THYROID Volume 2, Number
2, 1992 Liebert, Inc., Publishers
Transforming Growth Factor Beta, (TGF-ß,) mRNA in Sheep Thyroid Cells
EGGO,3 JUDY MEINKOTH,1 GERARD N.
ABSTRACT We examined TGF-ß mRNA levels in primary sheep thyroid cell cultures to determine whether the inhibitory effects of iodide on thyroid cells could be explained by an induction of TGF-ß mRNA and if this induction was mediated by iodine organification. Thyroid cells were incubated with TSH and five additives (insulin, somatostatin, growth hormone, transferrin, and glycyl-L-histidyl-L-lysin) for 2-3 weeks and then were exposed to sodium iodide (Nal) or l-methylimidazole-2-thiol (methimazole, MMI), or both for 72 h. Iodide at 10 6M and 10~4 M significantly increased the amount of TGF-ß mRNA as determined by Northern blot analysis with a rat TGF-ß, cDNA probe. This increase in TGF-ß, mRNA was abolished'by the addition of methimazole, an inhibitor of organification. These data indicate that the effects of iodide on thyroid growth and function may be mediated by a process that involves organification of iodide and increases in TGF-ß, mRNA levels.
ADDITION TO THE BIOSYNTHESIS OF THYROID HORMONE, iodine also plays a role in the regulation of thyroid growth and differentiation (1,2). In vivo excess iodide is known to inhibit thyroidal iodine transport and organification, leading to decreased thyroid hormone formation in normal thyroid cells (3). This also has been shown to occur in vitro, using primary cultures of sheep thyroid cells (4). Administration of iodide has an inhibitory effect on the hyperplastic iodine-deficient goiter that is characterized by follicular cell hyperplasia as well as angiogenesis and capsular growth due to fibroblast proliferation (5). We showed that iodide markedly inhibited growth in the FRTL-5 rat thyroid cell line cultures and that the inhibition was prevented by methylmercaptoimidazole (MMI) (6). The ability of MMI to prevent this inhibitory effect of iodine suggests a possible role of organic iodine in mediating the inhibitory action of sodium iodide (Nal) on thyroid cell growth. Several compounds containing covalently bound iodine, e.g., iodolactone (7) and iodolipid (8), which could have the potential to regulate thyroid growth function, have been described (9).
Recent analysis of cell growth control mechanisms have revealed that stimulatory factors, such as EGF or IGF-I, are counteracted by negative regulators, such as transforming growth factor-beta (TGF-ß) (10-12). We have shown that TGF-ß is able to inhibit growth stimulation by these factors in sheep thyroid cells in culture (4). The thyroid gland might, therefore, autoregulate its growth, depending on the balance between inhibitory and stimulatory growth factors (13,14). The purpose of this present study was to determine whether the inhibitory effect of iodide could be mediated by changes in a specific TGF-ß homolog (TGF-ß,) as an autocrine or a paracrine hormone, and if so, whether this induction is mediated by iodine organification. To examine if inhibitory effects of iodine may be mediated by changes in TGF-ß, we determined the influence of iodine on TGF-ß mRNA levels in sheep thyroid cells in culture. The determination of TGF-ß protein levels is difficult due to the low abundance and complicated processing of the TGF-ß protein (11). Results from our studies suggest that iodide may modulate thyroid growth and function by inducing TGF-ß | mRNA and that this induction involves the formation of an ¡odocompound whose synthesis is inhibited by methimazole.
Department of Medicine, University of California, San Diego, California. 2Presenl address: First Department of Internal Medicine, Toho University, School of Medicine, Tokyo, Japan. 'Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, UK. 141
YUASA ET AL.
MATERIALS AND METHODS Materials
Chemicals, hormones, and reagents were purchased from the Chemical Co. (St. Louis, MO) unless otherwise indi-
The statistical difference in relative ratios of mRNA was assessed by analysis of variance (ANOVA) using a randomized block design to correct variability between different autoradiograph exposure times and specific activities of cDNA probes. Statistical significance was determined atp < 0.05.
Sheep thyroid cell culture Sheep thyroid follicles for primary cell culture were prepared by treatment with collagenase as previously described (15) and plated at a density of 5 x 104 cells/cm2 on 100-mm size plates
Experiments were performed on confluent cells 2-3 weeks after plating.
To examine whether the inhibitory effect of iodide could be due to TGF-ß stimulation, we determined TGF-ß, mRNA levels in sheep thyroid cells by Northern blot analysis. Cells were treated with Nal at a concentration of 10-6 M, 10r4 M, and without added Nal as a control. As previously described, optimum iodide concentration for thyroid hormone synthesis and secretion is 10b M, and higher concentrations are known to inhibit iodothyronine synthesis, thyroid hormone secretion, and growth (2,4,6). In order to prevent iodide organification, 2 mM MMI was added to the cells in the same experiments.
Effects of iodide GF-ß, mRNA
(Becton Dickinson, Lincoln Park, NJ) in the modified F12 medium of AmbesiTmpiombato et al. (16) without serum, except for the initial 3-4 days of culture. This medium (6H) contains six additives: insulin (10 p-g/mL), cortisol (10 nM), transferrin (5 p.g/mL), glycyl-L-histidyl-L-lysin (10 ng/mL), somatostatin (10 ng/mL), and bovine TSH (300 p-U/mL).
incubated with Nal at concentrations of 10-6 M, 10~4 M, and without added Nal as a control in the complete medium with 6H for 72 h. In some experiments, 1-methylimidazole-2-thiol (MMI) at concentrations of 2 mM or in combination with Nal also was added to the cells to prevent organification. No difference in cell morphology or growth characteristics was noticeable between cells grown in the presence or absence of MMI. Cells
RNA extraction and
Total RNA was extracted by a guanidinium hydrochloride method from cell cultures. Total RNA, 10 u,g/lane, was loaded and electrophoresed on a denaturing 1 % formaldehyde gel ( 17), stained with ethidium bromide, transferred to nytran filters (Schleicher & Schuell, Inc., Keene, OH) and baked for 1 h at 80°C under vacuum.
Hybridization We used a rat TGF-ß, cDNA (from Dr. Michael B. Sporn) as probe on Northern blots. The rat cDNA TGF-ß, probe has been described (18) and contains 985 bp of coding region. Aqueous hybridization conditions were used (0.5 M sodium phosphate, pH 7.2, 7% SDS, 1% bovine serum albumin, 1 mM EDTA, pH 8) according to Church and Gilbert (19). Filters were prewetted in 25 mM NaH2P04, pH 6.8, 1 mM EDTA, prehybridized in hybridization solution at 65°C-70°C for at least 1 h, and hybridized with [32P]-dCTP random prime labeled (Amersham, Arlington Heights, IL) rat TGF-ß, cDNA at 65°C-70°C for 20-36 h. Filters were washed in 40 mM sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA, pH 8.0 at 65°C-70°C for 2 h. Relative mRNA ratios were determined by scanning densitometry of the autoradiograms and corrected for loading differences using a rat 28S rRNA oligonucleotide probe (20).
Ten micrograms of total RNA isolated from sheep cells was subjected to Northern blot analysis. Nonspecific binding of the cDNA probe to 28S and 18S rRNA was prevented when an aqueous hybridization method was used. The TGF-ß, 2.5 kb mRNA species gave a clearly detectable signal with the rat TGF-ß, probe. A representative autoradiograph of a Northern blot probed with a rat TGF-ß, cDNA is shown in Figure 1.
Addition of 10~6 M and 10~4 M Nal to the incubation medium of the sheep thyroid cells increased the predominance of the 2.5 kb TGF-ß, mRNA species. The same filter subsequently was probed with a 28S RNA oligonucleotide probe to assess total RNA loading. A summary of these results is given in Table 1. At a concentration of 10~6 M, the ratio of the 2.5 kb TGF-ß, mRNA species to 28S RNA was significantly increased from 0.55 ± 0.11 (control) to0.68 ± 0.30 (p < 0.05), corresponding to a 23% increase; 10~4 M Nal also showed a significant increase in the ratio of 0.72 ± 0.17 (p < 0.05), representing a 31% increase. The increases induced by 10~6 M and 10~4 M Nal were not significantly different from each other. However, effects at both Nal concentrations were significantly different from controls.
Effects of MMI on
To determine whether iodide was effective in increasing mRNA when iodide organification was blocked, 2 mM MMI was added to prevent organification. In sheep cells treated with MMI, iodide did not increase TGF-ß, mRNA levels, indicating that iodine organification is required for an effect on TGF-ß, mRNA levels (Fig. 1, Table IB).
DISCUSSION Iodide increased TGF-ß, mRNA levels in these studies, which is compatible with the hypothesis that iodine-induced
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