Thyroid hormone inhibits thyrotropin gene expression via a position-independent negative L-triiodothyronine-responsive element.

We have previously identified a 57-bp DNA fragment encompassing exon 1 of the beta-subunit gene of rat thyrotropin (rTSH beta) that mediates the negative response to L-triiodothyronine (T3). To determine the specific motif that confers this negative regulation, we tested the T3 sensitivity of various segments of this 57-bp gene fragment in transiently transfected pituitary tumor cells, GH3. The suppressive effects were mediated by a 17-bp motif (+11/+27, CGCCAGTGCAAAGTAAG) located at the 3' end of exon 1. The inhibitory effects mediated by the sequence were evident when a single copy of the motif was inserted 125 bp upstream or 11 bp downstream of the transcriptional start site. These findings indicate that the suppressive effect of T3 is an intrinsic property of the T3-responsive element and not dependent on position relative to the promoter. The T3 receptor (T3R) extracted from GH3 cells or expressed in vitro bound specifically to this sequence. Specific mutations introduced into this region result in a selective loss of nuclear protein binding and a corresponding loss of T3 sensitivity. Additional studies showed that the 17-bp sequence was not responsive to T3 in COS cells which lack endogenous T3R. Cotransfection of a T3R restored the T3 responsiveness of the TSH beta motif. In summary, we have identified an element in the rTSH beta gene that mediates negative regulation by T3 and binds to the T3R.

Thyrotropin-releasing hormone regulation of thyrotropin beta-subunit gene expression involves intracellular calcium and protein kinase C.

Our previous studies demonstrated TRH stimulation of TSH beta gene expression in rat pituitary cell cultures and GH3 tumor cells in a transient expression assay. To begin to characterize the gene-proximal elements of the pathways involved in TRH stimulation of TSH beta gene transcription, we examined the effects of factors that increase intracellular calcium concentration, [Ca2+]i, or activate protein kinase C on TSH beta promoter activity in transfected GH3 cells. TPA, a tumor-promoting phorbol ester, stimulated a dose-dependent increase in TSH beta promoter activity at 8 h similar to TRH (2-3-fold). TPA did stimulate protein kinase C activation without [Ca2+] mobilization. The calcium ionophore ionomycin increased cytoplasmic free [Ca2+] by stimulating both calcium influx and release from internal stores without affecting protein kinase C. Ionomycin also stimulated a dose-dependent increase (2-fold) in TSH beta promoter activity at 8 h. However, the voltage-dependent Ca2+ channel agonist Bay K 8644, which increased influx of extracellular calcium, had little or no effect on TSH beta gene expression until 48 h (5-fold). Similar effects on prolactin/mRNA levels were observed in these cells. Effects of these factors were not additive, suggesting a common pathway(s) to stimulate gene expression. Inhibition of intracellular calcium mobilization by treatment with 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) inhibited ionomycin effects on gene expression without affecting phorbol ester activity, and, conversely, inhibition of protein kinase C activity by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) or TPA desensitization blocked TPA effects without affecting ionomycin activity.(ABSTRACT TRUNCATED AT 250 WORDS)