Absence Of TSH Research Articles

Characterization of Apparently Paradoxical Thyrotropin Binding Inhibitory Immunoglobulins With Neutral Bioactivity.

ContextThe thyrotropin (TSH) receptor (TSH-R) autoantibody activity is clinically measured by inhibition of labeled ligand (TSH or M22) binding to the TSH-R (TSH-binding inhibitory immunoglobulin [TBII]) or by stimulation (TSH-R stimulating antibody [TSAb]) or inhibition (TSH-R blocking antibody [TSBAb]) of 3′,5′-cyclic adenosine 5′-monophosphate (cAMP) production in isolated cells.ObjectiveWe experienced a patient with hypothyroid Graves disease (GD) having strong positive TBII but with almost neutral bioactivities on the TSH-R. The aim of this study is the characterization of this apparently paradoxical TBII (serum sample S).MethodsWe first compared the TBII, TSAb, and TSBAb activities of serum sample S with mixtures of stimulating (S-mAb) and blocking monoclonal Ab (B-mAb). Next, we serially measured cAMPs stimulated by various serum samples in the presence or absence of TSH.ResultsMixtures of S-mAb and B-mAb did not reproduce the characteristics of serum sample S. Instead, serum sample S had a unique feature that blocked the TSH-stimulated cAMP initially but disappeared the blocking activity thereafter to reach the control level.ConclusionWe present here the TBIIs with neutral bioactivities found in the patient with autoimmune thyroid disease, which strongly inhibit TSH binding to the TSH-R but exerts neither TSAb nor TSBAb activity. Differences in the methods of detecting TRAb between TBII in vitro and bioassay may cause the discrepancy. Although serum sample S may be an extreme example, a variety of TRAb that not only stimulates or blocks but also interferes with TSH-R binding for only a short time may exist in the serum samples of GD patients.

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2.

The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Overall, the thyrocyte responses following exposure to β or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

Inhibitory effects of methimazole and propylthiouracil on iodotyrosine deiodinase 1 in thyrocytes.

Methimazole (MMI) and propylthiouracil (PTU) are commonly used for the treatment of Graves' disease. They share similar inhibitory effects on thyroid hormone biosynthesis by interfering with thyroid peroxidase (TPO)-mediated oxidation and organification of iodine. However, their potential effects on other thyroid functional molecules have not been explored in depth. To identify novel effects of MMI and PTU, DNA microarray analysis, real-time PCR, Western blotting, immunofluorescence staining and confocal laser scanning microscopy were performed using FRTL-5 rat thyroid cells. DNA microarray analysis indicated that both MMI and PTU suppress iodotyrosine deiodinase 1 (Iyd, Dehal1) mRNA levels. Further studies revealed that Dehal1 mRNA levels was stimulated by TSH, insulin and serum, while it was suppressed by iodine and a follicular concentration of thyroglobulin. MMI and PTU significantly suppressed Dehal1 expression induced by TSH, insulin and serum. On the other hand, although MMI suppressed Dehal1 expression in the absence of TSH, PTU only weakly suppressed Dehal1 without TSH. These results suggest that PTU and MMI may use different mechanisms to regulate Dehal1 expression, and TSH may play essential and differential roles in mediating PTU and MMI signals in thyrocytes. The drugs also inhibited re-distribution of Dehal1 protein into newly formed lysosomes following thyroglobulin endocytosis. These findings imply complex and multifaceted regulation of Dehal1 in the thyroid and suggest that MMI and PTU modulate Dehal1 expression and distribution of the protein in thyrocytes to exert their effect.

Thyroglobulin Increases Thyroid Cell Proliferation via the Suppression of Specific MicroRNAs

Thyroglobulin (Tg), stored in the follicular lumen, has also been shown recently to perform two unexpected roles: as an autocrine negative-feedback suppressor of thyroid function in the presence of TSH and as a potent inducer of thyroid cell growth in the absence of TSH. However, the underlying molecular mechanism(s) remain unclear. To elucidate a molecular pathway linking Tg to increased cell proliferation, we examined the regulation of microRNAs (miRNAs) by Tg using an miRNA microarray. We identified 21 miRNAs whose expression was significantly suppressed by Tg in rat thyroid FRTL-5 cells. Using specific miRNA analogs, we determined that miR-16, miR-24, and miR-195 mediate the induction of thyroid cell growth by Tg. The expression of miR-16 and miR-195 target genes, Mapk8, Ccne1, and Cdc6, which were previously shown to be essential for TSH-stimulated thyroid cell growth, were also induced by Tg. Moreover, the Tg-induced expression of these genes was reduced by overexpression of miR-16 and miR-195. Similarly, the induction of c-Myc by Tg was reduced by miR-24 overexpression. These results suggest that Tg could alter thyroid cell proliferation by increasing the expression of cell division-related genes such as Mapk8, Ccne1, Cdc6, and c-Myc through its suppression of specific microRNAs (miR-16, miR-24, and miR-195). In addition, we identified phosphatidylinositol 3-kinase as a key signaling pathway, linking Tg with cell proliferation. The present data support an important role for miRNAs as effectors for the effect of Tg on cell proliferation and perhaps other functions of Tg in the thyroid cell.

Dominant negative effect of mutated thyroid stimulating hormone receptor (P556L) causes hypothyroidism in C.RF-Tshr(hyt/wild) mice.

C.RF-Tshrhyt/hyt mice have a mutated thyroid stimulating hormone receptor (P556L-TSHR) and these mice develop severe hypothyroidism. We found that C.RF-Tshrhyt/wild heterozygous mice are also in a hypothyroid state. Thyroid glands from C.RF-Tshrhyt/wild mice are smaller than those from wild-type mice, and 125I uptake activities of the former are significantly lower than those in the latter. When TSHR (TSHR(W)) and P556L-TSHR (TSHR(M)) cDNAs were cloned and co-transfected into HEK 293 cells, the cells retained 125I-TSH binding activity, but cAMP response to TSH was decreased to about 20% of HEK 293 cells transfected with TSHR(W) cDNA. When TSHR(W) and TSHR(M) were tagged with eCFP or eYFP, we observed fluorescence resonance energy transfer (FRET) in HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP in the absence of TSH, but not in the presence of TSH. In contrast, we obtained FRET in HEK 293 cells expressing TSHR(W)-eCFP and TSHR (M)-eYFP, regardless of the presence or absence of TSH. These results suggest that P556L TSHR has a dominant negative effect on TSHR(W) by impairing polymer to monomer dissociation, which decreases TSH responsiveness and induces hypothyroidism in C.RF-Tshrhyt/wild mice.

AMP-Activated Protein Kinase Upregulates Glucose Uptake in Thyroid PCCL3 Cells Independent of Thyrotropin

Glucose is transported into cells by specific glucose transporter proteins (GLUTs) that are widely expressed in a tissue-specific manner. The mechanisms that regulate glucose uptake and metabolism in thyroid cells are poorly defined. Recently, our group showed that AMP-activated protein kinase (AMPK) plays a pivotal role in the rat thyroid gland, downregulating iodide uptake by thyroid cells even in the presence of its main stimulator thyrotropin (TSH). Since AMPK increases glucose uptake in different tissues, and taken into consideration that in pathophysiological conditions such as thyroid cancer a negative correlation between iodide and glucose uptake occurs, we hypothesized that AMPK might modulate glucose uptake in thyroid cells. Rat follicular thyroid PCCL3 cells cultivated in Ham's F-12 supplemented with 5% calf serum and hormones were exposed to the AMPK pharmacological activator 5-aminoimidazole-4 carboxamide ribonucleoside (AICAR) or AMPK antagonist compound C for 24 hours either in the presence or absence of TSH. Glucose uptake was assessed in vitro using 2-deoxy-D-[(3)H]glucose. AMPK activation by AICAR induced a significant increase in glucose uptake by PCCL3 cells, an effect that was completely reversed by the AMPK inhibitor compound C. Also, the AICAR mediated increase in glucose uptake was detected either in the presence or absence of TSH. The mechanism by which AICAR increases glucose uptake is related to higher levels of GLUT 1 protein content and hexokinase (HK) activity in thyroid cells. Our results show that AMPK activation significantly upregulates GLUT 1 content and glucose uptake, and it also stimulates hexokinase activity, the first step of glycolysis.

Propylthiouracil increases sodium/iodide symporter gene expression and iodide uptake in rat thyroid cells in the absence of TSH

Propylthiouracil increases sodium/iodide symporter gene expression and iodide uptake in rat thyroid cells in the absence of TSH

Triiodothyronine [T3]-induced hypothyroxinemia: Response and survival in a compassionate care cancer patient population.

e19573 Background: Increased survival under chemically induced hypothyroidism has been reported in cancer patients with poor prognosis, but the associated morbidity can deter patient compliance. Acting via a receptor site upon the plasma membrane avβ3 integrin, L-thyroxine (T4) in physiological concentrations non-genomically activates mitogenesis and neo-angiogenesis of cancer cells and proliferating vascular endothelium. Occlusion of the receptor by tetraiodothyroacetic acid (tetrac), a T4 derivative, induces apoptosis, inhibits angiogenesis and enhances chemotherapy and radiation. 3, 5, 3’-Triiodo-L-thyronine (T3) in physiological concentrations is significantly less mitogenic than T4 and inhibits pituitary release of thyrotropin (TSH). Absence of TSH, but with T3 support, depletes circulating T4 without resultant hypothyroidism. Methods: 21 cancer patients at dispersed clinical sites with stage 4 solid tumors and projected limited survival received T3 [n=10] only or with methimazole (MT) [n=9]. Serum TSH, free T4 (FT4) and T3 concentrations were obtained initially, then monthly. FT4 levels below lower normal range were achieved in all patients within 3 to 12 weeks. Results: See Table. Conclusions: T3 treatment with resultant hypothyroxinemia in poor prognosis cancer patients was associated with favorable response rates in 17/19 subjects. Two others with aggressive tumors had extended survival on long-term T3. [Table: see text] [Table: see text] [Table: see text]

Role of thyroglobulin on negative feedback autoregulation of thyroid follicular function and growth

Thyroid function is tightly regulated by TSH. Although individual follicles are exposed to the same blood supply of TSH and express relatively homogenous levels of the TSH receptor, the function of individual follicles is variable. It was shown that thyroglobulin (Tg), stored in the follicular lumen, is a potent negative feedback regulator of follicular function. Thus, physiological concentrations of Tg significantly suppress thyroid-specific gene expression and antagonize the TSH-mediated stimulation that induces expression of thyroid-specific genes. Tg coordinately regulates both basal and apical iodide transporters in thyroid follicular cells. Recently, it was also reported that Tg could induce thyroid cell growth in the absence of TSH. These results indicate that Tg is an essential autocrine regulator of physiological thyroid follicular function that counteracts the effects of TSH.

Resveratrol Increases Iodide Trapping in the Rat Thyroid Cell Line FRTL-5

Resveratrol, a polyphenol found in grapes, exhibits several beneficial health effects by its antioxidant, antiinflammatory, and chemopreventive properties. The aim of the present study was to determine the effect of resveratrol on iodide trapping and efflux as well as its mode of action using FRTL-5 cells, having in mind the pivotal role of the natrium iodide symporter (NIS) in the treatment of differentiated thyroid cancers. Cells were treated with resveratrol for various times and doses, in the presence or absence of thyrotropin (TSH). Iodide trapping, iodide efflux, rat NIS (rNIS) protein expression, and cyclic AMP (cAMP) production were evaluated. Resveratrol increased iodide trapping in a time-dependent (optimal 6 hours) and dose-dependent (100 microM) way in the presence of TSH. It showed an additive effect when concomitantly added with an optimal dose of TSH. Resveratrol (50 microM) increased (threefold) rNIS protein expression. In TSH-deprived cells, resveratrol also provoked an increase in rNIS protein (>3-fold in 6 hours) with an optimum at 40 microM. Resveratrol did not inhibit iodide efflux from FRTL-5 cells. It neither increased intracellular cAMP nor induced the arborization of living cells, two TSH-induced effects. A non-cAMP mode of action is highly suspected. Resveratrol increases iodide trapping in FRTL-5 cells, increasing iodide influx and rNIS protein level even in the absence of TSH. It has an additive effect with TSH. Consequently, resveratrol could be a promising molecule for radioiodide therapy in follicular and papillary differentiated thyroid carcinoma in association with recombinant human TSH.

Uptake of amiodarone by thyroidal and non-thyroidal cell lines

Amiodarone perturbs thyroid function, causing overt hypothyroidism or hyperthyroidism in 15% of patients. Changes in thyroid function are likely due, at least in part, to amiodarone and/or desethylamiodarone (DEA) concentration into the thyroid gland, but mechanisms whereby the drug uptake occurred are not known. Thyroidal (FRTL-5) or non-thyroidal [Chinese hamster ovary wild-type (CHOwt) or CHO stably transfected with NIS (CHO-NIS)] cells were exposed to 10 microM amiodarone or DEA. Cellular content of both drugs was measured by HPLC and normalized by protein concentration. Cellular concentration of the two drugs was higher in FRTL-5 (mean +/- SD 17.2 +/- 1.2 microg/mg protein of amiodarone and 18.9 +/- 0.7 microg/mg protein of DEA) than in CHO-NIS and CHOwt cells (10.8 +/- 0.8 microg/mg protein and 12.8 +/- 0.2 microg/mg protein, respectively, of amiodarone (p < 0.004); 11.9 +/- 0.1 microg/mg protein and 11 +/- 0.2 microg/mg protein, respectively, of DEA (p < 0.0002). DEA concentration was higher than that of amiodarone in all cell lines (p < 0.002). Differences between FRTL-5 and CHO cell lines were not dependent on TSH: in fact, cellular content of either drug did not change in the presence or absence of TSH in the culture medium. NIS did not intervene in amiodarone or DEA entry into thyroid cells, since amiodarone and DEA content was the same in CHOwt and CHO-NIS cells; in addition, KClO4 inhibited NIS function, but had no effect on drug uptake by the cells. At variance, 80 microM DEA reduced 125I uptake by CHO-NIS cells by 40% at 30 min without affecting cell viability. In conclusion, mechanisms whereby amiodarone is taken up by thyroid cells remain largely unknown, but the two main factors involved in thyroid-specific cellular transport, ie, NIS and TSH, seem to be excluded.

Requirement of Thyrotropin-Dependent Complex Formation of Protein Kinase A Catalytic Subunit with Inhibitor of κB Proteins for Activation of p65 Nuclear Factor-κB by Tumor Necrosis Factor-α

We previously demonstrated that TNF-alpha-dependent activation of p65 nuclear factor kappaB in rat thyroid FRTL-5 cells requires TSH. In the present study, we investigated the mechanism of this TSH action. Western blot analysis revealed that, in both the presence and absence of TSH, degradation of a cytosolic kappaB inhibitor (IkappaBalpha) occurred in response to TNF-alpha, resulting in nuclear translocation of p65 in both conditions. However, no DNA binding of p65 was detected in the absence of TSH, suggesting that posttranslational modification of p65 by TSH is required for its binding. Treatment of the cells cultured in the presence of TSH with a protein kinase A (PKA) inhibitor, H89, markedly reduced p65 binding and its transcriptional activity. However, transient block of TSH/cAMP-dependent activation of PKA catalytic subunit (PKAc) by adenylate cyclase inhibitor, SQ22536, had no effects on the p65 activation. Interestingly, it was found that PKAc formed a complex with IkappaBalpha and beta only in the presence of TSH, and this PKAc could be activated by TNF-alpha. TNF-alpha-dependent p65 activation was temporally associated with PKAc/IkappaBalpha complex formation. More than 3 h exposure of TSH was required for the complex formation and p65 activation. These results demonstrate that TSH induces the formation of PKAc/IkappaB complex in FRTL-5 cells and that this PKAc bound with IkappaB plays a critical role in TNF-alpha-dependent activation of p65.

Conditional expression of RET/PTC induces a weak oncogenic drive in thyroid PCCL3 cells and inhibits thyrotropin action at multiple levels.

Chromosomal rearrangements linking the promoter(s) and N-terminal domain of unrelated gene(s) to the C terminus of RET result in constitutively activated chimeric forms of the receptor in thyroid cells (RET/PTC). RET/PTC rearrangements are thought to be tumor-initiating events; however, the early biological consequences of RET/PTC activation are unknown. To explore this, we generated clonal lines derived from well-differentiated rat thyroid PCCL3 cells with doxycycline-inducible expression of either RET/PTC1 or RET/PTC3. As previously shown in other cell types, RET/PTC1 and RET/PTC3 oligomerized and displayed constitutive tyrosine kinase activity. Neither RET/PTC1 nor RET/PTC3 conferred cells with the ability to grow in the absence of TSH, likely because of concomitant stimulation of both DNA synthesis and apoptosis, resulting in no net growth in the cell population. Effects of RET/PTC on DNA synthesis and apoptosis did not require direct interaction of the oncoprotein with either Shc or phospholipase Cgamma. Acute expression of the oncoprotein decreased TSH-mediated growth stimulation due to interference of TSH signaling by RET/PTC at multiple levels. Taken together, these data indicate that RET/PTC is a weak tumor-initiating event and that TSH action is disrupted by this oncoprotein at several points, and also predict that secondary genetic or epigenetic changes are required for clonal expansion.

Effects of mutations involving the highly conserved S281HCC motif in the extracellular domain of the thyrotropin (TSH) receptor on TSH binding and constitutive activity.

A model has been proposed in which, in the absence of TSH, the extracellular domain of the TSH receptor would exert a silencing effect on the serpentine domain involved in activation of the G(alpha)(s) protein. Mutation of S281 in the ectodomain is supposed to release this constraint, thereby causing receptor activation. This defines S281 and its neighbors as a segment important in intramolecular signal transduction. The functional importance of this segment was explored by site-directed mutagenesis experiments involving S281, as well as the two cysteine residues (C283, C284) present immediately downstream. S281 was mutated to N, T, G, and A in this study, and the functional characteristics of the mutants were compared. We found that S281N, S281T, and S281G display stronger constitutive activity than S281A mutant, suggesting that increase in constitutive activity is related to the extent of disruption of the local structure of the ectodomain. C283 and C284, the two consecutive cysteines that are highly conserved in glycoprotein hormone receptors, were mutated to serine, either alone (S281HSC or S281HCS) or in combination (S281HSS) and were studied in two different TSH receptor backgrounds. The mutated cysteine ectodomains were either linked to a glycosylphosphatidylinositol anchor or the serpentine domain of the wild-type holoreceptor. Glycosylphosphatidylinositol-anchored ectodomain receptors showed good cell surface expression in CHO cells, but only S281HCS was able to bind TSH specifically, illustrating the importance of C283, or the putative disulphide bond, in maintaining the conformation of the ligand binding site. In contrast, cysteine mutants on an extracellular domain-holoreceptor background displayed severely impaired membrane targeting and were poorly expressed in COS cells. However, basal cAMP production, normalized to expression at the plasma membrane, indicated significant increase in constitutive activity of all three mutants, compared with the wild-type receptor. Altogether, these findings support a model in which the ectodomain would act as a silencer of the basal activity of the serpentine portion of the receptor.

Pax-8 is essential for regulation of the thyroglobulin gene by transforming growth factor-beta1.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that is thought to play a major role in the regulation of growth and differentiation of thyroid cells. However, little is known of its detailed mechanisms of action in thyrocytes. We have therefore studied the molecular mechanisms of TGF-beta1 action on thyroglobulin (TG) gene expression by focusing our attention on TGF-beta1 regulation of thyroid-specific transcription factors. TGF-beta1 decreased TG messenger RNA (mRNA) expression both in the presence and in the absence of TSH in rat thyroid FRTL-5 cells. Transfected into FRTL-5 cells, the activity of reporter plasmids containing the rat TG promoter ligated to a luciferase gene was significantly suppressed by the addition of TGF-beta1. When the nuclear extracts prepared from TGF-beta1-treated FRTL-5 cells were used in gel mobility shift assays, the amount of protein-DNA complex formed by Pax-8 was reduced, both in the presence and in the absence of TSH, but protein-DNA complexes formed by thyroid transcription factor-1 (TTF-1) and TTF-2 were not. The suppressive effect of TGF-beta1 on Pax-8/DNA complex formation is in part due to the suppression of Pax-8 mRNA and protein levels by TGF-beta1. Expressions of Pax-8 mRNA and protein, which were assessed by Northern blot and Western blot analyses, respectively, were decreased by TGF-beta1 treatment of FRTL-5 cells in a concentration-dependent manner. In a transfection experiment, mutation of the Pax-8-binding site caused a loss of both TGF-beta1- and TSH-responsiveness in TG promoter activity. Overexpression of Pax-8 abolished the TGF-beta1 suppression of TG promoter activity. These results indicate that TGF-beta1 decreases Pax-8 mRNA levels as well as Pax-8 DNA-binding activity, which, at least in part, seems to be involved in the TGF-beta1-induced suppression of TG gene expression.

Regulation of thyroid cell volumes and fluid transport: opposite effects of TSH and iodide on cultured cells.

Cell volume regulation by thyrotropin (TSH) and iodide, the main effectors involved in thyroid function, was studied in cultured thyroid cells. The mean cell volume, determined by performing 3-D reconstitution on confocal microscopy optical slices from living octadecylrhodamine-labeled cells cultured with both TSH and iodide (control cells), was 3.73 +/- 0.06 pl. The absence of iodide resulted in cell hypertrophy (136% of control value) and the absence of TSH in cell shrinkage (81%). These changes mainly affected the cell heights. The effect of TSH on cell volume was mediated by cAMP. The proportion of cytosolic volume (3-O-methyl-D-glucose space vs. total volume) decreased in the absence of iodide (85% of control value) and increased in the absence of TSH (139%), whereas protein content showed the opposite changes (121 and 58%, respectively). The net apical-to-basal fluid transport was also inversely controlled by the two effectors. Iodide thus antagonizes TSH effects on cell volumes and fluid transport, probably via adenylylcyclase downregulation mechanisms. The absence of either iodide or TSH may mimic the imbalance occurring in pathological thyroids.

Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture.

The proliferation of most normal cells depends on the synergistic interaction of several growth factors and hormones, but the cell cycle basis for this combined requirement remains largely uncharacterized. We have addressed the question of the requirement for insulin/IGF-1 also observed in many cell culture systems in the physiologically relevant system of primary cultures of dog thyroid epithelial cells stimulated by TSH, which exerts its mitogenic activity only via cAMP. The induction of cyclin A and cdc2, the phosphorylation of cdk2, the nuclear translocation of cdk4 and the assembly of cyclin D3-cdk4 complexes required the synergy of TSH and insulin. Cyclin D3 (the most abundant cyclin D) was necessary for the proliferation stimulated by TSH in the presence of insulin as shown by microinjection of a neutralizing antibody. Cyclin D3 accumulation and activity were differentially regulated by insulin and TSH, which points out this cyclin as an integrator that ranks these comitogenic pathways as supportive and activatory, respectively. Paradoxically TSH alone strongly repressed cyclin D3 accumulation. This inhibition was overridden by insulin, which markedly stimulated cyclin D3 mRNA and protein accumulation, but failed to assemble cyclin D3-cdk4 complexes in the absence of TSH. TSH unmasked the DCS-22 epitope of cyclin D3 and assembled cyclin D3-cdk4 in the presence of insulin. These data demonstrate that cyclin D synthesis and cyclin D-cdk assembly can be dissociated and complementarily regulated by different agents and signalling pathways.

Thyrotropin prevents apoptosis by promoting cell adhesion and cell cycle progression in FRTL-5 cells.

Apoptosis has been shown to be involved in endocrine tissue homeostasis as well as regression due to hormone deprivation. The goal of this study was to induce apoptosis and to investigate a potential role of TSH as a survival factor in thyroid follicular cells (FRTL-5) in vitro. Our results indicated that FRTL-5 cells underwent anchorage-dependent apoptosis when plated in the absence of serum and hormones, but when the cells became attached to the substrate by addition of TSH in the medium, apoptosis was prevented. The apoptosis was evaluated by positive terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling staining, typical apoptotic bodies by electron microscopy, DNA ladder by gel electrophoresis, and subdiploidy by propidium iodide-stained flow cytometry. TSH was shown to prevent apoptosis and maintain cell viability. cAMP partly mimicked this effect, which was inhibited by a specific inhibitor of protein kinase A, H-89. While investigating the mechanisms of apoptosis, we observed that the phosphorylated focal adhesion kinase was strengthened by TSH. Furthermore, FRTL-5 cells were found to undergo growth arrest in the G1 phase in the absence of TSH, accompanied by an elevated level of cyclin-dependent kinase inhibitor, p27, and a decreased level of cyclin D. In contrast, TSH promoted transition from G1 to S phase by decreasing P27 protein and increasing cyclin D expression. We concluded that in addition to regulating growth and differentiation, TSH may function as a survival factor in thyroid cells by preventing anchorage-dependent apoptosis in FRTL-5 cells partly via the cAMP pathway.

Influence of Cortisol on the Larval Bullfrog Thyroid Axis in Vitro and in Vivo and on Plasma and Ocular Melatonin

Adrenal (interrenal) steroids have an important role in amphibian development, antagonizing the metamorphic changes induced by the thyroid at first and then synergizing with the thyroid hormones as their level rises during metamorphosis. Because most of the studies of corticoids at metamorphosis have focused on peripheral tissues, we investigated the effect of cortisol (hydrocortisone; HC) in vitro and in vivo on the thyroid of Rana catesbeiana (bullfrog) tadpoles on 12:12 light/dark (LD) cycles. Plasma and ocular melatonin, which is altered by changes in thyroxine (T4) levels, were also assayed in some experiments. Thyroids from premetamorphic tadpoles secreted less T4 into culture media when incubated with 10 μg/ml HC and 0.2 μg/ml ovine thyrotropin (TSH) than with TSH alone and when cultured in the absence of TSH following 5 days of 10-μg HC injections, indicating that HC inhibited the thyroid at young stages. The effect of 10 μg/ml HC at older stages was investigated by culturing thyroids and pituitaries separately on the first day in control or HC media and then incubating the thyroids on the second day in homologous pituitary-conditioned media as a bioassay for pituitary TSH. HC had no effect on baseline T4 secretion by the thyroids of prometamorphic or climax tadpoles on the first day but increased T4 secretion over the control on the second day. Thyroids cultured with TSH and HC showed no increase in T4 secretion over the control TSH group on the second day, indicating that, in the previous experiments, HC had enhanced pituitary secretion of TSH, rather than the response of the thyroid to TSH. In vivo, 5 days of injections of 10 μg HC increased plasma T4 at prometamorphosis and decreased it at climax. There was no marked effect of HC on plasma or ocular melatonin levels. The findings showed that the nature of the effect of HC on the thyroid axis changes during metamorphosis from inhibition at early stages to a positive influence at prometamorphosis and finally to a negative effect on the T4 level in the plasma at climax.

The C-terminal domain of armadillo binds to hypophosphorylated teashirt to modulate wingless signalling in Drosophila.

Wnt signalling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts to stabilize Armadillo inside cells where it binds to at least two DNA-binding factors which regulate specific target genes. One Armadillo-binding protein in Drosophila is the zinc finger protein Teashirt. Here we show that Wingless signalling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Finally, we present evidence that the serine/threonine kinase Shaggy is associated with Teashirt in a complex. We discuss these results with respect to current models of Armadillo/beta-catenin action for the transmission of the Wingless/Wnt pathway.