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Regulation of Thyrotropin Production by Mouse Pituitary Thyrotropic Tumor Cells in Vitro by Physiological Levels of Thyroid Hormones*
Abstract
Primary suspension cultures from serially transplanted mouse pituitary thyrotropic tumors were shown to be regulated by physiological levels of thyroid hormones. TSH release was linear for up to 48 h in control cultures and was inhibited progressively after 10, 24, and 48 h in cultures exposed to triiodothyronine (T3), 4.0 nM. TSH release was inhibited up to 55% of control and glucose consumption was stimulated up to 2.6-fold in a dose-dependent fashion by T3 between 0.2 and 4.0 nM. A biphasic dose-dependent relationship for T3 and thyroxine (T4), and TSH production was demonstrated in two series of cultures. TSH production was stimulated progressively by T3 up to 0.1 nM and T4 up to 5 nM. At higher T3 and T4 levels, TSH production was progressively inhibited. Half-maximal inhibition occurred at total medium concentrations of 0.2 nM T3 and 15 nM T4 or free hormone levels of 8 x 10-12 M and 14 x 10-11 M, respectively. High affinity, low capacity nuclear binding sites were demonstrated for T3 and T4. The apparent equilibrium dissociation constants were 0.16 nM for T3 and 1.7 nM for T4 in serum-free medium. There were approximately 14,000 molecules of T3 or T4 bound per cell nucleus at saturation. These data suggest that T3 is approximately 15-fold more potent than T4 in regulating TSH production and cellular metabolism in these cells, and that these effects may be initiated by interaction with nuclear binding sites. Also, that within the physiological range, low levels of thyroid hormones stimulate and higher levels inhibit TSH production.
... Subsequently, similar values for L-T3 were reported in different cell culture systems. Gershengorn (38) recently reported that 0.2 nM L-T3 resulted in half-maximal inhibition of thyroid-stimulating hormone produc- (39) recently reported that L-T3 induces ,B-adrenergic receptors in cultured rat myocardial cells, and a half-maximal increase in f3-adrenergic receptor levels was induced by 0.3 nM L-T3. Therefore, the dose-response characteristics for L-T3 induction of growth hormone synthesis appear to be a general response characteristic for L-T3-inducible functions in cell culture and are not unique for the growth hormone response. ...
... It remains possible that the L-T3 reduction in receptor in dispersed pituitary cells reflects an absolute reduction in the thyrotroph population. This seems unlikely, however, because thyrotropic cells represent <11% of the pituitary cell population (41); have less nuclear receptor than somatotrophic cells (38); and, as estimated by immunoperoxidase staining, grow poorly under culture conditions (44). If thyroid hormone controlled the level of thyroid hormone nuclear receptors in the somatotroph in vivo, extrapolation ofour observations on hormone-mediated reduction of the receptor and the growth hormone response in GH, cells (6, 7) may allow prediction of the growth hormone response in different thyroidal states in vivo. ...
We have previously demonstrated that L-triiodothyronine (L-T3) induces an increase in growth hormone synthesis and messenger RNA in cultured GH1 cells, a rat pituitary cell line. In addition to regulating the growth hormone response, L-T3 elicits a time- and dose-dependent reduction in the level of its nuclear receptor, which is a direct function of the occupancy of the receptor binding site. In this study we have compared the relative affinity of L-T3, triiodothyroacetic acid, D-triiodothyronine (D-T3), and L-thyroxine (L-T4) for the receptor with the induction of the growth hormone synthesis and the ability of these compounds to elicit a reduction in thyroid hormone nuclear receptor levels. Triiodothyroacetic acid and D-T3 were specifically examined because the biologic effect of these compounds in the intact rat is significantly lower than predicted by their affinity for the receptor using isolated rat liver nuclei in vitro. In intact cells each compound demonstrated an excellent relationship between the relative receptor affinity, the induction of growth hormone production, and the concentration-dependent reduction in nuclear receptor levels. With the exception of D-T3, the relative affinity of iodothyronine was identical for the receptor using intact cells in serum-free media, or isolated GH1 cell nuclei in vitro. The apparent receptor affinity of D-T3 with intact cells was 5.5-fold lower than with isolated nuclei, which suggests a decrease in cell entry of D-T3 relative to the other iodothyronines. Quantitation of the [125I]iodothyronine associated with the receptor in GH1 cells after a 36-h incubation with L-125I-T4 was 90% L-T4 and 10% L-T3, which indicates that the major effect of L-T4 in GH1 cells is a result of intrinsic L-T4 activity. Studies with dispersed rat anterior pituitary cells demonstrated that L-T3 induces growth hormone synthesis and elicits a reduction in nuclear receptor levels in the same fashion as GH1 cells. The observation that thyroid hormone influences dispersed rat pituitary cells in a fashion qualitatively similar to GH1 cells may have implications for the growth hormone response of the somatotroph cell in vivo to different thyroidal states.
... We report here the first demonstration of successful transient expression of the mouse TSHp gene promoter in thyrotrope cells. TtT 97 tumors (Furth, 1955; Condliffe et al., 1969) contain a pure population of thyrotropes and represent a relatively untransformed model system which still responds in vivo and in culture to physiological regulators such as T3 and thyrotropin-releasing hormone (Gershengorn, 1978; Caccicedo et al., 1981). Therefore, any effects observed in gene transfer studies using these cells will reflect the situation in thyrotropes. ...
In TtT 97 cells, a thyrotropin-producing mouse pituitary tumor, thyroid hormone rapidly inhibits the transcription rate of both the thyrotropin α- and β-subunit (TSHβ) genes, and this closely parallels the increase in nuclear thyroid hormone receptor occupancy. In this study, we have identified regions of the mouse TSHβ gene which are involved in mediating tissue-specific and thyroid hormone-regulated expression. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the firefly luciferase gene. Following transfection by electroporation, efficient expression of TSHβ 5'-flanking luciferase constructs occurred only in cells derived from TtT 97 tumors which express the endogenous TSHβ gene. Deletion analysis demonstrated that the region of the 5'-flanking DNA between positions -271 and -80 relative to the major transcriptional start site is important for TSHβ promoter activity in thyrotropes. No expression was measurable in mouse L cells, a fibroblast line, whereas a low level of expression was seen in MGH 101A cells derived from a thyrotropic tumor which no longer expresses the TSHβ gene. Reduced expression of TSHβ constructs was also found in GH3 and GH4 pituitary tumor lines. Addition of thyroid hormone effectively inhibited the level of transient TSHβ promoter activity in TtT 97 cells in a dose-dependent manner. The inhibitory effect was more pronounced and more accurately reflected the transcription rate data when transfected cells were derived from tumors treated with thyroid hormone for 5 days prior to transfection. Deletion of all but 46 base pairs of TSHβ gene 5'-flanking DNA and 3 base pairs of the first exon had no effect on thyroid hormone inhibition. This indicates that signals sufficient for transcriptional regulation of the TSHβ gene by thyroid hormone reside in the vicinity of the proximal promoter and may act by interfering with basal transcriptional factors.
... Synthetic human a 1-39 ACTH was used for iodination and as standard. Alpha (7), thyrotropin (TSH) (7,8,20), and prolactin (21) were measured by radioimmunoassays using reagents for rat hormones from the NIAMDD Hormone Distribution Program (Dr. A. F. Parlow) as previously described. ...
ACTH-producing mouse pituitary tumor cells in culture (AtT-20/NYU-1 cells) were found to have binding sites for thyrotropin-releasing hormone (TRH). These putative receptors bound TRH with high affinity; the apparent equilibrium dissociation constant was 3.7 nM. The affinity of the receptors for a series of TRH analogues was similar to those previously reported for TRH-receptor interactions on thyrotropic and mammotropic cells in culture. Like some human pituitary tumors in situ, AtT-20/NYU-1 cells were found to produce the alpha subunit of the glycoprotein hormones (alpha). Alpha accumulation in the medium was constant (3.1 ng/mg cell protein per h) and was not affected by TRH. In contrast, TRH increased the amount of ACTH accumulated in the medium from AtT-20/NYU-1 cells to 190 and 420% of control at 1 and 24 h, respectively. TRH induced a dose-dependent increase in ACTH release during a 30-min incubation; half-maximal stimulation occurred at approximately 0.1 nM. TRH had no effect on ACTH release in vitro from anterior pituitary cells derived from normal rats. Because TRH stimulates release of ACTH in some untreated patients with Cushing's disease and Nelson's syndrome as well as pathological states associated with pituitary tumors (but not in normal subjects), AtT-20/NYU-1 cells may serve as an important in vitro model for human pituitary ACTH-secreting adenomas. Moreover, these findings suggest that the primary abnormality in Cushing's disease and Nelson's syndrome, allowing TRH stimulation of ACTH release, may be intrinsic to neoplastic adrenocorticotrophs rather than in neuroregulation of ACTH release.
Radioimmunoassay (RIA) systems have been developed to quantitate virtually every hormone available in pure form. This exquisitely sensitive technique has revolutionized the fields of endocrine physiology and clinical endocrinology. Bioassay techniques which have been employed for many years are not sufficiently sensitive to measure accurately all the anterior pituitary hormones in plasma; the development of RIAs in biologic fluids and tissues has permitted studies which have greatly expanded our knowledge of the factors involved in anterior pituitary hormone synthesis, metabolism, and action. A chapter on the general principles of RIAs for anterior pituitary hormones would have the disadvantage of being repetitive, several excellent reviews on this topic being already available in the literature.
For decades, the mouse has been actively studied for its own endocrine physiology and for problems in mammalian endocrinology. This chapter focuses on the mouse endocrinology. The mouse pituitary is similar in shape to the pituitary of the rat and weighs about 2 mg. The mouse pituitary gland is compressed dorsoventrally with the anterior lobe most ventral and accounting for the shape of the gland. The intermediate lobe and neural lobe lie dorsally in a scooped out depression in the center of the anterior lobe, and it can be separated easily from the anterior lobe because of the hypophyseal cleft between the two lobes. The anterior lobe contains at least five cell types, distinguishable by their hormone products: (1) growth hormone, (2) prolactin, (3) adrenocorticotropin, (4) thyroid-stimulating hormone, (5) luteinizing hormone, (6) and follicle-stimulating hormone. The intermediate lobe of the mouse pituitary contains two major cell types, those that secrete adrenocorticotropin (ACTH) and those that secrete melanocyte-stimulating hormone. Those secreting ACTH seem to be localized in a rostral zone of the pars intermedia where they have direct contact with the nervous tissue of the pituitary stalk.
The pituitary glycoprotein hormones, thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (PSH), and the placental hormone chorionic gonadotropin (CG) constitute a family of hormones, each of which consists of two dissimilar, non-covalently bound subunits, α and β (1). In a particular species, these hormones share a common α subunit, whereas each β subunit is unique and confers biologic specificity to the complete dimeric hormone. Each subunit is glycosylated, the α and β subunits of TSH having two and one oligosaccharide side chains, respectively, which are N-linked to the protein through asparagine residues (1). Glycosylation is required for subunit combination and protection from intracellular degradation, but not for secretion (2,3). Both subunits are required for biologic activity (1).
Production of pituitary thyrotropin (thyroid-stimulating hormone; TSH), the major modulator of secretion of L-triiodothyronine (L-T3) and L-thyroxine (L-T4) from the thyroid gland, is, in turn, regulated by the circulating thyroid hormone level. Superimposed on the pituitary-thyroid feedback system is a modulation by the central nervous system that appears to be mediated, in large part, by hypothalamic thyrotropin-releasing hormone (TRH). The studies described herein were undertaken to define the intracellular events that mediate TRH stimulation of the thyrotroph, in particular, TRH stimulation of TSH secretion.
This chapter explains the effect of thyroid hormone on the growth, development, and metabolism of virtually all tissues of higher organisms. These diverse effects of thyroid hormones in various tissues may be explained by several possible mechanisms. The first is that T3 and T4 control a wide variety of biochemical processes in the cell by completely independent mechanisms. The second possibility is that all of these diverse effects of thyroid hormones are controlled by a single primary biochemical action common to all cells and tissues which, in turn, mediates the wide variety of tissue effects resulting from thyroid hormone administration. By this formulation, the biological effects of thyroid hormone in cells and tissues can be separated into primary and secondary responses. The primary response would represent the initial biochemical event regulated by thyroid hormone, which in turn, modulates the secondary biological processes, which are quantitated as the tissue effects induced by thyroid hormone.
We have recently described a mouse pituitary tumor line, MGH 101A which is derived from a TSH-producing thyrotropic tumor line and now produces only the α-subunit of the glycoprotein hormones. In these studies, we have investigated the mechanism for the lack of TSHβ subunit expression in MGH 101A, as well as the failure of triiodothyronine (T3) to regulate α-subunit. Southern blot analysis of restriction endonuclease-digested DNA from MGH 101A tumors indicates the presence of a TSHβ gene and an α-subunit gene indistinguishable from those in a TSH-producing tumor (TtT 97). In MGH 101A tumors, however, TSHβ gene transcription was minimal (4 ± 2 ppm) relative to α-subunit (283 ± 29 ppm) and there was no significant difference in transcription after T3 treatment. In contrast, TtT 97 tumors had nearly equal rates of α-subunit (375 ± 25 ppm) and TSHβ (386 ± 31 ppm) gene transcription, and T3 suppressed the transcription of α-subunit and TSHβ genes by 76% and 87%, respectively. The MGH 101A tumor contained T3 receptors with a binding affinity (1.54 × 10−10 M) similar to receptors on TtT 97 tumors (1.78 × 10−10 M), but at a lower concentration (2800 vs. 4000 sites/cell). We conclude that the absence of TSHβ production in MGH 101A tumors is not due to the absence of the TSHβ gene, but perhaps to some other modification of the gene structure. This could also explain the failure of MGH 101A tumors to respond to T3, since they do contain T3 receptors of normal affinity.
Thyroid hormone (T3) negatively regulates TSH b-subunit (TSHb) messenger RNA (mRNA) gene expression in whole rat pituitary, in part at the level of mRNA stability. However, the regulation of TSHb mRNA turnover by T3 in pure populations of thyrotropes and in other species is unknown. To further investigate this, we used murine thyrotropic TtT97 tumor cells. Using primary cultures of TtT97 cells, T3 down-regulated TSHb mRNA to ;35% of the control level by 8 h. Actinomycin D chase revealed that T3 destabilized TSHb mRNA, reducing the half-life from ;24 to 7 h, and was accompanied by a decrease in TSHb mRNA size. Ribonuclease H analysis revealed that this T3-induced decrease in size was due to a shortening of poly(A) tail from ;160 to ;30 nucleotides and was specific for TSHb mRNA. Cycloheximide mimicked the poly(A) tail effect observed with T3 .I n the absence of T3, actinomycin D deadenylated TSHb mRNA without inducing rapid decay. We conclude that T3 reduces the steady state half-life of TSHb mRNA in murine TtT97 thyrotropic tumor cells accompanied by a reduction in poly(A) tail length. However, in the absence of T3, deadenylation alone is not sufficient to induce TSHb mRNA decay. Together with the high degree of sequence conservation in the 39-untranslated region of murine and rat TSHb mRNA se- quences and the similarities of the T3 effect, these data provide the first evidence for a highly conserved posttranscriptional mechanism operative across species. We propose a model in which T3 coordinately regulates shortening of the poly(A) tail and the activity of a trans- acting RNA-binding protein and/or an exonuclease to accelerate TSHb mRNA turnover. (Endocrinology 139: 1093-1100, 1998)
Five hypothyroid patients are reported with increased pituitary TSH response to TRH during administration of T3. In one patient treated with intravenous T3, 50 μg daily for 10 days, the peak serum TSH and total pituitary TSH reserve after TRH increased coincident with increases in serum T3 and T4 levels and a decrease in the basal TSH concentration. In four patients treated with oral T3, the peak serum TSH and total pituitary TSH reserve after TRH increased during administration of subphysiological doses of T3. Peak serum T3 levels occurred 4 h after ingestion and increased progressively with increasing T3 doses. Serum TSH levels decreased modestly with the nadir at 4 h after T3 ingestion and then returned to basal levels at 24 h. Augmentation of TSH responses to TRH occurred simultaneously with decreases in serum cholesterol, a swell as increases in the pituitary prolactin response to TRH, and increases in the GH and cortisol response to insulin induced hypoglycaemia where these responses could be studied. These data demonstrated a positive effect of subphysiological T3 therapy in these hypothyroid patients on the TSH response to TRH as well as increases in the responses of other pituitary hormones to stimulation.
Receptors for thyrotropin-releasing hormone (TRH) are present on mouse pituitary thyrotropic tumor cells. Incubation of thyrotropes with 100 nM TRH or 4 nM L-triiodothyronine (T3) for 48 h decreased the number of TRH receptors to approximately equal to 50 and 20% of control, respectively. There was no effect on the equilibrium dissociation constant which was 3-5 nM. The depletion in the number of available TRH receptors was time- and dose-dependent. TRH, 100 nM, decreased the receptor number to 70% after 24 h, 50% after 48 h, and 45% of control after 72 h. T3, 4 nM, decreased the receptor number to 52% after 24 h, 20% after 48 h, and 17% of control after 72 h. After 48 h, half-maximal depletion occurred with 1-2 nM TRH and approximately equal to 0.15 nM T3. Incubation with 100 nM TRH and 4 nM T3 caused a significantly greater reduction in the receptor level than either hormone alone. The decrease in the receptor level was reversible within 72 h after removal of TRH, 100 nM, but was only partially reversed, from 20 to 40% of control, after removal of T3, 4 nM, after 120 h. By regulating the number of available TRH receptors on the thyrotrope. TRH and T3 interact to control thyrotropin release.
The nuclear receptor affinity for L-triiodothyronine (L-T3), L-thyroxine (L-T4), L-triiodothyroacetic acid (triac), and D-triiodothyronine (D-T3) was compared to the potency of these thyroid hormone analogues in regulating thyrotropin (TSH) production and the number of membrane receptors for thyrotropin-releasing hormone (TRH) in mouse thyrotropic tumor cells in culture. L-T3 and triac were equally potent and D-T3 was one-sixth to one-fifth as potent in binding to the receptor and in regulating TSH production and TRH receptor number. L-T4 was the least potent analogue in each instance, but its relative receptor-binding affinity, measured after 3 h, was significantly less than its somewhat variable relative biological potency, measured after 48 h. The cells were shown to monodeiodinate L-[125I]T4 to L-[125I]T3 in a time-dependent manner, and the enhanced biological potency of L-T4 was ascribed to its conversion to L-T3. Thyroid hormones appear to regulate TSH production and the number of receptors for TRH in thyrotropic cells in culture through interaction with a nuclear receptor.
Our recent in vivo studies have suggested that intrapituitary l-thyroxine (T(4)) to 3,5,3'-triiodo-l-thyronine (T(3)) conversion with subsequent nuclear binding of T(3) is an important pathway by which circulating T(4) can inhibit thyrotropin release. The present studies were performed to evaluate various physiological and pharmacological influences on these two processes in rat anterior pituitary tissue. Intact pituitary fragments were incubated in buffer-1% bovine serum albumin containing 0.14 ng/ml [(131)I]T(3) and 3.8 ng/ml [(125)I]T(4). Nuclei were isolated after 3 h of incubation and the bound iodothyronines identified by paper chromatography. There was 0.3-1% [(125)I]T(3) contaminating the medium [(125)I]T(4), and this did not change during incubation. Nuclear [(125)I]T(4) was not decreased by 650-fold excesses of medium T(3) or T(4), suggesting that it was nonspecifically bound. The ratio of nuclear to medium [(131)I]- and [(125)I]T(3) were expressed as nuclear counts per minute per milligram wet weight of tissue:counts per minute per microliter medium. Intrapituitary T(4) to T(3) conversion was evidenced by the fact that the nuclear:medium (N:M) ratio for [(131)I]T(3) was 0.45+/-0.21, whereas that for [(125)I]T(3) was 2.23+/-1.28 (mean+/-SD, n = 51). A ratio (R), the N:M [(125)I]T(3) divided by the N:M [(131)I]T(3), was used as an index of intrapituitary T(4) to T(3) conversion. Increasing medium T(3) concentrations up to 50 ng/ml caused a progressive decrease in the N:M ratio for both T(3) isotopes, but no change in the value for R, indicating that both competed for the same limited-capacity nuclear receptors. Increasing concentrations of medium T(4) caused no change in the N:M [(131)I]T(3) but did cause a significant decrease in R in three of four experiments. These results suggest saturation of T(4)-5'-monodeiodination occurred at lower T(4) concentrations than saturation of nuclear T(3) binding sites. In hypothyroid rats, the N:M ratios for both [(131)I]T(3) and [(125)I]T(3) were increased (P < 0.005), but R was three-fold higher than in controls (P < 0.005). Animals given 10 mug T(4)/100 g body wt per d for 5 d had significantly decreased N:M ratios for both [(131)I]T(3) and [(125)I]T(3), as well as a decreased value for R. In fasted rats, neither N:M ratio was depressed, although hepatic T(4) to T(3) conversion in the same animals was 50% of control (P < 0.005). Iopanoic acid (13 muM), but not 6-n-propylthiouracil (29 muM), decreased the N:M [(125)I]T(3) with a significant decrease in the value for R (P < 0.025 or less). Neither sodium iodide (6 muM) nor thyrotropin-releasing hormone (7-700 nM) affected the T(3) N:M ratios. These results indicate that intrapituitary T(4) to T(3) conversion is stimulated in hypothyroidism and depressed in T(4)-treated animals, whereas opposite changes occur in hepatic T(4)-5'-monodeiodination. Unlike liver, anterior pituitary T(4)-5'-monodeiodination is not affected by fasting or incubation with 6-n-propyl-2-thiouracil, but T(4) to T(3) conversion is inhibited in both by iopanoic acid. These results indicate that there are important differences between anterior pituitary and other tissues in the regulation of T(4)-5'-monodeiodination.
High-affinity, limited-capacity, 3,5,3'-triiodo-L-thyronine (T3)-binding sites were established by in vitro saturation analysis in cell nuclei of the pituitary gland of arctic charr. The sites were extracted from the purified nuclei using 0.4 M NaCl and incubated with [125I]T3 in the presence of 0.2 M NaCl. T3 saturable binding attained equilibrium after 18-24 hr of incubation at 4 degrees. The association constant ranged from 6.7 to 20.1 liters.mol-1 x 10(9), indicating a T3 affinity greater than that for T3-binding sites in rainbow trout liver. The maximal binding capacity ranged from 0.93 to 2.05 10(-13) mol.mg DNA-1, representing a mean site abundance corresponding to 60% of that for nuclei from trout liver. Thyroxine (T4) completely displaced [125I]T3 in the pituitary nuclei of arctic charr and T3 completely displaced [125I]T4 in the pituitary nuclei of rainbow trout, suggesting that in salmonids both T4 and T3 bind to the same single class of sites. However, the site affinity for T4 was approximately 20-50x less than that for T3. The possible roles of these sites in pituitary function as well as their relationship to other nuclear T3-binding sites in salmonid fish are discussed.
The gene encoding the common alpha subunit of the rat pituitary glycoprotein hormones was isolated from a rat genomic DNA library. The gene spans approximately 8 kb, and contains four exons and three intervening sequences of 5.4 kb, 1.1 kb and 0.6 kb. Blot hybridization of restriction enzyme digests of rat genomic DNA suggests that the alpha gene is present in a single copy. The coding region and 424 bp of the 5'-flanking region of the gene were sequenced. Primer extension and S1 nuclease analyses revealed a single transcriptional start point downstream from consensus promoter elements. The organization of the rat alpha-subunit gene is similar to that of the human and bovine genes including the sizes and locations of the four exons and three introns. In addition, a region of strong sequence similarity has been identified in the 5'-flanking region of the rat, human and bovine genes. This region includes sequences which are similar to a putative triiodothyronine regulatory element and the previously identified cAMP regulatory region; such sequences may mediate the known effects of these factors on alpha-subunit gene expression.
An important physiological control of the glycoprotein hormone alpha-subunit is the negative feedback by thyroid hormones in the thyrotrope. A region of the rat glycoprotein hormone alpha-subunit gene that is involved in transcriptional regulation by thyroid hormone has been identified by transient transfection studies, and sequence-specific binding of the thyroid hormone receptor to a site within this region has been demonstrated. Deletion-mutation studies using plasmid expression vectors containing either 246, 170, or 80 base pairs of the 5'-flanking region of the rat alpha-subunit gene fused to the coding region of the bacterial chloramphenicol acetyltransferase gene demonstrate 3,5,3'-triiodo-L-thyronine (T3)-regulated expression in GH3 cells, a T3-responsive somatotrophic cell line. In order to investigate the possibility of thyroid hormone receptor interaction with this segment of the rat alpha-subunit gene, the binding of the thyroid hormone receptor to synthetic oligodeoxynucleotides was analyzed using an avidin-biotin complex DNA binding assay. An oligodeoxyribonucleotide representing a fragment of the alpha-subunit gene from -74 to -38, relative to the transcriptional start site, shows significant binding to [125I]T3-receptor complex present in nuclear extracts of GH3 cells. This fragment binds receptor to a degree similar to that seen with a fragment of the rat growth hormone gene which contains a putative thyroid hormone-responsive element. In addition, this fragment of the rat alpha-subunit gene binds to the in vitro synthesized human c-erbA beta protein, which has been identified as a member of the family of putative T3 receptors. These data demonstrate that a cis-active thyroid hormone-responsive element resides in the 5'-flanking region of the rat alpha-subunit gene and that the mechanism involved in the suppression of expression of this gene by T3 could involve specific binding of the thyroid hormone receptor to this region of the gene.
Thyroid-stimulating hormone (TSH) is a pituitary-derived glycoprotein of molecular weight 28,000 that is composed of two noncovalently linked subunits, α and β. TSH is chemically related to the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as to placental chorionic gonadotropin (CG). TSH is synthesized by thyrotropes of the anterior pituitary and stored in secretory granules. TSH is released into the circulation in a regulated manner, binds to thyroid cells and activates them to release thyroid hormones.
The ability of cells that produce growth hormone (GH) and cells that produce adrenocorticotropic hormone (ACTH) to differentiate in various culture media was analyzed by means of ultrastructural immunocytochemistry on 13-day embryonic mouse pituitaries that were maintained in organ culture for 3-11 days. At the time of culture, relatively undifferentiated nongranulated or poorly granulated cells that were unreactive with anti-growth-hormone serum (anti-GH) and anti-adrenocorticotropic-hormone serum (anti-ACTH) were present in the pituitary. After 10-11 days in culture, immunoreactive GH cells were obtained only in media that were supplemented with cortisol, whereas ACTH cells were obtained in all media tested, including Medium 199 alone. In cortisol-supplemented media, the GH cells showed ultrastructural features typical of those that occur in vivo, and anti-GH immunoreactivity was obtained after as little as 3 days in culture, i.e., at a stage comparable to that which occurs in vivo. The results indicate that mouse GH cells are capable of differentiating in Medium 199 supplemented only with cortisol, without the addition of fetal calf serum or insulin; cortisol therefore appears to be an essential component of the embryonic milieu for the production of GH-secretory granules.
Elevation of the concentration of free calcium ion in the cytoplasm of cells ([Ca²⁺]i) is an early event following the binding of many stimuli to plasma membrane receptors. The free (or ionized) calcium then plays an important role as a coupling factor leading to the activation of the final cellular response (“stimulus-response coupling”). The elevation of [Ca²⁺]i is caused by the mobilization (or redistribution) of cellular calcium, enhanced influx of extracellular Ca²⁺, inhibition of the efflux of intracellular calcium, or a combination of these processes. The chapter describes the methods that can be employed to study these various aspects of cellular calcium homeostasis, including the direct measurement of [Ca²⁺]i. It discusses studies of two pituitary cell types in vitro––mammotropic (GH3) and thyrotropic (TtT) cells in culture. The examples presented to illustrate the use of these methods are from studies that attempt to define the mechanism of the action of thyrotropin releasing hormone (TRH) in these two cell types.
We studied the effect of thyroid hormone on the transcription of the genes for the alpha- and beta-subunits of thyrotropin (TSH) in thyrotropic tumors (IAK 109D and 109F) carried in hypothyroid mice. Gene transcription was measured in isolated nuclei by allowing completion of RNA chains initiated in vivo in the presence of [alpha-32P]UTP and by hybridization of labeled RNA transcripts to filter-bound plasmids containing alpha or TSH-beta cDNA sequences. Treatment of animals carrying tumor IAK 109D with 3,5,3'-triiodo-L-thyronine (T3) (5 micrograms/100 g body weight) for 2 hr reduced TSH-beta gene transcription to less than 10% of control levels, whereas alpha RNA synthesis was reduced to 59% of control. The inhibition of TSH-beta gene activity was maintained after 6 hr of T3 treatment, whereas alpha gene transcription rose slightly to 77% of control. The tumor content of alpha and TSH-beta mRNA, determined by dot blot hybridization with 32P-labeled plasmid probes containing alpha or TSH-beta cDNAs, was unchanged after 2 hr of T3 treatment, and each was reduced by approximately 25% at 6 hr. These untreated tumors contained approximately equal amounts of alpha and TSH-beta mRNA. However, the basal rate of TSH-beta gene transcription was threefold greater than that of alpha gene transcription. Treatment of animals bearing tumor IAK 109F with the same dose of T3 for 30 min did not significantly affect alpha or TSH-beta gene transcription, but at 2 hr alpha and TSH-beta RNA synthesis had decreased to 50% and 10% of control values, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Thyrotropin releasing hormone (TRH) acutely stimulates release of thyrotropin (TSH) and prolactin from anterior pituitary cells. A considerable number of studies have been performed with neoplastic and nonneoplastic pituitary cells in culture to elucidate the sequence of intracellular events involved in this action. Although cyclic AMP was suggested as an intracellular messenger, it has been demonstrated that TRH stimulation of hormone release can be dissociated from changes in cyclic AMP concentration, thereby supporting the contention that cyclic AMP is not a required mediator. In contrast, stimulation of hormone release by TRH requires Ca2+ and it seems likely that Ca2+ is the intracellular coupling factor between TRH stimulation and hormone secretion. TRH has been shown to stimulate 45Ca2+ efflux from preloaded pituitary cells. Enhanced 45Ca2+ efflux is thought to reflect an increase in the free intracellular Ca2+ concentration which leads to hormone release; however, the source of this Ca2− is uncertain. Results are reviewed from a series of experiments in pituitary cells which attempt to determine the pool (or pools) of Ca2+ that is affected by TRH. These include the following: the effects of decreasing the extracellular Ca2− concentration on hormone release stimulated by TRH; the effect of TRH on cellular Ca2+ as monitored by chlortetracycline; the effects of TRH on Ca2+ influx; the effects of the organic Ca2+ channel blocking agents, verapamil and methoxyverapamil, on TRH-stimulated hormone release; and the effects of TRH on plasma membrane potential difference and on Ca2+-dependent action potentials. Based on these data, separate hypotheses of the early events in TRH stimulation of hormone release in mammotropes and thyrotropes are proposed. In mammotropes, TRH is thought to stimulate prolactin release optimally by elevating the free intracellular Cat+ concentration by mobilizing cellular Ca2− only. In contrast, in thyrotropes under normal physiological conditions, TRH is thought to stimulate TSH release by mobilizing Ca2 from a cellular pool (or pools) and to augment this effect by also inducing influx of extracellular Ca2+ through voltage-dependent channels in the plasma membrane.
A subclone of the FU5-5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein alpha 2u-globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 X 10(-6) of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4-8 X 10(-3). Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of alpha 2u-globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum-free medium raised the RSR to only 3 X 10(-5); exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6-20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t 1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 X 10(-8)M and 3 X 10(-11)M, respectively. Higher concentrations of insulin, although still in physiological range (10(-9)M), inhibited induction. At yet higher insulin levels, beyond the physiological range, alpha 2u-globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for alpha 2u-globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with alpha 2u-globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.
We have studied the regulation of the biosynthesis of thyrotropin (TSH) and its alpha and beta subunits by thyroid hormone in thyrotropic tumors carried in hypothyroid mice. Treatment with 3,5,3'-triiodo-L-thyronine (T3) (20 micrograms/100 g, body weight) daily for 4 or 10 days reduced serum TSH to 3 and 0.3% of control, respectively. Serum levels of free alpha subunit were reduced to 60 and 11% of control at 4 days and 10 days, respectively, and serum free TSH-beta was undetectable at both time points. There was no significant decrease in tumor TSH content after 4 days of treatment and, after 10 days, TSH content was reduced to 15% of control levels. There was no significant effect of T3 on tumor alpha subunit levels at either 4 or 10 days. In contrast, tumor TSH-beta content was markedly reduced after 4 days and 10 days of T3 treatment, to 29 and 10% of control levels, respectively. Translation of tumor poly(A) mRNA in a rabbit reticulocyte lysate system showed that thyroid hormone decreased translatable TSH-beta mRNA to undetectable levels at both 4 and 10 days, whereas translatable alpha mRNA was reduced strikingly only at 10 days in one of two tumors. RNA blot hybridization with 32P-labeled plasmid probes containing alpha or TSH-beta cDNAs showed that TSH-beta mRNA was reduced to less than 10% of control after both 4 and 10 days of T3 treatment, whereas, again, alpha mRNA was only reduced in one of two tumors at 10 days. Our data thus show that thyroid hormone affects alpha and TSH-beta mRNA and protein levels discordantly and suggest that regulation of TSH biosynthesis may occur predominantly at the level of TSH-beta mRNA.
Thyrotropin (TSH), a glycoprotein hormone of the pituitary consisting of two subunits (α and β), regulates thyroxine (T4) production by the thyroid gland. T4, in turn, regulates TSH biosynthesis and release. We have studied the regulation of the messenger RNA encoding the α subunit of TSH by T4 in pituitaries and in a transplantable thyrotropic tumor in mice. Hypothyroid male LAF1 mice bearing the TtT 97 thyrotropic tumor were injected daily with T4 for either 0, 1, 5, 12, or 33 days. Levels of TSH and its unassociated α (free α) and TSH-β subunits in the plasma of these animals fell to less than 5% of control values after 33 days. Concentrations of TSH and TSH-β in both tumor and pituitary also fell to low levels (<2% of control), while intracellular concentrations of free α subunit remained unchanged. Cellular levels of the mRNA encoding the precursor of the α subunit or pre-α (α mRNA) were measured by cell-free translation followed by electrophoretic analysis of immunoprecipitates of pre-α subunit and by nucleic acid hybridization to a radiolabeled cDNA probe specific for the α mRNA. In the pituitary, translatable and hybridizable α mRNA was decreased slightly after 1 day of T4 and decreased 40-50% after 5 and 12 days. In thyrotropic tumors, both translatable and total α mRNA showed a 60% decrease by 1 day and a maximum 85% decrease after 5, 12, and 33 days of T4. Therefore, T4 acts rapidly in vivo to decrease steady state α mRNA levels in the thyrotrope, and this decrease is maintained for the duration of treatment with thyroid hormone. This regulatory process is reflected in the sharp decreases in levels of TSH and free α subunit in plasma and in lower concentrations of the intact TSH in tissue. In contrast, the maintenance of high tissue concentration of free α subunit after T4 treatment may be a reflection of alterations in a post-translational process specific for the free α subunit, as opposed to that of the intact TSH.
This chapter discusses the regulation and organization of thyroid stimulating hormone (TSH) genes. The chapter discusses the development of highly sensitive and specific radioimmunoassays for each subunit after the dissociation of human TSH and purification of the α and β subunits. In an experiment described in the chapter, subunit secretion and regulation in clinical studies in humans were observed. In addition, extracts of normal human pituitary glands obtained postmortem were prepared, and the extracts were fractionated by gel chromatography on Sephadex G-100. In spite of variability in subunit content among normal pituitaries, a consistent excess of free α subunits relative to the sum of all free pituitary β subunits was demonstrated in each normal pituitary, with an α-to-β subunit ratio ranging from 1.3 to 8.3. These studies supported the concept that biosynthesis of the unique β subunits was limiting in the production of complete glycoprotein hormones.
Thyroid dysfunction is common in older individuals, yet the diagnosis is often complicated by atypical clinical presentations and difficulty in interpretation of laboratory tests. An understanding of the alterations in thyroid function occurring normally as a consequence of the aging process is necessary for correct laboratory diagnosis of thyroid dysfunction in the elderly. There are subtle alterations in hypothalamic and pituitary function but normal feedback control of TSH secretion persists. In the thyroid itself, morphologic changes develop with age, but have little impact on thyroid hormone economy. Thyroidal secretion of thyroxine decreases, but parallels the decrease in thyroxine degradation rate, resulting in unaltered plasma thyroxine levels. Decreased peripheral conversion of thyroxine to triiodothyronine causes a fall in triiodothyronine concentrations. Nonthyroidal illnesses in the elderly may perturb the laboratory assessment of thyroid function by producing isolated high or low thyroxine levels in euthyroid individuals.
Chlortetracycline (CTC), a probe of membrane-bound divalent cations, was used to study the action of thyrotropin-releasing hormone (TRH) in mouse pituitary thyrotropic tumor (TtT) cells in culture. Cellular fluorescence of CTC was caused by both Ca2+- and Mg2+-CTC complexes and was influenced by the concentration of these cations in the incubation medium. TRH, but not other peptides, caused a rapid, transient, and concentration-dependent decrease in the CTC fluorescence intensity; half-maximal effect occurred with 10--30 nM TRH. The decrement in fluorescence intensity caused by TRH was not due to enhanced loss of CTC from the cells. The decrease in fluorescence elicited by TRH was specific for Ca2+-CTC complexes because preincubation of the cells with 1 mM EGTA or 1 mM EDTA plus 2.05 mM Mg2+ abolished the response, whereas preincubation with 1 mM EDTA plus 2.05 mM Ca2+ permitted the usual TRH response. Antimycin A and carbonyl cyanide m-chlorophenylhydrazone decreased cellular ATP content to 37 +/- 1 and 32 +/- 1% of control, respectively, and abolished the TRH-induced decrease in CTC fluorescence. We conclude that TRH displaced Ca2+ from an energy-dependent, membrane-bound pool(s) within TtT cells and that this may be one mechanism by which the concentration of intracellular free Ca2+ is raised so that is couples stimulation by TRH to TSH secretion.
We have studied the contribution of thyroxine (T4) itself to the feedback regulation of thyrotropin (TSH) secretion. Thyroidectomized rats received T4 replacement by one of two methods: 1) 5 or 10 micrograms T4 . 100 g-1 . day-1 in drinking water or 2) continuous subcutaneous infusion of 0.5, 1.0, or 2.0 micrograms T4 . 100 g-1 . day-1. Replacement of 5 micrograms T4 . 100 g-1 . day-1 resulted in elevated plasma T4 and TSH while triiodothyronine (T3) was decreased. Replacement of 10 micrograms T4 . 100 g-1 . day-1 resulted in greater elevation of plasma T4, a transient decrease in T3, and no change in plasma TSH. Continuous replacement of T4 resulted in a dose-dependent elevation of plasma T4, little plasma T3 generation, and inhibition of the postthyroidectomy rise in plasma TSH. The pituitary responsiveness to thyrotropin-releasing hormone (TRH) was increased in groups receiving 1.0 or 2.0 micrograms T4 . 100 g-1 . day-1. It was concluded that 1) the plasma T4 exerts a negative feedback on basal TSH secretion in addition to that due to plasma T3 and 2) small amounts of T4 replacement enhance the TSH response to exogenous TRH in short-term hypothyroid rats.
Previous tracer studies have suggested that 5'-monodeiodination of l-thyroxine (T(4)) in anterior pituitary may contribute a substantial portion of specifically bound nuclear 3,5,3' l-triiodothyronine (T(3)) in this tissue in rats. To evaluate this possibility, a radioimmunoassay for nuclear T(3) in individual anterior pituitaries was developed. Animals received [(125)I]T(3) 60 min before removal of the anterior pituitary and isolation of the nuclei by differential centrifugation. This allowed calculation of the nuclear:serum T(3) ratio and comparison of expected with measured T(3). T(3) was extracted in ethanol, dried, and reconstituted in assay buffer. In untreated hypothyroid rats, anterior pituitary nuclear T(3) was 0.18 +/- 0.06 pg/mug DNA which was 0.13 pg/mug DNA greater than expected from the serum T(3) concentration and the pituitary nuclear:serum [(125)I]T(3) ratio. In 10 hypothyroid rats given a single bolus of 400 ng T(3)/100 g body wt., the nuclear T(3) by radioimmunoassay was 1.0 +/- 0.06 pg/mug DNA, whereas that expected from the T(3) specific activity calculations was 0.85 pg/mug DNA (P < 0.025). Serum T(4) concentrations in these rats were < 0.25 mug/dl but the nuclear T(3) derived from as little as 0.2 mug/dl T(4) could explain a large portion of these small discrepancies between observed and measured nuclear T(3). In 29 normal rats, anterior pituitary nuclear T(3) was 0.63+/-0.04 pg/mug DNA, whereas that expected from the serum T(3) concentration (55+/-2 ng/dl) was 0.23+/-0.02 pg/mug DNA (P < 0.001). Total pituitary T(3) based on this measurement was 92+/-6 pg. Because the maximal nuclear binding capacity for T(3) in rat anterior pituitary is 0.77 pg/mug DNA, these results suggest there is 82% occupancy of these nuclear receptors. The requirement for normal serum concentrations of both T(4) and T(3) to achieve normal nuclear T(3) saturation in anterior pituitary is in marked contrast to the situation in liver, kidney, and heart muscle which appear to require only a normal serum T(3). As a consequence, the anterior pituitary can monitor both serum T(4) and T(3) and respond appropriately to changes in their concentrations.
The effect of triiodothyronine (T3) on somatostatin (SS) mRNA levels in cultured fetal rat cerebrocortical cells was studied. Two different experimental approaches were sought. They differed in the length of time in which cells were deprived of thyroid hormones prior to the addition of exogenous T3. When the cells were not deprived of thyroid hormones, T3 caused a dose-related decrease in SS mRNA content at all doses tested. However, when the cells were deprived of T3 for 24 h, a biphasic effect was observed. These findings suggest that T3 regulates SS gene expression in fetal cultured cerebrocortical cells.
Transthyretin (TTR) is the principal carrier of thyroid hormones in rodent plasma and the major protein synthesized by the choroid plexus. Mice lacking TTR generated by targeted disruption (Episkopou, V., Maeda, S., Nishiguchi, S., Shimada, K., Gaitanaris, G. A., Gottesman, M. E., and Robertson, E. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2375-2379) had a 50% decrease in total thyroxine (T4) plasma levels but had normal free hormone levels as compared to wild-type mice. In the mutant serum there was increased T4 binding to thyroxine-binding globulin. Thyroxine-binding globulin mRNA levels were the same in mutant and wild-type animals. Wild-type serum depleted of TTR also presented increased T4 binding to thyroxine-binding globulin, suggesting that TTR competes with thyroxine-binding globulin for T4 binding. Total and free triiodothyronine and thyrotoropin-stimulating hormone levels were not affected by the absence of TTR. Liver deiodinase-I activity, mRNA levels, and brain deiodinase-II activity were normal in the mutant mice, suggesting that the absence of TTR does not affect tissue thyroid hormone content. The low T4 levels found in the mutant mice sera cannot be accounted for by increased glucuronidation because the liver activity of UDP-glucuronosyltransferase was not affected in the TTR-deficient mice. We concluded that transthyretin-deficient mice are euthyroid in the absence of the major plasma T4 carrier. We ascribed this to the normal free hormone levels in the serum of the mutant mice. Our data, therefore, strongly supported the free hormone hypothesis for thyroxine uptake (Mendel, C. M. (1989) Endocr. Rev. 10, 232-274).
Thyrotropic tumors (TtT97) contain mRNA transcripts for three T3-receptor (TR) isoforms, alpha 1, beta 1 and beta 2, and a non-receptor alpha 2-variant. We administered T4 (5 mg/l of drinking water) for one month to TtT97-bearing mice, to examine its effect on tumor growth and tumor TR isoform steady-state mRNA levels. Baseline mice were killed at the start of the experiment, and placebo mice were maintained hypothyroid. The treated tumors were 30-35% smaller than the baseline tumors (p = NS), while the placebo tumors were 2- to 7-fold larger than the baseline tumors (p < 0.05). TR beta 1 mRNA increased 5- to 6-fold, while TR beta 2 mRNA decreased by 76%. TR alpha 1 and the alpha 2-variant decreased by 52% and 70%, respectively. Therefore, the tumors decreased their growth rate in response to T4 administration, and increased the ratio of TR beta 1 to TR beta 2 mRNA. This raises the intriguing possibility of a correlation between the relative abundance of the TR beta isoforms and tumor growth.
Following the protracted hypothyroid state, treatment with thyroid hormone will induce a decline in TSH and reduce thyrotrope hyperplasia. Somatostatin is a hypothalamic peptide that has been implicated in the negative regulation of TSH secretion in the thyrotrope. Moreover, analogs of native somatostatin have potent TSH-reducing and growth-retarding effects on human thyrotropinomas. The TtT-97 tumor is an in vivo murine thyrotropic model that has retained its physiological response to thyroid hormone. This study investigates the regulation of somatostatin receptor subtypes in this tumor. TtT-97 tumors, actively growing in hypothyroid mice, did not express any significant somatostatin receptor messenger RNA (mRNA) or protein. T4 administration resulted in a reduction in TSH beta mRNA expression and a marked degree of tumor involution. Analysis of residual tumors from thyroid hormone-treated mice showed the specific up-regulation of SSTR1 and SSTR5 mRNA subtypes and the appearance of abundant, high affinity SSTR receptor-binding sites within the tumor. Thus, the TtT-97 tumor provides a thyrotrope-specific model in which to study the regulation of somatostatin receptor subtypes by thyroid hormone and correlate this expression with both antisecretory and antiproliferative effects.
Porcine TRF stimulates the release of TSH from rat anterior pituitaries in vitro at doses as small as 0.01 nanog. By increasing the doses of TRF, greater amounts of TSH are released into the incubation media. The pituitary response to TRF is inhibited by small amounts of T3 and T4. Act D does not abolish the response to TRF, indicating that de novo synthesis of TSH is not required for TRF to exert its effect, a- and β-MSH do not stimulate the release of TSH in vitro and did not reverse the inhibitory effect of T4 on TSH release in vitro. Preincubation with Act D reverses the inhibition of TSH release induced by T3 and T4. This may indicate that Act D interferes with the formation of inhibitory substances, induced by T3 and T4, which suppress the release of TSH after TRF.