Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin beta-subunit gene promoter transfected into pituitary cells

In order to investigate the molecular mechanism(s) by which TRH regulates the biosynthesis of TSH, we are studying the effects of TRH on the expression of the TSH subunit genes (alpha and TSH beta). To study the structure-function relation of TRH stimulation of the activity of the single rat TSH beta gene, chimaeric plasmids were constructed. The 5'-flanking region of the rat TSH beta gene including exon 1 (5'-untranslated region) was inserted into a promoterless, modified pBR, chloramphenicol acetyltransferase (CAT) expression vector. After transfection, specific TSH beta promoter activity was evident in both TRH-responsive pituitary-derived GH3 and primary pituitary cell cultures. To determine potential regulation of TSH beta promoter-directed activity in these cells by TRH, cells were incubated with media containing TRH (10(-7) to 10(-11) M) for 1 to 48 h. TRH stimulated a 1.5- to 3-fold increase in TSH beta promoter activity. Concomitant with an increase in CAT activity was an anticipated increase in PRL synthesis in the GH3 cells in response to TRH. The TRH effect on the TSH beta gene was specific; no increase in CAT activity was detected for TKCAT (thymidine kinase of herpes simplex virus promoter), pBRCAT (no promoter), or TSH beta CAT (3'-5'-orientation). Similar results were obtained using primary pituitary cell cultures. Deletion mutation analysis indicated that TRH sensitivity was detected in a 1.1 kilobase, but not in a 0.38 kilobase TSH beta gene fragment suggesting that the TRH responsive element(s) resides at least in part within the 700 base pairs of the 5'-flanking sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)