In vitro and in vivo regulation of thyrotropin receptor mRNA levels in dog and human thyroid cells.

Regulation of thyrotropin (TSH) receptor (TSHr) mRNA accumulation as compared with two other thyroid differentiation markers (thyroglobulin and thyroperoxidase (TPO] has been investigated by Northern blot. In dogs in vivo, chronic stimulation of the thyroid TSHr mRNA although it increased the levels of thyroglobulin and TPO mRNA. In dogs treated with thyroxin, the quiescent thyroids expressed normal levels of TSHr and TPO mRNA but depressed levels of thyroglobulin mRNA. In primary cultures of dog thyrocytes, dedifferentiation of the cells by treatment with epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate led to decreased TSHr mRNA levels and nearly abolished thyroglobulin and TPO gene expression. However, TSHr mRNA was always present, compatible with the fact that these cells, when treated by TSH, reexpress differentiation. Treatment of the cells with TSH or forskolin transiently increased the TSHr mRNA level after 20 h, an effect inhibited by cycloheximide. This up-regulation was confirmed at the protein level: forskolin-treated cells showed an enhanced cAMP response to TSH and an increased binding of labeled TSH to their membranes. Long term TSH treatment led to a slight down-regulation of TSHr mRNA in dog thyrocytes, but in human thyroid cells no marked down-regulation was observed.     

Modulation of thyroglobulin messenger RNA level by thyrotropin in cultured thyroid cells.

To examine the influence of thyrotropin (TSH) on the thyroglobulin (Tgb) mRNA content, the latter was evaluated in the cytoplasm of hog thyroid cells cultured in the absence (control cells) or presence of TSH. The Tgb mRNA levels were determined by, (i) kinetics of hybridization to sheep Tgb cDNA, (ii) capacity of coding for peptides immunologically related to Tgb in reticulocyte lysate. In cells cultured for 4 days in the absence of TSH, the content of Tgb mRNA sequences decreased to 30% of its initial value and the messenger activity to 15%. Conversely, TSH maintained the initial Tgb mRNA level in cells cultured in its presence, and TSH concentrations 50 micronU/ml or 5 mU/ml gave identical results. At each period tested poly (A) content was the same in TSH-treated and control cells. When TSH was added to media after 4 or 8 days culture without TSH, the Tgb mRNA level was partially restored. These results suggest that TSH exerts a positive control on Tgb gene expression through modulation of Tgb mRNA content of thyroid cells.     

Hormonal regulation of thyroid peroxidase in normal and transformed rat thyroid cells

The hormonal induction of thyroid peroxidase (TPO) mRNA is studied in the functional rat thyroid cell line FRTL-5 and compared to the induction of thyroglobulin (TG) mRNA and I- uptake. TPO and TG mRNAs are regulated by TSH and by insulin-like growth factor I (IGF-I) and/or insulin. However, while TPO is more sensitive to TSH regulation (5- to 6-fold increase vs. 2- to 3-fold increase by IGF-I), TSH and IGF-I are equally potent in increasing TG mRNA levels (3- to 4-fold). Regulation of I- uptake appears to be different: thus TSH greatly (15-fold) increases I- uptake, while IGF-I or insulin are completely ineffective. TPO and TG mRNAs and I- transport display different sensitivity to transformation of rat thyroid cells. Thus, when another differentiated rat thyroid cell line, the PC cells, are transformed by human c-myc (PC myc), TPO and TG mRNAs are both present at normal levels, while I- uptake is slightly decreased; in the PC cells transformed by polyomavirus middle-T-antigen (PC PyMLV) TPO mRNA is undetectable and I- uptake is greatly decreased, while TG mRNA is present at normal levels. All three differentiated functions are switched off in PC cells transformed by the cooperation of c-myc and polyomavirus middle-T-antigen (PC myc + PyMLV).     

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.     

Regulation of thyroperoxidase, thyroglobulin and iodide levels in sheep thyroid cells by TSH, tumor promoters and epidermal growth factor

Using sheep thyroid cells in culture, we have studied the effects of thyroid stimulating hormone (TSH), epidermal growth factor (EGF) and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on the activity and expression of both thyroglobulin (Tg) and thyroid peroxidase (TPO) and on the ability of cells to trap and organify iodide. Using Western blotting techniques, we found that TSH increased the absolute cellular levels of Tg. The optimum TSH concentration for Tg mRNA production was between 0.1-1.0 mU/ml. Thyroglobulin mRNA levels were stimulated by TSH but detectable levels were also present in cultures grown in its absence containing cortisol, insulin, transferrin, somatostatin and glycyl-lysyl-histidyl acetate. Unlike Tg, TPO protein levels were found to be completely dependent upon TSH. A time course of TSH stimulation of TPO mRNA showed increases after 8 h of TSH stimulation, whereas induction of Tg mRNA by TSH was seen at 24 h. Iodide trapping and organification were also TSH-dependent processes, showing maximum activities at 300-500 muU/ml of TSH. The addition of 10 nM TPA caused a biphasic decrease in radiolabeled pertechnetate uptake, with complete inhibition being seen at 14 h. Inhibition of iodide organification occurred more rapidly. TPA and EGF (1 nM) reduced the amount of newly synthesized Tg in TSH-stimulated cells by 50% but the absolute amount of Tg within the cells was not markedly inhibited at these early times.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells

Both thyrotropin (TSH) and epidermal growth factor (EGF) are potent mitogenic agents when added to dog thyroid cells in primary culture [Roger, P. P. and Dumont, J. E. (1984) Mol. Cell. Endocrinol. 36, 79-93]. The concomitant effect of these agents on the differentiation state of the cells was appreciated using cell morphology, iodide trapping, thyroglobulin synthesis and cytoplasmic thyroglobulin mRNA content as markers. Together with previous results [Mol. Cell. Endocrinol. 36, 79-93 (1984)] it is shown that cells cultured in the continuous presence of TSH maintain all the parameters at a near normal level. In the absence of TSH, thyroglobulin mRNA decreased to very low, though still detectable levels. Addition of TSH restored subnormal mRNA levels. Culture of cells in the presence of EGF for 4-6 days affected profoundly their morphology, abolished iodide trapping and decreased thyroglobulin synthesis and cytoplasmic mRNA content to undetectable levels. Addition of TSH to cells previously exposed to EGF reversed the growth factor effect on all four indexes. The redifferentiating effect of TSH was well observed within 3-4 days and was mimicked by the adenylate cyclase activators, forskolin and cholera toxin. When administered simultaneously, TSH and EGF achieved an intermediate situation, EGF antagonizing partially the effect of TSH on the expression of thyroglobulin gene. Another growth factor, fibroblast growth factor, while promoting thyroid cell proliferation also, did not interfere at all with TSH effects on cytoplasmic thyroglobulin mRNA content. Our results make the dog thyroid cell in primary culture an appropriate model to study the mechanisms involved in gene regulation by cyclic AMP and growth factors.     

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.     

Regulation by thyroxine of the mRNA encoding the alpha subunit of mouse thyrotropin

Thyrotropin (TSH), a glycoprotein hormone of the pituitary consisting of two subunits (alpha and beta), regulates thyroxine (T4) production by the thyroid gland. T4, in turn, regulates TSH biosynthesis and release. We have studied the regulation of the messenger RNA encoding the alpha subunit of TSH by T4 in pituitaries and in a transplantable thyrotropic tumor in mice. Hypothyroid male LAF1 mice bearing the TtT 97 thyrotropic tumor were injected daily with T4 for either 0, 1, 5, 12, or 33 days. Levels of TSH and its unassociated alpha (free alpha) and TSH-beta subunits in the plasma of these animals fell to less than 5% of control values after 33 days. Concentrations of TSH and TSH-beta in both tumor and pituitary also fell to low levels (less than 2% of control), while intracellular concentrations of free alpha subunit remained unchanged. Cellular levels of the mRNA encoding the precursor of the alpha subunit or pre-alpha (alpha mRNA) were measured by cell-free translation followed by electrophoretic analysis of immunoprecipitates of pre-alpha subunit and by nucleic acid hybridization to a radiolabeled cDNA probe specific for the alpha mRNA. In the pituitary, translatable and hybridizable alpha mRNA was decreased slightly after 1 day of T4 and decreased 40-50% after 5 and 12 days. In thyrotropic tumors, both translatable and total alpha mRNA showed a 60% decrease by 1 day and a maximum 85% decrease after 5, 12, and 33 days of T4. Therefore, T4 acts rapidly in vivo to decrease steady state alpha mRNA levels in the thyrotrope, and this decrease is maintained for the duration of treatment with thyroid hormone. This regulatory process is reflected in the sharp decreases in levels of TSH and free alpha subunit in plasma and in lower concentrations of the intact TSH in tissue. In contrast, the maintenance of high tissue concentrations of free alpha subunit after T4 treatment may be a reflection of alterations in a post-translational process specific for the free alpha subunit, as opposed to that of the intact TSH.     

Differential expression of Atlantic salmon thyrotropin β subunit mRNA and its cDNA sequence

To study the regulation of the thyroid system, an Atlantic salmon Salmo salar cDNA clone was isolated for thyroid stimulating hormone (TSH) β subunit gene. A cDNA (866 bp) was isolated from an adult Atlantic salmon pituitary cDNA library, this clone was sequenced and shown to be highly conserved when compared to other teleost β TSH subunit sequences. The cDNA was used as a probe for Northern blot analysis of total pituitary RNA from the different life cycle stages of Atlantic salmon. Northern blot analysis demonstrated that β TSH mRNA is expressed at all life cycle stages studied, including parr, smolt, immature fish at sea and sexually mature male fish. Densitometry of Northern blots showed that sexually mature male salmon had low levels of salmon β TSH mRNA compared to non-mature fish. Stunts, fish performing poorly in salt water, were shown to have elevated levels of β TSH mRNA when compared to healthy fish.     

In vivo effect of thyrotropin on intracellular translocation of thyroid peroxidase in rat thyroid cells by an indirect immunofluorescence method

The in vivo effect of thyrotropin (TSH) on the intracellular localization of thyroid peroxidase (TPO) in rat thyroid epithelial cells was examined by an indirect immunofluorescence method. The staining for TPO in the epithelial cells of normal rats appeared all over the cytoplasm, especially in the apical region. The injection of propylthiouracil for 3-10 days increased the staining in the apical region. The administration of L-thyroxine for 7-10 days to normal rats abolished the relatively high localization of TPO in the apical region, and resulted in TPO staining all over the cytoplasm. Six hours after TSH was injected into the thyroxine-treated rats, localization of TPO staining in the apical region was observed. These results suggest that TSH may play a role in the translocation of preexisting TPO to the apical region before TSH-induced biosynthesis becomes evident.     

Evidence for Somatostatin receptor 2 in thyroid tissue

Somatostatin receptor scintigraphy has found considerable interest for imaging thyroid tumours. Recently, also therapeutic application of Somatostatin analogues labelled with beta-emitting radionuclides has been suggested as treatment option for thyroid tumours with absent radioiodine uptake. Most of the radiolabelled analogues available show a predominant affinity for Somatostatin receptor subtype 2. This study reports on the in vitro characterisation of Somatostatin receptor subtype mRNAs in thyroid tumours and normal thyroid tissue by means of RT-PCR. Surgical samples of 21 patients were collected, and mRNA of 16 tumour and 17 control specimen was isolated. mRNA expression for Somatostatin, SSTR subtype 1-5, thyroid markers (NIS, TSH, Tg, TPO) and control markers (GAPDH, beta-actin) was determined. PCR results were correlated with immunohistochemistry staining using SSTR2 receptor specific antibodies. 94% of all samples expressed Somatostatin receptor mRNA with predominant expression of subtype 2, less predominant of subtype 5 and subtype 3. Somatostatin receptor subtype 2 mRNA expression correlated well with immunohistochemical staining pattern in 13/16 samples, SSTR2 immunohistochemistry was positive in 87% of the samples. Our results show that Somatostatin receptor 2 is predominantly expressed on thyroid tissue and is a valid target for treatment of thyroid tumours. Octreotide derivatives currently used in Nuclear medicine seem to be well suited to target receptors expressed in thyroid tumours.     

RXR receptor agonist suppression of thyroid function: central effects in the absence of thyroid hormone receptor

High-affinity agonists for the retinoic acid X receptors (RXR) have pleotropic effects when administered to humans. These include induction of hypertriglyceridemia and hypothyroidism. We determined the effect of a novel high-affinity RXR agonist with potent antihyperglycemic effects on thyroid function of female Zucker diabetic rats and nondiabetic littermates and in db/db mice. In both nondiabetic and ZFF rats, AGN194204 causes a 70-80% decrease in thyrotropin (TSH), 3,3',5-triiodothyronine, and thyroxine (T(4)) concentrations. In the db/db mouse, AGN194204 causes a time-dependent decrease in thyroid hormone levels with the fall in TSH that was significant after 1 day of treatment preceding the fall in T(4) levels that was significant at 3 days of treatment. Treatment with AGN194204 caused an initial increase in hepatic 5'-deiodinase mRNA levels which then fell to undetectable levels by 3 days of treatment and continued to be low at 7 days of treatment. After treatment for 5 days with AGN194204, both wild-type and thyroid hormone receptor beta (TR beta(-/-))-deficient mice demonstrated a nearly 50% decrease in serum TSH and T(4) concentrations. The results suggest that a high-affinity RXR agonist with antihyperglycemic activity can cause central hypothyroidism independently of TR beta, the main mediator of hormone-induced TSH suppression.