ABSTRACT

The interrelationship between the roles of Ca²⁺ and cyclic AMP (cAMP) in the control of intracellular iodide accumulation was investigated in the continuous rat thyroid cell strain FRTL-5. Using incubation conditions designed to allow independent study of iodide uptake and efflux into the incubation medium, dibutyryl cAMP (dbcAMP) mimicked the effect of TSH in enhancing iodide uptake, but the responses to both stimulators were decreased in the presence of the Ca²⁺ ionophore A23187, although the ionophore was ineffective when addition was delayed until after withdrawal of the stimulators. l-Adrenaline, an α-adrenergic agonist inactive with respect to cAMP accumulation in this cell strain, was also inactive with regard to iodide uptake. The rapid efflux of iodide from FRTL-5 cells seen in response to TSH was not mimicked by dbcAMP, but was reproduced by A23187, providing evidence for the involvement of Ca²⁺ in the release process. Under the latter conditions, no additional effect of TSH was obtained. l-Adrenaline also stimulated iodide efflux, in support of a possible role of α-adrenergic agonists in controlling the apical membrane presentation of iodide in vivo.

The observed differential effects of dbcAMP and α-adrenergic agonists on iodide uptake and efflux respectively, provide further evidence for the existence of two functionally distinct mechanisms controlling the available level of intracellular iodide. Opposing effects of free intracellular Ca²⁺ may, however, serve to control the activities of both influx and efflux processes since, whilst experimental increments in Ca²⁺ appear to promote iodide efflux through a non-cAMP-mediated pathway, inhibition by intracellular Ca²⁺ of thyroid cell cAMP accumulation may explain both the decreased response of iodide uptake observed in the presence of A23187, and the reduced stimulation effected by TSH under such conditions.

J. Endocr. (1987) 112, 51–56

Abstract

The present study has investigated the relative levels and interconversion of latent and active forms of transforming growth factor-β1 (TGF-β1) in human thyroid follicular cell cultures derived from sporadic non-toxic goitres. Northern blotting of RNA extracted from 72-h cultures revealed a 2·5 kb mRNA transcript hybridizing with a cDNA probe for latent TGF-β1, the intensity of which was doubled in cells exposed to Nal (10 μmol/l). Radioreceptor assay of follicular cell-conditioned medium for TGF-β1 content confirmed a similar enhancing effect of iodide. The endogenous active component of TGF-β1 present in conditioned medium represented only a minor fraction of the total TGF-β1 content, this fraction was not enhanced by exposure of follicular cells to iodide. The low level of endogenous active TGF-β1 in medium conditioned by either control or iodide-treated cells was confirmed by immunoadsorption with a precipitating antiserum against active TGF-β1, when such cells failed to show a reversal of the iodide-induced decrease in [methyl-³H]thymidine incorporation into trichloroacetic acid-precipitable material. In contrast to the inhibitory effect of iodide on de novo DNA synthesis, which appeared not to reflect an increase in active TGF-β1, the inhibitory effects of plasminogen (100 mg/l) or plasmin (2000 U/l) on [methyl-³H]thymidine incorporation into thyroid cells were reversible by TGF-β1 immunoadsorption. This provides evidence that the plasmin-mediated inhibition of DNA synthesis in thyroid follicular cells may be attributed to the growth-regulating action of an increased level of activated TGF-β1. The findings of this study therefore provide evidence that (i) human thyroid follicular cells are potentially capable of activating locally derived latent TGF-β1, (ii) an increase in thyroidal TGF-β1 mRNA and latent TGF-β1 peptide availability, following exposure of cells to iodide, is not accompanied by a corresponding increase in active TGF-β1, and (iii) within the thyroid gland, as in other epithelial tissues, activation of endogenous TGF-β1 may be dependent upon the proteolytic actions of plasmin.

Journal of Endocrinology (1994) 141, 183–190

Abstract

The release of latent transforming growth factor-β1 (TGFβ1), and conversion to the biologically active peptide, has been investigated in porcine thyroid follicular cells maintained in primary monolayer culture. Analysis by radioreceptor assay of medium conditioned for 72 h by subconfluent thyroid monolayers showed that a high proportion of the expressed TGF-β1 peptide was in the active form. Medium conditioned by iodide (10 aμmol/l)treated follicular cells contained higher levels of both active and total TGF-β1 than were present in medium conditioned by untreated cells. Exposure of cells to iodide also led to a marked decrease in [methyl-³H]thymidine incorporation that was relieved by immunoadsorption with a neutralizing antiserum against the active form of TGFβ1. Inclusion of a low dose (80 units/l) of porcine plasmin led to a small increase in incorporation of [methyl ³H]thymidine, while higher doses of plasmin (1250–5000 units/l) or plasminogen (100 mg/l) significantly reduced [methyl-³H]thymidine incorporation. This inhibition was effectively reversed by immunoadsorption of TGF-β1 from the medium during the test incubations. The study therefore provides direct evidence for a stimulatory role of thyroidal iodide in enhancing the release of latent TGF-β1 peptide, and suggests that in normal thyroid follicular cells, as in other TGF-β1 producing epithelia, post-secretory processing to the biologically active molecule occurs through an endogenous cellular mechanism. It appears likely that plasmin, generated locally within the thyroid follicular microenvironment, may play a fundamental role in effecting this conversion.

Journal of Endocrinology (1995) 144, 67–73

Slice preparations of normal human thyroid tissue have been used to investigate the effect of normal immunoglobulin G (IgG) on thyrotrophin (TSH)-induced accumulation of cyclic AMP. Incubation of slices in the presence of both TSH and normal IgG for 20 min reduced the stimulation of cyclic AMP accumulation elicited by TSH alone by approximately 30%. However, preincubation of slices with IgG for 100 min before addition of TSH virtually abolished the response to TSH. The latter effect of normal IgG was reversible, and removal of IgG before exposure to TSH allowed an unimpaired cyclic AMP response to TSH. The implications of these observations with respect to the application of this system to the functional bio-detection of thyroid-stimulating antibodies in IgG fractions from thyrotoxic sera are discussed.

ABSTRACT

The regulation of thyroid follicular cell growth in vitro involves autocrine or paracrine actions of insulin-like growth factor-I (IGF-I), which are partially suppressed by transforming growth factor-β (TGF-β). Using subconfluent monolayers of porcine thyroid follicular cells, the aims of this study were to establish whether the actions of TGF-β involve changes in the synthesis of, or response to, IGF-I. We also investigated the extent to which inhibitory actions of iodide on IGF-I-dependent proliferation of thyroid follicular cells may be attributable to the production of TGF-β by follicular cells, as opposed to iodide-mediated autoregulation events.

Exposure of porcine thyroid follicular cells in subconfluent monolayer culture to TGF-β over a 7-day period reduced both IGF-I release and the incorporation of [met hyl-³H]thymidine into trichloroacetic acidprecipitable cellular material, while preincubation of cells with NaI (0 ·1 mmol/l) for 24 h prior to the addition of TSH reduced the stimulatory effect of the latter on IGF-I release over the following 7 days. Preincubation of cells with iodide also reduced basal (i.e. autonomous) [methyl-³H]thymidine incorporation. This effect was partially reversed when, following initial exposure to follicular cells, iodide-containing preincubation medium was immunoadsorbed with a neutralizing TGF-β antiserum, and subsequently readded to the cells. Furthermore, similar immunoadsorption of iodide-free preincubation medium resulted in an enhancement of the control level of [methyl-³H]thymidine incorporation when the treated medium was returned to the original cultures. The results of this study are consistent with the hypothesis that IGF-I and TGF-β are both produced by subconfluent thyroid follicular cells in vitro, and that the inhibitory action of TGF-β on follicular cell growth may involve a decrease in the thyroidal production of IGF-I. While the attenuating action of iodide on follicular cell proliferation may, in part, reflect an increased autocrine production of TGF-β, and a reduction by TGF-β of the growth response to IGF-I, these studies also provide evidence that the intrathyroidal actions of TGF-β include an attenuation of IGF-I biosynthesis.

Journal of Endocrinology (1991) 130, 3–9

ABSTRACT

The role of cyclic AMP (cAMP) attenuation in mediating the autoregulatory actions of iodide on thyroid cell iodide uptake and surface morphological responses to TSH was investigated in the rat thyroid cell strain FRTL-5. Preincubation of cells for 6 h with up to 1 mmol sodium iodide/1 led to a progressive reduction in both accumulation of cAMP and iodide uptake responses to TSH. Forskolin-mediated accumulation of cAMP and iodide uptake responses were similarly reduced after preincubation with iodide, whilst the iodide accumulation response to dibutyryl cAMP (dbcAMP) was unaffected. The inhibitory effects of iodide on TSH or forskolin-responsive iodide accumulation were not seen if preincubation was limited to 3 h, and were also abolished by the thionamide drug methimazole (1 mmol/l). Medium containing 1 μmol iodide/l prevented the appearance of the surface microvilli and pseudopodia normally observed after re-addition of TSH or forskolin, although cytoplasmic retraction was still apparent under such conditions. In contrast, iodide was without effect on the ability of dbcAMP (1 mmol/l) to induce cytoplasmic retraction and the formation of microvilli and pseudopodia. Inclusion of 1 mmol sodium perchlorate/l together with iodide during preincubation failed to prevent or reduce the suppression by iodide of either iodide uptake or surface morphological differentiation, suggesting that both aspects of autoregulation may involve surface actions of organified iodide. These observations indicate that in FRTL-5 cells, autoregulation by iodide of both the functional and surface morphological actions of TSH principally reflects the attenuating activities of organified iodide on intracellular cAMP generation.

Journal of Endocrinology (1990) 124, 19–25

Department of Medicine and Department of Cell and Structural Biology, University of Manchester, Stopford Building, Oxford Road, Manchester m139pt

*Department of Clinical Biochemistry, Clinical Sciences Building, Hope Hospital, Eccles Old Road, Salford m6 8hd

received 14 April 1988

ABSTRACT

A clonal strain of rat thyroid cells (FRTL-5) has been used to investigate the biological activity of a Research Standard preparation of long-acting thyroid stimulator (LATS-B). Using the accumulation of intracellular cyclic AMP as a response parameter, significant stimulation was attained at a LATS-B dose of 0·75 mu./ml. The inter-bioassay coefficient of variation in response to a fixed dose of LATS-B (1·25 mu./ml) was 20·5%, as determined using eight sequential subcultures. Cells cultured directly from frozen stocks responded to both bovine TSH and LATS-B in a manner indistinguishable from cells subjected to regular subculturing. Cyclic AMP responses to incremental doses of LATS-B were potentiated after the inclusion of a low dose of forskolin (0·1 μmol/l). However, forskolin addition had no effect on the time-course of LATS-B-stimulated cyclic AMP accumulation, half-maximal responses being attained after 60 min in either the presence or absence of the diterpene. In the presence of 0·1 μmol forskolin/l, intracellular cyclic AMP responses to LATS-B were demonstrably parallel with those to human TSH (Second International Reference Preparation, 80/558), whilst parallel incremental cyclic AMP responses were also observed in respect of TSH and serial dilutions of a potent thyroid-stimulating immunoglobulin (TSIg) preparation, indicating that for this particular Graves' disease patient, TSIg bioactivity may be expressed in terms of a convenient and reproducible standard, as TSH microunit equivalents.

J. Endocr. (1985) 105, 7–15

ABSTRACT

Thyroid-stimulating hormone (TSH) has been shown to stimulate mitosis in cultures of the continuous thyroid cell strain FRTL-5, and this system may be used to quantify the growth-promoting effects of thyroid stimulators. Removal of TSH from the culture medium led to a progressive decline in the metaphase index (MI) to zero, after 7 days. Thus the cell culture conditions may be manipulated so that metaphases are absent in control cultures, i.e. in the absence of TSH. Restimulation with TSH caused an increase in mitosis only after a lag-phase of 20–24 h. A maximum MI was observed between 40 and 50 h, with a secondary peak between 70 and 75 h. An immunoglobulin G (IgG) preparation from a thyrotoxic patient with a small goitre which was a potent stimulator of adenylate cyclase in these cells produced a similar time-course. A dose–response relationship to TSH was obtained 47 h after addition of the hormone. Significant stimulation was observed with 10 mu. TSH/1, and maximal stimulation with 1 unit TSH/1; the highest dose tested (10 units TSH/1) slightly decreased the MI below the maximum. Stimulation of these cells appeared to be TSH specific, since FSH, human chorionic gonadotrophin, LH and isoproterenol did not induce mitosis. Epidermal growth factor under the experimental conditions employed was unable to induce mitosis. However, an increase in mitosis was observed with the adenylate cyclase stimulator forskolin. These experiments confirm the mitogenic properties of TSH and we describe a metaphase index assay for the detection of thyroid growth promotors.

J. Endocr. (1985) 106, 203–210

SUMMARY

Follicular cells isolated from normal human thyroid tissue have been cultured for up to 140 h with bovine thyrotrophin (TSH) or dibutyryl cyclic AMP (DBcAMP). Both compounds induced marked reorganization of the cells into three-dimensional follicular structures, whilst non-supplemented cells assumed a monolayer form.

Cultures treated initially with TSH or DBcAMP showed a greater iodide uptake capacity, in comparison with unsupplemented cultures, in which iodide uptake was markedly diminished after 24 h.

The release of tri-iodothyronine (T₃) and thyroxine (T₄) into the medium was determined by radioimmunoassay. Both TSH- and DBcAMP-treated cells showed a significant increase in iodothyronine output compared with unsupplemented control cells.

In contrast to the 'classical' TSH-induced depression of the T₄:T₃ ratio in vivo, an increase in the ratio was observed for both TSH- and DBcAMP-supplemented cells in vitro. The ratio was also significantly greater after TSH than after DBcAMP, and possible implications of this finding are discussed.