Regulation of thyroid hormone receptors and responses by thyrotropin-releasing hormone in GH4C1 cells

The present study was undertaken to test the effects of TRH on thyroid hormone receptors and responses in GH4C1 rat pituitary tumor cells. TRH caused a loss of up to 32% of specific nuclear thyroid hormone binding sites with an ED50 of approximately 1 nM, and this loss was additive to the receptor down-regulation caused by T3 itself. Scatchard analysis of nuclear T3 binding revealed that 10 nM TRH decreased the concentration of T3 receptors from Bmax (femtomoles per mg protein) of 110 to 50 while receptor affinity in serum-free medium changed from dissociation constant (Kd) 110 to 50 pM with TRH. TRH lowered the GH response to 0.5 nM T3 from 215% to 127% of control. The concentrations of TRH required to decrease T3 receptors and T3 responses were similar and indicated that these TRH effects are mediated by the TRH receptor. In the absence of added thyroid hormone TRH had little effect on the rate of GH synthesis. TRH did not affect the binding of 0.5 nM [125I]T3 to receptors during the first 8 h but reduced T3 receptor occupancy up to 25-50% in different experiments after 24 h. TRH blocked the induction of GH by T3 only after 48 h or longer. When cells were incubated for 2 weeks with or without 2 nM T3 and 10 nM TRH, the stimulation of cell growth by T3 was decreased by TRH (2- vs. 5-fold increase in cell number) as was stimulation of GH by T3 (5- vs. 13-fold). As expected, T3 blunted the PRL response to TRH from 19- to 3-fold. The effects of TRH on the density of thyroid hormone receptors could be mimicked by the calcium channel agonist BAY K8644 plus a protein kinase C-activating phorbol ester which together caused a 53% reduction in thyroid hormone binding. The dose-response and temporal relationships suggest a causal relationship between the TRH-mediated decrease in thyroid hormone receptors and the decrease in thyroid hormone responses in GH4C1 cells. It has previously been shown that thyroid hormones decrease the concentration of TRH receptors and TRH responsivity in pituitary cells. The results shown here for GH4C1 cells suggest that TRH regulation of T3 responses may also be important in feedback control at the pituitary level.     

Attenuation of thyroid hormone action by 1,25-dihydroxyvitamin D3 in pituitary cells

Interactions between thyroid hormone and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] were examined in a rat pituitary tumor cell line, GH4C1. Cells were incubated in thyroid hormone-depleted medium for 2 days, and specific nuclear binding of [125I]T3 was measured. 1,25-(OH)2D3 decreased nuclear [125I]T3 binding without changing total cellular uptake of [125I]T3. This 1,25-(OH)2D3 effect required 2-3 h to become evident and 24 h to reach a maximum (40-50% of control) and was reversible. Treatment with 1,25-(OH)2D3 for 8 h changed the maximal binding capacity for [125I]T3 from 80.2 +/- 2.9 to 50.3 +/- 6.3 fmol/10(6) cells, whereas Kd was not significantly altered. The decrease in [125I]T3 binding was dose dependent, with an IC50 for 1,25-(OH)2D3 of 1 nM in thyroid hormone-depleted medium. 1,25-(OH)2D3 caused little change in [125I]T3 binding to isolated nuclei, i.e. 1,25-(OH)2D3 does not compete directly with [125I]T3 for binding. It is unlikely that 1,25-(OH)2D3 decreased [125I]T3 binding by increasing the concentration of intracellular free calcium ([Ca2+]i), since 1,25-(OH)2D3 did not change [Ca2+]i in Indo-I-loaded GH4C1 cells. Two major species (6 and 2.6 kilobases) of mRNA for c-erb-A, which have been reported to encode nuclear thyroid hormone receptors, were found by Northern blot analysis, and both were decreased by treatment with 1,25-(OH)2D3 for 8 h. T3 (2 nM) caused a 3-fold increase in GH production over 72 h and 1,25-(OH)2D3 inhibited GH induction by T3, with an IC50 at approximately 1 nM. 1,25-(OH)2D3 stimulated PRL synthesis 5-fold when 10 nM T3 was present, but not when T3 was absent. In summary, 1,25-(OH)2D3 caused a dose-dependent down-regulation of nuclear thyroid hormone receptors at a pretranslational level and diminished GH induction by T3. These results suggest that 1,25-(OH)2D3 inhibits GH synthesis indirectly, at least partly, by attenuating endogenous thyroid hormone action.     

Mechanism of thyroid hormone inhibition of thyrotropin-releasing hormone action

The addition of thyroid hormone to cultures of GH3 or GH4C1 pituitary tumor cells maintained in medium with hypothyroid serum decreased the concentration of specific receptors for TRH. The relationship between thyroid hormone effects on TRH receptors and TRH responses was examined by testing the concentration dependence, time course, and specificity of these changes. The concentrations of T3 giving half-maximal decreases in [3H]TRH binding and inhibition of the PRL response to TRH were 0.20 and 0.24 nM, respectively. TRH stimulated the rate of [3H]uridine uptake by 50% in cultures incubated without added T3 but did not increase [3H]uridine uptake in cells incubated with thyroid hormone. The PRL response to TRH was substantially inhibited 12 h after the addition of T3, and the uridine uptake response was completely blocked in 8 h. Two other stimuli of PRL secretion, sodium butyrate and isobutylmethylxanthine, were effective in the presence or absence of T3. Thyroid hormone did not reduce the specific binding of either [125I-Tyr1]somatostatin or [125I]iodoepidermal growth factor. Somatostatin decreased the secretion of GH and PRL by pituitary tumor cells grown with or without T3. The data show that the effects of thyroid hormones on TRH receptors are specific and suggest that regulation of receptor concentrations may be the direct cause of thyroid hormone regulation of pituitary responsiveness to TRH.     

Differential regulation of thyroid hormone receptor messenger ribonucleic acid levels by thyrotropin-releasing hormone

In addition to its well known actions in stimulating TSH and PRL synthesis and secretion, TRH has been shown to decrease the concentration of thyroid hormone receptors (TRs) in GH4C1 cells as measured by nuclear thyroid hormone (T3) binding. In the present study we have investigated the effects of TRH on the levels of mRNA encoding the different forms of TR, TR beta-1, TR beta-2, and TR alpha-1 as well as that of the non-T3-binding variant, c-erbA alpha-2. GH3 cells were incubated with 100 nM TRH in the presence or absence of 1 nM T3 for 48 h, and mRNA levels were determined by Northern blot analysis. Results revealed that there is differential regulation of the individual TRs by TRH at the pretranslational level. The mRNA for the pituitary-specific form of TR, TR beta-2, was down-regulated by 60% by TRH in GH3 cells, while that of its alternative splice product, TR beta-1, was unchanged. A modest change was observed in TR alpha-1 mRNA levels, which were down-regulated by 20%; there was no change in c-erbA alpha-2 mRNA levels. Levels of nuclear T3 binding were assessed under the same conditions, and 100 nM TRH was found to decrease binding by 40% from 0.78 to 0.46 fmol/micrograms DNA. A similar change in nuclear T3 binding was seen after incubation with 1 nM T3. The effect of TRH on the GH mRNA response to T3 was investigated. In the absence of TRH there was a 4-fold induction of GH mRNA after incubation with 1 nM T3. In the presence of 100 nM TRH, no significant induction in GH mRNA by T3 was seen, indicating that T3 responsiveness as well as receptor concentration are diminished by TRH under these conditions.     

Characterization of thyroid hormone stimulation of uridine uptake by rat pituitary tumor cells

T3 caused a dose-related increase in the rate of [3H]uridine uptake into GH4C1 rat pituitary tumor cells. T3 increased uridine uptake to 130-180% of the control value, with a half-maximal effect at approximately 1 nM. T3 exerted a half-maximal effect at 1 h and a maximal effect at 2 h. In contrast, epidermal growth factor also increased uridine uptake by 75%, with an ED50 of 0.6 ng/ml (0.1 nM), but a half-maximal response required 4 min and a maximal effect required 20 min. T3 increased the rate of uptake at all uridine concentrations from 30 nM to 130 microM. Equilibrium binding of [125I]T3 to nuclear receptors required from 15 min at 50 nM [125I]T3 to 1 h at 0.5 nM, indicating that occupancy of nuclear receptors precedes maximal stimulation of uridine uptake. T3 did not stimulate the rate of uridine uptake at 20 C, when binding to nuclear receptors does not occur. Various thyroid hormones caused an increase in uridine uptake, with the rank order of potency 3,3',5-triiodothyroacetic acid greater than T3 greater than L-T4 greater than D-T4 approximately equal to 3,3',5,5'-tetraiodothyroacetic acid; rT3 was inactive. This order parallels the affinities of these compounds for nuclear thyroid hormone receptors.     

Relationships between thyroid hormone and glucocorticoid effects in GH3 pituitary cells

The interrelationships between thyroid hormone and cortisol actions were investigated in GH3 pituitary tumor cells. When GH3 cells were grown in thyroid hormone-deficient medium, cortisol did not affect the concentration of TRH receptors. Both thyroid hormones and TRH normally decrease the number of TRH receptors, and cortisol inhibited down-regulation by both hormones. TRH caused a greater increase in PRL synthesis when TRH receptors were high in the presence of cortisol and T3 than when TRH receptors were low (T3 alone). In the presence of cortisol, higher concentrations of T3 were required to decrease TRH receptors, while lower concentrations were necessary to stimulate GH synthesis. Cortisol and T3 alone stimulated GH synthesis 6- and 10-fold, respectively, while together they caused an 830-fold increase. In contrast, T3 did not alter the inhibition of PRL synthesis by the glucocorticoid. Cortisol did not significantly affect the amount of [125I]T3 bound to nuclei from cells incubated in thyroid hormone-deficient or T3-supplemented medium (approximately 100 and approximately 25 fmol/mg cell protein). The data suggest that cortisol modifies thyroid hormone action at a step subsequent to T3 receptor binding.     

Effects of iopanoic acid on thyroid hormone receptor, growth hormone production, and triiodothyronine generation from thyroxine in pituitary GH1 cells

We have studied the effect of iopanoic acid (IOP), a radiographic contrast agent which inhibits T4 to T3 conversion, on thyroid hormone nuclear receptors, GH response to T4 and T3, and T4 5'-monodeiodination in GH1 cells, a rat pituitary cell line. IOP at concentrations higher than 10 microM inhibits iodothyronine binding to the nuclear receptor without changing the dissociation constant (Kd) (0.1 nM for T3 and 1 nM for T4), and reduces the GH response to 50 nM T4, 5 nM T3, or the combined effect of T4 and glucocorticoids. These results could be explained by an inhibition of protein synthesis which was reduced by more than 50% by 50 microM IOP. By contrast, nontoxic concentrations of IOP did not change the GH response to different doses of T4 ranging from 1 nM to 50 nM. We also examined T3 generation from T4 and found that the intracellular T3 levels of cells incubated with 50 nM T4 were almost as high as those of cells incubated with 5 nM T3 which induces a full GH response. Intracellular T3 levels were markedly reduced in the cells incubated with T4 and IOP, but GH production was not reduced despite these differences in T3 levels. Additionally, more than 40% of the nuclear receptor was occupied by T3 in cells incubated with T4, whereas more than 90% was occupied by T4 in cells receiving the same amount of T4 with 5 microM IOP. Our results suggest that the effect of T4 on GH production by GH1 cells could be attributed to an important extent to the T3 generated from it, whereas when T4 monodeiodination is strongly inhibited, most of the biological activity is a result of intrinsic T4 activity.     

Down-regulation of thyroid hormone nuclear receptor levels by L-triiodothyronine in cultured glial C6 cells

L-Triiodothyronine (T3) produced a time- and dose-dependent depletion of nuclear thyroid hormone receptor levels in C6 cells, a rat glioma cell line. Receptor number diminished by 30-40% after a 48 h incubation with concentrations of T3 that saturate the nuclear receptor. The nuclear binding curve obtained in cells incubated for 48 h with T3 was shifted leftward of the curve obtained after a 3 h incubation, which indicates an apparent increase in receptor affinity after long-term incubation with T3. However, this change probably represents a further equilibration of the hormone, since the dissociation rate from the nuclei was similar in C6 cells after long- and short-term incubation with T3. The effect of T3 was further demonstrated in C6 cells incubated with short-chain fatty acids. Butyrate and isobutyrate increased receptor levels, and T3 partially decreased the response to these compounds. These findings suggest the existence of a desensitization process by which C6 glial cells would be protected against an excess of thyroid hormone.     

Epidermal growth factor decreases the concentration of thyrotropin-releasing hormone (TRH) receptors and TRH responses in pituitary GH4C1 cells.

Treatment of pituitary GH4C1 cells with epidermal growth factor (EGF) caused up to a 60% reduction in the amount of [3H]MeTRH bound to specific TRH receptors. The effects of EGF were first detectable after a 2-h incubation and maximal by 24-72 h. EGF elicited a half-maximal response at 0.03 nM. Equilibrium binding analysis was performed on intact cells that had been incubated with or without 10 nM EGF for 96 h. EGF decreased the apparent number of TRH receptors (maximum binding = 0.36 vs. 0.58 pmol/mg protein for EGF-treated and control cells, respectively) without altering the apparent affinity (dissociation constant = 6.4 vs. 7.4 nM). The effects of EGF on TRH receptors were reversible. When EGF was removed from the medium, TRH receptors returned to control levels within 48 h. To assess whether the reduction of TRH receptors was functionally important, the ability of TRH to stimulate phospholipid turnover was measured in cells with a normal complement of TRH receptors and in cells that had been treated with EGF for 72 h to reduce TRH receptor density. EGF significantly blunted the ability of TRH to stimulate release of inositol phosphates from metabolically labeled cells. TRH increased inositol monophosphate accumulation 6.3-fold in control cultures and 2.0-fold in EGF-treated cells. These data show that EGF regulates the concentration of TRH receptors on pituitary GH4C1 cells and the responsiveness of the cells to TRH.     

L-triiodothyronine (T3) regulates cellular growth rate, growth hormone production, and levels of nuclear T3 receptors via distinct dose-response ranges in cultured GC cells

Cultured rat somatotrophic cells have been useful models for the study of thyroid hormone action. A consensus of previous reports has indicated that approximately 0.2 nM T3 results in 50% occupancy of T3 nuclear receptors as well as half-maximal stimulation of several T3 responses. To characterize the nature of thyroid hormone responses in GC cells, we studied in detail the T3 dose relationships between nuclear receptor occupancy and three thyroid hormone responses (cell growth, GH production, and T3 nuclear receptor regulation). The dose response to T3 for each parameter was unique, and none was identical to the dose response for receptor occupancy. Respective T3 concentrations and percentage of T3 nuclear receptor occupancy resulting in 50% of the maximal response for GC cell growth were 0.05 +/- 0.02 nM and 15 +/- 3% (four experiments), 0.15 +/- 0.04 nM and 27 +/- 3% for GH production (three experiments), and 2.1 nM and 69% for down-regulation of T3 nuclear receptors (two experiments). We conclude that the dose response for occupancy of the T3 nuclear receptor covers a wide range of T3 concentrations. Within the wide dose-response range for nuclear occupancy a spectrum of biological responses are regulated by distinct thyroid hormone dose ranges. These data suggest that the impact of T3 nuclear receptor occupancy on T3 responses might be variable and that the mechanisms involved may be clarified through studies in GC cells.     

Existence of nuclear thyroid hormone receptors in the porcine thyroid gland and the rat thyroid cell line FRTL-5

We have investigated whether nuclear T3 receptors exist in the thyroid cell. Nuclear proteins extracted from porcine thyroid nuclei with 0.4 mol/l KCl were incubated with [125I]T3. The mixture was then analysed by sucrose density gradient ultracentrifugation which revealed that the T3-binding proteins migrated at the same position of 3.6 S as rat liver nuclear T3 receptors. Fractionation by high performance liquid chromatography using a size exclusion column and an ion exchanger column also demonstrated elution patterns of T3-binding similar to those of the rat liver receptor. Scatchard plots of crude nuclear extracts from porcine thyroid represented a curvilinear pattern. However, when the nuclear proteins partially purified by a DEAE column chromatography were analysed, a single binding component was found; the association constant was 4.1 x 10(10) l/mol and the maximal binding capacity was 602 fmolT3/mg protein. Displacement study with several T3 analogues showed a highly selective affinity for L-T3. Cultured rat thyroid cells of the FRTL-5 line also contained a single class of saturable, high affinity T3-binding site. Subconfluent cells in 100-mm dishes were incubated with increasing amounts of [125I]T3 at 37 degrees C for 3 h and radioactive T3 in isolated nuclei was counted. Scatchard analysis of data showed that the association constant and the maximal binding capacity were 3.44 +/- 0.63 x 10(10) l/mol and 63.7 +/- 17.8 fmolT3/mg protein, respectively. These results strongly suggest that there are nuclear T3 receptors, indistinguishable from the hepatic T3 receptors, in the porcine thyroid and rat FRTL-5 cells.     

Thyroid hormone regulates rat pituitary insulin-like growth factor-I receptors

As we previously obtained evidence that insulin-like growth factor-I (IGF-I) inhibits T3-induced GH secretion and GH mRNA expression without affecting basal GH secretion in thyroidectomized rat pituitary cells grown in hypothyroid medium, we examined changes in IGF-I receptors in the pituitary gland, as induced by thyroid hormone. Thyroidectomized rats and a quantitative receptor autoradiographic method were used. The density of [125I]IGF-I-binding sites in the anterior pituitary gland decreased 4 weeks after thyroidectomy; that is a significant decrease in the number of the receptors compared to findings in control rats (P less than 0.01). The affinity (Kd) remained unchanged. There were no changes in binding parameters in the ventroposterior thalamic nucleus in the brain, renal cortex, and liver parenchyma. The ip administration of T4 once a day (48 micrograms/kg) for 1-2 weeks compensated for the decrease in the binding capacity of [125I]IGF-I-binding sites to that of the control values (P less than 0.01). We propose that IGF-I receptors in the anterior pituitary gland may be regulated by thyroid hormone.     

Characterization of nuclear thyroid hormone receptors of cultured skin fibroblasts from patients with resistance to thyroid hormone

Nuclear thyroid hormone receptors of patients with the syndrome of resistance to thyroid hormone were investigated in cell lines from seven patients in four affected families and compared to results from six normals. Fibroblasts cultured from skin biopsies were used. When binding affinity and capacity for L-triiodothyronine (T3) were examined by incubating whole cells or isolated nuclei, no significant differences were found. The amount of receptor released during the incubation of nuclei (9.3% to 19.0% of total nuclear receptors) was also within the normal range in these patients. When T3 binding assays were performed on 0.3 mol/L KCl extracted receptor, a significant decrease in binding capacity (MBC) without a difference in binding affinity (Ka) was observed in four patients and a lower Ka with normal MBC was found in two patients. Recovery of receptors in saline extracts, from patients' fibroblasts showing a low MBC, was low in comparison to normals. Lability of salt extracted receptors at 38 degrees C was normal and salt extractability of T3 occupied receptors, examined by incubation of [125I]-T3 labeled nuclei with various concentrations of KCl, was only slightly decreased. This lower salt extractability of receptors was insufficient to account for the low MBC obtained by Scatchard analysis of T3 binding to nuclear extracts. Gel filtration and density gradient sedimentation of salt-extracted receptors showed Stokes radius of 34 A, and sedimentation coefficient of 3.4 S in all patients and normals. From these values, molecular weight of 49,000 and total frictional ratio (f/fo) of 1.4 were calculated for nuclear receptors from patients and normals, suggesting a somewhat asymmetrical shape of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

5,5'-Diphenylhydantoin (phenytoin) attenuates the action of 3,5,3'-triiodo-L-thyronine in cultured GC cells

We have previously reported that 5,5'-diphenylhydantoin (DPH) inhibits total cellular and specific nuclear T3 binding by cultured GC cells, a rat pituitary tumor cell line that produces GH. DPH decreased competitively the rate of T3 accumulation by GC cells and noncompetitively inhibited specific nuclear T3 binding as well. To determine the biological consequences of these DPH effects on cellular and nuclear T3 binding, we studied the effect of DPH on the growth rate and GH production of GC cells cultured in Dulbecco's Modified Eagle's Medium with 10% serum. Incubation with T3 stimulated the GC cell growth rate in a dose-dependent manner. The half-maximal growth rate was achieved at a T3 concentration of 0.18 nM, and the maximal effect was observed at 0.4 nM T3. Addition of DPH to GC cells cultured with 0.15 nM T3 resulted in a dose-dependent decrease in the GC cell growth rate. The half-maximal depression of the rate of GC cell growth occurred at 185 microM DPH, a concentration that results in an approximately 50% decrease in cellular and nuclear T3. The DPH-induced decrease in GC cell growth was abolished by the addition of increasing concentrations of T3 (maximal concentration, 1.0 nM). Similarly, DPH effected a dose-dependent decrease in GH production in cells cultured with physiological concentrations of T3 (0.15 nM). The decrease in GH production of cells incubated with 200 microM DPH was associated with a decrease of similar magnitude in GH mRNA. These findings suggested that the DPH effect on GH production was mediated at a pretranslational level. Addition of increasing concentrations of T3 up to 5 nM completely abolished the DPH-associated decrease in GH production. Finally, studies of the effects of DPH on cell growth and GH production in cultures maintained with T3-depleted conditions were carried out to detect putative agonist activity of DPH. In the present investigation, we were unable to detect agonist activity of DPH that was more than 10-15% of the effect of maximal doses of T3. The data suggest that DPH attenuates the action of T3 in GC cells, probably because of a decrease in the steady state concentrations of cellular and nuclear T3. These attributes of DPH suggest that the drug or related analogs may serve as prototypes of agents that may decrease thyroid hormone activity at target tissues.     

Growth hormone regulation in primary fetal and neonatal rat pituitary cell cultures: the role of thyroid hormone

GH is first detectable in the fetal rat pituitary between gestational days 18 and 19. The reasons for the GH surge soon after birth and subsequent postnatal decline to adult levels remain unclear. We therefore determined whether GH gene regulation in the developing pituitary could be distinguished from adult rat somatotroph function. In primary cultures of fetal and neonatal rat pituitary cells, GH secretion was detected by the 20th gestational day. These cells were stimulated by GH-releasing hormone (GHRH), but not by T3 or the morphogen retinoic acid. The stimulatory effect of T3 (0.25 mM) on GH secretion was detected only on the 2nd neonatal day and was similar to that seen in mature rat pituitary cell cultures. GHRH (10 nM) treatment for 24 h caused a 5-fold induction of GH secretion in pituitary cells derived from 2-, 5-, and 12-day-old neonatal rats. The presence or absence of T3 in the culture medium did not alter the response to GHRH. In contrast, only 2-fold induction of GH was observed in adult male pituitary cells during the same time course. Insulin-like growth factor-I (IGF-I; 6.5 nM), the peripheral target hormone for GH, resulted in a modest (20%) attenuation of GH secretion from pituitary cells derived from 20-day-old fetuses. IGF-I, however, produced a 70% reduction in GH levels in adult male pituitary cells grown under similar conditions. The effects of IGF-I on adult pituitary cells grown in T3-depleted medium were blunted. Addition of T3 partially restored the responsiveness of these cells to IGF-I. The results suggest that the high circulating GH levels in the fetal and neonatal rat may be secondary to relative insensitivity of the immature somatotroph to the inhibitory actions of IGF-I in addition to enhanced responsiveness to GHRH compared with the adult rat pituitary. Relative thyroid hormone deficiency in the immature rat may be contributory to this early transient state of pituitary IGF-I resistance.     

Heat shock of cultured GC cells enhances the level of triiodothyronine induced growth hormone (GH) and GH messenger ribonucleic acid

We have previously proposed that the effects of heat shock on thyroid hormone-responsive rat pituitary tumor (GC) cells may be a model relevant to the in vivo effects of nonthyroidal disease on thyroid hormone action. To determine the effects of heat shock on thyroid hormone responses, GC cells (normally cultured at 37 C) were studied after incubation at 41 C. After 18 h at 41 C there was enhanced synthesis of proteins (mol wt, 70,000 and 90,000) considered to be universal markers of the cellular response to heat shock. Incubation at 41 C also resulted in a significant decrease in GC cell viability and (after 24 h) arrest of GC cell growth. However, the induction of GH synthesis by T3 was significantly enhanced in GC cells stressed by incubation at 41 C. The addition of 5 nM T3 to thyroid hormone-depeleted GC cells resulted in a significantly greater (P less than 0.001) accumulation of GH (2642 +/- 280 ng/18 h) during 41 C incubation than during 37 C incubation (1223 +/- 175 ng/18 h). The enhanced T3-induced production of GH was coincident with a proportional increase (P less than 0.05) in cellular GH mRNA determined by dot hybridization analysis. Thus, the stress of 41 C incubation elicits a heat shock response in GC cells characterized by decreased viability and growth arrest, but enhanced accumulation of GH mRNA in response to T3. Our recent report on the identical effects due to the stress of implantation of the Walker 256 carcinoma on T3-induced rat pituitary GH mRNA in vivo suggests that heat shock of cultured GC cells is a valid in vitro model of nonthyroidal disease.     

Characterization of thyroid hormone receptors in human IM-9 lymphocytes

Although putatively identified more than 10 years ago, thyroid hormone receptors in human tissues remain poorly characterized. As a first step towards understanding the mechanism of thyroid hormone action in man we have characterized T3 binding sites in nuclei of the human lymphoblastoid line, IM-9 cells. In whole cell experiments at 37 degrees C, nuclear binding of [125I]T3 was saturable (Kd 34 +/- 6 pmol/l) and of finite capacity (approximately equal to 350 sites/cell). The binding sites were extracted from a nuclear pellet by treatment with 0.4 mol/l KCl and sonication. Separation of bound from free [125I]T3 in the extracts was achieved using the calcium phosphate matrix, hydroxyapatite at a concentration of 0.3 ml of a 150 g/l slurry. Rectilinear Scatchard plots were obtained only when the hydroxyapatite was washed with a buffer containing 0.5% Triton X-100. Under these conditions T3 binding sites in the nuclear extracts were present at a concentration of 22.4 +/- 8.6 fmol/mg protein and showed an affinity of (Kd, room temperature) 140 +/- 10 pmol/l. The same assay system was used to determine the hierarchy of affinities for a range of natural and synthetic analogues. Calling T3 100, the order of potencies observed was: Triac, 500; 3,5-diiodo-3'-isopropylthyronine, 89; T4, 32; 3,5-dimethyl-3'isopropylthyronine 2; 3,5-T2, 0.7, rT3, 0.4; 3'5'-T2, less than 0.01. These results suggest that the T3 binding sites present in human IM-9 lymphocyte nuclei and extracts thereof are thyroid hormone receptors. These cells may be a useful tool to increase our understanding of human T3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential binding of thyroid hormone receptors to mouse glandular kallikrein gene promoters: evidence for multiple binding regions in the mGK-6 gene

We have used a DNA-cellulose competition assay to investigate the binding of thyroid hormone receptors to fragments of the mouse glandular kallikrein genes and the human and rat GH genes. Nuclear extracts from human lymphoblastoid IM-9 cells were incubated with [125I]tri-iodothyronine [( 125I]T3) and DNA-cellulose. The ability of cloned gene fragments to compete for radiolabelled receptors bound to DNA-cellulose was compared with that of DNA from pBR322. As previously observed, a 900 bp fragment from the human GH gene showed preferential binding to the thyroid hormone receptor. High-affinity binding was observed with a synthetic fragment of the rat GH gene encompassing positions -163 to -192 but not with a similar fragment from positions -224 to -192. Preferential binding was also observed with fragments of the mouse glandular kallikrein gene, mGK-6. Binding to the entire gene and fragments containing 2300 and 776 bp of the promoter region was identical. Detectable but reduced binding was seen with a shorter fragment. These results suggest that the T3 receptor binds to multiple sites within the first 776 bp of the mGK-6 gene promoter. Potential thyroid hormone response elements can be identified within this region of the gene. In contrast, the kallikrein gene mGK-3, which shows a different response to thyroid hormone from that of mGK-6, showed no significant binding in the comparable promoter region.     

Thyroid hormone metabolism in neuron-enriched primary cultures of fetal rat brain cells

The metabolism of thyroxine (T4) by cultures of embryonic-rat brain cells grown in a chemically defined medium was studied. Cells in these cultures were predominantly neurons, characterized by the developmental increase of the binding of [3H]flunitrazepam to the high-affinity (0.67 nM) benzodiazepine neuronal receptors. The cultures also contained astrocytes, characterized by immunological studies using an anti-glial fibrillary acidic protein (GFAp) and by the increase in glutamine synthetase (GS). Incubation of the cells, in situ, with 125I-labelled 3,5,3'-triiodothyronine (T3) showed the presence of a single class of high-affinity nuclear receptors for T3 with a maximal binding capacity of 270-470 fmol T3/mg DNA and a Kd of 63 +/- 13 pM. Cells incubated in situ with 50 pM [125I]T4 actively metabolized the hormone. The major metabolite, 3,3',5'-triiodothyronine (rT3) (159 +/- 43 fmol/4 h/mg DNA), was almost completely released into the medium. T3 was a minor metabolite (77 +/- 3 fmol/4 h/mg DNA), 75% of which accumulated in the cells. Of this T3, 35% was bound to the nuclear receptors after 4 h of incubation. In vitro assays showed that the 5'-deiodinase activity increased during culture and the 5-deiodinase decreased slightly. Cytosine-arabinoside (ARAc) treatment of the cultures reduced the DNA content per culture dish, corresponding to a fall in the number of GFAp-positive cells (astrocytes) and to a decrease in GS. A small increase in the number of benzodiazepine sites was observed. ARAc treatment markedly reduced the T3 production (14.5 +/- 0.7 fmol/4 h/mg DNA) and did not change the rT3 production. We suggest that T4 is metabolized to T3 in astrocytes, taken up by neurons and binds to their nuclear receptors.