeligs JD, Ericson LE 1978 Blood capillary enlargement during the development of thyroid hyperplasia in the rat. Endocrinology 103:2306-2314 2. Folkman J, Klagsbrun M1987 Angiogenic factors. Science 235:442447 3. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G 1987 Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 8:95-114 4. Baird A, Walicke PA 1989 Fibroblast growth factors. Br Med Bull 45:438-452 5. Goodman AL, Rone JD 1987 Thyroid angiogenesis: endotheliotropic chemoattractant activity from rat thyroid cells in culture. Endocrinology 121:2131-2140 6. Geil W, Rafferzeder M, Bechtner G, Gartner R 1989 Release of an endothelial cell growth factor from cultured porcine thyroid folli-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
64
FGF IN THE THYROID
cles. Mol Endocrinol 3:858-867 7. Sporn MB, Roberts AB 1988 Peptide growth factors are multifunctional. Nature 332:217-219 8. Isozaki 0 , Tsushima T, Shizume K, Saji M, Ohba Y, Emoto N, Sato K, Sato Y, Kusakabe K 1985 Thyroid-stimulating antibody bioassay using porcine thyroid cells cultured in follicles. J Clin Endocrinol Metab 61:1105-1111 9. Saji M, Tsushima T, Isozaki 0 , Murakami H, Ohba Y, Sato K, Arai M, Shizume K 1987 Interraction of insulin-like growth factor I with porcine thyroid cells cultured in monolayer. Endocrinology 121:749-756 10. Tsuchiya Y, Saji M, Isozaki O, Arai M, Tsushima T, Shizume K 1990 Effect of lithium on deoxyribonucleic acid synthesis and iodide uptake in porcine thyroid cells in culture. Endocrinology 126:460-465 11. Saji M, Isozaki O, Tsushima T, Arai M, Miyakawa M, Ohba Y, Tsuchiya Y, Sano T, Shizume K 1988 The inhibitory effect of iodide on growth of rat thyroid (FRTL-5) cells. Acta Endocrinol (Copenh) 119:145-151 12. Emoto N, Gonzalez A-M, Walicke PA, Wada E, Simmons DM, Shimasaki S, Baird A 1989 Basic fibroblast growth factor (FGF) in the central nervous system: identification of specific loci of basic FGF expression in the rat brain. Growth Factors 2:21-29 13. Isozaki O, Kohn LD, Kozak CA, Kimura S 1989 Thyroid peroxidase: rat cDNA sequence, chromosomal localization in mouse, and regulation of gene expression by comparison to thyroglobulin in rat FRTL-5 cells. Mol Endocrinol 3:1681-1692 14. Shimasaki S, Emoto N, Koba A, Mercado M, Shibat F, Cooksey K, Baird A, Ling N 1988 Complementary DNA cloning and sequencing of rat ovarian basic fibroblast growth factor and tissue distribution study of its mRNA. Biochem Biophys Res Commun 157:256-263 15. Church GM, Gilbert W 1984 Genomic sequencing. Proc Natl Acad Sci USA 81:1991-1995 16. Schweigerer L, Neufeld G, Friedman J, Abraham JA, Fiddes JC, Gospodarowicz D 1987 Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature 325:257-259 17. Presta M, Moscatelli D, Joseph-Silverstein D, Rifkin DB 1986 Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration. Mol Cell Biol 6:4060-4066 18. Moscatelli D, Joseph-Silverstein J, Manejias R, Rifkin DB 1987 Mr 25,000 heparin binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc Natl Acad Sci USA 84:5778-5782 19. Presta M, Rusnati M, Maier JAM, Ragnotti G 1988 Purification of basic fibroblast growth factor from rat brain: identification of an Mr 22,000 immunoreactive form. Biochem Biophys Res Commun 155:1161-1172 20. Iberg N, Rogelj S, Fanning P, Klagsbrun M 1989 Purification of 18 kDa and 22 kDa forms of basic fibroblast growth factor from rat cells transformed by the ras oncogene. J Biol Chem 264:1995119955 21. Florkiewicz R, Sommer A 1989 Human basic fibroblast growth factor encodes four polypeptides: three initiate translation from non-AUG codons. Proc Natl Acad Sci USA 86:3978-3981 22. Prats H, Kaghad M, Prats AC, Klagsbrun M, Lelias JM, Leauzun P, Chalon P, Tauber JP, Amalric F, Smith JA, Caput D 1989 High molecular weight forms of basic fibroblast growth factor are initi-
23.
24.
25.
26.
27. 28.
29.
30.
31.
32.
33.
34.
35.
36.
Endo«1991 Voll28«Nol
ated by alternative CUG codons. Proc Natl Acad Sci USA 86:18361840 Quinkler W, Maasberg M, Bernotat-Danielowski S, Luthe N, Sharma HS, Schaper W 1989 Isolation of heparin-binding growth factors from bovine, porcine and canine hearts. Eur J Biochem 181:67-73 Sommer A, Brewer MT, Thompson RC, Moscatelli D, Presta M, Rifkin DB 1987 A form of human basic fibroblast growth factor with an extended amino terminus. Biochem Biophys Res Commun 144:543-550 Baird A, Mormede P, Ying SY, Wehrenberg WB, Ueno N, Ling N, Guillemin R 1985 A non-mitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proc Natl Acad Sci USA 82:5545-5549 Adashi EY, Resnick 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 Endocrinol 55:7-14 Baird A, Hsueh AJW 1986 Fibroblast growth factor as an intraovarian hormone: differential regulation of steroidogenesis by an angiogenic factor. Regul Pept 10:309-317 Raeside JI, Berthelon M-C, Sanchez P, Saiz JM 1988 Stimulation of aromatase activity in immature porcine Leydig cells by fibroblast growth factor (FGF). Biochem Biophys Res Commun 151:163-169 Gospodarowicz D, 111 CR, Hornsby 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 Roger PP, Dumont JE 1982 Epidermal growth factor controls the proliferation and the expression of differentiation of canine thyroid cells in primary culture. FEBS Lett 144:209-212 Westermark K, Karlsson FA, Westermark B 1983 Epidemal growth factor modulates thyroid growth and function in culture. Endocrinology 112:1680-1686 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 Endocrinol 38:141-150 Bacharach LK, Eggo MC, Mak WW, Burrow GN 1985 Phorbol esters stimulate growth and inhibit differentiation in cultured thyroid cells. Endocrinology 116:1603-1609 Tsushima T, Arai M, Saji M, Ohba Y, Murakami H, Ohmura E, Sato K, Shizume K 1988 Effects of transforming growth factor-/? on deoxyribonucleic acid synthesis and iodine metabolism in porcine thyroid cells in culture. Endocrinology 123:1187-1194 Grubeck-Loebenstein B, Buchan G, Sadeghi R, Kissonerghis M, Londei M, Turner M, Pirich K, Roka R, Niederle B, Kassal H, Werner W, Feldmann M 1989 Transforming growth factor-/? regulates thyroid growth. J Clin Invest 83:764-770 Toramontano D, Cushing GW, Moses AC, Ingbar SH 1986 Insulin-
like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Grave's-IgG. Endocrinology 119:940-942 37. Folkman J, Klagsbrun M, Sasse J, Wadsinski M, Ingber D, Vlodavsky I 1988 A heparin-binding angiogenic protein—basic fibroblast growth factor—is stored within basement membrane. Am J Pathol 130:393-400 38. Baird A, Ling N 1987 Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implication for a role of heparinase-like enzymes in the neovascular response. Biochem Biophys Res Commun 142:428-435
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 October 2015. at 13:19 For personal use only. No other uses without permission. . All rights reserved.
