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 triiodothyronine (rT3) attenuates ischemia-reperfusion injury.

Hypothyroidism has been associated with better recovery from cerebral ischemia-reperfusion (IR) injury in humans. However, any therapeutic advantage of inducing hypothyroidism for mitigating IR injury without invoking the adverse effect of whole body hypothyroidism remains a challenge. We hypothesize that a deiodinase II (D2) inhibitor reverse triiodothyronine (rT3) may render brain specific hypometabolic state to ensue reduced damage during an acute phase of cerebral ischemia without affecting circulating thyroid hormone levels. Preclinical efficacy of rT3 as a neuroprotective agent was determined in rat model of middle cerebral artery occlusion (MCAO) induced cerebral IR and in oxygen glucose deprivation/reoxygenation (OGD/R) model in vitro. rT3 administration in rats significantly reduced neuronal injury markers, infarct size and neurological deficit upon ischemic insult. Similarly, rT3 increased cellular survival in primary cerebral neurons under OGD/R stress. Based on our results from both in vivo as well as in vitro models of ischemia reperfusion injury we propose rT3 as a novel therapeutic agent in reducing neuronal damage and improving stroke outcome.

Harnessing the skin-thyroid connection for wound healing: a prospective controlled trial in guinea pigs.

BACKGROUND: Different elements of the hypothalamic-pituitary-thyroid axis have been found to be implicated in the normal physiology of the human skin. Their effects on wound healing and hair growth in rats have been described previously. There is close homology between the thyroid hormone receptors in humans and guinea pigs. AIM: To assess the effect of different doses of topical 3,3',5-triiodo-L-thyronine (T3) and recombinant human thyroid-stimulating hormone (TSH) on wound healing in guinea pigs. METHODS: Wounds were dressed every other day for 7 days, during which clinical measurements of the wounded areas were performed. Histological examination was performed at the end of the study. RESULTS: Application of high and low concentrations of topical T3 but not TSH demonstrated a significant dose-dependent reduction in the wound surface area through a process of contraction. The main significant histological result was an increase in the hair-follicle count. CONCLUSION: Topical T3 enhances wound healing in guinea pigs, primarily by wound contraction. As this is a critical stage in healing of chronic ulcers, topical T3 could be a useful treatment for wounds.

Type II iodothyronine deiodinase provides intracellular 3,5,3'-triiodothyronine to normal and regenerating mouse skeletal muscle.

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T(3) under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T(4)-to-T(3) conversion increases during differentiation in C(2)C(12) myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given (125)I-T(4) and (131)I-T(3), the intracellular (125)I-T(3)/(131)I-T(3) ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the (125)I-T(3)/(131)I-T(3) ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in (125)I-T(3)/(131)I-T(3) ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of (125)I-T(3) is doubled after skeletal muscle injury. Thus, D2-mediated T(4)-to-T(3) conversion generates significant intracellular T(3) in normal mouse skeletal muscle, with the increased T(3) required for muscle regeneration being provided by increased D2 synthesis, not by T(3) from the circulation.

Thyroid and bone.

The hypothalamic-pituitary-thyroid axis plays a key role in skeletal development, acquisition of peak bone mass and regulation of adult bone turnover. Euthyroid status is essential for maintenance of optimal bone mineralization and strength. In population studies, hypothyroidism and hyperthyroidism have both been associated with an increased risk of fracture. Furthermore, recent studies in healthy euthyroid post-menopausal women indicate that thyroid status in the upper normal range is also associated with low bone mineral density and an increased risk of non-vertebral fracture. Studies in mutant mice have demonstrated that thyroid hormone receptor α is the major mediator of T3 action in bone and that thyroid hormones exert anabolic actions during growth but have catabolic effects on the adult skeleton. Nevertheless, TSH has also been proposed to be a direct negative regulator of bone turnover, although the relative importance of T3 and TSH actions in the skeleton has yet to be clarified.

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.

Metabolic effect of 3,3',5'-triiodothyronine in cultured growth hormone-producing rat pituitary tumor cells. Evidence for a unique mechanism of thyroid hormone action.

Physiologic levels of 3,3',5'-triiodothyronine (rT3) are generally believed to have minimal metabolic effects in the pituitary gland and other tissues. In the present studies, the regulatory role of rT3 and other thyroid hormones on iodothyronine 5'-deiodinase (I5'D) activity was studied in a growth hormone-producing rat pituitary tumor cell line (GH3 cells). I5'D activity was thiol-dependent and displayed nonlinear reaction kinetics suggesting the presence of two enzymatic processes, one having a low Michaelis constant (Km for thyroxine [T4] of 2 nM) and a second with a high Km value (0.9 microM). Growth of cells in hormone-depleted medium resulted in a two- to 3.5-fold increase in low Km I5'D activity (P less than 0.001). The addition of thyroid hormones to the culture medium resulted in a rapid, dose-dependent inhibition of low Km I5'D activity with the following order of analogue potency: rT3 greater than or equal to T4 greater than 3,5,3'-triiodothyronine (T3). Using serum-free culture conditions, rT3 was approximately 50 times more active than T3. These inhibitory effects were noted within 15 min of hormone addition and could not be attributed to substrate competition with T4. These findings suggest that the control of T4 to T3 conversion by thyroid hormones in the anterior pituitary gland is mediated by a unique cellular mechanism that is independent of the nuclear T3 receptor; and under some circumstances, rT3 may play a regulatory role in controlling this enzymatic process.

Dual mechanisms of regulation of type I iodothyronine 5'-deiodinase in the rat kidney, liver, and thyroid gland. Implications for the treatment of hyperthyroidism with radiographic contrast agents.

Alterations in thyroid hormone status and the administration of radiographic contrast agents can markedly influence iodothyronine metabolism and, in particular, the activity of type I 5'-deiodinase (5'DI). In the present studies, the mechanisms responsible for these effects have been reassessed. As previously reported, the addition of iopanoic acid (IOP) to broken cell preparations resulted in a competitive pattern of 5'DI inhibition. However, the in vivo administration to rats of IOP or 3,3',5'-triiodothyronine (rT3) resulted in a noncompetitive pattern of inhibition of 5'DI in the liver, kidney, and thyroid gland, whereby marked decreases in maximal enzyme velocity (V max) were noted, with no change in the value of the Michaelis-Menten constant. In rats rendered hyperthyroid by the injection of 3,5,3'-triiodothyronine (T3), 5'DI activity was significantly increased in the liver and the kidney. The administration of IOP to these thyrotoxic animals resulted in a rapid loss of enzyme activity characterized by an approximate 80% decrease in 5'DI V max values in both tissues. Furthermore, this inhibitory effect persisted for longer than 60 h after a single IOP injection. IOP administration also decreased 5'DI V max levels in the thyroid gland by 52%. In other experiments, treatment of intact Reuber FAO hepatoma cells with IOP or rT3 induced a rapid decrease in 5'DI V max levels. In cells treated with cycloheximide, these agents enhanced the rate of disappearance of enzyme activity by greater than 12-fold, indicating a predominant effect on accelerating the rate of enzyme inactivation and/or degradation. These studies demonstrate that iodothyronines and other iodinated compounds have complex regulatory effects on 5'DI that entail alterations in the rates of both enzyme activation and inactivation. The previously accepted concept that rT3 and IOP impair thyroxine (T4) to T3 conversion in vivo by acting as competitive inhibitors is an oversimplification. Rather, the clinically beneficial effects of administering these agents to patients with hyperthyroidism may result primarily from the rapid and prolonged inactivation of 5'DI which occurs in the thyroid gland and peripheral tissues.

Ontogenesis of iodothyronine-5'-deiodinase. Induction of 5'-deiodinating activity by insulin, glucocorticoid, and thyroxine in cultured fetal mouse liver.

To elucidate the regulatory mechanism of ontogenetic development of iodothyronine-5'-deiodinase in the fetal and neonatal period, fetal mouse liver of the 19th day of gestation, in which no iodothyronine-5'-deiodinating activity was detectable, was cultured in Dulbecco-Vogt medium supplemented with 10% thyroid hormone-depleted fetal calf serum, insulin, hydrocortisone, and thyroid hormones. Iodothyronine-5'-deiodinating activity of the homogenate was assessed by the amount of iodide released from outer-ring-labeled reverse T3 and expressed as picomoles of 127I- per milligram of protein per minute. The enzyme activity was induced in a dose-dependent manner; optimal concentrations for insulin, hydrocortisone, and thyroxine were 1 microgram/ml, 0.4 microgram/ml, and 10(-6) M, respectively. Without supplementation of either hydrocortisone or thyroxine, no 5'-deiodination was detected. The enzyme activity was observed after 3 d of culture, peaked at days 14-20, and then gradually decreased. Lineweaver-Burk analysis revealed that the increase in activity was primarily due to an increase in Vmax (day 3, 0.2 pmol/mg protein per min; day 20, 2.5 pmol/mg protein per min). Half maximal thyroxine (T4) and triiodothyronine (T3) concentrations were 1 X 10(-7) M (free T4: 4 X 10(-10) M), and 2 X 10(-9) M (free T3: 5.0 X 10(-11) M), respectively, whereas reverse T3 did not elicit any activity at 10(-8)-10(-6) M. These results suggest that ontogenetic development of iodothyronine-5'-deiodinase in the liver of the fetal and neonatal mouse is induced by physiological concentrations of glucocorticoid and thyroid hormones, and that insulin plays a permissive role in enhancing T3 formation from T4 in the liver.

Interrelationships among thyroxine, growth hormone, and the sympathetic nervous system in the regulation of 5'-iodothyronine deiodinase in rat brown adipose tissue.

Thyroxine (T4) and reverse triiodothyronine are potent inhibitors of brown adipose T4 5'-deiodinase (BAT 5'D). This effect does not require protein synthesis and is due to an acceleration of the rate of disappearance of the enzyme. Growth hormone (GH) also inhibits BAT 5'D but by a mechanism mediated through a long-lived messenger that correlates with growth rate. This explains the failure of BAT 5'D to increase abruptly after thyroidectomy as does the type II 5'-deiodinase in pituitary and central nervous system or the BAT 5'D itself after hypophysectomy. Although virtually inactive when given acutely, triiodothyronine replacement partially reduces BAT 5'D in hypophysectomized and thyroidectomized (Tx) animals probably as a result of improvement of systemic hypothyroidism and an increase in GH levels in the Tx rats. The fine balance between these inhibitory factors and the stimulatory effects of the sympathetic nervous system suggests an important physiologic role for the enzyme in this tissue.

3,3',5-triiodothyronine administration in vivo modulates the hormone-sensitive adenylate cyclase system of rat hepatocytes.

The ability of 10 muM epinephrine or isoproterenol to stimulate cyclic AMP accumulation was decreased in hepatocytes isolated from hyperthyroid (triiodothyronine treated) as compared to euthyroid rats. In the presence of methylisobutylxanthine, epinephrine or isoproterenol-stimulated cyclic AMP accumulation was approximately 65% lower in hyperthyroid as compared with euthyroid rat hepatocytes. The ability of glucagon to stimulate a cyclic AMP response was also decreased in the hyperthyroid state, when assayed in either the absence or presence of a methyl xanthine. The character of the catecholamine-stimulated cyclic AMP response was beta adrenergic in both the hyperand euthyroid states. No evidence for an alpha(2) adrenergic mediated component of catecholamine action on cyclic AMP levels was noted. Cyclic AMP phosphodiesterase activity of hepatocyte homogenates was not altered in the hyperthyroid state. Hormone-stimulated, guanine nucleotide- and fluoride-activatable adenylate cyclase activity was reduced in subcellular fractions obtained from hyperthyroid as compared with euthyroid rat hepatocytes. Beta adrenergic receptor binding was reduced approximately 35% and glucagon receptor binding reduced approximately 50% in the hyperthyroid as compared with euthyroid rat hepatocyte membrane fractions. The status of the regulatory components of adenylate cyclase were examined by in vitro treatment of subcellular fractions with cholera toxin. The ability of cholera toxin to modulate adenylate cyclase was not altered by hyperthyroidism. Cholera toxin catalyzed AD[(32)P]ribosylation of hyperthyroid and euthyroid rat hepatocyte proteins separated electrophoretically displayed nearly identical autoradiograms. Studies of the reconstitution of adenylate cyclase activity of S49 mouse lymphoma cyc(-) mutant membranes by detergent extracts from rat hepatocyte membranes, indicated that hyperthyroidism was associated with a reduced capacity of regulatory components to confer fluoride, but not guanine nucleotide activatability to catalytic cyclase. Thyroid hormones regulate the hormone-sensitive adenylate cyclase system of rat hepatocytes at several distinct loci of the system.

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.

Molecular dynamics simulations of ligand dissociation from thyroid hormone receptors: evidence of the likeliest escape pathway and its implications for the design of novel ligands.

Steered molecular dynamics simulations of ligand dissociation from Thyroid hormone receptors indicate that dissociation is favored via rearrangements in a mobile part of the LBD comprising H3, the loop between H1 and H2, and nearby beta-sheets, contrary to current models in which the H12 is mostly involved. Dissociation is facilitated in this path by the interaction of the hydrophilic part of the ligand with external water molecules, suggesting strategies to enhance ligand binding affinity.

3,5,3' Triiodo-L-thyronine stimulates 2-deoxy-D-glucose transport into L6 muscle cells through the phosphorylation of insulin receptor beta and the activation of PI-3k.

3,5,3' Triiodo-L-thyronine (T3) stimulated the uptake of 2-deoxy-D-glucose (2-DOG) into L6 cells, nongenomically, starting at subpicomolar concentrations and reaching a peak at concentrations of 1-10 nM. Stimulation at the peak was usually approximately 250%. The uptake of 2-DOG declined with higher concentrations of T(3). The dose-response curve of insulin is similar in shape to that of T(3), and its peak stimulation can even reach 600% over the control. Wortmannin, an inhibitor of the PI-3k, completely inhibited the stimulation of 2-DOG uptake by T(3), with no effect on the control cells. L6 cells exposed for 10 minutes to T3 resulted in a 200%-300% stimulation of PI-3k, as measured by the production of labeled (32)P-PI-3P. Similar results were obtained with insulin. After incubation for 5 minutes with L6 cells, T(3) increased phosphorylation of the insulin receptor beta subunit; this correlated significantly with the degree of stimulation of 2-DOG uptake at 90 minutes (r = 0.89, p

Transactivation activity of thyroid hormone receptors in fish (Conger myriaster) in response to thyroid hormones.

We previously isolated cDNAs encoding conger eel (Conger myriaster) thyroid hormone (TH) receptors (TRs). In the present study, we investigated the transactivation activities of conger eel TRs treated with THs (3,3',5-triiodo-l-thyronine [T3], l-thyroxine [T4], and 3,3',5'-triiodo-l-thyronine [rT3]), or ligands and activators of other nuclear receptors. Following transient transfection into the Japanese eel (Anguilla japonica) hepatocyte cell line Hepa-E1, the conger eel TRs (cTRs) showed TH-dependent activation of transcription from a TH-responsive promoter. However, no transactivation activity of any of the four cTRs was observed with ligands or activators of other nuclear receptors. Although T3 activation for cTRs is stronger than other THs (T3>T4>rT3), the transactivation sensitivity was different from the activity of cTRs with THs, respectively. Therefore, we conclude that cTRs can act in concert with THs in fish metamorphosis.

Inhibition of pituitary type 2 deiodinase by reverse triiodothyronine does not alter thyroxine-induced inhibition of thyrotropin secretion in hypothyroid rats.

OBJECTIVES: Intrapituitary triiodothyronine (T3) production plays a pivotal role in the control of TSH secretion. Its production is increased in the presence of decreased serum thyroxine (T4) concentrations and the enzyme responsible, deiodinase type 2 (D2), is highest in hypothyroidism. In order to document the role of this enzyme in adult rats we developed an experimental model that inhibited this enzyme using the specific inhibitor, reverse T3 (rT3). METHODS: Hypothyroidism was induced with propylthiouracil (PTU; 0.025 g/l in drinking water) which in addition blocked deiodinase type 1 (D1) activity, responsible for the rapid clearance of rT3 in vivo. During the last 7 days of the experiment, the hypothyroid rats were injected (s.c.) for 4 days with 0.4 or 0.8 nmol T4 per 100 g body weight (bw) per day. For the last 3 days, the same amount of T4 was infused via s.c. minipumps. In additional groups, 25 nmol rT3/100 g bw per day were added to the 3-day infusion of T4. RESULTS: Infusion of 0.4 nmol T4/100 g bw per day did not affect the high serum TSH levels, 0.8 nmol T4/100 g bw per day decreased them to 57% of the hypothyroid values. The infusions of rT3 inhibited D2 activity in all organs where it was measured: the pituitary, brain cortex and brown adipose tissue (BAT). In the pituitary, the activity was 27%, to less than 15% of the activity in hypothyroidism. Despite that, serum TSH levels did not increase, serum T4 concentrations did not change and the changes in serum T3 were minimal. CONCLUSIONS: We conclude that in partly hypothyroid rats, a 3-day inhibition of D2 activity, without concomitant change in serum T4 and minimal changes in serum T3 levels, is not able to upregulate TSH secretion and we postulate that this may be a reflection of absent or only minimal changes in circulating T3 concentrations.

Regulation of cerebellar neuronal migration and neurite outgrowth by thyroxine and 3,3',5'-triiodothyronine.

The timing of granule cell migration in the developing cerebellum is regulated by thyroid hormone. Granule cell migration depends on the recognition of extracellular neuronal guidance molecule(s), such as laminin, and this, in turn, requires cell surface adhesion molecules (integrins) that are anchored on the cell membrane by the actin cytoskeleton. While many of the actions of thyroid hormone, specifically 3,5,3'-triiodothyronine (T3), are mediated by regulated gene expression, both thyroxine (T4) and 3,3',5'-triiodothyronine (rT3) also exert direct, positive control of the quantity of polymerized actin in cultured astrocytes without affecting gene expression. T4-dependent actin polymerization has been shown to (i) participate in the immobilization of laminin to the cell surface, (ii) help deposit laminin in the molecular layer of the developing cerebellum, and (iii) anchor integrin(s) that recognize laminin present in the extracellular matrix. In this study, we show that both T4 and rT3, but not T3, directly regulate the F-actin content of elongating neurites of cerebellar neurons. T4 and rT3 also promoted extensive granule cell migration from cerebellar explants, as well as, dense cell clustering and extensive neuronal process formation when granule cells were grown on a laminin-coated surface. Both granule cell migration and neuronal process outgrowth were markedly attenuated by the addition of integrin-blocking antibodies or binding peptides, by the absence of thyroid hormone or the presence of T3. These data suggest that the T4-dependent actin polymerization in developing neurons is necessary for these migrating cells to recognize the laminin guidance molecule, thereby providing a novel molecular mechanism for the profound influence of thyroid hormone on brain development that is independent of regulated gene expression.

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.

Thyroid hormones increase insulin-like growth factor I content in the medium of rat bone tissue.

The mechanism of action of thyroid hormones on bone is still not clear. At low concentrations, they stimulate bone formation; at high concentrations, they elicit bone resorption in vitro and in vivo. In the present study we investigated the effect of T3 and T4 as well as their active and inactive analogs (TRIAC, SKF L-94901, rT3, and DIT) on the IGF-I and TNF-alpha content in the medium of UMR-106 rat osteoblastic cells and fetal rat limb bones. In the dose-response studies, a biphasic increase in medium IGF-I was observed in both cells and limb bones, with peak stimulatory concentrations of 10(-8) M for T3 and 10(-7) M for T4 in both systems. At higher concentrations, at which thyroid hormones elicit bone resorption, the stimulatory effect diminished and finally was no longer detectable. The active analogs TRIAC and SKF L-94901 also enhanced IGF-I release in UMR-106 cells. The inactive compounds rT3 and DIT failed to increase IGF-I in these cultures. The protein content of the cell culture wells exposed to high concentrations of thyroid hormones was similar to those containing low concentrations, indicating that the decrease in IGF-I content at high doses was not due to toxic effects. This was also confirmed by trypan blue exclusion. Time course studies with UMR-106 cells revealed a significant increase in medium IGF-I after 2 days of incubation. No significant further increase was observed after this up to 5 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Triiodothyronine repression of imidazole-induced tyrosinase expression in B16 melanoma cells.

Investigations with mouse melanoma B16/C3 cell cultures have suggested that imidazole or a derivative thereof can facilitate expression of the tyrosinase (EC 1.14.18.1) structural gene. The induction of tyrosinase expression by imidazole was inhibited by T3 about 4-fold. When T3 (10 nM) was present for 19 h in proliferating B16/C3 cultures, basal activity of this enzyme was inhibited by approximately 60%. Neither T3 nor imidazole directly affected tyrosinase enzymatic activity in broken cell preparations. Addition of T3 to imidazole-induced cultures rapidly decreased tyrosinase expression (within 30 min) which remained repressed for at least 4 h before recovering. Recovery of tyrosinase activity could be blocked by readdition of hormone. The hormone effect was detectable at 1 nM and was maximal at 10 nM. Removal of supplemental T3 from the medium rapidly reversed the repression of tyrosinase activity. The biologically inactive analog rT3 (10 nM) failed to inhibit basal enzyme activity or alter the imidazole effect on tyrosinase expression. The experimental results with protein and RNA inhibitors indicate that the T3 response is independent of destabilizing the putative transcript for tyrosinase or altering the posttranslational events responsible for its synthesis.