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.     

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.     

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)     

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.     

Sequences required for cell-type specific thyroid hormone regulation of rat growth hormone promoter activity

We have located sequences within the rat growth hormone (rGH) promoter region which are required for pituitary cell-type specific responsiveness to T3 (thyroid hormone, 3,5,3'-L-triiodothyronine). Transient transfections with a series of plasmids containing as few as 202 nucleotides upstream of the start site of the rat growth hormone mRNA showed specific induction by T3 in rat pituitary cell lines. Both the magnitude and the kinetics of this response were similar to those of the endogenous rGH gene, showing a strong early induction followed by a decline in T3 effect. Deletion of an additional 19 base pairs (to -183 relative to the start site) eliminated this induction. Plasmids containing sequences up to -237 or -202 showed significant promoter activity but no T3 responsiveness in transfections of mouse fibroblasts or monkey kidney cells. The presence of high affinity nuclear T3 binding proteins was demonstrated in both cell types. These results show that sequences between -183 and -202 are required for pituitary cell specific T3 regulation of the rGH promoter. The lack of T3 responsiveness in non-pituitary cells suggests that such regulation may be mediated by factors present in pituitary cells and absent in other cells.     

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)     

Induction of growth hormone release by glycyrrhizae radix on rat

Induction of growth hormone (GH) by Glycyrrhizae Radix (GR), one of the most popular herbal medicine, and its major ingredients were studied in rat pituitary cells in vitro and in vivo assay. The MeOH extract and the n-hexane (HX) fraction of GR induced rat GH (rGH) release up to 1.89 times (0.34 +/- 0.04 nM) and 4.59 times (0.83 +/- 0.03 nM), compared to the basal level (p < 0.05). Among many ingredients isolated and purified from GR both glycyrrhetinic acid and glycyrrhizin induced significantly rGH release compared to the control (p < 0.05). After an intravenous injection of rat growth hormone releasing hormone (rGHRH) (10 microg/kg) as positive control, in SD rats, Tmax of plasma rGH level was 10 min, C(max) was 3.84 +/- 0.01 nM (n = 3), and enhanced plasma rGH level returned to the baseline in 90 min. Both AUC(0-90) (area under the curve) of plasma rGH level after HX fraction and that after rGHRH administration were increased significantly from the basal level, respectively (p < 0.01). In conclusions, HX fraction is the most active fraction of MeOH extract of GR in rGH induction.     

Thyroid hormone-responsive elements of the prolactin gene: evidence for both positive and negative regulation

To analyze the regulation of PRL gene expression by thyroid hormone (T3), fusion gene constructs containing various lengths of the rat PRL gene 5'-flanking sequence linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected into the GH3 cell line. Thyroid hormone had no effect on basal or cAMP-stimulated CAT expression in constructs containing more than 1.7 kilobasepairs of the 5'-sequence. However, deletion to 1.5 or 0.6 kilobasepairs resulted in an inhibition of both basal and cAMP-stimulated expression by T3. A construct containing the proximal enhancer region (positions -292 to -38 basepairs) linked to the herpes simplex thymidine kinase promoter (TK) and the CAT reporter gene also responded to T3 with inhibition of basal and cAMP-induced CAT expression. The distal enhancer region (positions -1714 to -1495) linked to thymidine kinase promoter CAT responded to T3 with a stimulation of CAT expression, and the response was additive with the stimulatory response to cAMP. Deletion analysis of the distal enhancer region revealed that the sequence between positions -1530 and -1565 was required for the stimulatory response to T3. The stimulatory response to T3 was additive with the response to estradiol, suggesting distinct elements, but deletion to position -1565 abolished the response to estradiol and permitted an inhibitory response to T3. Mutation of the estrogen response element prevents the response to estradiol, but only blunted the response to T3. Mutation of the sequence GGTCA at positions -1555 to -1551 resulted in an inhibitory response to T3, implicating this sequence in the stimulatory response.(ABSTRACT TRUNCATED AT 250 WORDS)