Retinol has specific effects on binding of thyrotrophin to cultured porcine thyrocytes

Retinoids are potential candidates for the treatment of thyroid cancer. However, one of the disadvantages of these substances is their dedifferentiating effect on normal non-transformed thyrocytes. To identify conditions under which no dedifferentiating effect of retinol on normal thyrocytes can be observed, we determined iodide uptake, protein iodination, expression of sodium-iodide symporter (NIS) mRNA and protein, and the binding of iodine-125-labelled bTSH in cultured porcine thyrocytes. Combination of TSH and < or =6.5 microM retinol increased iodide uptake and protein iodination compared with TSH alone over the entire incubation time, whereas TSH plus > or =13 microM retinol increased the uptake of iodine-125 only during the first 12 h but decreased it after 30 h and longer. After > or =30 h incubation times with > or =13 microM retinol, the fraction of apoptotic cells was enhanced and proliferation decreased. The incubation with retinol enhanced the binding of [125I]bTSH to thyrocytes, but did not influence expression of the NIS. With low retinol concentrations, the effect on the binding of TSH apparently predominated and retinol increased thyroid function; with higher concentrations the pro-apoptotic effect of retinol overlapped and a two-phased time course resulted. It can be concluded that low concentrations of retinol also exert differentiating effects in normal thyrocytes.     

Studies of HLA-DR expression on cultured human thyrocytes: effect of antithyroid drugs and other agents on interferon-gamma-induced HLA-DR expression

There have been conflicting reports on whether antithyroid drugs (ATD) act as immunosuppressive agents in patients with autoimmune thyroid disease. While some have claimed that methimazole (MMI) affects the immune system directly, we and others have suggested that its apparent immunosuppressive activity is due to its ability to inhibit thyrocyte, rather than immunocyte, activity. To further address the question, we studied the action of ATD on interferon-gamma (IFN gamma)-induced HLA-DR expression on thyrocytes in tissue culture. We used a cytotoxicity assay, using chromium-51-labeled Graves' disease (GD) thyrocytes and normal thyrocytes incubated sequentially with a monoclonal antibody against HLA-DR and complement, with a cytotoxicity index as the measure of thyrocyte HLA-DR expression. MMI and propylthiouracil (PTU) were added along with 200 U/mL IFN gamma to thyrocytes cultured for 10-14 days. IFN gamma or supernatants from leukoagglutinin-stimulated peripheral blood mononuclear cells (PBMC) stimulated thyrocyte HLA-DR expression; however, the addition of MMI or PTU to either the PBMC or thyrocytes caused no inhibition of the IFN gamma or PBMC IFN gamma stimulation of thyrocyte HLA-DR expression, using either normal or GD thyrocytes. Potassium perchlorate and sodium iodide also had no effect on IFN gamma-induced thyrocyte HLA-DR expression. TSH (either bovine or human) did not induce HLA-DR expression on thyrocytes by itself, but did enhance IFN gamma-induced HLA-DR expression in normal, but not GD, thyrocytes; once again, the further addition of MMI or PTU did not inhibit the enhancing effect of TSH on thyrocyte HLA-DR expression. Low concentrations of TSH binding inhibitory immunoglobulin (TBII; 100 micrograms/mL) did not alter the cytotoxicity index, but at 400 micrograms/mL or more it enhanced HLA-DR expression on normal, but not GD, thyrocytes in a manner similar to TSH; like TSH, it did not induce thyrocyte HLA-DR expression by itself. Moreover, addition of MMI to the combination of IFN gamma and TBII did not inhibit the response of thyrocytes in terms of HLA-DR expression. We conclude that ATD do not alter thyrocyte HLA-DR expression in vitro; however, the ATD may still cause immune effects in vivo secondary to their influence on thyroid hormone formation or synthesis or by inhibition of thyroid antigen presentation which indirectly may result in an immunomodulatory effect. While TSH and TBII similarly enhanced the IFN gamma-induced expression of HLA-DR on normal thyrocytes, they did not do so in GD thyrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ghrelin enhances the proliferating effect of thyroid stimulating hormone in FRTL-5 thyroid cells

Ghrelin regulates cell proliferation through the growth hormone secretagogue receptor (GHS-R). We confirmed the expression of GHS-R in FRTL-5 thyroid cells and investigated the effects of ghrelin in thyrocytes using FRTL-5 cells. Ghrelin increased intracellular calcium levels but not intracellular cyclic AMP levels. Ghrelin activated Erk within 2min, then activated Akt and STAT3. Erk phosphorylation was inhibited by the calcium inhibitor cyclopiazonic acid (CPA). Ghrelin alone did not stimulate FRTL-5 cell proliferation but enhanced the effects of thyroid stimulating hormone (TSH). Pretreatment with TSH potentiates the growth effects of ghrelin in thyroid cells, and p66Shc, a growth factor receptor adaptor protein, might mediate these synergistic effects. Ghrelin phosphorylated TSH-induced p66Shc, which was inhibited by CPA. Ghrelin did not affect the proliferation of ARO cells, which showed no increased expression of p66Shc after TSH treatment. Thus, ghrelin-induced intracellular calcium signaling enhanced the TSH-induced proliferation of thyrocytes, possibly mediated by the p66Shc pathway.     

TSH-Dependent expression of the LDL receptor-associated protein (RAP) in thyroid epithelial cells.

The low density lipoprotein (LDL) receptor-associated protein (RAP) is an endoplasmic reticulum (ER)-resident molecular chaperone for several LDL receptor family members and it also binds to thyroglobulin (Tg), the thyroid hormone precursor. Disruption of the RAP gene in thyrocytes results in impaired Tg secretion. To gain further insights into the function of RAP in the thyroid, we investigated whether its expression in thyrocytes is regulated by thyroid-stimulating hormone (TSH), a feature common to all proteins involved in thyroid hormone secretion. We found by immunofluorescence that in FRTL-5 cells cultured in the presence of TSH, RAP is expressed intracellularly. The levels of expression increased after exposure to TSH, beginning at 48 hours, in a concentration-dependent manner as observed by immunofluorescence and Western blotting. Expression of RAP was also increased by TSH in primary cultures of human thyrocytes as observed by Western blotting. In hypothyroid mice with high serum TSH, RAP was markedly increased compared with euthyroid mice as observed by immunohistochemistry and Western blotting. Based on these findings, we concluded that RAP is expressed by thyrocytes in a TSH-dependent manner, both in cultured thyroid cells and in vivo.     

Direct effects of protirelin (TRH) on cultured porcine thyrocytes.

In several cases of thyroid adenoma in which no TSH was detectable in the serum, a paradoxical decrease of PB123I and its conversion rate Q (PB123I/serum total radioactivity) was observed after infusion of protirelin (TRH). The possibile direct effect of TRH on thyrocytes was therefore studied in vitro. Porcine thyroid cells were cultured in NCTC-135 medium supplemented with a serum substitute and insulin. TRH and some of its analogs enhanced the incorporation of [3H]thymidine into DNA and the uptake of radioiodide into thyrocytes. In the presence of TSH, TRH did not stimulate radioiodine uptake, whereas incorporation of [3H]thymidine into DNA was not antagonized by TSH. Thus, in this culture system, TRH had a direct effect on both growth and metabolism of thyrocytes. TSH can modulate these effects.     

Stimulation of iodide uptake by human chorionic gonadotropin in FRTL-5 cells: effects on sodium/iodide symporter gene and protein expression

BACKGROUND: Various clinical and experimental findings support the concept that human chorionic gonadotropin (hCG) can stimulate iodide uptake in thyroid cells. DESIGN: We investigated the molecular mechanisms underlying the effects of hCG on iodide uptake, and particularly its action on the expression of Na+/I- symporter (NIS) mRNA and protein. METHODS: Iodide uptake was analyzed in FTRL-5 cells by measuring (125)I concentrations in cells after a 30-min exposure to 0.1 microCi carrier-free Na (125)I in the presence or absence of hCG or, for control purposes, TSH. Expression of NIS mRNA and NIS protein synthesis were evaluated, respectively, with a semiquantitative 'multiplex' RT-PCR method and Western blot analysis. RESULTS: Iodide uptake was increased by hCG in a dose- and time-dependent manner: maximal effects were observed after 72 h of stimulation. The effect was cAMP dependent and paralleled that of TSH, although it lacked the early cycloheximide-independent component seen with TSH, and its peak effect was lower. Semiquantitative multiplex RT-PCR revealed that hCG produced a significant increase in NIS mRNA levels that was detectable after 4 h and peaked after 48 h. In contrast, in TSH-stimulated FRTL-5 cells, maximum NIS mRNA expression was observed after 24 h of stimulation. Western blot analysis demonstrated that hCG also caused a 2.5-fold increase over basal values in NIS protein levels, which was similar to that observed after TSH stimulation although the peak effects of the latter hormone were less marked and occurred earlier. CONCLUSION: Our data demonstrated that hCG stimulates iodide uptake in FRTL-5 cells by increasing NIS mRNA and protein levels. Thus, the functional status of the thyroid may be influenced by hCG-dependent changes in NIS expression occurring during pregnancy.     

Similar and divergent patterns in the regulation of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of MMP-1 gene expression in benign and malignant human thyroid cells.

An imbalance between the activity of matrix metalloproteinases (MMPs) (proteolytic enzymes that degrade protein components of the extracellular matrix) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), may be one of the mechanisms responsible for tumor cell invasion. We have investigated the regulation of MMP-1 and TIMP-1 gene expression in benign and malignant (follicular, anaplastic, and papillary) human thyroid cells. As expected of cells with invasive potential, detectable MMP-1 messenger RNA (mRNA) levels were observed in malignant cells under basal conditions, in contrast to undetectable levels in benign cells. Exposure of these cells, for 1 h, to the active phorbol ester, phorbol 12-myristate 13-acetate (TPA, 100 nmol/L), acting via protein kinase C (PKC), elicited an increase in MMP-1 mRNA, with a peak stimulation after a 3- to 4-h culture period. Epidermal growth factor (EGF, 25 ng/mL), however, acting via protein tyrosine kinase (PTK), stimulated such gene expression in malignant cells but failed to do so in benign cells. TIMP-1 mRNA was not significantly altered by the TPA-PKC, EGF-PTK, or TSH-protein kinase A (PKA) pathways in malignant cells. In benign cells, however, TPA induced a small, though significant, increase in TIMP-1. The MMP-1 stimulation by EGF and lack of TPA-induced rise in TIMP-1 in malignant cells, in sharp contrast to the effects obtained in benign thyrocytes, seems to indicate that the MMP: TIMP balance favors a more extensive extracellular matrix protein breakdown by malignant thyrocytes, as expected of cells exhibiting invasive capacity. TSH (10-500 microU/mL) failed to significantly influence basal MMP-1 or TIMP-1 mRNA levels, but it caused a dose-dependent inhibition in TPA- and EGF-induced MMP-1 mRNA in malignant cells, and TPA-stimulated MMP-1 and TIMP-1 in benign cells. The repressive action of TSH on MMP-1 mRNA was mimicked by forskolin and 8-bromo-cAMP and was abrogated by the PKA inhibitor, H-89, suggesting that the TSH inhibitory action is PKA-mediated. In conclusion, the present study provides novel data on MMP-1 and TIMP-1 gene expression and their modulation by the major signal transduction pathways operating in human thyroid cells. Similar and divergent patterns have emerged in the regulation of such gene expression in benign and malignant human thyrocytes, in many instances in accord with the concept of MMP playing the role of stimulating, and TIMP inhibiting, cell invasion. Although MMP-1 may be just one of the many factors responsible for tumor cell invasion, the present findings demonstrating the possibility, at least in vitro, of repressing MMP gene expression may have important clinical ramifications.     

Inhibitory effect of interferon-gamma on the response of human thyrocytes to thyrotropin (TSH) stimulation: relationship between the response to TSH and the expression of DR antigen

Thyroid epithelial cells (thyrocytes) in autoimmune thyroid disease have been found to express DR antigens on their surfaces, and interferon-gamma (IFN gamma) induces DR antigen expression. This study was undertaken to determine the effect of IFN gamma on the response of human thyrocytes to TSH stimulation and the relationship between the response to TSH and the expression of DR antigen induced by IFN gamma, using monolayer cultures of Graves' thyrocytes. When confluent thyrocyte monolayers were incubated with TSH or Bu2cAMP (DBcAMP) for 7-9 days, T3 and thyroglobulin concentrations in the culture medium increased gradually in a dose-dependent manner. However, when TSH or DBcAMP was added after the cells had been cultured for 4 days with IFN gamma, T3 and thyroglobulin secretion in response to both 10 mU/mL TSH and 1 mM DBcAMP was significantly inhibited. The inhibition by IFN gamma was dose dependent and correlated with the number of DR antigen-positive thyrocytes present on the last day of culture. IFN alpha and -beta did not affect the response of thyrocytes to TSH or DBcAMP stimulation. These results suggest that DR antigen-positive thyrocytes fail to respond to TSH stimulation at a site located distal to cAMP formation.     

Studies on the mechanism of desensitization of the cyclic AMP response to TSH stimulation in a cloned rat thyroid cell line

We examined aspects of the mechanism of desensitization of adenylate cyclase activation by TSH in a cloned line of rat thyroid cells (FRTL). Increasing FRTL intracellular cAMP concentrations by preincubation for 6 h in either 1 mM dBcAMP or 100 microM forskolin did not induce TSH desensitization. Forskolin stimulation was unimpaired in TSH-desensitized cells, indicating 'uncoupling' of the adenylate cyclase catalytic unit from the TSH receptor. Stimulation by the Ni inhibitory pathway of the adenylate cyclase by epinephrine (10(-6) M-10(-4) M in the presence of 10(-4) M propranolol) was unaltered in cells previously desensitized to TSH. That is, Ni-mediated inhibition of adenylate cyclase was additive to TSH desensitization. Pre-exposure of FRTL cells for 18 h to 50 ng/ml pertussis toxin did not prevent the induction of TSH desensitization. TSH desensitization was prevented by cycloheximide or actinomycin D added during the last 3-4 h of a 6 h period of TSH stimulation. The rates of turnover of the putative desensitization protein and its mRNA therefore appear to be similar.     

Human recombinant interleukin 1 inhibits TSH-stimulated morphological changes in thyroid follicles cultured as semi-organs

To study the effects of human recombinant interleukin-1 on thyrocytes, we cultured thyroid follicles as semi-organs, each consisting of approximately 10-20 follicles, in the presence or absence of IL-1 alpha or beta. Semi-organ culture reproduces the in vivo environment well. After culture for 2 or 4 days, the follicles were incubated with TSH (10 U/l) for 4 h and fixed for light and electron microscopical examinations. Regardless of the presence or absence of IL-1, follicular structure, polarity, and luminal colloid did not change during culture. In thyroid epithelial cells cultured without IL-1, TSH markedly induced elongation of microvilli and formation of reabsorbed colloid droplets. On the other hand, both IL-1 alpha and beta inhibited these TSH-stimulated changes. The degree of inhibition correlated with the concentration of exposure to IL-1. We conclude that IL-1 inhibits TSH-stimulated morphological changes in thyroid follicles cultured as semi-organs, depending on the concentration of IL-1.