Thyroid hormone receptor binds to a site in the rat growth hormone promoter required for induction by thyroid hormone.

Abstract

Transcription of the rat growth hormone (rGH) gene in pituitary cells is increased by addition of thyroid hormone (T3). This induction is dependent on the presence of specific sequences just upstream of the rGH promoter. We have partially purified T3 receptor from rat liver and examined its interaction with these rGH sequences. We show here that T3 receptor binds specifically to a site just upstream of the basal rGH promoter. This binding site includes two copies of a 7-base-pair direct repeat, the centers of which are separated by 10 base pairs. 7.85/2 mM EDTA/5 mM 2-mercaptoethanol/10% glycerol).Fractions of 8 ml were collected. Unoccupied T3 receptor was located by incubating 0.2-ml aliquots of each fraction with 5 x 104 cpm of [1251]T3 with and without 100 nM nonradioactive T3 for 1 hr at room temperature. Bound and free hormone were separated on 1-ml bed volume Sephadex G-25 columns at 40C. Fractions containing receptor were pooled, and a Scatcharc analysis of binding of T3 was performed as described by Samuels et al. (10), except that incubation was at 40C for 18 hr.The unoccupied receptor pool was fully occupied with hormone by incubating overnight at 40C with 3 nM T3 containing 5 x 104 cpm of [1251]T3 per ml. After dialysis against buffer A at 4TC, the material was rerun on a Mono Q column and the fractions containing T3-occupied receptor were identified by the peak of radioactivity. Analytical experiments showed that [1251]T3 binding to this peak was saturable with nonradioactive T3. T3 itself does not elute from the column with the NaCl gradient used. As reported for DEAE-Sephadex chromatography (9), occupied T3 receptor elutes ahead of unoccupied receptor. Under the conditions described, near-baseline separation of the two species was achieved. During incubation with 3 nM T3, >90% of the unoccupied receptor from the first column bound T3 and was shifted to the earlier eluting position during the second chromatography. The occupied receptor pool eluting from the second Mono Q column was concentrated 50-fold by ultrafiltration (Amicon) and stored in aliquots in liquid nitrogen.As a control for the specificity of the receptor shift, in other experiments the unoccupied receptor pool was incubated with 3 nM reverse T3 together with tracer T3. Due to its i103 lower binding affinity, at this concentration the T3 receptor does not bind significant amounts of reverse T3 but does bind the [1251]T3. The eluates of the second chromatography of this preparation were assayed for T3 receptor activity and no decrease in unoccupied receptor was observed. The fractions containing the tracer ['25I]T3 receptor complexes from this column were pooled and concentrated as described above. Protein concentrations were determined according to Bradford (11).T3 Receptor-DNA Binding. Binding reactions of T3 receptor to DNA were performed in a final volume of 20 ,ul containing 10 mM Hepes (pH 7.8), 40 mM KCI, 1 mM dithiothreitol, 2 mM MgCl2, 5% glycerol, 2-5 ,g of poly-(dIdC), 10 fmol of 32P-labeled DNA fragment, and 20-120...

Relationship between Thyroid Functions and Urinary Growth Hormone Secretion in Patients with Hyper- and Hypothyroidism.

“…The observation that hyperthyroid patients exhibited an increase in urinary GH is consistent with recent studies demonstrating that spontaneous GH secretion is increased in thyrotoxic men [9]. Considerable evidence indicates that T3 stimulates the GH gene transcription and subsequently increases GH secretion [1][2][3]. Although it is also established that pulsatile GH secretion is mediated by hypothalamic GRH and somatostatin secretion [15][16][17], the stimulatory action of thyroid hormone on GH secretion occurs at the pituitary level rather than at the hypothalamic level [18,19].…”

Insulin-like growth factor I reduces thyroid hormone receptors in the rat liver. Evidence for a feed-back loop regulating the peripheral thyroid hormone action

“…T 3 is required for normal GH gene expression and thyroid hormone responsive elements have been identified in the GH gene promoter (Koenig et al 1987, DeGroot et al 1988, Williams & Brent 1995. Pituitary GH secretion and GH mRNA level are reduced in hypothyroid rats and restored to normal after treatment with T 3 (Hervas et al 1975, Nyborg et al 1985.…”

Novel aspects of T3 actions on GH and TSH synthesis and secretion: physiological implications

“…The increase in the Gh transcription rate triggered by T 3 requires the interaction of the THR/T 3 complex with sequences located close to the transcription start site (TSS) of Gh and the presence of specific transcriptional factors expressed in somatotrophs, since the insertion of Gh promoter in fibroblast or kidney cells did not evoke Gh transcription in response to T 3 (Larsen et al 1986, Koenig et al 1987.…”

“…In the anterior pituitary gland, THs downregulate the expression of genes that encode CGA (glycoprotein hormones alpha chain) and TSHB subunits of thyroidstimulating hormone (TSH) (Franklyn et al 1988) and upregulate Gh transcription (Crew & Spindler 1986, Koenig et al 1987, Lavin et al 1988. The THRB2 isoform participates in the positive and negative regulations of Gh and Tshb expressions, respectively (Abel et al 1999, Barra et al 2004.…”