Structural features of thyroid hormone response elements that increase susceptibility to inhibition by an RTH mutant thyroid hormone receptor

The chicken lysozyme silencer F2 (F2) thyroid hormone response element (TRE) contains an unusual everted palindromic arrangement, has a high affinity for thyroid hormone receptor (TR) homodimers, and is especially sensitive to dominant negative inhibition by, the T3 resistance (RTH) mutant TR beta P453H. We used various TREs and TR mutations to determine the mechanisms for this sensitivity. Changing the F2 orientation from an everted palindrome to a direct repeat with a 4-bp gap (DR+4) (F2-DR) decreased the sensitivity to inhibition at high T3 concentrations, while a loss of this sensitivity occurred with a palindromic arrangement of these same half-sites. F2 contains the dinucleotide TG 5' to each consensus half-site conforming to the optimal TR-binding octamer, YRRGGTCA. A T to A change in position 1 of both F2 half-sites markedly reduced T3-induction, yet only slightly reduced TR homodimer or TR-retinoid X receptor (RXR) heterodimer binding. The TR beta ninth heptad mutation, L428R, prevents TR heterodimerization with RXR and eliminates the inhibitory effect of the P453H mutant TR on the F2-DR, but not the F2 element. Structural features of a TRE that favor strong TR binding of both TR homodimers and TR-RXR heterodimers containing the mutant TR, such as the everted palindromic conformation or the optimal TR-binding consensus octamer, enhance the sensitivity of a TRE to inhibition by the mutant TR. Thus, both half-site orientation and sequence contribute to the sensitivity of a given TRE to dominant negative inhibition by a mutant TR.     

Thyroid hormone receptor binds with unique properties to response elements that contain hexamer domains in an inverted palindrome arrangement

Thyroid hormone receptor (T3R)-accessory protein heterodimers preferentially bind to thyroid hormone response elements (TREs), which contain hexamer domains arranged as a direct repeat separated by a 4-basepair gap (DR + 4). T3R homodimers, however, preferentially bind to elements that consist of an inverted palindrome. We now report on unique T3R binding patterns and functional characteristics of two such elements that mediate T3 regulation. We performed mutational analysis of the chicken lysozyme silencer F2 (LysF2) TRE and demonstrated that the two functional binding domains are arranged as an inverted palindrome separated by 6 basepairs. Both the LysF2 TRE and the similarly arranged myelin basic protein TRE bind T3R dimers at very low T3R concentrations. Despite the high relative affinity for T3R dimer binding, the T3 induction conferred by these elements is low compared to that of previously characterized TREs with a DR + 4 arrangement. The laminin-B1 gene element, previously shown to bind retinoic acid receptor, contains at least four hexameric binding domains. All of the domains can bind T3R simultaneously and are involved in conferring T3 induction, but bind with a different pattern than that reported for retinoic acid receptor. T3R homodimer binding to a series of mutant laminin-B1 elements and T3 induction were significantly correlated (r = 0.82; P < 0.05). T3R homodimer binding to LysF2 element mutants was not correlated with T3 induction (r = 0.32; P > 0.05); however, T3R-nuclear protein heterodimer binding was significantly correlated (r = 0.67; P < 0.05). T3R-nuclear protein heterodimers, but not homodimers, bound consistently to mutations of the LysF2 element that altered the gap between hexamers. The overall discordance between strong T3R binding to these elements and weak T3 induction indicates that the unusual hexamer arrangement places the T3R complex in an unfavorable configuration for maximal T3-dependent transactivation. The differential T3 sensitivity of generalized resistance to thyroid hormone-associated T3R mutants to the LysF2 element compared with the DR + 4 arrangement suggests that these unique features may have physiological significance.     

Thyroid hormone action on liver, heart, and energy expenditure in thyroid hormone receptor beta-deficient mice

Thyroid hormone (TH) responsive genes can be both positively and negatively regulated by TH through receptors (TR) alpha and beta expressed in most body tissues. However, their relative roles in the regulation of specific gene expression remain unknown. The TR beta knockout mouse, which lacks both TR beta1 and TR beta2 isoforms, provides a model to examine the role of these receptors in mediating TH action. TR beta deficient (TR beta-/-) mice that show no compensatory increase in TR alpha, and wild-type (TR beta+/+) mice of the same strain were deprived of TH by feeding them a low iodine diet containing propylthiouracil, and were then treated with supraphysiological doses of L-T3 (0.5, 5.5, and 25 microg/day/mouse). TH deprivation alone increased the serum cholesterol concentration by 25% in TR beta+/+ mice and reduced it paradoxically by 23% in TR beta-/- mice. TH deprivation reduced the serum alkaline phosphatase (AP) concentration by 31% in TR beta+/+ mice but showed no change in the TR beta-/- mice. Treatment with L-T3 (0.5 to 25 microg/mouse/day) caused a 57% decrease in serum cholesterol and a 231% increase in serum AP in the TR beta+/+ mice. The TR beta-/- mice were resistant to the L-T3 induced changes in serum cholesterol and showed increase in AP only with the highest L-T3 dose. Basal heart rate (HR) in TR beta-/- mice was higher than that of TR beta+/+ mice by 11%. HR and energy expenditure (EE) in both TR beta+/+ and TR beta-/- mice showed similar decreases (49 and 46%) and increases (49 and 41%) in response to TH deprivation and L-T3 treatment, respectively. The effect of TH on the accumulation of messenger RNA (mRNA) of TH regulated liver genes was also examined. TH deprivation down regulated spot 14 (S14) mRNA and showed no change in malic enzyme (ME) mRNA in both TR beta+/+ and TR beta-/- mice. In contrast treatment with L-T3 produced an increase in S14 and ME but no change in TR beta-/- mice. From these results, it can be concluded that regulation of HR and EE are independent of TR beta. With the exception of serum cholesterol concentration and liver ME mRNA accumulation, all other markers of TH action examined during TH deprivation exhibited the expected responses in the absence of TR beta. Thus, as previously shown for serum TSH, TR beta is not absolutely necessary for some changes typical of hypothyroidism to occur. In contrast, except for HR and EE, the full manifestation of TH-mediated action required the presence of TR beta.     

Glucocorticoids increase retinoid-X receptor alpha (RXRalpha) expression and enhance thyroid hormone action in primary cultured rat hepatocytes

A dynamic model of glucose overflow metabolism in batch and fed-batch cultivations of Escherichia coli W3110 under fully aerobic conditions is presented. Simulation based on the model describes cell growth, respiration, and acetate formation as well as acetate reconsumption during batch cultures, the transition of batch to fed-batch culture, and fed-batch cultures. E. coli excreted acetate only when specific glucose uptake exceeded a critical rate corresponding to a maximum respiration rate. In batch cultures where the glucose uptake was unlimited, the overflow acetate made up to 9. 0 +/- 1.0% carbon/carbon of the glucose consumed. The applicability of the model to dynamic situations was tested by challenging the model with glucose and acetate pulses added during the fed-batch part of the cultures. In the presence of a glucose feed, E. coli utilized acetate 3 times faster than in the absence of glucose. The cells showed no significant difference in maximum specific uptake rate of endogenous acetate produced by glucose overflow and exogenous acetate added to the culture, the value being 0.12-0.18 g g-1 h-1 during the entire fed-batch culture period. Acetate inhibited the specific growth rate according to a noncompetitive model, with the inhibition constant (ki) being 9 g of acetate/L. This was due to the reduced rate of glucose uptake rather than the reduced yield of biomass.     

Mutation in the thyroid hormone receptor beta gene (A317T) in a Thai subject with resistance to thyroid hormone

Analysis of the thyroid hormone receptor beta (TRbeta) gene of a Thai female with the syndrome of resistance to thyroid hormone (RTH) revealed a missense mutation at codon 317, changing the guanine in nucleotide 1234 to an adenine that results in the replacement of the normal alanine (GCT) with a threonine (ACT). The proposita was heterozygous, and this mutation was not present in her parents and her sister, compatible with a neomutation. This is the first report of TRbeta gene mutation causing RTH in an individual of Thai origin.     

Circulating thyroid hormone concentrations and placental thyroid hormone receptor expression in normal human pregnancy and pregnancy complicated by intrauterine growth restriction (IUGR)

Thyroid hormones are critical to growth and development of the human fetus. Abnormal placental development, a major cause of intrauterine growth restriction (IUGR), is associated with a high perinatal mortality and morbidity. Thyroid status has been postulated to play a role in the pathogenesis of such morbidity. In the present study, we have investigated fetal thyroid function and placental expression of thyroid hormone receptor (TR) alpha and beta variants during normal human pregnancy and in pregnancy associated with IUGR. Measurement of free thyroid hormones and TSH concentrations revealed significant rises in free T4 and free T3 between the second and third trimesters of normal pregnancy. Serum concentrations of free T4 and free T3 were lower in fetuses affected by IUGR, although serum TSH levels were not significantly different. Immunocytochemistry demonstrated the presence of TR alpha1, alpha2, and beta1 proteins within the nuclei of trophoblast and stromal placental cells. Immunostaining for these TR variants increased with increasing gestation in normal placenta. Comparison of IUGR placental samples with normal samples revealed greater immunostaining for TR alpha1, alpha2, and beta1 variants in IUGR. Examination of pretranslational expression of TR alpha1, alpha2, beta1, and beta2 variants by semiquantitative RT-PCR revealed increasing expression of TR alpha1, alpha2, and beta2 messenger RNAs with increasing gestation in normal pregnancy, which "mirrored" post-translational expression. However, and in contrast, there were no significant differences in expression of TR messenger RNAs in normal and IUGR placenta. The present findings of reduction in serum free thyroid hormones and increased expression of TR alpha and beta proteins in association with IUGR highlight the potential importance of thyroid status in influencing long-term fetal outcome in this condition.     

Regulation of gonadotropin-releasing hormone (GnRH) receptor gene expression in sheep: interaction of GnRH and estradiol

GnRH and estradiol are important regulators of GnRH receptors. When delivered to the anterior pituitary gland continuously, GnRH decreases numbers of GnRH receptors on gonadotropes. Treatment with estradiol consistently increases numbers of GnRH receptors. Because estradiol acts via intracellular receptors while GnRH exerts its effects through a membrane receptor, it is likely that these hormones influence GnRH receptor expression via different mechanisms. In this experiment, we tested two hypotheses: 1) continuous infusion of GnRH will decrease expression of the GnRH receptor gene; and 2) estradiol will override the negative effects of continuous infusion of GnRH on GnRH receptor expression. Ovariectomized ewes were administered either GnRH (10 microg/h, n = 10) or saline (n = 10) continuously for 136 h. At 124 h, 5 ewes in each group were administered estradiol (25 microg i.m.) and anterior pituitary glands were collected 12 h later. Treatment with GnRH caused an abrupt increase in circulating concentrations of LH, and the maximal mean concentration was observed 4 h after the start of GnRH infusion. Following this increase, concentrations of LH in GnRH-treated ewes declined and were similar to those in saline-treated ewes from 8 h to 124 h. After injection of estradiol at 124 h, circulating concentrations of LH increased in both GnRH- and saline-treated ewes. However, this response occurred within 6 h in ewes treated with GnRH compared with 9 h in ewes treated with saline (P < 0.05). Compared with saline-treated controls, treatment with GnRH decreased mean steady-state amount of GnRH receptor messenger RNA (mRNA) (P < 0.01) and concentration of GnRH receptors (P < 0.05). Treatment with estradiol caused an increase in concentrations of GnRH receptor mRNA (P < 0.05) and GnRH receptors (P < 0.01). Amounts of GnRH receptor mRNA and numbers of GnRH receptors in ewes treated with both GnRH and estradiol were not different from those in the control group but were higher (P < 0.002) relative to ewes treated with GnRH alone. Treatment with GnRH and estradiol also influenced the expression of genes encoding the LHbeta and FSHbeta subunits. Compared with saline-treated controls, treatment with GnRH reduced steady-state amounts of mRNA encoding LHbeta subunit (P < 0.005) and FSHbeta subunit (P < 0.05). Treatment with estradiol caused a decrease in concentrations of FSHbeta subunit mRNA (P < 0.01) but did not affect amounts of LHbeta subunit mRNA. The combined treatment of GnRH and estradiol reduced concentrations of mRNA encoding LHbeta subunit (P < 0.01) and FSHbeta subunit (P < 0.005). From these data we conclude that 1) reduced numbers of GnRH receptors during continuous infusion of GnRH are mediated in part by decreased expression of the GnRH receptor gene; and 2) estradiol is able to override the negative effect of GnRH by stimulating an increase in GnRH receptor gene expression and GnRH receptor concentrations. Therefore, although the gonadotrope becomes refractory to GnRH during homologous desensitization, this desensitization does not affect the cell's ability to respond to estradiol.     

Rat somatostatin sst2(a) and sst2(b) receptor isoforms mediate opposite effects on cell proliferation

We have investigated the actions of somatostatin (SRIF) and angiopeptin on cell proliferation of CHO-K1 cells expressing the recently cloned rat sst2(b) receptor (CHOsst2(b)) and compared these to their effects in cells expressing the sst2(a) receptor (CHOsst2(a)). In contrast to the sst2(a) receptor, the sst2(b) receptor did not mediate inhibition of bFGF (10 ng ml(-1))-stimulated re-growth and cell proliferation. Rather, SRIF (0.1-1000 nM) and angiopeptin (0.1-1000 nM) stimulated basal re-growth and proliferation of CHOsst2(b) cells in a concentration-dependent manner (estimated pEC50 values of 7.8 and 7.9, respectively). The opposite effects of SRIF on cell proliferation mediated through the two sst2 receptor isoforms were both abolished by 18 h pre-treatment with pertussis toxin. The proliferative effect via the sst2(b) receptor was also abolished by the tyrosine kinase inhibitor, genistein. In conclusion, the present study shows that the rat sst2(a) and sst2(b) receptor splice variants mediate opposite effects on cell proliferation.     

Distinct domains of M-T2, the myxoma virus tumor necrosis factor (TNF) receptor homolog, mediate extracellular TNF binding and intracellular apoptosis inhibition

The myxoma virus tumor necrosis factor (TNF) receptor homolog, M-T2, is expressed both as a secreted glycoprotein that inhibits the cytolytic activity of rabbit TNF-alpha and as an endoglycosidase H-sensitive intracellular species that prevents myxoma virus-infected CD4+ T lymphocytes from undergoing apoptosis. To compare the domains of M-T2 mediating extracellular TNF inhibition and intracellular apoptosis inhibition, recombinant myxoma viruses expressing nested C-terminal truncations of M-T2 protein were constructed. One mutant, deltaL113, containing intact copies of only two cysteine-rich domains, was not secreted and was incapable of binding rabbit TNF-alpha yet retained full ability to inhibit virus-induced apoptosis of RL-5 cells. Thus, the minimal domain of intracellular M-T2 protein required to inhibit apoptosis is distinct from that required by the extracellular M-T2 for functional TNF-alpha binding and inhibition. This is the first report of a virus-encoded immunomodular protein with two distinct antiimmune properties.     

Sites of volatile anesthetic action on kainate (Glutamate receptor 6) receptors

Molecular mechanisms of anesthetic action on neurotransmitter receptors are poorly understood. The major excitatory neurotransmitter in the central nervous system is glutamate, and recent studies found that volatile anesthetics inhibit the function of the alpha-amino-3-hydroxyisoxazolepropionic acid subtype of glutamate receptors (e.g. glutamate receptor 3 (GluR3)), but enhance kainate (GluR6) receptor function. We used this dissimilar pharmacology to identify sites of anesthetic action on the kainate GluR6 receptor by constructing chimeric GluR3/GluR6 receptors. Results with chimeric receptors implicated a transmembrane region (TM4) of GluR6 in the action of halothane. Site-directed mutagenesis subsequently showed that a specific amino acid, glycine 819 in TM4, is important for enhancement of receptor function by halothane (0. 2-2 mM). Mutations of Gly-819 also markedly decreased the response to isoflurane (0.2-2 mM), enflurane (0.2-2 mM), and 1-chloro-1,2, 2-trifluorocyclobutane (0.2-2 mM). The nonanesthetics 1, 2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane had no effect on the functions of either wild-type GluR6 or receptors mutated at Gly-819. Ethanol and pentobarbital inhibited the function of both wild-type and mutant receptors. These results suggest that a specific amino acid, Gly-819, is critical for the action of volatile anesthetics, but not of ethanol or pentobarbital, on the GluR6 receptor.     

Demonstration of complimentarity between monoclonal antibodies (MAbs) to human chorionic gonadotropin (hCG) and polyclonal antibodies to luteinizing hormone/hCG receptor (LH-R) and their use in better understanding hormone-receptor interaction

The concurrent influence of multiple neurotransmitter systems in mediating cocaine-induced convulsions is predicted by the results of previous receptor binding studies. The present results demonstrate that pharmacological manipulations of these predicted neurotransmitter systems alters the occurrence of cocaine-induced convulsions. The 5-HT reuptake inhibitor fluoxetine enhanced the occurrence and severity of convulsions produced by 100 mg/kg (-) cocaine, while the 5-HT2 receptor antagonists cinanserin, ketanserin and pirenperone antagonized cocaine-induced convulsions in a dose-dependent manner. Further, the M1 receptor antagonist pirenzepine antagonized cocaine-induced convulsions, but atropine did not. In addition, both the (+) and (-) stereoisomers of the sigma ligand SKF 10047 significantly attenuated cocaine-induced convulsions. (+)SKF 10047 was more potent than (-)SKF 10047 in this effect, suggesting a stereoselective effect at sigma receptor sites. In constrast, DA and NE neurotransmission do not appear to modulate the proconvulsant effects of cocaine in a specific, dose-dependent manner. Thus, of the CNS binding sites with which cocaine is known to interact, the results are consistent with the conclusion that 5-HT transporters and 5-HT2 receptor sites appear to be direct and primary sites related to cocaine-induced convulsions, while M1 and sigma binding sites appear to play important but secondary and modulatory roles in this response.     

Evidence for an inhibitory effect of physiological levels of insulin on the growth hormone (GH) response to GH-releasing hormone in healthy subjects

It has been previously reported that in healthy subjects, the acute reduction of free fatty acids (FFA) levels by acipimox enhances the GH response to GHRH. In the present study, the GH response to GHRH was evaluated during acute blockade of lipolysis obtained either by acipimox or by insulin at different infusion rates. Six healthy subjects (four men and two women, 25.8 +/- 1.9 yrs old, mean +/- SE) underwent three GHRH tests (50 micrograms iv, at 1300 h) during: 1) iv 0.9% NaCl infusion (1200-1500 h) after oral acipimox administration (250 mg) at 0700 h and at 1100 h; 2) 0.1 mU.kg-1.min-1 euglycemic insulin clamp (1200-1500 h) after oral acipimox administration (250 mg at 0700 h and at 1100 h); 3) 0.4 mU.kg-1.min-1 euglycemic insulin clamp (1200-1500 h) after oral placebo administration (at 0700 and 1100 h). Serum insulin (immunoreactive insulin) levels were significantly different in the three tests (12 +/- 2, 100 +/- 10, 194 +/- 19 pmol/L, P < 0.06), plasma FFA were low and similar (0.04 +/- 0.003, 0.02 +/- 0.005, 0.02 +/- 0.003, not significant), and the GH response to GHRH was progressively lower (4871 +/- 1286, 2414 +/- 626, 1076 +/- 207 micrograms/L 120 min), although only test 3 was significantly different from test 1 (P < 0.05). Pooling the three tests together, a significant negative regression was observed between mean serum immunoreactive insulin levels and the GH response to GHRH (r = -0.629, P < 0.01). Our results indicate that in healthy subjects, acipimox and hyperinsulinemia produce a similar decrease in FFA levels and that at similar low FFA, the GH response to GHRH is lower during insulin infusion than after acipimox. These data suggest that insulin exerts a negative effect on GH release. Because the insulin levels able to reduce the GH response to GHRH are commonly observed during the day, for instance during the postprandial period, we conclude that the insulin negative effect on GH release may have physiological relevance.     

Temperature effect on the action of corticotropin-releasing hormone (CRH) on secretion of immunoreactive beta-endorphin (IR beta EP) in TM3 and AtT-20 cell lines

Testicular function is sensitive to chemical and thermal stresses. To investigate the effects of small temperature changes on CRH-stimulated beta EP release, we employed TM3 cells, a mouse prepubertal Leydig cell line that secretes ir beta EP. To monitor beta EP secretion from these cells we used the reverse hemolytic plaque assay. After 3.5 hr incubation of cells with hormone, the EC50 of the CRH dose-response curve at 34 degrees C and 37 degrees C were 0.1 nM and 1 nM, respectively. For comparison, we also investigated the effect of temperature on CRH-stimulated beta EP release from a non-testicular cell line, AtT-20, a mouse anterior pituitary cell line. Using radioimmunoassay to measure ir beta EP levels in the media of AtT-20 cells, the EC50s for the CRH dose-response curve at 34 degrees C and 37 degrees C were 0.2 nM and 2 nM, respectively, at 1 h. After 3.5 h this temperature dependent difference in EC50 was still observed. These results suggest that CRH receptors or post-receptor actions in Leydig cells and anterior pituitary corticotropes are sensitive to small temperature changes.