Action of Reverse T3 on Cancer Cells.

Background: Reverse T3 (rT3; 3,3',5'-triiodo-L-thyronine) is widely regarded as an inactive naturally occurring analog of thyroid hormone. rT3 is known to bind to the thyroid hormone analog receptor on plasma membrane integrin αvß3. This integrin is generously expressed by tumor cells and is the initiation site for the stimulation by L-thyroxine (T4) at physiological free concentrations on cancer cell proliferation. Results: In the present studies, we show that rT3 caused increases of proliferation in vitro of 50% to 80% (P < 0.05-0.001) of human breast cancer and glioblastoma cells. Conclusion: rT3 may be a host factor supporting cancer growth.

Reverse T3 interacts with αvß3 integrin receptor and restores enzyme activities in the hippocampus of hypothyroid developing rats: Insight on signaling mechanisms.

In the present study we provide evidence that 3,3',5'-triiodothyronine (reverse T3, rT3) restores neurochemical parameters induced by congenital hypothyroidism in rat hippocampus. Congenital hypothyroidism was induced by adding 0.05% propylthiouracil in the drinking water from gestation day 8 and continually up to lactation day 15. In the in vivo rT3 exposure, hypothyroid 12-day old pups were daily injected with rT3 (50 ng/kg body weight) or saline until day 14. In the ex vivo rT3 treatment, hippocampal slices from 15-day-old hypothyroid pups were incubated for 30 min with or without rT3 (1 nM). We found that ex vivo and/or in vivo exposure to rT3 failed in restoring the decreased 14C-glutamate uptake; however, restored the phosphorylation of glial fibrillary acidic protein (GFAP), 45Ca2+ influx, aspartate transaminase (AST), glutamine synthetase (GS) and gamma-glutamate transferase (GGT) activities, as well as glutathione (GSH) levels in hypothyroid hippocampus. In addition, rT3 improved 14C-2-deoxy-D-glucose uptake and lactate dehydrogenase (LDH) activity. Receptor agonists/antagonists (RGD peptide and AP-5), kinase inhibitors of p38MAPK, ERK1/2, CaMKII, PKA (SB239063, PD98059, KN93 and H89, respectively), L-type voltage-dependent calcium channel blocker (nifedipine) and intracellular calcium chelator (BAPTA-AM) were used to determine the mechanisms of the nongenomic rT3 action on GGT activity. Using molecular docking analysis, we found rT3 interaction with αvß3 integrin receptors, nongenomically activating signaling pathways (PKA, CaMKII, p38MAPK) that restored GGT activity. We provide evidence that rT3 is an active TH metabolite and our results represent an important contribution to elucidate the nonclassical mechanism of action of this metabolite in hypothyroidism.

In vitro transdifferentiation of human cultured CD34+ stem cells into oligodendrocyte precursors using thyroid hormones.

The extent of myelination on the axon promotes transmission of impulses in the neural network, any disturbances in this process results in the neurodegenerative condition. Transplantation of oligodendrocyte precursors that supports in the regeneration of axons through myelination is an important step in the restoration of damaged neurons. Therefore, in the present study, the differentiation of human CD34+ stem cells into oligodendrocytes was carried out. The pure human CD34+ culture developed from the stem cells obtained from a peripheral blood of a donor were subjected to oligodendrocyte differentiation medium (ODM). The ODM at a concentration of 40ng/ml thyroxine, 40ng/ml 3,3',5-tri-iodo-thyronine showed distinct morphological changes from day 6 to 9 with cells exhibiting conspicuous stellate morphology and extensive foot processes. The real-time PCR analysis showed prominent expression of Olig2, CNPase, PDGFRα and PLP1/DM20 in the differentiated cells confirming the formed cells are oligodendrocyte precursors. The expression of these genes increased from days 6 to 9 corresponding to the morphological changes observed with almost no expression of GFAP+ cells. The distinct CNPase activity was observed in these differentiated cells compared to normal CD34+ stem cells correlating with results of real-time PCR conclusively explains the development of oligodendrocytes from human CD34+ stem cells.

Rapid responses to reverse T3 hormone in immature rat Sertoli cells: calcium uptake and exocytosis mediated by integrin.

There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T3 (rT3) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT3 are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT3 was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT3-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT3 may be mediated by integrin αvß3. In addition, it was demonstrated that calcium uptake stimulated by rT3 is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT3 also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT3 modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology.

3,5,3'-triiodothyronine (T3) stimulates cell proliferation through the activation of the PI3K/Akt pathway and reactive oxygen species (ROS) production in chick embryo hepatocytes.

Thyroid hormones (THs) have a wide variety of essential roles in vertebrates, ranging from the regulation of key metabolic processes to cell proliferation and apoptosis. The classical mechanism of action of THs is genomic; 3,5,3'-triiodothyronine (T3) binds to specific nuclear receptors (TRs) and modifies the expression of specific genes. Recently, a new category of mechanisms, termed nongenomic, has been discovered for T3. These mechanisms include, among others, the rapid activation of signal transduction pathways, such as PI3K/Akt and MAPK, which eventually lead to cell proliferation. These effects are mediated in some cell types by a plasma membrane receptor, identified as integrin αvß3, and in other cell types by cytoplasmic TRß1. The aim of this work was to analyze the effect of T3 on the cell growth of chick embryo hepatocytes at two different stages of development, 14 and 19 days, and to determine the activation of the signal transduction pathways, focusing on the potential involvement of a plasma membrane receptor and the possible participation of PI3K/Akt and reactive oxygen species (ROS). Our results clearly show that T3 stimulates cell proliferation at both stages of development through the activation of the PI3K/Akt pathway and the production of small amounts of ROS, which operate as effective second messengers. Moreover, we prove that these effects are not initiated at the plasma membrane receptor for T3.

3, 3'5 Triiodo L thyronine induces apoptosis in human breast cancer MCF-7 cells, repressing SMP30 expression through negative thyroid response elements.

BACKGROUND: Thyroid hormones regulate cell proliferation, differentiation as well as apoptosis. However molecular mechanism underlying apoptosis as a result of thyroid hormone signaling is poorly understood. The antiapoptotic role of Senescence Marker Protein-30 (SMP30) has been characterized in response to varieties of stimuli as well as in knock out model. Our earlier data suggest that thyroid hormone 3, 3'5 Triiodo L Thyronine (T(3)), represses SMP30 in rat liver. METHODOLOGY/PRINCIPAL FINDINGS: In highly metastatic MCF-7, human breast cancer cell line T3 treatment repressed SMP30 expression leading to enhanced apoptosis. Analysis by flow cytometry and other techniques revealed that overexpression and silencing of SMP30 in MCF-7 resulted in decelerated and accelerated apoptosis respectively. In order to identify the cis-acting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected hSMP30 promoter deletion reporter vectors in MCF-7 cells. As opposed to the expected epigenetic outcome, thyroid hormone down regulated hSMP30 promoter activity despite enhanced recruitment of acetylated H3 on thyroid response elements (TREs). From the stand point of established epigenetic concept we have categorised these two TREs as negative response elements. Our attempt of siRNA mediated silencing of TRß, reduced the fold of repression of SMP30 gene expression. In presence of thyroid hormone, Trichostatin- A (TSA), which is a Histone deacetylase (HDAC) inhibitor further inhibited SMP30 promoter activity. The above findings are in support of categorisation of both the thyroid response element as negative response elements as usually TSA should have reversed the repressions. CONCLUSION: This is the first report of novel mechanistic insights into the remarkable downregulation of SMP30 gene expression by thyroid hormone which in turn induces apoptosis in MCF-7 human breast cancer cells. We believe that our study represents a good ground for future effort to develop new therapeutic approaches to challenge the progression of breast cancer.

Non-receptor-mediated actions are responsible for the lipid-lowering effects of iodothyronines in FaO rat hepatoma cells.

Iodothyronines influence lipid metabolism and energy homeostasis. Previous studies demonstrated that 3,5-l-diiodothyronine (T(2)), as well as 3,3',5-L-triiodothyronine (T(3)), was able to both prevent and reverse hepatic steatosis in rats fed a high-fat diet, and this effect depends on a direct action of iodothyronines on the hepatocyte. However, the involvement of thyroid hormone receptors (TRs) in mediating the lipid-lowering effect of iodothyronines was not elucidated. In this study, we investigated the ability of T(2) and T(3) to reduce the lipid overloading using the rat hepatoma FaO cells defective for functional TRs. The absence of constitutive mRNA expression of both TRα1 and TRß1 in FaO cells was verified by RT-qPCR. To mimic the fatty liver condition, FaO cells were treated with a fatty acid mixture and then exposed to pharmacological doses of T(2) or T(3) for 24 h. Lipid accumulation, mRNA expression of the peroxisome proliferator-activated receptors (PPAR-α, -γ, -δ) the acyl-CoA oxidase (AOX), and the stearoyl CoA desaturase (SCD1), as well as fuel-stimulated O(2) consumption in intact cells, were evaluated. Lipid accumulation was associated with an increase in triacylglycerol content, PPARγ mRNA expression, and a decrease in PPARδ and SCD1 mRNA expression. The addition of T(2) or T(3) to lipid-overloaded cells resulted in i) reduction in lipid content; ii) downregulation of PPARα, PPARγ, and AOX expression; iii) increase in PPARδ expression; and iv) stimulation of mitochondrial uncoupling. These data demonstrate, for the first time, that in the hepatocyte, the lipid-lowering actions of both T(2) and T(3) are not mediated by TRs.

Hypothyroidism in rats decreases peripheral glucose utilisation, a defect partially corrected by central leptin infusion.

AIMS/HYPOTHESIS: The aims of this work were to determine the effect of hypothyroidism on insulin-stimulated glucose turnover and to unravel the potential mechanisms involved in such an effect. METHODS: Hypothyroidism was induced by administration of propylthiouracil, with partial T4 substitution. Euglycaemic-hyperinsulinaemic clamps, associated with the labelled 2-deoxy-D-glucose technique for measuring tissue-specific glucose utilisation, were used. To assess a possible involvement of leptin in the modulation of glucose metabolism by hypothyroidism, leptin was infused intracerebroventricularly for 6 days. A group of leptin-infused rats was treated with rT3 to determine a potential role of T3 in mediating the leptin effects. RESULTS: Compared with euthyroid rats, hypothyroid animals exhibited decreased overall glucose turnover and decreased glucose utilisation indices in skeletal muscle and adipose tissue. Leptinaemia in hypothyroid rats was lower while resistin mRNA expression in adipose tissue was higher than in euthyroid animals. Intracerebroventricular leptin infusion in hypothyroid rats partially restored overall, muscle and adipose tissue insulin-stimulated glucose utilisation and improved the reduced glycaemic response observed during insulin tolerance tests. The leptin effects were due neither to the observed increase in plasma T3 levels nor to changes in the high adipose tissue resistin expression of hypothyroid rats. The administration of leptin to hypothyroid animals was accompanied by increased expression of muscle and adipose tissue carnitine palmitoyl transferases, decreased plasma NEFA levels and reduced muscle triglyceride content. CONCLUSIONS/INTERPRETATION: Hypothyroidism is characterised by decreased insulin responsiveness, partly mediated by an exaggerated glucose-fatty acid cycle that is partly alleviated by intracerebroventricular leptin administration.

Effects of acute lindane intoxication and thyroid hormone administration in relation to nuclear factor-kappaB activation, tumor necrosis factor-alpha expression, and Kupffer cell function in the rat.

Nuclear factor-kappaB (NF-kappaB) DNA binding, tumor necrosis factor-alpha (TNF-alpha) expression, and parameters related to liver oxidative stress and Kupffer cell function were assessed in control rats and in animals given 3,3',5-triiodothyronine (T3) (0.1 mg T3/kg) and/or lindane (50 mg/kg; 4 h after T3). Liver NF-kappaB DNA binding and serum TNF-alpha levels were enhanced by the combined T3-lindane administration after 16-22 h, effects that were lower than those elicited by the separate treatments and coincided with increased hepatic TNF-alpha mRNA levels. Thyroid calorigenesis occurred independently of lindane, whereas T3, lindane and T3-lindane groups showed liver glutathione (GSH) depletion, with higher protein carbonyl levels in lindane and T3-lindane groups. Carbon-induced O2 consumption/carbon uptake ratios were not altered by T3 or lindane compared to controls, whereas combined T3-lindane administration elicited a 92% diminution with enhancement in the sinusoidal efflux of lactate dehydrogenase (LDH). In conclusion, depression of T3- or lindane-induced liver NF-kappaB activation and TNF-alpha expression occurred after their combined treatment, effects that correlate with the impairment of the respiratory burst activity of Kupffer cells and exacerbation of liver injury.

3,3',5'-triiodo-L-thyronine reduces efficiency of mRNA knockdown by antisense oligodeoxynucleotides: a study with pyroglutamyl aminopeptidase II in adenohypophysis.

The impact of hormones on the efficacy of antisense oligodeoxynucleotides (ASOs) is a poorly analyzed subject. We designed, based on the identification of potentially favorable local elements of mRNA secondary structure, eight phosphorothioate ASOs to knock down the expression of an ectopeptidase, pyroglutamyl aminopeptidase II (PPII), in primary cultures of adenohypophysis. Two of the PPII ASOs were very efficient, sequence-specific, and target-specific. Because the expression of PPII is upregulated by 3,3',5'-triiodo-L-thyronine (T3), we studied the impact of varying the protocol of PPII induction on the knockdown efficacy. Hormone removal at transfection increased markedly the ability of (1) PPII ASOs to reduce PPII mRNA levels or PPII activity in adenohypophyseal cells or in C6 rat glioma cells and (2) a thyrotropin-releasing hormone (TRH) receptor-1 (TRH-R1) ASO to reduce TRH-R1 mRNA levels in adenohypophyseal cells. There was no effect of hormone removal on transfection efficacy and no correlation between target mRNA levels and ASO efficacy. These data demonstrated that ASO efficacy could depend on T3 levels; this might be due to regulation of a step generally critical for ASO efficiency.

Effects of thyroid hormone on food intake, hypothalamic Na/K ATPase activity and ATP content.

The effects of thyroid hormone on whole body energy metabolism and compensatory effects on food intake are well established. However, the hypothalamic mechanisms that translate perceived whole body energy demands into subsequent appetitive behavior are incompletely understood. In order to address this question, we tested the effects of T3 on food intake and body weight in rats and measured neuronal Na/K ATPase activity and ATP content in the hypothalamus. Intraperitoneal T3 (100 microg/kg BW) administered for 6 consecutive days increased 24-h rat food intake from control, 26.6+/-1.2, to T3-treated 33.2+/-1.6 g (P<0.01). In T3-treated rats, rubidium-86 (86Rb) uptake (measured as a marker of Na/K ATPase activity) in ex vivo hypothalamic tissue increased (P<0.01) while the content of ATP in the ventral hypothalamus declined following T3 treatment (P<0.01). In another model of energy deficit, which was induced by a very low calorie diet, ATP content was also reduced in the hypothalamus compared to rats fed ad libitum. In summary, increased food intake in response to T3 may be secondary to decreased hypothalamic ATP content, perhaps resulting from both increased Na/K ATPase activity in the hypothalamus and metabolic signaling induced by whole body caloric deficit.

Effects of ligand and thyroid hormone receptor isoforms on hepatic gene expression profiles of thyroid hormone receptor knockout mice.

Little is known about the overall patterns of thyroid hormone (Th)-mediated gene regulation by the main Th receptor (Tr) isoforms, Tr-alpha and Tr-beta, in vivo. We used 48 complementary DNA microarrays to examine hepatic gene expression profiles of wild-type and Thra and Thrb knockout mice under different Th conditions: no treatment, treatment with 3,3',5-triiodothyronine (T(3)), Th-deprivation using propylthiouracil (PTU), and treatment with a combination of PTU and T(3). Hierarchical clustering analyses showed that positively regulated genes fit into three main expression patterns. In addition, only a subpopulation of target genes repressed basal transcription in the absence of ligand. Interestingly, Thra and Thrb knockout mice showed similar gene expression patterns to wild-type mice, suggesting that these isoforms co-regulate most hepatic target genes. Differences in the gene expression patterns of Thra/Thrb double-knockout mice and Th-deprived wild-type mice show that absence of receptor and of hormone can have different effects. This large-scale study of hormonal regulation reveals the functions of Th and of Tr isoforms in the regulation of gene expression patterns.

Myosin V plays an essential role in the thyroid hormone-dependent endocytosis of type II iodothyronine 5'-deiodinase.

In astrocytes, thyroxine modulates type II iodothyronine 5'-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormone-dependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC(50) levels approximately 1 nm) were >100-fold more potent than 3,5,3'-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH(2)-terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxine-dependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormone-dependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.

Substrate-induced down-regulation of human type 2 deiodinase (hD2) is mediated through proteasomal degradation and requires interaction with the enzyme's active center.

Type 2 iodothyronine deiodinase (D2) catalyzes the first step in thyroid hormone action, the deiodination of T4 to T3. Endogenous D2 activity is posttranslationally regulated by substrate that accelerates its degradation through the ubiquitin-proteasome pathway. To understand how D2 activity correlates with D2 protein during its normal decay and rT3-induced down-regulation, HEK-293 cells, transiently expressing human D2, were labeled with Na75SeO3 and then treated with 100 microM cycloheximide (CX), 30 nM rT3, and/or 10 microM MG132, a specific proteasome inhibitor, for 2-4 h. D2 protein and enzyme activity changed in parallel, disappearing with a half-life of 2 h in the presence of CX, or 1 h when CX + rT3 were combined. Treatment with MG132 blocked these effects. We created selenocysteine (Sec) 133 to cysteine (Cys) or alanine (Ala) D2 mutants, without changing Sec 266. The CysD2 activity and protein levels were also parallel, with a similar half-life of approximately 2 h, whereas the rT3-induced D2 down-regulation required approximately 1000-fold higher rT3 concentration (30 microM) due to a proportionally higher Michaelis constant of CysD2. In similar experiments, the AlaD2 mutant retained the short half-life but was not catalytically active and not susceptible to rT3-accelerated degradation. We conclude that substrate-induced loss of D2 activity is due to proteasomal degradation of the enzyme and requires interaction with the catalytic center of the protein.

Thyroid hormone acts centrally to programme photostimulated male american tree sparrows (Spizella arborea) for vernal and autumnal components of seasonality.

Thyroid hormone and long days interact to programme American tree sparrows (Spizella arborea) for seasonality (i.e. thyroid hormone-dependent photoperiodic gonadal growth, photorefractoriness, and postnuptial moult). This study explored in radiothyroidectomized (THX) males given thyroid hormone replacement therapy whether thyroid hormone acts within the brain and, additionally, the identity of the putative tissue-active thyroid hormone. The minimum dose (30 ng) of L-thyroxine (T4) that restored all components of seasonality when given i.c.v. daily during the first 21 days of photostimulation restored no component of seasonality when given s.c. The same dose of L-triiodothyronine (T3) also was ineffective when administered s.c., but restored photoperiodic testicular growth (though neither photorefractoriness nor postnuptial moult) when admiministered i.c.v. Three of seven birds given a 10-fold lower dose of T4 (3 ng) exhibited thyroid hormone-dependent photoperiodic testicular growth, albeit damped. The other four birds given 3 ng T4 and all birds given 3 ng T3 responded like THX controls, exhibiting only slight thyroid hormone-independent photoperiodic testicular growth. The highest dose (300 ng) of T3 restored all components of seasonality only when administered i.c.v. daily during the first 49 days of photostimulation. This demonstration in American tree sparrows is the first in any species that the thyroid-dependent transition from the breeding season to the non-breeding season can be effected by T3. The same dose of reverse T3 administered daily over the same 49 days restored photoperiodic testicular growth in only half of 10 subjects and photorefractoriness and moult in none. Collectively, the data support the hypothesis that thyroid hormone acts centrally to programme photostimulated male American tree sparrows for all components of seasonality. The most parsimonious interpretation of the data, including the threshold-like effect of 3 ng T4, favours T4 as the tissue-active thyroid hormone for vernal as well as autumnal events, but does not entirely exclude T3.

Altered response to thyroid hormones by breast and ovarian cancer cells.

BACKGROUND: In this study, L-thyroxine (T4), 3',3,5-triiodo-L-thyronine (T3), 3,5-diiodo-L-thyronine (T2), reverse T3; 3',5',3-triiodo-L-thyronine (RT3) and transferrin were added to breast cancer cell lines Hs 578T, MDA-MB-231, MDA-MB-468, and T-47D and ovarian cancer cell line OVCAR-3 to test the response to cell proliferation. MATERIALS AND METHODS: Breast and ovarian cancer cell lines were placed in serum-free medium prior to addition of effector. Proliferation was determined by thymidine incorporation. For Northern analysis, RNA was isolated and c-fos, cjun and TIEG expression assessed. RESULTS: No compound provided uniform results across all cell lines. T2 inhibited proliferation in Hs 578T and MDA-MB-468, had no effect in MDA-MB-231 and OVCAR-3, and stimulated proliferation in T-47D cells. T3 inhibited proliferation in all cell lines except T-47D in which two-state behavior occurred, with increased proliferation at low concentrations (< or = 10(-6) M) and decreased proliferation at high concentrations (> or = 10(-5) M). RT3 inhibited proliferation in Hs 578T, MDA-MB-231, and T-47D but had no effect in MDA-MB-468 and OVCAR-3. T4 inhibited proliferation in Hs 578T, MDA-MB-231, and MDA-MB-468 and had two-state behavior in T-47D and OVCAR-3. Finally, transferrin increased proliferation only in OVCAR-3 cells. Protooncogene expression was increased by both transferrin and T4 in the cell lines tested. CONCLUSIONS: Correlation of iodines and proliferative responses were used to determine "essential" iodines necessary to produce the observed effect. Interaction between these cancer cells and non-physiological concentrations of thyroid hormone can be explained by thyroid hormone receptors with altered binding properties. Thus, interaction of thyroid hormones and cancer cells may differ from what occurs with normal cells.

Type 2 iodothyronine deiodinase in rat pituitary tumor cells is inactivated in proteasomes.

The goal of these studies was to define the rate-limiting steps in the inactivation of type 2 iodothyronine deiodinase (D2). We examined the effects of ATP depletion, a lysosomal protease inhibitor, and an inhibitor of actin polymerization on D2 activity in the presence or absence of cycloheximide or 3,3', 5'-triiodothyronine (reverse T3, rT3) in rat pituitary tumor cells (GH4C1). We also analyzed the effects of the proteasomal proteolysis inhibitor carbobenzoxy- L-leucyl-L-leucyl-L-leucinal (MG132). The half-life of D2 activity in hypothyroid cells was 47 min after cycloheximide and 60 min with rT3 (3 nM). rT3 and cycloheximide were additive, reducing D2 half-life to 20 min. D2 degradation was partially inhibited by ATP depletion, but not by cytochalasin B or chloroquine. Incubation with MG132 alone increased D2 activity by 30-40% for several hours, and completely blocked the cycloheximide- or rT3-induced decrease in D2 activity. These results suggest that D2 is inactivated by proteasomal uptake and that substrate reduces D2 activity by accelerating degradation through this pathway. This is the first demonstration of a critical role for proteasomes in the post-translational regulation of D2 activity.

Studies of the hormonal regulation of type 2 5'-iodothyronine deiodinase messenger ribonucleic acid in pituitary tumor cells using semiquantitative reverse transcription-polymerase chain reaction.

We developed a sensitive competitive RT-PCR technique for quantitating the ratio of D2 to cyclophilin messenger RNA (mRNA) and used this to study type 2 deiodinase (D2) mRNA regulation. Hyperthyroidism in rats causes a 2- to 3-fold reduction in anterior pituitary and medial basal hypothalamus (MBH). Thyroid hormone (T3) withdrawal increased the D2/cyclophilin ratio 2- to 3-fold over 48 h in both GC and GH4C1 cells. T3 additional reduced D2 gene transcription by 50% over 2 h and about 30% over the next 2 h. D2 mRNA half-life is 2 h and is not affected by T3, indicating that its effect is due to suppression of D2 gene transcription. The T3 effect did not require new protein synthesis. Longer treatment with T3 led to a maximum decrease of 70% in D2 mRNA, indicating that there is also a T3-independent transcriptional component of the D2 gene. 3,3',5'-Triiodothyronine (reverse T3) caused a slight increase D2 mRNA over 24 h but an 80-90% decrease in D2 activity, indicating that it acts posttranscriptionally. Dexamethasone, 8 Br-cAMP, and TRH also caused modest increases in D2 mRNA in pituitary tumor cells. We conclude that D2 gene transcription has both T3-dependent and T3-independent components. Thus, posttranscriptional effects of D2 substrates such as T4 will be required for complete feedback inhibition of D2 activity. The short half-life of D2 mRNA and D2 protein explains the rapid response of D2 activity to thyroid hormone administration.

Adverse effects of reverse triiodothyronine on cellular metabolism as assessed by 1H and 31P NMR spectroscopy.

Effects of 3,3',5'-triiodothyronine (rT3) in connection with 3,3',5-triiodothyronine (T3) on 3T3 cells were studied in vitro by means of 1H and 31P NMR spectroscopy. In the cells incubated with 5 nM T3 for 3 h at pH 7.4, the ATP/ADP ratio was elevated from 6.9 to 8.4, whereas it was reduced to 6.1 in cells incubated with rT3. When the cells were incubated at pH 6.7, the ATP/ADP ratio was reduced to 6.6 and 5.2 at 1 and 2 h, respectively. In the presence of 5 nM of T3, however, the ratio was maintained above the control level. A 1-h preincubation with rT3 dramatically augmented the reductions caused by elevated acidity. These reductions were completely reversed when the cells were incubated with T3.

The direct effects of 3,5,3'-triiodo-L-thyronine (T3) on myocyte contractile processes. Insights into mechanisms of action.

Administration of 3,5,3'-triiodo-L-thyronine (T3) has recently been suggested to acutely improve left ventricular performance. However, the cellular and molecular mechanisms responsible for this improvement in left ventricular function with T3 remained unknown. Accordingly, the present study examined the direct effects of T3 administration on myocyte contractile function and the sarcolemmal systems that might potentially contribute to these effects. In isolated porcine left ventricular myocytes (n = 81), velocity of shortening increased in the presence of 80 pmol/L T3 compared with that in untreated myocytes (117.0 +/- 5.0 versus 77.3 +/- 3.3 microns/sec, p < 0.05). In a separate series of experiments (n = 29), myocyte velocity of shortening increased in the presence of both T3 and beta-adrenergic receptor stimulation (25 nmol/L isoproterenol) to greater than that with beta-adrenergic receptor stimulation alone (274.3 +/- 16.9 versus 203.7 +/- 16.2 microns/sec, p < 0.05). Cyclic adenosine monophosphate generation was next examined in isolated myocyte preparations (n = 9). In the presence of T3, no significant increase in cyclic-adenosine monophosphate generation was observed compared with that in untreated myocytes (39.1 +/- 8.3 versus 24.7 +/- 5.8 fmols/myocyte, p = 0.17). However, in the presence of both T3 and beta-adrenergic receptor stimulation, cyclic-adenosine monophosphate generation increased significantly to greater than that with beta-adrenergic receptor stimulation alone (224.4 +/- 61.1 versus 120.1 +/- 35.5 fmoles/myocyte, p < 0.05). Because cyclic-adenosine monophosphate modulates intracellular Ca2+ processes, L-type Ca+2 channel current (patch clamp methods; -picoamp/picofarad, n = 15) and peak intracellular Ca+2 levels (fura 2 ionic measurement, n = 47) were next measured. In the presence of T3, a shift in the activation voltage at peak L-type Ca+2 channel current was observed from baseline (5.5 +/- 1.4 versus 9.0 +/- 1.0 mV, p < 0.05). Furthermore, in the presence of both T3 and beta-adrenergic receptor stimulation, peak L-type Ca+2 channel current (8.9 +/- 0.7 versus 6.3 +/- 1.0 mV, p < 0.05) and peak intracellular Ca+2 levels (189.9 +/- 8.4 versus 171.7 +/- 8.3 nmol/L, p < 0.05) increased compared with values obtained with beta-adrenergic receptor stimulation alone. Important findings from the present study were twofold: (1) T3 improved myocyte contractile processes through a cyclic-adenosine monophosphate-independent mechanism and (2) T3 potentiated the effects of beta-adrenergic receptor stimulation transduction by increasing cyclic-adenosine monophosphate production, L-type Ca+2 channel current, and Ca+2 availability to the myocyte contractile apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)