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G A Brent

Publications and source records attributed to G A Brent.

At least 19 recordsLinked to original sources
PubMed1992

Differential capacity of wild type promoter elements for binding and trans-activation by retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are structurally similar and can bind as homodimers or T3R-RAR heterodimers to a single synthetic DNA response element. The interaction of these two types of receptors with wild type elements, however, has not been systematically investigated. Promoter elements from genes regulated by retinoic acid (RA) or thyroid hormone (T3) were tested for response to T3 and RA in transient transfections in both JEG and COS cells. The elements were classified as primarily responsive to RA or to T3 or responsive to both ligands. Binding of highly purified RAR alpha and T3R alpha to the various elements was assessed using the gel shift assay. Those elements predominantly responsive to one ligand showed preferential binding to the appropriate receptor. A series of point mutations were introduced into the rat GH T3 response element to further define sequence requirements for response to both RA and T3. Down-mutations in any of the three hexamers (previously demonstrated to be required for full response to T3 and full binding of T3R) also decreased RA induction and RAR binding. However, only one of two sets of up-mutations for T3 response also increased RA induction, demonstrating differences in hexamer preference between RAR and T3R. Variation in spacing of the three hexamers did not influence RA vs. T3 induction or RAR vs. T3R binding according to the predictions of a simple hexamer spacing model. There was a strong correlation between the extent of T3R dimer binding and strength of T3 induction for a subset of elements studied in JEG cells (r = 0.97, P < 0.01) and a weaker but significant correlation in COS cells (r = 0.65, P < 0.05)). In contrast, RAR dimer binding by the wild type elements did not quantitatively correlate with RA induction in either JEG (r = 0.13, P > 0.05) or COS cells (r = 0.21, P > 0.05). These results suggests that RAR interacts with a heterodimer partner(s) which influences binding site specificity, whereas T3R heterodimer partner(s) is less likely to alter binding site recognition. The observed difference in COS and JEG cells as well as the weak T3R binding-function relationship of the malic enzyme element, however, suggest that the influence of T3R heterodimer partner(s) on binding site specificity is likely to vary with cell type and the specific element tested.

Animals↗
PubMed1992

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.

Animals↗
PubMed1991

Oligomeric binding of T3 receptor is required for maximal T3 response.

Receptors in the thyroid-steroid hormone superfamily bind preferentially as dimers to palindromic response elements containing two hexameric half-sites. The 23-base pair rat growth hormone (rGH) T3 response element (T3RE), however, contains three hexameric binding domains, all of which are required for maximal T3 response. We examined the binding of purified T3 receptor alpha (T3R alpha), overexpressed in Escherichia coli, to wild-type and up and down mutations of the rGH T3RE to evaluate whether transcriptional potency correlates with changes in T3R binding. T3R binds to the rGH T3RE as a monomer, dimer, or higher order oligomer. Cooperative T3R dimer binding was demonstrated to two hexameric domains of the rGH T3RE arranged as either direct or inverted repeats. Decreased binding was seen with point mutations in each domain as well as with mutations which altered hexamer orientation and spacing within the site. These results demonstrate that all three hexamers of the rGH T3RE are involved in binding T3R. Occupancy of all three hexamers by T3R in the gel shift assay was observed with functional up mutations of the T3RE, increasing receptor concentration or addition of nuclear extract. The transcriptional response potencies of T3RE up or down mutants in a transient transfection assay correlated closely with T3R binding. These results confirm our earlier hypothesis that all three hexamers of the rGH T3RE bind T3R in a novel binding arrangement and provide a model for the interaction of T3R and other nuclear proteins with the DNA sequences of thyroid hormone-regulated genes.

Animals↗
PubMed1991

Effects of varying the position of thyroid hormone response elements within the rat growth hormone promoter: implications for positive and negative regulation by 3,5,3'-triiodothyronine.

The thyroid hormone response element (T3RE) of the rat GH (rGH) promoter is located at -188 to -165 relative to the mRNA start site (TSS). Similar sites have been identified in other genes regulated by T3. We have investigated some of these T3REs in positions within the rGH promoter to assess the relative influences of DNA-binding site and position on positive and negative regulation by T3. Synthetic oligonucleotides were used with sequences from the rGH T3RE and proposed negative T3REs (nT3RE) from the rat and human alpha-subunit and rat beta TSH genes. The nT3REs were placed in the background of the wild-type rGH promoter in two positions, at -55 and down-stream of the TSS, with up- and down-mutations of the rGH T3RE. Rat GH T3RE elements were placed 700 basepairs up-stream of a basal rGH promoter and some also at the -55 and TSS positions. Constructions were tested in a transient transfection assay in rat pituitary tumor cells. Two copies of the rGHPAL (palindromic T3RE) placed 700 basepairs up-stream of the rGH promoter conferred 10-fold T3 induction. In the -55 position, the rGHPAL increased T3 induction compared to that in controls, whereas a fragment from the rat and human alpha-subunit gene in the same position reduced induction. Negative T3REs from rat beta TSH and human alpha-subunit reduced T3 induction 50% when placed at the TSS position of a rGH promoter containing an up-mutant T3RE. The T3REPAL placed at the same site increased T3 induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Thyroid hormone: half-sites and insights. Molecular Approaches to the Study of Thyroid hormone Action: a Keystone Symposium, Tamarron, CO, USA, March 8-14, 1991.

As is evident from the range of topics addressed in this review, T3 has important and interesting effects on virtually every biological system in higher organisms. Many of these effects are beginning to be understood at the molecular level, while others are not far behind. It is striking that the diversity of biological activities of T3 is reflected in a broad spectrum of molecular mechanisms. Even within the relatively circumscribed paradigm of receptor activation by hormone binding, a surprisingly large number of possible interactions and complications has been discovered. This richness of alternative interactions leads directly to complex and unanticipated regulatory possibilities. A major strength of this meeting was that it encompassed the broad range of T3 effects and thereby was able to provide something new for every attendee. From this perspective, it is easy to predict that study of T3 will continue to provide new and surprising insights into major biological questions for years to come.

Animals↗
PubMed1990

Increased need for thyroxine during pregnancy in women with primary hypothyroidism.

BACKGROUND AND METHODS: Women with hypothyroidism have been thought not to require an increase in thyroxine replacement during pregnancy. To evaluate the effects of pregnancy on thyroxine requirements, we retrospectively reviewed the thyroid function of 12 women receiving treatment for primary hypothyroidism before, during, and after pregnancy. RESULTS: In all patients, the serum thyrotropin level increased during pregnancy. The mean (+/- SE) serum free-thyroxine index decreased from 111.0 +/- 5.8 before pregnancy to 86.5 +/- 5.2 during pregnancy (normal, 64 to 142; P less than 0.05), and the mean serum thyrotropin level increased from 2.0 +/- 0.5 mU per liter before pregnancy to 13.5 +/- 3.3 mU per liter during pregnancy (normal, 0.5 to 5.0 mU per liter; P less than 0.01). Because of high thyrotropin levels, the thyroxine dose was increased in 9 of the 12 patients. Among the three patients who did not require an increased thyroxine dose were two with low serum thyrotropin levels before pregnancy, suggesting excessive replacement at that time. The mean thyroxine dose before pregnancy was 0.102 +/- 0.009 mg per day; it was increased to 0.148 +/- 0.015 mg per day during pregnancy (P less than 0.01). The mean postpartum serum free-thyroxine index was 136.6 +/- 11.4 (P less than 0.05 as compared with values before and during pregnancy), and the mean postpartum serum thyrotropin level was 1.4 +/- 0.4 mU per liter (P less than 0.01 as compared with levels during pregnancy), demonstrating a decrease in the thyroxine requirement. The mean postpartum thyroxine dose was decreased to 0.117 +/- 0.011 mg per day (P less than 0.01 as compared with the dose during pregnancy). CONCLUSIONS: Our results indicate that the need for thyroxine increases in many women with primary hypothyroidism when they are pregnant, as reflected by an increase in serum thyrotropin concentrations. Although the effects of this modest level of hypothyroidism are not known, we think it prudent to monitor thyroid function throughout gestation and after delivery and to adjust the thyroxine dose to maintain a normal serum thyrotropin level.

Female↗
PubMed1990

Direct repeats.

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Animals↗
PubMed1989

Inhibition of thyroid hormone action by a non-hormone binding c-erbA protein generated by alternative mRNA splicing.

Thyroid hormone (T3) binds to a nuclear receptor protein which regulates gene expression by binding to specific DNA sequences near hormone-responsive genes. Proteins encoded by two cellular proto-oncogenes, c-erbA alpha and beta, bind T3 and can act as functional T3 receptors. In rats, alternative splicing of the alpha-gene transcript generates at least two distinct protein products, termed r-erbA alpha 1 and r-erbA alpha 2. Although these proteins bind to the same DNA sequence, r-erbA alpha 2 does not bind T3. We show here that expression of r-erbA alpha 2 inhibits the T3-dependent inductive effect of either r-erbA beta or r-erbA alpha 1 on expression of a T3-responsive test gene. Alternative splicing of the erbA alpha transcript thus generates products with opposing biological activities, suggesting a novel mechanism for the modulation of hormonal responsiveness.

Animals↗
PubMed1989

Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected beta type thyroid hormone receptor.

We have extensively characterized the sequences of the rat growth hormone (rGH) promoter required for induction by T3 (thyroid hormone, 3,5,3'-L-triiodothyronine) in a transient transfection system. Oligonucleotides containing portions of the rGH promoter sequence with various deletions and point mutations were placed upstream of the first 137 base pairs of the rGH promoter or the heterologous herpes virus thymidine kinase promoter in chloramphenicol acetyltransferase expression vectors. The rGH137 and thymidine kinase promoters show no or minimal response to T3 in the basal state. The constructs were tested in GH4C1 rat pituitary cells and COS cells (functionally deficient in thyroid hormone receptor) with and without a co-transfected plasmid expressing a beta type c-erbA gene coding for a functional T3 receptor. Oligonucleotides containing the T3 receptor binding site confer hormone-dependent induction in a manner that is independent of either orientation or variation in position on the helix relative to the promoter. Point mutations in the sequence -189 to -173 result in loss of T3 induction, and bases between -173 and -167 were also required for a full T3 response. The minimal length to confer T3 induction to the rGH promoter was 23 base pairs (-190 to -167). Point mutations creating a perfect duplication of 7 base pairs within the receptor binding site conferred 12-fold T3 response to the rGH137 promoter, 3-fold greater than the wild type rGH237 construct. T3 inductibility was also transferred to the thymidine kinase promoter by an oligonucleotide containing the sequence -200 to -157, demonstrating that cell type specific elements located 3' to 157 of the rGH promoter are not required for thyroid hormone responsiveness.

Animals↗
PubMed1989

Reversible alteration of red cell lithium-sodium countertransport in patients with thyroid disease.

Thyroid hormones may alter red blood cell (RBC) sodium content and transport. The functional importance of lithium-sodium (Li-Na) countertransport in regulating sodium (Na) transport in vascular smooth muscle and kidney by Na-H countertransport and the potential effect of thyroid hormone on these processes led us to study Li-Na countertransport and other sodium transporters in RBCs of patients with thyroid dysfunction. Patients with untreated hypothyroidism (10) and hyperthyroidism (10) were studied, along with normal subjects (10). The mean value for Li-Na countertransport was significantly higher in the hypothyroid group [0.46 +/- 0.08 (+/- SE) mmol/L cell.h; P less than 0.05] and lower in the hyperthyroid group (0.15 +/- 0.04 mmol/L cell.h; P less than 0.05) compared to that in the normal subjects (0.25 +/- 0.03 mmol/L cell.h). When all groups were combined, significant negative correlations were found between Li-Na countertransport and serum T4 (r = -0.48; P less than 0.01), free T4 index (r = -0.42; P less than 0.05), and serum T3 (r = -0.38; P less than 0.05). Li-Na countertransport was positively correlated with serum triglyceride (r = 0.57; P less than 0.01), but not with serum cholesterol levels (r = 0.28; P = NS). The values became normal in subsets of the hypothyroid (n = 5) and hyperthyroid groups (n = 5) during treatment. We found a bidirectional effect of thyroid status on RBC Li-Na countertransport, which was reversible when serum thyroid hormone levels became normal. Changes in Li-Na countertransport, a pathway of Na-H exchange, may influence renal sodium handling and vascular tone in patients with thyroid disease and contribute to abnormalities such as hypertension that occur in patients with hypothyroidism.

Biological Transport↗
PubMed1989

Adrenocorticotropin stimulation of aldosterone: prolonged continuous versus pulsatile infusion.

Continuous iv administration of ACTH leads to a sustained stimulation of cortisol but a transient stimulation of aldosterone followed by a decline to prestimulation levels by 72 h. Since CRH and ACTH are released in a pulsatile pattern in man, this study sought to investigate whether pulsatile administration of alpha-cosyntropin-(1-24) would lead to the maintenance of aldosterone stimulation over time. Eight normal male subjects on a 10-meq sodium, 100-meq potassium diet received both a continuous and a pulsatile (0.33 U ACTH/pulse over 15 min, pulsed every 2 h) infusion of cosyntropin (4 U/24 h) for 48 h (n = 4) or 72 h (n = 4). Aldosterone and cortisol were sampled every 6 h, and PRA and angiotensin-II every 24 h. Continuous infusion led to a stimulation of aldosterone followed by a progressive decline to preinfusion levels by 72 h [preinfusion 29 +/- 5 ng/dL (810 +/- 139 pmol/L); 72 h, 38 +/- 10 ng/dL (1054 +/- 277 pmol/L); P = 0.40]. Pulsatile infusion led to a stimulation of aldosterone which was maintained up to 72 h [preinfusion 33 +/- 7 ng/dL (915 +/- 194 pmol/L); 72 h, 85 +/- 13 ng/dL (2358 +/- 361 pmol/L); P less than 0.05]. Regression analysis of aldosterone (y) over time (x) from the peak level at 18 h for the continuous infusion showed a significant negative relation (r = 0.63; P = 0.001), indicating a progressive decline in aldosterone. However, for the pulsatile infusion, there was no relation (r = 0.02; P = 0.85), indicating maintenance of aldosterone levels. There were no significant differences in sodium, potassium, PRA, angiotensin-II, or cortisol between infusions to explain these differences in aldosterone levels. Therefore, pulsatile infusion of cosyntropin maintains aldosterone secretion over time.

Adrenocorticotropic Hormone↗
PubMed1989

Mutations of the rat growth hormone promoter which increase and decrease response to thyroid hormone define a consensus thyroid hormone response element.

We have previously identified sequences required for thyroid hormone (T3) induction of the rat GH (rGH) promoter, which lie in a region from -188 to -164 upstream of the mRNA start site. Within this region, Domains A, -189 to -184 and B, -179 to -174, are imperfect direct repeats, and domain C, -172 to -167, is a divergent inverted copy that matches the A domain at 4/6 positions. A series of synthetic mutant versions of this sequence were inserted upstream of a truncated rGH promoter, or as a replacement for wild-type sequences in a synthetic 237 base pair rGH promoter or upstream of the heterologous thymidine kinase promoter. Mutations changing the B domain to a perfect copy of the A domain significantly increased T3 induction (21.3-fold) relative to the wild type (3.6-fold). A single point mutation making the C domain a better match to the A domain also increased T3 induction to 16.2-fold. Combining this up-mutation with any of three down-mutations in the A, B, or C domains strongly decreased response, showing that all three domains contribute to the amplified T3 response. Binding affinity of the various mutant oligonucleotides was assessed using in vitro translated receptor and affinity paralleled the functional responses for most binding site mutations. Requirements for in vitro binding were, however, less rigorous than those for functional T3 induction. Based on these results, we propose a consensus T3 receptor binding half-site, AGGT(C/A)A, at least two copies of which are required for a T3 response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Thyroid hormone aporeceptor represses T3-inducible promoters and blocks activity of the retinoic acid receptor.

In the presence of its ligand, thyroid hormone receptor (T3R) binds specifically to DNA sequences near a number of genes and induces their expression. We show that in the absence of the hormone, a T3R binding site acts in cis to decrease expression from such genes. The endogenous T3 receptors in rat pituitary cell lines are sufficient to mediate this effect, as shown by comparisons of basal levels of expression directed by transiently transfected plasmids containing the rat growth hormone promoter with wild-type or point-mutated T3 response elements (T3RE). The magnitude of the negative effect is increased by increasing the strength of the T3RE or by raising intracellular levels of T3R by appropriate transfections. T3REs exert a similar negative effect on the herpes virus thymidine kinase (TK) promoter; this effect is dependent on expression of functional T3 aporeceptor (apoT3R). Analysis of a set of T3REs of increasing strength inserted upstream of the TK promoter showed a strong correlation between the level of induced expression in the presence of hormone and the level of repressed expression in the absence of hormone. These results show that, unlike other members of the nuclear hormone receptor family, T3R binds to specific DNA sequences in the absence of hormone and exerts a negative effect on expression of linked genes. The apparent affinity of apoT3R and hormone-bound T3R for a T3RE was assessed by using varying amounts of T3R expression vector in a transfection dose response assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor.

We have isolated a c-erbA cDNA clone from a GH3 cell library. The clone, denoted erb62, is 4.5 kilobases long and encodes a 461-amino acid beta-type c-erbA protein. This c-erbA protein binds 3,5,3'-triiodothyronine (T3) and T3 analogs with affinities similar to those of the authentic T3 receptor. By RNA gel blot analysis, erb62 hybridizes to a 6-kilobase RNA found in organs that express T3 receptors--e.g., heart, kidney, and brain. A COS-cell transient cotransfection system was used to show that erb62 encodes a biologically active T3 receptor. An oligonucleotide, corresponding to a portion of the rat growth hormone gene 5'-flanking region that contains a T3 response element, was inserted on the 5' side of the herpes simplex virus thymidine kinase promoter in a chloramphenicol acetyltransferase-expressing plasmid. Reporter gene expression directed by this hybrid promoter was T3 inducible only if this plasmid was cotransfected with an erb62-expressing plasmid.

Amino Acid Sequence↗
PubMed1988

Relationship between the concentration and rate of change of calcium and serum intact parathyroid hormone levels in normal humans.

We employed variable rates of infusion of EDTA or calcium gluconate to examine the relationships between the concentration and rate of change of serum total and plasma ionized calcium and serum immunoreactive intact PTH concentrations in normal subjects. Use of a sensitive immunoradiometric assay specific for PTH-(1-84) made it possible to determine the full range of PTH responses to perturbations in the extracellular calcium concentration. By progressively increasing the rate of administration of calcium gluconate or EDTA during rapid or slow infusions, linear rates of change in the serum calcium concentration were achieved in eight men. The slopes were 2-fold greater during the rapid infusions. Both the calcium infusions decreased serum PTH-(1-84) levels from a mean of 23.2 +/- 2.0 (+/- SE) to 6.4 +/- 1.0 and 5.6 +/- 1.0 ng/L for the rapid and slow infusions, respectively, with no obvious rate dependence. The rapid or slow EDTA infusions increased serum PTH levels to the same maximal extent (95.0 +/- 20.2 and 99.9 +/- 14.5 ng/L, respectively), also with no significant rate dependence. Thus, there was no effect of the rate of change of calcium on the PTH response, which was reflected in similar inverse sigmoidal relationships between PTH and serum total and plasma ionized calcium when the data for the slow and rapid infusions were pooled separately. Similar sigmoidal curves were found in normal women. These data suggest the feasibility of using calcium and EDTA infusions combined with an intact PTH assay to define the relationships between circulating levels of PTH-(1-84) and calcium in states of normal and deranged parathyroid physiology.

Adult↗
PubMed1988

Multihormonal regulation of the human, rat, and bovine growth hormone promoters: differential effects of 3',5'-cyclic adenosine monophosphate, thyroid hormone, and glucocorticoids.

We have analyzed the effects of a variety of hormones on activity of the rat GH (rGH), human GH, (hGH), and bovine GH (bGH) promoters. After transient transfection of rat pituitary tumor cells, all three promoters are induced by addition of 8-bromo-cAMP. Sequences required for the cAMP responsiveness of the hGH and rGH promoter lie within 183 base pairs of the mRNA start site. Although the rGH promoter is thyroid hormone (T3) responsive in this system, a construct containing 2.7 kilobases of the hGH promoter 5'-flanking sequences is not. Since we also found that the bGH promoter is T3 responsive in these cells, the hGH results are not likely to be due to a species specific factor required for induction in rat pituitary cells. The hGH promoter is weakly induced by dexamethasone whereas the rGH promoter does not respond to glucocorticoids. The hGH and rGH promoters are not responsive to TRH. These results illustrate the potential heterogeneity in hormonal responses of the same gene in different species.

8-Bromo Cyclic Adenosine Monophosphate↗