triiodothyronine--reverse and Pituitary-Neoplasms

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.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Blotting, Northern; Dexamethasone; Hormones; Iodide Peroxidase; Isoenzymes; Male; Pituitary Neoplasms; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thyrotropin-Releasing Hormone; Triiodothyronine; Triiodothyronine, Reverse; Tumor Cells, Cultured

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.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chloroquine; Cycloheximide; Cysteine Endopeptidases; Cytochalasin B; Iodide Peroxidase; Iodothyronine Deiodinase Type II; Isoenzymes; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Pituitary Neoplasms; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Rats; Thyroxine; Triiodothyronine, Reverse; Tumor Cells, Cultured; Ubiquitins

The current consensus is that iodothyronines down-regulate type II T4 monodeiodinase (5'-DII) by an extranuclear acceleration of enzyme inactivation. We have investigated 5'-DII regulation in cultured GC cells, in which thyroid hormone responses are mediated by nuclear thyroid receptor (TR). GC cells actively converted T4 to T3, independent of propylthiouracil and with a Km of 1.4 nM, which are characteristics of 5'-DII. When GC cells were incubated with 10 nM T3, the Km was not affected. However, the maximum velocity was significantly down-regulated by 10 nM T3, from 0.15 to 0.018 pmol/mg protein.min. Dose-response studies showed that a 50% reduction in enzyme activity was achieved with either 0.25 nM T3 or 12 nM rT3. Time-course studies showed that a 50% reduction in enzyme activity occurred after 40 min of incubation with 100 nM rT3 and after 160 min of incubation with 10 nM T3. The down-regulation of 5'-DII by physiological concentrations of T3 has the characteristics of an effect that is mediated by nuclear TR. Our studies, therefore, suggest that down-regulation of 5'-DII by these iodothyronines in GC cells may occur by different mechanisms: enzyme inactivation for rT3, in agreement with the current consensus, and decreased enzyme production for T3, probably mediated by TR.

Topics: Animals; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression Regulation, Enzymologic; Iodide Peroxidase; Iopanoic Acid; Neuroglia; Pituitary Neoplasms; Propylthiouracil; Rats; Time Factors; Triiodothyronine; Triiodothyronine, Reverse; Tumor Cells, Cultured

Thyroid hormones rapidly inhibit type II iodothyronine 5'-deiodinase (5'DII) activity in the rat central nervous system, anterior pituitary gland, and GH3 pituitary tumor cells. To gain insight into the cellular mechanisms responsible for this down-regulation, the effects of substrates, inhibitors, and alterations in the cellular thiol-disulfide balance on 5'DII regulation were investigated. The results demonstrate that in vitro competitive inhibitors, such as iopanoic acid, as well as iodothyronine substrates induce a rapid and irreversible loss of enzyme activity in rat cerebral cortex and anterior pituitary tissue. The potency of these agents in down-regulating this enzymatic process in intact GH3 cells is directly related to their competitive inhibitory effects on 5'DII activity in vitro. Additional studies demonstrated that treatment of intact GH3 cells with the sulfhydryl-oxidizing agent diamide mimicks the effect of substrate and results in the rapid inactivation of 5'DII. In contrast, preincubation of cells with the sulfhydryl-reducing agent dithiothreitol renders the enzyme less susceptible to the down-regulatory effects of substrate. Sulfhydryl-reducing agents thus appear to play a dual role in the 5'DII process by serving as cosubstrates and by modulating the enzymes susceptibility to substrate-induced down-regulation. These findings suggest that the in vivo inhibition of 5'DII by thyroid hormones involves a unique mechanism of enzyme regulation whereby the binding of ligand to the active site induces the rapid and irreversible inactivation and/or degradation of the enzyme. This inactivation of 5'DII initiated by substrate binding may be mediated by alterations in the sulfhydryl state of the enzyme as it progresses through the catalytic cycle.

Topics: Animals; Cell Line; Cerebral Cortex; Cycloheximide; Diamide; Dithiothreitol; Hypothyroidism; Iopanoic Acid; Kinetics; Male; Parathyroid Glands; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Thyroidectomy; Thyroxine; Triiodothyronine, Reverse

The conversion of T4 to T3 in the brain and anterior pituitary gland contributes significantly to the T3 content of these tissues and appears to be an important modulator of thyroid hormone action. In the present study, the antimanic agent lithium was demonstrated in cultured neural and pituitary tissue to have a significant inhibitory effect on the activity of low Km (type II) iodothyronine 5'-deiodinase (I5'D), the enzyme mediating T3 formation. At medium lithium concentrations of 3.3-5 mM, 15'D activity was decreased 44 +/- 3% (P less than 0.001) in the NB41A3 mouse neuroblastoma cell line and 48 +/- 2% (P less than 0.001) in the GH3 rat pituitary tumor cell line. This inhibitory effect was only observed in intact cells. Significant inhibition of this enzymatic process was also noted in the anterior pituitary gland of thyroidectomized rats injected 3-24 h earlier with either 4 or 10 mmol/kg BW LiCl. This decrease in low Km I5'D activity was accompanied by significant decreases in the serum T3 concentration and the pituitary nuclear T3 content. Renal high Km (type I) I5'D activity was unaffected by lithium administration. These studies demonstrate that lithium, an agent of proven therapeutic benefit in patients with manic-depressive illness, can affect changes in T4 metabolism and cellular T3 content in neural and anterior pituitary tissue. Given the prominent mood changes that occur in patients with disordered thyroid function, this finding suggests that the therapeutic benefits of lithium in affective illness may be derived in part from alterations in thyroid hormone economy in the brain.

Topics: Animals; Cell Line; Cerebral Cortex; Chlorides; Iodide Peroxidase; Kidney; Kinetics; Lithium; Lithium Chloride; Male; Mice; Neuroblastoma; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Thyroidectomy; Thyroxine; Triiodothyronine; Triiodothyronine, Reverse

The effects of thyroid hormones on morphology and growth were studied in rat pituitary tumor GH3 cells using medium containing serum depleted of thyroid hormones. T3 and T4 induce the cells to change from a flattened fibroblastic morphology to a rounded or spindle-shaped morphology. The induction in morphological changes is T3 and T4 specific and dose dependent. Thyronine and rT3 are ineffective in inducing morphological changes; the half-maximal effective concentrations for T3 and T4 are 0.3 and 2 nM, respectively. Concomitantly, T3 stimulates cell growth, as indicated by a 2-fold reduction in doubling time and a 2-fold increase in mitotic rate. The growth-stimulating effect has the same analog specificity and dose dependency as the morphological changes. The morphological changes could be potentially useful for evaluating the biological effects of T3 and its analogs and in studying the mechanism of thyroid hormone action.

Topics: Animals; Cell Division; Cell Line; Mitosis; Pituitary Neoplasms; Rats; Thyroxine; Time Factors; Triiodothyronine; Triiodothyronine, Reverse

The regulation of T4 5'-deiodinase activity was studied in cultured GH4C1 cells. Enzyme activity was measured in cell sonicates as the release of radioiodide from [125I]T4. Enzyme activity was stimulated 2- to 3-fold by hypothyroid serum and activators of protein kinase C, such as TRH and phorbol esters. The hypothyroid serum effect was maximal by 3 h, whereas TRH and phorbol esters required 6 h to achieve a maximal effect. The hypothyroid serum effect was gone within 4 h of returning the cells to control medium. In contrast, the TRH and phorbol ester effects persisted 24-48 h after removal of those agents. Both T4 and rT3 were at least as potent as T3 in blocking the effect of hypothyroid serum. The stimulation of 5'-deiodinase induced by hypothyroid serum was additive with that induced by kinase C activators. Trifluoperazine blocked the effect of TRH and phorbol esters, but not that of hypothyroid serum. It is concluded that stimulation of 5'-deiodinase activity can occur by at least two independent mechanisms: one involving hypothyroidism and another involving activation of protein kinase C. The relative potencies of various iodothyronines for abolishing the hypothyroid effect differ markedly from the relative binding affinities of these agents for the nuclear T3 receptor, suggesting that this thyroid hormone effect may not be mediated by the classical nuclear thyroid hormone receptor.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcimycin; Cell Line; Colforsin; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Hypothyroidism; Iodide Peroxidase; Phorbol Esters; Pituitary Neoplasms; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Thyroid Hormones; Thyrotropin-Releasing Hormone; Thyroxine; Trifluoperazine; Triiodothyronine; Triiodothyronine, Reverse

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.

Topics: Animals; Cell Line; Dose-Response Relationship, Drug; Growth Hormone; In Vitro Techniques; Iodide Peroxidase; Kinetics; Peroxidases; Pituitary Gland; Pituitary Neoplasms; Rats; Triiodothyronine, Reverse