TSH-induced Growth Research Articles

Regulation of FRTL-5 thyroid cell growth by phosphatidylinositol (OH) 3 kinase-dependent Akt-mediated signaling.

Thyrotropin (TSH)-initiated cell cycle progression from G1 to S phase in FRTL-5 thyroid cells requires serum, insulin, or insulin-like growth factor 1 (IGF-1) and involves activation of 3-hydroxy-3-methylglutaryl-CoA reductase, geranylgeranylation of RhoA, p27Kip1 degradation, and activation of cyclin-dependent kinase (cdk) 2. In the present report, we show that the serine-threonine kinase Akt is an important mediator of insulin/IGF-1/serum effects on cell cycle progression in FRTL-5 thyroid cells. The phosphoinositol (OH) 3 kinase inhibitors, Wortmannin (WM) and Ly294002 (LY), block the ability of insulin/IGF-1 to reduce p27 expression, to induce expression of cyclins E, D1, and A as well as cdk 2 and 4, and to phosphorylate retinoblastoma protein. They also inhibit insulin/IGF-1-increased DNA synthesis and cell cycle entrance (S+G2/M). Insulin/IGF-1 rapidly induced activation of Aktl in a PI3 kinase-dependent manner, and increased Aktl RNA levels. Most importantly, FRTL-5 cells transfected with a constitutively active form of Aktl have higher basal rates of DNA synthesis and no longer require exogenous insulin/IGF-1 or serum for TSH-induced growth. In sum, Aktl appears to have an important role in insulin/IGF-1 regulation of FRTL-5 thyroid cell growth and cell cycle progression.

Regulation and metabolic role of phospholipase D activity in human thyroid and cultured dog thyrocytes.

The actions of TSH, ATP, the ionophore A23187, the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin, and phorbol dibutyrate (PDBu) on 3H-cytidine-monophosphate phosphatidic acid (3H-CMP-PA) accumulation were studied in human thyroid slices to evaluate PA generation and inositol recycling towards phosphatidyl-inositol synthesis. The effects of the same agonists also were measured on phosphatidylbutanol (PtdBut) generation in 3H-palmitate or 3H-myristate prelabeled slices to assess the activity of phospholipase D (PLD). The phospholipid target of this PLD was determined on 3H-choline prelabeled human thyroid slices by measuring 3H-choline release in incubation medium and slices and 3H-choline incorporation in phospholipids. TSH (10 U/L) stimulated 3H-CMP-PA accumulation in an LiCl-and propranolol-insensitive way, as well as 2H-fatty acids incorporation into PA, diacylglycerol, and phosphatidylcholine (PtdCho) with on evidence of dose-dependent effects and had no detectable action on PLD activity. The effects of TSH were not reproduced by Bu2cAMP or forskolin. Thapsigargin and A23187 both increased CMP-PA accumulation and PtdBut generation, whereas ATP only stimulated PLD activity. The phorbol ester PDBu (5 x 10(-7) mol/L) increased PtdBut formation and 3-H-fatty acid incorporation into PtdCho, but had no effect on CMP-PA generation. Staurosporine (STSP) (5 x 10(-6) mol/L), a nonspecific inhibitor of protein kinase C, unexpectedly reproduced the effects of PDBu. The increase of 3H-choline in slices' supernatant and the decrease of 3H-choline-labeled PtdCho induced by PDBu, ATP, thapsigargin, and STSP indicate that the activated PLD hydrolyzed PtdCho. We suggest that the PA generation induced by PLD stimulation could contribute to the stimulated H2O2 formation and iodide organification observed with the agonists inducing PtdBut accumulation. Indeed, Bu2cAMP and forskolin, known to decrease iodide organification in human thyroid, inhibited the PLD stimulation induced by ATP and PDBu. In cultured dog thyrocytes, phorbol esters, and STSP induced DNA synthesis and dedifferentiation, whereas thapsigargin inhibited TSH-induced growth and killed phorbol esters stimulated cells, suggesting a positive role of PLD stimulation towards dedifferentiated growth and of simultaneously raised [Ca2+)i and stimulated protein kinase C-PLD towards growth arrest and cellular death.

Thyroid-stimulating hormone inhibits Fas antigen-mediated apoptosis of human thyrocytes in vitro.

The mechanisms of TSH-induced growth stimulation of thyrocytes in vivo have yet to be elucidated. We examined the antiapoptotic effect of TSH toward Fas antigen-mediated apoptosis of thyrocytes. Fas antigen was expressed on approximately 40% of unstimulated thyrocytes, and the expression was significantly inhibited by the addition of TSH in a dose-dependent manner. Treatment of thyrocytes with 8-bromo-cAMP mimicked the effect of TSH, suggesting that the inhibitory effect of TSH on Fas antigen expression was mediated by activating protein kinase A. In contrast, treatment of thyrocytes with either interleukin-1 beta (IL-1 beta) or interferon- gamma (IFN gamma) markedly increased Fas antigen expression on thyrocytes, and these effects were inhibited in the presence of TSH. The expression of the protooncogene product Bcl-2 did not change after the addition of TSH, 8-bromo-cAMP, IL-1 beta, IFN gamma, or a combination of TSH and IL-1 beta or IFN gamma. When thyrocytes stimulated with either IL-1 beta or IFN gamma were treated with anti-Fas IgM mAb, the cells were committed to apoptosis, whereas this apoptotic process was significantly inhibited by the addition of TSH. These results indicate that the Fas antigen is functionally expressed on the surface of thyrocytes, and TSH inhibits Fas antigen-mediated apoptosis of thyrocytes through the inhibitory effect of Fas antigen expression, resulting in the promotion of growth of the thyroid gland.

Thyroid-stimulating hormone inhibits Fas antigen-mediated apoptosis of human thyrocytes in vitro

The mechanisms of TSH-induced growth stimulation of thyrocytes in vivo have yet to be elucidated. We examined the antiapoptotic effect of TSH toward Fas antigen-mediated apoptosis of thyrocytes. Fas antigen was expressed on approximately 40% of unstimulated thyrocytes, and the expression was significantly inhibited by the addition of TSH in a dose-dependent manner. Treatment of thyrocytes with 8-bromo-cAMP mimicked the effect of TSH, suggesting that the inhibitory effect of TSH on Fas antigen expression was mediated by activating protein kinase A. In contrast, treatment of thyrocytes with either interleukin-1 beta (IL-1 beta) or interferon- gamma (IFN gamma) markedly increased Fas antigen expression on thyrocytes, and these effects were inhibited in the presence of TSH. The expression of the protooncogene product Bcl-2 did not change after the addition of TSH, 8-bromo-cAMP, IL-1 beta, IFN gamma, or a combination of TSH and IL-1 beta or IFN gamma. When thyrocytes stimulated with either IL-1 beta or IFN gamma were treated with anti-Fas IgM mAb, the cells were committed to apoptosis, whereas this apoptotic process was significantly inhibited by the addition of TSH. These results indicate that the Fas antigen is functionally expressed on the surface of thyrocytes, and TSH inhibits Fas antigen-mediated apoptosis of thyrocytes through the inhibitory effect of Fas antigen expression, resulting in the promotion of growth of the thyroid gland.

Thyroid Tumorigenesis

Thyroid tumorigenesis is discussed in the context of the thyroid as a stable tissue, composed of differentiated cells, with a greater dissociation of control of growth from control of differentiation than is found in stem cell tissues. Experimental thyroid carcinogenesis regimes usually use mutagen exposure followed by induced growth. The normal thyroid follicle cell has a limited growth capacity, so loss of one tumour suppressor gene followed by growth-associated loss of heterozygosity would allow escape from this growth limitation, and the formation of a neoplastic clone. In man, there are two pathways of tumour formation, one through follicular adenoma to follicular carcinoma, and one to papillary carcinoma. These two pathways show differing aetiology, and differing oncogene involvement. In the follicular carcinoma pathway TSH-induced growth is relevant as it is in experimental animals. Mutagenesis is important for both papillary and follicular carcinomas. Radiation mutagenesis is of particular current importance because of the occurrence of thyroid carcinoma in children exposed to fallout from Chernobyl. The greater capacity for post-mutagen growth in children than adults is likely to explain the increased radiosensitivity of children, both to external and internal radiation.

Cell proliferation and thyroid neoplasia

The classic experimental model of thyroid neoplasia using radiation as a mutagen followed by long-term goitrogen treatment gives rise to multiple tumours in rats. Radiation alone, with suppression of TSH produces no tumours, TSH-induced growth alone causes a low level of tumorigenesis. Cell proliferation in this model is therefore critical. Epimutation as well as mutation is important. Rodent and human thyroid tumours show a clear stepwise progression, associated with both morphological and oncogene changes. In experimental animals the finding that monoclonal adenomas and carcinomas induced in the presence of long-term high TSH retain TSH dependency suggests that the step in the tumour progression requiring the development of TSH independent growth is bypassed, explaining the frequency of tumour development in this model. Normal thyroid follicular cell growth is limited, and a genotoxic effect before the growth plateau is more effective in carcinogenesis than a genotoxic effect after a period of growth. These observations will be interpreted in relation to the importance of thyroid tumours in regulatory toxicology and the pathobiology of thyroid tumours in man.

Maternal Thyroid-Blocking Immunoglobulins in Congenital Hypothyroidism*

We evaluated 24 mothers whose babies had congenital hypothyroidism (CH) for the presence of immunoglobulins (Igs) that inhibited [125I]bovine TSH binding and blocked TSH-induced growth and function of FRTL-5 cells. Results were compared with those from 2 mothers with known primary myxedema (atrophic thyroiditis) whose babies had transient CH and with normal controls. Only 1 prospectively evaluated CH mother had potent TSH binding inhibitory, growth inhibitory, and function inhibitory IgGs. Further study of this discordant mother's serum indicated that she was hypothyroid, probably due to atrophic thyroiditis. Both mothers with known primary myxedema had blocking IgGs. The thyroid growth-blocking activity was verified by cell count, could be absorbed by and eluted from Staphylococcal protein-A, indicating that it was an IgG, and was not an anti-TSH idiotype. Half-maximal inhibition was similar in the three different assays for thyroid-blocking activity, suggesting that TSH binding inhibitory, growth inhibitory, and function inhibitory IgGs in some patients with primary myxedema may be the same antibody population. There was no correlation with the titer of antimicrosomal antibodies. These data suggest that maternal thyroid-blocking IgGs interacting with the TSH receptor do not play a role in most cases of sporadic CH. Determination of TSH binding inhibitory IgGs, but not antimicrosomal antibodies, is a sensitive screening test for the presence of TSH receptor-blocking antibodies.

Hyperthyroidism following primary hypothyroidism in association with polyendocrine autoimmunity

A 37 year old male with a strong family history of autoimmune disease presented with typical symptoms of hyperthyroidism. He had exophthalmos but no goitre. Hyperthyroidism was confirmed by failure of 131I neck uptake to suppress after 7 days treatment with triiodothyronine. Six years previously a diagnosis of primary hypothyroidism has been made. At diagnosis of hyperthyroidism, thyroglobulin antibodies, thyroidal microsomal antibodies and thyroid stimulating immunoglobulins were detected. The absence of thyroid growth stimulating immunoglobulins and presence of immunoglobulins blocking TSH-induced growth may account for the absence of goitre throughout. HLA -B8, -B, -DR3 and -DR4 genotypes, low C4 complement concentrations and islet cell autoantibodies were detected at the time of diagnosis and 1 year later diabetes mellitus developed.